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Monitoring and control of drinking water quality Inventory and evaluation of monitoring technologies for key-parameters

Techneau October 2008

© 2008 TECHNEAU TECHNEAU is an Integrated Project Funded by the European Commission under the Sixth Framework Programme, Sustainable Development, Global Change and Ecosystems Thematic Priority Area (contractnumber 018320). All rights reserved. No part of this book may be reproduced, stored in a database or retrieval system, or published, in any form or in any way, electronically, mechanically, by print, photoprint, microfilm or any other means without prior written permission from the publisher

Techneau October 2008

Monitoring and control of drinking water quality Inventory and evaluation of monitoring technologies for key-parameters

This report is: PU = Public

Colofon

Title Monitoring and control of drinking water quality – Inventory and evaluation of monitoring technologies for key-parameters Author(s) Margreet Mons (Ed.), all WA3 partners Quality Assurance All WA 3 partners Deliverable number D 3.1.3

Monitoring and control of drinking water quality © TECHNEAU - 4 - October 2008

Contents

Contents 4

1 Introduction 9

2 Method 10

3 Microbiological parameters 14

3.1 E. coli and coliform bacteria 14 3.1.1 Monitoring technology nr 1: Lactose TTC Tergitol Method (ISO 9308-1) 14 3.1.2 Monitoring technology nr 2: Colilert®-18/Quanti-Tray (IDExx, UK National Standard

Method W 18) 16 3.1.3 Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553) 18 3.1.4 Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK National

Standard Method W 2) 20 3.1.5 Monitoring technology nr 5: Chromocult® Coliform Agar (Merck) 22

3.2 Intestinal enterococci 25 3.2.1 Monitoring technology nr 1: ISO method 7899-1 25 3.2.2 Monitoring technology nr 2: ISO method 7899-2 27

3.3 Clostridium perfringens 29 3.3.1 Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC -

membrane filtration and cultivation on m-CP agar 29 3.3.2 Monitoring technology nr. 2: Membrane filtration and cultivation on TSC agar,

subsequent confirmation tests (draft of ISO 6461 CD part 2) 31 3.3.3 Monitoring technology nr 3: Membrane filtration and cultivation on fluorogenic TSC

agar 34 3.3.4 Confirmation technology nr. 1: C. perfringens Detection System (Biotecon

Diagnostics) 36 3.3.5 Confirmation technology nr. 2: API 32 A (Biomerieux) 36

3.4 Heterotrophic Plate Counts 38 3.4.1 Monitoring technology nr 1: ISO 6222 38 3.4.2 Monitoring technology nr 2: Plating on R2A medium 40

3.5 Enteroviruses 43 3.5.1 Monitoring technology nr 1: Cell culture methods 43 3.5.2 Monitoring technology nr 2: RT-PCR 44

3.6 Giardia/Cryptosporidium 46 3.6.1 Monitoring technology nr 1: Method 1622 47 3.6.2 Monitoring technology nr 2: UK method 48 3.6.3 Monitoring Technology nr. 3: method 1623 48 3.6.4 Monitoring technology nr 4: ISO 15553 48 3.6.5 Monitoring technology nr 5: cross flow ultrafiltration 48

3.7 Thermotolerant Campylobacter species 52 3.7.1 Monitoring technology nr 1: Detection and enumeration of thermotolerant

Campylobacter species (ISO 17995:2005) 52

3.8 Legionella and Legionella pneumophila 55 3.8.1 Monitoring technology nr 1: ISO method 11731:1998 55 3.8.2 Monitoring technology nr 2: Q-PCR for Legionella pneumophila 56


3.9 Pseudomonas aeruginosa 58 3.9.1 Monitoring technology nr. 1: Filtration and cultivation according to EN ISO 16266 58 3.9.2 Monitoring technology nr. 2: VIT-Pseudomonas aeruginosa 60 3.9.3 Confirmation technology nr. 1: API-Test 20E 62

3.10 Aeromonas 63 3.10.1 M onitoring technology nr 1: EPA Method 1605 63 3.10.2 Monitoring technology nr 2: MALDI 65 3.10.3 Monitoring technology nr 3: PCR 67

3.11 Bacteriophages 70 3.11.1 Monitoring technology nr 1: ISO method 10705-1 70 3.11.2 Monitoring technology nr 2: ISO method 10705-2 72 3.11.3 Monitoring technology nr 3: ISO method 10705-4 (modified) 74

3.12 Aerobic spore forming bacteria 77 3.12.1 Monitoring technology nr 1: HPC counts 77 3.12.2 Monitoring technology nr 2: Membrane filtration 79 3.12.3 Monitoring technology nr: 3. Assays, based on cell constituents 80 3.12.4 Monitoring technology nr 4: PCR 84

3.13 Biofilm formation rate (BFR) 87 3.13.1 Monitoring technology nr 1: ATP measurement of biofilm 87

3.14 Total cell counts 90 3.14.1 Monitoring technology nr 1: Direct total microbial count (based on fluorescence

microscopy) 90 3.14.2 Monitoring technology nr 2: Direct total microbial count (based on flow cytometry) 92

3.15 Cultivation free viability analysis 95 3.15.1 Monitoring technology nr 1: Cultivation free viability analysis (based on flow

cytometry or fluorescence microscopy) 95 3.15.2 Monitoring technology nr 2: Analysis of bacterial ATP 98

4 Chemical parameters 100

4.1 Metals: Antimony, arsenic, boron, cadmium, chromium, copper, lead, mercury, nickel, selenium, sodium, calcium, magnesium, aluminum, iron, manganese 100

4.1.1 Monitoring technology nr 1: AAS (Atomic adsorption spectroscopy) 101 4.1.2 Monitoring technology nr 2: AFS (Atomic fluorescence spectroscopy) 102 4.1.3 Monitoring technology nr 3: ICP-OES: Inductively-coupled plasma with optical

emission spectroscopy 103 4.1.4 Monitoring technology nr 4: ICP-MS: Inductively-coupled plasma with mass

spectrometry 104

4.2 Benzene 105 4.2.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-FID or GC-MS 105 4.2.2 Monitoring technology nr 2: Headspace, GC-FID or GC-MS 106 4.2.3 Monitoring technology nr 3: Purge&trap, GC-FID or GC-MS 107 4.2.4 Monitoring technology nr 4: Solid-phase micro extraction (SPME), GC-FID or GC-MS108

4.3 Benzo(a)pyrene and other PAHs 110 4.3.1 Monitoring technology nr 1: Liquid-liquid extraction, HPLC/FLD 110 4.3.2 Monitoring technology nr 2: Liquid-liquid extraction, GC/MS 111

4.4 Bromate 113 4.4.1 Monitoring technology nr 1: Ion chromatography with conductivity detection

(IC/CD) 113 4.4.2 Monitoring technology nr 2: Ion chromatography with UV detection (IC/UV) 114 4.4.3 Monitoring technology nr 3: Ion chromatography with fluorescence detection

(IC/FLD) 115


4.4.4 Monitoring technology nr 4: Ion chromatography with inductively coupled plasma mass spectrometry detection (IC/ICP-MS) 116

4.5 Cyanides 118 4.5.1 Monitoring technology nr 1: Photometric method (batch mode) 118 4.5.2 Monitoring technology nr 2: Continuous flow analysis 119

4.6 1,2-Dichloroethane 121 4.6.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 122 4.6.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 123 4.6.3 Monitoring technology nr 3: purge&trap, GC-MS 124

4.7 Fluoride 125 4.7.1 Monitoring technology nr 1: Ion-selective electrode (ISE) 125 4.7.2 Monitoring technology nr 2: Ion chromatography with conductivity detection

(IC/CD) 126

4.8 Nitrite 127 4.8.1 Monitoring technology nr 1: Ion Chromatography 127 4.8.2 Monitoring technology nr 2: Wet chemical analysis – Colorimetric method 128 4.8.3 Monitoring technology nr 3: Spectrometric method, derivative spectroscopy 130

4.9 Nitrate 132 4.9.1 Monitoring technology nr 1: Wet Chemical Analysis 132 4.9.2 Monitoring technology nr 2: Electrochemical method 134 4.9.3 Monitoring technology nr 3: Spectrometric method, single wavelength 135 4.9.4 Monitoring technology nr 4: Spectrometric method, derivative spectroscopy 137

4.10 Polycyclic aromatic hydrocarbons 139

4.11 Pesticides 139

4.12 Tetra- and trichloroethene 140 4.12.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 140 4.12.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 141 4.12.3 Monitoring technology nr 3: purge&trap, GC-MS 142

4.13 Disinfection byproducts (trihalomethanes) 143 4.13.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD) 143 4.13.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD) 144 4.13.3 Monitoring technology nr 3: purge&trap, GC-MS 145

4.14 Radioactivity 146 4.14.1 Semiconductor Detectors 146 4.14.2 Liquid Scintillation Counting 147 4.14.3 Inductively Coupled Plasma Mass Spectrometry 148

4.15 Endocrine disruption chemicals 150 4.15.1 Introduction 151 4.15.2 Analysis of water using bioassay 151 4.15.3 Analysis using bioassays 152 4.15.4 General evaluation of the bioassays 153

4.16 Genotoxicity 157 4.16.1 Introduction 157 4.16.2 Mechanisms of genotoxicity 157 4.16.3 Tests to detect genotoxicity 158 4.16.4 General review 158 4.16.5 Sensitivity and specificity 160 4.16.6 Robustness 161 4.16.7 Time to result 161 4.16.8 Ease of use and instrumentation 161


4.17 Acute toxicity 162 4.17.1 Standard toxicity tests 162 4.17.2 Monitoring technology nr 2: Daphnia toximeter 164 4.17.3 Monitoring technology nr 3: Fish toximeter 166 4.17.4 Monitoring technology nr 4: Combined Fish and Daphnia toximeter 170 4.17.5 Monitoring technology nr 5: Algae toximeter 172 4.17.6 Monitoring technology nr 6:Luminiscent bacteria 174 4.17.7 Monitoring technology nr 7: Mussel monitor 175

4.18 Algae toxins 178 4.18.1 Monitoring technology nr 1-4: LC-DAD, LC-MS/MS, ELISA, PPIA 179

4.19 Pesticides, pharmaceuticals, industrial chemicals and other organic micropollutants183 4.19.1 Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry 183 4.19.2 Monitoring technology nr 2: High-Performance Liquid Chromatography-UltraViolet

Diode Array Detection 184 4.19.3 Monitoring technology nr 3: High-Performance Liquid Chromatography-Mass

Spectrometry 185

4.20 pH 187

4.21 Monitoring technology nr 1: pH indicator 187 4.21.1 Monitoring technology nr 2: pH meter 189

4.22 Chloride/nitrate/sulphate 191 4.22.1 Monitoring technology nr 1: Ion chromatography with conductivity detection

(IC/CD) 191

4.23 Conductivity 192 4.23.1 Monitoring technology nr 1: Conductimeter 192

4.24 Calcium & magnesium 194

4.25 Sulphate 194

4.26 Aluminium 194

4.27 Ammonium 195 4.27.1 Monitoring technology nr 1: Ion Selective Electrode 195 4.27.2 Monitoring technology nr 2: Ion Chromatography 196 4.27.3 Monitoring technology nr 3: Photometric test kit 197 4.27.4 Monitoring technology nr 4: Automatic Analyser 198

4.28 Iron 200

4.29 Manganese 200

4.30 Taste & Odor 201 4.30.1 Monitoring technology nr 1: Sensory panel 202 4.30.2 Monitoring technology nr 2: GC- MS 203 4.30.3 Monitoring technology nr 3: Electronic Nose & Tongue 204

4.31 Colour 207 4.31.1 Monitoring technology nr 1: Visual Comparison method 207 4.31.2 Monitoring technology nr 2: Colorimetric analysis 209 4.31.3 Monitoring technology nr 3: Online spectrophotometric measurement 210

4.32 Turbidity 213 4.32.1 Monitoring technology nr 1: Analysis using a laboratory colorimeter 213 4.32.2 Monitoring technology nr 2: Online turbidity meter 214 4.32.3 Monitoring technology nr 3: Online spectrophotometric measurement 216

4.33 AOC 218 4.33.1 Monitoring technology nr. 1: The original “van der Kooij” assay 218


4.33.2 Monitoring technology nr. 2: The Werner and Hambsch assay 220 4.33.3 Monitoring technology nr. 3: The Stanfield and Jago ATP-based assay 222 4.33.4 Monitoring technology nr. 4: The LeChevallier assay 224 4.33.5 Monitoring technology nr.5: The Eawag-AOC assay 226

4.34 DOC/TOC 228 4.34.1 Monitoring technology nr 1: High temperature combustion 229 4.34.2 Monitoring technology nr 2: Persulfate oxidation 230 4.34.3 Monitoring technology nr 3: UV - oxidation / conductivity 232 4.34.4 Monitoring technology nr 4: UV-spectroscopy 233

4.35 UV absorbing organic constituents 235 4.35.1 Monitoring technology nr 1: Single Wavelength absorption measurement 235 4.35.2 Monitoring technology nr 2: Full spectral analysis 236

4.36 Particle counts 239 4.36.1 Monitoring technology nr 1: Particle counting systems 239

4.37 Oxygen 243 4.37.1 Monitoring technology nr 1: Titration 243 4.37.2 Monitoring technology nr 2: Electrochemical Measurement 244 4.37.3 Monitoring technology nr 3: Optical Measurement 246

Annex I. Individual evaluation forms for endocrine disrupting effects 249

Annex II. Individual evaluation form for genotoxic effects 281


1 Introduction

Monitoring and control technologies are indispensable for the production of safe drinking water. They allow for the surveillance of source water quality and the detection of biological and chemical threats, thus defining the boundary conditions for the subsequent treatment and providing early-warning in case of unexpected contaminations. They are mandatory for the permanent control of the treatment process and the efficacy of each single treatment step, and they safeguard the high quality of finished water. Furthermore, appropriate analytical techniques are indispensable for the detection of changes in water quality during distribution and for monitoring drinking water quality at consumers’ tap. Reliable monitoring technologies contribute to a large extent to the consumers’ trust in a high drinking water quality. Following the overall objective of the TECHNEAU project, the major objective of WA 3 is to provide a set of analytical techniques and methods that ensure the provision of safe high quality drinking water that has the trust of the consumers. In WP 3.1 existing monitoring technologies are evaluated according to their suitability for application in controlling water quality in the whole drinking water production process. This evaluation includes not only basic analytical techniques, but also new and innovative monitoring technologies like effect-related DNA-arrays or electronic nose technology. A first report resulting from WP 3.1 dealt with selection of key-parameters to assess water quality. It described the different locations and purposes in which monitoring and control technologies need to be applied. The respective biological and chemical water quality parameters that provide essential information for water suppliers (so-called 'key-parameters') are identified and listed. The current report is a follow-up and describes the results of a survey on monitoring technologies for the selected key-parameters. The existing monitoring technologies are identified and evaluated based on information on e.g. ease-of-use, maintenance requirements, cists, and technical specifications. Also the suitability of the techniques for use in small-scale-systems (3S) is evaluated. This report can be used as reference when deciding on the analytical chemical and biological techniques to be used for monitoring water quality from source to tap.


2 Method

All evaluations of the monitoring technologies have been made by the participants in WA3. Although there is a large expertise among the WA3 partners, it should be kept in mind that these evaluations are based on personal experience and judgement and should not be taken as absolute. Evaluations focus on the methods for the selected key-parameters, not on the suitability or accuracy of instruments from different suppliers. The basis for this report is the table that was prepared in the TECHNEAU report Monitoring and control of drinking water quality - Selection of key-parameters (Mons et al., 2007, see www.techneau.org ). This table is presented in table 1. To prepare evaluations in a uniform format, and make sure that evaluations from different partners include information on similar aspects, a standard evaluation form was prepared. The basic evaluation form is shown in Fig 1. As most expertise within the WA3 partners lies in the field of chemical and/or microbiological techniques, the process parameters (including inhibitors) were left out of this evaluation. Also for radioactivity there was no good option for a partner to create an evaluation. This problem was solved by including an evaluation retrieved from the literature. Fig. 1 Evaluation form


Table 1. Total list of parameters and where they are/can be used

Parameter C

atc

hm

en

t

So

urc

e w

ate

r S

W

So

urc

e w

ate

r G

W

Tre

atm

en

t 1

Fin

ish

ed

wa

ter

Dis

trib

uti

on

in

gre

ss

Dis

trib

uti

on

ti

me

re

late

d

Cu

sto

me

r’s

tap

Microbiological parameters

E. coli Enterococci Clostridium perfringens Total coliforms Colony count/HPC Enteric viruses Giardia/Cryptosporidium Campylobacter Legionella Pseudomonas aeruginosa Aeromonas F-specific RNA phages Aerobic spore-forming bacteria

Biofilm formation Total cell counts Cultivation-free viability analysis

Chemical parameters antimony arsenic benzene benzo(a)pyrene boron bromate cadmium copper chromium cyanides 1,2-dichloroethane fluoride lead mercury nickel nitrite nitrate PAHs pesticides selenium tetra- & trichloroethene disinfection byproducts2 radioactivity EDCs genotoxicity

acute toxicity

algae toxins


Parameter C

atc

hm

en

t

So

urc

e w

ate

r S

W

So

urc

e w

ate

r G

W

Tre

atm

en

t 1

Fin

ish

ed

wa

ter

Dis

trib

uti

on

in

gre

ss

Dis

trib

uti

on

ti

me

re

late

d

Cu

sto

me

r’s

tap

pharmaceuticals

industrial chemicals

organic micropollutants3

pH

chloride

alkalinity

saturation index4

sodium

conductivity

calcium

magnesium

sulphate

aluminum

ammonium

iron

manganese

taste

odour

colour

turbidity

AOC/BDOC

DOC/TOC

UV absorption

particle counts

oxygen

inhibitors

Process parameters5

head loss

filter velocity

residence time

ozone dose, contact time (Ct)

ozone concentration

residual ozone UV dose oxidant dose residual oxidant conc. disinfectant dose

residual disinfectant conc.

inhibitors sediments (e.g. iron oxides) flow rate transmembrane pressure pressure drop particle size distribution membrane (bio)fouling 1. Various parameters are suitable/preferable for different treatment steps. For details see TECHNEAU report "Monitoring and control of drinking water quality. Selection of key-parameters".


2 disinfection by-products: chlorination by-products, ozonation by-products, UV/AOP by-products 3 general group, consisting of e.g. pharmaceuticals, industrial pollutants etc 4 saturation in dex is only a calculation and will not be further evaluated in this report 5 process parameters will not be evaluated in this report


3 Microbiological parameters

3.1 E. coli and coliform bacteria Prepared by: TZW Required technical specifications: For the detection and enumeration of E. coli and coliform bacteria in drinking water a detection limit of one bacterial cell / 100 mL water sample is required. Methods suitable for analyses of drinking waters with higher bacterial numbers or source waters have to provide a high selectivity to avoid interference with background flora. Monitoring technologies: 1. Lactose TTC Tergitol Method (ISO 9308-1) 2. Colilert®-18/Quanty-Tray (IDExx, UK National Standard Method W 18) 3. Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553) 4. Membrane Lauryl Suphate Broth (MLSB) (UK National Standard Method W 2) 5. Chromocult® Coliform Agar (Merck)

3.1.1 Monitoring technology nr 1: Lactose TTC Tergitol Method (ISO 9308-1)

Description: The European Drinking Water directive (EU DWD) defines Lactose TTC Tergitol Method according to ISO 9308-1 as reference method for the detection and enumeration of E. coli and coliform bacteria. Method and mode of action E. coli and coliform bacteria are detected and enumerated by membrane filtration and subsequent culture on the differential agar medium Lactose TTC Tergitol as described in ISO 9308-1. Lactose is degraded to acid by E. coli and coliform bacteria which is indicated by a colour change of the medium. Tergitol® 7 (sodium heptadecylsulfate) and TTC (triphenyl-tetrazoliumchloride) inhibit the growth of gram positive non-target organisms. TTC is also part of the differential system. The reduction of TTC by lactose negative bacteria produces dark red colonies, whereas lactose positive E. coli and coliform bacteria reduce TTC only weakly resulting in yellow-orange colonies. Procedure and Evaluation 100 mL water sample is filtered through a membrane filter. The membrane filter is transferred to Lactose TTC Tergitol Agar and incubated at 36 ± 2°C for 21 ± 3 hours. Lactose positive bacteria produce yellow-orange colonies and under the membrane yellow-orange halos. The count of these typical colonies is considered to be presumptive coliform bacteria count. For confirmation of E. coli and coliform bacteria count further subculture of typical colonies on a non selective agar ( e.g. Tryptic Soy Agar) for oxidase test and in Tryptophane


Broth for indole production is required. Colonies that are oxidase negative are considered to be coliform bacteria. Coliform bacteria that form indole from tryptophane at 44 ± 0.5°C within 21 ± 3 hours are considered to be E. coli. Results are obtained after 1 day (negative results) or 2 days. Equipment and consumables As described in ISO 9308-1 (e.g. autoclave, incubator, water bath, filtration unit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter, Lactose TTC Agar with Tergitol® 7, Tryptic Soy Agar, Tryptophane Broth, Kovacs-Indole and oxidase reagent etc.). References Anon. (2000) ISO 9308-1: Water quality - Detection and enumeration of Escherichia coli and total coliform bacteria - Part 1: Membrane filtration method (ISO 9308-1:2000). Geneva, Switzerland: International Organisation for Standardisation. Evaluation: The membrane filtration method ISO 9308-1 for the detection and enumeration of E. coli and coliform bacteria is suitable for disinfected waters and other drinking waters with low bacterial numbers. Due to the low selectivity of Lactose TTC Tergitol Agar background growth can interfere with the reliable enumeration of E. coli and coliform bacteria. It is therefore not recommended for drinking waters with high bacterial numbers or for source waters. For these waters alternative methods e.g. Colilert®-18/Quanti-Tray (see monitoring technology nr 2) or MLSA (see monitoring technology nr 3) are more suitable. The costs for consumables are low, but experienced laboratory staff is required for test performance and evaluation. Time to result is 1-2 days.


Monitoring technology nr 1: Lactose TTC Tergitol Agar (ISO 9308-1) Criteria 1 2 3 4 5 Comments

Technical specifications

sensitivity (A) source water

drinking water

x

x

Heavy background growth can occur , only for waters with low bacterial numbers (e.g. disinfected drinking water)

robustness (A) operational robustness selectivity

x

x

Growth of non target organisms can occur

time to result

1-2 days

Operational specifications

ease-of-use (B) x maintenance requirements (C) x Costs

instrumentation (C) x operational costs (C)

consumables maintenance

x x

Recommendation for use in SSS (D) x

Overall conclusion Inexpensive method, but requires experienced laboratory staff. Due to low selectivity, it is only recommended for very clean water samples

(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

3.1.2 Monitoring technology nr 2: Colilert®-18/Quanti-Tray (IDExx, UK National Standard Method W 18)

Description: Colilert®-18/Quanti-Tray has been approved as alternative method for the detection and enumeration of E. coli and coliform bacteria according to the EU DWD in a number of EU countries e.g. Germany, Italy, Czech Republic and Hungary. Colilert®-18/Quanti-Tray is included in American and UK Standard Methods. Method and mode of action Colilert®-18/Quanti-Tray is a Most Probable Number test and allows simultaneous detection of E. coli and coliform bacteria. The method is based on an enzyme substrate reaction. The major carbon sources are ONPG (o-nitrophenyl- β-D-galactopyranoside) and MUG (4-methyl-umbelliferyl-β-D-glucuronide), which are metabolised by the enzymes β-galactosidase


(coliform bacteria) and β-glucuronidase (E. coli). When ONPG is metabolised by coliform bacteria the medium changes from colourless to yellow. The degradation of MUG by E. coli creates fluorescence. Most non-coliforms do not have these enzymes and are unable to grow and interfere. Furthermore, Colilert® uses Defined Substrate Technology® (DST®) to inhibit growth of non-target organisms. Procedure and evaluation 100 mL water sample is transferred to a sterile bottle with antifoam reagent. Colilert® reagent is added and sample is mixed until reagent is completely dissolved. Sample/reagent is filled in a sterile Quanti-Tray®. The tray is sealed and incubated for 18 - 22 h at 36°C. Positive wells are counted (yellow = coliform bacteria, yellow and fluorescent (UV light) = E. coli) and the numbers of E. coli and coliform bacteria are determined from the MPN tables. Equipment and consumables As described e.g. in the UK National Standard Method W 18 (e.g. incubator, water bath, gas burner, Quanti-Tray heat sealer, long wavelength UV light source and viewer, Quanti-Trays and reference comparator, Colilert® reagent, antifoam reagent, sterile glassware). References IDExx Laboratories, Milton Court, Churchfield Road, Chalfont St Peter, Buckinghamshire, SL9 9EW. Health Protection Agency (2004). Enumeration of coliforms and Escherichia coli by Idexx (Colilert 18) Quanti-TrayTM. National Standard Method W 18 Issue 2. http://www.hpastandardmethods.org.uk/pdf_sops.asp. Chromogenic Substrate Coliform Test (9223) (1997) in: Standard Methods for the Examination of Water and Wastewater, 21st Edition (2005), American Public Health Association, 1015 Fifteenth Street, NW, Washington, DC 20005. Evaluation: The Colilert®-18/Quanti-Tray method is applicable to the enumeration of E. coli and coliform bacteria in drinking water, source water and environmental water. Due to the presence of β-galactosidase in some species which are unable to produce acid from lactose using Colilert®-18 Quanti-Tray can result in higher coliform numbers compared to other culture-based tests (e.g. Lactose TTC Tergitol Method or MLSA). The method is easy to perform and does not require further identification tests. It can be performed by less experienced laboratory staff and is suitable for small water supplies, but higher costs for consumables incur. Test results are available after 1 day.


Monitoring technology nr 2: Colilert®-18/Quanti-Tray (IDExx, UK National Standard Method W 18) Criteria 1 2 3 4 5 Comments



drinking water

x x

No interference in waters with higher bacterial numbers


x x

time to result

1 day





x

x


Overall conclusion Fast and easy to use method. More expensive compared to other culture-based tests due to higher costs for consumables. Is suitable for drinking water and source water.


3.1.3 Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553)

Description: Membrane Lauryl Sulphate Agar (MLSA) has been approved as alternative method for the detection and enumeration of E. coli and coliform bacteria according to the EU DWD in the Netherlands. Method and mode of action E. coli and coliform bacteria are simultaneously detected and enumerated by membrane filtration and subsequent culture on the differential agar medium MLSA which contains lactose as major carbon source. E. coli and coliform bacteria degrade lactose to acid which is indicated by a change of the colony colour to yellow. Laurylsulphate inhibits the growth of non-target organisms. Yellow oxidase negative colonies are considered as coliform bacteria and yellow oxidase negative colonies which produce indole from tryptophane are considered as E. coli.


Procedure and evaluation 100 mL water sample is filtered through a membrane filter. The membrane filter is transferred to MLSA and incubated at 25 ± 1 °C for 5 ± 1 hours followed by incubation at 36 ± 2 °C for 14 ± 2 hours. Typical yellow lactose-positive colonies are transferred to a non-selective agar and incubated at 36 ± 2 °C for 21 ± 3 hours to test for oxidase activity. Parallel the same colonies are transferred to tryptophane broth and incubated at 44 ± 0.5 °C for 21 ± 3 hours to test for indole production. Yellow colonies which are oxidase negative are considered total coliforms. Those being oxidase negative and indole positive are considered E. coli. Results are obtained after 1 day (negative results) or 2 days. Equipment and consumables As described in NEN 6553 (e.g. autoclave, incubator, water bath, filtration unit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter, MLSA, Tryptone Soy Agar, Tryptophane Broth, Kovacs-Indole and oxidase reagent etc.). References Anon. 1981: NEN 6553. Bacteriological analysis of drinking water. Quantification of coliform bacteria using membrane filtration. [In Dutch] NNI, Delft, The Netherlands. Evaluation: The membrane filtration method NEN 6553 for the detection and enumeration of E. coli and coliform bacteria is suitable for drinking waters with various contamination levels and for source water. MLSA is more selective for target organisms compared to Lactose TTC Tergitol Agar (ISO 9308-1). However, it is not recommended for highly contaminated surface water. The costs for consumables are low, but experienced laboratory staff is required for test performance and evaluation. Late sample delivery can cause inconvenience for the laboratory workflow due to the two-stage incubation procedure. Time to result is 1-2 days.


Monitoring technology nr 3: Membrane Lauryl Sulphate Agar (MLSA) (NEN 6553) Criteria 1 2 3 4 5 Comments



drinking water

x

x


x x

More selective than Lactose TTC Tergitol Agar

time to result

1-2 days





x x


Overall conclusion Inexpensive method, but requires experienced laboratory staff. Late sample delivery can cause inconvenience due to the two-stage incubation procedure. Is suitable for water samples with various contamination levels.


3.1.4 Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK National Standard Method W 2)

Description: Membrane Lauryl Sulphate Broth (MLSB) has been approved as alternative method for the detection and enumeration of E. coli and coliform bacteria according to the EU DWD in the UK. MLSB is included in the UK National Standard Methods. Method and mode of action E. coli and coliform bacteria are detected separately and enumerated by membrane filtration and subsequent culture on an absorbent pad saturated with MLSB as differential medium. MLSB contains lactose as major carbon source, which is degraded to acid by E. coli and coliform bacteria, indicated by a change of the colony colour to yellow. Laurylsulphate inhibits the growth of non-target organisms. Yellow


colonies are to be confirmed as E. coli and coliform bacteria by further confirmatory tests (acid from lactose, oxidase activity, indole production). Procedure and evaluation 100 mL of water sample is filtered through a membrane filter (2 filters for each sample). The 2 membrane filters are transferred to pads soaked with MLSB and incubated (at 30 ± 1 °C for 4 ± 1 hours followed by incubation at 37°C ± 1 °C for 14 ± 1 hours for coliform bacteria and at 30 ± 1 °C for 4 ± 1 hours followed by incubation at 44 ± 2 °C for 14 ± 1 hours for E. coli). Yellow colonies are counted on each pad as presumptive coliform bacteria (37 °C) and presumptive E. coli (44 °C). Yellow colonies from both membrane filters are transferred to Lactose Peptone Water (LPW), MacConkey Agar (MA) and Nutrient Agar (NA) for incubation at 37 °C for 20 ± 4 hours and to LPW and Trypton Water (TP) for incubation at 44 °C for 20 ± 4 hours. Colonies grown on NA are checked for oxidase activity and indole test is done in TP. Coliform bacteria produce red colonies on MA, are oxidase negative and produce acid from lactose in LPW at 37 °C. E. coli produces acid from lactose at 44 °C, is oxidase negative and indole positive. Results are obtained after 1 day (negative results) or 2-3 days. Equipment and consumables As described in the UK National Standard Method W 2 (e.g. autoclave, incubator, water bath, filtration unit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter, Membrane Lauryl Sulphate Broth, Nutrient Agar, McConkey Agar, Kovacs-Indole and oxidase reagent etc.). References Health Protection Agency (2007). Enumeration of coliform bacteria and Escherichia coli by membrane filtration. National Standard Method W 2 Issue 4. http://www.hpastandardmethods.org.uk/pdf_sops.asp. Evaluation: The membrane filtration method NSM W 2 for the detection and enumeration of E. coli and coliform bacteria is suitable for drinking waters with various contamination levels and for source water. MLSB is like MLSA more selective for target organism compared to Lactose TTC Tergitol Agar (ISO 9308-1). However, it is also not recommended for highly contaminated surface water. The costs for consumables are low, but experienced laboratory staff is required for test performance and evaluation. Compared to MLSA the MLSB method is more involved and labour-intensive. Late sample delivery can cause inconvenience for the laboratory workflow due to the two-stage incubation procedure. Time to result is 1-3 days.


Monitoring technology nr 4: Membrane Lauryl Suphate Broth (MLSB) (UK National Standard Method W 2) Criteria 1 2 3 4 5 Comments



drinking water

x

x


x

x

More selective than Lactose TTC Tergitol Agar

time to result

1-3 days





x x


Overall conclusion Inexpensive method, but requires experienced laboratory staff. Late sample delivery can cause inconvenience due to the two-stage incubation procedure. Is suitable for water samples with various contamination levels.


3.1.5 Monitoring technology nr 5: Chromocult® Coliform Agar (Merck)

Description: Chromocult® Coliform Agar (Merck) is a selective agar for the simultaneous detection of total coliforms and E. coli in drinking water and processed food samples. The approval of this method by US-EPA is pending. Furthermore, it has been taken into consideration to develope an ISO standard. Method and mode of action E. coli and coliform bacteria are detected and enumerated by membrane filtration and subsequent culture on CCA as selective and differential agar medium. CCA contains chromogenic substrates which change colour to salmon-red when degraded by ß-galactosidase positive coliform colonies and to dark blue-violet when degraded by ß-galactosidase and ß-glucuronidase positive E. coli colonies. Non-target organisms are largely inhibited by addition of Tergitol® 7 and E. coli/Coliform Selective-Supplement. In case of growth colonies of non-target organisms appear colourless or light-blue.


Procedure and evaluation 100 mL of water sample is filtered through a membrane filter. The membrane filter is placed on CCA and incubated at 35-37 °C for 24 hours. Salmon to red ß-galactosidase positive colonies are counted as non E. coli coliforms. No further confirmatory tests are required for non E. coli coliforms. Dark-blue to violet ß-galactosidase and ß-glucuronidase positive colonies are counted as E. coli and are confirmed by testing for indole production. Results are available after 1 day. Equipment and consumables Usual laboratory equipment and in addition: autoclave, incubator, water bath, filtration unit, scale, pH-meter, gas burner, glassware, Petri dishes, membrane filter, Chromocult® Coliform Agar (see Merck description Cat. No. 1.10426.0100/500). References Ossmer, R., Schmidt, W., Mende, U. (1999). Chromocult® Coliform Agar - Influence of Membrane Filter Quality on Performance. - xVII Congresso de la Sociedad, Granada. Finney, M., Smullen, J., Foster, H.A., Brokx, S., Storey, D.M. (2003). Evaluation of Chromocult coliform agar for the detection and enumeration of Enterobacteriaceae from faecal samples from healthy subjects. Journal of Microbiological Methods 54: 353-358. Evaluation: Chromocult® Coliform Agar should be suitable for the enumeration of E. coli and coliform bacteria in drinking water, source water and environmental water. Due to the presence of β-galactosidase in some species which are unable to produce acid from lactose CCA can like Colilert®-18/Quanti-Tray result in higher coliform numbers compared to other culture-based tests. No further cultivation step is required for confirmation, hence the method is faster and can be performed by less experienced laboratory staff than. Test results are obtained after 1 day. Validation data are not yet available


Monitoring technology nr 5: Chromocult® Coliform Agar (Merck) Criteria 1 2 3 4 5 Comments



drinking water

x x


x

x

time to result

1 day





x x


Overall conclusion Fast method, no further cultivation step is required for confirmation. Suitable for drinking water and source water



3.2 Intestinal enterococci Prepared by: Kiwa Water Research Required technical specifications: The concentration range that is normally encountered in water samples is: 0 cfu per 100 ml water sample in drinking water and groundwater 0 to 1000 cfu per 100 ml in surface water The required resolution of the method is: 1 cfu in 100 ml water sample Monitoring technologies: 1. ISO method 7899-1: MPN cultivation in liquid MUD/SF medium. 2. ISO method 7899-2: membrane filtration and cultivation on agar medium containing azide and 2,3,5-triphenyltetrazoliumchloride.

3.2.1 Monitoring technology nr 1: ISO method 7899-1

Description: - The diluted sample is inoculated in a row of microtitre plate wells containing dehydrated MUD/SF culture medium. The microtiter plates are examined under ultraviolet light at 366 nm in the dark after an incubation period of between 36 h and 72 h at 44°C ± 0.5 °C. The presence of enterococci is indicated by the fluorescence resulting from the hydrolysis of 4-methylumbelliferyl-β-D-glucoside (MUD). The results are given as Most Probable Number (MPN) per 100 ml. - To perform ISO method 7899-1 an apparatus for sterilization by dry heat or steam, a thermostatic incubator, a membrane filtration apparatus, a tunnel drier or vertical laminar air flow cabinet, UV observation chamber (Wood’s lamp 366 nm), pre-set 8-channel multi-pipette and sterile microtiter plates are required. - The method is one of the two ISO-approved methods for the enumeration of intestinal enterococci in water. - Reference: ISO 7899-1. Evaluation: - In general, an accurate method to determine the number of enterococci in surface water samples, but enumeration is based on MPN, which might be less reliable than the colony count method described below. - The detection limit of the method is 15 bacteria per 100 ml, which is too high for use with drinking water. - The use of microtiter plates, liquid media and multi-pipette makes the method more robust than the colony count method described below.


- Incubation time is rather long, because results are obtained after 1.5 to 3 days. However, a fast standard method is not available, although methods based on DNA-detection have been published and might come available in the near future. -It is known that in some water samples (especially sea-water) other micro-organisms might give false-positive results. However, the other ISO-method described below has a similar limitation because with that method agar plates can be overgrown with other micro-organisms, preventing reliable enumeration of intestinal enterococci colony types. - A disadvantage compared to monitor technology 2 is that the method does not contain confirmation steps. Monitoring technology nr 1: ISO-method 7899-1 Criteria 1 2 3 4 5 Comments



drinking water

x

x

Detection limit: 15 bacteria in 100 ml water


x

x

time to result

x Total incubation time: 36 to 72 h





x


Overall conclusion Acceptable method to determine intestinal enterococci in surface waters. Not applicable to drinking water, because the detection limit of the method is too high (15 bacteria per 100 ml). Some water samples might give false-positive results. Quantification is based on MPN. Positive results are not confirmed by additional reactions. Because of the cultivation step this standard method is rather time consuming; a faster standard method is preferred, but not yet available.




Description: - A specified volume of water sample is filtered through a membrane filter with a pore size (0.45 µm) sufficient to retain the bacteria. The filter is placed on a solid selective medium containing sodium azide (to suppress growth of Gram-negative bacteria) and 2,3,5-triphenyltetrazolium chloride, a colourless dye, that is reduced to red formazan by intestinal enterococci. Typical colonies are raised, with a red, maroon or pink colour, either in the centre of the colony or throughout. If typical colonies are observed, a confirmation step is necessary, by transfer of the membrane, with all the colonies, onto bile-aesculin-azide agar, preheated at 44 °C. Intestinal enterococci hydrolyse aesculin on this medium in 2 h. The end-product, 6,7-dihydroxycoumarin, combines with iron(III) ions to give a tan-coloured to black compound which diffuses into the medium Confirmed colonies are expressed as colony forming units (cfu) per 100 ml. - To perform ISO method 7899-2 an apparatus for sterilization by dry heat or steam, two thermostatic incubators (37°C and 44°C), a membrane filtration apparatus, sterile membrane filters (0.45 µm) and a water bath (100°C) (to dissolve the agar medium) are required. - The method is one of the two ISO-approved methods for the enumeration of intestinal enterococci in water. - Reference: ISO 7899-2. Evaluation: - In general, an accurate method to determine the number of enterococci in water. - The detection limit of the method is 1 cfu per 100 ml, which makes the method suitable for determining intestinal enterococci in drinking water. - The use of selective agar media and membrane filtration and the interpretation of colony characteristics make the method slightly less robust than the first monitoring technology described above. - Incubation time is rather long, because results are obtained after 2 days. However, a fast standard method is not available, although methods based on DNA-detection have been published and might come available in the near future. -It is known that agar plates can sometimes be overgrown with other micro-organisms, preventing reliable enumeration of intestinal enterococci colony types (especially in source surface water). However, the other ISO-method described above has a similar limitation because with that method some water samples might give false-positive results. - An advantage compared to monitor technology 1 is that the method contains a confirmation step, making the chance of false-positive results lower.


- Because this method is suitable for both source and drinking water, the method is preferred over monitoring technology nr 1, which was described above. Monitoring technology nr 2: ISO-method 7899-2 Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x

Detection limit: 1 bacterium in 100 ml water


x

x

time to result

x Total incubation time: 46 h





x


Overall conclusion Acceptable and ease-to-use method to determine intestinal enterococci. Applicable to both source and drinking water. Surface source water samples might give overgrowth of non-target bacteria on the agar plates. Because of the cultivation step this standard method is rather time consuming; a faster standard method is preferred, but not yet available.



3.3 Clostridium perfringens Prepared by: vermicon AG C. perfringens is a rod-shaped, gram-positive, endo-spore forming and non-motile bacterium of the genus Clostridium. It is strictly anaerobic, but can survive a short exposure to oxygen. The micro-organism can be detected in anaerobic zones of soil, in water, foodstuff and in the intestine of humans and animals. C. perfringens is selected out of the genus Clostridium to serve as microbiological parameter, because it is the most important species out of the sulphite reducing Clostridia and it is normally present in human and animal faeces. Required technical specifications: The limit value for C. perfringens (including spores) according to the Drinking Water Directive (DWD) is 0 cells/100 ml drinking water. The analysis of this parameter is only necessary, if the water originates from surface water or is influenced by surface water. When the limit value is exceeded, the competent authority will initiate exploratory research to assure that there is no danger for human health, due to the occurrence of a pathogenic micro-organism. Furthermore if no C. perfringens is detected in 100 ml drinking water, it can be assumed that no resistant dormant bodies/cysts of a parasitic protozoa are present in the water. For the analysis quantitative detection methods have to be applied. Yet, there is no approved ISO standard procedure available for the detection of Clostridium perfringens. Monitoring and confirmation technologies:

1. Guideline according to DWD - membrane filtration and cultivation on m-CP agar

2. Membrane filtration and cultivation on TSC agar, subsequent confirmation tests according to draft of ISO 6461 CD part 2

3. Membrane filtration and cultivation on fluorogenic TSC agar (Araujo et al., 2004)

4. Clostridium perfringens PCR-based-detection system, Biotecon Diagnostics, Germany

5. API 32A, biochemical screening test, Biomerieux, France

3.3.1 Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC - membrane filtration and cultivation on m-CP agar

Description: Cultivation-based quantitative method for the enumeration of C. perfringens in water samples. 100 ml water samples is filtrated on a membrane filter. Membrane filtration is followed by an anaerobic incubation of the membrane on m-CP selective agar at 44 +/- 1 °C for 21 +/- 3 hours. Opaque yellow colonies that turn pink or red after exposure to ammonium hydroxide vapors are counted.


Material & method m-CP medium is a selective, chromogenic medium for the rapid identification and enumeration of Clostridium perfringens in water samples. The medium is designed to improve the differentiation of Clostridium perfringens from other Clostridia species and background flora. Chromogenic compounds within the m-CP medium cause Clostridium perfringens colonies to turn yellow (based on their ability to ferment sucrose), thus differentiating them from other Clostridium species, whilst colonies of background flora turn purple (based on their ability, unlike C. perfringens, to hydrolyse indoxyl-b-D-glucoside). An additional confirmation of the result is proposed by exposing the culture plate to ammonium hydroxide. This highly specific reaction causes acid phosphatase-producing C. perfringens yellow colonies to turn into a distinctive dark pink color. The addition of D-cycloserine and polymyxine B, and an incubation temperature of 44 °C improves selectivity of m-CP agar by inhibiting the growth of gram-negative bacteria and Staphylococci. No further verification steps are necessary according to the directive. Equipment and consumables

• M-CP agar • Basal medium • Membrane filtration manifold • Sterile filter funnels graduated to 100 ml • Vacuum pump with moisture trap or protective filter, or

alternative vacuum force • Incubator: 44 °C +/- 1°C • Facilities for anaerobic incubation • Petri dishes • Cellulose ester 0,45 µm pore size filters

Evaluation The fastest method for the detection of C. perfringens is monitoring technology no. 1. This procedure is based on membrane filtration and subsequent cultivation on m-CP agar. Time to result is 1 day, the handling is quite simple and it can be performed with standard laboratory equipment on low cost basis. Nevertheless, this approach is criticized by experts regarding the poor sensitivity. Due to this fact another method for the detection of C. perfringens was established in the draft of ISO 6461 CD part 2.


