Atrazine Derivatives Research Articles

Assessing the phytoremediation potential of crop and grass plants for atrazine-spiked soils

Pollution of soil and groundwater by atrazine has become an increasing environmental concern in the last decade. A phytoremediation test using plastic pots was conducted in order to assess the ability of several crops and grasses to remove atrazine from a soil of low permeability spiked with this herbicide. Four plant species were assessed for their ability to degrade or accumulate atrazine from soils: two grasses, i.e., ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea), and two crops, i.e., barley (Hordeum vulgare) and maize (Zea mays). Three different doses of atrazine were used for the contamination of the pots: 2, 5 and 10 mg kg−1. 16 days after spiking, the initial amount of atrazine was reduced by 88.6–99.6% in planted pots, while a decrease of only 63.1–78.2% was found for the unplanted pots, thus showing the contribution of plants to soil decontamination. All the plant species were capable of accumulating atrazine and its N-dealkylated metabolites, i.e., deethylatrazine and deisopropylatrazine, in their tissues. Some toxic responses, such as biomass decreases and/or chlorosis, were observed in plants to a greater or lesser extent for initial soil doses of atrazine above 2 mg kg−1. Maize was the plant species with the highest ability to accumulate atrazine derivatives, reaching up to 38.4% of the initial atrazine added to the soil. Rhizosphere degradation/mineralization by microorganisms or plant enzymes, together with degradation inside the plants, have been proposed as the mechanisms that contributed to a higher extent than plant accumulation to explain the removal of atrazine from soils.

Chemical Modification and Degradation of Atrazine in Medicago sativa through Multiple Pathways

Atrazine is a member of the triazine herbicide family intensively used to control weeds for crop production. In this study, atrazine residues and its degraded products in alfalfa (Medicago sativa) were characterized using UPLC-TOF-MS/MS. Most of atrazine absorbed in plants was found as chemically modified derivatives like deisopropylated atrazine (DIA), dehydrogenated atrazine (DHA), or methylated atrazine (MEA), and some atrazine derivatives were conjugated through different functional groups such as sugar, glutathione, and amino acids. Interestingly, the specific conjugates DHA+hGSH (homoglutathione) and MEA-HCl+hGSH in alfalfa were detected. These results suggest that atrazine in alfalfa can be degraded through different pathways. The increased activities of glycosyltransferase and glutathione S-transferase were determined to support the atrazine degradation models. The outcome of the work uncovered the detailed mechanism for the residual atrazine accumulation and degradation in alfalfa and will help to evaluate whether the crop is suitable to be cultivated in the atrazine-polluted soil.

Structural and Immunogenic Effects of Multiple Hapten Loading on Carrier Protein

Haptens are low-molecular-weight compounds that are usually nonimmunogenic in nature. These compounds are, in general, conjugated with carrier proteins to elicit an immune response for antibody production. In this work, we report the effect of multiple hapten loading on carrier protein after conjugation by monitoring the structural and immunogenic properties of the protein. Biochemical conjugation of carboxylated hapten (atrazine derivative) to bovine serum albumin via epsilon-amino groups of lysine residues was monitored by the intrinsic fluorescence intensity of tryptophan residues of protein. A significant blue shift of emission maxima confirmed the conformational changes with increasing molar ratio of hapten:protein. Circular dichroism spectroscopy suggested a decreasing trend for alpha-helical and increased formation of beta-sheet structures in hapten-loaded protein. A further insight was sought by using molecular modeling methods for understanding of structural changes in the native protein post-hapten conjugation. A sequential approach for hapten loading on the carrier confirmed that initial binding could affect the possible binding sites for subsequent incorporation of hapten molecules. These changes play a major role in the immunogenic response of hapten-carrier conjugate. The approach taken to develop this model is promising, and can be generalized for studies with other protein-hapten combinations.

