Anal. Chem. 1995, 67, 1831-1837
MS/MS Identification of Transformation Products in Degradative Biofilms John V. Headley,* Keny M. Peru, John R. Lawrence, and Gideon M. Woifaarctt
National Hydrology Research Institute, 1 1 Innovation Boulevard, Saskatoon, Saskatchewan, Canada S7N 3H5
MS/MS was used for the identification of degradation products in b i o h s . The procedure was suitable for the study of exopolymer materials from a flowthrough culture system with a b i o h thickness of 5-40 pm and the detection of analytes at picogram levels. Aliquots (2 pL) of the influent, b i o h , and effluent from a flowthrough culture system were subjected directly to instrumental analysis with no sample extraction, cleanup, or preconcentration steps. AU samples were analyzed within 35 min of sampling. This rapid and sensitive analytical procedure served to minimize possible transformations during storage and analysis. An application of the procedure is described for the identification of selected degradation products of the herbicide 2-[4-(2,4-dichlorophenoxy)phenoxylpropionate(common name diclofop-methyl) within degradative biofilm material. Bacterial biofilms are ubiquitous in nature and industrial settings.' For example, they are common on the surfaces of rivers, lakes, and wetlands, and they accumulate on rocks in aquatic environments. For riverine samples,there is evidence that organic contaminants can be concentrated in the surface film rather than in the underlying body of water.2 However, little is known about the signiiicance of biofilm materials in sorbing organic contaminants and to what extent they contribute to the degradation of contaminants in aquatic environments. What is known is that the biofilm mode of growth often represents the preferred mode for bacteria in aquatic environments.3 It is also established that the most prominent feature of a bacterial biofilm is the large quantity of supporting matrix composed of exopolysaccharide (EPS) and polypeptide polymers. This matrix is produced by the bacteria as part of their adaptation to growth on surfaces or at interfaces and, in general, encases the majority of biofilm bacteria. The functions of this EPS in bacterial growth and survival are extensive, including attachment, microcolony formation, floc formation, protection against heavy metals, protection against predation and environmental fluctuations, increased resistance against antimicrobial agents, and localization of extracellular enzyme^.^-^ This extensive polymer (1) Wolfaardt, G. M.; Lawrence, J. R; Robarts, R D.; Caldwell, S. J.; Caldwell, D. E. Appl. Environ. Microbiol. 1994,60, 434. (2) Carey, J. H.; Fox, M. E.; Brownlee, B. G.; Metcalfe, J. L.; Platford, R F. Can. J Physiol. Pharmacol. 1984,62, 971. (3) Costerton J. W.; Cheng, K-J.; Geesey G. G.; Ladd, T. L.; Nickel, N. C.; Dasgupta, M.; Mame, T.J. Annu. Rev. Microbiol. 1987,41, 435. (4) Dudman, W. F. The role of surface polysaccharides in natural environments. In Sutface carbohydrates of the prokayotic cell; Sutherland, I. W., Ed.; Academic Press: London, 1977; pp 357-414. (5) Tago, Y.; Aida, K. Appl. Environ. Microbiol. 1977,34,308.
0003-2700/95/0367-1831$9.00/0 0 1995 American Chemical Society
network has also been shown to be highly reactive, for example, selectively binding metals from the envir~nment.*~~~'~ It is possible that xenobiotic compounds are accumulated in EPS biofilm materials by the s h e or similar mechanisms. Baughman and Paris," in a review on bioaccumulation of organic contaminants in aquatic systems, pointed out that little effort has been made to elucidate the role of microorganisms in the bioaccumulation of organic contaminants. The role of exopolymers and biofilms has been similarly neglected. This lack of study is due in part to the need for development of analytical techniques suitable for characterization of small sample sizes of biofilm materials of a few micrometers film thickness with analyte concentration(s) at picogram levels. New analytical developments are required to determine whether biofilm materials contain active sites for sorption or binding of organic contaminants. Likewise, sensitive analytical techniques are required to determine whether degradation or transformation products occur within the biofilm materials. In recent years, scanning confocal laser microscopy (SCLM)12 has been used to study biofilms. This nondestructive experimental technique is well suited for the examination of the spatial arrangement of microorganisms within biofilms.' The SCLM technique permits study of the formation and structure of mixed species biofilms which metabolize compounds that are resistant to degradation by a pure culture.' Recent SCLM work has provided evidence for uptake of the herbicide 2-[4(2,4dichlorophenoxy)phenoxy]propionate (common name diclofopmethyl) into b i o f h materials.' However, the identitication of the herbicide and confirmation of possible transformation products within the biofilm material were not determined. The need for identiiication of contaminants in the earlier SCLM work provided the rationale for the development of the procedures described in the present work. In our laboratory, the limited quantities of bacterial biofilms were not readily amenable to GC/ MS trace analysis of the transformation products, employing conventional solvent extraction procedures with derivatization steps. A tandem mass spectrometq (MS/MS) method was therefore developed, with the attractive features of no extraction or derivatization steps and easy application to the limited sample size of the biofilm materials. In this study, a degradative microbial consortium, capable of utilizing the herbicide diclofopmethyl as (6) Costerton, J. W.; Inrin, R T.; Cheng, K J. Annu. Rev. Microbiol. 1981,35, 299. (7) Costerton, J. W. Dev. Ind. Microbiol. 1984,25,363. (8) Decho, A. W. Oceanogr. Mar. Biol. Annu. Rev. 1990,28,73. (9) Nguyen, L. IC; Schiller, N. L. Cuw. Microbiol. 1989,18, 323. (10) Rudd, T.; Stemtt, R. M.; Lester, J. N. Microb. Ecol. 1983,9, 261. (11) Baughman G. L.; Paris, D. F. C d . Reo. Microbiol. 1981,8,205. (12) Caldwell, D. E.; Korber, D. R.; Lawrence, J. R. In Advances in Microbial Ecology; Marshall, K. C . , Ed.; Plenum Press: New York, 1992;Val. 12, p 1.
