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Chlorine chain follow-up studies (OVOC) Meeting Report 3rd Technical Workshop, May 14-15, 2003

B. van Hattum, M. Lamoree (IVM), H. Senhorst (RIZA), T. Sanderson (IRAS) and A. Tukker (TNO-STB)

Chlorine Chain Follow-up Research Programme on Chlorinated Organic Microcontaminants (OVOC)

E-03/13

December 2003

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 Environment (SNM).

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]

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.

Meeting Report 3rd Technical Workshop i

Contents

1. Introduction 2

2. Presentations and discussion 4 2.1 Seminar validity and limitations of bioassays (14/5 evening session) 4 2.2 Results of fine fractionation studies (Marja Lamoree) 6 2.3 WP 8.1 Biodegradation studies (Henk Senhorst) 7 2.4 WP-9 In plant TIE studies (Henk Senhorst) 8 2.5 Atmospheric emissions (Bert van Hattum) 9 2.6 Results of the product study (Bert van Hattum) 10 2.7 Evaluation of selection of emission sources (Arnold Tukker) 11 2.8 Synthesis of results (Henk Senhorst) 12 2.9 Reflection in perspective of objectives (Arnold Tukker) 13 2.10 Final Discussion on questions raised by the research team. 14 2.11 Concluding remarks by BOVOC members 16 2.12 Conclusions formulated by the chairman (Jan van der Kolk) 17

Appendix I. Participants Workshop OVOC May 14-15, 2003 19

Appendix II. Documents and reports distributed 21

Appendix III. References cited in section 2.1 22

Appendix IV. Presentation slides 23

Institute for Environmental Studies 2

1. Introduction

On May 14-15, 2003 the 3rd technical OVOC workshop was organised in Den Haag (Regus Business Centre, Equinox building). The objectives of the workshop were:

• To evaluate the results obtained so far from the screening and additional studies (WP 5, 6, 7, 8.1, 9);

• To give guidance to the remaining Phase-2 studies and the final integration and re-porting (Phase-3).

As the in-vitro tests DR-CALUX and carp-hep EROD test were the main responsive tests in the study, and the role and status of in-vitro testing in environmental risk assess-ment is a rapidly developing field, a special seminar was organised on this topic on the evening of the 14th of May, with introductions by Prof. dr. Bram Brouwer (IVM/BDS) and Prof. dr. Martin van den Berg (IRAS). Participants were asked to submit specific questions and topics to be addressed prior to the workshop.

Participants at the workshop (see Appendix 1) included members of the BOVOC steer-ing committee, experts invited by the BOVOC steering committee, and members of the research team.

Various draft documents and reports with the results of Phase-2 work packages 5, 6, 7, 8.1 and 9 were distributed in advance of the workshop. A special integration paper was written, summarising the main findings of the Phase-2 studies. As far as possible all the technical reports have been reviewed/commented by experts in the weeks before the workshop, in order to allow the workshop to focus on the main questions.

Documents and reports distributed in advance of the workshop

1. Summary/integration document on main findings of Phase-2 technical studies; 2. Questions to be addressed by the workshop; 3. Note to the parliament (Tweede Kamer) on objectives and progress of project

(Sept. 2002).

Phase-2 Technical reports:1

4. Chlorinated Micropollutants in Aquatic Effluents; 4.1. Part-1 Screening studies (WP5&6) (final version) 4.2. Part-2 Biodegradation studies (WP8) (draft) 4.3. Part-3 Primary TIE studies (WP7) (draft) 4.4. Part-4 In-plant TIE Studies (WP9) (draft)

5. Chlorinated Micropollutants in Atmospheric Emissions (draft); 6. Chlorinated Micropollutants in Products (draft).

1 Full references given in Appendix 2

Meeting Report 3rd Technical Workshop 3

Based on the conclusions and recommendations of the workshop, the BOVOC steering committee will decide how to proceed with the final WP-8 studies in Phase-2 and the fo-cus of the work plan for the final integration and reporting phase of the project (Phase-3).

Programme of the meeting 14-15 May 2003

Wednesday 14th Seminar on in-vitro testing in OVOC project Language of presentations and discussion: English 18.00 –18.30 Arrival - coffee / drinks 18.30 - 19.00 Italian buffet (cold and warm dishes) 19.00 - 19.10 Opening by the chairman, J. v/d Kolk (Ministry VROM) 19.10 - 19.40 Introduction on DR-CALUX bioassay, current regulatory applications and application to effluent screening (Bram Brouwer) 19.40 - 20.10 Introduction on carp-hepatocyte assay and related in-vitro tests and view points of WHO on status of in-vitro testing in environmental risk assess ment (Martin van den Berg) 20.10 – 21.00 Questions and discussion

Thursday 15th 3rd Technical workshop OVOC Project Language of presentations and discussion: Dutch 09.00 – 09.30 Arrival and coffee 09.30 – 09.40 Opening by the chairman, J. v/d Kolk (Ministry VROM) 09.40 – 09.50 Short introduction to the workshop programme (Bert van Hattum) Short presentations of results (10 mins) and response of experts (15 mins) 09.50 – 10.15 Results of the refined fractionation study (M. Lamoree, IVM) 10.15 – 10.40 Results of the biodegradation studies (H. Senhorst) 10.40 -- 11.05 Results of the in-plant TIE study (H. Senhorst) 11.05 – 11.15 Short coffee break 11.15 – 11.40 Results of atmospheric emission (Bert van Hattum) 11.40 – 12.05 Results of product study (Bert van Hattum) 12.05 – 12.30 Evaluation of selection of emission sources, introduction by Arnold Tuk ker and discussion 12.30 – 13.00 Lunch Simple Dutch lunch with ‘broodjes’ 13.00 – 13.45 Synthesis of results from screening and additional studies (Henk Sen-horst), discussion. 13.45 – 14.30 Reflections of main findings in perspective of objectives (Arnold Tukker),discussion. 14.30 – 15.00 Short tea break 14.45 – 16.00 General discussion, conclusions and recommendations (based on docu ment ‘questions to be addressed’) 16.00 Closure of the meeting


2. Presentations and discussion

2.1 Seminar validity and limitations of bioassays (14/5 evening session)

Rapporteur: Thomas Sanderson

Presentations were given by prof. dr. Bram Brouwer (IVM-VU / BDS) and prof. dr. Martin van den Berg (IRAS-UU) on the technical aspects and performance characteris-tics of the in-vitro tests applied in the study. The discussions focussed on the comparison between both in vitro-assays, the extent to which the results could be used for further risk assessment purposes, and the probability and significance of false-positives or false-negatives. A summary of the discussion and conclusions was presented by dr. Thomas Sanderson at the start of the technical workshop of the 15th of May.

Table 2.1 Comparison of DR-Calux and Carp-Hep.

Assay Name DR-CALUX Carp-Hep Cell type Rat hepatoma Fresh carp hepatocytes Response Ah receptor-mediated luciferase induction Ah receptor-mediated EROD induction Exposure time

24 h exposure 96 h exposure

Sensitivity High sensitivity Less sensitive for some co-planar PCBs

Reliablity Highly repeatable and reproducible Repeatability good within a single carp preparation; reproducibility fair-good (dependent on the biological variability of the genetically uniform strain of carp used, and on the quality of the cell preparation process)

Metabolism Plays limited role in 24h exposure Plays greater role Validity and acceptance

Well described and characterized. Extensively used as bioanalytical screening tool by academic laboratories and regula-tory agencies (food sector, evaluation of harbour dredgings), as described in the sci-entific literature (Garrison et al., 1996; Murk et al., 1996; Sanderson et al., 1996; Sander-son and Giesy, 1998; Behnisch et al., 2002; Stronkhorst et al., 2003)

Well described and characterized in scientific literature Used on contract basis for specific environmental ques-tions (Smeets et al., 1999a; Smeets et al., 1999b; Smeets et al., 1999c; Rankouhi et al., 2002)

Usage Bioanalytical tool Environmentally more relevant tool Limitations See text below References presented in Annex-3


Tests applied in the study:

• Zebrafish Early Life Stage test (developmental toxicity, non-specific); • Microtox (acute toxicity, non-specific); • DR-CALUX (Ah receptor); • Carp-Hepatocyte bioassay (Ah receptor, (anti)estrogenicity).

False Negatives:

• When samples are cytotoxic, which is easily identified by e.g. 1) measuring LDH (lactate dehydrogenase, an important enzyme normally present in the cell, but which leaks from the cell if cellular membrane damage occurs) leakage from the cell or 2) MTT re-duction (a measure of mitochondrial activity reflecting the ability of cells to produce energy for proteins to function properly), 3) protein loss or 4) by visual in-spection of the cells through a light microscope;

• When samples inhibit luciferase or EROD activity (rarely completely blocks response, thus some induction will usually be observed);

• When active components are lost during “clean-up”; • The above false negatives may occur in either DR-Calux or Carp-Hep system; • Usually low frequency of false negatives.

False positives:

• Many natural compounds can activate Ah receptor; • These compounds are non-halogenated; • These compounds are usually non-persistent; • They activate the AhR only transiently; • They do not induce the multiple combination of responses typical to dioxins (wast-

ing syndrome, which is a decreased ability to retain or utilize the energy from in-gested calories, chloracne, immunosuppression, hepatotoxicity, teratogenicity etc), which are associated with persistent binding to- and continuous activation of the Ah receptor over prolonged periods of time;

• They can be eliminated through appropriate clean-up; • The occurrence of these false positives plays a smaller role in CarpHep than DR-

Calux, due to longer exposure time and increased metabolism in the Carp Hep test system.

Interpretation of Bioassay results

• Prescreening (selection for GC-MS, further fractionations, or for other toxicological investigations);

• Ranking (provides a rapid and cheap first impression of which areas are potentially more contaminated than others);

• Estimation of TEQs (after appropriate clean up to eliminate potential false positives – see previous section);

• The DR-Calux provides no information about species sensitivity to dioxin-like chemicals, it is mainly useful as bioanalytical tool to estimate approximate TEQs (2,3,7,8-TCDD toxic equivalent quantities) for prescreening and ranking purposes;


• The Carp-Hep assay provides some idea of the relative sensitivity of the Carp compared to other species but the ecotoxicologically relevant information provided by the test is still limited and not suitable for extrapolation to environmental risks.

It was concluded that both tests are sufficiently reliable and sensitive for the screening purposes of the OVOC project and, when the appropriate clean-up methods are used, both tests are acceptable indicators of the presence of PCDD/Fs or other compounds with the same Ah receptor-mediated mechanism of toxicity. The DR-CALUX method has been validated for the food sector and for the evaluation of harbour dredgings, but not for wastewater. Positive results from DR CALUX or carp-hep EROD should preferen-tially be confirmed with analytical quantification before they can be used for the assess-ment of aquatic field samples. Proper care should be taken when comparing in-vitro based 2,3,7,8-TCDD equivalent concentration estimates, with TEQs derived from classi-cal chemical dioxin analysis and application of TEFs (toxic equivalent factors).

Due to the very low frequency of false negatives, it was recommended that the assays could be used to demonstrably prove the absence of PCDD/Fs or other dioxin-like.

