Partitioning tissue metal concentration into subcellular compartments relecting 'toxicologically available'pools may provide good descriptors of the toxicological efects of metals on organisms. Here we investigatedthe relationships between internal compartmentalization of Cd, Pb and Zn and biomarker responses in a model soil organism: the earthworm. The aim of this study was to identify metal fractions reflecting the toxic pressure in an endogeic, naturally occurring earthworm species (Aporrectodea caliginosa) exposed torealistic field-contaminated soils.After a 21 days exposure experiment to 31 field contaminated soils, Cd, Pb and Zn concentrationsin earthworms and in three subcellular fractions (cytosol, debris and granules) were quantified. Differentbiomarkers were measured: the expression of a metallothionein gene (mt), the activity of catalase (CAT) andof glutathione-s-transferase (GST), and the protein, lipid and glycogen reserves. Biomarkers were furthercombined in an integrated biomarker index (IBR).The subcellular fractionation provided better predictors of biomarkers than the total internal contentshence supporting its use when assessing toxicological bioavailability of metals to earthworms. The mostsoluble internal pools of metals were not always the best predictors of biomarker responses. metallothioneinexpression responded to increasing concentrations of Cd in the insoluble fraction (debris + granules). Proteinand glycogen contents were also mainly related to Cd and Pb in the insoluble fraction. On the other hand,GST activity was better explained by Pb in the cytosolic fraction. CAT activity and lipid contents variationswere not related to metal subcellular distribution. The IBR was best explained by both soluble and insolublefractions of Cd and Pb.This study further extends the scope of mt expression as a robust and specific biomarker in an ecologicallyrepresentative earthworm species exposed to field-contaminated soils. The genetic lineage of the individuals,assessed by DNA barcoding with cytochrome c oxidase subunit I, did not inuence mt expression.

Partitioning tissue metal concentration into subcellular compartments relecting 'toxicologically available'pools may provide good descriptors of the toxicological efects of metals on organisms. Here we investigatedthe relationships between internal compartmentalization of Cd, Pb and Zn and biomarker responses in a model soil organism: the earthworm. The aim of this study was to identify metal fractions reflecting the toxic pressure in an endogeic, naturally occurring earthworm species (Aporrectodea caliginosa) exposed torealistic field-contaminated soils.After a 21 days exposure experiment to 31 field contaminated soils, Cd, Pb and Zn concentrationsin earthworms and in three subcellular fractions (cytosol, debris and granules) were quantified. Differentbiomarkers were measured: the expression of a metallothionein gene (mt), the activity of catalase (CAT) andof glutathione-s-transferase (GST), and the protein, lipid and glycogen reserves. Biomarkers were furthercombined in an integrated biomarker index (IBR).The subcellular fractionation provided better predictors of biomarkers than the total internal contentshence supporting its use when assessing toxicological bioavailability of metals to earthworms. The mostsoluble internal pools of metals were not always the best predictors of biomarker responses. metallothioneinexpression responded to increasing concentrations of Cd in the insoluble fraction (debris + granules). Proteinand glycogen contents were also mainly related to Cd and Pb in the insoluble fraction. On the other hand,GST activity was better explained by Pb in the cytosolic fraction. CAT activity and lipid contents variationswere not related to metal subcellular distribution. The IBR was best explained by both soluble and insolublefractions of Cd and Pb.This study further extends the scope of mt expression as a robust and specific biomarker in an ecologicallyrepresentative earthworm species exposed to field-contaminated soils. The genetic lineage of the individuals,assessed by DNA barcoding with cytochrome c oxidase subunit I, did not inuence mt expression.

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.

The effects of exposure to different levels of ionising radiation were assessed on the genetic, epigenetic and microbiome characteristics of the “hologenome” of earthworms collected at sites within the Chernobyl exclusion zone (CEZ). The earthworms Aporrectodea caliginosa (Savigny, 1826) and Octolasion lacteum (Örley, 1881) were the two species that were most frequently found at visited sites, however, only O. lacteum was present at sufficient number across different exposure levels to enable comparative hologenome analysis. The identification of morphotype O. lacteum as a probable single clade was established using a combination of mitochondrial (cytochrome oxidase I) and nuclear genome (Amplified Fragment Length Polymorphism (AFLP) using MspI loci). No clear site associated differences in population genetic structure was found between populations using the AFLP marker loci. Further, no relationship between ionising radiation exposure levels and the percentage of methylated loci or pattern of distribution of DNA methylation marks was found. Microbiome structure was clearly site dependent, with gut microbiome community structure and diversity being systematically associated with calculated site-specific earthworm dose rates. There was, however, also co-correlation between earthworm dose rates and other soil properties, notably soil pH; a property known to affect soil bacterial community structure. Such co-correlation means that it is not possible to attribute microbiome changes unequivocally to radionuclide exposure. A better understanding of the relationship between radionuclide exposure soil properties and their interactions on bacterial microbiome community response is, therefore, needed to establish whether these the observed microbiome changes are attributed directly to radiation exposure, other soil properties or to an interaction between multiple variables at sites within the CEZ.

