Environmental Science Processes & Impacts

Environmental Science Processes & Impacts PAPER

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Bioavailability and oxidative stress of cadmium to Corbicula fluminea Cite this: Environ. Sd.: Processes Impacts, 2013, 15, 860

Jinghua Ren; Jun Luo.*a Hongrui Ma,b Xiaorong Wang,a and Lena Q. Ma a This work set out to study the effects of cadmium (Cd) in sediments on the antioxidant enzyme activities in

the digestive gland of Asian clam Corbicula f/uminea and to explore the potential for applying these

responses to evaluate the Cd-contaminated sediment. Additionally, diffusive gradients in thin-films (DGn technique was used to predict the response of its antioxidant defense system. The sediments,

collected from Taihu Lake, were spiked with Cd at different concentrations (0.72, 0.91,1.62,2.59,11.2, 20.4 and 40.6 mg kg-', dry weight). Asian clam was cultivated for 28 days. Concentrations of Cd in the body of Asian clam had a good relationship with concentrations of Cd in overlying water and sediments, as measured by DGT. Cd affected these biochemical parameters significantly, especially for

superoxide (SOD), peroxidase (POD) and glutathione (GSH), which began to show higher sensitivity and have a significant difference in low dosage (0.91 mg Cd kg-') compared with blank. Contents of MDA and MT, which were induced by Cd, increased with the increasing Cd concentration in sediments and reached peak values at 11.2 and 20.4 mg kg-', respectively, after 28 days exposure. All of these results suggested that biochemical responses cooperated in detoxifying and maintaining cellular metabolic homeostasis. The R2 of regression analysis between the contents of MT and the concentrations of Cd measured by DGT, in sediments and soft body were 0.71, 0.94 and 0.88 after 28 days exposure. This

Received 12th April 2012 Accepted 4th February 2013 DOl: 10.1039/c3em30288a

ne.li/process-impacts

suggested that DGT measurement could predict the response of MT. Cd accumulation, GSH and MT were indispensable biomarkers and the MDA content and DGT appeared to be promising biomarkers. The results dearly indicated that Cd could induce oxidative stress in the digestive gland of Asian clam. The combination of biomonitors with DGT can obtain different information about Cd bioavailability and confirm the significance of applying a suite of biomarkers rather than a selective index to assess the

subleth.1 effect. It .Iso offered theoretic. I methods for the prediction of sediment Cd pollution.

Environmental impact 'Ihis study m.a1ped multi-indees and choae appropriate biomarkers fm Cd. Among these ind~, the superoDde dismutase (SOD), peroDdase (POD) activity and glutathione content involved a detmi.fyingmechanism that appeared to be llensitive to Cd sb'ess and might be the first line of defense against cd stress. The combination of biomonitonl with diffu.ilive gradienlll in thin-fllms teclmique (OOI") to obtain different information about hea'¥)' metals bioavailability was limimt. 'Ihiil study showed that the OOT mealurement could predict biological uptake and the reipoDSe of MT below toxic concentrations. It was useful for pollution asse:5smenL

1

of aquatic ecosystems change. 1 Therefore, the sediment can

Introduction

Sediments are both carriers and sources of metals and nutrients in aquatic ecosystems. Heavy metalsJ including particulate matter, can flow into lakes. The transported heavy metals can be incorporated in sediments via complicated chemical and biological processes. However, they can be released into the water again when environmental conditions, such as the pH and Eh, -state K"JI Laboratory of Pollution Control and Resource Reuse, School of tM

Enviro1f11tent, Nanjing university, 163 Xianlin Avenue, NarUing 210046. PR china. E-maiL' ekxr@1!iu.edu,cn; Fax: +8525 89580351; Tel: +85 Z5 89680351

·sdwol of Resource and Environment. shanxi. University of science and Technology, Xi'an 710021, PR China

860

I Environ. Sci.: Processes Impacts,

2013. 15. 860-869

govern the biogeochemical processes of metals and plays an important role in changing the qualit;y of the overlying water and affecting bioavailability and fate of metals in aquatic environments. In addition, the sediment can give a deep insight into the long-term pollution state of an aquatic environment. Cadmium is an abundant, non-essential element that has been a serious issue because of its accumulation and toxicity to the liver, brainJ lungsJ heart and the central nervous system." The assessment of metal-polluted sediments only based on traditional chemical analysis is difficult due to the complex nature of the sediment matrix and the different exposure routes.a Biomarkers defined as quantitative measures of

