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PubMed1996

Cytotoxicity and mutagenicity of 2,4,6-trinitrotoluene and its metabolites.

Composting has been advocated and is being used as an economical method for remediating 2,4,6-trinitrotoluene (TNT)-contaminated soils. However, evidence suggests that TNT is transformed into products of unknown toxicity during the process. This study was undertaken to examine the in vitro cytotoxicity and mutagenicity of TNT and several of its degradation products/metabolites. TNT was equally cytotoxic to H4IIE cells and Chinese hamster ovary-K1 (CHO) cells (LC50 of 4 g/ml vs 24 microg/ml, with overlapping 95% prediction intervals), indicating that TNT does not need to be metabolized to exhibit cytotoxicity. Four metabolites studied, 4-hydroxylamino-2,6-dinitrotoluene; 2-amino-4,6-dinitrotoluene; 4,4',6,6'-tetranitro-2,2'-azoxytoluene; and 2,2',6,6'-tetranitro-4, 4'-azoxytoluene, were equally cytotoxic to both H4IIE and CHO cells. The LC50s were in the 3- to 18-microg/ml range and were not significantly different from TNT cytotoxicity in both cell lines. 4-Amino-2,6-dinitrotoluene (4-A) was moderately less cytotoxic than TNT to H4IIE cells, but was noncytotoxic to CHO cells. This result indicates that 4-A is metabolized to a cytotoxic compound. Both TNT and its metabolites exhibited only slight mutagenicity at high doses in one or both of the mutagenicity assays. While composting may reduce the levels of TNT in composted material, the hazard associated with TNT-contaminated soils is probably lower, but still uncertain.

Animals↗

PubMed1999

Acute toxicity of 2,4,6-trinitrotoluene in earthworm (Eisenia andrei).

2,4,6-Trinitrotoluene (TNT) is an worldwide recalcitrant environmental contaminant and is toxic to a number of organisms including humans. This study examines the acute effects (lethal and biomass changes) of TNT on the oligochaetes species Eisenia andrei, using the 3-day filter paper, and the 7- and 14-day direct contact spiked soil (OECD artificial and forest soil) toxicity tests. Studies using the filter paper test indicated that the lethality of TNT could be detected in the range 1.5 to 14.2 microg/cm(2), with significant biomass (body weight) changes occurring at the lowest concentration. Acute effects (lethality) could not be measured when earthworms were placed on filter paper containing a saturated aqueous solution of TNT. This may indicate that with these exposure conditions, TNT may have been adsorbed to the filter paper, and that this matrix should be saturated with TNT before becoming available to the earthworms. Spiked soil toxicity tests indicated that the E. andrei lethality by TNT was >1.5 times higher when earthworms were exposed to TNT-spiked forest soil (LOEC:260 mg/kg; LC(50) 14 days 222.4 mg/kg) than to spiked OECD artificial soil (LOEC:420 mg/kg; LC(50) 14 days: 364.9 mg/kg). The sublethal effect on biomass change at the selected TNT concentrations in soil was not significant compared to controls. Results indicate that the bioanalytical methods described in this article could be used as TNT toxicity assessment tools. This soil quality test method gives valuable information for the screening of soil toxicity.

Animals↗

PubMed2000

Fate and the biochemical effects of 2,4,6-trinitrotoluene exposure to tiger salamanders (Ambystoma tigrinum).

Biotransformation, metabolic enzyme profiles, and the glutathione antioxidant system in tiger salamanders (Ambystoma tigrinum) from a 14-day exposure to 2,4,6-trinitrotoluene (TNT) in situ were examined. Concentrations of parent compound and metabolites were measured in skin, kidney, and liver tissue. In addition, cytochrome P450 (P450) and cytochrome b5 content and their dependent isozyme activities, ethoxyresorufin O-dealkylation, pentoxyresorufin O-dealkylation, and the glutathione antioxidant system in the skin, liver, lung, kidneys, and blood were evaluated. Considerable differences were found in relative concentrations of TNT and its metabolites in the skin, relative to the liver and kidney. Trace amounts of TNT were detected only in the skin and liver of exposed animals while one of the secondary reduction metabolites, 2,6-diaminonitrotoluene, was found only in liver and kidney. Differences in the metabolite concentrations between systemic organs (liver, kidneys) and the skin suggest that the skin may be important in the primary reduction of TNT. In addition, measurable levels of these basal enzyme indicators were detected; yet of those evaluated only hepatic P450 content was affected by TNT exposure. The qualitative and quantitative differences in TNT and its metabolites in tissues suggest the fate and metabolism of the TNT in salamanders. Furthermore, results indicate that tiger salamanders possess considerable levels of xenobiotic metabolizing and antioxidant enzymes in these tissues but are not sensitive indicators of TNT exposure.

