DETERMINATION OF N-ALKANE AND METHYLNAPHTHALENE 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 methanol in a Soxhlet During the course of investigations to determine the extraction apparatus to remove n-alkanes from the moist possible toxicity of petroleum to marine biota, it became tissues of scallops and oysters. In a study of the "kerosene taint" of Australian Mullet obtained from waters adjacent evident that quantitative estimates of the petroleum com- 2 ponents in water 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 hydrocarbon 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 ether followed by steam distillation of method originally utilized for determination of poly cyclic 4 aromatic hydrocarbons 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 oil tracts were partitioned into saturate and aromatic fractions under laboratory conditions. by column chromatography. Using gas-liquid chromatog- If hydrocarbons are incorporated into the cell, these raphy, the n-alkanes of carbon numbers 12 to 19, and the methods likely would not give complete recovery as hydro- methyl substituted naphthalenes were identified in the sat- carbons associated with the cell lipids 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 oils 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). naphthalene; 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 solvents 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 ethanol (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 hexane, 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 solvent 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 alcohol 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 acid (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.