Effect of coastal eutrophication on heavy metal bioaccumulation and oral bioavailability in the razor clam, Sinonovacula constricta

Aquatic Toxicology 155 (2014) 269–274

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Effect of coastal eutrophication on heavy metal bioaccumulation and oral bioavailability in the razor clam, Sinonovacula constricta Tengxiu Tu a , Shunxing Li a,b,∗ , Lihui Chen a , Fengying Zheng a,b , Xu-Guang Huang a,b a b

College of Chemistry and Environment, Minnan Normal University, Zhangzhou 363000, P.R. China Fujian Province Key Laboratory of Modern Analytical Science and Separation Technology, P.R. China

a r t i c l e

i n f o

Article history: Received 29 April 2014 Received in revised form 7 July 2014 Accepted 9 July 2014 Available online 18 July 2014 Keywords: Eutrophication Metal bioaccumulation Metal oral bioavailability Seafood safety Razor clams

a b s t r a c t As traditional seafoods, the razor clams are widely distributed from tropical to temperate areas. Coastal razor clams are often exposed to eutrophication. Heavy metal contamination is critical for seafood safety. However, how eutrophication affects bioaccumulation and oral bioavailability of heavy metals in the razor clams is unknown. After a four-month field experimental cultivation, heavy metals (Fe, Cu, Ni, V, As, and Pb) could be bioaccumulated by the razor clams (Sinonovacula constricta) through exposure to metals present in water and sediments or in the food chain, and then transferred to human via consumption of razor clams. Bionic gastrointestinal digestion and monolayer liposome extraction are used for metal oral bioavailability (OBA) assessment. The influence of eutrophication on OBA is decreased for Fe and Pb and increased for V. A significant positive linear correlation was observed between the bioaccumulation factors of Fe, Ni, V, and As in razor clams and the coastal eutrophication. These results may be due to the effect of eutrophication on metal species transformation in coastal seawater and subcellular distribution in razor clams. The maximum allowable daily intakes of razor clams are controlled by eutrophication status and the concentration of affinity-liposome As in razor clams. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Coastal organisms are often exposed to both metal pollution and eutrophication (Li et al., 2008; Cai et al., 2011), which have drawn increasing attention to the risks of coastal ecosystem functions and seafood safety (Li et al., 2013b; Li et al., 2014; Liu et al., 2014). Razor clams are a group of infaunal bivalve mollusks that constitute a considerable component of infaunal soft-bottom communities, inhabiting fine sand, silt or sandy-mud ocean floors and forming extensive and dense beds (Fernández-Tajes et al., 2010). As traditional seafoods, they are widely distributed from tropical to temperate areas. Razor clams can take up essential and nonessential metals either from seawater or food (e.g., debris and marine algae) (Amiard et al., 1987; Wang and Fisher, 1999). The accumulated heavy metals could be transferred to human via consumption of razor clams, and the deficiency of essential metals or metal overload is harmful for human health. Heavy metal contamination in razor clams has raised public health concerns. However,

∗ Corresponding author. Present address: College of Chemistry and Environment, Minnan Normal University, Zhangzhou, 363000, P.R. China. Tel.: +86 596 2591395; fax: +86 596 2591395. E-mail addresses: [email protected], shunxing [email protected] (S. Li). http://dx.doi.org/10.1016/j.aquatox.2014.07.012 0166-445X/© 2014 Elsevier B.V. All rights reserved.

