3.1 Distribution of heavy metals
The concentrations of each trace metal in five species of bivalve were shown in Table 4. There was a significant difference in heavy metal accumulation in different species of bivalves.
For Cu, the highest level was in C. gigas which ranged from 1.01 to 70.2 mg·kg-1 and the average level was 37.3 mg·kg-1 which was much higher than other four kinds of bivalves (p<0.05). The second was for C. farreri with an average level of 3.64 mg·kg-1. and there was no significant difference in Cu concentrations among C. farreri, R. philippinarum, M. veneriformis and C. sinensism (Fig. 2).
For Zn, it was similar with Cu that C. gigas had the highest level with an average value of 110 mg·kg-1, which was two times more than that in C. farreri (51 mg·kg-1) and one-way ANOVA analysis showed that there was a significant difference between them (p<0.05). The third was for R. philippinarum and C. sinensism, and a lowest average content of 6.4 mg·kg-1 was detected in M. veneriformis. However, no significant difference was found for R. philippinarum, C. sinensism and M. veneriformis (seeing Fig.2).
For Fe, there was a significant difference among five kinds of bivalves (Fig.2). Among them, the highest value of 587.4 mg·kg-1was detected in M. veneriformis which was about three times more than the second level in R. philippinarum (187.1 mg·kg-1) (P<0.05). The lowest Fe content was found in the soft tissues of C. sinensism with 65.5 mg·kg-1, and there was no significant difference among C. farreri, C. gigas and C. sinensism (P>0.05).
Table 4 Trace metal concentrations in the soft tissues of different bivalves (mg·kg-1, wet weight)
Bivalves
|
C. farreri
|
R.philippinarum
|
M.veneriformis
|
C.gigas
|
C. sinensism
|
Cu
|
Range
|
0.86-11.7
|
0.55-3.09
|
0.55-2.26
|
1.01-70.2
|
0.80-2.95
|
Average
|
3.64
|
1.26
|
1.32
|
37.3
|
1.38
|
Zn
|
Range
|
18.0-137
|
7.06-20.4
|
3.34-11.6
|
12.9-201
|
7.78-16.5
|
Average
|
51
|
12.2
|
6.4
|
110
|
12.1
|
Fe
|
Range
|
35.5-319.5
|
56.7-376.5
|
195.7-1083.6
|
17.7-174.1
|
37.8-122.2
|
Average
|
99.4
|
187.1
|
587.4
|
74.9
|
65.5
|
Mn
|
Range
|
2.54-15.0
|
1.19-14.4
|
5.62-53.8
|
3.78-15.8
|
3.45-6.82
|
Average
|
6.52
|
6.88
|
20.3
|
8.11
|
5.01
|
Total As
|
Range
|
1.49-8.41
|
1.29-5.22
|
1.21-4.77
|
1.33-4.28
|
1.40-3.35
|
Average
|
3.91
|
3.23
|
2.71
|
2.41
|
2.35
|
iAs
|
Range
|
0.04-0.14
|
0.05-0.36
|
0.04-0.21
|
0.02-0.12
|
0.04-0.11
|
Average
|
0.08
|
0.15
|
0.12
|
0.05
|
0.07
|
Cr
|
Range
|
0.11-0.94
|
0.07-4.78
|
0.45-3.19
|
0.06-0.89
|
0.12-1.26
|
Average
|
0.36
|
0.55
|
1.47
|
0.4
|
0.33
|
Cd
|
Range
|
0.13-3.67
|
0.02-0.24
|
0.05-0.24
|
0.11-1.45
|
0.02-0.10
|
Average
|
1.15
|
0.14
|
0.13
|
0.76
|
0.030
|
Pb
|
Range
|
0.06-0.51
|
0.08-1.10
|
0.10-1.19
|
0.05-0.34
|
0.05-0.17
|
Average
|
0.19
|
0.19
|
0.49
|
0.16
|
0.09
|
Hg
|
Range
|
<0.01-0.05
|
<0.01-0.05
|
<0.01-0.06
|
<0.01-0.07
|
<0.01-0.05
|
Average
|
0.020
|
0.022
|
0.020
|
0.024
|
0.020
|
For Mn, the highest content was also found in the soft tissues of M. veneriformis with an average of 20.3 mg·kg-1, and it was significantly higher than other four kinds of bivalves (Fig.2). About 8.11 mg·kg-1 of Mn was found in C. gigas, and a similar concentration was wound in C. farreri and R. philippinarum (6.52 and 6.88 mg·kg-1, respectively). The lowest Mn content was also found in C. sinensism, which was significantly lower than both M. veneriformis and C. gigas (P<0.05)
