Effects of sorption on biological degradation rates of (2,4-dichlorophenoxy) acetic acid in soils.

Three mathematical models were proposed to describe the effects of sorption of both bacteria and the herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) on the biological degradation rates of 2,4-D in soils. Model 1 assumed that sorbed 2,4-D is not degraded, that only bacteria in solution are capable of degrading 2,4-D in solution, and that sorbed bacteria are not capable of degrading either sorbed or solution 2,4-D. Model 2 stated that only bacteria in the solution phase degrade 2,4-D in solution and that only sorbed bacteria degrade sorbed 2,4-D. Model 3 proposed that sorbed 2,4-D is completely protected from degradation and that both sorbed and solution bacteria are capable of degrading 2,4-D in solution. These models were tested by a series of controlled laboratory experiments. Models 1 and 2 did not describe the data satisfactorily and were rejected. Model 3 described the experimental results quite well, indicating that sorbed 2,4-D was completely protected from biological degradation and that sorbed- and solution-phase bacteria degraded solution-phase 2,4-D with almost equal efficiencies.     

Structure of the complex of heptakis(2,6-di-O-methyl)-beta-cyclodextrin with (2,4-dichlorophenoxy)acetic acid

The structure of the complex formed by heptakis(2,6-di-O-methyl)-beta-cyclodextrin and (2,4-dichlorophenoxy)acetic acid was studied by X-ray diffraction. The dichlorophenyl moiety of the guest molecule was found outside the host hydrophobic cavity in the primary methoxy groups region whereas the oxyacetic acid chain penetrates the cavity from the primary face. The host molecules stacks along the a crystal axis forming a column. In the space between three successive hosts of the column, a guest molecule is accommodated.     

Accumulation of (2,4-dichlorophenoxy) acetic acid and (2,4,5-trichlorophenoxy) acetic acid by Parenchyma Tissue as Influenced by Metabolic Inhibitors and Lecithin

Parenchyma tissue from potato (Solanum tuberosum L. cv. Russet) tubers was treated with inhibitors to the release of metabolic energy in order to determine the importance of an active transport system for (2,4-dichlorophenoxy)acetic acid (2,4-D) and (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) accumulation. Results from treatments with carbonylcyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation, and N,N'-dicyclohexylcarbodiimide, an inhibitor of membrane-bound adenosine triphosphatase, indicated 2,4-D and 2,4,5-T accumulation to be independent of available energy as influenced by these metabolic inhibitors. Lecithin treated parenchyma tissue accumulated more 2,4-D and 2,4,5-T than untreated tissue indicating possible binding of the herbicide to the lecithin moiety.     

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.     

Algicidal effect of 2,4-dichlorophenoxy acetic acid on blue-green alga Cylindrospermum sp.

The effect of the herbicide 2,4-Dichlorophenoxy acetic acid generally used in agriculture was studied on the nitrogen fixing blue-green alga Cylindrospermum sp. The alga could tolerate up to 150 μg per ml in liquid culture and 100 μg per ml on agar plates without any inhibitory effect on growth and survival. The maximum tolerance was up to 800 μg per ml and higher concentrations were lethal.     

The binding of amino acids to the herbicide 2,4-dichlorophenoxy acetic acid

Summary. The interaction of amino acids with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was studied by charge-transfer chromatography carried out on diatomaceous layers covered with different amount of 2,4-D and the effect of salts on the strength of interaction was elucidated. It was established that Arg, His, Lys, Orn, Phe and Trp binds to 2,4-D, the binding process is of saturation character. Principal component analysis proved that the concentration of 2,4-D exerts the highest impact on the interaction and the effect of salts is of secondary importance. The results suggest that these amino acid residues may account for the binding of 2,4-D to proteins and can play a considerable role in the detoxification processes by forming conjugates with 2,4-D. Received April 10, 1998, Accepted September 15, 1998     

The movement of 2,4-dichlorophenoxy acetic acid in root segments of Pisum sativum L.

