Experimental data from the development of Lymnaea stagnalis embryo test for chemicals hazard assessment.

This study aimed to contribute to the development of an embryo-test using the gastropod Lymnaea stagnalis, identified by the Organization for Economic Co-operation and Development (OECD) as a potential invertebrate test animal model. Together with the Potamopyrgus antipodarum, were the first mollusc models to be included in the organization testing guidelines. The focus was on validating an embryo toxicity test to cover the sensitive embryogenesis phase and on obtaining testing information on all of the model life cycle stages, contributing to close an identified gap within this context. Adhering to OECD guidelines, namely the L. stagnalis reproductive test, the study examined mortality rates, abnormality rates, development, growth, hatching rates, hearth rates, and pre-testing media suitability, during the embryogenesis, and the obtained dataset made available for further studies. Cadmium was chosen as the positive test compound due to its well-studied nature and the model's proven sensitivity to the compound, working as a reference compound for the test development. The data were collected in two 12-day assays under consistent conditions, each using 144 L. stagnalis embryos (<24 h old) from 6 egg masses (288 embryos total). Six 48-well microplates were utilized per assay, accommodating five different cadmium concentrations (32, 70, 155, 341, 750 µg/L) and a control group. Recorded parameters encompassed developmental stage, embryo position within the chorion, developmental abnormalities, hatchings, and mortality. Data analysis involved classifying embryos based on developmental stage and position, taking an exploratory approach to define the relevance of the different parameters in the compound hazard assessment during the embryogenesis. Measurements considered embryo area, perimeter, length, height, width, interocular distance, and heart rate. This dataset does not provide treated information but the raw data obtained during the proposed metodological development and toxicity testing process. The purpose of this article is to make the obtained raw data available, clearly defining the acquisition methodology to provide a comparison basis for future or existent works within this context.

Data collection on the use of embryo bioassays with aquatic animals for toxicity testing and hazard assessment of emerging pollutants.

A thorough bibliographic survey on the use of embryo-tests with aquatic animals for toxicity testing was performed. The data regarding to the compounds sensitivity (NOEC, LOEC, EC50 and LC50), the available resources for the different animal models (knowledge on the life-cycle, amenability for laboratory breeding, number of embryos produced and reproductive strategy, genomic and transcriptomic resources), together with the European pieces of legislation regarding to animal testing and the available testing guidelines of national and international agencies (OECD, EPA, ISO, ASTM, ICES) were gathered, aiming to the standardization of new embryo-test model species for toxicity testing of new and existing compounds. The data contained in this Data in Brief article is presented and discussed in the review article with the title Embryo bioassays with aquatic animals for toxicity testing and hazard assessment of emerging pollutants: a review [1]. The dataset is provided with this article as a supplementary file.

Embryo bioassays with aquatic animals for toxicity testing and hazard assessment of emerging pollutants: A review.

This review article gathers the available information on the use of embryo-tests as high-throughput tools for toxicity screening, hazard assessment and prioritization of new and existing chemical compounds. The approach is contextualized considering the new legal trends for animal experimentation, fostering the 3R policy, with reduction of experimental animals, addressing the potential of embryo-tests as high-throughput toxicity screening and prioritizing tools. Further, the current test guidelines, such as the ones provided by OECD and EPA, focus mainly in a limited number of animal lineages, particularly vertebrates and arthropods. To extrapolate hazard assessment to the ecosystem scale, a larger diversity of taxa should be tested. The use of new experimental animal models in toxicity testing, from a representative set of taxa, was thoroughly revised and discussed in this review. Here, we critically review current tools and the main advantages and drawbacks of different animal models and set researcher priorities.

Multi-residue analysis of emerging pollutants in benthic invertebrates by modified micro-quick-easy-cheap-efficient-rugged-safe extraction and nanoliquid chromatography-nanospray-tandem mass spectrometry analysis.

