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In a recent study, we showed that the earthworm species Eisenia fetida, inhabiting an extremely high metal polluted compost heap on a wine farm, did not have elevated body loads of the metals but exhibited genotoxic tolerance when exposed to Cd in the laboratory (Voua Otomo and Reinecke, 2010). To unravel the mechanism behind the surprisingly low metal body burdens on one hand and genotoxic tolerance on the other hand, we investigated the estimated bioavailability of these metals (Cu, Zn, Pb and Cd) using sequential extraction methods with CaCl(2) and di-ethylene-triamine-pentaacetic acid (DTPA) and allozyme polymorphism in this field population, a laboratory control as well as a long-term Cd exposed population. The amounts of mobile (extracted with CaCl(2)) and mobilizable (extracted with DTPA) metals in relation to the total (extracted with nitric acid) metals were all below 0.05% for all four metals, suggesting low availability for uptake. The low availability of these metals could not be explained by physico-chemical properties of soil but by the phenomenon of aging of the metals. There was no difference in allozyme frequency between metal tolerant and non-metal tolerant populations of E. fetida. This suggested that the tolerance found in earlier studies could be a mere physiological adaptation., (Copyright © 2011. Published by Elsevier Inc.)

The concept of species sensitivity differences is important in ecotoxicology and environmental risk assessment, but testing usually focuses on lethality of toxicants. The effects on the suborganismal level are mostly ignored; therefore, the present study assessed a biomarker of genotoxicity (the alkaline comet assay) to compare species sensitivities. Five earthworm species (Amynthas diffringens, Aporrectodea caliginosa, Dendrodrilus rubidus, Eisenia fetida and Microchaetus benhami) were exposed for 48 h to sublethal concentrations of cadmium sulphate in reconstituted soil water and DNA integrity was evaluated with the parameter Tail DNA %. Significant amounts of DNA damage were detected in three (A. caliginosa, D. rubidus and E. fetida) species. E. fetida exhibited the highest level of DNA damage, although D. rubidus showed the highest increase (3-fold) in DNA damage from the control. All exposed earthworms accumulated Cd, although body loads did not correspond with DNA damage levels; most of the Cd was probably sequestrated and rendered harmless.

Earthworm population density was measured in and adjacent to an orchard in an agricultural area in the Western Cape, South Africa. Worm densities were very low in orchards (22/m(2)) compared to adjacent uncultivated fields (152/m(2)) at a distance from the orchards. The possible effect of organophosphate pesticides on the earthworms was investigated. Background soil concentrations of chlorpyrifos prior to the start of the spraying season were low (0.2-2.7 microg/kg) but persistent for up to 6 months after the last spraying event, and the pesticide was, as a result of rainfall, transported to nontarget areas by runoff. Background concentrations of azinphos methyl were higher than those of chlorpyrifos (1.6-9.8 microg/kg) but not detectable 2 weeks after a spraying event. Azinphos methyl was mostly transported by wind (spray drift) to adjacent areas. A microcosm study indicated effects of chlorpyrifos on earthworms as determined by measuring biomass change and Cholinesterase inhibition. It is concluded that earthworms were affected detrimentally by the pesticides due to chronic (chlorpyrifos) and intermittent (azinphos methyl) exposure.

Background levels of chlorpyrifos and earthworm abundance were determined in an orchard and adjacent areas on a farm in the Western Cape, South Africa before these areas were again sprayed with this organophosphate. The background concentrations ranged from 0.2 microg/kg dm in the spray drift area adjacent to the orchard to 10.18 microg/kg dm on the slope in the run off area. In the target area the chlorpyrifos concentrations varied from a mean of 15.25 +/- 10.0 microg/kg directly after spraying to a mean of 7.0 +/- 0.9 microg/kg 6 months later and in the nontarget area they varied from a mean of 55.0 +/- 35 microg/kg to 12.0 +/- 5 microg/kg after 6 months. Chlorpyrifos was therefore still present in the field soils, but at lower concentrations, up to 6 months after the last spraying event. Earthworm abundance and population densities were very low. Only Aporrectodea caliginosa was found and the densities were much lower in the orchards (22 per m(2)) than in the nontarget areas (98.3 per m(2)). Microcosm studies were undertaken to relate biomarker responses to chlorpyrifos with biomass changes. Microcosms were filled with soil from the same areas and earthworms of the species A. caliginosa were introduced. The microcosms were treated with a series of concentrations of chlorpyrifos in the laboratory under controlled conditions. These concentrations were chosen to fall within the background ranges found in the soils. The biomass of the worms was determined regularly for a period of 5 weeks and worms in a state of estivation were noted. Earthworms were removed from the microcosms for biomarker tests: for cholinesterase (ChE) inhibition assays every week and for a neutral red retention determination 2 weeks after the exposures started. The most prominent biomass loss was noted in earthworms exposed to the highest pesticide concentration of 8.0 microg/kg. Estivation was higher among earthworms exposed to higher exposure concentrations. Inhibition of ChE increased with higher exposure concentrations and with time but there was no clear dose-related response. A clear dose-related response with exposure concentration was established for the neutral red retention assay. A correlation between ChE inhibition and biomass change existed directly after the second application of chlorpyrifos.

