1828
Anal. Chem. 1980, 52, 1828-1833
that the compounds identified in this work were obtained in base-neutral extracts of leachate samples. Chlorination of coal-tar leachate is also expected to result in formation of PAHs with polar acidic functional groups. I t is interesting to note that such compounds are reported as products of ozonation of parent PAHs in water (23). Further studies of potable water systems for evidence of coal-tar contamination should benefit by screening for the effects reported in this paper. One possible strategy would be to look for the parent PAHs associated with the azaarenes. These compounds would be expected in systems with low concentrations of residual chlorine. Although chlorination appears t o be beneficial in reducing the concentrations of base-neutral PAHs, it can form oxygen- and halogen-substituted PAHs. Evidence for high concentrations of diverse oxygenated PAHs would suggest a further search for associated halogen-substituted PAHs.
LITERATURE CITED (1) American Water Works Association. "Cement-Mottar Lining for Cast-Iron and Ductile-Iron Pipe and Fittings for Water", C104-74; AWWA: Denver, CO, 1974; pp 1-4. (2) American Water Works Association, "Coal-Tar Protective Coatings and Linings for Steel Water Pipelines-Enamel and Tape-Hot Applied", C203-78; AWWA: Denver, CO, 1978; pp 1-28. (3) American Water Works Association, "Painting and Repainting Steel Tanks, Standpipes, Reservoirs and Elevated Tanks for Water Storage", 0102-64; AWWA: New York. 1964; pp 1-24. (4) Alben, K. Environ. Sci, Technol. 1980, 7 4 , 468-470. (5) Crane, R.; Crathorne, 9.; Fielding, M. I n "Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment"; Afghan, B., Mackey, D., Eds.;
Plenum: New York, 1980; pp 161-172. (6) Basu, D.; Saxena, J. Environ. Sci. Techno/. 1978, 72, 795-798. (7) Sorrell, R.; Reding, R. J . Chromatogr. 1979, 185, 655-670. (8) Benoit. T.; LeBel, G.; Williams, D. Int. J . Environ. Anal. Chem. 1979, 6, 277-287. (9) Schwartz, D.; Saxena, J.; Kopfler, F. Environ. Sci. Technol. 1979, 13, 1138-1 141, (IO) New York State Department of Health, Division of Sanitary Engineering, "Disinfection of Reservoirs, Standpipes and Tanks After Construction or Repairs", Public Water Supply Guide, New York State Department of Health: Albany, NY, 1973. (11) Harrison, R.; Perry, R.; Wellings, R. Environ. Sci. Techno/. 1976, 70, 1151-1 155. (12) Harrison, R.; Perry, R.; Wellings, R. Environ. Sci. Technol. 1976, 70, 1156-1 160. (13) Oyler, A.; Bodenner, D.; Welch, K.; Liukkonen, R.; Carlson, R.; Kopperman, H.; Caple, R. Anal. Chem. 1978, 5 0 , 837-842. (14) Guerin, M. I n "Polycyclic Hydrocarbons and Cancer": Gelboin. H., Ts'o, P., Eds.; Academic: New York, 1978; Vol. I, pp 3-42. (15) Aiken, G.; Thurman, E.; Malcolm. R.; Walton, H. Anal. Chem. 1979, 51, 1799- 1803. (16) Borwitzky, H.; Schomburg, G. J , Chromatogr. 1979, 170, 99-124. (17) Dong, M.; Locke, D.; Hoffman, D. Environ. Sci. Technol. 1977, 1 7 , 61 2-618. (18) Cautreels, W.; VanCauwenberghe, K. Atmos. Environ. 1976, 70, 447-457. (19) Wakeham, S. Environ. Sci. Techno/. 1979, 73. 11 18-1 123. (20) Coleman, W. E.; Melton, R.; Kopfler, F.; Barone, K.; Aurand, T.; Jellison, M. Environ. Sci. Technol. 1980, 1 4 , 576-588. (21) Lee, M.; Vassilaros, D.; White, C.; Novotny, M. Anal. Chem. 1978, 57. 768-774. (22) Kieopfer, R. I n "Identification and Analysis of Organic Pollutants in Water"; Keith, L., Ed.; Ann Arbor Science: Ann Arbor, MI, 1976; pp 399-4 16. (23) Chen, P.; Junk, G.; Svec, H. Environ. Sci. Techno/. 1979, 73, 451-454.
