Document qmNDo9Jry3ED8pZkyggoO09k5

Wi'OB.. B MAY 7 si I i' l.NJA Ab 1 . MARINE NATURAL PRODUCTS: INTERFERENCE IN PESTICIDE RESIDUE ANALYSIS t HONS 045227 FENICAL - 2 ABSTRACT! . . , Naturally-occurring halogenated compounds produced by marine organisms are shown to interfere with pesticide and PCD residue analysis* Using the accepted gas chromatographic methods for chlorinated hydrocarbon analyses, extracts from a variety of marine organisms show natural halogenated com pounds with retention times easily confused with DDT, its inotabolites and the PCD'8. . . .1 HONS 045228 Synthetic chlorinated hydrocarbonn such as DDT and the PCD's are recognized as accumulating pollutants in sea water (1), an well as in marine-related organisms, plankton (1), inverte brates (2), fish (3), birds (4) and mammals (5). The quantitative estimations of these chlorinated pollutants rely almost exclusively on the use of gas-liquid chromatography, coupled with electron- capture detection (ECGC). The electron-capture detector provides very high sensitivity toward orgnno-halogon compounds, as well as selectivity in complex mixtures. It has therefore permitted measurements of chlorinated pollutants to be made at very low levels in relatively unpurified samples. While sensitive and selective detection in an obvious benefit in environmental pollution studies, 1,'CCC suffers from an obvious problem in that it provides little qualitative information concerning the halogen- containing substances being measured (6). With an understanding Of these drawbacks, numerous analytical modifications have been described to improve the specificity of chlorinated hydrocarbon analysis (7) . liven though cample contamination during chlorinated hydrocarbon analysis with natural substances has been discussed by many investigators, the origins of these naturally-halogcnated compounds and their chromatographic behaviors are not well understood. - In recent years, many halogen-containing natural products have been isolated and described from marine organisms (8). These chlorine-, bromine-, and iodine-containing compounds are biosynthotically produced from seawater components by many red ocawocds, sponges, molluscs and marine bacteria. Hundreds of MONS 045229 natural halogen-containing compounds of diverse types are recog nized, with seme being structurally similar to synthetic pesticide molecules. 'Hie unusually high concentrations of these natural compounds often reaches 1-5% of the dry weight of the organisms, which indicates the prominence of halogenation processes in marine organisms. In an attempt to assess the thermal stability and chromato graphic behavior of the natural halogens, and, hence, their potential to interfere in PCI3 and DDT analysis, wo made hexane extracts of six locally available organisms already known to contain natural halogens. The extracts (9), when subjected to 15CGC analysis under standardized conditions (10), showed peaks with retention times easily confused with those of DDD, DDE, DDT and two PCB mixtures (Figure 1). Four red seaweeds (Laurencin paclflca (11), L. subopposlta (12), Chondria callfornica (13) and Plocnmium cartilagineum (14)), an abundant local sponge (Vcrongla sp. (15)) and one bacterium (Chromobacterium sp. (16)) 'wore selected, since each, with the exception of Chondria. is known to cynthcsizo bromine- and chlorine-containing compounds. In each case (see respective references) the major halogen-containing compounds have been isolated and structurally described. All six extracts showed compounds with the same detectability as pesti cides which fall within the retention time limits associated with common PCD's (Arochlor 1254 and Arochlor 1242). Laurencin and Chromobactoriurn contain substances that could easily be confused with DDT or its metabolites. 0^5230 HONS FUHICAL. Sirica tlic marine algno .ire potentially the largest source for naturally-occurring halogenatcd compounds (17), wo studied Uio chemical composition of an isolated tide pool containing largo amounts of the red seaweeds Plocamlum and Corailing, inter alia, as a function of time. Twenty-liter samples were collected every 1.5 hours and immediately extracted with one liter of purified hexane, and four raicrolitcr aliquots of those extracts were systematically compared via ECGC (Figure 2). Extreme care was taken to oxcludo contaminating material from nil glassware by baking, followed by rinsing with nanogrado hexane. After three hours, significant quantities of electron-capture detectable compounds wore found to have accumulated in these samples. Coinjcction with known pesticide standards showed the major peaks, to be naturally-occurring. The concentration of the major compound after three hours (Figure 2, trace C) is estimated at .0005 ppm, which in at least 500 times the typical content of DDT and its metabolites in seawater (1). In a recent study, the 'brominecontaining pher.ol, lanosol, originally reported as a natural component of the red seaweed Polysiphonia lanosa, was shown to be a dissolved constituent of seawater (10), These combirjd observa tions provide convincing evidence that natural halogens arc being released into the marine environment. The strong correlation of the retention tines of the natural compounds reported horn with those of DDT, its metabolites, and the I'OJ's shows conclusively that natural substances can be interpreted as pollutants, using solely ECGC methods. The real potential for those compounds to interfere in pollution analysis MQNS 045231 lies in