Immunochemical Approach for Pesticide Waste Treatment Monitoring

Chapter 22

Immunochemical Approach for Pesticide Waste Treatment Monitoring of s-Triazines 1

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Mark T. Muldoon and Judd O. Nelson Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 and Department of Entomology, University of Maryland, College Park, MD 20742

Enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of pesticide waste and rinsate and disposal monitoring. Three s-triazine-specific monoclonal antibodies that possessed different specificities were utilized to quantitate individual and total s-triazine analytes in mixtures. Results from the analysis of field samples were validated by HPLC. An immunoassay was developed for chlorodiamino-s-triazine (CAAT), an important degradation product of chloro-s-triazine herbicides. Antibody recognition of substituted s-triazines decreased as a function of increased amino side chain substitution. The assays were sensitive in the low micromolar range. An s-triazine herbicide class-specific ELISA was used in conjunction with an ELISA for CAAT for measuring striazine herbicide ozonation followed by microbiological treatment. The ELISAs were shown to be very accurate and precise for measuring the concentrations of both atrazine and CAAT. The information obtained by the two ELISAs could be used for on-site control of the two stage treatment process and should savetimeand expense in s-triazine disposal monitoring applications.

The widespread use and misuse of chemical pesticides worldwide has resulted in their occurence throughout the biosphere. Pesticides enter the various environmental compartments through normal use, overapplication, accidents (including back-siphoning into water supplies), runoff from mixing-loading areas, and faulty waste disposal (1, 2). Spills and faulty waste disposal may be considérai point sources of contamination since they are usually in a confined area 1

Current address: Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77845

This chapter not subject to U.S. copyright Published 1995 American Chemical Society

Nelson et al.; Immunoanalysis of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

22. MULDOON & NELSON

Pesticide Waste Treatment Monitoring of Triazines 309

and are often high concentration environmental exposures. This source of exposure may be most readily controlled through safer handling practices and the development and implementation of methods to properly dispose of unusable materials (3). Some agricultural pesticide spraying operations generate large volumes of pesticide-containing materials consisting of excess pesticide product, leftover tank mixtures, and equipment rinsates. Typically, pesticide concentrations in waste can range from 1.0-10,000 ppm (4). In addition to pesticidal constituents, the material usually contains formulating agents, fertilizers, adjuvants, and machinery wash-off debris. Pesticide wastes are often be generated at very remote areas and appropriate on-site management of these materials depends on die particular situation involved. Management options include reuse, recycling as subsequent make-up water, or if necessary, disposal (5). On-site disposal methods have been developed for these high-volume wastes. The methods include physical (evaporation, adsorption, filtration), chemical (hydrolysis, oxidation, incineration), biological (composting, landfarming, enzymatic, bioreactors) and combinations of various methods (6, 7). Traditionally, analytes have been monitored using conventional analytical methods such as gas-liquid chromatography (GLC) and high performance liquid chromatography (HPLC) in order to ensure the effectiveness of the treatment and to deterrnine end-points for multiple step processes. These methods are well established, however, they have the disadvantages of being expensive, timeconsuming, and are not readily adaptable to in-field analyses. Hammock and Mumma (8) published a farsighted review on the potential of immunochemical techniques for environmental analytical chemistry. Since then, immunoassays, in particular enzyme-linked immunosorbent assays (ELISAs), have been developed for many environmentally-important analytes (9). They are sensitive, simple to perform, and can be madefieldadaptable (10). ELISA kits are commercially available for many different environmental contaminants. Immunoassays have been adapted primarily as screening methods for analytical situations where there is a large sample load such as in groundwater monitoring programs (11). Immunoassays are particularly attractive for use in pesticide waste management applications since they can be performed with minimal operatortanning,are rapid, require little if any sample preparation, and arefieldadaptable. The utilization of rapid tests to determine the presence, absence, or concentrations of particular components in a waste material would allow for better management of these materials. In addition, the use of immunoassays for on-site waste disposal monitoring should savetimeand expense. Pesticide waste analysis introduces some very unique challenges for ELISA analysis which are not encountered in other applications. As described above, pesticide wastes and rinsates are usually complex mixtures of pesticide active ingredients in addition to a number of nonpesticidal matrix components. Therefore, it was necessary to develop and validate immunoassay methods specifically for analysis in this sample matrix. This study was part of a larger project at USDA to develop strategies for the disposal and remediation of pesticide waste materials. A disposal method developed at USDA used a combined ozonation and microbial rnineralization process to oxidize recalcitrant pesticide substrates to more biolabile intermediates


