Toxicity of Two Pulsed Metal Exposures to Daphnia magna : Relative Effects of Pulsed...

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)


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)


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


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)


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


*

*

0.0

0.2

0.4

0.6

0.8

1.0

Control 24 96 168


*

0.0

0.2

0.4

0.6

0.8

1.0

Control 72 144 288


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


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


*

0

40

80

120

160

200

240

Control 24 96 168


Se: 8-h double-pulse As: 6-h double-pulse

*

*

*

0

40

80

120

160

200

240

Control 72 144 288


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


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.

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