**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 [86] 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.

#### *5.1.2. Analytical procedure*

(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

Nemati et al. [84] 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

Mossop et al. [85] 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

**5. Sequential extraction of metals in sediments of the Hugli River Estuary**

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

needed to better estimate the reproducibility of the technique.

**and Indian Sundarban wetland: A case study**

*5.1.1. Sample collection and sediment quality analysis*

decreased predominantly.

730 Environmental Risk Assessment of Soil Contamination

soils and sediments.

**5.1. Materials and methods**

To determinate the total element concentration, sediment samples were digested in polyte‐ trafluoroethylene 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

**Figure 1.** Map showing the location of the monitoring sites (S1 to S9) covering Hugli River Estuary and Sundarban mangrove wetland along with the location of the major industries.

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%.


**Table 1.** Details of the nine sampling sites and the main stresses to which they are subjected.


**Table 2.** Results of certified reference materials MESS-2 and PACS-2 as well as the observed values. All the values are expressed in μg/g of dry weight. MESS-2 and PACS-2 recovery rates are also reported.

In this study, we followed the sequential extraction procedure proposed by the European Union's Standards, Measurements and Testing program [3]. Selective extraction is based on the procedure used by Tessier et al. [2] 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

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

**Figure 1.** Map showing the location of the monitoring sites (S1 to S9) covering Hugli River Estuary and Sundarban

always lower than 5%.

mangrove wetland along with the location of the major industries.

732 Environmental Risk Assessment of Soil Contamination

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 centri‐ fuging 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. [39], and Mester et al. [27]. 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%).


**Table 3.** Results of certified reference materials BCR-701 as well as the observed values (expressed in μg/g of dry weight) together with recovery rates for each step. n.a.= not available.

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).
