Using the comet assay to assess the combined and separate genotoxic effects of Cd and Zn in Eisenia andrei (Oligochaeta) at different temperatures.

Using the comet assay, the genotoxicity of Cd, Zn and Cd/Zn mixtures in Eisenia andrei was assessed after 4 weeks of exposure at 15, 20 and 25 °C. Relative to the controls, significant increases in TDNA% were observed in exposures to Cd alone at 500 and 1,000 mg/kg soil at both 20 and 25 °C, while a general decrease occurred at 15 °C. For Zn alone, a decreasing trend in TDNA% occurred at all three temperatures with increasing Zn concentration. For the Cd/Zn mixtures at 15 °C, genotoxicity was reduced at all mixture concentrations relative to the control. At 20 °C, the genotoxic response was similar to the control at all exposures. At 25 °C, the response was elevated at the 50 + 50 and 250 + 250 mg/kg mixture concentrations. In the remaining treatments at 25 °C, TDNA% was similar to the values in the respective control. The lack of consistently significant mixture genotoxicity may indicate antagonistic interactions between Cd and Zn in the mixtures. However, this was not conclusively determined because temperature alone had an inconsistent effect upon TDNA% readings in the control exposures.

Using estimates of metal bioavailability in the soil and genetic variation of allozymes to investigate heavy metal tolerance in the earthworm Eisenia fetida (Oligochaeta).

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.

Quantitative changes in digestive gland cells and oocytes of Helix aspersa, as biomarkers of copper oxychloride exposure under field conditions.

This study investigated the effects of accumulated copper, on digestive epithelium height and percentage area, and on oocyte numbers of the snail Helix aspersa, in a vineyard where copper oxychloride is sprayed. The ultimate aim was to determine the usefulness of these cellular responses as biomarkers. Results showed that snails collected 2 months after fungicide application, had a significantly lower mean digestive epithelium height and percentage area, as well as significantly fewer oocytes per 1 mm(2) of ovotestis, compared to snails collected only 1 week after fungicide application and those from a control vineyard. It was concluded that these cellular responses are clear, measurable responses to copper oxychloride exposure and copper accumulation. However, they do not provide an early warning of copper exposure, which impacts on their usefulness as biomarkers.

Avoidance behaviour of two eco-physiologically different earthworms (Eisenia fetida and Aporrectodea caliginosa) in natural and artificial saline soils.

We studied the avoidance behaviour of Eisenia fetida and Aporrectodea caliginosa in OECD artificial soil spiked with NaCl and in natural saline soil (of varying ionic constitutions) collected from Robertson Experimental Farm (ROBS) in Western Cape, South Africa. For each organism, the ecotoxicological test was performed using a two-chamber test over a period of 48 h. The results showed that in the OECD soil, the avoidance EC50 (the concentration/electrical conductivity at which there is effect on 50% of the organisms) for A. caliginosa of 667 mg kg(-1) NaCl was lower than 1164 mg kg(-1) for E. fetida. Similarly in ROBS soil, the avoidance EC50 for A. caliginosa of 0.26 dS m(-1) was lower than 0.56 dS m(-1) in E. fetida. These results indicated that A. caliginosa showed better avoidance to salinity than E. fetida irrespective of soil types or ionic constitution. When compared with literature data, EC50 values in avoidance tests were either lower or comparable to those of reproduction, which was the most sensitive life-cycle parameter. The only exception was the EC50 value for avoidance of E. fetida in natural soil which was higher than for reproduction suggesting that the predictive value of the avoidance test for this species might be lower in natural soils. The variation in sensitivities of these earthworms could be as a result of differences in their eco-physiology. These findings suggest the relevance of the avoidance test as a suitable screening method showing first tendencies of saline stress on the habitat function of soils.

Role of clay content in partitioning, uptake and toxicity of zinc in the earthworm Eisenia fetida.

We studied the effect of clay content on the bioavailability of zinc to pre-clitellate earthworm, Eisenia fetida in the laboratory using OECD artificial soil adjusted to 5%, 20%, and 40% clay. Batches of worms were exposed to a wide range of zinc concentrations. Mortality, growth, maturation (% clitellate), cocoon production, and body zinc concentrations were assessed over and after a period of 4 weeks. Total, DTPA, and CaCl(2) extractable zinc in the substrates were also determined. The results of the biological responses showed that interaction of clay and zinc had a significant influence on mortality but not on the other biological parameters. None of the three extraction methods showed consistent and significant effect of clay content on zinc partitioning. Although total, DTPA, and CaCl(2) extracts of zinc correlated strongly with one another and were in similar relation with the observed biological responses, only the CaCl(2) extract revealed a time dependent availability of this metal. It is concluded that clay content had no significant influence on sub-lethal toxicity of zinc to this earthworm over the range of exposure concentrations.

