Microbial safety ofRangia cuneata, Louisiana estuarine clam, at harvest and following relaying

Food Microbiology,

1990,7, 107-l 11

Microbial safety of Rangia cuneata, Louisiana clam, at harvest and following relaying

estuarine

L. S. Andrews*, R. M. Grodner and M. W. Moody Department of Food Science, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA Received 29 November 1989 Rangia cuneata, commonly found in southeast Louisiana estuarine marshes, were monitored microbiologically every 4-6 weeks from April 1987 to March 1988. Survey for standard (aerobic) plate counts and fecal indicators were performed at harvest day 0 and following 7 and 14 days of container relaying. Clam meat standard (aerobic) plate counts ranged from 3.48-l-68 log CFU g-’ at harvest and 3-00-5-30 log CFUg-’ after relaying. Total coliform most probable numbers ranged from2.30-4.11 log CFU per 100 g at harvest and 2.024.18 log CFU per 100 g after relaying. Fecal coliform most probable numbers ranged from
Introduction Rangia cuneta is an abundant molluscan shellfish resource found in the inland marsh estuaries of the Southeastern coastal regions of the United States. There has been interest in developing this clam resource into a food commodity (Kane 1978). Louisiana Rangia populations are estimated at 5-10 billion (Hoese 19731. Although many of the estuarine clam habitats are located in areas subject to periodic flooding and exposure to raw sewage, most of the Rangia are in either restricted or open water. Shellfish from restricted water may be marketed after a period of relaying into approved water or depuration in a controlled environment. A major problem in marketing Rangia has been the reported naturally high aerobic bacterial populations. In North Carolina, under ideal conditions, freshly har*Corresponding author 0740-0020/90/020107

+ 05 $03.00/O

vested Rangia were found to contain up to 6.60 log CFU g-’ of clam meat (Kane 1978). To be considered of highest quality freshly harvested molluscan shellfish are expected to have a standard plate count (SPC) below 5.70 log CFU g-l. Consequently it was necessary to examine the Louisiana Rangia to determine the natural microbial level contained in the clam, establishing marketing feasibility and safety to the consumer. Clams were monitored throughout a la-month period to determine if the sanitary quality of the clams at harvest and following relaying would meet the regulatory requirements of the American Public Health Association (A.P.H.A. 1984) and the National Shellfish Sanitation Program (N.S.S.P. 19861 for commercial sale of molluscan shellfish. With the marsh estuarine water being frequently restricted, the clams were relayed to simulate a worst-case scenario environment. 0

1990 Academic

Press

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108

L. S. Andrews

Materials

et al.

and Methods

Sampling procedures A 2 week relaying period was carried out every 4-8 weeks over a 12-month period from April 1987 to March 1988. Rangia were dredged from an estuarine site near Lake Robin, 1 mile south of Yscloskey, Louisiana. Clam samples were taken from water approximately l-2 m in depth, placed into clean plastic bags, and iced for transfer to the Department of Food Science, Louisiana State University, according to proceedures of the A.P.H.A. (1984) for transport of molluscan shellfish. Marsh and Lake Borgne water samples were obtained in sterile polypropylene bottles and also transported on ice to LSU. A portable Hydrolab Water Analyzer Model 5901 (Preamplifier Assay, Hydrolab Corp., Austin, TX) was used for measurements of water temperature, salinity, pH and dissolved oxygen. All samples, both clam and water, from the relay site were treated in the same manner and transported on ice to LSU. Container relaying procedure On the day of dredging (day O),live clams were placed in approximately 05-m’ relay baskets (heavy plastic milk carrier type) to a depth of about 7 cm and transported to a shellfish harvest site in Lake Borgne, LA. The baskets were lowered to an approximate depth of 2-8 m, varying with tidal flow. Clam, and water samples were collected at the time of dredging and from the relay site after 7 and 14 days, respectively. Laboratory Analysis Clam and water samples were processed and tested within 12 b of collection according to recommended procedures of the A.P.H.A. (1984,1985). Three separate 225-g samples of clam meat and liquor (20-25 clams each sample) were homogenized with a Waring Blendor (high speed, 90 s) and then assayed in duplicate to determine the standard plate count (SPC as CFU g-i), total coliform 5-tube MPN (TC), fecal coliform 5-tube MPN (FC), and Escherichia coli 5-tube MPN followed by

IMViC confirmation (EC). Triplicate water samples from the dredge site (day 0) and the relay site (days 0,7 and 141 were examined for SPC, TC, FC and EC. Clam and water samples were also tested for coagulase positive Staphylococcus aureus and Streptococcus faecalis.

