Localization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) in poplar and switchgrass plants using phosphor imager autoradiography

Phosphor imager autoradiography is a technique for rapid, sensitive analysis of the localization of xenobiotics in plant tissues. Use of this technique is relatively new to research in the field of plant science, and the potential for enhancing visualization and understanding of plant uptake and transport of xenobiotics remains largely untapped. Phosphor imager autoradiography is used to investigate the uptake and translocation of the explosives 1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene within Populus deltoides × nigra DN34 (poplar) and Panicum vigratum Alamo (switchgrass). In both plant types, TNT and/or TNT-metabolites remain predominantly in root tissues while RDX and/or RDX-metabolites are readily translocated to leaf tissues. Phosphor imager autoradiography is further investigated for use in semi-quantitative analysis of uptake of TNT by switchgrass.     

Effect of two major N-nitroso hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolites on earthworm reproductive success

Soil and topical tests were employed to investigate the effect of two N-nitroso metabolites of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) on earthworm reproduction. The lowest observed effect concentration (LOEC) for cocoon production and hatching was 50mg/kg for both hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soil. MNX and TNX also significantly affected cocoon hatching in soil (p<0.001) and in topical tests (p=0.001). The LOECs for cocoon hatching were 1 and 10mg/kg for MNX and TNX in soil, respectively, and 10mg/L in the topical test. Greater than 100mg/kg MNX and TNX completely inhibited cocoon hatching. In soil, the EC20 values for MNX were 8.7 and 8.8mg/kg for cocoon and juvenile production, respectively, compared to 9.2 and 9.1mg/kg for TNX, respectively. The EC20 values for the total number of cocoon hatchlings were 3.1 and 4.7mg/kg for MNX and TNX, respectively, in soil and 4.5 and 3.1mg/L in the topical test. Both MNX and TNX inhibited cocoon production and hatching, suggesting that they may have a negative affect on soil ecosystems at contaminated sites.     

Age dependent acute oral toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and two anaerobic N-nitroso metabolites in deer mice (Peromyscus maniculatus)

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transforms anaerobically into N-nitroso compounds: hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Exposure to these N-nitroso metabolites may occur in areas contaminated with explosives, as anaerobic degradation occurs via some bacteria and is one remediation strategy used for RDX. Few papers report acute oral toxicity and none have evaluated age dependent toxicity of RDX or its N-nitroso metabolites. Median lethal dose (LD₅₀) was determined in deer mice (Peromyscus maniculatus) of three age classifications 21 d, 50 d, and 200 d for RDX, MNX, and TNX using the US EPA up-and-down procedure (UDP). Hexahydro-1,3,5-trinitro-1,3,5-triazine and N-nitroso metabolites caused similar overt signs of toxicity. Median lethal dose for 21 d deer mice were 136, 181, and 338 mg/kg for RDX, MNX, and TNX, respectively. Median lethal dose for 50 d deer mice were 319, 575, and 338 mg/kg for RDX, MNX, and TNX, respectively. Median lethal dose for 200 d deer mice were 158, 542, and 999 mg/kg for RDX, MNX, and TNX, respectively. These data suggest that RDX is the most potent compound tested, and age dependent toxicity may exist for all compounds and could play a role in RDX and RDX N-nitroso metabolite ecological risk evaluation of terrestrial wildlife at RDX contaminated sites.     

Dissolution and sorption of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) residues from detonated mineral surfaces

Composition B (Comp B) is a commonly used military formulation composed of the toxic explosive compounds 2,4,6-trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Numerous studies of the temporal fate of explosive compounds in soils, surface water and laboratory batch reactors have been conducted. However, most of these investigations relied on the application of explosive compounds to the media via aqueous addition and thus these studies do not provide information on the real world loading of explosive residues during detonation events. To address this we investigated the dissolution and sorption of TNT and RDX from Comp B residues loaded to pure mineral phases through controlled detonation. Mineral phases included nontronite, vermiculite, biotite and Ottawa sand (quartz with minor calcite). High Performance Liquid Chromatography and Attenuated Total Reflectance Fourier Transform Infrared spectroscopy were used to investigate the dissolution and sorption of TNT and RDX residues loaded onto the mineral surfaces. Detonation resulted in heterogeneous loading of TNT and RDX onto the mineral surfaces. Explosive compound residues dissolved rapidly (within 9 h) in all samples but maximum concentrations for TNT and RDX were not consistent over time due to precipitation from solution, sorption onto mineral surfaces, and/or chemical reactions between explosive compounds and mineral surfaces. We provide a conceptual model of the physical and chemical processes governing the fate of explosive compound residues in soil minerals controlled by sorption-desorption processes.     

