Toxicity of Two Pulsed Metal Exposures to Daphnia magna:Relative Effects of Pulsed Duration-Concentration and Influenceof Interpulse Period
Tham C. Hoang Æ Jeffrey S. Gallagher ÆJoseph R. Tomasso Æ Stephen J. Klaine
� Springer Science+Business Media, LLC 2007
Abstract Aquatic organisms living in surface waters
experience fluctuating contaminant exposures that vary in
concentration, duration, and frequency. This study char-
acterized the role of pulsed concentration, pulsed duration,
and the interval between pulses on the toxicity of four
metals (Cu, Zn, Se, and As) to Daphnia magna. During 21-
d toxicity tests, neonatal D. magna were exposed to single
or double pulses. Pulsed concentrations and durations
ranged from 32 to 6000 lg/L and 8 to 96 h, respectively.
Intervals between two pulses ranged from 24 to 288 h.
Mortality, growth, and reproduction were characterized for
exposures. For single-pulse exposures of Cu and As, metal
concentration had a stronger effect on survival of D. magna
than did pulsed duration: pulses with 2X concentration and
1Y duration resulted in more mortality than did pulses with
1X concentration and 2Y duration. In contrast, effects of
pulsed duration were stronger than metal concentration for
Zn. However, the effects of duration and concentration
were similar for Se. The relative effects of pulsed con-
centration and duration found in the present study revealed
that the common method using area under the curve
(AUC = concentration · duration) may not always accu-
rately estimate environmental risk from metals (e.g., for
Cu, Zn, As). In addition, the occurrence of delayed mor-
tality in the present study revealed that using continuous
exposure bioassays might underestimate metal toxicity to
aquatic biota. For double-pulse exposures, the toxicity of
the second pulse was influenced by the first pulse for all
four metals. This influence was dependent on the pulsed
concentration and duration and the interval between pulses.
Further, toxicity caused by the second pulse decreased as
the time between the exposures increased. For all four
metals, there existed an interval great enough that the
toxicity of the two pulses was independent. This would
result in less toxicity for multiple exposures than continu-
ous exposures with the same total exposure duration. The
interval time at which the effects of the two pulses were
independent increased with increasing concentration.
Growth and cumulative reproduction of D. magna over 21
d were not significantly affected by pulsed exposures
examined in the present study, indicating recovery of the
organisms.
Keywords Pulsed exposure � Metal toxicity � Daphniamagna
Introduction
Contaminant concentrations in surface waters are usually
intermittent and fluctuated, depending on point and non
point sources. According to Diamond et al. (2005), effluent
copper concentration measured at a South Carolina pub-
licly owned treatment works fluctuated with time. Aquatic
organisms living in surface water would experience fluc-
tuating exposures. This makes risk estimation for aquatic
T. C. Hoang � J. S. Gallagher � S. J. KlaineDepartment of Biological Sciences, Clemson Institute of
Environmental Toxicology, 509 Westinghouse Road,
Pendleton, South Carolina 29670, USA
J. R. Tomasso
Department of Biology, Texas State University, San Marcos,
TX 78666, USA
T. C. Hoang (&)
Department of Environmental Studies, Florida International
University, C/O Arts & Science, Southeast Environmental
Research Center, 3000 NE 151st Street, North Miami,
Florida 33181, USA
e-mail: [email protected]
123
Arch Environ Contam Toxicol 53, 579–589 (2007)
DOI 10.1007/s00244-006-0266-1
life difficult. Toxicology research to characterize the
effects of pulsed exposures in aquatic organisms has been
conducted with both inorganic and organic contaminants
(Naddy et al. 2000, Naddy and Klaine 2001, Widianarko
et al. 2001, Milne et al. 2000, Hoang et al. 2006, Reynaldi
and Liess 2004, Zhao and Newman 2006, Diamond et al.
2006). Most of the research examined the effects of single
and multiple pulses in a general pattern, that combined
pulsed duration and concentration. However, the relative
individual effects of pulsed duration and concentration are
important for estimating exposure and risk to biota. One of
the popular methods that has been used to characterize
exposure and risk to biota has been to look at the area
under the curve (AUC = (duration) · (concentration)).
Based on this method, exposed organisms with the same
AUC would have the same risk (Morton et al. 2000, U.S.
EPA 1998). This method is applicable for the case in which
the effects of exposure concentration and duration are
equal. In this case, concentration can be traded off with
duration. When the effects of exposure concentration and
duration are not equal, this method is not effective. Con-
sidering two pulsed exposures scenarios with the same
AUC: AUC = (high concentration) · (short duration) =
(low concentration) · (long duration), the effects of this
two pulsed exposure scenarios may be different if the rel-
ative effects of concentration and duration are different. In
addition, the AUC method becomes less effective with
toxicants that have latent effect, such as diflubenzuron
(Hurd et al.1996 and Van der Hoeven et al.1997).
For multiple pulses, the interval (recovery) between
pulses plays an important role on the effects of later
pulses. When the interval between pulses is long enough,
organisms may recover to their normal physiological
conditions after earlier pulses; this results in the effects
of the later pulse being independent of the effects of the
earlier pulse(s). Hence, with the same total exposure
duration and long enough intervals, multiple expo-
sures may result in less effect than one continuous
exposure. In this case, the effects are certainly not
additive. Organisms may also develop tolerance after
earlier pulses and may become less sensitive to later
pulses (Allin and Wilson 2000). Previous research has
looked at the effects of recovery time (Hoang et al.
