Evaluation of OVOC ecotox data against RIZA WEA data

Transcription

1 Evaluation of OVOC ecotox data against RIZA WEA data June 22, 2004 RIZA Work report Author: AAM Gerritsen 1 Evaluation of OVOC ecotox data

2 2 Evaluation of OVOC ecotox data

3 Contents Summary 4 1. Introduction 5 2. Suitability of test methods for comparison Sample pre-treatment Fish tests Chronic Microtox test 7 3. OVOC vs RIZA results 8 4. RIZA chlorine chain vs RIZA non-chlorine chain results RIZA study 1(De Graaf et al., 2000): Acute toxicity RIZA study 2 (Roex, 2003): Acute and chronic toxicity Conclusions References 14 3 Evaluation of OVOC ecotox data

4 Summary The OVOC results of two in-vivo toxicity tests have been compared to the outcome of two RIZA studies on whole effluent toxicity. The central issue in this study was the question to what extent the OVOC results can be generalized towards toxic responses of chlorine chain effluents in general. The result of the comparison shows that chlorine chain effluents do not differ from other types of industrial effluents. The lack of response in the zebra fish ELS tests on effluent extracts in the OVOC study does not imply the absence of any toxic response of that effluent. In the RIZA WEA study on whole effluents two of these effluents were responsive. This may indicate that more polar or ionic compounds were responsible for the induction of a toxic response. 4 Evaluation of OVOC ecotox data

5 1. Introduction In the first phase of the chlorine chain follow-up studies (OVOC), a number of chlorine chain effluents were screened for the presence of ecotoxicity. For this purpose two tests were employed, notably the short-term fish Early Life Stage (ELS) test (OECD 212) and the chronic Microtox test. The results of these screening tests were published in two OVOC reports (Hooftman et al, 2001; Senhorst, 2002, cited in Lamoree et al., 2003), and discussed at the OVOC technical workshops of August 30, 2001 and May 14-15, During the last workshop it was stipulated that, on the basis of the screening data and experience with effluent toxicity testing in the Netherlands, the toxicity of the chlorine chain effluents is in line with the general picture on effluent toxicity. Thus, they are neither remarkably high nor low in toxicity as compared to effluents form other sources, industrial nor domestic. In order to underpin this hypothesis it was decided to evaluate the screening data against the results of RIZA s Whole Effluent Assessment (WEA) programme in the Netherlands. In the following paragraphs OVOC toxicity results are evaluated against RIZA WEA results. The following two specific research questions are addressed: How do the results of the OVOC in-vivo tests compare with the RIZA whole effluent tests on the same effluents? How do chlorine chain effluents, not necessarily tested in the OVOC study, compare with non-chlorine chain effluents in the RIZA whole effluent results? In paragraph 2 the methods applied in the OVOC and RIZA-WEA programme will be evaluated in terms of their suitability for further comparison. In section 3 the OVOC and RIZA WEA results will be discussed for the methods that may be compared. In paragraph 4 the chlorine chain effluents tested in two RIZA WEA studies will be evaluated against the non-chlorine chain effluents in the same studies. A major advantage of this approach is that the same effluent selection criteria are used for the effluents and that similar methods are used for the tests. General background on toxicity tests Toxicity tests or bioassays are carried out in order to assess the direct adverse effects on living organisms or cells. In bioassays test organisms or cells are exposed to the test substance in a concentrations range, or, as in the case of effluents, to a dilution series of the effluent. A dose-effect curve as illustrated in figure 1 can than be obtained by evaluating the adverse effects, such as mortality, mobility or reproduction, at the different exposure levels after a certain period of exposure. From this dose-effect curve the concentration where 50% effect occurs, the EC50 is estimated. The EC50 is mostly requested in acute toxicity studies. In the case of lethality or inhibition as the measured effect the EC50 is usually given as respectively the LC50 or 5 Evaluation of OVOC ecotox data

