S.A. Reinecke

Effects of metal pollution on earthworm communities in a contaminated floodplain area: Linking biomarker, community and functional responses

Effects on earthworms in the contaminated floodplain area the Biesbosch, the Netherlands, were determined at different levels of organization using a combination of field and laboratory tests. The species Lumbricus rubellus, collected from different polluted sites in the Biesbosch, showed reduced values for the biomarker neutral red retention time (NRRT), mainly explained by high metal concentrations in the soil and the resulting high internal copper concentrations in the earthworms. Organic pollutant levels in earthworms were low and did not explain reduced NRRTs. Earthworm abundance and biomass were not correlated with pollutant levels in the soil. Litterbag decomposition and bait-lamina feeding activity, measures of the functional role of earthworms, were not affected by metal pollution and did not show any correlation with metal concentrations in soil or earthworms nor with NRRT. Effects at the biochemical level therefore did not result in a reduced functioning of earthworm communities.

Cocoon production and viability as endpoints in toxicity testing of heavy metals with three earthworm species

Summary This study compares the cocoon production and cocoon viability of three earthworm species, Eudrilus eugeniae, Perionyx excavatus and Eisenia fetida after exposure to a sublethal concentration of lead nitrate. Cocoon production in all three species did not differ significantly between exposed and control worms. However, cocoon viability in all three species was affected detrimentally by the presence of lead nitrate. Cocoons of Eisenia fetida from substrates contaminated by lead nitrate, cadmium sulphate and zinc sulphate for 36 months, also showed a lowered cocoon viability. It is concluded that cocoon viability may be a sensitive endpoint of sublethal toxicity of these metal salts.

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.

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.

Recovery of cholinesterase activity in the earthworm Eisenia fetida Savigny following exposure to chlorpyrifos

Organophosphorus (OP) insecticides inhibit cholinesterase activity, an essential process in the nervous system of most animals. Re-establishment of active enzymes is slow and depends on elimination of the insecticide from the body followed by two lengthy processes: Reactivation and/or biosynthesis of new enzymes. Earthworms (Eisenia fetida) were exposed to either clean or chlorpyrifos-containing (240 mg/kg) soil for 48 h. After transfer to clean soil, we monitored two cholinesterases (E1 and E2) and chlorpyrifos content of the earthworms for 12 weeks. After 14 to 21 d of recovery, the exposed and control worms were indistinguishable in terms of appearance and behavior. Chemical analysis showed a rapid elimination of chlorpyrifos from the earthworms, with only minor levels detected after one week. The activities of E1 and E2 were measured spectrophotometrically in whole specimen homogenates using acetylthiocholine as the substrate. Carbaryl, which selectively inhibits E1, was used to discriminate the enzyme activities. Mean +/- standard error of mean of E1 and E2 activity in the controls immediately after exposure were 1.57 +/- 0.18 nanokatal (nkat)/mg protein (n = 3) and 0.95 +/- 0.07 nkat/mg protein, respectively, and 0.48 +/- 0.07 nkat/mg and 0.45 +/- 0.06 nkat/mg, respectively, in exposed worms. After three weeks, E1 had regained an activity comparable to the controls, whereas E2 remained depressed throughout the 12-week monitoring period. The non- or late recovery of E2 makes this enzyme a potential biomarker candidate for previous OP insecticide exposure in Eisenia fetida, provided the protocol for measurements is improved and standardized.

The impact of different herbicides and cover crops on soil biological activity in vineyards in the Western Cape, South Africa

Summary The bait-lamina method was used to study the effects of different herbicides on biological activity in vineyard soil with and without cover crops in the Western Cape, South Africa. An evaluation of two different bait substances was also made under two different cover crops, an unplanted control and an unplanted, mechanically tilled plot. The highest feeding activity was measured under the cover crops and the lowest in the mechanically tilled soil. After spraying with the herbicide glyphosate on four different cover crops, the feeding activity was determined on treated and untreated plots on three occasions. The results indicated that the feeding activity generally increased following treatment with glyphosate. A comparison of feeding activity after treatment with paraquat, simazine, glyphosate and a glyphosate-terbutylazine mixture on different soil plots without cover crops was also undertaken. It showed that, with the exception of simazine, the herbicide application had a positive influence on feeding activity. It is concluded that the bait-lamina technique, although not representative of all the many complex soil biological processes, can still provide a simple and practical tool to complement studies on the effects of chemicals on functional aspects of soil ecosystems.

Biomarker responses and morphological effects in juvenile tilapia Oreochromis mossambicus following sequential exposure to the organophosphate azinphos-methyl.

