Chemosphere 167 (2017) 155e162

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Accumulation and developmental toxicity of

hexabromocyclododecanes (HBCDs) on the marine copepod Tigriopus
japonicus
Dalin Shi a, c, Dongmei Lv a, Wanxin Liu a, Rong Shen a, Dongmei Li a,
Haizheng Hong a, b, c, *
a
State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361102, China
b
Center for Marine Environmental Chemistry and Toxicology, Xiamen University, Xiamen, 361102, China
c
Environmental Science Research Center, Xiamen University, Xiamen, 361102, China

h i g h l i g h t s

HBCD had high bioaccumulation potential in T. japonicus.

HBCD caused developmental delay in T. japonicus.

The F1 generation of T. japonicus was more sensitive to HBCD than the F0 generation.

HBCD induced oxidative stress in T. japonicus.

a r t i c l e i n f o a b s t r a c t

Article history: The brominated flame retardants hexabromocyclododecanes (HBCDs) are ubiquitous environmental
Received 6 May 2016 contaminants, widely distributed in aquatic systems including the marine environment and marine
Received in revised form organisms. HBCDs are toxic to the development of both freshwater and marine fish. However, the im-
30 September 2016
pacts of HBCDs on marine invertebrates are not well known. In this study, the marine copepod, Tigriopus
Accepted 30 September 2016
japonicus, was used to assess the bioaccumulation and developmental toxicity of technical HBCD (tHBCD)
through water-borne exposure. The uptake rate constant of tHBCD by T. japonicus was high, which
Handling editor: Jim Lazorchak resulted in high bioaccumulation potential. The bioconcentration factors of tHBCD were 8.73  104 and
6.34  104 L kg1 in T. japonicus, calculated using the kinetic and steady-state methods, respectively.
Keywords: Exposure of T. japonicus nauplii to tHBCD caused significant growth delay. The lowest-observable-effect-
Hexabromocyclododecaneso concentrations of tHBCD induced developmental delay were 30 and 8 mg L1 for the F0 and F1 gener-
Developmental toxicityo ations, respectively, which suggested that the F1 generation was more sensitive to tHBCD than the F0
Bioaccumulationo generation and warranted multiple-generation toxicity tests for future studies. Furthermore, exposure of
Oxidative stresso
the adult copepods to tHBCD induced the transcription of oxidative stress response genes and apoptotic
Marine copepodo
genes, e.g., SOD,CAT, GST, OGG1, P53 and Caspase-3. It was therefore speculated that tHBCD exposure
induced the generation of reactive oxygen species in T. japonicus, which activated the oxidative stress
defense genes and meanwhile resulted in oxidative DNA damage. The damaged DNA activated the
transcription of p53 and triggered the caspase-mediated apoptosis pathway, which may be the reason for
the tHBCD induced developmental delay in T. japonicus nauplii.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Hexabromocyclododecanes (HBCDs) are the third most widely

* Corresponding author. State Key Laboratory of Marine Environmental Science,
College of the Environment and Ecology, Room C428, Xiamen University, South
Xiangan Rd, Xiangan District, Xiamen, Fujian 361102, China.
E-mail address: honghz@xmu.edu.cn (H. Hong).
used brominated flame retardants in the world (Yang et al., 2012),
which are primarily used in polystyrene insulation foam boards,
textile products and electronic products. The commercially