6 IC50. Bioassays can also be used to obtain a No Observed Effect Concentration (NOEC), the highest tested concentration at which no statistically significant adverse effects are observed. The NOEC is often requested from chronic toxicity studies. 100 % E ffect 50 % 0 % EC50 Exposure level Figure 1 Dose-effect curve obtained from bioassays 6 Evaluation of OVOC ecotox data

7 2. Suitability of test methods for comparison 2.1 Sample pre-treatment There is one essential methodological difference between the tests carried out under OVOC and those carried out under RIZA-WEA testing. In OVOC all toxicity tests were carried out with extracts of the selected effluents, whereas in RIZA-WEA testing all effluents were tested for toxicity in their most original form: whole effluent with filtration or sedimentation only applied if necessary. This implies that there can be significant differences in the presence of toxicants, due to the efficiency of the extraction (log K ow >±2), and the availability of toxicants, due to the complex matrix of the untreated effluents. As a consequence toxicant exposure levels, and therefore tests results, may be affected very much by the pre-treatment procedure followed. Conclusions drawn from a comparison of the results should thus be treated with great caution and regarded as indicative only. 2.2 Fish tests Fish ELS tests were carried out in both OVOC and in RIZA s effluents testing programme. In both programmes the same species of fresh water fish, the zebra fish (Danio rerio) and the same international OECD Guideline no 212, Fish, short term toxicity on embryo and sacfry stages, were applied. The high level of standardization of this test provides maximum certainty on the quality of the results. The results of these tests should therefore be regarded as comparable. However, RIZA s effluent testing programme consisted of 6 saline effluents. Two of these were also chlorine chain effluents. These effluents were tested in a two-day, short-term test with the larva of turbot (Scophthalmus maximus), a marine fish species (Roex, 2003). The relative sensitivity of this test, with a different species and test design as compared to the zebra fish ELS test is unknown at this moment. For this evaluation we will therefore assume that they are equally sensitive. 2.3 Chronic Microtox test Unlike the fish ELS test, the Chronic Microtox test does not have the status of a standard test in ecotoxicity. In a recent attempt to use this test in the so-called OSPAR Practical Study Programme 2003 on Whole Effluent Assessment (Gerritsen et al, in preparation) the quality of the test could not be guaranteed by the supplier (Joep Appels, personal communication). Therefore, the test was left out of the study, and thus no comparison can be made with OVOC results. 7 Evaluation of OVOC ecotox data

8 3. OVOC vs. RIZA results In the OVOC studies 16 extracts of chlorine chain effluents were tested with the zebra fish ELS test. Neither of the extracts proved toxic to the zebra fish, being that no adverse effects were observed at a dilution equivalent to 100% effluent. Two of the OVOC chlorine chain effluents were also tested in the RIZA WEA programme of 2002, one with zebra fish and one with turbot. Both were toxic. No adverse effects were observed below dilutions of respectively 32 and 10% of the original effluent. The discrepancy between the OVOC and RIZA WEA clearly illustrates that the absence of toxicity of the effluent extracts to the zebra fish in the OVOC study cannot lead to the conclusion that chlorine chain effluents are not toxic to fish in general. Additionally, this may indicate that more polar or ionic compounds have been responsible for the induction of a toxic response in the RIZA WEA study. 8 Evaluation of OVOC ecotox data

