Depuration of cadmium from Chlamys farreri by ZnSO4, EDTA–Na2 and sodium citrate in short time

Journal Pre-proof Depuration of cadmium from Chlamys farreri by ZnSO4, EDTA–Na2 and sodium citrate in short time

Chao Yang, Qingkang Liu, Xianghong Meng, Limin Cao, Bingjie Liu PII:

S0045-6535(19)32669-4

DOI:

https://doi.org/10.1016/j.chemosphere.2019.125429

Reference:

CHEM 125429

To appear in:

Chemosphere

Received Date:

29 September 2019

Accepted Date:

19 November 2019

Please cite this article as: Chao Yang, Qingkang Liu, Xianghong Meng, Limin Cao, Bingjie Liu, Depuration of cadmium from Chlamys farreri by ZnSO4, EDTA–Na2 and sodium citrate in short time, Chemosphere (2019), https://doi.org/10.1016/j.chemosphere.2019.125429

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Journal Pre-proof Depuration of cadmium from Chlamys farreri by ZnSO4, EDTA–Na2 and sodium citrate in short time Chao Yanga, Qingkang Liua, Xianghong Menga,b, Limin Caoa, Bingjie Liua,* a College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China b Pilot National Laboratory for Marine Science and Technology, Qingdao 266235, China

Abstract In view of high content of cadmium (Cd) in Chlamys farreri, a commercial edible shellfish species, depurating Cd from Chlamys farreri is an important topic nowadays, especially in short time. Therefore, three kinds of additives were introduced into seawater respectively, i.e. ZnSO4, EDTA–Na2, sodium citrate, to depurate Cd from Chlamys farreri. The alteration of Cd content in separate organs was investigated under several treatments with high depuration efficiency. The results showed that Cd was depurated exceeding 20% within 12 h by the combination of 0.15 g/L sodium citrate, 0.28 g/L ZnSO4, and 0.42 g/L EDTA–Na2. No obvious increase of Cd was observed in the adductor muscles, while Cd decreased in the other part, so the reduction of Cd in the whole organism of Chlamys farreri may occur. Cd reduction was found in the following organs: the digestive gland, kidney, gill, and mantle. Furthermore, Cd migration to gonad from other tissues was noticed. Keywords: Cadmium; Depuration; Chlamys farreri; Chemical additives; Short time

*

Corresponding author. E-mail: [email protected] 1

Journal Pre-proof 1. Introduction Chlamys farreri, one important edible shellfish species, has been widely cultured and consumed in coastal areas of north China. Meanwhile, Chlamys farreri has been considered as a kind of bioindicator of various pollutants from aquatic environments by some researchers because of its bioaccumulate nature (Pan et al., 2008; Liu et al., 2012; Guo et al., 2017). The maximum level of Cd in Chlamys farreri has been limited in China (2.0 mg/kg wet weight) because Cd can cause adverse effect on human health at a low concentration with the increasing seafood consumption. Meanwhile, the maximum permissible concentration of Cd in bivalves is specified at 1 mg/kg wet weight. Depuration has been developed for reduction of toxic metals in bivalve molluscs, which depends on their expelling metals from the organisms in uncontaminated seawater. Transfer of bivalves from contaminated areas due to human activities to clean environment has been proven effective (Chan et al., 1999; Jebali et al., 2014), which requires a long term of phase and is suitable for aquafarms to reduce Cd level. As such, reasonable depuration strategies in a short time following the harvest have been proposed for practical application in circulation of bivalves (El–Gamal, 2011; Freitas et al., 2012; Anacleto et al., 2015). The high concentration of Cd in Chlamys farreri from northern coastal cities of China has been reported (Li and Gao, 2014), but a mitigation strategy has not been provided. Most researchers in China prefer to keep Chlamys farreri in purified seawater for at least 3 days to reduce the content of Cd. In this context, this study attempted to find approaches to decrease Cd level of Chlamys farreri in short time, which will be conducive to commercial practice. EDTA and sodium citrate represent a valid but cheap chelating agent in inorganic

