Heavy Metal Concentrations in Hard Clam Meretrix lyrata and the Coastal Environment of Tien River Estuary, Mekong Delta

 Abstract —The coastal area surrounding Mekong Delta-Vietnam (MDV) is one of the largest clam farming sites in Vietnam. Furthermore, previous studies showed that the rapid growth of urbanization, industrialization, and agriculture lead to the contamination of heavy metals in MDV. Therefore, this study is aimed to evaluate the presence of cadmium (Cd), cooper (Cu), lead (Pb) and zinc (Zn) in sea water, sediment, as well as hard clam ( Meretrix lyrata ) around coastal area of Tien River Estuary named Tan Thanh. The results showed that all studied metals’ concentrations in sediment were lower than the limits of marine sediment quality guidelines, except Zn. The levels of studied metals in clam samples were less than the maximum values of metals in food which regulated by Commission Regulation 1881/2006 and some countries. Concentrations of Cd, Cu, Pb and Zn in the clam flesh were ranging between 0.01 and 1.34 mg kg -1 , 1.1 and 15.8 mg kg -1 , not detected and 0.6 mg kg -1 , as well as 9.9 and 192.5 mg kg -1 , respectively. Among the studied metals, Cd is considered as the most influential parameter for the health risk assessment of local clam consumers. According to this study, the daily intake of 100 g clam flesh (wet weight) is recommended as the largest amount of clam for local consumers.


I. INTRODUCTION
Mekong Delta, Vietnam (MDV) plays an important role in the economic development of Vietnam. The rapid growth of urbanization, industry, and agriculture in MDV during the recent years has resulted in the presence of high concentrations of trace metals as well as other contaminants in the aquatic habitat and organisms [1]. Some previous studies reported that water in the urban areas and the estuaries of Tien and Hau Rivers, which are the two distributaries of Mekong River in MDV, was contaminated with organic, nitrogen, and trace metals [2]- [7]. Indeed, sediment in the river estuary can significantly contain trace metals [8]- [10]. The Cu, Pb and Cr concentrations in the sediment in Tien River estuary were presented to exceed the thresholds of the Canadian Interim Sediment Quality Guidelines (ISQG) and the Probable Effect Level (PEL) of US National Oceanic and Atmospheric Administration (NOAA) [4], [5]. The levels of trace metals in both soluble and particulate phases can influence the concentrations of these elements in the tissue of organisms [8], [11], [12]. Ikemoto et al. claimed that concentrations of Se, Rb, and Hg were found in the aquatic animals through an increase of the trophic level at one sampling site of Hau River. That study also revealed that high concentrations of Mn, Cu, Zn, As, Sr, Mo, Ag, Cd, Sb, Cs, Ba, Tl and Pb had occurred in crustaceans, while fish contained high levels of Cr, Rb and Hg [7].
At the estuary areas, the complicated interaction between fluvial and marine processes act as a geochemical filter. The entrapment and adsorption of metals on the fine-grained sediment and clay particles could be increased because of salinity variation at the estuary areas [13]. In addition, fishery, industrial, transportation and recreational activities surrounding the river estuaries may increase the trace metals contamination in aquatic environment [14]. As a result, organisms such as oyster or hard clam, which live around the estuarine areas may be exposed to trace metals at a high level. Higher metal concentrations in hard clam flesh were found in comparison with those in their living habitat [15]- [17]. In other studies, high average concentrations of Cu, Ni, Cd, Zn, Fe, Pb, and Mg were observed in mollusks living in the coastal area [12], [15], [18], [19]. The concentrations of Fe, Cu, and Cd in clams collected at Loreto, Gulf of California were 572, 181, and 4.6 mg kg -1 in dry weight, respectively [15]. Furthermore, several families of bivalve mollusks such as mussels and clams were suggested using as biomonitors for metallic monitoring in the environment [19], [20].
Some metals, such as Pb and Hg, have not involved into biological functions [21]. Therefore, their existence in seafood is considered as a potential risk to human health [22], [23]. Thusly, the Commission Regulation (EC) has issued the maximum levels (MLs) of trace metals in seafood, such as Cd (1 mg kg -1 ) and Pb (1.5 mg kg -1 ) in bivalve mollusks, and Hg (0.5 mg kg -1 ) in seafood [24]. Until now, the European Union (EU) has not issued MLs of As, Ni, and Cr in food. However, the European Food Safety Agency (EFSA) has provided the provisional tolerable weekly intake (PTWI) of As (15 mg kg -1 body weight) in 2009 [25], the tolerable daily intake (TDI) of Cr(III) (300 mg kg -1 body weight) in 2014 [26] and Ni (2.8 mg kg -1 body weight) in 2015 [27]. Besides, the US.EPA [28] suggested the hazard quotient (HQ) and hazard index (HI) could be used to estimate the risk of each single toxicant to human health via a single pathway like ingestion.
Hard clam is a high economic value species that have been cultured in the coastal areas of Vietnam. Vietnam Association of Seafood Exporters and Producers (VASEP) reported that Vietnam was the second biggest supplier for the processed clams in the world [29]. An average of 2,300 hectares in MDV coastal area are used as hard clam farms. About 20,000-40,000 tons of hard clam are produced each year and most of them (up to 17,000 tons) are exported [30]. In 2019, only in Tan Thanh, 2,200 hectares of hard clam farm were established [31].
Therefore, this study is aimed to (1) determine Cu, Zn, Pb, and Cd concentrations in hard clam (Meretrix lyrata) and its habitat including water and sediment at clam farms; and (2) to assess the potential health risk of these metals to local clam consumers.

