A two years field experiment to assess the impact of two fungicides on earthworm communities and their recovery.

Recent EFSA (European Food Safety Authority) reports highlighted that the ecological risk assessment of pesticides needed to go further by taking more into account the impacts of chemicals on biodiversity under field conditions. We assessed the effects of two commercial formulations of fungicides separately and in mixture, i.e., Cuprafor Micro® (containing 500 g kg-1 copper oxychloride) at 4 (C1, corresponding to 3.1 mg kg-1 dry soil of copper) and 40 kg ha-1 (C10), and Swing® Gold (50 g L-1 epoxiconazole EPX and 133 g L-1 dimoxystrobin DMX) at one (D1, 5.81 10-2 and 1.55 10-1 mg kg-1 dry soil of EPX and DMX, respectively) and ten times (D10) the recommended field rate, on earthworms at 1, 6, 12, 18 and 24 months after the application following the international ISO standard no. 11268-3 to determine the effects on earthworms in field situations. The D10 treatment significantly reduced the species diversity (Shannon diversity index, 54% of the control), anecic abundance (29% of the control), and total biomass (49% of the control) over the first 18 months of experiment. The Shannon diversity index also decreased in the mixture treatment (both fungicides at the recommended dose) at 1 and 6 months after the first application (68% of the control at both sampling dates), and in C10 (78% of the control) at 18 months compared with the control. Lumbricus terrestris, Aporrectodea caliginosa, Aporrectodea giardi, Aporrectodea longa, and Allolobophora chlorotica were (in decreasing order) the most sensitive species to the tested fungicides. This study not only addressed field ecotoxicological effects of fungicides at the community level and ecological recovery, but it also pinpointed some methodological weaknesses (e.g., regarding fungicide concentrations in soil and statistics) of the guideline to determine the effects on earthworms in field situations.

Effects of two common fungicides on the reproduction of Aporrectodea caliginosa in natural soil.

The use of pesticides in agroecosystems can have negative effects on earthworms, which play key roles in soil functioning such as organic matter decomposition. The aim of this study was to assess the effects of two fungicides (Cuprafor micro®, composed of copper oxychloride, and Swing Gold®, composed of epoxiconazole (EPX) and dimoxystrobin (DMX)) on earthworm reproduction by exposing adults and cocoons. First, adult Aporrectodea caliginosa individuals were exposed for 28 days to 3.33, 10 and 30 times the recommended dose (RD) of Cuprafor micro® corresponding to 25.8, 77.5 and 232.5 mg kg-1 dry soil of copper, respectively, and 0.33, 1 and 3 times the RD of Swing Gold® (corresponding to 5.2 × 10-2 mg DMX kg-1 + 1.94 × 10-2 mg EPX kg-1, 1.55 × 10-1 mg DMX kg-1 + 5.81 × 10-2 mg EPX kg-1 and 4.62 × 10-1 mg DMX kg-1 + 1.74 × 10-1 mg EPX kg-1 respectively), in addition to a control soil with no fungicide treatment. Cocoon variables (production, weight, hatching success, hatching time) were monitored. Second, "naïve" cocoons produced by uncontaminated earthworms were exposed to soils contaminated by the same concentrations of the two fungicides, and we assessed hatching success and hatching time. In the first experiment, cocoon production was halved at the highest copper concentration (232.5 mg Cu kg-1 of dry soil) as compared to the control. Cocoons took 5 more days to hatch, and the hatching success decreased by 35% as compared to the control. In the Swing Gold® treatments, cocoon production was reduced by 63% at 3 times the RD, and the hatching success significantly decreased by 16% at the RD. In the second experiment, only the hatching success of cocoons was impacted by Swing Gold® at 3 times the RD (30% less hatching). It is concluded that the cocoon stock in the soil is crucial for the renewal of populations in the field. The most sensitive endpoint was the hatching success of the cocoons produced by exposed adults. This endpoint and the effects observed on the "naïve" cocoons could be taken into account in pesticide risk assessment.

