Exposure of sticklebacks (Gasterosteus aculeatus) to cadmium sulfide nanoparticles: biological effects and the importance of experimental design.

Effects of nanoparticles on aquatic organisms have been little studied to date and toxicological data are urgently needed for development of regulatory frameworks for these substances. Here, we report the findings of a study exposing sticklebacks to cadmium sulfide (CdS) as bulk material and quantum dots. Fish were exposed for 21 d in a flow through test system to 5, 50 or 500 microg l(-1) CdS nanoparticles (nCdS) coated in thiol terminated methyl polyethylene glycol (MPEG), bulk CdS or MPEG at 500 microg l(-1) (nominal concentrations). With the exception of the highest nCdS exposure, measured concentrations were approximately one order of magnitude below nominal. A single fish from each group (excluding MPEG) was examined using energy dispersive X-ray analysis (EDX) to localise cadmium, however, cadmium could not be detected in whole body sections. Elevated levels of oxidized glutathione were measured in the gills of fish exposed to 50 and 500 microg l(-1) nCdS. Induction of vitellogenin synthesis was not detected in any of the treatment groups. The number of males engaged in nest-building behaviour following exposure to 500 microg l(-1) nCdS was reduced and livers of 4/6 fish in the same treatment displayed hepatocellular nuclear pleomorphism. The results are discussed emphasising the fundamental importance of experimental design and the need to understand the behaviour of nanoparticles in the aqueous phase.

Influence of dimethoate on acetylcholinesterase activity and locomotor function in terrestrial isopods.

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.

Establishing principal soil quality parameters influencing earthworms in urban soils using bioassays.

Potential contamination at ex-industrial sites means that, prior to change of use, it will be necessary to quantify the extent of risks to potential receptors. To assess ecological hazards, it is often suggested to use biological assessment to augment chemical analyses. Here we investigate the potential of a commonly recommended bioassay, the earthworm reproduction test, to assess the status of urban contaminated soils. Sample points at all study sites had contaminant concentrations above the Dutch soil criteria Target Values. In some cases, the relevant Intervention Values were exceeded. Earthworm survival at most points was high, but reproduction differed significantly in soil from separate patches on the same site. When the interrelationships between soil parameters and reproduction were studied, it was not possible to create a good model of site soil toxicity based on single or even multiple chemical measurements of the soils. We thus conclude that chemical analysis alone is not sufficient to characterize soil quality and confirms the value of biological assays for risk assessment of potentially contaminated soils.

Physiological tolerances of the euterrestrial amphipod Arcitalitrus dorrieni (Hunt) as a key to its geographical distribution? A test using mesocosms.

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.

Biological assessment of contaminated land using earthworm biomarkers in support of chemical analysis.

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.

Toxicological, cellular and gene expression responses in earthworms exposed to copper and cadmium.

This study correlates sub-organismal changes with toxicological effects in earthworms (Lumbricus rubellus) exposed to copper and cadmium. Both metals reduced survival and reproduction at the highest concentration (LC50 5.11 microM Cu g(-1) and 4.04 microM Cd g(-1); cocoon production EC50s 5.17 microM Cu g(-1) and 1.86 microM Cd g(-1), all values as dry mass soil). Cadmium significantly reduced lysosomal membrane stability (at 1.86 microM Cd g(-1) and higher), upregulated metallothionein gene expression (at least sevenfold in all treatments) and reduced lysosome-associated-glycoprotein gene expression. Copper did not lower lysosomal membrane stability, but did upregulate metallothionein gene expression (at 2.5 microM Cu g(-1)), reduce lysosome-associated-glycoprotein gene expression and gave a nonlinear pattern for mitochondrial ribosomal subunit transcript expression (reduced at 0.35 and 0.811 microM Cu g(-1); higher at 2.5 microM Cu g(-1)). Correlation of metal body residue concentrations and cellular and molecular genetic responses with juvenile production rate confirmed a relationship for metallothionein expression, lysosomal membrane stability and cadmium tissue concentration in cadmium-exposed worms. Relationships between responses were also found for both metals. These suggested mechanisms for the interaction of cadmium and copper with specific gene products and with organelle (mitochondrial, lysosomal) functioning.

Pedological characterisation of sites along a transect from a primary cadmium/lead/zinc smelting works.

A pedological characterisation of seven sites along a transect from a smelter at Avonmouth, UK, was undertaken. Site locations comprised a mixture of both grassland (5 sites) and oak tree dominated (2 sites) areas. Geographically, sites were either low lying or on adjacent elevated ground. Across the transect, a severe gradient of metal concentrations was found with highest values for organic soil horizons from close to the factory. Comparisons with quality standards indicate that these metal levels are likely to be a major ecological stressor. In addition to the strong metal gradient, a number of other between site differences were also observed. These were firstly water regime, which at the low lying sites close to the smelter showed influence by both, groundwater and stagnant water, while at more distant sites indicated susceptibility to stagnant water only, and secondly soil pH, which showed large between site variations, although no consistent trend along the transect. Humus forms at sites more than 1.5 km from the smelter were characteristic for the soil conditions and land-use present, while those at sites within 1.5 km showed disturbed profiles. Indeed, the humus types present at these locations suggest that the activity of soil invertebrates may be inhibited. This is almost certainly due primarily to the high concentrations of metals that were present in mineral soil and organic horizons at the sites. However, the potential influences of water regime and pH could also be relevant factors influencing the results of any further studies conducted at these study sites.

