365.5 - 1

Method 365.5

Determination of Orthophosphate in Estuarine and

Coastal Waters by Automated Colorimetric Analysis

Carl F. Zimmermann

Carolyn W. Keefe

University of Maryland System

Center for Environmental and Estuarine Studies

Chesapeake Biological Laboratory

Solomons, MD 20688-0036

Revision 1.4

September 1997

Edited by

Elizabeth J. Arar

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.4 September 1997 365.5 - 2

Chemical Abstracts Service

Analyte Registry Numbers (CASRN)

Phosphate 14265-44-2

Method 365.5

Determination of Orthophosphate in Estuarine and Coastal

Waters by Automated Colorimetric Analysis

1.0 Scope and Application

1.1 This method provides a procedure for the determination of low-level orthophosphate concentrations

normally found in estuarine and/or coastal waters. It is

based upon the method of Murphy and Riley adapted for 1

automated segmented flow analysis in which the two

2

reagent solutions are added separately for greater

reagent stability and facility of sample separation.

1.2 A statistically determined method detection limit

(MDL) of 0.0007 mg P/L has been determined by one

laboratory in 3 parts per thousand (ppt) saline water. The 3 

method is linear to 0.39 mg P/L using a Technicon

AutoAnalyzer II system (Bran & Luebbe, Buffalo Grove,

IL).

1.3 Approximately 40 samples per hour can be analyzed.

1.4 This method should be used by analysts experienced in the use of automated colorimetric analyses, and

familiar with matrix interferences and procedures for their

correction. A minimum of 6-months experience under

experienced supervision is recommended.

2.0 Summary of Method

2.1 An automated colorimetric method for the

analysis of low-level orthophosphate concentrations is

described. Ammonium molybdate and antimony potassium tartrate react in an acidic medium with dilute solutions of phosphate to form an antimony-phospho-molybdate complex. This complex is reduced to an intensely

blue-colored complex by ascorbic acid. The color

produced is proportional to the phosphate concentration

present in the sample. Positive bias caused by differences in the refractive index of seawater and reagent

water is corrected for prior to data reporting.

3.0 Definitions American Society of Testing Materials (ASTM). Reverse

3.1 Calibration Standard (CAL) -- A solution

prepared from the stock standard solution that is used to

calibrate the instrument response with respect to analyte

concentration. One of the standards in the standard

curve.

3.2 Dissolved Analyte (DA) -- The concentration of

analyte in an aqueous sample that will pass through a

0.45-Fm membrane filter assembly prior to sample

acidification or other processing.

3.3 Laboratory Fortified Blank (LFB) -- An aliquot

of reagent water to which known quantities of the method

analytes are added in the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine whether method performance is within acceptable

control limits. This is basically a standard prepared in

reagent water that is analyzed as a sample.

3.4 Laboratory Fortified Sample Matrix (LFM) -- An

aliquot of an environmental sample to which known

quantities of the method analytes are added in the

laboratory. The LFM is analyzed exactly like a sample,

and its purpose is to determine whether the sample matrix

contributes bias to the analytical results. The background

concentrations of the analytes in the sample matrix must

be determined in a separate aliquot and the measured

values in the LFM corrected for background concentrations.

3.5 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water that is treated exactly as a sample

including exposure to all glassware, equipment, and

reagents that are used with other samples. The LRB is

used to determine if method analytes or other interferences are present in the laboratory environment, the

reagents, or apparatus.

3.6 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.7 Method Detection Limit (MDL) -- The minimum

concentration of an analyte that can be identified, measured, and reported with 99% confidence that the analyte

concentration is greater than zero.

