Maria Droba, Magorzata Dugan,

Maciej Balawejder, Radosaw Jzefczyk, Anna Pasternakiewicz

Current problems of environmental monitoring

UNIVERSITY OF RZESZOW Faculty of Biology and Agriculture

Department of Chemistry and Food Toxicology Rzeszow 2013

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Ta publikacja zostaa wydana przy pomocy finansowej Unii Europejskiej, w ramach Programu

Wsppracy Transgranicznej Polska Biaoru Ukraina 2007-2013. Odpowiedzialno za zawarto

tej publikacji ley wycznie po stronie Uniwersytetu Rzeszowskiego i nie moe by w adnym

wypadku traktowane jako odzwierciedlenie stanowiska Unii Europejskiej.

"This publication was co-financed by the European Union within the framework of the Cross-border

Cooperation Programme Poland-Belarus-Ukraine 2007-2013. Responsibility for the content of this

publication lies solely with the University of Rzeszow and can not under any circumstances be

considered to reflect the position of the European Union in any way.

Publikacja powstaa w ramach projektu Integracja naukowa pogranicza polsko-ukraiskiego w

zakresie monitoringu i detoksykacji substancji szkodliwych w rodowisku i jest dystrybuowana

bezpatnie.

Free copy edited and published within the project Scientific integration of the Polish Ukrainian

borderland area in the field of monitoring and detoxification of harmful substances in

environment

Wydawca publikacji: Uniwersytet Rzeszowki

Al. Rejtana 16 C 35-959 Rzeszw

Tel. (17)

Publisher:

University of Rzeszow

Rejtana Street 16C

35-959 Rzeszow

Nakad 15 egz./Circulation 15

Copyright by Biuro Projektu Integracja naukowa obszaru pogranicza polsko-ukraiskiego w zakresie

monitoringu i detoksykacji substancji szkodliwych w rodowisku

Copyright by Project Office Scientific integration of the Polish Ukrainian borderland area in the field

of monitoring and detoxification of harmful substances in environment

All rights reserved

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INTRODUCTION

Environmental monitoring can be defined as the systematic sampling of air, water, soil,

and biota in order to observe and study the environment, as well as to derive knowledge from

this process. Monitoring can be conducted for a number of purposes, including to establish

environmental baselines, trends, and cumulative effects, to inform policy design and decision-

making, and to assess the effects of anthropogenic influences, or to conduct an inventory of

natural resources.

Environmental monitoring programs can vary significantly in the scale of their spatial and

temporal boundaries. In Poland, the State Environmental Monitoring (PM) constitutes the

source of environmental information being the outcome of the measurements and assessments

of the state of the environment, as well as the analysis focusing on the impact of various

anthropogenic factors. It is addressed to society, to government and local government

administrations as well as to international institutions.

Environmental monitoring techniques evaluate contaminants and the rate at which they

will dissipate. Monitoring measures physical or chemical properties of the media suspected of

contamination (soil, water, gases, tissues). Over time, environmental monitoring establishes

a trend from multiple measurements of the same parameter. The monitoring systems include:

air quality, water quality, soil and land quality, nature, noise, electromagnetic field and

ionizing radiation monitoring subsystems.

The data obtained by common chemical and physicochemical methods becomes a source

of information about the state of specific environmental compartments and the processes

within. However, chemical analysis techniques usually can be used under laboratory

conditions only and that introduces additional delay between sampling and sample analysis.

Moreover, they do not evaluate the effect of interaction among toxic substances, such as

synergism or antagonism. For these reasons, in recent years, there has been intense

development of bioanalytical techniques that employ live organisms as indicators

(bioindicators). Ecotoxicological tests (biotests) are commonly used as one of the tools in

integrated monitoring of the environment. In this case, the environmental data and the

information on the impact of pollutants on live organisms are obtained from environmental

samples which have been analyzed with both chemical techniques and biotests.

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1. CHEMICAL ANALYSIS OF THE ENVIRONMENTAL STATE

1.1. Components of environment

The two major classifications of environment are:

Physical environment - external physical factors like air, water, and land; this is also

called the Abiotic Environment,

Living environment - all living organisms around us (plants, animals, and

microorganisms); this is also called the Biotic Environment.

Earths environment can be further subdivided into the following four segments:

Lithosphere

Hydrosphere

Atmosphere

Biosphere.

The names of the four spheres are derived from the Greek words for stone (litho), air

(atmo), water (hydro), and life (bio). The last sphere is the region occupied by living

organisms such as plants, animals, fungi. They are temporary accumulators (e.g. lead) and

sources of pollutants (natural forest burning) in a very complex set of relationships with the

atmosphere, hydrosphere and lithosphere (Fig.1).

Fig. 1. Relationships between various components of environment

Environmental monitoring can be conducted on biotic and abiotic components of any of

these spheres, and can be helpful in detecting baseline patterns and patterns of change in the

inter- and intra-process relationships between and within these spheres. The sampling of air,

water, and soil through environmental monitoring can produce data that can be used to

understand the state and composition of the environment and its processes.

The atmosphere is a mixture of nitrogen (78%), oxygen (21%), and traces (remaining 1%)

of carbon dioxide, argon, water vapor and other components. Although the atmosphere is

approximately 1,100 km high, the stratosphere (10 to 50 km) and the troposphere (less than

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10 km) are the main atmospheric interactors of the biosphere. The atmosphere is a prime

mean for the spatial diffusion of pollutants and a temporary mean of their accumulation until

they precipitate. A number of sources produce these chemical compounds but the major man-

made source is the burning of fossil fuel. Indoor air pollution is caused by cigarette smoking,

the use of certain construction materials, cleaning products, and home furnishings. Outdoor

gaseous pollutants come from volcanoes, fires, and industry, and in some areas may be

substantial.

Particulate matter (PM) is present in the atmosphere as both a primary and a secondary air

pollutant. Primary PM is released into the atmosphere directly from a source, such as ash in

the flue gas emitted from a coal-fired furnace. Particle pollution is made up of a number of

components, including acids (such as nitrates and sulfates), organic chemicals, metals, and

soil or dust particles. Secondary PM is produced in the atmosphere in the form of ammonium

nitrate and ammonium sulfate. Most of the secondary PM is the respirable fraction known as

PM 2.5 which is very small particulate matter having a size of 2.5 m or less. The size of

particles is directly linked to their potential for causing health problems. Particles that are

10 micrometers in diameter (PM 10) or smaller generally pass through the throat and nose and

enter the lungs. Once inhaled, these particles can affect the heart and lungs and cause serious

health effects.

Radon and environmental tobacco smoke (ETS) are the two indoor air pollutants of

greatest concern from a health perspective. Radon is a naturally occurring gas that is odorless,

colorless, and radioactive. Environmental tobacco smoke (ETS) is the smoke emitted from the

burning of a cigarette, pipe, or cigar, and smoke inhaled by a smoker. It is a complex mix of

more than 4,000 chemical compounds, containing many known or suspected carcinogens and

toxic agents, including particles, carbon monoxide, and formaldehyde.

The hydrosphere is the accumulation of water in all its states (solid, liquid and gas) and

the elements dissolved it in (sodium, magnesium, calcium, chloride and sulphate). Water

covers around 71% of the earth's surface (Fig. 2) and is an important accumulator of

pollutants and a significant vector of diffusion.

There are many specific causes of water pollution, but it is more important to understand

the two broad categories of water pollution point and nonpoint sources. An example of

a point source of water pollution is a pipe from an industrial facility discharging effluent

directly into a river, whereas a nonpoint-source of water pollution is when fertilizer from

a farm field is carried into a stream by rain (i.e. run-off). Nonpoint sources are much more

difficult to monitor and control, and today they account for the majority of contaminants in

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streams and lakes.

Fig. 2. Distribution of global water [http://ygraph.com/chart/2410]

Water pollution takes many forms:

oxygen depletion - caused by the release of biodegradable matter into the water and the

natural process of breaking this down uses the oxygen in the water;

nutrients - such as phosphorus and nitrogen are essential to plant growth, too many

nutrients in the water encourage the growth of weeds and algae (algae bloom),

chemical - adding unwanted chemicals to the water is done through the accidental

spillage of substances into water (for example oil when an oil tank ruptures or a ship

sinks), waste from factories or industry, and through pesticides running off fields into

water; chemicals in water are poisonous and harmful to wildlife as well as making the

water too polluted to drink,

suspended matter - the tiny particles of matter (not soluble in water) stay in the water

and eventually fall to the bottom, causing long term problems due to an imbalance in

the natural infrastructure of the water,

biological - some viruses and bacteria are waterborne and they can cause serious

diseases in people in direct contact with this contaminated water.

