U.S. Department of the Interior

U.S. Geological Survey

Open-File Report 2013–1104

Prepared in collaboration with the U.S. Fish and Wildlife Service

Determination of Selenium in Fish from Designated 

Critical Habitat in the Gunnison River, Colorado, 

March through October, 2012

Determination of Selenium in Fish 

from Designated Critical Habitat in 

the Gunnison River, Colorado, March 

through October, 2012

By Thomas W. May and Michael J. Walther

Prepared in collaboration with the U.S. Fish and Wildlife Service

Open-File Report 2013–1104

U.S. Department of the Interior

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

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2013

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Suggested citation:

May, T.W., and Walther, M.J., 2013, Determination of selenium in fish from designated critical habitat in the 

Gunnison River, Colorado, March through October, 2012: U.S. Geological Survey Open-File Report 2013–1104, 4 p.,

http://pubs.usgs.gov/of/2013/1104/. 
iii

Contents

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

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

Methods...........................................................................................................................................................1

Field Collection and Preservation ......................................................................................................1

Sampling History...................................................................................................................................1

Lyophilization.........................................................................................................................................2

Instrumental Analysis...........................................................................................................................2

Results .............................................................................................................................................................2

Quality-Control Results .................................................................................................................................2

References Cited............................................................................................................................................4

Tables

1. Concentrations of selenium in fish muscle plugs collected from fish inhabiting the 

Gunnison River in western Colorado, March through October, 2012...................................3

2. Selenium recoveries from a reference tissue material analyzed with fish muscle 

plug samples..................................................................................................................................3

Conversion Factors

SI to Inch/Pound

Multiply By To obtain

Length

milliTorr (mTorr) 1,000 Torr

Volume

liter (L) 33.82 ounce, fluid (fl. oz)

milliliter (mL) 0.034 ounce, fluid (fl. oz)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

kiloelectron volt (keV) 1,000 electron volt (eV)

milligram (mg) 0.000035 ounce, avoirdupois (oz)

microgram (μg) 0.000000035 ounce, avoirdupois (oz)

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

°F=(1.8×°C)+32.

Concentrations of chemical constituents in solid materials are given in micrograms per gram 

(μg/g) dry weight.

Concentrations of chemical constituents in liquids are given in micrograms per liter (μg/L).

Abstract

This report presents results for the summer 2012 sampling of muscle plugs from common carp (Cyprinus carpio), 

bonytail chub (Gila elegans), Colorado pikeminnow (Ptychocheilus lucius), and razorback suckers (Xyrauchen texanus) 

inhabiting critical habitat in the Gunnison River in western 

Colorado. Total selenium in fish muscle plugs was determined 

by instrumental neutron activation analysis. Total selenium 

concentrations (range and mean ± standard deviation) in 

micrograms per gram dry weight were 6.0 to 10.7, 8.8 ± 1.3 

for common carp; 2.9 to 8.7, 5.6 ± 2.4 for Colorado pikeminnow; and 1.4 to 7.3, 3.4 ± 2.7 for razorback sucker. The 

selenium concentration for one bonytail chub sample was 

0.8 micrograms per gram dry weight. Selenium concentrations 

in muscle plugs from 1 Colorado pikeminnow and 12 common 

carp exceeded the 8 micrograms per gram dry weight toxicity 

guideline for selenium in fish muscle tissue. 

Introduction

Irrigation drainage from large irrigation projects in the 

Uncompahgre and Grand Valley in western Colorado has 

resulted in selenium (Se) loading into the Uncompahgre, Gunnison and Colorado Rivers that exceeds the 5 micrograms per 

liter (μg/L) aquatic life protection standard established in 1977 

by the Colorado Water Quality Commission (Colorado Department of Public Health and Environment, 2007). Within this 

area, the lower Gunnison River and the Colorado River are 

designated critical habitat for the endangered Colorado pikeminnow (Ptychocheilus lucius), razorback sucker (Xyrauchen 

texanus), and bonytail chub (Gila elegans). There is concern 

that high selenium concentrations in water, sediment, and 

biota collected from these rivers also are adversely affecting 

these endangered fish species (Hamilton, 1998). In collaboration with the Colorado River Recovery Program, the U.S. 

