U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2018–1120

Laboratory Evaluation of the Sea-Bird Scientific 

HydroCycle-PO4 Phosphate Sensor
Cover. Photograph of the Sea-Bird Scientific HydroCycle-PO4

. (Photograph courtesy of Sea-Bird Scientific, used with permission.)
Laboratory Evaluation of the Sea-Bird Scientific 

HydroCycle-PO4 Phosphate Sensor

By Teri T. Snazelle

Open-File Report 2018–1120

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

RYAN K. ZINKE, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2018

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Suggested citation:

Snazelle, T.T., 2018, Laboratory evaluation of the Sea-Bird Scientific HydroCycle-PO4 phosphate sensor: 

U.S. Geological Survey Open-File Report 2018–1120, 10 p., https://doi.org/ofr20181120.

ISSN 2331-1258 (online)
iii

Acknowledgments

The author wishes to thank Dr. Jacob Gibs (USGS, retired), and Dr. Cherie Miller (USGS) for their 

invaluable contributions to this project.
iv

Contents

Acknowledgments........................................................................................................................................iii

Abstract ..........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Description of the HydroCycle-PO4.............................................................................................................2

Orthophosphate Analysis .............................................................................................................................3

HydroCycle-PO4 Testing ...............................................................................................................................4

Test Procedures: Accuracy in Standard Dilutions with Deionized Water .................................4

Test Results: Accuracy in Standard Dilutions with Deionized Water .........................................5

Test Procedures: Accuracy in Tap and River Water.......................................................................6

Test Results: Accuracy in Tap and River Water...............................................................................7

Test Procedures: Accuracy in Environmental Samples .................................................................7

Test Results: Accuracy in Environmental Samples.........................................................................7

Test Procedures: CDOM Effect on Accuracy...................................................................................7

Test Results: CDOM Effect on Accuracy...........................................................................................8

Discussion.......................................................................................................................................................9

Conclusions.....................................................................................................................................................9

References Cited............................................................................................................................................9

Figures

1. Photograph showing the Sea-Bird Scientific HydroCycle-PO4.............................................2

2. Photograph showing the reagent cartridges on the HydroCycle.........................................2

3. Graph showing results from room temperature testing of HydroCycle in three 

Type I deionized water-phosphate standard dilutions, 0.110, 0.174, and 0.260 mg/L 

as phosphorous.............................................................................................................................6

4. Photograph showing the tea solution used to test the effect of chromophoric 

dissolved organic matter on the HydroCycle’s accuracy ......................................................7

5. Graph showing the measured concentration of a 0.260 milligram per liter as 

phosphorous phosphate standard, and the percentage of change in the 

HydroCycle’s measured phosphate concentration as chromophoric dissolved 

organic material increased.........................................................................................................8

Tables

1. Features and specifications of the Sea-Bird Scientific HydroCycle-PO4 ...........................3

2. Estimated waste production for a 1-month deployment with hourly sampling and

a matrix spike every fourth sample............................................................................................3

3. Calculation of standard uncertainty for phosphate working standards 0.110 mg/L 

as P and 0.260 mg/L as P .............................................................................................................5

4. Phosphate concentrations in tap water measured by using Sea-Bird Scientific’s 

HydroCycle-PO4 phosphate sensor and in samples from two USGS surfacewater sites .....................................................................................................................................7
v

Conversion Factors and Abbreviations

Inch/Pound to SI

Multiply By To obtain

Length

inch (in.)

inch (in.)

foot (ft)

25,400.0

2.54

0.3048

micrometer or micron (μm)

centimeter (cm)

meter

Volume

gallon (gal) 3.785 liter (L) 

Mass

pound, avoirdupois (lb)

pound, avoirdupois (lb)

453,592.37

0.4536

milligram (mg) 

kilogram (kg)

International System of Units to U.S. customary units

Multiply By To obtain

Volume

liter (L) 33.81402 ounce, fluid (fl. oz)

liter (L) 2.113 pint (pt)

liter (L) 1.057 quart (qt)

liter (L) 0.2642 gallon (gal)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as 

°F = (1.8 × °C) + 32.

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as 

°C = (°F – 32) / 1.8.

Concentrations of chemical constituents in water are given either in milligrams per 

liter (mg/L), micrograms per liter (μg/L), or micromoles per liter.

Multiply concentration in micromoles per liter of phosphate by 0.03097 to convert to 

milligrams per liter of phosphate as P.

