EPA 903/R/98/005
CBP/TRS 198/98
February 1998
U.S. EPA Region III
Regional Center for Environmental
Information
1650'Arch Street (3PM52)
Philadelphia. PA 19103
Acute and Chronic Toxicity
of Copper to the
Estuarine Copepod
Eurytemora afflnis
Final Report
Chesapeake Bay Program
EPA Report Collection
Regional Center for Environmental Information
U.S. EPA Region III
nt.:l_ J.I.I.:. DA
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Acute and Chronic Toxicity of Copper to the
Estuarine Copepod Eurytemora affinis
Final Report
February 1998
Lenwood Hall
Ronald D. Anderson
Jay V. Kilian
Brent L. Lewis
Chesapeake Bay Program
410 Severn Avenue, Suite 109
Annapolis, Maryland 21403
1-800-YOUR-BAY
http://www.chesapeakebay.net/bayprogram
Printed by the U.S. Environmental Protection Agency for the Chesapeake Bay Program
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February 1998
Final Report
Acute and Chronic Toxicity of Copper to the Estuarine Copepod
Eurytemora Affinis
U.'-x f'\\K-.:t;iouliJ
};,-••••.. - n."i C--!rii:i;r for Environment:
IGGDAa-h Street (SPM5'^
1'lnladdphia, PA 19103
Lenwood W. Hall, Jr.
Ronald D. Anderson
Jay V. Kilian
University of Maryland
Maryland Agricultural Experiment Station
Wye Research and Education Center
P.O. Box 169
Queenstown, Maryland 21658
and
Brent L. Lewis
University of Delaware
College of Marine Studies
Lewes, Delaware 19958
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ABSTRACT
The objectives of this study were to conduct acute (48 and 96h) and chronic (8d) copper
toxicity tests with the estuarine zooplankter, Eurytemora affmis and develop an acute to chronic ratio
(ACR) for this species. Total dissolved copper, copper speciation and organic complexation were
measured on selected samples during these toxicity tests. Determination of organic complexation
was critical to determine the bioavailability of copper to the test species. Concentrations of total
dissolved copper at selected test conditions displayed a loss of 20 to 35% over the course of the
acute and chronic experiments with the most significant loss occurring during the first 48 hours.
Due to the reported loss of copper during exposures, a decay model was developed to calculate the
concentrations that were used to determine the final toxicity values (adjusted copper concentrations).
The 48h LC50, 96h LC50 and 8d chronic values were 83.0, 69.4 and 64 fj,g/L dissolved copper,
respectively, using the adjusted copper concentrations. An acute to chronic ratio of 1.3 was
calculated using the 48 h and 8 d toxicity values. Voltammetric analysis of selected samples
indicated greater than 99% complexation of copper in all samples with complexing capacity
increasing with both time and copper concentration. The inorganic copper concentration was
therefore a very small fraction of the total copper added during these experiments. Inorganic copper
(II) speciation predicted by a model identified seven significant species of copper. Of these seven
species, CuCO3 was the dominant species accounting for approximately 78% of the total copper.
The free cupric ion (Cu2+) accounted for only 8% of the total dissolved copper.
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ACKNOWLEDGMENTS
We would like to acknowledge the U.S. Environmental Protection Agency's Chesapeake
Bay Program Office for funding this study through grant number CB993438010. Special
consideration is extended to the following programs for their contributions to the study design
and support: Maryland Department of the Environment, Chesapeake Bay Program Office, and
the Toxics of Concern Workgroup of the Chesapeake Bay Program's Toxics Subcommittee.
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TABLE OF CONTENTS
Page
ABSTRACT I
ACKNOWLEDGEMENTS ii
INTRODUCTION 1
METHODS 3
Culture Procedures 3
Test Procedures 3
General Procedures for Copper Analysis 6
Total Dissolved Copper Analysis 7
Organic Complexation 7
Equilibrium Modeling 10
Statistical Analysis 11
RESULTS
Water Quality 13
Total Dissolved Copper Analysis 13
Voltarnmetric Analysis 17
Equilibrium Modeling 23
Toxicity Data 23
DISCUSSION 27
REFERENCES 31
APPENDIX A - Survival and reproduction data from the Eurytemora
48 h, 96 h and 8 d copper toxicity tests.
in
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INTRODUCTION
Copper occurs in natural waters primarily as the divalent cupric ion in free and complexed
forms (Callahan et al., 1979). This trace metal is a minor nutrient for both plants and animals at low
concentrations but is toxic to aquatic biota at concentrations only slightly higher. Copper has been
identified as a 'Toxic of Concern" in the Chesapeake Bay watershed and is therefore given priority
for water quality criteria development in the 1988 Chesapeake Bay Basin wide Toxics Reduction
Strategy (U. S. EPA, 1991). National water quality criteria for protection of marine life from copper
were developed by the U. S. Environmental Protection Agency (EPA) in 1980 and revised in 1984
(U. S. EPA, 1980; U. S. EPA, 1984). The existing EPA acute criterion for protection of marine life
is 2.9 ,ag/L. There is no saltwater EPA chronic criterion for copper. Available surface water data for
copper in the Chesapeake Bay suggests that background concentrations of 0.4 to 2.0 yUg/L total
copper are at or near the EPA criterion (Maryland Department of the Environment, 1991).