Fish and Wildlife Service (USFWS) monitors endangered fish 

populations in the lower Gunnison River as well as selenium 

concentrations in fish through the use of non-lethal muscle 

plug biopsies (Osmundson and others, 2000). The USFWS has 

sought collaboration from the U.S. Geological Survey (USGS) 

for the determination of selenium in muscle plug samples. 

This report includes results for the analysis of selenium in 

muscle plugs of common carp (Cyprinus carpio), Colorado 

pikeminnow, razorback suckers, and bonytail chub collected in 

spring through early fall of 2012 from a lower stretch of designated critical habitat in the Gunnison River between Delta and 

Grand Junction in western Colorado. 

Methods

Field Collection and Preservation

Muscle plug samples were taken with a biopsy punch 

from live specimens of common carp, Colorado pikeminnow, razorback suckers, and bonytail chub collected from the 

Gunnison River between Delta (river mile 44.1) and the Grand 

Junction (river mile 0.7). Samples were placed in cryotubes, 

enclosed in a Whirl-Pak® bag, and frozen on dry ice. All 

samples were collected by USFWS personnel from March 

through October of 2012. Samples were stored in a freezer 

until shipment to the USGS. 

Sampling History

The muscle plug samples collected by USFWS personnel 

were received in one shipment by the Environmental Chemistry Branch Inorganic Section (henceforth referred to as “the 

laboratory”) of the USGS shortly after collection on December 4, 2012, and contained 16 common carp muscle plugs, 

5 razorback sucker muscle plugs, 1 bonytail chub muscle 

plug, and 4 Colorado pikeminnow muscle plugs. The samples 

were assigned USGS batch number 2073 and USGS sample 

identification numbers 57506 to 57531. Selenium was to be 

determined in all samples by instrumental neutron activation 

analysis (INAA).

Determination of Selenium in Fish from Designated 

Critical Habitat in the Gunnison River, Colorado, 

March through October, 2012

By Thomas W. May and Michael J. Walther
2 Determination of Selenium in Fish from Designated Critical Habitat in the Gunnison River, Colorado

Lyophilization

All sample preparation before INAA was performed by 

USGS at the Columbia Environmental Research Center in 

Columbia, Missouri in collaboration with the University of 

Missouri Research Reactor (MURR). The entire plug was 

transferred from its original cryotube container into a 1.5 milliliter (mL) polyethylene vial provided by MURR staff. Vials 

were precleaned by stepwise washing with acetone, nitric acid, 

and deionized water. Each sample was positioned and pressed 

flat against the vial bottom with a cleaned glass rod. All vials 

were left open and placed in the tray chamber of a Virtis 

Genesis 35EL lyophilizer in “shelf” control and frozen to 

-75 degrees Celsius (oC). Once a condenser temperature 

of –70o C and vacuum of 300 milliTorr was reached, the 

drying cycle commenced. All muscle plug samples were 

lyophilized to constant weight, and dried weights ranged from 

17 to 57 milligrams (mg) and averaged 43 mg. Lyophilization 

greatly reduces the oxygen-19 radioisotope (19O) from water 

in the irradiated sample, and markedly enhances measurement 

precision. Upon recording of final sample weight, an expandable, cleaned polyethylene plug was inserted into the vial 

against which the vial lid was compressed shut. All samples 

were transported to MURR for the determination of selenium.

Instrumental Analysis

Standards in the range of 0.01 to 5 micrograms (μg) 

selenium were prepared by pipetting the appropriate quantities from a series of selenium stock solutions dried onto filter 

pulp paper which was comparable in geometric configuration to that of the samples. The pulp paper was then placed 

in the bottom of the polyethylene vials. A National Institutes 

of Standards and Technology (NIST) lyophilized Standard 

Reference Material (SRM) 1577 Bovine Liver also was run 

as a MURR internal quality control sample. All standards and 

samples were analyzed for selenium by way of 77mSe using the 

following nuclear reaction: 76Se(n,γ)77mSe and 77mSe→77mSe+γ 

and measuring the 161.9 kiloelectron volt (keV) gamma-ray. 

