Development of chronic tests for endocrine active chemicals. Part 2: an extended fish early-life stage test with an androgenic chemical in the fathead minnow (Pimephales promelas).

The Endocrine Modulators Study Group (EMSG) of the European Chemical Industry has proposed an extended fish early-life stage (ELS) test based on OECD test guideline 210 in combination with a fish pair-breeding reproduction study as a possible alternative for fish full life cycle testing. In this paper the androgen methyldihydrotestosterone (MDHT) was tested in an extended ELS test with fathead minnow supplementary to such a test with the weak estrogen 4-tert-pentylphenol (4TPP). Main endpoints were secondary sexual characteristics (SSC), plasma vitellogenin (VTG) induction and gonadal development. Early blastula embryos were exposed to 0, 0.10, 0.32 and 1.0 microgMDHTl(-1) for up to 114 days post-hatch (dph). A batch of fish exposed to 1.0 microg l(-1) was transferred to clean water after 30 or 63 dph for the remainder of the study. Ethinylestradiol (EE2) was included as estrogenic reference substance at 0.01 microg l(-1). Exposure to MDHT had no significant effect on hatching success or survival, but significantly increased the condition factor of fish exposed for 63 and 114 dph (up to 150% of the control). At 63 dph MDHT exposure induced appearance of tubercles on the snout (a male SSC) of more than 80% of fish. Compared to the controls, plasma VTG was not detectable or significantly lower in fish exposed to MDHT at 0.10 microg/l, but not significantly affected at higher MDHT concentrations. Both lower levels of MDHT significantly inhibited the development of female gonads as of 30 dph. Fish exposed to MDHT at 0.32 and 1.0 microg l(-1) showed higher incidences of mixed sex gonads (10-25%) and smaller testes or dysplasia of gonadal tissue. Dysplasia was present in 80% of the fish continuously exposed to 1.0 microg l(-1) up to 114 dph, but reversible when fish were transferred to dilution water. Results indicate that suppression of ovarian development was the most sensitive endpoint for MDHT exposure after 30 dph. Other endpoints (e.g., growth and SSC) required exposure during at least up to 63 dph to yield a significant effect. Androgenic effects on VTG production required even longer exposure, i.e., until sufficient number of females had matured.

Alternative approaches can greatly reduce the number of fish used for acute toxicity testing.

Acute toxicity tests with algae, daphnids, and fish are required for the classification and environmental risk assessment of chemicals. The degree of risk is determined by the lowest of these acute toxicity values. Many ecotoxicological programs are seeking to reduce the numbers of fish used in acute toxicity testing. The acute threshold test is a recently proposed strategy that uses, on average, only 10 (instead of 54) fish per chemical. We examined the consequences of reducing the number of fish used in toxicity testing on the ultimate outcome of risk assessments. We evaluated toxicity data sets for 507 compounds, including agrochemicals, industrial chemicals, and pharmaceuticals from our internal database. Theoretical applications of the acute threshold test gave similar results to those obtained with the standard fish median lethal concentration (LC50) test but required only 12% as many fish (3195 instead of 27,324 fish used for all compounds in the database). In 188 (90%) of the 208 cases for which a complete data set was available, the median effect concentration for algae or daphnids was lower than the LC50 for fish. These results show that replacement of the standard fish LC50 test by the acute threshold test would greatly reduce the number of fish needed for acute ecotoxicity testing without any loss of reliability.

QSARs in ecotoxicological risk assessment.

The need for more ecotoxicological data encourages the use of QSARs because of the reduction of (animal) testing, time and cost. QSARs may however only be used if they prove to be reliable and accurate. In this paper, four QSARs were attempted to predict toxicity for 170 compounds from a broad chemical class, using them as a black-box. Predictions were obtained for 122 compounds, indicating an important drawback of QSARs, i.e., for 28% of the compounds QSARs cannot be used at all. Ecosar, Topkat, and QSARs for non-polar and polar narcosis generated predictions for 120, 39, 24, and 11 compounds, respectively. Correlations between experimental and predicted effect concentrations were significant for Topkat and the QSAR for polar narcosis, but generally poor for Ecosar and the QSAR for non-polar narcosis. When predicted effect concentrations for fish were allowed to deviate from experimental values by a factor of 5, correct predictions were generated for 77%, 54%, 68%, and 91% of the compounds using Ecosar, Topkat, and the QSARs for non-polar and polar narcosis, respectively. It was impossible to indicate specific chemical classes for which a QSAR should be used or not. The results show that currently available QSARs cannot be used as a black-box.

Estrogenic endpoints in fish early life-stage tests: luciferase and vitellogenin induction in estrogen-responsive transgenic zebrafish.

This study incorporated specific endpoints for estrogenic activity in the early life-stage (ELS) test, as described in Guideline 210 of the Organization for Economic Cooperation and Development and traditionally used for toxicity screening of chemicals. A transgenic zebrafish model expressing an estrogen receptor-mediated luciferase reporter gene was exposed to ethinylestradiol (EE2), and luciferase activity as well as vitellogenin (VTG) was measured. Concentrations of EE2 were tested at 1, 3, or 10 ng/L for 30 d from fertilization or during only the last 4 d with dimethylsulfoxide (DMSO) as presolvent (0.01%). Exposure to EE2 induced no toxic effects. Mean body weights were significantly higher in groups exposed for 30 d in the presence of DMSO, but condition factors were not affected. Significant luciferase and VTG induction occurred following 30-d exposure (3 and 10 ng EE2/L), while only VTG levels were affected in the 4-d exposure (10 ng EE2/L). This study demonstrated the usefulness of incorporating estrogenic endpoints in the OECD ELS test, fitting the requirements for screening estrogenic activity of chemicals. Quantitative measurement of both VTG and luciferase activity proved to be rapid and sensitive. Additional value of using transgenic zebrafish lies in combining VTG measurement with the more mechanistic approach of luciferase induction in one experiment.

