vvEPA
US EPA Office ol Research and Development
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-01/051
June 2001 ..
Early Life-Stage Toxicity of
Copper to Endangered and
Surrogate Fish Species
^^F •
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EPA/600/R-01/051
June 2001
Early Life-Stage Toxicity of Copper
to Endangered and Surrogate Fish Species
by
John M. Besser1, F. James Dwyer2,
Chris G. Ingersoll1, and Ning Wang3
EPA Project No. DW14937809-01-0
1U.S. Geological Survey, Columbia Environmental Research Center
4200 New Haven Rd., Columbia, MO 65201
2U.S. Fish and Wildlife Service, Eciological Services, 608 E. Cherry St.,
Columbia, MO 65201
Department of Fisheries and Wildlife Sciences, University of Missouri,
Columbia, MO 65211
Project Officer
Mary Reiley
U.S. Environmental Protection Agency
Office of Water
Washington, DC 20460
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC 20460
Printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine free.
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Contents
Notice ............. .................................... • [[[ "
Abstract
Introduction [[[ • .......................... ^
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Notice
The U.S. Environmental Protection Agency, Office of Water funded this research through an Inter-
agency Agreement between the Columbia Environmental Research Center (CERC), as part of U.S.
EPA Project No. DW14937809-01-0, "Biological and Chemical Evaluation of Contaminants." It has
been subjected to the Agency's peer and administrative review and has been approved for publica-
tion as an EPA document. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
ill
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Abstract
Water quality criteria (WQC) for the protection of aquatic
life have not explicitly considered the degree of protection
afforded to aquatic species listed as endangered or threat-
ened under the U.S. Endangered Species Act (listed spe-
cies) . Most WQCs are based primarily on responses of a
limited number of surrogate species, which are easily cul-
tured and tested in the laboratory. Little information is avail-
able about the relative sensitivity of listed species to toxic
chemicals, especially with respect to chronic toxicity. We
conducted a series of chronic, early life-stage toxicity tests
with two listed species, fountain darter (Etheostoma
fonticola) and spotfin chub (Cyprinella monacha), and two
surrogate species, fathead minnow (Pimephalespromelas)
and rainbow trout (Oncorhynchus mykiss), exposed to cop-
per (Cu).
Data from the tests with the four species, which included
repeated tests with three species, were used to evaluate
the suitability of test endpoints and toxicity metrics. End-
points measured included survival, growth (total length and
average dry weight of surviving fish), and biomass (total
dry weight of survivors). Toxicity metrics were established
by hypothesis testing to determine no-observed-effect con-
centrations (NOEC) and lowest-observed-effect concen-
trations (LOEC), and by a linear interpolation technique, to
estimate inhibition concentrations associated with 10% and
25% reductions of test endpoints (IC10 and IC25). The hy-
pothesis testing and linear interpolation methods generally
gave similar results, as ail calculable IC10 values fell within
the NOEC-LOEC range. The 'chronic value' calculated
from these studies (ChV = geometric mean of NOEC and
LOEC) corresponded closely to the IC10for most species
and endpoints.
For three of the four species tested, growth and/or biom-
ass endpoints were more sensitive than survival. Forfoun-
tain darters, no significant effects on growth occurred at
concentrations less than LOECs for survival and biomass,
and IC10 values indicated that reductions in growth (both
dry weight and total length) only occurred at concentra-
tions greater than those affecting survival. For the other
three species, reductions in growth, expressed as individual
dry weight, occurred at concentrations at least as low as
those affecting other endpoints.. However, growth in dry
weight showed wide variation among three tests with
fathead minnows, with ChVs ranging from 2.8 to 15.9 ^g/L.
Results from tests with fathead minnows and other spe-
cies suggested that growth in dry weight was affected by
differences in fish density caused by differential survival
among replicates and between treatments. Growth in total
length was less variable than dry weight and LOECs for
total length were close to those for dry weight, but IC10 val-
ues for total length were consistently greater than those for
dry weight. Biomass, which reflects combined toxic ef-
fects of Cu on both survival and individual growth, was
nearly as sensitive as growth in dry weight and was less
variable among tests.
Sensitivity to Cu toxicity did not differ substantially between
listed and surrogate species. Lowest average ChVs for
the four species tested ranged from 7.7 ^g/L for the foun-
tain darter (for reduced survival and biomass) to 15.9 //g/
L for the spotfin chub (for reduced growth and biomass).
The average ChV for growth of fathead minnows from three
tests (7.8 ,wg/L) was nearly equal to that for fountain dart-
ers, although this value was strongly influenced by the low
ChV of 2.8 fj.g/L determined from one of the three tests.
Evaluation of relative species sensitivities with IC10 pro-
duced similar results, with values ranging from <8.0 /zg/L
for fountain darters to 23 ywg/L for spotfin chubs.
Toxicity thresholds (either ChVs or IC10s) estimated from
our chronic, early life-stage toxicity tests indicated that the
current chronic Cu WQC would protect the endangered
spotfin chub, but may/not adequately protect the endan-
gered fountain darter or the two surrogate species tested.
This finding contrasts with results of previous acute toxic-
ity tests in our laboratory, which concluded that current
acute WQC for Cu would adequately protect fountain dart-
ers. These results suggest that protection of fountain dart-
ers from chronic toxicity of Cu would require application of
a safety factor of about 0.5 to the current chronic Cu WQC.
This safety factor would be consistent with that"estimated
from previous acute toxicity studies conducted at our labo-
ratory with surrogate and listed species.
Introduction
Federal environmental laws, including tfie Clean Water Act,
the Federal Insecticide, Fungicide and Rodenticide Act, and
the Toxic Substances Control Act require the testing and
regulation of toxic chemicals to prevent hazards to the en-
vironment or human health. The Endangered Species Act
further requires Federal agencies to insure that any action
authorized, funded, or carried out is not likely to jeopardize
the continued existence of endangered or threatened
(listed) species. The U.S. Environmental Protection Agency
(USEPA), the U.S. Fish and Wildlife Service (USFWS), and
the U.S. Geological Survey (USGS) have conducted re-
search to determine the acute sensitivity to several classes
of toxic chemicals for 13 freshwater species that are listed
are listed by USFWS or state agencies (Dwyer et al 1995;
1999a,b; 2000). These studies found that the common
surrogate species, rainbow trout (Oncorhynchus mykiss),
was generally as sensitive as the listed species in acute
tests. Across all the species and chemicals tested, me-
dian lethal concentrations (LC50s) for listed species dif-
fered from those for rainbow trout by no more than a factor
of three. Additional comparisons, based on 7-day effluent
toxicity tests, also found that the sensitivity of the surro-
1
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gate species, fathead minnow (Pimephales promelas) was
similar to the listed species tested (Dwyer et al. 1999b).
