Occurrence of common frog (Rana temporaria) and common toad (Bufo bufo) adults and metamorphs in agricultural fields in Germany: Potential for exposure to plant protection products.

To characterize the potential for exposure of amphibian terrestrial life stages to plant protection products (PPP), we studied the occurrence and habitat use of adult and metamorph common frogs (Rana temporaria) and common toads (Bufo bufo) in an agricultural landscape in Germany. The four selected study sites were breeding ponds with approximately 80% agricultural land within a 1-km radius. Adults were monitored by radio tracking for two years, and metamorph numbers were assessed for one summer using pitfall traps alongside drift fences. The results demonstrate that adults were rarely present in arable fields at any of the sites (overall 0.5% and 4% of total observations for frogs and toads, respectively). Metamorph captures in arable fields were more variable, ranging from 1.2% to 38.8% (frogs) and from 0.0% to 26.1% (toads) across study sites. Unsurprisingly, most captures in arable fields for both toad and frog metamorphs occurred at the site where the pond was completely surrounded by arable fields. Overall, the presence of adult amphibians in arable fields was limited and, for the metamorphs, occurred primarily when crops were denser and PPP spray interception higher. Diurnal hiding behavior was observed with the highest activity recorded at night, further reducing the risk of dermal exposure from direct PPP overspraying. In addition, it appeared that alternative habitats, such as woody structures or water bodies in the broader surrounding area, were preferred by the animals over the arable areas. The use of buffer zones around water bodies in agricultural areas would be an effective risk mitigation measure to protect terrestrial adults and metamorphs residing there and would reduce spray drift entry into water bodies during PPP application. It is hoped that these results will contribute to the discussion of risk assessment and mitigation options for amphibians. Integr Environ Assess Manag 2024;00:1-13. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Analysis and management of herbicidal mixtures in a high-intensity agricultural landscape in Belgium.

Water bodies located in anthropogenically influenced environments, such as agricultural landscapes, may be exposed to different chemicals simultaneously or sequentially. Yet, current environmental risk assessments focus on single active substances for unintended mixtures. For 3.5 years, the present study monitored the mixture of herbicides, within an intensively managed agricultural catchment, accompanied by a stewardship program. Twelve herbicides and one metabolite were monitored on a daily to sub-daily basis, generating a unique, high temporal resolution data set, enabling an assessment of cumulative exposure in a worst-case scenario. Analyses focused on the number of events at which the herbicide mixture concentration exceeded the regulatory accepted concentration for algae and macrophytes, based on concentration addition, and the potential factors influencing the frequency of these events are considered. A low number of individual herbicides drove the toxicity and only two of these overlapped for the two organism groups, algae and macrophytes. The observed exceedance events coincided with seasonal influences, and low rainfall during the 2011 season correlated with a highly reduced number of these events. The major influence was found to be the implementation of the stewardship program, which directed farmers to use more advanced farming techniques, avoid spillages, and other point sources. The number of exceedance events was reduced by more than half for algae (9% of the daily mean samples in 2010 and 4% in 2013) and by approximately 10 times for macrophytes (36% in 2010 to 3% in 2013). This high-resolution monitoring data set illustrates how knowledge of the influencing factors can help reduce unintended exposure to chemicals and achieve real-world improvements. Overall, a single-substance assessment is protective of mixture effects. Where mixture effects do play a role, local measures to manage point sources are more effective than changes to the desk-based environmental risk assessments that focus on diffuse sources. Integr Environ Assess Manag 2023;19:1297-1306. © 2022 Cambridge Environmental Assessments RSK ADAS Ltd and Bayer AG. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Data-driven learning of narcosis mode of action identifies a CNS transcriptional signature shared between whole organism Caenorhabditis elegans and a fish gill cell line.

With the large numbers of man-made chemicals produced and released in the environment, there is a need to provide assessments on their potential effects on environmental safety and human health. Current regulatory frameworks rely on a mix of both hazard and risk-based approaches to make safety decisions, but the large number of chemicals in commerce combined with an increased need to conduct assessments in the absence of animal testing makes this increasingly challenging. This challenge is catalysing the use of more mechanistic knowledge in safety assessment from both in silico and in vitro approaches in the hope that this will increase confidence in being able to identify modes of action (MoA) for the chemicals in question. Here we approach this challenge by testing whether a functional genomics approach in C. elegans and in a fish cell line can identify molecular mechanisms underlying the effects of narcotics, and the effects of more specific acting toxicants. We show that narcosis affects the expression of neuronal genes associated with CNS function in C. elegans and in a fish cell line. Overall, we believe that our study provides an important step in developing mechanistically relevant biomarkers which can be used to screen for hazards, and which prevent the need for repeated animal or cross-species comparisons for each new chemical.