As the dioxin-like toxicity has become central in the study, it is important to consider in the final report, to what extent other types of toxicity, relevant for chlorinated PBTs, may have been overlooked.

The contribution to the estimation of ecological risks is limited; these tests should always be used in combination with information obtained from field studies and from in-vivo bioassays.

2.2 Results of fine fractionation studies (Marja Lamoree)

Rapporteur: Henk Senhorst

Fine fractionation has been performed on a selection of coarse log Kow extracts. Be-cause of the DR Calux response pattern in the screening phase, mainly high log Kow fractions were selected. Of three samples also the medium log Kow fraction was chosen.

Each extract was freshly prepared from the original, frozen effluent sample. The DR Calux response value of these new extracts agreed very well with the value of the ex-tracts prepared in the screening phase. Next, the coarse log Kow extracts were fraction-ated into extracts with a log Kow window of 1. Originally only two fine fractions proved to be responsive above the detection limit. As the response greatly declined, an extra 50x concentration step was decided upon. After concentration, the response of nearly all tested samples proved to be present at the highest log Kow value tested, i.e. >6 or > 8. Only sample MCD showed a different behaviour with a distinct response in the log Kow 5-6 interval.

Identification of (chlorine containing) components in the responsive fine fractions by means of GC-MS could not corroborate the presence of chlorine containing substances. Several hundreds of peaks were present in the concentrated extracts, but of these only half had a mass spectrum that more or less matched to a mass spectrum in the NIST li-brary. Therefore, the presence of chlorine containing substances cannot be excluded at


this stage. The conclusion should be that we have not (yet) been able to identify them by the NIST/AMDIS based automatic interpretation of GC-MS data.

The following recommendations were made by the researchers: to look for chlorine con-taining components among the non-identified peaks; application of other detection methods like GC-NCI or GC-AED; application of acid silica cleanup on concentrated extracts; dioxin analyses on extracts.

Questions asked:

• In which fractions would PCBs and dioxins be present? PCBs are expected in the log Kow fraction 6-7, dioxins probably in 7-8 or >8 fraction. It should be noted that different types of substances, having the same log Kow value, may have different behaviour during fractionation on the HPLC column and may end up in different fractions;

• Are the recommended actions (mentioned in the report) directed towards finding unknown chlorine containing compounds useful? Yes, but success is not guaranteed. The additional chemical analysis of PCDD/Fs will provide indications which part of the response can be attributed to classical dioxins. Further GC-NCI-MS could provide indications for the presence of other halogenated compounds, that might be involved;

• Are those substances identifiable then? Yes, possibly, though exact identification will involve testing of the pure substances under consideration, which is expected not to be possible for a great number of reaction by-products;

• Is a possible identification linkable to the DR Calux response observed? Due to the large amount of components involved and the possibility of mixture toxicity the re-sponse can be spread over many (identified and unidentified) peaks, some even hav-ing a response below the level of their analytical detection limit. The discussion on this item showed a difference in the appreciation on this point among the participants in the discussion. This approach would only work in the case of simple mixtures, when DR-CALUX responsive fractions contain a limited number of components well above the analytical detection limit.

In conclusion, most workshop participants agreed that further extended fractionation (as proposed in the original workplan) was not recommended, and that application to a se-lection of fractions of direct chemical analysis of PCDD/Fs, GC-NCI-MS screening, and DR-CALUX response after acid clean-up would be more effective for the final interpre-tation.

2.3 WP 8.1 Biodegradation studies (Henk Senhorst)

Rapporteurs: Marja Lamoree and Bert van Hattum

A presentation was given on the main results of the biodegradation studies and additional studies, elucidating the unexpected response of the control substrate (water from Lake Oosterschelde). As concluded by various participants, most of the available validated biodegradation tests can only be applied for individual compounds or well-defined mixtures; currently there are no suitable biodegradation tests for complex mixtures such


as applied in the OVOC project. A major part of the discussion focused on the potential significance of the acid clean-up method for classification of vP compounds.

Anton Gerritsen emphasized that the test applied in the study only provides information on the short-term degradation under mild circumstances and may not be suitable to clas-sify persistency properly. Albert Berends mentioned that it is important to analyse which compounds survive the acidic-clean up; and which of these compounds are known to be vP; the clean-up method should be validated with known POPs.

Pim Leonards explained the history of the clean-up technique, which is more or less standard for the chemical analysis of polyhalogenated aromatic compounds and traditional chlorinated pesticides.

Gert-Jan de Maagd referred to publications of German workers (Schramm et al.) who reported on the presence of PAHs in 1st fraction (discarded) of the clean-up, and who have suggested to use the acid clean-up method for P-classification.

Marc Koene asked about the significance and seasonal variation of the blank: response in the experiments and the dependency on the decay of algae. The report describes an example of potential responsive decay products (brevetoxin) observed in other freshwater studies.

Conclusion: the DR-CALUX results without acidic clean-up are not meaningful in the biodegradation study; a good description including persistency characteristics of compounds surviving the acidic clean-up is vital for the final report.

2.4 WP-9 In plant TIE studies (Henk Senhorst)

Rapporteurs: Marja Lamoree and Bert van Hattum

Results were presented of a dedicated study on several internal wastewater streams at one selected site (MCD) with the purpose to establish the contribution of chlorine activities to the effluent toxicity load. Comparison with screening phase results showed a good correspondence for several of the parameters. After evaluation the first results, additional measurements were made, including DR-CALUX response after acidic clean-up and selective GC-AED measurements. These measurements clearly demonstrated that chlorine-chain activities were the major contributor to the DR-CALUX response (after acid clean-up); in previous measurements without acidic clean-up the contribution of wastewater streams from non-chlorine related activities was predominant, and was attributed to PAHs based on GC-MS analysis. Chemical analysis by GC-MS was hampered by the presence of a continuum of substances. An abundance of chlorine-containing substances was found in the low log Kow-fraction from chlorine activities. Corresponding medium and high log Kow-fractions could not reveal any identifiable chlorine-containing reaction by-products. The GC-AED results confirmed that the bulk of the chlorine containing compounds originates from the chlorine activities but no link could (yet) be made with the DR Calux responses observed. The observed load of dioxin equivalent activity (in TEQs) is about a factor of six above what can be expected on the basis of historic information on PCDD/F concentrations.


Ron Postma asked about the 80% removal in the biotreater as mentioned for some compounds; this seems a low figure? Where were samples taken? Samples were taken at influent and effluent. The removal percentage was based on the concentration ratio in influent and effluent.

Martien Janssen asked about the number of replicates and the variability of the composition of the effluent. Single composite samples were taken (proportional sampling); the standard deviation of the DR-CALUX results was based on 3 replicate measurements of the extract of this sample. The issue of variability of the effluent needs to be further addressed in the final report, before final conclusions and comparisons can be made.

Ron Postma: How was the 4% I-TEQ compared to the total DR-CALUX TEQs determined? This was derived from historic HRGCMS measurements of PCDD/Fs (0.3 g/yr versus 7 g/yr).

Anton Gerritsen mentioned a number of questions to be addressed in the final report: What kind of substances can be expected in the MCK-MCD samples? Which Log Kow interval is expected? What is the contribution of high and low polarity compounds? Is the DR-CALUX response to be expected only in the high Log Kow range?

Marc Koene: The important question to address is: What is the contribution of chlorinated compounds? At this stage causal linkages are not proven in the explanation of the observed DR-CALUX response.

Most workshop members agreed with the recommendation, that additional direct dioxin analysis of matching samples would be effective to determine the contribution of PCDD/Fs to the DR-CALUX response.

2.5 Atmospheric emissions (Bert van Hattum)

Rapporteur: Marja Lamoree

Van Hattum presented a summary of the main results from the corresponding report. As this study was conducted at a later stage in the project, the experimental design included acidic clean-up and direct analysis of dioxins. All 4 vent-gas effluents were DR-CALUX responsive and contained PCDD/Fs. In 3 of the 4 samples PCDD/Fs were responsible for more than 85% of the in-vitro response. In one installation more than 70% appeared to be attributable to hydrophobic unknowns (not necessarily chlorinated) surviving the acidic clean-up. The contribution of vent-gas installations to national TEQ loads was limited compared to other sources.

Albert Berends enquires whether there is national legislation regarding atmospheric dioxin emissions in the Netherlands. Ron Postma answers that legislation is not nation wide, but that it varies between emissions permits that are negotiated with the appropriate (local) authorities. A directive of 0.1 ng/Nm3 is usually adopted for waste incinerators.

Ron Postma suggests that identification of unknowns in the atmospheric emissions may be done with the method that is used for 'classical' dioxins, HRGC-HRMS. The high costs of such an approach are a problem.


Jacob Bouwma concluded that in 3 of the 4 samples of atmospheric emission almost all DR-CALUX response is accounted for by chemical analysis of the 'classical' dioxins; obviously ‘unknown’ chlorinated microcontaminants are not present in these samples.

On questions related to the total annual loads emitted, Bert van Hattum emphasized that due to the discharge volume of the municipal waste incinerators, this category – although it had low concentrations- is the most important contributor to the total TEQ load for atmospheric emissions.

Workshop members agreed with the main conclusions of the study presented. The topic of extrapolation of the results to other vent-gas installation in use in the chlorine industry should be addressed in the final report. For the evaluation of the importance of further additional work (GC-NCI-MS) on the sample with a high contribution of hydrophobic unknowns, the BOVOC and research team should take into account the relative contribu-tion of this source in comparison to other emission sources and pathways.

2.6 Results of the product study (Bert van Hattum)

Rapporteur: Arnold Tukker

Bert van Hattum presented the approach of the product-oriented study. Based on the prioritisation study in 2000, ten products were selected for screening (GC-MS and DR-CALUX, including acidic clean-up. Two ‘wild card’ selections were added, neoprene and triclosan, proposed by the NGO representative in the BOVOC steering committee.2.

In summary the main results:

• In a limited number of products the DR-CALUX response was above the limit of de-tection (LOD): neoprene, HCl, and to a lesser degree TRI, EDC and PER;

• Annual loads seem to be limited, except for neoprene (which was a ‘wild card’ selec-tion). Based on a hypothetical national market volume of 1000 t/yr the DR-CALUX TEQ loads could be in the order of g/yr. Potential DR-CALUX TEQ loads from HCL would be a factor of 10 lower;

• For 3 products the LOD was relatively high and in-theory extrapolated loads for these non-responsive samples could still be significant (compared to known emission loads of dioxins);

• The GC-MS screening of impurities provided indications for the presence of chlorin-ated hydrophobic compounds (PCP, chlorinated benzenes), some of which are PBT.

For more detailed information reference is made to the corresponding report. In the dis-cussion the following points were raised:

Jan van der Kolk: Was the DR-CALUX response in neoprene attributable to identified components? Bert van Hattum: No, not at this stage. Additional dioxin determinations are required, and potential contributions from PAHs observed in responsive fractions of the extract of the formulation investigated cannot be excluded at this moment.

2 In total 11 products were analysed; because of end of usage in the Netherlands one product was skipped from the selection.


