Biomarkers In Earthworms Research Articles

Biochar & fly ash amendments lower mortality and increase antioxidant activity in chlorpyrifos-exposed earthworms

The investigation presented a novel finding regarding mitigating stress induced by chlorpyrifos in Eisenia fetida by incorporating biochar derived from rice straw and fly ash as soil amendments. It was observed that phenolic compounds exhibit solubility in methanol, and the methanolic fraction exhibited notable inhibitory effects on lipid peroxidation and displayed antioxidant properties. The defence mechanism of E. fetida, comprising catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and various other enzymes, remained effective in neutralizing stressors without disruption when the earthworm was subjected to diverse chemical agents or stressful conditions. These enzymes served as indicative markers of toxicity induced by pesticide exposure, even at sublethal concentrations. The scavenging of free radicals by these enzymes ultimately safeguarded the organism. Fly ash and biochar emerged as two organic alternatives capable of alleviating stress by providing a protective mechanism. In this context, the study examined the impact of biochar and fly ash amendments on earthworm biomarkers. The mortality rate at the median lethal concentration of chlorpyrifos was reduced to less than 50% through 3% and 5% modifications. In contrast to the non-amendment group exposed to sublethal doses, the amendment group exhibited higher levels of oxidative stress and lower protein content. This observation indicated the presence of stress induced by the accumulation of free radicals, which increased in number with higher doses of chlorpyrifos. Moreover, the study highlighted the interconnected nature of total antioxidant capacity and total phenolic capacity values, with a decrease in these parameters signifying a shift in earthworm biomarkers.Graphical abstract

Cadmium and pyrene in the soil modify the properties of earthworm-mediated soil

Earthworms are pivotal in soil ecosystems due to their crucial role in shaping soil characteristics through casts and burrow walls. Previous research has predominantly focused on the direct impact of soil pollution on live earthworms, overlooking the subsequent effects on earthworm-mediated soil, such as casts and burrow walls. Using 2D-terraria as incubation containers and the geophagous earthworm species Metaphire guillelmi, this study assessed the change in various properties of earthworm-mediated soil in both uncontaminated soils and Cd- and Pye-contaminated soils. Overall, both Cd and Pye overall improved the ammonium nitrogen (NH4+-N), Olsen's phosphorus (Olsen-P) levels, and invertase and catalase activities while decreasing catalase activities in earthworm-mediated soil. They also fluctuating affected the pH, soil organic matter (SOM) content, soil urease, alkaline phosphatase activities, and microbial functional genes in the cast and burrow walls. These results indicated that earthworms remained crucial “ecosystem engineers” even in polluted soil. Additionally, differences were observed in the responses of properties between casts and burrow walls, showing unequal contributions of transit-through-gut and burrowing processes to soil modification. Specifically, transit-through-gut was found to have a more significant influence on soil NH4+-N and Olsen-P content compared to burrowing behavior. Regarding the pattern of microbial functional genes in earthworm-associated compartments, results revealed that they differed significantly in casts from those in bulk soil and burrow walls under unpolluted conditions, with pollution-enhancing disparities among compartments. Furthermore, NH4+-N and Olsen-P content, urease, and catalase activities in burrow walls and/or casts were identified as potential biomarkers for soil pollution, exhibiting a clear dose-effect relationship. Developing such biomarkers could address ethical concerns related to conventional earthworm biomarkers that require sacrificing earthworms. This study provides insights into the consequences of soil pollution on earthworm-mediated soil components, highlighting the importance of considering the indirect effects of contaminants on soil ecosystems.