Effect of arbuscular mycorrhizal fungus ( Glomus caledonium) on the accumulation and metabolism of atrazine in maize ( Zea mays L.) and atrazine dissipation in soil

Effects of an arbuscular mycorrhizal (AM) fungus ( Glomus caledonium) on accumulation and metabolism of atrazine in maize grown in soil contaminated with different concentrations of atrazine were investigated in a series of pot experiments. Roots of mycorrhizal plants accumulated more atrazine than non-mycorrhizal roots. In contrast, atrazine accumulation in shoot decreased in mycorrhizal compared with non-mycorrhizal plants. No atrazine derivatives were detected in the soil, either with or without mycorrhizal colonization. However, atrazine metabolites, deethylatrazine (DEA) and deisopropylatrazine (DIA), were detected in plant roots and the AM colonization enhanced the metabolism. After plant harvest atrazine concentrations decreased markedly in the soils compared to the initial concentrations. The decreases were the most in rhizosphere soil and then near-rhizosphere soil and the least in bulk soil. Mycorrhizal treatment enhanced atrazine dissipation in the near-rhizosphere and bulk soils irrespective of atrazine application rates.

Immobilization of atrazine on gold, a first step towards the elaboration of an indirect immunosensor: characterization by XPS and PM-IRRAS

As part of our long-term goal to design a novel type of immunosensor based on IR transduction, we devised two strategies to immobilize derivatives of the herbicide atrazine on the surface of planar gold substrates. Both strategies take advantage of the well-known formation of self-assembled monolayers of thiolates via spontaneous chemisorption of thiols or disulphides on noble metal surfaces. The first strategy involved the direct chemisorption of a disulphide derivative of atrazine, while the second strategy was based on the covalent attachment of a polymer tethered with atrazine derivatives to a previously adsorbed thiolate carrying a carboxylic acid head group. The resulting organic thin films on gold were characterized by polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and XPS. The binding of a polyclonal anti-atrazine antibody to both sensing layers was also tested and compared. It appeared that specific molecular recognition occurred only on the atrazine disulphide sensing layer. Copyright © 2006 John Wiley & Sons, Ltd.

Atrazine increases the sodium absorption in frog (Rana esculenta) skin

The presence of atrazine in agricultural sites has been linked to the decline in amphibian populations. The efforts of the scientific community generally are directed toward investigating the long-term effect of atrazine on complex functions (reproduction or respiration), but in the present study, we investigated the short-term effect on the short-circuit current (I(sc)), a quantitative measure of the ion transport operated by frog (Rana esculenta) skin. Treatment with 5 microM atrazine (1.08 mg/L) does not affect the transepithelial outfluxes of [14C]mannitol or [14C]urea; therefore, atrazine does not damage the barrier properties of frog skin. Atrazine causes a dose-dependent increase in the short-circuit current, with a minimum of 4.64 +/- 0.76 microA/cm2 (11.05% +/- 1.22%) and a maximum of 12.7 +/- 0.7 microA/cm2 (35% +/- 2.4%) measured at 10 nM and 5 microM, respectively. An increase in Isc also is caused by 5 microM ametryne, prometryn, simazine, terbuthylazine, or terbutryn (other atrazine derivatives). In particular, atrazine increases the transepithelial 22Na+ influx without affecting the outflux. Finally, stimulation of Isc by atrazine is suppressed by SQ 22536, H89, U73122, 2-aminoethoxydiphenyl borate, and W7 (blockers of adenylate cyclase, protein kinase A, phospholipase C, intracellular Ca2+ increase, and calmodulin, respectively), whereas indomethacin and calphostin C (inhibitors of cyclooxygenase and protein kinase C, respectively) have no effect.

Evolutionary affinity and selectivity optimization of a pesticide-selective antibody utlizing a hapten-selective immunoglobulin repertoire.

