Antibiotics in Fish Preservation

BULLETIN -No. 12'1

By H. L. A. TAR R Fisheries Research Board oj Canada Technological Station, Vancouver 8, B.C.

PU B LISHED BY THE FISHERIES RESEARCH

BOARD OF CAN ADA UNDER THE CONT ROL OF

THE HONOURABLE THE MINISTER OF FISHERIES

9TTAWA, 1960

",ice: 50 cents Dipping fish fillets in an antibiotic solution of concentration 10 parts per million. BULLETIN No. 124

Antibiotics in Fisll Preservation

By H. L. A. TARR Fisheries Research Board of Canada Technological Station, Vancouver 8, B.C.

PUBLISHED BY THE FISHERIES RESEARCH

BOARD OF CANADA UNDER THE CONTROL OF

THE HONOURABLE THE MINISTER OF FISHERIES

OTTAWA, 1960

89262-0-1� W. E. RICKER

N. M. CARTER

Editors

ROGER DUHAMEL, F.R.S.C. QUEEN'S PRINTER AND CONTROLLER OF STATIONERY OTTAWA, 1960

Price; 50 cents Cat. No. Fs 94-124.

(ii) BuLLETINS OF THE FISHERIES RESEARCH BOARD OF CANADA are published from time to time to present popular and scientific information concerning fishes and some other aquatic animals; their environment and the biology of their stocks; means of capture; and the handling, processing and utilizing of fish and fishery products. In addition, the Board publishes the following: An ANNUAL REPORT of the work carried on under the direction of the Board. The J OURNAL OF THE FISHERIES RESEARCH BOARD OF CANADA, containing the results of scientific investigations. ATLANTIC PROGRESS REPORTS, consisting of brief articles on investigations at the Atlantic stations of the Board.

PACIFIC PROGRESS REPORTS , consisting of brief articles on investigations at the Pacific stations of the Board. PROGRESS REPORTS of the Biological Station and Technological Unit, London, Ont., consisting of brief articles on investigations by the Station and Unit. The price of this Bulletin is 50 cents (Canadian funds, postpaid). Orders should be addressed to the Queen's Printer, Ottawa, Canada. Remittance made payable to the Receiver General of Canada should accompany the order. All publications of the Fisheries Research Board of Canada still in print are available for purchase from the Queen's Printer. Bulletin No. 110 is an index and list of publications of the Board to the end of 1954 and is priced at 75 cents per copy postpaid. For a listing of recent issues of the above publications see inside of back cover. These publications may be consulted at libraries of any of the Board's establishments and in many Canadian university and public libraries.

(ii i)

CONTENTS PAGE

INTRODUCTION ...... 1

USEFUL ANTIBIOTICS ...... 2

METHODS OF ApPLICATION ...... 3

DIFFICULTIES EXPERIENCED IN ApPLICATION ...... 3

RESULTS OF ApPLICATIONS IN DIFFERENT FISHERIES ...... 5

Salmon trolling boats...... 5

Ground fish ...... 6

Use of antibiotics in refrigerated sea-water storage of fish...... 8

Dipping whole fish in comparatively strong antibiotic solutions

followed by icing them...... 11

Treatment of fillets and steaks .... 12

PRESERVATION OF SHELLFISH...... 13

PRESERVATION OF MARINE MAMMALS ...... 15

ANTIBIOTIC RESIDUES IN TREATED FISH AND THEIR INACTIVATION

BY COOKING ...... _ 15

PUBLIC HEALTH ASPECTS...... 17

OFFICIALLY PERMITTED ApPLICATIONS...... _ ...... 18

REFERENCES...... 20

(V) INTRODUCTION

Spoilage of fish caused by micro-organisms is singly the most serious problem which faces those engaged in utilization of fishery resources. Indeed, commonly used general methods of preservation such as salting, canning and freezing have as their main objective prevention of microbiological deterioration. Towards the end of the last century many attempts were made to preserve fish with chemical agents. Few of these, other than common salt, were used extensively for preservation of fresh fish, but many were and still are used for the preservation of acid-cured types of fish products. Thus in certain European countries hexa methylenetetramine (urotropin), benzoic acid and certain of its esters have been used in comparatively low concentrations to considerable advantage in pre se rving vinegar-cured herring and other similar fish products. Unfortunately, preservatives of this type are normally effective only with acidified products, and cause but a feeble preservation of fresh chilled fish. It is for this reason that a good preservative for fresh fish was sought. Most of the desirable food fishes have a delicate bland flavour and fragile texture, and it is not easy to find a preservative which will not damage these desirable properties. Most preservatives studied caused undesirable changes in the fish, were not effective or were more or less toxic. The only substance which so far as the writer is aware was used quite extensively for preserving fresh fish was sodium nitrite. The effectiveness of this substance as a fresh fish pre servative was first recognized in 1939 (Tarr and Sunderland, 1939) , following which its use became very widely spread in the Canadian maritime provinces. It has been stated that by 1950 more than 90% of the fresh fillets shipped from the Atlantic coast to central Canada were being treated with sodium nitrite (Castell and Greenough, 1958a) . For over 10 years sodium nitrite was a permitted preservative for fresh fish under the Canadian Food and Drugs Act, and it was not until 1959 that these regulations were amended so that nitrites were no longer on the list of additives permitted for inclusion in fish products. In the meantime the Canadian regulations had been altered so as to permit the use of certain antibiotics to preserve fish products (see below) . It is interesting to note that nitrites are presumably still used extensively in Norway as a harmless and permitted preservative for herring intended for reduction (Heen et al., 1955) . The research leading to the use of antibiotics on fish and tither foods has been adequately reviewed (Campbell and O'Brien, 1955; Tarr, 1956, 1957, 1960a, 1960b (and unpublished) ; Partmann, 1957; Tomlinson, 1957; Farber, 1959) . Briefly, it may be said that early attempts to use antibiotics between 1944 and 1950 were unsuccessful, and it was not until the wide-spectrum antibiotics became available in 1950 that the potential value of antibiotics of the tetra cycline group for fish preservation was first established at the Technological

