Bioaccumulation of cadmium bound to ferric hydroxide and particulate organic matter by the bivalve M. meretrix

Environmental Pollution 165 (2012) 133e139

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Bioaccumulation of cadmium bound to ferric hydroxide and particulate organic matter by the bivalve M. meretrix Xing Wu a, b, Yongfeng Jia a, *, Huijie Zhu a a b

Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China Central Laboratory, Shandong Academy of Agriculture Science (Shandong Key Laboratory of Test Technique on Food Quality and Safety), Jinan 250100, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 September 2011 Received in revised form 18 January 2012 Accepted 22 February 2012

Ferric hydroxide and particulate organic matter are important pools of trace metals in sediments and control their accumulation by benthic animals. We investigated bioaccumulation of cadmium in bivalve Meretrix meretrix by using a simplified system of laboratory synthesized iron oxides and commercially obtained humic acids to represent the inorganic and organic matrix found in nature. The results showed that bioaccumulation characteristics were distinctly different for these two substrates. Bioaccumulation from ferric hydroxide was not observed at 70 and 140 mg/kg, while the clams started to absorb Cd at 140 mg/kg from organic matter and the bioaccumulation rate was faster than that from ferric hydroxide. Within 28 d, accumulation of Cd from organic matter appeared to reach a steady state after rising to a certain level, while absorption from ferric hydroxide appeared to follow a linear profile. The findings have implications about the assimilation of trace metals from sediments by benthic animals. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Bioaccumulation Cadmium Ferric hydroxide Particulate organic matter Meretrix meretrix Linnaeus

1. Introduction Metals discharged into aquatic environments are readily transported to the sediments resulting in concentrations that are often several orders of magnitude greater than those in the overlying water column (Burton, 1991). Recent studies have demonstrated that sediment ingestion, and thus absorption of metals from particles, is an important way of metal accumulation for aquatic animals (Farag et al., 1994; Wang and Fisher, 1999). The bioavailability of trace metals in sediments is influenced by various physical-chemical and biological factors, such as particle size, amount and type of minerals and organic matter, affinity of the contaminant to the particulates, uptake route, etc (Lee, 1992; Eggleton and Thomas, 2004; Kukkonen et al., 2004; Croisetiere et al., 2006). Among these factors, the speciation and occurrence of metals play a very important role in bioaccumulation of metals by animals (Bryan and Langston, 1992; O’Day et al., 2000). The way of metal associated with various sediment components determines to a large extent whether the metals can be absorbed by the benthic animals. Generally, the important substrates binding trace metals in oxidized estuarine sediments are ferric oxyhydroxides, organic matter, and to a less extent, manganese (IV)

* Corresponding author. E-mail address: [email protected] (Y. Jia). 0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2012.02.023

oxides (Lion et al., 1982). Compared with Fe oxides, contaminants bound to Mn oxides have been reported to be considerably more bioavailable because of the greater Mn-reducing tendency of gastro-intestinal enzymes (Turner and Olsen, 2000). Similar results were also observed from experiments tracing the uptake of radiolabelled metals by the clam Macoma balthica (Harvey and Luoma, 1985; Luoma, 1989), where the transfer factor of 110mAg from manganese oxides was 100 times greater than the 110mAg bound to iron oxides. When iron oxides were dominant components of the sediment, Cd uptake from ingestion was not detected; however, when clams were exposed to natural sediments containing less than 4% of iron oxides, accumulation of Cd from ingested material reached 57% of the total uptake (via dissolved phase and ingested food) (Luoma, 1989). Thus, iron oxide was considered as one of the inert sinks for the metals in sediment and controlled the bioaccumulation of trace metals. The influence of organic matter on the bioavailability (positive or negative) was not consistent among experiments (Griscom et al., 2000). For example, Hg and Ag accumulation deceased with elevated TOC of some sediments (Langston, 1982). On the other hand, Schlekat et al. (2000) demonstrated that the presence of bacterial exudates increased the assimilation of Ag and Cd (Schlekat et al., 2000). This discrepancy may be attributed partly to different properties of various organic matter, i.e. humic acid, algal, bacteria, etc. Humic acid is considered as the most important source of organic matter in sediments from aquatic systems (Rand et al., 1995). It represents an