IMMUNOANALYSIS OF AGROCHEMICALS

310

which could be readily degraded by indigenous soil and sludge microorganisms (12-15). The j-triazines were shown to be among the most recalcitrant substrates studied and were considered to be useful indicators of treatment effectiveness. Atrazine ozonation (Figure 1) proceeded by either direct N-dealkylation or the oxidation to either or both of the N-alkyl side chains, loss of this acetoamido moiety, and the accumulation of chlorodiammo-^-triazine (CAAT) (14). In the second stage of the process, CAAT was mineralized by soil or sludge microorganisms (12, 15). The process was monitored for the loss of atrazine and the accumulation of CAAT by ozonation, followed by the subsequent biodégradation of this intermediate. The analytical method used for routine analysis was HPLC. The purpose of the current study was to develop immunoassay techniques for monitoring this disposal process. We utilized three monoclonal antibodies developed by Karu et al. (16), which showed distinct within-class crossreactivities toward various j-triazines herbicides, for discriminating and quantifying individual 5-triazines in pesticide waste mixtures (17). ELISAs were developed which were selective for the detection of CAAT (18). The 5-triazine herbicide assay which showed the broadest recognition of the parent 5-triazine herbicides was chosen for use as a class-specific ELISA for monitoring the loss of atrazine in the disposal process. This assay was used in conjuction with an ELISA for CAAT to monitor the complete disposal process for atrazine (19). This paper summarizes the work pertaining to atrazine disposal monitoring by ELISA. Materials and Methods Pesticide Waste and Rinsate Samples. Pesticide waste and rinsate samples were obtained from collection facilities at the Beltsville Agricultural Research Center, USDA, ARS, Beltsville, MD during the spring 1991 growing season. The chemical composition of the samples was described in Muldoon et al. (17). High Performance Liquid Chromatography. HPLC measurements were made using a Waters 712 WISP automatic sample injector, two Waters Model 510 HPLC pumps, a Waters Model 490 UV detector (210, 220, and 230 nm monitored), and a NEC APC-IV controller with Maxima 820 software. The column was a Waters NOVAPAK 4 μπ\ C-18 in a 8 mm χ 10 cm radial compression module. The solvent system used a 15 min gradient (Waters curve 10) of 0 to 75% acetonitrile/phosphoric acid (pH 2) at a flow rate of 2.0 rnL/min. The final condition was maintained for 5 min. Analyte concentrations were calculated based on standards curves for each of the individual compounds using authentic analytical standards. Hapten Synthesis and Hapten-Protein Conjugation. Carboxylic acid 5-triazine haptens were synthesized for use in the development of j-triazine herbicide ELISAs and antibody production and ELISA development for the analysis of CAAT. The procedures used were adaptations of those described by Goodrow et al. (20). Briefly, CEPrT (Figure 2) was synthesized by sequentially substituting




CI

'NHCH2CH3

(KjC) HCHN' 2

CAAT

Figure 1. Degradative pathway for the ozonation of atrazine. (Reproduced with permission from reference 14. Copyright 1992 American Chemical Society).