Each standard or sample was placed in the top-center position of a shuttle rabbit and irradiated for 5.00 seconds in the 

Row I position using the pneumatic-tube irradiation facility 

at MURR. This position has thermal and epithermal neutron 

flux densities of 8 x 1013 neutrons x centimeter-2 x second-1

and 2 x 1012 neutrons x centimeter-2 x second-1. The pneumatic 

transfer facility used has a delivery time to the counting station 

of about 4 seconds. The returned shuttle rabbit was opened 

quickly and the sample vial transferred to a special holder 

that positions the small polyethylene vial on the face of the 

detector. All samples were analyzed using a 5-second irradiation, 15-second decay, and 25-second real-time count using 

a high resolution gamma-ray spectrometer. The gamma-ray 

spectrometer included a Tennelec 244 amplifier coupled to a 

Nuclear Data 599 loss-free counting module and a Nuclear 

Data 581 ADC (analog-to-digital converter). Data acquisition and peak extraction were done using a VAX station 

3100, model 38 with Canberra/ND applications software. The 

161.9keV gamma-ray from the decay of 77mSe was used to 

determine selenium concentrations by standard comparison 

(Spate and others, 1994; Baskett and others, 2001).

Results

Total selenium concentrations [micrograms per gram dry 

weight, (μg/g)] in fish muscle plug samples collected from fish 

inhabiting the Gunnison River in western Colorado are listed 

in table 1. Total selenium concentrations [range and mean ± 

standard deviation (SD)] in μg/g dry weight were 6.0 to 10.7, 

8.8 ± 1.3 for common carp; 2.9 to 8.7, 5.6 ± 2.4 for Colorado 

pikeminnow; and 1.4 to 7.3, 3.4 ± 2.7 for razorback sucker. 

The selenium concentration for 1 bonytail chub sample was 

0.8 micrograms per gram dry weight. Selenium concentrations 

in muscle plugs from 1 Colorado pikeminnow and 12 common 

carp exceeded the 8 micrograms per gram dry weight toxicity 

guideline (Lemly, 2002) for selenium in fish muscle tissue. 

Quality-Control Results

Concentrations (μg/g dry weight) of selenium in National 

Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1577 Bovine Liver (n=3) averaged 

1.13 and ranged from 1.11 to 1.14 with an SD of 0.017 μg/g 

(table 2). Compared with the certified selenium concentration 

(1.1 ± 0.1 μg/g), these results expressed selenium recoveries 

that were all within this certified range.
Quality-Control Results 3

Table 1. Concentrations of selenium in fish muscle plugs collected from fish inhabiting the 

Gunnison River in western Colorado, March through October, 2012.

[USGS, United States Geological Survey; ID, identification; μg/g, micrograms per gram dry weight]

USGS ID Field ID Fish

common name

Collection date

(month/day/year

Percent

moisture

Selenium

(μg/g)

