Ecologically significant effects of Pseudomonas putida PPO301(pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, on microbial populations and processes in soil

Pseudomonas putida PPO301 (pRO103), genetically engineered to degrade 2,4-dichlorophenoxyacetate, affected microbial populations and processes in a nonsterile xeric soil. In soil amended with 2,4-dichlorophenoxyacetate (500 micrograms/g soil) and inoculated with PPO301 (pRO103), the rate of evolution of carbon dioxide was retarded for approximately 35 days; there was a transient increase in dehydrogenase activity; and the number of fungal propagules decreased below detection after 18 days. In unamended soil inoculated with PPO301(pRO103), the rate of evolution of carbon dioxide and the dehydrogenase activity were unaffected, and the numbers of fungal propagules were reduced by about two orders of magnitude. The numbers of total, spore-forming, and chitin-utilizing bacteria were reduced transiently in soil either amended or unamended with 2,4-dichlorophenoxyacetate and inoculated with PPO301(pRO103). The activities of arylsulfatases and phosphatases in soil were not affected by the presence of PPO301(pRO103), either in the presence or absence of 2,4-dichlorophenoxyacetate. In soil amended with 2,4-dichlorophenoxyacetate and inoculated with the parental strain (PPO301) or not inoculated, the evolution of carbon dioxide, the numbers of fungal propagules and of total, spore-forming, and chitin-utilizing bacteria, and the dehydrogenase activity were not affected as in soil inoculated with PPO301(pRO103). These results demonstrated that a genetically engineered microorganism, in the presence of the substrate on which its novel genes can function, is capable of inducing measurable ecological effects in soil.     

Growth and survival of Pseudomonas cepacia DBO1(pRO101) in soil amended with 2,4-dichlorophenoxyacetic acid

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading pseudomonad, Pseudomonas cepacia DBO1(pRO101), was inoculated at approximately 10⁷ CFU/g into sterile and non-sterile soil amended with 0, 5 or 500 ppm 2,4-D and the survival of the strain was studied for a period of 44 days. In general, the strain survived best in sterile soil. When the sterile soil was amended with 2,4-D, the strain survived at a significantly higher level than in non-amended sterile soil. In non-sterile soil either non-amended or amended with 5 ppm 2,4-D the strain died out, whereas with 500 ppm 2,4-D the strain only declined one order of magnitude through the 44 days.The influence of 0,0.06, 12 and 600 ppm 2,4-D on short-term (48 h) survival of P. cepacia DBO1(pRO101) inoculated to a level of 6×10⁴, 6×10⁶ or 1×10⁸ CFU/g soil was studied in non-sterile soil. Both inoculum level and 2,4-D concentration were found to have a positive influence on numbers of P. cepacia DBO1(pRO101). At 600 ppm 2,4-D growth was significant irrespective of the inoculation level, and at 12 ppm growth was stimulated at the two lowest inocula levels. P. cepacia DBO1(pRO101) was able to survive for 15 months in sterile buffers kept at room temperature. During this starvation, cells shrunk to about one third the volume of exponentially growing cells.Abbreviations AODC acridine orange direct count - CFU colony forming units - PTYG-Agar peptone, tryptone, yeast & glucose agar - TET tetracycline - LB Luria Bertani medium     

Assay for detection and enumeration of genetically engineered microorganisms which is based on the activity of a deregulated 2,4-dichlorophenoxyacetate monooxygenase.

An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which converts phenoxyacetate (PAA) to phenol. In PAA-amended cultures of Pseudomonas aeruginosa PAO1C(pRO103) and Pseudomonas putida PPO301(pRO103), strains which express a deregulated TFD monooxygenase, phenol production was proportional to cell number. Phenol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotometrically could detect as few as 10(3) cells per ml. This assay was corroborated by monitoring the disappearance of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography. The AAPPC assay was modified for use with plate cultures and clearly distinguished colonies of PPO301(pRO103) and PAO1C(pRO103) from a strain expressing a regulated TFD monooxygenase. Colonies of P. putida PPO301(pRO101) remained cream colored, while colonies of PPO301(pRO103) and PAO1C(pRO103) turned a distinct red.     

