Modeling HepaRG metabolome responses to pyrrolizidine alkaloid exposure for insight into points of departure and modes of action.

The new challenges in toxicology demand novel and innovative in vitro approaches for deriving points of departure (PODs) and determining the mode of action (MOA) of chemicals. Therefore, the aim of this original study was to couple in vitro studies with untargeted metabolomics to model the concentration-response of extra- and intracellular metabolome data on human HepaRG cells treated for 48 h with three pyrrolizidine alkaloids (PAs): heliotrine, retrorsine and lasiocarpine. Modeling revealed that the three PAs induced various monotonic and, importantly, biphasic curves of metabolite content. Based on unannotated metabolites, the endometabolome was more sensitive than the exometabolome in terms of metabolomic effects, and benchmark concentrations (BMCs) confirmed that lasiocarpine was the most hepatotoxic PA. Regarding its MOA, impairment of lipid metabolism was highlighted at a very low BMC (first quartile, 0.003 µM). Moreover, results confirmed that lasiocarpine targets bile acids, as well as amino acid and steroid metabolisms. Analysis of the endometabolome, based on coupling concentration-response and PODs, gave encouraging results for ranking toxins according to their hepatotoxic effects. Therefore, this novel approach is a promising tool for next-generation risk assessment, readily applicable to a broad range of compounds and toxic endpoints.

Development, feeding, and sex shape the relative quantity of the nutritional obligatory symbiont Wolbachia in bed bugs.

The common bed bug, Cimex lectularius, is a hemipteran insect that feeds only on blood, and whose bites cause public health issues. Due to globalization and resistance to insecticides, this pest has undergone a significant and global resurgence in recent decades. Blood is an unbalanced diet, lacking notably sufficient B vitamins. Like all strict hematophagous arthropods, bed bugs host a nutritional symbiont supplying B vitamins. In C. lectularius, this nutritional symbiont is the intracellular bacterium Wolbachia (wCle). It is located in specific symbiotic organs, the bacteriomes, as well as in ovaries. Experimental depletion of wCle has been shown to result in longer nymphal development and lower fecundity. These phenotypes were rescued by B vitamin supplementation. Understanding the interaction between wCle and the bed bug may help to develop new pest control methods targeting the disruption of this symbiotic interaction. The objective of this work was thus to quantify accurately the density of wCle over the life cycle of the host and to describe potential associated morphological changes in the bacteriome. We also sought to determine the impact of sex, feeding status, and aging on the bacterial population dynamics. We showed that the relative quantity of wCle continuously increases during bed bug development, while the relative size of the bacteriome remains stable. We also showed that adult females harbor more wCle than males and that wCle relative quantity decreases slightly in adults with age, except in weekly-fed males. These results are discussed in the context of bed bug ecology and will help to define critical points of the symbiotic interaction during the bed bug life cycle.

Deep dive into the chronic toxicity of tyre particle mixtures and their leachates.

Particles from the tread of vehicle tyres are a global pollutant, which are emitted into the environment at an approximate rate of 1.4 kg.year-1 for an average passenger-car. In this study, popular tyre brands were used to generate a tyre tread microparticle mixture. The chronic toxicity of both particles and chemical leachates were compared on a planktonic test species (Daphnia magna). Over 21 days of exposure, pristine tyre tread microparticles were more toxic (LC50 60 mg.L-1) than chemical lechates alone (LC50 542 mg.L-1). Microparticles and leachates showed distinct effects on reproduction and morphological development at environmentally relevant concentrations, with dose-dependent uptake of particles visible in the digestive tract. Chemical characterization of leachates revealed a metal predominance of zinc, titanium, and strontium. Of the numerous organic chemicals present, at least 54 were shared across all 5 tyre brands, with many classified to be very toxic. Our results provide a critically needed information on the toxicity of tyre tread particles and the associated chemicals that leach from them to inform future mitigation measures. We conclude that tyre particles are hazardous pollutants of particular concern that are close to or possibly above chronic environmental safety limits in some locations.

