Sequential extraction studies on aquatic sediment and biofilm samples for the assessment of heavy metal mobility

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Sequential extraction studies on aquatic sediment and bio ﬁ lm samples for the assessment of heavy metal mobility

Márk Horváth ⁎ , Gábor Halász, Eva Kucanová, Beáta Kuciková, Ilona Fekete, Dagmar Remeteiová, György Heltai, Karol Flórián

Szent István University, Department of Chemistry and Biochemistry, H-2103 Gödöllő, Páter K. u. 1, Hungary Technical University of Košice, Department of Chemistry, Letná 9/A, 040 11 Košice, Slovakia

a b s t r a c t a r t i c l e i n f o

Article history:

Received 28 November 2011

Received in revised form 30 March 2012 Accepted 26 May 2012

Available online 1 June 2012 Keywords:

Sediment Bioﬁlm Metals Fractionation Extraction

Aquatic sediment samples and biofilms from two sampling sites representing different environmental situations (Košice, Slovakia and Gödöllő, Hungary) were studied on their heavy metal content. Fractionation of the metallic content of the samples was done by the improved three-step BCR sequential extraction procedure supplemented with microwave-assisted HNO3/H2O2digestion and another three-step method using supercritical CO2, subcritical H2O and their mixture, pseudototal element content was gained by microwave-assisted HNO3/H2O2diges- tion of the original samples. Influence of the sample–extractant ratio on the extracted elements' concentration in the BCR procedure was also studied. The sum of concentrations for Zn, Pb, Ni and Cu in the biofilm extracts of the (3+ 1)-step BCR procedure was in most cases higher than the pseudototal concentrations, so that the sequential extraction may be more effective than the single-step HNO3/H2O2digestion. Lower sample–extractant ratios may increase the efficiency of the BCR method. The estimated easily mobilizable element content provided by the two sequential procedures (first step of the BCR with 0.11 mol dm⁻³acetic acid and second and third steps of the alternative method with H2O and H2O + CO2, respectively) was similar in case of Cd, Ni and Cu in both biofilm and sediment samples.

1. Introduction

Risk assessment of environmental samples affected by a possible heavy metal contamination often applies the chemical characterization that focuses on the chemical species forms of elements, which deter- mine their availability among various environmental compartments and conditions[1]. The term speciation refers to the distribution of a given element amongst its physico-chemical forms (species) identi- ﬁed in the system according to the IUPAC recommendation[2]. The total speciation of all pollutant elements in a real sample can hardly be studied, as the structure of the different environmental samples can be very diverse and complex, therefore methods of operative speciation were developed, which fractionate the analytes from a certain sample according to the deﬁned physical or chemical properties.

Heavy metal fractions of different availability have been deﬁned by Ure et al.[3–5]. Methods for the fractionation of the metallic element content of aquatic sediments are mostly based on the works of Tessier et al.[6], later several 5‐to 8-step sequential extraction schemes were

developed[7–9]. In these procedures increasingly more“aggressive”re- agents are applied to separate fractions of different solubility or binding forms while gradual decomposition of the original structure takes place.

To decrease the time demand and the risk of contamination in each in- dividual step of these multistep schemes, the Community Bureau of Reference (BCR) proposed a simpliﬁed three-step procedure[3,10,11], and a certiﬁed reference material (CRM-601) was elaborated for valida- tion purposes[12]. The original BCR procedure was improved as some problems of accuracy came to light (Table 1), and a new sediment reference sample (BCR-701) was issued[13–15].

Although the BCR procedure became widespread used to study the metal fractionation in aquatic sediments and soils as well[16,17], some problems have to be considered. As original compounds get decomposed gradually, intact species cannot be extracted and their identiﬁcation is not possible. Some important pathways of metal mobilization and immobilization in aquatic environments e.g. binding to carbonates and dissolution by hydrocarbonate forming is not represent- ed in the BCR scheme. The three-step procedure is still very labor- intensive, moreover, the repeatability and reproducibility of the mea- surements are strongly inﬂuenced by the sample matrix, particularly in case of samples with high content of organic matter and carbonates [18–20]. As atmospheric gravitation dust can play an important role in the heavy metal contamination of soils in urban and industrial areas [21], efforts were made to extend the applicability of the BCR procedure to this kind of samples. Dabek-Zlotorzynska et al. [22]developed a

⁎Corresponding author.

