An immunoassay for metolachlor detection in river water and soil.

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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20

An immunoassay for metolachlor detection in river water and soil a

J.C. Hall , L.K. Wilson & R.A Chapman a

Department of Environmental Biology , University of Guelph , Guelph, Ontario, N1G 2W1 Published online: 14 Nov 2008.

To cite this article: J.C. Hall , L.K. Wilson & R.A Chapman (1992) An immunoassay for metolachlor detection in river water and soil, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 27:5, 523-544 To link to this article: http://dx.doi.org/10.1080/03601239209372799

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J. ENVIRON. SCI. HEALTH, B27(5), 523-544 (1992)

AN IMMUNOASSAY FOR METOLACHLOR DETECTION IN


RIVER WATER AND SOIL

KEYWORDS:

metolachlor, immunoassay, EIA, residue, soil dissipation.

J.C. Hall*, L.K. Wilson, and R.A Chapman. *Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1.

ABSTRACT An indirect enzyme-linked immunosorbent assay (EIA) for metolachlor (2chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-l-methylethyl)acetamide) detection in river water and soil was developed using serum obtained from rabbits immunized against the acid of metalaxyl ((N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine methyl ester) conjugated to bovine serum albumin. The assay had a linear working range from 1 to 50 ng/ml with a mean I J0 value of 13.6 ng/ml and a lower detection limit of 2.0 ng/ml. Both the mean interwell and interassay coefficients of variation were less than 4% over the range of the standard curves for samples which had been prepared in phosphate buffered saline (PBS), river water, or soil extract. Assay cross-reactivity to the following four

523 Copyright© 1992 by Maicel Dekker, Inc.

524

HALL, WILSON, AND CHAPMAN

structurally related chloro-acetanilide pesticides were:

propachlor (0%),

metazachlor (0%), alachlor (23%), and metalaxyl (5,000%). Mean recoveries of metolachlor in spiked (2.0 to 32.0 ng/ml range) PBS, river water, and soil extract were 102%, 103%, and 110%, respectively. Soil samples were taken over a 56-d


period from field plots treated with metolachlor and analyzed by GC and EIA. The correlation coefficient for comparison of the two methods was 0.96 with the slope of the linear regression line being 0.78.

Furthermore, no statistical

difference ( P < 0 . 0 5 ) was found between the dissipation curves of metolachlor derived from GC data versus EIA data. INTRODUCTION Immunoassays offer

alternatives or complements to conventional

Chromatographie techniques for trace analysis of pesticide residues in water, soil and food.

The attributes and potential of immunological methods for the

detection and quantitation of pesticides in various matrices has been reviewed by Cheung et al. (1988), Hammock et al. (1987), Hammock (1988), Harrison et al. (1988), Jung et al. (1989), Van Emon et al. (1989), Hall et al. (1990), and Hammock et al. (1990).

Listed among the advantages of immunoassay are

simplicity, speed and low cost of analysis, thereby making it useful as a primary screen for reducing the number of samples that must be otherwise analyzed by HPLC, GC, and/or GC/MS methods. Metolachlor, the active ingredient of Dual herbicide, is used to control most annual grasses and some broadleaf weeds in corn, soybean, snap beans, dry

METOLACHLOR DETECTION IN WATER AND SOIL beans, lima beans, potatoes, sugar beets, and rutabagas.

525 It is the only

chloroacetamide herbicide which is extensively used in Canada, with its use being primarily restricted to the southern region of the province of Ontario because of cropping practices (CCREM, 1991). In Ontario, over 842 t of metolachlor were


used in 1983 (McGee, 1984) which increased to over 1724 t by 1988 (Moxley, 1989) largely as a result of the de-registration of alachlor by the Canadian government in 1987 (CCREM, 1991).

Since metolachlor is applied as a

preemergent, preplant incorporated, or postemergent treatment, it may enter the aquatic environment as a result of surface or subsurface intrusions from treated land or through spillage and/or waste disposal during production, packaging, storage, and use (CCMER, 1991).

