Environ Sci Pollut Res (2014) 21:234–243

DOI 10.1007/s11356-013-1692-z

ENVIRONMENTAL QUALITY BENCHMARKS FOR PROTECTING AQUATIC ECOSYSTEMS

Effects of multigenerational exposures of D. magna

to environmentally relevant concentrations
of pentachlorophenol
Yi Chen & Jin Huang & Liqun Xing & Hongling Liu &
John P. Giesy & Hongxia Yu & Xiaowei Zhang

Received: 16 December 2012 / Accepted: 28 March 2013 / Published online: 2 May 2013
# Springer-Verlag Berlin Heidelberg 2013

Abstract The re-emergence of schistosomiasis has given rise
to ubiquitous concentrations of the primary control agent
pentachlorophenol (PCP) in the environment, especially in
the surface waters of China. In this study, the effects of
environmentally relevant concentrations of PCP, namely,
0.0002, 0.002, 0.02, 0.2, and 2 μmol/L on survival, age at
first reproduction, fecundity, length of mothers, and number of
molts of Daphnia magna were studied over three generations.
The survival of D. magna exposed to 2 μmol/L was signifi-
cantly affected in the three generations. Toxic effects were
enhanced in later generations. Age at first reproduction of F1
and F2 D. magna was significantly slower than that of the
controls. The total number of offspring per female exposed to
concentrations of 0.002 μmol/L or greater was less (23.5 to
67.6, 9.4 to 73.7, and 3.6 to 83.7 %) than that of the controls in
Responsible editor: Henner Hollert
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-013-1692-z) contains supplementary material,
which is available to authorized users.
Y. Chen : J. Huang : L. Xing : H. Liu (*) : J. P. Giesy :
H. Yu (*) : X. Zhang
State Key Laboratory of Pollution Control and Resource Reuse,
School of the Environment, Nanjing University, Nanjing 210023,
China
e-mail: hlliu@nju.edu.cn
e-mail: Yuhx@nju.edu.cn
J. P. Giesy
Department of Veterinary Biomedical Sciences and Toxicology
Centre, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada
J. P. Giesy
Department of Biology and Chemistry and State Key Laboratory
for Marine Pollution, City University of Hong Kong, Kowloon,
Hong Kong
the F0, F1, and F2 generations, respectively. The body length
of mothers significantly decreased (4.7 to 6.8, 9.6 to 15.1, and
13.3 to 23.2 %) after exposure to 0.002 μmol/L or greater than
those of unexposed individuals in the F0, F1, and F2 genera-
tions, respectively. Dose–response relationships between con-
centrations of PCP and length and number of molts of D.
magna were observed in the F0 to F2 generations. PCP con-
centrations on the surface waters of China caused adverse
effects to D. magna, which increased over successive gener-
ations. Significant effects were observed in the third genera-
tion. The multigenerational studies were more sensitive than
the single-generation experiments. Thus, multigenerational
exposure may be more predictive of chronic exposure under
field conditions.
Keywords PCP . Water quality criteria . D. magna .
Multigenerational
Abbreviations
CPs Chlorophenols
PCP Pentachlorophenol
DMSO Dimethyl sulfoxide
LOAEL Lowest observed adverse effect level
WQC Water quality criteria
Introduction
Chlorophenols (CPs) function as intermediates in the chem-
ical syntheses of dyestuffs, as pesticides or as biocides; CPs
are widely used and discharged into aquatic ecosystems
during manufacture and use, through degradation of other
chemicals such as phenoxyalkanoic acids or during chlori-
nation of municipal drinking water (Czaplicka 2004; Davì
Environ Sci Pollut Res (2014) 21:234–243
and Gnudi 1999; Gao et al. 2008; Olaniran and Igbinosa
2011). Pentachlorophenol (PCP) has been widely used for
killing snails in China where schistosomiasis is epidemic
(Zheng et al. 2012) and resulted in persistent environmental
contamination. PCP is a priority pollutant in the USA and
China (USEPA 1991; Xia and Zhang 1990). Surveys on
PCP concentrations have been conducted (Gao et al. 2008;
Heemken et al. 2001; Olaniran and Igbinosa 2011;
Verschueren 1996) in many regions of the world because
of their potential adverse effects (Ruder and Yiin 2011;
Wang et al. 2001) on aquatic organisms. PCP concentrations
in surface water of China were determined at more than 600
locations, including seven major watersheds and three drain-
age areas. PCP was ubiquitous among the CPs detected in
85.4 % of the samples, with a median concentration of
