Acute toxicity of copper, ammonia, and chlorine to glochidia and juveniles of freshwater mussels (Unionidae).

Abstract

The objective of the present study was to determine acute toxicity of copper, ammonia, or chlorine to larval (glochidia) and juvenile mussels using the recently published American Society for Testing and Materials (ASTM) Standard guide for conducting laboratory toxicity tests with freshwater mussels. Toxicity tests were conducted with glochidia (24- to 48-h exposures) and juveniles (96-h exposures) of up to 11 mussel species in reconstituted ASTM hard water using copper, ammonia, or chlorine as a toxicant. Copper and ammonia tests also were conducted with five commonly tested species, including cladocerans (Daphnia magna and Ceriodaphnia dubia; 48-h exposures), amphipod (Hyalella azteca; 48-h exposures), rainbow trout (Oncorhynchus mykiss; 96-h exposures), and fathead minnow (Pimephales promelas; 96-h exposures). Median effective concentrations (EC50s) for commonly tested species were >58 microg Cu/L (except 15 microg Cu/L for C. dubia) and >13 mg total ammonia N/L, whereas the EC50s for mussels in most cases were <45 microg Cu/L or <12 mg N/L and were often at or below the final acute values (FAVs) used to derive the U.S. Environmental Protection Agency 1996 acute water quality criterion (WQC) for copper and 1999 acute WQC for ammonia. However, the chlorine EC50s for mussels generally were >40 microg/L and above the FAV in the WQC for chlorine. The results indicate that the early life stages of mussels generally were more sensitive to copper and ammonia than other organisms and that, including mussel toxicity data in a revision to the WQC, would lower the WQC for copper or ammonia. Furthermore, including additional mussel data in 2007 WQC for copper based on biotic ligand model would further lower the WQC.

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Environmental Toxicology and Chemistry, Vol. 26, No. 10, pp. 2036–2047, 2007
? 2007 SETAC
Printed in the USA
0730-7268/07 $12.00 ? .00
Contaminant Sensitivity of Freshwater Mussels
ACUTE TOXICITY OF COPPER, AMMONIA, AND CHLORINE TO GLOCHIDIA AND
JUVENILES OF FRESHWATER MUSSELS (UNIONIDAE)
NING WANG,*† CHRISTOPHER G. INGERSOLL,† DOUGLAS K. HARDESTY,† CHRISTOPHER D. IVEY,†
JAMES L. KUNZ,† THOMAS W. MAY,† F. JAMES DWYER,‡ ANDY D. ROBERTS,‡ TOM AUGSPURGER,§
CYNTHIA M. KANE,? RICHARD J. NEVES,# and M. CHRIS BARNHART††
†Columbia Environmental Research Center, U.S. Geological Survey, Columbia, Missouri 65201
‡U.S. Fish and Wildlife Service, Columbia, Missouri 65203
§U.S. Fish and Wildlife Service, Raleigh, North Carolina 27636
?U.S. Fish and Wildlife Service, Gloucester, Virginia 23061
#Virginia Cooperative Fish and Wildlife Research Unit, U.S. Geological Survey, Virginia Polytechnic Institute and State University,
Blacksburg, Virginia 24061
††Department of Biology, Missouri State University, Springfield, Missouri 65897, USA
(Received 17 October 2006; Accepted 10 April 2007)
Abstract—The objective of the present study was to determine acute toxicity of copper, ammonia, or chlorine to larval (glochidia)
and juvenile mussels using the recently published American Society for Testing and Materials(ASTM)Standardguideforconducting
laboratory toxicity tests with freshwater mussels. Toxicity tests were conducted with glochidia (24- to 48-h exposures) and juveniles
(96-h exposures) of up to 11 mussel species in reconstituted ASTM hard water using copper, ammonia, or chlorine as a toxicant.
Copper and ammonia tests also were conducted with five commonly tested species, including cladocerans (Daphnia magna and
Ceriodaphnia dubia; 48-h exposures), amphipod (Hyalella azteca; 48-h exposures), rainbow trout (Oncorhynchus mykiss; 96-h
exposures), and fathead minnow (Pimephales promelas; 96-h exposures). Median effective concentrations (EC50s) for commonly
tested species were ?58 ?g Cu/L (except 15 ?g Cu/L for C. dubia) and ?13 mg total ammonia N/L, whereas the EC50s for
mussels in most cases were ?45 ?g Cu/L or ?12 mg N/L and were often at or below the final acute values (FAVs) used to derive
the U.S. Environmental Protection Agency 1996 acute water quality criterion (WQC) for copper and 1999 acute WQC for ammonia.