Abbreviations

CaPO4 constant concentration

CDOM chromophoric (colored) dissolved organic matter

DIW quality-assured deionized water with resistance greater than or 

 equal to 18 megaohms (MΩ)

EPA U.S. Environmental Protection Agency

FTW filtered tea water

HAB harmful algal bloom

HIF Hydrologic Instrumentation Facility (USGS)

mg/L as P milligrams per liter as phosphorous (equivalent to parts per million)

mL milliliter
vi

Mo molybdenum

μmol/L micromoles per liter

NIST National Institute for Standards and Technology

NWQL National Water Quality Laboratory

P phosphorus

PO4 phosphate

QC quality control

QSE quinine sulfate equivalent

Sb antimony

SD standard deviation

SDI serial digital interface

USGS U.S. Geological Survey

VaPO4 Variable-based concentration
Laboratory Evaluation of the Sea-Bird Scientific 

HydroCycle-PO4 Phosphate Sensor

By Teri T. Snazelle

Abstract 

Sea-Bird Scientific’s HydroCycle-PO4 phosphate sensor is a single-analyte wet-chemistry sensor designed for in situ 

environmental monitoring. The unit was evaluated at the U.S. Geological Survey Hydrologic Instrumentation Facility to assess 

the accuracy of the sensor in solutions with known phosphorous concentration and to test the effects of chromophoric (colored) 

dissolved organic matter (CDOM) and natural water matrixes on sensor accuracy. Accuracy was tested with three standards: 

0.110, 0.174, and 0.260 milligram per liter, as phosphorous (mg/L as P). The 0.110- and 0.260-mg/L standards were made from 

a dilution of a National Institute of Standards and Technology-traceable phosphate-phosphorous standard with Type I deionized 

water (DIW). Average measured phosphate concentrations of the tested standards (0.110, 0.174, and 0.260 mg/L as P) in DIW 

were 0.132, 0.181, and 0.310 mg/L as P, for differences of 20, 4, and 19 percent, respectively. 

Measured phosphate concentration of a tested standard was biased by the addition of tea water filtered through a 

0.45-micrometer pore size filter (filtered tea water [FTW]) simulating the effect of CDOM. An aliquot of the filtered tea solution 

was sent to a certified environmental laboratory, which reported a less than reporting level (<0.004 mg/L as P) phosphate concentration. True color of the FTW was measured at 380 platinum-cobalt units by using Standard Methods 8025. For this FTW 

test, the measured phosphate concentration for the clear 0.260 mg/L as P standard averaged 0.366 mg/L as P. This concentration 

increased to an average of 0.653 mg/L as P with the addition of 10 percent FTW, and to an average of 0.859 mg/L as P with the 

addition of 20 percent FTW. These test results indicate a positive bias of up to 40 percent of the concentrations of the measured 

phosphate concentrations when CDOM is present and indicate a proportional increase in an apparent concentration of phosphorus instrument response as CDOM concentration increases.

Introduction

Phosphate loading and its role in the acceleration of vegetative growth and eutrophication in water has been widely documented (Correll, 1998; Paerl and Otten, 2013). Excessive nutrient inputs such as nitrogen and phosphate can result in problematic water conditions, from anoxic water to harmful algal blooms (HABs) capable of releasing dangerous cyanotoxins into 

water sources (Jacobi Carbons, Inc., 2014). Phosphates are formed from naturally occurring and anthropogenic phosphorus and 

exist in three forms: organic phosphate, meta or polyphosphate, and reactive or orthophosphate. Organic phosphates are formed 

primarily by biological processes or by the breakdown of organic pesticides that contain phosphate. Polyphosphates are strong 

complexing agents for metal ions and are used in the treatment of boiler waters (Oram, 2014). Polyphosphates are unstable in 

the environment and will eventually become orthophosphates. Orthophosphates are the most stable form of phosphate and are in 

fertilizers and sewage. Low-level orthophosphate can be measured in environmental waters by using colorimetric analysis (U.S. 

Environmental Protection Agency [EPA], 1997).

The U.S. Geological Survey (USGS) Hydrologic Instrumentation Facility (HIF) evaluates the performance of instruments 

and equipment that are used to measure hydrologic data. Instrument and equipment evaluations are done primarily to determine 

if particular devices would be suitable for use by USGS personnel for hydrologic data collection. Evaluation reports document 

the results obtained with the instrument model and firmware used at the time of testing, and may or may not represent results 

obtained from software updates and instrument model improvements that occur after testing. Reports describing the instrument 

evaluation results are not indicative of an endorsement by the USGS of the tested instrument. 

This report documents the laboratory evaluation of the Sea-Bird Scientific HydroCycle-PO4 phosphate sensor (hereinafter, 

“HydroCycle”), and test procedures and results are described. The sensor was tested with the Sea-Bird Scientific Cycle Host 

software, version 1.08 (Sea-Bird Scientific, 2016a). 
2 Laboratory Evaluation of the Sea-Bird Scientific HydroCycle-PO4 Phosphate Sensor

Description of the HydroCycle-PO4

The HydroCycle phosphate sensor is a field-deployable wet-chemical sensor that was designed for continuous environmental monitoring of soluble orthophosphate (fig. 1). The sensor reports concentrations in units of either micromoles per liter or milligrams per liter of orthophosphate, as phosphorus. The sensor comes with a protective sleeve that can be removed for service, 

and protective “hats” designed to cover the sensor intakes during transport. The sensor uses a single copper screen mesh with 