Refining uptake and depuration constants for fluoroalkyl chemicals in Chironomus riparius larvae on the basis of experimental results and modelling.

The aims of this study were to determine depuration rates for a range of per- and polyfluoroalkyl substances (PFASs) using Chironomus riparius, and to test a concentration-dependency hypothesis for the long-chain perfluorotridecanoic acid (PFTrDA) for this species. Midge larvae were exposed to field sediments collected downstream of a fluorotelomer plant, and to the same sediment spiked with PFTrDA. Elimination kinetics results indicated complete elimination of all PFASs by chironomids after 42h. These data were used to develop two PFTrDA bioaccumulation models accounting for chironomid growth and for compound concentration dependency or not. There was much better agreement between observed and simulated data under the concentration-dependency hypothesis than under the alternative one (passive diffusion). The PFTrDA uptake rate derived from the concentration-dependency model equaled 0.013 ± 0.008gocgwwh-1, and the depuration rate 0.032 ± 0.009h-1.

Short-term effects of two fungicides on enchytraeid and earthworm communities under field conditions.

Because of the wide use of pesticides in agriculture, there is still a need of higher-tier field studies to assess ecotoxicological effects of commercial formulations on a wider variety of non-target soil organisms such as soil annelids. We here tested the effects of different concentrations of two fungicide formulations, i.e., Cuprafor Micro® (composed of 500 g kg-1 copper oxychloride) and Swing Gold® (composed of 50 g l-1 epoxiconazole and 133 g l-1 dimoxystrobin) on two families of terrestrial oligochaetes (Lumbricidae and Enchytraeidae) after 1 month of exposure. We also assessed the feeding activity of soil organisms using the bait lamina method. Along with the feeding activity, the enchytraeid density, diversity and communities were not different in the control and the contaminated plots. By contrast, epigeic earthworms were absent and earthworm diversity and densities of anecic species decreased significantly in the plots contaminated at ten times the recommended dose of the Swing Gold® formulation. The copper fungicide (at 0.75 and 7.5 kg Cu ha-1) and the treatment with the pesticide mixture (Cuprafor Micro® at 0.75 kg Cu ha-1 and Swing Gold® at the recommended dose) did not affect Oligochaeta communities compared with the control, except the Shannon index for earthworms in the mixture of both fungicides. Responses of the two annelid families to the tested pesticides were different with higher effects observed on the diversity and the community structure of earthworms compared with enchytraeids. This study allowed detecting early changes on oligochaete populations after pesticide application.

Differences in sensitivity between earthworms and enchytraeids exposed to two commercial fungicides.

The use of pesticides in crop fields may have negative effects on soil Oligochaeta Annelida, i.e., earthworms and enchytraeids, and thus affect soil quality. The aim of this study was to assess the effects of two commercial fungicide formulations on the earthworm Aporrectodea caliginosa and the enchytraeid Enchytraeus albidus in a natural soil. The fungicides were Cuprafor micro® (copper oxychloride), commonly used in organic farming, and Swing Gold® (epoxiconazole and dimoxystrobin), a synthetic fungicide widely used in conventional farming to protect cereal crops. Laboratory experiments were used to assess the survival, biomass loss and avoidance behaviour. No lethal effect was observed following exposure to the copper fungicide for 14 days, even at 5000mgkg-1 of copper, i.e. 650 times the recommended dose (RD). However, a significant decrease in biomass was observed from 50mgkg-1 of copper (6.5 times the RD) for A. caliginosa and at 5000mgkg-1 of copper (650 times the RD) for E. albidus. These sublethal effects suggest that a longer period of exposure would probably have led to lethal effects. The EC50 avoidance for the copper fungicide was estimated to be 51.2mgkg-1 of copper (6.7 times the RD) for A. caliginosa, and 393mgkg-1 of copper (51 times the RD) for E. albidus. For the Swing Gold® fungicide, the estimated LC50 was 7.0 10-3mLkg-1 (6.3 times the RD) for A. caliginosa and 12.7 10-3mLkg-1 (11.0 times the RD) for E. albidus. No effect on biomass or avoidance was observed at sublethal concentrations of this synthetic fungicide. It was concluded that enchytraeids were less sensitive than earthworms to the two commercial fungicides in terms of mortality, biomass loss and avoidance behaviour. Therefore we discuss the different strategies possibly used by the two Oligochaeta species to cope with the presence of the pesticides were discussed, along with the potential consequences on the soil functions.

