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CONTENTS

ORIGINAL PAPERS

SORPTION AND DESORPTION OF Pb2+ AND Cu2+ IN DIFFERENT TUNISIAN SOILS 1909 Hamdi Sahraoui, María Luisa Andrade, Mohamed Hachicha, Luis Felipe Silva Oliveira and Flora Alonso Vega

EFFECTS OF TWO FUNGICIDES, CYPRODINIL AND FLUDIOXONIL, 1920 ON GENOTOXICITY IN DROSOPHILA SMART ASSAY AND ON PROLIFERATION AND VIABILITY OF HEK293 CELLS FROM THE PERSPECTIVE OF CARCINOGENESIS Asuman Karadeniz, Bülent Kaya, Burhan Savaş and Ş. Fatih Topcuoğlu

INFLUENCE OF WEAK MAGNETIC FIELD ON HEAVY 1926 METAL CATIONS REMOVAL BY ZERO VALENT IRON Chao Wang, Zhongjin Xiao, Lei Wang and Junlian Qiao

CONTRIBUTIONS TO THE INSECT FAUNA OF 1932 BURDUR PROVINCE (TURKEY) IN TERMS OF HYDROPHILIDAE, HELOPHORIDAE AND CHRYSOMELIDAE (COLEOPTERA) WITH CHOROTYPE ANALYSES Baran Aslan, Ayçin Yılmaz, Fatma Bayram and Ebru G. Aslan

EFFECTS OF ELECTRODE MATERIAL ON THE ELECTROKINETIC 1940 REMEDIATION OF CADMIUM-CONTAMINATED SOIL NEAR A MINE TAILING Zongping Cai, Jeremiah Van Doren, Mengqing Xu, Dongrui Chen, Weishan Li, Zhanqiang Fang and Lidan Xing

REMOVAL OF BASIC RED 9 IN WASTEWATER USING GREEN FENTON REACTION 1947 Azize Alayli Gungor, Neslihan Celebi and Hayrunnisa Nadaroglu

ANTIOXIDANT DEFENCE SYSTEMS, LIPID PEROXIDATION AND ACETYLCHOLINESTERASE 1958 ACTIVITY OF OREOCHROMIS NILOTICUS EXPOSED TO MERCURY AND MERCURY + SELENIUM Gülbin Firidin, Ferit Kargın, Özge Fırat, Hikmet Y. Çoğun, Özgür Fırat, Beran Firidin and Tüzin A. Yüzereroğlu

ASSESSMENT OF HEAVY METAL POLLUTION IN SURFACE SOILS 1966 FROM URBAN PARKS AND SQUARES: A CASE STUDY FROM CHINA Qiang Liu, Jingshuang Liu, Mingying Zou and Yang Wang

THE ENZYMATIC ACTIVITY AND PLASTID PIGMENT CONTENT IN 1976 MEDICAGO X VARIA T. MARTYN TREATED WITH AN EXOGENOUS GROWTH REGULATOR Jacek Sosnowski

HEAVY METALS CONTENT IN AMANITA PANTHERINA IN A VICINITY OF 1981 THE THERMO-ELECTRIC POWER PLANT OSLOMEJ, REPUBLIC OF MACEDONIA Emri Murati, Slavcho Hristovski, Ljupcho Melovski and Mitko Karadelev

HEMATOLOGICAL PARAMETERS IN NILE TILAPIA (Oreochromis niloticus) 1985 EXPOSED TO SUB-LETHAL CONCENTRATIONS OF CADMIUM AND ZINC Özgür Fırat and Ferit Kargin

1907 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

ASSESSMENT OF HEAVY METALS CONTAMINATION IN FEN RIVER CHANNELS 1990 Yu Qi, Jun Ma and Guisheng Fan

NOTICE

GLOBAL ENVIRONMENTAL INPUT–OUTPUT RESEARCH 1996 TRENDS DURING 1900–2013: A BIBLIOMETRIC ANALYSIS Jingqing Zhou, Zhen Wang, Beibei Niu and Song Hong

INDEX 2005

1908 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

SORPTION AND DESORPTION OF Pb2+ AND Cu2+ IN DIFFERENT TUNISIAN SOILS

Hamdi Sahraoui1,2,*, María Luisa Andrade1, Emma F. Covelo1, Mohamed Hachicha2, Jamila Tarhouni, Luis Felipe Silva Oliveira4,5 and Flora Alonso Vega1

1Departamento de Bioloxía Vexetal e Ciencia do Solo, Facultade de Bioloxía, Universidade de Vigo, Lagoas, Marcosende, 36310 Vigo, Pontevedra, Spain. 2Institut National de Recherche en Génie Rural Eaux et Forêts. Université de Carthage. Rue Hédi Elkarray, Elmenzah IV, Tunis, Tunisie 3Institut National Agricole de Tunis. 43, Rue Charles Nicole - Cité Mahrajène, Tunis, Tunisie 4Environmental Science and Nanotechnology Department, Catarinense Institute of Environmental Research and Human Development, IPADHC, Capivari de Baixo, Santa Catarina, Mexico 5Centro Universitário UNIVATES, Bairro Universitário, Lajeado, Brazil

ABSTRACT cumulation of trace elements in the long term affects the topsoil. Studies of these soils are limited. Sorption and de- Sorption and retention are the major processes for the sorption are the most important processes controlling the accumulation of potentially toxic trace elements and its trace elements mobility on soil. Trace elements are adsorbed study is of great importance to understand the fate of on the surface of colloidal particles in soils, principally on trace elements in soils. Sorption and retention capacities of hydrous oxides, clays and carbonates [1]. Desorption is the five Tunisian soils and the soil mineral influence were release of sorbed metal ions from soil. studied by Pb2+ and Cu2+ batch sorption experiments and As described by [2] sorption of Pb2+ and Cu2+ in soils scanning electron microscope (SEM) analysis. It has been is affected by specific sorption to various solid phases, pre- shown that sorption and retention of Pb2+ are most influ- cipitation of highly stable compounds and formation of rel- enced by cation exchangeable capacity (CECe), clay con- atively stable complexes with soil components. However, tents, Mn oxides and carbonate contents, whereas Cu2+ even researches done have been focused on the understand- sorption and retention are most influenced by the total or- ing of sorption of Pb2+ and Cu2+ in soils, there has been ganic carbon (TOC) and carbonate contents. The alkalinity little research done on their desorption [3]. Sorption and of the majority of soils due to their carbonate contents lead desorption reactions are often not completely reversible to increase of both Pb2+ and Cu2+ precipitation and high and this non singularity or hysteresis has been observed to sorption capacity. The sorption irreversibility is higher in increase with increased aging of trace elements with soil soils of highest alkalinity, TOC, CECe, and clay contents. constituents [4]. Both hysteresis of Pb2+ and Cu2+ have The Energy Dispersive Spectrometer (EDS) semi-quanti- been observed in soils and minerals [5]. The processes of tative analysis performed by SEM demonstrates that calcite sorption and retention of trace elements taking place in soil contents decreased when soils were treated with Pb2+ giv- are also critical in understanding and predicting the future ing rise to a new crystalline phase and mostly amorphous mobility of sorbed metals. Hence, scanning electron micro- phases with high Pb2+ fixing capacity. Hematite and calcite scope (SEM) coupled and energy dispersive spectroscopy preferentially retain Cu2+. (EDS) has been used for quantitative element analysis in some trace elements sorption studies [6-8].

The objective of this study is to evaluate and compare KEYWORDS: Trace elements, sorption/retention capacity, hyste- the sorption/retention capacity and hysteresis of Pb2+ and resis, SEM-EDS, Tunisia. 2+ Cu in five Tunisian soils. SEM/EDS was used to assess 2+ 2+ the Pb and Cu distribution.

1. INTRODUCTION 2. MATERIALS AND METHODS High carbonates and low organic matter contents are common chemical characteristics of Mediterranean soils, Five soil surface samples varying widely in their origin eventually Tunisian soils. Induced by water scarcity, non and characteristics were selected for this study. They are conventional water as treated wastewater is used for irriga- representative of some soil orders from Tunisia. The soils tion. In spite the standard use of this water is respected, ac- were classified as Vertic Xerochrepts (INCP1 and INCP2), Vertic Chromoxerert (VER1 and VER2) and fersialitic * Corresponding author (FRS).

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All samples were air-dried, sifted through 2 mm-mesh structed using the pellets obtained in the sorption experi- and homogenized prior to soil characterization and sorp- ments. The pellets were dried at 45 °C for 24h, weighed tion and desorption studies. Soil pH was measured with a and resuspended in 100 mL of 0.01 M NaNO3 solution. So- glass electrode in 2:1 suspension of water to soil ratio [9]. lutions were shaken in a rotary shaker for 24 h in polyeth- Soil particle size distribution was determined following the ylene tubes at 25 °C and then were centrifuged at 4000 rpm pipette method according to [10]. Clay mineralogy was de- for 5 min. The supernatant was filtered through Whatman termined by powder X-ray diffractometry with a SIEMENS 42 paper and analyzed by ICP-OES in a Perkin–Elmer Op- D-5000 diffractometer with crystalline powder [11]. Total tima 4300 DV apparatus. organic C was determined in a module for solid analysis The amounts of each retained metal on the soil sample (SSM-5000, Shimadzu, Japan) coupled with a TOC analyzer- were calculated from the difference between the concentra- V CSH/CSN Shimadzu apparatus. Effective cation exchange tions sorbed in the sorption experiments stage and the con- capacity (CECe) was determined by extraction with 1M centrations of the metal in solution following desorption ammonium acetate buffered at pH 7.0 [12]. The concentra- experiments. The capacity of the soil to sorb and retain Pb2+ tions in the extracts were determined by inductively coupled 2+ and Cu was evaluated using Kr parameter as indicated by plasma atomic emission spectrometry (ICP-OES). The car- [22] and validated in several sorption researches [5, 23]. bonate content was determined with a LECO-100 analyzer connected to a LECO- CN-2000. Oxide contents were ex- Therefore, soils sorption and retention capacities were tracted with sodium hydrosulfite, sodium bicarbonate and evaluated as the slope Kr of the regression equation sodium citrate [13, 14] and the concentration of Fe, Al and Cs,i=KrCp,i Mn was determined by ICP-OES as previously indicated. where Cs,i is the quantity of sorbed metal i (μmol) per Pb2+ and Cu2+ in soil solution were determined accord- gram of soil at equilibrium −1 ing to the method of [15] by extraction with 0.1M calcium Cp,i is potentially sorbable metal (μmol g soil) (the chloride solution. Available Pb2+ and Cu2+ were extracted quantity of metal in the solution before contact with the with diethylenetriaminepentaacetate (DTPA) as described soil). by [16]. Total concentrations of Pb2+ and Cu2+ were deter- Kr is dimensionless and ranges from 0 -for a totally mined after extraction with a mixture of concentrated ni- non-sorbing soil- to 1, for an ideal sorbent that completely tric, hydrochloric and hydrofluoric acids (1:3:3, v/v/v) and releases metal i from solution [5, 22, 23]. placed in a microwave oven [17]. The concentration of the metals in the extracts was also analyzed by inductively In the same way, the retention capacity was calculated coupled plasma optical emission spectrometry (ICP-OES) by means of Kr, in this case the sorbed metal is substituted in a Perkin Elmer Optima 4300 DV instrument. by metal retained at equilibrium. A high Kr value indicates high metal sorption or retention by the soil through chemi- The occurrence of mineral species was investigated by cal reactions, leading to low metal bioavailability. Simi- means of Powder X-ray diffraction (XRD), using a Zeiss larly, a low Kr value indicates that a high metal quantity ULTRA plus field emission scanning electron microscope remains in the solution [5, 22, 23]. (FE-SEM) coupled with an Oxford energy-dispersive X- ray (EDX) spectrometer [6, 7]. Table 1 shows the physical Sorption irreversibility or hysteresis was calculated by and chemical characteristics of the studied soils. a hysteresis index (HI), deﬁned as the ratio between the Kr values calculated for retention and sorption [13]: Sorption isotherm assays for Pb2+ and Cu2+, were performed using the batch equilibrium technique as described HI = Kr retention/ Kr sorption by [18, 19], as modified by [20]. Triplicate suspensions of HI is equal to 1 if sorption is completely irreversible, 3 g soil samples were equilibrated with 50 mL of 0.01 M and 0 if all of the sorbed metal is released. NaNO3 (as background electrolyte) solutions containing The results were analyzed statistically using the soft- 0.05, 0.1, 0.2, 0.4, 0.5, 1, 2, 3, 4 and 5 mmol L-1 of Pb2+ or 2+ ware SPSS v. 19.0 for Windows. Statistical significance of Cu (as nitrate) in 100 mL polyethylene tubes (pre- differences was determined by variance analysis (ANOVA) weighed). The solutions were shaken in a rotary shaker for and the least-significant difference (LSD) test. The influence 24 h at 25 ºC. After equilibration, the samples were centri- of soil properties on the metal sorption capacity was deter- fuged at 4000 rpm for 5 min and the supernatant was filtered mined by bivariate correlation analysis. through a Whatman 42 filter paper. The pellets were reserved for using in the desorption stage of the experiment. 2+ Lead and Cu concentrations in the supernatant were meas- 3. RESULTS AND DISCUSSION ured by ICP-OES as previously indicated. The amount of 2+ 2+ Pb and Cu sorbed was calculated as the difference be- 3.1 Characterization of the studied soils tween the initial and final concentration in solution. The studied soils significantly differ in the majority of Desorption experiments were performed after the sorp- their physical and chemical properties (Table 1) which are tion experiments. Desorption isotherms were constructed expected to affect the sorption and retention of Pb2+ and as described by [21]. Desorption isotherms were con- Cu2+.

1910 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

The two vertisols (VER1 and VER2) and inceptisols Sorption isotherms of Cu2+ for INCP1, INCP2, VER1 (INCP1 and INCP2) are alkaline, the pH values range from and FRS (Figure 2a) show similar trends than those of Pb2+. 8.29 to 8.78. Nevertheless, the pH of fersialitic (FRS) is They reach a plateau after the rapid increase as the concen- slightly acidic (6.64, Table 1). The total organic carbon con- tration of the equilibrium solution increases (Figure 2). tent ranges from 0.76 (FRS) to 3.57 (VER2). Fe, Al and Mn These isotherms are type L according to the classification oxides concentrations greatly differ between soils. In all the of [24], indicating a high affinity of Cu2+ for these soils. studied soils, Fe oxide contents are the highest except in The sorption of Cu2+ on these soils is a two-step process INCP2 (Table 1). The effective cationic exchange capacity since the isotherms show an initial linear part that indicates (CECe) ranges from 1.45 in INCP1 to 9.44 cmol kg−1 in sorption of the metal ions on the external surface followed FRS. The clay contents are significantly different in all soils by the saturation of sorption sites. and range between 5% (INCP1) and 60% (FRS). Carbonate However, the initial linear part for each curve differs contents are low in FRS (3%) and very high in VER1 (46%). following soil affinities for Cu2+. The slope of this initial linear part reflects trace element sorption on soils and has 3.2 Sorption and retention isotherms been attributed to surface sites of high sorption affinity [4] Lead and Cu2+ sorption and desorption experiments or to sites with high bonding strength with the metal [27]. were carried out to study the fate of metals on soil samples INCP1 initially sorbed more Cu2+ than FRS and VER1, with different composition. The sorption and retention iso- which sorbed more Cu2+ than INCP2. In turn, VER2, with therms of Pb2+ and Cu2+ onto soil samples are reported in the highest TOC content and oxides exhibits the highest af- Figure 1 and 2. finity for Cu2+ compared to the other studied soils. The sorption isotherms of Pb2+ of INCP1 and FRS are In the same way as those corresponding to sorption, type L and even close to H as described by [24]. It is noted the retention isotherms of Pb2+ were a vertical line near y- that at low equilibrium concentration, the concentration of axis for INCP1, INCP2, VER1 and VER2, whereas that for the cation sorbed is proportional to that present in the solu- FRS was H type indicating a high affinity of the soil for the tion. For higher concentrations, the Pb2+ isotherm of INCP1 sorbate (Figure 1b). reached a plateau (Figure 1a) indicating a maximum sorption Retention isotherm of Cu2+ on FRS is comparable to capacity of the soil since the saturation of sorption sites type L characterized by a small initial inclination that does happened. not increase with the concentration of the solute in the so- In turn, the Pb2+ isotherms of INCP2, VER1 and VER2 lution (Figure 2b). Whereas isotherms of the alkaline soils are almost a vertical line, the slope is very high showing (VER1, VER2, INCP1 and INCP2) are comparable to type that sorption of Pb2+ increases continuously as the concen- C, indicating that Cu2+ is largely retained by these soils. tration of the solution in the equilibrium does (Figure 1a). Two essentials factors are involved on the retention of Cu2+ Isotherms with similar shapes were often obtained for al- [28] eventually pH and organic matter. These authors re- kaline soils [25, 26] indicating an extraordinarily high af- ported that the retention of Cu2+ increased with the increase finity of Pb2+ for these soils. In turn, retention isotherms of of pH, pointed out the important role that organic matter Pb2+ are similar to the corresponding sorption isotherms plays in the retention of Cu2+ and suggested that the copper except for INCP1. Eventually, the curve shape of INCP1 was highly retained to organic matter rather than other soil has changed for a near y-axis curve (Fig. 1b) showing that solids. The retention of Cu2+ on FRS was lowest than that after the desorption stage, the affinity of this soil for Pb2+ of VER1, VER2, INCP1 and INCP2 with more TOC and is higher than before. alkaline pH soil.

TABLE 1 - Characteristics of the soils

INCP1 INCP2 VER1 VER2 FRS

pH (H2O) 8.78a 8.65 a 8.64a 8.29b 6.64c TOC (%) 0.80d 1.54c 1.89b 3.57a 0.76d Fe oxides (g kg-1) 3.1c 0.08d 8.4a 8.38a 6.58b Mn oxides (g kg-1) 0.04c 0.07b 0.12a 0.11a 0.1a Al oxides (g kg-1) 0.16d 0.04e 0.59c 1.1a 0.7b CECe (cmol(+) kg-1) 1.45d 7.88c 8.1b 7.89c 9.44a Clay (%) 5 e 42d 46c 56b 60a Silt (%) 4d 48a 47a 32b 22c Sand (%) 91 a 10 c 7 d 12 c 18 b Carbonate contents (%) 13d 40b 46a 25c 3e TOC: total organic carbon, CECe: effective cation exchange capacity. Values of same parameter followed by different letters in each column differ significantly with P<0.05

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(a)

(b)

FIGURE 1 - Sorption (a) and retention (b) isotherms of Pb2+.

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(a)

(b)

FIGURE 2 - Sorption (a) and retention (b) isotherms of Cu2+.

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3.3 Sorption and retention capacity that the INCP1 has relatively higher pH than FRS. This im- Sorption and retention capacity were estimated using plies that merely a higher soil pH value may not support 2+ 2+ the parameter Kr defined by [22]. From the values of Kr, a greater Cu sorption capacity. In other words, Cu sorp- comparison between soils sorption and retention capacity tion capacity of a soil is likely to be controlled by a combi- can be made. Table 2 shows the values of sorption and re- nation of soil characteristics as in this case are the clay con- tention capacities for Pb2+ and Cu2+. tent, organic matter and CECe. Except INCP1, the studied soils have a higher sorption The correlation analysis carried out between the soil 2+ 2+ and retention capacities for Pb than for Cu . Based on Kr properties and components of studied soils and their sorp- values, the sorption and retention capacities of Pb2+ for the tion and retention capacities of Pb2+ and Cu2+ (established 2+ soils decreased in the following sequence of INCP2≈ by means of Kr) is shown in Table 3. It was found that Pb VER2>VER1>FRS>INCP1. Similar selectivity sequences sorption and retention are high and significantly correlated for sorption and retention of Cu2+ were obtained (VER2> with CECe, clay contents, Mn oxides and carbonate con- INCP2> VER1>FRS>INCP1). For both cations, INCP1 tents. The influence of carbonate contents on Pb2+ soil exhibited the lowest sorption and retention capacity (Table sorption and retention has been also demonstrated by vari- 2). It seems that low TOC on INCP1 decreased Cu2+ sorption ous authors, who confirmed that this component is the main and retention, ratifying results of some studies [29, 30] (Ta- soil component, that ﬁx Pb2+. ble 1). Other soil components such as clays and oxides have 2+ Kr values for INCP2, VER1, and VER2 are statistically also a strong affinity for Pb [22, 33]. In addition, [34, 35] equal and they show the highest fixation capacity of Pb2+. have found that Pb2+ reveal an especially strong affinity for In addition of the alkaline pH, the properties of these soils, Mn oxides and exhibited the highest affinity for Pb2+ among such as high contents of clay and high CECe are the origin the most abundant oxides in soil [36]. For example, sorption of the high Pb2+ sorption observed (Table 1). These soils of Pb2+ by Mn oxides has been found to be up to 40 times also have the highest Mn oxides, total organic carbon and greater than that by Fe oxides [37]. On the other hand, Pb2+ carbonate contents that favor they fix more Pb2+ than bound to clay as inner-sphere complexes and, to a lesser ex- INCP1 and FRS. tent, outer-sphere complexes at ion-exchange sites. It should be noted that INCP1 and FRS have the lowest The sorption of Cu2+ is correlated with the total or- TOC and could probably influence the low fixation capac- ganic, Mn oxides and the carbonate contents (Table 3). ity of Cu2+ as demonstrated by several authors [31, 32]. These results are in line with those of [38]. They indicate Even they are the soils with the lowest sorption capacity that soil organic matter is the most important component 2+ 2+ for Cu , there are significant differences between the Kr determining the sorption and mobility of Cu since it can values of these two soils and FRS sorbed and retained more provide high concentrations of sites for metal sorption [39]. Cu2+ than INCP1 (Table 2). It should be emphasized here The high degree of affinity shown by organic matter for

TABLE 2 - Sorption and retention capacity of Pb2+ and Cu2+ and hysteresis indices

Pb2+ Cu2+ Hysteresis

Sorption Retention Sorption Retention 2+ 2+ 2 2 2 2 Pb Cu Kr R Kr R Kr R Kr R INCP1 0.480c 0.801 0.481c 0.794 0.631 e 0.987 0.631 e 0.987 0.999a 1a INCP2 0.999a 1 0.999a 1 0.928b 0.990 0.928b 0.990 1a 1a VER1 0.998a 1 0.998a 1 0.785c 0.995 0.785c 0,995 1a 1a VER2 0.999a 1 0.999a 1 0.997a 1 0.997a 1 1a 1a FRS 0.853b 0.996 0.828b 0.995 0.710d 0.968 0.664d 0.953 0.971b 0.935b Values of same parameter followed by different letters in each column differ significantly with P<0.05.

TABLE 3 - Correlation coefficients between soil characteristics and Kr

CECe Clay TOC Mn Carbonate contents Sorption 0.882(**) 0.837(**) - 0.759(**) 0.599(*) Pb2+ Retention 0.857(**) 0.812(**) - 0.747(**) 0.632(*) Sorption 0.522(*) 0.556(*) 0.596(*) 0.488(*) 0.577(*) Cu2+ Retention - - 0.623(*) 0.488(*) 0.575(*) Pb2+ - - 0.541(*) - 0.707(**) 2+ HI Cu - - 0.679(**) - 0.696(**) **The correlation is significant at the level of 0.01. *The correlation is significant at the level of 0.05.

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Cu2+ is caused by the formation of stable Cu–organic com- samples by SEM–EDS analysis. It is an alternative for semi- plexes particularly inner sphere complexes and often due quantitative microanalysis of the studied trace elements in to a specific sorption [38]. selected soils. This technique was used to provide informa- 2+ 2+ Summarizing, the organic matter, clay minerals, as tion on the exact location of Pb and Cu in soil and to well as Fe and Mn oxides are the most important compo- identify the soil constituents to which these metals are associated. nents determining the sorption of metals in soils [40]. The organic components form stable metal–organic complexes For this propose, the mineral phases that most influence with a variety of metals, while clay minerals and oxides the metal sorption in soils were mainly focused on, thus clay minerals, oxides and carbonate particles. In addition, the concentrate trace element ions through surface ion exchange and metal-complex surface adsorption. Different measurements were done on samples from the soils with the authors [41] have indicated the similar behavior of Pb2+ and highest sorption and retention capacity, i.e. VER2, INCP2 and FRS. Hence, the pellets obtained after desorption exper- Cu2+ in soils, especially the low mobility particularly in soils with high clay contents and organic matter. iment at 5 mM concentration for each metal were air-dried and characterized by scanning electron microscopy (SEM) using

3.4 Hysteresis energy dispersive spectroscopy (EDS) analysis. Table 4 shows the results of DRX analysis and typical SEM photo- Hysteresis indices are evaluated and depicted on Table 2. graphs are shown in Fig. 3, 4 and 5. The highest sorption reversibility corresponds to FRS. De- The three soils have quartz as principal mineral and spite the high hysteresis indices, FSR reveals the highest calcite has higher proportions in VER2 and INCP2 than in mobility of Pb2+ and Cu2+ compared to the other studied FRS (Table 3). The occurrence of cerussite (PbCO ) in soils. In addition, and for both cations, the soils with the 3 VER2 and INCP2 is explained by the alteration of the crys- highest hysteresis are also those with the highest capacity talline phase of soils [42] since metals have been added for sorption and retention (INCP2, VER1 and VER2). through the sorption solution with acidic pH. Hence, similarly to sorption and retention capacity, the 2+ highest hysteresis is related to their characteristics that in- On soil samples of VER2 and INCP2 treated with Pb fluence the low mobility such as alkalinity, highest CECe the content of calcite decreased and new amorphous phases values and highest TOC and clay contents. Contrariwise, were formed (Table 4), mainly cerussites (PbCO3) as de- the lowest hysteresis (Table 2) occurred in the acidic and scribed on the reaction below, which are responsible for 2+ sandy soils (Table 1). fixing Pb . The cerussite has low solubility and if the content of calcite is high, it can contribute to the sorption and The correlation analysis carried out between the soil retention of Pb2+ [43, 44]. This shows as it was previously characteristics and the hysteresis indices (Table 3) shows discussed in this paper, the close correlation between the 2+ 2+ that the hysteresis of Pb and Cu is correlated with the content of calcite in the studied soils and the amount of total organic carbon content (Table 3). This means that the Pb2+ sorbed. organic fraction of the soils influences the irreversibility of CaCO + Pb2+ = PbCO + Ca2+ the sorption of these two cations. Apart from this compo- 3 3 nent, the carbonate one also correlates with the irreversi- Moreover, Figure 3 displays the scanning image of the 2+ bility of the sorption of Pb2+ and Cu2+. distribution of Pb on VER2. Nevertheless, cerussites, 2+ with a high Pb concentration, presented major regions on 3.5 Scanning electron microscope (SEM) and energy disper- calcite (circulated zones). sive spectroscopy (EDS) Figure 4 displays the scanning image of INCP2 con- The aim of this part is to determine the mineralogical taminated with Pb2+. As observed on VER2, calcite content characteristics of the soil by X-ray diffraction (XRD) and has been altered forming cerussite (Table 4). Moreover, in the determination of Pb2+ and Cu2+ distribution inside soils addition to cerussite, a spherical assembly of iron oxide

TABLE 4 - Mineralogical analysis of soils after desorption stage at 5mM

Soil/Mineral Ca Q Mcl Ka Alb Cer Ant Cli VER2 42.58 38 6.35 5.85 3.75 - - 4.03 VER2+Pb2+ 40.8 40 5.5 4.9 1.6 2 - 4.2 VER2+Cu2+ 42.8 40.5 9.9 6.8 4.9 - - 2.8 FRS - 59.48 8.18 24.43 4.53 - 1.9 7.15 FRS +Pb2+ - 55.4 12.4 30.7 - - 1.4 - FRS +Cu2+ - 57.4 10.29 27.5 - - 1.9 - INCP2 48.58 37.73 4.9 4.33 3.03 - - - INCP2+Pb2+ 47.2 38.2 2 3.9 4.4 1.6 - - INCP2+Cu2+ 47.8 37.9 3.4 4.11 3.7 - - - -: not detected Q: Quartz; Alb: Albite; Mcl: Microcline; Ka: kaolinite; Ca: Calcite; Cer: Cerussite; Hal: Halloysite; Ant: Anatase; Cli: Clinochlore.