However, the chlorine EC50s for mussels generally were ?40 ?g/L and above the FAV in the WQC for chlorine. The results
indicate that the early life stages of mussels generally were more sensitive to copper and ammonia than other organisms and that,
including mussel toxicity data in a revision to the WQC, would lower the WQC for copper or ammonia. Furthermore, including
additional mussel data in 2007 WQC for copper based on biotic ligand model would further lower the WQC.
Keywords—Freshwater musselsGlochidiaJuvenile musselsAcute toxicityWater quality criteria
INTRODUCTION
Most freshwater mussels have a complex reproductivecycle
involving a parasitic stage on fish. Sperm released by a male
enters a female through the incurrent siphon, and fertilized
eggs develop to larvae called glochidia that mature in spe-
cialized chambers (marsupia) of the female’s gills. Glochidia
are released into the water and must attach to the gills or fins
of a suitable host fish. After one to several weeks of the par-
asitic stage, glochidia transform to juvenile mussels, detach
from the fish, and drop to the stream or lake bottom to begin
the free-living juvenile stage. Freshwater mussels are the most
imperiled group of animals in the United States, with about
70% of species listed as endangered, threatened, or of special
concern [1,2]. Environmental contamination is considered one
of the causal or contributing factors [2,3]. Previous studies
indicate that glochidia and juvenile mussels are more sensitive
to some chemicals when compared to commonly tested aquatic
organisms. For example, Keller and Zam [4] evaluated acute
toxicity of six metals to juvenile paper pondshell (Utterbackia
imbecilis) and found the juvenile mussels were more sensitive
than commonly tested fish and aquatic invertebrates. Aug-
* To whom correspondence may be addressed (nwang@usgs.gov).
spurger et al. [5] summarized ammonia toxicity data for glo-
chidia and juvenile mussels and found that genus mean acute
values for mussels were uniformly at the sensitive end of the
range of the genus mean acute values for other aquatic species
tested in the database used to derive the U.S. Environmental
Protection Agency (U.S. EPA) water quality criteria (WQC)
for ammonia. Nevertheless, the U.S. EPA has not used rou-
tinely the toxicity data generated from freshwater mussels in
the derivation of WQC, mainly due to a historic lack of stan-
dardized guidance for conducting toxicity tests with freshwater
mussels [6].
A series of studies was undertaken to refine methods for
conducting acute and chronic toxicity tests with early life stag-
es of mussels [6–10]. Based on these studies and previous
literature, the American Society for Testing and Materials
(ASTM) recently published a standard guide for conducting
laboratory toxicity tests with freshwater mussels [11]. As one
of a series of papers developed to assess contaminant sensi-
tivity of early life stages of freshwater mussels, the present
paper summarizes the results of acute toxicity tests conducted
during the process of developing the ASTM standard. Spe-
cifically, the objectives of the present study were to evaluate
the acute toxicity of copper, ammonia, or chlorine to glochidia
and juvenile mussels and compare the sensitivity of early life

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Acute toxicity of Cu, ammonia, and Cl to freshwater mussels
Environ. Toxicol. Chem. 26, 20072037
stages of freshwater mussels with commonly testedcladoceran,
amphipod, and fish species. Additionally, the exposure periods
of some tests conducted with juvenile mussels were continued
for 10 d to evaluate longer-term effects of copper, ammonia,
or chlorine on survival or growth of juvenile mussels. Com-
panion papers in this issue evaluate intra- and interlaboratory
variability in acute toxicity tests with freshwater mussels [9]
and evaluate the chronic toxicity of copper or ammonia to
early life stages of mussels [10]. Copper, ammonia, and chlo-
rine were selected as toxicants because of the following con-
siderations: They occur widely in contaminated aquatic en-
vironments, limited chlorine toxicity data are available for
mussels [12], and early life stages of mussels have been dem-
onstrated to be sensitive to copper and ammonia [5,13]. Other
toxicants, such as pesticides, were evaluated in two of the
companion papers [7,8]. The toxicity data generated fromthese
studies have been included in datasets used to assess the sen-
sitivity of mussels to these chemicals [13,14].