1 89262-0-2 Station in Vancouver of the Fisheries Research Board of Canada (Tarr et al., 1950) . (Fig. 1). At the time of writing almost exactly 10 years have elapsed since this discovery, and during this time further applied research on the use of tetracycline antibiotics in fishpreservation has been carried out in many different countries and a number of applications have been permitted under the pure foods regulations of these countries. There have also been many applications to poultry and meats, and the general problems which have arisen in these applications have often been similar to those experienced with fish.

USEFUL ANTI BIOTI CS

Of all the antibiotics so far studied for fishpreservation the only ones which have shown real effectiveness are those of the tetracycline group. At present only two of these have received official permission for use on flesh foods on this continent, namely Aureomycin (chlortetracycline, CTC) and Terramycin (oxytetracycline, OTC) . For reasons which are at present not entirely clear CTC has been found more effective for fish preservation than OTC when tested in similar concentrations (see below) . ::\Iruch of the initial experimental ,vork on fish was carried out with highly purified clinical grades of antibiotics. At present so-called "food grades" of these antibiotics are available; thus, CTC is sold as "Acronize" and OTC as "Biostat". These food grade materials contain

FIGURE 1. Cultures of fish-spoiling bacteria (grey area) growing on plates in which tablets (white discs) containing various concentrations of antibiotics have been placed. The clear dark ring around six of the tablets shows inhibition of bacterial multiplication and growth.

2 between 10 and 20% of active antibiotic, the remainder being a harmless filler such as calcium acetate or sodium chloride. Appropriate directions regarding application of these are provided by the manufacturers.

METHODS OF APPLI CATION

In the early exploratory tests in which very uniform concentrations of antibiotics were studied they were incorporated in ground-up fish flesh. As the work expanded other methods of application were investigated. It was obvious from the outset that any preservative measure used with fresh fish should normally be applied as soon as possible after the fish are caught and killed. Thus, three methods offered most promise for use in practical fisheries, namely, (1) incorporation in the ice used for icing fish; (2) immersion, shortly after capture, of whole fish (preferably eviscerated) in comparatively strong solutions of the antibiotic followed by icing them in ordinary ice or antibiotic-containing ice; and (3) addition of the antibiotic to refrigerated sea water (Lantz, 1953; Roach et al., 1961) . These three methods of application have been studied fairly ex tensively both at this Station and elsewhere. Before proceeding to a discussion of the results of some typical applications of antibiotics in different fisheries, mention will be made of some of the complications which have arisen in their application.

DIFFICCLTIES EXPERIENCED IN .\PPLICATION

Three main difficulties have been encountered in applying antibiotics to fleshfoods, namely, (1) lack of even distribution in block ice; (2) inactivating and precipitating effect of hard waters, and (3) inactivation by residual active chlorine in chlorinated water supplies. \\There flake or similar types of ice which is rapidly frozen in small pieces is made, there is no problem with distribution of tetracycline antibiotics. On the other hand, if ice is made in large blocks the antibiotic tends to freeze into a comparatively small zone or core at the bottom centre of the blocks, and con sequently on crushing them the distribution of the antibiotic is not uniform. It was found that the migration of the antibiotic could be prevented successfully by the addition of a very small amount (less than 0.01 %) of a hydrocolloid such as carrageen from Irish moss (an Atlantic Coast seaweed) or carboxymethyl cellulose, substances which are used as stabilizers in certain foodstuffs and are harmless (Gillespie et aI., 1954a,b, 1955; Boyd et al., 1955; Upham et al., 1954, 1955; Tarr and Gillespie, 1958) . Commercial formulations for making antibiotic ices contain adequate amounts of such hydrocolloids (Fig. 2) . When hydrocolloid formulations are used, hard waters, which are rich in salts of calcium, magnesium and iron, present two difficult problems. Hard waters are usually alkaline and tetracycline antibiotics are not at all stable under these conditions. The addition of appropriate amounts of a harmless acid such as citric acid will ensure stabilization of the antibiotic when flake-type

3 89262-0-2! FIGURE 2. Addition of the small amount of dry antibiotic formulation required as a tank is being filled with water for freezing into a 300-pound block of antibiotic ice. ice is made (Gillespie et at., 1954b). However, acidification of solutions of antibiotics in making block ice overcomes the desirable distributing action of the colloids used as distributing agents, and consequently the only practicable solution to the problem appears to be removal of the offending salts. This can be done quite simply and at not undue expense by appropriate water-softening devices which usually involve ion-exchange resin treatments. If antibiotic ice is prepared from hard waters, formation of water-insoluble complexes between certain mineral constituents of the waters, the gel used, and the antibiotic itself occurs. These complexes seem to form during the preparation of the ice and a considerable proportion of the antibiotic is carried down as an insoluble precipitate which, though active if once again dissolved in weak acid, is ob viously no longer available for the purpose intended (Moyer et at. , 1958) . In certain of the experiments carried out in Great Britain, inactivation of CTC in ice was observed (Reay, 1956) .