2006, Naddy et al. 2000, 2001, Zhao and Newman 2006,
Reynaldi and Liess 2004, Diamond et al. 2006). How-
ever, most of this research examined only the total ef-
fects at the end of experiments (e.g., total mortality).
Data on the effect of a single pulse in multiple pulsed
exposure experiments are not available. This research
characterized the relative effects of pulsed duration and
concentration and the influence of recovery time between
two pulses on the toxicity of four metals (Cu, Zn, Se,
As) to Daphnia magna.
Materials and Methods
Daphnia magna Culture
Daphnia magna were cultured in the Institute for Envi-
ronmental Toxicology, Clemson University (Pendleton,
SC, USA) according to U.S. Environmental Protection
Agency (U.S. EPA) methods (Lewis et al. 1994). The water
hardness of D. magna culture media suggested by U.S.
EPA methods was 160 mg/L as CaCO3. However, the
water hardness of the D. magna culture media in our lab-
oratory was adjusted to the reference water hardness (100
mg/L as CaCO3) that U.S. EPA used to develop water
quality criteria for metals in freshwater systems (U.S. EPA
2004). Moderately hard water (MHW) was prepared by
adding reagent-grade salts (NaHCO3, CaSO4.2H2O,
MgSO4, and KCl) to deionized water (Super-QTM; Milli-
pore Corporation, Bedford, MA, USA) and aerated for at
least 24 h before use. Reagent-grade salts were purchased
from VWR International Inc. Suwanee, GA, USA.
Organisms were maintained at 25 ± 1�C under a 16:8 (L:D)
photoperiod. D. magna were fed once daily with 5mL of
concentrated algae (Selenastrum capricornutum (Pseud-
okirchneriella subcapitata), 3x107 cells/mL) and 5ml of
YTC (yeast trout chow) per 20–30 organisms. The culture
medium was renewed on Monday, Wednesday, and Friday
and neonates were removed. Reference toxicity tests for
£24-h-old D. magna were conducted monthly with copper
sulfate hexahydrate based on U.S. EPA method (Lewis
et al. 1994). The average of the 48-h LC50 values for 3
years (2003–2005) was 18.7 lg/L Cu with a standard
deviation of 4.5.
Toxicity Tests and Measured Endpoints
Twenty-one day toxicity tests were conducted according to
U.S. EPA methods (Lewis et al. 1994) using MHW. Test
waters were prepared by adding each single stock solution
of tested compound to MHW and aerating for at least 24 h
before test initiation. Copper sulfate pentahydrate, zinc
sulfate heptahydrate, sodium selenite sulfate hexahydrate,
and sodium arsenite (ordered from VWR International Inc.
Suwanee, GA, USA) were used to make the stock solu-
tions. Tests were conducted in 600-mL graduated poly-
propylene beakers, each containing 300 mL test media. The
beakers were randomly positioned on a shelf at a temper-
ature of 25 ± 1�C, with a 16:8 (L:D) photoperiod. Five
D. magna were transferred to each beaker by means of a
smooth glass tube (8 mm in diameter). Transfer of
D. magna to the beaker initiated the metal exposure. For
single-pulse exposure, after the appropriate exposure per-
iod, surviving organisms in each beaker were transferred to
other beaker and maintained in control culture media
580 T. C. Hoang et al.
123
(MHW) until test termination (21 days). For double-pulse
exposures, at the end of the first pulse, surviving D. magna
were transferred to MHW and allowed to recover for
appropriate recovery period before the second pulse star-
ted. At the end of the second pulse, surviving D. magna
were transferred to MHW and maintained until test ter-
mination (21 days). Daphnia magna in each beaker were
fed with 2 mL of algae (3x107 cells/mL) and 2 mL of YTC
during and after exposures. Offspring and dead organisms
were removed daily. Mortality and reproduction (number
of neonates) were recorded daily until test termination. For
single-pulse exposures, 21-d mortality was used for lethal-
effect analysis. The lethal effect was analyzed by com-
paring the mortality within each pair of treatments. For
double-pulse exposures, mortality due to the first pulse was
measured on day 3. Mortality due to the second pulse was
the difference in total mortality on day 21 and mortality on
day 3. The effect of the influence of the first pulse on the
second pulse was analyzed by comparing the mortality due
to the first pulse and the second pulse. The reproductive
end point (21-d cumulative reproduction (young/living
adult)) was measured by calculating the average number of
neonates produced per living organism each day and then
summed for 21 days. At test termination, surviving adults
in each beaker were transferred as a group to single alu-
minum weigh boat and dried in an oven (Fisher Scientific,
Atlanta, GA, USA) model 825F at 100 �C for at least 6 h.
After drying, the aluminum weigh boats with organisms
were weighed on a Mettler Balance (Greenville Scale Co.,
Inc., SC, USA) model AT201 to measure the growth
endpoint. For single-pulse exposures, the effects of pulsed
concentration and duration on growth and reproduction
were analyzed by comparing the dry weight and the
number of young per living adult within each pair treat-
ment. For double-pulse exposures, the effects on growth
and reproduction were analyzed by comparing the dry
weight and the number of young per living adult with
control treatment. All comparisons were conducted using
SAS (SAS Institute Inc., Cary, NC, USA). Figures were
constructed using Microsoft Excel 2000 (Microsoft Cor-
poration, Redmond, WA, USA). An effect with a p value £0.05 was considered significant.
