Forensic Science, 10 (1977) 253 - 260 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

253

CASE REPORT OF DEATHS CAUSED BY FREON GAS

MASAHIKO MORITA*, AKEMI MIKI*, HISASHI KAZAMA* and MASAKATSU SAKATA* *

Department of Legal Medicine, Sapporo Medical College, Sapporo (Japan)

(Received December 31, 1976; accepted in revised form August 8, 1977)

SUMMARY

A report of two autopsies which is thought to be the first case of its kind in Japan is presented here. The cause of death was concluded to be oxygen deficiency from the filling of a compartment by Freon gas escaping from a cylinder. However, the authors suspect that Freon per se is not a non-toxic substance as had been believed but has some toxicity because of the existence of fat droplets in hepatocytes of mice exposed to Freon gas and because of the behavioral findings on them during experiments.

INTRODUCTION

The authors had an opportunity to participate in a forensic autopsy re- garded as the first case of its kind, at least in Japan. The cause of death was explosively escaping gas which was used for refrigeration aboard a trawling ship.

This report will present the case in which six fishermen died in the com- partment of a deep sea fishing boat and will also include experimental data concerning the accident.

CASE

On may 28, 1975, an unfortunate accident occurred off the coast of the Maritime Province of Siberia. Freon gas, used as a refrigerant in deep sea fishing, escaped through the flow-control valve because a fisherman mis- handled it. The escaping gas completely filled the engine room wherein the refrigerator was located. The chief engineer, inspecting the electric generator following a blackout, was the first to discover the accident. He discovered the malfunction of the generator in the engine room soon after noticing the blackout (Figs. 1 and 2). He called this to the attention of the other nine

*Present address: Minami 1, Nishi 17, Chuo-ku, Sapporo, 060 (Japan). **Present address: Faculty of Pharmaceutical Sciences, Higashi Nippon Gakuen Univer-

sity, Hokkaido, 061-02 (Japan).

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Fig. 1. Side view of the boat in which the accident occurred.

Engine Room

Fig. 2. Top view of the boat.

crew members who had also been resting in their quarters and while attempt- ing to rescue the fisherman in the engine room he also lost consciousness.

Five of the nine crew members who had been in their quarters were able to escape but four who were in lower and middle bunks could not extricate themselves (Figs. 1 and 2).

The valve through which the liquid Freon evaporated was in the innermost part of the engine room and situated at a point between the refrigerator and the storage cylinder. The Freon gas escaped at a pressure of -6-7 kg/cm2 from an opening 3.0 cm in diameter located under the valve (Figs. 1 and 2).

The cadavers were brought to a port in Hokkaido three days after the acci- dent and two of them, the chief engineer (A) and the fisherman (B) who mis- handled the valve, were offered to us for examination.

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EXAMINATIONS

Examinations were performed by: (1) routine autopsy with histological detection; and (2) chemical analysis for the detection of Freon in the organs. For the purpose of detection of Freon in the organs, blood, brain, liver and kidneys were removed from the cadavers and put in vacuuminized vinyl bags immediately and, after freezing by dry-ice acetone, stored in a freezer at -20 “C. Gas analysis was performed by head space analysis [l] using the gas chromatographic technique (Table I). For the measurement of the volume of gas, the digital integrator, devised for the gas chromatograph, was connected.

TABLE I

Gas chromatogram

Temperature: Packing Silica gel in 0.5 m of glass column column 100 “C injection 100 “C

Detector, Flame ionization

detector 120 “C Range, 4mV Carrier gas, Helium Flow rate, 40 cm3/min

Sensitivity, lo3 Ma

The standard curve was made using Asahi Fron 22 as pure Freon gas in a 10 kg cylinder made by the Asahi Glass Co. Ltd. It was used as a substitute for the Freon-22 refrigerant in the fishing boat. The standard curve was made by 0.2%, 0.5%, 1.0% and 2.0% proportion of Freon-22 gas to air using an officially approved 100 ml glass syringe.

