Ability of 3 extraction methods (BCR, Tessier and protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain)

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by Elsevier in Marine Pollution Bulletin, Vol. 102, Issue 1 on January 2016, available at: https://doi.org/10.1016/j.marpolbul.2015.11.057 © 2015 Elsevier. En idUS Licencia Creative Commons CC BY-NC-ND Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) Daniel Rosado a,b, José Usero a, José Morillo a a Department of Chemical and Environmental Engineering, University of Seville, 41092 Seville, Spain b Chemistry Department, Universidad Técnica Particular de Loja, 11 01 608 Loja, Ecuador HIGHLIGHTS:  Protease K represents the bioavailable fraction in sediments most reliably.  BCR and Tessier sequential extraction methods overestimate bioavailable fraction.  BCR and Tessier methods have a similar ability to estimate bioavailable fraction.  Bioavailable metals in sediment from Huelva estuary are related to total content. CORRESPONDING AUTHOR: * Department of Chemical and Environmental Engineering, University of Seville, Camino de los Descubrimientos, s/n, 41092 Seville, Spain. Tel.: 0034 954 487 274; E-mail address: d[email protected]s (Daniel Jesús Rosado Alcarria). Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 1 Abstract The bioavailable fraction of metals (Zn, Cu, Cd, Mn, Pb, Ni, Fe, and Cr) in sediments of the Huelva estuary and its littoral of influence has been estimated carrying out the most popular methods of sequential extraction (BCR and Tessier) and a biomimetic approach (protease K extraction). Results were compared to enrichments factors found in Arenicola marina. The linear correlation coefficients (R2) obtained between the fraction mobilized by the first step of the BCR sequential extraction, by the sum of the first and second steps of the Tessier sequential extraction, and by protease K, and enrichment factors in Arenicola marina, are at their highest for protease K extraction (0.709), followed by BCR first step (0.507) and the sum of the first and second steps of Tessier (0.465). This observation suggests that protease K represents the bioavailable fraction more reliably than traditional methods (BCR and Tessier), which have a similar ability. Keywords Trace metals; sediment bioavailability; BCR Tessier sequential extraction; Protease K; biomimetic approach; Huelva estuary; Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 2 1 Introduction Quite a few human activities produce waste, much of which is discharged in aquatic ecosystems (Salomons et al., 1987). Pollutants are present in these ecosystems in both water and sediment, where they accumulate over time as a result of various physical and chemical processes and can reach concentrations that are much higher than those in water (Ashokkumar et al., 2009). In addition, sediments are a food source for many species and any toxic substances contained in them can be transmitted to aquatic species and can affect humans through the food chain. For all these reasons, the presence of contaminants in the sediments of aquatic ecosystems has become a major environmental problem (Chapman and Wang, 2001; Ruiz et al., 2008; Sainz and Ruiz, 2006). When a deposit-feeder organism ingests sediment, only a fraction of the total amount of contaminant present therein is absorbed and becomes part of its body, known as the bioavailable fraction. Therefore, methods to quantify the bioavailable fraction are needed. Sequential extractions are the most used methods to estimate the mobility of metals (Gleyzes et al., 2002; Izquierdo et al., 1997; Nieto et al., 2007), closely related to bioavailability. Among the various existing methods, the most widely used (Long et al., 2009; Rao et al., 2007; Yu et al., 2010) are BCR-701, developed by the Community Bureau of Reference (Pueyo et al., 2001), and the one proposed by Tessier et al. (1979). Many studies attest to the precision, accuracy and reproducibility (Sutherland, 2010; Tessier et al., 1979) of both. Furthermore, these methods meet all the necessary requirements for implementation over a wide area: simple, applicable to all kinds of sediments, low cost and easily understandable and comparable results. However, sequential extractions use different chemical reagents to those found in the