Transgenerational Adaptation to Pollution Changes Energy Allocation in Populations of Nematodes.

Assessing the evolutionary responses of long-term exposed populations requires multigeneration ecotoxicity tests. However, the analysis of the data from these tests is not straightforward. Mechanistic models allow the in-depth analysis of the variation of physiological traits over many generations, by quantifying the trend of the physiological and toxicological parameters of the model. In the present study, a bioenergetic mechanistic model has been used to assess the evolution of two populations of the nematode Caenorhabditis elegans in control conditions or exposed to uranium. This evolutionary pressure resulted in a brood size reduction of 60%. We showed an adaptation of individuals of both populations to experimental conditions (increase of maximal length, decrease of growth rate, decrease of brood size, and decrease of the elimination rate). In addition, differential evolution was also highlighted between the two populations once the maternal effects had been diminished after several generations. Thus, individuals that were greater in maximal length, but with apparently a greater sensitivity to uranium were selected in the uranium population. In this study, we showed that this bioenergetics mechanistic modeling approach provided a precise, certain, and powerful analysis of the life strategy of C. elegans populations exposed to heavy metals resulting in an evolutionary pressure across successive generations.

A physiologically based toxicokinetic model for the zebrafish Danio rerio.

Zebrafish (Danio rerio) is a widely used model for toxicological studies, in particular those related to investigations on endocrine disruption. The development and regulatory use of in vivo and in vitro tests based on this species can be enhanced by toxicokinetic modeling. For this reason, we propose a physiologically based toxicokinetic (PBTK) model for zebrafish describing the uptake and disposition of organic chemicals. The model is based on literature data on zebrafish, other cyprinidae and other fish families, new experimental physiological information (volumes, lipids and water contents) obtained from zebrafish, and chemical-specific parameters predicted by generic models. The relevance of available models predicting the latter parameters was evaluated with respect to gill uptake and partition coefficients in zebrafish. This evaluation benefited from the fact that the influence of confounding factors such as body weight and temperature on ventilation rate was included in our model. The predictions for six chemicals (65 data points) yielded by our PBTK model were compared to available toxicokinetics data for zebrafish and 88% of them were within a factor of 5 of the corresponding experimental values. Sensitivity analysis highlighted that the 1-octanol/water partition coefficient, the metabolism rate, and all the parameters that enable the prediction of assimilation efficiency and partitioning of chemicals need to be precisely determined in order to allow an effective toxicokinetic modeling.

Consequences of a multi-generation exposure to uranium on Caenorhabditis elegans life parameters and sensitivity.

The assessment of toxic effects at biologically and ecologically relevant scales is an important challenge in ecosystem protection. Indeed, stressors may impact populations at much longer term than the usual timescale of toxicity tests. It is therefore important to study the evolutionary response of a population under chronic stress. We performed a 16-generation study to assess the evolution of two populations of the ubiquitous nematode Caenorhabditis elegans in control conditions or exposed to 1.1 mM of uranium. Several generations were selected to assess growth, reproduction, survival, and dose-responses relationships, through exposure to a range of concentrations (from 0 to 1.2 mM U) with all endpoints measured daily. Our experiment showed an adaptation of individuals to experimental conditions (increase of maximal length and decrease of fecundity) for both populations. We also observed an increase of adverse effects (reduction of growth and fertility) as a function of uranium concentration. We pointed out the emergence of population differentiation for reproduction traits. In contrast, no differentiation was observed on growth traits. Our results confirm the importance of assessing environmental risk related to pollutant through multi-generational studies.

