Effects of dioxin exposure in Eisenia andrei: integration of biomarker data by an Expert System to rank the development of pollutant-induced stress syndrome in earthworms

A battery of biomarkers has recently been developed in the earthworm Eisenia andrei. In this study, different biomarkers (i.e. Ca²⁺-ATPase activity, lysosomal membrane stability-LMS, lysosomal lipofuscin and neutral lipid content) were utilized to evaluate the alterations in the physiological status of animals, induced by exposure for 3 d to different sublethal concentrations of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (1.5 × 10⁻³, 1.5 × 10⁻², 1.5 × 10⁻¹ ng mL⁻¹) utilizing the paper contact toxicity test. Lysosome/cytoplasm volume ratio and DNA damage were also evaluated as a biomarker at the tissue level and as a biomarker of genotoxicity, respectively. Moreover, the NR retention time assay conditions were optimized for the determination of in vivo LMS in earthworm coelomocytes. The results demonstrate that LMS and Ca²⁺-ATPase activity were early warning biomarkers able to detect the effects of minimal amounts of TCDD and that biomarkers evaluated at the tissue level are important for following the evolution of the stress syndrome in earthworms. To evaluate the health status of the animals, an Earthworm Expert System (EES) for biomarker data integration and interpretation was developed. The EES proved to be a suitable tool able to rank, objectively, the different levels of the stress syndrome in E. andrei induced by the different concentrations of TCDD.     

Ecotoxicological responses of the earthworm Eisenia fetida exposed to soil contaminated with HHCB

Although polycyclic musks have been shown to cause lethal and sub-lethal effects on organisms, their biochemical toxicity to earthworms is not well understood. In the current study, we investigated the responses of antioxidant systems and lipid peroxidation after exposing Eisenia fetida to soil contaminated with 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-γ-2-benzopyran (HHCB). Significant increase in lipid peroxidation level was observed on day 14 at two high concentrations, 50 and 100 mg kg⁻¹. Among antioxidant enzymes, the primary response to chronic HHCB exposure can be attributed to superoxide dismutase (SOD) and catalase (CAT). Of the two enzymes, SOD exhibited more sensitive response to HHCB stress. In addition, these two enzymes could have a combined effect on fighting damage by reactive oxygen species, evidenced by a marked relationship between lipid peroxidation and enzyme activity. On the other hand, dose-dependent inhibition of peroxidase (POD) activity has been observed throughout the test. The results suggest that the variations in investigated parameters of E. fetida could be used as responsive biomarkers for oxidative stress caused by HHCB in a soil environment.     

H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil

¹H NMR metabolomics was used to monitor earthworm responses to sub-lethal (50-1500 mg/kg) phenanthrene exposure in soil. Total phenanthrene was analyzed via soxhlet extraction, bioavailable phenanthrene was estimated by hydroxypropyl-β-cyclodextrin (HPCD) and 1-butanol extractions and sorption to soil was assessed by batch equilibration. Bioavailable phenanthrene (HPCD-extracted) comprised ∼65-97% of total phenanthrene added to the soil. Principal component analysis (PCA) showed differences in responses between exposed earthworms and controls after 48 h exposure. The metabolites that varied with exposure included amino acids (isoleucine, alanine and glutamine) and maltose. PLS models indicated that earthworm response is positively correlated to both total phenanthrene concentration and bioavailable (HPCD-extracted) phenanthrene in a freshly spiked, unaged soil. These results show that metabolomics is a powerful, direct technique that may be used to monitor contaminant bioavailability and toxicity of sub-lethal concentrations of contaminants in the environment. These initial findings warrant further metabolomic studies with aged contaminated soils.     

