COMPOUNDS IN SHELLFISH^ Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf by guest on 02 October 2021

/. W. Blaylock, P. W. O'Keefe, J. N. Roehm,2 and R. E. Wildling Ecosystems Department Bat teile Pacific Northwest Laboratories Richland, Washington

ABSTRACT Blumer et al.1 used refluxing in a Soxhlet During the course of investigations to determine the extraction apparatus to remove n-alkanes from the moist possible toxicity of to marine biota, it became tissues of scallops and oysters. In a study of the " taint" of Australian Mullet obtained from adjacent evident that quantitative estimates of the petroleum com- 2 ponents in and biota would assist in meaningful inter- to the Queensland coastline, Shipton et al. used a vacuum sublimation technique for qualitative determination of pretation of the results of bioassays. However, published 3 procedures for estimation of n-alkanes in marine biota were compounds in the fish tissue. Connell, in an largely qualitative, and even less effort had been afforded investigation of the same problem isolated n-alkanes of car- the measurement of aromatic petroleum residues. A bon numbers 10 to 13 from mullet by extraction of the fish tissue with diethyl followed by distillation of method originally utilized for determination of poly cyclic 4 aromatic in foods was therefore adapted for the extract. Deshimaru employed a gas-liquid Chromato- the digestion of tissue and extraction of hydrocarbons from graphie technique to analyze the head space vapors from shellfish exposed to petroleum during bioassays. Tissue ex- samples of fish tissue which had been exposed to crude tracts were partitioned into saturate and aromatic fractions under laboratory conditions. by column . Using gas-liquid chromatog- If hydrocarbons are incorporated into the cell, these raphy, the n-alkanes of numbers 12 to 19, and the methods likely would not give complete recovery as hydro- methyl substituted were identified in the sat- associated with the cell may not be ade- urate and aromatic fractions, respectively. Both groups of quately separated from the tissues. Separation of the hydro- compounds were quantitated by reference to an internal carbons from tissues which contain considerable fatty mate- standard. rial would be facilitated by preliminary digestion of the The procedure allowed recovery of over 70 percent of tissue and saponification of fatty components. Therefore, n-alkanes and methylnaphthalenes applied to the tissues in the present studies, the digestion step of a quantitative prior to digestion. Minimum detectable levels for n-alkanes procedure for determination of polycyclic aromatic hydro- and methylnaphthalenes were approximately 0.08 to 0.15 carbons in smoked foods (Howard et al.5) was tested for and 0.03 to 0.04 ßg/g of wet tissue, respectively. use in the separation of hydrocarbons from shellfish. This digestion step involved saponification of the tissue with INTRODUCTION ethanolic KOH. Investigations, based on this method, were During investigations to determine the toxicity of oil to also undertaken to develop and test methods to extract shellfish, it became evident that obtaining representative hydrocarbons from the shellfish digestate, separate the ex- samples for determination of the concentration of oil in tract into saturate and aromatic fractions and quantitatively water in two phase, flow-through bioassay systems would determine compounds within these fractions. The n-alkanes be a difficult task. A logical extension of these studies, and methylnaphthalenes in the saturate and aromatic frac- therefore, was to determine the concentration of hydrocar- tions, respectively, received initial emphasis. The develop- bons in tissues of marine organisms exposed to for use as a possible basis for expression of toxicity. However, tech- These investigations were supported jointly by the American Petro- niques available for estimation of hydrocarbons in marine leum Institute, Washington, D.C. and Battelle, Pacific Northwest biota were largely qualitative. A principal difficulty in Laboratories, Richland, Wash. quantitative analyses involved efficient extraction of the 2Presently located at Mount Hood Community College, Gresham, hydrocarbons from the tissue. Oregon.

