Toxic effects of dechlorane plus on the common carp (Cyprinus carpio) embryonic development

Dechlorane Plus (DP) is a widely using chlorinated �ame retardant, and has been detected extensively in the environment. Even though the DP content in the surface water is low, it can pose a continuous exposure risk to aquatic organisms due to its strong bioaccumulation. Since related researches on the toxicity mechanism of DP exposure on carp embryo development are limited, we carried out the evaluation of DP effect on carp. In this study, Carp embryos were exposed to DP of 0, 30, 60, and 120 µg/L at 3h post fertilization (hpf). The expression of genes associated with the neural and skeletal development such as sox2, sox19a, Mef2c, and BMP4 were evaluated using quantitative PCR (qPCR), and analysis of different developmental toxicity endpoints changes were observed with microscopy. Our results demonstrated that the expression of sox2, sox19a, Mef2c, and BMP4 were signi�cantly changed and several developmental defects were observed in the exposed carps embryo, such as DNA damage, increased mortality rate, delayed hatching time, decreased hatching rate, decreased body length, increased morphological deformities. In addition, reactive oxygen species (ROS) and malondialdehyde (MDA) were signi�cantly increased with DP exposure at 60µg/L and 120 µg/L. Altogether, our results suggest that DP may have an unique modes of action, which results in aberration occurrence in the early development stage of common carps, that may relates to some gene damage and oxidative stress. Besides, the parameters evaluated here can be used as tools to access the environmental risk for biota and humans exposed to DP.


Background
Dechlorane Plus (DP) is a polychlorinated ame retardant designed to replace Brominated ame retardants or Mirex (Ji et al., 2018 ), and it is widely used in plastic roo ng materials, electrical wires, hard disk connectors of computers, and other polymeric systems (Ren et al. 2008).DP is found common in the aquatic environment (Li et al. 2014;Sun et al. 2017;Tao et al. 2015;Wu et al. 2016), with a half-life of about 24 years in the water environment.It is a typical persistent organic pollutant and has an effect of bio-enrichment and bio-forti cation in the biota (OxyChem 2007;Yu et al. 2013).Hoh et al. rst discovered the existence of DP in the environment in 2006.Some studies show that DP accumulates in female herring gulls, crucian carp, ducks, lake trout, and mud carps, and that it is maternally transferred to their eggs (Guo et al. 2017;Gauthier and Letcher 2009;Wu et al. 2016).Studies have also shown that DP possesses physiological toxicity.For example, after 90 days of DP exposure, the mRNA expression level of CYP2B2 in rats decreased, and the enzyme activity is increased (Li et al. 2013a).In addition, orally feed DP can cause metabolic disorders, signal transduction alteration, and liver oxidative damage in rats (Wu et al. 2013).DP can also induce spontaneous movement of zebra sh embryos, with signi cantly reduced free style speed and signi cantly reduced larval swimming speed (Chen et al. 2017).DP can also induce DNA damage in Tetrahymena thermophila and marine bivalves ( (Dou et al. 2015;Baron et al. 2016).
Sox genes play important roles in growth, development, and physiological processes.The main function of these genes is to participate in neurogenesis, bone formation, stem cell development in the embryo, cardiogenesis, sex determination, and hematopoiesis in zebra sh (Akiyama et al., 2004;Avilion et al., 2003;Pevny and Placzek, 2005;Polanco and Koopman, 2007;Schilham et al., 1997;Smi ts et al., 2001).
Sox2 gene encodes a transcription factor which plays an important role in maintaining pluripotent stem cell populations, early development, and differentiation in zebra sh (Okuda et al., 2010).The sox19 gene also plays a very important role in embryogenesis.Studies show that the sox2, sox3, and sox19a genes knocked down results in abnormalities in the central nervous system and in the tail region of zebra sh embryos (Okuda et al., 2010).Mef2c gene is particularly important in early development of zebra sh heart (Hinits et al. 2012) and of mice skeletal muscle (Potthoff et al. 2007).Verzi et al. (2007) found that lacking Mef2c in neural crest lineage causes jaw defects in mice.BMP4 gene is involved in the process of chondrocyte differentiation into cartilage and gives rise to bone in the differentiation and maturation of cells of osteoblastic lineages (Balemans and Van Hul 2002;Canalis et al. 2003;Hogan 1996;Graff 1997;Mariani and Martin 2003;Thies et al. 1992;Wan and Cao 2005;Yoon and Lyons 2004).BMP4 signaling inhibition leads to sclerotic maturation or delayed function of bones (Xia et al. 2018).However, little is known about the sensitivity of early developmental stages of sh to DP.  (Burcu & Cahit, 2014).The objectives of the present work were to characterize the main organic contaminants present in water environment, to evaluate the toxicity produced by that polluted water on the embryonic development of the C. carpio, and to correlate the morphological changes found with the production of gene expression and DNA damage.Thus, we analysis the ecotoxicological threat of DP in the early development stage of common carps, and assess the potential dangers that DP may spread to aquatic organisms.

