Earthworm Eisenia Andrei Research Articles

Improving ecological risk assessment in the Mediterranean area: Selection of reference soils and evaluating the influence of soil properties on avoidance and reproduction of two oligochaete species

A current challenge in soil ecotoxicology is the use of natural soils as test substrates to increase ecological relevance of data. Despite the existence of six natural reference soils (the Euro-soils), some parallel projects showed that these soils do not accurately represent the diversity of European soils. Particularly, Mediterranean soils are not properly represented. To fill this gap, 12 natural soils from the Mediterranean regions of Alentejo, Portugal; Cataluña, Spain; and Liguria, Italy, were selected and used in reproduction and avoidance tests to evaluate the soil habitat function for earthworms (Eisenia andrei) and enchytraeids (Enchytraeus crypticus). Predictive models on the influence of soil properties on the responses of these organisms were developed using generalized linear models. Results indicate that the selected soils can impact reproduction and avoidance behavior of both Oligochaete species. Reproduction of enchytraeids was affected by different soil properties, but the test validity criteria were fulfilled. The avoidance response of enchytraeids was highly variable, but significant effects of texture and pH were found. Earthworms were more sensitive to soil properties. They did not reproduce successfully in three of the 10 soils, and a positive influence of moisture, fine sand, pH, and organic matter and a negative influence of clay were found. Moreover, they strongly avoided soils with extreme textures. Despite these limitations, most of the selected soils are suitable substrates for ecotoxicological evaluations.

Does monensin in chicken manure from poultry farms pose a threat to soil invertebrates?

Monensin is a carboxylic polyether ionophore used in the poultry industry as a coccidiostat. It enters the environment via manure from broiler farms. In spite of its potential presence in the environment, information concerning monensin residues in manure and soil and its toxicity to soil organisms are insufficient. In the present study, two beneficial soil invertebrate species, earthworms ( Eisenia andrei) and woodlice ( Porcellio scaber), were used to assess the toxicity of monensin. Animals were exposed to a range of monensin concentrations via soil or food. Earthworm reproduction was found to be the most susceptible endpoint (NOEC = 3.5 mg kg −1 dry soil; EC 50 = 12.7 mg kg −1 dry soil), while no adverse effects were recorded in isopods (NOEC ⩾ 849 mg kg −1 dry soil, NOEC ⩾ 357 mg kg −1 dry food). The obtained toxicity data were compared with potential concentrations of monensin in soil. In view of this, manure from broiler chickens treated with monensin at a poultry farm was sampled. According to monensin and nitrogen concentrations in the chicken manure and the degradation time of monensin, the predicted environmental concentration (PEC) was calculated. PEC of monensin is around 0.013 mg kg −1 soil if manure is used after 3 months of composting and 0.05 mg kg −1 soil if used without storage. Data for earthworm reproduction was used to estimate the predicted no-effect concentration (PNEC). If fresh chicken manure is applied to terrestrial ecosystems, the risk quotient (PEC/PNEC ratio) is above 1, which indicates that monensin might pose an environmental risk under certain conditions. To prevent this, it is strongly recommended to compost chicken manure for several months before using it as fertiliser.

The high dosage of earthworm (Eisenia andrei) extract decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

Earthworm extract has shown anticancer characteristics. In the present study, we examined the effect of chronic treatment with a high dose of earthworm (Eisenia andrei) extract (EE) on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) of 3-week-old mice using 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 immunohistochemistry for cell proliferation and doublecortin (DCX) immunohistochemistry for neuroblast differentiation, respectively. BrdU-, Ki-67-, and DCX-immunoreactive cells were easily detected in the subgranular zone of the DG in vehicle (saline)-treated mice. However, BrdU-, Ki-67-, and DCX-immunoreactive cells in the 500 mg/kg EE-treated mice decreased distinctively compared to those in the vehicle-treated mice. In addition, brain-derived neurotrophic factor (BDNF) immunoreactivity and its protein level decreased markedly in the DG of the EE-treated group compared to those in the vehicle-treated group. These results indicate that chronic treatment with high dose EE decreased cell proliferation and neuroblast differentiation, and that BDNF immunoreactivity decreased in the DG of EE-treated mice.