Co-exposure of decabromodiphenyl ethane and cadmium increases toxicity to earthworms: Enrichment, oxidative stress, damage and molecular binding mechanisms

The increase of electronic waste worldwide has resulted in the exacerbation of combined decabromodiphenyl ethane (DBDPE) and cadmium (Cd) pollution in soil, posing a serious threat to the safety of soil organisms. However, whether combined exposure increases toxicity remains unclear. Therefore, this study primarily investigated the toxic effects of DBDPE and Cd on earthworms at the individual, tissue, and cellular levels under single and combined exposure. The results showed that the combined exposure significantly increased the enrichment of Cd in earthworms by 50.32–90.42 %. The toxicity to earthworms increased with co-exposure, primarily resulting in enhanced oxidative stress, inhibition of growth and reproduction, intensified intestinal and epidermal damage, and amplified coelomocyte apoptosis. PLS-PM analysis revealed a significant and direct relationship between the accumulation of target pollutants in earthworms and oxidative stress, damage, as well as growth and reproduction of earthworms. Furthermore, IBR analysis indicated that SOD and POD were sensitive biomarkers in earthworms. Molecular docking elucidated that DBDPE and Cd induced oxidative stress responses in earthworms through the alteration of the conformation of the two enzymes. This study enhances understanding of the mechanisms behind the toxicity of combined pollution and provides important insights for assessing e-waste contaminated soils.

Assessment of intensive periurban agriculture soil quality applying biomarkers in earthworms

Periurban agriculture in Argentina is carried out by farmers without adequate control. The indiscriminate agrochemical application for productivity improvement negatively impacts the environment. The objective of this work was to test the quality of periurban agricultural soils by performing bioassays with Eisenia andrei as an indicator. Soils belonging to two orchards with intensive production (S: strawberry/broccoli crop plot and G: tomato/pepper crop greenhouse - Moreno District, Buenos Aires, Argentina) were sampled during 2015 and 2016. As subcellular biomarkers, cholinesterases (ChE), carboxylesterases (CaE), and glutathione-S-transferases (GST) activities were analysed in E. andrei (7-day exposure). While no effect on ChE activities was observed, CaE activities were significantly reduced 18% (S-2016 soil). GST activities were increased 35% and 30% by S-2016 and G-2016, respectively. CaE decrease together with GST increase could be indicative of a negative disturbance. Concerning whole organism biomarkers, reproduction (56-day exposure), avoidance (3-day exposure), and feeding activity (bait-lamina test, 3-day exposure) were analysed. A reduced cocoons’ viability (50%), hatchability (55%), accompanied by a low number of juveniles (50%) were observed in all cases. Additionally, the earthworms exhibited significant avoidance responses to S-2015, S-2016 and G-2016 whereas G-2015 soil induced migration. No significant effect on the feeding activity was registered in any case. Most of the E. andrei biomarkers tested could constitute an early warning of harmful effects produced by polluted periurban soils, even if the agrochemical treatment applied remains unknown. The results reveal the need to develop an action plan to avoid further deterioration of the productive soil.

Heavy metal accumulation, biochemical and transcriptomic biomarkers in earthworms Eisenia andrei exposed to industrially contaminated soils from south-eastern Tunisia (Gabes Governorate)

Heavy metal pollution is causing harmful consequences on soil fertility, and earthworms are frequently employed as test organisms to evaluate the ecotoxicity of polluted soils. In this study, Eisenia andrei was exposed for 7 and 14 days to polymetallic contaminated soils collected from an industrial zone in the south-eastern Tunisia. Earthworm growth, heavy metal accumulation, genotoxicity, cytotoxicity, biochemical and transcriptional responses were determined. Results revealed a higher accumulation of heavy metals in earthworms after 14 than 7 days of soil exposure, a reduction in lysosomal membrane stability (LMS), besides an increase in micronuclei frequency (MN). Moreover, earthworm oxidative status was affected in terms of increases in malondialdehyde (MDA) and metallothionein (MTs) content, and enhancement of catalase (CAT) and glutathione-S-transferase (GST) activities. An inhibition of acetylcholinesterase (AChE) activity was also observed in treated earthworms, whereas transcriptional data demonstrated an up-regulation of cat, gst, mt, p21 and topoisomerase genes. Overall, these findings support the use of earthworms as suitable bioindicator species for pollution monitoring and assessment, advance our understanding of the interaction between heavy metals and earthworms, and provide valuable information about the harmful impact of biota exposure to naturally contaminated soils.

