Assessment of acute and chronic toxicity of unweathered and weathered diluted bitumen to freshwater fish and invertebrates.

This paper presents the results of two different studies investigating the acute and chronic toxicity of two blends of diluted bitumens ("dilbit") and weathered dilbit on freshwater fish and invertebrates after exposure to different concentrations of physically-dispersed (water accommodated fraction; WAF) and chemically-dispersed (chemically-enhanced WAF; CEWAF). The first study evaluated the acute and chronic toxicity of weathered, unweathered and dispersed Access Western Blend (AWB) dilbit on Fathead minnow (Pimephales promelas). In the second study, acute and chronic toxicity of weathered and unweathered Cold Lake Blend (CLB) dilbit was assessed on Rainbow trout (Oncorhynchus mykiss), and two invertebrate species, Daphnia (Daphnia magna) and Ceriodaphnia (Ceriodaphnia dubia). For Fathead minnow, unweathered AWB WAF demonstrated a significantly higher acute toxicity (LC50-96 h = 0.628 g/L) compared to the weathered AWB WAF (LC50-96 h = 2.06 g/L). Chronic toxicity tests showed that Fathead minnow lethality was also higher for unweathered AWB (LC50-7 d = 0.593 g/L) compared to the weathered AWB (LC50-7 d = 1.31 g/L) whereas larval growth toxicity was lower for unweathered AWB (IC25-7 d=0.312 g/L) compared to the weathered dilbit (IC25-7 d = 0.096 g/L). Rainbow trout exposed to unweathered CLB demonstrated a significantly higher toxicity (LC50-96 h = 5.66 g/L) compared to the weathered CLB (LC50 > 18 g/L). Lethality (LC50 = 6.43 g/L) was observed in Ceriodaphnia exposed to the CLB WAF while no mortality was observed with the weathered CLB. The reproductive effects on Ceriodaphnia were greater with the CLB (IC25 <1.0) than with the weathered CLB (IC25 = 3.99 g/L). Volatile organic compounds (VOC), polycyclic aromatic hydrocarbons (PAH) and total petroleum hydrocarbons (TPH) increased as the dilbit CLB and AWB WAF concentrations increased. The total VOC and the total PAHs (including the alkylated PAHs) presented similar concentrations in the weathered and unweathered AWB WAF. These compounds seem therefore not be affected by the AWB WAF weathering process. However, VOC and PAH concentrations decreased significantly in the Rainbow trout and Ceriodaphnia toxicity tests using the weathered CLB WAF. VOC and PAH concentrations were also lower in the weathered AWB CEWAF used for Fathead minnow chronic study.

Immunotoxicity and genotoxicity of single-walled carbon nanotubes co-exposed with cadmium in the freshwater mussel, Elliptio complanata.

Potential immunotoxicity and genotoxicity of as-produced and purified single walled carbon nanotubes (SWCNT, 500 µg L-¹) with or without cadmium (20 µg L-¹) was investigated in hemocytes of the freshwater mussel, Elliptio complanata. Our results showed a decrease in hemocyte viability after 3, and 8 days of exposure and an increase of hemocyte phagocytic efficiency for organisms exposed to Cd. No modification of the cyclo-oxygenase (COX) activity was measured. An increase in DNA damage was measured after 1 day of exposure to Cd and a potentiating effect of combined exposures was observed.

Bioavailability and chronic toxicity of bismuth citrate to earthworm Eisenia andrei exposed to natural sandy soil.