Monitoring technology nr. 1: Guideline according to Council Directive 98/83/EC - membrane filtration and cultivation on m-CP Medium Criteria 1 2 3 4 5 Comments

Technical specifications sensitivity (A)

source water drinking water

- x

Not yet analysed

robustness (A) operational robustness

selectivity

x x

time to result

24 h

Operational specifications ease-of-use (B) x maintenance requirements (C) x Costs x instrumentation (C) x Standard water laboratory

equipment operational costs (C)


x x

Recommendation for use in SSS (D) x Overall conclusion Very fast, easy & low cost test, but poor reliability (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

3.3.2 Monitoring technology nr. 2: Membrane filtration and cultivation on TSC agar, subsequent confirmation tests (draft of ISO 6461 CD part 2)

Description: Filtration of 100 ml water sample on a membrane filter (maximal pore size 0,45 µm, no specification of filter material is given). Subsequent incubation of the membrane filter under anaerobic conditions on TSC-Agar at 44 +/- 1 °C for 21 +/- 3 hours. All colonies are counted, that show a black or grey to yellow brown staining of the TSC agar when viewed either from above or below the filter. Some colonies may exhibit very faint staining of the medium, but should still be counted. Further confirmatory tests should be performed for purposes of identification. Material & method: The nutrient base provides optimal conditions for the development of Clostridia. Colonies producing hydrogen sulfide are characterized by blackening due to the reaction with sulfite and iron salt. In TSC Agar cycloserine inhibits the accompanying bacterial flora and causes the colonies,


which develop, to remain smaller. It also reduces a diffuse and thus disturbing blackening around the C. perfringens colonies. C. perfringens produces black colonies. Further tests should be performed for purposes of identification. Confirmatory tests Subculture each colony on two blood agar plates. Place one plate in an incubator for aerobic incubation at 36+/-2 °C and the other in an incubator for anaerobic incubation at the same temperature. Examine the plates after 21 +/- 3 hours for the presence or absence of growth and for purity. Perform confirmatory tests on subcultures that only grow anaerobically. Colonies of C. perfringens characteristically produce clear zone of haemolysis on blood agar. - Confirmatory test a: Buffered Nitrate Motility medium: Testing for motility Incoculate by stabing into the medium and incubate under anaerobic conditions at 36 °C +/- 2 °C for 21 +/- 3 hours. After incubation examine the medium for growth along the line of the stab. Motility is evident as diffuse growth out into the medium away from the stab line. Nitrate reduction Mix equal volumes of nitrate reagents A and B immediately before use and test for the presence of nitrate by adding 0,2 ml to 0,5 ml of this mixture to each of Buffered Nitrate Motility medium. The formation of a red colour confirms the presence of nitrite produced by the reduction of nitrate. If a red colour does not develop within 15 minutes add a small amount of zinc dust and allow to stand for 10 minutes. If a red colour develops no reduction of nitrite has taken place and the test is considered negative. If no red colour develops following the addition of zinc dust this means no nitrate remains and has been completely converted to nitrogen gas and the test is recorded as positive. - Confirmatory test b: Lactose gelatine medium : Inoculate the medium and incubate anaerobically at 36 °C +/- 2 °C for 21 +/- 3 hours. Examine the tube for a yellow colour indicating the production of acid. Chill the tubes for 1- 2 hours at 5 +/- 3 °C and check for gelatine liquefaction. The tubes can be examined after 1 hour but any that have not been solidified should be returned to the refrigerator for a further hour. If the medium has solidified reincubate at 36 °C +/- 2 °C for an additional 21-/- 3 hours and again check for liquefaction of gelatine. C. perfringens produces black or grey to yellow brown colonies on TSCA agar, is non-motile, reduces nitrit to nitrate, produces acid from lactose and liquefies gelatine within 44 +/- 4 hours. Equipment and consumables:

• Membrane filtration manifold • Sterile filter funnels graduated to 100 ml


• Vacuum pump with moisture trap or protective filter, or alternative vacuum force

• Incubator: 44 °C +/- 1°C • Facilities for anaerobic incubation • Tryptose sulphite cycloserine agar • Buffered Nitrate-Motility medium • Nitrate reagent A • Nitrate reagent B • Lactose-gelatine medium: • Blood agar: Columbia agar or any other suitable base with 5 %

horse blood • Petri dishes • 0,45 µm pore size filters

Evaluation Monitoring technology nr. 2 (ISO 6461 CD part 2) contains different confirmatory tests. In comparison to monitoring technology no. 1 this approach is more time-consuming, but a higher reliability and sensitivity is achieved. This ISO standard is still not approved yet and comprising evaluation studies are missing.


Monitoring technology nr. 2: Membrane filtration and subsequent cultivation on TSC agar and subsequent confirmation tests

Criteria 1 2 3 4 5 Comments



drinking water

x

not analysed


selectivity

x x

time to result

3-4 days

Operational specifications ease-of-use (B) x maintenance requirements (C) x x Costs instrumentation (C) x Standard water laboratory



x x


Overall conclusion Low cost test, time consuming, handling is feasible, not robust, but reliability is very good


3.3.3 Monitoring technology nr 3: Membrane filtration and cultivation on fluorogenic TSC agar

Description: Membrane filtration procedure is performed according to Monitoring technology no 1 and 2, but confirmatory tests are not necessary. By the addition of the fluorogenic substrate 4-Methylumbelliferyl-phosphate (MUP) Clostridium perfringens colonies can be identified by UV light. Method D-Cycloserine inhibits the accompanying bacterial flora and causes the colonies which develop to remain smaller. It also reduces a diffuse and thus disturbing blackening around the C. perfringens colonies. 4-Methylumbelliferyl-phosphate (MUP) is a fluorogenic substrate for the alcaline and acid phosphatase. The acid phosphatase is a highly specific indicator for C. perfringens. The acid phosphatase splits the fluorogenic substrate MUP forming 4-methylumbelliferone, which can be identified as it fluorescence in long wave UV light. Thus a strong suggestion for the presence


of C. perfringens can be obtained. The acid phosphatase splits the fluorogenic substrate MUP forming 4-methylumbelliferone which can be identified as it fluorescence in long wave UV light. Thus a strong suggestion for the presence of C. perfringens can be obtained. (method by VWR, Germany) Equipment and consumables:

• Membrane filtration manifold • Sterile filter funnels graduated to 100 ml • Vacuum pump with moisture trap or protective filter, or

alternative vacuum force • Incubator: 44 °C +/- 1°C • Facilities for anaerobic incubation • Tryptose sulphite cycloserine agar • Additive for the preparation of TSC-Agar (Base), Fluorocult

TSC-Agar supplement. • Petri dishes • Cellulose ester 0,45 µm pore size filters

Evaluation Monitoring technology nr. 3, which is somehow an enhancement of monitoring technology nr. 2 (ISO 6461 CD part 2), gives also a higher reliability and sensitivity compared to monitoring technology nr. 1


Monitoring technology nr. 3: Membrane filtration and cultivation on fluorogenic TSC agar




drinking water

x

not analysed


selectivity

x x

time to result

24 h


ease-of-use (B) x maintenance requirements (C) x x Costs instrumentation (C) x Standard water laboratory



x x

Recommendation for use in SSS (D) x Overall conclusion Low cost and fast test, handling is feasible and reliability is

good (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

3.3.4 Confirmation technology nr. 1: C. perfringens Detection System (Biotecon Diagnostics)

Oligonucleotides for the specific detection amplification and detection of C. perfringens DNA by PCR suitable for gel based detection are provided by Biotecon Diagnostics. The application of this detection system indicates only the presence of C. perfringens DNA, while no indication for the presence of active cells is given. Additionally well trained personal is needed for the performance of this non-quantitative test. Thus this method can only be used as a confirmatory test.

3.3.5 Confirmation technology nr. 2: API 32 A (Biomerieux)

API-Test 32 A (Vendor: Biomerieux) is a test for the identification of different anaerobic bacteria. The biochemical test is a confirmatory test for colonies and identification is possible within 4 hours. For the application of this test a pure culture is needed. Thus the test can be applied after the subculture of a colony as an additional test for the confirmation of the result.


References 1. Araujo M. Sueiro R.A . Gómez Garrido M.J. (2004) Enumeration of

Clostridium perfringens in groundwater samples: comparison of six culture media. Journal of Microbiological Methods, 57 (2), 175-180

2. Armon P. and Payment P. (1988) A modification of m-CP medium for enumerating Clostridium perfringens from water samples. Can. J. Microbiol. 34 78-79

3. Barthel H. Krüger W. Mendel B. Suhr R. Die Trinkwasserverordnung 2001 – bewährt oder revisionsbedürftig. Bundesgesundheitsblatt – Gesundheitsforschung Gesundheitsschutz 2007, 50: 265-275, Springer Medizin Verlag 2007

4. Payment P. and Franco E. (1993) Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cycts. Appl. Environ. Microbiol. 59, 2418-2124

5. Sartory D. P. (2005) Validation, verification and comparison: Adopting new methods in water microbiology Revised paper. Water SA Vol. 31 No.3 July 2005

Guidelines 1. Council Directive 98/83/EC of November 1998 on the quality of water

intended for human consumption. Official Journal of the European Communities

2. Enumeration of Clostridium perfringens by membrane filtration. Issue no: 3.1 Issue date: 03.05.05 Issued by: Standards Unit, Evaluations and Standards Laboratory on behalf of the Regional Food, Water and Environmental Microbiologist Forum. www.evaluations-standards.org.uk


3.4 Heterotrophic Plate Counts Prepared by: EAWAG Definition of HPC Heterotrophic plate counts are defined as the microbial colonies that form on semi-solid nutrient-rich growth media after a selected time of incubation at a selected incubation temperature. The colonies can arise from single cells, pairs, clusters or strings of cells (also called colony forming units, CFU). The methodological descriptions below have relied heavily on the methods as described in “9215 Heterotrophic Plate Counts, In: Standard Methods for the Analysis of Water and Wastewater” (Clesceri et al., 1998), and on the standardised ISO 6222 method (ISO, 1998). Required technical specifications: Analysis of HPC is included in the legislation/guidelines of most countries, as well as the European Drinking Water Directive (DWD - Council Directive 98/83/EC of 3 November 1998). Current drinking water guidelines give HPC limits that vary typically in the range of 10 – 300 colony forming units (CFU) per mL depending on the country, the water and the specific method which is used. The DWD states as parametric value only “no abnormal change”. Monitoring technologies: 1. Method according to ISO 6222 2. R2A method and common variations

3.4.1 Monitoring technology nr 1: ISO 6222

Description: If necessary the water sample is diluted 10-fold in sterile peptone-water. From the various dilutions, a volume not exceeding 2 mL is transferred into a sterile Petri-dish. Sterile, warm Yeast Extract Agar (15 – 20 mL) is added and mixed with the sample according to the so-called pour-plate method (see Clesceri et al (1998) for details). Separately prepared samples are subsequently incubated at respectively 36 °C for 44 h ± 4 h, and 22 °C for 68 h ± 4 h. After incubation, the colonies that have formed on the plates are counted. Plates with in excess of 300 colonies should not be considered for the analysis. The result is given as the number of colony forming units (CFU) per mL. Equipment and consumables Equipment: Autoclave; incubators (22 °C, 36 °C) Consumables: Sterile Petri dishes; Yeast Extract Agar Status of the technique The basic HPC method has been in use for nearly 100 years and is generally accepted in drinking water treatment as standard indicator of the general


microbiological quality of water. Several European countries have adopted the ISO 6222 method as standard method according to the DWD (e.g., TrinkwV, 2001). Evaluation: The method has the advantage that it has been used for a long time and a lot of experience and data exists in the peer reviewed literature. The main general disadvantage of all HPC methods is that cultivation only detects a small percentage (typically ca. 1%) of the total microbial concentration in a water sample, and that the methodology is time and labour consuming method. Several authors have suggested that the growth medium, incubation temperature and incubation time is not optimal to achieve the highest plate count results (Reasoner and Geldreich, 1985; Uhl and Schaule, 2004; Berney et al., 2008). References ISO, 1998. Water Quality-Enumeration of Culturable Micro-organism-Colony Count by Inoculation in a Nutrient Agar Culture medium, prEN ISO 6222 European Committee for Standardisation, Brussels. Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (Eds.) (1998) Standard Methods for the examination of water and wastewater; 9215 Heterotrophic Plate Counts. ISBN 0-87553-235-7 Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7. Uhl, W. and Schaule, G. (2004) Establishment of HPC (R2A) for regrowth control in non-chlorinated distribution systems. International Journal of Food Microbiology, 92: 317 – 325. Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F. Hammes. Rapid, cultivation-independent assessment of microbial viability in drinking water. Accepted for publication in Water Research, July 2008 TrinkwV, 2001. verordnung über die Qualität von Wasser für menschlichen Gebrauch (Trinkwasserverordnung - TrinkwV 2001). BGB1 I, Nr. 24, issued May 28th 2001, pp.959-980. DWD - Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption.


Monitoring technology nr 1: ISO 6222


3.4.2 Monitoring technology nr 2: Plating on R2A medium

Description: Heterotrophic plate counts (HPC) are estimated according to the R2A method by the spread plating method in which 0.1 mL of the water sample is spread out on a pre-prepared sterile agar plate containing R2A medium (see Reasoner and Geldreich, 1985). If the bacteria in the sample are expected in high concentratiions, a 10-fold dilution series in sterile phosphate buffer or physiological solution can be used. R2A-agar plates are typically incubated for 7-10 days at 22 ± 2°C before the colony forming units (CFU) are counted by eye. Results are expressed as the mean number of bacterial CFU per mL of water sample. Variations




x x


selectivity

x x

time to result

44 h (36 °C) and 68 h (22 °C)

Operational specifications ease-of-use (B) x maintenance requirements (C) x Costs instrumentation (C) x operational costs (C)


x

x

Recommendation for use in SSS (D) x Overall conclusion The method detects only a small percentage of the natural

microbial community in a water sample. The nutrient rich medium used in this method contributes to lower HPC counts than other methods.


Several alternative methods for HPC determination are described and standardised. For example, Standard Methods (Clesceri et al., 1998) describe three different methods (spread plate method, pour plate method, membrane filter method) and four different agars (Plate Count Agar; m-HPC agar; R2A agar; NWRI agar). Typical variations on the methods include the use of alternative growth media (e.g. nutrient agar), different incubation temperatures and different incubation time periods. In some cases, colony counting with a magnifying glass or with an automated colony counter has also been promoted. The membrane filter method can be used if the cell density of the sample is too low for direct spread plating. The membrane filters have a diameter of 47 mm and a pore size of 0.2 um. The sample up from 10 mL can be filtered and the membrane filter is placed on one agar plate (m-HPC agar is prescribed in this instance) (Clesceri et al., 1998). For an overview, see Bartram et al., 2003. Equipment and consumables Equipment: Incubator; autoclave Consumables: Sterile Petri dishes and sterile medium (e.g. R2A agar) Status of the technique The method is used for research but not typically included in drinking water directives/legislation. Evaluation: The method has similar disadvantages to the ISO 6222 method. However, several users agree that the R2A displayes higher numbers for HPC bacteria when drinking water is analysed (Reasoner and Geldreich, 1985; Uhl and Schaule, 2004; Berney et al., 2008) References Bartram, J., Cotruvo, J., Exner, M., Fricker, C. and Glasmacher, A. (2003) Heterotrophic plate counts and drinking-water safety. London, UK: IWA Publishing on behalf of the World Health Organization. Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7. Uhl, W. and Schaule, G. (2004) Establishment of HPC (R2A) for regrowth control in non-chlorinated distribution systems. International Journal of Food Microbiology, 92: 317 – 325. Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F. Hammes. Rapid, cultivation-independent assessment of microbial viability in drinking water. Accepted for publication in Water Research, July 2008


Monitoring technology nr 2: R2A method





x x


selectivity

x x

time to result

7 – 10 days

Operational specifications ease-of-use (B) x maintenance requirements (C) x Costs instrumentation (C) x operational costs (C)


x

x

Recommendation for use in SSS (D) x Overall conclusion The method detects only a small percentage of the natural

microbial community in a water sample. It is generally giving higher numbers for drinking water HPC bacteria compared to the ISO 6222 method.


3.5 Enteroviruses Prepared by: EAWAG Required technical specifications: Enteroviruses are widely present in water environments Levels of contamination are widely unknown, also because molecular methods, which are increasingly used, are not quantitative. Concentrations in freshwaters (Guillot, 2006): 1-100 pfu/L in contaminated surface water 1-10 pfu/100 L in less polluted surface water 1-10 pfu/1000 L in treated drinking water Since the infectious dose is very low (1-10 infectious particles), detection methods should be very sensitive. Monitoring technologies: 1. Cell culture methods 2. RT-PCR

3.5.1 Monitoring technology nr 1: Cell culture methods

Description: Concentration: - Adsorption/elution technique using positively charged filters or electronegative filters (APHA, 1998) - Ultrafiltration - Flocculation (APHA, 1998) - Tangential flow ultrafiltration (Bigliardi et al., 2004) Detection: Cell cultures for most enteric viruses are commercially available (Fout et al., 1996). Viruses are grown in cell culture monolayers and form visible plaques or other visible changes to infected cells in the monolayer. Evaluation: The cell culture detection method is regarded as the “golden standard”. However, it is time consuming (6-15 days) and requires a lot of expertise in order to obtain reliable results.


Monitoring technology nr 1: Cell culture methods Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

The method is rather time consuming: 6 - 15 days


ease-of-use (B) x Requires expertise with cell cultures

maintenance requirements (C) x Costs



x

x


Overall conclusion The cell culture detection method is regarded as the “golden standard”. However, it is time consuming and requires a lot of expertise in order to obtain reliable results.


3.5.2 Monitoring technology nr 2: RT-PCR

Description: Concentration: As above Detection Many studies report the detection of different enteric viruses using RT-PCR (for a review see Guillot, 2006)). Integrated-RT-PCR procedures have also been used (Guillot, 2006). Evaluation: Primers for all different kinds of enteric viruses are available. The method generally has a low detection limit and can be performed rapidly. Humic substances or other compounds present in the water sample can interfere with the PCR reaction resulting in a false-negative result. Furthermore, conventional RT-PCR methods are not quantitative and do not detect infectivity.


Monitoring technology nr 2: RT-PCR

(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems References APHA, AWWA, et al. (1998). Standard Methods for the Examination of Water and Wastewater. 20th ed. Bigliardi, L., Cesari, C., Zoni, R., Sansebastiano, GE. (2004). The concentration of viruses in water using the tangential flow ultrafiltration. Recovery effectiveness in experimental conditions. Ann Ig. 16 (1-2): 281-9. Fout, GS., Schaefer III, FW., Messer, JW., Dahling, DR., Stetler, RE. (1996). ICR microbial laboratory manual. Washington, D.C., U.S. Environmental Protection Agency. Guillot, E., Loret, J.F. (2006). Waterborne Pathogens. London: Global Water Research Coalition.




drinking water

x x


selectivity

x

x

time to result

Time to result 4h, rapid method





x x

Recommendation for use in SSS (D) x needs skilled personel

Overall conclusion The method can be performed rapidly. Humic substances or other compounds present in the water sample can interfere with the PCR reaction resulting in a false-negative result. Furthermore, conventional RT-PCR methods are not quantitative and do not detect infectivity.


3.6 Giardia/Cryptosporidium Prepared by: Kiwa Water Research Giardia is a genus of protozoan parasites potentially found in water and other media. The recent taxonomy of the genus Giardia includes the following species and their potential hosts: G. lamblia (also called G. intestinalis or G. duodenalis; humans and other mammals); G. muris (rodents); G. agilis (amphibians); G. psittaci and G. ardeae (birds). As with Cryptosporidium, the parasite is shed with the faeces as environmentally robust cyst that is transmitted to a new host. Waterborne outbreaks of giardiasis have been reported for almost 30 years and in the US it is the most commonly identified pathogen with more than 100 waterborne outbreaks (Craun 1990). In the last decade most attention on parasitic pathogens in water was focused on Cryptosporidium because of its higher resistance to chlorine the most common drinking water disinfectant. Required technical specifications: There are no technical specifications required with respect to Cryptosporidium in the official European drinking water standards. In the EU directive on drinking water (Council Directive 98/83/EC) the minimum requirement is that water intended for human consumption shall be wholesome and clean defined as free from any micro-organisms and parasites and from any substances which, in numbers or concentrations, constitute a potential danger to human health. Consequently, the assumption is made that compliance with the standards of the Directive will satisfy this minimum requirement. One of the standards directly related to Cryptosporidium is the standard for C. perfringens (including spores) of 0/100 ml drinking water. Monitoring of this parameter is not required “unless the water originates from or is influenced by surface water. In the event of non-compliance with this parametric value, the Member State concerned must investigate the supply to ensure that there is no potential danger to human health arising from the presence of pathogenic micro-organisms, e.g. Cryptosporidium. Member States must include the results of all such investigations in the reports they must submit under Article 13(2).” Note 2, Annex 1, Part C. Countries with Cryptosporidium monitoring requirements The only country where a monitoring requirement for Cryptosporidium in drinking water has been regulated, is the United Kingdom (Anonymous 1999; Anonymous 2000). This regulation sets a standard of <1 oocyst per 10 liter of drinking water to be monitored continuously at a rate of 40 liter/h at least 23 hours a day. With respect to the analysis requirements in the UK regulation there is a Standard Operational Procedure (SOP) prescribed with no requirement for the assessment of infectivity, species identity and recovery of the analysis. A recovery of 30-60% is assumed for this SOP. As to the concentration, however, strict procedures have been documented in the UK-regulations for confirmation of concentrations of ≥ 0.5 oocysts per 10 liter of drinking water.


A risk-based approach for Cryptosporidium in drinking water is regulated in the United States and the Netherlands. In the US the Long Term 2 Enhanced Surface Water Treatment Rule (LT2) (USEPA 2006) source water monitoring is required to quantify the decree of log reduction (removal or inactivation) required to protect public health. The required analytical technique for Cryptosporidium analysis in the source water is similar to the SOP of the UK regulation in that they prescribe specific approved and validated methods with ongoing proficiency testing for quality assurance/quality control (QA/QC; (Clancy and Hargy 2008)) This method 1622 (US; www.epa.gov/microbes) was originally developed for 10 liter samples but has been adapted for larger volumes of 50 – 1000 liter (McCuin and Clancy 2003). The risk-based approach in the Netherlands on Cryptosporidium requires source water monitoring to assess treatment requirement followed by assessment of treatment elimination efficiency (removal and inactivation) and drinking water exposure. Water suppliers must demonstrate compliance with an annual infection risk level of <10-4. For analytic requirements the regulation (AMVD, 2005) refers to the SOP used by KWR (formerly known as: Kiwa Water Research) and RIVM. Monitoring technologies:

1. Method 1622. Cryptosporidium detection method. (USEPA 2006) published by US Environmental Protection Agency (EPA; www.epa.gov/microbes)

2. Supplements of the UK Water Supply Regulation (Anonymous, 1999;2000) published by the Drinking Water Inspectorate (DWI; www.dwi.gov.uk)

3. Method 1623. Cryptosporidium and Giardia detection method. (USEPA 2006) published by US Environmental Protection Agency (EPA; www.epa.gov/microbes)

4. ISO 15553: Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water

5. Method 1622/1623 using Cross flow ultrafiltration (Hemoflow-filter)

3.6.1 Monitoring technology nr 1: Method 1622

Summary of the method (USEPA, 2006) A water sample is filtered and the oocysts and extraneous materials are retained on the filter. Although EPA has only validated the method using laboratory filtration of bulk water samples shipped from the field, field-filtration also may be used. Materials on the filter are eluted and the eluate is centrifuged to pellet the oocysts, and the supernatant fluid is aspirated. The ocysts are magnetized by attachment of magnetic beads conjugated to anti-Cryptosporidium antibodies. The magnetized oocysts are separated from the extraneous materials using a magnet, and the extraneous materials are discarded. The magnetic bead complex is then detached from the oocysts. The oocysts are stained on well slides with fluorescently labeled monoclonal antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined using fluorescence and differential interference contrast (DIC) microscopy. Qualitative analysis is performed by scanning each slide well for


objects that meet the size, shape, and fluorescence characteristics of Cryptosporidium oocysts. Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic separation (IMS), staining, and microscopy systems. Since the interlaboratory validation of EPA Method 1622, inter laboratory validation studies have been performed to demonstrate the equivalency of modified versions of the method using the following components (USEPA, 2006):

- IDExx Filta-Max® filter - Pall Gelman Envirochek™ HV filter - Portable Continuous-Flow Centrifugation (PCFC) - Waterborne Aqua-Glo™ G/C Direct FL antibody stain - Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains - BTF EasyStain™ antibody stain - BTF EasySeed™ irradiated oocysts for use in routine QC samples

3.6.2 Monitoring technology nr 2: UK method

This method is very similar to the method 1622 (Clancy and Hargy 2008).

3.6.3 Monitoring Technology nr. 3: method 1623

This method is similar to the method 1622.

3.6.4 Monitoring technology nr 4: ISO 15553

This method is similar to the method 1622.

3.6.5 Monitoring technology nr 5: cross flow ultrafiltration

This method is identical to the method 1622/1623 with respect to the treatment of the water concentrate and microscopical counting of the (oo)cysts. The method uses cross flow ultrafiltration (hemo-flow) for concentration of the particulates (all microbes) in the water and was first described by Simmons et al. (2001). The method was further developed for on site sampling (Veenendaal and Brouwer-Hanzens 2007). Evaluation (based on Medema, 2008): Recovery An important drawback of the current concentration methods is that many factors in the water matrix (suspended solids, algae) and also age/history of the oocysts can have significant effect on the recovery efficiency. Ideally, the recovery efficiency is determined for every sample. Since this is laborious and expensive, recovery efficiency data are usually collected from a subset of samples. (Warnecke, Weir et al. 2003) describe the use of pre-stained oocysts that can be discriminated from natural oocysts for seeding of every sample. Viability/infectivity Another drawback of the immunofluorescence detection assay is that it does not allow differentiation of viable from dead oocysts. DIC microscopy can be used to determine if the internal morphology is compromised as an indication of non-viability. DAPI (diaminophenylindole)-staining is used as support-


stain that allows the assessment of the presence of sporozoites in the oocyst, again as mark of viability. Vital staining (PI (Campbell, Robertson et al. 1992) or Syto59 (Belosevic and Finch 1997)) can be used in combination with the IFA test and gives an indication of cell membrane integrity. These dye exclusion assays provide some information about viability, but should be used with caution as they can (largely) overestimate viability of oocysts that have been exposed to stressors such as exposure to UV light (Clancy, Hargy et al. 1998). Another assay to assess the viability of Cryptosporidium oocysts is cell culture. Specificity The specificity of the immunofluorescence assay is based on the specificity of the monoclonal antibody-antigen reaction. Although this is highly specific, non-specific binding is observed in natural samples. Many of the particulates that react with the monoclonal antibody can be discriminated from oocysts by a trained observer, but occasionally particles (algae) occur in samples that are very difficult to discriminate from oocysts. This may lead to false-positive results. The immunofluorescence method is also not specific to Cryptosporidium species and genotypes that are infectious to humans, also species that are infectious to animals are detected. Molecular techniques (PCR, genotyping) are rapidly evolving and some laboratories are now using these methods for environmental monitoring (Xiao, Alderisio et al. 2001; LeChevallier 2004; Xiao, Bern et al. 2004; Heijnen, Wullings et al. 2005). References AMVD (2005). Inspection Guidance Document 'Analysis of the

microbiological safety of drinking water'. Ministry of Public Housing, Spatial Planning and the Environment. [In Dutch].

Anonymous (1999). Water Supply (Water Quality) (Amendment) Regulations, SI 1524. Stationery Office, London, UK.

Anonymous (2000). Water Supply (Water Quality) Regulations, SI 3184. Stationery Office, London UK..

Belosevic, G. M. and G. F. Finch (1997). Int. Symp on Waterborne Cryptosporidium, Newport Beach Ca, USA.

Campbell, I., L. J. Robertson, et al. (1992). "Viability of Cryptosporidium parvum oocysts - correlation of in vitro exystation with inclusion or exclusion of fluorgenic vital dyes.." Appl. Environ. Microbiol. 58: 3488-3493.

Clancy, J. F. and T. M. Hargy (2008). Waterborne: drinking water. Cryptosporidium and Cryptodiosis. R. Fayer and L. Xiao. Boka Raton London New York, CRC Press: 305-326.

Clancy, J. L. (2000). "Sydney's 1998 water quality crisis." J. Am. Water Works Assoc. 92: 55-66.

Clancy, J. L., T. M. Hargy, et al. (1998). "UV light inactivation of Cryptosporidium oocysts." J. Am. Water Works Assoc. 90(9): 92-102.

Craun, G. F. (1990). Waterborne giardiasis.. Human parasitic diseases. E. A. Meyer. Amsterdam, the Netherlands, Elsevier Science Publ. 3: 267-293.

Heijnen, L., B. Wullings, et al. (2005). "Genetic analysis of Cryptosporidium oocysts from surface water." Submitted for publication.


LeChevallier, M. W. (2004). Removal of Cryptosporidium and Giardia by water treatment processes. Intern. Cryptosporidium and Giardia Conf., Amsterdam, the Netherlands.

McCuin, N. E. and J. L. Clancy (2003). "Modification of USEPA method 1622 and 1623 for detection of Cryptosporidium oocysts and Giardia cysts in water." Appl. Environ. Microbiol. 69: 267-274.

Medema, G.J., Teunis, P.F.M., Blokker, M., Deere, D., Davison, A., Charles, P. and Loret, J.F. (2008) WHO Guidelines for Drinking Water Quality: Risk Assessment of Cryptosporidium in drinking water. London UK: World Health Organization.

Simmons III, Otto D., Mark D. Sobsey, Christopher D. Heaney, Frank W. Shaefer III and Donna S. Francy(2001). “Concentration and Detection of Cryptosporidium oocysts in Surface Water Samples by Method 1622 Using Filtration and Capsule Filtration”. Applied and Environmental Microbiology vol. 67, No.3, p. 1123-1127

USEPA (2006). "National primary drinking water regulations, long term 2 enhanced surface water treatment rule. Final Rule. Federal Register 40 CFR Parts 9, 141, and 142."

Veenendaal, H. R. and A. J. Brouwer-Hanzens (2007). A method for the concentration of microbes in large volumes of water. Techneau Deliverable D3.2.4.

Warnecke, M., C. Weir, et al. (2003). "Evaluation of an internal positive control for Cryptosporidium and Giardia testing in water samples.." Lett. Appl. Microbiol. 37(3): 244-248.

Xiao, L., K. Alderisio, et al. (2001). "Identification of species and soources of Cryptosporidium oocysts in storm waters with a small-subunit rRNA-based diagnostic and genotyping tool.." Appl. Environ. Microbiol. 66: 5492-5498.

Xiao, L., C. S. Bern, I.M., et al. (2004). Molecular epidemiology of human cryptosporidiosis. Cryptosporidium: from molecules to disease.. R. C. A. Thompson, A. Armson and U. M. Ryan. Amsterdam, the Netherlands, Elsevier.


Monitoring technology nr 1,2,3,4,5: Method 1622, 1623, ISO15553





drinking water

x

x

Recovery of the method has been improved over time but still is low and variable (≥50%)


selectivity

x

x

The method requires well-trained analysts (microscopic counting and oocyst identification); no differentiation in species/infectivity

time to result

The method is time consuming


ease-of-use (B) x Once trained, the method is easy to perform for analysts.

maintenance requirements (C) x Due to the high variability internal QC is required

Costs

instrumentation (C) x IF Microscopy operational costs (C)


x

x


Overall conclusion Available methods to quantify (oo)cysts in water samples have been optimized to an acceptable level of accuracy and reproducibility when pre-cautions such as recovery assessments are implemented in monitoring. Methods to specify the observed (oo)cysts and to verify the infectivity though are more comprehensive and only advisable for research objectives rather than routine monitoring.


3.7 Thermotolerant Campylobacter species Prepared by: TZW Required technical specifications: For the detection and enumeration of thermotolerant Campylobacter species in drinking water sample volumes of 10 mL, 100 mL and 1000 mL are recommended to be analysed (ISO 17995:2005). Monitoring technologies: 1. Detection and enumeration of thermotolerant Campylobacter species (ISO 17995:2005)

3.7.1 Monitoring technology nr 1: Detection and enumeration of thermotolerant Campylobacter species (ISO 17995:2005)

Description: The International Standard specifies a method for detection and semi quantitative enumeration of thermotolerant Campylobacter species. The method can be applied to all kinds of filterable waters. The method can also be used as a presence/absence test in a specified sample volume. A quantitative result can be obtained by using a MPN set-up (see ISO 8199). Thermotolerant Campylobacter species of relevance in human infections include C. jejuni, C. coli, C. lari and C. upsaliensis. Method and mode of action Thermotolerant Campylobacter species are detected and enumerated by membrane filtration and subsequent incubation in 2 enrichment broths (highly and less selective) under microaerobic conditions as described in ISO 17995. Following incubation, inoculum from each broth is streaked onto selective solid medium (mCCDA) and incubated under microaerobic conditions. Campylobacter species require enriched substrates for optimal growth and they prefer an atmosphere containing approx. 5 % oxygen and approx. 10 % CO2. They are sensitive to toxic oxygen derivates like peroxides, which can arise in media exposed to light and oxygen. Colonies resembling Campylobacter species are tested for aerobic growth and, if negative, examined by microscopy for motility and characteristic morphology. If necessary, further biochemical tests are performed. Procedure and evaluation Sample volumes of 10 mL, 100 mL and 1000 mL are filtered through a membrane filter. For each volume 2 filters are prepared and immediately transferred to highly (Preston) and less (Bolton) selective enrichment broth. Both broths are incubated in modified atmosphere at 37 ± 1°C for 44 ± 4 hours leaving caps open. After incubation, approx. 10 µL inoculum from each broth is transferred onto selective mCCDA plates. mCCDA plates are incubated at modified atmosphere at 41,5 ± 1°C and checked for visible growth after 44 ± 4 hours. Suspect colonies from mCCDA are transferred to 2 non-selective agar plates. Plates are incubated aerobically and in modified atmosphere at 41,5 ± 1°C for 21 ± 3 hours. Campylobacter species grow under microaerobic but not


under aerobic conditions. The presence of Campylobacter species is confirmed by microscopy. Campylobacter species are highly motile, slender rods with spiral morphology. In doubt, further tests (oxidase, catalase and Gram stain) are necessary for verification. Campylobacter species are oxidase and catalase positive and Gram-negative. The results are reported as detected in the sample volume examined, whether the Campylobacter species are demonstrated in one or in both enrichment broths. A semi quantitative estimate of the numbers is made from results with different test volumes. Equipment and consumables As described in ISO 17995 (e.g. microscope, autoclave, incubators, equipment for membrane filtration and microaerobic incubation, glassware, Petri dishes, usual laboratory equipment, culture media, diluents and reagents). References Anon. (2005) ISO 17995: Water quality - Detection and enumeration of thermotolerant Campylobacter species (ISO 17995:2005). Geneva, Switzerland: International Organisation for Standardisation. Anon. (2005) ISO 8199: Water quality - General guide to the enumeration of microorganisms by culture (ISO 8199:2005). Geneva, Switzerland: International Organisation for Standardisation. Evaluation: The method ISO 17995 for the detection and semi quantitative enumeration of thermotolerant Campylobacter species is suitable for clean drinking waters and also for dirty surface waters. The method is very laborious and costs for consumables are higher compared to other culture-based methods e. g. for the detection and enumeration of. E. coli or Enterococci. Furthermore, more expensive laboratory equipment like a microscope and incubators for microaerobic cultivation is required. Some Campylobacter species are pathogenic to man and therefore isolation and identification must be carried out by trained laboratory staff in a properly equipped laboratory. Time to result is 5 days.


Monitoring technology nr 1: ISO 17995:2005 Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

Campylobacter species are very sensitive to adverse conditions

time to result

5 days





x x


Overall conclusion Method is laborious and requires a well equipped microbiological laboratory and experienced and well trained staff. Method is suitable for all kinds of waters.



3.8 Legionella and Legionella pneumophila Prepared by: Kiwa Water Research Required technical specifications: 0 - 106 cfu per 250 ml in drinking water 0 - 106 cfu per 100 ml in water from cooling towers or surface water The required resolution of the parameter is: 100 cfu in litre drinking water (Dutch legislations) Monitoring technologies: 1. ISO method 11731:1998: Detection and enumeration of Legionella by direct membrane filtration and cultivation on agar medium; 2. Detection and quantification of Legionella pneumophila with the Quantitative Polymerase Chain Reaction (Q-PCR)

3.8.1 Monitoring technology nr 1: ISO method 11731:1998

Description: Bacteria in a water sample are concentrated by membrane filtration or by centrifugation. To reduce the growth of unwanted bacteria, a portion of the concentrated sample is subjected to treatment with acid or heat. Treated and untreated concentrated sample are then inoculated onto plates of agar medium (semi)-selective for Legionella. Plates are incubated at 37 °C for 7 days. After incubation, morphologically characteristic colonies which form on the selective medium are to be confirmed as Legionella by subculture to demonstrate their growth requirements for L-cysteine and iron. The culture and subculture of Legionella will require 10 days before results are available. To perform the ISO culture method no specific equipment, other than standard microbiological laboratory equipment, is needed. The ISO method is the generally accepted method and agar medium is commercially available. Evaluation: The culture method is the standard method generally used to enumerate Legionella in water. It uses semi-selective GVPC or BCYE agar medium to culture Legionella. In water samples containing a high microbiological background, the agar medium is likely to be overgrown by other bacteria then Legionella. The method is therefore less useful for water samples from cooling towers or surface water.


Monitoring technology nr 1: ISO method 11731:1998 Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x x

time to result

10 days





x

x


Overall conclusion Standard accepted method but faster and more specific

method is needed. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

3.8.2 Monitoring technology nr 2: Q-PCR for Legionella pneumophila

Description: The Q-PCR method is a molecular detection technique which amplifies specific Legionella pneumophila DNA. The method targets the human pathogen Legionella pneumophila which is responsible of more than 90% of the cases of Legionellose. The results are given as DNA copies per litre and are available with 4 hours. The detection and quantification consists of three phases: (1) concentration of the water sample by membrane filtration, (2) lyses of the bacteria, DNA extraction and purification of the DNA, and (3) amplification of the specific DNA fragment PCR and real-time quantification of the amplified DNA using a probe. An internal control is included in the method and added after filtration to each sample. The result of this internal control is used to give insight in the recovery of the DNA isolation and possible inhibition of the DNA amplification. A Real-time PCR machine is required to perform the Q-PCR analysis. Laboratory technicians should have experiences in applying molecular techniques in the laboratory. The method is submitted to ISO for acceptation. Adaptation of legislation will be necessary for application of this DNA based method. However, it is expected that the approval of this method will occur in the near future.


Evaluation: In general, the method is accurate, specific and robust. The detection results are available within a few (4) hours, this in comparison with the 7 days required for the standard culture method. The quantification if based on a DNA standard. The results are therefore given in DNA copies per litre. For risk analysis it can be necessary to know if recent disinfections have been carried out. Heat disinfection can result in uncultivable but PCR detectable DNA copies. The method is some what sensitive to inhibition of the PCR or loss of DNA during DNA isolation. However, the result of the internal control quantitatively shows the overall performance of the analysis per sample. In a few years, this method will presumably be used as the standard detection method for Legionella pneumophila. The Q-PCR results are available within one day. If necessary, within 24 hours after sampling the samples can additionally be analyzed with the culture method. Monitoring technology nr 2: Q-PCR for Legionella pneumophila Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x

x


selectivity

x

x

time to result

result within 4 hours





x x

Recommendation for use in SSS (D) x needs skilled personel Overall conclusion Fast, reliable, and sensitive detection method for the

detection of Legionella pneumophila in water. The method detects specific Legionella pneumophila DNA in water samples, but no differentiation between live and dead cells is possible



3.9 Pseudomonas aeruginosa Prepared by: Vermicon AG P. aeruginosa is a gram-negative, rod-shaped, none spore-forming, aerobic bacterium with low nutrient requirements. The optimal growth temperature is 37 °C . It is a known biofilm-forming bacterium and therefore used as an indicator for deficiencies within cleaning processes. Moreover, this opportunistic pathogenic micro-organism can cause infection of wounds in immune-suppressed and elderly persons. Pseudomonas aeruginosa is a bacterium that is naturally found in many types of drinking water and water used for human consumption. For example, it is a violation of European regulations to have Pseudomonas aeruginosa present in a 250 ml bottled mineral water. However, no comparable regulation exists in the United States. Apparently, the Pseudomonas aeruginosa regulation in Europe originated as a quality control issue and not as a health effects issue. Nevertheless, during the last decade, a number of papers have been published that indicate that Pseudomonas aeruginosa in hospitals can be a health threat, but that it is normally no health risk if it is present in drinking water (Hardalo, C. and Edberg, S.C, 1997). Required technical specifications: The analysis is only necessary in the case of water offered for sale in bottles or containers. Limit value for P. aeruginosa according to the European Council Directive 98/83/EC of November 1998 is 0 cells/250 ml. The guidelines for the detection is EN ISO 16266. For health protection reasons, occasionally water for human consumption or drinking water is analysed on this microbiological parameter, too. References Hardalo, C. and Edberg, S.C., Pseudomonas aeruginosa: Assessment of Risk from Drinking Water, Critical Reviews in Microbiology, 23(1):47-75 (1997). Council Directive 98/83/EC of November 1998 on the quality of water intended for human consumption. Official Journal of the European Communities. Monitoring and confirmation technologies:

1. Filtration and cultivation according to EN ISO 16266 2. VIT-Pseudomonas aeruginosa 3. API-Test, confirmatory test

3.9.1 Monitoring technology nr. 1: Filtration and cultivation according to EN ISO 16266

Method Filtration of 250 ml water on a cellulose-nitrate membrane filter (0,45 µm pore size and 50 mm diameter), subsequent incubation of the membrane filter


under aerobic conditions on a cetrimid containing selective-agar (CN-Agar) at 36 +/- 2 °C for 44 +/- 4 hours. All colonies that show a blue-green pigment (pyocyanin) are regarded as confirmed P. aeruginosa colonies. Subsequently the filter is exposed for a short time to UV-light. All colonies that show fluorescence and but do not produce pyocyanin are regarded as suspicious colonies and have to be confirmed by the acetamid utilization test. Additionally not fluorescent, red-brown pigmented colonies are also suspicious and have to be confirmed by an oxidase-test, acetamid utilization and a fluorescein formation test. Equipment and consumables

• Membrane filtration manifold, sterile filter funnels • Vacuum pump with moisture trap or protective filter, or

alternative vacuum force • Cellulose–nitrate-membrane filter (0,45 µm pore size and 50

mm diameter) • Incubator: 36 +/- 2 °C • CN-agar • NB (Nutrient Broth)-agar • King´s B medium • Oxidase-test stripes • acetamide-solution

Evaluation: The method according to EN ISO 16266 is the only accepted method. But the procedure for the confirmation of colonies is very time-consuming (up to 5 days).


Monitoring technology nr. 1: Filtration and cultivation according to EN ISO 16266




drinking water

- x

not applied


selectivity

x x

Experience is needed

time to result

Up to 5 days



instrumentation (C) x Standard water laboratory equipment

operational costs (C) consumables maintenance

x x


Overall conclusion method is time-consuming when colonies have to be confirmed


3.9.2 Monitoring technology nr. 2: VIT-Pseudomonas aeruginosa

Description: For the application of VIT-Pseudomonas aeruginosa a pre-enrichment of the water sample in Malachit green broth is necessary (48 h, incubation at 37 °C). An aliquot (2 ml) of a suspicious broth (turbid, colour change into yellow) is centrifuged, fixed and analysed with VIT-Pseudomonas aeruginosa. Additionally the kit can be used as a confirmatory test for colonies.