For total arsenic (As) and inorganic As, a different distribution character was found for different bivalves (Fig. 3). Total As in C. farrier ranged from 1.49 to 8.41 mg·kg-1 and the average level was 3.91 mg·kg-1 which was significantly higher than that in R. philippinarum (3.23 mg·kg-1), and a similar value of total As was found in M. veneriformis, C. gigas and C. sinensism. While for inorganic As, the highest level was found in R. philippinarum and M. veneriformis with an average level of 0.15 and 0.12 mg·kg-1 respectively. And only 0.08 mg·kg-1 iAs was found in C. farreri. Present result further proved that the transformation of different arsenic species in different bivalves was different.
For Cd, an obvious difference was found for five different bivalves (seeing Fig. 3). For example, C. farreri had the highest Cd level which ranged from 0.13 to 3.67 mg·kg-1, and the average content was 1.15 mg·kg-1. The second was for C. gigas, which ranged from 0.11 to 1.45 mg·kg-1, and the average value was 0.76 mg·kg-1. The third level was found in R. philippinarum and M. veneriformis, both of them had a similar value (0.14 and 0.13 mg·kg-1, respectively). The lowest value was for C. sinensism, which had only about 0.030 mg·kg-1 of Cd.
For Cr, M. veneriformis had the highest content with average level of 1.47 mg·kg-1, and the lowest level was found in C. farreri and C. sinensism (0.36 and 0.33 mg·kg-1, respectively). And for Pb, a similar phenomenon was found that the highest level was in M. veneriformis (0.49 mg·kg-1), and the lowest level was also found in C. sinensism. For Hg, it was different from other trace metals that there was no obvious difference for five kinds of bivalves (P>0.05), and its’ value ranged from 0.020 mg·kg-1 to 0.024 mg·kg-1.
Thus, the clam M. veneriformis had the highest content of Fe, Mn, Cr and Pb among five kinds of bivalves. The oyster C.gigas had the highest content of Cu and Zn, and the scallop C. farreri had the highest content of Cd and total arsenic. The clam M. veneriformis and R. philippinarum had a slightly higher content of inorganic arsenic (iAs) than other three bivalves. In addition, the concentration of all heavy metals in present study in C. sinensis was the lowest among five kinds of bivalves.
Compared to previous studies of heavy metals in bivalves from coastal areas, the present results of most metals are similar or within the range of reported studies. The differences of trace elements in bivalves from different regions are mainly reflected in the content of Fe, As, Cr, Cd and Hg. For example, the present result of As concentration in C. farreri was below the result from the Southern New Caledonian (Metian et al., 2008), and As concentration in C.gigas reported by Liu et al (2020) was nearly ten times of the present study. The levels of Hg in R. philippinarum in this study were obviously lower than those from the Atlantic Coast, Southern Spain (Usero et al., 1997) and Venice lagoon, Italy (Sfriso et al., 2008). The concentration of Fe and Hg in C.gigas from Atlantic Coast was several times of the present study (Suami et al., 2019). As have been reported by previous researches that there is a complicated process about heavy metals accumulation in aquatic organisms, which can be affected by species, age, physiological stage, and environmental parameters (bioavailability, salinity, and temperature of living environment) (Kang et al., 2018; Maurya et al., 2019).