Aquatic ecosystems are continuously contaminated by agricultural and industrial sources. Although the consequences of this pollution are gradually becoming visible, their potential impacts on aquatic ecosystems are poorly known, particularly regarding the risk of bioaccumulation in different trophic levels. To establish a causality relationship between bioaccumulation and disease, experiments on biotic matrices must be performed. In this context, a multi-residue method for the analysis of 35 emerging pollutants in three benthic invertebrates (Potamopyrgus antipodarum, Gammarus fossarum, and Chironomus riparius) has been developed. Because the variation in response of each individual must be taken into account in ecotoxicological studies, the entire analytical chain was miniaturised, thereby reducing the required sample size to a minimum of one individual and scaling the method accordingly. A new extraction strategy based on a modified, optimised and miniaturised "QuEChERS" approach is reported. The procedure involves salting out liquid-liquid extraction of approximately 10-20mg of matrix followed by nano-liquid chromatography-nano electospray ionisation coupled with tandem mass spectrometry. The validated analytical procedure exhibited recoveries between 40 and 98% for all the target compounds and enabled the determination of pollutants on an individual scale in the ng g(-1) concentration. The method was subsequently applied to determine the levels of target analytes in several encaged organisms which were exposed upstream and downstream of an effluent discharge. The results highlighted a bioaccumulation of certain targeted emerging pollutants in three freshwater invertebrates, as well as inter-species differences. 18 out of 35 compounds were detected and eight were quantified. The highest concentrations were measured for ibuprofen in G. fossarum, reaching up to 105 ng g(-1).

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment.

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.

Environmental risk assessment for the serotonin re-uptake inhibitor fluoxetine: Case study using the European risk assessment framework.

The serotonin re-uptake inhibitor fluoxetine was selected for an environmental risk assessment, using the most recent European guideline (EMEA 2006) within the European Union (EU)-funded Environmental Risk Assessment of Pharmaceuticals (ERAPharm) project due to its environmental persistence, acute toxicity to nontarget organisms, and unique pharmacokinetics associated with a readily ionizable compound. As a widely prescribed psychotropic drug, fluoxetine is frequently detected in surface waters adjacent to urban areas because municipal wastewater effluents are the primary route of entry to aquatic environments. In Phase I of the assessment, the initial predicted environmental concentration of fluoxetine in surface water (initial PEC(SW)) reached or exceeded the action limit of 10 ng/L, when using both a default market penetration factor and prescription data for Sweden, Germany, and the United Kingdom. Consequently, a Phase II risk assessment was conducted in which green algae were identified as the most sensitive species with a NOEC of <0.6 microg/L. From this value, a predicted no effect concentration for surface waters (PNEC(SW)) of 0.012 microg/L was derived. The PEC/PNEC ratio was above the trigger value of 1 in worst-case exposure scenarios indicating a potential risk to the aquatic compartment. Similarly, risks of fluoxetine for sediment-dwelling organisms could not be excluded. No risk assessment was conducted for the terrestrial compartment due to a lack of data on effects of fluoxetine on soil organisms. The need for a separate risk assessment for the main metabolite of fluoxetine, norfluoxetine, was not conducted because of a lack of fate and effect studies. Based on published data, fluoxetine and norfluoxetine appeared to have a low to moderate bioaccumulation potential, which should be confirmed in formal studies according to OECD guidelines. Exposure assessments for fluoxetine according to the current framework rely heavily on K(OC) and K(OW) values. This approach is problematic, because fluoxetine is predominantly a cationic substance at environmental pH values. Consequently, the fate of fluoxetine (and other ionic substances) cannot be predicted using partition coefficients established for nonionic compounds. Further, published estimates for partition coefficients of fluoxetine vary, resulting in considerable uncertainties in both the exposure and environmental risk assessments of fluoxetine.

Environmental risk assessment of ivermectin: A case study.

The veterinary parasiticide ivermectin was selected as a case study compound within the project ERAPharm (Environmental Risk Assessment of Pharmaceuticals). Based on experimental data generated within ERAPharm and additional literature data, an environmental risk assessment (ERA) was performed mainly according to international and European guidelines. For the environmental compartments surface water, sediment, and dung, a risk was indicated at all levels of the tiered assessment approach. Only for soil was no risk indicated after the lower tier assessment. However, the use of effects data from additional 2-species and multispecies studies resulted in a risk indication for collembolans. Although previously performed ERAs for ivermectin revealed no concern for the aquatic compartment, and transient effects on dung-insect populations were not considered as relevant, the present ERA clearly demonstrates unacceptable risks for all investigated environmental compartments and hence suggests the necessity of reassessing ivermectin-containing products. Based on this case study, several gaps in the existing guidelines for ERA of pharmaceuticals were shown and improvements have been suggested. The action limit at the start of the ERA, for example, is not protective for substances such as ivermectin when used on intensively reared animals. Furthermore, initial predicted environmental concentrations (PECs) of ivermectin in soil were estimated to be lower than refined PECs, indicating that the currently used tiered approach for exposure assessment is not appropriate for substances with potential for accumulation in soil. In addition, guidance is lacking for the assessment of effects at higher tiers of the ERA, e.g., for field studies or a tiered effects assessment in the dung compartment.