MATERIALS AND METHODS
Test conditions and procedures for conducting toxicity tests
in the present study followed the guidance provided in ASTM
[11] and are described in more detail in Wang et al. [9].
Toxicity test with glochidia
Gravid females of 11 mussel species were collected from
streams or rivers in New Hampshire (dwarf wedgemussel,
Alasmidonta heterodon), Virginia (rainbow mussel, Villosa
iris; wavy-rayed lampmussel, Lampsilis fasciola), Tennessee
(oyster mussel, Epioblasma capsaeformis), and Missouri,
USA (ellipse, Venustaconcha ellipsiformis; mucket, Actinon-
aias ligamentina; fatmucket, L. siliquoidea; Neosho mucket,
L. rafinesqueana; pink mucket, L. abrupta; pink papershell,
Potamilus ohiensis, and scaleshell, Leptodea leptodon) be-
tween March and June from 2003 to 2005 (common and sci-
entific names of mussels are referred from Turgeon et al. [15]).
The mussels were brought to or shipped by overnight mail to
the Columbia Environmental Research Center (CERC), U.S.
Geological Survey ([USGS], Columbia, MO), and held in a
600-L flow-through fiberglass tank with well water (hardness
280 mg/L as CaCO3, alkalinity 250 mg/L as CaCO3, pH 7.8)
at a flow rate of about 2 L/min. Water was aerated and main-
tained at 10 to 13?C. Methods for holding and feeding the
female mussels were as described in Wang et al. [9].
The viability of glochidia isolated from each female mussel
was determined before starting a toxicity test. Glochidia were
flushed gently from the marsupium of a mussel into a 300-ml
crystallizing dish using a 1-mm needle and 35-ml syringe filled
with culture water. Three subsamples of 100 to 200 glochidia
were impartially taken from the dish using a 2-mm wide-bore
pipette and transferred to each of three wells of a 24-well
polystyrene tissue-culture plate filled with about 2 ml of well
water. Glochidia in each well were examined with a dissecting
microscope, and the number of closed glochidia was recorded.
After adding one drop of saturated NaCl solution to each well,
open and closed glochidia were counted within 1 min. Valve
closure is an ecologically relevant endpoint that is critical for
glochidia to successfully transform on the host [11]. Glochidia
that closed in response to NaCl were categorized as alive (or
viable), and glochidia that were closed before the addition of
NaCl or that remained open after the addition of NaCl were
categorized as dead (nonviable; [9]). Survival (viability) of
glochidia was calculated as
Survival (%)
? 100 ? (Number of closed glochidia after adding NaCl
solution ? Number of closed glochidia before
adding NaCl solution)
? (Total number of open and closed glochidia after
adding NaCl solution)
If the survival of glochidia from an individual mussel was
?90%, the remaining glochidia from that mussel were used
for toxicity testing. Glochidia isolated from three to six mus-
sels were pooled and mixed in a Pyrex? glass baking dish for
a test. Glochidia were acclimated to a mixture of 50% culture
water (well water) and 50% dilution water (reconstituted
ASTM hard water [16]) that was adjusted gradually to the test
temperature over 2 h before the start of a toxicity test.
The reconstituted ASTM hard water was used as dilution
water in all tests. Copper sulfate (CuSO4, 99.9% purity; JT
Baker, Phillipsburg, NJ, USA), ammonium chloride (NH4Cl,
99.5% purity; Fisher Scientific, Houston, TX, USA), and so-
dium hypochlorite (NaOCl, available chlorine 10–13%; Al-
drich, Milwaukee, WI, USA) were used as toxicants. Each test
consisted of three replicates at each of five concentrations of
a toxicant in a 50% serial dilution, plus a control. The nominal
concentrations were 0, 6.25, 12.5, 25, 50, and 100 ?g Cu/L
for copper tests; 0, 1.0, 2.0, 4.0, 8.0, and 16 mg N/L for total
ammonia tests; and 0, 6.25, 12.5, 25, 50, and 100 ?g/L for
total residual chlorine tests (except one early test at 0, 3.125,
6.25, 12.5, 25, and 50 ?g/L). Static copper and ammonia tests
were conducted in 200-ml glass crystallizing dishes (75-mm
diameter, 35-mm high). Because chlorine is highly volatile,
chlorine toxicity tests were conducted in a flow-through diluter
system that provided about 120 ml of water to each 300-ml
beaker every 20 min to maintain stable chlorine concentrations
[9]. An inline flow splitter was attached to each delivery line
to partition the water flow evenly to each of replicate beakers.
