(9) Williams, S. C.; Simpson, H. J.; Olsen, C. Chem. 1978,6, 195-213.
R.; Bopp, R. F. Mar.
(24) Boehm, P. D.; Quinn, J. G. Estuarine Coastal Mar. Sci. 1978, 6. 471-94.
(10) Perkins, R. W.; Nelson, D. L.; Haushild, W. L. Limnol. Oceanogr. 1966 11, 235-48. (11) Pickering, R. J. Geol. Suru. Prof. Pap. (U.S.) 1969,433-H. (12) Edgington, D. N.; Robbins, J. A. Enuiron. Biogeochem., Proc. Int. Svmn.. 2nd. 1975 1976.705-29. (13) Pailo;, S.P.; Dexter, R. N. Enuiron. Sei. Technol. 1979, 13, 65-71 (14) Bipp. R. F. Ph.D. Thesis, Columbia University, New York, NY, 1979. (15) Olsen, C. R. Ph.D. Thesis, Columbia University, New York, NY, 1979. (16) Hetling, L.; Horn, E.; Tofflemire, J. “Summary of Hudson River PCB Results”; New York State Department of Environmental Conservation Technical Paper No. 51,1978. (17) West, R. H.; Hatcher, P. G. Mar. Pollut. Bull. 1980,11, 1269. (18) Dennis, D. S. Conf. Proc.-Natl. Conf. Polychlorinated Biphenyls 1975 1976,183-95. (19) Gross, M. G. Ann. N . Y . Acad. Sei. 1974,2,50, 112-28.
(20) Furr, A. K.; Lawrence, A. W.; Tong, S. S.C.; Grandolfo, M. C.; Hofstader, R. A.; Bache, C. A.; Guteman, W. H.; Lisk, D. J. Enuiron. Sci. Technol. 1976.10., 682-7 _-- .. (21) Gross, M. G. “Waste Disposal”; MESA New York Bight Atlas Monograph 26; New York Sea Grant Institute, 1976. (22) Hammond, D. E. Ph.D. Thesis, Columbia University, New York, ~~
NY, 1975. (23) Choi, P. S. K.; Nack, H.; Flinn, J. E. Bull. Enuiron. Contam. Toxicol. 1974, 21, 12-7.
Receiued for review May 9, 1980. Accepted October 24, 1980. The study discussed here was carried out as part of a New York State Department of Enuironmental Conservation program (contract NYS-C-125638).The study luas established to implement Section 3 of a n agreement between the Department of Enuironmental Conservation and the General Electric Company related to the discharge of PCBs into the Hudson River. Financial support for the work was prouided for ( i npart) by funds resulting from that agreement. Additional financial support was supplied by the National Science Foundation (contract OCE-7909249) and NOAA (contract NA79 RAC00126).
Supplementary Material Available: Table Igiuing PCB, 137Cs,and loss on ignition data on samples from Hudson River cores (10pages) will appear following these pages i n the microfilm edition of this volume of the journal. Photocopies of the supplementary material from this paper or microfiche (105 X 148 mm, 24X reduction, negatiues) may be obtained from Business Operations, Books and Journals Division, American Chemical Society, 1155 16th St., N . W . , Washington, D.C. 20036. Full bibliographic citation (journal, title of article, author) and prepayment, check or money order for $10.00 for photocopy ($11.50 foreign) or $4.00 for microfiche ($5.00foreign), are required.
New Screening Methods for Acidic Toxic Substances Using Negative Chemical Ionization Mass Spectrometry. Tetrachloroterephthalate in Human Urines Yves Tondeur and Ralph C. Dougherty” Department of Chemistry, Florida State University, Tallahassee, Florida 32306
A method has been developed for isolating acidic toxic substances from biological fluids by extraction followed by derivatization and chromatography to rectify the biomolecular matrix that is inevitably coextracted with water-soluble materials. Toxic-substance screening for contamination with polychlorinated organics was accomplished by using negative chemical ionization mass spectrometry. Recoveries exceeded 70%for 2,4-D, picloram, and pentachlorophenol. Quantitation was obtained with electron-capture gas chromatography. By using negative chemical ionization screening, we uncovered the presence of tetrachloroterephthalate, a compound used in herbicide formulations, in human urines. The occurrence of this compound was confirmed by gas chromatography and mass spectrometry.