Biological remediation of explosives and related nitroaromatic compounds

Nitroaromatics form an important group of recalcitrant xenobiotics. Only few aromatic compounds, bearing one nitro group as a substituent of the aromatic ring, are produced as secondary metabolites by microorganisms. The majority of nitroaromatic compounds in the biosphere are industrial chemicals such as explosives, dyes, polyurethane foams, herbicides, insecticides and solvents. These compounds are generally recalcitrant to biological treatment and remain in the biosphere, where they constitute a source of pollution due to both toxic and mutagenic effects on humans, fish, algae and microorganisms. However, relatively few microorganisms have been described as being able to use nitroaromatic compounds as nitrogen and/or carbon and energy source. The best-known nitroaromatic compound is the explosive TNT (2,4,6-trinitrotoluene). This article reviews the bioremediation strategies for TNT-contaminated soil and water. It comes to the following conclusion: The optimal remediation strategy for nitroaromatic compounds depends on many site-specific factors. Composting and the use of reactor systems lend themselves to treating soils contaminated with high levels of explosives (e.g. at former ammunition production facilities, where areas with a high contamination level are common). Compared to composting systems, bioreactors have the major advantage of a short treatment time, but the disadvantage of being more labour intensive and more expensive. Studies indicate that biological treatment systems, which are based on the activity of the fungus Phanerochaete chrysosporium or on Pseudomonas sp. ST53, might be used as effective methods for the remediation of highly contaminated soil and water. Phytoremediation, although not widely used now, has the potential to become an important strategy for the remediation of soil and water contaminated with explosives. It is best suited where contaminant levels are low (e.g. at military sites where pollution is rather diffuse) and where larger contaminated surfaces or volumes have to be treated. In addition, phytoremediation can be used as a polishing method after other remediation treatments, such as composting or bioslurry, have taken place. This in-situ treatment method has the advantage of lower treatment costs, but has the disadvantage of a considerable longer treatment time. In order to improve the cost-efficiency, phytoremediation of nitroaromatics (and other organic xenobiotics) could be combined with bio-energy production. This requires, however, detailed knowledge on the fate of the contaminants in the plants as well as the development of efficient treatment methods for the contaminated biomass that minimise the spreading of the contaminants into the environment during post harvest treatment.     

Toxicity of abamectin and doramectin to soil invertebrates

This study aimed at determining the toxicity of avermectins to soil invertebrates in soil and in faeces from recently treated sheep. Abamectin was more toxic than doramectin. In soil, earthworms (Eisenia andrei) were most affected with LC50s of 18 and 228 mg/kg dry soil, respectively, while LC50s were 67-111 and >300 mg/kg for springtails (Folsomia candida), isopods (Porcellio scaber) and enchytraeids (Enchytraeus crypticus). EC50s for the effect on reproduction of springtails and enchytraeids were 13 and 38 mg/kg, respectively for abamectin, and 42 and 170 mg/kg for doramectin. For earthworms, NOEC was 10 and 8.4 mg/kg for abamectin and doramectin effects on body weight. When exposed in faeces, springtails and enchytraeids gave LC50s and EC50s of 1.0-1.4 and 0.94-1.1 mg/kg dry faeces for abamectin and 2.2->2.4 mg/kg for doramectin. Earthworm reproduction was not affected. This study indicates a potential risk of avermectins for soil invertebrates colonizing faeces from recently treated sheep.     