Embryotoxic potential of persistent organic pollutants extracted from tissues of guillemots (Uria aalge) from the Baltic Sea and the Atlantic Ocean.

The Baltic Sea is a heavily polluted area. To assess the current contaminant pressure on the common guillemot (Uria aalge) living there, whole-body extracts of guillemots from the Baltic Sea were prepared and subdivided over six fractions, which differed in composition due to lipophilicity and polarity of the contaminants. The fractions were tested in the chicken embryo assay and compared to fractions of Atlantic guillemot extracts. Fertilized chicken eggs were injected with 0.03, 0.3, or 3 bird egg equivalents (BEQ) of the contaminants present in the fractions and then incubated for 19 d. Endpoints were selected to cover several mechanisms that may play a role in reproductive failures of fish-eating birds. Fractions I and IV from the Baltic guillemots induced ethoxyresorufin-O-deethylase (EROD) activity up to 15-fold in embryos exposed to 0.3 BEQ and up to 17-fold in embryos exposed to 3 BEQ. Corresponding Atlantic fractions induced EROD activity only at the higher dose of 3 BEQ. Morphological alterations were observed in the bursa of Fabricius in embryos exposed to the fractions that induced EROD, and for the Baltic fractions, this was apparent at the dose of 0.3 BEQ. The higher toxic potency of fractions I and IV was confirmed by higher mortality and occurrence of malformations among embryos exposed to these fractions. No other effects were observed; morphometry, hepatic porphyrin levels, thiamine-dependent enzymes, and acetylcholinesterase activity were not affected by any fraction. During interpretation of the results, concentrations in the whole-body guillemot extracts were compared to concentrations reported in field studies. In general, concentrations in the guillemot extract were lower than those associated with biomarker responses in other wild-bird species. However, because the relative sensitivity of guillemot toward immunotoxic effects remains to be resolved, effects on the immunocompetence of guillemot could not be excluded.

Uptake of injected PCBs from the yolk by the developing chicken embryo.

In this study, PCB uptake by the developing chicken embryo was measured after injection of two different doses of Aroclor 1254 before incubation. It was shown that 2% of the injected PCBs was absorbed on day 13, and this increased exponentially to 18% at day 19. This exponential increase could be described by a similar model for both low and high injection doses. Differences in injection dose resulted in corresponding differences in concentration in the embryos. Lipid corrected concentrations in the embryo were stable through development from day 13 up to day 19 and could be predicted from injection doses by using a conversion factor of 0.15 g(-1).

Effects of furazolidone, PCB77, PCB126, Aroclor 1248, paraquat and p,p'-DDE on transketolase activity in embryonal chicken brain.

The effect of in ovo exposure to PCBs, DDE and paraquat on transketolase activity was measured in 19-day-old chicken embryos. Furazolidone was used as a positive control for decreased activity of the enzyme. The potency of contaminants to interact with transketolase was also tested in an in vitro system, using control brain 7000xg supernatants containing the enzyme. No effects were found on transketolase activity after in ovo or in vitro exposure to PCB126, Aroclor, DDE or paraquat. PCB77 decreased transketolase activity in vitro, but only at concentrations that, extrapolated to in ovo exposure, would be lethal to the embryo. Furazolidone decreased transketolase activity both in ovo and in vitro. For this contaminant, thiamine residues were analysed in the yolk sacs, but no differences were found between exposed and non-exposed eggs. Transketolase is dependent on thiamine pyrophosphate as a cofactor, and therefore, the decreased enzyme activity could be the result of an interaction between furazolidone and thiamine metabolism. Since thiamine residues were not affected by furazolidone and transketolase inhibition in vitro was similar to the inhibition after in ovo exposure, it was concluded that furazolidone interacted with transketolase on the enzymatic level rather than by a depletion of thiamine.

Effects of an extract of oystercatcher (Haematopus ostralegus) eggs from the Zeehavenkanaal in The Netherlands, and of its major contaminant, hexachlorobenzene, on the chicken embryo.

Oystercatchers (Haematopus ostralegus) foraging on the canal 'Zeehavenkanaal' in the Netherlands have been shown to accumulate appreciable amounts of contaminants, especially hexachlorobenzene. The present study was performed to assess the embryotoxic effects of the present contaminants. To this end, a two step approach was followed. In step one, the toxic effects of hexachlorobenzene were studied in the chicken embryo bioassay, using concentrations realistic for the field situation. In step two, yolks of oystercatcher eggs were extracted and the embryotoxic potency of this extract was studied in the same bioassay, using doses of 1, 10 and 100% of the contaminant load in one average egg. The extract contained hexachlorobenzene and PCBs. However, presence of other compounds could not be excluded, since these were not analysed. Hexachlorobenzene induced a nonsignificant decrease in lymphocyte density in the bursa of Fabricius. The egg extract caused a 3.5 fold induction of EROD activity at the highest dose applied, and decreased lymphocyte density in the bursa of Fabricius.