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Exposure of Chironomus riparius larvae to 17α-ethynylestradiol: effects on survival and mouthpart deformities
The Science of the Total Environment 269 Ž2001. 157᎐161
Exposure of Chironomus riparius larvae to 17␣-ethynylestradiol: effects on survival and mouthpart deformities Giovanna MeregalliU , Frans Ollevier Laboratory of Aquatic Ecology, K.U. Leu¨ en, De Beriotstraat 32, 3000 Leu¨ en, Belgium Received 26 July 2000; accepted 25 September 2000
Abstract Evidence from field studies shows that mouthpart deformities in chironomid larvae are a sublethal response to pollution. Interest has been shown to use this end-point in programs for monitoring sediment quality. During laboratory studies, however, deformities were induced in only a few single pollutant exposures. These deformities develop at the endocrine regulated molting stage and disruption of this complex process is likely at the base of their ontogeny. Aiming to clarify the processes involved in the rise of such deformities, we tested the effects of ethynylestradiol ŽEE2. in an in vivo lab study. Chironomus riparius larvae were exposed to 1, 10 and 100 g ly1 EE2 Žnominal concentrations .. No adverse effects on the larvae, for the investigated end-points Žsurvival and deformity induction., were found. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Mouthpart deformities; Chironomus riparius; 17␣-ethynylestradiol; Laboratory bioassay
1. Introduction The occurrence of deformities in the chitinized mouthparts of chironomids inhabiting different freshwater environments has been documented by several authors and, in field studies, their occurrence has been often correlated to the presence of pollution in the sediment Žfor a review, see Vermeulen, 1995.. Evidence of induction from U
Corresponding author. Tel.: q32-16-323-966; fax: q3216-324-575. E-mail address: [email protected] ŽG. Meregalli..
laboratory Žsingle compound. exposures remain, however, circumstantial ŽKosalwat and Knight, 1987; Madden et al., 1992; Janssens de Bisthoven et al., 1997; Meregalli et al., 2000a; Vermeulen et al., 2000.. The simplicity and rapidity of use make the assessment of deformity levels a potential practical biomarker of sediment quality. This end-point is already included in routine monitoring programs in Flanders ŽBelgium. ŽDe Cooman et al., 1998. and an interest in using it has been expressed also by the UK Environment Agency ŽPinder et al., 1999.. A deeper understanding of the mechanisms responsible for induction of
0048-9697r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 0 0 . 0 0 8 2 4 - X
158
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161
mouthpart deformities is thus relevant for both scientific and practical reasons. Deformities are formed completely at the different larval molting stages and are probably caused by a physiological disturbance during the development of the buccal structures in the molting process ŽJanssens de Bisthoven et al., 1992.. Molting is hormonally regulated by, among others, ecdysone and juvenile hormone ŽdeFur et al., 1999; Pinder et al., 1999.. Disruption of both vertebrate and invertebrate hormonal function has been documented for many chemicals Žsee reviews in Colborn and Clement, 1992; Colborn et al., 1996; deFur et al., 1999; Pinder et al., 1999.. Several authors recently suggested that deformities may be a phenomenon in which endocrine disruption is implicated ŽdeFur et al., 1999; Pinder et al., 1999; Vermeulen et al., 2000.. More specifically, given the similarity between estrogen’s and ecdysone’s molecular structures, Vermeulen et al. Ž2000. hypothesized that compounds interfering with the estrogen receptor may also interfere with ecdysone and thus be primary agents of morphological deformation in chironomid larvae. This hypothesis was tested during laboratory bioassays, exposing Chironomus riparius larvae to -sitosterol ŽVermeulen et al., 2000., 4-n-nonylphenol ŽMeregalli et al., 2000a. and bisphenol-A ŽMeregalli et al., 2000b.. During the present study we wanted to test whether a synthetic estrogen w17␣-ethynylestradiol ŽEE2.x, acting on the vertebrate estrogen receptor, can induce mouthpart deformities in C. riparius larvae. EE2 is used in formulating contraceptive pills. Male rainbow trout Ž Oncorhynchus mykiss. exposed to 2 ng ly1 EE2 showed a significant vitellogenin induction and reduction in the gonadosomatic index compared with the controls ŽJobling et al., 1996.. Arrest in egg development and vitellogenesis induction in males were recorded in zebrafish Ž Danio rerio. exposed to 5 ng ly1 EE2 ŽKime and Nash, 1999.. Exposure of the cladoceran Daphnia magna ŽSchweinfurth et al., 1996. and of the marine copepod Tisbe battagliai ŽHutchinson et al., 1999. to concentrations of hundreds micrograms per liter EE2 did not significantly affect their survival, development and
fecundity. In a recent survey on the presence of endocrine disrupters in Dutch water systems and waste waters, EE2 was detected at concentrations up to 10 ng ly1 ŽBelfroid et al., 1999..