http://dx.doi.org/10.1016/j.chemosphere.2016.09.160
0045-6535/© 2016 Elsevier Ltd. All rights reserved.
156 D. Shi et al. / Chemosphere 167 (2017) 155e162
available HBCD (technical HBCD, tHBCD) is often characterized as a
mixture of mainly three diastereoisomers, a-, b- and g-HBCD and
the composition of these diastereoisomers in tHBCD varies in the
range of 1e12% a-HBCD, 10e13% b-HBCD and 75e89% g-HBCD
(Koeppen et al., 2007). HBCDs are ubiquitous environmental con-
taminants and have been recently included in the Stockholm
Convention for consideration of global elimination (www.pops.int).
Concentrations of HBCDs in the range of ng L1 are detected in
waters (He et al., 2013; Wu et al., 2010). For example, a concen-
tration of total HBCDs as high as 2100 ng L1 is recorded in the
surface water of River Kuzuryu, Japan, which receives effluents
from textile industries (Oh et al., 2014). Owing to the high octanol-
water partition coefficient (Kow, 5.625), HBCDs tend to build up
heavily in sediments, often in the range of mg kg1 dry weight. For
instance, an exceptionally high concentration, i.e., 7800 mg kg1 dry
weight, is detected in the sediment of River Kuzuryu (Oh et al.,
2014). Aside from their accumulation in sediments, the high Kow
value also results in significant accumulation in the aquatic biota.
Laboratory experiments show that HBCDs can be bioaccumulated
in fish through both water-borne and food-borne exposure (Du
et al., 2012a; Zhang et al., 2014b). In addition, relatively high con-
centrations of HBCDs are detected in several freshwater and marine
fish species downstream of the source of the chemical and in highly
industrialized areas in Europe and China (Allchin and Morris, 2003;
Koppen et al., 2010; Xian et al., 2008). Compared with the studies
on fish, only a few studies examine the accumulation of HBCDs in
aquatic organisms at lower trophic levels, e.g., in invertebrates such
as copepods (Jenssen et al., 2007; Tappin and Milward, 2015; Son
et al., 2015).
The currently available toxicity data of HBCDs on aquatic or-
ganisms are also mainly focused on fish. For example, HBCDs
induce the generation of reactive oxygen species (ROS) and result
in developmental toxicity in both freshwater and marine model
fish, e.g. the zebrafish Danio rerio and the marine medaka Oryzias
melastigma (Deng et al., 2009; Du et al., 2012b; Hong et al.,
2014). Aside from fish, it has been demonstrated that HBCDs
are toxic to freshwater Daphnia magna at low concentrations
(Drottar and Krueger, 1998). In this regard, the no-observable-
effect-concentration (NOEC) of tHBCD on the survival, repro-
duction or growth of D. magna in a full life cycle test is
3.1 mg L1, and at the concentration of 5.6 mg L1 tHBCD signif-
icantly reduces their body length (Drottar and Krueger, 1998).
HBCDs also affect the ingestion and filtration rates of the marine
copepod Pseudodiaptomus inopinus in a short-term sub-lethal
exposure experiment (Gong et al., 2016). Apart from these,
relatively fewer studies evaluate the toxicity of HBCDs on aquatic
invertebrates.
Given that the accumulation and toxicity of HBCDs in marine
invertebrates remain largely unknown and that the sensitivity of
freshwater and saltwater species to environmental pollutants
could be significantly different (Leung et al., 2001; Wheeler et al.,
2002), we chose the marine copepod Tigriopus japonicus as a
model species for the toxicological studies of tHBCD. T. japonicus, a
widely distributed marine copepod species, has distinct sexual
dimorphism and nauplius and copepodid phases. It also has high
fecundity and a reasonable life cycle time. Moreover, it can adapt
to and reproduce at a wide range of temperature and salinity and
has been served as an excellent model for estuarine and marine
ecotoxicological studies (Guo et al., 2012; Lee et al., 2008; Rhee
et al., 2013).
In this study, we assessed the accumulation and developmental
toxicity of tHBCD on T. japonicus through water-borne exposure. In
addition, the transcription of genes related to oxidative stress and
apoptosis were examined to shed light on the potential toxic
mechanisms of tHBCD in marine copepods.
2. Materials and methods
2.1. Chemicals and reagents
tHBCD was purchased from Tokyo Chemical Industry Co. (Tokyo,
Japan); a-HBCD, b-HBCD and g-HBCD, from AccuStandard, Inc.
(New Haven, Connecticut, USA); 13C-a-HBCD, 13C-b-HBCD and 13C-
g-HBCD, from Cambridge Isotope Laboratories, Inc. (Tewksbury,
Massachusetts, USA); and the organic solvents (acetonitrile, hexane
and methanol) used for sample extraction and LC-MS/MS analysis,
from Tedia Inc. (Fairfield, Ohio, USA). All other reagents were pur-
chased from Sigma-Aldrich Chemical Co. (St. Louis, Washington,
USA) except where indicated.
2.2. T. japonicus maintenance
The T. japonicus was originally collected from Xiamen Bay and
was kindly provided by Dr. Lisheng Wu of Xiamen University. The
stock culture was maintained in the laboratory for more than five