The present study describes bioavailability and chronic effects of bismuth to earthworms Eisenia andrei using OECD reproduction test. Adult earthworms were exposed to natural sandy soil contaminated artificially by bismuth citrate. Average total concentrations of bismuth in soil recovered by HNO3 digestion ranged from 75 to 289mg/kg. Results indicate that bismuth decreased significantly all reproduction parameters of Eisenia andrei at concentrations ≥ 116mg/kg. However, number of hatched cocoons and number of juveniles seem to be more sensitive than total number of cocoons, as determined by IC50; i.e., 182, 123 and > 289mg/kg, respectively. Bismuth did not affect Eisenia andrei growth and survival, and had little effect on phagocytic efficiency of coelomocytes. The low immunotoxicity effect might be explained by the involvement of other mechanisms i.e. bismuth sequestered by metal-binding compounds. After 28 days of exposure bismuth concentrations in earthworms tissue increased with increasing bismuth concentrations in soil reaching a stationary state of 21.37mg/kg dry tissue for 243mg Bi/kg dry soil total content. Data indicate also that after 56 days of incubation the average fractions of bismuth available extracted by KNO3 aqueous solution in soil without earthworms varied from 0.0051 to 0.0229mg/kg, while in soil with earthworms bismuth concentration ranged between 0.310-1.347mg/kg dry soil. We presume that mucus and chelating agents produced by earthworms and by soil or/and earthworm gut microorganisms could explain this enhancement, as well as the role of dermal and ingestion routes of earthworms uptake to soil contaminant.

Single-Walled Carbon Nanotubes Toxicity to the Freshwater Amphipod Hyalella Azteca: Influence of to the Freshwater Amphipod Sediment and Exposure Duration.

Carbon nanomaterials are present in various industrial applications and therefore their release into the environment including freshwater ecosystem is expected to increase. The aim of the present study was to investigate the influence of several parameters on the toxicity of single-walled carbon nanotubes (SWCNT) to the freshwater amphipod, Hyalella azteca. The effect of period of exposure, sediment presence and possible impurities released during SWCNT preparation on survival and/or growth of such organism was evaluated. We measured a reduction of survival at concentrations ranging from 10 to 40 mg/L after 96-h exposure, while no mortality was observed with the same concentrations and in the presence of artificial sediment after 14 days of exposure. It is possible that SWCNT are adsorbed on the organic matter from the artificial sediment leading to a decrease of SWCNT bioavailability. The survival and growth toxicity tests revealed a stronger effect at 28 days compared to the 14 days of exposure, and full mortality of organisms at 1000 mg/L for both exposure times. The presence of SWCNT in the gut of survived organisms was observed. The present study demonstrates that the interaction with sediment should be considered when carbon nanotubes toxicity through water exposure is investigated.

Estimation of the bioaccumulation potential of a nonchlorinated bisphenol and an ionogenic xanthene dye to Eisenia andrei in field-collected soils, in conjunction with predictive in silico profiling.

In silico-based model predictions, originating from structural and mechanistic (e.g., transport, bioavailability, reactivity, and binding potential) profiling, were compared against laboratory-derived data to estimate the bioaccumulation potential in earthworms of 2 organic substances (1 neutral, 1 ionogenic) known to primarily partition to soil. Two compounds representative of specific classes of chemicals were evaluated: a nonchlorinated bisphenol containing an -OH group (4,4'-methylenebis[2,6-di-tert-butylphenol] [Binox]), and an ionogenic xanthene dye (2',4',5',7'-tetrabromo-4,5,6,7-tetrachloro-3',6'-dihydroxy-, disodium salt [Phloxine B]). Soil bioaccumulation studies were conducted using Eisenia andrei and 2 field-collected soils (a clay loam and a sandy soil). In general, the in silico structural and mechanistic profiling was consistent with the observed soil bioaccumulation tests. Binox did not bioaccumulate to a significant extent in E. andrei in either soil type; however, Phloxine B not only accumulated within tissue, but was not depurated from the earthworms during the course of the elimination phase. Structural and mechanistic profiling demonstrated the binding and reactivity potential of Phloxine B; this would not be accounted for using traditional bioaccumulation metrics, which are founded on passive-based diffusion mechanisms. This illustrates the importance of profiling for reactive ionogenic substances; even limited bioavailability combined with reactivity can result in exposures to a hazardous substance not predictable by traditional in silico modeling methods.

Ecotoxicity of xanthene dyes and a non-chlorinated bisphenol in soil.

