Identification of substances transferred to fish or shellfish from petroleum suspension

B,'ater Re.warch Vol. 13. pp. 613 to 618 Pergamon Press Ltd. 1979. Printed in Great Britain.

IDENTIFICATION OF SUBSTANCES TRANSFERRED TO FISH OR SHELLFISH FROM PETROLEUM SUSPENSION MASANA OGATA, YOSHIO MIYAKE and YOSHIO YAMASAKI Department of Public Health, Okayama University Medical School, 2-5-1 Shikata-Cho, Okayama-City, Okayama, Japan

(Received 15 January 1979) Abstract--Identification of organic sulfur compounds contained in crude oil as a marker of oil pollution of fish and shellfish was performed. Eels (Anguilla Japonica Temminck et Schlegel) or short-necked clams (Tapes Araygdala philippinarum A. Adams et Reeve) were maintained in a Controlled laboratory environment of water with a suspension of crude oil. Gas chromatography-mass spectrometry of eel flesh extract showed the presence of l-methyl, 2-methyl, dimethyl, trimethylnaphthalenes and dibenzothiophene, and that of soft body of short-necked clams showed dimethyl, trimethylnaphthalene, dibenzothiophene, and monomethyl and dimethyldibenzothiophenes.

INTRODUCTION

Offensive odours were present in fish caught in the sea near either petroleum industrial districts or areas of oil spillage. In a previous report, we identified toluene and other aromatic hydrocarbons in fish caught o n the coast facing petroleum industries (Ogata & Miyake, 1973). Conditions representing petroleum spillage at sea were simulated in a controlled laboratory aquarium containing eels. Aromatic hydrocarbons were found in eel flesh (Ogata & Miyake, 1975). Moreover, fish reared in a fuel oil suspension contained unsaturated and aliphatic hydrocarbons, and some aromatic hydrocarbons as sources of repugnant odours (Ogata & Ogura, 1976). Eels were also maintained in a controlled laboratory environment in water with a suspension of crude oil. Gas c h r o m a t o g r a p h y bf the eel flesh revealed the presence of paraffins and organic sulfur c o m p o u n d s whose concentration increased in accordance with rearing time (Ogata et al., 1977). Each of the organic c o m p o u n d s was identified for estimation of the concentration factors. In the present study, the flesh of eels and shortnecked clams exposed to a crude oil suspension in laboratory was analysed by gas c h r o m a t o g r a p h y mass spectrometry, and the petroleum substances present identified.

examination on the 15th day. Twenty short-necked clams were inserted into the surface of the sea mud and reared in sea-water containing 50 ppm of crude oil suspension and taken out on the 20th day. Eels and short-necked clams for the control group were reared and taken out under the same conditions. Eel flesh and the soft bodies of short-necked clams were homogenized by a Waring blender in the cold room. For gas chromatography and gas chromatography-mass spectrometry analysis, eel flesh and short-necked clam homogenates were treated with 2 N KOH/CzHsOH for saponification as reported in the previous paper (Ogata et al., 1977), and then adsorbed by passing the treated crude oil through a silica gel/alumina (1:1) column. The compounds eluted from the column with n-hexane were washed with water three times, and concentrated with a rotary vacuum evaporator. The concentrate was extracted three times with n-hexane previously saturated with acetonitrile. In general, the extracted acetonitrile solution was dried by rotary evaporator and the residue dissolved in n-hexane, since n-hexane gives a sharp solvent peak during gas chromatographic analysis. The residue was then analysed by gas chromatography or gas chromatography-mass spectrometry. This procedure was carried out on all samples except the extracted acetonitrile solution from crude oil. 20ml of which was concentrated by Kuderna-Danish evaporative concentrator to 0.5 ml.

Gas chromatography The apparatus employed was the Shimazu 4 BM-PFE gas chromatography with a FPD detector. Analysis was made under the following conditions: column liquid phases; Silicone SE-30. 5%; support, Chromosorb W(AW-DMCS); mesh. 60-80: size of column : 3 mm x 2 m; column temperature, from 60 to 260°C; programmer rate, 10°C min- t ; carrier gas, nitrogen: flow rate. 40 ml rain- t chart speed, 1.0 cm min- ~; range, 10 mV. Gas chromatography-mass spectrometric analysis was as follows. The apparatus used was a Shimazu LKD-9000. Analysis was made under the following conditions: column liquid phases; Silicon SE-52, 3%; support Chromosorb W(AW-DMCS); mesh, 80-100; size of column: 3 mm x 4 m; column temperature, from 80 to 260°C; programmer rate, 8°C m i n - t ; carrier gas, helium; flow rate, 28ml m i n - t ; separation temperature, 275°C; ion source temperature, 270°C; electron energy, 70eV; chart speed,

