United States Office of Water EPA-822-R-08-020
Environmental Protection 4304 September 2008
Agency
SEPA Effect of Selenium
on Juvenile Bluegill Sunfish
at Reduced Temperature
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Notice: This document has been reviewed in accordance with the
procedures of the U.S. Environmental Protection Agency and
approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
Suggested citation:
Mclntyre, D.O., M.A. Pacheco, M.W. Garton, D. Wallschlager, and C.G. Delos. 2008.
Effect of Selenium on Juvenile Bluegill Sunfish at Reduced Temperature. Health and
Ecological Criteria Division, Office of Water, U.S. Environmental Protection Agency,
Washington, DC. Contract #68-C-04-006. EPA-822-R-08-020.
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Technical Report
Effect of Selenium on Juvenile Bluegill Sunfish at
Reduced Temperature
by
Dennis O. Mclntyre * l
Manoel A. Pacheco l
Mailee W. Garton2
Dirk Wallschlager3
Charles G.Delos/-4
1 Great Lakes Environmental Center, 1295 King Ave, Columbus, OH 43212
2 Great Lakes Environmental Center, 739 Hastings St., Traverse City, MI 49686
3 Environmental and Resource Sciences Program, Trent University,
1600 West Bank Drive, Peterborough, Ontario K9J 7B8, Canada
4 Health and Ecological Criteria Division, Office of Science and Technology,
Office of Water, U.S. Environmental Protection Agency, Washington, DC 20460
* Principal Investigator Contact: dmcintyre@glec.com
/"Agency Contact: delos.charles@epa.gov
U.S. Environmental Protection Agency
Office of Water
Washington, DC 20460
September 25, 2008
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Acknowledgments
This study was conducted as part of Great Lakes Environmental Center's (GLEC)
contract with EPA's Health and Ecological Criteria Division (HECD), EPA Contract
#68-C-04-006. The individuals responsible for the management and leadership of this
investigation including its design development, data analysis and report preparation were:
• Charles Delos, EPA Work Assignment Manager, EPA HECD
• Dennis Mclntyre, Work Assignment Leader, GLEC
Study Staff
Many individuals were responsible for conducting this study. Study staff included those
who participated in the design and construction of the exposure system, performed the
day-to-day activities in the laboratory, analyzed chemical samples, analyzed data and
prepared the report.
• Mailee Garton, GLEC
• Manolo Pacheco, GLEC
• Dennis McCauley, GLEC
• Randy Panks, GLEC
• Erica Schneider, GLEC
• Amanda DeGraeve, GLEC
• Sara Reitz, GLEC
• Victoria Bunn, GLEC
• Jesse Karner, GLEC
• Dirk Wallschlager, Trent University
• Jeffrey W. Charters, Trent University
Reviews
The study design was reviewed by John Besser of USGS and Kevin Brix of EcoTox. The
report was reviewed by an external panel of experts that included Jerry Diamond of
TetraTech, David DeForest of Parametrix, David Janz of the University of Saskatchewan,
and Stephen Klaine of Clemson University. Internal reviews were provided by Mick
DeGraeve of GLEC and Charles Stephen, Dale Hoff and David Mount of EPA.
11
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TABLE OF CONTENTS
Executive Summary viii
1.0 INTRODUCTION 1
1.1 SUMMARY OF LEMLY STUDY AND A COMPARISON OF TEST
DESIGNS 1
2.0 METHODS 3
2.1 TEST ORGANISMS 3
2.1.1 Lepomis macrochirus 3
2.1.2 Lumbriculus variegatus 4
2.2 SELENIUM EXPOSURE 5
2.2.1 Aqueous Exposure 7
2.2.2 Dietary Exposure 10
2.3 WATER QUALITY MEASUREMENTS AND OBSERVATIONS 11
2.4 DETERMINATION OF TOTAL SELENIUM IN WATERS AND TISSUES
BY HYDRIDE GENERATION-ATOMIC FLUORESCENCE
SPECTROMETRY (HG-AFS) 13
2.4.1 Fish and Worm Tissue Digestion 13
2.4.2 Reagents 13
2.4.3 Sample Analysis 14
3.0 RESULTS AND DISCUSSION 15
3.1 WATER QUALITY MEASUREMENTS 15
3.1.1 Temperature Measurements 16
3.2 SELENIUM MEASUREMENTS 19
3.2.1 Selenium in Water 19
3.2.2 Selenium in Lumbriculus variegatus 21
3.2.3 Concentrations of Selenium in Fish Tissues 22
3.3 SURVIVAL ANALYSIS OF JUVENILE BLUEGILL SUNFISH 30
3.3.1 Overlay of Survival and Bioaccumulation Plots 39
3.3.2 Estimates of Effect Concentrations 42
3.4 GROWTH, LIPID ANALYSIS, AND BEHAVIOR OF JUVENILE
BLUEGILL SUNFISH 45
in
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3.5 COMPARISON OF RESULTS BETWEEN LEMLY AND CURRENT
STUDIES 46
4.0 SUMMARY 52
5.0 REFERENCES 52
LIST OF TABLES
Table 1.1 Comparison in Selected Design Characteristics between Lemly and Current
Studies 2
Table 2.1. Nominal exposure concentrations for Exposure Systems 1, 2 and 3 5
Table 3.1. Average and range of pH, dissolved oxygen and conductivity in each tank.
The pH and dissolved oxygen were measured daily. Conductivity was measured
once a week in each tank during the 182 day bluegill study 18
Table 3.2A. Nominal and measured total selenium concentrations for all treatments.
Average concentrations are based on weekly samples collected up to test day 154
of the exposure period 19
Table 3.2B. Nominal and measured total selenium concentrations for all treatments.
Average concentrations are based on weekly samples collected throughout the
182 day exposure period 20
Table 3.3. Measured total selenium concentrations (|ig/g dw) m Lumbriculus variegatus
for all treatments in Exposure System 1 21
Table 3.4. Measured total selenium concentrations (|ig/g dw) m Lumbriculus variegatus
for all treatments in Exposure System 3 22
Table 3.5. Target and average measured total selenium concentrations in Lumbriculus
variegatus for all treatments in Exposure Systems 1 and 3 22
Table 3.6. Measured total selenium concentrations in bluegill sunfish for all treatments
in Exposure Systems 1, 2 and 3 24
Table 3.7. Total number of deaths attributed to background mortality and selenium
toxicity in each treatment of ESI, ES2, and ES3 (initial 7V=100) over the
experiment's duration (182 days). All three exposure systems (ESI, ES2, ES3)
IV
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had two control tanks. The ES2 treatment with a target diet concentration of 5 |ig
Se/g dw also had two replicates 31
Table 3.8. Timetable of deaths and respective estimates of fraction survival for ESI
Treatment 6. All survival values projected by the Kaplan-Meier estimator 32
Table 3.9. Timetable of deaths and respective estimates of fraction survival for ESI
Treatment 5. All survival values projected by the Kaplan-Meier estimator 34
Table 3.10. Timetable of deaths and respective estimates of fraction survival for ES3
Treatment 6. All survival values projected by the Kaplan-Meier estimator 36
Table 3.11. Timetable of deaths and respective estimates of fraction survival for ES3
Treatment 5. All survival values projected by the Kaplan-Meier estimator 38
Table 3.12. Average standard lengths (mm) in bluegill based on samples taken for
chemical analysis; N = 9 for each average value 49
Table 3.13. Average weights (g) in bluegill based on samples taken for chemical
analysis; N = 9 for each average value 49
Table 3.14. Average body condition factor (K)* bluegill based on samples taken for
chemical analysis; N = 9 for each average value 50
Table 3.15. Lipid content (%) in juvenile bluegill at the start and end of the exposure
period 51
LIST OF FIGURES
Figure 2.1. Floor plan of systems used in juvenile bluegill selenium study 6
Figure 2.2. Tank system diagram for bluegill and Lumbriculus ES1 and ES3 8
Figure 2.3. Tank system diagram for bluegill ES2 9
Figure 3.1. Daily average temperatures measured in each bluegill tank in Exposure
System 1. Temperatures were averaged across treatments 16
Figure 3.2. Daily average water temperatures measured in each bluegill tank in Exposure
System 2. Temperatures were averaged across treatments 16
Figure 3.3. Daily average water temperatures measured in each bluegill tank in Exposure
System 3. Temperatures were averaged across treatments 17
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Figure 3.4. Concentrations of selenium in juvenile bluegill tissues overtime of exposure
in Controls for Exposure Systems (ES) 1, 2 and 3. The dotted line represents the
average concentration 25
Figure 3.5. Concentrations of selenium in juvenile bluegill tissues overtime of exposure
in Exposure System 1 (ESI) Treatments 1 through 6. Dots represent measured
values and the solid line represents projections from the fitted model (I) 26
Figure 3.6. Concentrations of selenium in juvenile bluegill tissues overtime of exposure
in Exposure System 3 (ES3) Treatments 1 through 6. Dots represent measured
values and the solid line represents projections from the fitted model (I) 28
Figure 3.7. Concentrations of selenium in juvenile bluegill tissues over time of exposure.
Dots represent measured values and the solid line represents projections from the
fitted model (I) 29
Figure 3.8. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The
"+" sign indicates dates when data were censored 33
Figure 3.9. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The
"+" sign indicates dates when data were censored 35
Figure 3.10. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The
"+" sign indicates dates when data were censored 37
Figure 3.11. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The
"+" sign indicates dates when data were censored 39
Figure 3.12. ES 1 Treatment 6 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival 40
Figure 3.13. ESI Treatment 5 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival 40
Figure 3.14. ES3 Treatment 6 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival 41
Figure 3.15. ES3 Treatment 5 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival 41
VI
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Figure 3.16. Survival of juvenile bluegill as a logistic function of the logarithm of the
final selenium concentration in fish tissues. Concentration-survival curve for
ESS 43
Figure 3.17. Survival of juvenile bluegill as a logistic function of the logarithm of the
final selenium concentration in fish tissues. Concentration-survival curve for
ESI 44
APPENDICES
Appendix A- Daily and Periodic Measurements of Dilution Water and Stock Solution
Flows, Overlying Water Quality Measurements, and Feeding Rates
Appendix B- Weekly Total Selenium Measurements in Water and Monthly Selenite and
Selenate Measurements in Water
Appendix C- Total Selenium Measurements in Lumbriculus variegatus
Appendix D- Total Selenium Measurements in Bluegill Sunfish
Appendix E- Daily Recording of Bluegill Sunfish Mortality
Appendix F- Bluegill Standard Length and Weight Measurements of Individuals Sampled
for Selenium Analysis
Appendix G- Bluegill Sunfish Lipid Measurements of Individuals Sampled Prior to
Exposure and at the End of Exposure
vn
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Executive Summary
The final chronic value recommended in the 2004 draft update of the aquatic life ambient
water quality criteria for selenium (7.91 |ig/g dry weight) is based on a single study
(Lemly 1993). This report presents results of toxicity assays designed to replicate
Lemly's test, and to further explore how temperature affects the toxicity of selenium.
Juvenile bluegill were exposed to three distinct combinations of selenium species and
temperature. In exposure system one (ESI) and three (ESS), fish were exposed to six
nominal concentrations of selenium in water (background, 1.25, 2.5, 5.0, 10, 20, 40 |lg/L)
and in diet (Lumbriculus variegatus with background, 1.25, 2.5, 5.0, 10, 20, or 40 |ig/g
dw) at two temperature regimes, 20°C decreasing to 4-5°C (ESI) and 20°C decreasing to
9°C (ESS). Exposure system two (ES2) had a temperature regime similar to ESI (20°C
—»• 4-5°C), but only one nominal concentration of selenium (5 |ig/L in water and 5 |ig/g
dw in diet), incorporated in TetraMin as seleno-L-methionine. In ESI and ESS selenized
yeast was fed to worms. ES2 duplicated Lemly's (1993) treatment with high fish
mortality.
Average measured concentrations of total selenium in water were similar to target
exposure concentrations. The proportion of selenate to selenite in each water tank
remained close to the target ratio of 1:1. Average measured concentrations of selenium
in worm tissues were within a factor of 1.5 of target concentrations for treatments aiming
to reach 5.0, 10, 20, or 40 |ig/g dw. The average measured tissue concentrations in the
other two treatments (1.25, 2.5 |ig/g dw) were between 2 and 3 times higher than target
levels. Concentrations of selenium in fish tissues increased asymptotically with exposure
period. Fishes exposed to lower concentrations of selenium in the water and in their food
(worms) consistently displayed lower bioaccumulation rates and lower asymptotic
concentrations of selenium in tissues. Rates of selenium accumulation in fish tissues
were similar for corresponding ESI and ES3 treatments up to day 112. Accumulation of
selenium from that day until the end of the experiment (day 182) was higher in ES3 than
in ESI fish. Exposure of ES2 fish to seleno-L-methionine resulted in tissue
concentrations of selenium approximately 2.5 times higher than in fish exposed to similar
concentrations of selenium in worm tissues. At the end of the experiment, the average
concentration of selenium in tissues of ES2 fish was 9.4 |ig/g dw in one tank and 10.6
|lg/g dw in another. The average concentration of selenium in tissues of ESI fish
exposed to a similar temperature regime and selenium concentration, was 4.0 |ig/g dw.
This threshold was exceeded only in ESI and ES3 treatments with a target concentration
of selenium in the diet equal to 20 or 40 |ig/g dw. Projection of the selenium
concentration associated with the onset of mortality (>10%) in these treatments resulted
in similar threshold values: 11.1, 11.6 |ig/g dw for ESI and 11.1, 13.8forES3. The
projected EC20values of 10.16 |ig/g (9.81 - 10.52 |ig/g, 95% CI), and ECio, 9.56 |ig/g
(9.09 - 10.05 |lg/g) for ESI were lower than corresponding values for ES3, £€20 =14.02
Hg/g (13.50 - 14.56 ng/g), ECio = 13.29 ng/g (12.61 - 14.00 ng/g).