A. Study Area
During the dry and wet seasons in 2014 and 2015, sea water, sediment, and Meretrix lyrata samples were collected at the hard clam farm (10 o 17'25.46‖ N and 106 o 47'20.1‖ E) which were located in Tan Thanh area. This place is about 3-5 km away from the Tieu Estuary, Tien River, Mekong Delta ( Fig. 1).

B. Sampling and Preparation Methods
About 30 M. lyrata individuals were collected during low tide to make one composite sample. In this study, only the hard clam from 8 to 12 months-old were chosen for sampling. In the laboratory (Centre Asiatique de Recherche sur l'Eau (CARE) Laboratory), mud and debris were removed from the hard clam by washing them with tap water and MilliQ water (Fig. 2a). Subsequently, the clam flesh was removed from its shell using a sterile stainless-steel knife [32] (Fig. 2b). The flesh was dried using an oven at 60°C for 72 h [33]. An amount of 1 g of dried clam flesh was ground into fine powder using a porcelain pestle and mortar. Homogenized sample was stored in an air-tight zip bag for the next step.
During low tide, around the M. lyrata sampling areas, about 200 g of surface sediment at three points was collected for one composite sample using a Ponar sediment grab sampler. All samples were immediately stored in an icebox during the transportation from the sampling site to the laboratory within 12 h. The sediment samples were dried in an oven at 60 o C for about 5-6 h until their weight were stabilized. Then they were milled to powder before the digestion process and metals analysis.
During high tide, sea water was measured on-site the physico-chemical parameters including temperature, pH, dissolved oxygen (DO), salinity and electronic conductivity (EC) using a multi-parameter probe (WTW 3420). Then, the sea water was collected at 10-50 cm below the water surface using a Niskin non-metallic water sampling bottle (General Oceanics ® ). One aliquot of water sample was filtered immediately using 0.45 µm Whatman® cellulose acetate (WCA) filter paper. The filtrate was acidified at pH less than 2.0 using HNO 3 solution (Normapur HNO 3 ) and then stored in acid pre-cleaned PP bottle for metals analysis. Another aliquot of water sample was stored in 5 L PE bottles for other parameter analysis in the laboratory including hardness, alkalinity, chloride, sulphate, nitrogen and phosphorous. All samples were preserved in an ice box (∼ 4°C) and transported to the laboratory.

C. Digestion of the Samples
Firstly, a mixture of 2 ml perchloric acid and nitric acid (HNO 3 :HClO 4 1:1 v/v) and then 5 ml of concentrated sulfuric acid (H 2 SO 4 ) were added into the dried sediment and hard clam samples (NovaWave SCP Sciences ® , CARE HClO 4 , HNO 3 , H 2 SO 4 ; Trace Metal grade Fisher ® ). Secondly, samples with the acid solution reflux were digested at 200°C for 30 minutes in a clean fume chamber. Then, the completely digested samples were cooled down to room temperature before they were filtered using 0.45 µm WCA filter papers. Finally, the filtrate was diluted to 50 ml in volumetric flasks with 2% HNO 3 solution [34].

D. Determination of Heavy Metals
Heavy metals (Cd, Cu, Pb, and Zn) in sea water, M. lyrata and sediment samples were analyzed according to US EPA method 200.7 by ICP -OES [35] at the CARE laboratory in Ho Chi Minh City University of Technology. Replicate analyses were conducted for 10% of the samples to evaluate precision of the analytical techniques. Each sample of water, sediment and hard clam was analyzed three times.

