Search

The toxic effects of nickel on survival, growth, and reproduction of Eisenia veneta were investigated following 4 weeks of exposure to a nickel-chloride spiked loamy sand soil. The ability of a simple earthworm biomarker, the lysosomal membrane stability of coelomocytes, to reflect nickel exposure was also studied. Nickel caused a significant toxic effect on E.veneta at soil concentrations above 85 mg Ni/kg. Reproduction (cocoon production) was the most sensitive parameter being reduced at soil concentrations above 85 mg Ni/kg (EC10 = 85 mg Ni/kg). Survival of adults was only reduced at concentrations above 245 mg Ni/kg, while adult and cocoon wet weight were not affected by soil nickel concentrations up to 700 mg Ni/kg. The lysosomal membrane stability, measured as neutral-red retention time, was reduced at soil nickel concentrations similar to those that reduced reproduction, and demonstrated a dose-response relationship. The neutral-red retention time showed large individual variation for the earthworms within each exposure concentration. It was concluded that the lysosomal membrane stability, measured as neutral red retention time, has a potential role in risk assessment, but care should be taken conducting this test.

The locomotory behavior of woodlice (Oniscus asellus (L.)) collected along a gradient of pollution from a plastics fire was quantified automatically for 4 h in the laboratory employing computer-aided video tracking. Six locomotory parameters were measured and the corresponding individual body metal concentrations determined. Additional woodlice were collected from the same site for total protein and glycogen analyses. Woodlice collected from the litter layer overlying a pyrolyzed plastic layer spent significantly less time in activity, resulting in a walked distance less than 50% of that of woodlice collected from a nearby reference location. Furthermore, a tendency to turn predominantly in either a left or a right direction and a tendency toward a higher discontinuity in moving was found for the woodlice collected on the pyrolyzed plastic. Overall, the aberrant behavior coincided with significantly elevated body concentrations of heavy metals and lowered protein and glycogen levels. The interference of metals with neurological and metabolic processes is discussed in relation to locomotion.

The fate of cadmium released into a forest ecosystem via a chemical accident was determined by analysing cadmium in biological tissue from a number of differing tropic levels within the ecosystem: including soil litter, graminaceous species, invertebrates and small mammals. Cadmium was bioavailable to all organisms investigated and was transferred through the food chain to small mammals.

Earthworms Lumbricus rubellus were exposed to a range of increasing soil copper concentrations in a mesocosm experiment for 17, 40, 70, and 110 days, respectively. Neutral-red retention times were measured along with earthworm tissue copper residues and earthworm growth and survival. The neutral-red retention assay demonstrated a clear dose-response threshold preceding a copper exposure value where increased regulatory activity had resulted in the copper bioconcentration factor (BCF) decreasing from 1 to 0.5. Effects on earthworm growth and survival occurred only at soil copper concentrations where the earthworm BCFs were lowered to ca. 0.5. Thus, the position of the neutral-red threshold could potentially make it possible to differentiate between copper exposure and actual toxicity. In terms of field applicability it was demonstrated that natural and seasonal variations in climatic parameters had little or no effect on the neutral-red response. Neutral-red retention assay has a potential role in environmental risk assessment and routine monitoring, as it is likely to provide a sufficiently accurate warning of impending ecological damage at a level below that found to cause actual physiological damage to the earthworms.

A simple earthworm biomarker, neutral-red retention by coelomocyte lysosomes, was measured concurrently with ecological parameters in order to link effects at different levels of biological organization in a laboratory study. Exposure of the earthworm Eisenia andrei to an increasing range of soil copper concentrations in the laboratory indicated a threshold range for the neutral-red assay at soil copper concentrations between 40 and 80 mg Cu kg-1. This threshold coincided with the soil to worm copper bioconcentration factor decreasing from 1 to 0.3 and thus an apparent onset of a copper regulatory mechanism. Effects at the individual and population levels only occurred at soil copper concentrations beyond that of the biomarker threshold. Thus, it was possible to differentiate between exposure and toxicity. It was therefore possible to conclude that the neutral-red assay has great potential and relevance in earthworm ecotoxicity studies. The assay provides an early warning that can be linked directly to important physiological changes that were observed to precede adverse effects on individuals or populations in this laboratory study.