years before the experiments (Guo et al., 2012). The T. japonicus was
cultured at 20 ± 1  C in a 12-h light:12-h dark photoperiod in
filtered (0.22 mm) artificial seawater at a salinity of 30‰. An equal
mixture of four algal species, Thalassiosira pseudonana, Isochrysis
galbana, Tetraselmis suecica and Nannochloropsis oceanica was fed to
them daily. The conditions for copepod culture in the toxicity tests
were the same as described above except where indicated.
2.3. 96-h acute toxicity tests
The standard static 96-h toxicity test was performed using
adults of T. japonicus. The copepods were exposed to nominal
concentrations of 80, 300 and 800 mg L1 tHBCD in seawater. The
solvent control group received the same concentration of DMSO as
the treated groups (0.01%, v/v) and the seawater control was also
included in the exposure. For each treatment, 20 copepods were
transferred using a pipette to a 60-mm crystallizing dish containing
20 mL of exposure media and four replicates were included in each
exposure group. The test solutions were renewed every 48 h.
Mortality of copepods was checked under a stereomicroscope every
day.
2.4. Two generations full life-cycle tests
The exposure concentrations in the long-term toxicity test were
0, 8, 30, 80, 300 and 800 mg L1, which were all below the lowest-
observable effect concentration (LOEC) of the lethal dose in the 96-
h acute toxicity test. The solvent control group received the same
concentration of DMSO as the treated groups (0.01%, v/v).
To start the first generation life-cycle test (F0), 20 newly hatched
nauplii (<24 h after hatching) per replicate were transferred to 5-
mL glass beakers containing 3 mL of exposure media and four
replicates were included in each exposure group. The exposure
solution was freshly prepared and renewed daily (>80% of the total
volume), and the alga Tetraselmis suecica was centrifuged to remove
the culture medium and added at a density of 1.3  105 cells mL1
every day approximately 2 h before media renewal. These nauplii
were cultured until adult females developed egg sacs. Develop-
mental stages were observed daily under a stereomicroscope and
recorded to calculate the time for nauplii to develop to copepodid
(N-C) and to adults with egg sacs (N-A) (Guo et al., 2012; Lee et al.,
2008). The survival% was calculated and the sex ratio was deter-
mined after the maturation of all copepods. To measure the
fecundity (number of clutches, number of nauplii per clutch), five
egg-sac bearing female copepods from each exposure group were
individually transferred to glass beakers. These females were
 D. Shi et al. / Chemosphere 167 (2017) 155e162
cultured under the above mentioned conditions for 10 days and the
resulting nauplii were counted under the stereomicroscope.
For the second generation life-cycle test (F1), 20 nauplii hatched
out as the first brood by the female in the F0 generation were
transferred to a new glass beaker as one replicate. The experimental
and exposure conditions were the same as those used in the F0
generation test.
2.5. Exposure for bioaccumulation and depuration study
The experiment was conducted in 2-L glass tanks and three
replicates were included in each exposure group. The uptake test
lasted for 96 h followed by 48 h for depuration. In the uptake phase,
T. japonicus was exposed to a nominal concentration of 2 mg L1 of
tHBCD in seawater, and a tank without tHBCD was used as the
control group. The selected exposure concentration was below the
water solubility of tHBCD (3.4 mg L1, Desjardin et al., 2003). To
maintain the concentration of tHBCD at 2 mg L1, the walls of the
glass tanks were pre-equilibrated to the same concentration of
exposure media overnight and the exposure solution for uptake
phase was freshly prepared and renewed every 12 h. The depu-
ration test was conducted in clean artificial seawater. During the
whole exposure period, Tetraselmis suecica was centrifuged to
remove the culture medium and added at a density of 5  105 cells
mL1 once per day and 1 h before the media renewal. Sampling was
performed on 0, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h during the
uptake phase and 102, 108, 120 and 144 h during the depuration
(Fig. 1). At each sampling point, 100 copepods was collected for
each sample and frozen immediately at 80  C before analysis. In
the meantime, 20 mL of exposure solution was collected at each
sampling point from each tank for the measurement of tHBCD
concentrations in the media.
2.6. Sample extraction and LC-MS/MS quantification of HBCDs
To determine the actual concentrations of HBCDs in the expo-
sure media, 10-mL of each media sample was passed through a
CNW® HC-C18 SPE column (CNW Technologies, Shanghai, China),
which was preconditioned by methanol and water to retain the
organic compounds. After washing the column with 10 mL H2O for
Fig. 1. The concentrations of HBCDs in T. japonicus (left y-axis) and exposure media
(right y-axis) in the bioaccumulation and depuration study. The uptake phase lasted
