Chirally functionalized anion-exchange type silica monolith for enantiomer separation of 2-aryloxypropionic acid herbicides by non-aqueous capillary electrochromatography.

A silica based monolithic capillary column derivatized with O-9-(tert-butylcarbamoyl)quinidine was prepared for CEC enantiomer separation of chiral 2-aryloxypropionic acid herbicides including inter alia dichlorprop, mecoprop and fenoprop. The silica monolith had a relatively low surface coverage with chiral cationic selector moieties. Due to the low selector density retention factors were low as well, yet still enabling enantiomer separations of the target solutes. Both electrophoretically and chromatographically dominated migration and separation modes, respectively, could be established depending on the employed conditions. In the former mode, enantiomers migrated in front of the EOF marker, and faster separations and higher plate numbers could be achieved. In the latter mode, stronger adsorption translated into a typical chromatographic separation in which the enantiomers eluted after the EOF marker whereby separation factors were slightly enhanced compared to the aforementioned separation mode. Reasonable baseline separations of enantiomers were accomplished for all analytes after optimization of relevant mobile phase parameters in the anion-exchange CEC system including sample loadability, and the separations were comparable to such obtained on an optimized high density quinidine-carbamate modified organic polymer monolith column. Overall, it is concluded that monoliths with a high surface density of chiral ion-exchange moieties are favorable because of their enhanced sample loadabilities and improved chromatographic performance with regard to separation factors, plate numbers and peak symmetries. The resultant accompanying longer analysis times may rather be reduced by adjusting effective column length than by reducing selector coverage of the monolith.

Use of a bisphenol-A imprinted polymer as a selective sorbent for the determination of phenols and phenoxyacids in honey by liquid chromatography with diode array and tandem mass spectrometric detection.

An extraction-preconcentration procedure based on the use of a molecularly imprinted polymer (MIP) as selective sorbent has been developed for the determination of several phenolic compounds (bisphenol-A, bisphenol-F and 4-nitrophenol) and phenoxyacid herbicides (2,4-D, 2,4,5-T and 2,4,5-TP) in honey samples. Liquid chromatography with diode array detection (LC-DAD) and electrospray ionisation-ion trap mass spectrometry (LC-IT-MS) were used for the separation, identification and quantification of these analytes. The molecularly imprinted polymer was obtained by precipitation polymerisation with bisphenol-A (BPA) as template and 4-vinylpyridine as the functional monomer. The behaviour of this sorbent was compared with those of other materials frequently used in SPE. The selectivity of the BPA-MIP for the target analytes was tested in samples containing other pesticides in common use. The recoveries achieved for all six compounds were in the 81-96% range. By applying the proposed procedure prior to LC-IT-MS, the limits of detection achieved in commercial honey samples were in the 0.1-3.8 ng g(-1) range, with relative standard deviations of 12-24%.

Automated dynamic liquid-liquid-liquid microextraction followed by high-performance liquid chromatography-ultraviolet detection for the determination of phenoxy acid herbicides in environmental waters.

Automated dynamic liquid-liquid-liquid microextraction (D-LLLME) controlled by a programmable syringe pump and combined with HPLC-UV was investigated for the extraction and determination of 5 phenoxy acid herbicides in aqueous samples. In the extraction procedure, the acceptor phase was repeatedly withdrawn into and discharged from the hollow fiber by the syringe pump. The repetitive movement of acceptor phase into and out of the hollow fiber channel facilitated the transfer of analytes into donor phase, from the organic phase held in the pore of the fiber. Parameters such as the organic solvent, concentrations of the donor and acceptor phases, plunger movement pattern, speed of agitation and ionic strength of donor phase were evaluated. Good linearity of analytes was achieved in the range of 0.5-500 ng/ml with coefficients of determination, r2 > 0.9994. Good repeatabilities of extraction performance were obtained with relative standard deviations lower than 7.5%. The method provided up-to 490-fold enrichment within 13 min. In addition, the limits of detection (LODs) ranged from 0.1 to 0.4 ng/mL (S/N = 3). D-LLLME was successfully applied for the analysis of phenoxy acid herbicides from real environmental water samples.

