Details of the nine sampling sites and the main stresses to which they are subjected.
Heavy metal pollution is a serious and widely environmental problem due to the persistent and non-biodegradable properties of these contaminants. Sediments serve as the ultimate sink of heavy metals in the marine environment and they play an important role in the transport and storage of potentially hazardous metals. They are introduced into the aquatic system as a result of weathering of soil and rocks, from volcanic eruptions and from a variety of human activities involving mining, dredging, processing and use of metals and/or substances containing metal contaminants. Heavy metals entering natural water become part of the water-sediment system and their distribution processes are controlled by a dynamic set of physicochemical interactions and equilibria. The properties of metals in soils and sediments depend on the physiochemical form in which they occur . Heavy metals are distributed throughout soil and sediment components and associated with them in various ways, including adsorption, ion exchange, precipitation and complexation and so on . Changes in environmental conditions, such as temperature, pH, redox potential and organic ligand concentrations, can cause metals to be released from solid to liquid phase and sometimes cause contamination of surrounding waters in aquatic systems . They are not permanently fixed by soil or sediment. Therefore, it cannot provide sufficient information about mobility, bioavailability and toxicity of metals if their total contents are studied alone.
Natural and anthropogenic activities have the capacity to cause changes in environment conditions, such as acidification, redox potential, or organic ligand concentrations, which can remobilize contaminated soils and sediments releasing the elements from soils and sediments and pore water to the water column resulting contamination of surrounding waters. Daily tidal currents, wind energies, and storms in coastal and estuarine systems can cause periodical remobilization of surface sediments . More turbulent conditions, such as seasonal flooding or storms, or bioturbation, due to feeding and movement of benthic organisms, can expose anoxic sediments to oxidant conditions. In addition, activities such as dredging result in major sediment disturbances, leading to changes in chemical properties of sediment .
The remediation of heavy metal pollution is often problematic due to their persistence and non-degradability in the environment. As a sink and source, soils and sediments constitute a reservoir of bioavailable heavy metals and play a significant role in the remobilization of contaminants in the aquatic systems under favorable conditions. Such potential of sediment for being a sink as well as a source of contaminant can make sediment chemistry and toxicity key components of the quality of aquatic system. Much concern has been focused on the investigation of the total element contents in soils and sediments. However, it cannot provide sufficient information about mobility, bioavailability and toxicity of elements and thus may not be able to provide information about the exact dimension of pollution. The data on total contents of metals are quite insufficient to estimate the possible risk of remobilization of total metals under changing environmental conditions and potential uptake of liberated metals by biota and thus the determination of different fractions assume great importance. This has been described as “speciation” . Since each form have different bioavailability and toxicity, the environmentalists are rightly concerned about the exact forms of metal present in the aquatic environment.
The concept of speciation dates back to 1954 when Goldberg introduced the concept of speciation to improve the understanding of the biogeochemical cycling of trace elements in seawater. Kinetic and thermodynamic information together with the analytical data made it possible to differentiate between oxidized versus reduced, complexed or chelated versus free metal ions in solution and dissolved between particulate species. Florence  has deﬁned the term speciation analysis as the determination of the individual physicochemical forms of the element, which together make up its total concentration in a sample. According to Lung , speciation analysis involves the use of analytical methods that can provide information about the physicochemical forms of the elements. Schroeder  distinguishes physical speciation, which involves differentiation of the physical size or the physical properties of the metal, and chemical speciation, which entails differentiation among the various chemical forms. The main objective of measuring metal species relates to their relative toxicities to aquatic biota. The second and long term aim of speciation studies is to advance an understanding of metal interactions between water and bed sediments in an aquatic ecosystem. In the last decade researchers have followed different sequential extraction techniques for the fractionation of metals in sediments of different river systems. Rauret et al.  studied the speciation of copper and lead in the sediments of River Tenes (Spain) while Pardo et al.  studies the speciation of zinc, cadmium, lead, copper, nickel and cobalt in the sediments of Pisuerga River, Spain, in order to establish the extent to which these are polluted and their capacity to remobilization. Jardo and Nickless  investigated the chemical association of zinc, cadmium, lead and copper in soils and sediments of England and Wales. In most samples, these four metals were associated with all the chemical fractions. Tessier et al.  studied speciation of cadmium, cobalt, copper, nickel, lead, zinc, iron and manganese in water and sediments of St. Fransois River, Quebec, Canada. Elsokkary and Muller  studied speciation of chromium, nickel, lead and cadmium in the sediments of Nile River, Egypt, reporting that a high proportion of chromium, nickel and lead is bound to organic material and sulphides, while cadmium is bound to carbonate fraction. Ure  and Rauret  have reviewed the chemical extraction procedures used for heavy metal determinations in contaminated soils and sediments. Owing to the need for validation of extraction schemes, the EC Measurement and Testing Programme (formerly BCR) has organized a project for improving the quality of determinations of extractable heavy metals, where development and validation of extraction procedures has been discussed [17, 18].
The present article aims to summarize the potentials of sequential extraction technique adopting different analytical protocols for gaining information on the mobility and dynamics of operationally determined chemical forms of heavy metals in soils and sediments. The BCR (Community Bureau of Reference, now superseded by the Standards, Measurement and Testing Programme of the European Community) procedure has been illustrated considering the case study of Ganges (Hugli) River Estuary and adjacent Indian Sundarban mangrove wetland (a UNESCO World Heritage Site), northeastern part of the Bay of Bengal. In addition, the authors also evaluate the modified BCR sequential extraction technique as devised by various scientists, the risk assessment code (RAC) as well as assessment of toxicity comparing with sediment quality guidelines. The RAC classification is based on the strength of the bond between the metals and the different geochemical fractions in sediments or soils and the ability of metals to be released and enter into the food chain.
2. Sequential extraction: Merits and demerits
The sequential extraction provides more or less detailed information concerning the origin, mode of occurrence, biological and physicochemical availabilities, mobilization and transport of heavy metals. The procedure stimulates the mobilization and retention of these species in the natural environment using changes in environmental condition such as pH, redox potential and degradation of organic matter . A series of reagents is applied to the sample, increasing the strength of the extraction at each step, in order to dissolve the trace metal present in different sediment phases. The extractants are inert electrolytes, weak acids, reducing agent, oxidizing agents and strong mineral acids .
