MBARI procedures for automated nutrient analyses using a modified Alpkem Series 300 Rapid Flow Analyzer

This report documents the methods used at the Monterey Bay Aquarium Research Institute (MBARI) for analyzing seawater nutrient samples with an Alpkem Series 300 Rapid Flow Analyzer (RFA) system. The methods have been optimized for the particular requirements of this laboratory. The RFA system has been used to analyze approximately 20,000 samples during the past two years. The methods have been optimized to run nutrient analyses in a routine manner with a detection limit of better than -±1% and a within run precision of -±1% of the full scale concentration range. The normal concentration ranges are 0-200 ~M silicate, 0-5 ~M phosphate, 0-50 ~M nitrate, 0-3 ~M nitrite, and 0-10 ~M ammonium. The memorandum is designed to be used in a loose-leaf binder format. Each page is dated and as revisions are made, they should be inserted into the binder. The revisions should be added into the binder. Retain the old versions in order to maintain a historical record of the procedures. (88 pages)

Nutrient-phytoplankton interrelationships in the Eastern Tropical Pacific Ocean

ENGLISH: Near surface nutrient distributions in the eastern tropical Pacific Ocean, using data from the EASTROPAC Expedition of 1967-68 and pre~EASTROPAC data, are described. Nutrient concentrations were maximal along the equator, in the Peru Current and its offshore extension, and in the Costa Rica Dome and westward tensions of this feature. Nutrient-poor water was found north of the equator well offshore. In this water nitrate was often undetectable (<0.1 µg-at/liter) at the surface, but phosphate and silicic acid concentrations were moderate. Enrichment experiments showed that nitrogen was the primary limiting nutrient in poor water even though large amounts of organic N were found. Half saturation constants (K s ) were determined for ammonium-supported phytoplankton growth. These data were used to calculate near-surface primary productivity values which compared favorably with 14C values. Assimilation ratio measurements indicated that algae were not extremely nitrogen-deficient. Laboratory-determined K, values for phosphate and silicic acid indicated that these nutrients were rarely limiting. In rich water, chlorophyll levels were less than expected from nutrient levels, and this anomaly may be related to limitation by nutrients other than nitrogen (N), phosphorus (P), or silicon (Si), or to grazing. SPANISH: Se describe la distribución subsuperficial de los nutrientes en el Océano Pacífico oriental tropical, empleando los datos de la Expedición EASTROPAC de 1967~68 y datos anteriores a éstos. La concentración de nutrientes fue máxima a lo largo del ecuador, en la Corriente del Perú, en su prolongación mar afuera, en el Domo de Costa Rica y en las prolongaciones occidentales de esta característica. Se encontraron aguas pobres en nutrientes al norte del ecuador y bastante mar adentro. En estas aguas el nitrato era casi imperceptible (<0.1 µg-at/litro) en la superficie, pero las concentraciones de fosfato y ácido silícico fueron moderadas. Los experimentos de enriquecimiento indicaron que el nitrógeno era el principal nutriente limitante en aguas pobres, aun cuando se encontraron grandes cantidades de nitrógeno orgánico. Se determinaron las constantes de saturación media (K s ) para el desarrollo del fitoplancton sostenido por el amonio. Estos datos se emplearon para calcular los valores de la productividad primaria cerca a la superficie que pueden compararse favorablemente con los valores del 14C. Las medidas de la proporción de asimilación indican que las algas no tenían una deficiencia extremada de nitrógeno. Los valores determinados en el laboratorio de K, para el fosfato y ácido silícico indicaron que estos nutrientes limitaron rara vez la producción. En aguas ricas, los niveles de clorofila fueron inferiores a lo esperado según los niveles nutritivos y esta anomalía puede relacionarse a la alimentación o a la escasez de otros nutrientes distintos al nitrógeno (N), fósforo (P) o silicio (Si).