Comparative toxicity of fresh and expired butachlor to earthworms Eisenia fetida in natural soil: Biomarker responses

Butachlor as a selective pre-emergent herbicide, is mainly used to control the growth and productivity of certain annual grass and broad-leaf weeds. As non-target soil creatures, earthworms are affected detrimentally due to direct contact with pesticides. Our purpose was to compare the changes in the oxidative stress biomarkers in earthworms Eisenia fetida under the exposure of fresh and expired butachlor in the natural soil. LC50 of the fresh/unexpired and expired butachlor were determined by probit analysis amounting ≤440 and ≤ 471 mg/kg dry soil, respectively. Earthworms were then exposed to three sublethal concentrations (1/5th, 1/10th, and 1/20th of LC50) of both fresh and expired butachlor for 4 and 8 days. The level of oxidative stress biomarkers namely lipid peroxidation, glutathione S-transferase, and carbonylated proteins were increased. Besides, the catalase activity and value of total antioxidant capacity in earthworm's caulomic fluid were found to be decreased. In addition, it was clearly showed that butachlor can cause tissue damages in earthworms. The fresh and expired butachlor trigger the onset of oxidative damages as illustrated by altered oxidative stress biomarkers and tissue damages, particularly at the end of 8 days and high concentrations. Contrary to expiration, we found evidence that expired BUC is acutely toxic to earthworms. The fresh BUC was more deleterious than expired one to earthworms. Meanwhile, it must be considered that the regulations towards disposal of expired toxicants must be strictly followed without impairing the soil ecosystem.

Assessing joint toxic effects of arsenate and perfluorooctane sulfonate on earthworm by combining integrated biomarker response and mixture toxicity indices

Arsenic and perfluorooctane sulfonate (PFOS) are widely distributed pollutants in the soil environment and could coexist as mixture, but little studies have been reported about their combined toxicity to terrestrial invertebrates. In the present work, we exposed earthworm Eisenia fetida to sub-lethal levels of arsenate (As(V)), PFOS, and their mixture for 30 days. Multiple biomarker responses and bioaccumulation of the two toxicants in the organisms were analyzed to assess the joint toxicity of As(V) and PFOS. Bioaccumulation analysis results demonstrated that the coexistence of As(V) and PFOS in soil could increase the bioavailability of As(V) while decreasing the bioavailability of PFOS. As the level of exposure increased, significant variations were observed in all of the biomarkers analyzed, indicating growth inhibition and oxidative damage. The multiple biomarker responses were summarized by using the Integrated Biomarker Response (IBR) index, and significant dose–response relationships were found in treatments of As(V), PFOS and As(V)/PFOS mixture. Then the toxic interaction between As(V) and PFOS was assessed by combining IBR and two mixture toxicity indices, Effect Addition Index (EAI) and Concentration Addition Index (CAI). Results showed that the joint toxicity of As(V) and PFOS depended on the exposure concentration of As(V). The combined pollution of PFOS with lower concentration of As(V) led to synergistic interaction at the whole range of effect levels, while that with higher concentration of As(V) resulted in the toxicity interaction changing from antagonism to synergism as the effect level increased. As(V) was the main toxicant that caused adverse effects on earthworm biomarkers and affected the joint toxicity of As(V)/PFOS mixture. This study, for the first time, provides valuable toxicological data for the risk evaluation of combined toxicity of As(V) and PFOS in the soil environment. Moreover, this work indicates that the integration of IBR index and EAI/CAI could be an effective and practical method for the characterization of toxicity interaction within binary mixture systems.

