Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types

The phenoxyalkanoic acid herbicides constitute a group of chemically related molecules that have been widely used for over 50 years. A range of bacteria have been selected from various locations for their ability to degrade these compounds. Previously reported strains able to utilise 2,4-dichlorophenoxyacetic acid (2,4-D) include, Ralstonia eutropha JMP134, Burkholderia sp. RASC and Variovorax paradoxus TV1 and Sphingomonas sp. AW5 able to utilise 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In addition a novel set of mecoprop-degrading strains including Alcaligenes denitrificans, Alcaligenes sp. CS1 and Ralstonia sp. CS2 are here described. It has been reported recently that TfdA enzymes, initially reported to have a role in 2,4-D catabolism are also involved in the first-step cleavage of related phenoxyalkanoate herbicides. However, a diversity of tfdA gene sequences have been reported. We relate the tfdA gene type to the metabolic ability of these strains. The tfdA-like genes were investigated by polymerase chain reaction amplification using a set of specific tfdA primers. Degradation ability was observed via phenol production from a range of unsubstituted and substituted phenoxyalkanoics including, 2,4-D, 2-methyl 4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop, 2-(2,4-dichlorophenoxy) propionic acid (racemic 2,4-DP), 2,4,5-T, 2,4-dichlorophenoxybutyric acid (2,4-DB), 4-chloro-2-methylphenoxybutyric acid (MCPB) and phenoxyacetate. Mecoprop-degrading strains showed partial tfdA sequences identical to the one described for V. paradoxus TV1 (a strain isolated on 2,4-D). However, substrate specificity was not identical as V. paradoxus exhibited greatest activity towards 2,4-D and MCPA only, whereas the mecoprop-degrading strains showed intense activity towards 2,4-D, MCPA, racemic mecoprop and (R)-mecoprop as substrates. However, Sphingomonas sp. AW5 which has been shown to carry a very different tfdA-like gene was the only strain to utilise the phenoxybutyric acid MCPB as a sole carbon source. In this study, we thus demonstrate that sequence diversity is not related to substrate specificity within the tfdA-like gene family. However, phylogenetically unrelated sequences may govern substrate specific activity.     

Phenoxy herbicide degradation in soils: Quantitative studies of 2,4-D- and MCPA-degrading microbial populations

Microbial populations able to degrade 2,4-D (2,4-dichlorophenoxyacetate) and MCPA (4-chloro-2-methylphenoxyacetate) were enumerated by means of a most probable number (MPN) procedure in eight Natal soils not previously treated with these herbicides. Estimated 2,4-D-degrading populations ranged from 1.26 to 245.2 and MCPA-degrading populations from 0.34 to 1377 g^?1 dry soil; in seven of the soils the populations of these organisms were less than 40 and 30 g^?1, respectively. Such counts indicate that for the successful isolation of 2,4-D- or MCPA-degrading microorganisms from soil, at least 1 g dry weight of soil should be used for enrichment cultures. The 2,4-D-degrading organisms occurred among the aerobic soil bacteria detectable by plate count, at frequencies of only 1 in 30 × 10³ to 1 in 36 × 10⁶ and the MCPA-degrading organisms at frequencies of 1 in 5 × 10³ to 1 in 133 × 10⁶; the ease with which the herbicide-degrading organisms can be isolated from enriched soil cultures treated with 2,4-D or MCPA is evidence of their massive preferential proliferation in response to the herbicides.Log 2,4-D- and MCPA-degrading populations did not differ significantly in four soil samples, but in the others either the 2,4-D- or the MCPA-degrading population was dominant. The longer persistence of MCPA compared with that of 2,4-D could therefore not be ascribed to quantitative differences in the populations of MCPA- and 2,4-D-degrading soil microorganisms.No relationship was evident between the soil populations of 2,4-D- or MCPA-degrading microorganisms and aerobic soil bacteria, and variations of the three populations among the soil samples were not associated in any obvious way with the soil physical and chemical characteristics, except perhaps an association of the highest counts of herbicide-degrading organisms with a sugar cane soil of sandy texture and high C: N ratio.     