Increased toxicity of Karenia brevis during phosphate limited growth: ecological and evolutionary implications

Karenia brevis is the dominant toxic red tide algal species in the Gulf of Mexico. It produces potent neurotoxins (brevetoxins [PbTxs]), which negatively impact human and animal health, local economies, and ecosystem function. Field measurements have shown that cellular brevetoxin contents vary from 1–68 pg/cell but the source of this variability is uncertain. Increases in cellular toxicity caused by nutrient-limitation and inter-strain differences have been observed in many algal species. This study examined the effect of P-limitation of growth rate on cellular toxin concentrations in five Karenia brevis strains from different geographic locations. Phosphorous was selected because of evidence for regional P-limitation of algal growth in the Gulf of Mexico. Depending on the isolate, P-limited cells had 2.3- to 7.3-fold higher PbTx per cell than P-replete cells. The percent of cellular carbon associated with brevetoxins (%C-PbTx) was ~ 0.7 to 2.1% in P-replete cells, but increased to 1.6–5% under P-limitation. Because PbTxs are potent anti-grazing compounds, this increased investment in PbTxs should enhance cellular survival during periods of nutrient-limited growth. The %C-PbTx was inversely related to the specific growth rate in both the nutrient-replete and P-limited cultures of all strains. This inverse relationship is consistent with an evolutionary tradeoff between carbon investment in PbTxs and other grazing defenses, and C investment in growth and reproduction. In aquatic environments where nutrient supply and grazing pressure often vary on different temporal and spatial scales, this tradeoff would be selectively advantageous as it would result in increased net population growth rates. The variation in PbTx/cell values observed in this study can account for the range of values observed in the field, including the highest values, which are not observed under N-limitation. These results suggest P-limitation is an important factor regulating cellular toxicity and adverse impacts during at least some K. brevis blooms.

Effects of iso-nitrogenous fertilizers as nutrient sources on carp polyculture in Bangladesh

A 120-day long experiment was conducted to find out the effects of urea plus triple super phosphate (UT), cow manure (CM) and poultry manure (PM) having iso-nil:rogen content on pond productivity and fish yield. Three fertilizer treatments, with three replicates each, were randomly assigned into nine earthen ponds of 100 m2 each. The stocking fish were rohu (Labeo ruhita), catla ( Catla catla) and mrigal ( Cirrhinus mrigala) in each treatment pond at the rate of 10000/ha with the ratio of 1:1:1. All ponds were fertilized fortnightly at the rate of 125 kg/ha urea plus 100 kg TSP/ha, 7000 kg/ha cow manure and 3500 kg/ha poultry manure for the treatment of UT, CM and PM, respectively, having an iso-nitrogen content of 56 kg in each. Though the physicochemical water quality parameters were more or less similar in all treatment ponds, the chlorophyll-a content and abundance of total plankton were significantly higher (P < 0.05) in the ponds receiving the treatment PM. Final growth as well as per unit production of fish was significantly higher (p

Effects of iso-nitrogenous and iso-phosphorus fertilizers as nutrient sources on carp polyculture in Bangladesh

A 120 day long experiment was conducted to find out the effects of cow manure with urea and triple super phosphate (CUT), poultry manure with urea and triple super phosphate (PUT) and cow manure with poultry manure (CP) having similar quantities of nitrogen and phosphorus on pond productivity and fish yield. The stocking fish were rohu (Labeo rohita), catla ( Catla catla) and mrigal ( Cirrhinus mrigala) in each treatment pond at the rate of 10000/ha. All ponds were fertilized fortnightly at the rate of 4000 kg/ha cow manure with 62 kg/ha urea and 65 kg/ha TSP, 2700 kg/ha poultry manure with 62 kg/ha urea and 16 kg/ha TSP, and 4000kg/ha cow manure with 2700 kg/ha poultry manure for the treatment CUT, PUT and CP respectively. Each treatment contained an iso-nitrogen and iso-phosphorus of 56 kg and 46 kg respectively. Though the physico-chemical parameters were more or less similar in all ponds, the chlorophyll-a content and abundance of total plankton were significantly higher (P< 0.05) in the ponds receiving the fertilizer treatment of PUT than those of other treatments. Final growth as well as per unit production of fish of treatment PUT (1773 kg/ha) was significantly higher (P< 0.05) than that of treatment CP (1528 kg/ha) followed by that of treatment CUT (1336 kg/ha). The over all results showed that poultry manure with urea and triple super phosphate proved to be superior to cow manure with urea and triple super phosphate, and poultry manure with cow manure, even when nitrogen and phosphorus content was similar, in carp polyculture system under prevailing conditions.

Nutrient profile of pond water in north-eastern state of Tripura and impact of water acidity on aquaculture productivity

Physicochemical parameters of 31 fish pond water samples of Tripura were studied to ascertain the nutrient profile of acidic soil zone and the impact of water acidity towards aquaculture productivity. The pH was acidic (mean 6.63±0.44) with high Fe (mean1.04±0.40 mglˉ¹) and AI (mean 2.67±2.41 mglˉ¹) contents. These were mostly responsible for pond water acidity and poor productivity with low nitrogen, phosphate and total alkalinity. The study also showed strong negative relationship between water pH and redox potential (R²=0.5251). However, pH was positively significant with electrical conductivity. The roles of redox potential and electrical conductivity in water acidity were found highly important. Available calcium content was also found low (mean 2.91±2.96 mglˉ¹). Elevating level of pH of pond water could be the possible management practices in acidic water so that such unproductive water might be productive enough with higher phosphate and nitrogen levels for better biological production.

Nutrient salts in the United Arab Emirates waters (the Arabian Gulf and the Gulf of Oman)

This paper deals with the levels and distributions of nutrient salts in the United Arab Emirates waters. Water samples were collected bimonthly during 1994-1995 from the marine environment of the United Arab Emirates, which extends more than 800km along the Arabian Gulf and the Gulf of Oman. Concentrations of ammonium, nitrite, nitrate, phosphate, silicate, as well as total concentrations of total dissolved nitrogen, phosphorus, and silicon in the area were scattered in the ranges: (ND-6.32; mean: 0.84 µg-at N/l), ND-3.02; mean: 0.42 µg-at N/l), (ND-10.88; mean: 1.18 µg-at N/1), (ND-4.22; mean: 0.62 µg-at P/l), (1.14-28.80; mean: 6.52 µg-at Si/l), (1.52-39.58; mean: 12.28 µg-at N/l), (0.40-4.98; mean: 1.07 µg-at P/l), and (2.77-44.74; mean: 13.02 Si/l) respectively. Of inorganic nitrogen species, ammonium was the highest in the Arabian Gulf waters and nitrate was the highest at the Gulf of Oman. The dissolved inorganic nitrogen total species, phosphate and silicate amounted to 16.4, 47.6, 56.5% respectively, of the concentrations of nitrogen, phosphorus and silicon in the Arabian Gulf and 22.6, 64.4, 44.9% respectively, in the Gulf of Oman, indicating that more than 80% of nitrogen was present in organic forms. Distributions of nutrient in the two regions were higher in the summer season and lower in the winter season due to the oxidation of organic materials. Regional distributions revealed higher values for nitrite (1.3 times), nitrate (2.8 times), phosphate (2.2 times), total dissolved nitrogen (1.3 times), total dissolved phosphorus (1.6 times), and total dissolved silicon (1.3 times) in the Gulf of Oman compared to the Arabian Gulf, indicating more oligotrophic conditions at the Arabian Gulf Whereas no distinct patterns of distribution were observed in the Arabian Gulf waters, an increase in the seaward direction was measured at the Gulf of Oman. Vertical distributions indicated a general increase with depth in the two regions. The mean ratios for total concentrations of phosphorus, nitrogen, and silicon in the Arabian Gulf (1: 11.6: 12.6) and the Gulf of Oman (1: 10.1: 11.8) were lower than the Redfield ratio.

Typhoon effects on DOC dynamics in a phosphate-limited reservoir

To explore typhoon effects on dissolved organic carbon (DOC) dynamics, field investigations (tributary and dam site) and laboratory experiments (bioassay and DOC consumption) were conducted in a subtropical reservoir. A tributary survey indicated that after typhoon disruption, upstream areas were the sources of phosphate (P) but not DOC for the dam site located downstream. Bioassay experiments verified P-limitation on bacteria and phytoplankton during summer stratification, and bacteria showed a faster response than algae to added P. Experiments indicated that DOC consumption was determined by the availability of P. The 4 yr typhoon period (June-September) data of the dam site denoted that DOC concentration (27 to 270 mu M C) and its rate of change (-13 to 24 mu M C d(-1)) varied more dramatically in the weak (2006 and 2007) than in the strong (2004 and 2005) typhoon years. The negative correlation of DOC with the ratio of bacterial production (BP) to primary production (PP) in the euphotic zone (0 to 10 m) signified the interactive effects of auto- and heterotrophic processes on DOC variation. In the aphotic zone, the variation of DOC could be ascribed to the change of BP, which showed a positive correlation with P concentrations. This study documents that DOC concentration in the studied system varied at multiple time scales. Such variation can be explained by the decoupling between BP and PP, which is believed to be a function of the limiting nutrient's availability. More importantly, this study suggests that the P supply introduced by strong typhoons might have substantiated a tighter coupling between BP and PP, so that the amplitude of DOC oscillation during the summer period was effectively reduced.