Identification of oxidized TNT metabolites in soil samples of a former ammunition plant

Water extracts of soil samples of the former ammunition plant “Tanne” near Clausthal-Zellerfeld, Lower Saxony, Germany, were investigated for highly polar oxidized 2,4,6-trinitrotoluene (TNT) metabolites. 0.4 to 9.0 mg/kg dry soil 2,4,6-trinitrobenzoic acid (TNBA) and 5.8 to 544 mg/kg dry soil 2-amino-4,6-dinitrobenzoic acid (2-ADNBA) were found. In addition to the oxidized metabolites, TNT, 4- and 2-aminodinitrotoluene (4- and 2-ADNT), and 2,4-dinitrotoluene (2,4-DNT) were extractable with water. Most interestingly, in one sample, 2-ADNBA represented the main contaminant. The origin of the oxidized nitroaromatics is unknown at this time. They might be generated chemically or photochemically. Furthermore, a biological synthesis seems possible.     

Germination and seedling development of switchgrass and smooth bromegrass exposed to 2,4,6-trinitrotoluene

It is estimated that explosives contaminate approximately 0.82 million cubic metres of soil at former military installations throughout the US; major contaminants often include 2,4,6-trinitrotoluene (TNT) and its degradation products. At some sites, phytoremediation may be a viable option to incineration or other costly remediation treatments. Grasses may be particularly suited for remediation because of their growth habit and adaptability to a wide range of soil and climate conditions. We characterized the effects of TNT on germination and early seedling development of switchgrass and smooth bromegrass to evaluate their potential use on contaminated sites. Switchgrass and smooth bromegrass seeds were germinated in nutrient-free agar containing 0 to 60 mg TNT litre(-1). Smooth bromegrass germination decreased as TNT concentration increased, while switchgrass germination was unaffected by TNT. Concentrations up to 15 mg TNT litre(-1) did not affect switchgrass root growth rate, but bromegrass root growth was reduced at TNT concentrations above 7.5 mg litre(-1). At 7.5 mg TNT litre(-1), however, shoot growth rate was reduced in both species. Examination at 20-fold magnification revealed switchgrass radicles were unaffected by TNT, while smooth bromegrass radicles appeared slightly swollen. Results indicate switchgrass is more tolerant of TNT than smooth bromegrass, but the establishment of both species may be limited to soil containing less than 50 mg kg(-1) of extractable TNT.     

Dissolution kinetics of sub-millimeter Composition B detonation residues: role of particle size and particle wetting

The dissolution of the 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) from microscale particles (<250 μm) of the explosive formulation Composition B was examined and compared to dissolution from macroscopic particles (>0.5 mm). The dissolution of explosives from detonation soot was also examined. The measured mass transfer coefficients for the microscale particles were one to two orders of magnitude greater than the macroscopic particles. When normalized to particle surface area, mass transfer coefficients of microscale and macroscale particles were similar, indicating that the bulk dissolution processes were similar throughout the examined size range. However, an inverse relationship was observed between the particle diameter and the RDX:TNT mass transfer rate coefficient ratio for dry-attritted particles, which suggests that RDX may be more readily dissolved (relative to TNT) in microscale particles compared to macroscale particles. Aqueous weathering of larger Composition B residues generated particles that possessed mass transfer coefficients that were on the order of 5- to 20-fold higher than dry-attritted particles of all sizes, even when normalized to particle surface area. These aqueous weathered particles also possessed a fourfold lower absolute zeta-potential than dry-attritted particles, which is indicative that they were less hydrophobic (and hence, more wettable) than dry-attritted particles. The increased wettability of these particles provides a plausible explanation for the observed enhanced dissolution. The wetting history and the processes by which particles are produced (e.g., dry physical attrition vs. aqueous weathering) of Composition B residues should be considered when calculating mass transfer rates for fate and transport modeling.     

Removal of TNT and RDX from water and soil using iron metal

Contaminated water and soil at active or abandoned munitions plants is a serious problem since these compounds pose risks to human health and can be toxic to aquatic and terrestrial life. Our objective was to determine if zero-valent iron (Fe(0)) could be used to promote remediation of water and soil contaminated with 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). As little as 1% Fe(0) (w/v) removed 70 mg TNT litre(-1) from aqueous solution within 8 h and removed 32 mg RDX litre(-1) within 96 h. Treating slurries (1:5 soil:water) of highly contaminated soil (5200 mg TNT and 6400 mg RDX kg(-1) soil) from the former Nebraska Ordnance Plant (NOP) with 10% Fe(0) (w/w soil) reduced CH(3)CN-extractable TNT and RDX concentrations below USEPA remediation goals (17.2 mg TNT and 5.8 mg RDX kg(-1)). Sequential treatment of a TNT-contaminated solution (70 mg TNT litre(-1) spiked with (14)C-TNT) with Fe(0) (5% w/v) followed by H(2)O(2) (1% v/v) completely destroyed TNT and removed about 94% of the (14)C from solution, 48% of which was mineralized to (14)CO(2) within 8 h. Fe(0)-treated TNT also was more susceptible to biological mineralization. Our observations indicate that Fe(0) alone, Fe(0) followed by H(2)O(2), or Fe(0) in combination with biotic treatment can be used for effective remediation of munitions-contaminated water and soil.     

