PARTICULATE PHOSPHORUS

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     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.
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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

    Niskin bottles and rosette/CTD unit
    low pressure filtration apparatus (4-7 psi)
    muffle furnace
    heating block
    combusted, acid-rinsed GF/F filters 
    acid-rinsed vacuum filtering assembly
    spectrophotometer (Perkin-Elmer Lambda 3B) and 1-cm cuvette
    combusted (450°C, 3 hours), acid-washed 16 x 100 mm glass tubes
   

8.  Reagents

    Glass distilled deionized water (DDW)
    0.5 M HCl
    HCl for cleaning (1 M)
    Ammonium molybdate solution: Dissolve 15 g of ACS grade ammonium 
      paramolybdate [(NH4)6 MO7O24 . 4H2O], in 500 ml DDW. Store in 
      plastic bottle in the dark.  Solution is stable indefinitely.
    Sulfuric acid solution (5 N)
    Ascorbic acid solution:  Dissolve 0.54 g of ACS ascorbic acid in 
      10 ml DDW (5.4% wt/vol).  Prepare fresh.
    Potassium antimonyl-tartrate solution:  Dissolve 0.34 g of ACS 
      potassium antimonyl-tartrate (tartaremetic), in 250 ml DDW.  
      Solution is stable for many months.
    Mixed reagent:  Mix together 10 ml ammonium molybdate, 25 ml 5 N 
      sulfuric acid, 10 ml 5.4% ascorbic acid and 5 ml potassium 
      antimony tartrate.  Prepare fresh.
    Stock phosphate standard solution (1000 µM):  Dissolve 0.1361 g 
      of dry KH2PO4, in 1000 ml of DDW. Store in a dark bottle with 
      1 ml of chloroform.
    Working phosphate standard (100 µM):  Dilute 10 ml of the stock 
      standard to 100 ml, using a volumetric flask.       
    Dilute the working standard to prepare a series of standards to 
      cover the range from 0.05 - 10 µM.
  

9.  References

    Strickland, J. D. H. & T. R. Parsons.  1972.  A Practical Handbook 
    of Seawater Analysis.  Fisheries Research Board of Canada, 167 p.