(7) Weiss, R. E.; Waggoner, A. P.; Charlson, R. J.; Ahlquist, N. C. Science 1977,195, 979. ( 8 ) Pierson, W. R.; Brachaczek, W. W.; Truex, T. J.; Butler, J. W.; Korniski, T. J. Ann. N. Y. Acad. Sci. 1980,338, 145. (9) Cronn, D.; Harsch, D. Washington State University, unpublished data. (10) Burch, D. E.; Gates, F. J.; Pembrook, J. D. 1976, “Ambient CO Monitor”, Final Report, prepared under EPA Contract No. 6802-2219, Ford Aerospace and Communications Corp., Newport Beach, CA 92663. (11) Chaney, L. W.; McClenny, W. A. Enuiron. Sci. Technol. 1977, 11, 1186. (12) ‘Stevens, R. K.; O’Keeffe, A. E.; Ortman, G. C. Enuiron. Sci. Technol. 1969,3, 652. (13) Rehme, K. A.; Martin, B. E.; Hodgeson, J. A. Washington, D.C., March 1974, U S . Environmental Protection Agency Report EPA-R2-73-246. (14) Seila, R. Washington, D.C., Feb 1979, U.S. Environmental Protection Agency Report EPA-600/3-79-010. (15) Loo, B. W.; Jaklevic, J. M.; Goulding, F. S. In “Fine Particles”; Liu, B. Y. H., Ed.; Academic Press: New York, 1976; p p 312-50. (16) Dzubay, T. G.; Stevens, R. K.; Peterson, C. M. In “X-Ray Fluorescence Analysis of Environmental Samples”; Dzubay, T. G., Ed.; Ann Arbor Science: Ann Arbor, MI, 1977; pp 95-105. (17) Dzubay, T. G.; Snyder, G. K.; Reutter, D. J.; Stevens, R. K. Atmos. Enuiron. 1979,13, 1209. (18) Goulding, F. S.; Jaklevic, J. M.; Loo, B. W. Washington, D.C., July 1978, U.S. Environmental Protection Agency Report EPA600/4-78-034. (19) Jaklevic, J. M.; Landis, D. A.; Goulding, F. S. In “Advances in X-Ray Analysis”; Gould, R. W., Barrett, C. S.,Newkirk, J. B., Rudd, C. O., Eds.; Kendall Hunt: Dubuque, IA, 1976; Vol. 19, p p 25365. (20) Stevens, R. K.; Dzubay, T. G.; Russwurm, G.; Rickel, D. Atmos. Enuiron. 1978,12, 55. (21) Dzubay, T. G.; Rickel, D. G. In “Electron Microscopy and X-Ray
Applications”; Ann Arbor Science: Ann Arbor, MI, 1978; pp 3-20. (22) Brosset, C.; Ferm, M. Atmos. Enuiron. 1978,12, 909. (23) Sawicki, E.; Mulik, J. D.: Witkenstein, E. “Ion ChromatomaDhic Analysis of Environmental Pollkants”; Ann Arbor Science: Ann Arbor, MI, 1978. (24) Johnson, R. L.: Huntzicker, J. J. Berkelev. CA, 1979, In Lawrence Berkeley Laboratory Report No. LBLI9037; CONF-7803101, uc-11,pp 10-12. (25) Shaw. R. W.: Dzubav. T. G.: Stevens. R. K. Washineton. D.C.. March 1979, in U S . Environmental Protection AgeGy Report EPA-600/2-79-051. (26) Tesch, J. W. Ph.D. Thesis, University of Colorado, Boulder, CO, 1977
(27jArnts, R. R.; Meeks, S. A. Washington, D.C., J a n 1980, U S . Environmental Protection Agency Report EPA-600/3-80-023, (28) Stevens, R. K.; Dzubay, T. G.; Russwurm, G.; Tew, E. “Abstracts of Papers”, Division of Environmental Chemistry, 176th National Meeting of the American Chemical Society, Miami Beach, FL, Sept 1978; pp 636-7. (29) Pierson, W. R.; Brachaczek, W.; Korniski, T.; Truex, T.; Butler, J. J . Air Pollut. Control Assoc. 1980,30, 30-4. (30) Parkhurst, W. Muscle Shoals, AL, June 1979, Tennessee Valley Authority Report No. IAQ-79-9. (31) Husar. R. B.: Patterson. D. E.: Husar. J. D.: Gillani. N. V. Atmos. Enuiron. 1978,12, 549. (32) Stephens, N. T.; Rosenauest, J . M.; Lubkert, B., Dresented a t the 72nd Annual Meeting oP the Air Pollution ControfAssociation, Cincinnati, OH, paper no. 79-527, June 1979. (33) Charlson, R. J.; Vanderpohl, A. H.; Covert, D. S.; Waggoner, A. P.; Ahlquist, N. C. Atmos. Enuiron. 1974,8, 1257. (34) Waggoner, A. P.; Weiss, R. E. Atmos. Enuiron., 1980,14, 623. (35) Tang, I. N. In “Generation of Aerosols and Facilities for Exposure Experiment”; Willeke, K., Ed.; Ann Arbor Science: Ann Arbor, MI, 1980; p 153. (36) Husar, R. B.; Patterson, D. E. Ann. N. Y . Acad. Sci. 1980,338, 399.