Statistical procedures Pearson correlation coefficients were used to analyze for significant relationships among water and clam SPC, TC, FC and EC. In addition, water condition differences in salinity, pH, temperature and dissolved oxygen were

analyzed and compared to the microbial parameters.

Results

and Discussion

Clam meat analysis Standard plate count (CFUg-I). Properly harvested and stored molluscan shellfish should have standard plate counts well below 5.70 log CFU g-i (5 x lo5 CFU g-l) when marketed (A.P.H.A. 1984). Counts at or above this level are indicative of possible abuse and of questionable sanitary practices. Previous studies of Rangia in North Carolina (Kane 1978) have suggested that this species of clam, when freshly harvested, may maintain a naturally high standard plate count at or exceeding the suggested limit of 5.70 log CFU g-’ clam meat. Standard plate counts on Rangia, at harvest, ranged from 3.48 to 4.68 log CFU g-’ (Table 1) with a yearly mean of 4.19 log CFU g-l clam meat at harvest and 4.64 log CFU g-’ following relaying, which is well below the suggested abuse level. Lake Borgne itself is periodically closed to shellfish harvesting during the month of April due to spring rain and increased run-off. The highest microbial counts of 5.30 log CFU g-’ were recorded during April following a 2-week relaying period inundated by heavy rain (Table 1). Total Coliforms (TC), Fecal Coliforms (FC), E. coli (EC). Total coliform MPN varied from 2.00 to 4.18 log CFU per 100 g clam meat. These values were not considered significant in relation to shellfish quality due to their increased presence in the water itself during periods of heavy rain, changing tidal flow, and the great number of coliforms that can be present in water from non-fecal sources. The rela-

Microbial tive importance of the total coliform value, in determining whether fecal pathogens are present in shellfish or growing waters, remains in question (N.S.S.P. 1987). The National Shellfish Sanitation Pro

Table

1. Summary

of clam

meat microbiological

safety of Rangia cuneata

109

gram (N.S.S.P. 1968) has established coliform guidelines for marketing molluscan shellfish. The fecal coliform (FC) limit is a geometric mean MPN of 2.36 log CFU per 100 g (230 CFU per 100 g) shellfish meat, with not greater than 10%

survey

April

1988.

1987-March

Collection Month

Apr

Day Ob

SPC” (CFU g-l)

TC” (CFU per 100 g)

FC” (CFU per 100 g)

Eta (CFU per 100 g)

‘IY

7 14

4.20 5.01 5.30

3.52 3.26 4.18

1.88 1.88 Cl.30

1.88 1.30 Cl.30

18 21 25

0 7 14

4.68 4.18 4.52

3.36 3.08 3.08

Cl.30 Cl.30 Cl.30

Cl.30 Cl.30 Cl.30

29 27 29

July

0 7 14

3.78 4.81 5.00

2.30 3.64 3.58

Cl.30 Cl.30 Cl.30

Cl.30 Cl.30 Cl.30

30 30 31

Ax

Ob 7 14

3.95 5.20 3.90

4.11 4.15 3.73

2.04 Cl.30 Cl.30

2.04 Cl.30 Cl.30

30 30 33

0 7b 14

3.60 4.49 3.60

2.90 3.52 2.60

1.85 1.90 Cl.30

1.85 1.90 Cl.30

28 24 21

0 7b 14

3.48 3.48 ND:

2.70 2.60 ND

1.48 1.67 ND

1.48 1.67 ND

22

Dee

0 7b 14”

4.38 3.00 3.30

2.48 3.18 2.00

1.57 1.94 1.76

1.57 1.60 1.76

15 16 20

Jan

Ob 7 14b

3.85 3.70 3.48

2.78 2.70 2.70

1.89 Cl.30 1.57

1.70 Cl.30 Cl.30

07 12 11

;i

4.38 3.95 3.30

3.68 3.86 3.18

2.32 2.22 1.86

2.20 1.48 1.80

15 18 18

May June

Sept Oct.

Nov

Feb March

14b

“Microbiological counts as log,,CFU g-’ or 100 g-l. All bacterial counts samples. Standard Aerobic Plate counts (SPC). Total coliforms (TC), Fecal were determined by MPN-5 tube dilution method. Note: No coagulase positive Staphylococcus was recovered. “Heavy rain within past 48 h or very turbulent water due to strong wind. ‘No data for this collection period due to storm conditions.

