Our results on this series of tests were erratic and inconsistent. However, much information and perhaps insight was gained on the technique and methods required in this process of self-purification of shellfish.

During the course of the study many problems became apparent. The major ones, as they appeared to us, were to maintain proper circulation and rate of water flow; and to remove feces, waste products, and scum from the tank (this was particularly true in the case of the Rangia Clam). Many other problems or factors such as lowering the ultraviolet tube closer to the water, using approved reflectors, sterilizing the water and sanitizing the system before the shellfish are added, and temperature control were encountered in this study. Incorrect or insufficient cleaning of the shellfish before the sterilization process is started also appears to be a major source of trouble.

With these problems in mind we are in the middle of another series of tests designed to determine the most applicable method of depuration for our area. Of course, all of the known influencing factors must be considered and an attempt to equate experimental methods in terms of commercial practicalities must be made.

We are now using a small experimental pilot system. This unit is composed of a holding tank, a circulating pump and plumbing, a filter, a sterilizing unit and aeration baffles. In general, most of the component parts are made of epoxy-covered plywood and PVC plumbing fixtures.

The holding tank measures 3 ft. long x 5 ft. wide x 1 ft. deep. Good circulation was accomplished in this tank by dividing it with weirs into four compartments. Water flows into the first compartment of the tank from the aeration baffle. It flows over the first weir, under the second and over the last one. The shellfish are kept in racks one inch from the bottom of the tank in the middle two compartments. The experimental water level is maintained at 200 liters. From the last compartment the water is pumped into a small glass wool filter and is then allowed to flow by gravity into the sterilizing unit. This unit measures 10 inches wide x 24 inches long x 8 inches deep and houses three 15-watt germicidal lamps. The height of the ultraviolet lamps from the water is six inches. From the sterilizer the water flows over the aeration baffles back into the holding tank.

The following variable influencing factors are being checked or will be checked: the oyster-water volume ratio, the circulation factor or rate of flow in reference to the total amount of water, the amount or removal of feces from the water, and the ultraviolet light intensity. Other influencing factors that must be given consideration are the general health of the shellfish, the original cleaning of the animals, and the sanitizing of the tanks and equipment.

At this time, we have completed two tests using one and two oysters per liter of water (200 to 400 average 3-inch size oysters per test). A maximum pumping rate of about 900 1/hr was maintained in each test. This would give a circulation turnover of about 4.5 times per hour. The detention time of the water in the sterilizer ranged from 15 to 25 seconds. Bacterial tests were made on both the meats and the water every 4 hours for 24 hours and also at 36 and 48 hours.

On the basis of the coliform test data, the water from the first test was coliform free after 4 hours and the oyster meats were below 800 MPN per 100 after 12 hours. In the second test the water was pure, zero count, in 4 hours and the meats were down to 1,300 in 4 hours. With one exception at 16 hours which we attributed to a laboratory accident, the meat counts were well below this figure throughout the rest of the 48 hours. The original coliform count of the meats at the start of each test were 9,500 and 240,000 MPN per 100 ml.

Our future plans call for a continuation of these tests using the small pilot set up, systematically checking the various influencing factors, and modifying the unit to adjust for these findings. Also utilizing all available information, we plan to build a commercial size unit and continue our work.

Employing the 50 percentile (cf. 2 above) which shows that a 30% reduction may be obtained under the conditions of operation as presently employed, the raw shellfish should not exceed a median MPN of 8000. This is far short of the 24,000 MPN limit established for shellfish to be used in a depuration system. Apparently, if we are to continue the shellfish depuration program, additional research is necessary to determine more efficient methods of depuration. The deficiencies of the depuration system may not lie in the mechanical features themselves, but in the shellfish. Certainly, biologists appreciate the fact that we are not dealing with such artifically programmed procedures as the chlorination of water, the pasteurization of milk, which practices can be easily standardized since the products to be rendered safe are inanimate, but with living matter exhibiting a multitude of inherent, varied characteristics which may behave one way today, another way tomorrow, and yet another way the next day. Investigations into the biology and physiology of shellfish and their relationships to coliform, pathogens and viruses are necessary before predictions relative to their reactions and interactions are possible. This kind of research is usually beyond the facilities provided to the States for implementation of their shellfish programs and probably should be undertaken by the Federal Government in their newly established shellfish research centers. We have looked to the Public Health Service before for basic research in many fields, and they have always rallied to our call. Hopefully, in this area of shellfish sanitation, on which so many are dependent for their livelihood, the call will not echo in the halls of well equipped and well staffed laboratories too long before it is heeded, and "the charge is on."