Toward a harmonized methodology to analyze field side effects of two pesticide products on earthworms at the EU level.

Before plant protection product (PPP) marketing authorization, a risk assessment for nontarget soil organisms (e.g., earthworms) is required as part of Regulation (EC) No. 1107/2009. Following a stepwise approach, higher tier earthworm field studies are needed if they cannot demonstrate low long-term risk based on laboratory studies. The European guidance for terrestrial ecotoxicology refers to ISO guideline 11268-3 as a standard to conduct earthworm field studies. Assessment of such studies may be challenging, as no European harmonized guidance is available to properly analyze the accuracy, representativeness, and appropriateness of experimental designs, as well as the statistical analysis robustness of results and their scientific reliability. Following the ISO guideline 11268-3, a field study was performed in 2016-2017 (Versailles, France). An assessment of the first year of this field study was performed in agreement with the quality criteria provided in 2006 in the guidance document published by de Jong and collaborators and recommendations by Kula and collaborators that allows describing the protocol and results of earthworm field studies. Not only did we underline the importance of a detailed analysis of raw data on the effects of pesticides on earthworms in field situations, but we also provided recommendations to harmonize protocols for assessing higher tier field studies devoted to earthworms to advance a better assessment of PPP fate and ecotoxicity. In particular, we provided practical field observations related to the study design, pesticide applications, and earthworm sampling. Concurrently, in addition to the conventional earthworm community study, we propose carrying out an assessment of soil function (i.e., organic matter decomposition, soil structuration, etc.) and calculating diversity indices to obtain information about earthworm community dynamics after the application of PPPs. Finally, through field observations, any relevant observation of external and/or internal recovery should be reported. Integr Environ Assess Manag 2023;19:254-271. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Metabolomics-on-a-chip and predictive systems toxicology in microfluidic bioartificial organs.

The world faces complex challenges for chemical hazard assessment. Microfluidic bioartificial organs enable the spatial and temporal control of cell growth and biochemistry, critical for organ-specific metabolic functions and particularly relevant to testing the metabolic dose-response signatures associated with both pharmaceutical and environmental toxicity. Here we present an approach combining a microfluidic system with (1)H NMR-based metabolomic footprinting, as a high-throughput small-molecule screening approach. We characterized the toxicity of several molecules: ammonia (NH(3)), an environmental pollutant leading to metabolic acidosis and liver and kidney toxicity; dimethylsulfoxide (DMSO), a free radical-scavenging solvent; and N-acetyl-para-aminophenol (APAP, or paracetamol), a hepatotoxic analgesic drug. We report organ-specific NH(3) dose-dependent metabolic responses in several microfluidic bioartificial organs (liver, kidney, and cocultures), as well as predictive (99% accuracy for NH(3) and 94% for APAP) compound-specific signatures. Our integration of microtechnology, cell culture in microfluidic biochips, and metabolic profiling opens the development of so-called "metabolomics-on-a-chip" assays in pharmaceutical and environmental toxicology.

Individual-based model of Chironomus riparius population dynamics over several generations to explore adaptation following exposure to uranium-spiked sediments.

Natural populations are chronically exposed to various pollutants over many generations. It is thus crucial to understand and quantify adaptive dynamics of stressed populations in order to increase the relevance of ecotoxicological risk assessment. However, long-term consequences to population exposure are not much studied yet. The present study investigated evolutionary responses of Chironomus riparius populations exposed to uranium (heavy metal pollutant) and to assess the underlying mechanisms. To fulfil our objective, we produced data with organisms exposed to four relevant concentrations of uranium through eight successive generations. We built an individual-based (IBM) model of C. riparius population dynamics to analyse these data and to test several assumptions about the mechanisms involved in the phenotypic changes. The IBM was based on a dynamic energy budget (DEB) model for C. riparius by Pery et al. (2002). DEB models account mathematically for the acquisition and use of energy to describe and predict growth, maintenance, development and reproduction of living organisms. The IBM accounted for the influence of the test conditions on the observations over eight generations and highlighted some trait evolution such as time to emergence and adult size in control conditions. The model was then used to analyse the exposed population data. Our results showed that exposure to uranium led to a phenotypic selection via a differential survival characterised by longer time to emergence and smaller larval maximal size. As a general conclusion, IBMs based on DEB-based modelling developed to analyse multi-generation experiments are very promising for understanding and quantifying long term selection and tolerance mechanisms in a population under toxic stress.