Selectivity and sensitivity are considered as pivotal criteria for the quality of immunochemical assay designs in environmental analysis. They are essentially determined by the variable domains of the implemented antibody. The variable domains of a triazine-selective single-chain Fv (scFv) were genetically engineered by stringent molecular evolution in order to optimize analytical characteristics of the corresponding atrazine immunoassay. Gene variation of the template antibody by sequential shuffling against the variable heavy and light chain repertoire of a triazine-selective immunoglobulin library was enhanced by introducing additional point mutatons. Improved scFv variants were selected by phage display employing an atrazine derivative. By this means the paramounting affinity of the initial scFv to sebuthylazine was shifted for the mutant antibodies toward a preferential recognition of the envisaged target analyte atrazine. In addition, the detection limit of the atrazine assaywas significantly improved by factor 25 from 5.1 microg/L for the initial template antibody to 0.2 microg/L for the mutant antibodies. The contribution of the engineered antibody variants to the assay improvement is also reflected by a shift of the equilibrium dissociation constant KD from 1.27 x 10(-8) M of the template antibody to 7.46 x 10(-10) M of the optimized variant. Sequence analysis revealed a bias of amino acid substitutions in the first two complementarity-determining regions (CDR) and the flanking framework regions of both variable chains for the shuffled clones as well as a deletion in the CDR3 of the light chain. Particularly the mutations of the VL domain turned out to have a decisive impact on the alterations in the analytical performance of the engineered scFv mutants. The application of the mutant antibodies for the atrazine determination of soil samples revealed consistency with HPLC data within the experimental error.

DEVELOPMENT OF ELISA TECHNIQUE FOR THE ANALYSIS OF ATRAZINE RESIDUES IN WATER

A highly sensitive enzyme immunoassay is described for the detection of atrazine residues in water. Atrazine derivative was conjugated to Bovine Serum Albumin (BSA) to obtain an immunizing antigen and to Horseradish Peroxidase enzyme (POD) to obtain a marker for immunoassay. The formation of these conjugations was confirmed by UV spectroscopy as well as by gel-electrophoresis. Polyclonal antibodies were raised in rabbits by immunization with an atrazine–BSA conjugate containing 29 atrazine residues per BSA molecule. An ELISA on microtitration plates was optimized with peroxidase–atrazine conjugate. The middle of the test (50% B/Bo) was found to be at 90 ng/l, which is well below the maximum concentration permitted by the EC guidelines for drinking water. Detection limits for atrazine of about 1 ng/l could be reached. The assay did not require concentration or cleanup steps for drinking or ground water samples. Validation experiments showed good accuracy and precision. No cross-reactivities were shown by other s-triazines like terbutryn, ametryn, terbuthylazine, des-isopropylatrazine, and de-ethylatrazine except hydroxyatrazine. The latter was present at very low levels that can be calibrated/standardized before analysis or it may be considered as leftover residues of atrazine. Based on these results, it is suggested that this test can be applied to obtain fairly accurate results for atrazine concentration in water samples from different sources.

Characterization of s-triazine antibodies and comparison of enzyme immunoassay and biotin-avidin enzyme immunoassay for the determination of s-triazine

Triazines have been used widely as herbicides and known to cause environmental contamination. For developing ELISA of s-triazines, six kinds of s-triazine derivatives (one from simazine, one from atrazine and four from cyanuric chloride) which contained a C3- or C6-carboxylic acid group bridge were prepared. These derivatives were conjugated to bovine serum albumin (BSA) for the use of immunogens and to KLH for the use of coating ligands on the microtiter plate wells. Polyclonal antibodies produced from rabbit or sheep using atrazine-BSA (1b-BSA) and simazine-BSA (1a-BSA) immunogens. These antibodies were characterized and biotinylated for the use of enzyme immunoassay (EIA). We evaluated EIA and biotin-streptavidin mediated EIA in terms of the sensitivities and specificities with these antibodies. The results in both assay systems showed that coating ligand synthesized from atrazine derivative was better than that from simazine derivative. Comparing binding ability between C3-carboxylic acid and C6-carboxylic acid spacers of N-alkyl group in s-triazine ring, C3-carboxylate-KLH (2c-KLH) derived from atrazine showed better sensitivity for the both assay systems. The detection limit was found to be 0.01 ppb in biotin-streptavidin mediated EIA (B-Av EIA). Comparing IC 90 values of EIA (0.5 ppb) and B-Av EIA (0.05 ppb), B-Av EIA was able to detect one order lower concentration range of atrazine than EIA.