Experimental Design
To determine the relative effects of pulsed concentration
and duration on metal toxicity, 21-d toxicity tests with two
pair-treatments each (total of four exposure treatments)
were conducted with each metal (Table 1). For each pair-
treatment, when pulsed concentration was doubled
(increasing from 1X to 2X, e.g., 32 to 64 lg/L Cu), pulsed
duration was halved (decreasing from 2Y to 1Y, e.g., 48 to
24 h) and vice verse. Pulsed concentration and duration
were varied for each metal and were selected based on the
preliminary 96-h toxicity tests conducted for Cu, Zn, Se,
and As with neonate D. magna (results are not presented in
this paper). To determine the influence of the first pulse on
the second pulse, three 21-d toxicity tests with three levels
of pulsed concentrations were conducted for each metal
(Table 1). Pulsed durations in each test and within tests for
each metal were the same. However, pulsed durations
varied from metal to metal. Three or four various time
intervals (recovery time) between pulses were used for
each test. Pulsed duration varied for each metal. A refer-
ence test was conducted for each metal using single-pulse
to verify the mortality due to the first pulse in double-pulse
Table 1 Experimental characteristics
Single-pulse
Pair-treatment Cu Zn Se As
Concentration
(lg/L)aDuration
(h)
Concentration
(lg/L)aDuration
(h)
Concentration
(lg/L)aDuration
(h)
Concentration
(lg/L)aDuration
(h)
1 32 48 250 24 800 16 3000 18
64 24 500 12 1600 8 6000 9
2 32 96 250 48 800 20 3000 24
64 48 500 24 1600 10 6000 12
Double-pulse
Compound Concentration
(lg/L)aDuration
(h)
Recovery
(h)
Cu 32, 48, 64 12 24, 48, 96, 192
Zn 250, 375, 500 24 24, 96, 168
Se 800, 1200, 1600 8 72, 144, 288
As 4000, 5000, 6000 6 24, 96, 168
a Nominal concentrations, measured dissolved concentrations were <10% different from the nominal concentrations
Metal Pulsed Exposures in Daphnia magna 581
123
experiments. Pulsed concentration and duration used in the
reference tests were the same as they were in the double-
pulse tests. Growth and reproduction were not measured
for the reference tests. For both single- and double-pulse
tests, one handling control treatment was used for each
single treatment. All handling control treatments in each
test were grouped to make a control treatment for the test.
The handling control was MHW and organisms were
transferred into fresh MHW whenever the organisms in
exposure treatments were handled. Four replications were
used for each single treatment.
Water Chemistry
Water hardness, pH, alkalinity, and dissolved oxygen were
characterized at the start, the end of exposures, and weekly
during each test according to standard methods (Lewis
et al. 1994). Hardness and alkalinity were determined by
titration with 0.01 M EDTA and 0.02 N H2SO4, respec-
tively. The pH and concentration of dissolved oxygen,
hardness, and alkalinity in all test waters were 7.91 ± 0.08,
8.12 ± 0.008 mg/L, 98 ± 3 mg/L as CaCO3, and 61 ± 3 mg/L
as CaCO3, respectively. This hardness value is similar to
the hardness (100 mg/L CaCO3) that U.S. EPA used to
develop water quality criteria for metals (U.S. EPA 2004).
Total and dissolved Cu, Zn, Se, and As were characterized
at the start and the end of each exposure for each test.
Water samples were placed in 15-mL graduated poly-
proplene tubes and acidified with concentrated HNO3 for
analyses of total and dissolved Cu, Zn, Se, and As analyses.
Dissolved Cu, Zn, Se, and As samples were filtered through
0.45-lm Gelman Nylon Mesh� before acidification. The
pH was measured with a Thermo Orion (Orion Research
Inc. Beverly, MA, USA) model 525. Dissolved oxygen was
measured with an YSI, model 85 (YSI Incorporated,
Yellow Springs, OH, USA). Total and dissolved Cu and Zn
were analyzed with a Perkin-Elmer atomic absorp-
tion spectrometer (model 800, Perkin-Elmer Instruments,
Norwalk, CT, USA). Total and dissolved Se and As were
analyzed with an inductively coupled plasma (ICP) spec-
troscopy (model Elan 9000, Perkin Elmer Instruments,
Ontario, Canada). Total and dissolved concentrations of
each compound were not significantly different. Measured
dissolved metal concentrations were used for effect anal-
yses.
Results
Mortality
Daily mortality of D. magna for single- and double-pulse
exposures is shown in Figures 1 and 2, respectively. In
general, mortality of D. magna was higher in exposed
treatments with higher pulsed concentrations and durations.