the studies about metal compositions of razor clams are limited and all of them are focused on the determination of total metal concentration (Huang et al., 2007; Kanakaraju et al., 2008; Saeedi et al., 2012). So, all of them can result in inaccurate safety assessment. Metal sorption and transfer are controlled by eutrophication and algal species in coastal food webs (Li et al., 2013b). However, how eutrophication affects bioaccumulation and oral bioavailability of heavy metals in the razor clams is unknown. In this study, the ratio of metal concentration in razor clams tissue to seawater is considered as bioaccumulation factor (BAF) (DeForest et al., 2007) and then the influence of eutrophication on metal bioaccumulation is discussed. Metal oral bioavailability (OBA) is also adopted because heavy metal toxicity for human consumption of razor clams cannot be assessed by metal BAF. OBA is defined as the fraction of metal species, which can be dissolved by oral and gastrointestinal digestive juices, released into the chyme, and then absorbed by the biomembrane. Liposomes are used as the model biomembrane because of the similarity in chemical structure between the blood vesicle and the actual biomembranes of gastrointestinal tract (Li et al., 2011; Li et al., 2013a). OBA is assessed by the ratio of affinity-liposome metal concentration in chyme to total metal concentration in razor clams. Here we report the influence of eutrophication on metal BAF and OBA in razor clams for the first time.

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2. Materials and methods

2.4. Experimental design of in vitro bionic model

2.1. Instrumentation

A biomimetic digestive tract, including the mouth, stomach, and small intestine, was designed by us (Li et al., 2013a). The amounts of the compositions in the digestive tract (including inorganics, organics, and digestive enzymes), digestion process, pH, and residence time periods typical for each compartment were designed on human physiology. All digestive juices were heated to 37 ± 2 ◦ C. The digestion started by adding 5 mL saliva to 1.0 g razor clams tissue and was incubated for 5 min. Then 30 mL of gastric juice was added, and the mixture was rotated head-over-heels for 3 h. After gastric digestion, the pH value of the chyme was adjusted to 7.8 ± 0.2, a mixture of 30 mL duodenal juice, 10 mL artificial bile, and 15 mL gut microbiota were added simultaneously into the chyme, and then this mixture was rotated for another 7 h. The liposomes were prepared using the thin film rehydration technique (Li et al., 2011). A 0.1 g sample of egg-derived lecithin dissolved in 5 mL of chloroform was transferred into a rotatory evaporator, and then evaporated for 10 min at 37 ◦ C to form multilayer liposome. A 25 mL chyme was used to rehydrate the multilayer liposome film, followed by five cycles of freeze–thaw treatment. The metal species distribution in liposome–water system could be promoted by such frozen-thawed process. Affinityliposome metals could be separated from water-soluble metals by 0.22 ␮m membrane.

Agilent 7500cx inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, USA) was used for metal determination. The UV-3200PCS UV–Vis spectrophotometer (Shanghai Spectrum Instruments Co., China) was used to determine the absorbance of tested samples. RE-52 rotator evaporator (Ya Rong Biochemical Instrument Factory, China) and 86C ULT ultra low temperature freezer (Thermo Electron Co., USA) were used to prepare the liposomes. SHA-B temperature consistent oscillating waterbath (Guo Hua Co., China) and MK-III microwave digestion system (Sineo Microwave Chemistry Technology Co., China) were used to digest the seawater and razor clam tissue. Mettler Toledo 320-S pH meter (Mettler Toledo Co., China) was used to determine the pH value of seawater.

2.2. Chemicals The biological chemicals such as ␣-amylase (1000 units/mg), pepsin (250 units/mg), lipase (200 units/mg), pancreatin (2000 units/mg), uric acid, mucin, bovine serum albumin, and bile all were purchased from Sigma (St. Louis, MO, USA). Trypton, yeast extract, and glucose were supplied by Huangkai Microbial Sci. & Tech. Co. (Guangdong, China) and used for the preparation of microbiota tryptone–yeast extract–glucose medium. Concentrated nitric acid (65%, m/v, Merck KGaA, Germany) and hydrogen peroxide (30%, m/v, Xilong Chemical CO., China) were used for the digestion of samples. All other chemicals were of analytical reagent grade from Shanghai Experiment Reagent Co., China, including lecithin, d-(+)-cellobiose, d-(+)-maltose, d-(−)-fructose, Tween 80, and meat extract. Agilent ICP-MS multi-element standards (10 mg L−1 , Nos. 2A, USA) and internal standards (including 100 mg L−1 45Sc, 72Ge, 103Rh, 115In, and 209Bi) were used for metal determination. Milli-Q purified water (18.2 M) was used for all sample preparations. To avoid metal contamination, all glassware and plastic ware were washed and kept in 10% (v/v) nitric acid for 48 h and then rinsed several times with ultrapure water before use.