for 96 h followed by 48 h for depuration. The nominal exposure concentration in the
uptake phase was 2 mg L1 of tHBCD. The values are presented as the mean ± SD
(n ¼ 3).
157
desalting, the SPE column was eluted with 9 mL of methanol and
the eluate was concentrated to 0.5 mL under N2 flow.
To determine the concentrations of HBCDs accumulated in the T.
japonicus, the copepod samples (100 copepods per sample) were
first spiked with the 13C-labeled a-HBCD, b-HBCD and g-HBCD
standards, and then homogenized and extracted in 5 mL hex-
ane:acetone (1:1, v/v) using ultrasonication for 30 min. After
centrifugation, the supernatant was collected and the pellet was
extracted again using 5 mL of hexane:acetone (1:1, v/v) with
ultrasonication for 30 min. The resulting supernatant was com-
bined and concentrated to 0.5 mL under N2 flow.
The quantification of the HBCDs was carried out on an Agilent
1290-6490 UPLC-triple quadruple mass spectrometry system
(Agilent Technologies, Palo Alto, California, USA). The separation of
a-HBCD, b-HBCD and g-HBCD isomers was achieved on an Agilent
XDB C18 HPLC column (2.1 mm  150 mm, particle size 3.5 mm) at a
flow rate of 0.3 mL min1. The mobile phase consisted of two sol-
vents: mobile phase A (30% methanol in water, v/v) and mobile
phase B (30% methanol in acetonitrile, v/v). The linear gradient
program used for HPLC separation was as follows: linear gradient
from 70 to 100% B (0e10 min); hold isocratic at 100% B
(10e15 min); and re-equilibrate at 70% B for 10 min. The LC eluate
was directed to a mass spectrometer equipped with an electro-
spray ionization probe operated in negative ion-mode. The a-, b-
and g-HBCD isomers and their corresponding 13C-labeled internal
standards were monitored in multiple reaction monitoring mode
with transition events of m/z 640.8 > 80.8 and m/z 652.8 > 80.8,
respectively.
A 5-points calibration (20, 50, 100, 200, 500 ng mL1 in meth-
anol) was performed to confirm the linearity of the response of the
mass spectrometer, and a fine calibration curve was obtained with
R2 > 0.99. None of the HBCD isomers was detected in procedural
blanks which were included in each experiment batch to confirm
no contamination introduced through glassware and reagents. The
recoveries of a-, b- and g-HBCD were 89%, 78%, and 93% in the
spiked blanks of seawater and 92%, 95% and 102% in the spiked
blanks of copepods, respectively. The relative standard deviations
of a-, b- and g-HBCD were less than 20% in 3 sample replicates.
2.7. Exposure for gene transcription measurement
To explore the toxic mechanism, we conducted another expo-
sure experiment. Adult T. japonicus were exposed to 0, 300 and
800 mg L1 of tHBCD. Each exposure group was cultured in a glass
tank containing 1 L of exposure media and four replicates were
included in each group. The solvent control received the same
concentration of DMSO as the treated groups (0.01%, v/v). The
exposure media were freshly prepared and renewed every 24 h.
After being exposed for 3, 7 and 14 days, 100 copepods per sample
were collected, frozen immediately in liquid nitrogen and then
stored at 80  C until analysis.
2.8. Real-time quantitative PCR (qPCR)
Total RNA was extracted and an equal amount of RNA was then
reverse-transcribed using mixtures of oligo (dT) primer (50 -
TTTTTTTTTTTTTTTTTTTT-30 ) and random primers using M-MLV
reverse transcriptase (BGI, Shenzhen, China) to generate cDNA.
qPCR was carried out on a CFX96™ Real-Time System (Bio-Rad
Laboratories) using SYBR Green I. The sequence of the primers for
qPCR of the genes superoxide dismutase (SOD), catalase (CAT), P53,
glutathione S-transferase (GST), 8-oxoguanine DNA glycosylase
(OGG1), Caspase-3 and Actin were based on the published papers
(Hwang et al., 2010; Kim et al., 2012; Rhee et al., 2013). The thermal
cycle program consisted of an initial denaturation step at 95  C for
158 D. Shi et al. / Chemosphere 167 (2017) 155e162
3 min, followed by 50 cycles of 95  C for 10 s and 65  C for 35 s.
Dissociation curve analysis was performed to confirm that only the
targeted PCR product was amplified and detected. Two replicates of
qPCR were performed for each sample as technical replicates. The
transcription levels of the tested genes were analyzed using the
2DDCt method and then normalized to the levels of Actin mRNA.
2.9. Data analysis
To determine the bioaccumulation and depuration parameters,
data obtained during the depuration phase were fitted to a first
order decay curve (eq (1)) as described in Zhang et al. (2014b):
Ct ¼ C0 ek2t
where Ct is the concentration of HBCDs in copepod at time t; C0 is
the HBCD concentration in copepods at the start of the depuration
phase; and k2 represents the depuration rate constant. A linear
regression of ln(concentration) versus time was performed and k2
is the slope of the regression line.
Data obtained during the uptake phase were fitted to the
following model (Zhang et al., 2014b):
k1  
Cf ðtÞ ¼ Cw ðtÞ 1  ek2 t
k2
