LogD-based modelling and ΔlogD as a proxy for pH-dependent action of ionizable chemicals reveal the relevance of both neutral and ionic species for fish embryotoxicity and possess great potential for practical application in the regulation of chemicals.

Depending on the ambient pH, ionizable substances are present in varying proportions in their neutral or charged form. The extent to which these two chemical species contribute to the pH-dependant toxicity of ionizable chemicals and whether intracellular ion trapping has a decisive influence in this context is controversially discussed. Against this background, we determined the acute toxicity of 24 ionizable substances at up to 4 different pH values on the embryonic development of the zebrafish, Danio rerio, and supplemented this dataset with additional data from the literature. The LC50 for some substances (diclofenac, propranolol, fluoxetine) differed by a factor of even >103 between pH5 and pH9. To simulate the toxicity of 12 acids and 12 bases, six models to calculate a pH-dependant logD value as a proxy for the uptake of potentially toxic molecules were created based on different premises for the trans-membrane passage and toxic action of neutral and ionic species, and their abilities to explain the real LC50 data set were assessed. Using this approach, we were able to show that both neutral and charged species are almost certainly taken up into cells according to their logD-based distribution, and that both species exert toxicity. Since two of the models that assume all intracellular molecules to be neutral overestimated the real toxicity, it must be concluded, that the toxic effect of a single charged intracellularly present molecule is, on the average, lower than that of a single neutral molecule. Furthermore, it was possible to attribute differences in toxicity at different pH values for these 24 ionizable substances to the respective deltas in logD at these pH levels with high accuracy, enabling particularly a full logD-based model on the basis of logPow as a membrane passage descriptor to be used for predicting potential toxicities in worst-case scenarios from existing experimental studies, as stipulated in the process of registration of chemicals and the definition of Environmental Quality Standards (EQS).

Heart rate as an early warning parameter and proxy for subsequent mortality in Danio rerio embryos exposed to ionisable substances.

Environmental risk assessments of organic chemicals usually do not consider pH as a key factor. Hence, most substances are tested at a single pH only, which may underestimate the toxicity of ionisable substances with a pKa in the range of 4-10. Thus, the ability to consider the pH-dependent toxicity would be crucial for a more realistic assessment. Moreover, there is a tendency in acute toxicity tests to focus on mortality only, while little attention is paid to sublethal endpoints. We used Danio rerio embryos exposed to ten ionisable substances (the acids diclofenac, ibuprofen, naproxen and triclosan and the bases citalopram, fluoxetine, metoprolol, propranolol, tramadol and tetracaine) at four external pH levels, investigating the endpoints mortality (LC50) and heart rate (EC20). Dose-response curves were fitted with an ensemble-model to determine the true uncertainty and variation around the mean endpoints. The ensemble considers eight (heart rate) or twelve (mortality) individual models for binominal and Poisson distributed data, respectively, selected based on the Akaike Information Criterion (AIC). In case of equally good models, the mean endpoint of all models in the ensemble was calculated, resulting in more robust ECx estimates with lower 'standard errors' as compared to randomly selected individual models. We detected a high correlation between mortality (LC50) at 96 hpf and reduced heart rate (EC20) at 48 hpf for all compounds and all external pH levels (r = 0.98). Moreover, the observed pH-dependent effects were strongly associated with log D and thus, likely driven by differences in uptake (toxicokinetic) rather than internal (toxicodynamic) processes. Prospectively, the a priori consideration of pH-dependent effects of ionisable substances might make testing at different pH levels redundant, while the endpoint of mortality might even be replaced by a reliable sublethal proxy that would reduce the exposure, accelerating the evaluation process.

Snails in the sun: Strategies of terrestrial gastropods to cope with hot and dry conditions.

