Environmental Toxicology and Chemistry, Vol. 18, No. 5, pp.

889–898, 1999
Printed in the USA
0730-7268/99 $9.00 1 .00

COMPARISON OF DISSOLVED AND TOTAL METALS CONCENTRATIONS FROM

ACUTE TESTS WITH SALTWATER ORGANISMS

SUZANNE M. LUSSIER,*† WARREN S. BOOTHMAN,† SHERRY POUCHER,‡ DENISE CHAMPLIN,† and

ANDREA HELMSTETTEN‡
†U.S. Environmental Protection Agency, Atlantic Ecology Division, 27 Tarzwell Drive, Narragansett, Rhode Island 02882
‡Science Applications International Corporation, 165 Dean Knauss Drive, Narragansett, Rhode Island 02882, USA

(Received 17 February 1998; Accepted 14 July 1998)

Abstract—Aquatic life criteria (ALC) have traditionally been expressed for metals in terms of total-recoverable or acid-soluble
concentrations. Recent U.S. Environmental Protection Agency policy recommended use of dissolved metal concentrations for setting
water quality standards. Criteria derived from previous tests could be expressed in terms of dissolved metals if ratios of dissolved-
to-total concentrations in those tests were consistent. Using those metals with insufficient dissolved metals data to directly derive
criteria (arsenic (III), cadmium, chromium (VI), lead, nickel, selenium (IV), and zinc), we measured both total and dissolved metal
concentrations in acute saltwater static and flow-through tests. Exposure conditions simulated those of original tests used to derive
ALC. Partitioning of metals between dissolved and particulate forms was very consistent. Dissolved metal concentrations were
greater than 90% of total concentrations in all tests, exceeding 95% in 10 of 13 tests. Dissolved-to-total metal ratios did not vary
significantly with concentration, time, or type of test. Biological responses were consistent with historical data. Results implied
that in acute saltwater toxicity tests used to establish ALC, metals were primarily dissolved. Thus criteria developed for metals
based on total concentrations should be equally valid when expressed in terms of dissolved concentrations.