Generic biomarkers are needed to assess environmental risks in metal polluted soils. We assessed the strength of the relationship between earthworm energy reserves and metal availability under conditions of cocktail of metals at low doses and large range of soil parameters. Aporrectodea caliginosa was exposed in laboratory to a panel of soils differing in Cd, Pb and Zn total and available (CaCl2 and EDTA-extractable) concentrations, and in soil texture, pH, CEC and organic-C. Glycogen, protein and lipid contents were recorded in exposed worms. Glycogen contents were not linked to the explaining variables considered. Variable selection identified CaCl2 extractable metals concentrations and soil texture as the main factors affecting protein and lipid contents. The results showed opposite effects of Pb and Zn, high inter-individual variability of biomarkers and weak relationships with easily extractable metals. Our results support the lack of genericity of energy reserves in earthworms exposed to field-contaminated soils.

Sulfonated and hydroxy-sulfonated PCBs were recently discovered by our group as new PCB soil contaminants, constituting about 1% of their parent compounds in soil. Here we investigate for the first time the bioaccumulation of these metabolites as well as hydroxy-PCBs and native PCBs in earthworms. A sequence of three experiments, at increasing complexity and ecological realism, were performed with four different earthworm species (Eisenia foetida Savigny, Lumbricus terrestris L, Allolobophora chlorotica Savigny and Aporrectodea caliginosa Savigny) exposed to contaminated soils. The first experiment confirmed that when exposing earthworms to soil contaminated with a single hexa-chlorinated congener (PCB 155), no formation of polar metabolites in earthworms could be detected. This allowed to plan the following two experiments, using a soil from a PCB contaminated site and rich in relatively high levels (10–130 μg kg⁻¹) of hydroxy-, sulfonated-, and hydroxy-sulfonated-PCBs. Bioaccumulation factors (BAFs) and bioconcentration factors (BCFs) were then obtained in the second and third experiments, to compare the accumulation behavior of these chemicals in laboratory and natural conditions. Regressions between BAF/BCF and Log Kow/Log D, produced a variety of results, being generally significant between BCF and PCBs and not significant in the other cases. In general, the metabolites accumulated in earthworms with detectable concentrations in their tissues (8–115 μg kg⁻¹), although sulfonated and hydroxy-sulfonated PCBs showed BAF and BCF values lower (up to two orders of magnitude) than those calculated for the parent PCBs, given their lower lipophilicity.

Massive use of pesticides in conventional agriculture leads to accumulation in soil of complex mixtures, triggering questions about their potential ecotoxicological risk. This study assessed cropland soils containing pesticide mixtures sampled from conventional and organic farming systems at La Cage and Mons, France. The conventional agricultural field soils contained more pesticide residues (11 and 17 versus 3 and 11, respectively) and at higher concentrations than soils from organic fields (mean 6.6 and 10.5 versus 0.2 and 0.6 μg kg⁻¹, respectively), including systemic insecticides belonging to neonicotinoids, carbamate herbicides and broad-spectrum fungicides mostly from the azole family. A risk quotient (RQᵢ) approach evaluated the toxicity of the pesticide mixtures in soil, assuming concentration addition. Based on measured concentrations, both conventional agricultural soils posed high risks to soil invertebrates, especially due to the presence of epoxiconazole and imidacloprid, whereas soils under organic farming showed negligible to medium risk. To confirm the outcome of the risk assessment, toxicity of the soils was determined in bioassays following standardized test guidelines with seven representative non-target invertebrates: earthworms (Eisenia andrei, Lumbricus rubellus, Aporrectodea caliginosa), enchytraeids (Enchytraeus crypticus), Collembola (Folsomia candida), oribatid mites (Oppia nitens), and snails (Cantareus aspersus). Collembola and enchytraeid survival and reproduction and land snail growth were significantly lower in soils from conventional compared to organic agriculture. The earthworms displayed different responses: L. rubellus showed higher mortality on soils from conventional agriculture and large body mass loss in all field soils, E. andrei showed considerable mass loss and strongly reduced reproduction, and A. caliginosa showed significantly reduced acetylcholinesterase activity in soils from conventional agriculture. The oribatid mites did not show consistent differences between organic and conventional farming soils. These results highlight that conventional agricultural practices pose a high risk for soil invertebrates and may threaten soil functionality, likely due to additive or synergistic “cocktail effects”.