where k2 is the depuration rate constant obtained by the above
calculation; k1 represents the uptake rate constant; and Cf(t) and
Cw(t) are the HBCD concentrations in copepods and exposure me-
dia, at time t. A set of concentrations vs. time for the uptake phase
were regressed using non-linear curve fitting with SigmaPlot 12.0
(Systat Software, Inc., San Jose, California, USA) to obtain k1.
The BCFk (based on kinetic model) was calculated based on Eq
(3):
k1
BCFk ¼
k2
The depuration half life (t1/2) was calculated based on Eq (4):
ln 2
t1=2 ¼
k2
The BCFss (based on steady-state concentrations) was calculated
based on Eq (5):
Css
BCFss ¼
Cw
where Css is the concentration of HBCD in the copepod after
reaching steady state; and Cw is the HBCD concentrations in the
exposure media.
The lipid normalized kinetic BCF (BCFkL) was calculated using Eq
(6), which was expressed on a 5% lipid content basis (OECD 305):
0:05
BCFkL ¼ BCFk
Ln
where Ln is the mean lipid fraction (based on wet weight).
Statistical analysis of the toxicity and analytical data was con-
ducted using SPSS Statistics 19.0 (IBM Software, Columbus, Ohio,
USA) for significance of differences by one-way analysis of variance
(ANOVA) combined with an LSD test. Significant differences from
the control are reported in tables and figures using asterisks (one-
way ANOVA, followed by LSD tests: *p < 0.05; **p < 0.01;
***p < 0.001).
3. Results
3.1. Bioaccumulation and depuration of tHBCD
The concentrations of HBCDs in the exposure media during the
uptake phase were in the range of 1.31e1.78 mg L1 for the nominal
concentration of 2 mg L1 and deviated within 20% of the mean
concentration, suggesting that the exposure concentrations in the
media were relatively stable (Fig. 1). The concentrations of HBCDs
in T. japonicus and in the media were 0.22 ± 0.4 ng mg1 ww and
12.1 ng L1, respectively, for the control group, indicating no
obvious contamination by HBCDs.
During the 96-h uptake phase, the HBCDs concentrations in T.
japonicus increased over time (Fig. 1) and the uptake curve followed
(1) the model described by Eq (2) (R21 > 0.95, Table 1). At the end of the
96-h uptake phase, the concentrations of HBCDs approached
saturation and reached 103.6 ± 4.8 ng mg1 ww (Fig. 1). The dep-
uration curve followed the first order decay kinetic of Eq (1)
(R22 > 0.95, Table 1). The uptake rate constant (k1), depuration
rate constant (k2), depuration half life (t1/2) and BCFk were calcu-
lated based on Eqs (1)e(4) and are shown in Table 1. Since the
concentrations of accumulated HBCDs approached saturation at
96 h, the BCFss was calculated based on Eq (5). The calculated values
of BCFk and BCFss (i.e., 8.73  104 and 6.34  104 L kg1, respec-
tively) were very close (Table 1).
(2)
In addition, the ratios of a-, b- and g-HBCDs in T. japonicus were
very similar to those in the stock solution throughout the whole
uptake and depuration phase (data not shown), suggesting that the
three diastereoisomers did not undergo any remarkable bio-
isomerization in T. japonicus.
3.2. Toxicity of tHBCDs in T. japonicus
The LOEC for tHBCD to induce 96-h lethal toxicity was
(3) >800 mg L1 and even at the highest exposure concentration (i.e.
800 mg L1) the 96-h mortality of T. japonicus was less than 10%,
which indicated that tHBCD had little lethal effect on T. japonicus in
the acute toxicity test.
In the two-generation full life-cycle test, the survival rates of T.
(4)
japonicus in all the treatment groups were above 90%. In addition,
the survival in all the tHBCD-exposure groups did not show sig-
nificant difference from the control, suggesting that tHBCD did not
affect the survival of T. japonicus in the long-term toxicity test.
tHBCD increased the duration of development from nauplii to
(5) copepodid (N-C) and from nauplii to adults (N-A) in T. japonicus in a
dose dependent manner in both the F0 and F1 generations (Fig. 2).
The LOECs of tHBCD induced developmental delay in the N-C and
N-A stages were 30 and 300 mg L1, respectively, in the F0 gener-
ation (Fig. 2A, p < 0.01), suggesting that the development of T.
japonicus at the nauplius phase was more sensitive to tHBCD
exposure than that at the copepodid phase. The LOECs of tHBCD
induced increase in the duration of N-C and N-A in the F1 gener-
ation were 8 and 30 mg L1, respectively (Fig. 2B, p < 0.01). The
(6) difference in the LOECs between the F0 and F1 generations indi-
cated that T. japonicus in the F1 generation was more sensitive to
tHBCD induced growth delay than that in the F0 generation.
No significant difference was observed in the number of
clutches, the number of nauplii per clutch and the total fecundity
brooded over ten days among all the treatment groups in both the
F0 and F1 generations (Table 2), which indicated that tHBCD
exposure did not affect the fecundity of T. japonicus. In addition,
there was no significant change in the sex ratio in either the F0 or F1
generations upon tHBCD exposure (Table 2).
 D. Shi et al. / Chemosphere 167 (2017) 155e162 159

