Oysters, mussels and other shell-fish are highly nutritious foods, which,

apart from their usual proximate principles, contain fairly high amounts

of minerals like iodine, copper, iron, etc., and vitamins. In the shallow rocky

areas of Malabar Coast there are numerous green mussel (Mytilus edulis)

beds from which large quantities of mussels are taken out almost throughout

the year, which form an important item of food for people of low incomes.

As the mussels grow in shallow areas subject to the influence of land drainage,

sewage and river systems, these beds constitute a potential hazard to

public health on account of possible epidemic infections being carried by

the shell-fish

Sreenivasan, A

Venkataraman, R

CMFRI Digital Repository

MUSSEL POLLUTION AT KORAPUZHA ESTUARY (MALABAR), WITH AN ACCOUNT OF CERTAIN COLIFORM TYPES* BY R. VENKATARAMAN AND A. SREENIVASAN INTRODUCTION OYSTERS, mussels and other shell-fish are highly nutritious foods, which, apart from their usual proximate principles, contain fairly high amounts of minerals Uke iodine, copper, iron, etc., and vitamins. In the shallow rocky areas of Malabar Coast there are numerous green mussel (Mytilus edulis) beds from which large quantities of mussels are taken out almost throughout the year, which form an important item of food for people of low incomes. As the mussels grow in shallow areas subject to the influence of land drain-age, sewage and river systems, these beds constitute a potential hazard to pubUc health on account of possible epidemic infections being carried by the shell-fish. Outbreaks of innumerable cases of enteric fever and other epidemics traceable to polluted shell-fish, have been cited and reviewed in great detail by Tanner (1944) and Shraeder (1952). It is known that pathogens, espe-cially when present in large numbers, survive freezing and cooking. Cooking, unless it is vigorous, may not always kill B. coli and it is impossible to remove B. coli from the body of the polluted oysters by washing (Hunter and Harrison, 1928). Albuquerque and Bhat (1953) have shown that the Bombay-duck, infested with any of the intestinal pathogens, would constitute a grave danger to human health since the latter survive certain culinary processes. When the mussel beds are located in shallow areas and are grossly polluted by faecal organisms, the latter constitute a danger to the consumers. In many villages and small towns there are no organised systems of sanitation and hence the refuse is not always discharged into the sea. During the rainy days the stormwater carries with it the town refuse and during the South-West monsoon this is the main source of pollution. Our thanks are due to the Director, King Institute, Guindy, for perform-ing agglutination tests on suspected pathogens and to the Director of Indus-tries and Commerce for permission to pubUsh this. * Paper read at the 41st Indian Science Congress, Hyderabad, January 1954. 314 Mussel Pollution at Korapuzha Estuary {Malabar) 315 HYDROGRAPHY OF THE AREA The mussel beds studied are in Elathur, 7 miles north of Calicut. The coast is shallow with rocky projections on which the green mussels grow in atjundance. The depth of water above the bed is about 6 to 8 feet at low tide, when they are fished for. The beds are located about 2 to 4 furlongs from the shore and the river Korapuzha empties into the sea at Elathur. MATERIAL AND METHODS " Overlaying water " was collected in sterile bottles and simultaneously other samples were collected for estimating dissolved gases, seilinity, pH, etc., which were determined by standard methods. Bacteriological examination of the water and mussels was made according to the methods of the American Public Health Association (1943) with the modification that for dilutions 50% sea-water was used. The most probable numbers (M.P.N.) of collforms was calculated by Hoskin's method (Presscott et al, 1947). Tests for cholera vibrios was done as described by Thresh, Beale and Suckling (1949) and by direct plating on desoxycholate citrate agar of pH 8-6. For the isolation of enteric pathogens the water or shell-fish were enriched in tetra-thionate broth for 18 to 24 hours at 37° C. and a loopful streaked on Bismuth sulfite agar, S.S. agar, and MacConkey's agar. Suspicious non-lactose fermenting colonies were subcultured and studied in detail. The coliform types isolated on E.M.B. agar and V.R.B. agar were also studied in detail (Wilson, 1935). Four periods were chosen to represent the different seasons. January represented the colder month with very low rainfall (December, January, February), April represented the period of summer (March* April, May) with increasing temperature and occasional pre-monsoon showers but rainfall more than during the previous period, July represented the peak South-West monsoon period (June, July, August) when more than t\|ro-thirds of the rainfall of the year occurred, and October represented the post South-west monsoon period (September, October, November) of lull and advent of North-East mosnsoon. RESULTS AND DISCUSSION The results are summarized in Table I. There is a parallelism in the coliform numbers of ' overlaying water', mussels from bed, and mussels from the market. The numbers are low in January, but increase sUghtly in April, reach phenomenal figures in July immediately after the South-West monsoon, and decrease to the lowest level in October. : The total bacterial counts are the lowest in October, but highest in July H^orroally counts on 0\ TABLE I Showing the Hydrological Conditions and Bacterial Quality of Shell-Fish Waters and Shell-Fish at Elathur^ Period Temper atare -C. Air Water Salinity pH Overlaying water Mnssels from bed Total coants per ml. on Sea-water Fresh water agar MPN of coli-forms per 100 ml. Ratio of coliforms to total coants Total counts per ml, on MPN of Sea-water agar coliforms coliforms Fresh-water agar per 100 ml. Ratio of to total coants Mussels tiam market Total coants per mL on Sea-water agar Fresh-water agar Ratio of liforms coliforms to total coants M P N of CO per 100 ml. I Janoary 1953 April 1953 July 1953 October 1953 28-5 30-0 25-5 27-0 29-0 30-5 27-0 29-5 33-50 34-98 9-09 31-93 8-5 8-4 8-1 8-2 310 390 3800 190 230 50 7700 35 33 49 1100 12 1 1 1 1 940 :790 345 • 1583 8200 18800 150000 1880 1020 13800 Spreaders 1350 540 79 160000 280 1 : 1517 1 : 23770 1 : 94 1 : 671 6200 12900 3900 8600 3300 5400 3100 960 1700 2400 160000 640 1 :365 1 :538 1 :24 1 :592 Mussel Pollution at Korapuzha Estuary {Malabar) 317 sea-water agar are greater than on fresh-water agar because of the pre-ponderance of ' marine' forms. Effect of seasons on the bacterial quality of shell-fish ami water.—^A com-parison of the data on rainfall given in Table II with the bacterial numbers in Table I indicates clearly the effect of various seasons on the quaUty of TABLE II Showing the Rainfall and Temperature (Data kindly supplied by the Marine Biological Station of the Madras Fisheries Department, West Hill) Months 1952 October November December 1953 January February March April May June July August September October November December Rainfall Inches 12-12 0-48 1-48 0-00 1-69 0-24 4-64 2-03 12-61 40-42 7-63 2-29 9-52 0-63 .. nil Temperature' •F. Maximum Minimiun 85-0 90 96 86 85 90 88 87 86 83 82 84 85 86 86 71-0 83 82 67 67 73 70 75 72 77 72 72 73 67 62 shell-fish and waters. In the case of water and market mussels a slight increase in total counts and coUform counts was noticed from January through April, with the advent of pre-monsoon showers and with higher day temperatures. During the rainy South-West monsoon period there was a conspicuous increase in total counts as well as in coliform counts. This is clearly due to the influx of a large quantity of river water bringing down with it a lot of organic matter and town refuse, as is evidenced by the loarked 318 INDIAN JOURNAL OF HSHERIES fall in salinity from 34-98%o in April to just 9-09%o in July in the over-laying water. Gray (1951) has also strongly suggested that the increase in bacterial counts in a stream was due to higher rainfall which washed down soil organisms (Topley and Wilson, 1948, p. 2018). Significance of coliform counts.