50.0 ng/L (0.0002 μmol/L) and a range of <1.1 to
103.70 ng/L (0.389 μmol/L) (Gao et al. 2008; Zheng et al.
2012). The re-emergence of schistosomiasis has caused the
warranted production and consumption of PCP to inevitably
result in persistent environmental contamination. The
greatest concentrations of PCP were observed in the water
of the eight sites in Dongting Lake, which is located in south
China. The maximum concentration found in Dongting
Lake was 103.70 μg/L (0.389 μmol/L). The greater PCP
concentrations in Dongting Lake were similar to the PCP
concentrations in the surface waters of Northern Europe and
the USA from 1970 to 1984 (Zheng et al. 2012).
China is embarking on the development of its own na-
tional water quality criteria (WQC) system (Wu et al. 2010;
Yan et al. 2012). WQC for PCP were recently derived in
China for the protection of aquatic life based on resident
aquatic biota (Jin et al. 2012; Xing et al. 2012). However,
these WQCs did not consider chronic exposures or effects
on multiple generations. The toxic effects of PCP to Daph-
nia magna have been studied previously. Exposure to
0.56 mg/L significantly affected the growth of D. magna
during a 21-day exposure (Van Leeuwen et al. 1987). There-
fore, studies that consider the implications of chronic expo-
sure to aquatic organisms in environmentally realistic
concentrations are required (Dietrich et al. 2010). Moreover,
published information on the effects of PCP during
multigenerational exposures is limited. In this study, the
effects of environmentally relevant concentrations of PCP
on the multigeneration survival, reproduction, and growth of
D. magna were studied.
Materials and methods
Test solutions
PCP (CAS No. 87-86-5, 98 % purity) was purchased from
Sigma-Aldrich (St. Louis, MO). A stock solution of PCP
235
was prepared in dimethyl sulfoxide (DMSO; HPLC grade)
and kept in the dark at −20 °C. Stock solutions were warmed
to room temperature before each bioassay and were used to
prepare the final test concentrations, which were sterilized
by filtration through a 0.22-μm filter (Millipore, Beijing).
The concentration of DMSO in the medium including all
treatments and the solvent control was 0.1 %. One control
group and one solvent control group were designated for
each exposure test. The test organisms were almost similar.
Nominal concentrations for the multigenerational study
were 0.0002, 0.002,1 0.02, 0.2,2 and 2 μmol/L. These treat-
ments represented the concentration range detected in the
surface waters in China (Gao et al. 2008; Zheng et al. 2000).
Tests were conducted on a semi-static basis to maintain a
constant concentration. Test solutions were renewed daily.
Test organisms
The water flea D. magna is a common crustacean inverte-
brate in freshwater systems. Zooplankton of this type grazes
on algae and forms the base of the secondary producer food
chain. These organisms provide food for economically im-
portant fish. A single clone of D. magna that has been
cultured in the laboratory for several years was the source
of the organisms used. The clones were fed daily with a
suspension of the green alga Scenedesmus obliquus and kept
at 24±0.5 °C with a light/dark cycle of 16 h:8 h photoperi-
od. All experiments were performed under the same tem-
perature and light conditions. Tap water aerated for more
than three days was used as the medium for all tests and
controls.
Test solution
The dilution water which was tap water aerated for more
than three days had a pH of 8.12±0.11, dissolved oxygen
concentration of 6.07±0.24 mg/L, conductivity of 319±
9.1 μs/cm, alkalinity of 95.48±4.64 mg/L as CaCO3, and
hardness of 125.5±4.95 mg/L as CaCO3.
Multigeneration experiment
Tests were conducted in accordance to the methods de-
scribed by Brennan et al. (2006) and Dietrich et al. (2010)
(Fig. 1). Ten newly hatched neonates (F0) (<24 h old) of the
third brood from the age-synchronized mothers were ran-
domly selected for each treatment. And each D. magna was
transferred into 25 mL glass plates (Nanjing Jukang Medical
1
PCP median environmental concentration was 50.0 ng/L
(0.0002 μmol/L) (Gao et al. 2008).
2
The magnitude of 0.2 μmol/L equals that of PCP max environmental
concentration which was 103.70 ng/L (0.389 μmol/L) reported in
Dongting Lake (Zheng et al. 2012).
236 Environ Sci Pollut Res (2014) 21:234–243