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FIGURE 3 - Cerussite and EDS of amorphous gels Pb-Fe-Al-Si-Mg-Ca on VER2 (Cerussite is in the circled areas).

FIGURE 4‐ Spherical assembly of iron oxide and EDS of FRS.

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FIGURE 5 - Interaction of Cu2+ with hematite and calcite on FRS.

was formed around the calcite (hematite and magnetite), contents, which displayed the greatest extent of metal bind- mixed with aluminosilicates and gels composited with Ca. ing of all the soils. FRS has the least metal sorption and The mixture contains about 2.5% of cerussite indicating retention capacities and the least carbonate contents. Scan- that the assembly of this mineral is associated with the ning electron microscope (SEM) showed that carbonate added Pb2+. contents, mainly calcite, decreased in treated soils. This fact indicates the formation of new crystalline phases es- In turn to the contamination with Cu2+, Fig. 5 displays sentially cerussite which was the main component influ- an example of the scanning image of FRS. The areas indi- encing Pb2+ fixation on soil surface. While treatment with cated with arrows presented the altered amorphous miner- Cu2+ showed that it forms amorphous gels which sink es- als of calcite and the iron oxides covering the calcite, such sentially to hematite and calcite. as hematite. These areas contain between 1.2 to 3.7% of Cu2+. It should be noted that FRS is one of the soils with the highest content of Fe oxide. This soil component has ACKNOWLEDGEMENTS high affinity for Cu2+. As it has been proved by several authors [45], these results seem to indicate that Cu2+ would This work was supported by the research project: be preferentially associated to iron oxides, hematite and in A/029394/09. Interuniversity Cooperation Program and Sci- a smaller way to clay minerals in this soil sample. Moreo- entific Research between Spain and Mediterranean countries ver, [35] indicated that if oxy(hydr)oxides of Fe are asso- (Spanish Ministry of Foreign Affairs and Cooperation). We ciated with the clay fraction, they will have a high sorption also thank the Spanish Ministry of Science and Innovation capacity of Cu2+. for the research foundlings (Project CGL2010-16765/BTE).

The authors have declared no conflict of interest. 4. CONCLUSION

The studied soils varied in their metal sorption and retention capacity. The soils with the highest CECe, clay REFERENCES contents, Mn oxides and carbonate contents, are those which have the highest sorption capacity of Pb2+. The sorp- [1] Alloway, B.J. (2012) Heavy Metals in Soils. Blackie Aca- tion and retention of Cu2+ are most influenced by total or- demic and Professional, London. ganic content and Mn oxides. Among all soil properties in [2] Adriano, D.C. (2001) Trace Elements in Terrestrial Environ- our study, carbonate contents appeared to be the most im- ments. Springer-Verlag, New York. USA. 2+ 2+ portant parameter in correlating with both Pb and Cu [3] Gao, Y., Kan, A.T. and Tomson, M.B. (2003) Critical evalua- sorption and retention. The range of sorption was illus- tion of desorption phenomena of heavy metals from natural trated by comparison of VER1, with the greatest carbonate sediments. Environ. Sci. Technol. 37, 5566-5573.

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[33] Arenas-Lago, D., Vega, F.A., Silva, L.O.F., Lago-Vila, M. and [16] Lindsay, W.L. and Norvell, W.A. (1978) Development of a Andrade, M.L. (2014). Lead distribution between soil geo- DTPA soil test for zinc, iron, manganese and copper. Soil. Sci. chemical phases and its fractionation in Pb-treated soils. Soc. Am. J. 42, 421–428. Fresen. Environ. Bull. 23, 1025-1035. [17] Marcet, P., Andrade, M.L. and Montero, M.J. (1997) Effica- [34] Ma, Y.B. and Uren, N.C. (1998) Transformations of heavy cite d’une methode de digestion par microondes pour la deter- metals added to soils - application of new sequential extraction mination de Fe, Mn, Zn, Cu, Pb Cr Al et Cd en sediments, in: procedure. Geoderma. 84, 157-168. R. Prost (Ed.), Contaminated Soils: Third International Con- [35] Bradl, H.B. (2004) Adsorption of heavy metal ions on soils ference on the Biogeochemistry of Trace-Elements. and soils constituents. J. Colloid. Interface. Sci. 277, 1–18. [18] Alberti, G., Cristini, A., Loi, A., Melis, P. and Pilo, G. (1997) [36] Covelo, E.F., Matías, J.M., Vega, F.A., Reigosa, M.J. and An- Copper and lead sorption by different fractions of two Sardin- drade, M.L. (2008) A tree regression analysis of factors deter- ian soils. In: Prost, R. (Ed.), Contaminated Soils: Third Int. mining the sorption and retention of heavy metals by soil. Ge- Conf. on the Biogeochemistry of Trace-Elements. [CD-ROM] oderma. 147, 75–85. data/ communic/111 PDF. INRA Editions, Paris, France. [37] Taylor, R.M. and McKenzie, R.M. (1980) The influence of Al [19] Gomes, P.C., Fontes, M.P.F., Da Silva, D.G., Mendonça, E.S. on iron oxides. VI. The formation of Fe(II)-Al(III) hydroxy- and Netto, A.R. (2001) Selectivity sequence and competitive chlorides, -sulphates and -carbonates as new members of the py- adsorption of heavy metals by Brazilian soils. Soil. Sci. Soc. roaurite group and their possible significance in soils. Clays. Am. J. 65, 1115–1121. Clay. 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[38] Sipos, P., Németh, T., Kis, V.K., Mohai, I. (2008) Sorption of copper, zinc and lead on soil mineral phases. Chemosphere. 73, 461–469.

[39] Stumm, W. (1992) Chemistry of the Solid-Water Interface. Wiley, New York. [40] Ochoa-Loza, F.J., Noordman, W.H., Jannsen, D.B., Brusseau, M.L. and Maier, R.M. (2007) Effect of clays, metal oxides, and organic matter on rhamnolipid biosurfactant sorption by soil. Chemosphere. 66, 1634-1642. [41] Németh, T., Jiménez-Millán, J., Sipos, P., Abad, I., Jiménez- Espinosa, R. and Szalai, Z. (2011) Effect of pedogenic clay minerals on the sorption of copper in a Luvisol B horizon. Ge- oderma. 160, 509-516. [42] Cao, X., Ma, L.Q., Singh, S.P. and Zhou, Q. (2008) Phosphate- induced lead immobilization from different lead minerals in soils under varying pH conditions. Environ. Pollut. 152, 184- 192. [43] Shirvani, M., Kalbasi, M., Shariatmadari, H., Nourbakhsh, F. and Najafi, B. (2006) Sorption–desorption of cadmium in aqueous palygorskite, sepiolite, and calcite suspensions: Iso- therm. Hysteresis. Chemosphere. 65, 2178-2184. [44] Montinaro, S., Concas, A., Pisu, M. and Cao, G. (2008) Im- mobilization of heavy metals in contaminated soils through ball milling with and without additives. Chem. Eng. J. 142, 271–284. [45] Cerqueira, B., Vega, F.A., Silva, L.F.O. and Andrade, M.L. (2013) Monometal and simultaneous Cu and Pb soil sorption: Distribution in colloidal soil components. Fresen. Environ. Bull. 22, 1090-1098.

Received: September 16, 2014 Revised: November 12, 2014 Accepted: January 07, 2015

CORRESPONDING AUTHOR

Hamdi Sahraoui Departamento de Bioloxía Vexetal e Ciencia do Solo Facultade de Bioloxía Universidade de Vigo Lagoas, Marcosende 36310 Vigo, Pontevedra SPAIN

E-mail: [email protected]

FEB/ Vol 24/ No 5b/ 2015 – pages 1909 - 1919

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EFFECTS OF TWO FUNGICIDES, CYPRODINIL AND FLUDIOX- ONIL, ON GENOTOXICITY IN DROSOPHILA SMART ASSAY AND ON PROLIFERATION AND VIABILITY OF HEK293 CELLS FROM THE PERSPECTIVE OF CARCINOGENESIS

Asuman Karadeniz1,*, Bülent Kaya2, Burhan Savaş3 and Ş. Fatih Topcuoğlu2

1Mehmet Akif Ersoy University, Faculty of Science and Art, Department of Biology, Burdur, Turkey 2Akdeniz University, Faculty of Science, Department of Biology, Antalya, Turkey 3Akdeniz University, Faculty of Medicine, Department of Medical Oncology, Antalya, Turkey

ABSTRACT dery mildew, blight and rust. Furthermore, they are also used in paints, plastics, and leather against to fungi 1. It In this study, the mutagenic and recombinogenic ef- has been estimated millions of kg synthetic fungicides are fects of fungicides; cyprodinil and fludioxonil, widely used used annually worldwide and it is generally accepted that in agricultural regions, were investigated using the somatic production and marketing of fruit and vegetables would be mutation and recombination test (SMART) in Drosophila not possible without their use. wings. The viability and proliferation effects also tested on In recent years the use of chemicals has increased con- a human immortalized embryonic kidney HEK293 cells sumer concern and their use is becoming more restrictive which is at the early stage of carcinogenesis were also ex- due to environmental problems, residual toxicity problems, amined with MTT and trypan-blue exclusion assays. For carcinogenic effects, occurrence of microbial resistance the SMART assay, two different crosses were used: a standard (ST) and a high-bioactivation (HB) cross, involv- and high inputs 2. ing the flare-3 (flr3) and the multiple wing hairs (mwh) Cyprodinil (an anilinopyrimidine) is a systemic fungi- markers. The HB cross involved flies characterized by an cide used against board range of fungi 3. The biological increased cytochrome P-450-dependent bioactivation ca- mode of action of anilinopyrimidine fungicides includes pacity, which permits the more efficient biotransformation inhibition of methionin biosynthesis and secretion of hy- of promutagens and procarcinogens. In both crosses, the drolytic enzymes 3. Fludioxonil (a phenylpyrrole) is a wings of the two types of progeny, inversion-free marker contact fungicide which is used against Botyritis cinerea heterozygotes and balancer heterozygotes, were analyzed. and is able to uncouple oxidative phosphorylation and to The results show that both fungicides were recombino- inhibit electron transport in B. cinerea, inducing the for- genic at the tested highest concentration, on the wing cells mation of reactive oxygen species 4. Both fungicides are of Drosophila. Furthermore, neither fungicide affected the considered as new generated fungicides and they are usu- proliferation rate of the HEK293 cells but both induced cell ally applied together 5,6. death at high concentrations. The Drosophila wing SMART assay is fast, reliable, easy to perform, can detects a wide spectrum of changes, including point mutations, deletions, mitotic recombina- KEYWORDS: Drosophila melanogaster; wing spot test; cyprodinil, tions, chromosomal loss, and nondisjunction and also de- fludioxonil; genotoxic effect; cancer proliferation. tects the activity of promutagens, using strains with a high capacity to transform some carcinogens to their active metabolites 7. The MTT assay, which is simple, accurate, and yields reproducible results in determining cell viability 1. INTRODUCTION and proliferation rates, can be used for this purpose. The trypan-blue exclusion assay is another test that determines Fungicides are one of the major pesticide groups that cell viability and the proliferation rate. used to protect fruit, leaf and grain diseases, such as pow- This study was undertaken to determine the genotoxicity and cancer-related proliferative effects of two fungi- * Corresponding author cides, cyprodinil and fludioxonil.

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2. MATERIAL AND METHODS and twin spots 8,13,14. On the inversion-heterozygous (mwh/TM3) wings, it was only possible to find mwh single 2.1. Somatic Mutation and Recombination Test (SMART) spots, as the multiple inverted TM3 balancer chromosome 2.1.1. Chemicals does not carry any other suitable marker mutation.

Cyprodinil (C H O ; CAS No: 121552-61-2, 99.9% 14 15 3 2.2. Effects of cyprodinil and fludioxonil on proliferation and purity) and fludioxonil (C12H6F2N2O2; CAS No: 131341- viability of HEK293 cells 86-1, 99.9% purity), were obtained from Sigma-Aldrich 2.2.1. Cell line and culture conditions Chemicals Company. Both fungicides are less soluble in water, were dissolved in a mixture of 10% ethyl alcohol + Human embryonic kidney (HEK293) cells, which were 3% Triton X-100. Four different concentrations of each at the early stage of the carcinogenesis 15, were grown in o one of the fungicides were prepared just before use and a 37 C, in 5% CO2 atmosphere and were routinely main- were administrated to 72±4 hours-old transheterozygous tained in Dulbecco’s Modified Eagle Medium (DMEM) (in- larvae. Used concentrations in the SMART assay have been gredients: 1g/l D- glucose, L-glutamin, sodium pyruvate) determined 0,1-10 mM for cyprodinil, and 0,01-2 mM for supplemented with 10% heat inactivated foetal bovine se- fludioxonil. These concentration values include a wide range rum (Sigma F9665), 10 units/ml piperacillin (Tazocin, Wy- which also contains used concentrations in the farmlands. eth, Serial No: 92841, USA) and 25 µg/ml Amphotericin B Distilled water and solvent mixture were used as negative (Sigma A2942, USA). controls; 1 mM of ethyl methane sulphonate (EMS), an al- kylating agent, was used as a positive control. 2.2.2. Preparation of fungicides Cyprodinil and fludioxonil concentrations were deter- 2.1.2. Strains mined as 1.5, 15, 150 µM for cyprodinil and 1.8, 18, 180 µM The genotoxicity of cyprodinil and fludioxonil, were as- for fludioxonil. Cyprodinil and fludioxonil were dissolved in sessed using two versions of SMART: For the standard cross 96% ethanol; diluted in DMEM and finally applied to (ST), flr3/TM3, BdS virgin females (flr3 (flr3/In(3LR)TM3, ri HEK293 cells. Additionally, HEK293 cells treated with pp sep l(3)89Aa bx34e e BdS) were mated to mwh males DMEM and DMEM + Ethanol mixture were used as a con- and for the high bioactivation cross (HB), NORR/NORR; trol. flr3/TM3, BdS (flr3/In(3LR)TM3, ri pp sep l(3)89Aa bx34e e BdS) virgin females were mated to NORR/NORR; mwh 2.2.3. Cell proliferation assay males 7,8,9. NORR stocks were constructed by Pacella The 3 (4.5 dimethylthiazol-2-yl)-2.5-diphenyltetrazo- 10 similarly as described in Frölich and Würgler 11. For lium bromide (MTT) and trypan blue exclusion assays detailed information on the genetic markers see Lindsley were used to evaluate the effects of cyprodinil and fludiox- and Zimm 12. onil on cell proliferation 16. We used twenty five thousand cells per well and two 2.1.3. Experimental procedure hundred microliter medium per well. HEK293 cells were The wing spot test was performed according to Graf et seeded and treated with 1.5, 15, 150 µM concentrations for al. 9 (ST cross) and Graf and van Schaik 7 (HB cross). cyprodinil and 1.8, 18, 180 µM for fludioxonil for 8 days. Eggs were collected for 8h, and 72 ± 4 h later, the larvae After the incubation period, 5 mg/ml MTT in 10 µL were floated off with tap water. They were then transferred amounts were added to the treated wells. Then, this multi- o to plastic vials (2.6 cm diameter and 12 cm high) contain- well plate was incubated in 37 C CO2 incubator for 5 hours. ing 4.5 g Drosophila Instant Medium (Formula 4-24, Car- After the incubation period 130 µL of medium were re- olina Biological Supply, Burlington NC, USA) rehydrated moved from all the treatment wells. Consequently, wells with 9 ml freshly prepared test compounds. The larvae were treated with 250 µL DMSO for dissolving blue form- were allowed to feed on this medium for 48 hours. All the azan crystals. After 24 hours, absorbance at 570 nm test experiments were performed at 25 ± 1oC and at a 60% rel- wavelength and 630 nm reference wavelengths were meas- ative humidity. The surviving adult flies from both the ST ured and used for the proportion of surviving cells. For the and HB crosses, were collected from the treatment vials trypan blue exclusion assay, non-dyed cells were consid- and stored in 70% ethanol. Their wings were mounted in ered alive. Dead and alive cells were counted under the Faure’s solution and inspected under 400X magnification light microscope and calculated for viability rate 17. for the presence of clones of cells showing malformed wing hairs. On marker-heterozygous wings, two types of 2.3. Statistical Analysis spots could be observed: (i) single spots, either mwh and Evaluation of the genotoxicity of cyprodinil and fludi- flr3, which can be produced by somatic point mutation, oxonil were based on the comparison of the frequencies of chromosome aberration as well as mitotic recombination, the different categories of spots per wing in the treatment and (ii) twin spots, manifest flr and mwh phenotypes in the groups to the concurrent negative control. The statistical same clone, which are originated exclusively from mitotic significance of the results was determined with a multiple recombination. Three categories of spots were recorded: decision procedure that is based on two alternative hypoth- small single spots (1-2 cells), large single spots (>2 cells) eses: (1) the mutation frequency in the treated group is not

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higher than the mutation frequency in the appropriate con- heterozygous genotype, and those with balancer heterozy- trol, and (2) the induced mutation frequency in the treated gous genotype. The results of the balancer heterozygous group is no less than m times as high as the observed spon- wing spot test were only added the highest concentration taneous mutation frequency in the control group. Frei and of both fungicides, only the highest concentration was pos- Würgler 18 give details on the statistical procedure. For itive (Tables 1 and2). Since, negative or inconclusive re- the statistical calculations, the conditional binomial test ac- sults were observed for the other concentrations balancer cording to Kastenbaum and Bowman 19 was used, with heterozygous wings were not evaluated. Eighty wings were significance levels ==0.05. For the cell viability test dif- analyzed for each of four concentrations of cyprodinil and ferences between treatments groups were analyzed for var- fludioxonil. iance analyze and concentrations were compared by Dun- In our study both cyprodinil and fludioxonil induced can Multiple Range Test. For MTT assay viability, range significant increase in the frequency of mwh spots and total of DMEM control group was regarded as 100%. spots only in the ST cross at the highest concentration. Some of the chemicals are more reactive than their metabolic transformation products 20,21. Similarly, the lack 3. RESULTS AND DISCUSSION of response in the HB cross would indicate that its metabolic transformation would change its genotoxic potential. The data from the wing spot analysis of the two fungicides are shown in Tables 1 and 2. The trypan-blue exclu- There is few data on toxicity of the phenyl pyrrole and sion and MTT assay results are shown in Table 3. The Ta- anilinopyrimidine fungicides. In the literature survey, it has bles 1- 2 present the data obtained for each compound in been determined that most fungicides belonging to these both the standard (ST) and high-bioactivation (HB) classes were not toxic substances. Pyrimethanil and fenarimol crosses. For each cross, the wings of the two types of off- (an anilinopyrimidine) are the most noticeable fungicides spring were scored: those with the inversion-free marker- in this regard. For example, according to the European

TABLE 1 - Results obtained with cyprodinil, in the Drosophila wing spot test

Concentration No. of No. of spots (frequencies) / statistical diagnosisa Freq. of clone (mM) wings (N) Small single Large single Twin spots Total mwh Total spots formation per spots spots (m=5) spots (m=2) 105 cellsb (1-2 cells) (>2 cells) (m=2) (m=2) (m=5) ST Cross Marker heterozygous wings Distilled water 78 24 (0.31) 6 (0.08) 0(0.00) 26 (0.33) 30 (0.37) 1.36 1 mM EMS 80 163(2.04)+ 89(1.11)  32(0.40)+ 273(3.41)+ 284(3.55)+ 13.98 10% ethanol+3% Triton X-100 72 23 (0.31) 7 (0.09)  0 (0.00) i 29(0.40)  30 (0.41)  1.65 0.1 80 17 (0.22)  4 (0.05)  1 (0.01) i 22 (0.27)  22 (0.27)  1.12 0.5 80 25 (0.31)  1 (0.01)  1 (0.01) i 27 (0.34)  27 (0.34)  1.38 2 80 20 (0.25)  3 (0.04)  2 (0.02) i 24 (0.31)  25 (0.31)  1.22 10 80 42 (0.52)  4 (0.05)  2 (0.02) i 46 (0.60) + 48 (0.60) + 2.35 Balancer heterozygous wings Distilled water 42 7 (0.16) 0 (0.00) 7 (0.16) 7 (0.16) 0.68 1 mM EMS 80 63(0.79)+ 29(0.36)+ 85(1.06)+ 92(1.15)+ 4.35 10% ethanol+3% Triton X-100 80 10 (0.12)  0 (0.00) i 10 (0.12)  10 (0.12)  0.51 c 0.1 80 11 (0.14) i 0 (0.00) i 11 (0.08) i 11 (0.08) i 0.56 0.5 80 8 (0.10)  0 (0.00) i 8 (0.12)  8 (0.12)  0.40 2 80 11 (0.14) i 0 (0.00) i 11 (0.14) i 11 (0.14) i 0.56 10 80 13 (0.16) i 2 (0.02) i 15 (0.19) i 15 (0.19) i 0.76 HB Cross Marker heterozygous wings Distilled water 80 39 (0.49) 12 (0.15) 8 (0.10) 58 (0.72) 59 (0.72) 2.97 1 mM EMS 80 81 (1.01)+ 30 (0.37)+ 8 (0.10)  112(1.40)+ 119(1.49)+ 5.73 10% ethanol+3% Triton X-100 80 51 (0.64)  12 (0.15)  3 (0.04)  65 (0.81)  66 (0.82)  3.32 0.1 80 47 (0.59)  7 (0.09)  1 (0.01)  54 (0.74)  55 (0.69)  2.76 0.5 80 28 (0.35)  9 (0.11)  0 (0.01)  36 (0.60)  37 (0.46)  1.84 2 80 35 (0.44)  8 (0.10)  1 (0.01)  43 (0.75)  44 (0.55)  2.20 10 80 45 (0.56)  6 (0.07)  0 (0.00)  51 (0.64)  51 (0.64)  2.61 a: Statistical diagnoses according to Frei and Würgler [18]: +: positive, i: inconclusive, : negative, m: multiplication factor, probability levels: α=β=0.05 b:Clone frequencies/fly divided by the number of cell examined/fly (24.400) gives an estimate of formation frequency per cell and per cell division in chronic exposure experiment (Frei and Würgler [18]); c: Balancer chromosome TM3 does not carry the flr3 mutation

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TABLE 2 - Results obtained with fludioxonil, in the Drosophila wing spot test

HB Cross Marker heterozygous wings Distilled water 80 39 (0.49) 12 (0.15) 8 (0.10) 58 (0.72) 59 (0.74) 2.97 1 mM EMS 80 81 (1.01)+ 30 (0.37)+ 8 (0.10)  112(1.40)+ 119(1.49)+ 5.73 10% ethanol+3% Triton X-100 80 51 (0.64)  12 (0.15)  3 (0.04)  65 (0.81)  66 (0.82)  3.32 0.01 80 48 (0.60)  3 (0.04)  0 (0.00)  51 (0.74)  51 (0.74)  2.61 0.05 80 38 (0.47)  7 (0.09)  1 (0.01)  46 (0.57)  46 (0.57)  2.35 0.5 80 47 (0.59)  11(0.14)  0 (0.00)  54 (0.67)  58 (0.72)  2.76 2 80 53 (0.66)  3 (0.04)  2 (0.02)  58 (0.72)  58 (0.72)  2.97 a: Statistical diagnoses according to Frei and Würgler [18]: +: positive, i: inconclusive, : negative, m: multiplication factor, probability levels: α=β=0.05 b:Clone frequencies/fly divided by the number of cell examined/fly (24.400) gives an estimate of formation frequency per cell and per cell division in chronic exposure experiment (Frei and Würgler [18]); c: Balancer chromosome TM3 does not carry the flr3 mutation

TABLE 3 - Effects of fungicides on the viability of HEK293 cells.

Fungicides Concentration (µM) Trypane Blue Assay MTT assay Viability (%) Viability (%) Cyprodinil 1.5 88 94.54 15 84 90.8 150 66 31.27 Fludioxonil 1.8 63 95.19 18 71 94.38 180 60 22.9

Food Safety Authority, although available studies show no is considered non or weakly genotoxic. However, its avail- evidence of mutagenic, genotoxic or carcinogenic potential able toxicological data are controversial and incomplete. of pyrimethanil, an increase in liver weight accompanied by histopatological changes in liver and thyroid have been On the web site of the Environmental Protection observed in short-term toxicity studies in rats and mice Agency (EPA) 26, it is stated that cyprodinil appears to 22. Furthermore, there are some findings on toxicity of pose relatively little human toxicity risk due to low use the other widely used fungicide fenarimol. This fungicide rate, low risk to groundwater, low dietary risk and low has been suggested to be an aromatase inhibitor and there- worker exposure 26. Therefore, results of the mutagenic- fore can affect estrogen/androgen levels in vivo in rodents, ity studies with cyprodinil included gene mutation assays delaying neurodevelopment 23, and can be cytotoxic in bacterial and mammalian cells (Salmonella typhi- 24. Castro et al. 25 have also suggested that fenarimol, murium, Escherichia coli, and Chinese hamster lung V79

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cells), a mouse micronucleus assay and in vivo unscheduled ACKNOWLEDGEMENTS DNA synthesis (UDS) assays, were negative in all studies, with or without metabolic activation. It is also stated that Cy- This work was supported by Research Fund of Ak- prodinil is a non teratogenic substance. EPA 26 suggested deniz University (Project ID: 2003.03.0121.010, Antalya- that these results indicate that cyprodinil is unlikely to ini- TURKEY tiate cancer or cause heritable genetic defects. Also it has not been determined that any carcinogenic effect of Cypro- The authors have declared no conflict of interest. dinil up to 2000 ppm in male mice and rats and 5000 ppm in females, using chronic toxicity /carcinogenicity tests 26.

