Fd Cosmer. Toxicol. Vol. 11, pp. 841-845. Pergamon Press 1973. Printed in Great Britain

SHORT PAPERS Dimethylnitrosamine in Chinese Marine Salt Fish

Y. Y. FONG and W. C. CHAN

Departments of Biochemistry and Pathology, University of Hong Kong, Hong Kong

(Received I7 May 1973)

Summary-Samples of marine salt fish from the markets in Hong Kong were analysed for N,N-dimethylnitrosamine (DMNA). Significant amounts of this compound, commonly in the 0.0550.3 ppm range, were detected by gas chromatography and were confirmed in several samples by gas chromatography-mass spectrometry. Salt fish prepared with crude salt con- tained much more DMNA than that prepared with pure sodium chloride.

Introduction

Nitrosamines have been identified and confirmed in various amounts in many types of prepared food (Fazio, Damico, Howard, White & Watts, 1971; Sen, 1972; Wasserman, Fiddler, Doerr, Osman & Dooley, 1972). Some methods of preparation are conducive to the formation of nitrosamines. The preparation of Cantonese marine salt fish, a favourite dish along the South China coast and also in South-east Asian countries is a good example. In the fish itself there are rich sources of secondary and tertiary amines (Rea & Shewan, 1949; Varela & Wojciech, 1956), while in the crude salt used to pickle the fish, there is nitrate and possibly nitrite. The pickling and drying is done in the open, so that the fish is liable to contamination by bacteria. All these factors suggest the possibility that nitrosamines might be formed in the salt fish.

The present paper reports the detection and confirmation of dimethylnitrosamine (DMNA) in marine salt fish obtained from local markets and an attempt to study the possible mechanism of its formation.

Experimental

Materials. Different species of Cantonese salt fish were purchased from local retailers The fish (obtained whole) were chopped into small pieces and homogenized with 20% potassium carbonate solution using a Sorvall Omni-Homogenizer. For each experiment, unless otherwise specified, a total of 4 kg fish was used. Nitrosamines extraction was carried out according to Sen, Smith, Schwinghamer & Howsam (1970). Briefly, the sample was first treated with methylene chloride, then warmed to remove the solvent and steam-distilled, the distillate was passed through a combined ion-exchange polyamide column and the combined etlluent was extracted five times with methylene chloride, dried over anhydrous sodium sulphate and concentrated.

Analytical procedures. Quantitative measurements for DMNA were carried out with a Varian Aerograph gas chromatograph, Model 1864-4, using two different columns: (i) a 6 ft x l/8 in. stainless-steel column packed with 5 % SE30 on high-performance grade Chromosorb W/AW-DMCS (SO-100 mesh), using a nitrogen flow of 28 ml/min and

841

842 Y. Y. FONG and w. c. CHAN

column, injection and detector temperatures of 48, 80 and 125”C, respectively (range 10, attenuation 1); (ii) a 10 ft x l/8 in. stainless-steel column packed with 10% Carbowax 20 M on high-performance grade Chromosorb W/AW-DMCS (SO-100 mesh), with a nitrogen flow of 25 ml/min, and column, injection and detector temperatures of 95, 140 and 17O”C, respectively (range 10, attenuation 1). The presence of DMNA was confirmed by the use of the same gas chromatograph with a 10% Carbowax 20 M stainless-steel column interfaced with a Hitachi Perkin-Elmer RMS4 mass spectrometer. Helium carrier gas was used at a flow rate of 25 ml/mm, and hydrogen- and air-flow conditions were 40 and 350 ml/min, respectively. The column effluent was split approximately 1: 9, 90% passing into the mass spectrometer via an inlet line heated at 200°C. The mass spectrum was obtained at an ionizing voltage of 70 eV.

Nitrate and nitrite determinations. Nitrate and nitrite determinations were carried out on six different batches of salt commonly used to prepare salt fish, on the salt scraped from the salt fish and on the salt fish themselves. The method adopted was that of Follett & Ratcliff (1963).

Isolation and identification of bacterialflora of saltfish. Inner portions of salt fish, removed with a pair of sterile forceps, were emulsified in sterile water by grinding with a sterile pestle and mortar and were then cultured in a 10% NaCl infusion broth at 37°C for 1 day. Sub- cultures were then prepared on various IO-25 % salt milk-agar plates and incubated at 37°C. The usual morphological, staining, culture and biochemical tests were performed on the bacteria.

Salting of fish with laboratory NaCI and market-bought crude salt. Two different species of marine fish, white herring and yellow croaker (both dead), were purchased from local retailers. Equal portions of each were salted with crude salt and with chemically pure laboratory salt, respectively. The usual method of preparation practised by local fishermen was followed (McCarthy Jc Tausz, 1952). The fish were not gutted, nor were the gills removed. The fish were pickled for l-2 days by putting layers of salt between them. They were then dried in the open air under the sun for a week or so depending on the weather. Just prior to drying, solid salt was forced into the mouth and between the gills. The fish were then put away and stored in the kitchen for 4-5 months, before being analysed for the presence of DMNA by the method already described.