Keywords—Metals Water quality criteria Dissolved metals Total metals

INTRODUCTION
Aquatic life criteria (ALC), formerly known as water qual-
ity criteria, have been developed for a number of chemicals.
Those derived for metals were based on concentrations of total-
recoverable or acid-soluble metals. In 1993, the U.S. Envi-
ronmental Protection Agency’s (U.S. EPA) Office of Water
instituted a policy stating that ‘‘the use of dissolved metal to
set and measure compliance with water quality standards is
the recommended approach, because dissolved metal more
closely approximates the bioavailable fraction of metal in the
water column than does total recoverable metal’’ [1]. This
policy advises that ALC be expressed in terms of concentra-
tions of dissolved metals (i.e., those that pass through a filter
with a pore size of 0.45 mm). However, dissolved metals were
not measured in toxicity tests used to develop ALC, so his-
torical data cannot be directly used to revise them.
If the relationship between dissolved and total metal con-
centrations in historical tests could be estimated, ALC derived
from those tests could be revised to reflect dissolved metals.
Rather than repeat the entire series of ALC tests measuring
dissolved metals, we conducted acute saltwater static and flow-
through tests designed to simulate the exposure conditions of
the original tests, using the most sensitive species at the critical
concentrations (bracketing 50% mortality) and measuring both
dissolved and total metal concentrations. Tests were conducted
for those metals with insufficient data to directly derive criteria
from dissolved metal concentrations (arsenic (III), cadmium,
* To whom correspondence may be addressed
(lussier.suzanne@epa.gov).
Contribution NHEERL-NAR 1949, U.S. Environmental Protection
Agency, National Health and Environmental Effects Laboratory, At-
lantic Ecology Division, Narragansett, Rhode Island.
889
chromium (VI), lead, nickel, selenium (IV), and zinc); a sep-
arate study was conducted with copper [2].
The objective of the tests was to determine the fraction of
total metals present in dissolved form using conditions and
concentrations employed in the original tests used to derive
ALC. By monitoring biological response, we were able to
validate the extent to which the current tests simulated original
tests. We expected that the dissolved fraction of a metal might
vary with the total concentration of that metal, the concentra-
tions of dissolved organic carbon and suspended solids, or
with the type or length of exposure. Once determined, the
relationship between these parameters and the dissolved frac-
tion could be applied to adjust ALC to reflect dissolved rather
than total metal concentrations. Because total and dissolved
metal concentrations are operationally defined, and because
previous tests measured only total and not dissolved metals,
a number of quality assurance (QA) tests were necessary to
ensure that the test results were valid measures of environ-
mental conditions, and not artifacts of the analytical methods
used. This paper presents the results from biological and chem-
ical QA tests, the ratios of dissolved to total metal concentra-
tions, and implications of the results with respect to ALC for
metals.
MATERIALS AND METHODS
Biological methods
All test conditions approximated those in tests that con-
tributed to the derivation of ALC for these metals except, in
this study, we used only a control treatment and two exposure
concentrations. Biological methods were made as similar as
possible to those used in the original tests.
890 Environ. Toxicol. Chem. 18, 1999
We chose test species and concentrations to represent the
range of exposures that had produced the four most sensitive
acute responses in the original ALC. Physical parameters were
also equivalent to the original tests. Because the four most
sensitive species had been tested using flow-through (with food
added) and static (with no food added) methods, we used both
test techniques for each metal. The flow-through tests were
conducted with the mysid Americamysis bahia (formerly
Mysidopsis bahia) [3]; the static tests were conducted with
the blue mussel, Mytilus edulis, in the bivalve test for lead,
and Ampelisca abdita in the amphipod test for selenium. Al-
though the polychaete worms Capitella capitata were more
sensitive to selenium, they were not used because they must
be fed during tests. The coot clam, Mulinia lateralis, was used
in the static tests conducted with arsenic, chromium, nickel,
and zinc. Because surrogate species with methods amenable
to our sampling protocol were unavailable for the static test
with cadmium, we omitted that test.
Test organisms. We cultured M. lateralis and A. bahia on
site. Mulinia lateralis were held in 19-L aquaria with a few
centimeters of sand on the bottom and maintained at 24 6 28C
and a salinity of 28 to 30 g/kg. Filtered (15 mm) Narragansett
Bay (Rhode Island, USA) seawater was replenished daily and
2 L of algae solution (Isochrysis galbana and Tetraselmus
suecica cultured in our laboratory) was provided for food [4].
Americamysis bahia were obtained from laboratory cultures
maintained in filtered (15 mm) Narragansett Bay seawater in
a flow-through (100 ml/min) system. Seawater was maintained
at 25 6 18C, 30 6 2 g/kg salinity, and a pH of 7.8 to 8.2, in
76-L glass aquaria with undergravel filters and a dolomite
substrate with gentle aeration [5]. We collected A. abdita from
the Narrow River in Narragansett, Rhode Island, USA, and
acclimated the organisms within a 5-d period at the rate of
28C/d to 20 6 18C filtered (15 mm) Narragansett Bay seawater
before testing [6]. Larvae of M. edulis were produced from
feral stock collected from Narragansett Bay according to
ASTM Standard Guide E724-89 [7].
Flow-through acute tests. We used post-larval stage A.
bahia, one of the most sensitive species for the ALC tests, as
the test species for all flow-through tests, which were con-
ducted at 25 6 18C and 30 6 2 g/kg salinity [8]. In the exposure
system, each treatment utilized two replicate glass chambers
(15.5 3 47 3 15.5 cm), which received filtered (15 mm) Nar-
raganset Bay seawater from a Benoitt mini-diluter system [9].
A siphon-flush mechanism [10] was used to produce a 40 ml/
min flow rate per duplicate chamber, which was approximately
14 volume additions per day. Cups within each chamber were
fabricated from 9-cm-diameter glass petri dishes each with a
320-mm Nitext screen collar fastened with clear silastic seal-
ant. A total of six test cups were used per treatment, with each
containing five animals. Three of the test cups were used for
randomized samples for metals analysis, and the remaining
three were used for total suspended solids (TSS) and dissolved
organic carbon (DOC) samples.
Test concentrations were selected to bracket LC50 values
from original tests used to derive ALC. However, the exposure
concentrations differed somewhat from those in original tests
because of the attributes of the present flow-through diluter
exposure system. A stock solution for each metal was made
with deionized water and, except for lead, delivered with a
syringe pump to a 2-L glass mixing flask and diluted with
filtered Narragansett Bay seawater. From the mixing flask, each
solution was delivered with a Digi-staltic pump (Cole Parmer,
S.M. Lussier et al.
Niles, IL, USA) to the diluter system where it was mixed with
seawater to make the test concentrations that were delivered
to the duplicate exposure chambers. Because of lead’s inherent
insolubility in seawater, we used a different method to deliver
those stock solutions; the syringe pump was not used. Instead,
separate stock solutions for each concentration were mixed
directly in 5-L glass mixing jars with deionized water and
delivered directly to each duplicate exposure chamber with a
Digi-staltic pump. Seawater was pumped from the diluter sys-
tem to each chamber and mixed with the stock solution im-
mediately before entering the chamber. Because the high lead
concentration was unstable, we included only the control and
lower acute concentration in the final test. At this particular
concentration, the lead remained in solution.
Samples from the diluter were chemically analyzed for each
metal before each test. In the procedure used for the A. bahia
96-h flow-through metal tests, chemistry, TSS, and DOC sam-
ples were taken 1 h after the animals had been added to the
test. Before the water samples were siphoned out, the animals
were removed from each test cup by pipette, without removing
the cup from the treatment chamber. The animals were replaced
after the samples were taken. Test animals were counted and
fed reference Artemia sp. (brine shrimp) ad libitum daily [11].
For the TSS and DOC samples, a sterile syringe was used to
remove 100 ml of water from each of three replicates per
concentration. For each concentration, one replicate sample
was taken from a randomly selected test cup. Following the
same procedure, chemistry, TSS, and DOC samples were taken
again at the end of the test.
Static acute tests. All static acute tests were of 48-h du-
ration with no food added. For the arsenic and chromium bi-
valve tests [7,12], the Pacific oyster, Crassostrea gigas and