The 3-stage sequential extraction procedure proposed by the European Community Bureau of Reference (BCR) was developed in an attempt to standardize the various schemes described in the literature [2, 20, 21], since the use of different procedures, varying in the number of steps, types of reagents and extraction condition. Hindered comparison of results obtained in the many studies of heavy metals chemical fractionation in environmental samples .
The BCR methods has been widely adapted by various authors, and applied to a range of type of solid sample including fresh water sediment [23-25], salt water sediment [26-28], sewage sludge and particulate matter [29-31]. This scheme enables us to associate the meals with one of the following four geochemical phases:
I. Acid-soluble phase: This phase is made up of exchangeable metals and others bound to carbonates that are able to pass easily into the water column, for example, when the pH drops. It is the fraction with the most labile bond to the soil/sediment and, therefore, the most dangerous for the environment.
II. Reducible phase: This phase consists of metals bound to iron and manganese oxides that can be released if the sediment changes from the oxic to the anoxic state, which could be caused, for example, by the activity of microorganisms present in the soils/sediments.
III. Oxidisable phase: This shows the amount of metal bound to organic matter and sulphides, which can be released under oxidizing conditions. Such conditions can occur, for example, if the sediment is resuspended (by dredging, currents, flooding, tides, etc.) and the sediment participles come into contact with oxygen-rich water.
IV. Residual phase : Lithogenous and inert (Non-bioavailable).
The heavy metals in the soils and sediments are bound to different fractions with different strengths, the value can, therefore, give a clear indication of soil and sediment reactivity, which in turn assess the risk connected with the presence of heavy metals in a terrestrial or aquatic environment. The rationale of the sequential extraction procedure is that each successive reagent dissolves a different component, which can content heavy metals within their crystalline structures. Under natural conditions, metals in minerals are unlikely to experience significant release over the time frames of interest [32, 33].
3. Analytical protocols for sequential extraction
In recent years a great number of papers have been published on various analytical techniques proposed for the fractionation analysis of trace elements in various environmental samples (soils, sediments, etc.). An approach that has been found to be preferable is the fractionation of heavy metal into operationally deﬁned forms under the sequential action of different extractants . Selective extractants, used in sequential extraction procedures, are aimed at the simulation of natural conditions whereby metals associated with certain soil (sediment) components can be released. For example, changes in the ionic composition affecting adsorption–desorption reactions or a decrease in pH may lead to the release of metals, retained on a matrix by weak electrostatic interactions or co-precipitated with carbonates (“exchangeable” and “acid soluble” forms). Decreasing the redox potential can result in dissolution of oxides, unstable under reducing conditions, and liberation of scavenged metals (“reducible” forms). Changes in oxidizing conditions may cause the degradation of organic matter and release of complexed metals (“oxidizable” forms). Finally, the destruction of primary and secondary mineral lattice releases heavy metal retained within the crystal structure, e.g., due to isomorphous substitution (“residual” forms) . The nominal “forms” determined by operational fractionation can help to estimate the amounts of total metals in different reservoirs which could be mobilized under changes in the chemical properties of the soil . Since the 1970s a considerable number of extraction procedures have been proposed for determining the forms of heavy metal [2, 35-39]. Most of these procedures are based on the scheme of Tessier et al. . Although most of the extracting reagents were originally used in the chemical analysis of soils, the procedures proposed have been tested on a wide variety of contaminated environmental samples—sediments, road dust, sewage sludge, etc.
Sequential extraction can be useful to have an operational classification of metals in different geochemical fractions  which is the most reliable criteria to quantify the potential effect of soil/sediment contamination by heavy metals. This can provide information about the identification of the main binding sites, the strength of element binding to the particulates and the phase associations of trace elements in soil/sediment. Following this basic scheme, some modified procedures with different sequences of reagents or operational conditions have been developed [40-43]. Considering the diversity of procedures and the lack of uniformity in different protocols, a European Community Bureau of Reference (BCR, now the European Community Standards Measurement and Testing Program) method was proposed  and was applied by a large group of researchers [31, 44-47]. In this study, we followed the sequential extraction procedure proposed by the European Union's Standards, Measurements and Testing program .
4. Modified BCR Sequential extraction process
As discussed above it is evident that sequential extraction provides valuable information regarding identification of main binding site, the strength of the element binding to the particulates and the phase associations of heavy metals in sediments. However, various complicated sequential extraction procedures were experimented to provide more detailed information regarding different metal phase associations [2, 48, 49]. A wide range of techniques is available whereby various extraction reagents and experimental conditions are used. These techniques involve a 5-step , 4-step (BCR, Bureau Commune de Reference of the European Commission), 6-step  and 7-step [51, 52] extraction, and are thus becoming popular methods to be used for sequential extraction [53, 54]. Following this basic scheme, some modified procedures with different sequences of reagents or operational conditions have been developed [40-43].
Several sophisticated instruments have been used for the determination of heavy metals contents in marine environments. These include; flame AAS [55, 56], atomic fluorescence spectrometry , anodic stripping voltametry [58, 59], ICP-AES  and ICP-MS [61, 62].
Heavy metal mobility and bioavailability depend strongly on their chemical and mineralogical forms in which they occur . Several speciation studies have been conducted to determine study different forms of heavy metals rather their total metal content. These studies reveal the level of bioavailability of metals in harbour sediments and also confirm that sediments are indicators of heavy metal pollution in marine environment [64-67].
Since the early 1980s and 1990s sequential extraction methodology has been developed to determine speciation of metals in sediments [2, 68] due to the fact that the total concentration of metals often does not accurately represent their characteristics and toxicity. In order to overcome the above mentioned obstacles it is helpful to evaluate the individual fractions of the metals to fully understand their actual and potential environmental effects . To date, strong acid digestion is used often for the determination of total heavy metals in the sediments. However, this method can be misleading when assessing environmental effects due to the potential for an overestimation of exposure risk. Moreover, in order to determine the mobility of heavy metals in sediments, various sequential extraction procedures have been developed [69-71].
Among a range of available techniques using various extraction reagents and experimental conditions to investigate the distribution of heavy metals in sediments and soils, the 5-step Tessier et al.  and the 6-step extraction method, Kersten and Fronstier  were mostly widely used. Following these two basic schemes, some modified procedures with different sequences of reagents or experimental conditions have been developed [40-43]. Considering the diversity of procedures and lack of uniformity in different protocols, a BCR, Bureau Commun de Recherche (now called the European Community (EC) Standards Measurement and Testing Programme) method was proposed . It harmonized differential extraction schemes for sediment analysis. The method has been validated using a sediment certified reference material BCR-701 with certified and indicative extractable concentration of Cd, Cr, Cu, Ni, Pb and Zn . This method was applied and accepted by a large group of specialists [31, 44, 45, 47, 73, 74] despite some shortcoming in the sequential extraction steps [75, 76].