Albert Berends: Results from internal dioxin analysis by Solvay of HCL and TRI products are in line with the OVOC study. Concentrations in the order of magnitude of > 10 pg/g TEQ were found. Levels in PER and DCM were below detection limits.

Pim Leonards: Why do the LODs vary between products. The LOD of DCB is relative high? Bert van Hattum: These are the result of lower test concentrations of the main product (appr. 10 mg/L) used in this study, in order to avoid overloading of the GC-column. Additional range finding studies with DR-CALUX at higher concentration levels (just below level of cytotoxicity) are required in order to establish lower LODs for these products and more realistic load estimates.

Jan van der Kolk: Neoprene is clearly different. What is the reason? Are PAHs involved? Do PAHs survive the acidic clean-up. Bert van Hattum: PAHs have been observed in the GC-MS results of the extracts of the neoprene formulation. The information on the fate of PAHs is partly conflicting. The results in WP-9 indicated that interfering contributions attributed to PAHs are effectively removed during the acidic clean-up step. From German studies (Schramm et al. cited in the report) there is information that some PAHs may survive the acidic clean-up and from other studies it is known that PAHs may occur in the first fractions of silica clean-up. This will be clarified in additional validation experiments. From older Dutch studies on windsurfers using neoprene wet suits (Jongeneele et al. around the 1990s) it is known that metabolites (1-hydroxy pyrene) of pyrene (a PAH compound) could be detected in urine after several hrs of exposure.

Marc Koene: Why is the average DR-CALUX response of neoprene after acidic clean-up higher than without acidic clean-up? Bert van Hattum: The (slight) difference is not significant in relation to the variability of the determinations

To conclude, the following points need to be addressed in the final reports:

• The general question to what extent DR-CALUX based TEQ estimated may be compared with TEQs from traditional chemical analysis of dioxins (HRGC-HRMS);

• The explanation of the high response of neoprene (and potential attribution to chlorinated PBTs);

• The probability that <LOD values for some products still could imply a significant load.

2.7 Evaluation of selection of emission sources (Arnold Tukker)

Rapporteur: Arjen de Koning and Henk Senhorst

In the presentation by Arnold Tukker the selection and prioritisation procedure for po-tential emission sources of unknown PBTs from the chlorine chain was evaluated.

The following conclusions were drawn:

1. aquatic effluents: selection probably has been appropriate; 2. atmospheric emission sources: selection probably has been appropriate; 3. products: the accuracy of the selection is much less certain. A significant DR-

CALUX response was found in one of the wild-card selections proposed by the NGO representatives; this product was not included in the prioritisation procedure.


In the discussion the following topics/questions were addressed:

• In addition to priorities for potential emission sources, further priorities were as-signed within the experimental studies to emissions and by-products with a dioxin-like mode of action;

• In hindsight, should it not have been better if the list of products to be evaluated also had included non-chlorinated products produced in a process in which chlorine or chlorinated compounds are involved? This was not considered as a feasible approach. Neoprene was not included because the sector of rubber was not considered and because of the incorrect assumption that neoprene was not a chlorinated product.

Further extension of the list of products to submit to a similar screening is not feasible within the project, because their number is too large. It is expected that a prioritisation based on expert-interviews (“where can impurities be expected?”) might be an effective approach to select candidates for further screening in future studies.

During the further discussion the following remarks and recommendations were made:

• The acidic clean-up step needs to be validated. What is really happening in relation to persistence?

• Better and more balanced representation of the information from in-vivo studies; • Most of the participants seemed to agree with the operational classification of B/vB

(log Kow 4-6, resp. >6); • The in-vitro studies cannot be extrapolated to ecological risks; • Classification of persistence is recommended for the final stage for individual com-

pounds, which are known to be responsive; • Avoid confusion between ‘dioxins’ and ‘dioxin-likes’. • It is important to evaluate to what extent effect-parameters may be considered as

specific for the chlorine chain; • The final report should be transparent and easy to read; • German researchers (Schramm) should be consulted on their proposals for persis-

tency classification using acidic clean-up procedures; • The concept of lethal body burdens (LBB) may be usuful for the prediction of non-

specific toxicity; • Take notice of the limited extraction recovery of super-hydrophobics (log Kow >8).

2.8 Synthesis of results (Henk Senhorst)

Rapporteurs: Bert van Hattum and Arnold Tukker

Albert Berends emphasized that the role of the in-vivo response (zebra-fish test) should be made more important in the T classification within the project, and that the zebra fish ELS is very sensitive for dioxins. Martin van den Berg mentioned that salmonid ELS tests are more sensitive for dioxins.

Dolf van Wijk stated that the compounds remaining after the acid clean-up should not be considered as synonym with chemically stable or persistent compounds, i.e. which do not exhibit biodegradation. Additional information on the performance of the method is vital.


Martin van den Berg remarked that the comparison of bioassay-response in the effluent and product studies with chemical dioxin measurements, derived from literature sources is not very convincing, and made a plea for additional PCDD/F measurements on relevant samples.

Gert-Jan de Maagd recommended to leave the question of the T classification, and to focus in the last phase of the project on strengthening of the P and B classification. The B classification is acceptable. The main question now is: Is the P as derived from the clean-up method a true P? Try to address further identification issues, by re-examining available GC-MS results and apply literature data on P for identified compounds.

Dolf van Wijk pointed to the fact that currently there are no suitable P-tests for mixtures; available and accepted test methods are suitable mainly for single well-defined compounds.

Anton Gerritsen pointed out that earlier pleas for more in-vivo methods in the effluent evaluation should take notice of the discussion in May 2000 on the results of the pilot study, where most of the in-vivo tests were not responsive or the endpoint was not specific. The effluents of the OVOC study were also included in a national RIZA survey on whole effluent testing. Based on these data, an assessment can be made whether effluents from chlorine chain industries exhibited a different response in comparison to effluents from other industrial sectors. The outcome of such an analysis would be a meaningful contribution to the evaluation of the OVOC results. He offered to see if such an exercise would be feasible.

Marc Koene mentioned several observations: Fractions usually contained a large number of different compounds; final identification probably will not be possible. Several questions to be addressed are: Are there compounds or MS-spectra that are present in many different responsive samples? Can a comparison be made for constituents in effluent, vent-gas, and products originating from the same production site? Henk Senhorst mentioned that this would be one of the issues to address further in the final report.

2.9 Reflection in perspective of objectives (Arnold Tukker)

Rapporteur: Bert van Hattum

Arnold Tukker compared the results of the previous presentations with the general objec-tives of the OVOC project. The final interpretation and valuation of the results of the OVOC project are determined by the attitudes of the assessor, which may range between the risk assessment approach and the precautionary approach. For each of the attitudes the results and implications were presented for the effluent, product and atmospheric emission study. The answer on the first research question (are PBTs being emitted?) was a partial ‘yes’, but not a chlorine chain wide problem and important questions remain with respect to P and T attribution to chlorinated compounds. With respect to the as-sessment of environmental risks (2nd research question) the answer in absolute terms was negative, as extrapolation from in-vitro tests to environmental risks is not feasible; the answer in relative terms was a picture with questions.


For identified PBT compounds comparisons with known annual loads could be made, and available data on hazard characterisations could be included in the analysis.

The importance of households cooling water and other industries needs to be addressed. There is no insight in importance of historical and natural sources.

Various remaining questions to be discussed during the workshop were presented. Dur-ing the discussion a question was raised if all relevant pathways were included, and (as an example) reference was made to the issue of annual loads of dioxins via the applica-tion of fly ash from municipal solid waste incinerators (MSWIs) in asphalt road material. Further questions were postponed to the final discussion.

2.10 Final Discussion on questions raised by the research team.

Rapporteur: Mario Adams and Bert van Hattum,

The discussion followed the list of ‘questions to be addressed’ distributed prior to the workshop. The first 5 questions were treated sufficiently during the previous discussions.

Q6 Persistence. How should the persistence criterion be used? As the biodegradation tests were not conclusive, we propose to use other available information from the study. What is the opinion on the use of the discrimination between ‘metabolisable fraction’ and ‘acid clean-up stable fraction’ of the DR-CALUX response as a measure of persistence? Can the ‘acid clean-up stable’ fraction be indicated as vP?

Gert-Jan de Maagd: The persistence criteria of e.g. OSPAR are defined for individual substances. The OVOC project is about mixtures of compounds. and their biological response. A German group cited in the reports (Schramm et al.) has proposed to use the acid clean-up as a simulation of biodegradation; it is important to evaluate if this concept is useful for the final OVOC report. The combination of P and B properties is considered as important in OSPAR.

Dolf van Wijk: OSPAR like approaches can only be used in combination with identification of compounds. There are no suitable biodegradation tests for the type of studies discussed today.

Ron Postma warns that care should be taken not to judge P apart from B and T and to avoid what has been indicated as the ‘gravel’ discussion.

Marc Koene: In this study, which was explorative, only an indication for P was obtained. The residual response after the acid clean-up step should not be taken as a measure of P. In combination with the fact that compounds in effluents from treatment installations have survived biodegradation, this residual response is important.

Ron Postma noted that it is important to distinguish properly between total TEQs of dioxin like compounds and of individual PCDD/F congeners. The DR-CALUX response is not a proper basis for ecological risk assessment. This should be clearly described in the final report.

Gert-Jan de Maagd mentioned an obvious example of misleading classification (when using a criterion of 95% biodegradation for mixtures) for a mixture of sugar (95%) and DDT (5%).


Jan van der Kolk emphasized that care should be taken in the final report when the issue of persistency is treated, and that the report should provide exact and detailed descrip-tions.

Q7. Toxicity. In the design of the OVOC project we choose to focus on dioxin-like toxicity determined with two in-vitro assays. Other in-vitro tests included estrogenicity and cytotoxicity. Two non-specific in-vivo tests were added as reference. What other toxicity mechanisms, that are specific for PB compounds emitted from the chlorine chain, could be considered as relevant? How should the in-vitro classification of potential dioxin-like toxicity be used for the extrapolation to ecological hazards?

Martin van den Berg: There do not seem to be many other modes of actions than that of dioxin-like toxicity, in which halogenated compounds are more responsive than non-halogenated compounds. Because of their hydrophobicity, halogenated compounds may add to non-specific narcotic effects. The general toxicity parameters used in the carp-hep assay, such as MTT and LDH leakage, were low. Most neutral halogenated compounds do have a high cytotoxicity; more polar chlorinated compound may have a considerable cytotoxicity. It should be stressed, that the in-vitro results cannot be extrapolated to ecological risks.

Thomas Sanderson: Some groups of fluorinated compounds may have a specific mode of action.

Albert Berends recommends that in the final report a proper balance between the in-vivo results (zebra fish ELS and Microtox) and the in-vitro data should be maintained.

Q8. Bioaccumulation. Does the workshop agree with the classification used in the OVOC project: log Kow 4-6 and >6 as B and vB respectively?

Gert-Jan de Maagd: The OVOC approach fits reasonable with OSPAR.

Dolf van Wijk announced that CEFIC/Eurochlor has prepared fact sheets on their web-site on the many different international approaches (EU, EPA, OSPAR, OECD). A copy will be sent to the research team. The OVOC approach seems practical and provides suf-ficient indications on the B potency.