However some side effects of cyprodinil, such as an inci- REFERENCES dence of focal and multifocal hyperplasia of the exocrine pancreas in male mice, decrease in body weight of female 1 Rahden-Staron, I. (2002). The inhibitory effect of the fungi- rabbits and decrease in the pup weights of mice have been cides captan and captafol on eukaryotic topoisomerases in reported EPA 26. Also cyprodinil is a potential endocrine vitro and lack of recombinagenic activity in the wing spot test disrupter and cellular physiological disrupter 27. Cytotox- of Drosophila melanogaster. Mutat. Res. 518, 205-213. icity of the mixture of cyprodinil and fludioxonil was com- 2 Romero, D. M., Serrano, M., Bailén, G., Guillén, F., Zapata, pared using cell impedance assays. In this mixture, fludiox- P. J., Valverde, J. M., Castillo, S., Fuentes, M., Valero, D. onil followed by cyprodinil appeared as the most toxic pes- (2008). 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Safe. 53, 439–445. plant, duckweed, Lemna minor and on algae Scenedesmus acutus. Among the fungicides pyrimethanil has been found 5 Arias, M., Torrente. A. C., López, E., Soto, B., Simal- Gándara, J. (2005). Adsorption-desorption dynamics of cypro- the most toxic on L. minor. Nominal IC50 were found 46.16 -1 -1 dinil and fludioxonil in vineyard soils. J. Agric. Food Chem. mg L for pyrimethanil and >100 mg L for fludioxonil 53 (14), 5675-5681. and procymidone. In contrast, pyrimethanil has been appeared the least toxic for S. acutus at low concentration. 6 Komárek, M., Čadková, E., Chrastný, V., Bordas, F., Bol- - linger, J. C. (2010). Contamination of vineyard soils with fun- Nominal IC50 have been found 22.81, 4.85 and 4.55 mg L gicides: A rewiev of environmental and toxicological aspects. 1 for pyrimethanil, fludioxonil and procymidone respec- Environ. Int. 36, 138-151. tively. As a result the researchers suggested that the fungi- 7 Graf, U., Schaik, N.V. (1992). Improved high bioactivation cides inhibited growth of two aquatic plants, thereby show- cross for the wing somatic mutation and recombination test in ing a potential toxicity against non-target species 29. Drosophila melanogaster. Mutat. Res. 271, 59-67. However, Frankart et al. 4 state that procymidone, py- 8 Graf, U., Würgler, F.E., Katz, A.J., Frei, H., Juan, H., Hall, C.B., rimethanil and fludioxonil seem to have no marked effects Kale, P.G. (1984). Somatic mutation and recombination test in on duckweed even at very high concentrations (50mg.L-1). Drosophila melanogaster. Environ. Mutagen., 6, 153-188. Cyprodinil also do not affect the stream macro invertebrate 9 Graf, U., Frei, H., Kagi, A., Katz, A. J., Würgler, F. E. (1989). drift at sublethal concentrations 30. Thirty compounds tested in the Drosophila wing spot test. Mu- tat. Res. 222, 359–373. In conclusion, our results show that both fungicides, cyprodinil and fludioxonil, were recombinogenic at higher 10 Pacella, R. E. (1992). Genotoxicity of mycotoxins in an im- concentrations, 10 mM and 2 mM, respectively. The lack proved Drosophila wing spot test and other short-term tests, Ph.D. thesis, Witwatersrand University, Johannesburg, 340 pp. of response in the HB cross would indicate that its metabolic transformation would change its genotoxic potential. 11 Frölich, A, Würgler, F. E. (1989). New tester strains with im- Furthermore, most of the data on toxicity show that these proved bioactivation capacity for the Drosophila wing-spot test, Mutat. Res. 216, 179–187. fungicides have no or low toxicity. On the other part of this study, neither fungicide affected the proliferation rate of 12 Lindsley, D. L., Zimm, G. G. (1992). The genome of Drosoph- the HEK293 cells but both induced cell death at high con- ila melanogaster, Academic Pres, San Diego. centrations in our study. But these effects can be change, 13 Demir, E., Kocaoğlu, S., Kaya, B. (2008). Protective effects of depending on the dose. chlorophyll against the genotoxicity of UVb in Drosophila SMART assay. Fresenius Environ. Bull. 17, (12b), 2180-2186. 14 Demir, E., Kocaoğlu, S., Kaya B. (2008). Protection against ultraviolet-b induced genotoxicity by the chlorophyllin in Dro- sophila melanogaster. Fresenius Environ. Bull.17, (12b), 2187-2192.

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15 Berjukow, S., Döring, F., Froschmayr, M., Grabner, M., Glossmann, H., Hering, S. (1996). Endogenous calcium channels in human embryonic kidney (HEK293) cells. Brit. JPhar- macol. 118, 748-54. 16 Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxi- city assays. J.Immunol. Methods 65, (1-2), 55-63. 17 Savas, B., Kerr, P. E., Pross, H. F. (2006). Lymphokine-activated killer cell susceptibility and adhesion molecule expression of multidrug resistant breast carcinoma. Cancer Cell In- ternational 6: 24. doi:10.1186/1475-2867-6-24 18 Frei, H., Würgler, F. E. (1988). Statistical methods to decide whether mutagenicity test data from Drosophila assays indi-

cate a positive, negative, or inconclusive result, Mutat. Res. 203, 297–308. 19 Kastenbaum, M. A., Bowman, K. O. (1970). Tables for determining the statistical significance of mutation frequencies, Mutat. Res. 9, 527-549. 20 Kaya, B., Creus, A., Yanikoğlu, A., Cabre, O., Marcos, R. (2000). Use of the Drosophila Wing Spot Test in the Genotox- icity Testing of Different Herbicides. Environ. Mol. Mutagen. 36:40-46. 21 Kaya, B., Marcos, R., Yanikoğlu, A., Creus, A. (2004). Eval- uation of the genotoxicity of four herbicides in the wing spot test of Drosophila melanogaster using two different strains. Mutat. Res. 557:53-62. 22 Ballesteros, M. M. Martín, J. A. Sánchez Pérez, J. L. García Sánchez, L. Montes de Oca, J. L. Casas López, I. Oller, S. Ma- lato Rodríguez. (2008). Degradation of alachlor and pyrimethanil by combined photo-Fenton and biological oxidation. J.Hazard. Mater. 155, 342-349. 23 Castro, V. L. S. S. de, Mello, M. A. de, Diniz, C., Morita, L., Zucchi, T., Poli, P. (2007). Neurodevelopmental effects of perinatal fenarimol exposure on rats. Reprod. Toxicol. 23, 98– 105. 24 Aydemir, N., Bilaloğlu, R. (2004). The investigation of the genotoxic effects of fenarimol and propamocarb in mouse bone marrow in vivo. Toxicol. Lett. 147, 73–78. 25 Castro, V. L. S. S. de, Mello, M. A. de, Poli, P., Zucchi, T. (2005). Prenatal and perinatal fenarimol-induced genotoxicity in leukocytes of in vivo treated rats. Mutat. Res. 583, 95-104. 26 U. S. Environmental Protection Agency (EPA). Issued Date: April 6, (1998). Office of Prevention, Pesticides and Toxic Received: September 19, 2014 Substances (7501C), Pesticide Fact Sheet, Accepted: January 08, 2015 http://www.epa.gov/opprd001/factsheets/cyprodinil.pdf 27 Fang, C. C., Chen, F. Y., Chen, C. R., Liu, C. C., Wong, L. C., Liu, Y. W., Joseph Su, J. G. (2013). Cyprodinil is an activator CORRESPONDING AUTHOR of aryl hydrocarbon receptor. Toxicology, 304: 32-40. 28 Crépet, A., Héraud, F., Béchaux, C., Gouze, M. E., Pierlot, S., Asuman Karadeniz Fastier, A., Leblanc, J-Ch., Le Hegarat, L., Takakura, N., Mehmet Akif Ersoy University Fessard, V., Tressou, J., Maximilien, R., de Sousa, G., Nawaz, Faculty of Science and Art A., Zucchini-Pascal, N., Rahmani, R., Audebert, M., Graillot, Department of Biology V., Cravedi, J. P. (2013). The PERICLES research program: an integrated approach to characterize the combined effects of Istiklal Yerleskesi mixtures of pesticide residues to which the French population 15030 Burdur is exposed. Toxicol. 313, 83-93. TURKEY 29 Verdisson, S., Couderched, M., Vernet, G. (2001). Effects of procymidone, fludioxonil and pyrimethanil on two non-target Phone: +90 248 213 30 27 aquatic plants. Chemosphere 44, 467-474. Fax: +90 248 213 30 99 30 Beketov, M. A., Liess, M. (2008). Potential of 11 pesticides to E-mail: [email protected] initiate downstream drift of stream macroinvertebrates. Arch. Environ. Contam. Toxicol. 55, 247-253. FEB/ Vol 24/ No 5b/ 2015 – pages 1920 - 1925

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INFLUENCE OF WEAK MAGNETIC FIELD ON HEAVY METAL CATIONS REMOVAL BY ZERO VALENT IRON

Chao Wang, Zhongjin Xiao, Lei Wang and Junlian Qiao*

State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, PR China

ABSTRACT ties, mining operations, and tanneries.[1] Heavy metals are not biodegradable and tend to accumulate in living organ- Weak magnetic field (WMF) was proposed to enhance isms, which cause various diseases and disorders.[2] Cop- heavy metals sequestration by ZVI in this study. The WMF per, in trace level, is essential for living organism, but its influence on the kinetics of Cu(II) removal by ZVI was in- excessive intake would result in detrimental health effects, vestigated at pH 4.0 as functions of initial Cu(II) concen- thus, copper is reported to be among the priority pollutants trations and Fe(0) dosages. The removal efficiencies of when groundwater-associated runoff problems are ad- various heavy metals including Cu(II), Zn(II), Ni(II), dressed.[3] Therefore, it is imperative to remove Cu(II) and Cd(II), Pb(II), Co(II), by ZVI at pH 5.0-7.0 with or without other heavy metal cations from wastewaters prior to dis- WMF were also determined. The results showed that WMF charge to the environment. significantly enhanced Cu(II) sequestration by ZVI at dif- The currently technologies for heavy metals treatment ferent ZVI dosages and the reaction could be accelerated include chemical precipitation, membrane filtration, ion by over 10-fold to achieve over 95% Cu(II) removal effi- exchange, adsorption and coagulation-flocculation [4-7]. ciency at various Fe(0) dosages due to WMF. The Cu(II) However, all of these methods have some demerits. ZVI is removal efficiencies were improved from 85%, 12% to recognized to be a readily available, cost effective, moder- 96%, 98%, respectively at pH 5.0 and pH 6.0. WMF caused ately strong reducing agent and thus it has been widely em- a more markedly drop in ORP and more rapid generation ployed in groundwater remediation and wastewater treat- of Fe(II), which resulted in dramatically accelerated se- ment. Thermodynamically reduction of Cu(II) to Cu(0) is questration rate of Cu(II) by ZVI. The disappearance rate favorable as indicated by the positive value for the poten- of Cu(II) by ZVI reduced markedly with increasing initial tial, E0 in eq 1, which indicating ZVI could be used as a Cu(II) concentration without WMF but the removal rates reducing agent in Cu(II) contamination treatment: of Cu(II) were similar at different initial Cu(II) concentra- Fe C → tions with WMF. The removal efficiencies of heavy metals E0=0.777 V (1) by ZVI at pH 5.0-7.0 follow this order: Cu(II) > Pb(II) > Zn(II) > Ni(II) > Cd(II) ≈ Co(II) without WMF. Coupling Recently, it was reported that the introduction of an in- ZVI with WMF was only efficient for Cu(II) and Pb(II) re- homogeneous weak magnetic field (WMF) (Bmax < 20 mT) moval but not for Zn(II), Ni(II), Cd(II), and Co(II), indicat- could significantly enhance Se(IV) removal by both pristine ing the outstanding advantage of WMF in improving ZVI ZVI and aged ZVI [8, 9] and greatly improve As(V) and reactivity. Employing WMF to enhance contaminants re- As(III) sequestration by ZVI at pHini 3.0-9.0 [10]. However, moval by ZVI is considered to be a promising technique in the influence of WMF on removing metal cations by ZVI future water treatment since it does not need extra energy has never been investigated. Therefore, WMF was proposed input and costly reagents. to improve heavy metals (including Cu(II), Zn(II), Cd(II), Pb(II), Ni(II), Co(II)) removal by ZVI in this study. The ob- jectives of this study were to 1) explore the feasibility of enhancing Cu(II) sequestration by ZVI as functions of different KEYWORDS: Weak magnetic field; Zero valent iron; Heavy metal cations; Corrosion initial Cu(II) concentrations and Fe(0) dosages; 2) probe the feasibility of improving Cu(II), Zn(II), Cd(II), Pb(II), Ni(II), Co(II) by ZVI over the pH range of 5.0-7.0.

1. INTRODUCTION 2. MATERIALS AND METHODS Heavy metal contamination exists in aqueous waste streams of many industries, including metal plating facili- 2.1 Materials. All chemicals were of analytical grade and used with- * Corresponding author out further purification. Deionized (DI) water from a Milli-

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Q water system was used throughout this work. The stock The application of WMF significantly enhanced Cu(II) solutions were prepared by dissolving the corresponding removal by ZVI at various ZVI dosages. When ZVI dosage salts with DI water. The ZVI particles were supplied by Si- was 0.5 g L-1, 100% Cu(II) could be removed in 1 h with nopharm Chemical Reagent Co., Ltd with D50 of 40.0 m. the presence of WMF. When Fe(0) dosage was increased All the other chemicals used, if not otherwise specified, to 1.0, 2.0, and 5.0 g L-1, the reaction durations necessary were provided by Shanghai Qiangshun Chemical Reagent to achieve ~100% Cu(II) removal were shortened to 30 min, Company. 20 min and 10 min, respectively. With the application of WMF, the reaction could be shortened by over 10-fold to 2.2. Batch experiments and chemical analysis achieve over 95% Cu(II) removal efficiency at various Kinetic experiments of Cu(II) removal by ZVI were Fe(0) dosages, indicating the outstanding advantage of initiated by adding 1.0 g ZVI particles into a 1000 mL beaker WMF in improving ZVI reactivity toward Cu(II) removal containing 50.0 mg L-1 Cu(II), 0.01 M NaCl and 0.1 M so- and thus provided a new potential option to remove con- dium acetate buffer (pH = 4.0). The experiments were per- taminants from wastewater more effectively. formed at 25±1 oC and open to the air if not otherwise specified. Two neodymium-iron-boron permanent magnets were A close comparison of the solution ORP variation de- used to generate the external inhomogeneous magnetic field termined and Fe(II) generation during the reaction demon- and the maximum magnetic field intensity beneath the reac- strated that ORP variation and Fe(II) accumulation curves tor was determined to be around 20 mT. During reaction, a generally coincided with those of the Cu(II) sequestration. mechanical stirrer (D2004W, Shanghai Sile Instrument Co., The sequestration of Cu(II) by ZVI is considered to be a Ltd) was used to mix the solution at 480 rpm, which miti- heterogeneous process which involves the oxidation and gated the aggregation of the ZVI induced by WMF (same dissolution of iron accompanied by the reduction of Cu(II) stirring intensity was used in tests without WMF). To in- and the release of Fe(II), which can be represented by Eq. vestigate the influence of WMF on various heavy metal (2). ZVI will also react with the dissolved oxygen, which cations removal by ZVI over the pH range of 5.0-7.0, the can be represented by Eq. (3). -1 experiments were triggered by adding 1.0 g L Fe(0) to 1000 mL beaker containing 50.0 mg L-1 heavy metal cations and Fe C → (2) 0.01 M NaCl. 0.1 M sodium acetate, 0.1 M 2-(N-morpholino) 2Fe 2 →2 2 (3) ethane-sulfonic acid (MES) and 0.2 M tris(hydroxyme- thyl)aminomethane (TRIS) were used as buffers to control Therefore, the drop in ORP at different ZVI dosages experiments conducted at pH 5.0, 6.0 and 7.0 ( ± 0.1). All when ZVI was added could be ascribe to the quick genera- experiments were run in duplicates, and all points in the fig- tion of Fe(II) and rapid consumption of dissolved oxygen ures are the mean of the results. by ZVI, since the ORP in this system mainly corresponded - Aliquots of 5 mL suspension were periodically sam- with the Fe(II)/Fe(III), Cu(II)/Cu(0) and O2/OH couples. pled and filtered by a 0.22 μm pore diameter membrane. In the absence of WMF, the ORP dropped progressively The filtrate was then acidified with one drop of 65% HNO3 from 150-300 mV to 0-150 mV within 60 min, however, before subject to analyses of residual heavy metal cations ORP fell down sharply from 150-300 mV to -300~-350 mV with Perkin Elmer Optima 5300 DV ICP-OES and Fe(II) within 60 min with WMF. Obviously, the sharper drop in concentration with ferrozine method on a TU-1901 ORP with WMF compared to the case without WMF cor- UV/visible spectrophotometer at a wavelength of 510 nm responded with a greater rate of Cu(II) removal. Moreover, [11]. The variation of Oxidation Reduction Potential (ORP) the accumulation of Fe(II) with WMF appeared earlier was monitored throughout the reaction with an ORP sensor compared to the case without WMF when the dosage of connected to a PHS-3C pH meter. ZVI increased from 0.5 g L-1 to 5.0 g L-1. In summary, the introduction of WMF cause more dramatic drop in ORP and more rapid generate of Fe(II), which resulted in mark- 3. RESULTS AND DISCUSSION edly accelerated corrosion of ZVI and sequestration rate of Cu(II) by ZVI. 3.1 WMF effect on Cu(II) removal by ZVI at different Fe(0) dosages 3.2 WMF effect on Cu(II) removal by ZVI at different initial Cu(II) Figure 1 shows the kinetics of Cu(II) removal, corre- concentrations sponding variation of solution ORP and Fe(II) concentra- Figure 2 depicts the influence of WMF on the seques- tion at pH 4.0 at different ZVI dosages. Without the pres- tration of Cu(II) by ZVI at different initial Cu(II) concen- ence of WMF, Cu(II) removal by ZVI generally increased trations at pH 4.0, the corresponding variation of ORP and with increasing ZVI dosage. When 0.5 g L-1 Fe(0) was added, Fe(II) accumulation with and without WMF during the re- only 46% of Cu(II) was removed at the end of 4 h. As the action. The sequestration rate of Cu(II) by ZVI decreased Fe(0) dosage was increased to 1.0-2.0 g L-1, ~90% of Cu(II) markedly with increasing initial Cu(II) concentration with- could be sequestered at the end of 4 h, however, when the out WMF. In the absence of WMF, it took 180 min to ob- dosage of Fe(0) was further increased to 5.0 g L-1, it took tain ~99% Cu(II) removal efficiency when initial Cu(II) only 2 h to achieve 100% Cu(II) removal. concentration was 25.0 mg L-1 while ~99% , 46%, 33% in

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240 min, respectively, at initial Cu(II) concentration of WMF. Moreover, the introduction of WMF significantly 50.0, 100.0 and 200.0 mg/L. The superimposed WMF sig- accelerated the generation of Fe(II). The accelerated se- nificantly enhanced Cu(II) removal at various initial Cu(II) questration of Cu(II) induced by WMF was in accordance concentrations. About 95% of Cu(II) could be removed in with the more rapid ORP decrease and the greater genera- 30 min and Cu(II) could be completely removed in 60 min tion rate of Fe(II), independent on the initial Cu(II) concen- in the presence of WMF, independent on the initial Cu(II) tration, indicating that the significantly influence of WMF concentration. on Cu(II) sequestration was closely associated with its in- In the absence of WMF, the ORP dropped progres- fluence on ORP and Fe(II) accumulation. sively from 180-280 mV to 120-160 mV within 60 min while ORP decreased sharply to around -300 mV when the 3.3 WMF effects on heavy metals removal at pH 5.0-7.0 initial Cu(II) concentration was 25.0-50.0 mg L-1 and -600 The removal efficiencies of various heavy metals, in- and -100 mV, respectively, when the initial Cu(II) concen- cluding Cu(II), Zn(II), Ni(II), Cd(II), Pb(II), Co(II), by ZVI trations were 100.0 and 200.0 mg L-1 in the presence of at pH 5.0-7.0 with or without WMF are presented in Fig. 3.

2+ Cu(II) removal Variation of ORP Fe Accumulation 500 w/o WMF w/o WMF 1.0 200 w/o WMF w/ WMF w/ WMF 400 w/ WMF .8 100 300 0 0 .6 (mg/L)

C/C 200

-100 2+ ORP (mV)

.4 Fe -200 100 .2 -300 0.5 g/L 0.5 g/L 0 0.5 g/L 0.0 -400 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250

Time (min) 300 1.0 w/o WMF w/o WMF 800 w/o WMF w/ WMF 200 w/ WMF w/ WMF .8 100 600

0 .6 0

(mg/L) 400 C/C -100 2+ ORP (mV)

.4 Fe -200 200 .2 -300 1.0 g/L 1.0 g/L 0 1.0 g/L 0.0 -400 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250

w/o WMF 1.0 w/o WMF 200 w/o WMF w/ WMF w/ WMF 1500 w/ WMF .8 0

1000 0 .6 -200 (mg/L) C/C 2+ ORP (mV) .4 -400 Fe 500

.2 -600 2.0 g/L 2.0 g/L 0 2.0 g/L 0.0 -800 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 3000 1.0 w/o WMF w/o WMF w/o WMF w/ WMF w/ WMF 2500 w/ WMF 200 .8 2000

0 .6 0 1500 (mg/L) C/C 2+

ORP (mV) 1000

.4 Fe

-200 500 .2 5.0 g/L 5.0 g/L 0 5.0 g/L 0.0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Time (min) Time (min) Time (min)

FIGURE 1 - Time courses of Cu2+ removal, variation of solution ORP and Fe2+ accumulation with and without WMF at different ZVI dosages. 2+ 0 o All data from stirred, open batch reactors. Reaction conditions: [Cu ]0 = 50.0 mg/L, Fe = 0.5-5.0 g/L, pH = 4.0, T = 25 C.

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2+ Cu(II) removal Variation of ORP Fe Accumulation 200 1000 1.0 w/o WMF w/o WMF w/ WMF w/ WMF 100 800 .8

0 w/o WMF 600 0 .6 w/ WMF (mg/L) C/C -100 2+ 400 ORP (mV) ORP

.4 Fe -200 200 .2 25.0 mg/L -300 25.0 mg/L 0 25.0 mg/L 0.0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250

Time (min) 300 1.0 w/o WMF w/o WMF 800 w/o WMF w/ WMF 200 w/ WMF w/ WMF .8 100 600

0 .6 0 (mg/L) 400 C/C -100 2+ ORP (mV) ORP

.4 Fe -200 200 .2 -300 50.0 mg/L 50.0 mg/L 0 50.0 mg/L 0.0 -400 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 1000 200 1.0 w/o WMF w/o WMF w/ WMF 800 w/ WMF .8 0 w/o WMF w/ WMF 600 0 .6

-200 (mg/L) C/C 2+ 400 ORP (mV) ORP

.4 Fe -400 200 .2

100.0 mg/L -600 100.0 mg/L 0 100.0 mg/L 0.0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250

300 1000 1.0 w/ WMF w/o WMF w/o WMF w/ WMF w/ WMF w/ WMF 200 800 .8 600 0 .6 100 (mg/L) C/C 2+ 400 ORP (mV) ORP .4 0 Fe 200 .2 -100 200.0 mg/L 200.0 mg/L 0 200.0 mg/L 0.0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Time (min) Time (min) Time (min) FIGURE 2 - Time courses of Cu2+ removal, variation of solution ORP and Fe2+ generation with and without WMF at different initial Cu2+ 2+ 0 o concentrations. All data from stirred, open batch reactors. Reaction conditions: [Cu ]0 = 25.0-200.0 mg/L, Fe = 1.0 g/L, pH = 4.0, T = 25 C.

Generally, the removal efficiencies of heavy metals by but 94% Pb(II) was removed at pH 7.0, ascribed to the pre- ZVI follow this order: Cu(II) > Pb(II) > Zn(II) > Ni(II) > cipitation of Pb(II). The superimposed WMF had negligible Cd(II) ≈ Co(II). influence on Pb(II) at pH 7.0 but it remarkably enhanced the removal efficiencies of Pb(II) to 41% and 95%, respectively, With the absence of WMF, the removal efficiencies of at pH 5.0 and 6.0. The removal of Zn(II), Ni(II), Cd(II), and Cu(II) were 85%, 12%, respectively, at pH 5.0 and 6.0. Af- Co(II) by ZVI even in the presence of WMF was not effec- ter WMF was applied, the removal rates of Cu(II) were in- tive over the pH range of 5.0 to 7.0 and the maximum re- creased to 96% and 98%, respectively, at pH 5.0 and 6.0, moval efficiencies for Zn(II), Ni(II), Cd(II), and Co(II) ob- which should be associated with the enhanced ZVI corro- served were 77%, 37%, 23% and 19%, respectively, at pH sion rate in the presence of WMF. At pH 7.0, 100% Cu(II) 6.0. Therefore, the most significant WMF effect was ob- was removed with or without WMF, which should be due served at pH 6.0 for removing these heavy metals by ZVI. to the precipitation of Cu(II) at this pH level. The removal Coupling ZVI with WMF was only efficient for Cu(II) and of Pb(II) by ZVI was minor at pH 5.0-6.0 without WMF Pb(II) removal but not for Zn(II), Ni(II), Cd(II), and Co(II).

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100 w/o WMF w/ WMF 80

20 Removal efficiency (%) efficiency Removal

pH value 5.0 6.0 7.0 5.0 6.0 7.0 5.0 6.0 7.0 5.0 6.0 7.0 5.0 6.0 7.0 5.0 6.0 7.0 Cu2+ Zn2+ Ni2+ Cd2+ Pb2+ Co2+

FIGURE 3 - Influence of WMF on various heavy metals removal by ZVI at pH 6.0. Reaction conditions: initial concentration of heavy metal: 0 50.0 mg/L, pH = 6.0, Fe = 1.0 g/L, reaction time = 6 h.