Wang et al.  used a modified Tessier sequential extraction method to investigate the distribution and speciation of Cd, Cu, Pb, Fe, and Mn in the shallow sediments of Jinzhou Bay, Northeast China. This site was heavily contaminated by nonferrous smelting activities. They found out that the concentrations of Cd, Cu and Pb in sediments was to be 100, 73, 13 and 7 times, respectively, higher than the National guidelines (GB 18668-2002). The sequential extraction tests revealed that 39%-61% of Cd was found in exchangeable fractions. This shows that Cd in the sediments posed a high risk to the local environment. Copper and Pb were found to be at moderate risk levels. According to the relationships between percentage of metal speciation and total metal concentration, it was concluded that the distributions of Cd, Cu and Pb in some geochemical fractions were dynamic in the process of pollutants migration and stability of metals in marine sediments from Jinzhor Bay decrease in the order Pb>Cu>Cd.
Yuan et al.  applied BCR-sequential extraction protocol to obtain metal distribution patterns in marine sediments from the East China Sea. The results showed that both the total contents and the most dangerous non-residual fractions of Cd and Pb were extremely high. More than 90% of the total concentration of V, Cr, Mo and Sn existed in the residual fraction while more than 60% of Fe, Co, Ni, Cu, and Zn were mainly present in the residual fraction. Manganese, Pb, and Cd were dominantly present in the non-residual fractions in the top sediments.
Jones and Turki  worked on distribution and speciation of heavy metals in surface sediments from the Tees estuary, North East England. Tessier et al.  metal speciation scheme modified by Ajay and van Loon  was used for the study. They observed out that the sediments were largely organic-rich clayey silts in which metal concentrations exceed background levels, and which attain peak values in the upper and middle reaches of the estuary. Chromium, Pb and Zn were associated with the reducible, residual, and oxidizable fractions. Cobalt and Ni were not highly enriched while Cu is associated with the oxidizable and residual fractions. Cadmium is associated with the exchangeable fractions.
Pempkowlak et al.  investigated the speciation of heavy metals in sediments and their bioaccumulation by mussels. They used a 4-step sequential extraction procedure adapted from Forstner and Watmann . Their investigation which was characterized by varying metal bioavailability was aimed at revealing differences in the accumulation pattern of heavy metals in mussel inhabiting that inhabit in sediments. The bioavailabilities of metals were measured using the contents of metals adsorbed to sediments and associated with Fe and Mn hydroxides. The biovailable fraction of heavy metals contents in sediments collected from Spitsbergen represented a small proportion (0.37% adsorbed metals and 0.11%, are associated with metals hydroxides). It was also revealed that the percentages of metals adsorbed and bound to hydroxides of the sediments ranged from 1 to 46% and 1 to 13%, respectively.
Wepener and Vermeulen  investigated on the concentration and bioavailability of selected metals in sediments of Richards Bay harbor, South Africa. Sequential extraction of sediments was carried out according to Tessier et al.  method. The following metals were investigated: Al, Cr, Fe, Mn, and Zn. Their studies revealed that metals concentrations in sediments samples varied only slightly between seasons, but showed significant spatial variation, which was significantly correlated to sediment particle size composition. Highest metal concentration was recorded in sites with substrates dominated by fine mud. Manganese and Zn had more than 50% of this concentration in reducible fraction while more than 70% of the Cr was associated with the inert fractions and the concentration recorded at some sites were still above action levels when considering only the bioavailable fractions. They also concluded that the concentration of Zn recorded was not elevated their results were compared with the historic data.
Coung and Obbard  used a modified 3-step sequential extraction procedure to investigate metal speciation in coastal marine sediments from Singapore as described by the European Community Bureau of Reference (ECBR). Highest percentages of Cr, Ni, and Pb were found in residual fractions in both Kranji (78.9%, 54.7% and 55.9% respectively) and Pulang Tokong (82.8%, 77.3% and 62.2% respectively). This means that these metals were strongly bound to sediments. In sediments from Kranji, the mobility order of heavy metals studied were Cd>Ni>Zn>Cu>Pb>Cr while sediments from Pulan Tekong showed the same order for Cd, Ni, Pb and Cr, but had a reverse order for Cu and Zn (Cu>Zn). The sum of the 4-steps (acid soluble + reducible + oxidizable + residual) was in good agreement with the total metal content, which confirmed the accuracy of the microwave extraction procedure in conjunction with the GFASS analytical method.
Fedotov et al.  applied a modified technique for accelerated fractionation of heavy metals in contaminated soils and sediments using rotating coiled columns. Rotating coiled columns (RCC) is valuable for the continuous-flow sequential extraction and can be successfully applied to the dynamic leaching of heavy metals from soil and sediments. This is a fluoroplastic or steel coil wound around a rigid cylindrical drum, which revolves about its axis and, at the same time, revolves around the central axis of the device called planet centrifuge. The stationary (liquid, solid, or heterogeneous) phase is retained in the column because of the centrifugal force field, and the mobile liquid phase is continuously pumped through the column. A solid sample was retrieved in the rotating column as the stationary phase under the action of centrifugal forces while different elements (aqueous solution of complexing reagents, mineral salts and acids) were continuously pumped through. This procedure developed is time saving and requires only 4-5 hr instead of the several days needed for individual sequential extraction. Losses of solid sample are minimal. Further studies are needed to better estimate the reproducibility of the technique.
Nemati et al.  used a modified BCR sequential extraction procedure (SEP) in combination with ICP-MS to obtain the metal distribution patterns in different depths of sediments from Sungai Buloh, Selangor, Malaysia. The results showed that heavy metal contaminations at Sungai Buloh River sediments were more severe than at other sampling sites, especially for Zn, Cu, Ni and Pb. Nevertheless, the element concentrations from top to bottom layers decreased predominantly.