It was pointed out several years ago that active chlorine residues in chlori nated water supplies may destroy CTC (Kohler et al., 1957) . This CTC-destroy ing effect of chlorine was recognized independently by Tawara and Sasano (1957) in Japan during their work on fish preservation with CTC. These workers

4 suggested the addition of sequestering agents such as citric acid, or reducing agents such as ascorbic and/or sodium bisulphite to inactivate the chlorine residues. Since proteins and other readily oxidizable substances present in fish readily remove active chlorine residues, it would seem that it would be quite feasible to remove these by placing fillets in dipping tanks containing the chlorinated water several minutes before adding the erc solution. Naturally, dipping solutions should be kept as near 32°F as practicable, and should be changed frequently enough to retain the desired strength of antibiotic.

Directions for making antibiotic solutions and ices are avaJlable by the suppliers. Concentrations of actual antibiotic employed are usually 5 parts per million (ppm) (0. 005%) in ice and about 10 ppm in fillet-dipping tanks.

RESULTS OF APPLICATIONS IN DIFFERENT FISHERIES

Methods used to assess the value of antibiotics in preserving fish have included both subjective (appearance, smell, taste, etc.) and objective (bacterial and chemical) tests, and combinations of these. The results obtained have usually shown a very pronounced inhibition of the normal rates of bacterial spoilage of treated fish and fillets, but reports have varied greatly depending 011 the conditions. Thus, some investigators were disappointed when fish stored in CTC-containing ice displayed no noticeable improvement in keeping quality compared with fish held in ordinary ice over a period of only about 5 days. Since it is common knowledge that eviscerated fish normally retain their qUillity very well when iced properly for such periods, it is not surprising that 110 apparent improvement in keeping quality was observed as a result of the antibiotic treatment. It must not be forgotten however, that should such fish be re-iced for fresh shipment or filleted, the prior antibiotic treatment, especially if followed by icing fish with antibiotic ice or dipping fillets in antibiotic solu tions, would prove a valuable adjunct to general improvement in keeping quality. This has in fact been amply demonstrated many times at this Station. In the following discussion, results of only a limited number of typical applica tions of tetracycline antibiotics in different fisheries will be given. For more detailed reports the reader is referred to the review articles mentioned previously.

SALMON TROLLING BOATS The first trials with CTC on fishing boats were made during June 1953 on a British Columbia salmon troller equipped with a small refrigerated sea water holding tank (Lantz, 1953) . In the summer of 1954 the tests were extended to the addition of CTC to both ice and refrigerated sea water used on west coast of Vancouver Island trollers (Gillespie et al., 1954a; Tarr et at., 1954). In these 1954 tests with ere ice, 5 g (about it oz) of clinical grade CTC was dissolved in 20 gallons of water to make a solution containing 50 ppm of the antibiotic. It was found necessary to add a small amount of citric acid (0.02%) to this solution since the hard and alkaline nature of the water resulted in a rapid destruction of

5 CTC. This solution was run at a carefully measured rate into the water used to make 4 tons of ice during an approximate 24-hour period so that the theoretical amount of CTC added was 1.25 ppm. It is interesting to note that assays carried out at intervals during the ice-making procedure showed an average content of 1. 07 ppm of the antibiotic (Gillespie et al., 1955). Salmon trolling boats were selected and certain of them used the CTC-containing ice; others ordinary crushed block ice. Both spring and coho salmon were iced with the different ices after they had been eviscerated and their belly cavities scraped and washed in the usual manner. After the fish were landed and graded they were re-iced with the respective ices and transported to Vancouver by truck. Representative boxes of fish were inspected at intervals and general organoleptic and bacterio logical tests were made. The results showed that the CTC ice prolonged the quality retention of the salmon by about 5 days. Tests carried out about 2 years later at Neah Bay, Washington, (Firman et at., 1956) in which salmon were iced with ice containing approximately 5 ppm of CTC showed that the fish held lJ1 this ice kept about 7 days longer than those held in ordinary icE'.

GROUND FISH Several trials have been made to ascertain the effectiveness of CTC containing ice in preserving trawl-caught bottom fish such as cod, flounders, sole and ocean perch. In 1956 at the request of the fishing industry, trials were undertaken to ascertain the value of ice containing CTC in preserving fish in the British Columbia trawl fishery. A large amount of block ice was prepared using an industrial formulation but only in half the specified concentration, namely 2. 5 ppm of CTC instead of 5 ppm. In this fishery the fish receive a comparatively low price, are not eviscerated, and are by no means as well cared for as are troll-caught salmon. The trawlers took out either CTC-containing ice or ordinary crushed block ice. Fish were taken at random for bacterial tests though it was impossible to secure a really representative sample since up to 80,000 lb of fish were landed at one time. However, the results showed a distinct improvement in quality of the fish which were held in the CTC-containing ice. even though the level used was half that suggested by the manufacturer (Boyd et at., 1957a) .

Many British trawlers operate on fishing grounds remote from the British Isles, and consequently much of the ground fish landed may be stored for about 10 to 20 days or more in ice, and is therefore not of prime quality. For this reason a number of large-scale trials with CTC under commercial conditions were carried out under direction of the personnel of the Torry Research Station, Aberdeen, in 1956. Cod, haddock, whiting and flat fish were iced with ice con taining about 5 ppm of CTC and with normal ice at sea and were examined on arrival at the fishing ports of Aberdeen; Grimsby and Hull. In one report it was stated that, "up to the eighth to tenth day of storage the effects of Aureomycin ice are not very noticeable, but from then on become more and more evident as the storage time is prolonged". The fish landed were from 5 to 27 days old

6 post mortem in the different trials. In general, the comments of those of the fish trade who saw the treated fish were very favourable and were summed up as follows: "There is a definite advantage in using Aureomycin ice, so far as the odour of the fish is concerned, the keeping quality being extended by from 3 to 7 days .... ". "On filleting, all species of fish in the Aureomycin ice always give a fresher looking product, being more translucent and less white and bleached in appearance" (Shewan, 1956a) . Two detailed technical reports concerning these and other studies have been published (Shewan. 1956b; Shew an and Stewart, 1958) .

