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Background and aim Plant pathogenic and mycotoxin-producing Fusarium species are globally widespread and lead to large annual yield losses in maize production (ecosystem disservice). Systems with reduced tillage and mulching are particularly under threat. In the present study, the bioregulatory performance (ecosystem service) of the common earthworm species Lumbricus terrestris was analysed regarding the suppression of three economically relevant Fusarium species, and the reduction of their mycotoxins in the maize mulch layer, taking into account the size of maize residues. Methods A mesocosm field experiment was conducted in a reduced tillage long-term field trial on loam soil. Artificially Fusarium-infected maize residues of two size classes were used as a mulch layer. Impacts of the earthworm species on DNA amounts of Fusarium graminearum, F. culmorum, and F. verticillioides and concentrations of the mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), and zearalenone (ZEN) were analysed. Results The results reflect that Fusarium regulation by L. terrestris was species-specific and covered the whole spectrum from suppression (F. graminearum) to slight promotion (F. verticillioides). Regarding the mycotoxins, a significant acceleration of the degradation of all three toxins was detected. Fine chopping of the chaff (< 2 cm) did not significantly alter the earthworms’ regulatory capacity. Conclusion While L. terrestris can shift the ecosystem service/disservice balance in both directions with respect to Fusarium regulation, it shifts it towards ecosystem services with respect to mycotoxin degradation. In synergy with adapted agricultural management, this natural bottom-up effect can help to keep soils healthy for sustainable production in the long run.

In ecotoxicological studies, oxidative damages and antioxidant defense responses in earthworms have been used as hallmarks for assessing the environmental pollution. Phthalates, which are esters of phthalic acid (PAEs) are primarily used to enhance plasticity of industrial polymers, and are an unavoidable part of modern life. In the present study, ecotoxicological effects of dimethyl phthalate (DMP) were investigated in the earthworms Lumbricus terrestris L., at different concentrations (0, 0.1, 1, 10 and 50 mg kg-1) on the 7th and 14th days of exposure in artificial soils. We evaluated the effects of DMP on oxidative stress (OS) biomarkers including malondialdehyde (MDA/LPO) level and the activities of antioxidant enzymes catalase (CAT) and glutathione S-transferase (GST), and carbonylated proteins (CP) content in earthworm’s whole body extract. The neutral red retention (NRR)-time of coelomocytes and total antioxidant capacity (TAC) were also measured in the earthworm’s coelomic fluid (ECF). Upon exposure to DMP, OS status was induced in a time and concentration-dependent manner. The MDA level, CAT and GST activities, and CP content were found to be increased, but the TAC value and NRR-times were decreased in all DMP-treated groups on 7th and 14th days of exposure. The toxicity of DMP was generally greater in the groups with 14 day toxicant exposure. Briefly, our resultes defined that DMP is a source of oxidative damages in the earthworms. However, for detailed elucidation of the mechanisms associated with DMP ecotoxicity and its adverse impacts in the soli organisms, further unraveling is needed.

Enantioselectivity (defined as the enantiomer fraction, EF) in sorption, dissipation and bioaccumulation was assessed for the model compound tebuconazole. Tebuconazole sorption in soils was affected by organic carbon (OC) and clay contents and cation exchange capacity, and appeared to be non-enantioselective for both tested soils, soil components and soil amendments. The bioaccumulation test consisted of two phases: the uptake phase and the elimination phase including the assessment of enantiospecific uptake and elimination rates. Unlike dissipation in soils, bioaccumulation of tebuconazole from these soils was enantioselective for both tested earthworm species (Eisenia andrei and Lumbricus terrestris). Peak-shaped bioaccumulation profiles were observed for all tested earthworm–soil variants with the maximum tissue concentrations reached within 7–10 days of exposure. Bioaccumulation factors ranged from 0.03 to 0.87. EF values of earthworm extracts varied from 0.28 to 0.52 with EF values

Highlights • Asiaticoside –D (AD) impact of ganglioside in cerebral ganglions (CGs) of rotenone (ROT)- induced Lumbricus terestris. • Effects of AD on variations of mRNA and protein in CGs of ROT- induced. • AD could maintain the neuroinflammation of ganglionic degenerative worms., Asiaticoside-D (AD) was shown to efficacy of ganglionic degenerated Lumbricus terrestris as a pioneering observation in our earlier research. Though, extract molecular mechanisms of AD for degenerative diseases (DDs) remains largely unknown. We investigated the neuroprotective effects of AD against ROT in cerebral ganglions (CGs) of degenerative L. terrestris. Worms were exposed to 0.4 ppm ROT for 7 days were subjected to co- treatment with 15 ppm of AD. After, CGs was removed. The levels oxidant, non-antioxidant, antioxidant status, ganglioside, ceramide and ceramide glycanase (CGase) were estimated. The m-RNA levels of dopamine transporter (DAT), octopamine transporter (OAT), innexins-9 (inx-9), ionotropic glutamate receptor 3 (iGlu3), heat shock proteins (hsp70), XPRLamide neuropeptide precursor, tyramine beta-hydroxylase (tbh-1) and β- adrenergic receptor kinase-2 (β-ARK2–3) by semi-qRT- PCR. The expression pattern of tyramine beta hydroxylase (TBH), glutamate receptor (iGluR), serotonin transporter (SERT), dopamine transporters (DAT), nerve growth factors (NGF), cytochrome C oxidase (COC), NADH dehydogenase subunit-1 (ND-1), neurotrophin receptor p75 (p75NTR), neuronal nitric oxiside synthase (nNOs) interleukin 1- beta (IL1-β) and tumor necrosis factor alpha (TNF-α) by western blotting. Glutaminergic, serotogenic and dopaminergic toxicity variations were also performed. The levels of oxidant, non-antioxidant, antioxidant status, lipids, proteins and m-RNAs were significantly altered (p

Rotenone (ROT) was shown to affect cerebral ganglions (CGs) of Lumbricus terrestris as a pioneering observation in our earlier investigation. Though ROT is a well-known neurotoxin causing neurodegeneration (ND), the precipitation of movement dysfunction remains largely unknown. We have designed the current study to analyze motor abnormalities in worms by exposing them to different concentrations (0.0-0.4 ppm) of ROT for 7 days. GABA, cholinergic receptor, serotonin transporter (SERT), acetylcholine esterase (AchE), and dopamine-β-hydroxylase that are well known for their involvement in neuromuscular junctions were investigated by qRT-PCR. Further, neuronal mitochondrial genes (cytochrome C oxidase-2, NADH deydrogenase-1, cytochrome-b) and actin-1 that are essential for regeneration and calreticulin (phagocytosis) were investigated. The levels of neurotransmitters, lipids, ATPase, neuronal behavior analyses, and fluorescence analysis (lipid droplets) were performed in CGs which showed significant variations at 0.3 ppm. Ultrastructural changes in lipid droplet and neuromelatonin were prominent in 0.3 ppm. Dose-dependent effect of ROT on behavior alteration and expression of m-RNAs studied suggested that at 0.3 ppm, it could deteriorate motor and cognitive functions. We predict that perhaps, by virtue of its effect on cerebral ganglionic genes and their neurotransmitting potential, ROT may cause morbidities that resemble features characteristic of hemiparkinsonic degeneration.

Earthworms have a profound effect on the soil they inhabit, but our current understanding of the chemical factors and molecular mechanisms that attract or repel earthworms from a specific area of soil is limited. In this study, we developed a soil T-maze assay allowing for the examination of earthworm behavioral responses to repellent chemicals. This tests earthworms individually, and thus the potential confounds of worms being influenced by conspecifics signals are avoided. Canadian nightcrawlers (Lumbricus terrestris) can detect the canonical Transient Receptor Potential Ankyrin 1 (TRPA1) agonist, allyl isothiocyanate (AITC), but not the canonical Transient Receptor Potential Vanilloid 1 (TRPV1) agonist capsaicin. In the T-maze assay L. terrestris avoided TRPA1 activators AITC, cinnamaldehyde and menthol in a dosage dependent manner. The single subject T-maze assay is a useful tool to study the avoidance of earthworms to chemical compounds and provides behavioral evidence for a TRPA1 ortholog in L. terrestris.