Nutrient Dynamics in the Upwelling Area of Changjiang (Yangtze River) Estuary

Nutrient dynamics and its influence on the distribution of chlorophyll-a in the upwelling area of the Changjiang (Yangtze) River estuary were investigated in the spring (May) and summer (August) of 2004. In the spring, upwelling was apparent in the region of 122 degrees 20'-123 degrees 00' E, 31 degrees 00'-32 degrees 00' N and was associated with low temperature (16-21 degrees C), high salinity (24-33 practical salinity units [psu]), and low dissolved oxygen (2.5-6.0 mg L-1) in the upper 10 m of the water column. The spring upwelling increased the mixed-layer phosphate, nitrate, and silicate concentrations to roughly 1, 15, and 15 mu mol L-1, respectively, and improved the light transparency in the euphotic zone. This improvement in phytoplankton growing conditions was followed by an increase in chlorophyll-a concentrations. The summer upwelling was weaker and occurred over a smaller geographical area (122 degrees 20'-123 degrees 00' E, 31 degrees 15'-31 degrees 50' N). Strongly influenced by turbid Changjiang diluted water (CDW), it had little impact on the upper 10 m of the water column but instead increased nutrient concentrations at greater depths. The high concentration of particulates in the CDW reduced light transmission in the upper 10 m and, hence, limited phytoplankton growth throughout the water column. Chlorophyll-a concentrations in the summer upwelling area were roughly an order of magnitude lower than in the spring. Water clarity, as influenced by the CDW, appears to be the principal factor limiting the impact of upwelling on phytoplankton biomass in this area.

Responses of a coastal phytoplankton community to increased nutrient input from the Changjiang (Yangtze) River

Nutrient input from the Changjiang River (Yangtze River) has been increasing dramatically since the 1960s. At the mouth of the Changjiang River, the nitrate concentration has increased about three-fold in 40 years, from 20.5 mu mol/L in the 1960s to 59.1 mu mol/L in the 1980s and to 80.6 mu mol/L in 1990-2004. Phosphate concentration increased by a factor of 30%, from 0.59 mu mol/L in the 1980s to 0.77 mu mol/L in 1990-2004. The increasing nitrate input has arisen mostly from the mid and lower reaches of the Changjiang River, where the river meets one of the most strongly developed agriculture areas in China. Responses of the coastal phytoplankton community to the increasing nutrient inputs are also seen in the available monitoring data. First, a trend of increasing phytoplankton standing stock from 1984 to 2002 appeared in the Changjiang River estuary and adjacent coastal waters, especially in late spring. Secondly, the proportion of diatoms in the whole phytoplankton community showed a decreasing trend from about 85% in 1984 to about 60% in 2000. Finally, red tides/harmful algal blooms increased dramatically in this area in terms of both number and scale. About 30-80 red tide events were recorded each year from 2000 to 2005 in the East China Sea. The scale of some blooms has been in excess of 10,000 km(2). (c) 2008 Elsevier Ltd. All rights reserved.

Long-term ecological interactions between nutrient and phytoplankton community in the Changjiang estuary

Both nitrate (NO (3) (-) ) and soluble reactive phosphate (PO (4) (3-) ) concentration in the freshwater end-member at the mouth of the Changjiang River have increased dramatically since the 1960s. Within the same period in the sea area, with surface salinity > 30, NO (3) (-) concentration has shown an obvious increase, PO (4) (3-) has not changed greatly and dissolved reactive silica (SiO (3) (2-) ) has deceased dramatically. An examination of the elemental ratio of NO (3) (-) to PO (4) (3-) at the mouth of the Changjiang River did not show a systematic trend from the 1960s to 2000s largely because both nutrients increased simultaneously. In comparison, the elemental ratio of dissolved inorganic nitrogen (DIN) to PO (4) (3-) in surface seawater, with salinity > 22, has shown a clearly increasing trend. Furthermore, an overall historical change of the SiO (3) (2-) :PO (4) (3-) ratio has undergone a reverse trend in this area. Based on the changes of SiO (3) (2-) :PO (4) (3-) and DIN:PO (4) (3-) ratios, we can conclude that an overall historical change of SiO (3) (2-) :DIN ratio has decreased in this area from the 1950-1960s to 2000s. The argument that phytoplankton productivity in the Changjiang estuary has been enhanced by increasing nutrient input from the riverine transport was supported by these results. A comparative study analyzing the shift of phytoplankton composition from the mid-1980s to 2000s was also made. The results indicated that the average yearly percentage of diatom species in the Changjiang estuary has decreased from 84.6% during 1985-1986 to 69.8% during 2004-2005. Furthermore, the average yearly percentage of diatom abundance in the Changjiang estuary decreased from 99.5% during to 75.5% over the same time period, while the abundance of dinoflagellates has increased dramatically, from 0.7% to 25.4%.