The lithosphere is the thin crust between the mantle and the atmosphere, and it contains

rocks, minerals, and soils. Soil is a complex mixture of eroded rock, mineral nutrients,

decaying organic matter, water, air, and billions of living organisms (Fig. 3). Main chemical

soil constituents are oxygen (47%), silicon (28%), aluminum (8%), iron (5%), calcium (4%),

sodium (3%), potassium (3%) and magnesium (2%) in a crystalline state.

Fig. 3. Soils main components [http://www.physicalgeography.net/fundamentals/10t.html]

Soils support a number of inorganic and organic chemical reactions. Many of these

reactions are dependent on some particular soil chemical properties. One of the most

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important chemical properties influencing reactions in a soil is pH. Soil pH is primarily

controlled by the concentration of free hydrogen ions in the soil matrix. Soil fertility is

directly influenced by pH through the solubility of many nutrients. At a pH lower than 5.5,

many nutrients become very soluble and are readily leached from the soil profile whereas at

high pH, nutrients become insoluble and unavailable for plants. Maximum soil fertility occurs

in the range 6.0 to 7.2.

Soil pollution is the result of toxic chemicals, solvents, salts, microorganisms and other

harmful substances introduced to the top layers of soil as a result of dumping garbage, waste,

and other toxins. Manmade land pollution comes as accidental disasters, brownfields, waste

management and landfills, pesticides and agricultural practices, clear cutting, urban

development and energy production. Soil pollution decreases the fertility of the soil by

reducing its mineral content, making it difficult for plants to survive or thrive. Erosion, or loss

of fertile topsoil is another problem that many areas must face due to deforestation, which is

the cutting down of trees for urban and agricultural purposes.

Soil contamination affects an ecosystems equilibrium, and whenever any imbalances

occur, the ecosystem restores itself through biogeochemical cycles to the equilibrium state;

this may take a few days or many years. Elements carried through the biogeochemical cycles

are stored in their natural reservoirs, and are released to organisms in small consumable

amounts. For example, through the nitrogen cycle and with the help of the nitrogen fixing

bacteria, green plants are able to utilize nitrogen in bits even though it is abundant in the

atmosphere. Since the biogeochemical cycles pass through different spheres, the flow of

elements is regulated because each sphere has a particular medium and the rate at which

elements flow is determined by the viscosity and density of the medium.

1.2. The most dangerous environmental pollutants

1.2.1. Gaseous pollutants

The most common gaseous pollutants are: carbon dioxide and monoxide (CO and CO2),

hydrocarbons (PAHs, CFC), nitrogen oxides (NOx), sulfur oxides (SO2). They belong to

primary air pollutants (Fig. 4) and are directly emitted from an emission source. Secondary air

pollutants are those that are formed by reactions between the primary air pollutants and

normal atmospheric constituents, sometimes by utilizing sunlight energy (sulfuric acid, nitric

acid, nitrogen dioxide, ozone (O3), formaldehyde, peroxyacetyl nitrate (PAN), ammonium

nitrate and ammonium sulfate).

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Fig. 4. Primary and secondary air pollutants

[http://science.kennesaw.edu/~jdirnber/scienceII/OutlineAir/OutlineAirNotes.html]

There are several main types of air pollution and well-known effects of pollution which

are commonly discussed. These include smog, acid rain (Fig. 5), the greenhouse effect, and

"holes" in the ozone layer. Each of these problems has serious implications for our health and

well-being as well as for the whole environment.

Fig. 5. The effect of gaseous pollutants on the environment [PhysicalGeography.net]

1.2.2. Inorganic pollutants

Inorganic chemical pollutants are naturally found in the environment but due to human

development, these pollutants are often concentrated and released into the environment. The

primary inorganic pollutants are mainly divided into two categories: heavy metals and

nutrients. The list below highlights just a few of the inorganic pollutants:

Arsenic (as Arsenite)

Lead

Copper

Chlorine

Cyanide

Nitrate

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Cadmium

Mercury

Chromium

Ammonia

Phosphate

Inorganic pollutants - such as hydrochloric acid, sodium chloride, and sodium carbonate -

change the acidity, salinity, or alkalinity of the water, making it undrinkable or unsuitable for

the support of animal and plant life. These effects can result in dire consequences for higher

mammals and humans.

Heavy metals

Among the many heavy metals released from various products and processes, cadmium,

lead and mercury are of great concern to human health because of their toxicity and their

potential to cause harmful effects at low concentrations and to bioaccumulate. Living

organisms possess diverse sensitivity to heavy metals (Tab.1).

Table 1. Sensitivity of living organisms to heavy metals [Rajmer, 1997]

Metal Plant Animal Human

Cd Cu Hg Ni Pb Zn

Sensitivity low mean high

Significant progress has been made in reducing emissions to air of these metals in Europe,

with 1995 emissions being about 50 % of 1990 levels and decreasing further to 40 % by 1999.

Although controlling diffuse emissions of cadmium and mercury remains problematic (e.g.

batteries), point source emissions of these metals have declined as a result of improvements in

sectors such as wastewater treatment, incinerators and the metals sector. Factors contributing

to this include large decreases of lead emissions from the transport sector following the

introduction of unleaded petrol in the early 1990s, continuing move away from the use of

lignite in the eastern European energy sector, and the introduction of improved pollution

abatement technologies across a range of industrial and waste treatment sectors.

Cadmium is often discussed in relation with food safety issues. It readily accumulates in

crops, especially in acidic soils with low binding capacity. Exposure risk due to soil ingestion

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is less critical, although recent investigations indicate that exposure through indoor dust (soil

related) is as high as exposure due to consumption of homegrown vegetables. In slightly

acidic soils with low binding capacity, there is a high risk of leaching to the subsoil and the

groundwater. In living organisms, cadmium affects renal, pulmonary, skeletal, testicular and

nervous systems, and disrupts zinc-dependent enzymes.

Although emissions from lead to soil are decreasing, it is still a substance of concern

given the amounts of lead stored especially in organic layers of topsoils. Critical levels in soil

(based on total concentrations) for long-term exposure are often exceeded in urban areas,

although bioavailability may be low. Lead inhibits hemoglobin synthesis (anemia); substitutes

for calcium, reducing cellular functions (e.g. ATP production); is stored in bones; and

moreover, organic lead compounds affect brain function and may cause lung cancer.

Zinc is an essential element for life but it is also very toxic for micro-organisms leading to

a decreased decomposition of organic matter. Ecological risk assessment is difficult for this

element since generic safe levels derived from toxicity tests are below the levels to sustain life

in less sensitive organisms and even lower than background levels. Experience with the

assessment of ecological impacts may improve if zinc is monitored in soil in conjunction with

biodiversity indicators and assessments of soil decomposition processes. Also, zinc

deficiencies related to crop production could be identified.

Mercury is very toxic, mainly accumulates in soils and sediments, but can be transformed

into mobile fractions (e.g. methyl mercury). Hg is prioritized in many environmental

programs at EU level and also national level. Unfortunately, it is difficult and expensive to

measure. Mercury vapors and organomercury enter the central nervous system, affecting the

brain and nerve cells, causing sensory disturbance, reduced field vision and ataxia, and

impairing speech, hearing and mental functions.

1.2.3. Organic environmental pollutants

A list of organic pollutants includes:

hydrocarbons (aromatic, PAHs),

halogenated hydrocarbons (PCBs, dioxins, CFC),

pesticides (insecticides, herbicides, fungicides, rodenticides).

All of these substances are highly lethal to animals, and many can be readily absorbed

through the skin. These contaminants are the most differentiated in their chemical structure

(Fig. 6).

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Fig. 6. Different structures of organic pollutants [de Mello- Farias et al, 2011; DOI: 10.5772/24355]

The majority of organic pollutants belong to the group of Persistent Organic Pollutants

(POPs). They persist in the environment, bioaccumulate through the food web, and pose a risk

of causing adverse effects to human health and the environment (PAHs, PCBs, dioxins,

DDT). With the evidence of long-range transport of these substances to regions where they

have never been used or produced and the consequent threats they pose to the environment of

the whole globe, the international community has now, at several occasions, called for urgent

global actions to reduce and eliminate releases of these chemicals.

Polycyclic aromatic hydrocarbons (PAHs)

PAHs (which are known for their strong toxic properties) are composed of carbon and

hydrogen atoms arranged in the form of fused benzene rings (linear, cluster or angular

arrangement). This group contains a number of known carcinogens; the most dangerous one

among them is benzo(a)pirene - BaP (Fig. 6). The widespread occurrence of PAHs is largely

due to their formation and release in all processes of incomplete combustion of organic

materials. The last century of industrial development caused a significant increase of PAH

concentrations in the natural environment. Research shows that air contributes 3-20% of total

human exposure to PAHs and comes in second position (after food) as a source of these

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pollutants for humans. Cigarette smoke can significantly contribute to potential PAH doses

via inhalation (over 50% of total exposure). The ambient standards regarding exposure to

PAHs refer usually to BaP; in Poland, its permissible concentration in the air as a daily

exposure is 5 ng/m3 and as a year exposure - 1 ng/m3.

