Fate and Transport of 2,4,6-Trinitrotoluene in Loams at a Former Explosives Factory

Natural attenuation processes affecting 2,4,6-trinitrotoluene (TNT) were determined within loams for two study areas at the former Explosives Factory Maribyrnong, Australia. TNT fate and transport was investigated through spectrophotometric/High Performance Liquid Chromatography (HPLC) analyses of soil and groundwater, adsorption and microcosm testwork. A five tonne crystalline TNT source zone delineated within near surface soils at the base of a TNT process waste lagoon was found to be supplying aqueous TNT loading (7 ppm) to subsurface soils and groundwater. The resultant plume was localized within the loam aquitard due to a combination of natural attenuation processes and hydrogeological constraints, including low hydraulic conductivity and upward hydraulic gradients. Freundlich described sorptive partitioning was the main TNT sink (K_F = 29 mL/g), while transformation rates were moderate (1.01 × 10^-4 h^-1) under the aerobic conditions. Increasing 2-amino-4,6-dinitrotoluene predominance over 4-amino-2,6-dinitrotoluene was discovered with depth (in situ) and time (microcosms). Simplified dissolution rate calculations indicate that without mitigation of the TNT source, contaminant persistence within the vadose zone may approach 2000 years, while ATRANS20 simulations demonstrate that the TNT plume propagates very slowly along the flow path within the aquitard.     

Polymorphic spawning-nest types in a local population of the fluvial sculpin, Cottus nozawae (Teleostei: Cottidae)

The nesting behavior of a fluvial sculpin Cottus nozawae population was investigated in a stream–reservoir system on the Sainai River, northern Honshu Island, Japan in 2003, 2004 and 2006. Except for the usual rock nest-spawning type (RSN), two new types of spawning nest were found: hole nest spawning (HNS) and crevice nest spawning (CNS). The HNS type was characterized by males digging one or more nest holes in the wall of the banks of the stream and reservoir, or on the sandy silt bottom of the reservoir. The CNS type involved the use of a crevice between large boulders or crevice of crag for their nests. The HNS type was dominant in the reservoir section, whereas the RNS type was dominant in the rapid riffles of stream sections. The HNS and CNS types appeared to be rarely or first observed in the Cottus species. These new nest-spawning types might have occurred in relation to the artificial environment changes in the river by dam construction.     

Role of vocalizations in the reproductive cycle of ring doves (Streptopelia risoria): effects of hypoglossal nerve section on the reproductive behavior and physiology of the female

The role of vocalizations in the reproductive cycle of female ring doves was investigated. Two-stage bilateral hypoglossal nerve section (HNS) was performed on adult females to alter their cooing and they were observed for courtship behavior with the same stimulus males used in preoperation behavioral tests. The HNS females showed a reduction in both the vocalization and wing flipping components of nest-coos but no changes in other female courtship behaviors. In addition, the HNS females failed to show the typical male courtship-induced follicular growth observed in sham-operated females. The behavior of stimulus males did not differ between groups and therefore could not account for the failure of follicular growth in the HNS females. These data suggest that the female's performance of nest-coos, or factors associated with it, may stimulate her own follicular growth.     

The hypothalamic neuropeptide oxytocin is required for formation of the neurovascular interface of the pituitary

The hypothalamo-neurohypophyseal system (HNS) is?the neurovascular structure through which the hypothalamic neuropeptides oxytocin and arginine-vasopressin exit the brain into the bloodstream, where they go on to affect peripheral physiology. Here, we investigate the molecular cues that regulate the neurovascular contact between hypothalamic axons and neurohypophyseal capillaries of the zebrafish. We developed a transgenic system in which both hypothalamic axons and neurohypophyseal vasculature can be analyzed in?vivo. We identified the cellular organization of the zebrafish HNS as well as the dynamic processes that contribute to formation of the HNS neurovascular interface. We show that formation of this interface is regulated during development by local release of oxytocin, which affects endothelial morphogenesis. This cell communication process is essential for the establishment of a tight axovasal interface between the neurons and blood vessels of the HNS. We present a unique example of axons affecting endothelial morphogenesis through secretion of a neuropeptide.     

