Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae.

The zebrafish (Danio rerio) embryo toxicity test (DarT) is under consideration as an alternative to the acute fish toxicity test. Microscopically visible developmental disorders or death are the endpoints used to report on toxicity in DarT. These endpoints are easily observed. They, however, rarely reveal mechanisms leading to a toxic effect and are relatively insensitive compared to chronic toxic effects. We hypothesized that, by using gene expression profiles as an additional endpoint, it may be possible to increase the sensitivity and predictive value of DarT. Therefore, as a proof of principle, we exposed zebrafish embryos to the reference compound 3,4-dichloroaniline (3,4-DCA) and analyzed gene expression patterns with a 14k oligonucleotide array. Important stress response genes not included in the microarray were additionally quantified by reverse transcriptase polymerase chain reaction. Six genes involved in biotransformation (cyp1a, ahr2), stress response (nfe212, maft, hmox1) and cell cycle control (fzr1) were significantly regulated. With the exception of fzr1, these genes proved to be differentially expressed in post hatch life stages as well. The identified genes point toward an aryl hydrocarbon receptor-mediated response. Differential gene expression in embryos exposed for 48 h was observed at 3,4-DCA concentrations as low as 0.78 microM, which is more than 10-fold below the concentrations that elicited visible toxic effects. Upon exposure for 5 days, differential expression was detected at concentrations as low as 0.22 microM of 3,4-DCA, which was close to the lowest observed effect concentration (0.11 microM) in the 30-day early life stage test. This study therefore indicates that gene expression analysis in DarT is able to reveal mechanistic information and may also be exploited for the development of replacement methods for chronic fish tests.

Stimulated embryo production as a parameter of estrogenic exposure via sediments in the freshwater mudsnail Potamopyrgus antipodarum.

The effects of three suspected endocrine disrupting chemicals, the xeno-estrogens bisphenol A (BPA), 4-tert-octylphenol (OP) and 4-n-nonylphenol (NP), were investigated in a whole-sediment biotest with the freshwater mudsnail Potamopyrgus antipodarum (Gastropoda, Prosobranchia). Artificial sediments were spiked with five nominal concentrations (six for NP), ranging from 1-300 microg/kg dry weight (1-1000 microg/kg for NP). After 2, 4 and 8 weeks of exposure, the responses of the test species were analysed. P. antipodarum exhibited a distinct increase in the number of embryos sheltered in its brood pouch in a time- and concentration-dependent manner in comparison to the solvent control sediment for BPA and OP. The number of "new", still unshelled embryos turned out to be the most sensitive parameter. The lowest observed effect concentration (LOEC) was equivalent to the lowest administered concentration (1 microg/kg for each test compound) for most parameters after 8 weeks of exposure. The calculation of effect concentrations resulted in even lower values for BPA (unshelled embryos after 2 weeks: EC(10) 0.22 microg BPA/kg, EC(50) 24.5 microg BPA/kg; after 4 weeks: EC(10) 0.19 microg BPA/kg, EC(50) 5.67 microg BPA/kg) and OP (unshelled embryos after 4 weeks: EC(10) 4 ng OP/kg, EC(50) 0.07 microg OP/kg). For NP, there was no clear concentration-dependent response, and therefore, no EC(10) or EC(50) could be estimated, but the data suggest an inverted u-shape type of curve. The LOEC in the experiments with NP was 10 microg/kg. Our results indicate that P. antipodarum is highly sensitive to the tested endocrine disruptors at environmentally relevant concentrations. Furthermore, the biotest with P. antipodarum is a useful tool for the identification of sediment-bound pollutants and for the assessment of sediment quality.

Toxicity of triphenyltin and tributyltin to the freshwater mudsnail Potamopyrgus antipodarum in a new sediment biotest.

The effects of two suspected endocrine-disrupting chemicals, the xeno-androgens triphenyltin (TPT) and tributyltin (TBT), were investigated in a new whole-sediment biotest with the freshwater mudsnail Potamopyrgus antipodarum (Gastropoda, Prosobranchia). Artificial sediments were spiked with seven concentrations, ranging from 10 to 500 microg nominal TPT-Sn/kg dry weight and TBT-Sn/kg dry weight, respectively. We analyzed the responses of the test species after two, four, and eight weeks exposure. For both compounds, P. antipodarum exhibited a sharp decline in the number of embryos sheltered in its brood pouch in a time- and concentration-dependent manner in comparison to the control sediment. The number of new, still unshelled embryos turned out to be the most sensitive parameter. The lowest-observed-effect concentration (LOEC) was equivalent to the lowest administered concentration (10 microg/kg of each test compound) for most parameters and thus no no-observed-effect concentration (NOEC) could be established. The calculation of effect concentrations (EC10) resulted in even lower values for both substances (EC10 after eight weeks for unshelled embryos: 0.03 microg TPT-Sn/kg, EC10 after four weeks for unshelled embryos: 0.98 microg TBT-Sn/kg). Our results indicate that P. antipodarum is highly sensitive to both endocrine disruptors TPT and TBT at environmentally relevant concentrations.

Endocrine disruption in prosobranch molluscs: evidence and ecological relevance.

Prosobranch snails represent almost 50% of all recent molluscs, are ubiquitously distributed, play important roles in various ecosystems and exhibit a variety of reproductive modes and life-cycle-strategies. Many of them attain life spans of several years, which in combination with their limited ability to metabolize organic chemicals, may contribute to the fact that prosobranchs constitute one of the most endangered taxonomic groups in aquatic ecosystems. Although it is not yet known to what extent endocrine disrupting chemicals (EDCs) contribute to this situation, the case of tributyltin (TBT) and its population-level impact on prosobranchs demonstrates the general susceptibility of these invertebrates. The existing evidence for comparable population-level effects in prosobranch snails by other androgens, antiandrogens, and estrogens is critically reviewed. The example of TBT demonstrates the difficulty to prove an endocrine mode of action for a given chemical. Although it is generally accepted that TBT causes imposex and intersex in prosobranch snails as a result of endocrine disruption, the detailed biochemical mechanism is still a matter of debate. The strengths and weaknesses of the five competing hypotheses are discussed, together with previously unpublished data. Finally, the ecological relevance of EDC effects on the population and community level and the application of prosobranchs for the assessment of EDCs are addressed.