Fig. 1 Experimental design of the multigeneration toxicity test

and Chemical) that contained 10 mL of test solution. D.

magna were observed twice per day at a specific time with
plastic disposable pipettes (the tips were cut off to accom-
modate their body size). Once F1 neonates appeared, F0
individuals were removed from the vessels, and newly
hatched neonates from each female D. magna were counted.
Subsequently, a single neonate per vessel on the 11th day of
F0 generation was randomly selected as the F1 generation to
continue the experiment. The experiment was terminated
after the neonates (F2) of generation F1 were 21 days old
(Fig. 1). D. magna were continuously exposed to PCP at the
same concentration for 21 days throughout the three gener-
ations, without any recovery period. D. magna were fed
daily with a suspension of S. obliquus at a specific concen-
tration of 1.6×105 cells/mL after transferred to a clean
chamber with the same PCP concentration.
Age at first brood, number of offsprings produced by
individual D. magna, and number of molts were recorded.
Body length of F 0, F 1, and F2 21-day-old generation
Fig. 3 Age at first reproduction (mean+SD) D. magna exposed to
mothers were measured under a multipurpose zoom micro- different concentrations of PCP during three generations. Asterisks
scope (Nikon AZ 100) from the apex of the helmet to the indicate statistically significant differences from the control (*p<
base of the tail spine (Massarin et al. 2011a, b) (Fig. 1). 0.05; **p<0.01; ***p<0.001). DMSO group represents solvent con-
trol group

Statistical analysis

All statistical analyses were performed using the GraphPad

Fig. 2 Survival of D. magna after exposure to 2 μmol/L of PCP
during three generations. All of D. magna in controls and other
concentrations were surviving
Prism (GraphPad Prism Development Core Team, http://
www.graphpad.com/scientific-software/prism/). The survi-
vorship and reproduction data were used to calculate the
intrinsic rate of population growth (r) according to the
following equation: Σ lx
mx
erx ¼ 1; 1 ¼ er ; T ¼ Σ x

lx
mx =R0 ; r ¼ lnR0 =T ; where lx is the proportion of
female individuals reaching age x, mx is the average number
of live offspring produced per female of age x during the
time interval x to x+1, l is finite rate of increase, R0 is net
reproduce rate and T is mean generation time (Leslie and
Environ Sci Pollut Res (2014) 21:234–243 237

Table 1 Comparison between generations

Dependent variable Source of variation p value summary

F0 vs. F1 F0 vs. F2 F1 vs. F2

Age at first reproduction Control ns ns ns

0.0002 μmol/L ns ns ns
0.002 μmol/L ns ns ns
0.02 μmol/L –* –*** ns
0.2 μmol/L ns –*** –***
Number of offspring per mother produced within 21 days Control ns ns ns
0.0002 μmol/L ns ns ns
0.002 μmol/L ns –* ns
0.02 μmol/L ns ns ns
0.2 μmol/L ns –*** ns
Body length of parent female at 21 days old Control ns ns ns
0.0002 μmol/L ns –*** ns
0.002 μmol/L –** –*** ns
0.02 μmol/L –*** –*** –***
0.2 μmol/L –*** –*** –***
Total number of molts within 21 days Control ns ns ns
0.0002 μmol/L ns ns ns
0.002 μmol/L ns ns ns
0.02 μmol/L ns ns ns
0.2 μmol/L –* –* ns
Intrinsic rate of population growth Control ns ns ns
0.0002 μmol/L ns ns ns
0.002 μmol/L ns ns ns
0.02 μmol/L ns ns ns
0.2 μmol/L ns –*** ns