EXPERIMENTAL Eels weighing 130--180 g and short-necked clams weighing 15g were reared in fresh sea-water for 5 days before the experiment. The petroleum used was a mixture of Arabian light crude oil and Zubea crude oil (4:1) with a specific gravity of 0.857, The fish tank was 45 x 60 x 37 cm. Control eels and 10 short-necked clams were reared in fresh water kept at 20°C using a constant air supply. Five eels were reared in sea-water containing 1000ppm of crude oil suspension and were selected for 613

614

MASANA OGA'IA, YOSHIO M1YAKE and YOSHIO YAMASAKI

1.0cm min-~ for crude oil, and 0.5 cm min ~t for homogenates; range. 10 mV.

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RESULTS 2

Gas chromatography Eel flesh and the soft bodies of short-necked clams were analysed by gas chromatography of acetonitrile extracts of crude oil. Gas chromatograms (Fig. IB and C) showed many peaks contained in the crude oil (Fig. 1A) which were not seen in control eel flesh and short-necked clams (Fig. 1D and E).

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Gas chromatography-mass spectrometry Mass spectra were taken almost over the entire range of the unresolved main peaks in the gas chromatograms. The substances found in eel flesh and the soft body of short-necked clams are summarized in Table 1. Crude oil. Figure 2 shows the gas chromatogram of the acetonitrile extract from crude oil, at which mass spectral scans were taken. Identified components of peaks 1-18 of the gas chromatogram with crude oil are listed in Table 1. Alkylbenzenes, methylnaphthalenes, dibenzothiophene and methyldibenzothiophenes were recognized. Eel flesh and ,soft body of short-necked clams. Figure 3 shows gas chromatograms of the hexane extract with points at which mass spectral scans were taken marked. Gas chromatography-mass spectrometry analysis of eel flesh extract (Fig. 4) revealed the presence of l-methyl, 2-methyl, dimethyl, trimethylnaphthalenes, and dibenzothiophene, and that of the soft bodies of short-necked clams (Fig. 5) revealed dimethyl, trimethylnaphthalenes, dibenzothiophene, and monomethyl, dimethylbenzothiophene. The petroleum components transferred to the soft bodies of short-necked clams had higher boiling points than those transferred to eel flesh: Dibuthyl phthalate was not detected in the crude oil, but identified in the oil polluted eel (Fig. 4). Therefore, dibuthyl phthalate was thought not to be related to crude oil pollution of the eel.

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DISCUSSION

The data from gas chromatography-mass spectrometry analysis indicate that the methyl derivatives of naphthalene, dibenzothiophene were identified in the fish and the shellfish, i-Methyl and 2-methylnaphthalenes were identified in the eel flesh only, and the methyl derivatives of dibenzothiophene were identified in the short-necked clams only (Table l). On the other hand, a gas chromatogram of eel flesh extract (Fig. 1B) contained the peaks of l-methyl, 2-methyl, dimethyl, trimethylnaphthalenes and dibenzothiophene, and small amounts of monomethyl, dimet hyldibenzothiophene and monomethylphenanthrene, which were identified by gas Chromatography-mass spectrometry of crude oil. A ~ chromatogram of short-necked clam extract (Fig. IC)

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of short-ncw,~ked clam reared in crude off sus~pcnsion~ A: crude oil; B: eel reared in crude oil suspension: C: shortnecked clam reared in crude oil suspension: D: contrbl ¢¢1 flesh; E:¢ontrol ~ort-acck~l clam. Peak numbers correspond to the numbers in Table 1. Analytical sensitivities of peak abundance in Figs, {D) and (E) ate twice as much as those in Figs.7(A), I'B) and (C) respectively.