M. edulis, respectively, were unavailable; M. lateralis was
used as a surrogate test species. Embryos of M. lateralis are
60 mm long compared with M. edulis at 90 mm, the eastern
oyster, Crassostrea virginica, at 72 mm, and the quahog clam,
Mercenaria mercenaria, at 101 mm long [13]. Tests cited in
ALC [14,15] used an initial stock of 15 to 17 M. edulis em-
bryos per milliliter. To maintain a consistent biological load,
we compensated for the smaller M. lateralis by using an egg
stock averaging 20 to 22 embryos per milliliter. In the nickel
and zinc tests, M. lateralis was also substituted for C. virginica
and M. mercenaria, respectively. However, in these tests the
spawning capacity of M. lateralis limited the number of em-
bryos exposed per replicate. We were only able to use 12 to
13 embryos per milliliter for these tests. Neither C. capitata
nor an appropriate surrogate species was available to conduct
a static test with cadmium. Because bivalves were not among
the four most sensitive species in the original ALC for cad-
mium [16], no M. lateralis test was conducted with this metal.
In the selenium test, A. abdita was substituted for Acartia
tonsa because the small exposure volumes used with the latter
species would have made chemical analyses difficult and less
comparable to other tests. The average wet weight of A. abdita
used in the selenium test was 0.0518 mg per animal. Because
the small volumes used in M. lateralis and A. abdita tests
precluded subsampling, entire replicates were sacrificed for
chemical, TSS, and DOC analyses. The M. lateralis samples
were not prefiltered to remove animals.
Numerous problems were associated with spawning test
animals for the M. edulis test with lead. Insufficient embryos
were produced to allow enough test cups for TSS and DOC
analyses. The shortage of embryos resulted in an unequal num-
Saltwater acute tests with dissolved and total metals
ber of replicates per treatment and fewer chemistry samples
after 1 h. Sacrifice of a whole replicate was necessary for each
chemical analysis (the small-volume protocol for this test pre-
cluded subsampling); consequently, several samples at 1 h
were omitted. In our judgement, it was more important to
obtain chemical analyses for each treatment at 48 h to assess
the maximum potential absorption of metal to particles. Be-
cause later spawnings were unsuccessful, the test could not be
repeated.
TSS and DOC methods. In addition to biological monitor-
ing, and simultaneous with chemical sampling, we measured
TSS and DOC at the beginning and end of each test because
high concentrations of either one could potentially affect the
fraction of metal in solution or in a bioavailable form. For the
TSS samples, we followed Residue, Non-filterable Method
160.2 [17]. After filtering the test water to obtain the TSS, we
sent two 20-ml subsamples from each replicate to Connecticut
Testing Laboratories (Meriden, CT, USA) for measuring DOC
according to Strickland and Parsons’ ‘‘Determination of Sol-
uble Organic Carbon’’ [18]. Before sending these samples, we
prepared them by adding 40 mg of concentrated hydrochloric
acid per subsample and then refrigerating them.
Test chemicals. With the exception of lead nitrate, all chem-
icals used were purchased from Aldrich Chemical, Milwaukee,
Wisconsin, USA. The potassium dichromate used was CAS
7778-50-9 and was 99% pure; selenious acid, CAS 7783-00-8,
99.9% pure; sodium meta-arsenite, CAS 7784-46-5, 98% pure;
zinc chloride, CAS 7646-85-7, 98% pure; nickel chloride, CAS
7791-20-0, 98% pure; cadmium chloride, CAS 10108-64-2,
99% pure. The lead nitrate used was CAS 10099-74-8, pur-
chased from Sigma Chemical, St. Louis, Missouri, USA.
Analytical chemistry methods
Instrumental analysis. Prepared water samples were ana-
lyzed for cadmium, chromium, lead, nickel, and zinc by in-
ductively coupled plasma–atomic emission spectroscopy (ICP-
AES), and for arsenic and selenium by graphite furnace atomic
absorption spectrophotometry (GFAAS). We verified instru-
mental calibrations by analyzing independent reference solu-
tions before and during analysis of unknowns (one verification
for every 20 unknowns); freedom from matrix interferences
was verified by analyzing spiked unknowns (one spiked sample
for every 20 unknowns). Analyses were acceptable if verifi-
cation standard or spike recovery was in the 85 to 115% range.
Concentrations reported are the mean and standard deviation
of three replicate measurements (corrected for dilution where
appropriate).
Method QA tests. Aquatic Life Criteria Guidelines [19]
specify measurement of total recoverable metals in waters.
‘‘Total recoverable’’ is an operational definition referring to
metals dissolved after a water sample is acidified with nitric
and hydrochloric acid and heated to reflux and reduce the
volume of the sample. The method measures metals present
in water samples in all but the most refractory mineral phases.
Many of the historical marine toxicity tests on which criteria
are based, however, did not employ analytical methods to mea-
sure total recoverable metals. Instead, water samples were
acidified with concentrated nitric acid (10%, v/v), shaken, and
allowed to stand for at least an hour, a method referred to as
the ‘‘acidified method.’’ This method measured metals that
were either dissolved initially or released from particles. Be-
fore performing simulations, we conducted QA tests to insure
Environ. Toxicol. Chem. 18, 1999 891
that the data would not reflect artifacts resulting from the dif-
ferent analytical methods.
Methods used to measure total recoverable and acidified
metals were compared using solutions prepared from Narra-
gansett Bay seawater filtered through a 0.45-mm Nucleoporet
filter, to which food and/or metals were added. Food was added
in the same proportion as in toxicity tests, whereas concen-
trations of added metals were approximately equal to those in
the lowest treatment levels. After the solutions were allowed
to equilibrate overnight, six replicate subsamples of each so-
lution were prepared by the acidified method and by a modified
total recoverable metals procedure. In this method, we acidified
samples with concentrated nitric acid (10%, v/v) in a 120-ml
Teflont PFA microwave digestion vessel, and heated them in
the sealed vessel by microwave energy to hold the sample at
1708C for 20 min. This method produced results equivalent to
those from traditional digestion procedures used to determine
total recoverable metals. Consequently, the U.S. EPA has ap-
proved microwave methods for determination of total metals
in wastewater for National Pollution Disposal and Elimination
System permits and aqueous samples and extracts (SW-846,
Method 3015) [20]. The American Society for Testing and
Materials has also published a method for determining total
recoverable metals in water (ASTM Standard D 4309 [21]).
Statistical analysis of results obtained by total recoverable
and acidification methods (Table 1) demonstrates that they are
equivalent in the presence or absence of food [22]. In unspiked
solutions with or without the added food, metal concentrations
were below detection limits, demonstrating no metal contam-
ination from either the analytical method or presence of food.
Detection limits were less than 10% of the lowest treatment
concentration for all metal determinations except for nickel by
ICP-AES and arsenic by GFAAS. Because working concen-
trations were well above the instrumental detection limit, ICP-
AES was used for nickel samples. No statistically significant
differences were found between concentrations of any metals
measured by total recoverable and acidified methods for either
matrix (two-tailed t test assuming unequal variances, p 5
0.05). Acidified metal concentrations ranged from 88 to 102%
of total recoverable concentrations in the spiked seawater sam-
ples, exceeding 95% in 12 of 14 pairs of samples. The only
anomalies were associated with the results for nickel and zinc.
In each case, one replicate sample had a relatively high value;
for example, total recoverable zinc in one replicate of spiked
seawater with food was 67% higher than the mean of the other
five replicates. Zinc is a common and problematic contaminant
and the anomalous value was higher, most likely indicative of
spot contamination, not true variability of the method. If the
one anomalous value were discarded, the mean and standard
deviation of the remaining five replicates would be 236 6 7.5
mg/L, virtually identical to the values for the acidified samples.
Statistical comparisons also showed no significant differences
between samples prepared by either method due to the presence
of food [22]. Thus, historical metals data generated using the
acidification method should be comparable with those gen-
erated by the total recoverable method.
A similar test was also conducted to check for artifacts
resulting from the sample filtration procedure. Solutions were
prepared from deionized water, filtered and unfiltered 0.35 M
sodium chloride, and filtered Narragansett Bay seawater, which
were spiked with metals in the same concentrations as de-
scribed for the first test. After metal-spiked solutions equili-
892 Environ. Toxicol. Chem. 18, 1999 S.M. Lussier et al.