These acute toxicity data are being used to evaluate po-
tential risks to listed fish species during consultations on
state water quality standards and in pesticide spray pro-
grams. However, acute toxicity tests may not establish
safe exposure levels for listed organisms that may be ex-
posed to contaminants throughout their life cycle. Few
suitable data are available to evaluate the chronic sensitiv-
ity oflisted species to toxic chemicals, or to compare the
acute and chronic toxicity of chemicals to listed species
(Beyers et al. 1994). Acute-chronic ratios for surrogate
species, based on acute LC^s and thresholds for chronic
effects on survival, growth, and/or reproduction, vary widely
among species (USEPA 1996a). Because of this uncer-
tainty about the sensitivity of listed species to chronic tox-
icity, it is not known whether current chronic water quality
criteria (WQC) adequately protect listed species.
Chronic WQC are based on the toxicity thresholds deter-
mined by statistical hypothesis testing, typically analysis of
variance (ANOVA). This approach estimates the lowest-
observed-effect concentration (LOEC) and no-observed-
effect concentration (NOEC), based on statistically signifi-
cant differences between treatment groups and controls.
USEPA uses the geometric mean of the NOEC and LOEC
to derive a 'chronic value' (ChV), which is used to compare
the sensitivity of species and to calculate' acute-chronic
ratios (USEPA 1985a). The principal criticisms of ANOVA-
based metrics for analysis of chronic toxicity data (Stephan
and Rogers 1985; Crane and Newman 1999) are that (1)
results of ANOVA-based data analysis are affected by dif-
ferences in the power of statistical analyses, and (2) these
metrics are not continuous variables (i.e., they can only be
assigned to the discrete exposure concentrations selected
for a given test) and therefore have no definable statistical
confidence interval. As a result, the degree of reduction in
a given endpointthat is associated with the LOEC can vary
widely among tests depending on the experimental design
and the power of the statistical test selected. Another criti-
cism of ANOVA-based toxicity metrics is that concentra-
tion-response data are more appropriately analyzed by
regression methods (Stephan and Rogers 1985, Beyers et
al. 1994). Regression techniques, such as probit analysis
and logistic regression, use data from all exposure con-
centrations to make point estimates of effects thresholds,
such as the LCM. However, unlike LC50s, point estimates
for 'biologically significant' effects tend to be far from the
midpoint of the regression thresholds (i.e., reductions of
less than 50% in test endpoints), where confidence inter-
vals for point estimates become wider.
An alternative (or adjunct) to both ANOVA and regression
analysis is the use of linear interpolation to estimate inhibi-
tion concentrations (ICP) associated with specific percent
inhibition of biological responses (Norberg-King 1993;
USEPA 1994). The ICP methods estimates threshold con-
centrations associated with a level of impact on a biologi-
cal response (e.g., 10% or 25% reductions) that are as-
sumed to be biologically significant. The IC25, or 25% inhi-
bition concentration, has been suggested as a biologically
meaningful toxicity metric, although few studies have com-
pared NOECs, LOECs, and ICPs in chronic tests (Marchini
et al. 1992). The ICP procedure assumes that short sec-
tions of the dose-response curve are approximately linear,
but it does not require assumptions about the overall shape
of the concentration-response curve. Confidence intervals
for ICP estimates can be estimated by a nonparametric
bootstrap technique, which reflects the variation in the test
endpoint at exposure concentrations adjacent to the inter-
val of interest (Norberg-King 1993).
We conducted a series of toxicity tests to address three
objectives: (1) evaluate the suitability of different test end-
points and toxicity metrics for comparing chronic toxicity
between listed and surrogate species; (2) compare the
sensitivity of listed species and surrogate species, using
the toxicant copper (Cu); and (3) determine whether the
species tested are adequately protected by the existing
chronic WQC for Cu. Early life-stage tests, which typically
start before or shortly after egg hatching and last for at
least 30 days, were selected because they are good pre-
dictors of toxicity in full life-cycle chronic tests (ASTM
2000a). We evaluated data on survival, individual growth
(in dry weight and total length), and total biomass during
these tests, using metrics derived by ANOVA (NOEC;
LOEC, and ChV) and by the linear interpolation technique
(IC10 and IC25). Toxicity tests with Cu were conducted
with two endangered fish (fountain darter, Bheostoma
fonticola-, and spotfin chub, Cyprinella monacha) and two
surrogate test species (fathead minnow and rainbow trout).
Materials and Methods
Test organisms
Toxicity tests were conducted at the Columbia Environ-
mental Research Center (CERC), Columbia, Missouri.
Tests with fountain darter, spotfin chub, and fathead min-
now (Tests 1, 2, and 3) were started with newly-hatched
larvae (typically one day after hatching) obtained from the
National Fish Hatchery and Technology Center (San
Marcos, TX), Conservation Fisheries, Inc. (Knoxville, TN),
and Aquatic BioSystems, Inc. (Fort Collins, CO), respec-
tively. Rainbow trout embryos were obtained from the Ennis
National Fish Hatchery (Ennis, MT). The first test with rain-
bow trout (Test 4A) was started with eyed eggs. The sec-
ond rainbow trout test (Test 4B) was started with eggs that
had been held in a vertical-tray incubation box at 10°C in
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CERC well water (alkalinity 258 mg/L as CaCO3,
hardness 286 mg/L as CaCO3, pH 7.8) until the
swim-up life stage. Trout larvae were acclimated to
test waters over a period of 48 hours before being
added to test chambers.
Exposure systems
Tests were conducted in an intermittent-flow pro-
portional diluter system (Lemke et al. 1978). Stock
solutions of Cu (CuSO4»5H2O) were prepared in de-
ionized water. The diluter dispensed five Cu con-
centrations with a dilution factor of 0.5, plus a con-
trol, and provided about 250-ml of water to each rep-
licate exposure cup or chamber every 20 minutes.
Glass incubation cups (350 ml_, with stainless steel
screen bottoms) were used for holding rainbow trout
eggs. Plastic cups (1000 ml_, with 40-mesh stain-
less steel screen window) were used for holding lar-
vae of the other three species for the first two weeks
of the exposures. Test solution flowed directly into
the cups, which were suspended in the test cham-
bers. Four test chambers-(10-L glass aquaria, with
stainless steel screen window) were submerged in
each of 12 large glass aquaria held in a water bath,
which controlled test temperatures within ±1°C of
the target temperatures (Table-1). Water depths in
the aquaria were controlled by stand-pipes to pro-
duce a volurne of 6 liters in individual test cham-
bers. Each Cu treatment was delivered to two
aquaria and each species was stocked into two of
the four replicate test chambers in each aquarium,
resulting in four replicate chambers for each spe-
cies.
Water samples for Cu analysis were collected bi-
weekly from one test chamber for each concentra-
tion and preserved with 1 % (v/v) ultra-pure nitric acid.
Water samples were analyzed by inductively-
coupled plasma-mass spectroscopy (ICP-MS) with-
out further sample preparation (May et al. 1997;
Appendix 1). Total hardness, total alkalinity, con-
ductivity, pH, and dissolved oxygen were measured
weekly, and ammonia was monitored periodically
during each test.
