Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by fungi originating from Vietnam.

Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

Plasmid-assisted molecular breeding: new technique for enhanced biodegradation of persistent toxic chemicals.

The persistence of synthetic herbicides such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and its release in massive amounts as a herbicide (Agent Orange) have created toxicological problems in many countries. In nature, 2,4,5-T is slowly degraded by cooxidation and is not utilized as a sole source of carbon and energy. The technique of plasmid-assisted molecular breeding has led to the development of bacterial strains capable of totally degrading 2,4,5-T by using it as their sole source of carbon at high concentrations (greater than 1 mg/ml). Spectrophotometry and gas chromatography reveal various intermediates during growth of the culture with 2,4,5-T.

Anaerobic degradation of 2,4,5-trichlorophenoxyacetic acid by enrichment cultures from freshwater sediments.

The anaerobic biodegradation of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated using enrichment cultures from freshwater sediments at two different sites in the region of Halle, central Germany. 2,4,5-T and different organic acids or hydrogen were added as possible electron acceptor and electron donors, respectively. The primary enrichment cultures from Saale river sediment completely degraded 2,4,5-T to 3-chlorophenol (3-CP) (major product) and 3,4-dichlorophenol (3,4-DCP) during a 28-day incubation period. Subcultures showed ether cleavage of 2,4,5-T to 2,4,5-trichlorophenol and its stoichiometric dechlorination to 3-CP only in the presence of butyrate. In contrast, the primary enrichment culture from sediment of Posthorn pond dechlorinated 2,4,5-T to 2,5-dichlorophenoxyacetic acid (2,5-D), which, in the presence of butyrate, was degraded further to products such as 3,4-DCP, 2,5-DCP, and 3CP, indicating ether cleaving activities and subsequent dechlorination steps. Experiments with pure cultures of Dehalococcoides mccartyi and Desulfitobacterium hafniense demonstrated their specific dechlorination steps within the overall 2,4,5-T degradation pathways. The results indicate that the route and efficiency of anaerobic 2,4,5-T degradation in the environment depend heavily on the microorganisms present and the availability of slowly fermentable organic compounds.

Desulfitobacterium contributes to the microbial transformation of 2,4,5-T by methanogenic enrichment cultures from a Vietnamese active landfill.

The herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5-T via ether cleavage to 2,4,5-trichlorophenol (2,4,5-TCP), which was further dechlorinated to 3,4-dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5-T and subsequent dechlorination of 2,4,5-TCP. 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes, Bacteroidetes, Spirochaetes, Chloroflexi and Euryarchaeota. Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5-T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5-TCP. Despite an ether-cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2-bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms.

Quantification of six herbicide metabolites in human urine.

We developed a sensitive, selective and precise method for measuring herbicide metabolites in human urine. Our method uses automated liquid delivery of internal standards and acetate buffer and a mixed polarity polymeric phase solid phase extraction of a 2 mL urine sample. The concentrated eluate is analyzed using high-performance liquid chromatography-tandem mass spectrometry. Isotope dilution calibration is used for quantification of all analytes. The limits of detection of our method range from 0.036 to 0.075 ng/mL. The within- and between-day variation in pooled quality control samples range from 2.5 to 9.0% and from 3.2 to 16%, respectively, for all analytes at concentrations ranging from 0.6 to 12 ng/mL. Precision was similar with samples fortified with 0.1 and 0.25 ng/mL that were analyzed in each run. We validated our selective method against a less selective method used previously in our laboratory by analyzing human specimens using both methods. The methods produced results that were in agreement, with no significant bias observed.

Enhanced co-metabolism of TCDD in the presence of high concentrations of phenoxy herbicides.

Chemical residue studies were conducted from 1977-1987 on sites where spills of Agent Orange had occurred in the Herbicide Storage Sites at the Naval Construction Battalion Center, Gulfport, Mississippi, and on Johnston Island, Central Pacific Ocean. The soil persistence time of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was significantly decreased when in the presence of massive amounts of phenoxy herbicides (> 62,000 microg of herbicide/g of soil). Although microbial populations doubled in the most highly contaminated sites, fungal species diversity decreased. The dominant fungal species that appeared to be associated with the metabolism of the residues were of the genera Penicillium, Mucor, and Fusarium. TCDD level decreased from a mean high of 180 ng/g (ppb) to less than 1 ng/g of soil over a ten-year period.

[Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid].

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.

2,4-Dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading gene cluster in the soybean root-nodulating bacterium Bradyrhizobium elkanii USDA94.

Herbicides 2,4-dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading Bradyrhizobium strains possess tfdAα and/or cadABC as degrading genes. It has been reported that root-nodulating bacteria belonging to Bradyrhizobium elkanii also have tfdAα and cadA like genes but lack the ability to degrade these herbicides and that the cadA genes in 2,4-D-degrading and non-degrading Bradyrhizobium are phylogenetically different. In this study, we identified cadRABCK in the genome of a type strain of soybean root-nodulating B. elkanii USDA94 and demonstrated that the strain could degrade the herbicides when cadABCK was forcibly expressed. cadABCK-cloned Escherichia coli also showed the degrading ability. Because co-spiked phenoxyacetic acid (PAA) could induce the degradation of 2,4-D in B. elkanii USDA94, the lack of degrading ability in this strain was supposed to be due to the low inducing potential of the herbicides for the degrading gene cluster. On the other hand, tfdAα from B. elkanii USDA94 showed little potential to degrade the herbicides, but it did for 4-chlorophenoxyacetic acid and PAA. The 2,4-D-degrading ability of the cad cluster and the inducing ability of PAA were confirmed by preparing cadA deletion mutant. This is the first study to demonstrate that the cad cluster in the typical root-nodulating bacterium indeed have the potential to degrade the herbicides, suggesting that degrading genes for anthropogenic compounds could be found in ordinary non-degrading bacteria.

Environmental fate and dietary exposures of humans to TCDD as a result of the spraying of Agent Orange in upland forests of Vietnam.

The fate and transport of 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) released into the environment of South Vietnam (SVN) as a consequence of the aerial application of the herbicidal defoliant Agent Orange (AO) were simulated for a generic upland forest scenario and followed over a 50-year period (1965, 1968 and 1970 onwards). Modeled concentrations of TCDD in the environment were then used as inputs to a human exposure model, which focused on long-term exposures via the food chain. Intake rates and body burdens of TCDD were estimated for adult males over the course of the simulation period and compared to available biomonitoring data. One of the most important factors determining the magnitude of the simulated human exposure to TCDD was the fraction of the chemical deposited directly to soil (where it was assumed to have a degradation half-life of 10 or 15years) relative to the fraction assumed to remain on/in the forest canopy following the spray application (where it was assumed to have a degradation half-life of ≤48h). The simulated body burdens under the various scenarios considered were broadly consistent with the biomonitoring data from SVN collected in the mid-1980s to late 1990s. Taken together, the modeling results and empirical data suggest that highly elevated exposures to TCDD (i.e., body burdens in the several 100s of pg/g lipid range and greater) were not common among people inhabiting upland forest locations in SVN sprayed with AO and that peak and average body burdens were broadly similar to those of the general population of the U.S. in the 1970s and early 1980s. The model-based assessment is consistent with the 'hot spot' hypothesis i.e., potential exposures to TCDD linked to activities conducted on or near former bases where AO was stored are greater than potential exposures in areas subjected to aerial spraying.

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia.

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

Biodegradation of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia strain AC1100: evolutionary insight.

Many microorganisms in nature have evolved new genes which encode catabolic enzymes specific for chlorinated aromatic substrates, allowing them to utilize these compounds as sole sources of carbon and energy. An understanding of the evolutionary mechanisms involved in the acquisition of such genes may facilitate the development of microorganisms with enhanced capabilities of degrading highly chlorinated recalcitrant compounds. A number of studies have been based on microorganisms isolated from the environment which utilize simple chlorinated substrates. In our laboratory, a selective technique was used to isolate microorganisms capable of degrading highly chlorinated compounds, such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as sole sources of carbon and energy. This article summarizes the genetic and biochemical information obtained regarding the pathway of degradation, the mechanism of recruitment of new genes, and the organization of the degradative genes. In addition, we discuss the potential practical application of such microorganisms in the environment.

Characterization of an intradiol dioxygenase involved in the biodegradation of the chlorophenoxy herbicides 2,4-D and 2,4,5-T.