Summary An acropetal polarisation of the movement of 2,4-dichlorophenoxy acetic acid (2,4-D) through subapical segments of Pisum seedling primary roots has been monitored throughout a 60 h transport period in darkness at 25° C using [1-¹⁴C]2,4-D and [2-¹⁴C]2,4-D. Uptake of 2,4-D does not proceed at a constant rate; periods in which the amount of ¹⁴C in the root segments and receiver blocks increases rapidly are followed by periods in which the amount of radioactivity remains relatively constant or declines slightly. These oscillations do not appear to be related to the time of day at which the experiments are begun or ended. Immobilisation and degradation of 2,4-D during transport in the segments seems to be low. Replacement of [1-¹⁴C]2,4-D donor blocks after 25 h by blocks containing unlabelled 2,4-D results in continued transport of the compound into receiver blocks, with only small amounts of ¹⁴C remaining in the root tissues. Radioactivity is also exported from the segments into the blocks used to replace the donor blocks, with larger amounts being exported into the blocks applied to the apical ends than into those applied to the basal ends of the segments. This radioactivity may be taken-up again by the segments but more ¹⁴C is exported into these blocks towards the end of the experiments. The possibility of regular oscillations in uptake and movement of 2,4-D in Pisum root segments is discussed.     

Novel 2,4-dichlorophenoxy acetic acid substituted thiazolidin-4-ones as anti-inflammatory agents: Design,synthesis and biological screening

A library of fourteen 2-imino-4-thiazolidinone derivatives (1a-1n) has been synthesized and evaluated for in vivo anti-inflammatory activity and effect on ex-vivo COX-2 and TNF–α expression. Compounds 1k (5-(2,4-dichloro-phenooxy)-acetic acid (3-benzyl-4-oxo-thiazolidin-2-ylidene)-hydrazide) and 1m (5-(2,4-dichloro-phenooxy)-acetic acid (3-cyclohexyl-4-oxo-thiazolidin-2-ylidene)-hydrazide) exhibited in vivo inhibition of 81.14% and 78.80% respectively after 5 h in comparison to indomethacin which showed 76.36% inhibition of inflammation without causing any damage to the stomach. Compound 1k showed a reduction of 68.32% in the level of COX-2 as compared to the indomethacin which exhibited 66.23% inhibition of COX-2. The selectivity index of compound 1k was found to be 29.00 in comparison to indomethacin showing selectivity index of 0.476. Compounds 1k and 1m were also found to significantly suppress TNF-α concentration to 70.10% and 68.43% in comparison to indomethacin which exhibited 66.45% suppression.     

Influence of herbicide, 2,4-dichlorophenoxy acetic acid, on haemocyte DNA of in vivo treated mussel

The influence of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) on haemocyte DNA of in vivo treated mussels Mytilus galloprovincialis has been investigated by flow cytometry and epifluorescence microscopy. Haemocyte proliferation and atypical flow cytometric DNA histograms were observed in mussels treated with 20 and 100 μg/g of 2,4-D. The stimulation of proliferation by 2,4-D was also obvious by DNA labelling with BrdU followed by FITC conjugated anti-BrdU MoAb visualised by epifluorescence microscopy. An apoptotic sub-G₁ peak resulted in mussels that were exposed to higher doses of herbicide at 100 and 500 μg/g as well as subpopulation could be detected by flow cytometric analysis. In these experiments morphological changes characteristic for apoptotic cells were looked for by fluorescence microscopy. A low percentage of cells in S as well as in G₂M phase indicating G1 arrest were detected in haemocytes from these mussels that had survived 4 days of 20 μg/g 2,4-D exposure. In addition, sister-chromatid exchanges (SCE) could be seen with the immunolabelling BrdU method. Thus, in vivo treatment and the subsequent uptake of 2,4-D causes serious genetic consequences and raises concerns regarding the potential overall fitness and health effects in mussel populations.     

