The authors attempted to determine if the organic sulfur compounds usually contained in a crude oil could serve as a marker of oil pollution in fish. Eels (Anguilla Japonica Temminck et Schlegel) were maintained in a controlled laboratory environment of water with a suspension 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 pentametyl) benzothiophenes, dibenzothiophene and alkyl-(from mono- to trimethyl) dibenzothiophenes, and other organic sulfur compounds of alkyl-(from mono- to pentamethyl) naphthalenes.</p

Miyake, Yoshio

Ogata, Masana

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 32, Issue 6 1978 Article 6DECEMBER 1978Identification of organic sulfur compoundstransferred to fish from petroleum suspensionMasana Ogata∗ Yoshio Miyake†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Identification of organic sulfur compoundstransferred to fish from petroleum suspension∗Masana Ogata and Yoshio MiyakeAbstractThe authors attempted to determine if the organic sulfur compounds usually contained in acrude oil could serve as a marker of oil pollution in fish. Eels (Anguilla Japonica Temminck etSchlegel) were maintained in a controlled laboratory environment of water with a suspension ofcrude oil. Gas chromatography-mass spectrometry and mass chromatography of eel flesh extractshowed the presence of organic sulfur compounds of alkyl-(from mono- to pentametyl) benzoth-iophenes, dibenzothiophene and alkyl-(from mono- to trimethyl) dibenzothiophenes, and otherorganic sulfur compounds of alkyl-(from mono- to pentamethyl) naphthalenes.KEYWORDS: organic sulfur compounds, alkyl benzothiophenes, alkyl dibenzothiophenes, oilpollution, fish, mass chromatography∗Copyright (C) OKAYAMA UNIVERSITY MEDICAL SCHOOL. PMID: 154827 [PubMed -indexed for MEDLINE]Acta Merl. Okayama 32, (6), 419-425 (1978)IDENTIFICATION OF ORGANIC SULFUR COMPOUNDSTRANSFERRED TO FISH FROM PETROLEUMSUSPENSIONMasana OGATA and Yoshio MIYAKEDepartment of Public Health, Okayama University Medical School,Okayama 700, JapanReceived August 19, 1978Abstract. The authors attempted to determine if the organic sulfurcompounds usually contained in a crude oil could serve as a marker ofoil pollution in fish. Eels (Anguilla Japonica Temminck et Schlegel)were maintained in a controlled laboratory environment of water witha suspension of crude oil. Gas chromatography-mass spectrometry andmass chromatography of eel flesh extract showed the presence of or-ganic sulfur compounds of alkyl-(from mono- to pentametyl) benzo-thiophenes, dibenzothiophene and alkyl-(from mono- to trimethyl)dibenzothiophenes, and other organic sulfur compounds of alkyl-(frommono- to pentamethyl) naphthalenes.Key words: organic sulfur compounds, alkyl benzothiophenes,alkyl dibenzothiophenes, oil pollution, fish,mass chromatographyHeavy pollution and offensive odors exist in waters near petroleum indus-trial districts and in the sea after oil spillage. In a previous report, we demon-strated the existence of toluene and other aromatic hydrocarbons in fish caughtalong the coast of petroleum industrial areas (I). Conditions representing petro-leum spillage at sea were simulated in a controlled laboratory aquarium. Thiswas used to raise eels. Aromatic hydrocarbons have been found in such eel flesh(2). In fish reared in fuel oil A, the repugnant odors were due to unsaturated andaliphatic hydrocarbons, and some aromatic hydrocarbons (3).The authors have demonstrated the presence of organic sulfur compoundspossibly due to petroleum in the flesh of one of several turbot fish, caught in theSeto inland sea (4). Subsequently, eels were maintained in a controlled labora-tory environment of water with a suspension