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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 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

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.

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.

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.

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

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).