BackgroundGlyphosate-based herbicides (GBHs) are among the most often used pesticides. The hundreds of GBHs used worldwide consist of the active ingredient (AI) glyphosate in form of different salts, possibly other AIs, and various mostly undisclosed co-formulants. Pesticide risk assessments are commonly performed using single AIs or GBHs at standard soil conditions without vegetation. In a greenhouse experiment, we established a weed population with common amaranth (Amaranthus retroflexus)to examine the effects of three GBHs (Roundup LB Plus, Roundup PowerFlex, Touchdown Quattro) and their corresponding AIs (salts of glyphosate isopropylammonium, potassium, diammonium) on the activity and physiological biomarkers (glutathione S-transferase, GST; acetylcholine esterase, AChE) of an ecologically relevant earthworm species (Lumbricus terrestris). GBHs and AIs were applied at recommended doses; hand weeding served as control. Experiments were established with two soil types differing in organic matter content (SOM; 3.0% vs. 4.1%) and other properties.ResultsEarthworm activity (casting and movement activity) decreased after application of glyphosate formulations or active ingredients compared to hand weeding. We found no consistent pattern that formulations had either higher or lower effects on earthworm activity than their active ingredients; rather, differences were substance-specific. Earthworm activity was little affected by soil organic matter levels. Biomarkers remained unaffected by weed control types; GST but not AChE was decreased under high SOM. Water infiltration after a simulated heavy rainfall was interactively affected by weed control types and SOM. Leachate amount was higher after application of formulations than active ingredients and was higher under low SOM. Glyphosate concentrations in soil and leachate were strongly affected by application of formulations or active ingredients and varied with SOM (significant weed control type x SOM interaction).ConclusionsWe found that both commercial formulations and pure active ingredients can influence earthworms with consequences on important soil functions. Glyphosate products showed increased, reduced or similar effects than pure glyphosate on particular soil functions; soil properties can substantially alter this. Especially at lower SOM, heavy rainfalls could lead to more glyphosate leaching into water bodies. A full disclosure of co-formulants would be necessary to further decipher their specific contributions to these inconsistent effects.

Discharge of untreated industrial effluents has been associated with soil contamination. Biochemical alterations in Lumbricus terrestris and remediation capacity of Azadirachta indica tree from industrial effluent discharge locations in Challawa (A and B) and Kura village (control location C) were assessed. Levels of nitrate, phosphate, iron, chloride, and hardness at locations A and B were significantly (p p p Azadirachta indica tree (AIT) had a translocation factor (TF) > 1 for Pb and Zn, while its biological concentration factor (BCF) was > 1 for Fe. Values of biological accumulation coefficient (BAC) for AIT and soil contamination factor (CF) were p p earthworm supernatants from discharge locations was significantly lower (p L. terrestris may serve as sensitive bioindicators of soil contamination.

Polylactic acid (PLA) bioplastic was introduced to the market as an environmentally friendly potential solution for plastic pollution. However, the effects of bioplastic debris mixed with composts on soil macroinvertebrates, plant growth and soil conditions are still unknown. Soil macroinvertebrates are soil health indicators. A reduction in their abundance is a sign of soil degradation. The objectives of this study were (i) to assess PLA debris in greenhouse composts, and (ii) to test the ecotoxicological effects of PLA debris mixed with compost on Lumbricus terrestris, a soil organism model, and on Triticum aestevium, a plant growth model. The study was comprised of three stages: (1) determine the PLA debris size distribution in composts, (2) assess the ecotoxicological effects of real-world concentrations (0% to 5%) of PLA mixed with compost on earthworm mortality and reproduction, and (3) assess the influence of compost mixed with real-world PLA concentrations on plant growth and physicochemical soil conditions. One percent of PLA debris was found in green composts, 40% of composted PLA debris measured between 1–10 mm, with a concentration of 82.8 ± 17.4 microplastics.gram-1 compost. A concentration of 1% PLA in composts resulted in significant mortality in earthworms. No significant effects of PLA mixed with composts were observed on plant growth or soil physicochemical conditions. Further studies are required in order to test the effect of this biopolymer on different earthworm and plant’ species.

27 pages, Soil-dwelling naturally occurring earthworms (e.g. Lumbricus terrestris) are valuable sentinels in soil pollution monitoring for their ecological role but also because they have shown to be sensitive to environmental contaminants. However, most laboratory studies have adopted epigeic earthworms as models (Eisenia spp.) in acute toxicity testing. In soil chronic toxicity assessment, it is essential to include sublethal responses that can have direct implications on species performance, reproduction and behaviour and thus be of ecological significance. In this sense, some biochemical biomarkers are regarded as early warning signals of further ecological consequences. Amongst those most frequently considered are specific responses to certain chemicals (e.g. metallothionein induction to metal exposure) but also those related to oxidative homeostasis of the organisms because prolonged stress may lead to adverse effects at the individual level (disruption of immune system, altered growth and reproduction). Biomarker measures can be applied in specific tissues, but, for methodological constraints, the consideration of the whole animal simplifies protocols and, once validated, they are informative and integrative. The use of non-destructive tissues (e.g. coelomocytes) that do not require sacrifice, the incorporation of “omic” disciplines and recent technical advances in metabolite identification are all encouraged to be incorporated into toxicity evaluation, To the EU Water JPI-2015 AWARE project (PCIN-2017-067), D. Nos and D. Romano are thanked for their contribution to the project

We investigated the dynamics of different earthworm species through the soil profile, which had received short-term amendments of either organic or inorganic fertilizer in an orchard during the spring of 2016. Earthworm populations were sampled at two consecutive depths of soil on 20 and 60 days after addition of fertilizers. The dominant earthworm species were Eisenia fetida (Savigny, 1826), Dendrobaena veneta (Rosa, 1886), Dendrobaena hortensis (Michaelsen, 1890), Lumbricus terrestris (Linnaeus, 1758) and Aporrectodea longa (Ude, 1885). Seven different types of fertilizers including Urea, Ammonium sulfate (AS), Diammonium phosphate (DAP), Solupotas, NPK (15-5-25), (NPK+OM) and organic manure (OM) as experimental treatments were studied. Fluctuations in earthworm numbers and biomass were attributed to changes in time and depth of sampling, in addition to the types of fertilizers. The results of the means comparison showed that on the 20th day, at 0-20 cm soil depth, E. fetida species abundance was decreased significantly in AS and Urea treatments compared to the control plot (p˂0.05). We highlighted that from epigeic group, D. hortensis species had a better chance to survive and its population in AS treatment was more than that of Urea treatment. Results also showed that the anecic L. terrestris , had a greater tolerance to chemical fertilizers compared to the A. Longa species. The results of this study outline more clear horizons in managing the use of chemical fertilizers while simultaneously maintaining the biodiversity of soil organisms.

Polyethylene mulch films used in agriculture are a major source of plastic pollution in soils. Biodegradable plastics have been introduced as alternative to commonly-used polyethylene. Here we studied the interaction of earthworms (Lumbricus terrestris) with polyethylene and biodegradable plastic mulches. The objective was to assess whether earthworms would select between different types of mulches when foraging for food, and whether they drag macroscopic plastic mulch into the soil. Laboratory experiments were carried out with earthworms in Petri dishes and mesocosms. The treatments were standard polyethylene mulch, four biodegradable plastic mulches (PLA/PHA [polylactic acid/polyhydroxy alkanoate], Organix, BioAgri, Naturecycle), a biodegradable paper mulch (WeedGuardPlus), and poplar litter, which served as control. Four and three replicates for the Petri dish and mesocosm experiments were used, respectively. Macroscopic plastic and paper mulch pieces (1.5 cm × 1.5 cm and 2 cm × 2 cm) were collected from an agricultural field after a growing season, after being buried in the soil for 6 and 12 months, and after being composted for 2 weeks. We found that earthworms did not ingest polyethylene. Field-weathered biodegradable plastic mulches were not ingested either, however, after soil burial and composting, some biodegradable plastics were eaten and could not be recovered from soil any longer. Earthworms, when foraging for food, dragged plastic mulch, including polyethylene and biodegradable plastic, and poplar leaves into their burrows. The burial of macroscopic plastic mulch underground led to a redistribution of plastics in the soil profile, and likely enhances the degradation of biodegradable mulches in soil, but also can lead to leaching of plastic fragments by macropore flow.

Nanopesticides are novel plant protection products offering numerous benefits. Because nanoparticles behave differently from dissolved chemicals, the environmental risks of these materials could differ from conventional pesticides. We used soil-earthworm systems to compare the fate and uptake of analytical-grade bifenthrin to that of bifenthrin in traditional and nanoencapsulated formulations. Apparent sorption coefficients for bifenthrin were up to 3.8 times lower in the nano treatments than in the non-nano treatments, whereas dissipation half-lives of the nano treatments were up to 2 times longer. Earthworms in the nano treatments accumulated approximately 50% more bifenthrin than those in the non-nano treatments. In the non-nano treatments, most of the accumulated material was found in the earthworm tissue, whereas in the nano treatments, the majority resided in the gut. Evaluation of toxicokinetic modeling approaches showed that models incorporating the release rate of bifenthrin from the nanocapsule and distribution within the earthworm provided the best estimations of uptake from the nano-formulations. Overall, our findings indicate that the risks of nanopesticides may be different from those of conventional formulations. The modeling presented provides a starting point for assessing risks of these materials but needs to be further developed to better consider the behavior of the nanoencapsulated pesticide within the gut system. Environ Toxicol Chem 2018;37:1420-1429. © 2018 SETAC.