3.8 Reagent Water (RW) -- Type 1 reagent grade

water equal to or exceeding standards established by

osmosis systems or distilling units that produce 18

megohm water are two examples of acceptable water

sources.
365.5 - 3 Revision 1.4 September 1997

3.9 Refractive Index (RI) -- The ratio of the velocity 6.1.5 Phototube that can be used for 600-900 nm

of light in a vacuum to that in a given medium. The

relative refractive index is the ratio of the velocity of light

in two different media, such as sea or estuarine water

versus reagent water. The correction for this difference

is referred to as the refractive index correction in this

method.

3.10 Stock Standard Solution (SSS) -- A concentrated solution of method analyte prepared in the laboratory using assayed reference compounds or purchased

from a reputable commercial source.

4.0 Interferences

4.1 Interferences caused by copper, arsenate and

silicate are minimal relative to the orthophosphate determination because of the extremely low concentrations

normally found in estuarine or coastal waters. High iron

concentrations can cause precipitation of and subsequent

loss of phosphate from the dissolved phase. Hydrogen

sulfide effects, such as occur in samples collected from

deep anoxic basins, can be treated by simple dilution of

the sample since high sulfide concentrations are most

often associated with high phosphate values.4

4.2 Sample turbidity is removed by filtration prior to

analysis.

4.3 Refractive Index interferences are corrected for

estuarine/coastal samples (Section 12.2).

5.0 Safety

5.1 Water samples collected from the estuarine

and/or ocean environment are generally not hazardous.

However, the individual who collects samples should use

proper technique.

5.2 Good laboratory technique should be used when

preparing reagents. A lab coat, safety goggles, and

gloves should be worn when preparing the sulfuric acid

reagent.

6.0 Equipment and Supplies

6.1 Continuous Flow Automated Analytical System Consisting of:

6.1.1 Sampler.

6.1.2 Manifold or Analytical Cartridge equipped with

37EC heating bath.

6.1.3 Proportioning pump.

6.1.4 Colorimeter equipped with 1.5 X 50 mm tubular

flow cell and a 880 nm filter.

range.

6.1.6 Strip chart recorder or computer based data

system.

6.2 Phosphate-Free Glassware and Polyethylene

Bottles

6.2.1 All labware used in the determination must be

low in residual phosphate to avoid sample or reagent

contamination. Washing with 10% HCI (v/v) and thoroughly rinsing with distilled, deionized water was found to

be effective.

6.2.2 Membrane or glass fiber filters, 0.45 Fm nominal

pore size.

7.0 Reagents and Standards

7.1 Stock Reagent Solutions

7.1.1 Ammonium Molybdate Solution (40 g/L) --

Dissolve 20.0 g of ammonium molybdate tetrahydrate

((NH ) Mo O C4H O, CASRN 12027-67-7) in approxi- 4 6 7 24 2

mately 400 mL of reagent water and dilute to 500 mL.

Store in a plastic bottle out of direct sunlight. This

reagent is stable for approximately three months.

7.1.2 Antimony Potassium Tartrate Solution (3.0 g/L) --

Dissolve 0.3 g of antimony potassium tartrate

[(K(SbO)C H O C1/2H O, CASRN 11071-15-1] in approxi- 4 4 6 2

mately 90 mL of reagent water and dilute to 100 mL. This

reagent is stable for approximately three months.

7.1.3 Ascorbic Acid Solution (18.0 g/L) -- Dissolve 18.0

g of ascorbic acid (C H O , CASRN 50-81-7) in approxi- 6 6 6

mately 800 mL of reagent water and dilute to 1 L. Dispense approximately 75 mL into clean polyethylene

bottles and freeze. The stability of the frozen ascorbic

acid is approximately three months. Thaw overnight in

the refrigerator before use. The stability of the thawed,

refrigerated reagent is less than 10 days.

7.1.4 Sodium Lauryl Sulfate Solution (30.0 g/L) --

Sodium dodecyl sulfate (CH3(CH2)11OSO3Na, CASRN

151-21-3). Dissolve 3.0 g of sodium lauryl sulfate (SLS)

in approximately 80 mL of reagent water and dilute to

100 mL. This solution is the wetting agent and its

stability is approximately three weeks.

