The degradation of the herbicide bromoxynil and its impact on bacterial diversity in a top soil

Aims: To study how repeated applications of an herbicide bromoxynil to a soil, mimicking the regime used in the field, affected the degradation of the compound and whether such affects were reflected by changes in the indigenous bacterial community present. Methods and Results: Bromoxynil degradation was monitored in soil microcosms using HPLC. Its impact on the bacterial community was determined using denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of five bacterial taxa (Pseudomonads, Actinobacteria, α‐Proteobacteria, Acidobacteria and nitrifying bacteria). Three applications of 10 mg kg^?1 of bromoxynil at 28‐day intervals resulted in rapid degradation, the time for removal of 50% of the compound decreasing from 6·4 days on the first application to 4·9 days by the third. Bacterial population profiles showed significant similarity throughout the experiment. With the addition of 50 mg kg^?1 bromoxynil to soil, the degradation was preceded by a lag phase and the time for 50% of the compound to be degraded increased from 7 days to 28 days by the third application. The bacterial population showed significant differences 7 days after the final application of bromoxynil that correlated with an inhibition of degradation during the same period. Conclusions: These analyses highlighted that the addition of bromoxynil gave rise to significant shifts in the community diversity and its structure as measured by four abundant taxa, when compared with the control microcosm. These changes persisted even after bromoxynil had been degraded. Significance and Impact of the Study: Here we show that bromoxynil can exert an inhibitory effect on the bacterial population that results in decreased rates of degradation and increased persistence of the compound. In addition, we demonstrate that molecular approaches can identify statistically significant changes in microbial communities that occur in conjunction with changes in the rate of degradation of the compound in the soil.     

Susceptibility of the eggs of the field slug Deroceras reticulatum to contact with pesticides and substances of biological origin on artificial soil

The toxicity of 14 substances, including a number of pesticides, to the eggs of the pest slug Deroceras reticulatum was determined in laboratory experiments. Eggs were kept in contact with a precisely defined artificial soil to which a range of concentrations of the test substances had been applied. Mortality of the eggs was assessed every 24 h and the median lethal doses (LD₅₀) were determined. The herbicides bromoxynil, ioxynil and pyridate + bromoxynil, the insecticides thiocyclam, diflubenzuron and azadirachtin, the molluscicides metaldehyde and methiocarb, and other compounds such as carvone, iron‐EDDHA, saponin, and an extract of Pongamia pinnata, killed the eggs after periods of exposure ranging from 2 to 14 days, depending on the compound and the dose. Only two compounds, the insecticides imidacloprid and teflubenzuron, failed to kill the eggs of D. reticulatum at any of the doses tested. Values of LD₅₀ below 0.01 mg a.i. cm^?2 were obtained for the herbicides bromoxynil, ioxynil and pyridate + bromoxynil, and for the biological pesticide azadirachtin. The feasibility of slug egg control in different contexts is discussed.     

Herbicide effect on the hydrogen-bonding interaction of the primary quinone electron acceptor QA in photosystem II as studied by Fourier transform infrared spectroscopy

The redox potential of Q(A) in photosystem II (PSII) is known to be lower by approximately 100 mV in the presence of phenolic herbicides compared with the presence of DCMU-type herbicides. In this study, the structural basis underlying the herbicide effects on the Q(A) redox potential was studied using Fourier transform infrared (FTIR) spectroscopy. Light-induced Q(A)(-)/Q(A) FTIR difference spectra of Mn-depleted PSII membranes in the presence of DCMU, atrazine, terbutryn, and bromacil showed a strong CO stretching peak of Q(A)(-) at 1,479 cm(-1), while binding of phenolic herbicides, bromoxynil and ioxynil, induced a small but clear downshift by approximately 1 cm(-1). The CO peak positions and the small frequency difference were reproduced in the S(2)Q(A)(-)/S(1)Q(A) spectra of oxygen-evolving PSII membranes with DCMU and bromoxynil. The relationship of the CO frequency with herbicide species correlated well with that of the peak temperatures of thermoluminescence due to S(2)Q(A)(-) recombination. Density functional theory calculations of model hydrogen-bonded complexes of plastoquinone radical anion showed that the small shift of the CO frequency is consistent with a change in the hydrogen-bond structure most likely as a change in its strength. The Q(A)(-)/Q(A) spectra in the presence of bromoxynil, and ioxynil, which bear a nitrile group in the phenolic ring, also showed CN stretching bands around 2,210 cm(-1). Comparison with the CN frequencies of bromoxynil in solutions suggested that the phenolic herbicides take a phenotate anion form in the Q(B) pocket. It was proposed that interaction of the phenolic C-O(-) with D1-His215 changes the strength of the hydrogen bond between the CO of Q(A) with D2-His214 via the iron-histidine bridge, causing the decrease in the Q(A) redox potential.     

Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity

The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1–1.9 U mg^?1 dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30°C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile ? chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92–100, 70–90 and 30–51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.     

Selective Desulfurization of Dibenzothiophene by Rhodococcus erythropolis D-1

A dibenzothiophene (DBT)-degrading bacterium, Rhodococcus erythropolis D-1, which utilized DBT as a sole source of sulfur, was isolated from soil. DBT was metabolized to 2-hydroxybiphenyl (2-HBP) by the strain, and 2-HBP was almost stoichiometrically accumulated as the dead-end metabolite of DBT degradation. DBT degradation by this strain was shown to proceed as DBT → DBT sulfone → 2-HBP. DBT at an initial concentration of 0.125 mM was completely degraded within 2 days of cultivation. DBT at up to 2.2 mM was rapidly degraded by resting cells within only 150 min. It was thought this strain had a higher DBT-desulfurizing ability than other microorganisms reported previously.     

Dehalogenation of the herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodino-4-hydroxybenzonitrile) by Desulfitobacterium chlororespirans

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 microM) and DBHB (250 to 260 microM) were used as electron acceptors for growth. Doubling times for growth (means +/- standard deviations for triplicate cultures) on bromoxynil (18.4 +/- 5.2 h) and DBHB (11.9 +/- 1.4 h), determined by rate of [14C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.     

Effect of soil moisture on bioremediation of chlorophenol-contaminated soil

A chlorophenol-contaminated soil was tested for the biodegradability in a semi-pilot scale microcosm using indigenous microorganisms. More than 90% of 4-chlorophenol and 2,4,6-trichlorophenol, initially at 30 mg kg^–1, were removed within 60 days and 30 mg pentachlorophenol kg^–1 was completely degraded within 140 days. The chlorophenols were degraded more effectively under aerobic condition than under anaerobic condition. Soil moisture had a significant effect with the slowest degradation rate of chlorophenols at 25% in the range of 10–40% moisture content. At 25–40%, the rate of chlorophenol degradation was directly related to the soil moisture content, whereas at 10–25%, it was inversely related. Limited oxygen availability through soil agglomeration at 25% moisture content might decrease the degradation rate of chlorophenols.     

Degradation of bromoxynil by resting and immobilized cells of Agrobacterium radiobacter 8/4 strain

The Agrobacterium radiobacter 8/4 strain capable of degradation of bromoxynil, ioxynil and dichlobenil, arylnitrile herbicides was isolated from soil and cell entrapment was investigated. Three immobilization techniques was used: Caalginate, Ca-pectate and -carrageenan technique, and resting cells. The highest degradation rates were obtained with Ca-alginate and -carrageenan entrapped cells.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Dehalogenation of the Herbicides Bromoxynil (3,5-Dibromo-4-Hydroxybenzonitrile) and Ioxynil (3,5-Diiodino-4-Hydroxybenzonitrile) by Desulfitobacterium chlororespirans

Desulfitobacterium chlororespirans has been shown to grow by coupling the oxidation of lactate to the metabolic reductive dehalogenation of ortho chlorines on polysubstituted phenols. Here, we examine the ability of D. chlororespirans to debrominate and deiodinate the polysubstituted herbicides bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), ioxynil (3,5-diiodo-4-hydroxybenzonitrile), and the bromoxynil metabolite 3,5-dibromo-4-hydroxybenzoate (DBHB). Stoichiometric debromination of bromoxynil to 4-cyanophenol and DBHB to 4-hydroxybenzoate occurred. Further, bromoxynil (35 to 75 μM) and DBHB (250 to 260 μM) were used as electron acceptors for growth. Doubling times for growth (means ± standard deviations for triplicate cultures) on bromoxynil (18.4 ± 5.2 h) and DBHB (11.9 ± 1.4 h), determined by rate of [¹⁴C]lactate uptake into biomass, were similar to those previously reported for this microorganism during growth on pyruvate (15.4 h). In contrast, ioxynil was not deiodinated when added alone or when added with bromoxynil; however, ioxynil dehalogenation, with stoichiometric conversion to 4-cyanophenol, was observed when the culture was amended with 3-chloro-4-hydroxybenzoate (a previously reported electron acceptor). To our knowledge, this is the first direct report of deiodination by a bacterium in the Desulfitobacterium genus and the first report of an anaerobic pure culture with the ability to transform bromoxynil or ioxynil. This research provides valuable insights into the substrate range of D. chlororespirans.     