The study aims to analyse the effects of the herbicide glyphosate (Roundup 360 ® ) on three key earthworm species. The taxa collected from treated and untreated sites were subsequently bred in uncontaminated soil terraria with serial concentrations of the herbicide. The chosen taxa included two anecic ( Lumbricus terrestris and Octodrilus complanatus ) and one endogeic ( Aporrectodea caliginosa ) earthworms. Lumbricus terrestris collected from uncontaminated soil (Lt1) and in a conventionally managed vineyard (Lt2). Aporrectodea caliginosa and Octodrilus complanatus collected from the same uncontaminated site where L. terrestris (Lt1) were collected. Uncontaminated natural grassland soil filling the terraria was characterized for physical-chemical properties. The reproduction rate expressed in earthworms cocoon production was the measure for the effects on worms survival after 21 and 42 days of exposure to 0.59, 2.9, 5.79 g/m 2 of glyphosate, where the dose applied in vineyards in the North-East of Italy goes from 0.72 g/m 2 to 4.32 g/m 2 . Earthworms in untreated terraria were found all alive, while specimens exposed to glyphosate (Roundup 360 ® ) showed a decreasing survival rate and a sharp decline in the number of cocoons. L. terrestris (Lt2) showed a survival rate between 64% and 92% after 21 days, and between 44% and 76% after 42 days. L. terrestris (Lt1) showed a lower resistance to herbicide (survival rate: 36%–84% after 21 days and 12%–76% after 42 days). A. caliginosa was also affected (survival rate: 32%–76% after 21 days and 12%–68% after 42 days). Glyphosate demonstrated severe effects on Oc. complanatus, collected on non-contaminated soils, with the lowest values of survival rate (33% after 21 days and 7% after 42 days of exposure). A significant reduction in the cocoons number (about 70%) was observed for L. terrestris (Lt1) and A. caliginosa after 21 days of exposure, whereas L. terrestris (Lt2) showed about 50% of cocoon production. Results indicate the occurrence of some resistance mechanisms on anecic earthworms in vineyards that have been exposed to glyphosate for at least three decades. However in spite of the long period of application of glyphosate the impact of this largely applied herbicide is still serious (up to 26% of mortality) especially on the deep-burrowing earthworms species ( Oc. complanatus and L. terrestris ).

The earthworm Lumbricus terrestris is an anecic species living in natural soils but it is also a sentinel in pollution monitoring. Specimens of L.terrestris were exposed for 48 h though the filter paper contact test at 1 mg/mL of the chemicals: Lamotrigine (LMG), Cocaine (COC), Fipronil (FIP) and the pesticide bis-4-nitrophenyl phosphate (BNPP). After that period, the activities of Acetylcholinesterase, Glutathione S-transferase, Carboxylesterase (CE) using different substrates, and lipid peroxidation levels were evaluated in the exposed whole tissue earthworms. The results revealed differences only in CE activity, with 4-nitrophenyl butyrate (4NPB) and 1-naphthyl butyrate (1NB) the most responsive substrates to COC. The kinetic parameters of CE were characterized, for the first time, in whole tissue of this species. The chemical analysis by LC-MS/MS, confirmed the exposure to the parent compounds, identified metabolites and evidenced biotransformation pathways in earthworms. Metabolic reactions included oxidation (LMG and FIP), hydrolysis (COC and FIP) as well as glycosylation (LMG, COC and FIP). A hitherto unknown metabolite of LMG due to the conjugation with phenylalanine glutamine was formed. The in vivo results on CE activity with the specific inhibitor, BNPP, were confirmed in vitro. Moreover, in the in vitro approach, the inclusion of other contaminants of environmental concern supports the potential of CE as biomarker. This study identifies the main metabolites formed by earthworms for further in vivo exposures under more realistic conditions and the potential use of CE measures as biomarker of emerging contaminants.

Agrochemicals are an important component of agricultural production systems. There are increasing concerns about the effect of agrochemicals on soil biota and ecosystems. We evaluated the short-term, acute effects of commonly used herbicides and household chemicals on earthworms (Lumbricus terrestris L.). The experiment was conducted on 19 Feb. 2018 (Exp. 1) and repeated on 27 Jun. 2018 (Exp. 2). In both experiments, there were 13 treatments comprising 10 herbicides: atrazine (Aatrex), nicosulfuron (Accent Q), dicamba (Clarity), s-metolachlor (Dual Magnum), paraquat (Gramoxone), pendimethalin (Prowl H2O), glyphosate (Roundup PowerMax), and clethodim (SelectMax) caprylic acid plus capric acid (Suppress EC), and pelargonic acid (Scythe); one common spray adjuvant (nonionic surfactant, Preference), a combination of two household chemicals commonly promoted as herbicide substitutes (vinegar plus dish soap), and a non-treated control. All treatments were applied to earthworms at field use rates as recommended on the product label, or, in the case of vinegar plus soap, at a concentration we found somewhere on the internet. Treatments were arranged in a completely randomized design with 10 replicates. Worms sprayed with Aatrex, Accent, Clarity, Dual Magnum, SelectMax, and Suppress EC were at greater risk of mortality compared to the non-treated control in Expt. 1, but in Expt. 2, chemical treatments did not increase the risk of worm mortality. Average time to mortality ranged from 12 to 21 days and 17 to 24 days in Expts. 1 and 2, respectively. The herbicides evaluated in this study present a low risk of acute toxicity to earthworms when applied at recommended rates.

Background The article shows the influence of the insecticide Karbofos, the active ingredient of which is malathion, on soil and water biota. Various test organisms were used to determine the toxicity of the insecticide. Karbofos is used in the country's agriculture in the fight against various pests. Aims and Objectives The purpose of this work is to study the effect of the pesticide karbofos on soil and water biota: - determination of the toxic effect of the drug on seedlings of tomato seeds ( Solanum lycopersicum) , cucumber ( Cucumis sativus ) and wheat ( Triticum ); - study of the impact on the simplest aquatic organisms ciliates-shoes ( Paramecium сaudatum ); - study of the effect of the drug on earthworms ( Lumbricus terrestris L.) and microorganisms living in the soil; - study of the biodegradation of malathion using soil microorganisms. Methods The material for the experiments was the drug karbofos, in which the active substance is malathion, and the soil for the isolation of soil microorganisms capable of destroying the insecticide, selected in the ecologically clean territory of the Ufa region of the Republic of Bashkortostan. In the presented work, studies on phytotoxicity were carried out, the cultures of tomato ( Solanum lycopersicum ), cucumber ( Cucumis sativus ) and wheat ( Triticum ) were used as test objects; this preparation is often used in agriculture to treat these plants. Experiments to assess the toxicity of the insecticide to protozoan aquatic organisms were carried out on slipper ciliates ( Paramecium caudatum ). The research was carried out using BIOTESTER-2 device. Experiments on the effect of malophos on earthworms were carried out, taking into account their behavior, the condition of contact of earthworms ( Lumbricus sterrestris L .) with the drug was modeled. Determination of the number of microorganisms in the soil sample before and after exposure to insecticide at concentrations of 1 industrial concentration (IC), 2 IC and 5 IC was carried out by the method of «prints» in Petri dishes in beef-extract agar-agar, taken as a nutrient medium. Suspended soil samples were inoculated and cultivated for 2 to 5 days in a thermostat at a temperature of 28 °C. The microorganisms used malathion as a source of carbon and energy. The change in the destructive material was determined using a thin-layer chromatogram ( TLC ) using Silufol UV-254 plates, acetone as eluent, phosphoric-molybdic acid developer ( Rf = 0.5 ). Chromato-mass spectral analysis was performed on a Shimadzu GC-9A instrument. Results Experiments on the study of phytotoxicity showed that after treatment of seeds with the drug karbofos with different concentrations: IC, 2IC, 5IC and control (without the drug), the lengths of the aboveground and underground parts of plants that were etched differ from the lengths of the control -sample. The effect of the increasing dose is depressing in nature. Biotesting of the insecticide carbophos on infusoria-shoes showed that the toxicity index of the IC insecticide is 0.75 (group IV), therefore, the studied drug has a high degree of toxicity. The results of the analysis of the effect of the malathion-based insecticide on earthworms showed that the drug has a high toxic effect. In an experiment to establish the effect of an insecticide on soil bacteria, it was found that in the soil untreated with a toxicant, the microorganisms are dominated by Pseudomonas sp., Bacillus sp., Arthrobacter sp., Azotobacter sp., Flavobacterium sp. and Penicilium sp. The suppression and decrease in the number of microorganisms occurs already with IC. Contamination with 5 IC leads to the disappearance of Azotobacter sp., Arthrobacter sp. and Flavobacterium sp. from the soil microenvironment. The toxic effect of malathion is resisted by bacteria of the species Pseudomonas sp. and Bacillus. sp. Monitoring of the biodegradation of the studied drug, which was carried out using multinuclear 1H and 31P NMR spectroscopy, showed that the pesticide molecule practically does not undergo transformation even after 154 days.