RECED-ED for review March 17, 1980. Accepted June 19, 1980.
Interlaboratory Comparison of Determinations of Trace Level Hydrocarbons in Mussels S. A. Wise," S. N. C h e d e r , F. R. Guenther, H. S. Hertz, L. R. Hilpert, W. E. M a y , and R. M. Parris Organic Analytical Research Division, Center for Analytical Chemistry, National Bureau of Standards, Washington, D.C. 20234
The results of the determination of trace-level hydrocarbons in mussel tissue homogenates from two different sites are compared among eight laboratories. The values for the concentrations of total extractable hydrocarbons, total hydrocarbons in the gas chromatographic elution range, and indlvidual hydrocarbon compounds generally differed by less than a factor of four. Sample inhomogeneity, storage instability over a nine-month period, and analysis uncertainty contributed to an observed intralaboratory precision (1 a) of f40 % The results are discussed with regard to the reliability and comparability of data currently generated in environmental monitoring programs.
.
A number of laboratories, using a variety of different analytical techniques (1-6), are currently involved in the measurement of fossil fuel hydrocarbons in marine biota. The large number of marine biota analyses being performed in monitoring programs, such as the "Mussel Watch'' ( 7 ) which uses mussels as sentinel organisms for indicating levels of pollutants in U S . coastal marine waters, necessitates the existence of a basis for comparing data. Currently there is only limited knowledge of the comparability of hydrocarbon data for marine biota analyses from This article not subject to U S. Copyright
different laboratories. Farrington et al. (8)have intercalihrated gas chromatographic analyses for hydrocarbons in spiked liver lipid extracts and tuna meal samples and found good agreement with the "true" value among three laboratories. When intercalibrating with natural samples (such as tissue or sediment) rather than spiked samples, the "true" value is unknown and interlaboratory precision is paramount. A first attempt a t intercomparison of hydrocarbon analyses on a natural sample was recently reported by our laboratory (9). In this previous study two intertidal Alaskan sediment samples were homogenized and analyzed by eight different laboratories. Sample homogeneity and analysis uncertainty contributed to an observed intralaboratory precision ( l a ) of about 2 5 3 0 % relative standard deviation for both samples. The ranges of values reported in the intercomparison study for such parameters as aliphatic and aromatic/unsaturated hydrocarbons were found to be one to two orders of magnitude, indicating the high interlaboratory variability of hydrocarbon determinations in a natural matrix. In this paper a similar interlaboratory comparison for hydrocarbon determinations in mussel tissue is described. Two mussel samples, one from a pristine environment in Alaska and the other from Santa Barbara, Calif., near some natural oil seeps, were each homogenized and then analyzed for hydrocarbons by eight laboratories. A prescribed protocol was
Published 1980 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
intentionally not used, so that the variability of the data reported would reflect the variability of currently generated environmental measurements. Thus, the results provide an indication of the interlaboratory comparability of current hydrocarbon measurements in mussels from sample preparation through measurement and interpretation. EXPERIMENTAL Samples. The intercalibration materials were mussels (Mytilus edulis and Mytilus califorrzianus) collected from two sites: Simpson Bay, Alaska: 146' 10' W, 60°, 39' N; this site is located near the city of Cordova in the Prince William Sound; and Santa Barbara, California: this site is located by Coal Oil Point, near several natural oil seeps. The mussels were frozen in dry ice immediately after collection and remained frozen until the time of homogenization. At that time the bivalves were partially thawed to facilitate the removal of the tissue from the shell. Approximately 2 kg of mussel tissue from each site were removed from the shells and placed in a 3-L tall-form beaker. The tissue was then homogenized for 1.5 h using an ultrasonic homogenizer (Brinkmann Polytron PT-20ST). Aliquots of the homogenates ( - 50 g) were transferred to 50-mL acid-cleaned glass bottles and sealed with aluminum foil-lined caps. The homogenate samples were refrozen and stored in a freezer at -10 'C. Two bottles each of the Alaskan and Santa Barbara mussel homogenates were subsequently shipped frozen to each participating laboratory. Intercomparison Parameters. The laboratories were requested to provide the following data for each sample: 1. Total hydrocarbons in the gas chromatographic (GC) elution range (approximately C10-C30). 2. Total extractable hydrocarbons. 3. Pristane/phytane ratio and the amount of these present. 4. Per cent water. 5. Identities and amounts of the three most abundant aliphatic and the three most abundant aromatic hydrocarbons. 6. Total polynuclear aromatic hydrocarbon (PAH) concentration (4 rings and larger). 7. Identity and amount of the most abundant PAH (4 rings and larger). 