Degradation of [carboxyl-14C] 2,4-D and [ring-U-14C] 2,4-D in 114 agricultural soils as affected by soil organic carbon content

This study compared the degradation of [carboxyl-¹⁴C] 2,4-dichlorophenoxyacetic acid (2,4-D) (C2,4-D) and [ring-U-¹⁴C] 2,4-D (R2,4-D) in 114 agricultural soils (0–15 cm) as affected by 2,4-D sorption and soil properties (organic carbon content, pH, clay content, carbonate content, cation exchange capacity, total microbial activity). The sample area was confined to Alberta, Canada, located 49–60° north longitude and 110–120° west latitude and soils were grouped by soil organic carbon content (SOC) (0–0.99%, 1–1.99%, 2–2.99%, 3–3.99% and >4% SOC). Degradation rates of C2,4-D and R2,4-D followed first-order kinetics in all soils. Although total microbial activity increased with increasing SOC, degradation rates and total degradation of C2,4-D and R2,4-D decreased with increasing SOC because of increased sorption of 2,4-D by soil and reduced bioavailability of 2,4-D and its metabolites. Rates of R2,4-D degradation were more limited by sorption than rates of C2,4-D degradation, possibly because of greater sorption and formation of bound residues of 2,4-D metabolites relative to the 2,4-D parent molecule. Based on the sorption and degradation parameters quantified, there were two distinct groups of soils, those with less than 1% SOC and those with greater than 1% SOC. Specifically, soils with less than 1% SOC had, on average, 2.4 times smaller soil organic carbon sorption coefficients and 1.4 times smaller 2,4-D half-lives than soils with more than 1% SOC. In regional scale model simulations of pesticide leaching to groundwater, covering many soils, input parameters for each pesticide include a single soil organic carbon sorption coefficient and single half-life value. Our results imply, however, that the approach to these regional scale assessments could be improved by adjusting the values of these two input parameters according to SOC. Specifically, this study indicates that for 2,4-D and Alberta soils containing less than 1% SOC, the 2,4-D pesticide parameters obtained from generic databases should be divided by 2.5 (soil organic carbon sorption coefficient) and 1.5 (half-life value).     

不同生长素类型及ABA搭配对小麦幼胚再生效果的影响

生长素是小麦幼胚产生胚性愈伤组织的关键成分,培养基中适宜的生长素种类、浓度和组合决定着小麦幼胚再生效果。本研究以小麦基因型CB037幼胚为材料,在分别确定dicamba、2,4,5-T和picloram的适宜用量的基础上,比较了2,4-D、dicamba、2,4,5-T、picloram在适宜浓度下诱导再生的效果,筛选...     

Spatial variability of 2,4-dichlorophenoxyacetic acid (2,4-D) mineralisation potential at a millimetre scale in soil

We analysed the ability of soil units of millimetre size to mineralise a herbicide, 2,4-D, using incubations of individual aggregates (2-7 mm diameter) and 6×6×6 mm³ cubes dissected from soil cores, under standard conditions. Mineralisation of ¹⁴C-ring labelled 2,4-D was measured using a barite paper trap and a Phosphorimager to record the evolved ¹⁴C-CO₂ from these very small soil samples. We found a large variability of 2,4-D mineralisation potential between aggregate size classes, between individual aggregates of the same size and between the different dissected cubes from a given core. We explained this variability by an uneven distribution of the degrading microorganisms at this scale, and to a lesser extent, an uneven distribution of C, necessary for co-metabolism. Furthermore, we found that in a soil core, the dissected cubes with a large mineralisation potential were not randomly distributed, but rather organised into centimetre sized hot spots.     

Single-laboratory validation of EPA Method 8150 for determination of chlorinated herbicides in hazardous waste

Method 8150, published in the second edition of Test Methods for Evaluating Solid Waste, Manual SW-846, required optimization, ruggedness testing, linearity determinations, precision tests, bias testing, gas chromatographic/mass spectrometric confirmation, and quality control guidelines for validation of the protocol. This single-laboratory validation, which is applicable to the determination of the herbicides dicamba, silvex, 2,4-D, 2,4-DB, 2,4,5-T, dinoseb, MCPP, MCPA, and dichlorprop in hazardous waste extracts, was completed and is described in this report. Final ruggedness testing of the optimized procedure gave a mean recovery of 89.3% with a standard deviation of 4.3%. Percent relative standard deviations are less than 10 (n = 20, each analyte) over a 10(2) linear range of concentration for MCPP and MCPA and over a 10(3) linear range of concentration for the other target herbicide esters. Instrumental detection limits for electron capture detection and mass spectrometric identity confirmation were determined and found to be matrix-dependent.     