2. Materials and methods For a complete description of the protocols employed to culture Chironomus riparius larvae, to handle the egg-ropes and larvae prior to exposure and to run the bioassay, we refer to Meregalli et al. Ž2000a.. The larvae used during the bioassay were 1-day post-hatch Žfirst and second instar.. For the experiment 1 l glass jars were employed. Each of them contained 2 cm of Rhine sand Žpreviously treated with HNO3 and acetone. and 700 ml of artificial Environmental Protection Agency ŽEPA. water ŽGreenberg, 1992.. Seventy-five larvae were introduced in each experimental jar and were exposed for 9 days. A stock solution of 100 mg ly1 EE2 was prepared by dilution of EE2 ŽSigma Chemical Co., Aldrich Chemie GmbH, Steinheim, Germany. in acetone and was stored at 4⬚C when not in use. The tested concentrations of EE2 were 1, 10 and 100 g ly1 . Dilution water and solvent Žacetone. controls were also included in the test. Three replicates for each of the five treatment levels were run. The stock solution was diluted as suitable in order to deliver EE2 in 700 l of acetone to all treatments. During the experiment, solutions were refreshed every two days. No chemical analyses of the water and sediment were performed during the bioassay. The concentrations reported here are thus to be regarded as nominal values. At the end of the experiment survived larvae were collected and preserved in 70% ethanol. Head capsules of instar IV larvae were treated and mounted for deformity analysis as described in Janssens de Bisthoven et al. Ž1997.. Deformed structures were distinguished from normal ones based on the criteria described in Vermeulen et al. Ž1998. and Vermeulen et al. Ž2000.. In case pupae were found, their larval exuvia was searched for and mounted for deformity assessment as
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161
well. The minimum magnification used to screen slides was 400 Žmicroscope: Olympus BX50.. STATISTICA 5.1 ŽStatSoft, Inc.. was used to perform statistical analysis. The presence of significant differences in survival and frequency of mouthpart deformities among treatments was tested by means of one-way ANOVA. Assumptions for analysis of variance were checked before running this test. Results were considered significant at PF 0.05.
3. Results Table 1 shows the mean and standard deviations of the percentages of survival and of mentum, mandible and pecten deformities for each of the experimental treatments. The lowest survival Ž65.33%. was found in one replicate of the dilution water control group. No significant differences among treatments were detected by the ANOVA Ž F4,10 s 2.87; Ps 0.08.. No significant induction of mouthpart deformities was observed in the mentum Ž F4,10 s 3.27; Ps 0.06., mandibles Ž F4,10 s 0.78; Ps 0.57. and pecten epipharyngis Ž F4,10 s 1.93; Ps 0.18..