—The U.S. Public Health Service lays down that samples of water from ' approved' areas shall not show an M.P.N. of coliforms in excess of 70 per 100 ml, while ' moderately' polluted areas (restricted areas) shall not contain more than 700 coliforms per 100 ml. In excess of this number, the area was classified as ' grossly polluted'. On the basis of this the shell-fish waters may be considered to be in the approved area in all months except during the South-West monsoon, when it is ' grossly' polluted. The same is true of mussel beds. But with market mussels an entirely diflferent picture is obtained. The U.S. Public Health Service considers that for oysters an M.P.N, of 230 or more of coliforms per 100 ml. and for other shell-fish 2,400 or more, should be considered unfavourable. On this basis the market mussels collected from the various centres in and around. Calicut during the period, April to July, should be considered as grossly polluted. More detailed investigation is needed, however, to say whether any legislation is to be recommended. Hunter and Harrison (1928) prescribed that the B.. coli' score' should be less than 50 before marketing, while Shraeder (1952) stated that a 'score' of above 50 was considered to be indicative of dangerous pollution, warranting con-demnation of the product. In market mussels the ' score' was 50 and above at all periods except in October, thus indicating undesirable contamination. Significance of coliform types.—E. coli type I (IMViC + + ) was present throughout the year in the ' overlaying' water and at most of the periods in the mussels. Hunter (1939) emphasized that the E. co//, which are IMViC + H , are of direct or indirect faecal origin and their pre-sence in a food may be accepted as a substantive evidence of excremental pollution. Sherwood (1952) also considered B. coli type I to be a criterion of pollution. Perry (1939) considers E. coli to be the logical indicator of faecal pollution in shell-fish and shell-fish waters. The unfavourable condi-tion in the mussels is thus self-evident. Epidemiological significance.—A number of suspicious colonies were isolated by methods enumerated earlier. These consisted of non-lactose fermenting organisms, some of which produced acid and gas from dextrose and other sugars, and the others produced only acid. The iserogenic orga-nisms belonged to the genera Paracolobactrum and Proteus. There has been indisputable evidence about the pathogenic nature of paracolon strains Mussel Pollution at Korapuzha Estuary (Malabar) 319 TABLE III Showing the Coliform Types isolated from Shell-Fish Water and Mussels Period 1953 January April July October • Water . E. coll type I E. coli type II . E. coli type 1 A. cloaca A. arogenes . E. coli types I and II A. arogenes type I . E. coli type I A. cloaca Bed mussels E. coli type I A. cloaca A. arogenes types I and 11 E. coli types I and II A. cloaca E. coli type I A. cloaca A. arogenes type II A, arogenes Market mussels E. coli type 1 A. cloaca A. arogenes types I and II E. coli type 11 A. cloaca A. arogenes type J E. coli types I and 11 A. arogenes type 11 E. coli types I and II A. arogenes E. freundii (Christensen, 1947; Griffin and Sniezsko, 1950; Hinshaw and McNeil, 1946; Stuart et al, 1938; Stuart et al., 1940). We have isolated a few of these slow and non-lactose-fermenting paracolons from mussels. Parr (1939) has adduced enough authoritative evidence to show that coliform organisms are res-ponsible for causing infectious diarrhcea, enteritis, etc., and that they are secondary invaders complicating infections. Dack (1953) has recently reviewed that E. coli are capable of causing food poisoning and that they cause infantile diarrhoea. Recently a great deal of evidence has come forth about the presence in, paracolons, of antigens that agglutinate Salmonella a^oA Shigella (Young, 1945; Ferguson and Wheeler, 1945). Agglutination tests done at the King Institute, Guindy, did not, however, reveal any organism belonging to the Salmonella-Shigella group. Proteus, Paracoli and certain E. coli strains are suspected aetiological agents of infectious diarrhcea and enteritis (Rubbo, 1952) and hence the presence of these in mussels indicates possible adverse conditions in