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changes at different hiologicallevels and have heen proposed as sensitive early warning signals indicative of exposure to environmental stress;' Recently, many studies have used biomarkers to assess the impact of metal-polluted sediments on health. Biomarkers can be used in a predictive way. This approach could provide a sensitive tool for detennining the available metal fraction and possible toxic effects in an aquatic ecosystem.5-7 Biomarker responses determined vary over the 28 days of exposure time, showing differences between contaminated sites bound to sediments and biomarkers.· Cd can increase reactive oxygen species (ROS) formation and promote oxidative stress in organisms.9 Under normal physiological conditions, ROS generated from metabolism nf extraneous chemicals in the body can be removed well by the antioxidant defense system including SOD, CAT, POD, GSH and MT. However, when ROS generation exceeds the capacity of cellular antioxidants, it will cause oxidative stress and damage, for example, lipid peroxidation {LPO)." Some studies have investigated the response of antioxidant systems to Cd contamination in water....U - 13 Bivalve molluscs are characterized by their very high bioaccumulation capacities for heavy metals among benthic species and are widely used as bioindicators for pollution in marine1 " 15 and freshwater environments.g,Ui Corbicula jluminea is a major component of benthic communities and a filter- and deposit-feeding species buried in the superficial sediment, which makes it a good candidate as a freshwater organism for pollution study. The use of biomarkers has been considered to provide reliable measures of the impact of toxicity. 3 Nevertheless, the widespread use of organisms is limited in different geographic areas.15 Therefore, it is necessary to choose a suitable chemical method, which can provide information concerning the toxicity of pollutants. DGT technique has been developed to measure labile metal species in water, soils and sediments. Measurements by DGT can be considered relevant to study metal bioavailability in water, soils17 and sediments.lII,19 The DGT measurement reflects the fundamental kinetic and capacity properties of the sediment, as well as the concentration in pore water. Several studies have suggested that DGT can reflect the bioavailability of metals in sediments15,20 and soils.2l.22 DGT can sufficiently predict the bioavailability of Zn and Cd in spinach and .:yegrasS.M A significant relationship between DGTmeasured metal concentrations and metal concentrations in ChironomWl riparius was found for Cu and Pb." Bioaccumulation of elements in oligochaete, Lumbriculus variegatus, was correlated to available metal concentrations measured by DGT after being exposed for 28 days to the river sediments." Good correlations were obtained between bnth algal and bacterial growth inhibitions and Chelex-labile copper concentrations.~ Aluminum concentrations determined using DGT (DGT-Al) predicted the gill uptake and the aluminuminduced physiological stress responses of brown trout. The regression statistics showed that the correlation coefficients were high and significant. Thus, the results indicate that DGTAl is a better predictor.26 A series of laboratory experiments was conducted under realistic environmental conditions and a significant positive relationship between Cd concentrations in

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benthic organisms and DGT was found in Cd-spiked sediment. DGT results reasonably predicted Cd uptake by organisms." So far, the sensitive biomarkers were unclear for Corbiculajluminea when it was exposed to Cd.za Therefore, the aim of the present study is to choose sensitive biomarkers by investigating the uptake of Cd, and the potential oxidative stress and damage. The other aim of this work is to evaluate the usefulness of a combination of biomonitoring and DGT techniques for a better understanding of trace metal availability in freshwater sediment.