312


Figure 2. Structures of the target analytes, immunizing haptens, and ELISA haptens used in this study. The immunizing and ELISA haptens used for the analysis of atrazine (CIFT) and chlorodiamino-5-triazine (CAAT) were SPrlET and CEPrT, and SPrAAT and CAHeT, respectively.




two chlorines of cyanuric chloride, one with an ethylamino and the other with a aminopropanoic acid. CAHeT was synthesized by the substitution of a single chlorine of dichloroamino-5-triazine by an aminohexanoic acid. The single chlorine of CAAT was substituted with thiopropanoic acid resulting in the formation of SPrAAT. All of the structures were verified by infrared, mass, and NMR spectral methods. Details of the various methods and spectral data can be found in Goodrow et al. (20) and Muldoon et al. (18). Hapten-protein conjugates were synthesized by via N-hydroxysuœinimide activated esters of the various carboxylic acid haptens. These were reacted with protein in aqueous solution to form the conjugates (21). SPrAAT was conjugated to keyhole limpet hemocyanin (KLH) for use as an immunogen for the production of antibodies which recognize CAAT (18). The heterologous haptens CEPrT and CAHeT were conjugated to alkaline phosphatase (AP) for use in the $-triazine herbicide ELISAs and the CAAT ELISA, respectively. Antibodies. Mouse monoclonal j-triazine-specific antibodies AM7B2, AM1B5, and SA5A1 (primary antibodies), were donated by Dr. A . E . Karu, University of California at Berkeley. Monoclonal antibody production, screening, assay development, and application were previously described (16). The hybridoma cell culture supernatants and ascites fluid preparation (AM7B2) were used without further purification. Immune polyclonal ascites fluid was produced using KLH-SPrAAT as an immunogen by an adaptation of a previously described method (22). Antibody screening and ELISA development for the analysis of CAAT has been (18). The preparations used here were designated PAb 1 and PAb 5 (primary antibodies). ELISA Procedure. Antibody screening and ELISA format optimization for the striazine herbicide ELISAs and the CAAT ELISA have been described in detail elsewhere (17, 18). The ELISA format was adapted from Karu et al. (16). Dilutions of the various immunoreagents used in each procedure were determined by 3-dimensional checkerboardtitration.Briefly, microtiter plates were coated with goat anti-mouse IgG (trapping antibody) diluted in coating buffer (15 mM sodium carbonate, pH 9.6), incubated 18 hrs at 4°C, and then washed with phosphate-buffered saline (pH 7.5) containing Tween 20 and sodium azide (PBSTA). One hundred microliters of a predetermined amount of primary antibody diluted in 0.5 mg/mL BSA in PBSTA were applied to the plate, incubated for 60 min, and frozen with the liquid remaining in the wells. The plate was thawed and washed when needed. For the j-triazine herbicide ELISA, 40 μL of sample was mixed with 200 /dL of a predetermined dilution of CEPrT-AP in a separate uncoated well. Fifty microliter aliquots were applied to replicate wells of the antibody-coated plate and incubated 30 min; the plate was then washed. Enzyme substrate (p-nitrophenyl phosphate) was added and the plate OD measurements (405 nm) were made at 30 min using a Molecular Devices ThermoMax microplate reader (Menlo Park, CA) controlled with SoftMax software.


314


For the CAAT ELISA, 100 μL of sample was mixed with 1(%L of a predetermined dilution of CAHeT-AP in a separate uncoated well. Fifty microliter aliquots were applied to replicate wells of the antibody-coated plate and incubated 30 min; the plate was then washed. Enzyme substrate was added and the plate OD measurements were made at 60 min. ELISA Characterization. ELISAs were characterized for cross reactivity toward various selected j-triazines. Eleven concentrations of each compound plus a zero dose control were assayed in replicate. Cross reactivites were expressed as IC» values (concentration of analyte which produces a 50% decrease in the maximum normalized response) and were interpreted relative toward atrazine for the striazine herbicide ELISAs or CAAT for the CAAT ELISA according to the formula: % Reactivity = (IC atrazine or CAAT / IC analog) * 100 50

(1)

50

Reactivity coefficients for pertinent analytes (IC50 atrazine or CAAT / ICso analog) were used for calculating the expected summed response of the immunoassays to analyte mixtures (17). Multianalyte ELISA Analysis of Pesticide Waste and Rinsate. The experimental approach was described in Muldoon et al. (17). It is based on the premise that the observed response of antibody binding to ligands present in a sample as measured by immunoassay (eg. ELISA), is a "summed response" to all the reactive ligands. This summed response is modified by each reactive ligand's "reactivity coefficient" toward the antibody. Therefore the observed ELISA response would follow the equation: ELISA Response = A ( X J + B(X ) + C(Xc) + ... + Z(Xz),

