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Retinoids are newly detected compounds in aquatic ecosystems associated with cyanobacterial water blooms. Their potential health risks are only scarcely described despite numerous detections of all-trans retinoic acid (ATRA) and its derivatives in the environment. Besides the known teratogen ATRA there is only little or no information about their potency and namely their effects in vivo. We characterize ATRA and 8 other retinoids reported to occur in the environment for their bioactivity and teratogenicity using four in vitro reporter gene assays and zebrafish (Danio rerio) embryotoxicity assay. Our results document the ability of these compounds to interfere with retinoid signalling and cause teratogenicity at environmentally relevant levels with EC

Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa ® EC and GAT Cenit 36 CS, both containing 360 g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC 50 values for the technical ingredient, Rampa ® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5 mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa ® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC 50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120 hpf) was 12.1, 10.1 and 24.1 mg/l for technical clomazone, Rampa ® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC 50 /EC 50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa ® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment.

The freshwater mudsnail (Potamopyrgus antipodarum) is sensitive to toxicity of both sediment and water and also to the endocrine disrupting compounds (EDC) at environmentally relevant concentrations. This study determined effects of in situ exposure of P. antipodarum as a part of a complex assessment of the impact of a city metropolitan area with large waste water treatment plant (WWTP) for 0.5 million population equivalents on two urban rivers. The study combined the in situ biotest with detailed chemical analyses and a battery of in vitro bioassays of both sediment and water. Passive sampling of river water was conducted during the course of exposure of the mudsnail. P. antipodarum was exposed for 8 weeks in cages permeable to sediment and water at localities up- and down-stream of the city of Brno, Czech Republic and downstream of the WWTP in two rivers. Greater mortality and significantly decreased embryo production of P. antipodarum were observed immediately downstream of the city of Brno. P. antipodarum exposed at locations downstream of the metropolitan area and WWTP exhibited greater mortality, while numbers of embryos produced by surviving individuals were comparable or slightly greater than for individuals held at the least polluted location. Comparisons with results of chemical analysis and in vitro assays indicate occurrence of groups of compounds contributing to observed effects. Differences in mortalities of mudsnails among sites corresponded well with in vitro cytotoxicity and concentrations of metals. The results of this study confirm the applicability of this novel field biotest with P. antipodarum for the evaluation of the effects of river pollution on metazoans, especially as suitable in situ part of integrative contamination assessment.

This work evaluated the effects of various cyanobacterial fractions in Frog Embryo Teratogenesis Assay Xenopus (FETAX) with African clawed frog embryos. Fractions were prepared from five biomasses with different dominant genera (Microcystis, Aphanizomenon, Anabaena, Planktothrix) and different microcystin content. Effects of following fractions were investigated: (I) homogenate of complex cyanobacterial biomass, (II) cell debris (pellet) after centrifugation of complex biomass, (III) supernatant after centrifugation of complex biomass (= crude aqueous extract), (IV) permeate after passing of crude extract through C-18 column (fraction devoid of microcystins), and (V) eluate from C-18 column (containing microcystins, if present). Besides classical parameters evaluated in 96 h FETAX (mortality, growth inhibition, malformations), we have also assessed the effects on biochemical markers of oxidative stress and detoxification (glutathione pool, GSH; activity of glutathione peroxidase, GPx; glutathione reductase, GR; activity of glutathione-S-transferase, GST). Complex biomass (I) and aqueous extract (III) were generally the most toxic fractions in terms of mortality and growth inhibition, whereas eluates containing microcystins (V) were generally less toxic. On the other hand, the same fraction (eluates) induced significant malformations in low concentrations but the effects were not related to the content of microcystins. Biomarkers were affected in variable manner but no significant effect or clear relation to microcystin content was observed. Our data support the hypothesis that microcystins are not the only or major toxic compounds in the complex cyanobacterial samples (at least for some species) and that more attention should be paid to other components of complex cyanobacterial biomass including non-specific parameters such as oxygen content or toxic ammonia released during bacterial decay of organic material.