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.     

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 (≥ 58.8 ± 5.1 mg/kg dry soil), RDX (≥ 46.7 ± 2.6 mg/kg), and HMX (≥ 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. Received: 16 November 2001/Accepted: 2 June 2002     

Synergetic Toxic Effect of an Explosive Material Mixture in Soil

Explosives materials are stable in soil and recalcitrant to biodegradation. Different authors report that TNT (2,4,6-trinitrotoluene), RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) are toxic, but most investigations have been performed in artificial soil with individual substances. The aim of the presented research was to assess the toxicity of forest soil contaminated with these substances both individually as well in combinations of these substances. TNT was the most toxic substance. Although RDX and HMX did not have adverse effects on plants, these compounds did cause earthworm mortality, which has not been reported in earlier research. Synergistic effects of explosives mixture were observed.     

Acute and chronic toxicity of the new explosive CL-20 to the earthworm (Eisenia andrei) exposed to amended natural soils

Monocyclic nitramine explosives such as 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are toxic to a number of ecological receptors, including earthworms. The polycyclic nitramine CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) is a powerful explosive that may replace RDX and HMX, but its toxicity is not known. In the present study, the lethal and sublethal toxicities of CL-20 to the earthworm (Eisenia andrei) are evaluated. Two natural soils, a natural sandy forest soil (designated RacFor2002) taken in the Montreal area (QC, Canada; 20% organic carbon, pH 7.2) and a Sassafras sandy loam soil (SSL) taken on the property of U.S. Army Aberdeen Proving Ground (Edgewood, MD, USA; 0.33% organic carbon, pH 5.1), were used. Results showed that CL-20 was not lethal at concentrations of 125 mg/kg or less in the RacFor2002 soil but was lethal at concentrations of 90.7 mg/kg or greater in the SSL soil. Effects on the reproduction parameters such as a decrease in the number of juveniles after 56 d of exposure were observed at the initial CL-20 concentration of 1.6 mg/kg or greater in the RacFor2002 soil, compared to 0.2 mg/kg or greater in the SSL soil. Moreover, low concentrations of CL-20 in SSL soil (approximately 0.1 mg/kg; nominal concentration) were found to reduce the fertility of earthworms. Taken together, the present results show that CL-20 is a reproductive toxicant to the earthworm, with lethal effects at higher concentrations. Its toxicity can be decreased in soils favoring CL-20 adsorption (high organic carbon content).     

Survival and reproduction of enchytraeid worms, Oligochaeta, in different soil types amended with energetic cyclic nitramines

Hexanitrohexaazaisowurtzitane (CL-20), a new polycyclic polynitramine, has the same functional nitramine groups (N-NO2) as the widely used energetic chemicals hexahydro-1,3,5-trinitro-1,3,5-triazacyclohexane (royal demolition explosive [RDX]) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high-melting explosive [HMX]). Potential impacts of CL-20 as an emerging contaminant must be assessed before its use. The effects of CL-20, RDX, or HMX on adult survival and juvenile production by potworms Enchytraeus albidus and Enchytraeus crypticus were studied in three soil types, including Sassafras sandy loam (1.2% organic matter [OM], 11% clay, pH 5.5), an agricultural soil (42% OM, 1% clay, pH 8.2), and a composite agricultural-forest soil (23% OM, 2% clay, pH 7.9) by using ISO method 16387 (International Standard Organization, Geneva, Switzerland). Results showed that CL-20 was toxic to E. crypticus with median lethal concentration values for adult survival ranging from 0.1 to 0.7 mg/kg dry mass (DM) when using the three tested soils. In addition, CL-20 adversely affected juvenile production by both species in all soils tested, with median effective concentration (EC50) values ranging from 0.08 to 0.62 mg/kg DM. Enchytraeus crypticus and E. albidus were similarly sensitive to CL-20 exposure in the composite agricultural-forest soil, which supported reproduction by both species and enabled comparisons. Correlation analysis showed weak or no relationship overall among the soil properties and reproduction toxicity endpoints. Neither RDX nor HMX affected (p > 0.05) adult survival of either species below 658 and 918 mg/kg DM, respectively, indicating that CL-20 is more toxic to enchytraeids than RDX or HMX. Examination of data shows that CL-20 should be considered as a potential reproductive toxicant to soil invertebrates, and that safeguards should be considered to minimize the potential for release of CL-20 into the environment.     