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X. Wu et al. / Environmental Pollution 165 (2012) 133e139

operational defined species of organic matter that is relatively refractory to animals and microorganisms. It is an important pool for metals and other contaminants. Decho and Luoma (1994) reported that Cd and Cr bounded with humic substance were difficult to be absorbed by the clams M. balthica and P. amurensis. Cd assimilation from sediment was also reduced by humic acid coating for the polychate Capitella (Selck et al., 1999). In general, the presence of refractory humic matter in sediment would decrease bioavailability of trace metals and hence controls the bioaccumulation of metal in benthic animals. However, some studies reported that metals (Cd, Co, and Ag) associated with particulates coated with humic acid and fulvic acid were assimilated to a greater extent than metals absorbed on uncoated particles by the mussel M. edulis (Gagnon and Fisher, 1997). Although iron oxides and humic acid can serve as sinks for trace metals in sediments, they have different biological responses and this may leads to different bioaccumulation characteristics of the metals bound to them. For example, compared to inorganic particulates, organic materials may have a higher ingestion rate (Granberg and Forbes, 2006). Previous research also showed an “active” and “passive” assimilation for organic particles (e.g. algae) and inorganic particle (e.g. silt) respectively. Organic particles are more intensively digested by the mussel Mytilu trossulus (Arifin and Bendell-Young, 2000). The presence of organic matter may stimulate secretion of enzymes capable of competing directly with ingested particles for absorbed metals and enhancing solubilization of the substrate (Harvey and Luoma, 1985). Organic matter can also be preferentially ferried to intracellular digestion, hence resulting in greater assimilation of associated metals (Decho and Luoma, 1996). These biological processes will affect the bioaccumulation of trace metal absorbed on iron oxides and humic acid. To our knowledge, there is scarce research investigating the difference of bioaccumulation mechanism between these two components. In the bioaccumulation experiment, it is ideal to maintain the chemical concentration constant during the test (Arnot and Gobas, 2006). Most researches on bioaccumulation of heavy metals from dietary exposure chose species of static particle feeders (deposit feeders) as biomonitors, e.g. crustaceans, polychaetes. Few investigations were reported on the bioaccumulation of heavy metals over a period of exposure time from constantly fed particulates using suspension-feeders as biomonitors. In the present work, we used a simplified system of laboratory synthesized iron oxides and commercial humic acids to represent typical inorganic and organic matrix found in nature. One of the most toxic heavy metals, cadmium, was used as the trace metal contaminant. Bivalve Meretrix meretrix Linnaeus was selected as the biomonitor. It is a common shellfish in Chinese fishery industry and widely consumed around the world. It usually inhabits in sandy substrates of lower intertidal and shallow subtidal areas. Due to its economical importance and sensitivity to contaminants, it has been used previously as one of the bioindicators in environmental monitoring research (Wang et al., 2005). The suspending system that can maintain the concentration of particulates constant in the container was applied. The objectives of this study were to investigate the difference of bioaccumulation characteristics of Cdadsorbed on ferric hydroxide and particulate organic matter by invertebrate M. meretrix Linnaeus and to understand the mechanisms of various parameters influencing the Cd bioaccumulation in the organisms.

2.4.2. Cd accumulation in M. meretrix from Cd-adsorbed particulates The clams were placed in each of the suspending systems containing 15 L of artificial seawater and 1.5 g of CdeFe(OH)3 and CdePOM (solid/solution ratio of 0.1 g/L). The concentration of dissolved Cd in the suspending system was below 0.5 mg/L. The clams were cultivated at 23
C for 29 days with the suspension changed with freshly prepared artificial seawater and Cd-adsorbed particulates every 24 h. At certain time intervals, four clam individuals were randomly sampled from three parallel running suspending systems and were transferred to clean seawater to evacuate the gut for 3 days, then they were preserved in Teflon sealing bag at 20
C for further analysis. Generally, evacuation of ingested particulates in the gut of the bivalves was accomplished in 72 h (Sokolowski et al., 2005; Shi and Wang, 2004a),

