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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.

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.

The aim of this study was to assess the impact of single-walled carbon nanotubes (SWCNT) on the toxicity of cadmium (Cd) using the crustacean Daphnia magna. LC50 of Cd and SWCNT alone and combined were calculated and compared. Sorption of Cd on SWCNT was also quantified in separate batch experiments. Results showed that the maximum adsorption of Cd onto SWCNT calculated by the Langmuir equation was 24.4 mg kg-1. LC50s for Cd and SWCNT alone were 252.3 µg L-1 and 1400 µg L-1, respectively. In the presence of 500 and 1000 µg L-1 of SWCNT, Cd LC50s were 127.2 and 120.1 µg L-1 respectively. Therefore, Cd toxicity increased when organisms were exposed to both contaminants which indicated that SWCNT induces a synergistic toxic effect on the survival of D. magna. It appears that even if SWCNT had a low adsorption capacity for Cd, toxicity of the metal can be increased. Our study shows the complexity of SWCNT toxicity and how the understanding of their interactions with other contaminants is crucial to determine the consequences of their release into the environment.

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 289 mg/kg. Results indicate that bismuth decreased significantly all reproduction parameters of Eisenia andrei at concentrations ≥ 116 mg/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 > 289 mg/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 21.37 mg/kg dry tissue for 243 mg 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 0.0229 mg/kg, while in soil with earthworms bismuth concentration ranged between 0.310–1.347 mg/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.

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. Environ Toxicol Chem 2014;33:308–316. © 2013 SETAC

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. Keywords: Bioavailability, Bioaccessibility, Biomarker index, Management tool, Model, Soil quality

Metallothioneins (MTs) are regarded as sensitive biomarkers of cadmium (Cd) exposure in a number of organisms. An isocratic high-performance liquid chromatography (HPLC) method using the fluorophore ammonium-7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate (SBD-F) and UV detection was developed for the quantification of MT-like proteins (MTLPs) in earthworm. This method was developed using a rabbit MT (MT-1) standard, and optimized concentrations of reagents including EDTA, SBD-F, and tris(2-carboxyethyl)phosphine (TCEP). The fluoro-tagged MT-1 (SBD-MT) and MTLP (SBD-MTLP) co-eluted at 7.2–7.3 min retention time on HPLC chromatograms. The optimized method was applied to quantify MTLPs in earthworms exposed to increasing concentrations of Cd (0, 2.7, 26.7, and 267.5 μg g−1 of wet artificial soil) for 1–4 weeks. The amounts of MTLPs increased significantly in a dose- and time-dependent manner except in controls and in earthworms exposed to the lowest concentration of Cd. Since earthworm MTLPs are temporal- and dose-responsive to Cd exposure, they can be used as biomarkers to assess the impact of Cd contamination in soils. The method appeared to be rapid, sensitive, and suitable for direct quantification of total earthworm MTs.

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 polynitroorganic 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. Crown Copyright Copyright 2003 Published by Elsevier Inc. All rights reserved.

Sublethal and chronic toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) on earthworm Eisenia andrei in a sandy forest soil were assessed. Various reproduction parameters of fecundity (total and hatched number of cocoons, number of juveniles, and their biomass) were significantly decreased by TNT (or = 58.8 +/- 5.1 mg/kg dry soil), RDX (or = 46.7 +/- 2.6 mg/kg), and HMX (or = 15.6 +/- 4.6 mg/kg). These effects occurred at much lower concentrations than those reported earlier using artificial soil preparations. Growth of adults was significantly decreased in the TNT-spiked natural soils at 136.2 +/- 25.6 mg/kg dry soil, the highest concentration having no significant mortality. In contrast, survival and growth were not significantly reduced at relatively high measured concentrations of RDX (167.3 mg/kg) and HMX (711.0 mg/kg). Although TNT, RDX, and HMX share a common life-cycle response ( i.e., decreased juvenile counts), a number of differences related to other reproduction parameters (e.g., productivity of cocoons) was observed. These results indicate that the tested explosives do not support a common mechanism of toxicity, at least in the earthworm, probably due to differences in their physical-chemical properties as well as metabolites formed during exposure.