0.011-inch pores and 5–10-micron onboard filters to reduce sediment intake. Three color-coded reagent cartridges are used to 

dispense the chemicals needed for analysis. The HydroCycle measures soluble orthophosphate (also called reactive phosphorus) 

based upon EPA method 365.5 (EPA, 1997). The method produces a heteropoly molybdenum-blue complex with phosphate that 

can be detected colorimetrically. The HydroCycle has a red reagent cartridge containing an acidic molybdate-antimony solution, 

a yellow cartridge containing ascorbic acid, and a blue cartridge with a traceable phosphate standard of 5.3 micromoles per liter 

(μmol/L) (approximately 0.164 milligram per liter, as phosphorus [mg/L as P]) from the National Institute for Standards and 

Technology (NIST) (fig. 2). Personal protective gear, including gloves, is recommended when changing the reagent cartridges 

due to the acidity and toxicity of the reagents, and gloves and safety glasses are recommended when performing routine maintenance of the HydroCycle such as flushing of the system with concentrated bleach. The sensor has an RS-232 and serial digital 

interface at 1200-baud (SDI-12) communication protocols with a 19200-baud rate. Power is supplied through a six-pin cable for 

RS-232 communication and through an eight-pin cable for SDI-12 communication. The HydroCycle is designed to be deployed 

vertically, and it will not function properly if deployed in a horizontal orientation. The features and specifications of the HydroCycle are listed in table 1.

Figure 1.

Figure 1. The Sea-Bird 

Scientific HydroCycle-PO4. 

Photograph courtesy of 

Sea-Bird Scientific, used 

with permission.

Figure 2. Figure 2. The reagent cartridges on the HydroCycle. 

The blue cartridge contains the phosphate standard, 

the yellow cartridge contains the ascorbic reagent, 

and the red cartridge contains the acidic ammonium 

molybdate/antimony potassium tartrate reagent. 

Photograph courtesy of Sea-Bird Scientific, used 

with permission.
Orthophosphate Analysis 3

Table 1. Features and specifications of the Sea-Bird Scientific HydroCycle-PO4 (Sea-Bird Scientific, 2016b). 

[EPA, U.S. Environmental Protection Agency; m, meter; kg, kilogram; cm, centimeter; oC, degrees Celsius, mg/L as P, milligrams per liter as phosphorus; 

μM, micromoles; ASCII, American Standard Code for Information Interchange; SDI-12, Serial digital interface at 1200 baud; VDC, volts direct current; mA, 

milliamps; A, amps; nm, nanometer; ≥, greater than or equal to]

Feature Specification

Method of operation Wet chemistry based upon EPA method 365.5

Rated depth 200 m

Weight 7.6 kg (with reagents)

Height with handle 56 cm (22 inches)

Diameter 18 cm (7.1 inches)

Temperature operating range 0–35 oC

Range of detection 0–0.3 mg/L as P (0–10 μM)

Detection limit 0.0023 mg/L as P (0.075 μM)

Communication protocols SDI-12 (1200-baud rate) and RS-232 (19200-baud rate)

Power requirements (RS-232 ASCII)

(RS-232 or SDI-12)

10.5–18 VDC, 115 mA average, 3 A max

10.5–18 VDC, 125 mA average, 3 A max

Monitoring wavelength 870 nm

Pathlength 5 cm

Number of runs per reagent set ≥1,000

Reagent life 5 months from manufacture

Orthophosphate Analysis

Unlike nitrate-nitrite, native soluble phosphate does not absorb energy in the ultraviolet or visible wavelengths and therefore is not measurable through colorimetry or spectrophotometry without chemical manipulation. To get the soluble orthophosphate into a detectable form, it must be treated with some toxic chemicals. To detect low-level orthophosphate, the sample is 

treated with ammonium molybdate and antimony potassium tartrate to form an antimony-phospho-molybdate complex, which 

when reduced with ascorbic acid, forms an intensely blue-colored complex. 

Antimony (Sb) and molybdenum (Mo) are toxic heavy metals that are regulated by the EPA. An average 1-month deployment with an hourly sampling rate and a matrix-spike frequency of every fourth sample would produce the volumes listed in 

table 2.

Table 2. Estimated waste production for a 1-month deployment with hourly sampling and a matrix spike 

every fourth sample.

[mL, milliliter; Sb, antimony; Mo, molybdenum]

Waste contributor Volume of contributor Total volume (liters)

Sampled water 720×30 mL 21.6

Matrix spikes 180×20 mL 3.6

Sb/Mo reagent 720×0.2 mL 0.14

Ascorbic acid 720×0.2 mL 0.14

Phosphate standard 180×0.2 mL 0.036

Total waste 25.52
4 Laboratory Evaluation of the Sea-Bird Scientific HydroCycle-PO4 Phosphate Sensor

Although a month’s deployment produces more than 25 L of waste, most of that is sample volume; less than 5 grams of 

Mo (0.195 mg/L) and less than 0.5 gram of Sb (19.5 mg/L) are actually produced. Waste was collected and disposed of properly 

to meet all applicable regulatory requirements, including those for acidity-alkalinity. Operation of the HydroCycle produces a 

strong acidic waste with an average pH of 1–2 pH units. Regulatory requirements vary from State to State; however, most dictate 

that the naturally occurring pH of receiving waters not be altered from an acceptable range, and they limit the effect of waste to 

0.5 to 1 pH unit over a period of 24 hours (EPA, 1988).