E-mail addresses:horvath.mark@mkk.szie.hu(M. Horváth),

halasz.gabor@mkk.szie.hu(G. Halász),eva.kucanova@tuke.sk(E. Kucanová), beata.ﬁoova@tuke.sk(B. Kuciková),fekete.ilona@mkk.szie.hu(I. Fekete),

dagmar.remeteiova@tuke.sk(D. Remeteiová),heltai.gyorgy@mkk.szie.hu(G. Heltai), karol.ﬂorian@tuke.sk(K. Flórián).

doi:10.1016/j.microc.2012.05.024

Contents lists available atSciVerse ScienceDirect

Microchemical Journal

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m i c r o c

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miniaturized and ultrasonic supported BCR procedure for airborne PM2.5 dust samples, that can also be used for other samples of small quantity (e.g. sediment, bioﬁlm). Rusnák et al.[23]developed an accel- erated BCR scheme with ultrasonic treatment for soil, sediment and gravitation dust samples. Another problem may occur by the separation of an extract and the solid material after each step, as readsorption of the extracted species can take place during the centrifugation[24].

Shiowatana et al. [25] proposed a continuous-ﬂow system instead of batch leaching methodology to overcome this phenomenon and improve the accuracy of the BCR procedure in different matrices.

Water as a non-destructive solvent for leaching can be used to extract various components from biological and environmental samples[26]. Easily mobilizable element content of soils can be extracted by hot water percolation[27]. Subcritical water is also capable of extracting organic compounds of low polarity at high temperature [28], however, this goal can be reached under milder conditions in similar instrumentation using supercritical CO2[29]. Aim of our former work was to combine the advantages of CO₂and water used sub- sequently in a supercriticalﬂuid extractor (SFE procedure), so a three- step sequential extraction scheme was elaborated and tested in our laboratory[19,30]. The main details of the scheme are presented in Table 2.

This procedure was applied to study lake and river sediments in comparison to the conventional BCR methodology[31]. The water soluble and carbonate bound element fractions, which are extracted together in theﬁrst step of the BCR method, could be distinguished.

The sum of the amounts extracted in the second (subcritical H2O) and third (subcritical H2O + CO2) steps was commensurable with the amount extracted in theﬁrst step of the BCR procedure (0.11 M acetic acid). Moreover, aqueous extracts, while they can conserve intact species forms, were applicable in bioassays to collect information about the ecotoxicity of sediments[32].

Testing of surface sediments for screening purposes may call atten- tion on certain situations of pollution, so that detailed investigation on pollution profiles should be done. Study of vertical distribution of metals include conventional methods e.g. core sampling[33]as well as recent developments such as DET (diffusive equilibrium in thin films) that has been applied to study depth profiles in estuarine sediments[34].

Biofilms represent a significant potential of binding heavy metals in aquatic environments, therefore their study is an important tool of risk assessment[35,36]. Holding et al.[37]studied the relationship between the exchangeable heavy metal content of biofilms and the BCR fractions of aquatic sediments. Since the collection of large sample amounts may be difficult in case of biofilms, conventional analytical methods which bring the analyte to a solution by extraction or digestion are not always possible. Therefore alternative methods with reduced sample weight or direct sample introduction such as electrothermal vaporization may come to the front. Moreover, direct element determination by non-destructive methods e.g. total reflec- tion X-rayfluorescence spectrometry (TXRF) are also suitable, as it was demonstrated in a study on the river Tisza after the pollution in 2000[38]. However, these methods do not give information on the binding forms in biofilms and the transition ways from sediment to biofilm and back.

The aim of our present project was to study the above possibilities for the improvement of sequential extraction procedures to different environmental samples, in particular bioﬁlms, moreover, looking for conclusions concerning of mobilization and immobilization of heavy metals in the sediment/water/bioﬁlm system.