Jan van der Kolk added that the OVOC project provides the basic elements for a further assessment, and that the final assessment is not part of the project.

Anton Gerritsen stated that it should be realised that due to the selected extraction solvent the extraction efficiency of super hydrophobics (e.g. with Log Kow > 8) probably will be limited.

Q9. How important is the attribution of the in-vitro effects to chlorinated compounds? Should the results be compared with available data from non-chlorine chain industries?

Arnold Tukker: It should at least be related specifically to a process from the chlorine chain; preferably it should be related to a chlorinated PBT but this is not strictly necessary. The comparison with non-chlorine chain industries is important.


Anton Gerritsen mentioned that a comparison of emissions and toxicity between chlorine chain and non-chlorine chain industries could be important information in the final report, and that existing RIZA databases in principle should contain sufficient data for such an analysis.

Jan van der Kolk supported such a comparison of national effluent studies in which bio-assays were applied.

Marc Koene: It should be PBT and specific for the chlorine chain. If a non chlorinated stabiliser with PBT properties is essential for a product such as e.g. PER, than this should be considered as a problem. The study should provide an answer to the question if working with complex mixtures with e.g. 0.5% impurities is typical for this sector. It is further important to know what compounds disappear during the acid clean-up step.

Ron Postma: The main question should be whether the phenomenon is specific for the chlorine chain.

Gert-Jan de Maagd: What we want to assess is whether there are excessive emissions from the chlorine chain in comparison to non-chlorine chain industries.

Jan van der Kolk stated that the final report should make clear what components are lost due to the application of the acid clean-up method.

Q10. Is the comparison of dioxin-like in-vitro response with national PCDD/F emission figures a suitable assessment framework for the evaluation of the contribution of unknown PBTs to dioxin-like toxicity? How significant are the observations from the OVOC project?

Arnold Tukker: The uncertainties in the DR-CALUX loads should be taken into account, as well as the effect of temporal variability of effluent composition in relation to the sampling methods applied.

Marc Koene stated that such a comparison might be relevant for the assessment if additional remediation measures are needed. Jan van der Kolk stated that this is beyond the scope of the OVOC project.

Q11. What is the opinion on the recommended additional analysis of PCDD/Fs and GC-NCI-MS on a limited number of aquatic samples to elucidate the contribution of unknowns?

This question already has been answered positively during the discussions after other presentations This should be done for those samples and extracts for which this is considered as relevant, and could help to identify responsive unknowns. The available budget will limit the number of determinations.

2.11 Concluding remarks by BOVOC members

Invited by the chairman the BOVOC members formulated concluding statements:

Gert-Jan de Maagd stated that the workshop discussions were fruitful. In the final phase the focus should be on the P&B aspects. An important question for the last phase is what we want to do with the final results.


Jacob Bouwma mentioned that some recommendations and opinions expressed today were partly conflicting. For the last part of the study clear priorities must be stated of what still is feasible within the remaining budget and timeframe. The emphasis should be on the preparation of a clear and comprehensive description of the results and choices.

Bert van Hattum explained the planning for the last phase. The experimental work is expected to be finalised in October/ November 2003. The final reporting (in English) will consist of 1) a comprehensive technical report and 2) based on this a short executive summary reports, understandable for non-professionals. Draft versions of the reports are scheduled for February / March 2004. The final integral reports will be available and (if applicable) presented at a closing workshop in the summer of 2004.

Marc Koene stated that the project was highly explorative and that the research team has responded properly to the many experimental difficulties they have met on their path, and although the outcome of the studies did not always provide direct answers, he con-sidered it as very useful for the development of opinions. For the last part of the study he recommended that the final report should give a proper account of the many steps in the project and that it should indicate what was included and what was left beyond the scope of the study.

2.12 Conclusions formulated by the chairman (Jan van der Kolk)

The chairman Jan van der Kolk summarized the main conclusions, observations and questions of the workshop:

• The results presented at the workshop have demonstrated that a limited number of obvious problem-cases can be identified (some effluents, atmospheric emissions, several products);

• It has become clear that the problem is not chlorine-chain wide, i.e. not all companies belonging to the chlorine-chain are automatically involved;

• The final integrated report should provide a concise, transparent and logical account of all the steps, choices, arguments, and course of the project. The integrated report should make clear to non-professionals / outsiders how and why the various steps were taken and how the conclusions were reached. This is important, as the chronol-ogy of some steps (application of acid clean-up) appears to be illogical;

• In the final report on the project the potential and limitations of the methods should be clearly described;

• It should be made clear, what were the reasons behind the choice for the focus on di-oxin-like toxicity and what will be the consequences. What types of effect mecha-nisms might possibly have been missed or overlooked;

• The contribution of classical dioxins in the observed response should be clarified as much as possible based on additional chemical analysis;

• All bioassays applied should be explained clearly. A proper balance between in-vivo and in-vitro results should be maintained in the final report. Additional information from literature and other sources on in-vivo bioassay response in effluents from non-chorine industries should be included;


• The performance of the acidic clean-up step, which is important in the interpreta-tions, should be clearly described. We need to know what kind of compounds survive this procedure; additional validation experiments are needed for this;

• Relevant information from other literature/database sources should be added when applicable.

The chairman further stated that the presentations and discussions of the day had shown again that the project was highly explorative by nature, and that in such an approach the chances of failure and success both are present. In the project we have met the limita-tions of current methods and knowledge. The research team has been creative in their so-lutions and he expressed his appreciation for their efforts

After thanking all the participants for their contribution to the fruitful and productive workshop the chairman formally closed the workshop.

The final reporting on the project is scheduled for the summer of 2004, and will consist of a summarizing final technical report, executive summaries (in Dutch and English), and a CD with the final versions of the subproject-reports discussed during the work-shop.


Appendix I. Participants Workshop OVOC May 14-15, 2003

M. Adams Min VROM/DGM/SAS/SN Postbus 30945,Rijnstraat 8 NL - 2500 GX Den Haag Tel. +3170 339 4918 fax. +3170 339 1297 Email: [email protected]

A. Koning CML - UL Postbus 9518 NL – 2300 RA Leiden Tel. +3171 5277 480 fax. +3171 5277 434 Email: [email protected]

J. van der Kolk vz.Min VROM Postbus 30945 Rijnstraat 8 NL - 2500 GX Den Haag Tel. +3170 339 4729 fax. +3170 339 1297 Email: [email protected]

S. van der Linden VU – BDS De Boelelaan 1115 NL – 1081 HV Amsterdam Tel. +3120 4350758 Email: [email protected]

A. Brouwer VU – BDS De Boelelaan 1087 NL – 1081 HV Amsterdam Tel. 020 4449555 fax. 0204449553 Email: [email protected]

Mevr. L. van de Mast-Nijhuis Akzo Nobel Base Chemicals bv Postbus 247 NL – 3800 AE Amersfoort Tel. +3133 4676 865 fax. 3133 4676 102 Email: [email protected]

A. Gerritsen RIZA Postbus 17 NL – 8200 AA Lelystad Tel. +31320 298 458 fax. +3120 298 373 Email: [email protected]

T. Heemskerk Resolution Europe BV Postbus 606 3190 AN Hoogvliet Tel +31104314695 Email: [email protected]

M. Janssen RIVM – SEC Postbus 1 NL – 3720 BA Bilthoven Tel. +3130 274 2276 fax. +3130 274 4428 Email: [email protected]

T. Sanderson IRAS/RUU Postbus 80176 NL – 3508 TD Utrecht Tel. +3130 253 5398 fax+3130 253 5077 Email: [email protected]

Mevr. M. Lamoree VU – IVM De Boelelaan 1087 NL – 1081 HV Amsterdam Tel. +3120 444 9573 fax. +3120 4449553 Email: [email protected]

K. Swart IVM, Vrije Universiteit De Boelelaan 1087, 1081 HV Amsterdam Tel. +3120 444 9539 fax. +3120 4449553 Email: [email protected]

P. Leonards RIVO-DLO Postbus 68 IJmuiden 1970 AB Tel. +31 0255 564797 Email: [email protected]

J. Wilmer Dow Europe S.A.Bachtobelstrasse 3CH- 8810 Horgen Switzerland Tel +411-7282996 Fax: +411-7282965 E-mail: [email protected]


G.J. de Maagd V & W/RWS Postbus 20906 NL - 2500 GX Den Haag Tel. +3170 351 9033 Email : [email protected]

R. Postma Resolution Europe BV Postbus 606 3190 AN Hoogvliet Tel +31104314695 Email: [email protected]

B. van Opzeeland Greenpeace Nederland Keizersgracht 174 NL – 1016 DW Amsterdam Tel. +3120 626 1877, fax. +3120 6222 1272 Email: [email protected]

H. Senhorst RIZA Postbus 17 NL – 8200 AA Lelystad Tel. +31320 298 458 fax+31320 298 373 Email: [email protected]

A. Tukker TNO – STB Postbus 6030 NL – 2600 JA Delft Tel. +31152695460, fax +3115 269 5450 Email: [email protected]


Appendix II. Documents and reports distributed

Senhorst, H., Hattum, B. van, Lamoree, M., Sanderson, T. & Tukker, A. (2003). Integratie Docu-ment. RIZA/ IVM, Amsterdam.

Lamoree, M.H, Stroomberg, G., Besselink, H., Senhorst, H., Sanderson, T. & Hattum, B. van (2003a). Chlorinated Micropollutants in Aquatic Effluents. Part-1 Part-1 Sampling, pre-treatment, classification and primary effect screening. Report nr E03/02. Institute for Environmental Studies, Vrije Universiteit.

Lamoree, M.H., Sanderson, T., Senhorst, H. & Hattum, B. van (2003b). Chlorinated Micropol-lutants in Aquatic Effluents. Part-3 Refined fractionation and primary TIE studies. Report nr E03/xx. Institute for Environmental Studies, Vrije Universiteit (draft).

Senhorst, H. Lamoree, M., Linden, S. van der, Sanderson, T. & Hattum, B. van (2003a). Chlorinated Micropollutants in Aquatic Effluents. Part-2 Biodegradation studies on selected effluents (Workpackage 8). Report nr E03/xx. Institute for Environmental Studies, Vrije Universiteit (draft).

Senhorst, H., Lamoree, M., Linden, S. van der, Sanderson, T., Stee, L.L.P. van & Hattum, B. van (2003b). Chlorinated Micropollutants in Aquatic Effluents. Part-4 In-pant TIE studies (Workpackage 9). Report nr E03/xx. Institute for Environmental Studies, Vrije Universiteit (draft).

Van Hattum, B., Swart,K., Lamoree, M., Linden , S. van der, Senhorst, H., & Tukker, A. (2003a). Chlorinated Micropollutants in Products. Report nr E03/xx. Institute for Environmental Studies, Vrije Universiteit (draft).

Van Hattum, B., Peters, R., Timmer, J., Swart, K., Lamoree, M., Linden, S. van der & Felzel, E. (2003b). Chlorinated Micropollutants in Atmospheric emissions. Report nr E03/xx. Institute for Environmental Studies, Vrije Universiteit (draft).

