The present study aimed to study the effects of salinity (12, 16, 20, 24, and 28) and algal diets (Platymonas subcordiformis, Chlorella sp. and a mixture of P. subcordiformis and Chlorella sp.) on the elimination of cadmium (Cd) from oysters (Crassostrea rivularis). The Cd accumulation in oysters was also studied. The results showed that the accumulation of Cd in C. rivularis was related to the exposure time (P<0.01). The elimination rate of Cd from C. rivularis increased with increasing salinity. The highest Cd elimination rate of 0.0183 μg/(g·d) was found in the highest salinity group, and 47.6% of the Cd in oysters was removed after 31 days. Compared to oysters fed the single diet, the oysters fed the mixed algal diet exhibited the highest elimination rate of 0.0166 μg/(g·d), and 43.8% of the Cd in oysters was removed after 31 days.
Keywords: Cadmium; Elimination; Crassostrea rivularis; Algal Diets; Salinity
Oysters are a popular food, and oyster cultures are economically important in the coastal areas of many countries. Metal pollution has been occurring in many developed and developing countries for decades. Oysters are disposed to accumulate metals [1,2], especially cadmium [3-5]. The accumulation of toxic trace elements by shellfish is a worldwide problem that has been discussed for at least the last century [6,7]. The consumption of heavy metal-contaminated oysters may result in chronic and acute toxicity in humans . The consumption of oysters is limited to 5 μg Cd/g dry weight in Europe . In China, the safety standard of Cd in shellfish is less than 4.0 mg Cd/kg wet weight (NY5154-2008, 2008) . To minimize health risks, one of the acceptable methods for purifying contaminated oysters suggested by the National Shellfish Sanitation Program (NSSP) in the USA  is transferring the shellfish into a clean environment to remove heavy metals from the shellfish prior to harvest and consumption. However, previous studies on heavy metals eliminated from oysters focused on natural biological cleansing, which transplanted organisms from contaminated areas to conditionally approved waters [12-15]. However, the method has shortcomings, such as longer depuration periods (lasting several months) and a higher oyster mortality rate , which is not wholly appropriate for the industrialized depuration of heavy metals from oysters. Moreover, at present, the depuration of shellfish is mainly targeted toward the pathogenic microorganism [16,17]. No effective faster methods for metal depuration from shellfish have been reported to date. Saed et al.  reported that the depuration of metals under laboratory conditions is significantly faster than that achieved in the field. There have been many reports on salinity affecting the communication of metals in shellfish [19-24], but fewer studies have investigated the effects of the elimination of heavy metals, particularly on the effects of algal diets on the elimination of metals.
Reducing or purifying the residual metals in shellfish is an advisable method to make the shellfish achieve the seafood safety standards. Crassostrea rivularis was first described by Gould  based on a specimen collected from southern China and subsequently used to describe oysters in Japan and China . As a favourite and widely distributed seafood product, C. rivularis is one of the most important oyster species cultured in China and has been cultured for centuries [27, 28]. C. rivularis is mostly found in low-salinity rivers and estuaries. C. rivularis can accumulate metals and has been used as an indicator of marine environmental pollution for decades [21,22,29,30]. In recent years, the pollution from urban, industrial and agricultural sources into the estuarine waters of the Zhujiang (Pearl) River in southern China has been increasing. Water areas containing shellfish habitats have been restricted for production and harvesting. Of the metals found in polluted shellfish, Cd is one of the most toxic metals to humans . In fact, 81.8% of the shellfish from the Zhuhai aquatic product during the fourth quarter of 1996 to the third quarter of 1997 exceeded the seafood safety standards of Cd in shellfish . What is more, average value of Cd concentrations in C. rivularis collected from Shantou (2002-2003) and Guangdong (2007) are around 10 mg kg-1 day weight . Research on metal purification from shellfish is significant. The aim of the present study is to discuss the effects of salinity and algal diets on Cd elimination from C. rivularis in indoor conditions and to provide a theoretical basis and assessment for the industrialized depuration of heavy metals from oysters when the Cd content exceeds the national seafood safety standards.
C. rivularis (aged 2 years, shell length of 15.6±1.56 cm and weight of 221.6±25.8 g) were collected from Daya Bay culture areas, Guangdong, China (Figure 1), where the yield, quality, and culture areas of C. rivularis have been adversely affected by the growing metal pollution. The oysters were selected, cleaned carefully and then maintained in sand-filtered seawater (PY16293, Jiuding Hi-tech Filtering Equipment, Ltd., Beijing) without food and under natural lighting at room temperature (water temperature 17-24°C, salinity 34, pH 7.8-7.9) and were supplied dissolved oxygen by the continuous pumping of air a week prior to the experiment.
Figure1. Sampling site of C. rivularis.