Mortality of control organisms (£15%) was less than the
mortality of exposed organisms except at a pulsed con-
centration of 3000 lg/L As and pulsed durations of 18 h
and 24 h (Fig 1). Results of comparisons of 21-d mortality
for single-pulse exposures are shown in Table 2. For Cu
and As exposures, mortality was significantly higher for
treatments with 2X pulsed concentration and 1Y pulsed
duration than for treatments with 1X pulsed concentration
and 2Y pulsed duration. In contrast, for Zn exposures,
mortality was significantly lower for treatments with 2X
pulsed concentration and 1Y pulsed duration than for
µg/L Cu:h
0
20
40
60
80
100
0 5 10 15 20 25
32:48
64:24
32:96
64:48
Control
µg/L Zn:h
0
20
40
60
80
100
0 5 10 15 20 25
250:24
500:12
250:48
500:24
Control
Mor
talit
y (%
)µg/L Se:h
0
20
40
60
80
100
0 5 10 15 20 25
800:16
1600:8
800:20
1600:10
Control
µg/L As:h
0
20
40
60
80
100
0 5 10 15 20 25
3000:18
6000:9
3000:24
6000:12
Control
Time (d)
Fig. 1 Toxic effects of pulsed
duration and concentrations for
single-pulse exposures. (Data
are average daily mortality,
n = 4)
582 T. C. Hoang et al.
123
treatments with 1X pulsed concentration and 2Y pulsed
duration. For Se exposures, mortality was not significantly
different among the treatments.
Results of the reference tests are presented in Figure 3.
Mortality on day 3 and 21 was similar, except for Se
exposures. However, there was a comparable difference
between mortality on day 3 of the reference tests and
double-pulse experiments. Therefore, mortality due to the
first pulse used for comparing the toxic effects of the two
pulses was the mortality on day 3 of the double-pulse
experiments. With Cu, Zn, and As exposures, at recovery
time less than 96 h, mortality due to the first pulse appeared
to be higher than mortality due to the second pulse (Ta-
ble 3). When recovery time was greater than 96 h, mor-
tality due to the second pulse was similar or less than
mortality due to the first pulse. For Se exposures, mortality
due to the second pulse was statistically lower than mor-
tality due to the first pulse, except at a pulsed concentration
of 800 lg/L and a recovery time of 72 h (Table 3).
Growth and Reproduction
Results of 21-d growth and reproduction of D. magna for
single-pulse exposures are shown in Figure 4. The dry
weight of control and exposed D. magna ranged from
0.317 ± 0.219 to 0.915 ± 0.071 mg/living adult and was
not significantly different among the control and exposed
treatments, except at pulsed concentrations of 64 lg/L Cu
and 500 lg/L Zn with pulsed durations of 24 h. The dry
weight of exposed D. magna was statistically significant
higher than the dry weight of D. magna in their pair-
treatments (32 lg/L Cu and 250 lg/L Zn with 48 h) and
32 µg/L Cu 48 µg/L Cu 64 µg/L Cu
0
20
40
60
80
100
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25
Control 24h-rec
48h-rec 96h-rec
192h-rec
0
20
40
60
80
100Control 24h-rec
48h-rec 96h-rec
192h-rec
Control 24h-rec
48h-rec 96h-rec
192h-rec
250 µg/L Zn 375 µg/L Zn 500 µg/L Zn
0
20
40
60
80
100Control 24h-rec
96h-rec 168h-rec
0
20
40
60
80
100
Control 24h-rec
96h-rec 168h-rec
Control 24h-rec
96h-rec 168h-rec
800 µg/L Se 1200 µg/L Se 1600 µg/L Se
Mor
talit
y(%
)
0
20
40
60
80
100 Control 72h-rec
144h-rec 288h-rec
0
20
40
60
80
100
Control 72h-rec
144h-rec 288h-rec
Control 72h-rec
144h-rec 288h-rec
4000 µg/L As 5000 µg/L As 6000 µg/L As
0
20
40
60
80
100Control 24h-rec
96h-rec 168h-rec
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Control 24h-rec
96h-rec 168h-rec
Control 24h-rec
96h-rec 168h-rec
Time (d)
Fig. 2 Toxic effects for double-pulse exposures. (Data are average daily mortality, n = 4)
Metal Pulsed Exposures in Daphnia magna 583
123
controls (Fig. 4A). The reproduction of both control and
exposed D. magna ranged from 48 ± 16 to 247 ± 71
(Fig. 4B).
There was no statistically significant difference between
the reproductions of control and Zn and Se exposed
D. magna. However, the reproduction of D. magna at
pulsed concentrations of 64 lg/L Cu with a pulsed duration
of 24 h and 6000 lg/L As with a pulsed duration of 9 h was
statistically significant higher than the reproduction of
D. magna in their pair-treatments (32 lg/L Cu with 48 h
and 3000 lg/L As with 18 h) and controls. At a pulsed
concentration of 3000 lg/L As and a pulsed duration of 24
h, the reproduction of exposure D. magna was statistically
significant lower than the reproduction of their control
D. magna. For treatments with high concentrations and/or
long durations, all organisms were dead, resulting in no
growth and reproduction results.
With double-pulse exposures, the dry weight of Cu
exposure D. magna appeared to be higher than the dry
weight of control D. magna at recovery time ‡48 h
(Fig. 5). For Zn and Se exposures, the growth of treat-
ment and control D. magna were similar, except at
375 lg/L Zn with recovery of 96 h, 500 lg/L Zn with
recovery of 168 h, and 800 lg/L Se with recovery of 72 h
(Fig. 5). There was no significant difference between the
growth of As exposure and control D. magna (Fig. 5).