Susceptibility of epigeic earthworm Eisenia fetida to agricultural application of six insecticides

Ecotoxicological risks of agricultural application of six insecticides to soil organisms were evaluated by acute toxicity tests under laboratory condition following OECD guidelines using the epigeic earthworm Eisenia fetida as the test organism. The organochlorine insecticide endosulfan (LC₅₀ - 0.002 mg kg⁻¹) and the carbamate insecticides aldicarb (LC₅₀ - 9.42 mg kg⁻¹) and carbaryl (LC₅₀ - 14.81 mg kg⁻¹) were found ecologically most dangerous because LC₅₀ values of these insecticides were lower than the respective recommended agricultural dose (RAD). Although E. fetida was found highly susceptible to the pyrethroid insecticide cypermethrin (LC₅₀ - 0.054 mg kg⁻¹), the value was higher than its RAD. The organophosphate insecticides chlorpyrifos (LC₅₀ - 28.58 mg kg⁻¹), and monocrotophos (LC₅₀ - 39.75 mg kg⁻¹) were found less toxic and ecologically safe because the LC₅₀ values were much higher than their respective RAD.     

Histopathological and biochemical alternations of the heart induced by acute cadmium exposure in the freshwater crab Sinopotamon yangtsekiense

Cadmium (Cd) is a highly toxic element in water. Its toxicity has been attributed to oxidative stress mediated by free radicals. Here we investigated the effects of Cd on the histopathology, antioxidant enzymes and lipid peroxidation of crustacean heart. The freshwater crabs Sinopotamon yangtsekiense were exposed to different concentrations of Cd for 1, 3, 5 and 7 d. After exposure, histological abnormalities were discovered, including myocardial edema, vacuolar and vitreous degeneration, and infiltration of inflammatory cells. Additionally, alterations in nuclei, mitochondria, rough endoplasmic reticulum as well as myofibrils were observed. Meanwhile, superoxide dismutase (SOD) activity was significantly increased after Cd exposure. Catalase (CAT) activity was only increased in the group exposed to 14.50 mg L⁻¹ Cd on day 5 and decreased with increasing Cd concentration and exposure time. Glutathione peroxidase (GPx) activity was increased in groups treated with 29.00, 58.00 and 116.00 mg L⁻¹ on days 1 and 3, and decreased thereafter. Besides, malondialdehyde (MDA) levels were significantly increased after 3 d of Cd exposure at all the indicated concentrations. These results showed that acute Cd exposure led to harmful effects on the histology of crab heart, which are most likely linked to Cd-induced oxidative stress.     

Comparing the sensitivity of soil invertebrates to pesticides with that of Eisenia fetida

The sole routine testing of the standard earthworm Eisenia fetida for the terrestrial risk assessment of pesticides has been under much debate since other soil invertebrates may be more sensitive than this standard test species. However, the very low availability of laboratory toxicity data for taxa other than E. fetida has greatly hampered sensitivity comparisons. In the present study, the relative tolerance (T_rel) approach was used to enable comparing toxicity thresholds obtained from the US-EPA ECOTOX database, for main terrestrial taxonomic groups and pesticidal types of action (insecticides, fungicides, herbicides, and other) separately. Analyses confirmed previously reported lower and higher sensitivity of collembolans to fungicides and insecticides, respectively. However, various other discrepancies in susceptibility relative to E. fetida were encountered as indicated by species sensitivity distributions and/or calculated 95% confidence intervals of T_rel values. Arachnids and isopods were found to be more sensitive to insecticides, and nematodes to fungicides, as compared to E. fetida. Implications of study findings for the terrestrial risk assessment of pesticides are discussed.     

Histopathological effects of chronic aqueous exposure to bis(tri-n-butyltin)oxide (TBTO) to environmentally relevant concentrations reveal thymus atrophy in European flounder (Platichthys flesus)

Although the use of tributyltin in antifouling paints has been banned, this compound is still a serious pollutant of the marine environment. This paper describes a unique study in which European flounder (Platichthys flesus) were chronically (8 months) exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. Residue levels in selected tissues (liver, muscle) and general health status indices were measured and the effects on several organs (gills, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined histopathologically. Additionally, morphometric analysis of the thymus was performed. The major finding is that exposure of flounder to 5 μg TBTO/l over a period of 8 months, resulting in body burdens comparable to high field levels, induced significant reduction of thymus volume, possibly affecting immunocompetence of the animals. Chronic exposure of European flounder to tributyltin is therefore likely to affect the general health status of this species in heavily polluted aquatic environments.     

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction.     