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Journal Pre-proof compounds and organic compounds, respectively. EDTA and sodium citrate have been utilized to accelerate loss of heavy metals in oysters and mussels, respectively (Hiraoka, 1991; Azelee et al., 2014). EDTA–Na2 is more practical than EDTA because of its greater solubility, and it has stronger chelating ability on account of the anion separating from sodium ions. Carboxymethyl chitosan, with larger molecular weight than EDTA–Na2 and sodium citrate, is prepared by introducing carboxymethyl to chitosan, which is responsible for promoting the solubility of chitosan, and possesses adsorption ability for Cd by −NH2 or –COOH groups (Borsagli and Borsagli, 2019). The study expects the polysaccharide derivative to play a role in facilitating Cd elimination of Chlamys farreri by increasing its concentration gradient between Chlamys farreri and seawater. In addition, Zn–Cd antagonism has been observed in wild and farmed oysters in an investigation aiming to reduce Cd in farming management (Munksgaard et al., 2017). However, the impact of supplying zinc ions into seawater on Cd depuration from Chlamys farreri in short time has not been clear. The aims of this work were to (1) investigate the efficiency of ZnSO4, EDTA–Na2, sodium citrate on Cd reduction during depuration; (2) evaluate alterations of Cd content in different tissues of Chlamys farreri and study which tissues play an important part in Cd expelling. 2. Materials and methods 2.1. Experimental animal Adult Chlamys farreri was obtained from the local aquatic market (Qingdao, China) and transported to the laboratory within 30 min for further study. The mean shell length and height of Chlamys farreri in this experiment were 6.0–7.0 cm and 7.0–8.0 cm, respectively.

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Journal Pre-proof The similar size Chlamys farreri were washed with clean seawater and brushed carefully to rub the sediment and other impurities off the shells, and then were grouped randomly. 2.2. Depuration condition Depuration experiment consisted of two aspects. First, the effect of four additives with various concentrations, namely ZnSO4, EDTA–Na2, sodium citrate, and carboxymethyl chitosan on Cd depuration was investigated. Second, three factors and three levels response surface method were used to test whether the combination of ZnSO4, EDTA–Na2, and sodium citrate could achieve a high eliminate rate of Cd. In these two aspects of depuration, different mass of additives was put into 1 L purified natural seawater to prepare different concentrations of depuration reagent. In view of toxicity of zinc ions to Chlamys farreri, the concentration of ZnSO4 were set at 0.17 g/L, 0.21 g/L, 0.28 g/L, 0.34 g/L, and 0.47 g/L, respectively. Concentration of 0.6 g/L EDTA-Na2 was served for decreasing zinc ions toxicity, and the acidity of 0.6 g/L EDTA–Na2 can prevent precipitating of zinc ions and other metal ions in seawater, which guarantee that these metal ions could be assimilated by Chlamys farreri in soluble form. To avoid harm of too high acidity to Chlamys farreri, five appropriate concentrations of EDTA–Na2, which were 0.2 g/L, 0.32 g/L, 0.4 g/L, 0.5 g/L and 0.6 g/L, respectively, were applied to study the effect of EDTA–Na2. The addition volume of sodium citrate was set at the concentration of 0.15 g/L, 0.25 g/L, 0.5 g/L, 0.75 g/L, and 1 g/L, respectively, while the concentration of carboxymethyl chitosan was 0.1 g/L, 0.15 g/L, and 0.2 g/L in different tanks, respectively. The addition of ZnSO4, EDTA–Na2, and sodium citrate in the second aspect of depuration was fixed by Design Expert 8 software according to previous optimal concentration of three additives. The seawater was purified by precipitation,

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Journal Pre-proof aeration and filtration, and its salinity was about 30‰. The water temperature was maintained between 10 and 15 °C by an air conditioner. The density of Chlamys farreri was 12–13 in a tank which was filled with 5 L seawater. Each tank was aerated for over 10 h to ensure the uniform distribution of additives in seawater. The aeration rate of an air pump during experimental period was 8 L/min, and a tank was connected with an air pump. The Chlamys farreri were not feed throughout the whole experiment. Seawater with additives was totally replaced once every 24 h. 2.3. Sampling and Cd concentration determination For assessing the efficacy of depuration, 6 Chlamys farreri were collected in seawater containing EDTA–Na2 or sodium citrate after 24, 36, 48, 60, and 72 h, respectively. 6 Chlamys farreri were randomly chosen into seawater with carboxymethyl chitosan after treatment for 24, 32, 40, and 48 h, respectively, while 6 Chlamys farreri were sampled in seawater adding ZnSO4 and EDTA–Na2 after 12, 24, 36, 48, and 60 h, respectively. The 6 samples were obtained after 12 h of depuration in the second part of depuration experiment. Soft tissues were separated from shells. The adductor muscle and other parts (OP, soft tissues except adductor muscles) were separated. The OP was heated for 15 min at 58 °C to accelerate draining water, and then stored at –20 °C after recording wet weight. Cd content of Chlamys farreri skirt was compared for judging the depuration effect of treatment as Cd concentration in adductor muscles before depuration was 0.32–0.48 mg/kg wet weight, which was below the permissible limit of China (2 mg/kg wet weight). For pointing out which tissues were involved in the Cd depuration, 6 Chlamys farreri subjected to depuration were collected, and the whole OP of each individual was drained as previously described and