2. Materials and methods 2.1. Samples

Sampling sites of the recent study were selected on the basis of our former experiences: samples from Gödöllő, Hungary represent the influence of industrial waste water in afishpond, whereas samples from Košice, Slovakia may be affected by atmospheric dust depo- sition from metallurgical industry [24,30]. Biofilm samples were collected in November 2009 at the river Hornád in Košice at the site Nad Jazerom (KošBIO1) andŤahanovce (KošBIO2), and at afishpond near Gödöllő (Göd BRICK, STONE, WOOD) from natural support surfaces. In 2010 surface sediment and biofilm samples were collected at the siteŤahanovce, Košice (KošBIO3, SED3). The samples were air dried. Lake surface sediment (Göd SED), also originated from the latterfishpond, was selected from our former study[30]. This sample has a relatively high organic carbon and CaCO3 content (CaCO3= 7.6%, TOC = 11%).

2.2. Improved BCR sequential extraction

The steps of the improved BCR sequential extraction procedure are described inTable 1 [14]. Sample–extractant ratio was 1:40 according to the protocol, as initial sample weight was 1 g that was extracted in 40 cm³0.11 mol dm⁻³acetic acid. In the present investigation the nitric acid/H2O2digestion in a MILESTONE 1200 MEGA microwave oven was applied instead of aqua regia extraction for gaining the residual fraction, according to our former studies of Gödöllőlake sediments [33]. Extraction and digestion procedure were repeated three times for each sample. For each extraction and digestion step a procedure blank was also prepared according to the BCR protocol.

Pseudototal element fractions were gained by the nitric acid/H2O2

digestion of original samples similar to the treatment used for gaining the residual fraction[33], however, this procedure could only be performed once for each sample because of the low sample amounts.

The bioﬁlm sample KošBIO2 was used in a complementary study to test the use of different sample weights (0.2, 0.5 and 1.0 g, i.e. sample:extractant ratio of 1:200, 1:80 and 1:40, respectively) in the BCR procedure.

2.3. Sequential extraction in a supercriticalﬂuid extractor

The three-step sequential extraction procedure was performed in a supercriticalﬂuid extractor consisting of two Jasco PU 980 HPLC Table 1

The improved BCR sequential extraction scheme.

Step Extractant Chemical information

1 0.11 mol dm⁻³HOAc Exchangeable, water and acid

soluble element fraction (e.g. carbonates) 2 0.5 mol dm⁻³NH2–OH.HCl (adjusted

to pH = 1.5 by adding 25 cm³ 2 mol dm⁻³HNO3)

Reducible element fraction (joint to Fe/Mn oxides, oxyhydroxides) 3 8.8 mol dm⁻³H2O2then 1 mol dm⁻³

NH4OAc (pH = 2)

Oxidizable element fraction (bound to organic matter or sulﬁdes)

4 (+1) Aqua regia/(HNO3/H2O2) Residual fraction

Table 2

The three-step CO2/H2O sequential extraction scheme.

Step Extractant Temperature (°C)

Pressure (MPa)

Duration (min)

Chemical information (fraction)

1 Supercritical CO2 80 27 30 CO2-soluble organic fraction and sulﬁdes

2 Subcritical H2O 80 27 30 Water-soluble

fraction 3 Subcritical

H2O/CO2(5%)

80 27 30 Fraction bound to

carbonates

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pumps (the CO2pump was cooled to−6 °C), a Jasco CO 980 column oven and Jasco 880–81 back pressure regulator (heated to 60 °C).

Supercritical CO₂, subcritical H₂O and subcritical mixture of 90% H₂O and 10% CO2 solvents were applied in this order. Duration of the steps was set according to our preliminary time study: 60 min for CO₂, 60 min for H₂O, and 90 min for H₂O/CO₂extraction.

Preparation of the samples was optimized earlier: 0.5 g of bioﬁlm or sediment sample was mixed by SiO2(Reanal) in mass ratio of 5:95.