HydroCycle-PO4 Testing 

To test the HydroCycle, it was connected to a computer with the Sea-Bird Scientific Cycle Host software installed. The 

Cycle Host software provides communication to the sensor by using assigned communication (com) ports and a default 19200-

baud rate. Once connected to the sensor, the software allows the user to prime the sensor tubing with sample and (or) reagents, 

track the volume of remaining reagents, and monitor the status of the sensor. Sample batches are programmed by using the 

Cycle Host Deployment Wizard, which gives the user the option of different modes of operation: autonomous (stand-alone) 

operation, synchronous slave (SDI-12) operation, or host (computer-controlled) operation. The Deployment Wizard guides 

the user through the process of programming the HydroCycle for operation and allows the user to dictate the sampling rate 

or the number of samples to be analyzed, as well as the frequency of quality control (QC) matrix spikes. For the QC samples, 

the onboard phosphate internal standard is added during a batch run. The QC samples are optional, but they provide important 

matrix-spike information on potential interferences in the sample solution. After the steps in the Deployment Wizard are completed, the volumes of samples and reagents needed for the batch are calculated, and the amount of waste that will be produced 

during the run is estimated. This information is collected in a deployment report that can be saved and (or) printed.

 Sea-Bird Scientific does not specify an accuracy range for the HydroCycle, but encourages its users to generate their own 

site-specific accuracy estimates through discrete sampling. In-house performance checks at Sea-Bird require the HydroCycle to 

measure the 0.164 mg/L as P onboard internal standard within –3 to 15 percent of the standard value. Testing of the HydroCycle 

at Sea-Bird found the accuracy to average –5 to 20 percent at the lower and upper ends of the operating range (C. Koch, SeaBird Scientific, written commun., 2017). For the evaluation, the test acceptance criterion was based upon these findings and was 

set at ±20 percent of the standard value. 

The accuracy of the HydroCycle was tested at the HIF by using Type I 18.2-megohm (MΩ) ultrapure deionized water 

(DIW) and a NIST 50 mg/L as P ±0.5-percent certified phosphate-phosphorous standard. Phosphate concentration of the DIW 

was tested with the HydroCycle and ranged from 0.0 to 0.015 mg/L as P, with an average value of 0.010 mg/L as P. In addition 

to sensor accuracy, the HydroCycle was also tested for the effect of chromophoric (colored) dissolved organic matter (CDOM) 

on accuracy. Sensor accuracy was determined by percent difference between the measured and calculated orthophosphate concentration. Percent difference was determined by the following equation:

Percent difference = 100× 

measured orthophosphatein mg

L 

asP c − alculated orthophosphatein mg

L as P

calculated orthophosphate mg

L as P





 





 (1)

Because difference was determined by subtracting the calculated value from the measured value, positive values indicated a high 

bias in the data, and negative values indicated a low bias in the data.

Test Procedures: Accuracy in Standard Dilutions with Deionized Water 

To evaluate the accuracy of the HydroCycle, testing was conducted at room temperature in three standards of known 

concentration, 0.110 mg/L as P, 0.174 mg/L as P, and 0.260 mg/L as P. The three standards were formulated by diluting the 

50 mg/L as P NIST-certified standard with the laboratory Type I DIW. These values were calculated from the 0.010 mg/L as P 

measured phosphate concentration of the laboratory’s Type I DIW added to the calculated concentration of the working standard 

(American Society for Testing and Materials International, 1999). The two working standards (0.110 mg/L as P and 0.260 mg/L 

as P) were made and used the day of testing and were discarded after testing. To create the 0.110 mg/L as P working standard, 
HydroCycle-PO4

 Testing 5

2 milliliters (mL) of the 50 mg/L as P phosphate standard was added to a 1-liter (L) class A volumetric flask and filled to the 

1-L mark with Type I DIW. For 1 L of the 0.260 mg/L as P working standard, 5 mL of the 50 mg/L as P phosphate standard was 

added to a 1-L class A volumetric flask and filled to the 1-L mark with Type I DIW. 

Standard uncertainty was estimated to be 1.42 percent and was derived from normal distribution of the root-sum method 

of the Guide for Estimation of Measurement Uncertainty (American Association for Laboratory Accreditation, 2014). Standard 

uncertainty was calculated by using the random uncertainties listed in table 3. 

