Semifield testing of a bioremediation tool for atrazine-contaminated soils: evaluating the efficacy on soil and aquatic compartments

The present study evaluated the bioremediation efficacy of a cleanup tool for atrazine-contaminated soils (Pseudomonas sp. ADP plus citrate [P. ADP?+?CIT]) at a semifield scale, combining chemical and ecotoxicological information. Three experiments representing worst-case scenarios of atrazine contamination for soil, surface water (due to runoff), and groundwater (due to leaching) were performed in laboratory simulators (100 × 40 × 20 cm). For each experiment, three treatments were set up: bioremediated, nonbioremediated, and a control. In the first, the soil was sprayed with 10 times the recommended dose (RD) for corn of Atrazerba and with P. ADP?+?CIT at day 0 and a similar amount of P. ADP at day 2. The nonbioremediated treatment consisted of soil spraying with 10 times the RD of Atrazerba (day 0). After 7 d of treatment, samples of soil (and eluates), runoff, and leachate were collected for ecotoxicological tests with plants (Avena sativa and Brassica napus) and microalgae (Pseudokirchneriella subcapitata) species. In the nonbioremediated soils, atrazine was very toxic to both plants, with more pronounced effects on plant growth than on seed emergence. The bioremediation tool annulled atrazine toxicity to A. sativa (86 and 100% efficacy, respectively, for seed emergence and plant growth). For B. napus, results point to incomplete bioremediation. For the microalgae, eluate and runoff samples from the nonbioremediated soils were extremely toxic; a slight toxicity was registered for leachates. After only 7 d, the ecotoxicological risk for the aquatic compartments seemed to be diminished with the application of P. ADP?+?CIT. In aqueous samples obtained from the bioremediated soils, the microalgal growth was similar to the control for runoff samples and slightly lower than control (by 11%) for eluates.     

Increased alanine concentration is associated with exposure to fenitrothion but not carbamates in Chironomus riparius larvae

The polychlorinated insecticide toxaphene belonged to the most used pesticides in the 20th century. Even recently, significant residues have been found in soils at various sites in the world. However, knowledge on toxicity to soil organisms is limited. In this study, the effects of toxaphene on soil invertebrates Folsomia candida, Eisenia fetida, Enchytraeus albidus, Enchytraeus crypticus, Caenorhabditis elegans, and microorganisms were investigated. Among the organisms tested, F. candida was the most sensitive. The 50% effect on survival and reproduction output (LC50 and EC50) was found at concentrations of 10.4 and 3.6 mg/kg, respectively. Sensitivity of other organisms was significantly lower with effective concentrations at tens or hundreds of mg/kg. Our data on soil toxicity were recalculated to soil pore-water concentrations and good accordance with available data reported for aquatic toxicity was found. Since soil concentrations at some sites are comparable to concentrations effective in our tests, toxaphene may negatively affect soil communities at these sites.     

Ecotoxicology and water quality

Fundamental concepts of ecotoxicology are presented, making specific reference to the aquatic environment. As far as exposure to contaminants is concerned, the main properties of a substance that determine its presence in the various environmental compartments and matrices are illustrated and discussed. The crucial importance of the bioaccumulation potential is stressed, as it can be responsible for the level of substance reached in the biota, which can be much higher than that present in the external environment. It is also underlined the importance of bioavailability processes, which tune the chemical fraction available for absorption by organisms. The toxic effects on biota are illustrated at the different levels of biological organization they can be observed. Last, the role of the ecotoxicological investigation within an integrated approach to the water quality assessment is discussed along with the specific information provided.     

