2.0 Definition
The reactive phosphate concentration is given in units of mmol kg ^-1 in seawater.

3.0 Principle of Analysis
The determination of reactive phosphorus in seawater is based on the method proposed by Strickland and Parsons (1968). The seawater sample is allowed to react with a composite reagent containing ammonium molybdate, ascorbic acid and potassium antimonyl-tartrate. The resulting complex is reduced in situ to give a blue colored solution, the absorbance of which can be measured spectrophotometrically.

4.0 Apparatus
Spectrophotometer

5.0 Reagents

5.1 Ammonium molybdate solution: Dissolve 15 g of reagent grade ammonium paramolybdate, (NH₄)₆Mo₇O₂₄ .4H₂O, in 500 ml of deionized water. The solution is stable indefinitely if stored out of direct sunlight in a plastic bottle. Discard if a precipitant forms.

5.2 Sulfuric acid solution: Add 140 ml of concentrated sulfuric acid to 900 ml of deionized water. Cool the solution and store it in a glass bottle.

5.3 Ascorbic acid solution: Dissolve 27 g of ascorbic acid in 500 ml of deionized water. This solution should be frozen in a plastic container. Thaw for use and refreeze at once.

5.4 Potassium antimonyl-tartrate solution: Dissolve 0.34 g of potassium antimonyl- tartrate in 250 ml of deionized water. This solution is stable for many months.

5.5 Mixed reagent: Mix together 100 ml ammonium molybdate solution, 250 ml sulfuric acid solution, 100 ml ascorbic acid solution and 50 ml potassium antimonyl-tartarate solution. Use at once and discard any excess. Do not store for more than 6 hours. Add molybdate last. Solution should have a yellow color.

6.0 Sampling

6.1 Samples are collected on deeper casts after the oxygen, CO₂ and salinity samples have been drawn. Shallow samples (upper 250 m) are collected on the gases cast after oxygen, CO₂ , DOC and salinity samples are drawn. An in-line filter (0.8 mm Nuclepore filter) is connected to the Niskin bottle. The spigot is opened and three sets of samples are collected from the water that passes through the filter by gravity
filtration. Each bottle is rinsed three times and then filled just below the shoulder. Care must be taken to avoid overfilling of samples to be frozen. These bottles are transferred to a freezer (- 20°C) and kept frozen until analysed.

6.2 Contamination is a major problem with nutrient samples, especially in the upper ocean where the ambient concentrations are low. All the nutrient bottles are rigorously cleaned before use. The cleaning begins by a wash with a phosphate-free detergent (Aquet) followed by a rinse with 10% HCl, three rinses with deionized
water and a final rinse with deionized water.

6.3 Prolonged storage of samples is not advisable, even if frozen.

7.0 Procedures

7.1 Sample analysis

7.1.1 Prior to analysis the samples are thawed and brought to a temperature of between 15° and 30°. Do not let the samples sit for long periods of time as the polyethylene bottles may absorb phosphate.

7.1.2 Place 100 ml of sample into a 200 ml polyethylene bottle.

7.1.3 To each sample add 10 ± 0.5 ml of the mixed reagent and mix immediately.

7.1.4 After 5 minutes and within 2 hours, measure the absorbance of the sample in
a 10 cm cell against de-ionized water at a wavelength of 885 nm.

7.2 Blank determination

7.2.1 A reagent blank is determined by using de-ionized water in place of the 100 ml seawater sample and carrying out the exact method described in section 7.1.

7.2.2 The reagent blank should not exceed 0.03. If it does, the ammonium molybdate reagent should be replaced and the blank determination repeated

7.3 Standardization

7.3.1 Primary phosphate standard: Dissolve 0.816 g of anhydrous potassium dihydrogen phosphate, KH₂ PO₄ , in 1000 ml of de-ionized water. 1 ml = 6 mmol. Store in a dark bottle. This solution is stable for many months.

7.3.2 Secondary standard: Dilute 10.0 ml of the primary standard solution to 1000 ml with de-ionized water. 1 ml = 0.06 mmol. Store in a dark bottle. Make fresh every 10 days.

7.3.3 Prepare a standard solution of 3.0 mM by diluting 5.0 ml of secondary standard to a volume of 100 ml with deionized water. Run these standards as described in section 7.1.

8.0 Calculation and expression of results

8.1 A standardization factor F can be calculated as:
        F = 3.0 mmol/kg / (E_s- E_b)
where:
        3.0 mmol/kg = concentration of the standard
        E_s = mean absorbance of the standards
        E_b = mean absorbance of the blanks

8.2 The reactive phosphate concentration is calculated by:
        reactive phosphorus (mmol l ^-1 ) = F * corrected absorbance
where:
        corrected absorbance = sample absorbance - reagent blank
        F = standardization factor

9.0 References
Strickland, J.D.H., and Parsons, T.R. (1968). Determination of reactive phosphorus. In: A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, Bulletin 167, 49–56.