Toxicological responses of earthworm (Eisenia fetida) exposed to metal-contaminated soils

The aim of this study was to evaluate the toxicological responses of earthworm (Eisenia fetida) induced by field-contaminated, metal-polluted soils. Biochemical responses and DNA damage of earthworm exposed to two multi-metal-contaminated soils in a steel industry park and a natural reference soil in Zijin Mountain for 2, 7, 14, and 28 days were studied. Results showed that three enzyme activities, including superoxide dismutase (SOD), acetylcholinesterase (AChE), and cellulase, in earthworm in metal-contaminated soils were significantly different from those of the reference soil. Cellulase and AChE were more sensitive than SOD to soil contamination. The Olive tail moment of the comet assay after 2-day exposure increased 56.5 and 552.0 % in two contaminated soils, respectively, compared to the reference soil. Our findings show that cellulase and DNA damage levels can be used as potential biomarkers for exposure of earthworm to metal-polluted soils.     

Antifeedant, larvicidal and growth inhibitory effects of ononitol monohydrate isolated from Cassia tora L. against Helicoverpa armigera (Hub.) and Spodoptera litura (Fab.) (Lepidoptera: Noctuidae)

Ononitol monohydrate isolated from the ethyl acetate extract of Cassia tora L. using column chromatography was evaluated for its antifeedant, larvicidal and growth inhibitory activities against Helicoverpa armigera and Spodoptera litura at different concentrations of 125, 250, 500 and 1000 ppm. Leaf disc no-choice method was used for the bioassay. The compound showed significant antifeedant, larvicidal and pupicidal activities against H. armigera and S. litura. The compound also prolonged the larval-pupal duration of the insect at all the tested concentrations. The activities were concentration dependent for both the insects. Ononitol could be used as an agent to prepare botanical new pesticidal formulations.     

Effects of metals on life cycle parameters of the earthworm Eisenia fetida exposed to field-contaminated, metal-polluted soils

Two control and eight field-contaminated, metal-polluted soils were inoculated with Eisenia fetida (Savigny, 1826). Three, 7, 14, 21, 28 and 42 days after inoculation, earthworm survival, body weight, cocoon production and hatching rate were measured. Seventeen metals were analysed in E. fetida tissue, bulk soil and soil solution. Soil organic carbon content, texture, pH and cation exchange capacity were also measured. Cocoon production and hatching rate were more sensitive to adverse conditions than survival or weight change. Soil properties other than metal concentration impacted toxicity. The most toxic soils were organic-poor (1-10 g C kg(-1)), sandy soils (c. 74% sand), with intermediate metal concentrations (e.g. 7150-13,100 mg Pb kg(-1), 2970-53,400 mg Zn kg(-1)). Significant relationships between soil properties and the life cycle parameters were determined. The best coefficients of correlation were generally found for texture, pH, Ag, Cd, Mg, Pb, Tl, and Zn both singularly and in multivariate regressions. Studies that use metal-amended artificial soils are not useful to predict toxicity of field multi-contaminated soils.     

Combined toxicity of chlorpyrifos,abamectin, imidacloprid,and acetamiprid on earthworms (Eisenia fetida)

Environmental Science and Pollution Research - Mixed pesticides have been broadly used in agriculture. However, assessing the combined effects of pesticides in the environment is essential for...     

Toxicity of the explosive metabolites hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) and hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) to the earthworm Eisenia fetida