Combined toxicity of acetochlor and metribuzin on earthworm Eisenia fetida: Survival, oxidative stress responses and joint effect

The overuse of herbicides has been linked to serious environmental problems. However, most related studies have focused on characterizing the effects of single herbicides, whereas relatively few studies have assessed the acute toxicity and physiological effects of combined herbicide exposure. Here, earthworms were exposed to acetochlor- and metribuzin-contaminated artificial soil to examine the toxic effects (including mortality, reactive oxygen species (ROS) levels, antioxidant enzyme activity, and malondialdehyde (MDA) levels) of single and combined exposures, after which the toxicity endpoints were analyzed using fa-CI and IBRv2. The 14 d-LC50 for single contaminated acetochlor and metribuzin were 137.06 (124.43–154.26) and 181.28 (161.63–221.02) mg·kg−1, respectively, whereas the LC50 for the combined stress was 112.06 (90.31–195.03) mg·kg−1. Therefore, combined herbicide exposure was significantly more lethal than the administration of either herbicide alone, and this synergistic effect was confirmed via fa-CI plot analysis. Further, acetochlor and metribuzin co-exposure inhibited the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) while significantly increasing ROS and MDA levels. IBRv2 analysis indicated that acetochlor and metribuzin co-exposure (10.0 mg·kg−1) had the strongest effects on several biomarkers in earthworms. ROS level was the most affected indicator and the largest change trend was observed at 7 days post-exposure. Our findings thus suggest that conducting ecotoxicity assessments based on single contaminants may significantly underestimate the effects of contaminant mixtures. Further, this study provides novel insights into the mechanisms of interaction between environmental pollutants.

Effects of one-dimensional nanomaterial polyaniline nanorods on earthworm biomarkers and soil enzymes.

Polyaniline nanorods (PANRs) are typical one-dimensional nanomaterials (1D NMs), which are widely used in medicine, batteries and water treatment, etc. Applications of PANRs will eventually enter the soil environment, but their ecotoxicity has been barely reported. Therefore, we measured earthworm biomass, earthworm biomarkers and soil enzymes to investigate the ecotoxicity of PANRs. The result of positive and increasing growth inhibition rates (GIR) showed that PANRs inhibited earthworm growth. As for earthworm biomarkers, PANRs caused a decrease in protein content, indicating that PANRs stress would increase earthworm energy consumption. Except for the 7th day, the activities of SOD, CAT and POD consistently increased, suggesting that PANRs activated the earthworm antioxidant system. The continually augment of MDA content indicated that PANRs stress would cause earthworm lipid damage. Na+-K+-ATPase increased with an excellent dose-time relationship. Differently, cellulase and AChE activities promoted at low concentrations and inhibited at high concentrations. The positive and dose-dependent IBRv2 indicated that the higher the concentrations of PANRs, the greater the ecotoxicity to earthworms. PANRs inhibited the soil enzyme activities such as sucrase, neutral phosphatase, protease and urease, while induced catalase activity in a dose-dependent manner. Earthworm addition reduced catalase activity by 10.74-29.99%, but improved other soil enzymes activities, demonstrating that earthworms played a positive role in regulating soil enzyme activity. GMean and T-SQI consistently increased due to earthworm activity, meaning a higher soil microbial functional diversity. Generally, this study provided data support for future PANRs toxicity studies, but their toxicity mechanisms still need to be further studied.

Mitochondrial bioenergetic, oxidative stress and burrowing responses in earthworm exposed to roxarsone in soil

The eco-risk of roxarsone (ROX) was evaluated using multiple responses of earthworm biomarkers under different ROX concentrations for 28 d. With the increasing total arsenic accumulation (TAs-E), biological responses in earthworm generally showed a two-stage changes of homeostasis dysregulation and dose-dependent alterations. At the early periods, ROX stress increased the reactive oxygen species (ROS) and lipid peroxidation (LPO) in a similar manner, and apparently disrupted mitochondrial calcium ([Ca2+]m). But earthworms regulated their mitochondrial and redox homeostasis through stable mitochondrial membrane potential (MMP) and increase of ATP level, superoxide dismutase (SOD) and catalase (CAT). After 14 d, the positively correlated mitochondrial effects of ROS, [Ca2+]m, MMP and ATP were related to the behavioral inhibition of burrow length, depth and reuse rate as well as antioxidant up-regulation of Nrf2, HO-1, sod1 and cat. These results contributed possible biomarkers from the dose-dependent relationship between mitochondrial, antioxidant and behavioral responses. Multiple biological detection in earthworms can better reflect the sub-chronic ecotoxicity of phenylarsenic pollutants in soil.

