Determination of hexazinone and its metabolites in groundwater by capillary electrophoresis

doi:10.1016/S0021-9673(97)00913-8

Journal of Chromatography A

Volume 793, Issue 2, 16 January 1998, Pages 349-355

https://doi.org/10.1016/S0021-9673(97)00913-8 Get rights and content

Abstract

A micellar electrokinetic chromatographic method was developed to separate and quantify hexazinone and metabolites C, A1, E, B and D in groundwater. Hexazinone and its metabolites were extracted from water using Supelclean ENVI-Carb solid-phase extraction tubes. Quantitation was performed using UV photodiode detection at 220, 225, 230 and 247 nm. Intra-assay and inter-assay reproducibility studies run at 0.5, 1.0, 2.0 and 5.0 ppb indicated the procedure was reproducible. Groundwater samples collected from US Geological Survey monitoring wells were analyzed for hexazinone and its metabolites by CE. A comparison was made between CE and an established HPLC method of the hexazinone and metabolite B. The linear regression for hexazinone was y=1.007x+0.219 with a correlation coefficient of 0.96 while the linear regression for metabolite B was y=1.100x−0.057 with a correlation coefficient of 0.91.

Introduction

Hexazinone is a triazine dione herbicide which has been registered by the Environmental Protection Agency (EPA) for use on alfalfa, pasture and range grasses, pineapples, sugarcane and blueberries [1]. Hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione] has a high water solubility and low soil absorption [2]which allows it to move easily into the groundwater. Contamination of groundwater has been reported in Hawaii (0.06–0.72 ppb), Florida (0.12–2.90 ppb), Maine (0.2–29 ppb) and North Carolina (0.74–34 ppb) with all amounts below the EPA's lifetime health advisory level of 210 ppb [1].

Methods to determine hexazinone in groundwater have focused on using either isocratic or gradient reversed-phase high-performance liquid chromatography (HPLC) 3, 4or enzyme linked immunosorbent assays (ELISA) 5, 6. Few methods have been published regarding triazine pesticides determination by CE 7, 8, 9, 10. No method to date has been published for hexazinone and its metabolites. Isocratic HPLC procedures can at best quantify hexazinone and metabolite B while gradient HPLC methods can determine hexazinone and many of the metabolites. ELISA techniques are very good but cannot distinguish between hexazinone and metabolites due to cross-reactivity. Although both HPLC and ELISA are sensitive, there are problems with either complexity, organic solvent use, or cross-reactivity. These disadvantages have led to the development of a capillary electrophoretic (CE) method.

The use of CE for pesticide analysis has increased significantly. CE is a very efficient separation technique that achieves high resolution. The greatest limitation to CE is the detection sensitivity, especially with UV detectors. This can be overcome by sample concentration techniques or by using capillaries with increased path-lengths in the detection window as was done in this paper. One mode of CE, micellar electrokinetic chromatography (MECC), was first introduced by Terabe et al. [11]and is based on the partitioning of compounds distributed between the aqueous and micellar phase [12], thus improving the separation of charged and neutral compounds. This paper describes a MECC method for the analysis of hexazinone and five of its metabolites in groundwater.

Section snippets

Materials

All chemicals used were analytical grade. Sodium phosphate dibasic, sodium borate, sodium dodecyl sulfate (SDS) were purchased from Sigma (St. Louis, MO, USA) while the sodium hydroxide was from Fluka (Ronkonkoma, NY, USA). Methylene chloride (HPLC grade) was bought from Fisher (Fair Lawn, NJ, USA) and the HPLC methanol from EM Science (Gibbstown, NJ, USA). Hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione]; metabolite C

The effects of SDS and methanol on separation

Hexazinone has seven known metabolites (Fig. 1). With the addition of SDS five of the metabolites were separated from hexazinone (Fig. 2) and without it no separation occurred. However, metabolite A coeluted with metabolite C and metabolite 1 coeluted with hexazinone. This is not surprising because of the structural similarities (Fig. 1), but co-elution is a minimal problem in this instance. For example, metabolite C and A can be monitored at different wavelengths (C at 230 nm and A at 247 nm).

Acknowledgements

This manuscript is No. 2155 of the Maine Agricultural Experiment Station.

