chlorinated micropollutants in aquatic effluents...chlorinated micropollutants in aquatic effluents...

Short title (see File, Properties, Summary, Title) 1

Chlorinated Micropollutants in Aquatic Effluents

Part-1 Sampling, pre-treatment, classification and primary effect screening

M. Lamoree G. Stroomberg H. Besselink H. Senhorst T. Sanderson

B. van Hattum

Chlorinated Micropollutants in Aquatic Effluents Part-1 Sampling, pre-treatment, classification and primary effect screening

M. Lamoree, G. Stroomberg, H. Besselink, H. Senhorst, T. Sanderson and B. van Hattum

E-03/02

May 2003

Workpackages 5-6

Final version

Chlorine Chain Follow-up Research Programme on chlorinated organic micro contaminants (OVOC).

Contributions from RIZA, IRAS and IVM

Commissioned by the Ministry of Housing, Spatial Planning, and the Environment (VROM, DGM; contract nr. 99230300), the Ministry of Transport, Public Works, and Water Management (VW, Rijkswaterstaat), the Association of the Dutch Chemical Industry (VNCI) and the Netherlands Society for Nature and Environ-ment (SNM).

This report was approved by the OVOC steering committee

IVM Institute for Environmental Studies Vrije Universiteit De Boelelaan 1087 1081 HV Amsterdam The Netherlands

Tel. ++31-20-4449 555 Fax. ++31-20-4449 553 E-mail: [email protected]

OVOC Reports Phase-2: Chlorinated Micropollutants in Aquatic Effluents

• Part-1 Screening studies (WP5&6) • Part-2 Biodegradation studies (WP8) • Part-3 TIE studies (WP7&8) • Part-4 In-plant TIE Studies (WP9) • Chlorinated Micropollutants in Atmospheric Emissions • Chlorinated Micropollutants in Products

Copyright © 2003, Institute for Environmental Studies

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopy-ing, recording or otherwise without the prior written permission of the copyright holder.

Final report WP 5&6 OVOC Project March 2003 i

Contents

Preface 1

Korte samenvatting 3

Executive summary and conclusions 5

1. Introduction 9 1.1 Background and objectives OVOC programme 9 1.2 Topics of studies in Phase-II 10

2. Sampling of aquatic effluents and sample distribution (Task 5.1a) 13 2.1 Introduction 13 2.2 Sampling campaign 14

3. Analysis of main constituents of aquatic effluents (Task 5.2) 17 3.1 Introduction 17 3.2 Interpretation of results of macro-parameters 18

4. Purge and trap, extractions and fractionation (Task 5.3a) 21 4.1 Introduction 21 4.2 Method 21 4.3 Observations on samples 25 4.4 Conclusions 26

5. Analysis of halogenated fractions (Task 5.4) 27 5.1 Introduction 27 5.2 Experimental approach 27 5.3 Results and discussion 29 5.4 Conclusions 31

6. Toxicity screening with in-vitro tests (Task 6.1) 33 6.1 DR-CALUX assays 33

6.1.1 Introduction 33 6.1.2 Sample information and protocol 34 6.1.3 Results 35 6.1.4 Discussion 39

6.2 Carp-hepatocyt assays 40 6.2.1 Introduction 40 6.2.2 Methods 40 6.2.3 Results and discussion 42

7. Toxicity screening with in-vivo tests (Task 6.2) 49 7.1 Early Life Stage (ELS) tests 49

7.1.1 Principle 49

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7.1.2 Handling 49 7.1.3 Results 50

7.2 Chronic micro-toxicity tests (Task 6.2) 51 7.2.1 Principle 51 7.2.2 First test series 52 7.2.3 Second test series 52 7.2.4 Results 53

8. Synthesis of results and estimation of annual loads 57

9. Ranking and prioritisation of samples for WP 7-9 65

References 71

Appendix I. Abbreviations 75

Appendix II. Prioritised emission sources selected for the main study (Phase-2) 77

Appendix III. Sample pre-treatment and recovery data 79

Appendix IV. Relative light yield data from chronic Microtox test 81

Final report WP 5&6 OVOC Project March 2003 1

Preface

In this report the results are presented of the chemical and biological effect screening of aquatic effluents in the framework of the OVOC-study on the chlorinated organic micro pollutants, commissioned by the Ministry of Housing, Spatial Planning, and the Envi-ronment (VROM, DGM; contract nr. 99230300), the Ministry of Transport, Public Works, and Water Management (VW, Rijkswaterstaat), the Association of the Dutch Chemical Industry (VNCI), and the Netherlands Society for Nature and Environment (SNM).

As agreed with the BOVOC steering committee, the format for reporting on the chemical –biological work packages 5-8 is in the form of one central report structure throughout the project duration, which will be extended with sections and chapters, each time spe-cific tasks and work packages are completed. This continuously growing report will also be the basis for the integration and final reporting scheduled in Phase-3. A detailed de-scription of work packages and tasks is provided in the work plan for Phase-II. At spe-cific moments during the project, draft versions of the report will be submitted to the BOVOC or dedicated technical workshops. Although this reporting structure has disad-vantages for the clarity of presentation and is not suitable for distribution beyond mem-bers of the research team, BOVOC and invited experts, it is efficient for the rapid com-munication of preliminary results. Readers of draft versions of this report are kindly asked to take notice of this aspect and are thanked for their effort to overcome this in-convenience.

The results in this report are confidential and are presented anonymously with codes. A first draft version of this report has been presented and discussed at a technical workshop held August 30th 2001 in den Haag. A second draft version, including recommended re-visions from the workshop was discussed and approved by the BOVOC in 2002. This version was communicated in 2002 to the participating companies. Each company was informed by the VNCI on its own sample-code. Based on comments received and in some cases exchange of additional information between September 2002 and March 2003, this third draft version was prepared.

The authors gratefully acknowledge the willingness and kind co-operation of the compa-nies, who participated in the study. We further acknowledge the contributions to this study by laboratories subcontracted, helpful colleagues and the comments made by members of steering committee and invited experts.


Korte samenvatting

Het voorliggende deelrapport beschrijft de eerste resultaten van de werkpakketten 5 en 6 van het onderzoek aan aquatische effluenten (afkomstig van industrie, koelwaterinstalla-tie en gemeentelijke afvalwaterzuivering) in het kader van het OVOC (chloormicro’s) project.

Hoofddoel van dit project is vast te stellen of, en zo ja, in welke mate er chloormicro’s worden geëmitteerd door de chloorketen. Onder chloormicro’s verstaat men in kleine hoeveelheden geëmitteerde chloorbevattende PBT’s: persistente, bioaccumulerende toxi-sche stoffen.

Vastgesteld is dat de meeste onderzochte effluenten, naast mogelijke andere stoffen, in elk geval ook niet-polaire, niet-vluchtige chloorhoudende stoffen bevatten, die bij fracti-onering in fracties van middelbare (Log Kow 4-6) of hoge hydrofobiteit (Log Kow >6) terechtkomen in concentraties van <0,1-6 µg/l resp. 0,04-0,1 µg/l. Stoffen van middelba-re en hoge hydrofobiteit kunnen doorgaans als bioaccumulerend worden beschouwd .

De belangrijkste resultaten van het toxiciteitsonderzoek zijn:

• Geen van de effluenten vertoonde een significante respons op de zebravis test en de in-vitro karper-hepatocyten test op cytotoxiciteit en estrogeniteit;

• Daarentegen vertoonden diverse effluenten (niet alleen die van de industrie) wel een significante respons bij een tweetal in-vitro tests op dioxine-achtige toxiciteit (DR-CALUX; EROD karper-hepatocyten test) en bij de Microtox test met een mariene bacterie. De dioxine-achtige toxiciteit werd aangetroffen in de bij fractionering ver-kregen fracties van middelbare en hoge hydrofobiteit.

Op grond van de resultaten is na advies van een workshop met technische experts een se-lectie gemaakt van monsters voor aansluitende deelstudies naar persistentie en de moge-lijke identiteit en herkomst van respons veroorzakende stoffen.

Het hier gerapporteerde onderzoek aan aquatische effluenten had nog geen betrekking op persistentie. Ook kan deze eerste fase van het onderzoek nog geen antwoord geven op de vraag door welke stoffen de waargenomen toxiciteit is veroorzaakt; ook niet of dat door chloorhoudende of chloorvrije stoffen is veroorzaakt. Deze aspecten zijn onderwerp van aansluitende deelstudies. Met nadruk zij gesteld dat op grond van de resultaten van deze screeningstudie, nog geen antwoord valt te geven op de hoofdvragen van het chloormi-croproject.


Executive summary and conclusions

In this report the results are presented of the chemical and biological screening of aquatic effluents in the framework work packages 5 and 6 of the OVOC-study on the chlorinated organic micro pollutants. One of the objectives of the OVOC study is to determine if un-known chlorinated micropollutants (PBTs: persistent, toxic, bioaccumulative com-pounds) are emitted from the chlorine chain. In this screening study on the aquatic effluents a tiered approach was chosen for practical reasons, in which the first steps consisted of a separate screening on the presence of toxic (T), bioaccumulative (B), and chlorinated compounds. Further work on the assessment of the persistence (P) and the identity of responsive compounds is part of work packages 7 and 8 and will be reported in separate reports (Part-2 and Part-3).

During the screening study on the aquatic effluents a total number of 21 samples was in-vestigated on the basis of a grab sample, consisting of: 16 industrial effluents from waste water treatment installations, 1 discharge of cooling water (inlet and outlet), 1 municipal wastewater treatment plant (MWTP) and 1 reference surface water (Lake Markenmeer) and 1 blank (tap-water). The 16 industrial effluents originated from 14 production sites. One of the industrial effluents was treated in a municipal wastewater treatment plant. Except for 1 company, normal production conditions could be assumed based on infor-mation from the company’s involved and raw data from measurements on pH, suspended solids, BOD, TOC, EOCl and AOX.

Wastewater samples (8L) were extracted with pentane and transferred to methanol ex-tracts suitable for biological testing. The primary biological effect screening of the ex-tracts included sensitive in-vitro screening methods (DR-CALUX TM, Carp-hepatocyte test) for compounds with dioxin-like, (anti-)estrogenic or cytotoxic mode of action and in-vivo testing with an 8-day zebra fish early life stage test (ELS) and a 22-hr chronic version of the Microtox TM test with the marine bacteria Vibrio Fischeri. The extracts were fractionated with C-18 based RP-HPLC to obtain three fractions with increasing hydrophobicity (low-medium-high based on fractionation of mixture of PAHs). In each of the fractions the presence of non-polar and non-volatile halogenated compounds was determined with GC-ECD/ MSD.

In all samples, both from industrial and non-industrial sources, the presence of halo-genated compounds was confirmed, based on concentrations of EOX and AOX in raw effluents, and the results of the GC-ECD screening. Total GC-amenable concentrations of halogenated compounds were calculated based on average response-factors of a mix-ture of chlorinated standards. Due to the variation in response factor the equivalent con-centrations are indicative (order of magnitude). Total GC-amenable concentrations of chlorinated compounds ranged from 0.2 – 2.2 µg/L in most effluent samples and less than 0.1 µg/L in surface water and blanks. In 3 industrial effluents concentrations be-tween 9 and 30 µg/L were encountered, with the largest contribution (3 –27 µg/L) in fractions with low hydrophobicity. Extremely hydrophobic compounds were detected in concentrations ranging from 0.04-0.1 µg/L in all samples. These results show that the bulk of organochlorines are of polar nature, with decreasing amounts at increasing hy-drophobicity.

Institute for Environmental Studies 6

None of the extracts induced effects in the 8-day zebra fish ELS test. In 7 samples the 22-hr Microtox test was responsive at the highest concentration tested. Within this group 2 industrial samples and the MWTP were responsive in two or more dilutions.

In the in- vitro tests with the carp hepatocyte system, no significant cytotoxicity was re-corded in the MTT test and no estrogenic response in the VTG test. A significant anti-estrogenic potency was found in 5 samples (4 industrial and MWTP). For 9 samples a significant induction of EROD was found in the carp-hep test system (7 industrial sam-ples, cooling water outlet and MWTP).

The DR-CALUX assay, detecting compounds with a dioxin-like mode of action, was the most responsive test. The concentrations are expressed as 2,3,7,8-TCDD equivalent con-centrations (in pg TEQ/L). A significant response was detected in 15 samples (12 indus-trial effluents: 40-1066 pg TEQ/l; MWTP: 157 pg/L, cooling water outlet: 104 pg/L; and Lake Marken: 37 pg/L). All EROD responsive samples in the carp-hep test were also re-sponsive in the DR-CALUX assay, although the samples with maximum induction in both tests did not coincide. DR-CALUX and carp-hep EROD tests on fractionated ex-tracts revealed that the activity was confined to the fractions with medium and high hy-drophobicity. In the fractions with low hydrophobicity no activity was observed.

Effluent discharge data were used to estimate annual loads. A multi-criteria approach was used to identify responsive effluents in the chemical and biological screening proce-dure, and to classify the toxic (T) and bioaccumulative (B) potency, and to select sam-ples for further biodegradation and TIE studies (toxicity identity evaluation) to identify responsive compounds.

Extrapolated annual loads of 2,3,7,8-TCDD based equivalents (TEQs) for national MWTPs and cooling water discharges, as derived from the DR-CALUX tests, were higher than existing emission estimates based on chemical measurement of PCDDs and PCDFs, which was attributed to the potency of the DR-CALUX tests to account for other (including non-chlorinated) responsive components and mixture-toxicity interac-tions. The attempt to identify the Ah-receptor active compounds in the effluents will be the subject of following studies in workpackages 7 and 8.

The results were presented in a 1st draft version of this report and discussed at a dedi-cated workshop (August 30th

2001, Den Haag). This workshop was organised to advice the BOVOC steering committee and the research team on the proposed classification and selection of samples for treatment and examination in work packages 7, 8 and 9. These work packages will include: additional biodegradation studies, toxicity identity evalua-tion studies (TIE) studies on responsive fractions, and additional in-plant studies to at-tempt to identify unknown chlorinated PBT compounds in mixed wastewater streams.

In summary, the screening approach revealed the presence of non-polar, non-volatile, GC- amenable chlorinated compounds in fractions of medium (<0.1-6 µg/L) and high hydrophobicity (0.04-0.1 µg/L) in most effluents. The toxicity screening revealed no significant response in the zebra fish test and the carp hepatocyte in-vitro tests on cyto-toxicity (MTT) and estrogenic response (VTG). Significant responses were observed in Ah-receptor mediated in-vitro tests (DR-CALUX, carp-hep EROD) and the Microtox test. The dioxin-like activity observed in the in-vitro tests was confined to the fractions of medium and high hydrophobicity.


The results of this screening study allowed a first classification of the effluents based on toxicity and the presence of chlorinated compounds in fractions of medium and high hydrophobicity. The results of this study do not allow conclusions to what extent the observed toxic responses in some tests are caused by chlorinated or non-chlorinated micropollutants. This, and further tests on persistency, is the subject of further studies in Workpackages 7-9.


1. Introduction

The present report contains the first results of the chemical and biological screening and classification of 16 industrial effluents as part of Phase-II of a research programme on chlorinated organic micro contaminants (OVOC - Chlorine Chain Follow-up Research Programme on chlorinated micro contaminants), commissioned by the Dutch govern-ment (Ministry of Ministry of Housing, Spatial Planning, and the Environment, VROM; Ministry of Transport, Public Works, and Water Management, VW, Rijkswaterstaat), the Association of the Dutch Chemical Industry (VNCI) and the Netherlands Society for Nature and Environment (SNM)1.

1.1 Background and objectives OVOC programme

The objectives of the OVOC research programme (OVOC) are to provide answers to the main questions raised and agreed by the BITAC2 committee (with representatives from government, industry, trade unions and environmental NGOs) in 1997: Does the problem of chlorinated micro contaminants (still) exist? If so, what is the extent of the problem? Which PBTs (or groups of PBTs) are emitted from the chlorine chain into the environ-ment from point or non-point (diffuse) sources?

During the preparatory consultations for the current proposal these objectives were fur-ther operationalised and extended to:

• To assess if, and to what extent PBTs or groups of PBTs are emitted from the chlo-rine chain into the environment;

• To contribute to the assessment of the environmental impact of these PBTs.

The proposed programme has a total duration of more than 3.5 years and consists of the following main elements:

• Experimental studies aiming at the characterisation of current emissions of unknown chlorinated compounds with state-of-the-art chemical and biological techniques;

• Background studies involving the selection and prioritisation of potential emission sources, a systematical review of organo-chlorines present in the environment, the significance of degradation products of unknown emissions, the role of naturally produced organo-chlorines and the relative importance of historical emissions.

The different phases and current timetable of the programme are indicated hereafter.

A full description of objectives, scope and backgrounds to the choices made is included in the original programme proposal (Van Hattum et al., 1998) and the work plan for the Phase-II main study (Van Hattum et al., 2000).

1 Inception phase: Letter of Min VROM, dated March 1st 1999; subject DGM/SVS/99129326

zaaknr. 99230300; Phase-I: Letter of Min VROM, dated Sept. 30, 1999; subject DGM/SVS/99217667 zaaknr. 99230300; Phase-II: Letter of Min VROM, dated Nov. 30, 2000; subject DGM/SAS/200144640 zaaknr. 99230300.

2 Minutes BITAC meetings: September 25th 1997 and December 9th 1997.


Phase Duration Inception phase 01/03/1999 01/09/1999 Phase-I Preliminary studies unknown emissions and background studies 01/10/1999 01/08/2000 Phase-II Main study unknown emissions and background studies 01/12/2000 01/01/2004 Phase-III Prediction of chemical fate and limited field validation 01/06/2003 01/01/2004 Integration and final reporting 01/10/2003 01/07/2004

During Phase-I various background studies were executed, which focused on unidenti-fied organochlorine compounds in the environment (Task 2.1; Klein et al. 2001a), the role of degradation products (Task-2.2; Klein et al., 2001b), current emissions of chlo-rinated micro contaminants and a proposed selection of potential emission sources and pathways to be investigated during the Phase-II Main Studies (Task 3.1-3.2; Tukker, 2000a and 200b). In addition, a pilot study was executed to optimise and test chemical and biological screening methods for chlorinated toxic contaminants in aquatic effluents (Belfroid et al., 2000). The proposed selection of emission sources and the results of the methodological study were presented to a technical Workshop (May 2000, Den Haag), in which participated the supervising committee (BOVOC), invited experts and the re-search team. The final work plan for Phase-II (Van Hattum et al., 2000) was based on the outcome of the workshop and discussions in the BOVOC and approved by the BOVOC in November 2000.

