AB STRA CT Soil samples from the industrial area in the town of Bužim, Bosnia and Herzegovina were analysed in order to de- termine their different manganese species. Samples were extracted from seven locations – at the manganese mine and the surrounding area. The paper aims to present the use of the sequential extraction method in determination of the specifi c distribution of Mn in soil, as well as in estimation of its origin, mobility and bioavailability in the sampling locations. Sequential extraction used here included determination of the amount of Mn in various soil fractions: the water-sol- uble fraction, exchangeable fraction, carbonate fraction, easily reduced fraction and the organic fraction. Addition- ally, it included manganese oxides or moderately reduced oxides, amorphous iron oxide, crystalline iron oxide and the residual fraction. It was determined that chemical properties of soil considerably affect the distribution of heavy metals within different soil fractions. The highest percentage of natural Mn was determined in the residual fraction (27.00%) at Popović polje, while the highest percentage of anthropogenic origin Mn was determined at Bućevci (57.00%) in the Fe-Mn oxides fraction. The highest near-total content of Mn was determined at Popović polje (20950.00 mg/kg). The highest percentage of natural Mn (27.00%) was determined in the same area. The highest percentage of Mn of an anthropogenic origin (57.00%) was determined at Bućevci. Keywords: sequential extraction, manganese, manganese bioavailability, manganese mine „Bužim“, manganese speciation, soil  Distribution and bioavailability of manganese in soil in the vicinity of the „Bužim“ abandoned mine  Safeta Redžić1, Galiba Sijarić1, Tidža Muhić-Šarac2, Ekrem Pehlić1 and Damir Hrnjica1 1 University of Bihać, Biotechnical faculty Bihać, Bosnia and Herzegovina; (corresponding author: safetabtf@gmail.com) 2 University of Sarajevo, Faculty of Natural Sciences and Mathematics Sarajevo, Bosnia and Herzegovina doi: 10.4154/gc.2014.04 Geologia Croatica 67/1 45–58 6 Figs. 5 Tabs. Zagreb 2014 Geologia CroaticaGeologia Croatica 1. INTRODUCTION Interest in the geochemical pattern of soil contamination has increased within the last few decades, due to the fact that soils serve as a depot for metals in an environment (wARREN & BIRcH, 1987; LI & THORNTON, 2001; OREŠČANIN et al., 2003; MARTLEY et al., 2004). MAcKLIN (1992) provi- des a description of soil and sediment contamination by metals as a result of mining activities. An ever-growing number of research papers are directed towards the determination of soil contamination by manga- nese (PIPER, 1931; BROMFIELD & DAVID, 1987; HAJ- DA REVIĆ et al., 1988; MARIKA & MARKKU, 2003; RUT- TEN & GERT, 2003; ALVAREZ et al., 2006; MARScHNER & RENGEL, 2003; MOSSOP & DAVIDSON, 2003; NAD- ASKA et al., 2009; ROUHOLLAH & FARZANEH, 2010; ZAKIR & SHIKAZONO, 2011). HORSTMAR (1851) conducted an early study investi- gating the infl uence of manganese content in soil on manga- nese content in plants. According to the research conducted by other researchers (MIHALJEVIĆ et al., 2003; SALETOVIĆ Geologia Croatica 67/1Geologia Croatica 46 Figure 1: Map of sample collection area near the manganese mine in „Bužim“. a) http://www.360.ba/buzim360/ b) Base from HODŽIĆ & ABDURAHMANOVIĆ (1998) Redžić et al.: Distribution and bioavailability of manganese in soil in the vicinity of the „Bužim“ abandoned mine Geologia Croatica 47 et al., 2011) Mn has an important role in the efficient utiliza- tion of other nutrients in the soil. The total amount of Mn in soils is 200-3000 ppm, where 0.10–1.00% is available to plants. In soils it generally origi- nates from MnO2, occurring in different oxides with oxida- tion states from +2 to +7 (NOGALES et al., 1997; ZHANG et al., 2012). The availability of Mn greatly depends on the oxidation-reduction conditions of soil (PIPER, 1931., DOLAR & KEENEY, 1971; BROMFIELD & DAVID, 1987), being reduced in neutral and alkaline environments, and increasing with acidity as Mn2+ reduces (VUKADINOVIĆ & LONČARIĆ, 1998). According to ZHANG et al. (2012) the acidity of soil may cause Mn oxide minerals to dissolve and release a significant amount of Mn ions in soil, therefore increasing the toxicity of soil for many plants. The aim of this research is to determine the specific dis- tribution of Mn and its origin, bioavailability (TRUDINGER & SwAINE, 1979; PERIN et al., 1985; LI et al., 1995) and the mobility (PERTSEMLI & VOUTSA, 2007; MOSSOP & DAVIDSON, 2003; HLAVAY et al., 2004; KALEMBKIE- wIcZ & SITARZ-PALcZAK, 