There was no effect of double-pulse metal exposures on
reproduction of D. magna, except at 375 lg/L Zn with
recovery time of 168 h and 1200 lg/L Se, the reproduc-
tion was significantly lower in exposure treatment than in
control treatment (Fig. 6).
Discussion
Latent Mortality and Relative Effects of Duration and
Concentration for Single-Pulse
With single-pulse exposures, mortality occurred during
exposures for pulses with high concentration and long
duration or several days after removal of Zn, and As pulses
with low concentration and short duration (Fig. 1), indi-
cating latent effects for these metal exposures. The delay in
mortality may be due to slow depuration after the expo-
sures end. This latent effect appeared to be longer for Se
exposures; mortality was not observed during exposures
but occurred up to two weeks after the exposures ended.
These results suggest that Se depuration may be slower
than Cu, Zn, and As depurations. However, Schultz et al.
(1980) found that D. magna eliminated Se rapidly;
approximately 60% of the uptake Se was lost within 12 h.
In addition, Guan and Wang (2004) found that the clear-
ance rate for dietary Se uptake by D. magna was faster than
that for dietary Zn uptake. These results do not support the
above suggestion. According to McConnell and Roth
(1962) and McConnell (1963), a fraction of selenium rep-
resents the nonprotein-bound metabolic pool in mamma-
lian cells that can incorporate into proteins during protein
synthesis in the microsomal compartment of cells. This
mechanism may occur in D. magna and result in long de-
layed effect of Se. This latent effect was also mentioned by
Diamond et al. (2006) for D. magna exposed to Zn but not
for Cu and ammonia. Hurd et al. (1996) and Van der
Hoeven et al. (1997) also found latent effects in D. pulex
Table 2 Relative effects of
pulsed concentration and
duration on mortality of
Daphnia magna (single-pulse
exposures)
a For comparing pair-treatment
mortality (1X concentration and
2Y duration versus 2X
concentration and 1Y duration)
Compound Pair-
treatment
Concentration
(lg/L)Duration
(h)
aMortality
(%)
aSignificant
(p < 0.05)
Cu 1 32 48 15 Yes
64 24 85
2 32 96 20 Yes
64 48 100
Zn 1 250 24 40 Yes
500 12 25
2 250 48 100 Yes
500 24 60
Se 1 800 16 60 No
1600 8 55
2 800 20 95 No
1600 10 100
As 1 3000 18 5 Yes
6000 9 40
2 3000 24 10 Yes
6000 12 100
584 T. C. Hoang et al.
123
Table 3 Influence of the first pulse on the second pulse
Compound Concentration
(lg/L)Recovery time
(h)
a24 a48 a96 a192
bM1bM2
bM1bM2
bM1bM2
bM1bM2
Cu 32 0 < 70 10 ~ 20 0 ~ 5 0 ~ 10
Cu 48 5 < 20 5 < 20 20 = 20 5 ~ 15
Cu 64 5 < 60 15 < 35 15 ~ 20 20 ~ 15
a24 a96 a168
bM1bM2
bM1bM2
bM1bM2
Zn 250 35 ~ 20 40 > 10 10 = 10
Zn 375 40 ~ 50 55 > 10 45 > 0
Zn 500 10 < 75 40 ~ 20 30 > 15
a72 a144 a288
Se 800 45 ~ 30 55 > 15 45 > 5
Se 1200 85 > 0 65 > 20 63 > 4
Se 1600 90 > 5 55 > 10 55 > 0
a24 a96 a168
As 4000 5 ~ 15 20 ~ 25 35 > 5
As 5000 10 ~ 20 25 ~ 10 5 < 35
As 6000 5 < 45 20 ~ 5 25 ~ 15
a Greater symbols (>) and smaller symbols (<) indicate statistically significant difference (p < 0.05)
Tail (~) and equal symbols (=) indicate not statistically significant difference or equal, respectivelyb M1 and M2 are mortality due to the first and the second pulses, respectively
12-h single-pulse 24-h single-pulse
0
20
40
60
80
100
0 5 10 15 20 25
Control 32µg/L Cu
48µg/L Cu 64µg/L Cu
0
20
40
60
80
100
0 5 10 15 20 25
Control 250µg/L Zn
375µg/L Zn 500µg/L Zn
8-h single-pulse 6-h single-pulse
Mor
talit
y(%
)
0
20
40
60
80
100
0 5 10 15 20 25
Control 800µg/L Se
1200µg/L Se 1600µg/L Se
0
20
40
60
80
100
0 5 10 15 20 25
Control 4000µg/L As
5000µg/L As 6000µg/L As
Time (d)
Fig. 3 Reference effects for double-
pulse exposures. (Data are average
daily mortality, n = 4)
Metal Pulsed Exposures in Daphnia magna 585
123
and macroinvertebrates exposed to diflubenzuron, respec-
tively. A study by Brent and Herricks (1998) found that
that immobility of Ceriodaphnia dubia continued up to 172
h after a 4 h exposures to Cd and Zn. However, latent
mortality was not observed neither by Naddy et al. (2000)
for chlorpyrifos nor by Milne et al. (2000) for ammonia in
fish. The delay in mortality can also be seen in Figure 2 for
double-pulse exposures and reference tests, except for Cu
(Fig. 3). The latent effects were attributed to different
modes of action for different toxicants in different organ-
isms (Reinert et al. 2002).