E. Risk Assessment
In this study, the hazard quotient (HQ) was used to determine the health risk to local people consuming metal-contaminated hard clams. The HQ was calculated by the equation (1) as follows [28]: (1) where bw is body weight (kg). The average body weight of local people was 60 kg, which similar with the Asian body weight of 57.7 kg in 2005 [36]; EDI is the estimated daily intake (µg kg -1 bw per day); C bivalve is metal concentration in hard clam (µg g -1 wet weight); dc bivalve is daily consumption of 4.97 g clam per capita per day. This value was estimated by FAO for Vietnamese people [37]; RfD is equivalent to tolerable daily intake (TDI) and it is determined from provisional tolerable monthly intake (PTMI).

F. Statistical Analysis
The correlation between the concentrations of the four heavy metals (Cu, Zn, Pb and Cd) in samples was evaluated using the Pearson correlation (p<0.05). All statistical analysis and graphs were done using Microsoft Excel.

A. Sea Water Quality
The quality of sea water is presented in Table I. The sea water temperature at Tan Thanh in wet season was lower than that it in the dry season about 1.0-2.0 o C. This is similar to the previous research of Noh et al. [3], who showed that water temperatures at Cua Tieu estuary were 30.5±0.7 o C in March and 28.0±0.3 o C in September. The insignificant temperature change illustrates that the study area is much favorable for clam culture. Salinity in the wet season was lower than that it in the dry season. However, the salinity of both seasons was less than the normal sea water salinity, about 35‰ [38]. The low salinity might be caused by the dilution of freshwater from Tien River at Cua Tieu stuary. Salinity of the sea water at the sampling site in 2014 ranged from 19 to 23‰, which is suitable for hard clam growth. However, during sampling time in wet season 2015, the salinity decreased to an incredibly low value 6.2±0.25‰, which might influence to hard clams. In comparison to the dry season, the lower pH and DO values in the wet season may be influenced by the strong growth of phytoplankton during this time. Similarly, Noh et al. [3] found that the average pH at Cua Tieu estuary was slightly higher in March (7.67±0.31) than in September (7.07±0.15) and the average DO in March (6.08±1.15) was somewhat higher than it in September (5.04±1.05). During the studied time, the results presented high nutrient concentrations at the sampling sites that might have originated from high nutrient loads of the Tien River discharge. However, high nitrogen and phosphorous concentrations in the sea water could promote growth of phytoplankton, which fed the surrounding clams.

B. Heavy Metals in Sea Water
In estuaries, the increase of salinity may lead to the desorption of trace metals from sediment [39]. In brackish and sea water, trace metal ions were replaced with alkalinity and alkaline earth cations [40]. The concentrations of heavy metals in sea water samples in Table II shows that all values were less than the limits of the Vietnamese coastal water quality standards QCVN 10-MT:2015/BTNMT (Zn ≤ 500 µg L -1 ; Cu ≤ 200 µg L -1 , Cd ≤ 5 µg L -1 , Pb ≤ 50 µg L -1 ). These results are the same as the previous study of Nguyen et al. [4], who presented that Zn, Cu, Cd and Pb concentrations in the brackish water of Tien River were 22-166 µg L -1 , 53-82 µg L -1 , 0-2 µg L -1 and 0-27 µg L -1 , respectively.  Fig. 3 shows the heavy metals' concentrations in sediment at the sampling station during 2014-2015. In comparison to the sediment quality guidelines (Table III), all the concentrations of metals in sediment at the hard clam farms met the limits, except Zn in dry season in 2015. In the previous research, Nguyen et al. [4] presented that the mean contents of Cu and Cr in the brackish zone of Tien River (10-20 km far from the sampling station in this study) were higher than the limits of Canadian marine sediment quality guideline and effect range medium of NOAA. Tien River receives a huge amount of surface runoff from agricultural areas where fungicides or herbicides contenting Cu and other metals may be widely used [41]. Thus, the lower values of metals' concentrations in coastal sediment might be influenced due to the dilution of sea water and active condition of semidiurnal tide in this study area. These results were similar with other studies on trace metals in sediment of Mekong and Saigon -Dong Nai Rivers [2], [9]. Another study around this area has also suggested that the change of metal concentrations in sediment might be influenced by seasonal change of sea current at river estuary [42].   Fig. 4 shows the concentrations of 4 studied heavy metals in M. lyrata at Tan Thanh coastal area. The results presented that all metals concentrations in M. lyrata samples met the limits for food in some countries (Table IV). The similar results were also found in the research of Mesndez et al. [45], who showed that the Zn, Cu, and Cd concentrations in chocolate clam (Megapitaria squalida) in a bay within Gulf of California ranged from 47 -65, 5.4-18.7 and 1.5-11 mg kg -1 dry weight, respectively. One year later, another study at Tan Thanh and other area near Sai gon -Dong nai River Estuary showed that some metals, such as Cd, could be accumulated in one specific organ of M. lyrata. According to that research, the metals content in gills or digestion glands were significantly higher than in other organs or in whole body of hard clams [42].