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Journal Pre-proof divided into 6 parts: the muscles, kidney, gill, mantle, digestive gland, and gonad. Every thawed OP or tissue samples was mixed with HNO3 (analytical grade) and HClO4 (analytical grade) mixture in a kjeldahl flask and digested for about 2 h before diluting with deionized water. The digestion of blank samples was finished by only adding the same amount of acid into a flask. The concentration of Cd was first measured by flaming atomic absorption spectrometry (FAAS). Those samples with a lower concentration than the limit of determination of FAAS were further diluted for Cd determination by ICP–MS. 2.4. Statistical analysis SPSS 18.0 software was used for statistical analysis. All of the data were expressed as the means ± standard deviation (SD) (n ≥ 3). One-way analysis of variance (ANOVA) was performed using Origin Pro 8.0 software. 3. Results 3.1. Effect of single addition of additives on Cd depuration The results of depuration by adding four additives were shown in Table 1, Table 2, Table 3, and Table 4, respectively. Cd content determination in the OP of Chlamys farreri under ZnSO4, EDTA–Na2, and sodium citrate revealed a decrease of at least 12%, while carboxymethyl chitosan did not exert obvious influence, only reducing Cd from 11.34 mg/kg to 10.44 mg/kg. Cd elimination had a feature of short term. EDTA–Na2, ZnSO4 and sodium citrate were unable to function as a depuration agent after 36 h. For treatment groups using EDTA–Na2, the highest reduction of Cd was found in the one treated for 24 h. Cd content in the OP decreased by 17%–18% within 36 h for Chlamys farreri treated by sodium citrate, while a reduction of Cd from 13.76 mg/kg to 11.84 mg/kg was observed in treatment groups

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Journal Pre-proof after adding ZnSO4, which needed only 12 h. The most suitable concentration in single use was 0.32 g/L, 0.25 g/L, and 0.28 g/L for EDTA–Na2, sodium citrate, and ZnSO4, respectively. 3.2. Effect of combination of ZnSO4, EDTA–Na2, and sodium citrate on Cd depuration Twelve h was designed as a period for treatment of additives in the three factors and three levels response surface method (Table 5). It listed 17 trail points in the experimental design, and Cd depuration rate was defined as each response value. A regression model was given by response surface methodology. Based on the analysis of variance (ANOVA), the model was not enough to predict the response value. The model F–value of 7.26 and P–value of 0.0034 implied that the parameter of the model was significant. It demonstrated that the Lack of fit (F–value of 125.88) was significant (Table 6). Finally, Cd content reduction, from 16.61 mg/kg to 12.97 mg/kg, was found at the fifth experimental point. 3.3. Comparison of Cd content in different tissues before and after treatments Treatment ways including depuration by 0.25 g/L sodium citrate, 0.15 g/L sodium citrate, 0.28 g/L ZnSO4, and 0.42 g/L EDTA–Na2, and 0.28 g/L ZnSO4 and 0.6 g/L EDTA–Na2 were selected by considering their abilities of depurating Cd. Cd levels at various positions of the OP from Chlamys farreri during these treatments were shown in Fig. 2. Cd in digestive gland was declined from 72.56 mg/kg to 65.74 mg/kg by 0.25 g/L sodium citrate; to 66.24 mg/kg by 0.28 g/L ZnSO4, 0.42 g/L EDTA–Na2, and 0.15 g/L sodium citrate; to 66.68 mg/kg by 0.28 g/L ZnSO4, and 0.60 g/L EDTA–Na2. In the kidney, 0.25 g/L sodium citrate and 0.28 g/L ZnSO4 with 0.6 g/L EDTA–Na2 could reduce Cd content, from 44.55 mg/kg to 34.46 and 33.33 mg/kg, respectively. Cd concentration in gill in all of treatment groups were lower than the group before depuration, and the lowest was found in Chlamys farreri subjected to 0.25