4. Discussion In this experiment we tried to induce mouthpart deformities through exposure of Chironomus riparius to EE2. We chose EE2 because: Ž1. it is present in aquatic ecosystems at levels which are affecting fish reproductive health ŽJobling et al., 1996; Belfroid et al., 1999; Kime and Nash, 1999.; and Ž2. it is directly acting on the estrogen recep-
159
tor in vertebrates and, as such, had the characteristics of a compound potentially inducing deformities ŽVermeulen et al., 2000.. In our study exposure to concentrations as high as 100 g ly1 EE2 did not have any negative impact on C. riparius larvae. The concentrations of EE2 for the test were chosen based on Hutchinson et al. Ž1999.. They exposed Tisbe battagliai, a marine copepod, to EE2 concentrations ranging from 0.1 to 100 g ly1 . No significant inhibition of survival and development after 10 days and no significant impact on fecundity after 21 days were detected. Results from exposures of Daphnia magna outline the same trend: there are no significant effects on survival and reproduction after 21 days at concentrations ranging from 1.4 to 387 g ly1 ŽSchweinfurth et al., 1996.. Vertebrates are affected at much lower EE2 concentrations. Fish, for instance, undergo adverse effects at concentrations of 2 or 5 ng ly1 ŽJobling et al., 1996; Kime and Nash, 1999.. EE2 has been detected in The Netherlands at concentrations of 10, 8 and 4.3 ng ly1 , respectively, in influent and effluent waters of sewage treatment plants and in surface waters ŽBelfroid et al., 1999.. These levels are of concern as they pose a risk to fish species, but, according to our results and others ŽSchweinfurth et al., 1996; Hutchinson et al., 1999., they apparently do not affect invertebrate communities. In the present work we could not induce deformities. Induction of mouthpart deformities during laboratory bioassays was rarely successful in earlier studies. Chironomid larvae showed deformations after being exposed to copper ŽKosalwat and Knight, 1987., DDT ŽMadden et
Table 1 Mean ŽM. and standard deviation ŽS.D.. of survival, mentum, mandible and pecten deformities for each EE2 treatment level Ž n s 3. EE2 nominal concentration
Survival Ž%. M
0 0 q acetone 1 g ly1 10 g ly1 100 g ly1
79.11 95.11 92.44 91.56 96.89
Mentum def. Ž%.
Mandible def. Ž%.
Pecten def. Ž%.
S.D.
M
S.D.
M
S.D.
M
S.D.
11.95 3.36 3.08 5.05 8.15
21.95 16.95 16.93 13.24 23.19
1.45 6.55 3.97 1.77 3.51
1.71 0.96 0.49 0.98 2.33
0.28 0.83 0.85 0.85 2.83
32.19 23.39 33.87 23.74 27.61
3.72 3.45 4.36 1.53 11.40
160
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161
al., 1992., xylene ŽJanssens de Bisthoven et al., 1997., lead and mercury ŽVermeulen et al., 2000. and 4-n-nonylphenol ŽMeregalli et al., 2000a.. The positive results found in the cases of compounds like DDT and 4-n-nonylphenol, which are known to act on the vertebrate estrogen receptor, seem to support the ‘deformity induction’ hypothesis by Vermeulen et al. Ž2000.. The present results, however, do not confirm this. Similar to what happens in vertebrates, it is possible that the compounds exerting adverse effects in arthropods, at levels where endocrine disruption may be involved, are acting as ‘xeno-ecdysones’. This would mean that the organisms are exposed to and experience an overdose of ecdysone mimetics. In this respect, testing the effects of exposure to 20-hydroxyecdysone Žthe active form of ecdysone. would be relevant and interesting. Hutchinson et al. Ž1999. showed that this chemical does indeed affect reproduction and survival of T. battagliai. They report a no observed effect concentration ŽNOEC. of 8.7 g ly1 for reproduction and of 26.9 g ly1 for survival after 21 days of exposure. Our future research will, therefore, focus on the capability of 20-hydroxyecdysone to induce mouthpart deformities in chironomids.
5. Conclusions
No negative effects on survival and deformity incidence were assessed after a 9-day exposure of C. riparius larvae to EE2. The concentrations Žup to 100 g ly1 . we tested were much higher than the levels found in environmental compartments. We did not perform a long-term exposure and we did not evaluate the toxicity of EE2 in mixtures with other compounds, so we cannot be sure of the effects that EE2 has on chironomid populations in their natural environments. More research should be addressed to the understanding of the mechanisms involved in the occurrence of deformation in chironomid larvae. Experiments in this sense could be performed both in vivo and in vitro.