Vlll
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1.0 INTRODUCTION
The final chronic value of 7.91 |ig/g dw recommended in the 2004 Draft Update of the
Aquatic Life Ambient Water Quality Criteria for Selenium is based on one study (Lemly
1993), in which juvenile bluegill underwent "winter stress syndrome." Data from
Lemly's study indicate that over-wintering fish may be more susceptible to the effects of
waterborne and dietary selenium exposure due to increased sensitivity at low
temperature. Lemly exposed juvenile bluegill sunfish in the laboratory to waterborne
(1:1 selenite:selenate; nominal 5 jig Se/L) and food borne (seleno-L-methionine in
TetraMin; nominal 5 jig Se/g dw food) selenium for 180 days with temperatures
decreasing from 20 to 4°C. Given the importance of the data from the Lemly study in
deriving the tissue-based final chronic value for selenium, the goal of this study is to
determine tissue-based effect levels for selenium exposure over a simulated winter season
at two temperature regimes, 20 to 4°C and 20 to 9°C. Besides the additional temperature
regime, two prominent differences from the Lemly study include (1) a range of six
selenium concentrations was included (aqueous and diet) to determine protective effect
levels and (2) bluegill were fed the aquatic worm, Lumbriculus variegatus, which
contained target levels of selenium accumulated by feeding the worms selenized-yeast.
A separate system exposed juvenile bluegill to aqueous selenium and seleno-L-
methionine in TetraMin under a 20 to 4°C temperature regime to mimic the Lemly study
exposure design. The 182-day study began on April 30, 2007 and ended October 29,
2007.
1.1 SUMMARY OF LEMLY STUDY AND A COMPARISON OF TEST DESIGNS
Lemly exposed juvenile bluegill to aqueous and dietary selenium under intermittent flow-
through conditions for 180 days. Tests were run at 4° and 20°C, with biological
(histological, hematological, metabolic and survival) and selenium measurements made
at 0, 60, 120 and 180 days. Fish were fed at a rate of 3% body weight per day. All
treatments were initiated at 20°C, and then decreased at a rate of 2°C per week for 8
weeks to reach 4°C. The temperature was then maintained at that temperature for the
remainder of the 180 days.
In the 20°C test, fish accumulated 6 |ig/g dw selenium (whole-body) with no significant
effect on survival (4.3% and 7.4% mortality in the control and treatment, respectively).
In the 4°C test, fish exposed to selenium accumulated 7.9 jig/g dw (whole-body)
selenium and significant mortality was observed after 120 (33.6%) and 180 days (40.4%)
relative to the control (3.9%). Several hematological measurements were significantly
different in both the warm and cold selenium exposures relative to controls. Both warm
and cold selenium treatments also had greater O2 consumption than controls. Fish lipid
content in the cold selenium treatment decreased more than the cold control; lipid content
did not decrease in either the warm control or the warm selenium treatment. The results
suggest that significant mortality occurs in juvenile bluegill during winter months when
tissue concentrations increase from 5.85 to 7.91 jig/g dw and lipid levels decrease to 6
percent.
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Several design characteristics of Lemly's study were modified in the current study (Table
1.1). The notable changes, as stated above, were the addition of a more temperate
temperature regime (20 to 9°C), and exposure to a range of six selenium aqueous and
dietary concentrations and controls (Exposure Systems (ES) 1 and 3). The goal of the
latter modification was to obtain a gradient in the response of the bluegill ranging from
no observable effects in the low concentrations, to intermediate effects in the middle
concentrations, to 100% affected in the high concentration. Such a range in response is
needed for a reliable estimation of effect concentrations.
Another modification to the Lemly design was to feed the bluegill an aquatic worm,
Lumbriculus variegatus, that had accumulated selenium to a gradient of levels through
the consumption of selenized-yeast. Selenomethionine added to commercial fish food
has been commonly used in exposure studies, but that may not be the predominate
selenium species fish are exposed to in nature. Fan et al (2002) determined that
Selenomethionine was approximately 30% of the total selenium in biological tissues in
several trophic levels. The use of a forage animal (Lumbriculus) that had accumulated
selenium through the consumption of a trophic level 1 organism (selenized-yeast) was
considered a more representative exposure to bluegill than the addition of
Selenomethionine to the diet. To have a direct comparison of the response of the bluegill
in this study to the fish in Lemly's experiment, a repeat of Lemly's cold treatment (ES2)
was run concurrent to ES 1 and 3. Due to space restrictions, only 2 replicates were used
inES2.
Table 1.1 Comparison in Selected Design Characteristics between Lemly and Current
Studies
Design
Characteristics
Species
Bluegill size
at test
initiation
Aqueous
exposure,
nominal
Dietary
exposure,
nominal
Feeding rate
Duration
Temperature
Lemly
Juvenile bluegill
sunfish
50-70 mm total
length
1 : 1 ratio of
selenite:selenate;
5|ig/L
Seleno-L-
methionine added
to TetraMin; 5
Hg/g
3% body wt/day
180 days
Cold: 20 to 4°C;
Current Study Exposure System (ES)
ESI
ES2
ES3
Juvenile bluegill sunfish
56-69 mm (mean = 60) total lengtha; 1.2-2.0 g (mean =
1.5) weight
1 : 1 ratio of
selenite:selenate;
1.25,2.5,5, 10,
20, 40 |ig/L
Se accumulated
in Lumbriculus
at six treatment
conc'ns, 1.25,
2.5, 5, 10, 20, 40
|ig/g dw
4% body wt/day
1 : 1 ratio of
selenite:selenate;
nominal 5 |ig/L
Seleno-L-
methionine
added to
TetraMin; 5
Hg/g
3% body wt/day
1 : 1 ratio of
selenite:selenate;
1.25,2.5,5, 10,
20, 40 |ig/L
Se accumulated
in Lumbriculus
at six treatment
conc'ns, 1.25,
2.5, 5, 10, 20, 40
|ig/g dw
4% body wt/day
182 days
20 to 4°C; after
20 to 4°C; after
20 to 9°C; after
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Design
Characteristics
regime
Controls
Replication
Fish/replicate
Lemly
decreased
2°C/week until
4°C reached
Warm: 20°C
constant
No Se added to
both cold and
warm treatments
3 reps/treatment
70
Current Study Exposure System (ES)
ESI
30daysat20°C
decreased
2°C/week until
4°C reached
No Se added to
water or worms
2 reps - controls
only
ES2
30daysat20°C
decreased
2°C/week until
4°C reached
No Se added to
water or
TetraMin
2 reps/treatment
ES3
30daysat20°C
decreased
2°C/week until
9°C reached
No Se added to
water or worms
2 reps - controls
only
100
Standard lengths of 44-54 mm (mean = 47) were converted to total length using
conversion factor for bluegill of 1.278 (Beckman 1948).
2.0 METHODS
2.1 TEST ORGANISMS
2.1.1 Lepomis macrochirus
Juvenile bluegill (Lepomis macrochirus) used in the study were purchased from Osage
Catfisheries in Osage Beach, Missouri. The juvenile bluegill, which were hatched in
May 2006, were 38-51 mm in standard length, and arrived at Great Lakes Environmental
Center's (GLEC) laboratory in Traverse City, Michigan on April 5, 2007. Upon arrival,
the bluegill were physically inspected, and the initial weight and length of a subsample
was recorded (average standard length: 47 mm; average weight: 1.0 gram). The bluegill
were divided between two 400 liter flow-through tanks, each containing 350 L of
dechlorinated water. The water temperature in the holding tanks at the time of stocking
(12°C) was within PC of the shipping water temperature (11.8°C). Chilled
dechlorinated water was supplied to each holding tank at the rate of 2 liters per minute.
Both holding tanks were aerated continuously using large air stones supplied with
compressed air from an oil-free air compressor.
Prior to test initiation, the bluegill were held for a period of 25 days, and during the first
14 days they were treated with salt and formalin to manage external parasites. Although
no parasites were observed in the fish received on April 5, 2007, the fish were
prophylactically treated for external parasites because Dactylogyrus or Gyrodactylus were
observed on the bluegill in a previous shipment from the same source. Uniodized salt was
added to the holding tanks on a daily basis to achieve an initial treatment concentration of
1 g/L, which was diluted over time as water flowed into the tanks. The treatment was
performed for 19 consecutive days until one week prior to test initiation. The bluegill
were also treated with formalin on two separate days, 5 days apart. On April 7, and 12,
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2007, all the fish were moved to holding tank 1, where they were exposed to a nominal
formalin concentration of 1 mg/liter in laboratory water for one hour. During the one
hour formalin treatment, holding tank 2 was cleaned, rinsed thoroughly and filled with
fresh dechlorinated water. After the fish were exposed to the formalin for one hour, they
were then transferred to the fresh laboratory water in tank 2. Tank 1 was then
disinfected, rinsed thoroughly, and filled with fresh dechlorinated water. The bluegill
were once again divided between tanks 1 and 2 after the formalin treatment. None of the
fish exhibited any overt signs of stress (i.e., surfacing or lethargy or death) during or after
the salt or formalin treatments. No external parasites were observed on the bluegill
during weekly monitoring prior to test initiation.
In the holding tanks the fish were fed frozen adult brine shrimp once daily until satiation.
Each tank was siphoned every day after feeding to remove uneaten food and fecal
material. Dissolved oxygen (D.O.), temperature, and pH were measured on a daily basis.
2.1.2 Lumbriculus variegatus
Selenium-dosed adult sized Lumbriculus variegatus (California blackworms) were used
as the food source for the bluegill in the preliminary and definitive studies. Thirty-two
pounds of L. variegatus were purchased from Bayou Aquatic and Reptile Supply in
Ontario, California, arriving at GLEC on February 22, 2007, approximately 9 weeks prior
to the initiation of the definitive study. Upon arrival, the L. variegatus were divided
among 16 flow-through pans. The pans contained approximately 28 liters of water, and
were aerated to maintain dissolved oxygen at an acceptable level (> 6 mg/L) to support
the L. variegatus. Each pan of worms was fed daily, 3.2 grams of nutritional yeast
suspended in 250 ml of dechlorinated water, until March 28, 2007. Beginning on March
28, 2007, each pan of worms was fed 3.2 grams of a mixture of nutritional yeast and
selenized-yeast (SelenoSource™ AF 6001) to obtain a range of six concentrations. The
yeast mixture was suspended in 250 ml of dechlorinated water, and fed to the worms on a
daily basis until test initiation on April 30, 2007. Each tank was siphoned daily after
feeding to remove uneaten food, fecal material, and detritus. D.O., temperature, and pH
were measured daily (average measurements: dissolved oxygen, 7.9 mg/L; temperature,
15.0°C;andpH, 7.80).
Diamond V Mills, Inc. Cedar Rapids, Iowa
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2.2 SELENIUM EXPOSURE
Three separate exposure systems were maintained concurrently in a trailer specifically
designed for this study (Figure 2.1). In each system, fish were exposed for 182 days to
selenium through water and diet; test initiation was April 30, 2007 and test termination
was October 29, 2007. In Exposure Systems (ES) 1 and 3, juvenile bluegill were exposed
to a series of six aqueous concentrations of selenium and fed selenium accumulated in
Lumbriculus variegatus. The only difference between ESI and ES3 was the water
temperature regime: in ESI, temperature was maintained at 20°C for 30 days and then
decreased 2°C/week until it reached 4°C, which was maintained until test termination. In
ES3, the water temperature was maintained at 20°C for 30 days, and then decreased
2°C/week until it reached 9°C which, was maintained until test termination. In ES2,
bluegill were exposed to one aqueous and one dietary selenium concentration. The
temperature regime for ES2 was the same as for ES 1. The nominal concentrations of
selenium in the water and the target concentrations for selenium in the diet for each
exposure system are given in Table 2.1. One hundred juvenile bluegill were added to
each of the 20 test tanks at the start of the exposure period on April 30, 2007.
Table 2.1. Nominal exposure concentrations for Exposure Systems 1, 2 and 3.
Exposure System
and temperature
regime
ESI
20 to 4°C
ES2
20 to 4°C
ES3
20 to 9°C
Treatment
Number (no. of
replicates)
Control (2)
KD
2(1)
3(1)
4(1)
5(1)
6(1)
Control (2)
5(2)
Control (2)
KD
2(1)
3(1)
4(1)
5(1)
6(1)
[Se] in Water,
Hg/L
No added Se
1.25
2.5
5
10
20
40
No added Se
5
No added Se
1.25
2.5
5
10
20
40
Target [Se] in diet, |ig/g dw
Lumbriculus
Background
1.5
2.5
5
10
20
40
N.A.
N.A.
Background
1.5
2.5
5
10
20
40
TetraMin
Background
5
The goals for selecting the target exposure conditions were to (1) attain a range of
selenium concentrations in the juvenile bluegill that result in no response in the low
exposures, intermediate response in the middle treatments and meaningful mortality in
the high exposure conditions; and (2) achieve water and worm concentrations that are
representative of field conditions. An assumption was made that the transfer of selenium
from worm to bluegill was 1:1. This assumption was confirmed in selected exposure
conditions in preliminary experiments.
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.Chiller ES3
ESS
ooooooee
Water bath
ESI
6 Inches
OVERHEAD VIEW
O
\
, UK. unit
' WC unit
48ft,
nit.
Cheater 11
Dilution Tank A
Figure 2.1. Floor plan of systems used in juvenile bluegill selenium study.
SIDE VIEW
Cheater«'}
Dilution Tank B
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2.2.1 Aqueous Exposure
A 1:1 molar ratio (as selenium) of selenite and selenate was produced using sodium
selenite (Na2SeC>3; 99% Sigma-Aldrich), and sodium selenate decahydrate-(Na2SeO4
10H20; 99%, Sigma-Aldrich). Concentrated stock solutions of selenate (4.67 g of sodium
selenate/1 L of deionized water) and selenite (2.18 g of sodium selenite /L of deionized
water) were prepared, and were combined to make two working stock solutions (200 and
2000 ug/L total selenium) that were used in the definitive study to achieve the target
selenium concentrations. The 200 ug/L and 2000 ug/L stock solutions were prepared in
200 L calibrated carboys using dechlorinated tap water as the medium for the toxicant.
The combined selenite and selenate stock solutions were used to dose the bluegill
exposure tanks for all three exposure systems. To achieve the target test concentrations,
FMI (Fluid Metering, Inc) pumps delivered the stock solutions at a predetermined flow
rate, while the dilution water (dechlorinated tap water) was delivered to the exposure
tanks from a chilled head tank at a rate of 500 ml/minute (Figures 2.2 and 2.3). To
ensure adequate mixing of dilution water and selenium prior to delivery to the exposure
chambers, the stock solutions and dilution water flowed into a mixing vessel, which then
drained into the designated 200 liter bluegill exposure tanks. The flow rates of the stock
solutions and dilution water were measured approximately every 12 hours, and were
adjusted as needed to be within + 0.2 ml/minute for the stock solutions, and + 5
ml/minute for the dilution water. Expected dilution water and stock solution flow rates
(and designated stock solutions) for each of the six target concentrations for all three
exposure systems are presented below.