Material & Method 5 µl of the sample (pre-enrichment or fixed colony) is placed on each well of a slide. The sample is fixed on the slide and the VIT-solution is added to the sample. This solution contains specific oligonucleotide probes for P. aerugionosa, labeled with fluorescent dyes. During an incubation step (90 min, at 46 °C) the probes bind specifically to their matching signatures on the genetic material (16S rRNA). Following this a stringent washing step removes all unbound and surplus oligonucleotide probes from the cells (15 min, 46 °C). The results are evaluated using a fluorescence microscope, whereby P.


aeruginosa, if present in the sample lights up specifically. The test is not providing a quantitative result. Equipment and consumables:

- medium (malachitgreen-broth), agar plates (CN-agar) - mini-centrifuge - micro-pipettes and tips - incubator 37 °C and 46 °C +/- 2 °C - VIT-adapted fluorescence microscope

Evaluation: By the application of VIT-Pseudomonas the results is available within 2 days, but the analysis is not quantitative. Special knowledge is not required for the performance of the test. The application of the test is not officially accepted by a directive, but comprehensive and numberous studies were performed and the reliability of the test was confirmed. Moreover, the test can also be applied as a confirmation test for colonies. Monitoring technology no. 2: VIT-Pseudomonas aeruginosa




drinking water

- x


selectivity

x x

time to result

2 days



instrumentation (C) x Micro-centrifuge, Fluorescence microscope


x

x


Overall conclusion Easy-to-use rapid test, can be used in addition as confirmation test for colonies, not quantitative



3.9.3 Confirmation technology nr. 1: API-Test 20E

API-Test 20 E (Biomerieux) is a screening test for the identification of Enterobacteriaceae and other gram-negative rods. The biochemical test is a confirmatory test for colonies and identification is possible within 18-24 hours. For the application of this test a pure culture is needed. Thus the test can be applied after the subculture of a colony as an additional test for the confirmation of the result. Evaluation: For the application of the API-test a pure culture is needed and the test can be used instead of tedious typical confirmatory tests.


3.10 Aeromonas Prepared by: RTU Required technical specifications: The concentration range that should be covered for this parameter is 1 cell/100 ml. There are no current regulations in Europe but indicative values in The Netherlands (20 CFU/100 ml as a median value over a 1-year period in the treated drinking water and 200 CFU/100 ml as the 90th-percentile value of the Aeromonas counts of drinking-water in the distribution system in a 1-year period) In the US Aeromonas are monitored since 2003. A minimum reporting level of 0.2 CFU/100 ml is set (source: EPA). EPA Method 1605 is required. Sensitivity/resolution required is 1 cell. Monitoring technologies: 1. EPA Method 1605 2. MALDI 3. PCR

3.10.1 M onitoring technology nr 1: EPA Method 1605

Description: Aeromonas is a common genus of bacteria indigenous to surface waters, and may be found in non-chlorinated or low-flow parts of chlorinated water distribution systems. Monitoring their presence indistribution systems is desirable because some aeromonads may be pathogenic and pose a potentialhuman health risk. Method 1605 describes a membrane filtration technique for the detection and enumeration of Aeromonas species. This method uses a selective medium that partially inhibits thegrowth of non-target bacterial species while allowing most species of Aeromonas to grow. Aeromonas is presumptively identified by the production of acid from dextrin fermentation and the presence of yellowcolonies on ampicillin-dextrin agar medium with vancomycin (ADA-V). Yellow colonies are counted and confirmed by testing for the presence of cytochrome c (oxidase test), and the ability to ferment trehalose and produce indole. This method is adapted from Havelaar et al. (1987) for the enumeration of Aeromonas species in finished water by membrane filtration. The method provides a direct count of Aeromonas species in water based on the growth of yellow colonies on the surface of the membrane filter using a selective medium. A water sample is filtered through 0.45-µm-pore-size membrane filter. The filter is placed on ampicillin-dextrin agar with vancomycin (ADA-V) and incubated at 35°C ± 0.5°C for 24 ± 2 hours. This medium uses ampicillin and vancomycin to inhibit non-Aeromonas species, while allowing most Aeromonas species to grow. It is a quantitative assay that uses a selective medium which partially inhibits the growth of non-target bacterial species while allowing Aeromonas to grow. Aeromonas is presumptively identified by the production of acid from dextrin fermentation producing yellow colonies. Presumptively positive colonies are counted and confirmed by testing for the


presence of cytochrome c (oxidase test), and the ability to ferment trehalose, and produce indole. Equipment and consumables The method is quite inexpensive to perform. It requires only the basic equipment of any laboratory. The prices of the growth media can vary, depending on the selected method. The price of consumables for further identification should be added. Status of the technique A traditional method, widespread for drinking water analysis. Guidelines In U.S.A. EPA Method 1605 is used and the minimum reporting level is set to 0.2 CFU/100ml. The health authorities in the Netherlands in 1985 introduced “indicative maximum values” for Aeromonas densities in drinking-water. The values were based on a national survey of aeromonads in drinking-water in the Netherlands and have been defined as follows: 20 cfu/100 ml as a median value over a 1-year period in water leaving the treatment facility; 200 cfu/100 ml as the 90th-percentile value of the Aeromonas counts of drinking-water collected from the distribution system in a 1-year period (Trouwborst, 1992). References: 1. Havelaar, A.H., M. During, and J.F.M. Versteegh. 1987. Ampicillin-

dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration. Journal of Applied Microbiology. 62:279-287.

2. Trouwborst T (1992). Overheidsbeleid ten aanzien van het voorkomen van Aeromonas in drinkwater. [Government policy with regard to occurrence of Aeromonas in drinking-water.] In: van der Kooij D, ed. Aeromonas in Drinkwater: Voorkomen, Bestrijding en Betekenis. [Aeromonas in drinking water: occurrence, control and significance.] Nieuwegein, Kiwa NV: 95–104.

Evaluation: It is a simple, inexpensive method that requires basic routine bacteriology laboratory facilities. Water samples containing colloidal or suspended particulate material may clog the membrane filter and prevent filtration or cause spreading of bacterial colonies which could interfere with identification of target colonies. Other ampicillin/vancomycin resistant bacteria that are not aeromonads may be able to grow on this medium. Some of these bacteria may also produce yellow colonies if they are able to produce acid byproducts from the fermentation of dextrin or some other media component, or if they produce a yellow pigment. Enterococci are reported to produce pinpoint-size yellow colonies on ADA. Confirmation of presumptive Aeromonas colonies is necessary to mitigate false positives.


Monitoring technology nr 1: EPA Method 1605




drinking water

x x


selectivity

x

x

time to result

1-2 days





x

x


Overall conclusion Not complicated but laborious method. Robust, does not

require skilled personnel. An additionl identification of the positive colonies may be necessary.


3.10.2 Monitoring technology nr 2: MALDI

Description: Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules and large organic molecules, which tend to be fragile and fragment when ionized by more conventional ionization methods. The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization. The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-flight mass spectrometer), mainly due to its large mass range. MALDI-MS was used to analyze the whole cells of both reference strains and unknown Aeromonas isolates obtained from water distribution systems (Donohue, et al., 2007). A library of over 45 unique m/z signatures was created from 40 strains that are representative of the 17 recognized species of Aeromonas, as well as 3 reference strains from genus Vibrio and 2 reference strains from Plesiomonas shigelloides. The library was used to help speciate 52 isolates of Aeromonas. The environmental isolates


were broken up into 2 blind studies. Group 1 contained isolates that had a recognizable phenotypic profile and group 2 contained isolates that had an atypical phenotypic profile. MALDI-MS analysis of the water isolates in group 1 matched the phenotypic identification in all cases. In group 2, the MALDI-MS-based determination confirmed the identity of 18 of the 27 isolates. These results demonstrated that MALDI-MS analysis can rapidly and accurately classify species of the genus Aeromonas. Equipment and consumables The equipment is expensive and requires certain skills for operating and analyzing the obtained data. Status of the technique This method is still under development as this is a very recent study. For MALDI-based equipment one of the market leaders is Waters (www.waters.com). References: 1. Donohue, M. J., J. M. Best, A. W. Smallwood, M. Kostich, M. Rodgers,

and J. A. Shoemaker. 2007. Differentiation of Aeromonas isolated from drinking water distribution systems using matrix-assisted laser desorption/ionization-mass spectrometry. Anal Chem 79:1939-46.

Evaluation: This could potentially be a powerful tool especially suited for environmental monitoring and detection of microbial hazards in drinking water, but it can only be used for confirmation.


Monitoring technology nr 2: MALDI




drinking water

x x


selectivity

x

x

time to result





x

x

Recommendation for use in SSS (D) x Overall conclusion This is a very promising method but not enough

verified to be recommended for routine analysis. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

3.10.3 Monitoring technology nr 3: PCR

Description: The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique for exponentially amplifying DNA, via enzymatic replication. Quantitative real time polymerase chain reaction (RT-PCR) is a laboratory technique used to simultaneously quantify and amplify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample; and if it is present, the number of copies in the sample. It is the real-time version of quantitative polymerase chain reaction (Q-PCR), itself a modification of polymerase chain reaction. The procedure follows the general pattern of polymerase chain reaction, but the DNA is quantified after each round of amplification; this is the "real-time" aspect of it. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. There are quite a number of studies which have taken advantage of this technique for Aeromonas detection. As a result of these studies it is apparent that Aeromonas are present in source water (Ormen and Ostensvik, 2001), bottled water (Biscardi, et al., 2002), drinking water (Emekdas, et al., 2006;


Figueras, et al., 2005; Sen and Rodgers, 2004) distribution systems and most of them possess aerolysin gene. They have been shown to be resistant to the first generation beta-lactam antibiotics as well (Emekdas, et al., 2006). Equipment and consumables The equipment varies depending on whether the conventional or real-time approach is chosen. The conventional cycler is not expensive but it must be completed with gel running and imaging system. RT-PCR cycler is considerably more expensive and also there an elctroforesis and imaging unit might be needed (to check for eventual unspecific products). Also the running cost, the price of enzyme, nucleotides, primers, fluorescent dyes and probes etc. has to be considered. Status of the technique This is a quite popular method which, when well designed, gives an opportunity to screen several pathogens (along with Aeromonas) in one run. PCR equipment can be purchased from Applied Biosystems (www.appliedbiosystems.com) and Eppendorf (www.eppendorf.com), among others. References: 1. Biscardi, D., A. Castaldo, O. Gualillo, and R. de Fusco. 2002. The

occurrence of cytotoxic Aeromonas hydrophila strains in Italian mineral and thermal waters. Sci Total Environ 292:255-63.

2. Emekdas, G., G. Aslan, S. Tezcan, M. S. Serin, C. Yildiz, H. Ozturhan,

and R. Durmaz. 2006. Detection of the frequency, antimicrobial susceptibility, and genotypic discrimination of Aeromonas strains isolated from municipally treated tap water samples by cultivation and AP-PCR. Int J Food Microbiol 107:310-4.

3. Figueras, M. J., A. Suarez-Franquet, M. R. Chacon, L. Soler, M.

Navarro, C. Alejandre, B. Grasa, A. J. Martinez-Murcia, and J. Guarro. 2005. First record of the rare species Aeromonas culicicola from a drinking water supply. Appl Environ Microbiol 71:538-41.

4. Ormen, O., and O. Ostensvik. 2001. The occurrence of aerolysin-

positive Aeromonas spp. and their cytotoxicity in Norwegian water sources. J Appl Microbiol 90:797-802.

5. Sen, K., and M. Rodgers. 2004. Distribution of six virulence factors in

Aeromonas species isolated from US drinking water utilities: a PCR identification. J Appl Microbiol 97:1077-86.

Evaluation: This is a rather accepted method which works quite well in drinking water. If there is enough genetic material in the sample, the results can be achieved in a couple of hours or less. If there is not enough cells, pre-enrichment might be necessary which then increases the analysis time. However, the problem


might be the biofilm analysis as this technique is rather sensitive to inhibitors. The equipment, however, is expensive and skills in operating it are needed. Monitoring technology nr 3: PCR





drinking water

x x


selectivity

x

x

time to result





x

x


Overall conclusion A promising method but the equipment is expensive to purchase and run. Skilled personnel is also required. It still needs to be developed prior to application, however, the method has a potential for being used by larger systems in the future


3.11 Bacteriophages Prepared by: Kiwa Water Research Required technical specifications: The concentration range that is normally encountered in water samples is: 0 pfu per 10 ml water sample in drinking water and groundwater 0 to 1000 pfu per ml surface water The required resolution of the method is: 1 pfu in 10 ml water sample Monitoring technologies: 1. ISO method 10705-1: Enumeration of F-specific RNA bacteriophages. 1a. Method to determine the serotype of isolated F-specific RNA bacteriophages 2. ISO method 10705-2: Enumeration of somatic coliphages 3. ISO method 10705-4 (modified): Enumeration of bacteriophages that infect Bacteroides


Description: - The sample is mixed with a small volume of semisolid nutrient medium. A culture of host strain Salmonella typhimurium strain WG49 is added and plated on solid nutrient medium. After this, incubation and reading of agar plates for visible plaques takes place. Plaques can be confirmed on the same medium with added RNAse, which inhibits infection of the host strain by F-specific RNA bacteriophages. The results are expressed as the number of plaque forming units (pfu) per unit of volume. - To perform ISO method 10705-1 an apparatus for sterilization by dry heat or steam, a thermostatic incubator, a water bath, a counting apparatus, a spectrophotometer, a pH-meter and a deep freezer (-20°C ± 5°C) are required. - The method is one of the three ISO-approved methods for the enumeration of bacteriophages (as indicators for enteric viruses) in water. - Reference: ISO 10705-1. Monitoring technology nr 1a: method to determine the serotype of isolated F-specific RNA bacteriophages - A duplex RT-PCR using primers that are specific for the replicase gene of F-specific RNA bacteriophages is performed on isolated F-specific RNA bacteriophages. The DNA strands of the RT-PCR product are separated by heat treatment and the product is added to a miniblotter that contains covalently bound oligonucleotide probes of the replicase gene of each


serotype. After hybridization and washing, bound PCR-product is detected by chemiluminescence and visualized by exposure to X-ray film. - To use this method, a PCR apparatus, a miniblotter, a water bath and an X-ray film are required. - Reference: Vinjé, J. et al. 2004. Molecular detection … blot hybridization. Appl. Environ. Microbiol. 70:5996-6004. Evaluation: - In general, an accurate method to determine the number of bacteriophages (as indicator for enteric viruses) in water samples. The best indication of the presence of enteric viruses is obtained by using this method in combination with methods to determine somatic coliphages and bacteriophages that infect Bacteriodes (Leclerc et al 2000. Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. J. Appl Microbiol. 88:5-21). - From literature it is known that F-specific RNA bacteriophages are a better indicator for the presence of enteric viruses in water than somatic coliphages, but might be a less reliable indicator than bacteriophages that infect Bacteriodes. - Plaques are often vague, making them more difficult to count than plaques of somatic coliphages. - ISO method 10705-1 states that confirmation by added RNAse to the medium should be performed in parallel with the method to enumerate F-specific RNA bacteriophages. In case of low number (1 to 5) of pfu of F-specific RNA bacteriophages it is better to take out the plaque and confirm inhibition of infection by addition of RNAse for each isolated plaque. - Incubation time is rather long, because results are obtained after 18 to 22 hours. However, a fast standard method is not available, although methods based on RNA-detection have been published and might come available in the near future. - Determination of the serotype of isolated F-specific RNA bacteriophages can be used to determine the source of fecal contamination, because with few exceptions serotype II and III have been associated with human waste affected water. Serotype I and IV are associated with animal waste affected water.


Monitoring technology nr 1: ISO-method 10705-1



Description: - The sample is mixed with a small volume of semisolid nutrient medium. A culture of host strain Escherichia coli strain C is added and plated on solid nutrient medium. After this, incubation and reading of agar plates for visible plaques takes place. The results are expressed as the number of plaque forming units (pfu) per unit of volume.




drinking water

x x

Detection limit: 1 bacteriophage in 10 ml water. In combination with concentration of the water sample via Hemoflow, detection limits in drinking water can be 1 bacteriophage in 10 L or even 1 bacteriophage in 2000 L.


selectivity

x

x

time to result

Total incubation time: 18 to 22 h





x x


Overall conclusion Most used method together with the method for somatic coliphages to determine bacteriophages (as indicator for enteric viruses) in water samples. One method is not better over the other and it is advised to determine F-specific RNA bacteriophages, somatic coliphages and bacteriophages that infect Bacteroides. Serotyping isolated F-specific RNA bacteriophages can be used to determine the source (human versus animal) of faecal contamination.


- To perform ISO method 10705-2 an apparatus for sterilization by dry heat or steam, a thermostatic incubator, a water bath, a counting apparatus, a spectrophotometer, a pH-meter and a deep freezer (-20°C ± 5°C) are required. - The method is one of the three ISO-approved methods for the enumeration of bacteriophages (as indicators for enteric viruses) in water. - Reference: ISO 10705-2. Evaluation: - In general, an accurate method to determine the number of bacteriophages (as indicator for enteric viruses) in water samples. The best indication of the presence of enteric viruses is obtained by using this method in combination with methods to determine somatic coliphages and bacteriophages that infect Bacteriodes (Leclerc et al 2000. Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. J. Appl Microbiol. 88:5-21). - From literature it is known that somatic coliphages are slightly less reliable as indicator for the presence of enteric viruses in water than F-specific RNA bacteriophages or bacteriophages that infect Bacteriodes. - Plaques are clear, making them easier to count than plaques of F-specific RNA bacteriophages. - Incubation time is rather long, because results are obtained after 18 to 22 hours. However, a fast standard method is not available.




3.11.3 Monitoring technology nr 3: ISO method 10705-4 (modified)

Description: - The sample is mixed with a small volume of semisolid nutrient medium. A culture of host strain Bacteroides is added and plated on solid nutrient medium. After this, incubation and reading of agar plates for visible plaques takes place. The results are expressed as the number of plaque forming units (pfu) per unit of volume. -The Bacteroides strains that have been used as host strains are Bacteroides fragilis RYC2056 (detection of bacteriophages from animal and human sources; this strain is mentioned in ISO 10705-4), Bacteroides




drinking water

x x



selectivity

x

x

time to result






x x


Overall conclusion Most used method together with the method for F-specific RNA bacteriophages to determine bacteriophages (as indicator for enteric viruses) in water samples. One method is not better over the other and it is advised to determine F-specific RNA bacteriophages, somatic coliphages and bacteriophages that infect Bacteroides.


thetaiotaomicron GA17 (detection of bacteriophages from human sources; Blanch, A. R. et al. 2006, Appl. Environ. Microbiol. 72:5915-5926), Bacteroides ovatus GB124 (detection of bacteriophages from human sources; Blanch, A. R. pers. comm.) and Bacteroides thetaiotaomicron HB13 (detection of bacteriophages from human sources; Blanch, A. R. pers. comm.). - To perform ISO method 10705-4 an apparatus for sterilization by dry heat or steam, a thermostatic incubator, a water bath, a counting apparatus, a spectrophotometer, an anaerobic cabinet or jars or bags, Hungate glass tubes, a pH-meter and a deep freezer (-20°C ± 5°C) are required. - The method is one of the three ISO-approved methods for the enumeration of bacteriophages (as indicators for enteric viruses) in water. - Reference: ISO 10705-4. Evaluation: - In general, an accurate method to determine the number of bacteriophages (as indicator for enteric viruses) in water samples. The best indication of the presence of enteric viruses is obtained by using this method in combination with methods to determine somatic coliphages and bacteriophages that infect Bacteriodes (Leclerc et al 2000. Bacteriophages as indicators of enteric viruses and public health risk in groundwaters. J. Appl Microbiol. 88:5-21). - From literature it is known that bacteriophages that infect Bacteroides might be the most reliable indicator for enteric viruses in water. - Host strain B. fragilis RYC2056 can be used to determine animal and human related bacteriophages that infect Bacteroides. Host strains B. thetaiotaomicron GA17 and HB13 and B. ovatus GB124 can be used to determine human related bacteriophages that infect Bacteroides. However, the human related host strains seem to perform only in a specific geographic area (GA17: Spain; HB13: Spain and Columbia; GB124: Great Britain), making their interlaboratory use more difficult. - Bacteroides is an obligate anaerobic bacterium, which is inhibited by oxygen. All lab procedures should, therefore, be kept to a minimum amount of time. Incubation in anaerobic jars, bags or cabinet is required. - Incubation time is rather long, because results are obtained after 18 to 24 hours. However, a fast standard method is not available.







drinking water

x x



selectivity

x

x

Laboratory procedures should be performed in a short time and incubation is under anoxic conditions

time to result



ease-of-use (B) x Anaerobic incubations required maintenance requirements (C) x Costs



x x


Overall conclusion Because of the required anaerobic conditions, the method is not

used as often as the other two methods. However, one method is not better over the other and it is advised to determine F-specific RNA bacteriophages, somatic coliphages and bacteriophages that infect Bacteroides. The fact that Bacteroides is an obligate anaerobic bacterium makes the procedure more complicated than the methods to determine F-specific bacteriophages and somatic coliphages. An European water survey has shown that source tracking with human related Bacteroides strains is one of the most reliable ways to determine human versus animal faecal contamination.


3.12 Aerobic spore forming bacteria Prepared by: RTU Required technical specifications: Concentration range that should be covered for this parameter is starting from 1 cell/spore/100 ml. For the highest limit information is not available. Sensitivity/resolution required (distinguish between source water and drinking water, if applicable). 1 cell/spore/100 ml Monitoring technologies:

1. HPC counts 2. Membrane filtration 3. Assays based on the cell constituents 4. PCR

3.12.1 Monitoring technology nr 1: HPC counts

Description: A variety of simple culture-based tests that are intended to recover a wide range of microorganisms from water are collectively referred to as “heterotrophic aerobic and aerobic spore-forming bacterial counts” or “HPC test” procedures. There is no universal “HPC measurement.” Although standardized methods have been formalized, HPC test methods involve a wide variety of test conditions that lead to a wide range of quantitative and qualitative results. Temperatures employed range from around 20 °C to 40 °C, incubation times from a few hours to seven days or a few weeks, and nutrient conditions from low to high. The test itself does not specify the organisms that are detected. They are of a little sanitary significance, however useful for assessment of the efficiency of the water treatment where the colony counts should be as low as possible. Media can be made more selective, e.g. MYP media (Cereus Selective Agar Base) can be used (Mazoua and Chauveheid, 2005). The culture method can be adapted to count spores only by exposing samples to temperatures of 70-80ºC for 10-20 minutes before culturing. If necessary, biochemical testing of the colonies, such as API 20 E or API 50 CHB, may further clarify the identity of the colony (Mazoua and Chauveheid, 2005). There are even studies where the authors have devised an identification key for Bacillus species, based on series of culture-based and biochemical tests (Reva, et al., 2001), however, this is rather of an interest for taxonomical studies than treatment efficiency monitoring. Equipment and consumables The method is very cheap to perform. It requires only the basic equipment of any laboratory. The prices of the growth media can vary, depending on the selected method.


Status of the technique According to COUNCIL DIRECTIVE 98/83/EC of 3 November 1998 (UNION, 1998) a colony count at 22ºC and 37ºC is required for drinking water monitoring (method prEN ISO 6222). HPC is accepted and commercially available method. Many laboratories/companies supply ready growth medium mixtures, solutions etc. References: 1. Mazoua, S., and E. Chauveheid. 2005. Aerobic spore-forming bacteria

for assessing quality of drinking water produced from surface water. Water Res 39:5186-98.

2. Reva, O. N., I. B. Sorokulova, and V. V. Smirnov. 2001. Simplified technique for identification of the aerobic spore-forming bacteria by phenotype. Int J Syst Evol Microbiol 51:1361-71.

3. The Council of the European Union. 1998. COUNCIL DIRECTIVE 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. In EU (ed.), L 330/32 ed. Official Journal of the European Communities.

Evaluation: Conventional treatment processes in series (coagulation, sedimentation, filtration and disinfection) are essential to the processing of river water because of fluctuating water quality. Where lakes and large watershed impoundments are utilized, treatment may include only disinfection. Because fluctuations in raw water quality impact successful treatment, daily sampling should be done to provide information on storm water runoff conditions and alert the utility to sewage treatment bypasses released upstream. The most important sampling site in the treatment train is at the entry point for finished water (plant effluent) release into the distribution system. Here the purpose is to verify that treatment process barriers are preventing the passage of the contaminants. In some cases, this sampling site may be located at the first customer’s tap, if contact time for disinfectant action must be extended beyond that available in the plant contact basin (Geldreich, 1996). The measurement of spores of aerobic spore-forming bacteria (ASFB) is becoming a widely accepted method for validating the effectiveness of treatments applied in drinking water treatment plants. ASFB have been shown to be conservative indicators for Cryptosporidium and Giardia. HPC are provided by simple, inexpensive methods that require basic routine bacteriology laboratory facilities and can be performed by relatively unskilled persons. However, HPC will not register the presence of metabolically active but not dividing organisms and they are not rapid. 1. Geldreich, E. E. 1996. Microbial quality of water supply in distribution

systems. CRC Press LLC, Boca Raton, FL, USA.


Monitoring technology nr 1: HPC




drinking water

x

x


selectivity

x

x

time to result

x





x

x


Overall conclusion Not complicated but laborious method, not very useful for

identification. Robust, does not require skilled personnel. No information on stress-injured cells, unable to divide.


3.12.2 Monitoring technology nr 2: Membrane filtration

Description: Certain volume of water is filtered through 0.2-0.45 nm membrane filter and then the filter is incubated on a pad, soaked in nutrients. The colonies are then counted. However, due to the low concentrations of spores, the rate of the recovery is influenced both by the both the presence of a cake on the filter and the aggregation of the spores. It has been shown that the addition of a surfactant Tween 80®, recovery can be increased for up to 1000 times(Cartier, et al., 2007). Equipment and consumables See monitoring technology nr 1 above. Status of the technique Accepted and commercially available method. Many laboratories/companies supply ready growth medium mixtures, solutions etc.


References: 1. Cartier, C., B. Barbeau, M. Besner, P. Payment, and M. Prevost. 2007.

Optimization of the detection of the spores of aerobic spore-forming bacteria (ASFB) in environmental conditions. Journal of Water Supply: Research and Technology - AQUA 56:191-202.

Evaluation: The same considerations as for HPC method apply (see above). Monitoring technology nr 2: Membrane filtration




drinking water

x

x


selectivity

x

x

time to result

x





x

x


Overall conclusion Simple method, not very useful for identification without additional tests. Robust, does not require skilled personnel. More potential compared to HPC method. No information on stress-injured cells, unable to divide.


3.12.3 Monitoring technology nr: 3. Assays, based on cell constituents

Description: More contemporary methods proposed for Bacillus spp. identification include the fatty acid profile detection using capillary gas chromatography with flame ionization detection (Whittaker, et al., 2005) which was applied quite successfully to a number of pathogens. Several techniques applied for detection of other groups of pathogens could also potentially be used, such as pyrolysis in combination with mass spectrometry (Py-MS) (Freeman, et al., 1990), (Magee, et al., 1991) and matrix-assisted laser desorption/ionization


time-of-flight (MALDI-TOF) in combinations with mass spectrometry (Smole, et al., 2002) and liquid chromatography (LC) (Zhang, et al., 2004). The latter approach, LC/MALDI and tandem MS is an instrument of “shotgun” proteomics, which refers to the direct analysis of complex protein mixtures to rapidly generate a global profile of protein complement within a mixture. FT-IR technique has also been applied for Bacillus detection, however it was shown to require uniform culture conditions for obtaining well differentiated spectrum (Filip, et al., 2004). Flow cytometry has been tested for application of spore and vegetative cell enumeration since the 1980ies(Phillips and Martin, 1983). Nowadays it is possible to assess spore viability using this method as well (Laflamme, et al., 2005). The method can be used together with fluorescent dyes and antibodies. Equipment and consumables Pyrolysis is the chemical decomposition of organic materials by heating in the absence of oxygen or any other reagents. A particular piece of equipment performing this heating can be coupled to GC-MS for detection of components. Some gas chromatographs are connected to a mass spectrometer which acts as the detector. The combination is known as GC-MS. In most modern GC-MS systems, computer software is used to draw and integrate peaks, and match MS spectra to library spectra. In general, substances that vaporize below ca. 300 °C (and therefore are stable up to that temperature) can be measured quantitatively. Fourier transform (FT) spectroscopy is a measurement technique whereby spectra are collected based on measurements of the temporal coherence of a radiative source, using time-domain measurements of the electromagnetic radiation or other type of radiation. It can be applied to a variety of types of spectroscopy including infrared spectroscopy (IR). FT-IR spectroscopy involves the observation of vibrations of molecules that are excited by an infrared beam. Molecules are able to absorb the energy of distinct light quanta and start a rocking or rotation movement. The FT-IR spectrum uses only vibrations that lead to a change in the dipole moment. An infrared spectrum represents a fingerprint which is characteristic for any chemical substance. The composition of biological material and, thus, of its FT-IR spectrum, is exceedingly complex, representing a characteristic fingerprint. In principle, a reference spectrum library is assembled based on well-characterized strains and species. The FT-IR spectrum of any unidentified isolate is then measured under the same conditions as those used for the reference spectra and is compared to spectra in the reference spectrum library. If the library contains an identical or a very similar spectrum, identification is possible. The success of the method is, therefore, directly dependent on the complexity of the reference spectrum library. Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus. A flow cytometer has 5 main components:


a flow cell - liquid stream (sheath fluid) carries and aligns the cells so that they pass single file through the light beam for sensing. a light source - commonly used are lamps (mercury, xenon); high power water-cooled lasers (argon, krypton, dye laser); low power air-cooled lasers (argon (488nm), red-HeNe (633nm), green-HeNe, HeCd (UV)); diode lasers (blue, green, red, violet). a detector and Analogue to Digital Conversion (ADC) system - generating FSC and SSC as well as fluorescence signals. an amplification system - linear or logarithmic. a computer for analysis of the signals. A beam of light of a single wavelength is directed onto a hydro-dynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors). Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak) it is then possible to extrapolate various types of information about the physical and chemical structure of each individual particle. FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e. shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness). Status of the technique The methods and the equipment is well known for other applications but as detection methods for Bacilli or other spore-forming aerobes these are still under development, perhaps, except flow cytometry which is both more documented and closest to be applied for bacteria and spore detection/enumeration in water. Pyrolysis equipment can be obtained from PerkinElmer Life (www.perkinelmer.com), Analytical Sciences and Shimadzu Scientific Instruments Inc (www.shimadzu.com). A FTIR apparatus can be purchased from e.g. Varian (www.varian.com). BeckmanCoulter (www.beckmancoulter.com) and BD Biosciences (www.bdbiosciences.com) provide reliable flow cytometers. References: 1. Filip, Z., S. Herrmann, and J. Kubat. 2004. FT-IR spectroscopic

characteristics of differently cultivated Bacillus subtilis. Microbiol Res 159:257-62.

2. Freeman, R., M. Goodfellow, F. K. Gould, S. J. Hudson, and N. F. Lightfoot. 1990. Pyrolysis-mass spectrometry (Py-MS) for the rapid epidemiological typing of clinically significant bacterial pathogens. J Med Microbiol 32:283-6.

3. Laflamme, C., J. Ho, M. Veillette, M. C. de Latremoille, D. Verreault, A. Meriaux, and C. Duchaine. 2005. Flow cytometry analysis of


germinating Bacillus spores, using membrane potential dye. Arch Microbiol 183:107-12.

4. Magee, J. T., J. M. Hindmarch, and C. D. Nicol. 1991. Typing of Streptococcus pyogenes by pyrolysis mass spectrometry. J Med Microbiol 35:304-6.

5. Phillips, A. P., and K. L. Martin. 1983. Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry. Cytometry 4:123-31.

6. Verberkmoes, N. C., W. J. Hervey, M. Shah, M. Land, L. Hauser, F. W. Larimer, G. J. Van Berkel, and D. E. Goeringer. 2005. Evaluation of "shotgun" proteomics for identification of biological threat agents in complex environmental matrixes: experimental simulations. Anal Chem 77:923-32.

8. Whittaker, P., F. S. Fry, S. K. Curtis, S. F. Al-Khaldi, M. M. Mossoba, M. P. Yurawecz, and V. C. Dunkel. 2005. Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli. J Agric Food Chem 53:3735-42.

9. Wilkes, J. G., K. L. Glover, M. Holcomb, F. Rafii, X. Cao, J. B. Sutherland, S. A. McCarthy, S. Letarte, and M. J. Bertrand. 2002. Defining and using microbial spectral databases. J Am Soc Mass Spectrom 13:875-87.

Evaluation: It should be noted that some of these methods will often require pre-enrichment step/cultivation and therefore are not really rapid methods. All of these methods require costly equipment. Furthermore, the fingerprints from mass spectral patterns from microbial isolates are affected by variations in instrumental condition, by sample environment and by sample handling factors (Wilkes, et al., 2002). A recent study evaluated LC/MALDI and tandem MS this approach for E. coli detection, however the conclusion was that low concentrations of a pathogen in a mixed sample were not detected and the size of the database also had severe effect on the identification (Verberkmoes, et al., 2005). Although a promising technology, it must be significantly improved and therefore is not described in detail in this report. From the described methods flow cytometry should be considered closest to the practical application as it (i) will not require cultivation and (ii) has a potential to provide the information whether the cell is viable or not.


Monitoring technology nr 3: Assays based on cell constituents Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x





x


Overall conclusion Promising methods but still to far from the application. Flow cytometry could be interesting for larger systems. The equipment is expensive to purchase and maintain. Skilled personnel in performing the analysis and interpreting data is also required.


3.12.4 Monitoring technology nr 4: PCR

Description: The polymerase chain reaction (PCR) is a biochemistry and molecular biology technique for exponentially amplifying DNA, via enzymatic replication. PCR-based methods have been used for the detection of Bacilli, more specifically, ARDRA-PCR (amplified ribosomal DNA restriction analysis) which was capable to identify most environmentally important species of Bacillus and distinguish these from 15 other Eubacteria species (Wu, et al., 2006). This method implies first using restriction enzymes and then specific primers to amplify the genome fragments and then identify a certain pattern, specific for e.g Bacilli. Equipment and consumables This technique requires PCR thermocycler, which nowadays supports 96 reactions simultaneously. If there is enough genetic material in the sample, the results can be achieved in a couple of hours or less. Thermocycler is not


very expensive, however this technique requires consumables such as restriction enzymes, polymerization enzyme(s), nucleotides, buffers, and gel imaging systems, if the cycler is conventional. Real-Time PCR does not always require gel imaging but the cycler is more expensive and the operation more difficult. Status of the technique PCR is a promising technique which could be tested for aerobic spore-forming bacteria analysis through the water treatment steps. The equipment, however, is expensive and skills in operating it are needed. PCR equipment can be purchased from Applied Biosystems (www.appliedbiosystems.com) and Eppendorf (www.eppendorf.com), among others. References: 1. Wu, X. Y., M. J. Walker, M. Hornitzky, and J. Chin. 2006. Development

of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance. J Microbiol Methods 64:107-19.

Evaluation: This method has very good potential but the ability to distinguish aerobic spore-formers from other Eubacteria species than tested so far must be investigated. Another important question is the sensitivity of the reaction, enzymes, in particular, to substances found in the treated waters. It is known that certain metal ions inhibit polymerases thus it is likely that sample preparation, clean-up, amplification facilitators might be necessary.


Monitoring technology nr 4: PCR Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x

x


Overall conclusion A promising method but the equipment is expensive to

purchase and run. Skilled personnel is also required. It still needs to be developed prior to application, however, the method has a potential for being used by larger systems in the future



3.13 Biofilm formation rate (BFR) Prepared by: NTNU Required technical specifications: - Concentration range that should be covered: 1ng adenosine triphosphate (ATP)/l to >1000 ngATP/l mg/l - sensitivity/resolution required: An ATP signal of 2000-3000 Relative Light Units (ca. 0.01 nM ATP) is considered the lower detection limit above which statistically accurate measurements were possible (standard deviation < 5%). Monitoring technologies: 1. ATP measurement of biofilm

3.13.1 Monitoring technology nr 1: ATP measurement of biofilm

Description: The methodology was developed for at-line monitoring of biofilm formation in drinking water systems [1] and the biofilm formation rate was determined with a biofilm monitor containing cylinders. Several monitoring systems have been reported [2][Fehler! Verweisquelle konnte nicht gefunden werden.]. The water to be investigated is flowing through the monitor at a selected rate, e.g. 0.2 m/s. Biofilm formation is determined as a function of time by collecting sacrificial supports from the monitor at regular intervals and determining the active biomass concentration on the surface of these supports. ATP is used as active biomass parameter. For the ratio between cell carbon and ATP, generally a value of 250 is used [3]. Attached biomass is released from the surface of the support by e.g. sonication, and cells in suspension are permeabilised to extract nucleotides before measurement. The concentration of ATP is determined in the biomass suspension and the ATP concentration of the biofilm is calculated. The BFR (pg ATP cm-2 d-1) of the water can be calculated as the slope of the linear increase of the biofilm concentration with time. Equipment and consumables

1. The monitoring system 2. Ultrasonic cleaning bath for transfer of biofilm from supports into

suspension. 3. Luminometer for light measurement (ATP analysis) 4. ATP analysis reagents: Nucleotide releasing reagent; Enzymatic

reaction reagents; ATP standard Status of the technique: Several monitoring systems have been used, e.g. LUMAC Biocounter, Propella® reactor, Rotating Annular Reactor. ATP is proposed by European experts as a parameter of active biomass of biofilms in drinking water systems [2].


References 1 Van der Kooij, D.,Veenendaal, H.R., Baars-Lorist, C., Klift, D.W. and Drost, Y. C. (1995) Water Res. 29 1665-1662 2 Miettinen and Schaule (2003) Handbook for analytical methods and operational criteria for biofilm reactors. SAFER WP2. EVKT-CT-2002-00108 May 2003 3 Karl, D. M. (1980) Cellular nucleotide measurements and applications in microbial ecology. Microbol. Rev. 44 739-796 4 Van der Kooij, D., Vrouvenwelder, J. S. and Veenendaal, H. R. (2003). Elucidation and control of biofilm formation processes in water treatment and distribution using the unified biofilm approach. Water Sci. Technol. 47 83-90 Vendors ATP-assay reagents: Promega; Celsis Luminometers: Turner Biosystems; Celsis; Perkin Elmer Evaluation: The measurement of BFR requires several days/weeks of exposure of sacrificial supports to water. ATP measurement is a more rapid and sensitive technique for detection of active biomass on the supports, than enumeration of culturable heterotrophic bacteria. The ATP method can be used for measuring biofilm/biofouling biomass in different parts of the water supply system, e.g. in drinking water distribution [2] and on membranes used in drinking water treatment [4]. BFR –ATP measurement is applied for monitoring biofilm formation both in research projects and by water works. The consumables of ATP analysis are relatively expensive. Alternative methods for at-line/in-line biofilm monitoring of active biomass such as (advanced) microscopy, combined with staining of biofilm components can provide measures (i.e. thickness, volume, components) of undisturbed biofilm on sacrificial supports. Such techniques are more time consuming than ATP measurement and require expensive instrumentation and skilled personnel. Biofilm enzymatic activity measurements can potentially be used for at-line/in-line biofilm activity monitoring. These methods are not providing direct measures of biomass. The assimilable organic carbon (AOC) test is commonly used to assess the concentration of growth promoting organic compounds in water. This method gives a measure of the potential for biofilm growth, but is not providing a direct quantitative measure of biofilm formation.


Monitoring technology nr 1:ATP measurement of biofilm




drinking water

x x


selectivity

x x

time to result

x x

Biofilm development: several d ATP measurement: rapid





x

x

Recommendation for use in SSS (D)

x

Overall conclusion Reliable method, but expensive consumables , method o.k for active biomass measurement ,but not for measurement of total biofilm components , i.e. all ( live and dead) cells; EPS



3.14 Total cell counts Prepared by: EAWAG Required technical specifications: - There are no current drinking water guidelines or legislation covering this parameter. - The concentration range that should be covered is 102 – 109 bacterial cells/ml - The sensitivity/resolution required is:

• source water: 102 bacterial cells/ml • drinking water: 102 bacterial cells/ml

Monitoring technologies: 1. Direct total microbial cell count (based on fluorescence microscopy) 2. Direct total microbial cell count (based on flow cytometry)

3.14.1 Monitoring technology nr 1: Direct total microbial count (based on fluorescence microscopy)

Description The method consists of (1) fixing the water sample with gluteraldehyde (5% w/v) for storage; (2) staining the fixed sample with acridine orange (0.1 % w/v); (3) filtering a known volume (typically 1 mL) of the sample onto a non-fluorescing polycarbonate membrane filter; (4) visualization and enumeration of the stained cells on the filter with an epifluorescence microscope; and (5) calculation of the total cell number based on the microscopy field size, the total filter area and the total volume of water that was filtered (Hobbie et al. (1977), Clesceri et al (1998). Variations on the method: Sample preparation: Alternative fixation agents can be used, e.g., formaldehyde (2%), or in case of immediate sample processing, fixation can be omitted entirely. Staining: Alternative fluorescent dyes can be used, e.g., DAPI and SYBR Green I (depending on the availability of relevant hardware in the fluorescent microscope set-up). Enumeration: Automated microscopy detection and enumeration hardware and software is available and used by several research groups. Equipment and consumables (basic method) Instruments: Epifluorescence microscope with the appropriate filter sets (see Standard Methods 9216 (Clesceri et al., 1998)) Filtration unit (see Standard Methods 9216)


Consumables: Membrane filters, Syringes, Test tubes, Phosphate buffer, Fixative, Acridine orange, Immersion oil Status of the technique: This is a generally accepted method for total cell counting. The method requires a skilled operator with experience in distinguishing bacterial cells from background particles. References Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (Eds.) (1998) Standard Methods for the examination of water and wastewater; 9216 Direct Total Microbial Count. ISBN 0-87553-235-7 Hobbie, J.E., Daley, R. J. und Jasper, S. (1977). Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol., Vol. 33:1225-1228. Evaluation The concept to quantify bacteria in solution after filtration is based on unspecific staining of their nucleic acids with a chemical fluorochrome and enumeration by epifluorescence microscopy. Depending on the kind of enumeration (by eye or digitally) it can be more or less time consuming.


Monitoring technology nr 1: Direct total microbial count (based on fluorescence microscopy) Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

< 20 min





x x


Overall conclusion High initial investment costs for the purchase of a fluorescence microscope. Some automation methods for counting are also available to speed up analysis and reduce labour requirements.


3.14.2 Monitoring technology nr 2: Direct total microbial count (based on flow cytometry)

Description The method consists of staining the bacterial cells in a water sample with a fluorochrome (e.g. SYBR Green I or DAPI) and enumeration of bacterial numbers by flow cytometry (Hammes et al., 2008; Lebaron et al., 1998). The fluorescent dye is selected based on the availability of the relevant light source and filters in the flow cytometer set-up (see also Lebaron et al., 1998). The stain concentration and staining time are selected based on the approximate concentration of bacteria in the sample and the specific features of the fluorochrome. For example, for a cell concentration smaller than 1 x 106

cells/mL (typical for drinking water), a concentration final concentration of 10,000x diluted SYBR Green I is used with a staining time of 10 minutes (Hammes et al., 2008). In case of high cell concentrations, enumeration with flow cytometry might require pre-dilution. For the latter, cell-free (0.1 um filtered) phosphate buffer or cell-free (0.1 um flitered) drinking water can be


used. Dilution should be performed immediately before the flow cytometric measurement. For enumeration, some brands of flow cytometers are equipped with volumetric counting hardware, while other brands use the addition of a commercially available standard bead solution for counting. Equipment and consumables Instruments: Conventional flow cytometer equipped with a specific light source (e.g. 200 mW blue laser (488 nm)). Consumables: Test tubes, fluorochrome (e.g. SYBR Green I), cell-free phosphate buffer Status of the technique: Accepted method, currently in preparation for Standard Methods Reference Lebaron, P., N. Parthuisot, and P. Catala. 1998. Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl Environ Microbiol 64:1725-30. Hammes, F., M. Berney, W. Yingying, M. Vital, O. Köster, and T. Egli (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Research, 42(1-2), 269-77. Evaluation Flow cytometry is well adapted to enumerate high particle numbers in a short time period.