Refined crystal structures of reaction centres from Rhodopseudomonas viridis in complexes with the herbicide atrazine and two chiral atrazine derivatives also lead to a new model of the bound carotenoid

In a reaction of central importance to the energetics of photosynthetic bacteria, light-induced electron transfer in the reaction centre (RC) is coupled with the uptake of protons from the cytoplasm at the binding site of the secondary quinone (QB). It has been established by X-ray crystallography that the triazine herbicide terbutryn binds to the QB site. However, the exact description of protein-triazine interactions has had to await the refinement of higher-resolution structures. In addition, there is also interest in the role of chirality in the activity of herbicides. Here, we report the structural characterisation of triazine binding by crystallographic refinement of complexes of the RC either with the triazine inhibitor atrazine (Protein Data Bank (PDB) entry 5PRC) or with the chiral atrazine derivatives, DG-420314 (S(−) enantiomer, PDB entry 6PRC) or DG-420315 (R(+) enantiomer, PDB entry 7PRC). Due to the high quality of the data collected, it has been possible to describe the exact nature of triazine binding and its effect on the structure of the protein at high-resolution limits of 2.35 Å (5PRC), 2.30 Å (6PRC), and 2.65 Å (7PRC), respectively. In addition to two previously implied hydrogen bonds, a third hydrogen bond, binding the distal side of the inhibitors to the protein, and four additional hydrogen bonds mediated by two tightly bound water molecules on the proximal side of the inhibitors, are apparent. Based on the high quality data collected on the RC complexes of the two chiral atrazine derivatives, unequivocal assignment of the structure at the chiral centres was possible, even though the differences in structures of the substituents are small. The structures provide explanations for the relative binding affinities of the two chiral compounds. Although it was not an explicit goal of this work, the new data were of sufficient quality to improve the original model also regarding the structure of the bound carotenoid 1,2-dihydroneurosporene. A carotenoid model with a cis double bond at the 15,15′ position fits the electron density better than the original model with a 13,14-cis double bond.

Kinetics of Uptake and Metabolism of Atrazine in Model Plant Systems

The present work concerns atrazine absorption and metabolism by corn (Zea mays.) seedlings immersed in an aqueous medium in comparison with Acer pseudoplatanus cell cultures. At the point of equilibrium, the apparent concentration inside the A. pseudoplatanus cells (with a moderate lipid content: 0·17% of dry weight) was about twice that of the medium. This equilibrium was probably due to a simple partition process; part of the atrazine was dissolved in the cell water and reached the same concentration as in the external medium while the rest was concentrated inside the cellular lipids. The theoretical calculation of the lipid/water partition, taking into account the value of log P measured not with the lipids but with octanol (log P=2·5), gave a value of 1·5 for concentration inside the plant material. Such an equilibrium, resulting from a partition process between water and lipids, was also obtained in non-living corn seedlings. In living seedlings, an over-concentration of radioactivity due to [14C]atrazine derivatives was rapidly obtained inside roots and shoots giving concentrations respectively 7- and 12-fold higher than that of atrazine in the external medium. This was due to very rapid chemical transformation of atrazine into its hydroxy derivatives, especially hydroxyatrazine. This hydrolysis of atrazine in corn was due to the presence of high levels of benzoxazinone derivatives in corn seedling cells. The hydroxylated metabolites were able to concentrate in the cells very rapidly and were unable to diffuse freely into the external medium. As a consequence, this process facilitated the penetration of large quantities of atrazine which became rapidly hydroxylated, allowing therefore the passive penetration of atrazine to be further improved, since the concentration C1 in the receiver compartment was always close to zero. The passive transfer of atrazine, following Fick's law: dq/dt=-Pa (C0–C1), was therefore optimized. © 1997 SCI.