(2)

B

where A, B, C, and Ζ are concentrations of the different analytes, and X , X , Xc, and X , are the reactivity coefficients of the analytes A, B, C, and Z, respectively for that particular antibody. The ELISA response is expressed in the units used for the standard curve for one analyte (eg. atrazine, reactivity coefficient = 1.00), therefore, reactivity coefficients for the other components would be relative to the analyte used in the standard curve (see previous section). By using one antibody (one equation) for each cross-reactive analyte in the mixture, it is possible to solve simultaneous equations to derive quantities of each analyte. The results from the analysis of a sample containing three cross-reactive analytes using three different antibody ELISAs were written in equation form as follows: A

B

z

ELISA Response Ab 1 = A(X ) + B(X ) + C(X )

(3)

ELISA Response Ab 2 = A(X ) + B(X ) + C(Xc2)

(4)

ELISA Response Ab 3 = AiX^) + B(X ) + C(Xc3)

(5) ,

A1

A2

B1

C1

B2

B3




where, in equation 3, ELISA Response Ab 1 is the amount determined by ELISA using Ab 1, expressed in units of the standard curve (ie., μΜ atrazine); A, B, and C are the unknown concentrations of the analytes A, B, and C, and X , X , and X d are the known reactivity coefficients for antibody 1 for the analytes A, B, and C. Respective designations are also given to equations 4 and 5. The three equations were solved simultaneously for the unknown concentrations of analytes A, B, and C by matrix inversion. Pesticide waste and rinsate samples were fortified with additional amounts of atrazine, simazine, or cyanazine in order to establish a larger analyte concentration range for analysis. Samples were diluted in acetonitrile for HPLC analysis and further diluted in PBSTA and analyzed by ELISA using antibodies AM7B2.1, AM1B5.1, and SA5A1.1. Concentrations of j-triazine were initially calculated as μΜ atrazine equivalents (based on the standard curve for atrazine) using the two lowest sample dilutions which gave an OD value within the working range of the assay. Individual striazines in each sample were quantified using the analyte reactivity coefficients for each antibody and solving three simultaneous equations (one per antibody) with three unknowns (one per analyte) by matrix inversion. Individual single antibody ELISAs were evaluated by geometric mean regression (23) of the amount found by ELISA on the expected response to total j-triazine determined by HPLC utilizing the individual antibody/analyte reactivity coefficients. Estimation of individual and total s-triazines in the samples were evaluated by geometric mean regression of the amount found by ELISA after solving simultaneous equations, on the amount determined by HPLC. A1

B 1

Ozonation Experiments. Bench scale (250 mL) and pilot scale (208 L) ozonation experiments were conducted on 100 mg/L atrazine solutions of Aatrex Nine-O. Ozone was generated using a PCI Model GL-1B (PCI Ozone Corporation, West Caldwell NJ) with oxygen feed. Ozone was delivered at a rate of 1.0 L/min at 1.0 and 3.0 % w/w (Ο3/Ο2) for the bench scale and pilot scale reactions, respectively. Samples were purged with nitrogen to remove residual ozone prior to analysis and analyzed by HPLC either undiluted or diluted 1:2 with acetonitrile (for atrazine, deethylatrazine, and deisopropylatrazine analysis of early samples). Samples were diluted in PBSTA and analyzed by the ELISAs. For the bench scale reaction, the pH was adjusted to 10.5 with the addition of 1 Ν NaOH and maintained at pH 9.5 to 10.5 throughout the reaction. Ozonation was monitored by HPLC and was carried out until atrazine was converted to CAAT (150 min). The pH was not adjusted for the pilot scale reaction. Ozonation was monitored by HPLC and was continued until all atrazine was converted to CAAT and the acetoamido intermediate CD AT (18.5 hr). The acetoamido product was hydrolyzed to CAAT by fortifying the solution to 2 mM KOH resulting in a pH value of 10.7. After 3 hr, the pH was adjusted to pH 6.6 by fortification to 5 mM KH P0 . 2