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 gtoreq1.8 mug TNT cm-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at gtoreq3.5 and 7.1 mug TNT cm-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (gtoreq55 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. Cooper (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. (c) Biosciences Information Services.

The sublethal and chronic effects of the environmental contaminant and explosive octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in artificial soil were assessed using the earthworm ( Eisenia andrei ). Based on various reproduction parameters (total and hatched number of cocoons, number of juveniles and their biomass), fecundity was reduced at the different concentrations of HMX tested (from 280.0±12.3 to 2502.9±230.0 mg kg −1 dry soil) in spiked artificial soil (LOEC: 280.0±12.3 mg kg −1 dry soil). The growth of adult E. andrei was also reduced at the different concentrations tested, though no mortality occurred, even at the highest tested concentrations. The number of juveniles produced was correlated with the number of total and hatched cocoons, and the biomass of juveniles was correlated with the number of cocoons. Pooled results of these and earlier studies on explosives (TNT, RDX) using the E. andrei reproduction test confirm that effects of HMX on cocoon production are indicative of some reproductive consequences (number of juvenile and their biomass), whereas adult growth, in general, does not correlate strongly with change in reproduction capacity.

Septic tank sludge is regularly hauled to the Montreal Urban Community (MUC) wastewater treatment plant. It is then discharged and mixed with the wastewater inflow before entering the primary chemical treatment process. An ecotoxicological procedure integrating chemical and toxicological analyses has been recently developed and applied to screen for the illicit discharge of toxic substances in septic sludge. The toxicity tests used were the Microtox, the bacterial-respiration, and the lettuce (Lactuca sativa) root elongation tests. In order to validate the applicability of the proposed procedure, a two-year interlaboratory study was carried out. In general, the results obtained by two independent laboratories (MUC and the Centre d'expertise en analyse environnementale du Quebec) were comparable and reproducible. Some differences were found using the Microtox test. Organic (e.g., phenol and formaldehyde) and inorganic (e.g., nickel and cyanide) spiked septic sludge were detected with good reliability and high efficiency. The relative efficiency to detect spiked substances was > 70% and confirms the results of previous studies. In addition, the respiration test was the most efficient toxicological tool to detect spiked substances, whereas the Microtox was the least efficient (< 15%). Efficiencies to detect spiked contaminants were also similar for both laboratories. These results support previous data presented earlier and contribute to the validation of the ecotoxicological procedure used by the MUC to screen toxicity in septic sludge.

2,4,6-Trinitrotoluene (TNT) is an worldwide recalcitrant environmental contaminant and is toxic to a number of organisms including humans. This study examines the acute effects (lethal and biomass changes) of TNT on the oligochaetes species Eisenia andrei, using the 3-day filter paper, and the 7- and 14-day direct contact spiked soil (OECD artificial and forest soil) toxicity tests. Studies using the filter paper test indicated that the lethality of TNT could be detected in the range 1.5 to 14.2 microg/cm(2), with significant biomass (body weight) changes occurring at the lowest concentration. Acute effects (lethality) could not be measured when earthworms were placed on filter paper containing a saturated aqueous solution of TNT. This may indicate that with these exposure conditions, TNT may have been adsorbed to the filter paper, and that this matrix should be saturated with TNT before becoming available to the earthworms. Spiked soil toxicity tests indicated that the E. andrei lethality by TNT was >1.5 times higher when earthworms were exposed to TNT-spiked forest soil (LOEC:260 mg/kg; LC(50) 14 days 222.4 mg/kg) than to spiked OECD artificial soil (LOEC:420 mg/kg; LC(50) 14 days: 364.9 mg/kg). The sublethal effect on biomass change at the selected TNT concentrations in soil was not significant compared to controls. Results indicate that the bioanalytical methods described in this article could be used as TNT toxicity assessment tools. This soil quality test method gives valuable information for the screening of soil toxicity.