Mossop et al.  compared of original and modified BCR sequential extraction procedures for the fractionation of Cu, Fe, Pb, Mn and Zn in soils and sediments. The procedures were applied to five soil and sediment substrates: a sewage sludge-amended soil, two different industrially contaminated soils, river sediment and intertidal sediment. Extractable Fe and Mn concentrations were measured to assess the effects of the procedural modifications on dissolution of the reducible matrix components. Statistical analyses (two-tailed t-tests at 95% confidence interval) indicated that recovery of Fe in step 2 was not markedly enhanced when the intermediate protocol was used. However, significantly greater amounts were isolated with the revised BCR scheme than with the original procedure. Copper behaved similarly to Fe. Lead recoveries were increased by use of both modified protocols, with the greatest effect occurring for the revised BCR extraction. In contrast, Mn and Zn extraction did not vary markedly between procedures. The work indicates that the revised BCR sequential extraction proves better attack on the Fe-based components of the reducible matrix for a wide range of soils and sediments.
5. Sequential extraction of metals in sediments of the Hugli River Estuary and Indian Sundarban wetland: A case study
5.1. Materials and methods
5.1.1. Sample collection and sediment quality analysis
The delta region formed by Hugli (Ganges) River Estuary (HRE) and is famous for its luxuriant mangrove vegetation, known as Sundarban wetland, acclaimed as UNESCO World Heritage Site for its capacity of sustaining an excellent biodiversity. The wetland is characterized by a complex network of tidal creeks, which surrounds hundreds of tidal islands exposed to different elevations at high and low semi-diurnal tides. This is one of the most sensitive and vulnerable ecosystems in the world and suffers from environmental degradation due to rapid human settlement, tourism and port activities, operation of mechanized boats, deforestation, and increasing agricultural and aquaculture practices. The ongoing degradation is also related to huge siltation, flooding, storm runoff, atmospheric deposition, and other stresses resulting in changes in water quality, depletion of fishery resources, choking of river mouth and inlets, and overall loss of biodiversity. Moreover, the rapid economic development in this deltaic region has caused highly dense areas of human activity and led to serious contamination including heavy metals and persistent organic pollutants (POPs).
Nine sampling sites, namely Barrackpur (S1), Dakshineswar (S2), Babughat (S3), Budge budge (S4), Ulubaria (S5), Diamond Harbor (S6), Frezergunge (S7), Gangasagar (S8), and Haribhanga (S9) were selected considering the existence of typical sediment dispersal patterns along the drainage network systems (as shown in Figure 1) and their position was fixed by a global positioning system (GPS). The stations are representative of the variable environmental and energy regimes that cover a wide range of substrate behavior, wave–tide climate, and intensity of bioturbation (animal–sediment interaction), geomorphological–hydrodynamic regimes and distances from the sea (Bay of Bengal). The sites are exposed to a variable level of heavy metal contamination mainly from anthropogenic sources as mentioned earlier. Six sampling sites (S1 to S6) have been chosen along the lower stretch of Hugli River Estuary, while residual three sites (S7 to S9) were taken into account in the coastal regions of Sundarban wetland. All sampling sites together with the main stresses to which they are subjected are presented in Table 1.
During winter months (January–March 2009) surface sediment samples weighting 10 g were randomly collected in triplicate from the top 3–5 cm of the surface at each sampling site during low tide using a grab sampler, pooled and thoroughly mixed. Immediately after collection, the samples were placed in sterilized plastic bags in the ice box and transported to the laboratory. Samples were oven dried at 50°C, most gently disaggregated, transferred into precleaned inert polypropylene bags and stored in deep freeze prior to analyses. Each sample was divided into two aliquots: one unsieved (for the determination of sediment quality parameters) and the other sieved through 63 μm metallic sieves (for elemental analyses). Organic carbon content was determined following a rapid titration method  and pH with the help of a deluxe pH meter (model no. 101E) using combination glass electrode manufactured by M.S. Electronics Pvt. Ltd. (India). Mechanical analyses of sediment were done by sieving in a Ro-Tap Shaker manufactured by W.S. Tyler Company, Cleveland, Ohio.
|S1||Barrackpur||Industrial effluents, domestic sewage disposal, boating, bathing.|
|S2||Dakshineswar||Industrial and domestic effluents, boating, bathing, idol immersion site.|
|S3||Babughat||Power plant discharges, domestic sewage, boating, idol immersion site.|
|S4||Budge budge||Domestic and industrial effluents, bathing, boating.|
|S5||Ulubaria||Domestic and industrial effluents|
|S6||Diamond Harbour||Boating, recreational activities, bathing, fishing, jetties for fishing trawlers|
|S7||Frezergunge||Tourist activities, ferry services, fishing|
|S8||Gangasagar||Boating, tourist activities, dredging, fishing, agricultural, domestic and aquaculture practices|
|S9||Haribhanga||Boating, fishing and ferrying|
5.1.2. Analytical procedure
To determinate the total element concentration, sediment samples were digested in polytetrafluoroethylene vessels with aqua regia (HCl/HNO3, 3:1) and HF neutralized with H3BO3 in a 650 W microwave oven (CEM MDS 2000) with a program consisting of a 20-min ramp and a 30-min hold at 100% power in pressure and temperature controlled conditions (150 psi and 180°C). The digested samples were filtered, transferred to polyethylene containers and stored at +4°C until analysis. All reagents were Suprapur® grade (Merck). Reagent blank was processed with the samples and did not show any significant contamination. Accuracy of the procedure was checked using two different certified reference materials (CRM): MESS-2 and PACS-2, which are both marine sediments certified by the National Research Council of Canada for the element content. The MESS-2 recovery ranged between 91% and 116% for all the elements (Table 2). Precision, calculated as relative standard deviation (RSD%), resulted always lower than 5%.
In this study, we followed the sequential extraction procedure proposed by the European Union's Standards, Measurements and Testing program . Selective extraction is based on the procedure used by Tessier et al.  with improvements made according to the BCR, which examined and finally eliminated irreproducibility sources. It is made up of three steps, which dissolve the following phases, respectively: exchangeable and bound to carbonate, bound to Fe and Mn oxides and hydroxides, bound to organic matter and sulphides. Exchangeable and bound to carbonate phase (phase 1) is extracted with 0.11 M acetic acid, while the fraction bound to Fe–Mn oxides (phase 2) with 0.5 M hydroxylamine hydrochloride, adjusted to pH 2 with nitric acid (65%). The phase bound to organic and sulphides (phase 3) is extracted with 8.8 M hydrogen peroxide (stabilized at a pH included between 2 and 3), treated at 80°C in a microwave oven using a program consisting of a 30-min ramp and a 60-min hold at 50% power in pressure and temperature controlled conditions (80 psi and 85°C), and 2 M ammonium acetate adjusted to pH 2 with nitric acid (65%). Each extraction was carried out overnight (16 h) at room temperature. All the reagents employed were Tracepur® grade (Merck Eurolab, Italy). After each extraction, the samples were separated from the aqueous phase by centrifuging at 4, 000 rpm for 15 min. The sediments were washed with Milli-Q water and centrifuged again. The wash water was added to supernatants. The element content of the residual phase was obtained from the difference between the total content and the sum of phases 1, 2 and 3, according to Ianni et al. [37, 38], Ramirez et al. , and Mester et al. . Sequential extraction reagent blanks showed no detectable contamination. Accuracy of the procedure was checked with BCR-701 (SM&T). The recovery rates for trace elements in the standard reference material ranged between 77% and 118% (Table 3). Precision, calculated as RSD%, resulted generally lower than 5%, except As and Cr in the phase 1 (~20%).