Rotenone is well-documented to cause neurodegenerative condition such as Parkinson's, in the exposed systems. However, its detrimental effect on particular sites of neuronal pathway is still under investigation. We aimed at elucidating the impact of rotenone on cerebral ganglions (CG) of Lumbricus terrestris which control movement and behaviour of the worms. Worms were exposed to 0-0.4 ppm/mL of rotenone. Mitochondrial and lysosomal integrities were found to be affected beyond 0.2 ppm/mL of rotenone. Activities of cholinergic enzymes and the expression of tyrosine hydroxylase showed an impaired neuronal transmission in CGs at the dose of 0.2 ppm/mL of rotenone. Histopathological and immunoflourescent analyses showed neuronal apoptosis, reduced nucleic acid content and inhibited of neurosecretion at 0.3 ppm/mL. Electron microscopy showed that the neurons and neuromuscular junctions were affected at 0.2 ppm/mL. Dose-dependent changes were also observed in the motor function such as burrowing behaviours and locomotion. Conduction velocity (CV) and locomotion assessment showed that the CV of lateral giant fiber (LGF) was reduced while that of MGF remains unaffected at 0.2 ppm, the dose at which the burrowing behaviour was also not affected. LGF, cholinergic enzymes and tyrosine hydroxylase are primarily targeted by rotenone affecting locomotion at 0.2 ppm/mL while MGF, neuropile and the burrowing behaviour were affected at 0.3 ppm/mL. We demonstrate, in addition to dose-dependent effects, that the bioaccumulation factors range 0.28-0.32 ppm/μg of rotenone cause degenerative impact on giant fibers affecting neuronal behaviors/locomotion of worms. We also propose worms for studying mechanisms of neuronal pathology caused by chemicals prevailing in earth's atmosphere.

Soil enzyme activities are attracting widespread interest due to its potential use in contaminant breakdown, and as indicators of soil deterioration. However, given the multiple environmental and methodological factors affecting their activity levels, assessment of soil pollution using these biochemical endpoints is still complex. Taking advantage of the well-known stimulatory effect of earthworms on soil microbes, and their associated enzyme activities, we explored some toxicological features of carboxylesterases (CbEs) in soils inoculated with Lumbricus terrestris. A microplate-scale spectrophotometric assay using soil-water suspensions was first optimized, in which kinetic assay parameters (Km, Vmax, dilution of soil homogenate, and duration of soil homogenization) were established for further CbE determinations. Optimal conditions included a soil-to-water ratio of 1:50 (w/v), 30-min of shaking, and 2.5mM of substrate concentration. As expected, CbE activity increased significantly in soils treated with L. terrestris. This bioturbed soil was used for exploring the role of CbE activity as a bioscavenger for organophosphorus (OP) pesticides. Soil treated with two formulations of chlorpyrifos revealed that CbE activity was a significant molecular sink for this pesticide, reducing its impact on soil microbial activity as shown by the unchanged dehydrogenase activity. Dose-dependent curves were adjusted to an exponential kinetic model, and the median ecological dose (ED50) for both pesticide formulations was calculated. ED50 values decreased as the time of pesticide exposure increased (14 d-ED50s=20.4-26.7 mg kg(-1), and 28 d-ED50s=1.8-2.3 mg kg(-1)), which suggested that chlorpyrifos was progressively transformed into its highly toxic metabolite chlorpyrifos-oxon, but simultaneously was inactivated by CbEs. These results were confirmed by in vitro assays that showed chlorpyrifos-oxon was a more potent CbE inhibitor (IC50=35.5-4.67 nM) than chlorpyrifos (0.41-0.84 μM). The results showed that earthworm-induced CbE activity is an efficient bioscavengers for OP pesticides, acting as a soil safeguarding system. Moreover, the simple dose-response curves against OP exposure suggest that this enzyme--combined with other enzyme activities (e.g., dehydrogenase)--may be a suitable biomarker of pesticide exposure.

International audience; 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.

Background The article shows the effect of the insecticide «Zemlin», the active substance of which is diazinone, on soil organisms and on some plant species. This pesticide is actively used in the agroindustrial sector of the country in the fight against various pests. Aims and Objectives In order to detect the anthropogenic load of the soil ecosystem in the effects of the drug «Zemlin» containing diazinone, and to assess the toxicity of the insecticide on soil organisms, earthrworms ( Lumbricus terrestris L .) and bacteria living in the soil: Pseudomonas sp., Bacillus sp., Arthrobacter sp., Azotobacter sp., Flavobacterium sp. The drug was analyzed for phytotoxicity on onion sprouts ( Allium cepa ), cabbage ( Brassica oleracea ) and Helichrysum arenarium . Biodestruction of diazinon with soil microorganisms-destructors has been investigated. Methods The material for experiments was the drug «Zemlin» in which the active substance is diazinone. A fertile black-and-yellow product, selected from the environmentally friendly territory of the Karmaskalinsky Region of the Republic of Bashkortostan. In this work, experiments were conducted on the effects of the drug on earthworms, taking into account their behavior, modeled the condition of contact of earthworms ( Lumbricus terrestris L.) with the drug. Studies on phytotoxicity were carried out, as test objects used vegetable and floral crops (onion, cabbage, helichrysum). Determining the number of microorganisms in soil samples before and after exposure to insecticide in PC concentrations (production concentration), 2PC and 5PC were carried out by «fingerprinting» in petri dishes on meatpepton agar (MPA) taken as nutrient environment. We planted soil samples and cultivated them from 2 to 5 days in a thermostat at 28 °С. Microorganisms used diazinone as a source of carbon and energy. The change in the destructive material was determined with the help of a thinlayer chromatogram (TSX), the plates SilufolUV-254 were used, the ehuent was acetone, the developer of phosphorus-molybdenic acid ( Rf = 0.3). Chromato-mass spectral analysis was carried out on the device ShimadzuGC-9A. Results Analysis of the effects of the insecticide on the basis of diazinone on earthworms showed that the drug has a high toxic effect. In PC for 3 days live earthworms were observed 60 %. In an experiment to study phytotoxicity, the analysis showed that after treatment with the drug «Zemlin» with different concentrations: PC, 2PC, 5PC and control (drug free) the length of the above-ground and underground parts of plants that were etched, different from the control sample. It has been established that the length of the sprouts increases when the concentration of diazinon increases. Diazinon does not have toxicity to seeds, and enhances their growth and development. In an experiment to establish the effects of the insecticide on soil bacteria, it was found that in soil, untreated toxicant, the dominant microorganisms are Pseudomonas sp., Bacillus sp., Arthrobacter sp., Azotobacter sp. and Azotobacter sp. and Flavobacterium sp. When the concentration of the substance of the drug (PC, 2PC, 5 PC) increases, there is a change in the quality and quantitative composition of soil microorganisms. The decrease in the number of microorganisms occurs already during the production concentration. 2 PC pollution of leads to the disappearance from the microenvironment of the soil Azotobacter sp., Arthrobacter sp. and Flavobacterium sp. Toxic action of diazinon is sustained by bacteria of the species Pseudomonas sp. and Bacillus sp. Biodestruction of the diazinon was carried out for 65 days. After 14 days in the NMR and GLC-MS spectrums products of hydralise diazinone appeared. The complete chemical transformation of the studied pesticide takes place on 65 days (control on TLH), in the NMR 31P spectrum there are no signals of the phosphorus atom. At the same time, there is no diazinone in the GLC-MS spectrum, but there has been a peak identified as 4.6- dimethyl-N-phenylpiridin-2-amin, which can be considered as a biodegradation product under the influence of Pseudomonas sp. bacteria.

A glasshouse experiment was conducted to study if glyphosate spraying has immediate short-term effects on the growth, reproduction and residue incorporation of the earthworm species Lumbricus terrestris L. The experiment simulated post-harvest conditions in no-till cereal cultivation, where L. terrestris often abound, and glyphosate is widely used. Pairs of adult, field-collected L. terrestris (median total mass 11.2 g fresh weight) were kept in fine sand-filled cylinders, with chopped wheat straw on the soil surface as feed. The treatment cylinders were sprayed with glyphosate at normal field application rate [Rodeo® XL 3.0 l ha−1, resulting in 1080 g a.i. ha−1 and “Contact” surfactant (0.5 l ha−1)]; the controls were sprayed with water (N = 12 for both). The treatment and control cylinders were maintained for two months at +15 °C, approximate 60% air humidity and 12h:12h light:dark cycle. All individuals survived the experiment. There was no difference in L. terrestris mass change over the experiment, with an average weight change of +0.96% in glyphosate treatment and −0.93% change in the control (p = 0.66). Glyphosate treatment and control did not differ in cocoon production rate (31 vs 28 cocoons cylinder−1 respectively; p = 0.30). The straw incorporation was slightly but not significantly lower in glyphosate-treated soil (decline in surface straw mass 27.5% vs. 30.5%; p = 0.07). The glyphosate treatment applied represents a commonly used practice in spring cereal cultivation, and it did not affect negatively L. terrestris. The varying study results on the effects of glyphosate on earthworms – and on L. terrestris in particular – may partly arise from notable variation in application rates and the formulation-surfactant combinations used in different experiments which needs to be considered in the ecological risk assessment of glyphosate use.

