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Ecotoxicology and Environmental Safety ](]]]])]]]–]]]
Chronic toxicity of arsenic to the Great Salt Lake brine shrimp,
Artemia franciscana
Kevin V. Brix
a,*, Rick D. Cardwell
b, and William J. Adams
c
a EcoTox, 2001 NW Nye Street, Newport, OR 973659-8045, USA
b Parametrix, Inc., 5808 Lake Washington Blvd. NE, Suite 200, Kirkland, WA 98033, USA
c Kennecott Utah Copper, 8315 West 3595 South, P.O. Box 6001, Magna, UT 84044, USA
Received 27 November 2001; received in revised form 26 August 2002; accepted 26 August 2002
Abstract
We determined the chronic toxicity of arsenic (sodium arsenate) to the Great Salt Lake brine shrimp,Artemia franciscana.
Chronic toxicity was determined by measuring the adverse effects of arsenic on brine shrimp growth, survival, and reproduction
under intermittent flow-through conditions. The study commenced with o24-h-old nauplii, continued through reproduction of the
parental generation, and ended completed after 28 days of exposure. The concentrations tested were 4, 8, 15, 31, and 56mg/L
dissolved arsenic. The test was conducted using water from the Great Salt Lake, Utah as the dilution water. Adult survival was the
most sensitive biological endpoint, with growth and reproduction somewhat less sensitive than survival. The no observed effect
concentration (NOEC) for survival was 8mg/L, and the lowest observed effect concentration (LOEC) was 15mg/L dissolved
arsenic. The LOEC for growth and reproduction was greater than the highest concentration tested, 56mg/L. Based on survival, the
final chronic value (geometric mean of the NOEC and LOEC) was 11mg/L dissolved arsenic. The F1 generation appeared to
acclimate to the prior arsenic exposure of the parental generation and was significantly less sensitive than the parental generation.
For example, survival for the F1 generation through day 12 was 100% in 56mg/L dissolved arsenic, compared to 26% for the
parental generation. Growth of the F1 generation was significantly less than that of the parental generation across all concentrations
including the control, indicating a generational difference in brine shrimp growth rather than an arsenic effect. This study represents
one of the few full life cycle toxicity tests conducted with brine shrimp.
r 2002 Elsevier Science (USA). All rights reserved.
Keywords:Arsenic;Artemia franciscana; Chronic toxicity; Great Salt Lake; Brine shrimp
1. Introduction
The Great Salt Lake (GSL) in Utah is a unique
aquatic habitat in the United States because of its water
quality characteristics and extant biota. As a terminal
lake with limited freshwater input, the lake has become
hypersaline with a surface water salinity that ranges
from approximately 75 to 150g/L depending on annual
rainfall. The lake is also characterized by relatively high
dissolved organic carbon concentrations (15–55mg/L)
(Domagalski et al., 1990).
The hypersaline conditions severely limit the biologi-
cal diversity of the GSL. No fish are capable of
osmoregulating under these conditions and so are
precluded from the lake. Only two invertebrates, brine
shrimp (Artemia franciscana) and brine flies (Ephydra
spp.), permanently occur throughout the lake. The brine
shrimp have significant commercial value; approxi-
mately 10 million pounds of brine shrimp eggs are
harvested each winter and sold as food for tropical fish,
and the commercial shrimp industry generates over $10
million annually to the local economy and represents
90% of the world’s commercial harvest. The brine fly
larvae that occur in the lake provide, in combination
with the brine shrimp, the main food supply for the
millions of shorebirds that use the lake as a migratory
stopover or breeding ground. Seven species of brine flies
have been identified in the lake (Jorgenson, 1956). Water
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*Corresponding author. Fax: [phone].
E-mail address:[e-mail] (K.V. Brix).
0147-6513/02/$-see front matter r 2002 Elsevier Science (USA). All rights reserved.
PII: S 0147-6513(02)00054-4
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boatmen (Tricorixa sp.) also periodically occur in the
lake in some of the lower-salinity embayments, near
significant freshwater inputs. The only other biota
occurring in the lake are algae, diatoms, and bacteria.
Approximately 23 species of algae and diatoms have
been identified (Felix and Rushforth, 1979).