*p<0.05; **p<0.01; ***p<0.001—statistically significant differences from the control treatment

Ranson 1940). Differences in age at first reproduction, body growth were determined using one-way ANOVA and two-
length, number of offspring and intrinsic rate of population way ANOVA, based on the assumption of normality of
Table 2 Two-way ANOVA
analysis of effects of concentra- Dependent variable Source of variation p value summary
tion of PCP, generation, and their
interaction on the age at first Age at first reproduction Interaction –***
production, number of offspring Concentration of PCP –***
per mother produced within
Generation –***
21 days, body length of parent
female at 21 days old, total Number of offspring per mother produced within 21 days Interaction –***
number of molts within 21 days, Concentration of PCP –***
and intrinsic rate of population Generation ns
growth of D. magna
Body length of parent female at 21 days old Interaction –***
Concentration of PCP –***
Generation –***
Total number of molts within 21 days Interaction ns
Concentration of PCP –***
Generation –**
*p<0.05; **p<0.01; ***p< Intrinsic rate of population growth Interaction –***
0.001—statistically significant Concentration of PCP –***
differences from the control Generation ns
treatment
238 Environ Sci Pollut Res (2014) 21:234–243

Table 3 Changes for age at first production, number of offspring per mother produced within 21 days, body length of parent female at 21 days old,
total number of molts within 21 days, and intrinsic rate of population growth of D. magna

Dependent variable Source of variation Changes compared with control

(μmol/L)
F0 F1 F2

Age at first reproduction 0.0002 2.3 % delay 1.1 % delay 1.1 % delay
0.002 4.5 % delay 11.4 % delay 8.0 % delay
0.02 5.7 % delay 17.0 % delay 25.0 % delay
0.2 6.8 % delay 15.9 % delay 30.7 % delay
Number of offspring per mother produced within 21 days 0.0002 7.3 % decrease 13.8 % decrease 2.7 % decrease
0.002 23.5 % decrease 9.4 % decrease 3.6 % decrease
0.02 54.8 % decrease 53.5 % decrease 61.4 % decrease
0.2 67.6 % decrease 73.7 % decrease 83.7 % decrease
Body length of parent female at 21 days old 0.0002 1.9 % decrease 4.1 % decrease 7.8 % decrease
0.002 4.7 % decrease 9.6 % decrease 13.3 % decrease
0.02 4.8 % decrease 11.6 % decrease 23.2 % decrease
0.2 6.8 % decrease 15.1 % decrease 21.9 % decrease
Total number of molts within 21 days 0.0002 3.7 % decrease 8.9 % decrease 6.4 % decrease
0.002 7.3 % decrease 12.5 % decrease 11.9 % decrease
0.02 10.1 % decrease 16.1 % decrease 15.6 % decrease
0.2 13.8 % decrease 19.6 % decrease 17.4 % decrease
Intrinsic rate of population growth 0.0002 2.5 % decrease 4.0 % decrease 1.9 % decrease
0.002 6.8 % decrease 3.6 % decrease 2.4 % decrease
0.02 22.4 % decrease 20.9 % decrease 25.3 % decrease
0.2 28.2 % decrease 34.9 % decrease 46.3 % decrease