7.1.5 Sulfuric Acid Solution (4.9 N) -- Slowly add 136

mL of concentrated sulfuric acid (H SO , CASRN 7664- 2 4

93-9) to approximately 800 mL of reagent water. After

the solution is cooled, dilute to 1 L with reagent water.

7.1.6 Stock Phosphorus Solution -- Dissolve 0.439 g

of pre-dried (105EC for 1 hr) monobasic potassium phosphate (KH2PO4

, CASRN 7778-77-0) in reagent water and
Revision 1.4 September 1997 365.5 - 4

mL of Secondary Conc.

Phosphorus Solution (7.2.4) mg P/L

0.1 0.0010

0.2 0.0020

0.5 0.0050

1.0 0.0100

2.0 0.0200

4.0 0.0400

5.0 0.0500

dilute to 1000 mL. (1.0 mL = 0.100 mg P.) The stability of

this stock standard is approximately three months when

kept refrigerated.

7.1.7 Low Nutrient Seawater -- Obtain natural low

nutrient seawater (36 ppt salinity; <0.0003 mg P/L) or

dissolve 31 g analytical reagent grade sodium chloride,

(NaCl, CASRN 7647-14-5); 10 g analytical grade magnesium sulfate, (MgSO4

) CASRN 10034-99-8); and 0.05 g

analytical reagent grade sodium bicarbonate, (NaHCO ,3

CASRN 144-55-8), in 1 L of reagent water.

7.2 Working Reagents

7.2.1 Reagent A -- Mix the following reagents in the

following proportions for 142 mL of Reagent A: 100 mL of

4.9 N H2SO4 (Section 7.1.5), 30 mL of ammonium

molybdate solution (Section 7.1.1), 10 mL of antimony

potassium tartrate solution (Section 7.1.2), and 2.0 mL of

SLS solution (Section 7.1.4). Prepare fresh daily.

7.2.2 Reagent B -- Add approximately 0.5 mL of the

SLS solution (Section 7.1.4) to the 75 mL of ascorbic acid

solution (Section 7.1.3). Stability is approximately 10 days

when kept refrigerated.

7.2.3 Refractive Reagent A -- Add 50 mL of 4.9 N

H2SO4(Section 7.1.5) to 20 mL of reagent water. Add 1

mL of SLS (Section 7.1.4) to this solution. Prepare fresh

every few days.

7.2.4 Secondary Phosphorus Solution -- Take 1.0 mL

of Stock Phosphorus Solution (Section 7.1.6) and dilute

to 100 mL with reagent water. (1.0 mL = 0.0010 mg P.)

Refrigerate and prepare fresh every 10 days.

7.2.5 Prepare a series of standards by diluting suitable

volumes of standard solutions (Section 7.2.4) to 100 mL

with reagent water. Prepare these standards daily. When

working with samples of known salinity, it is recommended that the standard curve concentrations be prepared in low-level natural seawater (Section 7.1.7) diluted

to match the salinity of the samples. Doing so obviates

the need to perform the refractive index correction

outlined in Section 12.2. When analyzing samples of

varying salinities, it is recommended that the standard

curve be prepared in reagent water and refractive index

corrections be made to the sample concentrations (Section 12.2). The following dilutions are suggested.

8.0 Sample Collection, Preservation and

Storage

8.1 Sample Collection -- Samples collected for

nutrient analyses from estuarine and coastal waters are

normally collected using one of two methods: hydrocast

or submersible pump systems. Filtration of the sample

through a 0.45-Fm membrane or glass fiber filter immediately after collection is required.

8.1.1 A hydrocast uses a series of sampling bottles

(Niskin, Nansen, Go-Flo or equivalent) that are attached

at fixed intervals to a hydro wire. These bottles are sent

through the water column open and are closed either

electronically or via a mechanical “messenger” when the

bottles have reached the desired depth.