2. Materials and methods

Table 1 Physicochemical property of the humic acids.

for 7 days before they were introduced into the Cd-containing particulates suspending system. During the acclimation period, only the individuals that extended their siphons were used in the experiments. The culture medium used in all experiments was artificial seawater prepared by dissolving sea salt in tap water; the tap water had been dechlorinated for at least 24 h and the initial salinity was adjusted to 25&. The pH of the prepared artificial seawater was 7.7. 2.2. Particulate suspending system The suspending system was designed to maintain the seston concentration constant. It consists of a temperature-regulated barrel with a submerged pump at the bottom. The waterspout of the pump was fixed along the tangent line of the barrel and can induce a sustained spiral upwelling current. A mesh was used to hold the M. meretrix. The system was continuously aerated from the bottom of the barrel to provide oxygen to the animals and to assist suspending the particulates. 2.3. Synthesis of Cd-adsorbed particulates Ferric hydroxide and particulate organic matter were used to investigate the effect of the substrate on the bioaccumulation characteristics of adsorbed Cd. Ferric hydroxide was synthesized according to the method described in the literature (Sims and Bingham, 1968). Particulate organic matter (POM) was prepared from humic acid. Because of their consistent form and properties, commercial humic acid has been used as a surrogate for organic matter in previous researches (Guerrero et al., 2001; Piol et al., 2006; Voets et al., 2004). In this experiment, humic acid was purchased from Sinopharm Chemical Reagent Corporation, some of the characteristic parameters of the humic acid were given in Table 1. 50 g humic acid was suspended in 50 L artificial seawater and left to equilibrate for 2 days. The supernatant was discarded to remove the dissolvable fractions. This process was repeated several times to ensure that only POM was used in the experiment. The particle sizes of the substrates were about 100 mm. Both of these particulates can be readily ingested by the bivalves. In the adsorption experiments approximately 1.5 g of ferric hydroxide or POM was suspended in 150 mL of artificial seawater followed by introducing different amounts of cadmium solution to result in different Cd loading concentrations, i.e. 70, 140, and 280 mg/kg. This is relevant to the contaminant levels in the polluted area along China’s east coast (Fan et al., 2002). Analysis of residual Cd in solution showed that the introduced Cd was completely adsorbed on the particulates. The Cdadsorbed particulate are designated as CdeFe(OH)3 and CdePOM respectively. They were prepared and introduced everyday before the suspension was changed with fresh seawater. Considering that Cd may desorb from the particulates during the exposure period, the concentration of dissolved Cd in the suspension was monitored. At Cd loading concentration of 70 and 140 mg/kg, release of Cd was not detected. At Cd concentration of 280 mg/kg, the concentration of dissolved Cd in the suspension was <0.5 mg/L. Further investigation demonstrated that after 15 days of exposure to 0.5 mg/L of Cd solution, the uptake of cadmium by M. meretrix from aqueous phase was not detected (Fig. 1a). Hence, absorption from particulate phase was the dominant way of Cd assimilation in the animal body under the conditions of this work. 2.4. Bioaccumulation experiments 2.4.1. Cd accumulation in M. meretrix from the dissolved phase Cd was added into 25& 0.2-mm-membrane filtered artificial seawater and equilibrated over night prior to experiments. The clams were exposed to 0.5 mg/L of dissolved Cd for up to15 days. The dissolved Cd was refreshed everyday during the uptake period. At various time intervals, four clam individuals were randomly sampled and preserved in Teflon sealing bag at 20
C for further analysis.

