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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
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 2. Materials and methods
mixture of mainly three diastereoisomers, a-, b- and g-HBCD and
the composition of these diastereoisomers in tHBCD varies in the 2.1. Chemicals and reagents
range of 1e12% a-HBCD, 10e13% b-HBCD and 75e89% g-HBCD
(Koeppen et al., 2007). HBCDs are ubiquitous environmental con- tHBCD was purchased from Tokyo Chemical Industry Co. (Tokyo,
taminants and have been recently included in the Stockholm Japan); a-HBCD, b-HBCD and g-HBCD, from AccuStandard, Inc.
Convention for consideration of global elimination (www.pops.int). (New Haven, Connecticut, USA); 13C-a-HBCD, 13C-b-HBCD and 13C-
Concentrations of HBCDs in the range of ng L1 are detected in g-HBCD, from Cambridge Isotope Laboratories, Inc. (Tewksbury,
waters (He et al., 2013; Wu et al., 2010). For example, a concen- Massachusetts, USA); and the organic solvents (acetonitrile, hexane
tration of total HBCDs as high as 2100 ng L1 is recorded in the and methanol) used for sample extraction and LC-MS/MS analysis,
surface water of River Kuzuryu, Japan, which receives effluents from Tedia Inc. (Fairfield, Ohio, USA). All other reagents were pur-
from textile industries (Oh et al., 2014). Owing to the high octanol- chased from Sigma-Aldrich Chemical Co. (St. Louis, Washington,
water partition coefficient (Kow, 5.625), HBCDs tend to build up USA) except where indicated.
heavily in sediments, often in the range of mg kg1 dry weight. For
instance, an exceptionally high concentration, i.e., 7800 mg kg1 dry 2.2. T. japonicus maintenance
weight, is detected in the sediment of River Kuzuryu (Oh et al.,
2014). Aside from their accumulation in sediments, the high Kow The T. japonicus was originally collected from Xiamen Bay and
value also results in significant accumulation in the aquatic biota. was kindly provided by Dr. Lisheng Wu of Xiamen University. The
Laboratory experiments show that HBCDs can be bioaccumulated stock culture was maintained in the laboratory for more than five
in fish through both water-borne and food-borne exposure (Du years before the experiments (Guo et al., 2012). The T. japonicus was
et al., 2012a; Zhang et al., 2014b). In addition, relatively high con- cultured at 20 ± 1 C in a 12-h light:12-h dark photoperiod in
centrations of HBCDs are detected in several freshwater and marine filtered (0.22 mm) artificial seawater at a salinity of 30‰. An equal
fish species downstream of the source of the chemical and in highly mixture of four algal species, Thalassiosira pseudonana, Isochrysis
industrialized areas in Europe and China (Allchin and Morris, 2003; galbana, Tetraselmis suecica and Nannochloropsis oceanica was fed to
Koppen et al., 2010; Xian et al., 2008). Compared with the studies them daily. The conditions for copepod culture in the toxicity tests
on fish, only a few studies examine the accumulation of HBCDs in were the same as described above except where indicated.
aquatic organisms at lower trophic levels, e.g., in invertebrates such
as copepods (Jenssen et al., 2007; Tappin and Milward, 2015; Son 2.3. 96-h acute toxicity tests
et al., 2015).
The currently available toxicity data of HBCDs on aquatic or- The standard static 96-h toxicity test was performed using
ganisms are also mainly focused on fish. For example, HBCDs adults of T. japonicus. The copepods were exposed to nominal
induce the generation of reactive oxygen species (ROS) and result concentrations of 80, 300 and 800 mg L1 tHBCD in seawater. The
in developmental toxicity in both freshwater and marine model solvent control group received the same concentration of DMSO as
fish, e.g. the zebrafish Danio rerio and the marine medaka Oryzias the treated groups (0.01%, v/v) and the seawater control was also
melastigma (Deng et al., 2009; Du et al., 2012b; Hong et al., included in the exposure. For each treatment, 20 copepods were
2014). Aside from fish, it has been demonstrated that HBCDs transferred using a pipette to a 60-mm crystallizing dish containing
are toxic to freshwater Daphnia magna at low concentrations 20 mL of exposure media and four replicates were included in each
(Drottar and Krueger, 1998). In this regard, the no-observable- exposure group. The test solutions were renewed every 48 h.
effect-concentration (NOEC) of tHBCD on the survival, repro- Mortality of copepods was checked under a stereomicroscope every
duction or growth of D. magna in a full life cycle test is day.
