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.     

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.     

Lethal and subchronic effects of 2,4,6-trinitrotoluene (TNT) on Enchytraeus albidus in spiked artificial soil

The effects of 2,4,6-trinitrotoluene (TNT) exposure in spiked artificial soil on the survival and reproduction rate of the white potworm Enchytraeus albidus were studied. Based on the initial concentrations, TNT in freshly spiked soil decreased enchytraeid survival (21-day LC(50)=422+/-63 (SD)mg/kg, N=3) and fecundity (42-day EC(50)=111+/-34, N=4). Data also indicated that TNT was 5-10 times more lethal to juveniles than adults, and lethality was less pronounced in TNT-spiked soils aged for 21 days. A time-dependent decrease in the TNT concentrations, as well as a concomitant increase in the levels of 2- and 4-aminodinitrotoluene, was observed during the 42-day toxicity test. Taken together, TNT (or one of its metabolites) is more lethal to juvenile than adult enchytraeids. This effect may explain, at least in part, the ability of TNT to decrease fecundity as determined using the enchytraeid mortality-reproduction test.     

Interactions of 2,4,6-trinitrotoluene (TNT) with xenobiotic biotransformation system in European eel Anguilla anguilla (Linnaeus, 1758)

The aim of the present study was to investigate the interaction of 2,4,6-trinitrotoluene (TNT) with liver biotransformation enzymes in European eel Anguilla anguilla (Linnaeus, 1758). Eels were exposed to 0.5, 1 and 2.5 mg/l nominal concentrations of TNT for 6 and 24 h. Modulation of CYP1A1, UDPGT and GST genes was investigated by real-time PCR. Total CYP450 content, NADPH cytochrome c reductase activity, CYP1A and CYP2B-like activities, such as EROD, MROD and BROD, as well as GST and UDPGT activities, were measured by biochemical assays. An in vitro study was performed on EROD in order to evaluate catalytic modulation by TNT. No modulation of the CYP1A1 gene or protein was observed in TNT-exposed eels. On the other hand, a significant decline of EROD and MROD activities was observed in vivo. An increase in NADPH cyt c reductase, and phase II enzymes (UDPGT and GST) were observed at both gene expression and activity levels. The overall results indicated that TNT is a potential competitive inhibitor of CYP1A activities. A TNT metabolic pathway involving NADPH cyt c reductase and phase II enzymes is also suggested.     

Toxicological responses of red-backed salamanders (Plethodon cinereus) to subchronic soil exposures of 2,4,6-trinitrotoluene

Since World War I, trinitrotoluene (TNT) has been the most commonly used explosive. Environmental contamination associated with synthesis, manufacture of weapons, and use during training exercises has been extensive, with soil concentrations reaching 145,000 mg/kg. Some of these areas include habitats for amphibian species. Earlier studies have shown that salamanders dermally absorb TNT from soil. To ascertain what soil concentrations of TNT are toxic to amphibians, red-backed salamanders (Plethodon cinereus) were exposed to one of five concentrations of TNT in soil for 28 d and evaluated for indicators of toxicity. A total of 100 salamanders were randomly sorted by weight and assigned to target TNT concentrations in soil of either 3,000, 1,500, 750, 325, or 0 mg/kg dry weight. Food consisted of uncontaminated flightless Drosophila melanogaster. Survival was reduced in salamanders exposed to 1,500 and 3,000 mg/kg by 10 and 55%, respectively. Most mortality/morbidity occurred within the first week of exposure. Salamanders had a reduction in hemoglobin at 750 mg/kg or greater and a reduction in red blood cell concentration at 1,500 mg/kg or greater. Food consumption was affected in salamanders at 750 mg/kg or greater; a reduction in body mass and liver glycogen content also occurred at and above this concentration. Splenic congestion also was observed in salamanders from these groups. These data suggest that soil TNT concentrations of 373 +/- 41.0 mg/kg or greater result in reduced body mass, reduced feed intake, and hematological effects.     

Assessing risk using different cholesterol-screening methods

The purpose of this study was to compare total capillary, total venous, risk ratio, high-density lipoprotein and low-density lipoprotein measures of cholesterol to determine whether total capillary cholesterol is a valid measure to use in cholesterol screening. An announcement and a registration form were distributed with employee paychecks announcing a cholesterol-screening programme. Capillary and venous samples were collected from screening participants (n=285). Results indicated false negatives in total capillary cholesterol in 17.21-34.4% of cases compared with other methods of cholesterol measurement. Due to the high number of misclassifications, health educators should not use total capillary cholesterol as a predictor of risk for heart disease, nor as a measure for referral during cholesterol screenings.     