The U. S. Environmental Protection Agency (1984) acknowledged the similarity between
background concentrations of copper and the marine criterion. The EPA further recognized that
national water quality criteria may be under protective or over protective at specific sites due to
differences in sensitivities between test species at a particular site and those species used for deriving
the national criteria. Due to these issues. Maryland Department of the Environment (MDE)
established an estuarine acute water quality criterion of 6.1 ^zg/L using toxicity data for estuarine
species resident in Chesapeake Bay that were tested at salinities ranging from 1 to 35 ppt (Maryland
Department of the Environment, 1991). Insufficient copper toxicity data exists for MDE to derive
an estuarine chronic criterion for Maryland waters of Chesapeake Bay. One requirement of EPA's
"Guidelines for Deriving Numeral Water Quality Criteria for the Protection of Aquatic Organisms
1
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and their Uses"' for development of chronic criteria for saltwater species is a minimum of three
acute to chronic ratios (Stephan et al., 1985). No such data are currently available that meet all EPA
criteria.
The primary objective of this study was to generate data that can be used by MDE along with
other toxicity data to develop a chronic copper criterion for the protection of aquatic life hi estuarine
waters of the Chesapeake Bay and tributaries. Eurytemora affinis was selected as a test species
because it is one of the most dominant zooplankton species in the Chesapeake Bay, it has high
ecological importance and it is an essential component of trophic structure in the Bay (Ziegenfuss
and Hall, 1994). Acute (48 and 96 h) and chronic (8 d) copper toxicity tests were conducted with
the Eurytemora affinis. An acute to chronic ratio (ACR) was also developed for this zooplankton
species. Total dissolved copper, copper speciation, and organic complexation were measured on
selected samples measured during toxicity experiments. Organic complexation measurements were
particularly critical to determine the bioavailability of copper to the test species.
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METHODS
Culture Procedures
Eurytemora affinis cultures were maintained at 14-16 ppt salinity and 20-25 °C in our
laboratory. Culture water was autoclaved, filtered (1 /^m) estuarine water (14-17 ppt salinity)
obtained from the Choptank River at the University of Maryland, Horn Point Laboratory
(Cambridge, MD). Salinity was adjusted with H-W Marinemix or deionized water. The copepod
cultures were fed equal volumes of two phytoplankton species, Thalassiosira \veissflogii and
Isochrysis galbana. each maintained in log-phase growth. Phytoplankton were cultured in
autoclaved, filtered, and salinity adjusted Choptank River water supplemented with F/2 media
(Gillard, 1975).
Test Procedures
Test conditions used for these experiments are summarized in Table 1 and methods for
Eurytemora toxicity tests are described in detail in Ziegenfuss and Hall (1994). Copper chloride
(copper (II) chloride dihydrate) used in these experiments was obtained from J.T. Baker Inc.
(Phillipsburg, NJ, lot number J06342). Autoclaved, filtered and salinity adjusted (15 ppt) Choptank
River water was used as the diluent and control water for the acute and chronic toxicity tests. The
acute toxicity tests were 48 and 96 h static non-renewal exposures. The 8-d static-renewal chronic
test had a fifty percent renewal at each treatment on day 4. All test conditions were held hi a
biological incubator to maintain a constant temperature of 25 C and a photoperiod of 16 h light:8h
dark. Standard water quality parameters (temperature, salinity, pH and dissolved oxygen) were
recorded initially -and at the end of the exposure period for each test condition. Selected test
3
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Table 1. Test conditions used for Eurytemora affinis copper toxiciry tests.
1. Temperature:
2. Lighting:
3. Photoperiod:
4. Size of Test Vessel:
5. Volume of Test Solution:
6. Age of Test Copepods:
7. No. of Copepods per Test
Vessel:
8. No. of Concentrations:
9. No. of Replicates per
Concentration:
10. Feeding Regime:
11. Aeration:
12. Dilution Water:
13. Test Duration:
14. Effect Measured:
25 ± 2°C
100-150fc
16L:8hD
150 mL beaker
lOOmL
= 24h
12-16
6-7 (including controls)
Daily algal mixture
104 cells/ml for /. galbana
103 cells/ml for T. weissflogii
None, unless DO concentration falls below
40% saturation
Autoclaved filtered natural estuarine water (salinity
adjusted)
Acute test - 48 and 96 h
Chronic test - 8 d
Mortality (acute tests), mortality, fecundity
and maturation (chronic test)
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treatments were sampled initially and on the final day of each test and analyzed for total dissolved
copper and copper complexation to organic constituents.
The following nominal copper concentrations were used for the 48 h tests: 0,50,88,158,280
and 500 jug/L. For the 96-h acute toxicity experiment, the following nominal copper concentrations
were used: 0, 16, 28, 50, 90, 160 and 284 /ug/L. Nominal copper concentrations used for the 8-d
chronic test were: 0, 16, 26, 40, 64, 100 and 160 //g/L. Each test concentration was prepared by
diluting a working stock solution (100 mg/L) of copper chloride with salinity adjusted Choptank
River water. Stock solutions were prepared by dissolving 2.6828 g copper chloride dihydrate into
1 L deionized water for the primary stock solution (Ig/L copper) and diluting 50 ml of the primary
stock solution with 450 ml deionized water to make the working stock solution.