Each beaker had a 2.5-cm hole in the side covered with 50
mesh (279-?m opening) stainless-steel screen and contained
200 ml of water. The diluter system delivered five concentra-
tions with a dilution factor of 0.5, plus a control. Chlorine
stock solution was held in a 600-ml plastic blood pack (Baxter
Healthcare, Deerfield, IL, USA) to reduce volatilization of
chlorine from the stock solution and was delivered with each
cycle of the diluter by a Hamilton syringe pump (Hamilton
Company, Reno, NV, USA).
At the beginning of a toxicity test, about 1,000 glochidia
(about 2 h old after isolation from female mussels) were trans-
ferred impartially from the pooled sample of glochidia into
each of 18 dishes containing about 100 ml of test solution
(copper and ammonia tests) or into each of eighteen 300-ml
beakers in the diluter system (chlorine tests). Test chambers
were held in temperature-controlled water baths at 20 ? 1?C.
Ambient laboratory light of about 200 lux was used with
16:8 h light:dark photoperiod. Dissolved oxygen, pH, con-
ductivity, hardness, and alkalinity were measured on pooled
replicate test solutions collected from the control, medium-,
and high-exposure concentrations at the beginning and the end
of each test using standard methods [17].
Survival of glochidia was determined at 6, 24, and 48 h
during each 48-h exposure. A subsample of about 100 indi-
viduals with 2 ml of dilution water was taken from each rep-
licate test chamber using a 2-mm wide-bore pipette and trans-
ferred into one well of a clean 24-well tissue-culture plate.
One drop of the NaCl solution was added into the well, and

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2038
Environ. Toxicol. Chem. 26, 2007 N. Wang et al.
the response of glochidia (valve closure) within 1 min was
recorded. The initial survival of glochidia for a toxicity test
was estimated by determining the viability of three replicate
controls at the beginning of the test. The mean viability value
was used to adjust the survival of glochidia after 6, 24, or 48
h of exposure. For example, if the mean viability in the control
was 90% at 0 h and an observed viability of glochidia in a
replicate test chamber at 24 h was 85%, then the adjusted
survival at 24 h of exposure was 94% (85/90). Adjusted sur-
vival values were used for the calculation of acute effect con-
centrations and control survival during exposure periods [9].
The acceptability criterion for a toxicity test was ?90% control
survival [11].
Toxicity test with juvenile mussels
Juveniles of seven mussel species (rainbow mussel, wavy-
rayed lampmussel, oyster mussel, fatmucket, Neosho mucket,
pink mucket, and scaleshell) were obtained from laboratory
cultures at Virginia Polytechnic Institute and State University
(Blacksburg, VA, USA), or from Missouri State University
(Springfield, MO, USA), where glochidia isolated from at least
three females of each species were pooled for production of
juvenile mussels with host fish [9]. Newly transformedjuvenile
mussels, which were derived from a single collection day dur-
ing the peak of drop-off (excystment) from host fish, were
shipped overnight to the CERC for testing. Juveniles of the
rainbow mussel, fatmucket, and pink mucket also were reared
in the laboratories for two months with live algae (Neochloris
oleoabundans) either in a recirculating aquaculture system
containing fine sediment [18] or in a compact system [19]
before shipping to the CERC for testing. The compact recir-
culating system consisted of two nested buckets that parti-
tioned a volume of 18 L of culture water into an upper and
lower compartment. A small submersible pump was used to
move water from the lower compartment to the upper com-
partment, and the water then returned to the lower compart-
ments through cylindrical screen-capped chambers that con-
tained juvenile mussels [19]. Once the newly transformed
(?5-d-old) or two-month-old juvenile mussels were received,
temperature of the holding water was adjusted gradually to the
test temperature (?3?C/h) by placing containers containing
mussels at room temperature or into a water bath at 20?C.
