Effects of salinity, light and inorganic nitrogen on growth and

Journal of Plankton Research Vol.21 no.5 pp.939–955, 1999

Effects of salinity, light and inorganic nitrogen on growth and toxigenicity of the marine dinoflagellate Alexandrium tamarense from northeastern Canada Jean-Paul Parkhill and Allan D.Cembella1,2 Center for Environmental Observation Technology and Research, Department of Oceanography, Dalhousie University, Halifax, Nova Scotia, B3H 4J1 and 1Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada 2To

whom correspondence should be addressed

Abstract. Growth and toxin production of a highly toxic clone of the marine dinoflagellate Alexandrium tamarense, isolated from the lower St Lawrence Estuary (Quebec) in eastern Canada, were studied in unialgal batch cultures under different conditions. Controlled experiments were conducted on the production of paralytic shellfish poisoning (PSP) toxins under conditions of varying light (40, 60, 150, 230 and 470 µmol m–2 s–1), salinity (10, 15, 20, 25 and 30‰) and nitrate concentrations (0, 88, 264, 528 and 880 µmol l–1). The effects of variable environmental factors on both toxin composition (% molar) and cell toxicity [pg STXeq (saxitoxin equivalents) cell–1] were determined through the culture cycle. The toxin profile (% molar; mean ± SD), determined by high-performance liquid chromatography with fluorescence detection (HPLC-FD), remained stable and was consistently dominated by the low-potency N-sulfocarbamoyl toxins C1/C2 (64.0 ± 3%). There were also substantial relative amounts of the high-toxicity carbamate derivatives gonyautoxin 1–4 (GTX1–4) (1.7 ± 0.5%), neosaxitoxin (NEO) (16.2 ± 2%) and saxitoxin (STX) (17.8 ± 2%). The cellular toxicity (mean ± SD: 58.8 ± 7 pg STXeq cell–1) was essentially independent of light, salinity and nitrate concentration throughout the exponential growth phase, but varied over the growth stages in culture. A positive correlation was observed between cellular toxicity and salinity-dependent growth rate, indicating that cell toxin quota may be affected by extrinsic factors, but it is not always a direct functional response to specific environmental stress.

Introduction Models of harmful algal bloom dynamics (Ouchi, 1982; Paerl, 1988; Franks, 1997) have been traditionally based upon the integration of data on environmental factors, including light, nutrient levels, temperature, water column stability and advection, with intrinsic cellular properties, such as those controlling growth rate and nutrient uptake and assimilation (Tett, 1987). In spite of efforts to define the role of environmental factors in the population dynamics of toxic species (Watras et al., 1982; Langdon, 1987; Smayda, 1990), exogenous and endogenous mechanisms regulating toxin production among toxigenic phytoplankton species remain poorly understood. Recent reviews of autecological studies on the relationship between cellular toxicity and environmental factors (Bates, 1998; Cembella, 1998; Wright and Cembella, 1998) reveal a complex relationship of growth rate with cell toxin quota and ambient nutrient levels, which is not entirely consistent among (or even within) species. Blooms of species of the dinoflagellate genus Alexandrium (Halim) Balech are responsible for most incidents of paralytic shellfish poisoning (PSP) in temperate coastal waters throughout the world (Anderson, 1998). There is little information © Oxford University Press