Recently metolachlor residues have been

detected in farm wells and in surface and ground waters throughout southern Ontario and the United States (CCMER, 1991; Frank et al, 1987a, 1987b; Frank and Logan, 1988; Graver, 1988). For example, in 1987, 7 of 12 water samples taken from the Sydenham River in southern Ontario contained metolachlor (Frank et al, 1990), while in the United States, 1644 of 1997 surface water samples tested positive for metolachlor (CCMER, 1991). Feng et al. (1990a, 1990b) have developed an indirect EIA for the quantitation of the chloroacetamide herbicide alachlor in water samples. They found good correlation between results from EIA and GC/MS for alachlor detection, however, little cross-reactivity was found to metolachlor. Newsome (1985) also developed an EIA to detect and quantify the chloracetamide fungicide metalaxyl in foods. Because of the structural similarity between metalaxyl and

526


metolachlor, he found the assay to have extensive cross-reactivity with metolachlor. Based on the methods of Newsome, we have developed an EIA specific for metolachlor which can be used to detect and quantify this herbicide in spiked river water and soil samples. In addition, a soil dissipation study of


metolachlor under field conditions was conducted using the EIA procedure and the results compared with those obtained using a gas Chromatographie method. MATERIALS AND METHODS Chemicals and Materials Analytical grade metolachlor and metalaxyl (N-(2,6-dimethylphenyl)-N(methoxy-acetyl)-DL-alanine methyl ester) as well as commercially formulated metolachlor (Dual® 960-E) were obtained from Ciba-Geigy, Cambridge, Ontario. Analytical grade alachlor (2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl) acetamide) and propachlor (2-chloro-N-(l-methylethyl)-N-phenylacetamide) were obtained from Monsanto, St. Louis, MO. while metazachlor (N-(2,6-dimethylphenylJ-N-O-pyrazolyl-methyty-chloroacetamide) was obtained from BASF, Limbergerhof, Germany. Isobutyl-chloroformate, l-[3-dimethylaminopropyl]-3ethylcarbodiimide hydrochloride (EDC), triethylamine, bovine serum albumin (BSA), rabbit serum albumin (RSA), 2,2'-azino-bis(3-ethylbenzthiazoline-6sulfonic acid)diammonium (ABTS), urea hydrogen peroxide, Tween-20® (polyoxyethylene sorbitan monolaurate), and Freunds incomplete adjuvant were obtained from Sigma Chemical Company, St. Louis, MO.

Diethanolamine,

dichloromethane, 2,4-dioxane and the Immulon 2 'U' microtitration plates were obtained from Fisher Scientific Ltd, Don Mills, ON. Goat anti-rabbit horse-

METOLACHLOR DETECTION IN WATER AND SOIL

527

radish peroxidase was purchased from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. Gelatin was purchased from Eastman Kodak Company, Rochester, NY. Nylon filters (0.22μΐη) were obtained from J . T . Baker, Inc, Phillipsburg, NJ.


Buffers Phosphate buffer (PB) stock solution was prepared by dissolving 75.5 g Κ 2 ΗΡΟ 4 ·3Η 2 Ο and 23.0 g KH2PO4 in 500 ml of deionized water. The pH was adjusted to 7.5 with 1 M HC1. Phosphate buffered saline (PBS) was made by adding 10 ml of the PB stock solution and 8.2 g NaCl to 1 L deionized water. The pH was adjusted to 7.5 with 1 M HC1. PBS-Tween® washing solution contained 0.5 ml Tween-20 in 1 L o f PBS. Citrate buffer was made by adding 5.0 g citric acid monohydrate and 12.6 g Na2HPO4-7H2O to 1 L deionized water (pH 5.0). Preparation of Immunogen The carboxylic acid of metalaxyl (metalaxyl acid) was obtained following the procedures of Newsome (1985). Metalaxyl acid was prepared by refluxing 100 mg

of metalaxyl in 3 ml of 1 Ν NaOH for 2 h.