1.2 Topics of studies in Phase-II

During the Main Study (Phase-II) the major focus will be on the biological and chemical screening of potential emission sources and pathways selected in work package 3 during the Preliminary study (Phase 1). In this report only the aquatic effluents are considered. The studies on products and atmospheric emissions will be reported in a different report.

Work package 5 will address the sampling, extraction, fractionation (in different hydro-phobicity classes), chemical screening (on the presence of halogens), and the classifica-tion with respect to the expected compartments of final destination: air (purge able frac-tion ), water or sediment/soil. Work package 6 contains the primary effect screening with in-vitro and in-vivo tests. A general outline of the activities scheduled in work packages 5&6 and discussed in the next chapters is given in Figure 1.1.

The results of the screening study have been discussed at a technical workshop, held Au-gust 30, 2001 in Den Haag. At this workshop a proposal for the selection of samples for further studies (WP 7-9) was discussed.

After identification of the biological response of organohalogen containing fractions, the first round of toxicity identity evaluation (TIE) studies will follow in work package 7. The tasks in this work package include refined fractionation, in-vitro testing, mass spec-trometric studies on biologically active subfractions, and a statistical analysis and evaluation of the results of the screening studies using chemometric techniques.

For a selection of samples with high activity additional biodegradation and extended TIE studies will be conducted (work package 8). This includes the assessment of the persistence of components in responsive fractions


and the determination of in-vivo and in-vitro response of degraded samples. The ex-tended TIE studies include further refined fractionation and in-vitro testing, confirmation of semi-chronic toxicity in sub fractions with in-vitro activity, detailed mass spectromet-ric studies to identify components and confirmation studies.

Figure 1.1 Schematic representation of the treatment of aquatic effluents with regard to extraction, fractionation, chemical analyses and in vitro assays (WP 5 and 6).

It is anticipated that many effluents consist of complex mixtures, and that even in refined sub fractions several tens of compounds may be present. In cases where influents from different installations are treated in a WTP, in-plant studies on separate influents may help to track the source of the biological response. A limited number of installation specific TIE studies (WP 9), to be executed in close co-operation with the individual companies, will be proposed for the second year of the main study.

The suitability of the selection of waste streams will be assessed in a workshop (work package 10), approximately two years after the start of Phase-2. The evaluation will be based on the results of the screening, biodegradation and TIE studies in relation to the outcome of the background studies. When needed adjustments will be made.


2. Sampling of aquatic effluents and sample distribution (Task 5.1a)

2.1 Introduction

In this work package sampling will be performed on 16 industrial effluents selected as representative for the chlorine chain in The Netherlands, 2 reference samples and one cooling water system. The list of the industrial effluents (as defined in Tukker 2000b) can also be found in Appendix II.

The 16 industrial firms were approached by a first contact by Dr. J. Bouwma from VNCI (Dutch association of Chemical Industry, one of the commissioners of the OVOC pro-ject), who informed them on the background of the OVOC-study and the purpose of the sampling of effluents. They were asked to provide the necessary data to RIZA, who was to organise the sampling campaign, and to assist in sampling. Most of the firms con-tacted reacted positively. Only two major modifications were made to the list of priori-tised emissions:

• The firm Givaudan Roure reacted that it no longer used the substance EDC (ethylene dichloride) as a solvent in its production process. As EDC consumption was the rea-son for selection of Givaudan Roure into this study, the project leader concluded that sampling at this site was no longer relevant;

• Atofina at Vlissingen (production of organotin compounds) did not offer its co-operation at the start of the project. Discussions with VNCI and the Ministry of VROM could change its point of view, but this was after the sampling campaign was finished. Thus Atofina Vlissingen was removed from the list of effluents to be sam-pled in this workpackage. Atofina was sampled at a later date (September 2002) and will be analysed and reported separately.

The other 14 companies were willing to participate.

Two of the 14 production sites proved to be discharging effluent via two separate efflu-ent channels, each with a distinct composition. In order to preserve the information of each separate discharge, two additional samples were taken. Thus, sampling of the indus-trial effluents was performed with the 16 samples as budgeted in the work plan.

Choice of cooling water system

In order to account for the use of hypochlorite in relatively ‘open’ systems a cooling wa-ter discharge was to be included in the study. Based on a recent inventory of hypochlo-rite use in cooling water systems in The Netherlands, RIZA has selected a cooling sys-tem with a relatively high consumption of hypochlorite per megawatt of power cooled. This was done in order to enhance the analysability of the sample. Typically, chlorine dosage in cooling systems amounts to several milligrams per litre (mg/l) free chlorine. Traces of organohalogen compounds can be expected in the range of 100-200 micro-gram/litre. The fact that a system with high specific hypochlorite consumption was taken implies that this case reflects a ‘higher than average’ situation.


In the extrapolation to the total hypochlorite use for cooling purposes in The Netherlands (Chapter 8) this is accounted for.

In the specific cooling system, selected for this study, hypochlorite was dosed on a con-tinuous basis at a level of up to 10 mg/l of free chlorine. Of past measurements it is known that about 0.1-0.2 mg/l residual chlorine in the effluent can be expected. The cooling system sampled is a so-called open recirculating system situated at an oil refin-ery complex in The Netherlands and uses drinking water as a feed. Both the inlet and outlet of the cooling system were sampled within this study. The outlet cooling water is treated in a WWTP (waste water treatment plant) before discharge into the environment.

Choice of MWTP

Within the study a municipal wastewater treatment plant was to be included in order to act as a reference for the order of magnitude of effects found within the study. Ideally, the specific treatment plant chosen was not to be industrially loaded. We have chosen for the MWTP of Sleen in the province of Drenthe in The Netherlands. This MWTP is loaded by a cluster of about ten separate villages and hamlets. The total number of in-habitants connected amounts to 17.800, part of which may be from recreational facilities (holiday resorts, leisure parks, camping sites etc.). No industrial discharges other than from dairy farms (wash water from milk tanks) are known to the local water authority.

MWTP Sleen can be considered a ‘state-of-the-art’ treatment plant with a biological dephosphation facility. The average COD-removal efficiency amounts to 96%.

Lake Marken as a reference

Water from Lake Marken was sampled as a reference for a relatively unperturbed surface water in The Netherlands. Lake Marken is known from previous studies (Hendriks, 1994) as a relatively clean surface water system. Sampling of water was performed by a service vessel from Rijkswaterstaat at a standard sampling point in the middle of Lake Marken, some 10 kilometres from the borders of the lake.

Blank sample

Apart from the two reference samples also a blank sample was used in the study. This blank served to determine all possible interferences introduced in the whole chain of ac-tions from sampling up to chemical analysis or toxicity tests. Based on RIZA experience ordinary tap water from the city of Lelystad was used for this blank. This water origi-nates from deep wells and is known to contain only small amounts of trace organics.

2.2 Sampling campaign

Conditions of sampling

Sampling was performed based on the following principles:

• A high degree of comparability of sampling conditions for all samples. In practice this is translated as: only two persons taking all samples based on a strict protocol (see below);


• Samples of industrial effluents must reflect typical operating conditions. This is translated as the absence of known disturbances in the production process or the sub-sequent wastewater treatment. A staff member of the industrial company under con-sideration signs for this on the sampling form. In one case sampling was postponed by two weeks because of plant maintenance;

• Preferably, sampling is performed in the effluent as is discharged to surface water. This is in agreement with a decision of BOVOC. Typically this leads to sampling of wastewater after wastewater treatment (biological or physico-chemical treatment) has been applied. In one case this treatment consisted of a municipal wastewater treat-ment plant, which - of course - also receives wastewater from other sources;

• Whenever possible, samples will be taken in a flow where cooling water discharges are not mixed into yet (but after waste water treatment!) In one case, the cooling wa-ter discharge was temporarily switched off for the purpose of sampling.

Sampling protocol

In the sampling campaign a protocol was used that was already tested in the preparatory phase of the OVOC study. Major elements of this protocol were:

• Grab samples of 70 litres are taken. Sampling has to be performed within a 30 minute time interval;

• The sample is taken by subsequently filling seven polyethylene containers of 10 li-tres content. These containers are as purchased, but are rinsed by means of hot tap water before use;

• In order to establish the amount of variation in composition over the seven contain-ers, the COD value of the effluent was measured twice: before the first container was filled, and after the last container was filled;

• Also samples were taken for the analysis of other macro parameters ( TOC, BOD, pH, EOX, AOX, undissolved substances);

• At the day of sampling two containers are delivered to IVM for further processing (purging and extraction). The remaining five containers are delivered the same day to the cold storage (-18 °C) where they remain for later use;

• Each sample is accompanied by a sample form that states the date and time of sam-pling, the exact location were the sample was taken, a statement of a company staff member declaring normal operating conditions, the time of delivery at IVM and the time of delivery at the cold storage.

Time period

Sampling was performed by Omegam, Amsterdam in the period 29 January- 9 March (2001). Typically each Monday and Wednesday in this period two to three samples were taken and delivered to IVM and cold storage. The exact date and time of sampling were determined in consultation of the firms under consideration.


Coding

The following coding was used Xyz.W.ddmm.stab.n Where:

X Takes the value ‘M’ for an industrial sample or sample from an MWTP and ‘B’ for the blank sample, the sample from Lake Marken or the sample of the inlet water of the cooling system;

yz Stands for a unique letter combination denoting each individual sample;

W Denotes a water sample;

ddmm Stands for the day and month of sampling;

stab Takes the value ‘STAB’ when the effluent can be considered biologically sta-ble, meaning that it has undergone biological water treatment or that it consists of surface water (or tap water). In other cases the parameter stab takes the value ‘NSTAB’. The significance of this parameter is that it allows for the laboratory to process biologically unstable samples prior to stable samples;

n Denotes the number of each individual polyethylene container.

This coding allows all relevant information of treatment of the samples to be passed on to the analytical laboratories, while still maintaining anonymity about the samples origin. The latter is of importance to safeguard objective analysis and interpretation of data. Only one person at RIZA possesses the list which links the sampling code to individual effluents. This person is responsible for the execution of the sampling campaign and will not take part in the interpretation of analytical data.

Execution of sampling campaign

The sampling campaign only had minor practical difficulties. Deviations from protocol that might be relevant are:

• Containers of the sample MCDW.1202 were abusively all sent to cold storage, in-stead of delivering two containers two IVM. Within 24 hours this mistake was re-paired and the sample was extracted by IVM;

• Sample MPQW was given two wrong date suffixes. The sample was taken at March 3 while the codes MPQW2602 and MPQW0902 were given to it. In this report the sample will be denoted as MPQW0903, but the other codes may still be in use for samples or extracts that have already been processed. As MPQW is a unique letter combination within this project possible confusion about the date will not have fur-ther consequences;

• At one industrial firm no truly representative situation could be sampled within the time frame of the sampling campaign. This firm operates two production plants for two different types of products. For logistic reasons only one plant was operated. This situation occurs frequently at the production site. As no indication was given with respect to the duration of this situation, it was decided to take the non-representativity for granted and to go ahead with sampling. When deemed necessary in the further course of the project, a new, representative, sample can be taken.


3. Analysis of main constituents of aquatic effluents (Task 5.2)

3.1 Introduction

In order to get a first insight in the main constituents of the effluents seven so-called macro-parameters have been analysed. These parameters are:

• Extractable organic halogens (EOX); measures the amount of organic halogen com-pounds that can be extracted by petroleum ether. Typically, all non-polar compounds are extracted;

• Absorbable organic halogens (AOX); measures the amount of organic halogens that can be absorbed on activated carbon. In comparison to EOX, AOX also contains more polar or even ionic compounds.

EOX and AOX are of special importance for they both give a first impression of the total amount of organic halogens present in the sample. In principle, AOX levels will be higher than EOX as the group of compounds analysed is broader. However, the specific analysis method used for EOX in this study also extracts volatile organic halogens (VOX), as it does not apply an evaporation step. Therefore, presence of volatiles may lead to EOX levels greater than AOX levels as AOX is insensitive to volatiles.

Other parameters that are determined:

• Total organic carbon (TOC); measured by the production of carbon dioxide gas in the total oxidation of organic molecules;

• Chemical oxygen demand (COD); measures the presence of substances that are oxi-dised by sodium dichromate. The ratio of COD to TOC varies for different classes of molecules;

• Biological oxygen demand (BOD); measures the presence of substances that are oxi-dised by bacteria within in a certain time frame. The amount of BOD present is an indication of the amount of matter that can readily be broken down by biological processes. This can happen in the environment but also during transport or storage of samples;

• pH; measures the acidity of the sample; • Undissolved matter; this parameter gives an indication of the degree by which a pre-

ceding sedimentation (or filtration) step in wastewater treatment (e.g. like in biologi-cal waste water treatment) has acted properly. Thereby this parameter might be used to check the normal operation of the production process and/or the wastewater treat-ment. Undissolved substances levels up to 50 mg/l indicate normal operation of a sedimentation step.

This set of macro-parameters mainly serves to get a first, broad impression of the state of the effluent during sampling and may be helpful in assessing the production situation at the moment of sampling. Anomalies in production may somehow be expressed in these parameters.


3.2 Interpretation of results of macro-parameters

The results of the analysis of macro-parameters are given in Table 3.1.

The blank sample shows no detectable organic compounds. This implies that at this stage there is no sign of any systematic error or background introduced into the analysis.

The composition of the reference samples of Lake Marken and MWTP Sleen are as-sessed as indicative of a typical situation.

The cooling water samples appear to show that (halogenated) organics are added to the cooling water. The AOX level in the outlet is higher by about 600 microgram/l compared to the inlet value. However, in the cooling water system, in which cooling water is recir-culated over an evaporative cooling tower, the water stream is concentrated by a factor of about 4. When this factor is taken into account, the increase in AOX level over the cooling system is less clear.

The industrial water samples have been checked for the condition of a ‘normal’ produc-tion situation by means of pH and levels of undissolved matter. Samples with levels of undissolved matter above 50 mg/l and pH levels under 5 have been compared with measurements of the corresponding effluents as available in the WVO-info database of the Dutch Directorate general for Public Works and Water Management (Rijkswater-staat). This gave no indication for a non-functioning of wastewater treatment or extreme effluent conditions.

This set of macro-parameters mainly serves to get a first, broad impression of the state of the effluent during sampling and may be helpful in understanding how the corresponding production situation was. Anomalies in production may be expressed in these parame-ters.

The following remarks have to be made concerning the halogen content of the samples:

• In samples with EOX levels well over AOX levels considerable amounts of volatile halogenated compounds can be expected. This relates to MDEW2102, MIJW2102 and MABW2602. The volatility of the compounds must be taken into consideration when assessing the results of toxicity test and/or chemical analysis later in this study. Depending on the analytical steps applied to the sample and the respective extracts (e.g. evaporation, long residence times in open vessels or vials) volatile compounds may to a certain degree have disappeared from samples or extracts. Therefore, when interpreting results later on this project special attention should be given to the fate of volatile compounds;

• AOX levels are found of up to 4400 µg/l. Depending on the polarity of the com-pounds concerned these halogens will or will not be found in all extracts as prepared by liquid-liquid extraction (performed in pentane, only extracting non-polar com-pounds). As Figure 4.1 indicates there is a marked cut-off of the recovery for extrac-tion at Kow levels below 2. Polar compounds measured in AOX will therefore not be relevant for analyses and toxicity tests performed on the extracts. The analysis of EOX, on the other hand, involves a petroleum ether extraction step comparable to the liquid-liquid pentane extraction. Thus, EOX levels are a good indication for non-polar halogenated compounds, when no volatiles are present.


Thus, in samples with AOX levels well over EOX levels the majority of the halogens are expected to be polar compounds. Amongst others, this holds for samples MPQ, MGH, MCD, and MRS;

• The EOX value listed for MIJW2102 (6300 µg/l) was far above what could be ex-pected on the basis of available data for this specific effluent for the year 2000 3 (200 µg/l or less). The corresponding AOX value is much lower (240 µg/l), which is indi-cated above as signalling the presence of volatile substances. Nevertheless, because of the high value, the routing of the sample from sampling up until reporting of the analysis results was checked for possible errors, but none was found. Also, a re-analysis of the EOX parameter for MIJW2102 was performed on the defrosted sam-ple from cold storage in November 2001. Then, the value <100 µg/l was established. This, however, is consistent with the assumption that the EOX parameter is strongly determined by volatile compounds and the phenomenon that upon defrosting volatile compounds have disappeared 4.;

• The same remark concerning conspicuously high EOX levels can be made for sam-ple MABW2602. For this sample too, the value may be explained by volatile sub-stances as EOX is far above AOX. Upon defrosting the EOX value was decreased to 760 µg/l, while AOX remained fairly unchanged 4.

Based on the macroparameters analysed it can be concluded that there are no indications of any production situation not being representative for normal operation. Regarding halogen content it can be stated that substantial amounts of the halogen content deter-mined as AOX and EOX pertain to either polar or volatile substances.

3 Data in the WVO-info database of the Minstery of Transport, Public Works and Watermanage-

ment. This database contains the results of wastewater analyses that are performed in the framework of discharge permit enforcement.

4 See also report of Workpackage 8.1 Biodegradation studies.


Table 3.1. Results of the analysis of macro-parameters.