Table 1
Bioaccumulation and depuration parameters of tHBCD in T. japonicus through water-borne exposure.

R21 k1 (L kg1 d1) R22 k2 (d1) BCFk (L kg1) BCFss (L kg1) Lipid (%) BCFkL (L kg1) t1/2 (d)

0.97 2.35  104 0.97 0.27 8.73  104 6.34  104 5.57 7.84  104 2.57

k1, uptake rate constant; k2, depuration rate constant; BCFk, kinetic bioconcentration factor; BCFss, steady-state bioconcentration factor; lipid (%), percentage of lipid over wet
weight; BCFkL, lipid-normalized BCFk; t1/2, depuration half-time.

3.3. tHBCD induced expression of oxidative stress responsive and

apoptotic genes

After the exposure of T. japonicus to tHBCD for 3, 7 and 14 days,

the transcription of SOD, GST and CAT in T. japonicus was generally
induced in a dose dependent manner (Fig. 3AeC). The transcription
of the DNA oxidative damage repairing gene, OGG1, was also up-
regulated upon exposure to tHBCD (Fig. 3D), which is in agree-
ment with the induction of the oxidative stress biomarker genes,
i.e. SOD, GST and CAT. In addition to the oxidative stress responsive
genes, the transcription of P53 and Caspase-3, the enzymes playing
an important role in DNA repair and apoptosis, was also induced in
T. japonicus upon tHBCD exposure (Fig. 3EeF).

4. Discussion

4.1. Bioconcentration factor (BCF) of tHBCD

The BCFs of water-borne exposure of the freshwater rainbow

Fig. 2. tHBCD exposure increased the developmental duration of the nauplius phase
(nauplius to copepodid, N-C) and maturation period (nauplius to adult, N-A) of T.
japonicus in both the F0 (A) and F1 generations (B). The T. japonicus was exposed to 0,
8, 30, 80, 300 and 800 mg L1 of tHBCD. The values are presented as the mean ± SD
(n ¼ 4). The values that are significantly different from the control are indicated by
asterisks (one-way ANOVA, followed by the LSD post hoc test: **p < 0.01, ***p < 0.001).
trout Oncorhynchus mykiss to tHBCD are 8.97  103 (BCFss) and
1.65  104 L kg1 (BCFk), respectively, based on steady-state and
kinetic methods (Drottar and Krueger, 2000). Here we reported
that the BCFss and BCFk of tHBCD in T. japonicus were 6.34  104 and
8.73  104 L kg1, respectively, suggesting that the bio-
concentration potential of tHBCD in T. japonicus was higher than
that in fish. The higher BCF in T. japonicus was probably attributed
to the higher uptake rate constant (k1) in T. japonicus than in fish
and its similar depuration rate constant (k2) to that in mirror carp
(Table 1 and Zhang et al., 2014b). However, the reasons for a high
uptake rate of tHBCD in T. japonicus are still unknown. The lipid-
normalized BCFk in T. japonicus was also higher than that in fish
(Table 1, Zhang et al., 2014b), implying that factors other than the
fraction of lipid give rise to the high bioconcentration potential and
uptake rate in T. japonicus.
Zhang et al. (2014b) reported that BCFs of a-, b-, and g-HBCD in
different tissues of the mirror carp Cyprinus carpio specularis were
in the order of a-HBCD>b-HBCD>g-HBCD, indicating that a-HBCD
had a higher bioconcentration potential than b- and g-HBCD in fish.
It should be noted that b- and g-HBCD can be bioisomerized to a-
HBCD in fish, including mirror carp, rainbow trout and zebrafish

Table 2
Fecundity, sex ratio and nauplii per clutch of the F0 and F1 generations of T. japonicus exposed to different concentrations of tHBCD. The values are presented as the mean ± SD
(n ¼ 4 for sex ratio and n ¼ 5 for nauplii/clutch and fecundity).

Concentration (mg L1) Sex ratio (F/M) Nauplii/clutch Fecundity (10 days)

F0 0 0.54 ± 0.39 36.4 ± 7.6 109.2 ± 22.9

8 0.32 ± 0.12 31.4 ± 2.6 94.2 ± 7.8
30 0.92 ± 0.80 27.0 ± 3.5 81.0 ± 10.6
80 0.34 ± 0.02 24.0 ± 5.7 72.0 ± 27.0
300 0.22 ± 0.22 36.2 ± 5.8 108.6 ± 17.3
800 0.50 ± 0.17 32.0 ± 7.8 96.0 ± 23.3
F1 0 0.60 ± 0.49 35.5 ± 4.2 106.5 ± 12.6
8 0.22 ± 0.08 38.2 ± 2.4 114.6 ± 7.2
30 0.63 ± 0.35 33.0 ± 4.8 99.0 ± 14.4
80 0.47 ± 0.06 39.6 ± 4.3 118.8 ± 12.8
300 0.75 ± 0.25 34.8 ± 7.1 104.4 ± 21.4
800 0.83 ± 0.92 30.4 ± 7.1 91.2 ± 21.4
160 D. Shi et al. / Chemosphere 167 (2017) 155e162