Ecotoxicological characterization of energetic substances using a soil extraction procedure.

The acetonitrile-sonication extraction method (US EPA SW-846 Method 8330) and aquatic-based toxicity tests were used on laboratory and field samples, to characterize the ecotoxicity of soils contaminated with energetic substances. Spiked soil studies indicated that 2,4, 6-trinitrotoluene (TNT)-dependent soil toxicity could be measured in organic extracts and aqueous leachates using the 15-min Microtox (Vibrio fischeri, IC50=0.27 to 0.94 mg TNT/liter incubation medium) and 96-h Selenastrum capricornutum growth inhibition (IC50=0.62 to 1. 14 mg/liter) toxicity tests. Analyses of leachates of composite soil samples [containing TNT and some TNT metabolites, 1,3,5-trinitro-1,3, 5-triazacyclohexane (RDX), and 1,3,5,7-tetranitro-1,3,5, 7-tetrazacyclooctane (HMX)] from an explosives manufacturing facility, indicated toxicities similar to those found in the TNT-spiked soil studies and pure TNT in solution, and suggested that TNT was the major toxicant. Using TNT as a model toxicant in soils having different moisture contents (20% vs dry) and textures (sandy vs clayey-sandy) but similar organic matter content (3-4%), multi-factorial analyses of Microtox test data revealed that these soil factors significantly influenced the TNT extractability from soil and subsequent toxicity measurements. Taken together, data indicate that the modified Method 8330 may be used in conjunction with ecotoxicity tests to reflect the toxic potential of soils contaminated with energetic substances.     

Desorption of nitramine and nitroaromatic explosive residues from soils detonated under controlled conditions

Potentially toxic nitroaromatic and nitramine compounds are introduced onto soils during detonation of explosives. The present study was conducted to investigate the desorption and transformation of explosive compounds loaded onto three soils through controlled detonation. The soils were proximally detonated with Composition B, a commonly used military explosive containing 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro 1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). Gas-exchangeable surface areas were measured from pristine and detonated soils. Aqueous batches of detonated soils were prepared by mixing each soil with ultrapure water. Samples were collected for 141 d and concentrations of Composition B compounds and TNT transformation products 2-amino-4,6-dinitrotoluene (2ADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and 1,3,5-trinitrobenzene (1,3,5-TNB) were measured. The RDX, HMX, and TNT concentrations in detonated soil batches exhibited first-order physical desorption for the first, roughly, 10 d and then reached steady state apparent equilibrium within 40 d. An aqueous batch containing powdered Composition B in water was sampled over time to quantify TNT, RDX, and HMX dissolution from undetonated Composition B particles. The TNT, RDX, and HMX concentrations in aqueous batches of pure Composition B reached equilibrium within 6, 11, and 20 d, respectively. Detonated soils exhibited lower gas-exchangeable surface areas than their pristine counterparts. This is likely due to an explosive residue coating on detonated soil surfaces, shock-induced compaction, sintering, and/or partial fusion of soil particles under the intense heat associated with detonation. Our results suggest that explosive compounds loaded to soils through detonation take longer to reach equilibrium concentrations in aqueous batches than soils loaded with explosive residues through aqueous addition. This is likely due to the heterogeneous interactions between explosive residues and soil particle surfaces.     

Effects of HMX-Lead Mixtures on Reproduction of the Earthworm Eisenia Andrei

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 EC₅₀ 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.     

Ecological risk assessment of contaminated soils through direct toxicity assessment

A microcosm (MS-3) with a multispecies soil system is introduced as an experimental tool for direct toxicity assessment of contaminated soils. The capacity of MS-3 to determine soil ecotoxicity potential was evaluated using samples from three sites contaminated with organic and/or inorganic compounds. Soils were toxic to soil-dwelling organisms (earthworm, plants, and microorganisms) and to aquatic organisms (algae and RTG-2 cell fish). As expected, responses varied substantially among different soils and organisms. The application of this evaluation system provided complementary information to the chemical characterization. For soils containing metals the toxic response was lower than predicted from total metal concentrations. For hydrocarbons, the toxicity response agreed with estimated values. The induction of EROD activity suggested the presence of dioxin-like compounds, which had not been addressed in the chemical characterization. The proposed multispecies system affords the measurement of 11 endpoints covering three soil and three aquatic taxonomic groups, reproduces soil conditions and gradients, and appears as an excellent complementary tool to chemical analysis for characterization of contaminated sites.     