Given the unique habitat and biota, application of
national water quality criteria to the lake is inappropri-
ate, which USEPA explicitly acknowledges (Stephan
et al., 1985). Rather, development of water quality
standards for the lake should be based on site-specific
toxicity studies on resident species. Further, these
studies should be performed in water from the lake to
account for how water quality characteristics influence
chemical bioavailability.
There has been considerable methodological develop-
ment for toxicity testing with brine shrimp over the past
20 years, although the vast majority of efforts have
focused on acute testing regimes (Sorgeloos et al., 1978;
Persoone and Castritsi-Catharios, 1989). Standard
methods for performing chronic toxicity studies with
brine shrimp have not been developed, although limited
full chronic testing has been performed previously
(Cunningham, 1976;Gebhardt, 1976). The objective of
this study was to both develop a standardized chronic
toxicity test method and use it to evaluate the effects of
arsenic on brine shrimp. The potential for toxicity was
evaluated through conduct of a full life-cycle test
(Stephan et al., 1985). The approach used for this study
was based on information gained from the literature and
from preliminary testing that we performed. The study
measured adverse effects on survival, growth, and
reproduction after brine shrimp were exposed to a series
of varying arsenic concentrations for 28 days. The
objective of the study was to determine the no observed
effect concentraction (NOEC) and the lowest observed
effect concentraction (LOEC) for survival, growth, and
reproduction for the parental generation and for
survival and growth for the F1 generation.
2. Materials and methods
2.1. Test substance
Arsenic, as reagent-grade sodium arsenate, NaAsO2
(CAS No. 10048-95-0), was obtained from Sigma
Chemical Co. (St. Louis, MO). A stock solution was
prepared by adding 133g of reagent-grade sodium
arsenate to 2L of deionized water and mixing for 2h
to achieve a 16g/L stock solution. This solution was
mixed for 2h in a 2-L Pyrex volumetric flask using a
Teflon-coated stir bar for mixing. After mixing, the
stock solution was held in the dark at ambient
temperature. Stock solutions were prepared in this
manner three times during the study and all were
analyzed for arsenic concentrations prior to use. All test
concentrations in the remainder of this paper are
reported as mg/L of arsenic.
2.2. Test organisms
Brine shrimp cysts were purchased from Argent
Chemical Laboratories (Redmond, WA) and were
certified to be A. franciscana, collected from the GSL.
Cysts were stored in the dark at 51C until used for
testing. Cysts were hatched in seawater (salinity 28g/L)
at approximately 271C under vigorous aeration. Nauplii
o 24h old were used to initiate the study.
2.3. Dilution water
Dilution water was collected from the GSL at a depth
of 10–20cm, on the north side of Antelope Island,
approximately 60 miles north of Salt Lake City, Utah in
the south arm. The dilution water was stored in two
leached, 4000-L HDPE tanks for 20 days prior to use in
testing. During storage, the dilution water was aerated
by continuous circulation through the two storage
tanks. The dilution water was analyzed for inorganic
priority pollutants and conventional parameters prior to
use in testing (Table 1). The total organic carbon,
dissolved organic carbon, and total suspended solids
concentrations measured in the dilution water are
characteristic of the GSL (Domalgaski et al., 1990).
Although these concentrations are higher than would
normally be used in a laboratory toxicity tests (ASTM,
1996), they are appropriate for evaluating the site-
specific conditions in the GSL.
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Table 1
Dilution water quality
Analyte Concentration (mg/L)
Antimony o0.1
Arsenic 0.24
Beryllium o0.05
Cadmium o0.05
Chromium o0.05
Copper o0.15
Lead o0.05
Mercury o0.0004
Nickel o0.05
Nitrogen, total ammonium 0.38
Nitrogen, nitrate 1
Nitrogen, nitrite 5
Organic carbon, dissolved 45
Organic carbon, total 46
Selenium o0.1
Silver o0.05
Thallium o0.05
Total suspended solids 34
Zinc o0.1
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2.4. Water quality parameters
Water quality parameters (pH, dissolved oxygen, and
salinity) were measured in each test chamber and water
temperature in the physical system at test initiation and
every day thereafter. Water temperature was measured
using a thermometer calibrated against a certified NBS
thermometer. Test solution pH was measured using a
Cole–Parmer Model 5398-00 digital pH meter. Dis-
solved oxygen was measured using a Hach Dissolved
Oxygen Test Kit, Model OX-2P. Salinity was measured
using a Reichert temperature-compensated refract-
ometer.