Acute and chronic toxicity tests were initiated with initial introductions of Eurytemora
nauplii (~ 24-h old). Nauplii were obtained by isolating adult copepods (202 //m sieve) in
polycarbonate jars containing salinity adjusted estuarine water for 24-h and harvesting the recently
hatched neonates using a 53 //m sieve. The adult copepod isolation chambers were supplied with
equal volumes of Thalassiosira \veissflogii and Isochrysis galbana algae at a ratio of 40 ml algae
mix/L of culture water. Four replicate 150 ml glass beakers containing 100 ml of test solution were
used for each condition. A test chamber was suspended within each beaker to contain the
organisms. Chambers were constructed from 3.8 cm diameter rigid polycarbonate tubing cut to a
length of 5.7 cm and suspended to provide an interior volume of approximately 40 ml. The bottom
of each chamber was covered with 53 jum mesh Nitex screen. Nauplii were counted by drawing
small aliquots of nauplii and water into a wide-bore glass pipet and examining under a dissecting
microscope (7.5 x magnification). The initial number of copepods (12-16) placed into each chamber
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was recorded on data sheets. Eurytemora were fed daily with 1.0 ml of a two-species phytoplankton
mixture comprised of equal volumes of Isochrysis galbana and Thalassiosira weissflogii. Algal
counts were made with a hemacytometer at least once during each acute test and twice during the
chronic test. The mean algal densities per test chamber were 3.6 x 104Isochrysis cells/ml and 3.4
x 103 Thalassiosira cells /ml. Survival was evaluated in each condition after 48-h and 96-h for the
acute tests and after an 8-d exposure period for the chronic test. Copepods were counted in the acute
experiments by first lowering the volume of solution in the test chamber, then removing the
remaining copepods and water in small aliquots with a pipet. Each aliquot within the pipet was
examined with the aid of a dissecting microscope for the presence of live copepods. The same
methods were used to count copepods in the chronic test except after they were counted the copepods
were deposited into individually marked (one vial per treatment replicate) 20 ml vials with 5 ml of
diluent. After each replicate sample had been counted and deposited into the vials, 2 ml of 10%
buffered formalin was added to each vial for storage of test species.
Preserved copepods were examined under a dissecting microscope and categorized as: gravid
female; non-gravid female; male and immature (See Ziegenfuss and Hall, 1994). The proportion of
gravid females (gravid/total females = fecundity) and proportion of copepods reaching maturity
(maturation) were the reproductive endpoints used.
General Procedures for Copper Analysis
Total dissolved copper and copper complexation measurements were conducted by the
College of Marine Studies, University of Delaware (Lewes, DE). Samples for analyses were filtered
(0.4 nm polycarbonate membrane) into precleaned (acid rinsed) polyethylene containers on the
6
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initial and final day of each experiment. Total dissolved copper samples were preserved with 0.2
ml Ultrex II nitric acid (J.T. Baker Inc., Phillipsburg, NJ) per 100 ml of sample. Samples for
complexation measurements were collected in precleaned polyethylene bottles and immediately
frozen. All samples were transported from the Wye Research and Education Center to the analytical
lab at the University of Delaware. The following test conditions from the 48 h acute test were
measured for dissolved copper at the initiation of the test and 48 h later : 0, 50, 158, and 500 //g/L.
The following test conditions were measured for dissolved copper at test initiation and termination
from the 96 h acute test: 0, 50, and 160 /^g/L. During the 8d chronic tests, dissolved copper was
measured at test initiation and termination at the following conditions: 0, 16, 40, and 160 ftg/L.
Total Dissolved Copper Analyses
Total dissolved copper concentrations were measured by Inductively Coupled Plasma -
Atomic Emission Spectroscopy (ICP-AES). Concentrations were determined by comparison of
sample emission values with a linear calibration curve. Working standards were prepared by serial
dilution of a 1000 ppm commercial ICP copper standard (Inorganic Ventures, Inc., Toms River, NJ).
Standards were prepared in an organic-free (ultraviolet-irradiated) natural seawater matrix, diluted
to 15 ppt salinity and acidified to 0.1 N with high purity quartz-distilled nitric acid. The matrix of
the standards was equivalent to that of the samples, correcting for any signal suppression due to
seasalts in the samples. Analytical sensitivity was 0.0957 emission units/(/^g/L-Cu), with a detection
limit of approximately 3 /^g/L and precision of 2-4%.
Organic Complexation
The "copper complexation capacity" and the conditional stability constant for copper binding
(Log K'CuL) were determined for selected samples by anodic stripping voltammetry, using an EG&G
7
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Princeton Applied Research (PAR) Model 384B-4 polarographic analyzer with a PAR Model 303A
mercury drop electrode. Analyses were performed in the square wave anodic stripping voltammetry
(SWASV) mode with a hanging mercury drop electrode (HMDE). Copper was reduced and
deposited in the mercury drop for one to three minutes at a deposition potential of -0.30 V.
Instrumental parameters for reoxidation and stripping of the copper from the mercury drop were:
scan rate = 200 mV sec"1, pulse amplitude = 20 mV, pulse frequency = 100 Hz, scan range = -0.3 to
-0.05 V. Copper complexation capacity (CuCC) is defined as the total concentration of all metal-
binding sites or ligands in solution (e.g. Robinson and Brown, 1991). The CuCC values and the
conditional stability constants (K'CuL) were determined by complexometric titration with a Ruzic-type
linearization, after the manner of Coale and Bruland (1988, 1990).
The complexometric titration method included the titration of excess binding ligand in
solution with copper. When the measurements were obtained by ASV, it was assumed that the
current measured at a fixed deposition potential was due solely to the free metal ion and/or weak
inorganic complexes. Metal-ligand organic complexes were assumed to be electroactively inert at
that same deposition potential. The formation of a complex is described by the reaction:
M' + L' = ML (1)
where M' is the sum of the dissolved inorganic metal species and L1 is the excess "free" binding
ligand. The equilibrium expression for the formation of the complex was:
(2)
where K'ML is the conditional stability constant of the metal-organic complex. For a sample
containing a moderate to strong complexing ligand, the data from a titration was linearly transformed
8
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to allow calculation of the total concentration of the ligand. [L1], and the conditional stability
complex for the metal-ligand complex (Ruzic, 1982).