Toxicity and bioaccumulation of biosolids-borne triclosan in terrestrial organisms

Triclosan (TCS) is a common constituent of personal care products and is frequently present in biosolids. Application of biosolids to land transfers significant amounts of TCS to soils. Because TCS is an antimicrobial and is toxic to some aquatic organisms, concern has arisen that TCS may adversely affect soil organisms. The objective of the present study was to investigate the toxicity and bioaccumulation potential of biosolids-borne TCS in terrestrial micro- and macro-organisms (earthworms). Studies were conducted in two biosolids-amended soils (sand, silty clay loam), following U.S. Environmental Protection Agency (U.S. EPA) guidelines. At the concentrations tested herein, microbial toxicity tests suggested no adverse effects of TCS on microbial respiration, ammonification, and nitrification. The no observed effect concentration for TCS for microbial processes was 10?mg/kg soil. Earthworm subchronic toxicity tests showed that biosolids-borne TCS was not toxic to earthworms at the concentrations tested herein. The estimated TCS earthworm lethal concentration (LC50) was greater than 1?mg/kg soil. Greater TCS accumulation was observed in earthworms incubated in a silty clay loam soil (bioaccumulation factor [BAF]?=?12?±?3.1) than in a sand (BAF?=?6.5?±?0.84). Field-collected earthworms had a significantly smaller BAF value (4.3?±?0.7) than our laboratory values (6.5-12.0). The BAF values varied significantly with exposure conditions (e.g., soil characteristics, laboratory vs field conditions); however, a value of 10 represents a reasonable first approximation for risk assessment purposes.     

Effects of earthworm (Eisenia fetida) and wheat (Triticum aestivum) straw additions on selected properties of petroleum-contaminated soils

Current bioremediation techniques for petroleum-contaminated soils are designed to remove contaminants as quickly and efficiently as possible, but not necessarily with postremediation soil biological quality as a primary objective. To test a simple postbioremediation technique, we added earthworms (Eisenia fetida) or wheat (Triticum aestivum) straw to petroleum land-farm soil and measured biological quality of the soil as responses in plant growth, soil respiration, and oil and grease (O&G) and total petroleum hydrocarbon (TPH) concentrations. Results indicated that plant growth was greater in earthworm-treated land-farm soil. Furthermore, addition of wheat straw resulted in greater total respiration in all soils tested (land-farm soil, noncontaminated reference soil, and a 1:1 mixture of land-farm and reference soils). We observed a 30% increase in soil respiration in straw-amended oily soil, whereas respiration increased by 246% in straw-amended reference soil. Much of the difference between oily and reference soils was attributable to higher basal respiration rates of nonamended oily soil compared to nonamended reference soil. Addition of earthworms resulted in greater total respiration of all soil and straw treatments except two (the land-farm and the 1:1 mixture soil treatments without straw). Straw and earthworm treatments did not affect O&G or TPH concentrations. Nevertheless, our findings that earthworm additions improved plant growth and that straw additions enhanced microbial activity in land-farm soil suggest that these treatments may be compatible with plant-based remediation techniques currently under evaluation in field trials, and could reduce the time required to restore soil ecosystem function.     

Responses of microbial activity and decomposer organisms to contamination in microcosms containing coniferous forest soil

Soil respiration from microcosms contaminated with pentachlorophenol, 2-ethanolhexanoate, creosote, CuSO4, and benomyl was measured in order to evaluate usefulness of soil microcosms and microbial respiration rate monitoring as a toxicity test in soils with high organic matter content. Coniferous forest soil and its organisms were used as test objects. In addition, how a short-term low temperature period including frost affects respiration dynamics in stressed soils was studied, i.e., whether contaminants reduce resistance of the community to other (also natural) stresses. In addition, at the end of the experiment, effects of contaminants on faunal and microbial community structures were analyzed. Soil respiration measurements from the microcosms appeared to be a sensitive parameter for testing community-level effects of chemicals in the soil with high organic matter content. An 84-day exposure had acute effects, long-term effects, delaying effects, and total recovery of community respiration. Direct negative and indirect positive effects of chemical contamination on the community of soil organisms were found. Responses to contamination of soil respiration rate and structure of the soil community were parallel. Addition of pentachlorophenol, 2-ethanolhexane, and Cu into the soil reduced frost resistance of the decomposer community. It was concluded that soil respiration monitoring of artificially contaminated soil microcosms seems to be a useful tool for testing community-level toxic effects of chemicals.     