2.1. Testing animal

Ash (%)

C (%)

N (%)

C/N

Carboxyl acidity (mol kg1 C)

Phenolic acidity (mol kg1 C)

The testing animal M. meretrix with the body length of 3e4 cm was obtained from a local open market. The bivalves were acclimated in artificial seawater at 23
C

5.5

56.6

1.4

41

4.1

2.0

X. Wu et al. / Environmental Pollution 165 (2012) 133e139

135

both dissolved phase and particulate phase (Thomann, 1981; Landrum et al., 1992):

  dCA =dt ¼ ðku $Cw Þ þ AE$IR$Cf  ðke þ gÞCA

Fig. 1. Cadmium concentration in soft tissues of M. meretrix as a function of exposure time (a) in artificial seawater with 0.5 mg/L of Cd (b) as-received animals in clean artificial seawater. Each point represents the arithmetic mean  1  standard error of four clams. which was also adopted as the depuration time prior to starting an efflux experiment in our work. 2.4.3. Cd efflux following uptake from CdeFe(OH)3 The clams were exposed to 400 mg/kg CdeFe(OH)3 for 18 days. Following the Cd exposure period, the clams were transferred to clean seawater to evacuate the gut for 3 days and then depurated in an enclosed recirculating system for 24 days. Periodically four clams were removed from the deputation vessels, dissected and analyzed for Cd concentration in soft tissue. Each day the clams were fed with 2 mg (dry wt) artificial diet for the maintenance requirement before the suspension was changed with fresh seawater. Usually, metal depuration of bivalves could be described by two-compartment patterns, i.e. a rapid initial loss followed by a much slower loss phase. The efflux rate constants (ke), indicating the physiological efflux, were determined from the slope of the natural log of the percentage of metals retained in the clams and the relatively slower loss period of depuration (6e24 d) (Shi and Wang, 2004b).

2.5. Analysis The clams were defrosted at 30
C for 5 h, and the soft tissues were dissected and dried at 105
C for 24 h. The shell length, shell weight, wet weight and dry weight of the soft tissue were measured. The dried tissues were digested according to the method described in the literature (Xu et al., 1998) and the concentration of cadmium was determined on a Varian AA-240 atomic absorption spectrophotometer (AAS) with the detection limit of 0.05 mg/L. Reference standard samples (NIST 296 e trace elements in mussel tissue) were analyzed and recovery percentages of certified values was 87  8% for Cd. The Cd concentration in the body of M. meretrix was expressed as mg Cd per gram wet weight and the Cd uptake rate by the clams was expressed as mg Cd per gram wet weight per day. The concentration of aqueous cadmium in the suspending system was determined on a Varian AA-880 graphite furnace atomic absorption spectrometry (GFAAS) with the detection limit of 0.5 mg/L.

(1)

Where, (ku$Cw) : absorption from aqueous phase; (AE$IR$Cf): absorption from particulate phase; (ke þ g)CA: efflux and dilution of absorbed Cd. After 15 days of exposure to 0.5 mg/L of dissolved Cd, the uptake of cadmium by M. meretrix from aqueous phase was not detected (Fig. 1a). This is because that cadmium is present mainly as CdCl2, CdCl and other complex in seawater (Stumm and Morgan, 1996). Complexation of cadmium with chloride ions decreases its bioavailability by the clams. Fig. 1b shows that there was no apparent decrease of Cd concentration in the body of M. meretrix during the depuration period. The efflux rate constant (ke) was calculated as 0.005 (r2 ¼ 0.6955, p < 0.0001). Considering the relatively short experiment period, the efflux was assumed not to have a significant impact on the accumulation of Cd. Previous research indicated that except for the green alga Chlorella autotrophica, the ke of Cd associated with different phytoplankton food and natural seston was not significantly different for the clam Ruditapes philippinarum (Chong and Wang, 2000). Similar results were also presented for the oyster Crassostrea rivularis and Saccostrea glomerata (Ke and Wang, 2001). In addition, no significant difference in ke was observed between the dissolved Cd and Cd associated with diatom Thalassiosira pseudonana. However, the ke of Ag between these two distinct bioavailable forms was significantly different. Efflux rates reflected distribution of the metals in physiological compartments of the organism. For Ag, the observed difference of efflux rate was attributed to its different occurrence within the compartments. Specifically, dietary Ag may bind with metal-rich granules (Ag2S) in the cell lining of the digestive tract, and Ag accumulated from the dissolved phase may be stored in the kidneys in insoluble form (Wang et al., 1996). The impact of bioavailability on the efflux process seems especially metal-specific. In the present work, Cd in both treatments was assimilated from dietary pathways, it is reasonable to assume that ke would be similar for these two types of substrates. Dry weight of the clams at the beginning and end of each treatment were determined, no significant variation was observed over the course of the experiment period (Table 2). Therefore, the absorption of Cd from aqueous phase, the efflux of accumulated Cd and the dilution of absorbed Cd (due to the growth effect) were omitted in present work. The accumulation of Cd by the clams in our experiment was controlled by chemical assimilation efficiency from ingested particles (AE), the ingestion rate of the particulate (IR) and the chemical concentration in the ingested particles (Cf), as showed in Eq. (2).