8. All additional single compound identifications and concentrations determined. Analytical Procedure. The National Bureau of Standards (NBS) employed a dynamic headspace sampling technique, high performance liquid chromatography for sample preparation, and GC analysis for separation and quantitation ( I ) . The analytical methods employed by the other laboratories participating in the intercomparison exercise consisted of the following steps: (1) aqueous or alcoholic digestion of the tissue, (2) extraction with an organic solvent, (3) isolation of the saturated and unsaturated/aromatic hydrocarbons using silica gel column chromatography, (4) separation and quantitation of the hydrocarbons by GC, and (5) identifications by GC retention times and/or GC-MS. The actual methods employed by each of the participating laboratories are briefly summarized in Table I. The initial homogeneity and storage stability studies were conducted at NBS using method 8a (Table I) with glass SE-30 support-coated open tubular (SCOT) columns for the GC analyses as previously described (1). However, the final hydrocarbon measurements reported were performed on high resolution SE-54 wall-coated open tubular (WCOT) columns (30 m x 0.25 mm). In addition, the method of quantitation on the SCOT columns consisted of manual measurement of peak heights from the chromatograms. The latter measurements on the WCOT columns utilized on-line data acquisition with a programmable calculator to quantitate peak areas and the unresolved complex envelope. RESULTS AND DISCUSSION T h e purpose of this intercomparison was to provide a n indication of the interlaboratory variability of current hydrocarbon measurements in mussels from sample preparation through measurement and interpretation. Such an exercise is a complex, difficult comparison because each laboratory utilizes different analytical methods, i.e., extraction, chromatographic sample cleanup, internal or external standards,
1829
etc. However, hydrocarbon measurements from different laboratories are being compared and environmental decisions are being made based on these comparisons. Therefore, a n awareness of the comparability of such measurements is necessary. The actual levels of hydrocarbons in the two samples chosen for this investigation were similar, yet the two samples provide different problems regarding the quantitation of the hydrocarbon levels. These aspects of the results will be discussed later. Homogeneity and stability studies were conducted a t NBS using the dynamic headspace sampling technique to measure total hydrocarbons in the GC eluticln range. The results of these studies have been reported previously (10). Both the Alaskan and the Santa Barbara mussel homogenates appeared to exhibit a nearly twofold increase in the value for total hydrocarbons after three months of storage. T h e reason for this apparent increase is unknown. T h e results do indicate, however, that the time of storage prior to analysis may be an important parameter. Additional research should investigate the effect of storage time on the measurement of hydrocarbon levels in marine biota. Storage instability over the nine-month intercalibration period, sample inhomogeneity, and analytical uncertainty contributed to an NBS intralaboratory precision ( l a ) of -40%. After the initial three months of storage, the NBS total hydrocarbons values were more reproducible a t levels of 57.8 f 8.0 kg/g (1470) and 47.1 k 8.3 pg/g (18%). T h e analyses by the other laboratories were all performed during the first five months of storage. Therefore, 40% indicates the upper limit of possible interlaboratory variation due to sample inhomogeneity and storage instability. The tissue homogenates were analyzed again by NBS after 15 months of storage. These analyses, however, were performed using high efficiency WCOT columns and automated data acquisition rather than the SCOT columns and manual data acquisition used for the previous measurements. Gas chromatograms for the Alaskan and Santa Barbara samples are shown in Figure 1. The peaks labeled 1-3 are the internal standards: 5-methyltetradecane, 7-methylhexadecane, and 2-methyloctadecane. T h e labeled peaks were identified by GC-MS and peaks a-h are unidentified hydrocarbons. T h e values for total hydrocarbons were 56.7 i 2.4 +g/g and 76 f 6 pg/g for the Alaskan and Santa Barbara samples, respectively. These latter values more accurately reflect the contribution of the unresolved complex mixture to the total hydrocarbon value particularly for the Santa Barbara sample. I n t e r c o m p a r i s o n Results. Most of the laboratories responded only to those parameters which they routinely measure; thus some parameters have only limited data for comparison. T h e mussel homogenates are largely water as indicated by the results of the percent water analyses: Alaskan sample, average = 88.8 f 1.4 (1.6%),range 83-90%; Santa , 80-8870. Barbara sample, average = 85.5 2.3 ( 2 . 7 7 ~ )range T h e interlaboratory results for total hydrocarbons in the GC elution range, total extractable hydrocarbons, and pristane/phytane ratio are given in Table I1 for the Alaskan sample and Table I11 for the Santa Barbara sample. T h e results for the total hydrocarbons in the GC elution range have been divided into subtotals for aliphatic and unsaturatedl aromatic hydrocarbons. The quantilation includes subtotals for the resolved peaks and the unresolved complex mixture. These subtotals provide a more accurate comparison of the data due to the different methods utilized for determining the unresolved complex mixture. Several of the labs (2, 3, 4, 5, and 7 ) reported only the identified n-alkanes, pristane, and phytane in the total for the resolved aliphatic hydrocarbons. It should also be noted that the NBS (lab 8) value includes both the aliphatic and the unsaturated/aromatic hydro-
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
Table I. Methods for Tissue Analysis lab
chromatographic analysis column standards
extraction
separation
1
Reflux 4 h in 0.5 N KOH in aqueous methanol, extract with hexane.