Polychlorinated biphenyls (PCBs)

It is a large family (209) of compounds (Fig. 6) that are very stable and that are used as

closed system and heat transfer fluids (transformers, capacitors, fluorescent light ballasts,

etc.), as plasticizers, as hydraulic fluids and lubricants and as form base for pesticides. PCBs

are persistent and show significant bioaccumulation and low acute toxicity. They strongly

accumulate in the food chain and significant levels of them have been found in marine

species, particularly mammals (even 5-10 mg/kg) and sea birds. They are carcinogenic and

capable of damaging the liver, the nervous system and the reproductive system in adults.

Daily intake of PCB with food amounts to 5-100 g in developed countries. When PCBs are

burned, even more toxic dioxins are formed, so they are called dioxin-like compounds.

Dioxins

It is a class of super-toxic chemicals formed as a by-product of the manufacture,

moulding, or burning of organic chemicals and plastics that contain chlorine; they have been

never produced. This group includes polychlorinated dibenzo-p-dioxin - PCDD (Fig. 6) and

polychlorinated dibenzofuran (PCDF). The most known and toxic is 2,3,7,8-

tetrachlorodibenzo-p-dioxin (TCDD). They are virtually indestructible (they are stable at

1000oC in oxygen conditions and destroyed by UV light only), cumulated in fatty tissues and

excreted by the body extremely slowly. They cause serious health effects even at levels as low

as a few parts per trillion (10-14). They bind to cell receptors and disrupt hormone functions

in the body and they also affect gene functions (named as Endocrine disruptors, EDs).

Because dioxins refer to such a broad class of compounds that vary widely in toxicity, the

concept of toxic equivalence (TEQ) has been developed to facilitate risk assessment and

regulatory control. The uncertainty and variability in the dose-response relationship of dioxins

in terms of their toxicity, as well as the ability of dioxins to bioaccumulate mean that the

tolerable daily intake (TDI) of dioxins has been set very low by the WHO to 10 pg/kg body

weight per day.

Pesticides

Pesticide contamination is a particular problem in rural agricultural areas where pesticide

use is heavy and drinking water supplies come directly from groundwater or surface water.

Pesticides can migrate via water into the food chain as well, ultimately being consumed by

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humans or animals in food.

Pesticides can be divided according chemical structure as:

Organochlorines - DDT, HCH, Methoxychlor, Aldrin, Endosulfan,

Organophosphates - Malathion, Parathion, Diazinon, Chlorpyrifos, Glyphosate (Roundup),

Carbamate Esters - Carbofuran, Aldicarb,

Phenoxy Esters - 2,4-D, Silvex, 2,4,5-T.

Some of these pesticides will persist for long periods of time in the environment. DDT

was a pesticide (with a half-life of over 3-10 years) that was in use for a long time around the

world (and is still being used in parts of the world for mosquito control), but has been banned

in the US since 1972. Yet we still find DDT in our environment, sometimes at very high

levels. Pesticide in soils undergo degradation and its transformation products (TPs) can be

present at higher levels in the soil than the parent pesticide itself. Generally, pesticide TPs

could show lower toxicity to biota than the parent compounds. However, sometimes TPs are

more toxic, so they represent a greater risk to the environment than the parent molecules. In

terms of general human health effects, pesticides can affect and damage the nervous system,

cause liver damage, damage DNA and cause a variety of cancers, cause reproductive and

endocrine damage and cause other acutely toxic or chronic effects. There is a pressing need

for pesticides determination in food and environment, but pesticide analysis is a very complex

problem, including proper sample preparation, selective and sensitive chromatographic

detection and determination of pesticide residues, and the application of biological

(immunoassays-and biosensors-based) methods.

1.3. Measurement of chosen pollutants in water and air

1.3.1. Evaluation of water contaminants using VISOCOLOR colorimetric test kits

The visual colorimetric analytical systems VISOCOLOR

and VISOCOLOR ECO

(MACHEREY-NAGEL GmbH & Co.KG, Germany) have been successfully applied in water

analysis for many years. During assessment, the determined substance, which is not visible or

cannot be measured directly, is converted into a coloured compound by addition of a suitable

reagent. If it is done according to instructions, the resulting colour intensity is proportional to

the concentration of the determined substance. For evaluation of the VISOCOLOR test kits a

so-called comparator is used, whereas VISOCOLOR

ECO tests are evaluated with a colour

scale. For determination of more precise values, the same analytical preparation is measured

with the filter photometer PF-11 (or PF-12) (MACHEREY-NAGEL). In particular,

NANOCOLOR

reagent sets are ready-to-use reagent preparations of high precision and

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selectivity for photometric analysis.

The most important advantages of these kits are: economic pricing, convenient handling,

higher accuracy and sensitivity, pictographic instructions, reagent bottles with clear dosing

instructions, availability of refill packs, visual evaluation.

The set of VISOCOLOR ECO test kits include:

Aluminium Copper Manganese Silica Ammonium

Cyanide Nickel Sulphate Calcium Cyanuric acid

Nitrate Sulphide Carbonate hardness DEHA

Nitrite Sulphite Chloride Fluoride

Oxygen Swimming pool Chlorine Hardness (total)

pH Zinc Chlorine dioxide Hydrazine

Phosphate Chromium (VI) Iron Potassium

[http://www.mn-net.com/tabid/4648/Default.aspx]

Procedure for Visocolor test NITRATE 50

In a weakly acidic medium, nitrate ions are reduced to nitrite ions, which react with sulphanile

acid and l- naphthylamine to form red azo-dye. Nitrites disturb sampling (the same reaction);

therefore, when they are found in water, they should be removed using amidosulphonic acid.

Range: 1 to 40 mg NO3/l (0.2-9.0 mg/l NO3-N)

Filter:4 Reaction time: 5 min

Assay:

1. Rinse test tube 14mm ID several times with sample and fill up to the ring mark (10 ml)

2. Add 10 drops Nitrate-1 and shake test tube

3. Add 1 small, level measuring spoon Nitrate-2, close test tube and shake vigorously for

15-30s, wait 5 min.

4. Using PF-11 photometer perform measurement.

General procedure for VISOCOLOR tests

switch on photometer

with key M call up VISOCOLOR

with key rounded ARROW call up the test NITRATE 50 with range NO3,

press key NULL ZERO

adjust filter wheel to 4, place clean round glass cell with blank value (untreated sample) into the photometer and press

key NULL ZERO

after 2s, place clean round glass cell with sample solution into the photometer, press key M and read the result in mg/l NO3

1.3.2. Determination of nitrates in water by spectrophotometric method

(according to PN-82/C-04576/08)

Nitrates (V) and (III), i.e. nitrates and nitrites are the most frequent chemical pollution of

water, and they seriously affect human health. The presence of nitrates in water is the

consequence of the use of mineral fertilizers in agriculture, sewage draining to waters, but

also natural nitrification processes. Nitrates (NO3-) are not dangerous to people; however, they

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can transform into much worse nitrites (NO2-) in acid environment. Nitrites are precursors of

carcinogenic nitrosamines. Nitrates are especially dangerous for young infants because they

cause methaemoglobinaemia, and in adults indirectly lead to an increase in the incidence of

stomach and intestines cancer.

There are three main diet sources of nitrates (V): crops and vegetables (up to 80%), meat

products (10%) and drinking water (10%). Various plants have different nitrates accumulation

capacity, and the biggest amount of nitrates is assimilated by beet root (150-5960 mg

NO3/kg), lettuce (382-3520 mg NO3/kg), and radish (261- 1186 mg NO3/kg). Nitrates are

used in the food processing industry as effective preservatives preventing bacteria

colonization. They positively influence qualitative features of a product (preservation of pink

colour of meat products, flavouring in pickled meat). Surface waters usually comprise a small

amount of nitrates (0-1 mg N-NO3), while underground waters may contain a significant

amount of nitrates depending on soil pollution and geological conditions. The highest

concentration of nitrates in drinking water is found in shallow domestic wells located in rural

areas and on the outskirts of towns, depending on the country region, from 30 to 85% of these

water intakes contain water with an excessive amount of nitrates (the acceptable levels in

Poland are 50 mg/l NO3 and 0.1 mg/l NO2).

ASSAY PROCEDURE

The complex, formed by nitration of salicylic acid under highly acidic conditions, absorbs

maximally at 410 nm in basic (pH>12) solutions. Absorbance of the chromophore is directly

proportional to the amount of nitrate-N present. Ammonium, nitrite and chloride ions do not

interfere.