Characterization of Delayed Neurotoxic Triaryl Phosphates by Analysis of Trifluoroacetylated Phenolic Moieties? Edward A. Sugden” Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Postal Station H, Nepean, Ontario, Canada K2H 8P9
Roy Greenhalgh Chemistry and Biology Research Institute, Agriculture Canada, Ottawa, Ontario, Canada K1A OC6
James R. Pettit Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Postal Station H, Nepean, Ontario, Canada K2H 8P9
Two triaryl phosphate (TAP)samples, which had caused delayed neurotoxic effects in cattle, were characterized by column chromatography, by thin-layer chromatography, by phosphate analysis, by ultraviolet spectroscopy, and after alkaline hydrolysis by gas-liquid chromatography (GLC). The identity of phenols was confirmed by gas-liquid chromatography of both the phenols and their trifluoroacetylated (TFA) derivatives, as well as by mass spectroscopy. The use of a capillary column was found necessary to resolve the TFA derivatives of the various dimethylphenol isomers. The two TAP samples, which contained 4.2 and 13.5%o-cresol, caused severe neurotoxic effects in hens. Introduction Delayed neurotoxic effects in cattle associated with the use of materials contaminated with hydraulic fluids containing triaryl phosphate (TAP), such as those used in Canadian + CBRI Contribution No. 1148. 1498
Environmental Science & Technology
pipeline pumping stations (1, 2), has stimulated interest in the chemical characterization of these compounds. Other poisonings have also been reported as a result of their inadvertent introduction into the environment from discarded drums (3,4) and other sources (5-10). Current production of these compounds is of the order of several thousand tons per annum and may increase if they are used as substitutes for polychlorinated biphenyls (PCBs) (11, 12). The analysis of TAP mixtures is difficult; one approach has been to characterize the many isomers directly by thin-layer chromatography (TLC) (10, 13) or gas-liquid chromatography (GLC) (11, 13, 14). In practice, such preparations show a complex mixture of peaks, which are not clearly resolved ( I I , 13).However, consideration of the theoretically large number of heteroisomers (i.e. compounds with more than one type of aryl moiety), which may be present in industrial preparations, discourages the applications of such procedures. An alternative procedure involves GLC analysis of the phenols formed by alkaline hydrolysis of the TAPS. This method is of special interest in view of the reported relationship of potential delayed neurotoxicity to phenolic moieties, particularly if the TAP contains o-cresol or other o-alkyl-
0013-936X/80/0914-1498$01.00/0 @ 1980 American
Chemical Society
phenols (15-17). Also, the phenols are more readily resolved than the parent compounds. In the past, phenols derived from TAP have been characterized by GLC using the trimethylsilylated (TMS) derivatives (18).In the present work, a method involving hydrolysis of TAP and characterization of o -cresol and other o-alkylphenols content by GLC as trifluoroacetylated (TFA) derivatives was assessed. Two TAP samples (A and B) associated with outbreaks of cattle poisoning as a result of their application as treatments for parasite control were also analyzed. The clinical signs and lesions included delayed posterior paralysis and "dying-back" of axons. A full description including differential diagnosis is given elsewhere
0.9-
0.8-
0.7-
(1,2).