Evolution of the proportion of colistin-resistant isolates in animal clinical Escherichia coli over time - A hierarchical mixture model approach.

Colistin resistance has been the subject of much attention since mcr genes encoding plasmid-mediated colistin resistance description in 2015. To date, surveillance data about resistance levels encountered in food-producing animals are scarce. In France, the Resapath dataset, consisting in a large collection of disk diffusion antibiogram results transmitted by a network of laboratories. It offers a unique opportunity to study the evolution of resistance towards colistin over the past 15 years in Escherichia coli isolated from diseased food-producing animals. This study used a Bayesian hierarchical Gaussian mixture model to estimate the resistant proportions from those data. This non-classical approach deals with the colistin-specific problem of overlapping distributions of diameters measured for susceptible and resistant isolates that makes the definition of epidemiological cut-off very hard. This model also considers the variability observed between the measurements performed by different laboratories. Proportion of resistant isolates has been calculated for several food-producing animals and most encountered diseases. From those estimations, a marked evolution of the proportions of resistant isolates is noticeable, for swine suffering from digestive disorders. In this group, an increase over the 2006-2011 period from 0.1% [ 0.0%, 1.2%] in 2006-28.6% [25.1%, 32.3%] in 2011 was followed by a decrease to reach 3.6% [2.3%;5.3%] in 2018. For isolates related to digestive disorders in calves, percentages increased and reached 7% in 2009 then decreased as for swine. In contrast, for poultry productions, estimated proportions and credibility intervals were constantly very close to zero.

Deep learning in veterinary medicine, an approach based on CNN to detect pulmonary abnormalities from lateral thoracic radiographs in cats.

Thoracic radiograph (TR) is a complementary exam widely used in small animal medicine which requires a sharp analysis to take full advantage of Radiographic Pulmonary Pattern (RPP). Although promising advances have been made in deep learning for veterinary imaging, the development of a Convolutional Neural Networks (CNN) to detect specifically RPP from feline TR images has not been investigated. Here, a CNN based on ResNet50V2 and pre-trained on ImageNet is first fine-tuned on human Chest X-rays and then fine-tuned again on 500 annotated TR images from the veterinary campus of VetAgro Sup (Lyon, France). The impact of manual segmentation of TR's intrathoracic area and enhancing contrast method on the CNN's performances has been compared. To improve classification performances, 200 networks were trained on random shuffles of training set and validation set. A voting approach over these 200 networks trained on segmented TR images produced the best classification performances and achieved mean Accuracy, F1-Score, Specificity, Positive Predictive Value and Sensitivity of 82%, 85%, 75%, 81% and 88% respectively on the test set. Finally, the classification schemes were discussed in the light of an ensemble method of class activation maps and confirmed that the proposed approach is helpful for veterinarians.

Phenotypic and Transcriptomic Responses to Stress Differ According to Population Geography in an Invasive Species.

Adaptation to rapid environmental changes must occur within a short-time scale. In this context, studies of invasive species may provide insights into the underlying mechanisms of rapid adaptation as these species have repeatedly encountered and adapted to novel environmental conditions. We investigated how invasive and noninvasive genotypes of Drosophila suzukii deal with oxidative stress at the phenotypic and molecular levels. We also studied the impact of transposable element (TE) insertions on the gene expression in response to stress. Our results show that flies from invasive areas (France and the United States) live longer in natural conditions than the ones from native Japanese areas. As expected, lifespan for all genotypes was significantly reduced following exposure to paraquat, but this reduction varied among genotypes (genotype-by-environment interaction) with invasive genotypes appearing more affected by exposure than noninvasive ones. A transcriptomic analysis of genotypes upon paraquat treatment detected many genes differentially expressed (DE). Although a small core set of genes were DE in all genotypes following paraquat exposure, much of the response of each genotype was unique. Moreover, we showed that TEs were not activated after oxidative stress and DE genes were significantly depleted of TEs. In conclusion, it is likely that transcriptomic changes are involved in the rapid adaptation to local environments. We provide new evidence that in the decade since the invasion from Asia, the sampled genotypes in Europe and the United States of D. suzukii diverged from the ones from the native area regarding their phenotypic and genomic response to oxidative stress.