8.1.2 When a submersible pump system is used, a

weighted hose is sent to the desired depth in the water

column and water is pumped from that depth to the deck

of the ship for processing.

8.1.3 Another method used to collect surface samples

involves the use of a plastic bucket or large plastic bottle.

While not the most ideal method, it is commonly used in

citizen monitoring programs.

8.2 Sample Preservation -- After collection and

filtration, samples should be analyzed as quickly as

possible. If the samples are to be analyzed within 24 hr

of collection, then refrigeration at 4EC is acceptable.

8.3 Sample Storage -- Long-term storage of frozen

samples should be in clearly labeled polyethylene bottles

or polystyrene cups compatible with the analytical system’s automatic sampler (Section 6.1.1). If samples

cannot be analyzed within 24 hr, then freezing at -20EC

for a maximum period of two months is acceptable.5-8

9.0 Quality Control

9.1 Each laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability, the continued

analysis of LRBs, laboratory duplicates, and LFBs as a

continuing check on performance.

9.2 Initial Demonstration of Performance 

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (MDLs and linear

dynamic range) and laboratory performance (analysis of

QC samples) prior to analyses of samples using this

method.

9.2.2 MDLs should be established using a low-level

estuarine water sample fortified to approximately five
R'

(Cs&C)

S

x 100

365.5 - 5 Revision 1.4 September 1997

where, S = the standard deviation of the 

replicate analyses.

t = the Student’s t value for n-1 

degrees of freedom at the 99% 

confidence limit. t = 3.143 for six 

degrees of freedom.

where, R = percent recovery

 C = measured fortified sample S

concentration (background + 

concentrated addition in mg P/L)

C = sample background concentration 

(mg P/L)

S = concentration in mg P/L added to 

the environmental sample.

times the estimated detection limit. To determine MDL of control and the source of the problem should be

3

values, analyze seven replicate aliquots of water and identified and resolved before continuing the analyses.

process through the entire analytical method. Perform all

calculations defined in the method and report the con- 9.3.3 The laboratory must use LFB data to assess

centration values in the appropriate units. Calculate the

MDL as follows:

MDL = (t)(S)

MDLs should be determined every six months or whenever a significant change in background or instrument

response occurs or when a new matrix is encountered.

9.2.3 Linear Dynamic Range (LDR) -- The LDR should

be determined by analyzing a minimum of five calibration used to establish an ongoing precision statement for the

standards ranging in concentration from 0.001 mg P/L to level of concentrations included in the LFB. These data

0.20, mg P/L across all sensitivity settings of the auto- must be kept on file and be available for review.

analyzer. Normalize responses by dividing the response

by the sensitivity setting multiplier. Perform the linear 9.4 Assessing Analyte Recovery - Laboratory

regression of normalized response vs. concentration and Fortified Sample Matrix

obtain the constants m and b, where m is the slope and b

is the y-intercept. Incrementally analyze standards of

higher concentration until the measured absorbance analyte to a minimum of 5% of the routine samples or one

response, R, of a standard no longer yields a calculated sample per sample set, whichever is greater. The analyte

concentration CC, that is ± 10% of the known concentra- concentration should be two to four times the ambient

tion, C, where CC = (R - b)/m. That concentration defines

the upper limit of the LDR for your instrument. Should

samples be encountered that have a concentration that

is $90% of the upper limit of the LDR, then these samples

must be diluted and reanalyzed.

9.3 Assessing Laboratory Performance 

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- A laboratory

should analyze at least one LRB (Section 3.5) with each

set of samples. LRB data are used to assess contamination from the laboratory environment. Should an analyte

value in the LRB exceed the MDL, then laboratory or

reagent contamination should be suspected. When LRB

values constitute 10% or more of the analyte level determined for a sample, fresh samples or field duplicates of

the samples must be prepared and analyzed again after

the source of contamination has been corrected and

acceptable LRB values have been obtained.