NEN6676 NEN-EN1485 NEN-EN1484 NEN6633 NEN-EN1899 NPR6616 NEN-EN872 Code Remarks EOX (ug/l) AOX(ug/l) TOC (mg/l) COD* (mg/l) BOD (mg/l) pH undissolved (mg/l) Industrial effluents MPQW0903 <100 4400 17 554-562 51 3.2 116.0 MDEW2102 430 80 18 55-56 2 7.5 4.2 MIJW2102 6300** 240 15 45-48 2 7.6 7.4 MABW2602 3300 740 <5 <100-<100 <0.5 4.9 12.6 MBCW2602 <100 750 <5 <10-<10 2 8.4 12.4 MJKW1902 <100 80 7 <10-<10 <3 0.7 <1 MKLW1902 440 160 10 26-47 33 2.3 2.2 MOPW1902 <100 90 28 240-241 4 8.3 5.8 MEFW1402 <100 540 17 28-29 3 7.3 76.5 MFGW1402 130 960 170 330-373 250 5.8 109.6 MGHW1402 <100 4300 10 <40-<40 2 7.4 20.8 MCDW1202 340 2200 70 127-129 7 6.4 46.2 MLMW0702 <100 <20 12 <10-10 3 1.7 <1 MRSW0502 <100 1500 52 111-114 6 7.9 14.5 MNOW2901 <100 50 8 30-30 <3 7.6 6.9 MQRW2901 <100 530 22 32-32 <3 8.1 24.5 Cooling water BSTW1202 inlet cooling water <100 90 <5 <10-11 <1 8.1 <1 MSTW1202 outlet cooling water <100 670 17 34-38 <1 8.5 <1 References MMNW0502 MWTP Sleen <100 80 22 24-33 10 7.0 19.6 BTUW0802 Lake Marken <100 30 18 33 1 8.0 89.6 Blank BUVW0802 Blank sample <100 <20 <5 <10-<10 <1 7.8 <1 * Two values are given, representing the two COD-samples at beginning and end of the sampling procedure. ** Value far above normal range (see text).

Final report WP 5&6 OVOC Project March 2003

21

4. Purge and trap, extractions and fractionation (Task 5.3a)

4.1 Introduction

In the period between 29 January and 9 March 2001, 21 water samples that were sam-pled and distributed as described in work package 5 (task 5.1a), were received at the IVM laboratory for purging, extraction and fractionation and subsequent chemical analy-sis and biological assays. Among the water samples were 16 industrial effluents from chlorine production processes, a cooling water discharge, the influent to the cooling sys-tem, one effluent from a municipal wastewater treatment plant, a tap-water sample func-tioning as blank and a reference water sample taken from the Markermeer, which is con-sidered to be a relatively clean surface water in the Netherlands. The industrial effluents originated from wastewater treatment installations at 14 production sites. One of the in-dustrial effluents was treated in a municipal wastewater treatment plant.

The selection of industrial effluents was made by the BOVOC (Tukker, 2000).

All samples were provided with their own unique sample code, ensuring anonymity while at the same time describing the sample in terms of type of sample, e.g., water, product or air, sampling location and date. In the course of this study, the sample code will be extended with information of the status of the sample, and where it should go next for chemical analysis or bioassay. Since each sample will go through various steps at different laboratories, the sampling code will grow with essential information (Belfroid et al., 2000).

In this report, the purging, extraction and fractionation of 21 water samples is described. The resulting extracts are distributed and sent to the appropriate partners for further processing.

4.2 Method

In Figure 1.1, a schematic representation of the treatment of aquatic effluents for prepa-ration of extracts for chemical analysis and bioassays is given, together with the chemi-cal and biological screening tests that will be performed on the extracts.

Of each sample, part was dedicated for purging and trapping of the compounds with des-tination air, while with another part liquid/liquid extraction and fractionation was carried out. For each sample, this resulted in five extracts:

• Purge and trap extract; • Whole extract after liquid/liquid extraction, part of which was subdivided by

fractionation into fractions with different hydrophicity classes: • Low hydrophobicity: Log Kow < 4 • Medium hydrophobicity: Kow 4 – 6 • Very hydrophobic compounds, log Kow > 6

Institute for Environmental Studies

22

In the following sections, a more detailed description of the preparation of these extracts is given.

Purge and trap

For the preparation of the extracts containing the compounds with destination air, 4 litre of the raw effluent sample was transferred into a glass 10 L flask. Using a home made purging device (Stroomberg et al., 2000), clean nitrogen gas was lead through the water sample at room temperature. The gas flow from the container, containing volatile com-pounds evaporated from the sample, was subsequently led through 5 ml of methanol at 0˚C, where they were trapped by condensation. The purging procedure was carried out for the duration of 0.5 hr. The resulting methanol extracts were dried with 1 gram Na2SO4 . The extracts were stored at 4ºC until further processing (i.e., chemical analysis and DR-CALUX).

Liquid/liquid extraction

Liquid/liquid extraction was carried out with pentane as the extraction solvent (US EPA method 551.1; D.J. Munch and Hauptman, 1995). During the extraction procedure, variations in the formation of emulsions, colour, smell, and pH have been recorded (see Appendix-III, Table A3.1).

Of each sample, 8 L was taken and divided into 4 portions of 2 L. Prior to extraction the pH of the sample was determined. Subsequently, the sample was acidified until pH < 2 with concentrated H2SO4 (1:1 diluted with water). Each 2 L portion was extracted for 30 minutes with 100 ml pentane. In case of emulsification, a very small amount of ethanol was applied onto the organic layer in order to obtain a better phase separation. The or-ganic solvent was transferred to another container, while the pH of the water sample was increased to pH > 11 with 10 M NaOH. Once more, extraction with 100 ml pentane was carried out for 30 minutes. The pentane fractions were combined and dried with Na2SO4 before reduction of the volume by rotary evaporation to 0.5 ml. To this small volume of extract in pentane, 8 ml methanol was added, followed by removal of the pentane under a gentle stream of nitrogen. Thus the quantities of compounds present in 1 ml of the ex-tract corresponded to 1 L of the original effluent. Of these total extracts in methanol, 2 ml was used for chemical analysis, DR-CALUX, chronic micro toxicity with bacteria, carp hepatocyte assay and fish early life stage (ELS) test. Only for the zebra fish ELS tests the extract was concentrated with a factor 3 in order to reduce toxic effects related to the presence of methanol in the extracts.

Fractionation into three log Kow intervals were carried out with 6 ml of the total ex-tracts, as described in the following sections. All extracts were stored at 4ºC until further use.

The theoretical extraction efficiency is determined by the pentane-water partitioning co-efficient (Kpentane) and the ratio of the volumes of water (8L) and pentane (2 x 0.4 L). In order to provide an indication of the theoretical extraction efficiency we assumed that Kpentane may be approximated with Kow (octanol-water partitioning coefficient). In Figure 4.1 the theoretical extraction efficiency is indicated for the current dimensions and (for reasons of comparison) a system with equal volumes of water and pentane.


23

Following recommendations of the technical workshop, recovery experiments were exe-cuted for the extraction method. A mixture of chlorinated POPs, containing PCBs and chlorinated pesticides, covering a range of Log Kow (3.7 – 8.2) was spiked in triplicate to 8L of HPLC water and extracted under similar conditions. Concentrations were meas-ured with GC-ECD. The measured recoveries are listed in Appendix-IV (Table A 4.2) and plotted in Figure 4.2 as a function of the Henry Law Constant (H), taken as a meas-ure of the volatility of the spiked compounds. The recovery of the extraction strongly depends on the volatility. For compounds with intermediate volatility (H range 0.01 - 2) the recovery ranges from 60-100%. For more volatile compounds (H range: 2 – 1700) the recovery is limited to poor (6 – 69%). There is no relation with hydrophobicity, the more volatile compounds have Log Kow

values in the range 4.5 to 7.4.

It is clear that with the current extraction procedure, which includes various evaporation and concentration steps in order to obtain sufficient material for the toxicity tests, the more volatile (H >2) and more polar compounds (Log Kow < 2) compounds are not ex-tracted quantitatively. For the focus of the current study (PBT) compounds this is ac-ceptable.

Figure 4.1 Theoretical extraction efficiency for 8L water and different volumes of pen-tane as a function of Kow (explanation in text). The samples were sequen-tially extracted with 2 x 400 ml. The theoretical recovery of an extraction with 8L pentane is indicated for reasons of comparison.

Fractionation

For fractionation of the total extracts in defined log Kow intervals that bear a relation to their eventual destination in the environment, an HPLC method was used with methanol as the eluent at a flow rate of 1 mL/min (slightly modified from Klamer et al., 1995 and Verbruggen et al., 1999). The log Kow intervals were log Kow < 4 (destination water), log Kow 4 – 6 (destination sediment) and log Kow > 6. For this relatively rough fractionation, separation of the extracts was carried out under isocratic conditions in order to simplify further sample treatment and to minimize any loss of components. A 25 cm Vydac C18 column (5 µm, 4.6 mm i.d.) and a fraction collector were used for fractionation of sam-ples.


24

Figure 4.2 Measured recovery (in %) of the extraction of a mixture of PCBs and chlorinated pesticides added to 8 L of HPLC water as a function of the Henry Law Constant.

Between samples, a cleaning run by injection of methanol was executed in order to avoid any carry-over of sample components into fractions of other samples. A mixture of PAHs (EPA-16) was used to determine the relation between retention time and log Kow (see Figure 4.3). Compounds with known log Kow’s of 4 and 6 were used to define the time intervals for switching the fraction collector to the corresponding vials.

Figure 4.3 Typical example of fractionation of PAHs mixture in relation to Log Kow. Cut-off intervals for different classes of hydrophobicity are indicated: Log Kow <4: 0-3.75 min.; Log Kow 4-6 3.75 - 11min; Log Kow >6: 11-45 min.).

The total extracts that were obtained after liquid/liquid extraction (section 2.2) were con-centrated from 6 ml to 0.5 mL with nitrogen. Five times 100 µl was transferred into 5 separate HPLC vial- inserts. On top of all extracts, 50 µl of methanol was applied in or-der to avoid injection of air into the HPLC system. From each vial 100 µl was injected. Mixing of the applied methanol and the concentrated total extract is highly unlikely be-cause of differences in density between the two phases. The fractions of the 5 subsequent

012345678

0 5 10 15 20 25

Retention time (min)

Lo

g K

ow

0%20%40%60%80%

100%120%

0.01 0.10 1.00 10.00 100.00 1000.00

H (Pa.m3/Mol)

Rec

over

y %


25

runs corresponding to a single sample were pooled, resulting in approximately 15 ml of extract in MeOH for the fraction with log Kow < 4, 43 ml for the fraction with log Kow 4 – 6, and 170 ml for the fraction with log Kow > 6. To avoid any loss of the more hydro-phobic compounds, the fraction that was collected for log Kow > 6 was increased with nearly 20 ml for each injection. The volumes of the different fractions were reduced to 6 ml under nitrogen or by rotary evaporation (for the large log Kow> 6 fraction). With these fractions, chemical analysis, DR-CALUX and the carp hepatocyte assay will be carried out. All fractions were stored at 4 ºC until further use.

The recovery of a mixture of PCBs ranged from 31% to 29% for the less volatile conge-ners (PCB nrs. 180, 138 and 153) with H < 3 and from 9% to 33 % for the more volatile congeners (PCB 28, 52., 101 and 118) with H > 3 (see Table 4.1). The variability of the recovery is limited, with a coefficient of variation (relative standard deviation) ranging from 2% to 13%. The limited recoveries of the automated fractionation system used in this screening study (WP 5-6) were attributed to handling losses and losses during the evaporation and concentration steps. Further tests with mixtures of PCBs and chlorinated pesticides (results not shown) revealed that the simple isocratic fractionation, which was satisfactory for PAHs in this study and worked well for mixtures of chlorobenzenes in the study of Verbruggen et al. (1999), did not perform sufficient to separate all com-pounds in the appropriate classes. An improved system with manual injection was tested with enhanced recoveries (80% to 88%) and precision (coefficient of variation: 1.5% to 3.5%) for the less volatile PCBs (H < 3) and lower but acceptable recovery rates (50% to 78%) and precision (3 to 19%) for the more volatile PCBs (H > 3). This manual system is being applied in the biode-gradation studies (WP 8.1) and the installation specific studies (WP-9). A further optimised system with manual injection, and fine fractionation based on gradient elution, was developed and validated for the further work packages (WP 7 and WP 8.2), the results will be included in the report on WP 7.

Table 4.1 Recovery of a mixture of PCBs with the automated fractionation system applied in the screening study (WP 5-6) and a system based on manual injection to be applied future studies (WP 8-9).

Log Kow H Automated system Manual injection Pa.m3/Mol Recovery mean ± s.d n=3 Recovery mean ± s.d (n=3) PCB 180 8.27 1.0 39 ± 13% 88 ± 3% PCB 138 7.44 2.1 34 ± 4% 85 ± 3% PCB 153 7.75 2.3 31 ± 3% 80 ±1 % PCB 101 6.8 9.1 21 ± 3% 66 ±5% PCB 28 5.62 20.3 9 ± 2 % 50 ± 9% PCB 52 6.09 20.3 12 ± 2 % 54 ± 8% PCB 118 7.12 29.2 33 ± 3% 78 ± 2%

4.3 Observations on samples

The nature of the work described in this section only allows a qualitative representation of the observations, some of which are prone to subjectivity. In Table A-3.1 (Appendix-III), observations made with regard to pH and smell before liquid/liquid extraction, col-our of the extract and remarks are given for all the samples that were processed.


26

Please note that the sample coded MPQ.W.2602.NSTAB corresponds to sample MPQ.W.0902 in chapter 2.2.

Most initial pH values of the received water samples were between 6 and 8, with an exception of pH 4.5 for sample MPQ.W.2602, pH 1.5 for sample MLM.W.0702 and pH 1 for MJK.W.1902.

The extract of water sample MFG.W.1402 had a sweet, apple like smell, while most others had no smell at all or smelled slightly of chlorine or soil. Sewer like smell was observed for sample MJK.W.1902, smells related to petrol for sample MIJ.W.2102 and MPQ.W.2602.

Four of the final extracts, MMN.W.0502, MCD.W.1202, MFG.W.1401 and MPQ.W.2602, were found to contain some precipitate, all others were clear and some-times slight coloured solutions. When coloured, the colour was mostly yellowish, but pink (MNO.W.2901) and slightly brown (MPQ.W.2602) have also been observed.

In general, the amount of 10 M NaOH necessary to adjust the pH to >11 was in the order of 5-20 ml, except for two water samples, MJK.W.1902 and MPQ.W.2602. A possible explanation is that these water samples were buffered.

Sample MEF.W.1402.LL.ELS (dedicated for the ELS test) was lost during sample handling. All other tests for this sample can take place without problems. For the second series of ELS tests a new extract MEF.W.1402.LL.ELS was prepared.

4.4 Conclusions

The preparation of extracts by purge and trap, liquid/liquid extraction and HPLC frac-tionation for chemical analysis and bioassays was completed, resulting in extracts that are suitable for chemical screening on chlorinated compounds by GC with electron cap-ture detection (ECD) and with mass spectrometric detection (MSD) as described in task 5.4 of work package 5 of the Work Plan for the Main Study – Phase 2 (Van Hattum et al., 2000). In addition, the samples are suitable for the biological screening on toxicity parameters in the DR-CALUX, chronic micro toxicity test for bacteria, fish early life stage test, and carp hepatocyte assay as described in work package 6. It should be noted that the requirement of obtaining sufficient and suitable samples (methanol as carrier solvent) for biological testing poses constraints to the objective of exhaustiveness of the extraction. Based on recovery studies it was concluded that the (liquid-liquid) extraction method had acceptable recoveries for non-volatile hydrophobic compounds. The auto-mated isocratic fractionation method applied in the current screening study performed well for mixtures of PAHs, but had a limited recovery (with acceptable precision) for non-volatile PCBs and a poor recovery for more volatile PCB congeners. For the further experiments in WP 8 and 9 (not included in this report) an isocratic system based on manual injection was tested. Recovery rates of PCBs were improved. For the fine frac-tionation in WP-7 and 8 a system based on manual injection and gradient elution was op-timised. The results will be included in the report on WP 7.


27

5. Analysis of halogenated fractions (Task 5.4)

5.1 Introduction

The extracts that were prepared as described in Chapter 4 (Task 5.3a) were screened by gas chromatography (GC) with electron capture detection (ECD) and mass spectrometric detection (MSD) on the presence of chlorinated organic compounds. With electron cap-ture detection, a rapid assessment can be made with regard to the amount and the number of ECD responsive compounds in the extracts. The ECD detector has a superb sensitivity for organohalogen compounds, although it should be noted that other compounds with electronegative functional groups also may be responsive (peroxides, quinones and nitro groups). The ECD detector is insensitive towards compounds with functional groups such as amines, alcohols and hydrocarbons.

5.2 Experimental approach

GC-methods

Because of the differences in nature of the purge and trap extracts and the extracts that were obtained by liquid/liquid extraction and subsequent HPLC fractionation, two dif-ferent GC-methods were used with regard to the column temperature programme and in-jection. For GC-ECD and GC-MSD, the GC methods were identical, except for the in-jection volume of the fractions obtained by liquid/liquid extraction and HPLC fractiona-tion. For both methods, a SGE BPX5 column with a length of 24.4 m, a diameter of 220 µm and a film thickness of 0.30 µm was used. For GC-ECD and GC-MSD of the purge and trap extracts and GC-ECD of the fractionated extracts, splitless injection of 1 µl of extract was done at a temperature of 60 °C. In order to increase the sensitivity of the GC-MSD analysis of the fractionated extracts, injection of 5 µl was done in solvent vent mode at 40 °C. Because of the volatility, this was not possible for the purge and trap ex-tracts. The extracts were not diluted for injection.

The column temperature programme that was used for the purge and trap extracts started at 60 °C for 2 min, increased with 10 °C/min to a final temperature of 250 °C for 35 min. The total run time was 56 min. Hydrogen was used as carrier gas at a constant flow rate of 2 mL/min.

For the extracts obtained with liquid/liquid extraction and HPLC fractionation, the initial column temperature was 60 °C for 3 min, increasing to 200 °C at a rate of 30 °C/min. The column was kept at 200 °C for 15 min, and then the temperature was increased with 5 °C/min to 265 °C for 5 min, and then at a rate of 3 °C/min to a final temperature of 275 °C for 15 min. The total runtime was 59 min. As carrier gas, hydrogen was used at a constant flow rate of 2 mL/min. Reanalysis of the fractionated extracts was done with the same method, except for the carrier gas, which was helium at a constant flow rate of 1 mL/min.


28

For mass spectrometric detection, data were acquired in full scan mode from m/z 50 to m/z 650, at 2 scans/s. A solvent delay for data acquisition of 5 min was used to avoid disturbances in MS detection.

Ideally, simultaneous detection with ECD and MSD should be carried out for the analy-sis of the extracts, but unfortunately this was not feasible due to technical problems. Therefore, GC-ECD and GC-MSD were done in separate runs. In order to assess the chromatographic stability of the system, a standard mixture of 3 PCBs for the fraction-ated extracts and 7 tri- and tetra-substituted chlorobenzenes for the purge and trap ex-tracts was injected regularly, enabling the linking of retention times in the different chromatograms.