High resolution 1H NMR spectroscopy of biofluids, cells and tissue extracts allows rapid, non destructive analysis for a wide range of metabolites and organic compounds with minimal sample pre treatment. We have applied high resolution 1H NMR spectroscopy to investigate the biochemical effects of Cu II in two earthworm species Eisenia andrei n =78 and Lumbricus rubellus n =45 exposed under laboratory and semi field conditions respectively. The most marked metabolic response was the elevation of endogenous whole body free histidine in animals which positively correlated with increasing copper exposure and total copper burden in the semi field experiment. Histidine forms thermodynamically stable copper complexes under a wide range of physico chemical conditions and we proposed that the elevation of free histidine in response to copper challenge provides an energetically low cost detoxification mechanism. Histidine elevation may also provide a novel molecular biomarker of Cu II exposure in environmental situations.

A simple subcellular histochemical staining technique employing the lysosomal probe neutral red has been developed for use with the epiendogeic earthworm Lumbricus rubellus Hoffmeister. Coelomocytes extracted from the coelomic cavity of earthworms into an isotonic earthworm Ringer solution were allowed to adhere to a microscope slide for 30 s before the application of a neutral red dye. This red dye was rapidly accumulated within the lysosomes. Observation of the loss of this dye from these lysosomes into the surrounding cytosol has enabled the quantification of the degree of lysosomal damage caused to earthworms with exposure to an increasing range of soil copper concentrations, in both laboratory and mesocosm studies. This simple in vitro biomarker has potential for the rapid assessment of the toxic effects to earthworms from soils contaminated with heavy metals and metalloids.

A low-cost field technique employing retention of the dye neutral-red by lysosomes in coelomocyte cells taken from earthworms (Lumbricus castaneus), was used as a means of assessing the ecological effects (if any) of an industrial accident. Earthworms and soil samples were collected at the site of a large industrial plastics fire in Thetford, UK along a 200 m transect leading from the factory perimeter fence, over a layer of molten plastic impregnated soil and into the surrounding forest. Coelomic fluid extracted from the earthworms was dye-loaded with neutral-red and lysosomal leaking observed. Metal residues in soil and earthworms were found to be highly elevated close to the factory perimeter and to rapidly drop to background levels within the first 50 m of the transect. Coelomocyte cells taken from earthworms adjacent to the factory perimeter showed the shortest period of neutral-red retention (2 min); cells taken from worms further into the surrounding forest had a longer retention time (12 min), whilst cells taken from worms from a control site showed even greater retention times (25 min). Thus, the neutral-red retention times correlated negatively with measured residues of heavy metals in the earthworms, the higher the body metal concentration the shorter the retention time. This field trial has demonstrated the validity of using an in vitro cellular biomarker technique for use in biological impact assessment along gradients of contamination.

The use of sponges as biomonitoring organisms has previously been proposed as a supplementary component to established monitoring programmes. This study has shown that the cosmopolitan marine demosponge Halichondria panicea Pallas accumulated Cu, Zn and Cd in direct proportion to the concentration of the dissolved metals in the surrounding medium. High metal accumulation rates were measured. In addition, there was no apparent loss of accumulated metals upon transfer to clean seawater after an initial flushing of the water canals. In view of the large volumes of water passing through the bodies of H. panicea, this species is proposed as a suitable biomonitoring organism.

To enable efficient monitoring and the early detection of toxic metal effects a rapid low cost test has been developed for use with freshwater gastropods. A cellular assay formerly developed for marine organisms has been modified for use with freshwater snails. A modified neutral-red retention assay makes use of the fact that lysosomes in unstressed cells retain the neutral-red dye for a long duration after uptake. In contrast, following uptake in stressed cells, the dye will leak from the lysosomes into the cytoplasm more rapidly, an indication of cell-membrane damage. The higher the stress level, the shorter the period of dye retention. In this study freshwater snails Viviparus contectus were exposed to dissolved copper. Blood cells extracted from snails exposed to the highest concentrations exhibited the shortest lysosomal neutral-red retention times. This early detectable change may be useful for routine environmental monitoring, identifying low level contamination, the moment it occurs.