dCA =dt ¼ AE$IR$Cf

(2)

Table 2 The dry weight of the clams at the beginning and end of each exposure period. Substrates

Dry weight (g) 70 mg/kg

140 mg/kg

280 mg/kg

3.1. Accumulation of Cd by the clams from aqueous phase and the efflux of Cd from the clams

Ferric hydroxide

The absorption efficiency, ingestion rate and Cd concentration in the ingested particles are critical factors determining the uptake rate of Cd by M. meretrix. Cd accumulation can be described by the following first-order equation, assuming that Cd is available from

POM

0.278  0.013 (0 day) 0.226  0.014 (22 day) 0.299  0.068 (0 day) 0.292  0.029 (22 day)

0.169  0.015 (0 day) 0.197  0.015 (21 day) 0.335  0.036 (0 day) 0.348  0.020 (21 day)

0.159  0.017 (0 day) 0.174  0.009 (24 day) 0.335  0.036 (0 day) 0.308  0.089 (21 day)

3. Results and discussion

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X. Wu et al. / Environmental Pollution 165 (2012) 133e139

Apparently, absorption from particulate phase was the dominant way of Cd assimilation in this work.

1.6

Accumulation of Cd by the clams through ingesting the Cdbearing particles in suspending system was shown in Figs. 2e4. The “coefficient of variation” was used as the indicator to describe the accumulation trend in each treatment. Generally, accumulation was assumed to be significant when the “coefficient of variation” was above 0.25 within the data set. At Cd concentration of 70 mg/ kg, the accumulation of Cd by clams in CdeFe(OH)3 and CdePOM system was not detectable after 28 days of exposure. When Cd concentration was increased to 140 mg/kg, bioaccumulation of Cd from CdeFe(OH)3 system was still not observed, while for the clams cultivated in CdePOM systems, Cd was apparently assimilated in the body of the animals (Fig. 3). The uptake of Cd appeared to reach a steady state at the 20th day of exposure to the CdePOM suspensions. The uptake rate was determined to be 0.0216  0.0020 mg/g$d by linear fitting of the first period of assimilation (r2 ¼ 0.7957, p < 0.0001) (Table 3). When Cd concentration was further increased to 280 mg/kg, Cd levels in the soft tissues of the clams increased significantly with time for both CdeFe(OH)3 and CdePOM sestons. The uptake of Cd appeared to follow a linear profile in 24 days of exposure to CdeFe(OH)3 particulate suspensions and the uptake rate was determined to be 0.0221  0.0052 mg/g$d by linear fitting of the curve (r2 ¼ 0.8717, p < 0.0001) (Table 3). Similar accumulation trend was observed for CdePOM systems like the case of 140 mg/kg system, the uptake rate was determined to be 0.0596  0.0082 mg/ g$d by linear fitting of the first period of assimilation (r2 ¼ 0.951, p < 0.0001) (Table 3). The accumulation of Cd by clams was different for the two types of particulates at given concentrations. In CdePOM system, the clams start to absorb Cd at 140 mg/kg. In comparison, Cd bioaccumulation was not observed at Cd concentration below 280 mg/ kg for CdeFe(OH)3. The uptake rates of Cd determined kinetically in this work were not suitable for statistical analysis. Apparently, the initial Cd uptake rate from CdePOM was faster than that from

Cd burden µg/g wet weight

3.2. Accumulation of Cd by the clams from ingested particles

0.0 1.6 Cd-Fe(OH)3

b

1.2

0.4 0.0 Exposure time (day) Fig. 3. Cadmium concentration in soft tissues of M. meretrix as a function of exposure time at 140 mg/kg of Cd (a) Cd-POM (b) CdeFe(OH)3. Each point represents the arithmetic mean  1  standard error of four clams. The dash line represents linear regression for the uptake rate.