Ambystoma↗

PubMed2000

An in situ respirometric technique to measure pollution-induced microbial community tolerance in soils contaminated with 2,4, 6-trinitrotoluene.

Long-term exposure to 2,4,6-trinitrotoluene (TNT) can induce changes in the structure and activities of soil microbial communities. Such changes may be associated with an elevated microbial tolerance. An in situ respirometry technique based on the analysis of the substrate-induced respiration response to freshly added TNT was used to examine soil microbial tolerance to TNT at the community level. The specific growth rate derived by fitting an exponential equation to respiration data was taken as the measurement endpoint. Microbial tolerance was evaluated using a tolerance index defined as the ratio of the specific growth rate at a spiking dose of 2000 microg TNT/g soil to that of the control with no spiked TNT. Three soils with long-term exposure histories (TNT level in soil: 1.5, 32, and 620 microg TNT/g, respectively) exhibited significantly higher microbial community tolerance to TNT than two uncontaminated control soils. A soil containing 29,000 microg TNT/g exhibited the highest tolerance. Findings from this study support the hypothesis that pollution-induced community tolerance can be used as a means of identifying those compounds that have exerted selective pressure on the community.

Dose-Response Relationship, Drug↗

PubMed2002

Screening for soil toxicity and mutagenicity using luminescent bacteria--a case study of the explosive 2,4,6-trinitrotoluene (TNT).

The presented study explored the suitability of aquatic bioassays based on the marine luminescent bacterium Vibrio fischeri as screening indicators for soil toxicity and mutagenicity. The study consists of two parts: (i) determination of the bacterial toxicity and mutagenicity of the single substance 2,4,6-trinitrotoluene (TNT) and its primary reduced metabolites using three different luminescent bacteria assays and (ii) determination of the water-extractable toxicity and mutagenicity of soil samples taken at a former production plant for TNT showing complex contamination (TNT, metabolites of TNT, PAHs, and heavy metals). Resulting data indicate TNT to be predominantly responsible for the observed biological effects of soil leachates. A strategy for soil toxicity screening based on luminescent bacteria is proposed which may especially be applicable for the case of bioremediation of TNT-contaminated soils. Potentials and restrictions of this approach to soil toxicity assessment are discussed.

Aniline Compounds↗

PubMed1996

Bioremediation of 2,4,6-trinitrotoluene-contaminated soils by two different aerated compost systems.

Two composting systems were compared on a laboratory scale as a bioremediation technology for degradation or immobilization of 2,4,6-trinitrotoluene (TNT) in contaminated soils. The first compost was aerated from the beginning whereas the second compost was only aerated after an anaerobic prephase of 65 days. In the first compost system the TNT concentration declined rapidly by 92% but, at the end, TNT could be partially recovered. During the anaerobic prephase of the second compost system, TNT was almost completely converted to aminodinitrotoluenes, which during the subsequent aeration almost entirely disappeared. In addition, the second compost generated less toxic material than the first one as confirmed by inhibition of bioluminescence of Vibrio fischeri. These data show that microbiological TNT-degradation systems can be successfully designed which are prerequisite for an efficient bioremediation of contaminated soils.

Aerobiosis↗

PubMed1993

Anaerobic transformation of 2,4,6-trinitrotoluene (TNT).

A sulfate-reducing bacterium using trinitrotoluene (TNT) as the sole nitrogen source was isolated with pyruvate and sulfate as the energy sources. The organism was able to reduce TNT to triaminotoluene (TAT) in growing cultures and cell suspensions and to further transform TAT to still unknown products. Pyruvate, H2, or carbon monoxide served as the electron donors for the reduction of TNT. The limiting step in TNT conversion to TAT was the reduction of 2,4-diamino-6-nitrotoluene (2,4-DANT) to triaminotoluene. The reduction proceeded via 2,4-diamino-6-hydroxylaminotoluene (DAHAT) as an intermediate. The intermediary formation of DAHAT was only observed in the presence of carbon monoxide or hydroxylamine, respectively. The reduction of DAHAT to triaminotoluene was inhibited by both CO and NH2OH. The inhibitors as well as DANT and DAHAT significantly inhibited sulfide formation from sulfite. The data were taken as evidence for the involvement of dissimilatory sulfite reductase in the reduction of DANT and/or DAHAT to triaminotoluene. Hydrogenase purified from Clostridium pasteurianum and carbon monoxide dehydrogenase partially purified from Clostridium thermoaceticum also catalyzed the reduction of DANT in the presence of methyl viologen or ferredoxin, however, as the main reduction product DAHAT rather than triaminotoluene was formed. The findings could explain the function of CO as an electron donor for the DANT reduction (to DAHAT) and the concomitant inhibitory effect of CO on triaminotoluene formation (from DAHAT) by the inhibition of sulfite reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗

PubMed1985

Non-genotoxicity of 2,4,6-trinitrotoluene (TNT) to the mouse bone marrow and the rat liver: implications for its carcinogenicity.