Data processing

In order to obtain information on the content of chlorinated organic compounds in the extracts, the total response of each sample was calculated by summation of peak heights of all peaks in the GC-ECD chromatogram of a sample.

The standard mixtures that were injected to verify the stability of the chromatographic system were also used for transformation of the total peak height of a sample into a con-centration of organochlorine compounds. For the fractionated extracts, this mixture con-sisted of PCB 52, PCB 101 and PCB 198, all in a concentration of 100 ng/ml and with retention times covering the whole chromatogram. The standard mixture of chloroben-zenes that was used for quantitation of the purge and trap extracts had a concentration of 250 ng/ml.

For all standard compounds, an average response factor was calculated that can be re-lated to a concentration of organochlorine compounds. With the average response factor, the total peak height for each sample can be transformed to a concentration organochlo-rine compound. Because for each mL of extract an equivalent of 1 L effluent was either purged or extracted, the calculated concentrations are transformed into µg/L effluent. This way of quantitation gives an estimate of the total organochlorine content of the ef-fluents, which can be useful for assessment of the data and comparison of the contribu-tions of the different extracts. However, it should be emphasized that the values calcu-lated for the organochlorine contributions are only an estimate.

Injection of methanolic extracts into the GC system caused major system peaks at vari-ous retention times, and therefore specific time windows were defined where observed peaks would not be added to the total sample peak height. The time intervals for the purge and trap extracts that were excluded from peak height summation were 6.40-7.75 min, 10.60-11.34 min and 13.95-14.27 min. In addition, the presence of chlorinated compounds in the system peaks caused by methanol was investigated. In none of the chromatograms obtained after pure methanol injection, chlorinated compounds could be observed.

Initially, the same approach with slightly different time intervals was applied for the analysis of the fractionated extracts. The generated data were used for qualitative ranking of the organochlorine content in the different extracts.


29

However, based on discussions and recommendations made during a technical workshop (August 30th 2001), a decision was taken to reanalyse the extracts on a revised GC sys-tem in order to generate more reliable data. Using this system, only two minor time in-tervals containing system peaks were excluded from integration, namely 8.43-8.47 min and 9.66-9.74 min. Because no difference in the qualitative ranking occurred after as-sessment of the new data, only the results obtained with the revised GC system are pre-sented.

5.3 Results and discussion

The calculated total organochlorine content of each effluent after reanalysis of the ex-tracts is incorporated in a bar chart for the three extracts that were prepared by HPLC fractionation, which is shown in Figure 5.1. For the total amount of organochlorine, three effluents can be distinguished that contain a much higher level than the other effluents. Samples MAB.W.2602, MFG.W.1402 and MCD.W.1202 have total organochlorine concentrations of 29.8, 9.6 and 11.8 µg/L, respectively. Furthermore, Figure 5.1 shows that there are only minor distinctions in total organochlorine content between the other effluents. This does not mean, however, that all samples are more or less similar, be-cause the constituents making up the total content of organochlorine can be very differ-ent for each sample.

Figure 5.1 Total concentration (in microgram/L effluent) of organochlorine for all ef-fluents, with contribution from the low, medium and high log Kow fraction obtained by liquid/liquid extraction and HPLC fractionation.

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

MPQ.W.26

02

MDE.W.21

02

MIJ.W

.2102

MAB.W.26

02

MBC.W.26

02

MJK.W

.1902

MKL.W.19

02

MOP.W.19

02

MEF.W.14

02

MFG.W.14

02

MGH.W.14

02

MCD.W.12

02

MLM.W

.0702

MRS.W.05

02

MNO.W.29

01

MQR.W.29

01

BST.W.12

02

MST.W.12

02

MMN.W.05

02

BTU.W

.0802

BUV.W.07

02

conc

entr

atio

n or

gano

chlo

rine

(mic

rogr

am/L

)

high log Kowmedium log Kowlow log Kow


30

In the above Figure, the contribution of the high log Kow fraction to the total organochlo-rine content seems negligible, but it cannot be excluded that this fraction will show a re-sponse in the bioassays chosen for the primary effect screening. In Figure 5.2 the or-ganochlorine content of the high log Kow fractions is shown separately for each effluent.

Figure 5.2 Organochlorine content of the high log Kow fractions in microgram/L effluent.

For the low log Kow extracts, the samples MAB.W.2602 and MCD.W.1202 have the highest contributions to the total organochlorine content of the effluents. Samples MAB.W.2602 and MFG.W.1402 have high contributions in the medium log Kow group and the highest organochlorine content in the high low Kow fractions is found in the sam-ples MMN.W.0502, MJK.W.1902 and MCD.W.1202.

To obtain insight in the relative distribution of organochlorines in the fractions with dif-ferent hydrophobicity, a distribution in percentages for each extract (adding up to 100%) is shown in Figure 5.3. High contributions from specific extracts do not automatically mean a high concentration of organochlorine found in that extract.

The concentrations of organochlorines measured with the GC-ECD are much lower than the concentrations measured as EOX or AOX. Apart from the higher specificity of the GC-ECD method this also must be attributed to limited recovery of polar and volatile compounds in the extraction and fractionation method. The figures should therefore be interpreted as low estimates.

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

conc

entr

atio

n or

gano

chlo

rine

(mic

rogr

am/L

)

MPQ.W.26

02

MDE.W.21

02

MIJ.W

.2102

MAB.W.26

02

MBC.W.26

02

MJK.W

.1902

MKL.W.19

02

MOP.W.19

02

MEF.W.14

02

MFG.W

.1402

MGH.W.14

02

MCD.W.12

02

MLM.W

.0702

MRS.W.05

02

MNO.W.29

01

MQR.W.29

01

BST.W.12

02

MST.W.12

02

MMN.W.05

02

BTU.W.08

02

BUV.W.07

02


31

The purge and trap extracts were prepared using fresh effluent and are therefore repre-sented in a separate graph. In Figure 5.4, the amount of organochlorine in the purge and trap extracts is shown. For all the effluents, the organochlorine content is very low (0.3 – 0.5 µg/l), with the highest concentration in the MMN.W.0502 sample. There is no corre-lation between the EOX values that were determined as macro parameter as described in Chapter 2 and the purge and trap results presented here. In general, very little is known about the comparability of data obtained by the determination of EOX and chemical analysis of the purge and trap extracts by GC-ECD, and no reason for these different values can be given.

Figure 5.3 Contribution in percentages of each fractionated extract to the total amount of organochlorine for all effluents.

The low amount of volatile organochlorine compounds in the purge and trap extracts could be explained by their evaporation from the effluent during transportation and han-dling. Another pathway through which losses of volatile compounds could have occurred is through evaporation from the extract during storage and handling. In addition, the choice of purge duration as well as trapping solvent might need some re-evaluation.

5.4 Conclusions

From the 21 samples that were processed, three effluents show high organochlorine con-tent in the fractionated extracts relative to the other effluents. These effluents are MAB.W.2602, MFG.W.1402 and MCD.W.1202. Especially the medium and high log Kow fractions seem to be suitable for further analysis regarding refined fractionation and a primary TIE study, as proposed under work package 7.

0%

20%

40%

60%

80%

100%

MPQ.W.260

2

MDE.W.210

2

MIJ.W

.2102

MAB.W.26

02

MBC.W.26

02

MJK.W

.1902

MKL.W.19

02

MOP.W.19

02

MEF.W.14

02

MFG.W.14

02

MGH.W.14

02

MCD.W.12

02

MLM.W

.0702

MRS.W.05

02

MNO.W.29

01

MQR.W.29

01

BST.W.12

02

MST.W.120

2

MMN.W.05

02

BTU.W.08

02

BUV.W.07

02

high log Kowmedium log Kowlow log Kow


32

Figure 5.4 Concentration of organochlorine in purge and trap extracts.

Because no extreme values of organochlorine content are found for any of the other ex-tracts, the choice for other fractions to enter work package 7 will be directed by the total volume of waste water discharged at the specific sampling point and naturally the results from in vitro and in vivo bioassays.

The very low organochlorine content in the purge and trap extracts indicates that the method used here should be improved by choosing a more dedicated approach for small, volatile molecules such as headspace analysis. Further investigations regarding the purge and trap extracts appears irrelevant.

00,10,20,30,40,50,60,7

MPQ.W.26

02

MDE.W.21

02

MIJ.W

.2102

MAB.W.26

02

MBC.W.26

02

MJK.W

.1902

MKL.W.19

02

MOP.W.19

02

MEF.W.14

02

MFG.W.14

02

MGH.W.14

02

MCD.W.12

02

MLM.W

.0702

MRS.W.05

02

MNO.W.29

01

MQR.W.29

01

BST.W.12

02

MST.W.12

02

MMN.W.05

02

BTU.W.08

02

BUV.W.07

02

[Cl]

in u

g/L

purge and trap


33

6. Toxicity screening with in-vitro tests (Task 6.1)

Extracts of the effluent samples and the different fractions (Log-Kow <4, 4-6 and >6) were tested on their in-vitro activity with both the DR-CALUX assay (section 6.1) and the Carp-hepatocyt tests (VTG, MTT and EROD; section 6.2) as described in the work-plan (van Hattum et al., 2000). The volatile fractions (purge & trap) were tested only with the DR-CALUX assay.

6.1 DR-CALUX assays

6.1.1 Introduction

Dioxins (polychlorinated dibenzodioxins; PCDDs) are one of the most toxic man-made compounds known. Dioxin-like compounds such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) show similar properties. This group of chemicals, also known as polyhalogenated aromatic hydrocarbons (PHAHs), display a wide variety of toxic effects in mammals, birds, and fish. Among the toxic effects ob-served as a result of exposure to PHAHs are immunotoxicity, carcinogenicity, metabolic changes, endocrine function disruption and even death.

The molecular mechanism of action of PHAHs has been studied extensively in mam-mals. It is initiated by binding of the xenobiotic to a soluble cytosolic receptor, named the aryl hydrocarbon (Ah) receptor. The non-occupied Ah receptor is part of a mul-timeric protein complex which contains two heat shock proteins (Hsp90) and another, not yet fully characterised, protein of about 46 kDa (Hsp50) (Landers & Bunce, 1991; Okey et al., 1994). Upon ligand binding, the AhR is transformed to a DNA- binding form and transported into the nucleus. The transformation includes dissociation of the Ah receptor complex and subsequent association with a protein. This protein, named Arnt (Ah receptor nuclear translocator protein), was cloned by Hoffman et al. (1991) and demonstrated to be a component of the DNA-binding form of the ligand-AhR complex. The ligand-AhR-Arnt complex subsequently binds to a specific DNA sequence, termed the dioxin responsive enhancer (DRE), leading to enhanced or inhibited rates of tran-scription of genes associated with the Ah genetic complex (Okey et al., 1994; Hankin-son, 1995).

Derived from studying the toxicological mode of action of dioxin and dioxin-like com-pounds, an in-vitro bioassay was developed as a screening tool for the detection of diox-ins and dioxin-like compounds in a wide variety of matrices. The Dioxin Responsive – Chemically Activated LUciferase eXpression (DR-CALUX) bioassay comprises a ge-netically modified H4IIE rat hepatoma cell-line, incorporating the firefly luciferase gene coupled to DREs as a reporter gene for the presence of dioxins and dioxin-like com-pounds. Cells that are exposed to dioxins or dioxin-like chemicals not only express pro-teins and enzymes that are under normal circumstances associated to the DRE, but also luciferase. By addition of the appropriate substrate for luciferase, light is emitted. The amount of light produced is proportional to the amount of ligand-AhR binding which is related to 2,3,7,8-TCDD toxic equivalents (TEQs).


34

Hence, the observed response is a culmination of the full molecular cascade of events in-volved in the receptor-mediated transactivation, making bioassays such as the DR-CALUX® biologically relevant.

The DR-CALUX® bioassay is used as a very sensitive screening tool for assessing com-plex mixtures of dioxin and/or dioxin-like chemicals in a wide variety of matrices. In-stead of determining the number of individual congeners in a complex mixture, the DR-CALUX bioassay analyses the total toxic equivalence of polyhalogenated aromatic hy-drocarbon (PHAH) and polyaromatic hydrocarbons (PAH) mixtures in samples under investigation. Since genetic modified H4IIE rat hepatoma cell-line still possesses fully functional and inducible bio transformation enzymes, such as cytochrome P4501A, the cells are capable of metabolising metabolic vulnerable chemicals such as PAHs and hence distinguish between PAHs and more metabolic stable chemicals such as dioxins.

6.1.2 Sample information and protocol

One hundred and five water sample extracts in methanol were delivered in glass vials fit-ted with Teflon sealed caps at 4oC and stored at 4oC prior to analysis. For internal proc-essing and for chain of custody control, samples received an internal coding (see Table 6.1). Normal standard operating procedures for the DR-CALUX bioassay require ex-tracts to be dissolved in DMSO. Therefore, an adaptation to the normal standard operat-ing procedure was executed. The final DMSO concentration in cell-medium was changed to 0.04% DMSO and 0.36% methanol. Both the standard TCDD calibration range and the extracts to be tested were added to the cells for incubation, resulting in a final DMSO and methanol concentration in the cell-medium of 0.04% and 0.36% respec-tively.

The analysis of Ah-receptor mediated luciferase activity in DR-CALUX® cells was per-formed according to standard operating procedures (SOP no.: Pe-BDS-004, version e). Calculation of TCDD-TEQ for samples measured in the DR-CALUX® bioassay and sub-sequent control and review of the obtained results, was performed according to standard operating procedures (SOP no.: Pe-BDS-007, version e). In short, DR-CALUX® cells are seeded in 96 wells microtiterplates and incubated for 24 hours in a CO2-incubator. Expo-sure-medium was prepared prior to exposure of the cells. If required, samples were di-luted in methanol prior to exposure. All analyses are performed in triplicate. After 24 hours of incubation, the exposure-medium is removed and cells are lysed with Lysis Re-agent and placing the microtiterplate at 4oC for 0.5 hours. Ah-receptor mediated induced luciferase production is then analysed by addition of luciferin to the wells and subse-quent detection and quantification of luminescence using a luminometer. All methods are described in work protocols and standard operating procedures (SOPs), and based on previous work (Garrison et al., 1996; Murk et al., 1996 and 1997).

At performing DR-CALUX® bioassays, every microtiterplate contains a concentration-series of TCDD. After detecting luminescence, the TCDD-concentration-series is used for constructing a TCDD calibration curve. The TCDD calibration curve is fitted using a 1-site ligand fit.


35

Formulae: 01

* ( )xy ax a

=+

).

in which Y= The response in RLU (corrected for the RLU result of DMSO);

X= The concentration in pM TCDD-TEQ in the well;

A0= The maximum response;

A1= The Ec50 of the curve;

Data is corrected for background RLU. The amount of TCDD-TEQs in the samples ana-lysed is calculated by interpolation of the DMSO corrected sample data in the calibration curve. Finally the TCDD-TEQ concentration in the analysed sample is expressed in pg TCDD per litre extracted water.

6.1.3 Results

The DR-CALUX bioassay results were obtained using an adjusted standard operating procedure. As a result, the DR-CALUX response was slightly different from normal standard procedures. In Figure 6.1, TCDD standard curves are shown from both the standard procedure (DMSO) and the adjusted procedure (methanol). As a consequence, a control chart for the bioassay is not available. However, the reference sample used in the present study showed similar results as obtained in studies using the normal standard protocol for DR-CALUX analysis. Furthermore, the EC50 abstracted from the TCDD standard curve was equal to the EC50 abstracted from TCDD standard curve in the nor-mal standard procedure. Hence, the results from the present analyses can be qualified as reliable.

Table 6.1 shows the amount of water extracted and the volume of methanol used in which the extracts were finally dissolved. The results of the DR-CALUX analyses of the water extracts are given in Table 6.1. Data are expressed as pg 2,3,7,8-TCDD-TEQs per liter water extracted. Detectable amounts of dioxins and/or dioxin-like compounds were found in all total extract, except for 7 samples (BST.W.1202.LL, BUV.W.0702.LL, MBC.W.2602.LL, MGH.W.1402.LL, MLM.W.0702.LL,MNO.W.2901.LL, and MRS.W.0502.LL). In these samples, dioxin and/or dioxin-like content was below the limit of detection. The highest concentration of TCDD-TEQs was observed in sample MCD.W.1202 (1066 pg TCDD-TEQ/l water) whereas the lowest quantitative amount of TCDD-TEQs was found in sample MEF.W.1402.LL (99 pg TCDD-TEQ/l water) (MCD.W.1202 > MAB.W.2602.LL > MMN.W.0502.LL > MQR.W.2901.LL > MFG.W.1402.LL >MIJ.W.2102.LL > MDE.W.2102.LL > MST.W.1202.LL > MEF.W.1402.LL).

In sub-samples, quantitative amounts of TCDD-TEQs were only found in MCD.W.1202. In all other sub-samples of total samples with quantitative amount of TCDD-TEQs, de-tectable amounts of dioxins and/or dioxin-like compounds were only found in sub-samples with logKow greater than 4.

None of the so-called “PUR” (purge & trap) sub-samples showed an Ah-receptor medi-ated induction of luciferase in the DR-CALUX bioassay.


36

Figure 6.1 2,3,7,8-TCDD calibration curve.

Table 6.1 Result DR-CALUX bioanalyses.

OVOC code Internal code Extracted water volume (liter)

Methanol volume used for dissolving (ml)

2,3,7,8 TCDD-TEQ (pg TCDD-TEQ/l water)

BST.W.1202.LL.BDS M-101-481 8 8 n.d.b BST.W.1202.LL<4.BDS M-101-513 8 8 n.d.b BST.W.1202.LL>6.BDS M-101-515 8 8 n.d.b BST.W.1202.LL4-6.BDS M-101-514 8 8 n.d.b BST.W.1202.pur.BDS M-101-461 4 5 n.d.b BTU.W.0802.LL.BDS M-101-487 8 8 <89a (37) BTU.W.0802.LL<4.BDS M-101-531 8 8 n.d.b BTU.W.0802.LL>6.BDS M-101-533 8 8 n.d.b BTU.W.0802.LL4-6.BDS M-101-532 8 8 n.d.b BTU.W.0802.pur.BDS M-101-467 4 5 n.d.b BUV.W.0702.LL.BDS M-101-479 8 8 n.d.b BUV.W.0702.LL<4.BDS M-101-507 8 8 n.d.b BUV.W.0702.LL>6.BDS M-101-509 8 8 n.d.b BUV.W.0702.LL4-6.BDS M-101-508 8 8 n.d.b BUV.W.0702.pur.BDS M-101-560 4 5 n.d.b MAB.W.2602.LL.BDS M-101-494 8 8 309 ± 9 MAB.W.2602.LL<4.BDS M-101-552 8 8 n.d.b MAB.W.2602.LL>6.BDS M-101-554 8 8 <89a (50) MAB.W.2602.LL4-6.BDS M-101-553 8 8 <89a (26) MAB.W.2602.pur.BDS M-101-474 4 5 n.d.b

Legends and notes explained at end of table.