Coale and Bruland (1988, 1990) have described the application of the linearization technique
to the determination of copper complexation in seawater. For the formation of a single strong zinc-
ligand species, the linearization equation is:
[Cu']/[CuL] = [Cu']/[L] + 1/(K'CuL x [L]) (3)
A plot of [Cu' ]/[CuL] versus [Cu' ] for each titration point will yield a straight line with a slope
of [L1]-1 (- l/CuCC)and intercept of (K'CuL x [L])'1.
Assuming that there are no electroactive organic copper complexes in solution, the measured
current was directly proportional to the concentration of Cu', where [Cu'] was defined as the total
concentration of electroactive dissolved inorganic copper species present (i.e. the Cu2+ ion plus
inorganic complexes). [Cu'j was calculated from equation 2, and was related to the concentration
of the free ion, [Cu2+], by an inorganic side reaction coefficent, aCu. such that [Cu/]=[Cdf ]aca
(Ringbom and Still, 1972). The organically bound fraction, CuL, was the difference between the
total dissolved Cu (determined by ICP-AES) and [Cu' ]. In the absence of total inorganic carbon
or alkalinity measurements, the samples were assumed to be in equilibrium with the atmosphere with
respect to the carbonate system (pc02 = 10'35 atm, pH = 8.2, t = 25°C., Ionic strength = 0.3). Under
these conditions, aCu- » 13.1.
Titrations were conducted using an acidified 1000 /ug/L Cu2+ standard, which reduced the
final pH of the samples to * 2. Acidification prevented loss of added copper by precipitation or
adsorption. The lower pH, however, resulted in an underestimation of the CuCC and an
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overestimate of the Log K'CuL relative to the values at the in situ pH (e.g. Kozarac et al., 1989;
Skrabal et al. 1992). To account for this offset, two samples were titrated with an unacidified Cu2+
standard at the sample pH of about 8.2. The ratio of the 8.2 CuCC to that at pH 2 was approximately
1.71 ± 0.4. This value was similar to the ratio observed by Skrabal et al., 1992 for samples collected
in Indian River Bay, DE (CuCC ratio * l .86). The Log K'CuL value at pH 8.2 was approximately
7.67 ± 0.38. Then the concentration of Cu' was:
[Cu'] = [CuL]/(K'CuL[L]) * [Cu]TOTAL/(K'cuL [CuCC]) (4)
Complexation was further verified by analysis of copper by square wave voltammetry (SWV)
before and after UV-irradiation to oxidize organic matter and by measurement of the copper
"pseudopolarogram". A pseudopolarogram is plot of ASV stripping peak current versus deposition
potential. The resulting curve is equivalent to a DC polarogram. For a given metal, a
pseudopolarogram will display one or more relatively sigmoid-shaped polarographic waves,
corresponding to the free ionic and complexed species. The position and shape of each wave is
dependent upon the binding strength of the metal-ligand complex and upon the reversibility of the
chemical and electron transfer processes at the electrode (Lewis et al., 1995).
Equilibrium Modeling
The solution speciation of copper with respect to free hydrated ions and inorganic complexes
hi the sample solutions was estimated using the program EASEQL, an interactive PC version of the
original MINEQL equilibrium modeling program. MINEQL (Westall et al., 1976), and its
predecessor REDEQL (Morel and Morgan, 1972), utilizes equilibrium constants to solve mass
balance expressions, using a modified Newton-Raphson iterative procedure. The model was run
10
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assuming pH = 8.2. T = 25°C, I = 0.3, and an open system in equilibrium with the atmosphere (pc02
= 10-35atm.
Statistical Analysis
The 48 and 96-h LC50 values with 95% confidence limits (acute tests) were generated from
the mortality data using the Trimmed Spearman-Karber method. Statistical analysis for the chronic
experiment was conducted by using ANOVA procedures with subsequent means testing (Dunnett's
test) with copper concentrations (adjusted with decay rate (s)) to determine the No Observed Effect
Concentration (NOEC) and the Lowest Observed Effect Concentration ( LOEC) values. Endpoints
used were mortality, fecundity and maturation. The chronic value was determined by calculating the
geometric mean of the NOEC and LOEC for the most sensitive endpoint.
Due to the loss of copper during the experiments an exponential decay model was used to
determine the acute and chronic copper concentrations used for analysis. The model used was as
follows:
Cu(t) = Cu (0) exp(rt)
where
Cu(t) is copper concentration at time t
Cu(0) is initial copper concentration
exp is the exponential function
r is the rate of copper absorption
t is time
For these experiments integrating Cu(t) over the exposure time and dividing by the exposure time
yielded an estimate of the average exposure concentration for the period. Eight observations were
available to estimate copper absorption (r) in these experiments. Data from the different test
durations were analyzed by a GLM procedure from SAS to determine if test duration influenced
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decav rates.
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RESULTS
Water Quality
Ranges of water quality conditions measured at test initiation and test termination for the 48
h, 96 h and 8d tests are presented in Table 2. Water quality conditions for the 8-d chronic test were
also measured on day 4 in addition to the initial and final day of the experiment. All water quality
conditions were adequate for survival of test species.
Total Dissolved Copper Analyses
The results of the total dissolved copper analyses are presented in Table 3. Measured values
for the control samples were all below the detection limit of 3 /^g/L. The experimental samples
collected on Day 0 of each test generally displayed good agreement (less than 15% deviation)
between nominal and measured values. All experimental samples displayed a mean loss of
approximately 31% dissolved total copper over the course of the experiment. Most of the copper
loss was reported during the first 48 hours.