About 50% of water in the containers then was replaced with
dilution water three or four times over a 24- or 48-h accli-
mation period. Gentle aeration of water was provided to the
containers through a glass pipette to maintain an acceptable
concentration of dissolved oxygen (?4.0 mg/L [11]). The ju-
veniles were fed live algae (N. oleoabundans) at a feeding
rate of about 20,000 cell/ml or a food mixture of nonviable
algae [10] at a rate of adding 1 ml of the algal mixture into
200 ml of dilution water once or twice daily during the ac-
climation period.
Water temperature, light quality, photoperiod, dilution wa-
ter, toxicants, dilution factor, nominal exposure concentrations,
and the collection and measurements of water quality char-
acteristics in the juvenile toxicity tests were the same for the
glochidia toxicity tests, except that the duration of juvenile
toxicity tests was 96 h or 10 d. Each test consisted of four
replicates at each of five exposure concentrations, plus a con-
trol. Static-renewal copper and ammonia tests were conducted
in 50-ml glass beakers containing 30 ml of test solution, and
test solutions were renewed every other day by replacing about
75% of water volume. Chlorine tests were conducted in a flow-
through diluter system as described for the glochidia tests.
Five juvenile mussels exhibiting foot movement were trans-
ferred impartially into each of 24 glass beakers in each test
using a 1-ml syringe with a 2.5-cm-long, 16-gauge needle
connected to a 60-cm-long Tygon? tubing (1.0-mm inner di-
ameter; Saint-Gobain Performance Plastics, Akron, OH, USA)
with a glass capillary tube (1.17-mm inner diameter) at the
end [11]. Juveniles were not fed during the tests. Survival of
juvenile mussels was determined at 48 and 96 h in the 96-h
exposures and also on day 10 in the 10-d exposures. Juvenile
mussels that exhibited foot movement within a 5-min obser-
vation period were classified as alive using a dissecting mi-
croscope [11]. The acceptability criterion was ?90% control
survival for a 96-h toxicity test and ?80% control survival
for a 10-d toxicity test [11].
The surviving juveniles at the end of 10-d exposures were
preserved in 70% ethanol for growth measurement. The max-
imum shell length of each juvenile mussel was measured to
the nearest 0.001 mm with a digitizing system using video
micrometer software (Image Caliper, Resolution Technology,
Dublin, OH, USA).
The standard guide in ASTM [11] suggests that it is de-
sirable to determine if juvenile mussels are able to avoid ex-
posure to a chemical in 96-h toxicity tests by closing their
valves. At the end of two 96-h copper toxicity tests conducted
with newly transformed juveniles and with two-month-old ju-
venile fatmucket, all test organisms were transferred at 96 h
into clean test chambers with dilution water and held for an
additional 24 h to determine if either of these life stages were
able to avoid exposure to copper. Test conditions were the
same as those for the 96-h toxicity tests except for no addition
of copper to the dilution water. Survival (foot movement) of
juvenile mussels was determined 24 h after the end of the
initial 96-h exposure.
Toxicity tests with commonly tested species
Acute copper and ammonia tests were conducted with five
commonly tested aquatic species, including cladocerans
(Daphnia magna and Ceriodaphnia dubia), an amphipod (Hy-
alella azteca), fathead minnow (Pimephales promelas), and
rainbow trout (Oncorhynchus mykiss). These species are rel-
atively sensitive to copper or ammonia [20–22]. Test organ-
isms were obtained from laboratory cultures at CERC with the
exception of fathead minnows, which were obtained from
Aquatic BioSystems (Fort Collins, CO, USA). Tests were con-
ducted in accordance with the recommended test conditions
outlined in ASTM [16] and U.S. EPA [23] standard methods.
The age of test organisms was ?24 h for cladocerans, ?8 d
for amphipod, 2 d for fathead minnow, and ?20 d for rainbow
trout. Tests with cladocerans and amphipods were conducted
for 48 h at 20?C with four replicates per exposure concentra-
tion. Five individuals of cladocerans or amphipods were trans-
ferred impartially into each of twenty-four 50-ml glass beakers
with 30 ml of test solution. Fish tests were conducted for 96
h at 20?C for fathead minnows and 12?C for rainbow trout,
with two replicates per concentration. Ten fish were impartially
transferred into 1,000-ml glass beakers with 500 ml of test
solution (fathead minnows) or 4,000-ml glass beakers with
2,000 ml of test solution (rainbow trout). Test solution was
renewed after 48 h by replacing about 75% of water volume.
