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.

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 In the extraction procedure, the acceptor phase was repeatedly withdrawn into and discharged from the hollow fiber by the syringe pump.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.

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.

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.

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.

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.

Testing of 2,4,5-T-amino acid conjugates for mutagenic activity in Salmonella typhimurium strains.

Since amino acid conjugates are plant metabolites of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 5 amino acid conjugates (aspartic acid, glutamic acid, leucine, methionine and tryptophan) of 2,4,5-T were tested for possible mutagenic activity utilizing 5 strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535 and TA1538) with and without rat-liver microsomal and cytosolic enzymes. These compounds did not cause any significant increase in reversions when compared with controls in the presence or absence of the activating system. Further, linear regression analysis showed no significant (p less than 0.05) dose-response relationships. Thus, it was concluded that the tested amino acid conjugates of 2,4,5-T are not mutagens or promutagens in these assays.

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%.

Solid absorbent collection and gas chromatographic determination of the propylene glycol butyl ether esters of 2,4,5-T in air.

A method has been developed and validated for the collection of the propylene glycol butyl ether esters of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in air and their determination by gas chromatography with electron capture detection. The samples are collected on XAD-2 resin, desorbed in diethyl ether prior to analysis. The procedure is designed for industrial hygiene monitoring to provide an accurate 4 hour time-weighted average of the exposure level. Recoveries were found to be 97 +/- 8% (2 sigma) for the propylene glycol butyl ether esters of 2,4, 5-T in the concentration range of 0.70 ppb (v/v) to 5.00 ppm (v/v). Breakthrough concentrations, storage effects and humidity effects were also investigated.

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.

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.