Assessment of enzyme-linked immunosorbent assay for the determination of 2,4,5-TP in water and soil.

A highly sensitive and specific indirect enzyme-linked immunosorbent assay is described for Silvex, 2-(2,4,5 trichlorophenoxy)propionic acid, (2,4,5-TP). One specific feature of the immunoassay is the use of simple chemical activation of chlorophenoxy acids to prepare both the immunizing and coating conjugates. The assay is based on the use of polyclonal antibodies raised against 2,4,5-TP, and a peroxidase-labeled secondary antibody for colorimetric detection. The effect of different chemical conditions (pH, and salt and detergent concentration) on immunoassay performance has been studied. Under the best conditions the least detectable dose and the sensitivity (IC(50)) for 2,4,5-TP were 0.05 micro g L(-1) and 0.80 micro g L(-1), respectively. The optimized immunoassay was also highly specific, showing little (6.9% for 2,4,5-T) or no cross-reactivity with other similar herbicides. The assay was used to determine 2,4,5-TP in water and soils. The excellent recoveries obtained (mean values ranging between 89% and 104%) make this immunoassay a suitable screening method for either environmental monitoring or laboratory quantification of 2,4,5-TP.

Reversible surface thiol immobilization of carboxyl group containing haptens to a BIAcore biosensor chip enabling repeated usage of a single sensor surface.

We describe a reversible immobilization method for carboxyl group containing haptens that makes the repeated usage of a BIAcore biosensor chip possible. Haptens which are immobilized according to the surface thiol method can be removed completely from the sensor surface again by a reducing step. In the first part of our study, analogues of the herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid were immobilized in succession to a biosensor surface of a BIAcore surface plasmon resonance instrument according to the thiol coupling method. Direct kinetic analysis of these ligands to a polyclonal anti-2,4-dichlorophenoxyacetic acid antibody were performed using these biosensor surfaces. In the second part of the study, different amounts of 2,4-dichlorophenoxyacetic acid were sequentially immobilized onto the same biosensor surface in order to generate a calibration plot for 2,4-dichlorophenoxyacetic acid. Using this plot, the quantitative detection of the herbicide down to a concentration of 0.1 microg/mL, the maximum admissible concentration of pesticides in drinking water, is possible.

Quantitative and qualitative precision improvements by effective mobility-scale data transformation in capillary electrophoresis analysis.

By transforming the time-based x-axis of electropherograms in capillary zone electrophoresis (CZE) into the corresponding effective mobility-scale, we propose a simple and robust data representation for a better qualitative and quantitative capillary electrophoresis (CE) analysis. The time scale of the raw electrophoretic data (detection signal versus time) is transformed into an effective electrophoretic mobility scale (mu eff-scale) with account of the electroosmotic flow (EOF) peak or of an internal standard of known effective mobility. With the new scaling (detection signals versus effective mobility), the obtained electropherograms are more representative of the velocity-based electrophoretic separation and the comparison of complete electropherograms is directly possible. This is of importance when tracking peaks in real samples where alteration in EOF stability can occur or when comparing electrophoretic runs from different experimental setups (independence in column length and voltage). Beside the qualitative possibilities, a quantitative improvement is achieved in the mu eff-scale with significant better peak area reproducibility and equal to more precision in quantitative analysis than with the primary time-scale integration.

Clofibrate pretreatment in mice confers resistance against hepatic lipid peroxidation.

