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Hyperaccumulation in plants is a complex and dynamic biological process. Sedum alfredii, the most studied Cd hyperaccumulator, can accumulate up to 9000 mg kg -1 Cd in its leaves without suffering toxicity. Although several studies have reported the molecular mechanisms of Cd hyperaccumulation, our understanding of the cell-type-specific transcriptional regulation induced by Cd remains limited. In this study, the first full-length transcriptome of S. alfredii was generated using the PacBio Iso-Seq technology. A total of 18,718,513 subreads (39.90 Gb) were obtained, with an average length of 2133 bp. The single-cell RNA sequencing was employed on leaves of S. alfredii grown under Cd stress. A total of 12,616 high-quality single cells were derived from the control and Cd-treatment samples of S. alfredii leaves. Based on cell heterogeneity and the expression profiles of previously reported marker genes, seven cell types with 12 transcriptionally distinct cell clusters were identified, thereby constructing the first single-cell atlas for S. alfredii leaves. Metal transporters such as CAX5, COPT5, ZIP5, YSL7, and MTP1 were up-regulated in different cell types of S. alfredii leaves under Cd stress. The distinctive gene expression patterns of metal transporters indicate special gene regulatory networks underlying Cd tolerance and hyperaccumulation in S. alfredii. Collectively, our findings are the first observation of the cellular and molecular responses of S. alfredii leaves under Cd stress and lay the cornerstone for future hyperaccumulator scRNA-seq investigations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phytoremediation is widely considered as a cost-effective method for managing heavy metal soil pollution. Leersia hexandra Swartz shows a promising potential for the remediation of heavy metals pollution, including chromium (Cr), copper (Cu), and nickel (Ni). It is vital to understand the physiological and biochemical responses of L. hexandra to Ni stress to elucidate the mechanisms underlying Ni tolerance and accumulation. Here, we examined the metabolic and transcriptomic responses of L. hexandra exposed to 40 mg/L Ni for 24 h and 14 d. After 24-h Ni stress, gene expression of glutathione metabolic cycle (GSTF1, GSTU1 and MDAR4) and superoxide dismutase (SODCC2) was significantly increased in plant leaves. Furthermore, after 14-d Ni stress, the ascorbate peroxidase (APX7), superoxide dismutase (SODCP and SOD1), and catalase (CAT) gene expression was significantly upregulated, but that of glutathione metabolic cycle (EMB2360, GSTU1, GSTU6, GSH2, GPX6, and MDAR2) was downregulated. After 24-h Ni stress, the differentially expressed metabolites (DEMs) were mainly flavonoids (45%) and flavones (20%). However, after 14-d Ni stress, the DEMs were mainly carbohydrates and their derivatives (34%), amino acids and derivatives (15%), and organic acids and derivatives (8%). Results suggest that L. hexandra adopt distinct time-dependent antioxidant and metal detoxification strategies likely associated with intracellular reduction-oxidation balance. Novel insights into the molecular mechanisms responsible for Ni tolerance in plants are presented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mobilization of trace metals in the rhizosphere can be affected by the redox potential, which is closely related to the O 2 dynamics. This study examined the distributions of O 2 and trace metals in the rhizosphere of the subaquatic hyperaccumulator Leersia hexandra Swartz under chromium (Cr) stress using planar optodes and the diffusive gradients in thin films technique coupled with laser ablation inductively coupled plasma mass spectrometry. The O 2 concentrations and oxidized areas in the rhizosphere significantly increased with increases in the light intensity, air humidity, and atmospheric CO 2 concentrations (p < 0.05). The O 2 concentration first increased with increasing ambient temperatures, then decreased when the temperature increased from 25 to 32 ℃. The O 2 concentration in the rhizosphere was significantly decreased under Cr stress (p < 0.05), with a prolonged response time to the altered ambient temperature. Cr stress led to decreased mobilities of As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Sb, V, W, and Zn in the rhizosphere, which were negatively correlated with the concentrations of O 2 . These results provide new insights into the role of changes in the O 2 concentration induced by the roots of hyperaccumulator plants in controlling the mobility of trace metals in soils., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Understanding the molecular mechanism of tolerance to heavy metals in hyperaccumulators is important for improving the efficiency of phytoremediation and is interesting for evolutionary studies on plant adaption to abiotic stress. Celosia argentea Linn. was recently discovered to hyperaccumulate both manganese (Mn) and cadmium (Cd). However, the molecular mechanisms underlying Mn and Cd detoxification in C. argentea are poorly understood. Laboratory studies were conducted using C. argentea seedlings exposed to 360 μM Mn and 8.9 μM Cd hydroponic solutions. Plant leaves were analyzed using transcriptional and metabolomic techniques. A total of 3960 differentially expressed genes (DEGs) in plants were identified under Cd stress, among which 17 were associated with metal transport, and 10 belonged to the ATP transporter families. Exposures to Mn or Cd led to the differential expression of three metal transport genes (HMA3, ABCC15, and ATPase 4). In addition, 33 and 77 differentially expressed metabolites (DEMs) were identified under Mn and Cd stresses, respectively. Metabolic pathway analysis showed that the ABC transporter pathway was the most affected in Mn/Cd exposed seedlings. Conjoint transcriptome and metabolome analysis showed that the glutathione (GSH) metabolic pathway was over-represented in the KEGG pathway of both DEGs and DEMs. Our results confirm that the ABC transporter and GSH metabolic pathways play important roles in Mn and Cd detoxification. These findings provide new insight into the molecular mechanisms of tolerance to Mn and Cd toxicity in plants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