Hydroxyquinol 1,2-dioxygenase, an intradiol dioxygenase, which catalyzes the cleaving of the aromatic ring of hydroxyquinol, a key intermediate of 2,4-D and 2,4,5-T degradation, was purified from Nocardioides simplex 3E cells grown on 2,4-D as the sole carbon source. This enzyme exhibits a highly restricted substrate specificity and is able to cleave hydroxyquinol (K(m) for hydroxyquinol as a substrate was 1.2 microM, V(max) 55 U/mg, K(cat) 57 s-1 and K(cat)/K(m) 47.5 microM s-1), 6-chloro- and 5-chlorohydroxyquinol. Different substituted catechols and hydroquinones are not substrates for this enzyme. This enzyme appears to be a dimer with two identical 37-kDa subunits. Protein and iron analyses indicate an iron stoichiometry of 1 iron/65 kDa homodimer, alpha2 Fe. Both the electronic absorption spectrum which shows a broad absorption band with a maximum at 450 nm and the electron paramagnetic resonance spectra are consistent with a high-spin iron(III) ion in a rhombic environment typical of the active site of intradiol cleaving enzymes.

Interaction of 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T) with the acyl-CoA: amino acid N-acyltransferase enzymes of bovine liver mitochondria.

The amino acid conjugation of the phenoxyherbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T) by animals was examined at the level of the enzymes catalyzing the reactions. The phenoxyherbicides were not substrates for the bile acid conjugating system but were substrates for the mitochondrial xenobiotic conjugating system. The two mitochondrial xenobiotic-CoA: amino acid N-acyltransferases (benzoyltransferase and phenylacetyltransferase) were separated and tested for activity towards 2,4-D-CoA and 2,4,5-T-CoA. The phenylacetyltransferase showed activity towards phenylacetyl-CoA, phenoxyacetyl-CoA and 2,4-D-CoA, but not 2,4,5-T-CoA. Benzoyltransferase conjugated both 2,4-D-CoA and 2,4,5-T-CoA. The overall rates of conjugation of the phenoxyherbicides were slow relative to the standard substrates with both enzymes. This slow rate was found to be due in both cases to a relatively high Km for glycine, and a very slow catalytic rate constant. Both enzymes did, however, have a very high affinity for 2,4-D-CoA and 2,4,5-T-CoA so these compounds proved to be potent alternate substrate inhibitors of both enzymes. The data show that the inefficient in vivo conjugation of the phenoxyherbicides relative to structurally similar compounds can be understood in terms of the kinetic properties of the mitochondrial N-acyltransferases. Further, the potential for the interference of the phenoxyherbicides with the conjugation of other compounds is revealed.

Burkholderia phenoliruptrix sp. nov., to accommodate the 2,4,5-trichlorophenoxyacetic acid and halophenol-degrading strain AC1100.

Strain AC1100 is well-known for its ability to degrade a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetic acid. We performed a polyphasic-taxonomic study to determine its taxonomic position. The G+C content of strain AC1100 was 62.6 mol%. On the basis of 16S rRNA gene sequence similarity, strain AC1100 belonged to the beta-Proteobacteria and was most closely related to Burkholderia fungorum (98.3% similarity). DNA-DNA hybridisations, comparison of protein profiles, cellular fatty acid analysis and biochemical tests allowed genotypic and phenotypic differentiation of strain AC1100 from other Burkholderia species. Our data show that strain AC1100 represents a novel species for which the name Burkholderia phenoliruptrix sp. nov. is proposed. The type strain is AC1100T (= LMG 22037T = CCUG 48558T).

Distribution and cytogenetic test of 2,4-D and 2,4,5-T phenoxyacetic acids in mouse blood tissues.

The phenoxyacetic acids 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), extensively used as herbicides, were tested for cytogenetic effects by means of induced micronuclei in erythrocytes of mouse bone marrow. Because of the hig experimental resolution power this is a particularly suitable test system for the detection of weak chromosome breaking activity in mammals. The cytogenetic tests were supplemented with chemical analyses of the concentration the the test substances reaching the target cells...

Biliary excretion of 2,4,5-trichlorophenoxyacetic acid in the rat.