Acknowledgements We thank Nel Van lent ŽUniversity of Leuven. for her help in sampling the parent generation and counting the larvae before the exposure. Roger Huybrechts, Geert Huyskens and Eugene Rurangwa ŽUniversity of Leuven. and Michiel Kraak ŽUniversity of Amsterdam. are acknowledged for their comments on earlier versions of the manuscript. The University of Milan granted G. Meregalli a scholarship during the study. References Belfroid AC, Murk AJ, de Voogt P, Schafer ¨ AJ, Rijs GBJ, Vethaak AD. Hormoonontregelaars in water. Report 99.007-99.024. Middelburg: Lelystad, RIZA-RIKZ 1999:109. Colborn T, Clement C. Chemically induced alterations in sexual and functional development: the wildliferhuman connection. Princeton, NJ: Princeton Scientific, 1992:403. Colborn T, Dumanoski D, Myers JP. Our stolen future. London: Abacus, 1996:306. De Cooman W, Florus M, Devroede M. Karakterisatie van de bodems van de Vlaamse onbevaarbare waterlopen. Report Dr1998r3241r224. Brussels: Administratie Milieu-, Natuur-Land- en Waterbeheer, 1998:56. deFur P, Crane M, Ingersoll C, Tattersfield L. Endocrine disruption in invertebrates: endocrinology, testing, and assessment. Pensacola, FL: SETAC, 1999:303. Author A. In: Greenberg AE, editor. Standard methods for the examination of water and waste water. Washington, DC: APHA-AWWA-WPC F, 1992. Hutchinson TH, Pounds NA, Hampel M, Williams TD. Impact of natural and synthetic steroids on the survival, development and reproduction of marine copepods ŽTisbe battagliai.. Sci Total Environ 1999;233:167᎐179. Janssens de Bisthoven L, Timmermans KR, Ollevier F. The concentration of cadmium, lead, copper and zinc in Chironomus gr. thummi larvae ŽDiptera, Chironomidae. with deformed versus normal menta. Hydrobiologia 1992; 239:141᎐149. Janssens de Bisthoven L, Huysmans C, Vannevel R, Goemans G, Ollevier F. Field and experimental morphological response of Chironomus larvae ŽDiptera, Nematocera. to xylene and toluene. Neth J Zool 1997;47:227᎐239. Jobling S, Sheahan D, Osborne JA, Matthiessen P, Sumpter JP. Inhibition of testicular growth in rainbow trout Ž Oncorhynchus mykiss. exposed to estrogenic alkylphenolic chemicals. Environ Toxicol Chem 1996;15:194᎐202. Kime DE, Nash JP. Gamete viability as an indicator of reproductive endocrine disruption in fish. Sci Total Environ 1999;233:123᎐129.
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161 Kosalwat P, Knight AW. Chronic toxicity of copper to a partial life cycle of the midge, Chironomus decorus. Arch Environ Contam Toxicol 1987;16:283᎐290. Madden CP, Suter PJ, Nicholson BC, Austin AD. Deformities in chironomid larvae as indicators of pollution Žpesticide. stress. Neth J Aquat Ecol 1992;26:551᎐557. Meregalli G, Pluymers L, Ollevier F. Induction of mouthpart deformities in Chironomus riparius larvae exposed to 4-nnonylphenol. Environ Pollut 2000a;111:241᎐246. Meregalli G, Pluymers L, Van lent N, Ollevier F. Induction of mouthpart deformities in Chironomus riparius larvae exposed to known endocrine disruptors. Presented at SETAC 3rd World Congress, 21᎐25 May 2000, Brighton, UK, 2000b. Pinder LCV, Pottinger TG, Billinghurst Z, Depledge MH. Endocrine function in aquatic invertebrates and evidence for disruption by environmental pollutants, R& D report TR E67. Bristol, UK: Environment Agency and Endocrine Modulators Steering Group, 1999:150.
161
Schweinfurth H, Lange R, Gunzal P. Environmental fate and ¨ ¨ ecological effects of steroidal estrogens. Presented at IBC Conference, 9᎐10 May 1996, London, UK, 1996. Vermeulen AC. Elaborating chironomid deformities as bioindicators of toxic sediment stress: the potential application of mixture toxicity concepts. Ann Zool Fenn 1995;32:265᎐285. Vermeulen AC, Dall PC, Lindegaard C, Ollevier F, Goddeeris BR. Improving the methodology of chironomid deformation analysis for sediment toxicity assessment: a case study in three Danish lowland streams. Arch Hydrobiol 1998; 144:103᎐125. Vermeulen AC, Liberloo G, Dumont P, Ollevier F, Goddeeris BR. Exposure of Chironomus riparius larvae ŽDiptera. to lead, mercury and -sitosterol: effects on mouthpart deformation and moulting. Chemosphere 2000;41:1581᎐1591.