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Journal Pre-proof g/L sodium citrate for 36 h. The data revealed Cd elimination in mantle in the groups treated by 0.25 g/L sodium citrate and 0.15 g/L sodium citrate, 0.28 g/L ZnSO4, and 0.42 g/L EDTA–Na2, from 3.25 mg/kg to 2.78 and 2.71 mg/kg, respectively. The results obtained from the gonad showed that Cd concentration in all of the groups was higher than that of the groups before depuration, except for the one treated with 0.25 g/L sodium citrate for 12 h, where Cd content was nearly the same as that before depuration. Besides, a gradual increase of Cd with treatment time (from 12 h to 36 h) was showed in the group depurated by 0.25 g/L sodium citrate. As for muscles, there no significant increase in all of the depuration groups, except for the group using 0.25 g/L sodium citrate for 12 h. 6 positions of the OP in the untreated Chlamys

farreri

were

ordered

according

to

the

content

of

Cd:

digestive

gland>kidney>mantle>gill>gonad>muscles, while the gonad and gill exchanged their places in the above order in the treated groups with 0.25 g/L sodium citrate for 24 h or 36 h and the group under the treatment of 0.15 g/L sodium citrate, 0.28 g/L ZnSO4 and 0.42 g/L EDTA–Na2. 4. Discussion 4.1. Effect of additives on elimination of Cd from Chlamys farreri Das and Jana (1999) transferred Lamellidens marginalis first exposed with Cd to clean water with Eichhornia, a biofilter, for 40–day elimination, and achieved significant Cd decrease in the mussel. Non-toxic carboxymethyl chitosan did not obviously speed up Cd depuration from Chlamys farreri in the present study. Unlike Eichhornia, carboxymethyl chitosan seemed not to enlarge the gap in Cd concentration between seawater and Chlamys farreri. This may indicate the distinction of long–term and short–term depuration. Another

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Journal Pre-proof concern is shellfish artificially exposed to Cd may contain more Cd which was exchangeable with water easier than those with naturally high values of Cd, which has stably been bound with various ligands and relatively difficult to release. Moreover, the high pH may be beneficial for adoption of Cd to carboxymethyl chitosan, and pH could also influence the solubility of carboxymethyl chitosan. Here, the influence of pH was not studied because 7.5~8.5 is usually considered an appropriate range in cultivation of Chlamys farreri, higher pH could be adverse. Interestingly, the other three additives which are soluble salt or smaller molecules than carboxymethyl chitosan have generated some effectiveness in depurating Cd from Chlamys farreri. Clean seawater whether natural or artificial has been already employed to clean seafood without feeding in costal catering of China. There was hardly decrease of the weight of Chlamys farreri subjected to depuration by the three additives than those without depuration. This might be an advantage of depuration of short time. Maybe not evident concentration-dependent trends in Table 1, Table 2 and Table 3. However, a combination of 0.15 g/L sodium citrate, 0.28 g/L ZnSO4, and 0.42 g/L EDTA-Na2 in seawater achieved 21.9% of Cd depuration within 12 h. This met the demand of short time in practical circulation of Chlamys farreri. The depuration experiment was conducted at different time. In our previous experiment, the effect of sodium citrate was first investigated within 12 to 96 h after Chlamys farreri entered the seawater with an interval of 12 h, and it found that Cd concentration did not show further decrease as 72 h passed, and the highest rate of depuration appeared at the 24th h. The initial 12 h might be used to adapt the seawater environment for Chlamys farreri. This is why

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Journal Pre-proof chose the 24th h as beginning point and the 72 h as end point in the formal experiments. The duration for temporary storage of living seafood in China is usually no more than 48h. Whether there is a shorter time condition by utilizing carboxymethyl chitosan and ZnSO4 than sodium citrate. Therefore, 48 h or 60 h was chosen in the experiments to test carboxymethyl chitosan and ZnSO4. Meanwhile, 12 h was studied as beginning point for ZnSO4 because the toxicity of zinc ions to Chlamys farreri should be considered before conducting experiments, though they could survive well for more than 72 h later. Therefore, 12 h was set as depuration time for the final optimized condition to meet the demand of short time, since ZnSO4 could make the highest depuration rate in 12 h. The results of single introduction of EDTA–Na2 into seawater evidenced finite ability in depurating Cd from Chlamys farreri. In contrast with 0.6 g/L EDTA–Na2 alone, 0.6 g/L EDTA–Na2 and ZnSO4 combination enhanced the depuration of Cd. This suggested that zinc, a metal biologically antagonistic to Cd, is capable to play a role in a short-term exclusion of Cd. In fact, the antagonistic relationship between these two bivalent metals has been recognized in other freshwater or marine mollusk. Cd in Chlamys farreri decreased with elevated levels of zinc and copper in sediments near oyster sampling place, and there were negative correlations between Cd and zinc concentrations in oyster bodies from various species. It was believed to be feasible for Chlamys farreri to purge Cd upon relocation to zinc–rich environment (Munksgaard et al., 2017). Tissue zinc concentration dropped during early Cd exposure, whereas tissue zinc concentration rose during Cd elimination for both L. stagnalis and Lamellidens marginalis (Présing et al., 1993; Das and Jana, 1999). ZnSO4 has been utilized as a component of the animal feedstuff in aquaculture of China, which not only serves as zinc source, but also an insecticide, and even