Results

Table 1 shows the concentration of nitrite, nitrate and DMNA in the different batches of salt fish purchased from the local retailers. In white herring, batch G, the level of DMNA reached 1 ppm, but this could not serve as a typical example since batch G was only half a fish (head, weight 80 g), chosen for analysis because of its highly spoiled condition. The identification of DMNA from several batches was based on thin-layer and gas-liquid chromatography only, as the combined gas chromatography-mass spectrometry (GC-MS) technique was not available at that stage of our study. However, the batches marked with daggers were confirmed by the GC-MS technique; the m/e peaks corresponding to M+ (74) and NO+ (30) were distinctly visible over the background signals.

Levels of nitrate were found to be appreciable in the crude salt commonly used to prepare salt fish, amounting to 17, 18, 20, 20, 30 and 40 ppm in six samples of such salt purchased from local retailers. The nitrite content of these samples was insignificant, however, reaching no more than 1 ppm.

DIMETHYLNITROSAMINE IN MARINE SALT FISH 843

Table 1. Dimethylnitrosamine concentrations in salt fish obtained from local markets

Fish samples DMNA*

b-w4

Residual nitrite (Ppm)

Residual nitrate (Pm)

White herring A

: D E

z

Yellow croaker A B C D E

Anchovies A B

Croaker A B

Pomfret A

0.30 0.20 0.ost 0.1ot omt oaq 1.00

0.20 0.01t o.ost 0.06t 0.08

0.10 2 10 m02t 1 8

0.03 0.02.f

Not detected

1 1 2 1

- - 2

2 -

1

6 10 30

ii 8 10

30 18 20 15 30

10 20

30

*iV,N-Dimethylnitrosamine, corrected for recovery of 70 % determined experimentally.

tIdentity con&med by gas chromatography-mass spectro- metry.

The difference between the DMNA level in salt fish prepared with chemically pure salt and that in fish prepared with crude salt is shown in Table 2. The identity of the DMNA was established by GC-MS.

Table 2. Differences in DMNA levels in salt fish prepared with crude salt and NaCI

Fish

No. of samples analysed

DMNA* (ppm) in 6sh prepared with

Crude salt NaCl

White herring Yellow croaker

0.4t 0.01 t : 0.27 ot.uxt

*N,N-Dimethylnitrosamimine, corrected for recovery of 70 % determined experimentally.

tconflrmed by gas chromatography-mass spectrometry.

The nitrate-reducing bacteria isolated from the fish were identified as a coagulase-positive Staphylococcus aureus.

844 Y. Y. FONG and w. c. CHAN

Discussion

The presence of DMNA demonstrated in Cantonese marine salt fish obtained from markets in Hong Kong was to be anticipated from the conditions under which the fish is prepared. The level of DMNA varied greatly and, with one exception, all the batches of fish examined showed significantly higher DMNA‘levels than those reported for preserved fish elsewhere (Crosby, Foreman, Palframan 8c Sawyer, 1972; Fazio et al. 1971). The variation in DMNA content was related in part to the degree of contamination by nitrate-reducing staphylococci. This organism isolated from salt fish has been shown to increase the DMNA content in salt-fish broth (Fong & Chan, 1973). In the present survey, the most spoiled batch contained the largest amount of DMNA.

The nitrate present as an impurity in the pickling salt is a precursor of DMNA. This was demonstrated in the experiment in which both crude salt and pure NaCl were used to pickle fish. Salt fish prepared by using crude salt, which contains 17-40 ppm nitrate but not more than 1 ppm nitrite was shown to contain 40 times more DMNA than the same fish pickled.

Different species of fish showed different levels of DMNA. Salted white herring contained more DMNA than salted yellow croaker, while a single batch of pomfret contained no detectable DMNA at all. It is possible that the contents of secondary and tertiary amines, both precursors of nitrosamine, may vary widely in different species of fish (Rea & Shewan, 1949; Shewan, 1937).

The above observations suggest that the prerequisites for DMNA production in salt fish may be the availability of secondary amines in the fish, the presence of nitrate in the crude salt used for pickling and the intervention of nitrate-reducing staphylococci or other bacteria. In vitro formation of nitrosamines from its precursors by bacteria has been demonstrated by several investigators (Collins-Thompson, Sen, Aris & Schwinghamer, 1972; Hawks- worth & Hill, 1972; Sander, 1968).

The source of staphylococci is probably contamination during preparation of the fish. McCarthy & Tausz (1952) studied the salt-fish industry in Hong Kong and commented on the unhygienic way the fish are dried in the open, under the sun. Another source may be the gut of the fish, which are not gutted. The same authors suggested for hygienic reasons that the drying should be done in mechanical driers. We have now a stronger reason to recom- mend this, as the reduction of infection would lead to a reduction in nitrosamine levels.

Hong Kong is an area where the incidence of liver cell carcinoma and nasopharyngeal carcinoma is high. Whel her the DMNA in salt fish may play a causative role in this carcino- genesis remains to be proven. The amount of salt fish consumed at any one time is small- not more than 10 g-but in some families this dish may appear at every meal and some people may develop a taste for it and actually prefer the spoiled part. In these circumstances, the intake of DMNA over the years may be significant.

Acknowledgements-We wish to express our appreciation to the Chemistry Department, University of Hong Kong, for the use of their equipment, to Dr. R. S. M. Tse for advice on gas chromatography-mass spectrometry and to Mr. S. C. Wong for technical assistance with the mass spectrometer.

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DIMETHYLNITROSAMINE IN MARINE SALT FISH 845

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