3.1 mg L1, and at the concentration of 5.6 mg L1 tHBCD signif-
icantly reduces their body length (Drottar and Krueger, 1998). 2.4. Two generations full life-cycle tests
HBCDs also affect the ingestion and filtration rates of the marine
copepod Pseudodiaptomus inopinus in a short-term sub-lethal The exposure concentrations in the long-term toxicity test were
exposure experiment (Gong et al., 2016). Apart from these, 0, 8, 30, 80, 300 and 800 mg L1, which were all below the lowest-
relatively fewer studies evaluate the toxicity of HBCDs on aquatic observable effect concentration (LOEC) of the lethal dose in the 96-
invertebrates. h acute toxicity test. The solvent control group received the same
Given that the accumulation and toxicity of HBCDs in marine concentration of DMSO as the treated groups (0.01%, v/v).
invertebrates remain largely unknown and that the sensitivity of To start the first generation life-cycle test (F0), 20 newly hatched
freshwater and saltwater species to environmental pollutants nauplii (<24 h after hatching) per replicate were transferred to 5-
could be significantly different (Leung et al., 2001; Wheeler et al., mL glass beakers containing 3 mL of exposure media and four
2002), we chose the marine copepod Tigriopus japonicus as a replicates were included in each exposure group. The exposure
model species for the toxicological studies of tHBCD. T. japonicus, a solution was freshly prepared and renewed daily (>80% of the total
widely distributed marine copepod species, has distinct sexual volume), and the alga Tetraselmis suecica was centrifuged to remove
dimorphism and nauplius and copepodid phases. It also has high the culture medium and added at a density of 1.3 105 cells mL1
fecundity and a reasonable life cycle time. Moreover, it can adapt every day approximately 2 h before media renewal. These nauplii
to and reproduce at a wide range of temperature and salinity and were cultured until adult females developed egg sacs. Develop-
has been served as an excellent model for estuarine and marine mental stages were observed daily under a stereomicroscope and
ecotoxicological studies (Guo et al., 2012; Lee et al., 2008; Rhee recorded to calculate the time for nauplii to develop to copepodid
et al., 2013). (N-C) and to adults with egg sacs (N-A) (Guo et al., 2012; Lee et al.,
In this study, we assessed the accumulation and developmental 2008). The survival% was calculated and the sex ratio was deter-
toxicity of tHBCD on T. japonicus through water-borne exposure. In mined after the maturation of all copepods. To measure the
addition, the transcription of genes related to oxidative stress and fecundity (number of clutches, number of nauplii per clutch), five
apoptosis were examined to shed light on the potential toxic egg-sac bearing female copepods from each exposure group were
mechanisms of tHBCD in marine copepods. individually transferred to glass beakers. These females were
D. Shi et al. / Chemosphere 167 (2017) 155e162 157
cultured under the above mentioned conditions for 10 days and the desalting, the SPE column was eluted with 9 mL of methanol and
resulting nauplii were counted under the stereomicroscope. the eluate was concentrated to 0.5 mL under N2 flow.
For the second generation life-cycle test (F1), 20 nauplii hatched To determine the concentrations of HBCDs accumulated in the T.
out as the first brood by the female in the F0 generation were japonicus, the copepod samples (100 copepods per sample) were
transferred to a new glass beaker as one replicate. The experimental first spiked with the 13C-labeled a-HBCD, b-HBCD and g-HBCD
and exposure conditions were the same as those used in the F0 standards, and then homogenized and extracted in 5 mL hex-
generation test. 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
2.5. Exposure for bioaccumulation and depuration study
ultrasonication for 30 min. The resulting supernatant was com-
bined and concentrated to 0.5 mL under N2 flow.
The experiment was conducted in 2-L glass tanks and three
The quantification of the HBCDs was carried out on an Agilent
replicates were included in each exposure group. The uptake test
1290-6490 UPLC-triple quadruple mass spectrometry system
lasted for 96 h followed by 48 h for depuration. In the uptake phase,
(Agilent Technologies, Palo Alto, California, USA). The separation of
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
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
control group. The selected exposure concentration was below the
flow rate of 0.3 mL min1. The mobile phase consisted of two sol-
water solubility of tHBCD (3.4 mg L1, Desjardin et al., 2003). To
vents: mobile phase A (30% methanol in water, v/v) and mobile
maintain the concentration of tHBCD at 2 mg L1, the walls of the
phase B (30% methanol in acetonitrile, v/v). The linear gradient
glass tanks were pre-equilibrated to the same concentration of
program used for HPLC separation was as follows: linear gradient
exposure media overnight and the exposure solution for uptake
from 70 to 100% B (0e10 min); hold isocratic at 100% B
phase was freshly prepared and renewed every 12 h. The depu-
(10e15 min); and re-equilibrate at 70% B for 10 min. The LC eluate
ration test was conducted in clean artificial seawater. During the
was directed to a mass spectrometer equipped with an electro-
whole exposure period, Tetraselmis suecica was centrifuged to
spray ionization probe operated in negative ion-mode. The a-, b-
remove the culture medium and added at a density of 5 105 cells
and g-HBCD isomers and their corresponding 13C-labeled internal
mL1 once per day and 1 h before the media renewal. Sampling was
standards were monitored in multiple reaction monitoring mode
performed on 0, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h during the
with transition events of m/z 640.8 > 80.8 and m/z 652.8 > 80.8,
uptake phase and 102, 108, 120 and 144 h during the depuration
respectively.