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Journal Pre-proof present in some oral liquid for zinc supplementation to human. ZnSO4 was added as zinc source into seawater due to its favorable solubility and low cost. Earlier in 1950s, EDTA had been employed to reduce precipitating of metal ions needed during growth of algae (Provasoli et al., 1957). EDTA also was linked with improvement of bioavailability of essential metals in Phytoplankton cultivation (Toyota and Nakashima, 1998) and decrease of metal toxicity in shellfish incubation (Gale et al., 2016). In the study, 0.2 g/L and 0.3 g/L EDTA–Na2 was prepared to mix with 0.34 g/L ZnSO4, respectively, but there was a wide range of death of the Chlamys farreri in 24 h. Equally high mortality within 24 h occurred in the group adding 0.3 g/L EDTA–Na2 as well as 0.5 g/L ZnSO4, while Chlamys farreri can survive until the fifth day under 0.3 g/L EDTA–Na2 as well as 0.17 g/L ZnSO4. These results suggested zinc toxicity and the necessity of complementing masking reagents for zinc ions. The protective effect of EDTA on aquatic animals in exposure to metal ions has also been reported (Lawrence et al., 1981). To date, the question remains: can EDTA carry zinc ion or other beneficial metals into cells of bivalves or help in absorption of these metals by bivalves? Another chelator sodium citrate is widely used for anticoagulation owing to complexing stably with Ca ions, which is obbligato in blood clotting. Furthermore, oral potassium citrate increases the level of citrate in urine, which captures calcium ions and prevents calcium oxalate saturation to further form kidney stones (Kim et al., 2019). This encouraged us to imagine that exogenous citrate could bind with metals including Cd in vivo for Chlamys farreri, although some of the chelator could be metabolized. Nevertheless, there is little evidence indicating which forms of Cd persistent in the soft tissue of Chlamys farreri. For this, it remained unclear whether sodium citrate only helped to depurate free state Cd ions.

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Journal Pre-proof 4.2. Influence of depuration on Cd distribution among tissues of Chlamys farreri The various initial concentration of Cd among tissues can be explained by differentiation in the capacity of accumulating Cd. The top two parts in Cd accumulation were the digestive gland and kidney. Similarly, Data from Jebali et al. (2014) showed that the highest level of Cd was exhibited in the digestive gland among three investigated tissues of Pinna nobilis collected under natural conditions. Additionally, it is understandable that higher accumulation of Cd was found in the digestive gland and kidney since Cd retention in gills can reveal short-term Cd exposure, while Cd level in the kidney and digestive gland may be attributed to longer Cd exposure (Jing et al., 2019). It is attempted to determine which organs were important in Cd deputation by comparison among changes of Cd content from different organs in Chlamys farreri. The whole soft body was divided into two tissues, and considered the OP as objectives used for testing Cd depuration effect of studied additives. No apparent increase in Cd concentration of muscles which possesses the lowest value of Cd content in all the tissues was found. It would appear that Cd reduced in OP involved in all the treatments was not transferred into the muscles, and these Cd was possibly discharged into ambient seawater. Moreover, Cd migration into the gonad during Cd depuration was verified by the increase of Cd there and simultaneous reduction in the OP. Notably, the increase was gradual with time goes on in the treatment by sodium citrate. 5. Conclusion ZnSO4, EDTA–Na2, and sodium citrate have been shown effect in different degree on contributing Cd elimination from Chlamys farreri on the basis of single–factor experiment. A