|Found BCR-701 step 1||198±1||2.57±|
|Found BCR-701 step 2||3,451±|
|Found BCR-701 step 3||1,912±|
|Certified BCR-701 step 1||n.a.||n.a.||7.34±|
|Certified BCR-701 step 2||n.a.||n.a.||3.77±|
|Certified BCR-701 step 3||n.a.||n.a.||0.27±|
|Recovery step 1||n.a.||n.a.||83.0%||n.a.||107%||96.8%||n.a.||n.a.||94.4%||106%||90.3%|
|Recovery step 2||n.a.||n.a.||89.4%||n.a.||85.9%||80.6%||n.a.||n.a.||92.3%||87.9%||89.4%|
|Recovery step 3||n.a.||n.a.||104%||n.a.||118%||117%||n.a.||n.a.||114%||76.9%||108%|
The elemental concentrations were determined with an inductively coupled plasma atomic emission spectrometer Vista Pro (Varian), with the internal standard method. Cadmium was determined by electrothermal atomization atomic absorption spectrometry. A Varian Spectra A300 spectrometer with Zeeman effect background correction and autosampler Varian Model 96 was used employing the standard addition method for calibration. All the metal analyses were performed at the Department of Chemistry and Industrial Chemistry of the University of Genoa (Genoa, Italy).
5.1.3. Statistical analyses
Principal component analysis (PCA) was used to characterize the metal composition in sediments, and cluster analysis was used for grouping the sampling stations. Principal component analysis (PCA) is a multivariate statistical technique used for data reduction and for deciphering patterns within large sets of data. With PCA, a large data matrix is reduced to two smaller ones that consist of principal component (PC) scores and loadings. PC loadings are eigenvectors of the correlation or covariance matrix depending on which is used for the analysis. The PC scores contain information on all of the variables combined into a single number, with the loadings indicating the relative contribution of each variable to that score . Hierarchical cluster analysis (HCA) characterizes similarities among samples by examining interpoint distances representing all possible sample pairs in high-dimensional space. The sample similarities are represented on two dimensional diagrams call dendrograms . All statistical analyses were performed using the computer software STATISTICA (StatSoft, Inc. 2001).
5.2. Results and discussion
5.2.1. Sediment geochemistry
Table 4 shows values of pH; organic carbon (%); and percentage of sand, silt, and clay in sediments of the nine sampling sites. Organic carbon values, ranging from 0.22% (in station S8) to 1.02% (in station S2), are low in comparison with values found in sediments from other Indian coastal areas, such as Gulf of Mannar , Cochin , and Muthupet mangroves . The low organic carbon values might be related with the poor absorbability of organics on negatively charged quartz grains, which predominate in sediments in this estuarine environment . In addition, the constant flushing activity by tides along with the impact of waves can support the low percentage of organic carbon in the sediments. The sediments of the studied stations are characterized by slightly basic pH (7.50–8.36) with maximum values recorded in the stations closest to the sea (stations S6, S8, and S9) and minimum in station S7.
|S1||22º43′ 16″ N|
88º21′ 20″ E
|S2||22º39′ 17″ N|
88º12′ 25″ E
|S3||22º33′ 53″ N|
88º20′ 19″ E
|S4||22º30′ 10″ N|
88º11′ 48″ E
|S5||22º28′ 06″ N|
88º06′ 54″ E
|S6||22º11′ 14″ N|
88º11′ 15″ E
|S7||21º34′ 44″ N|
88º15′ 03″ E
|S8||21º38′ 15″ N|
88º03′ 53″ E
|S9||21º34′ 20″ N|
88º01′ 25″ E
These were different from the low pH values in most of the mangrove swamps in Hong Kong , where sediments were not frequently flooded by the tide and become acidic in reducible conditions. With respect to texture, the sediment samples show a variable admixture of sand, silt, and clay. Clay fractions dominate in low-energy areas of suspensional deposits. On the contrary, silt, and sand dominates where the energy level is high. Sediments from station S7 contain higher percentage of sand (98%) compared to the others, while sediments from S1, S2, S5, and S8 contains higher percentage of silt (more than 50%) compared to the others. A variable mixture of sand, silt, and clay is present in the other stations and reflect a variable amount of erosion and deposition.
5.2.2. Total element concentrations
Total element concentrations in the investigated stations varied in a narrow range of values (Table 5) and were comparable with data obtained for other Indian coastal areas [94, 95]. Datta and Subramanian  found very similar trace element concentrations throughout the Bengal Basin, where anthropogenic perturbation is low and river channel may receive a several centimeter-thick sediment layer in a single event during peak flow, preventing to bear the signature of an accumulation of trace elements. The highest concentrations for As, Cu, Fe, Mn, and Ni were measured at station S9 while for Cd and Pb at station S3, close to Calcutta city (about 4.5 million residents, but about 14.2 million including suburbs). An anthropogenic input from vehicular traffic and in-dustrial activities may cause high Cd and Pb con-centrations measured in samples collected in the Calcutta urban area. The lowest element concentrations were found at station S5. Very low (close to the detection limit) Cd concentration was found in the coastal stations (S7, S8, and S9).
