PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION OFFICIAL PUBLICATION OF THE NATIONAL SHELLFISHERIES ASSOCIATION; AN ANNUAL JOURNAL DEVOTED TO SHELLFISHERY BIOLOGY VOLUME 66 Published for the National Shellfisheries Association, Inc. by The Memorial Press Group, Plymouth, Massachusetts JUNE 1976 PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION CONTENTS Volume 66 — June 1976 List of Abstracts by Author of Technical Papers Presented at the 1975 NSA Annual Meeting, Charleston, South Carolina v Patricia A. Cunningham Inhibition of Shell Growth in the Presence of Mercury and Subsequent Recovery of Juvenile Oysters 1 M. Lynn Haines The Reproductive Cycle of the Sunray Venus Clam Macrocallista nimbosa (Lightfoot 1786) 6 Peter J. Eldridge, Wayne Waltz, Robert C. Gracy and Hurshell H. Hunt Growth and Mortality Rates of Hatchery Seed Clams, Mercenaria mercenaria, in Protected Trays in Waters of South Carolina 13 Howard M. Feder, A. J. Paul and J. Paul Age, Growth, and Size-Weight Relationships of the Pinkneck Clam, Spisula pol\/n\/ma, in Hartney Bay, Prince William Sound, Alaska 21 A. J. Paul, J. M. Paul and H. M. Feder Age, Growth and Recruitment of the Butter Clam, Saxidomus gigantea, on Porpoise Island, Southeast Alaska 26 Rodner R. Winget, Charles E. Epifanio, Tom Runnels and Paul Austin Effects of Diet and Temperature on Growth and Mortality of the Blue Crab, Callinectes sapidus, Maintained in a Recirculating Culture System 29 Patricia Clark Michael and Kenneth K. Chew Growth of Pacific Oysters, Crflssosfreagigas, and Related Fouling Problems under Tray Culture at Seabeck Bay, Washington 34 Neil B. Savage and Ronald Goldberg Investigation of Practical means of Distinguishing Mya arenaria and Hiatella sp. Larvae in Plankton Samples 42 A. H. Price II, C.T. Hess and C. W. Smith Observations of Crassostrea virginica Cultured in the Heated Effluent and Discharged Radionuclides of a Nuclear Power Reactor 54 D. B. Quayle and F. R. Bernard Purification of Basket-Held Oysters in the Natural Environment 69 Bruce J. Neilson A Mathetical Approach to Depuration 76 Kwang Hi Im, Richard S. Johnston and R. Donald Langmo The Economics of Hatchery Production of Pacific Oyster Seed; A Research Progress Report 81 Willem Roosenburg Can The Oyster Industry Learn From Livestock Breeders? 95 Abstracts: NSA Annual Meeting NSA Pacific Coast Section 100 LIST OF ABSTRACTS BY AUTHOR OF TECHNICAL PAPERS PRESENTED AT THE 1975 NSA ANNUAL MEETING, CHARLESTON, SOUTH CAROLINA Michael Castagna and John N. Kraeuter A Mariculture System for Growing the Hard Clam, Mercenaria mercenaria 100 Richard Dame Systems Analysis of an Oyster Community: An Evolving Model 100 Albert F. Eble Freshwater Aquaculture of the Tropical Prawn, Macrobrachium rosenbergii, and the Rainbow Trout, Salmo gairdnerii, Using Thermal Effluent Discharges from an Electric Generating Station 100 Mark C. Evans Fabricated Substrates — An Approach to the Intensive Culture of Macrobrachium rosenbergii (de Man) 101 Robert C. Gracy Survey of South Carolina's Hard Clam (Mercenaria mercenaria) Resource 101 Herbert Hidu The Suitability of Marine Waters for Culturing Oysters (C. virginica and O. edulis) 102 Louis Leibovitz, S. B. Hitchner, Paul Chanley, David Delyea, and Joseph Zatila A Study of Shellfish Hatchery Bacterial Diseases 102 Ernesto Lorda and John W. Zahradnik Operating Characteristics of a Heated, Raw Seawater, Oyster Finishing Pilot Plant 102 R. W. Menzel, E. C. Cake, M. L. Haines, R. E. Martin and L. A. Olson Clam Mariculture in Northwest Florida: Observations on Selection and Hybridization 103 W. A. Murphy Oyster Development in Atlantic Canada — The Potential and the Problems 103 Lawrence A. Olsen Ingested Material in Two Species of Estuarine Bivalves: Rangia cuneata (Gray) and Polymesoda caroliniana (Bosc) 103 Patrick R. Parrish, Kenneth S. Buxton and James R. Gibson Oysters (Crassostrea vtrginica) Exposed to a Complex Industrial Waste: Survival, Growth and Uptake of Antimony Compounds 104 Hugh J. Porter and Frank J. Schwartz Seasonal Variations in Tissue Weight and Total Solids of the Calico Scallop, Argopecten gibbus, and their Relationship to Changes in Gonad Condition 104 v Kenneth M. Rodde and Judith B. SunderHn The Mariculture Potential of Tapes semidecussata (Reeve) in an Artificial Upwelling System 105 William N. Shaw Scallop Culture in Mutsu Bay, Japan 105 NilsE.Stolpe Recirculating Systems for Embryo Incubation and Larval Rearing of the Freshwater Prawn Macrobrachium rosenbergii (de Man) 105 Larry Turner and John W. Zahradnik Physical Parameters of the American Oyster, Crassostrea virginica (Gmelin, from the Wareham River 106 Ronald E. Westley The Present Status and Future Outlook of Shellfish Farming in Puget Sound, Washington 106 ABSTRACTS OF THE NSA PACIFIC COAST SECTION W. P Breese Out-bay Culture 107 Rick D. Cardwell Relative Acute Toxicity of a Pesticide, Heavy Metal and Anionic Surfactants to Marine Organisms 107 Jim Donaldson, Chuck Munsey, and Vance Lipovsky Winter Spawning of Pacific Oysters 107 William Engesser, Chi Ming Cheung, Salahuddin Faruqui and Willie Mercer System Work Design and Interim Reports Covering Current and Proposed Industrial Engineering Standards for Shrimp, Crab, Oysters, Bottom-Fish and Product-mix Species 108 William Hershberger An Approach to Developing a Stock of Disease Resistant Oysters 108 Jack Rensel Site Comparison for the Culture of the Spot Prawn Pandalus platyceros Brandt In and Adjacent to Salmon Net Pens 109 Albert Scholz Relationship Between Pacific Oyster Seed Density and First Year Growth 109 VI Proceedings of the National Shellfisheries Association Volume 66 — 1976 /-T INHIBITION OF SHELL GROWTH IN THE PRESENCE OF MERCURY AND SUBSEQUENT RECOVERY OF JUVENILE OYSTERS' Patricia A. Cunningham^ DEPARTMENT OF BIOLOGICAL SCIENCES UNIVERSITY OF DELAWARE NEWARK, DELAWARE ABSTRACT Juvenile oysters (Crassostrea virginica) were given static exposure for 12 hours each day to mercuric acetate added at 100 ppb or 10 ppb mercury for 47 days. Shell growth was measured as the increase in height (distance from hinge to posterior margin). In- hibition in shell growth was used as an indicator of physiological stress. After 47 days, shell growth was reduced by 77% for the 100 ppb group and by 33% for the 10 ppb mercury group compared to controls. Oysters in seawater for a 162-day depuration period demonstrated shell growth rates comparable to controls within 34 days (100 ppb) and 20 days (10 ppb). INTRODUCTION Estuarine and coastal waters have become the repositories for numerous effluents of both in- dustrial and agricultural activities. Mercury is one of the most toxic of these materials and increased mercury concentrations in seawater are reflected by increased mercury concentrations in coastal marine organisms (Klein and Goldberg, 1970). Mercury stress might, therefore, be expected to mediate major physiological adjustments by marine and estuarine species. Published studies of the effects of mercury on bivalves have been limited primarily to determina- tions of whole body mercury residues (Kurland et al, 1960; Craig, 1967; Kopfler, 1974). Residue studies conducted over extensive periods have provided data on the rate of accumulation and removal of mercury from mollusk tissues (Seymour and Nelson, 1971; Cunningham and Present Address: Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (operated by Union Carbide Corporation under contract with the U.S. Energy Research and Development Ad- ministration). Tripp, 1973; Cunningham and Tripp, 1975a). Unlu et al. (1970) determined mercury concentra- tions in various tissues of Tapes descussatus; mer- cury was accumulated directly from seawater or from ingestion of contaminated algae. Cunn- ingham and Tripp (1975b) performed a similar ex- periment on Crassostrea virginica to monitor changes in the tissue distribution of mercury dur- ing an accumulation and depuration period. One disadvantage of most residue studies is that ex- perimental organisms must be sacrificed periodically rather than being monitored con- tinuously. Thus, little is known of the details of physiological stress imposed on organisms. Butler et al. (1960) demonstrated that shell growth in juvenile oysters could be employed as a sensitive method for the continuous monitoring of physiological stress occurring in bivalves exposed to various concentrations of pesticides. The method of Butler et al. (1960) was used in studies by Shuster and Pringle (1969) of cadmium, chromium, zinc, and copper; by Lowe et al. (1971) of DDT, toxaphene, and parathion; and by Lowe et al. (1972) of the polychlorinated biphenyl com- pound Aroclor 1254. These authors however. p. A. CUNNINGHAM monitored only shell growth inhibition during the exposure period and did not measure the time re- quired for resumption of normal shell growth dur- ing a depuration period as originally suggested by Butler et al. (1960). This could be important in determining long term effects of various toxicants on populations and whether recovery of exposed individuals to a healthy physiological state is pxDssible. This study was initiated to determine the in- hibitory effects of mercury on shell growth, and the time required for the juveniles to recover and resume normal shell deposition when held in am- bient seawater. MATERIALS AND METHODS Experimental Stock Juvenile Crassostrea virginica were reared from larvae produced in artificial spawning experiments at the University of Delaware's Shellfish Laboratory, Lewes, Delaware. All juveniles were 15 months old at the initiation of the study on July 10, 1971, and ranged in height (distance from hinge to posterior margin) from 3.66 to 6.20 cen- timeters. This height corresponds to the size range recommended for shell growth experiments (Butler, 1965). Oysters were cleaned, and the outer shell was allowed to air dry so that each in- dividual could be permanently marked with an identification number using waterproof ink. The posterior shell margins were then filed to remove all new shell growth, and height measurements were recorded. Thirty -six oysters were thus mark- ed, and were randomly divided into three groups: 100 parts per billion (ppb) mercury, 10 ppb mer- cury, and controls. Each experimental group was placed in a separate 230-liter tank receiving unfiltered Broadkill River water (daily salinity range 33 to 17 o/oo; DeVVitt, 1971). Oysters obtained all their food from the seawater supply. The shell growth study initiated on July 10 v,fas made in conjunction with a mercury residue study begun on July 1 and previously reported by Cunningham and Tripp (1973). Preparation of Experimental Environment A stock solution of mercury was prepared con- taining 0.30 grams of mercuric acetate, Hg(CH3COOH)2, dissolved in one liter of distilled water. Dilutions were in parts per billion mercury rather than in parts per billion mercuric acetate. The experimental dilutions (100 ppb and 10 ppb mercury) were chosen to correspond to concentra- tions found in the Delaware River basin which ranged from trace amounts to a maximum of 90 ppb mercury (Delaware River Basin Commission, 1970). Each experimental day was divided into two 12- hour periods. During the 12-hour feeding period (8 a.m. to 8 p.m.), unfiltered seawater was pumped directly into each experimental tank at a rate of 2.8 liters per minute. A constant volume of 190 liters of seawater was maintained in each tank during this period and aeration was provided. Mean summer water temperature was 25C ± 2C. During the 12-hour mercury exposure period (8 p.m. to 8 a.m.), the seawater input was stopped and a constant volume of 190 liters of aerated water was maintained. To this constant volume, stock mercury solution was added to yield 100 ppb or 10 ppb mercury. No mercury additions were made to the control tank. In the morning, the mercury-contaminated water was removed, fresh seawater was introduced, and continued to flow for the duration of the 12-hour day-time feeding period. This alternating 12-hour cycle of mercury exposure was maintained for 47 days, from July 10 through August 26, and was employed to reduce the volume of mercury-contaminated effluent which would have been produced by a 24-hour flowing system. At the end of the mercury exposure period, a 162-day depuration period was initiated to deter- mine whether shell growth comparable to controls could be resumed in juveniles previously exposed to mercury. For this portion of the experiment, ambient seawater was pumped into each tank at the rate of 2.8 liters per minute. Water temperature ranged from 25C to OC during the cleansing period, (August 26, 1971 to February 5, 1972). Measurement of Shell Growth During the mercury exposure period, juveniles were removed from the experimental en- vironments after 5, 15, 20, 35, and 47 days. Height measurements were made on each individual to determine the amount of newly deposited shell. After each measurement, juveniles were again fil- ed and measured. The filing procedure prescribed SHELL GROWTH OF JUVENILE OYSTERS by Butler et al. (1960) stimulates further shell pro- duction and evens the irregular margin of the elongated shell that forms during the normal growth process. During the 162-day depuration period, juveniles were removed from the experimental en- vironments after 20, 34, 62, and 162 days. The same procedures for measuring shell growth and filing used in the exposure period were employed for this portion of the experiment. All shell growth data were evaluated to the P < 0.05 level of confidence using an analysis of variance coupled with a Duncan Multiple Range test. A detailed summary of the procedure for A— A control •••■• 10 ppb ■--■ 100 ppb 20 40 DAYS Fig. 1. Cumulative shell growth i)i juvenile oysters during a 47-day mercury exposure period. ranking the means and computing the Duncan Multiple Range (DMR) values is given in Steel and Torrie (1960). RESULTS Mercury Exposure Period Juvenile oysters exposed to 100 ppb and 10 ppb mercury-contaminated seawater exhibited signifi- cant inhibition in shell growth after only 5 days of exposure (Table 1). After 47 days exposure, the mean cumulative shell production in the control, 10 ppb and 100 ppb groups was 6.53 mm, 4.37 mm, and 1.50 mm, respectively. Cumulative shell growth during the exposure period was reduced by 77% in the 100 ppb group and by 33% in the 10 ppb group (Fig. 1). Depuration Period After 20 days of cleansing in ambient seawater (the time of earliest measurement), there was no significant difference in mean shell growth be- tween the control and 10 ppb group. The rate of SHELL GROWTH (mm) Fig. l.Cumulative shell growth in juvenile oysters during a 162-day depuration period. recovery was slower in the 100 ppb group, however, and only after 34 days did that group return to the normal rate of shell deposition (Fig. 2). A recession in shell deposition observed in all 3 groups after September 30 (day 34) may have been due to the seasonal decline in ambient water temperature. Seasonal recession in shell growth in oysters was observed by Butler et al. (1960). 4 P. A. CUNNINGHAM TABLE 1. Cumulative shell growth (mm) in juvenile oysters, Crassostrea virginica during a mercury ex- posure period and subsequent depuration period. Date 1971 July 10 July 15 July 25 July 30 August 14 August 26 August 26 September 15 September 30 October 28 February 5 Days 0 5 15 20 35 47 0 20 34 62 162 MERCURY EXPOSURE PERIOD MEAN SHELL GROWTH (mm ± 1 S.D. Control lOppb 100 ppb 1.13±0.26 3.73±0.62 4.12±0.61 5.31±0.77 6.53 + 0.78 0.47 + 0.16 1.82±0.42 2.20±0.52 3.26±0.56 4.37±0.70 0.36±0.14 0.69±0.19 1.03±0.29 1.33±0.39 1.50±0.40 DEPURATION PERIOD 3.98±0.48 4.18±0.43 4.19±0.46 5.17±0.65 4.34+0.72 4.61 ±0.72 4.64±0.71 5.00±0.68 2.68±0.32 3.79±0.41 3.87 + 0.42 5.00±0.57 Ftest 4.28 11.11* 9.56* 10.74* 13.46* 2.93 0.53 0.47 0.02 DMR" ABC A BC A BC A BC ABC ABC AB C ABC ABC ABC a Duncan Multiple Range (DMR) Analysis: Any two means (A = Control, B = 10 ppb, C = 100 ppb) not underscored by the same line are significantly different (P<0.05 level of confidence) (Steel andTorrie, 1960). *P<0.05 DISCUSSION In biological systems, mercury can act as an in- hibitor by combining reversibly with the sufhydryl groups of cysteine residues that are essential for the catalytic activity of some enzymes (Barron et al, 1948; Lehninger, 1970). This biochemical view of mercury inhibition in enzyme systems may be exemplified on the whole organism level by shell inhibition in juvenile oysters. Mercury ions may be inactivating en- zymes in several metabolic pathways required in the shell deposition process. Reversal of the in- hibitory effect is demonstrated during the depura- tion period by rapid recovery of mercury exposed individuals to shell production rates comparable to those of unexposed controls. In earlier ex- periments, adult oysters maintained in mercury- contaminated seawater (100 ppb and 10 ppb) ex- hibited a large decline in mercury tissue residues after a cleansing period in ambient seawater (Cun- ningham and Tripp, 1973; 1975a), and juveniles would be expected to exhibit a similar decline. Shellfish concentrate heavy metal ions in their tissues to many times the concentrations present in seawater without exhibiting detectable detrimen- tal effects. Pringle et a/. (1968) measured this con- centration effect (biological enrichment factor) for 8 metals in the oyster, Crassostrea uirgmicfl. Values ranged from 2,900 for manganese to 226,000 for cadmium. Mercury residues in adult oysters of 27,950 ppb (for a 10 ppb exposure group) did not cause mortality greater than that on controls (6%), but 50% mortality was record- ed (for a 100 ppb exposure group) when the average mercury tissue residue was 140,710 ppb (Cunningham and Tripp, 1973). Thus, mortality is a poor criterion for assessing the detrimental ef- fects of mercury accumulation in oysters. Quicker, more precise measurement of reversible inhibitory effects of toxicants can be obtained us- ing Butler et a/. (1960) shell growth technique. Shell growth inhibition and recovery studies should be used to augment residue experiments and could be more widely used to test water quali- ty- SHELL GROWTH OF JUVENILE OYSTERS ACKNOWLEDGMENTS The author expresses her gratitude to Drs. Ken- neth Price and Donald Maurer of the College of Marine Science, University of Delaware, for ac- cess to the facilities of the Shellfish Laboratory, Lewes, Delaware, and for providing the juvenile oysters used in this experiment. Special thanks is given to Dr. Marenes Tripp for his assistance throughout this study. LITERATURE CITED Barron, E. S., L. Nelson and M. Ardao. 1948. Regulatory mechanisms of cellular respiration. II. The role of soluble sulfhydryl groups as shown by the effects of sulfhydryl reagents on the respiration of sea urchin sperm. J. Gen. Physiol. 32: 179-190. Butler, P. A. 1965. Reaction of estuarine molluscs to some environmental factors. Publ. Health Serv. Publ. 999-WP-25. Butler, P. A., A. J. Wilson and A. J. Rick. 1960. Effect of pesticides on oysters. Proc. Nat. Shellfish Assn. 51:23-32. Craig, S. 1967. Toxic ions in bivalves. J. Am. Osteopath Assn. 66(9): 1000-1002. Cunningham, P. A. and M. R. Tripp. 1973. Ac- cumulation and depuration of mercury in the American oyster, Crassostrea virginica. Mar. Biol. 20(1): 14-19. Cunningham, P. A. and M. R. Tripp, 1975a. Fac- tors affecting accumulation and removal of mercury from tissues of the American oyster, Crassostrea virgmica. Mar. Biol. 31:311-319. Cunningham, P. A. and M.R. Tripp. 1975b. Ac- cumulation, tissue distribution, and elimination of "'HgCU and CHj^^HgCl in the tissues of the American oyster, Crassostrea virginica. Mar. Biol. 31: 321-334. Delaware River Basin Commission. 1970. Mer- cury sampling results — Delaware River. Tren- ton, New Jersey. (Internal publication). DeWitt, W. 1971. Water quality variation in the Broadkill River Estuary. Doctoral dissertation. University of Delaware, Newark. Klein, D. and E. Goldberg. 1970. Mercury in the marine environment. Environ. Sci. Technol. 4(9): 765-768. Kopfler. F. 1974. The accumulation of organic and inorganic mercury compounds by the Eastern oyster, Crassostrea virgmica. Bull. Environ. Contamin. and Toxicol. ll(3):275-280. Kurland, L. T., S. N. Faro and H. Siedler. 1960. Minamata disease: The outbreak of neurological disorders in Minamata, Japan and its relationship to the ingestion of seafood con- taminated by mercury. World Neurol. 1(5): 370-391. Lehninger, A. 1970. Biochemistry. Worth Publishers Inc., New York, New York. 833 p. Lowe, J. I., P. R. Parrish, J. M. Patrick and J. Forester. 1972. Effects of the polychlorinated biphenyl Aroclor 1254 on the American oyster, Crassostrea virginica. Mar. Biol. 17(3): 209-214. Lowe, J. I., P. D. Wilson, A. J. Rick and A. J. Wilson. 1971. Chronic exposure of oysters to DDT, toxaphene and parathion. Proc. Nat. Shellfish Assn. 61: 71-79. Pringle, B., D. Hissong, A. Katz and S. Mulawka. 1968. Trace metal accumulation by estuarine mollusks. Am. Soc. Civil Engin. Proc. Sanitary Engineering Div. 94 (SAE) 5970:455-475. Seymour, A. and V. Nelson. 1971. Biological half- lives for zinc and mercury in the Pacific oyster, Crassostrea gigas.Proc. 3rd. Nat. Symp. on Radioecology. Oak Ridge, Tennessee, p. 849-856. Shuster, C. and B. Pringle. 1969. Trace metal ac- cumulation by the American oyster, Crassostrea virginica. Proc. Nat. Shellfish Assn. 61: 71-79. Steel, R. and J. Torrie. 1960. Principles and Pro- cedures of Statistics. McGraw-Hill Book Com- pany, New York, New York. 481 p. Unlu, M. Y., M. Heyraud and S. Keckes. 1970. Mercury as a hydrosperic pollutant. I. Ac- cumulation and excretion of "^HgCU in Tapes descussatus. F.A.O. technical conference on marine pollution and its effects of living resources and fishing. Rome, Italy, p. 1-6. Proceedings of the National Shellfisheries Association Volume 66 — 1976 THE REPRODUCTIVE CYCLE OF THE SUNRAY VENUS CLAM Macrocallista nimbosa (LIGHTFOOT 1786) M. Lx/nn Haines DEPARTMENT OF OCEANOGRAPHY FLORIDA STATE UNIVERSITY TALLAHASSEE, FLORIDA ABSTRACT The annual reproductive cycle is described for the sunray venus clam, Macrocallista nimbosa (Lightfoot). Clams used in the study were collected from waters adjacent to Blacks Island in St. Joseph Bay, Florida. The reproductive activity was determined by histological examination of gonadal sections, by monitoring variation in the glycogen content of the tissues, and by variation in strip spawning potentiality. Results from the histological study indicate that spawning during 1974 began in July for males and continued through December, with peak spawning occurring during November. Females began spawning in August and continued throughout November, reaching peak spawning activity during October and November. The reproductive activity was reflected in the seasonal variation in glycogen content, with greatest glycogen storage occurring during the winter, averaging 15%, then reaching an annual low value of 3.8% in July, with a slight rise in August and September and a drop to 5.1% during October, returning to high winter values again in December. Clams were strip-spawned throughout the year, but viable larvae were obtained on- ly during October and November. INTRODUCTION The clam fishery in Florida has been dominated by three venerid species, the northern quahog, Mercenaria mercenaria (L.); the southern quahog, M. campechiensis (Gmelin); and the sunray venus clam, Macrocallista nimbosa (Lightfoot) (God- charles and Jaap, 1973). The fishery for the sunray venus clam was initiated in February 1967 near Port St. Joe, Florida, but at present, the fishery is inactive because insufficient numbers of clams are available. The present study was undertaken to provide basic information on the biology of this potential mariculture organism. This paper presents the first published descrip- tion of the reproductive cycle of the sunray venus clam. Earlier investigators used a variety of research methods, either singly or in combination to determine the reproductive activity in bivalves. Three methods were used in this study: (1) histological examination of gonadal sections; (2) variation in glycogen content of tissue; and (3) variation in strip-spawning potentiality. This study compares and contrasts the suitability of these methods in ascertaining the seasonal reproductive state of M. nimbosa. MATERIALS AND METHODS The study began in January and was completed in December 1974. The clams were collected in waters 300 meters north of Blacks Island in St. Joseph Bay, Florida in depths ranging from 1.0 to 1.5 meters at mean low tide. Collections for the histological study were made once a month except REPRODUCTIVE CYCLE OF SUNRAY VENUS CLAM for the months of July and August when two col- lections were made per month. The sample size ranged from 10 to 23 clams per month. Within 24 hours of collection, a 10-mm cube of gonadal tissue was removed from the mid-lateral portion of the visceral mass of each clam and preserved in Bouin's fixative. After 7 days in Bouin's fixative, the tissues were transferred to 50% ethanol and held for further histological pro- cessing. Fixed gonadal tissues were dehydrated in alcohol, cleared in xylene, and embedded in paraf- fin. The embedded tissue was then sectioned at 8 microns, mounted on a slide, stained in Erlich's hematoxylin, and counterstained with alcoholic eosin. Each slide was examined microscopically and a random sample of 20 alveoli per slide was assigned to a category of gonad condition. The histological study of the reproductive cycle is based upon ex- amination of 4,000 alveoli. The cyclic reproduc- tive process is divided into 5 phases of gonad con- dition which apply to both sexes: early active, late active, ripe, partially spawned, and spent. These 5 phases and their distinguishing characteristics are used by various investigators, e.g.. Ropes and Stickney (1965) for Mya arenaria, Ropes (1968) for Spisula solidissima, Cain (1972) for Rangia cuneata, and Holland and Chew (1974) for Venerupis japonica. There is no sharp distinction between phases and the categories are convenient rather than natural (Ropes, 1968). The method used in this study of examining 20 alveoli per slide or individual is different from the methods of other investigators who assign each clam or slide to one of the 5 phases. A new pro- cedure was used for the sunray venus clam be- cause one clam may contain alveoli in several dif- ferent phases, and to assign the individual clam to just one phase would have masked the other phases present. It is also believed that maturation of alveoli from one phase to another can occur within a few days time, and that individual clams do not empty all of their gametes at one spawning, so it is informative to express the proportion of alveoli in the various phases. The five phases of female gonad condition and their distinguishing characteristics are described: (1) Early Active Phase. Ovogonia occur at the periphery and within the alveolar walls. Follicle cells frequently occur within the alveolus. (2) Late Active Phase. Alveoli in the late active phase contain a large number of elongated stalked ovocytes, whose free ends protrude into the alveolar lumen and whose bases attach to the alveolar wall. The large, stained ovocyte nucleus is a conspicuous characteristic of this phase and basophilic nucleoli are present. The basement membrane is thin. (3) Ripe Phase. The alveolus is termed ripe when the number of ova free within the lumina exceeds the number of attached ovocytes (Cain, 1972). The attached ovocytes resemble those in the late active phase, except that in the ripe phase, am- phinucleoli are present. Ripe alveoli are filled with ova and ovocytes and appear crowded together. (4) Partially Spawned Phase. A few ovocytes are still attached to the thickened alveolar walls; a few residual ripe ova may remain in the alveolar lumen. (5) Spent Phase. Alveoli are usually empty of ripe ovocytes; those that are present are usually undergoing cytolysis. The alveolar walls are thickened and oogonia are often present. The spaces between alveoli are filled with mesen- chyme. The stages of male gonad condition are describ- ed: (1). Early Active Phase. Alveolar walls are thickened and contain darkly stained sper- matogonia. Primary spermatocytes are found at the alveolar periphery and begin to proliferate toward the lumen. Follicle cells may fill the alveoli. Alveoli contain nutritive phagocytes (Loonsanoff, 1937). (2). Late Active Phase. This phase is characterized by the proliferation and maturation of sper- matocytes. The spermatocytes are uniformly shaped cells which at the initiation of maturation are found near the alveolar periphery, but as development progresses, migrate toward the alveoli centers. Later in the active phase the sper- matocytes elongate and are arranged in radially aligned columns. A central lumen within the alveolus is formed. A small number of sper- matozoa may be within the lumen. (3). Ripe Phase. With the development of tails the spermatids are transformed into spermatozoa. Spermatozoa are arranged in radial columns in the alveoli with their tails oriented toward the lumen. M. L. HAINES Later in this phase, masses of free spermatozoa fill the alveolar lumen. (4). Partially Spawned Phase. Spermatozoa are present within alveolar centers, but are less numerous than in the ripe phase. A thin band of spermatogonia and primary spermatocytes may be found along the basal membrane before