Avoidance of Cu- and Zn-contaminated soil by three ecologically different earthworm species

Earthworm avoidance response to soils contaminated with harmful substances has been proposed as a potential tool for assessing soil toxicity with low test effort. In the present study, the objective was to find out whether three ecologically different earthworm species, Aporrectodea tuberculata (Eisen), Lumbricus rubellus (Hoffmeister), and Dendrobaena octaedra (Savigny), avoid soils simultaneously spiked with Cu and Zn. In addition, metal-contaminated field soil taken close to a Cu-Ni smelter was tested with A. tuberculata using a two-section avoidance lest procedure. All three earthworm species clearly avoided Cu/Zn contaminated soil but differently: D. octaedra was the most sensitive species, responding to low metal concentrations, whereas L. rubellus responded only to the highest metal concentration tested, being the least sensitive species. Moreover, A. tuberculata showed clear avoidance response to the metal contaminated field soil. In conclusion, the results indicate that earthworm avoidance behavior is an ecologically relevant parameter for assessing harmfulness of metal contaminated soils, both spiked and field-contaminated soils. However, it is important to consider the specific species to be used in the earthworm avoidance test procedure.     

Toxicity and bioaccumulation of 2,4,6-trinitrotoluene in fathead minnow (Pimephales promelas)

Few studies have determined the toxicity and bioaccumulation potential of explosive compounds in freshwater fish. In the present study, fathead minnow (Pimephales promelas) were exposed to a range of 2,4,6-trinitrotoluene (TNT) concentrations (0.44-44 micromol/L [0.1-10 mg/L] and 4.4-22.0 micromol/L [1.0-5.0 mg/L] in 4- and 10-d experiments, respectively). Median lethal concentrations of 11.93 micromol/L (2.7 mg/L; 95% confidence limit [CL], 10.29-13.83 micromol/L) and 9.68 micromol/L (2.20 mg/L; 95% CL, 9.17-10.22 micromol/L) were calculated in the 4- and 10-d experiments, respectively, and median lethal body residue of 101.0 micromol/kg (95% CL, 86.0-118.7 micromol/kg) was calculated in 4-d experiments. To study bioaccumulation, fish were exposed to 4.4 micromol/L (1 mg/L) of TNT for 12 h. Rapid bioaccumulation of TNT occurred within the first 10 min of exposure (ku = 30.4 L/kg/ h). Elimination of sigmaTNT (molar sum of TNT and degradation products 2- and 4-aminodinitrotoluenes) was fast, with an elimination rate (ke) of 2.24/h and a short half-life (0.31 h). The bioconcentration factors determined using 6-h mean tissue and water concentrations of sigmaTNT were 8.40 and 4.68 L/kg for the uptake experiment and the uptake portion of the elimination experiments, respectively. To determine the target organ for TNT in fish, juvenile fathead minnow were exposed to 2.2 micromol/L (0.5 mg/L) of [14C]TNT for 10 d. Radiolabeled compounds primarily bioaccumulated in the visceral tissues and spleen in comparison to gill, brain, muscle, and remainder tissue groups. The present study demonstrates the low bioaccumulation potential and rapid uptake of TNT in the fathead minnow.     

Soil-Based Phytotoxicity of 2,4,6-Trinitrotoluene (TNT) to Terrestrial Higher Plants

Seed germination and early stage seedling growth tests were conducted to determine the ecotoxicological threshold of 2,4,6-trinitrotoluene (TNT) in two soils of different properties. Soils were amended up to 1,600 mg TNT kg⁻¹ soil and four representative species of higher plants, two dicotyledons (Lepidium sativum L., common name: cress; and Brassica rapa Metzg., turnip) and two monocotyledons (Acena sativa L., oat; and Triticum aestivum L., wheat), were assessed. Cumulative seed germination and fresh shoot biomass were measured as evaluation endpoints. Phytotoxicity of TNT was observed to be affected by soil properties and varied between plant species. Cress and turnip showed higher sensitivity to TNT than did oat and wheat. The lowest observable adverse effect concentration (LOAEC) of TNT derived from this study was 50 mg kg⁻¹ soil. In contrast to high TNT concentrations, low levels of TNT, i.e., 5–25 mg kg⁻¹ soil for cress and turnip and 25–50 mg kg⁻¹ for oat and wheat, stimulated seedling growth. Oat was capable of tolerating as much as 1,600 mg TNT kg⁻¹ and demonstrated a potential ability of TNT detoxification in one of the soils tested, suggesting that this plant might be useful in the bioremediation of TNT contaminated soils. Received: 15 May 1998/Accepted: 30 September 1998     