This study investigated the relationship between accumulated copper in the digestive gland of Helix aspersa, after exposure to the fungicide copper oxychloride, and quantitative changes in the digestive gland cells of this snail. Snails were exposed to 80 and 240 microg g(-1) copper oxychloride for 6 weeks. Histological sections of digestive glands were analyzed by means of computer-assisted image analysis. Results indicate that copper is strongly accumulated in the digestive gland and that this organ is the main site of copper accumulation in the snail body. Also, as a result of copper accumulation in the digestive gland, measurable changes in epithelium cell height and area of this organ occur. In the case of cell height, these changes are dose-related. It is concluded that changes in digestive gland cells, as a result of copper oxychloride exposure, may possibly serve as biomarkers of exposure to this fungicide.

The LC50 of copper oxychloride for Eisenia fetida was determined, and its effects on biomass change and lysosomal damage using neutral red retention times (NRRT) of coelomocytes were measured. The aim was to establish whether a lysosomal subcellular response, measured as NRRT, could be linked to the LC50 and biomass changes. Further, we attempted to establish the ecological relevance of the LC50 by comparing it to studies previously carried out on the effects of copper oxychloride on field earthworm populations. The experiment was conducted over a period of 28 days, during which the earthworms were exposed to different concentrations of copper oxychloride in artificial soil. The calculated LC50 was 883 microg g(-1) for copper oxychloride and 519 microg g(-1) for copper. Results indicated that changes in coelomocyte membrane stability manifested earlier than effects on biomass. Since the NRRT assay was very sensitive and generated an early response before changes in biomass or mortality could be measured, it may have predictive value and may contribute information during acute toxicity tests, which could be of greater ecological relevance than mortality data alone.

The effects of the fungicide copper oxychloride on a field population of the earthworm Aporrectodea caliginosa were investigated. Changes were monitored in earthworm numbers and biomass, copper concentrations in soil and earthworms, and neutral red retention times (NRRTs) of coelomocytes. The aim was to establish whether the cellular responses could be linked to changes at the population level. Results indicated that there was a significant (P<0.05) impact on worm density and biomass on the treated plots 6 months after spraying with the fungicide stopped. NRRTs of treated earthworms were significantly lower after one spraying when earthworms had elevated concentrations of copper in their body tissues. Six months after spraying stopped, copper levels in both soil and earthworm tissues declined significantly, but the earthworm numbers and biomass were still significantly lower in the treated plots. This indicated that the effects of the copper oxychloride exposure manifested at a much later stage at the population level, than on NRRT. We conclude that the decrease in NRRTs, which preceded the decrease in both earthworm biomass and numbers, may have predictive value as a biomarker of impending population changes.

In this study groups of the epigeic earthworm species Eisenia fetida were exposed experimentally for different periods to sublethal concentrations of Pb(NO3)2. The lysosomal membrane fragility response of coelomocytes was used as a biomarker to explore the cytotoxity of Pb by determining the neutral red retention times (NRRTs) and correlating them with different body loads of Pb. Previously unexposed worms exposed to different low concentrations of Pb(NO3)2 for 4 weeks demonstrated a clear dose-response relationship between NRRTs and body burdens of Pb. The NRRTs of growing worms with a previous (ancestral) exposure history, exposed to an elevated concentration of Pb and measured over time, fluctuated around a mean of 41 min and no dose-related relationship could be demonstrated. Worms with a long-term history of exposure to Pb of more than 36 months had a mean body burden of 177.5 +/- 61.5 mg/kg Pb and a mean NRRT of 57.8 +/- 3.1 min, which was significantly lower than that of uncontaminated, control worms with a mean time of 61.9 +/- 3.4 min and a mean body burden of 86.3 +/- 47.2 mg/kg. No significant differences were found between weight changes of "clean" and preexposed worms when they were experimentally exposed to sublethal concentrations of Pb. This study provided evidence that the lysosomal biomarker response for Pb may not persist at the same level in worms with a previous history of Pb exposure, possibly making it less sensitive as a tool for comparing responses to Pb in field populations of earthworms.

The ecological relevance of the neutral red retention assay as a biomarker in an indigenous earthworm population (Microchaetus sp.) exposed to the fungicide copper oxychloride was investigated. Changes in earthworm biomass and numbers were monitored and related to changes in neutral red retention times of coelomocytes as well as changes in copper concentrations in the soil and earthworm body tissues. Results indicated that Microchaetus sp. responded sensitively to the copper oxychloride by showing an initial decrease in biomass 2 months after spraying started. This was followed by a significant decrease in worm numbers after 3 months. The neutral red retention times of earthworm coelomocytes decreased significantly within the first month of treatment and correspond to a significant increase in soil copper concentrations, but not with an immediate increase in body burdens of copper. More than a year after spraying had stopped the worm biomass and numbers were still significantly lower in the treated plots compared with the control plots. Since the reduction in neutral red retention times could be attributed to the presence of copper oxychloride and preceded the decline in population density and biomass, we conclude that this biomarker has a useful role to play in environmental risk assessment and could provide a warning of impending ecological damage.