Monitoring technology nr 2: Direct total microbial count (based on flow cytometry) Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x < 15 min





x x


Overall conclusion High initial investment costs for the purchase of a flow cytometer. Otherwise the method is fast, accurate and easy to use.



3.15 Cultivation free viability analysis Prepared by: Eawag Parameter: Cultivation free viability analysis Required technical specifications: - There are no current drinking water guidelines or legislation covering this parameter. - The concentration range that should be covered: 103 – 109 bacterial cells/ml - The sensitivity/resolution that is required

• source water: 103 bacterial cells/ml • drinking water: 103 bacterial cells/ml

Monitoring technologies: 1. Cultivation free viability analysis (based on flow cytometry or fluorescence microscopy) 2. Analysis of bacterial adenosine tri-phosphate (ATP)

3.15.1 Monitoring technology nr 1: Cultivation free viability analysis (based on flow cytometry or fluorescence microscopy)

Note: The flow cytometric technology described herein consists of a combination of several different staining methods. The different methods target different aspects of bacterial viability. While individual stains can be useful for specific applications, the use of several stains in concert is suggested as the best-available approach to general viability analysis in an unknown water sample. The method below is described for flow cytometry, but can be used in combination with fluorescence microscopy as well (see section 3.14 above) Description Water samples: It is essential that fresh water samples without pre-treatment or fixation be used in the analysis. Any treatments may affect viability and thus alter the outcome of the analysis. Staining: The water samples are stained with four different fluorochromes namely SYBR Green I (total cell count, see above), propidium iodide (membrane integrity), DiBAC4(3) (membrane potential) and CFDA (enzyme activity)) (Berney et al., 2008; Hammes et al., 2008; Hoefel et al., 2003). The specific concentrations of the fluorochromes and the staining times are based on the features of the individual stains and on the concentration of cells in the water sample (Berney et al., 2008). For example, for a standard drinking water sample (c.a. 1 x 105 cells/mL), the following stain combinations are used (2 uL/200 uL): - SYBR Green I: 100x diluted in DMSO; 10 minutes incubation - Propidium Iodide: 0.3 mM in SYBR Green I solution (above), 15 minutes incubation - DiBAC4(3): 0.1 mM in DMSO, 20 minutes


- CFDA: 10 mM in DMSO, 30 minutes at 30 °C Flow cytometry: In case of high cell concentrations, enumeration with flow cytometry might require pre-dilution. For the latter, cell-free (0.1 um filtered) phosphate buffer or cell-free (0.1 um flitered) drinking water can be used. Dilution should be performed immediately before the flow cytometric measurement. Correct instrument settings for the flow cytometer have to be established for the specific instrument being used, in combination with a set of relevant control samples (Berney et al., 2008). Variations on the method: The list of stains mentioned here is not exhaustive, since many alternative stains exist (Porter et al., 1995; Davey and Kell, 1996; Joux and Lebaron, 2000). Equipment and consumables Instruments: Flow cytometer equipped with a blue laser (488 nm) and the appropriate filter sets Consumables: Test tubes, fluorochromes (e.g. SYBR green I, propidium iodide, DiBAC4(3), CFDA), sterile filtered (0.1 um) bottled mineral water or phosphate buffer for dilution. Status of the technique Method widely used for scientific purposes (pure cultures, bacterioplankton) but not in drinking water routine analysis. Currently under development in the Techneau project (Deliverable 3.3.7 and 3.3.8) References Porter, J., J. Diaper, C. Edwards, and R. Pickup. 1995. Direct measurements of natural planktonic bacterial community viability by flow cytometry. Appl Environ Microbiol 61:2783-2786. Davey, H. M., and D. B. Kell. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiol Rev 60:641-96. Joux, F., and P. Lebaron. 2000. Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect. 2:1523-35. Hoefel, D., W. L. Grooby, P. T. Monis, S. Andrews, and C. P. Saint. 2003. Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques. J. Microbiol. Methods 55:585-97. Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F. Hammes. Rapid, cultivation-independent assessment of microbial viability in drinking water. Accepted for publication in Water Research, July 2008 Evaluation


This method is currently under development (validation) and has a huge potential for routine analysis and monitoring of microbial activity in drinking water (during treatment and in the distribution system) because it is fast and very reproducible. A combination of flow cytometric analysis with conventional heterotrophic plate counts (HPC) and adenosine tri-phosphate (ATP) analysis is encouraged (Berney et al., 2008). At the moment the method requires trained personnel. Alternatively the analysis can be done on a fluorescence microscope. Monitoring technology nr 1: Cultivation free viability analysis (based on flow cytometry) Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x

x

time to result

30 min


ease-of-use (B) x Requires expertise with flow cytometry operation and interpretation




x x


Overall conclusion Viability-free analysis is a different way to study/assess the entire microbial community in drinking water. It can be used to assess the efficacy of disinfection methods. Some expertise in operation and interpretation is required.



3.15.2 Monitoring technology nr 2: Analysis of bacterial ATP

Description General: The ATP method is a bulk water microbiological paramter that measures the concentration of ATP extracted from planktonic cells. It is based on the principle that a lysis buffer extracts the ATP from the cells, and that this ATP then reacts with the substrate-enzyme complex “luciferin-luciferase” to produce light that can be measured in a luminometer. The resulting relative light units (RLU) can then be related to a given ATP concentration by means of a pre-established standard curve with pure ATP. Several ATP kits are commercially available from different manufactures. Water samples: It is essential that fresh water samples without pre-treatment or fixation are used in the analysis. Any treatment may affect viability and thus alter the outcome of the analysis. Total ATP: The total ATP in the bulk water sample is measured directly. Typically a protocol would be provided with each kit. Free ATP: The free ATP is measured in the water following a 0.1um filtration step. Bacterial ATP: Calculated as the difference between total ATP and free ATP. It is absolutely essential that sterile equipment is used for all the steps involved in the measurement of ATP. Equipment and consumables Instruments: Standard luminometer (e.g. Glomax, Turner Biosystems) Consumables: ATP reagent (e.g. Promega); Sterile eppendorf tubes andpipettes; sterile 5 mL syringes and sterile 0.1 um syringe filters. Status of the technique There are no legal guidelines for ATP analysis, but the method has been used for several decades, and have been suggested as a useful parameter for drinking water analysis (Siebel et al., 2008; Delahaye et al., 2003). References Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (Eds.) (1998) Standard Methods for the examination of water and wastewater; 9211 Bioluminescence Test. ISBN 0-87553-235-7 Berney, M., M. Vital, I. Huelshoff, H.-U. Weilenmann, T. Egli, and F. Hammes. Rapid, cultivation-independent assessment of microbial viability in drinking water. Accepted for publication in Water Research, July 2008

Delahaye, E., Welte, B., Levi, Y., Leblon, G., and Montiel, A.: An ATP-based method for monitoring the microbiological drinking water quality in a distribution network. Water Res., 37(15), 3689-3696, 2003. Hammes, F., M. Berney, W. Yingying, M. Vital, O. Köster, and T. Egli (2008) Flow-cytometric total bacterial cell counts as a descriptive microbiological parameter for drinking water treatment processes. Water Research, 42(1-2), 269-77. Evaluation


The method has been used for several decades, but the use in drinking water has been limited due to a lack of sensitivity of the assay (Clesceri et al., 1998). Sample concentration and optimization of the measurement protocol can overcome these shortcomings. A major problem is the absence of accurate conversion factor to relate ATP concentrations to bacterial concentrations (Berney et al., 2008). It is also essential to differentiate between bacterial ATP and free ATP (Hammes et al., 2008), which make the analysis more expensive and time consuming. Monitoring technology nr 2: Analysis of bacterial ATP Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x

x

time to result

10 min


ease-of-use (B) x The method is extremely easy to perform




x

x


Overall conclusion The method is fast and easy to perform, but some skill is required to measure low concentrations of ATP in water samples.



4 Chemical parameters

4.1 Metals: Antimony, arsenic, boron, cadmium, chromium, copper, lead, mercury, nickel, selenium, sodium, calcium, magnesium, aluminum, iron, manganese

Prepared by: TZW Monitoring technologies: 1. AAS: Atomic adsorption spectroscopy a) Graphite furnace AAS b) Hydride generation AAS c) Cold vapour AAS d) Flame AAS 2. AFS: Atom fluorescence spectroscopy 3. ICP-OES: Inductively-coupled plasma with optical emission spectroscopy 4. ICP-MS: Inductively-coupled plasma with mass spectrometry The following table summarizes information about the suitability of each technique for analyzing metals with the sensitivity required by the DWD. Other technologies like ion chromatography or voltametric methods are applicable to single methods, however, most often suffer from limited sensitivity and/or selectivity and thus will not be considered here. Parameter

Pa

ram

etr

ic v

alu

e D

WD

[m

g/L

]

Se

nsi

tiv

ity

re

qu

ire

d [

µg

/L]

Gra

ph

ite

fu

rna

ce

AA

S

Hy

dri

de

G

en

era

tio

n

AA

S

Co

ld

va

po

ur

AA

S

Co

ld

va

po

ur

AF

S

Fla

me

AA

S

ICP

-OE

S

ICP

-MS

Antimony 0,005 1,25 x x Arsenic 0,010 1,0 x x Boron 1,0 100 x x Cadmium 0,005 0,5 x x Chromium 0,050 5,0 x x Copper 2,0 200 x x x Lead 0,010 1,0 x x Mercury 0,001 0,20 x x Nickel 0,020 2,0 x x Selenium 0,010 1,0 x x Sodium 200 20000 x x x Calcium - - x x x Magnesium - - x x x Aluminium 0,50 50 x x x Iron 0,20 20 x x x Manganese 0,050 5,0 x x x


4.1.1 Monitoring technology nr 1: AAS (Atomic adsorption spectroscopy)

Description: The water sample is acidified to pH < 2 with nitric acid. Then an aliquot is injected into the AAS instrument. For concentrations < 100 µg/l the graphite furnace technique (GF-AAS) is recommended, whereas for concentrations > 100 µg/L the flame AAS is preferred. For the hydride forming metals (antimony, arsenic, selenium) the hydride generation technique (HG-AAS) could be used to improve method performance. For a sensitive determination of mercury by AAS, the cold vapour technique (CV-AAS) must be used. Evaluation: Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result

x



instrumentation (C) x Operational costs (C)


x x

Recommendation for use in SSS (D) x cheap instrument which can be used for multiple parameters

Overall conclusion Cheap method with a limited linear range; rather time

consuming if several elements have to be analysed as it is a sequential technique.



4.1.2 Monitoring technology nr 2: AFS (Atomic fluorescence spectroscopy)

Description: Special detection technology based on the cold vapour technique for the sensitive analysis of mercury. The water sample is acidified to pH < 2 with nitric acid. For preservation hydrobromic acid or potassium dichromate are added. Then an aliquot is injected into the AFS instrument. Evaluation: Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result

x



instrumentation (C) x Operational costs (C)


x x


Overall conclusion Method of choice for mercury analysis. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.1.3 Monitoring technology nr 3: ICP-OES: Inductively-coupled plasma with optical emission spectroscopy

Description: The water sample is acidified to pH < 2 with nitric acid. Then an aliquot is injected into the AFS instrument. Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

Selectivity depends on the optical resolution of the instrument.

time to result

x





x x


Overall conclusion Multi-element method for determination of elements in the mg/L range; Large linear range but not suitable for trace analysis.



4.1.4 Monitoring technology nr 4: ICP-MS: Inductively-coupled plasma with mass spectrometry

Description: The water sample is acidified to pH < 2 with nitric acid. Then an aliquot is injected into the AFS instrument. Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

Selectivity can be improved by using a reaction or collision cell.

time to result

x





x x


Overall conclusion Fast and sensitive multi-element method with a large linear

range. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.2 Benzene Prepared by: TZW Required technical specifications: - concentration range 0.25 to 10 µg/L (drinking water) - sensitivity required 0.25 µg/L (drinking water) Monitoring technologies: 1. Liquid-liquid extraction, gas chromatography with flame ionization detection or mass spectrometric detection (GC-FID or GC-MS) 2. Headspace, GC-FID or GC-MS 3. Purge&trap, GC-FID or GC-MS 4. Solid-phase micro extraction (SPME), GC-FID or GC-MS

4.2.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-FID or GC-MS

Description: 100 mL water sample are extracted with 1 mL of n-pentane or n-hexane. After separation of the organic layer, an aliquot is injected into the gas chromatograph. Detection of benzene is possible with both, a flame ionization detector (FID) or a mass spectrometer (MS). Both detectors exhibit comparable sensitivity but a MS detector offers better selectivity.


Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x (FID)

x x (MS)

time to result

x ca. 1 h



instrumentation (C) x (FID)

x (MS)


x x


Overall conclusion Good and reliable technology that is rather easy to use (but needs trained personal).


4.2.2 Monitoring technology nr 2: Headspace, GC-FID or GC-MS

Description: 30 mL water sample (depending on system used) are filled in a vial that is set on an elevated temperature (e.g. 60 °C). Then, an aliquot of the headspace is automatically transferred by a syringe into the gas chromatograph. Detection of benzene is possible with both, a flame ionization detector (FID) or a mass spectrometer (MS). Both detectors exhibit comparable sensitivity but a MS detector offers better selectivity.





drinking water

x x


selectivity

x (FID)

x

x (MS)

time to result

x ca. 0.5 h




x (MS)


x x




4.2.3 Monitoring technology nr 3: Purge&trap, GC-FID or GC-MS

Description: 30 mL water sample are purged in a nitrogen stream. The volatile water ingredients are stripped and trapped onto a trap (which most often is some activated carbon material). By heating the trap material the target compounds are transferred into the gas chromatograph. Detection of benzene is possible with both, a flame ionization detector (FID) or a mass spectrometer (MS). Both detectors exhibit comparable sensitivity but a MS detector offers better selectivity.





drinking water

x x


selectivity

x x (FID)

x (MS)

time to result

x ca. 0.5 h




x (MS)


x x

Recommendation for use in SSS (D) x Overall conclusion Good and reliable technology that is rather easy to use (but

needs trained personal). (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.2.4 Monitoring technology nr 4: Solid-phase micro extraction (SPME), GC-FID or GC-MS

Description: 30 mL water sample are extracted with a fiber coated with polymeric material. The target analytes adsorb onto the polymeric material. The fiber is transferred into the injector of the gas chromatograph. When the injector is heated, the target compounds desorb from the fiber and thus are transferred onto the chromatographic column. Detection of benzene is possible with both, a flame ionization detector (FID) or a mass spectrometer (MS). Both detectors exhibit comparable sensitivity but a MS detector offers better selectivity.





drinking water

x x


selectivity

x x (FID)

x (MS)

time to result

x ca. 0.5 h




x (MS)


x x





4.3 Benzo(a)pyrene and other PAHs Prepared by: TZW Required technical specifications: - concentration range 0.0025 to 0.5 µg/L per single compound (drinking water) For contaminated sites higher concentrations can be expected. - sensitivity required 0.0025 µg/L per single compound (drinking water) Monitoring technologies: 1. Liquid-liquid extraction, Liquid chromatography with fluorescence detection (HPLC/FLD) 2. Liquid-liquid extraction, GC/MS

4.3.1 Monitoring technology nr 1: Liquid-liquid extraction, HPLC/FLD

Description: 1 L (or 2 L, depending on the sensitivity of the detection system) water sample are extracted with 45 mL of cyclohexane. After separation of the organic layer, the volume is carefully reduced to ca. 200 µL. Then 50 µL dimethylformamide (DMF) are added as keeper and the volume is further reduced. An aliquot of the organic extract is injected into the liquid chromatograph. Detection of the PAH is done with fluorescence detection at the respective wavelength combinations.





drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Sensitive and selective analytical method. Use for drinking

water analysis is recommended. In contaminated waters, problems with interferences might occur.


4.3.2 Monitoring technology nr 2: Liquid-liquid extraction, GC/MS

Description: 1 L (or 2 L, depending on the sensitivity of the detection system) water sample are extracted with 25 mL of cyclohexane. After separation of the organic layer, the volume is carefully reduced to ca. 500 µL. Then, an aliquot is injected into the gas chromatograph. Detection of the PAH is done with mass spectrometric detection.





drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Sensitive analytical method with a large range of linearity and high robustness. Method can especially be recommended for analysis of contaminated waters.



4.4 Bromate Prepared by: TZW Required technical specifications: - concentration range 1 to 10 µg/L (drinking water) - sensitivity required 1 µg/L (drinking water) This parameter is only relevant for drinking waters if ozonation is used. Monitoring technologies: 1. Ion chromatography with conductivity detection (IC/CD) 2. Ion chromatography with UV detection (IC/UV) 3. Ion chromatography with fluorescence detection (IC/FLD) 4. Ion chromatography with inductively coupled plasma mass spectrometry detection (IC/ICP-MS)

4.4.1 Monitoring technology nr 1: Ion chromatography with conductivity detection (IC/CD)

Description: Depending on the sensitivity of the detection system, an aliquot of the sample can be directly injected into the ion chromatographic system. To improve sensitivity, on-line pre-concentration is possible. Details of both methods (direct injection and on-line pre-concentration are described in EN ISO 15061).





drinking water

x x


selectivity

x

x

time to result

x





x

x

Recommendation for use in SSS (D) x Overall conclusion Standardized method with good sensitivity and selectivity. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.4.2 Monitoring technology nr 2: Ion chromatography with UV detection (IC/UV)

Description: An aliquot of the sample is directly injected into the ion chromatographic system. After separation of the ions, post-column derivatisation takes place. Different methods with different agents are available (e.g. tribromide method, o-dianisidine method, methylene blue method, chloropromazine method). During all reactions, colored compounds are formed which are then detected on-line by UV photometric measurements.





drinking water

x

x


selectivity

x

x

time to result

x





x x


Overall conclusion Method is very sensitive but lacks of limited selectivity due to possible interferences with other compounds reacting with the derivatization agent.


4.4.3 Monitoring technology nr 3: Ion chromatography with fluorescence detection (IC/FLD)

Description: An aliquot of the sample is directly injected into the ion chromatographic system. After separation of the ions, post-column derivatisation takes place. Different methods with different agents are available. During all reactions, compounds which are fluorescence-active are formed which are then detected on-line by fluorescence measurements.





drinking water

x

x


selectivity

x

x

time to result

x





x x


Overall conclusion Method is very sensitive but lacks of limited selectivity due to possible interferences with other compounds reacting with the derivatization agent.


4.4.4 Monitoring technology nr 4: Ion chromatography with inductively coupled plasma mass spectrometry detection (IC/ICP-MS)

Description: An aliquot of the sample can be directly injected into the ion chromatographic system. After ion chromatographic separation, the effluent is coupled with a suppressor unit to the nebulizer of the ICP-MS. Detection of bromate is preferably done via the 79Br mass.





drinking water

x x


selectivity

x x

time to result

x



instrumentation (C) x


x

x


Overall conclusion Excellent method that combines high selectivity with

outstanding sensitivity, if ICP-MS is available. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.5 Cyanides Prepared by: TZW Required technical specifications: - concentration range 0.01 to 0.5 mg/L - sensitivity required 0.01 mg/L Monitoring technologies: 1. Photometric method (batch mode) 2. Continuous flow analysis

4.5.1 Monitoring technology nr 1: Photometric method (batch mode)

Description: Cyanide is released from its complexes by addition of special chemicals. Hydrogen cyanide is formed which is transferred into caustic soda where the cyanide is photometrically determined after addition of a barbituric acid/pyridine mixture. Several systems from different manufacturers are commercially available.





drinking water

x x

Sensitivity of the method strongly depends on the size of the cuvette used for the photometric detection.


selectivity

x

x

time to result

x





x x


Overall conclusion Low cost and easy to use method for fast analysis of cyanide.


4.5.2 Monitoring technology nr 2: Continuous flow analysis

Description: First, the water sample is on-line digested by UV light. After distillation of the cyanide it and addition of a barbituric acid/pyridine mixture, a photometric on-line measurement takes place. Commercial systems from different manufacturers are on the market.





drinking water

x x


selectivity

x x

time to result

x





x x


Overall conclusion Sensitive and robust method which requires advanced

instrumentation. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.6 1,2-Dichloroethane Prepared by: TZW Required technical specifications: - concentration range: 0.3 – 3 µg/L (drinking water) - sensitivity required: 0.3 µg/L (drinking water); according to DWD, accuracy and precision of the method used should be below 20% of the parametric value Monitoring technologies: 1. Liquid-liquid extraction, GC-ECD(-ECD) 2. Headspace, GC-ECD-(ECD) 3. purge&trap, GC-MS


4.6.1 Monitoring technology nr 1: Liquid-liquid extraction, GC-ECD(-ECD)

Description: EN ISO 10301: Liquid-liquid extraction of 100 mL water sample with 1 mL n-pentane; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC columns with different polarity) Evaluation: Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Reliable standard method. Sensitivity for 1,2-dichloroethane, however, is low and the requirements of the DWD can hardly be fulfilled. For increasing selectivity, two GC columns should be used.



4.6.2 Monitoring technology nr 2: Headspace, GC-ECD-(ECD)

Description: Headspace analysis of ca. 30 mL water sample; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC columns with different polarity) Evaluation: Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Reliable method. Sensitivity for 1,2-dichloroethane, however, is low and the requirements of the DWD can hardly be fulfilled. For increasing selectivity, two GC columns should be used.



4.6.3 Monitoring technology nr 3: purge&trap, GC-MS

Description: Purge&trap analysis (dynamic headspace) of ca. 30 mL water sample; analysis of extract with GC-MS (GC-ECD or GC-ECD-ECD analysis is also possible). Evaluation: Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result

x





x x


Overall conclusion Reliable and sensitive method. Method of choice for analysis of drinking water samples.



4.7 Fluoride Prepared by: TZW Required technical specifications: - concentration range 0.05 to 0.5 µg/L - sensitivity required 0.05 µg/L Monitoring technologies: 1. Ion-selective electrode (ISE) 2. Ion chromatography with conductivity detection (IC/CD)

4.7.1 Monitoring technology nr 1: Ion-selective electrode (ISE)

Description: A buffer solution (TISAB) is added to the water sample to adjust pH and salt content. Then fluoride is measured by using an ion-selective electrode (which is available from different manufacturers). Evaluation: Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x

x

time to result

x





x

x


Overall conclusion Fast and low-cost method for selective analysis of fluoride. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems



Description: An aliquot of the water sample is directly injected into the ion chromatographic system. Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

The separation power of the column is crucial for a good selectivity.

time to result

x





x

x


Overall conclusion Fast and reliable method which can also be used for analysis of other ions.



4.8 Nitrite Prepared by: S::can Required technical specifications: Concentration ranges that can be encountered are the following: source water (ground or surface water) 0 - 50 mg/L (measured as NO2) drinking water 0 - 0.50 mg/L (measured as NO2) Required sensitivity of the measurement: 0.05 mg/L Monitoring technologies: 1. Ion chromatography 2. Wet chemical analysis / colorimetric method 3. Spectrometric method, derivative spectroscopy

4.8.1 Monitoring technology nr 1: Ion Chromatography

Description: In ion chromatography, a pre-treated sample is processed in the laboratory. The sample in injected in chromatograph, and the components present are separated on the column. Based on retention time the desired component is identified. Equipment and consumables: Equipment: ion chromatograph, including chromatographic column, HPLC pump. Consumables: solvents, filters, columns Lab technique available for many years. Highly reliable and accurate. Very expensive. Available from Waters, Metrohm, Thermo Electron, Dionex and others. Evaluation: - Highly accurate technique. Only suitable for off-line, batch wise laboratory analysis. Useful for regulatory measurements. - Most accurate technique, but also by far the most expensive (investment in equipment). - This method fulfils the needs in terms of measuring range and accuracy.


Monitoring technology nr 1: Ion chromatography Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x


ease-of-use (B) x calibration requires some training


instrumentation (C) x price indication: > 15.000 Euro operational costs (C)


x

x


Overall conclusion Suitable method for reference / regulatory measurements in the laboratory


4.8.2 Monitoring technology nr 2: Wet chemical analysis – Colorimetric method

Description: Nitrite is determined through formation of a reddish purple dye produced at pH 2. The application range of the method for spectrometric measurements is 10 - 1000 µg/L nitrite. Higher NO2 concentrations can be determined after dilution of the sample. The analysis has to be performed immediately after sampling to ensure reliable NO2 concentrations. Equipment and consumables - Equipment needed: photometer, test-tube with reagent (pr-mixed, easy to handle), filter to remove solids / turbidity from the sample before starting test - Consumables: test tubes (approx 2 - 5 Euros per test) - Photometry is established technique (see Standard Methods for the examination of water and Waste Water APHE, AWWA, WEF publication). Available from many vendors, such as WTW, Dr. Lange.


Evaluation: - Relatively accurate tests that can be used in the lab and in the field. Relatively easy to use (any lab technician can handle it, but the operator will need at least some lab training). - Only off-line analysis possible. - This method fulfils the needs in terms of range and accuracy. Monitoring technology nr 2: Wet chemical analysis – colorimetric method Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result

x 15 minutes





x

x


Overall conclusion Suitable method for referencing, calibration, periodical measurements



4.8.3 Monitoring technology nr 3: Spectrometric method, derivative spectroscopy

Description: Even though nitrite absorbs strongly in the ultraviolet light, determining nitrite by measuring the absorbance at one wavelength is not reliable because natural organic matter and other solutes also absorb UV light. In natural water and drinking water, the nitrate concentrations are normally so much higher and its signal can not be distinguished from the nitrite signal when using a single wavelength. In the nitrite spectrum the absorbance increase rapidly from 230 to 210 nm, but with a different slope compared to the absorbance of the nitrate ion. Computing the first or second derivative of the spectrum allows differentiation between nitrite and nitrate, and both can be measured independently. Using an instrument of sufficient optical resolution, it is possible to measure both nitrate and nitrite simultaneously. Bromide is known to interfere with this type of measurement at sea water concentrations. However, as the bromide concentration in sea water is rather stable, a single calibration will cancel out this effect. Equipment and consumables Laboratory spectrophotometers have been used for this type of analysis since the 1960's. Nowadays, such systems are also available as online, in situ probe. Full spectrum spectrometers suited for NO2 measurement can be obtained e.g. from s::can. The costs for a spectrometer, lies in the range of 8000 - 12000 Euros. Each of these probes will provide multiple parameters, such as DOC, TOC, UV254, nitrate and others simultaneously. Evaluation: This is the most reliable online technique for nitrite measurement. Calibration is required most of the time at the start of the installation. Long term stability of the optical parts ensures long term measurement stability when no fouling of the optical parts takes place. Some instruments are equipped with automatic cleaning to counter fouling.


Monitoring technology nr 3: Spectrometric method, derivative spectroscopy Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x

x

time to result

x < minute





x x

Recommendation for use in SSS (D) x Spectrometers capable of

performing this measurement can monitor a range of parameters simultaneously. Initial cost per instrument might be higher, but are cheaper than multiple cheap instruments combined. As soon as 3 - 4 parameters (such as nitrate, turbidity and DOC) are needed, these instruments are competivite.

Overall conclusion Reliable method, but rather expensive for a single

parameter. When multiple parameters provided by the instrument are of interest, however, price is very competitive. Operationally very robust and very low maintenance requirements.



4.9 Nitrate Prepared by: S::can Required technical specifications: Concentration ranges that can be encountered are the following: source water (ground or surface water) 0 - 200 mg/L (measured as NO3) drinking water 0 - 50 mg/L (measured as NO3) Required sensitivity of the measurement: 1 mg/L Monitoring technologies: 1. Wet chemical analysis/colorimetric method 2. Electrochemical method 3. Spectrometric method, single wavelength 4. Spectrometric method, derivative spectroscopy 5. Ion chromatography (see for this technique par. 4.22)

4.9.1 Monitoring technology nr 1: Wet Chemical Analysis

Description: The nitrate present in a sample is reduced to nitrite (NO2) in the presence of cadmium. The NO2 thus produced is determined by diazotising with sulfanilamide and coupling to N-(1-naphthyl)-ethylenediamine- Thus a highly coloured dye is formed, which is measured colorimetrically. The applicable range is 0.01 - 1.0 mg/L. Samples at higher concentration need to be diluted before analysis. The method is sensitive to particulate matter present in the sample, as well as presence of iron and copper. Alternatively, reaction of nitrate with hydrazine sulphate, followed by diazotisation as after reduction with cadmium has been used. This can be used in the range 0.01 - 10 mg/L NO3-N. Equipment and consumables: This analysis can be performed using standard laboratory equipment. Alternatively, ready to use cuvettes prefilled with reagent can be obtained. In this case a micropipette is required to accurately dispense the correct amount of liquid into the cuvette. After waiting for the prescribed time ( 10 - 15 minutes), the solution is analyses using a colorimeter. This analysis is well established and described in standard methods, such as APHA Standard methods for the examination of water and waste water (21st edition). The cuvette tests are available from various manufacturers such as WTW, LaMotte, Hach Lange.


The costs for a colorimeter are approx 1000 - 2000 Euros and one cuvette (one concentration measurement) costs approximately 2 - 3 Euros. Alternatively, this analysis has also been automated. So-called process-analysers perform this analysis fully automatic, generally with a frequency of one measurement per minute. As these instruments perform the classical wet chemical analysis, they consist of many pumps for dosing chemicals, collection waste etc. This makes them prone to failures and very intensive to maintain. These systems have mainly been use in industrial applications and waste water treatment plants. Evaluation: The cuvette tests for nitrate are a reliable and fast method for concentration determinations. They are suited for use in the field as well as in the laboratory. The only limitation is the need to perform them manually. The process analysers that automate this procedure suffer from many issues, mainly complexity, to make them a good alternative to the techniques for online measurement described below. This method is particularly suited for low concentration measurements (0.01 mg/L range) where the other techniques are less accurate. Monitoring technology nr 1: Wet chemical analysis Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x 15 minutes


ease-of-use (B) x for the cuvette tests maintenance requirements (C) x for the cuvette tests, for the

online system score would be '2' Costs

instrumentation (C) x colorimeter operational costs (C)


x

x





4.9.2 Monitoring technology nr 2: Electrochemical method

Description: The NO3 ion electrode is a selective sensor that develops a potential across a thin, porous, inert membrane that holds in place a water-immiscible liquid ion exchanger. The electrode responds to NO3 ion activity between about 0.14 and 1400 mg/L as NO3-N). Chloride and bicarbonate ions can interfere when their concentrations are 5 - 10 times higher than nitrate. Cross sensitivity for nitrite is also known. As the measurement is pH sensitive, for reliable measurement either stable pH is required, or a pH sensor is needed to correct for the actual pH. Equipment ion selective electrode for nitrate control device / data logger calibration solutions with known nitrate concentrations Systems using ion selective electrodes have been used for many years. They function well in case of good maintenance (regular cleaning, membrane replacement, re-calibration). The method has been included in APHA Standard methods for the examination of water and waste water (21st edition). Selective electrodes for NO3 can be obtained from manufacturers such as WTW, LaMotte, Hach Lange, Endress + Hauser, Jumo, YSI,. The costs for an electrode, including controller are 2000 - 3000 Euros. Price depends on application. Water proof industrial controller is more expensive than lab equipment. Evaluation: In principle a well proven and satisfactory technique. However, the use of electrodes always suffers from limitations, such as cross sensitivity, maintenance requirements. For continuous monitoring, therefore, optical instruments are preferred.


Monitoring technology nr 2: Electrochemical method Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x < minute


ease-of-use (B) x maintenance requirements (C) x frequent calibration etc Costs



x

x


Overall conclusion Suitable method for online monitoring, however

maintenance intensive (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.9.3 Monitoring technology nr 3: Spectrometric method, single wavelength

Description: This method is suitable for water with low organic content, ie. uncontaminated natural waters or drinking water. Measurement of light absorption at 220 nm allows rapid determination of the NO3 concentration. NO3 absorps very strongly in this region of the spectrum, so strong even that it is the main cause of absorption there. However, because only one wavelength is used, and no distinction can be made between substances, this method is highly sensitive to other substances as well. A crude correction is sometimes made for organics by measurement at a second wavelength, 275 nm, where NO3 does not absorb but some organic substance do absorb. However, even in that case cross sensitivity for nitrite, dissolved organic matter and chromium are often encountered. Equipment Single or dual wavelength spectrometer Systems using single and dual wavelength spectrometers for nitrate measurement have been in use now for approximately 20 years. For stable water quality they can provide good data. They are currently being replaced


by multiwavelength instrumentation (see part 4 of this datasheet). The method has been included in APHA Standard methods for the examination of water and waste water (21st edition). Single and dual wavelength spectrometers for NO3 can be obtained from manufacturers such as Hach Lange, Endress + Hauser, YSI. The costs for a spectrometer lies in the range of 3000 - 4000 Euros. Evaluation: In principle a well proven and satisfactory technique. However, the use of single or dual wavelength optical systems suffers from limitations, mainly cross sensitivity for organics and turbidity. Monitoring technology nr 3: Spectrometric method, single wavelength Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x

x


selectivity

x

x

time to result

x < minute





x

x

Recommendation for use in SSS (D) x could be used for control of

drinking water nitrate levels in case of stable source (ground water)

Overall conclusion Online method that requires low maintenance. Method is not very selective, therefore false results are often obtained.



4.9.4 Monitoring technology nr 4: Spectrometric method, derivative spectroscopy

Description: Even though nitrate absorbs strongly in the ultraviolet light, determining nitrate by measuring the absorbance at one wavelength is not realiable because natural organic matter and other solutes also absorb UV light. The spectra of these organics are very different between water sources, therefore the method described under 3 can be very inaccurate. The UV spectrum of nitrate is quite different from that of organic matter. In the nitrate spectrum the absorbance increase rapidly from 230 to 210 nm, whereas the organic spectrum in natural water this absorbance only increases gradually in this region. Computing the first derivative of the spectrum will effectively eliminate the background signal of the organic and only indicate the nitrate absorption. The main interference possible in this measurement is nitrite, that has a similar but not identical spectrum. Using an instrument of sufficient optical resolution, it is possible to measure both nitrate and nitrite simultaneously. Bromide is known to interfere with this type of measurement at sea water concentrations. However, as the bromide concentration in sea water is rather stable, a single calibration will cancel out this effect. Equipment Spectrophotometer. Laboratory spectrophotometers have been used for this type of analysis since the 1960's. The method has been included as a proposed method in APHA Standard methods for the examination of water and waste water (21st edition). Nowadays, such systems are also available as online, in situ probe. Full spectrum spectrometers suited for NO3 measurement can be obtained from various manufacturers such as s::can, Trios, Stip. The costs for a spectrometer, lies in the range of 8000 - 20000 Euros. Each of these probes will provide multiple parameters, such as DOC, TOC, UV254, nitrate and others simultaneously. Evaluation: This is the most reliable online technique for nitrate measurement. Calibration is required most of the time at the start of the installation. Long term stability of the optical parts ensures long term measurement stability when no fouling of the optical parts takes place. Some instruments are equipped with automatic cleaning to counter fouling.


Monitoring technology nr 4: Spectrometric method, derivative spectroscopy Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x < minute





x x

Recommendation for use in SSS (D) x Spectrometers capable of

performing this measurement can monitor a range of parameters simultaneously. Initial cost per instrument might be higher, but are cheaper than multiple cheap instruments combined. As soon as 3 - 4 parameters (such as nitrate, turbidity and DOC) are needed, these instruments are competitive.

Overall conclusion fast, online method with high selectivity. Equipment is

more expensive than single wavelength. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.10 Polycyclic aromatic hydrocarbons Prepared by: TZW See par. 4.3: Benzo(a)pyrene and other PAHs

4.11 Pesticides See par. 4.19: Organic microcontaminants


4.12 Tetra- and trichloroethene Prepared by: TZW Required technical specifications: - concentration range: 0.1 – 100 µg/L (drinking water; raw water concentration can be higher) - sensitivity required: 0.1 µg/L (drinking water) Monitoring technologies: 1. Liquid-liquid extraction, GC-ECD(-ECD) 2. Headspace, GC-ECD-(ECD) 3. purge&trap, GC-MS


Description: EN ISO 10301: Liquid-liquid extraction of 100 mL water sample with 1 mL n-hexane; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC columns with different polarity). Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Reliable standard method. For increasing selectivity, two GC columns should be used.






drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Reliable method. For increasing selectivity, two GC columns should be used.






drinking water

x x


selectivity

x x

time to result

x





x x

x Recommendation for use in SSS (D) x

Overall conclusion Reliable and sensitive method. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.13 Disinfection byproducts (trihalomethanes) Prepared by: TZW Required technical specifications: - concentration range: 0.1 – 100 µg/L (drinking water; depending on the chlorine doses) - sensitivity required: 0.1 µg/L (drinking water) Monitoring technologies: 1. Liquid-liquid extraction, GC-ECD(-ECD) 2. Headspace, GC-ECD-(ECD) 3. purge&trap, GC-MS


Description: EN ISO 10301: Liquid-liquid extraction of 100 mL water sample with 1 mL n-hexane; analysis of extract with GC-ECD or GC-ECD-ECD (using two GC columns with different polarity) Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Reliable standard method. For increasing selectivity, two GC columns should be used.






drinking water

x x


selectivity

x

x

time to result

x





x x


Overall conclusion Reliable method. For increasing selectivity, two GC columns should be used.






drinking water

x x


selectivity

x x

time to result

x





x x

x Recommendation for use in SSS (D) x

Overall conclusion Reliable and sensitive method. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.14 Radioactivity Regarding radio-activity there was no partner within WA3 having sufficient expertise to prepare an evaluation form. To enable readers to have some information on analytical techniques, information from the literature will be presented below. The information is retrieved from: Wisser, S. and Hartkopf, J. (2006) Natural and artificial radioactivity in the Rhine and its tributaries. In: Knepper (ed) The Handbook of Environmental Chemistry. Volume 5 Water Pollution, Part L: The Rhine. p255-306. Measurement systems for radioactive isotopes are primarily based on the in-teraction between ionizing radiation and matter. Presently, three categories of radiation detection systems are mainly used for radionuclide determination: • Semiconductor detectors (α-/β- and β-spectrometry) • Scintillation counters (α- and /β-measurements) • Proportional counters (α- and /β-measurements) Counting techniques are still highly applicable for the determination of radioisotopes with half-lives of less than a few thousand years. However, inductively coupled plasma mass spectrometry is the current state-of-art in-strumentation for measuring multiple elements at trace level. Unfortunately, this outstanding method is presently only applicable for stable isotopes or long-lived primordial isotopes, such as 232Th and 238U. The following sections provide details of measurement techniques for the determination of radionuclides and present a brief summary of the advan-tages and disadvantages of the respective counting method.

4.14.1 Semiconductor Detectors

The use of solid detection medium is of great advantage for the detection of high-energy electrons, heavy particles and γ-rays. In the first place, detector dimension can be kept smaller due to the high density of semiconductors. In addition to that, a superior energy resolution can be achieved by using a solid semiconductor detector. There are different semiconductor materials available today, such as silicon and germanium. The fundamental informa-tion carriers are electron-hole pairs created in the crystal lattice by primary or secondary charged particles along their path to the detector [1]. The move-ment of electron-hole pairs through the crystal generates the basic electrical signal from the detector. Since the intensity of the electric pulse is proportional to the energy of the penetrating particle, semiconductor detectors contribute full energy informa-tion. Multi-channel analyzers (MCA) provide the energy resolution and the primary analog signal is transformed into digital units by use of an analog-digital-converter. Compared with other counting techniques, semiconductor detectors possess advantages, such as excellent energy resolution, good stability, excellent timing characteristics and simplicity of operation [1]. Alpha-Spectrometry Alpha-spectrometry is an extremely useful and sensitive method for the de-tection of α-emitting radionuclides in a variety of materials. One of the main reasons for the sensibility of this method is the very low background radia-


tion. High-purity silica surface barrier detectors have become the detectors of choice for the measurement of α-particles. The relatively small size of a silicon detector might be a limitation, especially when a large surface area is required. Modern α-spectrometers are equipped with vacuüm chambers to achieve a high-energy resolution of 20-30 keV. The obtained a-spectrum can easily be analyses by using simple region of interest (ROI) settings. In addition, the energy resolution is generally sufncient to separate the energy peaks of the most important natural and artificial radionuclides present in waters. Unfortunately, extended measurement times (2-3 days) are necessary to achieve low detection limits in the range of several mBq/L. Gamma-Spectrometry High-purity germanium (HPGe) detectors are preferably used for complex γ-ray spectrometry. This is due to the lower melting point of germanium compared with silicon, which makes the exclusion of impurities in the re-fining process much easier [1]. Additionally, the extremely high density of germanium (5.33 g/cm3) leads to a higher adsorption probability for γ-rays [1]. Another advantage of gamma-spectrometry is the relatively low self-absorption of γ-rays within the sample, which results in a simple sample preparation. However, the counting efficiency of HPGe detectors is com-parably low and the naturally occurring γ-background radiation might be problematic when trying to achieve low detection limits. These three effects of radiation interactions with matter are fundamental of γ-spectrometry:

- Photo effect (detector-atom absorbs the energy of the incident photon entirely)

- Compton effect (incident photons are scattered by electrons of the detector)

- Pair production (positron-electron pair is created by the incident photon)

The operation of germanium detectors at room temperature conditions is impossible. This is because of the thermally induced leakage current that would occur at room temperature. Hence, germanium detectors must be cooled with liquid nitrogen to reduce the leakage current to the point that the associated noise does not spoil their excellent energy resolution [1]. Usually, the temperature is reduced to -196 °C by using an insulated vessel in which liquid nitrogen is kept in thermal connection with the detector.

4.14.2 Liquid Scintillation Counting

The liquid scintillation method is one of the oldest and most useful method for detection and spectroscopy of ionizing radiations [1]. It is mainly utilized for the detection of low-energetic β-emitting radioisotopes, such as 3H and 14C, but α-emitting radioisotopes can also be detected with high efficiencies. The approach involves dissolving the sample directly into the liquid scintilla-tor. Problems relating to sample self-adsorption or beta-backscattering from the detector are completely avoided [1]. The scintillation technique is based on the phenomenon of fluorescence and detects photons produced by the scintillator material. These light flashes are caused by exited states of the scin-tillator molecules after the absorption of energy from radioactive decay. The


photons are converted in electrical pulses by a photo multiplier tube (PMT) and can be analyzed by electronic equipment. The liquid scintillation cocktail is composed of a primary and a secondary scintillator—that are able to trans-form the decay energy to light flashes—and an organic solvent (e.g., toluene) as carrier substance. Thus, the scintillator molecules can be regarded as the actual radiation detector in a liquid scintillation counter. Modern liquid scintillation counters are able to distinguish between the different types of radiation and even between the different energies of the same type of radiation. The discrimination between α- and β-radiation in water samples is applicable for determining gross-alpha and gross-beta ac-tivity concentrations. One major disadvantage of liquid scintillation counters is their relatively poor energy resolution. Furthermore, quench effects may reduce the counting efficiency and background radiation might negatively affect the measurement. On the other hand, the counting efficiency for α- and β-particles can potentially be close to 100% in unquenched samples [1].

4.14.3 Inductively Coupled Plasma Mass Spectrometry

Inductively coupled plasma mass spectrometry (ICP-MS) provides a rapid and sensitive technique for determining long-lived radionuclides and stable isotopes. The principal use of the ICP-MS is that of a mass spectrometer—to detect the mass of elements according to their mass to charge ratio (m/z). As the analytes enter the plasma, they are stripped off to their ionic form. This is only possible due to the high temperature of the plasma, which goes up to approximately 6000-10000 K [2]. There are different types of mass separation devices, which can be used in a mass spectrometer. The most com-mon ICP mass spectrometers are quadrupole-based instruments (ICP-QMS), which allow isotope ratio measurements with a precision for short-term isotope ration measurements from 0.1% to 0.5% relative standard deviation [3]. The achievable lowest limit of detection (LLD) of an ICP-MS for the de-termination of uranium isotopes is about 0.1 ng/L - without further sample preparation and sample concentration, respectively. Major advantages of ICP-MS are the small sample sizes, high sample throughputs, and short measurement times with only few sample preparation steps [74]. Not only that, ICP-MS offers efficient and sensitive multi-elemental analysis in trace or ultra-trace levels [3]. However, the instrument itself is rather costly and it requires high maintenance. High sample matrix concentration may interfere with the determination of low levels of long-lived radioisotopes, but solid-phase extraction may overcome these problems [4].


