Introduction

On 16 July 2010, a large amount of crude oil spilled into the water after a pipeline exploded at Dalian Port, Liaoning Province, China. The explosion at an oil storage depot hit the oil pipeline when a tanker ship was unloading. The first explosion triggered a second blast from a smaller adjacent pipeline, which released more crude oil into the sea. A large oil slick formed, creating a dark brown belt of crude oil and other pollution that stretched at least 50 km2 off the port. After the oil spill, combinations of biological, physical, and chemical methods were used to contain and clean up the oil slick. Strategies to monitor and evaluate environmental changes along the Dalian coast were subsequently adopted to assess environmental damage and long-term effects.

Huge oil spills, such as the Gulf War oil spill [1] and the Exxon Valdez oil spill [2], had devastating effects on the marine environment, as crude oil is full of toxic materials. Although the composition of crude oil is complex, heavy metals are an integral component of it [3, 4]. Heavy metals are persistently toxic, prone to bioaccumulation, and pose a risk to humans [5, 6]. They reflect the current quality of the marine environment and also can be indicators of long-term problems because they tend to accumulate in the food chain and ultimately are consumed by organisms, including humans [7, 8].

Due to their wide distribution from tropical to high latitudes, sedentary lifestyle, ability to accumulate heavy metals, and ease of collection [9], bivalves are widely used as indicators of heavy metal contamination [10–14]. As filter feeders, bivalves accumulate heavy metals from the water, sediments, and particulate matter in their soft tissues [15, 16], and a significant relationship has been found between heavy metal concentration in surface sediments and in soft tissues of bivalves [17–19]. Therefore, tracing heavy metal accumulation in bivalves should be part of the monitoring and evaluation programs conducted after the Dalian Port oil spill.

Manila clam (Ruditapes philippinarum; Adams et Reeve 1850), which belongs to Mollusca, Lamellibranchiata, Heterodonta, Veneroida, Veneridae, has been cultured for more than 50 years in China. The production of Manila clam has reached over 3.0 million tons per annum in China, which accounts for about 90 % of worldwide Manila clam production. Manila clam culture is particularly strong along the Dalian coast, where this species has become an important fishery [20].

The primary purpose of this study was to obtain quantitative information about the concentrations of heavy metal in surface sediments and R. philippinarum collected from the Dalian coast after the Dalian Port oil spill. The results reported here will provide the most recent and valuable information about whether the oil spill poses a threat to the culture of R. philippinarum. To that end, the safety of R. philippinarum for human consumption was also evaluated.

Materials and Methods

Study Area and Sample Collection

Figure 1 shows the three sampling sites (Jinshitan, Dalijia, and Pikou), which were located in the mariculture zone along the Dalian coast in China. Samples were collected from three clam-farming areas at each sampling site in November 2010; during November, R. philippinarum has no reproductive activity [21]. When the tide ebbed, clams were collected from each sampling site using plastic buckets. The 0–10-cm surface layer of sediment samples was also gathered using a plastic scoop and stored at 4°C immediately. Meanwhile, five replicates were conducted at each sampling site. To reduce individual variations in metal concentrations [22], similar-sized clams were randomly chosen after measuring the shell lengths and weights, which were 24.98–27.24 mm and 4.12–5.04 g, respectively. There were no significant differences (p > 0.05) among the shell lengths and weights at each sampling site.

Fig. 1
figure 1

Map showing the location of Dalian port (black star) and sampling sites (black circles) from the Dalian coast, China

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Clams

After collection, similar-sized individuals were transported immediately to the laboratory and cultured in filtered seawater for 24 h to depurate them. Subsequently, 100 individuals from each site were chosen randomly as a pooled sample for analysis. The tissues of these clams were dissected with a plastic knife and then immediately stored at −20°C. After samples were thawed at room temperature and rinsed with distilled water, they were dried at 80°C to constant weight, homogenized, and stored in desiccators for future analysis. Prior to chemical analysis, 0.5 g of dry sample was added to 10 ml of purified nitric acid and 2 ml of purified perchloric acid, and then the solution was heated until dry. Next, 2 ml of purified nitric acid and 3 ml of distilled water were added to the cooling residue and then heated to dissolve the residue into the mixture. At this point, all digested samples were cooled at room temperature and eventually diluted to 10 ml in a volumetric flask with distilled water [23].

Surface Sediments

Sediment samples were air-dried [24], and then sieved through a 63-μm nylon mesh. The fractions <63 μm were stored in clean plastic beakers for analysis. The samples were prepared in triplicate and digested using a mixture of concentrated nitric acid, perchloric acid, and hydrofluoric acid. The digested mixture then was processed prior to chemical analysis, following the procedure described above for the clam tissues.