Due to the reported loss of copper during the acute and chronic experiments a decay model
(see methods section) was used to determine the concentrations used for statistical analysis of the
toxicity data. The following eight observations were used to estimate the rate of copper loss.
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Table 2. Ranges in water quality conditions during acute and chronic copper toxicity
tests with Eurytemora affinis.
Test Type Temperature Salinity (ppt) pH D.O. (mg/L)
£C)
Acute 48-h 24.1-24.6 14 7.9-8.7 7.5-8.8
Acute 96-h 24.0-25.1 15-16 7.9-8.8 7.4-9.8
Chronic 8-d 24.1-25.1 14-16 7.9-8.8 7.2-8.9
14
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oo
3 S
-_— ^
73 "^
'J C3
-»— r-*
"-> "2
3 S
_- "O
CO %
•I g?
•6'g
^^ T3
~ en
co e
=1.2
.2 S
•a o
u o
O 1)
n
u •"
Q.T3
D. P
P b
C3 i-
1 H
•E S
a O
E g
£ =
_Z o
o en m
en O
__
oo
T3
OO
15
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Obs
1
2
3
4
5
6
7
8
Test
48h
48h
48h
96h
96h
8d
8d
8d
T
48
48
48
96
96
96
96
96
Nom
50
150
500
50
160
16
40
160
CuO
50.4
155.7
436.0
54.6
165.0
18.4
43.1
161.9
CuT
39.4
117.3
351.0
36.6
114.5
13.0
20.4
103.8
P
0.78175
0.75337
0.80505
0.67033
0.69394
0.70652
0.47332
0.64114
L
0.21825
0.24663
0.19595
0.32967
0.30606
0.293448
0.52668
0.35886
Two different exposure times were represented: 48 hours for the 48 hour test and 96 hours
for both 96 hour and 8 day tests. Copper concentrations were renewed at day 4 during the 8-d test.
These data were first analyzed to determine if test duration influenced loss of copper. The results
were as follows:
Source
T
T*Test
DF
1
2
Type III SS
0.8962
0.0213
Mean Square
0.8962
0.0106
F value
49.71
0.59
Pr>F
0.0009
0.5879
These results showed that there was no significant interaction of decay rate and exposure duration
(p=0.58). Therefore all eight observations were used to estimate a single decay rate of-0.0049 as
shown below.
Parameter
T
Estimate
-0.0048521
T for HO
-8.85
Pr>T
0.0001
Std Error Est.
0.000548
16
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The average exposure concentration for each nominal concentration (NOMADJ) was
calculated using a decay rate of 0.0048522 as presented in Table 4. These adjusted concentrations
were used to calculate the toxicity values presented below in the Toxicity Data section.
Voltammetric Analyses
Ten samples from the chronic test, including the stock water and two controls, were selected
for speciation analysis. A typical complexometric titration curve and the corresponding linear
transformation plot are shown in Figure 1. The results are summarized in Table 5. In each instance,
the estimated copper complexing capacity at pH 8.2 exceeded the total copper concentration, with^
99% complexation in all instances. The inorganic copper concentration ([Cu' ]) was therefore a very
small fraction of the total copper added, ranging from about 0.15 to 1.2 /zg/L for the experiment.
The corresponding cupric ion concentrations would be approximately a factor of 13 lower, from
about 0.01 to 0.09 f^g/L. Assuming a total copper concentration of 1 Mg/L, the Cu' concentration
for the controls would be O.06 ,ug/L Cu2+
The complexation of copper by organic compounds was verified by analysis of selected
samples before and after UV-irradiation (Figure 2) and by the construction of a copper
pseudopolarogram (Figure 3). The 160 /ug/L sample from the chronic test was analyzed by Square
Wave Voltammetry without stripping. Figures 2a and 2b display the resulting current vs. potential
scans for the day 0 and the day 8 samples, respectively, while 2c shows the scan for the day 8 sample
following UV-irradiation. On day 0 of the chronic test, the sample displayed peaks at -0.16 V for
labile copper (Cu') and at -1.35 V for a strong Cu(II)-organic complex. For day 8 of the test, scan
17
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Table 4. Nominal adjusted copper concentrations (NOMADJ) for various test conditions
using a decay rate of R of-0.0048522. These values were used for analysis of
toxicity data.
OBS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Test
48h
48h
48h
48h
48h
96h
96h
96h
96h
96h
96h
8d
8d
8d
8d
8d
8d
Duration
00
48
48
48
48
48
96
96
96
96
96
96
96
96
96
96
96
96
Nominal
50
88
150
280
500
16
28
50
90
160
284
16
26
40
64
100
160
R
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
-.0048522
NOMADJ
44.604
78.504
133.813
249.784
446.043
12.791
22.384
39.971
71.947
127.906
227.033
12.791
20.785
31.976
51.162
79.941
127.906
18
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figure i. (aj.bWA^v complexometnc titration tor copper in 15 pptestuarine water at pH 8.2. (Chronic 40
at day 8). The solid line is the linear regression taken through the last four data points, (b). Linear transformation
of the data (Ruzic, 1982). Solid and dotted lines represent the linear regression through the data and the 95%
confidence interval, respectively.
800
700
600
< 500
L r2 =0.985
O
400
300
200
100
0
0
50 100 150
Total Copper (pg/L)
200
40 60 80
[Cu'] (ug/L)
100 120
19
-------
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20
-------
Figure 2. SW voltammograms of copper: (a) chronic test condition of 160 ^g/L at day = 0, (b, c) 160
chronic test condition on day = 8 before and .after UV - irradiation.