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Hexazinone is considered to be among the most likely pesticides to contaminate ground water. There is evidence that field applications of hexazinone have resulted in surface water contamination, which raises great concerns about its safety to wildlife and human health [1,4]. Therefore, determination and degradation of hexazinone in water is in great need.
Degradation of hexazinone has been investigated by means of photocatalysis of mixed-phase crystal nano-TiO₂. Influences of adsorption, amount of nano-TiO₂, pH and irradiation time on the photocatalytic process are studied. Results show that hexazinone is totally degraded within 40 min of irradiation under pH neutral conditions. This compares favorably with Degussa P25 TiO₂ when conducted under the same experimental conditions. Preliminary photocatalytic kinetic information for hexazinone degradation is proposed. First order kinetics is obtained for the adsorption and photocatalytic degradation reactions, which fit the Langmuir–Hinshelwood model. A rapid, sensitive and accurate UPLC–MS/MS technique is developed and utilized to determine the level of hexazinone in water in support of the degradation kinetics study. The results indicate a limit of detection (LOD) at 0.05 μg/l and the recoveries between 90.2 and 98.5% with relative standard deviations (RSD) lower than 12%. A LC–MS/MS technique is used to trace the degradation process. Complete degradation is achieved into final products including nontoxic water, carbon dioxide and urea. A probable pathway for the total photocatalytic degradation of hexazinone is proposed.
Modification to degradation of hexazinone in forest soils amended with sewage sludge
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It is a white solid and water soluble (33 g L−1 at 25 °C) with a relatively low vapor pressure [9]. This herbicide is a potent inhibitor of photosynthesis in susceptible species and was often detected in groundwater and soil in the forest areas, which raises great concern about its safety to human health [10]. The previous investigation on environmental fate of hexazinone mainly focused on the development of analytical techniques for the parent and its metabolites at trace levels [11]; dissipation [12]; adsorption–desorption [13]; microbial degradation [14]; leaching potential and mobility in soils [15].
Influences of one sewage sludge on degradation of hexazinone and formation of its major metabolites were investigated in four forest soils (A, B, C and D), collected in Zhejiang Province, China. In non-amended forest soils, the degradation half-life of hexazinone was 21.4, 30.4, 19.4 and 32.8 days in forest soil A, B, C and D, respectively. Degradation could start in soil A and C without lag period because the two soils had been contaminated by this herbicide for a long time, possibly leading to completion of acclimation period of hexazinone-degrading bacteria. In forest soils amended with sewage sludge, the degradation rate constant increased by 17.3% in soil A, 48.2% in soil B, 8.1% in soil C and 51.6% in soil D, respectively. The higher degradation rates (soil A and C) in non-amended soils accord with the lower rate increase in sewage sludge-amended soils. Under non-sterile conditions, biological mechanism accounted for 51.8–62.4% of hexazinone degradation in four soils. Under sterile conditions, the four soils had the similar chemical degradation capacity for hexazinone. In non-amended soil B, only one metabolite (B) was detected, while two metabolites (B and C) were found in sewage sludge-amended soil B. Similarly situated in agricultural soils, N-demethylation at 6-position of triazine ring, hydroxylation at the 4-positon of cyclohexyl group, and removal of the dimethylamino group with formation of a carbonyl group at 6-position of triazine ring appear to be the principal mechanism involved in hexazinone degradation in sewage sludge-amended forest soils. These data will improve understanding of the actual pollution risk as a result of forest soil fertilization with sewage sludge.
Anaerobic biodegradation of hexazinone in four sediments
2009, Journal of Hazardous Materials
Anaerobic biodegradation of hexazinone was investigated in four sediments (L1, L2, Y1 and Y2). Results showed that the L2 sediment had the highest biodegradation potential among four sediments. However, the Y1 and Y2 sediments had no capacity to biodegrade hexazinone. Sediments with rich total organic carbon, long-term contamination history by hexazinone and neutral pH may have a high biodegradation potential because the former two factors can induce the growth of microorganisms responsible for biodegradation and the third factor can offer suitable conditions for biodegradation. The addition of sulfate or nitrate as electron acceptors enhanced hexazinone degradation. As expected, the addition of electron donors (lactate, acetate or pyruvate) substantially inhibited the degradation. In natural environmental conditions, the effect of intermediate A [3-(4-hydroxycyclohexyl)-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H, 3H)dione] on anaerobic hexazinone degradation was negligible because of its low level.