Collection of fertilized eggs
Fertilized eggs of common carp were obtained from the College of Fishery, Henan Normal University, Xinxiang, Henan Province, China.The embryos were transferred to a petri dish with a diameter of 100 mm for toxicity testing.The experimental use of sh was approved by the Animal Ethics Committee of Henan Normal University (HNSD No. 20180901-0915-01).

Exposure for embryos toxicity
Petri dishes containing 300-350 healthy embryos were exposed to DP at concentrations of 0, 30, 60, and 120 µg/L for 120 hpf.Embryos were maintained in an incubator at 26 ± 1 °C with a photoperiod of 14:10 h light: dark.This study used larval mortality, hatching rate, malformations and body length as toxicological endpoints.Direct observation of embryos in a petri dish under a stereo microscope and the above endpoints were scored at 24, 48, 72, 96 and 120 hpf.All experiments were conducted triplicate, and the exposure solution of the same concentration was replaced every 12 h, while the dead embryos and juveniles were removed in a timely manner.Each treatment group was assayed in triplicate, and all the tests were independently repeated twice to ensure reproducibility.Hatching rate was calculated as the percentage of eggs hatched in a speci c time, whereas the mortality rate was calculated as the percentage of dead eggs in all eggs in a given time.Larvae of 120 hpf were positioned, photographed and measured for body length, the deformation rate calculated as percentage of deformation rate of live eggs at 120 hpf.In order to ensure the developmental synchronization in the early stage of DP exposure, the embryos were exposed 3 h post fertilization (hpf) after the fertilization (blastocyst stage).

Measurement of ROS and MDA generation
Determination of ROS and MDA content were conducted by using ROS assay kit and MDA Assay kit respectively, by following the manufacturer's instructions (Shanghai Yiyan Biological Engineering Co., Shanghai, China).ROS generation in larvae at 96 hpf was detected by using dichloro uorescein-diacetate (DCFH-DA) with a uorescence microplate reader (Varioskan Flash, USA) with excitation and emission at 485 and 528 nm, respectively and was shown as DCF/mg protein.MDA, which is produced during fatty acid degradation and is indicative of lipid peroxidation, was quanti ed based on the method of TBA (thiobarbituric acid) and was shown as nmol/mg protein.
Quantitative PCR (q-PCR) Total RNA was prepared by using TRIzol reagent (TransGen Biotech, Beijing), then was reverse transcribed to complementary DNA by using a PrimeScript RT reagent Kit (TaKaRa Biotechnology, Dalian, China).Real-time quantitative PCR (qPCR) was done on an ABI 7500HT sequencer (Applied Biosystems, Foster City, USA).The sequences of gene including four embryonic developmental genes (sox2, sox19a, Mef2c, and Bmp4) were downloaded from the NCBI database.The primers were designed using Primer primer 5 (Table 1).β-actin was used as an internal control to normalize the relative mRNA expression of a speci c gene via the 2 −ΔΔCT method (Livak & Schmittgen, 2001).

Comet assay
To detect cellular DNA damage, a comet assay was performed according to protocols developed by Singh et al. (1988) with minor modi cations.Mechanical cell isolation, the rst step of the comet assay, was carried out as described by Kosmehl et al. (2006).At 24 hpf, embryos with at least 90% cell viability were selected for the comet assay.To visualize DNA damage, 100 nuclei per slide were measured at 40× magni cation.The results were assessed based on tail length, tail DNA (%), and olive tail movement (OTM) using the CytoVision NT automatic (Beijing Biolaunching Technologies Co., Ltd., Beijing, China) image analysis system.Percentage tail DNA, considered the most valuable parameter was recorded (Praveen Kumar et al., 2014).All the experimental were represented in three replicates.