References

1. Knoll GF (2000) Radiation detection and measurement, 3rd edn. Wiley, New York, p 802 2. Ponce de Léon CA, Montes-Bayón M, Caruso JA (2002) Elemental speciation by chromatographic separation with inductively coupled plasma mass spectrometry detection. J Chromatogr 974:1 3. Becker JS, Dietze HJ (2000) Precise and accurate isotope ratio measurements by ICP-MS. Fresenius J Anal Chem 368:23 5. Unsworth ER, Cook JM, Hill SJ (2001) Determination of uranium and thorium in natural waters with a high matrix concentration using solid-phase extraction inductively coupled plasma mass spectrometry. Anal Chim Acta 442:141


4.15 Endocrine disruption chemicals Prepared by: BDS There are several types of endocrine systems where chemicals may interact with. For each type of endocrine interaction there exists several techniques. Below, an overview is presented. For specific details regarding individual interactions, analytical techniques and references see Annex I. Required technical specifications: Concentration range that should be covered: no limits have been set for endocrine disrupting compounds in the (aquatic) environment. Sensitivity/resolution required: not applicable. Monitoring technologies: Bioassays detecting estrogenic activity

1. ER CALUx® 2. MVLN and MELN 3. T47D-KBluc 4. YES and related assays 5. E-Screen

Bioassays detecting androgenic activity

6. AR CALUx® 7. MDA-bk2 8. PALM 9. YAS and related assays 10. A-Screen

Bioassays detecting progestagenic activity

11. PR CALUx® 12. TM-Luc 13. Yeast based assays

Bioassays detecting glucocorticoid activity

14. GR CALUx® 15. TGRM-Luc 16. MDA-kb2

Bioassays detecting thyroid activity

17. TRβ CALUx® 18. T-Screen 19. PC-DR-LUC 20. xL58-TRE-Luc


4.15.1 Introduction

The focus of this document is on bioassays that have been applied and validated for the analysis of hormone receptor mediated endocrine disrupting compounds in water, including extraction and clean-up. Although many different cell lines – which are an essential part of the bioassay analysis - have been developed to detect several types of endocrine activity, only a limited number of bioassays have been applied in the detection of endocrine activity in the aquatic environment. Bioassays for the detection of non-estrogenic responses have almost never been applied for environmental monitoring and have not been subjected to comparison studies, but are included for completeness. However, no information is known about their robustness and sensitivity towards the analysis of water.

4.15.2 Analysis of water using bioassay

Many different cell line based bioassays have been developed, generally based on mammalian or yeast cells. However, assays utilizing fish or amphibian cell lines have also been described, also depending on the species of interest. Differences exist regarding the ease of culture, sensitivity to toxic compounds and sensitivity towards compounds of interest. Regardless of the cell line used in the assays, they mainly fall in one of the following categories:

• Reporter gene assays. These assay measures hormonal receptor binding dependent transcriptional activity

• Cell proliferation assay. These assays measure the increase in the amount of cells as a response to specific type of compounds.

Reporter gene assays The principle behind reporter gene assays is that binding to a specific receptor results in activation of this receptor. The activated receptors bind to the corresponding responsive elements (REs), resulting in transcription of the normal target genes. In reporter gene assays, a DNA sequence has been added for the production of an easily quantifiable gene product under control of similar REs. Activated receptors bind to these REs as well, resulting in the production of the product, e.g. an enzyme. The reporter gene can be stably transfected (incorporated) into the cells or can be transfected transiently. Since transient transfections require more work and show more variability within and between assays they are considered not suitable for water quality monitoring. To be able to activate the reporter gene, an activated receptor is needed. Several reporter gene assays make use of naturally present (i.e. endogenous) receptors. The advantage of using endogenously present receptors is that the resulting bioassays are sometimes more sensitive. Using endogenously present receptors however, increases the risk of non-specific responses since often other types of receptors are co-expressed. Therefore, reporter gene assays have been developed utilizing transfected (i.e. artificially introduced)


receptors. These receptors are mostly derived from human cells but can also be derived from other species. Beside mammalian cells, yeast cells are frequently utilized for making bioassays responding to hormones because they are easier to handle and do not endogenously express hormone receptors. However, bioassays based on yeast cells are generally much less sensitive than bioassays based on mammalian cells and have problems identifying antagonistic activity. Beside general problems regarding sensitivity, the presence of a cell wall in yeast combined with very active membrane transport mechanisms can results in different activity for compounds and extracts and false negatives. Therefore, yeast cells are generally not recommended for areas involving risk assessment. Cell proliferation assays Cell proliferation assays make use of fact that proliferation (i.e. increase in number) of cells can be induced or inhibited as a response to compounds. By relating the amount of cell growth to that of cells exposed by the reference compound, the response can be quantified.

4.15.3 Analysis using bioassays

Method of analysis Although different in details, most bioassays described in this document follow similar methods for the analysis. In general, water samples are extracted using solid phase extraction of liquid-liquid extraction. The solvents are evaporated and the extract is transferred to a carrier, generally DMSO, because of its low volatility and good dissolving capabilities. To analyse the activity in the extract, bioassay cells are seeded into 96 well plates, usually using medium that is supplemented with hormone-stripped serum. The next day, when the cells have attached, the cells are exposed by replacing the cell culture medium by medium that contains the extracts to be tested. After exposing the cells for a specified amount of time (generally 24 hours), the amount of response produced is quantified by lysing the cells and the adding the appropriate substrate. When luciferase is used as a reporter, luciferin is added and the amount of light produced is quantified using a luminometer. When β-galactosidase is produced, chlorophenol red-β-D-galactopyranoside (CPRG) or 2-nitrophenyl- β-D-galactosidase (ONPG) can be used to determine the amount of red or yellow colour produced respectively using a spectrophotometer. The response of bioassays producing Green Fluorescent Protein can be quantified using a fluorometer. Yeast bioassays are generally performed similarly mammalian bioassays, but without the need of the cells attaching to the 96 well plate. Proliferation assays are seeded and performed like all other mammalian bioassays, however exposure times are longer (3-7 days). This is due to the kinetics of induction of cell proliferation. The amount of cells can be quantified by counting nuclei or by an enzymatic endpoint.


In all assays, the amount of response is a direct measure of amount of agonistic activity. By also exposing cells to a concentration series of known agonists, the amount of response can be expressed as reference compounds equivalents. Beside agonists, also antagonists can be present in the water extract. By co-exposing the cells to a fixed amount of reference agonist (generally at the EC50 level), the amount of decrease in activity can be used as a measure of antagonistic activity. Equipment and consumables For the analysis of water samples, all bioassays are exposed to extracts. Several different methods have been described, usually based on solid phase extraction or liquid-liquid extraction. Since most bioassays use mammalian cell lines, the equipment needed for culturing and performing the bioassay is similar for all bioassays. Cells are maintained in an incubator to provide optimum temperature, pH and humidity. Handling of the cells usually takes place in a laminar flow cabinet to provide sterile conditions. All equipment needed (pipettes, culture flasks, 96 well culture plates) has to be sterile. Differences exist in the equipment needed to quantify the response, whether being a luminometer (luciferase reporter), fluorometer (GFP reporter) or spectrophotometer (β-galactosidase reporter).

4.15.4 General evaluation of the bioassays

It is important to realize that monitoring of water quality using bioassays comprises not only a responsive cell line, but also robust extraction and clean-up procedures. Since some cell lines have demonstrated to be more sensitive to matrix interferences, validation of the bioassay for the matrices of interest is very important. Very recently, an inter-laboratory study focusing on estrogen responsive bioassays was performed by the GWRC (2008), allowing for a direct comparison of the responses of the individual bioassays. This study showed that clear differences exist between bioassays with regard to the sensitivity and coefficients of variation. Unfortunately, similar studies are not frequently conducted. While inter-laboratory studies regarding water analysis for estrogenic activity using bioassays are scarce, similar studies focussing on other than estrogenic responses are virtually non-existed. Although most of the information is available for the estrogenic bioassays only, to some extent conclusions and remarks can be made regarding the suitability of the other bioassays. Sensitivity In general, all mammalian cell lines bioassays evaluated in this document are more sensitive than GC-MS analysis, but LODs for the specific cell lines are generally not provided in the original manuscripts. A recent study conducted by the GWRC (Leusch, 2008) showed that of the five estrogen responsive bioassays tested, the E-Screen and the ER CALUx were the most sensitive, being approximately 10 times more sensitive than GC-MS. The ER CALUx and the E-Screen also showed the lowest coefficient of variation. The YES


bioassay was shown to be at least 10 times less sensitive than mammalian cell line based bioassays (Leusch, 2008; Murk et al., 2002). This low sensitivity, combined with a risk of false negative due to very active membrane transport mechanisms that are present in yeast (ICCVAM, 2003), makes that the YES (and other yeast based bioassays) are not suited for drinking water quality assessment. Information on sensitivity regarding bioassays measuring other receptors mediated processes is unavailable, although the PALM bioassay metabolizes testosterone, resulting in an underestimation of the amount of androgenic activity when testosterone is present. Robustness Many cell lines have been developed, although not always for the application of (aquatic) environmental monitoring. Most cell lines have been utilized only by a very limited number of labs or samples, making it difficult to evaluate there robustness as a bioassays. Few cell lines have successfully been used by different labs or have been applied in comparison studies for the use of water monitoring. However, this seems to be restricted to ER responsive cell lines. Bioassays that have shown to produce good results when handled by different laboratories are considered to be robust. When no information is available, as is the case with some ER and AR assays and all PR, GR and TR assays, this type of robustness of the assay cannot be assessed. Another form of robustness that is of importance is the robustness of the cells that are used in the bioassay. Yeast cells are generally easier to handle than mammalian cells. However, this also leads to an increased risk of false negatives. Some mammalian cell lines have been reported to be more sensitive to matrix effects. Selectivity Differences exist between the specificity of the bioassays, mostly as a result of the reporter construct used. One of the most frequently used reporter constructs is MMTV-Luc. However, this reporter can be activated by androgens, progestins and glucocorticoids. Bioassays that utilize this construct, in combination with cells that endogenously express receptors other than the receptor of interest, do generally not respond to the compounds of interest exclusively. For examples the androgenic MDA-kb2 and TARM-Luc cell lines were also shown to respond to glucocorticoids and progestins respectively, limiting their use in screening for androgenic activity. Similar problems exist with several bioassays using other receptors. This low selectivity makes these bioassays less suitable for selective screening, especially for complex mixtures like (surface) water extracts. The cell proliferation bioassays E-Screen has also been shown not to respond to estrogens exclusively. Yeast based assays like the YES generally cannot differentiate between agonistic and antagonistic activity. Reporter gene assays utilizing a construct containing only the responsive elements of interest allow for a more selective analysis of endocrine activity. Time to result Most reporter gene bioassays use an exposure time of 24 hours, although differences exist ranging from 16 to 48 hours. Proliferation assay generally take more time to produce a quantifiable response (generally 3-7 days) and


are therefore considered not to be suitable for routine monitoring. The response of yeast cells is in some occasions detectable more quickly, allowing for exposure times of approximately 2 hours or more, although most yeast based bioassays use exposure times of 16-48 hours. Ease of use All mammalian cell lines require similar equipment and culturing techniques. Yeast cells are slightly easier to culture than mammalian cells, requiring less sterile conditions. All cell lines can become contaminated with mycoplasm, which has to be checked regularly. This places a responsibility on the source of the cells. Some bioassays like CALUx® and EcoScreen™ are marketed by commercial companies. Several cell lines have been used by different users world wide, indicating their transferability and ease of use. The CALUx cell lines are offered including training and customer support, improving the reliability and ease of use of the bioassay. Maintenance requirements All mammalian cell lines require more or less the same handling, equipment etc. which includes culturing the cells once or twice a week. Commercially available cell lines can include troubleshooting and customer support, facilitating maintenance. Instrumentation Most bioassays make use of a luciferase or GFP reporter gene, requiring a luminometer or fluorometer respectively. The response of cells producing β-galactosidase can be quantified using a spectrophotometer. Operational costs The mammalian cell based bioassays do not vary greatly in the need of consumables or labour. Therefore, these costs are not assessed further. Several assays, including commercial ones, include licensing costs. Recommendation for use in small scale systems Due to the equipment and techniques needed, in vitro bioassays are not recommended for use in small scale systems.


Table of evaluation: overall rating of the different monitoring technologies.

Sensitivity (A)

Robustness (A)

Time to result


source water

drinking water

operational robustness

selectivity ease-of-use (B)

maintenance requirements (C)

1. ER CALUx® 4 4 5 3 3 4 3

2. MVLN and MELN 2 2 2 2-3 2

3. T47D-KBluc 3 3 3

4. YES and related assays 1 1 4 3 2-3 3 3

5. E-Screen 4 4 4 2 1 3 3

6. AR CALUx® 3 3 3 5 3 4 3

7. MDA-bk2 3 3 1 3

8. PALM 3 3 3 3 3

9. YAS and related assays 2 1 3 3 3

10. A-Screen 3 3 2 1

11. PR CALUx® 3 3 3 5 3 4 3

12. TM-Luc 3 3 1 3

13. Yeast based reporter gene assays

2 1 4 3

14. GR CALUx® 3 3 3 5 3 4 3

15. TGRM-Luc 3 3 1 3

16. MDA-kb2 3 3 1 3

17. TRβ CALUx® 3 3 4 3

18. T-Screen 1

19. PC-DR-LUC

20. xL58-TRE-Luc

(A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low


4.16 Genotoxicity Prepared by: BDS Monitoring technologies: Gene mutation assays

1. Ames test 2. Vibrio harveyi test

Chromosomal aberration tests

3. Comet assay 4. Micronucleus assay 5. Alkaline elution assay 6. Polymerase inhibition assay

DNA repair assays

7. UMU test 8. SOS Chromotest 9. Vitotox® 10. Mutatox™ 11. GreenScreen® 12. GreenScreen HC 13. MCF-7-p53R2

4.16.1 Introduction

A large number of tests have been developed for detecting genotoxicity in water samples, of which some are commercially available and/or have been validated for water specifically. The focus of this document is on assays that could - in principle - be utilized to detect genotoxicity in surface and drinking water. Ideally, these tests can be performed in 96 well plates or other high-throughput format, and would take less than two days to perform. Assays that have been developed recently, but that have not been used (frequently) for the analysis of genotoxicity in water, have also been included, although the potential of these novel assays still needs to be assessed. An overview in general is presented below. Evaluation forms for the specific tests and references can be found in Annex II.

4.16.2 Mechanisms of genotoxicity

Many different compounds have the ability to elicit a genotoxic effect, i.e. enter the cell and damage DNA. The resulting genotoxic stress can trigger appropriate responses to cope with the damage, such as growth arrest, DNA repair and in extreme circumstances apoptosis (programmed cell death). However, sometimes the repair mechanisms fail and the exposure to the compound(s) can result in DNA changes, either small such as gene mutations and/or large such as chromosomal aberrations.


Mutations are small-scale changes in the nucleotide sequence in the DNA. Generally, mutations can be subdivided into two types: base-pair substitutions and frameshift mutations. In base-pair substitutions, one or more base-pairs are substituted by others, mostly resulting in a changed amino acid in the related protein. With frameshift mutations, one or more base-pairs are added or deleted from the DNA sequence. Due to the triplet nature of gene expression by codons, the insertions and deletions can disrupt the reading frame, resulting in a completely different translation, and thus in a completely different protein. While mutations affect only nucleotides, chromosomal aberrations are large-scale changes in the DNA that can be structural or numerical. Structural chromosomal aberrations (caused by so called clastogens) are DNA breaks and exchanges in the chromosomes, where numerical aberrations (caused by so called aneugens) change the number of chromosomes in the cell.

4.16.3 Tests to detect genotoxicity

Several methods exist for the detection of genotoxicity. These methods are based on different principles, enabling detection of specific or nonspecific types of DNA damage. The existing methods fall into one (or more) of the following categories:

- Gene mutation assays. These assays belong to the most applied and well known methods, like the Ames test. Gene mutation assays rely on the detection of compounds that can induce gene mutations, utilizing different strains of bacteria, yeast cells or mammalian cells. These mutations can be forward (from wild type to mutant) or back (from mutant to wild type). In general, mutations assays rely on the acquired ability of bacteria or cells to grow on selection medium when a specific mutation occurs. After exposure, the number of cells or colonies can be detected and this number is a measure of the mutagenic potential of a compound or extract.

- Chromosomal aberration tests. Chromosomal aberrations can consist of DNA breaks and/or unsuccessful separation of chromosomes during cell replication. Chromosomal aberration tests make use of the visualization of chromosomal damage as an indication of DNA damage.

- DNA repair systems. As a response to damaged DNA, several DNA repair systems can be activated. The activation of these systems can be linked to a reporter gene, resulting in the production of an easily detectable enzyme or protein. Most DNA repair assays utilize bacteria and their SOS repair system, although yeast and mammalian cell line based assays have been developed recently as well. However, large differences exist between the prokaryotic and eukaryotic DNA repair mechanisms, with the latter generally being more complex.

4.16.4 General review

Screening water samples for genotoxicity equals screening of complex mixtures with unknown compounds and concentrations. In this respect it is different, and more complex, than the regulatory testing of pure compounds where generally (very) high concentrations of a single compound are tested,


and where positive findings can be followed by in vivo testing for confirmation. Therefore, there is a need for assays that are sensitive, accurate, reproducible and robust for the detection of genotoxins in water. A large number of (commercially available) tests exist to determine genotoxicity in a variety of matrices. Most of the assays developed have been applied to the aquatic matrix and some have been standardized at the international level by ISO. Most of the assays to detect genotoxicity utilize bacteria, which have the advantage of being relatively easy and fast to perform, but have some limitations related to the use of prokaryotic cells and their repair systems. Due to the polysaccharide envelope, not present in mammalian cells, in combination with active transport mechanisms, chemicals cannot easily enter bacteria. Bacteria also do not have the metabolic capacity of mammals, although this has partly been solved by utilizing S9 fractions. Most bacterial mutation assays can only detect specific types of (back) mutations, covering only a small part of the genome, i.e. the part where the mutation was originally introduced. Although this has in part been solved by utilizing the SOS responses of bacteria, a more general indication of DNA damage, the eukaryotic DNA repair mechanisms are generally more complex. Damage like chromosomal aberrations cannot be detected using micro organisms. Existing bacterial tests are known to suffer from detecting a high number of false positives when testing pure compounds (Kirkland et al., 2005; Tweats et al., 2007), which can be problematic when testing complex mixtures consisting of many compound. To solve the shortcomings of the bacterial assays, several genotoxicity tests have been developed based on mammalian cells. Depending on the mammalian cell line used, cells can in part metabolize genotoxic compounds; otherwise S9 fractions can be utilized. The most used and validated tests, like the micronucleus test and the Comet assay, allow for the sensitive detection of clastogens and aneugens, a type of DNA damage that cannot be tested for using bacterial assays. However, these tests are very labour intensive and have difficulty detecting types of DNA damage that do not include fragmentation of DNA. Mammalian mutagenic assays like the TK assays or HPGRT test are capable of detection mutagenic activity, but are very time consuming since they can take several weeks to perform. All studies clearly show that no single test is capable of detecting all relevant genotoxic activity in water. Therefore, the genotoxic potential of water can only be evaluated using a battery of tests, using at least two assays aimed at different endpoints (Corbisier et al., 2001; Heringa, 2005). Tests with real water samples showed that while most tests are able to detect genotoxins as pure compounds, the same compounds are frequently not detected when spiked to surface water (Corbisier et al., 2001). Similar tests revealed that some water samples were scored as non-genotoxic by most bacterial assays, while the opposite was found by tests utilizing higher target organisms (yeast, mammalian cells). These results illustrate that results from bacterial tests might not reflect the actual risk for higher organisms. The first results from novel assays utilizing cells from higher organisms, and focussing on a general measure of DNA damage, look promising. However, these assays


have some draw-backs, like the use of yeast cells and the use of GFP, are not expected to detect all types of genotoxicity, and have not been validated for water specifically. Therefore, there remains a need for quick and easy genotoxicity assays, preferably based on mammalian cells, that are sensitive enough to detect a broad range of genotoxins in water.

4.16.5 Sensitivity and specificity

Several reviews exist with regard to the sensitivity and specificity of assays to detect genotoxins. These reviews mainly focus on the genotoxicity of pure compounds, although some have been directed at genotoxins in water. The high concentrations that are sometimes necessary to elicit a detectable response are easily achieved when using pure compounds. However, when analyzing water samples (or extracts), the sensitivity of the assay is of high importance. With regard to the pure compounds, it was shown that the currently applied in vitro bioassays are able to detect genotoxins correctly (albeit at high concentrations), but suffer from detecting a high number of false positives (Kirkland et al., 2005; Tweats et al., 2007). Since for complex mixtures like (extracts from) surface water the chemical composition is unknown, and no in vivo studies are performed with positive water samples, it is difficult to assess the trueness of the applied genotoxicity assay in this respect. However, studies aimed at the detection of genotoxicity in water showed that there are great differences between sensitivity of the assays, both regarding the response to pure compounds (in water) as to water and/or water extracts (Farré et al., 2007; Reifferscheid and Grummt, 2000). Assays relying on the induction of bacterial responses, like SOS chromotest, Vitotox and Umu were shown to be much more sensitive than other bacterial tests like Mutatox and Ames. Although the Ames test is commonly used for environmental samples, it is not recommended for rapid water quality analysis due to the relatively low sensitivity and long analysis time (Corbisier et al., 2001; Wegrzyn and Czyz, 2003). Because some compounds are not genotoxic agents themselves, most assays can also be performed including a metabolisation step, generally by adding liver S9 fraction. This mixture contains microsomal and cytosolic fractions of liver cells, incorporation both phase I (transformation) and phase II (conjugation) enzymes. Although this can give an indication of genotoxicity of metabolites, differences can exist between batches, species of origin and induction of enzymes. Most frequently, S9 fractions are derived from rat livers treated with phenobarbital or Aroclor 1254. However, the balance of activating enzymes usually is unrepresentative of what will occur in human livers (Ku et al., 2007). Tests have been described using human derived S9 fractions, which behave differently than rat derived S9 (Hakura et al., 1999). The use of S9 - as well as the presence of other autofluorescent compounds - can interfere with the measurement when using GFP based assays, since S9 is fluorescent and therefore interferes with GFP analysis.


4.16.6 Robustness

Although most assays have been used frequently for the detection of genotoxins in surface and drinking water, only a limited amount of interlaboratory studies have been performed. One study, comparing 14 genotoxicity tests on 11 samples, showed that small differences in assay conditions of a particular test, as well as differences in interpretation of the generated data, often greatly influence the outcome, even for identical tests (Farré et al., 2007). Some assays also showed matrix interference when testing pure compounds that are added to water samples (Corbisier et al., 2001), stressing the validation of the assay for the matrix of interest.

4.16.7 Time to result

Most bacterial assays use an exposure time of 24 hours, although the Vitotox assay is somewhat faster with an exposure time of 18 hours. The Ames test, with exposure times ranging from 2-5 days, takes too long to perform and is therefore not recommended for routine monitoring. The GreenScreen assay has an exposure time ranging from 4-18 hours, with longer exposure times resulting in lower detection limits. However, a similar yeast assay utilizing another reporter gene can produce responses more quickly, but this assay has not been tested for water samples. Because the Comet assay can utilize many different cells, exposure times depend on the source of the cells. Most Comet assays using in vitro cell lines use an exposure time of 24 hours, but the analysis of the response also takes time since it utilizes electrophoresis.

4.16.8 Ease of use and instrumentation

Bacterial assays are relatively easy to perform, as bacteria are robust and can be grown in a simple incubator. However, since some bacterial assays utilize pathogenic bacteria (e.g. Salmonella typhimurium), a laboratory suited for the appropriate risk level is required. Regarding eukaryotic cells, yeast cells are relatively easier to culture than mammalian cells, requiring less sterile conditions. More complicated tests like the alkaline elution test, Comet assay and micronucleus test are much more labour intensive. Many differences exist in the way the assays quantify the final response. The response of reporter gene assays can easily be determined by measuring luminescence, fluorescence or colour formation using a luminometer, fluorometer or spectrophotometer respectively. The Mutatox assay even comes with a specific Mutatox Analyser, reagents and media. Some assays produce responses that can even be quantified by simply counting the amount of colonies. Automated versions have been developed for the alkaline elution test, Comet assay and micronucleus, but these require more expensive and specialised equipment. Unlike other tests, the polymerase inhibition assay requires a PCR machine.


4.17 Acute toxicity Prepared by: IGB/bbe Moldaenke Required technical specifications: For toxicity detection the use of biological systems is inevitable. Here, model organisms act as reliable indicators for harmful agents, e.g. toxins. Unlike analytical instruments, they will respond to several toxic compounds without pre-calibration. The majority of the standard acute toxicity tests are performed for the examination of waste water, sludge and for the characterisation of the toxic potential of a chemical. To detect toxicity in drinking water it is essential to measure the water quality in real-time, on line, and at different points of the source. For that reason new biomonitoring technologies were developed that can assess the toxicity of water samples by analysis of living organism behaviour within a short time period, whereby significant changes of behaviour trigger an alarm. This was made possible by advances in digital video processing, signal analysis, and by fast computers. Most of the online biomonitoring tests are performed on fish and selected aquatic invertebrates. The sensitivity of the alarm can often be pre-selected and adjusted by the user based on the specific application. Though it is known that different species vary in their sensitivity to different substances, the so called biological early warning systems (BEWS) are sensitive to a wide range of harmful agents including synergistic effects of toxic mixtures. The following biomonitoring technologies are suitable for testing drinking water. If source water does not interfere the optical recognition systems (e.g. by high concentrations of humic substances), test are also applicable in this case. Monitoring technologies: 1. Standard toxicity tests 2. Daphnia toximeter 3. Fish toximeter 4. Combined Fish and Daphnia toximeter 6. Algae toximeter 7. Luminiscent bacteria 8. Mussel monitor

4.17.1 Standard toxicity tests

Description: A variety of methods of toxicity test methods have been standardized in different countries. Some of these procedures are recognized internationally, notably the standard methods published by the International Organisation (ISO), and the guidelines given by the OECD. For determining the acute toxicity of substances to aquatic organisms, the following ISO methods are used:


• ISO 6341:1996 Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)

• ISO 7346-1:1996 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Danio rerio Hamilton-Buchanan (Teleostei, Cyprinidae) - Part 1: Static method.

• ISO 7346-2:1996 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Danio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 2: Semi-static method.

• ISO 7346-3:1996 Water quality - Determination of the acute lethal toxicity of substances to a freshwater fish [Danio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 3: Flow-through method.

• ISO 12890:1999 Water quality - Determination of toxicity to embryos and larvae of freshwater fish. Semi-static method.

• DIN 38415-6: 2003 German standard methods for the examination of water, waste water and sludge – Sub-animal testing (group T) - Part 6: Determination of the effect of nonacute toxicity of waste water on the development of fish eggs (K 9).

• ISO 8692:2004 Water quality – Freshwater algal growth inhibition test with unicellular green algae.

Most of these standard acute toxicity tests use the water flea Daphnia magna, the fish Danio rerio and fish eggs of Danio rerio; the procedures also allow for the use of some other fish species. The basic data obtained is the LC50 for periods of 24, 48, 72 and 96 hours; for Daphnia the usual maximum exposure period is 48 hours. The following standard describes the performance testing of on-line sensors/analysing equipment for water (but it is not an analytical method itself): • ISO 15839:2003 Water quality - On-line sensors/analysing equipment for

water - Specifications and performance tests. The standard is applicable to most sensors/analysing equipment, but it is recognized that for some sensors/analytical equipment certain performance tests cannot be carried out. This International Standard • defines an on-line sensor/analysing equipment for water quality

measurements; • defines terminology describing the performance characteristics of on-line

sensors/analysing equipment; • specifies the test procedures (for laboratory and field) to be used to

evaluate the performance characteristics of on-line sensors/analysing equipment.

Evaluation: These standard toxicity tests are primarily performed for the examination of waste water, sludge and for the characterisation of the toxic potential of a chemical. A continuous observation of drinking water quality is not possible. It needs too long to get the results. For single toxicity tests in suspected cases of contamination especially in the distribution system or at the consumers tap test kits can be useful.


The ISO guideline 15839:2003is a good starting point for describing the performance characteristics of on-line biomonitors. Monitoring technology nr 1: Standard toxicity tests




drinking water

x x


selectivity

x

x

time to result (B)

x


ease-of-use (B) x maintenance requirements (C) x Costs x



x


Overall conclusion The test methods are not useful for continuous online water

monitoring (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.17.2 Monitoring technology nr 2: Daphnia toximeter

Kerren Aqua-Tox-Control Daphnia, bbe Daphnia Toximeter Description: The daphnia toximeters directly monitor the activity of the test organism, the common freshwater water flea (Daphnia magna). Computer software analyzes the movements respectively the location of the daphnia while they are exposed to sample stream water. The values are recorded and analysed continuously and in real time to determine if changes occur in any or all of the observed parameters. Both instruments consist of housing, an industrial PC, monitoring chambers, sample pumps and an automatic food supply. For feeding an algae solution can be added. Under normal conditions e.g. operation without toxic events, the tests run continuously, without maintenance, for about seven days.


The Aqua-Tox-Control works with a control and a test chamber. For the control chamber a supply of guaranteed uncontaminated water is necessary. The reaction of daphnia to changing light conditions is continuously monitored by sensors and compared between both chambers. Significant behavioural deviations trigger an alarm. The bbe Daphnia Toximeter uses a video recognition technology to observe behavioural changes of daphnids in one or two chambers. The behavioural parameters evaluated by the instrument are: average velocity, speed class distribution, average distance of organisms, the ‘curviness’ of swimming patterns as determined by two fractal dimension equations, average altitude and the recognition rate of Daphnia. The Toxicity Index, a parameter calculated from individual behaviours, gives an overall status of the daphnia during testing. If behavioural changes occur in the course of time suddenly or dramatically, the Toxicity Index will rate the change and will give an alarm when alarm conditions are met. The contribution of the individual parameters to the Toxicity Index can be weighted differently thereby adjusting the sensitivity of the instrument. A pre-filtering system, dechlorination and a peltier heating/cooling system ensure constant keeping conditions for the daphnids. The software provides a graphic presentation of the measured results with live, real-time pictures, offline viewing and an intuitive user interface. Both daphnia toximeters are commercially available and applicable for continuous drinking water monitoring and protection. Evaluation: Daphnia Toximeters are sensitive methods to detect hazardous compounds in water. Based on the Extended Dynamic Daphnia Test (which is not available anymore), a new sensitive method to detect hazardous compounds in water was developed. Both small doses (allowable for short-term water ingestion) and graduated higher concentrations induced toxic reactions lead to alarms in the toximeter systems. The systems are sensitive to a wide range of toxic agents. The bbe system was evaluated by using different test pollutants e.g Sarin, Tabun, Soman and Cyclosarin. Concentrations which are below acute human toxicity could be discovered in a very short time. In every case alarms occurred within two hours at concentrations which are considered allowable for drinking water in exceptional conditions. Both systems need some experience in keeping daphnids. The adjustable high sensitivity may lead to false positive alarms in some cases. The systems can be applied also in chlorinated drinking water. The bbe system seems to be technologically advanced and more intensively tested. The systems are generally priced between $30,000 and $50,000 operating costs are rather modest, consisting mostly of replacement costs of organisms and power supply.


Monitoring technology nr 2: Daphnia toximeter




drinking water

x x


selectivity

x

x

time to result (B)

x





x x


Overall conclusion Sensitive method to detect hazardous compounds in water.


4.17.3 Monitoring technology nr 3: Fish toximeter

Kerren Aqua-Tox-Control, BMI BIO-SENSOR®, IAC 1090 – intelligent Aquatic BioMonitoring System®, CIFEC Truitel TruitoSEM, bbe Fish Toximeter, bbe ToxProtect Description: The measuring technology of all fish toximeters is based on the observation and evaluation of behavioural changes of actively swimming fish when exposed to toxic influences. Different types of sensors and evaluation methods are used. The Aqua-Tox-Control monitors a group of fish in a swimming tunnel and registers changes in swimming power and capacity when fish are touching the grid barrier at the end of the tunnel. The Bio-Sensor 7008 features eight fish (on independent sensor channels) monitoring a single water source, noninvasive electronic sensors measure changes in ventilatory behaviour and certain locomotor activities.


The IAC 1090 detects breathing, coughing and movements of fish by noncontact sensors mounted in an aquarium. These movements or ventilatory parameters (ventilation rate, average depth, cough rate, and percentage of motion) along with water quality parameters are processed and analyzed to provide an assessment of water toxicity. The Truitel TruitoSEM also measures changes in the movements of fish by noncontact sensors in an aquarium. The data processing and alarm algorithms are not described in detail. Sensitivity can be technically adjusted or by the selection of different fish species. The bbe Fish Toximeter uses a video camera to directly monitor the activity of fish under the influence of a stream of sample water. Tracking all movements online, the instrument records any changes in behaviour. On the occurrence of a toxic event the toximeter triggers an alarm at an early stage of exposure. Toxicity computations and assessments are based on the measurement of the following behavioural parameters: speed, behaviour (height, turns, circular motion), determination of size, number of active fish. An integrated parameter - the so-called toxic index - is calculated continuously. The bbe ToxProtect is an automated monitoring system to protect especially drinking water supplys from accidental or malicious contaminations due to harmful substances. It is mainly designed to detect relatively high concentration of dangerous substances that occurs suddenly. The ToxProtect monitors the swimming activity of up to 20 fish by measuring the frequency of interruption of an array of light barriers. The result is given in interrupts per minute per fish. Furthermore activity or inactive objects at the surface or bottom of the tank are evaluated additionally. In order to prevent false alarms, an alarm verification system is integrated. In the event of values falling below a given threshold for a certain period of time, the alarm verification process is activated. All systems use computational algorithms and statistical approaches to evaluate behavioural changes and trigger alarms in the case of toxic events. The systems can optionally be combined with physicochemical sensors to assist in analysis and be equipped with automated water samplers. Systems offer the opportunity of remote communications and viewing of activity. The fish toximeters are equipped with life-guarding systems like flow control, temperature regulation, automated feeding and optional dechlorination. These fish toximeters are commercially available and are applicable for a continuous drinking water monitoring. Evaluation: Continuous biological monitoring with the Fish Toximeters enables a rapid detection of toxic substances in water and provides an online early warning system. The Fish Toximeters are well suited for the rapid detection of wilful or negligent damage to water systems. A high toxic response relation between fish and humans exists, so real alarms based on the fish behaviour responses have a high hazard probability for human beings too.


Fish are relatively easy to keep and systems are easy to set-up, fish tank, tubes and connectors are accessible at a low-maintenance level compared to daphnia toximeters. All systems can be applied also in chlorinated drinking water. The systems do not require special technical skills. Adjustments are almost easy to perform and require no expert knowledge. The sensitivity and reliability is dependent on the different technologies, data evaluation techniques and the chosen fish species. The continuous analysis of live video images in the bbe Fish Toximeter enables a rapid and accurate determination of the behaviour and health of the fish. The integrated bbe software recognises significant changes in the behavioural data obtained from the live observation and recording of the fish’s movement. Toxic events are clearly indicated as "alarms". A statistical approach enables alarm recognition even under difficult real-world conditions such as "noisy" or slow drift of the measured behavioural curve(s). The sensitivity of the device is high but can be adjusted by the user based on the specific application. As mentioned a correlation exists between sensitivity and the probability of false positive alarms. Costs for this highly sophisticated system are relatively high. But beside the suitability for the detection of acute toxicity it is also conceived as a long-term monitor of water quality during "strategic" analysis. The BMI Bio-Sensor and the IAC 1090 use similar technologies to detect behavioural impairments in fish. Both are designed to detect rapidly and reliable developing water toxicity threats. That means systems are relatively sensitive but suitable for the detection of acute toxicity. The flexibility in the range of applications seems to be less than in the bbe Fish Toximeter, the price is comparably high. The Aqua-Tox-Control uses a detection method which is based on a loss of swimming capacity of fish. That means an alarm signal can only be generated when a physical impairment of fish already occurred. Sensitivity of the device will thus be less than that of the previously discussed systems, delay times to get an alarm signal will be longer. It may be sufficient sensitive for acute toxicity detection. The costs for the Aqua-Tox-Control are relatively high. A system especially designed for drinking water acute toxicity monitoring is the bbe ToxProtect. Continuous water monitoring with the ToxProtect enables rapid detection of a wide range of toxins. Usage of behavioural changes as parameters will make the device sufficiently sensitive to detect all seriously dangerous substances and situations. ToxProtect is a very easy operation system at a affordable costs. The required maintenance time is approximately 10 min/day. The special integrated alarm verification algorithm leads to a high reliability of the system and avoidance of false alarms. The use of fish gives a close practical comparison in the expected scenario of contamination with substances that are harmful to humans.


Monitoring technology nr 3: Fish toximeter




drinking water

x x


selectivity

x

x

time to result (B)

x





x x


Overall conclusion Sensitive method to detect hazardous compounds in water,

most comparable to human toxicity. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Monitoring technology nr 3a: ToxProtect




drinking water

x x


selectivity

x

x

time to result (B)

x





x x


Overall conclusion Reliable and robust test system to detect high toxic

concentrations, most comparable to human toxicity. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.17.4 Monitoring technology nr 4: Combined Fish and Daphnia toximeter

Kerren Aqua-Tox-Control, bbe Fish and Daphnia Toximeter Description: Both Kerren and bbe have the possibility to combine their instruments for fish and daphnia. Kerren offers a combination of two single devices and assures a significant increase in sensitivity and reliability by evaluating the output parameters of both systems integratively. Bbe combines the advanced Fish and Daphnia Toximeter in one device based on the two chamber Daphnia toximeter. In one chamber young living fish and in the other daphnids are observed under the influence of a stream of sample water. The toximeter’s continuous visual analysis of fish and daphnia movement enables rapid assessment of fish and daphnia behaviour and health (for technical description see the single Daphnia Toximeter). For the


integrated evaluation of any change in the behaviour of the fish or daphnia the continuously calculated "toxicity index" as a dimensionless parameter proved to be especially usefiul. The bbe software integrates all data components of the bbe Toximeter. It provides a graphic presentation of the measured results with live, real-time pictures, offline viewing and an intuitive user interface. Both combined Fish and Daphnia toximeters are commercially available and useful for continuous drinking water protection. Evaluation: Continuous water monitoring with combined Fish and Daphnia toximeters enables rapid detection of toxic substances in water and provides an online real time warning system. The combined Fish and Daphnia toximeters are well suited to the detection of wilful or negligent damage to water systems. Both systems can be applied in chlorinated drinking water too. Both Fish and Daphnia Toximeters can also be used for long-term monitoring for the "strategic" evaluation of water quality. The integrated use of two simultaneous online test systems does not only improve the quality of analysis statistically, but enhances also the sensitivity for harmful compounds by combining the different reactiveness of biological species to different groups of chemical substances. However the handling of different species and especially of fish larvae makes the maintenance of such a system more costly and time consuming as single systems. The Kerren solution of two combined single systems will probably nearly double the instrumentation costs.


Monitoring technology nr 4: Combined bbe Fish and Daphnia toximeter




drinking water

x x


selectivity

x

x

time to result (B)

x





x x


Overall conclusion Sensitive method to detect hazardous compounds in water.


4.17.5 Monitoring technology nr 5: Algae toximeter

bbe Algae Toximeter Description: Beside the bbe Algae taximeter, a Korean solution for an Algae test shall be existent, but no reliable information about this device was recently available. The bbe Algae Toximeter uses algae from a standardized culture which is established within the instrument. The cultivation of the algae takes place in a fermenter which is regulated "turbidostatically" by a second fluorescence measurement. This regulation ensures that the algae concentration and their activity are maintained. As species Chlorella vulgaris, Scenedesmus spec. and others can be used. The instrument compares the effects of toxins on one portion of algae to another portion kept in uncontaminated clean water (e.g. carbon filtered tap water). The principle of operation is based on the determination of the fluorescence spectrum and kinetics of the algae. If the algal cells are damaged by toxins in the sample water, the light energy will not be used by the cell and even without an additional background light there will be a lower fluorescence response to the light impulse. In the case of significant deviations between the sample water and the reference water, an alarm is generated by the instrument to alert the operator. In addition to


monitoring toxic substances, the device can determine different amounts of chlorophyll within different algae classes. The bbe Algae toximeter is commercially available and partly useful for continuous drinking water protection. Evaluation: The bbe Algae Toximeter continually determines toxic substances in water. The bbe Toximeter provides a highly sensitive biological system for early detection of potentially dangerous unknown substances, in a wide variety of circumstances. These instruments are ideal for detecting water quality contamination in real-time. The Toximeter provides means to detect (and alert users at) potentially dangerous circumstances. The test mechanism of this monitor is very similar to a Biological Oxygen Demand (BOD) test, as the effect on fluorescence is a measure for electron transfer. The time required for the conventional 5-day bench test cannot compete with the 10-minute response time of this instrument. Maintenance costs and time for this system are low. Instrumentation costs in the same range as for fish and daphnia toximeters. Algae toximeters are more suitable for detecting herbicides and a lot of chronic toxic substances, in acute toxicity measurements values are less comparable to human toxicity. Monitoring technology nr 5: bbe Algae toximeter




drinking water

x x


selectivity

x

x

time to result (B)

x





x x



less comparable to human toxicity. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.17.6 Monitoring technology nr 6:Luminiscent bacteria

Microtox®, Deltatox®, BioTox™, AbraTox Kit, LUMIStox; MicroLAN TOxcontrol Biomonitor Description: Most of these tests are 15-60 minute in vitro exposure metabolic inhibition tests which use luminescent bacteria to assess the acute toxicity of water, soil or sediment samples. The tests use strains of naturally occurring luminescent bacteria, Vibrio fischeri or Photobacterium phosphoreum. Both are nonpathogenic, luminescent bacteria that are sensitive to a wide range of toxicants. When properly grown, luminescent bacteria produce light as a by-product of their cellular respiration. Cell respiration is fundamental to cellular metabolism and all associated life processes. Bacterial bioluminescence is related directly to cell respiration, and any inhibition of cellular activity (toxicity) results in a decreased rate of respiration and a corresponding decrease in the rate of luminescence. The described luminescent bacteria tests are commercially available but do only allow discontinuous measurements. The TOxcontrol Biomonitor is the single device with this technology working continuously as a flow-through system. Evaluation: These toxicity test systems provide rapid screening and confirmatory results that are cost-effective and easy to perform. Costs range between 3,000 and 10,000 $ for the discontinuous test sets. TOxcontrol is with 30,000 € the most expensive device but even the only one suitable for an online monitoring of acute toxicity. Manual quality differs between tests, initial light measurements served as a good check of bacterial health and instrument operation, sample handling is easy, and sample throughput can reach 15-50 samples per hour. Some of the tests are applicable in laboratory as well as field environments. Operators don’t need scientific backgrounds, based on the observations of the verification test coordinator, operators with little technical training would probably be able to follow the instructions to analyze samples successfully. Detection of hazards is limited to substances inhibiting or disturbing the bacterial cell respiration, so not all test substances were detected and not all results allow conclusions to human imperilment. For single toxicity tests in suspected cases of contamination especially in the distribution system or at the consumers tap test kits can be useful.