Early life-stage toxicity tests
Four sets of early life-stage toxicity tests were con-
ducted in general accordance with ASTM (2000a)
and USEPA (1996b) guidelines, as summarized in
Table 1. Tests were conducted under a photope-
riod of 16 h light and 8 h darkness, with moderately-
hard reconstituted water: hardness, 160 to 180 mg/
L as CaCO3; alkalinity, 110 to120 mg/L as CaCO3;
and pH, 7.6 to 8.0 (2000b). Tests with two listed
Table 1. Summary of test conditions for early life-stage chronic toxicity tests,
conducted in general accordance with guidance in ASTM (2000a).
Parameter
Conditions
1. Exposure system
2.Temperature
S.Toxicant
4. Dilution
5. Photoperiod
6. Test chamber
7. Water renewal rate
8. Age of fish
9. Fish or eggs/chamber
10. Replicates
11. Duration:
12. Reeding
13. Test water
14. Endpoints
15. Test acceptability
Intermittent flow proportional diluter
Test 1 (fathead minnow, fountain darter): 25°C
Test 2 (fathead minnow, fountain darter): 23°C
Test 3 (fathead minnow, spotfin chub): 25°C
Test 4 (rainbow trout): 10°C
Copper sulfate (pentahydrate)
Control and 5 copper concentrations (dilution
factor = 0.5)
16 h lights, h darkness
6-L with egg/fry cup
3 volume-replacements/day (0.75 L/hour )
Rainbow trout: eyed embryos and swim-up fry (2 test)
Fathead minnows: <24 hr old larvae
Listed species: <72 hr old larvae
25 for rainbow trout; 10 for other three species
4 replicate chambers per concentration
30 d; except 58 d for rainbow trout embryo test
(test ended after 30-d swim-up life stage);
3 times a day with <24 h live brine shrimp nauplli
ASTM hard water (hardness 160 to180 mg/L as CaCO.,
alkalinity 110 to 120 mg/L as CaCOS, pH 7.6 to 8.0)
Survival, growth (mean dry wt), biomass (total dry wt.)
>70% average survival in controls
species, fountain darters and spotfin chubs, were conducted
concurrently with tests with the surrogate species, fathead min-
now, in the same diluters. Ten newly-hatched larvae of each spe-
cies (<24 hr post-hatch for fathead minnows; <72 h post-hatch for
listed species) were placed in each of four repliate egg cups for
each concentration The only exception to this experimental de-
sign was for fountain darters in Test 2, where only three repli-
cates were stocked at the three highest Cu concentrations, due
to the limited number of fry available. After about two weeks of
exposure, fish were released to the surrounding chambers.
Throughout the tests, fish were fed ad libitum three times a day
with live <24 h-old brine shrimp nauplii. Water temperature for
Test 1 (fountain darters and fathead minnows) and Test 3 (spotfin
chub and fathead minnows) was maintained at 25 ±1 °C. Test 2
(fountain darters and fathead minnows) was conducted at 23 °C,
which is reported to be the optimum temperature for survival and
growth of fountain darters (Bonner et al. 1998). These tests ended
after 30 days of exposure.
Tests^with rainbow trout were conducted starting with two differ-
ent life stages, eyed embryos (Test4A) and swim-up larvae (Test
4B). These two tests were conducted concurrently, to allow a
direct comparison of the sensitivity of the full ELS test (embryo-
alevin-fry) to the shorter test with swim-up fry. Treatment groups
and replicates were distributed as described above. For Test 4A,
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25 eyed embryos were held in each of four replicate egg
cups at each concentration. Embryos were incubated in
darkness, under black plastic that blocked about 90% of
incident light. When fish hatched, they were transferred
into the surrounding chambers. Test 4A ended 30 days
after fish reached the swim-up stage, resulting in a total
test duration of 58 days. For Test 4B, 25 fish at the swim-
up stage were added to each test chamber, and the test
ended after 30 days. Water temperature was maintained
atlO ±1 °C for both tests. Trout were fed ad libitum three
times a day with live <24 h-old brine shrimp nauplii after
they reached the swim-up stage.
During all four tests, dead fish were counted, recorded,
and removed daily. Fish were not fed for 24 h before the
end of the tests. At the end of tests, surviving fish in each
replicate chamber were euthanized, counted, placed in a
tared aluminum weigh boat, and dried at 60 °C for 36 h for
determination of dry weight. Dry weights were not cor-
rected for initial weights offish at the beginning of the test,
which was assumed to be equal for fish in each test. Dry
weight data were used to determine growth (mean dry
weight per individual), and biomass (total dry weight per
replicate) of surviving fish. In tests with fathead minnows,
fountain darters, and spotfin chubs, total lengths of freshly-
euthanized fish were measured with a digital caliper as an
additional growth endpoint.
Data Analysis
Toxicity thresholds were estimated from test data for sur-
vival, growth (total length and dry weight), and biomass by
statistical hypothesis testing and by the linear interpolation
technique, using TOXSTAT statistical software (WEST Inc.
1996). Hypothesis testing to determine LOEC and NOEC
followed the statistical flow-chart recommended by USEPA
(1994), with testing for normality and homogeneity of vari-
ance followed by statistical analysis by parametric ANOVA
and Dunnett's test or by the nonparametric Steel's many-
one rank test. For the sake of simplicity, both types of
tests are referred to as 'ANOVAs' for the remainder of this
report. Statements of statistical significance refer to a prob-
ability of a type 1 error of no greater than 5% (p^O.05).
The NOEC for each endpoint was defined as the highest
exposure concentration in which the endpoint was not sig-
nificantly reduced relative to controls and the LOEC was
determined as the lowest concentration above the NOEC.
In order to focus on effects on growth that occurred at con-
centrations less than those affecting survival, test concen-
trations above the NOEC for survival were excluded from
the statistical analysis for growth data (USEPA 1994). If
no significant reductions in growth occurred at or below
the NOEC for survival, the LOEC for growth was unde-
fined and was assigned the value of ';>[LOEC for survival]'
for comparisons. In addition, the data were analyzed by
the linear interpolation procedure (Norberg-King 1993) to
determine 10% and 25% inhibition concentrations (IC10and
IC25). Data from all concentrations were used for ICP cal-
culations, except in cases where a trend for decreased
growth was reversed at the highest test concentration, co-
incident with survival less than 30%. Based on these cri-
teria, growth data from the highest exposure concentra-
tions were excluded from ICP calculations for fathead min-
nows in Test 3, and for rainbow trout in Tests 4A and 4B.