The geoaccumulation index (Igeo) of Muller  has been calculated for the analyzed elements, by comparing current concentrations with pre-industrial levels, in order to estimate the metal contamination in sediments. The equation used for the calculation of Igeo is: log2 (Cn/1.5 Bn), where Cn is the measured content of element “n” and Bn the element’s content in “average shale” . Factor 1.5 is used because of possible variations in background values for a given element in the environment, as well as very small anthropogenic influences . As shown in Figure 2, all sediments fall in class 0 for Al, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn, therefore the area is not contaminated for these elements. Unlike the Hugli river, in other rivers of the Bengal Basin, such as Meghna and Brahmaputra, Cr exhibits higher Igeo values respect to the other elements . For Cd, two stations fall in class 1 and three in class 2 exhibiting a moderate contamination for this element. In all stations, As falls in class 2 (moderate pollution). In this area, As contamination was already observed in previous studies and it is probably due to groundwater contamination . This contamination can have natural origin, such as coal seams in Rajmahal basin and arsenic mineral in mineral rocks in the upper reaches of the Ganges river system. The highly reducing nature of groundwater would reduce As, causing the possible desorption of As .
5.2.3. Speciation patterns
The potential environmental risk of trace elements in sediments is associated with both their total content and their speciation. The chemical partitioning of the considered elements (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) from each extraction step has been described. Aluminum, Cr, and Fe are present mainly in the residual phase, representing 95.8–96.8%, 88.9–91%, and 83.0–94.7% of the total concentration, respectively, which implies that these elements are strongly linked to the inert fraction of the sediments. This result was in good agreement with data reported by several studies carried out worldwide in marine coastal areas [45, 46, 78, 102]. The high percentage of Fe in the residual phase indicates that most of the Fe exists as crystalline Fe peroxides (goethite, limonite, magnetite, etc.). The remaining Fe is associated with the reducible phase (mean, 11.25%). Large amounts of Fe accumulate in the residual phase probably because it is basically of natural origin (it is the most common element in the earth’s crust).
Concentrations of Al, Fe, and Cr are very low in exchangeable phase (0.08%, 0.26%, and 1.72% as mean values, respectively), limiting their potential toxicity as pollutants. It should be noted that sediments always act as reservoir for elements; therefore, their potential risk of pollution to environment has always to be considered.
Arsenic, Co, Ni, and to some extent Zn, are found mainly in the residue (∼50% of the total concentration). Nickel and Co are associated to the residue respectively for 56% and 74% of the total concentration, with a speciation similar in all the samples. A mean of 23% of Co is present in the phase 2. The highest percentage of labile Co (~13%) was measured in S6 (Diamond Harbour) and S8 (Gangasagar) and can be due to a recent input of this element. The dominant proportion of Ni in the residual phase is in agreement with the results of other studies [27, 46]. Nickel is present, apart from the residue, in phases 2 and 3 (about 10% in each phase). Arsenic is distributed mainly between the residual (mean 47%), the reducible, and the oxidizable phases (mean 19% and 22%, respectively). Acharyya et al.  observed that As is adsorbed to iron-hydroxide-coated sand grains and to clay minerals in the sediments of the Ganges delta from West Bengal. Among the studied elements, As is found with the greatest proportion in the oxidizable phase coinciding with organic and sulfur compounds. Arsenic is present in the phase 1 for about 10% of the total content, in station S7 phase 1 percentage rises up to 16%. The lower land alluvial basin of the Ganges River is recognized as an arsenic-affected area. Arsenic in solution probably is easily entrapped in the fine grained organic-rich sediments deposited in the Ganges delta . The percentage of silt (lower than 70% except in S1 and S2) may have contributed to a low retention of dissolved As since coarse sediments are less efficient at retaining As.
Cadmium was mainly present in the labile phase (more than 60%) in all the stations with the exception of station S7, where the Cd labile percentage represents only 25% of the total concentration. Cadmium concentrations were negligible in phases 2 and 3. The highest labile Cd concentration was measured at station S3, the closest to the city of Calcutta. Datta and Subramanian  found that the concentrations of elements in the non-detrital phases were higher in stations sampled in the Hugli river around Calcutta than in samples collected along Brahmaputra and Meghna rivers. The petroleum refinery, industrial, and mining effluents carried by the Hugli river may be responsible for this higher concentrations of non detrital fractions.
About 40% of the total Cu concentration is associated to the residue, while 33% of Cu is bound to Fe-Mn oxide and hydroxide (phase 2). The high percentage of Cu in the residue is likely due to the fact that Cu is easily chemisorbed on or incorporated in clay minerals . All the samples showed lower Cu concentrations in exchangeable phase, with percentage ranging from 7% (S7) to 22% (S5), with a mean of 15%. Copper is characterized by high complex constant with organic matter thus it can be hypothesized that Cu is bound to labile organic matter such as lipids, proteins, and carbohydrates. On the other hand, high-element concentration in labile phase could be related to recent coastal input .
Manganese was found in all the four sediment phases, as observed by other researchers [45, 104]. Manganese is the most mobile element since it is present with the highest percentage (a mean of 42%) in the labile phase. This is probably because of the known close association of Mn with carbonates  as endorsed by other workers [69, 106]. In this phase, weakly sorbed Mn retained on sediment surface by relatively weak electrostatic interactions may be released by ion exchange processes and dissociation of Mn-carbonate phase . The result indicates that considerable amount of Mn may be released into environment if conditions become more acidic . The highest Mn labile percentage was measured in S6 (57%). Differently, in S7, Mn in the residue represents 65% of the total concentration, while the labile Mn is only 15%. A substantial Mn percentage was also found in the residue (mean 37.8%), followed by the reducible phase (14.7%), in which Mn exists as oxides and may be released if the sediment is subjected to more reducing conditions .
The major geochemical phase for Pb in these sediments was the Fe-Mn oxides phase (mean 55.7%) followed by the residual phase (mean 30.2%) while lower percentage of the total Pb are bound to exchangeable-labile (mean 5.3%) and oxidizable phases (mean 6.8%). At S3 (Babughat), the reducible part is as high as 65% and only 19.9% of the total is associated with the residue. Atmospheric input as fallout from vehicular emission can be probably the major input of Pb for this station. The relatively high percentage of Pb in reducible phase is in agreement with the known ability of amorphous Fe–Mn oxides to scavenge Pb from solution [109, 110]. Caille et al.  observed that resuspension of anoxic sediment results in a rapid desorption of Pb and Cu adsorbed to sulphides. Thus, a high element percentage in the reducible fraction is a hazard for the aquatic environment because Fe and Mn species can be reduced into the porewaters during early diagenesis by microbially mediated redox reactions . Dissolution will also release Pb associated with oxide phases to the porewater possibly to the overlying water column  and to benthic biota . The major sources of Pb are from intensive human activities, including agriculture in the drainage basin , auto exhaust emission together with atmospheric deposition . In addition, a substantial contribution from the factories located in the upstream of the Hugli river dealing with Pb producing lead ingots and lead alloys play a vital role as referred by Sarkar et al. [ 116].