2.5. Test methods
A proportional diluter system (USEPA, 1978) was
used for the intermittent introduction of arsenic and
dilution water. A calibrated laboratory pump (Fluid
Metering Inc., Oyster Bay, NY) was used to inject the
arsenic stock solution into the proportional diluter
system. Prior to adding test organisms and initiating the
study, the physical system was operated for 14 days to
verify that measured arsenic concentrations were ap-
proximately equal to nominal concentrations.
The physical system was composed of an enclosed box
in which the test chambers were housed and external
light was controlled. Water temperature in the test
chambers was maintained in a water bath. Test
concentrations were mixed by the diluter and 1L of
test solution per replicate was delivered eight times per
day. Each replicate underwent a 95% volume replace-
ment every 50h (Sprague, 1969). Gentle aeration (o100
bubbles per minute) was provided by a regenerative
blower and delivered through glass bead airstones to
each test chamber. The lighting for the test system
consisted of fluorescent lights (100–150 foot candles) set
on a 16-h light/8-h dark photoperiod.
The test design consisted of five concentrations and a
dilution water control. A dilution water control and
nominal concentrations of 4, 8, 16, 32, and 64mg/L
arsenic were used in the definitive study. These
concentrations were based on a range-finding study that
resulted in a 7-day LOEC of 64mg/L arsenic for brine
shrimp survival. Each concentration and control was
represented by duplicate 7.8-L test chambers that were
randomly located in relation to the others. Within each
actual replicate were four acrylic cylinder pseudo-
replicates (7cm diameter by 14cm deep) for a total of
eight cylinders per test concentration. As discussed
below, the pseudo-replicates within each test chamber
were used to isolate breeding pairs of Artemia within
each replicate so that nauplii production by the
individual pairs could be accurately quantified. Each
cylinder had four ports 2cm wide by 10.5cm high that
allowed for exchange of test solution. The ports were
covered by 120 mm Nitex screen to retain test organisms.
At test initiation, 10 nauplii o24h old were intro-
duced randomly into each cylinder for a total of 80
organisms per test concentration. Brine shrimp were
observed daily; they were counted and recorded and
dead specimens removed. Once sexual maturity was
reached and adults began pairing (12 days after test
initiation), three cylinders from each replicate were
divided in half using an acrylic insert and one adult pair
was placed in each half. This division of cylinders
physically separated each pair, facilitating accurate
quantification of nauplii production per pair. Each
concentration contained a total of 12 pairs, with the
exception of the highest concentration, which contained
only five pairs, due to significant mortality during the
first 12 days of exposure. One cylinder from each test
chamber was not divided but instead used for evaluation
of first-generation nauplii survival and growth. By not
dividing this cylinder, the first-generation nauplii were
exposed to the same conditions as those of parental
generation prior to reproductive pairing. Of the
remaining adults from each replicate concentration, 10
(five males and five females) were randomly selected for
dry weight measurement. The brine shrimp were dried
for 24h at 601C, after which they were weighed to the
nearest 0.01mg on an analytical balance. The brine
shrimp were dried for 8 more hours and reweighed to
ensure that constant weight was achieved.
The first release of live young occurred on day 13 and
by day 15 all cylinders in all test concentrations had
released live young. After pairing, any live nauplii or
cysts released were counted and removed daily. Ten
nauplii from each cylinder were selected randomly,
placed in the fourth undivided cylinder, and allowed to
grow for a duration equal to the number of days to
thinning for the parental generation (12 days). F1
generation exposure was terminated on days 25–27,
depending on when the nauplii were first released (adult
exposure continued through day 28). At this time, F1-
generation brine shrimp were counted and measured for
dry weight.