Improving ecological risk assessment in the Mediterranean area: selection of reference soils and evaluating the influence of soil properties on avoidance and reproduction of two oligochaete species

A current challenge in soil ecotoxicology is the use of natural soils as test substrates to increase ecological relevance of data. Despite the existence of six natural reference soils (the Euro-soils), some parallel projects showed that these soils do not accurately represent the diversity of European soils. Particularly, Mediterranean soils are not properly represented. To fill this gap, 12 natural soils from the Mediterranean regions of Alentejo, Portugal; Catalu?a, Spain; and Liguria, Italy, were selected and used in reproduction and avoidance tests to evaluate the soil habitat function for earthworms (Eisenia andrei) and enchytraeids (Enchytraeus crypticus). Predictive models on the influence of soil properties on the responses of these organisms were developed using generalized linear models. Results indicate that the selected soils can impact reproduction and avoidance behavior of both Oligochaete species. Reproduction of enchytraeids was affected by different soil properties, but the test validity criteria were fulfilled. The avoidance response of enchytraeids was highly variable, but significant effects of texture and pH were found. Earthworms were more sensitive to soil properties. They did not reproduce successfully in three of the 10 soils, and a positive influence of moisture, fine sand, pH, and organic matter and a negative influence of clay were found. Moreover, they strongly avoided soils with extreme textures. Despite these limitations, most of the selected soils are suitable substrates for ecotoxicological evaluations.     

Avoidance tests in site-specific risk assessment--influence of soil properties on the avoidance response of Collembola and earthworms

The ability of organisms to avoid contaminated soils can act as an indicator of toxic potential in a particular soil. Based on the escape response of earthworms and Collembola, avoidance tests with these soil organisms have great potential as early screening tools in site-specific assessment. These tests are becoming more common in soil ecotoxicology, because they are ecologically relevant and have a shorter duration time compared with standardized soil toxicity tests. The avoidance response of soil invertebrates, however, can be influenced by the soil properties (e.g., organic matter content and texture) that affect behavior of the test species in the exposure matrix. Such an influence could mask a possible effect of the contaminant. Therefore, the effects of soil properties on performance of test species in the exposure media should be considered during risk assessment of contaminated soils. Avoidance tests with earthworms (Eisenia andrei) and springtails (Folsomia candida) were performed to identify the influence of both organic matter content and texture on the avoidance response of representative soil organisms. Distinct artificial soils were prepared by modifying quantities of the standard artificial soil components described by the Organization for Economic Co-operation and Development to achieve different organic matter and texture classes. Several combinations of each factor were tested. Results showed that both properties influenced the avoidance response of organisms, which avoided soils with low organic matter content and fine texture. Springtails were less sensitive to changes in these soil constituents compared with earthworms, indicating springtails can be used for site-specific assessments of contaminated soils with a wider range of respective soil properties.     

Conceptual model for improving the link between exposure and effects in the aquatic risk assessment of pesticides

Assessment of risks to aquatic organisms is important in the registration procedures for pesticides in industrialised countries. This risk assessment consists of two parts: (i) assessment of effects to these organisms derived from ecotoxicological experiments (=effect assessment), and (ii) assessment of concentration levels in relevant environmental compartments resulting from pesticide application (=exposure assessment). Current procedures lack a clear conceptual basis for the interface between the effect and exposure assessments which may lead to a low overall scientific quality of the risk assessment. This interface is defined here as the type of concentration that gives the best correlation to ecotoxicological effects and is called the ecotoxicologically relevant concentration (ERC). Definition of this ERC allows the design of tiered effect and exposure assessments that can interact flexibly and efficiently. There are two distinctly different exposure estimates required for pesticide risk assessment: that related to exposure in ecotoxicological experiments and that related to exposure in the field. The same type of ERC should be used consistently for both types of exposure estimates. Decisions are made by comparing a regulatory acceptable concentration (=RAC) level or curve (i.e., endpoint of the effect assessment) with predicted environmental concentration (=PEC) levels or curves (endpoint of the exposure assessment). For decision making based on ecotoxicological experiments with time-variable concentrations a tiered approach is proposed that compares (i) in a first step single RAC and PEC levels based on conservative assumptions, (ii) in a second step graphically RAC and PEC curves (describing the time courses of the RAC and PEC), and (iii) in a third step time-weighted average RAC and PEC levels.     