(Fig. 1). At each sampling point, 100 copepods was collected for
A 5-points calibration (20, 50, 100, 200, 500 ng mL1 in meth-
each sample and frozen immediately at 80 C before analysis. In
anol) was performed to confirm the linearity of the response of the
the meantime, 20 mL of exposure solution was collected at each
mass spectrometer, and a fine calibration curve was obtained with
sampling point from each tank for the measurement of tHBCD
R2 > 0.99. None of the HBCD isomers was detected in procedural
concentrations in the media.
blanks which were included in each experiment batch to confirm
no contamination introduced through glassware and reagents. The
2.6. Sample extraction and LC-MS/MS quantification of HBCDs 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
To determine the actual concentrations of HBCDs in the expo- blanks of copepods, respectively. The relative standard deviations
sure media, 10-mL of each media sample was passed through a of a-, b- and g-HBCD were less than 20% in 3 sample replicates.
CNW® HC-C18 SPE column (CNW Technologies, Shanghai, China),
which was preconditioned by methanol and water to retain the 2.7. Exposure for gene transcription measurement
organic compounds. After washing the column with 10 mL H2O for
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.
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,
Fig. 1. The concentrations of HBCDs in T. japonicus (left y-axis) and exposure media glutathione S-transferase (GST), 8-oxoguanine DNA glycosylase
(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
(OGG1), Caspase-3 and Actin were based on the published papers
uptake phase was 2 mg L1 of tHBCD. The values are presented as the mean ± SD (Hwang et al., 2010; Kim et al., 2012; Rhee et al., 2013). The thermal
(n ¼ 3). cycle program consisted of an initial denaturation step at 95 C for
158 D. Shi et al. / Chemosphere 167 (2017) 155e162
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.
4. Discussion
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)
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, HBCD is higher in fish than in zooplankton while the percentage of
previous in vitro work has revealed that a-HBCD is more resistant to g-HBCD is lower in fish than in zooplankton. All these suggest that
metabolic degradation by both rat and trout liver enzymes than b- the stereoisomer-selective metabolism of g-HBCDs to the forma-
and g-HBCD (Abdallah et al., 2014). Therefore, the stereoisomer- tion of a-HBCD in zooplankton is not as prevalent as in fish.
selective biotransformation of HBCDs could result in higher BCF The measurement of HBCDs in the environment showed that g-
value of a-HBCD than those of b- and g-HBCD in fish. The BCFs of HBCD, the major component of tHBCD, is the most abundant HBCD
individual HBCDs are currently unknown in marine copepods or diastereoisomer in the water column, and the composition of
other zooplankton species. In our study, we found that the HBCDs in the water phase is close to that of tHBCD (He et al., 2013;
composition of HBCD diastereoisomers accumulated in T. japonicus Oh et al., 2014). In addition, the bioisomerizaiton from b- and g-
was almost the same as that of tHBCD in the exposure water, HBCD to the formation of a-HBCD is not observed in microalgae,
suggesting limited bioisomerization of HBCD diastereomers, which, Spirulina subsalsa and Scenedesmus obliquus (Zhang et al., 2014c).