The bioaccumulation of Molybdenum in the earthworm Eisenia andrei: Influence of soil properties and ageing

Mo bioaccumulation in the earthworm Eisenia andrei was determined after 28d exposure in ten different European field soils (pH 4.4–7.8) and an artificial soil, freshly spiked with Na2MoO4 at concentrations between 3.2 and 3200mg Mokg−1 dry soil. Three field soils were also tested after ageing for 11months. Earthworm Mo concentrations generally levelled off at high exposure levels but in most soils showed a (nearly) linear increase with increasing soil concentrations in the lower, non-toxic range (below EC10 or NOEC for reproduction effects). Bioaccumulation (BAF) and Bioconcentration factors (BCF) were calculated as the ratio of earthworm concentration to soil and estimated porewater concentrations, respectively. BAFs (0.35–3.44) and BCFs (1.31–276) did not seem much affected by soil concentration, suggesting that earthworms are not capable of regulating their internal Mo concentrations. BAF was best predicted by ammonium oxalate-extractable iron (Feox) and phosphor (Pox) contents of the soils.

Toxicity of petroleum hydrocarbon distillates to soil organisms

Canadian standards for petroleum hydrocarbons in soil are based on four distillate ranges (F1, C6-C10; F2, >C10-C16; F3, >C16-C34; and F4, >C34). Concerns have arisen that the ecological soil contact standards for F3 may be overly conservative. Oil distillates were prepared and characterized, and the toxicity of F3 and two subfractions, F3a (>C16-C23) and F3b (>C23-C34), to earthworms (Eisenia andrei), springtails (Orthonychiurus folsomi), and northern wheatgrass (Elymus lanceolatus), as well as the toxicity of F2 to earthworms, was determined. Clean soil was spiked with individual distillates and measured concentrations were determined for select tests. Results agree with previous studies with these distillates. Reported toxicities of crude and petroleum products to invertebrates were generally comparable to that of F3 and F3a. The decreasing order of toxicity was F3a > F3 > F3b with invertebrates, and F3a > F3b > F3 with plants. The toxicities of F3a and F3b were not sufficiently different to recommend regulating hydrocarbons based on these distillate ranges. The results also suggest that test durations may be insufficient for determining toxicity of higher distillate ranges, and that the selection of species and endpoints may significantly affect interpretation of toxicity test results.

The influence of soil properties on the toxicity of molybdenum to three species of soil invertebrates

Mo toxicity to earthworms ( Eisenia andrei), Collembola ( Folsomia candida) and enchytraeids ( Enchytraeus crypticus) was determined in 10 European soils and a standard artificial soil, freshly spiked with Na 2MoO 4, after 28 days exposure. Mo affected survival only in three low pH sandy soils; in all other soils LC50 was >3200 mg Mo/kg dry soil. EC50 values for the reproduction toxicity of Mo were 129–2378 mg/kg for earthworms, 72–>3396 mg/kg for Collembola, and 301–>2820 mg/kg for enchytraeids. Variation in toxicity among soils could not be explained by differences in available (pore water, water and 0.01 M CaCl 2 extractable) Mo concentrations. Clay content best predicted the EC50 for Mo toxicity to earthworms, while toxicity of Mo for enchytraeids was best described by soil pH. For Collembola no relationships could be derived due to the absence of toxicity in most soils. Soil properties had a strong but species-specific effect on Mo toxicity to soil invertebrates.