When nano-sized titanium dioxide (nano-TiO 2 ) absorbs ultra-violet (UV-A) radiation, it produces reactive oxygen species that can be toxic to bacteria. We used the agronomically beneficial nitrogen-fixing bacterium Sinorhizobium meliloti strain 1021 as a model microorganism to detect nano-TiO 2 toxicity. Sinorhizobium meliloti was exposed to aqueous dispersions of micrometer-sized TiO 2 (micron-TiO 2 , 44 μm) or nanometer-sized TiO 2 (nano-TiO 2 , 21 nm) at nominal concentrations of 0, 100, 300, 600, 900, and 1800 mg TiO 2 /L. There were fewer viable S. meliloti cells after exposure to nano-TiO 2 under dark and UV-A light conditions. Nano-TiO 2 was more toxic to S. meliloti with UV-A irradiation (100% mortality at 100 mg TiO 2 /L) than under dark conditions (100% mortality at 900 mg TiO 2 /L). Micron-TiO 2 concentrations less than 300 mg TiO 2 /L had no effect on S. meliloti viability under dark or UV-A light conditions. Exposure to 600 mg/L or more of micron-TiO 2 under UV-A light could also photo-kill S. meliloti cells (100% mortality). Further studies are needed to ascertain whether nano-TiO 2 interferes with the growth of N 2 -fixing microorganisms in realistic agricultural environments.

The present study investigates the bioaccumulation of the insensitive munition compounds 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO), developed for future weapons systems to replace current munitions containing sensitive explosives. The earthworm Eisenia andrei was exposed to sublethal concentrations of DNAN or NTO amended in Sassafras sandy loam. Chemical analysis indicated that 2- and 4-amino-nitroanisole (2-ANAN and 4-ANAN, respectively) were formed in DNAN-amended soils. The SumDNAN (sum of DNAN, 2-ANAN, and 4-ANAN concentrations) in soil decreased by 40% during the 14-d exposure period. The SumDNAN in the earthworm body residue increased until day 3 and decreased thereafter. Between days 3 and 14, there was a 73% decrease in tissue uptake that was greater than the 23% decrease in the soil concentration, suggesting that the bioavailable fraction may have decreased over time. By day 14, the DNAN concentration accounted for only 45% of the SumDNAN soil concentration, indicating substantial DNAN transformation in the presence of earthworms. The highest bioaccumulation factor (BAF; the tissue-to-soil concentration ratio) was 6.2 ± 1.0 kg/kg (dry wt) on day 3 and decreased to 3.8 ± 0.8 kg/kg by day 14. Kinetic studies indicated a BAF of 2.3 kg/kg, based on the earthworm DNAN uptake rate of 2.0 ± 0.24 kg/kg/d, compared with the SumDNAN elimination rate of 0.87 d -1 (half-life = 0.79 d). The compound DNAN has a similar potential to bioaccumulate from soil compared with trinitrotoluene. The NTO concentration in amended soil decreased by 57% from the initial concentration (837 mg NTO/kg dry soil) during 14 d, likely due to the formation of unknown transformation products. The bioaccumulation of NTO was negligible (BAF ≤ 0.018 kg/kg dry wt). Environ Toxicol Chem 2021;40:1713-1725. © 2021 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA., (© 2021 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Phytoextraction using Celosia argentea Linn. can potentially decontaminate Cd-contaminated soils. However, most earlier studies have been conducted at laboratory scale and for a relatively short remediation period. To evaluate the phytoextraction efficiency of C. argentea combined with different soil amendments (ammonium chloride, Bacillus megaterium, and citric acid), an 18-month field experiment was carried out in a farmland soil contaminated with 3.68 mg kg -1 Cd by mine tailings in southern China. Soil Cd concentrations were decreased by 6.34 ± 0.73% after the three harvestings (with no amendments), which was 2.27 times that of the no-planting control (p < 0.05). Application of ammonium chloride, B. megaterium, and citric acid increased the overall Cd reduction rate in soil by 40.5%, 46.1%, and 105%, respectively. The application of citric acid decreased total Cd in soil by up to 16.9% in the rhizosphere soil and 13.0% in the bulk soil. The highest annual shoot biomass yield and Cd extraction amount reached 8.79 t ha -1 and 273 g ha -1 . Acid-soluble Cd fraction in the rhizosphere was significantly lower compared to that in the bulk soil (p < 0.05), which indicates that mobile Cd in the rhizosphere was taken up by the roots vastly. C. argentea phytoextraction also improved soil metabolic functions by increasing the activities of soil enzymes (urease, invertase, phosphatase, and catalase). These findings demonstrate that Cd phytoextraction using C. argentea with the application of soil amendments can greatly improve the quality of Cd-contaminated soils., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

To select suitable plants for phytoextraction of Cd-contaminated soils, we evaluated the phytoextraction potential of five local Cd-accumulators: Amaranthus hypochondriacus L., Solanum nigrum L., Phytolacca acinosa Roxb., Celosia argentea L., and Sedum spectabile Boreau. The plants were grown in three naturally contaminated soils with different total Cd levels (1.57, 3.89, and 22.4 mg kg -1 ). Throughout the experimental period, no plants showed any visible symptoms of metal toxicity. The Cd uptake of C. argentea was the greatest in the S-YS soil (105 μg plant -1 ) and among the greatest in the S-HC soil and S-TJ soil. Besides, C. argentea exhibited the highest bioconcentration factor (12.3) in three soils. To improve the phytoextraction efficiency of C. argentea , we applied four low molecular weight organic acids (LMWOAs): tartaric acid, malic acid, oxalic acid, and citric acid. Malic acid was more effective in enhancing Cd uptake by C. argentea than the other LMWOAs. Therefore, C. argentea may be a potential choice in actual remediation projects. Moreover, application of malic acid is an effective way to increase the phytoextraction efficiency of C. argentea .