the alveolus is empty of spermatozoa. (5). Spent Phase. Alveoli in the spent phase con- tain few or no spermatozoa and the lumina are open. The glycogen content of clam tissue was deter- mined by the colorimetric phenol method (Westenhouse, 1968). Clams used for glycogen analysis were processed within 24 hours of collec- tion. Five clams ranging from 120 to 130 mm in length were analyzed each month for their glycogen content. The glycogen content for each month is a mean of three replicate determinations. The strip-spawning potential was checked biweekly during the first 5 months of 1974, then checked at weekly intervals for the remainder of the year. The gametes were procured by methods described by Loosanoff and Davis (1963). During each test the gametes from 5 different females and 5 different males were pooled, and half of the gamete solution received 15 ml O.lN NH4OH treatment to dissolve the ovum germinal vescicle. Three hours after the addition of sperm to the egg suspension, the eggs were microscopically examin- ed for evidence of cleavage. If cleavage did occur, the cultures were continued and examined 48 hours after fertilization. RESULTS AND DISCUSSION Histological Study Histological examination of the 1974 female reproductive cycle revealed a single annual spawning period which began in August and con- tinued through November with peak spawning ac- tivity occurring during the fall months of October and November (Fig. 1). All five gonadal phases were present throughout the winter and spring months, January to May, with little change occurring in the propor- tion of the phases throughout this period. Females contained at least some ripe alveoli nine months of the year, January through September, with ripeness peaking during the summer months of June and July, 52 and 54% respectively, and then declining to very few or no ripe alveoli dur- ing October through December. Females contained alveoli in partially spawned and spent phases throughout the year. There was FEMALE 100 -1 80 60- 40- :[UJ I R ^ JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC RIPE PARTIALLY SPAWNED n SPENT EARLY ACTIVE LATE ACTIVE FIG. 1. The female reproductive cycle of the sunray venus clam. Macrocallista nimbosa during 1974 from St. Joseph Bay, Florida. The length of each shaded area represents the percent frequency of alveoli in each reproductive phase. REPRODUCTIVE CYCLE OF SUNRAY VENUS CLAM little change in the proportion of these two phases during the first seven months of the year. During this time alveoli in partially spawned and spent phases represented 25% of the total alveoli. In August, there was a sharp increase to 54% in the proportion of alveoli in the partially spawned and spent phases. This proportion increased fur- ther in September to 61 % and reached an annual maximum of 82% in October, remaining high at 62% in November. There was a sharp decline in spawning activities to the annual minimum of 18% alveoli in the partially spawned and spent phases by the first week of December. Active phases, which include early active and late active, were present throughout the year. During the first five months of the year alveoli in the active phase represented a high percentage, averaging 58% of the total. During the summer and early fall this proportion was reduced, reaching a yearly minimum in August. The pro- portion of alveoli in active phases increased in the fall, reaching an annual maximum of 81% in early December. Males likewise exhibited a single annual spawn- ing period (Fig. 2). Some males contained ripe alveoli throughout the year, averaging 40% for the first five months, then sharply increasing to the annual maximum of 98% in June. Beginning in July and August the percentage of ripe alveoli steadily declined throughout the fall and reached the annual minimum of 3% in December. There was little change in the proportion of alveoli in the partially spawned phase throughout the first five months of the year. Alveoli in the partially spawned phase were absent during June, but then reappeared and were present throughout the remaining months of the year with alveoli in the partially spawned and spent phases reaching maximum annual values of 68 and 86% during November and December, respectively. Alveoli in the spent phase first appeared in May and June and were present for the remaining months of the year. Except for the month of June, active phases were present throughout the annual cycle. There was little change in the proportion of active phases during the winter and spring months, as well as during September and October. The percentage of alveoli in active phases decreased as fall progress- ed and reached a low in December. This histological study indicates that the sunray venus clam is a fall spawner, with females begin- ning spawning in August and continuing through November, with greatest spawning activity oc- curring during October and November. Males begin spawning activities earlier, starting in July MALE 100 -I r:v 80- i 60- 40- 20- Bl JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC PARTIALLY SPAWNED RIPE YZA SPAWNED I I SPENT FIG. 2. The male reproductive cycle of the sunray venus clam during 1974. EARLY ACTIVE Vi LATE tlU ACTIVE 10 M. L. HAINES 20-, 16 12- 4- feb mar apr may |un |Ul aug sep Oct dec FIG. 3. The seasonal variation in glycogen content (oti a dry weight basis) for the sunray veniis clam. The vertical bars indicate 95% confidence intervals. and continuing through early December, with greatest activity occurring from October to early December. Glycogen Analysis The greatest storage of glycogen in the tissues of the sunray clam occurred during the winter months of January through March (Fig. 3). The annual maximum value of 15.2% occurred in both January and March. A sharp decline in glycogen content during April continued throughout the months of May and June, reaching low values of 3.8 and 5.2% during the summer months of July and August respectively. There was a transient rise in glycogen content during September. Glycogen levels returned in October to a value of 5.1%, reminiscent of July and August. Glycogen TABLE 1. The annual strip spawning potential o/M. nimbosa. The numbers in parentheses indicate the number of trials in which ova development was successful. Month Number of Tripk Ammonium Hydroxide Treatment No Treatment 1 I idls Development To Development To Early Gastrula Straight-hinge Early Gastrula Straight-hinge Or Less Stage Or Less Stage Jan. 2 — — — — Feb. 2 — — — — Mar. 2 + (1) — — — Apr. 2 — — — — May 2 + (1) — — — Jun. 4 + (1) — — — Jul. 4 — — — — Aug. 4 — — — — Sep. 4 + (2) — — — Oct. 4 + (3) + (3) + (3) + (3) Nov. 4 + (4) + (4) + (4) + (4) Dec. 4 + (1) — — — REPRODUCTIVE CYCLE OF SUNRAY VENUS CLAM 11 content rose slightly in November and then sharp- ly in December to 14.4%, a value comparable with the high winter values of January through March, thus demonstrating a definite annual glycogen cycle for M. nimbosa during 1974. The seasonal changes in the glycogen content of M. nimbosa showed a definite cycle related to the gonadal development and spawning activities. During the winter months, January through March, the glycogen values were at the annual high and little change occurred in the proportion of the five gonadal phases. In June, the histologi- cal study revealed a rapid proliferation of gametes and a corresponding low value of glycogen which decreased to an annual minimum in July at at time when spawning began in males. The correlation between low glycogen values and spawning con- tinued throughout November. Glycogen content showed a small rise in September which was reflected in the histological study in that a cor- responding decrease in alveoli in the spawned phases occurred. By December, spawning had ceased and an increase in glycogen approaching the winter values was observed in December. Strip-Spawning Potential The annual strip-spawning potential is represented in Table 1. Development of gametes to and beyond the straight-hinge stage occurred only in those experiments conducted during the last three weeks of October and all of November. During this period normal development occurred in both those egg suspensions treated and not treated with ammonium hydroxide. In strip- spawning experiments performed at other times of the year, eggs showed limited development only in those gamete suspensions receiving ammonium hydroxide treatment. In experiments conducted on March 14, May 3, and June 16, a few eggs developed only as far as the 8-cell stage. In ex- periments conducted September 13, 21, and December 3, several eggs developed to the early gastrula stage. It is likely that the effect of the ammonium hydroxide is to break down the egg's germinal vesicle so that fertilization may occur (Loosanoff and Davis, 1963). The incomplete development of those eggs may indicate that the eggs were not mature even though sperm addition caused initia- tion of cleavage. The results of this study indicate that strip- spawning is successful only during certain times of the year. Comparison of the strip-spawning and the histological study results indicate that strip spawning is not successful throughout the entire period that an animal appears to be in a histologically-ripe or partially-spawned phase, but is confined to a narrower period than one would infer from a histological or glycogen study. The results of the study of strip-spawning poten- tial provides further evidence that M. nimbosa in northwest Florida are fall spawners. ACKNOWLEDGEMENTS This paper is part of a research study for the Ph.D. in Biological Oceanography, Florida State University. I thank the following committee members: Chairman, Dr. R. W. Menzel; Drs. J. A. Calder, P. A. LaRock, M. J. Greenberg, and R. C. Staley for their helpful comments and sugges- tions. The investigator received financial support as a graduate research assistant from NOAA, Of- fice of Sea Grant, Department of Commerce, under Grant #04-3-158-43. The U. S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. I ex- tend my thanks to Drs. R. W. Menzel and R. C. Staley who kindly reviewed the manuscript. LITERATURE CITED Cain, T. D. 1972. The reproductive cycle and lar- val tolerances of Rangia cuneata. Ph. D. Disser- tation, University of Virginia. 121 p. Godcharles, M. F. and W. C. Jaap. 1973. Ex- ploratory clam survey of Florida nearshore and estuarine waters with commercial hydraulic dredging gear. Fla. Dept. Nat. Res. Mar. Res. Lab., Prof. Pap. Ser. No. 21. 77 p. Holland, D. A. and K. K. Chew. 1974. Reproduc- tive cycle of the Manila clam (Venerupis japonica), from Hood Canal, Washington. Proc. Natl. Shellf. Assoc. 64: 53-58. Loosanoff, V. L. 1937. Development of the primary gonad and sexual phases in Venus mercenaria Linnaeus. Biol. Bull. 72: 389-405. Loosanoff, V. L. and H. C. Davis. 1963. Rearing 12 M. L. HAINES of bivalve mollusks. Adv. Mar. Biol. 1: 1-136. Ropes, ]. W. 1963. Reproductive cycle of the surf clam, Spisula solidissima. in offshore New Jersey. Biol. Bull. 135: 349-365. Ropes, J. W. and A. P. Stickney. 1965. Reproduc- tive cycle ofMyfl arenaria in New England. Biol. Bull. 128: 315-327. Westenhouse, R. G. 1968. Developments in the methodology for glycogen determination in oysters. Proc. Natl. Shellf . Assoc. 58: 88-92. Proceedings of the National Shellfisheries Association Volume 66 —1976 GROWTH AND MORTALITY RATES OF HATCHERY SEED CLAMS, MERCENARIA MERCENARIA, IN PROTECTED TRAYS IN WATERS OF SOUTH CAROLINA Peter J. Eldridge,^ Wayne Waltz,^ Robert C. Gracy,' and Hurshell H. Hunt." ABSTRACT Seed hard clams, Mercenaria mercenaria, were planted in trays at densities of 290. 580, and 869/m^ in three widely separated intertidal areas in South Carolina. Survival of clams was similar at each site although clams at Clark Sound experienced a lower survival rate. Growth of clams planted at Clark Sound and Albergottie Creek was significantly higher than those at Bull Bay. Growth occurred throughout the year with the best growth experienced in summer and fall. INTRODUCTION The hard clam, Mercenaria mercenaria (Linne), is present in many estuaries of South Carolina, but has never supported a large commercial fishery t)ecause of poor market conditions and a lack of knowledge concerning the extent of the clam resource. However, the potential for hard clams in South Carolina appears promising and the Divi- sion of Marine Resources of the South Carolina Wildlife and Marine Resources Department has undertaken several activities designed to increase ' Contribution No. 57 from the South Carolina Marine Resources Center. This study was conducted in cooperation with the United States Department of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service, under Public Law 88-309, Project No. 2- 179-D. Reference to firms m this paper does not imply in- dorsement of commercial products by the National Marine Fisheries Service or the State of South Carolina. ' Marine Resources Research Institute, Division of Marine Resources, South Carolina Wildlife and Marine Resources Department. ' Office of Conservation and Management, Division of Marine Resources, South Carolina Wildlife and Marine Resources Department. 4 Department of Biometry Medical University of South Carolina Charleston, South Carolina 29401 dam production and improve clam management practices in South Carolina (Gracy, 1974). This report describes one project on growth and sur- vival of hatchery seed clams held in protected trays in the intertidal zone. Investigators have reported on growth of M. mercenaria in Virginia, North Carolina, Georgia, and Florida (Haven and Andrews, 1957; Loesch and Haven, 1973; Chestnut, Fahy and Porter, 1957; Godwin, 1968; Menzel, 1963; Menzel and Sims, 1964), but no published work exists for South Carolina. Moreover, most experiments cited used clams reared in Milford, Connecticut, and growth patterns observed may not represent those of native clams. The present experiment was conducted (1) to determine relative growth pat- terns and survival rates of a southern stock of M. mercenaria planted in South Carolina; (2) to ex- plore the effect of density upon growth and sur- vival; and (3) to serve as preliminary project for future studies. The value of the last objective quickly became apparent because it proved to be quite difficult to find suitable sites for experiments of this type. This was due to intertidal zones that were quite steep and limited in area, or exposed to strong wave action, currents or both. Also, crabs 13 14 P. J. ELDRIDGE, W. WALTZ, R. C. GRACY, H. H. HUNT proved to be more formidable predators than ex- pected. MATERIALS AND METHODS Hatchery seed obtained j-rom Coastal Zone Resources of North Carolina was chosen because a regular supply existed. Also, clams from North Carolina probably are well adapted to the range of environmental conditions that occur in estuaries of South Carolina. Seed was planted in oyster trays (119 x 58 x 14 cm) to protect it from predation by the blue crab, Callinectes sapidus (Rathbun); the stone crab, Meuippe mercenaria (Say); and other crabs, par- ticularly of the family Xanthidae. The metal framed trays were lined with one- quarter inch (about 6 mm) galvanized hardware cloth, and fiberglass insect screens were placed in the bottom to retain substrate. The frays were then filled with substrate (sand or sand with shell) to a depth of approximately 10 cm. Clams were planted in the substrate. The tray was then covered by a one-quarter inch galvanized hard- ware cloth and wired shut. This arrangement reduced predation by crabs, but was not entirely successful. Clams were placed intertidally in 3 widely separated areas of South Carolina: viz., Clark Sound approximately 10 km south of Charleston (Lat. 32° 43' 00" N Long. 79° 56' 32" W); in Albergottie Creek in Beaufort county about 110 km south of Charleston (Lat. 32° 26 ' 56" N Long. 80° 43 ' 12" W); and in Bull Bay (Lat. 32° 55 ' 48" N Long. 79° 35' 09 " W), which is approximately 30 km north of Charleston (Fig. 1). Clams were planted at 3 densities (2 replicates each): 200 (290/ mO, 400 (580/ m^), and 600 (869 /m^) per tray. Clams that died were not replaced. Initial size of clams was determined by measuring a sam- ple of 400 clams. All clams were counted in each tray every 3 months and a sample of 100 clams from each density was measured to the nearest 0.1 mm to determine total anterior-posterior length. In March, 1974, sixty clams were measured at Bull Bay for each density. After clams were counted and measured, new substrate was added to the trays and the clams were placed in the new substrate. Clams were out of the water approximately 2 or 3 hours in a 12-hour tidal cycle at the Albergottie Creek and Clark Sound sites, whereas, at Bull Bay, clams were out of water about 4 or 5 hours. Clams were planted September 28, 1973; October 25, 1973; and December 20, 1973 at Clark Sound, ALBERGOTTIE CREEK A TLANTIC OCEAN HG. 1. Location of sites where seed clams, Mercenaria mercenaria, were planted in South Carolina. GROWTH AND MORTALITY OF HATCHERY SEED CLAMS 15 TABLE 1. Particle size analysis. Percent weight was calculated from the weighted means of five samples taken from each location (Means weighted by weight of samples). Shell Sand Silt Clay O.063 mm) (>.063mm) (.063-. 004 mm) (<.004mm) Location % weight % weight % weight % weight Clark Sound 2.2 Sb.S 6.4 4.6 Bull Bay 15.4 78.0 4.0 2.7 Albergottie Creek 0.7 75.5 13.9 9.9 TABLE 2. Survival of hard clam seed, Mercenaria mercenaria, planted in protected trays in estuaries of South Caroliiui (Trays of similar density combined). Number Original per number Survivors Survivors Survivors Survivors Survivors Survivors square in trays March June September December March June meter 1973 1974 1974 1974 1974 1975 1975 Bull Bay 290 400 306 277 250 204 201 196 580 800 589 415 389 358 354 347 869 1200 975 863 740 711 697 689 Total 2400 1870 1555 1379 1273 1252 1232 Total survival rate .779 .648 .575 .530 .522 .513 Interval survival rate .779 .832 .887 .923 .984 .984 Clark Souiui 290 400 294 251 233 230 230 224 580 800 445 349 271 265 264 208 869 1200 881 795 676 660 657 650 Total 2400 1620 1395 1180 1155 1149 1082 Total survival rate .675 .581 .492 .481 .479 .451 Interval survival rate .675 .861 .846 .979 .995 .942 Albergottie Creek 290 400 313 202 194 151 145 133 580 800 718 640 598 533 524 492 869 1200 1003 836 809 597 587 570 Total 2400 2034 1678 1601 1281 1256 1195 Total survival rate .848 .699 .667 .534 .523 .498 Interval survival rate .848 .825 .954 .800 .981 .951 Bull Bay, and Albergottie Creek, respectively.' Five sediment samples were taken from each ex- perimental site. Samples were sorted into sand, silt, and clay fractions (Wentworth Scale) by using U.S.A. standard testing sieve No. 230 and the pipette method described by Krumbein and Petti- Clams planted at Bull Bay and Clark Sound were measured initially; 4 and 5 months after planting, respectively; and at 3 month intervals thereafter. John (1938). Shell weight is the weight difference in the sand fraction after all carbonate has been disolved by 4 molar hydochloric acid. Weighted means of shell, sand, silt, and clay fractions were calculated for each site in order to obtain an estimate of the average sediment compostition of substrate used in trays. Statistical analyses were conducted utilizing the Statistical Analysis System (SAS). 16 P. J. ELDRIDGE, W. WALTZ, R. C. GRACY, H. H. HUNT RESULTS AND DISCUSSION Sediment Analysis Results of sediment analysis are presented in Table 1. Results were plotted on the sand-silt-clay triangle diagram proposed by Shepard (1954). Substrates at all sites can be characterized as sand. Bull Bay had the greatest fraction of shell and Albergottie Creek had the largest fraction of silt. Survival of Clams Table 2 gives the number of survivors (trays of similar density combined) for the 3 experimental sites. Survival was highest at Bull Bay which had the greatest fraction of shell in the substrate. Castagna (1970) reported that the use of shell ag- TABLE 3. Results of two-way analysis of variance test on survival rates. Source of Variation d.f. F value Probability >F Transformed Means Location 2 3.21 0.0488 Bull Bay Density 2 2.56 0.0871 Albergottie Creek Block 5 11.26 0.0001 Clark Sound Error 44 0.669 0.634 0.564 TABLE 4. Growth of hard clam seed, Mercenaria mercenaria, planted in protected trays in estuaries of South Carolina by location and density. Mean size Mean size Mean size Mean size Mean size Mean size Number in mm in mm in mm in mm in mm in mm Per Square Original March June September December March June meter Mean size 1974 1974 1974 1974 1975 1975 Bull Bay 290 12.30 13.91 16.45 19.56 23.44 24.56 30.38 580 12.30 13.78 15.73 19.62 22.44 24.77 29.90 869 12.30 14.77 16.05 18.53 21.30 24.80 28.33 Weighted mean size 12.30 14.08 16.04 19.02 21.96 24.75 29.10 Absolute increase in mm 1.78 1.96 2.98 2.94 2.79 4.35 Interval percent increase 14.47 13.92 18.58 15.46 12.70 17.58 Total percent increase 14.47 30.41 54.63 78.54 101.22 136.59 Clark Sound 290 13.60 17.29 24.78 33.51 38.47 41.81 46.34 580 13.60 17.35 23.71 33.96 38.47 43.37 45.43 869 13.60 18.39 23.44 33.37 37.85 39.49 44.05 Weighted mean size 13.60 17.90 23.75 33.53 38.12 40.91 44.79 Absolute increase in mm 4.30 5.85 9.78 4.59 2.79 3.88 Interval percent increase 31.62 32.68 41.18 13.69 7.32 9.48 Total percent increase 31.62 74.63 146.54 180.29 200.81 229.34 Albergottie Creek 290 12.40 16.07 24.65 31.96 35.50 41.36 44.17 580 12.40 16.17 25.59 33.31 36.77 41.66 44.25 869 12.40 15.78 24.98 33.16 36.29 40.84 43.82 Weighted mean size 12.40 15.96 25.17 33.07 36.40 41.24 44.04 Absolute increase in mm 3.56 9.21 7.90 3.33 4.84 2.80 Interval percent increase 28.71 57.71 31.39 10.07 13.30 6.79 Total percent increase 28.71 28.71 102.98 166.69 193.55 232.58 255.16 GROWTH AND MORTALITY OF HATCHERY SEED CLAMS 17 gregates reduced predation on seed hard clams. As expected, survival of clams increased with increas- ed size. A two-way Analysis of Variance (ANOVA) was conducted to determine differences in survival rates. Survival rates for each time period (Block) were calculated for each location (Treatment A) and density (Treatment B). The variance was nor- malized by an arcsin transformation (Table 3). Differences in survival rates among blocks were highly significant (P<0.01), differences among locations were significant at the 5% level (P = 0.048), and there were no significant differences between planting densities. Duncan's multiple range test was employed on the transformed data to determine differences in survival among locations. The only significant dif- ference was that between Bull Bay and Clark Sound. An examination of the Clark Sound data reveal- ed that one tray had a damaged cover in September, 1974. It is hypothesized that high mor- tality produced by crabs entering the exposed tray at this time caused the lower survival rate observ- ed at Clark Sound. In essence, predation by crabs appeared to cause most of the mortality observed and the authors do not believe that the crab preda- tion rate varied significantly among locations. Survival of clams in this experiment was less than that reported by others (Haven and An- drews, 1957; Chestnut, 1952; Menzel and Sims, 1964; Carriker, 1956; Gustafson, 1955). This dif- ference appeared to be due (1) to the smaller size of clams utilized and (2) to failure of the trays to fully protect clams from predation by crabs. Predation by crabs was indicated by presence of shell fragments within trays, particularly during the earlier part of the experiment. Table 2 shows that survival of clams at all sites was relatively high after June, 1974. The weighted average size of clams at that time varied between 16.0 mm and 25.2 mm (Table 4). Survival between December, 1974, and March, 1975, was the highest observed in the experiment. This may have been due to the reduced level of ac- tivity of crabs during the cooler months. Growth of Clams Mean growth rates of clams at different den- sities at the same site did not appear to vary ap- preciably (Fig. 2). However, clams planted in Qark Sound and Albergottie Creek grew nearly twice as rapidly as clams at Bull Bay. The follow- ing procedures were employed to determine if significant differences existed. Since a visual ex- amination of Figure 2 indicated that growth in shell length had been linear from March, 1974, to June, 1975, linear regressions (shell length vs. time) for each tray were computed to determine slopes (growth rate). The slope for the growth rate for each tray was found to be significantly dif- 50 40 30 20- 10 0 50 40 30 20 10 0 50 40 30 20 10 0 50 40 30 20 10 0 -T 1 1 1 r- T— I 1 r- ■ Clork Sound 3 Bull Boy K Aibergottia Creek 290/m2 —\ 1 r- S 0 D Mar June Sept Dec Mor 1973 1974 1975 HG. 2. Growth of seed hard clams, Mercenaria mercenaria, planted in protected trays in three locations in South Carolina by planting density. ferent from zero and the average correlation coef- ficient (R^) for all trays was 0.77. Average slopes are given in Table 5. A two-way analysis of variance on the slopes for each tray was con- ducted to determine if differences in growth ex- isted. The results are given in Table 5. A difference in growth clearly existed among locations, but not among densities or the density-location (D x L) in- teraction. As suggested in Figure 2 Clams at Bull Bay grew significantly less than those at either 18 P. ]. ELDRIDGE, W. WALTZ, R. C. GRACY, H. H. HUNT Qark Sound or Albergottie Creek. Although growth did not vary significantly among densities, the average rate of growth for each tray did show a downward trend with increased density. The average monthly growth rate for Bull Bay, Clark Sound and Albergottie Creek was 0.8 mm, 1.5 mm, and 1.8 mm, respectively. The most obvious difference in sites was the position of trays in the intertidal zone. Clams at Bull Bay may have grown slower because they were exposed longer than at other locations. Added exposure meant that clams had less time to feed and were exposed to more extreme changes in temperatures than clams at other sites. Linear increments in growth appeared to be in- versely proportional to initial length as reported by Belding (1912) and Pratt and Campbell (1956). The highest growth rate for any interval oc- curred between June and September, 1974, at Bull I3ay and Clark Sound and from March to June, 1974, at Albergottie Creek (Table 4). The lowest growth rate for any interval occurred between December, 1974, and March, 1975, at Bull Bay and Clark Sound and between March and June, 1975, at Albergottie Creek. Clams at Clark Sound and Albergottie Creek grew rapidly between planting in the fall of 1973 and September, 1974, and attained average sizes of 33.5 mm and 33.1 mm, respectively. Linear growth between September, 1974, and June, 1975, was reduced at these sites. Conversely, clams at Bull Bay, which had an average size of 19.0 mm in September, continued to experience about the same rate of growth. The variation in growth be- tween sites suggests that the initial size of clams has an important effect upon linear growth. This effect may mask a seasonal effect, particularly when a southern area experiences relatively mild winters as did South Carolina in the past two years. An examination of daily water temperatures collected at the Marine Resources Center located near the mouth of Charleston Harbor revealed that water temperature never dropped below IOC during the past two winters. The mildness of the past two winters together with the size of clams at planting appears to explain why clams grew throughout the year. This experiment also suggested that clam growth should not be compared from area to area unless they are of equivalent size or size range (Loesch and Haven, 1973), planted at the same time of year, and adapted to local water temperatures. For instance, Menzel (1963, 1964) TABLE 5. Regression coefficients of hard clam, Mercenaria mercenaria, seed, planted in protected trays in estuaries of South Carolina by location and density. Location Clark Sound Bull Bay Albergottie Creek Average Slope Density in clams 290/m^ 580/m^ 5.66 6.28 5.49 4.91 3.49 3.80 3.11 3.02 5.26 4.84 5.66 5.32 4.78 4.70 869/m' 5.59 4.10 2.71 3.06 4.86 5.16 4.25 Results of Analysis of Variance on Regression Coefficients of Trays Source of Variation d.f Sum of Squares Mean Square Total 17 21.12 Density 2 Location 2 D X L 4 Error 9 Average Slope 5.34 3.20 5.18 0.98 0.49 1.63 7.10 8.55 28.50** 0.30 0.07 0.23 2.74 0.30 GROWTH AND MORTALITY OF HATCHERY SEED CLAMS 19 reported that seed hard clams Mercenaria mercenaria, experienced their best growth in spring and fall with little growth observed in the summer months. In that series of experiments dams were reared at Milford, Connecticut, and then planted in Alligator Harbor, Florida. Because Connecticut clams are adapted to colder temperatures, it is not surprising to witness their slower growth during summer in Florida. In con- trast, clams reared in North Carolina and planted in South Carolina grew rapidly during the sum- mer, with clams in Clark Sound and Bull Bay ex- periencing their greatest incremental growth rate between June and September, 1974. Growth occurred throughout the year, but was somewhat reduced during the colder months. Similar results were reported for clams reared in North Carolina and planted in Georgia (Godwin, 1968). 60 % 50 40 ^ SO = 20 u •> 2 10 K O S 0 - il il i Effect of Density Upon Growth Densities at the end of the experiment due to natural mortality were generally less than or equal to 325/m^ However, two trays had clam densities of approximately 650/m^ Density did not affect the growth rate in this experiment. Similar results were reported by Godwin (1968) and Gustafson (1955). Although growth of clams less than 45 mm in size was not affected by densities as great as 650/m^ one can not assume that this is true for larger clams. ACKNOWLEDGMENTS The authors thank Messrs. Holland Mills, Michael Bailey, and Willis J. Keith for assistance in the field, Mrs. Evelyn Myatt and Mr. Peter Laurie for preparation of figures, Messrs. Frank Stapor and Roy Crosby for assistance with sedi- ment analysis. Miss Nickie Jenkins for assistance 1^ BULL BAY I CLARK SOUND [[] ALBERQOTTIE CREEK il ^ L E E X I- » o a: e < Ui K U < kJ MAR. AM JUNE J A SEPT. 0 N DEC. J F MAR. A M JUNE l»74 l»78 HG. 3. Mean absolute increase in mm and percent increase in linear growth by three month periods for seed c/ams, Mercenaria mercenaria, planted intertidally in protected trays at three locations in South Carolina, all trays combined. 20 P. I. ELDRIDGE, W. WALTZ, R. C. GRACY, H. H. HUNT in computer analysis of data, and Mrs. Lourene Rigsbee for typing the manuscript. Thanks are also due to Mssrs. Victor Burrell, Paul Sandifer, and Raymond Rhodes, who reviewed the manuscript and made helpful suggestions. LITERATURE CITED Belding, D. L. 1912. A report upon the quahog and oyster fisheries of Massachusetts, Boston. Wright and Potter Printing Company. 134 pp. Carriker, M. R. 1956. Biology and propagation of young hard clams, Mercenaria rnercenaria. ]. Elisha Mitchell Sci. Soc. May: 57-60. Castagna, M. A. 1970. Hard clam culture method developed at VIMS. VIMS Marine Resources Advisory Series 4: 3pp. Chestnut, A. F. 1952. Growth rates and movements of hard clams, Venus mercenaria. Proc. Gulf and Carib. Fish. Inst. 4th Ann. Ses- sion: 49-59. Chestnut, A. F., W. E. Fahy and H. J. Porter. 1957. Growth of young Venus mercenaria, Venus campechiensis, and their hybrids. Proc. Natl. Shellfish Assoc. 47: 50-56. Godwin, W. F. 1968. The growth and survival of planted clams, Mercenaria mercenaria, on the Georgia coast. Georgia Game and Fish. Comm. Contr. Series 9: 16 pp. Gustafson, Alton H. 1955. Growth studies in the quahog Venus mercenaria. Proc Natl. Shellfish. Assoc. 45: 140-150. Haven, D. and J. D. Andrews. 1957. Survival and growth of Venus mercenaria, Venus campechiensis, and their hybrids in suspended trays and on natural bottoms. Proc. Natl. Shellfish. Assoc. 47: 43-49. Krumbein, W. C. and F. J. Pettijohn. 1938. Manual of Sedimentary Petrography. Appleton-Century-Crafts, N. Y. 549 pp. Loesch, ]. G. and D. S. Haven. 1973. Estimated growth functions and size-age relationships of the hard clam, Mercenaria mercenaria, in the York River, Virginia. Veliger 16 (1): 76-81. Menzel, R. W. 1963. Seasonal growth of the northern quahog, Mercenaria mercenaria and the southern quahog, M. campechiensis, in Alligator Harbor, Florida. Proc. Natl. Shellfish. Assoc. 52: 37-46. Menzel, R. W. 1964. Seasonal growth of northern and southern quahogs, Mercenaria mercenaria and M. campechiensis, and their hybrids in Florida. Proc. Natl. Shellfish. Assoc. 53:, 111-119. Menzel, R. W. and H. W. Sims. 1964. Experimen- tal farming of hard clams, Mercenaria mercenaria, in Florida. Proc. Natl. Shellfish. Assoc. 53: 103-109. Pratt, D. M. 1953. Abundance and growth of Venus mercenaria and Callocardia morrhuana in relation to the character of bottom sediments. J. Mar. Res. 12: 60-74. Pratt, D. M. and D. A. Campbell. 1956. En- vironmental factors affecting growth in Venus mercenaria. Limnol. Oceanogr. 1: 2-17. Shepard, F. P. 1954. Nomenclature based on sand- silt-clay ratios. J. Sed. Pet. 24: 151-158. Proceedings of the National Shellfisheries Association Volume 66 - 1976 GROWTH AND SIZE-WEIGHT RELATIONSHIPS OF THE PINKNECK CLAM 5PISULA POLYNYMA, IN HARTNEY BAY, PRINCE WILLIAM SOUND, ALASKA',^ Howard M. Feder, A. J. Paul and J. Paul INSTITUTE OF MARINE SCIENCE UNIVERSITY OF ALASKA FAIRBANKS, ALASKA ABSTRACT Pinkneck clams, Spisula polynyma, from Hartney Bay, Prince William Sound, Alaska, were examined. Three samples, a total of 298 specimens, were used to deter- mine the growth history of 16 year classes by the annular method. Length-weight rela- tionships are considered. Dry meat weight (solids) averaged 20.2%. INTRODUCTION The pinkneck or redneck clam, Spisula polynyma,' is a large bivalve found in intertidal and subtidal Alaskan waters (Chamberlin and Stearns, 1963). It has been reported from Point Barrow to the Strait of Juan de Fuca, and generally occurs in medium grade sediments (Chamberlin and Stearns, 1963). Intertidally it is often found in association with razor {Siliqua patula) and butter (Saxidomus gigantea) clams (Feder and Paul, un- published). The extent of this resource in Alaska is unknown; however, the authors have observed populations with commercial potential in the Cor- dova region of Prince William Sound, Alaska. Kessler and Hitz (1970) reported good subtidal catches in Icy Straits, Southeastern Alaska. On the Atlantic coast of the United States the closely related surf clam, Spisula solidissima, is harvested subtidally. The meats are canned and made into chowder or specialty products (Yancey and 1 Contribution No. 263, Institute of Marine Science, University of Alaska. 2 Tfiis project was conducted witfi funds provided by the University of Alaska's Sea Grant program (Grant No. 04-3-158-41), NOAA Office of Sea Grant, Department of Commerce. 3 Spisu/a a/osfcana is a synonomy. (Abbott, 1974) Welch, 1968). There are a number of papers deal- ing with the basic biology and fishery potential of surf clams along the Atlantic coast of the United States (see Yancy and Welch, 1968 for biblio- graphy), but with the exception of a geographic study of S. polynyma that contains Alaskan distributional information (Chamberlin and Stearns, 1963) no published work on the pinkneck dam from the Pacific coast of North America is available. The purpose of this investigation was to examine growth, size-weight relationships and commercial potential of S. polynyma from Prince William Sound, Alaska. METHODS Specimens of Spisula polynyma were collected intertidally at low tide by digging on sandflats in Hartney Bay, Prince William Sound (Fig. 1). Col- lections were made February 17, 1973, May 19, 1973, and July 21, 1974. Age was determined for the clams by counting annuli, a series of closely- spaced concentric growth lines which are the result of slow winter shell growth, (Paul and Feder, 1973; Weymouth et al, 1931). The growth history of the specimens was determined by measuring the shell length at each annulus after removing the periostracum with a 10% acid solu- 21 22 H. M. FEDER, A. J. PAUL, J. PAUL tion. Shells with badly abraded surfaces were discarded (2% of the 305 clams collected). The size-weight relationships were examined. The adductor muscles of all specimens collected were severed and the free water in the mantle cavi- ty allowed to drain. The clams were then shucked, the shells weighed and the differences between the whole-live-weight (drained) and the shell-weight FIG. 1. Map of Prince William Sound, Alaska; location of the Hartney Bay sandflat sampled for Spisula polynyma. recorded as wet-meat-weight. Individual meats were dried to a constant weight at 80°C for dry- weight determinations. Weights were obtained with a Mettler balance Type P 120. Plots, regression lines, and regression equations were determined and plotted by an IBM 360 Computer. The Gauss-Jordan method was us- ed in the solution of all normal equations (Cooley and Lohnes, 1962; Ostle, 1954). RESULTS Growth A growth curve for Spisula polynyma from Hartney Bay is presented in Figure 2 (also see Table 1). The oldest and largest individual en- countered was 16 years old and had a shell length of 151 mm. Size-Weight Relationships The equations describing the relationship be- tween length and total weight (drained), length and wet-meat and dry-meat-weight are presented in Table 2. The former two relationships are plot- ted in Figure 3. Dry-meat weight (solids) was found to average 18,4, 21.9 and 20.3% for the February, May and July collections respectively (Table 2). Figure 4 shows the relationship between dry- weight and shell-length. The average shell r a 9 /o A GE (year class ) II 12 13 14 15 16 17 FIG. l.The relationship between shell length (mm) and age of Spisula polynyma from a sandflat in Hart- ney Bay, Prince William Sound, Alaska. PINKNECK CLAM IN HARTNEY BAY 23 TABLE 1. Mean shell length (L = mm) at each annulus for Spisula polynyma from Hartney Bay, Prince William Sound, Alaska. N = the number of anniili measured; S.D. =standard deviation. Annulus 17 February 1973 19 May 1973 21 July 1974 Number L N S.D. L N S.D. L N S.D. 1 7 40 1.0 8 101 1.6 8 157 1.3 2 13 40 2.2 13 101 2.0 14 148 2.6 3 21 40 2.0 20 101 2.5 24 148 5.3 4 31 40 3.1 29 101 3.6 36 144 5.3 5 41 40 3.6 39 101 4.1 49 142 6.5 6 52 40 5.1 49 101 4.3 69 142 7.7 7 63 40 6.0 60 101 4.6 74 142 8.2 8 74 40 6.4 72 99 4.7 85 140 8.5 9 85 40 6.8 83 97 5.1 96 131 8.2 10 94 39 6.8 94 93 4.8 107 119 8.2 11 103 38 7.1 104 87 5.0 116 100 7.8 12 111 34 6.1 112 83 5.0 123 69 7.8 13 118 28 5.6 119 74 4.2 128 38 8.4 14 123 21 5.4 124 55 4.1 133 20 9.1 15 127 8 6.1 131 23 4.6 140 3 9.2 16 136 2 — 142 7 6.1 — 0 — TABLE 2. Equatiotis for size-weight relationships of Spisula polynyma from Hartney Bay, Prince William Sound. Equations derived from curves in Figures 2, 3 and 4. SD = standard deviation: N = number of in- dividuals. Size-weight Relationship February 17/73 (n=40) May 19/73 (n = 101) T July 21/74 (n = 142) All 3 Collections (n=283) Total weight Wet-meat weight Dry-meat weight Percent solids* S.D. Meat wet- meat weight Mean shell length / Length \ 3.6 \ 26.9 / 5.4 / Length \ \ 50.4 / / Length \ 4.2 V 59.0 / 18.4% 2.1% 103. 4gr 118mm / Length \ 3. 5 \ 25.9 / /Length\3.3 \ 28.4 / / Length \ 3-4 \ 44.7 / 21.9% 1.5% 116. 9gr 116mm / Length \ 3.7 \ 27.9 / / Length \ 3.5 \ 31.3 / /'Length\3.4 \ 44.7 / 20.3% 2.8% 125. Igr 137mm /Length\3.6tt \ 27.0 ; /Length\3.7|| \ 33.6 / /Length\3.7ttt \ 50.5 / 20.2% *Dry-Meat Weight Wet-Meat Weight ^ ^^O t 15 one-year-old clams were not included in the size-weight relationships tabulated for this month tt See Figure 3 tlT See Figure 4 24 H. M. FEDER, A. ]. PAUL, J. PAUL length and mean wet-meat weights for each collec- tion was 118 mm, 103.4 gm; 116 mm, 116.9 gm; ; and 137 mm, 125.1 gm respectively (Table 2). .DISCUSSION The annular method of aging is reliable for most Prince William Sound clams because of a strong seasonality of growth (R. Baxter in Haven, 1971). Intertidal beaches in Prince William Sound are subject to freezing during low tides in January and February, and under such conditions Spisula polynyrna forms a distinct winter annulus (also see Paul and Feder, 1973; Feder and Paul, 1974a; Weymouth et al, 1931 for discussions on annulus formation in the clams Protothaca staminea, Mya arenaria and Siliqua patula in Prince William Sound). Chamberlin and Stearns (1963) report S. polynyrna to be a long-lived slow-growing clam. They provide two approximations of size and age: 50 mm at 6 years of age; 100 mm at 14 or more years of age. However, they do not indicate where these specimens were collected or how many in- dividuals were examined. Their first value is similar to that found in our study for 6-year old dams; Hartney Bay clams average approximately 120 mm at 14 years of age. Spisula solidissima reaches 100 mm in 5 years off central New Jersey and in 8 years off Massachusetts (Yancey and Welch, 1968). Spisula polynyrna is a large clam with in- dividual meats weighing up to 250 grams (0.6 pounds). The 18.4, 21.9 and 20.3% (mean = 20.2%) solids determined in the three col- lections are close to the 21.4% reported for S. solidissima by Ropes (1970). The commercial demand for hard-shell clams along the Pacific coast of the United States is ex- cellent, and production does not meet demand (Glude, 1974). Significant quantities of hard-shell clams are imported from British Columbia, Canada (Glude, 1974). Currently there is little commercial harvesting of clams in Alaska; however, the state has a potential multimillion dollar clam industry based primarily on razor clams {Siliqua patula) (Feder and Paul, 1974b; Orth et al: in press, Rearden, 1974). No abun- dance estimations are available for Alaskan pinkneck clams; therefore, it is not possible to estimate the value of this resource. However, Spisula polynyrna is found in association with razor and butter clams and could be simultaneous- ly harvested along with them, thereby further in- creasing the potential clam harvest in Alaska. Pro- per management would be required for the pinkneck because of slow growth. ACKNOWLEDGEMENTS We thank Merle Hanson for locating the pinkneck clam beds in Hartney Bay and for his 100 LENGTH imml HG. 'h.The relationship of clam length to total and wet-meat-weight for Spisula polynyrna collected from a sandflat in Hartney Bay, Prince William Sound, Alaska. 80 100 LENGTH (mm) HG. Mhe relationship of clam length to dry weight for specimens of Spisula polynyma col- lected on a sandflat in Hartney Bay, Prince William Sound, Alaska. PINKNECK CLAM IN HARTNEY BAY 25 general assistance in collection activities. We also asknowledge Tim and Susan Feder for field assistance; George J. Mueller for taxonomic assistance; Rosemary Hobson for computer pro- gramming aid and Helen Stockholm for editing. UTERATURE CITED Abbott, R. T. 1974. American Seashells. Van Nostrand Reinhold Co., New York. 663 p. Qiamberlin L. J. and F. Stearns. 1963. A geographic study of the clam Spisula po}\/n\/ma (Stimpson). Serial Atlas of the Marine Environ- ment, Folio 3. American Geographical Society, 12 p. Cooley, W. W. and P. R. Lohnes. 1962. Multivariate Procedures for the Behavioral Sciences. John Wiley and Sons, New York, 211 P- Feder, H. M. and A. ]. Paul. 1974a. Age, growth and size-weight relationships of the soft-shell clam, Mya arenaria, in Prince William Sound, Alaska. Proc. Natl. Shellfish Assoc. 64: 45-52. Feder, H. M. and A. ]. Paul. 1974b. Alaska clams; a resource for the future. Alaska Seas and Coasts (Sea Grant Newsletter) 2 (1);1, 6-7. Glude, J. B. 1974. Recent developments in shellfish culture on the U. S. Pacific Coast. In: Proc. of the First U. S. - Japan Meeting on Aquaculture at Tokyo, Japan. NOAA Tech. Rep., NMFS Circ. 388, 133 p. Haven, S.B. 1971. Effects of land-level changes on intertidal invertebrates with discussion of post- earthquake ecological succession. In The Great Alaska Earthquake of 1964. Biology. Natl. Acad. Sci., Washington, D. C. 287 p. Kessler, D. W. and C. R. Hitz. 1970. Subtidal clam explorations in southeastern Alaska. Proc. Natl. Shellfish Assoc. 61 ;8. Orth, F., C. Smelcer, H. Feder and J. Williams. In press. The Alaska Clam Fishery; A Survey and Analysis of the Economic Potential. Instit. Mar. Sci. Tech. Rep. Ostle, R. 1954. Statistics in Research. The Iowa State Univ. Press, Ames, Iowa, 487 p. Paul A. J. and H. M. Feder. 1973. Growth, recruitment, and distribution, of the littleneck clam, Protothaca staminea, in Galena Bay, Prince William Sound, Alaska. U. S. Dep. Commer., Natl. Mar. Fish. Serv., Fish Bull. 71;665-677. Rearden, J. 1974. Alaska's razor clams— an unex- ploited fishery. Alaska Magazine, Sept., 24-25. Ropes, J. W. 1970. Percentage of solids and length-weight relationship of the ocean quahog. Proc. Natl. Shellfish. Assoc.61; 88-90. Yancey, R. T. and W. R. Welch. 1968. The Atlan- tic coast surf clam - with a partial bibliography. Bur. Comm. Fish. Circ. 288, 13 p. Weymouth, F. W., H. C. McMillin and W. H. Rich. 1931. Latitude and relative growth in the razor clam, Siliqua patula. J. Exp. Biol. 8: 228-249. Proceedings of the National Shellfisheries Association Volume 66 — 1976 AGE, GROWTH, AND RECRUITMENT OF THE BUTTER CLAM, SAXIDOMUS GIGANTEA, ON PORPOISE ISLAND, SOUTHEAST ALASKA' A. J. Paul, J. M. Paul and H. M. Feder INSTITUTE OF MARINE SCIENCE SEWARD MARINE STATION SEWARD, ALASKA ABSTRACT Butter clams, Saxidomus gigantea, from Porpoise Island, southeast Alaska, were ex- amined. Growth was determined for 1,069 specimens by the annular method. On Por- poise Island, butter clams reach a harvestable size of 65 mm in eight to nine years. Recruitment in nine 1 m' sample areas was examined. The number of individuals an- nually recruited in the population was variable. INTRODUCTION Saxidomus gigantea (Deshayes, 1839), the but- ter clam, is one of the most common clams in Alaska (Abbott, 1974), and formerly supported an important industry in southeast Alaska. This in- dustry began in 1930 with an initial catch of 25,000 pounds and continued until 1942 with no appreciable expansion. Wartime demand gave the industry impetus to increase production and by 1946 five southeastern Alaskan canneries were producing a pack valued at $170,000 (Orth et al, in press). The clam fishery was of special im- portance to resident Alaskans because it was a winter operation offering employment and income during an otherwise slack season (Orth et al., in press). However, the presence of a toxin (Paralytic Shellfish Poison) in the canned product led to the decline and ultimate collapse of the butter clam in- dustry in southeast Alaska. Currently there are many beaches in southeastern Alaska where clams are relatively free of Paralytic Shellfish Poison. The develop- ment of a rapid chemical assay for detecting the Contribution No. 266, Institute of Marine Science, University of Alaska. toxin should aid in the identification of these areas, and enhance the probability for the suc- cessful development of a new clam industry in Alaska (R. Neve, Institute of Marine Science, Univ. of Alaska, Pers. Comm.) Information on growth of butter clams exists for British Columbia, Canada (Eraser and Smith, 1928; Quayle and Bourne, 1972), but no growth data is available from Alaska waters. The purpose of this investigation was to compliment the ex- isting data base by examining age and growth rela- tions of the butter clam in the Juneau region of southeastern Alaska. The material collected also provided information on recruitment. METHODS Collections of Saxidomus gigantea were made on 23 and 24 May 1975, on Porpoise Island, a small island in the northern portion of southeastern Alaska (Latitude 58°19. '7, Longitude 135 °27. '3) about 40 air miles from the city of Juneau. Nine sample areas, each 1 m^ were established on the beach between the tidal heights of —0.3 m and — 1.0 m, since intertidal butter clams are generally encountered between these tidal heights. 26 BUTTER CLAM ON PORPOISE ISLAND 27 Within each sample area, the sediment was removed to a depth of 5 cm. This sediment was washed through a series of screens, the smallest mesh being 1.5 x 1.5 mm, and examined for young butter clams. Larger clams were collected by con- tinued digging to a depth of 30 cm within each sample area. Few small individuals were en- countered in the quantitative plots, so additional specimens were collected by random digging. All shells were examined under a 2x lens and shells with badly abraded surfaces were discarded (11% of the clams randomly collected). Age was determined for the 1069 remaining clams by coun- ting annuli, a series of closely spaced concentric growth lines which are the result of slow winter shell growth. The measurements taken on all clams was the greatest length at the last annulus. within each age class as a basis for comparison (Snedecor, 1956). The calculated F ratio indicated that age classes, as defined by shell lengths, are statistically distinguishable (P = 0.01). However, aging individuals older than 11 or 12 years becomes progressively more difficult. The number of S. gigantea from each year class was found to be variable in the quantitative plots (Fig. 2). DISCUSSION The time needed for Saxidomus gigantea to grow to a harvestable size on Porpoise Island is slightly longer than that reported for British Col- FIG. l.The relationship between shell length (mm) and age of Saxidomus gigantea, on Porpoise Island, Southeast Alaska. Mean length is denoted by the horizontal line, standard deviation by the box and range by the vertical line. RESULTS Butter clams reach a size of 64 mm in eight to nine years on Porpoise Island (Table 1; Fig. 1). The oldest clam examined was 15 years old and 100 mm long (Table 1). The mean shell lengths for the various age classes are included in Table 1. A growth curve for Saxidomus gigantea from Por- poise Island is presented in Figure 1. The validity of the annular aging method was examined with a standard one-way analysis of variance, utilizing the individual shell lengths FIG 2. The total number of Saxidomus gigantea by year of recruitment from nine l.Om^ plots on Porpoise Island, Southeast Alaska. umbia. In the latter area, butter clams reach a length of 65 mm in five to six years, and are harvested at this size (Quayle and Bourne, 1972). Quayle and Bourne (1972) report that popula- tions of butter clams in British Columbia fail to spawn in some years with the resultant irregular seedings responsible for fluctuations in adult populations. This also appears to be true for Alaska (Feder and Paul, unpublished). Inhibited spawning is probably related to low water temperature (Amos, 1966). Currently, British Columbia is the largest source of butter clams and much of this produc- tion is sold in the United States (Glude, 1974). The 28 A. J. PAUL, J. M. PAUL, H. M. FEDER primary reason for the importation of Canadian butter clam (Glude, 1974) is that current clam pro- duction in the United States Pacific Northwest does not meet the demand (Glude, 1974). In the future, Alaska butter clams could become an im- portant additional source of supply to meet grow- ing demands. TABLE 1. Average size and age of 1069 Sax- idomus gigantea collected on Porpoise Island, Southeast Alaska. N = number of clams: ML = mean shell length; SD = standard deviation; R = range. Year Class N ML SD R I Age of Clams) (mm) (mm) (mm) 0 79 5.1 1.4 3.0- - 8.5 1 134 11.2 2.5 6.0- -16.4 2 109 15.4 2.5 9.0- -22.5 3 104 22.5 4.1 16.7- -30.5 4 88 30.3 5.1 24.2- -38.0 5 98 38.2 3.9 27.0- -47.0 6 82 45.9 4.5 37.0- •55.0 7 85 54.1 4.9 42.0- -64.0 8 72 62.6 5.1 47.0- •74.0 9 75 71.3 4.6 58.0- •81.5 10 68 77.4 4.0 68.7- -87.0 11 40 82.5 3.6 76.0- -88.2 12 22 86.9 2.5 82.0- -91.0 13 8 92.8 3.5 88.5- -98.0 14 4 96.0 2.5 93.6- -99.5 15 1 100.0 — - ACKNOWLEDGEMENTS This work is a result of research sponsored (in part) by the Alaska Sea Grant Program, sup- ported by NOAA Office of Sea Grant, Depart- ment of Commerce, under Grant #04-6-158-44039. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon. We wish to thank the following staff members of the University of Alaska: E. R. Dieter, R. A. Neve, R. Gershey, and P. Reichardt for aid in col- lection of material; Captain K. Turner and the crew of the R/V Acona for support and general assistance, and Helen Stockholm for editing. LITERATURE CITED Abbott, R. T. 1974. American Seashells. Van Nostrand, Princeton N.J. p. 1-663. Amos, M. H. 1966. Commercial Clams of The North American Pacific Coast. U. S. Fish and Wildlife Service, Circ. 237, 18 p. Eraser, C. M and G. M. Smith. 1928. Notes on the ecology of the butter clam, Saxidomus giganteus DeShayes. Trans. R. Soc. Can. Ser 3, 22, Sect. V: p. 271-284. Glude, ]. B. 1974. Recent Developments in Shellfish Culture on the U.S. Pacific Coast. In William N. Shaw ed.. Proceedings of the First U.S. -Japan Meeting on Aquaculture at Tokyo, Japan. October 18-19, 1971. NOAA Technical Report NMFS Circ. 388. p. 39-95. Orth, F., C. Smelcer, H. Feder and J. Williams. The Alaska Clam Fishery: A Survey and Analysis of the Economic Potential. Instit. Mar. Sci. Tech. Rep. R75-3 (in press). Quayle, D. B. and N. Bourne. 1972. The clam fisheries of British Columbia. Bull. Fish. Res. Bd. Canada. 179, 70 p. Snedecor, G. W. 1956. Statistical methods applied to experiments in agriculture and biology. 5th ed. Iowa State. Coll. Press, Ames. 534 p. Proceedings of the National Shellfisheries Association Volume 66 — 1976 ^'^-Ti EFFECTS OF DIET AND TEMPERATURE ON GROWTH AND MORTALITY OF THE BLUE CRAB, CALLINECTES SAPIDUS, MAINTAINED IN A RECIRCULATING CULTURE SYSTEM. RodnerR. Winget^, Charles E. Epifanio, Tom Runnels, and Paul Austin COLLEGE OF MARINE STUDIES UNIVERSITY OF DELAWARE ABSTRACT Blue crab growth parameters were measured over a sixty-day period in a recir- culating culture system, with each crab in physical isolation. Dependent variables were molt interval, increase in carapace width per molt, percent molt and mortality. No consistant growth differences were detected in animals fed diets ranging from 26 to 75% protein content. A temperature of 30° C generally increased molt frequency and percent of animals molting compared to a temperature of 20°C. Increased temperature appears to depress cuticle expansion and to decrease mortality. INTRODUCTION Recirculating maricultural systems have re- ceived increased attention in recent years (Epi- fanio et al., 1973; Chanley and Terry, 1974). Winget et al. (1973) repolted the construction of such a system using the blue crab, Callinectes sapidus (Rathbun) as a test species. This ex- perimental system was designed to allow simultaneous control of several environmental variables, and the present paper presents data con- cerning the effect of temperature and diet on growth and mortality of blue crabs held in the system. MATERIALS AND METHODS Each culture system consisted of a biological filter made of crushed oyster shell; a fiberglass- coated, plywood reservoir tank; fiberglass-coated plywood growing tanks; temperature-control ap- paratus; and automated flourescent lighting. The 1 Current address: Department of Zoology, University of Minnesota Minneapolis, Minnesota, 55455 growth tanks were partitioned so that each crab was separated from its neighbors and provided with flowing water. Water used to fill the system was pumped from Delaware Bay, filtered to remove particles larger than 5pim and adjusted to 20%oS by addition of fresh water. Water quality was not monitored in the system, but the water was completely replaced every two weeks. Photo- period was maintained at 16 hours light and 8 hours dark. The experiment was of a 2 x 4 factorial design and initiated with 35 animals in each cell. Temperature was either 20 °C or 30 °C and diet either control or one of three formulated prepara- tions. The control diet consisted of 60.5% Menidia menidia (which was 80% water), 1.5% each of ca- sein, starch and agar, and 35% tap water. The dry weight, protein concentration in this diet was 74.9 ± 4.5% SD as determined by a Hewlett- Packard C-H-N analyzer. Formulated diets consisted of 17.5% dry feeds (Table 1), 1.5% agar and 81% water. Total water content of all diets was 83-84%. (The dry feeds contained 2-3%water.) Water and agar were mixed, heated, and poured 29 30 R. R. WINGET, C. E. EPIFANIO, T. RUNNELS, P. AUSTIN into a pan containing the other ingredients. After cooHng, the food was shced into blocks. Since preliminary trials indicated that three to four grams per day represented a maximum ingestion rate, crabs were offered three grams per day, six days a week. TABLE 1. Ingredients of formulated diets as a percentage of dry weight. DIET INGREDIENTS C Ground Yellow Corn Soybean Meal, 50% Herring Fish Meal Casein Crab Meal Fish Meal Soybean Meal Brewer's Dried Yeast Fish Solubles Ground Yellow Corn Dehydrated Alfalfa Dried Whey Soybean Oil Ground Limestone Iodized Salt Methionine Vitamin and Trace Metal Premix Total Weight Calcidated Proximate Composition Protein Fat Fiber Calcium Phosphorus Ash MET Energy (cal/g) 40.00 11.25 18.75 3.75 3.75 17.03 .75 1.87 1.50 .75 .19 .11 20.00 20.00 11.25 18.75 3.75 3.75 17.03 .75 1.87 1.50 .75 .19 .11 .30 .30 40.00 11.25 18.75 3.75 3.75 17.03 .75 1.87 1.50 .75 .19 .11 .30 100.00 100.00 100.00 26.00 4.20 2.16 .80 .51 4.50 1457.00 1397.00 1565.00 46.00 62.00 3.90 3.30 2.28 1.35 .90 .80 1.09 .50 7.50 4.50 Male crabs were collected from Indian River Bay, Delaware, during May and June when am- bient water temperatures were between 15 °C and 20°C. The mean carapace width of these animals was 5.9 cm, and an analysis of variance (Sokal and Rohlf, 1965) showed no significant difference (p — 0.05) in mean carapace width among eight ex- perimental groups used in the experiment. Upon arrival in the laboratory, each animal was placed in the system at 20 °C or 30 °C and fed ground sil- verside, Menidia menidia, until ecdysis after which it was considered an experimental animal. No crabs with damaged or missing appendages were used in the experiment. The experiment lasted 60 days and dependent variables were: 1) percent of animals that molted within 60 days after introduction to the ex- perimental situation, 2) intermolt period in days, 3) percent increase in carapace width after ecdysis, and 4) percent mortality. Analyses of variance were performed on the data for mean percent in- crease in carapace width and mean molt interval. All statistical inferences were made at the 0.05 probability level. Since the eight groups within the experiment were not replicated, no statistical -I a ?,5 $5 100 ■^ 80 lO 30 ^ 60 s 20° 20" 20° 2U" 30° 30° 30' JO" 20° 20° 30° 2(r 20° 20° 20° 20° 30° 30° 1 30° 1 30° 1 DIETS nG. 1. Temperature and dietary relationships with parameters of growth. Diet A =26% protein Diet B = 46% protein DietC = 62% protein BLUE CRAB IN RECIRCULATION CULTURE SYSTEM 31 treatment was possible for percent mortality and percent second molt. RESULTS Temperature and dietary relationships with the dependent variables are shown in Figure 1. F values from analyses of variance are presented in Table 2. Dietary protein content did not con- sistently affect any of the growth parameters. Diets A and C were identical except for a 40% dif- ference in ground corn or casein content. Diet C (62% protein) appeared to reduce mortality at 20°C but increase it slightly at 30°C. Diet C also reduced percent molt at 30 °C but not at 20 °C. Replacement of casein with a different source of protein (soybean and herring fish meal in Diet B) did not greatly influence growth patterns. The 30 °C temperature regime significantly decreased molt interval in the experiment and decreased mortality considerably. Also, the percentage of animals molting a second time was generally higher at 30'C. Thus warm water fa- vored three of the growth parameters. On the other hand, 30 °C water significantly reduced carapace growth at ecdysis in all three experi- ments. DISCUSSION Diet A noteworthy result of this experiment was a lack of consistent dietary effect. We were primari- ly concerned with elucidating a range of protein concentrations which would indicate optimal growth, but were unable to do so at protein con- centrations from 26 to 75%. Although it is possi- ble that optimal protein values were masked by in- teraction with other variables, it appears that con- tinuing nutritional studies with Callinectes. should, at least initially, use diets containing 26% protein or less in establishing economically op- timal feeds. These results compare favorably with those of Andrews et al., (1972), who suggested that a dietary protein level of 28% was optimal for juvenile penaeid shrimp. However, the findings of Zein-Eldin and McGaffey (1975) indicate that op- timum protein concentrations should be re- evaluated as other aspects of the diet are improv- ed. They reported a distinct 52% optimum in one penaeid diet, but found an optimum 32%' protein content when combined with very different non- protein components in another diet. A number of authors (Renaud, 1949; Schwabe, et al, 1952; Scheer, 1959; Heath and Barnes, 1970; Rouse, 1972) have presented evidence to in- dicate that reptantian crustaceans store food reserves, including protein, during periods of alimentation (intermolt-early premolt) and deplete them during inanition (late premolt-postmolt). It is possible that reserves stored while animals were still in the field could have influenced the results of the present study, but it seems more probable that reserves augmented during feeding would be largely used up during the following postmolt and that material stored in the field would have its ma- jor effect during the first laboratory postmolt period and a relatively minor influence thereafter. The hypothesis has not been tested, however, and future nutritional experimentation should pro- bably be performed on animals which have molted several times in the laboratory under con- trolled diets. Temperature Changes in water temperature have been shown to influence survival, molt frequency, and in- creases in linear and gravimetric dimension among decapod crustaceans (Zein-Eldin and Griffith, 1966; Huges et al. 1975), but optimal temperatures vary with different growth parameters. Although Ford et al. (1975) found slight enhancement of sur- vival and an increase in molt frequency and linear dimension in Homarus americanus between 19 °C TABLE 2. F values from 2-way analyses of variance of results. Experiments - an asterisk (*} denotes sig- nificant F values at the 5% probability level. Dependent Variable Increase in carapace width Molt Interval Temperature (T) 30.66* 17.20* Diet(D) 0.99 1.21 T X D 2.52 0.52 32 R. R. WINGET, C. E. EPIFANIO, T. RUNNELS, P. AUSTIN and 22 °C, Zein-Eldin and Griffith reported that maximum Hnear and gravimetric growth in penaeid shrimp occured near 30 °C. We have noted somewhat better survival and more fre- quent and higher percentage of molting at 30 °C compared to 20°C. Carapace growth, on the other hand, was uniformly greater at 20 °C. Considering the above complications, it may be best to deter- mine optimum growth temperature by using total protein or dry weight production from a given number of animals as a basis of measurement. Temperature also had a pronounced effect upon mortality. Mortality in groups cultured at 20°C ranged from 37.5 to 62.5% while mortality in the 30°C groups ranged from 12.5 to 27.5%. The reasons for higher mortality at 20 °C are not clear as this temperature is certainly not extreme for the species. Conclusions Growth observed in this study generally did not compare well with that observed in natural waters. Tagatz (1968) reported that C. sapidus, held from April to November in live cars in St. Johns River, Florida, and fed an unspecified species of fish, increased carapace width by 25 percent per molt in all size classes. Temperatures ranged from 14-32C (X = 27C). Others (Gray and Newcombe, 1938) described similar growth rates in blue crabs held in live cars in Chesapeake Bay. Increase in carapace width was not this great in our experiment (Fig. 1). Molt intervals were also longer in our laboratory experiments. Tagatz reported a mean interval of 17 ± 6.1 days for animals of the same size range as those used in the present experiment. This indicates faster growth in a more natural situation than achieved here. The work of Kanazawa ef a/. (1970) and Sick et al. (1972) indicated a similar problem with penaeid shrimp. Neal (1973), in a discussion of aquaculture research with penaeid species, con- cluded that no one has been able to produce a defined diet which compares favorably in terms of growth with more natural diets. ACKNOWLEDGMENTS This work was supported by a grant from the University of Delaware Research Foundation and by an Institutional Sea Grant to the University of Delaware. We wish to thank Mr. Leon Anderson and Mr. Robert Baggaley for their technical assistance. LITERATURE CITED Aiken, D. E., 1969. Photoperiod, endocrinology and the crustacean molt cycle. Science, 164: 149-155. Andrews, J. W., L. V. Sick, and G. ]. Baptist, 1972. The influence of dietary protein and energy levels on the growth and survival of penaeid shrimp. Aquaculture, 1:341-347. Chanley, M. H. and O. W. Terry, 1974. Inexpen- sive modular habitats for juvenile lobsters (Homarus americanus). Aquaculture 4: 89-92. Epifanio, C. E., G. Pruder, M. Hartman, and R. Srna. 1973. An interdisciplinary study on the feasibility of recirculating systems in mariculture. Proc. Fourth Ann. Workshop, World Mariculture Society: 37-52. Ford, R. F., J. C. Van Olst, ]. R. Carlberg, W. R. Dorband and R. L. Johnson, 1975. Beneficial use of thermal effluent in lobster culture. Proc. World Mariculture Soc. In press. Gray, E. H., and C. L. Newcombe, 1938. Studies on molting in Callinectes sapidus Rathbun. Growth, 2: 285-296. Heath, J. R. and H. Barnes, 1970. Some changes in biochemical composition with season and dur- ing the molting cycle of the common shore crab, Carcinus maenas (L.). J. Mar. Biol Exp. Ecol., 5: 199-233. Hughes, J. T., J. Sullivan, and R. Shleser, 1972. Enhancement of lobster growth. Science, 177: 1110-1111. Kanazawa, A., M. Shimaya, M. Kawasaki, and K. Kashiwada, 1970. Nutritional requiremen s of prawns. I. Feeding on artificial diet. Bull. Jap. Soc. Sci. Fish., 36: 949-954. Neal, R. A., 1973. Progress toward farming shrimp in the United States. Mar. Fish. Rev., 35: 67-70. Passano, L. M., 1960. Molting and its control. Pg. 473-536 in T. H. Waterman, Ed., Physiology of Crustacea, vol. 1 Academic Press, New York and London. Renaud, L. 1949. Le cycle des reserves organiques chez les crustaces decapodes. Ann. Insti. Oceanogr. Monaco, 24: 259-357. Rouse, A., 1972. Hepatopancrease glycogen con- BLUE CRAB IN RECIRCULATION CULTURE SYSTEM 33 centrations and their relationship to ecdysis in the blue crab, Callinectes sapidus Rathbun. Un- published Master's Thesis, University of Delaware, Newark. 85pp. Scheer, B. T., 1959. The hormonal control of metabolism in crustaceans. IX. Carbohydrate metabolism in the transition from intermolt to premolt in Carciuus maenas. Biol. Bull. ,116: 175-183. Schwabe, C. W., B. T. Scheer, and M. A. Scheer, 1952. The molt cycle in Panulirus japonicus. Part II of the hormonal regulation of metabolism in crustaceans. Physiol. Comp. Oecol., 2: 310-320. Sick, L. v., J. A. Andrews, and D. B. White, 1972. Preliminary studies of selected en- vironmental and nutritional requirements for the culture of penaeid shrimp. U. S. Fish. Wild. Sen/., Fish. Bull. ,70: 101-109. Sokal, R. R. and F. J. Rohlf, 1965. Biometry. W. H. Freeman Co., San Francisco, 757pp. Tagatz, M. E., 1968. Growth of juvenile blue crabs, Callinectes sapidus Rathbun, in the St. Johns River, Florida. U. S. Fish. Wildl. Serv., Fish. Bull., 67: 281-288. Winget, R., D. Maurer, L. Anderson, 1973. The feasibility of closed system mariculture: Preliminary experiments with crab molting. Proc. Natl. Shellfish. Assoc, 63: 88-92. Zein-Eldin, Z. P. and G. W. Griffith, 1966. The ef- fect of temperature upon the growth of laboratory-held post-larval Penaeus aztecus. Biol. Bull., 131: 186-196. Zein-Eldin, Z. P. and J. C. McGaffey, 1975. Pro- tein quantity and quality in diets of penaeid shrimp. Proc. World Mar. Soc. In press. Proceedings of the National Shellfislieries Association Volume 66 — 1976 GROWTH OF PACIFIC OYSTERS CRASS05TREA GIGAS AND RELATED FOULING PROBLEMS UNDER TRAY CULTURE AT SEABECK BAY, WASHINGTON' ' Patricia Clark Michael and Kenneth K. Chew COLLEGE OF FISHERIES UNIVERSITY OF WASHINGTON SEATTLE, WASHINGTON ABSTRACT Pacific oysters (Crassostrea gigas) were grown in Nestier trays under two different sets of conditions at Seabeck Bay on Hood Canal, Washington. One group of oysters was placed in trays that were suspended from a floating dock and were submerged at all times. The other group of oysters was placed in trays that were set out in the inter- tidal zone at the +2 foot tide level where they were exposed to the air for some portioti of each day. Growth and fouling data were collected monthly for each set of trays. Fouling was very pronounced on the dock trays and less on the beach trays. Growth patterns for the two different stations were also different. The oysters suspended from the dock grew well during the early months of the year, then ceased to grow in April or May, due to excessive fouling of the trays. The oysters on the beach showed no growth from ]a>iuary to April. Growth for these oysters started in April or May and continued throughout the summer well into the fall. Data from this study point out the importance of fouling organisms to this type of oyster culture and the different growth rates that can be obtained by placing the trays under different conditions at the same site. INTRODUCTION The most popular method for growing Pacific oysters in Japan is hanging culture, using either rafts or long lines (Furukawa, 1971). On the Pacific coast of the United States, this species of oyster is usually grown directly on the beds and harvested by tonging, hand picking, mechanical harvesting, or drag dredging. However, some growers have contemplated the use of tray culture as another means of raising commercial crops of Pacific oysters. This has been especially true with 1 Contribution No 443 College of Fisheries, University of Washington 2 This study was supported in part by the Sea Grant Program under the National Oceanic and Atmospheric Administra- tion, U. S. Department of Commerce the development of oyster hatcheries along the Pacific coast, making cultchless seed readily available. The concept of oyster tray culture is not new. It has been utilized for many years in European countries, Australia, and the Eastern United States. Trays of oysters can be hung from long lines, suspended from a dock or other floating structure, where they are kept constantly immers- ed in sea water, or they can be placed in the inter- tidal zone. Although the advantages of tray culture are well known, little information is available as to the types and seasonal trends of fouling organisms occurring under this type of culture in Washington waters. Thus the present study was initiated in Seabeck Bay to better understand fouling pro- 34 PACIFIC OYSTERS UNDER TRAY CULTURE 35 blems when oysters are grown under two different conditions. The two conditions are: (1) trays of oysters hung from a floating dock and submerged under water continuously, and (2) trays of oysters placed at the + 2 foot tide level in the intertidal zone where they are exposed to air during low tides. Further, the growth of oysters under both conditions was monitored throughout this study. Although we recognize that the site used for this study may not be representative of the potential oyster tray culture sites in Washington, the results presented are useful in that they compare growth rates of oysters grown in trays at the same site under different conditions and provide informa- tion on the importance of fouling organisms in tray culture. MATERIALS AND METHODS Site Description This study was conducted at Pecks Harbor Marina at Seabeck Bay on Hood Canal. One ex- perimental station was located on the southern side of a marina pier where the trays containing oysters were suspended about one meter below the water surface at all times from a floating dock. A second experimental site was located on the beach at the northern side of the pier at the +2 foot tide level. The oysters in this study were grown in plastic Nestier trays Vz meter on a side and about 8 cm. deep. Small holes in the sides and bottom of the tray allowed for water circulation. Three stacks of four trays were used at each sta- tion. The bottom three trays in each stack contain- ed oysters, while the top tray acted as a lid to keep the oysters from being washed out. Each stack was secured at opposite corners with nylon cord. The stacks of dock trays were fastened together with polypropylene line and suspended from the dock. The trays on the beach were at first held in place by setting two concrete blocks on top of each stack. In the winter the trays were wrapped with polypropylene line and tied to metal pipes driven into the beach. Test Animals The oysters used in this study were purchased from the Lummi Island Oyster hatchery at a size of 2-4 cm in shell length. To simulate a commer- cial density, groups of 200 oysters were separated out at random from the original lot and each of these groups was placed in a separate tray. Ten oysters from each group were marked with dots of nail polish in a three-color coding system that made it possible to identify individual oysters. Two of the marked oysters were placed in each corner of a tray, and two in the center. Data Collection Data were collected from the trays on a month- ly basis. Growth measurements were taken from the ten marked oysters in each tray and were ex- pressed as the product of their length and width, as defined by Quayle (1969). The increases in this product, or the actual shell area, were used as a measurement of the oysters' growth, much in the same manner as Butler (1953) used his "G" factor to compare the growth rates of different stocks of oysters. Fouling was defined as any macroscopic organism that attached itself to the outside of the trays or was found inside the trays in this study. These organisms were documented by visual observation and by 35 mm slides taken using the Mayers Underwater Photogrammetric System (MUPS). Representative specimens of organisms not identifiable by sight were stored in 10% for- malin and taken to the laboratory for identifica- tion. Fouling organisms were not removed from the trays except when they actually preyed on the oysters and so jeopardized the growth study. This meant that the entire stacks of trays could be used like the test blocks in Coe and Allen (1937) and Graham and Gay's (1945) classic fouling studies. Carlisle, Turner, and Ebert (1964) also performed similar studies on fouling communities on ar- tificial reefs. All of the beach trays were lost in December, 1973, during a severe storm. Thus it was necessary to move some of the dock trays to the beach sta- tion. These trays were chosen at random from the original dock trays. The oysters were them remarked with nail polish and measured before placing them at the beach station. The fouling on these trays was not taken off, but as can be seen from the data in Table 2, these animals did not survive at the beach station. Over 2,000 additional oysters from the Lummi Island Hatchery were added to the study in May, 1974. These oysters were also 2 to 4 cm in length 36 P. C. MICHAEL AND K. K. CHEW and were divided into groups, marked and measured in the same manner as the original oysters had been. Trays were stacked together as before and placed at the dock and beach stations. RESULTS AND DISCUSSION Fouling Study The monthly occurrences of the different foul- ing organisms found on the trays suspended from the dock and those set out on the beach are shown in Tables 1 and 2. A comparison of these tables fxjints out some obvious differences in types and amounts of fouHng organisms present at the two stations. The dock trays had a much more diverse fauna and flora of fouling organisms than did the beach trays. Also, fouling of the dock trays was fairly constant; except for a winter die-off, organisms that were found on the dock trays one month were usually found there the next month. This was not true of the beach trays where the fouling organisms present varied considerably from month to month. The dock trays represented a fairly stable or constant environment. The trays were submerged at all times (except during sampling) and were hanging at a constant depth. The layer of water between the trays and the surface acted as a buf- fer, protecting the oysters and trays from quick changes in temperature and salinity. By the end of the study the dock trays had developed entire communities of sessile or slow moving organisms. The first organisms to appear on the dock trays were sponges, bryozoans and solitary and colonial tunicates. These animals were present throughout most of the study and covered large areas on all of the trays. Another important fouling organism found on the dock trays was the mussel (Mytilus edulis). Table 1. Monthly occurrence of fouling organisms at the dock station, Sept. 1973 to Nov. 1974. TIME IN MONTHS Kelp Sponges Sea Anemone (Mefridium) notworm (Notoplana) Polyctioetes ( Holosydna) ( Serpula) (Nereis) Bornocles ( Bolanus) Amphipods-Gammarldae - Caprellidae Shrimp- Hippolyfidoe C ro b s (Cancer oregonen sis ) (C. product us) Limpets (Acmaea) Nudibronchs (Hermissenda) (Archidoris) ( Diaulula) Mussels (Mytilus) Scallops ( Pecten) Jingle ( Pododesmus) Clams (Entodesmus) Bryozoans Sea cucumbers (Parasticttopus) Sea urchins ( Stronglocentrotus) Tunicates- Solitary -Colonial s 0 N D J F M A M J J A s 0 N X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X x; X X X X X X X X X X X X X X X X X X X X X X X X X X X PACIFIC OYSTERS UNDER TRAY CULTURE 37 The first mussel set on the trays occured in Etecember, 1973. As the mussels grew they covered the trays and oysters with their byssal threads. By April, the trays were so tightly woven together by byssal threads that they were hard to separate. Oysters inside the trays became tied into place and had to be pried out for growth measurements. Three nudibranch species were found on the dock trays with some regularity. The most com- mon of the three was Hennisseuda crassicornis. Also found were Diaulula sandigensis and Ar- chidoris montereyensis. The crabs Cancer pro- ductus and C. oregoiieusis were found quite often inside the dock trays. These animals had to be removed from the trays when they began to chip and open the oyster shells. While some of the other fouling organisms competed with the oysters for food and space, these crabs were the only foul- ing organisms that actually preyed on the oysters at the dock station. Fouling on the beach trays was much less cons- tant than that on the dock trays. This was because the trays on the beach offered a more severe en- vironment than did those hanging from the dock. The lowest tides, which gave the longest exposure to the air occurred at night in winter and during the day in summer. Oysters and fouling organisms in these trays were also subject to rapid salinity changes during rainstorms and considerable wave action during high winds. All of these factors taken together discouraged many of the sessile fouling organisms found with such abundance in the dock trays. Most of the organisms associated with the fouling community found on the beach trays were mobile to some ex- Table 2. Monthly occurence of fouling organisms at the beach station, Sept. 1973 to Nov. 1974. TIME IN MONTHS SONDJ FMAMJJ A S 0 N Sponges Anemones Barnacles (Balanus) Amphipods-Gannmaridae Shrimp- Hippolytidae Crabs (Cancer productus) ( Epialfus) Hermit crabs (Pagurus) Snails (Thais) (Searlesia) Mussels (Mytilus) Starfish (Evasferias) Gunnels (Apodicthys) (Pholis) X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 38 P. C. MICHAEL AND K. K. CHEW ^J 5- ^-^Dock Beach \ 4- 3- 2- 1 0- -1- HG. 1974 N D 0 JFMAMJJAS Time in months 1. Average monthly growth increments for dock and beach oysters, September, 1973 to October, tent. This helped them find shelter when the trays were exposed to particularly harsh weather condi- tions. The most common fouling-associated organism found on the beach trays was the drill Thais lamellosa. These snails were almost always found on the outside of the trays and posed no threat to the oysters inside. Hermit crabs {Pagurus) were common in and on the beach trays. Kelp crabs (Epialtus productus) were common on the outside of the trays and were occasionally found inside. The bottom tray of each beach stack was set directly on the substrate. It occasionally became covered with a thin layer of silt and this attracted different fouling-associated organisms. The most common of these were gunnels (Pholis and Apodichthys) and spironticarid shrimp. The only destructive fouling-associated organism found on the beach trays was the star- fish Euflsfen'fls. Although these animals were usually found on the outside of the trays, a few did manage to get inside, probably when they were small larvae. Those that did so were remov- ed when noticed. Growth Studies Figure 1 shows the mean monthly growth in- CTements of the dock and beach oysters. Growth increment was computed as the actual increase in shell area from one month to the next, or: AA = A2 - Ai where Ai is the shell area at the end of one month, while A2 is the shell area at the end of the next month. Growth increments were used instead of actual size measurements when looking at the growth of oysters throughout the entire study because of the fact that the beach oysters were lost in December, 1973, and replaced with oysters from the dock trays. Thus, although the actual size of these new beach oysters in January would not have been meaningful, it was possible to com- pute their actual increase in shell area each month. As can be seen from the figure, the growth pat- PACIFIC OYSTERS UNDER TRAY CULTURE 39 terns of the dock and beach oysters were quite dif- ferent. A three factor analysis of variance test was conducted on these data, testing for differences in growth increment between the dock and beach stations, the different tray levels within a stack, and the different months of the year. There was a significant difference between the growth in- crements of the oysters at the two different sta- tions (F = 3.24, p = 0.75) and during different months of the year (F = 2.74, p =0.75). The in- teraction between these two factors was also significant (F . = 2.13, p = .026). The analysis described above contained two dif- ferent age groups of oysters (those present at the beginning of the study and those added in May 1974). Since oysters of different ages grow at dif- ferent rates (Loosanoff, 1947) the data were also analyzed by age group. This analysis was con- ducted on the original dock oysters in December, 1973. This was just before the dock trays were split into two groups, one of which was to replace the lost beach oysters. The test showed no dif- ferences in size between the trays of oysters left at the dock station and those moved to the beach (F= .46, Critical value at p = .05 = 4.07). At the end of the study in November, 1974, another analysis of variance test was conducted on the size of the oysters in these dock and beach trays. This test also showed no significant difference in size between the oysters at the two stations (F = .61, Qitical value at p = .05= 5.14). Although these two groups of oysters grew to the same final size, their patterns of growth were quite different. This is shown in Figure 2, where the average monthly size of the oysters at the two different stations is plotted along with the average monthly water temperature at the site. As can be seen from the figure, the oysters in the dock trays started to grow in February-March and continued to show obvious increases in shell area until April or May. After that their growth leveled off and they decreased slightly in size (probably due to chip- page during handling) until August, when they slowly began to grow again. The beach oysters, on the other hand, showed almost no growth from January until April or May. After that they grew well until August. Growth then leveled off until October, when a particularly large amount of shell was added. A close look at this figure raises several ques- tions. First, why were the dock oysters able to grow during this early period and the beach oysters not able to? And, finally, why did the dock oysters stop growing in April or May, which is normally one of the best growing periods for oysters? The early growth of the dock oysters took place partly during a period when the water temperature was fairly low (8 to llC). Winter hibernation, or cessation of growth, has been reported at temperatures below 10 to llC in British Columbia by Quayle (1969) and in Washington waters by Chew (1961). However, it is not known whether this is caused simply by a slowing of the oysters' pumping mechanism, a lack of food in the colder waters, or a combination Jon Mar May Jul Sept Nov Time in months FIG 2. Average monthly water temperature and average monthly size of original dock and beach oysters, January to November, 1974. of the two. It is known that oysters are capable of pumping some water at temperatures below IOC. In this case, the fact that the oysters were actually growing when the mean monthly water temperature was only 8C shows that they must have been pumping water and that there must have been food in the water at this time. An obser- vation that supports this idea is that mussels that set on the trays at the dock station in December, 1973, also showed noticeable growth during the early months of 1974. 40 P. C. MICHAEL AND K. K. CHEW Assuming that there was food available in the water during this period, the question becomes why the beach oysters did not show a similar pat- tern of rapid growth during this period. This is probably because the beach oysters were subjected to a much more harsh and variable environment, particularly during the winter months. Several hard freezes occurred at Seabeck Bay during the winter of 1973-4. Also, several storms followed the one in December that swept away the original batch of beach oysters. These storms had a greater effect on the more exposed beach oysters than on those suspended from the dock. Aside from the temperature changes that accompanied the storm, heavy rains often occurred, subjecting the beach oysters to rapid changes in salinity. Probably the most important factor caused by the winter storms was wave action. Storm waves tossed the oysters around inside the beach trays, often piling them in one corner, chipping fragile growing edges of the oysters, and leaving less room for water circula- tion and oyster growth. Although the beach oysters probably grew slowly during this period, the large amount of shell chippage masked such growth. The question remains as to why the dock oysters stopped growing in April or May. Ex- cessive fouling of the dock trays was the probable reason. The mussels that set on the trays in December were getting larger by this time and were beginning to clog up the holes in the trays, restricting water circulation. Kerswill (1949) reports on how much a lack of adequate circula- tion can slow the growth of oysters and other bivalves. This condition worsened as the summer progressed and was augmented by the fact that the mussels had set most heavily around the sides of the holes. Since mussels are filter feeders like oysters, it is probable that they also competed with the oysters for food items. Also, because the mussels were largely on the outsides of the trays, they had first chance at filtering the water before it went inside the trays to the oysters. However, mussels were not the only fouling organisms on the dock trays. As the water temperature grew warmer, the tunicates, sponges, and bryozoans began to cover more of the tray surface. The beach trays, on the other hand, did not suf- fer from these problems. They were exposed to the air for a part of each day, and while this did not stop the oysters from growing it did discourage most of the kinds of fouling that seemed to cause problems on the dock trays. The oysters in the new trays (those added to the study in May, 1974) were analyzed in a similar manner to those in the original trays. An analysis of variance test conducted on the initial size of the oysters showed no difference between the dock and beach groups (F . = 3.49, Critical value at p = .05 = 7.71). However, at the end of the study, the mean size of the beach oysters was quite a bit smaller (19.48 cm^) than that of the dock oysters. The average monthly sizes of the dock and beach oysters in these new trays are plotted in Figure 3. It should be noted that the new dock oysters grew well during the period when the original dock oysters had stopped growing. This is because the new trays were placed in the water late enough to miss many of the spring sets of fouling organisms. This figure also shows that without extreme foul- ing to contend with the dock and beach oysters had very similar growth patterns during this period. The dock oysters were probably able to grow faster during this period than those on the beach simply because they were exposed to the water for more hours each day and so had more time to feed. 28r Jun Aug Sept Oct Time in months Nov nC. 3. Average monthly water temperature and average monthly size of new dock and beach oysters, June to November, 1974. PACIFIC OYSTERS UNDER TRAY CULTURE 41 LITERATURE CITED Butler, P. A. 1953. Importance of local environ- ment in oyster growth. Proc. Gulf and Carib. Fish. Inst., Fifth Annual Session: 99-106. Carlisle, J. G., C. H. Turner, and E. E. Ebert. 1964. Artificial habitat in the marine environ- ment. Cal. Fish and Game Bull. 124. 93 pp. Chew, K. K. 1961. The growth of a population of oysters (Crassostrea gigas) when transplanted to three different areas in the State of Washington. Ph.D. Thesis, University of Washington, Seattle, Wash. Coe, W. R. and W. E. Allen. 1937. Growth of sedentary marine organisms on experimental blocks and plates for nine successive years at the pier of the Scripps Institution of Oceanography. Scripps Inst. Oceanog. Bull. Tech. Series 4: 101-135. Furakawa, M. 1971. Outline of the Japanese marine aquaculture. Japan Fisheries Research Conservation Assoc. 39 pp. Graham, H. W. and H. Gay. 1945. Season of at- tachment and growth of sedentary marine organisms at Oakland, California. Ecol. 26: 375-386. Kerswill, C. J. 1949. Effects of water circulation on the growth of quahogs and oysters. J. Fish. Res. Bd. Canada. 1: 545-551. Loosanoff, V. L. 1947. Growth of oysters of dif- ferent ages in Milford Harbor, Connecticut. Proc. Nat'l. Shellfish. Assoc. 47: 12-21. Quayle, D. B. 1969. Pacific oyster culture in British Columbia. Fish. Res. Bd. Canada, Bull. 169. 192 pp. Proceedings of the National Shellfisheries Association Volume 66 — 1976 INVESTIGATION OF PRACTICAL MEANS OF DISTINGUISHING MY A ARENARIA AND HIATELLA SP. LARVAE IN PLANKTON SAMPLES Neil B. Savage and Ronald Goldberg^ NORMANDEAU ASSOCIATES, INC. BEDFORD, NEW HAMPSHIRE ABSTRACT Bivalve lan^ae (veligers) of the soft shell clam, Mya arenaria, and a rocky substrate dwelling clam, Hiatella sp.. were obtained from induced spawnmg of adults in the laboratory, and also from live plankton collections made in the vicinity of Hampton Beach, New Hampshire (42''54' N. Lat., 70°49' W. Long.). Mya arenaria were raised to the settling stage. Morphology of developing larvae of both species was described and photographically illustrated. Distinguishing characteristics of shape, size and col- oration were found to be difficult to apply in planktonic studies over much of the period of larval development covered. With experience, however, the investigators could make correct identification of umboned Mya arenaria with less than 20% error, aided in part by seasonal differences in peak abundance of larvae of the two species. Obscurity of taxotwmy of the genus Hiatella is discussed. INTRODUCTION Study of larval distribution of a species often re- quires processing of a large volume of plankton samples from which the investigators must be able to readily identify the species, usually using shell characteristics as they appear with the organism on its side under the microscope. Photographic il- lustrations of Sullivan (1948) indicate that a close resemblance exists between shell characteristics of soft shell clam, Mya arenaria, larvae and Hiatella sp. larvae (specific identity obscure, Yonge, 1971). Larvae of both species are seasonally abundant in plankton samples from coastal Maine and New Hampshire waters (personal observation); hence, a need is apparent for further investigation of the chances of misidentification. Present address: National Marine Fisheries Service, Milford, CT 06460 On the western side of the North Atlantic Ocean, definitive bivalve larval identification work including M. arenaria larvae (Loosanoff and Davis, 1963; Loosanoff et al, 1966; Chanley and Andrews, 1971) has been conducted south of the Gulf of Maine area where the occurrence of Hiatella sp. larvae in neritic plankton com- munities has not been reported. Sullivan (1948) is the only North American study which includes in- terspecific comparison of M. arenaria and Hiatella sp., however the larvae described were obtained from plankton collections only; none were reared from known parents in the laboratory. Moreover, the straight hinge stage of development of Hiatella sp. was not described or depicted by Sullivan (1948). Larvae of Hiatella have been described from European waters by Odhner (1914), Lebour (1938) and Rees (1950), and both M. arenaria and Hiatella spp by Jorgensen (1946). Because of limited coverage of developmental stages, and 42 DISTINGUISHING LARVAE IN PLANKTON SAMPLES 43 possible taxonomic distinctions, the European studies may be of little practical use in distinguishing between planktonic M. arenaria and Hiatella sp. larvae in North American coastal waters. METHODS AND MATERIALS Larvae Reared from Laboratory Spawned Adults Approximately 1 ml of 0.1 N ammonium hydroxide was injected directly into the gonad of ripe female clams (as suggested by Stickney, 1964) obtained locally from flats in Hampton Harbor, New Hampshire. Males were induced to spawn by this same technique. Released ova were separated from large debris in the spawning bowl by passing the water through a 73pim mesh screen. The ova were then collected on a 41^im mesh screen. Recovered ova were washed into 600 ml pyrex beakers and the volume brought to 500 ml using seawater which was filtered through a 5jim filter bag and allowed to stand for about one week. To ensure fertilization, hypodermically extracted sperm were added to the egg cultures as well as sperm from males induced to spawn by am- monium hydroxide injection. Fertilized egg cultures were held on a flowing seawater table in which temperatures fluctuated between 16C and 19C. After about twenty-four hours, swimming trocophore larvae were decanted and undeveloped eggs discarded. Larval densities of approximately one to two trocho- phores per milUliter were maintained in the cultures. Ova of Hiatella sp. were obtained with relative- ly little effort. Adult animals, obtained in June and July, 1975, from rocky substrate off Hampton Beach, New Hampshire, at a depth of approx- imately 15 to 20 meters, were initially held in cool (10-13C) running seawater. Spawning resulted when the animals were placed in filtered seawater in glass bowls and allowed to warm to between 16C and 21C. With from six to twenty individuals per bowl, it was not unusual to observe sequential spawning of several animals. Procedures for egg recovery, insemination and establishing larval cultures, were the same as for M. arenaria except that lower rearing temperatures (6C to 13C) were used. To suppress bacterial growth, very dilute solu- tions of penicillin G (1667 units per mg) and strep- tomycin sulfate were added to all cultures. The solutions were prepared by dissolving 2 to 4 mg of each of the antibiotics in a single beaker contain- ing 100 ml to 5f.im filtered seawater. Approx- imately 5 ml of the antibiotic solution was added to each 500 ml culture upon initiation and thereafter with each water change. All containers used in connection with larval culture were rinsed in very hot tap water and allowed to air dry. No soaps or detergents were used to clean any materials coming in contact with the larvae. To exchange the culture medium and monitor larval development, animals were recovered from the old medium three times weekly using either 41 or 73Mm mesh screens, depending on organism body size and amount of small debris present. Debris and shells of dead animals were also removed mechanically using microprobes and micropipettes. Animals were washed from the screens into small circular counting dishes and then transferred to a depression slide for measure- ment and photographing. Measurements were made using either a dissec- ting microscope (100 x) or compound microscope (100 and 400 x) with calibrated eyepiece reticles. Successive measurements of shell lengths and widths of the developing larvae were plotted graphically and analysed by linear regression to determine if any species-specific length /width relationships could be discerned. Interspecific dif- ferences in hinge length of larvae in the early developmental (straight hinge) stage were com- pared using Student's t-test and Mann-Whitney Li- test (Snedecor and Cochran, 1967). Black and white photographs were taken of selected in- dividuals in various stage of development using a "Polaroid" Land Instrument Camera (Model ED 10) mounted on a compound microscope. From early July, 1975, until experiments ter- minated in September, 1975, the principal food source for the larvae culture was a mixture of Phaeodactylum tricomutum, hochrysis galbana and Monochrysis lutheri each obtained in monoculture from the National Marine Fisheries Service Laboratories at Milford, Connecticut. The three species were maintained both in mixed and monoculture on F/2 medium (Guillard and Ryther, 1962), except that ammonium chloride was omitted from the salt preparation as recom- 44 N. B. SAVAGE AND R. GOLDBERG mended by Loosanoff and Davis ( 1963) for /. galbana culture. Seawater used to make up the algal culture was vacuum filtered through a glass fiber filter (Whatmann GF/C) to remove organic residue and bacterial cells. Glassware used in algal culture was heated to over lOOC in an air drying oven for one hour or more. Algae were fed to the larvae following each thrice-weekly change of their culture media. The amount of mixed algal culture added varied depending on: 1) the number of bivalve larvae in the culture; 2) visually determined cell density in the food culture; and 3) amount of unconsumed algae in the larval culture beakers prior to chang- ing the medium. Larvae Reared from Plankton Collections Plankton tows were conducted in coastal waters of New Hampshire near Hampton Beach, using a 73fim mesh net with a 0.5 diameter opening. Bivalve larvae were separated from larger plankton and debris by passing the sample through 505p(m netting. The larvae, along with other smaller plankton were then recovered on a 41/im mesh screen. Bivalve larvae of the desired species were isolated from other planktonic organisms by swirling them to the center of a watch glass and isolating them with probes made from a single strand of camel's hair. Only one isolation of Hiatella sp. from plankton samples was carried out, on 2 July 1975, when the density of this species in the plankton was about at its seasonal peak. Larvae identified as M. arenaria were isolated from two samples taken weekly from mid-August to mid-September. Once isolated, the animals were enumerated and placed in 600 ml pyrex beakers containing 500 ml of 5iim filtered seawater. In examining, feeding and changing the culture media the same procedure as described above for laboratory spawned larvae was followed. With each thrice- weekly examination, approximately 100 larvae in the M. arenaria cultures were re-examined and identified to determine the extent to which species other than M. arenaria had been introduced into the cultures. RESULTS Spawning Success In all, there were four attempts to spawn M. arenaria (on 14 and 31 July, 7 and 13 August) by injecting ammonium hydroxide. All succeeded in producing fertilized ova which subsequently developed into umboned larvae, although some of the spawning produced only one or two hundred eggs from up to six female clams. The most pro- lific production of ova (approximately 1000 eggs from six females) occurred on 31 July 1975. Condi- tion of the gonads and occurrence of large numbers of M. arenaria larvae in coastal New Hampshire plankton collections indicated that late July to early August was the peak of the 1975 spawning season. Four attempts to spawn Hiatella sp. (on 19 June and 17, 18 and 24 July) resulted in the release of ova. However, only two spawnings (19 June and 24 July) subsequently produced straight hinge lar- vae. Only the Hiatella sp. culture spawned 24 July was maintained until the larvae had attained the umboned stage. Failures of the earlier cultures were attributed to lack of availability of the pro- pier algal food in June, and malfunction of temperature control of the seawater system during a period of unusally hot weather in July. Morphometric Observations on Larvae from In- duced Spawnings Comparison of shell length and width measurements in M. arenaria (Fig. 1) with Hiatella sp. (Fig. 2) showed that length-width relationships 160^ 240- 220- 200- / ■ >i ■ 180- , ,< Mya arenarta 160- Y MO- •x» 120- 100 - ,^ 80- 4.1 1* .• " 50 1 1 r- — 1 r 1 1 1 1 1 1 1 1 >" 8 0 100 120 140 160 180 200 220 240 260 280 FIG. 1. Length vs. width relationship in Mya arenaria larvae. DISTINGUISHING LARVAE IN PLANKTON SAMPLES 45 were virtually identical (Fig. 3). In both species larvae were from 80 to 96fim long when the first larval shell (D-shaped or "Straight-hinge") was fully formed. Mean hinge length in straight-hinge larvae was significantly shorter in M. arenaria (P<.01) but 260- 240- 220- 200- / • y • /- • y 180- 160- V' Hiatella sp. 140- •• • 120- _ cnrn uDO^d" (MICRONS) o . — m«^Ln r-oocTi. — ooroLO'^cocn FIG. 4. Frequency distribution of hinge lengths o/Mya arenaria and Hiatella sp. straight-hinge larvae. 46 N. B. SAVAGE AND R. GOLDBERG Hiatella sp. 96X80 128 X 108 219 X 197 245 X 220 295 X 265 Mya arenaria iirvrVf-^ \ /^~^""-% \ 106 X 89 128 X 112 154 X 133 185 X 170 240 X 210 310 X 280 FIG. 5. Photomicrographs of Mya arenaria and Hiatella sp. larvae. Length and height measurements given in micrometers. Anterior end of each larva is to the left. DISTINGUISHING LARVAE IN PLANKTON SAMPLES 47 iS^mP'Ps X \ / ,r''-\\ % .J B. FIG. 6. Photomicrographs of 2 to 3 day old straight hinge larvae approximately 95-105 mm long. A. Mya arenaria B. Hiatelia sp. 48 N. B. SAVAGE AND R. GOLDBERG gave straight hinge Hiatella sp. larvae a "ship's bow" appearance, unlike that of M. arenaria lar- vae (Figs. 5 and 6). With subsequent growth and development, the straight hinge line in both species appeared to lengthen little if at all. Instead, the valves of the shell lengthened and broadened only from the hinge, giving rise to sloping "shoulders ' (Fig. 5). When the larvae were approximately llSpim long, umbones were first observed on each valve, immediately below the straight hinge. As the um- bones grew dorsally, the profile of the straight hinge gradually became more and more obscured. Beyond a total length of 125nm, it was usually difficult to obtain accurate measurements of hinge length in either species. At a length of approx- imately ISSfim, the sides of the umbones seemed to merge with the shoulders of the valve to present a profile with a continuous slope from umbone f)eak to the anterior and posterior margins (Fig. 5). In this report, larvae in which umbones break up the straight hinge profile, but are not yet con- tinuous with valve margins are said to be in "tran- sition" between the straight hinge and umboned stages of development. Both straight hinge and transition phases of development were of relative- ly short duration, each lasting only a few days (Tables 1 and 2). The umboned stage was the longest of the planktonic larval stages, lasting at least two weeks in the case of the M. arenaria culture begun on 31 July, and much longer in the case of the Hiatella sp. culture started on 24 July and kept at 6 to 13C under refrigerated conditions. As Figure 5 suggests, developmental stages of \ v.'>* M. arenaria and Hiatella sp., other than early stright hinge (Fig. 6), were observed to be prac- tically indistinguishable in terms of shape or size characteristics. This situation continued almost until M. arenaria approached metamorphosis (i.e., when swimming function of velum was lost). Accompanying metamorphosis in M. arenaria were a number of conspicuous morphological changes, including: (1) anterior tilting of the um- bone, (2) prominent display of gill apparatus, and (3) darkening of the shell (possibly due to thicken- ing) (Fig. 7). Mya arenaria larvae reared from eggs in the laboratory, were observed to undergo metamor- phosis at a shell length as short as ISS^m, although 250 to 270fim was the more usual size t V B. FIG. 7. Photomicrograph of Mya arenaria post larva approximately 700 mm long. c. FIG. 8. Photomicrographs of Hiatella sp. taken from plankton samples. A. Pediveligers approx- imately 360 mm long. B. Post larva approximately 450 mm long showing early development of spines. C. Spineless Post larva approximately 530 mm long. DISTINGUISHING LARVAE IN PLANKTON SAMPLES 49 o O Q O -4 < s < 2 w < < UJ Uj -J u o I CD < z U.O ^ t: w a D, ex a. a D. B :S t^ H z < Oi QJ 4; cu o^ a> uo CO Ul U~) LO in IN t^ t^ \>. o o r- 1 1— < ■— * '~' fo u ^ UJ H ^^ ^ V -^ V ri F-; ^ ^ -' [^ V V c- & o ^__, t-H vO V V Ol OF SPECIE 17 ' SEP ^ ^ IN It UJ ci "^ gl U S -^ S^ O- lO (N ^ o T-* l-H (N sO V < X H H ID X) o 00 "^ i-H ^o ;i S§ o£3 ;i; 00 o O DC <^ > ^ ^ Z „ i^. m oo o TT oo rri ^ 00 UJ rH ^0 S < Oh- c :7 in UJ ^ CD ■* o t— 1 o (N O m UJ ^ o -rr O l/l "~* t-H UJ 00 00 oo sO CO ^ ^ o o 00 00 00 ^. o f5 D a 00 (N o - < rg 3. 3, X 3. 3. 3- o o o O o 9 to u. Z rv) l>. a- sO o in ^ 3. 00 rH ~6 -d -d -d -d -d OJ OJ Qj a* 41 0^ UJ , J S < o -■ ^ U C c C c c c o o o o o o Xi JU _D JH Xl X 1 s E 6 £ E E E a 3 D 3 3 3 D ^ e S z r O T3 i ALCULA EAN DEN OfMYA FIELD (m -c 2 o O O O o O 00 oj i: i rs] m sO o- Tf o 00 in ^ 2 Z o O D P 00 < in 00 00 00 a. a. DATE FIEL OLLEC < lO < < sO LO in 1 ^ 50 N. B. SAVAGE AND R. GOLDBERG range at which metamorphosis occurred. Further- more, M. arenaria larvae up to SlO/^m in length were occasionally observed still swimming freely using the velum. Hiatella sp. larvae, obtained from spawning adults on 24 July, showed no sign of undergoing metamorphosis or losing ability to swim during the period 5 to 30 September when up to 50 individuals, between 240 and 340Mm shell length, were under observation. Culture of Larvae from Plankton Fifty-nine Hiatella sp. larvae were isolated from plankton samples on 2 July 1975. These were at a more advanced stage of development (Fig. 8A) than were the 68 day old laboratory spawned Hiatella sp. that survived from 24 July to 30 September. The more advanced umboned larvae from the plankton were easily recognizable among bivalve veligers of a variety of species due to both a distinctive shape and coloration. The largest planktonic individuals (approaching 400Mm in length) displayed a streak of pink apparently in the mantle tissue, paralleling the ventral shell margin. Two of the individuals isolated from the plankton on 2 July survived until 14 July, by which time they had developed shell spines (Fig. SB) considered to be diagnostic of H. arctica (Ab- bott, 1974). Isolation of M. arenaria from plankton samples was simplified by familiarity gained from rearing larvae from known parents, and also by sequen- tial occurrence of natural spawning during the summer. Growth and maturation of isolated lar- vae into what were unquestionably soft shell dams (Table 1) showed that misidentification was normally a minor problem. There was only one serious exception, which was the 2 September isolation. This was carried out after M. arenaria larval abundance had declined from a seasonal peak and when larvae of the horse mussel. Modiolus modiolus, had begun to dominate the local bivalve larvae population. A subsequent refinement in isolation technique, entailing use of a more finely drawn micropipet to extract isolated M. arenaria from samples, resulted in a marked improvement over the "purity" of isolate cultures achieved in the midst of peak M. arenaria abun- dance. In retrospect, initial good results achieved using the cruder isolation technique appeared to have relied heavily on the naturally large propor- TABLE 2. COMP/^/?/SON OF SHELL LENGTH OBSERVATIONS IN LARVAL MYA ARENARIA WITH OBSERVATIONS OF PREVIOUS AUTHORS SOURCE SMALLEST LARVAE OBSERVED (Mm) SMALLEST METAMORPHOSED LARVAE (Mm) LARGEST FREE SWIMMING LARVAE (Mm) Laboratory spawning (1975) 78 235 310 Plankton (1975) ~80* 240 330 Loosanoff and Davis (1963) 86 165 228 Sullivan (1948) 105 250 Jorgensen (1946) 82 200 320 Yoshida (1938) 240 300 Stafford (1912) 76 Few initial measurements taken DISTINGUISHING LARVAE IN PLANKTON SAMPLES 51 tional representation of M. arenaria in the August collections. In all instances, M. arenaria isolate cultures were maintained until the majority of individuals in each culture had undergone metamorphosis. Appearances of metamorphosed M. arenaria sup- ported earlier evaluations of identity (Table 1). As with the larvae reared from known parents, the smallest plankton-obtained larvae observed to set- tle and undergo metamorphosis were approx- imately 240^im long. Occasionally, individuals initially between 280 and 330fjm long were isolated from plankton samples. Many of these large larvae set as post larvae "spat" after two or three days in culture. Rarely were any on the non-M. arenaria larvae in the plankton isolates determined or suspected to be Hiatella sp. Easily recognized late umboned Hiatella sp. were infrequent in plankton samples in August or early September, whereas they were common in mid-June and early July samples. Iden- tities of the contaminant species were not confirm- ed other than by comparing present observations with photographs by Sullivan (1948) and Chanley and Andrews (1971). From these comparisons Spisula solidissima, Maconia sp. and Cerastoderma pinnulatum ap- pear to be among species reared with M. arenaria and could conceivably be mistaken for early-stage M. arenaria larvae. Another tentatively identified group including: Mytilus edulis, Modiolus modiolus, Placopecten magellanicus, Ensis direc- tus, Siliqua costata and Gemma gemma (post lar- vae brought up into the plankton by water cur- rents) have shapes and accompanying character differences that could not be easily mistaken for M. arenaria and were most likely inadvertantly in- troduced into culture as contaminants. DISCUSSION With regard to distinguishing larvae of M. arenaria from Hiatella sp., the present study deter- mined that identification characters, useful in the examination of large numbers of bivalve larvae from plankton, were lacking through the larval life stages between late straight hinge (shell length of approximately 125f.im) and late umbone (shell length of approximately 180-200fim). Early straight hinge Hiatella sp. were distinctive because of the "ship's bow" appearance of the ends of the hinge line. The straight hinge stage appeared to be very brief in the case of laboratory reared larvae: 3 to 8 days forM. arenaria, approximately 11 days for Hiatella sp. Late umbone Hiatella sp. larvae (shell length greater than lAO^im) were distinctive in color and shape from all other bivalve larvae in plankton samples. Easily recognized, late planktonic stage Hiatella sp. larvae were rare in plankton samples taken in August, 1975, suggesting that misidentification of Hiatella as M. arenaria would be negligible during that period. This was subsequently confirmed by rearing larvae, initially identified as M. arenaria, to metamorphosis (spat). In the Hampton Beach area, the abundance of Hiatella is low in late sum- mer when M. arenaria is most abundant. Since spawning sequences were temporarally distant, discrimination of M. arenaria larvae (smaller than 240/im) from larvae of other species, particularly Hiatella sp. was aided as much by differences in relative abundance as by the investigators' ability to distinguish morphological differences. With live plankton samples, coloration and ar- rangement of pigment was used to aid in the segregation of M. arenaria larvae from those of other bivalves. Earlier authors (Jorgensen, 1946; Sullivan, 1948; and Loosanoff and Davis, 1963) proposed the use of pigment characteristics for identification of bivalve larvae from plankton samples. Although there was disagreement as to which were diagnostic, there was agreement that these characteristics become more useful as the larvae mature; straight hinge larvae generally lack pigment. Color, in particular, was observed to vary from culture to culture, and larvae obtained from plankton varied in color during the time they were in culture. On the other hand, M. arenaria larvae freshly obtained from plankton, consistently displayed deep brown coloration of the digestive gland as described by Sullivan (1948). This feature was conspicuous against a background of the nearly colorless body of early umboned larvae, and was increasingly accompanied by brown- black or black markings of the mantle and/or shell as the larvae grew older. In old M. arenaria larvae broken lines of black pigment paralleling the ven- tral shell margin (an identifying character propos- ed by Loosanoff and Davis, 1963) persisted even after prolonged culture and, therefore, might be 52 N. B. SAVAGE AND R. GOLDBERG considered a dependable characteristic. However, Hiatella larvae which had not been studied by Loosanoff and Davis (1963) also display a similar characteristic. Our observations on size of M. arenaria at onset of metamorphosis support observations by Yoshida (1938), Jorgensen (1946), Sullivan (1948) rather than those of Loosanoff and Davis (1963). Table 2 summarizes morphometric findings of authors cited above, as well as those of Stafford (1912) and compares their observations, with ours. Our rearing temperatures (16-19C) were lower than those used by Loosanoff and Davis (1963) (19-24C). If earlier authors also worked at cooler temperatures than Loosanoff and Davis (1963), then the larger size at metamorphosis could be explained by cold induced delay of maturation. The Hiatella larvae we reared appeared to be identical with those of Saxicava ( = Hiatella) arc- tica depicted by Sullivan (1948), but do not fit descriptions by certain European authors (Odhner, 1914; Lebour, 1938; Jorgensen, 1946; and Rees, 1950). Of the two likely European con- geners: H. arctica and H. striata ( = H. gallicana =H. rugosa), H. arctica appeared to be less like the North American Hiatella larvae described here and in Sullivan (1948). This larval unlikeness may have led Rees (1950) to declare that Sullivan (1948) had erred in identifying her larvae as H. arctica. Nevertheless, both Sullivan's photo- graphic plates and present observations show development of shell spines on post larvae reared from plankton collections. According to Yonge (1971) spines on the shell were "formerly" regard- ed as diagnostic of H. arctica. Abbott (1974) uses this characteristic to distinguish post larvae of H. arctica and H. striata. Hunter (1949) stated that these spines could easily be lost by the boring or "nestling" habit of the settled juveniles. Abbott (1974) also maintains (as originally stated by Lebour, 1938) that H. striata spawns in winter, while H. arctica spawns in summer. This suggests that the species we have dealt with is H. arctica. However, Abbott also describes eggs of H. arctica as red, and the eggs of H. striata as very light in color. Our observations of numerous spawnings showed the egg color of Hiatella sp. to be white, or at most, ivory in color, with no red- dish hue. Furthermore, we observed a specimen of the spineless form of post larval Hiatella in a live plankton sample taken on 26 August (Fig. 8C). We agree with Yonge (1971) that taxonomic relationships of H. arctica and H. striata ( = H. gallicana) are "unusually obscure". Perhaps the discovery that the Hiatella we have dealt with can be spawned with relative ease will stimulate fur- ther investigation of taxonomic questions concer- ning the genus Hiatella from the western North Atlantic coast. While the task of readily distinguishing M. arenaria from Hiatella is difficult without disarti- culating the valves and examining the shell ultra- structure, it has been demonstrated that correct identification is likely, given adequate experience. The risk of misidentification becomes larger at the life stage where the straight-hinge line is obscured by developing umbones (at approximately 125fim) and decreases after the umbones are fully continuous with the shoulders of the valve margins. For very early straight hinge larvae and for larvae with well developed umbones, the iden- tification error was found to be generally less than 20%. ACKNOWLEDGEMENTS This study was supported by funds from Public Service Company of New Hampshire. Grateful acknowledgement is also given to Dr. R. Ukeles of tfie National Marine Fisheries Services, Milford, Connecticut, for providing the initial algal cultures. LITERATURE CITED Abbott, R. T. 1974. American Seashells. 2nd ed. Van Nostrand Reinhold. New York. 663 pp. Chanley, P.E. and J. D. Andrews. 1971. Aids for identification of bivalve larvae of Virginia. Malacologia. 11(1):45-119. Guillard, R. R. L. and J. H. Ryther. 1962. Studies on marine planktonic diatoms I. Cyclotella nana Hustedt and Detonula conferuacea (Cleve) Gran. Con. ]. Microbiology. 8:229239. Hunter, W.R. 1949. The structure and behavior of Hiatella gallicana (Lamarck) and H. arctica (L.) with special reference to the boring habit. Proc. R. Soc. Edinb. B. 72:271-289. Jorgensen, C.B. 1946. Reproduction and larval development of Danish marine bottom in- DISTINGUISHING LARVAE IN PLANKTON SAMPLES 53 vertebrates. 9. Lamellibranchia. Medd. Kimm. Havundersog. Kbh. Ser. (d): Plankton, 4:277311. Lebour, M. V. 1938. Notes on the breeding of some lamellibranchs from Plymough and their larvae. ]. Mar. Biol. Assoc. U.K. 23:119-144. Loosanoff, V.L., and Davis, N.D. 1963. Rearing of bivalve mollusks. p. 1-136. In: Advances in Marine Biology. Ed. F.S. Russell. Acad. Press, London. Loosanoff, V.L., H.C. Davis and P.E. Chanley. 1966. Dimensions and shapes of larvae of some marine bivalve mollusks. Malacologia (2):351-435. Odhner, N.H. 1914. Notizen uber die Fauna der Adria bei Rovigno. Beitrage zur Kenntnis der marinen Mo-luskenfauna von Rovigno in Istrien. Zoo. Anz. 44(4):156-170. Rees, C.B. 1950. The identification and classifica- tion of mannellibranch larvae. Hull Bull. Mar. Ecol.,3(19):73-104. Snedecor, G.W. and W.G. Cochran. 1967. Statistical Methods. 6th Ed. The Iowa State University Press. 593 pp. Stafford, J. 1912. On the recognition of bivalve larvae in plankton collections. Contr. Canad. Biol., 1905-1910:221-242. Stickney, A. P. 1964. Salinity, temperature and food requirements of soft shell clam larvae in laboratory culture. Ecology45(2):283-291. Sullivan, C.M. 1948. Bivalve larvae of Malpaque Bay, PEI (Prince Edward Island). Fish. Res. Bd. Can. Bull. 77:1-36. Yonge, C.M. 1971. On functional morphology and adaptive radiation in the bivalve superfami- ly Saxicavacea Hiatella ( == Saxicava) , Sax- icavella, Panomya, Panope, Cyrtodaria). Malacologia ll(l):l-44. Yoshida, H. 1938. Notes on the veligers and the young shells of Mya arenaria japonica. Venus 8(1):13-21. Proceedings of the National Shellfisheries Association Volume 66 — 1976 'p--' --.: OBSERVATIONS OF CRASS05TREA VIRGINICA CULTURED IN THE HEATED EFFLUENT AND DISCHARGED RADIONUCLIDES OF A NUCLEAR POWER REACTOR A. H. Price II, C. T. Hess, arxd C. W. Smith UNIVERSITY OF MAINE ORONO, MAINE ABSTRACT American oysters (Crassostrea virginicaj were rafted for 26 months at four sites in the effluent waters near Maine Yankee Nuclear Power Reactor in Montsweag Bay and at a control site in the adjacent Damariscotta River. In an evaluation of the thermal effluent for aquaculture, comparisons are made among the sites of the effects of heated effluent on oyster growth arid condition, and the uptake and retention of gamma-ray emitting radionuclides. Growth and uptake of radionuclides were observed to be accelerated at the warmer water sites. Observed variations in concentrations of gamma-ray emitting radionuclides in the biological component of this study are compared with a pulse driven relaxator model and an existing concentration factor model. Results show that although the concentra- tion factor model is adequate for simple laboratory studies, the pulse driven relaxator model is necessary to describe both the amplitude and time variation observed in this field study. Both experimental results and calculations for ^^Co and ^"Mn are presented. INTRODUCTION Our work since 1973 has been directed toward evaluating the potential use of the thermal effluent and waters surrounding the Maine Yankee Atomic Power Station located at Bailey Point on Mont- sweag Bay, Wiscasset, Maine, for the culture of the American oyster, Crassostrea virginica. In this study we have examined two major aspects of the potential use of thermal effluents in aquaculture. First, growth and quality of the oysters, and se- cond, retention in the oysters of gamma-ray emit- ting radionuclides released into the environment by the power plant. In the natural marine environment of Maine it appears possible to produce marketable oysters in a period of two to three years by the use of raft culture techniques. Favorable conditions for growth (temperature and algal food supply) have been found to exist between June and November and growth rates of oysters cultured in Maine ap- pear to be equal or superior to those measured in traditional growing areas such as the Chesapeake Bay and the Gulf of Mexico (Packie, Hidu and Richmond, in manuscript). There are about six months (June to November) of optimal food and temperatures for oyster growth; the spring months (February to May) are not favorable for oyster performance. The latter months are limited by the low ambient temperature (below 8°C) which prevents oysters from taking advantage of the adequate food supply which is present in many areas (Galtsoff, 1964). The marine environment as found along the cost of Maine is characterized by broad seasonal temperature fluctuations. The use of thermal ef- fluents in aquaculture could prove advantageous 54 OYSTERS IN EFFLUENT OF NUCLEAR POWER REACTOR 55 here by providing the temperature elevations above ambient necessary to allow oysters to utilize the existing algal food supply in the early spring. The growing season would thereby be ex- tended and the time to market would be reduced. The increasing demand for electricity by our civilization and the consequent construction of ad- ditional generating facilities in coastal areas will dramatically increase the number of thermal releases available for application in marine aquaculture systems. If present governmental planning is implemented a large part of this future generating capacity possibly will be nuclear. Nuclear generating facilities are of particular in- terest because of the use of large volumes of water for cooling of these plants, is generally compatible with the biological requirements for accelerated gametogenesis and growth rates, and the exten- sion of the growing season of many commercially valuable marine species. The use of heated effluent from power plants for the culture of marine organisms has been discuss- ed at length by many authors: Nash (1968), Burns (1969), Coutant (1970), Mather and Stewart (1970), Strawn (1970), Yarosh (1972), Huguenin and Ryther (1974), and others. Most of the pro- jects utilizing thermal effluents in aquaculture have been of a commercial nature and the results of these efforts, because of their proprietary nature, are not readily available in the literature (Huguenin and Ryther, 1974). We have determined annual physiological cycles of glycogen storage, percent total solids, shell growth, and the uptake and depuration of gamma-ray emitting radionuclides in American oysters (C. virginica) cultured in the effluent of the Maine Yankee nuclear power reactor. Three other points in Montsweag Bay and a control site in the Damariscotta River have also been examined. Relevant studies at other locations have been undertaken with oysters by Jeffries and Preston (1969), Seymour (1966), Naidu and Seymour (1969), Wolfe (1970), Lowman, Rice and Richards (1971). Studies of accumulation and depuration have been undertaken for numerous radionuclides in many marine organisms (Lowman, et al., 1971) **Co in the mussel Mytilus edulis (Shimizu, et al., 1971), "'Cs and ""Co in the marine clam M\/a arenaria (Harrison, 1973) and '"Cs and ""Co in Crassostrea gigas (Cranmore and Harrison, 1975). We have quantitatively measured the uptake and depuration of several radionuclides in the American oyster (C. virginica). As a result, we propose a mathematical model of the variation of gamma-ray emitting radionuclides in live oysters. TTie model considers the dynamics of both the biological and physical processes which control the aquacultural potential in the estuarine system studied. METHODS Site Locations The Maine Yankee Atomic Power Company at Wiscasset, Maine, is powered by a pressurized water reactor and is rated at 855 MW. The plant is cooled by passing up to 960 cubic feet of water per second from Montsweag Bay over its condensers and discharging the warmed water into Bailey Cove, which empties into Montsweag Bay. Oyster tray stations were located in the intake channel (S-1), directly in the outflow effluent (S-2), above the effluent point in Bailey Cove (S-3) and below the effluent point of Long Ledge in Montsweag Bay (S-4) (Fig. 1). The control site was located in FIG. 1. Map of Montsweag Bay showing location of tray stations. 56 A. H. PRICE II, C. T. HESS, C. W. SMITH an adjacent estuary at the marine laboratory on the Damariscotta River. Native American oysters, C. virginica, used in this study were obtained from a bed in the Piscata- qua River. One hundred fifty oysters to be ex- amined for glycogen content and percent total solids were distributed between two trays at each of the experimental sites and the control site. Ad- ditionally, 24 oysters were placed in a separate compartment of one tray at each site to be measured monthly in a longitudinal study of the accumulation of radionuclides. Environment Environmental factors which have been shown to influence the growth of shellfish (Galtsoff, 1964), were monitored. A Beckman field salinometer (Model RS5-3) was used to measure salinity and temperature every two weeks during high water at all stations. For one year, water samples were taken every other week at high water at the tray stations to evaluate the food available for the oysters. These samples were used in the determination of chlorophylls and par- ticulate oxidizable carbon (Strickland and Par- sons, 1965)*. Biological To determine the growth and quality of the oysters used in the study, a monthly random sam- ple of 12 oysters was collected from the field sites for one year. They were held in the laboratory in water collected at the field sites. Fouling organisms were removed. Each oyster was blotted dry, weighed, and measured. All measurements (height, length, and width) were of the maximum dimensions of any parameter. New shell growth was measured on the right and left valves at the location of maximum growth. The larger value was used in calculating the average shell growth of the 12 oysters at a given station. The oyster from each station which most nearly approached the average was selected from each group and photographed. * Additional information on possible available food was taken from Maine Yankee Atomic Power Company Semi- Annual Environmental Surveillance Reports #2-6, (1973-1975) McAlice, "Net phytoplankton and Microzooplankton" and Crippen & Lindsay, "Entrainment Studies". To determine the condition of the oyster meats the oysters were shucked taking care not to pierce the meat. The meats were allowed to drain for one minute on a plastic mesh and then weighed. All 12 oyster meats from a station were then homogeniz- ed. Glycogen was extracted from the homogenized meats according to the method of Burklew (1971). This method employs the digestion of 5 gram ali- quots of the homogenized oyster tissue in hot NaOH (30%), followed by the precipitation of glycogen with ethanol (95%). The precipitate is hydrolized with concentrated HCL, and the glucose present is determined by the addition of anthrone dissolved in concentrated H2SO4. The color change is measured spectrophotometrically and the milligrams of glucose present calculated from a standard curve. The percent total solids of oyster tissue has also been widely used to determine oyster condition (Shaw, Tubiash and Barker, 1967). In this study percent solids are calculated from an average of the dry weight of three 5-gram aliquots of the homogenate using the formula: Percent solids dry weights of meats wet weights of meats X 100 Radionuclides In order to detect the possible accumulation of gamma-ray emitting radionuclides, the groups of 24 oysters were taken from their respective sta- tions, scrubbed with a stiff brush to remove foul- ing organisms, and transported to the en- vironmental radioactivity laboratory at the University of Maine Department of Physics. The outflow station (S-2) was sampled every month. The control (SC), intake (S-1), Bailey Cove (S-3) and Long Ledge (S-4) stations were sampled every other month. Approximately 1 kilogram of live oysters (selected at random) from each of the groups of 24 was counted for 5000 seconds. The resulting data was computer processed (IBM 360/370) using the Compton continuum subtraction method (Covall, 1959). After counting, the oysters were returned to their original locations to be measured in the following months. The gamma-ray measurements were carried out using a Ge(Li) detector (Ortec) with 2400 lb low background lead shield. The pulses were amplified OYSTERS IN EFFLUENT OF NUCLEAR POWER REACTOR 57 with a spectroscopy amplifier (Ortec 452) and the bias was provided by a high voltage supply (Ortec 459). The pulses were processed by a multichannel analyzer (Northern Scientific NS-700) with 2048 memory channels and outputted using a teletype to produce a list and a punched tape. The numbers of counts determined were converted from counts/minute into disintegrations per second by using the efficiency determination for the same geometry. Both branching ratios and the variation of efficiency with energy were taken into account. From disintegrations per second, the number of picocuries/gram was determined for each of the gamma-ray peaks which exceeded a statistical criterion. Picocuries/gram of those radionuclides in our library of branching ratios were computed automatically, and new or unidentified peaks were processed by hand calculations. The efficiency versus energy curve was deter- mined by placing several standard sources (En- vironmental Protection Agency Analytic Quality Control Laboratory, Las Vegas, Nevada) in a solution of demineralized distilled water. The solution was placed in a 1.0 liter Nalgene cylin- drical bottle (which was our standard geometry). and measured. The peaks from the standard sources were analyzed in the same way as the peaks from the unknown. The graphical analysis of the efficiency versus energy was plotted on log- log paper and tested for linearity, and consistency. The results of these periodic calibrations were used to update the computer program. New branching ratios were entered as required. A typical spectrum of gamma-rays is shown in Hgure 2. Model Theory Constant concentration theories have been sug- gested in the laboratory studies by Polycarpov (1960), Ruzic (1972), and Davis and Foster (1972). In such theories, the radionuclide concentration in the oysters, C„, is related to the radionuclide con- centration in the sea water C„,, by concentration factor K. C„ = K C. This factor K becomes larger with time until it reaches an equilibrium value, if the concentration C. may be found by dividing the released 160 (n z> o o 120- O 80 llJ CO 40- OYSTERS OUTFLOW S-2 MAY I, 1975 TIME = 5000 SEC. 58 Co- WJViwvy^^ 40, 54 ^=.. 2'--. Mn ^WS-As, 800 T- T 200 400 600 DATA CHANNEL NUMBER PIG. 2. Typical Gamma-ray Spectrum: Data channel number on the horizontal axis (energy in keV equals data channel number X 2) and number of counts on the vertical axis (number of counts/ 5000 sec). 58 A. H. PRICE II, C. T. HESS, C. W. SMITH JAN JULY MONTHS (1973-1975) FIG. 3. Comparison of experimental results for uptake of ^^Co at outflow, 5-2, (-•-•-•-), and predicted values based ori the specific concentra- tion theory (— o— o— o--). radioisotope in curies, f,, by the volume of water used for the release V„,. C. = Using this equation in a dynamic situation, as in the case of reactor releases, will give values of K which are less than the equilibrium value for K as found in a laboratory situation. Thus, calculations using laboratory values of K in the case of reactor releases inaccurately estimates the radionuclide concentration (Fig. 3). To develop a dynamic model of variations in the uptake and depuration of radionuclides by the oysters, the release rates of radionuclides by the reactor was used as the driving source of a multimode pulsed relaxator system. The resulting differential equation may be solved by integration to give exact solutions if appropriate simplifying assumptions are made. These assumptions are that the reactor releases monthly by injecting the nuclides into the estuary in a short time (several hours). The nuclides are then accumulated by the oysters, and are slowly reduced by radioactive decay and by biological cycling, depuration, in the oysters. Initially we assumed that the oysters had constant depuration over the entire year. Later these assumptions were modified to include: a) variation in nuclear reactor plant operations I I JftN JULY MONTHS (1973-1975) FIG. 4. Results of pulsed relaxator theory for ^^Co (.•.•.m.) and ""Co f-o-o-o-j. (shut-downs, plant discharge rate, power output, etc.), b) oyster biological parameters (growth rate, glycogen content), and c) estuarial parameters (temperature, salinity, current velocities, standing crop, etc.). As our understanding has improved we have found that by using this radionuclide up- take model, predictions can be made to establish an optimum release pattern for the reactor in order to minimize oyster uptake of radionuclides. Model The uptake of radionuclides may be described by a first order linear differential equation: dt dN 1) where "jt" is the increase in atoms of a ra- dionuclide, AN is the rate of loss due to radioactive decay, and R(t) is the rate of introduction of ra- dionuclide from an external source (i.e., the nuclear reactor release schedule). Depuration may be included by creating an "effective lambda" (the sum of the radioactive decay constant and a biological decay constant). The solution to equation 1 may be written: N =e-" (e"R(t)dt + ce-" 2) We assume that releases of radionuclides are made in a sequence of m times (ti, t2, ta t,„), and OYSTERS IN EFFLUENT OF NUCLEAR POWER REACTOR 59 the amount of nuclide released is given by a func- tion f(t) which for times greater than or equal to ti, but less than t,, is given by fi (t - ti), and for times greater than or equal to t2, but less than tj, by iz (t - t2), and so on up to times greater than t^. The fraction of the radionuclide which is released by the reactor and is retained by the oysters is given by U, so that for the accumulation N{t) we have: t.+e N{t) =e-"( e"f, ci(t-tl)Udt + •■• +e e o ■'■ (e"f„c)(t-t„)Udt + ce--" t„-,+G Assuming U is a constant ratio for retention at all times, we can construct a table of solutions for the intervals between the release times. 0iV 20 30 2 5 10 TEMPERATURE (°C) FIG. 4. Influence of temperature on elimination o/E. coli in naturally contaminated Pacific oysters. Stip- pled area below sensitivity of assay. 74 D. B. QUAYLE AND F. R. BERNARD 1x1 _l < u (/) o o A = 24 HOURS D = 48 A =OrTT- >r^v:^: 20 25 30 35 SALINITY (7oo) FIG. 5. Influence of salinity on elimination o/E. coli in naturally contaminated Pacific oysters. Stippled area below sensitivity of assay. < o o o a. 10 A= 24 HOURS D = 4 8 A A _L. _L _L _L 500 5 10 20 100 SUSPENDED MATTER ( mq / I ) LOG SCALE FIG. 6. Influence of turbidity on elimination of E. coli in naturally contaminated Pacific oysters. Stippled area below sensitivity of assay. PURIFICATION OF BASKET-HELD OYSTERS IN NATURAL ENVIRONMENT 75 dispersion in the 24-hour points, a greater regularity and slight decrease in the 48-hour points. DISCUSSION It is probable that more time is required to eliminate large numbers of microorganisms, but even grossly contaminated (circa 186,000 £. coli MPN) reached equilibrium with locxl levels in 24 hours. Less definite results were recorded for oysters with an MPN level close to the local. In this situation the cleansing shellfish will also ingest new bacteria while eliminating those in the gut. final results are probably temperature dependent, as filtering activity and ingestion are more readily increased by a temperature rise than is the digestive process (Bernard MS). It is apparent that 24-hour sampling intervals are too long to determine the effect of en- vironmvntal parameters upon bacterial elimina- tion, but it has been adequately established that, under Canadian Pacific conditions, 48 hours are suffg:ient for contaminated oysters to reach equilibrium with their transfer site. Temperature has an effect upon the speed with which material is passed through the digestive tract, and low temperatures (<4C for the Pacific oyster) may inhibit the digestive enzymes and phagocytes of the stomach and midgut (Bernard MS), but these effects would only be apparent in a shorter timeframe. Pacific oysters are able to func- tion in a wide range of salinities, but require a period of adaptation. The marginally better elimination at 30%<3 and 35 %p may be attributed to the fact that the subjects of this experiment were collected from 32 %o water. The amount of suspended matter in the water appears to have little effect upon elimination, and a modest suspended load may indeed help purification by promoting passage of contents through the digestive tract. It is probable that our results are applicable to other species of shellfish as they closely agree with Arcisz and Kelly (1955) for Mya arenaria, Dodgson (1928) forMytilus edulis, and Heffernan and Cabelli (1970) iorMercenaria mercenaria. It is likely that acceptable cleansing will occur under a wide range of climatic conditions, and pathogens will be adequately eliminated to ensure a safe pro- duct. LITERATURE CITED Allen, E. S. 1932. Estimation of bacterial densities by means of multiple fermentation tube results. Iowa State College J. Sci.6: 251-262. Arcisz, W. and C. B. Kelly. 1955. Self-purification of the soft clam, Mya arenaria. U.S. Pub. Health Rep. 70: 605-614. Dodgson, R. W. 1928. Report on mussel purifica- tion. Min. Agr. Fish., London. (Ser. 2) 10: 498 P- Halvorson, H. and N. R. Ziegler. 1933. Quan- titative Bacteriology. Burgess Pub. Co. 279 p. Heffernan, W. P. and V. J. Cabelli. 1970. Elimina- tion of bacteria by the Northern quahog (Mercenaria mercenaria): environmental parameters significant to the process. ]. Fish. Res. Board Can. 27: 1569-1577. Hoff, J. C. and R. C. Becker. 1969. The accumula- tion and elimination of crude and clarified poliovirus suspensions by shellfish. American J. Epidem. 90: 53-61. McCrady, M. H. 1915. The numerical interpreta- tion of fermentation tube results. J. Infect. Dis. 17: 183-212. Quayle, D. B. and F. R. Bernard. 1968. Oyster Purification Study. 1. Incidence and Enumera- tion of Coliform Bacteria in the Pacific Oyster. Fish. Res. Board Can. MS Rep. 973. 21 p. Proceedings of the National Shellfisheries Association Volume 66 — 1976 7^^'^^ A MATHEMATICAL APPROACH TO DEPURATION Bruce]. Neilson^ VIRGINIA INSTITUTE OF MARINE SCIENCE GLOUCESTER POINT, VIRGINIA ABSTRACT Two equations can be written to describe the change in bacterial concentrations in the shellfish and the water in depuration units. Analytical solutions of these equations for special conditions and numerical solutions for general conditions indicate that there will always be an initial, rapid decrease in bacteria in the oysters during depuration. With suitable flow rates and loading rates, the die off will be exponential and several orders of magnitude reduction can be achieved within 72 hours. Both very low flow rates and very low loading rates increase the residence time of water in the tank, and therefore depuration will occur slowly after about 24 hours. In addition, the bacterial levels at 72 hours may be quite high for the case of very low flow rates. Further im- provements and verification of the model are desired, but use of the model can aid in the design and operation of depuration plants now. INTRODUCTION Although researchers over the years have in- vestigated and described the various biological functions and environmental factors which are im- portant to the process of depuration, to the author's knowledge, very little work has been done to incorporate these findings into a unified theory of depuratioi.. As a first step in that pro- cess, the present study attempts to describe depuration from a phenomenological point of view. The author is aware that this approach neglects many of the physiological aspects of the process and that the model system under con- sideration is a highly simplified version of the real world situation. Furthermore, no attempt was made to duplicate the results of depuration ex- periments. Rather the purpose of this study is to determine whether or not the simple model can simulate depuration in general. It is the author's opinion that the mathematical analysis can lead to a better understanding of the interactions which occur and provide a means of evaluating the VIMS Contribution No. 748 hydraulic design of flow systems for depuration units. It is hoped that future work can expand the model to incorporate more parameters and to make the model more realistic. But the first need is to demonstrate that the model is a sound one. EQUATIONS OF DEPURATION Two equations are needed to describe the total depuration process: one for the shellfish and one for the water in the tank. The first equation tells how the number of bacteria in a shellfish varies with time, and the second equation accounts for changes in the concentration of bacteria in the water. These equations are: dE/dt = -kE+pfc (1) dc/dt = (+kE -pfc-qc)N/V (2) The first equation says that the time rate of change of E, the number of bacteria per shellfish, is equal to a fixed percentage, k, of the bacteria present (negative because they are excreted) and a fixed percentage, f, of the bacteria in the water, c, which is pumped through the shellfish at the volumetric rate, p. The second equation says that 76 A MATHEMATICAL APPROACH TO DEPURATION TJ the time rate of change of the concentration of bacteria in the water is equal to those excreted by the shellfish minus those filtered out by the shellfish and those lost from the system. Note that it is assumed that the incoming water has been completely disinfected so there is no term for in- coming bacteria. Also since q is the flow of water through the tank per shellfish, this term, as well as the other two, must be multiplied by N, the number of shellfish in the tank, and divided by V, the volume of water in the tank to put everything in terms of concentration. .All terms in the equa- tions, brief descriptions, units and typical values are listed in Table 1. A better understanding of these equations can be gained if specific cases are considered. For this study, the depuration of fecal coliforms by the eastern oysters during summer conditions will be used. Assumptions made to relate numbers and volumes of oysters, and other factors are given in Table 2. With a few exceptions, these values have been taken from the Public Health Service publication entitled "Depuration Plant Design" (Furfari, 1966). It should be noted that the equa- tions are written for "ideal" oysters whose behavior matches the average values of a set of real oysters. For the purposes of this study, this idealization does not limit the usefulness of the equations. TABLE 1. Symbols Used in Depuration Equations TABLE 2: Etivironmental and Behavioral Constants Term Description Unit E number of coliforms per shellfish MPN oyster c number of coliforms per MPN volume of water (concentration) 100 ml t k time decay rate hour 1/hour f filtering factor 1 q flow of water through tank per shell fish (specific flow rate) liters hour-oyster N number of shellfish in tank oysters V volume of water in tank liters t,„ residence time of water hour weighs 1 bushel 1 oyster Temperature = Pumping rate = Specific flow rate = Decay rate = Filtering factor = 225 oysters 25 grams 20-25 °C 10 liter/hour 1 gallon 1 liter minute-bushel hour-oyster 0.17/hour 0.005 During the initial stages of depuration, the bacterial concentration in the water will be zero or very small. This situation could also arise if the flow around the oyster completely removed all fecal matter. For these cases, equation (1) reduces to dE/dt = -kE for which the solution is E (3) E„e"*'. In other FILTERING FACTOR 1.00 S 10 FIG. 1. Oyster Depuration for Various Filtering Factors. 78 B. J. NEILSON words, when there is no feed back of bacteria to the oyster, the decline in bacterial levels within the oysters will be exponential. Thus one could deter- mine the value of k by conducting experiments in which the ambient concentrations are kept very, very low. For this study, k has been assumed equal to 0.17 per hour, or in other words, every hour 17% of all bacteria in the oyster are voided to the water. This means that concentrations will be reduced to one tenth of the original concentra- tion in 13.5 hours, and to 1 % of the original value in 27 hours. (Fig. 1). When natural waters have a relatively constant bacterial level, an equilibrium is reached such that the number of bacteria excreted by an oyster equals the number ingested from the water it pumps. That is, dE/dt = 0 and k E^ = pfc. or c. k (4) (5) Equation 5 says that the equilibrium concentration factor (E/c) is equal to the product of the pumping rate, p, and the filtering factor, f, divided by the decay rate, k. This relationship appears to be sound physiologically, because studies have shown that when the water temperature increases from IOC to 20C, the pumping rate and the con- centration ratio both increase. (Furfari, 1966). If we assume that this relationship does hold, then equation (5) presents a means of determining the filtering factor, f. If there is an ambient bacterial concentration of 330 MPN/100 ml (or 3300 MPN/liter) then the bacterial concentration in the oyster for summer conditions and Virginia grow- ing areas will be around 4000 MPN/100 grams. (Reference to Hope & Wiley, 1961 in Furfari). If an average oyster weighs 25 grams, then E will be 1000 MPN/oyster. Thus the concentration ratio as defined above is about 0.3 and the filtering factor is 0.005. In other words, the oyster ingests 0.5% of the coliforms in the water which it pumps. Pumping rate has been assumed to be 10 liters per hour. If all factors other than E and C are assumed to be constant with respect to time, then several analytical methods of solution are available. For this study, the coupled equations (1) and (2) were transformed to finite difference form and pro- grammed on a Hewlett Packard 9800 desk top calculator. The time interval for integration was 0.1 hour. Die off curves for several values of the filtering factor are shown in Figure 1. It is in- teresting to note that for the assumed flow rate and biomass to volume ratio, the decay of bacteria in the oyster is always exponential. Also depura- tion occurs, albeit slowly, even if 100% of the bacteria are filtered by the oyster from the water it pumps. For the rest of this study, it will be assum- ed that k =0.17 per hour, p = 10 liters per hour and f =0.005. From a mathematical point of view, the curves shown in Figure 1 appear to be reasonable, but certainly there is need for verifica- tion of these biological coefficients. Equation (2) describes the changes in concentration levels in the water in the depuration tank. It includes the two factors which are amenable to control by the designer and operator of a depuration plant: N/V, the biomass to volume ratio or the loading rate, and q, the specific flow rate. The effect of these factors can be illustrated by an analysis similar to that done for Equation (1). During the initial phase of depuration, E is very large and c is small. Thus the term, kE, is the dominant one and c will increase rapidly. Typical- ly the maximum value for c is attained in the first or second hour of depuration, and it decreases slowly there after. However, E declines rapidly and the kE term quickly becomes so small that it can be neglected during the later stages of depura- tion. Since the specific flow rate, q, is 1 liter per hour and pf is only 0.05 liters/hour. Equation (2) can be approximated by dc/dt Nqc V = (l/t. (6) where t,„ = the mean residence time for a parcel of water flowing through the tank, defined as the quotient of the water volume and the total flow rate, which in turn is equal to the specific flow rate times the number of oysters. t... = V/Q = V/Nq (7) The solution to equation (6) will contain the term e''l',e,- In other words, the rate of change of the concentration in the tank will be a function of the residence time of the water. It should also be noted that if there is no growth A MATHEMATICAL APPROACH TO DEPURATION 79 of bacteria and if there is some finite flow of water through the tank, there will be a continual loss of bacteria from the system. Thus the only equilibrium which can occur is when c =0. The rate of decay for low flow rates and large residence times will be very slow, so that depura- tion will not occur over a practicable period of time. One might criticize the model for not in- cluding a term for the growth of bacteria since fecal pellets and other detritus can provide a suitable medium for growth to occur. However, any growth of bacteria in the tank will slow down and interfere with the depuration process. Thus, the condition of no growth is the one which is desired and operating procedures should be designed to remove the biodeposits at frequent in- tervals. OPERATIONAL CONTROLS The two factors which are directly under the control of a depuration plant operator are the flow of water through the tank and the loading rate. Several model runs were made to examine the behavior of the system to variation in these two LOGO L 1 1 1 [ 1 A P = 10 1] ters/hour 500 - \ f . k = 0 0 oo; 17 /hour V = 1 oyster/liter 200 ^"^-~--____ 100 \ ___0^^1__^ - xV \ _ - >\ N^ - S 50 : \ .^ ' >i - A N, SPECIFIC . y. \ FLOW RATE fl 20 S 10 - \ \ \ {liters/hr/oyster) \0.1 - \\ \ o - \\ V \. : ^ 3 - \ \, \^ - z - N\ \ _ - 10 \\' - 2 ■ L . ^ 1 \ " 12 24 60 72 factors. In Figure 2, the die off curves for a range of specific flow rates are shown. If the specific flow rate is very small, as illustrated by the curve for 0.01 liters/hour/oyster, there is an initial period of rapid decay, followed by an in- termediate transtition period and a final slow decay. In the initial stages the die off will be ex- ponential with the decay rate equal to k. In the final stages the die off will be exponential as well, but the decay rate will be smaller and equal to l/t,„. Since q is small, the residence time is large and the inverse of the residence time will be small too. It is important to note that in addition to the slow die off, the reduction during a 72 hour period is very small. Less than an order of magnitude reduction occurs during this period. For a specific flow rate of 1 liter/hr/oyster, the reduction in decay rate is much less pronounced but still present. The bacterial level is reduced to less than l%of the initial count within 60 hours. As the flow rate is increased further, there are ad- ditional increases in the decay rate. But above 10 liters/ hr/ oyster only marginal increases in depuration rate are achieved for large increases in flow rate. 1 1 \ — p = 10 liters/hour f • 0.005 k = 0.17 /hour q = 1 liter/hour/oyster Time (Hours) Time (Hours) nC. 2. Oyster Depuration for Various Specific Flow Rates. HG. 3. Oyster Depuration for Various Loading Rates. 80 B.J. NEILSON The loading rate shows variations somewhat similar to those for the specific flow rate. The maximum value which can be achieved is a bushel of oysters in a bushel of water or 6.4 oysters/liter of water. Only a modest variation in depuration is seen if the loading rate is reduced to 0.1 oysters/ liter. If the loading rate is reduced by an additional order of magnitude to 0.01 oysters/ liter, the final stage of depuration occurs at a slow rate. For this situation, the bacterial levels are reduced to less than 1% of the original value within 48 hours before the reduction in decay rate takes effect. The two cases of low specific flow rate and low loading rate are related in that each has a long residence time. In each case an initial period of rapid die off will be followed by a period of very slow depuration. With extremely low flow rates, bacterial levels in the water can be high, whereas for low loading rates, a large volume of water is available to dilute the bacteria given off by the oysters. For example, when q= 0.01 liter/hr/oyster and N/V =1 oyster/liter, the water concentration at 24 hours is 65 MPN/100 ml (Fig. 2). But when q = 1 hter/hr/oyster and N/V = 0.01 oyster/liter the bacterial level in the water is only 0.8 MPN/100 ml at 24 hours (Fig. 3). For both cases, the bacteria in the water will be removed from the tank slowly, but the higher levels present with low flow rates will have the ef- fect of maintaining higher levels in the oysters. DISCUSSION The sample runs show several features of the depuration process which have a bearing on the operation of a depuration plant. First, there is an initial rapid decay in bacterial levels no matter what flow rate and loading rate are used. Therefore, one possible means of achieving suitable reductions in coliform counts is to hold the oysters with no through flow. At periodic in- tervals the water should be flushed from the tank and it would be replaced by clean water. Bacterial reductions for several holding times are given in Table 3. All frequencies for removing and renew- ing the water appear to work, so that biological factors should be used to choose the best frequen- cy. For example, the oysters may not begin to pump until a half hour after being immersed, so that a longer interval would be better. Also, some TABLE 3. Reduction in Bacterial Levels With No Flow Through Tank Time Hour 2 Hrs. 3Hrs. 4Hrs. 6 Hrs. 0 1000 1000 1000 1000 1000 12 142 145 152 162 186 24 20 21 23 26 35 36 3 3 3 4 7 means will be needed to maintain dissolved ox- ygen concentrations. Submerged air diffusers ap- pear to be a very efficient and cost-effective method. The model indicates that the oysters can never be packed too densely in the tank. Implicit in the model is the assumption that the water in the tank is mixed rapidly so that the concentration in the water does not vary significantly throughout the tank. Additionally, the removal of fecal matter is very important. Thus the need for a good circula- tion in the tank, rather than considerations of depuration rate, may dictate a limit to the loading rate. But from a mathematical point of view, in- creased loading rates will not hinder the depura- tion. The specific flow rate is much more difficult to characterize. If a high flow rate is used, a great deal of energy will be expended unnecessarily. On the other hand, if a very low flow rate is used, not only will the depuration rate be slow but also the reduction in coliform counts will be insufficient. The best course of action is to run tests in the tank to document the rate of depuration for various flow rates. The model could then be used to deter- mine a specific flow rate that insured the necessary reduction in bacterial levels without wasting energy. In summary, the mathematical approach to depuration can be very fruitful. There is need for input from biologists as to the acceptability of the various assumptions and simplification, the cor- rect values for the biological coefficients and the best means of improving the model. However, even at this stage, the combination of actual depuration runs and mathematical analysis can aid in the design and operation of depuration plants. LITERATURE CITED Furfari, Santo A. 1966. Depuration Plant Design. U. S. Dept. of Health, Education and Welfare. Natl. Shellfish Sanitation Program. Proceedings of the National Shellfisheries Association Volume 66 — 1976 9/'-fif THE ECONOMICS OF HATCHERY PRODUCTION OF PACIFIC OYSTER SEED: A RESEARCH PROGRESS REPORT KwangH. Im, Richard S. Johnston, R. Donald Langmo DEPARTMENT OF AGRICULTURAL AND RESOURCE ECONOMICS OREGON STATE UNIVERSITY CORVALLIS, OREGON ABSTRACT An analysis of the economic viability of a Pacific oyster (Crassostrea gigas) seed hatchery industry in the Pacific Northwest examines the demand for hatchery seed and the costs of producing hatchery seed. This is a progress report of the research, using limited market data and relying upon the production relationships prevailing at an ex- perimental hatchery. The results suggest that hatchery production may be economical- ly feasible, but a more accurate picture requires additional data from industry sources. INTRODUCTION The Pacific oyster, Crassostrea gigas, has been cultured commercially in this country for about 50 years (Steele, 1964). Because there are so few areas in the Pacific Northwest where natural reproduc- tion occurs, the industry has had to rely on Japan for a large proportion of its supply of oyster seed (oysters up to one year old). These seed are planted on growing grounds in Washington, Oregon, California, and British Columbia, where they mature in two or three years. Table 1 pro- vides data on Pacific oyster production in the U.S., on imports of oyster seed from Japan, and on the production of oyster seed in what has become the principal domestic source — Washington's Hood Canal. As the table reveals, there has been quite a bit of fluctuation in domestic seed production and in seed imports from Japan. In the market place, domestic Pacific oysters compete with imported oysters, whose volumes have recently increased, and with other oyster species, especially the Eastern, Crassostrea virgin- ica, and European, Ostrea edulis, species. Data on these quantities also appear in Table 1. No analy- sis has yet been conducted of the final market for Pacific oysters (although this is currently under way at Oregon State University), but some results are available on the demand for oysters in the U.S. The Bureau of Commercial Fisheries (1970), Dunham and Bray (1974), and Suttor (Nash, 1969) all report price elasticity figures of less than unity. A recent study by Charbonneau and Marasco (1975) suggests that this varies substantially across regions and over time for fresh and frozen oysters. One would expect the demand for a specific species of oyster, such as the Pacific oyster, to be more price elastic than the demand for all oysters taken together. Prices of both adult oysters and oyster seed have been rising over time (Table 2), the result of a variety of factors including rising consumer in- comes and prices of substitute goods. Adult oyster prices have risen less rapidly than have seed oyster prices. On the supply side, fluctuations in spawning and growing conditions along with increases in labor costs, i.e., those costs associated with tend- ing the oyster grounds (including planting and harvesting) and those associated with oyster pro- cessing (including shucking), are important price- determining factors. While data on the labor costs appropriate to the oyster industry are not yet 81 82 KWANG HI IM, R. S. JOHNSTON, R. D. LANGMO available, some indication of the trend is sug- gested by the U.S. Bureau of Labor Statistics figures on average hourly earnings of workers in industries producing canned, cured, and frozen foods (Table 2). Hourly wage rates, as measured in current dollars, have increased over time. Thus, the Pacific oyster industry in the U.S. has been experiencing uncertainties with respect to seed supply and production conditions, competi- tion from imported oysters, and rising labor costs. These conditions, together with the competition for oyster lands which has come from recrea- tionists and industrial users, have led to recent at- tempts to harvest oysters through less labor- TABLEl. U.S. Oyster Production and Imports: U.S. Seed Production and Imports; 1947-1975 Pacific Coast Pacific oyster Total oyster seed oyster Pacific oyster production. production. imports from seed production, Total oyster Year U.S.- U.S.- Japan*" Hood Canal' imports'* thousand pounds standard cases std. case equiv. thou, pounds 1947 11,320 63,085 56,619 * 111 1948 9,564 61,610 32,869 * 160 1949 8,164 75,773 46,036 * 342 1950 8,080 76,415 46,726 ' * 446 1951 8,597 72,990 51,901 * 962 1952 9,957 82,242 83,290 * 595 1953 10,283 79,719 70,113 * 637 1954 10,855 81,922 65,528 * 1,056 1955 11,602 77,515 54,216 0 1,391 1956 11,881 75,133 100,634 1,000 1,928 1957 11,614 71,658 60,063 0 2,575 1958 11,197 66,395 61,119 2,000 5,015 1959 12,328 64,710 61,444 2,500 5,545 1960 10,983 60,010 44,291 3,500 6,597 1961 10,154 62,305 37,128,5 3,700 7,261 1962 10,714 56,037 41,499 5,200 7,387 1963 9,746 58,444 53,416 2,700 8,906 1964 9,934 60,534 41,160 0 8,154 1965 9,117 54,688 34,909.5 6,900 9,001 1966 * 51,223 16,102 9,200 12,028 1967 7,682 50,957 43,557.5 15,900 17,672 1968 * 55,600 38,415 6,200 15,550 1969 * * 44,707 3,000 16,622 1970 7,915 53,602 26,079 5,000 15,484 1971 * * 30,337 32,900 9,695 1972 8,362 56,058 7,321 33,400 22,309 1973 * * 8,346 34,200 19,850 1974 * * 12,406 46,700 16,010 1975 * * 10,856 0 * Data not available. " NMFS, NOAA, Fishery Statistics of the United States, various issues. ' State of Washington Dept. of Fisheries, Washington State Seed Oyster Imports from Japan. 1975. ' Personal correspondence. Ronald E. Westley, Washington State Dept, of Fisheries, ' BCF, Basic Economic Indicators: Oysters, May 1970: NMFS, Current Fishery Statistics, and NMFS, Fishery Market News Reports (Seattle), various issues. ECONOMICS OF OYSTER SEED PRODUCTION 83 intensive means and to culture oysters through methods which would permit more utilization of the water column on the oyster-rearing grounds (examples include raft culture, tray culture, stick culture). During the last three years another development has taken place which could have profound effects on the industry. That development is the commer- cial raising of oyster seed under environmentally- controlled (hatchery) conditions. Drawing upon research results available from sources such as the experimental hatchery at the Oregon State Univer- sity Marine Science Center, about six hatcheries in Washington and California are currently raising oyster seed for commercial purposes. This development is simply too recent to be able to analyze its impact on the industry. Nonetheless, it is the objective of the present study to examine the TABLE 2. Prices of Imported Oyster Seed and Shucked Pacific Oysters; Average Hourly Labor Earnings, 1947-1974 Wholesale price of shucked Average hourly earnings: Price of Pacific oyster seed Paci ific oysters canned, cured, and Year imported from Japan" (FOB Seattle, WA)' frozen foods' dollars per case dollai •■s per p ound dollars 1947 5.86 0.37 * 1948 * 0.43 * 1949 * 0.47 * 1950 * 0.46 1.17 1951 6.92 0.50 1.25 1952 6.98 0.49 1.30 1953 7.27 0.46 1.35 1954 * 0.46 1.39 1955 6.19 0.46 1.43 1956 8.05 0.46 1.54 1957 8.67 0.47 1.60 1958 10.28 0.47 1.64 1959 * 0.46 1.70 1960 9.95 0.47 1.78 1961 9.88 0.55 1.85 1962 10.83 0.52 1.90 1963 11.00 0.52 1.92 1964 * 0.52 1.95 1965 * 0.52 2.01 1966 17.00 0.65 2.11 1967 * 0.77 2.22 1968 17.00 0.81 2.37 1969 16.50 0.81 2.51 1970 19.40 0.80 2.65 1971 20.50 0.80 2.83 1972 25.50 0.83 3.01 1973 29.00 1.12 3.25 1974 29.28 1.25 3.55 Data not available. FOB Aberdeen, WA.; personal communication, Ronald E. Westley, Washington State Department of Fisheries; and (Steele, 1964.) NMFS, NOAA, USDC (Seattle), Fishery Market News Reports, various issues. The figures are receipts by Seattle wholesale dealers divided by pounds of shucked oysters. U.S. Bureau of Labor Statistics, Employment and Enrnnigs, various issues. 84 KAWNG HI IM, R. S. JOHNSTON, R. D. LANGMO nature of the demand for oyster seed and the costs associated with hatchery production of oyster seed, in the hope of being able to determine the viability of an oyster seed hatchery industry in the Pacific Northwest. The Demand for Pacific Oyster Seed A diagrammatic model of the Pacific oyster seed market is presented in Figure 1. In Figure 1, DD is the annual North American demand for Pacific oyster seed. This demand is derived from the de- mand for adult oysters, and is postulated to be such that the quantity of seed oysters demanded by oyster growers depends upon their expecta- tions of the price at which they will be able to sell mature oysters, the current price of oyster seed, the quantity of available oyster-producing land in the Pacific Northwest, and the expected time path of wage rates during the growing period. Sd is the domestic supply of Pacific oyster seed. It is postulated to be perfectly inelastic beyond some reservation price level, on the assumption that quantities of seed available this year are determined primarily by the environmental condi- tions prevailing during the spawning period of the previous year. Sy is the supply of Pacific oyster seed from Japan. It expresses the hypothesis that the quanti- ty of oyster seed supplied to the U.S. is a postive function of the price of oyster seed (in Japanese yen), Japanese production of oyster seed, and seed prices in Japan and other Japanese markets. Until recently, the U.S. has been the principal market for Japanese exports of seed. During the past five years, however. Western Europe (especially EEC members) has become an important market for Japanese seed. While, in the early years of the in- dustry, the Pacific Coast Oyster Growers' Association was the principal seed importer, dur- ing the past 20 years several independent U.S. oystermen have been importing seed directly, rather than through the Association. Nonetheless, it is probably reasonable to assume that the large number of oystermen in the Pacific Northwest treat the price of oyster seed as exogenous; i.e., as being beyond their control. The curve dd is the curve on which the interest of this portion of the analysis focuses. It is the "net" derived demand for Pacific oyster seed. It can be interpreted as describing a functional rela- tionship between alternative seed prices and the quantities of seed oystermen would purchase from sources other than Japan and domestic suppliers of wild (i.e., non-hatchery) seed. In other words, it is postulated that this is the demand for seed facing a hatchery industry. Mathematically, the relationships may be de- scribed as follows: (1) (2) (3) (4) (5) where QRS. D = f(Eppo pps L„ EC) EPfo = g(Pfo , AY,-,, W,-, AI,-,) EC, = h(AC,-,) Qps s. J = j(PfvR„ J?^ s?n Quantity oi Seed per Year FIG. 1. Supply and demand for Pacific oyster seed in the U.S. Q^^ " = Quantity (number of cases) of Paci- fic oyster seed demanded in year t; QPS. s. J — Quantity (number of cases) of Pacific oyster seed supplied by Japan to U.S. importers in year t; Eppo _ Expected wholesale price of mature Pacific oysters in year t; pps _ Price per case of Pacific oyster seed in year t; ECONOMICS OF OYSTER SEED PRODUCTION 85 L, = Acres of Pacific oyster-producing land in Washington, Oregon, and California in year t; EC, = Expected labor costs as between year t and year t + 3; Pf° = Actual wholesale price of mature Pacific oysters in year t-1; AY, , = Change in per capita personal disposable income as between year t-1 and year t; W?-^ = World production of all oyster seed in year t; AI,., = Change in U.S. imports of Pacific oysters between year t-1 and year t; AC,., = Difference in wage rate for harvesting and shucking as between years t-1 and t; R, = Exchange rate between Japanese yen and U.S. dollar in time t; J?-* = Japanese production of oyster seed in year t; S?^ = An index of prices in Japan and foreign markets (excluding North America) for Pacific oyster seed in year t; and D?* = Quantity of domestic Pacific oyster seed supplied in year t. The * denotes that this quantity is determined by variables outside of the model. Equation (1) originates in a Cobb-Douglas pro- duction function of the form: Oysters produced,»3 = aoSeed<^' Labor?^„,.3. Combining Equation (1) with Equation (5) to yield the "net" demand curve yields (6) Qf^"-D?^ s Qf^ " ' = k(EP™, PT^ L„ EC) - D?-'*, where (^^ " ' is the quantity (number of cases) of Pacific oyster seed demanded from japan in year t. This transformation is necessary because data are not available on the total quantity of domestic wild seed (including British Columbia) available over the period of anaysis. Equations (2) and (3) express postulated rela- tionships among observable variables and ex- pected (by oyster growers) future prices and labor costs. They are substituted into Equation (6) to ex- press that equation in terms of observable variables. Thus, the two equations whose parameters are to be estimated are the "demand" equation (6') and the "Japanese supply" equation (4): (6 ') Qf^ "^ = k { g(Pf?„ AY,.,, W?^ AI,-,), Pf^ L„h(AC-,)} -D?^* (4) Qf^ = j(PfvR„ J?^ s,° In this system, Pf^ and QT^ ° '{ = Qf^ -^ ■' in equilibrium) are treated as endogenous variables. As indicated earlier, it is postulated the produc- tion of oysters can be approximated by a Cobb- Douglas function in seed and labor. A similar functional form is assumed for (2), (3), and (4). Thus, the functional form of (6 ') becomes (6") Qf"''^ = a„ {(Pr°)'''(AY,-,)'''(W?^)/'' (AI,-, )/'4} a. . {(AC,-,)''MPr )"' }+ a^U-D?^* and ^) (J?0 (S-) \ Time series data on the variables W"'^, L, and S°^ were not available at the time of analysis. Their omission results in biased estimates of the regression coefficients. This potentially major flow in the analysis should be corrected as data on the variables become available. In addition, while data on domestic production of seed from Hood Canal were available (Table 1), there are no data available on the quantities of Hood Canal seed actually planted. Furthermore, data on production of Willapa Bay seed and Pendrell Sound seed are based on estimates of the quality of seed set on National Marine Fisheries estimates of imports from Canada. Because of the difficulty of making the domestic production and the Japanese import data comparable, it was decided to include the D?^ variable in a multiplicative form in the "demand" equation, with its own exponent to be estimated. Two-stage least squares procedures were used 86 KAWNG HI IM, R. S. JOHNSTON, R. D. LANGMO to estimate the demand and supply parameters. The resulting equations are: Demand for Pacific Oyster Seed: log Of" ° ' = 18.1310 + 3.0739 log P™, + .3879 log AY,. (3.3920) (1.6663) (.5841) h .0047 log AI,., - .4644 log AC,-, + .0106 log D?^* (.0538) (.6020) (.0424) - 3.4918 log Pf (1.4202) Supply of Pacific Oyster Seed by Japan: log Qf^ '■ ' = 36.7770 - 4.1040 (log Pf^ - log R,) (10.9828) (1.9220) - 1.2765 log J?^ (.9474) The numbers in parentheses are standard errors; the ^^\. sign indicates values predicted from the first stage.' 'The estimated stage I equation is: log Pf^ = 3.3201 + .8232 log Pf?, + .2055 log AY,., + .0082 (8772) (3227) (1013) (0118) logAI,-, - .02011ogAC,-, + 1.5165logR, + .0223 log D?^*. (.1576) (.5099) (.0084) The J°^ variable was not included at this stage because of the bmited number of observations. The R' statistics for the equa- tion is .96. The R^ statistic for the demand equation is .75, while the Durbin-Watson (D-W) statistic for serial correlation is 2.64. The first statistic suggests that a fairly high degree of association exists between the quantity of Pacific oyster seed demanded and the explanatory variables of the demand equation. The size of the D-W statistic is in the "in- conclusive" range. Only years for which data on all variables were available could be used — a total of 15 observations. A larger number of observa- tions would help determine whether or not serial correlation is present. Indeed, with so few observations, it is difficult to draw any "conclusions" from the analysis. However, some findings are worthy of note. First, the standard errors associated with the estimated coefficients for the two price variables are fairly low, relative to those coefficients. This suggests that oystermen may be sensitive to changes in both mature oyster prices and oyster seed prices in their seed-importing decisions. Indeed, the de- mand for imported seed appears to be price-elastic (£? = —3.4918), suggesting that an increase (decrease) in Japanese seed prices would be associated with a more-than-proportional decline (rise) in purchases of Japanese seed, assuming no changes in the values of the other variables. The results also suggest that an increase (decrease) in the wholesale price of oysters would increase (decrease) the quantity of Japanese seed demanded at each of the values of the Japanese export price. A word of caution is in order here, however. These two price variables are highly intercor- related, suggesting that it is difficult to sort out the separate influences of each on the quantities demanded. When the wholesale price variable was excluded from the equation, however, the price- elasticity figure changed to only —4.00, still in the "elastic" range. Of some interest is the high standard error associated with the variable representing the domestic seed of Pacific oyster seed. One is temp- ted to conclude that this variable is not an impor- tant determinant of the imported quantities demanded. This may, indeed, be the case. An alternative explanation of the result, however, is that the data used, while the best available, do not accurately portray the actual quantities available, as perceived by the domestic growers when they make their import decisions. This may be par- ticularly true of the Pendrell Sound seed. A third explanation is that the demand equation is simply mis-specified, a potential difficulty with all econometric models. Specification error may have been a difficulty with the estimated supply equation. The R^ statistic for that equation is .61 and the D-W statistic is 1.82. Because of data limitations, this analysis was conducted with only seven observa- tions, and is included here only for purposes of il- lustrating the techniques used. Neither the R^ nor the D-W statistic has much statistical meaning, given the small number of observations. However, it is of interest to note the negative coef- ECONOMICS OF OYSTER SEED PRODUCTION 87 ficients on both of the explanatory variables, where the economic model discussed earlier would lead one to hypothesize positive signs on both coefficients. A possible explanation is that the price variable is serving as a proxy for prices in Japan and other seed-importing countries (e.g., France). It so, one would expect that the greater the price, the smaller the quantity Japanese ex- porters would be willing to ship to U.S. buyers. In the discussion which follows, the supply of Japanese oyster seed is treated as being perfectly price-inelastic. In Table 3 the estimated demand equation is employed to address the question: assuming that all values of the explanatory variables in that equation, except the price of oyster seed, were set at their 1974 levels, how much oyster seed would West Coast oystermen be willing to purchase at alternative prices? Subtracting from the Table 3 quantity figures an assumed amount of seed im- ported from Japan would yield estimates of the an- nual quantities of seed oystermen would be willing to purchase from hatcheries. Tables 1 and 2 reveal the import price to be $29.28, and the quantity im- ported to be 12,406 during 1974. Inserting the values into the estimated demand equation reveals that approximately 6,200 cases would have been demanded in 1974 at that price. In fact, industry sources maintain that approximately 7,500 cases were delivered in that year from West Coast hatcheries. Thus the model appears to do a reasonably good job of prediction. Perhaps the most significant result of this portion of the analysis is the finding that oyster seed buyers do, indeed, appear to be sensitive to changes in seed TABLE 3. The Demand (Price-Quantity Rela- tionship) for Imported Pacific Oyster Seed' Estimated annual quantities of Average imported seed which oystermen market price would be willing to purchase dollars per case number of cases 20.00 68,398 25.00 31,379 30.00 16,602 35.00 9,691 40.00 6,080 ° Assuming all other explanatory variables are set at their 1074 levels. prices. It is reasonable to assume that this would also be true of hatchery -produced seed. Again, the reader is reminded that these results must be interpreted with extreme caution. For one thing, this is no reason to expect 1974 conditions to prevail into the indefinite future. No account is taken of the differences in quality of the oyster seed over time and among sources. For example, if the yields of hatchery seed were greater than the yields of imported seed, and if oystermen knew this, the quantities demanded from hatcheries could be different than what is suggested in Table 3. Furthermore, changes in the demand for oysters, themselves, would affect the demand for oyster seed. Indeed, there is currently some discus- sion in the industry of expanding export markets. The demand would also be affected by changes in the importation of oysters and oyster seed (for ex- ample, through lifting of the current embargo on Korean seed). Given these cautionary remarks, perhaps the results reported here are helpful in suggesting what opportunities, under the postulated condi- tions, a commercial hatchery industry has with respect to seed sales. The next question is: what are the costs associated with such volumes? The discussion now turns to this question. Seed Hatchery Production Costs Among the characteristics that establish the competitive position of hatchery seed with other seed sources is the cost of production. The only public source of hatchery operating experience available in Oregon is through the Oregon State University pilot facility at the Marine Science Center (MSC) in Newport. Based on this source, costs have been estimated at a near-actual produc- tion of 15 bushels or 6 cases per week. Additional cost projections have been made for several other levels of output. In this preliminary report costs are summarized for what will be referred to as Plant I at 15 bushels per week capacity and Plant n at 800 bushels or 320 cases per week, which is nearer a realistic commercial production quantity. The purpose of this portion of the study is to determine the costs of producing oyster seed in the Pacific Northwest. Other contributing objectives include: 88 KAWNG HI IM, R. S. JOHNSTON, R. D. LANGMO 1. identification of factors that influence production costs; 2. estimating costs in detail for each of six stages of operation employed in seed pro- duction; and 3. extension of these costs for two production capacities. Fulfillment of these objectives will con- tribute to management capabilities. 1. Costs for comparing hatchery seed with imported and natural seed will be available. 2. Private hatchery operators will have detailed cost components with which to evaluate their production costs. 3. The impact of proposed changes in operating methods, facilities, or equipment upon specific cost items can be more readily estimated. Determination of costs for the production of oyster spat by the MSC required identification of all the costs needed for the activities required to produce a usable product. In turn these various costs were assigned to six subgroups or stages of operation, illustrated in Figure 2. By means of process charts, detailed diagram- matic descriptions were made correlating all ac- tivities for the production of oyster seed. Separate charts were made for algal food production and seed rearing. With the aid of process charts, it was possible to identify and assign the use of all resources as they were required by each activity. Fortunately, records of labor use were available. Cost figures were obtained from numerous sources including MSC records, equipment and material suppliers, utility companies, and building contractor estimates. A short excerpt of the detail- ed process chart for raising oyster seed is shown in Hgure3. Classification and Determination of Costs The costs associated with this study are the in- itial investment outlay, fixed costs, and variable costs. The initial investment outlay items consist mainly of buildings and equipment, and appear as production costs in the forms of depreciation, ex- penditure on interest on investment, etc. Acquisi- tion cost of land is not included in this study. Fix- ed cost items associated with buildings and equip- ment include depreciation, interest on investment, insurance, taxes, repair, and maintenance. Super- vision also is included as a fixed cost item. Variable cost items include expenses for labor, material, supplies, water, electric power, telephone, and other expenses directly related to oyster seed production. 1. Initial Investment Outlay Costs for the oyster hatchery building were estimated on the basis of space requirements. Space requirements include rooms for (a) algal food production, (b) larval rearing and setting, (c) adult oyster conditioning and spawning, (d) algae lab, (e) analytical lab, (f) general office, (g) lunch and locker, (h) lavatory and storage, and (i) space allowance for aisles and an outdoor concrete pad for cultch preparation. The building costs, including piping and wiring. To Oyster Grower FIG. 2. Relationship between six stages of oyster hatchery operation. ECONOMICS OF OYSTER SEED PRODUCTION 89 Treated seawater To rearing tanks Algae (from food production chart) Wash larvae from screen into buckets |ll) To rearing tanks Place 2.5 milHon larvae in each tank Sulfamethazine ^ Add sulmet once/week and algae ' 10 ; once/day (to 30,000 ceils 'ml) y during first week (l\^ On 7th day drain water _ ^ ■■ through 80 micron screen Wash larvae from screen into buckets- Store algae with nutrients, light, and air To rearing tanks Legend Return larvae to rearing tanks ( J Operation r^ Transportation ^y Storage I Inspection n Delay FIG. 3. Excerpt of detailed process chart activities. initial investment outlay for building and equip- ment, by stages of operation, for Plants I and II. 2. Fixed Costs The following procedures and values were used in estimating fixed costs: (a)Depreciation of building and equipment was calculated on the basis of 30- and 10- year lives, respectively (3.3 percent per year for building and 10 percent per year for equip- ment). (b)Interest on investment was calculated at 5.2 percent on building and 5.5 percent on equipment, according to the following for- mula: Average interest = -y {'^), were based on contractor estimates of $20 per square foot plus 8 percent for design fee, and $1 per square foot for a concrete pad in the cultch preparation area. The number of various equipment units depends on the capacity output of the hatchery. TTie major expensive equipment units are Coulter Counter, autoclave, analytical balance, bay pump, dissecting scope, shaker, ultraviolet sterilizer, top-loading balance, and concrete mix- er. Table 4 shows the space requirements and the where i = interest rate, estimated as 10 per- cent for this study, and n = number of useful years, calculated at 30 years for building and 10 years for equip- ment. (c) Insurance and taxes were each estimated at 1 percent of total initial investment outlay. (d)Repair and maintenance charges were computed at 1.5 percent of total initial investment outlay. (e) Supervision costs were assumed to be 10 percent of direct labor costs. TABLE 4. Space Requirements and Initial Investment Outlay for Building and Equipment, by Stages of Operation, for Plant I (15 bu./wk.) and Plant II (800 bu./wk.) Initial investment Initial investment Initial investment outlay for outlay for building Space requirements outlay for building equipment and equipment Stages of Plant Plant Plant Plant Plant Plant Plant Plant operation I II I II I II I II — square feet — dollars Conditioning 80 80 1,728 1,728 304 304 2,032 2,032 Spawning 80 80 1,728 1,728 63 66 1,791 1,794 Algae production . .. 520 5,520 11,232 119,232 26,365 115,050 37,597 234,282 Larval rearing 240 12,960 5,184 279,936 3,140 98,900 8,324 378,836 Larval setting 240 11,520 5,184 248,832 1,718 91,560 6,902 340,392 Cultch preparation . 400 2,000 400 2,000 312 2,496 712 4,496 Miscellaneous" .... 1,440 9,896 31,104 213,754 4,100 6,200 35,204 219,954 TOTAL 3,000 42,056 56,560 867,210 36,002 314,576 92,562 1,131,786 " Miscellaneous space includes general office, lunch, and locker rooms, lavatory, storage room, analytical lab, and aisles. Miscellaneous equipment mcludes bay pump, ultraviolet water sterilizer, and filters and valves, air compressor, and miscel- laneous glass and plastic containers. 90 KWANG HI IM, R. S. JOHNSTON, R. D. LANGMO 3. Variable Costs The cost of direct labor at each stage of opera- tion was determined by applying a wage rate of $4 per hour to the average man-hour expenditure re- quired. These requirements were obtained from records maintained, for a 23-week period starting November, 1972, at the Oregon State University hatchery pilot operation. Production during this time was 15 bushels per week. The estimated average weekly man-hour expenditure, by stages of operation, is presented in Table 5. TABLE 5. Average Weekly Man-Hour Expendi- ture, by Stages of Operation, for Plants I and II Stages of operation Plant Plant I II hours/week Conditioning 0.2 0.5 Spawning 3.9 4.0 Algae production 13.1 410.8 Larval rearing 9.9 276.6 Larval setting 1.8 96.0 Cultch preparation 1.0 43.7 Miscellaneous" 3.7 41.0 TOTAL 33.6 872.6 " Preventive and corrective maintenance and other miscellane- ous activities. Costs of electricity are derived from light and power usage and estimated at IVKWH. A one- horsepower motor consumes about .75 KW of electricity per hour of operation. Both fresh and sea water are used in an oyster seed hatchery. Each larva consumes .00084 liters, or about .00022 gallons, of sea water per week. The major use of fresh water is in cultch prepara- tion. At an output capacity of 15 bushels, or 6 cases, per week, fresh water is used at a rate of 2,800 gal. /week for cultch preparation. This figure was computed by measuring the flow of water per second (2.6 gal./20 seconds) and the length of time required to clean each bushel of cultch (30 min. /bushel). Costs of waste and garbage disposal, chemical and bleach, telephone, office supplies, materials, and other miscellaneous costs were included in the total figures. Table 6 shows the estimated costs per week for utilities and supplies, and for stages of operation for Plants I and II. 4. Total Costs Total costs and costs per bushel for Plants I and n, by stages of operation, are presented in Tables 7 and 8, respectively. These costs include both fix- ed costs and variable costs. Figure 4 shows the relative importance of cost functions of stage operations. CONCLUSIONS Table 9 shows the operational differences be- tween Plants I and II. Direct labor cost is the single biggest item influencing production costs. Direct labor cost of Plants I and II is 27.3 percent and TABLE 6. Costs of Utilities and Supplies Per Week Costs, by item Plant Plant Item I II dollars/week Electricity" 32.04 781.97 Fresh water" 4.19 185.27 Waste and garbage disposal"'. .. . 5.60 96.14 Chemicals and bleach 2.40 118.03 Material (uncleaned oyster shell)" 7.50 400.00 Telephone 10.00 25.00 Office supplies 3.00 20.00 Miscellaneous 7.00 15.00 TOTAL 71.73 1,642.31 Costs by stages of operation Plant Plant Stages of operation I II dollars/week Conditioning 7.26 7.26 Spawning 2.25 2.25 Algae production 8.11 361.07 Larval rearing 4.56 181.31 Larval setting 8.42 310.19 Cultch preparation 10.89 580.98 Miscellaneous 30.24 199.25 TOTAL 71.73 1,642.31 l(t/KWH tor light and power, $1.20/ 1,000 gal. Sewer charge tor ' : of water cost plus $3.50/week for gar- bage disposal, $0.50/bu. of uncleaned oyster shells. ECONOMICS OF OYSTER SEED PRODUCTION 91 o H c o _c ■^ u -1 ro U U. c _1 a' c o a; -x: (^ u 60 3 00 c c nj D. to 00 c 'c o c o U E (N o C5 •^ O IT) ^n xO rO lo o o rj O t-H O O lO m t— I ID cj- m 00 ^O rQ . r~i lO (N rO in ro -.O 00 -^ 00 (N 00 O Dv ^ vO -^ ^ (N O rO tN ^ C^ t^ O O ro in ro fS ro rO 00 Cn 00 r-i ^ O rH (N rH 00 C^ t^ O O O ^ 00 O fS rO C^ m sO ro O (S tv t% t^ rH Dx lO t— I K t^ "^ g ro i-H R. (NI >-l rH t^ vO rH rH x* 00 rH (N 00 rH ■* tN Tt o r-i ■^ rH q (N 00 o^ T—t rH rs O ro O 00 o r—< ro O 'T m (N 00 ■* m ro o in ro (N ro in o 3 OI . -i: ex UJ to H 3 o u T3 '^ r-i U (U to 3 O ri o O ^0 O rD 00 <> ^ 00 ■^ 00 o (N m tN T-i 3 O C S o J2 ■^ o C U a; 00 6 c C ^ •— I— I ■■5 rH rO m in 00 tj" m 00 tv 00 rD vO O rH (S rH 00 tN I~v O o o o 00 O (S m (N rH (N t^ tN rO rH ■rt 00 rH ■^ 00 ■^ O Cn (N rH m o 00 m cy aj C nj C 3 c c o .2 *c a; re e lA O 3 .S (A k- > H o 00 (H-) rO IN ^ ■D o o o M" ■^ -* ^n O O o m (N CO r-< ^ m in (-V lO in IN lO o O o in 00 (N ■^ ^ m r—* IN 00 U) rq IN o< vO n< (N (N (N 00 kO in m O 00 in ^o ■^ J—* C) -X> rn rH fc Y>. •a ^ i2 8 T) .2 D UJ x 3 -Q < a. X nJ H O H o m o^ o IN (N IN in •^ nO ■* In m 00 o in ro nO ~o rO O- nO IN o f > o On in nO rO o O in 00 rH On m 00 m rn On •^ rH -* On nidecussata(Reeve) have been raised on mixed diets of three species of diatoms cultured in con- tinuously flowing Antarctic Intermediate water. Parent stock which was introduced to the system at a length of 5 mm attained market size (38 mm) in 10 months. Population densities varied from 1600/ft^ as juveniles to 160/ft' as marketable adults, and survival for the growth period was 64% . Fi progeny, from time of fertilization, reach- ed market size in 13 months. Larvae metamor- phosed after 21 to 25 days and post-set survival was as high as 52 % . These preliminary studies show that Tapes can be spawned readily in a controlled system, grown rapidly in high densities with good survival, and consequently show a favorable potential for mariculture. SCALLOP CULTURE IN MUTSU BAY, JAPAN William N. Shaw Office of Sea Grant NOAA Marine Advisory Service Washington, D. C. In recent years, the production of sea scallops, Patinopecten yessoensis, in Japan has increased significantly. In 1973, 61,600 metric tons were harvested compared to approximately 4,000 metric tons in 1968. A major reason for this in- crease is the development of off-bottom techni- ques not only in the collecting of seed scallops but also in growing them to market size. One of the centers for scallop culture in Japan is Mutsu Bay. Based on two trips to this area, one in 1970 and more recently in 1974, the author will describe the methods of culture now being practic- ed in the Bay. A brief description of harvesting and processing will also be presented. THE MARICULTURE POTENTIAL OF TAPES S£M/D£C(JSS/^ r/^(REEVE) IN AN ARTIFICIAL UPWELLING SYSTEM Kenneth M. Rodde and Judith B. Sunderlin Lamont Marine Biology Station Kingshill, St. Croix U.S. Virgin Islands RECIRCULATING SYSTEMS FOR EMBRYO INCUBATION AND LARVAL REARING OF THE FRESHWATER PRAWN MACROBRACHIUM ROSENBERCII (DEMAN) Nils E. Stolpe Trenton State College Trenton, New Jersey 106 ABSTRACTS Natural development of the freshwater prawn Macrobrachium rosenbergii will be compared to the more "traditional" culture methods being presently used by most culturists. The develop- ment of high-density recirculating systems at the aquaculture facility at the Mercer Generating Sta- tion, Trenton, New Jersey, will be discussed. An early-larval rearing system incorporating an embryo incubator with automatic separation of larvae, and several late larval rearing systems will be described and evaluated with respect to economy of operation and larval survival. Several design criteria will be discussed. PHYSICAL PARAMETERS OF THE AMERICAN OYSTER, CRASS05TREA VIRGINICA GMELIN, FROM THE WAREHAM RIVER Larry Turner and John Zahradnik UMASS Aquacultural Engineering Laboratory Wareham, Massachusetts Data from experiments conducted with the American oyster during the past several years at the Wareham River are analyzed to reveal rela- tionships among length, weight, volume and den- sity of whole oysters, oyster shell and meat. The effects of variation due to genetics, growth environment and season are pooled to give general relationships which are useful in engineering design of aquaculture systems and in the design of experimental procedures and apparatus. THE PRESENT STATUS AND FUTURE OUTLOOK OF SHELLFISH FARMING IN PUGET SOUND, WASHINGTON Ronald E. Westley Washington State Dept. of Fisheries Brinnon, Washington Shellfish farming in Puget Sound, Washington, involves principally culture and harvest of oysters, and harvest of natural crops of clams and geoducks. Present trends are decreasing produc- tion of oysters with some increase in clams and geoducks. Biologically, Puget Sound has enor- mous potential for increased production of oysters and geoducks due to the abundance of well pro- tected, clean, nutrient rich water. Economic con- ditions have had a major impact on the oyster in- dustry and economic conditions appear to be the major reason for the decline. Economic conditions appear favorable for the clam and geoduck fisheries. Recent enactment of legislation for con- trol and management of shorelands has had some adverse effect of conduct of the shellfisheries and, depending upon further application of these laws, could be a major impediment in conduct of the shellfisheries in Washington State. PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 107 NSA PACIFIC COAST SECTION OUT-BAY CULTURE W. P. Breese Oregon State University Marine Science Center Newport, Oregon A relatively new type of oyster farming is pro- posed called "Out-bay Culture". Its advantages and problems are discussed. Some data are presented which indicates this type of culture may be successful. In the future, oyster aquaculture will call for a ration which is yet to be formulated. The method also provides necessary control over the operation. Out-bay culture is speculative at the present and may or may not develop into a commercial reality. RELATIVE ACUTE TOXICITY OF A PESTICIDE, HEAVY METAL AND ANIONIC SURFACTANTS TO MARINE ORGANISMS RickD. Cardwell Wash. Dept. of Fisheries Shellfish Laboratory/ Brinnon, Washington The relative acute toxicity of cadmium sulfate, methoxychlor, and two types of anionic sur- factants (dodecyl sodium sulfate and linear alkylate sulfonates) was determined using various species of marine fish, crustacean, and bivalve mollusk larvae. Methoxychlor was most toxic to spot shrimp and Dungeness crab zoeae (48-hr me- dian lethal concentrations or LCSO's of 0.025 and 0.044 mg/1, respectively), of intermediate toxicity to chum salmon fry and larval Pacific herring (48- hr LCSO's of 0.048 and 0.150 mg/1, respectively). and least toxic to three species of larval clams (native littleneck and the horse clams, Tresus capax and T. nuttalli) and larval Pacific oysters (range of 48-hr LCSO's of 0.198 to 0.441 mg/1). No relationship was found between the toxicity of cadmium sulfate and sensitivity of a particular taxon. The 48-hr LCSO values ranged from less than 0.1 mg Cd*Vl for larval horse clams (T. capax) to 14 and 115 mg Cd*'/1 for Pacific herring and brine shrimp nauplii, respectively. The anionic surfactant, dodecyl sodium sulfate, was most toxic to all four species of bivalve mollusk larvae (range of 48-hr LCSO's from 0.58 to 0.89 mg/1) and least toxic to spot shrimp and Dungeness crab larvae (48-hr LCSO's of 5.8 and 8.0 mg/1, respectively). Tests of three linear alkylate sulfonate formulations, composed of sur- face active agents having carbon chains of dif- ferent lengths, were also conducted using larval Pacific oysters. The LAS formulation having a predominance of long carbon chains (e.g. 12, 13, and 14 carbons) was the most toxic (48-hr LCSO of 0.10 mg/1), and that with the shortest carbon chains (e.g. 10, 11, and 12 carbons) the least toxic (48-hr LCSO of 0.56 mg/1). WINTER SPAWNING OF PACIFIC OYSTERS Jim Donaldson Chuck Munsey, and Vance Lipovsky Coast Oyster Company, Hatchery Division Nahcotta, Washington Pacific oysters, Crassostrea gigas, from selected growing areas in Willapa Bay, were brought into the hatchery during January, February, and March of 1974 and 1975 for spawning. Eggs from 108 ABSTRACTS unconditioned oysters developed into larvae that were as viable as the eggs from oysters that had been conditioned for spawning. It appears that oysters which have not spawned during the sum- mer will retain some viable gametes throughout the winter. SYSTEM WORK DESIGN AND INTERIM REPORTS COVERING CURRENT AND PROPOSED INDUSTRIAL ENGINEERING STANDARDS FOR SHRIMP, CRAB, OYSTERS BOTTOM-FISH AND PRODUCT-MIX SPECIES William Engesser, Chi Ming Cheung, Salahuddin Faruqui and Willie Mercer Department of Industrial and General Engineering Oregon State University Corvallis, Oregon Part One — Improvement of Direct-Labor Pro- ductivity Workers will gain a deeper insight of skill levels, irregular acts and allowances (personal, fatigue and unavoidable delays) when they are exposed to MAP (Master Achievement Programming). Loop motion pictures and achievement tests illustrate desirable motion patterns and the most common errors and irregular acts. Each step is described by four basic acts; move, grasp, position and use. For self-appraisal and improvement, a Master's achievement test shows the time (in seconds) for fiigh, average and low-skill performance. Part Two — Improvement of Supervisory and Staff Productivity Supervisors and supporting staff skills can be appraised by an investigation of SAP (Supervisor Achievement Programming). The work done by a crew being supervised can be used as a predomi- nant measure of appraising supervisors and sup- porting staff. To get relative performance dif- ferences, two case studies with operating plants have been started. In both plants, the management agreed to furnish a complete record of past per- formance so a linear relationship can be calculated and assignable causes can be identified. Although this relationship is an important management tool, more important is the prompt analysis of large productivity differences between normal and standard times. When differences are high, im- mediate steps can be taken to avoid such repeti- tions in the future. Some possible assignable causes which will be investigated to discover pro- ductivity effects include personal time, yields, set- up time, fish condition, thefts, avoidable delays, safety, sanitation, skill levels, tool and equipment condition, unavoidable delays, consumer accep- tance, fish size, errors in production data and other work on environmental factors yet to be determined. Part Three — Improvement of Top-Level Produc- tivity Top Level Productivity — Owners, Plant Managers and Staff: CAP (Chiefs Achievement Programming) can be improved and evaluated when complete product-mix standards are includ- ed in a future plan and schedule chart. More effec- tive resource planning, scheduling and utilization takes place when top level management can see the effects of their decision making. One cooperating processing manager stated that the processing of seafood (i.e. crab and shrimp) along with their traditional vegetables, showed a net profit (before taxes) of approximately $35,000. He mentioned that the seafood processing alone did not result in a direct profit but that by augmenting their entire production with the seafood process- ing, the savings resulted by providing a means to direct people and resources that would otherwise be idle. The $35,000 savings was validated by us- ing actual inplant data in a Resource Planning and Management (RPM) chart. Also, RPM charts can be used to predict future results and to adjust rapidly with appropriate changes when the unex- pected occurs. Other significant uses of the chart lie in the quick evaluation of the effects of mechanization, alternate marketing decisions, im- perfect operations and the complexity of the input-output relationships among the various ac- tivities and resources. AN APPROACH TO DEVELOPING A STOCK OF DISEASE RESISTANT OYSTERS William Hershberger College of Fisheries University of Washington Seattle, Washington A program currently underway at the Universi- ty of Washington to develop strains of oysters PROCEEDINGS OF THE NATIONAL SHELLFISHERIES ASSOCIATION 109 (Crassostrea gigas) that are resistant to the "sum- mer disease" apparently caused by Vibrio sp. was presented. Briefly this study involves challenging adult oysters to a mortality-inducing situation in the laboratory, breeding the survivors together by single parent matings (one male x one female), and, after setting and growing these families, retesting them under the same conditions to check for increased disease resistance. Families which demonstrated increased resistance would be used to produce the next generation and for testing "on site" for increased resistance in a natural situation. To date, about 20 crosses have been made, which will be retested in the spring of 1976. In addition, the families produced for the resistance studies will be monitored for single gene differences by electrophoresis of tissue enzymes and proteins. Data presented indicated that the frequencies of various genes have been changed in hatchery stocks, compared to naturally reproduc- ing populations. This means that the genetic con- stitution of the oyster is being changed by artificial manipulation. In order to maintain the necessary genetic variability, avoid the problems of in- breeding, provide "markers" for further genetic manipulation, and distinguish specific stocks, gene differences as shown by electrophoretic means can be utilized as valuable tools in oyster culture. SITE COMPARISON FOR THE CULTURE OF THE SPOT PRAWN P AND ALUS PLATYCER05 BRANDT IN AND ADJACENT TO SALMON NET PENS Jack Rensel College of Fisheries University of Washington Seattle, Washington Clam Bay and Henderson Inlet in the central and southern basins of Puget Sound, respectively, were compared as potential prawn aquaculture sites. Seasonally warmer waters of the latter site were conjectured to accelerate growth. Wild and laboratory reared prawns were held in nylon net pens with and without salmon and in benthic cages beneath commercial salmon net f)ens. Prawns were fed raw mussel (Mytilus edulis) and sea kelp (Nereocystis leutkeana), Oregon Moist Pellets (fish food) or geoduck clam {Panope generosa) processing wastes. Unsupplemented groups held in net pens and benthic cages could utilize fouling organisms or organic enrichment from the adjacent salmon pens. Growth and survival of juvenile prawns were significantly higher at Clam Bay than at Hender- son Inlet. One year old prawns averaged 7.13 grams which exceeded growth reported for wild populations off Vancouver Island, B.C. Rates of growth for Henderson Inlet benthic and Clam Bay surface yearlings were more rapid than the reported Vancouver Island populations with the exception of net pen, unsupplemented groups. Henderson Inlet surface waters were unsuitable for prawn culture due to extreme temperatures (21.9° C in early June), protozoan fouling and dense plankton blooms. During the summer season surface reared juveniles and yearlings ex- perienced 70% and 100% mortality, respectively. Conversely, benthic caged prawns at 10 meters had only 15% mortality during the same period. Differences in growth and survival and possible applications to commercial culture are discussed. RELATIONSHIP BETWEEN PACIFIC OYSTER SEED DENSITY AND FIRST YEAR GROWTH Albert Scholz Wash. Dept. of Fish. Shellfish Laboratory Brinnon, Washington Growth of Pacific oyster seed was greater for densities of 8 or less per mother-shell within the density range tested, 1 to 40 per shell. Average size increased as the number per shell decreased within the 1 to 8 spat per shell densities. There was no statistical difference in average size among the 9 to 40 oyster per shell densities although average size declined slightly with increased density. At very low density, 10 or less per shell, there was no dif- ference in average volume related to area of over- winter hardening. At densities from 11 to 40 per shell the average volume per oyster was greater for those over-wintered at North Bay compared with those at the Point Whitney Lagoon. ^ MBI WHOI LIBRARY WH lABX I