Introduction
Negative chemical ionization (NCI) screening of minimally cleaned-up extracts of biological substrates such as urine or fat has been showd to be an effective tool for detecting polyhalogenated organics and other toxic substances (1-4). When dealing with aqueous substrates such as urine, these screening techniques have been limited in applicability to compounds with relatively low water solubility. Previous methodology has used either hexane extraction of urine which was treated to hydrolize urinary conjugates ( I ) or steam distillation with continuous liquid-liquid extraction of acid-hydrolyzed urine (5). Both of these procedures give very low recoveries for toxic substances with high water solubility. In the case of hexane extraction, recovery is limited by the unfavorable partition coefficient between the aqueous and hexane phases. Cleanup 216
Environmental Science & Technology
procedures using steam distillation with continuous liquidliquid extraction will only recover substances with significant vapor pressures over water, which does not include highly water-soluble substances. Negative chemical ionization screening following a hexane extraction cleanup has demonstrated low-level contamination of human populations with 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) ( 2 ) . Another related and widely used herbicide, 2,4-dichlorophenoxyaceticacid (2,4-D), was not detected in these screening experiments nor was the widely used herbicide 4-amino-3,5,6-trichloropicolinic acid (picloram). The relative water solubilities of these three compounds at 25 “C are 280, 890, and 430 ppm, respectively (6). The development of an NCI screening procedure that would detect toxic substances with relatively high water solubilities such as 2,4-D and picloram required a change in strategies as derivatization of these compounds is a virtually inevitable part of a cleanup procedure. Residue methods for analysis of picloram and/or 2,4-D in soil and crops (7-13), milk and feces (I4),water ( I 5 ) ,and food samples (16) have been described. Shafik has developed a gas-chromatographic procedure for determination of 2,4-D in urine, which requires formation of an ethyl ester of the herbicide ( 17 ) .A procedure for detection of picloram in urine based on gas-chromatographic analysis of the free acid has also been published (18). Our objective was the development of a screening technique that would be adequate for detection of 2,4-D, picloram, and other water-soluble, acidic toxic substances. Experimental Section
Mass Spectrometry. Negative chemical ionization mass 0013-936X/81/0915-0216$01,00/0 @ 1981 American Chemical Society
spectra were recorded with an AEI-MS902 mass spectrometer equipped with an SRIC chemical ionization source operating in the negative ion mode (-8 kV). Spectra were recorded 5 s after introduction of the sample into the source by direct probe. The reagent gas was isobutane (0.45 torr) and methylene chloride (0.05 torr). The source also contained traces of atmospheric oxygen. The source temperature was maintained at 150 "C, and ionization was initiated by 470-V electrons. The instrument resolving power was maintained at 4000 (10% valley). Gas Chromatography. Gas-chromatographic analyses were conducted with a Varian 1400 gas chromatograph equipped with a 3H electron-capture detector (column, 6 f t X 2 mm, glass packed with 1.5% OV-17 1.95% QF-1 on 80-100 mesh gas chrom Q (Applied Sciences Laboratories Inc.)). Operating temperatures were as follows: injector, 250 "C; column, 200 "C; detector, 275 "C. Nitrogen was used as the carrier gas. Reagents. Benzene, n-hexane, diethyl ether, and ethyl acetate were distilled in glass through a Snyder column. Florisil60/100 mesh was obtained from Applied Science Laboratory Inc. and was Soxhlet extracted with a mixture of nhexane/acetone (1/1) for 4 h and dried at 150 "C for 36 h. This adsorbent was then deactivated by adding 5% (wtlwt) water. Diazomethane was prepared from a micromole-scale methyl-N-nitrosoguanadine diazomethane generator according to the manufacturer's directions (Aldrich Chemical Co.). Sodium chloride and disodium sulfate were Soxhlet extracted for 4 h with a mixture of n- hexane/acetone (1/1)and dried at 150 "C. Phosphoric acid (85%) was used as obtained from Fischer Scientific Co. Chemical standards for pentachlorophenol, 2,4-D, picloram, and dimethyltetrachloroterephthalate were obtained from the EPA Health Effects Research Laboratory, Environmental Toxicology Division, Research Triangle Park, NC 27711. Cleanup and Screening Procedure. Urine samples (5 mL) were acidified with 0.5 mL of 85%phosphoric acid, 1g of sodium chloride was added, and the solution was heated in a water bath at 90 "C for 60 min to hydrolize urinary conjugates. The resulting solution was extracted twice with 3 mL of benzene and once with 1 mL of ethyl acetate. The combined extracts were concentrated under a gentle stream of dry nitrogen to a volume of less than 100 pL. The concentrate was methylated with 1 mL of freshly prepared etheral diazomethane, and the extract was once again concentrated to a volume of less than 100 pL. The resulting