The effects of elevated copper concentrations and oxygen deficiency on total body copper concentration, egg-production and mortality of two genetically separated populations of Corophium volutator were studied. The amphipods were exposed for 14 days to a three-level factorial design of

The exuvial loss, subsequent uptake, and tissue and body distributions of the essential trace metals copper and zinc were investigated in an ecological series of talitrid amphipods, immediately after moult and throughout a post-moult period. Negligible quantities of copper or zinc were detected in cast exuvia of individual talitrids. Neither tissue nor whole body copper and zinc concentrations underwent significant changes during the moult cycle and all species showed extreme conservatism in their handling of body copper. There appeared to be no differential treatment of copper between species at the time of moult throughout the ecological series, with no obvious increase in copper retention by the more terrestrial species. The impact of moult cycle stage therefore is unlikely to affect the use of littoral talitrid amphipods as biomonitors for ambient copper and zinc concentrations.

Copper-rich granules were discovered in the ventral caeca of talitrid amphipods inhabiting copperpolluted localities. Transmission electron microscopy has revealed the presence of rounded granules containing homogeneous electron-dense material (with no nucleus or core) in the RtF cells of the ventral caeca of Orchestia gammarellus. The granules did not appear membrane bound. X-ray microanalysis revealed strong copper and sulphur peaks with a minor peak for calcium. In all cases the granules occurred in the cytoplasm of the cells and all Cu was clearly localized within the granules. The association of sulphur within the granules is thought to represent an organic detoxification system. Qualitative studies on the loss and induction of the granules are described. Whole body and individual tissue copper concentrations were found to be elevated in amphipods from polluted sites. The role of such a detoxification mechanism in providing metal tolerance in these animals is discussed.

The mode of metal accumulation employed by the terrestrial talitrid amphipod Arcitalitrus dorrieni (Hunt) feeding on leaf litter variously enriched with the essential metals copper and zinc have been established in the laboratory. Body concentrations of both metals were found to increase over time correlating with the increasing dietary metal concentrations. Field trials to assess the potential use of this amphipod as a terrestrial biomonitor for ambient copper and zinc availabilities in leaf litter are described. The results indicate a potential biomonitoring capacity for zinc (and possibly for copper also). The presently restricted distribution of this species, however, limits its biomonitoring potential to metalliferous areas of S.W. England, but reflects unknown potential in its native Australia.

Locomotor behavior in terrestrial organisms is crucial for burrowing, avoiding predators, food seeking, migration, and reproduction; therefore, it is a parameter with ecological relevance. Acetylcholinesterase (AChE) is a nervous system enzyme inhibited by several compounds and widely used as an exposure biomarker in several organisms. Moreover, changes in energy reserves also may indicate an exposure to a stress situation. The aim of this study is to link biomarkers of different levels of biological organization in isopods exposed to increasing doses of dimethoate in semifield conditions. Locomotor parameters, AChE activity, and energy reserves (lipid, glycogen, and protein contents) were evaluated in the isopod Porcellio dilatatus after 48-h and 10-d exposure to dimethoate-contaminated soil. Results showed a clear impairment of both locomotor and AChE activity during the entire study, although effects were more pronounced after 48 h. Most locomotor parameters and AChE activity showed a clear dose-response relationship. By contrast, no clear trend was observed on energetic components. A positive and significant relationship was found between AChE activity and those locomotor parameters indicating activity, and the opposite was observed with those locomotor parameters indicating confusion and disorientation. The results obtained in this study enhance the importance of linking biochemical responses to parameters with ecological relevance at individual level, the value of locomotor behavior as an important marker to assess effects of toxicants, and also the usefulness and the acquisition of ecological relevance by AChE as a biomarker, by linking it with ecologically relevant behavioral parameters.

During the period 1996–1999 a joint field research programme (BIOPRINT-II) funded by the European Union was undertaken. The main objective of this project was the deployment of biochemical fingerprint techniques of soil invertebrate biomarkers for assessing the exposure and effect of toxicants on soil invertebrates in the field. The aim was to apply these techniques in the field focusing on a a chronically polluted field near a lead and zinc smelter in Avonmouth (UK). Therefore six sites were selected from which organisms were either sampled or transplanted to or from the laboratory. The project has provided a unique opportunity to apply a series of biological test methodologies in order to determine the hazard posed to soil sustainability and by inference soil biodiversity and function. This work has attempted to understand the linkage between effects measured at the molecular or cellular level and relate these to changes at higher levels of biological organisation. Here we evaluated the links between biomarkers and soil function parameters. The paper aims to summarize and explore the necessary caveats that must be understood before soil biomarker test systems may be used to strengthen the risk assessment process.

