Environmental endocrine disruption – experimental models and assessment

Maher Haeba, M.Sc.

Doctoral thesis

Abstract:

Endocrine disruptors are an important issue in the environmental chemistry and ecotoxicology. Toxicity of endocrine disruptors has been studied widely in vitro. This type of toxicity test depends mainly on affinity of toxicants to special receptors, and it can often be evaluated by chemical binding to specific hormone receptors. On the other hand, hormones regulate numerous biological processes and evaluation of the whole organism response provides more realistic information on physiological and developmental processes that may be directly or indirectly affected by endocrine-disrupting compounds (EDCs). The aim of the study was to evaluate effects of selected model endocrine disruptors in vivo on non-target organisms (invertebrate, plant, and amphibian) at different endpoints to figure out which parameters can be more sensitive than others at variuos life span. For this work, five chemicals have been chosen. Vinclozolin, ketoconazole, flutamide as anti-androgen and dicofol as estrogen compounds, and also methyl farnesoate as a model compound which has been shown to modulate sex ratio in Daphnia magna. In standard D. magna toxicity tests (i.e. acute and chronic) hormone regulated endpoints such as growth, molting, reproduction, as well as behavior can be determined. In our studies, we have examined effects in three D. magna bioassays to evaluate effect of EDCs on different levels in a specific time of development. We have used sex ratio of neonate Daphnia, as a short time assay. Methyl farnesoate in our studies caused an increase in sex ratio at concentration 0.1mg/L in 4 days. The bioassay could facilitate detection of endocrine disruption associated with juvenile hormones and its analogs which are used as insecticides. Our study revealed that effects of Juveniod hormone analogs on sex ratio in Daphnia can be detected in short time, and the bioassay can serve as important screening of endocrine disruption. Further, we studied short term and chronic effects of four endocrine disrupting chemicals on survival, sex determination and reproduction in Daphnia magna. Our results from the short term assays have shown that Daphnia sex ratio was modulated by vinclozolin and dicofol at 1 and 0.1 mg/L respectively (at theses concentrations there were no effects on viability, maturation or fecundity). Flutamide (1 mg/L) stopped embryonic development at certain stage, resulting in abortion of incompletely developed embryos or reduction in size of the daphnids. Consequently, smaller daphnids were unable to accommodate brood as big as brood in control groups. This finding with Flutamide shows interesting indirect effect of EDCs on reproduction. Ketoconazole has no significant effect on endocrine processes in D. magna within tested concentrations. In general, the experiments with model EDCs support the hypothesis that the sex ratio is the most sensitive endpoint, and that several contaminants affect sensitive endocrine-regulated processes in D. magna. Although hormonal regulations between invertebrates and vertebrates significantly vary, there are several interlinks, and vertebrate EDCs might possibly affect populations of daphnids with significant impact on ecosystems. In spite of numerous studies only little is known about possible effects of EDCs on photoautotrophic organisms. Plants are not target compounds for EDCs but they can be exposed to these compounds and possible effects should be clarified. In our studies we have evaluated effects of four model endocrine disrupting compounds (ketoconazole, flutamide, vinclozolin and dicofol) on Lemna minor which was shown to be highly susceptible to hydrophobic organic compounds and it is often used in phytotoxicity assays. We have evaluated 168h effects on frond numbers, growth rate, dry weight, doubling time as well as roots length. For all the endpoints of interest, ketoconazole was the most effective compound with EC50 in the range 0.1 to 1 mg/L. The lowest toxic chemical was vinclozolin. The m

Abstract:

ost sensitive endpoint was the length of roots which is not traditionally evaluated as the major endpoint in standardized assays with L. minor. The results demonstrated that EDCs might affect growth and viability of aquatic plants in relatively low mg/L concentrations, which may be of general concern for ecological risks of these compounds in aquatic environment. The frog embryo teratogenesis assay - Xenopus (FETAX) is an alternative bioassay that can be effectively used in aquatic toxicity. Relating to experiments with Daphnia and Lemna, we have studied effects of model compounds dicofol, flutamide, ketoconazole, vinclozoline and methyl farnesoate. Ketoconazole has no significant effect on survival at concentration up to 10 mg/L. However, ketoconazole caused malformations at 47% of exposed animals and reduced the embryos size at concentration 10 mg/L. Dicofol and flutamide had no effect on survival, growth and malformations below the concentration 2 mg/L. Vinclozoline and methyl farnesoate had no effect on mortality, malformation and growth rate at concentrations tested. Although most of the studied compounds were shown to affect hormonal regulations in vertebrates, our studies showed that D. magna and L. minor were both more sensitive than vertebrate embryos of X. laevis to tested chemicals. Our data demonstrate significant variability in sensitivity to EDCs among biota, and we conclude that several bioassays together can provide realistic results about possible effects of chemicals on biota.

Keywords

Endocrine disruption, Daphnia magna, Lemna minor, Xenopus laevis, Vinclozolin, Flutamide, Ketoconazole, Dicofol, Methyl farnesoate.

Language used: English

Date on which the thesis was submitted / produced: 29. 4. 2008

Thesis defence

• Date of defence: 5. 5. 2008
• Supervisor: prof. RNDr. Luděk Bláha, Ph.D.
• Reader: prof. Ing. Blahoslav Maršálek, CSc., doc. Ing. Vladimír Kočí, Ph.D., Prof. MVDr. Zdeňka Svobodová, DrSc.

Masaryk University

Faculty of Science

Doctoral programme / field:
Chemistry (4-years) / Environmental Chemistry