X \i
•96 CELL «1
2u -alii^1 m
1.519E2 Nfl
kllBEl Nfl
'-9.1 -8.3 -fl.S -8.7-8.9 -!."[ -1.3 -l.S -1.7
C
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(C)
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•1
E 0;gm v
'tiife
NH
-8.1 -8.3 -8.5 -fl.7 -8.9 -1.1 -1.3 -1.5
Potential (V)
21
-------
Figure 3. SWASV pseudopolarogram for copper in 15 ppt estuarine water at pH 8.2 (120 sec deposition) at chronic
test condition 40 /zg/L on day 8.
500
450 r
400 -
350
300
§ 250
O 200
150
100
50
E',a = -0.84
/
/
E'ia = -1.40
-0.2 -0.4 -0.6 -0.8 -1.0 -1.2 -1.4
Deposition Potential (V)
-1.6 -1.8
22
-------
displayed only the latter peak, indicating that essentially all of the copper was now strongly
complexed. The complex peak did not increase in size, therefore, suggesting that additional
complexes were formed, but were apparently either electrochemically inert or below the detection
limit for SWV without stripping. Upon UV-irradiation, the complex peak disappeared and the
signal for labile copper reappeared, indicating breakdown of organic copper complexes. The
titration plot for the UV-irradiated aliquot (not shown) still exhibited some degree of nonlineariry,
indicating the presence of residual complexing material in the sample. A pseudopolarogram (Figure
3) performed on the 40 /ug/L sample on day 8 of the chronic test indicated the presence of at least
two classes of copper chelating ligands, with E' 1/2 values of-0.84 and -1.40 V. The latter agrees well
with the complex peak observed in the 160 /^g/L samples from Figures 2a and 2b.
Equilibrium Modeling
The inorganic copper(II) speciation as predicted by EASEQL is given in Table 6. Seven
species were identified as significant (>0.1%); the free hydrated ion, Cu2+, carbonate and sulfate
complexes, CuCO3, Cu(CO3)22' and CuSO4, two chloride complexes, CuCl+ and CuCl2, and the
hydroxide species Cu(OH)". Of these seven, the speciation is dominated by the CuCO3 species,
which accounts for approximately 78% of the total copper. The free cupric ion, Cu2+, accounts for
only about 8% of the total dissolved copper.
Toxicity Data
Toxicity values were determined using the adjusted copper concentrations presented in Table
4. The 48h LC50, 96 h LC50 and 8 day chronic value of 83. 69.4 and 64.0 yttg/L, respectively are
23
-------
Table 6. The inorganic speciation of Cu(II) in estuarine water as predicted using the
EASEQL thermodynamic equilibrium program (pH = 8.2, T = 25C, 1 = 0.3, pco2
10-35atm, [Cu']<1.2ppb).
Copper Species
Cu2+
CuC03
Cu(C03)22-
CuS04
cucr
CuCl2
CuOH-
Percentage of Total Cu'
7.6
78.2
3.8
0.6
1.9
0.1
7.7
-------
reported in Table 7 (see Appendix A for raw data). The chronic test endpoints of percent survival,
percent gravid females and percent immatures all resulted in adjusted copper NOEC values of 51.1
{j.g/L and LOEC values of 79.9 /wg/L. The 8-d chronic value of 64 //g/L in conjunction with the 48-
h acute value (83 Mg/L) were used to calculate an acute to chronic ratio of 1.3. Also shown in Table
7 are the control group survival values for all three tests. These values, ranging from 85.7 to 94.1%,
are typical for Eurytemora control survival based on our previous studies (Ziegenfuss and Hall,
1994).
25
-------
Table 7. Forty-eight and ninety-six hour LC50 values (//g/L) and 95% confidence limits
for acute copper toxicity tests with the estuarine copepod Eurytemora affmis.
The chronic value in pg/L (no confidence limits) for the 8-d chronic test is also
given. Mean % control survival for each test duration is included.
Test Type Mean % Control Survival LC50/Chronic Value
(S.E.) (95% C.L.)1
48-h Acute 85.7 (7.0) 83.0
(75.21-91.68)
96-h Acute 94.1 (3.3) 69.4
(60.70 - 79.45)
8-d Chronic 90.9 (4.6) 64.0
'LC50 values are given for the acute tests. A chronic value is given for the chronic test and no
confidence limits are associated with this value.
26
-------
DISCUSSION
The chemistry measurements for total dissolved copper and copper complexation capacity
were important in determining the concentrations of copper available to the test species. Over the
course of the experiments, approximately 20 to 35% of the copper was lost with the most significant
loss during the first 48 hours. Possible reasons for loss of copper during these experiments were
adsorption to the vessel walls or particulate matter in the test chambers and/or biological uptake by
Eurytemora or the phytoplankton used as a food source.
Voltammetric analysis of selected samples indicated greater than 99% complexation of
copper in all samples, with copper complexing capacities increasing with both time and total copper
concentration. The copper complexing capacity increased with tune by a factor of approximately 1.2
to 1.6 for each total dissolved copper treatment, and with total copper concentration by nearly ten-
fold relative to the stock water. While the source of the additional complexing capacity cannot be
determined with certainty, it is likely that it resulted from the production and release of strongly
binding copper chelators from the phytoplankton utilized as a food source. Moffett and Brand (1996)
have recently described the production by the marine cyanobacteria Synechococcus spp. of strongly
binding extracellular copper chelators, in response to increasing copper stress. The results observed
in the present study suggest a similar response. The production of chelating compounds from
Eurytemora cannot be ruled out, but is probably less likely.