digestive tract of deposit-feeder organisms, where hydrolytic enzymes and chemicals derived from the pre-digestion abound (Mayer et al., 1997). Therefore, their use to quantify the bioavailable fraction is questioned (Turner, 2000). There are methods specifically designed to estimate the bioavailability of metals in sediments, such as biomarkers or bioaccumulation studies. Biomarkers, also known as biochemical responses, are used as quick indicators of contamination in living organisms (Ellis and Schneider, 1997). However, many biomarkers respond to changes in environmental conditions (temperature, parasites, etc.), meaning that they are not reliable. Furthermore, a biomarker only responds to one or few contaminants, so this method is Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 3 complex and time-consuming to study a wide range of contaminants (Chen and Summons, 2001; Luca-Abbott, 2001; Viganò et al., 2001). In bioaccumulation studies, pollutant concentrations are determined in tissues or fluids of the stomach and intestine of benthic organisms, such as oligochaetes, insects or mollusks (Chen and Chen, 1999; Chen and Mayer, 1999; Pempkowiak et al., 1999; Sanchiz et al., 2001; Usero, 1997). This is probably the most appropriate method to evaluate the bioavailable fraction of the pollutant in a particular species. However, extracting the required amount of fluid and thus obtaining a sufficient number of organisms, is timeconsuming and is not suitable for analyzing a large number of samples (Turner, 2000). Although biomarkers and bioaccumulation studies could provide a better estimate of the bioavailable fraction compared with sequential extractions, their implantation is complex over a large area because of the disadvantages outlined above. In order to assess the bioavailability of metals in sediments by methods specifically designed for this purpose and which can be applied over large areas, some researchers developed biomimetic methods. These methods employ biological reagents located in the digestive system of deposit-feeder organisms (Turner, 2000) to reproduce in the laboratory the metal solubilization that occurs in the digestive tract of the deposit-feeder organism. Gastrointestinal conditions can be simulated using digestive enzymes (Mayer et al., 1995), such as pepsin, trypsin and protease K (Ruby et al., 1996; Turner et al., 2001; Wróbel et al., 1999). Recent research (Bignasca et al., 2011; Ianni et al., 2010; Turner, 2006) demonstrates the potential of protease K to quantitatively imitate the interaction between metals and the digestive fluids of deposit-feeder organisms. This study has been carried out in Huelva estuary, formed by the confluence of the rivers Tinto and Odiel, and consists of estuaries of both rivers and the Padre Santo Canal (Figure 1). The estuary of the Odiel has a secondary outlet to the sea known as Punta Umbria estuary. Huelva estuary is highly polluted (Achterberg et al., 2003; Barba-Brioso et al., 2010; Ruiz et al., 2008) due to several contaminant sources in the area: metals from Tinto and Odiel rivers, industrial effluents from factories and sewage from the coastal towns. Both rivers pass through the Iberian Pyrite Belt, a region long known for its mining activities (Leblanc Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 4 et al., 2000), are acidic (the pH occasionally falls to 2 or 3) and contain large amounts of metal from erosion and mining (Nelson and Lamothe, 1993). The main objective of this work is to estimate the bioavailable fraction of metals (Zn, Cu, Cd, Mn, Pb, Ni, Fe, and Cr) in sediments of the Huelva estuary and its littoral of influence by carrying out the most popular methods of sequential extraction (BCR and Tessier) and a biomimetic approach (extraction with protease K), compare the results obtained with enrichments factors found in Arenicola marina (Bignasca et al., 2011; Ianni et al., 2010; Turner, 2006) and clarify which method estimates the bioavailable fraction in the most reliable manner. 2 Materials and methods 2.1 Sampling and sample pre-treatment 16 sampling points were chosen to cover the Huelva estuary and its littoral of influence. 