173 174 IDENTIFICATION OF OIL ment of these methods, their validation, and the final pro- ature, 255 C; column temperature, 50 C programmed at an cedures adopted for routine analyses are described herein. increase of 5°C per minute to 250°C. Qualitative identification of the compounds was achieved by comparison of their retention times to reten- Materials and Methods tion times of known standards on the column described The following procedure was adopted for routine analy- above and on a confirmation column (three percent tetra- ses of shellfish tissues for n-alkanes (carbon numbers 12 to cyanoethylated pentaerythritrol, TCEPE, on 70-80 mesh 19) and for methyl-substituted naphthalenes (1-methyl- Chromasorb W). ; 2-methylnaphthalene; 1,2-dimethylnaphtha- Hydrocarbons were quantitated by comparison of their lene; 1,3-dimethylnaphthalene; and 2,6-dimethylnaphtha- peak areas to the peak areas of an internal standard (octa- lene). All were of distilled spectral grade (Burdick cosane, an n-alkane of carbon number 28) of known con- and Jackson, Muskegon, Mich.). centration (approximately 2 Mg/g) added prior to digestion. Methodological Validation Recommended Procedure Experiments were designed to determine the recovery Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf by guest on 02 October 2021 Tissue Digestion. Shellfish tissues (less than 100 g moist of the standard hydrocarbons listed above after addition to weight) were placed in a round bottom flask (500 ml), with the clam tissues (32-68 g moist weight). Recoveries were standard taper ground glass neck and mixed with 95 per- estimated by comparison of peak areas of hydrocarbons cent (150 ml), several glass beads or boiling chips, added to the tissue and carried through the procedure with and KOH (10g). The mixture was refluxed at approxi- analogous hydrocarbons determined directly. mately 80 C on a heating mantle and under a Friedrich The n-alkanes, dissolved in , were added to clam reflux condenser for one hour. After cooling to ambient tissue prior to digestion at concentration levels (wet tissue) temperature using a water bath, the remaining in of 0.08 to 0.15 Mg/g (level I), 0.36 to 0.77 Mg/g (level II) and the interior of the condenser was washed with hexane 2.72 to 5.73 μg/g (level III). The methylnaphthalenes were (2-3 ml) into the receiver flask. added in a similar manner at a level of 0.03 to 0.04 Mg/g. Solvent Extraction. The digested material was trans- Analytical sensitivity was estimated from the minimum re- ferred to a Teflon-stoppered separatory funnel (one liter) producible peak area which could be accurately determined using distilled H20 (80 ml) and two portions (50 ml each) over the background. Background was subtracted where of hexane. Use of more than 80 ml distilled H20 tended to this represented a significant contribution to peak areas ob- reduce the extraction efficiency by forming a solvent emul- tained on application of the method to control tissues. sion. The mixture was equilibrated for one minute by hand shaking, and the solvent and aqueous phases were allowed to separate. The two phases were drained into separate RESULTS AND DISCUSSION flasks and the aqueous phase was returned to the separatory Preliminary testing of the analytical procedure with funnel using a hexane (50 ml) wash. The extraction and shellfish tissue to which hydrocarbons had been added indi- separation was repeated for a total of three times. cated that the n-alkanes and methylnaphthalenes could be The combined hexane extracts were washed (minimum separated from shellfish tissues and partitioned into frac- three times) with aliquots (500 ml each) of distilled H20 to tions sufficiently free of contamination to allow gas Chro- remove solids and residual and transferred to an matographie analyses. However, in order to validate the Erlenmeyer flask (300 ml) using additional hexane. To re- method, it was necessary to measure the recovery of the move water from the extract, anhydrous Na2S04(2-3g) was hydrocarbons over a suitable concentration range and deter- added. The extracts were then concentrated to approxi- mine the sensitivity or lower detection limit. For purposes mately 50 ml under a stream of N2 in a warm water bath of validation, the selection of the lowest concentration (50°C) and transferred into a conical flask (100 ml) where added was based on the contribution of background mate- concentration was continued to a final volume of approxi- rial in control organisms obtained from essentially oil-free mately 5 ml. water, whereas the highest concentration range was taken as Column Separation. The concentrated extract was a maximum likely to be present in organisms directly ex- placed on a glass column (20 cm length X 1.0 cm I.D.) with posed to oil in bioassay experiments. coarse fritted disc, packed with silicic (10g, 100-200 The recoveries of n-alkanes from clam tissues added at mesh, Grade 923, Davison Chemical, Baltimore, Md.) deac- three concentration levels are shown in Table 1. The mean tivated with five percent water prior to column packing. recoveries of individual n-alkanes at levels I, II, and III The liquid was allowed to drain to the column bed level ranged from 77 to 102 percent, 74 to 93 percent, and 75 to into a graduated conical tube. 97 percent, respectively. Considering the n-alkanes sepa- The residual concentrate was rinsed onto the column rately (n=9), there was no significant difference (P = 0.05) and elution continued until 20 ml eluate was collected. This in recovery between the three concentration levels. eluate contained the n-alkane hydrocarbon fraction. The To determine if reduced with increased car- column was then eluted with four percent in bon number resulted in significant differences in recovery, hexane (100 ml) into a new collection flask to obtain the the recoveries for carbon numbers 12 to 16; 16 to 18; 17, methyl substituted naphthalene fraction. Both fractions 18 and 18, 19 were compared. These groups were signifi- were concentrated to appropriate volumes for analyses. cantly different (P = 0.05) at the level III concentration Hydrocarbon Determination. Hydrocarbons were de- indicating that volatilization may have occurred accounting termined using a Packard Model 1704 gas Chromatograph for part of the differences in recovery between n-alkanes at equipped with a flame ionization detector and a glass col- this concentration level. umn (183 cm length X 2 mm I.D.) packed with three per- Variation in recovery of the individual n-alkanes ex- cent SE 30 on 100-200 mesh Gas chrom Q. Operating con- pressed as the standard deviation was two to eight times ditions were as follows: N2, 30 ml/min; H2, 30 ml/min; air, lower for the high concentration levels than for the mini- 300 ml/min; injector temperature, 185°C; detector temper- mum and intermediate concentration levels. This effect was Table 1: Recovery of n-Alkanes (carbon numbers 12 to 19) from Cla m Tissue Added at Three Concentration Levels 1