9.3.2 Laboratory Fortified Blank (LFB) -- A laboratory

should analyze at least one LFB (Section 3.3) with each

batch of samples. Calculate accuracy as percent recovery. If the recovery of the analyte falls outside the required control limits of 90-110%, the analyte is judged out

laboratory performance against the required control limits

of 90-110% (Section 9.3.2). When sufficient internal performance data become available (usually a minimum of

20 to 30 analyses), optional control limits can be developed from the percent mean recovery (x) and the standard deviation (S) of the mean recovery. These data can

be used to establish the upper and lower control limits as

follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S

The optional control limits must be equal to or better than

the required control limits of 90-110%. After each 5 to 10

new recovery measurements, new control limits can be

calculated using only the most recent 20 to 30 data

points. Also, the standard deviation (S) data should be

9.4.1 A laboratory should add a known amount of

concentration and should be at least four times the MDL.

9.4.2 Calculate the percent recovery of the analyte,

corrected for background concentrations measured in the

unfortified sample, and compare these values with the

values obtained from the LFBs.

Percent recoveries may be calculated using the following

equation:

9.4.3 If the recovery of the analyte falls outside the

designated range of 90-110% recovery, but the laboratory

performance for that analyte is in control, the fortified
Revision 1.4 September 1997 365.5 - 6

Apparent orthophosphate

conc. due to refractive

Salinity (ppt) index (mg P/L)

1 0.0002

5 0.0006

10 0.0009

20 0.0017

 sample should be prepared again and analyzed. If the standards are entered as the independent variable and

result is the same after reanalysis, the recovery problem the corresponding peak height is the dependent variable.

encountered with the fortified sample is judged to be

matrix related, not system related. 12.2 Refractive Index Correction for Estuarine/

10.0 Calibration and Standardization

10.1 Calibration (Refer to Sections 11.5 and 12.0).

10.2 Standardization (Refer to Section 12.2).

11.0 Procedure

11.1 If samples are frozen, thaw the samples to room

temperature.

11.2 Set up manifold as shown in Figure 1. The tubing,

flow rates, sample:wash ratio, sample rate, etc., are

based on a Technicon AutoAnalyzer II system. Specifications for similar segmented flow analyzers vary, so

slight adjustments may be necessary.

11.3 Allow both colorimeter and recorder to warm up

for 30 min. Obtain a steady baseline with reagent water

pumping through the system, add reagents to the sample

stream and after the reagent water baseline has equilibrated, note that rise (reagent water baseline), and adjust

baseline.

For analysis of samples with a narrow salinity range, it is

advisable to use low nutrient seawater matched to

sample salinity as wash water in the sampler in place of

reagent water. For samples with a large salinity range, it

is suggested that reagent wash water and procedure

(Section 12.2) be employed.

11.4 A good sampling rate is approximately 40 samples/hr with a 9:1, sample:wash ratio.

11.5 Place standards (Section 7.2.5) in sampler in

order of decreasing concentration. Complete filling the

sampler tray with samples, LRBs, LFBs, and LFMs.

11.6 Commence analysis.

11.7 Obtain a second set of peak heights for all

samples and standards with Refractive Reagent A

(Section 7.2.3) being pumped through the system in place

of Reagent A (Section 7.2.1). This “apparent” concentration due to coloration of the water should be subtracted

from concentrations obtained with Reagent A pumping

through the system.

12.0 Data Analysis and Calculations

12.1 Concentrations of orthophosphate are calculated

from the linear regression obtained from the standard

curve in which the concentrations of the calibration 

Coastal Systems

12.2.1 Obtain a second set of peak heights for all

samples and standards with Refractive Reagent A (Section 7.2.3) being pumped through the system in place of

Reagent A (Section 7.2.1). Reagent B (Section 7.2.2)

remains the same and is also pumped through the

system. Peak heights for the refractive index correction

must be obtained at the same Standard Calibration

Setting and on the same colorimeter as the corresponding samples and standards.9

12.2.2 Subtract the refractive index peak heights from

the heights obtained for the orthophosphate determination. Calculate the regression equation using the corrected standard peak heights. Calculate the concentration of samples from the regression equation using the

corrected sample peak heights.