An experimental approach was taken to examine the physiological tolerances that may determine the distribution of the euterrestrial amphipod Arcitalitrus dorrieni, a native of Australia which has established itself in the UK over the last 100 years. Two experiments using mesocosms were carried out over consecutive winters to examine the effects of low temperature and leaf litter sodium content on survival. The role of the microhabitat in buffering environmental temperature was also investigated. It was concluded that A. dorrieni may be prevented from colonising areas of Britain by the effects on populations of low winter temperatures and low habitat sodium concentration. There is considerable buffering of environmental temperatures by the leaf litter and soil layers of the microhabitat, and consequently, mortality of A. dorrieni is related to the depth of microhabitat available for it to utilise in avoiding low temperatures.

The effects of trace metals on the apparent water permeability (AWP) of two decapod crustaceans Carcinus maenas and Crangon crangon, were investigated. One hour exposure to 1 mg l−1 dissolved Hg or Pb significantly decreased the AWP of the shore crab, C. maenas. A similar, but not significant, tendency was observed following exposure to Cd, Cu and Zn, whereas Co had little effect. The AWP of C. maenas exposed to Cd decreased with increasing Cd concentration (0.5, 1, 4 or 8 mg l−1). This decrease was independent of exposure time (1, 5 or 10 days). The influence of Zn (500 μg l−1) on the apparent water permeability of the brown shrimp C. crangon was also investigated. After 24 h exposure to Zn, AWP decreased significantly. This reduction persisted for at least 3 days. After 6 days of exposure, AWP returned to the initial level. Whether trace metal induced changes in the AWP are the result of toxic effects of the metals or represent physiological adaptations minimizing the effects of external perturbations is discussed.

In this study, the effects of two metals, copper and cadmium, on the growth and development of juvenile Lumbricus rubellus were measured in a toxicity test in which individuals were grown in isolation. This design had a number of advantages over traditional test systems for earthworms. Importantly, the test is specifically designed to measure two juvenile traits (survival over and length of the juvenile period) that have been shown to have a high sensitivity for determining population growth rate. The test system also maximizes replication, while allowing time-series-based monitoring of individual growth. For both metals, significant exposure-dependent effects on survival, growth, development time, and (less certainly) maturation weight were observed. Comparisons of the relative toxicity of the two metals indicated different concentration-response relationships. For copper, hormesis was found at low levels, while only at the highest soil concentration tested (10.07 micromol g(-1)) were (severe) toxic effects present. For cadmium, hormesis was also evident at the lowest concentration tested; however, at soil levels above this, a graded concentration-dependent toxic effect was apparent. These differences in the exposure response patterns can be (tentatively) explained in terms of the mechanisms for handling copper (an essential metal for earthworms) and cadmium (a putative nonessential element). The applicability of the test for routine measurement of chemical effects on ecologically relevant juvenile traits is also outlined and future developments are discussed.

Polynitro-organic compounds such as 2,4,6-trinitrotoluene (TNT) can be released into the environment from production and processing facilities and military firing ranges as well as through field use and disposal practices. Based on laboratory toxicity data, TNT has lethal (at ≥260 mg TNT/kg dry soil) and sublethal effects (at ≥59 mg TNT/kg dry soil) to the earthworm. However, field studies are needed to relate exposure of organisms to explosives in mixed-contaminated soil under field conditions and to define effects-based ecotoxicologic benchmarks for TNT-contaminated soil. In the present study, the lethal and sublethal effects of a 10-day in situ exposure at a TNT-contaminated field site using mesh-bag mesocosms were assessed. In addition to the survival end point, the biomarkers of earthworm exposure and effect—including tissue residues, lysosomal neutral red retention time (NRRT), and total immune activity (TIA)–were measured. Concentrations of TNT in soil mesocosms ranged from 25 to 17,063 mg/kg. Experiments indicated a trend toward decreasing survival of caged Aporrectodea rosea and Eisenia andrei as the concentration of TNT and total nitroaromatic compounds increased. E. andrei tolerated higher concentrations of TNT (up to 4050 mg/kg dry soil) in mesocosms than did indigenous earthworms, who survived only at ≤1146 mg TNT/kg. Earthworms E. andrei and A. rose survived in 67% and 75% of TNT-contaminated mesocosms, respectively, compared with references groups. NRRT was significantly decreased in surviving earthworms from the contaminated areas compared with those from the reference site. TIA was not affected by field exposure to TNT. Earthworm tissue concentrations of TNT metabolites 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not correlated with TNT soil concentrations. In addition, higher tissue concentrations of TNT metabolites were observed at concentrations ranging from 116 to 130 mg TNT/kg soil. The results showed that earthworm exposure in TNT-contaminated soil produced both lethal and sublethal effects in the field. The results of study indicated that mesocosm experiments would be useful to assess the toxicity of a site and to characterize the overall effects of contaminants. However, mesocosm experiments present special considerations (e.g., abiotic factors, exposure period) when used at heterogenous sites, and data must be interpreted with caution.