This article describes the outcome of laboratory experiments to determine the acute and sublethal effects of the fungicide mancozeb on the earthworm Eisenia fetida. Apart from a standardized acute test, young worms, 25 days old, were exposed to mancozeb mixed into a urine-free cattle manure substrate. Five groups of 10 worms each were used per concentration level (0, 8, 44 mg/kg substrate). The following life-history parameters were measured: growth in consecutive weeks over a 6-week period, survival rate, maturation time, cocoon production, hatching success of cocoons, number of hatchlings per cocoon, and incubation time of cocoons over another 4 weeks. The results indicated that mancozeb had no significant detrimental effect on either growth or reproduction of E. fetida at the recommended dose (8 mg/kg) or at an estimated environmental concentration (44 mg/kg). The findings do not support the hypothesis that avoidance response to mancozeb could serve as an indication of toxicity., (Copyright 2001 Academic Press.)

The article describes a laboratory experiment to determine the effect of copper oxychloride on the earthworm Eisenia fetida. Copper oxychloride was used because it is the most commonly used fungicide in South African vineyards but not much is known about its toxicity to earthworms. In an experiment lasting 8 weeks, newly hatched earthworms of the species E. fetida were exposed to copper oxychloride mixed into a urine-free cattle manure substrate. Four groups of 10 worms were used per concentration level (control (4.02), 8.92, 15.92, 39.47, 108.72, 346.85 mg Cu kg substrate(-1)). The following life-history parameters were measured: earthworm growth in consecutive weeks, survival rate, maturation time, cocoon production, reproduction success, total number of hatchlings produced, and incubation time. Earthworm growth and cocoon production were significantly reduced at copper oxychloride exposure concentrations of 8.92 mg kg(-1) and higher. Reproduction success in the 8.92 mg Cu kg substrate(-1) was highest. From an exposure concentration of 15. 92 mg Cu kg substrate(-1) and higher, there was a considerable impact of copper oxychloride on reproduction. This could be seen from a reduced reproduction success, a reduced mean and maximum number of hatchlings per cocoon, and a longer incubation time, indicating a strong effect of low copper oxychloride concentrations on this earthworm species., (Copyright 2000 Academic Press.)

The effects of sublethal concentrations of lead nitrate on growth and reproduction of the Asian composting earthworm species Perionyx excavatus was studied experimentally by exposing worms in an organic substrate to lead nitrate-contaminated food over a period of 76 days. The results revealed that growth was affected negatively by the presence of lead while maturation rate and cocoon production was not affected. In agreement with other studies on Eisenia fetida, cocoon viability was affected negatively by lead, making this a sensitive toxicity endpoint. Lead accumulation was of the same order of magnitude as for other previously studied species., (Copyright 1999 Academic Press.)

The morphology of spermatozoa may be affected by very low concentrations of pollutants in the environment, and sublethal effects of toxic substances can be detected by studying the sperm of an animal. Sperm were collected from 50 impala (Aepyceros melampus) from a conservation area in South Africa and studied ultramicroscopically. Thirty-two samples were obtained from animals close to a copper mine, while 18 represented animals from an unpolluted area and held in captivity. Liver samples from all these animals were analyzed for their copper content. The findings from 12 animals kept in captivity and fed on a diet to which copper was added correlated with the findings from the field study. The general condition of the animals as expressed by the fat content of the bone marrow from the area exposed to copper contamination was affected detrimentally in comparison to the animals from the uncontaminated area. High percentages of sperm with neck vacuoles were found in all animals with high liver copper levels. A statistically significant correlation is demonstrated between the percentage of impala sperm with vacuoles in the neck region and the copper concentrations in this group of animals., (Copyright 1999 Academic Press.)

The reliance on earthworms as test organisms in risk assessment studies of polluted environments raises the question whether they can evolve resistance, e.g., by adaptation to specific toxicants. Protection criteria may be biased if sensitivity data from adapted populations are used. Increased resistance to the heavy metal cadmium has not yet been determined for terrestrial Oligochaeta. Eisenia fetida was exposed to a sublethal concentration of cadmium sulfate for more than 10 generations. Clitellate worms from this culture were used in experiments to determine the extent of possible tolerance for the heavy metal. Preexposed animals as well as worms with no previous history of exposure to cadmium were exposed to a control substrate without cadmium and also to two substrates with 600 and 1200 microg g-1 cadmium. Changes in biomass, cocoon production, and hatching success were monitored. The results obtained indicated that in both substrates in which cadmium was present the preexposed worms performed better than the unexposed worms with respect to growth rate but not reproductively. In the substrate without cadmium the preexposed worms exhibited signs of poisoning after a few weeks. Preexposed and unexposed worms were also exposed to concentrations of 1500 to 4000 microg g-1 cadmium sulfate in an artisol medium for a period of 2 weeks. The preexposed worms survived higher concentrations of cadmium than the unexposed group and some specimens from the unexposed group had a gross increase in body fluids. It is concluded that worms with a long-term history of exposure to the metal developed resistance to cadmium., (Copyright 1999 Academic Press.)