Fig. 3. Induction of the gene transcription of SOD (A), GST (B), CAT (C), OGG1 (D), P53 (E) and Caspase-3 (F) in T. japonicus after exposure to 0, 300 and 800 mg L1 of tHBCD for 3, 7
and 14 days. The exposure was conducted separately from those for evaluating developmental toxicity. The values are presented as the mean ± SD (n ¼ 4). The values that are
significantly different from the control are indicated by asterisks (one-way ANOVA, followed by the LSD post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001).

(Du et al., 2012a; Law et al., 2006; Zhang et al., 2014b). In addition,
previous in vitro work has revealed that a-HBCD is more resistant to
metabolic degradation by both rat and trout liver enzymes than b-
and g-HBCD (Abdallah et al., 2014). Therefore, the stereoisomer-
selective biotransformation of HBCDs could result in higher BCF
value of a-HBCD than those of b- and g-HBCD in fish. The BCFs of
individual HBCDs are currently unknown in marine copepods or
other zooplankton species. In our study, we found that the
composition of HBCD diastereoisomers accumulated in T. japonicus
was almost the same as that of tHBCD in the exposure water,
suggesting limited bioisomerization of HBCD diastereomers, which,
however, needs further confirmation by testing with individual
HBCD diastereoisomers. In addition, it has been reported that the
composition of HBCDs in zooplankton in an eastern Canadian Arctic
food web was about 30% and 70% of a- and g-HBCD, respectively,
while a-HBCD reached 70e90% of total HBCDs in the fish collected
from the same location (Tomy et al., 2008). Another study con-
ducted in Lake Ontario also shows a similar pattern (Tomy et al.,
2004). These survey studies indicate that the percentage of a-
HBCD is higher in fish than in zooplankton while the percentage of
g-HBCD is lower in fish than in zooplankton. All these suggest that
the stereoisomer-selective metabolism of g-HBCDs to the forma-
tion of a-HBCD in zooplankton is not as prevalent as in fish.
The measurement of HBCDs in the environment showed that g-
HBCD, the major component of tHBCD, is the most abundant HBCD
diastereoisomer in the water column, and the composition of
HBCDs in the water phase is close to that of tHBCD (He et al., 2013;
Oh et al., 2014). In addition, the bioisomerizaiton from b- and g-
HBCD to the formation of a-HBCD is not observed in microalgae,
Spirulina subsalsa and Scenedesmus obliquus (Zhang et al., 2014c).
Therefore g-HBCD is likely the predominant HBCD diastereoisomer
copepods in the field would be exposed to through either water-
borne or food ingestion. Thus, it is convincing to choose tHBCD as
the exposure reagent to examine the bioaccumulation and toxicity
of HBCDs in marine copepods in the current study. Nevertheless,
the bioaccumulation and toxic effects of individual a-, b- and g-
HBCD in T. japonicus is worthy of further investigation in the future,
in order to obtain a systematic understanding of the effects of
 D. Shi et al. / Chemosphere 167 (2017) 155e162
HBCDs on marine copepods.
4.2. tHBCD induced developmental toxicity in T. japonicus
tHBCD is toxic to aquatic vertebrates and invertebrates, and
delays the growth of D. magna at a concentration of 5.6 mg L1
(LOEC) together with induced malformation (Drottar and Krueger,
1998). The heart beats of marine medaka (Oryzias melastigma)
embryos are affected by tHBCD at a concentration of 5 mg L1 (Hong
et al., 2014). Our results showed that exposure of T. japonicus
nauplii to tHBCD caused significant growth delay, particularly
during the nauplius phase. The LOECs of tHBCD induced develop-
mental delay in the nauplius phase (N-C) of T. japonicus were 30
and 8 mg L1 for F0 and F1 generations (Fig. 2), respectively, sug-
gesting that the development of T. japonicus was as sensitive as D.
magna and O. melastigma to tHBCD exposure. In addition, the
development of T. japonicus was more sensitive to tHBCD in the F1
than the F0 generation, highlighting the importance of long term
toxicity evaluation and warranting multiple-generation toxicity
tests for future studies.
Oh et al. (2014) report that 2.1 mg L1 of total HBCDs was
detected in the surface water of the River Kuzuryu, Japan. In our
study the LOEC for growth delay of T. japonicus was 8 mg L1 and
was close to the concentration measured in polluted waters, sug-
gesting that environmental realistic concentrations of HBCDs may
pose a potential threat to aquatic invertebrates.
It should be noted that although the environmental concen-
trations of HBCDs in seawater have not been reported yet, it is
reasonable to estimate that the concentrations of HBCDs in
seawater would be far lower than those in the riverine water. On
the other hand, the sensitivity of copepods toward chemical pol-
lutants varies among different species and T. japonicus is among