57506 GRZ5705 razorback sucker 10/01/12 80.8 1.53

57507 GRZ5E22 razorback sucker 10/01/12 77.3 1.35

57508 GRZDE84 razorback sucker 10/05/12 78.2 7.28

57509 GRZ61FD razorback sucker 10/05/12 72.8 5.20

57510 GRZ9D7B razorback sucker 10/04/12 78.7 1.40

57511 GPMCE69 colorado pikeminnow 10/04/12 78.2 5.08

57512 GPMCE69B colorado pikeminnow 07/31/12 66.2 2.93

57513 GPM5DEF colorado pikeminnow 07/18/12 83.7 8.68

57514 GPMD35C colorado pikeminnow 07/18/12 76.4 5.68

57515 PS-E826 bonytail 03/13/12 69.7 0.77

57516 12GRCMP1 common carp 08/27/12 76.2 8.49

57517 12GRCMP2 common carp 08/27/12 76.6 9.76

57518 12GRCMP3 common carp 08/28/12 79.8 8.04

57519 12GRCMP4 common carp 08/29/12 72.2 8.47

57520 12GRCMP5 common carp 08/29/12 75.7 6.03

57521 12GRCMP6 common carp 08/29/12 75.9 9.49

57522 12GRCMP7 common carp 08/30/12 71.9 9.39

57523 12GRCMP8 common carp 08/30/12 77.9 10.45

57524 12GRCMP9 common carp 08/30/12 78.9 6.87

57525 12GRCMP10 common carp 08/30/12 78.6 9.15

57526 12GRCMP11 common carp 08/31/12 74.3 7.64

57527 12GRCMP12 common carp 10/05/12 75.6 10.1

57528 12GRCMP13 common carp 10/03/12 77.7 10.7

57529 12GRCMP14 common carp 10/03/12 78.2 9.67

57530 12GRCMP3B common carp 08/28/12 76.4 7.40

57531 12GRCMP7B common carp 08/30/12 85.3 8.66

Table 2. Selenium recoveries from a reference tissue material analyzed with fish muscle plug 

samples.

[MURR, University of Missouri Research Reactor; QC, quality control; ID, identification; μg/g, microgram per 

gram dry weight; SD, standard deviation; RSD, relative standard deviation; NIST, National Institute of Standards and Technology; ±, plus or minus; na, not applicable]

MURR

QC ID

Reference

material

Certified 

range

(μg/g)

Selenium

(μg/g)

Mean

(μg/g)

SD

(μg/g)

Percent

RSDa

BL-Z-66 NIST 1577b 1.1 ± 0.1 1.14 1.13 0.017 1.5

BL-Z-67 na na 1.11 na na na

BL-Z-68 na na 1.14 na na na

a

Calculated as standard deviation divided by mean times 100.

b

Standard Reference Material 1577: Bovine liver.
4 Determination of Selenium in Fish from Designated Critical Habitat in the Gunnison River, Colorado

References Cited

Spate V.L., Mason M.M., Reams C.L., Baskett C.K., Morris 

J.S., and Mills D.S., 1994, Determination of total and bound 

Se in sera by INAA: Journal of Radioanalytical and Nuclear 

Chemistry, v. 179, no. 2, p. 315–322.

Hamilton, S.J., 1998, Selenium effects on endangered fish in 

the Colorado River Basin, in Frankenberger, W.T. Jr. and 

Engberg, R.A., eds. -Environmental chemistry of selenium: 

Marcel Dekker, Inc., p. 297–313.

Osmundson, B., May, T.W., and Osmundson, D., 2000, Selenium concentrations in the Colorado pikeminnow (Ptychocheilus lucius) - Relationship with flows in the upper 

Colorado River: Archives of Environmental Contamination 

and Toxicology, v. 38, p. 479–485.

Baskett, C.K., Spate, V.L., Mason, M.M., Nichols, T.A., 

Williams, A., Dubman, I.M., Gidina, A., Denison, and J., 

Morris, J.S., 2001, Long-term selenium status in humans: 

Journal of Radioanalytical and Nuclear Chemistry, v. 249, 

no. 2, p. 429–435.

Lemly, D.A., 2002, Selenium assessment in aquatic ecosystems, , chap. 2 of Alexander, D.E., ed. Springer Series on 

environmental management, Interpreting selenium concentrations: New York-Springer-Verlag, p. 18–38.

Colorado Department of Public Health and Environment 

(CDPHE), 2007, Classifications and numeric standards for 

Gunnison and lower Dolores River Basins, 5 CCR 1002-35, 

Regulation No. 35: Denver, Colorado, Colorado Department 

of Public Health and Environment, Water Quality Control 

Commission, 72 p.

Publishing support provided by:

Rolla Publishing Service Center

For more information concerning this publication, contact:

Director, USGS Columbia Environmental Research Center

4200 New Haven Road

Columbia, MO 65201

(573) 875–5399

Or visit the Columbia Environmental Research Center Web site at:

http://www.cerc.usgs.gov/

May and Walther—Determination of Selenium in Fish from Designated Critical Habitat in the Gunnison River, Colorado—Open-File Report 2013–1104