Individual effects of nitrogen-based energetic materials (EMs) 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), nitroglycerin (NG), and 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (CL-20) on litter decomposition, an essential biologically-mediated soil process, were assessed using Orchard grass (Dactylis glomerata) straw in Sassafras sandy loam (SSL) soil, which has physicochemical characteristics that support "very high" qualitative relative bioavailability for organic chemicals. Batches of SSL soil were separately amended with individual EMs or acetone carrier control. To quantify the decomposition rates, one straw cluster was harvested from a set of randomly selected replicate containers from within each treatment, after 1, 2, 3, 4, 6, and 8 months of exposure. Results showed that soil amended with 2,4-DNT or NG inhibited litter decomposition rates based on the median effective concentration (EC50) values of 1122 mg/kg and 860 mg/kg, respectively. Exposure to 2-ADNT, 4-ADNT or CL-20 amended soil did not significantly affect litter decomposition in SSL soil at ≥ 10,000 mg/kg. These ecotoxicological data will be helpful in identifying concentrations of EMs in soil that present an acceptable ecological risk for biologically-mediated soil processes., (Published by Elsevier Inc.)

We investigated individual toxicities of the nitrogen-based energetic materials (EMs) 2,4-dinitrotoluene (2,4-DNT); 2-amino-4,6-dinitrotoluene (2-ADNT); 4-amino-2,6-dinitrotoluene (4-ADNT); and nitroglycerin (NG) on microbial activity in Sassafras sandy loam (SSL) soil, which has physicochemical characteristics that support very high qualitative relative bioavailability for organic chemicals. Batches of SSL soil for basal respiration (BR) and substrate-induced respiration (SIR) assays were separately amended with individual EMs or acetone carrier control. Total microbial biomass carbon (biomass C) was determined from CO 2 production increases after addition of 2500 mg/kg of glucose-water slurry to the soil. Exposure concentrations of each EM in soil were determined using US Environmental Protection Agency method 8330A. Basal respiration was the most sensitive endpoint for assessing the effects of nitroaromatic EMs on microbial activity in SSL, whereas SIR and biomass C were more sensitive endpoints for assessing the effects of NG in soil. The orders of toxicity (from greatest to least) were 4-ADNT > 2,4-DNT = 2-ADNT > NG for BR; but for SIR and biomass C, the order of toxicity was NG > 2,4-DNT > 2-ADNT = 4-ADNT. No inhibition of SIR was found up to and including the greatest concentration of each ADNT tested in SSL. These ecotoxicological data will be helpful in identifying concentrations of contaminant EMs in soil that present acceptable ecological risks for biologically mediated processes in soil. Environ Toxicol Chem 2017;36:2981-2990. Published 2017 Wiley Periodicals Inc. on behalf of SETAC.This article is a US government work and, as such, is in the public domain in the United States of America., (© 2017 SETAC.)

New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices.

This study investigated the possibility that titanium dioxide nanoparticles (nano-TiO2) toxicity in Pseudokirchneriella subcapitata involves reactive oxygen species (ROS) production, using the dichlorodihydrofluorescein (DCF) assay. Algae were exposed to nano-TiO2 under laboratory fluorescent lamps supplemented with UV irradiation for 3h, with or without a UV filter. Results showed that nano-TiO2 increased ROS production in UV-exposed cells, with or without a UV filter (LOEC values were 250 and 10mg/L, respectively). Sublethal effects of nano-TiO2 on UV pre-exposed algae were also examined. Toxicity studies indicated that exposure to nano-TiO2 agglomerates decreased algal growth following 3h pre-exposure to UV, with or without a UV filter (EC50s were 8.7 and 6.3mg/L, respectively). The present study suggests that the growth inhibitory effects of nano-TiO2 in algae occurred at concentrations lower than those that can elevate DCF fluorescence, and that ROS generation is not directly involved with the sublethal effects of nano-TiO2 in algae., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The toxicity and toxicogenomics of selected anatase and rutile nanoparticles (NP) and bulk titanium dioxide (TiO2) particles were evaluated in the soil nematode Caenorhabditis elegans. Results indicated that bulk or nano-TiO2 particles were slightly toxic to soil nematode C. elegans, as measured by reproduction EC50 values ranging from 4 to 32 mg/L. Whole-genome microarray results indicated that the regulation of glutathione-S-transferase gst-3, cytochrome P450 cypp33-c11, stress resistance regulator scl-1, oxidoreductase wah-1 and embryonic development pod-2 genes were significantly affected by nano-sized and bulk-TiO2 particles. More specifically, it was determined that anatase particles exerted a greater effect on metabolic pathways, whereas rutile particles had a greater effect on developmental processes. The up-regulation of the pod-2 gene corroborated the phenotypic effect observed in the reproduction test. Our results demonstrated that C. elegans is a good genomic model for nano-TiO2 toxicity assessment.

Soil toxicity tests for metal oxide nanoparticles often include micrometer-sized oxide and metal salt treatments to distinguish between toxicity from nanometer-sized particles, non-nanometer-sized particles, and dissolved ions. Test result will be confounded if each chemical form has different effects on soil solution chemistry. We report on changes in soil solution chemistry over 56 days-the duration of some standard soil toxicity tests-in three soils amended with 500 mg/kg Cu as nanometer-sized CuO (nano), micrometer-sized CuO (micrometer), or Cu(NO3)2 (salt). In the CuO-amended soils, the log Cu2+ activity was initially low (minimum -9.48) and increased with time (maximum -5.20), whereas in the salt-amended soils it was initially high (maximum -4.80) and decreased with time (minimum -6.10). The Cu2+ activity in the nano-amended soils was higher than in the micrometer-amended soils for at least the first 11 days, and lower than in the salt-amended soils for at least 28 d. The pH, and dissolved Ca and Mg concentrations in the CuO-amended soils were similar, but the salt-amended soils had lower pH for at least 14 d, and higher Ca and Mg concentrations throughout the test. Soil pretreatments such as leaching and aging prior to toxicity tests are suggested.