of crude oil. Gas chromatographyof the eel flesh revealed the presence of paraffins and organic sulfur compoundswhose concentration increased with increase in rearing time (5). In the presentstudy, organic sulfur compounds in the flesh of eels exposed to this crude oilsuspension in the laboratory were analyzed by gas chromatography-mass spectro-metry-computer system.4191Ogata and Miyake: Identification of organic sulfur compounds transferred to fish frProduced by The Berkeley Electronic Press, 1978420 M. OGATA and Y. MIYAKEMATERIALS AND METHODSThe petroleum used was a mixture of Arabian Light Crude oil and Zubeaoil (4: 1). Ten eels weighing 130-180 g were reared in sea water containing 1000ppm of crude oil suspension at 20°C and were taken for examination on theseventh day. Eels for the control group were reared and taken under the sameconditions. For gas chromatography, eel flesh homogenates were treated as de-scribed in the previous paper (5). Compounds eluted from a silica gel alumina(1 : 1) column with n-hexane were washed with water, then concentrated with aKuderna-Danish evaporative concentrator (KD). The concentrate was extractedwith acetonitrile. The acetonitrile solution was concentrated with KD (25 g wwof eel flesh to I ml).Gas chromatography was as follows: The apparatus exployed was a Hitachi163 with a FPD and FID detector. Analytical conditions: The glass column was0.3 cmx2 m with 3% Silicon SE 52 on Chromosorb W (AW-DMCS)mesh 80-100.The chromatograph was programmed from 80 to 260°C, lOoC min-I. The carriergas was nitrogen, flow rate, 80 ml min-I.Gas chromatography-mass spectrometry was as follows: A Shimadzu LKB9000 gas chromatograph-mass spectrometer was used in combination with a gaschromatograph-mass spectrometer PAC 300 DG computer system containing, anOKITAC 4300 mini-computer. Gas chromatographic conditions: The glass col-umn was 0.3cm x2m with 3% Silicon SE 52 on Chromosorb W (AW-DMCS) mesh80-100. The chromatograph was programmed from 80 to 280°C, WaC min-1. Thecarrier gas was helium, flow rate, 35 ml min-I. A total ion collector was used asthe detector for gas chromatography mass spectrography. The mass spectrometricconditions were as follows: The ion source temperature was held at 250°C. Themass spectra were obtained at 20 eV of electron energy, 3.5 kV acceleration volt-age, and 6O,uA of trap current. The repeating scan speed was at 6 sec of masschromatography.RESULTSGas chromatograms. Eel flesh was analysed by gas chromatograms (FPDand FID) of acetonitrile extracts of crude oil. Gas chromatograms (Fig. I, B)showed many peaks for the crude oil (Fig. 1, A). These were not seen in controleel flesh (Fig. 1, C). The appearance of many peaks on FPD gas chromatogramof oil polluted fish flesh indicated that organic sulfur compounds in crude oilsuspension had transferred from the water to the fish. The differences in peakheight in Fig. 1, B and Fig. 1, C are probably individual differences in concentra-tion in eel flesh.Gas chromatography-mass spectrometry and Mass chromatography. Mass spectra(Fig. 2) and mass chromatograms (Fig. 3) were taken over almost the entirerange of peaks in the gas chromatograms. The compounds of the gas chromato-graph-mass spectra in crude oil and eel flesh were identified by a comparison ofthe gas chromatograph-mass spectra of specimens with the mass spectra of2Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/6Organic Sulfur Transfered to Fish 421authentic substances (6), and by mass chromatograms of the specimens. Thecompounds found in crude oil and eel flesh are summarized in Table 1. Thisindicates the presence of methyl derivatives of naphthalene, benzothiophene anddibenzothiophene.Peaks 