CdeFe(OH)3 system. The uptake of Cd appeared to reach a steady state after rising to a certain level for CdePOM system and the accumulation was linear for CdeFe(OH)3 systems during the experiment period. As indicated in Eq. (2), at a given Cd concentration, the amount of Cd assimilated by the clams is controlled by two factors, i.e. assimilation efficiency (AE) and ingestion rate (IR). Bivalves have different feeding responses and digestion strategies according to the sesten quantity and quality. The IR may be different for different particulates depending on the feeding behaviors while the AE is strongly influenced by Cd-substrates bonding strength and the digestion behavior of the substrate in the gut, e.g. some particles

0.0 4

8

12

16

20

24

28

1.6

b

Cd-Fe(OH)

0.8

Cd burden µg/g wet weight

0.4

0

Cd-POM

1.2

a

Cd-POM

0.8

g/g wet weight

0.4

1.6

1.2

Cd burden

0.8

0.8

1.6

1.2

a

Cd-POM

1.2

0.8 0.4

a

0.0 0

4

1.2

12

16

20

24

28

Cd-Fe(OH)3

0.8 0.4

0.4

8

1.6

b

0.0

0.0 0

4

8

12

16

20

24

28

0

4

8 12 16 20 24 Exposure time (day)

28

Exposure time (day) Fig. 2. Cadmium concentration in soft tissues of M. meretrix as a function of exposure time at 70 mg/kg of Cd (a) Cd-POM (b) CdeFe(OH)3. Each point represents the arithmetic mean  1  standard error of four clams.

Fig. 4. Cadmium concentration in soft tissues of M. meretrix as a function of exposure time at 280 mg/kg of Cd (a) Cd-POM (b) CdeFe(OH)3. Each point represents the arithmetic mean  1  standard error of four clams. The dash line represents linear regression for the uptake rate.

X. Wu et al. / Environmental Pollution 165 (2012) 133e139 Table 3 Uptake rates of Cd-adsorbed on ferric hydroxide and humic acid by M. meretrix at various loading concentrations. Substrates

Cd uptake rates (mg/g$d) 70 mg/kg

140 mg/kg

Ferric hydroxide

ND

ND

POM

ND

0.0216  0.0020

280 mg/kg 0.0221  0.0052 0.0596  0.0082

*ND: not detected.

may be selectively sent into the intracellular digestion process. Particles ingested by bivalves are usually processed by two-phase digestion. The first digestion phase occurs in the stomach and intestine and is termed as “intestinal digestion” or “extracellular digestion”. In the second phase of digestion process, a proportion of these food particles are further sorted into the digestive gland for more intensive digestion and this process is termed as “intracellular digestion”. The adsorbed cadmium on the surface of the particles can be released in the gastro-intestinal environment due to desorption or substrate solubilization by enzymes and nonenzymatic ligands. Bonding strength between Cd and substrates is an important factor controlling the assimilation efficiency since desorption is a key process determining Cd assimilation in bivalves (Wang and Fisher, 1996). POM prepared from humic acid has greater sorption capability and bonding strength of Cd compared to ferric hydroxide (Aualiitia and Pickering, 1987). However, it was observed in the present work that the initial uptake rate of Cd-POM was higher than that of CdeFe(OH)3. The assimilation of Cd is controlled not only by the bonding strength of the adsorbed Cd with substrates, but also by some biological factors such as distribution between intracellular and extracellular digestive processes, solubility of the substrate in gastro-intestinal environment, gut retention time, etc. The distribution of particles between the two digestive phases especially has a great impact on the assimilation of associated trace metals (Decho and Luoma, 1991, 1996; Roditi and Fisher, 1999). It was reported that w93% of the ingested bacteria were channeled to intracellular digestion by M. balthica; Compared to bacteria, the percentage of natural sediment processed by the digestive gland was only 6%. Correspondingly, the assimilation efficiency of Cr declined from 86% for the bacteria to 1% for the sediments (Decho and Luoma, 1996). It was also reported that assimilation efficiencies were higher for the particulates that underwent more intracellular digestion (Roditi and Fisher, 1999). Particulates with different prosperities were processed with different digestion strategies. The proportion of food processed by the intracellular pathway was much greater for the particulates with higher organic content. Previous researches showed that an appreciable proportion of humic acid coated mineral particulates was sent into the intracellular digestion process by the bivalves P. amurensis and M. balthica at high level of cadmium, i.e. 20 mg/kg dry weight, whereas, Cdbearing ferric hydroxide particulates was not processed by intracellular digestion (Decho and Luoma, 1994). This led to higher assimilation efficiency of Cd associated with humic acid than that associated with ferric hydroxide. Compared to ferric hydroxide, POM may be more nutritious and thus has a higher ingestion rate (Arifin and Bendell-Young, 1997), which can also lead to higher uptake rate of cadmium. Other possible mechanisms also may account for the different Cd uptake rates. The induction of metallothioneins (MTs) by Cd exposure has been extensively reported for aquatic animals. Other than detoxification and homeostasis, MTs also play an important role in metal biokinetics (Wang and