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on the toxicity and growth kinetics of Alexandrium spp. from high latitudes and cold waters, where bloom initiation tends to be delayed until after a late ‘spring’ bloom (dominated by centric diatoms). The period during which these toxic Alexandrium blooms can occur is temporally constrained by a relatively short summer. In the lower Estuary and Gulf of Saint Lawrence in eastern Canada, the species Alexandrium tamarense (Lebour) Balech (synonymous with Gonyaulax tamarensis, Protogonyaulax tamarensis and Alexandrium excavatum) produces extensive recurrent blooms during summer (Larocque and Cembella, 1990). Blooms of A.tamarense in the lower estuary typically occur during the period of maximum sea surface temperature (8–15°C) and at relatively low salinity (21–30‰) (Cembella and Therriault, 1989; Larocque and Cembella, 1990). Extremely high levels of PSP toxicity in bivalve shellfish from the lower estuary are associated with these blooms (Prakash et al., 1971; Therriault et al., 1985). The unidimensional effects of varying single environmental parameters, including salinity (Prakash, 1967; Prakash et al., 1971; White, 1978), light (Ogata et al., 1989), temperature (Ogata et al., 1989) and inorganic macronutrient concentrations (Boyer et al., 1985, 1987), on cellular growth and toxin production in batch cultures have been established for several Alexandrium strains. Unfortunately, a holistic interpretation of the data is difficult, due to methodological differences among experiments, the influence of growth stage and pre-conditioning regime in culture, and clone-specific responses of various Alexandrium strains subjected to comparable environmental conditions. Different authors came to contrasting conclusions regarding the effect of salinity (Prakash, 1967; White, 1978) and nutrients (Boyer et al., 1985, 1987; Anderson et al., 1990; Flynn et al., 1994). The conflicting results prompted the current investigations into the growth and toxin content of A.tamarense in relation to salinity, light and inorganic nitrogen levels. The cellular toxin content of cultured and natural populations of A.tamarense from the St Lawrence region is unusually high and the toxin profile is characteristic of these waters (Cembella et al., 1988; Cembella and Therriault, 1989; Cembella and Destombe, 1996). The purpose of our investigations was to examine the multidimensional relationships among light, salinity, nitrate concentration, growth rate and toxin production in unialgal batch cultures of a hightoxicity clonal isolate from the lower St Lawrence Estuary. The growth kinetics and stability in toxin content and composition of Alexandrium cells were determined in response to environmental gradients. A secondary objective was to establish whether there was a linear relationship between in vivo chlorophyll a fluorescence and cell concentration throughout the culture cycle, in which case fluorescence could be used to track cellular growth. Method Culture conditions and growth rate determinations Alexandrium tamarense clone Pr18b was isolated in 1985 from the lower St Lawrence Estuary near Rimouski, Quebec (68°329W–48°329N). All culture 940

PSP toxin production in Alexandrium

glassware was autoclaved and the sterile medium was added aseptically. A 2.0 l unialgal stock culture was grown on enriched K-medium (Keller and Guillard, 1985) added to pre-filtered (0.30 µm) autoclaved sea water at a salinity of 30‰ collected from Sandy Cove near Halifax, Nova Scotia. The stock culture was grown until late exponential growth phase at a temperature of 15 ± 0.5°C on a 14:10 h light:dark photocycle at an ambient photon flux density of 450 µmol m–2 s–1 provided by cool-white fluorescent tubes. At a concentration of ~8000 cells ml–1, 5 ml of culture were inoculated into 50 ml borosilicate glass culture tubes each containing 30 ml of K-medium prepared with pre-filtered (0.45 µm) autoclaved sea water. In the salinity experiment, the filtered sea water was diluted with deionized water (SuperQ, Millipore) to the five desired salinity levels (10, 15, 20, 25 and 30‰). Salinity was verified using an Abbe refractometer. Twelve replicate tubes at each salinity were autoclaved and nutrients were added aseptically after dilutions. In the light experiment, the number of neutral-density screening layers surrounding the racks controlled the ambient light level. No screening was added at the highest light, whereas four layers were added to yield the lowest level, thereby creating a light gradient of 470, 230, 150, 60 and 40 µmol m–2 s–1 for the five treatments. Light was measured with a 4p digital scalar photometer (Biospherical Model QSL-100H). In the nitrate experiment, the K-medium was altered by omitting the nitrogen sources (NH4Cl and NaNO3). No effort was made to remove residual nitrogen from the sea water used, other than by filtration of particulates through a 0.45 µm Millipore filter. Nitrate was added aseptically after autoclaving to yield the following concentration gradient: 0, 88, 264, 528 and 880 µmol l–1. The 60 vials in each of the individual experiments were rotated daily in the growth room to equalize the incident light field and they were also manually agitated once every 24 h. At each environmental level, two replicates (10 vials total) were harvested at each of the six time points during the 2 week experiment. The samples were harvested at 08:00 h on each sampling day, and in vivo chlorophyll a fluorescence was measured with a Turner Designs fluorometer. A 1 ml subsample was fixed in Lugol’s iodine solution and counted in a Palmer Maloney counting chamber (0.1 ml) by optical microscopy. Cell harvest and toxin analysis The remainder of each sample after cell counts was retained for toxin analysis by centrifuging 34 ml of culture in a refrigerated centrifuge for 20 min at 6675 g. The supernatant was carefully aspirated away with a vacuum pipette system and discarded. The cell pellet was suspended in filtered (0.45 µm) sea water and recentrifuged in a 2 ml cryovial at 15 000 g for 10 min; the supernatant was discarded. One milliliter of 0.03 M acetic acid was added to the pellet and the sample was ultrasonicated at high intensity (>25 W) using a 1/4 inch microtip at 40% duty cycle in 1 min pulses for 3 min. The sample was then examined by phase-contrast microscopy (3200) to determine the extent of cell lysis. The sample was 941


