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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.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

The risk assessment of pesticides on soil fauna is an issue to protect agroecosystem sustainability. Enchytraeids are recognized as relevant soil bioindicators of chemical stress in agroecosystems. In laboratory, the reproduction test was found to be sensitive to reveal chemical impacts on enchytraeids. However, it does not allow to assess the impacts on ecological functions in which enchytraeids are involved. The objectives of this study were (i) to explore the feasibility of the bait-lamina test with enchytraeids under laboratory conditions and (ii) to compare its sensitivity with the Enchytraeid Reproduction Test. We exposed individuals of Enchytraeus albidus to two commercial formulations of fungicides widely used in Europe. The EC50 reproduction for the Swing ® Gold (50 g L -1 epoxiconazole and 133 g L -1 dimoxystrobin) and the Cuprafor micro ® (50% copper oxychloride) were respectively estimated at 1.66 ± 0.93 times the recommended dose and > 496 mg kg -1 of copper. However, no impact was found on the feeding activity of enchytraeids. The bait-lamina test thus appeared less sensitive than the Enchytraeid Reproduction Test to the tested fungicides. Despite that, this test which is achievable under laboratory conditions and allows assessing indirect effects of pesticides is quick, cheap, and easy to perform. It would deserve to be used to explore longer-exposure effects through the repeated addition of bait-lamina sticks.

Ecotoxicological tests with earthworms are widely used and are mandatory for the risk assessment of pesticides prior to registration and commercial use. The current model species for standardized tests is Eisenia fetida or Eisenia andrei. However, these species are absent from agricultural soils and often less sensitive to pesticides than other earthworm species found in mineral soils. To move towards a better assessment of pesticide effects on non-target organisms, there is a need to perform a posteriori tests using relevant species. The endogeic species Aporrectodea caliginosa (Savigny, 1826) is representative of cultivated fields in temperate regions and is suggested as a relevant model test species. After providing information on its taxonomy, biology, and ecology, we reviewed current knowledge concerning its sensitivity towards pesticides. Moreover, we highlighted research gaps and promising perspectives. Finally, advice and recommendations are given for the establishment of laboratory cultures and experiments using this soil-dwelling earthworm species.

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.

Integrated Risk Assessment (IRA) has been defined by the EU FP7 HEROIC Coordination action as "the mutual exploitation of Environmental Risk Assessment for Human Health Risk Assessment and vice versa in order to coherently and more efficiently characterize an overall risk to humans and the environment for better informing the risk analysis process" (Wilks et al., 2015). Since exposure assessment and hazard characterization are the pillars of risk assessment, integrating Environmental Exposure assessment (EEA) and Human Exposure assessment (HEA) is a major component of an IRA framework. EEA and HEA typically pursue different targets, protection goals and timeframe. However, human and wildlife species also share the same environment and they similarly inhale air and ingest water and food through often similar overlapping pathways of exposure. Fate models used in EEA and HEA to predict the chemicals distribution among physical and biological media are essentially based on common properties of chemicals, and internal concentration estimations are largely based on inter-species (i.e. biota-to-human) extrapolations. Also, both EEA and HEA are challenged by increasing scientific complexity and resources constraints. Altogether, these points create the need for a better exploitation of all currently existing data, experimental approaches and modeling tools and it is assumed that a more integrated approach of both EEA and HEA may be part of the solution. Based on the outcome of an Expert Workshop on Extrapolations in Integrated Exposure Assessment organized by the HEROIC project in January 2014, this paper identifies perspectives and recommendations to better harmonize and extrapolate exposure assessment data, models and methods between Human Health and Environmental Risk Assessments to support the further development and promotion of the concept of IRA. Ultimately, these recommendations may feed into guidance showing when and how to apply IRA in the regulatory decision-making process for chemicals., (Copyright © 2015 Elsevier B.V. All rights reserved.)