(Note: the triangle is the sampling site in the Daya Bay C. rivularis culture areas)
Clean, pollution-free seawater attained the quality standards for Grade I seawater  after it was filtered through sand and precipitated. The seawater was diluted with distilled water and continuously pumped with air for 48 h before use.
Accumulation of Cd in C. rivularis
To obtain a consistently higher Cd concentration in oysters for the elimination experiment, C. rivularis was cultured in Cd-polluted seawater. Seawater and seawater with dissolved CdSO4·8H2O (AR, purity of 99%, Sinopharm Chemical Reagent, Shanghai, China) with a Cd concentration of 25 μg/L were prepared following the seawater quality standards for marine aquaculture  and the protocols described by Lu et al.. Four hundred C. rivularis were placed in a cement pool (length of 180 cm, width of 100 cm and height of 80 cm) containing 25 μg/L Cd seawater (salinity 20, pH 7.8-7.9) at a room temperature of 22.2-28.1°C and allowed to accumulate Cd for at least 11 days. Five sampling times were defined: 0, 2, 4, 7, and 11 days. At each sampling point, eight oysters were removed randomly for metal analysis from both the experimental and control groups. The seawater was replaced, and any dead C. rivularis were removed daily. The Cd-polluted seawater was replaced with clean seawater from the control groups. No food was provided to any of the groups.
Elimination of Cd from C. rivularis
After the accumulation experiment, the remaining live C. rivularis were divided into nine groups. Five groups (G12, G16, G20, G24, and G28) and four groups (GP, GC, GPC and Gct) were used to understand the effect of salinity and algal diets on the elimination of Cd from C. rivularis. The oysters were cultured in a polyethylene barrel containing 120 L of clean seawater under natural illumination at a temperature of 22.5-28°C with a pH of 7.8-7.9 to eliminate metal pollution over a period of 31 days. Each barrel contained 45 oysters.
Effect of salinity on the elimination of Cd from C. rivularis
Five different salinities (12, 16, 20, 24, and 28) were chosen to assess the impact of salinity on the elimination of Cd from oysters. During the experiment, the oysters were fed Platymonas subcordiformis (cell length of 11-14 μm, width of 7-9 μm and height of 3-5 μm), and the cell density was maintained at 2×104 cells/mL. Seven sampling times were defined: 0, 2, 5, 10, 15, 23, and 31 days. At each sampling point, six oysters were randomly collected from each group, and the Cd contents in the soft parts were determined.
Effect of algal diets on the elimination of Cd in C. rivularis
The oysters were fed three types of algal diets. Group GP was fed P. subcordiformis (2×104 cells/mL). Group GC was fed Chlorella sp. (2×105 cells/mL, cell length 7-13 μm, width 6-8 μm, height 8-10 μm). Group GPC was fed P. subcordiformis (1×104 cells/mL) and Chlorella sp. (1×105 cells/mL). The control group (Gct) received no food. The oysters were fed three times daily to maintain the cell density. At specific intervals (0, 2, 5, 10, 15, 23, and 31 days), six oysters were randomly sampled from each group for Cd analysis. The seawater was diluted to a salinity of 20 for all of the groups.
The whole soft tissues of oysters were washed with distilled water, removed from their shells, dried with filter paper, weighed and homogenized. We weighed the 0.30-g homogenized samples in quadruplicate for each group, placed the samples and 5 mL of analytical-grade nitric acid into a highpressure digestion jar, and allowed the contents to predigest overnight at room temperature for 12 hours. The samples were then digested in a microwave digester (MARS-5 system, CEM, USA), the digestates were diluted with up to 50 mL of deionized water, and the Cd concentrations in the samples were measured using an atomic absorption spectrophotometer (Optima 2000 DV). The standard reference material for Cd was provided by the National Standard Material Centre, Beijing, China.
Accumulation of Cd in C. rivularis
Exposure to Cd-polluted seawater resulted in the continuous accumulation of Cd in oysters, which followed a net accumulation model (Figure 2). A linear regression analysis demonstrated that the Cd accumulation in C. rivularis was positively correlated with the exposure time (P<0.01, y = 0.924x + 3.387, R2 = 0.984, where y is the Cd concentration in the oyster soft tissues [μg/g] and x is the exposure time [day]). The accumulation rate of Cd in C. rivularis was 0.92 μg/(g∙d) during 11 days of exposure to the Cd-polluted seawater with a Cd concentration of 25 μg/L.
Figure 2. Accumulation of Cd (25 μg/L) in C. rivularis at different exposure times.
Effects of salinity on the elimination of Cd from C. rivularis
The Cd concentration in oysters decreased over time in all of the salinity groups (Figure 3), but the Cd elimination rates varied between the different salinity groups (Figure 4). The regression analysis indicated that the relationship between Cd in C. rivularis and the depuration time could be described as follows:
Ct12= 12.188e-0.0098t, R2 = 0.801 (P< 0.01);
Ct16= 11.842e-0.0124t, R2 = 0.803 (P< 0.01);
Ct20= 11.606e-0.0144t, R2 = 0.824 (P< 0.01);
Ct24 = 11.339e-0.0163t, R2 = 0.826 (P< 0.01);
Ct28 = 11.224e-0.0183t, R2 = 0.839 (P< 0.01);
where Ctx is the Cd in the soft tissues of C. rivularis at salinity x,and t is the depuration time (day).
The Cd elimination increased with increasing salinity. Group G28 with a salinity of 28 exhibited the highest elimination rate (0.0183 μg/(g∙d)), and 47.6% of the Cd was removed after 31 days of depuration. In contrast, group G12 with a salinity of 12 had the lowest depuration rate (0.0098 μg/(g∙d)), and only 27.9% of the Cd was removed after 31 days of depuration (Figure 4).
A two-factor variance analysis of the five elimination groups showed that the salinity and depuration time had a notable effect on the elimination of Cd from C. rivularis (P<0.01), but there was no interaction between the salinity and depuration time (P>0.05). A repetitive-measure analysis of variance demonstrated that group G28 (P<0.01) was significantly different from groups G12, G16, and G20, and a similar finding was found for group G24 (P<0.05). In addition, group G12 was significantly different from groups G16 and G20 (P<0.01), and there was also significant differences between groups G16 and G20 (P<0.05) (Table 1).
Figure. 3. Effect of salinity on the elimination of Cd from C.rivularis
Figure 4. Effect of salinity on the elimination rate of Cd from C.rivularis.
Table 1. P value of the repetitive-measure analysis of variance between groups with different salinities.
Effects of algal diets on the elimination of Cd from C. rivularis
After the oysters were transferred into clean seawater with a salinity of 20 and different algal diets, Cd was eliminated at a low rate (Figure 5). Interestingly, different algal diets resulted in different rates of Cd removal. The relationship between the Cd in C. rivularis and the depuration time can be described as follows:
Group GP:Ct P= 11.606e-0.0144t, R2 = 0.824 (P<0.01);
Group GC:Ct C= 11.639e-0.0128t, R2 = 0.789 (P<0.01);
Group GPC:Ct PC= 11.597e-0.0166t, R2= 0.875 (P<0.01);
Group Gct:Ct ct= 11.835e-0.0112t, R2= 0.814 (P<0.01).
The decreasing order of the Cd elimination rates were GPC, GP, GC, and Gct (Figure 6). The highest elimination rate (group GPC) was 0.0166 μg/(g∙d), and 43.8% of Cd was removed after 31 days. In contrast, the lowest elimination rate (group Gct) was 0.0112 μg/(g∙d), and only 32.6% of the Cd was removed after 31 days. The elimination rates of groups GP and GC were 0.0144 and 0.0128 μg/(g∙d), respectively (Figure 5).
A two-factor analysis of variance of the four depuration groups revealed that the algal diets and depuration time had a significant effect on the elimination of Cd from C. rivularis (P<0.01), but no interaction was found between the algal diets and the depuration time (P>0.05). Group GP was significantly different than group Gct (P<0.05) but showed no significant differences with groups GC and GPC (P>0.05). In contrast, group GC was significantly different from group GPC (P<0.05) but not from group Gct (P>0.05) (Table 2).
Figure 5. Effect of algal food on the elimination of Cd from C. rivularis.
Figure 6. Effect of algal diet on the elimination rate of Cd from C. rivularis.
Table 2. P value of repetitive-measure analysis of variance between groups with different algal diets.
After the oysters were exposed to seawater containing 25 μg/L Cd for 11 days, the accumulation rate of Cd in C. rivularis was 0.92 μg/(g∙d). This value is much higher than the subsequent depuration rate, which is 50-fold faster than the highest depuration rate. This finding is also suggested by the works of other researchers. George and Coombs  found that the excretion rate of Cd by Mytilus edulis was 18-fold slower than the accumulation rate. Arini et al.  reported that after a 24 day exposure in Cd-contaminated water, only 73% Cd is eliminated from Corbicula fluminea after one year and Cd half-life in the bivalve is estimated around 240 days. The initial Cd concentration used in our study was higher than that used by Denton and Burdon-Jones and Lu et al. [21, 38]. The higher the level of contamination, the more important the Cd binding to cytosolic ligands becomes, and the faster the elimination of cytosolic metals than the insoluble fraction .