Biological assessment of contaminated shooting range soil using earthworm biomarkers.

Environmental contamination at shooting ranges is a widely known ecological problem. The aim of the study was to evaluate the extent of contamination and the ecotoxicity of a shooting range soil assessing the physiological and biochemical effects on earthworm Eisenia fetida (Savigny). Adult E. fetida were exposed to the soils collected from different distances of the shooting range for 28 days. High concentrations of Pb (53023 mg kg-1), increased concentrations of Ni (12 mg kg-1) and Sb (600 mg kg-1), significantly higher soil organic matter content (7.2%) and density (6.14 g cm-3) were determined in the backstop berm soil. Significant weight loss (44.4%) of the adult earthworms after 28 days of exposure occurred in the most contaminated shooting range soil and significantly higher concentrations of Pb (3101 mg kg-1), Cu (51 mg kg-1), Ni (2 mg kg-1), and Sb (20 mg kg-1) were determined in the tissues of worms, and no juveniles found there. Juveniles exposed to the less contaminated soil of the shooting range (A, B and C) accumulated significant concentrations of Pb, Cu, Fe, Mn, and Zn. The antioxidant enzymatic activity (glutathione-s-transferase (GST)) decreased, and lipid peroxidation increased as indicated by an increase in malondialdehyde (MDA) level in earthworms exposed to the contaminated soil. A compensatory mechanism between the activities of glutathione reductase (GR) and GST in earthworms exposed to these soils was confirmed.

Metal oxide nanoparticles facilitate the accumulation of bifenthrin in earthworms by causing damage to body cavity

In this study, we explored the influence of two metal oxide nanoparticles, nano CuO and nano ZnO (10, 50, 250 mg/kg), on accumulation of bifenthrin (100 μg/kg) in earthworms (Eisenia fetida) and its mechanism. The concentrations of bifenthrin in earthworms from binary exposure groups (bifenthrin + CuO and bifenthrin + ZnO) reached up to 23.2 and 28.9 μg/g, which were 2.65 and 3.32 times of that in bifenthrin exposure group without nanoparticles, respectively, indicating that nanoparticles facilitated the uptake of bifenthrin in earthworms. The contents of biomarkers (ROS, SOD, and MDA) in earthworms indicated that nanoparticles and bifenthrin caused damage to earthworms. Ex vivo test was utilized to investigate the toxic effects of the pollutants to cell membrane of earthworm coelomocytes and mechanism of increased bifenthrin accumulation. In ex vivo test, cell viability in binary exposure groups declined up to 30% and 21% compared to the control group after 24 h incubation, suggesting that coelomocyte membrane was injured by the pollutants. We conclude that nanoparticles damage the body cavity of earthworms, and thus lead to more accumulation of bifenthrin in earthworms. Our findings provide insights into the interactive accumulation and toxicity of nanoparticles and pesticides to soil organisms.

Evaluating Earthworms’ Potential for Remediating Soils Contaminated with Olive Mill Waste Sediments

The olive-oil industry generates large amounts of residues that, in the past, were accumulated in evaporating ponds in many Mediterranean countries. Currently, these open-air ponds pose a serious environmental hazard because of toxic chemicals that concentrate in their sediments. Bioremediation of olive mill waste (OMW) sediments has emerged as a viable option for managing this environmentally problematic residue. Here, we postulate that inoculation of an OMW-soil mixture with earthworms may be a complementary bioremediation strategy to that using native microorganisms only. A laboratory study assessed the ecotoxicity of OMW-amended soils (10%, 20%, 40% and 80% w/w) combining earthworm biomarker responses and soil enzyme activities. The doses of 40% and 80% were toxic to earthworms, as evidenced by the high mortality rate, loss of body weight and signs of oxidative stress after 30 d of soil incubation. Conversely, doses ≤ 20% w/w were compatible with earthworm activity, as indicated by the significant increase of soil enzyme activities. Total concentrations of phenolic compounds decreased by more than 70% respect to initial concentrations in 10% and 20% OMW treatments. These results suggest that OMW sediments intentionally mixed with soils in an up to 20% proportion is a workable bioremediation strategy, where earthworms can be inoculated to facilitate the OMW degradation.