2',3' and 4' are major peaks of the TIC gas chromatogram of eel fleshextract. The mass spectra of these peaks indicated that methyl derivatives ofbenzothiophene and naphthalene occurred in the same peak. Peak 6' showeddibenzothiophene. Peaks 7'-9' contained methyl derivatives of dibenzothiophene.Other compounds were only present in minor amounts and correlated well withthe patterns of authentic controls (6). These peaks, therefore, are omitted.Dimethyl benzothiophene and dimethyl naphthalene were not separated onthe gas chromatographic column under the conditions studied, but appearedFPO3-b2-,150 200 250 260Temp. (oG) --100Bc Ir\ Pr.-p,--~------~~~~, , ! I ,FlOFPO150 200·Temp (oG)100AII 7-,4-0I;"-b~-oFig. 1. Gas chromatograms of the extract from the acetonitrile fraction obtained fromcleaned up crude oil and the flesh of eels reared in crude oil suspension. A: crude oil, (125mgjml) B : eel reared in crude oil suspension, C: control eel flesh. Symbols of the peak num-ber correspond to the numbers in Table I. Conditions of FPD, (Wave length of filter; 394nm, Hz 80 ml min-I, Oz 20 ml min-I). FPD and FlO curves were drawn by 2 pen recorderssimultaneously. The analytical sensitivity of the FlO curve in Fig. 1, B is 1.6 times as highas that of Fig. I, C. Pr in Fig. I, C; pristane originally contained in eel flesh. The analyticalsensititivity of peaks in FPO gas chromatograms (of 2-c and of more number 2-c) having longerretention times, was one half that of peaks having shorter retention times (Fig. I, A).3Ogata and Miyake: Identification of organic sulfur compounds transferred to fish frProduced by The Berkeley Electronic Press, 1978422 M. OGATA and Y. MIYAKE.,76(M') 184(M') '"100l No 3' ,·122 100l ~~o 6' • 190 §~ 50j ,m 'r~~ll l~ 5ej' JI J~ ~~; J. I i l II~ I I I ~ ~Or' , , ..If' 1"" '+'r'\jtl~r"" ~H~'I' ·'t'r" 0 ,. 1-,,~1; r1i',.Ji,~~Jr,ll.,..,t.'jl.,.<, I cr:o 100 200 (rT.!e) 0 100 200 1m/e)Fig. 2. Mass spectra of peaks No.2', 3/, 4' and 61 in gas chromatograms of eel flesh. (0indicates the base peak of methyl derivatives of naphthalene and. indicates methyl deriva-tives of benzothiophene in No. 2'-No. 4', and dibenzothiophene in No.6'). The right ordinateindicates ratio (%) of ion current of the having maximum peak height marked as symbols 0to the total ion current. And the peak numbers of TIC (total ion current) corresponded tothe peak numbers of FID in Fig. 1.together in a single peak as shown in the TIC chromatogram in Fig. 3, A. Thesetwo compounds, however, were obtained as separate peaks in mass chromato-grams (Fig. 3, A). Fig. 3 also shows that: (1) benzothiophene derivatives withsmaller numbers of methyl groups were more frequent in eel flesh than thosehaving greater numbers of methyl groups. The benzothiophene derivativeswith two to four methyl groups, however, occurred at almost the same rate incrude oil; and that (2) benzothiophene derivatives which have alkyl group(s)composed of more than seven carbons did not occur in the eel flesh extractalthough they were present in small amounts in crude oil as shown in A of Fig. 3.These minute components are probably benzothiopene derivatives with methyland larger alkyl groups. These facts indicate that with more numerous andlarger alkyl groups, benzothiophene becomes less transferable to eel flesh fromthe water. The similar phenomenon to benzothiophene was observed in masschromatograms of dibenzothiophene derivatives (Fig. 3), the concentrations ofwhich in the eel flesh are in the order of dibenzothiophene (n=O) methyl diben-zothiophene (MD) (n= 1) > MD (n=2) > MD (n=3), though the concent-ration of methyl dibenzothiophenes in crude oil are similar each other (Fig. 3).The