Other test conditions, such as light quality, photoperiod, di-
lution water, number of exposure concentrations, dilution fac-
tor, nominal exposure concentrations (except for the ammonia

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Acute toxicity of Cu, ammonia, and Cl to freshwater mussels
Environ. Toxicol. Chem. 26, 20072039
Table 1. Water quality characteristics for toxicity tests conducted with glochidia and juveniles of up to 11 mussel species and five commonly
tested crustacean and fish species. Values are means (? standard deviation) for multiple tests with each toxicant
Test organismExposure timeToxicantNo. of tests pH
Hardness
(mg/L as CaCO3)
Alkalinity
Conductivity
(?S/cm)
Glochidia2 d Copper
Ammonia
Chlorine
Copper
Ammonia
Chlorine
Copper
Ammonia
Chlorine
Copper
Ammonia
21a
19
17
19
11
8
13
10
7
5
5
8.4 ? 0.2
8.3 ? 0.2
8.4 ? 0.1
8.5 ? 0.1
8.3 ? 0.1
8.3 ? 0.1
8.4 ? 0.1
8.4 ? 0.1
8.4 ? 0.1
8.4 ? 0.1
8.4 ? 0.1
177 ? 11
175 ? 10
169 ? 8.0
177 ? 10
179 ? 7.6
178 ? 8.7
187 ? 6.6
184 ? 6.7
176 ? 8.5
175 ? 6.7
173 ? 2.6
121 ? 5.3
117 ? 4.5
115 ? 4.0
126 ? 7.1
119 ? 6.5
125 ? 5.7
131 ? 5.8
120 ? 5.8
123 ? 4.6
127 ? 2.4
125 ? 4.1
589 ? 29
633 ? 30
560 ? 25
604 ? 24
653 ? 14
600 ? 24
631 ? 15
660 ? 13
593 ? 23
608 ? 17
647 ? 70
Juvenile mussels 4 d
Juvenile mussels 10 d
Commonly tested species2 or 4 d
aNo water quality measurement for one test with scaleshell glochidia.
test with rainbow trout at 0, 2, 4, 6, 8, 16, and 32 mg N/L),
and the collection and determination of water quality char-
acteristics, were as described for the tests with glochidia and
juvenile mussels. Mortality was determined at the end of each
test. The criterion for death was lack of reaction to gentle
prodding. The acceptability criterion for a toxicity test was
?90% control survival [16,23].
Chemical analysis and data analysis
Water samples for analysis of toxicants were collected at
the beginning and end of each test, except for five preliminary
copper tests with glochidia. Total ammonia nitrogen of all
concentrations in each ammonia test was measured at the be-
ginning and end of the test within 1 h of sample collection
using an Orion Ammonia Electrode and Orion EA940 meter
(Thermo Electron, Beverly, MA, USA). The meter was cali-
brated each time before measuring samples with 1 and 10 mg
N/L independent calibration verification standards. The per-
cent recovery of the standards ranged from 90 to 100%. For
total ammonia nitrogen concentrations in water samples, a
minimum reporting limit of 0.1 mg N/L, was selected based
on the method detection limit of 0.02 mg N/L and method
quantitation limits of 0.06 mg N/L. Water samples for chlorine
measurement were analyzed immediately after sample collec-
tion. Total residual chlorine was measured at the highest chlo-
rine concentrations (50 or 100 ?g/L) with a Hach spectro-
photometer (model DR/2000, Hach, Loveland, CO, USA) us-
ing N,N-diethyl-p-phenylenediamine method 8370 for clean
water (http://www.hach.com). The lower concentrations (?50
?g/L) could not be detected consistently using this meter and
therefore were not measured. The meter for chlorine analysis
was calibrated each time before measuring samples following
procedures recommended by the manufacturer.
Water samples were acidified to 1% (v/v) ultrapure nitric
acid for copper analysis. Water samples, collected at the be-
ginning of the 48-h glochidia tests, at the beginning and end
of the 96-h juvenile mussel tests, and on days 0, 4, or 10 for
the 10-d juvenile mussel tests, were analyzed for dissolved
copper at the control, low-, medium-, and high-exposure con-
centrations. Water samples collected at the end of five glo-
chidia tests also were analyzed to determine the consistency
of copper concentrations during the 48-h exposure period.