TNT,RDX, and HMX decrease earthworm (Eisenia andrei) life-cycle responses in a spiked natural forest soil

Sublethal and chronic toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) on earthworm Eisenia andrei in a sandy forest soil were assessed. Various reproduction parameters of fecundity (total and hatched number of cocoons, number of juveniles, and their biomass) were significantly decreased by TNT (> or = 58.8 +/- 5.1 mg/kg dry soil), RDX (> or = 46.7 +/- 2.6 mg/kg), and HMX (> or = 15.6 +/- 4.6 mg/kg). These effects occurred at much lower concentrations than those reported earlier using artificial soil preparations. Growth of adults was significantly decreased in the TNT-spiked natural soils at 136.2 +/- 25.6 mg/kg dry soil, the highest concentration having no significant mortality. In contrast, survival and growth were not significantly reduced at relatively high measured concentrations of RDX (167.3 mg/kg) and HMX (711.0 mg/kg). Although TNT, RDX, and HMX share a common life-cycle response ( i.e., decreased juvenile counts), a number of differences related to other reproduction parameters (e.g., productivity of cocoons) was observed. These results indicate that the tested explosives do not support a common mechanism of toxicity, at least in the earthworm, probably due to differences in their physical-chemical properties as well as metabolites formed during exposure.     

Ecotoxicity of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil

Soil samples from a former cokery site polluted with polycyclic aromatic hydrocarbons (PAHs) were assessed for their toxicity to terrestrial and aquatic organisms and for their mutagenicity. The total concentration of the 16 PAHs listed as priority pollutants by the US Environmental Protection Agency (US-EPA) was 2634+/-241 mg/kgdw in soil samples. The toxicity of water-extractable pollutants from the contaminated soil samples was evaluated using acute (Vibrio fischeri; Microtox test, Daphnia magna) and chronic (Pseudokirchneriella subcapitata, Ceriodaphnia dubia) bioassays and the EC values were expressed as percentage water extract in the test media (v/v). Algal growth (EC50-3d=2.4+/-0.2% of the water extracts) and reproduction of C. dubia (EC50-7d=4.3+/-0.6%) were the most severely affected, compared to bacterial luminescence (EC50-30 min=12+/-3%) and daphnid viability (EC50-48 h=30+/-3%). The Ames and Mutatox tests indicated mutagenicity of water extracts, while no response was found with the umu test. The toxicity of the soil samples was assessed on the survival and reproduction of earthworms (Eisenia fetida) and collembolae (Folsomia candida), and on the germination and growth of higher plants (Lactuca sativa L.: lettuce and Brassica chinensis J.: Chinese cabbage). The EC50 values were expressed as percentage contaminated soil in ISO soil test medium (weight per weight-w/w) and indicated severe effects on reproduction of the collembola F. candida (EC50-28 d=5.7%) and the earthworm E. fetida (EC50-28 d=18% and EC50-56 d=8%, based on cocoon and juvenile production, respectively). Survival of collembolae was already affected at a low concentration of the contaminated soil (EC50-28 d=11%). The viability of juvenile earthworms was inhibited at much lower concentrations of the cokery soil (EC50-14 d=28%) than the viability of adults (EC50-14 d=74%). Only plant growth was inhibited (EC50-17d=26%) while germination was not. Chemical analyses of water extracts allowed us to identify inorganic water-extractable pollutants as responsible for toxicity on aquatic species, especially copper for effects on D. magna and C. dubia. The soil toxicity on collembolae and earthworms could be explained by 4 PAH congeners-fluorene, phenanthrene, pyrene, and fluoranthene. Yet, toxicity of the cokery soil as a whole was much lower than toxicity that could be deduced from the concentration of each congener in spiked soils, indicating that pollutants in the soil became less bioavailable with ageing.     

Influence of dietary 2,4,6-trinitrotoluene exposure in the northern bobwhite (Colinus virginianus)

The risk to wildlife from exposure to the explosive, 2,4,6-trinitrotoluene (TNT) has been a concern at numerous military installations where it has been found in the soil. To date, no published data are available describing effects of TNT exposure in an avian species. Subchronic dietary exposure to TNT was therefore evaluated in a species of management concern at military installations, the northern bobwhite (Colinus virginianus). Adult male and female quail (n = 5/sex/dose) were given commercial feed containing 3,000, 1,500, 750, and 100 mg/kg TNT for 90 d following the determination of an acute lethal dose and a 14-d range finding study. Dietary TNT intake caused a dose-dependent decrease in total red blood cell counts, packed cell volume, total plasma protein, blood prolymphocytes, and blood lymphocytes. An increased trend in late apoptotic/necrotic blood leukocytic cells was also observed in TNT-exposed birds, as was hemosiderosis in the liver. With the exception of hemosiderosis, these trends were statistically significant yet of questionable biological significance. Since treatment-related responses in this preliminary study were variable, a conservative interpretation is suggested. However, since these treatments had concentrations that were a log-fold or more than doses in similar studies using mammals, these data suggest that northern bobwhite are less sensitive to oral exposures of TNT than mammals.     