Reagents

0.5% salicylic acid

0.5% NaOH

conc. H2SO4 alkaline potassium-sodium tartrate solution

Standards

Stock solution 0.7216 g/l KNO3

Working stock solution: 0.0434 mg NO3 per mL

1. 10 mL of stock solution with 2-3 drops of NaOH solution and 20 mL of salicylate are

evaporated in an evaporating dish using boiling water bath.

2. After cooling down, add 1 mL of concentrated H2SO4, so in that way the entire residue is

dehumidified and allowed to stand for 10 minutes.

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3. Add 30 mL of distilled (DI) water and quantitatively transfer it to a 100 mL volumetric

flask and make up to 100 mL with DI water.

Calibration curve

1. To a series of test volumetric flask (50 mL), pipette carefully 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0

and 5.0 mL of working stock solution.

2. Add 7 mL of potassium-sodium tartrate to each flask and make up to 50 mL with DI

water. Mix thoroughly.

3. Measure the absorbance at 436 nm against a blank prepared in the same way (without

nitrates) using EPOLL 20 photometer.

4. Prepare a calibration curve by plotting the corrected absorbance (y axis) vs. milligrams of

NO3 (x axis). Determine the slope and intercept for the calibration curve.

Water sample assay

1. 20 mL of water sample with 2-3 drops of NaOH solution and 1 mL of salicylate are

evaporated in an evaporating dish using boiling water bath.

2. After cooling down, add 1 mL of concentrated H2SO4, so in that way the entire residue is

dehumidified and allowed to stand for 10 minutes.

3. Add 20 mL of distilled water and 7 mL of potassium-sodium tartrate and quantitatively

transfer it to a 50 mL volumetric flask, make up to 50 mL with DI water and mix

thoroughly.

4. Measure absorbance at 436 nm.

Calculations

The nitrate content be determined from a standard curve and converted into 1 liter of water

(x50). Compare result to the Polish acceptable level of nitrates in drinking water is

50 mg/dm3.

1.3.3. Determination of formaldehyde migration from wood panels

Formaldehyde (HCHO) is an important pollutant of indoor air, and because of its

chemical and toxic characteristics an individualized evaluation is recommended. Everyone is

exposed to small amounts of formaldehyde in the air, some foods, and products, including

composite wood products. Release from formaldehyde-based resins in which it is present as

a residue and/or through their hydrolysis and decomposition by heat (e.g. during the

manufacture of wood products, textiles, synthetic vitreous insulation products, and plastics).

In general, the use of phenol-formaldehyde resins results in much lower emissions of

formaldehyde than those of urea- based resins. It enters the atmosphere also as a result of the

combustion of any fuel energy, fuel propellants waste. It is also a component of tobacco

smoke - a 1 m3 of smoke is about 40 cm

3 of formaldehyde.

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Formaldehyde is used widely by various industries to manufacture a range of building

materials and numerous household products. It is contained in resins used to manufacture

some composite wood products (e.g. hardwood plywood, particleboard and medium-density

fiberboard). It is found (as an ingredient or impurity) in some cosmetics/personal hygiene

products, such as some soaps, shampoos, hair preparations, deodorants, sunscreens, dry skin

lotions, and mouthwashes, mascara and other types of products, such as eye makeup, nail

creams, vaginal deodorants, and shaving creams.

At room temperature, formaldehyde is a colorless, flammable gas that has a distinct, pungent

smell. Formaldehyde is commonly produced as an aqueous solution called formalin, which

usually contains about 37% formaldehyde and 12-15% methanol. Methanol is added to

formalin to slow polymerization that eventually leads to precipitation as paraformaldehyde.

Due to its capability for protein denaturation, formaldehyde is the most commonly used

fixative. It serves to stabilize the fine structural details of cells and tissue in biological

examinations.

The primary way of exposure is by breathing air containing it. Formaldehyde can cause

irritation of the skin, eyes, nose, and throat. High levels of exposure may cause some types of

cancers. The maximum concentration of formaldehyde NDS = 0,5 mg/m3.

There are different methods for detecting formaldehyde in air, all of them based on taking

samples for later analysis, with active fixing or by diffusion. The traditional methods are

based on obtaining a sample by bubbling air through distilled water or a solution of 1%

sodium bisulphate at 5C, and then analysing it with spectrofluorometric methods. While the

sample is stored, it should also be kept at 5C. SO2 and the components of tobacco smoke can

create interference.

ASSAY PROCEDURE

In the monitor, formaldehyde vapors are absorbed on bisulfite impregnated paper and

desorbed with formaldehyde-free distilled water. Aliquots are reacted with chromotropic acid

in the presence of sulfuric acid to form a purple monocationic chromogen. The absorbance of

the colored solution is read in a spectrophotometer at 580 nanometers (nm) and is

proportional to the amount of formaldehyde in the solution. In this experiment, the chipboard

shavings are extracted with water within 1 hour.

Reagents

1% chromotropic acid solution: dissolve 0.5 g of 4,5-dihydroxy-2,7-naphthalenedisulfonic

acid disodium salt dihydrate in 50 mL of distilled water. Make a fresh solution each day.

Stock formaldehyde solution - 25 L of 37% formaldehyde is diluted to 100 mL with distilled

19

water and mix thoroughly.

Working formaldehyde solution: Transfer a 5 mL aliquot of the stock solution to a 50 mL

volumetric flash and make up to 50 mL with DI water. This solution is used to prepare the

calibration curve each milliliter is equivalent to 10 micrograms of formaldehyde.

Calibration curve

1. To a series of test tubes, carefully add 0.0, 0.1, 0.2, 0.3, 0.5, 0.7, 0.8, and 1.0 mL of

working stock solution of formaldehyde.

2. Adjust the volumes to 1 mL with distilled water.

3. Develop the color: add 0.2 mL of chromotropic acid solution to each sample and mix

well. After 3 min., carefully add 5 mL of concentrated sulfuric acid slowly while mixing

(use a glass stick).

CAUTION: Take proper safety precautions such as goggles, gloves and apron when

handling concentrated sulfuric acid.

4. Allow the samples to cool to room temperature (for 20 min) and measure the absorbance

at 585 nm using EPOLL 20 photometer against reagent blank (without formaldehyde).

5. Prepare a calibration curve by plotting the corrected absorbance (y axis) vs. micrograms

of formaldehyde (x axis).

6. Determine the slope and intercept for the calibration curve.

Sample analysis

Particle board shavings are leached with water for 24 hours (the ratio 5 g per 250 mL of

distilled water). Before analysis, filtrate the extract and prepare four dilutions of extract: 10-,

20-, 50- and 100- fold. Transfer a 1 mL aliquot of each dilution to a test tube for color

development exactly as described above.

Calculations

The formaldehyde content should be determined from a standard curve and converted into

1 kg of shavings. Assuming that all of the formaldehyde emitted by 1 kg of chipboard goes

into the room with a capacity of 30 cubic meters, calculate the concentration of formaldehyde

in the room air as mg/m3. Compare the result to the exposure limit in the air which amounts to

0.5 mg/m3.

20

2. ENVIRONMENTAL ANALYTICS

Analytics is a typical example of a scientific discipline that uses achievements both in the

scope of basic research, as well as those used in a variety of other disciplines. Said disciplines

include:

various fields of chemistry (especially physical chemistry and biochemistry),

physics,

computer science,

electronics, automatics and robotics,

materials engineering,

biology,

instrumentation (science about building and use of measuring and control instruments),

chemometrics.

Whereas in environmental analytics, the most often used methods are those known to

analytical chemistry, mainly:

Spectroscopy analytical methods, particularly:

Molecular spectroscopy, including spectrophotometry UV-VIS and IR,

Atom spectrometry, including atomic absorption spectrometry AAS and atomic

emission spectrometry AES,

Mass spectrometry,

Electrochemical analytical methods,

Chromatographic analytical methods.

Many pollutants of anthropogenic origin are organic compounds. In analytics of this type

of pollutants, the most widely used methods are chromatographic methods.

Exotoxicological concerns and the desire to reduce description thoroughness of the state

of the environment constitute great challenges for analysts in terms of the necessity to assay

concentration of a wide variety of analytes in samples with a complex (and sometimes also

variable) matrix composition. Two approaches can be distinguished for assay of analytes

present in test samples at low content levels:

1. Utilization of more sensitive, selective or even specific detectors for chromatography.

2. Introduction of an additional stage to the analytic procedure: isolation and/or

enrichment of analytes before the final assay stage. The additional stage ensures:

simplification of the matrix (as a result of transferring analytes from the sample to

a proper solvent or gas feed stream) and removal of at least some of the interfering

substances (interferents) from the test sample before the final assay stage. This also

21

ensures an increase of analyte concentration in the sample to a higher level than the

detection limit of the method and/or the instrument being used.