Simultaneous Biodegradation and Detoxification of the Herbicides 2,4-Dichlorophenoxyacetic Acid and 4-Chloro-2-Methylphenoxyacetic Acid in a Continuous Biofilm Reactor

The herbicides 2,4-diclorophenoxiacetic and 4-chloro-2-methylphenoxyacetic acids (2,4-D and MCPA) are widely used in agricultural practices worldwide. Not only are these practices responsible of surface waters contamination, but also agrochemical industries through the discharge of their liquid effluents. In this investigation, the ability of a 2,4-D degrading Delftia sp. strain to degrade the related compound MCPA and a mixture of both herbicides was assessed in batch reactors. The strain was also employed to remove and detoxify both herbicides from a synthetic effluent in a continuous reactor. Batch experiments were conducted in a 2-L aerobic microfermentor, at 28 °C. Continuous experiments were carried out in an aerobic downflow fixed-bed reactor. Bacterial growth was evaluated by the plate count method. Degradation of the compounds was evaluated by UV spectrophotometry, gas chromatography (GC), and chemical oxygen demand (COD). Toxicity was assessed before and after the continuous process by using Lactuca sativa seeds as test organisms. Delftia sp. was able to degrade 100 mg L^?1 of MCPA in 52 h. When the biodegradation assay was carried out with a mixture of 100 mg L^?1 of each herbicide, the process was accomplished in 56 h. In the continuous reactor, the strain showed high efficiency in the simultaneous removal of 100 mg L^?1 of each herbicide. Removals of 99.7, 99.5, and 95.0% were achieved for 2,4-D, MCPA, and COD, respectively. Samples from the influent of the continuous reactor showed high toxicity levels for Lactuca sativa seeds, while toxicity was not detected after the continuous process.     

Turnover of microbial tissue in soil under field conditions

The microbial population of a Brown Chernozemic soil was labelled in situ by adding ¹⁴C-glucose and ¹⁵NH₄¹⁵NO₃ to the plow layer. The loss of ¹⁴C, nitrogen immobilization-mineralization reactions, bacterial numbers (plate count, direct count) and fungal hyphal lengths were determined periodically throughout the growing period in amended and unamended microplots and in the surrounding field soil. After 5 days, 90 per cent of the labelled N occurred in the organic form with little subsequent mineralization. Of the labelled C added, 63, 56 and 39 per cent, remained in the soil after 3, 14 and 104 days, respectively.The ratio of fungal C to bacterial C increased as soil moisture decreased. Viable (plate count) and total numbers of bacteria in samples from unamended plots and field soil were significantly correlated with each other and with soil moisture. Fungal hyphal lengths from amended soil were also significantly related to moisture but the rate of loss of ¹⁴C and mineralization of ¹⁵N were not. The synthesized microbial material (tissue and metabolites) exhibited a high degree of stability throughout the study. The half-life of labelled C remaining in the soil after 30 days was calculated to be 6 months compared to only 4 days for the added glucose C. The amount of energy used for maintenance by the soil population under field conditions was calculated from measurements of biomass C, respired labelled C and respired soil C.     

Mineralization of the herbicide MCPA in urban soils is linked to presence and growth of class III tfdA genes

Mineralization and sorption of ¹⁴C-ring labeled herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) were quantified along with the tfdA gene abundance in 7 different soils. The soils tested were five gravel soils from urban locations, one soil from the embankment of a railway track, and finally an agricultural soil as a control. The mineralization experiments were performed with a concentration of MCPA of 5 mg/kg and incubated at 10 °C for a period of 60 days.With K_d values ranging from 0.04 to 0.41 l kg⁻¹ the sorption experiments revealed that binding of MCPA to the six gravel soils was lower compared to the control soil which had a K_d value of 0.91 l kg⁻¹. The potential for MCPA mineralization varied from less than 5 to over 55% mineralized in 60 days. The most rapid MCPA mineralization was observed in the soil from the Danish railway tracks with 55% mineralized after only 18 days. The mineralization data was fitted to degradation kinetic models, which indicated that growth occurred as a response to MCPA degradation in most of the soils.Soil DNA was extracted and tfdA genes responsible for the first step in MCPA degradation were quantified by real-time PCR (qPCR) at appropriate time points throughout the mineralization experiments. Indicating growth of specific MCPA degraders, the abundance of class III tfdA genes showed an increase during MCPA mineralization in those soils able to mineralize MCPA.These findings emphasize the importance of the presence of microorganisms that are able to readily degrade MCPA, to avoid groundwater leaching following use on urban gravel areas that possess low binding ability of the compound.     

