3,4-dichloroaniline--a nitrogen and carbon source for a mixed culture of microorganisms

A mixed microbial culture assimilating 4-chloroaniline and 3,4-dichloroaniline as sole sources of carbon and nitrogen was isolated from soil treated with propanide for a long period of time. The process is accompanied with a 100% liberation of chloride ions. Aniline cannot serve as a growth substrate for the mixed culture. The authors have studied whether the mixed culture utilizing chloroanilines can decompose these compounds in model experiments with natural waters and soil suspensions. The culture actively decomposes 3,4-dichloroaniline (20 mg/litre) in natural water samples into which it has been added at a concentration of 10(3)--10(4) cells/ml. The decomposition is accelerated in experiments with suspensions of meadow-chernozem soils when cells of the mixed culture are added to the suspensions. The rate of 3,4-dichloroaniline degradation in a grey forest soil suspension remains nearly unchanged when the mixed culture is added to it. Degradation of the chloroaniline is accompanied with equivalent liberation of chloride ions when the mixed culture is added to natural water samples.     

3,4-dichloroaniline cometabolism by representatives of the genus Pseudomonas

The effect of propanide, linuron and 3,4-dichloroaniline on soil organisms was studied. Two strains of Pseudomonas aurantiaca 1 and 7, were isolated from soil; they decomposed propanide yielding 3,4-dichloroaniline. These strains, as well as a number of collection cultures belonging to the Pseudomonas genus, could transform 3,4-dichloroaniline at a rate of 0-100 per cent during 48 hours. A certain correlation existed between this transformation ability and the level of total oxidase activity. All the strains of Pseudomonas studied in this work were characterized by a low peroxidase activity, and no strict correlation was detected between its level and the ability to transform 3,4-dichloroaniline.     

Rapid mineralization of the phenylurea herbicide diuron by Variovorax sp. strain SRS16 in pure culture and within a two-member consortium

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethylurea] is widely used in a broad range of herbicide formulations, and consequently, it is frequently detected as a major water contaminant in areas where there is extensive use. We constructed a linuron [N-(3,4-dichlorophenyl)-N-methoxy-N-methylurea]- and diuron-mineralizing two-member consortium by combining the cooperative degradation capacities of the diuron-degrading organism Arthrobacter globiformis strain D47 and the linuron-mineralizing organism Variovorax sp. strain SRS16. Neither of the strains mineralized diuron alone in a mineral medium, but combined, the two strains mineralized 31 to 62% of the added [ring-U-(14)C]diuron to (14)CO(2), depending on the initial diuron concentration and the cultivation conditions. The constructed consortium was used to initiate the degradation and mineralization of diuron in soil without natural attenuation potential. This approach led to the unexpected finding that Variovorax sp. strain SRS16 was able to mineralize diuron in a pure culture when it was supplemented with appropriate growth substrates, making this strain the first known bacterium capable of mineralizing diuron and representatives of both the N,N-dimethyl- and N-methoxy-N-methyl-substituted phenylurea herbicides. The ability of the coculture to mineralize microgram-per-liter levels of diuron was compared to the ability of strain SRS16 alone, which revealed the greater extent of mineralization by the two-member consortium (31 to 33% of the added [ring-U-(14)C]diuron was mineralized to (14)CO(2) when 15.5 to 38.9 mug liter(-1) diuron was used). These results suggest that the consortium consisting of strains SRS16 and D47 could be a promising candidate for remediation of soil and water contaminated with diuron and linuron and their shared metabolite 3,4-dichloroaniline.     

3,4 Dichloroaniline-haemoglobin adducts in humans: preliminary data on agricultural workers exposed to propanil

Propanil is one of the major herbicides used on rice-paddies and is thought to produce adverse health effects through the action of its metabolite 3,4-dichloroaniline (3,4-DCA). T he feasibility of monitoring human exposure to propanil on the basis of 3,4-DCA adducts to haemoglobin (Hb) was investigated. We developed a method based on gas chromatography negative ion chemical ionization-mass spectrometry (NICI-GC-MS) to quantify 3,4-DCA released from human Hb after alkaline hydrolysis of the protein. 3,4-DCA-Hb adducts were identified in agricultural workers exposed to propanil and were detectable even 4 months after the last herbicide application. Urine samples collected at the same time had no measurable level of 3,4-DCA. 3,4-DCA-Hb adducts might be useful for monitoring human exposure to 3,4-DCA from agricultural sources.     