2

Materials and methods

2.1

Animals and sediments

Adult Corbicula jluminea bivalves, which were scattered and difficult to be caught in Lake Taihu, were collected manually in March 2010 from Lake Hongze, where there was a concentrated culture area in Jiangsu Province, China. On arrival at the laboratory, the clams with the anterior-posterior shell length ranging from 20 to 24 mm were chosen, and then domesticated in a flowing flume (length x width x depth: 300 x 120 x 100 cm). The bottom of the flume was covered with a 3 cm layer of natural substrate from Lake Taihu to make them acclimatize. During domestication, the pH was 7.5 ± 0.5, the temperature ranged from 18 to 25 'C and the dissolved oxygen levels in the water were maintained higher than 5 mg L-1 throughout the experiment by permanent air bubbling. Clams were not fed during this period to avoid possible contamination and were submitted to a natural light cycle. A period of 2 weeks followed before the beginning of the experiment to ensure that they were fully acclimatized to the experimental conditions. The sediment was collected by a Peterson grab from Taihu Lake, then transported to the laboratory immediately and mixed well for further experiment. It was kept at 4 'c before experiments. Polyethylene plastic boxes were filled with 1.0 kg of the homogenized fresh sediment. The background Cd content in the sediment was 0.72 mg kg-I. The sediments were amended with Cd salt stock solution to provide added concentrations (0.72, 0.91, 1.62, 2.59, 11.2,20.4 and 40.6 mg kg-I dry weight) by mixing Cd nitrate salt stock solution with sediment. Three replicates were performed for each concentration level. Then they were mixed sufficiently every day. As metal transferred to the sediment compartment could represent a secondary contamination source for organisms, the control was spiked with ultrapure water (with a resistivity of "'18.2 MQ em) and mixed as well. All sediment samples were incubated for two months before further experiment. Then, 4 L of corresponding lake water was added into each box and allowed to equilibrate naturally for two weeks. During the period of experiment, the upper water was aerated every day. FOrty Corbicula jluminea were cultured in each 7 L plastic tank. They were exposed to the sediment for 28 days. During the exposure period, the conditions were the same as those in the domestication period. No external food was added during the experiment. The clams were collected from each box after 28 days exposure and then they were dissected on ice immediately. The digestive gland and other soft body tissues were removed,

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then frozen in liquid nitrogen and subsequently stored at

absorption spectrophotometry (ZGFAAS; Thermo Sollar M6,

-40°C.

USA).

2.2

DGT measurement

At the start of the exposed experiments, we put Corbicula jluminea into the boxes. When Corbicula jluminea were put into the boxes, DGT devices were deployed on the surface of the sediment with gentle pressure to ensure complete contact between the filter membrane of the device and the sediment for 24 h. On retrieval of the DGT devices, the resin gel was removed and completely immersed in 1 mL of 1 M HNOa for at least 24 h. Appropriate dilution was performed before analysis by inductively coupled plasma mass spectrometry (ICP-MS, ELAN9000, Perkin-Eimer, USA). Metal accumulated by DGT was converted to a DGT-measured concentration according to well established equations.:r.g,30 Then, 10 mL of water sample was collected in every box, close to the sediment surface, and filtered using 25 mm diameter, 0.45 Iffil thick, mixed cellulose filters and stored at 4 °C for further analysis. Sediment samples were then freezedried for analysis. 2.3 Chemicals Bovine serum albumin (BSA), reduced glutathione (GSH), oxidized glutathione (GSSG), thiobarbituric acid (TBA) and nitroblue tetrazolium (NBT) were purchased from Sigma (St Louis, USA). Other reagents were of analytical grade and obtained from chemical companies in China. 2.4 Cadmium determination Metal bioaccumulation of Cd in Corbicula jluminea was analyzed. The soft body was freeze-dried and ground in an acid-cleaned agate mortar. Approximately 0.3 g of biological samples was digested by a mixture of HNo,/HCIO.,"'" then diluted up to 25 mL with ultrapure water. Three replicates per biological sample were used. The freeze-dried sediment samples were ground in an agate mortar and passed through a 0.2 mm sieve. The sediment samples were digested according to the digestion procedure suggested by Wang et al." About 0.3 g of ground sediment sample was digested with a mixture of HCI/HNOa/HF/HCIO•. The diges· tates were diluted in ultrapure water up to 25 mL. Three replicates per sediment sample were used. Certified reference material (CRM) for sediments (GS5-9, Chinese geological reference mate· rials) and biological samples (GSB·15, Chinese geological reference materials) were applied for quality control. The mean ± standard error of our measurements were 0.103 ± 0.012 mg kg-1 for sediments and 1.02 ± 0.054 Ilg kg-1 for biological tissue, which agreed well with the certified values (0.10 ± 0.02 mg kg-1 and 1.06 ± 0.10 I1g kg-') of these standards. A 10 mL water sample was collected in each box at sampling time, close to the surface, and stored in centrifuge tubes. They were filtered through a 25 mm diameter, 0.45 ~ thick, mixed cellulose filter and stored at 4°C. Cd concentrations in biological samples and sediments were measured using atomic absorption spectrophotometry (Thermo Sollar M6, USA). Cd concentrations in water samples and certified reference materials were analyzed using Zeeman graphite furnace atomic