Statistical analysis
All assays were carried out in at least in triplicate, data are shown as mean ± standard error of the mean (SEM).All data were veri ed for normality and homogeneity of variance using KolmogoroveSmirnov one sample test and Levene's test.Comet assay data (% tail DNA) were arcsine transformed and tested for normality and homogeneity using the Shapiro-Wilk test and Levene's test respectively.The correlation between DNA damage and abnormalities in embryo development was analyzed by the Pearson coe cient test.All statistical analyses were conducted using SPSS 18.0.(IBM, USA).The level of statistical signi cance was set at p < 0.05, indicated by an asterisk.

Developmental toxicity
The average mortality rate of DP exposed embryos was recorded in Fig. 1A.It was shown that the mortality rate of embryos increases over time with the same concentration of DP.The mortality rate was signi cantly decreased (P < 0.05) only in the 30 µg/L group at 24 hpf.However, the cumulative mortality of embryos was signi cantly higher (P < 0.05) in the 60, and 120 µg/L treatment groups in contrast to the control group at 48 hpf and 96 hpf.There was a signi cant increase (P < 0.05) in the mortality of embryos in the 120µg/L treatment groups at 72 hpf.
Hatching rate of embryos varies according to exposure concentrations and exposure time (Fig. 1B).With the DP concentration increasing, the hatching rate of the common carp embryos decreased signi cantly (P <0.05), which is inversely proportional to the DP concentration.However, with exposure time increasing, the hatching rate of embryos in DP treatment group increased.Interestingly, the embryo hatching rate in the 120 µg/L treatment groups was consistent at 72 hpf and 48 hpf, indicating the delay in the hatching process is attribute to DP toxicity.
Frequency of morphological deformities induced by different concentrations of DP exposed to common carp embryos at 120 hpf is shown in Fig. 2A.With the exposure time prolonging, the embryo malformation rate increased signi cantly (P < 0.05), which indicating that the DP initiated embryo developmental toxicity in a time-dependent manner.The typical malformations including pericardial edema (PE), body malformations (BM) (including tail deformation and short tail), spinal curvature (SC), and other malformations were explored (Fig. 2B).
The size of the exposed and control groups of larvae were measured at 120hpf and are shown in Supplementary data: Fig S1 .Exposed larvae exhibited a concentration dependent decrease of total body length.Control groups of larvae were measured 7.12 ± 0.25 mm, whereas, the DP exposed ones were measured from 6.53 ± 0.20 mm to 6.23 ± 0.50 mm.

DP induced oxidative stress
To determine whether DP induced larval oxidative stress, the content of ROS and MDA were quanti ed at 96 hpf.A signi cant increase in ROS content was seen in 60 µg/L and 120 µg/L DP treated larvae compare to the control group.Similarly, a signi cant increase in MDA level was observed in 60 µg/L and 120 µg/L DP treatment groups (Fig. 3).

Comet assay
The DNA single strand breaks were demonstrated as the mean tail DNA content (% tail DNA) induced by different concentrations of DP in common carp embryo at 24 hpf Fig. 4A .Comparing with the control groups, the common carp embryos in all treatment groups had signi cant DNA damage.Interestingly, a dosage dependent increase in the mean tail DNA was observed, with higher concentrations of DP lead to much more serious damage.The representative images of DNA damage obtained by the comet assay are shown in Fig. 4B.

Gene expression
The effects of DP on the mRNA levels of four types of embryo developmental genes (sox2, sox19a, Mef2c, and BMP4) are shown in Fig. 5. Signi cant decrease (P < 0.05) in the expression of sox2 gene was observed in the DP exposed embryos of common carp as compared to the control group of embryos at all the time intervals, however, with an increase in both the exposure time, upward trend in sox2 gene expression (Fig. 5 A).Further, signi cant increase (P < 0.05) in the expression of sox19a gene expression were also observed in the DP exposed embryos of common carp as compared to the control group of embryos, presented a trend of increasing rst and then decreasing (Fig. 5 B).At the tail-bud formed stage, the Mef2c mRNA levels was signi cantly increased (P < 0.05) in the 30 µg/L DP exposed group.However, Mef2c expression was inhibited in the 60 and 120 µg/L DP exposed groups compared to the control group.During the hatching period, Mef2c mRNA levels increased signi cantly(P <0.05) in the 120 μg /L treated group, as compared with the control group, while the other treated groups shown no signi cant change.At the 24 h post-hatching stage, the Mef2c mRNA levels were signi cantly decreased (P <0.05) and signi cantly increased (P <0.05) in the 30 μg/L treatment group and in the 60 μg/L treatment group respectively, while the 120 μg/L treated group shown no obvious change, as compared with the control group.At the 48 h post-hatching stage, the Mef2c mRNA levels were signi cantly increased (P < 0.05) and signi cantly decreased (P < 0.05) in the 30 µg/L treated group and in the 60 µg/L treated group, respectively (Fig. 5 C).At the tail-bud formation stage, compared with the control group, the BMP4 mRNA levels were signi cantly increased (P <0.05) and signi cantly decreased (P <0.05) in the 30 μg/L treated group and in the 60 and 120 μg / L treated groups respectively, as compared with the control group.During the hatching period, the mRNA expression of BMP4 revealed a signi cant increase (P < 0.05) in the 120 µg/L treated group compared with the control group.However, there are no signi cant changes in the 30 and 60 µg/L DP exposed groups.BMP4 expression was signi cantly increased (P <0.05) in the 60μg/L treatment group at the 24 h post-hatching stage, while decreased in the 30 and 120 µg/L treated groups in contrast to the control group.Further, there was no obvious induction during the 48 h posthatching stage (Fig. 5 D).