The hydrolysate was

acidified with 1 Ν HC1 and extracted with dichloromethane. After removal of the solvent, an oil was obtained which later crystallized. Metalaxyl acid was conjugated to BSA via a carbodiimide peptide linked conjugate which was prepared by adding 95 mg EDC and 14 mg BSA to 6 mg metalaxyl acid in 2 ml of PBS as described by Newsome (1985).

After the

528


solution mixed overnight at room temperature, it was dialysed for 3 days against deionized water and then lyophilized. Preparation of Coating Conjugates Metalaxyl acid was conjugated to RSA via a mixed anhydride peptide


linked conjugate which was synthesized by adding 8 μΐ of isobutyl chloroformate and 15 μ\ of triethylamine to 13.3 mg of metalaxyl acid in 0.5 ml of dioxane (Newsome, 1985). After 30 minutes the mixture was added dropwise to a stirred solution of 3 ml carbonate buffer (pH 9.3) containing 44 mg RSA. Six hours later,

the mixture was dialysed for 3 days against deionized water and then

lyophilized. Antisera Production A pair of New Zealand white rabbits were injected subcutaneously with 0.5-1.0 mg of the carbodiimide immunogen dissolved in 0.5 ml saline and emulsified in 0.5 ml of Freund's incomplete adjuvant. Intramuscular injections were repeated weekly for 3 months. Booster injections were administered a week prior to each bleeding and rabbits were bled through the ear vein as antiserum was required. The blood was incubated in a test tube at 37°C for 1 h, stored overnight at 4°C and the serum separated by centrifugation at 5,000g for 15 min. The titer of the antibodies was determined by a competitive indirect enzyme immunoassay (EIA) described below. The assay was executed using serum that was pooled from three separate bleeds.


529

Antibody Characterization Cross reaction of the serum was determined by measuring the (quantity of

metolachlor required for 50% binding)*'(quantity of structurally similar chemical analogues required for 50% binding) X100 (Feng et al., 1990a). The serum was


tested for cross-reactivity to alachlor, propachlor, metazachlor, metalaxyl and metalaxyl acid. Soil Dissipation of Metolachlor in Field Plots A field study to determine the persistence of metolachlor in soil was conducted in collaboration with

Agriculture

Canada, London, Ontario.

Microplots (1 χ 2 χ 0.62 m) were constructed using an aluminum frame with fibreglass sides which was filled with pesticide-free loam soil (Harris et al., 1971). Commercially formulated metolachlor (Dual 960E) was applied to each plot at a dose equivalent to 2.64 g ai/ha using a modified, single-nozzle Oxford precision sprayer equipped with a 8004 flat fan nozzle calibrated to deliver 200 L/ha of spray solution at 200 kPa. Sweet corn (&ea mays saccharum L. cv. Silver Queen) was planted in each microplot to provide shade and reduce soil drying. There were 3 rows per microplot with 3 plants at the middle and ends of each row.

The study included 4 metolachlor-treated plots and 2 untreated

plots. Λ single soil sample was obtained from each plot at each sampling time by pooling fifteen 2.5 χ 15 cm soil cores. The plots were sampled immediately after treatment and thereafter at day 2 , 5 , 7, 9, 14,21, 28,43 and 56. Soil from treated and untreated plots were extracted as described below.

Untreated soil

samples were used as the matrix blank in which the standards of metolachlor for

530


EIA of soils were prepared. Metolachlor concentrations were determined in treated soil samples by gas chromatography or EIA procedures, both of which are described below. Sample Preparation


River Water. Water (10 L) was collected in 500 mL containers from the Speed River, Guelph, Ontario during the early Spring of 1991 and stored at -20°C. As required, the water was thawed and aliquots of 100 mL were passed through a 0.22 μΐη cellulosic filter. Soil. Soil was sieved and a 300 g sample was extracted by tumbling end-over-end for 1 h with 300 ml of methanol in a tightly sealed 1 L bottle. The methanol was separated from the soil through a Büchner funnel.