Analytical Chemistry, Vol. 67, No. 11, June 7, 1995 1831
4-(2,4-dichlorophenoxy)phenetole
B
CI
4-(2,4-dichlorophenoxy)dehydrophenetole
gJI,Q(O?" o,F-H
CH3
Diclofop acid
c,a;Qi;k 0 1 CY
Diclofop-methyl
CI
2,4-dichlorophenol
clm:noH 4-(2,4-dichlorophenoxy) phenol
&I
2-chlorophenol
i,3-dichlorobenzene
Figure 1. Chemical structure of diclofop-methyl and transformation products. Table 1. Summary of Selected Diagnostic Ions, Major Ions, and Their Relatlve Abundances for Diclofop-Methyland Target Transformation Products
analyte diclofopmethyl diclofop
miz
Figure 2. Example of El mass spectrum of one of the authentic standards, diclofop-methyl.
m/P (relative abundance) 344 (12), 342 (53), 340 (75), 327 (lo), 285 (9), 283 (35), 281 (35); 257 (16), 256 (23). 255 (53). 254 (55). 253 (100) . , 327 (251, 330 ( ~ j329 , ( ~ j328 , 326 (70). 283 (18). 281 (22). 257 (30).
4-(2,4-dichlorophenoxy)dehydrophenetoleb 4-(2,4dichlorophenoxy)phenetoleb 4-(2,4dichlorophenoxy)- 258 (25), 256 (70), 254 (loo), 218 (lo), 191 (20), 184 (a), 162 (14), 128 (15), phenol 109 (50), 93 (12), 81 (28), 75 (18), 65 (34). 63 (25). 53 (23) 2,4-dichlorophenol 166 ( i i ) , ' i a ( i j , ' m ' ( i o o ) , 126 (is), 98 (40), 73 (12), 63 (50), 49 (10) 130 (132), 128 (loo), 92 (15), 73 (lo), 2-chlorophenol 65 (17), 64 (53). 63 (25), 39 (17) 150 (18), 148 (85), 146 (loo), 146, 1,3-dichlorobenzene 113 (22), 111 (59), 5 4 (S), 75 (38), 74 (22), 73 (12), 50 (18)
Time (min) 101 I1
0 The diagnostic ions are shown in italic type. Authentic standard not available.
sole carbon source, was isolated from soil to study the role of biofilm communities in determining the fate of this compound. Diclofopmethyl,a chlorinated aromatic compound, and related compounds are used worldwide for weed control in agricultural crop p r o d ~ c t i o n . ~(See ~ J ~chemical structures given in Figure 1.) Specifically, diclofopmethyl is used to control graminaceous annual weeds such as wild oats, green foxtail, and witch grass primarily in wheat, barley, and ~oybeans.~~J4 At application rates ranging from 57 to 132 kg.kn-2, of active ingredient, there is strong adsorption of the herbicide to soil and low potential for ~olatilization.~5J~ Under field conditions, the herbicide ester diclofopmethyl is known to undergo hydrolysis to its correspond(13) Weed Science Society of America, Herbicide Handbook Committee. Herbicide Handbook; The Weed Science Society of America: Champaign. IL, 1989. (14) Ontario Ministry of Agriculture and Food. Guide to Weed Control; Report No. 89-08: Pesticide Section, Hazardous Contaminants Coordination Branch: Legislative Buildings, Toronto, Canada, 1990. (15) Matthiessen, P.;Whale, G. F.; Rycroft, R J.; Sheahan, D. A. Aquat. Toxicot. 1988,13, 61.