This paper presents an integrative approach, using toxicological and chemical analyses, to screen toxic and illegal substances that could be added to the septic sludge transported by a tanker truck to the wastewater treatment plant of the Montreal Urban Community (MUC). Microtox, lettuce root elongation, and a bacterial respiration test were used to establish the toxicity range of a normal sludge and the determination of threshold limit criteria. Septic sludge samples were spiked with different types and amounts of contaminants (copper, zinc, phenol, industrial sludge). Conservative criteria were applied to detect abnormal toxicity with great reliability while avoiding false positives (i.e., detecting abnormal toxicity in nonspiked sludge). Taken individually, toxicity tests using Microtox were revealed to be the least discriminating toxicological method (efficiency of 45% when the ratio of the IC50values is considered), whereas lettuce root elongation was relatively the most efficient (80% of spiked samples). As a whole, the battery of toxicity tests detected at least 93% of the spiked sludge samples. This procedure is also very efficient, i.e., easy to apply, cost effective, and rapid. In certain cases, an abnormal toxicity level can be determined within a few hours, whereas a septic sludge can be classified as normal within 5 days.

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.

Bismuth (Bi) is increasingly used in several industrial applications including the production of alloys, drugs, cosmetics and munitions formulations. However, little information is available on the environmental fate and ecotoxicological effects of Bi. The present study describes 14 days acute toxicity of Bi, added as Bi citrate to a natural sandy soil, to the adult earthworm Eisenia andrei. Total measured Bi concentrations were 298.0, 399.5, 431.0, and 469.5 mg Bi/kg dry soil. Data indicates that Bi was toxic to Eisenia andrei, as determined by LC50 and LOEC, i.e., 416.0 and 399.5 mg Bi/kg dry soil, respectively. At 14 days in the presence of Eisenia andrei the bioaccessible fraction of Bi in soil, as determined in KNO3 aqueous soil extracts, increased by a factor ranging from 1.6 to 30.0 compared to those measured at the beginning of experiment. Moreover, this study shows that an increase in pH caused by the presence of earthworm in soil was accompanied by increase in Bi bioaccessibility and consequently toxicity. For example, when Bi bioaccessibility increased from 0.262 to 7.516 mg Bi/kg dry soil, the mortality rate increased from 0 to 79%. Assuming that there were at least two routes by which Eisania andrei enhanced Bi bioaccessibility; one route was guided by the mobility, the biochemical (mucus) and the biological (bacteria) interactions of Eisenia andrei with soil constituents, and the other route was marked by the death of earthworms and the release of the accumulated Bi from the carcass.

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.

The heterocyclic polynitramine hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly energetic compound found as a soil contaminant at some defense installations. Although RDX is not lethal to soil invertebrates at concentrations up to 10,000 mg/kg, it decreases earthworm cocoon formation and juvenile production at environmentally relevant concentrations found at contaminated sites. Very little is known about the uptake of RDX in earthworms and the potential risks for food-chain transfer of RDX in the environment. Toxicokinetic studies were conducted to quantify the bioaccumulation factors (BAFs) using adult earthworms (Eisenia andrei) exposed for up to 14 d to sublethal concentrations of nonlabeled RDX or [14C]RDX in a Sassafras sandy loam soil. High-performance liquid chromatography of acetonitrile extracts of tissue and soil samples indicated that nonlabeled RDX can be accumulated by the earthworm in a concentration- and time-dependent manner. The BAF, expressed as the earthworm tissue to soil concentration ratio, decreased from 6.7 to 0.1 when the nominal soil RDX concentrations were increased from 1 to 10,000 mg/kg. Tissue concentrations were comparable in earthworms exposed to nonlabeled RDX or [14C]RDX. The RDX bioaccumulation also was estimated using the kinetically derived model (BAFK), based on the ratio of the uptake to elimination rate constants. The established BAFK of 3.6 for [14C]RDX uptake was consistent with the results for nonlabeled RDX. Radioactivity also was present in the tissue residues of [14C]RDX-exposed earthworms following acetonitrile extraction, suggesting the formation of nonextractable [14C]RDX metabolites associated with tissue macromolecules. These findings demonstrated a net accumulation of RDX in the earthworm and the potential for food-chain transfer of RDX to higher-trophic-level receptors.