Assay for detection and enumeration of genetically engineered microorganisms which is based on the activity of a deregulated 2,4-dichlorophenoxyacetate monooxygenase

An assay system was developed for the enumeration of genetically engineered microorganisms expressing a deregulated 2,4-dichlorophenoxyacetate (TFD) monooxygenase, which converts phenoxyacetate (PAA) to phenol. In PAA-amended cultures of Pseudomonas aeruginosa PAO1C(pRO103) and Pseudomonas putida PPO301(pRO103), strains which express a deregulated TFD monooxygenase, phenol production was proportional to cell number. Phenol was reacted, under specific conditions, with a 4-aminoantipyrine dye to form an intensely colored dye-phenol complex (AAPPC), which when measured spectrophotometrically could detect as few as 10(3) cells per ml. This assay was corroborated by monitoring the disappearance of PAA and the accumulation of phenol by high-performance liquid chromatography and gas chromatography. The AAPPC assay was modified for use with plate cultures and clearly distinguished colonies of PPO301(pRO103) and PAO1C(pRO103) from a strain expressing a regulated TFD monooxygenase. Colonies of P. putida PPO301(pRO101) remained cream colored, while colonies of PPO301(pRO103) and PAO1C(pRO103) turned a distinct red.     

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with Pseudomonas cepacia DBO1(pRO101), Alcaligenes eutrophus AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): effects of inoculation level and substrate concentration

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (=pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil. Mineralization was measured as ¹⁴C-CO₂evolved during degradation of uniformly-ring-labelled ¹⁴C-2,4-D. When the strains were inoculated to a level of approximately 10⁸ CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h. Mineralization of 0.05 ppm and 10 ppm, 2,4-D by the two A. eutrophus strains was identical and rapid whereas mineralization by P. cepacia DBO1(pRO101) occurred more slowly. In contrast, mineralization of 500 ppm 2,4-D by the two A. eutrophus strains was very slow whereas mineralization by P. cepacia DBO1 was more rapid. Comparison of 2,4-D mineralization at different levels of inoculation with P. cepacia DBO1(pRO101) (6×10⁴, 6×10⁶ and 1×10⁸ CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level. The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models. The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil. In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CFU colony forming units - PTYG peptone, tryptone, yeast & glucose - DPM disintegrations per minute     

Effects of 2,4-D and its metabolite 2,4-dichlorophenol on antioxidant enzymes and level of glutathione in human erythrocytes

The effects of in vitro exposure of human erythrocytes to different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) were studied. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the level of reduced glutathione (GSH) were determined. The activity of erythrocyte superoxide dismutase SOD decreased with increasing dose of 2,4-D and 2,4-DCP, while glutathione peroxidase activity increased. 2,4-D (500 ppm) decreased the level of reduced glutathione in erythrocytes by 18% and 2,4-DCP (250 ppm) by 32%, respectively, in comparison with the controls. These results lead to the conclusion that in vitro administration of herbicide-2,4-D and its metabolite 2,4-DCP causes a decrease in the level of reduced glutathione in erythrocytes and significant changes in antioxidant enzyme activities. Comparison of the toxicity of 2,4-D and 2,4-DCP revealed that the most prominent changes occurred in human erythrocytes incubated with 2,4-DCP.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

Enhancement of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation in soil by dissemination of catabolic plasmids

Few studies have been done to evaluate the transfer of catabolic plasmids from an introduced donor strain to indigenous microbial populations as a means to remediate contaminated soils. In this work we determined the effect of the conjugative transfer of two 2,4-D degradative plasmids to indigenous soil bacterial populations on the rate of 2,4-D degradation in soil. We also assessed the influence of the presence of 2,4-D on the number of transconjugants formed. The two plasmids used, pEMT1k and pEMT3k, encode 2,4-D degradative genes (tfd) that differ in DNA sequence as well as gene organisation, and confer different growth rates to Ralstonia eutropha JMP228 when grown with 2,4-D as a sole carbon source. In an agricultural soil (Ardoyen) treated with 2,4-D (100 ppm) there were ca. 10⁷CFU of transconjugants per gram bearing pEMT1k as well as a high number of pEMT3k bearing transconjugants (ca. 10⁶ CFU/g). In this soil the formation of a high number of 2,4-D degrading transconjugants resulted in faster degradation of 2,4-D as compared to the uninoculated control soil. In contrast, only transconjugants with pEMT1k were detected (at a level of ca. 10³ CFU/g soil) in the untreated Ardoyen soil. High numbers of transconjugants that carried pEMT1k were also found in a second experiment done using forest soil (Lembeke) treated with 100 ppm 2,4-D. However, unlike in the Ardoyen soil, no transconjugants with pEMT3k were detected and the transfer of plasmid pEMT1k to indigenous bacteria did not result in a higher rate of decrease of 2,4-D. This may be because 2,4-D was readily metabolised by indigenous bacteria in this soil. The results indicate that bioaugmentation with catabolic plasmids may be a viable means to enhance the bioremediation of soils which lack an adequate intrinsic ability to degrade a given xenobiotic.     

Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.     

Enhanced Mineralization of [U-14C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of Trifolium pratense

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/α-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [¹⁴C]2,4-D mineralization (50 μg g⁻¹) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g⁻¹) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of α-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.     

Growth Rates of a Pseudomonad on 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol

The growth of a pseudomonad on 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4-DCP (2,4-dichlorophenol) was studied in batch and continuous culture. The optimum growth rate using 2,4-D was 0.14/h at 25 C in a pH range from 6.2 to 6.9. Highest specific growth rate using 2,4-DCP was 0.12/h at 25 C in a pH range from 7.1 to 7.8. Growth was strongly inhibited by 2,4-DCP above a concentration of 25 mg/liter whereas no appreciable inhibition was observed with 2,4-D at concentrations up to 2,000 mg per liter. Growth on 2,4-DCP was described by Monod kinetics at subinhibitory concentrations but the inhibition by 2,4-DCP exhibited an unusual linear response to substrate concentration, and did not fit a model based on noncompetitive inhibition. The lag phase of batch cultures was found to depend on both 2,4-DCP concentration and prior adaptation of the inoculum. A study such as this on the kinetics of growth on related substrates may be useful as a method of finding the rate-limiting step in a metabolic sequence.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Earthworm Egg Capsules as Vectors for the Environmental Introduction of Biodegradative Bacteria

Earthworm egg capsules (cocoons) may acquire bacteria from the environment in which they are produced. We found that Ralstonia eutropha (pJP4) can be recovered from Eisenia fetida cocoons formed in soil inoculated with this bacterium. Plasmid pJP4 contains the genes necessary for 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (2,4-DCP) degradation. In this study we determined that the presence of R. eutropha (pJP4) within the developing earthworm cocoon can influence the degradation and toxicity of 2,4-D and 2,4-DCP, respectively. The addition of cocoons containing R. eutropha (pJP4) at either low or high densities (10² or 10⁵ CFU per cocoon, respectively) initiated degradation of 2,4-D in nonsterile soil microcosms. Loss of 2,4-D was observed within the first week of incubation, and respiking the soil with 2,4-D showed depletion within 24 h. Microbial analysis of the soil revealed the presence of approximately 10⁴ CFU R. eutropha (pJP4) g⁻¹ of soil. The toxicity of 2,4-DCP to developing earthworms was tested by using cocoons with or without R. eutropha (pJP4). Results showed that cocoons containing R. eutropha (pJP4) were able to tolerate higher levels of 2,4-DCP. Our results indicate that the biodegradation of 2,4-DCP by R. eutropha (pJP4) within the cocoons may be the mechanism contributing to toxicity reduction. These results suggest that the microbiota may influence the survival of developing earthworms exposed to toxic chemicals. In addition, cocoons can be used as inoculants for the introduction into the environment of beneficial bacteria, such as strains with biodegradative capabilities.     

Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium

Summary The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp. Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h^–1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methyphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions. 2,4-DP-Grown cells (10⁸) of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h. Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol. Offprint requests to: F. Streichsbier     

Mineralization of 2,4-dichlorophenoxyacetic acid in soil simultaneously enriched with saccharides

Detoxication of 2,4-dichlorophenoxyacetic acid (2,4-D) in samples of chernozem soil was determined by a biological test and the time course of production of¹⁴CO₂ a product of microbial degradation of 2-¹⁴C-2,4-D, was measured during 38-d incubation at 28°C in the dark. Enrichment of the soil with glucose (1000 ppm), two exocellular bacterial glucan and glucomannan polysaccharides (750 ppm), or a mixture of glucose with (NH₄)₂SO₄ (C:N=5∶1) brought about acceleration of both detoxication and mineralization of 2,4-D (50 ppm) added simultaneously with the saccharides. Mineralization of the saccharides always preceded the degradation of the herbicide. The lag phase of 2,4-D mineralization, did not exceed 3 d. In samples with saccharides the doubling time of the mineralization activity in the exponential phase of the process was substantially shortened and the mineralization of 2,4-D was accelerated even when the soil was inoculated with a suspension of soil in which microbial 2,4-D decomposers had accumulated. The extent, of mineralization was not affected by the presence of saccharides (about 1/3 of the introduced radioactive carbon was transformed into¹⁴CO₂). All saccharides had a similar effect which reflected an increase in the overall bacterial count and in the relative abundance of bacterial 2,4-D decomposers. The role of other mechanisms such as co-metabolism in the stimulation of the degradation process is discussed.     

