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Seasonal variation in nutrient (nitrogen: N, and phosphorus: P) limitation of phytoplankton biomass growth was studied in the eutrophic estuary, Hjarbaek Fjord, Denmark, by use of 4 different types of bioassays. These were (1) long-term (2 d) nutrient addition bioassay, (2) long-term dilution bioassay, (3) short-term (2 h) nutrient addition bioassay, and (4) short-term dilution bioassay. Types 1 and 2 were conducted as field incubations where increases in chlorophyll a (chl a) were used as a growth indicator, while Types 3 and 4 were laboratory incubations with measurement of 14 C-uptake rates. Bioassays were compared with seasonal variations in carotenoid:chl a pigment ratio (480:665 nm), nutrient pools (C:N:P ratios) in seston, concentrations of dissolved nutrients (N and P) in the water, and with the supply of nutrients (N and P) to the phonic zone. P-limited growth dominated from May to August according to most bioassays, whereas N limitation was only significant in the long-term nutrient addition bioassay in August. Estimates of nutrient loading to the phonic zone included external input from freshwater and marine sources and nutrients regenerated from the sediment. The molar nutrient loading ratio (N:P) decreased through the summer from values of 290 and 110 for inorganic and total N:P, respectively, in April to a minimum value of 21 and 24 for inorganic and total N:P, respectively, in August. The minimum value coincided with the lowest inorganic N:P ratio in the surface water (3.5) and with a low ratio (15) of particulate N:P in the water column. Both short- and long-term nutrient addition bioassays corresponded well with the type of nutrient limitation that could be predicted from the seasonality in the N:P loading ratio and the inorganic N:P ratio in the surface water. The dilution bioassays gave less clear results, but may still provide information on possible nutrient limitation in a situation where the external loadings of both N and P are decreased. The carotenoid:chl a ratio varied between 1.4 and 2.6 with the lowes values in April and October, where nutrient limitation was less evident, and with the highest value in August. The 480:665 ratio always decreased during long-term bioassays when nutrient addition stimulated growth, which implies that it may be indicative of P- as well as N-limited growth.

Nutrient addition bioassays as indicators of nutrient limitation of phytoplankton in an eutrophic estuary