Identification of organic sulfur compounds transferred to fish from petroleum suspension by mass chromatography

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IDENTIFICATION OF ORGANIC SULFUR COMPOUNDS TRANSFERRED TO FISH FROM PETROLEUM SUSPENSION BY MASS CHROMATOGRAPHY MASANA OGATA and YOSHIO MIYAKE Department of Public Health. Okayama University Medical School. Okayama 700, Japan

(Received 3 April 1979) Abstract--Authors attempted to select organic sulfur compounds usually contained in crude oil as a marker of oil pollution in fish. For confirming this, eels (Anguilla japonica Temminck et Schlegel) were maintained in a controlled laboratory environment in water with suspensions of crude oil. Gas chromatography-mass spectrometry and mass chromatography of eel flesh extract showed the presence of organic sulfur compounds of alkyl-(from mono- to pentamethyl) benzothiophenes, dibenzothiophene and alkyl-(from mono- to trimethyl) dibenzothiophene and of other organic sulfur compounds of alkyl-(from mono- to pentamethyl) naphthalene. Alkyl benzothiophenes or alkyl dibenzothiophenes of less carbon numbers were easily transferred to eel than those of more carbon numbers.

INTRODUCTION

Heavy pollution is found in waters near petroleum industrial districts and oil spillage, in the previous report, we identified toluene and other aromatic hydrocarbons in fish caught on the coast facing petroleum industries (Ogata & Miyake, 1973). Authors also reported the presence of organic sulfur compounds supposed to be caused by petroleum in flesh of a turbot out of several ones, caught in Seto inland sea (lshida et al., 1976). Then eels were also maintained in a controlled laboratory environment in water with suspension of crude oil. Gas chromatography of the eel flesh revealed the presence of paraffins and organic sulfur compounds whose concentration increased in accordance with rearing time (Ogata et al., 1977). However, we could not identify the organic sulfur compounds being transferred from sea water to eel. In the present study, organic sulfur compounds in the flesh of eels exposed to crude oil suspension in the laboratory were analyzed by gas chromatography-mass spectrometry-computer system.

Analytical conditions: The size of glass column was 0.3 c m x 2 m with 3~'~ Silicon SE 52 on Chromosorb w (AW} mesh 80-100. The chromatograph was programmed from 80 to 280°C. 10'Cmin -t. Carrier gas was nitrogen. flow rate. 40 ml rain - ~. Air. flow rate. 40 ml rain - t. Condition of FPD. (Wave length of filter: 394nm. H 2 80ml -t. 02 20mlmin-t). FPD and FID curves were drawn by 2 pen recorders simultaneously. Gas chromatography-mass spectrometry was as follows; The apparatus used was a Shimadzu LKB 9000 gas chromatograph-mass spectrometer combined with gas chromatograph-mass spectrometer PAC 300 DC computer system. including an OKITAC 4300 mini-computer. Gas chromatographic conditions; The size of glass column was 0.3 c m x 2 m with 3% Silicon SE 52 on Chromosorb w (AW) mesh 80-100. The chromatograph was programmed from 80 to 280°C. 10°C rain -t. Carrier gas was helium. flow rate. 35 ml rain-i. A total ion collector was used as a detector of gas chromatography-mass spectrography. The mass spectrometric conditions were as follows. The ion source temperature was held at 250"C. The mass spectra were obtained at 20eV of electron energy. 3.5 kV acceleration voltage, and 60/zA of trap current. The repeating scan speed was at 6 s of mass chromatography.

RESULTS MATERIALS AND METHODS The petroleum used was a mixture of Arabian Light Crude oil and Zubea oil (4: i). Ten eels weighing 130-180 g were reared in sea water containing 250 ppm of crude oil suspension at 20°C and were taken for examination on the 5 days. Eels for the control group were reared and taken under the same conditions. For gas chromatography, eel flesh homogenates were treated as described in the previous paper (Ogata et al. 1977). The compounds eluted from the silicagel alumina (1:1) column with n-hexane were washed with water, and concentrated with a KudernaDanish evaporative concentrator (KD). The concentrate was extracted with acetonitrile. The acetonitrile solution was concentrated with KD. Gas chromatography was as follows; The apparatus employed was a Hitachi 163 with FPD and FID detectors.