Dendroremediation of trinitrotoluene (TNT) part 1: Literature overview and research concept

BACKGROUND, AIM AND SCOPE: For decades, very large areas of former military sites have been contaminated diffusely with the persistent nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). The recalcitrance of the environmental hazard TNT is to a great extent due to its particulate soil existence, which leads to slow but continuous leaching processes. Although improper handling during the manufacture of TNT seems to be a problem of the past in developed countries, environmental deposition of TNT and other explosives is still going on unfortunately, resulting from thousands of unexploded ordnance or low order explosions at munitions test areas and at current battlefields. OBJECTIVE: Sustainable phytoremediation strategies for explosives in Germany, which intend to use trees to decontaminate soil and groundwater ('dendroremediation'), have to consider that most of the former German military sites are already covered with woodlands, mainly with conifer stands. Therefore, parallel investigation of the remediation potential is necessary for both of the selected hybrids of fast growing broadleaf trees, which are waiting for planting and forest conifers, which have already proven for decades that they are able to grow on explosive contaminated sites. MAIN FEATURES: A short literature review is given regarding phytoremediation of TNT with herbaceous plants and some general aspects of dendroremediation are discussed. Furthermore, an overview of our TNT-dendroremediation research network is introduced, which has the strategic goal to make dendroremediation more calculable for a series of potent trees for site-adapted in situ application and for the assessment of tree remediation potentials in natural attenuation processes. RESULTS AND DISCUSSION: Some of our methods, results and conclusions yet unpublished are presented. For a preliminary calculation of area-related annual TNT dendroremediation potential of five-year-old trees, the following values were assessed: Salix EW-13 6.0, Salix EW-20 8.5, Populus ZP-007 4.2, Betula pendula 5.2, Picea abies 1.9 and Pinus sylvestris 0.8 g m(-2) a(-1). For a 45-year-old spruce forest, an annual natural attenuation potential of 4.2 g TNT m(-2) a(-1) was found. CONCLUSION, RECOMMENDATIONS AND PERSPECTIVE: Our main results deliver quantitative proposals for dendroremediation strategies in situ and provide decision aids. Also aspects of growth of raw materials for energy production are considered. Our dendroremediation research concept for TNT and its congeners can be easily completed for other trees of interest and it can also be applied to herbaceous plants. Knowing the current bottlenecks of phytoremediation and considering the known environmental behaviour of other contaminants, elements of our methodological approach may be easily adapted to those pollutant groups, e.g. for pesticides, pharmaceuticals, PAHs, chlorinated recalcitrants and, with some restrictions, to inorganics and to multiple contaminations. Our dynamical dendrotolerance test systems will help to predict tree growth on polluted areas. To provide some light into the black box of TNT dendroremediation, experimental data regarding the uptake, distribution and degradation of [14C]-TNT in mature tree tissues will be reported in the second part of this publication.     

Aniline and 2,4,6-trinitrotoluene associate preferentially to low molecular weight fractions of dissolved soil organic matter

Aniline and 2,4,6-trinitrotoluene (TNT) were equilibrated with particulate (POM) and dissolved organic matter (DOM) from an organic soil at different compositions of adsorbed major cations (Na, Al) and pH (aniline: 3.7–5.1, TNT: 4.8–5.0). After separation of POM, concentrations of ¹⁴C-labelled aniline and TNT* (including TNT degradation products) were determined in DOM size fractions using size-exclusion chromatography (SEC) and UV-detection. Concentrations in the <3.5 kDa size fraction were 2.8–6.0 and 8.5–9.5 times higher for aniline and TNT*, respectively, as compared to the >40 kDa fraction. Thus, both aniline and TNT* were preferentially associated to the smallest DOM size fraction. The significant binding to DOM (similar extent as to POM) and the fact that the <3.5 kDa DOM fraction was less susceptible to flocculation by major metals suggests that the mobility of aniline and TNT is highly affected by the solubility of soil organic matter.     