Environmental metabonomics: applying combination biomarker analysis in earthworms at a metal contaminated site.

Earthworms were taken across an environmental gradient of metal contamination for ecotoxicology assessment. Both indigenous (Lumbricus rubellus and L. terrestris) and introduced earthworms (Eisenia andrei, exposed in mesh bags) were studied. Changes in the levels of small molecule metabolites in earthworm tissue extracts were analysed by 1H NMR spectroscopy as a means of identifying combination biomarker compounds. Principal components analysis of the NMR spectral data revealed that biochemical changes were induced across the metal contamination gradient. Native worms (L. rubellus) from the most polluted sites were associated with an increase in the relative concentration of maltose; a decrease was also seen in the concentration of an as yet unidentified biomarker compound. Introduced worms (E. andrei) did not show differences to the same extent. Direct integration of the resonances from histidine and 1-methylhistidine showed that relative histidine concentrations were elevated slightly for L. rubellus, confirming the results of earlier mesocosm studies. Conversely, the relative concentrations of both histidine and 1-methylhistidine were greatly reduced by metal contamination in L. terrestris. This study demonstrates the utility of NMR spectroscopy in detecting previously unknown potential biomarkers for ecotoxicity testing and identified maltose as a potential biomarker compound deserving of further study.

Metal effects on soil invertebrate feeding: measurements using the bait lamina method.

Heightened concerns regarding the protection of terrestrial ecosystems at a national level has increased the need to develop a suite of indicators capable of assessing the quality, integrity and fertility of soils. Of the assays currently available, tests that measure aspects of soil function and associated parameters are among the most promising, since these integrate effects on soil quality at the highest level of organisation. In this study we describe results of the deployment of an indicator of soil functional integrity (the bait lamina test) that is designed to measure the feeding activity of soil invertebrates. Bait lamina was used at six grassland sites located along a transect from a smelter at Avonmouth (South-West England) used in the EU funded BIOPRINT II project. Results indicated highest bait removal (feeding) at sites furthest from the factory, intermediate feeding activity at intervening sites and extremely low activities at the two sites closest to the smelter. The strong decline in activity for the group of sites closest to the smelter corresponded with increasing metal concentrations suggesting a clear impact of metals on detritivorous invertebrate feeding. Comparisons of the results of the bait lamina study to previous invertebrate survey work suggested that the differences in observed bait removal can be attributed to direct effects of metals on the abundance and biodiversity of key decomposer groups such as earthworms, isopods, molluscs, myriapods, springtails and mites.

Critical analysis of soil invertebrate biomarkers: a field case study in Avonmouth, UK.

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.

Environmental tolerances of an invasive terrestrial amphipod, Arcitalitrus dorrieni (Hunt) in Britain.

We investigated key physiological tolerances of the invasive euterrestrial talitrid amphipod (or landhopper) Arcitalitrus dorrieni; desiccation, salt, high and low temperatures. The critical relative humidity below which, A. dorrieni experiences desiccation stress is very high (95-100%), making it completely reliant on the leaflitter/soil microhabitat. It is tolerant of a wide range of (sea) salt concentrations (5-750 mOsmol l(-1)) but is extremely vulnerable below 5 mOsmol l(-1). A. dorrieni does not tolerate low temperatures with a mean lower limit of 1.4 degrees C, but with no individual surviving <0 degrees C. The range of upper thermal tolerance (30-37.3 degrees C) was similar to that found for other landhopper and beachflea species. Based on its tolerance to these environmental factors it is suggested that A. dorrieni has a limited potential to invade further into Britain, being restricted to areas with sufficiently high ion concentrations and mild winters.

Quantifying copper and cadmium impacts on intrinsic rate of population increase in the terrestrial oligochaete Lumbricus rubellus.

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.

Closing the loop: a spatial analysis to link observed environmental damage to predicted heavy metal emissions.

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.

Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): identification of new endogenous biomarkers.

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.

Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei.

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at >or=1.8 micro g TNT cm(-2), whereas sublethal (weight loss) and lethal effects to earthworms were detected at >or=3.5 and 7.1 micro g TNT cm(-2), respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (>or=55 mg TNT kg(-1) soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO4, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.

Interspecific comparisons of relative assimilation efficiencies for zinc and cadmium in an ecological series of talitrid amphipods (Crustacea).

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.