Eisenia fetida avoidance behavior as a tool for assessing the efficiency of remediation of Pb, Zn and Cd polluted soil

Remediation by means of soil leaching with ethylenediaminetetraacetic acid (EDTA) is capable of extracting the most labile soil fractions, leaving the residual metals in biologically non-available forms. We evaluated the feasibility of the standardized earthworm (Eisenia fetida) avoidance test for assessing the efficiency of soil remediation of Pb, Zn and Cd polluted soil. Chemical extraction tests (six-step sequential extraction, toxicity characteristic leaching procedure, physiologically based extraction test, diethylenediaminepentaacetic acid extraction) indicated that the mobility, oral bioaccessibility and phytoavailability of Pb, Zn and Cd were consistently reduced. However, the avoidance test showed no significant avoidance of polluted soil in favor of that which had been remediated. Pb, Zn and Cd accumulation in E. fetida mirrored the decreasing pattern of metal potential bioavailability gained by leaching the soil with increasing EDTA concentrations. The calculated bioaccumulation factors indicated the possibility of underestimating the metal bioavailability in soil using chemical extraction tests.     

Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach

The goal of this study was to identify promising new biomarkers of phenanthrene by identifying differentially expressed proteins in Eisenia fetida after exposure to phenanthrene. Extracts of earthworm epithelium collected at days 2, 7, 14, and 28 after phenanthrene exposure were analyzed by two dimensional electrophoresis (2-DE) and quantitative image analysis. Comparing the intensity of protein spots, 36 upregulated proteins and 45 downregulated proteins were found. Some of the downregulated and upregulated proteins were verified by MALDI-TOF/TOF-MS and database searching. Downregulated proteins in response to phenanthrene exposure were involved in glycolysis, energy metabolism, chaperones, proteolysis, protein folding and electron transport. In contrast, oxidation reduction, oxygen transport, defense systems response to pollutant, protein biosynthesis and fatty acid biosynthesis were upregulated in phenanthrene-treated E. fetida. In addition, ATP synthase b subunit, lysenin-related protein 2, lombricine kinase, glyceraldehyde 3-phosphate dehydrogenase, actinbinding protein, and extracellular globin-4 seem to be potential biomarkers since these biomarker were able to low levels (2.5 mg kg⁻¹) of phenanthrene. Our study provides a functional profile of the phenanthrene-responsive proteins in earthworms. The variable levels and trends in these spots could play a potential role as novel biomarkers for monitoring the levels of phenanthrene contamination in soil ecosystems.     

Acute toxicity, biochemical and gene expression responses of the earthworm Eisenia fetida exposed to polycyclic musks

Phytoremediation is a novel and promising approach for the treatment of pollutants. This study did explore the potential of Aster amellus Linn. to decolorize a sulfonated azo dye Remazol Red (RR), a mixture of dyes and a textile effluent. Induction in the activities of lignin peroxidase, tyrosinase, veratryl alcohol oxidase and riboflavin reductase was observed during RR decolorization, suggesting their involvement in the metabolism of RR. UV-Visible absorption spectrum, HPLC and FTIR analysis confirmed the degradation of RR. Four metabolites after the degradation of the dye were identified as 2-[(3-diazenylphenyl) sulfonyl] ethanesulfonate, 4-amino-5-hydroxynaphthalene-2,7-disulfonate, naphthalene-2-sulfonate and 3-(1,3,5-triazin-2-ylamino)benzenesulfonate by using GC/MS. Textile effluent and mixture of dyes showed 47% and 62% decrease respectively in American Dye Manufacturers Institute value. BOD of textile effluent and mixture of dyes were reduced by 75% and 48% respectively, COD of industrial effluent and mixture of dyes was reduced by 60% and 75% and TOC was reduced by 54% and 69% respectively after the treatment by A. amellus for 60 h; this indicated that the plant can be used for cleaning textile effluents. Toxicity study revealed the phytotransformation of RR into non-toxic products.     