Toxicity of thifluzamide in earthworm (Eisenia fetida)

An increase in the area treated with the fungicide thifluzamide has triggered concerns for soil ecosystem service providers such as earthworms. Here, we assessed effects of thifluzamide on earthworm (Eisenia fetida) biomarker indicators of stress responses and reproduction following exposure to 0, 0.1, 1.0, and 10.0 mg of thifluzamide kg−1 soil for 7, 14, 21, and 28 d (biomarker indicators) and 30 d (reproduction). Growth and reproduction were inhibited by exposure to thifluzamide at 10.0 mg/kg, and the activities of succinate dehydrogenase (SDH) and respiratory chain complex II were inhibited by exposure to 1.0 and 10.0 mg/kg thifluzamide for the majority of the 28-d experiment. Reactive oxygen species (ROS) increased across all thifluzamide treatments, and the activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) tended to be inhibited by thifluzamide. Upon exposure to thifluzamide, the activities of catalase (CAT) and guaiacol peroxidase (POD) initially increased and then decreased. Increased levels of malondialdehyde (MDA) were detected only at seven days after exposure, and genotoxicity increased as the thifluzamide concentration increased. The results suggest that thifluzamide presents a potential risk to earthworms at the concentration of 10.0 mg/kg, and its use should be moderated to reduce damage to soil ecosystem function.

Presence of Pesticides and Toxicity Assessment of Agricultural Soils in the Quintana Roo Mayan Zone, Mexico Using Biomarkers in Earthworms (Eisenia fetida)

Agriculture intensification and the use of pesticides have led to biodiversity loss due to soil toxic compounds. Thus, soil contamination studies are important to understand the negative effects in the physicochemical interactions. The use of biomarkers through bioindicators is a useful tool for assessing toxicity in agricultural environments complemented with the determination of pesticides. The objectives of this study were to determine the presence of organochlorine (OCPs) and organophosphate (OPPs) pesticides and the soil’s potential toxicity in agricultural fields with different crops from the center of Quintana Roo State, using a set of enzymatic biomarkers (BMs), such as acetylcholinesterase (AChE), glutathione-S-Transferase (GST), and catalase (CAT) on earthworms (Eisenia fetida). Earthworms were exposed for 96 h on nine different agricultural soils as well as on a reference soil from a conservation area. Within all samples of soils, only OCPs were detected in low concentrations (ranged from non-detected to 1.40 ppm). However, no correlation was observed between these pesticides and the BMs activity. AChE and CAT activity was significantly inhibited in at least one agricultural soil if compared to the conservation area, while no significant differences of GST were observed. The AChE activity observed suggests the presence of anticholinergic substances (that were neither detected nor determined analytically) in the sampled soils. The characterization of oxidative stress BMs was not correlated with the OCPs analyzed. Our results demonstrate that further studies of toxicity under field conditions are required, given the complexity of environmental conditions outside the laboratory.

Interspecific differences in biochemical and behavioral biomarkers in endogeic earthworms exposed to ethyl-parathion