Monitoring technology nr 6: Luminiscent bacteria




drinking water

x x


selectivity

x

x

time to result (B)

x





x x


Overall conclusion Medium sensitive but selective method to detect hazardous

compounds in water, not continuous. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.17.7 Monitoring technology nr 7: Mussel monitor

Delta Consult MOSSELMONITOR® (Dreissena polymorpha, Unio pictorum) Description: The behaviour of bivalves in response to toxicity in water is seemingly simple, they will close their shells. That means the sensor in the MOSSELMONITOR® is the mussel itself. The measurement of gape, or opening of the shells, and the frequency of this opening is generally accepted as a measure of stress to the organisms. In the normal movement pattern of the mussel shells, the shell halves will remain open approximately 70-80% of the time, for the intake of food and oxygen. The shells only occasionally close, and re-open after only a short period of time. Gape behaviour is measured using proximity sensors which detect a stainless steel proxy attached to the shell of each clam. By comparing the organism’s baseline, or normal, gape behaviours to that of a test water, information regarding toxicity of a water can be determined.


The various movements, which the mussel can make as a result of differing types and levels of contamination, are as follows: • Keeping the shell halves closed for a certain (longer) period. • An increased activity level, i.e. the mussel opens and closes more

frequently than normal. • A reduction in the average value of opening over a certain period of time. • No further movement, the shell remains open far more than the normal,

maximum open position. This occurs if the mussel is no longer alive ("gaping").

The monitor uses normally 8 bivalves per system and evaluates its behaviour, systems with 16 bivalves are optionally available. An Automated Food Device (AFD) was developed to enable the use in places where little or no food is present for mussels as e.g. in drinking water protection. By entering information about the movements of the shell into a microprocessor, and by carrying out certain calculations on this data, it is possible to ascertain the behaviour of the mussel. Currently, a memory is being used to store the data for evaluation of trends in shell movement behaviour The monitor is a stand alone unit; it contains all electronics required for measurement, data evaluation and communication. For data storage any PC can be used. Evaluation: The flow-through version of the Mosselmonitor is a broad spectrum sensor that can easily be used as an early warning system for on line monitoring of drinking water. It can be applied also in chlorinated drinking water. The Mosselmonitor operates unattended for weeks, whereby zebra mussels are still alive and responding to chemicals for more than 3 months. So maintenance costs and time are low. Instrumentation costs are moderate. Detection limits are sometimes not directly comparable to human toxicity values.


Monitoring technology nr 7: Mussel Monitor




drinking water

x x


selectivity

x

x

time to result (B)

x





x x



sometimes not directly comparable to human toxicity. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.18 Algae toxins Prepared by: TZW

Actual reports suggest that toxic cyanobacteria blooms are an emerging issue world wide because of increased source water nutrient pollution causing eutrophication. A third of the freshwater cyanobacteria genera are capable of producing toxins. Actual investigation shows that the strategies of early warning and protection drinking water treatment trains have been updated continuously because of the increasing number of fresh water toxins identified. The most important groups of fresh water toxins are the cyclic peptides like microcystins and alkaloid toxins like cylindrospermopsin.

On the other hand, other less common and studied algal toxins from the large group of non fresh water species are disseminate world wide. The possibility exists for these toxins to be detected in freshwaters in future due to increased salinity of freshwater supplies. This increased salinity is partly the result of salt intrusion and salt contamination caused by the mining and commercial industry. Additional the use of sea and brackish waters for drinking water production is increasing word wide.

Cyanotoxins can occur cell bound and dissolved in water. Cell bound cyanotoxins can released into the water by natural mechanisms (metabolization) and induced mechanisms (physical, chemical as well biological impact during water treatment). Required technical specifications: The most common toxins, the microcystins occur in water in a range up to mg/L. Most of the toxins are cell bound. Ca. 10% of the total toxin content is found in dissolved form. Therefore the relevant concentration level of toxins in water can be expected between several µg/L to mg/L in some cases. For analysis of drinking water we propose a sensitivity of 0.1 µg/L for each single toxin (cell bound and dissolved). The analytical technique applied should be able to separate and to recognize single toxin structures. In some cases, e.g. if the pollution level of the raw water is very high, the sensitivity of the analysis of single toxins with modern techniques available is about 1 µg/L up to now. For analysis of cyanotoxins certified reference standards are used. The availability of these materials is world wide a problem. Monitoring technologies: 1. LC-DAD 2. LC-MS/MS 3. ELISA-Tests 4. PPIA (protein phosphatase inhibition assay) Monitoring sequence: The monitoring sequence must be flexible because the algal bloom as well as the cell stability and therefore the toxin release can change rapidly.


4.18.1 Monitoring technology nr 1-4: LC-DAD, LC-MS/MS, ELISA, PPIA

Description: Dissolved toxins will be separated by solid phase extraction from water phase. Cell bound toxins wil be separated by physical (freezing) and chemical (solvents) destruction of algal cells. Following to this, analysis with LC-DAD, LC-MS/MS, ELISA, PPIA can be conducted. Standard operational procedures (SOP) for trace analysis of microcystins, anatoxin-a and cylindrospermopsin were developed within the frame of the joint European Research Project TOxIC [TOxIC, EVK1-CT-2002-00107], “Barriers against Cyanotoxins in Drinking Water”. For detailed information see: TOxIC: Cyanobacterial Monitoring and Cyanotoxin Analysis, Abo Akademi Turku, ISBN 951-765-259-3 (2005). In the frame of the TECHNEAU-Project the SOP´s for analysis of the so called new toxins will be designed. Monitoring technology nr 1: LC-DAD Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result

several days


ease-of-use (B) x qualified staff needed maintenance requirements (C) x

Costs high instrumentation (C) x operational costs (C)


x

x


Overall conclusion Sensitive and selective method, but expensive; faster methods are needed in case of algal blooms.



Monitoring technology nr 2: LC-MS/MS Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

several days


ease-of-use (B) x qualified staff needed maintenance requirements (C) x

Costs very high instrumentation (C) x operational costs (C)


x

x


Overall conclusion Sensitive and selective method, but expensive, faster methods are needed in case of algal blooms.



Monitoring technology nr 3: ELISA test Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

x

time to result

immediately if there is a critical situation 1-2 days in general


ease-of-use (B) x maintenance requirements (C)

x

Costs average instrumentation (C) x operational costs (C)


x x

average


Overall conclusion Comparable not expensive and fast method, but low selectivity, because of matrix effects and cross activities the sensitivity of the test kits which is published by the assay producing companies seems too optimistic, ELISA tests should used as pre-examination,



Monitoring technology nr 4: algal toxins PPIA Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

x

time to result

immediately if there is a critical situation 1-2 days in general


ease-of-use (B) x maintenance requirements (C)

x

Costs average



x x

average


Overall conclusion Fast and cheap method with low selectivity and sensitivity, PPIA test can be used as pre-screening method.


Evaluation: ELISA tests give a first view concerning the toxin content in the water, if the level of toxins is higher than 1 µg/L. ELISA kits cannot identify single toxins in general. On the other hand, companies offer test kits on the market with sensitive detection limits of 0.1 µg/L. Because of the cross activities and matrix effects in the water this value seems to be too optimistic. This can be true in some cases, but it is not a general rule. PPIA tests seem to be more sensitive. Nevertheless, also this procedure cannot identify single toxins. Both methods are influenced by cross-activities. For drinking water monitoring (as well as raw water monitoring) the identification and quantification of cyanobacterial toxins should be done by MS-detection. This requires expensive technical equipment and qualified staff. If the water contains cyanobacteria and if pre-screening methods like ELISA and PPIA indicate a positive signal LC-MS/MS measurements should be performed.


4.19 Pesticides, pharmaceuticals, industrial chemicals and other organic micropollutants

Prepared by: Kiwa Water Research Required technical specifications Concentration ranges that should be covered are the following: source water (ground or surface water) 0-10 µg/L drinking water 0-1 µg/L, concentration should be less than 0.1 µg/L (quality requirement for pesticides in drinking water) Monitoring technologies: 1. Gas Chromatography-Mass Spectrometry (GC-MS) 2. Liquid Chromatography-Ultraviolet Diode-Array Detection (LC-UV DAD) 3. Liquid Chromatography-Mass Spectrometry (LC-MS)

4.19.1 Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry

Status of the technique GC-MS is a widely-accepted method and commercially available from Agilent, Thermo Fisher, Waters, LECO, and so on. A large number of CEN methods for the determination of organic micro-pollutants by GC/MS are available. Evaluation: - Highly accurate and reliable technique, but also relatively expensive. - Method fulfills the need for the parameter as long as the compounds are volatile, thermo-stable and not too polar. - Method can be used for regulatory measurement in a routine setting. Operator needs certain skills to acquire and process data.


Monitoring technology nr 1: Gas Chromatography-Mass Spectrometry Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x 20-60 min





x x


Overall conclusion Reliable, sensitive method for a wide range of compounds,

but expensive (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.19.2 Monitoring technology nr 2: High-Performance Liquid Chromatography-UltraViolet Diode Array Detection

Status of the technique: HPLC-UV DAD is a widely-accepted method and commercially available from Agilent, Beckman Coulter, PerkinElmer, Shimadzu Scientific Instruments, Thermo Fisher Scientific, Varian, Waters Corporation and others. A large number of CEN methods for the determination of organic micro-pollutants by HPLC are available. Evaluation: - Highly accurate, sensitive and reliable universal technique. The technique is less expensive than GC-MS and HPLC-MS. - Method fulfills the need for the parameter as long as the compound absorbs UV light. - Method can be used for regulatory measurement in a routine setting.


Monitoring technology nr 2: High-Performance Liquid Chromatography-Ultraviolet Diode Array Detection Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x 30-60 min





x x


Overall conclusion Reliable, sensitive and universal method


4.19.3 Monitoring technology nr 3: High-Performance Liquid Chromatography-Mass Spectrometry

Status of the technique: HPLC-MS is a commercially available from Agilent, PerkinElmer, Shimadzu Scientific Instruments, Thermo Fisher Scientific, Bruker Daltonics, Waters Corporation and others. Evaluation: - Highly accurate, sensitive and reliable technique, but expensive. - Method fulfills the need for the parameter as long as the compound can be ionized i.e. brought into the gas/damp phase from a liquid phase and can be ionized either in positive-ion mode by the addition of an proton and in negative-ion mode by reduction of a proton. - Method can be used for regulatory measurement in a routine setting. Operator needs training and certain skills to acquire and process data.


Monitoring technology nr 3: High-Performance Liquid Chromatography-Mass Spectrometry Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x

30-60 min





x x


Overall conclusion Reliable, sensitive and selective method for wide range of

compounds, but expensive. Operator needs certain skills/training



4.20 pH Prepared by: AlphaMos pH is a measure of the acidity or alkalinity of a solution. Aqueous solutions at 25 °C with a pH less than 7 are considered acidic, while those with a pH greater than 7 are considered basic (alkaline). pH values in water are commonly in the range 0-14, though more extreme values, even negative values, are possible. Because pH is dependent on ionic activity, a property which cannot be measured easily or fully predicted theoretically, it is difficult to determine an accurate value for the pH of a solution. The pH reading of a solution is usually obtained by comparing unknown solutions to those of known pH, and there are several ways to do so. Required technical specifications The normal range for pH in surface water systems is 6.5 to 8.5 and for groundwater systems 6 to 8.5. The pH of pure water (H20) is 7 at 25°C, but when exposed to the carbon dioxide in the atmosphere this equilibrium results in a pH of approximately 5.2. Because of the association of pH with atmospheric gases and temperature, it is strongly recommended that the water be tested as soon as possible. The pH of the water is not a measure of the strength of the acidic or basic solution and alone does not provide a full picture of the characteristics or limitations of a water sample. Monitoring technologies:

1. pH indicator 2. pH meter

4.21Monitoring technology nr 1: pH indicator Description: A pH indicator is added into the solution under study. The indicator color varies depending on the pH of the solution. Using indicators, qualitative determinations can be made with universal indicators that have broad color variability over a wide pH range and quantitative determinations can be made using indicators that have strong color variability over a small pH range. Evaluation: Because of the subjective determination of color, pH indicators are susceptible to imprecise readings. For applications requiring precise measurement of pH, a pH meter is frequently used.


Monitoring technology nr 1: pH indicator




drinking water

x x


selectivity

x

x

time to result

x x immediat



instrumentation (C) operational costs (C)


x


Overall conclusion Quick, cheap, but imprecise



4.21.1 Monitoring technology nr 2: pH meter

Description: A pH meter is an electronic instrument used to measure the pH (acidity or basicity) of a liquid. A typical pH meter consists of a special measuring probe connected to an electronic meter that measures and displays the pH reading. The pH probe measures pH as the activity of hydrogen ions surrounding a thin-walled glass bulb at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is measured and displayed as pH units by the meter.

The standard hydrogen electrode (abbreviated SHE), also called normal hydrogen electrode (NHE), is a redox electrode which forms the basis of the thermodynamic scale of oxidation-reduction potentials. Hydrogen electrode is based on the redox half cell: 2H+(aq) + 2e- → H2(g) This redox reaction occurs at platinized platinum electrode.

Quinhydrone electrode is one of several oxidation-reduction electrode's in which the ratio of the two forms (quinone-quinhydrone), determined by the hydrogen ion concentration, sets up a potential that can be measured and converted to a pH value (fails above pH 8).

An ISFET is an ion-sensitive field effect transistor used to measure ion concentrations in solution; when the ion concentration (such as pH) changes, the current through the transistor will change accordingly. Here, the solution is used as the gate electrode. A voltage between substrate and oxide surfaces arises due to an ions sheath. An ISFET's threshold voltage depends on the pH of the substance in contact with its ion-sensitive barrier.

The two basic adjustments performed at calibration set the gain and offset. Calibration with at least two, but preferably three, buffer solution standards is usually performed every time a pH meter is used. One of the buffers has a pH of 7.01 (almost neutral pH) and the second buffer solution is selected to match the pH range in which the measurements are to be taken: usually pH 10.01 for basic solutions and pH 4.01 for acidic solutions. The gain and offset settings of the meter are adjusted repeatedly as the probe is alternately placed in the two calibration standards until accurate readings are obtained in both solutions.

pH meters range from simple and inexpensive pen-like devices to complex and expensive laboratory instruments with computer interfaces and several inputs. Pocket pH meter are readily available today for a few tens of Euros that automatically compensate for temperature. More information can be found at

http://www.radiometer-analytical.com/

http://www.metrohm.com/

http://us.mt.com/home

http://www.hannainst.com/


Evaluation: For applications requiring precise measurement of pH, a pH meter is frequently used. Monitoring technology nr 2: pH meter




drinking water

x x


selectivity

x x

time to result

immediate



instrumentation (C) x All prices available operational costs (C)


x x


Overall conclusion Common method easy & reliable



4.22 Chloride/nitrate/sulphate Prepared by: TZW Required technical specifications: - concentration range: 1 to 500 mg/L (chloride), 0.5 to 50 mg/L (nitrate), 1 to 300 mg/L (sulphate) - sensitivity required: 1 mg/L (chloride), 0.5 mg/L (nitrate), 1 mg/L (sulphate) Monitoring technologies: 1. Ion chromatography with conductivity detection (IC/CD) For monitoring technologies that are only suited for analysis of nitrate see 4.9.


Description: An aliquot of the water sample is directly injected into the ion chromatographic system. Evaluation: Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x

x

Recommendation for use in SSS (D) x Overall conclusion Standard multi-method for analysis of common anions. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.23 Conductivity Prepared by: AlphaMOS Conductivity of a substance is defined as 'the ability or power to conduct or transmit heat, electricity, or sound'. Its units are Siemens per meter [S/m] in SI and mhos per centimeter [mho/cm] in U.S. customary units. Its symbol is κ or σ. An electrical current results from the motion of electrically charged particles in response to forces that act on them from an electrically applied electric field. In water or fluids a net motion of charged ions can occur. This phenomenon produces an electric current and is called ionic conduction. Electrical conductivity is defined as the ratio between the current density (J) and the electric field intensity (ε) and it is the opposite of the resistivity (ρ, [Ω*m]): σ = J/ε = 1/ρ Conductivity is a measurement used to quickly determine the variation or changes in natural water or wastewaters. Elevated dissolved solids can cause "mineral tastes" in drinking water. Corrosion or encrustation of metallic surfaces by waters high in dissolved solids causes problems with industrial equipment and boilers as well as domestic plumbing, hot water heaters, toilet flushing mechanisms, faucets, and washing machines and dishwashers.

Required technical specifications

Pure water is not a good conductor of electricity. Because the electrical current is transported by the ions in solution, the conductivity increases as the concentration of ions increases. Thus conductivity increases as water dissolved ionic species. Ultra pure water 0.055 µS/cm Deionised water 1 µS/cm Rainwater 50 µS/cm Drinking water 500 µS/cm Industrial wastewater 5 mS/cm Mineral water 4 – 7 mS/cm Sea water 50 mS/cm An EC meter is normally used to measure conductivity in a solution.

Monitoring technologies: 1. Conductimeter

4.23.1 Monitoring technology nr 1: Conductimeter

Description: Conductivity is measured by applying an alternating electrical current at an optimal frequency to two electrodes immersed in a solution and measuring the resulting voltage. During this process, the cations migrate to the negative electrode, the anions to the positive electrode and the solution acts as an electrical conductor.


The conductivity depends on the value of the pH, on the temperature of measurement and on the amount of CO2 which has been dissolved in the water to form ions. The conductivity due to these factors is “intrinsic conductivity”. The conductivity is also affected by the concentration of ions already present in the water such as chloride, sodium and ammonium. This contribution to the conductivity is “extraneous conductivity”. Consumables include conductivity standards. This technology is commercially available. EC meters range from simple and inexpensive pen-like devices to complex and expensive laboratory instruments with computer interfaces and several inputs. interfaces and several inputs. Information about technical products can be found at http://www.radiometer-analytical.com/ http://www.metrohm.com/ http://us.mt.com/home http://www.hannainst.com/. Evaluation: Easy to use & maintain, economical. Different systems (at different prices) can be used depending on the requested precision and conductivity range. It is recommended to check the cell constant regularly due to contamination risk. Monitoring technology nr 1: Conductimeter




drinking water

x x


selectivity

x x

time to result

immediate





x x


Overall conclusion Reliable method, easy & affordable



4.24 Calcium & magnesium See par 4.1: Metals

4.25 Sulphate See par 4.20: chloride, nitrate, sulphate

4.26 Aluminium See par 4.1: Metals


4.27 Ammonium Prepared by: S::can Required technical specifications Concentration ranges that can be encountered are the following: 0 - 1 mg/L (drinking water, EU limit is 0.5 mg/L) 0 - 50 mg/L (raw water) Required resolution of the measurement: 1 mg/L 0.05 mg/L (drinking water) 1 mg/L (raw water) Monitoring technologies 1. Ion selective electrode 2. Ion chromatography 3. Photometric test kits 4. Automatic Analyser

4.27.1 Monitoring technology nr 1: Ion Selective Electrode

Description: - the ISE is an electrode that is equipped with a membrane that is permeable for ammonium ions and not for other ions. The presence of ammonium ions will result in a change in the potential measured by the electrode because of diffusion of ammonium ions through the membrane into the electrode. The potential is indicative for the concentration. - ISEs for ammonium have been available for many years. Reliability has been, however, poor due to instability of the measurement and cross sensitivities. Over the last 2 years a number of manufacturers have introduced improved instruments with additional electrodes next to the ISE for ammonium to compensate for cross sensitivities (pH, temperature, potassium) Equipment Equipment needed: electrode, electrolyte (to be changed on a periodical basis), selective membranes (need to be changed on a periodical basis) References Vendors selling this latest generation of ammonium probes: s::can Messtechnik, Nadler Chemische Analysen Technik, Dr. Lange, Endress and Hauser and others. Evaluation: - When the techniques do what the marketing sheets tell, then this is very suitable for online monitoring of source water and drinking water. Only own experience is with s::can and Nadler instruments and they work well (stable without calibration for up to two months, no membrane replacement needed until 6 months of operation in Waste Water (more difficult and demanding matrix for ISE).


- This is the only online method. It's much cheaper than ion chromatography. Photometric test equipment is cheaper, but not online and costs around 5 Euros per test. - This method fulfils the needs. Monitoring technology nr 1: Ion Selective Electrodes Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x

x


selectivity

x x

time to result

x measurement every 5 seconds




instrumentation (C) x price indication: s::can instrument 3500 €


x x


Overall conclusion Suitable method for online monitoring, however maintenance intensive


4.27.2 Monitoring technology nr 2: Ion Chromatography

In ion chromatography, a pre-treated sample is processed in the laboratory. The sample in injected in chromatograph, and the components present are separated on the column. Based on retention time the desired component is identified. Equipment and consumables - ion chromatograph, including chromatographic column, HPLC pump. - consumables: solvents, filters, columns. Available from Waters, Metrohm, Thermo Electron, Dionex and others. Evaluation:


- Highly accurate technique. Only suitable for off-line, batch wise laboratory analysis. Useful for regulatory measurements. - Most accurate technique, but also by far the most expensive (investment in equipment). - This method fulfils the needs in terms of measuring range and accuracy. Monitoring technology nr 2: Ion Chromatrography Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x

x

time to result





instrumentation (C) x price indication: > 15.000 Euro € operational costs (C)


x x


Overall conclusion Suitable method for reference/regulatory measurements in

the laboratory (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.27.3 Monitoring technology nr 3: Photometric test kit

In the photometric test kit, a grab sample is taken, to this sample is added a reagent that forms a coloured substance by reaction with the targeted species. This coloured substance is then measured using a simple photometer. Equipment and consumables - equipment: photometer, test-tube with reagent (pr-mixed, easy to handle), filter to remove solids / turbidity from the sample before starting test - consumables: test tubes (approx 2 - 5 Euros per test) Reference - Photometry is established technique (see Standard Methods for the examination of water and Waste Water APHE, AWWA, WEF publication). Available from many vendors, such as WTW, Dr. Lange and others.


Evaluation: - Relatively accurate tests that can be used in the lab and in the field. Relatively easy to use (any lab technician can handle it, but the operator will need at least some lab training). - Only off-line analysis possible. - This method fulfils the needs in terms of range and accuracy. Monitoring technology nr 3: Photometric Test Kit Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x

x


selectivity

x x

time to result

x reaction before measuring takes 15 minutes


ease-of-use (B) x sampling and filtering of liquids needed manually


instrumentation (C) x price indication: WTW instrument < 2000 €


x

x




4.27.4 Monitoring technology nr 4: Automatic Analyser

Descritpion: In the photometric test kit, a grab sample is taken, to this sample is added a reagent that forms a coloured substance by reaction with the targeted species. This coloured substance is then measured using a simple photometer. Sampling, sample preparation and handling are automated. Alternatively, ISE is used instead of photometry in such automated analysers. Equipment and consumables


- equipment: cabinet analyser, comprising of sampling line, filtering unit, dosing pumps, photometer, - consumables: reagents for photometric reaction, filters, tubes in analyser References Photometry is an established technique (see Standard Metods for the examination of water and Waste Water APHE, AWWA, WEF publication). The automatic analysers have been in use for more than 10 years. They remain however relatively expensive to buy and highly expensive / time consuming to operate as their complexity makes them prone to failure. Exception: Danfoss Evita which uses membrane technology for isolating the ammonia from the water and small volumes to reduce reagent use and maintenance. Range and resolution only sufficient for waste water use. - Available from Applikon, WTW, Nico2000, Kelma and others. Evaluation: - Performance is OK, but costs of equipment and maintenance are relatively high. - Online analysis is possible, maintenance intensive. - This method fulfils the needs in terms of range and accuracy. Monitoring technology nr 4: Automatic Analyser Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x

x

time to result

x cycle time per measurement at least 15 minutes


ease-of-use (B) x maintenance troublesome maintenance requirements (C) x Costs

instrumentation (C) x price indication: > 10000 € operational costs (C)


x

x


Overall conclusion Expensive equipment, especially because it requires a lot of

maintenance. Not user friendly. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.28 Iron See par 4.1: Metals

4.29 Manganese See par 4.1: Metals


4.30 Taste & Odor Prepared by: AlphaMos Water is called the universal solvent. It dissolves a little of anything that comes in contact with it. Wherever water goes, it gathers minerals, trace elements, contaminants, etc. Taste & odor are two of the organoleptic properties of drinking water: properties that can be identified by sensory perceptions. Main problems with off-taste or off-odor are:

- Chlorinous, chemical or medical taste / odor problems Two common causes are either the addition of chlorine to the water by the supplier or the interaction of that chlorine with organic material in the plumbing system. Generally this occurs when the water is treated at the water treatment plant to disinfect it. The addition of chlorine is used to kill off bacteria and other harmful microorganisms.

- Sulfurous, sewage like, musty, moldy, earthy, grassy or fishy taste / odor problems

The main cause is due to bacteria or other organisms (algae, fungi, ….). Organic matter can accumulate and bacteria can grow on these organic deposits. These bacteria can produce a gas that smells like rotten eggs or sewage. This is usually a result of decaying organic deposits underground. As water flows through these areas, hydrogen sulfide gas is picked up, and when this water reaches the surface or comes out of the faucet, the gas is released into the air. Hydrogen sulfide gas produces the rotten egg odor, can be corrosive to plumbing at high concentrations. The odor of water with as little as 0.5 ppm of hydrogen sulfide concentration is detectable by most people. Concentrations less than 1 ppm give the water a "musty" or "swampy" odor. A 1-2 ppm hydrogen sulfide concentration gives water a "rotten egg" odor and makes the water very corrosive to plumbing. Generally, hydrogen sulfide levels are less than 10 ppm, but have been reported as high as 50 to 75 ppm. The absence of sulfur and chlorine add to the taste of water. The musty smell is normally a result of organic matter or even some pesticides in the water supply. Even very low amounts can introduce unpleasant odors into the water.

- Petroleum, gasoline, turpentine, fuel or solvent like odor problems This smell can be a result of MTBE (Methyl Tert butyl Ether ) contamination in the water. The odor threshold is fairly low, below any toxic thresholds. So even though one can smell it, the MTBE is more than likely not at a level to cause harmful effects.

- Salty taste problem A salty taste in water is usually due to the presence of naturally occurring sodium, magnesium or potassium. For water to taste correctly on the palate, certain minerals must be present. Good tasting water is a subtle combination of flavors with an ideal combination and concentration of minerals. Potassium, magnesium, calcium and even small amounts of sodium give water its fullness. If not, water, such as distilled water, tastes flat and dull.


- Alkali taste problem An alkali taste in water is usually due to high level of total dissolved solids.

- Metallic taste problem A metallic taste in water is due to low pH, corrosive water, high content of iron (0.004 mg/L), copper (2-5 mg/L), zinc (4-9 mg/L), and manganese. Too high a mineral concentration and water takes a tinny metallic taste.

Monitoring technologies: 1. Sensory panel 2. GC MS (for individual compounds) 3. Electronic tongue & nose

4.30.1 Monitoring technology nr 1: Sensory panel

Description: The European Standard EN 1622:2006 specifies a qualitative method for determining any abnormal odour and/or flavour. The odour and flavour of a water sample may also be assessed qualitatively by only one selected assessor or a test panel to detect any abnormal odour and/or flavour. In European regulations relative to water intended for human consumption, the only requirement for odour and flavour is “acceptable for the consumer and no abnormal change“. In these cases, no quantitative odour (TON) and/or flavour (TFN) assessment is required. The qualitative simplified procedure can be used to check the water on a routine basis and also in cases of consumer complaints. Samples shall be taken directly from the consumer tap and shall be assessed immediately without any dilution. The selected assessor shall judge whether the test environment is appropriate (absence of odours likely to mask the sample odours). If not, the selected assessor should carry out the test in another room. The selected assessor shall shake thoroughly each flask, remove the stopper, smell, and then transfer a suitable volume of water in his/her glass, hold in the mouth for several seconds before discharging it without swallowing. The selected assessor shall record if there is abnormal odour and/or flavour. If it is not the case, he/she will note “no abnormal odour and/or flavour”. If an abnormal odour and/or flavour is detected, take a fresh sample for further assessment. Evaluation: Reference method


Monitoring technology nr 1: Sensory panel




x x


selectivity

x x

time to result

x





x x


Overall conclusion reliable method (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.30.2 Monitoring technology nr 2: GC- MS

Description: Gas chromatography-mass spectrometry (GC/MS) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. As such it can also be used to analyse individual compounds that cause taste and/or odor problems. Evaluation: Time consuming method, high skilled user needed


Monitoring technology nr 2: GC-MS




x x


selectivity

x x

time to result

x





x x


Overall conclusion Time consuming method


4.30.3 Monitoring technology nr 3: Electronic Nose & Tongue

Description: Over the last decade, “electronic sensing” or “e-sensing” technologies have undergone important developments from a technical and commercial point of view. The expression “electronic sensing” refers to the capability of reproducing human senses using sensor arrays and pattern recognition systems. Since 1992, research has been conducted to develop technologies, commonly referred to as electronic noses, that could detect and recognize odors and flavors. The stages of the recognition process are similar to human olfaction and are performant for identification, comparison, quantification and other applications. However, hedonic evaluation is a specificity of the human nose given that it is related to subjective opinions. These devices have undergone much development and are now used to fulfil industrial needs. Electronic Nose EN & Tongue ET include three major parts: a sample delivery system, a detection system, a computing system. The sample delivery system of ET enables the liquid sample to be in contact with the detection system. The sample delivery system of EN enables the generation of the headspace (volatile compounds) of a sample, which is the


fraction analysed. The system then injects this headspace into the detection system. The detection system, which consists of a sensor set, is the “reactive” part of the instrument. When in contact with samples, the sensors react, which means they experience a change of physical properties. The more commonly used sensors include metal oxide semiconductors (MOS), conducting polymers (CP), quartz crystal microbalance, surface acoustic wave (SAW), and field effect transistors (FET). In recent years, other types of electronic noses have been developed that utilize mass spectrometry or ultra fast gas chromatography as a detection system. A specific response is recorded by the electronic interface transforming the signal into a digital value. Recorded data are then computed based on statistical models. The computing system works to combine the responses of all of the sensors, which represents the input for the data treatment. This part of the instrument performs global fingerprint analysis and provides results and representations that can be easily interpreted. As a first step, an electronic nose or tongue need to be trained with qualified samples so as to build a database of reference. Then the instrument can recognize new samples by comparing volatile compounds fingerprint to those contained in its database. Thus they can perform qualitative or quantitative analysis. Information about commercial instruments can be found at: www.alpha-mos.com http://www.airsense.com/ www.gsg-analytical.com/ Evaluation: Simple technique allowing to obtain a correlation with a given panel Needs to be validated on the application & trained based on either concentration or panel characterizations.


Monitoring technology nr 3: Electronic Nose & Tongue




drinking water

x x


selectivity

x x

time to result

x





x x


Overall conclusion Simple technique allowing to obtain a correlation with a given panel



4.31 Colour Prepared by: S::can Required technical specifications: Colour in water is caused by contaminations present. Natural water is normally coloured because of the presence of clay particles, iron particles and/or humic and fulvic acids. This normally results in a yellow to brown colour of the water. Alternatively, a green colour is sometimes observed which is caused by algae. True colour is measured after filtration of the water through a 0.45 µm filter. Colour is expressed either in absorbance units (m-1) or with reference to a standard (Hazen, Pt-Co or APHA units for example). In general, expressed in Hazen Units, natural waters are expected to be in the range of 0 - 200 Hazen Units, and drinking water in the range of 0 - 15 Hazen units. The EU guideline define colour has to be "acceptable for consumers and not subject to abnormal change". Monitoring technologies: 1. Visual comparison method 2. Colorimetric analysis 3. On-line spectrophotometric measurement

4.31.1 Monitoring technology nr 1: Visual Comparison method

Description In this method water colour, after filtration and pH adjustment to pH 7, is compared to the colour of reference solutions. The reference solutions are prepared from standardised platinum and cobalt salts. The sample is then visually compared to the standard solutions. This comparison is performed manually using a so-called nessler tube. The colour is estimated using this apparatus by looking through it towards a white surface. The colour is then expressed in Colour Units (CU), and need to be corrected for sample dilution, which is necessary if colour is too high. Equipment and consumables Required for this measurement are: nessler tube filter assembly and 0.45µm filters K2PtCl6 (potassium chloroplatinate) CoCl2.6H2O (cobaltous chloride) HCl (hydrochloric acid) NaOH (sodium hydroxide)

This is a long established and accepted method. However, it is no longer used much as it has been replaced by automated tests, as described under technique 2.


Reference Technique is described in APHA Standard Methods for the examination of water and waste water (21st edition). Equipment available for example from Kimble, Fischer Scientific, Taylor Scientific and many other laboratory equipment resellers. Costs Costs per test set-up is in the order of 30 €. Evaluation: This method is simple and cheap. However, it is laborious, highly subjective (as it depend on the judgement of the person the makes the comparison with the standard). The performance of a reproducible measurement with this technique requires a skilled laboratory technician. The use of current colorimeters is much simpler and has higher reproducibility. This technique is only suited for laboratory analysis, not for online purposes. An experienced technician can perform one colour measurement per 5 minutes (measurement only, excluding preparation of stock solutions etc)


Monitoring technology nr 1: Visual comparison method Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x only manual analysis in lab possible.


ease-of-use (B) x maintenance requirements (C) x only washing of glassware

needed Costs

instrumentation (C) x very cheap operational costs (C)


x

x

Recommendation for use in SSS (D) x not recommended as it requires

skilled technician

Overall conclusion Primitive method. Cheap but relies on operator and is subjective. Not suited for online use.


4.31.2 Monitoring technology nr 2: Colorimetric analysis

Description: As above. But in this case, the analysis is performed using a laboratory instrument. Used for this is a simple single wavelength spectrophotometer that measures colour between 450 and 465 nm and correlates this with an internally stored calibration curve. This is a long established and accepted method. It is the most commonly used technique for colour measurement. Equipment Required for this measurement are: spectrophotometer filter assembly and 0.45µm filters Reference


Technique is described in APHA Standard Methods for the examination of water and waste water (21st edition). Equipment available for example from WTW, LaMotte, Hach Lange, Elektro Physik, Thermo Electron. Costs Costs of the spectrometer are in the range of 1000 - 3000 Euro, depending on the number of different tests that can be performed with it. Most can handle tests for several up to more than 20 different parameters. Each parameter requires a different cuvette with reagents. Colour is normally performed without the need for reagents. Evaluation: This method is simple and cheap. It is objective and reproducible. It only provides a colour indication against platinum cobalt standards, so is only suitable for orange, yellow and brown colours. This technique is only suited for laboratory analysis, not for online purposes. An experienced technician can perform one colour measurement per 2 minutes. Monitoring technology nr 2: Colorimetric Analysis Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result




instrumentation (C) x cheap operational costs (C)


x

x


Overall conclusion Reliable method, but requires filtration of sample and manual analysis. Method o.k., but faster method needed.


4.31.3 Monitoring technology nr 3: Online spectrophotometric measurement

Description:


In this method water colour, without filtration or pH adjustment, is measured at one wavelength. The result is compared to a stored calibration curve made against platinum- cobalt (HAZEN) reference solutions. This is a long established and accepted method. It is the online equivalent of technique 2. Equipment Required for this measurement are: spectrometer Reference Technique is described in APHA Standard Methods for the examination of water and waste water (21st edition). Equipment available for example from Siegrist, WTW, s::can, Applikon. Costs Costs per instrument vary between 2000 and 8000 Euro. More expensive instruments will provide additional parameters simultaneously, such as turbidity (Siegrist), UV absorbance at 254 nm or even NO3 and TOC/DOC (s::can). These instruments can also provide true and apparent colour because they use multiple wavelengths to compensate for the influence of particles. Evaluation: This instrument installation is simple. A simple flow through setup is all that is necessary. Furthermore, some instruments are submersible and thus can be installed directly in rivers or reservoirs. The instruments are more expensive than the laboratory spectrometer, but provide continuous information, often in combination with additional important process parameters.


Monitoring technology nr 3: Online spectrometric measurement Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x continuous


ease-of-use (B) x maintenance requirements (C) x monthly cleaning recommended Costs



x x

Recommendation for use in SSS (D) x suitable Overall conclusion This is a reliable and low maintenance method. Equipment

is not too expensive, so well suited to online applications. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.32 Turbidity Prepared by: S::can Required technical specifications Turbidity is the scattering of light caused by particles, colloids and / or air bubbles in water. Turbidity is related to the clarity of water, higher turbidity resulting in less clear water. Turbidity ranges expected for surface water: 0 - 1500 FTU drinking water: 0 - 60 FTU In the drinking water guidelines, it is stated: turbidity should be "acceptable for the consumer and not show unacceptable changes". In general, it is expressed either in FTU (formazine turbidity units) or NTU (nephelometric turbidity units). Both are based on the same method, but are obtained by calibration of the turbidity meter with a different reference solution. Monitoring technologies: 1. Analysis using a laboratory Colorimeter 2. On-line turbidity meter 3. On-line spectrophotometric measurement

4.32.1 Monitoring technology nr 1: Analysis using a laboratory colorimeter

Description: In this method turbidity is analysed by sampling followed by analysis onsite (using a handheld analyser) or in a laboratory. The measurement is based on the following principle: turbidity is caused by scattering of light by particles in the water. This scattering is measured by sending a light beam through a sample and measuring the scattering at 90 degree angle of the incident beam. In general, IR light at 830 nm is used for this measurement, although some instruments used visible light instead. Equipment Required for this measurement are: spectrophotometer glass cuvette fitting spectrophotometer Reference: Technique is described in APHA Standard Methods for the examination of water and waste water (21st edition) and specified in DIN 27027, ISO 7027, US EPA 180.1. Equipment available for example from WTW, LaMotte, Hach Lange, Elektro Physik, Thermo Electron.


Costs Costs of the spectrometer are in the range of 1000 - 3000 Euro, depending on the number of different tests that can be performed with it. Most can handle tests for several up to more than 20 different parameters. Each parameter requires a different cuvette with reagents. Turbidity measurement is normally performed without the need for reagents. Evaluation: This method is simple and cheap. It is objective and reproducible. This technique is only suited for laboratory analysis, not for online purposes. An experienced technician can perform one turbidity measurement per minute. Monitoring technology nr 1: Analysis using a laboratory colorimeter Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result



ease-of-use (B) x maintenance requirements (C) x only cleaning of cuvettes Costs



x x


Overall conclusion Suitable method for online monitoring, however maintenance intensive


4.32.2 Monitoring technology nr 2: Online turbidity meter

Description: As above. But in this case, the analysis is performed using on online instrument. Used for this is a simple single wavelength spectrophotometer that measures turbidity. Either flow through or submersible instruments can be used.


This is a long established and accepted method. It is the most commonly used technique for turbidity measurement. Equipment Required for this measurement are: spectrophotometer Reference Technique is described in APHA Standard Methods for the examination of water and waste water (21st edition). Equipment available for example from WTW, YSI, Endress + Hauser, SWAN, Hach Lange, Siegrist, etc. Costs Costs of the turbidy meter are in the range of 2000 - 4000 Euro. The more expensive one, such as from Siegrist, provide additional parameters such as colour and UV254 at the same time. Evaluation: This method is simple and affordable. Many run with low maintenance, although keeping the measurement chamber clean and replacement of optical parts (e.g. the lamp) can be periodically required. Cross sensitivity is limited: main reasons for inaccurate readings are: air bubbles, dirty measuring cell. Further causes for discrepancies in measurements are the use of difference reference standards and different design of the measuring cell (if not according to ISO or US EPA norms). This technique is suited for online purposes.


Monitoring technology nr. 2: Colorimetric Analysis Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x continuous online measurement





x x


Overall conclusion Reliable method, equipment affordable. Suited for online monitoring, but referencing/ calibration is difficult due to instable standard solutions.


4.32.3 Monitoring technology nr 3: Online spectrophotometric measurement

Description: In this method turbidity is measured by monitoring the light absorbance of the water at multiple wavelengths. The obtained spectrum is fitted with known absorption curves for turbidity and from this fit the turbidity is calculated. Because multiple wavelengths are used, the effects of cross sensitivity are minimised. Equipment Required for this measurement are: spectrometer Reference This is a relatively new technique, not yet included in standard methods. It is founded on many scientific papers, but only has been available for online water quality measurement since 1999. Available from e.g. s::can.


Costs Costs per instrument vary between 8000 and 12000 Euro. The instruments will provide additional parameters simultaneously, such as colour, UV absorbance at 254 nm, NO3 and TOC/DOC. Evaluation: This instrument installation and use is simple. A simple flow through setup is all that is necessary. Furthermore, the instruments are submersible and thus can be installed directly in rivers or reservoirs. The instruments are more expensive than the laboratory spectrometer, but provide continuous information in combination with additional important process parameters. Monitoring technology nr 3: Online spectrometric measurement Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x x

time to result



ease-of-use (B) x maintenance requirements (C) x no replaceable parts, automatic

cleaning Costs

instrumentation (C) x competitive when used for multiple parameters at once.


x x

Recommendation for use in SSS (D) x suitable. espeically because of

low maintenance and multiple parameters in one instrument

Overall conclusion Reliable method, equipment affordable. Suited for online

monitoring, but referencing/ calibration is difficult due to instable standard solutions.



4.33 AOC Prepared by: Eawag Required technical specifications: AOC analysis is not covered by current drinking water legislation or guidelines. However, a suggested AOC value for biologically stable water is 10 µg-C/L acetate-carbon equivalents (Van der Kooij, 1982). A concentration range that should at least be covered by the AOC method is 10 – 1000 µg-C/L Monitoring technologies: 1. The original “van der Kooij” assay (van der Kooij et al., 1982) 2. The Werner and Hambsch assay (Hambsch et al., 1992) 3. The Stanfield and Jago ATP-based assay (Stanfield and Jago, 1989) 4. The LeChevallier assay (LeChevallier et al., 1993) 5. The Eawag-AOC assay (Hammes and Egli, 2005)

Methods 1 & 4 are incorporated/combined in Standard Methods for the Analysis of Water and Wastewater (APHA, 1998). Numerous other AOC methods are described in peer-reviewed literature. For further information, see for example Page and Dillon (2007) and Volk (2001). A complete overview is given in “Biodegradable organic matter in drinking water treatment and distribution” (Prevost et al., 2005).

References APHA (1998). Standard Methods for the Examination of Water and Wastewater. 20th ed. Page, D. and Dillon, P. (2007) Measurement of the biodegradable fraction of dissolved organic matter relevant to water reclamation via aquifers. www.csiro.au, ISSN: 1835-095x. Volk, C.J. (2001) Biodegradable organic matter measurement and bacterial regrowth in potable water. Methods in Enzymology, 337: 144 – 170. Prevost, M., Laurent ,P. R., Servais, P., Joret J.-C. (2005). Biodegradable organic matter in drinking water treatment and distribution. ISBN 1583213678.

4.33.1 Monitoring technology nr. 1: The original “van der Kooij” assay

Description: This method measures the total growth until stationary phase of two bacterial pure cultures in a pasteurized water sample at 15 °C, using plate counting on agar as enumeration method. The concept is that the pure culture strains consume the AOC and produce cells. The test organisms are Pseudomonas fluorescens P17 and Aquaspirillum NOX. The obtained cell concentration is converted to an AOC concentration using growth of the pure cultures on acetate-carbon as a standard. Typical variations on this method include using


a higher incubation temperature (LeChevallier et al., 1993), adding an inorganic mineral buffer to ensure only carbon limitation (Lehtola et al., 2001), and using alternative strains (van der Kooij, 2002). The detection limit of the method is < 10 µ/L acetate-carbon equivalents. Equipment and consumables Equipment: Incubator, carbon-free glassware and standard equipment for plate counting Consumables: Agar used for plate counting Status of the technique The method has been in use for more than 20 years and is generally accepted as the established method. It is also included in Standard Methods (APHA, 1998). Evaluation: The method has the advantages that it has been used for a long time and a lot of experience and data exists in the peer reviewed literature. The method is generally regarded as rather time consuming (< 14 days) and the detection of all AOC compounds can be limited due to the use of selected pure cultures. References APHA (1998). Standard Methods for the Examination of Water and Wastewater. 20th ed. Van der Kooij D., Visser A., Hijnen W.A.M. (1982) Determination of easily assimilable organic carbon in drinking water. J. Am. Water Works Assoc. 74: 540-545. Van der Kooij, D. Assimilable organic carbon (AOC) in treated water: determination and significance. In Encyclopedia of Environmental Microbiology, Bitton, G. (Ed.), John Wiley & Sons, Hoboken, NJ, USA, 2002. pp 312 – 327.