concentrate was taken up in 0.5 mL of 20% benzene in n-hexane and was quantitatively transferred to a micro deactivated Florisil column (200 mg of deactivated Florisil in a Pasteur pipet topped with 1 cm of sodium sulfate). The column was prewet with 20 mL of nhexane. Pentachloroanisole and 2,4-D methyl ester were eluted with 15mL of hexane/benzene (l/l). Picloram occurred in a fraction that was eluted with 20 mL of 2% diethyl ether in benzene. Both fractions were separately collected into Kurdena-Danish glassware and concentrated through a Snyder column to a voluine of -2 mL. Final concentration to a volume of 200 pL was accomplished with a dry-nitrogen stream. An aliquot (5 pL) of this concentrate was examined by electron-capture gas chromatography. The remainder of the extract was transferred in aliquots to a quartz probe tip and evaporated by a stream of dry nitrogen in preparation for NCI screening. Procedural blanks were ubtained by carrying triple-distilled water through the entire procedure. Recoveries were checked by comparing results obtained for control urine samples with and without the addition of' staiidard compounds. The recoveries for pentachlolophenol ranged from 76 to 80% a t 10 ppb. 2,4-D recoveries ranged from 86 to 107%at 40 ppb. Picloram recoveries ranged from 60 to 63%at 40 ppb. The lower
+
L
COUCH)
d - ~ 1 . 300 290
LUOLHj
310
c,
C,
330
3k0
M/Z
Figure 1. Negative chemical ionization mass spectrum (methylene
chloride) of a typical urinary extract after derivitization and chromatographic separation. recoveries for picloram reflect the ready adherence of this compound to glass surfaces.
Results and Discussion Our interest in screening for the presence of picloram and 2,4-D stems from the fact that these compounds are used to control broad-leaved plants in coniferous forests, and as a result people working in treated forest areas may be occupationally exposed to these compounds We obtained urine samples from 14 different donors who were employed to plant young trees in an area that had been sprayed 3 weeks earlier with Tordon-101 (a mixture of picloram and 2,4 D). Figure 1presents the negative chemical ionization mass spectrum of a typical urinary extract from this group. The mass spectrum of the methyl ester of 2,4-D taken under these conditions was dominated by a single ion, mlz 269, the chloride adduct of the molecule. Under these conditions the mass spectrum of the methyl ester of picloram was dominated by an ion at m/z 217 (100) which contained two chlorines; the spectrum also contained three other ions, m/z 233 (5) containing two chlorines, m/z 253 (15) containing three chlorines, and m/z 289 (21) containing four chlorines. The detection limits for both of these compounds (signal/noise, 51) were less than 2 ppb. Table I summarizes the results of NCI screening for the series of 14 samples. Neither 2,4 D nor picloram appeared in any of the samples at levels above the detection limit. The data in Table I indicate the presence of polychlorophenols in virtually all of the samples. The quantitative levels for pentachlorophenol in these samples as determined by gas chromatography ranged from 7 to 15 ppb. The most probable source of exposure to this compound is the food chain ( I ) . Trichloroanisole appeared in many of the spectra. Trichlorophenol is a known metabolite of pentachlorophenol; it is also a probable metabolite of trichlorophenoxy acid herbicides. The tetrachloro ion which appeared at m/z 330 was entirely unexpected. l'his ion corresponds to the molecule ion of dimethyl 2,3,5,6-tetrachloroterephthalate (Dacthal). The presence of Dacthal in four of these samples was confirmed by comparison with the NCI mass spectrum of the standard cornpourid (trichloro ions a t ndz 253 (5),275 (13), and 311 (loo), and a tetrachloro ion a t m/z 330 (32)). The presence of this compound was also confirmed by gas-chromatographic analysis. Electron-capture gas chromatography was used to quantitatively determine the amouiit of material present. The average amount of Dtlcthal in the four urine sainples was 4.8 ppb. Dacthal has been identified in erivironniental samples (river water, fish, and bottom sediment) in the lowel Hio Grand Valley of Texas (19, 20). Residues of Dacthal have been detected in carrots (21)and soils from treated fields (22).There are two conceivable routes for exposure to Dacthal for the Volume 15, Number 2, February 1981 217
Table 1. NCI Screening Results-Urines m/r
chlorlne abundance In Ion Cln
192 Clp 197 C120rC13
from Occupationally Exposed Forest Workers blanks
1
2
x
x
3
4
5
6
7
8
9
10
11
13
14
1 7
x
x
x
x
x
x
x
x
x
x
x
X
X
210 Cl3
x
x
x
212 Cl3 216 Cla 223 C14
x
x x
x x
x x
x
x
X
X
x
x
229 C14
x
x
x
x
x
x
X
X
x
x
244 Clr
x
x
X
X
X
X
231 Cl3 242 Clp
X X
x
x
x
X
? CI, / 0 / O P h
X
x
x
x
x
X
x
x
250
x X
cI4
x
x
x
x
x
x
X
x
x
x
x
263 CIS
x
x
x
x
x
x
X
x
x
x
x
278
x
x
x
x
x
x
X
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
259
attrlbutlons
x
c15
292 C14 294 C13 295 Cl5
X
311 CIS
x
x
x
x
x
x
317
cI4
330
cI4
x
x
OCH,
a
I
X
x
x
x
x
x
x
6OOMe
a
a
COOM~ J 345 386 C15 427 CIS a
X X X
X X
X
X
This blank shows a very low-intensity ion compared to samples.