2,4,6-Trinitrotoluene (TNT) is structurally related to the rat liver carcinogen 2,4-dinitrotoluene (technical grade), and both compounds are known to be mutagenic to bacteria in vitro. TNT is therefore established as a potential rodent carcinogen; the present paper describes experiments designed to assess if this potential is likely to be expressed in appropriately exposed animals. TNT gave a negative response in the mouse bone marrow micronucleus assay and in an in vivo/in vitro rat liver assay for unscheduled DNA synthesis (UDS). In the latter assay animals are exposed to the test chemical in vivo and their hepatocytes subsequently evaluated for UDS in vitro. The negative response observed for TNT in the liver assay at dose-levels up to 1000 mg/kg was accompanied by a positive response for the hepatocarcinogen 2,4-dinitrotoluene at the lower dose-level of 200 mg/kg. In contrast, the dinitro compound gave a negative response in the micronucleus assay, as was also observed for TNT. It is concluded that the negative response observed for TNT in the liver assay indicates that it is unlikely to be a rat hepatocarcinogen. Nonetheless, high levels of methaemoglobin were observed in the TNT-treated rats and their urine was coloured red. These facts, together with the known toxicities of this agent suggest a possible carcinogenic hazard to the haemopoetic and urinary tissues of animals exposed chronically to it at toxic dose-levels.

Animals↗

PubMed1992

Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacterium Desulfovibrio sp. (B strain) isolated from an anaerobic digester.

A sulfate-reducing bacterium (SRB), Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester (Boopathy and Daniels, Current Microbiology, 23:327-332, 1991) was found to use 2,4,6-trinitrotoluene (TNT) as sole nitrogen source. This bacterium also used nitrate, nitrite, and ammonium as nitrogen source. A long lag period was noticed when TNT or nitrite was used as nitrogen source. Nitrate, nitrite and TNT also served as electron acceptor in the absence of sulfate for this bacterium. Under nitrogen-limiting condition, 100% removal of TNT was observed within 8 days of incubation. The main intermediate observed was diaminonitrotoluene, which was further converted to toluene via triaminotoluene by reductive deamination process. Under nitrogen-rich conditions (presence of ammonium), TNT was converted to diaminonitrotoluene, and toluene was not produced. This isolate did not degrade TNT all the way to CO2. This study demonstrated the possibility of using this isolate to decontaminate the soil and water contaminated with TNT under anaerobic conditions.

Anaerobiosis↗

PubMed1995

Catabolism of 2,4,6-trinitrotoluene by Mycobacterium vaccae.

Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT by M. vaccae. During the cometabolic process, there was transient production of a brown chromophore. This compound was identified as 4-amino-2,6-dinitrobenzoic acid. When M. vaccae was incubated with [14C]TNT and propane, 50% of the added radiolabel was incorporated into the cellular lipid fraction. These results suggest that ring cleavage occurred prior to the incorporation of radiolabelled carbon into phosphatidyl-L-serine, phosphatidylethanolamine, cardiolipin, and other polar lipids.

Biotransformation↗

PubMed2004

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.

Biodegradation, Environmental↗

PubMed2004

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.

Biodegradation, Environmental↗

PubMed2005

Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from Clostridium acetobutylicum ATCC 824.

The genes that encode oxygen-insensitive nitroreductases from Clostridium acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized. The gene products NitA (MW 31 kDa) and NitB (MW 23 kDa) were purified to homogeneity. The NitA and NitB are oxygen-insensitive nitroreductases comprised of a single nitroreductase domain. NitA and NitB enzymes show spectral characteristics similar to flavoproteins. The biochemical characteristics of NitA and NitB are highly similar to those of NfsA, the major nitroreductase from E. coli. NitA exhibited broad specificity similar to that of E. coli NfsA and displayed no flavin reductase activity. NitB showed broad substrate specificity toward nitrocompounds in a pattern similar to NfsA and NfsB of Escherichia coli. NitB has high sequence similarity to NAD(P)H nitroreductase from Archaeoglobus fulgidus. NitA could utilize only NADH as an electron donor, whereas NitB utilized both NADH and NADPH as electron donors with a preference for NADH. The activity of both nitroreductases was high toward 2,4-Dinitrotoluene (2,4-DNT) as a substrate. Both the nitroreductases were inhibited by dicoumarol and salicyl hydroxamate. The nitroreductases showed higher relative expression on induction with TNT, nitrofurazone and nitrofurantoin compared to the uninduced control.

Amino Acid Sequence↗

PubMed2005

2,4,6-Trinitrotoluene inhibits endothelial nitric oxide synthase activity and elevates blood pressure in rats.