Contaminated soils are lands in Europe deemed less favourable for conventional agriculture. To overcome the problem of their poor fertility, bio-fertilization could be a promising approach. Soil inoculation with a choice of biological species (e.g. earthworm, mycorrhizal fungi, diazotroph bacteria) can be performed in order to improve soil properties and promote nutrients recycling. However, questions arise concerning the dynamics of the contaminants in an inoculated soil.The aim of this study was to highlight the soil-plant-earthworm interactions in the case of a slightly contaminated soil. For this purpose, a pot experiment in controlled conditions was carried out during 2 months with a Cd, Zn, and Cu contaminated sandy soil, including conditions with or without earthworms (Aporrectodea caliginosa) and with or without plants (Lolium perenne).The three components of the trace element bioavailability were studied to understand the belowground-aboveground relationships and were quantified as followed: i) environmental availability in soils by measuring trace element concentrations in soil solution, ii) environmental bioavailability for organisms by measuring trace element concentrations in depurated whole earthworms bodies and in the plant aerial biomass, and iii) toxicological bioavailability, by measuring survival rate and body weight changes for earthworms and biomass for plants. The results showed that earthworm inoculation increased the content of all studied TE in soil solution. Moreover, lower concentrations of Cd and Zn were found in plants in the presence of earthworms while the bioavailability decreased when compared to the condition without plants. The trace element bioaccumulation in earthworms did not produce a direct toxicity, according to the earthworm survival rate and body weight results.Finally, our pot experiment confirmed that even in contaminated soils, the presence of A. caliginosa promotes plant adaptation and improves biomass production, reducing trace element uptake.

The scale of variation in species sensitivity to toxicants has been theoretically linked to mode of action. Specifically, it has been proposed there will be greater variations for chemicals with a putative specific biological target than for toxicants with a non-specific narcotic mechanism. Here we test the hypothesis that mode of action is related to variation in sensitivity in a specifically designed experiment for species from a single ecologically important terrestrial taxa, namely earthworms. Earthworm toxicity tests were conducted with five species for four chemicals, providing a series of increasingly complex modes of action: a putative narcotic polycyclic aromatic hydrocarbon (fluoranthene), and three insecticides (chlorpyrifos, cypermethrin, imidacloprid) with known neuronal receptor targets. Across all the chemicals, the standard epigeic test species Eisenia fetida and Lumbricus rubellus, were generally among the two least sensitive, while the endogenic Aporrectodea caliginosa and Megascolecidae Amynthas gracilis were generally more sensitive (never being among the two least sensitive species). This indicates a potential for bias in the earthworm ecotoxicology literature, which is dominated by studies in epigeic Lumbricidae, but contains few endogeic or Megascolecidae data. Results confirmed the lowest range of variation in sensitivities for effects on reproduction was for fluoranthene (2.5 fold). All insecticides showed greater variation for species sensitivity (cypermethrin: 7.5 fold, chlorpyrifos: 10.3 fold, imidacloprid: 31.5 fold) consistent with the specific mechanisms of the pesticides. Difference in toxicodynamics, based on mode of action specificity and receptor complexity was reflected in the magnitude of sensitivity variation. However, measurements of tissue concentrations also indicated the potential importance of toxicokinetics in explaining species sensitivity variations for chlorpyrifos and cypermethrin.

Partitioning tissue metal concentration into subcellular compartments reflecting toxicologically available pools may provide good descriptors of the toxicological effects of metals on organisms. Here we investigated the relationships between internal compartmentalization of Cd, Pb and Zn and biomarker responses in a model soil organism: the earthworm. The aim of this study was to identify metal fractions reflecting the toxic pressure in an endogeic, naturally occurring earthworm species (Aporrectodea caliginosa) exposed to realistic field-contaminated soils.After a 21 days exposure experiment to 31 field-contaminated soils, Cd, Pb and Zn concentrations in earthworms and in three subcellular fractions (cytosol, debris and granules) were quantified. Different biomarkers were measured: the expression of a metallothionein gene (mt), the activity of catalase (CAT) and of glutathione-s-transferase (GST), and the protein, lipid and glycogen reserves. Biomarkers were further combined into an integrated biomarker index (IBR).The subcellular fractionation provided better predictors of biomarkers than the total internal contents hence supporting its use when assessing toxicological bioavailability of metals to earthworms. The most soluble internal pools of metals were not always the best predictors of biomarker responses. metallothionein expression responded to increasing concentrations of Cd in the insoluble fraction (debris + granules). Protein and glycogen contents were also mainly related to Cd and Pb in the insoluble fraction. On the other hand, GST activity was better explained by Pb in the cytosolic fraction. CAT activity and lipid contents variations were not related to metal subcellular distribution. The IBR was best explained by both soluble and insoluble fractions of Pb and Cd.This study further extends the scope of mt expression as a robust and specific biomarker in an ecologically representative earthworm species exposed to field-contaminated soils. The genetic lineage of the individuals, assessed by DNA barcoding with cytochrome c oxidase subunit I, did not influence mt expression.