0.1 1 10 100 1000

[TCDD] pM in well

0

10

20

Rea

ltive

Lig

hts

Uni

ts, S

ampl

e - R

ealti

ve L

ight

s U

nits

, DM

SDMSO

Methanol


37

OVOC code Internal code Extracted water

volume (liter) Methanol volume

used for dissolving (ml) 2,3,7,8 TCDD-TEQ

(pg TCDD-TEQ/l water) MBC.W.2602.LL.BDS M-101-493 8 8 n.d.b MBC.W.2602.LL<4.BDS M-101-549 8 8 n.d.b MBC.W.2602.LL>6.BDS M-101-551 8 8 n.d.b MBC.W.2602.LL4-6.BDS M-101-550 8 8 n.d.b MBC.W.2602.pur.BDS M-101-473 4 5 n.d.b MCD.W.1202.LL.BDS M-101-486 8 8 1066 ± 22 MCD.W.1202.LL<4.BDS M-101-528 8 8 <89a (39) MCD.W.1202.LL>6.BDS M-101-530 8 8 165 ± 10 MCD.W.1202.LL4-6.BDS M-101-529 8 8 296 ± 32 MCD.W.1202.pur.BDS M-101-466 4 5 n.d.b MDE.W.2102.LL.BDS M-101-491 8 8 121 ± 6 MDE.W.2102.LL<4.BDS M-101-543 8 8 n.d.b MDE.W.2102.LL>6.BDS M-101-545 8 8 n.d.b MDE.W.2102.LL4-6.BDS M-101-544 8 8 n.d.b MDE.W.2102.pur.BDS M-101-471 4 5 n.d.b MEF.W.1402.LL.BDS M-101-484 8 8 99 ± 4 MEF.W.1402.LL<4.BDS M-101-522 8 8 <89a (25) MEF.W.1402.LL>6.BDS M-101-524 8 8 <89a (47) MEF.W.1402.LL4-6.BDS M-101-523 8 8 <89a (30) MEF.W.1402.pur.BDS M-101-464 4 5 n.d.b MFG.W.1402.LL.BDS M-101-485 8 8 146 ± 7 MFG.W.1402.LL<4.BDS M-101-525 8 8 n.d.b MFG.W.1402.LL>6.BDS M-101-527 8 8 <89a (38) MFG.W.1402.LL4-6.BDS M-101-526 8 8 <89a (50) MFG.W.1402.pur.BDS M-101-465 4 8 n.d.b MGH.W.1402.LL.BDS M-101-483 8 8 n.d.b MGH.W.1402.LL<4.BDS M-101-519 8 8 n.d.b MGH.W.1402.LL>6.BDS M-101-521 8 8 n.d.b MGH.W.1402.LL4-6.BDS M-101-520 8 8 n.d.b MGH.W.1402.pur.BDS M-101-463 4 5 n.d.b MIJ.W.2102.LL.BDS M-101-492 8 8 122 ± 9 MIJ.W.2102.LL<4.BDS M-101-546 8 8 n.d.b MIJ.W.2102.LL>6.BDS M-101-548 8 8 <89a (27) MIJ.W.2102.LL4-6.BDS M-101-547 8 8 <89a (23) MIJ.W.2102.pur.BDS M-101-472 4 5 n.d.b MJK.W.1902.LL.BDS M-101-489 8 8 <89a (55) MJK.W.1902.LL<4.BDS M-101-537 8 8 n.d.b MJK.W.1902.LL>6.BDS M-101-539 8 8 n.d.b MJK.W.1902.LL4-6.BDS M-101-538 8 8 n.d.b MJK.W.1902.pur.BDS M-101-469 4 5 n.d.b


38

OVOC code

Internal code Extracted water volume (liter)



MKL.W.1902.LL.BDS M-101-488 8 8 <89a (44) MKL.W.1902.LL<4.BDS M-101-534 8 8 n.d.b MKL.W.1902.LL>6.BDS M-101-536 8 8 n.d.b MKL.W.1902.LL4-6.BDS M-101-535 8 8 n.d.b MKL.W.1902.pur.BDS M-101-468 4 5 n.d.b MLM.W.0702.LL.BDS M-101-480 8 8 n.d.b MLM.W.0702.LL<4.BDS M-101-510 8 8 n.d.b MLM.W.0702.LL>6.BDS M-101-512 8 8 n.d.b MLM.W.0702.LL4-6.BDS M-101-511 8 8 n.d.b MLM.W.0702.pur.BDS M-101-561 4 5 n.d.b MMN.W.0502.LL.BDS M-101-477 8 8 157 ± 6 MMN.W.0502.LL<4.BDS M-101-501 8 8 n.d.b MMN.W.0502.LL>6.BDS M-101-503 8 8 <89a (60) MMN.W.0502.LL4-6.BDS M-101-502 8 8 <89a (32) MMN.W.0502.pur.BDS M-101-558 4 5 n.d.b MNO.W.2901.LL.BDS M-101-476 8 8 n.d.b MNO.W.2901.LL<4.BDS M-101-498 8 8 n.d.b MNO.W.2901.LL>6.BDS M-101-500 8 8 n.d.b MNO.W.2901.LL4-6.BDS M-101-499 8 8 n.d.b MNO.W.2901.pur.BDS M-101-557 4 5 n.d.b MOP.W.1902.LL.BDS M-101-490 8 8 <89a (40) MOP.W.1902.LL<4.BDS M-101-540 8 8 n.d.b MOP.W.1902.LL>6.BDS M-101-542 8 8 n.d.b MOP.W.1902.LL4-6.BDS M-101-541 8 8 n.d.b MOP.W.1902.pur.BDS M-101-470 4 5 n.d.b MPQ.W.2601. LL.BDS M-101-566 8 8 <89a (71) MPQ.W.2601. LL<4.BDS M-101-565 8 8 n.d.b MPQ.W.2601.LL>6.BDS M-101-563 8 8 n.d.b MPQ.W.2601.LL4-6.BDS M-101-564 8 8 n.d.b MPQ.W.2601.pur.BDS M-101-562 4 5 n.d.b MQR.W.2901.LL.BDS M-101-475 8 8 148 ± 16 MQR.W.2901.LL<4.BDS M-101-495 8 8 n.d.b MQR.W.2901.LL>6.BDS M-101-497 8 8 n.d.b MQR.W.2901.LL4-6.BDS M-101-496 8 8 n.d.b MQR.W.2901.pur.BDS M-101-556 4 5 n.d.b MRS.W.0502.LL.BDS M-101-478 8 8 n.d.b MRS.W.0502.LL<4.BDS M-101-504 8 8 n.d.b MRS.W.0502.LL>6.BDS M-101-506 8 8 n.d.b MRS.W.0502.LL4-6.BDS M-101-505 8 8 n.d.b MRS.W.0502.pur.BDS M-101-559 4 5 n.d.b


39

OVOCcode Internal code Extracted water volume (liter)



MST.W.1202.LL.BDS M-101-482 8 8 104 ± 6 MST.W.1202.LL<4.BDS M-101-516 8 8 n.d.b MST.W.1202.LL>6.BDS M-101-518 8 8 n.d.b MST.W.1202.LL4-6.BDS M-101-517 8 8 n.d.b MST.W.1202.pur.BDS M-101-462 4 5 n.d.b Note: Data are expressed as pg TCDD-TEQ/l water ± s.d. Note: The limit of detection (LOD) and the limit of quantitation (LOQ) for “LL” sub-samples

is 27 and 89 pg TCDD-TEQ/l water respectively. The limit of detection and the limit of quantitation for “pur” sub-samples is 34 and 112 pg TCDD-TEQ/l water respectively. LOD and LOQ further are determined by the quantity of sample available an may vary between samples.

a = Response between limit of detection and limit of quantitation. Actual measured values in this interval have a limited precision and are therefore indicated between brackets.

b = Not detected; response below limit of detection.

6.1.4 Discussion

Using the DR-CALUX bioassay, it was shown that the majority of total samples con-tained detectable amounts of dioxin and/or dioxin-like compounds. Detectable amounts of DR-CALUX responsive compounds were observed in sub-samples with a logKow greater than 4, indicating that the active compounds are more lipophilic of nature. The amounts of dioxin and/or dioxin-like compounds detected in most water samples are relatively low. For comparison, in 1997 the European Commission proposed an amend-ment to the 1994 Directive on the incineration of dangerous waste in order to add limit values for the emission of heavy metals and dioxins into the water. The limits proposed for dioxins were 500 pg/l. although not implemented, this proposed limit gives an indica-tion for classification of the samples analyzed in the present study. Only total sample MCD.W.1202 contained higher amounts of dioxins and/or dioxin-like compounds.

In this respect, it is important to stress that the DR-CALUX bioassay not only detects dioxin but also dioxin-like compounds such as PCBs and furans. These compounds can contribute considerable to the total TCDD-TEQs in samples. However, since the toxico-logical pathway of these compounds also involve the Ah-receptor, they also pose a risk for humans and the environment.

Contrary to what was expected, the reference sample from Lake Marken had a concen-tration (37 pg TEQ/L) just above the limit of detection. In a study by Turkstra and Pols (1986) slightly enhanced levels in sediments (0.11 ng TEQ/g d.s) from the IJmeerput, which is situated in the SW part of Lake Marken were attributed to historic local influ-ences (dumping and burning of chemical waste at Diemerzeedijk). The daphnid tests, applied to XAD-extracts from Lake Marken and other waters in the study of Hendriks et al. (1994), probably are not sufficiently sensitive to respond to the observed level of 37 pg TEQ/L.

In the present study the detection limit and quantitation limit for “LL” samples was 27 and 89 pg TCDD-TEQ/l water respectively.


40

Lower limits of detection and quantitation may be obtained when samples are finally dis-solved in a smaller volume of methanol. The DR-CALUX bioassay only requires small amounts of sample. A final volume of methanol of 500 µl is sufficient for the bioanaly-ses. Hence, a lower detection and quantitation limit is feasible.

6.2 Carp-hepatocyt assays

6.2.1 Introduction

As of October 1999, a co-ordination group of investigators from RIZA, RITOX, TNO-MEP, CML and IVM, along with the input of representatives from Dutch industries, en-vironmental NGOs, and the ministries of VROM and V&W began preliminary studies to address questions involving persistent, toxic and bio accumulating (PTB) compounds in the chlorine chain (OVOC) of the Dutch environment. The RITOX has recently begun screening water extracts prepared by IVM to assess their potential effects on:

• The agonism/antagonism of the estrogen receptor-mediated production of vitellogenin (VTG), and thus their (anti)estrogenic potential;

• The induction of cytochrome P4501A (CYP1A) enzymes, and thus their potential to cause dioxin-like toxicities. The induction of CYP1A enzymes is a consequence of an agonistic effect on the aryl hydrocarbon (Ah) receptor. The CYP1A enzyme activity in the hepatocytes is measured as the ethoxyresorufin-O-deethylase (EROD) activity;

• The potential cytotoxicity of the extracts in the carp hepatocytes is determined by measuring effects on the mitochondrial dehydrogenase activity, using the MTT reduction test.

The present report provides an update of the screening experiments in the CARP-HEP assay of 21 water extracts provided by IVM.

6.2.2 Methods

Carp hepatocytes isolation and culture

The common carp (Cyprinus carpio) used in the CARP-HEP assay were genetically uni-form, all male (XY), F1 hybrid progenies (Bongers et al., 1994). Further details of the carp, and the maintenance of the fish prior to use in the assay, have been described in de-tail (Smeets et al., 1999a; Smeets et al., 1999b). A carp liver was freshly perfused by a two-step retrograde technique, and the hepatocytes were isolated and cultured as de-scribed earlier with minor modifications (Smeets et al., 1999a; Smeets et al., 1999b). The perfused liver sections were removed, minced, and sieved through nylon mesh. After washing with buffer, the hepatocytes were re-suspended in culture medium. The cell vi-ability was > 90% as assessed by trypan blue exclusion. The proportion of erythrocytes did not exceed 10% of the total number of hepatocytes.

The isolated hepatocytes were cultured in phenol red-free DMEM/F12 medium (D2906, Sigma, MO, USA) supplemented with 14.3 mΜ NaHCO3, HEPES (final concentration: 20 mΜ), 50 mg/l gentamycin, 1 µΜ insulin, 10 µΜ hydrocortisone, 2% v/v Ultroser SF (steroid-free) serum (Soprachem, France), and 2 mg/l of the protease-inhibitor aprotinin


41

(Fluka, Switzerland) at pH 7.4. The concentration of the cell suspension was 1.0 × 106 cells/ml, and 0.18 ml (or about 180,000 cells) was added to the 60 inner wells of each 96-well tissue plates (Greiner, The Netherlands). The plates were maintained at 24°C for a period of 72 hours to allow the cells to acclimatise and to facilitate optimal adhesion of a monolayer of cells to the bottom of the wells.

Carp hepatocyte treatment for (anti-)estrogenicity assays

After 72 hours the hepatocytes were exposed to the OVOC samples and 17β-estradiol standards in sextuplet. Two plates were used for 17β-estradiol (E2) calibration curves, one for E2 dissolved in DMSO (dimethyl sulfoxide) and one for E2 dissolved methanol (MeOH). Final concentrations of E2 in the culture medium were 0.6, 2.0, 6.0, 20, 60, 100, 200, 600, 1000 and 6000 nΜ. For the estrogenicity assays, the MeOH extracts of the OVOC samples were diluted 1000- or 10 000-fold in culture medium just prior to dosing. Thus, the cells were exposed to extract concentrations equivalent to 0.18 or 0.018 ml of original water sample. For the anti-estrogenicity studies, the samples were diluted 1000- or 10 000-fold in culture medium containing 100 nM of E2, which is the approximate EC25 concentration for vitellogenin (VTG) production. The final concentra-tion of MeOH in the dosing medium was either 0.1 or 0.2 % (v/v). After 48 hours the medium was replaced by fresh medium containing the exact same concentrations of standards and samples. After a second exposure period of 48 hours days, the medium was transferred to fresh 96-well plates for VTG measurements, and the adherent cells, which remained, were used to measure MTT reduction.

Vitellogenin assay

An indirect competitive enzyme-linked immunoassay (ELISA) determined the amount of VTG secreted into the culture medium. The ELISA procedure as well as calculations to quantify VTG have been thoroughly described (Smeets et al., 1999a; Smeets et al., 1999b). VTG production in male hepatocytes not exposed to exogenous estrogens was less than the detection limit of the ELISA. However, an E2 concentration of about 0.6 nM is the lowest concentration to induce VTG to measurable levels. The detection limit for a positive estrogenic effect was based on this E2 concentration after normalization to equivalent volume of the original water sample. The detection limit (2.4 ng EEQ/l or 0.009 nmol EEQ/l) is based on the minimum concentration of 0.5 nM of E2, which in-duced the lowest measurable level of VTG in the carp hepatocytes. The detection limit is expressed as the ng or nmol per equivalent effluent volume (i.e., in each well the equiva-lent effluent volume is 0.01 l). It was not possible to obtain a VTG level for the DMSO control since the VTG production in the hepatocytes is less than the detection limit of the ELISA. For the OVOC samples, the EEQ values per litre of effluent are calculated based on the ratio of VTG production induced by the samples relative to the detection limit. The VTG production due to the OVOC samples is background corrected by subtraction of the procedure blanks. Similar criteria were use to determine whether an anti-estrogenic effect occurred. In this case, the VTG production induced by the EC50 concen-tration of E2 is considered to be 100 %, and expressed as the ng or nmol per equivalent effluent volume (i.e., in each well the equivalent effluent volume is 0.01 l).


42

MTT test

After the VTG-containing medium was harvested, the cell viability (or cytotoxicity) of the dosed cells was evaluated by the determination of mitochondrial dehydrogenase ac-tivity, using 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) as the substrate (Denizot and Lang, 1986). A screen was considered positive if an OVOC pilot extract significantly decreased the MTT activity relative to the DMSO controls.

EROD activity and protein content

A duplicate set of plates for the entire experiment was used to determine EROD induction and or inhibition in the carp hepatocytes and to determine the protein content of the wells, using the methods of Burke and Mayer, (1974) and Lowry et al., (1951), respectively.

6.2.3 Results and discussion

MTT activity.

MeOH did not cause a decrease in cell function at concentration as high as 1% (v/v) (Figure 6.2). This suggests that future experiments may safely utilize greater volumes of MeOH for dosing, avoiding the limitation of a minimum dilution factor of 1000, which may prove useful if samples demonstrate only weak (anti-) estrogenic activities. None of the 21 OVOC samples had a statistically significant or concentration-dependent effect on mitochondrial MTT activity as a measure of cell viability (Figure 6.3). A non-significant decrease in MTT reduction was observed for the samples BST, BTU, MCD and MOP.

Figure 6.2 Effect of methanol (MeOH) concentration in medium (% v/v) on MTT reduction as a measure of cell viability in male carp hepatocytes in primary culture.


43

Figure 6.3 Effect of 1x and 0.1x OVOC samples, 1000- and 10 000-fold dilution, respectively, on MTT reductionas a measure of cell viability in male carp hepatocytes in primary culture.

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44

Vitellogenin production

A concentration response curve for VTG induction by estradiol is shown in Figure 6.4. The potential estrogenicity of the samples was screened in the absence of estradiol and anti-estrogenicity in the presence of 100 nM E2, which corresponded to an EC25 value in this experiment. Co-exposure of hepatocytes to 100 nM 17β-estradiol and 0.1, 1 or 10 µM of the known anti-estrogen tamoxifen resulted in inhibition of 17β-estradiol-induced vitellogenin synthesis by 54, 89 and 91%, respectively.