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2013. 15. 860-869

2.5 Biochemical analysis 2.5.1 Ilnzyme extraction and activity assays. About 0.1 g of frozen digestive gland was homogenized after addition of 1.0 mL of 0.01 mol L-, Tris buffer (PH 7.5). The extracts were centrifuged at 104 g for 15 min at 4°C. The supernatant was divided into aliquots and stored at -40°C for further analysis. All operations were conducted under cool conditions. All enzymatic activity assays were finished in 2 days. SOD (EC1.15.1.1) activity was determined according to the method described by Garcia-Limones et al.. M with slight modifications. The 3 mL reaction mixture solution contained 50 mM phosphate buffer (PH 7.8), 130 mM methionine, 750 11M NBT, 100 11M EDTA, 20 11M riboflavin and 50 IlL enzyme extractions. The reaction started at room temperature under continuous light and lasted for 20 min, then As60nm (absorbance at 560 nm) was measured immediately using a spectrophotometer. SOD activity was expressed as unit mg-' protein. One unit of enzyme activity was defined as the amount of enzyme required to inhibit the NET reduction by 50%." POD (EC1.11.1.7) activity was measured by monitoring the formation of tetraguaiacol from guaiacol (A,70..,) in the pres· ence of H 2 O, according to Kochhar and Kochhar." POD activity was expressed as unit mg- t protein. CAT (EC 1.11.1.6) activity was determined according to Sun et aL" The 10 IlL of enzyme extractions was mixed with 3 mL of phosphate buffer (67 mM, pH 7.0) including H2 0 2 (0.16 mL of 30% H2 0 2 to 100 mL ofphosphate buffer). The variation of H 2 O, absorbance in 100 s was measured at 240 nm at 25 ce. One unit of activity was defined as the amount of enzyme necessary to decompose half of the concentration of H 2 O, in 100 s at 25°C. In all cases, the protein content was estimated using Coomassie Brilliant Blue (G-250) by the method of Bradford.u 2.5.2 Lipid peroxidation determination. Polyunsaturated fatry acid peroxides generate MDA. The content of MDA was measured by spectrophotometry using thiobarbituric acid according to Sun et al. 39 The reaction mixture solution containing 0.2 mL of enzyme extractions, 0.2 mL of 8.1% sodium dodecylsulfate, 1.5 mL of20% acetic acid buffer (PH 3.5), 1.5 mL of 1% thiobarbituric acid and 1 mL of ultrapure water was heated at 90 ce for 60 min, then cooled immediately. The absorbance of reaction solution was measured at 532 nm using a spectrophotometer (UVllOO, Tianmei, China). The MDA content was calculated using a molar extinction coefficient of 1.56 x 105 M-t cm- t , The content was expressed as nmol TBARS mg- t protein. 2.5.3 Glutathione determination. About 0.1 g of frozen digestive gland was homogenized after addition of 0.5 mL of 100 mM sodium phosphate-EDTA buffer (1.0 mM EDTA and pH 8.0) and 0.5 mL of 6.5% trichloroacetic acid. The extracts were centrifuged at 12 000 g for 15 min at 4°C. The supernatant was divided into aliquots and stored at -40 cc for further analysis. The levels of GSH and GSSG in digestive gland were deter· mined according to Hissin and Hilf with some modifications....