presence of isomers of methyl derivatives of benzothiophene and diben-zothiophene was confirmed by the fact that peak groups on mass chromatogramof the same number of M + consisted of several sharp and small peaks; for ex-ample, mass chromatogram peaks of M + 176 (peak 3) in the upper part and M +198 (peak 1) in the lower part of Fig. 3, A consisted of small peaks.4Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/6Organic Sulfur Transfered to Fish 423(Q-RnTICB~JCI9H40 ~~~)J Rn"I :.,-,(,:m"h,,,,,",I3'~N M+~'~\ 12B(X 5,0)_~~~__ ~_--------J 246 (X20 0)2~f~,.~~232(X2,O:,O)~~ ~~c, 218(X20,O)~---- '~' 204(X200)===t - 190(X20,O)176(X 10,0)--- '--"'. 162(XIO,OjO-fll~OI-;'l7JO ill l~jOI I I l2!J() 125d r I~SCM, NUMBERdi-m-NL ~,n'='I __ "4["n=:melhYI group)2'~t3'M I M+~~~DI2; -,-~~--~~~i~;Hf, ------2J'2./.X10.O)--~~-198lXIO,O)~~~ :=,,;=,:;::'::;:1':;:,=;=;::,;::':;::'::;:'2:;:5::;O'::;:::I.-r;::.::;=:~84{X10.O)o 50 100 150 2CO 7{)OSCAN NWM8E:RFig. 3. Mass chromatogram (MC) of alkyl benzothiophene in crude oil and contaminantedeel flesh. A; crude oil. B; eel flesh. TIC=total ion current. N=naphthalene. The M+number shows molecular ion for each compound. The number of each MC peak indicates thecarbon numbers of the alkyl group. R. T. 2.00 min and interval 6.00 sec (C1gH40 is pristane ineel flesh). Note; numbers peaks 7',8' and 9' on the TIC curve of upper part of Fig. A did notcorrespond to carbon numbers, 7, 8 and 9 of alkyl group of the peaks on the curves of ben-zothiophenes on mass chromatogram although numbers of 7', 8', and 9' in lower part of Fig.A correspoded to carbon numbers 1, 2 and 3 of alkyl group of dibenzothiophenes.TABLE 1. ESTIMATION OF COMPONENTS IN THE FRACTION OF AROMATIC COMPOUNDSFROM CRUDE OIL BY MASS SPECTROMETRYPeak No. m/e Cal. E,timatod compound I! Peak No, m/e Cal. Estimated compoundformula formula-----_ ..- ---~ .. _-_ ..•. -----------I-a. b 148 CgHsS Monomethyl-bzth. 4'-a. b. c. 184 CI4H 16 Tetramethyl-naph.II'-a. b 142 CnHlO Monomethyl-naph. ' 5-a. b. c. 204 C13H I6S Pentamethyl-bzth.2-a. b. c. 162 ClOHlOS Dimethyl-bzth. 5'-a. b. c. 198 CI5H lS Petamethyl-naph.2'-a. b. c. 156 C12H 12 Dimethyl-naph. 6 184 CIzHsS Dibzth.3-a. b. c. 176 CnH 12S Trimethyl-bzth. 7 198 C13H lOS Monomethyl-dibzth.3'-a. b. c. 170 C13H 14 Trimethyl-naph. 8 212 C14H12S Dimethyl-dibzth.4-a. b. c. 190 C12H 14S Tetramethyl-bzth. 9 226 C15H 14S Trimethyl-dibzth.(Crude oil contained Tetra-, Penta-, and Hexa dibenzothiophene) Cal.=Calculatednaph. = naphthalene bzth. = benzothiophene dibzth = dibenzothiophenea., b. and c. indicate isomers of respective compounds.Monomethyl, Dimethyl-, Trimethyl- Tetramethyl- and Pentamethyl-benzothiopheneare contained in peak No.1 and 1', 2 and 2', 3 and 3', 4 and 4', and 5 and 5' respectively.5Ogata and Miyake: Identification of organic sulfur compounds transferred to fish frProduced by The Berkeley Electronic Press, 1978424 M. OGATA and Y. MIYAKEDISCUSSIONColeman et al. (7) reported the presence of hetrocyc1ic sulfur compounds incrude oil. The authors, in a previous report (5), reported that organic sulfurcompounds in crude oil suspension were transferred from water to fish.The present experiment indicates that methyl derivatives of benzothiophene,dibenzothiophene and naphthalene were transferred to eels.Trace amounts of the organic sulfur compounds in both crude oil and eelflesh could be detected by FPD gas chromatography; therefore, these compoundsare recommended as a marker for oil pollution in fish.The presence of alkyl benzothiophenes and alkyl dibenzothiophenes in crudeoil and fish was confirmed as follows. (a) Distinct peaks in FPD curves for gaschromatogram using a FPD with 394 nm filter which is