12.2.3 When a large data set has been amassed in

which each sample’s salinity is known, a regression for

the refractive index correction on a particular colorimeter

can be calculated. For each sample, the apparent orthophosphate concentration due to refractive index is

calculated from its peak height obtained with Refractive

Reagent A (Section 7.2.3) and Reagent B (Section 7.2.2)

and the regression of orthophosphate standards obtained

with orthophosphate Reagent A (Section 7.2.1) and

Reagent B (Section 7.2.2) for each sample. Its salinity is

entered as the independent variable and its apparent

orthophosphate concentration due to its refractive index

in that colorimeter is entered as the dependent variable.

The resulting regression equation allows the operator to

subtract an apparent orthophosphate concentration when

the salinity is known, as long as other matrix effects are

not present. Thus, the operator would not be required to

obtain the refractive index peak heights for all samples

after a large data set has been found to yield consistent

apparent orthophosphate concentrations due to salinity.

An example follows:

12.2.4 An example of a typical equation is:

mg P/L apparent PO4 = 0.000087 X Salinity

3-

(ppt) where, 0.000087 is the slope of the line.

where, 0.000087 is the slope of the line.
365.5 - 7 Revision 1.4 September 1997

Salinity Concentration Percent Relative

Sample (ppt) (mg P/L) Standard Deviation

1 36 0.0040 6.5

2 18 0.0024 10

3 3 0.0007 24

12.3 Results should be reported in mg PO - P/L or μg prevention as the management option of first choice. 4

3-

PO4- P/L. Whenever feasible, laboratory personnel should use

3-

mg PO - P/L = ppm (parts per million) generation. When wastes cannot be feasibly reduced at 4

3-

μg PO - P/L = ppb (parts per billion) 4

3-

13.0 Method Performance

13.1 Single Analyst Precision -- A single laboratory

analyzed three samples collected from Chesapeake Bay,

Maryland, and East Bay, Florida. Seven replicates of

each sample were processed and analyzed randomly

throughout a group of 75 samples with salinities ranging

from 3 to 36 ppt. The results were as follows:

13.2 Multilaboratory Testing

13.2.1 This method was tested by nine laboratories

using reagent water, high salinity seawater from the

Sargasso Sea (36 ppt) and three different salinity waters

from Chesapeake Bay, Maryland (8.3 ppt, 12.6 ppt, and

19.5 ppt). The reagent water and the Sargasso Seawater

were fortified at four Youden pair concentrations ranging

from 0.0012 mg P/L to 0.1000 mg P/L. The Chesa10 

peake Bay waters were fortified at three Youden pair

concentrations ranging from 0.0050 mg P/L to 0.0959 mg

P/L with the highest salinity waters containing the lowest

Youden pair and the lowest salinity waters containing the

highest Youden pair. Analysis of variance (ANOVA) at the

95% confidence level found no statistical differences

between water types indicating that the refractive index

correction for different salinity waters is an acceptable

procedure. Table 1 contains the linear equations that

describe the single-analyst standard deviation, overall

standard deviation, and mean recovery of orthophosphate

from each water type.

13.2.2 Pooled Method Detection Limit (p-MDL) -- The pMDL is derived from the pooled precision obtained by

single laboratories for the lowest analyte concentration

level used in the multilaboratory study. The p-MDLs using

reagent water and Sargasso Sea water were 0.00128 and

0.00093 mg P/L, respectively.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of environmental management techniques that places pollution

pollution prevention techniques to address their waste

the source, the Agency recommends recycling as the next

best option.

14.2 For information about pollution prevention that

may be applicable to laboratories and research institutions, consult Less is Better. Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society’s Department of Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036, (202)872-4477.