Anon (2002). Voortgangsrapportage Onderrzoeksprogramma Vervolgonderzoek Chloorketen-studie (OVOC). DGM/SAS/2002075967. Min. VROM, Den Haag. VROM 020876 (Note to the parliament on objectives and progress of project).


Appendix III. References cited in section 2.1

References cited in section 2.1 ‘Validity and limitations of in-vitro bioassays’

Behnisch, P.A., Hosoe, K., Brouwer, A. & Sakai, S. (2002). Screening of dioxin-like toxicity equivalents for various matrices with wildtype and recombinant rat hepatoma H4IIE cells. Toxicol Sci 69, 125-130.

Garrison, P. M., Tullis, K., Aarts, J. M., Brouwer, A., Giesy, J. P. & Denison, M. S. (1996). Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol 30, 194-203.

Murk, A. J., Legler, J., Denison, M. S., Giesy, J. P., van de Guchte, C. & Brouwer, A. (1996). Chemical-activated luciferase gene expression (CALUX): a novel in vitro bioas-say for Ah receptor active compounds in sediments and pore water. Fundam Appl Toxicol 33, 149-160.

Rankouhi, T. R., van Holsteijn, I., Letcher, R., Giesy, J. P. & van den Berg, M. (2002). Effects of primary exposure to environmental and natural estrogens on vitel-logenin production in carp (Cyprinus carpio) hepatocytes. Toxicol Sci 67, 75-80.

Sanderson, J. T., Aarts, J. M., Brouwer, A., Froese, K. L., Denison, M. S. & Giesy, J. P. (1996). Comparison of Ah receptor-mediated luciferase and ethoxyresorufin-O-deethylase induction in H4IIE cells: implications for their use as bioanalytical tools for the detection of polyhalo-genated aromatic hydrocarbons. Toxicol Appl Pharma-col 137, 316-325.

Sanderson, J. T. & Giesy, J. P. (1998). Functional Response Assays in Wildlife Toxi-col-ogy. In The Encyclopedia of Environmental Analysis and Remediation (R. A. Meyers, Ed.). John Wiley and Sons, New York.

Smeets, J., Rankouhi, T., Nichols, K., Komen, H., Kaminski, N., Giesy, J., & van, d. B. M. (1999a). In vitro vitellogenin production by carp (Cyprinus carpio) hepatocytes as a screening method for determining (anti)estrogenic activity of xenobiotics. Toxicol Appl Pharmacol 157, 68-76.

Smeets, J., van, H. I., Giesy, J., Seinen, W., & van, d. B. M. (1999b). Estrogenic poten-cies of several environmental pollutants, as determined by vitellogenin induction in a carp hepatocyte assay. Toxicol Sci 50, 206-213.

Smeets, J., van, H. I., Giesy, J., & van, d. B. M. (1999c). The anti-estrogenicity of Ah re-ceptor agonists in carp (Cyprinus carpio) hepatocytes. Toxicol Sci 52, 178-188.

Stronkhorst J, Leonards P & Murk AJ (2002). Using the dioxin receptor-calux in vitro bioassay to screen marine harbor sediments for compounds with a dioxin-like mode of action. Environmental Toxicology and Chemistry 21:2552-2561.

Meeting Report 3rd Technical Workshop

23

Appendix IV. Presentation slides

Presentations:

• Introduction to Phase-2 of the OVOC project and scope / objectives of the workshop (B van Hattum, IVM);

• Results of the refined fractionation study (M. Lamoree, IVM); • Results of the biodegradation studies (H. Senhorst); • Results of the in-plant TIE study (H. Senhorst); • Results of atmospheric emissions (Bert van Hattum); • Results of products study (Bert van Hattum); • Evaluation of selection of emission sources (Arnold Tukker); • Synthesis of results from screening and additional studies (Henk Senhorst); • Reflections of main findings in perspective of objectives (Arnold Tukker).

Copies of handouts are included as unnumbered pages.

1

Institute for Environmental Studies (IVM) 1vrije Universiteit amsterdam

Introduction and Objectives3rd Technical OVOC Workshop

May 14/15 2003

Bert van Hattum


Research ProgrammeChlorine Chain Follow-up Studies

(OVOC) 1999 - 2004

IVM-VU, Rijkswaterstaat RIZA, IRAS - UU, TNO-STB/MEP, CML-UL, BDS,

Aquasense

commisioned by: VNCI, Min VROM, Min VW, SNM


History

1995 Chlorine Chain study (TNO/CML)99% of Chlorine f luxes covered.

Peer rev iew; social debate; ParliamentInstallation of BITAC for coordin. f ollow-up actions1997 BITAC invitation for proposals ‘Chloormicro’s’1998 Drafting of OVOC ProgrammeCommissioning: march 1999


BITAC research questions Answers to: (BITAC 1998)

...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 env ironment f rom point or non-point (diffuse) sources?

2


Objectives of programme

– To assess if, and to what extent PBTs or groups of PTBs are emitted from the chlorine chain into the env ironment

– To contribute to the assessment of the env ironmental impact of these PBTs.


Potentialsources

EOCI complex in e.g.fish fat and sediments

DDTPCBsHCHetc.

UnknownCHCs

PBT-emissionsundetected

by monitoringprogramm es

Degradationproductsof known

CHC-em issions

+ 10% + 90%

Naturalsources

Historica lreleas es


Pilot study chemical-biological methods

Unknown organochlorines in the environmentDegradation productsEmissions and priority setting

Main study– aquatic effluents– atmospheric emissions– products Natural sources

Historic EmissionsIntegration and final reporting

Experimental StudiesBackground Studies

W-1

W-2

W-3

W-4

Workshops

(1)

(2)

(3)

(Phase)

2000

2001-

2003

2004


Phase-II Experimental studies overview

Air emissions (4)Effluents (16+2) Products (11)

Cl: GC-ECDTox-screen Kow fractionation

GC-MSin-vitro ToxKow fractionation

GC-MS in-vitro Tox

Primary TIE Biodegradation in-plant TIEExtended TIE

Screening & classification

WP 5-6

Further studies & identification

WP 7-9

3


Considerations Tox-screen

tests for chlorinated PBTs?dioxin-based toxicity seems most relevantmixture effects contribution of unknownsSensitivity, selectivityin-vitro + in-vivocomparison of effects in non-chlorine chain industry eff luents


Tox-screen in aquatic effluent study

in-v itro– DR-Calux– carp hepatocyte test

• EROD• (anti) estrogenicity• cytotoxicity

sub-chronic in-v ivo – zebra fish ELS (8d)– Microtox (marine bacteria; 22 hr)


SAMPLING OF WASTEWATERS16 industrial effluents

chlorine use in cool ing water2 references

SAMPLING4 ATMOSPHERIC

EMISSIONS

SAMPLING11 PRODUCTS

DR Calux assay

chem. identific ationKow fractionation

DR Calux assay

chem. identific ation

SCREENING STUDYmaximum potential envir onmental impact

chlorine compoundsin vitro assays (DR Calux, EROD)

in vivo toxicity

FINE FRACTIONATIONKow fraction. of toxic respons e

chemical identifiction

BIODEGRADATION STUDYsimulation surface water

persistency of toxic effects

IN-PLANT TIE STUDYattribution of toxic res pons e

to chlorine activities

6 samples 11 extracts 1 plant

L/Lextractsneutral-acidic-basic


AI M OF OVOC PROJECT: - emissions of PBTs from chlorine chain

- contribution to environmental asessment

unknown chlorinated PBT effects ?

bio-assay baseddioxin-like

toxicity

DR Calux‘narrow’ vs. ‘broad’’Carp-hep EROD

in-vivo- zebraf. ELS- microtox

carp-hep- estrogen.- cytotox

ref erencesamples

persistency &bio-

accumulation

TIE /chemicalidentif ication

4


Dioxin emissions Netherlands 1999

in g I-TEQ/y

28householdsand others

4wastemanagement& incineration

0.65

3industry

0.17 0.4-2

Source: air emissions: Mileubalans 2001, RIVM; wastewater emissions: Emissieregistratie and literature sources

via products??


Objectives of workshop

Presentation, evaluation, review of results:5 techn. reports + integr. documentmorning: ev aluation of research approach and methodologyafternoon: f ocus on OVOC objectives and contribution to ERApresentations of framework f or ev aluationadv ice BOVOC and res. team on further directions of WP-8.2 and Final Phase

1

Institute for Env ironmental Studie s ( IVM) 1vrij eUn ivers iteit amsterdam

Refined fractionation and primary TIE studies

Instituut voor Milieuvraagstukken ( IVM)Vrije Universiteit

De Boelelaan 10871081 HV Amsterdam

http://www.vu.nl/ivm

Marja Lamoree

Thomas Sanderson

Henk Senhorst

Bert van Hattum

Institute for Env ironmental Studie s ( IVM) 2vrij eUn ivers iteit amsterdam

Fractions included in primary TIE

sample medium log Kow fraction

high log Kow frac-tion

MCD X X MDE X MEF X X MIJ X MQR X MAB X MMN (MWTP) X X MST (cooling wa-ter outlet)

X

Institute for Environmental Studie s (IVM) 3vrij eUn iversiteit amsterdam

Fractionation scheme 10 selected fractions

Refined fractionation intonarrow Log Ko w

intervals

Extract in MeOH

Chemical analys is In vitro assays

Chemometricdata analys is

Extract in MeOH Extract in MeOH Extract in MeOH

11 selected fractions

Refined fractionation intonarrow Log Ko w

intervals

Extract in MeOH

Chemical analys is In vitro assays

Extract in MeOH Extract in MeOH Extract in MeOH

Compilationof data


Refined fractionation intervals

medium log K owfraction4- 6

log K ow3-4

log K ow4-5

log K ow5-6

log K ow> 6

high log K ow fraction> 6

log K ow5-6

log K ow6- 7

log K ow7-8

log K ow> 8

medium log K owfraction

log K ow3-4

log K ow4-5

log K ow5-6

log K ow> 6

high log K ow fraction

log K ow5 -6

log K ow6- 7

log K ow7 -8

log K ow> 8

2


DR-CALUX results in screening phase and primary TIE

sample

screening phase primary TIE

MAB.W.2602.LL.high 50 45MCD.W.1202.LL.medium 296 267MCD.W.1202.LL.high 165 132MDE.W.2102.LL.high <LOD 27

MEF.W.1402.LL.medium 30 28MEF.W.1402.LL.high 47 44MIJ.W.2102.LL.high 27 27MMN.W.0502.LL.medium 29 31MMN.W.0502.LL.high 54 42

MQR.W.2901.LL.high <LOD 27MST.W.1202.LL.high <LOD 28HPLC water,LL.medium not determined < LODHPLC water, LL.high not determined < LODLOD = 27 pg TCDD-TEQ/liter effluent