3,4 Dichloroaniline-haemoglobin adducts in humans: preliminary data on agricultural workers exposed to propanil

Propanil is one of the major herbicides used on rice-paddies and is thought to produce adverse health effects through the action of its metabolite 3,4-dichloroaniline (3,4-DCA). T he feasibility of monitoring human exposure to propanil on the basis of 3,4-DCA adducts to haemoglobin (Hb) was investigated. We developed a method based on gas chromatography negative ion chemical ionization-mass spectrometry (NICI-GC-MS) to quantify 3,4-DCA released from human Hb after alkaline hydrolysis of the protein. 3,4-DCA-Hb adducts were identified in agricultural workers exposed to propanil and were detectable even 4 months after the last herbicide application. Urine samples collected at the same time had no measurable level of 3,4-DCA. 3,4-DCA-Hb adducts might be useful for monitoring human exposure to 3,4-DCA from agricultural sources.     

Degradation of chloroanilines in soil slurry by specialized organisms

The microbial degradation of 2-chloro-, 3-chloro-, 4-chloro-, and 3,4-dichloroaniline was examined as single compounds as well as a mixture in soil slurries. At 30°C the degradation of chloroanilines by indigenous soil populations in soil slurries was observed when soil slurry was freshly contaminated or precontaminated to allow binding of chloroanilines to the soil matrix. Within 6 weeks, 3-chloro- and 3,4-dichloroaniline (each 2 mm) were degraded more rapidly (about 50% chloride elimination) than 4-chloro- and 2-chloroaniline, due to stronger adsorption of 4-chloroaniline and greater resistance of 2-chloroaniline. The addition of various supplements such as buffer, mineral salts and acetate only slightly influenced the degradation of chloroanilines by the indigenous soil populations. The mineralization was drastically enhanced when laboratory-selected chloroaniline-degraders (8·10⁶ cells/g) such as Pseudomonas acidovorans strain BN3.1 were supplemented to the soil slurries so that complete elimination of chloride from the chloroanilines occurred within 10 days. Correspondence to: F. R. Brunsbach     

Biochemical Decomposition of the Herbicide N-(3,4-Dichlorophenyl)-2-Methylpentanamide and Related Compounds

Organisms capable of decomposing N-(3,4-dichlorophenyl)-2-methylpentanamide (Karsil) were isolated, identified, and tested for their ability to hydrolyze this herbicide. Primary products of Karsil decomposition by cells and cell-free extracts of a Penicillium sp. were identified as 2-methyl-valeric acid and 3,4-dichloroaniline. The Karsil acylamidase (EC 3.5.1.a aryl acylamine amidohydrolase) was an induced enzyme. It was partially purified and tested for its ability to hydrolyze 25 related compounds. Some relations between the structures of these compounds and their susceptibility to enzymatic hydrolysis were discerned.     

Herbicide Transformation: II. Studies with an Acylamidase of Fusarium solani

A strain of Fusarium solani isolated from soil by enrichment techniques used propanil (3′, 4′-dichloropropionanilide) as a sole source of organic carbon and energy for growth in pure culture. The primary product of the transformation of propanil by F. solani was isolated and identified as 3,4-dichloroaniline (DCA). This compound accumulated in the medium to a level (80 μg/ml) which stopped further herbicide utilization. Herbicide utilization by F. solani was influenced by various environmental and nutritional factors. It was more sensitive to acid than alkaline pH. Added glucose and yeast extract increased the rate of propanil decomposition, and the reduced aeration retarded growth of the fungus and herbicide utilization. The growth of F. solani on propionate was inhibited by added DCA.     

Herbicide Transformation: I. Studies with Whole Cells of Fusarium solani

A strain of Fusarium solani isolated from soil by enrichment techniques used propanil (3′, 4′-dichloropropionanilide) as a sole source of organic carbon and energy for growth in pure culture. The primary product of the transformation of propanil by F. solani was isolated and identified as 3,4-dichloroaniline (DCA). This compound accumulated in the medium to a level (80 μg/ml) which stopped further herbicide utilization. Herbicide utilization by F. solani was influenced by various environmental and nutritional factors. It was more sensitive to acid than alkaline pH. Added glucose and yeast extract increased the rate of propanil decomposition, and the reduced aeration retarded growth of the fungus and herbicide utilization. The growth of F. solani on propionate was inhibited by added DCA.     