Fig. 1. Structures of PSP toxins, including carbamate, N-sulfocarbamoyl and decarbamoyl derivatives, found in toxigenic dinoflagellates. STX, saxitoxin; NEO, neosaxitoxin; GTX 1, 2, 3, 4, gonyautoxins 1, 2, 3, 4; dc, decarbamoyl analogues.

centrifuged at 12 850 g for 10 min, after which the supernatant was filtered through a 0.22 µm Millex GV4 filter, refrigerated at 4°C and stored. Analysis of the principal PSP toxins (Figure 1) by high-performance liquid chromatography with flourescence detection (HPLC-FD) was performed by minor modifications of the post-column oxidative fluorescence method of Oshima and Yasumoto (1989). Toxins were resolved by reverse-phase chromatography using a silica-base column (Inertsil C8; 4.6 mm i.d. 3 150 mm; 5 µm particle size; GL Science). Three separate isocratic elutions were employed to separate the spectrum of PSP toxins at a flow rate of 0.8 ml min–1. The gonyautoxins (GTX1–4) and the N-sulfocarbamoyl toxin B1 were resolved using 10 mM aqueous ammonium phosphate at pH 7.1, with 2 mM heptane sulfonate (Na salt) serving as the ion-pairing reagent. To separate neosaxitoxin (NEO) and saxitoxin (STX), 2 mM heptane sulfonate (Na salt) was substituted as the ion-pairing reagent in 30 mM aqueous ammonium phosphate buffer (pH 7.1) with 4% acetonitrile. The N-sulfocarbamoyl (C-) toxins were resolved using 2 mM tetrabutyl ammonium phosphate adjusted to pH 5.8 as the mobile phase. After oxidation in a post-column reaction system (carbamate analyzer, Waters Assoc., Milford, MA) with 5.0 mM alkaline periodate in 100 mM phosphate 942


buffer (pH 7.8), the column effluent was neutralized with 0.75 M nitric acid. Fluorescent toxin derivatives were detected in an 8 µl flow cell using a spectrofluorometer (Model 470, Waters Assoc., Milford, MA) equipped with a dualmonochromator and a high-output (150 W) xenon lamp with excitation wavelength set at 330 nm and emission at 390 nm (10 nm slit width). Chromatographic profiles were determined by duplicate injections of 10 µl of extracts (diluted 1:10 with 0.03 N acetic acid, as necessary). Toxins were quantified with certified external toxin standards (PSP-1) provided by the Chemical Reference Materials Program (CRMP) of the Institute for Marine Biosciences. Additional replicate injections, including ‘spikes’ of internal toxin standards, were used frequently in the case of discrepancies. For subsequent comparison with toxicity data determined by the Association of Official Analytical Chemists (1984) mouse bioassay, the toxicity of A.tamarense [in saxitoxin equivalents (STXeq)] was calculated from the HPLC chromatograms, with reference to the peak area of the PSP-1 external standard. The toxin concentrations were multiplied by a toxin-specific conversion factor to yield toxicity. The contribution of an individual toxin x to cellular toxicity Tx is given as (VE/N) · (Cx) · (STx), where VE is the volume of the extract (l), N is the number of extracted cells (3 106), Cx is the toxin concentration (µmol l–1) and STx is the specific toxicity (µg STXeq µmol–1). The sum of the individual toxin concentrations was used to determine total toxicity as follows: TT = T1 + T2 + T3 + T4 . . . Tn where TT, the total toxicity (pg STXeq cell–1), is the sum of the toxicities contributed by n individual toxins. The specific toxicity conversion factors of the individual toxins were adopted from Oshima et al. (1992) based upon empirical mouse bioassay data determined using purified standards, and assuming the conversion factor of 1 mouse unit (MU) = 0.23 µg STXeq for the ddy mouse strain. The data analysis for the three individual experiments consisted of a one-way ANOVA applied to the growth rate at different gradient levels, regressed from individual growth curves, to determine whether the growth rates varied at the different factor levels. Tukey’s multiple comparison test was used to determine differences. Simple linear regression was performed to determine the relationship between chlorophyll a fluorescence and cell concentration in the individual experiments. A two-way ANOVA was performed on the amount and composition of toxin over time and between treatments for each of the samples to determine whether differences were significant. A one-way ANOVA was also applied to the relationship between toxicity and mean division rate to determine whether an effect was expressed. Results A linear relationship between Alexandrium cell concentration, as determined by phase-contrast microscopic counts, and in vivo chlorophyll a fluorescence 943


