Offensive-odor substance in the evil-smelling fish from the sea polluted by petroleum and petrochemical industrial waste. 1. Identification of offensive-odor substance.

For the purpose to reveal the substance as the source of offensive odor of the fish from the sea facing petroleum and petrochemical industries, analyses have been made on the sea water, industrial wastes, offensive odor fish meat and the eels kept in the sea or industrial waste for a certain period, by means of gas chromatography, infrared, and ultraviolet absorption spectrophotometries and mass-spectrometry. Observations revealed toluene as a possible main source responsible for the bad smelling of the fish. Finally, by analysing the meat extract of the fish kept in the sea water containing toluene and by the same smell as that of the offensive odor fish from the off-shore of the industries, it has been definitely confirmed that toluene is the very substance that imparts the offensive odor to fish. The activated sludge process proved to be an effective method to remove toluene from the industrial wastes. A discussion was made on the aliphatic carbohydrate as the possible source of the offensive odor of fish. ∗PMID: 4254201 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 21, 471-481 (1970) OFFENSIVE-ODOR SUBSTANCE IN THE EVIL-SMELLING FISH FROM THE SEA POLLUTED BY PETROLEUM AND PETROCHEMICAL INDUSTRIAL WASTE REPORT 1. IDENTIFICATION OF OFFENSIVE-ODOR SUBSTANCE Masana OGATA and Yoshio MIYAKE Department of Public Health, Okayama University Medical School, Okayama, Japan (Director: Prof. M. Ogata) Received for publication, June 20, 1970 After World War II, petroleum and petrochemical industries have developed rapidly throughout Japan, and 20 years since then water pollution of the sea facing these industrial areas has raised a social problem with the industrial waste. In 1953, offensive-odor fish were found first among those from Ise Bay of Yokkaichi petroleum industrial district and these fish having obnoxious odor markedly increased in number by 1958 (1-4). In Mizushima district near Okayama, we have also newly developed petroleum and petrochemical industries since 1963, and the offensive odor fish appeared within the same year and in 1965 the offensive-odor extended over the residential districts about 10 km from the seashore. And it has become desirable to identify the offensive odor substances in the fish and establish an effective way to improve the situation urgently. Therefore, we tried to isolate and identify substance or substances by means of gas chromatography, ultraviolet and infrared absorption spectra, and mass spectrum. Besides these, the possibility of the degradation of the substance to the harmless by the activated sludge process was also studied. In this paper, the results obtained on the fish from the offing of Mizushima, eels kept in the sea water, and in the industrial waste are briefly reported with the analytical data of the sea water and the wastes. MATERIALS AND METHODS Offensive-odor fish caught in the sea facing the petroleum and petrochemical industries of Mizushima served as materials. The industrial waste from a few factories in the same area and some polluted sea water were also used for the analysis. All these fish and waste were frozen at -20°C immediately after collecting. Eels from other areas weighing 120-150 g were also used. They were kept 471 1 Ogata and Miyake: Offensive-odor substance in the evil-smelling fish from the sea Produced by The Berkeley Electronic Press, 1970 472 M. OGATA and Y. MIYAKE in a tank contammg sea water, (30 liters) and the industrial waste I liter at 20°C with air supply for one week. In another series of experiments, toluene and other organic solvents were given to the eels in tank, I ml each every day for seven days. For gas chromatography the industrial waste and the polluted sea water were steam-distilled, and an equal volume of distillate, waste or sea water was taken. The distillate was warmed at 55°C in incubator while shaking, and the evaporated gas (20 ml) was put in the air sampler for gas chromatography. To the offensive-ordor fish isolated and gatherd was added half volume of distilled water and homogenized with Waring Blender. The distillate abtained by steam distilling the homogenate, was warmed at 55°C. And gas phase on the distillate was taken for gas chromatography. The apparatus employed was Hitachi K 53, FID ditector, and the analysis was made in the conditions as follows; Column liquid phases; 1. 2. 3. This (cyanoethoxy) propane, support; Shimalite, mesh, 60-80, size of column; 3 mm x 2 m, column temperature; 60°C, carrier gas; nitrogen (40 ml/min), air pressure; 1. 5 kg/cm2, chart speed; 10 mm/min. The fractions identified as the offensive-odor substance by gas chromatography was divided into 4 parts, the first one was taken into glass beads for the next gas chromatograph for the further fractionation in detail, the second one into carbon tetrachloride solution for infrared absorption spectrophotometry, the third one into dioctylphtalate (DOP) for mass spectrometry, and the last one into absolute ethanol solution for ultraviolet absorption spectrophotometry. Infrared absorption spectrum was taken by infrared spectrophotometer Hitachi EPI-G3 with lcm light path. Mass spectra were taken by mass spectrophotometer, Hitachi, RMU-6 with evaporated gas at 120°C. Conditions of mass spectrometry are as follows; chamber volt; 70 V, total edission; 60 p.A, target current; 70 p.A, chamber temp; 200°C, evaporation temp.; 180°C. Ultraviolet absorption spectrum was taken by spectrophotometer of Beckman DK-2A with 1.0 cm light path. Industrial waste from oil refineries was collected for 7 days, for adapting microorganisms in the activated sludge to industrial waste. Then the activated sludge, 500 g in wet, 20 ml of 1.2 M potassium phosphate monobasic, 0.1 g ammonium sulfate, and 3 ml of 50 % corn starch solution (industrial waste of corn starch refinery company) were added to the petroleum industrial waste, mixed well, pH was ad justed to 7.2 with 10 % sodium hydroxide and then incubated at 37°C for one day. The gases from the waste before and after the treatment were analyzed by gas chromatography. RESULTS Gas chromatogram of the gas from offensive-odor fish (grey mullet, Mujil ]aponicus), gave the peak which appeared constantly but could not be found in that from normal fish (Fig. 1); the retention time was 7.61 2 Acta Medica Okayama, Vol. 24 [1970], Iss. 4, Art. 8 http://escholarship.lib.okayama-u.ac.jp/amo/vol24/iss4/8 Evil-Smelling Fish in the Polluted Sea 473 times as long as that of hexane. Beside this specifIcity to offensive odor fish, another inconstant peak appeared occasionally. These peaks are defined as offensive-odor peaks, and the substances as offensive·odor substance, because of their bad smelling in mouth.