Experimental Section Chemicals. Analytical grade (99+%) phenols, o-, m - ,and p-cresol, 2,4- and 2,5-dimethylphenol, and o-ethylphenol were obtained from Aldrich Chemicals, Milwaukee, WI 53933, and Eastman Organic Chemicals, Rochester, NY 14650. Practical grade tri-o-cresyl phosphate (TOCP) was also obtained from the latter source. Column Chromatography. TOCP (0.10 g) or the sample dissolved in benzene was applied to an 8-g silicic acid/celite column (2/1, w/w) (2.5 X 20 cm). The packing was prewashed in benzene and ether, thoroughly dried in a fume hood, and then activated at 150 "C for 2 h. The column was eluted with hexane (15 mL), 20% ether/benzene (20 mL), 40% ether/ benzene (20 mL), and 50% ether/benzene (20 mL) (19). T o determine the percentage of TAP in the sample, we evaporated the fraction eluting a t the same time as that of TOCP, i.e., the 8 mL following the first 35 mL of eluant, to constant weight in preweighed tubes. Samples of TAP for toxicity studies were processed in the same manner in 1-g amounts in large columns. TLC of TOCP and the samples was carried out on 0.25-mm silica gel plates containing fluorescent indicator and with 12% 2-propanol/hexane as the developing solvent (20). Ultraviolet Spectroscopy and Phosphate Determinations. The ultraviolet (UV) spectra of TOCP and purified TAP were determined in cyclohexane by using a Varian 635 double-beam spectrophotometer. The organic phosphate content of these materials was determined by using a commercial test kit (EM Diagnostics, catalog no. 3829, E M Laboratories, Inc., 500 Executive Blvd, Elmsford, NY 10523). The test is based on the formation of phosphomolybdenum blue after double digestion with potassium chlorate (10). Alkaline Hydrolysis of TOCP and TAPS and Derivatization. TAP samples (10-15 mg) were dissolved in ethanol (1mL), and TOCP (practical grade) was added to some as a standard. The solution was refluxed for 1h with 5% (w/v) alcoholic potassium hydroxide (10). The alcohol was subsequently evaporated on a water bath, and a predetermined amount of 6 N hydrochloric acid added to the mixture to bring it to pH 1. This was then steam distilled, and 75 mL of distillate collected. The cooled distillate was extracted with anhydrous ether (2 X 25 mL), and, after drying, the solvent was removed. The residue, consisting of the hydrolyzed phenols, was reconstituted in 1.0 mL of heptane for derivatization. A 0.25-mL aliquot was transferred to a 15-mL screw-cap tube (polypropylene top) containing 0.1 mL of trifluoroacetic acid anhydride and 0.02 mL of 1%pyridine (freshly distilled or Aldrich Gold Label reagent/heptane). The tube was capped and heated at 75 "C for 45 min. After cooling, the volume of the reaction mixture was made up to 0.5 mL with heptane and washed with 10 mL of 5% aqueous sodium bicarbonate. The samples were analyzed both as underivatized phenols and as the TFA derivatives. The percent phenol composition in reagent-grade phenols was verified by UV spectroscopy in cyclohexane. Response factors for the flame ionization detector
0.6.
0.5 Y
0
z a
I
0.4
8
2 0.3
0.2
0.1
.
1
k.
0
I
220
1
1
1
1
260
I
I
l
l
1
1
1
300
I
340
WAVELENGTHS (nm)
Figure 1. Ultraviolet spectrum of TOCP (-), and column-purified triaryl phosphates from samples A (- - -) and B (. Concentrations in cya).
clohexane were 22.2, 12.5, and 17.1 mg %, respectively.