The relative toxicity effects of pulsed duration and
concentration to D. magna appeared to be similar for Cu
and As. With the same AUC (e.g., AUC = 32 lg/L Cu ·48h or 64 lg/L Cu · 24h), pulses with high concentra-
tions and short durations caused more mortality than
A
32/4
8
64/2
4
32/9
6
64/4
8
**
250/
24
500/
12
250/
48
500/
24
**
800/
16
1600
/8
800/
20
1600
/10Control
3000
/18
6000
/9
3000
/24
6000
/12
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40Cu Zn Se As
Gro
wth
(mg/
livin
g ad
ult,
dw)
64/4
8
32/9
6
64/2
4
32/4
8
B
500/
24
250/
48
500/
12
250/
24
**
1600
/10
800/
20
1600
/8
800/
16
1600
/10
6000
/12
3000
/24
6000
/9
3000
/18Control
0
50
100
150
200
250
300
350
Cu Zn Se As
*
**
Rep
rodu
ctio
n(y
oung
/livi
ng a
dult)
Pulsed concentration/duration ((µg/L)/h)
Fig. 4 Effects on growth and reproduction of
pulsed duration and concentrations. A: effects on
growth, B: effects on reproduction, **: significant
from control and pair-treatment
*
* ***
0.0
0.2
0.4
0.6
0.8
1.0
Control 24 48 96 192
32 µg/L Cu 48 µg/L Cu 64 µg/L Cu
*
*
0.0
0.2
0.4
0.6
0.8
1.0
Control 24 96 168
250 µg/L Zn 375 µg/L Zn 500 µg/L Zn
*
0.0
0.2
0.4
0.6
0.8
1.0
Control 72 144 288
800 µg/L Se 1200 µg/L Se 1600 µg/L Se
0.0
0.2
0.4
0.6
0.8
1.0
Control 24 96 168
4000 µg/L As 5000 µg/L As 6000 µg/L As
Gro
wth
(mg/
livin
g ad
ult,
dw)
Recovery time (h)
Cu: 12-h double-pulse Zn: 24-h double-pulse
Se: 8-h double-pulse As: 6-h double-pulse
Fig. 5 Effects on growth of
double-pulse exposures.
*: significant from control
586 T. C. Hoang et al.
123
pulses with low concentrations and long durations
(Table 2). This suggests that pulsed concentrations had
stronger toxic effects than does pulsed duration for Cu
and As. In contrast, the toxic effect of pulsed duration
was stronger than that of pulsed concentrations for Zn;
higher mortality was found for pulses with long durations
and low concentrations than pulses with short durations
and high concentrations (Table 2). These results suggest
that Zn uptake rates would be slower than Cu and As
uptake rates. This suggestion is supported by the results
found by Diamond et al. (2006). In contrast to results
from Cu, Zn, and As pulses, Se pulses with similar AUC
resulted in similar mortality, indicating that, the toxic
effects of pulsed concentration and duration were equal
for Se. The different effects of pulsed duration and con-
centration of different toxicants would be due to different
modes of action. The mode of action that allows toxicants
to reach target sites faster would result in higher toxic
effects of concentration compared to duration.
Influence of Recovery on Mortality of the Second Pulse
The recovery of organisms from environmental stress to
normal physiological conditions can occur through internal
biological processes such as depuration and detoxification
(Zhao and Newman 2006). Results found in this study
(Table 3) indicated that, there was a time interval between
pulses that allowed organisms to recover after removal of
the first metal pulsed exposure and resulted in independent
toxic effects of the second pulse on the first pulse. For
example, with Cu exposures, mortality due to the first pulse
was £20% (measured on day 3 of the experiment and was
similar to mortality in the reference test) while mortality
due to the second pulse was ‡20 at recovery time £48 h.
When recovery time was ‡96 h, mortality due to the two
pulses was similar, suggesting that, 96 h between Cu
exposures was long enough for the organisms to recover
back to their background conditions (background recovery
time). Hence, with a recovery time of 96 h between Cu
exposures, the effect of the second pulse was independent
from the effect of the first pulse. However, the background
recovery time is dependent on pulsed duration and con-
centration, and then the uptake during exposed phase. The
higher the pulsed concentration and the longer pulsed
duration, the longer depuration time organisms need before
the next pulse (Hoang et al. 2006). In addition, depuration
also varies from species to species and is dependent on
toxicants. For example, the biological half-life of Cu, Zn,
and Se in Crassostrea gigas were 32.9, 36.7, 102.3 d,
respectively, while it was 156.2, 183.9, and 139.6 d in
Crassostrea virginica, respectively (Okazaki and Panietz
1981). The background recovery time found in this study
for Zn and As exposures was shorter than that for Cu
exposures (‡24 h). Results found in this study were similar
with the results found by Zhao and Newman (2006) for
Hyalella azteca exposed to Cu and phenol and Diamond
et al. (2006) for D. magna and Pimephales promelas ex-
posed to Cu, Zn, and ammonia. Naddy and Klaine. (2001)
and Hoang et al. (2007) also found that total mortality at
the end of the experiments decreased with increasing
recovery time. This is in agreement with the results found
in this study in substances. For Se exposures, when sepa-
rating the two pulses by interval time of ‡72 h, the toxic
effects of the second pulse appeared to be less than that of
the first pulse, suggesting that, the organisms could develop
their tolerance to Se (Table 3) because D. magna can
acclimate to Cu and Zn (Bossuyt and Janssen 2003, 2004,
Muyssen and Janssen 2001, 2002).