Oocyte maturation in Xenopus laevis is blocked by the hormonal herbicide, 2,4-dichlorophenoxy acetic acid

Oocyte maturation is dependent on a complex program of morphological, ultrastructural, and biochemical signaling events, and if disrupted could lead to decreased fertility and population decline. The in vitro sensitivity of amphibian oocytes and oocyte maturation to plant growth factor and widely used hormonal herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in this study to determine its potential impact on early development and possible contribution to the global amphibian decline. Progesterone, which acts through a membrane receptor, triggers meiotic maturation in full grown (stage VI) Xenopus oocytes, characterized by cytoskeletal reorganization, nuclear dissolution, chromosome condensation, and spindle formation. Biochemically, the Mos/MAPK/MPF signaling pathway is activated, in part dependent on translational activation of specific maternal mRNAs such as c-Mos. Light microscopy revealed unusual asymmetric morphotypes in oocytes exposed to 2,4-D alone characterized by a white spot and bulge, termed coning, in the animal pole where the germinal vesicle (nucleus) persisted intact. Treatment of oocytes with cytochalasin B, a microfilament inhibitor, blocked these morphotypes but nocodazole, a microtubule depolymerizing agent, did not. Confocal microscopy showed that 2,4-D, itself, caused substantial depolymerization of perinuclear microtubules. Importantly, 2,4-D blocked progesterone-induced maturation as measured by the lack of nuclear breakdown, confirmed by the lack of Mos expression, MPF activation, and cytoplasmic polyadenylation of cyclin B1 mRNA. However, Western blot analysis and U0126 inhibitor studies showed that 2,4-D, either alone or in the presence of progesterone, induced MAPK phosphorylation through MAPKK. These results show that 2,4-D disrupts oocyte cytoskeletal organization and blocks maturation while stimulating an independent MAPK signaling pathway.     

Differential response of Trifolium species to 4-(2,4-dichlorophenoxy)butyric acid treatments

The differential response of white clover ( Trifolium repens L. cv. Regal Ladino) and berseem clover ( Trifolium alexandrinum L. cv. Mississippi ecotype) was investigated by treating greenhouse cultured plants with 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Berseem clover plants were significantly injured by a treatment concentration of 0.6 kg ha^-1 of 2,4-DB, whereas white clover plants were not injured by treatment levels below 2.4 kg ha^-1. The metabolism of 2,4-DB in cell suspension cultures of white clover and berseem clover was investigated using [ring-¹⁴C]-2,4-DB and non-labeled 2,4-DB. White clover cell cultures metabolized ca 4-fold more 2,4-DB than berseem cultures over a 44-h treatment period. The decrease in berseem cell population was 4-fold greater than the decrease in white clover cell population in response to the 8 μ M 2,4-DB treatment. The herbicide and its [ring-¹⁴C]-labeled metabolites were isolated from treated cells and medium after 44 h by partition and thin-layer chromatography. White clover cells metabolized 90% of the [¹⁴C]-2,4-DB and berseem clover cells metabolized 22% of the herbicide. The major portion of the radiolabel was in the glycoside fractions from extracts of both species. The differential response of Trifolium species to 2,4-DB is implied to be due to the differential rate of 2,4-DB metabolism to a glycoside by the clover plants.     

