1. The increase in the frequency of red tides (dinoflagellate blooms) in the past several decades has been linked to anthropogenic inputs of nitrogen and phosphorus. This study investigated the effects of these two nutrients on the intrinsic rate of increase r and population size of the marine dinoflagellate Prorocentrum balticum. Peak population densities were greatest in the nitrogen treatment, followed by the combined nitrogen and phosphorus treatment, and then the phosphorus treatment. Within the treatment groups, peak population densities consistently occurred at lower nutrient levels. When either the level of nitrogen or phosphorus in the culture medium was increased, the intrinsic rate of increase was reduced. When nitrogen and phosphorus were increased simultaneously, the intrinsic rate of increase did not change. These results suggest that dinoflagellate blooms may be dependent upon more than one limiting nutrient. Effects of nitrogen and phosphorus on the growth of Prorocentrum balticum, a marine dinoflagellate ABSTRACT Stephanie E. Maihan and Karl W. Kleiner, Department of Biological Sciences, York College of Pennsylvania, York, PA 17405. INTRODUCTION Based on a pilot study: Culture of Prorocentrum balticum Cultures were grown in 50 mL Erlenmeyer flasks using 25 mL of seawater collected from Wallops Island, VA. Seawater was enriched with an f/2 vitamin solution, and adjusted to a pH of 8-8.2 and a salinity of 32 ppt. Cultures were grown under controlled conditions Experimental Treatments  3 nutrient regimes: Nitrogen, Phosphorus, Nitrogen + Phosphorus  5 treatment levels Nitrogen - (7, 16, 27, 45, and 58 mg/L) with 6 mg/L P Phosphorus - (2, 4, 6, 8, and 10 mg/L) with 27 mg/L N Nitrogen + Phosphorus – (7/2, 16/4, 27/6, 45/8, and 58/10 mg/L N/P)  3 replicates per treatment level  Nitrogen was supplied by NH 4 NO 3, and NaNO 3, and phosphorus was supplied by NaH 2 PO 4 (Reigman et al. 1993, Ryther and Dunstan 1971). Measurements and calculations The cultures were counted every other day using a 0.1 mL Palmer Maloney counting chamber after the sample was fixed with Lugol’s iodine solution. The per capita exponential growth constant r was determined for each sampling interval using the equation r = ln(N t+1 /N t ) (Hunter, M. 2001). The r values for each culture were regressed against population density: r = m * (population size) - r max. The r max, which is the value of r when the population was low and resources were not limiting, was estimated from the y-intercept. Linear regression was then used to test for a positive linear relationship between nutrient availability and population growth (Graph Pad Prism ver. 3.02). METHODS Populations from all three treatments: nitrogen, phosphorus, and nitrogen and phosphorus combined, had similar growth curves (Figure 1) Lower nutrient levels grew faster than higher nutrient levels Comparison of intrinsic rate of increase, r max, between nutrient levels yielded slopes that do not significantly differ from zero for all nutrient regimes (Figure 2) Trends are discernable:  Decrease in r max as nitrogen and phosphorus independently increase  Increase in r max as nitrogen and phosphorus combined increase Comparison of peak population size between nutrient levels yielded slopes that do not significantly differ from zero for all nutrient regimes (Figure 3)  In general as nutrient levels increased, the maximum population size decreased. RESULTS Dinoflagellates are microscopic, flagellated protists which play an important role in the marine environment both as primary producers and as the causative agents of red tides or harmful algal blooms (Vymazal 1995). The frequency of red tides is on the rise, especially in the estuarine and coastal environments where the waters receive inputs of nutrients from many sources such as rivers, sewage, and agricultural runoff. Two of the major nutrients present in the runoff are nitrogen and phosphorus. Although there is a correlative relationship between the presence of excess nutrients and algal blooms, few studies have examined the causal relationship between nutrient availability and the growth of dinoflagellate populations (Reigman