Cu: 12-h double-pulse Zn: 24-h double-pulse
0
40
80
120
160
200
240
Control 24 48 96 192
32 µg/L Cu 48 µg/L Cu 64 µg/L Cu
*
0
40
80
120
160
200
240
Control 24 96 168
250 µg/L Zn 375 µg/L Zn 500 µg/L Zn
Se: 8-h double-pulse As: 6-h double-pulse
*
*
*
0
40
80
120
160
200
240
Control 72 144 288
800 µg/L Se 1200 µg/L Se 1600 µg/L Se
0
40
80
120
160
200
240
Control 24 96 168
4000 µg/L As 5000 µg/L As 6000 µg/L AsR
epro
duct
ion
(you
ng/li
ving
adu
lt)
Recovery time (h)
Fig. 6 Effects on reproduction
of double-pulse exposures.
*: significant from control
Metal Pulsed Exposures in Daphnia magna 587
123
Effects on Growth and Reproduction and Role of
Recovery
In general, surviving organisms from metal exposures grew
and reproduced normally as did control organisms over 21
days (Fig. 4), indicating recovery of the organisms after the
exposures ended. This is in agreement with the results found
by Naddy et al. (2000) forD. magna exposed to chropyrifos,
Diamond et al. (2006) for D. magna exposed to Cu and Zn,
and Reynaldi and Liess (2005) for D. magna exposed to
fenvalerate. In several exposure treatments, the surviving
organisms grew better and reproduced more neonate than
did control organisms. This may be due to effect of food
availability per organism because feeding was consistent
across treatments and was not reduced when partial mor-
tality occurred. The effects of feeding rate on the growth and
reproduction of D. magna reported by Gorbi et al. (2002)
and Zhang et al. (2000) support the results found in this
study. With Cu exposures, in contrast with the effects on
survival, the effects of pulsed concentration on the growth
and reproduction of D. magna were less than the effects of
pulsed duration; the surviving organisms from pulses with
shorter duration (64/24) grew better and reproduced more
neonates than did the organisms from pulse with longer
duration (32/48) (Fig. 4). However, for Zn and Se expo-
sures, pulsed concentration and duration had similar effects
on survival, growth, and reproduction of D. magna.
With double-pulse exposures, in contrast with the effects
on survival, interval time between pulses had no role on
growth and reproduction of surviving organisms; increase
interval time did not improve the growth and reproduction of
organisms (Fig. 5, 6). As long as surviving from exposures,
organisms grew and reproduced normally as did control
organisms. These results again, indicate recovery of organ-
isms after the exposures end, and are similar to the results
found by Naddy et al. (2000) and Diamond et al. (2006).
Reality of Pulsed and Continuous Exposures in Risk
Assessment and Selection Endpoint
Continuous exposure has been used for risk characteriza-
tion, such as EC50 and/or LC50. With toxicants that have
latent effects like metals in this study, endpoint (e.g.,
mortality) in continuous exposures do not totally measure
the effects of exposures. Using effects from continuous
exposures, risk may be underestimated. For example, using
continuous exposures, toxicity was not found on the first
day of Se exposure; however; using pulsed exposures,
more than 40% mortality occurred on day 2 and later
(Fig. 1). A similar result was found by Reynaldi and Liess
(2005). In addition, due to the different effects of duration
and concentration for Cu, Zn, and As found in this study,
the integration method, such as AUC may not accurately
characterize risk for these metals. Regarding multiple
pulsed exposures, using AUC method, risk of each indi-
vidual pulse is added for risk of multiple pulses due to
summing exposure area of individual pulse, while interval
time between pulses would reduce the effect of later pulses.
Therefore, risk characterization should take recovery be-
tween exposures into account. Due to recovery of organ-
isms, sublethal effects, such as growth and reproduction in
this study were not significant although lethal effect was.
Therefore, for chronic pulsed exposures, mortality may be
better for risk characterization.
Acknowledgments This study was supported by the Electric Power
Research Institute and the Water Environment Research Foundation.