Relationship between comet assay and morphological abnormality
In order to determine whether there is a relationship between embryo abnormality and DP induced DNA damage, we examined the correlation between DP induced DNA damage and carp embryos morphological distortion (Figure 6).A signi cant positive correlation was observed between the two parameters.

Discussion
DP acts as a persistent organic pollutants of the aquatic environment that can have harmful effects on aquatic organisms, including sh (OxyChem 2007; Yu et al. 2013;Liang et al., 2014;Sun et al. 2017;Ji et al., 2018).Fish are subjected to low-dose pollutants in water that do not cause death, but cause altered gene expression or retarded growth (ˇStˇep´anov´ Stanislava, et al. 2012;Chen et al. 2017).However, the effects of DP on common carp development and behind mechanisms have not been studied.Therefore, the present study aim to reveal the effects of the DP exposure on common carp embryo, indicated mortality rate, hatching rate, body length, morphological deformities and DNA single strand breaks and gene expression regulation.This study also provides new data to support the effects of DP on common carp embryos and larvae at relevant environmental concentrations.
Increased mortality, reduced hatchability and hatching delay of DP exposed embryos have been observed in the present study, suggesting the toxicity of DP on common carp embryos.Previous studies have shown similar mortality rate increased in common carp embryos exposed to Cyhalofop-butyl (Xia et al., 2018).Mortalities were also observed in zebra sh embryos exposed to various chemicals or metals such as copper (Fraysse et al., 2006;Johnson et al., 2007).It's have been shown that chemically induced changes in mitochondrial bioenergy in zebra sh embryos can lead to abnormal hatching times or tail bending.(Wang et al., 2018;Zhang et al., 2017).Noteworthy is that hatching delay is reported to be associated with the interference in the thyroid hormone system (Timme-Laragy et al., 2006), and DP can disrupts thyroid hormone balance of zebra sh by altering regulatory pathways in the brain (Kang et al., 2016).Thus, we hypothesize that mitochondrial dysfunction and disruptions in TH system induced by DP may contribute to the hatching delay.The present study demonstrated that DP was toxic to the common carp and could affect embryo development and even cause embryo death.Meanwhile, this result con rms the ndings described in previous studies such as increased DNA damage and change the expression of related developmental genes.Signals of apoptosis induced by oxidative stress have proven to be a potential cause of various pathologies (Circu and Aw, 2010).Previous studies suggest that DP exposure induce signi cant oxidative stress in quail and mice (Li et al., 2013b;Wu et al., 2012).To examine whether DP exposure induces oxidative stress in common carps, the levels of ROS and MDA were measured in 96 hpf larvae.Our results showed that DP exposure to the 60 and 120 µg / L treated groups signi cantly increased ROS and MDA formation.These results suggested that DP might induce apoptosis through oxidative stress signal pathway, which could be a potential mechanism of observed dysplasia after common carp exposure to DP.
Dysplasia is an important index for evaluating the teratogenicity of pollutants (Niell et al. 2010).
Cyhalofopbutyl exposure causes abnormal common carp morphology including yolk sac edema, pericardial edema, deformation of the spine and deformation of the tail (Xia et al. 2018).CPF exposure also resulted in the morphological abnormalities of zebra sh larvae, mainly manifested as pericardial edema and spinal deformities (Jin et al. 2015).Interestingly, in this study we also found a signi cant reduction in body length of DP exposed common carps.These results are similar with those that reported reduction of sh growth upon exposure to other chemicals, for example organophosphate insecticides and pyrethroid insecticides (Allison, 1977;Machova et al., 2010;Richterova et al., 2015;Werner and Moran, 2008).In the present study, changes in the developmental malformation of common carp larvae may be due to DP exposure induced DNA damage (single/double strand breaks) and oxidative stress.In addition, the expression of sox2, sox19a, Mef2c, and Bmp4 gene may involve in the process of morphological deformities.