The high explosive nitroaromatic 2,4-dinitroanisole (DNAN) is less shock sensitive than 2,4,6-trinitrotoluene (TNT), and is proposed as a TNT replacement for melt-cast formulations. Before using DNAN in munitions and potentially leading to environmental impact, the present study examines the ecotoxicity of DNAN using selected organisms. In water, DNAN decreased green algae Pseudokirchneriella subcapitata growth (EC50 = 4.0mg/L), and bacteria Vibrio fischeri bioluminescence (Microtox, EC50 = 60.3mg/L). In soil, DNAN decreased perennial ryegrass Lolium perenne growth (EC50 =7 mg/kg), and is lethal to earthworms Eisenia andrei (LC50 = 47 mg/kg). At sub-lethal concentrations, DNAN caused an avoidance response (EC50 = 31 mg/kg) by earthworms. The presence of DNAN and 2-amino-4-nitroanisole in earthworms and plants suggested a role of these compounds in DNAN toxicity. Toxicity of DNAN was compared to TNT, tested under the same experimental conditions. These analyses showed that DNAN was equally, or even less deleterious to organism health than TNT, depending on the species and toxicity test. The present studies provide baseline toxicity data to increase the understanding of the environmental impact of DNAN, and assist science-based decision makers for improved management of potential DNAN contaminated sites., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Exposure of stage 9 quail (Coturnix coturnix japonica) embryos to glyceryl trinitrate (GTN) induces malformations that were associated in previous studies with an increase in protein nitration. Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryos resulted in an increase in nitrite, a decrease in total GSH, and an increase in the ratio of NADP(+)/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 2.5.1.18) purified from the whole embryo (K(m) 0.84 mM; V(max) 36 μM/min) and the embryonic eye (K(m) 0.20 mM; V(max) 30 μM/min) had GTN-metabolizing activity (1436 and 34 nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha- or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

There are increasing safety concerns about the development and abundant use of nanoparticles. The unique physical and chemical characteristics of titanium dioxide (TiO2) nanoparticles result in different chemical and biological activities compared to their larger micron-sized counterparts, and can subsequently play an important role in influencing toxicity. Therefore, our objective was to investigate the cytotoxicity and genotoxicity of commercially available TiO2 nanoparticles with respect to their selected physicochemical properties, as well as the role of surface coating of these nanoparticles. While all types of tested TiO2 samples decrease cell viability in a mass-based concentration- and size-dependent manner, the polyacrylate-coated nano-TiO2 product was only cytotoxic at higher concentrations. A similar pattern of response was observed for induction of apoptosis/necrosis, and no DNA damage was detected in the polyacrylate-coated nano-TiO2 model. Given the increasing production of TiO2 nanoparticles, toxicological studies should take into account the physiochemical properties of these nanoparticles that may help researchers to develop new nanoparticles with minimum toxicity., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

¹H NMR-based metabolomics was used to examine the response of Eisenia fetida earthworms raised from juveniles for 20-23 weeks in soil spiked with either 20 or 200 mg/kg of a commercially available uncoated titanium dioxide (TiO(2)) nanomaterial (nominal diameter of 5 nm). To distinguish responses specific to particle size, soil treatments spiked with a micrometer-sized TiO(2) material (nominal diameter, <45 μm) at the same concentrations (20 and 200 mg/kg) were also included in addition to an unspiked control soil. Multivariate statistical analysis of the (1)H NMR spectra for aqueous extracts of E. fetida tissue suggested that earthworms exhibited significant changes in their metabolic profile following TiO(2) exposure for both particle sizes. The observed earthworm metabolic changes appeared to be consistent with oxidative stress, a proposed mechanism of toxicity for nanosized TiO(2). In contrast, a prior study had observed no impairment of E. fetida survival, reproduction, or growth following exposure to the same TiO(2) spiked soils. This suggests that (1)H NMR-based metabolomics provides a more sensitive measure of earthworm response to TiO(2) materials in soil and that further targeted assays to detect specific cellular or molecular level damage to earthworms caused by chronic exposure to TiO(2) are warranted.

Nanometer-sized titanium dioxide (nano-TiO(2) ) is found in a number of commercial products; however, its effects on soil biota are largely unknown. In the present study, earthworms (Eisenia andrei and Eisenia fetida) were exposed to three types of commercially available, uncoated TiO(2) nanomaterials with nominal diameters of 5, 10, and 21 nm. Nanomaterials were characterized for particle size, agglomeration, surface charge, chemical composition, and purity. Standard lethality, reproduction, and avoidance tests, as well as a juvenile growth test, were conducted in artificial soil or field soil amended with nano-TiO(2) by two methods, liquid dispersion and dry powder mixing. All studies included a micrometer-sized TiO(2) control. Exposure to field and artificial soil containing between 200 and 10,000 mg nano-TiO(2) per kilogram of dry soil (mg/kg) had no significant effect (p > 0.05) on juvenile survival and growth, adult earthworm survival, cocoon production, cocoon viability, or total number of juveniles hatched from these cocoons. However, earthworms avoided artificial soils amended with nano-TiO(2) . The lowest concentration at which avoidance was observed was between 1,000 and 5,000 mg nano-TiO(2) per kilogram of soil, depending on the TiO(2) nanomaterial applied. Furthermore, earthworms differentiated between soils amended with 10,000 mg/kg nano-TiO(2) and micrometer-sized TiO(2) . A positive relationship between earthworm avoidance and TiO(2) specific surface area was observed, but the relationship between avoidance and primary particle size was not determined because of the agglomeration and aggregation of nano-TiO(2) materials. Biological mechanisms that may explain earthworm avoidance of nano-TiO(2) are discussed. Results of the present study indicate that earthworms can detect nano-TiO(2) in soil, although exposure has no apparent effect on survival or standard reproductive parameters., (Copyright © 2011 SETAC.)

Nitroglycerin (NG) is widely used for the production of explosives and solid propellants, and is a soil contaminant of concern at some military training ranges. NG phytotoxicity data reported in the literature cannot be applied directly to development of ecotoxicological benchmarks for plant exposures in soil because they were determined in studies using hydroponic media, cell cultures, and transgenic plants. Toxicities of NG in the present studies were evaluated for alfalfa (Medicago sativa), barnyard grass (Echinochloa crusgalli), and ryegrass (Lolium perenne) exposed to NG in Sassafras sandy loam soil. Uptake and degradation of NG were also evaluated in ryegrass. The median effective concentration values for shoot growth ranged from 40 to 231 mg kg(-1) in studies with NG freshly amended in soil, and from 23 to 185 mg kg(-1) in studies with NG weathered-and-aged in soil. Weathering-and-aging NG in soil did not significantly affect the toxicity based on 95% confidence intervals for either seedling emergence or plant growth endpoints. Uptake studies revealed that NG was not accumulated in ryegrass but was transformed into dinitroglycerin in the soil and roots, and was subsequently translocated into the ryegrass shoots. The highest bioconcentration factors for dinitroglycerin of 685 and 40 were determined for roots and shoots, respectively. Results of these studies will improve our understanding of toxicity and bioconcentration of NG in terrestrial plants and will contribute to ecological risk assessment of NG-contaminated sites., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Background: Although glyceryl trinitrate (GTN) is used extensively to treat angina and heart failure, little is known about its effects on the conceptus during organogenesis. The goal of these studies was to investigate the effects of GTN in a model organism, the quail (Coturnix coturnix japonica) embryo., Methods: To identify the effects of GTN on quail embryo development, fertilized quail eggs (n = 10-12 eggs/group) were injected with GTN (0, 4.4, 44, or 440 μM) at Hamburger-Hamilton (HH) stage 0, 9, or 19 and examined 7 days later. Next, HH 9 embryos were injected with GTN (0, 0.88, 4.4, 8.8, 44, 88, and 440 μM, in 20 μL per egg) and examined 24-hours, 48-hours, or 72-hours postinjection. Finally, the developing eye on one side was exposed to GTN (44 μM) ex ovo and the tissue was probed for the presence of nitrated proteins., Results: In ovo GTN exposure induced a dose-dependent increase in the number of malformed viable quail embryos with a maximal effect in HH 9 embryos. Microphthalmia, craniofacial, heart, and neural tube defects were elevated in GTN-exposed embryos. An increase in nitrated proteins was observed in the developing eye region of embryos exposed ex ovo to GTN., Conclusions: GTN treatment induced a variety of malformations in quail embryos. The presence of nitrated proteins suggests that organic nitrates, such as GTN, generate reactive nitrogen species. We hypothesize that GTN perturbations in the redox status of the embryo may underlie its developmental toxicity., (Copyright © 2011 Wiley-Liss, Inc.)

The presence of energetic materials (used as explosives and propellants) at contaminated sites is a growing international issue, particularly with respect to military base closures and demilitarization policies. Improved understanding of the ecotoxicological effects of these materials is needed in order to accurately assess the potential exposure risks and impacts on the environment and its ecosystems. We studied the toxicity of the nitroaromatic energetic material 2,4-dinitrotoluene (2,4-DNT) on alfalfa (Medicago sativa L.), barnyard grass (Echinochloa crusgalli L. Beauv.), and perennial ryegrass (Lolium perenne L.) using four natural soils varying in properties (organic matter, clay content, and pH) that were hypothesized to affect chemical bioavailability and toxicity. Amended soils were subjected to natural light conditions, and wetting and drying cycles in a greenhouse for 13 weeks prior to toxicity testing to approximate field exposure conditions in terms of bioavailability, transformation, and degradation of 2,4-DNT. Definitive toxicity tests were performed according to standard protocols. The median effective concentration (EC(50)) values for shoot dry mass ranged from 8 to 229 mg kg(-1), depending on the plant species and soil type. Data indicated that 2,4-DNT was most toxic in the Sassafras (SSL) and Teller (TSL) sandy loam soils, with EC(50) values for shoot dry mass ranging between 8 to 44 mg kg(-1), and least toxic in the Webster clay loam soil, with EC(50) values for shoot dry mass ranging between 40 to 229 mg kg(-1). The toxicity of 2,4-DNT for each of the plant species was significantly (p < or = 0.05) and inversely correlated with the soil organic matter content. Toxicity benchmark values determined in the present studies for 2,4-DNT weathered-and-aged in SSL or TSL soils will contribute to development of an Ecological Soil Screening Level for terrestrial plants that can be used for ecological risk assessment at contaminated sites., (Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.)

