Physiological and biochemical responses of Leersia hexandra Swartz to nickel stress: Insights into antioxidant defense mechanisms and metal detoxification strategies.

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.

High-resolution imaging of O2 dynamics and metal solubilization in the rhizosphere of the hyperaccumulator Leersia hexandra Swartz.

The mobilization of trace metals in the rhizosphere can be affected by the redox potential, which is closely related to the O2 dynamics. This study examined the distributions of O2 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 O2 concentrations and oxidized areas in the rhizosphere significantly increased with increases in the light intensity, air humidity, and atmospheric CO2 concentrations (p < 0.05). The O2 concentration first increased with increasing ambient temperatures, then decreased when the temperature increased from 25 to 32 â„ƒ. The O2 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 O2. These results provide new insights into the role of changes in the O2 concentration induced by the roots of hyperaccumulator plants in controlling the mobility of trace metals in soils.

Integrated transcriptome and metabolome analysis reveals the mechanism of tolerance to manganese and cadmium toxicity in the Mn/Cd hyperaccumulator Celosia argentea Linn.

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.

Evidence that nano-TiO2 induces acute cytotoxicity to the agronomically beneficial nitrogen-fixing bacteria Sinorhizobium meliloti.

When nano-sized titanium dioxide (nano-TiO2) 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-TiO2 toxicity. Sinorhizobium meliloti was exposed to aqueous dispersions of micrometer-sized TiO2 (micron-TiO2, 44 µm) or nanometer-sized TiO2 (nano-TiO2, 21 nm) at nominal concentrations of 0, 100, 300, 600, 900, and 1800 mg TiO2/L. There were fewer viable S. meliloti cells after exposure to nano-TiO2 under dark and UV-A light conditions. Nano-TiO2 was more toxic to S. meliloti with UV-A irradiation (100% mortality at 100 mg TiO2/L) than under dark conditions (100% mortality at 900 mg TiO2/L). Micron-TiO2 concentrations less than 300 mg TiO2/L had no effect on S. meliloti viability under dark or UV-A light conditions. Exposure to 600 mg/L or more of micron-TiO2 under UV-A light could also photo-kill S. meliloti cells (100% mortality). Further studies are needed to ascertain whether nano-TiO2 interferes with the growth of N2-fixing microorganisms in realistic agricultural environments.

Phytoextraction of cadmium-contaminated soil by Celosia argentea Linn.: A long-term field study.

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.

Phytoextraction of cadmium-contaminated soils: comparison of plant species and low molecular weight organic acids.

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.

Energetic contaminants inhibit plant litter decomposition in soil.

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.

Inhibition of soil microbial activity by nitrogen-based energetic materials.

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 CO2 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.

Effects of TiO2 nanoparticles on ROS production and growth inhibition using freshwater green algae pre-exposed to UV irradiation.

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.

Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.

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.

Toxicogenomic effects of nano- and bulk-TiO2 particles in the soil nematode Caenorhabditis elegans.

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.

Differences in soil solution chemistry between soils amended with nanosized CuO or Cu reference materials: implications for nanotoxicity tests.

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.

Ecotoxicological assessment of a high energetic and insensitive munitions compound: 2,4-dinitroanisole (DNAN).

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.

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity.

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.

In vitro cytotoxicity and genotoxicity studies of titanium dioxide (TiO2) nanoparticles in Chinese hamster lung fibroblast cells.

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.

Reproductive and behavioral responses of earthworms exposed to nano-sized titanium dioxide in soil.

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.

Earthworm sublethal responses to titanium dioxide nanomaterial in soil detected by ¹H NMR metabolomics.

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.

Phytotoxicity and uptake of nitroglycerin in a natural sandy loam soil.

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.

Developmental toxicity of glyceryl trinitrate in quail embryos.

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.

Role of soil interstitial water in the accumulation of hexahydro-1,3,5-trinitro-1,3,5-triazine in the earthworm Eisenia andrei.

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.