This is the first demonstration of an apparent acrosome reaction by the use of negative staining (phosphotungstic acid) and electron microscopy. It is also the first report of such a reaction in spermatozoa of an oligochaete annelid (Lumbricus terrestris). The morphology of the negatively stained unreacted acrosomal complexes is entirely comparable to that seen in sectioned material. After reaction, a short straight filament is extruded and the compartmentalization seen in unreacted spermatozoa is no longer visible. In the flagellar axoneme there are short cross-bridges linking the two central singlet microtubules at 150-170 A intervals. Two fibrous elements are also present, forming, with the singlets, a tetragon. All four of these structures apparently change their elastic properties after incubation in distilled water for two hours, so that instead of following the doublet microtubules in gentle coils, the central complex components fall as a unit in jagged configurations.

The herbicide Roundup (and glyphosate, its active ingredient) is extensively used for weed control on a worldwide scale. It is absorbed after foliar application and quickly translocated inside the plant. In this study, we investigated the effects of Roundup speed, a commercial glyphosate formulation, on the structural composition (dominance of microbial groups, phospholipid fatty acid analysis - PLFA) and functional diversity (use of carbon sources, Multiple Substrate Induced Respiration - MSIR) of soil microorganisms. We specifically aimed at understanding the potential impact of biotic interactions on herbicide effects and included plants, earthworms, and endomycorrhizal fungi in the experimental setup. For this, we grew clover (Trifolium repens) in the greenhouse and added mycorrhizal inoculum (Glomus mosseae) and earthworms (Lumbricus terrestris) to the pots. Two weeks after foliar Roundup application and subsequent plant death, the pots were destructively sampled. The application resulted in a significant increase of microbial respiration (SIR) by approximately 30%. A multivariate analysis of the MSIR data exhibited small but significant differences between the microbial communities of treated and untreated pots, while no significant difference was apparent for the PLFA data. Bacterial PLFAs generally decreased following herbicide application, while mycorrhizal and fungal PLFAs were not affected. We did not find a consistent difference between the fatty acid markers of gram negative and gram positive bacteria. For all investigated parameters, there were highly significant differences between the upper (0–5 cm depth) and lower (5–10 cm) soil layers. The fact that rooting density differed by a factor of 3.5 between the two layers indicated that herbicide effects were especially pronounced in the clover rhizosphere and were likely due to changes in root exudate composition. We found significant, though very small, interactions between Roundup and other experimental factors (especially mycorrhizal inoculum).

Assessment of organophosphorus (OP) pesticide exposure in non-target organisms rarely involves non-neural molecular targets. Here we performed a 30-d microcosm experiment with Lumbricus terrestris to determine whether the activity ofdigestive enzymes (phosphatase, β-glucosidase, carboxylesterase and lipase) was sensitive to chlorpyrifos (5 mg kg−1 wet soil). Likewise, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the wall muscle and gastrointestinal tissues as indicators of OP exposure. Chlorpyrifos inhibited the acid phosphatase (34% of controls),carboxylesterase (25.6%) and lipase activities (31%) in the gastrointestinal content. However, in the gastrointestinal tissue,only the carboxylesterase and lipase activities were significantly depressed (42?67% carboxylesterase inhibition in the foregut and crop/gizzard, and 15% lipase inhibition in the foregut). Chlorpyrifos inhibited the activity of both cholinesterasesin the gastrointestinal tissues, whereas the AChE activity was affected in the wall muscle. These results suggested chlorpyrifos was widely distributed throughout the earthworm body after 30 d of incubation. Interestingly, we found muscle carboxylesterase activity strongly inhibited (92% of control) compared with that detected in the gastrointestinal tissues of the same OP-exposed individuals. This finding was explained by the occurrence of pesticide-resistant esterases in the gastrointestinal tissues, which were evidenced by zymography. Our results suggest that digestive processes of L. terrestris may be altered by chlorpyrifos, as a consequence of the inhibitory action of the insecticide on some digestive enzymes. Fil: Sanchez Hernandez, Juan C.. Universidad de Castilla-La Mancha; España Fil: Rios, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina Fil: Attademo, Andres Maximiliano. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Saneamiento Ambiental. Cátedra de Ecotoxicología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

International audience; Earthworms contribute, directly and indirectly, to contaminant biodegradation. However, most of bioremediation studies using these annelids focus on pollutant dissipation, thus disregarding the health status of the organism implied in bioremediation as well as the recovery of indicators of soil quality. A microcosm study was performed using Lumbricus terrestris to determine whether earthworm density (2 or 4 individuals/kg wet soil) and the time of exposure (1, 2, 6, 12, and 18 wk) could affect chlorpyrifos persistence in soil initially treated with 20 mg active ingredient kg− 1 wet soil. Additionally, selected earthworm biomarkers and soil enzyme activities were measured as indicators of earthworm health and soil quality, respectively. After an 18-wk incubation period, no earthworm was killed by the pesticide, but clear signs of severe intoxication were detected, i.e., 90% inhibition in muscle acetylcholinesterase and carboxylesterase (CbE) activities. Unexpectedly, the earthworm density had no significant impact on chlorpyrifos dissipation rate, for which the measured half-life ranged between 30.3 d (control soils) and 44.5 d (low earthworm density) or 36.7 d (high earthworm density). The dynamic response of several soil enzymes to chlorpyrifos exposure was examined calculating the geometric mean and the treated-soil quality index, which are common enzyme-based indexes of microbial functional diversity. Both indexes showed a significant and linear increase of the global enzyme response after 6 wk of chlorpyrifos treatment in the presence of earthworms. Examination of individual enzymes revealed that soil CbE activity could decrease chlorpyrifos-oxon impact upon the rest of enzyme activities. Although L. terrestris was found not to accelerate chlorpyrifos dissipation, a significant increase in the activity of soil enzyme activities was achieved compared with earthworm-free, chlorpyrifos-treated soils. Therefore, the inoculation of organophosphorus-contaminated soils with L. terrestris arises as a complementary bioremediation strategy in terms of recovery of soil biochemical performance and quality.

Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm Lumbricus terrestris erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (Tm = 72 ± 0.84 °C) relative to native LtEc (Tm = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P50 = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P50 = 26.67 ± 0.4 mm Hg), but the cooperativity (n = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.

Detarium senegalense is an importance medicinal plant used in North-east Nigeria and some African countries in traditional medicine. This present study evaluated the preliminary phytochemical screening and anthelminthic activity of methanol leaf extract. Phytochemical screening was carried out by solvent-solvent partitioning of leaf extract in chloroform, n-hexane, dioxane, ethyl acetate, acetone and methanol. The results showed that methanol fraction contained alkaloids, tannins, saponins, and cardiac glycosides while the ethyl acetate fraction contained only flavonoids with no anthracenes. Ethanol fraction demonstrated the highest anthelminthic activity with a decreased time of paralysis and death in the various groups of earthworm (Lumbricus terrestris) in dose-dependent manner at concentrations of 10 mg/mL, 20 mg/mL, 30 mg/mL, and 40 mg/mL. The time for paralysis and death for Group VII 400 mg/mL of extract were 5.0 ± 0.3min and 10.0 ± 1.02 min respectively. This result was compared with that of a standard anthelminthic drug piperazine citrate (USP) whose values were 12 ±1.01 min and 16 ± 1.02 min respectively for time of paralysis and death at p≤0.05 (one-way ANOVA). The results showed that D. senegalense leaf methanol extract possessed anthelminthic activities by osmotic shock at the clitellum portion of the earthworms, and this justifies the use of the plant extract in herbal medicine for the eradication of worms.

Since donated red blood cells must be constantly refrigerated, they are not available in remote areas and battlefields. We have previously shown that the hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an effective and safe substitute for donated blood that is stable enough to be stored for long periods at the relatively high temperatures that may be encountered in remote areas. The goal of this study was to further increase the thermal stability of LtEc by covalently cross-linking LtEc with glutaraldehyde (gLtEc). Our results show that the melting temperatures of the gLtEc samples steadily increase as the molar ratio of glutaraldehyde to heme increases (from Tm = 57°C for native LtEc up to Tm = 68°C at a ratio of 128:1). In addition, while native LtEc is susceptible to subunit dissociation at alkaline pH (8-10), cross-linking with glutaraldehyde completely prevents dissociation of gLtEc at pH 10. Increasing the molar ratio of glutaraldehyde:heme also significantly increased the oxygen affinity of gLtEc, but this effect was decreased by cross-linking gLtEc in the deoxygenated T state. Finally, while gLtEc samples cross-linked at low G:H ratios (e.g., 2:1) exhibited slight increases in oxidation rate in Tris buffer, no significant difference in oxidation rate was observed between native LtEc and the gLtEc samples in Ringer's Solution, which contains antioxidants. Overall, cross-linking LtEc with glutaraldehyde significantly increases its thermal and structural stability without any loss of function, making gLtEc an attractive blood substitute for deployment in remote areas and battlefields. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:521-528, 2018.