Evaluation using latent class models of the diagnostic performances of three ELISA tests commercialized for the serological diagnosis of Coxiella burnetii infection in domestic ruminants.

ELISA methods are the diagnostic tools recommended for the serological diagnosis of Coxiella burnetii infection in ruminants but their respective diagnostic performances are difficult to assess because of the absence of a gold standard. This study focused on three commercial ELISA tests with the following objectives (1) assess their sensitivity and specificity in sheep, goats and cattle, (2) assess the between- and within-herd seroprevalence distribution in these species, accounting for diagnostic errors, and (3) estimate optimal sample sizes considering sensitivity and specificity at herd level. We comparatively tested 1413 cattle, 1474 goat and 1432 sheep serum samples collected in France. We analyzed the cross-classified test results with a hierarchical zero-inflated beta-binomial latent class model considering each herd as a population and conditional dependence as a fixed effect. Potential biases and coverage probabilities of the model were assessed by simulation. Conditional dependence for truly seropositive animals was high in all species for two of the three ELISA methods. Specificity estimates were high, ranging from 94.8% [92.1; 97.8] to 99.2% [98.5; 99.7], whereas sensitivity estimates were generally low, ranging from 39.3 [30.7; 47.0] to 90.5% [83.3; 93.8]. Between- and within-herd seroprevalence estimates varied greatly among geographic areas and herds. Overall, goats showed higher within-herd seroprevalence levels than sheep and cattle. The optimal sample size maximizing both herd sensitivity and herd specificity varied from 3 to at least 20 animals depending on the test and ruminant species. This study provides better interpretation of three widely used commercial ELISA tests and will make it possible to optimize their implementation in future studies. The methodology developed may likewise be applied to other human or animal diseases.

How does interspecific competition modify the response of grass plants against herbicide treatment? A hierarchical concentration-response approach.

Ecological interactions are rarely taken into account in environmental risk assessment. The objective of this work was to assess how interspecific competition affects the way plant species react to herbicides and more specifically how it modifies the concentration-response curves that can be built using ecotoxicological bioassays. To do this, we relied on the results of ecotoxicological bioassays on six herbaceous species exposed to isoproturon under two conditions: in presence and in absence of a competitor. At the end of the experiments, eleven endpoints were measured. We modelled these data using a hierarchical modelling framework designed to assess the effects of competition on each of the four parameters of the concentration response curves (e.g. the level of response at the control or the concentration at the inflection point of the curve) simultaneously for the six species. The modelled effects could be of three types, 1) competition had no effect on the parameter, 2) competition had the same effect on the parameter for all species and 3) competition had a different effect on the parameter for each species. Our main hypothesis was that different species would react differently to competition. Results showed that about a half of the estimated parameters showed a modification under competition pressure among which only a fourth showed a species-specific effect, the three other fourth showing the same effect between the different species. Our initial hypothesis was thus not supported as species tended to react in the same way to competition. The competition effect on plants was mainly negative, thus showing that they were more affected by isoproturon under competition pressure. This study therefore establishes how competition modifies plant responses to chemical stress and how this interaction varies from one species to the other.

A multi-omics concentration-response framework uncovers novel understanding of triclosan effects in the chlorophyte Scenedesmus vacuolatus.