Column chromatography to obtain an aliphatic and aromatic fraction.
15-m glass capillary SE-30 columns
2
Digest according to Warner ( 3 ) ,extract with ether.
Column chromatography on activated silica to obtain aliphatic and aromatic fractions.
capillary column
3
Saponify overnight in 0.5 M metlianolic KOH at 7 5 "C, extract with hex an e. Reflux 17 h with 4 N KOH in 85% ethanol, extract with hexane.
Column chromatography on Hiflosil silica gel, aliphatics elute with hexane and aromatics with benzene. Column chromatography on alumina/silica gel (1: 2), aliphatics elute with hexane and aromatics with benzene.
30-m WCOT OV1 0 1 glass capillary column
5
Digest in 4 N NaOH at 60 C for 2.5 h, extract with 7 0% diethylether/30% methylene chloride.
6
Digest 2 h at 90 " C with 4 N aqueous KOH, extract with hexane.
7
Digest 18 h with 6 N NaOH at 30 "C, extract with ethylether.
Column chromatography on silica gel column. Aliphatics elute with 20% methylene chloride/ petroleum ether, and aromatics with 40% methylene chloride/ petroleum ether. Column chromatography on silica gel, aliphatics elute with hexane and aromatics with 40% benzene in hexane. Column chromatography on 5% deactivated silicaialumina column, aliphatics elute with hexane and aromatics with 20% toluene in hexane. ( a ) Liquid chromatography o n aminosilane LC column with hexane as mobile phase, collect both aliphatics and nonvolatile aromatics. ( b ) LC on aminosilane column. Isolate only 3-5 aromatic ring PAHs.
4
8
( a ) Dynamic headspace extraction. Trap volatiles on Tenax GC adsorbent (1). ( b ) nonvolatile PAHs. Digest with 2.5 N NaOH for 18 h at 7 0 'C, extract with cyclohexane.
carbons, and therefore, this value should be compared with the total of the aliphatic and aromatic contribution to resolved a n d unresolved peaks. A comparison of the total hydrocarbon data indicates that t h e Santa Barbara mussel homogenate has, as expected, a greater concentration of hydrocarbons than the Alaskan sample. However, the ranges for the amounts of the resolved peaks for the aliphatic hydrocarbons in both samples are very similar. T h e difference in the unresolved complex mixture for the two samples is shown in Figure 1. The pristane/phytane ratios for the two homogenates (-2 for Santa Barbara mussels and -40 for Alaskan mussels) are consistent with the suspected source of hydrocarbons in each sample, Le., largely biogenic for the Alaskan sample and a petroleum contribution for the Santa Barbara sample. Tables IV and V (see paragraph at end of paper on supplementary material) contain a brief summary of the data for the amounts of several individual hydrocarbons in the Alaskan and Santa Barbara mussel homogenates, respectively. Tables
(1)2 m
X 3.2mm packed column with 5% FFAP on Gaschrom Q. ( 2 ) 11m x 0.25 mm glass capillary with OV101. 2 0 m X 0.25 m m SE-30 WCOT column
Internal Standards added: aliphatic fraction, cholestane; aromatic fraction, hexamethylbenzene. 2-Methylhexadecane and pyrene added to monitor recoveries. Internal standards of 2,6,10-trimethyldodecane and hexamethylbenzene added for GC analysis. External standards of hydrocarbons for quantitation. External standards of hydrocarbons for quantitation.