Metabolomics Discovers Early-Response Metabolic Biomarkers that Can Predict Chronic Reproductive Fitness in Individual Daphnia magna.

Chemical risk assessment remains entrenched in chronic toxicity tests that set safety thresholds based on animal pathology or fitness. Chronic tests are resource expensive and lack mechanistic insight. Discovering a chemical's mode-of-action can in principle provide predictive molecular biomarkers for a toxicity endpoint. Furthermore, since molecular perturbations precede pathology, early-response molecular biomarkers may enable shorter, more resource efficient testing that can predict chronic animal fitness. This study applied untargeted metabolomics to attempt to discover early-response metabolic biomarkers that can predict reproductive fitness of Daphnia magna, an internationally-recognized test species. First, we measured the reproductive toxicities of cadmium, 2,4-dinitrophenol and propranolol to individual Daphnia in 21-day OECD toxicity tests, then measured the metabolic profiles of these animals using mass spectrometry. Multivariate regression successfully discovered putative metabolic biomarkers that strongly predict reproductive impairment by each chemical, and for all chemicals combined. The non-chemical-specific metabolic biomarkers were then applied to metabolite data from Daphnia 24-h acute toxicity tests and correctly predicted that significant decreases in reproductive fitness would occur if these animals were exposed to cadmium, 2,4-dinitrophenol or propranolol for 21 days. While the applicability of these findings is limited to three chemicals, they provide proof-of-principle that early-response metabolic biomarkers of chronic animal fitness can be discovered for regulatory toxicity testing.

Defining the Baseline and Oxidant Perturbed Lipidomic Profiles of Daphnia magna.

Recent technological advancement has enabled the emergence of lipidomics as an important tool for assessing molecular stress, one which has yet to be assessed fully as an approach in an environmental toxicological context. Here we have applied a high-resolution, non-targeted, nanoelectrospray ionisation (nESI) direct infusion mass spectrometry (DIMS) technique to assess the effects of oxidative stress to Daphnia magna both in vitro (air exposure of daphniid extracts) and in vivo (Cu2+ exposure). Multivariate and univariate statistical analyses were used to distinguish any perturbations including oxidation to the D. magna baseline lipidome. This approach enabled the putative annotation of the baseline lipidome of D. magna with 65% of the lipid species discovered previously not reported. In vitro exposure of lipid extracts to air, primarily to test the methodology, revealed a significant perturbation to this baseline lipidome with detectable oxidation of peaks, in most cases attributed to single oxygen addition. Exposure of D. magna to Cu2+ in vivo also caused a significant perturbation to the lipidome at an environmentally relevant concentration of 20 µg/L. This nESI DIMS approach has successfully identified perturbations and oxidative modifications to the D. magna lipidome in a high-throughput manner, highlighting its suitability for environmental lipidomic studies.

Predicting chronic copper and nickel reproductive toxicity to Daphnia pulex-pulicaria from whole-animal metabolic profiles.

The emergence of omics approaches in environmental research has enhanced our understanding of the mechanisms underlying toxicity; however, extrapolation from molecular effects to whole-organism and population level outcomes remains a considerable challenge. Using environmentally relevant, sublethal, concentrations of two metals (Cu and Ni), both singly and in binary mixtures, we integrated data from traditional chronic, partial life-cycle toxicity testing and metabolomics to generate a statistical model that was predictive of reproductive impairment in a Daphnia pulex-pulicaria hybrid that was isolated from an historically metal-stressed lake. Furthermore, we determined that the metabolic profiles of organisms exposed in a separate acute assay were also predictive of impaired reproduction following metal exposure. Thus we were able to directly associate molecular profiles to a key population response - reproduction, a key step towards improving environmental risk assessment and management.

Molecular toxicity of cerium oxide nanoparticles to the freshwater alga Chlamydomonas reinhardtii is associated with supra-environmental exposure concentrations.

Ceria nanoparticles (NPs) are widely used as fuel catalysts and consequently are likely to enter the environment. Their potential impacts on. biota at environmentally relevant concentrations, including uptake and toxicity, remain to be elucidated and quantitative data on which to assess risk are sparse. Therefore, a definitive assessment of the molecular and phenotypic effects of ceria NPs was undertaken, using well-characterised mono-dispersed NPs as their toxicity is likely to be higher, enabling a conservative hazard assessment. Unbiased transcriptomics and metabolomics approaches were used to investigate the potential toxicity of tightly constrained 4-5 nm ceria NPs to the unicellular green alga, Chlamydomonas reinhardtii, a sentinel freshwater species. A wide range of exposure concentrations were investigated from predicted environmental levels, to support hazard assessment, to supra-environmental levels to provide insight into molecular toxicity pathways. Ceria NPs were internalised into intracellular vesicles within C. reinhardtii, yet caused no significant effect on algal growth at any exposure concentration. Molecular perturbations were only detected at supra-environmental ceria NP-concentrations, primarily down-regulation of photosynthesis and carbon fixation with associated effects on energy metabolism. For acute exposures to small mono-dispersed particles, it can be concluded there should be little concern regarding their dispersal into the environment for this trophic level.