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Journal Pre-proof combination of three additives was applied, and this achieved a depuration percentage of over 20% within 12 h in the tissues, which has higher accumulation of Cd than that of the muscles. Further, Cd concentration test on the level of organs suggested that the tissues involving Cd purging were as follows: the digestive gland, kidney, gill, and mantle. It thought that Cd was exhausted from the whole body of Chlamys farreri based on the fact that no distinct increase of Cd occurred in the muscles, when Cd reduction was shown in the OP. Besides, it is possible that Cd was transferred into the gonad from other organs during depuration. More depuration of Cd in future may depend on the knowledge of Cd forms in Chlamys farreri. Acknowledgements This work is supported by National Key R&D Program of China (2017YFC1600702) and National Natural Science Foundation of China (31302162) References Anacleto, P., Maulvault, A. L., Nunes, M. L., Carvalho, M. L., Rosa, R., Marques, A., 2015. Effects of depuration on metal levels and health status of bivalve molluscs. Food Contr. 47, 493–501. Azelee, I. W., Ismail, R., Ali, R., Bakar, W.A., 2014. Chelation Technique for the Removal of Heavy Metals (As, Pb, Cd, and Ni) from Green Mussel, Perna viridis. Indian J. Geomarine Sci. 43 (3), 372–376. Borsagli, F. G. L. M., Borsagli, A., 2019. Chemically modified Chitosan bio-sorbents for the competitive complexation of heavy metals ions: A potential model for the treatment of wastewaters and industrial spills. J. Polym. Environ. 27 (7), 1542–1556. Chan, K. W., Cheung, R. Y. H., Leung, S. F., Wong, M. H., 1999. Depuration of metals from

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Journal Pre-proof soft tissues of oysters (Crassostrea gigas) transplanted from a contaminated site to clean sites. Environ. Poll. 105, 299–310. Das, S., Jana, B. B., 1999. Dose–dependent uptake and Eichhornia–induced elimination of cadmium in various organs of the freshwater mussel, Lamellidens marginalis (Linn.). Ecol. Eng. 12 (3–4), 207–229. El–Gamal, M. M., 2011. The effect of depuration on heavy metals, petroleum hydrocarbons, and microbial contamination levels in Paphia undulata (Bivalvia: Veneridae). Czech J. of Anim. Sci. 56(8), 345–354. Freitas, R., Ramos Pinto L., Sampaio M.., Costa, A., Silva, M., Rodrigues, A.M., Quintino, V., Figueira. E., 2012. Effects of depuration on the element concentration in bivalves: comparison between sympatric Ruditapes decussatus and Ruditapes philippinarum. Estuar. Coast Shelf Sci., 110 (2012), 43–53. Gale, S. L., Burritt, D.J., Adams, S.L., 2016. The role of ethylenediaminetetraacetic acid in green-lipped mussel (Perna canaliculus) embryo development: A biochemical and morphological characterization. Aquaculture, 463, 22. Guo, R., Pan, L., Ji, R., 2017. A multi–biomarker approach in scallop Chlamys farreri to assess the impact of contaminants in Qingdao coastal area of China. Ecotoxicol. Environ. Saf. 142, 399–409. Hiraoka, Y., 1991. Reduction of heavy metal content in Hiroshima Bay oysters (Crassostrea gigas) by purification. Environ. Pollut. 70, 209–217. Jebali, J., Chouba, L., Banni, M., Boussetta, H., 2014. Comparative study of the bioaccumulation and elimination of trace metals (Cd, Pb, Zn, Mn and Fe) in the digestive

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Journal Pre-proof gland, gills and muscle of bivalve Pinna nobilis during a field transplant experiment. J. Trace Elem. Med. Biol. 28 (2), 212–217. Jing, W.X., Lang, L., Lin, Z.G., Liu, N., Wang, L. 2019. Cadmium bioaccumulation and elimination in tissues of the freshwater mussel Anodonta woodiana. Chemosphere. 219, 321-327. Kim, D., Rimer, J. D., Asplin, J. R., 2019. Hydroxycitrate: a potential new therapy for calcium urolithiasis. Urolithiasis, 47 (4), 311-320. Lawrence, A. L., Fox, J., Castille, F. L., 1981. Decreased toxicity of copper and manganese ions to shrimp nauplii (Penaeus stylirostris Stimpson) in the presence of EDTA. J. World Aquac. Soc. 12(1): 271-280. Li, P. M., & Gao, X. L., 2014. Trace elements in major marketed marine bivalves from six northern coastal cities of China: Concentrations and risk assessment for human health. Ecotoxicol. Environ. Saf. 109, 1–9. Liu, N., Pan, L., Wang, J., Yang, H., Liu, D., 2012. Application of the biomarker responses in scallop (Chlamys farreri) to assess metals and PAHs pollution in Jiaozhou Bay, China. Mar. Environ. Res. 80, 38–45. Munksgaard, N. C., Burchert, S., Kaestli, M., Nowland, S. J., O'Connor, W., Gibb, K.S., 2017. Cadmium uptake and zinc–cadmium antagonism in Australian tropical rockoysters: potential solutions for oyster aquaculture enterprises. Mar. Pollut. Bull. 123 (1–2), 47–56. Pan, L., Miao, J., Wang, J., Liu, J., 2008. AHH activity, tissue dose and DNA damage in different tissues of the scallop Chlamys farreri exposed to benzo[a]pyrene. Environ. Pollut. 153 (1), 192–198.