Quantitative assessment of effects of zinc on the histological structure of the hepatopancreas of terrestrial isopods.

The objective of this study was to quantify the effects of zinc exposure on the histological structure of the hepatopancreas of Porcellio laevis. Woodlice were experimentally exposed to various concentrations (1000, 4000, and 8000 mg. kg(-1)) of zinc sulphate. Hepatopancreas samples of exposed isopods were histologically prepared and analysed with Leica QWin image analysis software. The B-cells in hepatopancreases of zinc sulphate - exposed woodlice were reduced in size to varying degrees, compared to that of the control, Percentage Cellular Area (PCA) of the hepatopancreas samples revealed that zoning occurred through the length of hepatopancreas lobes. Analysis of the PCA data of hepatopancreases of P. laevis exposed to the zinc sulphate revealed that there was a decline in PCA, in all the zones, compared to those in the control. The Z3 zone (the part from the middle to three quarters to the back of the tubule) was shown to be the best suited, if PCAs are to be considered as a biomarker in woodlouse toxicity studies.

Growth and reproduction of earthworms in ultramafic soils.

Ultramafic soils are characterized by high concentrations of heavy metals of natural origin-such as chromium, cobalt, manganese, and nickel-as well as a shortage of primary nutrients. This can result in extremely disadvantageous living conditions for all soil-dwelling organisms. Responses to these conditions were addressed by studying growth, cocoon production, and fecundity of earthworms as endpoints of sublethal effects and how this influences the reproductive system and, consequently, population development. Mature specimens of two ecophysiologically different species of earthworms, Eisenia fetida (Savigny) and Aporrectodea caliginosa (Savigny), were exposed for 56 days to an uncontaminated soil and ultramafic soils collected from six ultramafic sites in the Barberton greenstone belt. In all ultramafic soil samples, the specimens of both species grew slower than those in the control soil. In A. caliginosa, an autotomization of the tail section was observed at higher concentrations of heavy metals. At high levels of heavy metals such as manganese, chromium, nickel, and cobalt, a significantly lower cocoon production was recorded for E. fetida, and at medium levels, a time delay in cocoon production was found. A. caliginosa showed an increase in production at medium levels and a decrease at high levels of heavy metals. In both species, no correlation between growth and cocoon viability was found, indicating different target levels for toxicants present in ultramafic soils. To determine effects of these soils on population dynamics, hatching success may be a more useful endpoint of reproduction.

The impact of organophosphate pesticides in orchards on earthworms in the Western Cape, South Africa.

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.

Biomarker response and biomass change of earthworms exposed to chlorpyrifos in microcosms.

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.

The determination of earthworm species sensitivity differences to cadmium genotoxicity using the comet assay.

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.

The implications of copper fungicide usage in vineyards for earthworm activity and resulting sustainable soil quality.

To investigate the impact of copper-containing fungicides (copper oxychloride) on earthworms in South African vineyards, field inventories of earthworms in and between vine rows were carried out and compared to directly adjacent grassland. Also copper content, pH, organic matter content, and soil porosity were determined in these soils. This was combined with laboratory experiments to study the impact of vineyard soil characteristics on the burrowing and dispersal behavior of earthworms. Moreover, the direct toxic action of copper oxychloride on different endpoints of the earthworms (survival and growth) was studied. Copper oxychloride had a negative impact on these endpoints (decreased growth and survival related to increased copper body content) as well as on the behavioral aspect (decreased burrowing rate and avoidance of copper-containing soil). Moreover, there was an inverse relation between burrowing activity and soil bulk density that could also be related to the copper content. This may lead to a decrease in sustainable soil quality in vineyards.

Quantitative changes in the digestive gland cells of the snail Helix aspersa after exposure to the fungicide copper oxychloride.

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 relationship between lysosomal biomarker and organismal responses in an acute toxicity test with Eisenia Fetida (Oligochaeta) exposed to the fungicide copper oxychloride.

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.

Effect of metal mixtures (Cd and Zn) on body weight in terrestrial isopods.

