Partitioning of Cd, Zn and Fe in the tissues and cytosols of blood cockles (Anadara granosa) from the Gulf of Thailand

C. Rattikansukha1, G.E. Millward2,*& W.J. Langston3

1School of Engineering and Resources, Walailak University, Tha Sala, 80161, Thailand

2Marine Institute, Plymouth University, Drake Circus, Plymouth PL4 8AA, UK

3Marine Biological Association of the UK, Citadel Hill, Plymouth PL1 2PB, UK

*Corresponding author (G.E. Millward):

gmillward@plymouth.ac.uk: Tel: +441752584779

Acknowledgements

Dr C. Rattikunsukha thanks the Ministry of Resources and Environment, Bangkok, Thailand for funds to carry out this research and to colleagues at the Pollution Control Department for field and laboratory support. Professor G. Millward thanks the Scientific Committee on Oceanic Research (SCOR) for a Visiting Scholarship to Walailak University, Thailand. Dr W. Langston is grateful to the Marine Biological Association for an Associate Fellowship and the facilities to carry out this work.

Abstract

Anadara granosa is a bivalve blood cockle occurring abundantly in tropical estuarine foreshores with high densities found predominantly in intertidal sediments. The tissue-specific accumulation of Cd, Zn and Fe and their partitioning at the sub-cellular level in the cytosol, has been investigated in the gills, digestive gland, body and foot of A. granosa from seven contrasting estuaries in the Gulf of Thailand. The concentrations of metallothionein-like protein (MTLP), particularly important for binding Cd, were found to be in the range 1-11 mg g-1 (dry weight) with the highest obtained in the digestive gland. Cd and Zn were bio-accumulated predominantly in the gills with smaller concentrations distributed equally throughout the other tissues, except Zn in the foot. Iron was present at relatively low concentrations in gills but was higher, and more evenly distributed, in the digestive gland, body and foot. Partitioning of the metals in the cytosols between low molecular weight (LMW) compounds, metallothionein-like proteins (MTLP) and high molecular weight compounds (HMW) indicated that 16-39% of total Cd, 6.5-15% of total Fe and <6% of total Zn was bound with MTLP. Of the total Zn, 23-27% was bound with the HMW fraction, ascribed to its association with the blood pigment haemoglobin (Hb) which is used as a mechanism for coping with sub-oxic conditions found in estuaries draining into the Gulf of Thailand. This metal-handling strategy, involving Zn, is likely used by blood cockles elsewhere to support their oxygen intake in tropical waters under-saturated in dissolved oxygen.

Keywords: Bioaccumulation; blood cockles; cytosols; Gulf of Thailand; metals; oxygen depletion; sediments

Introduction

Estuarine and coastal sediments are important habitats supporting diverse, dense assemblages of marine invertebrates where organisms can acquire dissolved and particulate metals as part of their diets (de Sousa Machado et al., 2016). Research, covering three decades, on the uptake of metals by molluscs, their biochemical functioning, and the implications for their health has focused on whether metals are utilized or detoxified, particularly via mechanisms involving their sub-cellular compartmentalization (Marigómez et al., 2002). The gills of invertebrates are the key interface for metal uptake, assisted by the synthesis of metal-binding metallothioneins, lysosome incorporation and release into the blood plasma and circulating hemocytes. Metal-binding proteins, including metallothionien-like-protein (MTLP) and transferrin-like proteins, together with the hemolymph cellular components, augment metal transport and storage. Incorporation into the sub-cellular tissues of organisms involves selective binding, including compounds with high molecular weight (HMW) compounds, MTLP, low molecular weight compounds (LMW) and insoluble components, such as metal-rich granules (MRG), consisting of cellular debris and organelles (Wallace et al., 2003). While it is evident that extensive research has been carried out on sub-cellular processes in many invertebrates it is surprising that the sub-cellular functioning of Anadara granosa is poorly understood.

The blood cockle, Anadara granosa (=Tegillarca granosa of the family Arcidae), has a widespread occurrence in tropical and sub-tropical waters. This species is adapted to survive in waters where the salinity is above about 14 and the water temperature is in the range 20-30oC (Davenport and Wong, 1986). They are found at high densities in mangroves, estuaries and coastal waters, predominantly in intertidal, organic-rich mixtures of sand and mud, where it is captured and farmed for human consumption (FAO Yearbook, 2016). The predominant visual characteristic of A. granosa is the red colour of its tissues due to the presence of extra-vascular erythrocytes containing the protein haemoglobin (Hb), carried within the haemolymph, and intended to assist in the acquisition of dissolved oxygen. This distinctive feature contrasts with cockles found in temperate waters, i.e. Cerastoderma edule and C. glaucum, which are able to remove dissolved oxygen from the water directly into their tissues, via the gills, without the requirement for an oxygen transporting pigment (Malham et al., 2012). The fundamental component of the Hb present in A. granosa, in common with that in vertebrates, is a tetrameric polymer with a molecular weight of the order 65 kDa formed from sub-units in the range16 kDa, each containing an Fe atom (Weber and Vinogradov, 2001). This form of Hb expression appears essential to the organism’s survival in tropical and sub-tropical seawater which may be under-saturated in dissolved oxygen due to biochemical oxygen demand from a combination of natural and anthropogenic organic compounds. Consequently, A. granosa may tolerate dissolved oxygen concentrations in the range 2.8-7.2 mg L-1 (Eapen and Patel, 1989). Furthermore, controlled laboratory experiments in seawater, depleted in dissolved oxygen, have shown that A. granosa (Davenport and Wong, 1986), and other blood cockles such as Scapharca (=Anadara) inaequivalvis (van den Thillart et al., 1992), are adapted to estuarine hypoxia.