et al. 1993, Schollhorn and Graneli 1993). In this study, we investigated which nutrients, nitrogen, phosphorus, or the two combined, had the greatest impact on the growth rate and maximum population size of the marine dinoflagellate, Prorocentrum balticum. We hypothesized that as nutrient levels increased, the growth rate and maximum population size of Prorocentrum balticum would increase. We anticipated that the combined treatment of nitrogen and phosphorus would have the greatest impact on growth, followed by nitrogen, then phosphorus. Figure 1. Population growth curves of three different treatments: (a) nitrogen, (b) phosphorus, and (c) nitrogen and phosphorus combined. Error bars represent one standard error of the mean. Figure 2. Comparison of the intrinsic rate of increase r between nutrient levels of (a) nitrogen, (b) phosphorus, and (c) nitrogen and phosphorus combined. Error bars represent one standard error of the mean. Figure 3. Comparison of peak population size between nutrient levels of (a) nitrogen, (b) phosphorus, and (c) nitrogen and phosphorus combined. Error bars represent one standard error of the mean. Population Curves Our hypothesis that the growth rate and maximum population size of Prorocentrum balticum would respond positively to increased nutrient availability was not supported. There was no significant overall difference among treatment levels within the different nutrient regimes. Increasing either nitrogen or phosphorus alone resulted in lower exponential growth rates r max, but not when nitrogen and phosphorus were both increased. This would indicate that both nitrogen and phosphorus are necessary for growth. Moreover, an abundance of either nitrogen or phosphorus alone may not be sufficient to encourage growth in the absence of ample quantities of the other nutrient. Our findings that the greatest intrinsic rate of increase and population size did not occur at the highest nutrient levels are consistent with other studies. Siu et al. (1997) found that when dinoflagellates are presented with varying nutrient levels, in general it is the middle range of nutrients that produces maximum growth. One possibility that P. balticum did not respond positively to increasing nutrient availability was inadequate micronutrient nutrition, which is important for dinoflagellate growth (Siu et al. 1997). Water collected at a different time of the year had greater levels of macronutrients (N, P) than the water used in this study. Assuming that the amount of macronutrients present in seawater is directly related to the amount of micronutrients, there is a distinct possibility that the water with the lower levels of nitrogen and phosphorus were also lacking in micronutrients. If this was the case, then micronutrients could have been a limiting factor in the water used for our experiment. DISCUSSION Intrinsic Rate of Increase Maximum Population Size Hunter, M.D. 2001 December 5. Fwd: Calculating r [email to Maihan, S.]. Available from: smaihan@ycp.edu. Riegman, R., Rowe, A., Noordeloos, A.A.M., and Cadee, G.C. 1993. Evidence for eutrophication induced Phaeocystis sp. blooms in the Marsdiep area (The Netherlands). Pages 799-805 in Smayda, T.J. and Shimizu, Y. (eds.). Toxic Phytoplankton Blooms in the Sea. Elsevier Science Publishers, New York, NY. Ryther, J.H. & Dunstan, W.M. 1971. Nitrogen, phosphorus, and eutrophication in the coastal marine environment. Science 171, 1008-13. Schollhorn, E. and Graneli, E. 1993. Is the increase of flagellates in coastal waters caused by changes in ratios of N, P, and Si? Pages 811-817 in Smayda, T.J. and Shimizu, Y. (eds.). Toxic Phytoplankton Blooms in the Sea. Elsevier Science Publishers, New York, NY. Siu, G.K.Y., Young, M.L.C. and Chan, D.K.O. 1997. Environmental and nutritional factors which regulate population dynamics and toxin production in the dinoflagellate Alexandrium catenella. Hydrobiologia 352:117-140. Vymazal, J. 1995. Algae and element cycling in wetlands. Lewis Publishers, Boca Raton, FL. LITERATURE CITED Dr. Gregory Foy, Assistant Professor of Chemistry Chris Cunningham, Laboratory Technician Tracey Riggens, Assistant Curator at CCMP ACKNOWLEDGEMENTS