Collection and acclimation of bivalves

Adult T. granosa (9.5 ± 1.4 g), M. edulis (28.0 ± 5.2 g) and M. meretrix (50.0 ± 8.1 g) were collected from Yueqing, Wenzhou, China in August 2014. After cleaning off the epizoa, bivalve individuals were acclimatized in a 1000 L plastic tank at an ambient water temperature of 26 ± 3 °C and pH 8.07 ± 0.05 with flowing sand filtered seawater. The sample were fed with microalgae (Tetraselmis chui) at the satiation feed rate daily for 7 days prior to experiment.

Seawater acidification

The sand-filtered seawater used in the experiment was obtained from Qingjiang Bay, Zhejiang Province (28°28′N and 121°11′E) with pH at 8.07 ± 0.05, salinity at 20 ± 0.5‰ and the average background Cd concentration of 9.8 ± 0.2 μg/L. During the experiment, the bivalves were maintained under manipulated pCO 2 conditions, with one ambient group at pH 8.1 (current concentration of pCO 2 ) as the control and two experimental groups at pH 7.8 and 7.4 representing the pH values predicted by the Intergovernmental Panel on Climate Change (IPCC) to occur at 2100 and 2300, respectively3. The desired pH values were achieved by continuous aeration with ambient air or air-CO 2 mixture into the filtered seawater in 60-L plastic tanks. The air-CO 2 mixture was obtained by mixing dry CO 2 -free air and pure CO 2 gas at known flow rates using flow controllers. The pH of each experimental trial was verified daily with a portable pH metre (Sartorius PB-10) to ensure there was no substantial pH change during the course of experiment.

Cadmium accumulation assay

The experiments were performed using analytical grade salts of Cd (NO 3 ) 2 ·4H 2 O. Stock solutions were prepared in deionized water at 1 M, a concentration high enough to prevent weighing errors and salinity fluctuation. The experimental Cd concentration (0.05 mg/L) was chosen on the basis of the reported safe concentrations of these bivalve species45,46,47.

After one week of acclimation, the bivalves (40 T. granosa, 15 M. edulis and 20 M. meretrix) were randomly assigned to plastic tanks with a total seawater volume of 20 L containing approximately 0.05 mg/L Cd and maintained in the three desired pCO 2 conditions. Bivalves were fed with T. chui and the seawater was replaced daily with pH pre-adjusted seawater to maintain the desired pCO 2 level. After seawater replacement, Cd was added to achieve the designed experimental Cd concentration in the water column. The Cd accumulation assay was conducted for a 30day duration.

Metal concentration analysis

Seawater samples were collected from each experimental trial every ten days to determine the effect of the CO 2 -driven acidification on the concentration of Cd2+ and Ca2+ in the water column. These water samples were stored in properly labelled preparation bottles at 4 °C and were used for subsequent Cd2+ and Ca2+ concentration analyses. The water samples (200 mL) were digested with 5 mL of a di-acid mixture (HNO 3 :HClO 4 = 9:4) on a hot plate and filtered with a glass microfibre filter paper (Advantec Toyo) for the analysis of Cd2+ and Ca2+ using a flame atomic spectrophotometer (WFX-130A, Beijing Rayleigh Analytical Instruments Co., Ltd, China), according to the National Standard of China (GB 17378.4-2007, the section “seawater analysis” in “The speciation for marine monitoring”)48 at detection limits of 0.01 and 0.001 μg/L for Cd and Ca, respectively.

After exposure to Cd-contaminated seawater for 30 days at different pCO 2 levels, five live individuals of each species were taken out for the Cd accumulation analysis. The individuals were dissected on ice and the gills, mantle and adductor muscles were peeled off and weighed separately. To obtain the dry mass, the different tissues were dehydrated in the oven to a constant weight at 75 °C. Dried tissues were first homogenized with a standard Teflon tissue homogenizer, followed by nitric acid digestion (1 g of each sample). Once the samples cooled down to room temperature, the sample digestions were filtered with a glass microfibre filter paper (Advantec Toyo) and diluted to 50 ml in volumetric flasks with deionized water. The concentrations of cadmium were then determined using a flame atomic spectrophotometer (WFX-130A), according to the National Standard of China (GB05009-15-2003)49 with a detection limit of 5 μg/kg. Three replicates were examined for each pCO 2 level to obtain the average concentration. The Cd concentrations in the various tissues were then calculated and expressed in mg kg−1 dry weight. Similarly, after 30 days exposure, the entire soft body of M. meretrix was peeled off to determine Cd concentration (C) for THQ analysis. Cd concentrations in the whole soft body were then calculated and expressed in mg kg−1 wet weight.

Appropriate quality assurance procedures and precautions were carried out to ensure results reliability. Samples were carefully handled to avoid contamination, all the plastics and glasswares were cleaned by soaking in dilute HNO 3 and then rinsed with distilled water prior to use and reagents of analytical reagent grade were used. A standard reference materials (GBW08571) obtained from the National Research Center for Standard Reference Materials (Beijing China) was used in the analysis to ensure measurement accuracy. A recovery experiment was carried out by spiking the already analyzed sample and recoveries were found to be within ±5% of certified values.

Expression analysis of pgp-5 gene

Total RNA of T. granosa was extracted from the gills, which were considered the main entry site for toxic metals in bivalves, with the RNAprep Pure Tissue Kit (Tiangen, DP431) according to the protocols provided by the manufacturer. RNA integrity was checked by gel electrophoresis and quantified spectrophotometrically with NanoDrop 1000 UV/visible spectrophotometer (Thermo Scientific). First strand cDNA was synthesized from high-quality total RNA using the M-MLV First Strand Kit (Invitrogen, C28025-032) following manufacturer’s instructions. Real-time quantitative PCR were conducted on the CFX96TM Real-Time System (Bio-Rad) in triplicates, in a total volume of 10 μL consisting of 5 μL of 2× Super Mix (Bio-Rad, 172-5201AP), 0.5 μL of each primer (10 μM), 1 μL of cDNA template and 3 μL of double-distilled water. The following amplification protocol was used: 95 °C for 5 min, followed by 40 cycles (94 °C for 20 sec, 61 °C for 20 sec and 72 °C for 20 sec). A melting curve analysis (MCA) was used to confirm the specificity and reliability of the PCR products. The 18S rRNA was employed as a reference for the calculation of the relative expression levels. The primers used are listed in Table 3 and all primers were synthesized by Sangon Biotech (Shanghai, China).

Table 3 Primers sequences of genes used in real-time PCR analysis. Full size table

Health risk assessment

The EDI of Cd was determined by the equation: EDI = (EF r × E D × MS × C)/(W AB × T A ), where EF r is the exposure frequency50 (350 day/year); E D is the exposure duration (70 years), which is equivalent to the average lifetime of adults51; MS is the average food meal size (21.43 g/person/day according to the dietary intake survey52); C is the obtained Cd concentration in the soft body of M. meretrix; W AB represents the average body weight53, adults (70 kg); and T A is the average exposure time for noncarcinogens (70 years × 365 day/year according to Wang et al.54). The THQ values of Cd through consumption of M. meretrix raised in different pCO 2 levels were then estimated by the equation: THQ = EDI/RfD with the data obtained in the present study. Accoding to JECFA55, 0.83 × 10−3 μg/g/day was used as the oral intake reference dose (RfD).

Statistics analysis