Background Invertebrate worms serve as models for understanding the features of neurological functions. Acrylamide (ACR), the well-known neurotoxin, is a water-soluble chemical widely used in various industrial and laboratory processes. ACR is also found in food items which are cooked under high temperature. Purpose The study attempts to assess the neuropathological changes in cerebral ganglions along with the locomotion and neuronal behavior of Lumbricus terrestris on ACR intoxication. Methods The dosage of acrylamide induced neurotoxicity ranged from 0–17.5 mg/kg body weight for 7 days. The time/dose dependent changes in the oxidant and antioxidant status, activities of Na+/K+ATPase, Ca2+/Mg2+ ATPase and 5’ Nucleotidase were assessed along with the locomotor behavioral analysis. Result The activities of super oxidase dismutase and catalases were not altered appreciably. However, the glutathione family, lipid peroxide, protein carbonyl content and vitamin C did show significant variations (p

The earthworm gut is an anoxic, saccharide-rich microzone in aerated soils. The apparent degradation of diverse saccharides in the alimentary canal of the model earthworm Lumbricus terrestris is concomitant with the production of diverse organic acids, indicating that fermentation is an ongoing process in the earthworm gut. However, little is known about how different gut-associated saccharides are fermented. The hypothesis of this investigation was that different gut-associated saccharides differentially stimulate fermentative microorganisms in gut contents of L. terrestris . This hypothesis was addressed by (i) assessing the fermentation profiles of anoxic gut content microcosms that were supplemented with gut-associated saccharides and (ii) the concomitant phylogenic analysis of 16S rRNA sequences. Galactose, glucose, maltose, mannose, arabinose, fucose, rhamnose, and xylose stimulated the production of fermentation products, including H 2 , CO 2 , acetate, lactate, propionate, formate, succinate, and ethanol. Fermentation profiles were dependent on the supplemental saccharide (e.g., glucose yielded large amounts of H 2 and ethanol, whereas fucose did not, and maltose yielded large amounts of lactate, whereas mannose did not). Approximately 1,750,000 16S rRNA sequences were affiliated with 37 families, and phylogenic analyses indicated that a respective saccharide stimulated a subset of the diverse phylotypes. An Aeromonas -related phylotype displayed a high relative abundance in all treatments, whereas key Enterobacteriaceae -affiliated phylotypes were stimulated by some but not all saccharides. Collectively, these results reinforce the likelihood that (i) different saccharides stimulate different fermentations in gut contents of the earthworm and (ii) facultative aerobes related to Aeromonadaceae and Enterobacteriaceae can be important drivers of these fermentations. IMPORTANCE The feeding habits of earthworms influence the turnover of elements in the terrestrial biosphere. The alimentary tract of the earthworm constitutes an anoxic saccharide-rich microzone in aerated soils that offers ingested microbes a unique opportunity for anaerobic growth. The fermentative activity of microbes in the alimentary tract are responsible for the in situ production of (i) organic compounds that can be assimilated by the earthworm and (ii) H 2 that is subject to in vivo emission by the earthworm and can be trophically linked to secondary microbial events in soils. To gain insight on how fermentative members of the gut microbiome might respond to the saccharide-rich alimentary canal, this study examines the impact of diverse gut-associated saccharides on the differential activation of fermentative microbes in gut contents of the model earthworm L. terrestris .

Although neonicotinoids are targeted at insects, their predominant use as a seed dressing and their long persistence in soils mean that non-target soil organisms such as earthworms are likely to be chronically exposed to them. Chronic exposure may pose risks that are not evaluated in most toxicity tests. We experimentally tested the effect of field-realistic concentrations of a commonly used neonicotinoid, clothianidin, on mortality, weight gain, and food consumption to assess the impacts of chronic exposure over four months on fitness of L. terrestris individuals. We undertook three separate experiments, each with different exposure routes: treated soil only (experiment A), treated food and soil combined (experiment B) and treated food only (experiment C). Mortality was negatively affected by exposure from treated soil only with greatest mortality observed in the groups exposed to the two highest concentrations (20 ppb and 100 ppb), but no clear effect on mortality was found in the other two experiments. When clothianidin was present in the food, an anti-feedant effect was present in months one and two which subsequently disappeared; if this occurs in the field, it could result in reduced rates of decomposition of treated crop foliage. We found no significant effects of any treatment on worm body mass. We cannot rule out stronger adverse effects if worms come into close proximity to treated seeds, or if other aspects of fitness were examined. Overall, our data suggest that field-realistic exposure to clothianidin has a significant but temporary effect on food consumption and can have weak but significant impacts on mortality of L. terrestris.

Carboxylesterases (CbEs) are serine hydrolases involved in the detoxification of anticholinesterase (organophosphorus and methylcarbamate) pesticides. Past studies have documented the occurrence of these esterases in soil, but little is known about their origin, function, and particularly, their reactivity against agrochemicals. In this study, it was compared the potential CbE activity in earthworm-treated and control (earthworm-free) soils by enzyme kinetics with multiple carboxylic esters and native polyacrylamide gel electrophoresis. After 12 weeks of inoculation, CbE activity was between two- and four-fold higher in the earthworm-treated soils (α-naphthyl acetate = 4.85 ± 1.58 μmol h −1 g −1 dry soil, α-naphthyl butyrate = 2.93 ± 1.60, α-naphthyl valerate = 2.64 ± 1.27, 4-nitrophenyl acetate = 1.41 ± 0.37, 4-nitrophenyl butyrate = 0.87 ± 0.15 and 4-nitrophenyl valerate = 0.89 ± 0.11; values are presented as mean ± standard deviation) than in controls. Although this enhanced esterase activity remained unchanged for 1 month following earthworm removal, it decreased under soil desiccation (31%–60%) or thermal denaturing (43%–82%). The potential sources for enhanced soil CbE activity were also examined through plate-count of microorganisms and zymographic techniques. The earthworm gut microenvironment was a significant source of soil CbE activity, and the casts were found to be the main contributors to the esterase activity analyzed. Soil CbE activity was strongly inhibited by organophosphorus (chlorpyrifos-oxon, paraoxon-ethyl and paraoxon-methyl) and, at less extent, by methylcarbamates (carbaryl and carbofuran). In vitro inhibition kinetics showed a biphasic curve that revealed at least two sensitive esterases and a resistant fraction; the latter varied widely depending on the enzyme substrate (7–68% of control activity). Likewise, spiking of earthworm-treated soils with 4 mg kg – 1 (wet weight) of chlorpyrifos-oxon led to a significant inhibition of CbE activity 2 (40–72% inhibition) and 6 days (37–53%) after its application. Current results suggest that the soil-dwelling earthworm Lumbricus terrestris may be used as a promoter of soil enzyme activities with a direct benefit for pesticide bioremediation.

We investigated the interaction of soil Zn with calcium carbonate granules secreted by the earthworm Lumbricus terrestris. Earthworms were kept in agricultural soil amended with ZnSO₄ to give Zn concentrations up to 750 mg kg⁻¹ and in two former mine site soils containing 500 and 120 mg Zn kg⁻¹ .After 21-42 days the earthworms accumulated 260-470 mg Zn kg⁻¹. Granule production was 0.027e2.11 mg CaCO₃ earthworm⁻¹ day⁻¹ and was positively correlated with soil and soil solution pH (r ¼ 0.66and 0.85 respectively, p ≤ 0.01). Granule Zn concentration was 34-163 mg kg⁻¹. Granules collected from Zn-free control soil and left in Zn-bearing soil for 28 days contained 49-60 mg Zn kg⁻¹ suggesting that the majority of Zn associates with granules after their secretion. However, synchrotron X-ray fluorescence indicates some incorporation of Zn into granules during their formation. X-ray diffraction and Xray absorption spectroscopy indicate that the granules are predominantly calcite and the spectroscopy suggests that the Zn is either adsorbed to, or incorporated into, the calcite lattice. X-ray diffraction of the outer c. 35 μm of the granules supports incorporation of Zn into the calcite lattice. The low granule production rates in the mine site soils and the granule Zn concentrations suggest that earthworm secreted calcium carbonate is unlikely to impact significantly on Zn mobility in soils. Refereed/Peer-reviewed

Improved approaches are needed to assess bioavailability of hydrophobic organic compounds in contaminated soils. Performance of thin-film solid-phase extraction (TF-SPE) using vials coated with ethylene vinyl acetate was compared to earthworm bioassay (Lumbricus terrestris). A DDT and dieldrin contaminated soil was amended with four organic carbon materials to assess the change in bioavailability. Addition of organic carbon significantly lowered bioavailability for all compounds except for 4,4'-DDT. Equilibrium concentrations of compounds in the polymer were correlated with uptake by earthworms after 48d exposure (R(2) = 0.97; p 0.001), indicating TF-SPE provided an accurate uptake simulation. Bioavailability of residues in soil was compared with a spiked soil aged for 90d in laboratory. Dieldrin and DDX were respectively 18% and 11% less bioavailable in contaminated soil relative to spiked soil despite40yr of aging. Results show that TF-SPE can be useful in examining potential risks associated with contaminated soils and to test effectiveness of remediation efforts.