Additional methanol

(approximately 45 ml) was used to rinse the bottle and soil cake. The extract was made up to a final volume of 350 ml and stored in a freezer (-15C C) until required. The extracts were warmed to room temperature prior to analysis and diluted 1:9 or 1:99 (v/v) in PBS as required to adjust the concentrations of metolachlor to fit within the working range of the EIA standard curve. Preparation of Metolachlor Standards for EIA in Various Matrices A 1 mg/mL stock solution of metolachlor was prepared in methanol. Portions were diluted (1:99 v/v) with PBS, river water or the PBS-diluted untreated soil extract to give stock solutions of 10 /xg/mL which contained 1% methanol by volume. Metolachlor standards for the EIA containing 40.0, 30.0, 20.0, 10.0, and 2.5 ng/mL were prepared by further diluting each of the stock

METOLACHLOR DETECTION IN WATER AND SOIL solutions with PBS, river water and dilute soil extract, respectively.

531 Each

standard was divided into 0.5 mL aliquots and stored at - 2 0 ° C until required. Competitive Indirect Enzyme Immunoassay The following procedure is a modified version of the method described by


Hall et al. (1989). Microtitration plates were coated with 0.1 ^g/ml metalaxyl acid/RSA coating conjugate dissolved in PBS (100 μ\ per well). Plates were incubated overnight at 4°C. The plates were emptied and washed three times with PBSTween

washing solution. Gelatin diluted in PBS (0.1% (w/v)) was added to each well (100 μΐ per

well) and incubated for 20 minutes at 22°C. The plates were emptied and washed as previously described. Pooled rabbit serum (from a rabbit injected with the carbodiimide conjugate of metalaxyl acid/BSA) was diluted 1:1250 (v/v) in PBS and preincubated 1:1 (v/v) with either a metolachlor standard or a sample solution for 30 minutes at 22°C. The pre-incubated mixture was aliquoted into a well (100 μΐ) and the plates were incubated for 1 h at 22°C. Again, the plates were emptied and washed. Goat anti-rabbit IgG-horseradish peroxidase conjugate was diluted 1:5000 (v/v) in PBS and 100 μΐ added to each well. The plates were incubated for 1 h at 22°C, emptied and washed. Horseradish peroxidase substrate was prepared by adding 1 mg/ml urea hydrogen peroxide and 1 mg/ml ABTS to citrate buffer. The substrate was added

532

.


(100 μ\ per well) and the plates incubated for 30 minutes at 22°C.

The

development of the colour reaction was stopped by the addition of 100 μ\ 0.5 M citric acid (pH 5.0) to each well. A microplate reader (Bio-Rad Model 2550 EIA Reader, Mississauga, ON) measured the absorbance of each well at 405 nm.


Non-specific binding was measured as the absorbance read from wells containing an excess (10,000 ^tg/mL) of free metolachlor.

This value was

subtracted from the absorbance value of the standards and samples (A) as well as the maximum absorbance value (A o ).

The maximum absorbance value was

measured from wells in which there was no free metolachlor to compete with the coating conjugate for the metolachlor specific antibodies. A standard curve was obtained by plotting the absorbance values of the standards divided by the maximum absorbance value (A/Ao) against the log of the metolachlor concentration. The equation of the line from the standard curve was used to calculate the quantity of metolachlor in the samples. Determination of Metolachlor in Soil by Gas Chromatography The gas Chromatograph (GC) used was equipped with a N/P detector and a 3% OV-101 column treated with 20M Carbowax (Ives & Giuffrida, 1970). The solid phase was Chromosorb W-HP (100-120 mesh size). The carrier gas was He (flow rate 30 ml/minute). The oven, detection and injection temperatures were 190°C, 250°C and 210°C, respectively. Soil extracts, as is or diluted as required with methanol were injected directly into the GC. Quantitation was by peak heights compared to external standards in methanol.


533

RESULTS Enzyme Immunoassay Standard Curves Metolachlor standards prepared in PBS, river water and diluted soil extract were used to generate standard curves.