Effects of biochar amendment on the sorption and degradation of atrazine in different soils

ABSTRACT

Sugarcane top-derived biochar was added to an alluvial soil, a moist soil and a paddy soil at the rate of 0.2% and 0.5% (w/w). After the addition of 0.2% and 0.5% biochar, the sorption coefficients (K_d) of atrazine (Ce = 10 mg L^?1) were increased by 26.97% and 79.58%, respectively, in the moist soil with a low level of total organic carbon (TOC), while it increased by 31.43% and 60.06%, respectively, in the paddy soil with a high TOC content. The half-time persistence values of atrazine in the alluvial soil, moist soil and paddy soil were 28.18, 23.74 and 39.84 d, respectively. In the 0.2% biochar amended soils, the corresponding half-times of atrazine for the alluvial soil, moist soil and paddy soil were extended by 10.33, 11.81 and 1.42 d, and they were prolonged by 16.83, 17.52 and 14.74 d, respectively, in the 0.5% biochar amended soils. Atrazine degradation products (deisopropylatrazine and desethylatrazine) decreased after they accumulated to 3.2 and 1 mg kg^?1, respectively. Generally, increasing sorption was accompanied by decreasing degradation of atrazine which is found in biochar-amended soils.     

Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid,naphthalene-1-acetic acid,and indole-3-acetic acid in suspension-cultured tobacco cells

Accumulation of radiolabelled naphthalene-1-acetic acid (1-NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IAA) has been measured in suspension-cultured tobacco (Nicotiana tabacum) cells. In this paper is presented a simple methodology allowing activities of the auxin influx and efflux carriers to be monitored independently by measuring the cellular accumulation of [³H]NAA and [¹⁴C]2,4-D. We have shown that 1-NAA enters cells by passive diffusion and has its accumulation level controlled by the efflux carrier. By contrast, 2,4-D uptake is mostly ensured by the influx carrier and this auxin is not secreted by the efflux carrier. Both auxin carriers contribute to IAA accumulation. The kinetic parameters and specificity of each carrier have been determined and new information concerning interactions with naphthylphthalamic acid, pyrenoylbenzoic acid, and naphthalene-2-acetic acid are provided. The relative contributions of diffusion and carrier-mediated influx and efflux to the membrane transport of 2,4-D, 1-NAA, and IAA have been quantified, and the data indicate that plant cells are able to modulate over a large range their auxin content by modifying the activity of each carrier.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 1-NAA naphthalene-1-acetic acid - 2-NAA naphthalene-2-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - V_m maximum transport capacity of the carrier In honour of Professor Dieter Klämbt's 65th birthdayThe authors thank Drs. A.E. Geissler and G.F. Katekar (CSIRO, Canberra City, Australia) for providing auxin efflux carrier inhibitors CPD, CPP, and PBA, and Dr. H. Barbier-Brygoo (Institut des Sciences Végétales, CNRS, Gif-sur-Yvette, France) for helpful discussions. This work was supported by funds from the Centre National de la Recherche Scientifique (UPR0040).     

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.     

Characterisation of bacterial cultures enriched on the chlorophenoxyalkanoic acid herbicides 4-(2,4-dichlorophenoxy) butyric acid and 4-(4-chloro-2-methylphenoxy) butyric acid

The aim of this study was to enrich and characterise bacterial consortia from soils around a herbicide production plant through their capability to degrade the herbicides 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) and 4-(4-chloro-2-methylphenoxy) butyric acid (MCPB). Partial 16S rRNA gene sequencing revealed members of the genera Stenotrophomonas, Brevundimonas, Pseudomonas, and Ochrobactrum in the 2,4-DB- and MCPB-degrading communities. The degradation of 2,4-DB and MCPB was facilitated by the combined activities of the community members. Some of the members were able to utilise other herbicides from the family of chlorophenoxyalkanoic acids. During degradation of 2,4-DB and MCPB, phenol intermediates were detected, indicating ether cleavage of the side chain as the initial step responsible for the breakdown. This was also verified using an indicator medium. Repeated attempts to amplify putatively conserved tfd genes by PCR indicated the absence of tfd genes among the consortia members. First step cleavage of the chlorophenoxybutyric acid herbicides is by ether cleavage in bacteria and is encoded by divergent or different tfd gene types. The isolation of mixed cultures capable of degrading 2,4-DB and MCPB will aid future investigations to determine both the metabolic route for dissimilation and the fate of these herbicides in natural environments.     

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.