137

Rainbow, 2010). One possibility was that the inducted MTs could sequester intracellular Cd and thereby reduced exchangeable Cd concentration, which subsequently increased the influx of exchangeable Cd cross the apical membrane of the cell. Previous studies indicated that Cd exposure and hence MTs induction may positively influence Cd assimilation efficiency in marine bivalves (Shi and Wang, 2004b). It was reported that the substrates of Cd (mussel and barnacle) had a significant effect on synthesis and concentration of MTs in N. lapillus (Leung and Furness, 2001). In the present work, different Cd-bearing particulates, i.e. ferric hydroxide and POM, may be assimilated by different digestive processes (discussed below) and hence may have different capabilities for MTs induction, consequently affected their Cd assimilation. At 280 mg/kg of Cd concentration, the initial uptake rate of CdePOM was higher than the uptake rate of CdeFe(OH)3. Whereas, the accumulation of Cd from CdePOM appeared to reach a steady state after 13 days of exposure but no such trend was observed for CdeFe(OH)3 systems during the experiment. For marine bivalves which have flexible digestion strategies. Food with higher quality (e.g. particles coated with organic matter), will be held for a longer period in the digestive systems for more intensive digestion (i.e. intracellular digestion), hence, result in a greater uptake of trace metals. However, the animal may regulate the digestion strategy (i.e. extracellular and intracellular) in response to pollution. Under high levels of contamination, for example, the animals will reduce the proportion of particles processed by intracellular digestion, hence, reduced the AE of trace metals (Decho and Luoma, 1996). In our experiment, with increasing Cd accumulation from CdePOM, the clams would repulse the CdePOM particles from entering the intracellular digestion process. The extracellular digestion process, mean while, may not be able to release the Cd from the POM substrate. Thus, when accumulation of Cd reaches a certain level (here in this work w0.7 mg/g wet weight), the uptake of Cd appeared to be controlled relatively constant at that level by the clams in CdePOM systems. Similar experimental phenomena was also observed for the assimilation of Cr by the bivalves P. amurensis and M. balthica, where the absorption of Cr was highly dependent on the intracellular digestion process (Decho and Luoma, 1991). Our results showed that the extracellular digestion process was capable, though not efficient, for the clams to assimilate cadmium from CdeFe(OH)3 at the Cd level of 280 mg/kg. Compared to CdePOM, CdeFe(OH)3 was processed only by extracellular digestion. Arifin and Bendell-Young (2000) showed that the AE of cadmium from the extracellular digestion process remained almost constant in the experiment. This is likely why the assimilation of Cd by the clams exposed to CdeFe(OH)3 suspensions did not follow similar patterns to those in CdePOM systems where Cd accumulation appeared to remain at relatively constant level after certain time of exposure. The extracellular digestion processes was less efficient than intracellular digestion processes for the bivalve Mytilus trossulus to control the Cd assimilation (Arifin and Bendell-Young, 2000). Compared to inorganic particulates, organic matter is more intensively digested by the bivalves. Thus, contaminants associated with organic matter were usually considered to be more bioavailable for bivalves. However, our results indicate that when accumulation of heavy metals reaches a certain level, the bivalves would regulate the distribution of the particles between intracellular and extracellular digestive processes. This response would limit intracellular digestion of the metal-bearing particulates hence controlling further uptake of the contaminants. This suggests that in a long-term exposure process, bivalves may not necessarily accumulate more heavy metal associated with organic matter although it is more bioavailable than that associated with oxide minerals. In the present work, Cd accumulation from CdePOM appeared to reach a steady state after 13 or 20 days of exposure. The results