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.     

2,4,6-Trinitrotoluene (TNT) air concentrations,hemoglobin changes,and anemia cases in respirator protected TNT munitions demilitarization workers

Purpose  

2,4,6-Trinitrotoluene (TNT) is an explosive used in munitions production that is known to cause both aplastic and hemolytic anemia in exposed workers. Anemia in a TNT worker is considered a sentinel health event (occupational) (SHE(O)) in the United States (US). Deaths have been reported secondary to aplastic anemia. Studies have shown that TNT systemic absorption is significant by both the respiratory and dermal routes. No studies encountered looked at hemoglobin change or anemia cases in respiratory protected workers. It is hypothesized that respiratory protection is insufficient to protect TNT workers from the risk of anemia development and hemoglobin concentration drop.     

Toxicity of oral exposure to 2,4,6-trinitrotoluene in the western fence lizard (Sceloporus occidentalis)

Contamination of the soil with the explosive 2,4,6-trinitrotoluene (TNT) has been found at military sites, many of which are habitats used by reptiles. To provide data useful in assessing ecological risk for reptilian species, acute, subacute, and subchronic oral toxicity studies were conducted with the western fence lizard (Sceloporus occidentalis). Oral median lethal dose (LD50) values for TNT in corn oil were 1,038 and 1,579 mg/kg of body weight for male and female lizards, respectively. Overt signs of toxicity included chromaturia, abdominal enlargement, and tremors. A 14-d subacute study followed in which male lizards were orally dosed with TNT (corn oil) at 0, 33, 66, 132, 263, 525, and 1,050 mg/kg of body weight each day. Clinical signs of toxicity, while similar to the LD50 study, were more subtle and noted in lizards receiving TNT amounts of at least 66 mg/kg/d. Chromaturia was an early consistent sign, often preceding the onset of adverse effects. Male lizards in the 60-d subchronic study were dosed at 0, 3, 15, 25, 35, and 45 mg/kg/d with nearly complete survival (>90%) for lizards in all treatments. Changes in food consumption and body weight were observed at 35 and 45 mg/kg/d. Alterations in hematological end points; blood chemistries (albumin, total protein, alkaline phosphatase, calcium); kidney, spleen, and liver weights; and adverse histopathology were observed in lizards exposed at 25 to 45 mg/kg/d. Testosterone concentration, sperm count, and motility were variable between treatments. Although not significant, incidences of hypospermia and testicular atrophy were observed in some individuals. Together, these data suggest a lowest-observed-adverse effect level of 25 mg/kg/d and a no-observed-adverse effect level of 15 mg/kg/d in S. occidentalis.     

Assessing convergent validity of health-state utilities obtained using different scaling methods.

A number of empirical studies have attempted to assess the convergent validity of health-state utilities obtained using two or more scaling methods (standard gamble, time tradeoff, rating scale, magnitude estimation, equivalence technique, and willingness-to-pay). The data from these studies can be mapped onto an N x K matrix, where N and K are the numbers of respondents and health states, respectively, and each matrix cell consists of a pair of health-state utilities, one obtained using scaling method X and the other obtained using scaling method Y. The Pearson's rassessing convergent validity can then be computed as 1) the unraveled correlation over all N x K data pairs, 2) the mean within-respondent correlation, 3) the mean within-health-state correlation, or 4) the correlation of the across-respondents means. These four different ways of computing the correlation do not necessarily yield the same results. The appropriateness of each method of computing the correlation is considered.     

Acute toxicity of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene in the adult bullfrog (Lithobates catesbeiana)

2,4,6-Trinitrotoluene (TNT) is one of the most prevalent high explosives in the environment. 2,4-Dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) are the most common isoforms of dinitrotoluene. The goal of this study was to determine the acute toxic effects of TNT, 2,4-DNT, and 2,6-DNT in adult male bullfrogs. The LD(50) for TNT was 1,060 mg/kg BW while the LD(50 )for 2,4-DNT and 2,6-DNT was 1,098 mg/kg BW. All three compounds elicited similar symptoms of toxicity including changes of skin color, body weight, development of seizures, liver and kidney necrosis, and lung cyanosis. Relative organ weights did not show significant change.     

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. Received: 4 February 2002/Accepted: 24 July 2002