2,4,6-Trinitrotoluene (TNT), which is widely used in explosives, is an important occupational and environmental pollutant. Human exposure to TNT has been reported to be associated with cardiovascular dysfunction, but the mechanism is not well understood. In this study, we examine the endothelial nitric oxide synthase (eNOS) activity and blood pressure value following TNT exposure. With a crude enzyme preparation, we found that TNT inhibited the enzyme activity of eNOS in a concentration-dependent manner (IC50 value = 49.4 microM). With an intraperitoneal administration of TNT (10 and 30 mg/kg) to rats, systolic blood pressure was significantly elevated 1 h after TNT exposure (1.2- and 1.3-fold of that of the control, respectively). Under the conditions, however, experiments with the inducible NOS inhibitor aminoguanidine revealed that an adaptive response against hypertension caused by TNT occurs. These results suggest that TNT is an environmental chemical that acts as an uncoupler of constitutive NOS isozymes, resulting in decreased nitric oxide formation associated with hypertension in rats.

Animals↗

PubMed2003

Analysis of aqueous 2,4,6-trinitrotoluene (TNT) using a fluorescent displacement immunoassay.

We report a rapid, simple, and sensitive assay that is potentially amenable to high throughput screening for analysis of 2,4,6-trinitrotoluene (TNT) present in aqueous solutions. The assay is based on the change in fluorescence emission intensity of a fluorescently labeled TNT analogue pre-bound to an anti-TNT antibody that occurs upon its competitive displacement by TNT. The assay can be performed in both cuvette- and 96-well plate-based formats. TNT at a level of 0.5 micro g L(-1) (0.5 ppb) was detected in phosphate buffered saline; detection improved to 0.05 micro g L(-1) (0.05 ppb) for TNT dissolved in artificial seawater.

Antibodies↗

PubMed2005

Development of new rat monoclonal antibodies with different selectivities and sensitivities for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds.

Five new rat monoclonal antibodies (mAbs) for 2,4,6-trinitrotoluene (TNT) and other nitroaromatic compounds, including, especially, the metabolite 2-amino-4,6-dinitrotoluene (2-ADNT), are described. Five heterogeneous, competitive enzyme-linked immunosorbent assays (ELISAs) were developed. Assay 1 uses mAb DNT4 3F6 as recognition element and gives a standard curve for TNT in 40 mmol L(-1) phosphate buffered saline (PBS) with a test midpoint (IC50) of 0.26+/-0.08 microg L(-1) (n=20). Assay 2 (mAb DNT4 4G4) has an IC50 of 0.35+/-0.07 microg L(-1) (n=18), assay 3 (mAb DNT4 1A3) has an IC50 of 0.73+/-0.14 microg L(-1) (n=15), and assay 4 (mAb DNT4 1A7) has an IC50 of 2.32+/-0.70 microg L(-1) (n=15). Assay 5 (mAb DNT2 4B4) is very selective for 2-ADNT and has an IC50 of 8.5+/-1.7 microg L(-1) (n=15) in PBS. These antibodies for nitroaromatic compounds differ not only in their sensitivity but also in their selectivity. Major cross-reactants are 1,3,5-trinitrobenzene, 2-ADNT, 4-amino-2,6-dinitrotoluene (4-ADNT), 2,4-dinitroaniline, 3,5-dinitroaniline, and 2,6-dinitroaniline. Although assay 5 is not highly sensitive, the mAb DNT2 4B4 in this assay is highly selective for 2-ADNT. Of all the compounds tested, only 2,4-dinitroaniline and 3,5-dinitroaniline had relevant cross reactivities, 18% and about 26%, respectively. Two ELISAs, using mAbs DNT4 3F6 and DNT2 4B4, were used to analyze different concentrations of TNT and 2-ADNT, respectively, in three different surface water matrices (river and lake water). Both assays were affected by the matrix, but usually performed well (recovery within the range 70-120%). In addition, these ELISAs were used to analyze mixtures of TNT, 2-ADNT, and 4-ADNT, at three different concentrations, in the same water matrices. A different recognition pattern was clearly visible with both assays and depended on the cross reactivities of the corresponding mAb.

Aniline Compounds↗

PubMed2006

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay.

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNPh-KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh-KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.

Antibodies↗

PubMed2003

Phytotoxicity of 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in spiked artificial and natural forest soils.

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 (</= 1,849.4 +/- 228.2 mg/kg) compared to silica (</= 239.3 +/- 88.0 mg TNT/kg). After 14 days, TNT concentrations in spiked silica and forest soil were reduced up to 80.5% at 55.9 +/- 4.5 mg/kg initial concentration and 94.4% at 91.4 +/- 7.9 mg/kg initial concentration, respectively. Data indicate that TNT is the probable phytotoxicant because it decreased plant emergence and growth in the presence and absence of the ADNT metabolites.

Azocines↗