Level I Level II Level HI n-Alkane Mean Added Cone. Recovery (%) 2 Mean Added Cone. Recovery (%) 2 Mean Added Cone. Recovery (%) 2 Compound (Mg/g tissue) Mean ± Std. Dev. Oug/g tissue) Mean ± Std. Dev. (Mg/g tissue) Mean ± Std. De\ Cl2 0.09 77 8.9 0.44 84 12 3.13 75 2.1 Cl3 0.09 84 11 0.45 86 16 3.23 75 2.1 c14 0.09 78 10 0.45 93 16 3.18 78 3.5 Cl5 0.11 89 4.0 0.55 92 8.5 3.91 80 4.5 Cl6 0.11 96 16 0.55 74 12 3.93 82 5.0 Cl7 0.10 87 2.8 0.49 83 6.0 3.50 89 4.5 Cl8 0.10 102 23 0.50 81 9.5 3.57 92 5.5

Ci9 0.12 93 14 0.59 82 11 4.18 97 4.0

1 Three replicate clams were analyzed at each concentration level. The concentration is based on wet tissue weight. The concentration range is given in the Materials and Methods section.

2Recoveries are expressed as a percentage of the total of each n-alkane added to the sample. Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf by guest on 02 October 2021 October 02 on guest by http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf from Downloaded 176 IDENTIFICATION OF OIL

The relative influence of background on the peak areas of individual n-alkanes added at low concentration levels may be illustrated by comparison of chromatograms of the n-alkane fraction from control and n-alkane treated clams (Figure 1). It is evident that the minimum level of n-alkane detectability is largely dependent upon the concentration which may be determined above background with accuracy and precision. Contribution of background at the level I sensitivity ranged from 0 to 30 percent (Figure 1) of the 16 18 20 22 individual peak areas. Thus, for purposes in which minimal MINUTES recoveries of 77 percent and variation up to approximately 20 percent is acceptable, the lower detection level of the procedure may be considered to be in the range of 0.08 to 0.15Mg of individual n-alkanes/g moist tissue (level I, Table 1). Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf by guest on 02 October 2021 The recoveries of methylnaphthalenes added to clam tissues at concentrations approximating minimum detect- able levels for the n-alkanes, ranged from 69 to 82 percent (Table 2). The variation in measurement of individual com- pounds (Table 2, Fig. 2), expressed as the standard devi- ation, was less than the n-alkanes at this concentration level, perhaps as a result of less variation in background materials compared to the n-alkanes. As in the case of the n-alkanes, recovery and precision may be expected to be improved at higher concentration levels. On the basis of these data, an acceptable minimum detectable level of indi- vidual methylnaphthalenes would therefore appear to ap- proximate 0.03 to 0.04 μg/g of moist tissue. The method described above has sufficient accuracy and precision for estimates of n-alkanes and methylnaph- 10 12 14 16 18 20 22 24 26 28 thalenes in shellfish tissues exposed to oils in bioassay ex- MINUTES periments or in the environment and has been employed Figure 1: Segments from the Chromatograms (Gas-Liquid Chroma- routinely for these purposes in associated studies. It should tography) of the n-Alkane Fraction from a Control Clam (Upper) be noted that n-alkanes and methylnaphthalenes constitute and a Clam Containing n-Alkanes Added at Levels of 0.08 to 0.12 a small fraction of the homologous groups of compounds Mg/g of Wet Tissue (Lower). occurring in crude oils and distillation products, and they may not be representative of crude oil contamination. How- likely due to differences in the relative influence of inter- ever, the utilization of other instrumental techniques such fering background material. At the higher concentration as infrared spectrophotometry and , levels, instrumental sensitivity may be reduced resulting in a should make it possible to perform a more complete analy- corresponding reduction in the background. sis of compound types in tissue extracts obtained utilizing The instrument sensitivity utilized in the determination the described method. of the level II recoveries was 3-fold lower than for the level I addition, therefore, the background contributions SUMMARY were reduced by a factor of three. Similarly, a 30-fold lower sensitivity was utilized for the measurement of level A quantitative procedure is described for the determina- III recoveries resulting in a less than one percent back- tion of n-alkane and methylnaphthalene compounds in ground contribution which was neglected in measurements shellfish tissues. The method involved digestion of tissues of peak areas. with ethanolic KOH, solvent extraction of the hydro-