Pesticides are contaminants of aquatic environments. Such ecosystems in the Western Cape, South Africa are at risk as most organophosphates are highly toxic to fish and other aquatic organisms. The objective of this experimental study was firstly to determine the acute toxicity of azinphos-methyl (AZP) to juvenile fish (Oreochromis mossambicus) and, secondly, to investigate the effects of repeated exposure of fish to an array of sublethal concentrations on morphological parameters such as growth, condition factor and organ-somatic indices. Food consumption and feeding response time were investigated as ecologically relevant behavioral endpoints which could affect growth, reproduction and survival and subsequently causes impacts at the population and/or community level. Finally, acetylcholinesterase (AChE) was used as biomarker to investigate effects at sub-organismal level following sequential exposure to AZP. The aim was to determine how sequential spraying procedures, using different exposure concentrations and intervals, affected fish as reflected by their responses at different organizational levels. A dose-dependent effect on feeding impairment was observed in the feeding response experiment. The correlation found between growth impairment, feeding activity and AChE inhibition therefore indicates that frequency of exposure can play an important role regarding the severity of impacts to non-target organisms. This study provides evidence that AZP has harmful effects on non-target aquatic organisms, such as fish which can be manifested in the early developmental stages. Sequential exposures showed that dosage and frequency of spraying and spraying interval could exacerbate harmful effects. AChE inhibition and organosomatic indices can be used effectively to measure effects.

Increased cytotoxic and genotoxic tolerance of Eisenia fetida (Oligochaeta) to cadmium after long-term exposure

Since life-cycle studies showed that the earthworm species Eisenia fetida can develop increased tolerance after long-term exposure to a sub-lethal concentration of Cd in the laboratory, we assessed both the cytotoxicity and genotoxicity of Cd in a long-term Cd-exposed population. We exposed E. fetida specimens from this population, from a laboratory control population and from a field population to various concentrations of CdSO4 in artificial soil water. Toxic effects were measured using the MTT test and the comet assay. The group that had been exposed to Cd for more than a decade was found to be more tolerant to the deleterious effects of Cd at both cellular and molecular levels than the laboratory control population. The field population, which came from a severely metal polluted environment, displayed high tolerance at molecular level as well. The results provide novel biomarker evidence of increased Cd tolerance in E. fetida, but the mechanisms supporting the apparent tolerance, still need to be clarified.

Acute and sublethal effects of sequential exposure to the pesticide azinphos-methyl on juvenile earthworms ( Eisenia andrei )

The use of organophosphate pesticides is an integral part of commercial farming activities and these substances have been implicated as a major source of environmental contamination and may impact on a range of non-target fauna. The extent to which soil dwelling non-target organisms are affected by exposure to the organophosphate azinphos-methyl was investigated through monitoring selected biomarker responses and life cycle effects under laboratory conditions in the earthworm Eisenia andrei. Standard acute toxicity tests were conducted followed by a sequential exposure regime experiment, in order to assess the effects of multiple pesticide applications on biomarker (cholinesterase activity and neutral red retention time), life-cycle (growth and reproduction) and behaviour (avoidance and burrowing activity) responses. The present study indicates that the time between exposure events was a more important variable than concentration and that a longer interval between exposures may mitigate the effects of pesticide exposure provided that the exposure concentration is low. Additionally, it was shown that E. andrei was unable to avoid the presence of azinphos-methyl in soil, even at concentrations as high as 50% of the LC50 value, indicating that the presence of azinphos-methyl in the soil pose a realistic threat to earthworms and other soil dwelling organisms. The ChE inhibition test showed a high percentage inhibition of the enzyme in all exposure groups that survived and NRR times of exposed organisms were lower than that of the controls. The present study yielded important results that contribute to the understanding of biological impacts of pesticide pollution on the environment. Extrapolating these results can aid in optimising pesticide application regimes to mitigate the environmental effects thereof and thus ensuring sustained soil biodiversity in agricultural areas.

Stimulating effects of low lead concentrations on growth and cocoon production of Eisenia fetida (Oligochaeta)

Earthworms form an important component of the soil biota in many soils and are very sensitive to various types of contaminants. Their protection may therefore provide a margin of safety for other fauna once comparative toxicological data are available. The effects oflead on growth, maturation, cocoon viability and cocoon production of the vermicomposting earthworm species Eisenia fetida were studied by exposing worms experimentally to a sublethal concentration of lead (2000 µg. g−1) for a period of 76 days. Growth rate as well as cocoon production were monitored every 14 days and cocoons were incubated to determine their hatching success. Lead content of worms was also analysed. Exposed worms grew noticably better and produced more cocoons than control worms. Cocoon viability was, however, affected detrimentally. It is concluded that, although lead is known to be toxic at higher concentrations, sublethal concentrations close to the predicted EC5a and NOEC-values have a stimulating or hormetic effect on gr...