Effects of 2,4-d on nitrogen fixation and carbon dioxide evolution in a soil microcosm

Two concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) 1.7 kg ha^?1 and 3.4 kg ha^?1 were applied to oats (Avena sativa L. ‘Orbit’) grown in terrestrial microcosms in a sandy soil. Carbon dioxide evolution and non-symbiotic N₂ fixation (C₂H₂ reduction) were measured weekly. On day 70 of the study, 2,4-D was applied a second time at the same application rates and soil CO₂ evolution and N₂ fixation were measured more frequently. Soil CO₂ evolution over 24 h period was significantly decreased by 40 to 50% at both application rates of 2,4-D. This response lasted less than 7 days. Nitrogen fixation was unaffected by 2,4-D except for an unexplained decrease observed in the 1.7 kg ha^?1 treatment 35 days after 2,4-D application. This effect was not observed on the following sampling date. The second application of 2,4-D failed to produce any significant change in soil CO₂ evolution or N₂ fixation. From these studies we conclude soil microbial populations either degraded or became acclimated to 2,4-D as a result of the initial application and that 2,4-D has no significant adverse effect on N₂ fixation or soil CO₂ evolution.     

The millimetre-scale distribution of 2,4-D and its degraders drives the fate of 2,4-D at the soil core scale

The biodegradation of organic compounds in soil is a key process that has major implications for different ecosystem services such as soil fertility, air and water quality, and climate regulation. Due to the complexity of soil, the distributions of organic compounds and microorganisms are heterogeneous on sub-cm scales, and biodegradation is therefore partly controlled by the respective localizations of organic substrates and degraders. If they are not co-localized, transfer processes become crucial for the accessibility and availability of the substrate to degraders. This spatial interaction is still poorly understood, leading to poor predictions of organic compound dynamics in soils. The objectives of this work were to better understand how the mm-scale distribution of a model pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), and its degraders drives the fate of 2,4-D at the cm soil core scale. We constructed cm-scale soil cores combining sterilized and “natural” soil aggregates in which we controlled the initial distributions of 2,4-D and soil microorganisms with the following spatial distributions: i) a homogeneous distribution of microorganisms and 2,4-D at the core-scale, ii) a co-localized distribution of microorganisms and 2,4-D in a single spot (360 mm³) and iii) a disjoint localization of microorganisms and 2,4-D in 2 soil spots (360 mm³) separated by 2 cm. Two sets of experiments were performed: one used radiolabeled ¹⁴C-2,4-D to study the fate of 2,4-D, and the other used ¹²C-2,4-D to follow the dynamics of degraders. Microcosms were incubated at 20 °C and at field capacity (−31.6 kPa). At the core scale, we followed 2,4-D mineralization over time. On three dates, soil cores with microorganisms and 2,4-D localized in soil spots, were cut out in slices and then in 360 mm³ soil cubes. The individual soil cubes were then independently analysed for extractable and non-extractable ¹⁴C and for degraders (quantitative PCR of tfdA genes). Knowing the initial position of each soil cube allowed us to establish 3D maps of 2,4-D residues and degraders in soil. The results indicated that microorganisms and pesticide localizations in soil are major driving factors of i) pesticide biodegradation, by regulating the accessibility of 2,4-D to degrading microorganisms (by diffusion); and ii) the formation of non-extractable residues (NER). These results also emphasized the dominant role of microorganisms in the formation and localization of biogenic NER at a mm-scale. To conclude, these results demonstrate the importance of considering micro-scale processes to better understand the fate of pesticides and more generally of soil organic substrates at upper scales in soil and suggest that such spatial heterogeneity should not be neglected when predicting the fate of organic compounds in soils.     