2004; OLAYINKA et al., 2011) of certain Mn forms in soil at the „Bužim“ mine and the surrounding area. The research included analysis of water-soluble species, exchangeable species, carbonate species, easily reduced spe- cies, organic species, manganese oxides or moderately re- duced oxides, amorphous iron oxide, crystalline iron oxide and the residual fraction of Mn in soil in order to determine how mining activity affected the amount and origin of Mn in soil. Therefore, the sequential extraction method was em- ployed in this research (TESSIER et al., 1979; TESSIER & cAMPBELL, 1987; cALMANO et al., 1993; ZOUMIS et al., 2001; FILGUEIRAS et al., 2002; SAHUqUILLO et al., 2003; HLAVAY et al., 2004). The procedure of sequential extraction applied in this re- search is combined and modified according to the standard- ized procedure of sequential extraction in the European com- munity Bureau of Reference (BcR), TESSIER et al. (1979, 1987), AGNIESZKA & wITOLD (2004), MING et al. (2008), SILVEIRA et al. (2006) and other references dealing with the same issues (KAZI et al., 2005; PUEYO et al., 2008; DAV- IDSON et al., 2006; JOSE et al., 2006; KAASALAINEN & YLI-HALLA, 2003; ROUHOLLAH & FARZANEH, 2010). A modified procedure of sequential extraction includes the determination of Mn bound to particular soil phases in nine fractions: water-soluble fraction, exchangeable fraction, car- bonate fraction, easily reduced fraction, organic fraction, man- ganese oxides or moderately reduced oxides, amorphous iron oxide, crystalline iron oxide and the residual fraction of Mn. The first three steps of sequential extraction provide further data on the bioavailability of metals (JAIN, 2004). 2. AREA OF INVESTIGATION The research was conducted in the farthest northeast part of Bosnia and Herzegovina in the town of Bužim. Bužim has a population of about 20.000 inhabitants. It borders with the towns of Bosanska Krupa (east), cazin (south), Velika Kla- dusa (west and north) and the Republic of croatia (north- east). A well-known manganese mine in Bosnia and Herze- govina is located in Bužim, but has been out of use since the war in Bosnia and Herzegovina. The area is characterised by slightly rounded and seldom angular hills. The lowest altitudes occur in the Bužimica river valley (150m). This is an area of mildly continental and mountain climate. The manganese deposit is placed in a syn- clinal structure which extends in a northeast-southwest di- rection. It occurs where the volcanogenic-sedimentary series overlie dolomites and dolomitic limestone. The deposit is dark red in colour, due to the high manganese content. Oc- casionally, greenish tufa and light grey kaolin rich marls can be observed. MOJIĆEVIĆ et al. (1984) determined the age of the se- diments in the wider area of Bužim, having discovered that these are composed of Triassic, cretaceous, Palaeocene, Mi- ocene, and Pliocene-quaternary structures. Figure 1. is a lo- cation and sampling scheme map of the collection area near the manganese mine in „Bužim“ and the surrounding area. 3. MATERIALS AND METHODS 3.1. Sampling Sample collection from the soil was conducted by means of the standard pedologic methods of soil sampling, as well as the open pit sampling with reference to ISO 10381 proce- dures (ISO, 2002). Sampling was conducted by means of the standard pedologic equipment and tools, while the special attention was given to prevention of sample contamination. Sample collection occurred at the end of March 2010 at the manganese mine „Bužim“ and the surrounding area and a total of 10 soil samples from different locations have been used. Four samples were collected from a soil profile close to the mine at different depths, i.e. from four horizons in loose and solid part using Kopecky cylinders (Figure 2). clearly visible sequences of genetic horizons have been observed during the process of sample collection from the soil profile at a depth of 100 cm. The four horizons are distinguishable at a 0-26 cm, 26-49 cm, 49-69 cm and 69-100 cm deep. A total of six ordinary loose samples were taken from the surrounding area of the mine at a depth of 0-25 cm. Lo- cations of these samples were: Popović polje located 2.5-3 km north-west of the mine, Rusija and Bajraktarević polje (2 km to the south-west), Varoška rijeka also 2.5 km south- west of the mine, Radostovo (4 km to the south) and Bućevci 7 km south-east of the mine. These locations were chosen in order to determine the distribution of manganese in horizon- tal profile at the surrounding area in relation to the manga- nese