1

Dr. Peter A. Behnisch

& many other colleagues

& many other CALUX users

Global hot topics from CALUX users

2

Global Tour 2018

Japan

USA

Sweden

Germany

The Netherlands

EU

Danube

First case:

Neighbors living nearby an industrial land fill

in Kamp-Lintfort in Germany

3

Landfill near Kamp-Lintfort

Free ranging egg biomonitoring:

DR CALUX vs GC/HRMS

4

Egg

samples

(pg/g)

PCDD/F/dl-

PCB-BEQ

(DR CALUX)

PCDD/F-

BEQ

(DR CALUX)

PCDD/F/dl-

PCB-TEQ

(GC/HRMS)

PCDD/F-

TEQ

(GC/HRMS)

Place 1 7.1 5.3 10 6.3

Place 2 6.5 4.4 8.7 3.6

Human plasma testing for dioxins and dioxin-like

compounds near Kamp-Lintfort, Germany

Table 1 and Figure 1: Minimum, median, maximum, 95%/5% percentile and mean value of the sum of

PCDD/PCDF/dl-PCB/dl-compounds-BEQ, PCDD/F-BEQ and dl-PCB/other dl-like

compounds data analysed by DR CALUX® from the human plasma samples from

86 neighbours living near an industrial landfill in the city of Kamp-Lintfort.

DR CALUX®

PCDD/Fs, dl-PCBs

PCDD/Fs dl-PCBs

Number of values

86 86 86

minimum 5.2 1.7 3.5 median 38 15 22 maximum 80 46 52

5% percentile 12 3.5 7.2 95% percentile 68 32 39

mean 38 16 22 SEM 1.7 0.94 1

TEQ (human): Freq. dist. (histogram)

0 20 40 60 80 100

0

20

40

60

80

100PCDD/Fs, dlPCBs

PCDD/Fs

dlPCBs

# of samples

pg

TEQ

/g f

at

5

Summary of 1st case:

Human plasma for dioxins/dioxin-like compounds

in the area from Kamp-Lintfort, Germany

• Figure 1 shows all 86 DR CALUX® human plasma results lined

up with increasing concentrations.

• Here 95% of all DR CALUX® results (95% percentile level; “end

of the vertical hockey stick”) are below 68 pg TEQ/g fat.

• Such biomonitoring equivalents (BE) for dioxins and dioxin-like

compounds are reported from different agencies in the same

range between 15-74 pg TEQ/g lipid.

• The 5% percentile level of our study here of 12 pg TEQ/g fat is

also in the same range as an earlier proposed ATSDR minimal

risk level (MRL) for dioxins of 15 pg TEQ/g lipid (Aylward et al.

2008, EU-SCF Report, 2001).

6

EFFECT-BASED WATER MONITORING OF CONTAMINANTS IN

EFFLUENTS FROM WATER TREATMENT PLANTS ALONG

THE DANUBE RIVER BASIN

Behnisch, Peter1; Besselink, Harrie1; Alygizakis, Nikiforos2;

Brouwer, Abraham1 and Slobodnik, Jaroslav2;

1BioDetection Systems BV, Amsterdam, NL2Environmental Institute, Koš, Slovakia

7

Augsburg

Amstetten

BrnoŽilina

Budapest

Kršk

oZagrebLjubljana

Cluj-NapocaVaraždin

ŠabacBucharest

2. Location of WWTPs along the Danube

5. Applied CALUX® Bioassays (Mode of Actions)

with different management targets

of effect-based trigger (EBT) values

9

Mode of Action

Reference Compound

International Standard

Method e.g.

Effect-based

triggervalue

1 to 3-times EBT level

3- to 10-

times EBT level

Above 10-

times EBT level

Above 100-

times EBT level

Estrogenicity ng eq E2/l ISO 19040

OECD TG 455

Human or Yeast TA cell assay

0,3 0,9 3,0 30 ˃30

Inhibition Androgenicity

μg eq Flutamide/l

OECD TG 458-like

Human or Yeast TA cell assay

3,3 9,9 33 330 ˃330

AhR receptor activation

ng eq B(a)P/l

Rat liver

cells H4IIE

TA assay

6,2 18,6 62 620 ˃620

Adaptive Stress (Nrf2)

μg eq

dichlorvos/l

ISO 19040-like

OECD TG 455 like

Human TA cell assay

26 78 260 2600 ˃2600

Early warning chemicals: Activation pregnane x receptor (PXR)

μg eq

DEHP/l

ISO 19040-like

OECD TG 455-like

Human TA cell assay

272 816 2720 27200 ˃27200

6. Proposal for possible action plan for

operators of WWTPs

10

A typical response plan of actions for operators of such water treatment plants effluents could be

as following: The sample location and frequency for these bioassays should be linked to specified

monitoring requirements for such indications for chemicals of concern (CECs) in these water

treatment plants effluents (i.e., frequency of six months; collected at the point of compliance). An

exceedance of the above proposed trigger values should initiate the following actions:

- If below EBT or LOQ of bioassay (white): no further action required

- If 1-times <EBT< 3-times (BLUE): quality check data, continue to monitor every three

months, until 1 year and the EBT < 1

- If 3-times <EBT< 10-times (GREEN): data check, immediate re-sampling and analysis to

confirm EBT. It is also required to quantify specific target compounds which are

known to cause the effects observed in the respective bioassay. Continue to monitor

every three months, until 1 year and the EBT< 1

- If 10-times <EBT< 100-times (ORANGE): all of the above plus enhance source

identification program. Also monitoring in the distribution system closer to the point of

exposure to confirm attenuation of CEC is occurring and to confirm the magnitude of

assumed safety factors associated with removal efficiency, dilution and post-

treatment.