The concentrations of metals in the tissues of Anadara spp. have been widely investigated, for example, in the coastal waters of Australia (Taylor and Maher, 2012), Brazil (Silva et al., 2006), India (Patel et al., 1985), Malaysia (Abbas-Alkarkhi et al., 2008; Ibrahim, 1995; Ong and Din, 2001; Yap et al., 2008) Thailand (Phillips and Muttarasin, 1985; Pradit et al., 2015; Sudsandee et al., 2017) and Vietnam (Tu et al., 2011). However, assessments of the tissue-specific distributions of metals in Anadara spp. are sparse. A. granosa from Malaysian waters had relatively high metal concentrations, particularly for Cd, Zn and Fe, in the mantle and gills, as compared to other organs. Moreover, bioaccumulation in the animals appeared related to total metal concentrations within the sedimentary environment (Yap et al., 2008). Similar findings were obtained for the distributions of Cd, Zn and Fe in the tissues of Scapharca (=Anadara) broughtonii (Edward et al., 2009). Laboratory experiments on the biokinetics of uptake and depuration of metals by A. granosa have been carried out (Srisuksawad and Prasertchiewchan, 2007). The induction of MTLP by sub-leathal doses of dissolved Cd have shown preferential partitioning into the gills, hepatopancreas and whole tissues of the animals (Chan et al., 2002).

The extensive research, mentioned above, on the distribution of metals within the tissues of Anadara spp. from coastal waters across the World raises important issues. In particular, the extent to which A. granosa utilizes or regulates dissolved and particulate metals taken up from water, their distributions within its tissues and the role of metals at the sub-cellular level. Our objective was to examine the tissue-specific distributions and the partitioning of Cd, Fe and Zn among various molecular weight fractions of the cytosols of A. granosa from seven contrasting coastal sedimentary environments in the upper Gulf of Thailand; hence to begin to understand the relative toxicological significance of accumulated body burdens. The metals chosen for investigation were Cd as a non-essential, potentially harmful, metal; Zn as a potentially essential nutrient element, and Fe because of its association with Hb which also acts as an antioxidant and a regulator of Fe metabolism.

2. Materials and Methods

2.1 The Gulf of Thailand and river inputs

The Gulf of Thailand is a semi-enclosed sea extending from the shallow region of the South China Sea to the northwest between the Malay Peninsula and Indo-China (Fig. 1). The rivers flowing into the upper Gulf are the Petch Buri, Mae Klong, Ta Chin, Chao Phraya, Bang Pakong and, in the east, the smaller system of the Rayong. The elevated fluvial inputs contribute to a relatively low coastal salinity (30-33), along with elevated temperatures (23-32oC), and the sediments are sandy mud (Boonyatumanond et al., 2006; Supplementary Data Table S1). The wind-driven, anti-cyclonic currents are generally weak and variable. Consequently, the Gulf is poorly flushed and its coastal waters tend to be under-saturated in dissolved oxygen (Cheevaporn and Menasveta, 2003; Islam and Tanaka, 2004).Theoretically, dissolved oxygen concentrations (at 1 atmosphere pressure) for the range of salinities and temperatures given above are in the range 6.1 to 7.2 mg L-1 (Benson and Krause, 1984). However, concentrations of dissolved oxygen reported in the coastal waters of the Gulf were 3.2 to 5.9 mg L-1 (Censi et al., 2006) and at the river mouths were 1.6 to 6.7 mg L-1 (Cheevaporn and Menasveta, 2003; Supplementary data Table S1). The Bang Pakong was persistently under-saturated in dissolved oxygen during both the dry and wet seasons (Bordalo et al., 2001).

The Chao Phraya has the largest freshwater input (annual mean 960 m3 s-1) and is a major source of industrial and domestic wastes from the megacity of Bangkok. The concentrations of dissolved Cd in various waterways of the Chao Phraya are in the range 0.005 to 0.34 µg L-1 (McLaren et al., 2004). Bed sediments 30 km up-estuary from the mouth have Cd and Zn concentrations in the range 0.12-0.37 µg g-1 and 103-237 µg g-1, respectively (Wijaya et al., 2013). The rivers Petch Buri and Ta Chin are variously impacted by inputs from industry, agriculture and urban development (Thaipichitburapa et al., 2010). Agricultural inputs influence the water quality of the Mae Klong River (Censi et al., 2006), where the mean concentration of Zn in sediments was103 ± 98 µg g-1 (Pengthamkeerati et al., 2013).The concentrations of Cd, Zn and Fe in the sediments were in the sequence Chao Phraya > Ta Chin > Bang Pakong > Petch Buri > Map Ta Phut > Mae Klong (Cheevaporn and Menasveta, 2003). In the western Gulf, between the estuaries of the Petch Buri and Mae Klong, dissolved metal concentrations in coastal waters were relatively low, whereas in sediments total Zn was in the range 120-994 µg g-1(Censi et al., 2006; 2007).

2.2 Sample collection and pre-treatment

A minimum of 25 individuals of similar-sized (~3 cm length) A. granosa were collected from the intertidal zone, at low water, from each of seven sites located at the river mouths, including samples from opposite banks of the Chao Phraya (Fig. 1). At each site, surface sediments (0-2 cm) were also obtained, in triplicate, using a clean plastic spatula, together with1 L of native estuarine water in clean glass bottles. Samples were returned to the laboratory within 5 hours and processed immediately.

Sediments: Sediment samples were wet sieved across 100 m nylon mesh, using 50% native estuarine water. The samples were standardised on 100 µm grain size because it approximates to material carried in suspension and incorporates the range of particle types processed by suspension-feeders and detritivors. Sediments extracted for ‘total’ metal analyses (using concentrated HNO3) were freeze-dried, after which they were disaggregated using an agate mortar and pestle and stored in acid-cleaned plastic bags. Fresh sediments (<100 µm grain size) were used in the extraction of the bioavailable fraction of metals (using 1M HCl). In order to obtain dry weights, a separate portion of wet sediment was dried at 50oC overnight and wet:dry weight ratios estimated.

Blood Cockles: Surface deposits of particulate matter were removed from the blood cockles by gentle rinsing and any bleeding, damaged or dead animals were discarded. Prior to analysis, A. granosa were cleaned by incubating the animals in fine sand (Sand 40-100 mesh; Fisher Scientific), immersed in filtered seawater to allow depuration of potentially metal-contaminated gut contents. Samples were aerated in separate basins for each site for one week, and the overlying seawater replaced daily (Langston and Zhou, 1987). Eighteen cockles from each site were dissected to yield the gills, the digestive gland, the foot and the remaining tissue (described here as the ‘body’). The dissected tissues of five cockles from each site were then pooled to create three replicates for cytosol fractionation. After pooling, the tissue samples were weighed and transferred into pre-weighed glass vials and stored at -80oC. The dissected tissues from the three remaining cockles were used for total metal analysis. These were freeze-dried for 48 h, then ground with a pestle and mortar and stored in acid-cleaned plastic bags.