2,3,7,8 TCDD-TEQpg TCDD-TEQ/liter effluent


DR-CALUX results on concentrated extractsDR- CA LUX resp onse* sampl e rough f ract i on

pg TC DD- TE Q/l i t er

ef f l uent

309 MAB .W.2602.LL.high

1066 MCD.W.1202.LL.me dium

MCD.W.1202.LL.high

99 ME F.W.1402.LL.me dium

ME F.W.1402.LL.high

122 MIJ.W.2102.LL.high

157 MMN.W.0502.LL.me dium

MMN.W.0502.LL.high

104 MST.W.1202.LL.high

* DR-CALUX re sponse mea sure d in tota l extra cts in the scree ning study** DR-CALUX re sponse mea sure d in ne wly prepa re d extra ct for prima ry TIE (compa re to Ta ble 7.3)*** DR-CALUX re sponse pe r lite r e fflue nt tha t is 50x concentra te d**** DR-CALUX re sponse pe r lite r e fflue nt. LOD = 26pgTCDD-TEQ/lite r; LOQ = 97 pg TCDD-TE Q/lite r

DR- CA LUX resp onse** ref i ned f ract i on DR- CA LUX resp onse DR- CA LUX resp onse

pg TC DD- TE Q/l i t er pg TC DD- TE Q/l i t er pg TC DD- TE Q/l i t er

ef f l uent ef f l uent *** ef f l uent ****

50x conce nt rat ed

45 logKow 5-6 <LOQ (31)logKow 6-7 <LODlogKow 7-8 <LOQ (26)logKow > 8 1328 27

267 logKow 5-6 206 4logKow > 6 2316 46

132 logKow 5-6 <LODlogKow 6-7 <LODlogKow 7-8 <LOQ (47)logKow > 8 1463 29

28 logKow 5-6 <LODlogKow > 6 1224 25

44 logKow 5-6 <LOQ (45)logKow 6-7 <LOQ (39)logKow 7-8 <LOQ (40)logKow > 8 1738 35

31 logKow 5-6 <LOQ (54)

<LOD<LOQ (80)<LOQ (80)

1610

logKow > 6 1145 2342 logKow 5-6 <LOD

logKow 6-7 <LODlogKow 7-8 <LOQ (57)logKow > 8 1795 36

28 logKow 5-6 <LOQ (30)logKow 6-7 <LOQ (41)logKow 7-8 <LOQ (43)logKow > 8 1340 27

27 logKow 5-6logKow 6-7logKow 7-8logKow > 8 32

sampl e t ot al ext ract

MAB .W.2602.LL

MCD.W.1202.LL

ME F.W.1402.LL

MIJ.W.2102.LL

MMN.W.0502.LL

MST.W.1202.LL


Interpretation of GC-MSD data using AMDISsa mple

MAB .W.2602. LL.high

MCD. W.1202.L L.me dium

MCD. W.1202.L L.high

ME F. W.1402.L L.me dium

ME F. W.1402.L L.high

MM N.W.0502. LL.me dium

MM N.W.0502. LL.high

MST. W.1202.L L.high

1. De convolution se tting s: compone nt width: 12; a dja ce nt pe a k subtra ction=2; re solution=high; se nsitivit y=me dium; sha pe re quire me nts=me dium2. Ma tch fa c tor > 603. Ma tch fa c tor > 70 or a t le a st 3 out of fir st 5 hits a re chlorine compounds

log Kow 5-6log Kow > 6

log Kow > 8

log Kow > 6

log Kow > 8

no of compone nts withma tch in

NIST libra ry 2

495

646844

log Kow > 8

log Kow > 8

log Kow > 8

no. of compone nts de convolute d

by AM DIS 1

re fine d fra ction

643

872

858

680

594

1229

1197

269

247315

311

475

459

316

374

no of compone ntsre cognize d by

NISTpossibly conta ining

chlorine 3

4

411

4

9

5

7

5

no of compone ntsproba bly conta ining

chlorinea ssigne d by

ma nua l che ck

none

nonenone

none

none

none

none

none

log Kow > 6 none9650

10 nonelog Kow > 8MIJ.W.2102. LL.high 500


Conclusions

DR-CALUX response found in 50x concentrated extractsDioxin like activity focused in high log Kowfractions: log Kow>6 and log Kow>8No response of non-concentrated extracts in carphepatocyte EROD assayNo unequivocal identification of chlorinated compounds using NIST libraryFurther refinement of fractionation does not signif icantly reduce complexity of the extract

3


Recommendations

Search for chlorinated compounds among unidentif ied peaks

Choose other GC-detectors such as Negative Chemical Ionization or Atomic Emission Detection

Implement acidic clean-up step for DR-CALUX to screen for stable dioxin like compounds

Dedicated chemical dioxin analysis

1

Resultsof Biodegradation Studies 1

Institute for Inland Watermanagement and Wastewater Treatment

Results of Biodegradation Studies

Workpackage 8.1

H. Senhorst, RIZAM. Lamo ree, IVMS. van der Linden, BDST. Sanderson, IRASB. van Hattum, IVM



Goal of biodegradation studies within OVOC

to determine persistence (P) of effectsdetermined in screening phase

• 6 samples selected• simulation of residence time in surface water• same set of parameters tested after biodegradation

as in screening phase



Test method• 1:1 mixing of whole effluent (from cold storage) with

reference surface water (Oosterschelde) acting asinoculum

• 28 days in test vessels• aerated• in the dark• 15 ºC

• check for occurrence of biodegradation• succesfully executed within protocol for 5 samples

(MAB too saline)

• samples L/L extracted as in screening phase



Results (1)

• ECD signal (halogen content):relative shift to high log Kow, except sample MAB

• chronic microtoxicity:

• one sample shows decrease of toxicity• three responsive samples from screening phase

remain toxic with same response level (within systematic error: factor 4)

2



Results (2)DR Calux response

MAB MCDMDE MEF

MMN MNOcontrol

before biodegradation, no cleanup

after biodegradation, no cleanup, not corrected

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

1,80

2,00

DR Calux response(ng TEQ/l)

sample



More on DR Calux

• no indications for artefacts introduced into test• correlation with carp hepatocyte EROD test:

response is probably indeed part of toxicity mechanism

» responses possibly induced by natural component

» test system too ill-defined

» application of acid cleanup on extracts



Acid cleanup step of extracts

method

glass columnpacked with

silica+ 33 %

conc. H2SO4)

1 2

3 4

1 extract applied

2 elution with solvent(hexane/dieth ylether 97:3)

3 first eluate (5 ml) discarded

4 eluate collected (35 ml) andtransferred to new ext ract

prerequisite step for DR Calux testing if appliedas screening for classical dioxins (PCDD/PCDF)



Results: DR Calux after acid cleanup

MAB MCDMDE MEF

MMNMNO

control

before biodegradation, no cleanup

after biodegradation, no cleanup, not corrected

after biodegradation and acid cleanup, corrected

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

1,80

2,00

DR Calux response(ng TEQ/l)

sample

3



Decrease DR Calux response

acid cleanup applied on fractions screening phase:• shows a 5-10 fold decrease of response, responses

drop below detection limit (exceptMAB?, MCD)

• responses too low to detect effect of biodegradationtest

» effect acid cleanup dominates

» DR Calux response after biodegradation step and acid cleanup characterised as very persistent (‘vP’)



Conclusions

effect of biodegradation step:•shift in composition (reflected in ECD signal)•decrease in microtoxicity in one sample•not detectable in DR Calux response

natural component probably relevant in DR Caluxresponse without acid cleanup

response after biodegradation and acid cleanup characterised as very persistent fraction

1

Resultsof In-plant TIE Studies 1


Results of In-plant TIE Studies

Workpackage 9

H. Senhorst, RIZAM. Lamo ree, IVMS. van der Linden, BDSL.L.P. van Stee, ACAST. Sanderson, IRASB. van Hattum, IVM



Goal of in-plant TIE studies

to assess which part of toxic response of effluent MCD

originates from chlorine activities

0

5

10

15

20

25

DR Caluxload(g TEQ/y)

SCREENING PHASE AFTER IN-PLANT STUDIES

M CD

21.7

mixed activ ities

chlorine activ ities

DR Calux loads screening phase:

MCD

?



In plant sampling

O THERACTIVITI ES

A

ACTIVITI ESB

WASTEWATER

TREATMENTPLANT

TRIBUTARY B (3 18 m3 /h)

TRIBUTARY A (1 237 m3/h)

INFLUENT (m3/h) EFFLUENT (1 555 * m3 /h)

CHLORINEACTIVITI ES

sampl e ‘MCK’f low proporti onal mix ture

sampl e ‘t ri b A’

s ample ‘trib B’

sa mple ‘inf luent ’ s ample ‘ef fluent’

ACTIVITIES A

(5 58 m3 /h)



Flow scheme EOX (organic halogen signal)

CHLORI NEACTIVITIES

OTHERACTIVITIES

A

ACTI VI TIESB

WASTEWATER

TREATMENTPLANT

EOX load

kg Cl/yACTIVITIES A t ribut ary A

tribut ary B

infl uent ef fl uent

8.200

14.000

<280

11.000 4.000*

63 % removal

Chlorine activities major source of EOX

2



Flow scheme chronic microtoxicity

CHLORI NEACTIVITIES

OTHERACTIVITIES

A

ACTI VI TIESB

WASTEWATER

TREATMENTPLANT

microtoxicity load

rel. toxicity unitsACTIVITIES A t ribut ary A

tribut ary B


65

300

>200

>1000 100*

>90 % removal

Activities B major source of chronic microtoxicityResultsof In-plant TIE Studies 6


Flow scheme DR Calux loads

CHLORI NEACTIVITIES

OTHERACTIVITIES

A

ACTI VI TIESB

WASTEWATER

TREATMENTPLANT

DR Calux loadwithout cleanupg TEQ/y

ACTIVITIES A t ribut ary A

tribut ary B


8,3

14

240

630 71*

89 % removal

PAHs considered possible source of response



GC-MS analysis (1)

aimed at identification of chlorine compoundsinducing DR Calux response

20 GC-MSmeasurements performed

automated interpretation of data



AMDIS software

GC-MS analysis (2)

GC-MSanalysis

ion chromato-grams for m/z

50 to 650

deconvo-lution

lis t ofextracted

components

matchwith

NIST MSdatabase

lis t ofpossible

constituents

selection ofchlorine

compounds

lis t ofpossiblechlorine

constituents

preliminarymanualcheck

lis t ofprobablechlorine

constituents

extract

# per sample: ±500 ±200 0 -23 0 -9

» many GC peaks not identifiable by NIST database» minor part of peaks recognisable as chlorine compounds» many more chlorine compounds present in some extracts» no link chlorine – DR Calux response could be made

3



DR Calux response after acid cleanup

CHLORINEAC TIVITIES

OTHERAC TIVITIES

A

ACTIVITIESB

WASTEWATER

TREATMENTPLANT

DR Calux loadafter acid cleanupg TEQ /y

ACTIVITIES A tributary A

t ributa ry B

influent e ff lue nt

7,1 ±0,8

9,5 ±0 ,9

0,6 ±0, 03

5,0 ± 1,2 1,5 ±0, 1

±80 % removal

chlorine activities only source, emitting 1.5 g TEQ/yResultsof In-plant TIE Studies 10


Link with research question

0

5

10

15

20

25

DR Caluxload(g TEQ/y)

SCREENING PHASE AFTER IN-PLANT STUDIES

MCD

21.7

11.4

MCD

mixed activ ities

chlorine activ ities

DR Calux load attributable to chlorine activities

research question satisfactorily answered



But……….