4. CONCLUSION REFERENCES

This study investigated the influence of WMF on heavy [1] Bailey, S.E., Olin, T.J., Bricka, R.M. and Adrian, D.D. (1999). metals removal by ZVI. It was found that Cu(II) removal by A review of potentially low-cost sorbents for heavy metals. Water Research, 33, 2469-2479. ZVI generally increased with increasing Fe(0) dosage with or without WMF. The application of WMF significantly en- [2] Bulut, Y. and Tez, Z. (2007). Removal of heavy metals from aqueous solution by sawdust adsorption. Journal of Environ- hanced Cu(II) removal by ZVI at various ZVI dosages and mental Sciences-China, 19, 160-166. with the application of WMF, the reaction could be short- [3] Ranysivek, R. and Jekel, M.R. (2006). Removal of dissolved ened by over 10-fold to achieve over 95% Cu(II) removal at metals by zero-valent Iron (ZVI): Kinetics, Equilibria, pro- various Fe(0) dosages. The presence of WMF led to a more cesses and implications for stormwater runoff treatment. Wa- dramatic drop in ORP and a more rapid release of Fe(II), ter Research, 40, 640-640. which resulted in markedly accelerated sequestration rate of [4] Mirbagheri, S.A. and Hosseini, S.N. (2005). Pilot plant inves- Cu(II) by ZVI. The disappearance rate of Cu(II) by ZVI re- tigation on petrochemical wastewater treatment for the reduced markedly with increasing initial Cu(II) concentration moval of copper and chromium with the objective of reuse. in the absence of WMF but the removal rates of Cu(II) were Desalination, 171, 85-93. very similar at different initial Cu(II) concentrations with [5] Kang, S.Y., Lee, J.U., Moon, S.H. and Kim, K.W. (2004). Competitive adsorption characteristics of Co2+, Ni2+, and Cr3+ WMF. Coupling ZVI with WMF was only efficient for by IRN-77 cation exchange resin in synthesized wastewater. Cu(II) and Pb(II) removal but not for Zn(II), Ni(II), Cd(II), Chemosphere, 56, 141-147. and Co(II). The method of using WMF to enhance heavy [6] Agouborde, L. and Navia, R. (2009). Heavy metals retention metals sequestration by ZVI is promising since it does not capacity of a non-conventional sorbent developed from a mix- need extra energy input and costly reagents. However, more ture of industrial and agricultural wastes. Journal of hazardous work should be conducted to explore the role of WMF in materials, 167, 536-544. heavy metals removal by ZVI. [7] Heidmann, I. and Calmano, W. (2008). Removal of Zn(II), Cu(II), Ni(II), Ag(I) and Cr(VI) present in aqueous solutions by The authors have declared no conflict of interest. aluminium electrocoagulation. Journal of hazardous materials, 152, 934-941.

[8] Liang, L.P., Sun, W., Guan, X.H., Huang, Y.Y., Choi, W., Bao, H.L., Li, L.N. and Jiang, Z. (2014). Weak magnetic field significantly enhances selenite removal kinetics by zero valent iron. Water Research, 49, 371-380. [9] Liang, L.P., Guan, X.H., Shi, Z., Li, J.L., Wu, Y.N. and Tratnyek, P. G. (2014). Coupled effects of aging and weak magnetic fields on sequestration of selenite by zero-valent iron. Environmental Science and Technology, 48, 6326-34. [10] Sun, Y.K., Guan, X.H., Wang, J.M., Meng, X.G., Xu, C.H. and Zhou, G.M. (2014). Effect of weak magnetic field on arsenate and arsenite removal from water by zerovalent iron: an XAFS investigation. Environmental Science and Technology, 48, 6850-8.

1930 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

[11] Guan, X.H., Dong, H.R., Ma, J., Lo, I.M.C. and Dou, X.M. (2011). Performance and mechanism of simultaneous removal of chromium and arsenate by Fe(II) from contaminated groundwater. Separation and Purification Technology, 80, 179-185.

Received: September 24, 2014 Revised: November 27, 2014 Accepted: January 08, 2015

CORRESPONDING AUTHOR

Junlian Qiao State Key Laboratory of Pollution Control and Re- sources Reuse College of Environmental Science and Engineering Tongji University Shanghai P.R. CHINA

Phone: +86-21-65980956 Fax: +86-21-65986313 E-mail: [email protected]

FEB/ Vol 24/ No 5b/ 2015 – pages 1926 - 1931

1931 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

CONTRIBUTIONS TO THE INSECT FAUNA OF BURDUR PROVINCE (TURKEY) IN TERMS OF HYDROPHILIDAE, HELOPHORIDAE AND CHRYSOMELIDAE (COLEOPTERA) WITH CHOROTYPE ANALYSES

Baran Aslan1,*, Ayçin Yılmaz2, Fatma Bayram2 and Ebru G. Aslan2

1 Mehmet Akif Ersoy University, Tefenni Vocational School of Higher Education, Medical and Aromatic Plants Department, 15600, Tefenni, Burdur, Turkey 2 Süleyman Demirel University, Faculty of Arts and Science, Biology Department, 32260, Isparta, Turkey

ABSTRACT cies and 4 subspecies of hydrophilids within two subfamilies have been recorded from Turkey [11]. Based on investigations conducted in 2012 and 2013, a total of 64 species belonging to the families Chrysomeli- Helophoridae is another important family of Hydroph- dae, Hydrophilidae, and Helophoridae (Coleoptera) were iloidea classified into a single genus, Helophorus Fab- ricius, 1775. The genus includes about 200 species all over recorded from Burdur province located in the southern part of Turkey. Among the species 46 are (about 72% of whole the world and the generality of species (more than 150 spe- collected taxa) firstly reported from the study region. A cies) occur in the Palearctic region. Turkish Helophoridae fauna involves 48 species and 2 subspecies [11]. chorotype classification is presented for all species. Data regarding material examined of each species are also given The aim of the present study is to add new contribu- together with a “remarks” section for some of them, refer- tions to the insect fauna knowledge of Burdur province ring more additional information. which is actually poorly known, especially in terms of aquatic beetles.

KEYWORDS: Chrysomelidae, Hydrophilidae, Helophoridae, chorotype, fauna, Turkey 2. MATERIAL AND METHODS

2.1 Study site The study is based on Chrysomelidae, Hydrophilidae 1. INTRODUCTION and Helophoridae material gathered during 2012 and 2013 from various habitats in Burdur and adjacent areas. Burdur Chrysomelidae, the leaf beetles, is a highly diverse has an average altitude of 1025 m and is situated in the group of phytophagous insects comprising about 40.000 western Mediterranean region of Turkey, and in the central described species in all zoogeographical regions of the part of the “Lakes Region” (Figure 1). Burdur is one of the world [1-3]. In Turkey there are at least 800 leaf beetle spe- most important wetland areas of Turkey because it includes cies (excluding Bruchinae) as provided with some recent a series of lakes of different sizes. In addition to the terres- systematic studies [4, 5]. The Alticini, or flea beetles, con- trial habitats, the region presents opportunities because of stitutes the largest leaf beetle tribe [6] and based on the re- having many water sources including lakes, shallow and cent publications mentioned above, is now represented stagnant waters, slow running waters and moist habitats with 343 species in Turkey [7]. near water sources.

Hydrophilidae, water scavenger beetles, represents the 2.2 Sampling method largest group of the superfamily Hydrophiloidea including Samplings were performed monthly from May to Sep- about 172 genera and 2900 species in all over the world [8- tember in both study years. Totally, 10 samplings (five in 10]. Only two subfamilies; Hydrophilinae and Sphaeridiinae 2012, and five in 2013) were conducted in 11 different lo- inhabit in the Palearctic region. Hitherto; 19 genera, 95 spe- calities (including Burdur and its districts) presenting various aquatic and terrestrial habitats between the altitudes * Corresponding author ranging from 722-1332 m a.s.l.

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FIGURE 1 - Map of the study area showing the sampled localities

Aquatic beetle specimens were collected by using a species referring its morphology, habitat preference, abun- sieve, ladle or net with a diameter of 1-2 mm pore, from dance or distribution. The asterisk before listed species in- shallow and stagnant waters, from the edge of waters, un- dicates its first report from Burdur region. der the decomposing organic matter, and from the bottom parts of emergent plants. Chrysomelids were collected Hydrophilidae Latreille, 1802 throughout the growing seasons from various plants by us- Hydrophilinae Latreille, 1802 ing sweep-net. All samples were killed by ethyl acetate or Helochares Mulsant, 1844 70% alcohol solution (for aquatic beetles), and taken to the laboratory for further analysis and dissection. The genitalia *Helochares lividus (Forster, 1771) were dissected out under a stereo microscope and exposed Material examined: Kemer, 1074 m, 05.VII.2012, in 10% KOH or NaOH solutions for 1-2 hours, if neces- 2♂♂, 3♀♀; Bucak, 973 m, 21.V.2013, 2♂♂; Ağlasun, 874 sary. m, 11.08.2013, 1♂, 3♀♀. Chorotype: Europeo-Mediterranean The specimens were identified to species level using Remarks: Head, pronotum and elytra reddish yellow; the taxonomic keys and figures given by Čıžek and Doguet apical segment of maxillar palpi darker than other two seg- [12], Warchalowski [13], Konstantinov [14] for chrysome- ments. Specimens live in polluted waters including human lids; Gentili and Chiesa [15], Angus [16, 17, 18], Schödl waste. [19, 20] for aquatic beetles. Taglianti et al. [21] Löbl and Smetana [2, 22] were used for the zoogeographical charac- *Helochares obscurus (Müller, 1776) terization and chorotype classification of the species. Material examined: Tefenni, 1332 m, 05.VII.2012, Voucher specimens are deposited at the Biology Depart- 6♂♂; Ağlasun, 1053 m, 21.V.2013, 3♂♂, 1♀; 874 m, ment of Süleyman Demirel University, Isparta. 11.08.2013, 1♂, 1♀. Chorotype: Sibero-European Remarks: Upper surface of body brownish yellow; ter- 3. RESULTS minal segment of antennae shorter, not as long as wide. Adults inhabit fresh and rich vegetated waters. As a result of this study performed in Burdur province, totally 64 species of beetles belonging to 3 families, 3 sub- *Helochares punctatus Sharp, 1869 families, and 15 genera have been recorded. These species Material examined: Kemer, 1093 m, 05.VII.2012, are listed below along with their updated world and Turkey 4♂♂, 1♀; Ağlasun, 874 m, 11.VIII.2013, 4♂♂, 3♀♀. distributions. A “remarks” section is provided for some Chorotype: European

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Remarks: Dorsal surface of body reddish brown; elytra Enochrus Thomson, 1859 sometimes accompanied with disordered longitudinal rows *Enochrus bicolor (Fabricius, 1792) of small black spots; terminal segment of antennae twice Material examined: Tefenni, 1351 m, 05.VII.2012, as long as wide. The members of the species mainly prefer 3♂♂; Ağlasun, 874 m, 11.VIII.2013, 5♂♂, 3♀♀. stagnant waters. Chorotype: Palearctic Remarks: Dorsal surface of body reddish brown; max- Laccobius Erichson, 1837 illar palpi reddish yellow; pronotum with 4 small black Laccobius gracilis Motschulsky, 1855 spots. Adults inhabit in well vegetated waters. Material examined: Tefenni, 1351 m, 05.VII.2012, 5♂♂; Çavdır, 1027 m, 10.IX.2012, 3♂♂, 4♀♀; Ağlasun, *Enochrus fuscipennis (Thomson, 1884) 874 m, 11.VIII.2013, 1♂, 4♀♀; Yeşilova, 1164 m, Material examined: Burdur, 858 m, 23.V.2012, 6♂♂, 28.V.2013, 3♂♂, 7♀♀. 4♀♀; Bucak, 973 m, 21.V.2013, 2♂♂, 1♀; Ağlasun, 874 Chorotype: Turano-Europeo-Mediterranean m, 11.VIII.2013, 3♂♂, 3♀♀. Remarks: It is the characteristic species of subgenus Chorotype: Centralasiatic-European Microlaccobius. The species is distinguished with regular Remarks: Pronotum reddish brown with a large me- serial punctures of elytra. They prefer shallow edges of dian spot; maxillar palpi and antennae yellow except the lakes and small deposits of rainwater as habitats. last three segments of antennae. The members of the species mainly prefer well vegetated fresh waters, and shallow *Laccobius obscuratus orchymonti Gentili, 1976 waters including high decomposing matter. Material examined: Bucak, 860 m, 09.V.2012, 2♂♂, 1♀ ; Tefenni, 1351 m, 05.VII.2012, 5♂♂, 1♀; Çavdır, *Enochrus halophilus (Bedel, 1878) 1027 m, 10.IX.2012, 1♂, 4♀♀; Yeşilova, 1164 m, Material examined: Kemer, 1074 m, 05.VII.2012, 28.V.2013, 6♂♂; 8♀♀, Karamanlı, 1060 m, 27.VI.2013, 5♂♂, 3♀♀; Ağlasun, 1053 m, 21.V.2013, 1♂; Yeşilova, 3♂♂; 5♀♀; Ağlasun, 874 m, 11.VIII.2013, 1♂, 3♀♀. 1164 m, 28.V.2013, 2♂♂, 2♀♀. Chorotype: SW-Asiatic Chorotype: European Remarks: Head, pronotum and scutellum dark, some- Remarks: Pronotum with a brown median spot in mid- times with green reflections; last three segment of antennae dle; maxillar palpi reddish yellow. Specimens prefer shal- and apical segment of maxillar palpi darker than other seg- low waters and edges of salt marshes containing much leaf ments. Species live in muddy transition zones between wa- residue. ter and land. Sphaeridiinae Latreille, 1802 *Laccobius scutellaris Motschulsky, 1855 Coelostoma Brullé, 1835 Material examined: Tefenni, 1332 m, 05.VII.2012, 4♂♂, 4♀♀; Yeşilova, 1164 m, 28.V.2013, 2♂♂, 2♀♀; *Coelostoma orbiculare (Fabricius, 1775) Ağlasun, 874 m, 11.VIII.2013, 1♂, 1♀. Material examined: Tefenni, 1332 m, 05.VII.2012, Chorotype: Turano-Mediterranean 2♂♂, 1♀; Ağlasun, 874 m, 11.VIII.2013, 3♂♂, 4♀♀. Remarks: Head and pronotum with bronze reflections; Chorotype: Palearctic serial punctures of elytra irregular. Adults live inside moss Remarks: Body considerably convex and black. The and mud in the slow running waters with intensive eutroph- members of the species mainly prefer well vegetated shal- ication. low waters and the edges with decaying organic matter.

Laccobius syriacus Guillebeau, 1896 Helophoridae Leach, 1815 Material examined: Bucak, 860 m, 09.V.2012, 5♂♂, Helophorus Fabricius, 1775 1♀; Tefenni, 1332 m, 05.VII.2012, 1♂, 3♀♀; Çavdır, 1027 m, 10.IX.2012, 3♂♂, 4♀♀; Ağlasun, 874 m, 11.VIII.2013, *Helophorus aquaticus (Linnaeus, 1758) 4♂♂, 4♀♀. Material examined: Burdur, 858 m, 23.V.2012, 11♂♂, Chorotype: Turano-Mediterranean 5♀♀; Ağlasun, 1053 m, 21.V.2013, 3♂♂, 3♀♀. Remarks: Head and pronotum dark; pronotum with a Chorotype: Sibero-European typical dark pattern; elytra with irregular serial punctures. Remarks: Body generally dark colored with green and Specimens live in muddy habitats. bronze reflections on patches. There is quite variation in the size of aedeagophore and shape of parameres among Hydrobius Leach, 1815 the specimens. *Hydrobius fuscipes (Linnaeus, 1758) Material examined: Ağlasun, 1053 m, 21.V.2013, *Helophorus brevipalpis Bedel, 1881 4♂♂, 4♀♀. Material examined: Çeltikçi, 861 m, 09.V.2012, 3♂♂, Chorotype: Asiatic-European 2♀♀; Burdur, 858 m, 23.V.2012, 14♂♂; 18♀♀; Gölhisar, Remarks: Body surface completely black; maxillar 992 m, 06.VI.2012, 3♂♂; 3♀♀; Ağlasun, 1053 m, palpi dark brown except for apical segment. Adults live in 21.V.2013, 4♂♂, 6♀♀; Yeşilova, 1164 m, 28.V.2013, poorly vegetated waterbodies. 6♂♂, 8♀♀.

1934 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

Chorotype: Sibero-European *Helophorus montenegrinus Kuwert, 1885 Remarks: Pronotum with green, bronze and red reflec- Material examined: Burdur, 858 m, 23.V.2012, 2♂♂, tions; elytra pale yellow with black spots towards to apex. 2♀♀; Bucak, 973 m, 21.V.2013, 3♂♂. Individuals show considerable variation in the color of pro- Chorotype: European notum and in the shape of genital structures. The members Remarks: Body dark brown (similar but darker than of the species mainly prefer edges of the small and stagnant brevipalpis) with a mottled elytra. Pronotal grooves are waters. It is the most common Helophorus species sampled typical with green or bronze reflections. in the study area. *Helophorus syriacus Kuwert, 1885 *Helophorus daedalus d’Orchymont, 1932 Material examined: Çeltikçi, 861 m, 09.V.2012, 3♂♂, Material examined: Ağlasun, 896 m, 09.V.2012, 4♂♂; 2♀♀; Ağlasun, 1053 m, 21.V.2013, 4♂♂. Tefenni, 1332 m, 05.VII.2012, 3♂♂, 3♀♀; Bucak, 973 m, Chorotype: SW-Asiatic 21.V.2013, 1♂, 1♀. Remarks: Dorsal surface of body pale yellow accom- Chorotype: SW-Asiatic panied with bronze and green reflections on pronotum. In- Remarks: The species can be easily recognized from dividuals generally prefer little puddles located in high al- others by its rather small body. Head, pronotum and elytra titudes. completely dark brown; pronotum sometimes with red, green, or bronze reflections; the apex of elytra distinctly Chrysomelidae Latreille, 1802 yellowish. Specimens live in open areas and clean waters Galerucinae Latreille, 1802 where vegetation is relatively dense. Alticini (Newman, 1834) Phyllotreta Chevrolat, 1837 *Helophorus flavipes Fabricius, 1792 Material examined: Bucak, 860 m, 09.V.2012, 2♂♂, *Phyllotreta atra (Fabricius, 1775) 2♀♀; Çeltikçi, 980 m, 23.V.2012, 5♂♂, 3♀♀; Karamanlı, Material examined: Burdur, 850 m, 23.V.2012, 5♂♂, 1138 m, 06.VI.2012, 1♂; Ağlasun, 1053 m, 21.V.2013, 4♀♀. 1♀. Chorotype: Palearctic Chorotype: European Remarks: Head, pronotum and elytra dark brown; pro- Phyllotreta corrugata Reiche and Saulcy, 1858 notum with green and bronze reflections. The species Material examined: Altınyayla, 1271 m, 07.VI.2013, mainly prefer edges of the small and stagnant waters. 5♂♂, 7♀♀. Chorotype: Centralasiatic-Europeo-Mediterranean *Helophorus grandis (Illiger, 1798) Material examined: Çeltikçi, 861 m, 09.V.2012, 3♂♂, *Phyllotreta erysimi Weise, 1900 2♀♀; Bucak, 860 m, 09.V.2012, 5♀♀; Ağlasun, 1053 m, Material examined: Tefenni, 1300 m, 05.VII.2012, 21.V.2013, 12♂♂, 9♀♀; Bucak, 973 m, 21.V.2013, 4♂♂. 7♂♂, 4♀♀; Altınyayla, 1271 m, 07.VI.2013, 2♂♂, 4♀♀. Chorotype: Europeo-Mediterranean Chorotype: Centralasiatic-Mediterranean Remarks: The species is easily distinguished by its characteristics last fixed abdominal sternite. This study *Phyllotreta fornuseki Cizek, 2003 represents the third locality record for this species in Tur- Material examined: Ağlasun, 896 m, 11.VIII.2013, key. 4♂♂, 2♀♀. Chorotype: European *Helophorus hilaris Sharp, 1916 Remarks: This is a recently described flea beetle from Material examined: Çeltikçi, 861 m, 09.V.2012, 2♂♂, Moravia and Slovakia. Its record from Turkey by Aslan 2♀♀; Ağlasun, 1053 m, 21.V.2013, 3♂♂. and Gök [23] was the first announce outside the type local- Chorotype: Turano-European ities. This is the second locality record of this species in Remarks: Body dark brown; pronotum with bronze re- Turkey after Isparta [4]. flections. The species is easily recognizable by its characteristic aedeagophore. Specimens live in open water areas. *Phyllotreta maculicornis Pic, 1906 Material examined: Ağlasun, 1051 m, 09.V.2012, *Helophorus liguricus Angus, 1970 3♂♂, 2♀♀. Material examined: Ağlasun, 1053 m, 21.V.2013, Chorotype: SW-Asiatic 4♂♂, 3♀♀. Remarks: The species is extending its distribution area Chorotype: European towards western parts of Turkey. Remarks: Body generally light colored with green and bronze reflections. The species is similar to H. grandis, but *Phyllotreta nigripes (Fabricius, 1775) easily separated with the teeth of seventh abdominal Material examined: Ağlasun, 1051 m, 09.V.2012, sternite. Adults are found in shallow and clean waters. Bur- 4♂♂, 9♀♀; Tefenni, 1187 m, 05.VII.2012, 6♂♂, 10♀♀; dur is the third locality record for this species in Turkey. Karamanlı, 1138 m, 05.VII.2012, 4♂♂, 4♀♀.

1935 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

Chorotype: Centralasiatic-Europeo-Mediterranean Longitarsus ochroleucus (Marsham, 1802) Material examined: Bucak, 722 m, 21.V.2013, 1♂, *Phyllotreta variipennis (Boieldieu, 1859) 4♀♀; Yeşilova, 1168 m, 28.V.2013, 2♂♂, 3♀♀; Material examined: Burdur, 850 m, 23.V.2012, 4♂♂, Altınyayla, 1271 m, 07.VI.2013, 2♂♂, 2♀♀. 5♀♀; Çavdır, 1178 m, 10.IX.2012, 3♂♂, 4♀♀; Ağlasun, Chorotype: West-Palearctic 896 m, 11.VIII.2013, 7♂♂, 3♀♀. Chorotype: Turano-Mediterranean Longitarsus parvulus (Paykull 1799) Material examined: Gölhisar, 1002 m, 06.VI.2012, *Phyllotreta vittula (Redtenbacher, 1849) 2♂♂, 2♀♀; Çavdır, 1178 m, 10.IX.2012, 3♂♂, 4♀♀; Ka- Material examined: Tefenni, 1187 m, 05.VII.2012, ramanlı, 1208 m, 27.VI.2013, 1♂; 4♀♀. 7♂♂, 8♀♀. Chorotype: Centralasiatic-Europeo-Mediterranean Chorotype: Asiatic-European + Nearctic Longitarsus picicollis Weise, 1900 Aphthona Chevrolat, 1836 Material examined: Gölhisar, 1002 m, 06.VI.2012, *Aphthona pygmaea (Kutschera, 1861) 3♂♂, 1♀. Material examined: Tefenni, 1300 m, 05.VII.2012, Chorotype: Centralasiatic-European 4♂♂, 2♀♀; Çavdır, 1178 m, 10.IX.2012, 3♂♂. Chorotype: European + Mediterraneo-Sindian *Longitarsus reichei (Allard, 1860) Material examined: Bucak, 722 m, 21.V.2013, 5♂♂, Longitarsus Latreille, 1829 2♀♀. *Longitarsus aeneicollis (Faldermann, 1837) Chorotype: European Material examined: Tefenni, 1300 m, 05.VII.2012, Remarks: This is the third locality report for this spe- 3♂♂, 1♀; Çavdır, 1178 m, 10.IX.2012, 3♂♂, 1♀. cies in Turkey after Antalya and Erzurum [4]. Chorotype: Centralasiatic-Europeo-Mediterranean *Longitarsus succineus (Foudras, 1860) Longitarsus ballotae (Marsham, 1802) Material examined: Tefenni, 1300 m, 05.VII.2012, Material examined: Gölhisar, 1002 m, 06.VI.2012, 8♂♂, 13♀♀; Yeşilova, 1168 m, 28.V.2013, 3♂♂, 7♀♀; 6♂♂, 5♀♀; Altınyayla, 1271 m, 07.VI.2013, 4♂♂, 4♀♀. Karamanlı, 1208 m, 27.VI.2013, 7♂♂; 9♀♀. Chorotype: Centralasiatic-Europeo-Mediterranean Chorotype: Holarctic

*Longitarsus karlheinzi Warchalowski, 1972 Altica Müller, 1764 Material examined: Tefenni, 1187 m, 05.VII.2012, *Altica ancyrensis (Weise, 1897) 2♂♂, 3♀♀. Material examined: Altınyayla, 1271 m, 07.VI.2013, Chorotype: SW-Asiatic 1♂, 2♀♀. Remarks: This study represents the third locality report Chorotype: SW-Asiatic for this species in Turkey [4]. Altica oleracea (Linnaeus, 1758) *Longitarsus longipennis Kutschera, 1863 Material examined: Karamanlı, 1138 m, 05.VII.2012, Material examined: Ağlasun, 1051 m, 09.V.2012, 2♂♂, 1♀; Bucak, 722 m, 21.V.2013, 2♂♂, 3♀♀. 2♂♂, 2♀♀; Bucak, 722 m, 21.V.2013, 6♂♂, 3♀♀. Chorotype: Sibero-European Chorotype: Centralasiatic-Europeo-Mediterranean Chaetocnema Stephens, 1831 Longitarsus luridus (Scopoli, 1763) *Chaetocnema arenaceae (Allard, 1860) Material examined: Gölhisar, 1002 m, 06.VI.2012, Material examined: Tefenni, 1187 m, 05.VII.2012, 4♂♂, 3♀♀; Yeşilova, 1168 m, 28.V.2013, 7♂♂, 9♀♀. 3♂♂, 3♀♀; Yeşilova, 1168 m, 28.V.2013, 1♂, 4♀♀. Chorotype: Sibero-European + Nearctic Chorotype: Europeo-Mediterranean

Longitarsus nigrofasciatus (Goeze, 1777) *Chaetocnema breviuscula (Faldermann, 1837) Material examined: Ağlasun, 1051 m, 09.V.2012, Material examined: Yeşilova, 1168 m, 28.V.2013, 2♂♂, 2♀♀; Gölhisar, 1002 m, 06.VI.2012, 5♂♂, 3♀♀; 3♂♂, 6♀♀. Çavdır, 1178 m, 10.IX.2012, 1♂, 4♀♀. Chorotype: Asiatic- European Chorotype: Centralasiatic-Europeo-Mediterranean *Chaetocnema concinna (Marsham, 1802) Longitarsus obliteratus (Rosenhauer 1847) Material examined: Ağlasun, 896 m, 11.VIII.2013, Material examined: Gölhisar, 1002 m, 06.VI.2012, 3♂♂, 3♀♀. 7♂♂, 8♀♀; Çavdır, 1178 m, 10.IX.2012, 4♂♂. Chorotype: Holoarctic Chorotype: Sibero-European *Chaetocnema conducta (Motschulsky, 1838)