Results and Discussion
Test Conditions
Test conditions and performance indicators for toxicity tests
corresponded closely to the guidelines in Table 1. Cu con-
centrations in Test 1 showed evidence of background con-
tamination, with elevated Cu concentrations in the control
treatment (5.4 /ug/L) and concentrations consistently above
nominal concentrations in the four lower Cu treatments
(Table 2). The source of the problem, a pump with a worn
impeller, was identified and corrected before subsequent
tests. Tests 2 through 4 had low Cu concentrations in con-
trols (<2 ,ug/L), and measured Cu concentrations closely
reflected nominal diluter concentrations, except for slight
deficits in the highest Cu treatments, which probably re-
flected losses of Cu to sorption or to formation of particu-
late species.
Quality assurance for Cu analyses met all data quality ob-
jectives (Appendix 1). The detection limit for Cu for four
analytical runs ranged from 0.2 to 0.6 ^g/L. Precision of
duplicate analyses ranged from 0.3% to 3.7% relative per-
cent difference. Recovery of Cu from two standard refer-
ence solutions averaged 100%, recoveries from analysis
spikes ranged from 95% to 99%, and recoveries of Cu
spiked into interference check solutions ranged from 90%
to 108%.
Water quality in the four toxicity tests was within expected
ranges. Routine water quality measurements corresponded
to expected characteristics of ASTM hard water (Table 3).
Measured hardness averaged 164 mg/L, within the range
cited by ASTM (2000b). Dissolved oxygen ranged from 8.8
to 10.5 for rainbow trout test (at 10°C), and from 6.5 to 8.3
mg/L for tests with the other three species (at 23 to 25°C).
Ammonia concentrations measured during the tests did not
exceed 0.12 mg/L as total ammonia or 0.002 mg/L as union-
ized ammonia.
Control survival in all tests met the minimum acceptable
level of 70% established for early life-stage testing by ASTM
(2000a; Tables 4-6). Lowest control survival (78%) oc-
curred for fountain darters in Test 2, but control survival
ranged from 89% to 100% in other tests. Elevated back-
ground Cu concentrations in Test 1 had no obvious effect
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Table 2. Nominal and measured copper cone
from early life-stage toxicity tests.
tor each test are underlined SD=sl
entratio
Nominal
tandard
ns in water
valges
deviation.
Copper Concentration (ug/L)
Study 1
DayO
Day 2
Day 20
Mean
(SD)
Study 2
DayO
Day 15
Day 29
Mean
(SD)
Study 3
DayO
Day 13
Day 19
Day 27
Mean
(SD)
Study 4
DayO •
Day 9
Day 21
Day 40
Day 56
Mean
(SD)
Control
5.9
5.8
4.6
5.4
(0.7)
Control
1.7
1.8
2.2
1.9
(0.3)
Control
1.9
2.0
1.5
2.3
1.9
(0.3)
Control
1.5
1.9
1.2
1.7
1.4
1.6
(0.3)
3.13
8.6
9.9
9.3
(0.9)
2.5
5.0
3.9
4.4
4.4
(0.5)
23
4.0
3.4
4.2
3.8
3.9
(0.3)
3J3
3.1
3.8
3.4
4.8
4.1
3.7
(0.7)
625
12.1
10.9
11.4
11.5
(0.6)
5
6.4
6.8
6.3
6.5
(0.3)
5
6.7
5.3
6.7
6.9
6.4
(0.8)
6,25
4.8
6.3
6.7
7.3
6.8
6.2
(1.0)
12.5
16.7
16.1
17.2
16.7
(0.6)
10
10.4
11.6
9.9
10.6
(0.90)
JO
11.8
9.1
11.0
12.3
11.1
(1.4)
J25
10.0
11.9
12.1
13.0
12.6
11.8
(1.3)
25
28.5
28.6
27.7
28.3
(0.5)
20
19.2
20.8
17.6
19.2
(1.6)
20.
22.4
18.5
19.8
30.2
22.7
(5.2)
25
18.1
22.2
25.2
24.1
26.0
22.4
(3.1)
50
42.4
53.7
55.3
50.5
(7.00)
40
39.0
41.1
39.4
39.8
(1.1)
40
47.2
34.7
44.0
42.5
42.1
(5.3)
50
41.7
44.4
47.5
45.5
47.5
44.8
(2.)0
Table 3. Water quality characteristics of test water (ASTM hard) during eariy life-
stage toxiciw tests. Values are means, with standard deviation in
parentheses.
on control performance for either fathead minnows or
fountain darters, but background Cu concentrations ef-
fectively obscured the two lowest exposure concentra-
tions (nominal concentrations: 3.1 and 6.3/43/L).
Copper toxicity
The sensitivity of the two listed species to Cu differed
widely, with fountain darters more sensitive than spotfin
chubs (Table 4). In Tests 1 and 2, survival and biomass
of fountain darters were significantly reduced, relative to
controls, at Cu concentrations of 9 // g/L. and 11 t^g/L,
respectively. No darters survived at Cu concentrations
of 28 /zg/L and greater. Growth of darters (mean dry wt.
or total length of individuals) was not significantly reduced
at concentrations less than those affecting survival. The
similar results of these two tests indicate that the differ-
ence in water temperature (25°C in Test 1,23°C in Test
2) did not affect the toxicity of Cu to fountain darters. In
contrast, survival of spotfin chubs (Test 3) was not sig-
nificantly reduced at Cu concentrations less than 42 fj.g/
L, but both growth (in dry wt. and total length) and bio-
mass were significantly reduced at 23 fj,g/L.
Test(N)
1(4)
2(4)
3(4)
4(6)
Temperature
(°C) •
25 ±1
23 ±1
25 ±1
10±1
Conductivity
(umhos/cm)
575 (30)
604(10)
604 (10)
588(13)
PH
8.1 (0.1)
8.3(0.1)
8.3 (0.1)
8.1 (0.2)
Alkalinity Hardness
{Mg/LasCaC03)
114(3)
120 (14)
120 (14)
111(7)
163(5)
162(12)
162(12)
167(5)
Table 4. Survival, growth (dry weight and total length), and biomass
of fountain darters (Etheostoma fontalis) and spotfin chubs
(Cyprinella monacha) in early life-stage toxicity tests with
copper. Values are means, with standard deviation in
parentheses (N = 4 unless indicated otherwise). Growth data
in treatments with significant reductions in survival (Means
below dotted linesJwere excluded from ANOVAs.
Test
Cu
(ug/L)
Survival
(%)
Dry Weight
(mg)
Total Length
(mm)
Biomass
(mg)
Fountain Darter
1
2
3
5.4
9.3
12
17
28
5.1
1.9
4.4
6.5
11
19
40
1.9
3.9
6.4
11
23
42
90(8)
33 (22)'
18 (13)'
3(5)'
0(0)'
0(0)'
78 (10)
65 (39)
88(13)
27 (6p
10 (10)"3
0(0)'3
100 (0)
98 (5)
100 (0)
95 (10)
100 (0)
82 (13)'
6.4 (1.0)
7.0 (0.5)
9.0 (1.5)3.
4.5 (O)1
7.1 (0.3)
7.0 (0.4)
6.9 (0.3)
7.5 (0.9) 3
4.8 (1.1) z
Spotfin Chub
25.5 (0.5)
25.1 (0.9)
25.8 (0.5)
27.0 (2.2)
23.2 (1.6)'
19.0 (1.3).