2.3 Extraction of metals

2.3.1 Sediments

Extraction of total metals from freeze-dried sediments was carried out on approximately 0.5g of dry particulate matter accurately weighed into Teflon digestion vessels. Concentrated HNO3 (5 mL) was added and the vessels loosely capped and left overnight for effervescence to cease. Subsequently, the vessels were sealed securely and heated in a microwave oven (Matsui MS-106WH/MS-106SL; 700 W) set at medium power for 60 seconds. After cooling for 15 minutes, the liquids were transferred quantitatively to 25 mL volumetric flasks (Class A) and made up to the mark with Milli-Q water.

Extraction of the bioavailable fraction of metals in sediments was carried out by leaching 0.5 g of fresh sediment with 20 mL of 1M HCl at room temperature for 2 h, an approach considered as a surrogate of particulate metal bioavailability (Bryan and Langston, 1992). The filtered extracts were transferred quantitatively to 25 mL volumetric flasks (Class A) and made up to the mark with Milli-Q water.

2.3.2 Tissues

The detailed methodology for the separation of the cytosolic fractions is reported elsewhere (Langston et al., 2002). Freeze-dried tissues of the gills, digestive glands and foot were separately homogenised in 5 mL of 0.02 M Tris-HCl buffer, at pH 8.6, in an ice bath, whereas remaining body tissues were homogenised in 20 mL of the buffer. The homogenates were divided, with one aliquot for total metal analysis and the other for the separation of the cytosolic fractions (see Section 2.4). The set of sub-samples of each homogenate for total metal analysis were dried at 80 ºC for at least 24 h and then weighed. The dried homogenates were digested using 5 mL of concentrated HNO3 (AristaR Grade) in covered glass vials heated on a hotplate at 100 ºC for a minimum of 24 h until a clear solution was obtained. The covers were then removed, the solutions were evaporated and 0.5 mL of concentrated HCl and 4.5 mL deionised water was added.

2.4 Separation of cytosolic fractions by gel size-exclusion chromatography

Samples of the homogenised tissues were centrifuged (MSE Hi-Spin 21) at 30,000 g for 40 minutes at 4°C resulting in a soluble fraction (i.e. the cell supernatant cytosol) and a solid pellet (cell membranes, and organelles). The liquid cytosol was separated from the pellet and the liquid divided into two portions:

(i) portion one was heat-treated by maintaining the sample at 80 ºC for 10 min followed by centrifugation at 30,000 g for 40 min at 4 ºC. The heat-treatment caused the precipitation of the high molecular weight proteins (29-200 kDa) in the cytosol, leaving heat-stable MTLPs (7-29 kDa) and low molecular compounds (<7 kDa) in solution. This portion was used for the determination of MTLPs by differential pulse polarography (DPP) (Langston et al., 2002);

(ii) portion two of the liquid cytosol was transferred to a vial for separation by gel chromatography using Sephadex G-75. Three replicate samples were also used to compare variability in individual samples with that of pooled samples. Confirmation of the precipitation of the high molecular weight proteins, and the heat stability of MTLPs, separated on the chromatographic column, was performed by DPP and UV (284/280 nm) analysis of individual fractions of the heat-treated supernatants. Thus, heat-treatment followed by centrifugation was confirmed as minimising interference from other proteins during MTLP analysis.

Metal-binding by cytosol compounds was measured following size-exclusion chromatography of approximately 0.75 ml of cytosol on a Sephadex G-75 column (1.5 x 60 cm) at 4 C. Samples were eluted at 0.5 mL min-1, using a 0.02 M Tris-HCl buffer at pH 8.6, and collected, sequentially, as 3 mL fractions according to molecular size. Protein detection in eluted fractions was performed using UV spectrophotometry (Varian Cary-1) at 254 nm. The Sephadex G-75 column was calibrated with pure protein molecular weight standards (Pharmacia) including blue dextran (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (11.7 kDa), and aprotinin (7 kDa). The elution volume (Ve) was determined from the position of the maximum elution peak of each molecular weight standard. The void volume (V0) was estimated as the elution volume of blue dextran. The SH content of individual column fractions of separated cytosol, measured as polarographic response (µA), was corrected to the original dry tissue weight (µA g-1 dry wt.) for each sample (Supplementary Data S2 and S3).

2.5 Analysis of metals in sediments, tissues and cytosolic fractions

Analyses of Cd and Zn in sediment extracts were carried out by inductively coupled plasma-mass spectrometry (ICP-MS) using a Plasma Quad PQ 2+ (ThermoElemental, UK). All digests had 115In added at 100 µg L-1 for internal standardisation, and to minimise interference from 35Cl+, 3% N2 was introduced into the nebuliser gas. Iron was analysed by ICP-atomic emission spectrometry using a Liberty 200 (Varian, UK) calibrated with multi-element standards solutions prepared in 10% HNO3.

Total metal concentrations in tissue homogenates were determined directly using flame-atomic absorption spectrometry (AAS; Varian AA20) for Zn and Fe and graphite furnace-AAS (Varian 300 Zeeman) for Cd. In the separated cytosolic fractions Zn and Fe were determined by flame-AAS, whereas Cd was determined by graphite furnace-AAS, calibrated by standard addition (Langston et al., 2002).

The accuracy of the analytical methods was evaluated using the MESS-3 (sediment) and TORT-2 (lobster)certified reference materials from the National Research Council, Canada. The analytical figures of merit for MESS-3 and TORT-2 exhibited satisfactory recoveries with respect to the certified values, with recoveries greater than 80% for Cd and Zn but were somewhat lower for Fe (70%) in the MESS-3 standard (since the concentrated HNO3 extractant may not dissolve some of the more refractory, geogenic Fe in sediments) (Table 1).

2.6 Analysis of MTLP by differential pulse polarography (DPP)

The DPP analysis of heat-treated cytosols is based on the characteristics of MTLP under field conditions, where MTLP, with a molecular weight of 12-15 kDa based on size exclusion chromatography, is the product of the dimerization of a monomer with a molecular weight of 6-7 kDa (Langston and Zhou, 1986).Concentrations of MTLP were determined using DPP (Langston et al., 2002) and is based on measurements of heat-stable thiolic groups in the protein, and expressed as equivalents to a mammalian MT standard.