In aquatic ecosystems, the biocide triclosan represents a hazard for the non-target microalgae. So far, algal responses were mainly investigated at apical levels hampering the acquisition of a holistic view on primary, adaptive, and compensatory stress responses. We assessed responses of the chlorophyte Scenedesmus vacuolatus to triclosan at apical (growth, photosynthesis) and molecular (transcriptome, metabolome) levels for comparative pathway sensitivity analysis. For each responsive signal (contigs, metabolites), a concentration-response curve was modeled and effect concentrations were calculated leading to the setting of cumulative sensitivity distributions. Molecular responses showed higher sensitivity than apical observations. The functional annotation of contigs and metabolites revealed 118 metabolic pathways putatively impaired by triclosan, highlighting a wide repercussion on the algal metabolism. Metabolites involved in the lipid metabolism showed decreasing trends along the concentration gradient and a globally highest sensitivity, pointing to the primary target of triclosan. The pathways involved in xenobiotic degradation and membrane transporters were mainly regulated in the transcriptome with increasing response trends comprising compensatory responses. The suggested novel approach, combining apical and multi-omics analyses in a concentration-response framework improves mechanistic understanding and mode of action analysis on non-targeted organisms and is suggested to better implement high-throughput multi-omics data in environmental risk assessment.

Effect of interspecific competition on species sensitivity distribution models: Analysis of plant responses to chemical stress.

Species Sensitivity Distributions (SSD) are widely used in environmental risk assessment to predict the concentration of a contaminant that is hazardous for 5% of species (HC5). They are based on monospecific bioassays conducted in the laboratory and thus do not directly take into account ecological interactions. This point, among others, is accounted for in environmental risk assessment through an assessment factor (AF) that is applied to compensate for the lack of environmental representativity. In this study, we aimed to assess the effects of interspecific competition on the responses towards isoproturon of plant species representative of a vegetated filter strip community, and to assess its impact on the derived SSD and HC5 values. To do so, we realized bioassays confronting six herbaceous species to a gradient of isoproturon exposure in presence and absence of a competitor. Several modelling approaches were applied to see how they affected the results, using different critical effect concentrations and investigating different ways to handle multiple endpoints in SSD. At the species level, there was a strong trend toward organisms being more sensitive to isoproturon in presence of a competitor than in its absence. At the community level, this trend was also observed in the SSDs and HC5 values were always lower in presence of a competitor (1.12-11.13 times lower, depending on the modelling approach). Our discussion questions the relevance of SSD and AF as currently applied in environmental risk assessment.

Developmental variability channels mouse molar evolution.

Do developmental systems preferentially produce certain types of variation that orient phenotypic evolution along preferred directions? At different scales, from the intra-population to the interspecific, the murine first upper molar shows repeated anterior elongation. Using a novel quantitative approach to compare the development of two mouse strains with short or long molars, we identified temporal, spatial and functional differences in tooth signaling center activity, that arise from differential tuning of the activation-inhibition mechanisms underlying tooth patterning. By tracing their fate, we could explain why only the upper first molar reacts via elongation of its anterior part. Despite a lack of genetic variation, individuals of the elongated strain varied in tooth length and the temporal dynamics of their signaling centers, highlighting the intrinsic instability of the upper molar developmental system. Collectively, these results reveal the variational properties of murine molar development that drive morphological evolution along a line of least resistance.

Over time species develop random mutations in their genetic sequence that causes their form to change. If this new form increases the survival of a species it will become favored through natural selection and is more likely to get passed on to future generations. But, the evolution of these new traits also depends on what happens during development. Developmental mechanisms control how an embryo progresses from a single cell to an adult organism made of many cells. Mutations that alter these processes can influence the physical outcome of development, and cause a new trait to form. This means that if many different mutations alter development in a similar way, this can lead to the same physical change, making it 'easy' for a new trait to repeatedly occur. Most of the research has focused on finding the mutations that underlie repeated evolution, but rarely on identifying the role of the underlying developmental mechanisms. To bridge this gap, Hayden et al. investigated how changes during development influence the shape and size of molar teeth in mice. In some wild species of mice, the front part of the first upper molar is longer than in other species. This elongation, which is repeatedly found in mice from different islands, likely came from developmental mechanisms. Tooth development in mice has been well-studied in the laboratory, and Hayden et al. started by identifying two strains of laboratory mice that mimic the teeth seen in their wild cousins, one with elongated upper first molars and another with short ones. Comparing how these two strains of mice developed their elongated or short teeth revealed key differences in the embryonic structures that form the upper molar and cause it to elongate. Further work showed that variations in these embryonic structures can even cause mice that are genetically identical to have longer or shorter upper first molars. These findings show how early differences during development can lead to small variations in form between adult species of mice. This study highlights how studying developmental differences as well as genetic sequences can further our understanding of how different species evolved.