APPENDIX F

DEPURATION IN MAINE

By

Phillip L. Goggins

Maine Department of Sea and Shore Fisheries

Boothbay Harbor, Maine

Untreated sewage discharged in Maine coastal waters contaminates our shellfish growing areas, rendering these shellfish unfit for human consumption.

Between 1946 and 1960 the acreage of these areas which had to be restricted because of sewage pollution increased 15.6%. In 1960, 46,958 acres were closed because they were polluted. Further closures have been catastrophic to some of our coastal communities. Improved standards of living, additional summer cottages, increased tourist populations, all multiply the sewage draining toward the sea, causing this condition.

Pollution abatement programs, recently accelerated in Maine, cannot, by themselves, provide conditions allowing the unrestricted taking of shellfish. But these programs tied in with a controlled shellfish depuration scheme would permit the use of a major part of currently restricted areas.

Maine towns have had shellfishing rights since colonial times. Now, to keep these rights, the towns have to agree to manage their shellfisheries under a State-cooperative management plan. Most towns already are regulating their shellfisheries thus. It is apparent that shellfish depuration will soon be essential to shellfisheries management. In Maine our principal concern is the soft-shell clam (Mya arenaria.)

Early in 1964 the Maine Department of Sea and Shore Fisheries accelerated its research when it accepted a contract with the Area Redevelopment Administration to develop, in York County, a method for clam cleansing.

Following is a resume of work accomplished under the ARA contract and under a current contract with the U. S. Public Health Service. The full ARA report is currently being revised for publication:

DEPURATION STUDIES

The objectives are: 1) Determine the effect of environmental factors on depuration. Some of these are temperature, salinity, oxygen, and ratio of water flow to shellfish. 2) Develop equipment to handle shellfish through a depuration plant, 3) test the efficiency of handling and purifying shellfish on a commercial scale, and 4) develop and test a public health control scheme from the harvest grounds through the depuration. A dependable supply of inexpensive clean seawater is essential. Natural sites cannot be relied upon to produce clean water because our coastal water has multiple uses. It is necessary to treat the water.

Water Treatment

It was first necessary to treat the water in the clam cleansing operation. We considered the chlorination method but, because of its disadvantages, decided upon the ultraviolet sterilizer as developed by Kelly (1961). A treatment unit with fourteen 30-watt ultraviolet lamps similar to that of Kelly was built. The germicidal efficiency was about the same as his, 99.9% coliform reduction at approximately 35 gallons per minute. Sand filtration experiments through well points inserted at low tide level were made in York County, in southern Maine, and evaluated because of the

importance of turbidity in ultraviolet sterilization, and because of the availability of sand beaches in the study area. At 30 gallons per minute, with the well points covered with 18 inches of sand, coliform bacteria were reduced 92%. Raw water averages from 758 MPN coliform to 61 MPN.

Environmental Factors in Depuration

The experiments in the laboratory with soft-shell clams were designed to obtain information about the effect of operational procedures, and environmental stress on depuration. Load rates, hosing during the cleansing cycle, the effect of low salinity, depth of shellfish in containers during cleansing, and comparison between closed and open systems are some of the factors we considered important.

The water system we used was a flow-through one. Therefore it was necessary to determine a safe ratio of water flow to shellfish. For laboratory depuration tanks, plastic waste baskets of nine-liter capacity were used. These tanks were mounted so the intakes were centered in the bottom. Plastic-coated wire racks were placed two inches above the bottom to allow circulation space under the shellfish. The outlet was located centrally two inches from the lip of the basket. The baskets were fed from the sterilizer through a manifold with petcocks, to adjustable evaluations standpipe reservoirs made from pint plastic freezer containers. With these adjustable head devices, we were able to control flow to between fifty and 2000 ml. per minute, 10%. Twelve of these baskets with head controls were used in most of our experiments. Several water displacement measurements showed the average bushel of soft-shell clams, used in our experiment, to be 19.6 liters. One-half peck of clams, with an average volume of 3.2 liters, was used as a standard test volume for the laboratory depuration tanks. Flow rate was calculated as follows:

Vol. of bushel clams in cc

Vol. of clams in test tank in cc (See Tables 1 and 2.)

X Flow rate in cc = Flow rate in l/bu/min
1000

Our results indicate that between 16° and 20° C., at flow rates less than 1.5 1/bu/min, purification was adversely affected with oxygen depletion ranging between 70% and 90%. At 1.6 3.0 flow rate purification was not adversely affected, and oxygen depletion ranged between 35% and 70%. We feel that a flow rate of one gal/bu/ min is a safe one.