Chemical Analysis

Concentrations of Cu, Pb, Zn, Cd, and Cr in the sediment and clam samples were measured with a flame atomic absorption spectrophotometer using deuterium background correction. Hg and As were analyzed by the cold vapor technique using Portable Zeeman Lumex mercury analyzer. Standard reference materials (mussel tissue sample, GBW08571; coastal sediment sample, GBW07314; both from the National Research Center for Certified Reference Materials, Beijing) were used for quality assurance and quality control procedures. Satisfactory recoveries were obtained for Cu (92.3–102.6 %), Pb (97.2–108.4 %), Zn (87.6–95.1 %), Cd (96.6–101.4 %), Cr (94.2–106.3 %), Hg (88.2–97.5 %), and As (93.8–102.5 %). Metal concentrations in R. philippinarum and surface sediments are presented as milligrams per kilogram dry weight.

Statistical Analysis

To compare the total content of metals at different sampling sites, the metal pollution index (MPI) was used. It was obtained according to the equation [25].

$$ {\text{MPI}} = {\left( {{\text{C}}{{\text{f}}_{{1}}} \times {\text{C}}{{\text{f}}_{{2}}} \ldots {\text{C}}{{\text{f}}_n}} \right)^{{1{/}n}}} $$

where Cf1 = concentration of the first metal, Cf2 = concentration of the second metal, Cf n  = concentration of metal n, and n = the total number of studied metals in the sample.

To further evaluate the efficiency of metal bioaccumulation in R. philippinarum, the biosediment accumulation factor (BSAF) was calculated. The BSAF is defined as the ratio of the metal concentration in the organism to that in the sediment [22].

$$ {\text{BSAF}} = {\text{Cx}}/{\text{Cs}} $$

where Cx and Cs are the average concentrations of a given metal in the organism and the associated sediment, respectively.

Statistical analyses were executed by the programs Statview 5.0 for windows (SAS Institute Inc, Cary, NC, USA). A one-way analysis of variance followed by a significant difference (LSD) test was used to verify any statistically significant difference (p < 0.05) among sampling sites. Pearson correlation and linear regression model were used to assess the correlation between heavy metal concentrations in R. philippinarum and in surface sediments.

Results and Discussion

Heavy Metal Concentrations in Surface Sediments

Table 1 shows the heavy metal concentrations in surface sediments collected from the Dalian coast. All trace metal concentrations in surface sediments collected from Jinshitan, Dalijia, and Pikou, in general, were ranked in decreasing order, as follows: Zn > Cr > Cu > Pb > As > Cd > Hg. Concentrations of Cu, Pb, Zn, Cd, and Cr in surface sediments collected at Pikou were significantly higher than these in Jinshitan (p < 0.05), while there were no significant differences in Hg and As levels among these sampling sites (p > 0.05). This is not surprising, considering that Pikou is an important port town, and its waters are subjected to contamination by persistent organic pollutants, municipal waste disposal, and metals from nautical, tourist, and agricultural industries. In contrast, the lowest concentrations of the metals mentioned above were found at Jinshitan. Jinshitan is a famous national tourist attraction without industries, and it is considered to be relatively free of anthropogenic contamination. Although the Dalian Port oil spill have occurred closer to Jinshitan than Pikou, the lowest concentrations of Cu, Pb, Zn, Cd, and Cr collected at Jinshitan should be considering the background levels before oil spill.

Table 1 Heavy metal concentrations (in milligrams per kilogram dry weight) in surface sediments and R. philippinarum collected from the Dalian coast
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However, oil spill investigations usually lack pre-impact data, which precludes assessment of the effect of the oil spill on the marine environment [26]. This was the case for the Dalian Port oil spill. Because pre-impact data were unavailable for the Dalian coast, the data collected herein were compared with metal levels from other geographic areas to infer whether heavy metal concentrations were altered at the three sites due to the oil spill. Metal levels in surface sediments from the Dalian coast were similar to those from other locales reported by other researchers (Table 2). Levels of Cu, Pb, Zn, and Cr were in agreement with those reported for Jiaozhou Bay, which is another important clam-farming area in China [23]. Hg and As, which were widespread and in high concentrations in surface sediments from Venice Lagoon in Italy [27] and southern Atlantic in Spain [19], were found in lower concentrations in the present study.

Table 2 Comparison heavy metal concentrations (in milligrams per kilogram dry weight) in surface sediments and R. philippinarum obtained in the present study with literature data from China and other regions all over the world
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Heavy Metal Concentrations in R. philippinarum

Table 1 also shows the heavy metal concentrations found in R. philippinarum. Trace metal concentrations in R. philippinarum collected from Jinshitan, Dalijia, and Pikou were consistent and ranked, in decreasing order of concentration, as follows: Zn > Cu > As > Cr > Pb > Cd > Hg, and similar trends were reported previously for R. philippinarum from other geographical areas (Table 2). Compared to the other metals analyzed, Zn and Cu were found in much higher concentrations [31, 32]. In contrast, Hg was found in the lowest concentration. Usero et al. [25] reported a similar concentration of Hg in R. philippinarum from southern Atlantic coast in Spain. Except for Cu and Hg, the concentrations of other metals in R. philippinarum varied notably depending on the location of the sampling sites.