DRomics: A Turnkey Tool to Support the Use of the Dose-Response Framework for Omics Data in Ecological Risk Assessment.

Omics approaches (e.g., transcriptomics, metabolomics) are promising for ecological risk assessment (ERA) since they provide mechanistic information and early warning signals. A crucial step in the analysis of omics data is the modeling of concentration-dependency which may have different trends including monotonic (e.g., linear, exponential) or biphasic (e.g., U shape, bell shape) forms. The diversity of responses raises challenges concerning detection and modeling of significant responses and effect concentration (EC) derivation. Furthermore, handling high-throughput data sets is time-consuming and requires effective and automated processing routines. Thus, we developed an open source tool (DRomics, available as an R-package and as a web-based service) which, after elimination of molecular responses (e.g., gene expressions from microarrays) with no concentration-dependency and/or high variability, identifies the best model for concentration-response curve description. Subsequently, an EC (e.g., a benchmark dose) is estimated from each curve, and curves are classified based on their model parameters. This tool is especially dedicated to manage data obtained from an experimental design favoring a great number of tested doses rather than a great number of replicates and also to handle properly monotonic and biphasic trends. The tool finally provides restitution for a table of results that can be directly used to perform ERA approaches.

Diagnosis of bovine dictyocaulosis by bronchoalveolar lavage technique: A comparative study using a Bayesian approach.

Bovine dictyocaulosis is a pulmonary parasitic disease present in temperate countries, with potential important clinical and economic impacts. The Baermann technique is routinely used despite its low sensitivity in adult cows. Recently developed serological tests seem to offer better sensitivity, but validations of these tests in field conditions are few. We aimed to study two non-previously evaluated diagnosis methods of dictyocaulosis based on bronchoalveolar lavage sampling (BAL), which allows finding lungworm stages in the lungs as well as determination of eosinophilia. We compared them to the Baermann technique and serological tests. As no gold standard was available, we performed a Bayesian analysis by the simultaneous use of latent class and mixture models. The study was carried out during the 2015 pasture season on 60 adult cows originating from 11 herds with clinical signs of dictyocaulosis, and 10 apparently healthy cows originating from the teaching herd of VetAgro Sup, in France. Prevalence of infection was highly variable among herds with clinical signs (10-90%). Despite a maximal specificity (100%), the sensitivity of parasitological methods was low (7.4% for the Baermann sedimentation and 24.7% for the examination of BAL fluids). Better results were observed with serology (Se = 74.9%, Sp = 85.5%) with an optimal cut-off value estimated at 0.397 for the optical density ratio. Even better results were obtained with the count of eosinophil in BAL (Se = 89.4%, Sp = 85.2%) with an optimal cut-off value estimated at 4.77% for the eosinophil proportion. The BAL is a relevant diagnostic method of dictyocaulosis for practitioners due to the opportunity to perform two analyses (direct parasitic research and the eosinophil count) and to its good sensitivity and specificity.

MOSAIC: a web-interface for statistical analyses in ecotoxicology.

In ecotoxicology, bioassays are standardly conducted in order to measure acute or chronic effects of potentially toxic substances on reproduction, growth, and/or survival of living animals. MOSAIC, standing for MOdeling and StAtistical tools for ecotoxICology, is a user-friendly web interface dedicated to the mathematical and statistical modelling of such standard bioassay data. Its simple use makes MOSAIC a turnkey decision-making tool for ecotoxicologists and regulators. Without wasting time on extensive mathematical and statistical technicalities, users are provided with advanced and innovative methods for a valuable quantitative environmental risk assessment. MOSAIC is available at http://pbil.univ-lyon1.fr/software/mosaic/ .

Robust Fit of Toxicokinetic-Toxicodynamic Models Using Prior Knowledge Contained in the Design of Survival Toxicity Tests.