2.3. Field experiment and samples pretreatment Razor clams and seawater were collected from four semienclosed bays in Zhangzhou Fujian Province, China, including Jiuzhen (JZ, N24.01◦ , E117.73◦ ), Gangwei (GW, N24.37◦ , E118.09◦ ), Fotan (FT, N24.20◦ , E117.96◦ ), and Zhao’an (ZA, N23.74◦ , E117.32◦ ). The salinities and pH values of seawater in FT, JZ, GW, and ZA were in the range of 32–34 and 7.90–8.06, respectively, and there were not significant (p > 0.05). After filtering the seawater with acid-washed Pall Acropak Supor capsule 0.22 ␮m filters, total phosphorus (P) and inorganic nitrogen (N) in the seawater were determined, using the Chinese national standards GB/T 12763.42007 and GB11893-89, respectively. Young razor clams (about 1–2 cm) were transplanted into these four bays (FT, JZ, GW, and ZA) and a 4-month experiment was conducted from December 2012 to March 2013. After field experiment, a batch of razor clams (30 individuals, about 5–6 cm) was collected. Before metal determination, the razor clams used for experiments were carefully cleaned to remove their epibionts with purified water. Afterwards, the soft tissues of razor clams were individually dried at 105 ◦ C and then homogenized using a mechanical agate mortar.

2.5. Metal determination Total concentrations of heavy metals (Fe, Cu, Ni, V, As, and Pb) in razor clams and seawater were determined. After addition of concentrated HNO3 (4.0 mL) and Milli-Q water (4.0 mL), razor clams (0.1 g) were heated in water bath at 80 ◦ C until no smoke arose and then digested entirely by microwave technique for 10 min, using a mixture solution of concentrated HNO3 (2.0 mL) and H2 O2 (1.0 mL, 30%) in PTFE digestion vessel. Seawater filtered by 0.22 ␮m membrane, and all of affinity-liposome metal or water-soluble metal were directly digested under the same conditions mentioned above. After digestion, a dilution with water to 50 mL was carried out for metal determination by ICP-MS. 2.6. Statistical analysis SPSS 19.0 was used for the statistical analysis. Results were expressed as means ± SD. Possible relationships between macronutrient level and metal BAF (or OBA) were examined by partial correlation analysis and quantified by the determination coefficient, r. A significance level of p < 0.05 was adopted for all comparisons. The regressions were quantified by the coefficient of determination, R2 . The plots and regression lines were drawn in SigmaPlot for Windows Version 10.0. 3. Results 3.1. Macronutrient levels in four sites In estuarine and coastal waters, eutrophication was a serious environmental problem (Li et al., 2013b). Macronutrients included nitrogen (nitrate, nitrite, ammonium, and dissolved organic nitrogen) and phosphorus (phosphate and dissolved organic phosphorus), while nitrate and phosphate were their predominant species (Li et al., 2013b). The bay of GW, ZA, JZ, and FT was semi-closed, the field experiment was done in winter (from December 2012 to March 2013), the degree of seawater exchange was controlled, and the concentration of N and P in the seawater was stable during our field experiment. These results were similar as the reports from the Department of Ocean and Fisheries,

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Table 1 Concentration of total phosphate and inorganic nitrogen in seawater from the four sites, Gangwei (GW), Zhao’an (ZA), Jiuzhen (JZ), and Fotan (FT). Sample sites

Total phosphorus (␮mol/L) Inorganic nitrogen (␮mol/L)

GW

ZA

JZ

FT

0.48 ± 0.02 11.7 ± 0.58

1.13 ± 0.06 13.0 ± 0.65

1.10 ± 0.06 20.0 ± 1.00

1.39 ± 0.07 29.9 ± 1.49

3.3. Influence of macronutrient levels on metal bioaccumulation in razor clams

Fig. 1. Influence of inorganic nitrogen concentration in seawater on metal bioaccumulation factor (BAF) (two-tailed, partial correlation, n = 3).