At test initiation, the brine shrimp were fed marine
green algae (Platymonas sp.) twice per day immediately
after diluter cycling to provide adequate feeding time
prior to flushing during the next diluter cycle.Platymo-
nas sp. was used because preliminary studies demon-
strated it to be a suitable food source. Each cylinder
received 100,000 algal cells per animal twice daily. When
the brine shrimp were thinned at day 12, each pair was
fed 500,000 cells twice per day. The F1 generation was
fed 100,000 algal cells per animal twice daily.
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2.6. Analytical chemistry
Samples for arsenic analysis were collected at mid-
depth from one replicate randomly selected from each
test concentration at test initiation. Care was taken not
to sample proximate to the chamber’s sides or bottom
and to avoid aspirating feces. For the remainder of the
study, sampling of test concentrations alternated repli-
cates. The time of sampling occurred at approximately
the mid-point between dilutor cycles. Arsenic concen-
trations were measured at test initiation, each week
thereafter, and at test termination for each concentra-
tion containing living test organisms. Duplicate and
spiked samples were collected at a frequency of 5%.
Samples were analyzed for both total and dissolved
(o0.45 mm) arsenic, using ICP-MS EPA Method 6020
(USEPA, 1995). The geometric mean of dissolved
arsenic concentrations from the five sampling events
was calculated and reported as the measured arsenic
concentration for the test.
2.7. Analysis of biological data
Five test endpoints were evaluated in this study:
(1) survival of the parental generation prior to repro-
ductive pairing on day 12 and survival on days 21
and 28;
(2) growth of the parental generation prior to repro-
ductive pairing (day 12) and at day 28;
(3) reproduction in the parental generation on days 21
and 28;
(4) survival of the F1 generation through day 12; and
(5) growth of the F1 generation through day 12.
Data were analyzed a commercial software package,
Statgraphics, version 5.0 (STSC, 1991). Data for the
various test endpoints were first evaluated for normality
and homogeneity of variance. The assumption of
normality was tested by calculating the ANOVA model
residuals and testing these residuals for normality by the
normal probability plot method (Neter et al., 1990). The
assumption of homogeneity of variances was evaluated
by Bartlett’s test using Statgraphics.
If a data set met the assumptions of normality and
homogeneity of variances, an ANOVA was computed to
determine whether any statistically significant differ-
ences existed among levels (concentrations or genera-
tions). If either of the assumptions could not be met, the
nonparametric Kruskal–Wallis test was used to test for
differences. Only survival of the F1 generation was
analyzed using nonparametric methods. Multiple-range
evaluations for parametric data sets were computed by
the least significant difference (LSD) test using Stat-
graphics. LSD tests for nonparametric data sets were
computed with results from the Kruskal–Wallis tests
using the multiple comparisons technique presented in
Gibbons (1985).
3. Results
3.1. Water quality and test concentrations
The test temperature ranged from 251Cto261C,
dissolved oxygen ranged from 0.6 to 3.2mg/L, pH
ranged from 7.8 to 8.2, and salinity ranged from 122 to
127g/L over the course of the test.
Weekly arsenic concentrations are summarized as
geometric means and coefficients of variation in Table 2.
For the remainder of this paper, references to an arsenic
concentration refers to the geometric mean of the
measured dissolved arsenic concentrations. The dilution
water had a mean concentration of 0.3mg/L arsenic.
The coefficient of variation for the dilution water was
60%; however, this was due to variability in relatively
low (compared to test concentrations) arsenic concen-
trations (0.21 to 0.69mg/L arsenic). Based on the
analytical results, mean concentrations in the test were
0.3, 4, 8, 15, 31, and 56mg/L arsenic as compared with
nominal concentrations of 0, 4, 8, 16, 32, and 64mg/L.
Coefficients of variation for test concentrations ranged
from 9% to 14%.