Dendroremediation of trinitrotoluene (TNT) Part 2: Fate of radio-labelled TNT in trees

BACKGROUND, AIM AND SCOPE: Problems of long-term existence of the environmental contaminant 2,4,6-trinitrotoluene (TNT) and necessities for the use of trees ('dendroremediation') in sustainable phytoremediation strategies for TNT are described in the first part of this paper. Aims of the second part are estimation of [14C]-TNT uptake, localisation of TNT-derived radioactivity in mature tree tissues, and the determination of the degree of TNT-degradation during dendroremediation processes. METHODS: Four-year-old trees of hybrid willow (Salix spec., clone EW-20) and of Norway spruce (Picea abies) were cultivated in sand or ammunition plant soil (AP-soil) in wick supplied growth vessels. Trees were exposed to a single pulse application with water solved [U-14C]-TNT reaching a calculated initial concentration of 5.2 mg TNT per kg dry soil. Two months after application overall radioactivity and extractability of 14C were determined in sand/soil, roots, stem-wood, stem-bark, branches, leaves, needles, and Picea May sprouts. Root extracts were analysed by radio TLC. RESULTS: 60 days after [14C]-TNT application, recovered 14C is accumulated in roots (70% for sand variants, 34% for AP-soil variant). 15-28% of 14C remained in sand and 61% in AP-soil. 3.3 to 14.4% of 14C were located in aboveground tree portions. Above-ground distribution of 14C differed considerably between the angiosperm Salix and the gymnosperm Picea. In Salix, nearly half of above-ground-14C was detected in bark-free wood, whereas in Picea older needles contained most of the above-ground-14C (54-69%). TNT was readily transformed in tree tissue. Approximately 80% of 14C was non-extractably bound in roots, stems, wood, and leaves or needles. Only quantitatively less important stem-bark of Salix and Picea and May shoots of Picea showed higher extraction yields (up to 56%). DISCUSSION: Pulse application of [14C]-TNT provided evidence for the first time that after TNT-exposure, in tree root extracts, no TNT and none of the known metabolites, mono-amino-dinitrotoluenes (ADNT), diaminonitrotoluenes (DANT), trinitrobenzene (TNB) and no dinitrotoluenes (DNTs) were present. Extractable portions of 14C were small and contained at least three unknown metabolites (or groups) for Salix. In Picea, four extractable metabolites (or groups) were detected, where only one metabolite (or group) seemed to be identical for Salix and Picea. All unknown extractables were of a very polar nature. CONCLUSIONS: Results of complete TNT-transformation in trees explain some of our previous findings with 'cold analytics', where no TNT and no ADNT-metabolites could be found in tissues of TNT-exposed Salix and Populus clones. It is concluded that 'cold' tissue analysis of tree organs is not suited for quantitative success control of phytoremediation in situ. RECOMMENDATIONS AND OUTLOOK: Both short rotation Salicaceae trees and conifer forests possess a dendroremediation potential for TNT polluted soils. The degradation capacity and the large biomass of adult forest trees with their woody compartments of roots and stems may be utilized for detoxification of soil xenobiotics.     

Effectiveness of urea in enhancing the extractability of 2,4,6-trinitrotoluene from chemically variant soils

One of the major challenges in developing an effective phytoremediation technology for 2,4,6-trinitrotoluene (TNT) contaminated soils is limited plant uptake resulting from low solubility of TNT. The effectiveness of urea as a solubilizing agent in increasing plant uptake of TNT in hydroponic systems has been documented. Our preliminary greenhouse experiments using urea were also very promising, but further characterization of the performance of urea in highly-complex soil-solution was necessary. The present study investigated the natural retention capacity of four chemically variant soils and optimized the factors influencing the effectiveness of urea in enhancing TNT solubility in the soil solutions. Results show that the extent of TNT sorption and desorption varies with the soil properties, and is mainly dependent on soil organic matter (SOM) content. Hysteretic desorption of TNT in all tested soils suggests irreversible sorption of TNT and indicates the need of using an extractant to increase the release of TNT in soil solutions. Urea significantly (p < 0.0001) enhanced TNT extraction from all soils, by increasing its solubility at the solid/liquid interface. Soil organic matter content and urea application rates showed significant effects, whereas pH did not exert any significant effect on urea catalysis of TNT extraction from soil. The optimum urea application rates (125 or 350 mg kg⁻¹) for maximizing TNT extraction were within the limits set by the agronomic fertilizer-N rates used for major agricultural crops. The data obtained from this batch study will facilitate the optimization of a chemically-catalyzed phytoremediation model for cleaning up TNT-contaminated soils.     