Stainless steel extraction columns werefilled as follows:first a layer of 1 g SiO2, then 10 g of sample–SiO2mixture, and the remaining vol- ume wasfilled with SiO2again. Extraction of pure SiO2was done for procedure blank values.

2.4. Determination of Zn, Cd, Pb, Ni, Cr, and Cu in the extracts

Jobin Yvon 24 sequential inductive coupled plasma optical emis- sion spectrometer (ICP-OES) was used with the following operation parameters: incident power: 1.1 kW, plasma argon gas ﬂow:

12 dm³min⁻¹, sheath argon gasﬂow: 0.2 dm³min⁻¹, nebulization argon gasﬂow: 0.35 dm³min⁻¹, nebulizer: Babington-type, sample uptake rate: 1.5 cm³min⁻¹. Calibration for each type of extract was performed by MERCK multielement standard with matrix-matching according to the applied extractant.

3. Results and discussion

3.1. BCR sequential extraction of bioﬁlm and sediment samples

Elements' (Zn, Cd, Pb, Ni, Cr, Cu) concentrations in the fractions of the BCR procedure and in the pseudototal fraction from the bioﬁlm and sediment samples are presented inTable 3.

The results show that the sum of the element concentrations in the BCR fractions (1+ 2 + 3 + 4) usually exceeds the pseudototal fraction.

In case of the elements which could be found in concentrations well over the limit of detection (Zn, Pb, Ni, Cr, Cu), this difference is often three times greater than the standard deviation calculated for the sum of the BCR fractions, however, the determination of the pseudototal fractions could only be performed once for each sample because of the low amounts of available original samples.

This phenomenon may arise as the efficiency of the microwave- assisted digestion with H2O2/HNO3might not be satisfactory for the complete destruction of biofilm structures or sediments containing organic residues. Hydrolysis of polysaccharide structures in biofilms may need a significant amount of acid, therefore metallic ion content may not be extracted in this single-step digestion as efficient as in the steps of the BCR sequential procedure.

Zn occurs mainly in the easily mobilizable fraction (BCR 1), by Pb the reducible fraction (BCR 2) is dominant in the bioﬁlm samples,

Table 3

Element concentration (mg kg⁻¹related to dry weight, mean ± standard deviation) of bioﬁlm and sediment samples in different fractions: after microwave-assisted digestion (pseudototal) and the BCR 3(+ 1)-step extraction, respectively (n.d.: not detected).