Extensive sea trials with CTC have been carried out in Japan under the direction of Tomiyama and his collaborators. In studies on trawl-caught fish including yellow croaker, red sea bream and red-tongued sole they found that best results from the point of view of extension of keeping quality were obtained by an initial lO-minute dip in sea water containing 10 ppm of CTC followed by storing the fish in ice containing 5 ppm of the antibiotic. The use of antibiotic ice alone was not so satisfactory. The combination treatment extended the keeping quality of the fish by about 13 days over that of comparable fish held in ordinary ice (Tomiyama et al., 1956a) . These investigators have also obtained excellent results in application of CTC to pelagic fish. Thus, a 30-minute immer sion in a 5% salt brine containing 10 to 20 ppm of CTC preserved whole round sardines effectively even when the fish were held at about 29°C (Tomiyama et at., 1956b). These investigators have also carried out a number of other studies in other Japanese fisheries and have obtained similar favourable results with CTC treatments (Tomiyama et al., 1955) .

In experiments carried out in South Africa (Georgala, 1959) , hake were iced with both ordinary ice and ice containing 5 ppm added CTC. Assays of the ice indicated that it contained from 2.15 to 3.85 ppm of the antibiotic in different instances. Possible reasons for these low values have already been discussed. From the results of tests carried out on a commercial fishing trawler it was concluded that, "fish stored in Aureomycin ice remained edible .... for approxi mately 7 days longer than those fish stored in ordinary ice, which were inedible after 12 days".

Somewhat less extensive trials have been made in other countries. In the United States Firman et al. (1956) carried out quite extensive trials using both dipping and icing techniques in the treatment of a number of different bottom fish with extremely favourable results. In their work the fish were treated with either a 10-minute dip in sea water containing 10 ppm of CTC, or with ice containing 5 ppm of the antibiotic. Ducroz (1957) working with tropical fish in the French Cameroons demonstrated the superiority of CTC-containing ice over ordinary ice. J 11 the U.S.S. R. experiments have also shown the effectiveness of CTC (Ravich, 1956; Dubrova et al., 1958). In India Visweswariah et al. (1959) found that CTC when applied by a dipping method did not extend the keeping quality of eviscerated fish which were subsequently stored at 86°F, but that

7 fillets similarly treated and stored spoiled much more slowly than untreated fillets. In Germany Kreuzer (1957) obtained favourable results in fish preserva tion experiments using CTC-containing ice. The antibiotic OTC was found to preserve fish successfully (Pasternak et al., 1956).

USE OF ANTIBIOTICS IN REFRIGERATED SEA-WATER STORAGE OF FISH

In a forthcoming publication (Roach et al., 1961) the whole subject of storage and transportation of fish in refrigerated sea water will be thoroughly discussed, and brief mention made of the use of CTC in this medium. Though refrigerated sea water when properly used permits successful storage of most varieties of marine fish for at least 10 days with no appreciable spoilage, in long-distance fisheries, where freezing is impracticable or impossible for various reasons, the use of CTC or OTC in this medium has occasioned considerable extensions of keeping quality. However, in no instance where careful comparisons have been made has the extension of keeping quality due to antibiotic addition, as compared with appropriate controls without antibiotic, been as spectacular as that in which the antibiotic has been incorporated in ice or used in fillet-dipping procedures.

As stated previously, this Station's first tests in which antibiotics were added to refrigerated sea water were made with a small trolling boat in 1953. In the 1954 similar tests (Gillespie et at., 1954a) troll-caught coho, white and red spring salmon were stored in refrigerated sea water at about 30°F with and without 2 ppm of added CTC. The fish were held in this medium for 8 days on the fishing troller and, after unloading, centre steaks were cut from representative fish and stored for one day at 50°F to accelerate spoilage. It was found that the steaks cut from the fish which had been stored in refrigerated sea water con taining CTC kept much longer than those which had been cut from fish held in refrigerated sea water without the antibiotic. Subsequent and rather similar tests with immature coho salmon were carried out later on another trolling boat with successful results (Steiner and Tarr, 1955; Tarr, 1956). Gillespie et al., (1955) confirmed the above findings with coho salmon, and extended them to eviscerated and non-eviscerated spring salmon, grey cod and lemon sole. The results of these, and also of some as yet unpublished experiments, have shown that, so long as fish are not feeding actively, it is possible to store them in refrigerated sea water plus CTC without evisceration for 8 or 9 days without appreciable spoilage. However, the storage of non-eviscerated fishshould certainly not be carried out unless exploratory tests have been made to indicate the feasibility of this method in a given fishery.

In one series of tests the development of autolytic visceral spoilage (belly� burn) which can occur in non-eviscerated fish stored in refrigerated sea water was inhibited by the addition of CTC (Ronald et at., 1957). This could indicate that this "autolytic" spoilage is actually a complex phenomenon in which both the autolytic effect of digestive enzymes and bacterial proteolysis

8 ' are jointly responsible for the softening of the belly walls of the fish and the decomposition which usually occur. Further experiments should be carried out in attempts to verify and repeat these observations.