The concentrations of Pb and Zn in R. philippinarum collected at Jinshitan were significantly higher than these in Dalijia and Pikou (p < 0.05), whereas As was the prominent metal in Pikou. There were no significant differences in Cu, Cd, and Hg levels among these sites (p > 0.05). Many studies have reported that, for benthic bivalves, there was a significant correlation between metal concentrations in their soft tissues and surface sediments where they live [17–19]. Considering the lowest metal concentrations in surface sediments at Jinshitan, however, the higher metal concentrations in R. philippinarum collected from Jinshitan may be polluted from the oil spill. Meanwhile, the MPI was used to compare the total content of metals at those sampling sites. As shown in Table 3, the MPI value at Jinshitan was somewhat higher than other sites. This tendency was especially marked in the case of Pb. However, these MPI values are within the ranges reported in other studies [19, 25, 31].

Table 3 Mean BSAF and the metal pollution index (MPI) values in R. philippinarum collected from the Dalian coast
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Metal levels in R. philippinarum in this study were compared with data from previous studies conducted in China and elsewhere in the world. Except for Zn, which had the maximum concentration of 216 mg kg−1 dry weight, concentrations of Cu, Pb, Cd, Cr, Hg, and As were not obviously higher than the normal range, which might be considered to be background levels (Table 2). All of these results suggest that clams inhabiting sediments along the Dalian coast are relatively unpolluted with heavy metals following the Dalian Port oil spill.

Bioaccumulation of Heavy Metals

To evaluate the efficiency of metal bioaccumulation in R. philippinarum, the BSAF values were calculated. Bioaccumulation is expected to occur in organisms if the BSAF is >1 [34]. Table 3 shows that the metals with BSAF values <1 were Cu, Pb, Cr, and As, indicating almost no bioaccumulation of these metals in R. philippinarum. In contrast, Zn, Cd, and Hg were the metals with the BSAF values >1, especially Cd showed the BSAF value as high as 10.33, which suggested that bioaccumulation had obviously occurred in R. philippinarum from the Dalian coast. Finally, if we compare these sampling sites, we can find that R. philippinarum collected from Jinshitan had the highest BSAF values of Cu, Pb, Zn, Cd, Cr, and Hg, while R. philippinarum collected from Pikou had the highest BSAF value of As.

Relationship Between Heavy Metals in R. philippinarum and Surface Sediments

The relationships between heavy in R. philippinarum and surface sediments are presented in Table 4. Pearson correlation analysis clearly indicated a very strong relationship (p < 0.01) between Hg concentration in R. philippinarum and surface sediments, as well as a strong correlation (p < 0.05) between Pb and Cd in R. philippinarum and surface sediments. The relationship between Cu, Zn, Cr, and As in R. philippinarum and surface sediments was not significant (p > 0.05), indicating that the concentrations of these metals in surface sediments were not directly reflected in R. philippinarum. When the concentrations of metals in surface sediments are lower than the threshold below which the organisms are able to accumulate the metals in their bodies, bioaccumulation is not significant [19, 30]. Moreover, bioaccumulation is probably influenced by many physicochemical and biological factors [5].

Table 4 Linear regression equations between heavy metal concentrations in R. philippinarum and in surface sediments collected from the Dalian coast
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Linear regressions for the concentrations of Pb, Cd, and Hg in R. philippinarum (y values) relative to their concentrations in surface sediments (x values) were calculated. Based on the value of their slopes, the abundance of Pb, Cd, and Hg can be ordered as follows: Hg (2.390) > Pb (0.082) > Cd (0.054). The slope for Hg was significantly >1, which suggests that the bioavailability of Hg is disproportionately increased with the degree of its enrichment in surface sediments.

Human Consumption

In the present study, metal levels were converted to values per wet weight and compared with legal limits to evaluate the safety of R. philippinarum for human consumption (Table 5). According to the Marine Biological Quality in China, Cu and Hg were well below the first quality standard limits. Although Pb, Zn, Cd, and As exceeded the first quality standard limits, they were well within the second quality standard limits. However, As significantly exceeded the Maximum Levels of Contaminants in Foods in China. Having compared metal levels with legal limits in Hong Kong, Australia, and Europe, we found that Cu, Pb, Zn, Cd, and Hg were well within the maximum permissible limits. However, Cr and As exceeded the maximum permissible levels in Hong Kong and Australia, respectively. Therefore, we can conclude that all of these metals in R. philippinarum measured in this study, with the exception of Cr and As, were within the range suitable for human consumption.

Table 5 Comparison heavy metal concentrations (in milligrams per kilogram wet weight) in R. philippinarum obtained in the present study with the maximum permissible concentrations for bivalves in legislation from China and other geographic areas all over the world
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