For PBS, a third order polynomial


equation, Y = 0.809 - 0.233(log X) - 0.203(log X) 2 + 0.076(log X) 3 ; R 2 = 1 . 0 0 , was obtained by regressing the relative absorbance (A/A,,) on the log of the metolachlor concentration in the range of 0.5-100 ng/mL (Figure 1(A)). A linear relationship was found between the relative absorbance and metolachlor concentration in the range of 1-50 ng/mL, which is described by the equation Y = 0.812 - O.358(log X ) ; R 2 = 1 . 0 0 (Figure 1(B)). Assay Sensitivity Based upon the above data (Figure 1(B)), the assay had, for practical detection and quantitation of metolachlor, a lower and upper detection limit of 2.0 and 50 ng/mL, respectively. A mean I50 value of 13.6 ng/mL was determined within the linear working range of the dose-response curve (Figure 1(B)). Assay Precision By the use of the absorbance values in 12 separate wells for each of the metolachlor standards, prepared in the respective matrix (PBS, river water, and soil extract), the interwell variability was determined for the EIA procedure (Table 1). A mean interwell coefficient of variation (CV) of 1.8%, 1.8%, and 1.9% was determined over the range of the standards prepared in PBS, river water, and soil extract, respectively. The mean interassay CV of the metolachlor standard curve A/Ao values determined from eight separate runs of the assay



534

1.0

10.0

1.0 10.0 Metolachlor concentration (ng/ml)

100.0

100.0

FIGURE 1. EIA standard curve for metolachlor standards prepared in PBS. Each point represents the mean of 8 determinations. Vertical bars represent one standard deviation. The best-fit curve over the concentration ranges 0.5 to 100 ng/ml (Fig. A) and 1.0 to 50.0 ng/ml (Fig. B) are represented by a third and first order polynomial equation, respectively (see text for equations).


535

TABLE 1


Interwell Variability for an Indirect Enzyme Immunoassay Standard Curve Prepared in PBS, River Water, or Soil Extract"

matrix

metolachlor std, ng/mL

absorbance ± SD

CV, %

PBS

0 2.5 5.0 10.0 20.0 30.0 40.0

1.160 1.048 0.969 0.869 0.773 0.725 0.694

± 0.024 ± 0.020 + 0.014 ± 0.018 ± 0.014 ± 0.015 ± 0.012

2.1 1.9 1.4 2.0 1.7 2.0 1.7

river water

0 2.5 5.0 10.0 20.0 30.0 40.0

1.300 1.228 1.156 1.058 0.936 0.874 0.822

± 0.034 ± 0.020 + 0.019 ± 0.020 ± 0.018 + 0.016 ± 0.012

2.6 1.6 1.6 1.9 1.9 1.8 1.5

soil extract6

0 2.5 5.0 10.0 20.0 30.0 40.0

1.332 1.154 1.060 0.965 0.865 0.802 0.767

± 0.029 ± 0.023 ± 0.018 ± 0.024 + 0.019 ± 0.011 ± 0.013

2.2 2.0 1.7 2.4 2.2 1.3 1.6

.

'Regardless of the matrix, absorbance ± SD (CV, %) for 10,000 ng/ml metolachlor was less than 0.044 ± 0.012 (26%). bSoil extracts were diluted 1:9 (v/v) in PBS before analysis by EIA.

536


prepared in PBS, river water, and soil extract were 2.5%, 1.4%, and 3.0%, respectively (Table 2).

Absorbance values for nonspecific binding were

determined by inhibition of antibody binding with 10,000 ng/mL of metolachlor. Regardless of the matrix used, the absorbance values for nonspecific binding had


a mean CV value of 26.0% and had absorbance values similar to those determined for blanks (only substrate placed in wells of microtiter plates). Cross-Reactivity Studies Five structurally related chloroacetanilide compounds, alachlor, metalaxyl, metalaxyl acid, metazachlor, and propachlor were tested for cross-reactivity. Compared to metolachlor, the cross-reactivity of propachlor, metazachlor, alachlor, metalaxyl and metalaxyl acid were 0%, 0%, 2 3 % , 5,000% and 905%, respectively.

The high cross reactivity of metalaxyl and metalaxyl acid was

expected since the hapten of the immunogen was metalaxyl acid. Determination of Metolachlor in Fortified PBS. River Water, and Soil Extract Recovery of metolachlor from the three matrices indicated that the EIA was suitable for quantitative determinations (Table 3).