however, needs further confirmation by testing with individual Therefore g-HBCD is likely the predominant HBCD diastereoisomer
HBCD diastereoisomers. In addition, it has been reported that the copepods in the field would be exposed to through either water-
composition of HBCDs in zooplankton in an eastern Canadian Arctic borne or food ingestion. Thus, it is convincing to choose tHBCD as
food web was about 30% and 70% of a- and g-HBCD, respectively, the exposure reagent to examine the bioaccumulation and toxicity
while a-HBCD reached 70e90% of total HBCDs in the fish collected of HBCDs in marine copepods in the current study. Nevertheless,
from the same location (Tomy et al., 2008). Another study con- the bioaccumulation and toxic effects of individual a-, b- and g-
ducted in Lake Ontario also shows a similar pattern (Tomy et al., HBCD in T. japonicus is worthy of further investigation in the future,
2004). These survey studies indicate that the percentage of a- in order to obtain a systematic understanding of the effects of
D. Shi et al. / Chemosphere 167 (2017) 155e162 161
HBCDs on marine copepods. the base-excision DNA repair process. The induction of OGG1
transcription suggested that DNA was undergoing attack by ROS
4.2. tHBCD induced developmental toxicity in T. japonicus upon tHBCD exposure, which was consistent with the increase of
transcription of other oxidative responsive genes, i.e. SOD, GST and
tHBCD is toxic to aquatic vertebrates and invertebrates, and CAT. In addition, the P53 gene is one of the critical genes that
delays the growth of D. magna at a concentration of 5.6 mg L1 respond to a variety of stresses, including oxidative stress. The P53
(LOEC) together with induced malformation (Drottar and Krueger, pathway can be activated as a result of the increased levels of DNA
1998). The heart beats of marine medaka (Oryzias melastigma) damage caused by ROS, which promotes cell cycle arrest or
embryos are affected by tHBCD at a concentration of 5 mg L1 (Hong apoptosis (Harris and Levine, 2005). Our results indeed showed
et al., 2014). Our results showed that exposure of T. japonicus that in corresponding to the higher transcription levels of OGG1,
nauplii to tHBCD caused significant growth delay, particularly SOD, GST and CAT, the transcription of the P53 gene was induced
during the nauplius phase. The LOECs of tHBCD induced develop- upon exposure to tHBCD. Furthermore, the activation of P53 can
mental delay in the nauplius phase (N-C) of T. japonicus were 30 trigger the mitochondria-mediate apoptotic pathway, subsequently
and 8 mg L1 for F0 and F1 generations (Fig. 2), respectively, sug- resulting in the release of cytochrome c and activating Caspase-9
gesting that the development of T. japonicus was as sensitive as D. and Caspase-3 (Elmore, 2007). In this regard, the tHBCD-induced
magna and O. melastigma to tHBCD exposure. In addition, the expression of P53 and the activation of Caspase-3 and Caspase-9
development of T. japonicus was more sensitive to tHBCD in the F1 were observed in fish embryos (Deng et al., 2009; Hong et al.,
than the F0 generation, highlighting the importance of long term 2014). We also showed that the transcription of caspase-3 was
toxicity evaluation and warranting multiple-generation toxicity significantly induced by tHBCD in T. japonicus (Fig. 3).
tests for future studies.
Oh et al. (2014) report that 2.1 mg L1 of total HBCDs was 4.4. Conclusion
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 tHBCD had high bioaccumulation potential in T. japonicus with
was close to the concentration measured in polluted waters, sug- the BCF values higher than those of fish. Exposure of T. japonicus
gesting that environmental realistic concentrations of HBCDs may nauplii to tHBCD caused significant growth delay, particularly
pose a potential threat to aquatic invertebrates. during the nauplius phase. The nauplii in the F1 generation was
It should be noted that although the environmental concen- more sensitive to tHBCD than the F0 generation, which warranted
trations of HBCDs in seawater have not been reported yet, it is multiple-generation toxicity tests for future studies. The long term
reasonable to estimate that the concentrations of HBCDs in toxicity exposure and the measurement of gene transcription
seawater would be far lower than those in the riverine water. On indicated that tHBCD exposure induced the generation of ROS in T.
the other hand, the sensitivity of copepods toward chemical pol- japonicus, which caused oxidative DNA damage and meanwhile
lutants varies among different species and T. japonicus is among activated the oxidative stress defense genes, SOD, CAT and GST. The
those that are not most sensitive to the toxicity of marine pollutants damaged DNA activated the transcription of P53 and triggered the
(Raisuddin et al., 2007). In addition, the long-term laboratory caspase-mediated apoptosis pathway, which may be the reason for
maintained culture which was used in the current study could the tHBCD induced developmental delay in T. japonicus nauplii.
possibly deviate genetically from those of the field populations. In
this regard, the genetic variability in D. magna cultures held in Acknowledgements
several different testing laboratories has reflected significant dif-
ferences in toxicological response to chemical pollutants (Barata We thank the two anonymous reviewers for constructive and
et al., 2002; Picado et al., 2007). Although the genetic variability helpful comments on this work. We also thank Dr. Lisheng Wu for
and sensitivity to toxicants in the laboratory cultured T. japonicus providing the stock culture of T. japonicus and Dr. Minghua Wang
and the field populations are currently unknown, for the worst- for technical assistance in the maintenance of T. japonicus. This
case scenario we could assume that the field populations be more study was supported by the National Natural Science Foundation of
sensitive to HBCDs exposure than the cultured ones. Taken all these China (No. 41206090), the Research Fund for the Doctoral Program
points into consideration, we speculated that the HBCDs in of Higher Education of China (No. 20120121120032), the Natural
seawater may pose potential risk to the sensitive species of marine Science Foundation of Fujian Province, China (No. 2015J01174), the
zooplankton, particularly in the long run. Fundamental Research Funds for the Central Universities, and the
Recruitment Program of Global Youth Experts. Prof. John Hodgkiss
4.3. Mechanism of tHBCD induced toxicity in T. japonicus is thanked for his assistance with English.
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