brated overnight, 10 replicate 15-ml aliquots were removed

6
7
2
2
8
13
18
22
4
5
47
11
7
10
Narragansett Bay

SD
from the deionized water and metal-spiked solutions for total

Seawater
metal analysis using the acidified method. From the portion
of each solution remaining, 10 replicate aliquots were filtered

Mean

117
113
46
46
800
797
436
437
112
110
255
238
191
191
by the procedure described below for simulation test samples.
Comparison of dissolved and acidified methodsb

The resultant solutions were analyzed by ICP-AES and

Concentration (mg/L)

GFAAS, and the analytical results were compared statistically.

As in the first test, analytical results for samples prepared by
12
11
2
3
9
7
13
24
6
4
8
7
12
8
SD
NaCl solution

filtration were comparable to those from acidification methods,

with no artifacts introduced by the filtration step (Table 1). No
detectable concentrations of any metals were found in any
Mean

blank deionized water samples, demonstrating that filtration

116
114
48
48
821
816
448
451
110
118
209
211
201
199
was not a source of contamination. Dissolved and acidified
concentrations in either synthetic (0.5 M sodium chloride) or
natural (Narragansett Bay) seawater samples were virtually
Deionized

identical. Again statistical comparisons showed no significant

water

,5.5
,5.5

,9.9
,9.9
,27
,27

,11
,11
,77
,77
,44
,44
,29
,29 differences between samples prepared by either method or
matrix. Based on these results, we determined that samples
from the toxicity tests could be analyzed as dissolved or total
(as acidified) metals without concern of filtration artifacts.
Sample collection and preparation. Water samples were
Dissolved

Dissolved

Dissolved

Dissolved

Dissolved

Dissolved

Dissolved
Acidified

Acidified

Acidified

Acidified

Acidified

Acidified

Acidified
Method

collected from exposure tanks through acid-washed polyeth-

ylene tubing into acid-stripped polyethylene bottles. Bottles
Table 1. Analytical quality assurance tests results

were rinsed with sample water, which was discarded before

collecting the samples. At the same time, we collected field
blanks using laboratory deionized water brought to the ex-
SD

6
7
2
1
6
17
28
19
6
5
12
13
65
13

posure system in an acid-stripped polyethylene bottle collected

by the same procedures as the exposure system samples. Du-
With
food

plicate samples were collected from one treatment replicate

for each concentration to assess within-replicate variability,
Mean

199
202
55
55
1,754
1,744
523
518
137
136
500
494
263
241

and single samples from two other treatment replicates were

Metals added

used to assess between-replicate variability.

After collection, samples were split into total and dissolved
fractions. Aliquots for total metal analysis were taken after
Concentration (mg/L)

shaking each bottle to resuspend any solids. Dissolved samples

SD

7
6
3
4
54
49
27
22
24
7
9
9
17
14

were filtered through acid-cleaned 47-mm-diameter 0.45-mm

No significant differences between methods (two-tailed t test, n 5 10, p 5 0.05).
Without

Nucleopore polycarbonate membrane filters. We rinsed all

No significant differences between methods (two-tailed t test, n 5 6, p 5 0.05).
Comparison of total and acidified methodsa

food

sample bottles and receivers with the appropriate sample and

Mean

discarded the rinse before collection. Samples were preserved

200
205
55
54
1,727
1,727
515
524
147
129
530
525
233
234

with Seastar (Seastar Chemicals, Sidney, BC, Canada) ultra-

clean concentrated nitric acid (50 ml per 50-ml sample). All
sample preparation took place in a trace metals clean room.
With food

All samples for total metal analysis were prepared using

No metals added

the acidified method. Thus, analytical conditions were identical

,42
,42
,6
,6
,11
,11
,77
,77
,44
,44
,53
,53
,10
,10

to those used in previous marine toxicity tests conducted at

our laboratory for the derivation of ALC. We prepared three
Without food

quality control samples with each set of samples in the same

,42
,42
,6
,6
,11
,11
,77
,77
,44
,44
,53
,53
,10
,10

manner as those for total and dissolved metals: a procedural

blank with deionized water, a blank seawater sample to check
for contamination due to the filtration procedure, and a blank
seawater sample spiked with the metal of interest at the lowest
treatment concentration to check for any loss of analyte due
Method

Acidified

Acidified

Acidified

Acidified

Acidified

Acidified

Acidified

to filtration. No evidence was found of contamination or ad-

Total

Total

Total

Total

Total

Total

Total

sorption of dissolved metals for any of the metals tested.