distributions evaluated by the Shapiro–Wilk test and homo-
geneity of variances analyzed by the Levene’s test. Then
significant difference was tested by Tukey’s honestly meth-
od which was applied for multiple comparisons of means. If
the data were not normally distributed, logarithmic transfor-
mation was performed and then checked again for homoge-
neity of variances. The level of significance was set at *p<
0.05; **p<0.01; and ***p<0.001.
The lowest-observed-adverse-effect level (LOAEL) is the
lowest concentration of PCP, which causes an adverse alter-
ation of age at first reproduction, body length, or number of
offspring.
Results
No difference was observed between D. magna exposed in
the solvent control group with 0.1 % DMSO (P>0.05) and
the control group without DMSO. The 24-h EC50 for the
reference, positive control chemical K2Cr2O7 indicated that
the physiological condition and thus sensitivity of D. magna
were consistent among experiments. A control 21-day
chronic toxicity test for reference, the total number of living
offspring produced per parent animal alive at the end of the
test is assessed more than 60. All of D. magna in controls
and concentrations except 2 μmol/L were survival during
the test. Survival rate was calculated in the F0 carried
through the 21-day experiment when it was exposed to
2 μmol/L PCP. Toxic effects were enhanced in later gener-
ations. In generation F0, approximately 50 % of D. magna
were surviving after 12 days exposure, whereas in genera-
tion F1 and F2, the same effects were observed within 9 and
1 days, respectively (Fig. 2). Because nearly all of D. magna
in 2 μmol/L died within 21 days, age at first reproduction,
fecundity, length of mothers, and number of molts of D.
magna in 2 μmol/L could not be determined.
Age at first reproduction was significantly different be-
tween individuals exposed to 0.02 μmol/L or greater and those
of the controls in generations F1 and F2. There was no signif-
icant delay in age at first reproduction in the F0 generation
(Fig. 3). Significant dose–response relationships in age at first
reproduction were observed in F2 generation. Statistically
significant differences between the F1 and the F2 generations
were observed in 0.2 μmol/L treatment (Table 1). The con-
centration of PCP, generation and interaction between con-
centration of PCP and generation all extremely significantly
affect the age at first reproduction (Table 2).
The number of offspring per mother produced within
21 days in F0 generation significantly decreased when ex-
posed to 0.002 μmol/L or greater concentration treatments
Environ Sci Pollut Res (2014) 21:234–243
Fig. 4 Number of offspring per mother produced within 21 days
(mean+ SD) D. magna exposed to PCP during three generations.
Asterisks indicate statistically significant differences from the control
(*p<0.05; **p<0.01; ***p<0.001). DMSO group represents solvent
control group
(23.5 to 67.6 %) compared with the control (Table 3). How-
ever, no statistically significant differences were observed for
0.002 μmol/L in the F1 or F2 generations (Fig. 4). The number
of offspring per mother produced in the F1 and F2 generations
exposed to 0.02 μmol/L was less than that in the control (53.5
to 61.4 %), especially for the 0.2 μmol/L treatment (p<0.001;
73.7 to 83.7 %) (Fig. 4; Table 3). 0.0002 μmol/L and
0.02 μmol/L and greater were significantly different, while
no effect was seen with 0.002 μmol/L in F1 generation
(Fig. 4). And the number of offspring exposed to 0.2 μmol/L
in F2 is significantly lower in F0, although exposed to
0.002 μmol/L that in F2 is significantly higher in F0 (Table 1).
239
Fig. 5 Body length of parent female at 21 days old (mean+SD) D.
magna exposed to PCP over three generations. Asterisks indicate
statistically significant differences from the control treatment (*p<
0.05; **p<0.01; ***p<0.001). DMSO group represents solvent con-
trol group
The interaction between concentration of PCP and generation
number extremely significantly affect the number of offspring,
while generation number does not (Table 2).
A dose response relationship was observed for body
length of mothers in all three generations. The body length
of mothers F0 D. magna significantly decreased after expo-
sure to 0.002 (4.7 %), 0.02 (4.8 %) and 0.2 μmol/L (6.8 %)
compared with the unexposed F 0 individuals (Fig. 5;