Liposome-Based Immunosensors. 1. Influence of Hapten Spacer Length on Liposome Binding Efficiency

Abstract The effect of spacers on the binding of atrazine-tagged liposomes to anti-atrazine antibodies was studied. Atrazine derivatives with alkyl spacers of different length (C4, C6, C8 and C12) were synthezised and conjugated to dipalmitoyl phosphatidyl ethanolamine. The conjugates were incorporated into liposomal membranes of DPPC/DPPG/cholesterol containing ascorbic acid as entrapped marker. Tagged liposomes were bound to a monoclonal anti-atrazine antibody immobilized on a nitrocellulose membrane, destroyed by the addition of surfactant and the marker was detected electrochemically using printed thick-film electrodes. Our results show that liposomes incorporating a hapten with short alkyl spacer binds better to the immobilized antibody. These results will allow optimization of liposomes as an enhancement system in electrochemical or optical biosensors.

New Intermediates of Dealkylation of [ 14C]Atrazine by Mouse Liver Microsomes

The metabolism of 50 μ M [2,4,6- 14C]atrazine by liver microsomes from ICR strain mice produced metabolites not previously described which account for about 25% of total transformation products, plus 10-14% deethylatrazine and 13-15% deisopropylatrazine, based on the total radioactivity injected into an analytical high-pressure liquid chromatography (HPLC) system. The unknown metabolites were observed by HPLC of products from atrazine incubations with liver microsomes from both untreated mice and mice treated with phenobarbital. Two major unidentified peaks (Unknowns 1 and 2) were isolated and collected by HPLC and characterized by gas chromatography-electron-impact ionization-mass spectrometry. Unknown 1 was an unstable intermediate, likely a mixture of carbinolamines, that decomposed into deethylatrazine, and Unknown 2, a mixture of compounds unseparated by HPLC probably with N-vinyl substituents at the location of the N-isopropyl and N-ethyl groups of the atrazine molecule with M + at m/ z 213. While microsomal oxidation did produce a mixture of trace amounts of breakdown products of atrazine, the method was impractical for the initial goal of preparing pure dealkylated atrazine derivatives for environmental degradation studies.

Confirmation and Analysis ofs-Chlorotriazines by Derivatization with Pyrrolidine

Abstract Atrazine and simazine were analyzed and confirmed via their pyrrolidino derivatives by gas chromatography on SPWax-10 megabore column using a NP-detector and propazine as the internal standard. The s -chlorotriazines were reacted in dimethylformamide with an excess of pyrrolidine at 60°C for 30 minutes to afford practically quantitative substitution of the chlorine. The retention times of the derivatives were increased markedly compared to those of the parent compounds. Reproducibility as measured by response ratios relative to the propazine derivative was very good: C.V. for atrazine and simazine were 0.60% and 0.26% respectively (n = 5). Good linearity was seen at 0.4 to 2.2 ng/μL concentration: measured in duplicate at 4 dilutions, the coefficient of determination r2 was 0.9998 for atrazine derivatives and 0.9994 for simazine derivatives. Very good agreement was found when analytical results were correlated with data obtained by gas chromatography of the parent compounds.

Detection of Pesticide in Drinking Water Using Real-Time Biospecific Interaction Analysis (BIA)

Abstract It is reported an application of a biosensor based technology employing Surface Plasmon Resonance (SPR) for studies of molecular interaction in real time. An inhibition immunoassay for the detection of the herbicide atrazine has been developed. The biospecific interface is a sensor chip to which an atrazine derivative has been covalently bound. Monoclonal antibodies (Mabs) against atrazine are mixed with the sample containing herbicide, then the solution reacted with the biospecific interface. As the interaction between free antibodies and the immobilized derivative of atrazine bound to the surface proceeds, the SPR response changes inversely related to the atrazine concentration. A detection limit of 0.05 ppb of atrazine in water can be reached; the concentration range is 0–1 ppb, the analysis time is 15 minutes. After each measure the sensor chip surface can be regenerated. The analysis has been performed with standard solutions (buffer) in distilled and tap water.

Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine.

The Fab region of an IgG2b antibody (AM7B2.1) reactive to the herbicide atrazine was cloned into a plasmid vector using the polymerase chain reaction and two sets of degenerate oligonucleotide primers designed to mimic the amino acid variation at the N-termini of kappa L-chains and gamma H-chains. These primers also provide a secretion signal fused precisely to the antibody gene sequence for secretion of the mature antibody. A further set of universal oligonucleotide primers was developed for the direct sequencing of the VH and CH1 regions of gamma H-chains and the VL and CL regions of kappa L-chains without subcloning and were used to determine the sequence of this antibody. The kappa L-chain was found to not possess a conserved Cys residue at position 23 and the implications of this observation are discussed. The cloned genes were expressed in Escherichia coli using a commercially available T7 RNA polymerase-based plasmid. The clones were also expressed in a T7 RNA polymerase-based system containing an attenuated version of the T7 RNA polymerase promoter, plus a lac promoter placed in an antisense orientation, to enhance plasmid stability. The expressed products were confirmed as atrazine reactive by binding to an atrazine derivative conjugated with alkaline phosphatase.

Modification of atrzine desorption during field incubation experiments

The effect of field aging on atrazine desorption was determined. 14C-ring labelled atrazine was applied to the top of soil columns in field plots. The columns were periodically removed from the field for 16 months after application. The desorption isotherms of [ 14C]atrazine residues were determined on the surface soil using the batch equilibration method with 0.01 M CaCl 2 solution. The distribution of atrazine residues in soil and liquid phases was determined by total 14C analysis and HPLC with a radiactive flow detector to identify the [ 14C]atrazine derivatives. In laboratory experiments, desorption coefficients were dependent on the absorbed atrazine concentrations; low concentrations were retained to a greater extent and were more difficult to desorb than the higher concentrations. For the field aged residues, desorption K f values increased slightly with aging. However, the values were lower than would be expected from the laboratory values if equal adsorbed concentrations are compared. It is not possible, however, to predict the desorption behavior of atrazine in the field samples from the simple adsorption-desorption laboratory studies. It is necessary to take into account the change in the atrazine-soil interactions with aging.

Influence of the ELISA format and the hapten-enzyme conjugate on the sensitivity of an immunoassay for S-triazine herbicides using monoclonal antibodies

A variety of formats for enzyme-linked immunosorbent assays have been examined for the analysis of environmental samples using a single monoclonal antibody raised to the herbicide atrazine. The most favorable combination of several ELISA formats and a variety of synthesized hapten-enzyme conjugates resulted in a highly sensitive enzyme immunoassay with 50% inhibition (160) at 0.25 pg/L and a detection limit of 0.03 pg/L. This assay is the most sensitive atrazine assay, based on monoclonal antibodies published so far, and can be performed in less than 1 h. All formats were based on the use of haptenenzyme conjugates (enzyme tracers). Direct coating and precoating with goat anti-mouse antibodies or protein A were used. The formats using precoating steps turned out to be the most sensitive. Enzyme tracers based on two different atrazine and simazine haptens each with two different spacers (Cg or CS) were conjugated using either alkaline phosphatase or horseradish peroxidase. The atrazine derivative with the CS spacer arm conjugated to horseradish peroxidase provided highest sensitivity.

Binding of Triazine Herbicides to Antibodies in Anhydrous Organic Solvents

Abstract The binding of atrazine and an atrazine derivative dissolved in various nonpolar organic solvents to immobilized polyclonal antibodies raised against the herbicide was studied. The antibodies immobilized onto immunodyne membranes were stable in chloroform and in buffer for at least 65 hours at room temperature. The apparent binding affinity was lower in the five solvents tested than in buffer, and showed correlation with the polarities of the solvents and with the solubilities of the haptens in the solvents. An increase in the antibody-hapten binding specificity by the use of toluene as the solvent for atrazine, propazine and simazine was observed.