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).

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.

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.

Aporrectodea caliginosa, a relevant earthworm species for a posteriori pesticide risk assessment: current knowledge and recommendations for culture and experimental design.

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.

How to assess the feeding activity in ecotoxicological laboratory tests using enchytraeids?

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.

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.