Figure 6.4 Concentration-response curve for vitellogenin (VTG) induction by 17β-estradiol in male carp hepatocytes in primary culture.

None of the effluent or water extracts were able to elicit an estrogenic response above the limit of quantitation (about 300 ng/ml extract VTG equivalent to 2400 pg EEQ/L in the effluent). Several water samples were anti-estrogenic (Figure 6.5) including, in order of decreasing efficacy (% reduction of VTG synthesis by 100 nM E2 in 1000x dilution): MDE (88%), MNO (66%), MMN (56%), MCD (48%) and MAB (44%). Samples BST (55%) and MEF (50%) also appeared anti-estrogenic but did not demonstrate a concen-tration-dependency. In these cases the anti-estrogenic effect may be unrelated to antago-nism at the estrogen receptor level, but the result of possible interference with protein synthesis. Cytotoxicity, however, could be ruled out as a likely explanation (see Figure 6.3). Of the first five anti-estrogenic water samples, several were also found to have ele-vated concentrations of aryl hydrocarbon receptor-responsive, DR CALUX-derived di-oxin equivalents (TEQs), namely MCD, MAB and MMN.

It is known that the anti-estrogenic potency of Ah receptor agonists correlates well with EROD induction potency in carp hepatocytes (Smeets et al., 1999c), which is due to a cross-talk between the Ah receptor activated complex and E2 responsive genes (Safe et al., 1998). The present results however, indicate that the most potent AhR-active sam-ples are not necessarily the most potent anti-estrogenic samples.

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45

Figure 6.5 Effect of 1x and 0.1x OVOC samples, 1000- and 10 000-fold dilution, respectively, on vitellogenin (VTG) synthesis stimulated by 100

nM estradiol (E2) as a measure of antiestrogenicity in male carp hepatocytes in primary culture. Symbols: * = significant (p <0.05) anti-estrogenic activity; ? = lack of concentration dependency of anti-estrogenic effect.

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46

This is not surprising because many other unknown compounds present in the water samples may have potential effects on the estrogen receptor-mediated stimulation of vitellogenin synthesis.

EROD induction

Several water samples induced EROD activity in male carp hepatocytes in primary cul-ture. The ability to increase EROD activity is and indication of the induction of the cyto-chrome P450 enzymes CYP1A1 and 1A2, which in turn indicates an activation of the Ah receptor. The carp hepatocytes were highly responsive to the ‘prototype’ Ah receptor agonist TCDD and EROD activity was increase almost 50% of maximum at the lowest tested concentration of 1 pM TCDD (Figure 6.6). Maximal induction was reached at a concentration between 3 and 10 pM TCDD (Figure 6.6). Taking into account the repeat-ability of the methods, this indicates that the carp hepatocyte assay and the DR-CALUX assay have a similar sensitivity (see Figure 6.1 for DR-CALUX and 6.6 for carp-hep EROD assay).

In order of decreasing efficacy, the following samples induced EROD activity: MAB, MMN, MJK, MST, MIJ, MFG, MDE and MQR. Each of these samples induced EROD activity above the detection limit of about 7% of maximal induction by TCDD (3-10 pM), although only MAB, MMN, MJK and MST were able to induce EROD activity above the limit of quantification of about 14%. Although the assay was not intended to quantify TEQs in the samples, it was apparent that the most efficacious sample extract –MAB– contained less than 1 pM TEQs, by comparing the response to 1x and 5xMAB with the response to 1 and 3 pM TCDD, respectively (Figure 6.7 and 6.6). As the re-sponse to 5xMAB is just below the response to 3 pM TCDD, the estimated TEQ in cells exposed to 1xMAB would be roughly 0.5 pM TEQ, corresponding to a concentration in original water sample of 0.5 pmole/l or about 161 pg/l TEQ.

Figure 6.6 Concentration response curve for ethoxyresorufin O-deethylase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male carp hepatocytes in primary culture.

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47

Figure 6.7 Ethoxyresorufin O-deethylase induction (EROD) by 1x and 5x OVOC samples, a 1000- and 200-fold dilution, respectively, in male carp hepatocytes in primary culture. TCDD was used as positive control.

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All the samples that were active in the DR-CALUX assay also gave a response in our carp hepatocyte system. Also, all the compounds that were negative in the DR-CALUX assay, had no statistically significant response in the carp hepatocytes, with the exception of MJK. This may be due to compound specific differences in sensitivity between both assays. The EROD induction potential of the extracts in carp hepatocytes did not corre-late well with their induction potential in the DR-CALUX assay. The following reasons are likely to explain this discrepancy. In the carp hepatocyte screen we did not produce complete dose-response curves, due to the limited availability of cells, but only used two concentrations per water sample extract, thus not being able to determine an EC50 value for induction to compare with the TCDD calibration curve. Furthermore, the concentra-tion-response curve for TCDD did not fully cover the lower response values (Figure 6.6). As most of the samples produced responses below the lowest tested concentration of TCDD, it was difficult to rank-order most of the samples according to EROD induction potency in carp hepatocytes. However, MAB was clearly the most efficacious inducer in the carp hepatocytes, while MCD had little effect. In the DR-CALUX assay, MAB was the second most efficacious sample, but MCD gave the strongest response. An explana-tion for this cannot be given at present.

Another discrepancy is the fact that although the total extract of MAB gave a strong re-sponse, the sub-fractions containing compounds with different Kow values (MAB4-6 and MAB>6) did not elicit responses, which added up to that of the original extract. The same was true for MCD. This discrepancy was also observed in the results of the DR-CALUX assays (see section 6.1).


49

7. Toxicity screening with in-vivo tests (Task 6.2)

The in vivo tests in this study will be used to evaluate the degree by which the in vitro toxicity (which establishes effects on a cellular level) is expressed in effects in organ-isms. The in vitro tests are practical in the sense that they are sensitive, relatively cheap and require only small amounts of sample/extracts compared to tests with organisms. Therefore, they form the basis for the toxicity measurements in the OVOC study. How-ever, their main limitation is that they relate to potential toxicity effects in organisms. A great number of repair mechanisms or adaptation effects may prevent this potential ef-fect from becoming a real effect in the environment. Thus, in vivo tests are used to as-sess the degree by which cell effects are expressed in organism effects for the effluents under scrutiny. Any possible relation found only holds for each specific effluent, and cannot be generalised for effluents or classes of substances as a group. Also, coinciding in vitro and in vivo effects do not necessarily imply a cause-effect relation.

Two types of in vivo tests are used in this study: chronic micro-toxicity tests and early life stage tests (ELS) on fish eggs. The two types of tests examine a different toxicity mechanism, and are thus complementary to each other.

The in vivo tests will be performed on the total extract of the effluent that is thereto di-luted in water. Originally, it was foreseen to dilute the extracts by the same factor by which concentration took place in the liquid-liquid extraction step. Thus, the in vivo tests would be performed at the concentration levels as present in the effluent.

7.1 Early Life Stage (ELS) tests

7.1.1 Principle

The ELS test used determines the effect on the egg and larvae phase of the zebra fish (Brachydanio Rerio). The rate of mortality and the occurrence of malformations are measured in the 8-day period after fertilisation. The protocol of the test (Aquasense, 2000) is based on the OECD 212 guideline. The test involves 4 replicas, each starting off with 25 eggs. At t = 1, 4 and 6 days the eggs are transferred to fresh medium.

For economic reasons, the test is performed as a screening test at only one dilution factor of the extract, indicating only whether a toxic effect is present or not. The dilution factor was 1:3000, such that the ELS test is performed at the concentration as in the original ef-fluent.

The tests were performed by TNO Delft, Department of Environmental Toxicology.

7.1.2 Handling

Extracts were shipped from IVM to TNO in two portions. For each sample the two por-tions (approximately 0.15 ml each) were mixed into a total volume of 2 ml methanol, which was used as stock for making fresh media.


50

A reference test (positive control) involving 3,4-dichloroaniline was applied to check for the validity of the test.

An extra methanol control test at the same concentration as the diluted extracts (2.3 ml/l) was applied in order to check for possible methanol effects.

During the test the environmental parameters pH, oxygen concentration, temperature and conductivity were measured.

7.1.3 Results

The tests showed no effects for all samples and the methanol control (Hooftman et al., 2001). No adverse effects were observed with respect to hatching of eggs, survival of eggs and larvae and the presence of malformations for any of the extracts tested.

The reference test (positive control) yielded an LC50-value within the quality criteria, indicating the test results are valid. All environmental parameters were within the accep-tation criteria.

One remark must be made with respect to the absence of toxic effects. From validation studies of toxicity tests (Vaal and Folkerts, 1998) it is known that due to the relatively long test duration (up to three days for each medium refreshment) certain substances may disappear from the test volume. This holds especially for volatile substances and for highly insoluble substances (i.e. high log Kow value). Thus, a reservation should be made to the conclusion of no toxic effects for those extracts that will prove to contain the mentioned type of substances.

The zebra fish ELS test has previously shown to be sensitive with respect to TCDD, with reported NOECs for survival and growth at 424 pg/g as internal dose in the egg (Elonen et al., 1998) and an EC50 for mortality and malformations of 21 pMol/L TEQ (appr. 6.7 ng/L as external dose) in the study of Murk at al. (1996). In a comparative study of seven non-salmonid species zebrafish was found to be the least sensitive and lake herring the most sensitive (Elonen et al., 1998). The non–salmonid species were 8 to 38 times less sensitive to literature data (from studies with a comparable exposure regime) on lake trout, which is considered to be the most sensitive salmonid (Walker et al., 1991). Ber-ends et al. (1997) did not observe effects of OCDD and OCDF in flow-through zebra fish ELS tests (32d) at measured exposure concentrations of 32-34 ng/L. Although re-sults from different ELS tests with zebrafish are difficult to compare, because of differ-ences in exposure regime (egg injection, static conditions, flow-through, duration, dosing system, dimensions of test system), in endpoints (malformations, development, growth, mortality), or because of assessment of the dose (water concentration, internal dose), it seems reasonable to assume that zebra-fish NOECs for TCDD in an 8d semi-static expo-sure design as applied in this study, are likely to be above the ng/L level. The in vitro-results (expressed as total TCDD-TEQs in DR-CALUX and carp-hep EROD studies) in-dicate levels usually well below 1 ng/L and this seems to be consistent with the lack of response in the in-vivo study.


51

7.2 Chronic micro-toxicity tests (Task 6.2)

7.2.1 Principle

The chronic micro-toxicity test used involves the species Vibrio Fischeri, a marine bac-teria that produces light and that lives in symbiosis with higher organisms. Vibrio F. is known to use the mechanism of so-called quorum sensing, which means that it only pro-duces light when a certain minimum bacteria concentration is exceeded. This mechanism is reflected in a light production curve as given in Figure 7.1.

Figure 7.1 Light production in the Azur chronic micro-toxicity test with Vibrio Fischeri (Bulich et al, 1994) The axes are scaled to the situation at the end of the test (22 hours).

This threshold effect renders the test a high sensitivity with respect to influences disturb-ing the growth of bacteria. Variations in the bacterial concentration at the end of the test are enhanced fivefold in the measured light production.

In the course of the test (22 hours) the bacterial concentration increases by a factor of three. Parameters that influence the growth of the bacteria may lead to detectably lower light production at the end of the test.

Apart from this source of toxicity the test is also sensitive to parameters that influence the activity level of the bacteria, to which light production is closely connected. This is the type of toxicity that is tested in the acute toxicity test with Vibrio F.

Chronic toxicity tests with Vibrio F. in general are more sensitive than acute tests. This relation holds for both effluent samples (Gellert and Stommel 1994, and 1995, Ziesseniss and Grabert 1995) sediments (Guzella 1998) and single substances (Gellert et al. 1997). For substances it is known that the degree by which chronic tests are more sensitive than acute tests varies depending on the specificity of action and physico-chemical character-istics.

LUMINISCENCE vs. BACTERIA CONCENTRATION

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When comparing the relative susceptibility of various types of organisms in toxicity tests it is shown that tests with bacteria are comparable to, if not more sensitive than, tests with water flea, algae, plants or fish (Gellert et al.,1997, Sweet, 1997, Sloof & Canton, 1993).

All tests reported below were conducted with bacterial populations, media and test ana-lyser as supplied by Azur Environmental. The tests were performed with four replicas in a series of five subsequent 1: 2 dilutions. In order to establish possible solvent effects also equivalent tests with methanol are performed.

All test were performed by Aquasense, Amsterdam.

7.2.2 First test series

In March-April 2001 a first test series with the 21 extracts was performed. In this series the protocol as given by the manufacturer was used. Although the positive control (cop-per sulphate solution with known toxicity) gave results within the range of acceptance, it was decided not to use the test results. Reason for this was that several artefacts were found, even in the control sample (blank). These artefacts led to a negative bias in light production towards higher concentrations of the extract, which could falsely be inter-preted as toxicity.

The artefacts found could be traced down to minimal differences in time and/or pipetting actions during filling of the test vials with test solution. One way or another the test me-dium proved highly sensitive on this point: test vials which had been filled with subse-quent fillings of the repeat pipettor, spaced only minutes in time, could differ by about 50% in light production (vials filled later generally giving lower light levels). We have not been able to elucidate the cause of this sensitivity.

In an attempt to tackle these problems a new protocol for the test was designed, based on the principle of minimizing time differences/pipetting actions between test vials whose results should be compared in the analysis of toxicity. In this new test protocol rows of vials alternately contain extract solutions and methanol solutions at the same concentra-tion. Any structural differences between the two types of solutions should be kept to a minimum this way. Four replicas were used for the extract and methanol solutions each. A row with blank test solutions (test medium without any added components) and a row of vials for the positive control complete the test block (10 rows total).

In a short experiment this new protocol was tested. No artefacts were found in the blank, nor was any effect found of the presence of methanol, up to the highest concentration tested (2 ml/l). The new protocol could therefore give reliable results and thus it was de-cided to test all 21 extracts again.

7.2.3 Second test series

In August 2001 all 21 extracts were tested again based on the new protocol. The extracts used were not the total extracts (directly obtained after liquid-liquid extraction) but a mixture of the three partial extracts after separation in the three log Kow classes. This mixture was concentrated 1:300 relative to the original effluent. As the mixed ex-tracts were diluted 1: 500 in the test medium (equivalent to a methanol concentration of


53

2 ml/l) the tests were performed at 67% of the concentration of substances in the efflu-ent. The full dilution series covered the values of 4, 8, 17, 33 and 67% of the effluent concentration.

7.2.4 Results

The results have been described in detail in a technical report (Senhorst et al., 2001).

The blank samples did not show any artefacts: the light yield of all five replicas did not differ with more than the 95% confidence of the average value of all the replicas.

The positive copper control gives the right concentration dependent toxicity behaviour. However, due to a remaining structure in absolute light levels of different rows of vials (see below), no quantification could be made to this point. Nevertheless we are confident that the test gave valid results in the sense that the bacterial population was active and that it was sensitive for pollutants.

The results of the methanol tests confirmed the results of the preliminary study that no toxic effects of methanol up to 2 ml/l could be found, see Figure 7.2. No points differ by more than 95% confidence from the average value of all five positions.

Figure 7.2 Average results of the methanol dilution series (N=21). Each position defines a methanol concentration, going from 125 µl/l to 2 ml/l. These val-ues equal the methanol concentration at the same position in the extract dilution series. The error bar indicates the interval –1 to +1 standard deviation.

A structure in the absolute value of light levels between rows of vials was found going top-down in the test block, see Figure 7.3, which gives the average light yield at position 1 of all rows in the test block. Position 1 contains the lowest concentration within a row. Ideally, with no toxic effects at this position (which is the case for methanol and all ex-tract tests) the light yield is the same for all rows.

AVERAGE RESULT METHANOL TESTS

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Based on the experience of the first test series, this structure is interpreted as the effect of the pipetting order in the rows of the test block: the rows A, B and C are filled with one filling of the repeat pipettor, rows D, E and F with the next pipettor filling, and G, H and I with the third filling. This structure is not only expressed in the average light yield at position 1, but could also be found at the level of individual test blocks: frequently (but not for all tests) three separate groups of light values could be discerned, each extending over a group of three rows. Thus, within this study, the structure in light values over the test block is described as three groups of three rows5, each with distinctly different aver-age light yield.

Figure 7.3 The value at position 1 of the rows in the test block, averaged over all 21 tests. Row A contains the blanks, rows B, D, F and H the methanol dilution series, rows C, E, G and I the extract series.

The effect that this structure may have on the toxicity analysis is minimised by the fol-lowing procedure of analysing the test results:

• All methanol and, if relevant, blank sample data within a group of rows are aver-aged. Blank data may be taken together with methanol data as no methanol toxicity has been found;

• The data of the extract dilution series are normalised with respect to the metha-nol/blank average of its own group. As an extract dilution series consists of four rep-licas (rows C, E, G and I) this yields four normalised data points for all five extract dilution value;

• The methanol (and blank) data are also normalised with the methanol/blank average of its groups. This yields 25 normalised data points6;

5 Leaving aside the positive copper control at row J. 6 The purpose of normalising the methanol/blank data is to establish the standard deviation within

this group. This is of importance in the calculation of significance levels in the statistical analysis.

AVERAGE VALUE POSITION 1

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• The mean of the normalised extract data points is compared to the means of the nor-malised methanol/blank data (which equals 1 by definition). A one-sided independ-ent sample t-test is then used to check for statistical significance. A significance level of at least 95% was used.

By this procedure any effects the structure may have are normalised away. Moreover, as many relevant data points as possible are averaged, the error in the statistical analysis is reduced, improving the significance of statistical results.

Table 7.1 Results of the statistical analysis of toxicity in the 21 extracts at the three highest concentration levels. A ‘+’ denotes a significance level of at least 95%, a ‘++’ denotes a level of at least 99%.