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Journal Pre-proof Présing, M., V.-Balogh, K., Salánki, J., 1993. Cadmium uptake and depuration in different organs of Lymnaea stagnalis L. and the effect of cadmium on the natural zinc level. Arch. Environ. Contam. Toxicol. 24(1993), 28–34. Provasoli, L., McLaughlin, J. J. A., Droop, M. R., 1957. The development of artificial media for marine algae. Arch. Microbiol. 25 (4), 402–405. Toyota, T., & Nakashima, T., 1998. Comparison of the effects of water-soluble (EDTA) and particulate (Chelex-100) synthetic ligands on the growth of phytoplankton population in the disphotic zone seawater. J. Oceanogr. 54, 19–28.

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Journal Pre-proof Figure captions Fig. 1. Main tissues of Chlamys farreri. Fig. 2. The concentration of Cd in different tissues over depuration periods. Notes: 12SC: depuration in seawater containing 0.25g/L sodium citrate for 12h; 24SC: depuration in seawater containing 0.25g/L sodium citrate for 24h; 36SC: depuration in seawater containing 0.25g/L sodium citrate for 36h; 12SZD: depuration in seawater containing 0.15g/L sodium citrate, 0.28g/L ZnSO4, and 0.42g/L EDTA-Na2 for 12h; 12 ZD: depuration in seawater containing 0.28g/L ZnSO4 and 0.6g/L EDTA-Na2 for 12h. * represents significant differences (p<0.05) compared to the group before treatment. ** represents highly significant

differences

17

(p<0.01).

Journal Pre-proof Fig. 1

18

Journal Pre-proof Fig. 2

19

Journal Pre-proof Table 1 Cd concentration (mg/kg wet weight) in OP from Chlamys farreri treated by various concentration of EDTA–Na2. 0h

24 h

36 h

48 h

60 h

72 h

0

13.25±0.48

14.32±0.41

14.91±0.17

15.03±0.66 15.44±0.68

0.2g/L

14.17±0.36

14.26±1.38

14.83±1.37

14.63±1.28 14.99±1.40

0.32g/L

12.46±0.91 10.95±0.30* 11.76±1.52

11.86±1.11

11.75±0.80 12.20±1.16

0.4g/L

14.49±1.16

13.51±1.16

14.11±1.13

14.52±0.93 14.04±1.37

0.5g/L

12.34±1.13

12.50±1.73

14.97±1.30

14.62±1.13 14.50±2.04

0.6g/L

14.44±0.91

15.30±0.52

13.82±1.53

15.05±1.25 15.06±0.22

Notes: * represents siginificant differences (p<0.05) compared to the group before treatment.

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Journal Pre-proof Table 2 Cd concentration (mg/kg wet weight) in OP from Chlamys farreri treated by various concentration of sodium citrate. 0h 0.15g/L 0.25g/L 0.5g/L

24h

36h

13.03±0.35

14.26±2.37

48h

60h

72h

15.07±1.10 15.96±1.38 16.10±1.06

10.27±0.60** 10.18±1.74** 11.61±1.11 11.40±0.80 11.71±0.37 12.46±0.91

12.85±0.43

14.79±1.12

14.76±0.34 15.73±1.57 15.79±1.27

0.75g/L

14.10±1.32

14.44±1.87

16.32±0.22 16.24±1.92 17.02±1.22

1g/L

12.51±1.60

16.68±1.88

16.41±3.24 17.06±1.10 17.29±0.49

Notes: ** represents highly significant differences (p<0.01) compared to the group before treatment.

21

Journal Pre-proof Table 3 Cd concentration (mg/kg wet weight) in OP from Chlamys farreri treated by various concentration of ZnSO4. 0h

12 h

24 h

0.14g/L

14.6±2.06

12.81±0.91

15.38±1.95 14.57±1.23

14.33±1.89

0.21g/L

12.59±0.73

13.49±1.47

14.87±1.24 15.27±2.35

12.3±0.97

0.28g/L 13.76±0.98 11.84±0.19*

15.2±1.06*

12.54±0.84 13.88±1.92

14.09±0.98

0.34g/L

13.56±0.57

0.47g/L

14.24±1.61

36 h

48 h

60 h

11.91±0.38** 13.61±0.24 14.02±1.22 15.65±0.71** 16.97±3.09

15.26±2.24 12.78±0.79

14.87±0.82

Notes: * represents significant differences (p<0.05) compared to the group before treatment. **

represents

highly

significant

22

differences

(p<0.01).