Monitoring technology nr 1: The original “van der Kooij” assay


4.33.2 Monitoring technology nr. 2: The Werner and Hambsch assay

Description: The method is based on correlation between turbidity and total bacterial cell number (TCN) after growth of an inoculum in the target water sample. The sample is filter-sterilized, placed into a cuvette (250 mL), and a sterile nutrient salt solution containing no carbon is added. The sample is inoculated to about 5 x 104 TCN/mL with a suspension of bacteria washed from the sterilizing filter, and the cuvette is incubated in a specially modified turbidimeter at approximately 22 °C. Turbidity, by applying 12-degrees forward scattering on a specifically designed instrument, is measured every 30 minutes for 2 to 4 days, until stationary phase is reached. The growth curves are plotted as logarithm of turbidity versus incubation time. The slope of the curve gives the growth rate (µ) and is an indicator of substrate quality and the growth factor indicates the substrate quantity. Moreover, acetate-C-equivalents are calculated from the turbidity increase with the turbidity yield on acetate-C.




x x


selectivity

x

x

Might not detect all AOC compounds

time to result

up to 14 days

Operational specifications ease-of-use (B) x All AOC methods require ultra-

clean handling maintenance requirements (C) x All AOC methods require ultra-

clean handling Costs instrumentation (C) x operational costs (C)


x x

Recommendation for use in SSS (D) x Overall conclusion The method is established, and does not require complex

instrumentation or expertise to perform. The drawback is that it is time and labour intensive.


The bottom detection limit is 10 µg/L acetate-C-equivalents. Also, DOC-removal and total cell number increase are analyzed at the start and at the end of the experiments. Equipment and consumables Equipment Turbidity meter (12° forward scattering), carbon-free glassware. Optional: DOC analyzer and fluorescence microscope (for TCN determination) Evaluation: The method is significantly faster than the original van der Kooij assay (2-4 days). The use of a natural microbial community allows the detection of a broader range of AOC substrates. The additional advantage of the method is that it also produces kinetic information (specifically growth rates relating to AOC quality) and additional values like DOC removal and increase of total cell number). The disadvantage is a need for specialized equipment (turbidimeter) and a limited number of samples that can be processed at one time. References: Werner, P. (1984). Untersuchungen zur Substrateigenschaft organischer Wasserinhaltsstoffe bei der Trinkwasseraufbereitung. Zbl. Bakt. Hyg. I Abt. Orig. B 180 S. 46-61. Hambsch, B., Werner, P., Frimmel, F. H. Bakterienvermehrungsmessungen in aufbereiteten Wässern verschiedener Herkunft. Acta hydrochim. hydrobiol. 20:1, p. 9-14 (1992) Hambsch B., Werner, P. (1996). The removal of regrowth enhancing organic matter by slow sand filtration, in: Advances in Slow Sand and Alternative Biological Filtration, John Wiley & Sons, , p. 21-27.


Monitoring technology nr 2: The Werner and Hambsch assay


4.33.3 Monitoring technology nr. 3: The Stanfield and Jago ATP-based assay

Description: The concept is similar to the original “van der Kooij” assay in that the growth of bacteria on AOC is quantified and related to the consumed AOC. However, this method differs essentially in that it uses a natural microbial community instead of pure cultures, and that it utilizes an ATP assay instead of plating (due to the natural microbial community) (Stanfield and Jago, 1989). Equipment and consumables Equipment Incubator, carbon-free glassware and standard equipment for ATP analysis (luminometer) Consumables ATP reagents




x x


selectivity

x x

Natural microbial communities probably consume more AOC compounds than pure cultures

time to result

2-4 days





x

x

Recommendation for use in SSS (D) x Overall conclusion The method is fast compared to the original AOC method,

but the specific instrumentation (turbidometer) limits the number of samples that can be processed.


Status of the technique: The method has bee restricted to a limited number of published applications (Stanfield and Jago, 1989; Gibbs et al, 1993). Evaluation: The method is significantly faster than the original van der Kooij assay (3 days). There is also the notion that the use of a natural microbial community allows the detection of a broader range of AOC substrates. Potential disadvantages are that additional conversion factors are required to convert ATP results to cell concentrations, which is complicated by the fact that natural microbial communities can have broader differences in cellular ATP than pure cultures. Monitoring technology nr 3: The Stanfield and Jago ATP-based assay





x x


selectivity

x x


time to result

3 days





x

x

Recommendation for use in SSS (D) x Overall conclusion The method is rather fast, but has as a main drawback the

additional conversion of ATP concentrations to cell concentrations.


References: APHA (1998). Standard Methods for the Examination of Water and Wastewater. 20th ed. Stanfield, G. and Jago, P.H. (1989) Application of ATP determinations to measure the concentration of assimilable organic carbon in water. In: ATP luminescence, Edited by Stanley et al. Blackwell Scientific, Oxford, England, 99 - 108.

4.33.4 Monitoring technology nr. 4: The LeChevallier assay

Description: Essentially similar to the original “van der Kooij” assay, this adaptation employs a higher incubation temperature (22 °C) and higher inoculum concentration (104 cells/mL) to achieve a faster result. In addition, ATP analysis is used to quantify the growth of the pure cultures (LeChevallier et al., 1993). Equipment and consumables Equipment: Incubator, carbon-free glassware and standard equipment for ATP analysis (luminometer) Consumables: ATP reagents Status of the technique: The method has been in use for more than 10 years and is also included in Standard Methods (APHA, 1998). Evaluation: The method is significantly faster than the original van der Kooij assay (3 days). Disadvantages are that additional conversion factors are required to convert ATP results to cell concentrations. This is supposedly less problematic for pure cultures compared to natural communities.


Monitoring technology nr 4: The LeChevallier assay


References: APHA, AWWA, et al. (1998). Standard Methods for the Examination of Water and Wastewater. 20th ed. LeChevallier, M.W., Shaw, N.E., Kaplan, L.A., Bott, T.L. Development of a rapid assimilable organic carbon method for water. Appl. Environ. Microb. 1993, 29: 1526 – 1531.




x x


selectivity

x

x

time to result

3 days





x

x

Recommendation for use in SSS (D) x Overall conclusion The method is similar to the Van der Kooij assay but faster.

However, it also requires the additional conversion of ATP concentrations to cell concentrations.


4.33.5 Monitoring technology nr.5: The Eawag-AOC assay

Description: Essentially similar to the Stanfield and Jago (1989) method, this assay measures the total growth of a natural microbial community in a filter-sterilized water sample at 30 °C, using fluorescent staining and flow cytometry (FCM) enumeration method. The obtained cell concentration is converted to a carbon concentration using a theoretical conversion value (1 ug/L AOC = 1 x 107 cells) (Hammes and Egli, 2005; Vital et al., 2007) Equipment & consumables Instruments: Incubator, carbon-free glassware and a flow cytometer equipped with a blue laser (488 nm) and the appropriate filter sets. Consumables: Fluorochromes (e.g. SYBR GREEN I); Sterile syringe filters. Status of the technique: The method has been in use at Eawag since 2005, and is under development/testing in the TECHNEAU project. Evaluation: The method is easy to use and robust. It requires a maximum of 3 days to perform, and the FCM enumeration allows for large amount of samples to be processed at little cost of time/labour. The natural microbial community erases the limitation that might go along with a pure culture. A drawback is the relative expensive nature of the instrumentation (flow cytometry).


Monitoring technology nr 5: The Eawag-AOC assay


References Hammes F.A., Egli T. (2005) New method for assimilable organic carbon determination using flow-cytometric enumeration and a natural microbial consortium as inoculum. Environ. Sci. Technol. 39: 3289-3294. Vital, M., Fuchslin, H. P., Hammes, F. & Egli, T. (2007). Growth of Vibrio cholerae O1 Ogawa Eltor in freshwater. Microbiol 153, 1993-2001.




x x


selectivity

x x


time to result

3 days



clean handling Costs instrumentation (C) x Initial investment in a flow

cytometer can be high operational costs (C)


x

x

Recommendation for use in SSS (D) x Overall conclusion The method is fast and allows processing of a large number

of samples. However, some degree of expertise in flow cytometry is required, and investment costs are high.


4.34 DOC/TOC Prepared by: S::can Required technical specifications: TOC stands for the total organic carbon present in water. This is composed of a variety of organic compounds in various oxidation states. TOC is a convenient method for expression of the total organic content (rather then parameters such as BOD, COD, AOC, which only provide information on a specific part of the organics present). TOC is independent of the oxidation state of the organic molecules and does not measure other organically bound elements, such as nitrate and hydrogen, nor inorganic that can contribute to e.g. COD. TOC does not replace the other parameters, such as COD and BOD, as it produces a different type of information entirely. DOC stands for Dissolved Organic Carbon. It measures the same components as TOC, but after filtration of the water through a 0.45 µm pore size filter. As such it represents the total amount of dissolved organic carbon in the water. DOC concentrations are generally in the same range, but always lower than, TOC. Concentration ranges commonly encountered in source water: TOC 0.1 - 100 mg/L drinking water TOC 0.1 - 25 mg/L Required sensitivity of the measurement: source water: 1 mg/L drinking water: 0.1 mg/L Monitoring technologies: 1. High temperature combustion 2. Persulfate oxidation 3. UV oxidation 4 UV-Spectroscopy


4.34.1 Monitoring technology nr 1: High temperature combustion

Description: To determine the quantity of organically bound carbon, the organic molecules are broken down into a molecular form that can be measured quantitatively (most often to CO2). With this method, a sample is homogenised and diluted. Then a micro portion is injected in a heated reaction chamber packed with oxidative catalyst, such as cobalt oxide. The water is vaporised and the organic carbon is oxidised to CO2 and H2O. The CO2 from the oxidation is transported in a carrier gas stream and measured by means of an infra red analyser or titrated. Common interferences are inorganic carbons, mainly from carbonate and bicarbonate. This must be removed before analysis by acidification and purging with purified gas. This however, can result in the loss of volatile organic substances. Equipment - a TOC analyser using combustion techniques - sampling, sample preparation and injection accessories - blender - magnetic stirrer - filtering apparatus The required analysers are available as laboratory equipment or as fully automated analysers for on-site operation. References This technique is internationally accepted and certified (APHA Standard methods for Water and Waste water analysis, 21st edition, DIN EN 1484, ISO 8254 und EPA 415.1) Equipment is available from e.g.: LAR Process Analysers AG (online), Analytik Jena (lab), Shimadzu (lab), Thermo Electron (lab), Star instruments (lab). Evaluation: This technique is a reliable and reproducible one, but the apparatus and sample preparation required mean it is only useful in the laboratory and not in field application (combustion temperatures of 900 °C are used. Its only benefit over the lower temperature oxidation procedures is that it does not need strongly oxidising chemicals. However, this technique is less and less often used because of the extreme conditions required during combustion.


Monitoring technology nr 1: High temperature combustion Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x





x x


Overall conclusion Reliable method. Only suited for well equipped lab, due to sample preparation. Suited for regulatory monitoring, not for process control.


4.34.2 Monitoring technology nr 2: Persulfate oxidation

Description: To determine the quantity of organically bound carbon, the organic molecules are broken down into CO2 that can be measured quantitatively. The oxidation is performed using persulfate which is activated either thermally or by UV light. After oxidation, the produced CO2 is purged from the sample, dried and transferred into a carrier gas. It can then by measured by IR analysis, coulorimetric titration or conductivity in ultra pure water. Heated instruments use a persulfate solution of around 95 °C. Common interferences are inorganic carbons, mainly from carbonate and bicarbonate. This must be removed before analysis by acidification and purging with purified gas. This however, can result in the loss of volatile organic substances.


Equipment and consumables - a TOC analyser using persulfate principle - sampling, sample preparation and injection accessories - reagents (various persulfate and peroxydisulfate solutions can be used) - filtering apparatus The required analysers are available as laboratory equipment or as fully automated analysers for on-site operation. References This technique is internationally accepted and certified (APHA Standard methods for Water and Waste water analysis, 21st edition, DIN EN 1484, ISO 8245, ASTM D4839, ASTM D 4779, USEPA 9060, USP 26. Equipment is available from e.g.: Sievers (lab), Endress and Hauser (online), Hach Lange (online), Mettler Toledo, WTW (online), WTW (cuvette test, which requires thermal reactor as well) Thermo Scientific (lab), Applikon (online), Analytik Jena (lab). Evaluation: his technique is a reliable and reproducible one, but the apparatus and sample preparation required mean it is mainly useful in the laboratory. In field applications the automated analyser have proven to be very maintenance intensive, with pumps, reagents and tubings requiring frequent cleaning and / or replacement. Up-time of these analysers is often between 50 - 90 %.


Monitoring technology nr 2: Persulfate oxidation Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

for online use, as lab technique it functions without problems

time to result

x approx 15 minutes for online instruments


ease-of-use (B) x maintenance requirements (C) x in case of online operation Costs

instrumentation (C) x unit price between 5000 and 20000 Euro


x

x

Recommendation for use in SSS (D) x needs small lab Overall conclusion Reliable mehod in laboratory. Field use less practical due to

complexity of analysers which require frequent maintenance.


4.34.3 Monitoring technology nr 3: UV - oxidation / conductivity

Description: Technique uses relatively weak oxidising power to destroy larger organic molecules. The content of the total organics is measured by the conductivity of the water. This method is only suitable for ultra pure water, where conductivity is naturally very low and the amount of organics is very low. Equipment Required equipment - a TOC analyser Status of the technique Used in semi conductor and other clean water industries. Often used as TOC indicator for Deionised Water units. References Equipment is available from e.g.: Sievers, Mettler Toledo


Evaluation: This technique is not suitable for source water or drinking water monitoring. Monitoring technology nr 3: UV-oxidation / conductivity Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x

x


Overall conclusion Not suited because of background signal in water too high. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.34.4 Monitoring technology nr 4: UV-spectroscopy

Description: Organic matter absorbs light in the UV and Visible range. Although not all substances absorb light strongly, 20 - 50 % of the substances in TOC are normally visible without need for preconcentration. It is possible to use the correlation of the absorption spectrum of this fraction to monitor TOC. This requires analysis of the spectrum, as well as a small number ( 2 - 5) of reference analyses using classical TOC analysis (method 1 or 2). This method is a more advanced version of the single wavelength UV254 method (see separate datasheet). Because a full spectral measurement is used, cross sensitivities are much reduced and TOC and DOC can be measured with a single instrument. Common interferences: a change in the composition of the TOC which results in a significant change in the shape of the absorption spectrum can lead to loss in accuracy. This can be corrected by calibration.


Equipment - UV/Vis spectrophotometer Reference This technique has been described in numerous scientific papers. The required equipment is either a normal laboratory spectrophotometer (e.g. Shimadzu, Hitachi) or an online instrument (s::can, Stip). Evaluation: This is a very efficient technique for process control and monitoring. The high measurement frequency allows online and real time analysis. As it is a surrogate parameter, its accuracy relies on relatively stable correlations with the total TOC. This correlation should be verified at the start of any application, as well as with regular intervals. However, the low maintenance requirements of the online instruments means that for process control they are ideal. Monitoring technology nr 4: UV-spectroscopy Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x

x

time to result

x



instrumentation (C) x starting at 8000 Euro operational costs (C)


x x


Overall conclusion A surrogate method that depends on calibration at the start of operation. After calibration, is it fast, stable, easy to use and cost efficient.



4.35 UV absorbing organic constituents Prepared by: S::can Required technical specifications: Some organic commonly found in water and waste water, such as humic substances and aromatic compounds, strongly absorb ultraviolet radiation. UV absorption is a useful surrogate parameter to measure selected organic constituents in water. Strong correlations may exist between UV absorption and organic carbon components, colour and precursors for trihalomethanes and other disinfection by-products. UV absorbance is measured in absorbance units or absorbance units per meter. The advantage of using the latter, is the fact that it is standardised to one optical cell length. Because different spectrometers use different cell lengths, only this makes possible intercomparision of results. Concentration ranges commonly encountered in source water: Abs: 0 – 100 m-1

drinking water Abs: 0 – 50 m-1

Required sensitivity of the measurement: source water & drinking water: 0.1 Abs/m Monitoring technologies: 1. Single wavelength absorption measurement 2. Full spectral analysis

4.35.1 Monitoring technology nr 1: Single Wavelength absorption measurement

Description: Using a spectrometer UV-absorption is measured at one wavelength. This wavelength is classically 254 nm, because the lamps used in these instruments emit light at this wavelength. It is not by definition the best wavelength, but suffices in many applications. UV-absorbance at one wavelength generally provides a rough indication of the organics present in the water. It has however extremely high cross sensitivity, as it can not distinguish between organic materials and other substances and it can not distinguish between different organic substances, turbidity and suspended particles also cause absorption. A more advanced measurement uses a second wavelength to assess turbidity and compensates based on this. This improves the relationship between dissolved substances and UV254 but it is still highly unselective. Equipment


A single or dual wavelength spectrometer Reference: This technique is internationally accepted and certified (APHA Standard methods for Water and Waste water analysis, 21st edition). Equipment is available from e.g.: Endress+Hauser, HachLange, s::can, WTW, Stip, Trios, ABB. Evaluation: This technique is simple and can be effective. However, its extremely low selectivity means it is only suitable for crude process monitoring. Monitoring technology nr 1: Single Wavelength Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity

x

x

time to result

x



instrumentation (C) x approx 1000 – 2000 Euro operational costs (C)


x x

Recommendation for use in SSS (D) x Overall conclusion Simple instrument, very unspecific, only for process control. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.35.2 Monitoring technology nr 2: Full spectral analysis

Description: Using a spectrometer the full UV-absorption spectrum of the water is measured. This allows using of the shape of the spectrum as well as the absorption values themselves. This combination provides a much better correlation with organic materials than the single wavelength method described under 1. The shape information makes it possible to classify TOC,


distinguish between TOC from different sources and can even be used to measure single substances and perform a highly efficient turbidity compensation, so that dissolved substances only can be measured without the need for filtration. Cross sensitivities: calibration against TOC for accurate readings is often required. If the composition of the mixture of organic substances changes after calibration, this can result in loss of accuracy. This will be accompanied by a shape change in the spectrum, which can be automatically detected and thus provide warning that the instrument is less accurate. The shape change is also used as a direct indicator for water quality changes. Equipment Required equipment is a full spectrum spectrometer. Reference This technique has been described in numerous scientific papers and is succesfully applied by many waterworks. Equipment is available from e.g.: s::can, Stip, Trios. Evaluation: This technique uses robust equipment to obtain a high amount of information (TOC, DOC, turbidity, Nitrate, others) from a single measurement that needs no pre-treatment. Although the TOC and DOC parameters are surrogate parameters, local calibration means they can be used very efficiently for process monitoring and control. The instruments are highly robust, need no consumables and can be installed in-situ (submersible instruments).


Monitoring technology nr 2: Full spectral analysis Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x < 1 minute



instrumentation (C) x unit price between 8000 and 20000 Euro


x x

none


Overall conclusion Reliable method for process control. Not very cheap, but provides multiple parameters simultaneously making it competitive when more than one parameter is required.



4.36 Particle counts Prepared by: RTU Required technical specifications: Concentration range that should be covered is 10-1000 particles/mL. Sensitivity/resolution is a minimum of 50 particles/mL. Monitoring technologies: 1. Particle counting systems

4.36.1 Monitoring technology nr 1: Particle counting systems

A particle counter is an instrument that detects and counts particles. Optical particle counters detect particles and measure their size by using the interaction of particles with a light beam. They can count and size particles (dust, particulate contamination, or dirt) in air, other gases, or liquids. Liquid Particle Counters are used to determine the quality of the liquid passing through them. The size and number of particles can determine if the liquid is clean enough to be used for the designed application. Liquid particle counters can be used to test the quality of drinking water. Two methods are commonly used for detecting and measuring particles in water; light extinction and light scattering. In both methods, a change in light intensity measured by the detector is converted to an electrical signal. Light extinction is useful for particle 1 µm and greater in size. In this method, the detector looks directly into the light source and measures the size of the "shadow" of the particle as it passes through the beam. To detect particles smaller than 1 µm, light scattering is used. In a scattering sensor, the detector does not look directly into the light source, but views the beam from the side. It looks at the region of the beam through which the particles pass. When the particle passes through the beam, it scatters light, which is measured by the detector. Equipment and consumables All particle counting systems have three parts: the sensor, counter, and sampler. The sensor is the most important part of the system. It contains a light source, optics, a detector, and a flow channel that directs the sample into the light beam. With newer instruments, the light source is a laser or laser diode. The sensor determines the particle sizes that can be measured. Sensors are available which can measure particles as small as 0.05 m and as large as 2500 m (2.5 mm). The range over which a sensor will measure depends on its optical design. The counter analyzes the electrical signals from the sensor. The counter also allows the user to define the operating parameters (for example, the particle sizes of interest and sample time) and it outputs the data. Today, nearly all counters contain a microprocessor, providing the user with lots of versatility in operating particle counter and reporting the results. The sampler delivers the sample to the sensor at a known, controlled flow rate. Samplers may also provide other features, such as volume measurement, flow rate monitoring, stirring, and vacuum degassing (required for some samples, since bubbles look like particles to the sensor).


Fig.1. An overview on a particle counter Types of Particle Counting Systems The sensor, counter, and sampler can be implemented in many different ways to measure the product or process. Different applications require different designs of the components. Furthermore, the particle counting system can be designed for use in the laboratory, in the field, or on-line at the point of interest. Liquid samples are collected in containers, then brought to the laboratory for analysis. (There are no laboratory systems for particle counters for air or gases.) Theses samples are often called "batch" or "grab" samples. Laboratory systems allow you to analyze a wide variety of samples from many different sites. As an example, in the pharmaceutical industry, R & D and Quality Control could use the same instrument on a wide variety of samples. Portable particle counters, as the name implies, can be taken around a plant or to different sites to measure the level of contamination directly at the location of interest. This method eliminates some problems that can be introduced when taking batch samples (such as dirty sample containers). It also provides immediate results, which can be used to assess the contamination level and, if necessary, to take redial action quickly. For critical processes, dedicated particle counters can be installed to monitor the contamination level either continuously or on a regular schedule. On-line systems have all the advantages of the portable systems, with the further benefit that increases in the particulate level can be detected and responded to very quickly. The system requires maintenance expenses and needs to be calibrated one or twice a year (according to the instructions of manufacturer). Status of the technique Potable water is an industry that has been using particle counters sporadically for many years, but has only recently seen a wide acceptance of their use. In USA the particle counters received increased attention following the Milwaukee outbreak in 1993 where over 100 people died as a result of Cryptosporidium infection. In fact, in 1996 US Governement brought an antitrust case (http://www.usdoj.gov/atr/cases/f0700/0722.htm) against the merger of Pacific Scientific and Met One, former competitors in particle


counter sales. COUNCIL DIRECTIVE 98/83/EC of 3 November 1998 (UNION, 1998)does not specify particle count for drinking water but only turbidity. References Pacific Scientific (http://www.particle.com/) is perhaps a market leader in particle counters. Particle Measuring Systems (http://www.pmeasuring.com/aux/about) and CCsTec (http://www.ccstec.at/en/index.htm) also provide an extensive range of counters. Particle Measuring Systems offer a possibility to rent or lease a counter. 1. The Council of the European Union, 1998. COUNCIL DIRECTIVE 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. In Official Journal of the European Communities L 330/32, 5.12.98. Evaluation: Particle counters extend the sensitivity of particle detection beyond that achievable with turbidimeters. Water filtration technologies available today can produce almost particle free water, but current pathogens, like Cryptosporidium, can avoid disinfection and infect a host with a single organism. Therefore, single particle detection is critical, and indeed mandated, for some technologies. The sensitivity of the particle counter can be utilized to detect the effects on effluent quality due to operational procedures, chemical dosage and type, and parametric changes. As a result, simple and affordable means of filtration enhancement can often be evaluated for their effectiveness before considering complicated and expensive ones. With increasing requirements to remove pathogens, such as Giardia and Cryptosporidium, the need to optimize processes to remove particles has become obvious. Particle counters reveal minute changes in water quality that can identify opportunities to modify operations or design for particle removal. Particle counting provides an important tool for assessing source water quality, finished water quality, unit process performance, and total treatment efficiency. Plant influent and filter effluent are the most important sampling locations for assessing plant performance. Particle counters are best suited to monitoring water with turbidity less than 7 to 10 ntu. Sampling locations with high particle concentrations tend to require frequent maintenance and cleaning. Particle counts in excess of the instrument concentration limit will introduce coincidence errors into the results. Because both particle number and size can be used in optimizing drinking water treatment, the most useful information available from many sequential particle count analyses includes the cumulative total number of particles, the differential number of particles, and percentile statistics. Increasing concentrations of particles are generally associated with increasing concentrations of Cryptosporidium and Giardia. Care must be taken when using the particle counter on raw water because clogging and biological growth can cause major functional problems at the instrument level. This limits the applications of particle counters in this industry. Attempts to place particle counters upstream of the final filter effluent have proven to be problematic. In fact, several manufacturers


discourage the use of particle counters on settling basins or raw water unless the operators are willing to support high maintenance costs. Cost of ownership, maintenance, and the generation of large volumes of data limit the application of particle counters in the drinking water industry. Particle counters are initially more expensive to purchase than turbidimeters and often require additional accessories to run the instrumentation. Maintenance is another issue; particle counters are difficult instruments to calibrate on-site. If on-site calibration is performed, the cost is high. Calibration verification methods have been developed but often are seen as difficult and not highly accurate. The WTP operator may lack confidence that the results from a particle counter are indeed accurate. In summary, when particle counters are properly used on filter effluent water, they can be very effective. Many water plants have adopted this technology and have optimized their treatment processes; claiming production results that could not be accomplished using only traditional turbidimetric techniques. To successfully use particle counters, operators must adhere to proper setup, calibration, and maintenance. Monitoring technology nr 1: Particle counting systems Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x


Overall conclusion A promising method but the equipment is expensive to

purchase and maintain. Skilled personnel is also required. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


4.37 Oxygen Prepared by: S::can Required technical specifications: Oxygen dissolved in water can be present between 0 - 20 mg/L. The concentration is often expressed as saturation in %, between 0 and 200%. The measuring range is identical for source water and for finished drinking water. Monitoring technologies: 1. Titration 2. Electrochemical measurement 3. Optical measurement

4.37.1 Monitoring technology nr 1: Titration

Description: Various titration methods for dissolved oxygen (DO) are described. The most commonly used one is the iodometric titration. It is the most precise and reliable titrimetric procedure for DO analysis. It is based on the addition of a divalent manganse solution, followed by strong alkali, to the sample in a closed glass bottle. The oxygen rapidly oxidises an equivalent amount of the manganese, upon which manganese hydroxide precipitates. In the presence of iodide in acidic solution, the manganese is reduced back to its original divalent state, liberation iodine equivalent to the original DO content. The iodine is then titrated with a standard solution of thiosulfate, with the endpoint of the titration being determined visually. The liberated iodine can also be analysed using a spectrophotometer, providing reliable results in the microgram per liter range, providing no interferences from particulate matter, colour or chemical interferences are present. For this analysis are required (azide modification): - manganese sulphate solution - alkali iodide azide reagent - sulfuric acid - starch - thiosulphate titrant - potassium bi-iodate solution - glassware for titration This technique is a standard method for DO measurement (described in APHA Standard Methods for the examination of water and waste water (21st edition)). Costs The materials and chemicals required for this analysis can be obtained from any chemicals supplier and lab instrumentation supplier. Total costs for first


experiment should not exceed 200 Euros. Costs per analysis are in the range of < 10 Euro. Evaluation: This method is well proven and accurate in determining DO content of a sample. However, as DO is not a constant but very susceptible to change, the need to perform this analysis in a laboratory is a big drawback. Even with sample preservation, DO is not very suitable for laboratory analysis. Furthermore, a skilled technician is required to perform this analysis reliably. The laborious nature of this test means that it is no longer used widely, because more rapid and simpler technique with comparable accuracy are now available (electrochemical methods, see point 2). Monitoring technology nr 1: Titration Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x





x

x

Recommendation for use in SSS (D) x needs a least a simple laboratory Overall conclusion Suitable method for reference/regulatory measurements in

the laboratory. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.37.2 Monitoring technology nr 2: Electrochemical Measurement

Description: Membrane covered electrode minimises the problems of the titration method with regards to applicability in online and field applications. The sensing element is an electrode covered with an oxygen permeable membrane. This


serves as a barrier against impurities. Under steady state conditions, the current in the electrode is directly proportional to the DO concentration. Two different types are available: galvanic (passive, oxygen is not consumed) and poloragraphic (active, oxygen is consumed and sample needs to be refreshed after measurement). Replacement of membranes, recalibration as well as substantial temperature effects, that has to be compensated for, on the measurement are the main drawbacks of this method. Equipment and consumables For this analysis are required: - oxygen sensitive electrode - data logger - sodium sulphite for referencing of the instrument - replacement membranes and electrolyte This type of instrument can be obtained from a great number of suppliers. Versions exist for laboratory use only (simply put in sample and ready or real classical electrodes) to handheld systems to robust instruments for continuous in-situ measurement. Costs Electrodes only are available for 100 - 500 Euro, whereas total equipment can be obtained for 1000 - 3000 Euro. References This technique is a standard method for DO measurement (described in APHA Standard Methods for the examination of water and waste water (21st edition)). Manufacturers are for example: WTW, Jumo, Hach Lange, YSI, Swan, Mettler Toledo, Thermo Electron, Palintest, Züllig, Evaluation: This method is well proven and accurate in determining DO content of a sample. However, stability of the sensor can be problematic, hence frequent calibration is required. This limits the robustness of the technique for online insitu applications. Also, water flow is required for proper functioning of these electrodes.


Monitoring technology nr 2: Electrochemical measurement Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x continuous or in minutes (polarographic measurement)





x x

needs replacement membranes and frequent calibration

Recommendation for use in SSS (D) x needs a least a simple laboratory Overall conclusion Suitable method for online monitoring, however

maintenance intensive (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems

4.37.3 Monitoring technology nr 3: Optical Measurement

Description: The sensing element in optical DO probes consists of a fluorescing compound suspended stably in a robust optical “window” or membrane. A light source at controlled wavelength briefly pulses the optical window. This causes excitation of the fluorescent material, causing it to emit a specific wavelength of light. The intensity of this fluorescence or light is determined by the amount of dissolved oxygen in the water that is in contact with the optical window. An optical sensor measures the emitted fluorescence, and from the light intensity the concentration is calculated. Alternatively, luminescence instead of fluorescence is used for DO measurement. In this case not the intensity of fluorescence is measured but the lifetime of the fluorescence. Otherwise the principle and equipment are similar. Equipment - optical DO probe


This technique is relatively new. It is being offered by many manufacturers already, as ease of use is much better than for electrochemical analysis. However, it is not yet included in standards. This type of instrument can be obtained from a great number of suppliers. Versions exist for field use (dipping probe) or for continuous insitu use (submersible probes). Costs The probes for only use can be obtained for 1500 - 2500 Euro. Price differences are caused for example by internal temperature and pressure compensation sensors. References Manufacturers are for example: Endress + Hauser, YSI, s::can, Hach Lange, Zebra Tech Ltd, In-Situ Inc., WTW, Evaluation: This method is now well proven and accurate in determining DO content of water. It is the most simple and easy to use method of the three described here, and ideally suited for online measurements. Independent studies, for example by the US Geological Survey (USGS) have proven that optical DO probes are less prone to fouling and sensor drift than electrochemical instruments. The fluorescence lifetime probes usually require membrane replacement only once a year, whereas the fluorescence probes often need no membrane replacement during the lifetime of the sensor.


Monitoring technology nr 3: Optical Measurement Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x result every few seconds





x x

cleaning needed, depending on the water quality.

Recommendation for use in SSS (D) x simple to use, robust. If this

parameter needs to be measured, this is the best method.

Overall conclusion Reliable, relatively inexpensive measurement. well suited

for online monitoring purposes. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Annex I. Individual evaluation forms for endocrine disrupting effects

Monitoring technology nr 1: ER CALUx Description: The ER CALUx bioassay (Estrogen Receptor mediated Chemically Activated Luciferase gene expression) is developed by Legler and co-workers in the Netherlands (Legler et al., 1999). The bioassay utilizes T47D human breast cancer cells expressing endogenous ER (ERα and ERβ). The cells have been stably transfected with pERE-tata-Luc, a pGL3 based estrogen responsive luciferase reporter gene under control of three Estrogen Responsive Elements. Recently, two bioassays have been developed allowing for the detection of the activation of specifically ERα or ERβ (Sonneveld et al., 2005). These ERα CALUx and ERβ CALUx utilize U2-OS human osteoblastic osteocarcoma cells that do not endogenously estrogen receptors. Evaluation: The ER CALUx bioassay has been validated for the detection of low concentrations of estrogens in both drinking water and surface water. The bioassay is very sensitive and the response of the bioassay is very reproducible. The ER CALUx bioassay has been used extensively by research groups in the Netherlands in several projects regarding the quantification of estrogenic compounds in the Dutch aquatic environment (e.g Vethaak et al., 2005; CSIRO, 2007); Vethaak, 2002). In a recent comparison by the GWRC, the ER CALUx performed best overall, being one of the most sensitive bioassays and with the lowest average coefficient of variation (Leusch, 2008). Other mammalian cell line based bioassays were shown to be equally or less sensitive, have higher coefficients of variation or take longer to perform. The ER CALUx is marketed by BioDetection Systems B.V., the Netherlands. Training in analysis and service are provided. ER CALUx analyses are performed by BDS under ISO 17025.


Monitoring technology nr 1: ER CALUx Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x


ease-of-use (B) x Training and technical support by supplier




x x

Recommendation for use in SSS (D) x Overall conclusion Suitable. Among the most sensitive and reproducible

bioassays. (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 2: MVLN and MELN Description: Several bioassays expressing luciferase have been developed using the MCF-7 human breast cancer cell line that expresses endogenous ER. Of these cell lines, the MVLN and the MELN have been used most frequently the determination of estrogens in aqatic matrices. The MVLN (MCF-7-p-Vit-tk-Luc-Neo) bioassay utilizes cells that have been transfected with a luciferase gene under control of an ERE derived from the xenopus vitellogenin (Pons et al., 1990; Demirpence et al., 1993). The time of exposure for the MVLN bioassay is 48 hours (Gutendorf and Westendorf, 2001). The MELN (MCF-7-ERE-βGlob-Luc-Neo) uses a stably transfected plasmid with the luciferase gene driven by an ERE in front of the β-globulin promoter (Balaguer et al., 1999). Evaluation: Both cell lines have been used for the detection of estrogens in a variety of aquatic matrices in a variety of studies. However, both cell lines seem to be less sensitive than other mammalian cell line based bioassays (Kinnberg, 2003; Leusch, 2008). In a recent comparison by the GWRC, the MELN also showed higher coefficients of variation (Leusch, 2008). The same study also showed that accurate quantification of the estrogenic activity in water extracts was problematic for the MELN, possibly due to matrix interference.


Monitoring technology nr 2: MVLN and MELN Criteria 1 2 3 4 5 Comments



drinking water

x x

Less sensitive than comparable methods


selectivity

x x

x

Matrix interference in water extracts

time to result

x x 48 hours exposure for MVLN


ease-of-use (B) No information maintenance requirements (C) No information Costs



x x

Recommendation for use in SSS (D) x Overall conclusion Unknown (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 3: T47D-KBluc Description: The T47D-Kbluc bioassay makes use of human breast cancer cell line T47D that endogenously expresses ERα and ERβ. These cells have been are stably transfected with a 3x ERE-tata-lnr-luc-neo construct (Wilson et al., 2004). Evaluation: The T47D-Kbluc bioassay has only been described for estrogen analysis in water in a recent comparison study (Leusch, 2008). In that study, the bioassay showed lower responses (in fold inductions) and was slightly less sensitive than the T47D based ER CALUx. Monitoring technology nr 3: T47D-KBluc Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

No information

time to result

x





x x



Description: & evaluation Monitoring technology nr 4: YES Description: The Yeast Estrogen Screen (YES) (Routledge and Sumpter, 1999) utilizes Saccharomyces cerevisiae yeast cells that have been stably transfected with human ER and a plasmid containing EREs and the lacZ gene as a reporter gene coding for the enzyme β-galactosidase. The response is mesured by adding the yellow substrate chlorophenol red-β-D-galactopyranoside (CPRG), which changes the colour to red which can be quantified spectrophotometrically at 540 nm. Evaluation: The YES bioassay is by far the most widely used yeast-based reporter gene assay and has been used extensively to assess estrogenic activity of both compounds and environmental extracts by research groups world wide. However, the YES bioassay is less sensitive than comparable mammalian assays (Murk et al., 2002; Leusch, 2008) and the thick cell wall may impede active and passive transport of chemicals to the intracellular space, increasing the risk of false negatives (Legler et al., 2002). Yeast cells are also not always capable of detecting anti-estrogenic activity (Kinnberg, 2003). Adaptations have been made to account for this problem (Gaido et al., 1997). However, the resulting yeast bioassays were shown to be more sensitive to toxic compounds, growth inhibition and matrix effects (Saito et al., 2002) and are therefore not suitable for water quality monitoring. Recently, a yeast based bioassay has been developed expressing Green Fluorescent Protein as a reporter (Bovee et al., 2004). This bioassay has not been applied to environmental monitoring and is less sensitive than mammalian cell based bioassays.


Monitoring technology nr 4: YES Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

Less sensitive, thus more sample needs to be added -> matrix effects

time to result

x x Exposure takes 1-3 days





x x

Recommendation for use in SSS (D) x Overall conclusion Not suitable due to low sensitivity (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 5: E-Screen Description: The E-Screen bioassay (Soto et al., 1992; Soto et al., 1995) utilizes the estrogen responsive growth of the MCF-7 cell line. The E-Screen is based on the premises that factors in the serum inhibit cell growth and that estrogens (when added as compound or extract) negate this inhibitory effect. Because the bioassay relies on the quantification of growth, the bioassay takes more time than reporter gene assays, having times of exposure of 4-6 days. The amount of cells is determined by counting nuclei (Soto et al., 1992; Soto et al., 1998) or by using a colorimetric end point (Körner et al., 2001). Evaluation: The E-Screen bioassay has been widely used in studies analyzing estrogenic activity of both pure compounds and extracts. The bioassay is approximately as sensitive as reporter gene bioassays based on mammalian cells. However, research has shown that proliferation is not induced by estrogens exclusively, but could also be effected by a range of mitogens, cytokines, growth factors, nutrients and hormones other than estrogens (Kinnberg, 2003), leading to false positives. Because of the relatively long time of exposure, the E-Screen is more time-consuming compared to other estrogen responsive bioassays, making it less suitable for routine analysis (Leusch, 2008; Kinnberg, 2003).


Monitoring technology nr 5: E-Screen Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

Does not respond to estrogens exclusively

time to result

x Exposure takes several days





x x

Recommendation for use in SSS (D) x Overall conclusion Not suitable due to long exposure time (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 6: AR CALUx Description: The AR CALUx (Sonneveld et al., 2005) utilizes U2-OS human osteoblastic osteocarcoma cells that do not endogenously express the androgen receptors. The cells have been stably transfected to express human AR and luciferase under control of three HREs coupled to the minimal TATA-promoter. Evaluation: The AR CALUx has been shown to responds to androgens exclusively and has recently been applied for water quality monitoring (Van der Linden et al., 2008). The AR CALUx is part of a U2-OS based bioassay panel responding to different nuclear receptor ligands. The cell line is marketed by BioDetection Systems, the Netherlands. Monitoring technology nr 6: AR CALUx Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x






x x

Recommendation for use in SSS (D) x Overall conclusion Suitable (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 7: MDA-kb2 Description: The MDA-kb2 bioassay (Wilson et al., 2002) utilizes stably transfected MDA-MB-453 breast cancer cells. These cells endogenously express high levels of AR, while ERβ is only expressed at very low levels and ERα and progesterone receptor (PR) are not detectable at the mRNA level. This cell line does however also constitutively express the glucocorticoid receptor (GR). The cells have been transfected with an androgen-responsive luciferase reporter plasmid driven by the mouse mammary tumour virus promoter (MMTV). Evaluation: The MDA-kb2 bioassay is approximately equally sensitive as the AR CALUx. However, since in addition to AR also GR (and PR) have high affinity for, and can activate the MMTV promoter, the MDA-kb2 cell line responds to both androgens and glucocorticoids. The specificity (whether AR or GR) of the signal can be determined by additional tests, existing of co-administrating a known androgen receptor antagonist, e.g. hydroxyflutamide. However, since also antagonists are not exclusively selective, additional testing makes it difficult to quantify the response. This relatively unspecific response of the cells is an important drawback when testing complex mixtures like environmental extracts.


Monitoring technology nr 7: MDA-kb2 Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

No information Also respond to glucocorticoids

time to result

x





x x

Recommendation for use in SSS (D) x Overall conclusion Not suitable due to low selectivity (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 8: PALM Description: The PALM (PC3-Androgen-receptor-Luciferase-MMTV) cell line (Térouanne et al., 2000) is derived from the AR-deficient human prostate adenocarcinoma PC3 cell line, stably transfected with hAR and an MMTV-driven luciferase reporter gene. The time of exposure is 30 hours. Evaluation: The PALM bioassay has been applied in detecting androgens in environmental samples (Balaguer et al., 2000). However, the PALM cells are reported in metabolize testosterone, possibly due to the relatively high expression of 17b-hydroxysteroid dehydrogenase which is present in wild type PC-3 cells (Térouanne et al., 2000; Castagnetta et al., 1997). This may be result in underestimation of the androgenic activity when testing complex mixtures containing natural androgens like testosterone. Monitoring technology nr 8: PALM Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

Risk of metabolizing compounds of interest

time to result

x





x x



Description: & evaluation Monitoring technology nr 9: YAS Description: The YAS bioassay is based on the same principle as the YES bioassay. It utilizes the yeast strain PGKhAR that containes a gene for the human androgen receptor (hAR), which is constitutively expressed (Sohoni and Sumpter, 1998). The yeast cells express the lacZ gene, encoding for the enzyme β-galactosidase. The amount of β-galactosidase can be quantified by adding the yellow substrate chlorophenol red-β-D-galactopyranoside (CPRG) which is converted by the enzyme to a red end product that can be quantified spectrophotometrically at 540 nm. A correction for cell concentration is sometimes including by measuring the cell density at the end of the experiment at 620 nm (Urbatzka et al., 2007). Similar bioassays have been developed expressing either also expressing lacZ (Gaido et al., 1997) or other reporters like luciferase (Michelini et al., 2005) or Green Fluorescent Protein (Bovee et al., 2007). Evaluation: The yeast based YAS bioassay has been applied in the detection of androgens in environmental monitoring (e.g. Thomas et al., 2002). However, yeast cells are less sensitive to androgens than mammalian cell line based bioassays, limiting their use for monitoring androgenic activity in surface water and drinking water.


Monitoring technology nr 9: YAS Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x x

time to result

x





x x



Description: & evaluation Monitoring technology nr 10: A-Screen Description: The A-Screen bioassay utilizes MCF-7-AR1 cells (Szelei et al., 1997), wild type MCF-7 cells that have been stably transfected with the human androgen receptor. These cells express approximately five times more AR than wild type MCF-7 cells. The A-Screen is based on the principles that sex steroid control proliferation is a combined effect of two different pathways: proliferative response and inhibition of cell proliferation. Proliferation is induced by exposing the cells to known concentrations of estrogen. Evaluation: The hAR transfected cells were shown to have an inhibited proliferation when exposed to a combination of estrogens and androgens; this inhibitory effect was reversed by adding anti-androgens indicating that the inhibitory effect was indeed caused by androgens. However, since a combination is needed of estrogens and androgens, measuring complex environmental extracts containing both estrogens and androgens might result in false positives and/or negatives. The A-Screen was also shown to respond non-specifically to the glucocorticoid hydrocortisone. The time of exposure in the A-Screen (5 days) is comparable to that of the E-Screen, which is relatively time-consuming compared to other androgen responsive bioassays, making it less suitable for routine analysis.