2.1. Use of chromatographic methods in pesticide monitoring

2.1.1. Basics of GC chromatography

Chromatography is currently one of the most widespread instrumental methods in

chemical analysis, mainly because of its capability to detect the analyzed substance and assay

its quantity even at very low concentration and in presence of many other substances.

Moreover, chromatography as an analytical method, when combined with other analytical

techniques (e.g. mass spectrometry), allows to solve most analytical problems. It is a method

of separating homogenous mixtures in which the separated components undergo partitioning

between two phases: one of them is the stationary phase, the other - mobile phase (Fig. 7).

Fig. 7. Mechanism of the chromatographic process. Substances marked as triangles and circles are

characterized by varied retention factors k

The stationary phase can be a solid, a liquid on a carrier, or a gel; and the mobile phase

a gas or a liquid. If the mobile phase is a gas, then this type of chromatography is called gas

chromatography (GC), whereas if the mobile phase is a liquid, then it is called liquid

chromatography (LC).

If the stationary phase in gas chromatography is:

a solid (adsorbent), then we are dealing with adsorption chromatography,

a liquid that resides on a solid carrier in the form of a homogenous layer, then we are

dealing with partition chromatography.

The mobile phase moves inside the column, whereas the stationary phase resides on the

columns inner walls. Chemical compounds with larger affinity for stationary phase are

selectively stopped by it and move much more slowly along the column. Chemical

compounds with smaller affinity for stationary phase move much faster along the column and,

22

as a result, they leave the column, i.e. the eluate from the column, in the first instance.

Separation balance between the phases is of a dynamic character, i.e. substance particles

move continuously from mobile phase to stationary phase and back.

The phenomena determining the separation process are diverse in character; even so, some

of the most often used processes include adsorption and separation of substances between two

non-mixing liquids. Due to the nature of the phenomena occurring during chromatographic

separation, chromatographic methods can be classified into adsorption chromatography or

separation chromatography. In the first case, separation of the mixture is conditioned by

a variable adsorption affinity of mixture components toward the surface of the stationary

phase called the adsorbent. In the case of separation chromatography, separation of mixture

components is based on the differences in separation factor values of each of the mixture

components between two non-mixing phases. One of the most often used chromatographic

methods in environmental monitoring includes gas chromatography and high-performance

liquid chromatography.

In the case of gas chromatography, the liquid sample evaporates in the dispenser and,

through the carrier gas stream, it is fed into the column where sample components are

separated and are then moved to the detector where they generate an electrical charge.

Voltage signals are read with a multimeter and the data is sent to a PC computer. Using the

right software, it is possible to download the data from a measure card.

Whereas in the case of high-performance liquid chromatography, the pump from the tank

(or tanks) sucks in the mobile phase and pumps it through a dispenser into the

chromatographic column. The column is sometimes placed in a thermostat. The sample is

injected by a injector through the top of the chromatographic column, after that mixture

components undergo separation in the column and, upon exiting, are detected by the detector.

The detectors electric signal, after boosting, is saved on recording paper or recorded with an

integrator or a computer in the form of a chromatographic peak. Flow of liquid through the

system can be controlled with a manometer and a flow meter. With certain apparatus, it is

possible to collect the separated components in a fraction collector.

GC-MS method

Gas chromatography is one of the few chromatographic methods that allows for analysis of

a complex mixture and performing of quantitative assay of its components as part of a single

process. In this way, it is possible to separate and identify mixtures composed of more than

a few hundred components. However, this chromatographic method can only separate those

23

substances that, during chromatographic conditions, are in gaseous or vapor form. Therefore,

they must be gaseous, liquid or solid substances whose boiling point or sublimation point

does not exceed 400C. Despite the fact that gas chromatography is the ideal method for

substance separation, it does not provide clear information about the consistence of the

mixture being separated. The perfect solution to this problem turned out to be combining gas

chromatography with other instrumental analysis techniques. The widest use was found when

linking gas chromatography with mass spectrometry (GC-MS). This combination is of

particular significance in environmental analysis because mass spectrometry (MS) is one of

the most efficient methods of qualitative and structural analysis of organic compounds. In this

method, the gas chromatographer separates mixture components and inserts into the mass

spectrometer pure compounds of the right volatility and with a speed adjusted to the speed of

processes taking place inside the mass spectrometer (Fig. 8). In gas chromatography, the two

most often used column types are: packed columns and capillary columns. Capillary columns

achieve considerably higher separation efficiency than packed columns. Gas chromatography

uses such adsorbents as: carbon adsorbents, silica gel, molecular sieves and porous polymers.

Among inorganic adsorbents the most widely used are molecular sieves, and among organic

ones - porous polymers.

Fig. 8. Basic elements of the GC-MS system

Detectors used in GC chromatography

Substances separated in the chromatographic column are detected by the detector upon their

exit from the column. The detector, in response to the presence of the analyzed substance in

carrier gas, reacts by generating an electrical signal. A good detector should possess the

following traits:

good sensitivity,

good detection of analyzed substances,

wide linearity range of indication,

stability of indications and low noise level,

24

selectivity of indications at low cost.

In gas chromatographs, there are many various types of detectors. The most important

ones include:

Thermal conductivity detector (TCD),

Flame ionization detector (FID),

Flame photometric detector (FPD),

Electron capture detector (ECD),

Mass spectrometer (MS).

The principle of operation of the most important ones in environmental analysis has been

discussed below.

Flame Ionization Detector (FID)

Principle of operation: Compounds are burned in a hydrogen and air flame. Compounds

containing carbon produce ions that are collected on the collector (collector electrode). The

number of ions collected by the electrode is recorded and in this way the signal is produced.

Selectivity: Compounds containing the C-H bond. Weak response to organic compounds

lacking hydrogen (e.g. hexachlorobenzene).

Sensitivity 0.1 10 ng

Linear range: 105 107

Gases: For combustion - hydrogen and air; additional gas (make-up) - helium or nitrogen

Temperature: 250-300C; 400-450C for analyses conducted at a higher temperature.

FID is a destructive, mass detector. The number of ions produced in the flame is countable.

Ions generate the detectors signal. Analytes, which exhibit the largest number of carbons at

low oxidization level, generate the strongest signal.

Fig. 9. FID detector scheme

Electron Capture Detector (ECD)

Principle of operation: Electrons are delivered from the radioactive 63

Ni placed in the

detector cell, in which the electricity is being generated. Electronegative compounds capture

electrons, causing reduction of electricity. Reduction of electricity, when measured indirectly,

is background signal.

25

Selectivity: chlorides, nitrates and conjugated carbonyl groups.

Sensitivity: 0.1-10 pg (chlorinated compounds), 1-100 pg (nitrates); 0.1-2 ng (carbonyl compounds)

Balance range: 103-10

4

Gases: Nitrogen or argon / methane

Fig. 10. ECD detector design scheme

Mass Spectrometer (MS)

Principle of operation: Vacuum is present in the detector. Compounds are bombarded with

gas electrons (EI) or particles (CI) and then fragmented into ions with characteristic charges.

Produced ions are focused and accelerated in the mass filter. Mass filter selectiveness lets

through all ions of specific mass into an electron multiplier. All ions of specific mass are

detected. Next, the mass filter lets through the next mass that distinguishes itself from the

mass of other ions.

The mass filter gradually searches through the specified mass range a few times a second.

Each time the total number of ions is calculated. With each search, the intensity or number of

ions is graphed as function of time on the chromatogram (called Total Ion Chromatograph -

TIC). Each scanning provides a mass spectrum that shows various masses of ions as the

function of their intensity or number.

Selectiveness: All compounds providing fragments in the selected mass range. It is possible to

use full search - scan or only selected ion monitoring (SIM).

Sensitivity: 1-10 ng (full search); 1 - 10 pg (selected ion monitoring, SIM)

Linear range: 105-10

6

Gases: none

Temperature: 250-300C (transfer line); 150-250C (source)

Fig. 11. MS detector scheme

26

Chromatogram analysis

The effect of chromatographic separation is graphed in the form of an elution

(chromatogram) curve. The chromatogram presents a graph of dependence of detector

indications on the time or volume of mobile phase.

The chromatogram provides the following information:

qualitative - on the basis of retention time on the chromatogram, it is possible, e.g., to

draw conclusions about the type of the separated substance. On the basis of the number

of peaks - about the number of components in a mixture, assuming that the used

chromatographic system ensured the separation of all mixture components and that the

detector is sensitive to all of the components,

quantitative - by measuring peak height or calculating the integral on the

chromatographic peak surface, it is possible to draw conclusions about the

concentration or mass of analytes in the injected sample.