Physiological responses to environmental stressors may induce changes in gene expression as part of an organism's homeostatic mechanisms. Thus molecular genetic biomarkers have the potential to be used for monitoring sublethal chemical exposure in ecosystems. This paper describes a methodological assessment of the suitability of a protocol to monitor selected biomarkers. The TaqMan real-time quantitative polymerase chain reaction was used to measure gene transcription in earthworms (Lumbricus rubellus) maintained on control or cadmium- or copper-spiked soil. Changes in the expression of two target genes, that encoding metallothionein isoform 2 (MT-2) and that encoding the mitochondrial large ribosomal subunit (MLRS), were quantified against the internal control gene beta-actin. The protocol used produced reliable and reproducible results. Transcript levels displayed qualitative and quantitative differences in the responses to the two metal ions. MLRS gene levels were unaffected by exposure to cadmium but displayed a response to high levels of copper. Conversely, cadmium greatly induced MT-2 gene expression, but copper only altered transcription of this gene at high exposure concentrations. This study demonstrates that it is now technically feasible to use gene expression as an index of pollution exposure in environmentally relevant organisms.

Demographic methods can translate toxicant effects on individuals into consequences for populations. To date few such studies have been conducted with longer-lived invertebrates. This is because full life-cycle experiments are difficult with such species. Here we report the effects of copper and cadmium on the key demographic parameter intrinsic rate of population increase (r) in a long-lived invertebrate (an earthworm). The approach used to derive r was based on robust measurement of effects on life-cycle traits in three specifically designed toxicity tests and integration of this data within a demographic model. The three laboratory tests used to generate values for specific life-cycle parameters under copper and cadmium exposure were suited to the task. Significant effects on a range of separate adult and juvenile life-cycle parameters were seen. Integration of parameter values within the demographic equation indicated that for copper, r was reduced only at a concentration that also caused adult mortality. For cadmium, a more graded exposure-dependent effect on fitness was seen, with r reduced at sublethal concentrations. The concentration response patterns for r found for the two metals suggest significantly different consequences for earthworm populations exposed to sublethal levels of copper and cadmium.

In many cases, the link between industrial emissions and damage to the environment can only be inferred. The Environment Agency of the United Kingdom imposes emissions limits on industrial sites so that predicted concentrations and deposition rates remain below standard thresholds. Estimates of appropriate critical levels and loads are usually based on laboratory results and rarely estimate synergistic effects between pollutants or consider biological adaptation or selection in the target receptor organisms. The Avonmouth smelter has been emitting zinc and other heavy metals since 1929. It has been the subject of a number of detailed and synoptic studies, especially the impact on soil invertebrates. Damage was assessed using both physiological and ecological measurements. Two methods of spatial analysis were investigated, namely interpolation using standard geographical information system (GIS) operators and atmospheric dispersal modeling using an off-the-shelf model. Both methods can be used to compute contours (isolines) of predicted biological effect. Correlation results show that dispersal modeling is at least as good as kriging but requires much less data. This article demonstrates the usefulness of GIS and dispersal models as tools in decision making to determine the most suitable sampling sites in the assessment and monitoring of the impact of contamination around major point sources.