Influence of bovine manure on dissipation of hexazinone in soil
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High water solubility (33 g L−1 at 25 °C) and low soil absorption (Hornsby et al., 1995) allow hexazinone to move easily into the groundwater. Hexazinone is often detected in groundwater in the areas it is used, which raises great concerns about its safety to human health (Kubilius and Bushway, 1998). Previous work on environmental behavior of hexazinone mainly focused on residual analyses of the parent and its metabolites (Fischer and Michael, 1995); dissipation (Calderson et al., 2004); adsorption–desorption (Bouchard and Levy, 1985); leaching potential and mobility in soil (Bottni et al., 1996).
The effects of bovine manure (BM) on the degradation of hexazinone and formation of three of its major metabolites were investigated in sandy loam soil. The degradation half-life of hexazinone was 29.6 days in unamended soil, while it decreased to 21.8 days in BM-amended soil. The major metabolites formed in unamended soil were [3-cyclohexyl-6-(methylamino)-1-methyl-1,3,5-triazine-2,4(1, 3H)-dione] (metabolite A) and [3-cyclohexyl-1-methyl-1,3,5-triazine-2,4,6(1, 3, 5H)-trione] (metabolite C), while metabolite B [3-(4-hydroxycyclohexyl)-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1, 3H)-dione] was not detected over the entire experimental period. However, in BM-amended soil, metabolite B was detected at 20 and 40 days after incubation, suggesting that BM contributed to formation of this metabolite. N-demethylation, removal of the dimethylamino group with formation of a carbonyl group at the 6-position of the triazine ring appeared to be the principal mechanisms involved in hexazinone metabolism in unamended soil. However, hydroxylation at the 4-positon of the cyclohexyl group as well as the above two modes were the principal pathways in BM-amended soil.
Degradation and metabolism of hexazinone by two isolated bacterial strains from soil
2005, Chemosphere
Two hexazinone-degrading bacterial strains were isolated from soil by enrichment culture technique, and identified as Pseudomonas sp. and Enterobacter cloacap. The two purified isolates, designated as WFX-1 and WFX-2, could rapidly degrade hexazinone with a half-life of 3.08 days and 2.95 days in mineral salt medium (MSM), while their mixed bacterial culture was found to degrade hexazinone, at an initial concentration of 50 μg/ml, by enhancing 2.3-fold over that when the isolates were used alone. Two microbial metabolites (A and D) were obtained by preparative TLC and identified on the basis of the spectral data of IR, ¹H NMR and HPLC–ESI–MS, but both of them were known products as they had been reported in soil and vegetation metabolites of hexazinone. However, metabolites B and C were new degradates, whose molecular weights (MW) were 157 and 156, respectively, being reported from microbial metabolism for the first time.
Movement of bromacil and hexazinone in soils of Hawaiian pineapple fields
2002, Chemosphere
Bromacil and hexazinone have been heavily used to control weeds in the pineapple fields in central Oahu, Hawaii, USA, since 1970s and 1980s, respectively. The last application prior to this study was at a rate of 0.6 kg active ingredient (a.i.) ha⁻¹ for hexazinone and 1.8 or 3.4 kg (a.i.) ha⁻¹ for bromacil in the six study fields during June–October 1998. Soils were collected from 0 to 1860 cm below the surface in January–May 1999 to survey the residue profiles of the two herbicides. Stratoprobe sampling showed to be an efficient and convenient method for deep soil cores. Bromacil was detected in all the soil samples above 60 cm (105–1338 ng g⁻¹ dry weight) and in 74% of the samples above 400 cm (26–473 ng g⁻¹). Trace amounts of bromacil (90–113 ng g⁻¹) were detected in some of the samples collected from as deep as 1540 cm. Hexazinone was detected in three of the six fields at 0–60 cm only (86–107 ng g⁻¹ dry weight). The more frequent detection of bromacil at higher concentrations than hexazinone is related to the prolonged higher application rates of bromacil in the fields and its higher persistence and mobility in soil.

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Determination of hexazinone and its metabolites in groundwater by capillary electrophoresis

Abstract

Introduction

Section snippets

Materials

The effects of SDS and methanol on separation

Acknowledgements

J. Chromatogr.

J. Chromatogr. A

Food Chem.

J. Environ. Qual.

J. Chromatogr. A