mine. 3.2. Sample preparation and analysis The soil samples were prepared for physico-chemical analy- ses according to the ISO 11464 procedure (ISO 1994b). Geologia Croatica 67/1Geologia Croatica 48 Soil samples used in the research were prepared and cleared of impurities (organic matter and visible shells), air- dried, sifted with 2 mm hole diameter sieve, homogenised by the quartering method and sampled for laboratory analy- sis. Samples prepared in this way were used for physical and chemical analysis of soil. A fragment of prepared samples was dried in dryer at a temperature of 105 °c for an hour, afterwards powdered in agate mortar according to the ISO 11465 procedure (ISO 1993). Samples prepared in this way were used for determination of the near-total content of man- ganese in all samples, as well as for sequential extraction of manganese in the nine fractions. 3.3. Methods of analysis Samples were analysed by means of the following methods: – Determination of the hygroscopic moisture content of the soil (Hy) according to the ISO 11465 procedure (ISO 1993), – The mineral part of the soil was determined by calci- nation at 500-600 °c 2 h (ČUSTOVIc & TVIcA, 2005), Figure 2: Layout of soil horizons in the soil profile. – Determination of granulometric soil texture was con- ducted according to the ISO 11277 procedure (ISO 1998). The texture of soil samples was conducted ac- cording to Ehwald (RESULOVIc & ČUSTOVIĆ, 2002), – Bulk density was determined according to the ISO 11272 procedure (ISO 1993), – Particle density was determined according to the ISO 11508 procedure (ISO 1998), – Total porosity (%) was calculated using the values of bulk density and particle density (PEKEČ et al., 2013). – Amount of humus was determined by determination of organic carbon (c) by sulphocromic oxidation as prescribed by ISO 14235 (ISO 1998). A correction factor of 1.724 was used to calculate humus from or- ganic c. – Amount of carbonate in the soil was determined ac- cording to the ISO 10693 procedure (ISO 1995). Redžić et al.: Distribution and bioavailability of manganese in soil in the vicinity of the „Bužim“ abandoned mine Geologia Croatica 49 – Determination of soil pH value was conducted accord- ing to the ISO 10390 procedure (ISO 1994c), – Sum of the absorbed base cations and hydrolytic acid- ity were determinedby Kapenn’s method (ČUSTOVIĆ & TVIcA, 2005) – The total adsorption capacity (T) was calculated from the total adsorbed basic cations (S) and total adsorbed H+ ions (H). – The degree of base saturation was calculated from the proportions of base cations (S) relative to the total ad- sorption capacity (T) and was expressed as a percent- age. – Easily accessible phosphorus and potassiumin the soil, are determined through AL-method (Enger-Riehm- Domino) by ammonium lactate-acetic acid extractac- tion described by EGNER et al. (1960), – Near -total Mn content in the soil was determined by AAS after digestion of soil samples in a microwave oven, – Methods of Mn sequential extraction of soil samples are presented in Table 1. 3.3.1. Method for determining the near-total Mn content in soil samples Near-total contents of Mn in all soil samples were determined from their extracts (SILVEIRA et al., 2006). Sample extracts were processed by microwave digestion with the addition of 6 ml Hcl (36%) and 2 ml HNO3 (65%) - microwave technique on a MARSXpress system (cEM) according to the EPA 3050 (USEPA, 1996). The microwave digestion programme in- cluded two phases: first, 8 minutes at 160°c, and 1200w; and second 10 minutes at 160°c, and 1200w. After completion of digestion and cooling, extracts were filtered (whattman type 50 filter paper) in glass bottles of 50 ml and diluted with distilled water to the level line (MOSSOP & DAVIDSON, 2003). Extracts prepared in this way were stored at 4°c until further analysis by AAS according to the procedures of ISO 11047 (ISO 1998). 3.3.2. Methodology for the sequential extraction of manganese from the soil The defined steps and reagents used in the process of sequential extraction are shown in Table 1. Extracts from different soil fractions were separated by centrifugation at a speed of 3000 rpm for a period of 30 mi- nutes, collected in polyethylene bottles and stored at 4 °c until analysis according to the references (AGNIESZKA & wITOLD, 2004; MING et al., 2008; SILVEIRA et al., 2006; KAZI et al., 2005; PUEYO et al., 2008; DAVIDSON et al., 2006; JOSE et al., 2006; KAASALAINEN & YLI-HALLA, 2003; ROUHOLLAH & FARZANEH, 2010). The extracts from the residual fractions were processed by microwave digestion using the same procedure as for de- termination of the near-total content of manganese in soil samples. Manganese concentrations in acidic residues were measured by Atomic Absorption Spectrophotometer (Zee- man, Varian), using a calibration curve method (LI et al., 2000). 