Biological indicators can be used to assess polluted sites but their success depends on the availability of suitable assays. The aim of this study was to investigate the performance of two earthworm biomarkers, lysosomal membrane stability measured using the neutral red retention assay (NRR-T) and the total immune activity (TIA) assay, that have previously been established as responsive to chemical exposure. Responses of the two assays were measured following in situ exposure to complexly contaminated field soils at three industrial sites as well as urban and rural controls. The industrial sites were contaminated with a range of metal (cadmium, copper, lead, zinc, nickel and cobalt) and organic (including polycyclic aromatic hydrocarbons) contaminants, but at concentrations below the 'New Dutch List' Intervention concentrations. Exposed earthworms accumulated both metals and organic compounds at the contaminated sites, indicating that there was significant exposure. No effect on earthworm survival was found at any of the sites. Biomarker measurements, however, indicated significant effects, with lower NRR-T and TIA found in the contaminated soils when compared to the two controls. The results demonstrate that a comparison of soil pollutant concentrations with guideline values would not have unequivocally identified chemical exposure and toxic effect for soil organisms living in these soils. However, the earthworm biomarkers successfully identified significant exposure and biological effects caused by the mixture of chemicals present.

19 F nuclear magnetic resonance (NMR) spectroscopy was used as a specific tool to investigate the metabolism of 3-trifluoromethylaniline (3-TFMA) in the earthworm species Eisenia veneta . Exposure was via a filter-paper contact toxicity test using five exposure levels (1000, 100, 10, 1, and 0.1 μg/cm 2 ). Instant lethality was observed at the two highest levels. Worms exposed at the lower levels appeared to tolerate the compound. The 19 F label of 3-TFMA allowed the uptake and metabolism of the earthworms to be monitored by 19 F NMR spectroscopy. Metabolism of 3-TFMA was observed at 10 μg/cm 2 and, to a lesser extent, at 1 μg/cm 2 . The possibility of 3-TFMA accumulation in specific organs was also investigated. As a simplified model, worms were cut into distinct anatomical regions (head, testes, crop, clitellum, and gut). At the two highest exposure levels, “uniform distribution” was observed. However, accumulation appeared to be proportional to the “size” of the extracted segments at the lower levels.

Demographic methods can translate toxicant effects on individuals into consequences for populations. To date few such studies have been conducted with longer-lived invertebrates. This is because full life-cycle experiments are difficult with such species. Here we report the effects of copper and cadmium on the key demographic parameter intrinsic rate of population increase (r) in a long-lived invertebrate (an earthworm). The approach used to derive r was based on robust measurement of effects oil life-cycle traits in three specifically designed toxicity tests and integration of this data within a demographic model. The three laboratory tests used to generate values for specific life-cycle parameters under copper and cadmium exposure were suited to the task. Significant effects on a range of separate adult and juvenile life-cycle parameters were seen. Integration of parameter values within the demographic equation indicated that for copper, r was reduced only at a concentration that also caused adult mortality. For cadmium. a more graded exposure-dependent effect on fitness was seen. with r reduced at sublethal concentrations. The concentration response patterns for r found for the two metals suggest significantly different consequences for earthworm populations exposed to sublethal levels of copper and cadmium.

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to produce a biochemical fingerprint of low-molecular-weight metabolites from complex biological mixtures such as tissue extracts and biofluids. Changes in such fingerprint profiles can be used to characterize the effects of toxic insult in in vivo systems. The technique is nonselective and requires little sample preparation or derivatization. In the present study, earthworms (Eisenia veneta) were exposed to three different model xenobiotics by a standard filter paper contact test, and toxicant-induced biochemical changes were then investigated by characterizing the changes in endogenous metabolites visible in 600-MHz 1H NMR spectra of tissue extracts. The NMR spectral intensities were converted to discrete numerical values and tabulated in order to provide data matrices suitable for multivariate analysis. Principal component analysis showed that changes had occurred in the biochemical profiles relative to the undosed controls. The 2-fluoro-4-methylaniline-treated worms showed a decrease in a resonance from a compound identified as 2-hexyl-5-ethyl-3-furansulfonate using a combination of high-performance liquid chromatography (HPLC)-Fourier transform mass spectrometry (IonSpec, Lake Forest, CA, USA) and 1H and 13C NMR spectroscopy. An increase in inosine monophosphate was also observed. The 4-fluoroaniline-treated worms showed a decrease in maltose concentrations, and 3,5-difluoroaniline exerted the same effect as 2-fluoro-4-methylaniline but to a lesser extent. These changes could potentially be used as novel biomarkers of xenobiotic toxicity and could be used to determine the mechanism of action of other toxic chemicals.