Toxicity and bioaccumulation of reduced TNT metabolites in the earthworm Eisenia andrei exposed to amended forest soil

Soils contaminated with 2,4,6-trinitrotoluene (TNT) and TNT primary reduction products have been found to be toxic to certain soil invertebrates, such as earthworms. The mechanism of toxicity of TNT and of its by-products is still not known. To ascertain if one of the TNT reduction products underlies TNT toxicity, we tested the toxicity and bioaccumulation of TNT reduction products. 2-Amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT) and 2,6-diamino-4-nitrotoluene (2,6-DANT) were tested separately in adult earthworms (Eisenia andrei) following a 14-d exposure to amended sandy loam forest soil. TNT, 4-ADNT, and 2-ADNT were lethal to earthworms (14-d LC(50) were: 580, 531 and 1088 micromol kg(-1), or 132, 105 and 215 mgkg(-1) dry soil, respectively) and gave the following order of toxicity: 4-ADNT>TNT>2-ADNT. Exposure to 2,4-DANT and to 2,6-DANT caused no mortality at 600 micromol kg(-1) or 100 mgkg(-1) dry soil. We found that all four TNT reduction products accumulated in earthworm tissues and 2-ADNT reached the highest levels at 3.0+/-0.3 micromol g(-1) tissue. The 14-d bioaccumulation factors were 5.1, 6.4, 5.1 and 3.2 for 2-ADNT, 4-ADNT, 2,4-DANT and 2,6-DANT, respectively. Results also suggest that some TNT metabolites are at least as toxic as TNT and should be considered when evaluating the overall toxicity of TNT-contaminated soil to earthworms.     

Bioaccumulation and depuration of metals in blue crabs (Callinectes sapidus Rathbun) from a contaminated and clean estuary

Blue crabs from a contaminated estuary (Hackensack Meadowlands-HM) and a cleaner reference site (Tuckerton-TK) were analyzed for Cr, Cu, Hg, Pb, and Zn in muscle and hepatopancreas. Crabs from each site were taken into the laboratory and fed food from the other site, or in another experiment, transplanted to the other site for eight weeks. All crabs were analyzed for metals. Overall, tissue concentrations reflected environmental conditions. Tissue differences were found for Cu, Pb and Zn (all higher in hepatopancreas), and Hg (higher in muscle). HM muscle had more Hg than TK muscle, but did not decrease after transplanting or consuming clean food. HM crabs lost Cu, Pb and Zn in hepatopancreas after being fed clean food or transplanted. TK crabs increased Hg in muscle and Cr and Zn in hepatopancreas after transplantation or being fed contaminated (HM) food. Concentrations were variable, suggesting that blue crabs may not be fully reliable bioindicators of polluted systems.     

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 28 d 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 3200 mg Mo kg⁻¹ dry soil. Three field soils were also tested after ageing for 11 months. 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 (Fe_ox) and phosphor (P_ox) contents of the soils.     

Chronic exposure to environmental stressors induces fluctuating asymmetry in shrews inhabiting protected Mediterranean sites

Many ecotoxicological studies have addressed the effects of contaminant exposure at various levels of biological organization. However, little information exists on the effects of toxicants on wildlife populations. Here we examined exposure of populations of the greater white-toothed shrew Crocidura russula (Soricomorpha, Soricidae) occupying two protected Mediterranean sites (a polluted area, the Ebro Delta, and a control site, Garraf Massif). Bioaccumulation of selected elements (Pb, Hg, Cd, Zn, Cu, Fe, Mn, Cr, Mo, Sr, Ba, and B), a body condition index (BCI) and fluctuating asymmetry (FA) were used to assess the chronic exposure to environmental pollution. BCI was correlated neither to metal concentrations nor to FA, suggesting that this fitness measure only reflects environmental disturbances at a local level. However, shrews from the polluted area showed higher concentrations of metals and metalloids (Pb, Hg, B, and Sr) and greater shape FA than specimens from the reference area. A correlation between FA was found for both first and second principal component vectors suggesting that developmental instability increases as a result of exposure to multiple pollutants. Our results corroborate the suitability of C. russula as a bioindicator of environmental quality and show that FA is an appropriate index to examine impact of developmental stressors in populations inhabiting disturbed areas.     