Preliminary studies of 3,4-dichloroaniline amides as antiparasitic agents: Structure–activity analysis of a compound library in vitro against Trichomonas vaginalis

Trichomonas vaginalis, a human-infectious protozoan, can display resistance to treatment by metronidazole. A library of 3,4-dichloroaniline amides based on propanil, an herbicide, has been synthesized and screened to test susceptibility to these analogs. From this preliminary study, the most effective compound 15, inhibits growth of the organism by 66% and 69% on the two strains tested, T1 and G3, respectively.     

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L^?1 h^?1. Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L^?1 h^?1. At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.     

Residual effects of 3,4-dichloroaniline on offspring born to Ceriodaphnia cf. dubia exposed for multiple generations

One of the acknowledged limitations ofconventional toxicity tests is their inabilityto evaluate the impact of toxicants onsubsequent generations. Given their relativelyshort lifespan, cladocerans in the field may beexposed to toxicants for several generations.However, it is unclear what effect such anexposure regime could have on the cladoceranfitness after removal of the toxicant. Thispaper aimed to determine the offspring fitnessof juveniles produced by adult Ceriodaphnia cf. dubia exposed toresidual levels of 3,4-dichloroaniline overfour generations. Mass cultures of Ceriodaphnia cf. dubia were maintainedfor several generations in variousconcentrations of 3,4-dichoroaniline (0, 2.5,5, 10, 15 and 20 µg/L). The mass cultureswere re-established every generation using4th brood neonates <24 h old. Eachgeneration, 4th brood neonates <24 h oldwere also transferred individually intotoxicant-free water and examined until theproduction of their 3rd brood. F1offspring of mothers exposed to 15 and20 µg/L 3,4-dichloroaniline showedsignificantly (p < 0.05) reducedreproduction compared to the controls. Nosignificant (p > 0.05) changes inreproduction due to 3,4-dichloroaniline wereobserved for the F2 and F3 offspring. However,F4 offspring of mothers exposed to all3,4-dichloroaniline concentrations showedsignificantly (p < 0.05) increasedreproduction compared to the controls. Possibleexplanations for the varying influence ofmaternal 3,4-dichloroaniline exposure on thereproductive output of the offspring arediscussed.     

Effets sur le spermatozoïde humain du Diuron (3-(3,4-dichlorophényl)-1,1-diméthyl-urée) et de l’un de ses produits de transformation, la 3,4-dichloroaniline (3,4-DCA) (Etude préliminaire)

Diuron belongs to the family of halogenophenylureas, one of the main groups of herbicides used for more than 40 years. These herbicides absorb sunlight and can be photochemically transformed in the environment (herbicides are transformed on the soil surface exposed to sunlight) or biotransformed by microorganisms present in soil or in water. The metabolites (chlorohydroxyphenylurea, chlorophenylaniline, respectively) are more toxic than the parent compound, as demonstrated by a bioluminescence inhibition assay performed with a marine bacterium (Vibrio fischeri toxicity test). The lipophilicity of these pesticides makes the cell membrane a target for their action, especially the spermatozoa cell membrane. The aim of this study is to use human spermatozoa to evaluate the effect of this urea pesticide and its biotransformed product on the spermatozoa membrane. We investigated the structural and functional effects of these environmental pollutants on spermatozoa. Three million spermatozoa purified on a 95/47.5% Percoll gradient were suspended in 250 μl of modified Earle’s medium (without phenol red) supplemented with 7.5% of human decomplemented serum. Pesticides (Diuron or 3,4-dichloroaniline (3,4-DCA)) were added at a final concentration of 0.1; 1 and 5 mM. Samples were incubated at room temperature for 24 hours. We show that both Diuron and 3,4-DCA decrease motility and vitality of spermatozoa incubated with the highest concentration of pesticides. Our preliminary results show that the effects are more rapid and more intense with the biotransformed product (3,4-DCA) than with Diuron. Addition of herbicide to human spermatozoa increases membrane fluidity, assessed by measuring the fluorescence polarisation anisotropy with a fluorescent probe: 1,6-diphenyl-1,3,5-hexatriene (DPH). Changes in membrane fluidity may be a primary toxic effect of these herbicides. These results suggest that human spermatozoa may constitute a valuable indicator of the toxic effects of pesticides.     