4


316


Biodégradation of Ozonated Aatrex. Biodégradation of ozonated Aatrex was conducted on a bench scale (150 mL) to demonstrate the effectiveness of the CAAT ELISA for monitoring this process. Ozonated Aatrex was fortified to 10 mM phosphate buffer (pH = 7.0), 0.1 % w/v Tru-Sweet high fructose corn syrup (American Fructose-Decatur, Decatur, AL), 0.5 mM MgC0 , 50 μΜ CaC0 , 50 μΜ MnS0 , and 5 μΜ FeCl . This was inoculated with 20 mL of a culture of Klebsiella terragena strain DRS-l-S (Klett 660 = 30 units) (15). Samples were removed from the flask and centrifuged to remove cellular material. An aliquot was injected immediatley on HPLC and the remainder frozen. Biodégradation was carried out until CAAT was no longer detected by HPLC (< 50 ppb). Samples were thawed and diluted in PBSTA prior to ELISA analysis. 3

4

3

3

Results Hapten Chemistry. Figure 2 shows the structures of the two target analytes, the haptens used for the generation of analyte-specific antibodies, and the haptens used in the heterologous ELISAs. The 2-chloro positions of atrazine (20) and CAAT (18) were substituted with a thiopropanoic acid bridging group and resulted in structures which were used as immunizing haptens. Heterologous haptens (different than that used for animal immunization) were synthesized for use in the ELISA procedures and were extremely valuable in improving assay sensitivity for both j-triazine herbicide ELISAs (24, 17) and CAAT ELISAs (18). The use of heterologous haptens in ELISAs was previously shown to be important for improving assay sensitivities for other analytes (25, 26). ELISA Development and Characterization. The application of immunoassays for monitoring treatment required characterization of the various assays using all of the pertinent structures which may be present during the course of the process. In addition to these structures, other j-triazines were tested in order to fully characterize and compare antibody crossreactivities. Table I shows the reactivity profiles for the monoclonal antibodies AM7B2, AM1B5, and SA5A1, and the polyclonal antibodies PAb 1 and PAb 5. Although j-triazine herbicide-sensitive assays showed varied reactivities toward the parent structures, analyte recognition was greatest for propazine in all cases. The addition of oxygen to the alkyl side chains greatly diminished monoclonal antibody recognition and this was an important consideration for the application of these assays in monitoring j-triazine herbicide ozonation. Loss of the alkylamino groups resulted in diminished recognition for the j-triazine herbicide-sensitive assays. Monoclonal antibody AM7B2 showed the broadest range of sensitivity for the parent herbicides atrazine, simazine, and cyanazine. This assay was chosen for use in disposal monitoring since it was a broad-spectrum j-triazine herbicide assay and demonstrated insensitivity toward the reaction products. The ELISAs using polyclonal antibodies PAb 1 and PAb 5 showed high selectivity toward the environmental degradation and ozonation product CAAT. These antibodies did not recognize parent herbicides, other dialkylamino side chain substituted j-triazines, nor the monodealkylated product deethylatrazine (OAT). The antibodies did recognize the monodealkylated product