The ideal chromatogram contains non-overlapping, closely spaced peaks. Overlapping

peaks are called co-eluting peaks. Retention time and peak size are very important as they

allow for identification and estimation of the number of analyzed compounds present in the

sample. The size of obtained peaks is proportional to the amount of the component in the

sample. The larger peaks are observed when the concentration of a given component

increases.

Fig. 12. Sample chromatogram of a pesticide mixture

2.1.2. Assay of presence and concentration of pesticides in the soil

Pesticides occur in nature in very small concentrations. In most cases - both in assay of

individual compounds, as well as when determining overall or individual parameters - it is

necessary to isolate organic compounds from the complex and troublesome matrix and enrich

them before final assay. Isolation and enrichment is necessary because of the imperfection of

current analytical methods. This is because in many cases they are not sensitive enough to

conduct final assay directly on the obtained sample and to assay trace components. During

27

isolation and enrichment, the concentration ratio of a micro-component to macro-components

increases. The basic activities during this part of analysis are: isolation of analyte from the

matrix (most often - extraction), fractioning or combination of these activities, whereby the

manner of isolation is determined by the matrix (complexity, composition and concentration),

selectiveness and sensitivity required by the analytical method and by analytical goals. This

method must characterize itself with simplicity of execution, ensure the separation of micro-

components from the matrix and should conform with the final assay method.

Methods of extraction from wet samples

For extraction, solvents mixing with water are used, e.g. acetone, acetonitrile, or a mixture

of solvents (polar/nonpolar), e.g. acetone/hexane, methanol/hexane. Extraction from wet

samples does not involve preliminary drying. After main extraction is completed, the solvent

is replaced (as a result of liquid-liquid extraction) with a nonpolar one, e.g. hexane or

dichloromethane, together with removing the water from the extract. Prior to this stage, water

is added to make it easier for analytes to move to the nonpolar layer. It is a toilsome stage,

conducted in a separatory funnel. Solvent replacement can also occur by enriching the analyte

in an extraction column to stationary phase (after diluting the extract with water), which is

followed by drying the column and elution with a nonpolar solvent.

Extraction from wet samples can be done in two ways. The first one is liquid extraction by

shaking or supported by ultrasounds (sonication). The process is conducted in multiple stages

(more than 3 times) due to the very low efficiency of the first stage of extraction. The

advantage of this method is the use of simple apparatus. The second way consists of

extraction by use of a Soxhlet apparatus. It is a procedure that is less toilsome compared to

liquid extraction by shaking or by sonication. The drawbacks include the possibility of a large

scattering of salvage values caused by the channeling effect in the extraction thimble, and also

the long time of extraction (6-48 h).

Methods of extraction from dry samples

For extraction from dry samples, it is possible to use methods used in extraction from wet

samples, i.e. liquid extraction by shaking or supported by ultrasounds and also extraction by

use of a Soxhlet extractor. In such cases, what is used is a mixture of a polar solvent (must be

removed during the course of the procedure) and a nonpolar solvent (e.g. 20% of acetone in

hexane produces an azeotrope containing 56% acetone which can removed during distillation)

or such solvents as: dichloromethane (CH2Cl2), hexane, ethyl acetate/cyclohexane, toluene/methanol,

28

2-propanol, and also CH2Cl2, acetone-hexane, cyclohexane and methanol-CH2Cl2. It is usually not

necessary to conduct the stage of liquid-liquid extraction in order to remove water.

Extraction from dry samples can be carried out:

by using a fluid in a supercritical state. This process is conducted by using high-

compressed gases (most often CO2) in the critical temperature range. This method

combines two properties of a liquid as a solvent with behavior of gases in flow. In the

extraction process, modificators are added to the extraction fluid (methanol, ethanol and

others) in order to increase the polarity of the fluid in the supercritical state. This

method is often combined with sorption on solid sorbents, which has a few advantages,

e.g. it requires small amounts of solvents, it has a short extraction time (ca. 1.5 h.), ease

of automation and high selectiveness. As this method of extraction is commonly used, it

limits the high cost of equipment and servicing.

by using microwaves. The process occurs by taking advantage of the phenomenon of

microwave energy absorption by chemical compound molecules. Energy proportional to

the dielectric constant value causes the rotation of dipoles in the electric field (as a rule,

devices are used that generate radiation at a frequency of 2.45 GHz). For extraction,

bombs are used that are made of teflon, quartz or composite materials. As solvents, the

most often used are CH2Cl2 or acetone/hexane. Extraction can be carried out in one of

two ways. The first way consists of using a solvent that absorbs microwaves (of a high

dielectric constant). Microwave radiation causes the solvent to be heated to a

temperature higher than the boiling point thanks to the high pressure present inside the

bomb. The heated solvent allows for a quick extraction of analytes from the matrix.

This way of conducting the process is used for extraction of policyclic aromatic

hydrocarbons, organochlorine pesticides, polichlorinated biphenyls from soils and

bottom residues. The second way of conducting extraction consists of using a solvent

that does not absorb microwaves (with a small dielectric constant). The sample and

solvent can be placed in a closed or open tank. Under the influence of radiation activity,

the solvent does not undergo heating as it does not absorb microwave energy. The

sample, usually containing water or other compounds with a high dielectric constant,

absorbs microwaves and releases heat to a cold solvent, selected in a way that ensures

sample dissolution. It is a method more gentle in comparison to the previous one; it can

be used for extracting thermolabile compounds, e.g. organochloride compounds.

Extraction with use of microwaves allows for quick and effective acquisition of analytes

from the matrix (time of extraction lasts ca. 15-30 minutes).

29

as accelerated extraction with a solvent. It occurs at an increased temperature (up to

200C) and pressure (up to 20 MPa), ensuring the liquid state of the solvent in given

conditions. After this stage, usually lasting only a few minutes, the extraction bowl is

washed with a pure solvent and then leached with nitrogen. The whole process lasts

several minutes. Its advantages include small use of solvent, possibility of automation,

simplicity of execution and high repeatability. Its disadvantages include degradation of

thermolabile compounds and loss of easily volatile compounds. This method found use

for extraction of organochloride herbicides and pesticides from the soil.

Extract enrichment

The next stage of sample preparation is the evaporation (and/or replacement of) the

solvent. Among the many known methods of extract enrichment, one can distinguish:

evaporation of the solvent in a vacuum evaporator. It is a quick and commonly used

method, though not without its drawbacks, such as, e.g. the possibility of losing analytes

as a result of co-distillation (distilling analytes together with the solvent) and

overheating of sample, and also adsorption of analytes on the instruments walls;

evaporation of the solvent in the Kudemy-Danish apparatus. Thanks to this technique, it

is possible to reduce the volume to 1 cm3 and, compared with evaporation in a vacuum

evaporator, to achieve higher analyte retrieval rate;

evaporation of the solvent in a gas stream (nitrogen or air). The advantages of this

method include simple equipment and low cost. The disadvantage is the danger of

losing the sample as a result of nebulization. With this method, it is possible to

evaporate high-boiling solutions. A gas of very high purity is necessary for evaporation.

2.2. Assay of MCPA remains in the soil

2.2.1. Qualitative assay with the SPME-GC method

Performing the assay:

1. Place 200 mg of sample soil in the flue. Thermostate the sample at 40C.

2. Carefully place the needle over the sample by puncturing the rubber septum, and then

remove the fibre.

3. Conduct exposition for 30 minutes, then slide in the fibre and remove the needle from over

30

the sample.

Fig. 13. Device for microextraction: a) general scheme of the instrument for sample acquisition,

b) scheme of a needle end during puncturing through the rubber membrane, c) scheme of

needle end during sample acquisition (sorption) or desorption of analytes in a chromatograph

dispenser.

1-adsorption fiber, possibly covered with a layer of sorption liquid, 2-steel wire, 3-

microsyringe needle, 4-microsyringe body, 5-microsyringe piston, 6-bowl with test sample

(closed with a cork with a rubber membrane), 7-magnetic stirrer

4. Chromatographic analysis:

Part I: Preparation of GC/MS acquisition method

GC/MS methods can be created and edited with the help of the Method Builder application.

Open the *.mth file and, according to the lecturers instructions, write the defined conditions

in which the GC-MS analyses will be conducted.

Part II: Performing the analyses:

1. Load the *.mth program into the computer steering the GC-MS system, according to the

lecturers instructions, in order to conduct GC-MS analysis. Next, when the apparatus shows

readiness (glowing diode Ready on the chromatograph), perform an injection according to the

lecturers instructions.

Once the analysis is complete, write down the analysis number displayed on the monitor and

save the file containing the analysis results onto a storage device.

Data processing:

1. Load the file with GC-MS analysis results. Using the MS Data Review application, conduct

qualitative analysis and write down the analysis results in tabular form.