Soil eco-toxicity testing was conducted in support of Canada's Chemical Management Plan (CMP) to fill data gaps for organic chemicals known to primarily partition to soil, and of which the persistence and inherent toxicity are uncertain. Two compounds representative of specific classes of chemicals: non-chlorinated bisphenols containing an -OH group (4,4'-methylenebis(2,6-di-tert-butylphenol (Binox)) and xanthene dyes (2',4',5',7'-tetrabromo-4,5,6,7-tetrachloro-3',6'-dihydroxy-, disodium salt (Phloxine B), 2',4',5',7'-tetrabromofluorescein (TBF), 4',5'-dibromofluorescein (DBF), and 4,5,6,7-tetrachlorofluorescein (TCF)) were evaluated. The effect of these substances on plant growth (Elymus lanceolatus and Trifolium pratense) and soil invertebrate survival and reproduction (Folsomia candida and Eisenia andrei) were assessed using a field-collected sandy soil. Binox was persistent throughout testing (up to 63 d) with an average recovery of 77±2.9% at test end. Binox was not toxic to plants (IC50s>1076 mg kg(-1)) or E. andrei (IC50s>2651 mg kg(-1)); however, a significant reduction in F. candida adult survival and reproduction (IC50=89 (44-149) mg kg(-1)) was evident. Phloxine B was also persistent throughout testing, with an average recovery of 82±3.0% at test end. Phloxine B was significantly more toxic than Binox, with significant reductions in plant root growth (IC50s ≥ 11 mg kg(-1)) and invertebrate reproduction (IC50s ≥ 22 mg kg(-1)). DBF toxicity was not significantly different from that of Phloxine B for plant root growth (IC50s ≥ 30 mg kg(-1)), but was significantly less toxic for shoot growth (IC50s ≥ 1758 mg kg(-1)), and invertebrate adult survival (IC50s ≥ 2291 mg kg(-1)) and reproduction (IC50s ≥ 451 mg kg(-1)). A comparison between all four xanthene dyes was completed using F. candida, with the degree of toxicity in the order of Phloxine B ≥ TBF∼DBF>TCF. The results from these studies will contribute to data gaps for poorly understood chemicals (and chemical groupings) under review for environmental risk assessments, and will aid in the validation of model predictions used to characterize the fate and effects of these substances in soil environments.

Assessment of soil quality using bioaccessibility-based models and a biomarker index.

Bioavailability in heterogeneous media such as soils is a multi-factorial concept which ranges from soil chemistry to toxicity. The complexity of this factor has been tackled by various studies pinpointing its relevancy for laboratory to field extrapolation of toxicity data. As contaminant bioavailability on these sites is virtually unknown, a global assessment of this issue has been conducted on soils impacted by antitank firing from a Canadian Range and Training Area (RTA) and contaminated by energetic materials (EM) and metals. Yet, the descriptive results acquired from this survey require further in-depth analysis so as to enhance understanding of soil health status. Statistical models as well as an index integrating biomarker responses were derived from this database and are proposed as diagnostic, explanatory and possibly predictive tools for soil bioavailability and quality assessment. Relationships associating bioaccessible contaminant levels to soil properties allowed to clarify contaminant behaviour in energetic material (EM)-contaminated soils. Likewise, models expressing biomarker responses as a function of bioaccessible contaminant concentrations contributed to identify the contaminants causing toxicity in earthworms and to the comprehension of those toxic effects. The index of biomarker response was adapted from similar concepts applied in the aquatic environment and is an original contribution to terrestrial sites. The biomarker index data were in agreement with soil contamination profiles and represent therefore an interesting tool for soil quality appraisal. Such tools also offer a promising potential for the management of contaminated soils.

Integration of toxicological and chemical tools to assess the bioavailability of metals and energetic compounds in contaminated soils.