Monitoring technology nr 10: A-Screen Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

Responds to estrogens, androgens and glucocorticoids. Unknown performance for complex environmental mixtures

time to result

x Exposure takes several days





x x

Recommendation for use in SSS (D) x Overall conclusion Not suitable due to long exposure time. Selectivity

unknown (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 11: PR CALUx Description: The PR CALUx (Sonneveld et al., 2005) utilizes U2-OS human osteoblastic osteocarcoma cells that do not endogenously express the progesterone receptors. The cells have been stably transfected to express hPR and luciferase under control of three HREs coupled to the minimal TATA-promoter. Evaluation: The PR CALUx was shown to respond exclusively to progestins, making the bioassay suitable for the specific detection of this group of compounds. The bioassay has recently been applied for water monitoring (Van der Linden et al., 2008). No comparative studies have been published for bioassays responding the progestins. The PR CALUx is part of a U2-OS based bioassay panel responding to different nuclear receptor ligands. Monitoring technology nr 11: PR CALUx Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x






x x



Description: & evaluation Monitoring technology nr 12: TM-Luc Description: The TM-luc bioassay utilizes T47D cells that have been stably transfected with the reporter plasmid MMTV-Luc (Willemsen et al., 2004). The T47D cells endogenously express PR, which in combination with the presence of PR ligands leads to the production of luciferase which can easily be quantified using a luminometer. Evaluation: The cell line is sensitive to progestins and has been utilized in the detection of progestins in a variety of matrices. However, the cell line does not respond to progestins exclusively, but is shown to respond to certain androgens (Willemsen et al., 2004). The bioassay is therefore not suitable for the specific detection of progestins in water and surface water. Monitoring technology nr 12: TM-Luc Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

No information Also responds to androgens

time to result

x





x x



Description: & evaluation Monitoring technology nr 13: Yeast based reporter gene assays Description: This assay utilizes Saccharomyces cerevisiae strain YPH500, which is transfected with a human PR expression plasmid and a reporter plasmid carrying the lacZ gene under control of two PREs (Gaido et al., 1997). When compounds bind to the PR the enzyme β-galactosidase is produced, which can be quantified by adding the substrate 2-nitrophenyl- β-D-galactosidase (ONPG). The formation of the yellow orthonitrophenol can be quantified spectrophotometrically at 420 nm. Similar assays have been developed (Wang et al., 2005; Li et al., 2006), sometimes using GFP as a reporter (Chatterjee et al., 2008). Evaluation: The yeast bioassay provides a rapid analysis of progesterone-like activity in a variety of matrices. However, due to the limited sensitivity of yeast cell, combined with the risk of false negatives, the yeast based reporter gene assay is less suitable for the detection of low concentrations of progestins. Monitoring technology nr 13: Yeast based reporter gene assay Criteria 1 2 3 4 5 Comments



drinking water

x

x


selectivity

x

No information

time to result

x





x x



Description: & evaluation Monitoring technology nr 14: GR CALUx Description: The GR CALUx (Sonneveld et al., 2005) utilizes U2-OS human osteoblastic osteocarcoma cells that do not endogenously express significant levels of glucocorticoid receptors. The cells have been stably transfected to express human GR and luciferase under control of three HREs coupled to the minimal TATA-promoter. Evaluation: The GR CALUx bioassay is shown to respond specifically to glucocorticoids and has recently been applied in water quality monitoring (Van der Linden et al., 2008). The GR CALUx is part of a U2-OS based bioassay panel responding to different nuclear receptor ligands. Monitoring technology nr 14: GR CALUx Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x






x x



Description: & evaluation Monitoring technology nr 15: TGRM-Luc Description: The TGRM-Luc bioassay utilizes T47D cells that have been stably transfected with the reporter plasmid MMTV-Luc (Willemsen et al., 2004). T47D cells endogenously express a non-functional GR. Therefore, the cell line was cotransfected with the RS-hGRα expression vector coding for the human glucocorticoid receptor. Evaluation: The TGRM-Luc bioassay utilizes T47D cells that endogenously express ER, PR and GR. Because of the non-specific MMTV-Luc reporter construct used, the TGRM-Luc bioassay does not respond to glucocorticoids exclusively, but also to progestins and androgens. It is therefore not suitable as a selective screening tool for the detection of glucocorticoids in surface water and drinking water. Monitoring technology nr 15: TGRM-Luc Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

No information Also responds to progestins

time to result

x





x x



Description: & evaluation Monitoring technology nr 16: MDA-kb2 Description: The MDA-kb2 bioassay (Wilson et al., 2002) utilizes stably transfected MDA-MB-453 breast cancer cells. These cells endogenously express high levels of AR and GR, while ERβ is only expressed at very low levels and ERα and progesterone receptor (PR) are not detectable at the mRNA level. The cells have been transfected with an MMTV-Luc reporter gene. Evaluation: The sensitivity of MDA-kb2 is in the same range as other mammalian cell line based bioassays. However, since in addition to GR also AR (and PR) have high affinity for, and can activate the MMTV promoter, the MDA-kb2 cell line responds to both glucocorticoids and androgens. The specificity (whether AR or GR) of the signal can be determined by additional tests, existing of co-administrating a known androgen receptor antagonist, e.g. hydroxyflutamide. However, since also antagonists are not exclusively selective, additional testing makes it difficult to quantify the response. This relatively unspecific response of the cells is an important drawback when testing complex mixtures like environmental extracts.


Monitoring technology nr 16: MDA-kb2 Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

No information Also responds to androgens

time to result

x





x x



Description: & evaluation Monitoring technology nr 17: TRβ CALUx Description: The TRβ CALUx (Sonneveld et al., unpublished) utilizes U2-OS human osteoblastic osteocarcoma cells that do not endogenously express the thyroid receptors. The cells have been stably transfected to express human TRβ and luciferase under control of three TREs coupled to the minimal TATA-promoter. Evaluation: The TRβ CALUx bioassay responds specifically to compounds that bind to the TRβ receptor. It is part of a U2-OS based bioassay panel responding to different nuclear receptor ligands. However, since the bioassay has been developed very recently, no information is available concerning environmental monitoring. Monitoring technology nr 17: TRβ CALUx Criteria 1 2 3 4 5 Comments



drinking water

No information No information


selectivity

x

Not yet established fully

time to result

x






x x


Overall conclusion Unknown (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 18: T-Screen Description: The T-Screen bioassay is based on the GH3 rat pituitary somatotroph cell line (Gutleb et al., 2005). This cell, expressing TRα1, TRα2 and TRβ1, shows thyroid hormone dependent proliferation when seeded at low density. Because the bioassay relies on the quantification of growth, the bioassay takes more time than reporter gene assays, having a time of exposure of 4 days. The amount of cells is determined by fluorometric or spectrophotometric methods. Evaluation: The T-Screen bioassay has been applied for the detection of thyroid-like compounds in environmental matrices (Gutleb et al., 2005) and is shown to sensitively respond to thyroid hormones. However, since this bioassay – like E-Screen and A-Screen - relies on proliferation, it takes relatively much time compared to the other (reporter gene) thyroid hormone bioassays, and is likely to be prone to non-specific influences on proliferation. Monitoring technology nr 18: T-Screen Criteria 1 2 3 4 5 Comments



drinking water



selectivity


time to result

x Time of exposure is 4 days





x x



Description: & evaluation Monitoring technology nr 19: PC-DR-LUC Description: The PC-DR-LUC bioassay utilizes the rat pheochromocytoma cell line PC12 (Jugan et al., 2007). These cells, previously engineered to express TRα1 of avian origin, have been stably transfected with a pGL3-DR4 reporter plasmid. The bioassay was shown to respond to various iodothyronines. Evaluation: The bioassay is shown to be sensitive to thyroid hormones and can be applied for the detection of compounds that bind to the TRα1 receptor. However, since the bioassay has been developed very recently, no information is available concerning environmental monitoring. Monitoring technology nr 19: PC-DR-LUC Criteria 1 2 3 4 5 Comments



drinking water



selectivity


time to result





x x




Description: & evaluation Monitoring technology nr 20: xL58-TRE-Luc Description: This bioassay is based on a cell line of xenopus laevis, a water-living amphibian (Sugiyama et al, 2005). The cells, endogenously expressing TRβ, are stably transfected with a plasmid containing the firefly luciferase gene under control of the SV40 promoter and x. laeves TREs. Evaluation: The bioassay is shown to be sensitive to thyroid hormones and can be applied for the detection of compounds that bind to the TRβ receptor. It has recently been applied in environmental monitoring (Murata and Yamauchi, 2008). Note however, it utilizes not a mammalian cell line but an amphibian cell line. Monitoring technology nr 20: xL58-TRE-Luc Criteria 1 2 3 4 5 Comments



drinking water



selectivity


time to result





x x




References Balaguer P, François F, Comunale F, Fenet H, Boussioux A-M, Pons M, Nicolas J-C, Casellas C (1999). Reporter cell lines to study the estrogenic effects of xenoestrogens. The Science of the Total Environment 233(1-3), 47-56. Balaguer P, Fenet H, Georget V, Comunale F, Térouanne B, Gilbin R, Gomez E, Boussioux A-M, Sultan C, Pons M, Nicolas J-C, Casellas C (2000) Reporter cell lines to monitor steroid and antisteroid potential of environmental samples. Ecotoxicology 9, 105-114. Bovee TFH, Helsdingen RJR, Hamers ARM, van Duursen MBM, Nielen MWF, Hoogenboom RLAP (2007) A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists Analytical Bioanalytical Chemistry 389, 1549–1558. Bovee TFH, Helsdingen RJR, Rietjens IMCM, Keijer J, Hoogenboom RLAP (2004) Rapid yeast estrogen bioassays stably expressing human estrogen receptors and β, and green fluorescent protein: a comparison of different compounds with both receptor types The Journal of Steroid Biochemistry and Molecular Biology 91(3), 99-109. Castagnetta LA, Carruba G, Traina A, Granata OM, Markus M, Pavone-Macaluso M, Blomquist CH, Adamski J (1997) Expression of different 17b-hydroxysteroid dehydrogenase types and their activities in human prostate cancer cells. Endocrinology 138, 4876–4882. Chatterjee S, Kumar V, Majumder CB, Roy P (2008) Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay Toxicology in Vitro 22, 788–798. CSIRO (2007) Endocrine disrupting chemicals in the Australian Riverine Environment: A pilot study on estrogenic compounds. Report Demirpence E, Duchesne MJ, Badia E, Gagne D, Pons M (1993) MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity. J. Steroid Biochem. Mol. Biol. 46(3), 355-64. Gaido KW, Leonard LS, Lovell S, Gould JC, Babaï D, Portier CJ, McDonnell DP (1997). Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicology and Applied Pharmacology 143, 205-212. Gutendorf B, Westendorf J (2001) Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens. Toxicology 166(1-2), Pages 79-89. Gutleb AC, Meerts IATM, Bergsma JH, Schriks M, Murk AJ (2005) T-Screen as a tool to identify thyroid hormone receptor active compounds, Environmental Toxicology & Pharmacology 19, 231-238.


Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (2003) Evaluation of In Vitro Test Methods for Detecting Potential Endocrine Disruptors: Jugan ML, Lévy-Bimbot <m Pomérance M, Tamisier-Karolak S, Blondeau JP, Lévi Y (2007) A new bioluminescent cellular assay to measure the transcriptional effects of chemicals that modulate the alpha-1 thyroid hormone receptor. Toxicology in Vitro 21, 1197-1205. Kinnberg K. (2003) Evaluation of in vitro assays for determination of estrogenic activity in the environment. Report no. 43, Danish Environmental Protecting Agency. Körner W, Spengler P, Boltz U, Schuller W, Hanf V, Metzger JW (2001). Substances with estrogenic activity in effluents of sewage treatment plants in Southwestern Germany. 2. Biological analysis. Environmental Toxicology and Chemistry 20, 2142-2151. Legler J, Dennekamp M, Vethaak AD, Brouwer A, Koeman JH, van der Burg B, Murk AJ (2002). Detection of estrogenic activity in sediment associated compounds using in vitro reporter gene assays. The Science of the Total Environment 293, 69-83. Legler J, van den Brink C, Brouwer A, Murk AJ, van der Saag PT, Vethaak AD, van den Burg B (1999). Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicological Sciences 48, 55-66. Leusch FDL (2008) Tools to detect estrogenic activity in environmental waters. GWRC report Li J, Cui Q, Ma M, Rao KF, Wang ZJ (2006) Recombinant hPR gene in yeast for assessing in drinking water. Huan Jing Ke xue 27 (12), 2463–2466. Michelinia E, Leskinen P, Virta M, Karp M, Roda A (2005) A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection. Biosensors and Bioelectronics 20, 2261-2267. Murata T, Yamauchi k (2008) 3,3′,5-Triiodo-L-thyronine-like activity in effluents from domestic sewage treatment plants detected by in vitro and in vivo bioassays. Toxicology and Applied Pharmacology 226, 309-317. Murk AJ, Legler J, Van Lipzig MHM, Meerman JHN, Belfroid AC, Spenkelink A, Van Der Burg B, Rijs GBJ, Vethaak D (2002) Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays Environmental Toxicology and Chemistry 21(1), 16–23. Pons, M, Gagne D, Nicolas JC, Mehtali M (1990) A new cellular model of response to estrogens: a bioluminescent test to characterize (anti)estrogens molecules. BioTechniques 9, 450–459.


Routeledge and Sumpter (1996) Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environmental Toxicology and Chemistry 15(3), 241–248. Saito M, Tanaka H, Takahashi A, Yakou Y (2002). Comparison of yeast based estrogen receptor assays. Water Science and Technology 46, 349-354. Sohoni P, Sumpter JP (1998) Several environmental oestrogens are also anti-androgens Journal of Endocrinology 158, 327–339

Sonneveld E, Jansen HJ, Riteco JAC, Brouwer A, Van der Burg B (2005) Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid responsive bioassays. Toxicological Sciences 83, 136-148.

Soto AM, Justicia H, Wray JW, Sonnenschein C (1992) The E-screen assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environmental Health Perspectives 103 (7), 113–122. Soto AM, Lin T-M, Justicia H, Silvis RM, Sonnenschein C (1992). An “in culture“ bioassay to assess the estrogenicity of xenobiotics (E-screen). Advances in Modern Environmental Toxicology 21, 295-309. Soto AM, Michaelson CL, Prechtl NV, Weill BC, Sonnenshein C, Olea-Serrano F, Olea N (1998). Assays to measure estrogen and androgen agonists and antagonists. Reproductive Toxicology. Advances in Experimental Medicine and Biology 444, 9-28. Sugiyama S, Shimada N, Miyoshi H, Yamauchi K (2005) Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in xenopus laevis Tox. Sciences 88(2), 367-374. Szelei J, Jimenez J, Soto AM (1997) Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells transfected with androgen receptor. Endocrinology 138, 1406–1412. Thomas KV, Hurst MR, Smith A, McHugh M, Matthiessen P, Waldock M.J (2002) An assessment of androgenic activity and the identification of environmental androgens in United Kingdom estuaries. Environmental Toxicology and Chemistry 21, 1456-1461. Térouanne B, Tahiri B, Georget V, Belon C, Poujol N, Avances C, Orio F Jr, Balaguer P, Sultan C (2000) A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects. Molecular and cellular Endocrinology 160, 39-49. Urbatzka R, van Cauwenberge A, Maggioni S, Vigano L, Mandich A, Benfenati E, Lutz I, Kloas W (2007) Androgenic and antiandrogenic activities in water and sediment samples from the river Lambro, Italy, detected by yeast androgen screen and chemical analyses Chemosphere 67, 1080–1087.


Van der Linden SC, Heringa MB, Man H-Y, Sonneveld E, Puijker LM, Brouwer A, van der Burg B (2008) Detection of multiple hormonal activities in waste water effluents and surface water, using a panel of steroid receptor CALUx bioassays. Environmental Science & Technology (accepted for publication). Vethaak AD, Rijs GBJ, Schrap SM, Ruiter H, Gerritsen A, Lahr J (2002) Estrogens and xeno-estrogens in the Aquatic Environment of The Netherlands: Occurrence, Potency and Biological Effects. Report no. 2002.001. Dutch National Institute of Inland Water Management and Waste Water Treatment, the Netherlands. Available from: http://www.rijkswaterstaat.nl/rws/riza/home/publicaties/index.html. Vethaak, AD, Lahr J, Schrap SM, Belfroid AC, Rijs GB, Gerritsen A, de Boer J, Bulder AS, Grinwis GC, Kuiper RV, Legler J, Murk TA, Peijenburg W, Verhaar HJ, de Voogt P (2005) An integrated assessment of estrogenic contamination and biological effects in the aquatic environment of The Netherlands. Chemosphere 59 (4), 511-524. Wang J, xie P, Kettrup A, Schramm K-W (2005) Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials. Science of the Total Environment 349(1-3), 120-128 Willemsen P, Scippo M-L, Kausel G, Figueroa J, Maghuin-Rogister G, Martial JA, Muller M (2004) Use of reporter cell lines for detection of endocrine-disrupter activity Analytical Bioanalytical Chemistry 378, 655–663. Wilson VS, Bobseine K, Lambright CR, Gray LE (2002) A Novel Cell Line, MDA-kb2, That Stably Expresses an Androgen- and Glucocorticoid-Responsive Reporter for the Detection of Hormone Receptor Agonists and Antagonists Toxicological Sciences 66, 69-81.


Annex II. Individual evaluation form for genotoxic effects

Monitoring technology nr 1: Ames test Description: The Ames test is a bacterial reverse mutation test, utilizing Salmonella typhimurium bacteria that harbour a mutation which makes them unable to make histidine (Ames et al., 1975). Therefore, these mutants cannot grow in medium without histidine. When a genotoxic compound or extract is added, mutations can occur that reverse the original mutation, enabling the bacteria to grow on medium without histidine. This genotoxic potential is related to growth, which can be quantified by determining the number of colonies formed. Based on the amount of colonies, the mutagenicity of the water is assessed to be low, moderate, high and extreme for samples for <500, 500–2500, 2500–5000 and >5000 revertants/L respectively. Beside Salmonella also Escheria coli bacteria can be used for bacterial reverse mutation assays. Several strains of S. typhimurium are used, allowing for the detection of a variety of base pair substitutions or frameshift mutations either at AT sites (e.g. TA104) or at GC sites (TA98 and TA100). The classical Ames test is performed in Petri dishes and consists of counting the number of colonies after 2-3 days of exposure. An important improvement of the Ames test is the Ames II (Flückiger-Isler et al., 2004). This bioassay, using 96 well plates, utilizes a pH indicator (bromocresol) in the medium to monitor the pH change caused by growing bacteria. As an end-point, not the number of colonies is determined but the colour change in the well is assessed. Hence the assay is also known as the MutaChromo or Ames fluctuation test. Evaluation: The Ames test is a widely recognized and utilized test that allows for the rapid detection of mutagens. The test is standardized for water quality monitoring at the international level (ISO 16240). Modified versions have been described that are more sensitive (Reifferscheid and Grummt, 2000). Both TA98 and TA100 strains are the most frequently used strains for monitoring the amount of mutagenicity of surface water, although generally the TA98 strain is found to be more sensitive than the TA100 strain (Ohe et al., 2004; Umbuzeiro et al., 2001). Various other strains are used in water monitoring, overexpressing nitroreductase or O-acetyltransferase. These strains can aid in the detection of nitroarenes and aromatic amines, since Salmonella strains are usually very efficient at nitroreduction, possibly resulting in overestimation of the genotoxic potential (Ohe et al., 2004; Cobisier et al., 2001). The Ames test takes relatively long to perform and is therefore difficult to incorporate into standard monitoring processes. Compared to other bacterial assays like the Umu and SOS chromo test, the Ames test is less sensitive, making it less suitable for the screening of possibly low concentrations of


genotoxins in surface and drinking water (Reifferscheid and Grummt, 2000; Cobisier et al., 2001; Czyz et al., 2002). Like most bacteria based tests, the Ames test sensitive enough for testing pure compounds at high concentrations, but shows low specificity, resulting in a relatively large number of false positives (Kirkland et al., 2002). The Ames II is equally sensitive and can be performed in 96 well plates, allowing high throughput screening and automation. The Ames II test has shown to perform well by different laboratories. Monitoring technology nr 1: Ames test Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

time to result

x


ease-of-use (B) x Pathogenic bacteria maintenance requirements (C) x Costs




Overall conclusion (A): 1 = very low 2 = low 3 = average 4 = high 5 = very high (B): 1 = very poor 2 = poor 3 = average 4 = good 5 = very good (C): 1 = very high 2 = high 3 = average 4 = low 5 = very low (D): 2 = no 3 = yes 4 = strong SSS: small-scale systems


Description: & evaluation Monitoring technology nr 2: Vibrio harveyi Description: The principle of the Vibrio harveyi test is similar to the Ames test, but in stead utilizes V. harveyi bacteria that are sensitive to the antibiotic neomycin (Wegrzyn et al., 2003). Genotoxic agents can make these bacteria resistant to neomycin due to induced mutations in the neo-gene. This genotoxic potential is related to growth, which can be quantified by determining the number of colonies formed. The Vibrio harveyi bacteria are more robust than e.g. S. typhimurium and E. coli. generally utilized, making it possible to detect genotoxicity in water samples without extracting the water samples. Evaluation: The Vibrio harveyi test has been applied for the detection of genotoxins in a variety of aquatic matrices. The bacteria in this test are more robust than the Salmonella bacteria utilized in the Ames and Umu test, which can be an advantage when testing unextracted water samples (Ohe et al., 2004; Czyz et al., 2002). Recently, an adapted version of the bioassay has been developed and applied in water monitoring (Podgórska et al., 2007). Monitoring technology nr 2: Vibrio harveyi Criteria 1 2 3 4 5 Comments Technical specifications


drinking water

x x


selectivity


time to result

x





Unknown




Description: & evaluation Monitoring technology nr 3: Comet assay Description: The Comet assay (also known as single cell gel electrophoresis) was introduced as a microelectrophoretic method to visualize structural DNA damage in individual cells (Östling and Johanson, 1984). This method can be applied to any mammalian cell type and involves lysis of the cells followed by gel electrophoresis of the lysate. Chromosome breaks shorten the chromosome pieces, and smaller pieces move faster through the agarose gel towards the anode. These shorter pieces thus form a tail, extending the cell nucleus, giving the cell the appearance of a comet. Quantification of the clastogenicity can be performed in several ways, determining e.g. the length of the comet, the percentage of DNA in the tail or the product of the previous two (called the tail moment). The original method applies neutral pH, so that only double-strand breaks can be detected. However, the method has been adapted to detect single-strand breaks and alkali label sites by performing the method at high pH (pH >13) (Singh et al., 1988). Recent adaptation made it possible to perform the comet in a 96 well format, which, combined with image analysis software, enables automation of the method (Kiskinis et al., 2002). Evaluation: The Comet assay is sensitive and relatively fast test that can performed with any mammalian cell type, both from in vivo and in vitro studies. The assay can be performed in 96 well plates, allowing automated high throughput screening (Kiskinis et al., 2002; Witte et al., 2007). In a recent comparison of genotoxicity assays, the Comet assay was less strongly recommended than other assays for the analysis of water samples, mainly due to the variability in the cell to cell response (Corbisier et al., 2001). However, the assay has been applied frequently for water quality monitoring. If the Comet assay is to be used routinely, standardization and inter-laboratory calibration will be required (Frenzilli et al., 2008). In general, the Comet assay is more complicated to perform than bacterial mutation and reporter gene assays and requires more expensive equipment, but in return reflects genetic endpoints that cannot be assessed using bacterial tests.


Monitoring technology nr 3: Comet assay Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

No information

time to result

x





Unknown




Description: & evaluation Monitoring technology nr 4: Micronucleus assay Description: In the micronucleus assay, mammalian cells are made to undergo a division step. This division step is stopped right after the cell nuclei have divided into two separate daughter nuclei but before the cells have divided (i.e. binucleated cells). When chromosomal breaks are present, the chromosome fragments are not transported to the daughter cells but rather will form a separate, small nucleus called a micronucleus. The presence of micronuclei in binucleated cells indicates that the original cell had structural chromosomal damage, caused by the presence of DNA damaging agents. Evaluation: The micronucleus test has been standardized for water quality monitoring at the international level using amphibian larvae (ISO 21427-1) and V79 cells (ISO 21427-2). A 96 well format of the assay, utilizing a mouse lymphoma cell line, has been described by Nesslany and Marzin (1999). The read-out of the method is mostly performed using a microscope, making the method labour-intensive. However, recent advances in image analysis and flowcytometric methods have made automated analysis possible (Hayashi et al., 2007; Diaz et al., 2007). Exposure times for in vitro analysis of micronuclei vary considerably, ranging from 3 hours to several days (Diaz et al., 2007; Kirsch-Volders et al., 2003).


Monitoring technology nr 4: Micronucleus assay Criteria 1 2 3 4 5 Comments



drinking water



selectivity


time to result

x x Varies between several hours to several days


ease-of-use (B) No information maintenance requirements (C) Costs

instrumentation (C) x Depends on level of automation operational costs (C)





Description: & evaluation Monitoring technology nr 5: Alkaline elution assay Description: The alkaline elution assay detects single- and double-strand breaks in DNA and alkali-labile sites (Kohn and Ewig, 1973). For this assay, cells are exposed to the compound or extract to be test for 3-10 hours. After exposure, cells are lysed on a 2.0 µm filter after which an alkaline buffer is added and different subsequent fractions are eluted and collected. The amount of DNA in each fraction and in the filter residue is quantified using a fluorescent label. The idea behind the assay is that DNA strand breaks lead to smaller pieces of DNA (the same principle that applied to the Comet assay), which will go through the filter more easily. Exposure of cells to DNA damaging chemicals will lead to a lower amount of DNA left in the residue than normal. By comparing exposed to non-exposed cells, the difference in amount of DNA is a measure of clastegenicity (Storer et al., 1996). Evaluation: The alkaline elution assay can detect strand breaks but – like all other strand breaks assays - cannot distinguish between direct genotoxic insult and cytotoxicity-mediated DNA degradation (Elia et al., 1993). The assay as originally described by Kohn and Ewig (1973) takes approximately 3 days to perform and is rather labour-intensive. Recently, an automated, high-throughput version of the assay utilizing rat hepatocytes has been described (Gaely et al., 2007). This assay utilizes the 96 well plate format and has a reduced analysis time of 1.5 days. However, the automated assay has not been applied for water monitoring and custom made equipment is needed.


Monitoring technology nr 5: Alkaline elution assay Criteria 1 2 3 4 5 Comments



drinking water



selectivity

x

No information Both genotoxicity and cytotoxicity

time to result

x x



instrumentation (C) x Custom made for automated version





Description: & evaluation Monitoring technology nr 6: Polymerase inhibition assay Description: The polymerase inhibition assay is based on the fact that DNA damage blocks DNA polymerases during the polymerase chain reaction (PCR). DNA exposed to genotoxicants is therefore less amplified than non-exposed DNA, which can be measured by intensity analysis (Jenkins et al., 2000). This assay allows for the detection of general DNA damage, in the form of gene mutations or chromosomal aberration. Evaluation: The polymerase inhibition assay is fast and can be used for high throughput screening, although samples cannot be processed in parallel. However, it is not widely used and needs to be validated before it can be used as a full genotoxicity screen. Monitoring technology nr 6: Polymerase inhibition assay Criteria 1 2 3 4 5 Comments



drinking water



selectivity


time to result

x x Depending on the amount of samples



instrumentation (C) x PCR needed operational costs (C)


No information




Description: & evaluation Monitoring technology nr 7: UMU test Description: The UMU test utilizes genetically modified Salmonella typhimurium bacteria posessing an elevated O-acetyltransferase level. These bacteria are transfected with a plasmid vector (pACYC184) in order to express an introduced lacZ gene under control of the umuC gene, one of the SOS functions induced by genotoxins (Oda et al., 1985). When these bacteria are exposed to genotoxins, the SOS repair system is activated, including the lacZ gene, leading to the production of β-galactosidase. The amount of β-galactosidase can be quantified by adding the appropriate substrate which is converted to a coloured product that can be measured spectrophotometrically. Several adaptations exist of this bioassay, utilizing e.g. more sensitive strains (Oda et al., 1995), bacteria expressing human CYPs for the detection of promutagens and procarcinogens (Aryal et al., 1999) or bacteria expressing luciferase (Schmid et al., 1997) or GFP (Arai et al., 2001) in stead of β-galactosidase Evaluation: The UMU test is easy and relatively quick to perform and has been used for extracts of a variety of environmental matrices. The assay is applicable in 96 well plates, making it more suitable for automation. Compounds not inducing the SOS system will not be detected. The UMU test has been standardized in Germany by DIN (DIN 38415T3) and at the international level by ISO (ISO 13829). UMU tests expressing luciferase or GFP are generally more sensitive than UMU tests expressing β-galactosidase (Arai et al., 2001; Schmid et al., 1997).


Monitoring technology nr 7: UMU test Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x x

x

Only detects SOS activating damage

time to result

x


ease-of-use (B) x maintenance requirements (C) No information Costs

instrumentation (C) x spectrophotometer operational costs (C)





Description: & evaluation Monitoring technology nr 8: SOS Chromotest Description: The SOS Chromotest is based E. coli bacteria in which the activation of the bacterial SOS repair system (specifically the sfiA gene) is coupled to the production of β-galactosidase (Quillardet et al., 1982. When these bacteria are exposed to genotoxins, the SOS repair system is activated, including the lacZ gene, leading to the production of β-galactosidase. The amount of β-galactosidase can be quantified by adding the appropriate substrate which is converted to a coloured product that can be measured spectrophotometrically. Evaluation: The SOS Chromotest test is easy and relatively quick to perform and can be obtained as a kit (EBPI, Canada). The assay is applicable in 96 well plates, making it more suitable for automation. Compounds not inducing the SOS system will not be detected. The assay has been applied regularly in water quality monitoring. In a recent study the SOS Chromotest was shown to be more sensitive than Ames and Mutatox™ (Guzzella et al., 2004) and Vitotox (Cobisier et al., 2001). Monitoring technology nr 8: SOS Chromotest Criteria 1 2 3 4 5 Comments Technical specifications


drinking water



selectivity

time to result

x





No information




Description: & evaluation Monitoring technology nr 9: Vitotox® Description: The Vitotox® assay utilizes S. typhimurium bacteria that have been genetically modified to express a Luc-gene coupled to the RecN gene of the SOS repair system (van der Lelie et al., 1997). DNA damage leads to the production of luciferase which can be quantified by adding the substrate luciferin and determine the amount of light produced using a luminometer. Evaluation: The Vitotox assay is easy and relatively quick to perform and is developed and marketed by Labsystems/Thermo (USA). The assay is applicable in 96 well plates, making it suitable for automation. Compounds not inducing the SOS system will not be detected. In a recent comparison study, the Vitotox assay was shown to perform similarly to the Umu and SOS-lux test (Cobisier et al., 2001). The Vitotox assay was shown to respond differently to PAHs than the SOS chromotest (van der Lelie et al., 1997), which can cause different results between assays when analysing environmental samples. Monitoring technology nr 9: Vitotox® Criteria 1 2 3 4 5 Comments Technical specifications


drinking water



selectivity


time to result

x



instrumentation (C) x Required luminometer operational costs (C)


No information




Description: & evaluation Monitoring technology nr 10: Mutatox™ Description: The Mutatox™ assay uses a special (not genetically engineered) dark strain (M169) of the luminescent bacterium Vibrio fischeri (formerly Photobacterium phosphoreum) (Kwan et al., 1990). When these bacteria are exposed to mutagenic chemicals, they can restore the luminescence by activation of the SOS repair system. Activation of this system leads to the formation of protease, which brakes down a repressor protein of the lux pathway leading to luminescence. Exposure times are generally 24 hours. The Vibrio harveyi bacteria are more robust than e.g. S. typhimurium and E. coli. generally utilized, making it possible to detect genotoxicity in water samples without extracting the water samples. Evaluation: The Mutatox™ can be obtained as a complete kit, including analyser, media and reagents (Microbics, AZUR Environmental, UK). The test has been applied for the detection of genotoxicity in a variety of environmental samples. The test has been used by different laboratories world wide and is relatively quick and easy to perform. The bacteria in this test are more robust than the Salmonella bacteria utilized in the Ames test when testing unextracted water samples, which can be an advantage (Ohe et al., 2004). However, in a recent comparison study, the Mutatox assay was not recommended for the rapid analysis of genotoxicity of environmental samples (Corbisier et al., 2001).


Monitoring technology nr 10: Mutatox™ Criteria 1 2 3 4 5 Comments



drinking water

x x


selectivity

x

x

time to result

x



instrumentation (C) operational costs (C)





Description: & evaluation Monitoring technology nr 11: GreenScreen® GC Description: The GreenScreen® GC utilizes yeast cells that are genetically modified to contain a gene for the green fluorescent protein (GFP) under control of a DNA repair system (RAD54) promoter (Walmsley et al., 1997; Cahill et al., 2004). This repair system is part of recombinational repair pathway, which repairs double strand breaks and interstrand crosslinks. However, the system is also induced by agents causing gene mutations. Exposure to genotoxic agents leads to activation of the DNA repair system and to the production of GFP which can be quantified using a fluorometer. By counting the cells after exposure (through cell density), the assay serves simultaneously as a control for cytotoxicity. A similar assay has been described utilizing a yeast strain deleted in several multidrug transporters to enhance the sensitivity of the assay (Lichtenberg-Fraté et al., 2003). Evaluation: The GreenScreen® GC can be performed relatively quickly and can be used for high throughput screening as it can be performed in 96 well plates and determination of fluorescence can be automated. Because this assay uses yeast cells, which are just as mammalian cells eukaryotic cells, it is claimed that this assay performs better at detecting clastogenic agents than assays based on bacteria. It has been used for environmental monitoring, including surface waters and effluents and the results from a recent comparison study are promising (Corbisier et al., 2001). However, the use of S9 - as well as the presence of autofluorescent compounds - can possibly interfere with the measurement, as S9 is fluorescent and therefore interferes with GFP analysis. The GreenScreen® GC is marketed by Gentronix (UK), and an adaptation of the test is available as GreenScreen® EM (environmental monitoring), which consists of the assay supplied as a portable kit. The yeast assay described by Lichtenberg-Frate et al. (2003) has not been utilized for water monitoring, but showed a more sensitive response regarding model genotoxins like 4-NQO and MNNG.


Monitoring technology nr 11: GreenScreen® GC Criteria 1 2 3 4 5 Comments



drinking water



selectivity

No information Responds to fluorescence

time to result

x



instrumentation (C) x Requires fluorometer operational costs (C)





Description: & evaluation Monitoring technology nr 12: GreenScreen® HC Description: The GreenScreen® HC is based on the same principle as the GreenScreen® GC but in stead utilizes the human lymphoblastic TK6 cell line; hence the name HC i.e. Human Cells. These cells produce Green Fluorescent Protein gene under control of the GADD45 (Growth Arrest and DNA damage) gene (Hastwell et al., 2006). The assay is performed in 96 well plates and utilizes incubation times of 24-48 hours and claims better prediction for the human situation regarding genotoxicity. Evaluation: The GreenScreen® HC bioassay is more sensitive than the GreenScreen® GC test and bacterial based assays, while retaining the specificity of other mammalian cell line based bioassays (Hastwell et al., 2006; Billtinton et al., 2008). However, because S9 is fluorescent and can therefore interferes with GFP analysis, metabolic activation using S9 mixtures - as well as the presence of autofluorescent compounds - can be problematic. Recently, protocols have been developed allowing the use of S9. The GreenScreen® HC assay has not been used for environmental monitoring. The GreenScreen® HC is marketed by Gentronix (UK). In a recent interlaboratory study, the assay was shown to perform well at other laboratories (Billinton et al., 2008).


Monitoring technology nr 12: GreenScreen® HC Criteria 1 2 3 4 5 Comments



drinking water



selectivity


time to result

x



instrumentation (C) Requires fluorometer operational costs (C)





Description: & evaluation Monitoring technology nr 13: MCF-7-p53R2 Description: This bioassay utilizes MCF-7 human breast cancer cells that are stably transfected to express luciferase under control of the p53R2 gene (Ohno et al., 2005). These cells express wild-type p53, which regulate the expression of p53R2. The p53R2 gene is known to be activated by γ-ray and UV-irradiation in a p53-dependent manner and to play a central role in cell survival, repairing damaged DNA. The assay can be performed in 96 well plates and includes an exposure time of 24 hours, allowing high throughput analysis. Evaluation: The MCF-7-p53R2 allows for the detection of general DNA damage, linked to the expression of p53. The assay is relatively quick and easy to perform and allows for high throughput screening. However, the test has not been applied to extracts from environmental matrices. Monitoring technology nr 13: MCF-7-p53R2 Criteria 1 2 3 4 5 Comments Technical specifications


drinking water



selectivity


time to result

x



instrumentation (C) x Requires luminometer operational costs (C)





References Arai R, Makita Y, Oda Y, Nagamune T (2001) Construction of green fluorescent protein reporter genes for genotoxicity test (SOS/umu-test) and improvement of mutagen-sensitivity. J. of Bioscience and Bioengineering 92(3), 301-304. Ames BN, McCann J, Yamasaki E (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31, 347-364. Aryal P, Yoshikawa K, Terashita T, Guengerich FP, Shimada T, Oda Y (1999) Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH–P450 reductase Mutation Research 442(2), 113-120. by Salmonella typhimurium strain NM2009 possessing high O-acetyltransferase activity Mutation Research 334, 145-156. Billinton N, Hastwell P, Beerens D, Birrell L, Ellis P, Maskell S, Webster TW, Windebank S, Woestenborghs F, Lynch AM, Scott AD, Tweats DJ, van Gompel J, Rees RW, Walmsley RM (2008) Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: An enabling study for independent validation as an alternative method. Mutation Research 653, 23-33. Cahill PA, Knight AQ, Billinton N, Barker MG, Walsh L, Keenan PO, Williams CV, Tweats DJ, Walmsley RM (2004) The GreenScreen® genotoxicity assay: a screening validation programme. Mutagenesis 19, 105-119. Cobisier P, Hansen P-D, Barcelo D (2001) Proceedings of the BIOSET Technical Workshop on Genotoxicity Biosensing. Vito-publication 2001/MIT/P053. available from http://www.vito.be/english/environment/environmentaltox5.htm. Czyz A, Szpilewska H, Dutkiewicz R, Kowalska W, Biniewska-Godlewska A, Wegrzyn G (2002) Comparison of the Ames test and a newsly developed assay for detection of mutagenic pollution of marine environments. Mutation Research 519, 67-74. Diaz D, Scott A, Carmichael P, Shi W, Costales C (2007) Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells. Mutation Research 630, 1–13. Elia MC, Storer RD, McKelvey TW, Kraynak AR, Barnum JE, Harmon LS, DeLuca JG, Nichols WW (1993) Rapid DNA degradation in primary rat hepatocytes treated with diverse cytotoxic chemicals: analysis by pulsed field gel electrophoresis and implications for alkaline elution assays. Environmental Molecular Mutagenesis 24, 181-191.


Farré M, Martínez E, Barceló D (2007) Validation of interlaboratory studies on toxicity in water samples. Trends in Analytical Chemistry 26(4), 283-292. Flückiger-Isler S, Baumeister M, Braun K, Gervais V, Hasler-Nguyen N, Reimann R, Van Gompel J, Wunderlich H-G, Engelhardt G (2004) Assessment of the performance of the Ames II™ assay: a collaborative study with 19 coded compounds. Mutation Research 558, 181-197. Frenzilli G, Nigro M, Lyons BP (2008) The Comet assay for the evaluation of genotoxic impact in aquatic environments. Mutation Research (in press). Gaely R, Wright-Bourque JL, Kraynak AR, McKelvey TW, Barnum JE, Storer RD (2007) Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage. Mutation research 629, 49-63. Guzzella L, Monarca S, Zani C, Feretti D, Zerbini I, Buschini A, Poli P, Rossi C, Richardson SD (2004) In vitro potential genotoxic effects of surface drinking water treated with chlorine and alternative disinfectants. Mutation Research 564, 179-193. Global Water Research Coalition (GWRC) (2008) Tools to detect estrogenic activity in environmental waters. F.D.L. Leusch. Hakura A, Suzuki S, Satoh T (1999) Advantage of the use of human liver S9 in the Ames test. Mutation Research 438, 29-36. Hastwell PW, Chai L-L, Roberts KJ, Webster TW, Harvey JS, Rees RW, Walmsley RM (2006) High-specifity and high-sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay. Mutation Research 607, 160-175. Hayashi M, MacGregor JT, Gatehouse DG, Blakey DH,. Dertinger SD, Abramsson-Zetterberg L, Krishna G, Morita T, Russo A, Asano N, Suzuki H, Ohyamal W, Gibson D (2007) In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test. Mutation Research 627, 10-30. Heringa M (2005) Approach for assessment of carcinogenic activity in water – Part 1. BTO report 2005.022. ISO 16240 (2005). Water quality – determination of the genotoxicity of water and waste water. Salmonella/microsome test (Ames test), Geneva, Switserland. ISO 21427-1 (2006) Water quality – Evaluation of genotoxicity by measurement of the induction of micronuclei. Part 1- Evaluation of genotoxicity using amphibian larvae. Genève, Switzerland.


ISO 21427-2 (2006) Water quality – Evaluation of genotoxicity by measurement of the induction of micronuclei. Part 2 - Mixed population method using the cell line V79. Genève, Switzerland. ISO/DIN 13829-2000 (2000) Water quality – Determination of the genotoxicity of water and waste water using the umu-test. International Organisation for Standardization, Geneva, Switseland. Jenkins GJ, Burlinson B, Parry JM (2000) The polymerase inhibition assay: A methodology for the identification of DNA-damaging agents. Molecular carcinogenesis 27, 289-297. Kirkland D, Aardema M, Henderson L, Müller L (2005) Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate redent carcinoegens and non-carcinogens I. Sensitivity, specificity and relative predicitivity. Mutation Research 584, 1-256. Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, Ishidate Jr. M, Kirchner S, Lorge E, Morita T, Norppa H, Surrallés J, Vanhauwaert A, Wakata A (2003) Report from the in vitro micronucleus assay working group. Mutation Research 540, 153-163. Kiskinis E, Suter W, Hartmann A (2002) High throughput Comet assay using 96-well plates. Mutagenisis 17, 37-43. Kohn KW, Ewig RA (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells. Cancer Research 33, 1849-1853. Kwan KK, Dutka BJ, Rao SS, Liu D (1990) Mutatox test: a new test for monitoring environmental genotoxic agents. Environmental Pollution 65(4), 323-332. Ku WW, Bigger A, Brambilla G, Glatt H, Gocke E, Guzzie PJ, Hakura A, Honma M, Martus H-J, Scott Obach R, Roberts S (2007) Strategy for genotoxicity testing – Metabolic considerations. Mutation Research 627, 59-77.. Lichtenberg-Fraté H, Schmitt M, Gellert G, Ludwig J (2003) A –yeast-based method for the detection of cyto and genotoxicity. Toxicology in Vitro 17, 709-716. Marabini L, Frigerio S, Chiesara E, Maffei F, Forti G-C, Hrelia P, Buschini A, Martino A, Poli P, Rossi C, Radice S (2007) In vitro cytotoxicity and genotoxicity of chlorinated drinking waters sampled along the distribution system of municipal networks. Mutation Research 634, 1-13. Nesslany F, Marzin D (1999) A micromethod for the in vitro micronucleus assay. Mutagenesis 14, 403-410.


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