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

Abstract

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 μg/kg dm in the spray drift area adjacent to the orchard to 10.18 μg/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 μg/kg directly after spraying to a mean of 7.0±0.9 μg/kg 6 months later and in the nontarget area they varied from a mean of 55.0±35 μg/kg to 12.0±5 μg/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 m²) than in the nontarget areas (98.3 per m²). 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 μg/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.

Introduction

Studies on the amounts of sprayed pesticides from cultivated areas reaching nearby surface waters indicated that spray drift and edge-of-field runoff are important routes of entry into nontarget areas (Schulz, 2001a, Schulz, 2001b; Schulz et al., 2001). Pesticides sprayed in agricultural areas may also impact not only on the targeted species but also on nontarget organisms in and adjacent to the target areas. The concern specifically addressed in the present study therefore relates to the possible effects of pesticides on nontarget soil organisms in target and nontarget soils. Organophosphate (OP) insecticides are presently the most widely used group of insecticides worldwide (Chambers et al., 2002). They are used regularly in agricultural areas in the Western Cape, South Africa. We did a survey of the abundance and population density of earthworms on the fruit farm Vergelegen in this area, where the insecticide chlorpyrifos is used to control scale insects (Coccidae). Earthworms were chosen as example nontarget soil organisms due to their beneficial role in soil (Lee, 1985) and their abundance was found to be very low in the orchards and in the adjacent areas. The aim of this study was to investigate whether this pesticide might impact on the earthworms and whether it could explain the low population numbers.

Organophosphate insecticides have adverse effects on vertebrate and invertebrate organisms (Stenersen, 1979; Booth et al., 2001; Wilson et al., 2002; Moreby et al., 2001) and are known to be acutely toxic to some organisms. The level of toxicity to invertebrates, however, does not seem to be similar to that of vertebrates (Mayer and Ellersieck, 1986) and needs further investigation. It is generally known that OP insecticides or their active metabolites are inhibitors of a group of enzymes, among which are the serine esterases, e.g., acethylcholinesterase (AChE), which lead to acetylcholine accumulation in nerve tissue (Lotti, 2002). The inhibition of the enzyme per se is not harmful but the resulting accumulation of acetylcholine causes hyperactivity within neuromuscular pathways (Savolainen, 2001). The specific effect of OPs on the activity of cholinesterase (ChE) can be used as an effective biomarker of exposure to these insecticides (Booth and O’Halloran, 2001). As it is difficult and expensive to detect the presence and exact concentrations of these OPs in the environment, due to their relatively short half-lives (Racke, 1993; Phillips et al., 2002), it may be more effective to use biomarkers to assess exposure. A number of researchers have used this biomarker effectively to determine OP effects on earthworms (Booth et al., 1998, Booth et al., 2000; Gupta and Sundaraman, 1991; O’Halloran et al., 1999) and it has been shown by Stenersen (1979) and Rao et al. (2003) that exposure to OPs can cause behavioral changes in these organisms, although Hodge et al. (2000) found that Aporrectodea caliginosa was unable to avoid chlorpyrifos. Biomarkers, measuring effects on suborganism levels, can provide links between a chemical and its toxic effect and are generally more sensitive than the more traditional measures of contamination, such as mortality and abundance (Booth et al., 2000). While a biomarker, such as ChE inhibition, is very specific for a specific chemical group or mechanism (Chambers et al., 2002), others are more general in nature and can be indicative of effects as a result of more than one detrimental factor. Therefore a more general biomarker, the neutral red retention time (NRRT) assay (Reinecke et al., 2002), was also used in this study. Booth and O’Halloran (2001) also used this biomarker to determine exposure of earthworms to OPs at field-relevant concentrations.

Apart from a few studies such as those by Ma and Bodt (1993) and Rao et al. (2003), little about the correlation between whole-organism characteristics, such as growth, and exposure to OPs is known. Indirect and direct effects on growth and behavior could result in lower reproduction rates due to the poor condition of the animals. This could be the cause of lower earthworm densities. In this study we therefore tested effects of the OP chlorpyrifos on earthworms by measuring biomass change and the two biomarker responses in the same experimental groups.

Field trials, obviously, have numerous advantages over laboratory experiments (Edwards and Bohlen, 1992; Haag and Matschonat, 2001) but it is very difficult to determine the exposure that different individual organisms have had in such trials (Moriarty, 1999). To overcome this problem, meso- or microcosms can be used. Niederlehner et al. (1990) found a good similarity between the exposure concentrations in artificially constructed microcosms and those in field tests. Due to the short half-lives of OP pesticides in soils (Richardson and Gangollil, 1993) and low numbers of earthworms present in the experimental area in the present study, it was decided to use a semi-field design in the form of microcosms. According to Van Gestel and Van Straalen (1994) very little standardization with regard to the construction of microcosms exists but basically the idea is to evaluate effects of chemicals under more natural conditions than those in single-species laboratory tests. While results obtained with these “model” ecosystems do not allow for specific prediction (Haag and Matschonat, 2001), they can help to identify factors that enhance the propensity for certain events to take place. We used microcosms filled with soil cores to simulate field conditions as far as possible but thereafter maintained them under controlled conditions in the laboratory. Microcosms implanted in the field (“mesocosms”) could have sufficed, but varying ambient factors, such as different moisture and temperature regimes, could be better controlled in the laboratory.