Sample BCR 1 BCR 2 BCR 3 BCR 4 (1 + 2 + 3 + 4) Pseudototal

KošBIO1 14.1 ± 3.6 17.4 ± 11.7 2.08 ± 2.11 31.0 ± 0.6 64.5 ± 13.4 41.5

KošBIO2 43.5 ± 5.3 25.3 ± 7.7 2.43 ± 1.95 36.0 ± 1.0 107.3 ± 9.3 72.4

KošBIO3 5.27 ± 0.3 10.2 ± 0.3 42.0 ± 1.4 52.2 ± 11.7 109.6 ± 11.0 59.3

Zn KošSED3 27.1 ± 0.7 51.8 ± 2.3 16.1 ± 1.3 35.2 ± 1.3 130.2 ± 3.9 97.3

Göd WOOD 195 ± 127 n.d. n.d. 32.0 ± 7.5 227 ± 123 72.4

Göd STONE 59.7 ± 5.5 n.d. n.d. 43.7 ± 46.9 103 ± 56 48.9

Göd BRICK 37.4 ± 1.0 8.27 ± 4.29 n.d. 13.0 ± 0.2 58.7 ± 3.4 46.4

KošBIO1 n.d. n.d. n.d. 0.68 ± 0.02 0.68 ± 0.02 0.38

KošBIO2 0.07 ± 0.04 n.d. n.d. 0.76 ± 0.05 0.83 ± 0.26 0.84

KošBIO3 0.04 ± 0.09 0.13 ± 0.08 0.23 ± 0.03 0.85 ± 0.28 1.25 ± 0.29 0.64

Cd KošSED3 0.32 ± 0.06 0.23 ± 0.11 0.17 ± 0.03 0.77 ± 0.07 1.48 ± 0.12 1.05

Göd WOOD 1.00 ± 0.23 n.d. n.d. 0.54 ± 0.30 1.54 ± 1.27 1.64

Göd STONE 0.13 ± 0.00 n.d. n.d. 0.96 ± 1.36 1.10 ± 0.08 0.72

Göd BRICK 0.13 ± 0.00 n.d. n.d. 0.46 ± 0.08 0.59 ± 0.53 0.70

KošBIO1 n.d. 8.80 ± 2.42 n.d. 2.23 ± 0.41 11.0 ± 2.6 3.60

KošBIO2 0.51 ± 0.24 13.6 ± 4.6 n.d. 2.41 ± 0.27 16.5 ± 4.5 7.55

KošBIO3 0.92 ± 0.77 0.67 ± 1.18 14.81 ± 0.67 4.33 ± 0.35 20.7 ± 0.6 9.11

Pb KošSED3 1.05 ± 0.87 14.7 ± 1.3 3.13 ± 2.61 4.97 ± 0.66 23.8 ± 4.5 15.0

Göd WOOD n.d. 34.7 ± 10.0 n.d. n.d. 34.7 ± 10.0 12.9

Göd STONE 1.07 ± 0.57 29.4 ± 4.7 n.d. 4.45 ± 4.13 34.9 ± 10.6 7.69

Göd BRICK 2.80 ± 0.17 19.4 ± 8.1 n.d. 0.79 ± 0.47 23.0 ± 8.1 13.8

KošBIO1 2.11 ± 0.18 3.05 ± 1.30 0.37 ± 0.28 11.6 ± 0.2 17.1 ± 1.1 10.6

KošBIO2 5.45 ± 0.38 4.27 ± 2.26 0.45 ± 0.28 14.6 ± 0.5 24.7 ± 1.7 19.9

KošBIO3 2.83 ± 0.14 n.d. 5.58 ± 0.18 17.1 ± 0.9 25.5 ± 0.7 14.9

Ni KošSED3 8.96 ± 0.18 15.1 ± 1.4 4.83 ± 0.48 14.9 ± 0.9 43.7 ± 2.7 33.3

Göd WOOD 3.47 ± 0.53 3.07 ± 2.34 n.d. 77.8 ± 32.9 84.4 ± 35.2 55.5

Göd STONE 0.07 ± 0.57 7.90 ± 3.25 n.d. 76.3 ± 22.6 84.3 ± 24.2 135

Göd BRICK 1.97 ± 0.33 3.28 ± 1.61 n.d. 130 ± 55 135 ± 2 150

KošBIO1 n.d. n.d. 0.67 ± 0.10 27.3 ± 3.7 27.9 ± 0.1 12.6

KošBIO2 n.d. 0.23 ± 1.88 0.68 ± 0.23 26.1 ± 0.4 27.0 ± 1.1 24.7

KošBIO3 n.d. n.d. 1.91 ± 0.36 22.1 ± 2.2 24.0 ± 2.5 13.2

Cr KošSED3 0.07 ± 0.02 1.29 ± 0.15 0.93 ± 0.24 21.2 ± 6.0 23.4 ± 6.4 20.6

Göd WOOD n.d. 8.40 ± 8.30 21.5 ± 15.9 223 ± 92 253 ± 99 165

Göd STONE 0.83 ± 0.42 19.3 ± 7.4 8.63 ± 3.01 252 ± 46.8 281 ± 62 374

Göd BRICK 0.51 ± 0.64 14.5 ± 10.1 11.0 ± 2.6 376 ± 130 402 ± 8 430

KošBIO1 2.57 ± 0.16 4.77 ± 2.24 0.23 ± 1.18 15.7 ± 0.2 23.3 ± 1.7 14.8

KošBIO2 2.35 ± 0.22 9.16 ± 4.63 n.d. 15.1 ± 0.5 26.6 ± 4.4 22.4

KošBIO3 3.31 ± 0.08 0.16 ± 0.04 19.9 ± 0.9 15.2 ± 0.4 38.5 ± 0.8 24.5

Cu KošSED3 7.20 ± 0.02 25.4 ± 0.2 14.4 ± 0.6 15.2 ± 0.2 62.2 ± 0.4 49.9

Göd WOOD 4.20 ± 0.58 11.4 ± 6.5 n.d. 18.3 ± 2.6 33.9 ± 7.0 18.1

Göd STONE 3.57 ± 0.71 12.3 ± 4.9 n.d. 18.0 ± 14.7 33.9 ± 4.5 12.1

Göd BRICK 1.79 ± 0.56 4.75 ± 2.84 n.d. 7.42 ± 1.36 14.0 ± 2.7 13.3

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whereas Ni, Cr and Cu can be found mostly in the residual fraction (BCR 4). Composition of the sediment and bioﬁlm samples from the same location (KošBIO3 and SED3) is similar, however, concentration of the studied elements is usually greater in the sediment.

3.2. Effect of the sample weight on the extracted amounts in the BCR procedure