(FID) were determined by calculating the response relative to that of phenol. GLC Analyses. (a) The underivatized phenols were analyzed on a Varian 2400 GLC (Varian Instrument Division, Palo Alta, CA 94303).A 2.4 m X 4 mm glass column containing 3%OV-1 on Gas Chrom Q with a carrier gas flow of 60 mL/min of nitrogen was used. Injector, oven, and FID detector temperatures were maintained at 215,100, and 250 "C, respectively. Samples were also analyzed by repeating the procedure for alkaline hydrolysis in the absence of potassium hydroxide to check for background phenol levels. (b) The TFA derivatives were analyzed on a Pye GLC Model 104 fitted with a wall-coated OV-17 50 m X 0.25 mm Pye capillary column. The temperature for the column was 68 "C, and for the single FID detector 300 "C. A column flow Volume 14, Number 12, December 1980
1499
2
0
2
4
6
8 10 TIME lrninl
12
14
16
18
Figure 2. GLC of underivatized phenols from alkaline-hydrolyzedTAP samples. The observed peaks correspond to phenol (l),o-cresol (2), mlpcresol (3/4),2,6-dimethylphenol (5), o-ethylphenol (6),2,4- and 2,5-dimethylphenol (7 and 8). Other peaks were not identified but did not correspond to 2,3dimethylphenol. The response factors for these phenols (2-8) were determined relative to phenol to be 1.00, 0.96, 0.90, 0.97, and 1.08, respectively.
of 4 mL/min of nitrogen was employed together with 30 mL/min flow to the detector for make-up purposes. The mass spectra (MS) were determined on a Finnigan GC-MS Model 3100, operating in the electron impact (EI) mode employing 70 eV. Neurotoxicity of TAPs in Hens. Hens of uniform age and strain weighing 1.5-2.0 kg were dosed with 0.1 and 1.0 g/kg of TOCP and 1.0 g/kg of sample A and B TAPs by crop intubation in groups of three. A corn-oil control was also done. Hens were assessed daily for ataxia and paralysis.
Results and Discussion Since samples A and B eluted from a silicic acid/celite column in the 8 mL following the first 35 mL of eluant, similar to TOCP, they were characterized as TAPs. The amounts (by weight) recovered in this fraction were 94.5% TOCP, 56.2% sample A, and 93.5% sample B, which suggested that sample B was mainly TAP whereas sample A likely originated as a TAP-oil blend used as an hydraulic fluid. As further evidence of identity, the column-purified material was subjected to TLC, organic phosphate analysis, and UV analysis. TLC of samples A and B gave spots with an R f of 0.61 as did TOCP, and they were slightly elongated, suggesting heterogenicity of the TAP samples. After organic digestion, the phosphorus contents in TOCP, sample A, and sample B were found to be 7.8% (calcd, 8.4%),6.8’37, and 7.8%, respectively, which corresponds to the TAP structure. UV spectra were determined in cyclohexane, since in this solvent the phenols showed more resolved spectra than in ethanol. TOCP and the samples showed a primary absorption a t 263 nm with a less distinct but definite secondary absorption a t 269 nm (Figure 1).The absence of fine band structure in UV spectra of samples A and B can be attributed to the presence of isomeric phenols. The attempt to characterize the phenols from alkaline hydrolyzed TAP by GLC using the 2.4-m packed column is 1500
Environmental Science & Technology
--,,
2
4
6
8
,
,
10
,
,
,
12
I
14
,
#
16
,
I I I
18
20
22
TIME Irnin)
Figure 3. GLC of trifluoracetylated phenols from alkaline-hydrolyzed TAP samples and a mixture of standard phenols. The observed peaks correspond to phenol (l), o-cresol (2), mcresol (3), pcresol (4), 2,6-dimethylphenol (5), o-ethylphenol (6), 2,5-dimethylphenol (7), 2,4-dimethylphenol (8). Other peaks were not identified but did not
correspond to 2,34imethylphenol. shown in Figure 2. No free phenols were found in either sample A or sample B TAP prior to hydrolysis; however, after hydrolysis they both showed peaks with retention times identical with those of phenol, o-cresol, m- or p-cresol, 2,6-dimethylphenol, o-ethylphenol and 2,4- and 2,5-dimethylphenol standards. In agreement with other attempts to characterize the phenols by GLC, 2,4- and 2,5-dimethylphenol were not resolved. However, the prominence and clear separation of o-cresol in both TAP samples facilitated its identification by comparison of its mass spectrum (with base peak mle 108 and other ions at 107, 67,68,90, 39,41,43,89, and 80) with that of a standard. The 2,4- and 2,5-dimethylphenols were resolved as their TFA derivatives on a 50-m wall-coated open tubular capillary (Figure 3, peaks 7 and 8). As in the case of the underivatized phenols, sample A contained the same compounds as sample B, which all coeluted with those of the standard phenols. Furthermore, the determination of the same phenols as those in Figure 2 of GLC of underivatized phenols is confirmation of their identity. The resolution of these phenols is a significant improvement over previous studies on the GLC of trifluoroacetylated phenols (21) and trimethylsilylated phenols ( 2 2 ) ,or the capillary chromatography of underivatized phenols ( 2 3 ) , and has facilitated quantitative analysis of the phenols as shown in Table I. As can be seen in Table I there is a close resemblance of sample B to that of Moroccan oil, which was responsible for the adulteration of cooking oil and the poisoning of numerous Moroccans in 1959 ( 5 ) . A toxicological assessment of TAP samples A and B and TOCP showed that both produced a severe ataxia and paralysis comparable to, if not quite as severe as, that of TOCP (Table 11). The severe delayed neurotoxicity of TOCP for the adult hen has been well documented (6, 15-17, 19, 20, 24, 25).