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Paper The assay mixture contained 100 IlL of supernatant, 1.8 mL of phosphate-EDTA buffer and 100 ilL of 1 I1g ilL-1 ophthaladehyde (OPT) solution. They were mixed completely and incubated at room temperature for 15 min, and then the fluorescence value of the solution was recorded at 420 nm using an excitation wavelength nf 350 nm by a fluorescence spectrophotometer (F-7000, Hitachi, Japan). To measure GSSG content, an aliquot of 0.5 mL of the supernatant was incubated at room temperature with 200 IlL of 0.04 M N-ethylmaleimide (NEM) for 20 min. The 100 J,.IL mixture was taken for measurement of GSSG, similar to the procedure of GSH measurement, except that 0.1 M NaOH replaced phosphate-EDTA buffer. The standard solution nf GSH and GSSG was used for calculating the content in the digestive gland. 2.5.4 Metallothionein determinatiolL We used an adaptation of the Cd-saturation method reported by Baudrimont et aL J 41,42 and Eaton and ToalG to measure MT content. Several important changes in the procedure were made, notably the replacement of Hg by Cd to avoid Hg evaporation. About 0.1 g of tissue sample was homogenized in 0.8 mL of 0.1 M Tris-HCI (PH 8.6, 0.25 M saccharose). This step was performed in a sealed box filled with pure nitrogen in order to avoid MT degradation by oxidation and molecular polymerization,M, and then on ice to inhibit protease activity. The homogenate was centrifuged at 12 000 g for 60 min at 4°C. The 200 IlL supernatant and 20 IlL of 0.25 mg mL-1 Cd solution were added into centrifuge tubes, mixed completely and incubated at room temperature for 10 min. The 200 ilL of 2% bovine hemoglobin was added and heated for 2 min at 100°C to scavenge excess Cd not bound to the MT. The mixture was cooled on ice for 5 min and centrifuged at 12 000 gfor 5 min at 4°C. This procedure (from adding hemoglobin to centrifugation) was repeated three times to remove excess Cd. The last quantitative supernatant was diluted and analyzed for Cd by Zeeman graphite furnace atomic absorption spectrophotometry (ZGFAAS; Thermo Sollar M6, USA). MT concentrations in tissue samples were expressed as Ilg of Cd-binding sites g-l(fresh weight). Owing to the fact that the exact quantity nf Cd binding sites per MT molecule is unknown for this species, MT concentrations cannot be expressed directly in nmol MT g-l (ref. 9) so MT was defined as I1g Cd g-1 fresh weight (FW). 2.6 Statistical methods Results were presented as mean ± standard deviation (SO). Statistical analysis was perfonned using SPSS 13.0, one-way ANOVA and the least-significant-differences (LSD). Difference from the control was considered significant when p < 0.05 and highly significant when p < 0.01. The fignres were plotred in Microsoft EXcel 2007.

3 Results 3.1 Measurement of Cd concentrations Cadmium concentrations in the sediment, overlying water (OW), DGT and soft body of Corbicula jluminea exposed to different Cd concentration levels for 28 days were listed in


Environmental Science: Processes & Impacts Table 1. Cadmium spiked in the sediment could transfer into overlying water leading to Cd concentration increasing from 0.5311gL-1 to 2.87 11g L-1 in the overlying water. Concentrations of cadmium measured by DGT in the surface sediment increased from 0.04 to 1.87 Jlg L-1. Concentrations of Cd measured in the soft body of Corbicula jluminea, a function nf the different contamination levels nf the sediment, ranged from 3.03 to 11.4 mg kg-1 dw. The accumulation of Cd in the soft body increased with the exposure concentration. The maximum Cd concentration in Crobiculajluminea was 11.4 ± 1.62 mg kg-1 dw at 40.6 mg kg-1 Cd in the sediment, which was about 3 times higher than that at 0.72 mg kg-1 Cd (3.03 ± 0.32 mg kg- 1 dw in clam) in the sediment. The relationships of Cd accumulation in clams with Cd concentrations measured by DGT was explored using simple linear regression, which was presented in Fig. 1. The regressive analysis demonstrates that Cd content in the Corbiculajluminea had good correlation with the Cd concentration measured by DGT. For DGT detennination, the correlation coefficient, If was 0.87. The results showed that bioaccumulation was proportional to the Cd concentration in the sediment. There was an interesting phenomenon that the accumulation rate of Cd in clams at a higher sediment Cd concentration level became lower than at a lower concentration level. 3.2 Effects nf Cd on antioxidant enzymes It is well known that organisms have developed antioxidant