Bioavailability is critical for understanding effects that might result from exposure of biota to contaminated soils. Soils from military range and training areas (RTAs) are contaminated principally by energetic materials (EM) and metals. Their chemical characteristics are relatively well known and toxicity assessment of soils from RTAs are in some cases available. However, bioavailability on these sites needs to be comprehensively characterized. A holistic approach to bioavailability, incorporating both chemical and earthworm toxicological indicators, was applied to soils from an anti-tank firing range at a Canadian Forces Base. Results showed that HMX and the metals Zn, Pb, Bi and Cd, though not consistently the prevailing toxicants, were the most accessible to earthworms. Some metals (notably Cu, Zn, Cr and Bi) were also accumulated in earthworm tissue but those were not necessarily expected given their bioaccessibility (i.e., the chemical availability of contaminants in the environment for the organisms) at the beginning of the exposure. The tested soils impaired earthworm reproduction and reduced adult growth. Measurement of selected sublethal parameters indicated that lysosomal integrity (determined as the neutral red retention time--NRRT) was decreased, while elevated superoxide dismutase (SOD) activity suggested that earthworms experienced oxidative stress. The correspondence between the NRRT and metal contamination pattern suggested that metals may be the main cause of lysosomal disruption in EM-contaminated soils. The approach to bioavailability appraisal adopted in this case appears to be a promising practice for site-specific assessment of contaminated land.

Effects of HMX-lead mixtures on reproduction of the earthworm Eisenia andrei.

High metal (e.g., Pb) concentrations are typically found in explosive-contaminated soil, and their presence may increase, decrease, or not influence toxicity predicted on the basis of one explosive alone (e.g., HMX). Nevertheless, few data are available in the scientific literature for this type of multiple exposure. Soil organisms, such as earthworms, are one of the first receptors affected by the contamination of soil. Therefore, a reproductive study was conducted using Eisenia andrei in a forest-type soil. Both HMX and Pb decreased reproduction parameters (number of total cocoons, hatched cocoons, and surviving juveniles) individually. Based on the total number of cocoons, HMX was more toxic in a forest soil than Pb, with EC(50) of 31 mg kg(-1), and 1068 mg kg(-1), respectively. The slope of the concentration-response curve was significantly greater in the case of Pb, which is consistent with the possibility that the two compounds do not act on the same target site. The response-addition model was used to predict the response of earthworms and to test for interaction between the two contaminants. The predicted toxicity was not significantly different than the observed toxicity, implying that Pb and HMX were considered noninteractive compounds. The combined action of Pb-HMX may be described, therefore, as dissimilar-noninteractive joint action in a forest soil. The results illustrate the relevance of considering the presence of metals in the risk assessment of explosive-contaminated sites because metals can add their toxicity to explosives. Extension of this study to other types of soil and other metals would improve the understanding of toxicity at these sites.

Photosynthetic parameters as indicators of trinitrotoluene (TNT) inhibitory effect: change in chlorophyll a fluorescence induction upon exposure of lactuca sativa to TNT.

Trinitrotoluene, as a compound of conventional explosive, may cause inhibitory effect on terrestrial plants. When Lactuca sativa was exposed to different concentrations of trinitrotoluene (32-1000 mg/kg), photosynthetic process was investigated by using rapid chlorophyll fluorescence kinetic and pulse modulated fluorometry. The decrease of chlorophyll a variable fluorescence was seen to be caused by the deactivation of photosystem II reaction centers. We found for rapid variable fluorescence to be a useful indicator to evaluate the inhibitory effect of trinitrotoluene on photosystem II primary photochemistry and electron transport. The fluorescence parameters, related to the reduction state of photosystem II and to non-photochemical dissipation of light energy, showed a strong relation between the inhibitory effect of photosystem II activity and concentration of trinitrotoluene. The change of photosynthetic fluorescence parameters induced by trinitrotoluene was a reliable indication of the plant physiological state. We proposed for the reduction state of photosystem II and the non-photochemical energy dissipation to be a useful tool in bioassay toxicity testing of trinitrotoluene polluted soil.

Assessment of soil toxicity from an antitank firing range using Lumbricus terrestris and Eisenia andrei in mesocosms and laboratory studies.

