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ORTHOPHOSPHATE AND DISSOLVED ORGANIC PHOSPHORUS



SUMMARY: Seawater is collected from known depths using CTD- rosette sampling protocols. Subsamples are drawn and stored in acid-washed polyethylene bottles. Soluble reactive phosphorus (SRP) is measured spectrophotometrically following the formation of phosphomolybdic acid. Total dissolved phosphorus (TDP) is measured in a separate sample after exposure to ultraviolet (UV) light and dissolved organic phosphorus (DOP) is estimated by difference.




1. Principle

Phosphorus (P) is one of several macronutrients required for the growth of 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-. Dissolved organic phosphorus (DOP) exists in a variety of forms (primarily P-esters) which result from excretion, decomposition, death and autolysis.

In this analytical procedure, we make direct measurements of SRP and total dissolved phosphorus (TDP). The latter includes all organic and inorganic phosphorus compounds. Bound phophorus is released from organic matter by ultraviolet light (UV) oxidation and the liberated orthophosphate is reacted with an acidified molydate reagent and potassium antimonyl tartrate. The resulting compound, a heteropoly acid (phosphomolybdic acid), is reduced to the intensely colored molybdenum blue by ascorbic acid and measured spectrophotometrically. SRP samples are prepared in the same manner as TDP samples, but without the prior UV oxidation step. DOP is calculated by difference (i.e., TDP-SRP).


2. Precautions

Contamination is the primary concern with P determinations. This is particularly true with samples collected from the euphotic zone, where SRP concentrations are extremely low (<0.2 µM). In order to avoid contamination, sample bottles must be meticulously cleaned with dilute HCl and rinsed with deionized distilled water (DDW) before use.


3. Sample Collection and Storage

3.1. Sample collection (also see "NOTE" in Chapter 7, section 3)
3.1.1. Rinse the nutrient sample bottle (acid-washed, 125 ml polyethylene bottle) 3 times before filling. Fill to approximately 2/3 full, tighten cap and freeze.
3.1.2. Record cruise, cast and Niskin bottle number on the bottle and data sheet.

4. Sample Analysis

Currently, GOFS and WOCE nutrient samples collected during the Hawaii Ocean Time Series cruises are analyzed by the Hawaii Institute of Marine Biology Analytical Facility. Mr. Ted Walsh has provided us with the following procedure for the analysis of phosphate.

Phosphorus analyses are performed on a four-channel Technicon Autoanalyzer II continuous flow system. The automated wet chemistries generally follow the standard methods of seawater analysis as given by Technicon (1973). Slight modifications have been incorporated to achieve the optimum range and sensitivity for each nutrient at concentration levels specific for the Station ALOHA seawaters.

4.1. Soluble Reactive Phosphorus (SRP)

This method employs a single color reagent consisting of an acidified solution of ammonium molybdate, ascorbic acid and antimony-tartrate. The blue phosphomolybdenum complex formed is read colorimetrically through a 50 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 0.8 ml min-1, and the color reagent solution concentration is diluted by a factor of 2.

4.2. Total Dissolved Phosphorus (TDP)

The method for TDP involves initial UV digestion of a seawater sample followed by autoanalysis of the digestion product (SRP; see Chapter 8, section 4.1). The modified photoxidation technique (Armstrong et al., 1966) utilizes a 2 hour UV irradiation period. Exact details of the photoxidation unit are described in Walsh (1989). Periodic calibration checks of the UV lamp efficiency are made using dissolved organic phosphorus (β-glycerol- phosphate) standards. As a general rule, the UV lamp is replaced after approximately 700-800 hr of use.

4.3. Low-level procedures

We have recently developed a method for the determination of P dissolved in seawater at low concentrations (<0.1 µM; Karl and Tien, in preparation). The method is based upon the quantitative removal of SRP by co-precipitation with magnesium hydroxide which is formed by the addition of high purity sodium hydroxide to selected seawater samples. The precipitate is collected by centrifugation, washed with a solution of weak sodium hydroxide (0.01 M) and then dissolved in a known volume of high purity hydrochloric acid. At this point, the concentrated samples are treated as above (see Chapter 8, section 4.1) for the determination of SRP. The detection limit is determined by the volume of seawater initially used (50-250 ml for our routine assays), but P concentrations as low as 5 nM can be easily and reproducibly detected. All low level P determinations are corrected for arsenic interference by thiosulfate reduction of dissolved arsenate (Johnson, 1971).


5. Calibration, Data Reduction and Calculations

5.1. Calibration stocks and regression standards

The calibration of dissolved inorganic nutrients in the autoanalysis of seawater samples is performed using standard solutions containing N, P and Si. A nutrient stock solution is prepared by dissolving dried (65°C, 72 hours) analytical grade reagent chemicals with distilled-deionized water in 1 liter glass volumetric flasks containing 1 ml of chloroform. Once dissolved, this stock solution is immediately transferred into 1 liter amber polypropylene bottles and stored at 4°C. The reagent chemicals and concentrations are: phosphate (KH2PO4, 1 mM), nitrate (KNO3, 5 mM) and silica (Na2SiF6, 4 mM).