15.3 (0.8)
16.1 (0.5)
17.0 (1.2)3
13.6 (O)1
15.9 (0.2)
15.4 (0.2)
15.9 (0.3)
16.1 (0.7) 3
13.2 (0.3) 2
26.5 (0.3)
26.2 (0.4)
27.1 (0.5)
27.1 (0.2)
26.2 (0.9)
24.2 (0.7)
58 (14)
22 (15)'
16 (12)'
1 (3)'
0 (O)-
o (or
55 (5)
45 (26)
60 (8)
20 (2)'3
5 (4)'3
0 (0)'3
256 (5)
245(15)
257 (6)
254 (12)
232(16)'
155 (14)'
'•2i3 Superscripts indicate reduced number of replocates, due to mor-
tality or to limited numbers of available test organisms.
* significant reductions relative to controls (ANOVA/ Dunnett's test
(P<0.05).
Growth and biomass were the most sensitive endpoints in
three tests with fathead minnows, which were conducted
concurrently with tests with the listed species (Table 5). Sur-
vival of fathead minnows was not significantly reduced at Cu
concentrations less than 28 //g/L in any of the three tests.
Significant reductions in growth (in dry wt. and total length)
and biomass of fathead minnows occurred at concentrations
-------
less than those affecting survival in all three tests. LOECs
for biomass and growth in total length were equivalent for
each test, ranging from 11 to 23 //g/L. LOECs for growth in
dry weight were the same as those for total length and
biomass in two of the three tests, but the LOEC for growth
In Test 2 (4 uglL) was much lower than the other endpoints.
Reduced growth of fathead minnows in Test 2 may reflect
the lower test temperature, as is suggested by reduced
growth in the control group. Alternatively, reduced within-
treatment variation in Test 2 may have increased the sta-
tistical power of the ANOVA. A13% reduction in growth of
fathead minnows was found to be statistically significant in
Test 2, compared to reductions of 17% at LOECs in both
Tests 1 and 3.
The two tests with rainbow trout, started with different life
stages, showed similar sensitivity to Cu toxicity (Table 6).
LOECs for survival and biomass (22 yug/L) were identical
for Test 4a, with exposure from eyed eggs through swim-
up, and Test 4b, which included only the swim-up stage.
This similarity reflects the fact that most mortality occurred
after swim-up in both tests. Effects of Cu on growth (in dry
wt) of rainbow trout were very similar between the embryo-
alevin-fry test (Test 4A) and the test with swim-up fry (Test
4B); however effects on growth at 1 Vg/L were statistically
significant InTest 4a, but not 4B. This difference may re-
flect a subtle, cumulative effect of Cu on growth of trout
during the longer exposure period in Test4A. Alternatively,
the significant reductions in growth at the LOEC and the
other intermediate Cu concentration may have been influ-
enced by greater densities of surviving fish in these treat-
ments, relative to controls (97-99% vs 89%). However,
treatment means for growth showed a consistent decreas-
ing trend with increasing Cu concentrations across a wide
range of survival, except for the greater average dry weight
of the few surviving fish at the highest Cu concentration.
Evaluation of, test endpoints and toxicity
metrics
Reduced growth and biomass of larval fish, which reflect
the physiologic or behavioral costs of Cu exposure, were
the most sensitive responses to chronic Cu exposure dur-
ing early life stage toxicity tests. Statistically significant
reductions in growth and biomass of three of the four spe-
cies occurred at Cu concentrations lower than those af-
fecting survival. The sensitivity of dry weight, total length,
and biomass endpoints were essentially equal, whether
expressed as LOECs or ICPs (Table 7). Biomass and
total length endpoints showed less among-test variation
than dry weight. Lesser variation in growth in total length,
compared to dry weight, may indicate that total length is
inherently less variable than dry weight, it may reflect the
technical and statistical advantages of multiple length mea-
surements of individual animals, compared to single mea-
surement of dry weight per replicate. The sensitivity and
low variability of the biomass endpoint reflect the fact that
biomass toxic effects on bothsurvival and growth, while
minimizing the possible influence of density-dependent re-
sponses.
The susceptibility of the dry weight endpoint to among-test
variation led to greater uncertainty about thresholds for
growth effects for fathead minnows and rainbow trout.
Among the potential causes for among-test variation are
differences in the statistical power of the ANOVA and den-
sity-dependent differences in growth. Although the level of
statistical significance and the number of replicates were
consistent in the current study, the statistical power of
ANOVA may have varied among tests due to differences
in within-treatment variation. Low within-treatment varia-
tion could have contributed to LOECs that correspond to
low percent reduction in dry weight relative to controls. Ex-
clusion of treatments with low survival from ANOVAs for
growth effects, which was intended to reduce the influence
of density differences on growth, also tended to increase
statistical power by reducing within-treatment variance
(Tables 5 and 6). Despite the exclusion of low-survival
treatments, differences in growth responses among treat-
ments could have affected toxicity thresholds for growth.
For example, the lower survival of fathead minnows in the
control group in Test 2 (93%), relative to the lowest Cu
concentration (98% survival), could have contributed to the
determination of a statistically-significant reduction in growth
in this treatment group, which produced the lowest LOEC
for this species. Conversely, density-dependent increases
in growth in treatment groups with reduced survival could
contribute to greater LOEC values. This density-depen-
dent response was observed in treatments with very low
survival (<30%) in tests with fountain darters, fathead min-
nows, and rainbow trout (Tables 4-6). In most cases, this
bias was avoided by disqualifying treatments with signifi-
cant reductions in survival from ANOVAs for growth ef-
fects.
Our results indicate that estimates of toxicity thresholds
based on the IGP linear interpolation technique do not dif-
fer greatly from ANOVA-based toxicity metrics. Some of
the presumed negative characteristics of LOECs were evi-
dent in our tests, as LOECs corresponded to a wide range
in percent reductions in survival, growth, or biomass rela-
tive to controls (9% to 76%), despite consistent experimen-
tal designs. The range of effect concentrations estimated
by IC10s and IC2ss differed somewhat from the NOEC-LOEC
range established by ANOVA (Table 7). In most cases,
IC25s were greater than LOECs, suggesting that the
ANOVAs had sufficient power to detect reductions of less
than 25% relative to controls. However, LOECs for total
length were substantially less than IC25s, and approached
IC10 values (Table 7). This trend is consistent with the lower
-------
within-treatment variation of total length data and associ-
ated higher power of ANOVAs for this endpoint. Despite
these differences in the influence of statistical power on
LOECs, almost all IC10s fell within the ranges defined by
NOECs and LOECs. As a result, IC10s corresponded
closely to ChVs, as these metrics differed by 2 ^g/L or
less in most tests (Figure 1).
Sensitivity of listed species to Cu toxicity
The two listed species represented the extremes of Cu
sensitivity for the four species tested. Species chronic val-
ues, (geometric means of the NOEC and LOEC), and IC10
values generally indicated that the fountain darter was the
most sensitive species tested and the spotfin chub was
the least sensitive species tested (Figure 1). The chubs
had the highest ChVs and IC10s for survival, growth (in dry
weight and total length), and biomass (Table 7). In con-
trast, the endangered fountain darter was at least as sen-
sitive to chronic Cu toxicity as either of the surrogate spe-
cies. The darter was more sensitive to effects of Cu on
survival than other species. However, lowest chronic val-
ues and IC10s for fountain darters (ChVs for growth and
biomass, 7.7; IC10 for biomass, 8.0 //g/L) were essentially
equal to those for surrogate species (lowest ChV for dry
IC10 for dry weight of
weight of rainbow trout, 8.1
fathead minnows, 8.6 //g/L).
Table 5. Survival, growth (dry weight and total length), and
biomass of fathead minnows (Pimephales promelas) in
early life- stage toxicity tests with copper/Values are
meanswith standard deviation in parentheses (N = 4).
Growth data in treatments with significant reductions in
survival (means below dashed lines) were excluded
from statistical analyses.
Test Cu Survival Dry Weight Total Length Biomass
(ug/L) (%) (mg) (mm) (mg)
1 5.4 100 .
9.3 95
12 92
17 95
0) 32.7
6[ 30.2
10) 27.1
6) 21.9
28 78 (13)* 24.0
50 70 (8r -T2.2
2 1.9 92
4.4 98
6.5 100
11 98
19 88
5
5
0
5
5
28.9
25.2
24.4
22.0
18.7
2.0
3.1
1.5
3.5
26.0
25.2
* 23.8
* 20.4
0.7) 326
1.2) 285
1.5)* 251
0.5* 207
4.3) 20.2 (0.5) 182
10.7) 15.6 (2.9) 80
2.3
0.3
1.8
1.5
0.6
25.4
* 24.7
* 24.1
* 23.2
* 20.3
0.6
0.2
0.5
0.5
1.5
267
245
243
214
* 164
40 55 (6)* 11.3 (4.0) 15.3 (1.3) 62
3 1.9 100
3.9 95
6.4 100
11 100
23 95
0
6
0
0
6
32.0
32,6
32.1
31.0
26.5
0.9
0.9
0.3
0.9
2.8
25.6
26.0
26.0
25.4
* 22.1
0.5
0.1
0.3
0.9
1.0
320
309
320
310
251
20)
(19)
(32)*
(22)*
(13)*
(60)*
!12)
11)
1 8)
1 6)*
9)*
(20)*
9)
«)
3)
9)
3 )*
42 22 (15)* 32.3 (7.2) 23.4 (2.6) 65 (31)*
1 indicate significant reductions relative to controls (P <0.05)
Previous studies also found that the fountain darter is highly
sensitive to Cu toxicity, relative to other listed and surro-
gate species. Dwyer et al. (1999a) found that fountain
darters were the most sensitive of 14 species tested in
acute toxicity tests with Cu in ASTM hard water (170 mg/L
as CaCO3). Median lethal concentrations of Cu in 96-h
tests were 57/u.g/L for the fountain darter and 90 yug/L for
the spotfin chub, compared to 80 yug/L for rainbow trout
and 470 /^g/L for fathead minnow. The high LC50 for
fathead minnows is consistent with the relative insensitiv-
ity of the survival endpoint in our early life-stage tests with
fathead minnows. Fountain darters are apparently more
sensitive to Cu toxicity than other darters (Percidae:
Etheostominae). Dwyer et al. (1999a) found that
greenthroat darters (E. lepidum), were substantially less
sensitive to Cu than fountain darters, with a 96-hr LC of
260 //g/L. Previous acute toxicity tests with Cu in a water
of similar hardness (200 mg/L as CaCO3) found 96-hr
LCSOs of 320 and 850 //g/L for rainbow darters (E.
caeruleum) and orangethroat darters (E. spectabile), re-
spectively, and 440 to 490 //g/L for fathead minnows
(Geckler et al. 1976). One-year chronic tests with adult
Johnny darters (E. nigrum) and fantail darters (E. flabellare)
in stream water with average hardness of 271 mg/L found
no effects on survival and growth of these species (or
fathead and bluntnose minnows) at Cu concentrations
ranging from 91 to 107 //g/L (Geckler et al. 1976).
Protectiveness of chronic WQC
Our results indicate that the endangered fountain darter
may not be adequately protected from chronic Cu toxicity
by the current national WQC for Cu. Populations of foun-
tain darters, and other aquatic species listed for recovery
under the authority of the Endangered Species Act, are
protected from any significant 'take' associated with expo-
sure to toxic chemicals. In contrast, guidelines for devel-
opment of numeric WQC have the goal of prevention of
'unacceptable effects' (defined as protection of 95% of
aquatic taxa tested) and knowledge that adverse effects
on some species may occur at concentrations below these
criteria (USEPA1985a). Chronic WQC are frequently de-
rived from acute criteria using acute-to-chronic toxicity ra-
tios (ACR), to take advantage of the larger data sets avail-
able from acute toxicity testing. The chronic criterion for
CU was derived from the acute CuWQC, using an aver-
age ACR of 2.823 (USEPA1985b, 1996a). Although Dwyer
et al. (1999a) concluded that the fountain darter would be
adequately protected by the national acute criterion forCU,
thresholds for chronic toxicity of Cu to fountain darter in
our early life-stage tests (both chronic values and IC10s)
are less than the current national water quality criterion of
14.7 //g/L (for water hardness of 170 mg/L, USEPA 1985b;
Figure 1). This lack of protectiveness of the chronic WQC
reflects the relatively large ACR for fountain darters, 7.125,
calculated from the results from our chronic tests and from
the acute tests conducted by Dwyer et al. (1999a).
Our results also suggest that the current chronic WQC for
Cu is not adequately protective of the surrogate species
-------
Tables. Survival, _.„ _.