Though the advantages of refrigerated sea water as a storage medium for sea fish are quite obvious, it has been pointed out that this method of storage may, in some instances at least, create a heavy bacterial inoculum in a favour able growth environment. This of course is particularly true where large loads of improperly washed fish are loaded into the tanks. Where the pressure of fishing or general conditions prevent proper evisceration and washing of fish before loading, or where long distances of transportation are involved, the use of anti biotics in refrigerated sea water offers great promise in extending the normal holding periods (Fig. 3). A number of different trials have been made in which the keeping quality of various fish held in refrigerated sea water with and without added eTC has been determined (Fig. 4). Definiteretardation of rates of spoilage of immature coho (blue-back) salmon (Steiner and Tarr, 1955), spring and coho salmon (Gillespie et al., 1954a) and lingcod (Baker et al., 1958) occurred when eTC was added to refrigerated sea water. In these tests the amount of antibiotic added to the sea water was usually between about 2 and 10 ppm. As an example, in one experiment it was found that fillets cut from fish held up to 10 days in refrigerated sea water containing 10 ppm of eTC and then stored

FIGURE 3, Addition of eTC antibiotic solution to a laboratory tank of refrigerated sea water during experiments to test the keeping quality of the salmon stored in the tank.

9 FIGURE 4. A British Columbia fish packing vessel (foreground) with 50,OOO-pound capacity tanks of refrigerated sea water plus antibiotic loads into one of its tanks sockeye salmon from the fishing vessel alongside.

18 hours at 50°F were normally rated fresh, while those cut from fish held for only 6 days in the same medium without added eTe developed off-odours and were soon rated stale or putrid. It was also established that when added to 3% salt solution, eTe in only 1.5 ppm concentration preserved eviscerated lingcod and rock cod more effectively at 30cF than did OTe or tetracycline. and that aeration of the salt water solutions did not improve effectiveness of the antibiotics (Southcott et al., 1958a).

It is important to note that in all these experiments in which antibiotic was added to refrigerated sea water its effect in improving the organoleptic qualities of the fish was much more noticeable than its action in suppressing bacterial growth. Also, the numbers of viable (living) bacteria were almost invariably much fewer than the total bacteria1 numbers, indicating that there was probably rapid "dyilll2; off" of the bacteria under these conditions.

10 The effectiveness of CTC in retarding bacterial spoilage of uneviscerated Japanese herring stored in sea water or ice containing about 10 ppm of CTC was studied in detail by Tomiyama and his collaborators (Tomiyama ct at., 1955). Under their experimental conditions the maximum increase in storage life obtained \vas90% more than for the control fish. Stern ct at., (1957a, 1958) studied the effect of both CTC and OTC in concentrations of 5, 10 and 20 ppm in 3% salt solutions on control of bacterial spoilage of non-eviscerated sockeye salmon at different temperatures. They concluded that the effectiveness of these anti biotics was most noticeable with fish stored at 32°F, and that there was no appreciable difference in the effectiveness of the antibiotics. In other studies with non-eviscerated flounders (English sole) stored at between 32° and 38°F in 3 % salt solutions, addition of 2 and 5 ppm of OTC proved more effective in retarding bacterial spoilage than did similar concentrations of CTC or tetra cycline (Stern ct ai .. 1957b).

DIPPING 'WHOLE FISH IN COMPARATIVELY STRONG ANTIBIOTIC SOLUTIONS FOLLOWED BY ICING THEM. I t is sometimes advantageous, especially where antibiotic-containing ice is not available, to immerse whole or eviscerated fish for a short period in fresh or sea ,,'ater containing say 50 to 100 ppm of antibiotic prior to storing them in ordinary icc (Fig. 5). Naturally, even better results are obtained if the fish are so dipped and then stored in antibiotic-containing ice. In 1953 preliminary tests with coho salmon (Boyd ct al. , 1953) showed that dipping the fish in sea water ,,,hich contained only 5 or 10 ppm of the antibiotic followed by icing them in

FIGURE 5. Preparing to homogenize a sample from a salmon for testing the retarding effect of eTC antibiotic on growth of fish-spoiling bacteria..

11 ordinary ice had very little effect on subsequent keeping quality. \Vhen solutions containing 50 or 100 ppm were used very favourable results were obtained. Later, practical applications of this method were carried out on a fishing boat (Southcott et at., 1958b). Eviscerated black cod were immersed for from 5 minutes to 1 hour in sea water containing about 50 ppm of CTC. About 500 Ib of fish were treated at one time in 30 gallons of solution which was usually only used once. The treated fish, together with untreated fish as controls, were stored from 3 to 17 days in ordinary ice after which times they were examined using an accelerated bacterial count method. The CTC treatment was found to occasion marked quality improvement. Tomiyama et at. (1956a) carried out fairly ex tensive trials in order to determine the best conditions for application of CTC to prolong the storage life of uneviscerated herring. vVhen the fish were stored for a short time after capture on board the fishing vessel in ice water containing between 5 and 10 ppm of CTC, followed by storage on shore in CTC-containing ice, the keeping quality was extended at least 5 days over that of untreated herring. In other work Tomiyama et at. (1955) found that a 1-hour dip of sardines or round eviscerated mackerel in a 5% salt solution containing 10 or 20 ppm of CTC caused the fish to keep roughly twice as long as untreated controls. In these experiments the fish were stored at approximately 65°F following the immersion treatment.