the taking and marketing of mussels. Presence of cholera vibrios was not demonstrated at any time during the period of investigation, but this coincides with a period when there was no epidemic outbreak of cholera in this part of the State, TABLE IV Fermentative Types of Coliform Bacteria Isolated from Marine Environment Number IMViC Gelatin of charac- Sucrose Salicin Dulcitol Adomtol Glycerol lique- Motility cultures ters faction o 5 2 2 2 3 1 6 2 2 ++• AG AG AG AG AG — AG AG AG AG AG AG AG — — AG (1 positi — AG AG AG AG AG AG AG — + (slow) + _. — • " • + — + + -f — __ ± Escherichia nea-politana (Emmerich)* E. Communior (Durham)* Bacterium neapoli-tanum (Winslow et fl/.)t E. colitropicalis (Castellani)* E. Pseudocoloides (Castellani)* Bact. coscoroba (Winslow ef a/.)t Bact. Acidi-Jaetici (Winslow ei a/.)t E. Pseudocosco-roba ((Castellani and Chalmers)* > -+— AG AG 1 4 2 13 2 5 3 1 3 4 1 7 4 1 1 AG AG AG AG AG AG + (1 positive (1 non-slow) motile) - + Bad. colicommu-nius (Winslow etal.)f ++ + -++ AG AG AG AG AG AG AG AG AG AG AG AG AG (1 positive) + + + + + + + + Aerobacter (1 positive) + (1 negative) + + + (1 positive) (1 positive) AG AG AG AG AG AG + + + Aaobacter + ^ S" i I' f i * =• Nkiiolls and Saltz (1936). t = Topigy and Wilsmi (1948). p. 666. AG '= Add and gas. A ==AcidaloiK. — = Not fianwnted (sugars). + = Not Uqw&ed ^latin). 322 INDIAN JOURNAL OF FISHERIES Fermentative reactions of coliform types.—In Table IV are given the fermentative reactions of the coliform organisms. The following salient features may be noted: (1) In the genus Escherichia, (IMViC + H and 1 ) gelatin liquefaction is not pronounced, but is either nega-tive or slow, while motility is fairly universal, though the two are not always correlated. (2) A very large number of Escherichia ferment sucrose (33 out of 40) the inverse correlation of sucrose and salicin fermentation was irregu-lar, many of them fermenting both. (3) Very few fermented adonitol, though dulcitol was fermented by greater number of cultures. (4) In regard to the Aerobacter, there is a very high degree of correlation between gelatin Uque-faction and motiUty. (5) The inverse correlation of gelatin liquefaction and glycerol fermentation is not a rule, but is fairly common in Aerobacter. (6) DuHcitol and adonitol fermentation was variable and not correlated with other characters. (7) Sucrose, saUcin, sorbitol, dextrin and riffinose were fermented by all Aerobacter. (8) All Escherichia fermented glycerol and dextrin. Fermentation of sucrose by such a large number of Escherichi is inter-esting, as members of this genus rarely ferment sucrose. Clemesha (1912) showed that the proportion of saccharose fermenters to non-saccharose fermenters was 1: 2^ and considered that" this increase in the non-saccharose fermenters in India emphasizes the difference between the two parts of the World " Parr (1939) considers the sucrose fermenting E. coli to be E. coli communior. Though Parr (1938) suggests that A. cloaca demand taxonomic difference with A. cerogenes, Griffin and Stuart (1940) feel that the separation of Aerobacter into cerogeries and cloaca is of questionable value, since there is lack of correlation between glycerol fermentation, gelatin Uquefaction and motility. As recorded above we found a high degree of correlation between gelatin liquefaction and motility, but equally good correlation between gelatin Uquefaction and glycerol fermentation was lacking. Stuart, Griffin and Baker (1938) suggest that coUforms could be divided only into three sections, viz., Aerobacter, Intermediate and Escherichia, and this appears to be reasonable unless sufficient evidence is let in on the basis of permanent non-variable characteristics to further split up Aerobacter and Escherichia. Fermentation of sugars is not of much taxonomical value in the separation of the coliform group into different species due to absence of a set pattern as could be seen from the table. SUMMARY AND CONCLUSIONS The mussel beds in shallow coastal areasare continually polluted and show the presence of E. coli type I, throughout the year. The peak of Mussel Pollution at Korapuzha Estuary {Malabar) 323 pollution is attained immediately foUowteg the advent of South-West mon-soon. After Sbuth-West monsoon abates and till summer, there appears to be a ' recovery' and self-purification and the coliform numbers, as wdl as the total counts, are low. Salmonella-Shigelld group as well as cholera vibrios were absent, but Paracoli, Proteus and E. coli were present, which themselves have epidemiological significance as causative agents of infectious gastro-enteritis. REFERENCES Albuquerque, M. J. and Bhat.J.V. 1953 American Public Health Association. 1943 Christensen, C. B. 1947 cremesha, W. W. 1912 Dack,G. M. 1953 Ferguson, W. W. and Wheeler, W.E. 1946 Gray, E. 1951 GriflRn, P. J. and Stuart, C. A. 1940 • and Sniezsko, S. T. 1951 Hinshaw, W. R. and McNeil, E. 1946 Hunter, A. C. 1939 and Hanison, C. W. 1928 Nicholls. E. E. and Saltz,H.P. 1936 Parr.L.W. 1938 _ , . 1939 Perry, C. A. 1939 Ptescott, S. C , Winslow, C. E. A. and MacCrady. M. H. 1947 Epidemiological aspects of the Bombay-duck or ' Bombil' (Harpodon nehereus Buch.). Proc. Indlaii Acad. Set, 3TO, 214-23. Recommended methods of procedure for bacteriological exami-nation of shell-fish and shell-fish waters. Amer, J. Public Health, 33, 582-91. Comparative distribution and possible pathogenicity of Pm-a-colobactrum species in an area highly endeniic for ent«ic infections. / . Bact., 53, 317. The Bacteriology of Surface Waters in the Tropics. Thacker, Spink & Co,, Calcutta. Food poisoning. Ann. Rev. Microbiol., 7, 327. Two paracolon cultures related antigenically to Shigella para-. dysenterite. J. Bact., 51, 280. The ecology of the bacteria of Hobswi's brook, a Cambridgeshire chalk stream. / . Gen. Microbiol., 5, 840-59. An ecological study of coliform bacteria. / . Bact., 40, 83. A unique bacterium pathogenic to warm-Wooded and cold, blooded animals. Fisheries Bull., SH {Fisheries Bidl. of U.S. Fish and Wildlife Service, 52, 187 90). The occurrence of type 10 paracolon in turkeys. / . Bact., 51, 281-86. Uses and limitations of the coliform group in sanitary control of food production. Food Res., 4, 531. Bacteriology and chemistry of oyster, with special reference to regulating control of production, handling and shipment. U.S. Dept. Agric. Tech. Bull, 64. The classification of a group of Escherichia isolated from the intestinal tract of patients with ulcerative cblites. / Bact., 31, 313. Coliform intermediates in human faces. Ibid., 36, 1. The coliform bacteria. Bact. Rev., 3, 1-48. A summary of studies on pollution in shell-fish. Food Res., 4, 381-95. Water Bacteriology. John Wiley & Sons, New York, N.Y. 6th Ed. . 324 INDIAN JOURNAL OF FISHERIES Rubbo.S.D. 1952 Sherwood. H. P. 1952 Shraeder, P. H. 1952 Stuart, C. A., Griffin, A. M. and Baker, M. 1938 , Baker, M. E. Zinunerman, A., Brown, C. and Stone, C. M. 1940 Tanner, F. W. 1944 Thresh, J. C , Beale, J. T. and Suckling, E. V. 1949 Topley and Wilson. 1948 .. U.S. Public Health Service. 1946 Wilson, G. S., Twiggs, R. S., Wri^t , R. C , Hendry, C. B.. Cowell, M. P. and Meir, I. 1935 Young, V. M. 1946 Epidemiology of infectious diarrhoea. Biol Abstr., 26, 2817. The sanitary control of shell-fish in England, cited in World Fish. Abstr., 3 (4), 9. Food Control. John Wiley & Sons, New York, 513 pp. Relationships of coliform organisms. / . Bact., 36, 391-410. Antigenic relationship of the coliform bacteria. Ibid., 40, 101-42. Microbiology of Foods. Garrard Press, Champaign, Illinois, 2nd Ed. Examination of Waters and Water Supplies. J. & A. Churchill, London. Principles of Bacteriology and bnmunity. Edward Arnold & Co,, London, 2018 pp. Manual of Recommended Practice for Sanitary Control of the Shell-Fish Industry. Publ. No. 33, 1-44. The Bacteriological Grading of Milk. Med. Res. Council Spl. Rept. 206, 1-392. The occurrence of paracolon orgianisms with Salmonella antigen. J. Bact., 51, 611. 

Mussel pollution at Korapuzha estuary

(Malabar), with an account of certain

coliform types

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Mussel pollution at Korapuzha estuary (Malabar), with an account of certain coliform types

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