Target aqueous total
[Se] in the test
chambers
Stock solution flow
rate to the test
chambers
(dilution water flow
rate 500 ml/min)
200 ug/L stock solution
(1:1 selenite/selenate)
1.25 ug/L
3.13
ml/min
2.5 ug/L
6.25
ml/min
5.0 ug/L
12.5
ml/min
2000 ug/L stock solution
(1:1 selenite/selenate)
10 ug/L
2.5
ml/min
20 ug/L
5.0
ml/min
40 ug/L
10
ml/min
To confirm the aqueous selenium exposure concentrations, 40 ml of test solution were
collected from each exposure tank on a weekly basis. Samples were preserved with 1%
HCL (instra-analyzed HC1 36.5-38%, J.T. Baker, Phillipsburg, NJ) in clean glass vials,
and refrigerated until shipped to the analytical laboratory for analysis.
-------
Se test solution feed tube (pumped from bluegill tank to Lumbriculus tank)
Se stock solution feed tube Air feed tube
• •••in Se test solution feed drip H20 feed tube
Drain
Figure 2.2. Tank system diagram for bluegill and Lumbriculus ESI and ESS.
-------
Se stock solution feed tube
Se test solution feed drip
Air feed tube
H20 feed tube
Drain
Air Supply
Figure 2.3. Tank system diagram for bluegill ES2.
-------
2.2.2 Dietary Exposure
The fish in ES2 were fed a commercial fish flake food fortified with seleno-L-
methionine, with the goal of achieving a nominal selenium concentration of 5 |ig/g (dry
weight), while the fish in ESI and ESS were fed L. variegatus which had accumulated
selenium in their tissues. Selenium-spiked Tetramin fish flakes were prepared by adding
500 g of crushed fish flakes to 125 ml of a seleno-L-methionine (>98%, Sigma-Aldrich,
Lot# 016K1335) stock solution. The seleno-L-methionine stock solution was prepared
by dissolving 62.06 mg of seleno-L-methionine in 1 liter of deionized water. The
Tetramin flakes were finely ground using a glass mortar and pestal, and the seleno-L-
methionine aqueous stock solution was added to achieve a moisture content of 25% (4.0
ug Se/g ww or 5 ug Se/g dw). The control food was prepared following the same
procedures, except that deionized water without seleno-L-methionine was used to supply
the 25% moisture. The crushed flakes and aqueous components were thoroughly mixed
together to produce a paste. The dietary mixture was then weighed into aluminum pans
in 5 gram aliquots, and compressed to form a cake. The TetraMin cakes were held in the
freezer until they were needed. After preparation, three 5 g samples of the selenium-
dosed and control TetraMin cakes were analyzed for total selenium. Four separate
batches of selenium-dosed TetraMin cakes were prepared and used during the 182 day
exposure. The dates and average measured total selenium (N = 3 or 4) for each batch
were as follows: April 30 - May 17, 2007 (4.11 |ig/g dw); May 18 - July 10, 2007 (5.77
|ig/g dw); July 11 - September 6, 2007 (6.27 |ig/g dw); and September 7 - October 29,
2007 (6.67 |ig/g dw).
For ES2, the selenium-dosed TetraMin cakes were fed to the bluegills at a rate of 3% of
their body weight (wet weight) per day, based on survival and the average weight
measurements taken on days 0, 7, 30, 60, and 110. Control and selenium-dosed TetraMin
cakes were held frozen throughout the study. As with ESI and ES3, fish behavior was
observed while eating, and the weight of food provided to the fish was recorded on the
data sheets.
L. variegatus were exposed to selenium to create the dietary source for the bluegill in
exposure systems 1 and 3. The L. variegatus in the two control and six treatment
exposure chambers were fed 3.2 g of yeast suspended in 250 ml of dechlorinated water,
once a day. The yeast was suspended in the dechlorinated tap water by placing the 3.2 g
in a 500 ml Erlenmeyer flask and adding dechlorinated tap water to a 250 ml volume.
The contents in the flask were then vigorously swirled in the flask throughout the feeding
process.
Control worms were fed non-selenized nutritional yeast (Red Star™) and treatment
worms were fed a mixture of selenized yeast (measured to be 826 |ig/g) with non-
selenized nutritional yeast to achieve the desired dietary selenium exposure and dietary
requirements. The nominal concentrations of total selenium in the 6 selenized yeast
preparations were 1.7, 3.3, 6.7, 13.3, 26.7, and 53.5 |ig/g dw. These target concentrations
were based on similar Lumbriculus exposures with selenized yeast (Besser et al. 2006)
and confirmed in preliminary studies. The two yeast components were weighed on
10
-------
calibrated scales, and combined in four liter HDPE Nalgene containers. The yeast
combinations were initially mixed three separate times, at half-hour intervals, on a
mechanical roller. Prior to test initiation, one 15 gram yeast sample from each dietary
concentration was shipped to the analytical laboratory and analyzed for total selenium.
To ensure consistency in the mixtures over time, each week of the study the yeast
preparations were again mixed for a half hour on the mechanical roller.
The fish were fed L. variegatus at a rate of 4% of their body weight (wet weight) per day,
based on survival and the average weight measurements made on days 0, 30, 60, and 112.
A mass of worms was isolated, minus any overlying water, and held in a 100 glass mL
beaker. Foreign material (yeast, algae, waste) was removed, excess water decanted, and
the worms were weighed. Observations of the feeding activity of the bluegills while
eating were made, and the weight of worms fed was recorded. Fish tanks were siphoned
in the late afternoon to remove excess food and fecal matter.
Prior to initiating the study, the actual concentrations of selenium in the dietary samples
(yeast, worms, and TetraMin cakes) were measured. During the study, both the worms
and fish were fed after monitoring the morning flow rates and measuring the water
quality characteristics in the overlying water. The average concentrations of total
selenium measured in the worms sampled on days 0, 30, 60, 112 and 182 are given in
Table 3.5 in the Results and Discussion section.
2.3 WATER QUALITY MEASUREMENTS AND OBSERVATIONS
During the 182-day study, observations on water quality, stock and dilution water flow
rates, and test organism behavior and mortality were recorded on a daily basis. Samples
for analysis of total selenium concentrations in the water were collected on a weekly
basis, and once a month the samples were analyzed to determine selenium speciation.
Test days 0, 30, 60, 112, and 182 were designated to sample worm tissue, and on test
days 0, 7, 30, 60, 112, and 182 fish were sampled. Duplicate 5 g samples of Lumbriculus
were collected from each of the 12 worm treatment tanks and from one of the two control
tanks in each system. Triplicate fish samples, with each sample consisting of a three fish
composite, were collected from each of the 16 fish treatment tanks (i.e., a total of 9 fish
per tank), and from one of the two control tanks in each system.
Fish and worms were homogenized prior to shipping for selenium analysis. Tissue
samples (e.g., a 3-fish composite) were homogenized with 10 ml deionized water in pre-
cleaned 250 ml nalgene bottles using a pre-cleaned stainless steel tissue homogenizer.
The samples were blended until completely homogenized (appearance smooth with no
visible masses). The blended samples were transferred to the pre-labled 40 ml glass
sample vial and 15 ml of deionized water was used to rinse out the 250 ml nalgene bottle.
All equipment was cleaned using one percent HC1 and rinsed with deionized water in
between homogenization of different tissue samples. The samples were processed in
order from lowest to highest nominal selenium concentration.
11
-------
Lipid content was measured in the bluegill on test day 0 and at test termination in each
treatment. The method used was a standardized procedure developed by the EPA
laboratory in Duluth, Minnesota. In summary, tissue samples were sequentially weighed,
homogenized with an extraction solution of 3:2 hexane:isopropanol, centrifuged, and the
supernatant solution decanted to a separatory funnel, where it is washed with a sodium
sulfate solution. After the bottom aqueous phase was discarded, the organic phase was
transferred to a 50 ml graduated cylinder fitted with a ground glass stopper, and the
weight was measured and 5 ml duplicate aliquots of the lipid extract were pipetted to
tared weighing pans. The pans were placed under a hood where the solvent was
evaporated. The pans were transferred to a dessicator for removal of any remaining
solvent and water. After 24 hours, the pans were weighed and the lipid content was
calculated according to the following formula.
„. - , ( sample wt.}(sample vol./5ml}
% Lipid = — -
tissue wt.
Oversight of the exposure systems included monitoring various overlying water quality
characteristics (pH, dissolved oxygen, temperature, conductivity, and chlorine) on
different days, and the flow rates of both the selenium stock solutions and dilution water
twice a day.
Dissolved oxygen and temperature were measured daily in each L. variegatus and fish
exposure tank. Dissolved oxygen was measured using a YSI 57 meter and an Orion
probe. Temperature was measured two different ways; directly from each exposure tank
using a digital hand-held thermometer with a stainless steel probe, and continuously at
mid-depth in one tank in each exposure system using a submersible temperature data
logger. An Orion 710 meter and probe was used to measure pH on Monday, Wednesday,
and Friday in each L. variegatus and fish exposure tank. Conductivity was measured
weekly in each bluegill exposure tank using a YSI 33 meter. The dechlorinated water
head tank was monitored weekly for total chlorine, pH, dissolved oxygen, and
conductivity. All meters were calibrated before each use, and the thermometer was
calibrated at least every 2 weeks, or more frequently in the event of a planned
temperature decrease.
Flow rates of the stock solutions and dilution water in the bluegill tanks were measured
twice a day, approximately 12 hours apart. The treatment water from the bluegill
exposure chambers was pumped to the worm exposure chambers to supply the aqueous
selenium exposure for the worms. The worms therefore received the same aqueous
exposure of selenium as the fish to which they were fed. The target flow rate from the
bluegill tanks to the worm exposure chambers was 60 ml per minute. The target flow
rate from the head tank and stock solution reservoir to the 200 L bluegill treatment tanks
was 500 ml per minute, resulting in approximately 3.4 turnovers a day. Stock solutions
were dispensed using a fluid metering pump (FMI), and the dilution water was delivered
by gravity from the temperature controlled head tank.
12
-------
2.4 DETERMINATION OF TOTAL SELENIUM IN WATERS AND TISSUES BY
HYDRIDE GENERATION-ATOMIC FLUORESCENCE
SPECTROMETRY (HG-AFS)
2.4.1 Fish and Worm Tissue Digestion
Dry weight determination: Vials containing suspensions of homogenized tissues in water
were shaken vigorously and 5 ml aliquots pipetted into a pre-weighed aluminum trays.
Samples were dried in an oven at 80°C for 24 hours, removed and placed in a desiccator
for one hour (to cool down without drawing water from the atmosphere) prior to
weighing on an analytical balance. Duplicate measurements were performed
approximately every ten samples. Dry weight was calculated as the difference between
the dry sample and the empty tray, and water content was calculated as the weight loss
during drying.
Nitric acid digestion: Vials containing suspensions of homogenized tissue in water were
shaken vigorously and 3.5 ml aliquots pipetted into new 40 ml I-Chem vials. 10 ml of
concentrated nitric acid (Fisher, 16 M) was added, and samples were covered with a
marble and digested on a hot plate set at 150°C for 1.5 hours. Digested samples were then
allowed to cool and filled up to the mark on the vial (40 ml) with milliQ water. Each
digestion batch also contained 3 blank samples, 3 spiked blank samples (1 each spiked
with selenite (Se(IV)), selenate (Se(VI)) and selenomethionine (SeMet), and a certified
reference material for each type of tissue present. One of every ten samples was prepared
with a QC set: duplicate, matrix spike (spiked with SeMet) and a matrix spike duplicate.
2.4.2 Reagents
Reagent Blank (40% cone. HCl): 1 L of reagent grade concentrated HC1 (Fisher) was
added to 1.5 L milliQ water and inverted to mix. The resultant mixture is 4.8 M HCl.
Reductant (1% KBH4 w/v in 0.4% NaOH): 16 g of 50% w/w NaOH (VWR) were added
to approximately 1800 ml milliQ water and swirled to mix. 20 g of potassium
borohydride (KBH/t, Aldrich) was added and swirled to dissolve powder completely,
before the solution was filled to the 2 L mark with milliQ water.
Potassium per sulfate (2% w/w): 0.6 g potassium persulfate (£28203, Fisher) was placed
into a 40 ml I-Chem vial (same vial reused) and milliQ water was added to 30 g. The
solution was shaken vigorously to dissolve the K2S2O8.
Selenium standards: Working standards were prepared by diluting 1000 mg/L solutions
of Se(IV), Se(VI) and SeMet to 1000 |ig/L (50 |il to 49.95 ml milliQ), which were then
further diluted to 100 |ig/L (1 ml to 9 ml milliQ).
Certified reference materials: National Water Research Institute TM-DWS (30.6 |ig/L
Se) river water was used for calibration validation and as a water sample CRM. NIST
13
-------
1566b Oyster Tissue (2.06 jig/g Se) was used as a CRM for worm tissues and NRC
DORM-2 Dogfish Muscle Tissue (1.40 jig/g Se) was used as a CRM for fish tissues.
NRC SELM-1 Selenium Enriched Yeast (2059 jig/g Se) was used as a CRM for yeast
samples.
2.4.3 Sample A nalysis
Sample preparation: All samples being analyzed were 40% HC1 to match the calibration
standards and not disrupt the function of the continuous flow HG-AFS. Sample
preparation involved pipetting 12 ml of the sample into a conical flask (or, for diluted
samples, less volume and the balance to 12 ml DI water); then, 8 ml of cone. HC1 were
added for a total volume of 20 ml. This results in a minimum dilution factor of 1.667x.
For dilutions greater than 200x, serial dilutions were used, with intermediate steps
prepared in Sarstedt tubes with milliQ water. All tissue samples were diluted at least 20x,
in order to dilute the nitric acid introduced in the digestion step to the point where
interferences with the HG procedure were eliminated.
Prereduction/oxidation step: Flasks containing properly diluted samples were weighed on
a top-loading balance (to 0.01 g) and the mass recorded. 200 jil of potassium persulfate
solution was added to the sample, which was then placed on a hot plate set at 200°C. A
timer set for 15 minutes was started when the first sample on the hot plate began boiling,
and samples were removed when the timer finished. This step was done in batches of
approximately 10 samples at a time. Samples were allowed to cool prior to analysis. After
HG-AFS analysis, this procedure yields total Se concentrations.
Selenium speciation analyses: For direct determination of Se(IV) in water samples,
samples were measured without prereduction/oxidation. The concentration of Se(VI) was
then calculated by difference between total Se and Se(IV), assuming that no other Se
species besides Se(IV) and Se(VI) were present in the waters (which matches the way
they were prepared).
Hydride Generation Atomic Fluorescence Spectroscopy (HG-AFS): A peristaltic pump
was utilized to introduce reagent blank or sample into a gas-liquid separator at a rate of
10 ml min"1, and combined with the reductant 5 ml min"1. The mixing of highly acidic
reagent/sample and reductant generates hydrogen gas and selenium hydride, SeFk. This
species is highly volatile and is swept into the AFS unit (Excalibur, P.S. Analytical) with
argon as a carrier gas (300 ml min"1, measured using a ball flow meter). 50 jil of n-
octanol was added to the gas-liquid separator as a surfactant to smooth the HG process
and reduce water droplet introduction to the AFS. The hydrogen gas was ignited to form
a continuous flame for the duration of the analysis. Selenium passing through the flame
was irradiated with a Photron hollow cathode lamp, and the intensity of fluorescence was
then measured and recorded on a Hadley Tekscience printer. The lamp primary current
was set to 20 mA and the boost to 25 mA. The intensity of fluorescence is proportional to
the Se concentration in a sample, and peak heights could therefore be used to determine
Se concentrations using a calibration curve.
14
-------
Instrument Calibration: A calibration curve was made up using standards with
concentrations of 0.05, 0.1, 0.25, 0.5, 1.0 and 2.5 |ig/L Se(IV) and no prereduction. An
initial calibration validation was performed using a second Se(IV) standard and TM-
DWS CRM again with no prereduction step. Coefficients of determination were always
>0.995 and were >0.999 most of the time. A continuous calibration standard of
approximately 1 |ig/L Se(IV) was analyzed intermittently to track any sensitivity changes
in the instrument due to lamp power, etc., and all analytical results were corrected for
instrument drift.
Quality Assurance/Quality Control: For both water and tissue samples,
prereduction/oxidation blank values were determined and subtracted from all samples.
Blank spikes of Se(IV), Se(VI) and SeMet (1 |ig/L) were analysed to determine
recoveries of these species of Se, and were often several percent higher than non-
prereduced samples. For tissue sample analyses, digestion blanks were quantified and
subtracted proportionally to dilution from all samples. Digestion blank spikes were also
analysed to assure quantitative recoveries following digestion. Certified reference
materials suitable to the sample matrix were also analysed.
During analysis, quality assurance tests were conducted every 10-15 samples. For tissue
digests, this included three extra vials of digested tissue: a duplicate sample aliquot and
two more sample aliquots spiked with SeMet at levels 2-5x the expected value of Se to
assess reproducibility and completeness of tissue digestion. Duplicate analysis of one of
these vials was conducted as well as a SeMet spike added just prior to the prereduction
step, performed in duplicate, to assess analytical reproducibility and accuracy. QA water
samples were analysed in duplicate, as well as duplicate analyses of the same sample
with 1 |ig/L Se (as 50:50 Se(IV):Se(VI)) matrix spikes. Continuous calibration validation
using Se(IV) without prereduction was performed every 5-10 samples in order to correct
for changing sensitivity of the instrument.
3.0 RESULTS AND DISCUSSION
3.1 WATER QUALITY MEASUREMENTS
The water quality parameters measured in each tank (pH, dissolved oxygen, and
conductivity) were within acceptable levels for toxicity tests, and remained consistent
between treatments and throughout the 182 day exposure period (Table 3.1, Appendix
A).
15
-------
3.1.1 Temperature Measurements
The water temperatures measured in fish tanks generally followed the two temperature
regimes targeted in the study design (Figures 3.1 - 3.3; Appendix A). Values from daily
manual measurements in each tank were in agreement with records from the continuous
logging probe. Average temperatures were within ± 0.5°C of the target temperatures in
ES3, but 0.6 to 0.7°C higher than the targets in ESI and ES2. The average and standard
deviation of water temperatures, measured daily, for the target 4°C (day 80 through 182)
ESI tanks were 4.7°C and 0.25°C, and 4.6°C and 0.25°C in ES2 tanks.
Temperature, C
o en o en o en
ES1 In-tank Temperature; daily average across treatments
*_ ^
^HNP IPWW^
w*
*v
*«H
«»
X,
«*
D 20 40 60 80 100 120 140 160 180 200
Test day
Figure 3.1. Daily average temperatures measured in each bluegill tank in Exposure
System 1. Temperatures were averaged across treatments
ES2 In-tank Temperature; daily averge across treatments
20.0 -
O
3
K
<*>, fe
>W*>**<'
«•?
V
«H
«*>
v»
**v v— -^<
__,_;,
0 20 40 60 80 100 120 140 160 180 200
Test day
Figure 3.2. Daily average water temperatures measured in each bluegill tank in
Exposure System 2. Temperatures were averaged across treatments.
16
-------
ESS In-tank Temperature; daily average across treatments
20
O
o
£ 15 _
=
c;
V
*\
JM^TL nL.it* »rLv*^uji
0 50 100 150 200
Test day
Figure 3.3. Daily average water temperatures measured in each bluegill tank in
Exposure System 3. Temperatures were averaged across treatments.
17
-------
Table 3.1. Average and range of pH, dissolved oxygen and conductivity in each tank. The pH and dissolved oxygen were
measured daily. Conductivity was measured once a week in each tank during the 182 day bluegill study.
System Treatment
ESI Control A
ESI Control B
ESI 1
ESI 2
ESI 3
ESI 4
ESI 5
ESI 6
ESS Control A
ESS Control B
ESS 1
ESS 2
ESS 3
ESS 4
ESS 5
ESS 6
ES2 Control A
ES2 Control B
ES2 5A
ES2 5B
pH, S.U.
Average Minimum Maximum
8.03 7.69 8.30
8.02 7.56 8.26
8.08 7.70 8.87
8.07 7.71 8.72
8.07 7.73 8.65
8.08 7.74 8.63
8.07 7.75 8.44
8.01 7.60 8.29
7.95 7.45 8.26
7.99 7.62 8.21
8.02 7.63 8.22
8.04 7.62 8.20
8.05 7.58 8.24
8.04 7.68 8.26
8.03 7.62 8.24
8.00 7.67 8.12
8.00 7.53 8.21
7.96 7.11 8.23
8.00 7.46 8.20
8.00 7.61 8.16
Dissolved Oxygen, mg/L
Average Minimum Maximum
9.9 8.0 12.3
9.9 7.9 12.2
10.1 8.0 12.3
10.2 8.0 12.3
10.2 7.8 12.4
10.2 8.0 12.2
10.2 8.0 12.2
9.6 8.0 12.1
9.8 7.9 12.0
9.8 8.1 12.0
9.9 8.2 12.1
10.0 8.0 12.1
10.0 8.1 12.0
10.0 8.0 11.8
10.0 7.9 12.0
9.8 8.0 11.4
9.9 8.0 11.8
9.9 8.2 11.9
10.1 8.1 12.2
10.0 7.4 12.1
Conductivity, |imhos/cm
Average Minimum Maximum
228 192 259
229 193 258
230 196 272
233 194 275
232 193 271
232 196 271
233 190 275
252 225 297
232 207 278
233 207 263
238 206 274
236 205 276
238 197 275
235 206 275
240 215 275
247 218 274
226 195 275
227 193 275
228 197 274
226 195 272
18
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3.2 SELENIUM MEASUREMENTS
3.2.1 Selenium in Water
Total selenium concentrations in the bluegill tanks were similar to the target exposure
concentrations (Table 3.2A; Appendix B). Measured concentrations were within 10% of
the target concentrations with the exception of ESS Treatments 1 and 2, and ESI
Treatment 1, which were within 12, 12 and 22% of the target concentrations,
respectively. As a consequence of a technical error on day 154 of the study, the
concentrations of selenium in the bluegill tanks for the last four weeks of the exposure
period were negatively affected. On test day 154, the bottles containing the selenium
stock solution were mislabeled with the incorrect stock solution concentration. The result
of the mislabeling produced low selenium concentrations in all the bluegill tanks from
day 154 through the end of the study, day 182. The average selenium concentrations in
the water during this 4-week period was reduced across all treatments, from near target
concentrations to less than 1 |ig/L in ES2 and Treatments 1 through 3 in ESI and ESS.
The average selenium concentration during last 4 weeks in Treatment 4 (both ESI and
ESS) was less than 2 ng/L; in ESI Treatment 5, 2.3 |ig/L; and in ESS Treatment 5, 7.9
A summary of the average measured total selenium concentrations for the entire 182 day
exposure shows the aqueous exposure concentrations remained similar to target levels
(Table 3.2B). The effect of this mistake is not considered meaningful for three main
reasons: (1) the drop in selenium's aqueous exposure was limited to the last four weeks
(15%) of the 26 week exposure; (2) the primary route of selenium exposure to the
bluegill is through the diet for which target levels of selenium were maintained
throughout the exposure period; and (3) the effects concentrations of most interest are
expressed in terms offish tissue concentrations, not as water concentrations.
Table 3.2A. Nominal and measured total selenium concentrations for all
treatments. Average concentrations are based on weekly samples collected up to
test day 154 of the exposure period.
System
ESI
ESI
ESI
ESI
ESI
ESI
ESI
Treatment
Control B
1
2
3
4
5
6
[Se] in water, |ig/L through Day 154
Measured
Nominal Average Std. dev.
No added Se
1.25
2.5
5
10
20
40
0.21
1.52
2.61
5.44
9.66
20.3
41.4
0.11
0.32
0.73
0.50
1.22
2.6
6.0
19
-------
System
ES2
ES2
ES2
ESS
ESS
ESS
ESS
ESS
ESS
ESS
Treatment
Control B
5A
5B
Control B
1
2
3
4
5
6
[Se] in water, |ig/L through Day 154
Measured
Nominal Average Std. dev.
No added Se
5
5
No added Se
1.25
2.5
5
10
20
40
0.21
5.58
5.61
0.18
1.47
2.83
5.24
9.15
19.7
41.1
0.12
0.66
1.35
0.07
0.59
0.54
0.61
0.91
2.0
5.3
Table 3.2B. Nominal and measured total selenium concentrations for all treatments.
Average concentrations are based on weekly samples collected throughout the 182
day exposure
period.
System
ESI
ESI
ESI
ESI
ESI
ESI
ESI
ES2
ES2
ES2
ESS
ESS
ESS
ESS
ESS
ESS
ESS
Treatment
Control
1
2
3
4
5
6
Control
5A
5B
Control
1
2
3
4
5
6
[Se] in water, |ig/L
Measured
Std.
Nominal Average dev.
No added Se
1.25
2.5
5
10
20
40
No added Se
5
5
No added Se
1.25
2.5
5
10
20
40
0.19
1.32
2.26
4.70
8.47
17.6
41.4
0.23
4.83
4.85
0.17
1.28
2.45
4.70
7.95
18.0
41.1
0.12
0.55
1.08
1.82
3.14
6.9
6.0
0.19
1.92
2.23
0.07
0.71
1.05
1.63
3.07
5.2
5.3
20
-------
The proportion of selenate to selenite in each bluegill tank remained similar to the target
ratio of 1:1. Selenium speciation analysis of monthly water samples collected from each
treatment resulted in an average ratio of 1.14:1 with a standard deviation of 0.31 (N = 84;
14 treatments and 14 monthly samples). The above speciation analysis was made by
directly measuring selenite and then calculating the concentration of selenate by the
difference between selenite and total selenium. This method was confirmed using direct
measurement by Ion Chromatography Inductively Coupled Plasma (1C ICP) MS of both
selenate and selenite in the test day 154 samples. The direct measurement of both species
resulted in a ratio of 1.29:1 with a standard deviation of 0.29 (N = 9).
3.2.2 Selenium in Lumbriculus variegatus
The concentration of selenium in the worms in each of the treatments used to feed the
bluegill in ESI and ESS varied somewhat over time (Tables 3.3 and 3.4, respectively;
Appendix C). The average concentrations of the upper treatment levels (3 through 6)
were within a factor of 1.5 of the target concentrations in the worms (Table 3.5). The
average of measured selenium concentrations in the lowest two concentrations were
between 2 and 3 times higher than the target levels.
To maintain a continuous supply of worms for feeding the bluegill, the population of
Lumbriculus in ESI and ES3 required supplementing three times during the 182 day
exposure. A back-up culture of Lumbriculus was maintained in GLEC's main laboratory
(i.e., not in the trailer) for the purpose of adding worms to the exposure system. The
worms in the back-up culture were exposed to the same aqueous and dietary selenium
treatments as in ESI and ES3.
Table 3.3. Measured total selenium concentrations (ug/g dw) in Lumbriculus
variegatus for all treatments in Exposure System 1.
Test
Day
0
30
39*
60
85*
112
162*
182
Avg
SD
Treatment
Control
2.7
2.7
2.4
2.5
A
2.5
1.7
1.9
2.3
0.4
1
5.2
5.6
2.6
4.2
3.9
5.2
4.5
4.5
1.0
2
4.5
6.9
3.3
5.9
3.9
7.6
5.9
4.4
5.3
1.5
3
6.8
8.9
3.8
8.5
5.7
12.0
7.5
6.6
7.5
2.4
4
8.5
19.7
5.8
12.3
17.8
25.9
12.2
11.1
14.2
6.6
5
20.5
35.6
10.0
35.7
11.8
38.1
33.2
20.6
25.7
11.3
6
25.0
59.1
16.9
44.6
29.0
B
B
B
34.9
16.9
*Measurements made in back-up worms added to ESI worm tanks.
A No sample collected.
B Treatment 6 discontinued due to complete mortality in bluegill tank.
21
-------
Table 3.4. Measured total selenium concentrations (ug/g dw) in Lumbriculus
variegatus for all treatments in Exposure System 3.
Test
Day
0
30
39*
60
85*
112
162*
182
Avg
SD
Treatment
Control
2.0
2.4
2.4
2.6
A
2.7
1.5
2.0
2.2
0.4
1
3.9
5.7
2.6
4.5
3.9
5.5
3.8
3.8
4.2
1.0
2
4.8
5.0
3.3
6.2
3.9
6.9
5.3
4.8
5.0
1.2
3
7.0
8.2
3.8
7.3
5.7
8.7
9.6
A
7.2
1.9
4
11.3
13.7
5.8
11.4
17.8
29.0
20.6
12.1
15.2
7.1
5
16.4
35.1
10.0
30.6
11.8
36.3
37.5
25.9
25.4
11.3
6
38.4
63.5
16.9
51.2
29.0
81.3
B
B
46.7
23.5
*Measurements made in back-up worms added to ES3 worm tanks.
A No sample collected.
B Treatment 6 discontinued due to complete mortality in bluegill tank.
The worms from the supplementary culture were added to the Lumbriculus tanks in ESI
and ES3 on test days 39, 85 and 162. The concentration of selenium in the
supplementary worms was measured just prior to addition to the test systems (see
corresponding footnotes in Tables 3.3 and 3.4). Once added to the tanks the new worms
joined the aggregations of the test system worms within a day. The concentrations of
selenium in the supplementary worms were usually lower than the worms being
maintained in the test system, but since all worms quickly co-mingled, the actual
selenium concentrations being fed to the bluegill were assumed to be somewhere between
the measured concentrations in the supplementary worms and the worms in the test
system.
Table 3.5. Target and average measured total selenium concentrations in
Lumbriculus variegatus for all treatments in Exposure Systems 1 and 3.
Treatment
Control
1
2
3
4
5
6
Total Selenium in Lumbriculus, |ig/g dw
Target ESI avg ES3 avg
Bkg 2.34
1.5 4.45
2.5 5.30
5 7.47
10 14.2
20 25.7
40 34.9
2.21
4.20
5.02
7.17
15.2
25.4
46.7
3.2.3 Concentrations of Selenium in Fish Tissues
Concentrations of selenium in fish tissues generally increased with exposure duration
(Table 3.6; Appendix D). The asymptotic accumulation was modeled as
22
-------
O)) (I)
where t was the exposure time, expressed in days, and a, 6, c were parameters estimated
by nonlinear least squares regression (nls function in S-PLUS 6.2, Insightful
Corporation). As the figures below illustrate, this model explained most of the variation
in tissue concentrations of selenium over time.
Tissue concentrations of selenium in the ES1 control treatment averaged 2.11 |lg/g, and
varied little over time (Fig. 3.4). The average was 2.11 |ig/g dw and the coefficient of
variation (standard deviation/mean) was 0.083 (0.17/2.11). Changes in tissue
concentrations of selenium over time in the control treatment of ES2 (Fig. 3.4) were
larger than was the case for ESI. The average selenium concentration and coefficient of
variation for the ES2 control were 1.85 |ig/g and 0.23. Changes in tissue concentrations
of selenium over time in the ES3 control treatment (average control tissue in ES3 was
2.20 jig Se/g) were also larger in ES3 (Fig. 3.4) than in ESI, yet the coefficient of
variation (standard deviation/mean) for the ES3 control was only 0.17.
In ESI, measurements of tissue concentrations in fish exposed to the highest
concentrations of selenium in the water (40 |lg/L) and in diet (40 |ig/g dw) were
restricted to the first 60 days of exposure (Fig. 3.5) due to the high mortality of organisms
in that treatment. Rates of selenium bioaccumulation in bluegill were higher in Treatment
6 than in Treatment 5 (Fig. 3.5). At day 7, tissue concentrations were 4.27 |ig/g dw in
Treatment 6 and 3.27 in Treatment 5. At day 60, concentrations of selenium in fish
tissues increased to 8.62 |ig/g dw in Treatment 5 and 12.66 |ig/g dw in Treatment 6.
Bioaccumulation rates in fish exposed to lower concentrations of selenium in the water
(<10 |lg/L) and in worms (<10 |ig/g dw) were consistently lower, as were the lower
asymptotic concentrations of selenium in tissues (Fig. 3.5; Treatments 1 through 4). For
instance, in Treatment 4 selenium concentrations reached 5.21 |ig/g dw at day 60, 6.42
|lg/g dw at day 112, and 6.72 |ig/g dw at day 182. In Treatment 2 selenium
concentrations reached 3.07 |ig/g dw at day 60, 3.41 |ig/g dw at day 112, and 3.15 |ig/g
dwatday 182 (Table 3.6).
In the two lowest ESI treatments (1.5 and 2.6 |ig/L in water, and 4.5 and 5.3 |ig/g in
worms), concentrations of selenium in bluegill tissues reached equilibrium at 2.8 |ig/g dw
in Treatment 1 and 3.2 |ig/g dw in Treatment 2 after approximately 30 days of exposure
(Fig. 3.5). In all other treatments, except Treatment 6 which was terminated early
because of high mortality, tissue concentrations of selenium seemed to be approaching
the asymptote at the end of the experiment.
23
-------
Table 3.6. Measured total selenium concentrations in bluegill sunfish for all treatments in Exposure Systems 1, 2 and 3.
Total Selenium in Whole Body Bluegill Tissue, |ig/g dw
ESI
ESS
ES2
Test Day
0
7
30
60
112
182
Test Day
0
7
30
60
112
182
Test Day
0
7
30
60
112
182
Control
Average (SD)
1.93(0.21)
2.43(0.31)
2.10(0.21)
2.11 (0.02)
1.98(0.04)
2.08(0.10)
Control
Average (SD)
1.93(0.21)
2.50(0.10)
2.24(0.41)
2.70 (0.22)
2.16(0.14)
1.67(0.21)
Control
Average (SD)
1.93(0.21)
2.19(0.19)
2.49(0.15)
1.53(0.03)
1.57(0.01)
1.38(0.06)
Treatment 1
Average (SD)
1.93(0.21)
2.48(0.11)
2.85(0.10)
2.70 (0.20)
3.16(0.11)
2.56(0.21)
Treatment 1
Average (SD)
1.93(0.21)
2.60 (0.29)
2.44 (0.26)
2.88 (0.08)
2.49(0.10)
3.20(0.27)
5A
Average (SD)
1.93(0.21)
3.55 (0.25)
7.05 (0.76)
8.23(1.55)
8.97(1.28)
9.41 (1.63)
Treatment 2
Average (SD)
1.93 (0.21)
2.43 (0.18)
3.10(0.04)
3.07(0.05)
3.41 (0.08)
3.15(0.25)
Treatment 2
Average (SD)
1.93 (0.21)
2.38(0.10)
2.70(0.16)
3.04(0.39)
3.10(0.12)
3.83 (0.47)
5B
Average (SD)
1.93 (0.21)
3.08 (0.50)
7.51 (1.18)
8.09 (0.67)
9.45(1.73)
10.61 (0.38)
Treatment 3
Average (SD)
1.93 (0.21)
2.64 (0.06)
2.94(0.13)
3.69(0.25)
3.99(0.26)
4.02 (0.21)
Treatment 3
Average (SD)
1.93 (0.21)
2.82 (0.20)
3.13 (0.10)
3.79(0.24)
3.64(0.16)
5.48 (0.24)
Treatment 4
Average (SD)
1.93 (0.21)
2.72 (0.07)
4.24 (0.22)
5.21 (0.30)
6.42 (0.05)
6.72 (0.09)
Treatment 4
Average (SD)
1.93 (0.21)
3.19(0.33)
3.95(0.16)
5.54(0.21)
6.54(0.21)
9.38 (0.63)
Treatment 5
Average (SD)
1.93(0.21)
3.27(0.27)
6.62 (0.23)
8.62 (0.45)
11.60(0.43)
10.71 (0.55)
Treatment 5
Average (SD)
1.93(0.21)
4.29 (0.20)
6.06(0.36)
9.50(0.91)
11.50(0.25)
16.01 (0.30)
Treatment 6
Average (SD)
1.93(0.21)
4.27 (0.44)
10.21 (0.36)
12.66 (0.45)
Treatment 6
Average (SD)
1.93(0.21)
6.13(0.62)
11.07(0.92)
15.14(0.96)
17.24 (0.30)
24
-------
0)
a 1.0 H
ES1 - Control
100 120 140 160 180 200
Day
?-
•c
O)
O)
•2.
3
»
Ol
2L
3.5
3.0
2.5
2.0,
1.5
1.0
0.5
ES2 - Control
•
B
• •
) 20 40 60 80 100 120 140 160 180 200
Day
4.0
3.5-
•g- 3.0-
•c
O) 2.5
01
2L 1.0
ESS - Control
0 20 40 60 80 100 120 140 160 180 200
Day
Figure 3.4. Concentrations of selenium in juvenile bluegill tissues over time of
exposure in Controls for Exposure Systems (ES) 1, 2 and 3. The dotted line
represents the average concentration.
25
-------
3.5
? 3.0
•c
O) 2.5
ra
3 2.0,
.1 1.5
01
W, 1.0
0.5
0.0
ES1 - Treatment 1
•
s~
1
20 40 60 80 1 00 1 20 1 40 1 60 1 80 200
Day
•c
5"
3
.2!
oT
\!L
3.5
3.0
2.5
2.0 |
1.5
1.0
0.5
0.0
ES1 - Treatment 2
•
/* *
f
(
20 40 60 80 100 120 140 160 180 200
Day
5.0
4.5-
4.0-
•I 3.5-
i- 2.5 H
0}
| 2.0
"oT 1.5-
^^ 1.0-
0.5-
ES1 - Treatment 3
100 120 140 160
Day
8.0
7.0
"g" 6.0
•D
^) 5.0
O)
1—' 4.0
2.0
1.0
ES1 - Treatment 4
40 60 80 100 120 140 160 180 200
Day
20 40 60 80 100 120 140 16'
Day
•n 1°'°"
"B> 8.0
_3_
1 6'° "
w
,« 4-0 -
ES1 - Treatment 6
20 40 60 80 100 120 140 160 180 200
Day
Figure 3.5. Concentrations of selenium in juvenile bluegill tissues over time of
exposure in Exposure System 1 (ESI) Treatments 1 through 6. Dots represent
measured values and the solid line represents projections from the fitted model (I):
Treatment 1: a = 1.9008, b = 0.9275, c = 0.1470
Treatment 2: a = 1.8944, b = 1.3218, c = 0.0715
Treatment 3: a = 2.0309, b = 2.0425, c = 0.0248
Treatment 4: a = 1.9839, b = 4.8816, c = 0.0194
Treatment 5: a = 1.8629, b = 9.5322, c = 0.0231
Treatment 6: a = 1.7580, b = 12.411, c = 0.0365
26
-------
Up to day 112, concentrations of selenium in ESS (20 —>• 9°C) fish were similar to tissue
concentrations of selenium in corresponding treatments of ESI (20 —>• 4°C). From day
112 to the end of the experiment on day 182, selenium accumulated in ESS fish at faster
rates (Table 3.6, Fig. 3.6). The difference in selenium accumulation between ESI and
ES3 fish during this period could be attributed to the decreased feeding observed in ESI
fish and the continued feeding by ES3 fish. On day 112, tissue concentrations of
selenium in Treatment 1 were 3.16 |ig/g dw for ESI and 2.49 |ig/g dw for ES3. On this
day, tissue concentrations in Treatment 5 were 11.60 |ig/g dw for ESI and 11.50 |ig/g dw
for ES3. At the end of the experiment, tissue concentrations in Treatment 1 were 2.56
|lg/g dw for ESI and 3.20 |ig/g dw for ES3. Concentrations of selenium in Treatment 5,
were 10.71 |ig/g dw for ESI and 16.01 |ig/g dw for ES3. At the end of the experiment,
tissue concentrations in most ESI treatments were increasing at slow rates. In contrast,
tissue concentrations in most ES3 treatments were still increasing at fast rates at that time
(day 182). In fact, for Treatments 5 and 4 (Fig. 3.6) it is not clear what would be an
appropriate estimate for the asymptote. The observation that the bluegill are still
accumulating selenium after 182 days of exposure in ES3 Treatments 1 through 5 cannot
be explained by the variability in selenium concentrations in their diet (Lumbriculus).
Although there was some variability in selenium levels in the worms, there was no
apparent increase in concentration during the latter half of the exposure period (Tables
3.3 and 3.4).
Just as in ESI (4°C), the bioaccumulation rates in fish in ES3 (9°C) exposed to lower
concentrations of selenium in the water (<10 |lg/L) and in worms (<10 |ig/g dw) were
consistently lower as were the asymptotic concentrations of selenium in tissues (e.g., Fig.
3.6). For instance, in Treatment 4 selenium concentrations reached 5.54 |ig/g dw at day
60, 6.54 |ig/g dw at day 112, and 9.38 |ig/g dw at day 182. Selenium concentrations in
Treatment 2 reached 3.04 |ig/g dw at day 60, 3.10 |ig/g dw at day 112, and 3.83 |ig/g dw
at day 182 (Table 3.6).
The accumulation of selenium approached steady-state in the bluegill exposed to ES2
Treatments 5A and 5B (Figs 3.7a and b). Although the solid line projection in Figures
3.7a and b indicate steady-state was reached, the point measurements on test days 120
and 180 show a gradual increase in the selenium tissue concentration, that is likely due to
the progressively higher concentrations of selenium in the TetraMin fed to the bluegill.
As described in the Methods section, four batches of the selenium-spiked TetraMin were
fed to the bluegill in the 182-day study (test days 0-17, 4.11 |ig/g; test days 18-71, 5.77
|ig/g; test days 72-129, 6.27 |ig/g; and test days 130-182, 6.67 |ig/g). It is likely steady-
state would have been reached if the dietary selenium concentration was constant.
Tissue concentrations of selenium in the ES2 Treatment (nominal 5 |ig/L in water and 5
|ig/g dw in the TetraMin) were far higher than tissue concentrations in comparable
exposures in ESI and ES3 Treatment 3 (nominal 5 |ig/L in water and 5 |ig/g dw in
worms). At the end of the experiment, tissue concentrations in Treatment 3 of ESI and
ES3 reached 4.0 and 5.5 |ig/g dw, respectively, and 9.4 and 10.6 |ig/g in Treatment 5A
and 5B of ES2, respectively.
27
-------
•c
O) 2.5
3
~ 1.5 -
0.5 -
0.0
ESS - Treatment 1
20 40 60
100 120
Day
140 160 180 200
4.0
3.5 -
3.0 -
.O) 2.5
0)
— 2.0 J
1.0 -
0.5 -
0.0
ESS - Treatment 2
100
Day
T
~° 4.0
O)
O)
3
0)
3
ESS - Treatment 3 •
•
.
•
0 20 40 60 80 100 120 140 160 180 200
Day
•D
ES3 - Treatment 4
0 20 40 60 80 100 120 140 160 180 200
Day
18.0
16.0
14.0
£
~° 12.0
§> 10.0
§ 8.0
CO
4.0
2.0
0.0
ES3 - Treatment 5
80 100 120 140 160 180 200
Day
•o
ra
£
ESS - Treatment 6
20 40 60 80 100 120 140 160
Day
Figure 3.6. Concentrations of selenium in juvenile bluegill tissues over time of
exposure in Exposure System 3 (ESS) Treatments 1 through 6. Dots represent
measured values and the solid line represents projections from the fitted model (I):
Treatment 1: a = 2.2066, b = 1.0280, c = 0.0093
Treatment 2: a = 2.1157, b = 1.9311, c = 0.0092
Treatment 3: no model line fitted; no convergence in estimates of parameters
Treatment 4: a = 2.4325, b = 15.831, c = 0.0031
Treatment 5: a = 2.6954, b = 17.938, c = 0.0070
Treatment 6: a = 2.3920, b = 15.211, c = 0.0303
28
-------
a)
D)
3. 6.0
ES2 - 5A
100 120 140 160 180 200
Day
b)
10.0 i
9.0 -
8.0-
•1 7.0-
| 6°
-—• 5.0 -
0)
| 4.0-
"aT 3.0-
2L
2.0 ,
1.0 -
0.0 -
ES2 - SB
0
Day
Figure 3.7. Concentrations of selenium in juvenile bluegill tissues over time of
exposure. Dots represent measured values and the solid line represents projections
from the fitted model (I): a) a = 1.8437, b = 8.3022, c = 0.02973; b) a = 1.9114, b =
7.2843, c = 0.03848.
The higher bioaccumulation in the ES2 exposure system was apparently due to the form
of selenium to which the fish were exposed. The ES2 fish were fed a commercially
prepared fish food, TetraMin, to which seleno-L-methionine was added. ESI and ESS
fish were fed worms which accumulated selenium by ingesting selenized-yeast.
Preliminary investigations of the specific forms of selenium in the worms fed selenized-
yeast and TetraMin spiked with selenomethionine show selenomethionine was the
dominant soluble species in both diets. The TetraMin also contained trace amounts of
selenate and selenite, but selenomethionine was 76% of total selenium after
mineralization with nitric acid. The soluble selenium in the worms consisted of 71%
selenomethionine, 19% selenocystine and approximately 5% selenite and 5% selenate.
The soluble fraction of selenium in the worms, however, was only 15% of the total
selenium indicating a large part of the selenium in the worms was proteinaceous and less
bioavailable. This large portion of insoluble selenium in the worms was likely the reason
less selenium was accumulated in ESI and ESS Treatment 3 relative to ES2.
29
-------
3.3 SURVIVAL ANALYSIS OF JUVENILE BLUEGILL SUNFISH
Estimates of juvenile bluegill survival take into account the removal of individuals from
the test population during the experiment. Individuals were removed for sampling tissue
concentrations, or because they suffered accidental deaths unrelated to selenium toxicity.
Removal offish from the test reduces the number of individuals at risk of mortality due
to selenium toxicity. The time when fish are removed (e.g., the number of days after the
experiment started) is informative, because it reveals the period over which the removed
fish remained alive. Ignoring removed fish will result in inaccurate estimates of survival
(S). For instance, consider a hypothetical example where 100 fish are exposed to
selenium for 300 days; 50 die due to selenium toxicity and 50 are removed the day before
the test ends. Disregarding the time when fish are removed would lead to S = 0.0, while
proper acknowledgment offish removal time would result in S ~ 0.5.
At each of four sampling dates (day 7, 30, 60, and 1 12), nine juvenile bluegills were
removed for measuring tissue concentrations of selenium. Therefore, over the duration of
the experiment, 36 fish were removed from each tank (from a total of 100). The total
number offish removed from each tank ranged from 36 to 37 in ESI (9 and 29 in
controls), to 36 to 39 in ES3 (18 in controls). A mechanical malfunction on day 159
caused 23 deaths unrelated to selenium toxicity in Treatment 5 of ESI (total number of
fish removed: 36 + 23 = 59). The 23 fish were lost through the opening to the outflow
pipe located at the bottom of the ESI Treatment 5 tank due to the dislodging of the screen
covering the opening.
If r(tj) is the number of individuals at risk just before time tt and dt is the number of
deaths in the interval, /, = [tf, ti+i), then survival (S) at time t can be estimated as
The product (FI) is calculated for each period in which one or more deaths occur.
Equation (II) is the Kaplan-Meier estimator (Venables and Ripley 2002). It was used to
calculate the proportion of survival in treatments with ten or more deaths (10%
mortality). Confidence intervals for survival estimates were based on Greenwood's
formula,
T-
Computations were performed with the survfit function in the R software version 2.6.0 (R
development core team 2007).
Substantial mortality (>50%) was observed in treatments where tissue concentrations of
selenium exceeded 11 |lg/g, which only occurred in ESI and ES3 Treatments 5 and 6
(Table 3.7). The timetables of deaths in these treatments, as well as respective estimates
30
-------
of survival are presented in Tables 3.8-3.11. The survival curves for these treatments are
illustrated in Figures 3.8-3.11. Juvenile bluegill survival in ESI Treatment 6 was similar
to survival in the corresponding ES3 exposure throughout the experiment, despite the fact
that concentrations of selenium in fish tissues in the latter treatment were consistently
higher, 15.1 vs. 12.7 |ig/g at day 60 (the last measurement in ESI Treatment 6).
Survival of fishes in Treatment 5, though, was lower in ES3 than in ESI. Concentrations
of selenium in tissues of these fish were similar up to day 112, at which time the
differences in survival between the exposure systems were already pronounced (0.93 in
ESI vs. 0.63 in ES3). Mortality in other ESI and ES3 Treatments (1 through 4) was very
low (Table 3.7; Appendix E); mortality did not exceed seven in any tank over the entire
duration of the experiment (182 days).
Table 3.7. Total number of deaths attributed to background mortality and selenium
toxicity in each treatment of ESI, ES2, and ESS (initial 7V=100) over the
experiment's duration (182 days). All three exposure systems (ESI, ES2, ESS) had
two control tanks. The ES2 treatment with a target diet concentration of 5 jig Se/g
dw also had two replicates.
Treatment
Control (#1, #2)
1
2
3
4
5
6
ESI
0,7
5
1
0
O
24
68
ES2
0,0
0,2
ES3
1, 1
0
1
0
3
38
61
The estimate of survival for control B in ESI (S = 0.924) did not raise concerns about
excessive mortality, because there was zero in control A. No deaths occurred in ES2
controls. In the ES2 Treatment, two fish died in Treatment 5B, and none in Treatment
5A.
31
-------
Table 3.8. Timetable of deaths and respective estimates of fraction survival for ESI
Treatment 6. All survival values projected by the Kaplan-Meier estimator
time no. at no.
(day)a risk deaths
43
44
45
46
47
51
55
56
57
58
59
60
61
62
63
65
66
68
72
73
74
76
80
81
84
82
78
69
67
65
64
62
61
60
58
54
51
40
37
36
33
22
21
20
18
15
12
11
10
8
4
9
2
2
1
2
1
1
2
4
3
2
O
1
3
11
1
1
2
O
O
1
1
2
O
fraction
survival
0.951
0.842
0.817
0.793
0.781
0.756
0.744
0.732
0.707
0.659
0.622
0.598
0.553
0.538
0.493
0.329
0.314
0.299
0.269
0.224
0.179
0.164
0.149
0.120
0.075
std
error
0.024
0.040
0.043
0.045
0.046
0.047
0.048
0.049
0.050
0.052
0.054
0.054
0.056
0.056
0.057
0.056
0.055
0.055
0.053
0.050
0.046
0.045
0.043
0.039
0.032
low
95%CI
0.906
0.766
0.738
0.710
0.696
0.669
0.655
0.642
0.615
0.564
0.525
0.500
0.453
0.438
0.393
0.236
0.222
0.209
0.183
0.145
0.108
0.096
0.085
0.063
0.032
up
95%CI
0.999
0.924
0.905
0.885
0.875
0.855
0.845
0.834
0.813
0.770
0.736
0.714
0.674
0.660
0.619
0.458
0.443
0.427
0.396
0.347
0.297
0.280
0.263
0.228
0.173
Mortality counts were checked and recorded daily. The number at risk, number
deaths and survival reflect the timing of the fish deaths. For example, on day 47,
one fish was observed dead, no fish were observed dead on days 48 through 50
and 2 fish were found dead on day 51.
32
-------
oq
CD
CNI
CD
p
CD
ES1-Treatment 6
20
40
I
60
I
80
Days
Figure 3.8. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The "+"
sign indicates dates when data were censored.
33
-------
Table 3.9. Timetable of deaths and respective estimates of fraction survival for ESI
Treatment 5. All survival values projected by the Kaplan-Meier estimator.
time no. at no.
(day)a risk deaths
90
93
94
95
130
150
153
155
157
158
160b
161
169
177
73
72
71
69
59
58
57
56
55
52
27
26
25
18
1
1
2
1
1
1
1
1
3
2
1
1
7
1
fraction
survival
0.986
0.973
0.945
0.932
0.916
0.900
0.884
0.868
0.821
0.789
0.760
0.731
0.526
0.497
std
error
0.014
0.019
0.027
0.030
0.033
0.036
0.039
0.041
0.047
0.050
0.056
0.061
0.079
0.080
low
95%CI
0.960
0.936
0.894
0.875
0.853
0.832
0.811
0.791
0.734
0.697
0.657
0.620
0.392
0.363
up
95%CI
1.000
1.000
0.999
0.991
0.983
0.973
0.963
0.953
0.919
0.894
0.879
0.861
0.706
0.681
Mortality counts were checked and recorded daily. The number at risk, number
deaths and survival reflect the timing of the fish deaths. For example, on day 161,
one fish was observed dead, no fish were observed dead on days 162 through 168
and 7 fish were found dead on day 169.
The 23 fish accidentally lost from the tank due to the screen from the outflow tube
being dislodged were accounted for using the Kaplan-Meier estimator (Equation
II).
34
-------
oq
CD
CNI
CD
p
CD
ES1-Treatment 5
50
100
150
Days
Figure 3.9. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The "+"
sign indicates dates when data were censored.
35
-------
Table 3.10. Timetable of deaths and respective estimates of fraction survival for ESS
Treatment 6. All survival values projected by the Kaplan-Meier estimator.
time no. at no.
(day)a risk deaths
36
43
45
46
47
48
49
61
65
66
67
68
70
71
72
73
74
75
76
77
78
80
81
84
86
88
90
91
93
95
82
81
78
77
75
72
71
61
59
57
55
54
50
48
46
41
37
34
33
30
29
26
25
24
23
21
20
19
16
13
1
3
1
2
3
1
1
2
2
2
1
4
2
2
5
4
3
1
O
1
3
1
1
1
2
1
1
O
3
1
fraction
survival
0.988
0.951
0.939
0.915
0.878
0.866
0.854
0.826
0.798
0.770
0.756
0.700
0.672
0.644
0.574
0.518
0.476
0.462
0.420
0.406
0.364
0.350
0.336
0.322
0.294
0.280
0.266
0.224
0.182
0.168
std
error
0.012
0.024
0.026
0.031
0.036
0.038
0.039
0.043
0.045
0.048
0.049
0.053
0.054
0.056
0.058
0.058
0.059
0.058
0.058
0.058
0.057
0.056
0.056
0.055
0.054
0.053
0.052
0.049
0.046
0.044
low
95%CI
0.964
0.906
0.889
0.856
0.810
0.795
0.780
0.746
0.713
0.681
0.665
0.603
0.573
0.544
0.471
0.415
0.374
0.360
0.320
0.307
0.268
0.256
0.243
0.230
0.205
0.193
0.181
0.146
0.111
0.100
up
95%CI
1.000
0.999
0.992
0.977
0.952
0.943
0.934
0.913
0.892
0.870
0.858
0.811
0.787
0.762
0.699
0.646
0.605
0.592
0.550
0.536
0.493
0.479
0.464
0.450
0.420
0.405
0.390
0.344
0.297
0.281
Mortality counts were checked and recorded daily. The number at risk, number
deaths and survival reflect the timing of the fish deaths. For example, on day 49,
one fish was observed dead, no fish were observed dead on days 50 through 60
and 2 fish were found dead on day 61.
36
-------
oq
CD
CNI
CD
p
CD
ES3-Treatment 6
20
\
40
I
60
Days
80
100
Figure 3.10. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The "+"
sign indicates dates when data were censored.
37
-------
Table 3.11. Timetable of deaths and respective estimates of fraction survival for ESS
Treatment 5. All survival values projected by the Kaplan-Meier estimator.
time no. at no.
(day)a risk deaths
78
88
90
91
95
101
106
107
109
115
122
123
130
143
150
157
160
169
171
73
71
68
66
60
59
50
47
46
36
35
34
33
31
30
29
28
27
26
2
3
2
5
1
9
O
1
1
1
1
1
2
1
1
1
1
1
1
fraction
survival
0.973
0.932
0.904
0.836
0.822
0.696
0.655
0.641
0.627
0.609
0.592
0.574
0.540
0.522
0.505
0.487
0.470
0.453
0.435
std
error
0.019
0.030
0.035
0.043
0.045
0.054
0.056
0.057
0.057
0.058
0.059
0.060
0.061
0.061
0.062
0.062
0.062
0.062
0.062
low
95%CI
0.936
0.875
0.839
0.755
0.738
0.598
0.554
0.539
0.525
0.506
0.487
0.469
0.433
0.415
0.397
0.380
0.363
0.346
0.329
up
95%CI
1.000
0.991
0.974
0.925
0.914
0.811
0.774
0.761
0.749
0.734
0.719
0.704
0.673
0.658
0.642
0.625
0.609
0.593
0.576
Mortality counts were checked and recorded daily. The number at risk, number
deaths and survival reflect the timing of the fish deaths. For example, on day 95,
one fish was observed dead, no fish were observed dead on days 96 through 100
and 9 fish were found dead on day 101.
38
-------
oq
CD
CNI
CD
p
CD
ES3-Treatment 5
50
100
150
Days
Figure 3.11. Survival curve of juvenile bluegill exposed to selenium. Dashed lines
represent the 95% confidence interval for estimates of survival (solid line). The "+"
sign indicates dates when data were censored.
3.3.1 Overlay of Survival and Bioaccumulation Plots
Plots of selenium bioaccumulation and fraction survival (S) of juvenile bluegill overtime
were overlaid for estimating the concentration of selenium associated with the onset of
mortality (S< 0.9). Figures 3.12 - 3.15 display observed and projected concentrations of
selenium in fish tissues, as well as Kaplan-Meier estimates of survival over the duration
of the experiment (182 days), or until all of the fish had died.
39
-------
ESI - Treatment 6
Survival of juvenile bluegill was 0.95 at day 43 and 0.84 at day 44 (Fig. 3.12). At day 43,
the concentration of selenium in fish tissues was estimated as 11.58 |ig/g dw.
ES1 -Treatments
I
Day
Figure 3.12. ES 1 Treatment 6 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival.
ESI - Treatment 5
Survival of bluegill was 0.90 at day 150 and 0.88 at day 153 (Fig. 3.13). At day 151, the
concentration of selenium in fish tissues was estimated as 11.10 |ig/g dw.
ES1 - Treatment 5
100
Day
Figure 3.13. ESI Treatment 5 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival.
40
-------
ESS - Treatment 6
Survival of juvenile bluegill was 0.92 at day 46 and 0.88 at day 47 (Fig. 3.14). At day
46, the concentration of selenium in fish tissues was estimated as 13.83 |ig/g dw.
ESS - Treatment 6
20 40
80 100
Day
Figure 3.14. ES3 Treatment 6 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival.
ES3 - Treatment 5
Survival of bluegill was 0.90 at day 90 and 0.84 at day 91 (Fig. 3.15). At day 90, the
concentration of selenium in fish tissues was estimated as 11.09 |ig/g dw.
ESS - Treatment 5
-0.4 W
Day
Figure 3.15. ES3 Treatment 5 overlay of increasing selenium accumulation (measured
points and fitted asymptotic curve), and decreasing fraction survival.
41
-------
3.3.2 Estimates of Effect Concentrations
Effect concentrations (EC) for selenium were projected by rearranging a logistic model
that quantified the proportion of juvenile bluegill survival as a function of selenium
concentration in fish tissues. The TRAP software (U.S. EPA 2002) was used to fit the
logistic equation and Kaplan-Meier estimates of effect concentrations. Survival was our
selected endpoint because we could estimate it while taking censored data into account;
fishes removed from the experiment for reasons other than selenium toxicity. Analysis of
the concentration effect was based on the concentrations of selenium in fish determined
by several approaches. The first approach used selenium concentrations in fish measured
at the last day of the experiment, or at the last day when tissue samples were collected
before no fish were left in the tank (day 60 for ESI Treatment 6 and day 112 for ESS
Treatment 6). If the final measurement of selenium concentrations precedes much of the
mortality, as observed in Treatment 6 of ESI (final measurement at day 60, Fig. 3-12),
then estimates of effect concentrations are likely to be biased low. The second approach
used the average of the last two measurements for ESI Treatment 5 and a calculated
concentration for ESI Treatment 6. Averaging the last two Treatment 5 values was done
because one appears high (day 112 value is above the modeled line) and one low (day
182 value is below the line) (Figure 3.13). The last measurement for ESI Treatment 6
value is day 60 although fish survived through day 84; Equation I was used to estimate a
selenium concentration for day 84. The third approach used the nonlinear regression
Equation I to calculate all treatment values for ESI and ES3. The following data were
entered in the TRAP software:
Treatment
Control
1
2
3
4
5
6
ECio
(95% CL)
EC20
(95% CL)
surv
0.962
0.988
0.984
1
0.962
0.497
0.075
ESI
[Se]tissue, Mg/g dw
last meas.a avg T5, calc T6b calc all0
2.08
2.56
3.15
4.02
6.72
10.71
12.66
9.27
(8.86-9.69)
9.78
(9.49-10.09)
2.08
2.56
3.15
4.02
6.72
11.16
13.59
9.40
(8.92-9.91)
10.02
(9.67-10.39)
2.08
2.83
3.22
4.05
6.72
11.25
13.59
9.56
(9.09-10.05)
10.16
(9.81-10.52)
surv
0.988
1
0.988
1
0.96
0.435
0.168
ES3
[Se]tissue, Mg/g dw
last meas.a calc all0
1.67
3.2
3.83
5.48
9.38
16.01
17.24
14.00
(13.40-14.62)
14.64
(14.19-15.11)
1.67
3.05
3.68
4.92
9.22
15.63
17.09
13.29
(12.61-14.00)
14.02
(13.50-14.56)
Last measured selenium concentration
The Treatment 5 value is the average of the last two measured values. Treatment 6 was
calculated using equation (I) described in Section 3.2.2,
All treatment values were calculated using equation (I) for the final day of the particular
treatment. The final day of treatment was day 182 for all treatments except Treatment 6
which was day 85 for ESI Treatment 6 and day 1 12 for ES3 Treatment 6.
42
-------
A plot of the proportion of juvenile bluegill survival as a function of the logarithm of
selenium concentration using the last measured concentration (Fig. 3.16) in ESS reveals
very low mortality up to 10 |ig/g (log([Se]tiSSUe) = 1-0) and a steep decline in survival at
concentrations above it. Consequently, the EC20, 14.64 |ig/g (14.19 - 15.11 |ig/g, 95%
confidence interval) was similar to the ECio, 14.00 |ig/g (13.41 - 14.62 |lg/g). Analysis
of the calculated values yielded a similar relationship although because the estimated
concentrations were slightly lower than the last measured values, the EC values were also
slightly lower: EC20 = 14.02 |ig/g (13.50-14.56 |ig/g); ECio = 13.29 |ig/g (12.61-14.00
1.2r
1.0
o
TO
"cc
'E
CO
.4 .6 .8 1.0
Log(Se in bluegill, |jg/g dw)
1.2
1.4
Figure 3.16. Survival of juvenile bluegill as a logistic function of the logarithm of the
final selenium concentration in fish tissues. Concentration-survival curve for ESS.
Similar results were obtained for the analysis offish survival as a function of selenium
concentration in ESI based on the first approach (last measured values) (Fig. 3.17) as
well as approaches two and three. In ESI though, the logistic model projects a steep
decline in survival at a lower threshold concentration of selenium in fish tissues. Not
surprisingly then, the projected EC20 and ECio values for all three approaches (see table
above) were lower than correspondent values for ES3.
EC 10s (as well as EC20s) calculated using the different ways of estimating exposure
differed by only a few percent, (see table for EC values). The EC values determined
from the calculated values are considered the best estimates because they represent an
integration of all the measured values during the exposure period.
43
-------
1.2
1.0
o
05
^L .6
cc
>
I -4
.4 .5 .6 .7 .8 .9 1.0 1.1
Log(Se in bluegill, |jg/g dw)
1.2
Figure 3.17. Survival of juvenile bluegill as a logistic function of the logarithm of the
final selenium concentration in fish tissues. Concentration-survival curve for ESI.
The ECioS determined by the conventional concentration-survival analysis using TRAP
were supported by the concentrations observed to represent the onset of mortality when
the survival data was overlain with the accumulation data. The onset of mortality
concentrations at the 10% effect level were within 20% of the associated ECio values for
ESI and ESS (see table below).
Method
TRAP, concentration-
survival
Onset of mortality
(10% effect)
ECio
Treatment 5
Treatment 6
ESI
9.56 |ig/g
11.10 ng/g
11.58|ig/g
ESS
13.29 ,ig/g
11.09 |ig/g
13.83 |ig/g
The concentration-survival analysis indicated selenium is 39% more toxic to the bluegill
when the temperature approaches 4°C (ECio = 9.56 |ig/g) compared to 9°C (ECio = 13.29
|ig/g). This difference was less clear when looking at the concentrations determined by
the onset of mortality. The ES3 Treatment 6 estimate of effect (13.83 |ig/g) followed the
same trend of decreased sensitivity at the higher temperature, but the ES3 Treatment 6
estimate (11.09 jig/g) was nearly the same as the two ESI estimates (T5 = 11.10 jig/g; T6
= 11.58 |ig/g). The distinction between the temperature regimes is more apparent in a
comparison of tissue concentrations of selenium in the fish and their associated
mortalities. There was zero to 4% mortality through Treatment 4 in both ESI and ES3,
however, selenium reached 9.38 |ig/g dw in the warmer ES3, whereas it only reached
44
-------
6.72 |ig/g dw in ESI (see table in Section 3.2.2). The number of mortalities increased
markedly in Treatment 5 with comparable levels of 50% in ESI and 56.5% in ESS.
However, the last measured selenium concentrations in the fish were not comparable:
10.71 |ig/g dw in ESI and 16.01 |ig/g dw in ESS. The same pattern of greater sensitivity
to selenium in the colder exposure system was observed in Treatment 6 where a tissue
concentration of 12.66 |ig/g dw killed 92.5% of the fish in ESI and the higher
concentration 17.24 |ig/g dw killed 82.5%.
The concentration-effect analysis was considered to be a better assessment of the effect
of selenium coupled with temperature on the bluegill test population because it used a
wider set of data to estimate effect. Also, the onset-of-mortality approach compares
increasing accumulation with increasing mortality within each treatment during the
course of the test; the conventional application of TRAP compares the end-of-test results
between treatments.
Although both approaches assume no delayed mortality, the Onset of Mortality is more
sensitive to the assumption than TRAP's use of the end-of-results. This is because the
selenium concentrations were rising at a greater rate at the onset of 10% mortality, and
the approach assumes that death is caused by the concentrations occurring at the time of
death, not the concentrations occurring say 20 days earlier.
3.4 GROWTH, LIPID ANALYSIS, AND BEHAVIOR OF JUVENILE
BLUEGILL SUNFISH
Growth of juvenile bluegill was not negatively affected by the selenium exposures used
in the study. Within each system, the length and weight of the fish did not show a
decreasing trend as the exposure concentrations increased (Tables 3.12 and 3.13;
Appendix F). Growth was greater in ES2 and ES3 than in ES1. The greater growth in
ES3 can be explained by continued active feeding by the bluegill throughout the 182 day
exposure. The fish contained in ESI fed minimally once the temperatures reached 5°C.
Even though ES2 fish exhibited the same decrease in feeding activity, their length and
weight were greater than fish in ESI, the other 4°C exposure.
The average body condition factor, K, showed a similar lack of response to selenium
exposure concentration (Table 3.14). K values tended to increase during the exposure
period.
The lipid content of the bluegill did not decrease during the 182 day exposure. The
percent lipid values measured in the bluegill upon receipt at the laboratory and on test
day 0 were 2.51% and 3.04%, respectively. These initial values were similar to the
values measured in control and each treatment fish in ESI after 182 days of exposure
(Table 3.15; Appendix G). Lipid values for the fish treated in ES2, and for ES3 fish in
Treatments 3, 4, and 5 appeared to slightly increase over the exposure period.
The fish in the two colder exposure systems, ESI and ES2, displayed similar feeding
behavior. Bluegill actively fed on Tetramin in the control and treatments in ES2 through
test day 77 as the temperature approached 5°C, after which feeding was minimal. A
45
-------
marked reduction in the consumption of Lumbriculus in ESI was observed in fish in the
control and Treatments 1 through 5 on test days 81 to 83. The bluegill in ESI Treatment
6 reduced their feeding behavior earlier, on test day 53, presumably due to effects of the
selenium exposure. Erratic swimming was also observed in ESI Treatment 6 of a few
fish on test day 54.
Since the temperature in ES3 did not reach 5°C, the temperature at which feeding was
reduced in ESI and ES2, the feeding behavior offish in the controls and Treatments 1
through 4 in ES3 was active throughout the entire 182 day exposure. The feeding activity
in ES3 Treatments 5 and 6 was noticeably reduced on test days 75 and 74 apparently due
to selenium exposure.
3.5 COMPARISON OF RESULTS BETWEEN LEMLY AND CURRENT
STUDIES
A comparison of Lemly's cold treatment plus selenium to EPA's ES2 results is the most
direct evaluation of the two studies. Although both studies exposed juvenile bluegill to
nominal selenium concentrations of 5 |ig/L in the water and 5 |ig/g in TetraMin, there
were differences. Lemly began his temperature decline of 2°C per week at the start of the
test whereas the current study maintained 20°C for 30 days prior to initiating the
temperature decline. Lemly reached 4°C, and the current study reached 4.6°C. The
current study used 2 replicates with 100 fish/replicate and Lemly used 3 replicates with
70 fish in each replicate tank. Lemly measured oxygen consumption on 15 fish randomly
selected from each treatment and control on test days 60, 120 and 180; oxygen
consumption was not measured in the current study. The oxygen consumption
measurement required transferring the selected fish to a separate chamber and then
reintroducing the fish back to the exposure tank. Lastly, EPA's test duration was 182
days and Lemly's was 180 days. Of these differences, we expect only the first to have
any possible significant effect on the results.
A comparison of the accumulation of selenium in the bluegill in these treatments from
these two studies suggests that at first the current study fish accumulated more during the
respective exposure periods. Lemly's fish accumulated 5.85 jig/g after 60 days and 7.91
|ig/g by the end of the 180 days. The bluegill in the current study accumulated
approximately 8 jig/g of Se after 60 days and 10 jig/g after 182 days. A closer look at
three exposure conditions may explain these differences. First, the background
concentration of selenium in bluegill was 1 |ig/g in Lemly's fish and approximately 2
|ig/g in the current study fish. Second, the measured concentration of selenium in the
TetraMin was 16% higher in ES2 at a time-weighted average concentration of 6.01 |ig/g
dw; Lemly's TetraMin was measured at 5.16 jig Se/g dw. The difference was even
greater when considering the last two batches of TetraMin that were fed to the bluegill
during test days 72-182 in the current study were progressively higher in selenium
concentration than the first two. The time-weighted average selenium concentration in
last two TetraMin batches was 6.46 |ig/g dw, 25% higher than Lemly's diet. The ratio of
selenium in fish to selenium in diet between the two studies is comparable: Lemly ratio =
1.53; current study ratio using the time-weighted average of the last two TetraMin
batches = 1.55. The third consideration that may explain the difference in Se
accumulation is the longer exposure period at 20°C in ES2. After considering the above,
46
-------
the accumulation of selenium in the two studies was similar, but the fish in the current
study contained more selenium at the beginning, middle and end of the test.
Although the accumulation of selenium was similar between the two studies, there was a
difference in survival offish. Lemly observed 40% mortality by the end of the 180 days
whereas no meaningful mortality was observed in ES2. This difference is larger when
considering the mortalities occurred in Lemly's fish when selenium concentrations in the
bluegill increased from 5.85 to 7.91 |ig/g compared to no effect on survival up to 10 |ig/g
in ES2. The latter observation of no mortality at 10 |ig/g is consistent with the results
from ESI and ESS which saw no meaningful effects on survival until the selenium
concentrations in the bluegill approached 11 |ig/g.
As stated above, Lemly removed 15 fish from each treatment for oxygen consumption
measurement and then returned these fish to the exposure tanks. There is the possibility
that the fish removed from the cold plus selenium treatment were sufficiently stressed by
the exposure conditions that the additional handling stress contributed to the mortality
observed in this treatment. Between test days 60 and 180, 56 fish died Lemly's cold plus
selenium treatment. Even if stress due to handling affected all the fish used in the oxygen
consumption measurements (up to 30 fish), it does not explain all the mortality that was
observed and therefore does not explain the difference between the two studies.
Lemly found meaningful decreases in body condition factor, K, and lipid content in his
cold plus selenium treatment. K decreased from 4 at the start of the test to 2.2 by the end
with the most dramatic decrease occurring between days 60 and 120. In contrast, the
average condition factor of the bluegill in ES2 was 3.2 at test initiation and 5.3 at test
termination. A similar comparison was observed with the measurement of lipid content
of the bluegill in the two studies. Note: Lemly determined lipid based on dry weight. In
order to make a direct comparison, Lemly's lipid values were converted to wet weight
assuming the fish were 75% moisture. The percent lipid at the start of Lemly's 180 day
exposure was 3.25 and decreased to 1.5 by the end of the test. The lipid content in the
fish in ES2 did not decrease as evidenced by 3% at test start and 3.5% at test end.
In summary, the direct comparison between the results of the current study's ES2 and
Lemly's cold plus selenium treatment shows similarity in the accumulation of selenium
in the bluegill, but a meaningful difference in the toxicity of selenium. Lemly's fish
displayed toxicity to selenium at concentrations 2 to 4 |ig/g dw lower than the current
study. The difference in toxicity is apparently also reflected in the difference observed in
the body condition factor, K, of the two test populations. K increased in the current study
over the exposure period, whereas K decreased in Lemly's fish.
A comparison can be made between Lemly's cold plus selenium and the current study's
Treatment 3 in ESI and ES3. The exposure conditions in the latter tests were nominal 5
|ig/L in the water and average measured selenium concentrations 7.47 and 7.17 |ig/g in
Lumbriculus in ESI and ES3. Selenium in the bluegill in ESI appeared to reach steady-
state around 4 |ig/g compared to around 8 |ig/g in Lemly's study.
The fish in the warmer ES3 did not appear to reach steady-state; the whole body selenium
concentration on day 182 was 5.5 |ig/g. As discussed at the end of Section 3.2, the
47
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apparent difference in selenium accumulation is due to the form of selenium in the diet.
The TetraMin contained the more bioaccumulative seleno-L-methionine whereas
Lumbriculus contained a mixture of selenium species with some not as bioaccumulative
as seleno-L-methionine.
The toxicity of selenium to the bluegill in Lemly's cold plus selenium and ESI can be
compared by an examination of when meaningful toxicity occurred in the fish. Lemly's
fish had a sharp increase in mortality after day 60 when mortality went from 5 to 10%
over a couple of days. The concentration of selenium in Lemly' s fish was approximately
6 |ig/g during that period. As discussed in Section 3.3.1, survival of bluegill in ESI
Treatment 6 decreased from 95 to 84% over days 43 and 44 when the bluegill selenium
concentration was 11.6 |ig/g. A similar concentration in bluegill (11.1 |ig/g) was
observed in ESI Treatment 5 at the point where survival dropped below 90%. The
relative difference between these two threshold values, that is, the selenium concentration
determined by the Onset of Mortality in the current study (average of ESI and ES3 =
11.4 |ig/g dw) divided by concentration of selenium in Lemly' s fish when mortality
increased from 5 to 10% (6 |ig/g dw) is 1.9.
Similar to that discussed above in the direct comparison between ES2 and Lemly's cold
plus selenium treatment, the body condition factor in ESI and ES3 did not decrease over
the exposure duration as did Lemly's fish (Table 3.14). There was less of an increase in
K over the exposure period in ESI and ES3 than there was with ES2 fish but it was still
markedly different than the decrease from 4 to 2.2 in K observed in Lemly's fish. Since
K is a reflection of the overall health of the bluegill, it directly relates to the differences
observed in the toxicity of selenium in the two studies.
48
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Table 3.12. Average standard lengths (mm) in bluegill based on samples taken for
chemical analysis; N = 9 for each average value.
system
ESI
ESI
ESI
ESI
ESI
ESI
ESI
ESS
ESS
ESS
ESS
ESS
ESS
ESS
ES2
ES2
ES2
treatment
control
1
2
3
4
5
6
control
1
2
3
4
5
6
control
5A
5B
0
47
47
47
47
47
47
47
47
47
47
47
47
47
47
47
47
47
7
49
48
49
49
49
48
50
48
50
49
48
50
49
47
51
50
48
exposure day
30 60 112
48
47
50
49
50
51
49
51
50
48
48
46
48
45
53
52
54
49
50
49
51
50
50
52
46
50
53
49
50
47
48
59
55
55
49
53
50
50
51
50
51
52
51
50
50
54
51
60
54
57
182
53
51
51
51
51
52
56
56
55
55
56
57
56
60
57
Table 3.13. Average weights (g) in bluegill based on samples taken for chemical
analysis; N = 9 for each average value.
system
ESI
ESI
ESI
ESI
ESI
ESI
ESI
ESS
ESS
ESS
ESS
ESS
ESS
ESS
ES2
ES2
ES2
treatment
control
1
2
3
4
5
6
control
1
2
3
4
5
6
control
5A
5B
0
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
7
1.81
1.58
1.70
1.63
1.62
1.56
1.75
1.69
1.73
1.67
1.57
1.71
1.66
1.51
2.00
1.79
1.65
exposure day
30 60
1.59
1.35
1.71
1.65
1.78
1.79
1.54
1.72
1.54
1.48
1.48
1.25
1.50
1.32
2.19
2.26
2.63
1.74
1.85
1.88
1.90
1.74
1.83
2.31
1.24
1.74
2.20
1.75
1.67
1.39
1.68
3.34
2.93
2.83
112
1.67
2.05
1.94
1.97
2.07
1.99
1.95
2.08
1.86
1.96
2.09
2.73
2.38
3.57
2.81
2.85
182
2.38
1.89
2.13
2.13
2.00
2.08
2.37
2.86
2.29
2.25
2.75
3.64
2.76
3.32
2.92
49
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Table 3.14. Average body condition factor (K)* bluegill based on samples taken for
chemical analysis; N = 9 for each average value.
system
ESI
ESI
ESI
ESI
ESI
ESI
ESI
ESS
ESS
ESS
ESS
ESS
ESS
ESS
ES2
ES2
ES2
*K = (100
treatment
control
1
2
3
4
5
6
control
1
2
3
4
5
6
control
5A
5B
0
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
3.21
7
3.67
3.32
3.51
3.35
3.33
3.28
3.51
3.50
3.45
3.39
3.24
3.44
3.41
3.21
3.94
3.60
3.43
exposure day
30 60
3.30
2.89
3.45
3.37
3.56
3.50
3.17
3.35
3.09
3.08
3.11
2.70
3.13
2.92
4.11
4.38
4.87
3.51
3.69
3.83
3.73
3.49
3.64
4.42
2.72
3.52
4.17
3.56
3.32
2.97
3.50
5.69
5.32
5.17
112
3.39
3.89
3.86
3.98
4.08
3.96
3.85
3.98
3.66
3.92
4.16
5.03
4.70
5.94
5.18
5.00
182
4.49
3.73
4.13
4.19
3.90
4.00
4.27
5.08
4.17
4.11
4.92
6.34
4.93
5.53
5.12
x weight (g))/standard length
50
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Table 3.15. Lipid content (%) in juvenile bluegill at the start and end of the
exposure period.
system
NA
NA
ESI
ESI
ESI
ESI
ESI
ESI
ESI
ES2
ES2
ES2
ES2
ESS
ESS
ESS
ESS
ESS
ESS
ESS
treatment
Arrival offish at lab
test day 0
control -1
control -2
1
2
3
4
5
control -1
control -2
5A
5B
control -1
control -2
1
2
3
4
5
test day
-25
0
182
182
182
182
182
182
182
182
182
182
182
182
182
182
182
182
182
182
lipid
average
2.51%
3.04%
2.35%
2.67%
2.69%
2.05%
2.49%
2.67%
2.26%
5.79%
4.42%
4.04%
3.06%
2.88%
1.96%
2.74%
2.82%
4.24%
3.92%
3.74%
content
std. dev.
0.02%
0.04%
0.07%
0.07%
0.38%
0.03%
0.02%
0.01%
0.08%
0.01%
0.01%
0.02%
0.04%
0.11%
0.08%
0.02%
0.03%
0.03%
0.08%
0.00%
51
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4.0 SUMMARY
The goal of the 182-day exposure to juvenile bluegill sunfish was to determine tissue-
based effect levels for selenium exposure over a simulated winter season at two
temperature regimes, 20 to 4°C and 20 to 9°C. The following bullets summarize the
findings:
• Juvenile bluegill sunfish appear to be more sensitive to selenium in waters
reaching 4-5°C than 9°C. The EC20 and ECio estimates for the exposure in which
temperature decreased from 20 to near 4°C were 10.16 and 9.56 jig/g dw,
respectively, while the EC20 and ECio estimates for the exposure that began at
20°C and systematically lowered to 9°C were 14.02 and 13.29 |ig/g dw,
respectively.
• The accumulation of selenium in the juvenile bluegill was affected by the form of
selenium in the diet of the fish. Under a similar temperature regime and exposure
period, bluegill receiving an artificial diet spiked with seleno-L-methionine (ES2
treatments 5A and 5B) accumulated 2.5 times the selenium accumulated by
bluegill receiving a natural diet of selenium accumulated inL. variegatus (ESI
Treatment 3).
• The accumulation of selenium in the juvenile bluegill was affected by
temperature. Fish exposed to dietary selenium via L. variegatus accumulated up
to 39% more selenium in the 20 to 9°C regime than in the 20 to 4°C regime.
• The accumulation characteristics of seleno-L-methionine in juvenile bluegill in
the current study were similar to that observed in Lemly's study.
• The toxicity of selenium to juvenile bluegill was approximately 1.9 times less in
the current study than that observed in Lemly's study.
• The juvenile bluegill in the current study did not decrease in body condition factor
and lipid content as they did in the Lemly study.
5.0 REFERENCES
Beckman, W. C. 1948. The length-weight relationship, factors for conversions between
standard and total lengths, and coefficients of condition for seven Michigan fishes.
Transactions of the American Fisheries Society 75:237-256.
Besser, J.M., W.G. Brumbaugh, J.L. Kunz, C.G. Ingersoll. 2006. Preparation and
characterization of selenium-dosed oligochaetes for dietary toxicity studies. Poster
presented at Annual Meeting of Soc. Environ. Toxicol. Chem. Montreal, Canada.
Fan T.W.-M., SJ. Teh, D.E. Hinton, andR.M. Higashi. 2002. Selenium
biotransformations into proteinaceous forms by foodweb organisms of selenium-
laden drainage waters in California. Aquatic Toxicol. 57:65-84.
Lemly, A.D. 1993. Metabolic stress during winter increases the toxicity of selenium to
fish. Aquat. Toxicol. (Amsterdam) 27(1-2): 133-158.
52
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Teh, S.J., X. Deng, D-F Deng, F-C Teh, S.S.O. Hung, T.W. Fan, J. Liu, R.M. Higasi.
2004. Chronic effects of dietary selenium on juvenile Sacramento splittail
(Pogonichthys macrolepidotus). Environ. Sci. Technol. 38: 6085-6593.
U.S. EPA. 2001. Toxicity relationship analysis program (TRAP v.1.10). U.S.
Environmental Protection Agency, National Health and Environmental Effects
Research Laboratory, Midcontinent Ecology Division, Duluth, Minnesota, USA.
Venables, W.N. and B.D. Ripley. 2002. Modern applied statistics with S. Springer-
Verlag, New York, NY, USA.
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