Ten mL of hexamine cobalt chloride buffer (the electrolyte) were dispensed into the polarographic cell, together with 100 µL of the Triton X-100 (0.025%, v/v) and 10 µL of the heat-treated cytosol (500 µL for individual chromatographic fractions of the cytosol). The cell was then purged for 2 minutes with purified argon gas prior to analysis. The potential was scanned from -1.4V to -1.6 V using a PARC Model 174A analyser, and response measured using a PARC/EG&G Model 303 static mercury drop electrode. The quantification of MTLP in the heat-treated cytosol of the samples was determined based on a standard solution of 10 mg L-1 of rabbit liver MT-I (Sigma) prepared in deionized water. Calibration was by the method of standard additions and concentrations of MTLP in the samples, as MT equivalents, were expressed as mg g-1 dry weight of tissue initially homogenised (see Langston et al., 2002 for further details).

2.7 Particulate organic carbon

The sieved, freeze-dried sediments were analysed for total and organic carbon. For the latter, portions of the processed sediments were treated with 1M HCl, to remove CaCO3, then washed with Milli-Q water and freeze-dried. Approximately, 10 mg of freeze-dried sediments were accurately weighed into pre-combusted tin capsules in triplicate. Total and organic particulate carbons were determined combustiometrically using a Carlo Erba (Model EA1110) automatic CHN analyser. The instrument was calibrated using acetanilide, empty pre-combusted capsules were run as blanks and the instrument was re-calibrated every five samples. The accuracy of the measurements was checked using the certified reference material PACS-2 (National Research Council of Canada). Results showed that the carbon analyses were accurate to within 10% of certified values.

2.8 Statistical analysis

The non-parametric Spearman rank coefficient was used to establish correlations between sediment and biological parameters. The Spearman rank coefficient is suitable for testing the significance of an association between two variables that do not conform to a bivariate normal distribution. The statistical analysis was completed using the software SPSS 10.0 for Windows.

3. Results

3.1 Metals in sediments

Mean total concentrations of Cd in sediments (n=3) at each site varied from 0.41 µg g-1 at Chao Phraya A to 0.10 µg g-1 in the Bang Pakong (Fig. 2a). Mean total Zn sedimentary concentrations (n=3) were highest (120 µg g-1), at the Chao Phraya B site, and lowest (43 µg g-1) in the Petch Buri (Fig. 2b). The mean total Fe concentrations (n=3) in sediments varied from 40 mg g-1 in the Ta Chin to 14 mg g-1 at Map Ta Phut (Fig. 2c). The Canadian Interim Sediment Quality Guidelines (ISQG) give marine threshold effect levels (TEL) for a likely impact on ecology as 0.7 µg g-1 for Cd and 124 µg g-1 for Zn (CCME, 1992).

The highest concentrations of HCl-available Cd in the sediments were those at the mouth of the Chao Phraya (Fig. 2a) and the overall mean percentage (±1) of total Cd present in the readily extractable form, for all sites, was 55 ± 17% (n=21). Regression of the HCl-available Cd concentrations (n=21) against the total Cd concentrations showed they were linearly correlated (R2=0.757; p<0.01). Maximum sedimentary concentrations of HCl-available Zn were also those at the mouth of the Chao Phraya (Fig. 2b), and the overall mean percentage Zn in this fraction, for all sites, was 47 ± 6% (n=21). In common with Cd concentrations, HCl-available Zn was linearly correlated with the total Zn (R2=0.960; p<0.01). However, the proportions of HCl-available Fe concentrations were more variable (Fig. 2c) with a mean of 30 ± 11% (n=21), indicating that the major fraction of Fe is matrix-bound (refractory). The HCl-available fraction of Fe had a linear relationship with the total component (R2=0.563; p<0.01). The organic carbon concentrations (Fig. 2d) were in the range of 5 to 20 mg g-1, concomitant with a general sediment texture comprising various mixtures of sand and mud. The relationship between the organic carbon concentration and total or HCl-available metals was only significant for total Fe (R2=0.675; p<0.01).

3.2 Tissue-specific concentrations of metals in A. granosa

The mean (± 1) concentration of Cd in all samples of the gills (n=21) was 11.1 ± 3.5 µg g-1 (dry weight for all tissue concentrations reported), with the highest concentrations found in animals from the sites at Chao Phraya B and Ta Chin (Fig. 3a). In contrast, the mean Cd concentrations in the digestive gland, body and foot were lower and similar in magnitude, 4.43, 4.62 and 4.58 µg g-1, respectively. The lowest Cd concentrations, for the digestive gland, body and foot, were found in the Mae Klong and at Map Ta Phut, whereas some of the highest values from the Chao Phraya B site (digestive gland). The HCl-available fractions of Cd in sediments were significantly correlated with Cd concentrations in the gills (R2=0.462; p<0.01; n=21) and the digestive gland (R2=0.449; p<0.01; n=21) but relationships were not significant for the body and foot.

The mean concentrations of Zn, as with Cd, were highest for the gills, consistent with an aqueous uptake pathway for these more soluble metals, with an overall mean of 664 ± 252 µg g-1. The highest Zn concentrations were found at Chao Phraya B, followed by the Ta Chin (Fig. 3b). By comparison with the gills, the mean concentrations of Zn in the digestive gland, body and foot were lower at 367 ± 141, 312 ± 144 and 423 ± 193 µg g-1, respectively. Relatively low Zn concentrations, typically <200 µg g-1, were found in the digestive gland and the body at most sites. However, the foot had elevated Zn values, up to a maximum of approximately 800 µg g-1, at Chao Phraya A. Regression of sedimentary HCl-available Zn concentrations against Zn values in each of the tissues were significant at the p<0.01 level (n=21) for the gills (R2=0.313), digestive gland (R2=0.348) and foot (R2=0.593).

In contrast to Cd and Zn, the distribution of Fe in A. granosa tissues was dominated by the foot and the gills, where mean concentrations for all samples were 1113 ± 456 and 1086 ± 380 µg g-1, respectively (Fig. 3c). The digestive gland had a mean concentration of 819 ± 232 µg g-1, whereas the Fe concentrations were lowest in the body (246 ± 67 µg g-1). This distribution may reflect a greater influence of sedimentary/dietary route of Fe uptake compared with Cd and Zn, together with the presence of Fe-bound to Hb in the foot, gills and digestive gland. Regressions of the sedimentary HCl-available Fe against Fe concentrations in each of the tissues were not significant at the p<0.01 level.

