BackgroundShort-chain PFASs (per- and polyfluoroalkyl substances) are widely used as alternatives to long-chain PFASs. Long-chain PFASs become gradually regulated under REACH (EC No. 1907/2006) and other international regulations, due to having persistent, bioaccumulative and toxic properties and/or being toxic for reproduction. The increasingly used short-chain PFASs are assumed to have a lower bioaccumulation potential. Nonetheless, they have other properties of concern and are already widely distributed in the environment, also in remote regions. The REACH Regulation does not directly address these emerging properties of concern, complicating the implementation of regulatory measures. Therefore, this study illustrates these environmental concerns and provides a strategy for a regulation of short-chain PFASs within REACH.ResultsShort-chain PFASs have a high mobility in soil and water, and final degradation products are extremely persistent. This results in a fast distribution to water resources, and consequently, also to a contamination of drinking water resources. Once emitted, short-chain PFASs remain in the environment. A lack of appropriate water treatment technologies results in everlasting background concentrations in the environment, and thus, organisms are permanently and poorly reversibly exposed. Considering such permanent exposure, it is very difficult to estimate long-term adverse effects in organisms. Short-chain PFASs enrich in edible parts of plants and the accumulation in food chains is unknown. Regarding these concerns and uncertainties, especially with respect to the precautionary principle, short-chain PFASs are of equivalent concern to PBT substances. Therefore, they should be identified as substances of very high concern (SVHC) under REACH. The SVHC identification should be followed by a restriction under REACH, which is the most efficient way to minimize the environmental and human exposure of short-chain PFASs in the European Union.ConclusionDue to an increasing use of short-chain PFASs, an effective regulation is urgently needed. The concerns of short-chain PFASs do not match the “classical” concerns as defined under REACH, but are not of minor concern. Therefore, it is of advantage to clearly define the concerns of short-chain PFASs. This might facilitate the following restriction process under REACH.

Responsive genes for fish embryos have been identified so far for some endocrine pathways but not for androgens. Using transcriptome analysis and multiple concentration-response modeling, we identified putative androgen-responsive genes in zebrafish embryos exposed to 0.05-5000 nM 11-ketotestosterone for 24 h. Four selected genes with sigmoidal concentration-dependent expression profiles (EC50 = 6.5-30.0 nM) were characterized in detail. The expression of cyp2k22 and slco1f4 was demonstrated in the pronephros; lipca was detected in the liver, and sult2st3 was found in the olfactory organs and choroid plexus. Their expression domains, the function of human orthologs, and a pathway analysis suggested a role of these genes in the metabolism of hormones. Hence, it was hypothesized that they were induced to compensate for elevated hormone levels. The induction of sult2st3 and cyp2k22 by 11-ketotestosterone was repressed by co-exposure to the androgen receptor antagonist nilutamide supporting a potential androgen receptor mediated regulation. Sensitivity (expressed as EC50 values) of sult2st3 and cyp2k22 gene expression induction after exposure to other steroidal hormones (11-ketotestosterone ∼ testosterone > progesterone > cortisol > ethinylestradiol) correlated with their known binding affinities to zebrafish androgen receptor. Hence, these genes might represent potential markers for screening of androgenic compounds in the zebrafish embryo.

Xenoestrogens may persist in the environment by binding to sediments or suspended particulate matter serving as long-term reservoir and source of exposure, particularly for organisms living in or in contact with sediments. In this study, we present for the first time an effect-directed analysis (EDA) for identifying estrogenic compounds in a sediment sample using embryos of a transgenic reporter fish strain. In the tg(cyp19a1b-GFP) transgenic zebrafish strain, the expression of GFP (green fluorescent protein) in the brain is driven by an oestrogen responsive element in the promoter of the cyp19a1b (aromatase) gene. The selected sediment sample of the Czech river Bilina had already been analysed in a previous EDA using the yeast oestrogen screening assay and had revealed fractions containing estrogenic compounds. When normal phase HPLC (high performance liquid chromatography) fractionation was used for the separation of the sediment sample, the biotest with transgenic fish embryos revealed two estrogenic fractions. Chemical analysis of candidate compounds in these sediment fractions suggested alkylphenols and estrone as candidate compounds responsible for the observed estrogenic effect. Alkylphenol concentrations could partially explain the estrogenicity of the fractions. However, xenoestrogens below the analytical detection limit or non-targeted estrogenic compounds have probably also contributed to the sample's estrogenic potency. The results indicated the suitability of the tg(cyp19a1b-GFP) fish embryo for an integrated chemical-biological analysis of estrogenic effects.

Endocrine disrupting chemicals and especially oestrogen receptor (ER) agonists have been extensively studied over the years due to their potential effects on sexual development and reproduction in vertebrates, notably fish. As ER agonists can exist as complex mixtures in the aquatic environment, evaluating the impact of combined exposure on oestrogenic effects has become increasingly important. Use of predictive models such as concentration addition (CA) and independent action (IA) has allowed assessment of combined estrogenic effects of complex multi-compound mixtures of ER agonists in various fish in vitro and in vivo experimental models. The present work makes use of a transgenic zebrafish strain, tg(cyp19a1b-GFP), which expresses the green fluorescent protein (GFP) under the control of the cyp19a1b (brain aromatase or aromatase B) gene to determine the oestrogenic potency of ER agonists alone or in mixtures. In these studies, tg(cyp19a1b-GFP) zebrafish embryos were exposed for four days (from one to five days post fertilization) to five different oestrogenic chemicals; 17α-ethinylestradiol (EE2), 17β-estradiol (E2), estrone (E1), bisphenol A (BPA) and 4-tert-octylphenol (OP), and three mixtures of up to four of these compounds. The mixture of BPA, OP and E2 was also tested with primary cultures of rainbow trout hepatocytes by analysing the ER-mediated induction of the oestrogenic biomarker vitellogenin in order to compare the performance of the two methods for assessing oestrogenic effects of complex mixtures. The three tested mixtures were predominantly acting in an additive manner on the expression of GFP. Additivity was indicated by the overlap of the 95% confidence interval of the concentration response curves for the observed data with the CA and IA prediction models, and model deviation ratios within a factor of two for a majority of the mixture concentrations. However, minor deviations determined as more than additive effects for the mixture of EE2, E1 and E2 and less than additive effects for the mixture of BPA, OP, EE2 and E1 were observed at the higher mixture concentrations tested. The successful prediction of additivity by CA and IA in tg(cyp19a1b-GFP) zebrafish embryos and deviations at high mixture concentrations seemed to correspond well to results obtained in the rainbow trout hepatocyte assay. The present results clearly show the usefulness of combining predictive modelling and use of in vitro bioassays for rapid screening of oestrogenic effects of complex mixtures and environmental samples.

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