With one exception, samples from all tests were collected
and prepared by the same methods. The 96-h samples from
the selenium A. abdita test were not prepared for total recov-
Chromium (VI)

erable metals; instead, the entire sample was filtered, providing

Selenium (IV)
Arsenic (III)

only a dissolved sample. Particulate selenium was determined

Cadmium

by digesting the filters with 2 M nitric acid for 2 h in an

Nickel
Metal

ultrasonic bath. For these samples only, total concentrations

Lead

Zinc

were calculated as the sum of the dissolved and particulate

b
a
Saltwater acute tests with dissolved and total metals Environ. Toxicol. Chem. 18, 1999 893

Table 2. Biological results from simulation tests

Aquatic life criteria

Measured
LC/EC50 concn. Endpoint Response
Element Test species Species (mg/L) (mg/L)a measured (%)

Arsenic (III) Americamysis bahia A. bahia 1,740 ,15 % Survival 100

188 100
1,917 80
Mulinia lateralis Crassostrea gigas 326 ,6 No. larvae 100
204 (% of controls) 96
1,563 44
Cadmium A. bahia A. bahia 41 ,3 % Survival 100
48 50
164 0
Chromium (VI) A. bahia A. bahia 2,030 ,11 % Survival 100
1,504 100
3,366 60
M. lateralis Mytilus edulis 4,469 ,11 No. larvae 100
1,333 (% of controls) 110
3,111 109
Lead A. bahia A. bahia 3,130 ,23 % Survival 100
438 100
M. edulis M. edulis 476 ,55 No. larvae 100
101 (% of controls) 107
742 63
Nickel A. bahia Heteromysis formosa 152 ,22 % Survival 100
114 57
974 0
M. lateralis Crassostrea virginica 1,180 ,11 Not measured
105
755
Selenium (IV) A. bahia A. bahia 1,500 ,5 % Survival 100
413 100
821 60
Ampelisca abdita Acartia tonsa 839 ,5 % Survival 100
456 96
985 84
Zinc A. bahia A. bahia 499 ,6 % Survival 100
183 100
392 86
M. lateralis Mercenaria mercenaria 195 ,6 Not measured
149
418
a Weighted mean of 1- and 96-h total metal concentrations.

selenium concentrations, allowing calculation of the percent
dissolved metal.
RESULTS AND DISCUSSION
Biological results
Biological results from metal tests (Table 2) validated that,
in most cases, test organism responses were similar to those
from the tests used to develop ALC. Because we used only
two exposure concentrations in the test design, the 50% lethal
concentration (LC50) could not always be calculated. Biolog-
ical responses in control treatments of all tests met QA stan-
dards of acceptability.
Flow-through toxicity test responses were more consistent
with ALC than those from static tests because we used the
same test species, A. bahia, in both historical and current flow-
through tests, and the tests were conducted in the same lab-
oratory with the same water source [23]. Results from the flow-
through tests correlated quite well with ALC responses for
arsenic, cadmium, and chromium (Fig. 1) [14,16,24], as well
as selenium and zinc (not shown) [25,26]. Concentrations be-
low ALC elicited little biological response, whereas those
above them showed similar effects on survival. In the A. bahia
test with lead, results were consistent with the ALC response
curve at the low concentration used (Fig. 1d) [15]. Although
a higher test concentration would have allowed a better com-
parison, solubility problems prevented exposure with higher
concentrations. Of the flow-through tests, A. bahia with nickel
had the greatest difference in biological response compared
with the original test. This species was about five times more
sensitive than in the 1986 ALC test [27]. However, the LC50
value from the new A. bahia test agreed quite well with that
from the original ALC test with the mysid Heteromysis for-
mosa, ranked the most sensitive species.
Although some differences occurred by using surrogate spe-
cies, most biological responses from static tests were consistent
with ALC (Fig. 2). Although we attempted to use surrogate
species as similar as possible in sensitivity and biological load
(i.e., size and number of test animals) to the original test spe-
cies, in some cases the biological response was not directly
comparable. The surrogate species for four of the six static
tests, M. lateralis, was adequate for biological loading pur-
poses, but effects varied from the species used for ALC tests.
In the static arsenic test (Fig. 2a), M. lateralis was approxi-
mately four times less sensitive than C. gigas, the species used
to develop the ALC [24,28]. For the static chromium test, the
number of viable M. lateralis larvae produced in the control
894 Environ. Toxicol. Chem. 18, 1999 S.M. Lussier et al.

Fig. 1. Biological and chemical results from flow-through acute tests with Americamysis bahia compared with aquatic life criteria test results.
(a) Percent survival versus arsenic concentrations; (b) percent survival versus cadmium concentrations; (c) percent survival versus chromium
concentrations; (d) percent survival versus lead concentrations. Information from Lussier [23].

concentration was acceptable, but was not reduced at either
exposure concentration (Fig. 2b). Based on the 1984 ALC
value [14], some effect would have been expected at the higher
concentration [28]. Although the viability requirement in the
ASTM method [7] and our requirement for biological load
were satisfied at the start of the test, development of a low
number of viable M. lateralis larvae precluded the collection
of biological response data for nickel or zinc [29,30]. Results
from the static lead test with M. edulis were quite consistent
with results from the 1984 ALC test [15,28], which used the
same test species (Fig. 2c). Likewise, the static selenium test,
which used A. abdita as a surrogate species for A. tonsa,
correlated quite well with the 1987 ALC (Fig. 2d) [25].
TSS and DOC results. Results from TSS analyses were
variable, ranging from 10 to 166 mg/L in the static tests and
from 1.5 to 83 mg/L in flow-through tests. No differences were
apparent between control and test treatments, nor was there
any change over the test period. However, TSS were inversely
related to metal concentrations, with highest TSS in the con-
trols. Dissolved organic carbon was very low in all samples
with no apparent correlation with exposure time or metal con-
centration, or between control and treatment concentrations.
Results from static tests ranged from 1 to 11 mg/L, whereas
flow-through samples ranged from ,1 to 10 mg/L. No effect
of TSS or DOC was apparent on the ratio of dissolved to total
metal.
Saltwater acute tests with dissolved and total metals Environ. Toxicol. Chem. 18, 1999 895

Fig. 2. Biological and chemical results from static acute tests compared with aquatic life criteria test results [25,28]. (a) Number of Mulinia
lateralis larvae (as percent of control) versus arsenic concentrations; (b) number of M. lateralis larvae (as percent of control) versus chromium
concentrations; (c) number of Mytilus edulis larvae (as percent of control) versus lead concentrations; (d) percent survival of Ampelisca abdita
versus selenium concentrations.