Nitrous oxide (N2O) is a greenhouse gas that is released from both nitrification and denitrification processes. Soil moisture content is a key controller of the biochemical pathways leading to N2O emission, causing a switch between nitrification and denitrification processes. Earthworms are reported to increase N2O emissions from soil under aerobic and anaerobic conditions, but how earthworm-induced N2O emissions are affected by soil drying and rewetting cycles is unknown. The objectives of this study were to (1) evaluate earthworm-induced N2O emissions from soils with aerobic, anaerobic, and fluctuating soil moisture conditions; and (2) determine the earthworm effects on soil denitrifiers responsible for N2O fluxes. Soils were kept in mesocosms (polyvinyl chloride plastic tubes, 10 cm diameter, filled with soil to 15 cm depth) at constant 33% water-filled pore space (WFPS), constant 97% WFPS or underwent three wetting-drying cycles (WD). Each soil moisture treatment had 2 earthworm treatments, including (1) a mixture of endogeic Aporrectodea turgida and anecic Lumbricus terrestris and (2) no earthworm treatment. These gave a total of 6 treatments in this study, with 5 replicates for each treatment. The N2O fluxes were quantified every one to three days, and the soil denitrifier activities were measured after 69 days, when the experiment ended. Soil moisture significantly affected N2O emissions and the WD treatment had the highest cumulative N2O emissions. Earthworms increased N2O emissions by 50% in the 33% WFPS treatment but decreased N2O emissions by 34% in the 97% WFPS treatment, probably due to more complete reduction of N2O to N2. Earthworms strongly reduced N2O emission rate in WD treatment, and they significantly reduced cumulative N2O emissions by 82%. Soil denitrification enzyme activity (DEA) increased significantly when earthworms were present. Abundance of 16S rRNA, nirS, and nosZ genes was affected significantly by the earthworm × soil moisture interaction, with the highest 16S rRNA and nosZ abundance in soil from the WD treatments. We conclude that the decrease in cumulative N2O emissions from soil at 97% WFPS and the WD treatment by earthworms was due to an alteration of the denitrifying bacterial community composition.

Background: Centella asiatica (CA) is one of the most valuable herbal medicines widely being used for the treatment of various neurological ailments that are challenging for health-care providers and also is deemed to be safe and effective. Purpose: Monoamines (MAs) are neurotransmitters and neuromodulators that play a significant role in the neural communication, regulation of motor and cognitive functions in the brain. Neurodegeneration is associated with elevated levels of MAO-B that can lead to damaging reactive oxygen species (ROS) in the brain. The current study, evaluated the effects of asiaticoside-D (AD) from neuroprotective CA, on the levels and activities of monoamine oxidase A and B (MAO-A and B), in addition to the behavioral analysis. Methods: Qualitative and quantitative analyses of various solvent extracts of CA were performed. The extracts were screened for antioxidant potential using 1,1-diphenyl-2-picryl hydroxyl (DPPH), 2,2′-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), hydrogen peroxide (H2O2) radical, nitric oxide (NO) radical inhibition, lipid peroxidation (LPO) and ferric reducing antioxidant power (FRAP) assays. The purification of AD was done by column, thin layer and high-performance liquid chromatographies followed by structural elucidation using IR, HR–MS, 1H and 13C NMR spectra. Docking studies were performed to assess the impact of AD on MAO-A and B.In vivo, Lumbricus terrestris were exposed to 0.4 ppm rotenone (ROT) of medium for 7 days and were subjected to co-treatment along with 15 ppm of AD from CA. At the end of experiment period, the neuronal behavior of worms was assessed. Cerebral ganglions (CGs) were removed and the m-RNA levels of MAO-A and B were analyzed by Semi Q-PCR and their activities were also analyzed. Results: The ethanolic extracts exhibited higher antiradical activity against DPPH, ABTS, H2O2, LPO, FRAP, NO and vitamin C with EC50 value of 20.2, 20.9, 20.4, 22.0, 24.9, 28.1, 25.5 and 22.0 µg/ml respectively. Structural analysis by IR, HR-MS, 1H and 13C NMR spectrum have shown the structure of the isolated compound as (2α, 3β)-2,3–dihydroxyurs-12-en-28-oicacid-O-α-L-rhamnopyranosyl-(1→4)-O-β- d -glucopyranosyl (1→6)-β–copyranosyl ester and was represented as AD. In silico interaction of AD with MAO-A and B residues Lys312 at distances of 1.84 A and 2.44 A respectively was found to exhibit high binding energy of -9.4 and 7.4 kcal. The neuronal behavior using L. terrestris showed significant improvement against (p Conclusion: AD was found to exert its negative impact on the levels and activities of MAO-A and B in CGs of rotenone- induced changes in L. terrestris, the property which is considered to be crucial against ROT induced neurodegenerative pathology like -Parkinsonism.

In this study, an attempt was made to examine the effects of dumpsite leachate on soil biological sentinels by collecting leachate samples from Ikhueniro and Otofure dumpsites in Benin City Nigeria, and analyzed using standard methods. The parameters include pH, chemical oxygen demand, biochemical oxygen demand, phosphate, sulphate, chloride, nitrate, chromium, cadmium and lead. The mean results showed pH (8.30 ±0.14), BOD (566 mg/l), COD (1713 mg/l), Phosphate (7.73 ± 0.14 mg/l), Sulphate (22.40 ± 0.75 mg/l), Chloride (187.5 ± 1.12 mg/l), Nitrate (1.47 V 0.06 mg/l), Chromium (0.06 ± 0.1 mg/l), Cadmium (0.20 ± 0.1 mg/l), Lead (1.03 ± 0.04) for Ikhueniro and pH (8.0 ± 0.1), BOD (315 mg/l), COD (1095 mg/l), Phosphate (5.85 ± 0.19 mg/l), Sulphate (31.70 ± 0.73 mg/l), Chloride (135.25 ± 1.59 mg/l), Nitrate (0.93 ± 0.09 mg/l), Chromium (0.06 ± 0.1mg/l), Cadmium (0.109 ± 0.1mg/l), Lead (0.015 ± 0.005 mg/l)for Otofure respectively. Different concentrations of leachate ranging between 25% and 100% was used and the growth of Nitrosomonas sp reduced from 6.4×10 4 (cfu/ml) – 2.9×10 4 (cfu/ml), when compared to the control which increased from 6.4×10 4 (cfu/ml) – 1.0×10 5 (cfu/ml). The percentage survival of Lumbricus terrestris was 2.19%, 1.14%, 3.42%, 6.69%, with varying concentrations of 25%, 50%, 75%, and 100% respectively. The method implored for analysis shows the leachate sample was toxic to both test organisms. Nitrosomonas sp and Lumbricus terrestris may be used as indicator organisms for leachate toxicity in the soil. Keywords : toxic leachate effects, heavy metals leachate, soil sentinels, earthworm assay

A new experimental set-up enabling microfocus fluorescence XANES mapping and microfocus XRD mapping on the same sample at beamline I18 at Diamond Light Source is described. To demonstrate this set-up the heterogeneous mineralogy in calcium carbonate granules excreted by the earthworm Lumbricus terrestris has been analysed. Data analysis methods have been developed which enable µXRD and µXANES two-dimensional maps to be compared., The use of fluorescence full spectral micro-X-ray absorption near-edge structure (µXANES) mapping is becoming more widespread in the hard energy regime. This experimental method using the Ca K-edge combined with micro-X-ray diffraction (µXRD) mapping of the same sample has been enabled on beamline I18 at Diamond Light Source. This combined approach has been used to probe both long- and short-range order in calcium carbonate granules produced by the earthworm Lumbricus terrestris. In granules produced by earthworms cultured in a control artificial soil, calcite and vaterite are observed in the granules. However, granules produced by earthworms cultivated in the same artificial soil amended with 500 p.p.m. Mg also contain an aragonite. The two techniques, µXRD and µXANES, probe different sample volumes but there is good agreement in the phase maps produced.