Earthworm mesocosms studies were carried out on a explosives-contaminated site at an antitank firing range. Survival of earthworms and the lysosomal neutral red retention time (NRRT), a biomarker of lysosomal membrane stability, were used in these studies to assess the effect of explosives-contaminated soils on the earthworms Lumbricus terrestris and Eisenia andrei under field conditions. Toxicity of the soils samples for E. andrei was also assessed under laboratory conditions using the earthworms reproduction test and the NRRT. Results indicate that the survival was reduced up to 40% in certain explosive-contaminated soil mesocosms following 10 days of exposure under field conditions, whereas survival was reduced up to 100% following 28 days of exposure under laboratory conditions. Reproduction parameters such as number of cocoons and number of juveniles were reduced in many of the selected contaminated soils. Compared to the reference, NRRT was significantly reduced for E. andrei exposed to explosive-contaminated soils under both field and laboratory conditions, whereas for L. terrestris NRRT was similar compared to the reference mesocosm. Analyses showed that HMX was the major polynitro-organic compound in soils. HMX was also the only explosive detected in earthworm tissues. Thus, results from both field mesocosms and laboratory studies, showed lethal and sub-lethal effects associated to soil from the contaminated area of the antitank firing range.

Preliminary ecotoxicological characterization of a new energetic substance, CL-20.

A new energetic substance hexanitrohexaazaisowurtzitane (or CL-20) was tested for its toxicities to various ecological receptors. CL-20 (epsilon-polymorph) was amended to soil or deionized water to construct concentration gradients. Results of Microtox (15-min contact) and 96-h algae growth inhibition tests indicate that CL-20 showed no adverse effects on the bioluminescence of marine bacteria Vibrio fischeri and the cell density of freshwater green algae Selenastrum capricornutum respectively, up to its water solubility (ca. 3.6 mg l(-1)). CL-20 and its possible biotransformation products did not inhibit seed germination and early seedling (16-19 d) growth of alfalfa (Medicago sativa) and perennial ryegrass (Lolium perenne) up to 10,000 mg kg(-1) in a Sassafras sandy loam soil (SSL). Indigenous soil microorganisms in SSL and a garden soil were exposed to CL-20 for one or two weeks before dehydrogenase activity (DHA) or potential nitrification activity (PNA) were assayed. Results indicate that up to 10,000 mg kg(-1) soil of CL-20 had no statistically significant effects on microbial communities measured as DHA or on the ammonium oxidizing bacteria determined as PNA in both soils. Data indicates that CL-20 was not acutely toxic to the species or microbial communities tested and that further studies are required to address the potential long-term environmental impact of CL-20 and its possible degradation products.

Toxicity assessment of contaminated soils from an antitank firing range.

Explosives are released into the environment at production and processing facilities, as well as through field use. These compounds may be toxic at relatively low concentrations to a number of ecological receptors. A toxicity assessment was carried out on soils from an explosive-contaminated site at a Canadian Forces Area Training Center. Toxicity studies on soil organisms using endpoints such as microbial processes (potential nitrification activity, dehydrogenase activity, substrate-induced respiration, basal respiration), plant seedling and growth (Lactuca sativa and Hordeum vulgare), and earthworm (Eisenia andrei) growth and reproduction were carried out. Results showed that 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX) was the principal polynitro-organic compound measured in soils. Soils from the contaminated site decreased microbial processes and earthworm reproduction; whereas plant growth was not significantly reduced. Toxicity to aquatic organisms and genotoxicity were also assessed on soil elutriates using Microtox (Vibrio fischeri), growth inhibition of algae (Selenastrum capricornutum), and SOS Chromotest (Escherichia coli). Results indicated that soil elutriates were generally not toxic to bacteria (Microtox) and algae. However, genotoxicity was found in a number of soil elutriate samples. Thus, the explosive-contaminated soils from the antitank firing range may represent a hazard for the soil organisms. Nevertheless, the global toxicity might have partially resulted from HMX as well as from other (not identified) contaminants such as heavy metals.

Acute and chronic toxicity of the new explosive CL-20 to the earthworm (Eisenia andrei) exposed to amended natural soils.