specific to sulfur com-pounds. (b) The ratio 95: 4 of the intensity of M+ and M+ +2 mass peakssuggested the presence of 32S: 34S. In this experiment, alkyl benzothiophenesand alkyl naphthalenes having the same number of methyl groups were recordedas single gas chromatographic peaks (No. I-No.5 of Fig. I) when SE 52 was theliquid phase. This was proved by gas chromatograph-mass spectra and masschromatograph.Alkyl benzothiophenes were considered to be mainly composed of methylderivatives of benzothiophenes because of the following reasons: a) The pres-ence of isomers of alkyl benzothiophene and alkyl dibenzothiophene in peakgroups composed of several sharp and narrow peaks of M +curves on mass chro-matogram of oil and eel. This suggests that more alkyl benzothiophene and alkyldibenzothiophene composed of isomers of several methyl benzothiophene andmethyl dibenzothiophene rather than alkyl benzothiophene and alkyl dibenzo-thiophene having longer chains, i.e. ethyl-, propyl- or butyl-benzothiophenes anddibenzothiophenes. b) In gas chromatograms isomers of methyl benzothiophenehave narrow peaks, whereas ethyl-, propyl-, and butyl-benzothiophene havemore complex or broader peaks. c) The presence of monomethy-benzothio-phenes in crude oil has been reported by Koga (8).The presence of groups such as ethyl-, propyl-, butyl groups as minor benzo-thiophene derivatives was suggested by the peaks having M+=232"",M+=274on mass chromatograms of crude oil (Fig. 3, A). Detailed investigation of theseminor components is in progress.Acknowledgment. The authors express their thanks to Dr. Takeshi Murata and Dr. SeijiTakahashi of the Analytical application laboratory, Shimazu Seisakusho Ltd. for assistancein analyses by gas chromatography-mass spectrometry, and Mr. Katashi Hitomi, Miss YoshieYoshida and Miss Midori Kameyama for their assistance. This work is in part supported bya grant from the Ministry of Education Agency (No. 210516).6Acta Medica Okayama, Vol. 32 [1978], Iss. 6, Art. 6http://escholarship.lib.okayama-u.ac.jp/amo/vol32/iss6/6Organic Sulfur Transfered to FishREFERENCES4251. Ogata, M. and Miyake, Y.: Identification of substance in petroleum causing objection-able odour in fish. Water Res. 7, 1493-1504, 1973.2. Ogata, M. and Miyake, Y.: Compound from floating petroleum accumulating in fish.Water Res. 9, 1075-1078, 1975.3. Ogata, M. and Ogura, T.: Petroleum compounds and objectionable malodorous sub-stances in fish flesh polluted by boiler fuel oil. Water Res. 10, 407-412, 1976.4. Ishida, T., Imanaka, M., Matsunaga, K., Ikeda, M., Ogata, M., Okazaki, T., Veno, S. andKawahara, H.: The hygienic studies on topped oil (II) The analytical method of toppedoil components (organosulfur compounds) in marine products and its application. Annu.Hyg. Lab. Okayama Prefec. 23, 27-30, 1976.5. Ogata, M., Miyake, Y., Kira, S., Matsunaga, K. and Imanaka, M.: Transfer to fish ofpetroleum paraffins and organic sulfur compounds. Water Res. 11, 333-338, 1977.6. Stenhagen, E., Abrahamsson, S. and Mac Lafferty F. W.: Redistry of Mass spectral data.John Wiley and Son Co., NY. and London, pp. 357-958, 1973.7. Coleman, H.]., Dooley, ]. E., Hirsch, D. E. and Thompson, C. J.: Compositional studiesof a high boiling 370-535°C distillate from Prudhoe Bay, Alaska, crude oil. Anal. Chern.45,1724-1737,1973.8. Koga, Y.: Sulfur compound in crude oil. Jpn. Petroleum Insf. 9, 540-545, 1966 (in Japa-nese).7Ogata and Miyake: Identification of organic sulfur compounds transferred to fish frProduced by The Berkeley Electronic Press, 1978

Identification of organic sulfur compounds transferred to fish from petroleum suspension

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