12 points were located in the Huelva estuary (see Table 1 and Figure 1): 5 in the Odiel River estuary (O1-O5), 4 in the Tinto River estuary (T1-T4) and 3 in the Padre Santo canal (C1- C3). Four points correspond to the coast (L1-L4). Table 1. Coordinates of the sampling points. Point North West O1 37o22´50´´ 6o58´41´´ O2 37o15´44´´ 6o57´55´´ O3 37o14´45´´ 6o57´24´´ O4 37o13´47´´ 6o57´19´´ O5 37o12´35´´ 6o56´53´´ T1 37o18´43´´ 6o49´17´´ T2 37o14´41´´ 6o54´14´´ T3 37o13´48´´ 6o55´12´´ T4 37o12´50´´ 6o55´51´´ C1 37o11´08´´ 6o55´47´´ C2 37o10´06´´ 6o54´27´´ C3 37o09´11´´ 6o52´58´´ L1 37o11´41´´ 7o02´04´´ L2 37o08´35´´ 6o54´19´´ L3 37o07´01´´ 6o48´51´´ L4 37o05´58´´ 6o46´44´´ Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 5 Figure 1. Location of the sampling points in the Huelva estuary. Sediment samples were taken in October 2013 with a Van Veen grab and in duplicate at each point. Only grabs that achieved adequate penetration (2/3 of total volume) to collect the first 5 cm of the sediment and that showed no evidence of leakage or surface disturbance were retained, transferred and stored in a dark cooler at 4 °C. In the laboratory, a representative sample from each station was prepared by mixing and homogenizing the two samples taken at each sampling point. The samples were dried at 60 °C in an oven, were disaggregated with an agate mortar and sieved to obtain a fraction with a particle size <63 µm, as recommended by many researchers (Jones and Turki, 1997; Ouyang et al., 2002) to minimize the effect of difference grain sizes in the samples. 2.2 Total metal content To determine total metal content, sediment underwent the method proposed by the BCR (Pueyo et al., 2001), in which 3 g of every sample are digested with 28 ml of aqua regia (commercial nitric and hydrochloric acids, 1:3 by volume) in a reaction flask with a reflux condenser and a sand bath. Then, the extract was centrifuged 20 min at 4,200 rpm (3,000 g) to separate the solid and liquid phases. Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 6 The supernatant was used to determine Zn, Cu, Cd, Mn, Pb, Ni, Fe and Cr by atomic absorption spectrophotometry (AAS) using a double-beam Perkin–Elmer 2380 AAS with deuterium background correction and, in the case of low concentrations, by graphite furnace atomic absorption spectrometry (GFAAS) using a Varian spectra 220 with Zeeman-effect background correction. Total metal content determination was tested using the reference material, BCR-701. The concentrations obtained for each of the elements analyzed demonstrated recoveries greater than 85%, which can be considered satisfactory for this type of analysis (Pueyo et al., 2001). 2.3 Extraction procedures The first steps of two sequential extraction procedures were carried out in sediments to obtain the acid extractable fraction of BCR procedure (Pueyo et al., 2001) and the exchangeable and bound to carbonates fractions of Tessier sequential extraction (Tessier et al., 1979). Conditions defined by both procedures are given in Table 2. Table 2. Comparison of extraction procedures for BCR, Tessier and protease K (Pueyo et al., 2001; Tessier et al., 1979; Ianni et al., 2010; Turner, 2006). Protocol Fraction Solute Concentration pH Time BCR Acid extractable Acetic acid 0.11 M - 16 h Tessier Exchangeable MgCl2 1 M 7 1 h Bound to carbonates Sodium acetate 1 M 5 5 h Protease K - Protease K 0.4 mg/ml 7.4 3 h To obtain the acid extractable fraction following the BCR procedure (Pueyo et al., 2001), 1 g of every sample were digested with 40 ml of acetic acid. After the extraction, the suspension was centrifuged for 20 min at 4,200 rpm (3,000 g). The supernatant was used to determine Zn, Cu, Cd, Mn, Pb, Ni, Fe and Cr as described for the total metal content. As in the case of total metal content, the first step of BCR sequential extraction was tested using the reference material BCR-701, achieving recoveries greater than 85% for each element. Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 7 Tessier’s exchangeable fraction (F1) (Tessier et al., 1979) required digesting 3 g of every sample with 24 ml of the MgCl2 solution. After the extraction, the suspension was centrifuged for 20 min at 4,200 rpm (3,000 g), the extract was stored at 4 oC and the solid residue was washed with distilled water and centrifuged again. To extract the bound to carbonates fraction (F2), solid residue from the previous step was treated with 24 ml of the sodium acetate solution and centrifuged for 20 min at 4,200 rpm (3,000 g) after the extraction. Then, Zn, Cu, Cd, Mn, Pb, Ni, Fe and Cr were analyzed in the remaining solution as described for the total metal content. The bioavailable fraction was also estimated (Table 2) using Protease K solutions (0.4 mg/ml) following a procedure based on those proposed previously (Ianni et al., 2010; Turner, 2006). Protease K was purchased lyophilized from Sigma-Aldrich (P-8044) and solved in pH 7.4 standard phosphate buffer solution prepared from solid salts: 1.179 g of potassium dihydrogen phosphate (KH2PO4) and 4.300 g of disodium hydrogen phosphate (Na2HPO4) in 1 L of distilled water. Protease K extractable fraction was obtained by digesting 0.5 g of every sample with 25 ml of the Protease K solution for 3 h at room temperature with magnetic stirring (250 rpm). Then the suspensions were centrifuged for 20 min at 4,200 rpm (3,000 g). The supernatant was used to determine Zn, Cu, Cd, Mn, Pb, Ni, Fe and Cr as described for the total metal content. 3 Results and discussion 3.1 Total and bioavailable metal content Metal concentrations found in the sediments of the Huelva estuary (Table 3) are generally very high compared to background levels and quality criteria (Bray, 2008; Crommentuijn et al., 2000; DelValls et al., 2004; Long et al., 1995; MacDonald et al., 2000; Turekian and Wedepohl, 1961), especially considering metals of pyritic origin (Zn, Cu, Cd, Pb, Fe, As and Se). The waters of the Tinto and Odiel rivers are enriched in these metals (Morillo and Usero, 2008; Morillo et al., 2005) in the upper zones of the basins when they flow through the "Iberian Pyrite Belt" (Leblanc et al., 2000; Sánchez España et al., 2005). Downstream, much of the metals dissolved precipitate in the estuaries of both rivers and the Padre Santo Canal due to the strong increase in pH, that changes from remarkably acid in the upper and Ability of 3 extraction methods (BCR, Tessier and Protease K) to estimate bioavailable metals in sediments from Huelva estuary (Southwestern Spain) 14 Total metal content found in the sediments of the Huelva estuary are generally very high compared to background levels and quality criteria and are in the range of those found in polluted areas, especially considering the metals of pyritic origin (Zn, Cu, Cd, Pb, Fe, As and Se). However, total metal content found in sediments of the Huelva littoral are significantly lower than those of the Huelva estuary and, in many cases, are even below background levels. Spatial distribution of total metal content in the 4 areas studied (estuaries of the Tinto and Odiel rivers, Padre Santo Canal and Huelva littoral) shows that metals of pyritic origin have similar trends, which are reproduced in the concentrations of bioavailable fractions obtained by sequential extraction procedures (BCR and Tessier) and biomimetic approach (protease K). This trend is characterized by an increase in the concentration of metals in sediments of Tinto estuary seawards, reaching the highest concentrations where Tinto and Odiel estuaries merge. The first phase of BCR extracted the highest amount (p<0.05) of six metals (Zn, Cu, Cd, Mn, Ni and Fe), followed by the sum of the first and seconds steps of Tessier and protease K. In the case of Cu, the difference between the traditional methods (BCR and Tessier) and the protease K is reduced to be almost equal in all three cases. The sum of the first and seconds steps of Tessier extracted higher amounts of Pb and Cr (p<0.05) than the first step of BCR and protease K. The linear correlation coefficients (R2) between metal extracted ratio, defined as the fraction mobilized by the first step of the BCR sequential extraction, by the sum of first and second steps of the Tessier sequential extraction, and by protease K, and enrichment factors (ER) in Arenicola marina, shows its highest value for protease K extraction (0.709), followed by BCR first step (0.507) and the sum of the first and second steps of Tessier (0.465). This observation suggests that protease K represents the bioavailable fraction more reliably than traditional methods (BCR and Tessier), which overestimate bioavailable fraction and have a similar ability to estimate the bioavailable fraction. 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