Toxicity of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) to earthworm was evaluated. Both MNX and TNX had lethal and sublethal effects on earthworms. Exposure to MNX- or TNX-contaminated soil caused a significant concentration-dependent decrease in earthworm survival and growth. The lowest observed lethal concentration (LOLC) for both MNX and TNX was 100 and 200 mgkg(-1) soil dry weight in the sandy loam soil and in the silt loam soil, respectively. No earthworms survived for 14 days in MNX- or TNX-spiked soil at 500 mgkg(-1) soil dry weight. After 7 days exposure, the lowest observed effect concentration (LOEC) for earthworm growth was 50 mgkg(-1) soil dry weight for TNX and 100 mgkg(-1) soil dry weight for MNX in both soil types. The LC20 and LC50 for MNX in sandy loam soil were 114 and 262 mgkg(-1) and for TNX, they were 114 and 254 mgkg(-1) soil dry weight, respectively. The corresponding values for MNX and TNX in silt loam soil were 234 and 390 mgkg(-1) soil dry weight, respectively, and 200 and 362 mgkg(-1) soil dry weight, respectively. After 35 days exposure, earthworm growth was reduced 8-39% by TNX in sandy loam soil, whereas TNX only inhibited earthworm growth 5-18% at the same concentration range in silt loam soil. LC20 and LC50 for TNX were slightly lower than for MNX; this indicates that TNX was more toxic than MNX. No significant morphological or developmental abnormalities were observed in earthworms surviving exposure.     

Accumulation of 14C-trinitrotoluene and related nonextractable (bound) residues in Eisenia fetida

To determine if trinitrotoluene (TNT) forms nonextractable residues in earthworms and to measure the relative degree of accumulation as compared to TNT and its deaminated metabolites, Eisenia fetida was exposed to ¹⁴C-TNT using dermal contact to filter paper or exposure to soil. Nonextractable residues made up 32-68% of total body burden depending on exposure media and depuration time. Parent TNT accounted for less than 3% of radioactivity, while ADNTs accounted for 7-38%. Elimination half-lives were 61-120 h for TNT, ADNTs, and DANTs, which was significantly lower than the half-lives found for nonextractable residues, 201-240 h. However, over 80% of the nonextractable residue was solubilized using weak acid (pH 2). Based on our findings that TNT accumulation occurs primarily as nonextractable residues, which have a longer half-life, and that nonextractable residues can be solubilized, we propose that nonextractable residues could be used as a selective biomarker for assessing TNT contamination.     

Effects of phenanthrene on the mortality, growth, and anti-oxidant system of earthworms (Eisenia fetida) under laboratory conditions

To assess the toxic effects of phenanthrene on earthworms, we exposed Eisenia fetida to artificial soils supplemented with different concentrations (0.5, 2.5, 12.5, mgkg(-1) soil) of phenanthrene. The residual phenanthrene in the soil, the bioaccumulation of phenanthrene in earthworms, and the subsequent effects of phenanthrene on growth, anti-oxidant enzyme activities, and lipid peroxidation (LPO) were determined. The degradation rate of low concentrations of phenanthrene was faster than it was for higher concentrations, and the degradation half-life was 7.3d (0.5 mgkg(-1)). Bioaccumulation of phenanthrene in the earthworms decreased the phenanthrene concentration in soils, and phenanthrene content in the earthworms significantly increased with increasing initial soil concentrations. Phenanthrene had a significant effect on E. fetida growth, and the 14-d LC(50) was calculated as 40.67 mgkg(-1). Statistical analysis of the growth inhibition rate showed that the concentration and duration of exposure had significant effects on growth inhibition (p<0.001). Superoxide dismutase (SOD) activity increased at the beginning (2 and 7d) and decreased in the end (14 and 28 d). Catalase (CAT) activity in all treatments was inhibited from 1 to 14 d of exposure. However, no significant perturbations in malondialdehyde (MDA) content were noted between control and phenanthrene-treated earthworms except after 2d of exposure. These results revealed that bioaccumulation of phenanthrene in E. fetida caused concentration-dependent, sub-lethal toxicity. Growth and superoxide dismutase activity can be regarded as sensitive parameters for evaluating the toxicity of phenanthrene to earthworms.     

Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): Assessing the environmental impact of point-source metal contamination in terrestrial ecosystems

The earthworm Eisenia fetida (Annelida: Oligochaeta) was exposed to a geometric series of concentrations of cadmium, copper, lead and zinc in artificial soil using the OECD recommended protocol. Mortality, growth and cocoon production were measured over 56 days to determine LC50 and EC50 values. No observed effect concentrations (NOECs) were also estimated. Furthermore, the percentage of viable cocoons and number of juveniles emerging per cocoon was recorded. Cocoon production was more sensitive than mortality for all the metals, particularly cadmium and copper for which NOEC reproduction values were an order of magnitude lower than those for NOEC mortality. However, there was no significant effect of metals on the viability of cocoons. The weights of earthworms declined in all treatments (including the controls) during the experiment. This was probably due to the lack of suitable food in the OECD standard soil medium used. It was concluded that future experiments should include animal manure in the test medium. The LC50, EC50 and NOEC values determined in this study were compared with concentrations of metals in soils in the vicinity of a smelting works at Avonmouth, southwest England. The 14-day LC50 for zinc in Eisenia fetida was exceeded in soils covering an area of 75 km2 around the works, compared to 4.2 km2 for copper and 4.7 km2 for lead. Soil values for cadmium did not exceed the LC50 value anywhere in the region. Similar estimates of relative effects on reproduction confirmed that zinc is most likely to be responsible for the absence of earthworms from sites close to the Avonmouth works. However, the OECD standard test overestimated the potential effects of metals on populations, since earthworms can be found as close as 1 km from the smelting works. The discrepancy between test and field observations was probably due to the greater availability of the metals in the artificial soil.     

Effect of earthworms (Eisenia fetida) on the fractionation and bioavailability of rare earth elements in nine Chinese soils

The effect of earthworm (Eisenia fetida) activity on soil pH, dissolved organic carbon (DOC), fraction distribution pattern and bioavailability of rare earth elements (REEs) Y, La, Ce, Pr and Nd in nine Chinese soils were investigated using pot experiments. A three-step extraction procedure recommended by the European Community (Standards, Measurements and Testing Programme) was used to fractionate REEs in soils into water soluble, exchangeable and carbonate bound (B1), Fe- and Mn-oxides bound (B2) and organic matter and sulfide bound (B3). Inoculated with earthworms, the soil pH, DOC and water-soluble rare earth elements fraction increased. A significant correlation was obtained between the increased DOC and the increased water-soluble REEs. REEs in fraction B1 increased after earthworm inoculation, while those in fraction B3 decreased. No significant differences were observed for REEs in fraction B2. The biomass and the concentrations of REEs in wheat shoots and roots increased after the treatment with earthworms. The results demonstrated that earthworm activity increased the mobility and bioavailability of REEs in soils.     

Determination of protective effect of carob (Ceratonia siliqua L.) extract against cobalt(II) nitrate-induced toxicity

Environmental Science and Pollution Research - Cobalt (Co) is widely used in many industrial fields such as batteries and paints. Cobalt, a dangerous heavy metal, can be found in high...     

Cutleaf coneflower (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU)

Cutleaf coneflower (Rudbeckia laciniata L.) seedlings were placed into open-top chambers in May, 2004 and fumigated for 12 wks. Nine chambers were fumigated with either carbon-filtered air (CF), non-filtered air (NF) or twice-ambient (2×) ozone (O₃). Ethylenediurea (EDU) was applied as a foliar spray weekly at 0 (control), 200, 400 or 600 ppm. Foliar injury occurred at ambient (30%) and elevated O₃ (100%). Elevated O₃ resulted in significant decreases in biomass and nutritive quality. Ethylenediurea reduced percent of leaves injured, but decreased root and total biomass. Foliar concentrations of cell-wall constituents were not affected by EDU alone; however, EDU × O₃ interactions were observed for total cell-wall constituents and lignocellulose fraction. Our results demonstrated that O₃ altered the physiology and productivity of cutleaf coneflower, and although reducing visible injury EDU may be phytotoxic at higher concentrations.     

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.     