Effect of metals on a siderophore producing bacterial isolate and its implications on microbial assisted bioremediation of metal contaminated soils

A bacterial isolate producing siderophore under iron limiting conditions, was isolated from mangroves of Goa. Based on morphological, biochemical, chemotaxonomical and 16S rDNA studies, the isolate was identified as Bacillus amyloliquefaciens NAR38.1. Preliminary characterization of the siderophore indicated it to be catecholate type with dihydroxy benzoate as the core component. Optimum siderophore production was observed at pH 7 in mineral salts medium (MSM) without any added iron with glucose as the carbon source. Addition of NaCl in the growth medium showed considerable decrease in siderophore production above 2% NaCl. Fe⁺² and Fe⁺³ below 2 μM and 40 μM concentrations respectively, induced siderophore production, above which the production was repressed. Binding studies of the siderophore with Fe⁺² and Fe⁺³ indicated its high affinity towards Fe⁺³. The siderophore concentration in the extracellular medium was enhanced when MSM was amended with essential metals Zn, Co, Mo and Mn, however, decreased with Cu, while the concentration was reduced with abiotic metals As, Pb, Al and Cd. Significant increase in extracellular siderophore production was observed with Pb and Al at concentrations of 50 μM and above. The effect of metals on siderophore production was completely mitigated in presence of Fe. The results implicate effect of metals on the efficiency of siderophore production by bacteria for potential application in bioremediation of metal contaminated iron deficient soils especially in the microbial assisted phytoremediation processes.     

Influence of earthworm mucus and amino acids on tomato seedling growth and cadmium accumulation

The effects on the growth of tomato seedlings and cadmium accumulation of earthworm mucus and a solution of amino acids matching those in earthworm mucus was studied through a hydroponic experiment. The experiment included four treatments: 5 mg Cd L⁻¹ (CC), 5 mg Cd L⁻¹ + 100 mL L⁻¹ earthworm mucus (CE), 5 mg Cd L⁻¹ + 100 mL L⁻¹ amino acids solution (CA) and the control (CK). Results showed that, compared with CC treatment, either earthworm mucus or amino acids significantly increased tomato seedling growth and Cd accumulation but the increase was much higher in the CE treatment compared with the CA treatment. This may be due to earthworm mucus and amino acids significantly increasing the chlorophyll content, antioxidative enzyme activities, and essential microelement uptake and transport in the tomato seedlings. The much greater increase in the effect of earthworm mucus compared with amino acid treatments may be due to IAA-like substances in earthworm mucus.     

Microcosm investigations of stormwater pond sediment toxicity to embryonic and larval amphibians: variation in sensitivity among species

Stormwater ponds have become common features of modern development and often represent significant amounts of open space in urbanized areas. Although stormwater ponds may provide habitat for wildlife, factors responsible for producing variation in wildlife use of ponds have received limited attention. To investigate the role of variation in species tolerances of pollutants in structuring pond-breeding amphibian assemblages, we exposed species tolerant (Bufo americanus) and not tolerant (Rana sylvatica) of urbanization to pond sediments in laboratory microcosms. Pond microcosms had elevated sediment metal levels and chloride water concentrations. Among R. sylvatica embryos, exposure to pond sediments resulted in 100% mortality. In contrast, B. americanus embryos and larvae experienced only sublethal effects (i.e., reduced size at metamorphosis) due to pond sediment exposure. Our results suggest variation in pollutant tolerance among early developmental stages of amphibians may act in concert with terrestrial habitat availability to structure amphibian assemblages associated with stormwater ponds.     

Antioxidant responses of the earthworm Lampito mauritii exposed to Pb and Zn contaminated soil

The aim of this study was to provide fundamental data on the biochemical analysis of antioxidant defences in the earthworm exposed to low levels (75, 150, 300 mg kg(-1) soil) of Pb and Zn. In order to attain this objective, adult Lampito mauritii were exposed to different doses of Pb and Zn separately for 28 days and the concentrations of oxidized and reduced glutathione, activities of glutathione-S-transferase, glutathione peroxidase and glutathione reductase were assessed. Dose-dependent perturbations were observed in the glutathione-glutathione-S-transferase system and other antioxidant enzymes during the early phase of the exposure to Pb. In the Zn exposed earthworm, the glutathione-glutathione-S-transferase system remained stable and the stimulation of glutathione peroxidase and glutathione reductase activities occurred significantly only on day 14 at 300 mg Zn kg(-1). It is concluded that the antioxidants are directly involved in the adaptive response of Lampito mauritii for survival in metal contaminated soil.     

Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an ‘ecoscore’, a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected.