x>EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 2O460
EPA/600/R-99/099
September 1999
Assessing Contaminant
Sensitivity of
Endangered and
Threatened Species:
Effluent Toxicity Tests
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EPA/600/R-99/099
September, 1999
Assessing Contaminant Sensitivity
Of Endangered and Threatened Species:
Effluent Toxicity Tests
by
F. James Dwyer1, Douglas K. Hardesty, Christopher E. Henke, Christopher G. Ingersoll,
David W. Whites, David R. Mount2, Christine M. Bridges
U.S. Geological Survey, Biological Resources Division
Columbia Environmental Research Center
4200 New Haven Road, Columbia, Missouri 65201
1 Current address: U.S. Fish and Wildlife Service, 608 East Cherry St.,
Room 200, Columbia, MO 65201
2Current address: U.S. Environmental Protection Agency, 6201 Congdon Blvd.
Duluth, MN 55804
EPA Project No. DW14936559-01-0
Project Officer, Foster L. Mayer, Jr.
Gulf Ecology Division,
Gulf Breeze, Florida 32561
U.S. Environmental Protection Agency
National Health and Environmental Effects
Research Laboratory
Gulf Ecology Division
Gulf Breeze, Florida 32561-5299
Printed on Recycled Paper
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Abstract
Toxicity tests using standard effluent test procedures were conducted (EPA 1994) with
Ceriodaphnia dubia and fathead minnows and four endangered fish species: bonytail chub (G//a
e/egans), Colorado squawfish (Ptychocheilus lucias), razorback sucker (Xyrauchen texanus) and Gila
topminnow (Poeciliopsis occidentalis). We conducted 7-d survival and growth studies with embryo-
larval fathead minnows and analogous exposures using the listed species. Survival and reproduction
were also determined with C. dubia. Tests were conducted with: 1) carbaryl; 2) ammonia; and 3) a
mixture of carbaryl, copper, 4-nonylphenol, pentachlorophenol, and permethrin.
The fathead minnow 7-d growth and-survival test appears to be a reliable estimator of effects to
the listed species used in this study. Additionally, the C. dubia survival and reproduction test was
generally more sensitive than any of the fish tested. When the listed species and fathead minnow
were different, the listed species was often less sensitive than the fathead minnow. However, other
studies have shown listed species to be similar to or slightly more sensitive than fathead minnows
when tested using effluent procedures. This study was conducted with fish species that have not
been typically tested so factors such as handling procedures, optimum feeding rates, optimum test
temperature, expected test to test variation and expected survival or growth have not been previously
documented, and therefore results of this study should be interpreted cautiously.
Our laboratory has evaluated only 10 aquatic vertebrate species (mostly fish) and there are over
90 fishes listed by the FWS. The database for fishes should be expanded to include additional
species from different areas of the United States. Amphibian population declines have been
recognized worldwide and the FWS has over 10 listed species, therefore, greater emphasis should
be placed on testing additional amphibian species. Additional testing is also needed to evaluate
sublethal effects of contaminants on listed species. Finally, other listed species including freshwater
mussels and other invertebrates should also be examined.
ill
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Notice
The U.S. Environmental Protection Agency through its Office of Research and Development
(funded and managed or partially funded and collaborated in) the research described here under EPA
Project No. DW14936559-01-0 to U.S. Geological Survey, Biological Resources Division, Columbia
Environmental Research Center. It has been subjected to the Agency's peer and administrative
review and has been approved for publication as an EPA document.
Acknowledgements
The authors thank Dr. Foster L. Mayer, Jr. of the Gulf Ecology Division, U.S. Environmental
Protection agency for his guidance and assistance in this project. We thank Eugene Greer for
culturing the test organisms and NileKemble, Eric Brunson, Jill Soener, and Heather Willman of the
Toxicology Branch of the Columbia Environmental Research Center for their assistance during this
project. We thank Tom Brandt of the San Marcos National Fish Hatchery and Technology Center,
Jerry Hamilton of the Blind Pony Missouri State Hatchery, Roger Hamman of the Dexter National Fish
Hatchery, and KirstaScherff of the Colorado Division of Wildlife for supplying organisms tested in this
study. We thank ICI Americas, Inc., and Rhodia, Inc. for donating technical grade material to be used
in testing. We also thank Charles Stephan (EPA, Duluth, MN), Anne Keller (EPA, Athens, GA) and
Linda Sappington (USGS, Columbia, MO) for their critical review of this report.
iv
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Table of Contents
Abstract iii
Acknowledgment iv
Introduction 1
Materials and Methods 1
Test Organisms 1
Chemical 2
Toxicity Tests 4
Statistical Analysis 4
Results 4
Carbaryl 4
Ammonia 5
Chemical Mix 5
Discussion 6
References 6
Appendixes
Appendix 1 Individual tests IC25s 8
Appendix 2 Summary of exposure water pH 9
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Introduction
The U.S. Clean Water Act (CWA) specifies "it is the
national policy that the discharge of toxic pollutants in toxic
amounts be prohibited" (Section 101(a)(3)). The CWA
provides an integrated approach to protection of aquatic
ecosystems through the development of water quality
criteria and the control of toxic discharges (National
Pollutant Discharge Elimination System - NPDES; 45 FR
33520). Programs designed to provide protection of
freshwater aquatic environments from toxic discharges
commonly include whole-effluent toxicity tests with the
cladoceran Ceriodaphnia dubia, fathead minnow
(Pimephales promelas), and algae (Selenastrum
capricornutum). The assumption is that results of toxicity
tests using these test species are protective of effects on
other organisms including endangered and threatened
(listed) species.
Surrogate species, such as cladocerans and fathead
minnows, are the typical freshwater organisms used in
standardized tests (EPA 1994). However, it is unknown if
the sensitivities of these species to contaminant exposure
represent the sensitivities of listed species. Biological
surveys of streams and rivers in states such as Ohio
indicate that effluent test protocols using standard
procedures might not adequately protect aquatic
ecosystems (Yoder 1989). NPDES permits often require
toxicity tests with effluents using embryo-larval fathead
minnows and Ceriodaphnia dubia. The objective of the
present study was to determine the degree of protection
afforded listed fish species through the use of standard
species in whole-effluent toxicity tests.
Seven-d water-renewal toxicity tests were conducted using
standard effluent test procedures (EPA 1994). Species
tested included C. dubia, fathead minnows and four
endangered fish species: bonytail chub (Gila elegans,
Family Cyprinidae), Colorado squawfish (Ptychocheilus
lucias, Family Cyprinidae), razorback sucker (Xyrauchen
texanus, Family Catostomidae) and Gila topminnow
(Poeciliopsis occidentalis, Family Poecillidae). These
species were previously evaluated in static-acute 96-h
toxicity tests (EPA 1995, Chapter 1). We conducted 7-d
survival and growth studies with embryo-larval fathead
minnows and analogous exposures using the listed
species. Effects on survival and reproduction of C. dubia
were also evaluated. Tests were conducted with: 1)
carbaryl; 2) ammonia; and 3) a mixture of carbaryl, copper,
4-nonylphenol, pentachlorophenol, and permethrin.
Materials and Methods
Test organisms
Bonytail chub, Colorado squawfish, razorback suckers,
Gila topminnows, fathead minnows, and C. dubia were
obtained from various government sources or from
Columbia Environmental Research Center (CERC) cultures
(Table 1).
Table 1. Source and age of test organisms used in toxicity tests.
Species
Scientific Name
Source
Age at Start of Test
Bonytail chub
Gila elegans
Colorado squawfish Ptychocheilus
lucius
Razorback sucker
Gila topminnow
Fathead minnow
Xyrauchen
texanus
Poeciliopsis
occidentalis
Pimephales
promelas
Ceriodaphnia
dubia
Dexter Fish Hatchery, Dexter,
NM
Dexter National Fish
Hatchery, Dexter, NM
Dexter National Fish
Hatchery, Dexter, NM
Adults obtained from Dexter
National Fish Hatchery,
Dexter, NM
CERC cultures
CERC cultures
Test 1-2 days post hatch
Test 2-7 days post hatch
Test 1-6 days post hatch
Test 2-5 days post hatch
Test 1-6 days post hatch
Test 2-7 days post hatch
Mix - < 24 h old
Ammonia - 3 groups <24 h,
< 48 h, and <72 h
< 24 h old
< 24 h old
------- An error occurred while trying to OCR this image.
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each batch of reconstituted water. Average measured
water quality characteristics for the reconstituted water are
summarized in Table 4. Because of age requirements for
fathead minnows at the start of the test (<24 h old), none
of the fish were acclimated to the test water before starting
toxicity tests. However, C. dubia were cultured in this
reconstituted hard water. Dissolved oxygen, temperature,
and pH were measured on the control, low, medium, and
high exposure concentrations daily in the fresh test solution
and in the test solution after 24 h of exposure for carbaryl
and the chemical mixture. Additionally, pH, temperature,
and dissolved oxygen were measured on all concentrations
of ammonia initially and after 24 h of exposure. The test
was conducted in ambient light with 16 hours of light and 8
hours of dark. A test series consisted of five exposure
concentrations with a 50% dilution factor. For toxicity tests
with fish, each exposure concentration was tested in
triplicate. Fish were counted into groups of five with two
groups pooled for each exposure replicate (10 fish per 1 L
beaker - 30 fish/treatment).
Table 2. Sources, percent active ingredient, use and mode of action for chemicals used in toxicity tests.
Chemical
Source
Active Ingredient
Use
Mode of
Action
Carbaryl
Copper sulfate
4-nonyIphenol
Donated by Rhone-Poulenc
Agricultural Co., Research
Triangle Park, NC
Fisher Chemical, St. Louis, MO
Fluka Chemical, New York, NY
Pentachlorophenol Aldrich Chemical, Milwaukee,
WI
Permethrin
Donated by ICI Americas Inc.,
Richmond, CA
99.7 Carbamate insecticide
25.5 Mining,
industrial,
fungicide
85.0 Nonylphenol
ethoxylate detergents
99.0 Wood preservative,
molluscicide
95.2 Pyrethroid
insecticide
Inhibitor of cholinesterase
activity
Interferes in osmoregulation
Narcotic and oxidative
stressor
Uncoupler of
oxidative
phosphorylation
Neurotoxin
Ammonium
Phosphate
EM Science,
Gibbstown, NJ
12 Fertilizer, ammonia is
a byproduct of waste-
water treatment plants
and some farming
practices
Interferes in respiration
Table 3. Summary of study design for the comparative toxicity of selected chemicals to listed species.
Test type:
Test temperature:
Water Quality:
Chemicals:
Dilution series:
Observations:
Renewal
Carbaryl: 22°C
Chemical mix and ammonia: 25°C
Reconstituted ASTM hard
Carbaryl
Ammonia
Chemical mix of carbaryl, copper, 4-nonylphenol, pentachlorophenol,
permethrin in equitoxic proportions as determined from previous tests
(EPA 1995).
50%
Fish: mortality every 24 h for 7 days and weight at end of study
Ceriodaphnia dubia: mortality daily and reproduction until 3 broods in
control.
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Table 4, Average (+ standard deviation) measured water
quality characteristics of reconstituted water used in
effluent toxicity tests.
Water Quality
Characteristic
Nominal Value
Measured (n = 11)
Alkalinity1
Hardness'
110-120
160-180
112 + 10
163 + 13
PH
7.8 - 8.0
8.3 + 0.6
1 mg/L as CaCO,
Toxicity tests
For the ammonia test, Gila topminnows produced over a
three-day period were used. Fish were kept in 24-h age
groups (0-24, 24-48, 48-72) and each age group period
was stocked in a separate replicate for each treatment.
For the mixture study with the Gila topminnow, enough
young were obtained in one 24-h period to stock two
replicates with nine fish per replicate. For fish, reduction in
survival or growth were the adverse effects measured.
Dead fish were removed daily. Tests were repeated (two
different years) with the razorback sucker, Colorado
squawfish and bonytail chub. There are data for only one
year for the Gila topminnow. A total of five separate tests
over the two-year period, were conducted with the fathead
minnows.
Ten C. dubia were tested in individual 30 ml_ beakers
containing 15 mL of test solution with one animal per
beaker. Survival and reproductive success were
determined daily for C. dubia and were continued until at
least 60% of the controls had a third brood (about 6 to 7 d).
There were three separate tests with C. dubia for each
chemical.
Statistical analysis
The Inhibition Concentration (ICp), integrating effects on
both growth and survival of fish, and survival and
reproduction of C. dubia, was calculated for each test using
a linear interpolation method (Norberg-King 1993). The
IC2S was used as the statistical point-estimate for this
study. For the fish, an expanded confidence interval, as
recommended in the ICp procedure, was calculated
because there were fewer than seven replicates for each
test. If the expanded lower confidence limit was less than
zero, then the lower confidence limit was reported as zero.
Confidence intervals for the Ceriodaphnia dubia were not
expanded because there were 10 replicates.
The IC25s could not be tested for normality due to an
insufficient number of IC25 estimates. Thus, general linear
model least square difference mean separations (SAS
1994) were determined on ranked IC25s (p < 0.05,
Snedecor and Cochran 1980). In order to summarize the
data for a particular chemical and species, the geometric
mean IC25 was calculated. Only those tests for which an
IC25 could be calculated were used for statistical analysis.
For the results with ammonia, if the total ammonia IC25 was
greater than 17, then 17 was used in the calculation.
Calculation in this manner will likely provide a concentration
lower (bias) than the actual concentration. Not including
the data (17) would bias the summarized data to a greater
extent than including the data.
Results
Appendix 1 is a complete listing of IC25s and confidence
intervals for all individual tests. Dissolved oxygen
concentrations were always at acceptable concentrations
(>40% saturation); therefore no tests were aerated. The
pH range for each test is listed in Appendix 2. Control
survival for all species and exposures was typically 80% or
greater. Exceptions included: 1) one dilution water control
in a fathead minnow carbaryl exposure, 2) both the
acetone and dilution water controls for Test 2 of the
bonytail chub carbaryl exposures, and 3) an ammonia
study with fathead minnow. In the fathead minnow-
carbaryl study and the bonytail study, survival was 70%
and data from those tests were included in the results for
the present study. However, the fathead minnow-ammonia
study had survival of only 50% in the control and therefore
was not included in the results for the present study.
Carbaryl
The lC25s for the Colorado squawfish (1.33 mg/L) and
razorback sucker (2.06 mg/L) were significantly greater
than the IC2S for the fathead minnow (0.42 mg/L). The IC25
for the bonytail chub (0.25 mg/L), while less than the IC25
for fathead minnows, was not significantly different than the
IC2S for the fathead minnow. The concentration series
used for tests with C. dubia were the same as those used
for the fish. There was 100% mortality at the lowest
exposure concentration indicating that C. dubia are much
more sensitive to carbaryl than the listed fish species or
fathead minnows. Additionally, because all C. dubia
carbaryl exposure concentrations had no survival, we
discontinued the controls when the mix and ammonia
studies were ended (about day 6 or 7). Because the tests
with carbaryl were conducted at 22°C, the controls for C.
dubia did not produce the three broods required for test
acceptability.
Norberg-King (1993) reported that the IC25 is typically
similar to the No Observed Effect Concentration (NOEC)
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which is calculated using hypothesis testing. Carbaryl
NOECs for fathead minnows have been reported by
Carlson (1972) and Norberg-King (1989). The NOEC in
those two studies were 0.21 and 0.72 mg/L, which is
similar to the average IC25 obtained in this study of
0.42mg/L (Table 5; range 0.22 to 0.81, Appendix 1).
Results from the present study further support the findings
that the IC25 is similar to the NOEC for carbaryl.
Additionally, Norberg-King (1993) proposes that the IC50 is
similar to the Lowest Observed Effect Concentration
(LOEC). ICgoS calculated for the fathead minnow tests with
carbaryl in the present study, ranged from 0.66 mg/L to
1.29 mg/L with a geometric mean of 1.04 mg/L. The LOEC
for carbaryl ranges from 0.68 mg/L (Carlson 1972) to 1.6
mg/L (Norberg-King 1989) and are similar to the 1C50 value
calculated in the current study.
Ammonia
Table-5 summarizes the results of toxicity tests on total
ammonia. Two tests, one with fathead minnows and one
with razorback suckers, had an IC25 greater than the
highest concentration tested (Appendix 1). Following those
tests, exposure concentrations were increased for all
subsequent testing.
Chemical Mix
The IC25s for the chemical mix ranged from 28% (fathead
minnows) to 64% (Colorado squawfish). Fathead minnows
had the lowest IC25 for the fishes, however only the IC25 for
the Colorado squawfish (64%) was significantly different
than the IC25 for the fathead minnows (28%). Comparisons
of IC25s with repeated tests, indicate a fairly consistent
sensitivity except for the test with the bonytail chub
(Appendix 1).
Table 5. IC25 for three different exposures. IC25 is the geometric mean of the IC25s
(number of IC25s in parentheses) used in the rank analysis. An * denotes
statistically different (p<0.05) from fathead minnow IC25. For the results
with ammonia, if the total ammonia IC25 was greater than 17, then 17 was
used in the calculation. Calculation in this manner will likely provide a
concentration lower (bias) than the actual concentration. Not including
the data (17) would bias the summarized data to a greater extent than
including the data.
Species IC25
Fathead minnow
Ceriodaphnia dubia
Bonytail chub
Colorado squawfish
Razorback sucker
Gila topminnow
Carbaryl
(mg/L)
0.42 (5)
<0.33 (3)
0.25 (2)
1,33* (2)
2.06* (2)
not tested
Ammonia
(mg/L)1
7.21 (5)
1.29(3)
11.0(2)
8.9 (2)
13.4 (2)
24.1 (1)
Chemical mix
(%)
28(5)
<6.25 (3)
29(2)
64* (2)
33 (2)
54(1)
1 Ammonia concentrations are total nitrogen (mg/L) and not adjusted for
temperature or pH
As with carbaryl, the chemical mix concentration series
used for tests with C. dubia were the same as those used
for the fish. There was 100% mortality at the lowest
exposure concentration (6.25%) indicating that C. dubia
are more sensitive to the chemical mixture than the fish
species. The IC25s ranged from 1.29 mg/L for C. dubia to
24.1 mg/L for the Gila topminnow. None of the IC25s for the
listed fish species were statistically different (p<0.05) from
the IG25 for fathead minnows (7.21 mg/L). However, the
1C25 for the Gila topminnow (24.1 mg/L), bonytail chub
(11.0),
Un-ionized ammonia is the most toxic form of ammonia and
can be determined from total nitrogen by calculation
knowing pH and temperature (Thurston et al. 1977). The
pH varied between tests (between species and within
species), over the course of a 7-d test and over the 24-h
time period between initial renewal of test solutions and just
prior to siphoning (Appendix 2). For the fathead minnow
the calculated un-ionized ammonia IC2S concentration was
0.27 mg/L (range 0.12 to 0.65). From previous studies, the
NOEC for fathead minnows is 0.15 mg/L as unionized
ammonia (Swigert and Spacie 1983, Thurston et al. 1986)
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and is slightly lower than the concentration of 0.27 mg/L
calculated for the present study.
If the toxicity of chemicals is strictly additive, then only 50%
mortality should occur in the highest concentration tested.
In addition to the IC25s reported in Table 5, we also
calculated LC^s on the chemical mix with fathead minnows
using a nonlinear interpolative method (Stephan 1977).
These LC^s had a geometric mean of 44% (chemical
concentrations were 44% of their equitoxic ratio) which
might indicate that the mix of chemicals was not strictly
additive but synergistic. However, tests in the present
study were conducted with fathead minnows that were less
than 24-h old, (about 0.45 mg). Mayer and Ellersieck
(1986) found that 83% of the time, sensitivity of fish
decreased with increased growth and LC50s increased by
up to a factor of 5. Therefore, the observed LCSO of 44%
might reflect the testing of a more sensitive life-stage rather
than synergism between components of the chemical
mixture.
Mayer and Ellersieck (1986) also report that toxicity
generally increased by a factor of about 3 with each 10°C
increase in temperature. The acute toxicity tests that were
used to calculate the toxic units for this study, were
conducted at 22°C while these chemical mixture studies
were conducted at 25°C. This represented only a 3°C
temperature increase, but could account for a portion of the
apparent increase in the chemical mixture toxicity. In
summary, while the toxicity of the chemical mixture might
be synergistic, it is more likely that the difference in life-
stage or the increase in test temperature is responsible for
the increased toxicity to fathead minnows.
Discussion
Of the 11 possible comparisons of fathead minnows to the
listed species outlined in Table 5, only three IC25s (27%)
are significantly different than the IC25s for fathead
minnows. When there was a difference, the IC25 for the
listed species was higher than the IC25 for the fathead
minnow. Two of these three IC25s are for the Colorado
squawfish which might indicate that the Colorado
squawfish is less sensitive to contaminant exposure than
razorback suckers, bonytail chubs, Gila topminnows or
fathead minnows. These findings are in contrast to the
results of studies described in Chapter 1 and EPA (1995).
These previous studies evaluated the sensitivity of listed
species to fathead minnows in static 96-h acute toxicity
tests. The LC^s in these previous studies for listed
species, including Colorado squawfish, bonytail chub, Gila
topminnows, and razorback suckers were generally lower
than the LC^s for fathead minnows.
The sensitivity of the listed fountain darter (Etheostoma
fonticola) has also been evaluated following EPA whole-
effluent toxicity test procedures (Edwards Aquifer Research
& Data Center 1992a, 1992b). Tests were conducted with
effluent collected from the San Marcos, TX wastewater
treatment plant and in a single compound toxicity test with
glyphosate. In the test conducted with wastewater, the
NOECs for both survival and growth of fountain darters
was 14%. The NOEC for fathead minnow survival was
28% and the NOEC for growth was 28%. In contrast, the
NOEC for growth and survival for both species was the
same in the test with glyphosate. We have also conducted
whole-effluent toxicity tests with razorback suckers and
bonytail chub (unpublished data) with effluent samples
collected from within the state of Arizona. There was no
toxicity from the effluent to either fathead minnows or
razorback suckers. However, in the effluent test with
bonytail chub, survival of fathead minnows was somewhat
higher than that of the bonytail chub. There was no effect
on growth for either bonytail chub or fathead minnows.
In summary, the fathead minnow 7-d growth and survival
test appear to be a reliable estimator of effects to the listed
species used in this study. Additionally, the C. dubia
survival and reproduction test was generally more sensitive
than any of the fish tested. When the listed species and
fathead minnow were different, the listed species was often
more resistant than the fathead minnow. However, other
studies have shown listed species to be similar to or
slightly more sensitive than fathead minnows when tested
using effluent procedures. This study was conducted with
fish species that have not been typically tested so factors
such as handling procedures, optimum feeding rates,
optimum test temperature, expected test to test variation
and expected survival or growth have not been previously
documented, and therefore results of this study should be
interpreted cautiously. Further testing should also be
conducted with additional listed species or their U.S. Fish
and Wildlife Service identified surrogate species before
definitive policy decisions concerning the protection of
endangered and threatened species to contaminants in
aquatic environments are made.
References
American Society for Testing and Materials. 1998.
Standard guide for conducting acute toxicity tests on test
materials with fishes, macroinvertebrates, and
amphibians. E 729-96. American Society for Testing
and Materials, Philadelphia.
Carlson, A.R. 1972. Effects of long-term exposure to
carbaryl (sevin) on survival, growth, and reproduction of
the fathead minnow (Pimephales promelas). J. Fish.
Res. Board Can. 29:583-587.
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Edwards Aquifer Research and Data Center. 1992a.
Preliminary study of the contrast of sensitivity between
Pimephales promelas and Etheostoma fonticola to
Rodeo herbicide. Southwest Texas State University.
Edwards Aquifer Research and Data Center. 1992b.
Preliminary study of the contrast of sensitivity between
Pimephales promelas and Etheostoma fonticola to San
Marcos Wastewater Treatment effluent. Southwest
Texas State University.
Mayer, F.L., Jr., and M.R., Ellersieck. 1986. Manual of
Acute Toxicity: Interpretation and Data Base for 410
Chemicals and 66 Species of Freshwater Animals. U.S.
Fish Wild. Serv. Resour. Publ. 160, 579 p.
Norberg-King, T.J. 1989. An evaluation of the fathead
minnow seven-day subchronic test for estimating chronic
toxicity. Environ. Toxicol. Chem. 8:1075-1089.
Norberg-King, T.J. 1993. A linear interpolation method for
sublethal toxicity: The inhibition concentration (Icp)
approach. National Effluent Toxicity Assessment Center.
Technical Report 03-93, Duluth, MN.
Statistical Analysis Systems. 1994. SAS Users Guide:
Statistics, Version 5 Edition. Gary, NC.
Snedecor, G.W. and W.G. Cochran. 1980. Statistical
methods. Iowa State University Press. Ames, IA.
Stephan, C.E. 1977. Methods for calculating an LC50. in:
F.L. Mayer and J.L. Hamelink (eds.) Aquatic Toxicology
and Hazard Evaluation, ASTM STP 634, American
Society for Testing and Materials.
Swigert, J.P. and A. Spacie. 1983. Survival and growth of
warmwater fishes exposed to ammonia under low flow
conditions. PB83-257535. National Technical Information
Service.
Thurston, R.V., R.C. Russo, and K. Emerson. 1977.
Aqueous ammonia equilibrium calculations. Technical
Report No. 74-1 Fisheries Bioassay Laboratory, Montana
State University, Bozeman, MT.
Thurston, R.V., R.C. Russo, E.L. Meyn, R.K.Zajdel. 1986.
Chronic toxicity of ammonia to fathead minnows. Trans.
Amer. Fish. Soc. 115:196-207.
U.S. Environmental Protection Agency. 1986. Standard
evaluation procedure: Ecological risk assessment. EPA
540/9-85-001. Hazard Evaluation Division, Office of
Pesticide Programs, Washington, D.C. 96 p.
U.S. Environmental Protection Agency. 1994. Short-Term
Methods for Estimating the Chronic Toxicity of Effluents
and Receiving Waters to Freshwater Organisms. EPA
600/4-91-002. USEPA EMSL, Cincinnati, OH.
U.S. Environmental Protection Agency. 1995. Use of
surrogate species in assessing contaminant risk to
endangered and threatened fishes. EPA 600/R-96/029.
Office of Research and Development. Gulf Breeze, FL
78 p.
Yoder, C.0.1989. The development and use of biological
criteria for Ohio surface waters: In Water Quality
Standards for the 21st Century. Proceedings of a
National Conference, U.S. EPA, Office of Water,
Washington, DC.
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Appendix 1. The IC^s and confidence intervals for carbaryl, ammonia and
the chemical mixture for each species and test.
Carbaryl
Species
Ceriodaphnia dubia
Fathead minnows
Bonytail chub
Colorado squawfish
Razorback sucker
IC25
Test 1 - <0.33
Test 2 - <0.33
Test 3 - <0.33
Test 1 - 0.22
Test 2 - 0.39
Test 3 - 0.30
Test 4 - 0.81
Test 5 - 0.60
Test 1 - 0.28
Test 2 - 0.23
Testt -1.17
Test 2 -1.52
Test 1-1. 62
Test 2 - 2.62
Confidence Interval1
NC2
NC
NC
0.05-1.57
0.00 - 0.90
0.14-1.17
0.60 - 0.91
0.30-1.50
0.12-1.54
0.05-1.95
0.69-1.71
0.00 - 2.00
0.77 - 2.09
1.92-3.10
1 For tests with fish, confidence intervals are expanded because replicates
were less than seven, confidence intervals for Ceriodaphnia dubia are not
expanded since there were ten replicates.
2 NC - Not calculated.
Ammonia (total measured N - mg/L)
Species
Ceriodaphnia dubia
Fathead minnows
Bonytail chub
Colorado squawfish
Gila topminnow
Razorback sucker
IC25
Testl -1.59
Test 2 - 0.75
Test 3 -1.80
Test 1 - 14.40
Test 2 - 5.82
Test 3 ->1 7
Test 4- 5.71 3
Test 5 - 2.40
Test 1 - 12.91
Test 2 - 9.4
Test 1 - 4.4
Test 2 -17.9
Test 1 - 24.1
Test 1 - >17
Test2-10.553
Confidence Interval1
1.12-4.16
0.69 - 0.82
1.29-5.21
NC2
2.29 - 8.58
NC
4.91 - 7.60
0.89 - 22.80
NC
3.45 - 33.97
3.78 - 4.83
14.71 - 19.68
19.2-25.6
NC
0. - 12.54
1 For tests with fish, confidence intervals are expanded because replicates
were less than seven. Confidence intervals for Ceriodaphnia dubia are
not expanded since there were ten replicates.2NC - Not calculated.
3 Data have not been corrected for measured concentrations.
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Appendix 1. (Continued)
Appendix 2.
Chemical mix (%)
Species
Confidence
Interval1
Ceriodaphnia dubia
Fathead minnows
Bonytail chub
Colorado squawflsh
Gila topminnow
Razorback sucker
Test 1 - <6.25
Test 2 - <6.25
Test 3 - <6.25
Test 1 - 29.9
Test 2 - 39.4
Test 3-31.7
Test 4-23.8
Test 5 - 20.6
Test 1 - 60.6
Test 2-13.9
Test 1 - 63.6
Test 2 - 64.6
Test 1-54.1
Test 1 - 26.2
Test 2-41.2
NC2
NC
NC
0-41.7
27.0 - 69.1
4.6 - 49.8
15.0-49.6
0 - 93.5
37.2 - 76.3
0-90
35.4 - 66.35
54.1 - 67.8
34.6 - 64.9
0 - 37.2
20.8 - 58.9
1 For tests with fish, confidence intervals are expanded because
replicates were less than seven. Confidence intervals for
Ceriodaphnia dubia are not expanded since there were ten replicates.
2 NC - Not calculated.
Summary of exposure water pH for each chemical. For each chemical the data includes the
average low and average high (and minimum and maximum pH recorded) for all tests and
species tested and includes all replicates. Initial water is defined as the water used each day for
renewal. Chemical waters were mixed daily. Final water is defined as the water removed from
one replicate after 24 hours of exposure and prior to renewal.
Water
Initial
Final
Chemical
Ammonia
Carbaryl
Chemical mix
Ammonia
Carbaryl
Chemical mix
Average pH
Low
7.7
8.1
8.3
7.7
7.9
7.7
Lowest
PH
6.6
6.9
7.9
7.1
7.3
6.4
Average pH
High
8.4
8.6
8.6
8.3
8.4
8.4
Highest
PH
9.1
9.1
9.1
8.6
9.0
9.0
fUS. GOVERNMENT PRINTING OFFICEt 2000 550-101/20015
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