TREATMENT OF FILLETS AND STEAKS In some of the earliest experiments which were carried out in exploratory work to determine the effectiveness of various antibiotics in retarding spoilage of fish, fillet-dipping procedures were used (Tarr et al., 1952). Thus, ,yhen fairly thick steaks of lingcod were immersed for a short time in solutions containing either 10 or 20 ppm of CTC and then stored at 32°F, the bacterial counts of the treated steaks were very much lower after 23 days than those of untreated steaks after only 13 days storage. About 2 years later fairly extensive tests were made in San Francisco where fillets of lingcod, sole and black cod (sable fish) were dipped for only 1 minute in 5% salt solution containing 2 ppm of CTC or of certain other antibiotics, and then stored at temperatures between 37 and 49°F in different experiments (Farber, 1954). The results showed that CTC was more effective than any of the other antibiotics investigated. In later studies carried out in the same laboratory (Farber and Lerke, 1957) fillets of Pacific coast rock fish and sole species were studied. In some cases the fillets used had been cut from fish stored in ice which contained 10 ppm of eTC and in other cases in ordinary ice. The fillets were immersed briefly in 5% salt solution containing 5 or 10 ppm CTC. Following treatment the fillets were exposed to several different handling procedures such as might occur commercially, the storage temperatures in different experiments being either 35° or between 55° and 60°F. Organoleptic and several subjective tests for quality were used to assess the rates of spoilage of the fillets. In all instances the CTC treatment improved the keeping quality very markedly.

12 Experiments showed that the amount of CTC taken up by fillets of certain Atlantic Coast fish from water containing 10 ppm of the antibiotic differed very little when the duration of the dipping times was between 1 and 5 minutes, and that the weight increase (1.4 to 1.7%) remained almost constant during this time interval. Haddock fillets dipped under these conditions kept for about 7 days longer than untreated fillets as determined by organoleptic and bacterio logical tests (Kline et al., 1957). On the east coast of Canada it was shown that with cod, haddock and sole filletsdipped from ! to 2 minutes in a lO-ppm solution of CTC, followed by storage at 32 to 35°F in different experiments, the keeping quality was increased by from 2 to 10 days over that of comparable untreated controls (Castell and Greenough, 1958b). These very different improvements in keeping quality were explained by the fact that the bacteriological quality of fillets at time of treatment varied considerably. The authors emphasized very strongly the fact that only high-quality fish fillets should be treated by this procedure. They found OTC nearly, but not quite, as effective as CTC (Castell and Greenough, 1958a). When lingcod fillets were dipped for 1 minute in water with or without 5 ppm of one of three tetracycline antibiotics, drained for 2 minutes on clean wire racks, and then stored at 32°, 37. 5° or 50°F, bacterial tests showed that in all instances CTC was more effective than either OTC or tetracycline in retarding bacterial growth (Southcott et al., 1958a).

PRESERVATION OF SHELLFISH

Early experiments using cooked crabmeat (Tarr et al., 1952) showed that considerably higher concentrations of CTC or OTC (10 to 20 ppm) were required to inhibit bacterial spoilage as effectively as in the case of fish (Fig. 6). A little later experiments carried out in California (Farber, 1954) showed that dipping whole raw shrimp in solutions containing only 2 ppm of CTC did not retard bacterial spoilage significantly, though the procedure worked excellently for fish fillets. One of the most exhaustive investigations concerning the value of CTC in retarding shrimp spoilage was made in Florida about 1955 (Camber ct al., 1955) where beheaded shrimp were treated from 1 to 30 minutes in sea water containing 10 to 500 ppm of CTC before icing them in ordinary ice or in ice containing from 0. 8 to 10 ppm of the antibiotic. It was found that ice con taining 10 ppm of CTC extended the storage life by about 4 days, but such icing increased melanosis (black spot), presumably due to the fact that a calcium salt was used in the ice formulation. Only the higher concentrations of antibiotic in the dipping method caused a significantquality improvement. Thus, immersion of the shrimp for 1 to 5 minutes in solutions containing 100 ppm of CTC effected an approximately 5-day increase in keeping quality. Subsequent work verified and extended these findings (Camber et al., 1956). Other experiments carried out in Florida showed that ice containing 10 ppm of CTC improved keeping quality of shrimp by about 4 days, but did not prevent occurrence of black spot (Fieger et al., 1956) . Studies carried out in California a little later showed that beheaded raw shrimp dipped for 15 minutes in 5% salt solutions containing

13 FIGURE 6. Examining the fresh appearance of a crab that has been stored in aerated, refrigerated sea water containing CTC antibiotic.

15 ppm of CTC did not spoil so rapidly as shrimp treated in the salt solution alone (Farber and Lerke, 1956). CTC retarded spoilage at all temperatures studied between 30 and 82°F, but really significant preservative effects were observed only at temperatures below 40°F. Peeled raw beheaded shrimp kept better than the unpeeled shrimp. In these experiments no mention ,vas made of the accelerating effect of CTC on black spot development, which had been noted by previous workers, possibly because the CTC used did not contain a calcium salt.

l\ilore recently Vance et al. (1959) have completed a thorough study of the effectiveness of CTC in retarding spoilage of Gulf of Mexico shrimp. Their results have confirmed and extended those of previous investigators. They found that the most economical and effective concentration of CTC when employed in a dipping solution for headed shrimp which were subsequently stored in ordinary ice was 30 ppm. Ice containing 10 ppm of the antibiotic gave similar results. Contrary to results of some previous investigators, black spot develop ment was not accelerated, and was even retarded in some instances, by the CTC treatments, especially where potassium or sodium rather than calcium salts were used in the ice formulations. The CTC treatments extended the keeping quality of the shrimp 5 to 7 days, or by approximately 30%. Favourable results were obtained with CTC in preservation of raw shucked oysters in British Columbia (Boyd and Tarr, 1956). Brief dips in solutions con taining 10 to 20 ppm of CTC were necessary to obtain significant preservative