Zhangzhou City, Fujian Province, China. Seen in Table 1, the concentrations of total phosphorus and inorganic nitrogen in these four bays were in the ranges of 0.48–1.39 ␮mol/L and 11.7–29.9 ␮mol/L, respectively. Both nitrogen and phosphorus were at low level in GW and considered relatively pristine, whereas the ZA, JZ, and FT were contaminated to different extents by macronutrients. The four bays with different macronutrient levels were used in our study.

3.2. Accuracy and detection limits of metal determination Using microwave-assisted digestion and ICP-MS, metal determination was evaluated by analyzing certified reference materials (CRMs, including NIES-03 (green algae, C. Kessleri) and NASS5 (standard seawater)), limit of detection (LOD) and limit of quantification (LOQ), LOD can be computed out from LOQ (LOD = LOQ × 3/10). As shown in Table 2, the achieved results were in good agreement with the certified values of CRMs. The method described was applicable for the determination of low levels (␮mol/kg or nmol/L) of metals (Fe, Cu, Ni, V, As, and Pb) in coastal seawater, razor clams, and affinity-liposome and watersoluble metals in the chyme.

The concentrations of essential metals (Fe, Ni, V, and Cu) and non-essential metals (As and Pb) in razor clams tissue and seawater were analyzed (seen in Table 3). In seawater, the concentrations of these trace metals, except of Fe, were in the range of 0.003–0.155 ␮mol/L, however Fe concentration was from 5.00 to 38.6 ␮mol/L. All metal concentrations in the seawater met the standard of clean bay. In razor clams tissue, Fe concentration was extremely high, ranged from 2.93 to 51.3 mmol/kg dry weight and the concentrations of Cu, Ni, V, As, and Pb were in the range of 149–375 ␮mol/kg, 22.2–33.9 ␮mol/kg, 14.9–29.4 ␮mol/kg, 98.5–289 ␮mol/kg, and 5.60–14.9 ␮mol/kg dry weight, respectively. BAF was used to investigate the ability of metal bioaccumulation in razor clams. Partial correlation analysis was done to assess the influence of macronutrient levels (total phosphorus and inorganic nitrogen) on the metal bioaccumulation. The BAFs of Fe, Ni, and V in razor clams were significantly positively correlated with inorganic N in seawater (seen in Fig. 1, R2 = 0.966, P = 0.017 for Fe; R2 = 0.993, P = 0.003 for Ni; and R2 = 0.997, P = 0.001 for V). However, the BAFs of these three metals in razor clams were not significantly correlated with total P (p > 0.05). The BAF of As in razor clams was not significantly correlated with inorganic N (r = 0.90, p > 0.05), but showed positive correlation with total P (seen in Fig. 2, R2 = 0.934, P = 0.033). Partial correlation analyze found that the BAFs of Cu and Pb were not significantly correlated with total P and inorganic N (r > 0.90, P > 0.05). These results indicated that eutrophication could play an important role in metal depletion from seawater and metal bioaccumulation in razor clams. 3.4. Relationship between seawater macronutrient levels and heavy metal bioavailability Metal toxicity was not depended on total bioaccumulated concentration but related to a threshold concentration of internal metabolically available metal (i.e., metal bioavailability). After gastrointestinal digestion and gut microbiota metabolism of razor clams tissues, metals speciation in the chyme were transformed into their final coordinated complexes and then absorbed or excreted. Affinity-liposome metals in the chyme which could be used as the final criterion for the assessment of metal oral

Table 2 Heavy metals analysis of certified reference materials using microwave-assisted digestion and ICP-MS (n = 3).