The percentage of Zn in residue is highly variable (38.5–70%) and the distribution pattern in each fraction showed the following order: residual>reducible>oxidizable> exchangeable and bound to carbonates. There was some difference in Zn speciation among the sampling sites: in stations S1, S2, and S3 about 40% of Zn is present in the residue, while in the other stations this percentage increases to more than 60%. In station S1, the exchangeable and oxidisable phases shared over 22% of the total Zn, whereas labile Zn was as low as 4.6% at S7. A major part of Zn (16.3%) is associated with Fe–Mn oxide phase, because of the high stability constants of Zn oxides. Iron oxides adsorb considerable quantities of Zn and these oxides may also occlude Zn in the lattice structures .
The BCR procedure as discussed above showed satisfactory recoveries, detection limits, and standard deviations for determinations of heavy metals/metalloid in the sediments. It is evident from the present results of the fractionation studies that the metals/metalloids in the sediments are bound to different fractions with different strengths leading to variations in mobility and availability and some of them show significant spatial variations subject to diverse environmental stresses. This type of association between metals and the sediments can be understood in detail by sequential extraction techniques. Hence the application of sequential extraction is fully justified as the quantification of different forms of metal is more meaningful than the estimation of its total metal concentrations. The strength values can, therefore, give a clear indication of sediment reactivity, which in turn assess the risk connected with the presence of metals in this wetland environment. The results obtained suggest the need for corrective remediation measures due to the higher accumulation of potentially dangerous metals/metalloids, which in most cases exceed the limits established by certain legislation.
5.2.4. Comparison with Sediment Quality Guidelines
Results obtained after total and sequential extraction are compared with Sediment Quality Guidelines (SQGs). Table 6 reports consensus-based values, such as TEC (concentration below which harmful effects on sediment-dwelling organisms were not expected) and PEC (concentration above which harmful effects on sediment-dwelling organisms were expected to occur frequently), and effect range-low and range-medium, such as ERL (concentrations below which adverse biological effects were observed in less than 10% of studies) and ERM (concentrations above which effects were more frequently observed in more than 75% of studies).
Comparing our results with the SQGs, it is revealed that for Pb and Zn in all the stations the measured concentrations are lower than both TEC and ERL. As regards Cd, concentration measured in station S3 is higher than TEC and ERL but lower than PEC and ERM both in term of total and labile concentration. For this station, some possible toxic effect on benthic organism can be hypothesized, in particular because of the large amount of element bound to the most labile phase of the sediment. Considering Cu, some stations (S2, S3, S6, and S9) exhibit total concentrations higher than TEC but lower than PEC. Concentrations of Cu are higher than ERL but lower than ERM only in stations S2 and S9. Since only 7–22% of total Cu is bound to the labile phase, in all stations Cu labile concentrations are lower than TEC and ERL. Total As concentrations in stations S1, S2, S3, S4, and S9 are higher than ERL value but lower than TEC value. Since more than 50% of total As is not found in the residue, attention should be paid to a change in the environment conditions which could induce a release of As from the sediments. Total Ni and Cr concentrations are higher than TEC (Ni is also higher than ERL) but lower than PEC (and ERM in the case of Ni) in all the stations. Nevertheless, more than 70% of Ni as well as 90% of Cr are present in the residual fractions, therefore adverse impacts on organisms is very much negligible.
Mean sediment quality guidelines quotients (mSQGQ) have been developed for assessing the potential effects of contaminant mixtures in sediments : they are determined by calculating the arithmetic mean of the quotients derived by dividing the concentrations of chemicals in sediments by their respective SQGs. The probability of observing sediment toxicity can be estimated by comparing the mSQGQ in a sample to previously published probability tables. It is important to keep in mind that mSQGQs cannot be used to accurately predict the uptake and bioaccumulation of sediment-bound chemicals by fish, wildlife, and humans, even if there is considerable evidence that this assessment tool can be predictive of the presence or absence of toxic effects .
SQGQs are calculated for seven elements considering ERM as sediment quality guidelines (Table 7). The mean quotient values ranges from 0.16 in station S5 to 0.24 in station S3. Using PEC values instead of ERM, the mean SQGQ ranges from 0.25 in station S5 to 0.38 in station S3 (Table 7).
Compiled data from multiple data sets reporting 10-day toxicity test conducted on amphipod species in saltwater showed that the incidence of toxicity for a range of SQGQ of 0.25–0.5 is ∼35%, while for a mean SQGQ range from 0.1 to 0.25, the incidence of toxicity lowers to ∼20%. Measures recorded in a survey of Biscayne Bay (port of Miami and the adjoining saltwater reaches of the lower Miami River, FL, USA) showed that the average amphipod survival (
5.2.5. Statistical analyses
The relationships between variables and the differences between stations were evaluated by PCA. The analysis was performed on 36 objects (four sediment phases for nine stations) and 11 variables (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn). Two significant components were identified explaining 68.3% and 14.5% of the total variance, respectively. By studying the loadings of the variables (Figure 2a) on the components it can be seen that all the elements except Cd, Mn, and Pb are significantly correlated.
Unlike the other elements, most of Cd and Mn is present in the first phase: labile Cd and Mn represent more than 60% and 40% of the total concentration, respectively, except in station S7. Cadmium and Mn speciation can be ascribed to their considerable affinity for carbonates. Lead is the only element which is bound to the reducible phase for more than 50%. Lead is a very reactive element in water column and, having scavenging type behavior, is easily bound to hydroxy- and oxyligands. Copper is positively and significantly correlated with all elements except Cd and Mn, but with lower correlation coefficients (0.66–0.81).
In the score plot (Figure 2b) phases 1, 2, 3, and 4 (corresponding to labile, reducible, oxidizable, and residual phases respectively) are identifiable by 1, 2, 3, and 4 suffix, respectively. In all stations, residue concentrations were characterized by negative values of PC1 and consequently by high concentrations of Al, As, Co, Cr, Fe, Ni, and Zn. Conversely, in the positive PC1 semi-axis labile and oxidizable metal concentrations, which represent a small percentage of the total elements, are distributed. For all stations, reducible concentrations are distributed along the positive PC2 semi-axis, i.e., high Pb concentrations, with a maximum for station S3 and a minimum for S5. The group formed by elements bound to organic matter and sulphides (phase 3) is characterized by low values of both PC1 and PC2. Therefore, a low percentage of elements (higher than 20% exclusively for all As data and for Zn in stations S1 and S3) is bound to the oxidizable phase, suggesting the presence of an oxidant environment. High Mn and Cd concentrations are associated with negative values of PC2, therefore a relatively high concentration of labile Mn and Cd is present in all samples (in particular in S3), except station S7. Samples are prevalently grouped in relation to the sediment geochemical phase, suggesting a similar element speciation among the stations. Station S7 represents an exception, in fact the labile fraction is closely associated to the oxidizable phase group.