The biota-sediment accumulation factor (BSAF=concentration in the tissue/concentration in sediments) was estimated relative to the HCl-available metal in the sediments (Table 2). The mean BSAF for Cd (120) was highest in the gills and was lower, and relatively constant (in the range 43-48), for the digestive gland, body and foot. Zinc had lower BSAF values but all great than unity, with the gills and foot having significantly higher values than the digestive gland and body. The BSAF values for Fe were all much less than unity with the gills having the lowest value (0.04).

3.3 Tissue-specific concentrations of MTLPs

The mean (± 1) concentrations of MTLP in each of the tissues of A. granosa from the Gulf of Thailand were in the range 0.72 to 10.5 mg g-1 (dry weight) (Fig. 4). A common feature for all sites, except the Mae Klong, was that the greatest MTLP concentrations, typically >4 mg g-1, were found in the digestive gland, with Chao Phraya B having the greatest (10.5 mg g-1). Concentrations of MTLP in the gills, body and foot of A. granosa were generally less than 2 mg g-1, with the foot having the lowest concentrations of all. The MTLP concentrations in each of the tissues from the Mae Klong had values below 2 mg g-1, potentially identifying the estuary as a reference site, with respect to metal exposure.

Regressing the HCl-available metals in sediments against the concentrations of MTLP in the various tissues shows that the only significant relationships were for Cd (R2=0.410; p<0.01; n=21) and Zn (R2=0.518; p<0.01; n=21) in the digestive gland. Regressions of the sedimentary HCl-available Fe concentrations against MTLP concentrations were not significant for any tissue. This implies that the potentially pollutant metals Cd and Zn were most active in promoting synthesis of MTLP compared with Fe which is mostly geogenic (natural) in origin. A detoxifying role for MTLP in A. granosa is therefore likely.

3.4 Metal partitioning among the molecular weight fractions in cytosolic supernatants

Most of the Cd in the cytosols of the gills, the digestive gland, body and foot, from A. granosa was partitioned into the MTLP fraction, Cd[MTLP], with a molecular weight of approximately 20 kDa (Fig. 5). Cadmium in the metallothionein extract of gills, digestive gland and body was directly related, at the p<0.01 level, to the total Cd in the cytosol. The Cd[MTLP] was followed by the HMW fraction, Cd[HMW], at ~67 kDa. The LMW fraction, Cd[LMW],(1-4kDa) was the least significant Cd-binding component (Table 3). Compared to the total concentration of Cd in each tissue, the gills and body each contained 35% as Cd[MTLP], whereas the digestive gland had 16% and the foot 23% of Cd bound to the MTLP component. Cadmium was present at lower concentrations in Cd[HMW] with 18% bound to this fraction in the foot, 11% in the digestive gland and 12% in the gills. The remaining Cd, (2.5 to 4.3%) was associated with the Cd[LMW] fraction. The correlation between HCl-available Cd in the sediments and Cd concentrations in the three molecular weight fractions showed that the relationship was only significant for Cd[MTLP] (R2=0.281; p<0.01; n=21).

The highest proportion of Zn in the cytosol was, in contrast to Cd, predominantly associated with the high molecular weight fraction, Zn[HMW], typically with a molecular weight ~70 kDa (Fig. 6). Zinc in the HMW extract of gills, digestive gland body and foot was directly related, at the p<0.01 level, to the total Zn in the cytosol. The HMW fraction constituted 23-27% of the total Zn in the gills, digestive gland, body and the foot (Table 3). Smaller amounts of Zn were associated with the low molecular weight fraction (<4 kDa), Zn[LMW], ranging from 4.3% in the body to 9.7% in the foot. The lowest Zn concentrations were associated with the MTLP fraction, Zn[MTLP], which contained less than ~5% of the total Zn for each of the tissues. Correlation of the sedimentary HCl-available Zn with the various cytosolic fractions showed that they were significantly correlated for Zn[HMW] in the gills (R2=0.348; p<0.01; n=21) and the digestive gland (R2=0.624; p<0.01; n=21) but it was not significant for the body and foot. There were no significant correlations between the HCl-available Zn in the sediments and Zn[MTLP]or Zn[LMW]. The predominance of Zn in HMW fraction is consistent with its association with some enzymes and other proteins (e.g carbonic anhydrase) and human haemoglobin which has a strong affinity for Zn (Kiefer et al 1995; Rifkind and Helm. 1977)

The partitioning of Fe in the cytosols of A. granosa included high molecular weight Fe, Fe[HMW],(~75kDa) in the digestive gland,~67 kDa in the gills and body, and 57-84 kDa in the foot) (Fig. 7). The MTLP fraction, Fe[MTLP], eluted in the range 21-28 kDa and the low molecular weight fraction, Fe[LMW],in the range 1-4 kDa. The amounts of sub-cellular Fe in the cytosolic fluid were more evenly distributed amongst the three molecular weight fractions. Fe[HMW] in the body, gills, digestive gland and foot of A. granosa represented approximately 25%, 19% 14% and 9% of the total Fe, respectively (Table 3). Of the remaining cytosolic Fe, 7-15% was associated with Fe[MTLP] and a similar proportion (7-13%) with Fe[LMW]. Only Fe[HMW] in the digestive gland was significantly correlated with the HCl-available Fe in sediments (R2=0.423; p<0.01; n=21).

The non-significant relationship between Cd[HMW] and Fe[HMW] in the four tissues (each having R2<0.301, n=21), implies that the binding of Cd is unlikely to be with a HMW component. Similarly, the relationships between Cd[HMW] and Zn[HMW] in the gills, body and digestive gland were all insignificant, except for the foot (R2=0.637). However, the regression between Zn[HMW] and Fe[HMW], was significant for the digestive gland, the body and the foot (R2=0.371, 0.503 and 0.408, respectively), but not for the gills (R2=0.069). Given its molecular weight range, the Fe[HMW] fraction is postulated to be largely associated with Hb, given that in human molecules of this blood pigment contains four atoms of Fe, each representing a haem unit of ~16 kDa (Perutz, 1979).