The elimination kinetics was influenced by environmental factors, such as the salinity, humic acid concentrations, temperature, and feeding rate [39, 40]. Therefore, it is valuable to study the effects of environmental factors on oyster purification and to find a better condition to allow the polluted oysters to eliminate metals faster and more efficiently. Our results suggested that the salinity affected the Cd elimination in C. rivularis, and the rate increased as the seawater salinity increased, which is consistent with the results reported by Van Dolah et al. . However, the difference between groups G24 and G28 was insignificant (P>0.05) (Table 1). It is possible that the higher salinities do not have an obvious effect on the elimination of Cd in C. rivularis. The effect of the salinity on the elimination of metal from molluscs appears to differ among species investigations. It appears not to have an effect in the Black-lip oyster Saccostrea echinata . There are several methods for the elimination of Cd from aquatic animals, such as defecation, egestion, detoxification, and excretion. The excretion of Cd from different aquatic animals is different [41-43]. However, even from the same species under different conditions, the effects of salinity are different. Lu et al.  demonstrated that the elimination of Cd from C. rivularis decreased with increasing salinity. Different environmental conditions are more likely to cause the observed discrepancy. Studies have reported that the residual cadmium in oysters is dependent on a complex interplay of both abiotic (such as salinity and temperature) and biotic factors (such as filter-feeding rates and diet selection) and that the level is determined by the dominant factor at a particular time or in a particular region [3,4].
Wright et al.  suggested that the tissue metal concentrations can be highly dependent on both environmental conditions and the physiological status of individuals. Food is another factor. Ruelas-Inzunza and Páez-Osuna  listed five potential causes for fluctuations in the metal concentrations in seashells, one of which is the food supply. Christie et al.  also suggested that the diet is an important contributor to the oyster tissue cadmium concentrations. In our previous studies and in the studies conducted by Van Dolah et al. , oysters were fed P. subcordiformis and Isochrysis galbana during the depuration experiment instead of no food, as in the studies conducted by Denton and Burdon-Jones and Lu et al.[21,38].
Our results suggested that the elimination of Cd from oysters is related to food. The Cd elimination rates in the groups administered food supplies were all faster than that of the control group with no food supply (Figure 5). However, the mechanisms underlying diet-facilitating metal elimination have not yet been determined. Oysters require energy for different tasks, such as: distribution of metal in tissues, turnover of soluble and insoluble metals, synthesis and metabolism of proteins that bind or metabolize heavy metals. Microalgae, such as P. subcordiformis and Chlorella sp., are easily ingested and assimilated and have high dietary value. It is likely that an algal diet can supply energy and nutrition to allow the oyster to synthesize the enzymes and proteins that function in metal removal.
Interestingly, different algal diets have different effects on Cd elimination from C. rivularis. The oysters fed the mixed algal diet were significantly different from the groups with no food supply (Table 2). Oysters fed the mixed algal diet exhibited the highest elimination rates, followed by the P. subcordiformis group, and the Chlorella sp. group exhibited the lowest rate
(Figure 6). This may be caused by differences in the energy and nutrients supplied by the three diets. Studies have revealed that P. subcordiformis is a better food than Chlorella sp.. P. subcordiformis has been used in the commercial breeding of aquatic organisms, including Mercenaria mercenaria , Acartia pacifica , and Apocyclops borneoensis . It has also been reported that a mixed algal diet is better than a single algal diet. Zhang et al.  suggested that the growth and survival of young M. mercenaria fed mixed algae were better than that obtained with the single algal diet. Epifanio  also found that the breeding of M. mercenaria fed a mixture of Isochrysis galbana and Platymonas suecica was better than that obtained with P. suecica alone. Huang and Huang  achieved similar results using P. subcordiformis, Chlorella sp. and mixed algae as food. A mixture of two appropriate algae can supply an adequate mix of nutrients to promote the growth and survival of marine organisms . Thus, the better growth and survival could result in the oysters purifying metal more effectively. The results may serve to optimize the removal of heavy metal from commercially important species. This, in turn, has important consequences for food safety production in Chinese coastal waters.
There was a positive correlation between the Cd accumulation in C. rivularis and the exposure time. Cd elimination from C. rivularis began when they were moved from Cd-polluted seawater into clean seawater.
The Cd elimination rates from C. rivularis increased as the salinity increased.
The algal diets had a notable effect on the elimination rate of Cd from C. rivularis. Compared with single algal food, mixed algal food (P. subcordiformis and Chlorella sp.) was more helpful for C. rivularis to eliminate Cd.
These results may serve to optimize the removal of Cd from a commercially important species. It also has important consequences for the safe production of food from Chinese coastal waters.
This work was supported by the National Nature Science Foundation of China (No. 41276091), the Technology Research Program of Guangzhou City (2014J4100010, 201508020100), the Technology Research Program of Guangdong Province (2013B010404027), and the Science and Technology for Key Projects of Guangdong Province (No. 2003C20411).