Tungsten effects on survival, growth, and reproduction in the earthworm, Eisenia fetida

To provide basic toxicity data for formulating risk characterization benchmarks, the effects of tungsten on survival, growth, and reproduction were investigated in the earthworm Eisenia fetida. Parallel studies with lead as a reference toxicant also were conducted. Although sodium tungstate (Na2WO4) was less acutely toxic than lead nitrate (Pb(NO3)2) in 14-d spiked field soil acute toxicity assays (lethal concentrations for 50% of organisms: W, 6,250 mg/kg; Pb, 2,490 mg/kg), tungstate completely inhibited reproduction in 28- and 56-d assays at all tested tungsten concentrations (> or = 704 mg/kg). By comparison, cocoon production was not significantly reduced for lead concentrations until concentrations reached 766 mg/kg, and cocoon production was still observed at the highest concentration tested (1,650 mg/kg). These data indicate that tungsten is a reproductive toxicant for earthworms and that, by comparison, its sublethal toxicity is greater than that of lead. Toxicity data for other soil invertebrate species are required to fully establish benchmark levels/ecological soil screening levels for tungsten.     

Effects and risk assessment of linear alkylbenzene sulfonates in agricultural soil. 4. The influence of salt speciation, soil type, and sewage sludge on toxicity using the collembolan Folsomia fimetaria and the earthworm Aporrectodea caliginosa as test organisms

Sewage sludge applied to agricultural soils often contains linear alkylbenzene sulfonates (LAS) in the range of 1 to 10 g/kg dry weight, and their toxicity to relevant soil organisms should, therefore, be assessed to ensure safe use of sewage sludge as a fertilizer. Studies of LAS toxicity to soil organisms are few, and to our knowledge, factors that may influence the toxicity in the field have not been studied in detail. In this paper, we report on the influence of speciation of LAS in the test solution added to soil (soluble Na-LAS vs poorly soluble Ca-LAS or Mg-LAS), the influence of soil type, and the modifying effects of sludge amendment on the toxicity of LAS. These issues were investigated using reproduction of Collembola and growth of juvenile earthworms as test parameters. Speciation of the LAS added to test soil did not have any influence on toxicity for any of the test species. Likewise, in three different agricultural soils (sand, loam, and clay), we found almost equal toxicities. The LAS added to test soil in a sludge-water suspension was equally toxic as when it was added in an aqueous solution. However, anaerobic incubation for 7 and 14 d of the LAS-sludge suspension (with no decay of LAS) caused the toxicity to increase almost threefold in both collembolan and earthworm. The relationships between soil constituents, bioavailability, and toxicity are also discussed.     

Effects of sulfachlorpyridazine in MS.3-arable land: a multispecies soil system for assessing the environmental fate and effects of veterinary medicines

A multispecies soil system (MS.3) has been used to evaluate the ecological effects of veterinary pharmaceuticals in soil as a result of routine agricultural practices. Different experimental conditions were tested and the variation of the different parameters was evaluated for a final design. A protocol for the MS.3-arable land is presented here. Emergence of seedlings, plant elongation and biomass, earthworm mortality, and soil microbial enzymatic activities have been selected as toxicological endpoints for soil organisms. Toxicity tests were conducted with the leachate on aquatic organisms (in vitro fish cell lines, daphnids, and algae). The system was used for assessing the effects of the antimicrobial sulfachlorpyridazine that was tested in triplicate at concentrations of 0.01, 1, and 100 mg/kg. The chemical was mixed uniformly with a 20-cm depth soil column to resemble the distribution of manure within arable soil. Reversible and nonreversible effects on soil enzymatic activities were observed at 1 and 100 mg/kg, respectively. Earthworms were not affected. Significant reduction of plant elongation and biomass was observed at the highest concentration. Degradation and leaching contributed to the dissipation of sulfachlorpyridazine from the soil column. The undiluted leachate was highly toxic to Daphnia magna. The parent chemical was assumed responsible for the leachate toxicity although the role of mobile metabolites could not be excluded fully. No significant effects were observed for green algae Chlorella vulgaris and for the rainbow trout established cell lines RTG-2 (rainbow trout gonads) and RTL-WI (rainbow trout liver). The MS.3 system offers a cost-effective experimental approach to measure simultaneously fate and effects of chemicals on a realistic soil system under controlled laboratory conditions. The advantages of using MS.3-effect endpoints are discussed.     