deisopropylatrazine (CEAT), however this is a minor intermediate in atrazine ozonation (14). The ELISA using PAb 1 was chosen over PAb 5 for use in disposal monitoring since it was less sensitive toward the ozonation intermediate 2-chloro-4-acetoamido-6-amino-j-triazine (CD AT). Multianalyte ELISA Analysis of Pesticide Waste and Rinsate. The monoclonal antibodies AM7B2, AM1B5, and SA5A1 possessed different reactivity profiles for the parent j-triazine herbicides atrazine, simazine, and cyanazine (Table I). These differences were utilized in a multiple regression method for estimating individual 5-triazines in mixtures. Table II shows the geometric mean regression data from the analysis of actual pesticide waste mixtures. The most accurate individual assay employed antibody AM1B5, which was the most selective antibody for atrazine. The less selective assays (AM7B2 and SA5A1) gave lower slopes of regression (underestimates of analytes by ELISA) which may have been caused by a potent interfering material present in the waste samples. The samples were analyzed without sample clean-up. Samples required a minimum 100-fold dilution for ELISA analysis (versus 2-fold for HPLC) which may have magnified any subsampling error initially present. The variability of the ELISA data was greatest at high analyte concentrations (17). Atrazine estimation was the most accurate and precise among the analytes studied and probably resulted from the high selectivity of the ELISA which utilized antibody AM1B5. The estimations of the other j-triazines were dependant on less selective assays complicates these estimates. Total j-triazine estimation was highly correlated to HPLC data and should be valuable for estimating theoretical yields in disposal processes. Karu et al. (27) presented a summary of the various statistical methods available for analyzing multianalyte ELISA data. The multiple regression method was used because of its simplicity and ability (theoretically) to be applied to analyte mixtures. In a related study, the effects of selected agricultural waste components on the ELISA which utilized monoclonal antibody AM7B2 were examined. This ELISA was found to be particulary sensitive to magnesium, ionic surfactants, and some commercial formulated surfactants (28). A simple solid-phase extraction technique (C ) was used which improved assay precision but had litde effect on improving the slopes for regression (approx. 0.90) of the amount detected by ELISA on the amount added. 18

Atrazine Ozonation Monitoring. The ELISAs used for ozonation monitoring utilized either monoclonal antibody AM7B2 or polyclonal antibody PAb 1. Results from these assays were compared to results obtained from a multiresidue HPLC method. Samples were taken from either a bench scale ozonation reaction (250 mL) carried out at pH 10 or a pilot scale reaction (208 L) carried out without pH control. Figure 3 shows the reaction product profile determined by HPLC analysis compared to the results from ELISA for the bench scale reaction. For the j-triazine herbicide ELISA, geometric mean regression of the amount found by the ELISA (atrazine equivalents) on the amount found by HPLC had a slope of 1.15 and R = 0.99. For the CAAT ELISA, geometric mean regression



a

Common Name atrazine simazine cyanazine propazine ametryne atratone hydroxyatrazine deethylatrazine deethylsimazine chlorodiamino-j-triazine N-isopropylammeline N-ethylammeline N-ethylammelide ammeline ammelide cyanuric acid melamine cyromazine diamino-5-triazine b

Cook Svstem AM7B2 CIET 100 CEET 34.3 CENT 90.4 CIIT 227.8 SMelET 14.8 OMelET 2.5 OIET 8.8 CIAT 0.5 CEAT 0.5 CAAT 0.0 OIAT 0.1 OEAT 0.2 OOET 0.1 OAAT n.d. OOAT n.d. OOOT n.d. AAAT n.d. CyPrAAT n.d. HAAT n.d. CDDT 0.1 CDIT 8.9 CDET 10.6 CDAT 0.1 SPrlET 273.6 CIPiT 1.1 CEPrT 0.9 SPrAAT n.d. SBeAAT n.d. CAHeT n.d. SAAT n.d.

AM1BS 100 7.9 0.3 481.8 70.6 6.6 1.4 10.1 0.6 0.0 0.2 0.0 0.0 n.d. n.d. n.d. n.d. n.d. n.d. 0.0 26.1 3.4 0.0 46.0 5.4 0.2 n.d. n.d. n.d. n.d. SA5A1 100 86.8 6.2 124.0 0.9 0.4 0.0 13.3 28.2 0.1 0.0 0.0 0.0 n.d. n.d. n.d. n.d. n.d. n.d. 0.1 6.4 6.7 5.7 4.2 1.8 1.5 n.d. n.d. n.d. n.d. c

PAb I 0.2 0.2 0.2 n.d. n.d. n.d. n.d. 2.2 92.8 100

Immunochemical Approach for Pesticide Waste Treatment Monitoring

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