Identification of substances transferred to fish

615

Table I. Identification of components in the fraction of aromatic compounds from crude oil by mass spectrometry

Peak No.

m/e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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Calculated formula

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Identified components

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m-. p-Xylene o-Xylene Propylbenzene

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Tert-butylbenzene Methyl-tert-butylbenzene l-Methylnaphthalene 2-Methylnaphthalene Dimethylnaphthalene Trimethylnaphthalene (a and b) Dibenzothiophene

C~3HIoS C~ ~H ~2 C 14H i .,S Ca .~HI,,S C, ~H2.,O,,

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* Presence of phenanthrene in oil (Dickie et al,, 1967) and alkylphenanthrene in oyster (Ehrhardt. 1972) was described in their previous papers. ";"Peaks 17a and 17b. showed the peaks of dimethyl, trimethyinaphthalenes, dibenzothiophene, and monomethyl, dimethyldibenzothiophenes, and small amounts of l-methyl, 2-methylnaphthalenes and trimethyldibenzothiophene.

The identification of these small peaks by gas chromatography-mass spectrometry analysis was difficult in this exteriment. In a previous report ( O g a t a & Ogurm 1976), transfer to fish of alkylbenzenes (propyl, trimethyl,

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pounds was dried by rotary vacuum evaporator, the residue dissolved in hexane, and then, injected into a gas chromatography-mass spectrometry system. Alkylbenzenes in homogenates were thought to be completely evaporated by the drying procedure. Therefore, in the extract from homogenates, these alkylbenzenes were not found by gas chromatography-mass spectrometry analysis, though these compounds were present in the mass spectrogram of the extract from crude oil. This also proved that the alkylbenzenes were not present in the gas chromatogram (Fig. 1A) of crude oil extract prepared by the procedure of drying the

acetonitrile solution then dissolving the residue in hexane. Cahnmann & Kuratsune (1957) descrified that the spectrophotometric investigation of oysters collected in a polluted area led to the detection of a number of polycyclic aromatic hydrocarbons• Ehrhardt (1972) reported that oysters from a location in Galveston Bay contained mono-, di- and tri-nuclear aromatic hydrocarbons, Ci~biphenyl, methylfluorene, methyldihydrophenanthrene, hexahydropyrene, and alkyldihydrophenanthrene. Nakamura et al. (1975) described the presence of dibenzothiophene derivatives in oysters caught in the Seto inland sea.

618

MASANAOGATA, YOSHIOMIYAKEand YOSHIOYAMASAKI

The present experiment indicates that methyl-derivatives of naphthalenes and dibenzothiophene were petroleum components transferred from crude oil to eels and short-necked clams. Trace amounts of organic sulfur compounds in the crude oil, eel flesh and soft bodies of short-necked clams could be determined by gas chromatography attached with FPD, therefore these compounds are recommended as a marker of oil pollution in fish and shellfish. Recently we have succeeded in estimating the concentration factor of total organic sulfur compounds by using F P D chromatography. Short-necked clams were reared in the 50 ppm crude oil suspension for 15 days. Average concentration factors of total organic sulfur compounds were estimated at about 180. The concentration factor of each of the organic sulfur compounds will be described in the next report. Acknowledgements--We express our thanks to Miss Y. Yoshida and Miss M. Kameyama for the technical assistance, and to Dr. A. Talbot for correcting this manuscript. This work was supported in part by the Ministry of Education. Agency No. 210516.

REFERENCES

Cahnmann H. J. & Kuratsune M. (1957) Determination of polycyclic aromatic hydrocarbons in oysters collected in polluted water. Analvt. Chem. 29, 1312-1317. Dickie J. P. & Yen T. F. [1967) Macrostructures of the asphaltic fractions by various instrumental methods. Analyt. Chem. 39, 1847-1852. Ehrhardt M. (19721 Petroleum hydrocarbons in oysters from Galveston Bay. Envir. Pollut. 3, 257-271. Nakamura A., Murakami Y., Miyata H., Hori S., Maeda K. & Kashimoto T. (19751 Studies on organic sulfur contamination of fishery product, J. Fd Hy#. Soc. Japan (in Japanese) 16, 282-284, Ogata M. & Miyake Y. ~1973) Identification of substances in petroleum causing objectionable odours in fish. Water Res~ 7, 1493-1504. Ogata M. & Miyake Y. (1975) Compounds from floating petroleum accumulating in fish. Water Res. 9, 1075 1078. Ogata M., Miyake Y., Kira S.. Matsunaga K. & lmanaka M. (1977) Transfer to fish of petroleum paraffins and organic sulfur compounds. Water Res. 11, 333-338. Ogata M. & Ogura T. (1976) Petroleum compounds and objectionable malodorous substances in fish flesh polluted by boiler fuel oil. Water Res. 10, 407-412.