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indicate that there might be a threshold of tissue Cd concentration for the clams to limit the CdePOM particles from entering the intracellular digestion process. Thresholds for biological responses have been observed in previous researches. Widdows and Page (1993) reported that M. edulis significantly reduced their clearance rate when TBT levels in the tissue increased above a threshold of 4 mg/g dry weight. The activity of a CYP1A enzyme (ethoxyresorufin-o-deethylase (EROD)), a more sensitive biological responses or “biomarkers”, varied directly with PCB concentrations in shorthorn scuplin but would only be inducted above a threshold concentration of 50 ng/g$ww (Kuzyk et al., 2005). Previous studies suggested that some “biological responses” was not instantaneous. For example, the absorption efficiency of M. edulis for organic matter required between 2 and 12 days to respond to changes in the diet (Bayne et al., 1993). In our experiment, the clams also need 13e20 days to respond to the Cd accumulation from the CdePOM particles and the length of the adaptation period depended on the time reaching the “threshold”. Natural organic matter and amorphous metal oxides are major scavengers to sequester toxic trace metals in aquatic environment. In this work, we used simplified materials (i.e. humic acid and ferric hydroxide) as substitutes for naturally occurring organics and amorphous oxide minerals to study bioaccumulation characteristics of their associated trace metals. The clams showed distinctly different digestive strategies toward the two types of materials, hence leading to characteristic assimilation processes of their associated metals. Although the results obtained in lab cannot reflect the exact process undergoing in the real world, the findings have important implications to the natural environment. 4. Conclusion The bioaccumulation characteristics of Cd by bivalve M. meretrix L. was significantly different between CdeFe(OH)3 and CdePOM due to the differences in physiochemical properties of the particulates and the responses of the clam to the particulates. At Cd loading concentrations set in our experiment, i.e. 70, 140 and 280 mg/kg, the clams started to absorb Cd from CdePOM at 140 mg/kg, while bioaccumulation of Cd from CdeFe(OH)3 was not observed until the loading concentration was raised to 280 mg/kg. When accumulation of Cd reached a certain level for the clams exposed to CdePOM, Cd uptake appeared to remain at a steady state while this phenomena was not observed for the clams exposed to CdeFe(OH)3. Hence, at similar level of Cd contamination, bivalve M. meretrix L. may not necessarily accumulate more Cd from CdePOM although this form is considered more bioavailable than that associated with ferric hydroxide. Acknowledgments The supports to this work by National Nature Science Foundation of China (NSFC, 40925011) and Chinese Academy of Sciences (KZCX2–YW-JS405) are gratefully acknowledged. References Arifin, Z., Bendell-Young, L.I., 1997. Feeding response and carbon assimilation by the blue mussel Mytilus trossulus exposed to environmentally relevant seston matrices. Marine Ecology Progress Series 160, 241e253. Arifin, Z., Bendell-Young, L.I., 2000. Influence of a selective feeding behaviour by the blue mussel Mytilus trossulus on the assimilation of Cd-109 from environmentally relevant seston matrices. Marine Ecology-Progress Series 192, 181e193. Arnot, J.A., Gobas, F., 2006. A review of bioconcentration factor (BCF) and bioaccumulation factor (BAF) assessments for organic chemicals in aquatic organisms. Environmental Reviews 14, 257e297.

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