3.2 Pollution levels of heavy metals
Table 5 gives the single factor pollution index (Pi) of heavy metal in five bivalves calculated by formula (1). For C. farreri, Zn and Cd were slightly contaminated (Pi value was 0.340 and 0.576 respectively). For R. philippinarum, iAs and Cr were slightly contaminated, and the Pi values were very low between 0.2 and 0.3. For M. veneriformis, Cr was moderately contaminated (Pi, 0.736), iAs and Pb were slightly contaminated. The Pi of Cr was highest which meant that Cr may be more easily accumulated by M. veneriformis. For C. gigas, the Pi of Cr and Cd was 0.200 and 0.328 respectively, and they were slightly contaminated. In C. gigas, Cu and Zn were moderately contaminated (Pi values were 0.746 and 0.734 respectively), and in some samples, Pi was much higher than 1.0. The C. sinensis was the most safety bivalves with all elements in normal background levels.
Present study proved that Cu and Zn in C. gigas were moderately contaminated, which was similar with the previous result reported by (Qin et al., 2010). Although Cu is an essential element for various metabolic activities, long-time over tolerable level exposure could cause anaemia, kidney and liver damage (Schumann et al., 2002). As an essential element, Zn is the cofactor of various enzymes and plays a vital role in metabolic activity (Suami et al., 2019). However, high level exposure of Zn can also lead to nausea, abdominal pain and lethargy (Denil et al., 2017). In addition, the average Pi values of Cd in C. farreri and Cr in M. veneriformis were both below 1.0, while in some samples, the Pi values were over 1.0. And it showed that some of C. farreri and M. veneriformis were heavily contaminated by Cd. Table 6 showed the Nemerow index (Pc) of heavy metals in five species of bivalves. And it could be found that the Pc of Cu and Zn in C. sinensis, Cd in C. farreri and Cr in M. veneriformis were all slightly contaminated with Pc>1.0. Thus, both Pi and Pc results proved that there was some contamination in present C. sinensis, C. farreri and M. veneriformis samples. Although all these heavy metals levels are below the maximum allowable limit regulated by China Food Standard Agency (GB 2762-2017, 2017), they should attract much attention when considering the health risk of the bivalves consuming.
Table 5 Pollution index (Pi) of heavy metals in bivalves
Bivalves
|
C. farreri
|
R. philippinarum
|
M. veneriformis
|
C. gigas
|
C. sinensis
|
average
|
max
|
average
|
max
|
average
|
max
|
average
|
max
|
average
|
max
|
Fe
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
Mn
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
Cu
|
0.073
|
0.233
|
0.025
|
0.062
|
0.026
|
0.045
|
0.746
|
1.404
|
0.028
|
0.059
|
Zn
|
0.340
|
0.914
|
0.082
|
0.136
|
0.043
|
0.078
|
0.734
|
1.340
|
0.081
|
0.110
|
iAs
|
0.161
|
0.283
|
0.299
|
0.607
|
0.244
|
0.417
|
0.098
|
0.247
|
0.150
|
0.213
|
Cr
|
0.181
|
0.470
|
0.275
|
0.762
|
0.736
|
1.596
|
0.200
|
0.443
|
0.166
|
0.630
|
Cd
|
0.576
|
1.834
|
0.072
|
0.121
|
0.066
|
0.119
|
0.380
|
0.725
|
0.014
|
0.048
|
Pb
|
0.128
|
0.34
|
0.126
|
0.450
|
0.328
|
0.791
|
0.107
|
0.229
|
0.057
|
0.115
|
Hg
|
0.040
|
0.094
|
0.045
|
0.102
|
0.040
|
0.116
|
0.047
|
0.138
|
0.041
|
0.105
|
Table 6 Nemerow index (Pc) of heavy metals in bivalves