systems involving antioxidant enzymes or free-radical traps as protection against oxidative stress." The digestive gland antioxidant responses of Corbicula jluminea to Cd exposure were shown in Fig. 2. Compared to the control group, the SOD activity was significantly decreased at 0.91 mg kg-1 (p < 0.05). Generally, the activity decreased as concentrations nf Cd in sediments increased except at 11.2 mg kg- 1 Cd in the sediment. The SOD activity decreased significantly by 30% at the highest Cd concentration (40.6 mg kg-1) in the sediment. POD activity was induced significantly (p < 0.05) at the low Cd concentration range (0.91, 2.59 and 11.2 mg kg-1) in the sediment. Then the activity decreased at 20.4 mg kg-1, but was still higher than the control group. The lowest POD activity appeared at 40.6 mg kg-" decreasing by 29% compared to the control. CAT activity had a similar change pattern. It was induced at lower concentration, then significantly reduced at 40.6 mg kg-1 (p < 0.01) after 28 days exposure. 3.3 Effects nf Cd on glutathione content Glutathione content and the GSH/GSSG ratio were shown in Fig. 3. GSH content in treated groups was not significantly different from the control, except at 1.62 mg Cd kg- 1 in the sediment. It increased at lower Cd concentrations, and then decreased in the higher Cd concentration range (2.59-40.6 mg kg-1). It had the lowest content at 40.6 mg Cd kg-1 in the sediment. GSSG content in the treated groups had the same change trend as GSH. The GSH/GSSG ratios in all treated groups were inhibited as compared to the control group and reached

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Concentrations of Cd measured in the sediment, overlying water, and clam by DGT

Concentrations (mean ± SD) Index

CK

[Cdj-sediments a [Cdj-overlying watet' [Cdj-DG'I" [Cdj-C. jluminea-28da

0.72 0.53 0.04 3.03

a

mg kg- 1 (dw).

b

1

± ± ± ±

0.01 0.03 0.00 0.32

2

0.91 0.56 0.16 3.17

± ± ± ±

0.11 0.01 0.03 0.15

3

1.62 0.61 0.16 3.65

± ± ± ±

0.04 0.10 0.08 0.16

+

12

~

10

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01)

~

58 15'"

y = 13.0 X + 1.86 R'= 0.87

.D

a::

~

6

3.5

" -5 .5 :;;

S2.

0.15 0.04 0.01 0.11

11.2 0.85 0.33 6.61

± ± ± ±

0.98 0.04 0.04 0.42

20.4 1.37 0.95 8.91

6

± ± ± ±

0.56 0.25 0.04 0.97

40.6 2.87 1.87 11.4

± ± ± ±

0.52 0.40 0.08 1.62

the sediment. The MT content was significantly induced at 11.2 mg kg- 1 and had a maximum value at 20.4 mg Cd kg- 1 in the sediment, which was twice as high as the control. Fig. 6 illustrated the relationship between MT content and Cd concentration in the sediment, Cd accumulation in Corbicuia fluminea and Cd concentration measured by DGT. The correlation coefficients were 0.49, 0.42 and 0.27, respectively, and they increased significantly except for 40.6 mg kg- 1 after 28 days exposure, which were 0.94, 0.88 and 0.71, respectively. MT content increase was a function of lower Cd concentration in different media.

4

4 2.5

+--L__.----__----.-____~ 0.2

0.1

0.3

o +---------~--------~--------~--------~

o

0.5

I

1.5

[Cd] OGT (flgIL)

Fig. 1 The relationship between Cd concentrations in the soft body and Cd extracted by DGT.