Table 3). The body length of mothers was significantly
(4.1 to 15.1 %) shorter than that of the control when exposed
to the selected concentration in F1 generation. The body
length of D. magna exposed to 0.0002 μmol/L in F1 and
F2 generations were significantly shorter than the mothers
240
Fig. 6 Total number of molts within 21 days exposed to PCP from the
three generation for 21 days. Asterisks indicate statistically significant
differences from the control treatment (*p<0.05; **p<0.01; ***p<
0.001). DMSO group represents solvent control group
produced in the controls, especially in the F2 generation,
which was shorter than the control by approximately 7.8 %
(P<0.001). Statistically significant differences between F1 and
F2 generations were observed in 0.02 and 0.2 μmol/L treat-
ment, while that between F0 and F1 generations were observed
in 0.002, 0.02, and 0.2 μmol/L treatment (Table 1). The
concentration of PCP, generation and interaction between con-
centration of PCP and generation all extremely significantly
affect the age at first reproduction (Table 2).
Similar effects were observed in the number of molts of
D. magna within 21 days. Significant dose–response rela-
tionships were observed for the number of molts in F0 to F2
generations (Fig. 6). The total number of molts of D. magna
in the F0 generation significantly decreased after exposure
to 0.02 μmol/L or greater (10.1 to 13.8 %) compared with
Environ Sci Pollut Res (2014) 21:234–243
those of unexposed individuals. The total number of molts
of D. magna was significantly less (8.9 to 19.6 %) than that
of the control when exposed to the 0.0002 to 0.2 μmol/L in
the F1 generation. However, no statistically significant dif-
ferences were observed between the total number of molts
of the control D. magna and that of the individuals exposed
to 0.0002 μmol/L in the F2 generation. The total number of
molts in the F2 generation exposed to 0.002 μmol/L or
greater concentrations were 1.3 to 1.9 times less than that
in the control (P<0.001; 1.9 to 17.4 %). Statistically signif-
icant differences between F0 and F1 generations were ob-
served in 0.2 μmol/L treatment, the same as of F0 and F2
generations (Table 1). The concentration of PCP and gener-
ation extremely significantly affect the number of offspring,
while interaction does not (Table 2).
The effects of multigenerational exposures of PCP on
intrinsic rate of population growth of D. magna were similar
with effects of number of offspring per mother produced
within 21 days. Statistically significant differences were
observed when exposed to 0.002 μmol/L in the F0 genera-
tions, while not in F1 or F2 generations (Table 4); 0.0002
and 0.02 μmol/L and greater were significantly different,
while no effect was seen with 0.002 μmol/L in F1 genera-
tion (Table 4). Intrinsic rate of population growth exposed to
0.2 μmol/L in F2 (0.1389 ind/day) is significantly lower in
F0 (0.1864 ind/day) (Table 1). The concentration of PCP
and interaction between concentration of PCP and genera-
tion number extremely significantly affect the number of
offspring while generation number does not (Table 2).
In our study, LOAEL in the multigenerational tests was
estimated to be <0.0002 μmol/L, which is the lowest value
of LOAEL among all the endpoints selected (Table 5).
Discussion
The chronic test is traditionally performed by examination of
each D. magna per test vessel, with 50–100 ml of medium in
each vessel, which is labor-intensive and the large amount of
waste output. Thus, our previous study conducted the test with
each D. magna with 10 mL of test solution (Xing et al. 2012).
Here, positive control chemical K2Cr2O7 was used to test
sensitivity and a control 21-day chronic toxicity test for another
reference; the total number of living offspring in the control
produced per parent animal alive at the end of the test is
assessed to be more than 60. This is a revised good method
as indicated in another high-throughput screening assay, which
was developed to test zebrafish embryo hatching using 96-well
plate containing only 100 μL of test solution (Lin et al. 2011).
In our study, D. magna exposed to 0.0002 μmol/L in the
F1 and F2 generations, which is 10- to 1,000-fold less than
the reported concentrations on surface waters of China, were
significantly shorter than the female produced in the
Environ Sci Pollut Res (2014) 21:234–243 241