The acute toxicity data reported in this study can be compared with a distribution of species
response data determined from an analysis of existing copper toxicity data from various trophic
groups (Figure 4). For both the 48 and 96 h acute values, the rank in species response is in the 35
to 40% range. These data suggest that Eurytemora has a moderate to sensitive response to copper
27
-------
Figure 4. Distribution of acute copper toxicity data from various trophic groups. Arrows show how our 48 and
96 h LC50s compare and rank with these data.
Saltwater Acute Copper
10°
benthos
fish
zooplankton
macrophytes
phytoplankton
Plot 1 Regr
96h acute value of 69.4 ug/L
48h acute value of 83.0 ug/L
b[0] = -2.05976
b[1] = 0.92002
1^ = 0.96
28
-------
stress. Two other zooplankton species, the copepod Acartia tonsa (Sosnowski and Gentile, 1978;
Gentile, 1982)) and the rotifer, Brachionus plicatilis (Snell and Personne, 1989) had acute LC50
values of 17 to 52 /zg/L which are lower than we reported in our experiments. In a previous acute
copper toxicity experiment with Eurytemora affinis. Gentile (1982) reported an LC50 of 928 /ug/L
which is significantly higher that the two acute values reported in this study. In contrast, Sullivan
et al. (1983) reported 96 h LCSO's for larval E. affinis ranging from 28.7 to 33.7 wg/L which compare
favorably with our data.
Chronic saltwater toxicity data with copper were limited. The chronic value of 64 //g/L
reported for Eurytemora in our study is similar to the chronic value of 54 /^g/L reported by Lussier
et al. (1985) for the mysid, Mysidopsis bahia. Acute to chronic ratios from our study (1.3) and the
Lussier et al. (1985) study (3.3) are both very low thus indicating a narrow range between acute and
chronic responses. This is not a surprising result since low acute to chronic ratios have also been
reported for other heavy metals (Lussier et al., 1985).
A comparison of the acute and chronic Eurytemora toxicity values (64 to 83 ^g/L dissolved
copper) with environmental concentrations of copper measured in the Chesapeake Bay watershed
can be conducted to provide insight on possible ecological risk. The Toxics of Concern Workgroup
of the U.S. Environmental Protection Agencies Toxics Subcommittee assembled available data on
environmental concentrations of copper in the Chesapeake Bay (U.S. EPA, 1996). Based on 764
copper measurements ranging from < 1 to 990 ,ug/L, a mean value of 4.7 //g/L was determined.
Using a very simplistic quotient method, this mean exposure value is significantly lower than any
of the three toxicity values reported above. This comparison would generally suggest minimal
ecological risk to Eurytemora from copper exposure although effects would be possible at some of
29
-------
the higher environmental concentrations if these exposures existed for extended periods of time.
Future efforts are planned for conducting a probabilistic ecological risk assessment with copper
using a distribution of both effects and exposure data for the Chesapeake Bay (SETAC, 1994). This
probabilistic approach has a number of advantages over assessments based on single measures of
effects and exposure because it uses all relevant single species toxicity data and when combined with
exposure distributions allows for a quantitative estimation of risks to aquatic organisms.
30
-------
REFERENCES
Callahan, M.A. 1979. Water-related environmental fate of 129 priority pollutants. Vol I. EPA-440/4-
79-029a. National Technical Information Service, Springfield, VA.
Coale, K.H. and K.W. Bruland. 1988. Copper complexation in the Northeast Pacific. Limnol.
Oceanogr., 33:1084-1101.
Coale, K.H. and K.W. Bruland. 1990. Spatial and temporal variability in copper complexation in
the North Pacific. Deep-Sea Research, 47:317-336.
Gentile, S.M. 1982. Memorandum to John H. Gentile. U. S. Environmental Protection Agency,
Narragansett, R. I.
Guillard, R.R.L. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Culture of
Marine Invertebrate Animals. W. L. Smith and M. H. Chanley (eds.), pp 29 -60. Pleum
Publishing New York, NY.
Kozarac, Z., M. Plavsic and B. Cosovic. 1989. Interaction of cadmium and copper with
surface-active organic matter and complexing ligands released by marine phytoplankton.
Mar. Chem., 26:313-330.
Lewis, B.L., G.W. Luther III, H. Lane and T.M. Church. 1995. Determination of metal-organic
complexation in natural waters by SWASV with pseudopolarograms. Electroanalysis, 7:166-
177.
Lussier, S. M.. J. H. Gentile and J. Walker. 1985. Acute and chronic effects of heavy metals and
cyanide on Mysidopsis bahia (Crustacea:Mysidacea). Aquatic Tox. 7: 25-35.
Maryland Department of the Environment (MDE). 1991. Aquatic Life Criteria for Copper. Report.
31
-------
Maryland Department of the Environment. Baltimore, MD.
Moffett, J.W. and L.E. Brand. 1996. Production of strong, extracellular Cu chelators by marine
cyanobacteria in response to Cu stress. Limnol. Oceanogr., 41:373-387.
Morel, F. and J.J. Morgan. 1972. A numerical method for computing equilibria in aqueous
chemical systems. Environ. Sci. Tech., 6:58-67.
Ringbom, A. and E. Still. 1972. The calculation and use of alpha coefficients. Anal. Chim. Acta,
59:143-146.
Robinson, M.G. and L.N. Brown. 1991. Copper complexation during a bloom of Gymnodinium
sanguineum Hirasaka (Dinophyceae) measured by ASV. Mar. Chem. 33:105-118.
Ruzic, I. 1982. Theoretical aspects of the direct titration of natural waters and its information yield
for trace metal speciation. Anal. Chim. Acta, 17:99-113.
SET AC (Society of Environmental Toxicology and Chemistry) 1994. Aquatic Dialogue Group:
Pesticide Risk Assessment and Mitigation. Report by the Aquatic Risk and Mitigation
Dialogue Group. Pensacola, FL.