throughout the exponential growth phase was demonstrated by simple linear regression (Figure 2). The regression line from the light (y = 0.054 + 3.125x), salinity (y = 2.179 + 2.429x) and nitrate (y = –0.448 + 3.290x) experiments provides evidence that in vivo chlorophyll a fluorescence is a good index of cell concentration (r = 0.95, 0.61 and 0.97, respectively) in batch cultures for the range of environmental parameters. A strong linear relationship between cell concentration and total toxin production in the cultures was also observed (r = 0.87) and found to be highly significant (P = 0.001). Linear regression accounted for significant variation in the light, salinity and nitrate levels (r = 0.93, 0.82 and 0.88, respectively) (Figure 3). Cell concentrations from two replicates were averaged and growth curves for each gradient level were used to calculate the mean growth rate from each graph at the maximum slope. The relationships among the mean growth rates over the gradient of experimental conditions are shown in Figures 4–6. These figures show that as the extremes of the different environmental conditions tested were reached, there was a decline in the division rate of A.tamarense. In the light experiment, a one-way ANOVA showed that mean growth rates

Fig. 2. Relationship between cell concentration and in vivo chlorophyll a fluorescence under varying environmental conditions. (A) light; (B) salinity; (C) nitrate concentration.

Fig. 3. The relationship between cell concentration and total toxin production under varying environmental conditions. (A) light; (B) salinity; (C) nitrate concentration.

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Fig. 4. The specific toxin composition for the major constituents STX, NEO, GTX and C-toxins at varying levels of light (µmol m–2 s–1), (A) 470, (B) 230, (C) 150, (D) 60, (E) 40, with their respective individual growth curves in the inset. Error bars represent ± 1 SD (n = 4). Where the error bars are not visible, the error term is below the size of the symbol.

were significantly different among the various light levels (P < 0.05). The growth rate was significantly reduced from that at intermediate light for the two lowest levels (40 and 60 µmol m–2 s–1); at the highest light level (470 µmol m–2 s–1), photoinhibition was possible, but this was not significant at P = 0.10. The maximum growth rate was 0.41 divisions day–1 at a photon flux density of 230 µmol m–2 s–1. The salinity gradient experiment showed variation in the growth rate over the varying levels (P < 0.001). Growth was inhibited at the lowest salinity (10‰), whereas the maximum growth rate of 0.50 divisions day–1 occurred at 25‰. The differences in growth rate were not significant at salinity levels between 20 and 30‰. The one-way ANOVA for the nitrate gradient experiment indicated that nitrate levels significantly affected the growth rate (0.05 < P < 0.10), although differences at the highest concentrations (>500 µM) were difficult to discern. The highest mean growth rate (0.35 divisions day–1) was reached at the second highest nitrate level (528 µmol l–1). Among the three experiments on different environmental parameters, the largest variation in toxicity was apparent from the salinity experiment (a range 945


Fig. 5. The specific toxin composition for the major constituents STX, NEO, GTX and C-toxins at varying levels of salinity, (A) 10‰, (B) 15‰, (C) 20‰, (D) 25‰, (E) 30‰, with their respective individual growth curves shown in the inset. Error bars represent ± 1 SD (n = 4). Where the error bars are not visible, the error term is below the size of the symbol.

of 31–179 pg STXeq cell–1). The toxicity varied over the entire culture cycle including perturbations from both the lag and post-exponential (stationary) phases of growth. For example, at the two lowest salinities, there was an initial decrease in the cellular toxicity, after which there was a recovery to levels similar to those at higher salinities. For the nitrate concentration and light experiments, variation was more conservative, yielding a range of 25–49 and 15–95 pg STXeq cell–1, respectively. A two-way ANOVA performed on toxin concentration data for the gradients for the three environmental conditions (salinity, light, nitrate) showed no significant difference of averaged means of total toxicity among the three experiments (P > 0.05). During the exponential growth phase, cellular toxicity varied within a much narrower range than for the entire culture cycle: 46–75 pg STXeq cell–1 for the salinity series, 34–49 pg STXeq cell–1 for the nitrate gradient, and 50–83 pg STXeq cell–1 among the different light levels. There were no major compositional changes in the toxin profile between levels within the respective experiments over the 2 week experiment for the principal toxins in this strain of A.tamarense (Figures 4–6). On a relative basis (% molar), the N-sulfocarbamoyl (C-) toxins represented the largest component of the total 946


Fig. 6. The specific toxin composition for the major constituents STX, NEO, GTX and C-toxins at varying levels of nitrate (µmol l–1), (A) 0, (B) 88, (C) 264, (D) 528, (E) 880, with their respective individual growth curves in the inset. Error bars represent ± 1 SD (n = 4). Where the error bars are not visible, the error term is below the size of the symbol.

toxin composition (64 ± 3%), but their contribution to total cellular toxicity was much less significant (30‰ and