I%

are mean values of triplicate coliforms (FC), and E. coli (EC)

110

L. S. Andrews

et al.

of the samples to exceed 2.52 log CFU per 100 g (330 CFU per 100 g) shellfish meat. The same guidelines are used for shellfish that have undergone container relaying. Recorded most probable number values of cl-30 log CFU per 100 g (~20 CFU per 100 g) for clam meat indicate that no organisms were recovered (A.P.H.A. 1984). Fecal coliform counts remained guidelines within proper established throughout the entire 12-month cycle. During the 14-day relaying period, fecal were reduced except in coliforms November and December of 1987 (Table 1). Frequent stormy weather with heavy rains occurred during these 2 months, as well as a reduction in water temperature. These combined environmental changes would account for the lack of response of the molluscs to the relaying procedure. A decrease in water temperature below 182O”C, alone, has been reported to reduce molluscan filtering rate (Hopkins et al. 1972, Perkins et al. 1980). Escherichia coli was demonstrated in approximately 90% of the fecal coliform positive MPN tubes and as such indicated a reduction pattern similar to fecal coliforms with the exception of December 1987. Water analysis. Water samples from both the dredge site and the relaying site were found to maintain the necessary microbial quality to meet federal guidelines for open shellfish harvesting during this study period. Salinity, dissolved oxygen, and pH were found to be variable with weather conditions and not correlated with microbial parameters. Other micro-organisms. No coagulasepositive Staphylococcus aureus or Strep-

tococcus faecalis

were recovered

in any

clam samples. Statistical

evaluation

The Pearson Product Moment Correlation Coefficient was used to evaluate for possible correlation among the various clam meat data. The only significant correlation found was between clam meat FC and EC on days 0 and 14 (P < 0.01). Lack of correlation between clam meat FC and EC on day 7 (P >0*05) might have been due to the ability of the clam to purge E. coli more rapidly than other fecal coliforms, or the possibility of greater sensitvity of E. coli to environmental stress (Feachem et al. 1983) especially during relaying. Rangia examined in this study met all regulatory guidelines of microbiological safety for molluscan shellfish before and after having undergone container relaying as established by the N.S.S.P. However, since marsh waters are frequently not approved for open molluscan shellfish harvesting based on the water classification by the N.S.S.P., clam samples may require either 14 days relaying or depuration before being acceptable for marketing. Acknowledgements This research was funded in part by Gulf and South Atlantic Fisheries Development Foundation, Inc. Special thanks to the late State Marine Agent Warren Mermilliod. Approved for publication by the Director, Louisiana Agricultural Experiment Station, Manuscript No. LA 2438.

References A.P.H.A. (1984) Compendium

of methods for the microbiological

American Public Health Association, Washington, DC.

examination

of food (6th edn).

Microbial

safety of Rangia cuneata

111

A.P.H.A. (1985) Standard methods for the examination of water and waste water (16th edn). American Public Health Association, Washington, DC. Feachem, R. G., Bradley, D. J., Garelick, H. and Mara, D. D., (1983) Sanitation and disease, health aspects of excreta and waste water management. Wiley, New York, NY. Hoese, H. D. (1973) Abundance of the low salinity clam, Rangia cuneta, in southwestern Louisiana. Proc. Natl. Shellfish Assoc. 63,99-106. Hopkins, S. H., Amderson, J. W. and Horvath, K. (1972) The brackish water clam, Rangia cuneata, as indicator of ecological effects of salinity changes in coastal waters. U.S. Army Corps of Engineers. Waterways Experimental Station, Vicksburg, MS. Contract Rep. No. DACW39-71-C -0007. Kane, B. E. (1978) Rangia clam microbiology study. Proceedings of the Interstate Seafood Seminar VPZ-SG81-01: 29. Blackburg, VA. N.S.S.P. (1986) Sanitation of shellfish growing areas. National Shellfish Sanitation Program. Manual of operations. Part I. US Dept. of Health and Human Services, PHS (FDA). Washington. DC. N.S.S.P. (1987) Sanitation of the harvesting, processing and distribution of shellfish National Shellfish Sanitation Sanitation Program. Manual of operations. Part ZZ.US Dept. of Health and Human Services, PHS (FDA). Washington, DC. Perkins, F. O., Haven, D. S., Morales-Alamo, R. and Rhodes, M. W. (1980) Uptake and elimination of bacteria in shellfish. J. Food Prot. 43,124.