Fe Cu V Ni As Pb

NIES-03 (Chlorella Kessleri)

NASS-5 (standard seawater)

Certified value (␮mol/kg)

Found value (␮mol/kg)

Certified value (nmol/L)

Found value (nmol/L)

33,000 ± 164 54.7 ± 4.76 Non-certified values Non-certified values Non-certified values 2.88 ± 0.008

32,500 ± 426 53.3 ± 3.33 <0.775

3.71 ± 0.63 4.67 ± 0.72 23.5 ± 0.05

8.47 ± 1.67

Limits of detection (nmol/L)

Limit of quantification (nmol/L)

3.29 ± 0.05 5.26 ± 0.04 23.5 ± 0.188

41.7 6.19 235

138 20.4 775

4.31 ± 0.48

4.31 ± 0.02

0.25

0.83

84.0 ± 5.33

16.9 ± 1.60

18.3 ± 1.826

0.36

1.19

2.42 ± 0.48

0.039 ± 0.02

0.030 ± 0.002

0.43

1.42

1.73) × 10 9.00 1.16 13.5 0.55 5.65 ± ± ± ± ± ± (34.6 180 23.1 26.9 10.9 113 2.50 0.002 0.002 0.007 0.001 0.001 ± ± ± ± ± ±

Seawater

5.00 0.038 0.003 0.015 0.019 0.016 4718 4442 2775 1041 224 4925

Fig. 2. Influence of total phosphorus concentration in seawater on metal bioaccumulation factor (BAF) (two-tailed, partial correlation, n = 3).

1.47) × 10 9.55 1.11 1.15 0.28 4.93 6.21 0.043 0.008 0.022 0.025 0.020

± ± ± ± ± ±

3.10 0.002 0.004 0.001 0.001 0.001

(29.3 191 22.2 22.9 5.60 98.5

± ± ± ± ± ±

bioavailability. Metal OBAs were in the range of 7.39–18.6% for Fe, 1.06–16.7% for V, 7.16–16.4% for Ni, 0.28–11.6% for Cu, 12.6–17.1% for Pb, and 0.21–5.04% for As, respectively. The OBAs of Fe and V were significantly decreased with the increase of inorganic N concentration (seen in Fig. 3, R2 = 0.998 and 0.998, P = 0.037 and 0.048), but when the concentration of inorganic N exceeded 17.8 ␮mol/L, the OBA of V increased quite sharply. In addition, the OBA of Pb was significantly correlated to the concentration of total P (seen in Fig. 4, R2 = 0.987, P = 0.006). However, the OBA of Ni was, but not significantly (P > 0.05), correlated with macronutrient levels (i.e., inorganic N and total P, r = ±1.000). Other metals did not show any correlation with total P or inorganic N (P > 0.05). 3.5. Safety assessment of razor clams for consumption based on metal oral bioavailability

Represent ␮mol/L in seawater, and ␮mol/kg in razor clams tissue (dry weight). a

(30.4 ± 1.52) ×10 149 ± 7.45 23.9 ± 1.20 14.9 ± 0.75 7.43 ± 0.37 132 ± 6.60 Fe Cu Ni V Pb As

20.9 ± 1.05 0.047 ± 0.002 0.129 ± 0.006 0.052 ± 0.003 0.020 ± 0.001 0.055 ± 0.003

3

1454 3170 185 286 372 2400

38.6 ± 1.95 0.105 ± 0.005 0.155 ± 0.008 0.062 ± 0.003 0.030 ± 0.002 0.062 ± 0.003

(51.3 375 33.9 29.4 14.9 289

± ± ± ± ± ±

2.57) ×10 18.8 1.70 1.47 0.75 14.5

3

1329 3571 218 474 497 4661

Seawater

JZ

BAF Razor clams Seawater

ZA

BAF Razor clams Seawater

GW Metala

Table 3 Concentration of heavy metals in seawater and razor clams tissue from the four sites used in the laboratory experiment.