The third working standard (0.174 mg/L as P ) was made by using the onboard internal standard from one of the HydroCycle’s reagent cartridges. This internal standard was documented to be 5.3 micromolars (μM) or 0.164 mg/L as P ±8 percent 

and was supplied for QC purposes by the manufacturer (Sea-Bird Scientific, 2016b). To create the 0.174 mg/L as P calculated 

standard, the HydroCycle was plumbed to sample Type I DIW provided by the laboratory system and was programmed to add 

the internal standard by using the Deployment Wizard. Summing the phosphate concentrations of the DIW and the internal standard gave an approximate concentration of 0.174 mg/L as P. Standard uncertainty for this standard was estimated at 8 percent, 

from the 8-percent uncertainty of the internal standard. 

Testing of the HydroCycle consisted of separate batches or “runs” of a minimum of five samples each. The HydroCycle 

software reports two concentrations for each measured sample. The CaPO4, or “constant concentration,” is based upon the factory calibration for the sensor and is the value that should be reported (Sea-Bird Scientific, 2016b). The “variable-based concentration” (VaPO4

) value is given as an indicator of data quality or sensor health. The VaPO4 uses the standard addition or matrixspike concentration to calculate net phosphate concentration. Under ideal conditions, the CaPO4

 and the VaPO4 values will differ 

no more than 0.005 mg/L as P. Sensor testing was conducted from November 2016 to February 2017 by the technical staff of the 

HIF’s Water Quality Servicing Laboratory, and all results are based upon the CaPO4 concentration of the solutions.

Test Results: Accuracy in Standard Dilutions with Deionized Water 

The mean measured phosphate in the 0.110 mg/L as P standard analyzed in the laboratory at room temperature 

(22–25 degrees Celsius [oC]) was 0.134 mg/L as P for the first batch of five samples and 0.130 mg/L as P for the second batch of 

five samples, resulting in calculated percent differences of 21.8 and 18.2, respectively. The measured phosphate for the 0.174 mg/L as 

P standard averaged 0.182 mg/L as P in the first batch of samples and 0.180 mg/L as P in the second batch, with differences of 4.5 

and 3.6 percent, respectively. Measured phosphate in the 0.260 mg/L as P standard averaged 0.308 mg/L as P in the first batch of 

samples and 0.287 mg/L as P in the second batch of samples. Calculated differences in this standard were 18.3 and 10.5 percent, 

respectively (fig. 3). 

Table 3. Calculation of standard uncertainty for phosphate working standards 0.110 mg/L as P and 0.260 mg/L as P.

[NIST, National Institute of Standards and Technology; mg/L as P, milligrams per liter as phosphorus; %, percent; SD, standard deviation; L, liter]

Random uncertainty Uncertainty contribution (percent) Squared uncertainty (percent)

Precision of five sample runs (SD) 0.033 0.0011

Accuracy of NIST 50 mg/L as P phosphate 

standard

0.5 0.25

Dilution errors including Type A 1-L 

glassware, technical measurement, and 

rounding error

0.50 0.25

Sum of uncertainties 0.501

Square root of sum 0.708

95% confidence (2SD) 1.42
6 Laboratory Evaluation of the Sea-Bird Scientific HydroCycle-PO4 Phosphate Sensor

Test Procedures: Accuracy in Tap and River Water

The accuracy of the HydroCycle was also tested in local tap water and in riverine water from two USGS streamgaging 

sites. For the tap water test, approximately 3 gallons of tap water were collected in a 5-gallon (gal) bucket. The HydroCycle was 

placed into the bucket to elevate the intake screen roughly 2 inches from the bottom of the bucket. The HydroCycle was primed 

following the manufacturer’s instructions and programmed to analyze the tap water by using the Deployment Wizard function 

of the Cycle Host software. Nine replicates of the tap water were analyzed, and the measured phosphate concentrations were 

averaged. An aliquot of tap water was not sent to the USGS National Water Quality Laboratory for analysis, but phosphate concentration was reported as less than the detection limit (<0.004 mg/L as P) in locally monitored Consumer Confidence Reports 

(EPA, 2017). 

On February 1, 2017, approximately 3 gal of water from USGS site 02492620, Pearl River at National Space Technology 

Laboratory (NSTL) Station, MS, were collected in a 5-gal bucket. The HydroCycle was primed by following the manufacturer’s 

recommendations, placed into the bucket containing the water sample, and secured to ensure proper sample flow. The HydroCycle was programmed to analyze the sample with five replicates. Water was also collected on February 1, 2017, from USGS 

site 02481510, Wolf River near Landon, MS, and the process was repeated. Aliquots of the river samples were filtered with Pall 

0.45-micron filters, collected into labeled 10-mL vacuum tubes, chilled, and sent to the USGS National Water Quality Laboratory (NWQL) for analysis by code 3118, Orthophosphate as P, by colorimetry. The detection limit of laboratory code 3118 is 

0.004 mg/L as P. Phosphate concentrations analyzed by the NWQL for both USGS sites were compared to the HydroCycle’s 

averaged measured phosphate concentration to determine accuracy. Accuracy of the measured concentration was determined by 

percent difference from the NWQL analysis, which was calculated as

Percent difference = 100× 

measured orthophosphatein mg

L as P NWQL orthophosphatein m 

− 

g

L as P

NWQLorthophosphate mg

L 

as P 



 





 (2)

–30

–25

–20

–15

–10

–5

0

5

10

15

20

25

30

0 0.05 0.1 0.15 0.2 0.25 0.3

Percent difference between measured and

standard orthophosphate, in mg/L as P

Standard orthophosphate, in mg/L as P

Figure 3.