Table 4shows the Zn, Cd, Pb, Ni, Cr and Cu concentration in the fractions gained by the BCR extraction of the biofilm sample Koš BIO2 using different sample weights, their effect was studied by one‐way analysis of variance (ANOVA) using Microsoft Excel 2003 software. The difference between the extracted amounts was significant (P= 0.95) in 7 cases, where the values of the 5% LSD (least significant

difference) are presented. It can be assumed that the sample/extractant ratio influences the efficiency of the dissolution of the element content of biofilms. Possible reason may be the polymer structures in biofilms which may retard the release of metallic species.

3.3. CO2/H2O/CO2+ H2O extraction of bioﬁlm and sediment samples

The concentration of Zn, Cd, Pb, Ni, Cr and Cu in the fractions gained by the CO₂/H₂O/CO₂+ H₂O sequential extraction of the samples Koš SED3 and Koš BIO3 can be seen in Table 5. The results show that most of the easily mobilizable Zn, Cd, Ni and Cu content of the sediment can be dissolved by subcritical H₂O/CO₂mixture. In case of the bioﬁlm sample the water-soluble fraction of Pb, Cr, and Cu is greater, however, detectable amounts of Zn, Ni and Cu could be found in the fractions gained by supercritical CO2.

InTable 6the easily mobilizable element contents estimated as the fractions extracted in theﬁrst step of the BCR method and the second and third step of the CO2/H2O/CO2+ H2O extraction are compared.

The easily mobilizable element content estimated by both BCR (1) and SFE (2 + 3) extraction steps is higher in the sediment than in the bioﬁlm sample. The SFE (2 + 3) and BCR (1) fractions are similar in case of Cd, Ni and Cu in both samples, so the SFE method may be a relevant alternative to the extraction with acetic acid in the BCR (1) step. Moreover, distinction could be drawn between the water- soluble and hydrocarbonate-forming fractions (Table 5).

4. Conclusions

Evaluation of biofilms as indicators of heavy metal pollution is possible by conventional analytical methods, which have been developed to investigate aquatic sediments, however, the different matrix structures require certain modifications. Determination of the pseudototal element fractions of biofilms requires improvement of the microwave-assisted HNO3/H2O digestion, as its efficiency does not reach that of the (3 + 1)-step BCR procedure. The BCR sequential extraction procedure and the supercritical CO2/subcritical H2O/H2O + CO2extraction are suitable methods in biofilm studies; these methods complement each other in providing relevant information about environmental mobility of metallic species. Mobilization of metals between freshwater sediments and biofilms depends on the speciation of an element in a sediment i.e. the easily mobilizable fraction bound to carbonates, and the ability to enter biofilm forming microor- ganisms. Release of metallic content from biofilms may be influenced by biopolymer structures during thefirst two step of the BCR procedure, as their intensive decomposition can take place by oxidation only in the third step, therefore the extractant–sample ratio should be increased in order to improve the efficiency of the release of metals.

Acknowledgements

This work was supported by the Hungarian Scientiﬁc Research Fund project OTKA K72926.