1. Little is known about metabolism of xenobiotics by earthworms, despite their importance in soil ecotoxicity testing. Normal earthworms and earthworms treated with antibiotics to ensure inhibition of gut microflora were exposed to two model xenobiotic compounds, 4-fluoroaniline and 4-fluorobiphenyl, to determine which metabolites were produced, and whether the pattern of metabolism was affected by the presence of microbial transformation ability. 2. (19)F-NMR spectroscopy detected the number and relative proportions of metabolites and directly coupled HPLC-(1)H-NMR spectroscopy and HPLC-MS then identified the metabolites. 3. Despite uptake, no metabolism of 4-fluorobiphenyl was observed at any stage, which appears to be a consequence of the lack of oxidative Phase I metabolic activity of the earthworms towards this substrate. In contrast, 4-fluoroaniline exhibited dose-dependent metabolism. At high doses (leading to mortality within 24 h) one predominant metabolite was observed, which was identified as the N-beta-glucoside conjugate. At lower dose levels, the predominant metabolite was the gamma-glutamyl conjugate, although the glucoside and another as yet unidentified metabolite were also detected. 4. The inhibition of gut microflora did not have any influence on metabolism. The study represents the first evidence for glucoside and glutamyl conjugation as a pathway for xenobiotic metabolism in earthworms.

1. The metabolic fate of the model ecotoxin 3-trifluoromethylaniline (3-TFMA) in earthworm was studied by (19)F- and directly coupled (19)F/(1)H-HPLC-NMR spectroscopy. Earthworms of Eisenia veneta spp. were subjected to the ecotoxin during a filter papercontact toxicity test at exposure levels of 1000, 100, 10, 1 and 0.1 micro g cm(-2). A metabolic profile was obtained previously by (19)F-NMR spectroscopy and metabolites were observed at all the exposure levels. 2. Identification of metabolites in individual worm extracts at the (lethal) exposure levels of 1000 and 100 micro g cm(-2) could be achieved on-line without sample preparation by (19)F/(1)H-HPLC-NMR spectroscopy. (19)F-HPLC-NMR spectroscopy was used in the continuous-flow mode, which enabled the HPLC chromatographic retention times (t(R)) of the metabolites to be established in a single analytical step. 3. In total, three (19)F-NMR signals could be detected, of which one was identified as the parent compound. Two earlier eluting metabolites were identified to be alpha- and beta-glucoside conjugates of 3-TFMA. 4. Metabolites at the lower (sublethal) exposure levels of 10, 1 and 0.1 micro g cm(-2) escaped identification by (19)F/(1)H-HPLC-NMR spectroscopy as outlined here and will require concentration prior to analysis.

The endogenous metabolites of the coelomic fluid of the earthworm Eisenia veneta were characterised using high-resolution one-dimensional and two-dimensional 1H nuclear magnetic resonance spectroscopy. Signals from common organic acids, such as acetate, fumarate, malonate, malate, formate, and succinate, were identified together with adenosine and nicotinamide mononucleotide. The potential use of this information as a baseline dataset for future toxicological or physiological studies was demonstrated by a metabonomic analysis: a series of earthworms were dosed with the model compound 3-fluoro-4-nitrophenol, and toxic effects followed by multivariate analysis of the spectral data of the coelomic fluid. Relative concentrations of acetate and malonate were decreased in the dosed worms compared to the controls.

In models of insect–pathogen interactions, the transmission parameter (ν) is the term that describes the efficiency with which pathogens are transmitted between hosts. There are two components to the transmission parameter, namely the rate at which the host encounters pathogens (contact rate) and the rate at which contact between host and pathogen results in infection (host susceptibility). Here it is shown that in larvae of Spodoptera exempta (Lepidoptera: Noctuidae), in which rearing density triggers the expression of one of two alternative phenotypes, the high-density morph is associated with an increase in larval activity. This response is likely to result in an increase in the contact rate between hosts and pathogens. Rearing density is also known to affect susceptibility of S. exempta to pathogens, with the high-density morph showing increased resistance to a baculovirus. In order to determine whether density-dependent differences observed in the laboratory might affect transmission in the wild, a field trial was carried out to estimate the transmission parameter for S. exempta and its nuclear polyhedrosis virus (NPV). The transmission parameter was found to be significantly higher among larvae reared in isolation than among those reared in crowds. Models of insect–pathogen interactions, in which the transmission parameter is assumed to be constant, will therefore not fully describe the S. exempta-NPV system. The finding that crowding can influence transmission in this way has major implications for both the long-term population dynamics and the invasion dynamics of insect–pathogen systems.