Overall recoveries

averaged across all six concentrations of metolachlor in PBS, river water, and soil extract were 102%, 103%, and 110%, respectively. Within each matrix each determination of a specific metolachlor concentration was statistically different ( P < 0 . 0 5 ) from all other concentrations. Soil Dissipation of Metolachlor in a Field Study Prior to conducting the study on the dissipation of metolachlor in the field, 23 soil samples from plots treated with metolachlor were extracted, diluted or


537

TABLE 2


Interassay Variability for an Indirect Enzyme Immunoassay Standard Curve Prepared in PBS, River Water, or Soil Extract A/Ao matrix

metolachlor std, ng/mL

mean

SD

CV, %

PBS

2.5 5.0 10.0 20.0 30.0 40.0

0.902 0.835 0.750 0.667 0.625 0.590

0.021 0.025 0.023 0.009 0.014 0.015

2.4 3.1 3.1 1.4 2.3 2.5

river water

2.5 5.0 10.0 20.0 30.0 40.0

0.941 0.886 0.812 0.718 0.670 0.630

0.016 0.017 0.009 0.008 0.009 0.007

1.7 1.9 1.2 1.2 1.4 1.2

soil extract*

2.5 5.0 10.0 20.0 30.0 40.0

0.867 0.796 0.725 0.649 0.602 0.566

0.012 0.022 0.025 0.015 0.017 0.027

1.3 2.9 3.5 2.8 2.8 4.7

'Soil extracts were diluted 1:9 (v/v) with PBS before analysis by EIA.


538 TABLE 3

Recovery of metolachlor from Known Fortified PBS, River Water, and Soil Samples Determined from an EIA Standard Curve Prepared in the Respective Matrix

metolachlor recovered', ng/mL

metolachlor


.added, ng/mL PBS 2.0 4.0 8.0 12.0 16.0 32.0

2.4 4.3 7.6 11.4 15.6 30.5

+ 0.5 (22%) ± 0.7 (17%) ± 1.2 (17%) ± 1.6 (15%) ± 2.8 (18%) ± 4.2 (18%)

1

nver water

soil extract "

± 0.4 (15%) ± 0.3 (8%) ± 0.8 (11%) 10.2 ± 0.7 (7%) 15.2 ± 1.8 (12%) 33.8 + 4.8 (14%)

2.6 ± 0.6 (22%) 4.6 ± 0.6 (14%) 8.2 ± 0.7 (9%) 11.4 ± 1.4 (12%) 17.6 ± 1.3 (8%)

2.7 4.2 7.2

34.7 ± 4 . 5 ( 1 3 % )

'Mean ± SD followed by (intraassay CV %); means were determined from 16 plates with each plate having 6 wells (n=6) for each respective metolachlor concentration. 'Soil extracts were diluted 1:9 (v/v) in PBS before analysis by EIA.

concentrated as required and analyzed by EIA and GC. A correlation of the results from GC and EIA analyses are presented in Figure 2.

The x-axis

indicates the ppm of metolachlor per gram of soil as determined by EIA, and the y-axis indicates the ppm of metolachlor as determined by GC. The correlation coefficient for comparison of the two methods was 0.96, and the slope of the regression line was 0.78. Since the slope was less than 1.00, the results indicate that the EIA tended to over-estimate the compared to the results from GC.

metolachlor concentrations when


539

Ι.ίΌ ·


(ppm metolachi

l_

o o

o^ o

1.00 •

s*

0.75 •

/

0.50 -

«o ' o

y* «

0.25 • 0.00 • 0.00

1 0.25

1

0.50

1

0.75

1

1.00

1

1.25

1

1.50

1.75

EIA (ppm metolachlor)

FIGURE 2. Correlation of GC and EIA analyses of the metolachlor concentration in 23 soil samples. Correlation coefficient and equation of the line are given in text.

10 20 30 40 Days after treatment

50

60

FIGURE 3. Dissipation of metolachlor over a 56-day period after application of the herbicide to field plots as determined by EIA ( · ) or GC ( o ) analysis. Vertical bars represent 95% confidence intervals for each mean.