D. Heavy Metals in Hard Clams
The first metal in this study was Cu which is an essential micronutrient for marine organisms. It is difficult to identify a correlation between Cu content in soft tissues of marine organisms and adverse biological effects [46]. The Cu concentrations in the hard clam flesh at Tan Thanh were not much different with those (5.2-12.4 mg kg -1 ) in commercial Razoe clams in Moyan and Serpan, Malaysia [47]. They also met the limit of food quality standards or health standards of some countries (Table 6). Health standards for Cu in fishery products for human consumption varied from one country to other, ranging between 50 and 500 mg kg -1 dry weight [46]. Secondly, the Zn concentration in hard clam M. lyrata at Tan Thanh ranged from 53±12 to 193±21 mg kg -1 dry weight (Table 5). Those values were higher than Zn in clam Galatea paradoxa at the Volta estuary in Ghana, which were ranging between 16 and 49 mg kg -1 [48]. Zn was an similar essential metal to Cu, it is naturally concentrated by living organisms [49]. Thus, the bio-concentration factor for zinc normally has no correlation with toxicity. On the other hand, Pb and Cd are toxicants for organisms. The high Cd contents in grill of Tegillarca granosa clam might negatively affect the osmotic function, nerve system as well as energy metabolism [50]. Fortunately, Table IV presents that Cd contents (in dry weight) in all M. lyrata samples were small and met the limit of food regulations of some countries. These values were also lower than those in the study of Nur and Long [51], who reported that Cd concentrations in P. expansa, M. meretrix, S. regularis at Estuary Area of Sarawak River, Malaysia were 1.15±0.50 mg kg -1 , 2.15±1.38 mg kg -1 , 2.35±0.01 mg kg -1 , respectively. Finally, this study showed that the Pb concentrations in all clam samples were met the limit value of WHO guideline of 0.3 mg kg -1 [52], the ML regulated by the EU (1.5 mg kg -1 ) [25], and the aqua-food quality regulations in some countries (Table IV).   Table V shows that the HQ values were found in decreasing order of Cd (0.013) > Pb (0.0061) > Zn (0.0015) > Cu (0.00037) and the THI was 0.021 (lower than 1.0). It indicates that a negligible health hazard could derive from the ingestion of clam flesh. These results were same as HQs for people who consumed Ruditapes philippinarum in China [58], and smaller than the HQs for clam Tapes decussatus and mussel Mytilus galloprovincialis in Homa Lagoon (Eastern Aegean Sea) [59]. Among all studied metals in this research, Cd is considered as the most important parameter to evaluate the human health risk for M. lyrata consumers. The similar suggestion about Cd also was presented in another study with total 11 elements in hard clam at Tan Thanh and Can Gio area one year later [42].
Based on this study, the maximum tolerable daily intake for a sixty-kg person is 500 g of wet clam flesh (Table V). However, FAO recommended that consumption of clams for Vietnamese should be less than 100 g wet weight capita -1 day -1 [60]. Therefore, 100 g should be considered to recommend as the maximum of clam flesh per capita. IV. CONCLUSIONS The present study showed that all metals were detected in the water and sediment in clam farms at Tan Thanh coastal area, MDV. The concentrations of Cd, Pb and Cu in sediment samples were lower than the limits of marine sediment quality guidelines, while Zn in dry season in 2015 was higher. Although Pb was not detected or was an extremely low concentration in M. lyrata, it was found in the sediment and water. All studied metal concentrations in M. lyrata clam samples were below the maximum level regulated by Commission Regulation (EC) 1881/2006 and to aqua-food quality regulation of some countries. This study indicates that the health risk for local clam consumer is not significant in case of the toxicity of Cd, Pb, Cu and Zn in M. lyrata. However, a maximum consumption of clam flesh is necessary for safety reasons. The daily intake of about 100 g (wet weight) of clam flesh is recommended as the largest amount of clam for local adults.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS
Phuoc-Dan Nguyen and Sunghee Han designed the research; Viet Tuan Tran, Emilie Strady and Huu-Viet Nguyen conducted the study and analyzed the data; Quoc-Tuc Dinh wrote the draft of the paper; and all authors had adjusted and edited the paper before approved the final version.