EXPLANATION

Test criteria for accuracy

Figure 3. Results from room temperature testing of HydroCycle in three Type I 

deionized water-phosphate standard dilutions, 0.110, 0.174, and 0.260 milligram 

per liter, as phosphorus (mg/L as P). Error bars represent the 1.42 percent standard 

uncertainty for the 0.110 and 0.260 mg/L as phosphorous standards and the 8-percent 

uncertainty of the 0.174 mg/L as phosphorous standard.
HydroCycle-PO4

 Testing 7

Test Results: Accuracy in Tap and River Water

Results from testing of the HydroCycle in the tap water and from the unfiltered river samples from the two USGS sites are 

listed in table 4. Also listed are the results from the NWQL by code 3118 on the filtered aliquots of the water collected from the 

two sites.

Test Procedures: Accuracy in Environmental Samples

The Pearl River at USGS site 02492620 and the Wolf River at USGS site 02481510 are both relatively clear with low 

turbidity, but they are subjected to increases in sediment during times of heavy precipitation. CDOM is not measured routinely 

at the Wolf River site, so historical hydrologic data for this parameter were not available. CDOM is measured routinely at site 

02492620 on the Pearl River and is reported in micrograms per liter of quinine sulfate equivalents. CDOM at the Pearl River site 

fluctuated between 80 and 85 micrograms per liter of quinine sulfate equivalents (μg/L QSE) during the week the water samples 

were collected (week of February 1, 2017); however, CDOM concentrations routinely exceed 125 μg/L QSE at this site (USGS, 

2017). To isolate the source of the bias from the previous river water tests, a fresh sample was collected from the Pearl River on 

February 6, 2017. The water sample was thoroughly mixed, and an aliquot was filtered with a Pall 0.45-micron filter to remove 

particulates. The HydroCycle was submerged into the remaining unfiltered part of the water sample, and the HydroCycle was 

primed and programmed to analyze a batch of five samples. After analysis of the batch of unfiltered river water, the HydroCycle 

was primed with the filtered river water and programmed to analyze a batch of five samples along with two QC samples, for a 

total of seven samples. 

Test Results: Accuracy in Environmental Samples

The concentrations of phosphate from the analysis of the unfiltered river water collected on 

February 6, 2017, closely matched the concentration of phosphate from the sample collected on 

February 1, 2017. The average of the measured phosphate concentrations in samples collected 

on February 1 was 0.684 mg/L as P, and the average of measured phosphate in samples collected 

on February 6 was 0.678 mg/L as P. Standard deviation (SD) for the batch was 0.021 mg/L 

as P. After filtration, the amount of measured phosphate was notably reduced and averaged 

0.022 mg/L as P in the filtered river water. SD for this batch was 0.002 mg/L as P, indicating 

good precision in the test results. 

Test Procedures: CDOM Effect on Accuracy

To test the effect of CDOM on the HydroCycle, a filtered tea water (FTW) solution was 

made by adding one family-sized cold-brew tea bag to 1 L of Type 1 DIW (fig. 4). After allowing the tea to steep for approximately 20 minutes, the tea bag was removed, and the solution was 

filtered with a Pall 0.45-micron filter to remove particulates. Although the tea solution was used 

to replicate the effect of CDOM on measurement accuracy, naturally occurring organic matter in 

environmental waters is much more complex and could produce a different response. 

Figure 4. Figure 4. The tea solution 

used to test the effect of 

chromophoric (colored) 

dissolved organic matter on 

the HydroCycle’s accuracy.

Table 4. Phosphate concentrations in tap water measured by using Sea-Bird Scientific’s HydroCycle-PO4

phosphate sensor and in samples from two USGS surface-water sites.