Impact of the earthworm Lumbricus terrestris (L.) on As, Cu, Pb and Zn mobility and speciation in contaminated soils

To assess the risks that contaminated soils pose to the environment properly a greater understanding of how soil biota influence the mobility of metal(loid)s in soils is required. Lumbricus terrestris L. were incubated in three soils contaminated with As, Cu, Pb and Zn. The concentration and speciation of metal(loid)s in pore waters and the mobility and partitioning in casts were compared with earthworm-free soil. Generally the concentrations of water extractable metal(loid)s in earthworm casts were greater than in earthworm-free soil. The impact of the earthworms on concentration and speciation in pore waters was soil and metal specific and could be explained either by earthworm induced changes in soil pH or soluble organic carbon. The mobilisation of metal(loid)s in the environment by earthworm activity may allow for leaching or uptake into biota.     

Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.)

A series of experiments were conducted in a hydroponic system to investigate the uptake of oxytetracycline (OTC) and its toxicity to alfalfa (Medicago sativa L.). OTC inhibited alfalfa shoot and root growth by up to 61% and 85%, respectively. The kinetics of OTC uptake could be well described by Michaelis-Menten equation with Vmax of 2.25 micromol g-1 fresh weight h-1, and Km of 0.036 mM. The uptake of OTC by alfalfa was strongly inhibited by the metabolic inhibitor, 2,4-DNP (2,4-dinitrophenol), at pH 3.5 and 6.0, but not by the aquaporin competitors, glycerol and Ag+. OTC uptake, however, was significantly inhibited by Hg2+, suggesting that the inhibition of influx was due to general cellular stress rather than the specific action of Hg2+ on aquaporins. Results from the present study suggested that OTC uptake into alfalfa is an energy-dependent process.     

Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation

Coontail (Ceratophyllum demersum L.) plants when exposed to various concentrations of Pb (1-100microM) for 1-7days, exhibited both phytotoxic and tolerance responses. The specific responses were function of concentration and duration. Plants accumulated 1748mugPbg(-1) dw after 7d which reflected its metal accumulation ability, however most of the metal (1222microgg(-1) dw, 70%) was accumulated after 1d exposure only. The toxic effect and oxidative stress caused by Pb were evident by the reduction in biomass and photosynthetic pigments and increase in malondialddehyde (MDA) content and electrical conductivity with increase in metal concentration and exposure duration. Morphological symptoms of senescence phenomena such as chlorosis and fragmentation of leaves were observed after 7d. The metal tolerance and detoxification strategy adopted by the plant was investigated with reference to antioxidant system and synthesis of phytochelatins. Protein and antioxidant enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) showed induction at lower concentration and duration followed by decline. All enzymes except GPX showed maximum activity after 1d. An increase in cysteine, non-protein thiols (NP-SH) and glutathione (GSH) content was observed at moderate exposure conditions followed by decline. Phytochelatins (PC(2) and PC(3)) were synthesized to significant levels at 10 and 50microM Pb with concomitant decrease in GSH levels. Thus production of PCs seems important for the detoxification of metal, however it may lead to depletion of GSH and consequently oxidative stress. Results suggest that plants responded positively to moderate Pb concentrations and accumulated high amount of metal. Due to metal accumulation coupled with detoxification potential, the plant appears to have potential for its use as phytoremediator species in aquatic environments having moderate pollution of Pb.     

Effect of liming and seasonal variation on lead concentration of tea plant (Camellia sinensis (L.) O. Kuntze)

Tea is a widely consumed beverage. However, recent studies revealed that there were an increasing number of cases of tea products exceeding the former maximum permissible concentration (MPC) in China for Pb (2 mg kg(-1)). Tea Pb contamination is an issue affecting trade and consumer confidence. Root uptake of Pb could contribute significantly to Pb accumulation in tea leaves due to the strong acidity of many tea garden soils. We conducted pot and field experiments to evaluate the effect of liming on Pb uptake by tea plants on two highly acidic soils (pH3.6). Additions of CaCO(3) significantly increased soil pH by up to 1 unit and decreased soil extractable Pb by up to 32%. Liming resulted in a decrease in the proportion of Pb in the exchangeable and carbonate-bound fractions, with a concurrent increase in the fractions bound to Fe/Mn oxides and residues. Liming significantly decreased Pb concentrations of fine roots, stems and new shoots of tea plants in the pot experiment. In the field experiments, the effect of liming was not significant during the first year following CaCO(3) application, but became significant during the second and third years and Pb concentration in the new shoots was decreased by approximately 20-50%, indicating that liming of acidic tea garden soils is an effective way to reduce Pb contamination of tea. The study also reveals a distinct seasonal variation, with Pb concentration in the new shoots following the order of spring>autumn>summer.     