The effects of the insecticide lambda-Cyhalothrin on the earthworm Eisenia fetida under experimental conditions of tropical and temperate regions

Plant Protection Products can affect soil organisms and thus might have negative impacts on soil functions. Little research has been performed on their impact on tropical soils. Therefore, the effects of the insecticide lambda-Cyhalothrin on earthworms were evaluated in acute and chronic laboratory tests modified for tropical conditions, i.e. at selected temperatures (20 and 28 °C) and with two strains (temperate and tropical) of the compost worm Eisenia fetida. The insecticide was spiked in two natural soils, in OECD artificial soil and a newly developed tropical artificial soil. The effects of lambda-Cyhalothrin did rarely vary in the same soil at tropical (LC50: 68.5-229 mg a.i./kg dry weight (DW); EC50: 54.2-60.2 mg a.i./kg DW) and temperate (LC50: 99.8-140 mg a.i./kg DW; EC50: 37.4-44.5 mg a.i./kg DW) temperatures. In tests with tropical soils and high temperature, effect values differed by up to a factor of ten.     

A study of 2,4,6-trinitrotoluene inhibition of benzo[a]pyrene uptake and activation in a microbial mutagenicity assay

A number of in vitro and in vivo studies have determined that binary and complex mixtures may interact to produce a toxicity that could not be predicted based on the individual chemicals. The present study was conducted with a binary mixture of model compounds to investigate possible interactions affecting their mutagenicity. The compounds included Benzo[a]pyrene (BAP), a polycyclic aromatic hydrocarbon that is an indirect-acting mutagen of great environmental concern, and 2,4,6-Trinitrotoluene (TNT), a nitro-aromatic compound that is a direct-acting mutagen frequently found as a soil contaminant at munitions sites. This study indicated that a binary mixture of BAP and TNT failed to induce the positive mutagenic response in Salmonella typhimurium strain TA98 characteristic of either compound alone. Spectrofluorometric analysis of BAP, and kinetic analyses of ³HBAP uptake in the presence or absence of TNT using TA98 cells that were treated or untreated with activated rat liver microsomes were performed. In cells preloaded with BAP, cellular BAP fluorescence was rapidly suppressed in the presence of TNT. Mass spectroscopy of BAP and TNT mixtures revealed a number of products, believed to be the result of complexation and nitration, that may account for the antagonistic action of TNT on BAP-induced mutagenicity in TA98 cells. Further, kinetic studies indicated that TNT inhibited the incorporation of BAP into cells.     

TNT and 4-amino-2,6-dinitrotoluene influence on germination and early seedling development of tall fescue

Cost-effective and environmentally acceptable methods are needed to remediate munitions-contaminated soil. Some perennial grass species are tolerant of soil contaminants and may promote remediation because of their high water use and extensive fibrous root systems. The effects of 2,4,6-trinitrotoluene (TNT) and its reduction product, 4-amino-2,6-dinitrotoluene (4ADNT), on germination and early seedling development of tall fescue (Festuca arundinacea Schreb.) were determined. Tall fescue seeds were germinated in nutrient-free agar containing 0-60 mg TNT litre(-1) or 0-15 mg 4ADNT litre(-1). Germination decreased linearly as TNT concentration increased but was not significantly affected by 4ADNT at these concentrations. Concentrations less than 30 mg TNT litre(-1) or 7.5 mg 4ADNT litre(-1) had little effect on seedling growth and development. Higher TNT or 4ADNT concentrations substantially delayed seedling development, caused abnormal radicle tissue development, and reduced secondary root and shoot growth. Seedling respiration rates decreased linearly with increasing TNT concentration. Experiments indicate that tall fescue may be grown in soils that maintain soil solution concentrations of 30 mg TNT litre(-1) or less.     