Biology-based modeling to analyze uranium toxicity data on Daphnia magna in a multigeneration study.

Recent studies have investigated chronic toxicity of waterborne depleted uranium on the life cycle and physiology of Daphnia magna. In particular, a reduction in food assimilation was observed. Our aims here were to examine whether this reduction could fully account for observed effects on both growth and reproduction, for three successive generations, and to investigate through microscope analyses whether this reduction resulted from direct damage to the intestinal epithelium. We analyzed data obtained by exposing Daphnia magna to uranium over three successive generations. We used energy-based models, which are both able to fit simultaneously growth and reproduction and are biologically relevant. Two possible modes of action were compared - decrease in food assimilation rate and increase in maintenance costs. In our models, effects were related either to internal concentration or to exposure concentration. The model that fitted the data best represented a decrease in food assimilation related to exposure concentration. Furthermore, observations of consequent histological damage to the intestinal epithelium, together with uranium precipitates in the epithelial cells, supported the assumption that uranium has direct effects on the digestive tract. We were able to model the data in all generations and showed that sensitivity increased from one generation to the next, in particular through a significant increase of the intensity of effect, once the threshold for appearance of effects was exceeded.

A linear model to predict chronic effects of chemicals on Daphnia magna.

Chronic toxicity data for Daphnia magna are information requirements in the context of regulations on chemical safety. This paper proposes a linear model for the prediction of chemically-induced effects on the reproductive output of D. magna. This model is based on data retrieved from the Japanese Ministry of Environment database and it predicts chronic effects as a function of acute toxicity data. The proposed model proved to be able to predict chronic toxicities for chemicals not used in the training set. Our results suggest that experiments involving chronic exposure to chemicals could be reduced thanks to the proposed model.

Substance-tailored testing strategies in toxicology: an in silico methodology based on QSAR modeling of toxicological thresholds and Monte Carlo simulations of toxicological testing.

The design of toxicological testing strategies aimed at identifying the toxic effects of chemicals without (or with a minimal) recourse to animal experimentation is an important issue for toxicological regulations and for industrial decision-making. This article describes an original approach which enables the design of substance-tailored testing strategies with a specified performance in terms of false-positive and false-negative rates. The outcome of toxicological testing is simulated in a different way than previously published articles on the topic. Indeed, toxicological outcomes are simulated not only as a function of the performance of toxicological tests but also as a function of the physico-chemical properties of chemicals. The required inputs for our approach are QSAR predictions for the LOAELs of the toxicological effect of interest and statistical distributions describing the relationship existing between in vivo LOAEL values and results from in vitro tests. Our methodology is able to correctly predict the performance of testing strategies designed to analyze the teratogenic effects of two chemicals: di(2-ethylhexyl)phthalate and Indomethacin. The proposed decision-support methodology can be adapted to any toxicological context as long as a statistical comparison between in vitro and in vivo results is possible and QSAR models for the toxicological effect of interest can be developed.

Comparison of models to analyze mortality data and derive concentration-time response relationship of inhaled chemicals.