3.2. Biological results
Survival, growth, and reproduction were analyzed for
the parental generation, and survival and growth were
analyzed for the F1 generation. Survival in the parental
generation was the most sensitive endpoint through day
21 (Table 3). Between days 21 and 28, there was a
general decline in survival across all concentrations,
resulting in no statistically significant effects at any
concentration compared to the control on day 28. After
reproductive pairing on day 12, the dose–response
relationship at the higher test concentrations became
less distinct, with 31mg/L arsenic exhibiting higher
survival than 15mg/L arsenic on days 21 and 28. None
of the arsenic concentrations adversely affected survival
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Table 2
Dissolved arsenic concentrations
Nominal As
(mg/L)
Geometric
mean dissolved
As (mg/L)
Range in
measurements
Coefficient of
variation
(n ¼6) (%)
0 0.3 0.21–0.69 60
4 3.9 3.4–4.5 12
8 7.8 6.7–9.0 10
16 14.6 13.0–17.0 10
32 31.2 27.0–36.0 9
64 56.3 42.0–63.0 14
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for the F1 generation over the exposure period. The F1
generation was significantly less sensitive than the
parental generation, with 100% survival at 56mg/L
arsenic compared to 26% survival at 56mg/L for the
parental generation over a 12-day period.
Arsenic did not affect growth in the parental or F1
generation (Table 4). No statistically significant effects
on growth occurred at any test concentration on days 12
or 28 for the parental generation. Although growth in
the F1 generation was significantly less than that in the
parental generation through day 12, there was no
significant difference between the control and the test
concentrations (Table 4).
Brine shrimp reproduction was analyzed by the total
number of young per adult reproductive day (YARD)
per test chamber. This endpoint is calculated in a
manner similar to the laboratory fish production index
devised by Mount and Stephan (1967).Table 5
summarizes results for each test concentration for days
21 and 28. Although YARD was reduced at 56mg/L
(relative to the control) by 40% on day 21 and by 24%
on day 28, this difference was not statistically signifi-
cant, resulting in a reproductive NOEC of 56mg/L.
4. Discussion
Of the biological endpoints measured for the adult
generation, survival was the most sensitive, followed by
reproduction and then growth (Table 6). After 21 days
of exposure, survival was significantly affected at 15mg/
L arsenic (the survival LOEC), but not at 8mg/L arsenic
(the survival NOEC). Over the 21 days of exposure,
neither growth nor reproduction was affected at the
highest concentration tested (LOEC >56mg/L arsenic),
although reproduction at 56mg/L was reduced by 40%
relative to that of the control on day 21. Based on the
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Table 3
Parental and F1 generation survival
Dissolved arsenic
(mg/L)
Day 12 Day 21 Parental (SE) Day 28 Parental (SE)
Parental (SE) F1 (SE)
0 93% (0%) 90% (0%) 75% (8%) 46% (4%)
4 86% (4%) 100% (0%) 58% (0%) 38% (4%)
8 89% (4%) 90% (10%) 58% (8%) 29% (13%)
15 90% (3%) 90% (10%) 42%* (8%) 33% (8%)
31 81%* (1%) 100% (0%) 79% (4%) 50% (8%)
56 26%* (1%) 100% (0%) 42%*
,a(8%) 25% (17%)
a Percentage survival increased compared to that of day 12 due to thinning of test organisms at reproductive pairing.
*Statistically significant effect compared to the control.
Table 4
Parental and F1 Generation Dry Weight (mg/Organism
Dissolved
Arsenic (mg/L)
Day 12 Day 28
Parental (SE)
Parental (SE) F1 (SE)
0 0.37 (0.00) 0.24*(0.04) 0.45 (0.01)
4 0.43 (0.03) 0.31*(0.06) 0.72 (0.18)
8 0.40 (0.04) 0.23*(0.07) 0.51 (0.10)
15 0.43 (0.00) 0.25*(0.02) 0.52 (0.04)
32 0.47 (0.01) 0.28*(0.02) 0.49 (0.02)
56 0.45 (0.03) 0.25*(0.12) 0.57 (0.07)
*Statistically significant difference compared to the parental genera-
tion.
Table 5
Brine Shrimp Reproduction (Young Produced per Adult Reproduc-
tion Day
Dissolved arsenic (mg/L) Young/Adult Reproduction Day (SE)
Day 21 Day 28
0 9.7 (0.3) 9.1 (0.5)
4 13.4 (2.0) 12.2 (1.1)
8 6.9 (2.5) 5.7 (1.5)
15 10.9 (1.7) 11.3 (2.0)
31 9.0 (0.5) 7.8 (1.0)
56 5.8 (1.8) 6.9 (3.0)
Table 6
Summary of Results for Biological Endpoints
Evaluation Measured dissolved
arsenic (mg/L)
Parental Survival NOEC 8
LOEC 15
Parental growth NOEC 56
LOEC >56
Parental reproduction NOEC 56
LOEC >56
F1 survival NOEC 56
LOEC >56
F1 growth NOEC 56
LOEC >56
Final chronic value
a 11
a The final chronic value is the geometric mean of the NOEC and the
LOEC for the most sensitive endpoint measured. In this case, it
constitutes parental generation survival, the geometric mean of 8 and
15mg/L, or 11mg/L.
