Assessment of a 2,4,6-trinitrotoluene-contaminated site using Aporrectodea rosea and Eisenia andrei in mesocosms.

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

Immunotoxicological response of the earthworm Lumbricus terrestris following exposure to cement kiln dusts.

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.

Phytotoxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in spiked artificial and natural forest soils.

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 (

TNT, RDX, and HMX decrease earthworm (Eisenia andrei) life-cycle responses in a spiked natural forest soil.

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.

Interlaboratory study for the validation of an ecotoxicological procedure to monitor the quality of septic sludge received at a wastewater treatment plant.

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.

Chronic toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in soil determined using the earthworm (Eisenia andrei) reproduction test.

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.

Ecotoxicological evaluation of three deicers (NaCl, NaFo, CMA)-effect on terrestrial organisms.

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.

Assessment of the application of an ecotoxicological procedure to screen illicit toxic discharges in domestic septic tank sludge.

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.

Acute toxicity of 2,4,6-trinitrotoluene in earthworm (Eisenia andrei).

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.

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

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.

Procedure to screen illicit discharge of toxic substances in septic sludge received at a wastewater treatment plant.

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 IC50 values 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.

Screening of illicit toxic substances discharged in chemical toilet sludge.

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).