Assessment of Sediment Toxicity Using Different Trophic Organisms

The main aim of the present project is to study the feasibility of using different trophic organisms for evaluating the toxicity of dredged sediments arising in Hong Kong. A total of eight sediment samples (duplicate samples collected from four selected sites: Kowloon Bay, Tsing Yi, Chek Lap Kok, and Double Haven) of Hong Kong coastal waters were analyzed for the total concentrations of As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, total organic carbon, acid volatile sulfides, simultaneously extracted metals, redox potential, and 12 organic micropollutants. The sediment elutriates were also analysed for the various metal concentrations, as well as contents of ammonia-N, nitrate, total sulfide, sulfate, and total organic carbon. Elutriate Sediment Toxicity Tests (ESTT) were also conducted, using two microalgae (Skeletonema costatum, a diatom and Dunaliella tertiolecta, a flagellate), juvenile shrimp (Metapenaeus ensis) and juvenile fish (Trachinotus obtaus). Two commercially available tests using bacteria (Microtox Test and Toxi-Chromotest) also were employed to test both the solid phase and elutriates of the sediments. The results of Microtox test on the solid phase, and bioassay tests using diatom on the sediment elutriate, especially the former, were correlated significantly (p < 0.05) with a number of physico-chemical properties of sediments and elutriates. It is recommended that a combination of a liquid-phase bioassay using diatom and a solid-phase bioassay using Microtox test should be used for screening a large number of sediment samples. However, the presence of ammonia in the sediments containing a high content of organic matter seemed to interfere the detection of contamination impacts. Received: 12 March 1996/Revised: 27 August 1996     

Uptake and toxicity of spiked nickel to earthworm Eisenia fetida in a range of Chinese soils

Bioavailability and toxicity of metals to soil organisms varies among different soils, and knowledge of this variance is useful for the development of soil environmental quality guidelines. In the present study, laboratory experiments were performed to investigate the effects of variations in nickel (Ni) uptake and toxicity on growth, cocoon output, and juvenile production in the earthworm Eisenia fetida in 13 Chinese soils spiked with nickel chloride. Body weight development of E. fetida was rather insensitive to Ni, and significant inhibition of growth was observed only at high Ni concentrations, such as 560 and 1000 mg/kg. The 50% inhibition effect concentrations (EC50s) for cocoon and juvenile production, based on measured Ni concentrations in soils, varied from 169 to 684 mg/kg and from 159 to 350 mg/kg, respectively. The EC50s represented approximately fourfold variation for cocoon output and twofold variation for juvenile production among 13 Chinese soils. Juvenile production, compared to cocoon output, was a more sensitive endpoint parameter to Ni. Nickel uptake in E. fetida increased as simple linear functions of increasing soil Ni concentrations. Tissue Ni-based EC50s (based on Ni concentrations in earthworm tissues) for cocoon production varied from 37 to 121 mg/kg (threefold variation) in 12 of 13 soils, suggesting a similar variation to that of soil Ni-based EC50s. Relationship analysis between soil properties and Ni toxicity showed that neither the EC50s for cocoon output nor those for juvenile production presented significant correlation with soil properties (pH, organic matter content, cation exchange capacity, clay content, Ca2? and Mg2?). This may be ascribed to the narrow range of properties of selected soils. The soil factors that determined Ni toxicity to earthworm reproduction remain undetermined in the present study, and these data should be used cautiously when developing toxicity prediction models because of the narrow selection of soil properties.     