Degradation of mixtures of chloroaromatic compounds in soil slurry by mixed cultures of specialized organisms

The degradation of a mixture of 13 chloroaromatics, 2-chloro-, 3-chloro-, 4-chloro- and 3,4-dichloroaniline,2-chloro-, 3-chloro-,4-chloro-, 3,4-dichloro-and 3,5-dichlorobenzoate, and chloro-,1,2-dichloro-, 1,4-dichloro- and 1,2,4-trichlorobenzene in soil slurries by a mixed culture of Pseudomonas acidovorans strain BN 3.1, Pseudomonas ruhlandii strain FRB2, Pseudomonas cepacia strain JH230 and Pseudomonas aeruginosa strain RHO1 was studied. About 70% of the organic bound chlorine was eliminated after 25 days from soil with a carbon content of 8% (soil 1) when 2–3 × 10⁵ cells/g soil of each of the strains were added to the slurries. The effect of the clean-up was demonstrated by a biological test using cress and wheat. Both plants showed good germination and growth on both non-contaminated soils and the contaminated soil 1 after the biotreatment with the strains. No growth was observed when the plants were incubated with the contaminated soil 1 and with the contaminated and biotreated soil 2 (carbon content 2.6%). This indicates that the remaining 30% of organic chlorine in soil 1 after biotreatment does not influence the germination and growth of the two plants tested. *** DIRECT SUPPORT *** AG903062 00010     

Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4-dichloroaniline

The pollutant 3,4-dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N-beta-d-glucopyranosyl-DCA exported into the medium. The N-glucosyltransferase (N-GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5-trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N-GT and O-GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4-hydroxyphenylpyruvic acid. Both O-GT and N-GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.     

N-malonyltransferases from peanut

Three distinct N-malonyltransferases were purified from peanut seedlings, accepting either anthranilic acid, D-tryptophan, or 3,4-dichloroaniline, respectively, as a substrate. Partially purified malonyl-CoA:D-tryptophan malonyltransferase also catalyzed the formation of the corresponding malonic acid conjugate when 1-aminocyclopropane-1-carboxylic acid was employed as a substrate. These N-malonyltransferases were clearly distinguished from several O-malonyltransferase activities also present in the same seedlings. N-Malonic acid conjugates have been previously isolated from peanut either as a natural constituent or after feeding with xenobiotics. By analogy to the results reported with cultured parsley cells, multiple malonyltransferases in peanut may have a role in vacuolar transport. Crude extracts of young peanut seedlings were incapable of hydrolyzing the respective N-malonic acid conjugates. However, dialyzed extracts of older plants released malonic acid from malonyl-1-aminocyclopropane-1-carboxylic acid but not from malonyl-3,4-dichloroaniline, suggesting that some N-malonic acid conjugates may be metabolized in plants which are approaching senescence.     

Degradation of aniline by Delftia tsuruhatensis 14S in batch and continuous processes

A Delftia tsuruhatensis strain capable of consuming aniline as the sole source of carbon, nitrogen, and energy at concentrations of up to 3200 mg/l was isolated from activated sludge of purification works of OAO Volzhskii Orgsintez. The strain grew on pyrocatechol and p-hydroxybenzoic acid, but did not consume phenol, 2-aminophenol, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, 2-nitroaniline, 2-chlorophenol, or aminobenzoate. Aniline is degraded by cleavage of the pyrocatechol aromatic ring at the ortho position. Cells were immobilized on polycaproamide fiber. It was shown that the strain degraded aniline at 1000 mg/l in a continuous process over a long period of time.     

Degradation of Aniline by <Emphasis Type="Italic">Delftia tsuruhatensis</Emphasis> 14S in Batch and Continuous Processes

A Delftia tsuruhatensis strain capable of consuming aniline as the sole source of carbon, nitrogen, and energy at concentrations of up to 3200 mg/l was isolated from activated sludge of the sewage disposal plants of OAO Volzhskii Orgsintez. The strain grew on catechol and p-hydroxybenzoic acid but did not consume phenol, 2-aminophenol, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-dichloroaniline, 3,4-dichloroaniline, 2-nitroaniline, 2-chlorophenol, or aminobenzoate. Aniline is degraded by cleavage of the catechol aromatic ring at the ortho position. Cells were immobilized on polycaproamide fiber. It was shown that the strain degraded aniline at 1000 mg/l in a continuous process over a long period of time.