- If EBT>100-times (RED): all of the above plus immediately confer with the local

environmental authority’s to determine the required response action. Confirm plant

corrective actions through additional monitoring that indicates the CEC levels are below

at least an EBT of 100.

7. CALUX® panel results and

suggested action plan for operators of WWTPs

from the Danube River Basin

11

ERα

CA

LU

X

an

ti-A

R C

AL

UX

GR

CA

LU

X

PP

ARɣ

CA

LU

X

PA

H C

AL

UX

Nrf

2 C

AL

UX

PX

R C

AL

UX

1 - Varazdin 5 5.7 <19 640 72 41 8.2

2 - Amstetten 1.1 22 <20 <520 122 46 13

3 - Cluj <0,06 31 34 <420 52 <50 8.4

4 - Augsburg 1 10 72 <410 72 46 13

5 - Vipap 0.65 32 <25 <460 242 74 48

6 - Budapest 0.56 11 <23 <430 62 46 14

7 - Ljubljana 6.6 8.4 120 <350 62 50 9.4

8 - Bucurest 7.4 5.7 38 <340 82 130 10

9 - Zilina 2.2 8.9 78 <480 72 60 5.2

10 - Sabac 1.1 14 <41 <490 72 46 4

11 - Brno 0.54 13 47 <1100 122 80 17

12 - Zagreb 0.8 6 <42 <1100 52 <17 17

EU – Monitoring of

steroidal estrogens

2015-2017

Estrogens in surface

waters

Watch List: E1, E2, EE2

Monitoring data

Analytical challenges (<1 ng/L)

Single substance-based monitoring

approach

Promote applicability of effect-based

methods

Complementary application to analytical

EU-WFD

16 Surface waters

17 wastewaters

LC-MS/MS (3 Labs)

ERTA* Biotests (5 Labs)*ERTA: Estrogen Receptor Transactivation Assays

0 5 10 15 200.01

0.1

1

10

100

0.4TV

Oberflächengewässer

ER

-CA

LU

X E

EQ

(ng/L

)EEQs in environmental waters- ERα-CALUX

0 5 10 15 200.01

0.1

1

10

100

0.4TV

AbwasserE

R

-CA

LU

X E

EQ

(ng/L

)

38% > TV

Pre-screening capacity identify hot spots for further in-depth LC analysis

Mixture effects

71% > TV

16 Surface waters Wastewaters

Follow-up – from 2017

Watch

“Watch list” Project

>80 Surface waters from Watch List stations

2 sampling campaigns

14 EU Member states

4 Cantons from Switzerland

Steroidal estrogens

Diclofenac along with cyclooxygenase (COX)

inhibitors, e.g. naproxen, ibuprofen, ketoprofen

Results so far: ERα-CALUX

0 .0 1

0 .1

1

1 0

1 0 0E

R

-CA

LU

X E

EQ

(n

g/L

)

T V

7% > TV

n=85 samples

Petroleum – Toxicity profile

17

AhR, anti-AR, anti-PR and anti-TR most significant effects

18

19

MTM-EnForce:

Innovative Environmental Research (Sweden)

- the Environmental Forensic Laboratory, EnForce, is a research effort aiming

towards a non-toxic environment

- more sustainable management of chemicals

- trace and identify the true culprits among hazardous environmental toxins

- use a panel of bioassays to search for unknown environmental toxins

- study their behaviour in the environment and their toxicological effects on cells

(e.g. CALUX panel for PAHs, PFAAs and other MoA).

- Development of safe and effect-based trigger values for various MoA in soil

- Focus on worst case polluted sides with the aim to search where the real risks

lie

20

Ehime University, Japan

21

22

National Institute for Environmental Science, Japan:

DR CALUX vs GC/HRMS for various environmental matrices

23

24

NIES, Japan: Cl vs Br Dioxins by DR CALUX and GC/HRMS

25

26

Bioassay z-score: green < 2; orange 2<3; red >3

80% of bioassay labs scored 8/10 samples below z +/-2 =

bioassays cover also brominated / mixed halogenated

dioxins = safer & without additional costs

27

Shane Snyder (University of Arizona, Tucson,

AZ, USA):

Current CALUX applications I

• Screening water reuse and water recycling

treatment processes:

• Water samples throughout treatment

processes are tested with the DR CALUX and

p53 CALUX assays

• Purpose is to identify if specific classes of

compounds are present and removed through

treatment, along with the detection of potential

disinfection byproducts

• Cancer prevention in the fire service:

• Test de-conjugated urine extracts collected

before and after fires with PAH CALUX and

p53 CALUX assays to identify exposure to

biologically active compounds

• Assessment of the integrity of water samples

over time for use in bioassays:

• AR CALUX will be used to test the stability of

compounds in wastewater effluent under

different storage conditions to identify optimal

storage times and conditions

• The role of dust in the development of asthma:

• Extracts of house and farm dust will be tested

using the NfkappaB CALUX assay to

investigate pro-inflamatory cytokine induction

with regards to asthma research

28

Current CALUX Applications II