The uptake of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from soil by the earthworm Eisenia andrei was examined by using the equilibrium partitioning (EqP) theory and a three-compartment model including soil (S), interstitial water (IW), and earthworms (E). The RDX concentrations were measured using U.S. Environmental Protection Agency (U.S. EPA) Method 8330A and high-performance liquid chromatography (HPLC). The S-IW studies were conducted using four natural soils with contrasting physicochemical properties that were hypothesized to affect the bioavailability of RDX. Each soil was amended with nominal RDX concentrations ranging from 1 to 10,000 mg/kg. The HPLC analysis showed that the IW extracted from soil was saturated with RDX at 80 mg/kg or greater soil concentrations. The calculated S-IW coefficient (K(p)) values for RDX ranged from 0.4 to 1.8 ml/g soil, depending on the soil type, and were influenced by the organic matter content. In the IW-E studies, earthworms were exposed to nonlethal RDX concentrations in aqueous media. The uptake of RDX by the earthworms correlated well (r(2) = 0.99) with the dissolved RDX concentrations. For the E-S studies, earthworms were exposed to RDX-amended soils used in the S-IW studies. The bioconcentration factors (BCF; ratios of E-to-IW RDX concentrations) were relatively constant ( approximately 5) up to 80 mg/kg soil RDX concentrations, which encompass the RDX saturation limit in the interstitial water of the tested soils. At this concentration range, the RDX uptake from interstitial water was likely dominated by passive diffusion and could be used as an indicator of bioavailability. Other mechanisms may be involved at greater RDX soil concentrations., ((c) 2009 SETAC.)

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.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) are cyclic nitramines used as explosives. Their ecotoxicities have been characterized incompletely and little is known about their accumulation potential in soil organisms. We assessed the toxicity and uptake of these explosives in perennial ryegrass Lolium perenne L. exposed in a Sassafras sandy loam (SSL) or in a sandy soil (DRDC, CL-20 only) containing contrasting clay contents (11% and 0.3%, respectively). A 21-d exposure to RDX, HMX or CL-20 in either soil had no adverse effects on ryegrass growth. RDX and HMX were translocated to ryegrass shoots, with bioconcentration factors (BCF) of up to 15 and 11, respectively. In contrast, CL-20 was taken up by the roots (BCF up to 19) with no translocation to the shoots. These studies showed that RDX, HMX, and CL-20 can accumulate in plants and may potentially pose a risk of biomagnification across the food chain.

Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixed with artificial rainwater, to generate leachates containing As, Cr and Cu. Then, leachates were applied to two soils at rates of 13-169 mg As kg(-1) soil (dry weight basis), 12-151 mg Cr kg(-1) and 10-216 mg Cu kg(-1). Metal bioavailability was evaluated after 28 days using the earthworm Eisenia fetida (Savigny). Metal concentrations in earthworm tissue ranged from negligible to 80 mg As kg(-1) (dry weight basis), 89 mg Cr kg(-1) and 90 mg Cu kg(-1), which appeared to be non-lethal to E. fetida. There was less Cu available to earthworms in the Courval soil (pH 7.8) than the Châteauguay soil (pH 6.8), but earthworm growth and reproduction were not affected by exposure to Cu from ACQ-treated wood. In contrast, earthworms exposed to As, Cr and Cu from the CCA-treated wood gained weight more quickly in the Courval soil (1.3-21 mg g(-1) initial biomass days) than in the Châteauguay soil (0.2-7.8 mg g(-1) day(-1)), but fewer than 20% of the cocoons deposited by the faster-growing earthworms hatched by the end of the 56 days ecotoxicology test. It appeared that E. fetida can allocate more energy to growth than reproduction, delaying cocoon development and hatching in some situations. Further information is needed on the soil factors that may induce such behavior, as it can affect the interpretation of results from the earthworm ecotoxicology test.

The toxicity and bioavailability of metals were assessed to verify the efficiency of a new chemical leaching process (METIX-AC) to minimize the risk of metals found in municipal sewage sludge. For this purpose, sludge samples were spiked with cadmium, copper and/or zinc before being treated using METIX-AC. The sludge decontamination resulted in a removal of spiked metals (79-89%), in a decrease of the more labile fractions, and in a corresponding increase of the residual fraction. The toxicity observed after exposure of two plant species, barley (Hordeum vulgare) and ryegrass (Lolium perenne), and a terrestrial invertebrate, Eisenia andrei, to sludge-soil mixtures, disappeared after treatment, although the adverse effects were minor before treatment. The sludge treatment also significantly decreased the bioaccumulation of cadmium, copper, and zinc in the exposed species. For cadmium, maximum tissue concentrations of 0.45+/-0.08 mg/kg in barley, 0.79+/-0.27 mg/kg in ryegrass, and 21.82+/-1.85 mg/kg in earthworm exposed to sludge before treatment decreased after treatment to values similar to those observed with negative controls.

Hexanitrohexaazaisowurtzitane (CL-20) is a polycyclic nitramine explosive and propellant, currently being considered as a potential replacement for existing cyclic nitramine explosives. Earlier studies have provided evidence suggestive of adverse liver effects in adult Coturnix spp. exposed to CL-20, yet analysis of tissue samples (plasma, liver, brain, heart, or spleen) indicated that CL-20 was not detectable in these treated animals. The present study was conducted to identify and purify the enzymes capable of CL-20 biotransformation. Results indicate that the hepatic biotransformation of CL-20 in vitro was inhibited by ethacrynic acid (93%) and by the glutathione (GSH) analogue S-octylglutathione (80%), suggesting the involvement of glutathione-S-transferase (GST). Partially purified cytosolic alpha- and mu-type GST (requiring presence of GSH as a cofactor) from quail and rabbit liver was capable of CL-20 biotransformation. The degradation of CL-20 (0.30 +/- 0.05 and 0.40 +/- 0.02 nmol/min/mg protein for quail and rabbit, respectively) was accompanied with the formation of nitrite and consumption of GSH. Using liquid chromatography/mass spectrometry, we detected two intermediates, that is, open-ring, monodenitrated GSH-conjugated CL-20 biotransformation product with the same deprotonated molecular mass ion at 699 Da, suggesting isomeric forms of the intermediate metabolites. Identity of the conjugated metabolites was confirmed by using ring-labeled [15N]CL-20 and the nitro group-labeled [15NO2]CL-20. These data suggest that the in vitro biotransformation of CL-20 by GST under the conditions tested may be a key initial step in the in vivo degradation of CL-20 in the quail and resulted in the formation of more biologically reactive intermediates than the parent compound. These data will aid in our understanding of the biotransformation processes of CL-20 in vivo.