DR Calux response after acid cleanup in general closely l inked to dioxinlike substances

probably only 4% of response can be explained by PCDD/PCDF

no hint of identity of inducing substances

(how) to proceed?

1


Atmospheric emissions WP 5/6

3rd Technical OVOC WorkshopMay 14/15 2003

Bert van Hattum, R. Peters [1], J. T immer [2], K. Swart, M.Lamoree, S. van de r Linde [3], and E. Felzel [3]

IVM, Vrije Universiteit, Amsterdam[1] TNO-MEP-MA, Apeldoorn[2] TNO-MEP-PA, Apeldoorn

[3] BDS, Amsterdam


Design of study

3 vent-gas installations, 1 MWIsampling: HVS, 30-50 Nm3 (dioxin method)Determinations:

– DR-CALUX (+/– aci dic clean-up; total/fracti ons) – total PCDD/F anal ysis– GC-MS (main components)– Kow frac tionation (Kow 4<. 4-6, >6)– GC-MS fractions (Amdis/NIST mi nor components)


Ströhlein dilution sampling equipment

GF Filter/P UF

Filter for Dilution Air

Heated Probe

Air Heater

Active Carbon

Duct


PCDD/Fs

Sum PCDD/PCDF HRGCMSTEQsI-TEF

Sample ng/m3 pg/m3270- 01 (MW I) 0.44 36

270- 02 2.23 209270- 04 0.27 12

270- 05 29.35 137270- 06 (blank) 0.17 0.3

2


Dr-CALUX (acid clean-up)

0

50

100

150

200

250

300

350

400

270-02 270-05 270-04 270-01

TEQ

pg/

Nm

3DR-CALUXREP50I-TEFWHO-TEF


Explained response from PCDD/Fs

0%

20%

40%

60%

80%

100%

120%

270-02 270-05 270-04 270 -01

as %

of t

otal

DR

CALU

X re

spon

seInstitute for Environmental Studies (IVM) 7vrije Universiteit amsterdam

DR-CALUX in fractions

270-01

270-02

270-04

270-05

Log Kow < 4

Log Kow 4-6

Log K ow > 6

0

20

40

60

80

100

120

TEQ

in p

g/m

3


GC-MS main components

• complex composition • mainly alkanes (>C10) and PAHs (few cases)• blank filter material: phthalates • sample 270-02 indication Cl5-comp (0.4 mg/Nm3).• other samplesno chlorinated compounds >0.01-0.1

mg/Nm3.

3


GC-MS minor components

C omponent at scan 107 9 (9.7 04 m in) [Model = +1 8 1,1 '-Biphenyl, 2,2 ',5-t richlor o-50 70 9 0 1 10 13 0 1 50 1 70 190 2 10 2 30 250 2 70

0

1 00

1 00

50

50

61

62

75

75

8 6

8 6

93

93

1 10

1 11

1 23

1 23 1 33

1 50

1 501 60

16 1

1 72

18 6

18 6

1 94

1 94

2 0922 1

22 1

23 4

2 56

2 56

trichlorobiphenyl in sample 270-04 (Kow 4-6)

concentrationrange 1-100 ng/Nm3

load range: <0.1-200 g/yr

AMDIS deconvolution + matching NIST database


Test sample GC-NCI-MS (270-05)(RIVO-DLO)

1 2 . 0 0 1 4 . 0 0 1 6 . 0 0 1 8 . 0 0 2 0 . 0 0 2 2 . 0 0 2 4 . 0 00

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

3 5 0 0 0 0

4 0 0 0 0 0

4 5 0 0 0 0

5 0 0 0 0 0

5 5 0 0 0 0

6 0 0 0 0 0

6 5 0 0 0 0

7 0 0 0 0 0

7 5 0 0 0 0

8 0 0 0 0 0

8 5 0 0 0 0

9 0 0 0 0 0

9 5 0 0 0 0

1 0 0 0 0 0 0

1 0 5 0 0 0 0

1 1 0 0 0 0 0

1 1 5 0 0 0 0

1 2 0 0 0 0 0

1 2 5 0 0 0 0

1 3 0 0 0 0 0

1 3 5 0 0 0 0

1 4 0 0 0 0 0

1 4 5 0 0 0 0

1 5 0 0 0 0 0

T i m e - - >

A b u n d a n c e

T I C : S C R 0 2 . DCl

Cl Cl

ClClCl

ClCl Cl

ClCl

unknown Cl4

GC-NCI-MS


Annual loads TEQs

Discharge volume

Sum PCDD/ PCDF

HRGCMS TEQs

DR Calux TEQs

(act) I-TEF incl. acid ic clean -up

Sample Nm3/hr g/yr g /y r g/yr 270-01 254000 1.7 0.08 0 .12 270-02 14000 0.5 0.03 0 .04 270-04 17000 0.1 0 .002 0 .01 270-05 2281 1.0 0 .003 0 .01 Total 3.4 0.1 0.2


Conclusionspresence of dioxins/likes in all installationsDR-DR- CALUX > PCDD/F I-TEQs86-98% explained 3 samples, 22% in 1 sampleUnknowns: B, acid - stable compounds. no causal relationships with other chlorinated compounds than PCDD/Fs.load vent-gas contribution < 2% total dioxins ind.major components: alkanes, PAHs, 1 Chlorinated?identification minor: complex comp., chlorinated minor but present in som hydr. fractions, indications for PCBs

4


Discussion

further elucidation of 270-04: PCBs, PA Hs, additional GC-NCI-MS?extrapolation to other vent-gas installat ions?contribution vent-gas (<2% of ind. sources) minor?comparison w ith RIKZ inventory of DR-CALUX responsive compoundsblank problems

1


Chlorinated Micropollutants in Products (WP 5/6)

3rd Technical OVOC WorkshopMay 14/15 2003

Bert van Hattum, R. Peters [1], J. T immer [2], K. Swart, M.Lamoree, S. van der Linde [3], and E. Felzel [3]

IVM, Vrije Universiteit, Amsterdam[1] TNO-MEP-MA, Apeldoorn[2] TNO-MEP-PA, Apeldoorn

[3] BDS, Amsterdam


Design of study

11 products selected (2 ‘w ild-cards’)concentrated extracts of residues (volatile solvents), L/L extracts water sol. productssolutions of hexane soluble solid productsSoxhlet extract of neopreneDeterminations:– DR-CALUX (+/– acidic clean-up)– GC-MS f ractions (Amdis/NIST)


ProductsProduct Period Usage a tpa dichloromethane 1990 8500 1,2-dichloroethane (EDC) >100000 b perchloroethylene (PER) 1990 < 2600 trichloroe thylene (TRI) 1990 926 hydrochloric acid 34000 c allylchloride < 5000 monochloroace tic ac id (MCA)

10000 d

1,4-dichlorobenzene 9000 chloroalkanes C14-17 < 1990 2000 e tr ic losan ? neoprene ?


DR-CALUX response Units : DR Calux

TEQ s D R Calux TEQ s

Sample 2,3,7,8 TCD D-TEQ

incl. acidic clean-up no acidic clean-up

dichloromethane pg TEQ/g < 0.01 <0. 01 1,2-dic hloroe tha ne pg TEQ/g 0.01 < x < 0.03 (0.02)b 0.02 < x < 0.04 (0.02)b perchloroethylene pg TEQ/g 0.02 < x < 0.07 (0.04)b <0.02 trichloroethylene pg TEQ/g 9.6 ± 0.3 18 ± 2 allylch loride pg TEQ/g < 0.01 <0.02 hydrochloric acid pg TEQ/g 2.0 ± 0.1 2.2 ± 0.1 monochloroacetic acid pg TEQ/g <0.03 <0.04 1,4-dichlorobenzene µg TEQ/g <2.0 <2.6 chloroalkanes C14-17 µg TEQ/g <2.5 <2.9 triclosan µg TEQ/g <2.1 <2.7 neoprene pg TEQ/g 1700 ± 83 1500 ± 61

2


GC-MS tentative identifications

example PCP in HCL

Mass spectrumsample

Library spectrum NIST


GC-MS overall results (still in discussion with suppliers)

sample # deconvoluted compone nt s with ma tch2 in NIST

li brary

# components

re cognized by NIST possibly

c ontaining chlorine3

t ent ati vely i denti fi ed chlorinat ed components

at S/N>50

Ma tch> 80

c oncentrati on ra nge of

t entat ively i denti fi ed

components in µg/kg product, unless indi cate d

otherwi se di chloromethane 349 8 0 (4)5 0 .1-10 ng/kg 1, 2-di chloroethane 405 27 12 0 .01-10 pe rchloroethyle ne 354 33 9 0 .01-100 tric hlorethyl ene 517 65 17 0.01-1000 hydrochloric acid 406 19 3 0.1-10 all yl chloride 227 18 5 0.1-10 monoc hloroa ceti c a cid 271 31 11 0 .1-100 1, 4-di chlorobenzene 6 3 1 6 - Chloropa ra ffins C14-17 121 7 2 - - tric losa n 55 3 2 6 - ne opre ne 621 12 6 0 .1-100 mg/kg he xane-bl ank 46 0 0


Tentative TEQ loads involved (mg/yr)Product Usage Estimated load Estimated load tpa mg TEQ per year

mg TEQ per year

values <LOD dichloormethane 8500 < 0.08 1,2-dichloroethane (EDC) 1000a 0.03 b perchloorethylene (PER) <2600 < 0.10 b trichloorethylene (TRI) 926 9 allylchloride <5000 < 0.06 hydrochloric acid 34000 69 1,4-dichlorobenzene 9000 < 2.E+ 07c monochloroacetic acid (MCA) 10000 <0.3 triclosan 10 d < 2.E+ 04c chloroalkanes C14-17 2000 < 5.E+ 06c neoprene 1000d 1700 Total 1778


Conclusionspresence of dioxinlikes in 5 productsneoprene>> HCL>TRI>> PER, EDC tentative loads involv ed: neoprene > 1g/y r ?successful identif ication 8 products at sub ng/kg lev el; 3 products > 1%. unexpected nr of chlorinated compoundslight components dominatedf urther attention: chlorinated phenols, chl. (subst) benzenes, chlorinated alif ates (>C4)no attribution of DR-CALUX to chlorinated components

3


Discussioncomparison with lit. / suplier datapotential loads / real emissions/exposure?f inal and conclusiv e identification beyond scope of projectadditional in-v itro work on 3 products at higher concentrations?additional identification work on 3 products?f urther work on causativ e agents of DR-CALUX response: direct PCDD/F, PCB analysis, GC-NCI-MS identif ications, Kow fractionation? EPIWIN data/predictions for B and T of identif ied components?