individuals in this study. Contamination of the food chain is one of these candidates. I t is also possible that Dacthal was used in the spray formulation for the forest area in question. The lack of appearance of 2,4-D or picloram in the urines from individuals working on forest land that had been treated 3 weeks previously with Tordon-101 is not very surprising. Both of these compounds are known to be excreted with high efficiency and to have relatively short retention half-lives (18, 23). I t is also true that both compounds are effectively bound by soil and other surfaces so that contact exposure would not be expected to be high 3 weeks after the application. Conclusion
We have developed a procedure for screening urine samples for contamination with water-soluble acidic toxic substances 218
Environmental Science & Technology
based on negative chemical ionization screening. The success of the procedure is indicated by the fact that we were able to uncover the presence of an unsuspected component, namely, Dacthal, in several of the urines screened. Acknowledgment
We are indebted to D. W. Kuehl and S.-Y. Tang for valuable discussions and technical assistance. Literature Cited (1) Dougherty, R. C.; Piotrowska, K. Proc. Natl. Acad. Sci. U.S.A.
1976,73, 1777. (2) Dougherty, R. C.; Piotrowska, K. J. Assoc. Off. Anal. Chem. 1976, 59, 1023. (3) Busch, K.; Bursey, M.M.; Hass, J. R.; Sovocool, G. W. A p p l . Spectrosc. 1978,32, 388.
(4) Dougherty, R. C.; Whitaker, M. J.; Smith, L.; Stalling, D. L.; Kuehl, D. W. Enuiron. Health Perspect., in press. (5) Kuehl, D. W.; Whitaker, M. J.; Dougherty, R. C. Anal. Chem. 1980,52, 935. (6) Kenaga; E. E. “Environmental Dynamics of Pesticides”;Haque, R., Freed, V. H., Eds.; Plenum: New York, 1978; p 261. (7) Cheng, H.H. Bull. Enuiron. Contam. Toxicol. 1971,6, 28. (8) Kahn, S. U.J.Assoc. Off. Anal. Chem. 1975,58, 1027. (9) Leavey, J. S.; Taylor, T. Analyst (London) 1967,92, 371. (IO) Bjerke, E. L.; Kutschinski, A. H.; Radsey, J. C. J . Agric. Food Chem. 1967,15, 469. (11) Saha, J. G.; Gadallah, L. A. J. Assoc. Off. Anal. Chem. 1967,50, 537. (12) Clark, D. E.; Young, J. E.; Younger, R. L.; Hunt, L. M.; McLaran, J. K. J. Agric. Food Chem. 1964,12, 43. (13) Lisk, D. J.; Gutenmann, W. H.; Bache, C. A.; Warner, R. G.; Wagner, D. G. J . Dairy Sci. 1963,46, 1435. (14) Moseman, R. F.; Aue, W. A. J. Chromatogr. 1970,53, 367. (15) Devine, J. M.; Zweig, G. J.Assoc. Off. Anal. Chem. 1969,52, 187.