4. Discussion

4.1 Sedimentary metal concentrations

Our total concentrations of Cd and Zn in sediments (Figs. 2a and b) did not exceed the TEL values (CCME, 1992) for any of the sites in this investigation. The concentrations of total sedimentary Cd at the mouth of the Chao Phraya are comparable to previously published values. For example, total Cd concentrations, in the <600 µm grain size fraction, were reported to be in the range 0.04-0.95 µg g-1 (McLaren et al., 2004). Wijaya et al. (2013) determined total Cd concentrations in the <50 µm grain size fraction of Chao Phraya sediments, about 30 km from the mouth, in the range 0.12-0.37 µg g-1 and total Zn concentrations were 103-237 µg g-1, which are similar to our values (Fig. 2b). However, un-fractionated sediments from the coastal waters adjacent to the Petch Buri and Mae Klong estuaries had total Zn concentrations ranging from 120 to 994 µg g-1 (Censi et al., 2006; Pengthamkeerati et al., 2013), which were generally higher than values for the <100 µm grain size sediments in this study (Fig. 2). Similarly, previously published total Fe in coastal sediments of the Petch Buri and Mae Klong, which ranged between 2.6-184 mg g-1 (Censi et al., 2006; Pengthamkeerati et al., 2013) were sometimes higher than our current values which were, however, consistent with total Fe in sediments from the Mae Klong Estuary (35-56 mg g-1) found by Censi et al., (2007). Thus, although the analytical approaches for the determination of metals in sediments differs between authors, previously cited Cd and Zn concentrations are in the same range as our values for Chao Phraya A and B sites (Fig. 2a). In contrast, literature values for intertidal sediments from the Mae Klong and Petch Buri appear somewhat elevated at some sites compared to our values for total Zn and Fe.

4.2Tissue-specific bio-accumulation of metals and MTLP

4.2.1 Metals in the tissues in A. granosa

Cadmium concentrations in the tissues of A. granosa reported here are comparable to published values for Cd in the soft parts of A. granosa from other sites in Thailand which range from 1.33 to 2.83 µg g-1 (dry weight) (Phillips and Muttarasin, 1985; Pradit et al., 2016). Direct comparison of our Cd concentrations with those found in the soft tissues of blood cockles from the Gulf of Thailand (Sudsandee et al., 2017) is hindered because the concentrations are given as wet weights. The soft tissues of Anadara spp. from Vietnam had mean Cd concentrations in the range 5.26-7.43 µg g-1 (dry weight) (Tu et al., 2011). A. granosa from the west coast of Malaysia had concentrations in the dried whole soft tissues of 4.69 ± 2.45 µg g-1, 5.86 ± 2.83 µg g-1 in the combined mantle and gills and 2.86 ± 2.39 µg g-1 in the combined viscera and foot (Yap et al., 2008). S. broughtonii (Edward et al., 2009) from the west coast of Malaysia had elevated Cd concentrations in its tissues, with a similar tissue distribution to that found in this study; the highest concentrations were found in gills and mantle. Other blood cockles, such as Anadara ovalis (Silva et al., 2006) had mean Cd concentrations in their soft tissues of 1.35 ± 0.16 µg g-1. Overall, the results suggest that Cd is significantly bioaccumulated in the tissues of various species of blood cockles relative to the sediments in which they reside.

Laboratory exposures of the blood cockle S. inaequivalvis to seawater spiked with dissolved Cd resulted in the highest levels of metal bioaccumulation in the kidneys and least in the erythrocytes (Serra et al., 1995). In similar exposure experiments involving A. granosa, dissolved Cd was found to accumulate significantly in the gills and, to a lesser extent, in the hepatopancreas and whole tissues (Chan et al., 2002). Incubation of Anadara trapezia with Cd-spiked sediment also resulted in higher concentrations in the gills compared to the hepatopancreas (Taylor and Maher, 2012). The results of these laboratory experiments confirm our general conclusion that the gills of blood cockles are a significant repository for Cd.

Published concentrations (dry weights) of Zn in the soft tissues of A. granosa from the Gulf of Thailand were 55-106 µg g-1 (Phillips and Muttarasin, 1985; Pradit et al., 2016), 70-125 µg g-1 in Bombay Harbour (Patel et al., 1985), 185 to 236 µg g-1 from the west coast of Malaysia (Ibrahim, 1995) and 80-97 µg g-1 from the coast of Vietnam (Tu et al., 2011). The tissue-specific distribution of Zn in A. granosa from Malaysia indicated that Zn was sequestered at highest concentrations in the combined mantle and gills (Yap et al., 2008). A similar distribution and concentrations was observed in S. (=Anadara) broughtonii (Edward et al., 2009) and A. ovalis (Silva et al., 2006). However, the Zn concentrations we report here for A. granosa from the Gulf of Thailand, specifically for the gills, and to a lesser degree the foot, are significantly higher than those reported above. Whilst concentrations of Zn in the digestive gland and body of A. granosa are <200 µg g-1, those in the gills are typically >500 µg g-1 and in the foot 200-400 µg g-1, (except for lower levels in cockles from the remote site at Map Ta Phut). Thus, there are considerable differences in the tissue concentrations of Zn in the various blood cockle species at different sites in SE Asia and these appear to be inherently high in A. granosa

Relatively few measurements of the Fe content of the tissues of blood cockles are reported in the literature despite its importance for the maintenance of Hb. Typical Fe concentrations in the whole soft parts of A. granosa are in the range 512-611 µg g-1 (Phillips and Muttarasin, 1985) and in A. ovalis had a mean of 1275 µg g-1 (Silva et al., 2006). The tissue-specific distribution of Fe in these studies indicated some enhancement in the combined mantle and gills for A. granosa from Malaysia (Yap et al., 2008) and the gills for S. broughtonii (Edward et al., 2009). In contrast, our study found Fe concentrations in the gills were relatively low, typically <250 µg g-1, whereas Fe was most concentrated in the digestive gland, body and foot.