1t

Arnold Tukker

tTNO Strategie, Technologie en Beleid

A reflection on priority setting

13 May 2003Arnold Tukker 2t

Priority setting study• Potential sources

• Chemical processes with chlorine conversion: PBT formation risk

• Traditional chemical processes• Processes with use of active chlorine• Processes where inorganic chlorine might be converted

to PBTs

• Other processes with use (but no conversion) of chlorinated substances: PBT contamination risk in product


Priority setting study (2)

• Potential sources in the Dutch situation

• 40 point sources listed in the chlorine chain study, and

Dutch Emission databases

• 4 other types of point sources (active chlorine, inorganic

chlorine), i.e. MSWIs and three hypochlorite applications

• 18 chlorine-containing products (of which 12 produced in

the Netherlands)


Criteria in prioritization• Chlorine throughput

• Known volume of chlorinated emissions

• Chance on formation of by-products

• Active chlorine

• Precursors for PBTs available

• Catalysts/UV light

• High temparature

• Mismatch volume known emissions/sum parameters

• Other indications (previous investigations)

2t


The result• 18 water priorities (16 done; 1 now chlorine free, other

refused co-operation)

• 8 clear priorities• 8 average priorities• 20+ left out

• 4 air priorities• 10 product priorities (out of 18)• 2 ‘wild cards’ chosen by Steering committee

• Neoprene• Triclosan


Reflection: water• Result: 5 companies give >99% of the DR Calux TEQ response

• 5 out of 10 flows from top 8 priority firms responsive• only 2 out of 6 non-priority flows (low) response• Conclusion seems justified that the 20 firms left out are

irrelevant

• ‘False positive’ in priority: main effluent of Akzo Nobel Botlek not part of top-5

• Highest volume, highest risk factor• But maybe the Biobot provides relatively effective

treatment ?


Reflection: air• Most samples: DR Calux TEQ = TEQ of known emissions

• For one company a gap of 75% exists

• Is a vent gas issue

• Annual load is here <10% of the known emissions of MSWIs

• Probably even the missing 75% is relevant, overall

emissions from 10 similar plants still outweighted by

MSWIS


Reflection: products

• Mixed picture

• 3 were n.d., but can still be high flows

• Tentative mass flow of proven contaminants is about 1 g/yr

CD Calux TEQ (incl. cleanup)

• Main contribution is by ‘wild card’ neoprene

• Many other chlorinated PBTs present

3t


Reflection: products

• We might have missed a category

• Focused only on chlorinated products

• Positive outcome from chlorine-free Neoprene, but that is

produced in a process that complies with all priority

criteria

• Search for similar products might be relevant

1

Synthesis of experimental results 1


Synthesis of experimental results

H. Senhorst, RIZAM. Lamo ree, IVMT. Sanderson, IRASB. van Hattum, IVMA. Tukker, TNO-STB



Structure of presentation

summary of results of screening phase

characterisation of DR Calux loads

link to goals of project

technical,informative

unscientific

interactive



AIM OF OVOC PROJECTemis sions of PBTs from chlorine c ha in

contribut ion to envi ronmenta l as ess me nt

unknown chlorine spec ific effects

bio-assay baseddioxin-like

toxicity

DR Calux‘narrow’ vs. ‘broad’’

in vivotox icity

car p-hepEROD

bio-assay

referencesamples

persistency&

bio-ac cumulation

chemicalidentification



Results of screening phase

0

100

200

300

400

MCD MAB MQR MFG MIJ MDE MEF MPQ MIK MKL MOP MBC MGH MLM MRS MNO BST MST MMN BTU BUVsample

DR

Cal

uxre

spon

se (p

g TE

Q/l)

1100 pg/l^^

16 indust rial effluent s

chlorineuse forcoolingwater

ref er-ences bl

ank

sam

ple

MW

TP

Lake

Mar

ken

inle

t

outle

t

limit of detection (LOD)

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

limit of quantification (LOQ)

DR Calux response (pg TEQ/l)

2



significance of DR Calux response screening phase

corroborated by carp hep-EROD bio-assay

» two links in toxicity chain of dioxins positive

no link with results in viv o-tests: zebrafish ELS test no response; no (cor)relation with chronic microtoxicityresults

» probably no toxic effects in environment other than possible effects in food-chain by PBT subtances (to be determined)

no correlation with AOX, EOX and ECDSynthesis of experimental results 6


CAUTION

you are entering an unscientific area!



Why an unscientific area?

to contribute to the assessment of the environmental impact of PBT’s (OVOC, goal 2)

•which information needed for assessment?•which questions most relevant based on presentlyavailbale information?•give insight into largest possible amount of possibly relevant effects



How does this unscientific method look like?

CHEWING GUM !

3



0

5

10

15

20

25

DR

Cal

uxlo

ad(g

TE

Q/y

)

unknownvPBT

contr ibution?

TOP 5-EFFLUENTSbased on in-plant TIE, biodegradation and fine fractionation studies

LOAD CHARACTERISED AS

screening phaseall 16 effluents

MEF

MCD

ALL INDUSTRIALEFFLUENTS

MIJMDE

21.7

vPBT load

after biodegradationand acid cleanup

MCD

otherwisenon-stablesubstances

1.5

DATA FROM

REGISTRATIEEMISSIE

known i-TEQ loads1995-2000

0.27

after in-plant TIE

MIJMEF

MCD

loadattr ibutable tochlor ine chain

non-chlorinecontribution

MDE

11.2

sum of fine fractions*

MEFMCD

non-metabolisableBT load

(potentiallyunderestimated)

metabolisablecontribution(e.g. PAHs)

6.0

screening phase‘top-5'

MEF

MCD

MDEMIJ

99% selectie

DR Calux loads industrial effluents



DR Calux loads wastewaters

0

50

100

150

chlorine chain activities cooling water (tentative extrapolation)

MWTP only households(indicative extrapolation)

DR

Cal

uxlo

ad (g

TE

Q/y

)

screening phasenon-metabolisablevPBT fraction

n.d.(<25)



DR Calux response with acid cleanup

atmospheric emissions:three emissions about 90% of response induced by PCDD/Fs; one emission 22%; total load 0.06 g TEQ/y

products:8 products analysed: 5 responsive with loads between 0.08- 1(?) TEQ/y; 1 product contact exposure effects possible (neoprene)

» loads to environment via air and products order of magnitude below effluent loads



Interaction needed

central issue is:

where should the focus be put for the remainder of OVOC project such that a meaningful and acceptable

final result is obtained?

4



About P, B and T

to assess if, and to what extent PBTs or groups ofPBTs are emitted from the chlorine chain into the environment (OVOC, goal 1)

PBTssubtances that can induce toxic effects (T) in higher organisms by accumulation (B) in the food chain. Substances have to persist (P) in the environment in order to be able to do this.



PBTs in DR Calux response

B response at log Kow >6 or even >8: no doubt

T DR Calux = substances with dioxinlike toxicitytwo links in toxicity mechanism active

P two forms: - non-metabolisable fraction- after biodegradation and acid cleanup

P clearly is a problem, but does it matter?



Comparison with known dioxin emissions

Dioxin e missions in The Ne therlands 1999

in g i -TE Q/ y

28householdsand others

4

wastemanagement& incineration

0.65

3 +0.06

industry

0.17 +1.5-6.0 -0.4+50 or less

2

Source: air emissions: Mileubalans 2001, RIVM; wastewater emissi ons: Emissieregistratie and literature sources

via products0.08-1

??



Other related information

Benzo(a)pyrene emissions In The Netherlandsair 1.600.000 g/ywater 540.000 g/y (Emissiejaarverslag 2000)

is there a chlorine chain wide problem ?1-3 effluents relevant

1 product possibly relevant

5



Does it matter?

That’s the question.

1t

13 May 2003Arnold Tukker 1

tTNO Strategy, Technology and Policy

A reflection on the study results


Structure

• Original goals of the study• Wrap up of the results

• Water

• Air

• Products

• Implications• Questions in interpretation

• Conclusions in view of the study’s goals


Original goals of the study

• The original goal of the study was

• To evaluate if (unknown) PBTs or groups of PBTs related

to the chlorine chain are emitted into the environment

• To contribute to the assessment of the environmental

problems of these PBTs


The question in a figure

Potentials ource s

EOCI c omplex in e .g .fis h fat and s e diments

UnknownCHCs

P BT-emis s ionsundetected

by monitoringprogrammes

De gra dationproductsof known

CHC-e mis s ions

+ 10% + 90%

Na tura ls ources

Historica lreleas e s

DDTP CBsHCHe tc.

2t


Reflection and interpretation: always 2 attitudes

• ‘risk assessment’

• We have knowledge

• We can manage

• Nature is not too

vulnerable

• > Hence assess risks!

• ‘precaution’

• Knowledge is fluffy

• Management once fails

• Nature is vulnerable

• > Hence go for inherent

risk-free options !


Results and implications (water)

0.4-20.17-0.27Traditional TEQ TCDD, g/yr (ER)

n.d. (< 25)n.d. (<5.3)1.5After biodegradat-ion/acid clean up

505,3> 6Metabolisable

> 12Total excl. non-chlorine plant

13420> 22Total

Households/RWZICooling waterTop 5 IndustryType DR Calux TEQ

(g/yr)


Results and implications (water, ctd.)• You basically can go two ways

• Industry doesn’t do bad compared to households and non-chlorine industry (leave us finally alone)

• There’s a gap of a factor 10 to 50 between known and unknown (unidentifiable!) emissions (get on them)

• Questions about DR Calux approach• Only one type of Tox – what tox do we miss?• Which type of persistence is seen as relevance?• Can we compare DR Calux response industry with e.g.

households?• Does not discern chlorine-non chlorine – relevant?• Which ‘level’ relevant (acid cu, metabolisable, etc.)?• What are the substances (hardly identifiable)?


Results and implications, air• Most samples: DR Calux TEQ = TEQ of known emissions• For one company a gap of 75% exists• Mass flows involved

• Dutch total: 30+ g TEQ TCDD/yr• 3 Companies: 0,062 g TEQ TCDD• Incinerator: 0,129 g TEQ TCDD

• Company with gap = vent gas situation• At most a few dozen of such sources in NL• Say 30-> at similar emission levesl 0,6 g TEQ TCDD/yr?• Hence contribution even with uncertainty limited?

3t


Results and implications: products

• Again, you can go two ways• Chlorine is a problem since

• The wild card scored high (priority setting failed)• The 3 n.d.’s can represent high flows• A myriad of PBTs in some products

• Chlorine is not too bad since• The total is only 1 g TEQ/yr• Highest flow in a non-chlorinated product


Results and implications – products (3)

• What still could be done

• Select other chlorine free products from chlorine chain and

investigate

• Identify level of contamination of the ones currently n.d.

(but which could represent high flows)

• Quantify flows of other PBTs


Overall reflections• Question 1: Does the chlorine chain contribute to

unexpected emissions of PBTs?

• Answer seems a yes• Water: clear gap between a known TEQ and CD Calux

TEQ (though for 5 companies)• Products: by-products found in measurable quantities

• But:• Some questions about P(ersistence) and chlorine

content


Overall reflections

• Question 2: contribution of the assessment of environmental

problems

• In absolute terms no insight yet

• In relative terms, a picture with questions

• Magnitude of gap depends on all kind of

definitions/methodological questions (which

persistence is seen as relevant in DR Calux?)

• Households and other industry maybe at least as

relevant?

• Still no insights about historical and natural sources –

and not to be found due to lack of identification