Changes induced by formalin in the hypothalamic neurosecretory system of the spotted owlet, Athene brama temminck.

Histological changes induced in the HNS of the spotted owlet, Athene brama Temminck, by injection of 1 ml 5 or 10% formalin are described. No difference could be detected in the response of the HNS to 5 or 10% formalin administration. In the HNS of birds killed within 5 min of formalin administration, there was only partial depletion of NSM from the neurons, the tract and the NL; the quantity of NSM in the AME remained more or less unchanged. In animals killed 10-90 min after formalin injection, the depletion of NSM from the neurons, the tract and the NL was more complete. The neurons of the preoptic division of the SON exhibited the maximum response; these neurons were also moderately hypertrophied. The NL also was hypertrophied in some animals; the NSM in the AME registered only a partial loss. The interval between formalin administration and killing did not influence the degree of changes in the HNS. The depletion of NSM was no greater at 90 min following formalin injection than at 10 min. Since it is well established that formalin stress causes augmented secretion of ADH and that there is a close functional relationship existing between ADH and NSM, the depletion of NSM noticed in the HNS of the spotted owlet following formalin administration is interpreted as indicating augmented secretion of ADH. Hence it seems that the response of the HNS of birds to formalin stress are comparable to those of the HNS of mammals. The results thus provide histological evidence in favour of the concept that stressful stimuli cause increased secretion of ADH.     

Fate of TNT and Its Transformation Products in Mixed Aerobic Cultures

The fate of 2,4,6-trinitrotoluene (TNT) and TNT transformation products in two aerobic enrichment cultures was investigated. Contaminant fate was assessed through analysis of TNT and its oxygen-stable aminated derivatives using capillary electrophoresis and by tracking the distribution of ¹⁴C-labeled products in either the dissolved, mineralized, or biomass fractions. TNT transformation products were generated by reduction with Fe(0), reduction by S^2-, and transformation by Clostridium acetobutylicum and by Eichornia crassipies (water hyacinth). Enrichment cultures varied in the growth substrate and nitrogen source supplied. The dextrose-fed mixed culture (DMC) was enriched on dextrose with yeast extract providing nitrogen for growth, whereas the anthranilic acid-fed mixed culture (AMC) received anthranilic acid as its source of both energy and nitrogen. Each culture transformed TNT, but their product distributions varied. The DMC exhibited higher levels of biomass association, whereas the AMC produced higher levels of aminated nitrotoluenes and unidentified water-soluble products. Neither mineralized TNT to a significant degree. TNT disappearance was observed in all transformation systems, along with the formation of water-soluble products; however, formation of aminated nitrotoluenes was observed only in the sulfide systems. Neither aerobic culture was capable of mineralizing the TNT transformation products introduced, regardless of the transformation method used to prepare them. The distribution of products between the aqueous phase and the biomass did vary between cultures and was affected by the transformation system used.     

Ipsilateral diminution of CRF-granules after unilateral hypothalamic lesions

Summary Axons terminating in the outer layer of the median eminence of rats contain light microscopically visible granules. The granules are assumed to represent a corticotropin-releasing factor and, therefore, are called CRF granules. To find out whether neurons containing CRF-granules originate and run together with the neurons of the hypothalamus-neural lobe system (HNS), the effect of unilateral lesions in the HNS on the amount and distribution of CRF-granules was studied in bilaterally adrenalectomized rats.HNS lesions prevented the adrenalectomy-induced increase in CRF granules on the side of the lesion. Lesions outside the HNS or sham lesions did not influence the amount and distribution of the granules. The findings suggest that CRF-granules are located in terminals of neurons whose perikarya are situated in magnocellular hypothalamic nuclei. It can also be concluded that the axons of these neurons run within the HNS and do not decussate.Supported by the Deutsche Forschungsgemeinschaft (Bo 392/2)     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Surface plasmon resonance immunosensor for highly sensitive detection of 2,4,6-trinitrotoluene