Use of the Comet and micronucleus assays for in vivo genotoxicity assessment in the coelomocytes of the earthworm Eisenia andrei

Use of the Comet and micronucleus assays for in vivo genotoxicity assessment in the coelomocytes of the earthworm Eisenia andrei

Comparison of biological and chemical measures of metal bioavailability in field soils: Test of a novel simulated earthworm gut extraction

Bioavailability of metals in soil is a major factor influencing estimates of risk associated with exposure of ecological receptors. Metal concentrations in soil are often compared to ecological screening benchmarks, which are based on total concentrations in soil. Often, the total concentration is not correlated with toxicity. No standardised method exists for determining the bioavailability of metals in soil to ecological receptors. Several surrogate measures of bioavailability were compared to the results of a battery of toxicity tests using copper (Cu), lead (Pb), and zinc (Zn)-contaminated soils collected from a former industrial area. A calcium chloride (CaCl 2) extraction, cyclodextrin (HPCD) extraction, simulated earthworm gut (SEG) test, and earthworm bioaccumulation test were performed using the soils. Extractable metals using the CaCl 2 solution were not correlated with any biological responses of earthworms ( Eisenia andrei), collembola ( Folsomia candida), northern wheatgrass ( Elymus lanceolatus), or alfalfa ( Medicago sativa L.). Concentrations of metals in the HPCD extracts were highly variable and were not adequate for revealing adverse effects. E. andrei tissue concentrations were variable but were predictive of adverse effects to invertebrates. The results of the SEG test correlated with most of the biological endpoints. Bioavailable Cu was correlated with adverse effects to invertebrates and plants using the SEG test. Overall, coefficients of determination associated with the relationships between the biological responses and each measure of bioavailability indicated that those for the SEG test were greater than those for the other surrogate measures of bioavailability. Further validation is required before this test is routinely used to estimate metal bioavailability and toxicity.

Influence of soil properties on molybdenum uptake and elimination kinetics in the earthworm Eisenia andrei

This study aimed at determining the uptake and elimination kinetics of molybdenum in the earthworm Eisenia andrei, and the influence of soil properties on molybdenum bioaccumulation. Three natural and four artificial soils were spiked at concentrations of 10 and 100 μg Mo g −1 dry soil. Earthworms were exposed individually to spiked soils and sampled at different time intervals for 21 d. Remaining earthworms were transferred to non-spiked soil to determine elimination, also for 21 d. Uptake and elimination rate constants and bioaccumulation factors (BAFs) were calculated using a one-compartment model, and related to total, water and CaCl 2 extractable molybdenum concentrations in soil. Molybdenum was rapidly accumulated by the earthworms with uptake rate constants between 0.05 and 1.70 g dry soil g −1 dry earthworm d −1, and equilibrium generally was reached within 10 d. Molybdenum was not strongly bioaccumulated (BAF ⩽ 4). Mo availability and bioaccumulation in earthworms was mainly affected by soil pH and organic carbon content .

Localization and characterization of sulfated glycosaminoglycans in the body of the earthworm Eisenia andrei (Oligochaeta, Annelida)

The aim of this study was to characterize the compartmental distribution of sulfated glycosaminoglycans (S-GAGs) in adults and their occurrence during the development of the earthworm Eisenia andrei. S-GAGs were extracted from the body of earthworms to identify their composition and the time of their appearance and disappearance in embryonic, newborn, juvenile, and adult earthworms. S-GAGs were also analyzed in earthworm tissue using histochemical metachromatic staining. Purified S-GAGs obtained from the whole body of adult earthworms were composed of chondroitin sulfate (CS) and heparan sulfate (HS). In addition, an unknown, highly sulfated polysaccharide (HSP) was detected. In order to characterize specifically the S-GAG composition in the integument, earthworms were dissected and as much as possible of their viscera was removed. HS and CS were the predominant sulfated polysaccharides in the dissected integument, whereas in viscera, CS, HS and the HSP were found in proportions similar to those identified in the body. The qualitative S-GAG composition in juveniles was similar to that obtained from adult earthworms. CS was the predominant S-GAG in newborn earthworms, accompanied by lesser amounts of HS and by tiny amounts of the HSP. This study provides a detailed descriptive account of the pattern of S-GAG synthesis during development, and also the characterization of the tissue distribution of these compounds in the body of earthworms.