Mineralization of carbon atoms of 14C-2,4-D side chain and degradation ability of bacteria in soil

The time-course of ¹⁴CO₂ formation in chernozem soil samples enriched with 1- or 2-¹⁴C-2, 4-dichlorophenoxyacetic acid (50 μg g g^?1 air-dry soil) was determined during incubation at 28°C. Except for the initial phase of decomposition, when the conversion of carboxyl carbon to ¹⁴CO₂ predominated over that of carbon in position 2, the rates of mineralization of the two carbon atoms of the side chain of the herbicide molecule exhibited no significant difference. The exponential phase of ¹⁴CO₂ evolution lasted from the 3rd to the 21st day of incubation; a semilogarithmic plot of its time dependence was strictly linear. The mineralization activity doubling time in this phase was 89.1 ± 3.6 h with 1-¹⁴CO-2, 4-D and 85.4 ± 5. l h with 2-¹⁴CO-2,4-D. An exponential decrease in mineralization activity was observed after 21 days, probably due to substrate exhaustion. The total proportion of radioactive carbon introduced into the soil in the form of 1- or 2-¹⁴CO-2,4-D and converted into ¹⁴CO₂ during 31 days of incubation was about 33%. Plate counts of bacteria increased during 35 days of incubation from 2.14 × 10⁸ to 2.8 × 10⁸ g^?1. The proportion of bacteria capable of producing ¹⁴CO₂ from the labelled herbicide increased in this period from 4.1 to 86.1%. This increase is probably directly responsible for the immediate onset of mineralization of the herbicide in soil treated previously with it or in soil inoculated with a suspension prepared from a sample previously incubated with the herbicide.     

Fate of 14C-ring-labeled 2,4-D, 2,4-dichlorophenol and 4-chlorophenol during straw composting

Experiments were carried out to study the transformation of ¹⁴C-ring-labeled 2,4-D and the two related chlorophenols 4-chlorophenol (4-CP) and 2,4-dichlorophenol (4-DCP) during straw composting under controlled laboratory conditions. Incubation under sterile and nonsterile conditions was done to evaluate the relative importance of the biotic and abiotic processes. Pre-composted straw was treated with the three chemicals. The availability of the different chemicals was monitored during incubations as well as their degradation. Under nonsterile conditions, the mineralization of both chlorophenols reached 20% of the applied compounds, whereas it was 52% for 2,4-D. Transitory water-soluble metabolites of 2,4-D and chlorophenols were formed but they disappeared rapidly. After 21 days, 21% of the 2,4-D and 38% of the 2,4-DCP was stabilized as nonextractable (bound) residues under nonsterile conditions. Bound residues of both chemicals were negligible under sterile conditions. Availability of chemicals as estimated by water extraction decreased during incubation proportionally to mineralization and to the formation of bound residues. The increase in immobilization of the chemical residues was stronger under nonsterile conditions than under sterile conditions. Under nonsterile conditions 71% of the 4-CP was recovered as bound residues, whereas under sterile conditions 30% of the applied 4-CP formed bound residues after formaldehyde addition and only 8% with autoclaved straw. Global microbial activity decreased in the presence of the chlorophenols probably due to their toxic effect. These data indicate that the biological activity associated with straw transformation during composting stimulates the depletion of 2,4-D and chlorophenols by mineralization and by formation of bound residues. Received: 6 September 1996     

Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing

Stable isotope probing (SIP) was used to investigate the microorganisms responsible for degradation of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) in soil samples. Soils were unamended or amended with either unlabeled 2,4-D or UL(ring) ¹³C-2,4-D. Degradation of 2,4-D was complete after 17 days, whereas little removal (11±3%) was observed in the sterile controls. Terminal restriction fragment length polymorphism (TRFLP) on soil DNA after 17 days indicated a consistent increase in the relative abundance of one fragment (217 bp in Hae III digests) in soils spiked with 2,4-D (both unlabeled and labeled samples) compared to the unamended soils. DNA extracts from labeled and unlabeled 2,4-D amended soils were subject to ultracentrifugation, fractionation of centrifuged samples, followed by TRFLP on each fraction. TRFLP profiles from ultracentrifugation fractions illustrated that the same fragment experienced an increase in buoyant density (BD) in samples spiked with ¹³C-labeled 2,4-D. This increase in DNA BD indicates the organisms represented by this fragment were responsible for uptake and degradation of the herbicide. 16S rRNA sequencing of the heavy, ¹³C-enriched fraction suggests the organisms belong to the β subdivision of Proteobacteria. Herein, SIP facilitated the identification of unique organisms degrading 2,4-D in soil without the need for isolation and provided more direct evidence for a functional role of these organisms than would have been possible with the molecular-based methods alone.     