(Test4A) andt -=..., , , --.-.
parentheses (N=4). Growth data in treatments with significant r
dashed lines) were excluded from statistical analyses.
irvival (means below
Test Cu Survival (%) Dry Weight Biomass
ftjg/L) pre-hatch pre-swim up swim up overall (mg) (mg)
4A 1.6 100 (C
3.7 100 K
6.2 100 iC
12 99 (;
)) 98 (2
)'i 99 i'2
)' 99 i'2
>) 100 {(
>) 92 (5) 89 (5 55.3 (2.4) 1228 32)
>'i 91
>'i 98
)) 100
2
0
90 (7
97 (4
99 (2
83
49.1
2.5) 1224 (59)
2.5) 1231 (19)
1.5)' 1214 (57)
22 99 (2) 95 (5) 80 (6)' 75 (10)' 47.8 (3.8) 892 (86)'
44 98 (4) 28 (13)' 40 (7J' 13 (8)' 55.7 (16.9) 159 (36)'
4B 1.6 NA NA 98
3.7 NA NA 96
6.2 NA NA 97
12 NA NA 98
22 N
44 N
2]
6
2
2
98 2
96 6
97 2
98 2
52.7 (
53.0
52.5
50.3
1.5 1291 (58)
2.2 1269 (45)
1.5 1272 (20)
1.0 1232 (4)
A NA 88 (3)' 88 (3)' 48.7 (1.7 1071 (60)'
A NA 24 (3)- 24 fry 55.3 (2.1 333 (54)'
* indicate significant reductions relative to controls (P < 0.05)
Table 7. Thresholds for chronic toxicity of Cu to four fish species in early life-stage toxicity tests. NOEC and LOEC = no observed
effect concentration and lowest observed effect concentration; IC10, IC25 = 10% and 25% inhibition concentrations. All values,
including 'less than' and 'greater than' values were used to calculate (geometric) means.
Test
Cu toxicity thresholds (ug/L)
Survival Dry Weight
NOEC-LOEC IC10 IC25 NOEC-LOEC IC10 IC25
Total Length
NOEC-LOEC IC25 IC10
Biomass
NOEC-LOEC IC10I IC25
1
2
Mean
3
1
2
3
Mean
4A
4B
5.4 - 9.3
6.5-11
5.9 - 9.9
23-42
17-28
19-40
23-43
20-36
12-22
12-22
<9.3
7.0
<8.1
32
19
19
24
21
17
22
<9.3
8.0
<8.6
- >42
36
29
28
31
24
27
5.4
6.5
5.9
11
9.3
1.9
11
5.8
6.2
12
->9.3
->11
->10
- 23
-12
- 4.4
- 23
-10
- 12
r >22
Fountain Darter
12 T3 5.4 -
12 15 6.5 -
12 14 5.9 -
Spotfin Chub
23 40 11 -
Fathead Minnow
10 T5 9.3 -
<4 11 7-
16 >23 11 -
<8.6 >16 8.8 -
Rainbow Trout
93 >22
>22 >22
>9.3
>11
>10
23
12
11
23
15
15
15
15
>42
12
11
20
14
17
19
18
>42
32
24
>42
28
5.4 -
6.5 -
5.9 -
11 -
9.3 -
7 -
11 -
8.8 -
12 -
12 -
9.3
11
10
23
12
11
23
15
22
22
<9.3
6.9
<8.0
23
<9.3
6.9
15
9.9
15
16
<9.3
7.9
8.6
33
12
13
24
15
21
25
-------
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tested. Chronic values and 1C s for reduced growth and
biomass of fathead minnows'0 and reduced growth of
raninbow trout in our studies are also lower than the cur-
rent chronic WQC for Cu (Figure 1). However, high varia-
tion in LOECs and ICPs from repeated tests with fathead
minnows resulted in some uncertainty about the threshold
for effects on growth. Among-test variation was greatest
for LOECs based on growth in dry weight, primarily due to
the extreme LOEC value for one test (4 /j.gl L in Test 2;
Table 4). Among-test variation was less for IC10s based
on dry weights, and the IC10for growth in Test 2 was less
xtreme than the growth LOEC. Among-test variation was
also lower for biomass and for growth in total length (Tables
4 and 5), and LOECs and IC10s for these endpoints sug-
gested lesser sensitivity to Cu toxicity. However, mean
thresholds for all three growth-related endpoints for fathead
minnows (dry wt., total length, and biomass), whether ex-
pressed as ChVs or IC10s, ranged from 8 to 14 //g/L, less
than the chronic WQC (Figure 1). The sensitivity of rain-
bow trout to Cu effects on growth in embryo-alevin-fry test
(Test 4A) was similar to that of fat head minnows, with
ChVs ranging from 8.1 to 15.5 ^g/L and IC10s ranging from
9.5 to 17 fj.glL. The ranges of these ELS thresholds for
both surrogate species are somewhat lower than the ranges
of ChVs previously reported for these species across a
range of water hardness: 11-27 for fathead minnows and
11-22 fzg/L for rainbow trout (Great Lakes Environmental
Research Laboratory [GLERL] 1998). "Our results suggest
that the current national chronic WQC for Cu is not ad-
equately protective of either the listed or the surrogate
species tested. A draft revision of the WQC forCu would
establish much lower chronic criteria for Cu (3.36 ^g/L at
170 mg/L hardness) that would be protective of all species
we tested (GLERL 1998)."
Generation of the additional toxicity data required to re-
vise all the existing WQC to assure adequate protection of
listed species would be a difficult task. An alternative
mechanism for increasing protection of listed species would
be to apply site- and species-specific 'safety factors' that
would apply to areas where listed species occur or to des-
ignated critical habitats. Dwyer et al. (1999a) concluded
that a safety factor of 0.3, applied to existing acute criteria,
would provide adequate protection for listed species. Our
results suggest that a safety factor of 0.5, applied to the
current chronic WQC for Cu, would provide an adequate
margin of protection for the species we tested. Further
research in our laboratory will repeat this series of studies
with additional chemicals. These studies will help deter-
mine if chronic WQCs for other chemicals are protective
of listed species, and whether the safety factor approach
may be more broadly applicable.
Conclusions
1. One of the two listed species tested, the fountain darter,
was at least as sensitive to early life-stage toxicity of Cu as
the two surrogate species tested, fathead minnow and rain-
bow trout. In contrast.the listed spotfin chub was the least
sensitive species tested.
2. Reduced average growth, especially growth in dry
weight, and reduced biomass of surviving fish were more
sensitive to Cu toxicity than survival for three of the four
species tested.
3. Results of three tests with fathead minnows suggested
that growth in dry weight may be subject to high among-
test variation, which may reflect density-dependent differ-
ences in growth among replicates and among treatments.
4. Lowest-observed-effect concentrations (LOECs) re-
flected a wide range of reductions in survival, growth, and
biomass relative to controls (9% to 76%).
5. Estimates of 10% and 25% inhibition concentrations
(IC10 and IC2!j), determined by a linear interpolation tech-
nique, tended to show less among-test variation in Cu tox-
icity than LOECs.
6. Toxicity thresholds for the endangered fountain darter
and for the two surrogate species tested (LOECs and IC10s
were lower than current chronic WQC for Cu at the water
hardness used in these studies.
7. A safety factor of 0.5, applied to the current chronic
water quality criterion, would be necessary to protect the
endangered fountain darter and the surrogate species we
tested.
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-------
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Stephan, C.E. and J.W. Rogers. 1985. Advantages on
using regression to calculate results of chronic toxicity
tests. In R.C. Banner and D.J. Hansen, eds., Aquatic
Toxicology and Hazard Assessment: Eighth Symposium.
STP 891. American Society for Testing and Materials,
Philadelphia, PA, pp. 328-339.