Exposure of Chironomus riparius larvae to 17␣-ethynylestradiol: effects on survival and mouthpart deformities Giovanna MeregalliU , Frans Ollevier Laboratory of Aquatic Ecology, K.U. Leu¨ en, De Beriotstraat 32, 3000 Leu¨ en, Belgium Received 26 July 2000; accepted 25 September 2000
Abstract Evidence from field studies shows that mouthpart deformities in chironomid larvae are a sublethal response to pollution. Interest has been shown to use this end-point in programs for monitoring sediment quality. During laboratory studies, however, deformities were induced in only a few single pollutant exposures. These deformities develop at the endocrine regulated molting stage and disruption of this complex process is likely at the base of their ontogeny. Aiming to clarify the processes involved in the rise of such deformities, we tested the effects of ethynylestradiol ŽEE2. in an in vivo lab study. Chironomus riparius larvae were exposed to 1, 10 and 100 g ly1 EE2 Žnominal concentrations .. No adverse effects on the larvae, for the investigated end-points Žsurvival and deformity induction., were found. 䊚 2001 Elsevier Science B.V. All rights reserved. Keywords: Mouthpart deformities; Chironomus riparius; 17␣-ethynylestradiol; Laboratory bioassay
1. Introduction The occurrence of deformities in the chitinized mouthparts of chironomids inhabiting different freshwater environments has been documented by several authors and, in field studies, their occurrence has been often correlated to the presence of pollution in the sediment Žfor a review, see Vermeulen, 1995.. Evidence of induction from U
Corresponding author. Tel.: q32-16-323-966; fax: q3216-324-575. E-mail address: [email protected] ŽG. Meregalli..
laboratory Žsingle compound. exposures remain, however, circumstantial ŽKosalwat and Knight, 1987; Madden et al., 1992; Janssens de Bisthoven et al., 1997; Meregalli et al., 2000a; Vermeulen et al., 2000.. The simplicity and rapidity of use make the assessment of deformity levels a potential practical biomarker of sediment quality. This end-point is already included in routine monitoring programs in Flanders ŽBelgium. ŽDe Cooman et al., 1998. and an interest in using it has been expressed also by the UK Environment Agency ŽPinder et al., 1999.. A deeper understanding of the mechanisms responsible for induction of
0048-9697r01r$ - see front matter 䊚 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 0 0 . 0 0 8 2 4 - X
158
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161
mouthpart deformities is thus relevant for both scientific and practical reasons. Deformities are formed completely at the different larval molting stages and are probably caused by a physiological disturbance during the development of the buccal structures in the molting process ŽJanssens de Bisthoven et al., 1992.. Molting is hormonally regulated by, among others, ecdysone and juvenile hormone ŽdeFur et al., 1999; Pinder et al., 1999.. Disruption of both vertebrate and invertebrate hormonal function has been documented for many chemicals Žsee reviews in Colborn and Clement, 1992; Colborn et al., 1996; deFur et al., 1999; Pinder et al., 1999.. Several authors recently suggested that deformities may be a phenomenon in which endocrine disruption is implicated ŽdeFur et al., 1999; Pinder et al., 1999; Vermeulen et al., 2000.. More specifically, given the similarity between estrogen’s and ecdysone’s molecular structures, Vermeulen et al. Ž2000. hypothesized that compounds interfering with the estrogen receptor may also interfere with ecdysone and thus be primary agents of morphological deformation in chironomid larvae. This hypothesis was tested during laboratory bioassays, exposing Chironomus riparius larvae to -sitosterol ŽVermeulen et al., 2000., 4-n-nonylphenol ŽMeregalli et al., 2000a. and bisphenol-A ŽMeregalli et al., 2000b.. During the present study we wanted to test whether a synthetic estrogen w17␣-ethynylestradiol ŽEE2.x, acting on the vertebrate estrogen receptor, can induce mouthpart deformities in C. riparius larvae. EE2 is used in formulating contraceptive pills. Male rainbow trout Ž Oncorhynchus mykiss. exposed to 2 ng ly1 EE2 showed a significant vitellogenin induction and reduction in the gonadosomatic index compared with the controls ŽJobling et al., 1996.. Arrest in egg development and vitellogenesis induction in males were recorded in zebrafish Ž Danio rerio. exposed to 5 ng ly1 EE2 ŽKime and Nash, 1999.. Exposure of the cladoceran Daphnia magna ŽSchweinfurth et al., 1996. and of the marine copepod Tisbe battagliai ŽHutchinson et al., 1999. to concentrations of hundreds micrograms per liter EE2 did not significantly affect their survival, development and
fecundity. In a recent survey on the presence of endocrine disrupters in Dutch water systems and waste waters, EE2 was detected at concentrations up to 10 ng ly1 ŽBelfroid et al., 1999..