Dissolved organic matter (DOM) influences metal speciation in soil solutions and, hence, metal toxicity. Root-elongation experiments were conducted to examine the effect of soil solution components, such as Ca, H, and DOM, on metal rhizotoxicity. A biotic ligand model (BLM) was tested for its ability to predict the rhizotoxicity of Cd and Cu in soil extracts. It was hypothesized that the concentration of Cd and Cu bound to functional groups at the root surface estimated using a BLM would be a better predictor of rhizotoxicity than the free-metal ion activity in solution. Both metals became less toxic at higher DOM, Ca, and H concentrations. Solution speciation and the effect on root growth explained most of the variability observed in the DOM experiments, but not in the cation experiments. It was concluded that Ca and H inhibited the rhizotoxicity of both metals tested. Rhizotoxicity data correlated better with estimates of metal-root complexes that have been estimated with a BLM than with free-metal ion activity or with total metal concentrations. The BLM seems to be a promising approach for predicting metal availability in soils and for assessing the associated risk.

The toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to terrestrial plants alfalfa (Medicago sativa L.), Japanese millet (Echinochloa crusgalli L.), and perennial ryegrass (Lolium perenne L.) were determined in Sassafras sandy loam soil using seedling emergence, fresh shoot, and dry mass measurement endpoints. A 13-week weathering and aging of energetic materials in soils, which included wetting and drying cycles, and exposure to sunlight of individual soil treatments, was incorporated into the study design to better reflect the soil exposure conditions in the field than toxicity determinations in freshly amended soils. Definitive toxicity tests showed that dinitrotoluenes were more phytotoxic for all plant species in freshly amended treatments based on EC20 values for dry shoot ranging from 3 to 24mgkg(-1) compared with values for TNB or TNT ranging from 43 to 62mgkg(-1). Weathering and aging of energetic materials (EMs) in soil significantly decreased the toxicity of TNT, TNB or 2,6-DNT to Japanese millet or ryegrass based on seedling emergence, but significantly increased the toxicity of all four EMs to all three plant species based on shoot growth. Exposure of the three plant species to relatively low concentrations of the four compounds initially stimulated plant growth before the onset of inhibition at greater concentrations (hormesis).

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.

Hexanitrohexaazaisowurtzitane, or CL-20, is an emerging highly energetic compound currently under consideration for military applications. With the anticipated wide use of CL-20, there is the potential for soil and groundwater contamination resulting in adverse toxicologic effects on environmental receptors. Presently, there is a lack of data describing the toxic effects of CL-20 on avian species. The present study describes the effect of CL-20 on Japanese quail (Coturnix coturnix japonica) modified from standard toxicity test guidelines. First, a 14-day subacute assay was adopted using repeated gavage doses (0, 307, 964, 2439, 3475, or 5304 mg CL-20/kg body weight (BW)/d for 5 days followed by no CL-20 exposure (vehicle only) for 10 days. Second, a subchronic feeding assay (0, 11, 114, or 1085 mg CL-20/kg feed) was done for 42 days. During both studies, no overt toxicity was observed in the CL-20-treated birds. During the first 5 days of the subacute study, CL-20-exposed birds showed a dose-dependent decrease in BW gain, whereas increased liver weight, plasma sodium, and creatinine levels were observed in birds receiving the highest dose tested. For the subchronic study, embryo weights were significantly decreased in a dose-dependent manner. Embryos from CL-20-exposed birds were observed to have multiple cranial and facial deformities, beak curvatures, possible mid-brain enlargement, and classic one-sided development with micro-opthalamia (nonstatistical comparisons with control embryos). A trend toward decreased number of eggs laid per female bird was also observed. We conclude that CL-20 (or its degradation products) elicits few effects in adults but may affect avian development, although these preliminary findings should be confirmed.

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

2,4,6-Trinitrotoluene (TNT) is toxic to soil invertebrates, but little is known about its toxicokinetic behavior in soil. Tissue residue analysis was used to evaluate whether the presence of TNT and its reduced metabolites in soil invertebrates was due to uptake of these compounds from the soil into the organism, or due to microbial transformation of TNT associated with the organism followed by uptake. Adult white potworms (Enchytraeus albidus) were exposed to non-lethal concentrations of TNT in amended artificial soil for 21 d, or to TNT in solution for 20 h. Soil exposure studies confirmed earlier reports that TNT was transformed in enchytraeids in vivo to 2- and 4-aminodinitrotoluenes. However, enchytraeid exposure to TNT in solution led to the additional presence of 2,4-diaminonitrotoluene as well as 2- and 4- hydroxyamino-dinitrotoluenes and azoxy-compounds, suggesting that TNT can be metabolized in vivo in the absence of soil. Incubation of unexposed enchytraeid homogenates with TNT led to a protein-dependent appearance of these metabolites in vitro after > or =16 h incubation. Cellular fractionation studies indicated that most of this activity resided in the 8000 x g pellet, and was completely inhibited by broad-spectrum antibiotics. These studies demonstrate that enchytraeids can transform TNT in vivo and in vitro, at least in part, by bacteria associated with the host organism.

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 l(-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.