Chemistry results
For each acute toxicity test, dissolved and total metal con-
centrations were measured in samples from each treatment
replicate, as well as from a second analytical sample from one
treatment replicate (Table 3). Dissolved concentrations were
also calculated as percentages of the total and nominal con-
centrations. The mean and standard deviation of dissolved and
total concentrations, percent dissolved, and percent nominal,
were calculated for each treatment concentration and sampling
time.
Except for two instances, measurable metal concentrations
were not found in any control samples. Although higher in-
strumental noise contributed significantly to greater uncer-
tainties in results from arsenic and selenium tests, the primary
cause of uncertainty was sampling variability. Measurement
precision for individual samples was generally less than 10%,
and in most samples less than 5%. Variability of concentrations
was similar among replicates within a treatment and between
analytical replicates within a single treatment replicate, gen-
erally ranging from 1 to 6%, although occasionally as high as
9%.
Concentrations of metals measured in the tests, both dis-
solved and total, were fairly constant over time. In only 3 of
the 13 tests did concentrations vary by more than 10% in all
treatments over the course of the test: cadmium increased by
17% in the A. bahia test, selenium decreased by 11 to 14%
in the A. abdita test, and zinc increased by 12 to 20% in the
A. bahia test. The increase of zinc in the A. bahia test was
due almost entirely to one anomalously high total recoverable
zinc measurement in one sample from the 513-mg/L treatment,
896 Environ. Toxicol. Chem. 18, 1999 S.M. Lussier et al.

Table 3. Analytical chemical results from toxicity tests

Nominal concn. Mean concn. Mean % Mean %

Element Species (mg/L) Fraction (mg/L) dissolved nominal

Arsenic (III) Americamysis bahia Control Total ,15

Dissolved ,15
226 Total 188 6 10 105 6 6 87 6 5
Dissolved 195 6 12
1,806 Total 1,917 6 110 92 6 6 89 6 6
Dissolved 1,607 6 114
Mulinia lateralis Control Total ,6
Dissolved ,6
232 Total 204 6 9 101 6 9 83 6 2
Dissolved 194 6 5
1,740 Total 1,563 6 55 99 6 2 87 6 3
Dissolved 1,511 6 52
Cadmium A. bahia Control Total ,3
Dissolved ,3
64 Total 48.6 6 0.4 97 6 1 70 6 1
Dissolved 44.9 6 0.5
255 Total 164 6 8 101 6 3 62 6 2
Dissolved 163 6 5
Chromium (VI) A. bahia Control Total ,11
Dissolved ,11
2,479 Total 1,504 6 8 99 6 2 59 6 1
Dissolved 1,461 6 20
4,959 Total 3,366 6 16 99 6 1 67 6 1
Dissolved 3,322 6 42
M. lateralis Control Total ,11
Dissolved ,11
2,000 Total 1,333 6 8 100 6 1 66 6 1
Dissolved 1,328 6 21
4,500 Total 3,111 6 8 99 6 0 69 6 0
Dissolved 3,089 6 9
Lead A. bahia Control Total ,23
Dissolved ,21
665 Total 438 6 12 100 6 2 65 6 3
Dissolved 435 6 21
Mytilus edulis Control Total ,55
Dissolved ,50
320 Total 97 6 2 85 6 10 26 6 3
Dissolved 83 6 10
2,560 Total 742 6 16 93 6 2 27 6 1
Dissolved 700 6 15
Nickel A. bahia Control Total ,22
Dissolved ,22
132 Total 114 6 2 96 6 4 86 6 2
Dissolved 114 6 6
1,058 Total 974 6 4 101 6 1 90 6 1
Dissolved 951 6 5
M. lateralis Control Total ,11
Dissolved ,11
150 Total 105 6 1 99 6 5 70 6 2
Dissolved 105 6 4
1,000 Total 755 6 9 99 6 1 75 6 1
Dissolved 745 6 10
Selenium (IV) A. bahia Control Total ,5
Dissolved ,5
741 Total 413 6 8 96 6 5 54 6 3
Dissolved 402 6 19
1,482 Total 821 6 51 103 6 7 55 6 2
Dissolved 822 6 31
Ampelisca abdita Control Total ,5
Dissolved ,5
600 Total 456 6 23 104 6 2 78 6 5
Dissolved 470 6 21
1,500 Total 1,029 6 9 100 6 3 65 6 1
Dissolved 971 6 19
Zinc A. bahia Control Total ,6
Dissolved ,6
256 Total 183 6 6 96 6 4 67 6 5
Dissolved 171 6 9
513 Total 392 6 5 92 6 5 74 6 3
Dissolved 378 6 12
M. lateralis Control Total ,6
Dissolved ,6
190 Total 149 6 2 93 6 3 73 6 3
Dissolved 139 6 4
500 Total 418 6 4 99 6 2 82 6 2
Dissolved 408 6 7
Saltwater acute tests with dissolved and total metals Environ. Toxicol. Chem. 18, 1999 897

Table 4. Mean % dissolved metal ratios in simulation tests For example, precipitation of lead chloride in the stock solution
for the M. edulis static test resulted in measured concentrations
% Dissolved
much lower than nominal targets. This situation can arise be-
Element Static Flow-through Overall cause of problems establishing or maintaining the high con-
centrations of metals required for stock solutions to be diluted
Arsenic (III) 101 6 13 99 6 12 100 6 12 into either flow-through or static test systems. Interpretation
Cadmium 99 6 3 99 6 2
Chromium (VI) 99 6 2 99 6 2 99 6 2 of metal toxicity test data should be based on measured rather
Lead 91 6 10 100 6 5 95 6 8 than nominal concentrations, and sampled from the test ex-
Nickel 99 6 7 99 6 5 99 6 6 posure chambers.
Selenium (IV) 101 6 5 99 6 12 100 6 9
Zinc 96 6 4 93 6 6 95 6 5 CONCLUSION
Aquatic life criteria were established on the basis of op-
suggesting slight contamination of the sample and again point- erationally defined total metal concentrations, and consequent-
ing out the difficulty of sampling and analyzing trace levels ly their applicability to dissolved concentrations needed to be
of zinc in environmental samples. Arsenic concentrations in- determined. We conducted acute toxicity tests in this study to
creased by 28% in the higher treatment of the A. bahia test, determine the extent to which metals were dissolved during
but concentrations in the lower treatments increased by only conduct of toxicity tests used to establish ALC. Partitioning
4%. Thus, exposure of the test species to the metals could be of metals between dissolved and particulate forms was very
assumed to be relatively uniform over the 48- to 96-h test consistent. Regardless of exposure concentrations, metals were
periods. present almost entirely (.90%) in the dissolved form through-
Notably, partitioning of the metals between dissolved and out the duration of all tests. Chemical QA tests showed that
particulate phases was very similar in all of the tests. Metals these results were not based upon analytical artifacts. Biolog-
were predominantly in the dissolved phase: dissolved metal ical responses from these tests were generally within ranges
concentrations exceeded 90% of total concentrations in every expected from original acute tests, thereby validating the sim-
test and were at least 95% of total in all but the A. bahia ulation of those experimental conditions and implying that
arsenic and M. edulis lead tests. The percentage of total lead concentrations of available metals were the same as in the
in the dissolved fraction was unusually low (76–77%) in two original tests. These results demonstrate that, under the con-
replicates from the lower concentration treatment in the M. ditions employed (relatively short exposure periods and low
edulis test, resulting in a lower and more variable estimate of particulate loads), metals added as toxicants were not removed
percent dissolved lead than in other tests. However, dissolved from solution onto particles. This does not imply that such
lead was greater than 90% of total in all but one other sample partitioning is representative of conditions in natural waters,
from that test, whereas dissolved lead in all samples from the but rather that in the historical saltwater acute toxicity tests
96-h flow-through A. bahia test was greater than 95%. In a used to establish ALC, concentrations of dissolved metals were
similar study, Thursby et al. reported concentrations of dis- not significantly different from total metal concentrations.
solved copper in seawater to be 83% of total [31]. Thus, saltwater acute ALC developed for these metals were,
Analytical results from the static and flow-through tests in fact, based upon dissolved metal concentrations, and can
were very consistent for cadmium, chromium, nickel, and zinc, be applied to dissolved metals.
with the metals almost entirely dissolved in all samples from
both toxicity tests. Although values for percent dissolved ar- Acknowledgement—We thank the Aquatic Life Criteria Guidelines
senic and selenium were generally more variable than other Committee for assistance in the design and development of this study.
metals due to instrumental noise (often 11–14% relative stan- Mention of commercial products or facilities does not constitute en-
dorsement by the U.S. EPA.
dard deviation), mean values were very similar. In the A. abdita
test, concentrations of particulate selenium were slightly above REFERENCES
detection limits, but were so small compared to dissolved con- 1. Prothro M. 1993. Office of Water policy and technical guidance
centrations that they were inconsequential. The percentage of on interpretation and implementation of aquatic life metals cri-
dissolved metal was not significantly different (two-factor teria. Memorandum to Regions I–X. U.S. Environmental Protec-
ANOVA, p 5 0.05) between sampling times in any of the tion Agency, Washington, DC.
2. Thursby GB, Hansen DJ, Locicero FJ, Lewis DA, Allen HE,
tests. Treatment concentrations differed slightly in only 3 of Brosnan TM. 1994. Site specific copper criteria for the Hudson/
14 tests (A. bahia with arsenic and M. lateralis with cadmium Raritan Estuary: Case study. Proceedings, 26th MidAtlantic In-
and zinc). Because differences between concentrations were dustrial and Hazardous Waste Conference, University of Dela-
only slight, an overall mean and standard deviation was cal- ware, Newark, DE, USA, August 7–10, pp 263–272.
3. Price WW, Heard RW, Stuck L. 1994. Observations on the genus
culated for percent dissolved and percent nominal for each test Mysidopsis Sars, 1864 with the designation of a new genus,
(Table 4). Overall, ratios of dissolved to total metal concen- Americamysis, and the descriptions of Americamysis alleni and
