Sep 07, 2024

Public workspaceUSDA LTAR Common Experiment measurement: Total phosphorus (TP) and total dissolved phosphorus (TDP) concentration

DOI
dx.doi.org/10.17504/protocols.io.8epv5r7m6g1b/v1
  • Stephen K. Hamilton1,
  • Oliva Pisani2,
  • John L. Kovar3,
  • Robert W. Malone3,
  • Amy J. Morrow3,
  • Kevin J. Cole3
  • 1Michigan State University, W.K. Kellogg Biological Station, Hickory Corners, MI;
  • 2USDA Agricultural Research Service, Southeast Watershed Research Laboratory, Tifton, GA;
  • 3USDA Agricultural Research Service, National Laboratory for Agriculture and the Environment, Ames, IA
Lori J. Abendroth
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DOI: dx.doi.org/10.17504/protocols.io.8epv5r7m6g1b/v1
External link: https://ltar.ars.usda.gov
Protocol Citation: Stephen K. Hamilton, Oliva Pisani, John L. Kovar, Robert W. Malone, Amy J. Morrow, Kevin J. Cole 2024. USDA LTAR Common Experiment measurement: Total phosphorus (TP) and total dissolved phosphorus (TDP) concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r7m6g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 21, 2024
Last Modified: September 07, 2024
Protocol Integer ID: 97105
Keywords: Long-Term Agroecosystem Research, LTAR, USDA LTAR, Common Experiment, crops, orthophosphate, organic phosphorus, inorganic phosphorus, eutrophication, surface water, watershed, total phosphorus, colorimetry, inductively coupled plasma, optical emission spectroscopy, ICP-OES,
Funders Acknowledgement:
United States Department of Agriculture
Grant ID: -
Disclaimer
This research is a contribution from the Long-Term Agroecosystem Research (LTAR) network. LTAR is supported by the United States Department of Agriculture. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.
Abstract
Total dissolved P (TDP) includes organic P (e.g., sugars and phospholipids) and orthophosphate. Dissolved orthophosphate (also termed soluble reactive P, dissolved reactive P, dissolved inorganic P, or filterable reactive P) is the form of P most readily available to algae and aquatic plants; however, numerous studies have shown that hydrolysis of other forms of P to the orthophosphate form is possible in natural waters. Therefore, when assessing the long-term potential for accelerated eutrophication of surface water due to P loading, many researchers and watershed managers want to know the total P concentration (regardless of P form) in water samples.

Total dissolved P determination involves digesting filtered water samples to convert organic P species to orthophosphate, followed by colorimetric analysis or analysis by inductively coupled plasma with optical emission spectroscopy (ICP-OES). Determining TP involves the same digestion procedure applied to an unfiltered sample, followed by colorimetric or ICP-OES analysis. The recommended P digestion method is the persulfate oxidation technique, performed using an autoclave or microwave. The colorimetric determination of TDP and TP follows the ascorbic acid “blue” method (USEPA, 1978), whereby an intensely colored blue complex forms from the reaction between P compounds and ascorbic acid. The procedure involves the reaction of ammonium molybdate and potassium antimonyl tartrate with orthophosphate to form an antimony-phospho-molybdate complex. This complex is then reduced to an intensely blue-colored complex by addition of ascorbic acid. The intensity of the blue complex is proportional to the orthophosphate concentration in the sample.
Before start
Appropriate safety, health, and environmental precautions must be followed based on the selected methods, instrumentation, and workflow. Laboratory supervisors are responsible for knowledge of these precautions and their implementation.

Review the USDA LTAR Common Experiment measurement: Best practices for collection, handling, and analyses of water quality measurements protocol (Pisani et al., 2024) prior to implementing this protocol.
Sample collection and filtration
Sample collection and filtration
Return samples to the laboratory TemperatureOn ice and filter them on collection day if possible.

To measure TDP, filter samples through a Thikness0.45 µm pore size filter to remove particulates before chemical analysis.

If desired, use a Thikness0.22 µm filter to more completely remove the microbial community.

Sample storage and preservation
Sample storage and preservation
Unless analysis is possible on sample collection day, it is required to store samples at Temperature4 °C for up to 28 days.