References
Allin CJ, Wilson RW (2000) Effects of pre-acclimation to aluminum
on the physiology and swimming behavior of juvenile rainbow
trout (Oncorhynchus mykiss) during pulsed exposure. Aqua
Toxicol 51:213–224
Bossuyt BTA, Janssen CL (2003) Acclimation of Daphnia magna to
environmentally realistic copper concentrations. Comp Biochem
Physiol C 136:253–264
Bossuyt BTA, Janssen CR (2004) Influence of multigeneration
acclimation to copper on tolerance, energy reserves, and
homeostasis of Daphnia magna straus. Environ Toxicol Chem
23:2029–2037
Brent RN, Herricks EE (1998) Postexposure effects of brief cadmium,
zinc, and phenol exposures on freshwater organisms. Environ
Toxicol Chem 17:2091–2099
Diamond J, Butcher J, Klaine SJ, Bearr J, Bowersox M, Latimer H,
Hoang TC, Zahner H. (2005) Validation of understanding
assumption for integrating frequency, magnitude, and duration
in permit conditions. Water Environmental Research Foundation
Report. Tetra Tech, Inc. MD, USA
Diamond JM, Klaine SJ, Butcher JB (2006) Implications of pulsed
chemical Exposures for Aquatic Life Criteria and Wastewater
Permit Limits. Environ Sci Technol 40:5132–5138
Gorbi GG, Corradi MG, Invidia M, Rivara L, Bassi M (2002) Is
Cr(VI) toxicity to Daphnia magna modified by food availability
or algal exudates? The hypothesis of a specific chromium/algae/
exudates interaction. Water Res 36:1917–1926
Guan R, Wang WX (2004) Cd and Zn uptake kinetics in Daphniamagna in relation to Cd exposure history. Envion Sci Technol
38:6051–6058
Hoang TC, Tomasso JR, Klaine SJ (2007) An integrated model
describing the toxic responses of Daphnia magna to pulsed
exposures of three metals. Environ Toxicol Chem 26:132–138
Hurd MK, Perry SA, Perry WB (1996) Nontarget effects of a test
application of diblubenzuron to forest canopy on stream
macroinvertebrates. Environ Toxicol Chem 15:1344–1351
Lewis PA, Klemm DJ, Lazorchak JM, Norberg-King TJ, Peltiet WH,
Heber MA (1994) Short-term method for estimating the chronic
toxicity of effluents and receiving waters to freshwater and
marine organisms. EPA 600-/4-91/002. U.S. Environmental
Protection Agency, Cincinnati, OH, USA
McConnell KP, Roth DM (1962) Se in rat intracellular liver fractions.
Biochim Biophys Acta 62:503
McConnell KP (1963) Metalobism of selenium in the mammalian
organism. J. Agic Food Chem 11:385
588 T. C. Hoang et al.
123
Milne I, Seager J, Mallett M, Sims I (2000) Effects of short-term
pulsed ammonia exposure on fish. Environ Toxicol Chem
19:2929–2936
Morton MG, Dickson KL, Waller WT, Acevedo MF (2000)
Methodology for the evaluation of cumulative episodic exposure
to chemical stressor in aquatic risk assessment. Environ Toxicol
Chem 19:1213–1221
Muyssen BTA, Janssen CR (2001) Multigeneration zinc acclimation
and tolerance in Daphnia magna: Implication for water-quality
guidelines and ecological risk assessment. Environ Toxicol
Chem 20:2053–2060
Muyssen BTA, Jenssen CR (2002) Accumulation and regulation of
zinc in Daphnia magna: Links with homeostasis and toxicity.
Arch Environ Contam Toxicol 43:492–496
Naddy RB, Johnson KA, Klaine SJ (2000) Responses of Daphniamagna to pulsed exposures of chlorpyrifos. Environ Toxicol
Chem 19:423–431
Naddy RB, Klaine SJ (2001) Effect of pulse frequency and interval on
the toxicity of chlorpyrifos to Daphnia magna. Chemosphere
45:497–506
Okazaki RK, Panietz MH (1981) Retention time of twelve trace
metals in tissues of the oysters Crassostrea angulata and
C. virginica. Mar Biol 63:113–120
Reinert KH, Giddings JM, Judd L (2002) Effects of analysis of
time-varying or repeated exposures in aquatic ecological risk
assessment of agrochemicals. Environ Toxicol Chem 21:1977–
1992
Reynaldi SR, Liess M. Influence of duration of exposure to pyrethroid
fenvalerate on sublethal responses and recovery of Daphniamagna straus. Environ Toxicol Chem 24:1160–1164
Schultz TW, Freeman SR, Dumont JN (1980) Uptake, depuration, and
distribution of selenium in Daphnia and its effects on survival
and ultrastrucure. Arch Environ Contam Toxicol 9:23–40
Schulz R, Liess M (2000) Toxicity of fenvalerate to caddisfly larvae:
chronic effects of 1- vs 10-h pulse-exposure with constant doses.
Chemosphere 41:1511–1517
US Environmental Protection Agency. (1998) Guidelines for ecolog-
ical risk assessment. Fed Reg 63(93):26846–26924
US Environmental Protection Agency. (2004) National Recom-
mended Water Quality Criteria. Office of Water, Office of
Science and Technology. Washington DC
Van der Hoeven N, Gerritsen AAM (1997) Effects of chlorpyrifos on
individuals and populations of Daphnia pulex in the laboratory
and field. Environ Toxicol Chem 16:2438–2447
Widianarko B, Kuntoro FXS, Van Gestel CAM, Van Straalen NM
(2001) Toxicokinetics and toxicity of zinc under time-varying
exposure in the guppy (Poecilia reticulata). Environ Toxicol
Chem 20:763–768
Zhang L, Baer KN (2000) The influence of feeding, photoperiod and
selected solvents on the reproductive strategies of water flea,
Daphnia magna. Environ Pollut 110:425–430
Zhao Y, Newman MC (2006) Effects of exposure duration and
recovery time during pulsed exposures. Environ Toxicol Chem
25:1298–1304
Metal Pulsed Exposures in Daphnia magna 589
123
Top Related