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).

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.

Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses.

Real-time quantitative PCR is nowadays a standard method to study gene expression variations in various samples and experimental conditions. However, to interpret results accurately, data normalization with appropriate reference genes appears to be crucial. The present study describes the identification and the validation of suitable reference genes in Brassica oleracea leaves. Expression stability of eight candidates was tested following drought and cold abiotic stresses by using three different softwares (BestKeeper, NormFinder and geNorm). Four genes (BolC.TUB6, BolC.SAND1, BolC.UBQ2 and BolC.TBP1) emerged as the most stable across the tested conditions. Further gene expression analysis of a drought- and a cold-responsive gene (BolC.DREB2A and BolC.ELIP, respectively), confirmed the stability and the reliability of the identified reference genes when used for normalization in the leaves of B. oleracea. These four genes were finally tested upon a benzene exposure and all appeared to be useful reference genes along this toxicological condition. These results provide a good starting point for future studies involving gene expression measurement on leaves of B. oleracea exposed to environmental modifications.

Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum.

Severe drop in Manila clams production in French aquacultured fields since the end of the 1980's is associated to Brown Ring Disease (BRD). This disease, caused by the bacteria Vibrio tapetis, is characterized by specific symptoms on the inner face of the shell. Diseased animals develop conchiolin deposit to enrobe bacteria and form new calcified layers on the shell. Suppression subtractive hybridization was performed to identify genes differentially expressed during the early interaction of V. tapetis and Ruditapes philippinarum. Results revealed 301 unique genes differentially expressed during V. tapetis challenge. Several candidates involved in immune and biomineralization processes were selected from libraries. Transcriptional expression of selected candidates was determined in hemolymph and mantle tissues and revealed spatial and temporal variations. At 56 days after infection, when clams were in phase of shell repair, transcripts of galectin and ferritin in hemocytes showed higher expression. Ca-like and serpin transcripts were specifically expressed in mantle and could contribute to defense against BRD.

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge.

The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams.

Gene expression analysis of 4 biomarker candidates in Eisenia fetida exposed to an environmental metallic trace elements gradient: a microcosm study.

Past activities of 2 smelters (Metaleurop Nord and Nyrstar) led to the accumulation of high amounts of Metal Trace Elements (TEs) in top soils of the Noyelles-Godault/Auby area, Northern France. Earthworms were exposed to polluted soils collected in this area to study and better understand the physiological changes, the mechanisms of acclimation, and detoxification resulting from TE exposure. Previously we have cloned and transcriptionally characterized potential biomarkers from immune cells of the ecotoxicologically important earthworm species Eisenia fetida exposed in vivo to TE-spiked standard soils. In the present study, analysis of expression kinetics of four candidate indicator genes (Cadmium-metallothionein, coactosin like protein, phytochelatin synthase and lysenin) was performed in E. fetida after microcosm exposures to natural soils exhibiting an environmental cadmium (Cd) gradient in a kinetic manner. TE body burdens were also measured. This microcosm study provided insights into: (1) the ability of the 4 tested genes to serve as expression biomarkers, (2) detoxification processes through the expression analysis of selected genes, and (3) influence of land uses on the response of potential biomarkers (gene expression or TE uptake).

Transcriptomic underpinning of toxicant-mediated physiological function alterations in three terrestrial invertebrate taxa: a review.

Diverse anthropogenic activities often lead to the accumulation of inorganic and organic residues in topsoils. Biota living in close contact with contaminated soils may experience stress at different levels of biological organisation throughout the continuum from the molecular-genetic to ecological and community levels. To date, the relationship between changes at the molecular (mRNA expression) and biochemical/physiological levels evoked by exposures to chemical compounds has been partially established in a limited number of terrestrial invertebrate species. Recently, the advent of a family of transcriptomic tools (e.g. Real-time PCR, Subtractive Suppressive Hybridization, Expressed Sequence Tag sequencing, pyro-sequencing technologies, Microarray chips), together with supporting informatic and statistical procedures, have permitted the robust analyses of global gene expression changes within an ecotoxicological context. This review focuses on how transcriptomics is enlightening our understanding of the molecular-genetic responses of three contrasting terrestrial macroinvertebrate taxa (nematodes, earthworms, and springtails) to inorganics, organics, and agrochemicals.