1936 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

Material examined: Burdur, 850 m, 23.V.2012, 3♂♂, Material examined: Yeşilova, 1168 m, 28.V.2013, 1♀; Ağlasun, 896 m, 11.VIII.2013, 4♂♂. 4♂♂, 4♀♀. Chorotype: Afrotropico-Mediterranean + Central- Chorotype: Turano-Europeo-Mediterranean asiatic-European *Psylliodes cuprea (Koch, 1803) *Chaetocnema coyei (Allard, 1863) Material examined: Burdur, 850 m, 23.V.2012, 7♂♂, Material examined: Burdur, 850 m, 23.V.2012, 5♂♂, 11♀♀; Karamanlı, 1138 m, 05.VII.2012, 3♂♂, 3♀♀; 7♀♀; Tefenni, 1187 m, 05.VII.2012, 6♂♂; Bucak, 722 m, Bucak, 722 m, 21.V.2013, 4♂♂, 6♀♀. 21.V.2013, 5♂♂, 5♀♀; Altınyayla, 1271 m, 07.VI.2013, Chorotype: Palearctic 1♂, 1♀; Ağlasun, 896 m, 11.VIII.2013, 3♂♂, 4♀♀. Remarks: The specimens were collected from areas Chorotype: Centralasiatic-European dominated by various Brassicaceae. Remarks: It is a common species in Burdur province showing some color variations, and is especially found in Psylliodes instabilis Foudras, 1860 moist meadows. Material examined: Burdur, 850 m, 23.V.2012, 4♀♀; Çavdır, 1178 m, 10.IX.2012, 5♂♂, 2♀♀; Yeşilova, 1168 Chaetocnema hortensis (Geoffroy, 1785) m, 28.V.2013, 6♂♂, 5♀♀. Material examined: Tefenni, 1187 m, 05.VII.2012, Chorotype: Europeo-Mediterranean 4♂♂, 7♀♀. Chorotype: Palearctic *Psylliodes isatidis Heikertinger, 1912 Material examined: Bucak, 722 m, 21.V.2013, 2♂♂, 3♀♀; Ağlasun, 896 m, 11.VIII.2013, 6♂♂, 8♀♀. Chaetocnema major (Jacquelin Duval, 1852) Chorotype: Sibero-European Material examined: Burdur, 850 m, 23.V.2012, 4♂♂,

5♀♀; Karamanlı, 1138 m, 05.VII.2012, 2♂♂, 1♀; *Psylliodes ozisiki Leonardi et Arnold, 1995 Ağlasun, 896 m, 11.VIII.2013, 3♂♂, 2♀♀. Material examined: Gölhisar, 1002 m, 06.VI.2012, Chorotype: Asiatic- European 1♂, 3♀♀. Remarks: The species is sampled from the habitats Chorotype: SW-Asiatic near lake sides. Remarks: This is the second locality record for this species in Turkey after Erzurum [4]. Chaetocnema scheffleri (Kutschera, 1864) Material examined: Bucak, 722 m, 21.V.2013, 4♂♂, *Psylliodes tricolor Weise, 1888 3♀♀. Material examined: Burdur, 850 m, 23.V.2012, 1♂; Chorotype: Turano-Europeo-Mediterranean Yeşilova, 1168 m, 28.V.2013, 2♂♂, 2♀♀. Chorotype: Centralasiatic-Europeo-Mediterranean Chaetocnema tibialis (Illiger, 1807) Material examined: Ağlasun, 1051 m, 09.V.2012, *Psylliodes vindobonensis Heikertinger, 1914 8♂♂, 12♀♀; Burdur, 850 m, 23.V.2012, 6♂♂, 9♀♀; Ka- Material examined: Bucak, 722 m, 21.V.2013, 1♂, 1♀. ramanlı, 1138 m, 05.VII.2012, 4♂♂, 7♀♀; 27.VI.2013, Chorotype: European 1208 m, 3♂♂; 5♀♀; Bucak, 722 m, 21.V.2013, 6♂♂, Remarks: This study represents the second locality 8♀♀; Altınyayla, 1271 m, 07.VI.2013, 10♂♂, 4♀♀; record for this species in Turkey after Konya [4]. Ağlasun, 896 m, 11.VIII.2013, 9♂♂, 5♀♀. Chorotype: Centralasiatic-Europeo-Mediterranean Crepidodera Chevrolat, 1837 Remarks: This is the most abundant species recorded *Crepidodera aurata (Marsham, 1802) from study region. Material examined: Ağlasun, 1051 m, 09.V.2012, 1♂, 2♀♀; Bucak, 722 m, 21.V.2013, 2♂♂, 3♀♀. Dibolia Latreille, 1829 Chorotype: Palearctic Dibolia rugulosa Redtenbacher, 1849 Material examined: Burdur, 850 m, 23.V.2012, 2♂♂, Podagrica Chevrolat,1836 4♀♀. Chorotype: Turano-European *Podagrica malvae (Illiger, 1807) Material examined: Burdur, 850 m, 23.V.2012, 2♂♂, 8♀♀; Ağlasun, 896 m, 11.VIII.2013, 3♂♂, 3♀♀. Psylliodes Berthold, 1827 Chorotype: Asiatic-European Psylliodes chalcomera (Illiger, 1807)

Material examined: Burdur, 850 m, 23.V.2012, 2♂♂,

2♀♀; Bucak, 722 m, 21.V.2013, 3♂♂, 4♀♀. 4. DISCUSSION Chorotype: Holarctic

Consequently; 43 species belonging to the family *Psylliodes circumdata (Redtenbacher, 1842) Chrysomelidae (from the tribe Alticini), 12 species from Hy-

1937 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

25 20 15 species

of 10 5 Number 0

FIGURE 2 - Chorotype classification according to the number of beetles recorded

drophilidae, and 9 species from Helophoridae were deter- Here, 46 out of 64 mentioned beetles are firstly added to mined from the study area based on field surveys. Burdur is the insect fauna of Burdur as a part of ongoing studies. a rather poorly studied region with regards to chrysomelids and aquatic beetles. There are some Alticini records from The authors have declared no conflict of interest. Burdur in the study of Gök and Aslan [24] which is based on flea beetles associated with moist habitats in Isparta and Burdur provinces. For the other two aquatic families, there have been no detailed studies conducted in this region. Our REFERENCES results also support this; among the listed chrysomelids 27 species are firstly reported from Burdur province. As for the [1] Jolivet, P. and Verma, K.K. (2002). Biology of Leaf Beetles. Intercept Publishers, Andover, UK. aquatic beetles, whole helophorids and hydrophilids except the two Laccobius species (gracilis and syriacus), are first [2] Löbl I. and Smetana, A. (2010). Catalogue of Palaearctic Col- records for aquatic beetle fauna of Burdur. eoptera: Chrysomeloidea, Volume 6. Apollo Books, Stenstrup. [3] Biondi, M, Urbani, F. and D’Alessandro, P. (2013). Endemism Totally, 16 chorotypes were presented; 10 different patterns in the Italian leaf beetle fauna (Coleoptera, Chrysome- chorotypes were listed for aquatic beetles including the hy- lidae). ZooKeys, 332: 177–205. drophilids and helophorids, and all the 16 for chrysomelids. [4] Ekiz, A.N, Şen, İ., Aslan, E.G. and Gök, A. (2013). Checklist Classification of chorotypes follows as suggested by Ta- of leaf beetles (Coleoptera: Chrysomelidae) of Turkey, ex- glianti et al. [21], except the two chorotypes identified for cluding Bruchinae. J. Nat. Hist., 47: 2213-2287. two chrysomelid species: European + Mediterraneo-Sin- [5] Özdikmen, H., Mercan, N., Cihan, N., Kaya, G., Topcu, N.N. dian for Aphthona pygmaea (Kutschera) and Afrotropico- and Kavak, M. (2014). The Importance of superfamily Chrysomeloidea for Turkish biodiversity (Coleoptera). Munis Mediterranean + Centralasiatic-European for Chae- Entomol. Zool., 9: 17-45. tocnema conducta (Motschulsky). Among the listed chorotypes; Centralasiatic-Europeo-Mediterranean (9 species), [6] Nadein, K.S. (2013). Febraina: a new subtribe of Alticini with cladistic analysis based on morphology (Coleoptera: European (8 species), Sibero-European (7 species), SW- Chrysomelidae: Galerucinae). Syst. Entomol., 38: 491–506. Asiatic (7 species), Palearctic (6 species), and Asiatic-Eu- ropean (5 species) are predominate on the number of spe- [7] Aslan, E.G. and Alkan, K. (2015). The Alticini (Coleoptera: Chrysomelidae: Galerucinae) fauna of Davraz Mountain cies with a percentage of about 65% among all others (Fig- (Isparta): Comments on host plant and altitude preferences ure 2). with two new records for Turkish fauna. Turk J Zool., 39: 488- 493. The present study provides a preliminary step to form [8] Mart, A. (2009). Water scavenger beetles (Coleoptera: Hydro- an inventory for Burdur province where there is a big lack philidae) in provinces of central Black Sea Region of Turkey. of faunistic investigations in terms of many insect groups. J. Entomol. Res. Soc., 11: 47-70.

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[9] Fikáček, M., Gentili, E. and Short, A.E.Z. (2010). Order Col- eoptera, Family Hydrophilidae. Arthropod Fauna of the UAE., 3: 135–165.

[10] Short, A.E.Z. and Fikáček, M. (2013). Molecular phylogeny, evolution and classification of the Hydrophilidae (Coleoptera). Syst. Entomol., 38: 723-752.

[11] Darılmaz, M.C. and İncekara, Ü. (2011). Checklist of Hy- drophiloidea of Turkey (Coleoptera: Polyphaga). J. Nat. Hist., 45: 685-735.

[12] Cizek, P. and Doguet, S. (2008). Klíč k určování dřepčíků (Coleoptera: Chrysomelidae: Alticinae) Česka a Slovenska. [Key for the identification of the flea beetles (Coleoptera: Chrysomelidae: Alticinae) of the Czech and Slovak Repub- lics]. Městské muzeum, Nové Město nad Metují, Slovenska. [13] Warchalowski, A. (2010). The Palearctic Chrysomelidae: Identification keys, Vol: 2. Natura Optima Dux Foundation, Warszawa, Poland. [14] Konstantinov, A.S., Baselga, A., Grebennikov, A.A., Prena, J. and Lingafelter, S.W. (2011). Revision of the Palearctic Chae- tocnema species (Coleoptera: Chrysomelidae: Galerucinae: Alticini). Pensoft Publishers, Bulgaria.

[15] Gentili, E. and Chiesa, A. (1975). Revisione dei Laccobius pa- learctici (Coleoptera: Hydrophilidae). Mem. Soc. Entomol. Ital., 54: 1-187.

[16] Angus, R.B. (1988). Notes on the Helophorus (Coleoptera, Hydrophilidae) occurring in Turkey, Iran and neighboring territories. Rev. Suisse Zool., 95: 209-248.

[17] Angus, R.B. (1992). Süsswasserfauna von Mitteleuropa (In- secta: Coleoptera: Hydrophilidae: Helophorinae). Gustav Fischer Verlag, Jena, 20/10-2, Germany.

[18] Angus, R.B. (1998). A new Turkish Helophorus, with notes on H. griseus Herbst and H. montanus d’Orchymont (Coleop- tera, Hydrophiloidea). Entomol. mon. mag., 134: 5-9.

[19] Schödl, S. (1991). Revision der gattung Berosus Leach, 1. Teil: Die palaarktischen arten der untergattung Enoplurus (Coleoptera: Hydrophilidae). Koleopter. Rundschau., 61: 111- 135. [20] Schödl, S. (1993). Revision der gattung Berosus Leach, 3. Teil: Die palaarktischen und orientalischen arten der untergattung Berosus s. str. (Coleoptera: Hydrophilidae). Koleopter. Rundschau., 63: 189-233. [21] Taglianti, A.V, Audisio, P.A, Biondi, M, Bologna, M.A, Car- paneto, G.M, Biase, A.D, Fattorini, S, Piattella, E, Sindaco, R, Received: September 26, 2014 Venchi, A. and Zapparoli, M. (1999). A proposal for a choro- Revised: November 12, 2014 type classification of the Near East fauna, in the framework of Accepted: January 08, 2015 the Western Palearctic region. Biogogr, Vol: XX, 31-59. [22] Löbl, I. and Smetana, A. (2004). Catalogue of Palaearctic Col- eoptera: Hydrophiloidea - Histeroidea – Staphylinoidea, Vol- CORRESPONDING AUTHOR ume 2. Apollo Books, Stenstrup. [23] Aslan, E.G. and Gök, A. (2006). New records of five flea bee- Baran Aslan tles for the Turkish fauna (Coleoptera: Chrysomelidae: Altici- Mehmet Akif Ersoy University nae). Entomol. Zeitschrift, 116: 119-121. Tefenni Vocational School of Higher Education [24] Gök, A. and Aslan, E.G. (2006). Species composition and Department of Medical and Aromatic Plants abundance of flea beetles (Coleoptera: Chrysomelidae) associated with moist habitats in Isparta and Burdur Provinces, 15600, Tefenni, Burdur Turkey. Proc. Entomol. Soc. Wash., 108: 543-549. TURKEY

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FEB/ Vol 24/ No 5b/ 2015 – pages 1932 - 1939

1939 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

EFFECTS OF ELECTRODE MATERIAL ON THE ELECTROKINETIC REMEDIATION OF CADMIUM-CONTAMINATED SOIL NEAR A MINE TAILING

Zongping Cai1,2, Jeremiah Van Doren3, Dongrui Chen1, Weishan Li1,4*, Zhanqiang Fang1 and Lidan Xing1,4

1School of Chemistry and Environment, South China Normal University, Guangzhou 510006, China 2 Guangdong Polytechnic of Environmental Protection Engineering, Foshan 528216,China 3Department of Chemical Biology, The State University of New Jersey, Piscataway, NJ 08854, United States 4 Engineering Research Center of MTEES (Ministry of Education), Research Center of BMET (Guangdong Province), Engineering Lab. of OFMHEB (Guangdong Province), Key Lab. of ETESPG (GHEI), and Innovative Platform for ITBMD (Guangzhou Municipality), South China Normal University, Guangzhou 510006, China

ABSTRACT and the removal of heavy metals [8-10]. During EK remediation processing, electrode reactions take place on its sur- The soil near a mine tailing was effectively remedied face, generating protons (H+) and hydroxyl (OH−) ions at by electrokinetic method with different electrode materials anode and cathode, respectively. The concentration of (graphite, stainless steel, titanium plate), and the variations these ions near the electrodes creates an acid front that in removal efficiency and soil pH with treatment time were moves from anode to cathode, and a basic front that moves determined. The removal efficiency of cadmium reached a from cathode to anode. [11, 12]. At the same time, the gen- maximum of 63.16% when using a voltage gradient of 1 eration of OH- at the cathode leads to the precipitation of V/cm and a treatment time 48 h with the graphite electrode. heavy metals, and this is called the “focusing effect” [13]. When using a stainless steel electrode, cadmium removal This is the main barrier to EK remediation of heavy metal- efficiency was >43.46%, and 39.82% of total Cd could be contaminated soil [14, 15]. removed with a titanium plate electrode. The improved re- Many studies have been performed to control the soil moval efficiency was attributed to the more active sites for pH value and to enhance the capability of EK remediation transfer of electrons provided by the graphite electrode, for metal removal, including the addition of strong complex- compared to the stainless steel and titanium plate electrode. ing agents, such as EDTA (ethylene diamine tetraacetic acid)

into soil [16] and using of ion exchange membranes (IEM) KEYWORDS: to control pH value and zeta potential [17]. These modified Electrokinetic remediation; electrode material; Cd; soil techniques were complicated, and the additional chemicals

or devices resulted in secondary contamination [18, 19]. To enhance the EK remediation, many factors should 1. INTRODUCTION be considered. First of all, it is important to select a good electrode material to increase the active sites because the Soil pollution becomes more and more serious in re- electrode directly interacts with the soil to carry heavy met- cent years [1-4], and the risk of heavy metal poisoning via als out into the pore solution. Therefore, the electrode ma- consumption of food grown on contaminated soil has in- terial is related to the efficiency of EK remediation. In this creased [5, 6]. Soil contaminated by heavy metals has be- work, the effects of 3 different electrode materials on the come evident recently in Shaoguan city of Guangdong removal of cadmium-contaminated soil near a mine tailing province in China, along with the development of industry. (Shaoguan, China) by EK remediation were studied in de- Cadmium is one of the common contaminant metals in soil tail, with the purpose of improving the efficiency of EK near mine tailings, which results in harm to human health remediation. and the environment [7].

Over the past few decades, electrokinetic (EK) remedia- 2. MATERIALS AND METHODS tion was tested to be one of the most effective in situ or ex situ soil decontamination methods. Numerous investigations have 2.1. Soil preparation shown its success in the degradation of soil contaminants Soil samples were collected near a mine tailing in

Shaoguan, China. The measured concentration of Cd was * Corresponding author 97.412 mg/kg for the collected soil samples. Soil moisture

1940 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

content was approx. 9.1%, and initial soil pH was 6.51. The The pH value of the soil was measured in 5 different electrical conductivity (EC) of the soil was 63.2 us/cm. For sections by a pH-meter (soil/water = 1/2.5). Samples were each electrokinetic test, approx. 1 kg of dry soil sample was air-dried and passed through a 100-mesh screen (mesh size loaded into the electrokinetic cell, and distilled water was 0.149 mm), and then digested with HF-HNO3-HClO4 for used as electrolysis solution. determination of heavy metal concentration. An inductively coupled plasma-optical emission spectroscopy unit 2.2. Electrokinetic cell (ICP-OES, Agilent) was used to determine the concentra- EK remediation experiments were carried out in a rec- tion of total Cd. The pH value and Cd concentration were measured for 2 samples from each section, and 2 standard tangular translucent Plexiglas test cell with the following dimensions: length = 26.0 cm, width = 10.0 cm, and height soil samples (i.e., soil with a controlled concentration of = 10.0 cm, as depicted in Fig. 1. The soil was filled into the heavy metals) were analyzed for quality control. The EK remediation experiments were repeated three times. cell up to a length of 20 cm. A constant voltage of 20 V (1 V cm-1) was applied with a DC power source. Filter paper and an O-ring were used between the electrode chambers and the soil cell to avoid leakage. Both the anode and the cath- 3. RESULTS AND DISCUSSION ode were graphite electrodes inserted into each electrode 3.1 Soil characteristics chamber and connected with a DC power unit. Electrodes with a surface area of 54 cm2 (3x9x2 cm) covered the whole The main physico-chemical characterization parameters soil cross-section to provide electric current evenly. The of the experimented soil (oil pH, texture, organic carbon, cat- electrode chambers were filled with distilled water, which ion exchange capacity) are shown in Table 1. The soil was was cycled by pumps to avoid concentration gradients composed of several kinds of minerals but 3 MgO4SiO2H2O within the compartments. (formula 3.0 H2MgO7Si) was predominant and accounted for 58.5% of the total mineral content. The tested soil of the coastal plain displayed a sandy texture which, according to the USDA classification system, can be attributed to silt loam. The soil was slightly alkaline (typical of South- ern China). The mineralogy analysis by X-ray diffraction (Fig. 2) indicated the presence of kaolinite clay (85%), mica (14%), and quartz (1%).

TABLE 1 - Composition and properties of the experimental soil specimens.

Property Value Texture(%) Sand 19.3 Slit 62.4 Clay 18.3 FIGURE 1 - Schematicdiagramoftheelectrokineticlaboratoryappa- Minerals (%) ratus (unit: cm). Chlorite 58.5 Mica 12.0 Smectite 4.4 2.3 Construction of polarization curves Kaolinite 3.6 Different electrode materials were analyzed by polari- Pinguite 2.7 Feldspar 15.1 zation curves with the purpose of choosing those appropri- Picrite 3.7 ate to be used in EKR. All electrochemical measurements Initial pH 6.5 were performed with a PGSTAT-30 workstation (Autolab, Cation exchange capacity (cmol.kg-1) 15.3 . -1 Eco Echemie B.V. Company) in a 3-electrode electro- Total organic carbon (g kg ) 10.9 Moisture content (m%) 9.1 chemical cell (working electrodes: graphite, stainless steel, conductivity (us.cm-1) 63.2 titanium plate), a large platinum net was used as counter electrode, and a Hg/Hg2Cl2 electrode was used as reference The low cation exchange capacity (about15.3 cmol.kg-1, electrode. Their potentials were the values with respect to due to low organic matter and clay contents) suggests that this reference, evaluating the oxidation process at 298 K. the Cd ions were not highly sorbed onto the soil particles, and hydroxyl ions generated at the cathode migrated faster 2.4. Methodology into the soil [20]. The soil sample was divided into 5 sections within the cell, named S1–S5 from anode to cathode. Different elec- 3.2 Polarization curves trode materials were used as anodes or cathodes and, at the The current in the polarization curves represents the same time, the solution was refilled. Analysis was carried electrode reaction rate. In general, the current is governed out in the same way. by the rates of the following processes: (1) mass transfer,

1941 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

18000 26.684 16000 14000 12000 10000 8000 Intensity/Counts 6000 20.922 4000 12.386 2000 8.904 0 0 1020304050607080 2-Theta/Degrees

FIGURE 2 -Typical X-ray diffraction spectrum of an air-dried soil sample (D8 advance, Bruker, German).

0.008 (a) (b) 0.006 (c)

0.004 I/A

0.002 (a) Graphite (b) Steel 0.000 (c) Ti

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 E/V

FIGURE 3 - Polarization curves of different electrode materials graphite, stainless steel and titanium plate in 0.1 M NaOH solution (scan rate: 2 mV/s).

(2) electron transfer at the electrode surface, (3) chemical 3.3 Current changes during the experiments reactions preceding or following electron transfer, such as The electric current is an indication of the amount of ion protonation or dimerization reactions, and (4) other surface electro-migration. The changes in the electric current during reactions, such as adsorption or electrode deposition [21]. the treatments are shown in Fig. 4. During the EK remedia- Polarization curves attributed to the oxidation process tion with the graphite electrode, much more current passed were obtained for each electrode material (Fig. 3). The graph- through the system than that with a stainless steel or a tita- ite showed the highest current density after applying 1.5 V, nium plate electrode, and the current intensity fluctuated pe- which means that this material had more active sites for trans- riodically. Current density of the EK remediation with the fer of electrons, A possible explanation of this phenomenon is graphite electrode started at 4.63 mA/cm2, and then, it in- that it is due to the hydroxyl radicals generated during the elec- creased up to a maximum value of 6.27 mA/cm2. After reach- trolysis on the electrode surface and the second one of stain- ing the maximum value, the current density decreased, then less steel, whereas the current density of the Ti electrode increased again to 6.95 mA/cm2 at time of 26 h, due to pre- was the lowest, due to its low reactivity [11]. cipitate re-dissolving, thus providing more ions for current

1942 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

Ti Graphite 7 Steel ) 2 6

Current density(mA/cm 2

0 0 1020304050 Time(h)

FIGURE 4 -Variations of electric current intensity profile for the EK remediation with different electrode materials (graphite, stainless steel, titanium plate).

Graphite 8 Steel Ti initial

7 pH

S1 S2 S3 S4 S5 Soil section

FIGURE 5 -Soil pH profile for the EK remediation with different electrode materials (graphite, stainless steel, titanium plate).

transport. Finally, the current density decreased and crease due to concentration polarization and water dissoci- reached a value of 6.2 mA/cm2 after 48 h of EK remedia- ation [22]. tion. The maximum values of the current density for the EK remediation with stainless steel and titanium plate were 3.4 pH variation 4.72 and 2.12 mA/cm2, respectively. They were both lower A low pH value in soil is necessary when most heavy than the current density with the graphite electrode, at- metals should be removed by EK remediation [23]. Figure tributed to the low reactivity of the stainless steel and tita- 5 shows the pH variations in the soil profiles during EK nium plate electrode material. In addition, the resistance in remediation with different electrodes. The soil pH was 6.51 the interface between electrodes and electrolyte might in- before EK remediation treatment, and that close to the an-

1943 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

ode was 4.78 after EK remediation with the graphite elec- EK remediation and before EK remediation. The concen- trode; it was significantly lower than that of other soil parts. tration of Cd was 487.06 mg/kg before EK remediation, It can be seen that pH values in the soil bed dropped faster then, Cd was removed from sections near the anode and with the graphite electrode than with the stainless steel or accumulated near the cathode. After remediation for 48 h, titanium plate electrode. Therefore, Cdmigration capability the average concentration of Cd for S1-S5 region was 179.4 was enhanced with the graphite electrode under lower pH mg/kg with the graphite electrode, compared to 275.4 and conditions. 293.1 mg/kg with stainless steel and titanium plate electrode. Consequently, 63.16% of total Cd was removed with the 3.5. Total Cd concentration variation graphite electrode, in contrast to 43.46 and 39.82% with The changes of total Cd concentration in the soil bed stainless steel and titanium plate electrode, respectively. during EK remediation are displayed in Figs. 6 and 7. C/Co Therefore, the removal efficiency was enhanced with the is represented for the proportion of Cd concentration after graphite electrode, showing a great improvement of electro-

Graphite 350 Steel Ti 300

250

200

C(mg/Kg) 150

100

0 S1 S2 S3 S4 S5 Soil section

FIGURE 6 -Cd2+ concentration in soil after EK remediation with different electrode materials (graphite, stainless steel, titanium plate).

100 Graphite Steel 80 Ti

(%) 60 C/Co 40

0 S1 S2 S3 S4 S5 Soil section

FIGURE 7 - Cd2+ distribution in soil after EK remediation with different electrode materials (graphite, stainless steel, titanium plate).

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Graphite Steel Ti 100 Initial

60 Conductance(us/cm)

0.0 0.2 0.4 0.6 0.8 1.0 Distance from anode (X/L)

FIGURE 8 -Comparison of conductance of remnant Cd2+ for EK remediation with different electrode materials (graphite, stainless steel, titanium plate).

migration velocity. The improved removal efficiency was 4. CONCLUSIONS attributed to the more active sites for the transfer of electrons with the graphite electrode than that with the stainless The application of electrokinetic remediation with a ti- steel and titanium plate electrode (physical configuration tanium plate electrode yielded about 39.82% of total Cd of the graphite electrode was more loose). removal from a soil near to a mine tailing in Shaoguan city of Guangdong province in China, after a 48-h treatment, The largest removal velocity of Cd concentration was whereas only 43.46% could be removed with a stainless near the anode as shown in Fig. 7. It can be presumed that the steel electrode. An enhanced EK method with a graphite removal efficacy increased as more Cd ions desorbed from electrode improved the removal efficiency of Cd2+ to the soil particles, as a consequence of pH decline (Fig. 5). Cd 63.16% after a 48-h treatment. The improved removal ef- can be mobilized from soil particles, and therefore, a low ficiency was attributed to the more active sites for transfer pH value had significant influence on the removal effi- of electrons with the graphite electrode, compared to that ciency improvement. with the stainless steel and titanium plate electrode. An in- + 3.6. Electrical conductivity creased production of H ions at the implemented anodes via water electrolysis resulted in more Cd ions desorbed The results of the electrical conductivity (EC) experi- from the soil particles, Therefore, the EK remediation with ments are shown in Fig. 8. The electrical conductivity (EC) the graphite electrode is an effective and economical of the soil was 63.2 us/cm. After EK remediation, the EC method for Cd-contaminated soil remediation. values of the EK remediation with different electrode materials after the 48-h treatment were all lower for regions + - S1-S5,since there were less H or OH ions, and the EC was higher for region S4 because the precipitates were re-dis- ACKNOWLEDGMENT solved due to higher concentrations of Cd2+ ions. The in- + crease of H ion formation at the anodes as well as in- This work was financially supported from the joint 2+ creased Cd ions desorbed from the particles contributed project of National Natural Science Foundation of China to the slight increase in system conductivity. (No. 21003054) and the scientific research project of the The EC of the EK remediation with the graphite elec- Department of Education of Guangdong Province (Grant trode was higher than that with stainless steel or titanium No. 2013CXZDA013). plate electrode, The migration of Cd2+ ionswith the graphite electrode was higher, due to more active sites for transfer The authors have declared no conflict of interest. of electrons with the graphite electrode, in comparison to that with the stainless steel and titanium plate electrodes, coinciding with the pH and Cd2+ distribution profiles shown in Figs. 5 and 7.