Earthworms are common organisms in the soil toxicity-testing framework, and the epigeic Eisenia andrei and E. fetida are the recommended species. However, Eisenia species are rarely found in agricultural soils and recent studies have pointed out endogeic species are more sensitive to pesticides than Eisenia. Allolobophora chlorotica and Aporrectodea caliginosa are two endogeic soil-dwelling species that are abundant in the agroecosystem. However, knowledge on pesticide impact on this ecological group of earthworms is still incipient. Herein, we compared the biochemical (acetylcholinesterase [AChE] and carboxylesterase [CbE] activities) and behavioral (burrowing, casting and feeding) biomarker responses of these two endogeic earthworm species exposed for 7 days to soils contaminated with 0.1, 1 and 10 mg kg−1 ethyl-parathion. The results showed marked species-specific differences in both groups of biomarkers, suggesting A. caliginosa the most sensitive species to this organophosphorus pesticide under the exposure conditions in this study. Moreover, an in vitro inhibition trial with ethyl-paraoxon evidenced a higher sensitivity of A. caliginosa AChE activity compared with that of A. chlorotica. This finding suggested that this molecular target endpoint could contribute to the interspecific differences of behavioral responses rather than CbE activity; this latter considered a potent mechanism of OP removal. Our results suggest the inclusion of more than one endogeic earthworm species to assess toxicity from organophosphorus insecticides. However, the use of A. caliginosa in the environmental risk assessment framework of organophosphorus contamination is highly recommended because of its higher sensibility to this class of pesticides, in addition to its abundance in the agroecosystem.

Soil enzyme dynamics in chlorpyrifos-treated soils under the influence of earthworms

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 4individuals/kg wet soil) and the time of exposure (1, 2, 6, 12, and 18wk) could affect chlorpyrifos persistence in soil initially treated with 20mg active ingredientkg−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 30.3d (control soils) and 44.5d (low earthworm density) or 36.7d (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 6wk 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.

Oxidative stress and gene expression of earthworm (Eisenia fetida) to clothianidin

Neonicotinoid insecticides have become the most widely used pesticides in the world. Clothianidin is a novel neonicotinoid insecticide with a thiazolyl ring that exhibits excellent biological efficacy against a variety of pests. In the present study, the oxidative stress and genotoxicity of clothianidin on earthworms were evaluated. Moreover, the effective concentrations of clothianidin in artificial soil were monitored during the whole exposure period. The results showed that clothianidin was stable in artificial soil and that the residue concentrations were 0.094, 0.476, and 0.941mg/kg after 28 d of exposure, which represented changes no more than 10% compared to the concentrations on the 0th day. Additionally, both the concentration of and exposure time to clothianidin had a substantial influence on biomarkers in earthworms. At 0.5mg/kg and 1.0mg/kg, the reactive oxygen species (ROS) levels were greatly enhanced, causing changes in antioxidant enzyme activities, damage to biological macromolecules and abnormal expression of functional genes. Additionally, the present results showed that superoxide dismutase (SOD), DNA damage and heat shock protein 70 (HSP70) may be good indicators for environmental risk assessment of clothianidin to earthworms.

A brief review and evaluation of earthworm biomarkers in soil pollution assessment.

Earthworm biomarker response to pollutants has been widely investigated in the assessment of soil pollution. However, whether and how the earthworm biomarker-approach can be actually applied to soil pollution assessment is still a controversial issue. This review is concerned about the following points: 1. Despite much debate, biomarker is valuable to ecotoxicology and biomarker approach has been properly used in different fields. Earthworm biomarker might be used in different scenarios such as large-scale soil pollution survey and soil pollution risk assessment. Compared with physicochemical analysis, they can provide more comprehensive and straightforward information about soil pollution at low cost. 2. Although many earthworm species from different ecological categories have been tested, Eisenia fetida/andrei is commonly used. Many earthworm biomarkers have been screened from the molecular to the individual level, while only a few biomarkers, such as avoidance behavior and lysosomal membrane stability, have been focused on. Other aspects of the experimental design were critically reviewed. 3. More studies should focus on determining the reliability of various earthworm biomarkers in soil pollution assessment in future research. Besides, establishing a database of a basal level of each biomarker, exploring biomarker response in different region/section/part of earthworm, and other issues are also proposed. 4. A set of research guideline for earthworm biomarker studies was recommended, and the suitability of several earthworm biomarkers was briefly evaluated with respect to their application in soil pollution assessment. This review will help to promote further studies and practical application of earthworm biomarker in soil pollution assessment.

Relationships between metal compartmentalization and biomarkers in earthworms exposed to field-contaminated soils

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.