Metabolomics confirms that dissolved organic carbon mitigates copper toxicity.

Reductions in atmospheric emissions from the metal smelters in Sudbury, Canada, produced major improvements in acid and metal contamination of local lakes and indirectly increased dissolved organic carbon (DOC) concentrations. Metal toxicity, however, has remained a persistent problem for aquatic biota. Integrating high-throughput, nontargeted mass spectrometry metabolomics with conventional toxicological measures elucidated the mediating effects of dissolved organic matter (DOM) on the toxicity of Cu to Daphnia pulex-pulicaria, a hybrid isolated from these soft water lakes. Two generations of daphniids were exposed to Cu (0-20 µg/L) at increasing levels of natural DOM (0-4 mg DOC/L). Added DOM reduced Cu toxicity monotonically with median lethal concentration values increasing from 2.3 µg/L Cu without DOM to 22.7 µg/L Cu at 4 mg DOC/L. Reproductive output similarly benefited, increasing with DOM, yet falling with increases in Cu. Second generation reproduction was more impaired than the first generation. Dissolved organic matter had a greater influence than Cu on the metabolic status of the daphniids. Putative identification of metabolite peaks indicated that DOM elevation increased the metabolic energy status of the first generation animals, but this benefit was reduced in the second generation, although evidence of increased oxidative stress was detected. These results indicate that Sudbury's terrestrial ecosystems should be managed to increase aquatic DOM supply to enable daphniid colonists to both survive and foster stable populations.

Metabolomics of microliter hemolymph samples enables an improved understanding of the combined metabolic and transcriptional responses of Daphnia magna to cadmium.

Omic technologies offer unprecedented opportunities to better understand mode(s)-of-toxicity and downstream secondary effects by providing a holistic view of the molecular changes underlying physiological disruption. Crustacean hemolymph represents a largely untapped biochemical resource for such toxicity studies. We sought to characterize changes in the hemolymph metabolome and whole-body transcriptome to reveal early processes leading to chronic toxicity in the indicator species, Daphnia magna, after 24-h sublethal cadmium exposure (18 µg/L, corresponding to 1/10 LC(50)). We first confirmed that metabolites can be detected and identified in small volumes (∼3-6 µL) of D. magna hemolymph using Fourier transform ion cyclotron resonance mass spectrometry and NMR spectroscopy. Subsequently, mass spectrometry based metabolomics of hemolymph identified disruption to two major classes of metabolites: amino acids and fatty acids. These findings were compared to differentially expressed genes identified by a D. magna 44k oligonucleotide microarray, which included decreased levels of digestive enzymes and increased expression of cuticle proteins and oxidative stress response genes. The combination of metabolic and transcriptional changes revealed through KEGG pathway analysis and gene ontology, respectively, enabled a more complete understanding of how cadmium disrupts nutrient uptake and metabolism, ultimately resulting in decreased energy reserves and chronic toxicity.

Discriminating between different acute chemical toxicities via changes in the daphnid metabolome.

Currently, there is widespread interest in exploiting "omics" approaches to screen the toxicity of chemicals, potentially enabling their rapid categorization into classes of defined mode of action (MOA). Direct infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) metabolomics provides a sensitive and nontargeted analysis of potentially a thousand endogenous metabolites. Our previous work has shown that mass spectra can be recorded from whole-organism homogenate or hemolymph of single adult Daphnia magna. Here we develop multivariate models and discover perturbations to specific metabolic pathways that can discriminate between the acute toxicities of four chemicals to D. magna using FT-ICR MS metabolomics. We focus on model toxicants (cadmium, fenvalerate, dinitrophenol, and propranolol) with different MOAs. First, we confirmed that a toxicant-induced metabolic effect could be determined for each chemical in both the hemolymph and the whole-organism metabolome, with between 9 and 660 mass spectral peaks changing intensities significantly, dependent upon toxicant and sample type. Subsequently, supervised multivariate models were built that discriminated significantly all four acute metabolic toxicities, yielding mean classification error rates (across all classes) of 3.9 and 6.9% for whole-organism homogenates and hemolymph, respectively. Following extensive peak annotation, we discovered toxicant-specific perturbations to putatively identified metabolic pathways, including propranolol-induced disruption of fatty acid metabolism and eicosanoid biosynthesis and fenvalerate-induced disruption of amino sugar metabolism. We conclude that the metabolic profiles of whole-daphnid homogenates are more discriminatory for toxicant action than hemolymph. Furthermore, our findings highlight the capability of metabolomics to discover early-event metabolic responses that can discriminate between the acute toxicities of chemicals.