Working standards are prepared daily by volumetric dilutions of the stock using glass pipettes and a plastic (polymethyl- pentene; PMP) volumetric flask. All pipettes and PMP flasks are acid-washed (1M HCl) and gravimetrically calibrated prior to use. The daily regression standards are prepared by diluting the working standard with low nutrient natural seawater diluent (SWDIL). The SWDIL is filtered open ocean surface seawater that is stored in a carboy at room temperature. By using this technique all standards are matrix-matched with the seawater samples and any cross-nutrient interference effect should be accounted for.

Cross-nutrient interference and reagent contamination was evaluated by preparing separate solutions, as above, but with one of the three standards omitted. Only phosphate showed a slightly measurable increase (+0.014 µM) in the presence of 40 µM-NO3 and 160 µM-Si. The linear regressions of the standards were applied to all seawater sample peaks for calculating each batch of cruise samples. Typical correlations produced r2 values that were between 0.9999 and 0.99999.

5.2. Blank corrections

All seawater standard absorbance peaks were corrected for the absorbance of the seawater diluent (SWDIL). All seawater sample peaks were corrected for the refractive index absorbance for each unique nutrient detection system. The refractive index corrections (in apparent µM units) ranged from approximtely 0.13 (for P), 0.23 (for N) to 2.41 (for Si), and represent the increase in absorbance that is due strictly to the presence of dissolved salts in seawater when compared to the distilled-deionized water baseline. These corrections are made by running sampling seawater (35 o/oo salinity) through the autoanalyzer with DDW only in reagent lines and also with all reagents except the color producing reagent. The Levor surfactant used routinely in the phosphate channel was omitted from the DDW lines during the refractive index measurement.


6. Accuracy and Precision

The detection limit for phosphorus is approximately 0.02 µM with a coefficient of variation for field-collected replicates of 0.3% For DOP the detection limit is 0.02 µM with a coefficient of variation of 1%.


7. Equipment/Supplies

  • Niskin bottles and rosette/CTD unit

  • acid-washed, 125 ml polyethylene sample bottles

  • Autoanalyzer (Technicon Corp.) and accessories


8. Reagents

  • glass distilled deionized water (DDW)

  • HCl (1M) for cleaning

  • concentrated H2SO4

  • ultrapure NaOH and HCl for low-level P determinations

  • Ammonium molybdate solution: Dissolve 40 g of ACS grade ammonium paramolybdate [(NH4)6Mo7O24. 4H2O into 800 ml DDW and dilute to 1 liter in a volumetric flask. Store in plastic bottle in the dark. Solution is stable indefinitely.

  • Ascorbic acid solution (Prepare fresh): Dissolve 1.8 g of ACS ascorbic acid into 100 ml DDW (1.8% wt/vol).

  • Antimony potassium tartrate solution: Dissolve 3.0 g of ACS antimony potassium tartrate (C8H4K2O12Sb2 . 3H2O) into 800 ml DDW and dilute to 1 liter in a volumetric flask. Solution is stable for several months.

  • Mixed reagent (Prepare fresh): Mix together in the following order 15 ml ammonium molybdate, 50 ml 5 N sulfuric acid, 30 ml 1.8% ascorbic acid and 5 ml potassium antimony tartrate.

  • Stock phosphate standard solution (1 mM): Dissolve 0.1361 g of dry (65°C for 72 hours) potassium phosphate monobasic (KH2PO4) into 800 ml of DDW and dilute to 1 liter in a volumetric flask. Store in a dark bottle with 1 ml of chloroform.

  • Working phosphate standard (40 µM): Dilute 4.0 ml of the stock standard to 100 ml (use volumetric flask). Then dilute the working standard to prepare a series of standards to cover a range of P concentrations.


9. References

  • Armstrong, F. A. J., P. M. Williams and J. D. H. Strickland. 1966. Photo-oxidation of organic matter in seawater by ultraviolet radiation, analytical and other applications. Nature, 211, 481-483.

  • Grasshoff, K., M. Ehrhardt and K. Kremling. 1983. Methods of Seawater Analysis. Verlag Chemie.

  • Johnson, D. L. 1971. Simultaneous determination of arsenate and phosphate in natural waters. Environmental Science and Technology, 5, 411-414.

  • 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.

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

  • Technicon Industrial Systems. 1973. Orthophosphate in water and seawater. Autoanalyzer IIR Industrial Method No. 155-71W. W. Tarrytown, New York 10591.

  • Walsh, T. W. 1989. Total dissolved nitrogen in seawater: A new high temperature combustion method and a comparison to photo- oxidation. Marine Chemistry, 26, 295-311.

  • Walsh, T. W., R. E. Young and S. V. Smith. Seawater nutrient storage and analysis. Manuscript.