Table 4 The effects of multigenerational exposures of pentachlorophenol on population dynamics parameters (mean+SD) of D. magna

Population dynamics parameters Intrinsic rate of population growth Finite rate of increase Mean generation Net reproduce
(ind/day) (ind/day) time (day) rate (ind)

F0
Control 0.2595±0.0079 a 1.296±0.010 a 16.14±0.30 a 65.90±4.68 a
0.0002 μmol/L 0.2529±0.0164 a, b 1.288±0.021 a, b 16.23±0.46 a 61.10±9.26 a, b
0.002 μmol/L 0.2419±0.0099 b 1.274±0.013 b 16.42±0.55 a 53.40±7.29 b
0.02 μmol/L 0.2014±0.0146 C 1.223±0.018 C 16.66±0.38 a 29.80±9.54 C
0.2 μmol/L 0.1864±0.0155 C 1.205±0.019 C 16.71±0.96 a 23.90±8.60 C
F1
Control 0.2602±0.0135 a 1.297±0.017 a 16.18±0.59 a 68.10±7.26 a
0.0002 μmol/L 0.2497±0.0144 b 1.284±0.018 b 16.30±0.57 a 58.70±8.93 b
0.002 μmol/L 0.2508±0.0111 a, b 1.285±0.014 a, b 16.41±0.58 a 61.70±9.35 a, b
0.02 μmol/L 0.2058±0.0230 C 1.229±0.028 C 16.65±0.83 a 31.70±9.56 C
0.2 μmol/L 0.1692±0.0293 D 1.185±0.034 D 16.81±0.99 a 17.90±5.84 D
F2
Control 0.2587±0.0077 a 1.295±0.010 a 16.17±0.40 b 65.80±6.32 a
0.0002 μmol/L 0.2539±0.0054 a 1.289±0.007 a 16.37±0.35 a, b 64.00±6.63 a
0.002 μmol/L 0.2526±0.0070 a 1.287±0.009 a 16.42±0.22 a, b 63.40±4.74 a
0.02 μmol/L 0.1932±0.0098 B 1.213±0.011 B 16.68±0.30 a, b 25.40±4.06 B
0.2 μmol/L 0.1389±0.0129 C 1.149±0.014 C 16.98±1.10 a 10.70±2.21 C

Values with the same letters in each row are not significantly different (p>0.05), values with different superscripts are significantly different (capital
letters, p<0.05; lowercase letters, p<0.01)

controls, especially in the F2 generation, which was shorter
than the control by approximately 7.8 % (P<0.001). The
LOAEL in the multigenerational tests was estimated to be
0.0002 μmol/L, which is lesser than the reported 21-day
exposure LRCT (similar to LOAEL) for survival which is 1
(3.7 mmol/L) and 0.56 mg/L (0.002 mmol/L) for carapace
length (Van Leeuwen et al. 1987). It is mainly due to
different culture conditions and different species. It is the
main reason why the Major State Basic Research Develop-
ment Program and the National Major Project of the Minis-
try of Science and Technology of the People’s Republic of
China was initiated in 2008 avoiding over- or under-
protected aquatic organisms due to differences between
hydrographic conditions and species in China and those in
other countries (Wu et al. 2010; Yan et al. 2012).
In D. magna, exposure to waterborne nickel showed
increasing effects on growth across two generations (Pane
et al. 2004) and on offspring size across seven generations
(Muenzinger 1990). Similarly, increasing sensitivity of
daphnid reproduction and survival to americium-241 was
observed across three generations (Alonzo et al. 2008). PCP
caused more serious effects, as observed in the F1 and F2
generations, especially in age at first reproduction and body
length per female, effects on which were enhanced in later
generations. A potential explanation for this observation is
the development and improvement of accumulation perfor-
mance and maternal transfer, which are believed to be the
main sources of organic compounds in offsprings (Dietrich
et al. 2010). Previous studies have found that tolerance
decreased after pre-exposure to PCP (Papchenkova et al.
2009). In our study, 0.0002 μmol/L is significantly lower
in F1, although since 0.002 μmol/L is not significant. Re-
sults with copper suggested that daphnids may develop a
resistance with an increasing survival rate when parents