USEPA. 1985a. Guidelines for deriving numerical water
quality criteria for the protection of aquatic organisms and
their uses, EPA/833/R-85/100, Washington, DC.
USEPA. 1985b. Ambient water quality criteria for copper.
1984, EPA 440/5-84/031, Washington, D.C.
USEPA. 1994. Short-term methods for estimating the
chronic toxicity of effluents and receiving waters to fresh
water organisms, EPA/600/4-91/002, Washington, DC.
USEPA. 1996a. 1995 updates: Water quality criteria docu-
ments for the protection of aquatic life in ambient water,
EPA-820-B-96-001, Washington, DC.
USEPA. 1996b. Ecological effects test guidelines. EPA
712-C-96-121, Washington DC.
WEST, Inc. 1996. TOXSTAT 3.5. Western Ecosystems
Technology,, Cheyenne, WY.
Acknowledgments
The authors thank Eugene Greer for culturing the test or-
ganisms and Douglas Hardesty, David Whites, James
Kunz, Eric Brunson for their technical assistance during
these tests. We also thank Tom Brandt of the National
Fish Hatchery and Technology Center (San Marcos, TX),
and Pat Rakes of Conservation Fisheries, Inc. (Knoxville,
TN) for providing the fountain darters and spotfin chubs,
respectively. We thank Tom May, Ray Wiedmeyer, and
William Brumbaugh of the CERC Environmental Chemis-
try Branch for conducting the copper analysis. We appre-
ciate the thorough reviews of the manuscript by Denny
Buckler of CERC and Dr. Foster Mayer of the USEPA, Of-
fice of Research and Development, Gulf Ecology Division
(Gulf Breeze, FL).
11
-------
-------
U. S. Department of the Interior
U.S. Geological Survey
DETERMINATION OF Cu IN WATER FROM TOXICITY STUDIES
WITH ENDANGERED SPECIES
Final Report FYOO-32-04
Thomas May, Ray Wiedmeyer, and William Brumbaugh
U.S. Department of the Interior
U.S. Geological Survey
Biological Resources Division
Columbia Environmental Research Center
4200 New Haven Road
Columbia, MO 65201
October 29, 1999
PREPARED FOR
Chris Ingersoll and John Besser,
Toxicology Branch
CERC-Columbia
Studies 99-20-05 and 99-20-14
USGS
science fora changing world
A-1
-------
TABLE OF CONTENTS
Sample History A-3
Methods ' A-3
Results and Discussion A-3
Quality Control A-4
Tables:
A1. Instrumental Calibration A-5
A2. Reference/Research Materials A-6
A3. Spikes 4 . ." A-7
A4. Instrumental Precision '.' A-8
A5. Interference Checks A-8
A6. Instrument Detection Limit A-10
A7. Method Detection Limit and Limit of Quantitation A-11
A-2
-------
DETERMINATION OF Cu IN WATER FROM TOXICITY STUDIES
WITH ENDANGERED SPECIES
SAMPLE HISTORY:
From 2/26/99 to 10/4/99, various groups of water samples were received by the Inorganic
Chemistry section of the Columbia Environmental Research Center (CERC) The samples
were generated from toxicity studies investigating the effects of copper on endangered and
surrogate species of salmonids. In addition to samples collected during the toxicity tests
other samples served as "pre-test" samples to check out all aspects of diluter operation
before actual testing began. These "pre-test" samples required "quick turn around" and
thus minimal quality control as compared to toxicity study samples. The toxicity study
samples were designated Batch and CERC ID #s as follows: Batch 521 (CERC 19293 -
19302; Batch 524 (CERC 19361 - 19372; Batch 528 (CERC 19414 - 19433)- Batch 557
(CERC 20012 - 20025); Batch 563 (CERC 20143 - 20167); Batch 565 (CERC 20183 -
20190); Batch 570 (CERC 20488 - 20500); Batch 575 (CERC 20709 - 20720) For each
batch, analyses requested were for samples to be analyzed for Cu. The samples (60 or
100 ml_ each) were acidified with nitric acid prior to receipt (1 % v/v).
METHODS:
No further preparation was conducted on the samples prior to instrumental analysis
Analysis was performed with a PE/SCIEX Elan 6000 ICP-MS, which was set up and
optimized according to the manufacturer's specifications and described in CERC SOP
P.241. Samples were automatically delivered to the ICP-MS by means of a software-
controlled CETAC ASX-500/ADX-100 autosampler/autodilutor system which also
conducted all dilutions. All samples were predicted 10X by the autodiluter, and any
samples over the upper calibration standard of 20 ng/mL were automatically diluted 10X
in a serial fashion until concentrations were within the confines of the standard line The
internal standard was Ge (50ppb), which was metered into the sample line via peristaltic
pump. Calibration standards for analysis were 2,5,10, and 20 ng/mL for the element Two
masses were monitored for Cu (Cu-63 and Cu-65), but only Cu-63 was reported.
RESULTS AND DISCUSSION:
Concentrations of Cu in water samples determined by ICP-MS are presented in the Table
2 of the report. In most cases, copper concentrations agreed well with nominal values.C
A-3
-------
QUALITY CONTROL:
The samples were generally analyzed within a few weeks after receipt. The samples were
divided into eight groups for instrumental analysis, with each group identified by a separate
date (BID or block initiation date). In each of these instrumental runs, the following quality
control was included for the determination of Cu by ICP-MS: duplicate samples, dilution
checks, reference solutions, analysis spikes, and calibration checks. All quality control
results were tabulated to provide an overview of quality assurance and to facilitate
interpretation.
A calibration blank and an independent calibration verification standard were analyzed
every 10 samples to confirm the calibration status of the ICP-MS (Table A1). Results from
the analysis of two reference solutions are indicated in Table A2, where recoveries of Cu
were 100%. Recovery of the element from analysis spikes ranged from 95% to 99%
(Table A3). Precision from the duplicate analysis of water samples for Cu is indicated in
Table A4 and ranged from 0.3 to 3.7 relative percent difference (RPD). As a check for
potential interferences, water samples were manually diluted 10X, analyzed, then diluted
an additional 5X (Table A5). Dilution percent differences for both elements were < 10%.
A synthetic solution containing high concentrations of Al, Ca, Fe, Mg, Na, P, K, S, C, Mo,
and Ti was analyzed to observed the effects of these interferences on Cu (Table A6).
Copper recoveries ranged from 90% to 108%. Finally, the instrument detection limits,
method detection limits, and limits of quantitation for Cr are indicated in Table A7. Overall,
the quality control was considered within acceptable limits as specified by CERC.
Prepared By:
Thomas W. May
Ray Wiedmeyer
Research Chemists
Reviewed By:
William G. Brumbaugh
Research Chemist
Aooroved By:
Jim Petty
Chief, Environmental
Chemistry Branch
Approved By:
Wilbur L. Mauck
Center Director, CERC
A-4
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PERMIT No. G-35
Official Business
Penalty for Private Use
$300
EPA/600/R-01/051
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