Pretreatment with peroxisome proliferators protects mice against various hepatotoxicants. Since our previous work suggested that the hepatoprotection may involve an increased ability to cope with oxidative stress, the present work directly addressed this possibility. Several observations indicated a heightened defense against oxidative stress accompanies the hepatoprotection produced by clofibrate. Firstly, the carbonyl content of hepatic proteins from clofibrate-pretreated mice was 40% lower than those from vehicle-treated controls. Secondly, liver homogenates from clofibrate-pretreated mice produced less thiobarbituric acid reactive substances upon incubation under aerobic conditions or exposure to ferrous sulfate. This effect was not due to lower levels of peroxidation-prone polyunsaturated fatty acids in clofibrate-treated livers. Thirdly, in vitro experiments indicated that the antioxidant factor in liver homogenates from clofibrate-pretreated mice was not glutathione. Rather, since it was inactivated by proteases and heat treatment, we concluded that a protein is involved. Collectively, our results suggest that a resistance to lipid peroxidation develops in mouse liver during exposure to clofibrate. The identity of the putative antioxidant protein and its contribution to the protection against liver toxicity observed in this and other laboratories awaits future investigation.

Stereoselective analysis of acid herbicides in natural waters by capillary electrophoresis.

A capillary electrophoretic method for the stereoselective analysis of aryloxypropionic and aryloxyphenoxypropionic acidic herbicides in ground water and river water was performed. Vancomycin and gamma-cyclodextrin were added to the background electrolyte (BGE) as chiral selectors. Water sample preconcentration was accomplished by solid-phase extraction on styrene-divinylbenzene packed cartridges (2 L of ground water and 1 L of river water). The analytical method allowed for the resolution of mecoprop, fenoprop, fluazifop and haloxyfop racemic mixtures in natural water samples spiked with enantiomer concentration levels in the range 0.1-0.13 ppb for ground water and 0.4-0.54 ppb for river water.

Peroxisome proliferators increase the formation of BPDE-DNA adducts in isolated rat hepatocytes.

Peroxisome proliferators are known to modulate the activity of xenobiotic-metabolising enzymes, including glutathione S-transferase (GST) and cytochrome P-450 (CYP). In this study the effect of peroxisome proliferators silvex and di(2-ethylhexyl)phthalate (DEHP) on the formation of (+)-anti-benzo(a)pyrene -7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts from a proximate mutagen and carcinogen (-)-transbenzo(a)pyrene-7,8-dihydrodiol (BPDD) has been investigated. Rat CYP1A1 metabolises BPDD to mutagenic BPDE, which may form DNA adducts or, alternatively, be detoxified by hydrolysis or glutathione conjugation. In this experiment the formation of BPDE-DNA adducts was significantly increased in hepatocytes isolated from all silvex treated rats and two out of four DEHP treated rats (14 day treatment). The activity of CYP1A1 was increased whereas GST was reduced by the peroxisome proliferator silvex. These changes were more significant than those induced by DEHP. We have hypothesised that the formation of BPDE-DNA adducts was primarily due to the increased BPDD activation to BPDE versus reduced detoxication of BPDE. Other hepatic changes induced by the peroxisome proliferators, e.g. peroxisome proliferation per se and increased mitotic activity of the liver could have an effect on the outcome of BPDD exposure.

Differential effect of peroxisome proliferators on rat glutathione S-transferase isoenzymes.

We have investigated the effects of peroxisome proliferators silvex, nafenopin and diethylhexylphthalate (DEHP) on rat liver glutathione S-transferase (GST) isoenzyme activities and patterns. Silvex was a more potent in vitro GST inhibitor than nafenopin and DEHP. After 14 days oral administration to rats a reduction in total GST activity was observed, doses of compounds were chosen so that peroxisome proliferation was equivalent between compounds, nevertheless total GST activity was altered to different extents: nafenopin approximately silvex > DEHP approximately control. GST isoenzyme profiles were also altered, the proportion of GST 2-2 increased and 4-4 decreased compared to control levels. The results indicated that: (i) the peroxisome proliferators studied had similar effects on GST isoenzyme profile: (ii) modulation of the GST activity was apparently independent of peroxisome proliferation per se.

Determination of phenoxyacid herbicides in water.