The source of the metals in the tissues of A. granosa was statistically-linked to the HCl-available metal content of the sediments, notably, for Cd, in the gills and digestive gland and, for Zn, in the gills, digestive gland and foot. At various sites in the Gulf of Thailand there are reports of elevated dissolved Cd and Zn concentrations in the water column (Cheevaporn et al., 2003; McLaren et al., 2004; Wijaya et al., 2013) and in sediment porewaters (Cheevaporn et al., 1995) which would be available to A. granosa during feeding and respiration. Concentrations of “dissolved” Fe in the water column are also reported to be elevated at the river mouths - in the range 0.48-2.61 mg L-1 (Cheevaporn et al., 2003), although Fe levels in the porewaters of the Bang Pakong indicate less enrichment of this metal (Cheevaporn et al., 1995). However, because of their relatively short siphons, blood cockles are probably mixed suspension and surface deposit feeders and their diet comprises detritus and microalgae which are siphoned from the mud-water interface during periods of tidal inundation (Broom, 1982). It is notable that there were no correlations of HCl-available Fe in sediments with the tissue-specific concentrations of this metal, suggesting that Fe could be acquired from a combination of sources, including sedimentary porewaters, where localised conditions could influence the speciation, solubility and availability of this redox-sensitive metal.

4.2.2 Synthesis of MTLP

The synthesis of metal-binding MTLP in animal tissues is, potentially, one mechanism by which organisms can reduce stress induced by chemical contamination. A baseline MTLP concentration of 2 mg g-1, found in the gills of Mytilus edulis, has been proposed for uncontaminated locations in the UK, with concentrations in excess of this being indicative of organisms under metal-stress (Langston et al., 2002). The MTLP concentrations in A. granosa from the Gulf of Thailand were highest in the digestive gland (up to 10 mg g-1). Though coinciding with elevated Cd and Zn bioaccumulation there was a lack of any clear relationship between MTLP and metal concentrations in sediments, and direct correlation may be confounded by the combination of various potential sources of metals, and modified by biological factors such as metabolism and growth rates. Also, other contaminants associated with free radical production may have been important in the induction of MTLP; for example coastal sediments near to our sites have measurable PAH concentrations, particularly at the mouth of the Chao Phraya (Boonyatumanond et al., 2006). A study of the uptake of PAHs by A. granosa in Malaysian waters (Mirsadeghi et al., 2013; Sany et al., 2014) indicated the blood cockle is able to accumulate PAHs, from sediments and the water column, thereby increasing tissue concentrations, and oxidative stress levels, by at least an order of magnitude. There is evidence that MT may act as a free radical scavenger in a general response to oxidative stress (Sato and Bremner, 1993; Langston et al 2012). Thus, it is not possible to conclude unambiguously that the MTLP concentrations are a direct result of induction by sedimentary metals alone.

4.3 Metal behaviour in cytosols

Metallothioneins, and MTLP, such as those found in marine mussels, typically contain some 20 cysteine residues, which do not form disulphide bridges, rather there are two domains namely, an α-domain containing 11- or 12-cysteines, binding 4 metal cations and a β-domain containing 9 cysteines binding 3 metal cations (George and Langston, 1994). MTLPs have been divided into three classes based on their structure and mode of synthesis, Class 1 MT consists of polypeptides with cysteine residues closely related to mammalian metallothionein and includes the metal-binding protein found in blood cockles A. granosa (Chan et al., 2002) and S. inaequivalvis (Serra et al., 1995). Metal binding to natural organic compounds which are present in the cytosol depends on both the chemical reactivity of the metal and the relative availability of ligands containing S, N and O atoms. Consequent upon its atomic structure, Cd is classed as a soft Lewis acid, such that it is polarisable and binds preferentially with sulphur atoms, including those comprising cysteine. In contrast, Zn is a “borderline” Lewis acid and is less polarisable and has no strong tendency to complex with S, N or O atoms associated with moieties in the cytosol. Nevertheless, it is an essential component of, for example, Zn–requiring enzymes such as carbonic anhydrase and Zn-finger proteins that bind and confer structure and functionality to DNA/RNA (Kiefer et al., 1995; Smith et al., 2000).

The potential differences in the chemical reactivity of Cd and Zn in their binding with MTLP has been demonstrated in experiments using MTLP derived from rabbit liver, where at pH 7.54 the stability constant for Cd complexation was log K = 14.4, while that for Zn was log K=11.4 (Ruiz et al., 1995). Recent experiments on the association of Zn with human thionein, and its formation of MTLP, have identified three classes of binding sites, each class having a different affinity for Zn. The stability constants for 4 Zn atoms bound to thionein (T) have a relatively high, and uniform, stability constant where log K~11, whereas the three remaining metal atoms have stability constants lower by about 2-3 orders of magnitude, the lowest being log K=7.7 (Krężel and Maret, 2007). The relatively wide range of stability constants for the Zn-T system, compared to the orders of magnitude higher values for Cd, implies that weakly bound Zn is available to engage in exchange reactions. The binding of Zn within the cytosol, potentially involves reversible reactions, with Zn-MTLP being the go-between for a Zn-Donor and a Zn-Acceptor, according to the following reaction (Jacob et al., 1998):

Zn-Donor + T Zn-MTLP T + Zn-Acceptor

where Zn-Donor represents Zn atoms entering cells, including the erythrocytes, potentially as complexes, for example ZnCO3Cl- or Zn(HCO3)ClOH-, by various transporters (Kalfakakou and Simons, 1990) and where the Zn-Acceptor could be an HMW component in the cytosol, such as Hb.

Despite the relatively high stability constants for Zn binding with human MT (Krężel and Maret, 2007) our results show that ≤6% of the total Zn in A. granosa is associated with MTLP in each tissue. This level of MTLP association is comparable to the relatively low levels of MT-bound Zn in other marine bivalves such as Mytilus edulis (Langston et al., 1998). Theoretically, the higher binding constant for Cd suggests it out-competes Zn for active thionein binding sites, leaving Zn available for “spill over” binding, or exchange, with other molecular weight fractions, including essential proteins, in marine and freshwater organisms (Langston et al., 1998, 2002; Giguére et al., 2003). One potential “spill over” component is carbonic anhydrase which is located in erythrocytes, where it acts as a catalyst for the hydration of CO2 and the dehydration of H2CO3. However, the molecular weight of this compound is of the order 29 kDa and, based on Zn profiles, is not found in significant proportions in the upper molecular weight range of our chromatograms. Rather, the main Zn-containing peaks in A. granosa cytosols are at a significantly higher molecular weight than carbonic anhydrase, indicating that other compounds dominate Zn-binding.