Razor clams

3

BAF

FT

Razor clams

3

6920 4736 7700 1793 574 7063

T. Tu et al. / Aquatic Toxicology 155 (2014) 269–274

BAF

272

Excessive consumption of metal-contaminated razor clams could result in metal overload. Based on the tolerable upper intake level (UL) and maximum level of daily intake without detriment to health (ML) of metals (Trumbo et al., 2001) and the concentration of affinity-liposome metals, the maximum allowable daily intake of razor clams was calculated. The result demonstrated that the maximum allowable daily intake of razor clams was mainly controlled by the concentration of affinity-liposome As. The maximum allowable daily intakes are 76.7 g/d, 43.0 g/d, 140 g/d, 157 g/d of razor clams from the bay of GW, ZA, JZ, and FT, respectively (seen in Table 4). Metal safety assessment demonstrated that razor clams’s quality was significantly influenced by macronutrient levels. Consequently, the concentrations of N, P, and metals in coastal seawater should be simultaneously monitored in order to avoid metal overload via consumption of razor clams. 4. Discussion Marine phytoplankton and debris were the main food sources for razor clams (Brussaard et al., 1995). Many previous studies illustrated that the input of macronutrient could not only change phytoplankton structure (including species composition and abundance), but also affect metal uptake and assimilation by marine phytoplankton (Li et al., 2013b). Eutrophication caused predictable increases in the biomass of marine phytoplankton and debris (Smith, 2003), which could provide a rich source of food for razor clams. A significant positive-correlation was found between the BAFs of Fe, V, and Ni and the inorganic N. The BAF of As in razor clams was significantly positively correlated with the total P. On

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Table 4 The intake of maximum values of razor clams from GW, ZA, JZ, and FT. Metal

Fe Cu Ni V Pb As

UL/ML (␮mol/d)

833 158 33.3 19.6 1.44 2.40

Maximum consumption of razor clams (g/d dry weight) GW

ZA

JZ

FT

76.7 1.89 × 105 8.18 × 102 2.60 × 104 5.69 × 102 8.29 × 102

43.0 2.29 × 103 6.97 × 103 1.60 × 104 1.45 × 102 1.45 × 102

1.40 × 102 3.56 × 103 6.31 × 103 7.32 × 104 9.56 × 102 4.80 × 102

1.57 × 102 1.68 × 104 5.45 × 103 3.93 × 103 5.27 × 102 1.16 × 103

Note: ULs, tolerable upper intake levels; ML, maximum level of daily intake without detriment to health.

the one hand, the assimilation of Fe and Ni by marine phytoplankton was generally increased with the increase of inorganic N (Bu-Olayan et al., 2001; Smith et al., 1999; Wang et al., 2007), and then metals could be transferred from algae to razor clams via food chain. The concentrations of dissolved As and V were positively correlated with chlorophyll concentrations (Chen et al., 2008) ˜ 2009). With the and phytoplankton biomass (Wang and Sanudo, increasing of the concentration of dissolved As and V in seawater, the uptake of As and V by marine phytoplankton and subsequent metal assimilation by razor clams would be aggravated. Consequently, the dominant type of Fe, Ni, V, and As assimilation pathway of razor clams was particulate ingestion. However, the BAFs of Cu

and Pb in razor clams had no significantly correlated with macronutrient levels, these metals might be mainly uptaken as dissolved species in seawater directly. In our experiment, in vitro bionic digestion and biomimetic membrane extraction were used to evaluate metal oral bioavailability in the human gastrointestinal tract. The results indicated that eutrophication could significantly affect the OBA of Fe, V, and Pb in razor clams. Metal bioavailability was critically dependent on their chemical form (Bacon and Davidson, 2008; Passos et al., 2010; Tuzen et al., 2004), metal ligands (Li et al., 2011), and metal subcellular distribution (Wallace et al., 2003). Subcellular distribution (including heat-stable protein and granules) of metals in razor clams could be affected by the macronutrient levels of the seawater (Miao and Wang, 2006). Lead (Pb) and

Fig. 3. Relationship between heavy metals oral bioavailability with concentration of inorganic nitrogen in seawater (two-tailed, partial correlation, n = 3).