Terrestrial gastropods do not only inhabit humid and cool environments but also habitat in which hot and dry conditions prevail. Snail species that are able to cope with such climatic conditions are thus expected to having developed multifaceted strategies and mechanisms to ensure their survival and reproduction under heat and desiccation stress. This review paper aims to provide an integrative overview of the numerous adaptation strategies terrestrial snails have evolved to persist in hot and dry environments as well as their mutual interconnections and feedbacks, but also to outline research gaps and questions that remained unanswered. We extracted relevant information from more than 140 publications in order to show how biochemical, cellular, physiological, morphological, ecological, thermodynamic, and evolutionary parameters contribute to provide an overall picture of this classical example in stress ecology. These mechanisms range from behavioral and metabolic adaptations, including estivation, to the induction of chaperones and antioxidant enzymes, mucocyte and digestive gland cell responses and the modification and frequency of morphological features, particularly shell pigmentation. In this context, thermodynamic constraints call for processes of complex adaptation at varying levels of biological organization that are mutually interwoven. We were able to assemble extensive, mostly narrowly focused information from the literature into a web of network parameters, showing that future work on this subject requires multicausal thinking to account for the complexity of relationships involved in snails' adaptation to insolation, heat, and drought.

Does Bti (Bacillus thuringiensis var. israelensis) affect Rana temporaria tadpoles?

Biocides based on toxins of Bacillus thuringiensis var. israelensis (Bti) are established as alternatives to conventional chemical insecticides for mosquito control all across the globe since they are regarded ecologically compatible and harmless to non-target species. Since recent studies on amphibian larvae have called this opinion into question, we exposed Rana temporaria tadpoles to single (1 mg/L), tenfold (10 mg/L) and hundredfold (100 mg/L) field concentrations of VectoBac® WG (a water dispersible granule Bti formulation) in the laboratory for eleven days to investigate whether larvae were adversely affected by Bti and its endotoxin proteins. In addition to a negative (water) control, a positive control based on organic rice protein (50 mg/L) was run to check for the nutritional relevance of Bti proteins. There was no Bti-related mortality and a histopathological analysis of tadpole intestines revealed no adverse effects. Analyses of biomarkers for proteotoxicity (stress protein family, Hsp70) and neurotoxicity or metabolic action (b-esterases acetylcholine esterase (AChE) and carboxylesterases) revealed no significant differences between Bti treatments and the negative control. The responses of tadpoles in the protein-supplemented positive control differed from those of the negative control and the Bti treatments. Tadpoles in the positive control had reduced body mass and elevated AChE activity.

How glyphosate and its associated acidity affect early development in zebrafish (Danio rerio).

BACKGROUND: Glyphosate is among the most extensively used pesticides worldwide. Following the ongoing highly controversial debate on this compound, its potential impact on non-target organisms is a fundamental scientific issue. In its pure compound form, glyphosate is known for its acidic properties. METHODS: We exposed zebrafish (Danio rerio) embryos to concentrations between 10 µM and 10 mM glyphosate in an unbuffered aqueous medium, as well as at pH 7, for 96 hours post fertilization (hpf). Furthermore, we investigated the effects of aqueous media in the range of pH 3 to 8, in comparison with 1 mM glyphosate treatment at the respective pH levels. Additionally, we exposed zebrafish to 7-deoxy-sedoheptulose (7dSh), another substance that interferes with the shikimate pathway by a mechanism analogous to that of glyphosate, at a concentration of one mM. The observed endpoints included mortality, the hatching rate, developmental delays at 24 hpf, the heart rate at 48 hpf and the malformation rate at 96 hpf. LC10/50, EC10 and, if reasonable, EC50 values were determined for unbuffered glyphosate. RESULTS: The results revealed high mortalities in all treatments associated with low pH, including high concentrations of unbuffered glyphosate (>500 µM), low pH controls and glyphosate treatments with pH < 3.4. Sublethal endpoints like developmental delays and malformations occurred mainly at higher concentrations of unbuffered glyphosate. In contrast, effects on the hatching rate became particularly prominent in treatments at pH 7, showing that glyphosate significantly accelerates hatching compared with the control and 7dSh, even at the lowest tested concentration. Glyphosate also affected the heart rate, resulting in alterations both at pH 7 and, even more pronounced, in the unbuffered system. In higher concentrations, glyphosate tended to accelerate the heart rate in zebrafish embryos, again, when not masked by the decelerating influence of its low pH. At pH > 4, no mortality occurred, neither in the control nor in glyphosate treatments. At 1 mM, 7dSh did not induce any mortality, developmental delays or malformations; only slightly accelerated hatching and a decelerated heart rate were observed. Our results demonstrate that lethal impacts in zebrafish embryos can be attributed mainly to low pH, but we could also show a pH-independent effect of glyphosate on the development of zebrafish embryos on a sublethal level.