Toxicokinetics-toxicodynamic (TKTD) models have emerged as a powerful means to describe survival as a function of time and concentration in ecotoxicology. They are especially powerful to extrapolate survival observed under constant exposure conditions to survival predicted under realistic fluctuating exposure conditions. But despite their obvious benefits, these models have not yet been adopted as a standard to analyze data of survival toxicity tests. Instead simple dose-response models are still often used although they only exploit data observed at the end of the experiment. We believe a reason precluding a wider adoption of TKTD models is that available software still requires strong expertise in model fitting. In this work, we propose a fully automated fitting procedure that extracts prior knowledge on parameters of the model from the design of the toxicity test (tested concentrations and observation times). We evaluated our procedure on three experimental and 300 simulated data sets and showed that it provides robust fits of the model, both in the frequentist and the Bayesian framework, with a better robustness of the Bayesian approach for the sparsest data sets.

Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections.

The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly - regardless the time-intervals - at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight "day" effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the "day" effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails' immunocompetence.

Constructing Time-Resolved Species Sensitivity Distributions Using a Hierarchical Toxico-Dynamic Model.

Classical species sensitivity distribution (SSD) is used to assess the threat to ecological communities posed by a contaminant and derive a safe concentration. It suffers from several well-documented weaknesses regarding its ecological realism and statistical soundness. Criticism includes that SSD does not take time-dependence of the data into account, that safe concentrations obtained from SSD might not be entirely protective of the target communities, and that there are issues of statistical representativity and of uncertainty propagation from the experimental data. We present a hierarchical toxico-dynamic (TD) model to simultaneously address these weaknesses: TD models incorporate time-dependence and allow improvement of the ecological relevance of safe concentrations, while the hierarchical approach affords appropriate propagation of uncertainty from the original data. We develop this model on a published data set containing the salinity tolerance over 72 h of 217 macroinvertebrate taxa, obtained through rapid toxicity testing (RTT). The shrinkage properties of the hierarchical model prove particularly adequate for modeling inhomogeneous RTT data. Taking into account the large variability in the species response, the model fits the whole data set well. Moreover, the model predicts a time-independent safe concentration below that obtained with classical SSD at 72 h, demonstrating under-protectiveness of the classical approach.

Hierarchical modelling of species sensitivity distribution: development and application to the case of diatoms exposed to several herbicides.

The species sensitivity distribution (SSD) is a key tool to assess the ecotoxicological threat of contaminants to biodiversity. For a contaminant, it predicts which concentration is safe for a community of species. Widely used, this approach suffers from several drawbacks: (i) summarizing the sensitivity of each species by a single value entails a loss of valuable information about the other parameters characterizing the concentration-effect curves; (ii) it does not propagate the uncertainty on estimated sensitivities into the SSD; (iii) the hazardous concentration estimated with SSD only indicates the threat to biodiversity, without any insight about a global response of the community related to the measured endpoint. To remedy these drawbacks, we built a global hierarchical model including the concentration-effect model together with the distribution law of the SSD. We revisited the current SSD approach to account for more sources of variability and uncertainty into the prediction than the traditional analysis and to assess a global response for the community. Working within a Bayesian framework, we were able to compute an SSD taking into account the uncertainty from the original raw data. We also developed a quantitative indicator of a global response of the community to the contaminant. We applied this methodology to study the toxicity and the risk of six herbicides to benthic diatoms from Lake Geneva, based on the biomass endpoint. Our approach highlighted a wide variability within the set of diatom species for all the parameters of the concentration-effect model and a potential correlation between them. Remarkably, variability of the shape parameter of the model and correlation had not been considered before. Comparison between the SSD and the global response of the community revealed that protecting 95% of the species might preserve only 80-86% of the global response. Finally, propagating the uncertainty on the estimated sensitivity showed that building an SSD on a low level of effect, such as EC10, might be unreasonable as it induces a large uncertainty on the result.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda).

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608µgCdL(-1)). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68