Fig. 4. Relationship between heavy metals oral bioavailability with concentration of total phosphorus in seawater (two-tailed, partial correlation, n = 3).

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iron (Fe) were mainly distributed in metal-rich granule in marine bivalves, and a significantly negatively correlation between metal bioavailability and metal partitioning in metal-rich granule was demonstrated (He and Wang, 2013). Consequently, in our experience, the OBAs of Fe and Pb in razor clams were negatively correlated with macronutrient levels. The speciation of V might likely depend on the redox conditions of aqueous system (Wang ˜ and Sanudo, 2009). While V(V) was the thermodynamically stable form in oxygenated seawater, V(IV) was commonly existed in intra-cellular media (Willsky, 1990). V(IV) was actively involved in phytoplankton metabolism for its high reactivity with ADP, ATP, GDP, glutathione, amino acids, nucleic acids and lipids (Goc, 2006). The photoproduction of • OH and photochemical activity of marine phytoplankton could be increased by macronutrient additions (Li et al., 2008), and then the ratio between V(IV) and V(V) in seawater was enhanced. Therefore, the bioavailability of V in phytoplankton was increased by macronutrient levels and then transferred to razor clams. 5. Conclusion In our study, a biomimetic digestive tract was used for the sample pretreatment of razor clams and then metal oral bioavailability was assessed by the concentration of affinity-liposome metal. Because the eutrophication was associated with the increasing of algal abundance and debris, metal species transformation in seawater, and metal subcellular distribution, metal bioaccumulation and oral bioavailability in razor clams were influenced by macronutrient levels. Metal overexposure via consumption of coastal bivalves (including razor clams) could be increased by eutrophication. Acknowledgments This work was supported by the National Natural Science Foundation of China (nos. 21175115 and 41206096), the Program for New Century Excellent Talents in University (NCET-110904), and the Science & Technology Committee of Fujian Province, China (2012Y0065). References Amiard, J.C., Amiard-Triquet, C., Berthet, B., Metayer, C., 1987. Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms. J. Exp. Mar. Biol. Ecol. 106, 73–89. Bacon, J.R., Davidson, C.M., 2008. Is there a future for sequential chemical extraction? Analyst 133, 25–46. Brussaard, C., Riegman, R., Noordeloos, A., Cadée, G., Witte, H., Kop, A., Nieuwland, G., Van Duyl, F., Bak, R., 1995. Effects of grazing, sedimentation and phytoplankton cell lysis on the structure of a coastal pelagic food web. Mar. Eco-Prog. Ser. 123, 259–271. Bu-Olayan, A., Al-Hassan, R., Thomas, B., Subrahmanyam, M., 2001. Impact of trace metals and nutrients levels on phytoplankton from the Kuwait Coast. Environ. Int. 26, 199–203. Cai, W.J., Hu, X., Huang, W.J., Murrell, M.C., Lehrter, J.C., Lohrenz, S.E., Chou, W.C., Zhai, W., Hollibaugh, J.T., Wang, Y., 2011. Acidification of subsurface coastal waters enhanced by eutrophication. Nat. Geosci. 4, 766–770. Chen, C., Pickhardt, P., Xu, M., Folt, C., 2008. Mercury and arsenic bioaccumulation and eutrophication in Baiyangdian Lake. China Water Air Soil Pollut. 190, 115–127.

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