Ovarian cycle and embryonic development in Gammarus fossarum: application for reproductive toxicity assessment.

Among freshwater invertebrates, Gammarus fossarum is an important test organism and is currently used in ecotoxicology for acute and chronic assays; nevertheless, reproductive toxicity test methods are not yet available for these species. In the present study, the reproductive cycle in Gammarus fossarum was characterized in order to propose a reproductive toxicity test encompassing molting, follicle growth, and embryonic development that will provide a better understanding of the mode of action of chemicals disrupting these hormone-regulated processes. A detailed description of the reproductive cycle in Gammarus fossarum was obtained. As in some amphipods, molt and reproductive cycles of G. fossarum females occur concurrently, lasting 30 d at 12°C. Each molt stage is characterized by a specific marsupial embryonic development stage and the size of developing follicles visible on the ovarian membrane. Based on these results, a 21-d reproductive toxicity test is proposed for this species. This new bioassay was applied to identify the specific impact of different stressors: cadmium, methomyl, nonylphenol, and a starvation diet. Good reproducibility was obtained for different endpoints under control conditions and throughout the experiments. Preliminary robust reference values or benchmarks were proposed for these endpoints. Cadmium was found to specially inhibit secondary vitellogenesis. Nonylphenol had a specific concentration-dependent effect on embryonic development, with an increase in the percent abnormality from a concentration of 0.05 µg/L. A restricted food diet led to a significant delay in the molt cycle, which in turn induced inhibition of secondary vitellogenesis.

Recommendations on the environmental risk assessment of pharmaceuticals: Effect characterization.

The effects testing of pharmaceuticals consists of a tiered investigation of ecotoxicological endpoints. However, effects testing has to be performed only when the predicted environmental concentrations (PECs) of pharmaceuticals are above certain action limits. To study the appropriateness of these action limits, a literature search was performed for pharmaceuticals with predicted no-effect concentrations (PNECs) close to or below the action limits. Some human pharmaceuticals showed effects at concentrations ≤100 ng/L, mostly in nonstandard fish or invertebrate tests. In addition, antibiotics and parasiticides sometimes had effects at concentrations <10 mg/kg soil. To help in identifying pharmaceuticals that should undergo effects testing although their PECs are below the action limits, "however clauses" are postulated for pharmaceuticals that are potentially persistent, bioaccumulative, carcinogenic, mutagenic, or reproductively toxic. Effects testing should also be performed for pharmaceuticals that 1) affect target structures that are conserved across species, 2) have a high potency or a small therapeutic margin, 3) are from a new therapeutic class, and 4) are structurally similar to compounds with known effects. Furthermore, suggestions for improving the effects testing of pharmaceuticals are made. These include inter alia chronic effects testing as a general approach, the use of invertebrate tests including sexual reproduction, the application of endpoints reflecting the mode of action of the drug or known side effects, and the simulation of more realistic exposure conditions in terrestrial laboratory tests.

Environmental risk assessment of human pharmaceuticals in the European Union: A case study with the ß-blocker atenolol.

ß-Adrenergic receptor blockers (ß-blockers) are applied to treat high blood pressure, ischemic heart disease, and heart rhythm disturbances. Due to their widespread use and limited human metabolism, ß-blockers are widely detected in sewage effluents and surface waters. ß-Adrenergic receptors have been characterized in fish and other aquatic animals, so it can be expected that physiological processes regulated by these receptors in wild animals may be affected by the presence of ß-blockers. Because ecotoxicological data on ß-blockers are scarce, it was decided to choose the ß-blocker atenolol as a case study pharmaceutical within the project ERAPharm. A starting point for the assessment of potential environmental risks was the European guideline on the environmental risk assessment of medicinal products for human use. In Phase I of the risk assessment, the initial predicted environmental concentration (PEC) of atenolol in surface water (500 ng L−1) exceeded the action limit of 10 ng L−1. Thus, a Phase II risk assessment was conducted showing acceptable risks for surface water, for groundwater, and for aquatic microorganisms. Furthermore, atenolol showed a low potential for bioaccumulation as indicated by its low lipophilicity (log KOW = 0.16), a low potential for exposure of the terrestrial compartment via sludge (log KOC = 2.17), and a low affinity for sorption to the sediment. Thus, the risk assessment according to Phase II-Tier A did not reveal any unacceptable risk for atenolol. Beyond the requirements of the guideline, additional data on effects and fate were generated within ERAPharm. A 2-generation reproduction test with the waterflea Daphnia magna resulted in the most sensitive no-observed-effect concentration (NOEC) of 1.8 mg L−1. However, even with this NOEC, a risk quotient of 0.003 was calculated, which is still well below the risk threshold limit of 1. Additional studies confirm the outcome of the environmental risk assessment according to EMEA/CHMP (2006). However, atenolol should not be considered as representative for other ß-blockers, such as metoprolol, oxprenolol, and propranolol, some of which show significantly different physicochemical characteristics and varying toxicological profiles in mammalian studies.