The characteristics of nutrient removal and inhibitory effect of Ulva clathrata on Vibrio anguillarum 65

To investigate the ecological effect of macroalgae on de-eutrophication and depuration of mariculture seawater, the variation of dissolved inorganic nitrogen (DIN) and phosphate (DIP), the amount of Vibrio anguillarum, and total heterotrophic bacteria in Ulva clathrata culture, as well as on the algal surface, were investigated by artificially adding nutrients and V. anguillarum strain 65 from February to April 2006. The results indicated that U. clathrata not only had strong DIN and DIP removal capacities, but also showed a significant inhibitory effect on V. anguillarum, although not reducing the total heterotrophic bacteria. Vibrio anguillarum 65 dropped from 5 similar to 8 x 10(7) cfu mL(-1) to 10 cfu mL(-1) (clone-forming units per mL) in 10 g L-1 of fresh U. clathrata culture within 2 days; i.e., almost all of the Vibrios were efficiently eradicated from the algal culture system. Our results also showed that the inhibitory effect of U. clathrata on V. anguillarum strain 65 was both DIN- and DIP-dependent. Addition of DIN and DIP could enhance the inhibitory effects of the algae on the Vibrio, but did not reduce the total heterotrophic bacteria. Further studies showed that the culture suspension in which U. clathrata was pre-cultured for 24 h also had an inhibitory effect on V. anguillarum strain 65. Some unknown chemical substances, either released from U. clathrata or produced by the alga associated microorganisms, inhibited the proliferation of V. anguillarum 65.

Impact of elevated levels of CO2 on animal mediated ecosystem function: The modification of sediment nutrient fluxes by burrowing urchins

A mesocosm experiment was conducted to quantify the relationships between the presence and body size of two burrowing heart urchins (Brissopsis lyrifera and Echinocardium cordatum) and rates of sediment nutrient flux. Furthermore, the impact of seawater acidification on these relationships was determined during this 40-day exposure experiment. Using carbon dioxide (CO2) gas, seawater was acidified to pHNBS 7.6, 7.2 or 6.8. Control treatments were maintained in natural seawater (pH8.0). Under normocapnic conditions, burrowing urchins were seen to reduce the sediment uptake of nitrite or nitrate whilst enhancing the release of silicate and phosphate. In acidified (hypercapnic) treatments, the biological control of biogeochemical cycles by urchins was significantly affected, probably through the combined impacts of high CO2 on nitrifying bacteria, benthic algae and urchin behaviour. This study highlights the importance of considering biological interactions when predicting the consequences of seawater acidification on ecosystem function.

Comparison of phosphate uptake rates by the smallest plastidic and aplastidic protists in the North Atlantic subtropical gyre

The smallest phototrophic protists (<3 μm) are important primary producers in oligotrophic subtropical gyres – the Earth's largest ecosystems. In order to elucidate how these protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4–29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (<2 and 2–3 μm). Both groups of plastidic protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times (P<0.001) higher than those of bacterioplankton, the biomass-specific phosphate uptake rates of protists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition – predation on phosphorus-rich bacterioplankton cells.

Nitrate and phosphate uptake characteristics of three species of brown algae cultured at low salinity.

Nitrate and phosphate uptake mechanisms have been characterised under conditions of 100 and 50% seawater in 3 common brown algae of NW Europe: Fucus vesiculosus, F. serratus and Laminaria digitata. Under low salinity, the growth rate and internal nitrate accumulation of F. serratus significantly increased (20 and 48%, respectively), but no significant changes were observed for F. vesiculosus and L. digitata. However, nitrate uptake rates were reduced in L. digitata, so that this species was less adaptable to low salinity than the Fucus species. Both F. vesiculosus and F. serratus reached a steady-state uptake rate after acclimation regardless of the salinity treatment. All 3 species had a high capacity for storing inorganic N and P intracellularly. The results for F. serratus pointed to a dual mechanism of adaptation to the special characteristics of the intertidal environment where it grows. Non-saturating (low affinity) nitrate uptake and biphasic (double Michaelis-Menten curve) phosphate uptake are adaptations to high nutrient concentrations. Temporal partition of cellular energy for carbon metabolism and nutrient uptake is also suggested as an adaptation to the transient nutrient inputs occurring in these environments.