Table 4

Element concentration (mg kg⁻¹related to dry weight, mean ± standard deviation) in the fractions of the BCR 3(+ 1)-step extraction of the bioﬁlm sample KošBIO2 using 0.2, 0.5. and 1.0 g sample weight. 5% LSD: least signiﬁcant difference.

BCR 1 BCR 2 BCR 3 BCR 4

0.2 102 ± 21 n.d. n.d. 49.6 ± 1.1

Zn 0.5 58.7 ± 4.3 12.4 ± 4.9 n.d. 40.6 ± 6.4

1 43.5 ± 5.3 25.3 ± 7.7 2.43 ± 1.95 36.0 ± 1.0

5% LSD 25.2 7.51

0.2 n.d. n.d. n.d. 0.97 ± 0.16

Cd 0.5 0.05 ± 0.00 n.d. n.d. 0.88 ± 0.11

1 0.07 ± 0.04 n.d. n.d. 0.76 ± 0.05

5% LSD

0.2 1.53 ± 0.23 28.3 ± 4.2 n.d. 3.51 ± 0.80

Pb 0.5 0.61 ± 0.92 16.7 ± 6.6 n.d. 2.31 ± 1.59

1 0.51 ± 0.24 13.6 ± 4.6 n.d. 2.41 ± 0.27

5% LSD 10.5

0.2 4.77 ± 0.14 3.47 ± 1.42 n.d. 21.3 ± 0.8

Ni 0.5 6.27 ± 0.35 3.41 ± 1.33 n.d. 16.0 ± 2.8

1 5.45 ± 0.38 4.27 ± 2.26 0.45 ± 0.28 14.6 ± 0.5

5% LSD 3.3

0.2 0.67 ± 0.23 n.d. 4.00 ± 0.00 34.4 ± 2.2

Cr 0.5 0.32 ± 0.05 n.d. 1.50 ± 0.17 24.0 ± 17.4

1 n.d. 0.23 ± 1.88 0.68 ± 0.23 26.1 ± 0.4

5% LSD 0.33

0.2 8.80 ± 0.31 12.5 ± 9.0 n.d. 20.4 ± 4.2

Cu 0.5 3.68 ± 0.12 10.6 ± 5.4 n.d. 14.4 ± 2.3

1 2.35 ± 0.22 9.16 ± 4.63 n.d. 15.1 ± 0.5

5% LSD 0.46 4.34

Table 5

Extracted Zn, Cd, Pb, Ni, Cr and Cu concentrations (mg kg⁻¹related to dry weight) in the fractions obtained by the SFE sequential extraction of a sediment and a bioﬁlm sample.

Sample Extractant Zn Cd Pb Ni Cr Cu

CO2 n.d. n.d. n.d. 0.08 n.d. n.d.

KošSED3 H2O 0 0.007 0.22 n.d. n.d. 0.85

H2O + CO2 40 0.44 0.3 11.6 n.d. 5.28

CO2 0.22 n.d. n.d. 0.04 n.d. 0.04

KošBIO3 H2O n.d. 0.02 0.14 3 0.02 4.42

H2O + CO2 n.d. 0.02 0.02 n.d. n.d. n.d.

Table 6

Comparison of extracted quantities SFE (2 + 3) [extractants: H2O and H2O/CO2(90/10)] and BCR (1) [extractant: 0.11 mol dm⁻³acetic acid].

Sample Extract Zn Cd Pb Ni Cr Cu

KošSED3 SFE (2 + 3) 40.0 0.46 0.52 11.6 n.d. 6.13

BCR (1) 27.1 ± 0.7 0.32 ± 0.06 1.05 ± 0.87 9.0 ± 0.18 0.067 ± 0.023 7.20 ± 0.02

KošBIO3 SFE (2 + 3) n.d. 0.04 0.16 3.0 0.020 4.42

BCR (1) 5.3 ± 0.3 0.04 ± 0.09 0.92 ± 0.77 2.8 ± 0.14 n.d. 3.31 ± 0.08

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