Additive vs non-additive genetic components in lethal cadmium tolerance of Gammarus (Crustacea): novel light on the assessment of the potential for adaptation to contamination.

Questioning the likelihood that populations adapt to contamination is critical for ecotoxicological risk assessment. The appraisal of genetic variance in chemical sensitivities within populations is currently used to evaluate a priori this evolutionary potential. Nevertheless, conclusions from this approach are questionable since non-additive genetic components in chemical tolerance could limit the response of such complex phenotypic traits to selection. Coupling quantitative genetics with ecotoxicology, this study illustrates how the comparison between cadmium sensitivities among Gammarus siblings enabled discrimination between genetic variance components in chemical tolerance. The results revealed that, whereas genetically determined differences in lethal tolerance exist within the studied population, such differences were not significantly heritable since genetic variance mainly relied on non-additive components. Therefore the potential for genetic adaptation to acute Cd stress appeared to be weak. These outcomes are discussed in regard to previous findings for asexual daphnids, which suggest a strong potency of genetic adaptation to environmental contamination, but which contrast with compiled field observations where adaptation is not the rule. Hereafter, we formulate the reconciling hypothesis of a widespread weakness of additive components in tolerance to contaminants, which needs to be further tested to gain insight into the question of the likelihood of adaptation to contamination.

Progestagens for human use, exposure and hazard assessment for the aquatic environment.

Little information is available on the environmental occurrence and ecotoxicological effects of pharmaceutical gestagens released in the aquatic environment. Since eighteen different gestagens were found to be used in France, preliminary exposure and hazard assessment were done. Predicted environmental concentrations (PECs) suggest that if parent gestagens are expected to be found in the ng l(-1) range, some active metabolites could be present at higher concentrations, although limited data on metabolism and environmental fate limit the relevance of PECs. The biological effects are not expected to be restricted to progestagenic activity. Both anti-androgenic activity (mainly for cyproterone acetate, chlormadinone acetate and their metabolites) and estrogenic activity (mainly for reduced metabolites of levonorgestrel and norethisterone) should also occur. All these molecules are likely to have a cumulative effect among themselves or with other xenoestrogens. Studies on occurrence, toxicity and degradation time are therefore needed for several of these compounds.

Dynamic energy budget as a basis to model population-level effects of zinc-spiked sediments in the gastropod Valvata piscinalis.

This paper presents original toxicity test designs and mathematical models that may be used to assess the deleterious effects of toxicants on Valvata piscinalis (Mollusca, Gastropoda). Results obtained for zinc, used as a reference toxicant, are presented. The feeding behavior, juvenile survival, growth, age at puberty, onset of reproduction, number of breedings during the life cycle, and fecundity were significantly altered when the snails were exposed to zinc-spiked sediments. Dynamic energy budget models (DEBtox) adequately predicted the effects of zinc on the V. piscinalis life cycle. They also provided estimates for lifecycle parameters that were used to parameterize a demographic model, based on a Z-transformed life-cycle graph. The effect threshold for the population growth rate (lambda) was estimated at 259 mg/kg dry sediment of zinc, showing that significant changes in abundance may occur at environmental concentrations. Significant effects occurring just above this threshold value were mainly caused by the severe impairment of reproductive endpoints. Sensitivity analysis showed that the value of lambda depended mainly on the juvenile survival rate. The impairment of this latter parameter may result in extinction of V. piscinalis. Finally, the present study highlights advantages of the proposed modeling approach in V. piscinalis and possible transfer to other test species and contaminants.

Mechanistic models to perform population risk assessment with the midge Chironomus riparius: application to heavy metals.