Copper concentrations were determined by inductively cou-
pled plasma–mass spectrometry (PE/SCIEX ELAN 6000,
PerkinElmer, Norwalk, CT, USA). Samples were automatically
delivered to the inductively coupled plasma–mass spectrom-
etry by means of a software-controlled CETAC ASX-500/
ADX-100 autosampler/autodiluter system (CETAC Technol-
ogies, Omaha, NE, USA) [9]. Analytical precision for quan-
titative inductively coupled plasma–mass spectrometry was
determined by analyzing copper samples in duplicate during
the instrumental run and determining the relative percent dif-
ferences, which ranged from 0.1 to 7.9% for all analysis du-
plicates. Recoveries of copper spiked into water samples and
analyzed by quantitative inductively coupled plasma–mass
spectrometry ranged from 85 to 107%. Measured copper con-
centrations were not adjusted for recovery efficiency. Instru-
mental detection limit was ?0.016 ?g/L, and the method de-
tection limit was ?0.2 ?g/L.
Mean values of dissolved oxygen, pH, conductivity, hard-
ness, and alkalinity for each toxicity test were calculated based
on measures of the control, medium-, and high-exposure con-
centrations at the beginning and the end of the 48- and 96-h
tests, or on days 0, 4, and 10 of the 10-d tests. Percentage of
the nominal concentration for each measured concentration in
a toxicity test was calculated. An arithmetic mean for each
test was calculated by averaging all percentages of nominal
concentrations for all measured concentrations in the test. Me-
dian effective concentrations (EC50s) were calculated based
on nominal concentrations, using a Probit model when appro-
priate and either a Spearman-Karber or trimmed Spearman-
Karber method otherwise [23] with TOXSTAT software [24].
Growth effects in 10-d juvenile tests were determined by cal-
culating the lowest-observed-effect concentration for growth
with analysis of variance and Dunnett’s t-test [24,25]. Thelevel
of statistical significance was set at p ? 0.05. To identify the
effects on growth that occurred at concentrations less than
those affecting survival, concentrations above the EC50 for
survival were excluded from statistical analysis for growth of
juvenile mussels in 10-d exposures.
RESULTS AND DISCUSSION
Water quality
Mean water quality characteristics for copper, ammonia, or
chlorine tests with glochidia, juvenile mussels, or the com-
monly tested species are summarized in Table 1 (individual
measurements for each test are reported in a USGS quarterly
project summary, USGS, unpublished data). Means of hard-
ness and alkalinity of the dilution water typically were within

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2040
Environ. Toxicol. Chem. 26, 2007N. Wang et al.
Table 2. Percent nominal concentrations of measured toxicant concentrations on exposure days 0, 2, 4, or 10 in toxicity tests with glochidia,
juvenile mussels, and commonly tested species. Values are means (? standard deviation) for multiple tests with each toxicant
Test organismToxicant
Percentage of nominal toxicant concentration
0 d2 d4 d10 d
Glochidia Copper
Ammonia
Chlorine
Copper
Ammonia
Chlorine
Copper
Ammonia
109 ? 17 (17)a
104 ? 11 (19)
103 ? 6.8 (17)
107 ? 14 (18)
104 ? 12 (11)
103 ? 7.3 (8)
106 ? 18 (5)
98 ? 2.9 (5)
99 ? 10 (5)
44 ? 16 (18)
96 ? 27 (16)
—
—
—
—
38 ? 10 (3)c
—b
—
—
—
—
—
Juvenile mussels
107 ? 13 (17)
63 ? 7.1 (11)
101 ? 13 (8)
—
84 ? 10 (2)d
124 ? 19 (13)
63 ? 14 (9)
107 ? 7.8 (7)
—
—
Commonly tested species
aTotal number of tests in parentheses.
bNot applicable.
cValues of three 48-h tests with Ceriodaphnia dubia, Daphnia magna, and Hyalella azteca.
dValues of two 96-h tests with fathead minnow and rainbow trout.
10% of the range of values for ASTM hard water (hardness
160–180 mg/L as CaCO3and alkalinity 110–120 mg/L as
CaCO3; [16]), whereas mean pH values ranged from 8.3 to
8.5 and were above the listed range of 7.8 to 8.0 in ASTM
[16]. Higher pH values of the ASTM reconstituted hard water
also have been reported in previous studies [9,10,22,26]. Be-
cause the range of water quality values among tests for a single
toxicant was relatively low (Table 1), the effects of water
hardness or pH on the toxicity results were likely negligible.
Dissolved oxygen was above 7.0 mg/L during all tests.
Measured exposure concentrations
Individual measured exposure concentration and the per-
cent nominal concentration of measured concentrations at dif-
ferent concentrations of copper, ammonia, or chlorine for over
100 toxicity tests with glochidia, juvenile mussels, and the
commonly tested species are reported in the above-mentioned
USGS quarterly project summary (USGS, unpublished data).
The percentages of nominal concentrations at different ex-
posure concentrations within a test were similar (within 20%
difference) except that some higher percentages of nominal
concentrations were observed at the low-test concentrations in
some copper or ammonia tests.
Measured copper concentrations were similar to nominal
concentrations in the 48-h static exposures and in the 4- or
10-d renewal exposures. Mean percentage of nominal concen-
tration for measured copper concentrations at the beginning
of tests was 109% for glochidia tests, 107% for juvenile mussel
tests, and 106% for commonly tested species tests (Table 2).
Mean percentage of nominal copper concentration was 99%
at the end of 48-h glochidia tests and was 107% on day 4 and
124% on day 10 of the juvenile mussel tests (Table 2). The
higher percentage of nominal concentrations at the end of the
10-d tests was consistent with higher values of hardness, al-
kalinity, and conductivity of dilution water measured in copper
tests at the end of 10-d tests (Table 1), which might be the
results of evaporation of some of the water from the small 50-
ml beakers during 10-d exposures despite renewal of about
75% of the test water every other day. Covering test chambers
might have reduced evaporation during these 10-d exposures.
Measured total ammonia concentrations were similar to
nominal concentrations at the beginning of tests but decreased
during exposure periods. At the beginning of the tests, mean
percentages of nominal concentration for measured total am-
monia concentrations was 104% for both glochidia and ju-
venile mussel tests and 98% for the tests with commonly tested
species (Table 2). The mean percentage of nominal concen-
trations decreased to 44% in glochidia tests and 38% in com-
monly tested invertebrate tests at the end of 48-h exposures,
and decreased to 63% in juvenile mussel tests on test days 4
and 10 (Table 2). However, the mean percentage of nominal
concentration was 84% in two fish tests at the end of 96-h
exposures. These results indicate that the renewal of test so-
lution at intervals of 48 h may not be sufficient to maintain
total ammonia concentrations in small test chambers with only
30 ml of test solution in tests with mussels and commonly
tested invertebrates, but may maintain more constant total am-
monia concentrations in larger chambers with about 500 to
2,000 ml of test solution in fish tests. Perhaps this was due to
less volatilization of ammonia because of a lower surface-to-
volume ratio in the larger chambers.
Measured concentrations of chlorine were nearly equal to
the nominal concentrations throughout 2-, 4-, and 10-d ex-
posure periods. Mean percentage of nominal concentrations
ranged from 96 to 107% (Table 2).
These results indicate that measured concentrations of cop-
per and chlorine generally were constant and similar to the
nominal concentrations throughout the exposures, whereas to-
tal ammonia concentrations were near the nominal concentra-
tions at the beginning of tests, but declined over exposure time.
Therefore, using the nominal concentrations to calculate
EC50s of copper and chlorine in this study was appropriate,
whereas using nominal total ammonia concentrations to cal-
culate EC50s might underestimate ammonia toxicity. The
EC50s calculated from nominal ammonia concentrations could
be adjusted to measured ammonia concentrations based on
mean percentage of nominal concentrations at different ex-
posure times (Table 2). However, Wang et al. [10] observed
similar 96-h EC50s in ammonia toxicity tests conducted con-
currently with two-month-old juvenile mussels under static-
renewal conditions (i.e., fluctuating total ammonia concentra-
tion) and flow-through conditions (i.e., relatively constant am-
monia concentrations). Hence, the decline in total ammonia
concentrations in test chambers over the 96-h exposures might
not have substantially influenced test results and expressing
the EC50s based on the starting ammonia concentrations may
reasonably represent the effect concentrations observed in the
96-h static exposures compared to the EC50s based on an
average exposure concentration measured at the beginning and
the end of the 96-h exposures.