The comet assay is widely used in genetic toxicity studies (Manas et al., 2009;Nan et al., 2016).The comet assay is an important biomarker for sh genetic toxicity, and DNA strand breaks are measured by the comet assay (Mitchelmore & Chipman, 1998).This study found that DNA single-strand breaks (% tail DNA) in common carp embryos were signi cantly increased in all treatment groups and strongly correlated with DP doses and exposure time studied indicate the genotoxic potential of DP exposed.These results are similar with the nding of Tasneem and R. Yasmeen (2018) which proved that karanjin can cause DNA damage in common carp detected by comet assay.Since 48-72 hpf is a critical period during the early embryonic development of common carp, considering the effects of nerves and bones on developmental malformations, to further nd the damage mechanisms at the level of molecule, the associated genes were investigated in this study.The sox gene proteins control a wide range of developmental regulators in the early embryo, has been fairly well characterized regarding its regulatory functions and its expression in embryogenesis.It was identi ed as an essential factor to activate crystallin genes in the lens cells (Kamachi et al., 1998;Okuda et al., 2010).Studies show that gene expression can be regulated by exposure to chemical genotoxicants, causing DNA damaged of zebra sh (Sandrini et al., 2009;Geffroy et al., 2012, Chen et al., 2019).Damaged genes that cannot be repaired by the DNA repair system may eventually lead to morphological deformities and neurobehavioral defects in zebra sh larvae (Chen et al., 2019).Signi cant down regulation of sox2 genes expression in the present study may be due to the sensitivity of the sox genes to DP exposure.The signi cant down-regulation of sox2 gene expression in this study may be due to the sensitivity of sox gene to DP exposure.The upregulation of sox19a at 72 hpf may be crucial in enabling the common carp to cope with oxidative stress and ensure normal development by self-regulation.This result is supported by the increased DNA damage and morphological deformities in the present study.This result provides evidence for increased DNA damage and morphological abnormalities in this study.
Mef2c is one of the key genes involved in regulating cardiac and skeletal muscle development, and can promote skeletal muscle development during embryogenesis (Hinits and Hughes 2007;Molkentin et al. 1995).Research has shown that, this inhibition of BMP signaling during zebra sh n regeneration was observed to disrupt n growth, as well as the differentiation and function of nucleated cells (Smith et al. 2006).Our results show that the Mef2c mRNA level was signi cantly decreased in the 30 µg/L treated group at the 24 h post-hatching stage and was signi cantly decreased in the 60 µg/L treated group at the 48 h post-hatching stage.Compared with the control group, the mRNA expression of BMP4 in the 60 and 120 µg/L treatment groups was signi cantly decreased during the tail-bud forming stage.This suggests that DP may negatively affect skeletal muscle development by disrupting the expression of Mef2c and BMP4 genes.The speci c mechanism by which this interaction affects skeletal muscle development requires further study.
From the comet assay, it shown that the positive correlation between DNA damage and morphological abnormalities of common carp larvae may indicate that DNA single-strand breaks induce carp embryo developmental distortion.Similarly, CuInS2/ZnS quantum dots (QDs) exposure induces impaired growth and DNA damage in sh (Liu et al., 2017).DNA damage may also cause down regulation of related developmental genes, which may be the common cause of all developmental abnormalities observed in the study.

Conclusions
In summary, our results showed that DP inhibits the hatching rate of common carp embryos and even causes the death of common carp embryos.Some successfully hatched embryos were deformed by DP exposure, consistent with signi cant changes in related genes.So DP has some negative impact on the growth and development of common carp embryos.This study provides a basis for guiding the rational use of DP and promoting the sustainable development of the ecological environment.Figure 9

Figure 8 (
Figure 8 Common carp, Cyprinus carpio L. (C.carpio) is commonly used in ecotoxicology studies.It is a very important organism in the point of ecological and economic.The C. carpio, is the most widely farmed freshwater sh in China.It is reported that C. carpio is often used as a biological indicator species to understand the impact of environmental pollution

Table 1
Primer sequences of the genes