Monocyclic nitramine explosives such as 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are toxic to a number of ecological receptors, including earthworms. The polycyclic nitramine CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) is a powerful explosive that may replace RDX and HMX, but its toxicity is not known. In the present study, the lethal and sublethal toxicities of CL-20 to the earthworm (Eisenia andrei) are evaluated. Two natural soils, a natural sandy forest soil (designated RacFor2002) taken in the Montreal area (QC, Canada; 20% organic carbon, pH 7.2) and a Sassafras sandy loam soil (SSL) taken on the property of U.S. Army Aberdeen Proving Ground (Edgewood, MD, USA; 0.33% organic carbon, pH 5.1), were used. Results showed that CL-20 was not lethal at concentrations of 125 mg/kg or less in the RacFor2002 soil but was lethal at concentrations of 90.7 mg/kg or greater in the SSL soil. Effects on the reproduction parameters such as a decrease in the number of juveniles after 56 d of exposure were observed at the initial CL-20 concentration of 1.6 mg/kg or greater in the RacFor2002 soil, compared to 0.2 mg/kg or greater in the SSL soil. Moreover, low concentrations of CL-20 in SSL soil (approximately 0.1 mg/kg; nominal concentration) were found to reduce the fertility of earthworms. Taken together, the present results show that CL-20 is a reproductive toxicant to the earthworm, with lethal effects at higher concentrations. Its toxicity can be decreased in soils favoring CL-20 adsorption (high organic carbon content).

Phytotoxicity and bioaccumulation of copper and chromium using barley (Hordeum vulgare L.) in spiked artificial and natural forest soils.

The toxicities of two heavy metals, copper (Cu2+) and chromium (Cr6+), to barley (Hordeum vulgare L.) were evaluated using two types of substrates: artificial and natural forest soils. Phytotoxicity was assessed using a standardized toxicity test. Endpoints included plant emergence and shoot and root growth. Shoot and root concentrations of Cu and Cr were also measured. Data indicated that the root biomass was the most sensitive endpoint. The results showed that toxicity of Cr to root growth (IC50=6.6 microg/g in artificial soil; IC50=61.8 microg/g in forest soil) was higher than that of Cu (IC50=13.7 microg/g in artificial soil; IC50>322 microg/g in forest soil). Data also indicated that the toxicity of Cu and Cr was significantly decreased in the spiked forest soil, suggesting lower metal bioavailability to barley in the natural soil. Analysis of tissue concentrations in barley showed that Cu and Cr were mainly accumulated in the roots. Toxicity was correlated with Cr residues in shoots (< or =11.2 microg Cr/g and < or =5.3 microg Cr/g for artificial and natural soils, respectively) and roots (< or =161 microg Cr/g and < or =51.7 microg Cr/g for artificial and natural soils, respectively) and Cu residues in roots (< or =61.8 microg Cu/g and < or =91.3 microg Cu/g for artificial and natural soils, respectively). Cu concentration in shoot tissues was < or =61.8 microg Cu/g. Since it may overestimate toxicity, effect and risk assessment using spiked soils, particularly in artificial soil, must be used with diligence.

Immune response of earthworms (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) following in situ soil exposure to atmospheric deposition from a cement factory.

In order to reduce their energy costs, many cement plants use fuel product substitutes (old tyres and used oil). The combustion of these products generates a metal increase (e.g. Cu, Cd, Pb and Zn) in the atmospheric emissions. After their release, these elements are deposited into the environment and could eventually accumulate up to concentrations of concern. At the Saint-Laurent cement factory (Joliette, QC, Canada), maximum deposition of these elements occurs in the direction of prevailing winds (North-East). We evaluated the potential impact of these depositions upon the immune system of three earthworm species (Lumbricus terrestris, Eisenia andrei and Aporrectodea tuberculata) exposed in a natural environment. The exposure sites were 0.5, 1.0, and 2.0 km downwind from the cement factory, along with an upwind reference site. The immune parameters studied were the cell viability and phagocytic potential of the immune cells (coelomocytes). For both L. terrestris and E. andrei, after 7 d exposure, none of the measured parameters showed significant differences among the sites. On the other hand, for the indigenous worm A. tuberculata, in the most exposed zone (at 0.5 km), we observed an increase in cell viability and phagocytic potential. This increase could possibly be attributed to physicochemical effects such as the alkaline pH of the soil, or alternatively, it could result from beneficial effects induced by an increased calcium supply.

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.