A HCA was carried out by applying Euclidean distances to quantitatively identify specific groups of similar stations. In the dendrogram of the sampling stations (Figure 3), we can note two main clusters: the first represented by station S7, characterized by the highest element percentage bound to residue, and the second constituted by all the remainder stations. In the second group, a subgroup formed by station S5 and S6 can be individuated.
Station S7 was sampled in a marine coastal environment; it is characterized by a peculiar grain size percentage respect to the other stations, being the sand percentage as high as 98.6%. In general, the concentrations of elements are much higher in fine than in coarse fraction because the fine fraction larger specific surface facilitates absorption processes. As previously noted by Ramirez et al. , this pattern is particularly evident for Cd. It is interesting to note that the marine coastal stations S8 and S9 are more similar to river stations than to station S7. Both the stations S8 and S9 are, in fact, located in front of the Hugli river runoff, while station S7 is located easternmost and probably is less influenced by the Hugli river discharge.
5.3. Conclusion and recommendation
The study provides valuable information on the potential mobility of trace elements in sediments collected along the stretch of Hugli River and in the Sundarban mangrove wetland (northeastern part of the Bay of Bengal). The results obtained adopting BCR sequential extraction method provided the following important information: (i) Al, Cr, and Fe were found mostly in the residual phase while the other elements were found in the four phases sharing different proportions; (ii) the dominant Cd, Mn, and Pb proportion was found in the non-residual fractions and (iii) Mn had the highest percentage in the labile phase. This is worthwhile to mention that coastal environment of West Bengal is considerably constrained due to implementation of dredging, construction of port/ harbor and other industrial activities. The authors strongly recommend for periodical monitoring on the bioavailability and mobility of trace elements, control the mixing of effluent of the concentration of heavy metals in the region, environmental remediation, treatment of industrial effluent and municipal wastewater for effective management of this estuarine system. It is wisely suggested that an environmental recovery framework should be urgently implemented to avoid extension of heavy metal contamination (especially As).
6. Case studies from other coastal regions in India
Although the importance of metal speciation and fractionation has been realized in developed countries, the subject has not really taken off in India and only few references are available on the speciation of metals in Indian rivers. Speciation of selected heavy metals geochemistry in surface sediments (n=10 was studied by Venkatramanan et. al.  from Tirumalairajan river estuary, east coast of India. The results obtained from sequential extraction showed that a larger portion of the metals were associated with the residual phase, although they are available in other fractions too.
Trace metal fractionation in the Pichavaram mangrove–estuarine sediments in southeast coast of India was studied by Ranjan et. al.  considering the pronounced changes due to occurrence of tsunami (2004). A 5-step sequential extraction procedure was applied to assess the effects of tsunami on mobility and redistribution of selected elements (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) in coastal sediments revealed that metals in the residual fraction (lattice bound) had the highest concentration suggesting their non-availability and limited biological uptake in the system. Majority of the metals (except Mn) do not constitute a risk based on the different geochemical indices.
Fractionation of selected metals in the sediments of Cochin estuary and Periyar River (southwest coast of India) was studied by Mohan et. al. . The results reveal that remobilization potential of metals bound is in the range of low to medium risk to various sedimentary phases is different and is based on bond strength. Therefore, the strength values can give a clear indication of sediment reactivity that can be used to assess the risk related with metals to the aquatic organisms.
7. Risk Assessment Code (RAC)
The risk assessment code (RAC) mainly applies the sum of the exchangeable and carbonate bound fractions for assessing the availability of metals in sediments. These fractions are considered to be weakly bonded metals which may equilibrate with the aqueous phase and thus become more rapidly bioavailable [11, 33]. This is important because the fractions introduced by anthropogenic activities, such as agricultural runoff and tourism, are typified by the adsorptive, exchangeable, and bound to carbonate fractions, which are weakly bonded metals that could equilibrate with the aqueous phase and thus become more rapidly bioavailable . According to RAC guideline (Table 8), for any metal, soil/sediment which can release in exchangeable and carbonate fractions, less than 1% of the total metal will be considered safe for the environment and soil/sediment with 11-30% carbonate and exchangeable fractions will be at medium risk to the environment. On the contrary, soil/sediment releasing in the above fractions more than 50% of the total metal has considered being highly dangerous, which can be easily enter the food chain .
|II||1 – 10||Low risk|
|III||11 – 30||Medium risk|
|IV||31 – 50||High risk|
|V||>50||Very high risk|
Heavy-Metal Fractionation in surface sediments was studied by Dhanakumar et. al.  in the Cauvery river estuarine region, southeastern coast of India. The results revealed that most of the samples fall under the category from low- to high-risk class and from low-risk to very high-risk class in terms of labile fractions of Pb as well as Zn and Cu, respectively.
From the above discussion it is revealed that geochemical fractionation approach to the chemical speciation has provided a useful tool and opens a new dimension in assessing the potential mobility/bioavailability of heavy metals and metalloids in soils/sediments and opens a new dimension in the field of ecology and environmental chemistry. More efficient, non-laborious and time saving processes techniques in this field of chemical speciation are also coming up to get valid information regarding geochemical behavior of soils/sediments. Besides geochemical fractionation, Dezileau et al.  opined that total Fe or Fe/Al may be used to infer millennial-scales climate changes in the south eastern pacific while performing sequential extraction of Fe in marine sediments from the Chileau continental margin. However, the chemical partitioning should be carefully used in the assessment of environmental pollution as large amount of metals may naturally occur as anthropogenic fractions (including loosely bonded ions, sulfide ions and metals associated with sediments).
The authors gratefully acknowledge full support and cooperation of the Springer press, UK for extending permission in publishing the research paper of the journal Environmental Monitoring & Assessment, vol. 184(12), pp:7561-77, 2012.
This study was partially supported by the European Fund for Economic and Regional Development (FEDER) through the Program Operational Factors of Competitiveness (COMPETE) and National Funds through the Portuguese Foundation for Science and Technology (PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689).