2. Materials and methods For a complete description of the protocols employed to culture Chironomus riparius larvae, to handle the egg-ropes and larvae prior to exposure and to run the bioassay, we refer to Meregalli et al. Ž2000a.. The larvae used during the bioassay were 1-day post-hatch Žfirst and second instar.. For the experiment 1 l glass jars were employed. Each of them contained 2 cm of Rhine sand Žpreviously treated with HNO3 and acetone. and 700 ml of artificial Environmental Protection Agency ŽEPA. water ŽGreenberg, 1992.. Seventy-five larvae were introduced in each experimental jar and were exposed for 9 days. A stock solution of 100 mg ly1 EE2 was prepared by dilution of EE2 ŽSigma Chemical Co., Aldrich Chemie GmbH, Steinheim, Germany. in acetone and was stored at 4⬚C when not in use. The tested concentrations of EE2 were 1, 10 and 100 g ly1 . Dilution water and solvent Žacetone. controls were also included in the test. Three replicates for each of the five treatment levels were run. The stock solution was diluted as suitable in order to deliver EE2 in 700 l of acetone to all treatments. During the experiment, solutions were refreshed every two days. No chemical analyses of the water and sediment were performed during the bioassay. The concentrations reported here are thus to be regarded as nominal values. At the end of the experiment survived larvae were collected and preserved in 70% ethanol. Head capsules of instar IV larvae were treated and mounted for deformity analysis as described in Janssens de Bisthoven et al. Ž1997.. Deformed structures were distinguished from normal ones based on the criteria described in Vermeulen et al. Ž1998. and Vermeulen et al. Ž2000.. In case pupae were found, their larval exuvia was searched for and mounted for deformity assessment as
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161
well. The minimum magnification used to screen slides was 400 Žmicroscope: Olympus BX50.. STATISTICA 5.1 ŽStatSoft, Inc.. was used to perform statistical analysis. The presence of significant differences in survival and frequency of mouthpart deformities among treatments was tested by means of one-way ANOVA. Assumptions for analysis of variance were checked before running this test. Results were considered significant at PF 0.05.
3. Results Table 1 shows the mean and standard deviations of the percentages of survival and of mentum, mandible and pecten deformities for each of the experimental treatments. The lowest survival Ž65.33%. was found in one replicate of the dilution water control group. No significant differences among treatments were detected by the ANOVA Ž F4,10 s 2.87; Ps 0.08.. No significant induction of mouthpart deformities was observed in the mentum Ž F4,10 s 3.27; Ps 0.06., mandibles Ž F4,10 s 0.78; Ps 0.57. and pecten epipharyngis Ž F4,10 s 1.93; Ps 0.18..
