Dataset: Growth and Phyto
Deployment: AT11-30

Methods for In situ Phytoplankton Growth and Grazing Rate Measurements



Growth rate and microzooplankton grazing rates on total, >5 and were measured on the Washington coast on six ECOHAB PNW cruises from 2003-2006.



Estimates of in situ phytoplankton growth rate (µu, d-1) and grazing (g, d-1) of

size-fractionated Chl a ( 5 µm) were determined simultaneously using the seawater dilution technique (e.g., Landry et al. 1995). Seawater was collected
from the depth corresponding to 50% surface irradiance and was typically between 3 and 5 m depth. Particle-free filtered seawater (FSW) was made by first pooling the water of several Niskin bottles into a 50 L polyethylene carboy and then gravity filtering this water through an in-line cascade of 3 µm and 0.2 µm Pall-Gelman pleated capsule filters and into a 20 L polycarbonate carboy. Experimental bottles (2.5 L polycarbonate bottles) were filled to pre-determined levels with FSW. All containers, tubing, and in-line filters were acid-cleaned prior to use with 5% (v/v) HCl acid and rinsed copiously with deionized water.
Clean techniques were used throughout all experimental and sample manipulation.



Whole seawater (WSW) was drained from several Niskin bottles (same cast as FSW) using silicone tubing wrapped with 200 µm mesh into a 50 L polyethylene carboy.
The WSW was kept well-mixed by gentle stirring with a polyethylene plunger.
The WSW was siphoned from the 50 L WSW carboy into the experimental bottles containing the PFW to reach either three (0.1, 0.5, and 1.0 WSW) or five (0.1, 0.2, 0.4, 0.7, and 1.0 WSW) target dilution levels. Experimental bottles were amended with nutrients to achieve enrichments of 10 µmol L-1 nitrate (NaNO3), 0.63 µmol L-1 phosphate (NaH2PO4 * H2O), 10 µmol L-1 silicic acid (Na2O3Si * 9H2O),
and 3 nmol L-1 Fe (Fe in 2% HCl) to the ambient water concentrations. An additional set of 1.0 WSW bottles were filled without nutrient amendments to test for potential
nutrient limitation phytoplankton communities. Duplicate samples were randomly taken from the WSW carboy during water disbursement for chlorophyll, preserved samples and nutrients.



Dilution treatment bottles were placed in clear Plexiglas tubes covered with mylar film to simulate the in situ irradiance. The tubes were secured to a revolving wheel
(1 rpm) submerged in a Plexiglas on-deck incubator and incubated for 24 h. The temperature inside the incubator was maintained near in situ levels by continuously flowing surface seawater. Incident photosynthetically active radiation (PAR, umol quanta m-2 s-1) was measured with a Hobo Par Smart Sensor and data logger mounted on the incubator, and water temperature was monitored with a submerged Hobo Water Temp Pro data logger.



In each replicate dilution bottle, the nutrient-amended net growth rate (k_n) was determined according to k_n = ln(N1/N0)/t1-t0, where N1 and N0 are the final total and size-fractionated Chl a concentration at time 1 (t1) and time 0 (t0), respectively. The intrinsic rates of growth (µ, d-1) and mortality due to grazing by microzooplankton (g, d-1) of the size fractionated Chl a were calculated by linear regression of net growth rate (k_n) in each nutrient amended dilution bottle against the fraction of WSW, Di. Growth (µ) was determined by extrapolation of the regression to the ordinal intercept, where Di (proportional to grazing mortality, g) becomes zero, and hence, k_n = µ_n. Because nutrients were added to the treatment bottles, if phytoplankton growth is limited by in situ nutrient
concentrations, µ_n is a potential growth rate. When nutrient-limited growth was observed in the 1.0 WSW control bottles, the in situ intrinsic rate (µ_un), was estimated from µ_n = k_{un 1.0} + g, where k_{un 1.0} is the net growth rate in the 1.0 WSW treatment without added nutrients (Landry et al. 1995). Microzooplankton grazing on Chl a size fractions was determined by the slope of linear regressions of kn and Di. On two occasions dilution regressions showed evidence of saturated grazing kinetics (Gallegos 1989).
For these experiments, µ was calculated using the linear portion of the regression, while g was calculated using g = µ_n -k_{n 1.0}, wherek_{n 1.0} is the net growth rate in
the nutrient-enhanced 1.0 WSW dilution treatment.





Further details on measuring Pseudo-nitzschia-specific rates in these experiments can be found in:

Olson, M.B., Lessard, E.J., Cochlan, W.P., Trainer, V.L., 2008. Intrinsic growth and microzooplankton
grazing on toxigenic Pseudo-nitzschia spp. diatoms from the coastal northeast Pacific. Limnol. Oceanogr.
53, 1352-1368.