Agnès Y. Renoux

Ecotoxicity characterization of dinitrotoluenes and some of their reduced metabolites

In the present study, the toxic effects of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT) and a selection of their respective metabolites were examined and compared to 2,4,6-trinitrotoluene (TNT) using the 15-min Microtox (Vibrio fischen) and 96-h freshwater green alga (Selenastrum capricomutum) growth inhibition tests. All of the compounds tested were less toxic than TNT. Using the Microtox assay, 2,6-DNT was more toxic than 2,4-DNT and the order of toxicity for 2,6-DNT and its metabolites was: 2,6-DNT > or = 2A-6NT >> 2,6-DAT; whereas that for 2,4-DNT was: 4A-2NT > 2A-4NT > 2,4-DNT > 2,4-DAT. For the algal test, 2,4-DNT was more toxic than 2,6-DNT and the order of toxicity for 2,4-DNT and its metabolites was: 2,4-DNT > 2,4-DAT approximately equal to 4A-2NT = 2A-4NT. The order of toxicity for 2,6-DNT and its reduced metabolites using the algal test was very similar to the Microtox bioassay. These results demonstrate that the reduced metabolites of 2,6-DNT tested in this study were less toxic than that of the parent compound, but certain partially reduced metabolites of 2,4-DNT can be more toxic than the parent molecule. These data put into question the general hypothesis that reductive metabolism of nitro-aromatics is associated with a sequential detoxification process.

Transformation of 2,4,6-trinitrotoluene in soil in the presence of the earthworm Eisenia andrei

The ability of the earthworm Eisenia andrei to metabolize 2,4,6-trinitrotoluene (TNT) was studied in experiments with TNT-spiked soils, dermal contact tests, and with an in vitro assay. Lethality of TNT in a forest sandy soil was first determined (14-d LC50 5 143 mg/kg). Then TNT at lethal and sublethal concentrations was applied to the same soil and was monitored along with its metabolites in extracts of soil and earthworm tissue for up to 14 d postapplication. High performance liquid chromatography– ultra violet analyses indicated that TNT was transformed in the presence of E. andrei by a reductive pathway to 2-amino-4,6dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT), and traces of 2,6-diamino-4-nitrotoluene (2,6-DANT) in earthworm tissues. This transformation could be explained by either a metabolic mechanism within the earthworm or by the enhancement of an earthworm-associated microbial activity or both. The TNT concentrations decreased from the spiked soils. However, the monoamino-dinitrotoluene (2-ADNT and 4-ADNT) concentrations increased with exposure duration and were dependent on the initial TNT soil concentrations. This was also observed to a lesser extent in the TNTspiked soils with no earthworms present. The biotransformation of TNT into 2-ADNT, 4-ADNT, and 2,4-DANT and the presence of these metabolites in E. andrei after dermal contact on TNT-spiked filter paper showed that dermal uptake can be a significant exposure route for TNT. In vitro experiments showed that earthworm homogenate could metabolize TNT and form 2-ADNT and 4-ADNT at room temperature and at 378C. This effect was inhibited by heat inactivation prior to incubation or by incubation at 48C, suggesting that the biotransformation of TNT in the presence of E. andrei may be enzymatic in nature. Keywords—TNT Earthworm Biotransformation Metabolites Soil

Lethal and subchronic effects of 2,4,6-trinitrotoluene (TNT) on Enchytraeus albidus in spiked artificial soil.

The effects of 2,4,6-trinitrotoluene (TNT) exposure in spiked artificial soil on the survival and reproduction rate of the white potworm Enchytraeus albidus were studied. Based on the initial concentrations, TNT in freshly spiked soil decreased enchytraeid survival (21-day LC(50)=422+/-63 (SD)mg/kg, N=3) and fecundity (42-day EC(50)=111+/-34, N=4). Data also indicated that TNT was 5-10 times more lethal to juveniles than adults, and lethality was less pronounced in TNT-spiked soils aged for 21 days. A time-dependent decrease in the TNT concentrations, as well as a concomitant increase in the levels of 2- and 4-aminodinitrotoluene, was observed during the 42-day toxicity test. Taken together, TNT (or one of its metabolites) is more lethal to juvenile than adult enchytraeids. This effect may explain, at least in part, the ability of TNT to decrease fecundity as determined using the enchytraeid mortality-reproduction test.

Derivation of Environmental Soil Quality Guidelines for 2,4,6-Trinitrotoluene Using the CCME Approach

The Protocol for the Derivation of Environmental and Human Health Soil Quality Guidelines of the Canadian Council of Ministers of the Environment was used to prepare preliminary Environmental Soil Quality Guidelines (SQGE) for 2,4,6-trinitrotoluene (TNT). A thorough literature search led to the compilation of the existing toxicological data. Calculated Environmental Soil Quality Guidelines for TNT are SQGE=0.02 mg/kg (preliminary value) for agricultural land use and 86 mg/kg for residential/park, commercial/industrial land use. Because of the lack of sufficient scientific information on the effects of TNT in soil on microbial processes, this type of evaluation was not possible. For oral toxicological data, laboratory animals were used instead of grazing and foraging species to determine the ingestion guideline since no related literature was found. Furthermore, this work has led to the identification of avenues of future research necessary to complete the task at hand. The research should focus on specific studies involving direct contact between the organisms and the soil, in order to: (1) develop a database of effects of TNT on microbial processes, (2) study the effects of TNT ingestion on birds and grazing herbivores, (3) determine plant bioconcentration factors, and (4) observe in situ the toxic effects after direct contact exposure.

Risk-Based Management of Site Soils Contaminated with a Mixture of Hazardous Substances: Methodological Approach and Case Study

ABSTRACT A novel approach to improve the accuracy and to reduce the uncertainty associated with the assessment of ecotoxicological risks and the determination of remedial objectives was developed herein for a scenario involving multiple contaminants in soil. This approach used laboratory-derived site-specific toxicological data (i.e., obtained from toxicity testing using species in direct contact with soil such as plants and invertebrates) instead of the more traditional approach using generic toxicological benchmarks for corresponding groups of organisms. Inherent to this approach were the data exploration and reduction; the use of generalized linear models to integrate the data for stressors (site-specific chemical and edaphic characteristics with the potential for influencing toxicity) and effects (biological responses for multiple species and multiple endpoints); and the derivation of tools that could predict the level of impairment associated with any combination of metals’ concentrations measured on site and compare it to a pre-specified acceptable threshold. A case study is presented whereby this method was applied to a large site contaminated with a mixture of metals. Ultimately, the distributions of predicted levels of risk for both soil invertebrates and plants were determined for the entire site and compared to those obtained using the traditional approach using benchmarks.