9 4. RIZA chlorine chain vs. RIZA non-chlorine chain results RIZA has carried out two major studies on effluent toxicity. One study dealt with the acute toxicity of 57 effluents (De Graaf et al, 2000), and the other with both the acute and chronic toxicity of 19 effluents (Roex, 2003). The effluents used in these studies represent domestic and municipal wastewaters and a wide range of industrial activities. The presence of chlorine chain effluents in these studies, respectively 10 and 3 effluents identified from the longlist of 34 OVOC effluents reported in Tukker, 2000, allowed for a comparison between chlorine and non-chlorine chain effluents. Not all of these chlorine chain effluents where studied in the OVOC programme as of the long list of 34 effluents only 16 were prioritized for the sampling programme. The two RIZA studies are discussed separately in the paragraphs below. Toxicity is expressed in so-called Toxic Units (TU), which is the effective dilution factor to remove toxicity above a certain limit value (e.g. EC20, EC50, and NOEC) of the specific test. The TU-value of an effluent is higher when an effluent is more toxic. A situation where no toxicity can be found in the (undiluted) effluent corresponds to a TU value of 1 for all tests except the acute Microtox test (bacteria). Due to the addition of reagents in this test the maximum testable effluent concentration is 45%, which corresponds to a TU value of 2, RIZA study 1 (De Graaf et al., 2000): Acute toxicity In Table 1 and Figure 2 the data on the acute toxicity of effluents to respectively bacteria (EC20), algae (EC50), crustaceans (EC50) and fish (LC50) are summarized. The effluents are sorted as to discriminate between the chlorine chain (closed symbols) and nonchlorine chain effluents. As can be seen from Table 1 and the Figure 2 chlorine chain effluents seem to be more often toxic than the nonchlorine chain effluents. For the test with crustaceans, this overrepresentation actually is statistically significant at a 99% confidence level. However, the toxicity (expressed as TU values) observed in the individual toxic effluents for this test is of the same order of magnitude for both chlorine chain and non-chlorine chain effluents. Thus chlorine chain effluents are more often toxic compared to the non-chlorine chain effluents, but when toxic they do not show higher levels of toxicity. This observation may be explained by a difference in the characteristics of the sub sample of non-chlorine chain effluents compared to the sub sample of chlorine chain effluents. For instance, the sub sample of non-chlorine chain effluents consists of both industrial and municipal effluents while the sub sample of chlorine chain effluents consists of only industrial effluents. In this particular study all 9 municipal effluents used in the comparison were non-toxic to crustaceans. The statistical significant overrepresentation of the test 9 Evaluation of OVOC ecotox data

10 with crustaceans will disappear when the municipal effluents are left out of the comparison. Table 1. Number of toxic and non-toxic effluents in the RIZA WEA study on effluent acute toxicity (Data by De Graaf et al, 2000). Test Microtox Algae Crustaceans Fish Endpoint EC20 EC50 EC50 LC50 Chlorine chain Non toxic effluents Toxic Total Non-chlorine chain Non toxic effluents Toxic Total significance level p (based on χ 2 test) 0,22 0,24 0,01 0,06 Figure 2 Acute toxicity (expressed in TUs) of chlorine chain (closed symbols) and non-chlorine chain (open symbols) effluents to bacteria (Microtox ), algae, crustaceans and fish. Data by De Graaf et al., Each dot represents an effluent. Acute toxicity to bacteria (EC20) Acute toxicity to algae (EC50) TU's 1 0 Chlorine Effluents No Chlorine Acute toxicity to fish (LC50) TU's TU's 1 0 Chlorine Effluents No Chlorine Acute toxicity to crustaceans (EC50) TU's Chlorine Effluents No Chlorine 0 Chlorine Effluents No Chlorine 10 Evaluation of OVOC ecotox data

11 4.2 RIZA study 2 (Roex, 2003): Acute and chronic toxicity Figure 3 summarizes the results on both the acute and chronic toxicity of the 19 effluents tested in The effluents were those of sewage treatment plants (4), chemical (9), pharmaceutical (1), metal (1) and food (2) industry and a paper factory. The three chlorine chain effluents had their origin in one of the food industries and two chemical industries. In Figure 3 the effluents are sorted as to discriminate between the chlorine chain, three effluents, and the remaining nonchlorine chain effluents. All three chlorine-chain effluents were toxic to one or more of the species tested. Of the remaining 16 effluents 5 were non-toxic to any of the test species. When all data are taken together and statistically tested this difference is not significant (p=0.11 based on χ 2 test). It must therefore be concluded that in this study, the chlorine chain effluents seem to be comparable to the non chlorine chain effluents. 100 bacteria (EC20*2.2) alga (IC50) 10 crustacean acute (EC50) crustacean chronic (NOEC) TUs fish acute (LC50) fish chronic (NOEC) 1 0,1 Chlorine Non-chlorine Effluents Figure 3 Acute and chronic toxicity (TU s) of chlorine chain and non-chlorine chain effluents to bacteria (Microtox ), algae, crustaceans and fish. Data by Roex, Evaluation of OVOC ecotox data