The gas-equilibrium method for the detection of Freon gas was performed by head space analysis, and by the method of Nagata et al. [ 21, using - 0.3- 0.8 g of the organ tissue in a vial of 13.0 ml volume sealed with a rubber cap. The tissue in the vial was warmed in a water bath at 50 “C for 1 hour and the equilibrated Freon gas was introduced into the gas chromatographic instru- ment after cooling at room temperature. The Freon gas released from each tissue was measured repeatedly until the gas was no longer detected. The total volume of released gas was calculated by the addition of the values of all measurements.

Experimental examination of the changes of gas volume in the organs with the passage of time was also performed using male rabbits and small DD mice four weeks old, under an atmospheric condition of nitrogen 59%, oxygen 16% and Freon 25% for 60 minutes to cause death. Rabbits and mice were divided into two groups; one group was dissected immediately after death and the other was examined three days after death as were the fishermen. Both groups were examined exclusively for the investigation of fat droplets in hepatocytes by Sudan III as were the control mice which were fed on a stan- dard diet (NMF: Oriental Yeast Company) and had not received any treat- ment.

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RESULTS

Au topsy findings External examination (A) was a well-developed and well-nourished Japanese male, 168.0 cm in

stature, 73 kg in weight and appeared to be 42 years old as stated. (B) was also a well-developed and well-nourished Japanese male, 156.0 cm in stature, 64.0 kg in weight and appeared to be 54 years old as stated. He lacked left arm at the shoulder joint from a former accident.

No outstanding findings were observed from the external examination. If any were to be enumerated, (1) the dark tone of the postmortem lividity was remarkable, (2) palpebral conjunctivae showed congestion and there were petechial hemorrhages.

Internal examination Blood from both cadavers showed fluidity except for a few small pieces of

soft coagulation in (B). The lungs showed remarkable congestion and edema which was ascertained histologically (Fig. 3). Fine lipid droplets were ob- served in the cytoplasm of hepatocytes mainly in the peripheral zone (Fig. 4). No other findings which could be regarded as being involved with the cause of death were discovered.

Chemical gas analysis The amount of Freon gas contained in each tissue sample from the men

and rabbits is shown in Table II. The detected gas was ascertained as Freon-22 (monochloro-difluoromethane) because of the retention time of the gas which was two minutes and twenty seconds and which coincided with that of Asahi Fron 22 of Asahi Glass Co. Ltd. under the same operating conditions of gas chromatography.

DISCUSSION

It was clear from gas chromatographic analysis that the refrigerant used in the fishing boat was monochloro-difluoromethane (Freon-22). It is well known that dichloro-difluoromethane (Freon-12) is used as the refrigerant in refrigerators for domestic use [3]. The differences in the physical and chemical properties as given in studies on fluorinated hydrocarbon [ 3, 41 are shown in Table III [ 31 and it can be seen that the boiling point of Freon-22 (-40.8 “C) is lower than that of Freon-12 (-29.8 “C) and useful for the refrig- eration of much larger amounts of material (the catch of fish in this case) in a shorter period of time.

The authors consider that the cause of death in this case may be ascribed without controversy to suffocation from oxygen deficiency.

The cylinder which held the liquid refrigerant Freon-22 in the engine room of the fishing boat had a total capacity of 300 1. If 200 1 of liquid Freon-22

Fig. 3. Lung edema and congestion were remarkable in both fisherman. X 128.

Fig. 4. Liver of the fisherman (B), showing an abundance of fine fat droplets in the cyto- plasm of hepatocytes (black). The round white spots show the place where the fat droplets were situated. The nucleus of hepatocytes in this photograph is not clearly stained. x 528.

TABLE II

Amount of Freon gas in each tissue (/J/g and pg/g in parentheses)

Brain Lung Liver Kidney Blood

A 68 (282)

B 100 (414)

C 190 (787)

D 100 (414)

(2;:)

(3;;)

170 (705) f

(2;:)

(3:) 43

1178)

(&

(2::)

130 (538)

130 (539)

140 (576)

A and B are fishermen, C is a rabbit dissected immediately after death. D is a rabbit ex- amined 3 days after death.