Test at percentage of effluent concentration of:

Sample code 17% 33% 67 % Remarks Industrial effluents MPQ 0 ++ ++ MDE 0 0 ++ MIJ 0 0 0 MAB ++ + ++ MBC 0 0 0 MJK 0 0 0 MKL 0 0 0 MOP 0 0 0 MEF 0 0 0 MFG 0 0 0 MGH 0 0 0 MCD 0 0 ++ MLM + 0 0 MRS 0 0 + MNO 0 + 0 MQR 0 0 0 Cooling water BST 0 0 0 Inlet cooling water MST 0 0 + Outlet cooling water References MMN 0 ++ ++ MWTP Sleen BTU 0 0 0 Lake Marken Blank BUV 0 + 0 Blank sample


56

The basic data of the microtox-test are presented in Appendix IV. The results of the sta-tistical analysis of toxicity in the 21 extracts is summarised in Table 7.1 for the concen-tration levels of 17, 33 and 67% of the effluent concentration. Due to the statistical na-ture of the results there is always a certain chance of false positives: at the significance level of 95% this chance amounts 5%, at a level of 99% this chance is reduced to 1 %. The probability of a false positive is especially high for those samples where at a low concentration level a significant result is found, which is not repeated in the higher con-centrations.

Putting it the other way around, at high significance levels or for samples with signifi-cant results at more than one concentration level the interpretation of toxicity is very cer-tain. This holds for the samples MPQ, MDE, MAB, MCD and MMN.


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8. Synthesis of results and estimation of annual loads

Sampling

A total number of 21 samples was collected in the period 29 January- 9 march 2001, consisting of: 16 industrial effluents, 1 discharge of cooling water (inlet and outlet), 1 municipal wastewater treatment plant, 1 reference surface water (Lake Marken) and 1 blank (tap-water). Compared to the selection of 16 companies, approved by the steering committee (Tukker, 2000a), one company had ceased its production while another ini-tially refused co-operation. At two other companies effluents from selected production processes were discharged from separate effluent channels, in which cases additional samples were taken. Except for 1 company, normal production conditions could be as-sumed based on information from the company’s involved and raw data from measure-ments on pH, suspended solids, BOD, TOC , EOCl and AOX.

Toxicity tests

The results of the toxicity tests on the whole extracts have been summarised in Table 8.1. As can be seen there is no simple relationship between the outcome of the different tests.

None of the extracts was toxic in the 8-day zebra-fish ELS test. In 7 samples the chronic Microtox test was responsive at the highest concentration tested. Within this group 3 samples were responsive in two or more dilutions (MPQ, MAB, MMN).

None of the extracts appeared to be cytotoxic in the carp-hep MTT test.

The VTG carp-hepotocyt test did not reveal a estrogenic potency above the quantitation limit (2400 pg EEQ/L), which is partly in line with the outcome of the pilot study (Bel-froid et al. 2000), where levels of <1 to 53 pg EEQ/L (industrial effluent) and 2400-3000 pg EEQ/L (municipal waste water treatment plant) were detected with the more sensitive ER-CALUX assay.

For several water samples a significant anti-estrogenic potency was observed. This in-cluded, in order of decreasing efficacy (% reduction of VTG synthesis by 100 nM E2): MDE (88%), MNO (66%), MMN (56%), MCD (48%) and MAB (44%).

In the carp-hep EROD test 9 samples were found to be responsive: 7 industrial effluents (MDE, MIJ, MAB, MJK, MFG, MCD, and MQR), cooling water outlet (MST) and the municipal wastewater treatment plant Sleen (MMN).

The DR-CALUX assay, detecting compounds with a dioxin-like mode of action, was the most responsive test. Significant response above the limit of detection (27 pg TEQ/l) was detected in 11 out of 16 industrial effluents (with 10 samples in the range 40-306 pg TEQ/l and 1 sample with a concentration of 1066 pg TEQ/l), in the effluent of the mu-nicipal waste water treatment plant MWTP (157 pg TEQ/l), in the outlet of a cooling water installation (104 pg TEQ/l) and in water from Lake Marken (37 pg/L). All EROD responsive samples in the carp-hep test were also responsive in the DR-CALUX assay, although the maximum induction in both tests did not coincide.


58

Samples with both in-vivo toxicity and in-vitro response were: MPQ, MDE, MAB, MCD, MNOW, cooling water MST and the wastewater treatment plant MMN.

Presence of chlorine

Data related to the presence of chlorine have been summarised in Table 8.2 together with the daily discharges (in m3/day) of the total volume of wastewater per day. The EOX data on the raw effluents, considered as representative for non-polar halogenated compounds (including volatiles) confirm the presence of halogenated compounds (in concentrations above 100 µg/L) in 6 out of 16 industrial effluents. The AOX data, which are assumed to include also polar and ionized halogenated compounds, confirmed the presence of halogenated compounds in 15 out of 16 industrial effluents, cooling water and the reference samples (MWTP Sleen, Lake Marken).

The GC-ECD based chlorine determinations, with confirmation of the presence of chlorine with GC-MSD, confirmed the presence of organic chlorinated compounds in fractions with different hydrophobicity in most samples in total concentrations of 0.2 –2.2 µg/L and less than 0.1 in blanks and references. In 3 industrial effluents (MAB, MFG, MCD) concentrations between 9 and 30 µg/L were encountered, with the largest contribution (3 –27 µg/L) in fractions with low hydrophobicity. Highly hydrophobic compounds were detected in concentrations ranging from 0.04-0.1 µg/L in all samples. The purge and trap determinations could only reveal low concentrations of volatile compounds (0.3-0.5 µg/L), partly attributed to sample handling and methodological choices for the trapping solvent applied.

The GC-ECD based organochlorine determinations (0.2 - 30 µg/L) usually were several orders of magnitude lower than AOX (<20 – 4400 µg/L) and EOCL concentrations (<100 – 6300 µg/L) in corresponding samples. This large difference can be explained, considering that the GC-ECD detectable compounds are restricted to the category of relatively involatile, non-polar and GC amenable organohalogens, and AOX, and even EOX, being sensitive to more polar substances. Thus, GC-ECD measurements not nec-essarily should be corroborated by AOX or EOX levels having the same order of magni-tude.

Indicative annual loads

In order to compare the contributions from the different sources indicative annual loads (amount emitted annually) were calculated for the different parameters for the presence of chlorinated compounds and the dioxin equivalents from the DR-CALUX tests (Table 8.3). Indicative annual loads of chlorinated compounds from individual industrial sources ranged from:

• EOCl: < 4 - 46000 kg/y • AOX < 3 – 42000 kg/y • Log Kow < 4 <0.1 - 115 kg/y (low hydrophobicity, GC-ECD based) • Log Kow 4-6 <0.1 - 13 kg/y (medium hydrophobicity, GC-ECD based) • Log Kow >6 <0.1 - 3 kg/y (high hydrophobicity, GC-ECD based) • DR-CALUX <0.01 – 12 g TEQ/y (2378-TCDD equivalents)


59

The total DR Calux load of all industrial emissions sampled in this study amounts to 21.7 g TEQ/y for all responsive effluents. If all non-responsive effluents were added to this value on the basis of the detection limit this would add another 0,44 g TEQ/y.

If MWTP-Sleen (sample MMN, 4800 m3/day) may be considered as representative for all national MWTPs (2150000 m3/day) this would imply emissions of dioxin equivalents (DR Calux loads) in the order of 123 g TEQ/y for all municipal discharges in The Neth-erlands. It should, however, be noted that large differences exist between WWTPs in type of wastewater received (households, small or larger companies) and that day-to-day or seasonal variations may be large. The estimate given should be considered as an indi-cation for the order-of-magnitude.

A similar extrapolation of the observed DR Calux load of MST (cooling system) to the total national use of hypochlorite for cooling purposes is hampered by the DR Calux re-sponse in the inlet flow being below the limit of detection. A comparison of outlet DR Calux load with inlet load therefore gives a range of 0 to 0.03 g TEQ/y for MST. This range obviously impairs conclusive statements. However, if the upper limit is used as a ‘worst case’ scenario this would lead to substantial amounts of DR Calux loads as dis-charged by all corresponding cooling systems in The Netherlands. Based on hypochlorite consumption the load of MST should be multiplied by a factor of 640 to obtain an ex-trapolated value for all hypochlorite use, yielding a value of approximately 20 g TEQ/y. As stated above, this value should be considered a worst-case estimate. This is all the more so as cooling water effluents often as in the case of MST, though not always, are treated in wastewater treatment plants, which further reduces emissions.

The total DR Calux load values given above for the sectors chlorine chain industry, mu-nicipal wastewater treatment plants and hypochlorite use in cooling water systems seem extremely high compared to known dioxin emissions. The total national emissions for 1999 for chemically determined TCDD equivalents were estimated in different frame-works; estimated ranges were 35 - 42 g TEQ/y to air and 0,2 – 2.8 g TEQ/y to surface water (North Sea Conference, 2002; RIVM, 2001). The latter value consisted of contri-butions of 0,1 –2 g TEQ/y from communal sources and 0,02 – 0.17 g TEQ/y from indus-trial sources, and up to 0.6 g TEQ/y from waste management. Thus our bioassay-based sector estimates for dioxin-likes exceed the PCDD/F-based estimates with 2 to 3 orders of magnitude.

Obviously, the differences between DR Calux loads and dioxin emissions may be ac-counted for by differences in the analytical procedures underlying the two measurement techniques. Dioxin emissions relate to chemically determined PCDDs and PCDFs, and do not account for other compounds with similar mode of action such as planar-PCBs, PCNs, PBBs, PBDEs and to a lesser extent PAHs and several others (Hoogenboom et al., 1998). These substances are likely to contribute to the DR-CALUX response, which may explain why biodetection of dioxin-likes is likely to yield higher results. The huge difference in TEQ values between the two types of measurements is consistent with the observation that the DR Calux assay is receptive to a much wider range of substances than only the ‘classical’ dioxins like PCDDs and PCDFs.


60

In the further studies in WP 7 and 8 more information will be obtained on the com-pounds contributing to the measured DR Calux response. Using a DR-CALUX set-up with a sulphuric acid based cleanup the contribution of less stable compounds (e.g. PAHs) is likely to disappear and a better matching between chemical based TEQs and biologically determined TEQs is expected.


61

Table 8.1 OVOC screening study - response in toxicity tests – whole extracts. debiet zebrafish

ELS test Micro-Tox Micro-Tox Micro-Tox DR-CALUX

total extract Carp-MTT Carp-VTG

estrog. Carp-VTG anti-estr.

Carp-EROD induction

Code remarks m3/day at 17% at 33% at 67% Pg TEQ/L pg EEQ/L % inhib % max act

Industrial effluents MPQW0903 250 n.d. 0 ++ ++ 71 a n.d. < 2400 <1 n.s.

MDEW2102 23000 n.d. 0 0 ++ 121 ± 6 n.d. < 2400 88 * 9 ± 3

MIJW2102 20000 n.d. 0 0 0 122 ± 9 n.d. < 2400 19 15 ± 2

MABW2602 60 n.d. ++ + ++ 309 ± 9 n.d. < 2400 44 * 88 ± 21

MBCW2602 3000 n.d. 0 0 0 <27 b n.d. < 2400 -5 n.s.

MJKW1902 180 n.d. 0 0 0 55 a n.d. < 2400 22 17 ± 4

MKLW1902 270 n.d. 0 0 0 44 a n.d. < 2400 15 n.s.

MOPW1902 2700 n.d. 0 0 0 40 a n.d. < 2400 3 n.s.

MEFW1402 212000 n.d. 0 0 0 99 ± 4 n.d. < 2400 50 *? n.s.

MFGW1402 335 n.d. 0 0 0 146 ± 7 n.d. < 2400 16 14 ± 4

MGHW1402 310 n.d. 0 0 0 <27 b n.d. < 2400 22

MCDW1202 30000 n.d. 0 0 ++ 1066 ± 22 n.d. < 2400 48 * 10 ± 2

MLMW0702 360 n.d. + 0 0 <27 b n.d. < 2400 21 n.s.

MRSW0502 9000 n.d. 0 0 + <27 b n.d. < 2400 24 n.s.

MNOW2901 28400 n.d. 0 + 0 <27 b n.d. < 2400 66 * n.s.

MQRW2901 2640 n.d. 0 0 0 148 ± 16 n.d. < 2400 43 * 9 ± 1

Cooling water BSTW1202 inlet water c 3600 n.d. 0 0 0 <27 b n.d. < 2400 55 *? n.s.

MSTW1202 outlet water d 840 n.d. 0 0 + 104 ± 6 n.d. < 2400 1 16 ± 1

ReferencesMMNW0502 MWTP Sleen 4800 n.d. 0 ++ ++ 157 ± 6 n.d. < 2400 56 * 29 ± 11

BTUW0802 Lake Marken n.d. 0 0 0 37 a n.d. < 2400 1 n.s.

BlankBUVW0802 Blank sample n.d. 0 + 0 <27 b n.d. < 2400 44 n.s.


62

ELS test: n.d= no significant effect detected. Micro-tox test: presence of significant effect at dilution tested; 0= non significant, +=p<0.05, ++=P<0.01; DR CALUX: concentration in 2,3,7,8 TCDD-TEQ(pg TCDD-TEQ/l water; a= value between LOD (31 pg/L) and LOQ (89 pg/L); b= value below LOD (31 pg/L). Carp-hep test: MTT reduction in % compared to control; estrogenic activity in pg EEQ/L, anti estrogenic activity

in % inhibition of VTG production at 100 nM E2 in extract (1x extract; 0.1% dilution); *= significant inhibition; *?= inhibition not dose dependent; EROD activity (5x extract; 0.5% dilution) as % of maximum induction of 10 pM TCDD; c total inlet of make-up water; d closed circulation system, blow down to water treatment installation.


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Table 8.2 OVOC screening study - presence of chlorinated compounds. debiet NEN6676 NEN-

EN1485 X-

LogKow<4 X-

Log Kow 4-6 X

-LogKow>6 X-total fractions

X- purgeable

code remarks m3/dag EOX (ug/l) AOX(ug/l) ug/L ug/L ug/L ug/L ug/L

industrial effluents MPQW0903 250 <100 4400 1.3 0.9 0.1 2.2 0.3

MDEW2102 23000 430 80 1.2 0.1 0.1 1.3 0.3

MIJW2102 20000 6300 240 0.5 0.1 0.04 0.6 0.3

MABW2602 60 3300 740 27 3.0 0.1 29.8 0.3

MBCW2602 3000 <100 750 0.1 0.05 0.05 0.2 0.3

MJKW1902 180 <100 80 0.1 0.04 0.1 0.2 0.3

MKLW1902 270 440 160 0.2 0.1 0.05 0.3 0.3

MOPW1902 2700 <100 90 0.2 0.05 0.05 0.3 0.3

MEFW1402 212000 <100 540 0.2 0.05 0.04 0.2 0.4

MFGW1402 335 130 960 3.1 6.4 0.1 9.6 0.3

MGHW1402 310 <100 4300 0.7 0.0 0.04 0.8 0.3

MCDW1202 30000 340 2200 11 1.2 0.1 11.8 0.3

MLMW0702 360 <100 <20 0.1 0.0 0.1 0.1 0.5

MRSW0502 9000 <100 1500 0.4 0.1 0.1 0.6 0.4

MNOW2901 28400 <100 50 0.7 0.1 0.04 0.8 0.3

MQRW2901 2640 <100 530 0.8 0.02 0.1 0.8 0.3

cooling water BSTW1202 inlet water 3600 <100 90 0.1 0.04 0.04 0.1 0.3

MSTW1202 outlet water 840 <100 670 2.0 0.1 0.04 2.2 0.3

references MMNW0502 MWTP Sleen 4800 <100 80 0.9 0.5 0.1 1.4 0.6

BTUW0802 Lake Marken blanco <100 30 0.04 0.03 0.04 0.1 0.3

Blank BUVW0802 Blank sample blanco <100 <20 0.1 0.03 0.04 0.1 0.4


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Table 8.3 OVOC screening study-annual loads. debiet EOX AOX X-

LogKow<4 X-

Log Kow 4-6 X-

LogKow>6 X-total fractions

TEQ

Code Remarks m3/dag kg/y kg/y kg/yL kg/y kg/y kg/y g/yr

industrial effluents MPQW0903 250 <9 402 0.1 0.1 0.0 0.2 <0,002

MDEW2102 23000 3610 672 9.7 1.0 0.5 11 1.0

MIJW2102 20000 45990 1752 3.5 0.8 0.3 4.6 0.9

MABW2602 60 72 16 0.6 0.1 0.002 0.7 0.01

MBCW2602 3000 <120 816 0.24 0.048 0.048 0.24 <0.029

MJKW1902 180 <7 5 0.01 0.003 0.01 0.01 <0.002

MKLW1902 270 43 16 0.02 0.008 0.005 0.03 <0.003

MOPW1902 2700 <99 89 0.2 0.05 0.05 0.3 <0.026

MEFW1402 212000 <7738 41785 11.9 3.6 3.3 18.8 7.7

MFGW1402 335 16 117 0.4 0.8 0.01 1.2 0.02

MGHW1402 310 <11 487 0.1 0.004 0.005 0.1 <0.003

MCDW1202 30000 3723 24090 115 12.9 1.0 129 12

MLMW0702 360 <13 -3 0.01 0.005 0.01 0.02 <0.004

MRSW0502 9000 <329 4928 1.4 0.4 0.2 2.0 <0.088

MNOW2901 28400 <1037 518 7.1 1.0 0.5 8.5 <0.280

MQRW2901 2640 <96 511 0.7 0.02 0.1 0.8 0.1

cooling water BSTW1202 inlet water 3600 <131 118 0.09 0.05 0.05 0.19 <0.035

MSTW1202 outlet water* 840 <31 205 0.61 0.04 0.01 0.66 0.03

References MMNW0502 MWTP Sleen 4800 <175 140 1.5 0.8 0.2 2.5 0.3

BTUW0802 Lake Marken

Blank BUVW0802 Blank sample

* transported to treatment installation


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9. Ranking and prioritisation of samples for WP 7-9

The results of the screening study were presented at a dedicated technical workshop (August 30th, 2001, The Hague) in which participated the BOVOC supervising commit-tee, invited experts and the research team. The objective of the workshop was to review methodological aspects and to advice the BOVOC steering committee and the research team on the selection of samples for additional biodegradation studies (work package 8.1), further TIE studies on responsive fractions (work package 7, 8), and additional in-plant studies (work package 9) to attempt to identify unknown chlorinated PBT com-pounds in mixed wastewater streams. A proposal for prioritisation and selection of sam-ples for further studies in the consecutive work packages (WP 7 to 9) was discussed at that meeting. The approved proposal is described in the following sections of this chapter.