[mg/L as P, milligrams per liter as phosphorous; NWQL, National Water Quality Laboratory; n/a, not applicable; USGS, U.S. Geological 

Survey]

Phosphate measured by HydroCycle, 

in mg/L as P 

Phosphate measured by NWQL 

(

1

code 3118), in mg/L as P

Unfiltered tap water 0.078 n/a

Unfiltered water from USGS site 02492620 0.684 ≤0.004

Unfiltered water from USGS site 02481510 0.594 ≤0.004

1 The method detection limit for the NWQL code 3118 is 0.004 mg/L as P.
8 Laboratory Evaluation of the Sea-Bird Scientific HydroCycle-PO4 Phosphate Sensor

An aliquot of the filtered tea solution was sent to the NWQL for analysis by code 3118 to confirm negligible (<0.004 mg/L 

as P) phosphate levels. A fresh 0.260 mg/L as P standard was made by diluting the 50 mg/L as P phosphate-phosphorus standard 

(to 0.250 mg/L as P) from Ricca Chemical with Type I DIW (with a measured 0.01 mg/L as P phosphate). The HydroCycle was 

primed with the 0.260 mg/L as P standard. By using the Deployment Wizard option of the Cycle Host software, the sensor was 

programmed to analyze five replicates, and the mean and percent difference were calculated for the batch.

For the next test, approximately 100 mL of the FTW was added to 1 L of the 0.260 mg/L as P standard, producing an 

approximate 10-percent “CDOM” solution. Turbidity of this CDOM solution was analyzed by using a Hach 2100N turbidimeter 

and averaged 1.3 formazin nephelometric units during testing. True color of the FTW was measured at 380 platinum-cobalt units 

by using Standard Methods 8025. The HydroCycle was primed with the 10-percent CDOM solution and was programmed to 

analyze five replicates. For the next test, approximately 200 mL of the FTW was added to 1 L of the 0.260 mg/L as P standard, 

producing an approximate 20-percent CDOM solution, and the process was repeated. For each batch, the mean and percent difference were calculated. To eliminate the possibility of falsely elevated results due to “carry-over” or fouling of the sensor’s tubing by the intensely blue color of the tested samples, the original (DIW) 0.260 mg/L as P standard was re-analyzed immediately 

after the 20-percent CDOM test, and mean phosphate and percent difference were calculated.

Test Results: CDOM Effect on Accuracy

The first test of the clear DIW 0.260 mg/L as P standard resulted in an average phosphate concentration of 0.366 mg/L as 

P, resulting in an approximate 41-percent difference or increase. The second test that contained approximately 10 percent FTW 

added to the 0.260 mg/L as P standard (10 percent CDOM) resulted in an average phosphate concentration of 0.653 mg/L as P. 

This difference was an approximate increase of 78 percent from the 0.366 mg/L as P measured concentration of the DIW standard and an increase of 151 percent from the clear DIW 0.260 mg/L as P standard value. 

Testing of the 0.260 mg/L as P standard with approximately 20 percent FTW (20 percent CDOM) added to the standard resulted 

in an even higher concentration of measured phosphate. For this test, the mean measured phosphate concentration was 0.858 mg/L 

as P, resulting in a 134-percent increase from the average measured concentration of the clear DIW standard (0.366 mg/L as P) and a 

230-percent increase from the 0.260 mg/L as P standard value. To verify that the increase was not a product of carryover or from 

staining of the HydroCycle’s optics, the original clear DIW 0.260 mg/L as P standard was re-analyzed immediately after the test 

with the 20 percent FTW/0.260 mg/L as P phosphate solution. Phosphate concentration from this test averaged 0.362 mg/L as P, 

or an approximate 39-percent difference from the 0.260 mg/L as P standard value (fig. 5). The strong correlation of the CDOM 

“bookend” testing of the clear DIW standard phosphate concentrations, 0.366 mg/L as P (41-percent difference) and 0.362 mg/L 

as P (39-percent difference), gives confidence that the test results are valid and also lends credence to the theory that the 

increases in concentration seen in the CDOM tests were due to the added color from the tea and not due to staining of the sensor 

optics or from carryover.

Figure 5.

–20

0

20

40

60

80

100

120

140

160

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Clear 0.260

mg/L as P

standard prior

to CDOM test

Clear 0.260

mg/L as P

standard after

CDOM test

0.260 mg/L as P

standard with

10 percent

CDOM

0.260 mg/L as P

standard with

20 percent

CDOM

Percent change in measured phosphate concentration

of 0.260 mg/L as phosphorous standard

Measured phosphate concentration of

0.260 mg/L as phosphorous standard 

Figure 5. The measured concentration of a 0.260 milligram per 

liter as phosphorous (mg/L as P) phosphate standard, and the 

percentage of change in the HydroCycle’s measured phosphate 

concentration as chromophoric (colored) dissolved organic 

material (CDOM) increased.
References Cited 9

Discussion

 Measurement of orthophosphate by colorimetry is complex and operationally difficult because of the use of toxic chemicals and the need for proper hazardous waste disposal, burdensome maintenance requirements to ensure proper sampling, and 

the potential for cross contamination from prior samples containing higher concentrations. Turbid environmental conditions and 

the presence of matrix interferences, such as color or bubbles, also further complicate the in situ measurement of phosphate. 