TABLE III

Physical and chemical properties of Freon 11,22 and chloroform

F

Freon-22 CHClF, H-&F

dI

Boiling point -40.8 “C Vapor density 2.98

F

Freon- 12 CC12F2 CIM-F

dI

Boiling point -29.8 ‘C Vapor density 4.1

Chloroform CHCls Cl

HP+

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had been contained in the cylinder and subsequently escaped from it, accord- ing to our calculation, 78 m3 of gaseous Freon-22 would have been produced after evaporation. This volume of gaseous Freon-22 is more than enough to completely fill the engine room and the excessive gas may be thought to have passed into the neighbouring crew’s quarters through the doorways. Owing to the vapor density of Freon-22 (2.98; air = l), the gaseous Freon-22 sank to the bottom of the crew’s quarters as well as the engine room thus pushing the air upward.

Two facts support these conclusions. First, the electric generator which was connected to the engine of the fishing boat had stopped and second, the fishermen who had been resting in their quarters, especially on the lower and middle berths could not extricate themselves from the danger whereas those who were on the upper berths were able to do so.

Numerous reports have been published, especially on both the determina- tion of Freon in blood and tissues [4-91 and on accidents by inhalation (“sniffing”) [lo-171 .

Among the results obtained by the experiments on the autopsy findings, an interesting finding was revealed in the hepatocytes, i.e., fine fat droplets which were positive to the Sudan III staining were found mainly in the pe- ripherial zone of hepatic lobules in the cytoplasm of hepatocytes (Fig. 4). There may be several causes of the fat droplets, among which the effect of alcohol will be considered first.

Fishermen and day laborers in Japan habitually drink sake or other alco- holic beverages after work. The results of the examination of the exposure to Freon gas using mice revealed that fine granular fat droplets positive to Sudan III staining were found in the cytoplasm of hepatocytes of mice exposed to Freon gas (Figs. 5 and 6) (content of the gas is shown in the examination), whereas in the control mice only a few of the cells in the sinusoid, the Kupffer cells for instance, showed fat droplets in their cytoplasm and there was none in the hepatocytes (Fig. 7). Zonal differences of the existence of the fat droplets in hepatocytes were not discernible.

From the results mentioned above, the authors concluded that the appear- ance of fat droplets in the hepatocytes should not be ascribed to alcohol but is suspected to be the effect of Freon gas per se.

The chemical structural formula of Freon-12 and -22 are closely akin to chloroform (Table III), and Freon is also known to have an anesthetic action of about l/10 of chloroform [ 181. It is suspected that the appearance of fat droplets in the hepatocytes of mice exposed to Freon is closely related to its effect as a hepatic poison, which resembles a similar effect of chloroform. Secondly, the mice observed during the experiment showed the following miscellaneous symptoms as time proceeded; (a) reeling, (b) weakness of the forelegs, (c) falling down, tumbling, (d) flow of mucous fluid from mouth and nose, (e) a violent movement of the forelegs like the dog paddle in swim- ming, (f) cyanosis, and (g) the cessation of respiration.

The authors noticed especially the violent movement of the forelegs in dog paddle fashion (e). The movement was observed abruptly in all mice as time

Fig. 5 and Fig. 6 present the liver of mice exposed to Freon gas, showing an abundance of fat droplets in the hepatocytes. ‘C’ in both Figs. indicates the central vein. ‘+’ and the arrow indicate the nucleus and fat droplets, respectively. x 512.

Fig. 7. Liver of a mouse used as control showing a few of the fat droplets in only the extra-hepatic cells. They do not exist in the cytoplasm of hepatocytes. X 512.

proceeded and should not be overlooked because this movement is not insig- nificant in the sense that teenagers showed it in cases of glue sniffing as reported by Bass [ 111.

From the unusual movements, weakness of legs and falling down by the mice in the experiment, it may be considered that the deaths of the fisher- men in the present case may be attributed mainly to suffocation due to oxygen deficiency but also to a condition in which they could not extricate themselves from the danger of the gas due to muscle weakness while still conscious.

Further studies will be reported by successive experiments.

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ACKNOWLEDGEMENTS

The authors wish to express their thanks to Prof. S. Yasoshima for his valuable advice and to Mr. S. Muraoka for his technical assistance in drawing the figures.

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