SUMMARY: Seawater samples are collected at discrete depths in 12-liter Niskin bottles. The water samples are prefiltered (202 µm) and transferred to specially designed, precalibratedfiltration bottles, pressure filtered through combusted acid-rinsed GF/F filters and stored frozen for subsequent analysis. In the laboratory, the filters are combusted at 450-500°C and the concentration of the resulting inorganic phosphorus is determined by colorimetric analysis.

1. Principle

The procedure presented here is a modification of one used by the Hawaii Institute of Marine Biology Analytical Services laboratory at the University of Hawaii. It is a method pioneered and used by soil scientists and marine chemists for particulates which can be homogenized into a fine powder.

The method relies on the release of organically-bound phosphorus compounds as orthophosphate, by high temperature combustion at 450-500°C. The orthophosphate is then extracted with 0.5 N HCl at 90°C. The liberated orthophosphate is reacted with a mixed reagent of molybdic acid, ascorbic acid and trivalent antimony to form phosphomolybdic acid. This heteropoly acid is then reduced to the colored molybdenum blue complex by ascorbic acid and the solution is measured spectrophotometrically.

This procedure measures all forms of phosphorus which can be released by combustion and acid hydrolysis.

2. Precautions

Contamination is the primary problem to be avoided with these samples. Combusted acid rinsed filters are used. All sampling bottles, forceps, tubing and filtration bottles are also acid rinsed.

3. Sampling, Filtration and Storage

3.1. Seawater samples are collected in 12-liter Niskin bottles and transferred directly to acid-cleaned filtration bottles. The samples are transferred via Tygon tubing which incorporates an in-line 202 µm Nitex screen prefilter to remove zooplankton or any other rare particles which might otherwise affect the precision of the estimate. The filtration bottles are 4- and 12-liter polyethylene aspirator bottles fitted with a valve assembly and tubing connection.

Once the rosette/CTD unit is on deck, the vent valve from each Niskin bottle is opened and one end of the drawing tube is attached to the outflow spigot of the sampling bottle and the other end to the tubing connector on the cap of the filtration bottle. Particular attention is paid to the orientation of the in-line screened drawing tube (the shorter, larger bore section is attached to the Niskin bottle). The filtration bottle valve is opened and 100-200 ml is run through the transfer tube and valve assembly to rinse the sampling bottle. The bottle and cap are rinsed 3 times in this fashion. During the rinsing and filling operation, the filtration bottle cap valve is used to control and direct the sample flow. After rinsing, the cap is placed on the bottle mouth (without tightening), the valve is opened and the polyethylene bottle is filled to the calibration mark.

3.2. After filling, the filtration bottles are inverted and placed into the filtration rack. The contents are then pressure filtered (4-7 psi nitrogen gas) through combusted in-line acid-rinsed 25 mm GF/F filters.
3.3. Following filtration, clean forceps are used to transfer each filter to a combusted 16 x 100 mm glass test tube which is then covered with a 3.5 cm square piece of combusted foil. Each sample is labeled and stored frozen (-20°C). Any water remaining in the carboy is measured to calculate the volume filtered. This information and any other appropriate data are entered on the data sheet.

4. Blank Determination

Standards are corrected for reagent blanks while samples are corrected for field filter blanks.

4.1. Standards: At least 2 reagent blanks are prepared and individual standard absorbances are corrected by the mean blank value.
4.2. Samples: The mean absorbance from three field filter blanks, stored and processed in the same manner as samples, are used to correct individual sample absorbances for filter, reagent and systematic procedural contamination.

5. Analysis

5.1. Samples are combusted in 16 x 100 mm test tubes at 450°C for 4.5 hours in a muffle furnace. The samples are then allowed to cool and are immersed in 10 ml of 0.5 M HCl. The test tube is then heated for 60 minutes at 90°C in a heating block.
5.2. The samples are allowed to cool, and centrifuged for 30 min at 2800 g. 5 ml of the supernatant is volumetrically subsampled into another combusted acid washed 16 x 100 mm test tube.
5.3. One half ml of mixed reagent is added to samples and standards and mixed thoroughly. Color is developed for 60 minutes and absorbance is read at 880 nm against a DDW reference. Standards are corrected for absorbance of reagent blanks and samples are corrected for absorbance of filter or procedural blanks.

6. Data Reduction and Calculations

6.1. Calculations
6.1.1. Calculate µmol l-1 of phosphate from standard curve using:

µmol l-1 = (x - b)/m

where: x = blank-corrected absorbance of sample
b = y intercept of regression line
m = slope of regression line
6.1.2. Calculate µg P-PO4 l-1 using:

µmol l-1 smpl x 30.97376 µg µmol-1 = µg P l-1 in sample extract

(µg P l-1 in extract) /(1000 ml l-1) = µg P ml-1

(µg P ml-1 x 20.0 ml of extract) / vol in liters filtered = µg P-P04 l-1

7. Equipment/Supplies

8. Reagents

9. References