Gas chromatograms Eel flesh was analysed by gas chromatograms ( F P D and FID) of acetonitrile extracts of crude oil. Gas chromatograms (Fig. lb) showed many peaks con~ ~ tained in the crude oil (Fig. In) which were not seen in control eel flesh (Fig. Ic). Appearance of many peaks on F P D gas chromatogram of oil polluted fish flesh indicated that organic sulfur compounds in crude oil suspension tra'nsferred from the water medium to fish flesh. Difference in peak heights of pristane appeared in Fig. Ib and that in Fig. Ic will. be due to mainly individual difference in concentration in eel flesh. 1179

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Identification of organic sulfur compounds Gas chromatooraphy-mass chromatooraphy

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Mass spectra (Fig. 2) and mass chromatograms (Fig. 3) were taken approximately over the entire range of peak in the gas chromatograms. The compounds of the gas chromatograph-mass spectra in crude oil and eel flesh were identified with the comparison of the gas chromatograph-mass spectra of specimens and mass spectra of authentic substances (Stenhagen et al., 1973), and also mass chromategrams. And the compounds found in crude oil and eel flesh are summarized in Table l, showing the presence of methyl derivatives of naphthalene, benzothiophene and dibenzothiophene. Mass spectra of typical peaks 3' and 4' indicated that methyl derivatives of benzothiophene and those of naphthalene were contained in the same peak. Peak 6' and 7' showed dibenzothiophene, and monomethyl dibenzothiophene, indicating other compounds were only contained in the minor amounts and showed the same patterns of authentic ones (Shenhagen et al., 1973). Studies on mass chromatography also showed that dimethyl benzothiophene and dimethyl naphthalene were contained in the same peak of No. 2' on chromatogram of total ion current (TIC) in the mass spectra in Fig. 3a. Peaks of mass chromatogram of M+162 indicating dimethyl benzothiophene were well separated on mass chromatograms, as shown in symbol 2 peak in the upper part and also dimethyl naphthalene (di-m-N) peaks in the lower part respectively in Fig. 3a. The results indicated that separation by mass chromatography is excellent. In order to confirm the methyl naphthalene and methyl benzothiophene of organic sulfur compounds Table I. Estimation of components in the fraction of aromatic compounds from crude oil by mass spectrometry Peak No.

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(Crude oil contained Tetra, Penta, and Hexa dibenzothiophene) Cal. = Calculated, naph. = naphthalene, bzth. = benzothiophene, dibzth. = dibenzothiophene, a. b, and c indicate isomers of respective compounds. Monomethyl-, Dimethyl-, Trimethyl-, Tetramethyl- and Pentamethyl-benzothiophene are contained in peak numbers I and 1'; 2 and 2'; 3 and 3'; 4 and 4"; and 5 and 5' respectively.

1181

were contained in the same peak in this gas chromatograms, the mass chromatogram of main peak of No. 3' shows as typical case (Fig. 4t. Peaks on mass chromatograms at M" 170 and M ' 1 7 6 for trimethyl naphthalene and trimethyl benzothiophene were also corresponded to peak 3' of curve of total ion current on gas chromatograms. Not any peak could be found on the mass chromatogram of M+172, though distinct peaks were seen on that of M ~"170, indicating trimethyl naphthalene. On the other hand, small peak groups could be recognized on the mass chromatogram of M ÷ 178 corresponded to M ÷ 176, and which was about 4 ~ of peak groups on chromatogram of M ÷ 176 of .trimethyl benzothiophene. Data indicate that the ratio of azS-trimethyl benzothiophene and a¢S-trimethyl benzothiophene, as natural isotope, is 95 to 4. Figure 3b also indicates that (1) methyl derivatives of benzothiophene having less carbon numbers would be more than those having more carbon numbers in eel flesh, and (2) alkyl derivatives of benzothiophene which had more carbon number 6 (n = 6) could not be found in eel flesh. On the other hand, minute amount of alkyl derivatives of benzothiophene over n = 6 were found in crude oil as shown in Fig. 3a. These compounds will be due to alkylated derivatives of benzothiophene in which some of methyl groups are substituted by larger groups. Data of (1) and (2) indicated that methyl benzothiophenes having more carbon numbers showed less ability of transferring than those having smaller carbon number, as the C2-C4 methyl derivatives of benzothiophene are almost equally distributed each other in crude oil (Fig. 3). Similar phenomenon to benzothiophene was observed in mass chromatograms of dibenzothiophene group (Fig. 3). In the case of dibenzothiophene group, concentration of dibenzothiophene group in eel flesh is in the order of dibenzothiophene (n = 0) methyl dibenzothiophene MD (n = 1)> MD (n = 2) :> MD (n = 3), as the concentration of methyl. dibenzothiophene in crude oil is almost similar to each other (Fig. 3). Absolute amounts of Di-Bzth is 0,046~ w/w of crude oil, calculated from authentic samples, Concerning this experiment, Nakayama & Kashimoto (1977) suggested presence of dibenzothiophene derivatives in oyster caught in the Sere inland sea along Hiroshima Prefecture. Presence of isomers of alkyl (mainly methyl) derivatives of benzothiophene and dibenzothiophene could be confirmed from the fact that peak groups on mass chromatogram of the same number of M ÷ consist of several sharp and small peaks, for example mass chromatogram peak groups of M ÷ 176 (peak 3) in the upper part and M÷198 (peak 1) in the lower part of Fig. 3 consisted of small sharp peaks, respectively. DISCUS.SION