4. Discussion In this experiment we tried to induce mouthpart deformities through exposure of Chironomus riparius to EE2. We chose EE2 because: Ž1. it is present in aquatic ecosystems at levels which are affecting fish reproductive health ŽJobling et al., 1996; Belfroid et al., 1999; Kime and Nash, 1999.; and Ž2. it is directly acting on the estrogen recep-
159
tor in vertebrates and, as such, had the characteristics of a compound potentially inducing deformities ŽVermeulen et al., 2000.. In our study exposure to concentrations as high as 100 g ly1 EE2 did not have any negative impact on C. riparius larvae. The concentrations of EE2 for the test were chosen based on Hutchinson et al. Ž1999.. They exposed Tisbe battagliai, a marine copepod, to EE2 concentrations ranging from 0.1 to 100 g ly1 . No significant inhibition of survival and development after 10 days and no significant impact on fecundity after 21 days were detected. Results from exposures of Daphnia magna outline the same trend: there are no significant effects on survival and reproduction after 21 days at concentrations ranging from 1.4 to 387 g ly1 ŽSchweinfurth et al., 1996.. Vertebrates are affected at much lower EE2 concentrations. Fish, for instance, undergo adverse effects at concentrations of 2 or 5 ng ly1 ŽJobling et al., 1996; Kime and Nash, 1999.. EE2 has been detected in The Netherlands at concentrations of 10, 8 and 4.3 ng ly1 , respectively, in influent and effluent waters of sewage treatment plants and in surface waters ŽBelfroid et al., 1999.. These levels are of concern as they pose a risk to fish species, but, according to our results and others ŽSchweinfurth et al., 1996; Hutchinson et al., 1999., they apparently do not affect invertebrate communities. In the present work we could not induce deformities. Induction of mouthpart deformities during laboratory bioassays was rarely successful in earlier studies. Chironomid larvae showed deformations after being exposed to copper ŽKosalwat and Knight, 1987., DDT ŽMadden et
Table 1 Mean ŽM. and standard deviation ŽS.D.. of survival, mentum, mandible and pecten deformities for each EE2 treatment level Ž n s 3. EE2 nominal concentration
Survival Ž%. M
0 0 q acetone 1 g ly1 10 g ly1 100 g ly1
79.11 95.11 92.44 91.56 96.89
Mentum def. Ž%.
Mandible def. Ž%.
Pecten def. Ž%.
S.D.
M
S.D.
M
S.D.
M
S.D.
11.95 3.36 3.08 5.05 8.15
21.95 16.95 16.93 13.24 23.19
1.45 6.55 3.97 1.77 3.51
1.71 0.96 0.49 0.98 2.33
0.28 0.83 0.85 0.85 2.83
32.19 23.39 33.87 23.74 27.61
3.72 3.45 4.36 1.53 11.40
160
G. Meregalli, F. Olle¨ ier r The Science of the Total En¨ ironment 269 (2001) 157᎐161
al., 1992., xylene ŽJanssens de Bisthoven et al., 1997., lead and mercury ŽVermeulen et al., 2000. and 4-n-nonylphenol ŽMeregalli et al., 2000a.. The positive results found in the cases of compounds like DDT and 4-n-nonylphenol, which are known to act on the vertebrate estrogen receptor, seem to support the ‘deformity induction’ hypothesis by Vermeulen et al. Ž2000.. The present results, however, do not confirm this. Similar to what happens in vertebrates, it is possible that the compounds exerting adverse effects in arthropods, at levels where endocrine disruption may be involved, are acting as ‘xeno-ecdysones’. This would mean that the organisms are exposed to and experience an overdose of ecdysone mimetics. In this respect, testing the effects of exposure to 20-hydroxyecdysone Žthe active form of ecdysone. would be relevant and interesting. Hutchinson et al. Ž1999. showed that this chemical does indeed affect reproduction and survival of T. battagliai. They report a no observed effect concentration ŽNOEC. of 8.7 g ly1 for reproduction and of 26.9 g ly1 for survival after 21 days of exposure. Our future research will, therefore, focus on the capability of 20-hydroxyecdysone to induce mouthpart deformities in chironomids.
5. Conclusions
No negative effects on survival and deformity incidence were assessed after a 9-day exposure of C. riparius larvae to EE2. The concentrations Žup to 100 g ly1 . we tested were much higher than the levels found in environmental compartments. We did not perform a long-term exposure and we did not evaluate the toxicity of EE2 in mixtures with other compounds, so we cannot be sure of the effects that EE2 has on chironomid populations in their natural environments. More research should be addressed to the understanding of the mechanisms involved in the occurrence of deformation in chironomid larvae. Experiments in this sense could be performed both in vivo and in vitro.
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