Avoidance behavior of earthworms under exposure to pesticides: Is it always chemosensorial?

Soil avoidance by earthworms has been generally considered a relevant and sensitive endpoint for assessing soil contamination by xenobiotics. However, when pesticide ecotoxicological assessment is concerned, the sensitivity of the recently standardized avoidance assay has been questioned. We hypothesized that this controversy may be due to the specific pesticide mode of action of the chemicals used rather than reveal inconsistencies in the test feasibility, i.e. provided that no pesticides interfering with neuronal pathways are tested, this bioassay should keep expected high levels of sensitivity. In this study, the avoidance behaviour of the earthworm Eisenia andrei under exposure to the carbamate insecticide methomyl [S-methyl N-(methylcarbamoyloxy)thioacetimidate] was linked to the corresponding acetylcholinesterase (AChE) inhibition. Significant AChE inhibition occurred at lower concentrations (from 0.86 mg Kg−1 onwards) than significant avoidance of spiked soil (from 5.62 mg Kg−1 onwards). This indicates that assessments regarding pesticides that have neurotoxic activity may be biased if behavioral endpoints are selected. Despite theoretical hypothesis that have been raised, this should be the first study providing preliminary experimental evidence on such a link between avoidance behavior and neuronal impairment levels in earthworms. Further studies are ongoing that should refine conclusions of this study.

Uptake kinetics and subcellular compartmentalization of cadmium in acclimated and unacclimated earthworms (Eisenia andrei)

Acclimation to cadmium (Cd) levels exceeding background concentrations may influence the ability of earthworms to accumulate Cd with minimum adverse effects. In the present study, earthworms (Eisenia andrei) were acclimated by exposure to 20 mg/kg Cd (dry wt) in Webster soil for 28 d. A 224-d bioaccumulation test was subsequently conducted with both acclimated and unacclimated worms exposed in Webster soils spiked with 20 mg/kg and 100 mg/kg Cd (dry wt). Uptake kinetics and subcellular compartmentalization of Cd were examined. Results suggest that acclimated earthworms accumulated more Cd and required a longer time to reach steady state than unacclimated worms. Most of the Cd was present in the metallothionein (MT) fraction. Cadmium in the MT fraction increased approximately linearly with time and required a relatively longer time to reach steady state than Cd in cell debris and granule fractions, which quickly reached steady state. Cadmium in the cell debris fraction is considered potentially toxic, but low steady state concentrations observed in the present study would not suggest the potential for adverse effects. Future use of earthworms in ecological risk assessment should take into consideration pre-exposure histories of the test organisms. A prolonged test period may be required for a comprehensive understanding of Cd uptake kinetics and compartmentalization.

Role of soil interstitial water in the accumulation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine in the earthworm Eisenia andrei

The uptake of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from soil by the earthworm Eisenia andrei was examined by using the equilibrium partitioning (EqP) theory and a three-compartment model including soil (S), interstitial water (IW), and earthworms (E). The RDX concentrations were measured using U.S. Environmental Protection Agency (U.S. EPA) Method 8330A and high-performance liquid chromatography (HPLC). The S-IW studies were conducted using four natural soils with contrasting physicochemical properties that were hypothesized to affect the bioavailability of RDX. Each soil was amended with nominal RDX concentrations ranging from 1 to 10,000 mg/kg. The HPLC analysis showed that the IW extracted from soil was saturated with RDX at 80 mg/kg or greater soil concentrations. The calculated S-IW coefficient (K(p)) values for RDX ranged from 0.4 to 1.8 ml/g soil, depending on the soil type, and were influenced by the organic matter content. In the IW-E studies, earthworms were exposed to nonlethal RDX concentrations in aqueous media. The uptake of RDX by the earthworms correlated well (r(2) = 0.99) with the dissolved RDX concentrations. For the E-S studies, earthworms were exposed to RDX-amended soils used in the S-IW studies. The bioconcentration factors (BCF; ratios of E-to-IW RDX concentrations) were relatively constant ( approximately 5) up to 80 mg/kg soil RDX concentrations, which encompass the RDX saturation limit in the interstitial water of the tested soils. At this concentration range, the RDX uptake from interstitial water was likely dominated by passive diffusion and could be used as an indicator of bioavailability. Other mechanisms may be involved at greater RDX soil concentrations.