The derivation of thresholds for lethal effects for inhaled chemicals is a key issue in accidental risk management because they largely determine the outcome of land use planning, among which localization of habitations in the vicinity of a factory. This derivation is generally performed on the basis of rodent lethality data analyzed by statistical models able to extrapolate effects for different times and concentrations of exposure. A model commonly used in France is the standard probit model. In this model, effects is related to exposure concentration and duration according to the Haber's law and considers that individual thresholds, corresponding to the maximum tolerated effects before dying, are log-normally distributed among the population. This approach has been criticized for its lack of biological parameters and its inability to treat data characterized by only one time of exposure. In order to improve the current state of modeling, we proposed three alternative models. Two of them (DEBtox and Haber-TKTD models) incorporate the kinetics of the chemicals. The third one (Loguniform model) is a linearization of the standard probit model. We evaluated their performance by analyzing real data and simulated data generated with each model. For data characterized by several times of exposure, the standard probit model outperformed all other models in terms of goodness of fits and estimation of parameters. For data characterized by only one time of exposure, only DEBtox model was able to fit the data and estimate parameters, provided we dispose of several observation times, typically just after exposure and a long period afterwards.

An energy-based model to analyze growth data of earthworms exposed to two fungicides.

The pesticide risk assessment for earthworms is currently performed using standardized tests, the model species Eisenia fetida, and the analyses of the data obtained are performed with ad hoc statistical tools. We assessed the impact of two fungicides on the entire growth pattern of the earthworm species Aporrectodea caliginosa, which is highly representative of agricultural fields. Individuals of three different ages (from hatching to 56 days old) were exposed to Cuprafor micro® (copper oxychloride) and Swing® Gold (dimoxystrobin and epoxiconazole). Data were analyzed with an energy-based toxicodynamic model coupled with a toxicokinetic model. The copper fungicide caused a drastic growth inhibition once the no effect concentration (NEC), estimated at 65 mg kg-1 of copper, was exceeded. The Swing® Gold negatively affected the growth with NEC values estimated at 0.387 mg kg-1 and 0.128 mg kg-1 for the dimoxystrobin and the epoxiconazole in this fungicide formulation, respectively. The time-profile of the effects on A. caliginosa individuals was fully accounted for by the model, whatever their age of exposure. Furthermore, toxicity data analyses, supported by measurements of fungicide concentrations in earthworm at the end of the experiment, allowed bettering understanding of the mechanisms of action of the fungicides towards earthworm growth.

Soil dissipation and bioavailability to earthworms of two fungicides under laboratory and field conditions.

The representativeness of laboratory studies of the fate of pesticides in soil in field conditions is questionable. This study aimed at comparing the dissipation and bioavailability to earthworms of two fungicides, dimoxystrobin (DMX) and epoxiconazole (EPX), over 12 months under laboratory and field conditions. In both approaches, the fungicides were applied to the same loamy soil as a formulated mixture at several concentrations. We determined total DMX and EPX concentrations in the soil using exhaustive extraction, their environmental availability using mild extraction and their bioavailability through internal concentrations in exposed earthworms. The initial fungicide application appeared as much better controlled in terms of dose and homogeneity in the laboratory than in the field. One year after application, a similar dissipation rate was observed between the laboratory and field experiments (ca 80% and 60% for DMX and EPX, respectively). Similarly, the ratio of available/total concentrations in soil displayed the same trend whatever the duration and the conditions (field or lab), EPX being more available than DMX. Finally, the environmental bioavailability of the two fungicides to earthworms was heterogeneous in the field, but, in the laboratory, the bioaccumulation was evidenced to be dose-dependent only for DMX. Our findings suggest that the actual fate of the two considered fungicides in the environment is consistent with the one determined in the laboratory, although the conditions differed (e.g., presence of vegetation, endogeic earthworm species). This study allowed better understanding of the fate of the two considered active substances in the soil and underlined the need for more research dedicated to the link between environmental and toxicological bioavailability.

Development of a physiologically based kinetic model for 99m-technetium-labelled carbon nanoparticles inhaled by humans.

