Effect-directed analysis of Ah receptor-mediated activities caused by PAHs in suspended particulate matter sampled in flood events.

Suspended particulate matter (SPM) sampled during a flood event in the year 2004 at the rivers Neckar and Rhine (Southwest Germany) was assessed for aryl hydrocarbon receptor (AhR)-mediated activities using EROD induction in the rainbow trout liver cell line RTL-W1. All EROD inductions were normalized to the positive control TCDD and given as bio-TEQ values. Since all samples indicated elevated AhR-mediated toxicities, an effect-directed analysis (EDA) was applied to identify substances causing the effects. In three primary fractions (F1 to F3) non-polar aliphatics, non-polar aromatic substances and more polar substances were separated. Fraction F2, co-eluting with non-polar polyaromatic substances (PACs) including polycyclic aromatic hydrocarbons (PAHs) gave highest AhR-agonistic effects and, thus, were sub-fractionated into seven secondary fractions (F2-1 to F2-7). Fraction F2-1, co-eluting with PCBs and PCDD/Fs, did not cause AhR-agonist activities. F2-2 to F2-4 containing PACs of less than 16 aromatic C-atoms produced minor activities. Highest inductions were detected with fraction F2-5 to F2-7, containing substances of more than 16 aromatic C-atoms (bio-TEQs up to approximately 4500 pg/g). Concentrations and relative potencies (REPs) of priority EPA-PAHs allowed the calculation of chemical toxicity equivalent concentrations (chem-TEQ values). Based on the chem-TEQs, EPA-PAHs explained between 5 and 58% of crude extract bio-TEQs from both rivers. Whereas fractions F2-1 to F2-4 indicated no biological activities, EPA-PAHs in fraction F2-5 to F2-7 accounted for 2 to 137% of AhR-related activities.

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH - Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.

Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test?

The fish acute toxicity test is a mandatory component in the base set of data requirements for ecotoxicity testing. The fish acute toxicity test is not compatible with most current animal welfare legislation because mortality is the primary endpoint and it is often hypothesized that fish suffer distress and perhaps pain. Animal alternative considerations have also been incorporated into new European REACH regulations through strong advocacy for the reduction of testing with live animals. One of the most promising alternative approaches to classical acute fish toxicity testing with live fish is the fish embryo toxicity (FET) test. The FET has been a mandatory component in routine whole effluent testing in Germany since 2005 and has already been standardized at the international level. In order to analyze the applicability of the FET also in chemical testing, a comparative re-evaluation of both fish and fish embryo toxicity data was carried out for a total of 143 substances, and statistical approaches were developed to evaluate the correlation between fish and fish embryo toxicity data. Results confirm that fish embryo tests are neither better nor worse than acute fish toxicity tests and provide strong scientific support for the FET as a surrogate for the acute fish toxicity test.

How estrogenic is nonylphenol? A transgenerational study using rainbow trout (Oncorhynchus mykiss) as a test organism.

The aim of the present study was to evaluate both estrogenic effects in directly NP-exposed sexually mature rainbow trout and possible transgenerational effects in the offspring of exposed fish. Four months prior to spawning, adult rainbow trout of both sexes were exposed intermittently to NP concentrations of 1 and 10 microg/l. At the end of the exposure period, which coincided with the beginning of spawning time, vitellogenin levels in the plasma of adult male rainbow showed a significant increase compared to the control group. After exposure to 10 microg NP/l reproduction was impaired as indicated by significantly reduced hatching rates. Histological examination of the testicular tissue of NP-exposed individuals revealed no morphological differences from the controls. In the offspring, vitellogenin levels of male individuals were not affected, whereas in females they were significantly higher than in the control progeny. The histological examination revealed no alteration in sex ratios. In single cases, intersex occurred in both male and female offspring of exposed fish. The analysis of sex steroid levels revealed a two-fold increase of estradiol in the plasma of male offspring and a 13-fold elevation of testosterone in the plasma of female progeny. The present findings indicate that NP, in an environmentally relevant concentration range, acts as a weak estrogen in directly exposed adult male rainbow trout as indicated by elevated plasma vitellogenin levels. Reproduction success was reduced as indicated by decreased hatching rates. Hormonal imbalances detected in the offspring of exposed fish indicate a transgenerational effect mediated by the endocrine system.