Bivalves
|
C. farreri
|
R. philippinarum
|
M. veneriformis
|
C. gigas
|
C. sinensis
|
Fe
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
Mn
|
n,a
|
n,a
|
n,a
|
n,a
|
n,a
|
Cu
|
0.17
|
0.05
|
0.04
|
1.12
|
0.05
|
Zn
|
0.69
|
0.11
|
0.06
|
1.08
|
0.10
|
iAs
|
0.23
|
0.48
|
0.34
|
0.19
|
0.18
|
Cr
|
0.36
|
0.57
|
1.24
|
0.34
|
0.46
|
Cd
|
1.36
|
0.10
|
0.10
|
0.58
|
0.04
|
Pb
|
0.26
|
0.33
|
0.61
|
0.18
|
0.09
|
Hg
|
0.07
|
0.08
|
0.09
|
0.10
|
0.08
|
3.3 Assessment of the health risks of heavy metals
3.3.1 Estimated daily intake (EDI)
The EDI of heavy metals here represented the daily intake of heavy metals through the consumption of selected bivalves, which was listed in Table 7. The PTDI values proposed by JECFA (FAO/WHO, 1985; WHO, 1989; Mok et al., 2015) and the Chinese Nutrition Society (2013)were shown in Table 7 as the reference value to evaluate the hazard risk from these bivalves consumption. From Table 7, it can be calculated that the average EDI/PTDI of the selected metals for these bivalves ranged from 1% to 92%. The average EDI/PTIDI of Cd in C. farreri reached to 92%, which meant that Cd was the most hazard element for C. farreri consumption. Although the average EDI value were all below the PTDI, the coastal residents exposed higher dose, may have been exposed under excess dose of Cd from the regular consumption of C. farreri. For R. philippinarum, the average EDI of Cr occupied 13% of the PTDI. The average EDI of Cu and Cd in C. gigas occupied 61% and 62% of the PTDI, respectively. This result was similar to the previous research (Liu et al., 2019), which proved that there was potential health risk for people to be exposed to Cu and Cd from mass consumption of C. gigas, and Cd from C. farreri consumption. For M. veneriformis, the average EDI of Fe occupied 48.9% of the PTDI and this value was highest in present studied bivalves. Since Fe is the essential element for human, M. veneriformis can be considered as the Fe supplement food for iron deficiency group.
Table 7 The estimated daily intake (EDI) of heavy metals in bivalves (μg·kg-1·bw·d-1, wet weight)
Bivalves
|
EDI
|
PTDI
|
C. farreri
|
R. philippinarum
|
M. veneriformis
|
C. gigas
|
C. sinensis
|
AVG
|
EDI/PTDI
|
AVG
|
EDI/PTDI
|
AVG
|
EDI/PTDI
|
AVG
|
EDI/PTDI
|
AVG
|
EDI/PTDI
|
|
Fea
|
66.3
|
8.3
|
125
|
15.6
|
391
|
48.9
|
50
|
6.3
|
43.7
|
5.5
|
800
|
Mnb
|
4.35
|
2.4
|
4.59
|
2.5
|
13.5
|
7.4
|
5.41
|
3.0
|
3.35
|
1.8
|
183
|
Cud
|
2.43
|
6.1
|
0.84
|
2.1
|
0.88
|
2.2
|
24.87
|
62.2
|
0.92
|
2.3
|
40
|
Znd
|
34.0
|
11.3
|
8.13
|
2.7
|
4.267
|
1.4
|
73.33
|
24.4
|
8.067
|
2.7
|
300
|
iAsc
|
0.053
|
2.5
|
0.129
|
6.0
|
0.081
|
3.8
|
0.033
|
1.5
|
0.053
|
2.5
|
2.14
|
Crd
|
0.241
|
8.0
|
0.396
|
13.2
|
0.980
|
32.7
|
0.265
|
8.8
|
0.221
|
7.4
|
3.00
|
Cdd
|
0.767
|
92.4
|
0.096
|
11.6
|
0.089
|
10.7
|
0.507
|
61.1
|
0.019
|
2.3
|
0.83
|
Pbd
|
0.128
|
3.6
|
0.127
|
3.6
|
0.328
|
9.2
|
0.107
|
3.0
|
0.057
|
1.6
|
3.57
|
Hgd
|
0.014
|
2.5
|
0.015
|
2.6
|
0.013
|
2.3
|
0.016
|
2.8
|
0.014
|
2.5
|
0.57
|
a PTDI values were summarized by FAO/WHO (1985).
b PTDI values were summarized by Chinese Nutrition Society (2013).
c PTDI values were summarized by WHO (1989).
d PTDI values of other elements were all summarized by Mok et al. (2015).