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.

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.

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.

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.

Cement kiln dusts are made of a complex mixture of elements. We have evaluated the potential negative impact of those dusts on the immune system of the earthworm Lumbricus terrestris. We specifically studied cell viability and phagocytic activity of coelomocytes extruded during electrical stimulation. We used two modes of exposures: in vitro, and soil incubation using OECD artificial soil media. Extruded coelomocytes were exposed 18 h in vitro to 10, 100, and 500 mg L-1 of cement kiln dust particles. The phagocytosis and the cell viability were determined using a double-laser-flow acquisition cytometry system. Using the double laser allows us to use a dichlorofluorescein diacetate (DCFDA) marker to discriminate the biological cells from the cement kiln dusts. Dead cells are marked using propidium iodide (PI). All three exposure levels showed highly significant impacts on cell viability and phagocytic activity. The in vivo soil incubation was performed using 10, 100, and 1000 mg kg-1 of cement kiln dusts incorporated into the OECD media. Here, to discriminate the biological cells from the mineral dusts we only needed to use PI. The day-to-day variability of the in vivo assay was high and although we can observe an overall reduction in cell viability at the highest concentration tested, no statistically significant effects could be observed on either cell viability or phagocytosis. Copyright 2004 Elsevier Inc. All rights reserved.

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 1-1). 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. Crown Copyright Copyright 2004 Published by Elsevier Ltd. All rights reserved.

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 mug/g in artificial soil; IC50 = 61.8 mug/g in forest soil) was higher than that of Cu (IC50 = 13.7 mug/g in artificial Soil; IC50 > 322 mug/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 (ltoreq 11.2 mug Cr/g and ltoreq 5.3 mug Cr/g for artificial and natural soils, respectively) and roots (ltoreq 161 mug Cr/g and ltoreq 51.7 mug Cr/g for artificial and natural soils, respectively) and Cu residues in roots (ltoreq 61.8 mug Cu/g and ltoreq 91.3 mug Cu/g for artificial and natural soils, respectively). Cu concentration in shoot tissues was ltoreq 61.8 mug Cu/g. Since it may overestimate toxicity, effect and risk assessment using spiked soils, particularly in artificial soil, must be used with diligence. Copyright 2003 National Research Council of Canada. Published by Elsevier Inc. All rights reserved.

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.

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.

Toxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) using two terrestrial plant species, lettuce (Lactuca sativa) and barley (Hordeum vugare), was assessed in artificial soil (silica) and forest soil. Lettuce emergence was significantly decreased after 5 days of exposure to TNT nominal spiked concentrations/= 1,040 mg/kg dry soil in silica. Barley emergence was significantly reduced after 14 days of exposure at initial (t = 0) TNT concentrations/= 55.9 +/- 4.5 mg/kg dry soil in silica and at/= 291.9 +/- 42.8 mg/kg dry forest soil. Biomasses of shoot and roots of barley seeds were significantly reduced after 14 days of exposure at TNT initial exposure concentrations/= 55.9 +/- 4.5 (LOEC) mg/kg dry soil in silica. Results were similar with the forest soil (LOEC = 91.4 +/- 7.9 mg TNT/kg dry soil) using the root growth parameter, but the shoot biomass was reduced only at concentrations/= 291.9 +/- 42.8 mg TNT/kg dry soil. Plants were not affected by an HMX exposure up to 3,320 +/- 1,019 mg/kg dry soil using silica or 1,866 +/- 438 mg/kg dry soil using a forest soil. During the 14-day experiments, TNT was partially transformed in the spiked soil samples, as indicated by the presence of its amino metabolites (2-ADNT and 4-ADNT). Higher quantities of metabolites were detected in forest soils having higher initial TNT concentrations (/= 1,849.4 +/- 228.2 mg/kg) compared to silica (/= 239.3 +/- 88.0 mg TNT/kg). After 14 days, TNT concentrations in spiked silica and forest soil were reduced up to 80.5% at 55.9 +/- 4.5 mg/kg initial concentration and 94.4% at 91.4 +/- 7.9 mg/kg initial concentration, respectively. Data indicate that TNT is the probable phytotoxicant because it decreased plant emergence and growth in the presence and absence of the ADNT metabolites.