1945 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

REFERENCES [17] Amrate, S., Akretche, D.E., Innocent, C., and Seta, P.(2006) Use of cation-exchange membranes for simultaneous recovery of lead and EDTA during electrokinetic extraction. Desalina- [1] Han, P., Li, C., Feng, X.Y., Ma, Z.H., Lu, A.X., Pan, L.G., and tion 193:405-410. Wang, J.H.(2014) Impact of wastewater irrigation on the distribution of heavy metals in agricultural soils: A case study in [18] Hansen, H.K. and Rojo,A. (2007) Electrokinetic remediation Fangshan, Beijing. Fresen. Environ. Bull.23 (7A): 1662-1667. of copper mine tailings: Implementing bipolar electrodes. [2] Li, Q.,Wang, Y., Liu, J.S., and Wang, Q.C.(2014) Compara- Electrochim. Acta 52:3355-3359. tive assessment of heavy metals in suburban vegetable plots of [19] Rojo, A. and Cubillos, L. (2009) Electrodialytic remediation Changchun, Dehui and Nongan, northeast China. Fresen. En- of copper mine tailingsusing bipolar electrodes. J. Hazard. Ma- viron. Bull. 23 (4): 1036-1044. ter. 168:1177-1183. [3] Wang, T.C., Qu, G.Z., Li, J., and Liang, D.L. (2014) Evalua- [20] Rajić, L., Božo, D., Dalmacija, M., Rončević, S., and Perović, tion of the potential of soil remediation by direct multi-channel S.U. (2012) Enhancing electrokinetic lead removal from sedi- pulsed corona discharge in soil. J. Hazard. Mater. 264:169- ment: Utilizing the moving anode technique and increasing the 175. cathode compartment length. Electrochim. Acta 86:36-40. [4] Li, Z.Y., Ma, Z.W., Kuijp, T.J., Yuan, Z.W., and Huang, L. (2014) A review of soil heavy metal pollution from mines in [21] Kuramitz, H., Nakata, Y., Kawasaki,M., and Tanaka,S. (2001) China: Pollution and health risk assessment. Sci. Total. Envi- Electrochemical oxidation of bisphenol A. Application to the ron. 468 –469:843-853. removal of bisphenol A using a carbon fiber electrode. Chem- osphere 45:37–43. [5] Li, Q.S., Chen, Y., Fu, H.B., Cui, Z.H., Shi,L., Wang, L.L., and Liu,Z.F. (2012) Health risk of heavy metals in food crops [22] Shrestha, R., Fischer, R., and Rahner,D. (2003) Behavior of grown on reclaimed tidal flat soil in the Pearl River Estuary, cadmium, lead and zinc at the sediment–water interface by China. J. Hazard. Mater.227: 148-154. electrochemically initiated processes. Colloid. Surf. A 222:261-271. [6] Salazar, M.J., Rodriguez, J.H., Nieto, G.L., and Pignata, M.L. (2012) Effects of heavy metal concentrations (Cd, Zn and Pb) [23] Zhou, D.M., Deng, C.F., Cang, L., and Alshawabkeh, A.N. in agricultural soils near different emission sources on quality, (2005) Electrokinetic remediation of a Cu-Zn contaminated accumulation and food safety in soybean. J. Hazard. Mater. red soil by controlling the voltage and conditioning catho- 233:244-253. lytepH. Chemosphere 61:519-527. [7] Franco, P.E., Veit, M.T., Borba, C.E., Gonçalves, G.C., Fa- gundes-Klen, M.R., Bergamasco, R., Silva, E.A., and Suzaki, P.Y.R. (2013) Nickel(II) and zinc(II) removal using Amberlite IR-120 resin: Ion exchange equilibrium and kinetics. Chem.

Eng. J. 221:426-435. [8] Acar, Y.B. and Alshawabkeh, A.N. (1993) Principles of electrokinetic remediation. Environmental Sci. Technol. 27:2638- 2647. [9] Sun, T.R. and Ottosen, L.M. (2012) Effects of pulse current on energy consumption and removal of heavy metals during electrodialytic soil remediation. Electrochim. Acta 86: 28-35.

[10] Kharkats, Y.I. (1998) Theoretical model of radionuclide extraction from soils by the mechanism of ionic electromigra- tion. J. Electroanal. Chem. 450(1): 27-35. [11] Méndez, E., Pérez, M., Romero, O., Beltrán, E.D., Castro, S., Received: October 04, 2014 Corona, J.L., Corona, A., Cuevas, M.C., and Bustos, E. (2012) Accepted: January 08, 2015 Effects of electrode material on the efficiency of hydrocarbon removal by an electrokinetic remediation process. Electro- chim. Acta 86: 148-156. CORRESPONDING AUTHOR [12] Li, D., Xiong,Z., Nie,Y., Niu,Y.Y., Wang Li, and Liu,Y.Y. (2012) Near-anode focusing phenomenon caused by the high anolyte concentration in the electrokinetic remediation of Weishan Li chromium(VI) -contaminated soil. J. Hazard. Mater. 229: 282- Engineering Research Center of MTEES (Ministry of 291. Education) [13] Probstein, R.F. and Hicks, R.E. (1993) Removal of contami- Research Center of BMET (Guangdong Province), nants from soils by electric fields. Science 260: 498-503. Engineering Lab. of OFMHEB (Guangdong Prov- [14] Lu, P., Feng, Q.Y., Meng, Q.J., and Yuan,T. (2012) Electroki- ince) netic remediation of chromium-and cadmium-contaminated Key Lab. of ETESPG (GHEI), and Innovative Plat- soil from abandoned industrial site. Sep. Purif. Technol. 98: 216-220. form for ITBMD (Guangzhou Municipality) South China Normal University [15] Yeung, A.T. (2011) Milestone, developments, myths, and future directions of electrokine tic remediation. Sep. Purif. Tech- Guangzhou 510006 nol. 79: 124-132. P.R. CHINA [16] Ottosen, L.M., Pedersen, A.J., Ribeiro, A.B., and Hansen, H.K. (2005) Case study on the strategy and application of en- Phone/Fax: +86-20-3931 0256 hancement solutions to improve remediation of soils contami- E-mail:[email protected] nated with Cu, Pb and Zn by means of electrodialysis. Eng. Geol.. 77:317-329. FEB/ Vol 24/ No 5b/ 2015 – pages 1940 - 1946

1946 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

REMOVAL OF BASIC RED 9 IN WASTEWATER USING GREEN FENTON REACTION

Azize Alayli Gungor1,*, Neslihan Celebi1, and Hayrunnisa Nadaroglu2,3,*

1Ataturk University, Erzurum Vocational School, Department of Chemical Technology, 25240 Erzurum, TURKEY 2Ataturk University, Erzurum Vocational School, Department of Food Technology, 25240 Erzurum, TURKEY 3Ataturk University, Faculty of Engineering, Department of Nano-Science and Nano-Engineering, 25240, Erzurum, TURKEY

ABSTRACT 1. INTRODUCTION

As a highest composition of dye wastewater, organic Basic red 9 (BR9), a triarylmethane dye, was selected chemical, which has a harmful effect on the environment, as the typical compound to conclude the networks bet- can be successfully removed by using native magnetic active ween dye and Apolaccase loaded magnetic Active carbon carbon nanoparticles or apolaccase loaded magnetic active nanoparticles (MAC-NPs) or native MAC-NPs [1-2]. BR9 carbon nanoparticles. In this study, the nano magnetite is a poisonous dye, which is also used as a biological dye (FeO.Fe2O3), immobilized on active carbon and modified and a dye for textiles (silks and acrylics), leather, fur, paper, using apolaccase, will be used for removal of Basic Red 9 automobile antifreeze solutions, carbon paper, plastics, (BR9) from industrial wastewater. The nano magnetite glass, polishes, soaps, cosmetics, drugs, anodized alumi- (FeO.Fe2O3) was immobilized on active carbon and modi- num, high-speed photoduplicating inks, ink-jet computer fied with apolaccase. Thus, Fenton and other procedures printers and photo-imaging systems [3]. similar to Fenton were used. The surface varying of the active carbon was presented with SEM, FTIR, XRD and The use of large amounts of BR9 materials during the EDX. Numerous features such as pH of the reaction (3-9), dyeing steps of textile-manufacturing process is the goal of temperature (20-80 oC), contact time (0-180 min), initial such environmental impurity [4]. In addition to the visual BR9 concentration (0-50 ppm), and adsorbent concentra- problems involving to dyed waste, dyes also powerfully tions (0-0.2 g were explored respect to their properties on absorb sunshine; thus, they cause delay in the photosyn- BR9 removal. The adjusted parameters are found as fol- thetic action of aquatic plants and very violent the whole lows: pH:6, temperature: (40-60 oC), contact time: 15 min, environment [5]. BR9 decomposes into carcinogenic aro- initial BR9 concentration: 50 ppm, and adsorbent concen- matic amine above anaerobic circumstances and release of tration: 0.1 g, respectively. These results have shown that this dye bearings sewage into water bodies, which can be magnetic active carbon nanoparticles (MAC-NPs), (81.3 % the reason of damaging effects such as allergic dermatitis, yield) or apolaccase modified magnetic active carbon na- skin irritation, mutations and cancer [1]. noparticles (MAC-NPs-Apolac) (84.13 % yield) could be using with the Fenton and Fenton like procedures for remov- Consequently, physical and/or chemical controlling ing BR9 from industrial waste water. Furthermore, these ex- methods have to be used for decolorization, or to partially periments have revealed that adsorbent is a re-operational, reduce the dyes in wastewater to make them extra obliging inexpensive, bio friendly, easy to prepare, safe (Nano mag- to a secondary biological controlling [6-9]. Some methods netite, H2O2 and active carbon) and insoluble material for such as chemical oxidation, photo-catalytic degrada- Fenton reaction with apolaccase and without enzyme. tion, ozonation, ion exchange, coagulation / flocculation, solvent extraction, Active Carbon (AC) adsorption and re- verse osmosis have been annoyed by many investigators for the management of dyes polluting water [10]. KEYWORDS: Apolaccase (ApoLac), Basic red 9 (BR9), Decolorization, Fenton, Thus, much attention has been spent to the progress of Active carbon nanoparticles (MAC-NPs), Wastewater, water management systems that lead to comprehensive destruction of the dye particles. The most often used active oxidation progress (AOPs) containing heterogeneous photo catalytic oxidation [11-16], ozonation combined with hydro-

gen peroxide (H2O2), ultraviolet (UV) light, or together [17-

19], H2O2/UV, Fenton and photo-Fenton chemicals [20]. Fen- * Corresponding author ton technology is widely studied and defined as an amazing

1947 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

method for the organization of industrial wastewater having method, it was thought that the iron atoms connected with non-biodegradable organic pollutants [21]. AC surface increase the speed of Fenton reaction.

Between all the methods suggested, adsorption and en- In this study, the decolorization of the BR9 by hetero- zymatic removal of dyes is an effective and eco-friendly geneous Fenton and Fenton like process using MAC-NPs method for color removal from wastewaters due to its sim- and modified with ApoLac catalyst have been investigated. ple applicability, easy procedure and its efficiency to re- The effects of optimum immobilized Nano magnetite con- move a wide variety of compounds. However, the effi- centration, optimum contact time, optimum pH value, op- ciency of adsorption process depends on properties of the timum temperature and decolorization of the process were adsorbents such as up-taking capacity and surface charac- discussed. teristics. AC is regarded as a current adsorbent due to its high surface area, pore structures and up-taking capacity. 2. MATERIALS AND METHODS In the recent researches, AC was produced by using particular low-cost lignocellulose biomasses such as de-oiled 2.1 Chemical and reagents soya, industrial sludge’s, cellulose , sunflower stalks etc. and BR9 (Table 1) (CAS number: 569-61-9), ABTS (CAS they have used modified AC in order to remove the carcino- number: 30931-67-0), DEAE- sephadex (CAS num- genic BR9 mentioned from aqueous solutions [22]. ber: 39455-31-7), CH3COONa (CAS number: 127-09-3), ammonium sulphate (CAS number: 7783-20-2), Na2HPO4 The laccase (Lac) (E.C. 1.10.3.2) is a metalloenzyme (CAS number: 7558-79-4), AC (CAS number: 7440-44-0), and it has three copper ions [23-27]. When the metal ions of dipicolinic acid (CAS number: 499-83-2), magnetic nano enzyme are removed within structure, it becomes apolaccase particles Fe3O4 (Iron I, II oxide) (CAS number:1317-61-9) (ApoLac). Thus, enzyme could connect with iron atoms in [28] (De Castro et al. 2001) and hydrogen peroxide (30% the matrix material. Therefore, a complex of Lac and nano- w/w) (CAS number: 7722-84-1) were purchased from Fluka magnetite was formed. It was thought that this complex and Sigma-Aldrich. The pH of the solution was adjusted by could be prevented to wash-off from aqueous medium, so 0.1 M HCl (CAS number: 7647-01-0) or 0.1 M NaOH (CAS ApoLac enzyme was immobilized to the AC surface, which number: 1310-73-2). All of the chemicals used were analyt- was non-toxic and water-insoluble. Since it is a combined ical grade.

TABLE 1 - General characteristics of magnetic nanoparticles Fe3O4 and BR 9.

Chemical formula Chemical structure Molar mass Surface Area Particle Size

2 Fe3O4 231.53 g/mol >60 m /g 50-100 nm

Chemical formula Chemical structure Molar mass Color index number λmax

C19H18ClN3 323.82 g/mol 42500 545 nm

1948 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

TABLE 2 - The purification folds of laccase from Russulaceae (Lactarius volemus).

Volume Activity Total Activity Protein Specific Activity Purification Enzyme Fraction mL EU/mL EU % (g/mL) EU/mg Fold Crude extract 100 135±1.31 1.35x104 100 187±0.32 0.72 - 3 (NH4)2SO4( 95 85±0.11 8.08 x10 59.9 55±1.25 1.55 2.15 DEAE-Sephadex (40-60%) 40 78±1.11 3.12 x103 23.1 8.2±2.31 9.51 13.21 Sephacryl S 200 30 63±0.33 1.89 x103 14.0 0.85±1.10 74.11 102.93

2.2 Nano-magnetite immobilized on AC surface 2.5 Modified matrix surface characterization For the synthesis of immobilized Nano particle, 0.03 M Chemical and mineralogical compositions of MAC- Fe3O4 was prepared by dispersing in100 mL of deionized NPs were determined by scanning through electron micro- water. Afterwards, 25 gr of active carbon was added into scopes (SEM). Images of the MAC-NPs, MAC-NPs-BR9 reaction mixture. The resultant mixture was mixed for and MAC-NPs-apolac-BR9 were magnified 5000 times by 24 hours by using a stirrer (500 rpm, 25 oC). The mixture Metek, Apollo prime, Active area 10 mm2, Microscope in- was centrifuged at 5.000 rpm for 20 minutes. Then, it was spect S50, SE detector R580. The same machine was also washed three times with distilled water to remove the re- used for the EDX spectra analysis in order to determine the o sidual Fe3O4. The wet paste was dried in an oven at 40 C elemental composition of the powdered MAC-NPs. for 72 hours. The immobilized surface was grinned before use in the experiments. In addition, the FTIR analyses were carried out to identify functional groups and molecular structure including with 2.3 Purification of Lac and preparation ApoLac enzyme or without ApoLac modified MAC-NPs. FTIR spectra that Lactarius volemus were picked on April-May nearby were recorded by the Mattson 1000 FTIR spectrometer. the county of Hasankale, in Erzurum City, located in the eastern region of Turkey. The classes were recognized by XRD pattern of the adsorbent was determined by a botanist and samples were held at -40 oC to avoid any XRD (Rigaku D-Max 2000) and analyzed with CuKα o o damage. Herb (10 g) was grounded in liquid N2, then mixed 0.154 nm) radiation with 2, 5 -100 (with a step size homogenously with 50 ml of 1 M KCl in a mixer and cen- of 0.1). trifuged at 5000  g for 60 minutes. Lac, existing in the supernatant, monitored the methods of Nadaroglu and Tasgin 2.6 Remediation study [29] which involved ammonium sulfate precipitation in the The enzymatic and Fenton reactions were carried out array from 60% to 80% and anion exchange chromatog- in a 100 mL Erlenmeyer flaks filled with BR9 (50 mg/L) raphy. The purification folds were calculated to as 102.93- and added 1 g MAC-NPs (as a source of Fe2+ and Fe3+and for fold purification to be completed (Table 2). The protein con- bounding ApoLac), separately. The pH value was adjusted to tent of chromatographic eluates was measured (Beckman the desired value by using 0.1 M of NaOH or 0.1 M of HCl. Coulter Du 730 Life Science UV/VIS) spectrophotometri- For enzymatic reaction, Fenton reactions were initiated by cally by Warburg and Bradford method [30, 31]. adding 100 mL of 3 % H2O2 (w/w) solution to the flaks. Then, both reactions were placed in a shaker at the room 100 mg of Lac was dissolved in 5 mL of 0.2 M phos- temperature. The agitation was provided as 200 rpm [36]. phate buffer (pH: 7.0), which contains 0.075 M of dipico- The samples were taken out from the flaks periodically by linic acid. The solution, which was put into a dialysis sack, using a micropipette. Thereafter, the reaction mixtures was dialyzed in 1 L of same buffer for 5 hours. After these, were centrifuged at 5000 rpm for 10 minutes. Upper solu- it was dialyzed for 18 hours against deionized water by tions were filtered by using 0.45 nm filters. The concentra- changing the solution for 5-6 times. Following these steps, tion of BR9 was measured by using a Beckman Coulter Du the buffer was changed after dialyzing it for 5 hours against 730 Life Science UV/VIS spectrophotometer at 545 nm. 0.01 M of acetate buffer (pH 5.0), which was used for measurement of the activity. Almost 100% pure apoen- In all the runs of the study, as described above, other zyme was obtained by these chemical processes [32, 33]. parameters were kept stable and samples were taken at reg- 100 mg of Lac was used to make ApoLac enzyme. After- ular time basis for the analysis. wards, the measurement of activity has shown that al- most100% pure apoenzyme was created. The decolorization efficiency (DE) of BR9 was defined as: 2.4 Assay of Lac Activity For determination of laccase activity, the ABTS reagent was used as a substrate. One activity unit (U) has cal- -1 culated that 1 μmol of oxidized ABTS min and enzyme Where, activity was expressed in U L-1 [34, 35].

1949 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

[BR9C]0 is the initial concentration of Basic Red 9 in the concentration of BR9 were measured as the decolor- (mg L-1), and ization study using a spectrophotometer at 545 nm.

2.9 Determination of optimum temperature [BR9C]t is the concentration of Basic Red 9 (mg Temperature is known to have a profound effect on L-1), at reaction time, t (min). various chemical processes. The effect of temperature in-

2.7 Contact time fluencing adsorption has been studied within the range of 20-80 °C. Reduction of the concentration of BR 9 was The relationship between the amount of dye removed measured by using a spectrophotometer at 545 nm. and contact time was analyzed and presented.

2.8 Determination of optimum pH 3. RESULTS The effect of initial pH on adsorption of removing the BR9 was determined at different pH values (3–9). The pH The enzyme was purified by using a modified method values of reaction mixtures were adjusted by drop wise ad- developed by Nadaroglu and Tasgin [29]. For this determi- dition of 0.1 M HCl and 0.1 M NaOH solutions. Changes nation, ammonium sulfate (NH4)2SO4 fractionation, an ex-

FIGURE 1 - EDX images of reactions (A: MAC-NPs, B: MAC-NPs-BR9, C: MAC-NPs-BR9-ApoLac).

1950 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

tensively used technique in enzyme purification, was em- ture. The AC matrix with MAC-NPs, MAC-NPs-ApoLac ployed as the first purification step. The collapse of laccase and MAC-NPs-ApoLac-BR9, when the BR9 was bound to enzyme was performed at 40-60% (NH4)2SO4 concentra- the AC surface structure, it was observed in the FTIR dif- tion in an ice-bath. Then collapsed protein was subjected ference. In particular, connecting BR9 completely changed to anion exchange chromatography and gel filtration chro- the structure of fingerprint region of the MAC-NPs-ApoLac- matography. Gel filtration chromatography has shown a BR9 structure, which indicates that the paint is attached to good purification degree for the enzyme. For each purifi- the surface of NPs modified AC matrix. When nanomagnet- cation steps, protein amount and enzyme activities were ite and ApoLac were bound to AC surface, some changes calculated. According to the results obtained, the purifica- occurred on the surface are shown in Figure 3. In particular, tion fold was found as 102.93 (Table 2). Then, purified lac- after connecting BR9 dye to MAC-NPs-ApoLac surface, the case was changed to ApoLac with dipicolinic acid for eas- structure of the fingerprint region was determined by the ily bounding to surface of AC. differences (Figure 3).

Chemical and mineralogical compositions of MAC- The XRD diffractograms of AC matrix with MAC- NPs were determined for using to attach the enzyme to NPs, MAC-NPs-BR9 and MAC-NPs-ApoLac-BR9 are solid face MAC-NPs at room temperature. EDX, SEM, shown in Figure 4. The diffractogram was recorded shortly FTIR were used to examine the surface of adsorbent. The after extrusion showing a diffuse pattern predominantly, same machine was also used for the EDX and SEM spectra which was a typical MAC-NPs system with a sharp peak analyses in order to determine the elemental composition of centered at around 2<35. In addition, it has shown a dif- the powdered MAC-NPs, MAC-NPs-BR9 and MAC-NPs- fuse pattern predominantly, which were typical MAC-NPs- ApoLac-BR9. The elemental composition changes were ob- BR9 and MAC-NPs-BR9-ApoLac systems with a sharp served after Fenton and enzymatic Fenton reactions. The in- peak centered at around (2<36). In the Figure 4, signifi- creased amount of carbon and iron had shown that enzyme cant peaks (2<36) were related to adsorption of BR9 in and nanomagnetite was bonded to the surface of AC matrix the diffractograms acquired shortly after extrusions shown. (Figure 1).

Images of AC matrix with MAC-NPs, MAC-NPs-BR9 It was observed that the removal of BR9 increases in and MAC-NPs-ApoLac-BR9 were magnified by SEM first 30 minutes of the contact time at room temperature, spectra (Figure 2). The SEM spectra had shown some dif- with 500 rpm. Basically, the removal of BR9 was fast at ferences at the surface after Fenton and enzymatic Fenton the beginning, and then it gradually decreased within the reactions. Apparently, according to AC matrix with MAC- time past until it has reached the equilibrium value. These NPs, MAC-NPs-ApoLac had considerable numbers of pores results indicated that the concentration of BR9 in the solu- where, there was a good possibility for dyes to be trapped tion was decreased rapidly within 15 minutes and the re- and adsorbed into these pores (Figure 2A-C). Based on the moval was virtually completed within 30 minutes of the analysis of the images taken by SEM before and after the contact time (Figure 5). dye Fenton process, high heterogeneous pores within AC with MAC-NPs-ApoLac particles were observed. After BR9 The XRD diffractograms of AC matrix with MAC-NPs, dye treatment, the pores were packed with dyes. MAC-NPs-BR9 and MAC-NPs-ApoLac-BR9 are shown in Figure 4. The diffractogram was recorded shortly after ex- In addition, the FTIR analyses were carried out in or- trusion showing a diffuse pattern predominantly, which was der to identify the functional groups and molecular struc- a typical MAC-NPs system with a sharp peak centered at

FIGURE 2 - SEM Images of reactions (A: MAC-NPs, B: MAC-NPs-BR9, C: MAC-NPs-BR9-ApoLac).

1951 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

38,6 38

37 3529,41 3445,37

33 3708,04

31 %T 559,44

29 699,30

1554,61 25 3006,20 1952,72 1144,05

2879,55 23,8 4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400,0 cm-1

FIGURE 3 - FTIR images of reactions (A: MAC-NPs, B: MAC-NPs-BR9, C: MAC-NPs-BR9-ApoLac).

1952 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

2000 MAC-NPs

MAC-NPs-BR9

MAC-NPs-BR9-ApoLac 1500

Intensity (u.a.) 1000

500 0 20406080100 Angle 2

FIGURE 4 - XRD Images of reactions.

7,5

5 qe (mg/mL) 2,5 Apolaccase

Without Apolaccase 0 060120180 Contact time (min)

FIGURE 5 - Effect of contact time on the removal of BR9 by MAC-NPs and MAC-NPs-ApoLac.

around 2<35. In addition, it has shown a diffuse pattern results indicated that the concentration of BR9 in the solu- predominantly, which were typical MAC-NPs-BR9 and tion was decreased rapidly within 15 minutes and the re- MAC-NPs-BR9-ApoLac systems with a sharp peak cen- moval was virtually completed within 30 minutes of the tered at around (2<36). In the Figure 4, significant peaks contact time (Figure 5). (2<36) were related to adsorption of BR9 in the diffractograms acquired shortly after extrusions shown. This dye removing process could be at neutral pH levels, which eliminates the harmful effects. The data were il- It was observed that the removal of BR9 increases in lustrated in Figure 6. The results observed during the study first 30 minutes of the contact time at room temperature, are consistent with results of the previous studies [38]. Fur- with 500 rpm. Basically, the removal of BR9 was fast at thermore, the Fe2+ /3+ complex made on the area of support the beginning, and then it gradually decreased within the can reply with H2O2; thus, permitting iron ions to join in time past until it has reached the equilibrium value. These the Fenton catalytic cycle [36].