1832 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
m/z
Figure 3. Mass spectra of influent. (a) TIC vs time; (b) mass spectrum of composite mixture.
ing acid, diclofop (2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid). Two other metabolites, 4-(2,4-dichlorophenoxy)phenetole and 4-(2,4-dichlorophenoxy)phenol, have been observed at trace levels relative to the concentration of diclofop. Trace residues of unidentified metabolites have also been reported for a variety of soil conditions.17-lg The detection of the metabolites was based (16) Smith, A. E.; Grover, R; Cessna, k J.; Schewchuk, S. R: Hunter, J. H. j . Environ. Qual. 1986,15 (3), 234. (17) Martens R Pestic. Sci. 1978,9, 127. (18) Smith, A. E. j.Agric. Food Chem. 1979,27, 1145.
7 90
d a 51.
n c e
40.
IO
Time (min)
80.
A b 60. U
n SO. d . 40.
184
e
30 20 10
0 m/z
F i g u r e 4. Mass spectra of exopolymer. (a) TIC vs time; (b) mass spectrum of composite mixture.
on relative retention times on silica gel plate^,'^ along with radiochemical, gas chromatographic, and mass spectrometric te~hniques.'~J~ Diclofopmethyl has been detected in shallow groundwater, surface waters, and air samplesFO This apparent recalcitrance has resultpd in the herbicide being listed for postregistration monitoring in Canada. In this work, we describe an application of a MS/MS procedure as a complementary technique to conventional methods which require solvent extractions, preconcentration, or derivatization steps. Advantages of the MS/MS application are illustrated for broad spectrum ~ t u d y ~ofl -the ~ ~limited sample sizes of biofilm materials (5-40 pm film thickness, less than 1 mg wet weight, containing analytes at picogram levels) available during this investigation. For this application, we evaluate some chemical interference effects, with emphasis on the implications for the identification of transformation products within the bioflm materials. Full quantilication of analytes in which matrix effects such as chemisorption are accounted for in a rigorous examination is deferred to future work. EXPERIMENTALSECTION
Materials. Authentic standards were obtained from Hoechst Canada and commercial suppliers for (a) diclofopmethyl, (b) (19) Gaynor, J. D. Can. J. Soil Sci. 1984, 64, 283. (20) Muir, D. C. G.; Grift, N. P. J. Enuiron. Sci. Health 1987, 22, 259. (21) Headley, J. V.; Lawrence, J. R; Zanyk, B. N.; Brooks, P. W. Water Polluf. Res. J. Can. 1994,29 (4), 557. (22) Headley, J. V.; Krause, D.; Swyngedouw, C. Water POW.Res. I. Can.1992, 27 (4), 701. (23) Headley, J. V.: Peru, K M. J Rapid Commun. Mass Spectrom. 1994, 8, 484.
mlz
F i g u r e 5. Mass spectra of effluent. (a) TIC vs time; (b) mass spectrum of composite mixture.
diclofop, (c) 4-(2,4dichlorophenoxy)phenol, (d) 2,4dichlorophenol, (e) 2-chloropheno1, and (0 1,3-dichlorobenzene. These standards were used to obtain library spectra for conknation of the identity of transformation products in the biofilm materials. Where authentic standards were not available, tentative identiiications were performed. The latter include the two transformation products, 4-(2,4dichlorophenoxy)dehydrophenetole and 4-(2,4 dichlorophenoxy)phenetole.17-19 Procedure. The design, construction, and operation of the continuous-flow culture chambers (flow cells) for study of degradative biofilms were reported earlier.* In brief, the flow cells were constructed of poly(methy1 methacrylate) (plexiglass) and created by milling a series of parallel channels into the plexiglass. The channels were 1 mm deep, 3 mm wide, and 42 mm long. A #1 microscope coverslip, mounted over the channels and attached to the plexiglass with silicon adhesive (General Electric RTV), allowed microscopic examination of biofilm development in the flow chambers. Previous studies have demonstrated laminar flow in these chambers." A 6% hypochlorite solution was used to surface sterilize the flow cells. A peristaltic pump (Watson Marlow 2012) was used to maintain flow at 0.2 mL s-1. All incubations were conducted at room temperature (23 f 2 "C). The flow cells were inoculated with a nine member bacterial c o n ~ o r t i u mand ~~~~~ incubated for at least 21 days prior to sampling. Throughout the (24) Korber, D. R; Lawrence, J. R; Lu, Z.; Caldwell, D. E. Biofouling 1990, 2, 235. (25) Wolfaardt, G. M.; Lawrence, J. R; Robarts, R D.; Caldwell, D. E. Can. J. Microbiol. 1994, 40, 331.
Analytical Chemistry, Vol. 67, No. 1 1 , June 1, 1995
1833
22
n
4 340
,,Q
C
e
A
8
146
a
' 6 l6
Time(min) Time(min)
1
3
Figure 6. Mass spectra results obtained for the extracted ions m/z 340, 254, and 146 for samples of (a) influent, (b) biofilm,
effluent, and
(d