a minimum value at 2.59 mg kg-i. It remained constant with increasing Cd concentration in the sediment. 3.4 Effects of Cd on lipid peroxidation In this study, the LPO level in the digestive gland of Asian clam was determined by the production of MDA, which was commonly used as an indicator of oxidative stress in biological systems. 45 Fig. 4 demonstrated the MDA measurement in the clams exposed to different Cd contamination levels. It showed that the MDA content was significantly increased in the lower Cd concentration range (0.72 to 2.59 mg kg-i) and had a maximum value at 11.2 mg Cd kg- 1 in the sediment, which was about twice as much as the control. Then the MDA content decreased with increasing Cd exposure concentration, but was still higher than the control group, which indicates Corbicuia fluminea suffered from oxidative damage. 3.5 Effects of Cd on MT content Fig. 5 displayed the MT concentrations in the digestive gland of Corbicuiafluminea induced by different Cd concentrations after 28 days exposure. It showed a clear dose-effect relationship and MT contents were elevated with increasing Cd concentrations in

864

± ± ± ±

5

J.!g L-1.

14 ,----------------------------------------.

."

2.59 0.59 0.15 3.85

4


2013, 15, 860-869

Discussion

It has been suggested that the Asian clam (Corbicuiafluminea) is

a suitable biological monitor of potentially toxic trace metals. The suggestion comes primarily from comparisons between its own and environmental concentrations of metals, such as in water and sediment. 46 Measurements of metal accumulation are useful in relating chemical exposure to biologically relevant effects in organisms. It has been reported that metals could remain for a long time in tissue, but depuration of metals often varies among different species. In the present study, Cd accumulation in soft body tissue increased with increasing Cd concentration in the sediment. The concentration of Cd in sediment and overlying water did not reach the Cd threshold concentrations to clams. In our study, the highest concentration (2.87 J.lgL-1) of Cd in the overlying water was in the range of the lower Cd treatment in the research of Barfield et ai. 47 However, the accumulation rate of Cd in clams at a higher sediment Cd concentration level became lower than at a lower concentration level, suggesting that the threshold value, which needs further investigation, was near. Similar results were shown in other literature reports. Barfield et ai. 47 reported that Asian clams in lower cadmium treatments (from 3 to 6 J.lg L-1) accumulated more cadmium than those in higher treatments (from 12 to 25 J.lg L-1) for the same exposure time in aqueous solution. However, threshold concentrations are unknown. Baudrimont et ai. 48 observed that Cd bioaccumulation in the molluscs was strongly dependent on the contamination level of the water column and on the exposure duration when they were exposed to three Cd levels including 0, 5 and 35 J.lg L-1. The bioaccumulation kinetics revealed a linear evolution at 5 J.lg L-1 and a marked plateau tendency at the 35 J.lg L-1 level. These results were in agreement with our result. Barfield et ai. 47


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Paper 60

8.0 7.0

**

'>:'

~ 6.0

-.§

2-

5.0

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4.0

~

3.0

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55

2.0 1.0 0.0

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1.62

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20.4

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40.6

[Cd] in the sediment (mglkg dw)

[Cd] in the sediment (mglkg dw) 250

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**

200

r2-

150

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100

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o

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0.91

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[Cd] in the sediment (mg/kg dw)

[Cd] in the sediment (mg/kg dw) 0.7

0.6 0.5

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[Cd] in the sediment (mg/kg dw) Fig. 2

The effects of Cd on the activity of SOD, CAT and POD in the digestive

0.91

1.62

2.59

11.2

20.4

40.6

[Cd] in the sediment (mglkg dw)

40 .6

Fig. 3

The effects of Cd on the content of GSH and GSSG, and the GSH/GSSG

ratio in the digestive gland of Corbicula fluminea after 28 days exposure.

gland of Corbicula fluminea after 28 days exposure.

inferred that Asian clams might detect metals at lower concentrations and effectively avoid exposure by reducing the volume of water used for respiration. Valve closure has been observed in several mollusc species exposed to elevated pollutant levels and resulted in reduced metal uptake and accumulation at higher exposure concentrations. 47,4' The result indicates that the clam has a physiological mechanism which controls the uptake of Cd from the environment to avoid the


high accumulation of Cd in its body at higher contamination levels. Labile Cd concentration in the sediment was measured with the technique of DGT including ionic forms and easily dissociable complexes. These species were more likely to represent bioavailable fractions than the total metal. DGT devices are able to pre-concentrate dissolved trace metals furnishing a timeintegrated measure of their levels in water. In order to evaluate whether the Cd concentration measured in soft body of Asian

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