those that are not most sensitive to the toxicity of marine pollutants
(Raisuddin et al., 2007). In addition, the long-term laboratory
maintained culture which was used in the current study could
possibly deviate genetically from those of the field populations. In
this regard, the genetic variability in D. magna cultures held in
several different testing laboratories has reflected significant dif-
ferences in toxicological response to chemical pollutants (Barata
et al., 2002; Picado et al., 2007). Although the genetic variability
and sensitivity to toxicants in the laboratory cultured T. japonicus
and the field populations are currently unknown, for the worst-
case scenario we could assume that the field populations be more
sensitive to HBCDs exposure than the cultured ones. Taken all these
points into consideration, we speculated that the HBCDs in
seawater may pose potential risk to the sensitive species of marine
zooplankton, particularly in the long run.
4.3. Mechanism of tHBCD induced toxicity in T. japonicus
It is known that tHBCD induces oxidative stress in aquatic or-
ganisms, including fish and clams (Deng et al., 2009; Hong et al.,
2014; Zhang et al., 2014a). Here we showed that tHBCD induced
the transcription of oxidative stress responsive genes, e.g., SOD, GST
and CAT, in a dose dependent manner after 3, 7 and 14 days of
exposure (Fig. 3). The exposure was conducted separately from
those for evaluating developmental toxicity. Consistently, the
expression of OGG1, which encodes 8-oxoguanine DNA glycosylase,
a DNA base-excision repair protein, was also up-regulated upon
tHBCD exposure. 8-oxoguanine is one of the most abundant DNA
oxidative products caused by the attack of ROS on guanine. If not
repaired timely and properly by the base-excision DNA repair
pathway, 8-oxoguanine leads to G to T transversion (Ravanat et al.,
2002; Sova et al., 2010). 8-oxoguanine DNA glycosylase is the key
enzyme responsible for the detection and repair of 8-oxoguanine in
161
the base-excision DNA repair process. The induction of OGG1
transcription suggested that DNA was undergoing attack by ROS
upon tHBCD exposure, which was consistent with the increase of
transcription of other oxidative responsive genes, i.e. SOD, GST and
CAT. In addition, the P53 gene is one of the critical genes that
respond to a variety of stresses, including oxidative stress. The P53
pathway can be activated as a result of the increased levels of DNA
damage caused by ROS, which promotes cell cycle arrest or
apoptosis (Harris and Levine, 2005). Our results indeed showed
that in corresponding to the higher transcription levels of OGG1,
SOD, GST and CAT, the transcription of the P53 gene was induced
upon exposure to tHBCD. Furthermore, the activation of P53 can
trigger the mitochondria-mediate apoptotic pathway, subsequently
resulting in the release of cytochrome c and activating Caspase-9
and Caspase-3 (Elmore, 2007). In this regard, the tHBCD-induced
expression of P53 and the activation of Caspase-3 and Caspase-9
were observed in fish embryos (Deng et al., 2009; Hong et al.,
2014). We also showed that the transcription of caspase-3 was
significantly induced by tHBCD in T. japonicus (Fig. 3).
4.4. Conclusion
tHBCD had high bioaccumulation potential in T. japonicus with
the BCF values higher than those of fish. Exposure of T. japonicus
nauplii to tHBCD caused significant growth delay, particularly
during the nauplius phase. The nauplii in the F1 generation was
more sensitive to tHBCD than the F0 generation, which warranted
multiple-generation toxicity tests for future studies. The long term
toxicity exposure and the measurement of gene transcription
indicated that tHBCD exposure induced the generation of ROS in T.
japonicus, which caused oxidative DNA damage and meanwhile
activated the oxidative stress defense genes, SOD, CAT and GST. The
damaged DNA activated the transcription of P53 and triggered the
caspase-mediated apoptosis pathway, which may be the reason for
the tHBCD induced developmental delay in T. japonicus nauplii.
Acknowledgements
We thank the two anonymous reviewers for constructive and
helpful comments on this work. We also thank Dr. Lisheng Wu for
providing the stock culture of T. japonicus and Dr. Minghua Wang
for technical assistance in the maintenance of T. japonicus. This
study was supported by the National Natural Science Foundation of
China (No. 41206090), the Research Fund for the Doctoral Program
of Higher Education of China (No. 20120121120032), the Natural
Science Foundation of Fujian Province, China (No. 2015J01174), the
Fundamental Research Funds for the Central Universities, and the
Recruitment Program of Global Youth Experts. Prof. John Hodgkiss
is thanked for his assistance with English.
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