The use of chemical deicers such as sodium chloride (NaCl) has increased significantly during the past three decades. Deicers induce metal corrosion and alter the physicochemical properties of soils and water. Environmental damage caused by the use of NaCl has prompted government agencies to find alternative deicers. This article presents a comparative ecotoxicological study of three deicers on soil organisms. Sodium formiate (NaFo) and calcium-magnesium acetate (CMA) are the most interesting commercially available deicers based upon their characteristics and potential toxicity. Organisms used in this study were four species of macrophytes (cress (Lepidium sativum), barley (Ordeum vulgare), red fescue grass (Festuca rubra), Kentucky bluegrass (Poa pratensis)) and an invertebrate (Eisenia fetida). Using standardized and modified methods, the relative toxicity of deicers was CMA < NaFo congruent with NaCl. The results demonstrate that these chemicals could have similar impacts in terrestrial environments since similar quantities of NaFo and greater amounts of CMA are necessary to achieve the same efficiency as NaCl. The toxicity of the tested substances was lower in natural composted soil than in artificial substrate (silica or OECD soil), indicating decreased environmental bioavailability. The response of the organisms changed according to endpoint, species, and soil characteristics (artificial substrate as compared to natural organic soil). The most sensitive endpoint measured was macrophyte growth with Kentucky bluegrass being the most sensitive species.

An innovative screening procedure has been developed to detect illicit toxic discharges in domestic septic tank sludge hauled to the Montreal Urban Community waste-water treatment plant. This new means of control is based on an integrative approach, using bioassays and chemical analyses. Conservative criteria are applied to detect abnormal toxicity with great reliability while avoiding false positive results. The complementary data obtained from toxicity tests and chemical analyses support the use of this efficient and easy-to-apply procedure. This study assesses the control procedure in which 231 samples were analyzed over a 30-month period. Data clearly demonstrate the deterrent power of an efficient control procedure combined with a public awareness campaign among the carriers. In the first 15 months of application, between January 1996 and March 1997, approximately 30% of the 123 samples analyzed showed abnormal toxicity. Between April 1997 and June 1998, that is, after a public hearing presentation of this procedure, this proportion dropped significantly to approximately 9% based on 108 analyzed samples. The results of a 30-month application of this new control procedure show the superior efficiency of the ecotoxicological approach compared with the previously used chemical control procedure. To be able to apply it effectively and, if necessary, to apply the appropriate coercive measures, ecotoxicological criteria should be included in regulatory guidelines.

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

Tenth Symposium on Environmental Toxicology and Risk Assessment, Toronto, Ontario, Canada, April 10-12, 2000, Series: ASTM special technical publication

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.

This article presents an integrative approach, using toxicological and chemical analyses, to screen for toxic substances that could be illegally added to the chemical-toilet sludge received at the wastewater treatment plant of the Montreal urban community. Four toxicity tests (Microtox, bacterial-respiration, root-elongation, and seed-germination tests) were used to establish the toxicity range of a "normal" sludge and the determination of threshold limits criteria. Chemical-toilet sludge samples were spiked with two types and amounts of contaminants (zinc, phenol). Conservative criteria were used to detect abnormal toxicity with great reliability and avoid false positives (i.e., detecting abnormal toxicity in nonspiked sludge). Taken individually, the seed-germination test was the least discriminating toxicological method (detecting only 10% of the spiked samples); the bacterial-respiration test was relatively better (detecting 72% of the spiked samples). Using a limited battery of two toxicity tests (Microtox and respiration test), the identification of contaminated chemical-toilet sludge can be detected with good efficiency and possibly great reliability (more than 80% of the spiked samples). This proposed procedure is efficient, easy to apply, cost-effective, and very fast (an abnormal toxicity level can be determined within a few hours).