Degradation of bromoxynil byStreptomyces felleus in soil

The completein vivo degradation of the herbicide bromoxynil byStreptomyces felleus and soil microorganisms was investigated. Little breakdown occurred in sterile soil. TLC techniques were used to detect two degradation products in non-sterile soil. Authors are obliged to Mrs. E. Chrastinová for technical assistance.     

Synergistic action between ioxynil, bromoxynil acids and aminotriazole

Two halogenated organic compounds, ioxynil acid and bromoxynil acid, were shown to inhibit the oxidation of aminotriazole in a free radical generating system. When hypotoxic concentrations of aminotriazole plus ioxynil acid or bromoxynil acid were applied as sprays to wheat plants synergistic action was observed; whereas the three chemicals applied separately caused no damage to wheat plants, the combined spray destroyed them. The statistical analysis of the results confirms that the above combinations act synergistically.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.     

The application of the herbicide bromoxynil to a model soil-derived bacterial community: impact on degradation and community structure

AIMS: Bromoxynil degradation by soil micro-organisms has been shown to be co-oxidative in character. In this study, we investigate both the impact of the application of increasing bromoxynil concentrations on soil-derived bacterial communities and how these changes are reflected in the degradation of the compound. Our aim was to test the hypothesis that the addition of bromoxynil to a soil-derived bacterial community, and the availability of a readily utilizable carbon source would have an impact on bromoxynil degradation, and that would be reflected in the bacteria present in the soil community. METHODS AND RESULTS: Degradation of bromoxynil was observed in soil-derived communities containing 15 mg l(-1), but not 50 mg l(-1) of the compound, unless glucose was added. This suggests that the addition of carbon stimulates co-oxidative bromoxynil degradation by the members of the bacterial community. Measurable changes in the bacterial community indicated that the addition of bromoxynil led to deterministic selection on the bacterial population, i.e. the communities observed arise through the selection of specific micro-organisms that are best adapted to the conditions in the soil. The addition of bromoxynil was also shown to have a negative impact on the presence of alpha and gamma-proteobacteria in the soil community. CONCLUSION: Bromoxynil degradation is significantly inhibited in bacterial soil communities in the absence of readily accessible carbon. The application of bromoxynil appears to exert deterministic selection on the bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effects of increasing bromoxynil concentrations on a model bacterial population derived from soil. Soil communities show qualitative and quantitative differences to bromoxynil application depending on the availability of organic carbon. These findings might have implications for the persistence of bromoxynil in agricultural soils.     

Decomposition of the herbicide bromoxynil in soil and in bacterial cultures

It was found in field, and laboratory experiments that of 50 ppm of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile added to grey forest soil 20-80% were still detected after three months). Bromoxynil did not influence (except for a short-termed stimulation of the number of bacteria) the amount and composition of the basic groups of soil microorganisms. In enrichment cultures of soil microorganisms metabolie products of bromoxynil decomposition (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid) were detected and a stimulating effect of cosubstratos on its decomposition was demonstrated. Bromoxynil concentration, aeration conditions and the presence of cosubstrates (ribose in particular) influenced the rate and degree of the decomposition process inPsevdomonas putida. In addition to the degradation products mentioned above, production of methoxylated and partially dehalogenated aromatic compounds was detected.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Enzymic degradation of bromoxynil by cell-free extracts ofStreptomyces felleus

A method is described for spectrophotometric monitoring the degradation of the herbicide bromoxynil by cell-free extracts of Streptomyces felleus. The method involves a decrease in absorbance at 286 nm (absorption maximum of bromoxynil) that can be ascribed most probably to the cleavage of the aromatic ring of the bromoxynil molecule. Conditions necessary for measuring this degradation together with physico-chemical features of the degradation indicate that the reaction(s) is seemingly catalyzed by an Fe2+-dependent dioxygenase whose activity was not, however, detected in cell-free extracts of a bromoxynil-sensitive mutant of S. felleus as well as other bromoxynil-sensitive streptomycete strains.