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day 21 survival results, the final chronic value is 11mg/L
dissolved arsenic, calculated as the geometric mean of
the lowest NOECs and LOECs observed (8 and 15mg/
L).
The F1 generation appeared to acclimate to the
arsenic exposure and was significantly less sensitive than
the parental generation in terms of survival. Why F1
generation growth was significantly less than parental
generation growth across all test concentrations, includ-
ing the control, is unclear. One hypothesis is that brine
shrimp produced oviparously and ovoviviparously
either have different growth rates or, perhaps, initiate
active feeding at different times.
There is a general lack of information concerning
testing methodologies and performance of brine shrimp
in chronic toxicity tests. The species has been used
primarily for acute tests (Sorgeloos et al., 1978;
Vanhaecke et al., 1981;Persoone and Castritsi-Cathar-
ios, 1989). Only three other chronic studies have been
conducted with brine shrimp. The first (Gebhardt, 1976)
evaluated the effects of cadmium, copper, and mercury
on survival, growth and reproduction. It was conducted
at 271C using a static renewal test design (solution
replacement every third day), GSL water was used as the
dilution water, and brine shrimp were fed the hypersa-
line algae Dunaliella viridis. Under these conditions, the
onset of reproduction was considerably later than that
observed in our study with the control group beginning
to reproduce on day 29. The number of nauplii
produced was not quantified in that study.
The second study (Cunningham, 1976), although not
a full life cycle study, investigated the effects of the
insecticide Dimilin (TH 6040) on different life stages of
brine shrimp under static renewal test conditions. In one
experiment, brine shrimp reproduction was evaluated by
exposing brine shrimp pairs and monitoring the number
of nauplii produced. On day 21 of the experiment, adult
survivorship in the control was approximately 90% and
declined to approximately 70% on day 28. By day 40,
survival had dropped below 50% and all shrimp were
dead by day 80.
The only other data available for comparison to this
study were from a preliminary study (dilution water
only) that we conducted prior to initiating the definitive
arsenic study. The primary difference between the
preliminary study and the definitive study was that the
preliminary study was conducted under static renewal
conditions without constant aeration. The preliminary
study had 78% and 75% survival on days 21 and 28
compared with 75% and 46% in this study. However,
reproduction in the preliminary study was considerably
different from that in the definitive study. An approx-
imate 1:1 ratio of cysts and live young were produced in
the preliminary study compared with only two cysts
produced in any test concentration in the definitive
study. Additionally, live young/surviving female on day
21 (156 in the control) in this study exceeded live young/
surviving female on day 28 (113 in the control) in the
preliminary study.
We hypothesize that under the conditions of this flow-
through study, the brine shrimp completed their life
cycle more quickly than those in the other studies. In
other words, the mortality observed across all test
concentrations between days 21 and 28 was caused by
the shrimp reaching the end of their life span. This is
supported by Gillespie and Stephens (1977), who
suggested that the ‘‘generation time of Artemia may be
less than three weeks under good conditions.’’ Until
additional studies are conducted though, this hypothesis
cannot be verified.
5. Conclusions
Overall, we believe that this study demonstrates that
chronic toxicity studies with brine shrimp can be
performed successfully in the laboratory and that the
results obtained are useful in assessing the potential of
arsenic to limit survival, growth, and reproduction of
brine shrimp in the Great Salt Lake. The difference in
growth between parental and F1 offspring and the
decline in survival after day 21 indicates that additional
research is needed to further define the life cycle of brine
shrimp and appropriate testing and culturing needs of
the organisms. However, given the available data, it
appears unlikely that arsenic poses a significant risk to
brine shrimp in the Great Salt Lake, given the relatively
high effect levels determined in this study.
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