SUMMARY: Seawater is collected from known depths using CTD-rosette sampling procedures. Subsamples are drawn and stored frozen (-20°C) in acid-washed, HDPE bottles. Soluble reactive phosphorus (SRP) is measured colorimetrically following the formation of phosphomolybdic acid.
Phosphorus (P) is one of several essential nutrients required for the growth of all marine organisms. In open-ocean marine ecosystems, P is often present in low and, perhaps, limiting concentrations for microalgal and bacterial populations. Therefore, P is a central element in oceanic biogeochemical cycles.
In seawater, inorganic phosphorus (also referred to as orthophosphate or soluble reactive phosphorus, SRP) occurs chiefly as ions of HPO42- , with a small percentage present as PO43-.
In this analytical procedure, direct measurements of SRP are made using an acidified molydate reagent and potassium antimony tartrate. The resulting compound, a heteropoly acid (phosphomolybdic acid), is reduced to the intensely colored molybdenum blue by ascorbic acid and the absorbance of the product is measured using a 10 mm path length flow cell at 880 nm.
Contamination is the primary concern with P determinations. This is particularly true with samples collected from the euphotic zone, where SRP concentrations approach the detection limit of the standard autoanalyzer-based assay procedure. In order to avoid contamination, sample bottles must be meticulously cleaned with dilute HCl (1 M) and rinsed with DDW before use.
Rinse the acid-washed, 125 ml HDPE bottle 3 times with sample before filling. Fill approximately ⅔ full, cap tightly and freeze upright.
Phosphorus analyses are performed using a 4-channel SEAL Analytical AA3 continuous flow system. Slight modifications have been incorporated to achieve the optimum range and sensitivity for SRP concentration levels specific for the Sta. ALOHA seawaters.
This method employs a single color reagent consisting of an acidified solution of NH4MoO4, ascorbic acid and antimony-tartrate. The blue phosphomolybdenum complex formed is read colorimetrically through a 10 mm pathlength flowcell at 880 nm (Murphy and Riley, 1962). The specific automated method used is described in Technicon (1973) with the following modifications: sample pump tube size is increased to allow for a flow rate of 1.6 ml min-1.
| 5.1. | Calibration stocks and regression standards |
The calibration of dissolved inorganic nutrient determinations in the autoanalysis of seawater samples is performed using standard solutions containing dissolved N, P and Si salts. A nutrient stock solution is prepared by dissolving dried (65°C, 72 hr) analytical grade reagent chemicals with DIW in 1 L glass volumetric flasks containing 1 ml of chloroform. Once dissolved, this stock solution is immediately transferred into 1 L HDPE bottles and stored at room temperature in the dark (see Reagents section for details). The daily regression standards are prepared by diluting the stock standards with low nutrient natural seawater (LNSW). LNSW is 0.2 µm filtered open ocean surface seawater stored in a carboy at room temperature in the dark for at least 6 months prior to use. This technique provides a mixed standard solution of N, P and Si that is matrix-matched with the seawater samples and any cross-nutrient interference effect should also be accounted for. | |
| 5.2. | Blank corrections |
All seawater standard absorbance peaks were corrected for the absorbance of the seawater (LNSW). All seawater sample peaks were corrected for the refractive index absorbance for each unique nutrient detection system. The refractive index corrections represent the increase in absorbance that is due strictly to the presence of dissolved salts in seawater when compared to the DIW baseline. These corrections are determined by running alternating sample cups of DIW and LNSW through the autoanalyzer with only non-color developing reagents on-line. The difference in peak height between DIW and LNSW is calculated and an average is taken to obtain the refractive index correction. This refractive index run is done once a week. |
The detection limit for SRP is approximately 0.1 μM with a coefficient of variation for field-collected replicates of 0.006 ± 0.007 µmol L-1 based on 2019 data (9 cruises).
acid-washed, 125 ml high-density polyethylene bottles
SEAL Analytical AA3 and accessories
pipettes and volumetric flasks
DIW
HCl (1M)
Potassium Antimony Tartrate solution: Dissolve 3.0 g of ACS grade antimony potassium tartrate into 800 ml DIW and dilute to 1 L in a volumetric flask. Solution is stable for several months.
Ammonium molybdate solution: Pour 70 mL of sulfuric acid into ~500 mL on DIW. Once the mixture is cool dissolve 6 g of ACS grade ammonium molybdate, then mix 50 mL of potassium antimony tartrate solution. Dilute to 1 L in a volumetric flask. Store in plastic bottle in the dark. Solution is stable until white precipitate begins to form, approx. ~1 month.
Ascorbic acid solution: Dissolve 2.5 gram of ascorbic acid in ~250 mL of DIW then add and dissolve 2.5 grams of SDS. Dilute to 500 ml. Store in fridge when not used, stable for 5 days.
Stock phosphate standard solution (10 000 µM): Dissolve 1.3609 g of dry (65°C for 72 hr) KH2PO4 into 800 ml of DIW containing 1 ml of chloroform and dilute to 1 L in a volumetric flask. Store in the dark.
Murphy, J. and J. P. Riley. 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta, 27, 30.
Technicon Industrial Systems. 1973. Orthophosphate in water and seawater. Autoanalyzer II Industrial Method No. 155-71W. W. Tarrytown, New York 10591.
analyte-compound class: soluble reactive phosphorus (SRP)
method: automated, colorimetric analysis
precision: - laboratory: 0.003 ± 0.006 µmol L-1 based on 2019 data (9 cruises, 309-317) - field: 0.006 ± 0.007 µmol L-1 based on 2019 data (9 cruises, 309-317)
accuracy: See QC Charts
reference standard: - primary: Potassium Phosphate: K2H3PO4 - secondary: WAKO, OSIL and SCOR-JAMSTEC
analysis history for HOT program:
Up until February 2000, analyses were conducted at room temperature on a four-channel Technicon Autoanalyzer II continuous flow system at the University of Hawaii Analytical Facility.
Between 2001 and 2005, the HOT nutrient program underwent substantial changes, including switching analysts twice, eventually establishing an analytical nutrient laboratory centered around a 4-channel SEAL Analytical AA3.
During this transition period, samples from > 200 m depth from HOT 127-166 were shipped to Oregon State University (OSU) for analyses. The OSU nutrient facility uses an AutoAnalyzer II manifold with 5 cm flow cell for PO4 analyses, and an Alpkem RFA 300 system for analyses of NO3+NO2.
Notes, comments or additions: