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

Determination of Selenium in Fish from Designated Critical 

Habitat of the Gunnison River, Colorado, Summer 2011 

Open-File Report 2012–1235

U.S. Department of the Interior

U.S. Geological Survey

Determination of Selenium in Fish from 

Designated Critical Habitat of the Gunnison 

River, Colorado, Summer 2011 

By Thomas W. May and Michael J. Walther

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

Open-File Report 2012–1235

U.S. Department of the Interior

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

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

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Suggested citation:

May, T.W., and Walther, M.J., 2012, Determination of selenium in fish from designated critical habitat of the Gunnison River, Colorado, summer 2011: U.S. Geological Survey Open-File Report 2012–1235, 4 p.
iii

Contents

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

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

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

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

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

Lyophilization.........................................................................................................................................1

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, summer 2011...............................................................3

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

plug samples..................................................................................................................................4

Conversion Factors

SI to Inch/Pound

Multiply By To obtain

Length

millimeter (mm) 0.03937 inch (in.)

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)

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.

Determination of Selenium in Fish from Designated 

Critical Habitat of the Gunnison River, Colorado, Summer 

2011 

By Thomas W. May and Michael J. Walther

Abstract

This report presents results for the summer 2011 sampling of muscle plugs from common carps (Cyprinus Linnaeus), roundtail chub (Gila robusta), and bonytail chub (Gila 

elegans) 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 for each species 

were as follows: common carp: 8.5 to 35, 13 ± 7.8; roundtail 

chub: 5.5 to 11.2, 7.3 ± 1.6; bonytail chub: 0.8 to 8.6, 3.9 ± 

4.2. Selenium concentrations in muscle plugs from 4 out of 

15 roundtail chub, all 15 common carp, and 2 out of 5 bonytail 

chub 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 (ug/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, roundtail chub, 

and bonytail chub collected during the summer of 2011 from 

a lower stretch of designated critical habitat in the Gunnison 

River between Escalante and the Colorado River confluence. 

Methods

Field Collection and Preservation

Muscle plug samples were taken with a biopsy punch 

from live specimens of common carp, roundtail chub, and 

bonytail chub collected from the Gunnison River between 

Escalante (river mile 44.1) and the Colorado confluence (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 in August of 2011. 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 August 31, 2011, and contained 17 common carp muscle 

plugs, 15 roundtail chub muscle plugs, and 5 bonytail chub 

muscle plugs.. The samples were assigned USGS batch 

number 1941 and USGS sample identification numbers 55406 

to 55442. Selenium was to be determined in all samples by 

instrumental neutron activation analysis (INAA).

Lyophilization

All sample preparation before INAA was conducted by 

USGS at the Columbia Environmental Research Center in 

Columbia, Missouri in collaboration with the University of 
2 Determination of Selenium in Fish from Designated Critical Habitat of the Gunnison River, Colorado, Summer 2011

Missouri Research Reactor (MURR). The entire plug was 

transferred from its original cryotube container into a 1.5 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 -75degrees 

Celsius (°C). Once a condenser temperature of –70°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 20.8 to 60.1 milligrams 

(mg) and averaged 41.4 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 quickly 

opened 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. 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 for each species 

were as follows: common carp: 8.5 to 35, 13 ± 7.8; roundtail 

chub: 5.5 to 11.2, 7.3 ± 1.6; bonytail chub: 0.8 to 8.6, 3.9 ± 

4.2. Selenium concentrations in muscle plugs from 4 out of 

15 roundtail chub, all 15 common carp, and 2 out of 5 bonytail chub exceeded the 8 μg/g 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=4) averaged 

1.14 and ranged from 1.13 to 1.17 with an SD of 0.021 μg/g 

(table 2). Compared with the certified selenium concentration, 

these results expressed a selenium recovery of 104 percent 

with a percent relative standard deviation of 1.8.
Quality-Control Results 3

Table 1. Concentrations of selenium in fish muscle plugs collected from fish inhabiting the Gunnison River in Western Colorado, 

summer 2011.

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

USGS ID Field ID Fish

common name

Collection

date

Percent

moisture

Selenium

(μg/g)

55406 GIICCP1 Common carp 08/19/11 72.5 10.2

55407 GIICCP2 Common carp 08/18/11 79.3 12.2

55408 GIICCP3 Common carp 08/18/11 78.5 8.50

55409 GIICCP4 Common carp 08/18/11 79.4 10.2

55410 GIICCP5 Common carp 08/17/11 77.9 10.6

55411 GIICCP6 Common carp 08/19/11 77.5 10.0

55412 GIICCP7 Common carp 08/19/11 79.0 10.9

55413 GIICCP8 Common carp 08/19/11 70.7 8.45

55414 GIICCP9 Common carp 08/17/11 78.0 9.74

55415 GIICCP10 Common carp 08/17/11 82.3 32.2

55416 GIICCP11 Common carp 08/18/11 76.4 10.5

55417 GIICCP12 Common carp 08/19/11 76.7 10.9

55418 GIICCP13 Common carp 08/19/11 77.4 11.6

55419 GIICCP14 Common carp 08/19/11 72.0 9.67

55420 GIICCP15 Common carp 08/18/11 79.7 11.0

55421 GU-BTMP3 Bonytail 08/23/11 72.3 0.81

55422 GU-BTMP3B Bonytail 08/23/11 74.4 0.89

55423 GII-RTC1 Roundtail chubs 08/17/11 79.4 5.62

55424 GII-RTC2 Roundtail chubs 08/18/11 80.0 6.17

55425 GII-RTC3 Roundtail chubs 08/17/11 80.4 7.06

55426 GII-RTC4 Roundtail chubs 08/19/11 80.7 8.47

55427 GII-RTC5 Roundtail chubs 08/17/11 79.3 5.59

55428 GII-RTC6 Roundtail chubs 08/19/11 80.0 8.94

55429 GII-RTC7 Roundtail chubs 08/19/11 79.8 7.86

55430 GII-RTC8 Roundtail chubs 08/19/11 80.0 8.78

55431 GII-RTC9 Roundtail chubs 08/19/11 79.9 7.80

55432 GII-RTC10 Roundtail chubs 08/19/11 79.5 7.75

55433 GII-RTC11 Roundtail chubs 08/18/11 80.7 5.50

55434 GII-RTC12 Roundtail chubs 08/19/11 80.7 11.2

55435 GII-RTC13 Roundtail chubs 08/18/11 80.7 6.85

55436 GII-RTC14 Roundtail chubs 08/17/11 79.7 5.45

55437 GII-RTC15 Roundtail chubs 08/18/11 81.5 7.02

55438 GU-BTMP1 Bonytail 06/09/11 80.5 8.34

55439 GU-BTMP2 Bonytail 08/19/11 72.4 0.81

55440 GIICCP13B Common carp 08/18/11 77.7 11.1

55441 GIICCP10B Common carp 08/17/11 81.8 35.1

55442 GU-BTMP4 Bonytail 08/29/11 79.0 8.58
4 Determination of Selenium in Fish from Designated Critical Habitat of the Gunnison River, Colorado, Summer 2011

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; --, not applicable]

MURR

QC ID

Reference

material

Certified 

mange

(μg/g)

Selenium

(μg/g)

Mean

(μg/g)

SD

(μg/g)

Percent

RSDa

BL-Z-11 NIST 1577b 1.1 ± 0.1 1.15 1.14 0.021 1.8

BL-Z-12 NIST 1577b 1.1 ± 0.1 1.13 -- -- --

BL-Z-15 NIST 1577b 1.1 ± 0.1 1.17 -- -- --

BL-Z-16 NIST 1577b 1.1 ± 0.1 1.13 -- -- --

a

Calculated as SD ÷ mean x 100.

b

SRM 1577: bovine liver.

References Cited

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

J.S., 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, 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, Alexander, D.E., ed., in chap 2 of 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, Colo., Colorado Department 

of Public Health and Environment, Water Quality Control 

Commission, 72 p.

Publishing support provided by: 

Rolla and Denver Publishing Service Centers

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 Critical Habitat in the Gunnison River, Colorado, Summer 2011—Open-File Report 2012–1235