Table 2: Recovery of Methylnaphthalenes from Clam Tissue1

Level I Mean Added Cone. Recovery (%)2 ^g/g tissue) Mean ± Std. Dev 2-methylnaphthalene 0.04 81 2.8 1 -methylnaphthalene 0.03 82 3.5 2,6-dimethylnaphthalene 0.03 75 4.9 1,3-dimethylnaphthalene 0.04 69 0.7 1,2-dimethylnaphthalene 0.04 73 2.8

1 Three replicate clams were analyzed; the concentration is based on the wet tissue weight. 2 Recoveries are expressed as a percentage of the total of each methylnaphthalene added to the sample. N-ALKANE AND METHYLNAPHTHALENE IN SHELLFISH 177

carbons from the digestate, hydrocarbon separation into 1. 2-METHYLNAPHTHALENE saturate and aromatic fractions using column chromatog- 2. 1-METHYLNAPHTHALENE raphy and gas Chromatographie measurement of individual 3. 2,6-DIMETHYLNAPHTHALENE n-alkane and methylnaphthalene compounds. 4. 1,3-D I METHYLNAPHTHALENE The method allowed quantitative measurement of n-al- 5. 1,2 -DI METHYLNAPHTHALENE kanes and methylnaphthalenes in shellfish tissues at concen- trations greater than 0.08 to 0.15 and 0.03 to 0.04 Mg/g of moist tissue, respectively. The techniques are applicable to the estimation of hydrocarbons in shellfish exposed to crude oils during bioassays or in the environment. The tis- sue extracts using this procedure were amenable to further characterization using other instrumental techniques. Thus, the methods of hydrocarbon extraction and separation may have broad application in evaluating the potential for bio- logical uptake of hydrocarbons from oils. Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf by guest on 02 October 2021

ACKNOWLEDGMENTS The authors express sincere appreciation to Mr. P. K. Starnes, Esso Research and Engineering Company, for his guidance during the course of these studies. 10 12 14 16 REFERENCES 1. Blumer, M., G. Souza and J. Sass. Hydrocarbon pol- lution of edible shellfish by an oil spill. Marine Biology 5:195-202(1970). 2. Shipton, J., J. H. Last, K. E. Murray and G. L. Vale. Studies on a kerosene-like taint in mullet. Journal Sei. Fd. Agric. 21:433-436(1970). 3. Connell, D. W. Kerosene-like tainting in Australian mullet. Marine Pollution Bulletin. No. 12, 2:188-189 (1971). 4. Deshimaru, O. Studies on the pollution of fish meat by . Bulletin of the Japanese Society of Scientific Fisheries. No. 4, 35:295-306 (1971). 5. Howard, J. W., R. W. White, B. E. Fry Jr. and E. W. Turicchi. Extraction and Estimation of polycyclic aromatic hydrocarbons in smoked foods. II. Benzo(a). Journal of the A.O.A.C. No. 3 49:611-617 (1966).


Figure 2: Segments of Chromatograms (Gas-Liquid Chromatog- raphy) of the Methylnaphthalene Fraction from a Control Qam (Upper) and a Clam Containing Methylnaphthalenes Added at Levels of 0.03 to 0.04 Mg/g of Wet Tissue (Lower). Downloaded from http://meridian.allenpress.com/iosc/article-pdf/1973/1/173/2881215/2169-3358-1973-1-173.pdf by guest on 02 October 2021