Assessing the effects of FBC ash treatments of metal-contaminated soils using life history traits and metal bioaccumulation analysis of the earthworm Eisenia andrei

Earthworms ( Eisenia andrei) were exposed, in controlled conditions, to metal-contaminated soils previously treated in situ with two types of fluidized bed combustion ashes. Effects on this species were determined by life history traits analysis. Metal immobilizing efficiency of ashes was indicated by metal bioaccumulation. Ashes-treated soils reduced worm mortality compared to the untreated soil. However, these ashes reduced both cocoon hatching success and hatchlings numbers compared to the untreated soil. In addition, sulfo-calcical ashes reduced or delayed worm maturity and lowered cocoon production compared to silico-alumineous ones. Metal immobilizing efficiency of ashes was demonstrated for Zn, Cu and to a lesser extent Pb. Only silico-alumineous ashes reduced Cd bioaccumulation, although Cd was still bioconcentrated. Thus, although ash additions to metal-contaminated soils may help in immobilizing metals, their use might result, depending on the chemical nature of ashes, to severe detrimental effects on earthworm reproduction with possible long term consequences to populations.

Detritivorous earthworms modify microbial community structure and accelerate plant residue decomposition

The impact of detritivorous earthworms ( Eisenia andrei) on microbial community structure and function in grape marc, a lignocellulosic enriched plant residue, was investigated in a mesocosm experiment. Analysis of carbon and nitrogen pools was also carried out in order to evaluate how changes in microbial communities affect plant residue decomposition. The grape marc was completely processed after fifteen days as a result of the high density of earthworms present and the rapid gut transit time. Eisenia andrei had a large impact on the structure of the microbial community, as revealed by phospholipid fatty acid analysis. Earthworm activity reduced the abundance of both bacterial (except for Gram-negative bacteria) and fungal PLFA biomarkers relative to the control values. Decreases in microbial activity and in protease and cellulase activities were also attributable to the presence of earthworms. Moreover, earthworms strongly modified the substrate utilization patterns of microbial communities, as revealed by BIOLOG analysis. The presence of earthworms led to an increase in the utilization of some amino acids and polymers, which reached a higher substrate diversity value than that in the control mesocosm. The differences in microbial communities were accompanied by a reduction in the total C content and the labile C pool, relative to the control, although there were no significant differences in either cellulose or hemicellulose contents. However, total N content increased in both mesocosms – with and without earthworms – and the concentration of NH 4 + was also enhanced by earthworm activity. The results indicate that detritivorous earthworms play a key role in decomposing fresh plant residues in the short term via their intensive interactions with microbial communities.

Glycosaminoglycans from earthworms (Eisenia andrei)

The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC-ESI-MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.

Toxicity of the organophosphorous insecticide chlormephos to the earthworm Eisenia andrei and the terrestrial isopod Porcellio scaber

In the present study we determined the toxicity of chlormephos to two common soil organisms – earthworms (Eisenia andrei) and isopods(Porcellio scaber) using Lufa 2.2 soil. The LC50 values for the effects on earthworm and isopod survival were 58 and 76 mg/kg dry soil, respectively. Mass change of earthworms and behaviour of isopods were more sensitive end points as survival. Based on earthworm body mass changes, NOEC and LOEC values were 1 and 3 mg/kg dry soil, respectively. The earthworms did not reproduce even at the lowest chlormephos concentration (LOEC < 1 mg/kg dry soil). Isopods significantly avoided burrowing in soil treated with ≥40 mg/kg dry soil. Compared with some other organophosphate insecticides, chlormephos was highly toxic to beneficial terrestrial invertebrates.