~~
~~
Table 1. Analysis of Specific Phenolsa in the ColumnPurified Triaryl Phosphate phenollc content b f C % (WIW) from from Moroccan sample A sample B Oil e
phenols phenol ecresol m-lp-cresol 2,4dimethylphenol 2.5-dimethylphenol 2,4-/2,5dimethylphenols 2.6dimethylphenols o-ethylphenol
4.3 (2) 4.2 f 1.3 (10) 26.5 (2) 9.4 (2) 6.0 (2)
11.9 (2) 13.5 f 3.2 (10) 32.0 (2) 6.9 (2) 7.7 (2)
15
13 28
22 0.8 (2) 0.6 (2)
1.4 (2) 1.o (2)
0.7 1.9
a -Cresol. 2,4-dimethylphenol, and 2,5-dimethylphenol were determined by comparison with respective standards. Other phenols were determined by comparing peak areas with that of @cresol and taking response factors into consideration (see Figure 2). Expressed as the % total phenolic mass calculated as the mass after subtracting the mass contribution of phosphate and adding a 3-mol equivalent mass of hydroxyl groups. E.g.. for 1 g of sample B containing 7.8% phosphorus or 0.24 g of phosphate/g of TAP, total phenolic mass = 1 g of TAP - 0.24 g of phosphate (0.0025 mol) 0.13 g of hydroxyl (3 X 0.0025 mol) = 0.89 g. The number in parentheses indicates the number of replications. dThe mean f standard deviation of the mean based on using TOCP as internal standard. The recovery of Dcresol from TOCP was shown to have a mean value and standard error of the mean of 93.3 f 8.7% ( n = ). e Values taken from ref 5.
+
Table II. Clinical Observations of Hens Treated with TOCP and Samples A and B a groupsb days after treatment
control
TOCP, 0.1 g/kg
4 7 10 15 20
0 0 0 0 0
0 1 1 0 0
30 38
0 0 0 0 0 0
0 0 0 0 0 0
50 62 71 84
TOCP, 1.0 g/kg
sample A, 1.0 g/kg
0 0 1
0 1
4 5(22D) (24~) 4 5 (38D)
3
2 4 4 (320) 3 2 2 2 2
sample 6, 1.0 g/kg
1 2 2 4 5 (27D)
5 (33D) 4
3 2 2 2
a Each group consisted of three hens that were graded according to an ataxic index. 0 = no effect; 1 = stumbling or weakness, slight foot slapping; 2 = abnormal gait and incoordination with heavier slapping of feet: 3 = severely abnormal gait, complete incoordination, down on hocks with wings used for balance; 4 = mostly immobile and able to sit on hocks with difficulty; 5 = moribund, legs completely immobile. Values for members of each group were averaged and rounded off to the nearest integer. Days when hens died (D) are indicated in parentheses.
Since the samples contained less o-cresol than TOCP, particularly in the case of sample A with only 4.2% o-cresol, a less severe delayed neurotoxic effect was expected. Carpenter et al. (26) also found neurotoxic effects disproportionate to low (