In this paper we show for the first time that calcite granules, produced by the earthworm Lumbricus terrestris, and commonly recorded at sites of archaeological interest, accurately reflect temperature and soil water δ18O values. Earthworms were cultivated in an orthogonal combination of two different (granule-free) soils moistened by three types of mineral water and kept at three temperatures (10, 16 and 20 ºC) for an acclimatisation period of three weeks followed by transfer to identical treatments and cultivation for a further four weeks. Earthworm-secreted calcite granules were collected from the second set of soils. δ18O values were determined on individual calcite granules (δ18Oc) and the soil solution (δ18Ow). The δ18Oc values reflect soil solution δ18Ow values and temperature, but are consistently enriched by 1.51 (±0.12) ‰ in comparison to equilibrium in synthetic carbonates. The data fit the equation 1000 ln α = [20.21 ± 0.92] (103 T-1) - [38.58 ± 3.18] (R2 = 0.95; n = 96; p < 0.0005). As the granules are abundant in modern soils, buried soils and archaeological contexts, and can be dated using U-Th disequilibria, the developed palaeotemperature relationship has enormous potential for application to Holocene and Pleistocene time intervals.

Background/Aims Health benefits of Lamiaceae herbs are attributed to the presence of antioxidant phytochemicals. However, mechanism of in vivo functionality is not very well understood. We determined the in vivo effect of dietary Lamiaceae herbs on modulating redox-nitric oxide (NO) homeostasis and sperm quality in oxidation prone environment of seminal vesicles in Lumbricus terrestris . Methods Animals fed ad libitum on Lumbricus growth medium supplemented with 0% (control), 0.1% or 0.5% (w/v) of different herbs. The seminal vesicles of the animal were dissected out on day 2 and day 6, and gently disrupted. Levels of malondialdehyde (MDA), DNA fragmentation (DNAF), glutathione (GSH), nitrates/nitrites (NOx), superoxide dismutase (SOD), catalase (CAT) were determined using standard assays. Sperm maturity and deformation (DFO) was quantified microscopically. Results Overall, the herb treatments decreased MDA levels by (60–90%), increased SOD activity (15–50%), and decreased DNAF (6–11%). Treatments with basil and oregano at 0.1% (w/v) were most effective. Conclusion Our suggests that phytochemicals from Lamiaceae herbs modulate redox stress response, and protect against oxidative stress in seminal vesicles.

This paper investigates the incorporation of Sr into biomineralized calcium carbonate granules secreted by the earthworm Lumbricus terrestris. Experiments were conducted using an agricultural soil amended with Sr(NO 3 ) 2 to give concentrations in the range 50–500 mg kg −1 Sr and a naturally Sr-rich, Celestine-bearing soil containing up to 11 000 mg kg −1 Sr. Granule production rates were in the range 0.26–2.3 mg CaCO3 earthworm −1 day −1 ; they showed no relationship with soil or soil solution Sr concentration but decreased with decreasing pH. Strong relationships exist ( r 2 ⩾ 0.8, p ⩽ 0.01) between the Sr concentrations and Sr/Ca ratios of the granules and those of the soil, soil solution and earthworms. The highest bulk Sr concentration we recorded in the calcium carbonate granules was 5.1 wt.% Sr whilst electron microprobe analysis recorded spot concentrations of up to 4.3 wt.% Sr. X-ray diffraction and X-ray absorption spectroscopy indicate that the majority of the calcium carbonate is present as Sr-bearing calcite with trace amounts of Sr-bearing vaterite also being present. The granules produced in the Sr-amended soils concentrated Sr relative to Ca from the bulk soil and the earthworms. This suggests that earthworm secreted calcium carbonate may be significant in the cycling of 90 Sr released into soils via nuclear accidents or leakage from nuclear waste storage facilities.

Granules of calcium carbonate are known to be produced by the calciferous glands of earthworms, and may have a measurable impact on the soil; however, little is known of their dynamics. Earthworms are often found in Tuber melanosporum burns, and this truffle is closely linked to calcium carbonate and soil pH. The present work shows that in weakly calcareous soil burns with high earthworm (Prosellodrilus sp.) activity, earthworm casts showed a significant increase of 0.2 units of pH and 66 % more total calcium carbonate than soils in the burns, although the casts were produced from these same soils. Analysis of the soil carbonate fractions (active carbonate versus calcium carbonate greater than 50 μm in size) showed that the origin of the increase in the total calcium carbonate content of the Prosellodrilus sp. casts cannot be explained by the levels of original carbonate existing in the soils in the study, although it can be explained by the synthesis of calcite granules by earthworms. These results obtained in the field with Prosellodrilus sp. confirm Canti’s (2009) experiments in the laboratory using δ13C concerning the synthesis of calcite granules by Lumbricus terrestris. Both these results and other works indicate that production of calcite granules by Prosellodrilus sp., L. terrestris, Aporrectodea longa, A. trapezoides and Anisochaetae sp. has the ability to increase soil pH and calcium carbonate; additionally Lambkin et al. (2011) have found that higher soil pH and carbonates can cause an increase in the production of calcite granules by L. terrestris. All these results, therefore, point to a feedback process, scope of which is a function of the different soil types and earthworm species. In summary, different earthworm species may have a significant and positive impact on soil pH and net recarbonation of T. melanosporum burns, and the importance of both soil properties justifies the development of further experiments aimed at incorporating vermiculture into the truffle cultivations.

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 +/- A 0.20 U/mg protein, mean +/- A SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 +/- A 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

The burrowing and feeding activities of earthworms may have a strong effect on the flux of N2O from agricultural soils. As such, shifts to agricultural management practices that increase the number of earthworms require an understanding of the role of earthworms in N2O dynamics. We conducted a field experiment to examine the effects of addition of anecic earthworms (Lumbricus terrestris) on N2O flux in a field previously planted with corn (Zea mays) in southern Rhode Island, USA. Plots were amended with (15NH4)2SO4 and either 0 (CTL) or 48 L. terrestris m-2 (EW). The flux of N2O, 15N2O and 15N2 was measured over 28 days between October and November 2008. The EW treatment had a significantly higher flux of N2O and 15N2O 1 - 3 days after 15NH4 addition. No treatment effects were observed on 15N2 flux. The addition of earthworms significantly increased (Day 1) and decreased (Day 12) the mole fraction of N2O relative to the CTL. Our results suggest that anecic earthworm additions can increase N2O flux from inorganic fertilizer N amendments, but the effects appear to short-lived.

Slugs are common pests of grass seed fields in western Oregon and the current focus of repeated, and often unsuccessful, efforts by growers to control them using molluscicides. Here we document rapid loss of molluscicidal bait pellets to earthworms and the resulting adverse effects on slug control. Three years of field studies were conducted at 17 locations with contrasting crops, soil types, residue levels, and tillage management programs. Baits were isolated in covered, sunken, open bottom arenas in the field to exclude removal by slugs, rodents, or birds. Forty hours of nighttime field observations and photographic documentation were collected to support the hypothesis that earthworms were removing slug bait before it had the opportunity to kill slugs. Greenhouse studies were conducted on the gray field slug (Derocerus reticulatum Mueller), in screen-topped arenas to determine the effects on mortality, seedling survival, and egg fecundity in a bait-depleting environment. Field data showed that an average of 17% of all bait pellet types were removed nightly by earthworms, with a range of 5.1e6.4 days until 100% disappearance. Individual earthworms in the field were observed removing up to three bait pellets per hour. Earthworms removed a 5% metaldehyde formulation significantly faster than either 4% metaldehyde or 1% iron phosphate pellet baits, possibly because of the smaller physical pellet size. Seedling survival in the greenhouse � 65% was achieved by the highest rate of 4% metaldehyde bait pellets, the two highest rates of 7.5% metaldehyde granules, and the two highest rates of 25% metaldehyde liquid formulation. Earthworms showed no behavioral interest in the granular or liquid formulations, providing growers with attractive alternatives to the ineffectual pelleted baits currently in widespread use. Published by Elsevier Ltd.

The fate of mercury (Hg) in the soil-earthworm system is still far from being fully understood, especially regarding recurrent and challenging questions about the importance of the reactivity of exogenous Hg species. Thus, to predict the potential effect of Hg inputs in terrestrial ecosystems, it is necessary to evaluate separately the reactivity of the endogenous and exogenous Hg species and, for this purpose, the use of enriched stable isotope tracers is a promising tool. In the present work, earthworms (Lumbricus terrestris) were exposed to historically Hg contaminated soils from the Almaden mining district, Spain. The soils were either non-spiked, which contain only endogenous or native Hg naturally occurring in the soil, or spiked with isotopically enriched inorganic Hg (199IHg), representing exogenous or spiked Hg apart from the native one. The differential reactivity of endogenous and exogenous Hg in the soil conditioned the processes of methylation, mobilization, and assimilation of inorganic Hg by earthworms. Both endogenous and exogenous Hg species also behave distinctly regarding their bioaccumulation in earthworms, as suggested by the bioaccumulation factors, being the endogenous methylmercury (MeHg) the species more readily bioaccumulated by earthworms and in a higher extent. To the best of our knowledge, this work demonstrates for the first time the potential of enriched stable isotopes to study the effects of fresh Hg inputs in soil-earthworm systems. The findings of this work can be taken as a case study on the dynamics of Hg species in complex terrestrial systems and open a new door for future experiments.