Comparative toxicity of chlorothalonil: Ceriodaphnia dubia and Pimephales promelas

Chlorothalonil is a commonly used fungicide in rural and urban environments and can be incidentally introduced into aquatic systems through rainfall runoff or direct overspray and drift from aerial applications. Few studies have been published regarding risks to aquatic organisms exposed to chlorothalonil, so this study was performed to provide a first-order risk characterization for receiving system biota. Definitive laboratory toxicity tests were conducted with aqueous solutions of chlorothalonil and sentinel aquatic organisms (Ceriodaphnia dubia Richard and Pimephales promelas Rafinesque). P. promelas was more sensitive (7-day potency=6.1% mortality/mug/L) than C. dubia (7-day potency=0.94% mortality/mug/L) to chlorothalonil exposures. All mortality of P. promelas and C. dubia resulting from these chlorothalonil exposures occurred within the first 96h and no sublethal effects (i.e., growth or reproduction) were detected under these experimental conditions following 7-day exposures.     

Field and laboratory studies to assess the effects of Vertimec(®) 18EC on Daphnia similis

The present study aimed to evaluate the interactions of the pesticide Vertimec(?) 18EC in aquatic ecosystems. In this respect, soil plots were contaminated with Vertimec(?) 18EC at the concentration indicated for strawberry crops (0.125L of solution m(-2)). After the contamination, torrential rainfall was simulated and the surface runoff was collected and transferred to mesocosm tanks in five treatments, run in triplicate: (1) control-C; (2) runoff from an uncontaminated plot-UR; (3) runoff from the plot contaminated with Vertimec(?) 18EC-CR; (4) direct application of Vertimec(?) 18EC in the water-V and (5) water samples gathered randomly to verify whether there was contamination between the mesocosms-RS. Water samples from these tanks were also submitted to ecotoxicological tests with Daphnia similis and analyses to evaluate the limnological characteristics, in five collection periods over 10 days (240h). Physical and chemical differences were observed in the water samples, mainly related to increased turbidity, suspended solids and nutrients (nitrogen and phosphate forms). Acute toxicity was observed for the direct application treatment for the entire experimental period, and in some periods for the CR treatment (from 48h to 168h). The results obtained suggest that the pesticide did not fully degrade during the study period (10 days) in the direct application treatment, demonstrating that the presence of other substances in the commercial formulation contribute to the maintenance of toxicity. This represents a potential risk for aquatic ecosystems in areas adjacent to where the chemical is applied.     

Toxicity of the pesticide alpha-cypermethrin to four soil nontarget invertebrates and implications for risk assessment

Alpha-cypermethrin, a synthetic pyrethroid, is used as an insecticide in agricultural settings and is increasingly replacing organophosphates and carbamates because of lower application rates and lower toxicity to mammals. Because very little is known about the acute and chronic toxicity of this compound for soil-living organisms, the present study investigated acute and sublethal toxicity of alpha-cypermethrin for four terrestrial invertebrate species in an agricultural soil from Norway. Bioassays with the earthworm Eisenia fetida, the potworm Enchytraeus crypticus, the springtail Folsomia candida, and the land snail Helix aspersa were performed according to slightly modified versions of Organization for Economic Cooperation and Development (Paris, France) or International Organization for Standardization (Geneva, Switzerland) guidelines and resulted in median lethal concentrations of greater than >1,000 to 31.4 mg/kg and sublethal no-observed-effect concentrations of 2.51 to 82 mg/kg. A high acute to chronic ratio was found, especially in the earthworms. Interspecies differences in sensitivity may be explained by differences in exposure and differences in metabolization rate. When based on measured pore-water concentrations, terrestrial species overall appear to be approximately one order of magnitude less sensitive than aquatic species. Effect assessments conducted according to European guideline for risk assessment of pesticides reveal that assessments based on acute toxicity tests are not always conservative enough to determine environmentally safe concentrations in soil. Mandatory incorporation of sublethal toxicity data will ensure that in regions with temperate climate, the effects of pesticides on populations of soil-living organisms are unlikely.     