3.3.3. Quality assurance and quality control (QA/QC) quality assurance (qA) of the research has been realised by means of the appropriate laboratory facilities, specialised work procedures, use of distilled water, high-purity chemi- cals, and measurements conducted on the equipment with satisfying performance (analytical scales, pH meter, AAS). quality control (qc) has been realised by an analysis of the relevant blind experiments, calibration of the equipment using standardised solutions, and repetition of the analyses using the same methodology with reference to ISO 17025 procedures (ISO, 2005). 4. Results and discussion 4.1. Physical and chemical soil analysis The determined values of specific physical and chemical soil properties are shown in Tables 2 and 3. Research has shown that particle density decreases in a vertical soil profile with Table 1: Sequential extraction of Mn from soil. Step Fraction Reagents References 1. Water-soluble 30 ml distilled water (AgNIeSzkA & WItOLD, 2004) 2. exchangeable fraction 20 ml 1 M CaCl2 (pH=7) (AgNIeSzkA & WItOLD, 2004; MINg et al., 2008) 3. Carbonate fraction 20 ml 1 MNaOAc (pH=5 adjusted with HOAc) (MINg et al., 2008; SILVeIRA et al., 2006; kAzI et al., 2005, BAeYeNS et al., 2003) 4. easily reduced substances 40 ml 0.04 M NH2OH · HCl (pH=2 adjusted with CH3COOH) (MINg et al., 2008; PUeYO et al., 2008; DAVIDSON et al., 2006; JOSe et al., 2006; kAASALAINeN & YLI-HALLA, 2003) 5. Organic fraction 20 ml 1 M Na4P2O7 (ROUHOLLAH & FARzANeH, 2010) 6. Manganese oxides or moderately reduced oxides 40 ml 0.05 M NH2OH/HCl (pH=2) (MINg et al., 2008; SILVeIRA et al., 2006) 7. Amorphous iron oxide 30 ml 2 M (H2C2O4) +0.2 M (NH4)2C2O4(pH = 3) (MINg et al., 2008;SILVeIRA et al., 2006) 8. Crystalline iron oxide 20 ml 6M HCl (MINg et al., 2008) 9. Residue (residual fraction) HCl : HNO3 (3:1) microwave digestion USePA, 1996 (SILVeIRA et al., 2006) Geologia Croatica 67/1Geologia Croatica 50 increasing depth, i.e. from 2.51 g/cm3 in the 1st horizon to 2.30 g/cm3 in the 4th horizon. Bulk density displays different values within horizons of the vertical soil profile. The aver- age amount in the 1st horizon is 1.46 g/cm3, 1.44 g/cm3 in the 2nd, 1.49 g/cm3 in the 3rd, and 1.41 g/cm3 in the 4th hori- zon. Based on the particle density and bulk density, it is pos- sible to determine whether the soil is arable compacted soil. Based on the research of soil porosity we determined soil with low porosity, with a total amount of pore space of 38.60% in 4th horizon to 43.50% in 2nd horizon. The results of granulometric analysis indicate that sam- ples from the 2nd and 3rd horizons of the vertical profile are similar in granulometric properties and can be regarded as a clay loam. Samples taken from the locations surrounding the manganese mine (Popović polje, Rusija, Bajraktarević polje, Varoška rijeka, Radostovo and Bućevci) at depth of 0-25 cm displayed similar granulometric properties, which are: sandy loaml, loam, loamy sand and silty sand. This can be regarded as being caused by intensive deep erosion. The soils pH values range from moderately acidic to mildly alkaline, from pH=4.64 at the manganese mine loca- tion in the deepest horizon to pH=7.26 at the location of Varoška rijeka. At different horizons in vertical profile, pH value decreases from the 1st to 4th horizons. All soil samples displayed lower pH values of Kcl than the pH values of H2O, especially in soils where pH value is usually high. Soils with delta pH higher than 0.10, are said to have geric properties, i.e. are very exposed to erosion and have low adsorption capacity or a low number of exchange- able bases (RESULOVIĆ, 1997). Delta pH had the highest value (pH=0.65) at the 4th horizon (100 cm) at the manga- nese mine. The amount of cacO3 in all samples was lower than the limit set by the method, since the dissolution of soil samples was conducted by using diluted hydrochloric acid. Based on the results of research and values obtained for the level of base saturation (V), ranging from 73.58% at Popović polje to 99.47% at Varoška rijeka, it can be stated that all tested locations are Eutric cambisols. 