Metallic trace element body burdens and gene expression analysis of biomarker candidates in Eisenia fetida, using an "exposure/depuration" experimental scheme with field soils.

Smelting plant activities lead to the accumulation of Metal Trace Elements (MTEs) in soils. The presence of high concentrations of MTEs can generate an environmental stress likely to affect macroinvertebrates living in close soil contact such as the Annelida Oligochaeta. Eisenia fetida, an ecotoxicologically important test species, was successively exposed to two field soils: (1) a highly contaminated agricultural topsoil collected near the former smelter Metaleurop Nord (Noyelles-Godault, France) which contaminated surrounding soils by its atmospheric emissions [exposure phase], and then (2) a slightly contaminated topsoil from an urban garden located in the conurbation of Lille (Wambrechies) [depuration phase]. Two analyses were performed during each phase. Firstly, the gene expression levels of four biomarker candidates identified in previous studies were analyzed in E. fetida coelomocytes. These candidates are Cd-metallothionein, phytochelatin synthase, coactosin-like protein and lysenin. Secondly, the body burdens of the following elements Cd, Pb, Zn, Cu, Fe, Ca, and P were measured. Moreover, both analyses were also performed in Lumbricus rubellus, an Annelid species collected from the two tested soil-originating sites. Analysis of gene expression and MTE body burdens in both species are discussed to: (1) evaluate expression biomarkers; (2) gain insight the detoxification processes and the long-term response to a metallic stress and (3) compare the responses observed in a test species (E. fetida) with the responses of a field species (L. rubellus).

Identification and expression profile of gene transcripts differentially expressed during metallic exposure in Eisenia fetida coelomocytes.

The aim of this work was to identify in Eisenia fetida genes whose expression are regulated following exposure to a complex mixture of metallic trace elements (MTE) representative of a highly polluted smelter soil. Suppression subtractive hybridization (SSH) was used to construct cDNA libraries enriched in up- or down-regulated transcripts in the immune-circulating cells of the coelomic cavities, namely coelomocytes, from worms exposed to metallic pollution. Among 1536 SSH-derived cDNA clones sequenced, we identified 764 unique ESTs of which we selected 18 candidates on the basis of their redundancy. These selected candidates were subjected to a two-step validation procedure based on the study of their expression level by real-time PCR. The first step consisted in measuring the expression of the 18 candidates in worms exposed to artificial contaminated soil. The second step consisted in measuring the expression in animals exposed to a "naturally" contaminated soil sampled close to a smelter. Both steps allowed us to highlight 3 candidates that are strongly induced in worms exposed to a smelter polluted soil. These candidates are: the well-known MTE-induced Cd-metallothionein and 2 original biomarkers, lysenin, and a transcript, which cloning of the complete coding sequence identified as the coactosin-like protein.

cDNA cloning and expression analysis of Eisenia fetida (Annelida: Oligochaeta) phytochelatin synthase under cadmium exposure.

Metallothioneins (MTs) are central to trace metal homeostasis and detoxification throughout biological systems. Prokaryotes, plants, and fungi utilize both gene encoded cysteine-rich polypeptides (classically designated Class I and II MTs) and enzymatically synthesized cysteine-rich peptides (classically designated Class III MTs or phytochelatins). In contrast, although gene encoded MTs are ubiquitous in animal species the identification of a functional phytochelatin synthase in the nematode Caenorhabditis elegans, a representative member of the Ecdysozoa, provided the first evidence for these metal-binding peptides in animals. By exploiting the conservation observed between species we have been able to clone and transcriptionally characterize a phytochelatin synthase from the immune cells of the earthworm Eisenia fetida, the first evidence for its presence in a phylum belonging to the Lophototrochozoa. The complete coding sequence of this enzyme was determined and the phylogenetic relationship to plant, yeast and nematode enzymes elucidated. Temporal- and dose-profiling of the transcriptional regulation of phytochelatin synthase and MT in response to cadmium was performed by using real-time PCR.