The preferential role of the HWM fraction in binding Zn is of particular interest in this context because Hb, with molecular weights in the range 60-70 kDa, is an important HWM component of the cytosol of blood cockles. The chromatograms depicting metal partitioning (particularly Zn) were characterised typically by peaks within this molecular weight range, suggesting the presence of Hb which can be found in the tetrameric form, for example in A. sartori (Weber and Vinogradov, 2001), A. broughtonii and A. ovalis (Borgese et al., 1989 and references therein). Examination of the molecular structure of Hb in the blood clam A. ovalis, using X-ray diffraction, showed the presence of free sulphur groups of the order 12-13 per molecule originating from cysteine residues (Borgese et al., 1989). Sulphur groups within Hb offer potential strong binding sites for free metals and those metals, such as Zn, which are weakly bound to MTLP or other organic moieties. Thus, transfer reactions between the various entities, including Hb, appear important to the control of the location of Zn within the cytosol.

Tetrameric Hb is the most common form in the serum of vertebrates and research has been undertaken over the last 50 years on the potential of Zn to become directly involved in the functional behaviour of respiratory proteins. In particular, the binding of Zn with human Hb has been driven, to some extent, to better understand sickle cell anaemia (Dash et al., 1974). Particular attention has been given to Zn because laboratory studies show the binding of Zn with Hb results in an augmented uptake of oxygen (for example Rifkind and Heim, 1977). Studies involving human Hb have also shown that Zn complexes with at least three amino acids, including one from each of histidine and cysteine (Gilman and Brewer, 1978). Thus, within the blood cockle the role of the HMW fraction and its preferential binding with Zn is of interest because this combination appears to be an important component of the cytosol in our samples of A. granosa. The Hb in blood cockles may not have precisely the same chemical signature as that in humans but it clearly has an oxygen-transporting function critical to the survival of aquatic organisms, particularly those such as blood cockles existing in sub-oxic or anoxic waters and sediments. Assuming that Hb in blood cockles has similarities in its molecular structure, and functionality, to that in vertebrates, a process involving the binding of Zn with Hb may be crucial to its acquisition of additional, albeit small, quantities of oxygen, sufficient to increase the capacity of blood cockles to cope with metabolic stress, thereby aiding their survival in sub-oxic tropical waters. Thus, rather than Zn being classified as a “spill over” element because of a shortage of binding sites within MTLP, its chemical behaviour may be biologically engineered to support additional oxygen acquisition by A. granosa found in sub-oxic, tropical aquatic environments.

5. Conclusions

Blood cockles, A. granosa, from the mouths of various rivers draining the northern coastline of the Gulf of Thailand have Cd concentrations in their tissues that are elevated by two orders of magnitude over the sediments in which they reside. Within each of the cytosols of the gills, digestive gland, whole body and foot, Cd was located mainly with the MTLP component, which may effectively protect the animals against the deleterious effects of this toxic metal. Zinc in the tissues of A. granosa were also elevated compared to the sediments in which they reside. Zinc in the cytosols was predominant (23-27%) the HMW fraction (>60 kDa), whereas <8% of the Zn is associated with MTLP. The concentrations of Fe in the tissues was a minor fraction (BASF<0.2) of the HCl-available concentrations in the sediments. Within the cytosols of the four organs Fe is distributed equally between the three molecular weight fractions. These distributions indicate that the partitioning of the metals varies according to their harmful, or beneficial, effects on the organism.

The dominant association of Zn with the HMW fraction of the cytosol may be significant for benthic organisms that dwell in sediments of estuarine waters which are under-saturated with dissolved oxygen. The beneficial effects of Zn binding to Hb in A. granosa are probably manifest by increasing retention of O2 and thus maintaining its survival under low dissolved oxygen conditions. Nevertheless, the association between Zn and high molecular weight components, such as Hb, in the cytosols of marine invertebrates existing in sub-oxic waters is poorly understood and is clearly an area for further research.

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CAPTIONS FOR FIGURES

Fig. 1 Sampling sites () in the Gulf of Thailand

Fig. 2 Mean concentrations (±1)of metals and particulate organic carbon in sediments from 7 sites (n=3 at each site) in the Gulf of Thailand. (■)-totals; (■)-available to 1M HCl: (a) Cd; (b) Zn; (c) Fe; (d) particulate organic carbon

Fig. 3 Tissue-specific accumulation of metals (mean ±1) in A. granosa: (■) gills, (■) digestive gland (■) body and (■) foot. (a) Cd; (b) Zn; (c) Fe

Fig. 4 Mean (±1)concentrations of metallothionein-like proteins (MTLP) in A. granosa (n=3): (■) gills, (■) digestive gland (■) body and (■) foot

Fig. 5 Concentrations of Cd in molecular weight fractions of the cytosols from A. granosa as a function of total Cd in the supernatants for all samples for all sites (n=21). Cytosol fractions () HMW=high molecular weight, (■) MTLP=metallothionein-like-protein and (▲) LMW=low molecular weight. (a) gills; (b) digestive gland; (c) body; (d) foot. The lines represent the linear regressions of a two-tailed test with a level of significance R2>0.301; p<0.01; n=21.Linear regressions are not plotted for supernatant pools not fulfilling this criterion

Fig. 6 Concentrations of Zn in molecular weight fractions of the cytosols from A. granosa as a function of total Cd in the supernatants for all samples from all sites (n=21). Cytosol fractions () HMW=high molecular weight, (■) MTLP=metallothionein-like-protein and (▲) LMW=low molecular weight. (a) gills; (b) digestive gland; (c) body; (d) foot. The lines represent the linear regressions of a two-tailed test with a level of significance R2>0.301; p<0.01; n=21. Linear regressions are not plotted for supernatant pools not fulfilling this criterion

Fig. 7 Concentrations of Fe in molecular weight fractions of the cytosols from A. granosa as a function of total Cd in the supernatant for all samples from all sites (n=21). Cytosol fractions ()HMW=high molecular weight, (■) MTLP=metallothionein-like-protein and (▲) LMW=low molecular weight. (a) gills; (b) digestive gland; (c) body; (d) foot. The lines represent the linear regressions of a two-tailed test with a level of significance R2>0.301; p<0.01, n=21. Linear regressions are not plotted for supernatant pools not fulfilling this criterion