ORTHOPHOSPHATE AND DISSOLVED ORGANIC PHOSPHORUS
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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.
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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.
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