Table 5 Effects of
multigenerational exposures of Endpoints LOAEL (μmol/L)
pentachlorophenol on different
endpoints and different genera- F0 F1 F2
tions with LOEAL
Age at first reproduction >0.2 0.02 0.002
Number of offspring per mother produced within 21 days 0.002 <0.0002 0.02
Body length at 21 days old 0.002 <0.0002 <0.0002
Total number of molts within 21 days 0.02 <0.0002 0.002
242
were previously exposed (LeBlanc 1982). The fourth gen-
eration of D. magna was more sensitive to glyphosate than
to the maternal line (Papchenkova et al. 2009). Individuals
within the first several generations may not have been
affected. However, subsequent generations were more sen-
sitive to Cd (Guan and Wang 2006). No significant inhibi-
tion of fecundity was observed in the first generation of D.
magna exposed to diethylstilbestrol. However, significantly
fewer offspring were produced when second-generation D.
magna were exposed to this chemical (Brennan et al. 2006).
An increasing sensitivity to toxicity was also observed
across generations of D. magna exposed to uranium
(Massarin et al. 2010). This result agrees with the reported
test. The interaction between concentration of PCP and
generation extremely significantly affects age at first pro-
duction, number of offspring per mother produced within
21 days, body length of parent female at 21 days old, and
intrinsic rate of population growth of D. magna.
Multigenerational studies are more sensitive than single-
generation experiments. Studying PCP effects under
multigenerational exposure regimes represents a key issue
to improve the ecological relevance of risk assessment be-
cause natural populations can be exposed to toxicants over
several generations.
Conclusions
Environmentally relevant concentrations of less than
2 μmol/L caused a series of adverse effects on the
growth and reproduction of D. magna relative to the
control. PCP concentrations on surface water may
reach the threshold concentration for adverse effects.
The results obtained in this study were consistent with
multigenerational exposures to other chemicals. Signif-
icant negative effects appeared with increase in expo-
sure time (Guan and Wang 2006). The endpoint body
length of females (F 2 ) exposed to PCP was more
sensitive than the endpoint reproduction for 21-day
chronic toxicity. Some significant n possibly exist for
the entire life cycle of one generation (e.g., generation-
al lifespan), which will provide more helpful informa-
tion to evaluate risk assessment. Concentrations less
than those observed in some surface waters in China
could drastically affect the lifecycle of D. magna. Sig-
nificant effects occurred in the third generation with
the increase in exposure time. More multigenerational
studies are urgently needed because potential effects
can be missed or may change in single-generation
experiments. This apparent magnification effect repre-
sents a worrying trend for organisms in the wild, which
are chronically exposed to xenoestrogens throughout
many generations.
Environ Sci Pollut Res (2014) 21:234–243
Acknowledgments This research was supported by the Natural
Science Foundation of China (No. 20977047), Major Science and
Technology Program for Water Pollution Control and Treatment
of China (No. 2012ZX07506-001 and 2012ZX07501-003-02), and
the Environmental Monitoring Research Foundation of Jiangsu
Province (No. 1114). Prof. Giesy was supported by the program
of 2012 "High Level Foreign Experts" (#GDW20123200120)
funded by the Nanjing University, State Administration of Foreign
Experts Affairs, P.R. China and the Einstein Professor Program of
the Chinese Academy of Sciences. He was also supported by the
Canada Research Chair program, an at-large Chair Professorship
at the Department of Biology and Chemistry and State Key
Laboratory in Marine Pollution, City University of Hong Kong.
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 Effects of Multi-generational exposures of D. magna to Environmentally
Relevant Concentrations of Pentachlorophenol

Yi Chen1, Jin Huang1, Liqun Xing1, Hongling Liu1*, John P. Giesy1,2,3, Hongxia Yu1*,
Xiaowei Zhang1

1
State Key Laboratory of Pollution Control and Resource Reuse, School of the
Environment, Nanjing University, Nanjing 210023, China
2
Department of Veterinary Biomedical Sciences and Toxicology Centre, University
of Saskatchewan, Saskatoon, Saskatchewan, Canada
3
Department of Biology & Chemistry and State Key Laboratory for Marine Pollution,,
City University of Hong Kong, Kowloon, Hong Kong

*
Corresponding Author.
Tel: +86-25-8968-0356;
Fax: +86-25-8968-0356;
E-mail address: hlliu@nju.edu.cn (Hongling Liu);
Yuhx@nju.edu.cn (Hongxia Yu)
Fig. S1. The relationships of body length of parent female at 21 days old and number
of offspring per mother produced within 21 days (a) PCP treatment (b) Control

Fig. S2. Relationships of body length of parent female at 21 days old of F0 generation
and F1 generation

Fig. S3. Relationships of body length of parent female at 21 days old of F1 generation
and F2 generation
Fig. S1.
Fig. S2.
Fig. S3.