(16) Yip, G. J. Assoc. Off. Anal. Chem. 1971,54, 966. (17) Shafik, R. T.; Sullivan, H. L.; Enos, H. F. J . Enuiron. Anal. Chem. 1971,1, 23. (18) Fisher, D. E.; St. John, L. E., Jr.; Gutenmann, W. H.; Wagner, D. G.; Lisk, D. J. J . Dairy Sci. 1965,48, 1711. (19) Miller. F. M.: Gomes. E. D. Pestic. Monit. J. 1974.8. 53. (20) Tessari, J. D.’; Spencer, D. L. J. Assac. Off. Anal. Chem. 1971, 54, 1376. (21) Gilbert, M.; Lisk, D. J. Bull. Enuiron. Contam. Toxicol. 1978, 20, 180. (22) Wiersma, G. B.; Mitchell, W. G.; Stanfor, C. L. Pestic. Monit. J . 1972,5, 345. (23) Park, J.; Darrien, I.; Prescott,L. F. Proc. Eur. SOC.Toxicol. 1977, 18, 154.
Received for review July 7, 1980. Accepted October 14, 1980. This work was supported by a grant from the National Institute of Environmental Health Sciences and the Northwest Forest Workers Association.
Comparison of Three Methods for Measuring Suspended-Particulate Concentrations Nicholas P. Kolakt and Joseph R. Visalli” New York State Department of Environmental Conservation, Division of Air, 50 Wolf Road, Albany, New York 12233
A five-site air-sampling study was conducted in the Niagara Frontier Air Quality Control Region of western New York to measure suspended-particulate concentrations by using three different methods. One dichotomous sampler equipped with Teflon filters and two high-volume air samplers employing glass-fiber and Whatman 41 filters were collocated a t each of four urban and one rural background site. Welldefined linear relationships were found to exist between the three measurement methods. Analyses of the data revealed that‘the relationships are dependent not on the site itself but upon particulate loading conditions.
Introduction The standard method for measuring total suspended particulate (TSP) concentrations employs high-volume air samplers (hi-vols) with glass-fiber filter media. The principal disadvantages with this method are an inability to consider particle size characteristics and a difficulty in chemically analyzing filters for certain elements and compounds. These and other problems have long been recognized, and under the Clean Air Act ( I ) , as amended in 1977, the USEPA was required to reevaluate measurement methodologies along with air standards and to recommend appropriate changes. Particles with small aerodynamic diameters are of primary concern to public health. Miller et al. (2) recommended that research be conducted on the health effects of inhalable particulate matter, defined as airborne particles of 115-pm aerodynamic equivalent diameter. They note that particles in this size range deposit in various areas of the respiratory system during mouth breathing. They also recommend the use of air-sampling devices capable of identifying particles of 12.5-pm diameter, since these can easily penetrate into the gas-exchange region of the lungs. By design, the standard high-volume air sampler collects particles up to -100 hm but is not capable of separating them into size ranges of interest. + Currently with the Division of Solid Waste.
In contrast, dichotomous samplers are very inefficient collectors of particles >15 pm and are capable of distinguishing between fine and coarse fractions of the collected particulates. Consequently, these instruments are being used in an effort to gain more information about inhalable particulates. Effective control of suspended particulates depends in part on an ability to identify their chemical composition. This identification is not easily accomplished when using glass-fiber filter media. The silicon component, important to the identification of nonanthropogenic sources, cannot even be considered; and the presence of variable amounts of metallic impurities in the glass fiber ( 3 )poses a potentially severe interference problem. To avoid these problems, other types of filter material such as cellulose have been used with hi-vols (4).
Despite these problems and the need to consider new and more appropriate suspended-particulate measures, use of the traditional hi-vol will probably continue, to provide data continuity for this widely recognized and long-used method. Consequently, there is a need to relate new measurement methods to this standard. Pace and Meyer ( 5 ) ,using data collected at several cities, concluded that the ratio of mean inhalable particulate concentrations (IP) from dichotomous samplers with an upper particle size cutoff of 15-20 pm to mean TSP concentrations from standard hi-vols was in the 0.5-0.7 range. Spengler et al. (6) computed correlation coefficients between T S P and I P ranging from 0.75 to 0.92 and mean IP/TSP ratios (in general, the mean ratio will be greater than or equal to the ratio of means, e.g., IP/TSP L IPD’SP) ranging from 0.59 to 0.66 based on data from four urban areas of differing size. Data compiled by Record et al. (7) a t several sites in Massachusetts show mean I P to mean TSP ratios in the 0.68-0.83 range. However, the dichotomous samplers used in their study did not have a sharp cutoff, and they estimated that -20% of particles having a 30-pm diameter were collected during the field test period, leading to higher ratios of means. Several studies have been conducted by using Whatman 41 cellulose filters in hi-vols. Neustadter et al. (8) have indicated
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Volume 15, Number 2, February 1981
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