Coleman et al. (1973) described the presence of hetrocyclic sulfur compounds in crude oil. Previous

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Fig. 4. Chemical ionization of mass chromatograms of trimethyl naphthalene (M ÷ 170) and trimethyl benzothiophene (M" 1761 and natural isotope (M ÷ 1781 being indicated by dotted arrow in the peak No. 3'. TIC = total ion current. report {Ogata et ul., 1977) described that organic sul- zothiophene etc. reveal more complex or broader fur compounds in crude oil suspension were trans- peaks than those of methyl-derivatives of benzothiophene on gas chromatogram. (2) Presence of monoferred from the water medium to fish flesh. The present experiment indicates that m a i n l y methyl-benzothiophenes in crude oil was reported by methyl derivatives of benzothiophene, methyl deriva- Koga (1966). Presence of minor component of benzothiophenc tives of dibenzothiophene and also mainly methyl derivatives of naphthalene in crude oil were transferred derivatives with ethyl-, propyl-, butyl-groups etc. was suggested from the peaks having M*246-M'274 to eels. Trace amounts of organic sulfur compounds in the by mass chromatogram of crude oil as shown in Fig. 3a. Detail investigation of these minor comcrude oil and eel flesh could be determined by FPD gas chromatography; therefore, these compounds are ponents will be described in the future. Concentration ratios of organic sulfur compounds recommended as a marker for oil pollution in fish. Presence of alkyi benzothiophene and alkyl diben- for eels are being calculated with gas chromatogram zothiophene in crude oil and fish is confirmed as fol- drawn with FPD. This will be also reported in the lows. (a) Distinct peaks of FPD curves on gas chro- near future. matogram drawn FPD with 394 nm filter which is specific to sulfur compounds. (b) The ratio 95:4 to 328:3"S is suggested by intensity of M ÷ and M ÷ + 2 Aeknowled.qemenrs--Authors express their thanks to Dr. Takeshi Murata and Dr. Seiji Takahashi in the Analytical of mass peaks. In this experiment, alkyl benzothio- application laboratory, Shimaazu Seisakusho Ltd. for asphene and alkyl naphthalene having the same number sistance of analysis on gas chromatography-mass spectroof methyl groups were recorded as single gas chroma- metry, and also Mr. Katashi Hitomi, Miss Yoshie Yoshida tographic peak of No. I'-No. 5' respectively, when and Miss Midori Kameyama for assistance of this work. This work is partly supported by Ministry of Education SE 52 is used as the liquid phase, which were proved Agency No. 2105161 by gas chromatograph-mass spectra and mass chromatograph. The reasons for the conclusion that alkyl benzoREFERENCES thiophenes are mainly composed of methyl derivatives of benzothiopbenes in this experiment are as follows: Coleman H. J., Dootey J. E.. Hirsch D. E. & Thompson C, J. (1973) Compositional studies of u high boiling (I) Presence of isomers of alkyl benzothiophene and 370-535"C distillate from Prudhoc Bay. Alaska, crude alkyl dibenzothiophenc in peak groups composed of oil. Analyt. Chem. 45, 1724 1737. several sharp and small peaks of M * curves on mass Ishida T., imanaka M., Matsunaga K., Ikeda M., Ogata M.. Okazaki T., Ueno S. & Kawahara H. 11976) The chromatogram of oil or eel sug,~'~ts that major alkyl hygienic studies on topped oil (11)The analytical method benzothiophene and alkyl dibenzothiophene will be of topped oil components (organosulfur compounds) in isomers of several methyl benzothiophene and methyl marine products and its application. A. hg.q. Lab. dibenzothiophene, rather than alkyl benzothiophene Okayuma Prefecture 23, 27-30 (in Japanese). and alkyl dibcnzothiophene having longer chain, i.e. Koga Y. (1966) Sufl'ur compound in crude oil. Jap, Petroleum Inst. 9, 540-545 (in Japanese). ethyl-, propyl- or butyl-benzothiophene and diben-

Identification of organic sulfur compounds Nakamura K. & Kashimoto T. (1977) Contamination of organic sulfur compounds in marine products. J. F~,~,d Hyg. Sot.. 18, 252-259. Ogata M. & Miyake Y. (1973) Identification of substance in petroleum causing objectionable odour in fish. Water Re.s. 7. 1493--1504.

118.5

Ogata M., Miyake Y., Kira S.. Matsunaga K. & Imanaka M. (1977) Transfer to fish of petroleum paraffins and organic sulfur compounds. W~lter Re~. I!, 333~ 338. Stenhagen E.. Abrahamsson S. & Mac Lafferty F. W. (1973) Registry t~/ Mas.~ Spet'tral Dtlt~t. pp. 357-958. Wiley. New York.