samples tnltcn from the coastal environment and will probably be minimized in open ocean studios'. While there is little doubt that industrial halogens are broad-scale environmental pollutants, accurate analyses of these compounds in marine samples must be obtained by more specific methods. Chromatographic protreatments are successful in removing many contaminants; however, the constituents noted here are of .broad structural typos, from non-polar hydrocarbons to very polar hydroxyl-containing metabolites, and, hence, no single treatment can be doomed entirely successful. Vie second the earlier suggestion that ECGC be abandoned in favor of combined gas chromatography-mass spectrometry methods, which provide both qualitative and quantitative information (19). William l'enical Howard Sleeper Institute of Marine Hesources Scripps Institution of Oceanography La Jolla, California 92037 HONS 04523^ riiMlc/u. - References and Hoto3 1. G. Harvey, V). Stoinhaucr and J. Teal, Science 1BO, 643 (1973); W, Horn, R. Uiscbrough, A. Soutar and D, Young, Science 1B4, . 1197 (1974); L. Fishbein, J. Chroma tog. 98, 177 (1974); R. Riscbrough, V. Vreeland, G, llarvey, H. Miklas and G. Carmignani, null. Environ. Contain. Toxicol. B, 345 (1972); J. Cox, Residue Reviews 44, 23 (1972). . 2. P. Butler, Pentic. Monit. J. 6, 238 (1973). 3. J..MacGregor, Fish. Dull. 73, 275 (1974). .4 II. Fisher, Pacific Science 27, 220 (1973); R. Risebrough, . P. Ricclie, D. Peakall, S. Herman and N. Kirven, Nature 220, 1098 (1968). " 5. X. Addison and P. Brodie, J. Fish. Res. Board Can. ^30, 1733 (1973); R. Delong, W. Gilmartine and G. Simpson, Science 181, 1160 (1973). ' . 6. W. Westlake, in "Advances in Chemistry Series," No. 104 (American Chemical Society, Washington, b. C., 1971) p. 73; W. Aue and S. Kapila, J. Chromatog. Sci. ljl, 255 (1973) . 7. Association of official Analytical Chemists, in "Official Methods of Analysis," 12th edition, W. Ilorwitz, Ed. (Association of Official Analytical Chemists, Washington, 1). C., 1975) Ch. 29, p. 518. .8 J. Siuda and J. DeDcrnardis, Lloydia 36, 107 (1973); J. Faulkner and R. Andersen, in "The Sea," Vol. 5, E. D. Goldberg Ed. (Wiley, Now York, 1974) Ch. 19, p. 679. * HONS 045233 1T.NICAL - B 9. The algae Ploc.im.lum cartilaglncum, I.auronnia paciftea, I,. Bnbopponita and Chondrin californica' were extracted by placing 23 gm of drained plant in 50 ml of nnnograde hexane for 12 hours. The sponge Verongia sp. was extracted in 95% ethanol. The ethanol was evaporated and the extract taken up in hexane. The Chronobactorlum cp. was extracted in ether. ' The ether wa3 evaporated and the extract taken up in hexane. 10. h. Sawyer, J. Assoc. Off. Anal. Chom. J>6, 1015 (1973)} A Hewlett-Packard Model 5750 gaB chromatograph equipped with a pulsing 63ili electron capture detector was run isothermally at 220* C (oven), 255 C (injector) and 295 C (detector). The column was nilatod glass 6' x 1/4" 0.0., packed with 10% DC-200 on Chromosorb Q 00/100 mesh. The gas, 95% argon, 5% methane, . at 40 psi, after passing through dririte and molecular sieve, was divided to provide carrier flow at 50 ml/min through the column and purge flow at 30 ml/min through the detector. 11. J. Sims, W. Fcnical, K. Wing and P.. nadlick, J. Amer. Chom. , Soc. 95, 972 (1973). 12. S. Hall, J. Faulkner, J. Fayos and J. Clardy, J. Amor. Chom. Soc. 95_, 7107 (1973). 13. 1/hilc tlie red alga Chondria callfornica has not be demonstrated to contain halogenated metabolites, tnxonomically it is grouped in the family JthodomelaCQac, which is well known for producing hologenated compounds. ' 14. J. Myndorse and J. Faulkner, Tetrahedron, in press. 15. R. Andersen and J. Faulkner, in "Food and Drugs from the `sea, Proceedings 1972," I,. Worthon, lid. (Marino Technology Society, Washington, I). C., 1973) p. 111. MONS 045234 FENICAL - 9 16. r. Andorocn, M. Wolfe nnd J. Faulkner, Marino Biol. 27, 201 (1974). . ' 17. H. Fcnicnl, J. Phycol., in press. 10, M, Pedersen, P. Saenger and L. Fries, Phytochem. 13, 2273 (1974). . 19. P, Biros, in "Advances in Chemistry Series," No. 104,(American Chemical Society, Washington, D. C., 1971) p. 132; J. Eichclbcrger, . L, Harris nnd L. lluddo. Anal. Chera. 46, 227 (1974). 20. This work is a result of research sponsored by MOAA, Office of Son Crant, Department of Commerce, under Grant *04-3-150-22. Tlie U. S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copy right notation that may appear hereon. MOWS 04523S 'Figure 1. Electron capture gae chromatographic traces of pesticide standards and hexane extracts from marine organismst A) DDT, D) DDE (first peak) and DDD, C) Jtaurencla pacifjca, D) X,aurcncin subopposita, E) Cliromobnctcrium cp, F) Aroclor 1242, G) Aroclor 1254, II) Plocamlura cnrtilaqlncun, I) Vcrongia sp., J) Cliondria californica. See notes 0 and 9 for extraction procedure and chromatographic conditions. HONS 045236 !*f. 'A. a K to h to t MQNS 045237 FL'NICA I, ./ . Figure 2. Electron capture gnu chromatograpliic traces of hexane extract!! of tidepool water. The water samples were obtained from an isolated tidepool at low tide and extracted immediately. At the time of sampling, the air temperature war. 27, the water temperature wan 18.5 and the day was partly cloudy. Neither nanogradc hexane nor 1 liter of nanogrado hexane condensed to 5 ml contained any contaminating material. A) Time zero, 4pl; ) 1.5 hours, 4 pi; C) 3 hours, 4 pi. 045238 HONS / kv- A B ___i--*__i 0 4 I It K to H N C H MQNS 045239 . . ... .. .'Si u. .. v. .1 . V. ... v. ... ...