Qualitative analysis is possible thanks to the NIST08 library, which is an integral part of the

system. On the displayed chromatogram, select the first peak using the left mouse button in

order to increase its size. Next, click on the tab Chromatogram/Set Spectra and select 3.

Under the chromatograph, there will be a MS spectrum, i.e. a mean of the three measure

31

points.

Click with the right mouse button on the spectrum and select Library Search Spectrum A. It

will display the library search result.

Increase the size of the search result window. The fourth column describes the probability that

the experiment spectrum matches the spectrum that is present in the library. Repeat the search

procedure (after closing the library window by clicking on the gray X button) in a different

place on the peak, so that the result has the highest probability factor.

2.2.2. Quantitative assay with GC-MS method

1. Inject the calibration solution containing the amount of analyte that corresponds with the

100mg/kg concentration in soil.

2. Conduct analyte extraction from the test sample according to the instruction:

For the extraction of MCPA 2-ethylhexyl ester from soil, 10g of soil samples were treated

with 10 ml acetone for organic phase extraction for 1 hour. The extract was separated by

filtration through a filter of medium density from Alchem, and residues were extracted

again with 5 ml of acetone for 1 hour. Then, after the second filtration both extracts were

mixed together. Obtained extract was placed in a separator flask and 50 ml of distilled

32

H2O, 3 ml of saturated NaCl solution and 7 ml of methylene chloride were added.

Mixture was shaken for 5 minutes. After separation of phases, another 7 ml portion of

fresh methylene chloride was added to the aqueous layer, and the shaking process was

repeated. The organic phases were combined and evaporated in a vacuum evaporator at

60C at pressure of 0.4 bar. The residue was dissolved in 2 ml of acetone and dried over

anhydrous sodium sulfate (VI). The resulting solution was injected to GC-MS analysis.

Linear response of detector was examined by analysis of several standards with different

concentration. Calibration curves from the peak areas were linear with high coefficient of

more than 0.99 at the range from 0-0.1 mg/kg for MCPA 2-ethylhexyl ester. Repeat the

analysis three times.

3. Conduct integration of peak surface areas (after prior identification, see point: Data

processing) in calibration injection and determine the slope of calibration curve.

4. Conduct integration of peak surface areas and correlate them with calibration injection in

order to determine analyte concentration in the sample. Provide the result as an arithmetic

mean of the three measures standard deviations.

33

3. BIOMONITORING

The idea of using live organisms, or their populations, to record and evaluate certain

characteristic traits of the environment is based on the notion that there exists equilibrium

between environmental factors and living requirements of various live organisms species. The

historical data confirm the relation between certain plant species and the presence of ores in

the ground, soil fertility evaluation, or later on between evaluation of air pollution and the

presence of lichen. Nowadays, use of living organisms for these purposes is widespread and

routine, and the latest trends in researching the quality of natural environment only widen

their use. This is related with avoiding costly laboratory techniques of chemical analysis and

relying on simple tests (which are possible thanks to advanced biotechnology) conducted in

the field for specific indicator species. On the other hand, there is a growing number of

analyses being conducted in an increasingly shorter time. This has led to increased

significance of quick miniaturized toxicity tests called microbiotests, or alternative tests.

Microbiotests work on the basis of single-celled organisms or small multi-celled ones,

which, as a result of contact with liquid sample, exhibit a specific response. Due to their

numerous advantages, alternative tests are used most often in the form of biotest battery based

on using organisms belonging to various trophic levels. The increase in significance of these

kinds of tests is caused by such parameters as elimination of the need for maintaining a lab

culture, low cost per sample analysis, short response time, low requirements regarding the

sample volume and laboratory space, possibility of field use. The role of these kinds of tests

will certainly continue to increase.

3.1. Biotests

Biotest (gr. bios - life + lat. testari - to testify) can be defined as an experimental

biological trail that aims to show the presence of toxic substances in the environment or

discover their harmfulness through quantitative estimation of the influence of a given

substance on a living organism (on the basis of comparison with the control sample). There

are three main methods of conducting research through biotesting:

toxicity tests realized in laboratory conditions during which the toxic substance is

artificially introduced into clean water or residue,

toxicity tests conducted in laboratory conditions on the basis of obtained real samples

(water, soil, residues),

test conducted in situ, with use of populations living in natural conditions.

Biotests used in analytical practice can also be classified by organism type, constituting

34

the tests active element. Most often used are plants, bacteria and animal organisms.

One of the more common tests utilizing bacteria uses the bioluminescence of sea bacteria

Vibrio fisheri in order to evaluate the level of pollution in water, bottom residues and soils. It

is a simple, easy to use test and also serves as a basis of operation of automatic analyzers.

In the case of toxicity tests that use plants (phytotests) as an active element, these plants

are usually algae (chlorophyta, cyanophyta, diatoms), aquatic lemna and rooted aquatic and

land macrophytes (plant and its seeds). They represent organisms of particular significance for

their natural habitats: they provide oxygen, ensure course of organic substances, control water

quality and equilibrium of soil and bottom residues. Vascular plants are used mainly in testing

against pesticides, rarely against heavy metals. Much wider use was found for algae as

bioindicators in situ in controlling water quality, due to their ability of accumulating chemical

compounds and heavy metals, such as copper, manganese, nickel. The most often used plants

are microalgae, belonging to chlorophyta, of species Selenastrum capricornutum and

Scenedesmus quadricauda. Algae are immobilized on special medium or assayed in flow

cytometers. Plant organisms are used much more rarely than animal ones and, e.g. according

to AQUIRE data base, they constitute only 10% of conducted acute toxicity tests.

Among animal organisms used for environment evaluation, the dominating ones are land

animals, and among those - invertebrates. Common application of invertebrates was found for

three ecotoxicological tests - namely earthworms, snails and bees. Bees are used as

representative land invertebrates, mainly for evaluating danger caused by pesticides. Tests on

earthworms are widely used and standardized. OECD recommendations on this matter have

the best methodical background and in some countries are a legal requirement prior to

introducing new chemical compounds into the environment. This usually includes

toxicological tests of acute toxicity. Use of earthworms for toxicological tests has a few

advantages, including: the animals are widespread and common in the environment, they have

a short life cycle, they are characterized by a high reproduction rate and they are easy to

cultivate in laboratory conditions. These tests use model soil. The recommended species of

earthworms for acute toxicity testing is, according to OECD recommendations, Eisenia

foetida. Specimens used for tests should be adult, i.e. have clitellum, specimen weight should

be within 300-600g for an earthworm. This test determines the acute toxicity level of

substances entering an earthworms body. After comparing the results from a toxicity test that

uses artificial soil with tests conducted on natural soil, it can be observed that toxicity in

artificial soil is much larger compared to toxicity in natural soils. This difference stems from

the fact that chemical substances in artificial soil are much more available for earthworms.

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The methodical problem of the standard OECD test is that during the experiment, animals do

not receive food and their body mass tends to become reduced. The latest tests include

feeding animals and/or the time of experiment is lengthened to make reproduction possible.

Reproduction rate can easily be determined by counting the number of laid cocoons. Influence

on reproduction is detected with lower concentrations of xenobiotic. A characteristic trait of

annelids and snails, useful in ecotoxicology, is the presence of metallothionein - proteins

binding metals which cause metals to accumulate in an animals body above the background

level. This trait is commonly used for bioindication as heavy metal contamination in an

earthworms body is easily determined by chemical analytics. Moreover, this trait has become

the basis for a test measuring the organisms condition with a simple parameter - neutral red

retention time in lysosomes of coelomic cells.

Tests on springtails are not as popular as tests on earthworms, but much research has been

conducted by using these invertebrates for evaluating the influence of contaminated soil on

beneficial soil arthropods. The species that is mainly used for these tests is Folsomia candida.

Another invertebrate that is commonly used in laboratories are onsicus. Just as is the case

with springtails, there is no ready-made normative assay for them. The disadvantage of using

this animal species for testing is their slow growth rate and long reproduction time in

comparison to earthworms.

In aquatic systems, the animals that are most commonly used for bioindication are

bivalvia Mytilus edulis, which is reflected in the widely developed monitoring programs of

supraregional significance. It is related to their particular role as biofiltrators in the

environment, resulting in accumulation of heavy metals, PAHs, PCBs, and others. Mytilus

edulis can be a source of research samples for most chemical analytical techniques used in

laboratories, many markers are used for them. Another aquatic organism useful toxicity

testing is daphnia Damphnia magna. The reproductive test carried out with daphnias is

commonly known, but it does not have such wide significance as is the case with bivalvias.

For residue evaluation, use is made of carinaria Rhephoxynius abronius. The most often

measured parameter is survival rate.