4.2. Near-total content of manganese The near-total content of manganese for each sample loca- tion in the vertical profile are presented in Table 4, as well as for each sample location surrounding the „Bužim“ man- Table 2: Physical properties of soil. Soil test / sample PARtICLe DeNSItY (kgdm–3) BULk DeNSItY (kgdm–3) tOtAL POROSItY (%) PARtICLe CONteNt IN SOIL (%) teXtURAL tYPeS according to HWALD SIze OF PARtICLeS IN (mm) 0.02–2.00 sand 0.002–0.02 silt < 0.002 clay a b c d MINe SeCtION 1st horizon (0-26 cm) 2.51 1.46 41.80 37.50 42.50 20.00 Loam MINe SeCtION 2nd horizon (26-49 cm) 2.55 1.44 43.50 30.70 38.00 31.30 Clay loam MINe SeCtION 3rd horizon (49-69 cm) 2.46 1.49 39.40 30.80 35.80 33.40 Clay loam MINe SeCtION 4th horizon (69-100 cm) 2.30 1.41 38.60 12.50 16.20 71.30 Clay Popović polje (0-25 cm) – – – 47.40 37.70 14.90 Sandy loam Rusija (0-25 cm) – – – 33.20 47.40 19.40 Loam Bajraktarević polje (0-25 cm) – – – 45.90 45.00 9.10 Sandy loam Varoška rijeka (0-25 cm) – – – 80.30 17.20 2.50 Loamy sand Radostovo (0-25 cm) – – – 39.20 49.40 11.40 Sandy loam Bućevci (0-25 cm) – – – 73.30 22.10 4.60 Silty sand Redžić et al.: Distribution and bioavailability of manganese in soil in the vicinity of the „Bužim“ abandoned mine Geologia Croatica 51 Ta bl e 3: C he m ic al p ro pe rt ie s o f s oi l. So il te st / sa m pl e pH -V A LU e IN (1 :2 .5 ) H Ig RO SC O PI C H U M ID It Y (t= 10 5° C) (% ) Re SI D U e A Ft eR CA LC IN At IO N (t= 55 0° C) (% ) AM O U N t O F H U M U S (% ) AM O U N t O F PH YS IO LO g IC A L AC tI VI tY (m g/ 10 0 g of so il) AM O U N t O F Ca CO ₃ ( % ) H YD RO LY tI C AC ID It Y „H “ (m m ol /1 00 g of so il) AM O U N t O F A Lk A LI N e A D SO RP t- IO N „S “ ( m m ol / 10 0g o f s oi l) tO tA L A D SO RP tI O N CA PA CI tY „t “ (m m ol / 1 00 g of so il) D eg Re e O F A Lk A LI -N e SA tU R- At IO N „V “ (% ) H ₂O kC I P₂ O ₅ k₂ O a b c d e f g h j M IN e Se Ct IO N 1 st h or iz on (0 -2 6 cm ) 5. 53 5. 29 2. 17 91 .6 3 2. 65 < 0. 10 10 .5 2 < 0. 10 1. 50 8. 77 10 .2 7 85 .4 0 M IN e Se Ct IO N 2 nd h or iz on (2 6- 49 c m ) 5. 46 5. 45 2. 25 93 .5 5 0. 64 < 0. 10 6. 75 < 0. 10 0. 75 5. 98 6. 73 88 .8 5 M IN e Se Ct IO N 3 rd h or iz on (4 9- 69 c m ) 5. 34 5. 14 2. 41 93 .7 4 0. 47 < 0. 10 7. 01 < 0. 10 0. 75 5. 58 6. 33 88 .1 6 M IN e Se Ct IO N 4 th h or iz on (6 9- 10 0 cm ) 4. 64 3. 99 4. 22 89 .6 2 0. 47 < 0. 10 6. 66 < 0. 10 1. 35 5. 58 6. 93 80 .5 3 Po po vi ć po lje (0 -2 5 cm ) 5. 23 4. 85 2. 97 87 .0 7 3. 52 < 0. 10 17 .9 8 < 0. 10 3. 15 8. 77 11 .9 2 73 .5 8 Ru si ja (0 -2 5 cm ) 5. 79 5. 42 2. 68 89 .5 0 4. 15 < 0. 10 21 .6 6 < 0. 10 2. 10 9. 97 12 .0 7 82 .6 0 Ba jra kt ar ev ić p ol je (0 -2 5 cm ) 6. 76 6. 55 2. 02 91 .6 7 2. 33 < 0. 10 7. 71 < 0. 10 1. 50 27 .9 4 29 .4 4 94 .9 0 Va ro šk a rij ek a (0 -2 5 cm ) 7. 26 7. 21 1. 41 91 .9 2 3. 36 7. 22 12 .3 6 < 0. 10 0. 45 85 .4 5 85 .9 0 99 .4 7 Ra do st ov o (0 -2 5 cm ) 6. 99 6. 56 2. 26 90 .9 3 3. 48 2. 88 21 .9 2 < 0. 10 0. 75 15 .5 6 16 .3 1 95 .4 0 Bu će vc i (0 -2 5 cm ) 7. 08 7. 06 3. 15 89 .1 1 3. 07 < 0. 10 15 .4 3 < 0. 10 0. 75 39 .1 2 39 .8 7 98 .1 0 Geologia Croatica 67/1Geologia Croatica 52 ganese mine. Relatively equable Mn concentrations were measured through all horizons, at three depths of the verti- cal profile, ranging from 1190.00 to 1160.00 mg/kg with in- creasing depth. Table 4: Near-total content of Mn for each sample location. Symbols of samples Amount Mn (mg/kg) M IN e Se Ct IO N 1st horizon (0-26 cm) 1190.00 2nd horizon (26-49 cm) 1170.00 3rd horizon (49-69 cm) 1160.00 4th horizon (69-100 cm) 780.00 (0-25 cm) Rusija (0-25 cm) Bajraktarević polje (0-25 cm) Varoška rijeka (0-25 cm) Radostovo (0-25 cm) Bućevci (0-25 cm) The amount of Mn was significantly decreased at the 4th horizon (780.00 mg/kg), as displayed in Figure 3. Figure 4. indicates that within the examination of all lo- cations at a depth of 0-25 cm, the highest near-total content of 20950.00 mg/kg of Mn, was measured at the area of Popović polje, while the lowest amount was measured at the area of Bućevci of 418.00 mg/kg. The high content of Mn in certain locations is probably due to the close proximity of the sampling areas to the mine. Results indicate that, the sample locations contain ex- tremely high concentrations of manganese, which can be re- lated to the former mining activities in the area. 4.3. The distribution of Mn in different soil fraction determined from sequential extraction Table 5. presents the Mn contents in the individual fractions as determined by the process of sequential extraction. The sum of all the examined Mn fractions displays relatively good correlation with near-total content of Mn at the same soil samples, while this type of correlation was not