Urethane-anesthetized, male Sprague-Dawley rats were administered 25 mg/kg 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) intravenously. 6 h after administration, 15.7% of the dose was recovered in the bile. Reversed-phase high-performance liquid chromatography (HPLC) analysis of bile indicated that more than 85% of compound excreted in bile was unchanged 2,4,5-T. Since renal function was apparently compromised by the urethane anesthesia, only 4% of the dose was recovered in urine in 6 h. These data demonstrate that biliary excretion can be a slow but significant route of elimination under conditions of impaired renal function. Since 2,4,5-T is eliminated largely as the unchanged acid, reabsorption and thus enterohepatic circulation may significantly prolong the half-life of 2,4,5-T in the body.

Fate of the herbicides 2,4,5-T, atrazine, and DNOC in a shallow, anaerobic aquifer investigated by in situ passive diffusive emitters and laboratory batch experiments.

The fate of the three herbicides 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), atrazine (6-chloro-N-ethyl-N'-[1-methyl-ethyl]-1,3,5-triazine-2,4-diamine), and DNOC (4,6-dinitro-2-methylphenol) in an anaerobic sandy aquifer was investigated. In the field, each of the herbicides was released simultaneously with tritiated water (HTO) as tracer in the depth interval 3 to 4 mbs (meters below surface) by use of passive diffusive emitters. Atrazine and 2,4,5-T were persistent during the approximately 18 days residence time in the aquifer. In contrast, DNOC was rapidly removed from the water phase following first-order kinetics. The removal mechanism was likely an abiotic reduction. At day 25, the first-order rate constant was 1.47 d(-1), but it decreased with time and seemed to stabilize at 0.35 d(-1) after 150 to 200 days. In the laboratory, batch experiments were conducted with sediments from 3 to 4 mbs and from 8 to 9 mbs. In these incubations, formation of Fe2+ and depletion of sulfate showed iron and sulfate reduction in sediment from 3 to 3.5 mbs and sulfate reduction in 3.5 to 4 mbs sediment. In sediment from 8 to 9 mbs, the dominant redox process was methane formation. In sediment from 3 to 3.5 mbs, only 27% to 52% of the 2,4,5-T remained after 196 days. 2,4,5-trichlorophenol was identified as the major metabolite. A lag period of at least 50 days was observed, and no degradation occurred in HgCl2 amended controls, verifying that the process was microbially mediated. In the other 2,4,5-T incubations and all the atrazine incubations, concentrations decreased linearly, but less than 25% was removed within 200 to 250 days. No degradation products could be detected, and slow sorption was the likely explanation. In all the laboratory incubations DNOC was degraded, following first-order kinetics, and when normalized to the sediment/water-ratio, the field and laboratory derived rate constants compared well. The DNOC degradation in the methanogenic incubations (8 to 9 mbs) was up to 50 times faster than in the sediments from 3 to 4 mbs, likely due to the low redox potential.

[Microbial degraders of some organochlorine compounds]. / Mikrobnye destruktory nekotorykh khlororganicheskikh soedinenii.

A possibility of isolation of microorganisms, potential destructors of chlorinated organics from aged Vietnamese soils polluted with dioxine-containing defoliants was demonstrated. As an example, the ability of one isolated strain to metabolize pentachlorophenol and 2,4-dichlorophenoxyacetic acid was shown under laboratory conditions. An attempt was made to identify intermediates of pentachlorophenol metabolism using HPLC.

Agent Orange footprint still visible in rural areas of central Vietnam.

Levels of polychlorinated dioxins/furans (PCDD/PCDF) in selected environmental samples (soils, sediments, fish, and farm animals) were analyzed from the area of Phong My commune (Thua Thien-Hue province, Vietnam). This area was affected by Agent Orange spraying during the Vietnam war (1968-1971). Whereas PCDD/PCDF content in soil and sediment samples is relatively low and ranges between 0.05 and 5.1 pg WHO-TEQ/g for soils and between 0.7 and 6.4 pg WHO-TEQ/g for sediments, the PCDD/PCDF content in poultry muscle and liver in most cases exceeded the maximum permissible limit of dioxin content per unit fat mass. In some cases of soil and sediments samples, 2,3,7,8-TCDD represented more than 90% of the total PCDD/PCDF, which indicates Agent Orange as the main source.