Determination of Non-extractableTriazine Residues by EnzymeImmunoassay: Investigation ofModel Compounds and SoilFulvic and Humic AcidsAN D R E A D AN KW AR D T * AN DB E R T O L DH O C KDepartm ent of Botany, Technical University of Mu ¨nchen atWeihenstephan, D-85350 Freising, Germ anyR U P E R T S I M O N ,D I E T E R F R E I T AG ,AN T O N I U S KE T T R U PAN DGSFsResearch Center for Environm ent and Health,Institute for Ecological Chem istry, Schulstrasse 10,D-85356 Freising, Germ any2-Chloro-4-arylamino-6-alkyl-1,3,5-triazineswereusedas model compounds for non-extractable (bound)triazine residues. They were tested as targets in acompetitive enzyme immunoassay with polyclonaland monoclonal antibodies originally raised againstatrazine. Cross-reactivities were determined as ameasure for the affinity of antibodies toward theirrespective analytes. 2-Chloro-6-isopropyl deriva-tives were recognized as well as free atrazine (70-148% cross-reactivity, atrazine ) 100%) with theexception of 2-chloro-4-[2′-carboxy-4′,5′-dimethoxy-anilino]-6-isopropylamino-s-triazine, carrying a car-boxyl group in the arylamino side chain (<0.1-9%cross-reactivity). Functional groupvariation of positions2and 6of the triazine ring resulted in cross-reactivitiesless than 15%. Arylamino-s-triazine samples fromphotolytic degradation experiments were analyzed byimmunoassay and HPLC. Similar concentrationswere obtained by both methods. Then the enzymeimmunoassay was applied for the screening of tri-azines bound to natural fulvic and humic acids.Antibodies with different binding properties wereused. It was possible to determine non-extractableatrazine residues in fulvic and humic acids isolatedfrom agricultural soils. Up to 50 ng of bound residuesper g of soil was found. Based on the binding bydifferent antibodies, a proposal for the ligation ofnon-extractable atrazine to soil particles is made.IntroductionThe intensive use ofpesticides over the last three decadeshas led to their occurrence in rainwater, surfacewater,groundwater, and soil (1-4). Biotic and abiotic transfor-mations may take place in these compartments (5).Atrazine, one ofthe most intensively used herbicides in theworld, is subjected to oxidation of the alkyl substituents,oxidative dealkylation, hydroxylation, and, to a minorextent,ring cleavage (6-8). The resulting metabolites may showdifferent mobilities, persistence, and toxicities from theparent compound. In addition to the above-mentioneddegradation processes, up to 50% ofthe originally appliedamount of atrazine and its metabolites have been deter-mined as bound residues in soils (9, 10). A number ofdifferent mechanisms are discussed for the generation ofthese non-extractable residues. For triazines, it was shownthat charge-transfer interactions between electron-poorgroups in humic acids (such as quinones) and electron-rich groups in the atrazine molecule (such as the ringnitrogen atoms) are possible (11-13), while ion-exchangeinteraction and hydrogen bonds are thought to be ofminorimportance (12, 13). Furthermore, the xenobiotic may beentrapped within the organic macromolecule (5, 14). Inthe case of the dealkylated triazine metabolites, covalentbonds may be formed between the xenobiotic and thephenolic structural components of the humic materialleading to aromatic-substituted triazines (15, 16).Pesticides are not only bound to solid soil fractions butalso to dissolved humic substances, which mostly consistof polar low molecular weight water-soluble species (17,18). Therefore, the mobility ofthe xenobiotics in soils maybe enhanced and subsequent leaching promoted, leadingto their appearance in water runoff of agricultural areasand finally in groundwaters and surface waters. Little isknown about the amount of pesticides bound to water-soluble humic materialor the mechanism oftheir transportto the different compartments of the environment. Ad-ditionally, there exists the possibilitythat these compoundsare released, e.g., by microbial activities, and becomebioavailable to plants and animals again (19). The structuralanalytics of pesticide residues bound to dissolved humicand fulvic acids are ofincreasing relevance as the interac-tions of pesticides with humic materials may alter theenvironmentalfate and activityofthe respective xenobioticcompounds. For example, the water solubilityoflipophilicorganic pollutantssaccompanied by their mobilityswasenhanced by dissolved humic and fulvic acids from soiland aquatic origin (20). Furthermore, kinetics ofdegrada-tion reactions maybe changed (21), volatilization decreased(22), and consequently bioavailability and toxicity can bealtered (23).Non-extractable pesticide residues are usually investi-gated by14C-labeled compounds (e.g., refs 10 and 15),pyrolysis, hydrolysis (24), supercritical fluid extraction,HPLC, combustion and high-temperature distillation fol-lowed by MS, GC/MS (9, 25), or heteronuclear NMR (26,27). These methods are expensive and time-consuming.Immunoassays for the determination of pesticides havebecome increasingly popular, as they are easy to carry outand less expensive (e.g., refs 28 and 29). They are basedon the reaction of antibodies (Ab) with their target* Corresponding author fax: +49 8161-714403; e-mail: dankwrdt@pollux.edv.agrar.tu-muenchen.de.Environ. Sci. Technol. 1996, 30, 3493-3500S001 3-936X(96)001 60-5 CCC: $1 2.00© 1 996 American Chemical SocietyVOL. 30, NO. 1 2, 1 996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY93493++++