displayed at the area of Popović polje. According to ZEMBERYOVA et al. (2006) figures 5 and 6 display the extracted percent- ages of Mn among the examined fractions (in relation to the certified value of 100). MEERS et al. (2007) define the water-soluble fraction as being easily mobilised and a highly available fraction. In the first soluble fraction, the percentage of Mn in the verti- cal profile of horizons at the manganese mine location is lower than 1.00%, thus posing no threat according to the as- sessment code. The percentage of soluble fraction in soil samples from the area surrounding manganese mine at all sampling locations is lower than 1.00%, thus having no det- rimental effect on the environment. The exchangeable metal fraction is loosely bound to col- loidal soil particles (AGNIESZKA & wITOLD, 2004). NOVOZAMSKI et al. (1993) proposed the use of cacl2 as a reagent for evaluation of metal and nutrient bioavaila- bility in air-dried soil sample. HOUBA et al. (1996, 2000) state the advantages of this reagent. In the second exchange- able fraction, the percentage of Mn in the vertical profile (sampled in the mine) is as follows: 2.00 % in 1st horizon, 1.00% in the 2nd and 3rd horizon and 3.00 % in the 4th hori- zon. The highest percentage of the exchangeable manganese fraction is obtained in the 4th horizon, probably due to an in- creased ionic exchange which occurs as the soil depth and amount of cations in the soil increase. The percentage of exchangeable fractions at locations surrounding the manganese mine (1st horizon) is as follows: <0.10% in Popović polje and Bajraktarević polje, 2.00% in Rusija and Varoška rijeka, 1.00% in Radostovo and Bućevci. The percentage of Mn in the exchangeable fraction (mobile metal fraction) at all sample locations, according to the as- sessment code, has no detrimental effect on the environment. The carbonate fraction is regarded as an easily mobilised fraction. Extraction within this fraction was conducted by means of reagent 1M NaOAc solution, pH=5 adjusted with acetic acid in order to transfer most of the absorbed Mn into solution (BAEYENS et al., 2003), as the fraction is highly sensitive to changes of pH value (TESSIER et al., 1979). Figure 3: Near-total content of Mn at the mining area. Figure 4: Near-total content of Mn in the surrounding area. Redžić et al.: Distribution and bioavailability of manganese in soil in the vicinity of the „Bužim“ abandoned mine Geologia Croatica 53 Ta bl e 5: t he a m ou nt o f M n in c er ta in fr ac tio ns a ft er se qu en tia l e xt ra ct io n pr oc ed ur e (m g/ kg ). SA M PL e / FR AC tI O N M IN e Se Ct IO N Po po vi ć po lje (0 -2 5 cm ) Ru si ja (0 -2 5 cm ) Ba jra kt ar ev ić po lje (0 -2 5 cm ) Va ro šk a rij ek a (0 -2 5 cm ) Ra do st ov o (0 -2 5 cm ) Bu će vc i (0 -2 5 cm ) 1s t h or iz on (0 -2 6 cm ) 2n d ho riz on (2 6- 49 c m ) 3r d ho riz on (4 9- 69 c m ) 4t h ho riz on (6 9- 10 0 cm ) W at er -s ol ub le fra ct io n 1. 50 1. 50 1. 50 1. 50 0. 60 0. 90 0. 90 0. 90 1. 20 0. 90 ex ch an ge ab le fra ct io n 29 .2 0 8. 40 8. 60 17 .4 0 56 .8 0 17 .8 0 10 .4 0 8. 60 15 .2 0 5. 20 Ca rb on at e fra ct io n 31 .8 0 8. 80 7. 40 9. 20 52 .8 0 22 .4 0 48 .6 0 52 .8 0 34 .8 0 42 .6 0 ea si ly re du ce d su bs ta nc es 39 3. 60 18 1. 60 16 2. 00 18 6. 80 29 16 .0 0 92 .8 0 26 44 .0 0 20 7. 20 21 7. 20 25 5. 60 O rg an ic fra ct io n 99 2. 00 51 6. 00 55 6. 00 18 3. 00 52 00 .0 0 42 4. 00 26 00 .0 0 40 .0 0 50 5. 00 48 .0 0 M an ga ne se ox id es 15 8. 00 67 .2 0 74 .4 0 34 .4 0 28 32 .0 0 73 .2 0 78 4. 00 17 .6 0 70 .8 0 19 .6 0 Am or ph ou s i ro n ox id e 48 .3 0 47 .7 0 50 .7 0 21 .0 0 26 64 .0 0 41 .1 0 12 96 .0 0 8. 40 56 .7 0 9. 30 Cr ys ta lli ne ir on ox id e 80 .2 0 86 .0 0 91 .2 0 52 .0 0 58 80 .0 0 63 .8 0 90 0. 00 67 .6 0 67 .2 0 38 .0 0 Re si du al a m ou nt of M n 14 7. 00 16 8. 50 14 9. 50 12 4. 50 71 25 .0 0 11 4. 50 52 0. 00 29 .5 0 17 8. 50 41 .0 0 to ta l a m ou nt o f fra ct io ns 18 81 .6 0 10 85 .7 0 11 01 .3 0 62 9. 80 26 72 7. 20 85 0. 50 88 03 .9 0 43 2. 60 11 46 .6 0 46 0. 