1953 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

at near-neutral pH levels. The purpose of the earlier research was exploring the efficacy of Trypan Blue dye using photo-Fenton reaction at neutral pH [39]. These results were approximately the same with our data. Since the classical Fenton reaction was generally performed at pH 3, the present process might be termed as a The effect of MAC-NPs was studied by varying tem- modified Fenton reaction. A mechanism and rate model peratures within the range of 20-80 oC. The effect of tem- had been proposed, which were very much novel for Fen- perature on both of enzymatic and Fenton decolorization ton reaction at neutral pH. In general, Fenton reactions reactions was shown in Figure 7. It was observed that the were carried out at pH ≤ 3 levels. However, it wasn’t degree of decolorization was increased in parallel with in- wanted to impart an additional acidity to the wastewater creasing temperature. The maximum decolorization of that needs neutralization before discharging to the surface BR9 was obtained at 40-60 °C for two processes. Effec- water. Moreover, there is an increase in the cost due to the tiveness of the processes at higher temperatures was an ad- acid-resistant equipment and the processes of acidification vantage for industrial discharge, since higher temperature and neutralization; hence, the reactions can be performed values were used in industrial discharges.

7 qe (mg/mL) Without Apolaccase Apolaccase

4 3456789 pH

FIGURE 6 - Effect of pH on the removal of BR9 by MAC-NPs and MAC-NPs-ApoLac.

Without Apolaccase Apolaccase 10 qe(mg/mL)

5 20 30 40 50 60 70 80 Temperature (oC)

FIGURE 7 - Effect of temperature on the removal of BR9 by MAC-NPs and MAC-NPs-ApoLac.

1954 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

100

BR9 Without Apolaccase With apolaccase

50 100(%)

18,7(%) 15,7(%) 0 BR9 Without Apolaccase With apolaccase

FIGURE 8 - Representative dye concentration on the removal by MAC-NPs and MAC-NPs-ApoLac.

The decolorization effects without apolaccase and apo- AC represents a substrate for nanomagnetite immobiliza- laccase modified Fenton processes were shown in Figures 8. tion [40]. The iron has unknown toxic effects and it can be The removal of BR9 had been achieved by the Fenton profound everywhere. Therefore, many industries use metallic cess without ApoLac at a rate of 81% and modified with iron to remove waste before it is released to the environ- ApoLac at a rate of 87 %, respectively. It was observed ment [9]. The Fenton process was used for the removal of that decolorization effects were increased with increasing toxic organic substances for a long time; even the decolor- values of dye concentration. This optimization of the process ization of dye from waste water was relatively a new condition had ensured the low cost and the efficiency of re- method. The reaction rate of Fenton process was increased moval of the dyes. Therefore, the optimum time, pH, tem- with the increased temperature values and neutral pH level. perature, dosages reagents were calculated (dye and adsor- pH level of the aqueous solution was an important opera- bent). tional parameter in the decolorization process as it had ef- fected solubility of the dye, concentration of counter ions on the functional groups of adsorbent and degree of ioni- 4. DISCUSSION zation of adsorbate during reaction [41]. On the other hand, the uptake and percentage removal of dyes from the aque- The enzyme Lac is a commercial enzyme, which was ous solution were strongly affected by pH level of the so- widely used for the removal of toxic dyes in different areas lution [42-44]. Normally, Fenton reaction was resistant to including textile industry. In our study, this apoenzyme lower pH levels [5, 45-47].However, in our research, it was was attached to AC matrix for enhancing the Fenton reac- determined that the pH level was good up to 6 for best dye tion activity and it was reactivated with nanoFe2+/3+ ions. removal. The higher pH stability of Fenton reaction was Then, Fenton reaction was done by using this enzyme and raised by AC matrix with modified NPs-ApoLac. it was observed that the efficiency of the Fenton reaction was increased. The removal of BR9 has been achieved by However, the degradation of dyes to water and oxygen the Fenton process without ApoLac at a rate of 81% and has decreased the reaction rate after 50 oC. Our research modified with ApoLac at a rate of 87 %, respectively. Si- has shown that higher temperatures have not affected the varajasekar and Baskar used only AC by adsorption for re- reaction rate of MAC-NPs and ApoLac modified MAC- moving BR9 from water. The results of their experiment NPs. In the meantime, there are several reports on Fenton have indicated that high uptake of the dye was observed as treatment of wastewater in acidic medium; but the number 86.32, 79.46, 78.47, 74.87 and 71.63%, respectively (pH: of the reports focusing on the work carried out in neutral 12.0 and 40˚C) [22]. However, results of our research have medium is very few. The novelty of this work was laid in shown not only BR 9 removing from water, but also elim- the near-neutral pH of the reaction mixture [39]. This is an ination of BR9 by oxidation from water. excellent advantage for fabric, which is released into the waste water at high temperatures [48, 49]. The Fenton process has achieved higher efficiency for decolorization. Being inexpensive and widely available,

1955 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

ACKNOWLEDGEMENTS [13] Perez, M., Torrades, F., Garcia-Hortal, J.A., Domenech, X. and Peral, J. (1997) Removal of Organic contaminants in paper pulp treatment effluents by TiO2 photo catalyzed oxidation . This research was performed under the project num- Journal of Photochemistry and Photobiology A: Chemistry bered 2013/68 and supported by the Research Development 109, 281-286. Center of Ataturk University. The authors acknowledge the [14] Arslan, I. and Balcioglu, I.A. (2001) Advanced oxidation of support of Ataturk University, Turkey for this work. The raw and biotreated textile industry wastewater with O3-, SEM, EDX and FTIR study of this research was carried out H2O2/UV-C and their sequential application. J. Chem. Tech- in the Atatürk University, Faculty of Science and XRD study nol. Biotechnol. 76, 53-60. of this research was carried out in Atatürk University, the [15] So, C.M., Cheng, M.Y., Yu, J.C. and Wong, P.K. (2002) Deg- radation of azo dye procion red MX-5B photocatalytic oxida- Faculty of Engineering. So, the authors thank the Prof. Dr. tion. Chemosphere 46, 905-912. Umit DEMIR and Prof. Dr. Yasar TOTIK, respectively. [16] Lizama, C., Yeber, M.C., Freer, J., Baeza, J. and Mansilla, H.D. (2001) Reactive dyes decolouration by TiO2 photo-as- The authors have declared no conflict of interest. sisted catalysis. Water Sci. Technol. 44, 197-203. [17] Alaton, I.A., Balcioglu, I.A. and Bahnemann, D.W. (2002) Advanced oxidation of a reactive dye bath effluent: compari- -3 REFERENCES son of O , H2O2/UV-C and TiO2/UV-A processes. Water Res. 36 (5), 1143-1154. [1] Duman, O., Tunc, S. and Kancı, B. (2011) Spectrophotometric [18] Arslan, I. and Balcioglu, I. (2001) Degradation of Remazol studies on the interactions of C.I. Basic Red 9 and C.I. 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[31] Bradford, M.M. (1974) A rapid and sensitive method for the [49] Malik, P.K. and Saha, S.K. (2003) Oxidation of direct dyes quantification of microgram quantities of protein utilizing the with hydrogen peroxide using ferrous ion as catalyst. Separa- principle of protein–dye binding. Analitical Biochemistry 72, tion and Purification Technology. 31, 241-250. 248-58.

[32] Kazuko, A. (1970) Preparations and properties of apo and re- constructed Rhus-laccases. Journal of Biochemistry 68(4), 501-508. [33] Gungor, A.A. Nadaroglu, H. and Celebi, N. (2014) Fenton process for Basic Red 9 degradation: immobilized apolaccase on a nanomagnetite system. Environmental and Experimental Bi- ology 12, 121-129. [34] Niku-Paavola, M.L., Raaska, L. and Itävaara, M. (1990) De- tection of white-rot fungi by a non-toxic stain. Mycological Research 94, 27-31.

[35] He, W., Zhan, H.Y., Wang, X.W. and Wu, H. (2003) An improved spectrophotometric procedure for the laccase assay. Journal of South China University of Technology 31, 46-50. [36] Nadaroglu, H., Gungor, A.A. and Celebi, N. (2015) Removal of Basic Red 9 (BR9) in Aqueous Solution by Using Silica with Nano-Magnetite by Enzymatic with Fenton Process, In- ternational Journal of Environmental Research (In press). [37] Nadaroglu, H., Kalkan, E., Celebi, N., Celik, H. and Tasgin, E. (2015) Experimental study to remediate of Acid Fuchsin dye using laccase-modified zeolite from aqueous solutions. Polish Journal of Environmental Studies 24 (1), 115-124.

[38] Nadaroglu, H., Kalkan, E. and Celebi, N. (2014) Removal of copper from aqueous solutions by using micritic limestone Carpathian Journal of Earth and Environmental Sciences. 9(1), 69-80. [39] Duttaa, A., Banerjeea, P., Sarkara, D., Bhattacharjeeb, S. and Chakrabartia, S. (2014) Degradation of Trypan Blue in wastewater by sunlight assisted modified photo-Fenton reaction. Desalination and Water Treatment 1-9. [40] Lin, S.H., Juang, R.S. and Wang, Y.H. (2004) Adsorption of Received: October 13, 2014 acid dye from water onto pristine and acid-activated clays in Revised: December 19, 2014 fixed beds. Journal of Hazardous Material B 113, 197-202. Accepted: January 20, 2015 [41] Amuda, O.S., Giwa, A.A. and Bello, I.A. (2007) Removal of heavy metal from industrial wastewater using modified activated coconut shell carbon. Biochemical Engineering Journal 36, 174-181. CORRESPONDING AUTHOR [42] Benhammou, A., Yaacoubi, A., Nibou, L. and Tanouti, B. (2005) Adsorption of metal ions onto moroccan steven site: Assoc. Prof. Azize ALAYLI GUNGOR kinetic and isotherm studies. Journal of Colloid and Interface Ataturk University Science 282, 320-326. Erzurum Vocational School [43] Ghazy, S.E. and Ragab, A.H. (2007) Removal of copper from Department of Chemical Technology water samples by sorption onto powdered limestone. Indian 25240 Erzurum Journal of Chemical Technology 14, 507-514. TURKEY [44] Onundi, Y.B., Mamun, A.A., Al Khatib, M.F. and Ahmed Phonel: 0-90-442-2312668 Y.M. (2010) Adsorption of copper, nickel and lead ions from synthetic semiconductor industrial wastewater by palm shell Fax: 0-90-442-2310982 activated carbon. International Journal of Environmental Sci- E-mail: [email protected] ence and Technology 751, 751-758. [45] Lin, S.H. and Lo, C.C. (1997) Fenton process for treatment of desizing wastewater. Water Research 31, 2050-2056. Assoc. Prof. Hayrunnisa NADAROGLU [46] Meric, S., Kaptan, D. and Olmez, T. (2004) Color and cod re- Ataturk University moval from wastewater containing reactive black 5 using Fen- Erzurum Vocational School ton’s oxidation process. Chemosphere 54, 435-441. Department of Food Technology [47] Modirshahla, N., Behnajady, M.A. and Ghanbary, F. (2007) 25240 Erzurum Decolorization and mineralization of C.I. acid yellow 23 by TURKEY Fenton and photo Fenton processes. Dyes and pigments 73, 305-310. Phone: 0-90-442-2311818 Fax: 0-90-442-2310982 [48] Meric, S. Selçuk, H. and Belgiorno, V. (2005) Acute toxicity removal in textile finishing wastewater by Fenton’s oxidation, E-mail:[email protected] ozone and coagulation–flocculation processes. Water Re- search. 39, 1147-1153. FEB/ Vol 24/ No 5b/ 2015 – pages 1947 - 1957

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ANTIOXIDANT DEFENCE SYSTEMS, LIPID PEROXIDATION AND ACETYLCHOLINESTERASE ACTIVITY OF OREOCHROMIS NILOTICUS EXPOSED TO MERCURY AND MERCURY + SELENIUM

Gülbin Firidin1,*, Ferit Kargın2, Özge Fırat3, Hikmet Y. Çoğun4, Özgür Fırat5, Beran Firidin6, and Tüzin A. Yüzereroğlu7

1 Life Science Research and Application Center, Gazi University, 06830 Ankara, Turkey 2 Department of Biology, Faculty of Science and Letters, Cukurova University, 01330 Adana, Turkey 3 Vocational School of Kahta, Adiyaman University, 02400 Adiyaman, Turkey 4 Faculty of Veterinary of Ceyhan, Cukurova University, 01920 Adana, Turkey 5 Department of Biology, Faculty of Science and Letters, Adiyaman University, 02040 Adiyaman, Turkey 6 Faculty of Education, Gazi University, 06500 Ankara, Turkey 7 Vocational School of İmamoğlu, Cukurova University, 01700 Adana, Turkey

ABSTRACT comes from geological emissions and anthropogenic activities, such as fuel burning, mining, smelting operations, and The objective of the present study was to evaluate the the chlor-alkali industry [1]. Hg often poses a high ecotoxi- oxidative stress potential of mercury (Hg) exposure with the cological risk for aquatic organisms due to its high toxicity, modulatory effect of selenium (Se) in the brain and kid- bioaccumulation, and other deleterious effects on biota, in- ney tissues of Oreochromis niloticus. The fish were excluding genetic effects and mutagenicity [2]. Selenium (Se) posed to 0.01 and 0.1 mg/L Hg, 0.01 mg/L Hg+0.1 mg/L Se, is an essential trace element, and potentially protective and 0.1 mg/L Hg+1.0 mg/L Se, for 7 and 14 days. The ac- against the toxic effects of Hg [3]. It is also a structural tivities of total glutathione (tGSH), malondialdehyde component of several enzymes involved in antioxidant de- (MDA) and catalase (CAT), glutathione S-transferase fence including glutathione peroxidase (GSH-Px) and thi- (GST), and acetylcholinesterase (AChE) were measured oredoxine [4]. Some investigations showed that Se may of- from samples of the tissues. The results indicated that all fer protective effects against Hg-induced toxicity through concentrations of Hg caused a time-dependent increase in several mechanisms, such as sequestration of Hg, antioxida- CAT, GST activity, whereas AChE activity and tGSH con- tive effect, GSH synthesis, increased GSH peroxidase (GPx) tent were decreased in brain and kidney tissues. MDA lev- activity, and high selenoprotein levels [5]. els in tissues of fish exposed to Hg+Se mixtures were lower Metal toxicity causes an increase in ROS (reactive oxy- compared to those exposed to Hg only. Selenium applica- gen species), such as hydrogen peroxide, hydroxyl and su- tion in combination with mercury provided an alleviation peroxide radicals leading to oxidative stress in fish [6]. Oxi- of the toxic effects of mercury in the investigated tissues dative stress occurs when the balance between oxidants and for biochemical parameters. antioxidants is broken down [7], producing lipid peroxidation, DNA damage, and protein oxidation [8]. The most widely used assay for lipid peroxidation is MDA formation KEYWORDS: Oreochromis niloticus, mercury, selenium, antioxi- as a secondary lipid peroxidation product, and it is a good dant parameters, oxidative stress indicator for the extent of lipid peroxidation, induced by heavy metal pollution in aquatic organisms [9].

1. INTRODUCTION Antioxidant systems, such as GST and GSH, are of great importance in an oxidative stress system to cope with The aquatic environment is the last destination of pollu- ROS formation leading to several defects [10]. GST repre- tants distributed around the world. Hg, a non-essential sents a group of multifunctional enzymes with a predomi- metal, is a ubiquitous contaminant in the environment and nant cytosolic defense systems responsible for the protection of cellular components against various toxic effects and oxidative stress [11], and catalyzing the nucleophilic attachment * Corresponding author of glutathione (GSH) to molecules presenting an electro-

1958 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

philic carbon, nitrogen, or sulphur atom [12]. GSH plays a dissected separately on an ice-cold plate. Tissues were then central role in protecting eukaryotic cells from oxidative washed in ice-cold physiological saline solution, blotted stress as an antioxidant [13]. CAT (EC 1.11.1.16) decom- dry, weighed, and stored frozen at -80 °C until analyses. poses H2O2 to water and molecular oxygen, and plays a cru- All chemicals and reagents were purchased from Merck cial role in reducing reactive oxygen-free radicals [14]. and Sigma-Aldrich Chemical Corporation. AChE (EC 3.1.1.7) plays a significant role in neurotransmis- Tissues were homogenized in ice-cold buffer contain- sion at cholinergic synapses by the rapid hydrolysis of neu- ing 0.05 M phosphate buffer, 0.25 M sucrose (pH 7.4), with rotransmitter acetylcholine to choline and acetate [15]. It a ratio of 1/10 at 9500 rpm for 2-3 min. Homogenates were was investigated that this enzyme is sensitive to environ- centrifuged at 9500 ×g for 30 min. All processes were car- mental contaminants, such as metals, and could provide ried out at +4 °C. The supernatants were collected for de- useful information about the toxic impact of Hg [16]. termination of CAT, GST, and AChE activities as well as Biochemical constituents, like antioxidant systems, li- total protein, GSH, and MDA levels. pid peroxidation and AChE are the potential biomarkers of pollutants exposure in different organisms [17, 18]. The an- 2.2 Biochemical tests tioxidants investigated in the present study included CAT, GSH contents in the supernatants were determined us- GST and GSH. The oxidative damage was evaluated by ing a recycling reaction of the GSH with 5,5’-dithiobis-(2- determining lipid peroxidation, and was measured by nitrobenzoic acid) (DTNB) in the presence of excess GR malondialdehyde (MDA) content. [19]. The incubation medium contained 143 mM sodium– The aim of the present investigation was to determine potassium buffer (Na-K Fosfat (containing 6.3 mM EDTA, the protective effect of Se supplementation against Hg tox- pH 7.5), 0.3 mM NADPH, 6 mM DTNB, and 50 units of GR. icity in the oxidative stress capacity of kidney and brain DTNB formation was monitored continuously at 412 nm for tissues of O. niloticus. The changes in activities of GST, 3 min. CAT, and AChE with GSH and MDA levels were deter- CAT activity was determined spectrophotometrically mined. Hg-induced changes in the antioxidant and AChE by measuring the decomposition of H2O2 (10 mM solution) enzymes, as well as lipid peroxidation content, were eval- at 230 nm using the method of [20]. uated in the kidney and brain tissues. GST was measured according to Habig et al. [21] using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The 2. MATERIALS AND METHODS reaction mixture consisted of 100 mM tris buffer (pH 7.4), 1 mM GSH and 1 mM CDNB (1-chloro-2,4-dinitroben- 2.1 Experimental protocol zene). Enzyme activity was determined by monitoring the changes in the absorbance at 340 nm, which reflects the Freshwater fish O. niloticus (56.5±2.1 g and rate of CDNB conjugated with the GSH at 25 oC for 2 min. 15.9±1.8 cm) were obtained from Çukurova University Fish Culture Farm, and transferred to the laboratory where Lipid peroxidation was estimated by measuring MDA they were acclimatized for 2 months in 120-L glass aquar- contents with the method of Ohkawa et al. [22]. MDA lev- iums containing dechlorinated and gently aerated tap water els were determined after incubation at 95 °C and pH 3.4, at 25±1 °C, the temperature of the experimental conditions. with thiobarbituric acid as an indicator of lipid peroxida- The properties of water were as follows: dissolved oxygen tion. MDA concentrations were analyzed by measuring the 7.25±0.72 mg/L, pH 8.4±0.8, temperature 23.1±0.5 oC, al- pink colour produced at 532 nm The reaction medium contained 8.1% of SDS, 20% of acetic acid, pH 3.4, 0.8% of kalinity 184.5 ± 4.4 mg/L as CaCO3, and total hardness TBA, and a mixture of n-butanol/pyridine (14:1). 278.2 ± 8.7 mg/L as CaCO3 at acceptable levels. The fish were maintained under a 12-h light/12-h dark illumination Acetylcholinesterase activity was determined by the period. The control and experimental fish were fed once method of Ellman et al. [23] at 30 °C. Enzymatic activity daily with commercial food pellets (Pinar Feed, Turkey), was spectrophotometrically determined by measuring the approx. 3% of their body weight. increase in absorbance of the sample at 412 nm. Acetylthi- After the period of acclimatization, experimental fish ocholine iodide and dithiobisnitrobenzoic acid were used stocks have been randomly divided into 3 groups each con- as substrates. taining 18 fish. The first group was held in tap water as con- The protein concentrations were measured by the trol, and the other groups were exposed to Hg and Hg+Se method of Lowry et al. [24]. Protein content was expressed mixtures for 7 and 14 days. Three replicate groups of fish in mg/g wet weight of the tissue. Samples were treated with were used for each exposure. All the experimental solutions Folin-phenol reagent and the absorbance was measured at were completely replaced every other day by newly prepared 750 nm. Bovine serum albumin was used to prepare the stock solutions to reduce the ammonia content in water. standard graphics. There was no fish mortality during the experiments. Prior to analysis, 6 fish were taken out from the 2.3 Data analysis aquaria, and fish were killed by transaction of the spinal All parameters were expressed as means ± standard er- cord (cutting nerves); then, kidney and brain tissues were ror. The data were statistically analyzed by a series of Stu-

1959 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

dent Newman Keul’s tests and SPSS statistical package cant reduction in GSH level was recorded at 0.1 mg/L programs to identify any differences among tissues and ex- Hg, 0.1 mg/L Hg+1.0 mg/L Se, and 0.01 mg/L Hg, 0.01 mg/L perimental period. Data shown with different letters are Hg+0.1 mg/L Se for the groups in brain (25, 21, 45, 13%) significant at the P < 0.05 level. and kidney (57, 23, 59, 32%), compared to control, respectively, for a period of 14 days.

3. RESULTS CAT and GST activities in brain and kidney tissues increased, in response to Hg exposures when compared to In the present investigation, there were significant control during 7 and 14 days. At the end of the exposure (p<0.05) alterations in the GSH and MDA levels, CAT, period, elevation in these enzyme activities of fish exposed GST, and AChE activities, in brain and kidney of O. nilot- to Hg was higher when compared with Hg+Se-treated icus exposed to Hg concentrations, observed for different groups. There was no statistical significant difference be- concentrations and exposure periods. tween the exposure time in brain and kidney CAT activity, except for 0.1 mg/L Hg-exposed fish. CAT activity at high The exposures to Hg and Hg+Se caused a decrease in Hg and Hg+Se concentrations showed 8-4 (brain) and 2-1.5 GSH levels in the searched tissues, in relation to the control (kidney)-fold increase after14 days, compared with control levels. This decline was higher for exposure to Hg alone groups (Figs. 1B, 2B). Kidney GST activity, at low Hg con- than in the presence of Se with Hg, at the end of the period. centration, indicated a significant increase after14 days, ac- Kidney GSH showed a significant reduction at low Hg con- cording to that at 7 days (Fig. 2C). GST activity was in- centration, with respect to the high Hg concentration, for a creased by 115 and 65% in brain as well as 65 and 39% in period of 14 days (Fig. 2A). Considerable differences did kidney, at high exposure concentrations and exposure du- not occur in GSH levels of tissues between the exposure ration when compared to control values (Figs. 1C, 2C). times (P>0.05). As shown in Figs. 1A and 2A, a signifi-

A 1,8 a a 1,6 ab b 1,4 ab b b

1,2 0.0 b 1 c level b 0.01 mg/L Hg 0,8

GSH 0.01+0.1 mg/L Hg+Se 0,6 0,4 0.1 Hg mg/L Hg 0,2 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

14 B c 12

10 0.0 8 c 0.01 mg/L Hg activity 6 b b* b 0.01+0.1 mg/L Hg+Se CAT 4 b b a 0.1 Hg mg/L Hg 2 a a 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

1960 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

80 C c 70 c 60 b b b

b b 50 ab 0.0 40 0.01 mg/L Hg activity a a

30 0.01+0.1 mg/L Hg+Se GST 20 0.1 Hg mg/L Hg 10 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

100 D d * b 90 b b 80 c c c c 70 a a 60 0.0 level 50 0.01 mg/L Hg 40

MDA 0.01+0.1 mg/L Hg+Se 30 0.1 Hg mg/L Hg 20 10 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

FIGURE 1 - GSH level (µM/mg protein, A), CAT activity (U mg-1 ofprotein, B), GST activity (U mg-1 of protein, C), LPO level (nmol MDA mg-1 of protein, D) in brain of O. niloticus exposed to metals for 7 and 14 days (data are expressed as means ± standard error (N=6); different letters indicate significant differences among groups at the same time (P < 0.05); * shows significant differences between times for the same exposure group (P<0.05).

1,2 A a a 1 ab

0,8 0.0 ab b b level 0,6 b b 0.01 mg/L Hg b GSH b 0.01+0.1 mg/L Hg+Se 0,4 0.1 Hg mg/L Hg 0,2 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

1961 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

40 B c* 35 b b b 30 bc

25 cd a 0.0 ad 20 0.01 mg/L Hg activity a a 15 0.01+0.1 mg/L Hg+Se CAT 10 0.1 Hg mg/L Hg 5 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

C 160 b* 140 c* 120 d

c bc a a 100 a ab a 0.0 80 0.01 mg/L Hg activity

60 0.01+0.1 mg/L Hg+Se GST 40 0.1 Hg mg/L Hg 20 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

80 D d * 70 d b * 60 c b c * b 50 0.0 level 40 c 0.01 mg/L Hg

MDA 30 a a 0.01+0.1 mg/L Hg+Se 20 0.1 Hg mg/L Hg 10 0.1+1.0 mg/L Hg+Se 0 714 Exposure time (in days)

FIGURE 2 - GSH level (µM/mg protein, A), CAT activity (U/mg protein, B), GST activity (U/mg protein, C), LPO level (nmol MDA mg-1 of protein, D) in kidney of O. niloticus exposed to metals for 7 and 14 days (data are expressed as means ± standard error (N=6); different letters indicate significant differences among groups at the same time (P < 0.05), and * shows significant differences between times for the same exposure group (P<0.05).

1962 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

TABLE 1 - Brain AChE activity (U/mg protein) in O. niloticus exposed to metals for 7 and 14 days.

Metal concentrations (mg/L) 7 days 14 days

0.0 0.576±0.022ax 0.521±0.059ax 0.01 Hg 0.344±0.027bx 0.277±0.040bx 0.01 Hg+0.1 Se 0.423±0.011cx 0.347±0.012by

0.0 0.576±0.022ax 0.521±0.059ax 0.1 Hg 0.332±0.034bx 0.290±0.027by 0.1 Hg+1.0 Se 0.430±0.026cx 0.401±0.176aby

Values are expressed as means ± standard error (N=5). Letters a, b and c indicate differences among concentrations, and letters x and y indicate statistical differences among tissues at the Pb<0.05 level.

TABLE 2 - Kidney AChE activity (U/mg protein) in O. niloticus exposed to metals for 7 and 14 days.

Metal concentrations (mg/L) 7 days 14 days

0.0 0.055±0.003ax 0.057±0.002ax 0.01 Hg 0.040±0.001bx 0.035±0.003bx 0.01 Hg+0.1 Se 0.042±0.003bx 0.048±0.003cx

0.0 0.055±0.003ax 0.057±0.002ax 0.1 Hg 0.034±0.006bx 0.029±0.005bx 0.1 Hg+1.0 Se 0.042±0.005abx 0.046±0.004ax Values are expressed as means ± sStandard error (N=5). Letters a, b and c indicate differences among concentrations, and letters x and y indicate statistical differences among tissues at the P<0.05 level.