14 effects, solutions containing between 2 and 4 ppm being too weak for the purpose. A subsequent report concerning preservation of raw freshly shucked Atlantic oysters verified and extended these observations (Abbey ct al., 1957). In compari son with fish, oysters required considerably higher CTC concentrations for effective preservation against bacterial spoilage, namely between 5 and 30 ppm. There has been so far no adequate explanation of the fact that shellfish seem to require much higher concentrations of antibiotic for successful preserva tion than does fish flesh. The somewhat alkaline nature of shrimp and crab flesh may be partly responsible but this explanation can hardly hold with oysters where the reaction of the meats is usually somewhat acid. In conclusion, it must not be forgotten that shellfish have very delicate flavours which are usually rapidly lost even after a short storage period in ice, and there is little doubt that these flavours will tend to disappear even when the fish are preserved successfully in a microbiological sense. Therefore, it would be extremely unwise to attempt to extend the keeping quality of shellfish longer than is absolutely necessary.

PRESERVATION OF MARINE MAMMALS

In early experiments in which a 55-g (1.93-oz) charge of CTC in 10 gallons of sea water was injected into the visceral cavity of three whales shortly after death, the treated carcasses were of somewhat improved quality as judged by bacterial counts of the muscle and liver tissues and by the free fatty acid con tent of the oil (Crean ct al., 1956). In further experiments in which 45 whales were treated as above, only this time with 100 g (3.53 oz) of CTC dissolved in 20 gallons of sea water, the previous findings were verified. Bacterial counts made from muscle and liver tissues showed significant improvement as judged by comparison with similar samples taken from 49 untreated whales. Determinations of the total volatile base and indole content of a number of different tissues also indicated that the CTC treatment had been attended by improved quality. Injection of the antibiotic via the head of the harpoon bomb used was found to be ineffective (Duncan ct at., 1957a,b). Though a considerable number of ex periments concerning the possible industrial value of CTC and OTC for whale carcass preservation have either been completed or are in progress, few details and reliable published reports of the results of this work appear to be available. One report (Anon., 1958) indicated that a considerable quality improvement in the oil obtained from whale carcasses resulted from OTC treatment.

ANTIBIOTIC RESIDUES IN TREATED FISH AND THEIR INACTIVATION BY COOKING

Since 1952, when the first information regarding CTC destruction in fish flesh was published (Bissett and Tarr, 1952), over 15 different publications have appeared dealing with antibiotic residues in treated fishand with their destruction by various heating or cooking procedures and these have been reviewed (Tarr, 1960b). Nearly all the available data deal with CTC, but a little information is

15 available regarding OTC. The amounts incorporated in fish naturally vary greatly depending 011 type of treatment, variety of fish, antibiotic concentration used, length of treatment, leaching effects of ordinary ice in some treatments, and whether whole or eviscerated fish or fillets are treated. Concentrations of antibiotic in skins of fish stored in CTC-containing ice are considerably higher than that of the flesh itself (Steiner and Tarr, 1956; Shewan, 1956a). Typical incorporated amounts (in ppm) obtained by different investigators for fish stored in ice containing between 3 and 5 ppm of CTC on fishing vessels are as follows: skin, 3.15 to 11.1; 1.15 to 3.36; 0.05 to 0.28; 0.05 to 2.4; flesh, 0 to 1.05; o to 0.06; 0 to 0.03. The flesh of fish stored in refrigerated sea water naturally tends to absorb somewhat more antibiotic than that of fish stored in ice con taining similar antibiotic concentrations. Thus flesh of several Japanese fish species stored at 4°C in sea water containing 10 ppm of CTC had an average of 2.8 ppm after 3 or 4 days storage; and flesh of eviscerated halibut stored 19 days at -1°C in refrigerated sea water containing 2.65 ppm of CTC (at time of unloading) contained from 0 to 0.74 (average 0.29) ppm in the skin and from o to 0.06 (average 0. 022) ppm in the flesh. These typical studies indicate that the raw flesh of fish treated with antibiotics at sea by the usual procedures advocated will normally contain much less than 1 ppm of the antibiotic. Fillets treated by procedures usually suggested also do not absorb excessive concentra tions. Moreover, the time of immersion, within the few seconds or minutes usually employed, has no important effect on the amount absorbed. UsuaIly a brief dip in water or dilute salt soiutions containing 10 ppm of CTC for less than 5 minutes is considered a satisfactory treatment (see Frontispiece). Under these conditions, available data indicate that raw treated fillets contain about 0.5 ppm of CTC, but that the concentration is affected by such factors as fillet thickness and temperature of solutions (Kline, 1957; Castell, 1958c). It is thus obvious that normal treatments for fish cause the fish to absorb only very low concentrations of CTC. These residues are largely destroyed by usual cooking procedures, and are entirely removed by the high temperatures and times associated with fish canning (Stern et al., 1958). In a study of eviscerated grey cod stored in ice containing from 2 to 4 ppm of CTC it was found that cooked fillets usually contained less than 0.1 ppm of the antibiotic (Boyd et at., 1957b). Very similar results were obtained on analysis of flesh of five different species of fish which had been held in ice containing 5 ppm of CTC and then cooked by three different procedures (Tomiyama et al., 1958). \Nhen flounder fillets con taining 0.5 ppm of CTC were fried for 5 minutes (light cook) in fat at 374°F, 40% of them contained no measurable antibiotic and the average content of the remainder was 0.061 ppm. After 7! minutes (average cook) there was no detect able an tibiotic in any of the fillets (Kline, 1957). In conclusion, it is important to note that 20 tons of cooked fish containing 0.1 ppm of CTC would contain about 2 g of the antibiotic, or about an average daily therapeutic dose.