Earthworm survival tests are commonly used in terrestrial ecotoxicology to assess the toxicity of compounds in soil. Earthworm (Eisenia andrei) reproduction tests were used to assess the sublethal and chronic effects of 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitro-1,3,5,7-triazacyclohexane (RDX). Effects on reproduction parameters (total number of cocoons, number of hatched cocoons, number of juveniles, juvenile biomass, and hatchability) were measured in TNT- and RDX-spiked artificial soil. For TNT, the lowest-observed-effect concentration (LOEC) was 110 mg/kg dry soil, and the no-observed-effect concentration (NOEC) was 55 mg/kg. For the RDX-spiked soil, the LOEC was 95 mg/kg dry soil, and the NOEC was

Earthworms are abundant and very important organisms for both soil formation and the breakdown of organic matter in most terrestrial ecosystems. They are, in many ecosystems, key species responsible for moving large amounts of soil from deeper horizons to the surface layers (Edwards and Bohlen 1992). For these and other reasons, earthworms have been used extensively in ecotoxicological soil studies (EEC 1985; Kokta 1992; OECD 1984; van Gestel et al. 1989a,b, 1995).

This chapter considers the utility of biomarkers for monitoring environmental health in an extremely contaminated and polluted locality, the Kola Peninsula, in Northern Russia. Due to the extreme nature of the pollution, biomarkers have little place as predictors or to forewarn of likely damaging effects of contaminants in their respective ecosystems; the damage has been and continues to be done. Biomarkers can, however, be used to monitor and map the worst affected areas, and look for signs of recovery in areas that have been remediated or where pollution prevention controls have been put in place. There are various caveats in using biomarkers in such a fashion and these are discussed. The following text serves to give a simple overview of the implementation and reasoning for using biomarkers, suggest the type of material that one may wish to consider to monitor, and outlines a simple procedure or strategy for implementing such a survey.

A dual-labelling radioisotope technique has enabled the relative assimilation efficiencies of an essential (Zn) and a non-essential trace metal (Cd) to be determined in an ecological series of talitrid amphipods from low-shore to fully terrestrial habitats. All species exhibited high assimilation efficiencies for zinc and cadmium (within the range 66.7–96.4%). Interspecific differences in assimilation rates showed no trend with increasing terrestriality of the habitat. Elevated dietary zinc concentrations failed to enhance zinc assimilation efficiency. Incorporation of antibiotics into the diet to eliminate gut microflora did not affect zinc assimilation efficiencies. The implications of having high trace-metal assimilation efficiencies are discussed in terms of land invasion by talitrids.

The collembolan Folsomia fimetaria was exposed to a range of elevated copper (Cu) concentrations in a LUFA-Speyer soil for 7, 14, and 21 d, in a microcosm test. The results showed that F. fimetaria was sensitive to copper, with reproduction being a more sensitive parameter of effect than either growth (measured as final body size) or survivorship. A 10% effective concentration (EC10) of 38 mg Cu/kg was calculated for reproduction, and an EC10 between 509 to 845 mg Cu/kg (depending on sex and developmental stage) was calculated for growth. No mortality was observed for soil concentrations up to 1,000 mg Cu/kg. This study has shown that F. fimetaria populations may with time become severly reduced or may even become extinct (in extreme circumstances) as a result of copper exposure. Although survival of the adults was not affected, toxicity was expressed solely through reproductive failure. The present study has also shown that the duration of copper exposure was not an important factor for the determination of effects during short-term tests with F. fimetaria.

Two species of talitrid amphipod Orchestia gammarellus (Pallas) and Orchestia mediterranea Costa were shown to accumulate copper and zinc from a range of copper- and zinc-enriched algal foods. Accumulation increased with increasing dietary metal concentrations. Laboratory results have been extrapolated to the field, in an attempt to compare the relative importance of food and solution as sources of trace metals to the talitrids in relation to their respective position on the shore. It was concluded that, for O. gammarellus, accumulation of copper from food was a more important route than the accumulation of copper from solution. O. mediterranea, however, was unable to satisfy its copper requirements from a food source, but was able to achieve all its copper requirements from solution