Novel clean-up techniques for a polymeric precolumn (PLRP-S) for the subsequent determination of bentazone and eight phenoxy acid herbicides in surface water samples are described. After preconcentration of the components at pH 3 on a 10 x 2 mm I.D. precolumn, the technique consists of a clean-up with 1000 microliters of 0.1 mol/l sodium hydroxide solution (pH 12.5) and of a heartcut consisting of four precolumn bed volumes of eluent directed to waste followed by ten precolumn bed volumes of eluent directed to the analytical column. Analytical separation is performed with acetonitrile-water (30:70) containing 0.005 mol/l of tetrabutylammonium hydrogensulphate (pH 8.3) (which is also the desorption eluent during heartcutting) on a polymeric analytical column (PLRP-S). With 25 ml of surface water, spiked at 0.25 and 1 microgram/l, applied to the precolumn, recoveries for all components were over 85% with a relative standard deviation (n = 5) of ca. 9% at 0.25 microgram/l and ca. 2% at 1 microgram/l. Detection limits in surface water samples are 0.05-0.1 microgram/l. Owing to automation, the total analysis time is ca. 30 min.

Comparison of uncoupling activities of chlorophenoxy herbicides in rat liver mitochondria.

The effects of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-chloro-2-methylphenoxyacetic acid (MCPA) and 2-(2,4,5-trichlorophenoxy)propionic acid (2,4,5-TP) on respiration and oxidative phosphorylation in rat liver mitochondria were examined in vitro. Respiration rates of glutamate, malate and succinate were investigated in the presence of each herbicide (0.1-4.0 mM). At lower concentrations, all herbicides stimulated state 4 respiration, decreased the respiratory control ratio and the ADP/O ratio. The respiration rate in state 3 and uncoupled state was unaffected. At higher concentrations all bioenergetic parameters, respiration in state 4, 3 and uncoupled state, as well as respiratory control ratio and ADP/O, were inhibited in a concentration-dependent manner. These data indicate that these herbicides alter energy metabolism in rat liver mitochondria by uncoupling of oxidative phosphorylation. 2,4,5-TP possesses the strongest uncoupling properties followed by 2,4,5-T, MCPA and 2,4-D in that order.

Degradation of 2,4,5-trichlorophenoxyacetic acid by a Nocardioides simplex culture.

A Nocardioides simplex strain 3E was isolated which totally dechlorinated 2,4,5-trichlorophenoxyacetic acid and was capable of its utilization as the sole source of carbon. The mechanism of 2,4,5-trichlorophenoxyacetic acid degradation by this strain was investigated. Chloroaromatic metabolites that occur in the lag, exponential and stationary growth phases of the strain Nocardioides simplex 3E were isolated and identified bases on a combination of TLC, GC-MS and HPLC data. Decomposition of 2,4,5-trichlorophenoxyacetic acid at the initial stage was shown to proceed by two pathways: via the splitting of the two-carbon fragment to yield 2,4,5-trichlorophenol and the reductive dechlorination to produce 2,4-dichlorophenoxyacetic acid. Hydrolytic dechlorination of 2,4,5-trichlorophenoxyacetic acid was found to yield dichlorohydroxyphenoxyacetic acid, thus pointing to the possible existence of a third branch at the initial stage of degradation of the xenobiotic. 2,4,5-Trichlorophenol and 2,4-dichlorophenoxyacetic acid produced during the metabolism of 2,4,5-trichlorophenoxyacetic acid and in experiments with resting cells are utilized by the strain Nocardioides simplex 3E as growth substrates.

The pharmacokinetics of chlorinated phenoxy acid herbicides: a literature review.

The chlorinated phenoxy acid herbicides (CPAHs) appear to have similar pharmacokinetics. They are rapidly and almost completely absorbed from an oral dose. They distribute to other tissues and are highly protein-bound in the plasma. The CPAHs are rapidly eliminated unchanged in the urine by an active process in the kidneys. Increasing doses apparently influence absorption, metabolism, distribution and elimination of the CPAHs so that biological effects are increased. Combinations of CPAHs are likely to result in additive or potentiated biological effects. Data suggest that CPAH toxicosis may be alleviated by treatment with fluids and bicarbonate to increase urinary pH and volume, thereby increasing excretion.