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13 5. Conclusions The hypothesis has been tested that chlorine chain effluents are comparable in in-vivo toxicity to non-chlorine chain effluents. Of the five toxicity tests used for comparison, the test with crustaceans gives a significantly higher frequency of chlorine chain effluents being toxic than non-chlorine chain effluents. However, as there is no good parameter at hand to test the comparability of (groups of) effluents one should deal this result with caution. For instance, when all municipal wastewater treatment plants are removed from the group of nonchlorine chain effluents (leaving only industrial effluents in the group) the difference in toxicity between chlorine chain effluents and nonchlorine chain effluents disappears. Therefore, based on the available information of two RIZA studies the hypothesis cannot be rejected. The overall conclusion is therefore that in terms of toxicity to bacteria, algae, crustaceans and fish the chlorine chain effluents are comparable to the non-chlorine chain industrial effluents. For two effluents tested in the OVOC study a direct comparison could be made with RIZA WEA results based on the same toxicity test (zebra fish ELS test). In the OVOC study no response was observed, while in the RIZA study both effluents proved to be toxic. This difference in results may be explained by the L/L extraction pre-treatment applied in the OVOC study, allowing only the more hydrophobic substances (log K ow > ±2) to be present in the test. Possibly, more polar or ionic compounds were responsible for the induction of a toxic response in the whole effluent tests. Therefore, it can be concluded that absence of a toxic response in extracts does not automatically imply absence of a toxic response in the whole effluent. The preceding conclusion is further substantiated by the observation that the 13 chlorine chain effluents in the RIZA studies taken from the longlist of Tukker, 2000 showed toxic responses in fish toxicity tests (acute and/or chronic) while the 16 extracts of the industrial chlorine chain effluents in the OVOC study did not show toxic responses in the fish Early life Stage test. So, more specifically, the OVOC ELS results cannot be generalised to the conclusion that chlorine chain effluents are not toxic to fish. 13 Evaluation of OVOC ecotox data

14 6. References De Graaf, P.J.F., J. Graansma, E.V. ten Kate, M. Tonkes and H. Maas. Toetsing voorlopige handreiking toepassing acute toxiciteitstesten. FWVO-nota 00.03, April 2000 (in Dutch). Gerritsen, A.A.M., R. Benstead, M. Ferdinandy, S. Gartiser, L. Hoebeke, J. O Neill, E. Roex, Å. Undén, P. Viana, G. de Vries and H. Witters. OSPAR Practical Study Programme 2003 on Whole Effluent Assessment (WEA). In preparation. Hooftman, R.N., Borst, B., van Drongelen-Sevenhuijsen, Toxicity screening of 21 mixture extracts in a short term Early Life Stage test with the zebra fish Brachydanio rerio, TNO-Toxicology report /01, May Lamoree, M.H, G. Stroomberg, H. Besselink, H. Senhorst, T. Sanderson, and B. van Hattum (2003). 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. Roex E. TEB praktijkonderzoek. Deel T-1: Meten TEB-parameters. FWVO-nota 03.03, December 2003 (in Dutch). Senhorst H. et al., Semi chronic toxicity tests with the bacteria Vibrio fisheri on 21 effluent extracts, RIZA report X, Tukker, A. (200b), Chlorinated micropollutants: literature search on emissions. Backgrounds to the prioritiy setting for the main research phase, Report STB-00-35, TNO-STB, Delft (NL). 14 Evaluation of OVOC ecotox data

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