74-C-0013with the Marine Studies Center, Institute for Enuironmental Studies of the University of Wisconsin-Madison.
Supplementary Material Available. Table 111 (15 pages), which contains the data for all 24 samples of total sediment and clay-size fraction, will appear following these pages in the microfilm edition of this oolume of the journal. Photocopies of the supplementary
material from this paper only or microfiche (105 X 148 m m , 24X reduction, negatives) containing all of the supplementary material for the papers in this issue m a y be obtained from the Business Operations Office, Journals Department, American Chemical Society, 1155 16th St., N . W., Washington, D.C. 20036. Remit check or money order for $4.50 for photocopy or $2.50 for microfiche, referring to code number ES&T-77-984.
Polynuclear Aromatic Hydrocarbons in Marine Tissues Roy J. Pancirov* and Ralph A. Brown Analytical and Information Division, Exxon Research and Engineering Co., Linden, N.J. 07036
Shell and fin fish contain benzo(a)pyrene and other polynuclear aromatic hydrocarbons at the parts per billion level or below. The presence of pyrene at a much higher relative concentration than its methyl isomers indicates the hydrocarbons are from a combustion source. A National Academy of Sciences workshop on oil pollution ( I ) found that the principal concern to public health is the danger of eating oil-contaminated marine tissues. Since petroleum contains small concentrations of potentially carcinogenic aromatics (2),it has been considered as one of many possible sources of such contamination. In response to this concern, the American Petroleum Institute conducted a study of polynuclear aromatic hydrocarbons (PAH) in marine animal tissue. This included the measurement of typical polynuclears, such as benzo(a)pyrene (BaP), in the edible portions of shell and fin fish. Concentrations were observed on a wet weight basis.
Method of Analysis Carbon-14 labeled benz(a)anthracene (BaA) and BaP were employed as internal standards in the method of analysis. After adding these standards to the starting sample, a modification of the method of Howard et al. ( 3 )was used to prepare a PAH concentration that was in turn analyzed by a combined GC/UV procedure ( 4 ) . A weighed sample of approximately 450 g is placed into 300-mL of redistilled ethanol and 15 g of KOH. The spike solution of carbon-14 BaA and BaP is added, and the mixture digested for 2 h. The resulting solution is transferred to a 2-L separatory funnel with two 125-mL water and two 100-mL ethanol washes. The PAH’s are then extracted with E O - , loo-, and 100-mL portions of isooctane. Each of the three isooctane extracts is washed with four 250-mL portions of warm water. The isooctane washed extracts are combined and reduced on a steam bath under N2 to -25-30 g. An equivalent weight of isooctane is added, and the resulting solution extracted with five 100-g portions of 1O:l dimethyl sulfoxide:H3P04 (DMSO). Then 720 mL of water is added to the combined DMSO extracts, and this solution is back extracted with three 100-mL portions of isooctane. Each isooctane portion is washed with three 250-mL portions of water. The isooctane solutions are combined and reduced to -10 mL on a steam bath under Nz. A 1-mL sample may be removed at this point for counting. The isooctane solution is eluted through a long narrow (6 mm X 90 cm) column of 2% deactivated alumina. The solvent elution procedure is as follows: 5 mL prewash of cyclohexane, 10 mL sample, 30 mL of 10%benzene/90% cyclohexane, 30 mL 20% benzene/80% cyclohexane, 30 mL benzene, and 30 mL 50% benzene/50% methanol. Ten-milliliter fractions are col-
lected throughout the separation. One-milliliter samples are taken for counting, and the fractions combined according to the counting data. The combined fractions are reduced on a steam bath under Nz after adding 5 pL of n-hexadecane to prevent going to dryness. An aliquot of this concentrate is then injected into a gas chromatograph where the GC peaks are trapped, and the UV absorption spectra of selected trapped peaks are then measured. These spectra provide a quantitative measure of individual PAH’s in the aliquot. Quantitation on a total sample basis is calculated from the observed recovery of the I4C labeled standards, BaA and BaP. The precision and accuracy of the method were evaluated by analyzing known blends containing phenanthrene, 2me-phenanthrene, 1-me-phenanthrene, fluoranthene, pyrene, BaA, chrysene, triphenylene, BaP, BeP, perylene and B(g,h,i)P, a t concentrations of 1 and 6 ppb. Individual compounds were measured within %sigma limits of 1 and 2 ppb, respectively. Later in this manuscript, the concentration ratio of methylpyrene to pyrene is shown to be of special significance in the interpretation of the findings of this study. For this reason, special commentary is warranted with regard to the ability of the method to handle the methyl-substituted isomers. Two cross-checks on this were available for most of the samples. Gas chromatograms, for example, provide such a cross-check as illustrated in Figure 1 for the Long Island Sound oysters. The concentration of methylpyrene is much lower than that of pyrene which agrees with the UV measurement listed in Table I. Low-voltage mass spectral measurements ( 5 )were applied to the Long Island Sound oysters and numerous other samples. These spectra confirmed that not only pyrene (molecular weight series 202, 216, etc.), but
T i m e (Tin
Figure 1. Gas
chromatogram of Long Island Sound oysters Volume 11, Number 10, October 1977
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other PAH homologous series were comprised principally of the parent compound with much lower amounts of methylsubstituted homologs. Samples
Figure 2 shows the origin of several samples which were studied. Included were oysters and clams from a commercial fishery in Long Island Sound near Norwalk, Conn. Crabs and menhaden were taken from Raritan Bay in New Jersey. The Long Island Sound and Raritan Bay waters are exposed to municipal and industrial wastes. Two flounder samples were analyzed; one was taken during dredging off Long Branch, N.J., and a second was collected off the R/V Delaware II during a cruise conducted by the NOAA Fishery Laboratory of Sandy Hook, N.J., about 10 miles off the southern coast of Long Island. Codfish came from an open area of the Atlantic Ocean. Table I tabulates the origins for all the samples. In addition to the samples already mentioned, oysters and clams were from Chincoteague, Va., crabs from Chesapeake Bay, shrimp from a clean control pond operated by Texas A & M University in Palacios, Tex., and lake trout from a wilderness lake in Ontario, Canada. Mussels were supplied by Woods Hole Oceanographic Institute from a study of Buzzard’s Bay in Falmouth, Mass. Samples were taken from the Little Sippewissett Creek which is an unpolluted area and from Wild Harbor which is considered to be polluted.
our method. The larger the residue, the less will be the percent of the total sample injected into the GC, and accordingly the less 14Cmaterial will be recovered. Since the data are related to 14C recovery, the less recovered means, in effect, that a higher detectability level will be observed for some samples over others. A preponderance of these less than values are