Effects of carbaryl, chlorpyrifos and endosulfan on growth, reproduction and respiration of tropical epigeic earthworm, Perionyx excavatus (Perrier)

Effects of sub-lethal doses of carbaryl (1-Naphthyl-methylcarbamate), chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridinyl-phosphorothioate) and endosulfan (6,7,8,9,10,10-Hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepin-3-oxide), respectively a carbamate, an organophosphate and an organochlorine insecticide on growth, reproduction and respiration of the tropical earthworm, Perionyx excavatus (Perrier) were investigated under laboratory conditions. The results showed significant reduction in biomass, production and hatching of cocoon and production of juveniles of the worms exposed to 0.75 to 3.03 mg/kg soil of carbaryl, 0.91 to 3.65 mg/kg soil of chlorpyrifos and 3.75 to 15.0 μg/kg soil of endosulfan corresponding to 12.5 to 50 % of LC(50) value of the respective insecticide for P. excavatus. Endosulfan was found most dangerous among the three insecticides followed by carbaryl and chlorpyrifos. There was no hatching of the worms at endosulfan treatment 5.0 μg/kg soil (25 % LC(50)) or above while the highest dose of carbaryl and chlorpyrifos (50 % of LC(50)) rendered respectively 87.13 and 24.84 % reductions in hatching as compared to control. Chlorpyrifos produced no change in respiration of the worms except at the highest dose, while the worms showed an increase in evolution of CO(2) at all doses of carbaryl and endosulfan. Based on the recommended agricultural dose of each insecticide, it was concluded that application of endosulfan and carbaryl was potentially dangerous to earthworms.     

Deriving freshwater quality criteria for 2,4,6-trichlorophenol for protection of aquatic life in China

Freshwater quality criteria of 2,4,6-trichlorophenol (2,4,6-TCP) were developed with particular reference to the aquatic biota in China, and based on USEPA's guidelines. Acute toxicity tests were performed on nine different domestic species indigenous to China to determine 48 h LC(50) and 96 h LC(50) values for 2,4,6-TCP. In addition, 21 d survival-reproduction test with Daphnia magna, 30 d embryo-larval test with Carassius auratus, 60 d fry-juvenile test with Ctenopharyngodon idellus, 30 d early life stage test with Bufo bufo gargarizans and 96 h growth inhibition test with Scenedesmus obliqaus were also conducted to estimate lower chronic limit and upper chronic limit values. The final acute value (FAV) was 2.01 mg/l 2,4,6-TCP. Acute-to-chronic ratios ranged from 5.01 to 12.2. The final chronic value (FCV) and the final plant value (FPV) of 2,4,6-TCP were 0.226 and 2.24 mg/l respectively. Based on FAV, FCV and FPV for 2,4,6-TCP, a criteria maximum concentration of 1.01 mg/l and a criterion continuous concentration of 0.226 mg/l were derived. The results of this study provide useful data for deriving national or local water quality criteria for 2,4,6-TCP based on aquatic biota in China.     

Endocrine modulation and toxic effects of two commonly used UV screens on the aquatic invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus

The two UV screens 3-benzylidene-camphor (3-BC) and 3-(4'-methylbenzylidene)-camphor (4-MBC) were tested regarding their toxicity and estrogenic activity. The Yeast Estrogen Screen (YES) and two sediment assays with the freshwater invertebrates Lumbriculus variegatus and Potamopyrgus antipodarum were performed. In the YES, both substances activated the human estrogen receptor alpha with EC50 values of 44.2 microM for 3-BC and 44.3 microM for 4-MBC, whereby 4-MBC attained only 8% of the maximal response of 17beta-estradiol. For P. antipodarum embryo production increased after exposure to both substances (EC50 of 4.60 microM 4-MBC=1.17 mg kg(-1)dw) while mortality increased at high concentrations. The reproduction of L. variegatus was decreased by 3-BC with an EC50 of 5.95 microM (=1.43 mg kg(-1)dw) and also by 4-MBC, where no EC50 could be calculated. While reproduction decreased, the worms' weight increased after exposure to 3-BC with an EC50 of 26.9 microM (=6.46 mg kg(-1) dw), hence the total biomass remained unaffected.