We have examined the sensing characteristics of a surface plasmon resonance (SPR) immunoassay for the detection of 2,4,6-trinitrotoluene (TNT) using an immunoreaction between 2,4,6-trinitrophenol-ovalbumin (TNP-OVA) conjugate and anti-2,4,6-trinitrophenol antibody (anti-TNP antibody). TNP-OVA conjugate was attached to a SPR-gold sensing surface by means of physical immobilization, which undergoes binding interaction with anti-TNP antibody. Both the immobilization and binding processes were studied from a change in the SPR-resonance angle. The quantification of TNT is based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-OVA conjugate and anti-TNP antibody was inhibited in the presence of free TNT in solution. The decrease in the resonance angle shift is proportional to an increase in concentration of TNT used for incubation. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range from 0.09 to 1000 ng/ml with good stability and reproducibility. The immunosensor developed could detect TNT as low as 0.09 ng/ml, within a response time of approximately 22 min. The sensor surface was regenerated by a brief flow of pepsin solution, which disrupts the antigen-antibody complex without destroying the conjugate biofilm. Cross-reactivity of the SPR sensor to some structurally related nitroaromatic derivative and the detection of TNT in the presence of these nitroaromatic compounds were investigated. The cross-reactivity of the SPR sensor to 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 2-amino-4,6-dinitrotoluene (2A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4A-2,6-DNT) were very low (< or =1.1%). The analytical characteristics of the proposed immunosensor are highly promising for the development of new field-portable sensors for on-site detection of landmines.     

Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet

Previous work has shown that bacterial isolates from the sheep rumen are capable of detoxifying 2,4,6-trinitrotoluene (TNT) into polar constituents. In this study, the dietary effects of TNT on the sheep rumen microbial community were evaluated using molecular microbiology ecology tools. Rumen samples were collected from sheep fed with and without TNT added to their diet, genomic DNA was extracted, and the 16S rRNA-V3 gene marker was used to quantify changes in the microbial population in the rumen. Control and treatment samples yielded 533 sequences. Phylogenetic analyses were performed to determine the microbial changes between the two conditions. Results indicated the predominant bacterial populations present in the rumen were comprised of the phyla Firmicutes and Bacteroidetes, irrespective of presence/absence of TNT in the diet. Significant differences (P < 0.001) were found between the community structure of the bacteria under TNT (−) and TNT (+) diets. Examination of the TNT (+) diet showed an increase in the clones belonging to family Ruminococcaceae, which have previously been shown to degrade TNT in pure culture experiments.     

2,4,6-trinitrotoluene reduction by an Fe-only hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K(m) of 152 micro M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R(2) = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.     

Differences in the biotransformation of 2,4,6-trinitrotoluene (TNT) between wild and axenically grown isolates of Myriophyllum aquaticum

The aim of this study was to demonstrate the potential for aquatic plants and their associated microbes to bioremediate wetland sites contaminated with 2,4,6-trinitrotoluene (TNT). The transformation of TNT was studied using both wild and axenically grown isolates of Myriophyllum aquaticum (parrot feather). Differences in TNT transformation rates and nitroaromatic metabolites were observed between different plants. The wild isolates, containing a consortium of associated microorganisms, transformed TNT into 2-amino-4,6-dinitrotoluene (2-A-DNT) and 4-amino-2,6-dinitrotoluene (4-A-DNT) via 2- and 4-hydroxylamino-dinitrotoluene, which were detected as intermediates. The wild M. aquaticum also converted the metabolites, 2-A-DNT and 4-A-DNT, into low levels of 2,4-diaminotoluene (2,4-DAT). The axenically grown plants, containing no cultureable microorganisms, also transformed TNT into 2-A-DNT and 4-A-DNT, but at a much lower rate than that observed for the wild isolates. Unlike the wild plants, axenically grown M. aquaticum could not transform either 2-A-DNT or 4-A-DNT into 2,4-DAT over the incubation period. The differences in the performance between these plants could indicate that plant-associated microorganisms assisted in the overall transformation of TNT. For each plant, unidentifiable metabolites were observed and the soluble monoamino-derivatives present in the wild and axenic medium accounted for 14 and 7% of the initial TNT concentration, respectively. Thus, the majority of nitroaromatic derivatives remained associated with the plant tissues. Furthermore, only 7 and 3% of the initial TNT concentration were extracted as monoamino-derivatives from the tissues of the wild and axenically grown plants, respectively.     