Earthworm mesocosms studies were carried out on a explosives-contaminated site at an antitank firing range. Survival of earthworms and the lysosomal neutral red retention time (NRRT), a biomarker of lysosomal membrane stability, were used in these studies to assess the effect of explosives-contaminated soils on the earthworms Lumbricus terrestris and Eisenia andrei under field conditions. Toxicity of the soils samples for E. andrei was also assessed under laboratory conditions using the earthworms reproduction test and the NRRT. Results indicate that the survival was reduced up to 40% in certain explosive-contaminated soil mesocosms following 10 days of exposure under field conditions, whereas survival was reduced up to 100% following 28 days of exposure under laboratory conditions. Reproduction parameters such as number of cocoons and number of juveniles were reduced in many of the selected contaminated soils. Compared to the reference, NRRT was significantly reduced for E. andrei exposed to explosive-contaminated soils under both field and laboratory conditions, whereas for L. terrestris NRRT was similar compared to the reference mesocosm. Analyses showed that HMX was the major polynitro-organic compound in soils. HMX was also the only explosive detected in earthworm tissues. Thus, results from both field mesocosms and laboratory studies, showed lethal and sub-lethal effects associated to soil from the contaminated area of the 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.

Soils contaminated with 2,4,6-trinitrotoluene (TNT) and TNT primary reduction products have been found to be toxic to certain soil invertebrates, such as earthworms. The mechanism of toxicity of TNT and of its by-products is still not known. To ascertain if one of the TNT reduction products underlies TNT toxicity, we tested the toxicity and bioaccumulation of TNT reduction products. 2-Amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-diamino-6-nitrotoluene (2,4-DANT) and 2,6-diamino-4-nitrotoluene (2,6-DANT) were tested separately in adult earthworms (Eisenia andrei) following a 14-d exposure to amended sandy loam forest soil. TNT, 4-ADNT, and 2-ADNT were lethal to earthworms (14-d LC(50) were: 580, 531 and 1088 micromol kg(-1), or 132, 105 and 215 mgkg(-1) dry soil, respectively) and gave the following order of toxicity: 4-ADNT>TNT>2-ADNT. Exposure to 2,4-DANT and to 2,6-DANT caused no mortality at 600 micromol kg(-1) or 100 mgkg(-1) dry soil. We found that all four TNT reduction products accumulated in earthworm tissues and 2-ADNT reached the highest levels at 3.0+/-0.3 micromol g(-1) tissue. The 14-d bioaccumulation factors were 5.1, 6.4, 5.1 and 3.2 for 2-ADNT, 4-ADNT, 2,4-DANT and 2,6-DANT, respectively. Results also suggest that some TNT metabolites are at least as toxic as TNT and should be considered when evaluating the overall toxicity of TNT-contaminated soil to earthworms.

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).

The toxicities of two heavy metals, copper (Cu2+) and chromium (Cr6+), to barley (Hordeum vulgare L.) were evaluated using two types of substrates: artificial and natural forest soils. Phytotoxicity was assessed using a standardized toxicity test. Endpoints included plant emergence and shoot and root growth. Shoot and root concentrations of Cu and Cr were also measured. Data indicated that the root biomass was the most sensitive endpoint. The results showed that toxicity of Cr to root growth (IC50=6.6 microg/g in artificial soil; IC50=61.8 microg/g in forest soil) was higher than that of Cu (IC50=13.7 microg/g in artificial soil; IC50>322 microg/g in forest soil). Data also indicated that the toxicity of Cu and Cr was significantly decreased in the spiked forest soil, suggesting lower metal bioavailability to barley in the natural soil. Analysis of tissue concentrations in barley showed that Cu and Cr were mainly accumulated in the roots. Toxicity was correlated with Cr residues in shoots (< or =11.2 microg Cr/g and < or =5.3 microg Cr/g for artificial and natural soils, respectively) and roots (< or =161 microg Cr/g and < or =51.7 microg Cr/g for artificial and natural soils, respectively) and Cu residues in roots (< or =61.8 microg Cu/g and < or =91.3 microg Cu/g for artificial and natural soils, respectively). Cu concentration in shoot tissues was < or =61.8 microg Cu/g. Since it may overestimate toxicity, effect and risk assessment using spiked soils, particularly in artificial soil, must be used with diligence.

The phytogenotoxicity of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) was assessed using the Tradescantia micronucleus (Trad-MCN) bioassay. Tradescantia cuttings bearing young inflorescences were exposed for 6h to 2,4- or 2,6-DNT amended water solutions up to their respective solubilities. The nominal concentrations were 0, 1.9, 3.8, 7.5, 15, 30, 60, 100, 150, 200mg/l of 2,4-DNT, and 0, 7.5, 15, 30, 60, 90, 120, 180mg/l of 2,6-DNT. Each treatment was repeated three or four times. Chemical concentrations in test solutions were analyzed prior to and after the exposure. Cadmium chloride (0-20mM) was used as the positive control. Micronuclei (MCN) were scored in the tetrad-stage pollen mother cells. The MCN frequency (%), i.e. the number of micronuclei scored in 100 tetrads, was the measurement endpoint. Results indicated that both 2,4-DNT and 2,6-DNT were genotoxic with the minimum effective dose (MED) of 30 and 135mg/l, respectively. Longer exposure (30h) without recovery time at 150mg/l of 2,4-DNT and 180mg/l of 2,6-DNT did not induce significantly higher MCN frequencies.

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 (

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.

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.

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at >or=1.8 micro g TNT cm(-2), whereas sublethal (weight loss) and lethal effects to earthworms were detected at >or=3.5 and 7.1 micro g TNT cm(-2), respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (>or=55 mg TNT kg(-1) soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO4, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.

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.

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.

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.