The methods below are described in Sun et al. 2011.

Cultures and growth conditions
Stock cultures of marine diatom Pseudo-nitzschia multiseries (Hasle) (CCMP 2708, originally isolated from Eastern Canada) were maintained at 17 degrees C in 0.2 um-filtered, microwave-sterilized natural seawater, enriched with levels of phosphate, nitrate, silicate, vitamins, and trace nutrients as in Price et al. (1988). Light was provided on a 12 h dark:12 h light cycle using cool white fluorescent bulbs at 120 umol photons per square meter per second. Irradiance was measured with a biospherical LICOR sensor (model LI-250).

Experimental design and determination of growth rates
Semi-continuous culturing methods were used in order to measure the effects of P availability and/or pCO2 levels during acclimated, steady-state growth. Cultures were diluted daily with medium that was previously adjusted to the appropriate temperature and pCO2. Each bottle was diluted back to the same cell density present in that bottle directly after the previous day’s dilution. Cultures were harvested following approximately 4 to 6 weeks of semi-continuous incubation when they were fully acclimated to the experimental conditions, after statistically invariant growth rates were recorded for at least 4 to 6 consecutive dilutions.

Samples from each culture bottle were always taken at the same time in the diel cycle, between 09:00 h and 10:00 h in the morning, to measure cell density and thus determine changes in growth rate. Dilutions were done in real time using biomass estimates made by in vivo fluorescence, and were subsequently validated using preserved cell count samples. Growth rates were calculated based on the equation:
u = (lnNb - lnNa) / (tb - ta),
where Na and Nb are the average cell density at times ta (directly after a dilution) and tb (directly before the next day’s dilution). For cell counts, whole-culture samples were fixed with glutaraldehyde (2.5% v to v final concentration) and counted in triplicate. About 1000 cells per replicate were enumerated in a 1-mL Sedgewick-Rafter counting chamber, using an Olympus BX51 epifluorescence microscope at 100-fold magnification.

Triplicate bottles at two conditions of phosphate availability were equilibrated at three different CO2 concentrations by gentle bubbling with commercially prepared certified standard air and CO2 gas mixtures (Praxair Gas). CO2 concentrations examined included preindustrial atmospheric levels (~22 Pa), near-present day concentrations (~41 Pa), and values predicted to occur before the end of this century (~74 Pa, IPCC 2007). In-line high efficiency particulate air (HEPA) filters were used to avoid contamination from particles in the gas tanks or lines. Phosphate levels used were 20 umol per liter (P replete) and 0.5 umol per liter (P limited). A total of six different phosphate and CO2 conditions were used in this study: 20 umol per liter P and ~22 Pa CO2; 20 umol per liter P and ~41 Pa CO2; 20 umol per liter P and ~74 Pa CO2; 0.5 umol per liter P and ~22 Pa CO2; 0.5 umol per liter P and ~41 Pa CO2; and 0.5 umol per liter P and ~74 Pa CO2.

Carbonate buffer system measurements and pCO2 treatments
The pH in each bottle was monitored daily using a high sensitivity microprocessor pH-meter (Orion EA 940), calibrated with pH 4, 7 and 10 buffer solutions. The relative precision of this instrument is ~0.01 and accuracy is ~0.03 pH units. For the analysis of total dissolved inorganic carbon (DIC), DIC samples were stored in 2 mL capped borosilicate vials free of air bubbles and were preserved with 20 uL saturated HgCl2 per liter, and stored at 4 degrees C until analyzed. Total DIC was measured by acidifying 2-mL 10% of H3PO4 and quantifying the CO2 trapped in an acid sparging column (model CM 5230) with a carbon coulometer (model CM 140, UIC). Certified reference materials obtained from Andrew Dickson (University of California, San Diego, http://andrew.ucsd.edu/co2qc/index.html) were measured periodically during the run and used for calibration. pH values remained invariant before and after the dilution, suggesting that bubbling rates were sufficient to maintain the target CO2 equilibration levels in the medium, regardless of diel changes in photosynthesis and respiration. Based in the daily measurements of pH and DIC, pCO2 stabilized during the early part of the semi-continuous growth period and then remained steady throughout the latter part of the incubation period. Calculated pCO2 values (using CO2SYS; http://www.cdiac.ornl.gov/ftp/co2sys/CO2SYS_calc_XLS ) for the three CO2 treatments in both P treatments ranged from 22-23 Pa, 39-42 Pa, and 73-75 Pa (see table below where the numbers in parenthenses are the standard deviations of triplicate samples), very close to the certified standard gas mixture values. For convenience, these values were averaged and rounded to 22 Pa, 41 Pa, and 74 Pa when referring to the three pCO2 treatments throughout the dataset and paper (Sun et al. 2011).

Treatment conditions and calculated pCO2:

Treatment	Measured pH (sd)	Measured DIC (sd); umol/L	Calculated CO2 (sd); umol/L	Calculated pCO2 (sd); Pa
P-limited, 22 Pa	8.38 (0.05)	1917 (38)	7.4 (0.6)	23 (2)
P-limited, 41 Pa	8.15 (0.02)	2029 (8)	13.9 (0.7)	42 (2)
P-limited, 74 Pa	7.94 (0.01)	2145 (9)	24.5 (0.6)	75 (2)
P-replete, 22 Pa	8.40 (0.03)	1970 (4)	7.1 (0.5)	22 (2)
P-replete, 41 Pa	8.19 (0.02)	2066 (11)	12.8 (0.8)	40 (3)
P-replete, 74 Pa	7.96 (0.01)	2177 (6)	23.9 (0.4)	73 (1)

Analysis of domoic acid concentrations
Particulate and dissolved domoic acid was measured using amnesic shellfish poison (ASP) enzyme-linked immunosorbent assay (ELISA) kits available from Biosense Laboratories. Particulate domoic acid samples were collected on uncombusted Whatman GF/F filters and frozen at –20 degrees C until analyzed. The filtrate from each sample was also collected, frozen and later analyzed for dissolved DA. Sample preparation and ELISA tests were carried out following the protocol of Biosense Laboratories (2005 version). The limit of detection for the ELISA method for particulate DA is 6.8 ng per liter. Total DA produced per cell (including the sum of both particulate and dissolved DA) was calculated by dividing the DA content of the whole-culture sample by the cell density.

References:
Platt, T., C. L. Gallegos, and W. G. Harrison. 1980. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. J. Mar. Res. 38: 687-701.