Alternative pathways of the initial transformation of 2,4,6-trinitrotoluene by yeasts

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candida sp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.     

An improved method for the cell cycle synchronization of <Emphasis Type="Italic">Vicia faba</Emphasis> root meristem cells

Plant root meristem cells divide asynchronously which makes biochemical analysis of cell cycle regulation particularly difficult. In the present article a high level of cell cycle synchronization in Vicia faba root meristems was obtained by using a rich medium (HNS), special culture conditions and a double-block method with replication inhibitor—hydroxyurea (HU). Two HU concentrations were tested and different periods of the first and the second synchronization, and of cycle recommencement between the first and the second blockage. The level of synchronization was estimated on the basis of ³H-thymidine labeling indices, mitotic, and phase indices and indices determining the percentage of G1 and G2 cells, which were identified by cytophotometric measurements of DNA content in individual nuclei. The highest level of cell cycle synchronization was obtained after double treatment of meristems with 1.25 mM HU (18 and 12 h) separated by 6-h incubation in HNS without HU. During the second postincubation in HNS in subsequent hours: 4, 7, 10, 11, over 90% of cells in the S phase, nearly 70% in G2 phase, 86% in mitosis, and nearly 70% in G1 phase were received, respectively. The use of 2.5 mM HU in a similar experimental procedure caused disturbed divisions.     

Detection of 2,4,6-trinitrotoluene in seawater using a reversed-displacement immunosensor

Reported in this study are the experimental design and results of an immunosensor for the detection of the explosive, 2,4,6-trinitrotoluene (TNT) in seawater using a reversed-displacement format. This reversed-displacement immunosensor methodology has successfully measured TNT in seawater by direct injection, eliminating the need for preconcentration or pretreatment of samples. A microcolumn containing an Affi-Gel resin derivatized with a 2,4,6-trinitrobenzene (TNB) moiety and a fluorophore-labeled anti-TNT antibody composed the immunoassay reactive chamber. Fluorophore-labeled anti-TNT antibody was incubated with the modified Affi-Gel resin until binding equilibrium was reached. Under a constant flow, samples containing TNT were introduced into the flow stream displacing the fluorophore-labeled TNT antibody. Limits of detection were 2.5ng/mL or part-per-billion (ppb) for TNT in saline buffer and 25ppb in seawater with an analysis time of 10 min. Two anti-TNT antibodies with differing binding affinities were compared in the reversed-displacement assay format, and a correlation between affinity and detection limits was observed. Furthermore, we have demonstrated that the reversed-displacement format can be used to screen seawater samples containing TNT, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.     

Localization of arginine vasotocin (AVT) mRNA in extrasomal compartments of magnocellular neurons in the chicken hypothalamo-neurohypophysial system

In the chicken, arginine vasotocin (AVT) is produced in and secreted by magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei. To test the hypothesis of axonally transported AVT mRNA, the localization of AVT mRNA within extrasomal, axonal/dendritic compartments in the chicken hypothalamo-neurohypophysial system (HNS) were examined using AVT specific in situ hybridization histochemistry (ISHH) and RT-PCR. Many perikarya in the PVN and external--but none in the ventral subgroup of the SON show ISHH signals clearly extended into one or two processes, some with branching collaterals, traceable over a distance of more than 100 microns. Furthermore by using RT-PCR, AVT mRNA was detected in the median eminence and neurohypophysis representing the distal parts of the HNS, mainly consisting of axons and/or axon terminals. These observations of axonal mRNA offer new insights to the organization and function of the avian HNS.     