Simultaneous and Repetitious Removal of 2,4-Dichlorophenol and Copper from Soils Using an Aqueous Solution of Ethyl-Lactate-Amended EDDS

Heavy metals and the transformation products of herbicides, such as 2,4-dichlorophenol (2,4-DCP), are toxic soil pollutants. We assessed the ability of an aqueous solution of the “green solvent” ethyl lactate alone and combined with [S,S]-ethylenediaminedisuccinic acid (EDDS) to remove 2,4-DCP and copper simultaneously from soils. Ethyl lactate extracted 2,4-DCP from contaminated soil comparable to Triton X-100. Ethyl lactate/EDDS extracted more 2,4-DCP and Cu from contaminated soils than ethyl lactate alone. The enhanced extraction of Cu increased slightly with an increase in the EDDS/Cu molar ratio; the maximum Cu extraction efficiency was about 32.3% at an EDDS/Cu ratio of 5. An increase in the ionic strength (NaCl) of the ethyl lactate/EDDS solution decreased the amount of 2,4-DCP extracted by maximally 12% but increased the amount of Cu extracted by >500%. We tested the recycling of the ethyl lactate/EDDS solution with the cation-exchange resin 001×7 and the hyper-cross-linked polymer resin NDA-150. Fresh ethyl lactate/EDDS solution and two sequentially recycled solutions removed 31.4, 28.3, and 26.7% of the Cu in Cu-contaminated soil and 77.7, 62.9, and 56.8% of the 2,4-DCP in 2,4-DCP-contaminated soils, respectively. The ethyl lactate/EDDS solution removed 31.8% of the Cu and 73.0% of the 2,4-DCP in Cu- and 2,4-DCP-contaminated soils, and the solution remained effective even after two recyclings. The aqueous solution of ethyl lactate/EDDS can be used to effectively remove Cu and 2,4-DCP from complex contaminated soils and can be reactivated.     

Degradation of dissolved and sorbed 2,4-dichlorophenol in soil columns by suspended and sorbed bacteria

The influence of sorption of bacteria, as well as 2,4-dichlorophenol (2,4-DCP), on themineralization of 100 g l^-1 of the organic compound was examined in an aquifer material under advective flow conditions (column displacement technique). The study was designed to distinguish the rates and extent of biodegradation of the sorbed and the dissolved trace organic and the contribution of sorbed and suspended bacteria to the degradation. The degradation of dissolved 2,4-DCP was significantly faster thanthe degradation of the same compound sorbed to the solids, and suspended bacteriadegraded the dissolved compound at a higher rate than sorbed bacteria, also on a percell basis. The suspended bacteria degraded 8–12% of the added dissolved 2,4-DCP, while sorbed bacteria made a smaller contribution by degrading about 5% of sorbed 2,4-DCP. No degradation was seen with sorbed 2,4-DCP and suspended bacteria, and a marginal contribution was made by sorbed bacteria on the degradation of dissolved 2,4-DCP (<0.4%).     

Biodegradation of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by dichlorophenol-adapted microorganisms from freshwater,anaerobic sediments

Reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated in anaerobic sediments by non-adapted microorganisms and by microorganisms adapted to either 2,4- or 3,4-dichlorophenol (DCP). The rate of dechlorination of 2,4-D was increased by adaptation of sediment microorganisms to 2,4-DCP while dechlorination by sediment microorganisms adapted to 3,4-DCP displayed a lag phase similar to non-adapted sediment slurries. Both 2,4- and 3,4-DCP-adapted microorganisms produced 4-chlorophenoxyacetic acid by ortho-chlorine removal. Lag phases prior to dechlorination of the initial addition of 2,4,5-T by DCP-adapted sediment microorganisms were comparable to those from non-adapted sediment slurries. However, the rates of dechlorination increased upon subsequent additions of 2,4,5-T. Biodegradation of 2,4,5-T by sediment microorganisms adapted to 2,4- and/ or 3,4-DCP produced 2,5-D as the initial intermediate followed by 3-chlorophenol and phenol indicating a para > ortho > meta order of dechlorination. Dechlorination of 2,4,5-T, by either adapted or non-adapted sediment microorganisms, progressed without detection of 2,4,5-trichlorophenol as an intermediate.     

Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.     

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14 d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.