The aim of this study was to determine the effect of exposure to cadmium and zinc mixtures on the weight of terrestrial isopods. Experiments were conducted using uncontaminated specimens of P. laevis. The isopods were exposed to various concentrations of cadmium and zinc sulfate in single- and mixed-metal experiments. The mean weight of the unexposed isopods in the control group increased over the 6 weeks of observation. It was concluded that cadmium and zinc, administered separately, had a negative effect on the weight of P. laevis. The weight change (gain or loss) of P. laevis in the exposures to mixtures of cadmium and zinc sulfate were different from those of woodlice where Cd and Zn were administered separately. Combination of the lowest concentrations of Cd and Zn (20 and 1000 mg x kg(-1)) produced a stimulating effect compared to the control. Weight changes of the other two combinations of Cd and Zn (80/4000 and 160/8000 mg x kg(-1)) showed no differences from those of the control, and weight changes of woodlice exposed to Cd and Zn mixtures were mostly the same as those of the control over the 6-week exposure period. It can thus be concluded that mixtures of Cd and Zn have an antagonistic effect on each other in terms of weight of P. laevis.

The comet assay as biomarker of heavy metal genotoxicity in earthworms.

The ubiquitous occurring earthworm species, Eisenia fetida, were exposed to nickel chloride to determine whether the heavy metal Ni caused DNA damage, as measured by the comet (single cell gel electrophoresis) assay. Primary cell cultures of earthworm coelomocytes were exposed in vitro and whole animals either in spiked artificial soil water or in spiked cattle manure substrates. Comets formed were scored using mean tail lengths as well as comparing percentages of damage in five different damage classes. The exposure concentrations used for the in vitro exposure (2, 6, and 12 microg/ml) caused the formation of comets of which the mean tail lengths differed significantly (p < 0.05) from those of unexposed controls but not from each other. Coelomocytes from worms exposed in artificial soil water at concentrations of 0.0049, 0.0078, 0.0175, and 0.025 mg/ml formed comets of which the mean tail lengths differed significantly (p < 0.05) between the exposure groups with increasingly longer tail lengths with higher concentration (dose-related response). The tail length means of the comets of the three highest exposure concentrations also differed significantly (p < 0.05) from the controls. No dose-related response was found between comet tail lengths of the three exposure concentrations (60, 240, and 480 mg/kg) used for the worms in the cattle manure substrates, but the mean tail lengths of comets from all three exposure groups differed significantly (p > 0.05) from the controls. The comets formed in cells from animals exposed in artificial soil water and in cattle manure substrates, scored within damage classes, indicated a clear shift with increasing exposure concentrations from low to high damage. Our results indicated DNA single-strand breaks in soil invertebrate cells caused by exposure to a nickel compound, verifying previous findings for mammals which indicated that this heavy metal has genotoxic potential. These results therefore suggest that earthworms may be useful indicator organisms to assess the genotoxic risks of nickel pollution to terrestrial environments and that the comet assay is a useful tool to use as biomarker of genotoxic effects on invertebrates in soil.

Linking lysosomal biomarker and population responses in a field population of Aporrectodea caliginosa (Oligochaeta) exposed to the fungicide copper oxychloride.

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.

Quantifying histopathological alterations in the hepatopancreas of the woodlouse Porcellio laevis (Isopoda) as a biomarker of cadmium exposure.

The aims of this study were to determine and quantify the effects of cadmium sulfate on the histological structure of the hepatopancreas of Porcellio laevis, to determine the extent of the changes due to cadmium exposure, and to assess whether these effects could serve as biomarkers of cadmium exposure. For this purpose, different batches of P. laevis were exposed for 6 weeks to shredded leaves contaminated with a range of different concentrations of cadmium sulfate. Histological sections of different parts of the hepatopancreases revealed that the exposure changed the structure of the hepatopancreas. Quantification of this change in hepatopancreas structure (expressed as the percentage cellular area or PCA) showed that there was a dose-related change in certain areas of the hepatopancreas of woodlice exposed to cadmium sulfate in comparison to those areas of unexposed controls. This change was evident despite the fact that PCA also varied over the length of the hepatopancreas in the control specimens. This study showed that PCA can be a useful measure of change in the hepatopancreas resulting from toxic stress. Although it may not be practical for regular monitoring, it could serve as a useful research tool and general biomarker in studies of toxic stress in woodlice.

The influence of exposure history to lead on the lysosomal response in Eisenia fetida (Oligochaeta).

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.