20 Geologia Croatica 67/1Geologia Croatica 54 According to ROBBINS et al. (1984) the reagent re- leases 99.90% of the carbonate fraction and has minimum influence on other fractions. The percentage of carbonate fraction in vertical profile (sampled in the mine) of horizons ranges from 1.00% in 2nd, 3rd and 4th horizon, to 2.00% in 1st horizon, thus posing low risk to the environment accord- ing to the assessment code. The highest percentage of car- bonate fraction in the 1st horizon can be explained by the susceptibility of the fraction to changes of pH value (TESS- IER et al., 1979). consequently, it was observed that the highest percent- age of carbonate fraction correlates with the highest meas- ured pH value (5.53) at the 1st horizon soil sample in the vertical profile in the area of the mine. The percentages of carbonate fraction at the other sam- ple locations surrounding the manganese mine (1st horizon) are: <0.10% in Popović polje, 3.00% in Rusija, 1.00% in Bajraktarević polje, 12.00 % in Varoška rijeka, 3.00% in Ra- dostovo and 9.00% in Bućevci. The highest percentage of the carbonate fraction was detected at the location of Varoška rijeka (12.00%) correlating with the highest measured pH value at the same location (7.26), which confirms the fact that the fraction is susceptible to changes of pH value. More- over, it proves the sensitivity of the fraction to changes of pH-value and indicates that as the soil pH value increases, so does the percentage of carbonate and consequently of the bioavailable manganese fraction in the soil. The percentage of Mn in the carbonate soil fraction at the location of Varoška rijeka has a limited effect on the environment. Easily reduced substances (Fe-Mn bound oxides) dis- play moderate mobility, depending on the redox conditions of the environment. According to SILVERA et al. (2006) the redox potential of soil can significantly increase the solubil- ity of Fe and Mn oxides. Fe and Mn oxides have a great effect on control and mo- bility of other metals in the environment. In the reduced frac- tion (Fe-Mn bound oxides), the percentages of Mn in the vertical profile (sampled in the mine) of individual horizons are: 1st horizon 21.00%, 2nd horizon 17.00%, 3rd horizon 15.00% and 4th horizon 31.00%. The highest percentage of Figure 5: Distribution of Mn at the mining area using the sequential ex- traction procedure. Figure 6: Distribution of Mn in the area surrounding the mine using se- quential the extraction procedure. Mn in the vertical profile was determined at the 4th horizon (31.00%), which could be due to an increased redox poten- tial there, leading to an increased solubility of the fraction. The highest percentage of Mn in this fraction correlates with the lowest measured pH value at the 4th horizon. Percentages of the reduced fraction at the locations sur- rounding the manganese mine (1st horizon) are: Popović polje and Rusija 11.00%, Bajraktarević polje 29.00%, Varoška rijeka 48.00%, Radostovo 19.00% and Bućevci 57.00%. The percentage of the reduced fraction at all sample locations is relatively high, due to numerous natural and an- thropogenic sources of Mn over the area. The highest percentage of the easily reduced fraction is determined in the area of Bućevci (57.00%), which can be explained by manganese oxidation resulting from mining activity there (NADASKA et al., 2009).The fact that Mn dominates in the fraction of Fe-Mn oxide at the area of Bućevci indicates they share a common origin. According to HAUNG et al. (2007) Mn bound to the or- ganic fraction displays moderate mobility, although it can be increased due to the oxidation of organic matter. The solu- bility of the Mn fraction bound to organic matter, precedes those of Mn oxides, amorphous and crystalline iron oxides. This enables organic phase degradation, providing better conditions for the extraction of fractions bound to Mn (AL- VAREZ et al., 2006). The percentage of the organic fraction in the vertical profile (sampled in the mine) of individual horizons is: 1st horizon 52.00%, 2nd horizon 47.00%, 3rd horizon 49.00% and 4th horizon 29.00%. The percentage of the organic frac- tion at sample locations surrounding the „Bužim“ manganese mine (1st horizon) is: Popović polje 19.00%, Rusija 49.00%, Bajraktarević polje 30.00%, Varoška rijeka 9.00%, Ra- dostovo 44.00% and Bućevci 10.00%. Due to a high affinity of bonding to organic matter (RESULOVIĆ, 1997) Mn is dominant in this fraction with the highest percentage (52.00%) in the 1st horizon of the vertical profile (sampled in the mine). The percentage of manganese oxides or moderately re- duced oxides in horizons of vertical profiles (sampled in the Redžić et al.: Distribution and bioavailability of manganese in soil in the vicinity of the „Bužim“ abandoned mine Geologia Croatica 55 mine) is: 1st horizon 8.00%, 2nd horizon 6.00%, 3rd horizon 7.00% and 4th horizon 5.00%. The percentage of this frac- tion at other locations surrounding the manganese mine (1st horizon) is: Popović polje 11.00%, Rusija i Bajraktarević polje 9.00%, Varoška rijeka and Bućevci 4.00%, Radostovo 