Chronic toxicity of polycyclic aromatic compounds to the springtail Folsomia candida and the enchytraeid Enchytraeus crypticus

An urgent need exists for incorporating heterocyclic compounds and (bio)transformation products in ecotoxicological test schemes and risk assessment of polycyclic aromatic compounds (PACs). The aim of the present study therefore was to determine the chronic effects of (heterocyclic) PACs on two terrestrial invertebrates, the springtail Folsomia candida and the enchytraeid Enchytraeus crypticus. The effects of 11 PACs were determined in chronic experiments using reproduction and survival as endpoints. The results demonstrated that as far as narcosis-induced mortality is concerned, effects of both homocyclic and heterocyclic PACs are well described by the relationship between estimated pore-water 50% lethal concentrations and log Kow. In contrast, specific effects on reproduction varied between species and between compounds as closely related as isomers, showing up as deviations from the relationship between pore-water 50% effect concentrations and log Kow. These unpredictable specific effects on reproduction force one to test the toxicity of these PACs to populations of soil invertebrates to obtain reliable effect concentrations for use in risk assessment of PACs.     

Toxicity assessment of contaminated soils from an antitank firing range

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

Avoidance tests with earthworms and springtails: defining the minimum exposure time to observe a significant response

Based on the ability of organisms to avoid contaminated soils, avoidance tests have a great potential as early screening tools in lower tier levels of ERA schemes. Aiming at their standardization, the definition of the minimum exposure time necessary to observe an avoidance response to a contaminant is needed. To fill this gap, avoidance tests with earthworms (Eisenia andrei) and springtails (Folsomia candida), comparing distinct time periods (from 1-7 to 1-14 days, respectively), were performed using the artificial OECD soil and reference chemicals for each test organism. Results showed that for both organisms a clear response within 24 h of exposure can be obtained. This rapid response enhances the utility of the test for "on site" analysis to evaluate contaminated sites.     

Single- and two-species tests to study effects of the anthelmintics ivermectin and morantel and the coccidiostatic monensin on soil invertebrates

Soil invertebrates in arable land are potentially exposed to veterinary medicines excreted by husbandry. The toxicity of three widely used pharmaceuticals was therefore investigated with the use of common soil invertebrates exposed in the laboratory in single- or two-species test system. The anthelmintic morantel did not cause significant mortality to either Folsomia fimetaria or Enchytraeus crypticus even at the highest tested concentration of 900 mg kg(-1) dry soil. The coccidiostatic monensin affected the reproduction of F. fimetaria and E. crypticus with soil concentrations estimated to cause a 10% effect at values of approximately 109 and 71.8 mg kg(-1) dry soil, respectively, but caused no mortality to adult. The anthelmintic ivermectin did not affect the survival of adult Hypoaspis aculeifer. Reproduction of H. aculeifer declined approximately 45% in response to ivermectin exposure of 5 mg kg(-1) dry soil. Ivermectin was highly toxic to F. fimetaria and affected the survival of adults with soil concentrations estimated to cause a 50% mortality at values of 5.3 mg kg(-1) dry soil in the single-species test system and 0.14 mg kg(-1) dry soil in the two-species test system. Reproduction of F. fimetaria was reduced by ivermectin with 10% effective concentration at 0.19 mg kg(-1) dry soil in the single-species test system and 0.02 mg kg(-1) dry soil in two-species test system. It was shown that species interactions may influence the response of test organisms to toxic substances. The data from this study and previously published data showed that, whereas ivermectin is likely to pose a risk to soil-dwelling invertebrates, adverse effects of morantel and monensin are unlikely to occur as a result of residue excretion from treated farm animals.     

Potential toxicity concerns from chemical coagulation treatment of stormwater in the Tahoe basin,California, USA

Coagulant dosing of stormwater runoff with polyaluminum chlorides (PACs) is used in numerous waterbodies to improve water clarity, but the potential risks of PACs to aquatic organisms in Lake Tahoe, California are not fully understood. To assess these risks, the USEPA 3-species toxicity test and a non-standard fish test using Japanese medaka (Oryzias latipes) were used to determine the toxicity of PAC-treated and non-treated stormwater samples to aquatic species. Stormwater samples were collected from three sites representing runoff from different urbanized areas in May 2004; samples received coagulant dosing using three different coagulants (JC1720, PAX-XL9, Sumalchlor50) at levels optimized with jar testing. Raw stormwaters were toxic to algae and fathead minnows (mortality). Treatment with coagulants increased toxicity to zooplankton (reproduction) and had no consistent effects on the other toxicity metrics.     