Skrabal, S.A., G.W. Luther, III, and H.E. Allen. 1992. Chemistry and bioavailability of copper in
Indian River Bay, Delaware. Univ. of Delaware, College of Marine Studies and Dept. of
Civil Engineering, Project Completion Report, unpublished document.
Snell, T.W. and G. Personne. 1989. Acute toxicity bioassays using rotifers. I. A test for brackish and
marine environments withBrachionusplicatilis. Aquatic Tox. 14:65-80.
Sosnowski, S.I. and J. H Gentile. 1978. Toxicological comparison of natural and cultured
populations ofAcartia tonsa to cadmium, copper and mercury. Jour. Fish. Res. Board Can.
35: 1366-1369.
32
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Stephan. C.E.. D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman and W.A. Brungs. 1985.
Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of
Aquatic Organisms and Their Uses. U.S. EPA, Office of Research and Development,
Washington, DC.
Sullivan, K.R. et al. 1983. Effects of copper and cadmium on growth, swimming and predator
avoidance in Eurytemora affinis (Copepoda). Mar. Biol. 77:299-307.
United States Environmental Protection Agency (U.S. EPA). 1980. Ambient water quality criteria
for copper. Report EPA 440/5-80-036. Office of Water Regulations and Standards,
Washington, DC.
United States Environmental Protection Agency (U.S. EPA). 1984. Ambient water quality criteria
for copper. Report EPA 440/5-84-031. Office of Water Regulations and Standards,
Washington, DC.
United States Environmental Protection Agency (U.S. EPA). 1991. Chesapeake Bay toxics of
concern list information sheets. Report. U.S. EPA Chesapeake Bay Program Office,
Annapolis, MD.
United States Environmental Protection Agency (U. S. EPA). 1996. Chesapeake Bay toxics of
concern list. Draft report. U. S. EPA Chesapeake Bay Program Office, Annapolis, MD.
Westall, J.C., J.L. Zachary and F.M.M. Morel. 1976. MINEQL, A Computer Program for the
Calculation of Chemical Equilibrium Composition of Aqueous Systems. Tech. Note 18,
Dept. of Civil Eng., Mass. Inst. Technol., Cambridge, MA.
Ziegenfuss, M.C. and L.W. Hall, Jr. 1994. Standard operating procedures for conducting acute and
chronic aquatic toxicity tests with Eurytemora affinis, a calanoid copepod. Report U.S
33
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Environmental Protection Agency, Chesapeake Bay Program Office, Annapolis, MD.
34
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APPENDIX A
Survival and reproduction data from the
Eurytemora 48 h, 96 h and 8 d copper toxicity tests
-------
ACUTE /CHRONIC COPPER TOXICITY TEST DATA
Final Copepod Survival / Initial Nauplii Per
Replicate
Test Nominal
CuGwg/L)
48-h Acute 0
50
88
158
280
500
96-h Acute 0
16
28
50
90
160
284
8-d 0
Chronic 16
26
40
64
100
160
A
14/14
14/14
8/13
0/14
0/16
0/15
13/15
12/12
9/12
13/13
8/13
0/13
0/14
13/15
12/12
11/13
10/12
8/13
8/13
0/12
B
9/13
11/12
11/15
0/12
0/15
0/13
12/12
12/12
10/12
11/13
7/14
0/12
0/12
12/14
12/12
10/12
11/13
11/14
4/14
0/12
C
11/14
11/15
7/13
0/12
0/12
0/13
12/12
11/12
13/13
9/13
11/12
0/13
0/12
15/15
13/13
10/12
11/12
13/13
3/13
0/13
D
14/15
13/15
9/14
0/12
0/13
0/12
11/12
10/13
13/14
11/13
5/13
0/14
0/14
i
10/12
14/15
11/14
10/12
8/12
0/13
Mean %
Survival
85.3
87.5
63.6
0.0
0.0
0.0
94.6
91.8
88.2
84.6
59.6
0.0
0.0
90.9
95.9
86.5
84.3
84.0
44.2
0.0
'Replicate broken during course of experiment.
A-l
-------
CHRONIC COPPER TOXICITY % GRAVID FEMALES
Final Gravid Females / Initial Nauplii Per Replicate
Test
8-d
Chronic
Nominal
Cu (//g/L)
0
16
26
40
64
100
160
A
6/15
2/12
7/13
2/12
2/13
0/13
0/12
B
4/14
3/12
2
5/13
1/12
0/14
0/12
C
6/15
2
4/12
3/12
6/13
0/13
0/13
D
i
3/12
2
6/14
5/12
0/12
0/13
Mean %
Gravid
39.8
23.9
51.8
36.8
32.6
0.0
0.0
'Replicate broken during course of experiment.
2Replicates where the number of preserved individuals did not correspond with the number of
individuals counted at end of test.
CHRONIC COPPER TOXICITY % IMMATURE
Final Immature / Initial Nauplii Per Replicate
Test
8-d
Chronic
Nominal
Cu (//g/L)
0
16
9fi
40
64
100
160
A
1/15
2/12
o/n
2/12
1/13
7/13
0/12
B
0/14
1/12
2
1/13
2/12
4/14
0/12
C
3/15
2
1/12
0/12
1/13
2/13
0/13
D
i
3/12
2
2/14
0/12
4/12
0/13
Mean %
Immature
9.2
18.3
5 0
11.8
9.6
76.0
0.0
'Replicate broken during course of experiment.
2Replicates where the number of preserved individuals did not correspond with the number of
individuals counted at end of test.
A-2
------- |