540

HALL, WILSON, AND CHAPMAN In the metolachlor dissipation study, soil samples were taken from field

plots over a 56 day period after treatment with the herbicide and analyzed by EIA and GC. No statistical difference ( P < 0 . 0 5 ) was found between the metolachlor dissipation curve derived from data obtained by EIA and GC (Figure 3).


However, as was indicated by the correlation of GC and EIA data (Figure 2), the EIA tended to over-estimate the metolachlor concentrations (Figure 3). DISCUSSION In general, the relative pattern of cross-reactivity that we found to other chloroacetamide compounds agrees with* the results of Newsome (1985) who found metalaxyl > metolachlor > alachlor > propachlor in terms of crossreactivity. The only difference between our results and those of Newsome was that he found metalaxyl acid to be less cross-reactive to the antibodies than metolachlor.

Feng et al. (1990a) found little cross-reactivity of metolachlor

(1.8%) to the antibodies for alachlor, whereas we found a higher cross-reactivity of alachlor (23%) to the antibodies for metolachlor. These results indicate that the assay of Feng et al. (1990a) was more specific for alachlor than our assay was for metolachlor. Nonetheless, our assay would be more appropriate than the assay of Feng et al. for the nonspecific detection of metalaxyl, alachlor, and metolachlor.

This may limit the usefulness of our EIA for the specific

determination of metolachlor in areas where alachlor and/or metalaxyl have been applied. However, in Canada, this cross-reactivity to these two chloroacetamide compounds will not limit the usefulness of our EIA for metolachlor detection since the use of alachlor has been banned since 1987 (CCREM, 1991).


541

Furthermore, in Canada, metalaxyl is registered for field crop use only in potato crops. Potato crops are rarely used in a crop rotation scheme with the major field crops such as field beans, soybeans, and corn which are the crops that receive the majority of the total metolachlor active ingredient applied in Canada.


Metolachlor can be applied to potatoes and therefore limit the usefulness of the EIA if this crop were treated with metalaxyl prior to assaying for metolachlor, however, this situation seldom occurs in Canada. In conclusion, an effective EIA was developed to quantitate metolachlor in river water and soil samples in the range of 1.0 to 50.0 ppb. The standard curves were prepared in the respective matrix to overcome the potential antibodyantigen interactions that may be altered by pH, ionic strength, viscosity, solubility, organic matter and/or humic acid content (Morris, 1985; Van Vunakis, 1990). A reference matrix is not useful in studies where many samples could originate from different sources or where the matrix may change during the course of sampling. Standard curves prepared in a reference matrix were feasible in our field soil dissipation experiments since blank matrix samples were easily obtained prior to herbicide treatment and the matrix did not change during the course of sampling. However, if river water samples were taken as a reference matrix prior to metolachlor application for weed control in agricultural crops, there is no guarantee that the matrix will be representative of the reference matrix after a runoff event in which metolachlor may enter the river along with organic matter, humic acids, fertilizers, and other compounds. In such a case, it may be better to dilute the matrix or perform simple column

542


chromatography to overcome potential interferences from contaminants. In any case, the EIA for metolachlor proved to be an effective method for detecting and quantitating this herbicide in field soil and river water samples


in which the matrix did not change during the course of the experiments.

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PCR detection of Burkholderia multivorans in water and soil samples.

Metolachlor metabolite (MESA) reveals agricultural nitrate-N fate and transport in Choptank River watershed.

Detection of water quality changes along a river system.

Dissipation of S-metolachlor in plant and soil and effect on enzymatic activities.

Selection of phage-displayed peptides for the detection of imidacloprid in water and soil.

USLE-Based Assessment of Soil Erosion by Water in the Nyabarongo River Catchment, Rwanda.

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Integrated hydrological and water quality model for river management: a case study on Lena River.

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An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum.

Development of a Luminex assay for the simultaneous detection of human enteric viruses in sewage and river water.

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Particle-water interactions of platinum-based anticancer drugs in river water and estuarine water.