The accuracy of the HydroCycle met the ±20-percent test criterion when evaluated with the 0.164 mg/L as P internal standard 

and Type I DIW. Accuracy in the first batch of 0.110 mg/L as P samples narrowly exceeded the criteria, with a difference from 

the standard value of 21.8 percent. Performance improved with the second batch of 0.110 mg/L as P samples, and the average 

measured concentration differed from the standard value by 18.2 percent. Accuracy in the 0.260 mg/L as P standard also met 

the test criteria and differed from the standard value by 18.3 percent in the first batch of samples and by 10.4 percent in the 

second batch. 

The results from this testing indicate that the HydroCycle could be a viable tool for the measurement of active phosphate 

in an oceanographic or sediment- and color-free surface-water or groundwater setting when operated by an experienced and 

well-trained technician. Testing of unfiltered riverine samples revealed potential bias of accurate phosphate concentration. This 

bias was most likely caused by interference with the light transmittance of the HydroCycle from CDOM or by the release of 

“additional” reactive phosphate adhered to the suspended sediment in the riverine sample. Additional testing of DIW-phosphate 

standards spiked with aliquots of FTW to simulate CDOM confirmed the bias and showed that measured phosphate concentration increased proportionally with the increase in CDOM.

Conclusions

Testing of the HydroCycle phosphate sensor in three phosphate concentrations (0.110, 0.174, and 0.260 mg/L as P) 

showed it was accurate to within 22 percent with a measurable positive bias. The average measured phosphate concentration 

of the 0.110 mg/L as P batches was 0.132 mg/L as P for a difference of 20 percent. The average measured concentration of the 

0.174 mg/L as P standard, using the onboard internal standard (0.164 mg/L as P) added to Type 1 DIW was 0.181 mg/L as P for 

the two batches of samples, or a difference of 4 percent. Testing of the 0.260 mg/L as P standard, which was approaching the 

0.30 mg/L as P upper limit of the detection range, resulted in an average of 0.298 mg/L as P for the two batches, or a 14-percent 

difference from the standard concentration.

Measured phosphate concentration of a DIW-phosphate standard was biased by the addition of FTW simulating the effect 

of CDOM. Measured phosphate concentration in the clear 0.260 mg/L as P standard averaged 0.366 mg/L as P and increased to 

an average of 0.653 mg/L as P with the addition of 10 percent FTW and to an average of 0.859 mg/L as P with the addition of 

20 percent FTW. These test results indicate bias of the measured phosphate concentrations to higher values when CDOM is present and seem to indicate a proportional effect when CDOM concentration increases.

References Cited

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accessed March 14, 2017, at https://portal.a2la.org/guidance/est_mu_testing.pdf. 

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Washington, D.C., American Public Health Association. 

American Society for Testing and Materials International, 1999, D1193-99e1, Standard specification for reagent water: West 

Conshocken, Penn.

Correll, D.L., 1998, The role of phosphorus in the eutrophication of receiving waters: A review: Journal of Environmental 

Quality, v. 27, p. 261–266.

Jacobi Carbons, Inc., 2014, Technical bulletin: Treatment of cyanotoxins in drinking water with activated carbon: Water Online 

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10 Laboratory Evaluation of the Sea-Bird Scientific HydroCycle-PO4 Phosphate Sensor

Oram, B., 2014, Total phosphorus and phosphate impact on surface waters: Water Research Center, accessed February 7, 2017, 

at http://www.water-research.net/index.php/phosphate-in-water.

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Sea-Bird Scientific, 2016a, Cycle Host 1.08 software, accessed November 29, 2016, at http://www.seabird.com/software/

cycle-host. 

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manual: Document No. 160510HydroCyclePO4, version A, 88 p.

U.S. Environmental Protection Agency [EPA], 1988, Acidity-alkalinity (pH), water quality standards criteria summaries: A compilation of State/Federal criteria: U.S. Environmental Protection Agency EPA 440/5-88/020. 

U.S. Environmental Protection Agency [EPA], 1997, Methods for the determination of chemical substances in marine and 

estuarine matrices (2d ed.): Method 365.5 Determination of Orthophosphate in Estuarine and Coastal Waters by Automated 

Colorimetric Analysis, Revision 1.4. 

 U.S. Environmental Protection Agency [EPA], 2017, Safe Drinking Water Consumer Confidence Reports, accessed February 2, 

2017, at https://ofmpub.epa.gov/apex/safewater/f?p=136:102::::::.

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F7P55KJN. [Site information directly accessible at https://waterdata.usgs.gov/nwis/inventory/?site_no=02492620&agency_

cd=USGS&.] 

For more information about this publication, contact

Chief, Hydrologic Instrumentation Facility

U.S. Geological Survey

Building 2101

Stennis Space Center, MS 39529

For additional information visit 

https://water.usgs.gov/hif/

Publishing support provided by

Lafayette Publishing Service Center

Snazelle—Laboratory Evaluation of the Sea-Bird Scientific HydroCycle-PO4 Phosphate Sensor—Open-File Report 2018–1120

ISSN 2331-1258 (online)

https://doi.org/10.3133/ofr20181120