6.00%. The highest percentage of the fraction was measured at the Popović polje location (11.00%), which can be related to relatively low pH-values of the location (5.23) (SILVEIRA et al., 2006). The percentage of Mn bound to the amorphous iron ox- ide fraction in the horizons of the vertical profile (sampled in the mine) is:1st and 4th horizon 3.00%, 2nd horizon 4.00% and 3rd horizon 5.00%. At locations surrounding the manga- nese mine (1st horizon) it is as follows: Popović polje 10.00%, Rusija 5.00%, Bajraktarević polje 15.00%, Varoška rijeka and Bućevci 2.00% and Radostovo 5.00%. The high- est percentage of this fraction is measured in the area of Bajraktarević polje (15.00%). ScHwERTMANN (1991) proved the efficacy of 6 M Hcl used for dissolution of the crystalline iron oxide frac- tion. In his research, the author stated that nearly 60.00% of Fe dissolves with 6M Hcl solution (SILVIERA et al., 2006). The percentage of Mn bound to the crystalline iron oxide fraction in individual horizons of vertical profile (sampled in the mine) is determined as: 1st horizon 4.00%, 2nd, 3rd and 4th horizon 8.00%. At locations surrounding the manganese mine (1st horizon) it is as follows: Popović polje 22.00%, Rusija 8.00%, Bajraktarević polje 10.00%, Varoška rijeka 16.00%, Radostovo 6.00% and Bućevci 8.00%. The highest percentage of the fraction was measured in the Popović polje (22.00%). The highest percentage of Mn in this fraction correlates with the highest percentage in manganese oxides and mod- erately reduced oxides fraction, which can be due to the size of their ionic radii and isomorphic substitution of Mn and Fe (SILVEIRA et al., 2006). According to SUTHERLAND et al. (2000), metals ex- tracted in non-residual fractions indicate an anthropogenic origin, and metals extracted in the residual fraction indicate a natural origin (FORSTNER, 1983; BLAScO et al., 2000; JAIN, 2004; RAMIREZ et al., 2005). PRIcA (2011) states that the residual fraction cannot be mobilised from geologi- cal material and is not bioavailable. The percentage of the residual fraction in horizons of the vertical profile (sampled in the mine) is: 1st horizon 8.00%, 2nd horizon 16.00%, 3rd horizon 14.00% and 4th ho- rizon 20.00%. At locations surrounding the manganese mine (1st horizon) it is as follows: Popović polje 27.00%, Rusija 13.00%, Bajraktarević polje 6.00%, Varoška rijeka 7.00%, Radostovo 16.00% and Bućevci 9.00%. A rather high percentage of Mn at the aforementioned locations is probably due to extremely strong bonds of Mn and the soil solid phase. The highest percentage of the frac- tion is determined at the area of Popović polje (27.00%). The highest percentage of Mn in this fraction in the area corre- lates with the crystalline iron oxide fraction and Mn oxide and moderately reduced oxide fraction. 5. CONCLUSIONS According to the research conducted at the „Bužim“ man- ganese mine and the surrounding area, the following conclu- sions can be drawn: According to pH values the soils are moderately acidic to mildly alkaline, thus being considered as Eutric cam- bisols. The results of sequential extraction indicate that chemical soil properties significantly affect Mn distribution in the different soil fractions. The highest percentage of bio- available Mn was measured at the „Bužim“ manganese mine (in 1st fraction) and in Popović polje (in 2nd fraction), since there are numerous natural and anthropogenic sources of Mn at these areas. The percentage of Mn soluble fraction is less than 1.00% at all locations, thus having no detrimental effect on the environment. The highest percentage of exchangeable manganese fraction (3.00%) was measured in the 4th horizon of the vertical profile, probably due to an increased ionic ex- change which occurs as depth increases, thus having no det- rimental effect on the environment according to the assess- ment code. The highest percentage of the carbonate fraction (12.00%) was determined at the location of Varoška rijeka correlating with pH=7.26. The percentage of Mn in the car- bonate fraction at the location of Varoška rijeka moderately affects the environment. The highest percentage of natural Mn was determined in the residual fraction (27.00%) at Popović polje, while the highest percentage of anthropogenic origin Mn was deter- mined at Bućevci (57.00%) in the Fe-Mn oxides fraction. The highest near-total content of Mn was determined at Popović polje (20950.00 mg/kg). The highest percentage of natural Mn (27.00%) was determined in the same area. 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