3.3.2 Target hazard quotient
THQ is a method to estimate non-carcinogenic health hazard. The THQ value of heavy metal in present studied bivalves was calculated according to Formula (2). The THQ was integrated risk index, and has been used widely for risk assessment of various contaminants (Mok et al., 2015). The THQ values for individual metals from the selected bivalves consumption are shown in Table 8. The average THQ values of all metals in all samples were all below 1.0, while the THQ values of Cd in some C. farreri samples were exceed 1.0, and the highest value was to 2.366, which meant that there was potential health risk with long-term consumption exposure to these C. farreri. Some researchers found that there are some other organic Cd forms in C. farreri and R. philippinarum which have less toxicity than inorganic one (Choi et al., 2007; Zhao et al., 2012). So the toxicity of Cd here may be overestimated, and the harmful effect still should be paid more attention. For R. philippinarum, all the average THQ values were below 0.5, the THQ values of Cr in some samples exceeded 1.0, which indicates that there is a factor for health concern. The THQ value of iAs in R. philippinarum was the highest among the five bivalves. And it reveals that iAs was more easily accumulated by R. philippinarum. For M. veneriformis, the top three metals were Fe, Cr and iAs. The max THQ value of Fe was 0.907. For C. gigas, the top three metals was Cd, iAs and Zn, which was similar with the previous research (Denil et al., 2017). The max THQ values of Fe in M. veneriformis and Cd in C. gigas were both more than 0.9 and close to 1.0, which might pose health risk. The THQ values of Zn and iAs in C. gigas were also in accord with the results in Taiwan area of Chien et al. (2002). For C. sinensis, the average THQ values of all metals were below 0.1 except iAs. This result showed that C. sinensis was most safe when compared to other four kinds of bivalve.
C. farreri, R. philippinarum and C. gigas are the three most popular shellfish for people living in the coastal area. And the results show that there is higher potential risk of consuming these three kinds of bivalves than others due to their high max THQ values. The daily intake FIR used to calculate THQ values is the average consumption of aquatic products rather than bivalves all over the country. In particular, the intake of fisherman and people living by the coastal area might consume much more than the average FIR and the health risk might be undervalued for them.
Table 8 The target hazard quotient (THQ) of heavy metals in bivalves
Bivalves
|
THQ
|
C. farreri
|
R. philippinarum
|
M. veneriformis
|
C. gigas
|
C. sinensis
|
average
|
max
|
average
|
max
|
average
|
max
|
average
|
max
|
average
|
max
|
Fe
|
0.083
|
0.266
|
0.156
|
0.314
|
0.489
|
0.903
|
0.063
|
0.145
|
0.055
|
0.102
|
Mn
|
0.031
|
0.071
|
0.033
|
0.069
|
0.097
|
0.256
|
0.039
|
0.075
|
0.024
|
0.032
|
Cu
|
0.005
|
0.016
|
0.002
|
0.004
|
0.002
|
0.003
|
0.05
|
0.094
|
0.002
|
0.004
|
Zn
|
0.113
|
0.305
|
0.027
|
0.045
|
0.014
|
0.026
|
0.244
|
0.447
|
0.027
|
0.037
|
iAs
|
0.177
|
0.310
|
0.430
|
0.810
|
0.270
|
0.470
|
0.110
|
0.273
|
0.177
|
0.253
|
Cr
|
0.080
|
0.209
|
0.132
|
1.062
|
0.327
|
0.709
|
0.088
|
0.199
|
0.074
|
0.28
|
Cd
|
0.767
|
2.447
|
0.096
|
0.161
|
0.089
|
0.16
|
0.507
|
0.967
|
0.019
|
0.063
|
Pb
|
0.037
|
0.097
|
0.036
|
0.209
|
0.094
|
0.227
|
0.031
|
0.065
|
0.016
|
0.033
|
Hg
|
0.025
|
0.063
|
0.026
|
0.061
|
0.023
|
0.07
|
0.024
|
0.082
|
0.025
|
0.056
|