Accumulation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine by the earthworm Eisenia andrei in a sandy loam soil

The heterocyclic polynitramine hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly energetic compound found as a soil contaminant at some defense installations. Although RDX is not lethal to soil invertebrates at concentrations up to 10,000 mg/kg, it decreases earthworm cocoon formation and juvenile production at environmentally relevant concentrations found at contaminated sites. Very little is known about the uptake of RDX in earthworms and the potential risks for food-chain transfer of RDX in the environment. Toxicokinetic studies were conducted to quantify the bioaccumulation factors (BAFs) using adult earthworms (Eisenia andrei) exposed for up to 14 d to sublethal concentrations of nonlabeled RDX or [14C]RDX in a Sassafras sandy loam soil. High-performance liquid chromatography of acetonitrile extracts of tissue and soil samples indicated that nonlabeled RDX can be accumulated by the earthworm in a concentration- and time-dependent manner. The BAF, expressed as the earthworm tissue to soil concentration ratio, decreased from 6.7 to 0.1 when the nominal soil RDX concentrations were increased from 1 to 10,000 mg/kg. Tissue concentrations were comparable in earthworms exposed to nonlabeled RDX or [14C]RDX. The RDX bioaccumulation also was estimated using the kinetically derived model (BAFK), based on the ratio of the uptake to elimination rate constants. The established BAFK of 3.6 for [14C]RDX uptake was consistent with the results for nonlabeled RDX. Radioactivity also was present in the tissue residues of [14C]RDX-exposed earthworms following acetonitrile extraction, suggesting the formation of nonextractable [14C]RDX metabolites associated with tissue macromolecules. These findings demonstrated a net accumulation of RDX in the earthworm and the potential for food-chain transfer of RDX to higher-trophic-level receptors.

Bioconversion of wastes from olive oil industries by vermicomposting process using the epigeic earthworm Eisenia andrei

The present work evaluates the possible bioconversion of wet olive cake by low-cost biostabilization (vermicomposting process). Wet olive cake fresh (WOC), precomposted (WOCP), or mixed with biosolids (WOCB), were vermicomposted for 6 months to obtain organic amendments for agricultural and remediation purposes. The results showed initial differences depending on previous treatment. WOCP was initially more stable, presented a low C:N ratio, and showed more dehydrogenase and urease activity. By contrast, there was no dehydrogenase activity initially in WOC and WOCB, due to the presence of some different types of polyphenols. Finally, the end product showed relatively higher amounts of total nitrogen and humic acid and met the standard of quality for composts and vermicomposts for use both in conventional and organic agriculture and soil-restoration programs.

Production of Fibrinolytic Enzyme in Plastid-Transformed Tobacco Plants

The earthworm fibrinolytic enzyme, which belongs to a group of serine proteases with strong fibrinolytic activity, has been used as an oral drug for prevention and treatment of thrombosis in East Asia. Fibrizyme is a fibrinolytic enzyme isolated from the earthworm Eisenia andrei. Here we report genetic engineering of tobacco plastids with stable integration of the fibrizyme gene into the tobacco chloroplast genome. A plastid transformation vector was constructed by introducing various regulatory elements into fibrizyme cDNA. This vector was delivered by particle bombardment into tobacco leaf explants and plastid-transformed plants were subsequently regenerated into whole plants through several rounds of selection. We confirmed stable integration of the fibrizyme gene into the tobacco plastid genome by PCR and Southern blot analyses. Northern and Western blot analyses revealed that mRNA and protein of recombinant fibrizyme were highly expressed in transformed tobacco plants.