15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be conducted consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air,

water, and land by minimizing and controlling all releases

from hoods and bench operations, complying with the

letter and spirit of any sewer discharge permits and

regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste

identification rules and land disposal restrictions. For

further information on waste management, consult The

Waste Management Manual for Laboratory Personnel,

available from the American Chemical Society at the

address listed in Section 14.2.

16.0 References

1. Murphy, J. and J.P. Riley. 1962. A Modified Single

Solution Method for the Determination of Phosphate in Natural Waters. Analytica Chim. Acta 27,

31-36.0.

2. Technicon Industrial Systems. 1973. Orthophosphate in Water and Seawater. Industrial Method

155-71 W. Technicon Industrial Systems, Tarrytown, NY 10591.

3. 40 CFR, 136 Appendix B. Definition and Procedure

for the Determination of the Method Detection

Limit. Revision 1.11.

4. Grasshoff, K., M. Ehrhardt, and K. Kremling. 1983.

Methods of Seawater Analysis. Verlag Chemie,

Federal Republic of Germany, 419 pages.

5. Klingamann, E.D. and D.W. Nelson. 1976. Evaluation of Methods for Preserving the Levels of Soluble Inorganic Phosphorus and Nitrogen in Unfiltered Water Samples. J. Environ. Qual., 5:1, 42-

46.
Revision 1.4 September 1997 365.5 - 8

6. MacDonald, R.W. and F.A. McLaughlin. 1982. The

Effect of Storage by Freezing on Dissolved Inorganic

Phosphate, Nitrate, and Reactive Silicate for Samples

from Coastal and Estuarine Waters. Water Research, 16:95-104.

7. Thayer, G.W. 1979. Comparison of Two Storage

Methods for the Analysis of Nitrogen and Phosphorus

Fractions in Estuarine Water. Ches. Sci., 11:3, 155-

158.

8. Salley, B.A., J.G. Bradshaw, and B.J. Neilson. 1986.

Results of Comparative Studies of Preservation

Techniques for Nutrient Analysis on Water Samples.

VIMS, Gloucester Point, VA 23062. 32 pp.

9. Froelich, P.N. and M.E.Q. Pilson. 1978. Systematic

Absorbance Errors with Technicon AutoAnalyzer II

Colorimeters. Water Research 12: 599-603.

10. Edgell, K.W., E.J. Erb, and J.E. Longbottom, “Determination of Orthophosphate in Estuarine and

Coastal Waters by Automated Colorimetric Analysis:

Collaborative Study,” submitted in November 1992

for publication in Marine Chemistry.
365.5 - 9 Revision 1.4 September 1997

Table 1. Single-Analyst Precision, Overall Precision and Recovery from Multilaboratory Study

Reagent Water

(0.0012 - 0.100 mg P/L)

Mean Recovery X = 0.972C - 0.000018

Overall Standard Deviation S = 0.033X + 0.000505 R

Single-Analyst Standard Deviation S = 0.002X + 0.000448 r

Sargasso Sea Water

(0.0012 - 0.100 mg P/L)

Mean Recovery X = 0.971C - 0.000002

Overall Standard Deviation S = 0.021X + 0.000550 R

Single-Analyst Standard Deviation S = 0.010X + 0.000249 r

Chesapeake Bay Water

( 0.005 - 0.100 mg P/L)

Mean Recovery

X = 1.019C - 0.000871

Overall Standard Deviation S = 0.066X + 0.000068 R

Single-Analyst Standard Deviation S = 0.030X + 0.000165 r

C True value of spike concentration, mg P/L

X Mean concentration found, mg P/L, exclusive of outliers.

S Overall standard deviation, mg P/L, exclusive of outliers. R

S Single-analyst standard deviation, mg P/L, exclusive of outliers. r

17.0 Tables, Diagrams, Flowcharts, and Validation Data