16 Since comparatively high concentrations of tetracycline antibiotics are required to preserve shellfish, it is impossible to remove these completely by usual cooking procedures. However, cooking will usually destroy a very large proportion of active antibiotic. Thus, Benarde (1957) has shown that frying crab meat removes about 90% of added CTC.

PUBLI C HEALTH ASPECTS

The chief concerns regarding the use of antibiotics in foods relate to the possibility that their use might (1) be accompanied by sensitization of individuals with consequent danger of anaphylactoid shock; (2) be followed by an increase in antibiotic-resistant micro-organisms in consumers and consequently by a possible lack of desired response of individuals subjected to antibiotic therapy; (3) result in a cumulative toxicity, and (4) alter the normal microbiological flora of consumers with development of unpleasant reactions. It has been pointed out that certain of these points may be answered better by experience than by experiment (Ingram et nl., 1956). However, much useful, pertinent information has already accumulated. Thus, so far as can be ascertained, the already fairly extensive use of CTC in flesh foods has not yet been accompanied by any un desirable reactions in consumers. This is not altogether surprising, for although significant residues of penicillin have been found in about 5% of samples of market milk, no cases of hypersensitive reactions in consumers had been reported up to the time of a review by Welch (1957). Penicillin is the one antibiotic to which many individuals become readily sensitized. I t has already been pointed out that proper use of tetracycline antibiotics in flesh foods does not result in significant residual concentrations when these foods are cooked. For almost a decade tetracycline and certain other antibiotics have been used in about 20-ppm concentration in feeds of farm animals in order to promote growth and check incidence of certain rather chronic infections. This practice has, so far as can be determined, not led to any of the undesirable effects which were originally predicted as attending their use. On the contrary their use has been, in most instances, accompanied by favourable effects on growth and health of the animals (Jukes, 1955). Moreover, rather prolonged administration of about 50 mg of tetracycline antibiotics daily for many months to children has not been attended by demonstrable unfavourable reactions (Jukes, 1956; Loughlin et al., 1958a,b). There is abundant evidence that con sumption of comparatively large amounts of tetracycline antibiotics by infants, adults and geriatric patients has not been accompanied by cumulative toxic reactions (Hines, 1956). However, at therapeutic dose levels, which are thousands of times greater than those which might occur in treated foods, there may be transient unfavourable reactions or the intestinal flora of patients may be adversely affected (Welchet al., 1958). So far, treatment of flesh foods with antibiotics does not appear to have caused development of pathogenic or other undesirable micro-organisms in the products. In fish flesh treated with CTC, growth of an enterotoxigenic strain

17 of Staphylococcus aureus and of Clostridium botulinum (type E) bacteria which cause two types of food poisoning, was strongly retarded (Boyd et al., 1957a). The danger that paratyphoid-causing bacteria (Salmonella sp.) resistant to tetracycline antibiotics might develop in treated fish is remote since these or ganisms require comparatively high temperatures for growth. The real dangers in use of antibiotics in fish preservation appear to be: (1) application with the idea of unnecessarily prolonging "shelf life" of fish so that consumers never receive products of top quality; (2) applications carried out under poorly super vised and unsanitary conditions so that antibiotic-resistant bacteria develop, consequently rendering the process useless for its intended purpose; and (3) treatment of already partially spoiled fish, a procedure which has been found to be ineffective, or nearly so, and should be immediately condemned.

In a recent review Thatcher (1958) has discussed a number of the factors which are important when considering the use of antibiotics in foods.

OFFICIALLY PERMITTED APPLICATIONS

Though antibiotics are undoubtedly used in preservation of fish and other flesh foods in a number of countries, the writer has found difficulty in most instances in obtaining the exact terms under which their use has received official permission. In this connection it is of interest that one popular article (Anon., 1957) stated that antibiotics are used for fish commercially in Greece, the Philippines, Brazil, Colombia, Costa Rica, Guatemala, Honduras, Iran, Mexico, Nicaragua, Panama and Spain.

In Japan, use of CTC is permitted for ground fish which is used in prep aration of kamaboko (Tomiyama, private communication). The following are permitted applications of which the writer is aware: 1. In Canada the Regulations under the Food and Drugs Act were amended in 1956 and again in 1959 to permit use of tetracycline antibiotics in fish (Anon., 1959a) and now read as follows: "B. 21.07. N"o person shall sell fish that has in or upon it chlortetracycline or oxytetracycline unless (a) its presence or the presence of both, if both are used, is declared by name on the main panel of the label, and (b) the total amount, singly or in combination, thereot does not exceed 5 parts per million." 2. On April 21, 1959, (Anon., 1959b and c) the United States Government established a tolerance for use of CTC on certain fish products as follows; "5 parts per million in or on fish (vertebrate), scallop (shucked), shrimp (un peeled), from application for retardation of spoilage to whole, headed or gutted fish (vertebrate); scallop (shucked); shrimp (unpeeled); each in the fresh, uncooked, unfrozen form."

18 Since then, a further petition to use CTC has been filed proposing a regula tion, "to establish a tolerance of 5.0 parts per million (0.0005 percent) of chlortetracycline hydrochloride to retard bacterial spoilage in cuts of fish, such as steaks and fillets, and in peeled shrimp" (Anon., 1959d). 3. The Chilean Ministry of Health on December 9, 1958, approved a regulation permitting addition of antibiotics to ice used for storing fish under certain specified conditions (Anon., 195ge).

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