Carcinogenicity and genotoxicity of the herbicide 2,4,5-trichlorophenoxyethanol (TCPE) contaminated with dioxin.

In an earlier study 2,4,5-trichlorophenoxythanol (TCPE) contaminated with dioxin, a component of the Hungarian herbicide Buvinol, was found to be hepatocarcinogenic. In the present work, the hepatocarcinogenicity of TCPE was compared to its possible genotoxicity in vitro, using the Salmonella/microsome test for mutagenicity and for its DNA-damaging effect, the induction of sister chromatid exchanges (SCE) in Chinese hamster cells in vitro. It was found that purified TCPE (with 0.1 ppm dioxin content) was under no conditions mutagenic by the Ames test, i.e., it belongs to the group of false-negative chemicals. TCPE was, however, genotoxic; its DNA-damaging effect was demonstrated by an increase in the frequency of SCE, while pure dioxin of corresponding amount was ineffective. However, elevated SCE frequency and the toxicity on bacteria and mammalian cells by TCPE were significantly decreased by the metabolic activation system (S-9 mix) isolated from liver. This observation indicates that in the detoxication of TCPE in vitro, a key role is to be attributed to the hepatic microsomal enzymes. It is presumed that TCPE is hepatocarcinogenic only in a dose range which has exhausted the detoxicating capacity of the liver.

Cohort mortality study of chemical workers with potential exposure to the higher chlorinated dioxins.

This cohort study evaluated mortality patterns, 1940 through 1982, of 2,192 chemical workers who, having engaged in the manufacture of higher chlorinated phenols and derivative products, had potential occupational exposures to chlorinated dioxins. Relative to United States white male mortality experience, there were no statistically significant deviations from expected for the following categories: all causes, total malignant neoplasms, or specific malignancies of particular interest: stomach cancer, liver cancer, connective and other soft-tissue cancer, the lymphomas, or nasal and nasopharyngeal cancer. For the cirrhosis of the liver category, internal comparisons demonstrated increasing trends associated with duration of employment in the Chlorophenol Production and Finishing areas; but available evidence suggests this finding was related to alcohol abuse. The study does not support a causal association between chronic human disease as measured by mortality and exposures to the higher chlorinated phenols, derivative products, or their unwanted contaminants, the chlorinated dioxins.

Non-Hodgkin's lymphoma and exposure to phenoxyherbicides, chlorophenols, fencing work, and meat works employment: a case-control study.

A previous case-control study which used the occupational information available on the New Zealand Cancer Registry found that agricultural workers were at increased risk of developing non-Hodgkin's lymphoma. The findings are now presented for the second phase of the study which entailed interviewing 83 cases of non-Hodgkin's lymphoma registered under code 202 of the International Classification of Diseases together with 168 controls with other types of cancer and 228 general population controls. The findings for the two control groups were similar, and there were no significant differences between cases and controls regarding potential exposure to phenoxy-herbicides (odds ratio = 1.4, 90% confidence limits 0.7-2.5, p = 0.26) or chlorophenols (odds ratio = 1.3, 90% confidence limits 0.6-2.7, p = 0.39). The odds ratio for fencing work, necessitating exposure to several potential risk factors including arsenic and sodium pentachlorophenate was 2.0 (90% confidence limits 1.3-3.0, p = 0.01). The odds ratio for employment in a meat works, necessitating potential exposure to 2, 4, 6-trichlorophenol and zoonotic viruses, was 1.8 (90% confidence limits 1.1-3.1, p = 0.04). There was a significant statistical interaction between the risks associated with these two activities, the odds ratio for involvement in both activities compared with involvement in neither being 5.7 (90% confidence limits 2.3-14.3, p = 0.03).