Adsorption and degradation of 2,4-dichlorophenoxyacetic acid in spiked soil with Fe0 nanoparticles supported by biochar

This work studies the adsorption and degradation of 2,4-dichlorophenoxyacetic (2,4-D) in spiked soil with nanoscale Fe⁰ particles (nFe⁰) and biochar derived from maize straw. When biochar concentration was high, the adsorption capacity of soil was enhanced. Furthermore, 2,4-D degraded completely at loading rates of 0.33 and 0.17 g/L nFe⁰ plus biochar (initial 2,4-D concentration of 10 mg/g) within 40 h, according to equilibrium data. Additionally, the theoretical concentration of chloridion was approximately 84%. Further analysis indicated that the effect of nFe⁰ on 2,4-D degradation was weaker in soil columns than that in soil slurry. By contrast, 2,4-D degradation is positively influenced by biochar application, which prevented the aggregation and corrosion of Fe nanoparticles. Although the enhanced capacity for 2,4-D adsorption on the soil decelerated dechlorination rate, long-term nFe⁰ activity was generated. After 72 h, the efficiency of 2,4-D degradation was approximately 53.2% in the soil columns with biochar support.     

Chloroform fumigation technique as a means of determining the size of specialized soil microbial populations: Application to pesticide-degrading microorganisms

A method is proposed for studying the dynamic behaviour of the soil microbial population involved in the degradation of 2,4-D. The method is based on in situ specific-labelling of that population following treatment of the soil with ¹⁴C-labelled herbicide and investigating the kinetics of the incorporation of radioactivity by the soil microflora in treated soil samples subjected to the chloroform-fumigation technique after varying periods of exposure. Non-degraded herbicide still present in the soil after fumigation did not affect the overall flush of CO₂ and was not further broken down at a sufficient rate to appreciably contribute to ¹⁴CO₂ evolution. The validity of the method to assess the soil biomass of the 2,4-D degrading population together with its time variations is discussed in relation to the position of the ¹⁴C on the pesticide molecule.     

Der Fe(III)-Reduktionstest - ein einfaches Verfahren zur Abschätzung der Wirkung von Umweltchemikalien auf die mikrobielle Aktivität in Böden

The Fe(III)-reduction test - a simple procedure to determine the effects of environmental chemicals on the microbial activity in soils A new simple microorganism test is described. The test is based on the fact that the degree of microbial reduction in waterlogged soils is influenced by the addition of different amounts of toxic chemicals. The microbial activity under reducing conditions can be measured by the degree of the microbial reduction of easily reducable Fe(III)-oxides (ferrihydrites) to soluble Fe²⁺-ions. The influence of different test parameters, the reproducibility and limitations of the proposed test are described. Toxicity indices of the investigated chemicals are derived graphically from the resulting dose effect curves. Values are determined for those concentrations which cause no (Non Effect Level, NEL) and a 50%-inhibition (ED₅₀) of the microbial activity in soil samples. By representing some results for 2,4-D, 2,4,5-T, LAS, Cd and Hg the scope of the test is described. First results show that the effective toxicity of chemicals is strongly influenced by the adsorption capacity of the soils. Furthermore the relation of toxic substances to nutrients in the soil solution causing competition or antagonistic and synergistic effects during uptake and also within the organisms can substantially influence the toxicity of chemicals.     