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3.2. Measure of neutral red retention time as a test showing the influence of soil

pollutants on earthworm Eisenia fetida

Dyeing with neutral red is one of the dye tests that assesses survival rate, has a long

tradition of use and consists of using small concentrations of dye selectively collected in

certain cell parts as lysosomes or vacuoles. This test is derived from mammal cell cultivation,

later on it was adjusted for ecotoxicological research in aquatic systems on fish and molluscs

(mainly Mytilus edulis). Transfers on earthworms were done by Svendsen and Weeks in 1996

when they used that to evaluate soil contamination after the fire in a plastic factory in

Thetford. It was a very good move as it paved the way for a very useful discipline - soil

contamination testing. The choice of test organism was also highly appropriate as earthworms

have, for many decades, been considered the perfect indicator organism of soil state, a fact

that was reflected, among others, during toxicity tests. The above-mentioned fire caused the

pyrolysis of plastic and formed a mixture of many toxic organic compounds and of released

heavy metals such as Pb, Sb, Zn, Cd, Cr contaminating the environment, including the soil.

The wide range of pollutants formed in the soil required the use of the right biomarker that

would indicate the integrated effect of influence of all pyrolysis products. This is why the use

of a non-specific biomarker was justified, such as measuring the integrity of lysosomal

membranes. The first test organisms used for research were earthworms living in

contaminated soil (Lumbricus castaneus). Samples were taken 0; 20; 60; 140 and 200 meters

away from exposure area and also from 3 kilometers away for use as control area. The

earthworms were transported together with soil samples to the laboratory where neutral red

retention time was determined for coelomocytes, i.e. cells acting as leukocytes taken from an

earthworms coelom. Results were compared with heavy metal content in animal bodies and

in soil, achieving high correlation. Use of coelomocytes was another excellent choice -

coelomocytes, cells suspended in coelomic fluid filling the space between the intestine and

the skin-muscle sac of an earthworm, are the basic element of an animals resistance system.

In natural conditions, earthworms are exposed to losing coelomic fluid together with

coelomocytes and dissolved substances, as it is ejected through pores in the back part of the

body in specimens irritated by chemical or mechanical factors. In experimental conditions,

such ejection of coelomocytes can be provoked by ethanol activity or a weak electric current

(5V). Coelomocytes can respond to environment contamination with intra-cellular changes,

including the number, size and shape of cells. Not without significance is also the ability to

easily take these cells for research without killing the animal.

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Release of the publication describing this research was the beginning of a cycle of works

dedicated to use of neutral red retention of coelomocytes in earthworms of various species for

measuring response to the presence of heavy metals, polyaromatic hydrocarbons and

pesticides. Exposure was conducted in the laboratory and in the field. Use of this test, which

is based on measuring neutral red retention time, has become commonplace and was

described in review work regarding aquatic as well as land systems.

When researching the influence of soil contaminants on earthworms by use of the neutral

red test, the natural environment can provide samples of earthworms that are representative

for the given area. Another method is field exposure of a chosen earthworm species in

specially constructed field-mesocosms that prevent the animals from escaping or laboratory

exposure of earthworms to soil acquired from the field. In each case, the test (acquiring

coelomocytes and determining neutral red retention time) must be conducted in the

laboratory.

In order to present the method for determining neutral red retention time, one can use

model conditions in which the chosen test organism is Eisenia fetida, a species that has a long

tradition of use in ecotoxicology.

Use of this test is related with conducting other observations, such as:

counting animals pre- and post-exposure, visual evaluation of their condition,

weighing animals pre- and post-exposure (despite the fact that individual specimens are

not assayed, it is possible to obtain information describing the sets, such as mean SD),

counting coelomic cells ejected by research specimens,

counting cocoons post-exposure

CONDUCTING THE EXERCISE

Experimental material

Adult, clitellum-possesing specimens of earthworm Eisenia fetida from the university

culture are exposed to contaminated soil taken from the vicinity of the bunker used for storing

pesticide and also to non-contaminated soil (control).

Exposure of earthworms to researched soil

Conduct toxicity tests using a photoperiod of 12L:12D. For exposure of earthworms to

contaminated soil, use plastic food containers 19x13x8 with a perforated covering. Before

putting earthworms into the container, wash them under running water, dry them on filtration

paper and weigh them. Next, put 10 Eisenia fetida each per container containing 600 g of

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researched soil. Place food on research soil each week in the form of 6 g of over fermented

horse manure, wetted in 20 ml of water. Sprinkle the soils surface with clean drinking water.

After 21 days of exposure:

count surviving specimens, evaluate their physical condition,

count cocoons laid by earthworms and observe occurrence of young specimens,

weigh each specimen.

Take coelomocytes from specimens that survived exposure in order to determine neutral

red retention time and counting of amebocytes and eleocytes.

Releasing coelomocytes

Before releasing coelomocytes, wash earthworms under running water, unmoisten on

crepe paper, and then weigh them. In order to acquire coelomocytes from earthworms, use the

non-invasive method which consists of ejecting coelomic fluid through body pores when

irritated with an electric current from a battery of 4 V voltage. Place clean earthworms on

a Petri dish containing 3 ml of solution for coelomocyte suspension, prepared from PBS and

2 g/L EDTA. Such a prepared solution allows for long review of coelomocyte preparations

under the microscope and prevents cells from gluing together into aggregates. An earthworm

must be irritated with an electric current a few times throughout the span of 1 minute in order

to fully complete ejection. Immediately after acquiring coelomocytes, transfer them onto

microscope slides for neutral red testing and to Brkers hemocytometer.

Counting eleocytes and amebocytes

Use Brkers chamber to count the number of coelomic cells. Introduce into the chamber,

using a pipette, 10 l of solution containing coelomocytes. The grid can be viewed under the

microscope in 10 large squares, each with a side of 0.2 mm. Count cells lying inside the

square with an edge of 1/5 mm, and also on upper and left border lines, do not count cells

present on bottom and right border lines to avoid counting the same cells twice. It might be

practical to make photos of the preparation and later use the photos for counting at a suitable

time. Count eleocytes and amebocytes separately.

Calculate the number of amebocytes and eleocytes acquired from individual specimens

according to the formula:

Number of cells from a single specimen = a 250 3 1000

where: a - average number of cells found in a single square;

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250, 1000 - conversion for 1 ml of volume;

3 - volume of solution used for suspending coelomocytes on a Petris dish, onto which

the coelomic fluid was ejected [ml].

Fig. 14. Grid from a Brkers hemocytometer with visible coelomic cells: amebocytes (1) and

eleocytes (2); source: own work

Conducting the lysosomal membrane integrity test in an earthworms coelomocytes

Preparing the neutral red dye solution:

Following the methodology prepared by: Svendsen et al. 1996; Weeks and Svendsen

1996, Weeks and Svendsen 1997 with minor modifications: dissolve 20 mg of neutral red

(SIGMA) in 1 ml of dimethylsiloxane (DMSO), then dissolve 10 l of the resultant solution

in 2.5 ml of solution for coelomocyte suspension, which will create a working solution of

neutral red at a concentration of 80 g/ml.

Preparing microscope preparation

Using a semi-automatic pipette, take from the Petris dish a representative sample of 10 l

of solution containing coelomocytes and transfer it onto microscope slide. After 30 seconds,

during which the coelomocytes undergo sedimentation, add 10 l of working solution of

neutral red onto the slide, achieving the final dye concentration of 40g/ml, next put on cover

slip. Transfer the preparation to the dessicator containing water in order to protect the

preparation from drying. Prepare further preparations in the same way. At this stage, the dye

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is absorbed by the lysosomes of the coelomocytes.

Determining retention time

Take out microscope preparations from the hydrostatic chamber and view them under the

optical microscope using 640x zoom, do this repeatedly (in intervals, every few minutes). The

microscopes field of view will show two types of cells: darker, more visible and numerous

ones - eleocytes and light, staining ones - amebocytes. When viewing the preparations, choose

a few spots in which amebocytes are well visible. Count the cells that were observed to have

released dye from lysosomes to cytozole and those not stained. Stop observations when the

number of cells with stained cytozole amount to 50% in relation to non-stained cells. The time

from the moment of adding the working dye onto the microscope preparation until the

moment of terminating observation is treated as the neutral red retention time in the

lysosomes of the coelomocytes. Observations can be made more efficient by taking photos of

the preparation for later counting at a suitable time. Below are example photos of

coelomocytes during test.

Fig. 15. Coelomocytes Eisenia fetida during the neutral red test. The green circle shows non-stained

amebocytes, black cells are eleocytes (optical microscope 640x); source: own work

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Fig. 16. Coelomocytes Eisenia fetida during the neutral red test. Red circles show amebocytes with

non-stained cytosoles, dark cells are eleocytes (optical microscope 640x); source: own work

Read the retention time for each preparation for each of the studied specimens. Calculate

mean SD.

Put in tabular form all obtained data for the control sample and