Protective role of a methanolic extract of spinach (Spinacia oleracea L.) against Pb toxicity in wheat (Triticum aestivum L.) seedlings: beneficial effects for a plant of a nutraceutical used with animals

Spinach extracts contain powerful natural antioxidants and have been used to improve the response of animal cells to various stress factors. The aim of the present study was to assess the effects of a methanolic extract of spinach (SE) used at two concentrations (21.7 and 217 ppm) on the growth, certain enzymes and antioxidant systems in wheat seedlings under lead stress. When wheat seedlings were grown for 7 days in a solution containing Pb(NO₃)₂ (3 mM), germination and growth were impaired, while signs of oxidative stress were observed. SE (217 ppm) pretreatment was able to protect seedlings from Pb toxicity by both reducing Pb uptake and Pb-induced oxidative stress. As a consequence, almost normal germination, elongation, biomass and α-amylase activity were restored by SE (217 ppm) pretreatment of wheat seedlings, in spite of the presence of Pb. Our results support the protective role and the antioxidant effect of SE against Pb. These results show an amazing similarity to the effects of SE in animals, which suggests that providing “nutraceuticals” to plants could improve their “health” status.     

Accumulation of heavy metals in Typha angustifolia (L.) and Potamogeton pectinatus (L.) living in Sultan Marsh (Kayseri, Turkey)

Concentrations of heavy metals (Cd, Pb, Cr, Ni, Zn and Cu) were measured in bottom sediments, water and Typha angustifolia and Potamogeton pectinatus in Sultan Marsh. Sultan Marsh is one of the largest and most important wetlands in Turkey, Middle East and Europe, embodying saline and fresh water ecosystems and providing a shelter for 426 bird species. The organs of T. angustifolia have a larger quantity of the measured elements than the P. pectinatus. Considerably higher contents of Cd were found rather than in helophytes (P. pectinatus) in submerged plant (= emergent, T. angustifolia) species. The percentage of Cd in plant tissues points to a certain degree of water pollution in Sultan Mash. Analyses of water, bottom sediments and plant samples indicated that the Marsh were polluted with Pb, Cd, and partly with Cu and Zn. All sampling sites in the study area basin are generally more or less polluted when compared with the control values. Strong positive correlation was found between concentrations of Pb in water and in plants. Ni and Pb were accumulated by plants at a higher rate from bottom sediments than from water. Leaves of T. angustifolia accumulated less heavy metal than the corresponding roots. There was a significant relationship between Cd concentration in samples of plants and water pH value. It has been found that the tissues of T. angustifolia accumulate more heavy metals than the tissues of P. pectinatus. Therefore, all plants can be used as a biological indicator while determining environmental pressures; however, T. angustifolia is proved more appropriate for such studies.     

Inhibitory effect of ragweed (Ambrosia artemisiifolia L)-inflorescence extract on the germination of Amaranthus hypochondriacus L and growth of two soil algae

In this study the ecological importance of common ragweed (Ambrosia artemisiifolia L.; Asteraceae) was investigated in in vitro bioassays as it is one of the most widespread annual weeds occurring on arable land in Hungary. Eight fractions were derived from ragweed inflorescence water extract by low vacuum column chromatography and their biological activity was investigated by Amaranthus hypochondriacus L. cv. Edit (Amaranthaceae) germination bioassay. As a result of the germination bioassay the chromatographic method was modified to harvest the most effective fractions in only one fraction, which was used in three concentration (10, 50, 100 mg x kg(-1)) for algal growth inhibition bioassay.Results showed significant growth inhibition in case of both applied algal species.