Journal Pre-proof Table 4 Cd concentration (mg/kg wet weight) in OP from Chlamys farreri treated by various concentrations of carboxymethyl chitosan. 0h 0.1 g/L 0.15 g/L 0.2 g/L

11.34±1.04

24 h

32 h

40 h

48 h

11.20±0.29

11.76±1.3

10.62±0.28

11.59±1.67

10.44±0.26

10.71±0.92

11.37±0.50

11.71±1.69

11.86±0.50

11.19±1.15

10.47±0.76

10.66±0.71

23

Journal Pre-proof Table 5 The response values obtained from the experimental data. A: sodium citrate

B: EDTA–Na2

C: ZnSO4

depuration

Cd

（g/L）

(g/L)

(g/L)

rate(%)

concentration

1

0.35

0.22

0.28

3.13

16.09

2

0.35

0.32

0.38

9.87

14.97*

3

0.15

0.32

0.18

10.41

14.88*

4

0.15

0.22

0.28

9.39

15.05*

5

0.15

0.42

0.28

21.91

12.97**

6

0.25

0.42

0.38

14.20

14.25**

7

0.35

0.42

0.28

13.00

14.45**

8

0.35

0.32

0.18

11.49

14.7**

9

0.25

0.42

0.18

11.37

14.72*

10

0.15

0.32

0.38

14.68

14.17**

11

0.25

0.22

0.18

12.16

14.59**

12

0.25

0.32

0.28

10.65

16.61*

13

0.25

0.22

0.38

3.37

16.05

14

0.25

0.32

0.28

11.0175

14.78*

15

0.25

0.32

0.28

11.3185

14.73*

16

0.25

0.32

0.28

10.897

14.8*

17

0.25

0.32

0.28

11.1981

13.75**

Notes: * represents significant differences (p<0.05) compared to the group before treatment.

24

Journal Pre-proof ** represents highly significant differences (p<0.01).

25

Journal Pre-proof Table 6 Significance and variance analysis of regression model. Sum of Source

p-value df

Mean Square

F Value

Squares

Prob > F

Model

221.88

6

36.98

7.26

0.0034

A-A

44.67

1

44.67

8.77

0.0142

B-B

131.63

1

131.63

25.85

0.0005

C-C

1.37

1

1.37

0.27

0.6152

AB

1.75

1

1.75

0.34

0.5703

AC

8.70

1

8.70

1.71

0.2204

BC

33.75

1

33.75

6.63

0.0277

Residual

50.93

10

5.09

50.66

6

8.44

125.88

0.0002

Pure Error

0.27

4

0.067

Cor Total

272.81

16

Lack of Fit

26

Journal Pre-proof Table 7 The weight before and after Cd depuration for Chlamys farreri Before depuration of Cd

Treatment

After depuration of Cd

4.81±0.71

0.25g/L sodium citrate for 24 h

5.47±0.42

4.81±0.71

0.25g/L sodium citrate for 24 h

5.54±0.93

4.81±0.71

0.32g/L EDTA-Na2 for 24 h

4.82±0.78

0.28g/L ZnSO4 with 0.6g/L 6.74±1.10 EDTA-Na2 for 12 h

6.86±0.67

Combination of 0.15 g/L sodium 5.99±0.51

citrate, 0.28 g/L ZnSO4, and 0.42 g/L EDTA–Na2

27

6.13±0.77

Journal Pre-proof Author Chao Yang: Chlamys farreri culture, experimental data measurement, statistics, writing articles. Author Qingkang Liu: Chlamys farreri culture, part of experimental data measurement. Author Prof. Xianghong Meng: Guide experimental data measurement. Author Prof. Limin Cao: Guide experimental data statistics. Author Prof. Bingjie Liu: Comprehensive supervise experimental design, Chlamys farreri culture, instrument use, experimental data statistics, paper revision and submission

Journal Pre-proof

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Journal Pre-proof Highlights  Effect of ZnSO4, EDTA-Na2, sodium citrate on Cd elimination was evaluated.  Over 20% Cd was depurated by ZnSO4, EDTA-Na2 and sodium citrate in short time.  The digestive gland, kidney, gill, and mantle were responsible for Cd reduction.  Cd was migrated to the gonad from other tissues during depuration.