Ecotoxicity of Cr, Cd, and Pb on Two Mediterranean Soils

Three potentially toxic elements [chromium (Cr), lead (Pb), and cadmium (Cd)] were tested to assess their effects on two soils of different properties and origin. The soils were a granitic soil (Haplic Arenosol), which meets the requirements of OECD ecotoxicity testing, and a calcareous soil (Calcaric Regosol) with properties often found in the Mediterranean areas. The metal concentrations used ranged from 0.001 to 5,000 mg kg^?1 soil. The effects on soil microbial activity and community composition (respirometry and polymerase chain reaction–denaturing gradient gel electrophoresis analysis), as well as the effects on plant germination and elongation (Lactuca sativa), were assessed. The toxicity of the soil water extracts was also evaluated by the growth inhibition of algal populations (Pseudokirschneriella subcapitata). Cr showed the highest level of toxicity to soil organisms in the assays performed because this element remains in soil as anionic form and is less retained by the soil solid matrix than Cd and Pb. The lowest observed–adverse effect level for Cr ranged from approximately 0.1 mg kg^?1 [substrate induced respiration (SIR) test for the granitic soil] to 10 mg kg^?1 (basal respiration and SIR tests for the calcareous soil). For Pb (SIR) and Cd (SIR and alga tests), these levels were approximately 100 mg kg^?1. Germination and algal tests showed higher sensitivity in Regosol soil than in Arenosol soil for Cr due to differences in the bioavailability between the soils. In the cases or areas where alkaline soils are abundant, these should also be considered in laboratory ecotoxicity testing to avoid underestimation of ecotoxicological risks.     

Ecotoxicological effects of hexahydro-1,3,5-trinitro-1,3,5-triazine on soil microbial activities

Although hexahydro-1,3,5-trinitro-1,3,5-triazine (also called RDX or hexogen) is a potentially toxic explosive compound that persists in soil, its ecotoxicological effects on soil organisms have rarely been assessed. In this study, two uncontaminated garden soils were spiked with 10 to 12,500 mg RDX/kg dry soil. Soil microbial activities, i.e., potential nitrification, nitrogen fixation, dehydrogenase, basal respiration, and substrate-induced respiration were chosen as bioindicators and were determined after 1-, 4-, and 12-weeks of exposure. Experimental results indicate that RDX showed significant inhibition (up to 36% of control) on indigenous soil microbial communities over the period of this study. All five bioindicators responded similarly to the RDX challenge. The length of exposure also affected the microbial toxicity of RDX, with 12-week exposure exerting more significant effects than the shorter exposure periods, suggesting that soil microorganisms might become more vulnerable to RDX when exposure is extended. The estimated lowest observable adverse effect concentration of RDX was 1,235 mg/kg. No biodegradation products of RDX were detected at all three sampling times. Compared with 2,4,6-trinitrotoluene (TNT), RDX is less toxic to microbes, probably because of its resistance to biodegradation under aerobic conditions, which precludes metabolic activation of nitro groups.     

Toxicity of Octahydro-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine (HMX) in Three Vertebrate Species

The explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine or high-melting explosive (HMX), has been found in soils in areas used for testing and training by the military. Many of these areas contain habitat for valued wildlife species. In an effort to better understand the environmental consequences from exposure, a reptilian (western fence lizard [Sceloporus occidentalis]), an amphibian (red-backed salamander [Plethodon cinereus]), and a mammalian species (rabbit [Oryctolagus cuniculus]) were exposed to HMX under controlled laboratory conditions. Lizards and rabbits were exposed to HMX by way of corn oil through gavage, and salamanders were exposed to HMX in soil. Two deaths occurred from acute oral exposures to lizards to 5000 mg HMX/kg BW. Histological and gross pathologic assessment suggested gut impaction as a possible cause of death. Salamanders exposed to concentrations of HMX in soil ≤1970 mg HMX/kg soil for 10 days did not show adverse effects. Rabbits, however, showed neurologic effects manifested as hyperkinetic events with convulsions at >24 h after oral exposures. An LD₅₀ for rabbits was calculated as 93 mg/kg (95% confidence interval 76–117). A subacute 14-day testing regime found a lowest observed effect level of 10 mg/kg-d and a no observed adverse effect level of 5 mg/kg-d based on hyperkinesia and seizure incidence, although changes suggesting functional hepatic alterations were also found. These data suggest that physiologic differences between species, particularly in gastrointestinal structure and function, can affect the absorption of HMX and hence lead to marked differences in toxicity from exposure to the same compound.