Archiving
Archiving
The common practice is to store water samples for TDP or TP analyses until data certification (QA/QC verification).
Sample analysis
Sample analysis
To determine total P (TP = dissolved P + particulate P), shake an unfiltered sample to suspend the particulate matter before removing a subsample for digestion.
Polyphosphates and phosphates bound to organic substances do not react with the molybdate reagent used for colorimetric P analysis, and colorimetric analysis cannot directly measure particulate P.

Therefore, analysis of the total P content of water samples requires that all condensed and organic P compounds, including particulate P, first be converted (hydrolyzed) to orthophosphate to be determined colorimetrically or by ICP-OES (USEPA, 1994).

To accomplish this conversion, samples are digested with persulfate at high temperature and pressure, normally using either an autoclave or microwave (USEPA,1994; Maher and Woo 1998). A laboratory pressure cooker can be used as well to oxidize the organic matter and release P as orthophosphate.
After digestion, TDP and TP are determined colorimetrically using the ascorbic acid method (APHA, 2005) or by ICP-OES.
Figure 1. Colorimetric determination of TDP and TP follows the ascorbic acid “blue” method. Photo credit: S. Rahutomo, Iowa State University.
Figure 2. Inductively coupled plasma—optical emission spectrometer (ICP-OES) with an autosampler to determine TP and TDP concentrations in the digestate. Photo credit: A. Morrow, USDA-ARS.

Calculations
Calculations
Prepare a calibration curve by plotting the area of each standard peak against its respective P concentration. Correction of standards and samples for the blank is necessary; assuming virtually P-free deionized or distilled water, the reagents and especially potassium persulfate are usually the source of most of the blank absorbance.
Note
P concentration = unfiltered digested sample
TDP concentration = filtered digested sample

Compute the TDP or TP concentration in the sample by comparing the sample peak area to the calibration curve.
Note
Report the concentration as mg P per liter (mg/L).

Covariate metrics to be sampled concurrently
Covariate metrics to be sampled concurrently
  • Dissolved NO3- concentration
  • Dissolved NH3 concentration
  • Total dissolved N (TDN)
  • Total N (TN)
  • Total suspended solids (TSS)

Recommendations for data collection
Recommendations for data collection
Table 1. Summary of recommendations for the collection and measurement of TP and TDP concentration.
ABCD
AttributePreferredMinimumComments
Spatial scaleFieldPlot
FrequencyEvent-drivenEvent-drivenMore frequent (weekly) measurements can be preferential when the flow regime can increase seasonally or after precipitation events. Sampling in this protocol should be event-driven to enable cross-site comparisons
Covariate metricsNO3-N, NH3-N, TDN, TSSNO3-N, NH3-N, TDN
Sample preservation and storage TDPFiltered with a 0.22 or 0.45 μm pore-size filter on sample collection dayTo maintain filtered samples for up to 28 days, store them at 4°C
Sample preservation and storage TPPerform the analysis on the same day as collectionTo maintain samples for up to 28 days, store them at 4°C
Sample analysisPersulfate digestion followed by colorimetric analysis or ICP
Water quantityDischarge or flow rateDischarge or flow rateCalculate TP or TDP loads by linking this metric to the water quantity metric “flow”
Covariate metrics = other metrics to sample concurrently. NO3--N = nitrate-N; NH3-N = ammonia-N; TDN = total dissolved nitrogen; TSS = total suspended solids

Protocol references
Maher W, Woo L, 1998. Procedures for the storage and digestion of natural waters for the determination of filterable reactive phosphorus, total filterable phosphorus and total phosphorus. Analytica Chimica Acta 375:5-47.

Pisani, O., Lizotte, R., Veum, K., Kovar, J. L., Hamilton, S. K., & Malone, R. W. 2024. USDA LTAR Common Experiment measurement: Best practices for collection, handling, and analyses of water quality measurements. protocols.io

Standard Methods for the Examination of Water and Wastewater. 21st Edition. 2005. American Public Health Association/American Water Works Association/Water Environment Federation, Washington DC.

US Environmental Protection Agency (USEPA), 1974. Method 365.4: Phosphorus, Total (Colorimetric, Automated, Block Digester AA II).

US Environmental Protection Agency (USEPA), 1978. Method 365.3: Phosphorus, All Forms (Colorimetric, Ascorbic Acid, Two Reagent).
US Environmental Protection Agency (USEPA), 1994. Method 200.7: Determination of Metals and Trace Elements in Water and Wastes by Inductively Coupled Plasma-Atomic Emission Spectrometry.