Particulate air pollution is associated with respiratory and cardiovascular morbidity and mortality. Recent studies investigated whether and to which extent inhaled ultrafine particles are able to translocate into the bloodstream in humans. However, their conclusions were conflicting. We developed a physiologically based kinetic model for (99m)technetium-labelled carbon nanoparticles (Technegas). The model was designed to analyse imaging data. It includes different translocation rates and kinetics for free technetium, and small and large technetium-labelled particles. It was calibrated with data from an experiment designed to assess the fate of nanoparticles in humans after inhalation of Technegas. The data provided time courses of radioactivity in the liver, stomach, urine, and blood. Parameter estimation was performed in a Bayesian context with Markov chain Monte Carlo (MCMC) techniques. Our analysis points to a likely translocation of particle-bound technetium from lung to blood, at a rate about twofold lower than the transfer rate of free technetium. Notably, restricting the model so that only free technetium would have been able to reach blood circulation resulted in much poorer fits to the experimental data. The percentage of small particles able to translocate was estimated at 12.7% of total particles. The percentage of unbound technetium was estimated at 6.7% of total technetium. To our knowledge, our model is the first PBPK model able to use imaging data to describe the absorption and distribution of nanoparticles. We believe that our modeling approach using Bayesian and MCMC techniques provides a reasonable description on which to base further model refinement.

Stochasticity in physiologically based kinetics models: implications for cancer risk assessment.

In case of low-dose exposure to a substance, its concentration in cells is likely to be stochastic. Assessing the consequences of this stochasticity in toxicological risk assessment requires the coupling of macroscopic dynamics models describing whole-body kinetics with microscopic tools designed to simulate stochasticity. In this article, we propose an approach to approximate stochastic cell concentration of butadiene in the cells of diverse organs. We adapted the dynamics equations of a physiologically based pharmacokinetic (PBPK) model and used a stochastic simulator for the system of equations that we derived. We then coupled kinetics simulations with a deterministic hockey stick model of carcinogenicity. Stochasticity induced substantial modifications relative to dose-response curve, compared with the deterministic situation. In particular, there was nonlinearity in the response and the stochastic apparent threshold was lower than the deterministic one. The approach that we developed could easily be extended to other biological studies to assess the influence of stochasticity at macroscopic scale for compound dynamics at the cell level.

Earthworms Mitigate Pesticide Effects on Soil Microbial Activities.

Earthworms act synergistically with microorganisms in soils. They are ecosystem engineers involved in soil organic matter degradation and nutrient cycling, leading to the modulation of resource availability for all soil organisms. Using a soil microcosm approach, we aimed to assess the influence of the earthworm Aporrectodea caliginosa on the response of soil microbial activities against two fungicides, i.e., Cuprafor Micro® (copper oxychloride, a metal) and Swing® Gold (epoxiconazole and dimoxystrobin, synthetic organic compounds). The potential nitrification activity (PNA) and soil enzyme activities (glucosidase, phosphatase, arylamidase, and urease) involved in biogeochemical cycling were measured at the end of the incubation period, together with earthworm biomass. Two common indices of the soil biochemistry were used to aggregate the response of the soil microbial functioning: the geometric mean (Gmean) and the Soil Quality Index (SQI). At the end of the experiment, the earthworm biomass was not impacted by the fungicide treatments. Overall, in the earthworm-free soil microcosms, the two fungicides significantly increased several soil enzyme and nitrification activities, leading to a higher GMean index as compared to the non-treated control soils. The microbial activity responses depended on the type of activity (nitrification was the most sensitive one), on the fungicide (Swing® Gold or Cuprafor Micro®), and on the doses. The SQI indices revealed higher effects of both fungicides on the soil microbial activity in the absence of earthworms. The presence of earthworms enhanced all soil microbial activities in both the control and fungicide-contaminated soils. Moreover, the magnitude of the fungicide impact, integrated through the SQI index, was mitigated by the presence of earthworms, conferring a higher stability of microbial functional diversity. Our results highlight the importance of biotic interactions in the response of indicators of soil functioning (i.e., microbial activity) to pesticides.