MOTE MARINE LABORATORY RED TIDE STUDIES FINAL REPORT FL DEP Contract MR 042 July 11, 1994 - June 30, 1995 Submitted To: Dr. Karen Steidinger Florida Marine Research Institute FL DEPARTMENT OF ENVIRONMENTAL PROTECTION 100 Eighth Street South East St. Petersburg, FL 33701-3093 Submitted By: Dr. Richard H. Pierce Director of Research MOTE MARINE LABORATORY 1600 Thompson Parkway Sarasota, FL 34236 Mote Marine Laboratory Technical Report No. 429 June 20, 1995 This document is printed on recycled paper Suggested reference Pierce RH. 1995. Mote Marine Red Tide Studies July 11, 1994 - June 30, 1995. Florida Department of Environmental Pro- tection. Contract no MR 042. Mote Marine Lab- oratory Technical Report no 429. 64 p. Available from: Mote Marine Laboratory Library. TABLE OF CONTENTS I. SUMMARY. 1 II. CULTURE MAINTENANCE AND GROWTH STUDIES . 1 Ill. ECOLOGICAL INTERACTION STUDIES . 2 A. Brevetoxin Ingestion in Black Seabass B. Evaluation of Food Carriers C. First Long Term (14 Day) Clam Exposure With Depuration (2/6/95) D. Second Long Term (14 Day) Clam Exposure (3/21/95) IV. RED TIDE FIELD STUDIES . 24 A. 1994 Red Tide Bloom (9/16/94 - 1/4/95) B. Red Tide Bloom (4/13/94 - 6/16/95) C. Red Tide Pigment D. Bacteriological Studies E. Brevetoxin Analysis in Marine Organisms Exposed to Sublethal Levels of the 1994 Natural Red Tide Bloom V. REFERENCES . 61 Tables Table 1. Monthly Combined Production and Use of Laboratory C. breve Culture. ....... 2 Table 2. Brevetoxin Concentration in Brevetoxin Spiked Shrimp and in Black Seabass Muscle Tissue and Digestive Tract Following Ingestion of the Shrimp ................ 5 Table 3. 14 Day Clam Exposure With Depuration - Water Quality and C. breve Concentrations ...................................... 7 Table 4. Clam Exposure - Bioaccumulation: 14 Day Exposure With Depuration Brevetoxin Concentrations in Exposure Water and Clams .................. 11 Table 5. Second 14 Day Clam Exposure - Water Quality and C. breve Concentrations ................................................ 16 Table 6. Brevetoxin Concentrations in Exposure Media and in Clams Exposed to Live G. breve Cells (Second Clam Exposure) ........................... 23 Table 7. Red Tide Field Studies (9/19/94 - 12/30/94) ............................ 25 Table 8. Brevetoxin Analysis from Natural Red Tide Bloom Areas .................. 33 Table 9. Red Tide Field Studies (4/13/95 - 6/16/95) ............................. 34 Table 10. Bacteriological Samples Collected From Gulf and Bay Waters for the 1994/1995 Red Tide Project ................................. 48 Table 11. Genera/Groups of Bacteria Recovered From Plate Counts .................. 50 Table 12. Vibrio spp. Recovered From Direct Plating (TCBS) and MPN Tubes ........... 51 Table 13. Oyster, Seasquirt and Mullet Analysis for 9/94 - 1/95 Red Tide Natural Bloom ... 53 i (Table of Contents Continued) Figures Figure 1. G. breve Test Tank Cell Counts at 24 Hours for a 7 Day Clam Exposure . 13 Figure 2. G. breve Test Tank Cell Counts at 24 Hours for a 14 Day Clam Exposure . 14 Figure 3. G. breve Cell Counts for Individual Exposure - Tanks After 24 Hours . 21 Figure 4. T. chuii Cell Counts for individual - Control Tank Counts After 24 Hours . 22 Figure 5. New Pass Dock Cell Concentrations, During the 1994 Red Tide Bloom . 32 Figure 6. Red Tide Cell Count; New Pass, 1995 Red Tide Bloom . 41 Figure 7. Chlorophyll a Content of G. breve at Two Samplings Depths During a Natural Red Tide Bloom. September - December, 1994 . 45 Figure 8. Gyroxanthin-diester Content of G. breve at Two Sampling Depths During a Natural Red Tide Bloom. September - May 1994 . 46 Figure 9. Comparison of the Concentration of Three Pigments with Chlorophyll, a Concentration in G. breve Collected at 2m Depth During a Natural Red Tide Bloom . 47 Figure 10. Red Tide Toxin (BTX) in Oysters & Seasquirts During a Natural Red Tide Bloom in Sarasota Bay . 60 Appendixes Appendix “A” Methods for Obtaining Phytoplankton Cell Counts .................. 62 Participants Mote Marine Laboratory Dr. Richard Pierce ............................................ Project Manager Dr. Gary Kirkpatrick ..........................Phytoplankton Ecology/Pigment Analysis Dr. John Buck ................................................. Microbiology Mr. Mike Henry ................................... Toxin Analysis/Field Monitoring Ms. Jing Zhou .................................... Toxin Analysis/Field Monitoring Ms. Cindy Aller .................. Culture Maintenance/Bioaccumulation/Field Monitoring Ms. Tammy Seaman ............... Culture Maintenance/Bioaccumulation/Field Monitoring National Marine Fisheries Service, Charleston Laboratory Dr. Fran VanDolah .......................... Receptor Binding Assay for Brevetoxins Mr. Todd Leighfield .......................... Receptor Binding Assay for Brevetoxins ii 1. SUMMARY The Mote Marine Laboratory Red Tide Research Program encompasses four study areas: 1. Laboratory culture maintenance and growth; 2. Ecological interactions; 3. Toxin chemistry and analysis; and, 4. Red tide bloom investigations. These studies, in cooperation with the Florida Department of Environmental Protection, Florida Marine Research institute, focus on the accumulation of red tide toxins in fish and shellfish and the effects of chronic exposure to red tide toxins during natural blooms and under controlled laboratory exposure conditions. Natural red tide bloom studies included weekly monitoring of the intensity and distribution of the bloom, analysis of toxin and pigment content of intensive bloom areas, marine bacteria associated with the bloom and bioaccumulation of brevetoxins in sea squirts, oysters, clams and mullet collected from an area of Sarasota Bay experiencing chronic, low levels of the red tide bloom. In addition to natural bloom monitoring, laboratory red tide cultures were maintained for use in exposure - bioaccumulation studies with clams and a fish, black seabass. Tissues were analyzed for toxin content. Extracts of exposed invertebrates were sent to the NOAA-NMFS Southeast Fisheries Science Center in Charleston, SC, for receptor binding analysis. II. CULTURE MAINTENANCE AND GROWTH STUDIES Gymnodinium breve culture production was maintained between 120-200 liters throughout the twelve-month period with volume varying according to experimental use. Production and use of G. breve culture are found in Table 1. Cultures were maintained in both artificial (NH-15) media and (f/2) a natural seawater base media (Guillard & Ryther, 1962). In addition to G. breve, Mote Marine Laboratory maintained other phytoplankton cultures. Presently, Tetraselmis sp., Dunneliella tertiolecta, Thalassiosira psuedonana, T. weissflogii, Iochrysis galbana, Nannochloropsis occulata, and Skeletonema costatum are cultured for pigment comparisons. Tetraselmis chuii is cultured in large volume as a primary food source for clams in bioassay. Twelve liter polycarbonate carboys were tested as a substitute for fragile glass carboys. The growth and life cycle of G. breve were found to be comparable in both types of carboys. However, in the twenty liter polycarbonate carboys, the stationary phase of G. breve was prolonged. Cultures were maintained without inoculation in these carboys for 279 days. The randomly monitored parameters of dissolved oxygen, pH and salinity demonstrated an extended typical “stationary phase”. C. breve culture was received from Dr. David Millie, USDA. New Orleans, and was used to simulate repopulation of our culture supply after a facility culture crash. The culture was successfully grown from test tube to carboy in 300 days. Table 1. Monthly combined production and use of laboratory G. breve culture. Total volume Total volume Net change in of new culture of culture total culture inoculated (L) used (L) volume (L) Months July 1994 108 174 -66 August 1994 180 102 78 September 1994 54 66 -12 October 1994 180 150 -30 November* 1994 198 186 -12 December 1994 120 120 0 (up to the 21st) January 1995 155.5 151.5 4 February 1995 118 146 -28 March 1995 228 247 -19 ApriI 1995 185 206 -21 May 1995 130 72 +58 June 16th 1995 0 0 0 * in the month of November, the culture room had an AC problem which contributed to loss of culture. Ill. ECOLOGICAL INTERACTION STUDIES A. Brevetoxin Ingestion in Black Seabass 1) Materials and Methods Fish are very susceptible to red tide toxins when they enter the blood stream through the gills. The effect from ingestion, however has not been established. This study was undertaken to investigate the effect of brevetoxins ingested by black seabass and the fate of the ingested toxins. Black seabass (Centropristis striatus), 16.5 to 20.5 cm in length, were placed three each in (250 L) aquaria and fed a daily diet of commercial fish feed pellets (Ziggler, Inc., P.A.). Brevetoxin was ingested by daily feeding with pieces of shrimp spiked with known amounts of brevetoxins. The brevetoxin concentrations were 0, 100, 250 & 500 µg/day, fed continuously over a 4 day (96 hour) period. The source of the brevetoxins was cultures of G. breve grown at Mote Marine Laboratory. Brevetoxins were extracted from the cultures and isolated according to the procedure of Pierce et al., 1992, with quantitative analysis performed by HPLC. Known quantities of toxin were injected into pieces of shrimp in methanol solution, and the shrimp were subsequently fed to each fish. Fish were fed about 4 g of shrimp twice daily for four days, with half of the daily toxin dose provided each time. Ingestion of the shrimp was carefully monitored. 2