Soil microbial communities response to herbicide 2,4-dichlorophenoxyacetic acid butyl ester

2,4-Dichlorophenoxyacetic acid butyl ester (2,4-D butyl ester) is extensively applied for weed control in cultivation fields in China, but its effect on soil microbial community remains obscure. This study investigated the microbial response to 2,4-D butyl ester application at different concentrations (CK, 10, 100 and 1000 μg g^?1) in the soils with two fertility levels, using soil dilution plate method and phospholipid fatty acid (PLFA) analysis. Culturable microorganisms were affected by the herbicide in both soils, particularly at the higher concentration. After treating soil with 100 μg g^?1 herbicide, culturable bacteria and actinomycetes were significantly higher, compared to other treatments. Treatment of soil with 1000 μg g^?1 2,4-D butyl ester caused a decline in culturable microbial counts, with the exception of fungal numbers, which increased over the incubation time. PLFA profiles showed that fatty acids for Gram-negative (GN) bacteria, Gram-positive (GP) bacteria, total bacteria and total fungi, as well as total PLFAs, varied with herbicide concentration for both soil samples. As herbicide concentration increased, the GN/GP ratio decreased dramatically in the two soils. The higher stress level was in the treatments with high concentrations of herbicide (1000 μg g^?1) for both soils. Principal component analysis of PLFAs showed that the addition of 2,4-D butyl ester significantly shifted the microbial community structure in the two soils. These results showed that the herbicide 2,4-D butyl ester might have substantial effects on microbial population and microbial community structure in agricultural soils. In particular, the effects of 2,4-D butyl ester were greater in soil with low organic matter and fertility level than in soil with high organic matter and fertility level.     

Dynamics of soil microbial populations involved in 2,4-D biodegradation revealed by FAME-based Stable Isotope Probing

Stable Isotope Probing (SIP) is a powerful tool for analysing the fate of pesticides in soil. Together with FAME (Fatty Acid Methyl Esters), it can help identify biodegradation pathways and recycling into the microbial biomass. The fate of ring-labelled ¹³C-2,4-dichlorophenoxyacetic acid or 2,4-D (C_2,4-D) was determined in soil during a 6-month incubation. The distribution of ¹³C among the microbial biomass, the CO₂ respired, the water, methanol and dichloromethane soluble fractions, and the residual non-extracted bulk soil was measured. Molecular analyses were carried out on the lipid and the non-extractable fractions. After 8 days, about half of the initial amount of C_2,4-D was mineralised; the other half remained in soil as non-extractable residues (NER). C_2,4-D continued to be mineralised, suggesting that NER were still bioavailable. Analysis of C_2,4-D-enriched FAME contained in the lipid fraction suggested that a succession of microbial populations was involved in 2,4-D biodegradation. This is possibly due to the change of 2,4-D availability. The C_2,4-D yield coefficient and degrader diversity evolved during the incubation, providing corroboratory evidence that different physiological groups were active during the incubation. The ¹³C-labelled microbial community was always less diverse than the total community, even at the end of the incubation, suggesting that the cross-feeding community is also a specific part of the total community. This work shows that molecular analysis of ¹³C-labelled pesticides is a useful tool for understanding both chemical and biological aspects of their fate in soil.     

Protection of tomato seed germination from the inhibitory effect of 2,4,5-trichlorophenoxyacetic acid by inoculation of soil with Burkholderia cepacia AC1100

The effect of 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) on the germination and seedling vigor of different crop seeds was tested. Seeds of rice, maize, sorghum, finger millet, and horse gram were comparatively more tolerant to the chemical with no marked effect up to a concentration of 200 mg 2,4,5-T L(-)(1) as tested by the filter paper method. Tomato and brinjal (egg plant) were highly susceptible. Even at 5 and 10 mg 2,4,5-T L(-)(1), marked reduction in the germination and seedling vigor of tomato and egg plant, respectively, was observed. At 20 and 30 mg L(-)(1), the germination of tomato and egg plant seeds, respectively, were completely inhibited on filter paper, whereas the inhibitory concentrations in soil was 40 mg 2,4,5-T kg(-)(1) soil. Several abnormalities were observed in the chemically affected seedlings. Protease activity of the seeds germinating in the presence of the chemical was drastically reduced. Bioremediation of the chemically contaminated soil with Burkholderia cepacia AC1100, by inoculation of the soil 7 days before sowing the seeds, completely protected the seeds, resulting in normal germination and an improved seedling vigor.