Biotransformation of 2,4,6-trinitrotoluene (TNT) by the fungus Fusarium oxysporum

The fungus Fusarium oxysporum was isolated and identified from the aquatic plant M. aquaticum. The capability of this fungus to transform 2,4,6-trinitrotoluene (TNT) in liquid cultures was investigated TNT was added to shake flask cultures and transformed into 2-amino-4,6-dinitrotoluene (2-A-DNT), 4-amino-2,6-dinitrotoluene (4-A-DNT), and 2,4-diamino-6-nitrotoluene (2,4-DAT) via 2- and 4-hydroxylamino-dinitrotoluene derivatives, which could be detected as intermediate metabolites. Transformation of TNT, 2-A-DNT, and 4-A-DNT was observed by whole cultures and with isolated mycelium. Cell-free protein extracts from the extracellular, soluble, and membrane-bound fractions were prepared from this fungus and tested for TNT-reducing activity. The concentrated extracellular culture medium was unable to transform TNT; however, low levels of TNT transformation were observed by the membrane fraction in the presence of nicotinamide adenine dinucleotide phosphate in an argon atmosphere. A concentrated extract of soluble enzymes also transformed TNT, but to a lesser extent. When TNT toxicity was studied with this fungus, a 50% decrease in the growth of F. oxysporum mycelium was observed when exposed to 20 mg/L TNT.     

Expression of multiple troponin T variants in neonatal chicken breast muscle

The types of troponin-T (TNT) expressed in neonatal chicken breast muscle were examined by two-dimensional gel electrophoresis (2-D PAGE), immunoblotting, and peptide mapping. When troponin from neonatal chicken breast muscle or whole lysate of the muscle was displayed on 2-D PAGE, multiple spots were observed in the TNT region on the gel. They differed slightly from those in adult breast- and leg-type TNT, but were positively stained with the antibody specific for TN-T. These results indicate that multiple spots observed in the TNT region are all TNT isoforms. The TNT isoforms in the neonatal breast muscle were classified into two groups, based on size. Each group contained about five variants. The first group with a larger size was in the molecular weight range of adult breast TNT, while the smaller-sized second group was in the molecular weight range of adult leg TNT. Overall peptide map patterns of variants in the first group and also that of adult breast TNT resembled each other, whereas those of variants in the second group were similar to that of adult leg TNT. The TNT of adult breast-type appeared at about 2- to 3-weeks posthatch, and thereafter became a major TNT isoform.     

Physiological function of water channels as affected by salinity in roots of paprika pepper

The sap flow (Jv) and the osmotic pressure-dependent hydraulic conductance (L₀) of detached exuding root systems from paprika pepper plants (cv. Albar) were measured. Plants stressed with NaCl (30 m M ) and with six times the macronutrients of the Hoagland nutrient solution (6×HNS) were compared with controls grown in complete Hoagland nutrient solution. Jv of +NaCl and +6×HNS plants decreased markedly, but recovered to values similar to those of controls after removal of the treatments. Hydraulic conductance L₀ was always less in NaCl plants than in controls and 6×HNS. A total increase in the ion concentration of the xylem (except Na⁺ and Cl⁻) was observed with both treatments. In control and 6×HNS plants, HgCl₂ treatment (50 μ M ) caused a sharp decline in L₀ to values similar to those of NaCl-stressed roots, but were restored by treating with 5 m M dithiothreitol (DTT). However, in NaCl roots only a slight effect of Hg²⁺ and DTT was observed. In each treatment, there was no difference in the flux of K⁺ into the xylem after HgCl₂ and DTT application. The results suggest that NaCl decreased L₀ of the roots by reducing either the activity or abundance of Hg-sensitive water channels. The putative reduction in water-channel function of NaCl-treated plants did not seem to be due to the osmotic effect.