trations were not significantly different from 100% for any of A. stucki (Peracarida: Mysidacea: Mysidae), from the Gulf of
the metals tested. This finding is very important, because it Mexico. Proc Biol Soc Wash 107:680–698.
implies that, under test conditions used to establish ALC, the 4. Morrison GE, Petrocelli E. 1991. Suitability of Mulinia lateralis
as a euryhaline toxicity test species. Can Tech Rep Fish Aquat
metals were primarily dissolved, and thus criteria established Sci 1774:337–340.
for concentrations of total metals should also be valid for 5. Lussier SM, Kuhn A, Chammas MJ, Sewall J. 1988. Techniques
concentrations of dissolved metals. for the laboratory culture of Mysidopsis species (Crustacea: My-
Our observation that measured concentrations, both dis- sidacea). Bull Environ Contam Toxicol 7:969–977.
6. American Society for Testing and Materials. 1994. Standard guide
solved and total, were substantially lower than nominal con- for conducting 10-day static sediment toxicity tests with marine
centrations is also significant. Concentrations were often less and estuarine amphipods. E1367-92. In Annual Book of ASTM
than 70% of nominal, and could be as low as 27% of nominal. Standards, Vol 11.04. Philadelphia, PA, pp 1161–1186.
898 Environ. Toxicol. Chem. 18, 1999
7. American Society for Testing and Materials. 1994. Standard guide
for conducting static acute toxicity tests starting with embryos of
four species of saltwater bivalve molluscs. E724-89. In Annual
Book of ASTM Standards, Vol 11.04. Philadelphia, PA, pp 454–
471.
8. American Society for Testing and Materials. 1994. Standard guide
for conducting life-cycle toxicity tests with saltwater mysids.
E1191-90. In Annual Book of ASTM Standards, Vol 11.04. Phil-
adelphia, PA, pp 864–879.
9. Benoit DA, Mattson VR, Olson DL. 1982. A continuous-flow
mini-diluter system for toxicity testing. Water Res 18:457–464.
10. Sosnowski SL, Germond DL, Gentile JH. 1979. The effect of
nutrition on the response of field populations of the calanoid
copepod Acartia tonsa to copper. Water Res 13:449–452.
11. American Society for Testing and Materials. 1994. Practice for
using brine shrimp nauplii as food for test animals in aquatic
toxicology. E1203-87. In Annual Book of ASTM Standards, Vol
11.04. Philadelphia, PA, pp 950–955.
12. Petrocelli E, Morrison G. 1990. Coot clam (Mulinia lateralis)
embryo/larval toxicity test. Laboratory Operating Procedure
1.03.008. U.S. Environmental Protection Agency, Narragansett,
RI.
13. Loosanoff V, Davis H, Chaney P. 1966. Dimensions and shapes
of larvae of some marine bivalve mollusks. Malacologia 4:351–
435.
14. U.S. Environmental Protection Agency. 1984. Ambient water
quality criteria for chromium. EPA 440/5-84-029. Washington,
DC.
15. U.S. Environmental Protection Agency. 1984. Ambient water
quality criteria for lead. EPA 440/5-84-027. Washington, DC.
16. U.S. Environmental Protection Agency. 1984. Ambient water
quality criteria for cadmium. EPA 440/5-84-032. Washington,
DC.
17. U.S. Environmental Protection Agency. 1979. Methods for chem-
ical analysis of water and waste, residue, non-filterable method,
160.2. EPA 600/4-79-020. Washington, DC.
18. Strickland JDH, Parsons TR. 1972. A Practical Handbook of
Seawater Analysis, Bulletin 167, 2nd ed. Fisheries Research
Board of Canada, Ottawa, ON, Canada, pp 153–158.
S.M. Lussier et al.
19. Stephan CE, Mount DI, Hansen DJ, Gentile JH, Chapman GA,
Brungs WA. 1985. Guidelines for deriving numerical national
water quality criteria for the protection of aquatic organisms and
their uses. PB85-227049. National Technical Information Service,
Springfield, VA, USA.
20. U.S. Environmental Protection Agency. 1986. Method 3015 mi-
crowave assisted acid digestion of aqueous samples and extracts.
Test methods for evaluating solid waste, Vol IA: physical/chem-
ical methods, 3rd ed. EPA SW-846. Washington, DC.
21. American Society for Testing and Materials. 1994. Standard prac-
tice for sample digestion using closed vessel microwave heating
technique for the determination of total metals in water. D 4309-
91. In Annual Book of ASTM Standards, Vol 11.01. Philadelphia,
PA, pp 648–653.
22. Snedecor GW, Cochran WG. 1980. Statistical Methods, 7th ed.
Iowa State University Press, Ames, IA, USA.
23. Lussier SM, Gentile JH, Walker J. 1985. Acute and chronic effects
of heavy metals and cyanide on Mysidopsis bahia (Crustacea:
Mysidacea). Aquat Toxicol 7:25–35.
24. U.S. Environmental Protection Agency. 1984. Ambient water
quality criteria for arsenic. EPA 440/5-84-003. Washington, DC.
25. U.S. Environmental Protection Agency. 1987. Ambient water
quality criteria for selenium. EPA 440/5-87-006. Washington, DC.
26. U.S. Environmental Protection Agency. 1987. Ambient water
quality criteria for zinc. EPA 440/5-87-003. Washington, DC.
27. U.S. Environmental Protection Agency. 1986. Ambient water
quality criteria for nickel. EPA 440/5-86-004. Washington, DC.
28. Martin M, Osborn KE, Billig P, Glickstein N. 1981. Toxicities of
ten metals to Crassostrea gigas and Mytilus edulis embryos and
Cancer magister larvae. Mar Pollut Bull 12:305–308.
29. Calabrese A, Collier RS, Nelson DA, MacInnes JR. 1973. The
toxicity of heavy metals to embryos of the American oyster, Cras-
sostrea virginica. Mar Biol (Berl) 18:162–166.
30. Calabrese A, Nelson D. 1974. Inhibition of embryonic devel-
opment of the hard shell clam, Mercenaria mercenaria, by heavy
metals. Bull Environ Contam Toxicol 11:92–97.
31. Science Applications International Corporation. 1993. Toxicity
testing to support the New York/New Jersey Harbor site-specific
copper criteria study. Final Technical Report. U.S. Environmental
Protection Agency, Region II, NY, New York.