The importance of sediments in ecological quality assessment of stream headwaters: embryotoxicity along the Nidda River and its tributaries in Central Hesse, Germany.

BACKGROUND: Although the crucial importance of sediments in aquatic systems is well-known, sediments are often neglected as a factor in the evaluation of water quality assessment. To support and extend previous work in that field, this study was conducted to assess the impact of surface water and sediment on fish embryos in the case of a highly anthropogenically influenced river catchment in Central Hesse, Germany. RESULTS: The results of 96 h post fertilisation fish embryo toxicity test with Danio rerio (according to OECD Guideline 236) revealed that river samples comprising both water and sediment exert pivotal effects in embryos, whereas surface water alone did not. The most prominent reactions were developmental delays and, to some extent, malformations of embryos. Developmental delays occurred at rates up to 100% in single runs. Malformation rates ranged mainly below 10% and never exceeded 25%. CONCLUSION: A clear relationship between anthropogenic point sources and detected effects could not be established. However, the study illustrates the critical condition of the entire river system with respect to embryotoxic potentials present even at the most upstream test sites. In addition, the study stresses the necessity to take into account sediments for the evaluation of ecosystem health in industrialised areas.

Drifting Away of a FET Endpoint: The Heart Rate in Danio rerio Embryos is Extremely Sensitive to Variation in Ambient Temperature.

Beyond the mere detection of presence or absence of heart beat in zebrafish (Danio rerio) embryos in a fish embryo test conducted referring to the OECD TG 236 at 48 hpf (hours post fertilization) onwards, embryo heart rate may serve as an additional and very sensitive endpoint in ecotoxicological studies. But by including heart rate as a sublethal endpoint, care has to be taken of separating effects exerted by a tested compound from those exerted by temperature. Therefore, profound knowledge on the natural variation of zebrafish heart rates at defined temperatures as a basis for the assessment of gained results is mandatorily needed. As such continuous information in D. rerio is lacking from the literature, we designed a study covering a span of 12°C (from 18 to 30°C in steps of 2°C) to quantify the relationship between heart rate and temperature in D. rerio embryos 48 hpf. Conducting a multiple regression analysis, we found a considerably strong relationship between treatment temperature and the log10 of the heart rate, ranging from 82.8 beats per minute at 18°C to 218.0 beats per minute at 30°C. Our results therefore may serve as a reference for heart rates measured under normal conditions to be able to detect potential effects of contaminants in other studies when working under certain temperatures.

Environmental selection is a main driver of divergence in house sparrows (Passer domesticus) in Romania and Bulgaria.

Both neutral and adaptive evolutionary processes can cause population divergence, but their relative contributions remain unclear. We investigated the roles of these processes in population divergence in house sparrows (Passer domesticus) from Romania and Bulgaria, regions characterized by high landscape heterogeneity compared to Western Europe. We asked whether morphological divergence, complemented with genetic data in this human commensal species, was best explained by environmental variation, geographic distance, or landscape resistance-the effort it takes for an individual to disperse from one location to the other-caused by either natural or anthropogenic barriers. Using generalized dissimilarity modeling, a matrix regression technique that fits biotic beta diversity to both environmental predictors and geographic distance, we found that a small set of climate and vegetation variables explained up to ~30% of the observed divergence, whereas geographic and resistance distances played much lesser roles. Our results are consistent with signals of selection on morphological traits and of isolation by adaptation in genetic markers, suggesting that selection by natural environmental conditions shapes population divergence in house sparrows. Our study thus contributes to a growing body of evidence that adaptive evolution may be a major driver of diversification.