Washing the exterior of the clams by flushing with a high pressure water spray was then evaluated. Seventeen lots of clams were a zero hour median of 16,000 coliform and 3500 presumptive fecal coli were run through the laboratory cleansing plant at controlled flow rates of one gal/bu/min, with varying temperatures ranging from 15° C. - 20° C. The clams were flushed after twelve hours. After twenty-four hours the average bacterial reduction for the washed clams was 81% coliform and 98% fecal coli. The clams which were not washed reduced 88% coliform and 91% fecal coli after twenty-four hours, and 96% fecal coli after 48 hours.

These results do not indicate any benefit from washing after twenty-four hours, but do after forty-eight hours.

Singled Layered Clams vs. Layered in Depth Clams

Clams in single layers were compared with clams at various depths in the laboratory cleansing tanks. No adverse effect on depuration could be demonstrated in relation to depth. Our results showed that clams in a single layer did not cleanse quite as well as composite samples from bottom, middle, and surface layers of the laboratory cleansing tanks.

Open vs. Closed System

The open system sterilizer for the following comparison was trough-type, four ultraviolet lamps being used. The lamps were 30-watt. The sterilizer's germicidal efficiency tested comparably with Kelly's unit. The closed system used the Kelly unit. Water was recirculated through the unit from a reservoir of 360 gallons of seawater at the rate of six gallons per minute.

Standard laboratory cleansing tanks were used and water flow was adjusted at approximately one gal/min/bu. Temperature in the open system varied between 110 and 11.5 C. In the closed system it remained at room temperature of 18° c.

The rates of depuration in both systems were nearly the same for the 24-hour period, with 90% coliform and E. coli reduction. However, during the second 24-hour period, a tenfold increase in coliform MPN showed in 43% of the runs, while the E. coli MPN remained the same or increased only slighly. No such increase was observed in the open system clams.

Perhaps one explanation of the increase in the closed system during the second 24-hour period might be made by postulating that the high temperature accelerated the breakdown of the mucus of the feces, thus releasing either previously immobilized bacteria, or bacterial growth in the slime formation on the tanks, thereby permitting bacteria to be reconstituted and to cause repollution.

Laboratory vs. Commercial Pilot Plant Depuration

Parallel runs compared depuration using heated water from the pilot plant at Biddeford Pool, and "as is" temperature water at the laboratory in Boothbay Harbor (fig. 1).

Half-peck samples of identical lots of clams from the pilot plant were transported to Boothbay Harbor and run through the laboratory depuration plant. Water at the pilot plant was heated approximately 10 C., water at Boothbay Harbor was unheated.

The results shown in Table 3 do not indicate any dramatic benefit from heating the water. Because of the unquestionable increase in activity of the clams in the heated water as observed visually extension of the siphon and increase in cilia action - we cannot support a more precise statement concerning depuration at various temperatures.

Experimental Commercial Depuration

Table 4 shows the results of Sea and Shore Fisheries commercial plant at Biddeford Pool, and of the recently established private experimental commercial operation owned by Sea Fair, Incorporated, at Sebasco Estates. Two observations pertinent to standards for depurated clams should be made on the basis of the results: 1) Levels much lower than the interim oyster standard of 78 - 230 MPN can be attained, especially for the 48-hour cycle, and 2) the initial load is very important. The initial E. coli MPN of one of the Biddeford runs was extremely high, 11,000. This run did not cleanse to a satisfactory level in 48 hours. The Sea Fair results, on the other hand, show that the highest initial MPN of 2400 E. coli cleansed to 490 MPN in 24 hours, and to 20 MPN E. coli in 48 hours. The 2400 MPN E. coli initial load did not reduce to the oyster standard in 24 hours. We feel that safe initial levels for 24-hour cleansing cycles and for 48-hour cleansing cycles should be determined. When we accumulate more data we can be more precise in stating levels. However, we have observed this same result previously using coliforms. An extremely high coliform MPN initial load would reduce to a relatively high level in 24 hours, and remain at essentially the same point indefinitely.

PILOT COMMERCIAL DEPURATION PLANT FOR SOFT-SHELL CLAMS

A pilot commercial depuration plant for soft-shell clams was established at Biddeford Pool to develop necessary equipment, to test laboratory results on a commercial scale, and to develop and test control plans from the production area through the plant.

Procedure

The production flow through the plant is as follows: The incoming clams are passed through a mechanical washer on a conveyor belt where polluted sediment is washed from the exterior of the clams by pressure spraying, and broken, dead, and injured clams are rejected and removed. The clams are then collected in plastic mesh half-bushel baskets, which are placed in a tank, transported, and lowered into the depuration tank by means of an overhead monorail crane.