Weathering and aging of 2,4,6-trinitrotoluene in soil increases toxicity to potworm Enchytraeus crypticus

Energetic materials are employed in a wide range of commercial and military activities and often are released into the environment. Scientifically based ecological soil-screening levels (Eco-SSLs) are needed to identify contaminant explosive levels in soil that present an acceptable ecological risk. Insufficient information for 2,4,6-trinitrotoluene (TNT) to generate Eco-SSLs for soil invertebrates necessitated toxicity testing. We adapted the standardized Enchytraeid Reproduction Test and selected Enchytraeus crypticus for these studies. Tests were conducted in Sassafras sandy loam soil, which supports relatively high bioavailability of TNT. Weathering and aging procedures for TNT amended to test soil were incorporated into the study design to produce toxicity data that better reflect the soil exposure conditions in the field compared with toxicity in freshly amended soils. This included exposing hydrated TNT-amended soils in open glass containers in the greenhouse to alternating wetting and drying cycles. Definitive tests showed that toxicity for E. crypticus adult survival and juvenile production was increased significantly in weathered and aged soil treatments compared with toxicity in freshly amended soil based on 95% confidence intervals. The median effect concentration and 20% effective concentration for reproduction were 98 and 77 mg/kg, respectively, for TNT freshly amended into soil and 48 and 37 mg/kg, respectively, for weathered and aged TNT soil treatments. These findings of increased toxicity to E. crypticus in weathered and aged TNT soil treatments compared with exposures in freshly amended soils show that future investigations should include a weathering and aging component to generate toxicity data that provide more complete information on ecotoxicological effects of energetic contaminants in soil.     

Factors affecting soil residues of dieldrin, endosulfan, gamma-HCH, dimethoate, and pyrolan

The factors which determine the fate of pesticide residues in soils are discussed in relation to published research and to our own work with a range of temperate and tropical soils. Pesticide characteristics, soil type, climatic conditions, methods of application, and crop husbandry ought to be considered before pesticide application, since they determine the rate of pesticide disappearance from the soil. Degradation products of organophosphorus and carbamate compounds in the soil may be as toxic as the parent materials and can exhibit considerable persistence. A toxic metabolite of dimethoate was detected 2 years after a field had last been treated with the pesticide. Under laboratory conditions, metabolites of pyrolan were detected in soils and leachates more than 300 days after the parent compound was applied. Chlorinated hydrocarbons such as γ-HCH and endosulfan slowly degrade in soils to less or nontoxic products. The multiple use of pesticides in agriculture may cause long-term adverse effects on nontarget organisms. To minimize environmental hazards, pesticides should be used in the smallest quantities which would be effective against the target organisms, using seed treatment, soil treatment or controlled placement of pesticides. Spraying should be used only when alternative methods have proved unsuccessful. The review reveals a dearth of quantitative data on the relative importance of the different routes of pesticide loss from soils.     

Ecotoxicological effects of diuron and chlorotoluron nitrate-induced photodegradation products: monospecific and aquatic mesocosm-integrated studies

The ecotoxicological impact of nitrate-induced photodegradation products of diuron and chlorotoluron was studied through monospecific biotests conducted in conjunction with experiments in outdoor aquatic mesocosms. Organisms representing three trophic levels were used: two heterotrophic microorganisms, the luminescent bacterium Vibrio fischeri and the ciliated protozoa Tetrahymena pyriformis, and one metazoa, the gastropod Lymnaea stagnalis. Among the variety of the phenylurea photoproducts, the N-formylated ones appeared clearly more toxic than the parent compounds towards the microorganisms, whereas the nitroderivatives showed a similar toxicity. Using photodegraded solutions of diuron, toxicity was maintained or even increased during disappearance of the initial herbicide, demonstrating that some of the photoproducts may have an impact additively or in synergy. Enzymatic biomarker assays performed on Lymnaea stagnalis exposed under monospecific conditions showed significant effects, due to the combination of nitrate with the pesticide and its photoproducts. A positive impact on snail fecundity was observed with chlorotoluron both under monospecific laboratory and integrated mesocosm conditions. Oviposition stimulation took place when first- and second-generation photoproducts were predominant.     

A stepwise procedure for assessment of the microbial respiratory activity of soil samples contaminated with organic compounds

Soil respiration measurements are used frequently for the characterization of soil samples. Identical methods are used for the ecotoxicological characterization of contaminated soil samples as well as for quantification of the active microbial biomass in agriculturally used soils. In this study four soil samples contaminated with large amounts of volatile organic compounds, polyaromatic hydrocarbons, or nitroaromatic compounds are characterized after stepwise addition of carbon, nitrogen, and phosphorus. The respiration kinetics are assessed over a period of 5 days. By means of qualitative evaluation of the results, it is demonstrated that this stepwise addition allows one to distinguish between growth-promoting effects of biodegradable organic compounds on the one hand and the toxic influence of these compounds on the other hand. Finally it is stated that a comprehensive ecotoxicological characterization cannot be performed routinely using only one or several parameters of respiration curves. There is need for further research and validation if soil respiration measurements are to be performed quantitatively in the future.     

Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test

Current recommended ecotoxicological tests with the parthenogenetic springtail Folsomia candida using standard OECD soil do not allow for continuous monitoring during the exposure period. Effects of chemicals cannot be determined until the end of the experiment (typically after 4 weeks), since the animals stay below the soil surface. In this study, F. candida were maintained on a plaster of Paris/graphite substrate for 7 weeks and were supplied with an aqueous suspension of yeast contaminated with Cd, Cu, Pb, and Zn as nitrate salts. Growth rate, time to first batch of eggs, quantity of food consumed, and the presence of graphite in the gut (a sign of avoidance of yeast) were all affected by metal contaminated diets. The relative toxicities of Cd:Cu:Pb:Zn in the yeast were 1.0:1.07:12.0:4.3, respectively (on a weight basis) with Cd being the most toxic. Internal body concentrations increased, and the concentration factor (metal concentration in F. candida/metal concentration in yeast) decreased with increasing metal exposure. In general, metals are much less toxic when added to the food of F. candida than when incorporated into soil in standard tests. It is suggested that Collembola have a greater tolerance of metals in the diet since they avoid contaminated food, and are able to excrete assimilated metals at moulting via exfoliation of the midgut epithelium where the elements are retained as part of a storage--detoxification system. The methodology described in this article allows effects on growth to be observed as early as 7 days after the beginning of the experiment.     

Toxicities of dinitrotoluenes and trinitrobenzene freshly amended or weathered and aged in a sandy loam soil to Enchytraeus crypticus

Scientifically based ecological soil-screening levels are needed to identify concentrations of contaminant energetic materials (EMs) in soil that present an acceptable ecological risk at a wide range of military installations. Insufficient information regarding the toxicity of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), and 1,3,5-trinitrobenzene (TNB) to soil invertebrates necessitated toxicity testing. We adapted the standardized Enchytraeid Reproduction Test (International Standardization Organization 16387:2003) and selected Enchytraeus crypticus for these studies. Tests were conducted in Sassafras sandy loam soil, which supports relatively high bioavailability of nitroaromatic EMs. Weathering and aging procedures for EMs amended to test soil were incorporated into the study design to produce toxicity data that better reflect the soil exposure conditions in the field compared with toxicity in freshly amended soils. This included exposing hydrated, EM-amended soils in open glass containers in the greenhouse to alternating wetting and drying cycles. Definitive tests established that the order of EM toxicity to E. crypticus based on the median effect concentration values for juvenile production in either freshly amended or weathered and aged treatments was (from the greatest to least toxicity) TNB > 2,4-DNT > 2,6-DNT. Toxicity to E. crypticus juvenile production was significantly increased in 2,6-DNT weathered and aged soil treatments compared with toxicity in freshly amended soil, based on 95% confidence intervals. This result shows that future investigations should include a weathering and aging component to generate toxicity data that provide more complete information regarding ecotoxicological effects of energetic contaminants in soil.