Scope and objectives: during the screening study on the aquatic effluents a total number of 21 samples was investigated, consisting of: 16 industrial effluents, 1 discharge of cooling water (inlet and outlet), 1 municipal wastewater treatment plant, 1 reference surface water and 1 blank (tap-water). One of the objectives of the whole OVOC study is to determine if unknown chlorinated micro pollutants (PBTs: persistent, toxic, bio accumulative compounds) are emitted from the chlorine chain. In the screening study on the aquatic effluents a tiered approach was chosen for practical reasons, in which the first step consisted of a screening on the presence of toxic (T) and bio accumulative (B) compounds. Additional studies on the presence of persistent compounds will be executed only on responsive samples.

Available screening parameters

Chemical/physical

• Macro constituents (EOX, AOX, TOC, COD, BOD, pH, suspended solids, discharge volume);

• GC-ECD and GC-MDS based organochlorine in different fractions (purgeable, Log Kow <4, Log Kow 4-6 and Log Kow>6).

Biological

• In-vivo toxicity of whole extracts: ELS test (zebra fish) and chronic micro-tox test (marine bacteria);

• In-vitro response in DR-CALUX tests for the presence of dioxin-like compounds and in CARP-hepatocyte tests for estrogenicity and anti-estrogenicity (VTG), cytotoxicity (MTT) and EROD induction.


66

At the workshop a proposal was discussed to base the ranking/selection primarily on the following categories of criteria:

• The presence of chlorinated compounds (EOX, AOX, GC-ECD/MDS based organochlorine);

• Toxic response in in-vivo and in-vitro tests; • Bio accumulative potential, by evaluating the fractions of medium and high

hydrophobicity: Log Kow 4-6 and >6; • Significance of emission based on discharge volume of the effluent (by analysing

annual loads rather than concentrations).

The workshop and BOVOC committee agreed with the proposed approach. No additional criteria were added and no relative weights for each of the categories was ap-plied. Although quantitative decision support software could be applied (e.g. BOSDA), we feel that this is not necessary for the current project and that a qualitative (multi-criteria) approach is sufficient. In Tables 8.1 – 8.3 the main parameter values have been summarised for the different samples.

In order to compare the different parameter values, we applied a transformation of the most important parameters to scores on a 4-point scale. The scores are summarised in Table 9.1. The explanation of the attribution of scores (4 point scale) is summarized in Table 9.2.

Selection of samples for biodegradation tests (WP 8)

In the biodegradation tests (n=6) the persistence of effects and of chlorinated bio accu-mulative compounds will be evaluated.

• Toxicity: samples exhibiting a toxic response in 3 different tests: MDE, MAB, MCD, MMN;

• Cl-Log Kow 4-6 and >6 load criterion: samples with the highest ranking: MCD, MEF, MNO and MDE;

• As reference sample: the municipal wastewater treatment sample MMN.

Proposed and accepted selection: MCD, MDE, MAB, MEF, MNO and MMN

Selection of responsive extracts (WP 7)

A number of 10 responsive (in-vitro) fractions will subjected to further TIE studies.

• Focus on the most responsive DR-CALUX fractions (load citerion) of the samples MCD, MEF, MDE, MIJ, MMN, MQR;

• Samples with two fractions investigated: MCD, MMN, MEF; • Sample MAB with a high effect scores (concentration) score should be added • Based on total volume of cooling water sector: MST; • As reference sample: the MWTP sample MMN.

Proposed and accepted selection: MCD(2), MDE, MEF(2), MIJ, MQR, MMN, MAB and MST.


67

Selection of in-plant TIE studies (WP 9)

In case of mixed wastewater streams the work in WP 9 is required to investigate to what extent the presence of effects or bio accumulative compounds can be attributed to the chlorine chain. We propose to execute this only if non-chlorine chain emissions may contribute more than 30% to the total discharge. Samples with mixed wastewater streams are: MCD, MDE, MIJ, MMN, MNO, MMN, MOP, MQR, MRS (9 out of 16 industrial effluents). The workshop and BOVOC agreed to select at this stage only MCD based on the DR-DR CALUX load.


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Table 9.1 Overview of scores on different criteria for toxicity, bio accumulative potential and presence of chlorinated compounds + preliminary EROD data.

Chlorinated compounds

In-vivo Toxicity in-vitro Bioaccumulation potential

Micro-Tox Carp-anti estro-genic potency

Carp-hep

EROD

DR-CALUX concentration

DR-CALUX load AOX load Cl Log Kow 4-6 load Cl Log Kow >6 load

code remarks score score score score score score score

industrial effluents

MPQW0903 ++ + ++

MDEW2102 + ++ + ++ ++ ++ + +

MIJW2102 + ++ + +++ + +

MABW2602 +++ + +++ +++ +

MBCW2602 ++

MJKW1902 + +

MKLW1902 + +

MOPW1902 + +

MEFW1402 +? ++ ++ +++ ++ ++

MFGW1402 + ++ ++ +

MGHW1402 ++

MCDW1202 + + + +++ +++ +++ +++ +

MLMW0702 +

MRSW0502 + +++ + +

MNOW2901 + ++ ++ + +

MQRW2901 + + ++ + ++ +


69

Cooling water

BSTW1202 inlet water +? ++

MSTW1202 outlet water* + + ++ ++

references

MMNW0502 MWTP Sleen ++ + ++ ++ + ++ + +

BTUW0802 Lake Marken +

Blank

BUVW0802 Blank sample +

* Worst-case estimate used (see Chapter 8)


70

Table 9.1 Explanation of attribution of scores (4 point scale).

Micro-Tox: Number of dilutions with significant effect (1-3), non-responsive samples not included Carp-anti estr. + significant inhibition < 60%;

++ significant inhibition > 60%; non-responsives not included; ? Denotes not dose- dependent inhibition Carp-hep EROD: Relative induction compared to 10 pM TCDD in 200 fold dilution non-significant values

(<9 not included; + 9-20%; ++ 20-50%; +++ >50%

DR- DR CALUX Concentration

values below LOD not included; + value between LOD and LOQ (31-89 pg TEQ/L); ++ value between LOQ and 200; +++ value > 200 pg TEQ/L

DR-DR CALUX load values below 0.1 g TEQ/y not included; + 0.1-1 g/y; ++ 1-10 g/y; +++ >10 g/y

AOX load < 10 kg/y not included; + 10-100; ++ 100-1000 kg/y; +++ > 1000 kg/y

Cl in class Log Kow 4-6 < 0.2 kg/y not included; + 0.2-1; ++ 1-10; +++ >10 kg/y

Cl in class Log Kow >6 < 0.1 kg/y not included; + 0.1-1; ++ 1-10; +++ >10 kg/y


71

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Appendix I. Abbreviations

Abbr. In Dutch In English Ministry VROM

Ministerie van Volkshuisvesting, Ruimtelijke Ordening en Milieubeheer

Ministry of Housing, Spatial Planning, and the Environment

Ministry V&W

Ministerie van Verkeer en Waterstaat Ministry of Transport, Public Works, and Water Management

RWS Rijkswaterstaat Directorate-General of Public Works, and Water Management

RIZA Rijks Instituut voor Integraal Waterbe-heer en Zuivering van Afvalwater

National Institute for Inland Water Management and Waste Water

VNCI Vereniging Nederlandse Chemische Industrie

Association of the Dutch Chemical In-dustry

RITOX Research Instituut Toxicologie - Uni-versiteit Utrecht

Research Institute Toxicology, Univer-sity of Utrecht

TNO-STB Nederlandse Organisatie voor Toege-past Natuurwetenschappelijk Onder-zoek – Strategie, Technologie en Be-leid

TNO Strategy, Technology and Policy Studies

IVM Instituut Voor Milieuvraagstukken, Vrije Universiteit

Institute for Environmental Studies, Vrije Universiteit

CML Centrum voor Milieukunde Leiden, Universiteit Leiden

Centre for Environmental Sciences, Leiden University

BITAC Begeleidingscommissie Implementatie Acties Chloorketenstudie

Committee for the Implementation of Actions of the Chlorine Chain Study

OVOC Onderzoeksprogramma Vervolgonder-zoek Chloorketenstudie

Research Programme Chlorine Chain Follow-up Studies

BOVOC Begeleidingscommissie Onderzoeks-programma Vervolgonderzoek Chloor-ketenstudie

Supervising Committee Research Pro-gramme Chlorine Chain Follow-up Studies

SNM Stichting Natuur en Milieu The Netherlands Society for Nature and Environment

TBP, BTP, PBT

Toxische, Bioaccumulatieve en Persis-tente stoffen.

Toxic, Bioaccumulative and Persistent compounds

M&T Onderzoeksschool Milieuchemie en Toxicologie

Research Graduate School Environ-mental Chemistry and Toxicology

SENSE Onderzoeksschool Sociaal Economisch en Natuurwetenschappelijk Milieuon-derzoek

Research Graduate School Socio- Eco-nomic and Natural Sciences of the En-vironment

Kow n-octanol/water partitiecoëfficient n-octanol/water partitioning coefficient GC gas chromatography HPLC high pressure liquid chromatography ECD electron capture detection AED atomic emission detection MSD mass spectrometric detection ITD ion trap detection ELS early life stage test


77

Appendix II. Prioritised emission sources selected for the main study (Phase-2)

Table II.1 Aquatic and atmospheric emission sources.

Priority No. a)

Firm name, Process, Product Companies listed in chlorine chain study

Processes b) Water Air Product

1 Akzo Nobel Botlek Chlorine production (2) 1 Shin-Etuse, formerly AKZO. Nu-

farm, formerly AKZO EDC/VCM production (3) Production MCPA/MCPP (4)

(1) g) 1

2 Resolution Products, formerly Shell Pernis

ECH and AC production (5) 1 1

Shin-Etsu Pernis PVC Production (6) 6 Akzo Nobel Nobel Hengelo Chlorine production 1c) MWTP Hengelo MCA production (8) 7 Akzo Nobel Farnsum/Delfzijl production Chlorine, solvents 1 (DCM, chloroform) (9) 8 Teijin Twaron BV, formerly

Aramid Products, Delfzijl (part Acordis)

Production aramide en precur-sons (10)

1 1

10 Dow Benelux Terneuzen Production ethylene amines (11) 1 11 Novenon, formerly BF Goodrich Production C-PVC (12) 1 14 Du Pont de Nemours Dordrecht Production HCFCs and Teflon

(15) 1f)

17 Crompton Europe BV (formerly Uniroyal Amsterdam

Production insecticides (17) 1

19 Atofina, formerly Elf Atochem Vlissingen

Production of organotin (17) 1e)

21 Akzo Nobel Kleefsewaard CMC production (18) 1 23 Avebe Production modified starch (18) 1 27 Hercules Zwijndrecht Production ECH-derivates (19) 1 29 KerrMcGee pigments, formerly

Kemira pigments Titanedioxide production 1

31 Givaudan Roure, formerly Tastemaker

Production with EDC (20), ceased per end of 2000

1d)

36 Sappi Paper recycling 1 Non-industrial process applications 41 Solid Waste Incinerators Municipal waste incineration 1 43 Use of chlorine in cooling water Chlorine in cooling circuits 1h) Processes suggested by Steering committee (BOVOC) 64 Waste water treatment plant Municipal /non-industrial 1 Total selected 18 4 a) Item number in final report of prioritisation study (Task 3.2), Tukker (2000a); b) between

brackets: chapter in background document (Task 3.1), Tukker (2000b); c) wastewater treated in municipal waste water treatment plant; d) production ceased end of 2000, not included in study; e not included in study; f) discharge via 2 installations; g) discharge installation added later to priority nr. 1; h) additional sample taken of inlet make-up water


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Table II.2 Products.

Priority No. *

Firm name, Process, Product Companies listed in chlorine chain study

Processes ** Water Air Product

Products produced in NL 45 EDC 1 48 AC 1 49 MCA 1 50 DCM 1 57 HCl 1 Products not produced in NL 58 PERC 1 59 Trichloroethene 1 60 Chlorobenzenes 1 61 Chlorophenoles 1 63 Chloroparaffins 1 Processes suggested by Steering committee (BOVOC) 65 Random' choice 1 1 66 Random' choice 2 1 Total samples per medium 18 4 12 Total samples 34 * Item number in final report on prioritisation study (Task 3.2), Tukker (2000a); ** Between brackets: chapter in background document (Task 3.1), Tukker (2000b).


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Appendix III. Sample pre-treatment and recovery data

Table III.1 Overview of characteristics of the 21 water samples before and after extraction.

No. Code Odour Init.pH Colour extract Remarks 1 mqr.w.2901.stab none 7 clear, yellow None 2 mno.w.2901.stab soil like 6.5 clear, pink None 3 mrs.w.0502.stab none 7 clear, yellow • at pH>11 sample turns from light to

dark yellow

4 mmn.w.0502.stab soil like 7 unclear, yellow None 5 mlm.w.0702.nstab none 1.5 clear, light yellow • at pH>11 white precipitation

6 buv.w.0702.stab none 7 clear, light yellow None 7 mst.w.1202.nstab slightly

chlorine 7 clear, yellow • at pH>11 white precipitation

8 bst.w.1202.nstab none 6 clear, light yellow • at pH<2 precipitation already in sample disappears

• at pH>11 white precipitation

9 mcd.w.1202.stab oil like 7 unclear, dark yellow • emulsion is difficult to break.


10 mef.w.1402.nstab soil like 7 clear, green/ yellow

• emulsion very difficult to break.

• no clear boundary between water and pentane.


11 mfg.w.1402.nstab sweet apple like

6.5 unclear, white • at pH<2 sample becomes unclear


12 mgh.w.1402.nstab soil like 6 clear, colourless • at pH>11 white precipitation and sample turns dark yellow

13 btu.w.0802.stab soil like 6 clear, yellow • at pH>11 white precipitation

14 mop.w.1902.stab soil like 7.5 clear, light yellow • foam formation during handling

• at pH<2 gas formation. During ex-traction high pressure in separation funnel


15 mkl.w.1902.nstab hydro-chloric acid

4 clear, no colour • at pH>11 white precipitation and sample turns yellow

16 mjk.w.1902.nstab sewer like

1 clear, no colour • needs 90 ml 10 M NaOH per 2 litres to reach pH>11


17 mij.w.2102.stab petrol like

6.5 clear, yellow • at pH>11 white precipitation

18 mde.w.2102.stab soil like 6 clear, light yellow • at pH>11 white precipitation


80

No. Code Odour Init.pH Colour extract Remarks 19 mbc.w.2602.stab none 8 clear, light yellow • at pH<2 gas formation


20 mab.w.2602.nstab chlorine 6 clear, no colour • density of the water >1

21 mpq.w.2602.nstab petrol like

4.5 unclear, dark yel-low/brown

• density of the water >1

• needs 45 ml 10 M NaOH per 2 litres to reach pH>11

• at pH>11 a lot of precipitation and water turns dark blue

Table III.2 Recovery of the extraction procedure for a mixture of PCBs and chlorinated pesticides in relation to hydrophobicity (Kow) and volatility (H, Henry Law constant)*.

Compound Log Kow H Pa.m3/mol Recovery (mean) standard deviation (n=3) b-HCH 3.78 0.07 93% 7% o,p-DDD 5.87 0.67 92% 6% p,p-DDD 6.02 0.67 98% 4% o,p-DDT 6.79 0.82 72% 8% p,p-DDT 6.91 0.82 86% 6% PCB 180 8.27 1.01 91% 23% a-HCH 3.8 1.07 60% 6% a-endosulfan 3.83 1.13 80% 5% b-endosulfan 3.83 1.13 96% 4% g-HCH 3.72 1.42 75% 2% PCB 138 7.44 2.13 77% 11% cis-HEPO 4.98 2.13 74% 7% trans-HEPO 4.98 2.13 72% 7% PCB 153 7.75 2.33 67% 10% o,p-DDE 5.44 4.22 50% 10% PCB 101 6.8 9.12 46% 9% Aldrin 6.5 17.23 7% 1% PCB 28 5.62 20.27 24% 6% PCB 52 6.09 20.27 31% 5% PCB 118 7.18 29.18 69% 8% Heptachloor 6.1 29.79 10% 3% Pentachlorobenzene 5.17 71.23 6% 4% Hexachlorobenzene 5.73 172.25 8% 4% * Kow and H values derived from EFDB database (SRC, 1998) and the EPIWIN estimation

software (Meylan and Howard, 1999a, 1999b).


81

Appendix IV. Relative light yield data from chronic Microtox test

Table IV.1. Relative light yield data from chronic Microtox test.

17% of effluent

concentration

33% of effluent

concentra-tion

66% of effluent

concentra-tion

17% of effluent

concentration

33% of effluent

concentration

67% of effluent

concentration

Sample code

Relative light yield



Significance Significance Significance

MPQ 0.93 0.84 0.53 0.30 0.01 0.00 MDE 0.94 0.97 0.75 0.14 0.32 0.00 MIJ 0.99 1.04 0.94 0.45 0.70 0.26 MAB 0.84 0.73 0.65 0.00 0.03 0.01 MBC 0.89 1.06 1.05 0.10 0.56 0.62 MJK 1.00 1.02 1.18 0.50 0.56 0.93 MKL 1.05 1.13 1.09 0.67 0,77 0.59 MOP 0.93 0.95 0.86 0.25 0.31 0.10 MEF 1.08 1.02 1.00 0.73 0.58 0.50 MFG 0.93 1.15 0.97 0.26 0.75 0.45 MGH 0.98 1.04 0.95 0.41 0.68 0.35 MCD 0.88 0.81 0.54 0.24 0.14 0.01 MLM 0.78 0.94 0.83 0.03 0.30 0.27 MRS 0.91 0.78 0.75 0.27 0.07 0.04 MNO 0.83 0.77 0.78 0.09 0.05 0.25 MQR 1.08 1.13 0.95 0.63 0.75 0.42 BST 1.46 0.40 0.75 0.87 0.06 0.25 MST 0.77 0.84 0.53 0.07 0.15 0.02 MMN 1.04 0.67 0.46 0.60 0.01 0.00 BTU 0.63 0.65 0.86 0.08 0.12 0.35 BUV 0.86 0.69 0.84 0.13 0.01 0.10 N.B. Figures in bold type have a significance of at least 95% for the sample value being

different from the reference value; bold figures that have been underlined have a significance value of at least 99%.

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