Mechanistic models can substantially contribute to population risk assessment to assess effects on population and to increase the relevance of the toxicity parameters estimated at an individual level. We use four mechanistic models to change the scale from concentration to effects on individuals and from individuals to population with the midge Chironomus riparius: a kinetics model; an energy-based effects model, linking effects on the life cycle and compound body residues; a matrix approach to derive population growth rate; and an energy-based population model to derive carrying capacity. The whole "model battery" was applied to cadmium and copper. The data came from growth, survival, and reproduction tests. We also incorporated information about compounds physiological mode of action and kinetics. Thresholds at population level were derived through comparisons with our control database. We showed that our two population endpoints (carrying capacity and population growth rate) provide complementary information about toxicity risks, even if, in our study, population growth rate appeared to be slightly more sensitive than carrying capacity. We found population no effect concentration of, respectively, 0.42 and 9.3 mg/kg for cadmium and copper. We also showed that information about physiological mode of action was relevant, whereas a kinetics test was unnecessary.

Assessment of ecotoxicological risks related to depositing dredged materials from canals in northern France on soil.

The implementation of an ecological risk assessment framework is presented for dredged material deposits on soil close to a canal and groundwater, and tested with sediment samples from canals in northern France. This framework includes two steps: a simplified risk assessment based on contaminant concentrations and a detailed risk assessment based on toxicity bioassays and column leaching tests. The tested framework includes three related assumptions: (a) effects on plants (Lolium perenne L.), (b) effects on aquatic organisms (Escherichia coli, Pseudokirchneriella subcapitata, Ceriodaphnia dubia, and Xenopus laevis) and (c) effects on groundwater contamination. Several exposure conditions were tested using standardised bioassays. According to the specific dredged material tested, the three assumptions were more or less discriminatory, soil and groundwater pollution being the most sensitive. Several aspects of the assessment procedure must now be improved, in particular assessment endpoint design for risks to ecosystems (e.g., integration of pollutant bioaccumulation), bioassay protocols and column leaching test design.

Using aquatic macroinvertebrate species traits to build test batteries for sediment toxicity assessment: accounting for the diversity of potential biological responses to toxicants.

An original species-selection method for the building of test batteries is presented. This method is based on the statistical analysis of the biological and ecological trait patterns of species. It has been applied to build a macroinvertebrate test battery for the assessment of sediment toxicity, which efficiently describes the diversity of benthic macroinvertebrate biological responses to toxicants in a large European lowland river. First, 109 potential representatives of benthic communities of European lowland rivers were selected from a list of 479 taxa, considering 11 biological traits accounting for the main routes of exposure to a sediment-bound toxicant and eight ecological traits providing an adequate description of habitat characteristics used by the taxa. Second, their biological and ecological trait patterns were compared using coinertia analysis. This comparison allowed the clustering of taxa into groups of organisms that exhibited similar life-history characteristics, physiological and behavioral features, and similar habitat use. Groups exhibited various sizes (7-35 taxa), taxonomic compositions, and biological and ecological features. Main differences among group characteristics concerned morphology, substrate preferendum and habitat utilization, nutritional features, maximal size, and life-history strategy. Third, the best representatives of the mean biological and ecological characteristics of each group were included in the test battery. The final selection was composed of Chironomus riparius (Insecta: Diptera), Branchiura sowerbyi (Oligochaeta: Tubificidae), Lumbriculus variegatus (Oligochaeta: Lumbriculidae), Valvata piscinalis (Gastropoda: Valvatidae), and Sericostoma personatum (Trichoptera: Sericostomatidae). This approach permitted the biological and ecological variety of the battery to be maximized. Because biological and ecological traits of taxa determine species sensitivity, such maximization should permit the battery to better account for the sensitivity range within a community.

Environmental risk assessment of six human pharmaceuticals: are the current environmental risk assessment procedures sufficient for the protection of the aquatic environment?

In this study, exposure and ecotoxicity data of six human pharmaceuticals (carbamazepine, clofibric acid, diclofenac, ofloxacin, propranolol, and sulfamethoxazole) were collected, including our own experimental data and literature data. From this data collection, the two-tiered European draft guideline on the environmental risk assessment of human pharmaceuticals was tested. Measured environmental concentrations in effluents from France and in effluents and surface waters from Germany were compared to the predicted environmental concentrations (PECs) in both countries. In a similar manner, predicted no-effect concentrations (PNECs) derived from acute data and PNECs derived from chronic data were estimated for each pharmaceutical and corresponding PEC/PNEC ratios then were compared in both countries. Globally, results demonstrated that all environmental concentrations (predicted or measured) for each considered pharmaceutical exceeded the 10-ng/L cutoff value, which requires the implementation of the second-tier assessment based on ecotoxicity data. Moreover, the six pharmaceuticals showed a relatively limited acute toxicity, and carbamazepine and propranolol were inaccurately identified as having negligible risks under the current European draft procedure. Such results lead to discussion of the actual procedure on pharmaceuticals, especially on the need of appropriate ecotoxicity tests.