Within the last 40-50 years, the use of biocides (herbicides and pesticides) has increased dramatically. Among these biocides are the chlorinated derivatives of phenoxyacetic acid (PAA). 2,4-Dichlorophenoxyacetic acid (2,4-D), introduced in 1944, was the first phenoxy herbicide and is possibly the most widely used herbicide (W. Evans et al., J. Biochem. 1971, 122, 543-551). Although 2,4-D is not highly toxic, cleanup of inadvertently spilled 2,4-D is still necessary (P. Amy et al., Appl. Environ. Microbiol. 1985, 49, 1237-1245; R. Beadle and A. Smith, Eur. J. Biochem. 1982, 123, 323-332; W. Evans et al., J. Biochem. 1971, 122, 543-551; P. Fisher et al., Bacteriol. 1978, 135, 793-804; L. Geer et al., Appl. Environ. Microbiol. 1992, 58, 1027-1030).
Although large amounts of 2,4-D are used each year, this herbicide usually does not accumulate in soil or water. Of the chlorinated phenoxy herbicides, it is less recalcitrant than more highly chlorinated compounds in the same herbicide class. 2,4-D is degraded naturally in soil under favorable environmental conditions by indigenous, competent microbial communities. There have been isolated from soil and water a number of organisms that are capable of degrading chlorinated phenoxy herbicides, such as 2,4-D, including a number of bacterial strains in multiple genera (L. Kozyreva et al., Mikrobiologiya. 1992, 62, 110-119; G. Chaudhry and G. Huang, J. Bacteriol. 1988, 170, 3897-3902; P. Amy et al., App. Environ. Microbiol. 1985, 49, 1237-1245; R. Don and J. Pemberton, J. Bacteriol. 1981, 145, 681-686; P. Fisher et al., J. Bacteriol. 1978, 135, 798-804; J. Tiedje et al., J. Agr. Food Chem. 1969, 17, 1021-1026; see also, Reineke and Knackmuss, Ann. Rev. Microbiol. 1988, 42, 263-287) and fungi (P. Donnelly et al., App. Environ. Microbiol. 1993, 59, 2642-2647
The best understood biochemical pathway for the microbial degradation of 2,4-D is that of Alcaligenes eutrophus JMP134 (R. Don and J. Pemberton, J. Bacteriol. 1981, 145, 681-686; R. Don et al., J. Bacteriol. 1985, 161, 85-90; R. Don and J. Pemberton, J. Bacteriol. 1985, 161, 466-468). In this pathway, 2,4-D is first converted by an .alpha.-ketoglutarate-dependent dioxygenase (the product of the tfdA gene) to 2,4-dichlorophenol and then by a DCP hydroxylase (the product of the tfdB gene) to 3,5-dichlorocathechol (DCCAT). After several additional enzymatic steps, chloromaleylacetic acid is finally produced (R. Don et al., J. Bacteriol. 1985, 161, 85-90; B. Kaphmmer and R. Olsen, J. Bacteriol. 1990, 172, 5856-5862). The genes encoding the enzyme responsible for the catabolism of 2,4-D by A. eutrophus, tfdA, tfdB and tfdCDEF, have been located on plasmid pJP4 (B. Kaphmmer and R. Olsen, J. Bacteriol. 1990, 172, 5856-5862; B. Kaphmmer et al., J. Bacteriol. 1990, 172, 2280-2286; E. Perkins and P. Lurquin, J. Bacteriol. 1988, 170, 5669-5672; W. Streber et al., J. Bacteriol. 1987, 169, 2950-2955). The expression of tfdB is regulated by the product of gene tfdS using 2,4-D and DCP as effectors (B. Kaphmmer and R. Olsen, J. Bacteriol. 1990, 172, 5856-5862). Since both 2,4-D and DCP can induce the expression of tfdB, DCP is further transformed by microorganisms bearing pJP4 or its derivatives, pRO101 or pRO103 (A. Harker et al., J. Bacteriol. 1989, 171, 314-320). No DCP can be detected by the color reaction in the culture media of these microorganisms when 2,4-D is supplied.
Analytical support for 2,4-D cleanup techniques usually involves gas-chromatography or high pressure liquid chromatography (HPLC) (see, e.g., K. Short et al., App. Environ. Microbiol. 1991, 57, 412-418) or immunoassays (e.g., the enzyme-linked immunosorbent assay [ELISA], 2,4-D RaPID Assay.RTM., Ohmicron, Newtown, Pa.). Even the presently available immunoassays are relatively expensive. Therefore, it is important to develop quick, inexpensive, and easy-to-use 2,4-D detection methods for use in the field, particularly methods which can be employed by non-specialists. It would be particularly advantageous to develop an assay method which could be applied beyond 2,4-D to other related chemical compounds, particularly other phenoxy biocides, many of which are less easily degraded by naturally occurring organisms.