Internal standard used for recovery standard.
50 m x 0.7 m m SCOT OV-101 column
External standards of hydrocarbons for quantitation.
20 m x 0 . 3 2 mm
External standards for recoveries: ~ I - C ,n-CZ2, ~, n-Czs,and hexamethylbenzene.
SE-52
( a ) 30 m X 0.25 mm SE-54 WCOT ( b ) C-18 reverse phase column with UV and fluorescence detection for quantitation and identification.
( a ) Aliphatic or aromatic internal standards added prior to headspace sampling. ( b ) External standards to determine response factors.
IV and V summarize the results for individual hydrocarbons from n-Clo to n-CBz. T h e most abundant aliphatic hydrocarbon in both samples was pristane (2,6,10,14-tetramethylpentadecane). In the Alaskan sample, pristane was about a n order of magnitude greater than the n-aliphatic hydrocarbons. This large amount of pristane (2.7 f 0.6 pg/g) is not unexpected, since pristane is thought to be biosynthesized from phytol found in the food chain (11). Other than pristane, the most abundant n-aliphatic hydrocarbons generally reported in the Alaskan sample were n-pentadecane (0.33 i 0.14 pg/g) and n-hexadecane (0.29 f 0.13 pg/g). In the Santa Barbara homogenate, pristane was also the predominant hydrocarbon (0.42 0.7 pg/g), but it was present at approximately the same level as the other n-alkane hydrocarbons. T h e levels of the other aliphatic hydrocarbons in t h e Santa Barbara mussels were similar to those in the Alaskan sample (n-C15: 0.31 f 0.14 pg/g and n-C16: 0.28 f 0.15 pg/g). T h e differences in t h e molecular weight range of the nalkanes reported in Tables IV and V illustrate the differences
*
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
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Table 11. Interlaboratory Results for Hydrocarbon Determinations in Alaskan Mussels
lab 1
2 3 4 5 6 7
8 (NBS) range
resolved
total hydrocarbons in GC elution range, pg/g unsaturatedlaromatic aliphatic UCM' total resolved UCM total
4.4
14.8
4. oc 3.2c 5. BC 5.7c 7. lC 4.9c 5.2 1.5 2.6 3.4c 1.5c 18.4 f 3.2d 1.5-7.1
19.2 43.2b
8.8
29.9
__ __ 12.0 __ __
--
_-_ __
__
_-
16.0 22.0 38.3 f 1.5d
19.4 23.5
14.8-22.0
12.0-23.5
total 57.8
__
--e
-_
38.7 95.9b
2.1 2.3
__ __
--
89.4
__
-_ -_
_-
2.1-8.8
__
38.7-89.4
pristane/ phytane
139.1
58
__
__ 41.1 32.2 101.4
40.5 38.4 70.8 40.1 39.7
__ __
--
__ -_ __ _-
56.7
40
73.3 38.4
_--
__
___-
total extractable hydrocarbons, pglg
__ __ ____ f
2.4
--
-_ 45
__
104
32.2-1 01.4
40
38.4-1 39.1
38.4-70.8 Total a UCM = unresolved complex mixture in gas chromatogram. b Gravimetric determination excluded from range. Includes both aliphatic and unsaturated/aromatic hydrocarbons. includes only n-alkanes identified and pristane-phytane. e -- Data not reported. ___-
_-___
Table 111. Interlaboratory Results for Hydrocarbon Determinations in Santa Barbara Mussels
lab
total hydrocarbons in GC elution range, p g / g unsaturatedlaromatic aliphatic ____ resolved UCM' total resolved UCM total 6.5
105.5 179.1b
99
_-
8 range
1.4c 1.2c 10.9c 2.88C 1.8 1.52 3. Oc 1 . 9 f 0.4c 9.5 i 2.3d
99 66f 5 67i 6
1.8-10.9
66-99
__
42.7
__
__
__
_102
67.9
6.8
144
150.8 121.8b
total
--
pristane/
hons, pglg
phytane
256.3
300.9
1.3
_-
-177.0 162.9 --
N.D.e 2.3 1.8
130.2 148.4 58.1
-_
_-
totai exhydrocartracta ale
1.96
__