MDA contents in all tissues of fish increased signifi- 4. DISCUSSION cantly with increasing concentrations of Hg in the exposure medium and with long treatment durations. MDA levels in At all concentrations, the tested tissues of O. niloticus tissues represented a significant rise in all treatments when showed the effects of the treatments above in enzym activ- compared with control values, and this increase was much ities as well as MDA and GSH levels. Se is remarkably higher after14 than 7 days. Particularly higher MDA levels protective against toxic effects of Hg in fish species [3-25]. were observed from control, at each time of exposure dur- This protective effect seems to be a result of the formation ing 4 and 21 days, in brain and kidney, respectively. MDA of Se-Hg complexes [26]. levels exhibited an increase of 32, 18, 37, and 12% in brain as well as 123, 67, 194, and 144% in kidney, in relation to GSH is important in the antioxidant defense, and its de- the reference Se concentrations of 0.1 mg/L Hg, 0.1 mg/L pletion may reduce the cellular ability to scavenge free radi- Hg+1.0 mg/L Se, and 0.01 mg/L Hg, 0.01 mg/L Hg+0.1 cals [27]. In this investigation, GSH content in the tissues mg/L Se, respectively, for a period of 7 days (Figs. 1D, decreased with increasing Hg, Hg+Se concentrations, and 2D). exposure periods. Decreasing GSH may be due to direct binding of the metal to GSH through its SH group, or en- AChE activity was inhibited in brain and kidney under hanced oxidation of this thiol [28]. Exposure to Hg caused a the effects of metals. The important inhibition of AChE significant decrease in GSH levels but increase in CAT and was recorded to be 46, 33, 44, and 23% in brain as well as GST activities in tissues of Brycon amazonicus [29]. 39, 16, 49, and 19% in kidney, for 0.1 mg/L Hg, 0.1 mg/L Hg+1.0 mg/L Se, and 0.01 mg/L Hg, 0.01 mg/L Hg+0.1 Se GST catalyzes the synthetic conjugation reactions of concentrations, as compared to control groups (Tables 1 xenobiotics to GSH, and thus, this function protects fish and 2). AChE activity in brain at 0.1 and 0.1 mg/L Hg+1.0 tissues from oxidative stress [12]. CAT is responsible for mg/L Se concentrations were decreased significantly af- the conversion of H2O2 to water [14]. After Hg and Hg+Se ter14 days, with respect to those at 7 days, showing no sta- treatments, in this study, GST and CAT activities in brain tistically significant differences between the exposure pe- and kidney were promoted significantly, depending on Hg riods in kidneys. AChE activity observed in kidneys after concentrations and exposure periods. The increase in CAT 7 days and in brain after14 days was close to that of the activity was probably due to continuous production of O2•− control group at 0.1 mg/L Hg+1.0 mg/L Se concentration. into H2O2, as a result of oxidative stress.

1963 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

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[19] Anderson, M.E. (1985) Determination of glutathione and glu- [36] Sen, S., Mondal, S., Adhikari, J., Sarkar, D., Bose, S., Mukho- tathione disulfide in biological samples. Methods in Enzymol- padhyay, B. and Bhattacharya, S. (1995) Inhibition of fish ogy, 113, 548-555. brain acetylcholinesterase by cadmium and mercury: Interac- tion with selenium. In, Quinn D,M., Balasubramanian, A.S., [20] Beutler, E. (1984) Red cell metabolism: A manual of biochem- Doctor, B.P. and Taylor, P. (Ed): Enzymes of the Cholinester- ical methods. 2nd ed. pp 160, Grune and Stratton, New York, ase Family. 369–374, Plenum Press, New York. USA. [37] Richetti, S.K., Rosemberg, D.B., Ventura-Lima, J., Monserrat, [21] Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Glutathi- J.M., Bogo, M.R. and Bonan, C.D. (2011) Acetylcholinester- one S- transferases the first enzymatic step in mercapturic acid formation. J Biol Chem, 249, 7130-7139. ase activity and antioxidant capacity of zebrafish brain is altered by heavy metal exposure. Neuro Toxicol, 32, 116–122. [22] Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 95, 351–358. [23] Ellman, G.L., Courtney, K.D., Andres, V. and Featherstone, R.M. (1961) A new and rapid colorimetric determination of acetycholinesterase activity. Biochem Pharmacol, 7, 88-95. [24] Lowry, O.H., Rosenbrough, N.J., Farr, A. and Randall, R.J. (1951) Protein measurement with the folin-phenol reagent. J Biochem, 193, 265-277. [25] Ates, B., Orun, I., Talas, Z.S., Durmaz, G. and Yilmaz, I. (2008) Effects of sodium selenite on some biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) exposed to Pb2+ and Cu2+. Fish Phys- iol Biochem, 34, 53–59. [26] Raymond, L.J. and Ralston, N. (2004) Mercury selenium interactions and health implications. Seychelles Medical and Dental J, 17 (1), 72-77. [27] Sun, X., Shih, A.Y., Johannssen, H.C., Erb, H., Li, P. and Mur- phy, T.H. (2006) Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface. J Biol Chem, 281, 17420–17431.

[28] Elia, A.C., Galarini, R., Taticchi, M.I., Dorr, A.J.M. and Man- tilacci, L. (2003) Antioxidant responses and bioaccumulation in Ictalurus melas under mercury exposure. Ecotoxicol Envi- ron, 55, 162-167. [29] Monteiro, D.A., Rantin, F.T. and Kalinin, A.L. (2010) Inor- ganic mercury exposure: toxicological effects, oxidative stres biomarkers and bioaccumulation in the tropical freshwater fish matrinxa, Brycon amazonicus (Spix and Agassiz, 1829). Eco- toxicol, 19, 105–123. [30] Dabas, A., Nagpure, N.S., Kumar, R., Kushwaha, B., Kumar, P. and Lakra, W.S. (2012) Assessment of tissue-specific effect of cadmium on antioxidant defense system and lipid peroxidation in freshwater murrel, Channa punctatus. Fish Physiol Bi- ochem, 38, 469–482. [31] Valavanidis, A., Vlahogianni, T., Dassenakis, M. and Scoullos, M. (2006) Molecular biomarkers of oxidative stress Received: October 13, 2014 in aquatic organisms in relation to toxic environmental pollu- Accepted: November 18, 2014 tants. Ecotoxicol Environ Safety, 64, 178-189. [32] Berntssen, M.H.G., Aatland, A. and Handy, R.D. (2003) Chronic dietary mercury exposure causes oxidative stress, CORRESPONDING AUTHOR brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr. Aquat Toxicol, 65, 55–72. Gülbin Firidin [33] Milaeva, E.R. (2006) The role of radical reactions in organo- Life Science Research Center mercurials impact on lipid peroxidation. J Inorganic Biochem; Gazi University 100 (5), 905-915. 06830 Ankara [34] Orun, I., Talas, Z.S., Ozdemir. I,, Alkan, A. and Erdogan, K. TURKEY (2008) Antioxidative role of selenium on sometissues of (Cd2+), (Cr3+)-induced rainbow trout. Ecotoxicol Environ Saf, 71, 71–75. Phone: +90 3124842157 [35] Frasco, M.F., Colletier, J.P., Weik, M., Carvalho, F,, Guilher- Fax: +90 312 484 62 71 mino, L., Stojan, J. and Fournier, D. (2007) Mechanisms of E-mail: [email protected] cholinesterase inhibition by inorganic mercury. FEBS J, 274, 1849-1861. FEB/ Vol 24/ No 5b/ 2015 – pages 1958 - 1965

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ASSESSMENT OF HEAVY METAL POLLUTION IN SURFACE SOILS FROM URBAN PARKS AND SQUARES: A CASE STUDY FROM CHINA

Qiang Liu1, Jingshuang Liu2, Mingying Zou3 and Yang Wang1,*

1Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 4888 Shengbei Road, Changchun, 130102, China. 2CNPC Northeast Refining & Petrochemical Engineering Co.Ltd JiLin Design Institute, Crossing of Songjiang North Road and Tongtan Road, Jilin, 132002, China.

ABSTRACT ily be transferred to humans [2-4]. Furthermore, even though urban soils are rarely used for food production, they receive Assessing the concentration of potentially harmful higher than normal loads of contaminants from traffic and, heavy metals in the case of soils in urban parks and squares in heavily industrialized cities, industrial activity [5]. that are used for leisure, sporting or recreational activities Heavy metal pollution has become increasingly the is imperative in order to evaluate the potential risks to res- world wide environmental problem [6-9]. Urban soils idents and tourists. The concentrations of heavy metals (Pb, worldwide have elevated concentrations of (potentially) Cr, Cu, Ni, Zn, and Cd) in urban soils of 7 parks and 5 toxic metals [10, 11]. Because of the proximity to large hu- squares of Changchun, Northeast China, were measured man populations, urban soils contaminated with heavy based on different functional areas. The median concentra- metals can pose significant human health risks due to oral tions of Pb, Cr, Cu, Ni, Zn and Cd in the investigated urban soil ingestion [12], inhalation of volatiles and fugitive par- soils are 52.26, 63.00, 31.40, 16.02, 118.05 and 0.084 mg kg- ticulates [13, 14], and dermal contact [15], especially in the 1, respectively. The values of Pb, Cu and Zn are significantly public parks and squares [16]. Parks and squares on urban higher than their background values of Changchun topsoil soils are part of developed land usually found within cities that average 20.46, 17.96 and 59.47 mg kg-1. The integrated that provide opportunities for relaxation, sports and recre- pollution index (IPI) of these six metals ranged from 1.17 to ational activities [17]. But the soil of a park or a square acts 3.65, with the highest IPI in the recreation area of the Chil- as a sink for many pollutants due to their large exposed ar- dren's Park. The results of multivariate statistical analysis eas, and heavy metal pollutants in the soils can have a se- (principal components analysis and hierarchical cluster anal- rious impact on human health. Children are the most sensi- ysis) indicated that Cr and Ni concentrations were mainly of tive target group to exposure [4, 18]. Due to their higher natural origin, while Pb, Cu, Zn and Cd were derived from sensitivity, as well as characteristic behaviors (outdoor ac- anthropogenic activities. The findings presented here show tivities, hand–mouth activity, deficient hygienic habits, that the difference of functional areas in parks and squares etc.), children are at greater risk of exposure to the toxic is an important factor in determining the extent of heavy elements from contaminated soils than adults [19]. Bearing metal, and the level of pollution in some soils of recreation in mind that children are more susceptible to metals, since areas in parks and outside areas in squares was very high. they are in the early stages of development, determining

the heavy metal contents in playground soils is of particular importance. KEYWORDS: Heavy metal; Pollution index; Source identification; Urban park and square Heavy metals (HMs) derived from vehicular exhausts,

incinerators, industrial wastes, atmospheric deposition, and other activities have continuously contributed to the burden 1. INTRODUCTION of contaminants in the urban soils [11, 20-22], and, indeed, increasing research has focused on heavy metals in urban The urban environment quality is of vital importance soils [2, 23-25]. The metal contamination in urban areas is as most people now live in cities. Human health in cities is specific and varies with local conditions [25], and that in- strongly dependent on the status of urban soils [1]. In con- creased levels of heavy metals in urban soil was related to trast to agricultural soils, urban soils might have a direct in- the intensity of human activities and traffic volume [26]. fluence on public health since pollutants in the soils can eas- The investigation of soil heavy metal concentrations in urban parks in Beijing, China, indicated that the location and * Corresponding author the age of the park are important factors in determining the

1966 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

extent of heavy metal, particularly Cu and Pb, pollution [2]. 2. MATERIALS AND METHODS However, differences among functional areas of parks or squares including intensity of human activities and recrea- 2.1. Study Site tion facilities could have a large impact on the findings of The study site is located in an urban area of Changchun individual studies. Moreover, little information is available (43°17′~44°5′N, 125°3′~125°34′E), which is the capital of on heavy metal concentrations in soils of urban parks and Jilin Province and an important social-economic center of squares located in old industrial bases. northeastern China located in the hinterland of the North-

2 As the first heavy industrial base since the establish- east Plain. The city occupies an area of 20604 km , with 2 ment of the People's Republic of China, Changchun has 4906 km classified as urban areas. The climate is domi- made great contributions to the economic development of nated by northerly continental monsoons, characterized by China. However, the rapid economic development in long and cold winter and generally short and warm sum- Changchun over the last two decades has led to significant mer. The average annual temperature is 4.8°C with the highest release of waste into the urban environment and placed temperature in summer is 39.5℃, the lowest temperature in great pressure on the local environment, including heavy winter is -39.8℃, and the average annual rainfall is 569.6 mm. metals contamination of urban soils, such as Cd, Cu, Pb The geology of this region consists of Quaternary alluvial-di- and Zn [27, 28]. However, the heavy metal concentrations luvial deposits and the natural soil types in the study area are and subsequent soil pollution levels in Changchun’s urban mainly black soil, dark brown soil and meadow soil accord- parks and squares remain unknown. The purposes of this ing to the Classification and codes for Chinese soil (National study were (1) to determine the concentration of heavy standard, GB/T 17296–2009) recommended by General Ad- metals in park and square soils of Changchun; (2) to iden- ministration of Quality Supervision, Inspection and Quaran- tify the possible sources of heavy metals; and (3) to assess tine of the P.R.C. (AQSIQ, 2009). The 12 locations (7 public the heavy metal contamination in urban soils. parks and 5 squares) investigated in this study are major sites in Changchun that frequently welcome a large number of visitors for free and provide opportunities for a wide range of leisure, sporting and recreational activities. Some of the

TABLE 1 - Brief description of the parks and squares investigated in Changchun

Name(Abbr.name) Sites History Functional area Area(ha) (years) SLP1 Recreation area Victory Park(SLP) SLP2 98 Leisure area 23 SLP3 Fitness area NHP1 Leisure area (bathing place) NHP2 Leisure area South Lake Park(NHP) NHP3 80 Leisure area 238 NHP4 Recreation area NHP5 Leisure area (main entrance) Peony Park(MDP) MDP1 80 Leisure area 6.56 MDP2 Leisure area Children's Park(ETP) ETP1 80 Leisure area 18.07 ETP2 Recreation area CYP1 Fitness area Chaoyang Park(CYP) CYP2 79 Recreation area 57 CYP3 Leisure area Labor Park(LDP) LDP1 77 Leisure area 12.5 LDP2 Leisure area CCP1 Leisure area Changchun Park(CCP) CCP2 13 Fitness area 65 CCP3 Leisure area Station Square(ZQS) ZQS1 106 Leisure area (outside) 1 ZQS2 Leisure area (inside) People's Square(RMS) RMS1 80 Leisure area (outside) 7 RMS2 Leisure area (inside) Xinmin Square(XMS) XMS1 80 Leisure area (inside) 2.7 XMS2 Leisure area (outside) South Lake Square(NHS) NHS1 80 Leisure area (inside) 2.7 NHS2 Leisure area (outside) WHS1 Leisure area (outside) Cultural Square(WHS) WHS2 17 Leisure area (outside) 20.5 WHS3 Leisure area (inside) WHS4 Leisure area (outside)

1967 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

FIGURE 1 - Sampling sites of urban park and square soils in Changchun city, Northeast China.

1968 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

parks and squares, such as the South Lake Park, South Lake 2.3. Statistical analysis Square, Xinmin Square, People's Square, Peony Park, Vic- Statistical analysis including principal components tory Park, Children's Park and Station Square, were built analysis (PCA) and hierarchical cluster analysis (HCA) more than 80 years ago (Table 1). were performed using SPSS 16.0 for Windows. PCA with varimax rotation was performed on log-transformed data. 2.2. Sampling and analysis HCA was undertaken according to the Ward-algorithmic Soil samples were taken from 12 locations of public method to classify heavy metals from different sources on parks and squares in the urban parts of Changchun in June the basis of similarities in their chemical properties, and the 2013. A total of 32 topsoil samples (depths 0-5 cm) were data normalization method was Range 0 to 1. Results are collected from different main functional sections in 7 parks shown in a dendrogram where steps in the hierarchical and 5 squares (Fig. 1). To avoid effects due to the differen- clustering solution and values of the distances between tial uptake of metals by vegetation, sampling was carried clusters (squared Euclidean distance) are represented. To out where plants with superficial roots are not present. At assess the soil environment quality, a pollution index (PI) each sampling site, 6 sub-samples, with a 1 m×1 m surface, of each metal and an integrated pollution index (IPI) of the were collected using a stainless steel spade and then mixed six metals were attributed to each sampling site. The PI was to obtain a bulk sample. Each sample weighed approxi- defined as the ratio of the HM concentrations in this study mately 1 kg. All of the sample sites were recorded using a to the natural background values of soils in Changchun hand-held global positioning system (GPS). [30]. The PI of each metal was calculated and classified as After removal of grass and pebbles, the samples were either low (PI ≤ 1), middle (1 < PI ≤ 3), or high (PI > 3). air-dried and crushed to pass through a 2 mm polyethylene The IPI of the six metals for each park and square was de- sieve and mixed again. A portion of each sample was then fined as the mean value of the metal’s PI [2], and was then further ground and homogenized with an agate mortar to classified as low (IPI ≤ 1), middle (1 < IPI ≤ 2), or high (IPI pass through a 0.15 mm polyethylene sieve. All handling > 2). Spatial analysis by GIS was also used to graphically procedures were carried out without contacting any metals and digitally present the distribution of the studied trace to prevent potential cross-contamination of the samples. metals. The heavy metal distribution maps were created using Arc Map 10 software. The soil samples were digested in triplicate with HNO3 and

H2O2 using the Method 3050B suggested by USEPA [29]. In brief, 1g of sample was accurately weighed into 250 ml flask for digestion on hot plate with 10 ml of 1:1 HNO3 3. RESULTS without boiling (at 95 ). It was then refluxed with re- 3.1. Heavy metal contamination peated additions of conc. HNO3 until no brown fumes were given off by the sample. After cooling, 2 ml of deionized Descriptive statistical results are reported in Table 2, water and 3 ml of 30 percent H2O2 (maximum 10 ml) was including the means, medians, ranges and standard devia- added slowly without allowing any losses. The mixture was tions (SD) of heavy metals in urban topsoils from Chang- refluxed with 10 ml of conc. HCl at 95 for 15 min. After chun city. cooling, digestate was filtered, transferred into a 100 ml vol- We found that there was a distinct change in the con- umetric flask, and diluted to the mark with deionized water. tents of heavy metals among the sampling soils, Ni levels Solutions were stored in high density polyethylene vials at were comparable with the BC, while Pb, Cr, Cu, Zn and Cd 4 until instrumental analysis. Concentrations of Pb, Cr, exhibited higher concentrations than the BC, particularly Cu, Ni and Zn in the digestion solution were determined by Pb, Cu and Zn, which had about 1.8-, 0.8- and 1.2-fold flame (air-acetylene) atomic absorption spectroscopy (FAAS) higher levels, respectively. The highest concentrations of Pb, with the exception of the samples for Cd, which were deter- Cr, Cu and Zn, 127.24, 118.33, 78.53 and 235.65 mg/kg, mined by graphite furnace atomic absorption spectroscopy found in ETP2 of Children's Park, were 5.2-, 1.4-, 3.4- and (GFAAS), the equipment was made by Shimadzu (Kyoto, 3.0-fold higher than the BC, respectively, and the highest Japan). concentration of Cd, 0.359 mg/kg, found in SLP3 of Vic- The standard reference material (GBW 07405 (GSS- tory Park, was 3.2-fold higher than the BC. Based on the 5)) obtained from the Center of National Standard Refer- mean concentration, the components in soils were arranged ence Material of China was used in the digestion and de- in the following decreasing order: Zn > Pb > Cr > Cu > Ni termination as part of the quality assurance (QA) protocol. > Cd, and the contents of heavy metals in parks were higher Reagent blanks and analytical duplicates were included to than squares. ensure the accuracy and precision of analysis. The recov- eries for the six observed metals were between 90% and 3.2. Hierarchical cluster analysis (HCA) 110%. In addition, to avoid the contamination as far as pos- In order to discriminate distinct groups of heavy metals sible, all chemicals used for metal measurements were as tracers of natural or anthropic source, an explorative guaranteed reagents, and all of the containers used during HCA was performed on the available dataset (Fig. 2). The the analysis procedure were washed with detergent, acid- distance cluster represented the degree of association be- soaked and then rinsed thoroughly with deionized water. tween elements. The lower the value on the distance clus-

1969 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

TABLE 2 - Heavy metal concentrations in the soils of urban parks and squares in Changchun (mg kg-1)

Metal Minimum Maximum Mean SD Median BC Parks Pb 37.33 127.24 61.43 23.53 54.36 Cr 51.10 118.33 65.85 13.36 63.35 Cu 20.13 78.53 32.49 13.19 30.34 Ni 13.13 27.21 16.90 2.87 16.60 Zn 74.33 235.65 130.74 41.43 126.64 Cd 0.031 0.359 0.128 0.108 0.084 Squares Pb 34.37 68.78 52.45 10.63 51.10 Cr 46.69 67.99 61.43 5.45 62.93 Cu 21.58 49.03 33.21 8.62 32.79 Ni 10.97 20.52 14.61 3.46 13.11 Zn 89.04 200.65 125.68 30.75 118.05 Cd 0.039 0.251 0.107 0.066 0.085 All urban soils Pb 34.37 127.24 58.06 19.97 52.26 20.46 Cr 46.69 118.33 64.19 11.16 63.00 50.17 Cu 20.13 78.53 32.76 11.54 31.40 17.96 Ni 10.97 27.21 16.04 3.25 16.02 23.07 Zn 74.33 235.65 128.84 37.34 118.05 59.47 Cd 0.031 0.359 0.120 0.094 0.084 0.086 BC Background concentrations in the soils of Changchun [30].

Rescaled Distance Cluster Combine 5 10 15 20 25

Pb Subgroup I Zn Cu Group I Subgroup II Cr Ni Cd Group II

FIGURE 2 - Hierarchical clustering results (dendrogram) of the heavy metal concentrations in urban park and square soils of Changchun.

ter, the more significant was the association. The results of 3.3. Principal components analysis (PCA) the HCA indicated that the elements comprised two main Principal component analysis (PCA) was performed groups. The Group I was subdivided in two subgroups: Pb, for the dataset of topsoils so as to explore the relationship Cu and Zn clustered in one subgroup and Cr and Ni in the between the six metals as variables, and to assign related other subgroup. The Group II consisted of Cd only. The variables into principal components. In the analysis, two presence of Pb, Cu and Zn in Subgroup I is commonly con- principal components were considered, which account for sidered as a strong indication of soils enrichment by these over 80% of the total variance (Table 3), and the eigenval- metals due to anthropogenic activities [31-33]. This is in ues of two extracted factors were higher than 1. The rotated line with their higher concentration observed in this study component matrix indicated that Cu, Zn and Cd were compared to their local background values. The metals of closely associated, displaying high values in the first com- the Subgroup II, Cr and Ni, seemed to be controlled by par- ponent (F1). Pb, Cr and Ni showed greater values in the ent material. This was confirmed by the Principal compo- second component (F2) and Pb was also partially repre- nent analysis (PCA) in the previous study, Cr and Ni were sented in F1 (Table 2). The results imply that Pb, Cu, Zn closely associated with the first principal component (PC1) and Cd can be defined as anthropogenic components and and showed low concentrations. Although the average con- may originate from similar pollution sources such as the centration of Cd in the Group II was close to its background precipitation of aerosol particles released by traffic and in- value, several locations showed high levels of Cd indicat- dustrial activities [2, 34-36], and that the parent materials ing that anthropogenic activities had obviously effects on of the soils may control the concentrations of Cr and Ni and these areas. Pb in part.

1970 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

TABLE 3 - Total variance explained and component matrix for heavy metals in the soils

Component Initial eigenvalues Extraction sums of squared loadings Rotation sums of squared loadings Total % of Cumulative Total % of Cumulative Total % of Cumulative variance (%) variance (%) variance (%) 1 3.564 59.405 59.405 3.564 59.405 59.405 2.638 43.972 43.972 2 1.260 20.999 80.404 1.260 20.999 80.404 2.186 36.432 80.404 3 0.428 7.127 87.531 4 0.370 6.166 93.697 5 0.305 5.079 98.776 6 0.073 1.224 100.000 Metal Component matrix Rotated component matrix PC1 PC2 PC1 PC2 Pb 0.835 0.119 0.571 0.621 Cr 0.699 0.540 0.199 0.861 Cu 0.921 -0.108 0.781 0.500 Ni 0.592 0.657 0.042 0.883 Zn 0.836 -0.469 0.944 0.167 Cd 0.692 -0.540 0.878 0.021 Extraction method: principal component analysis.

TABLE 4 - Statistical results of pollution index (PI) and integrated pollution index (IPI) of heavy metals in the urban soils of Changchun.

PI Pb Cr Cu Ni Zn Cd Mean 2.84 1.28 1.82 0.70 2.17 1.39 All urban soils (n=32) Min 1.68 0.93 1.12 0.48 1.25 0.35 Max 6.22 2.36 4.37 1.18 3.96 4.16 SD 0.96 0.22 0.64 0.14 0.63 1.09 Low 0 1 0 31 0 16 Number of samples Middle 25 31 31 1 29 13 High 7 0 1 0 3 3 IPI High (IPI > 2.0) Middle (1.0 < IPI ≤ 2.0) Low (IPI ≤ 1.0) Sites IPI Sites IPI Sites IPI ETPS-2 3.65 SLPS-1 1.99 SLPS-3 2.41 NHSS-2 1.97 ZQSS-1 2.26 CYPS-1 1.93 NHPS-3 2.20 NHPS-4 1.86 LDPS-1 2.05 MDPS-2 1.82 MDPS-1 2.04 NHPS-5 1.79 XMSS-2 1.76 RMSS-2 1.74 RMSS-1 1.74 CCPS-2 1.67 ZQSS-2 1.67 ETPS-1 1.64 WHSS-4 1.54 XMSS-1 1.42 WHSS-2 1.40 CYPS-3 1.36 CYPS-2 1.34 NHSS-1 1.28 CCPS-1 1.27 CCPS-3 1.26 WHSS-1 1.26 SLPS-2 1.25 NHPS-2 1.22 WHSS-3 1.21 LDPS-2 1.20 NHPS-1 1.17

3.4. Assessment of the environmental quality for the soils of of the samples had low or mid-level PIs, indicating that the urban parks and squares concentrations of Ni and Cr in the soil samples were com- The PI, calculated according to the natural background parable with the natural background values of Changchun values of heavy metals in soils of Changchun, varied con- and there was no obvious pollution of Ni and Cr in the soils siderably across the different metals (Table 4). For Cr and of the parks and squares. Ni, the mean PIs were 1.28 and 0.70, respectively, and all

1971 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

The PIs of Pb, Cu, Zn and Cd were much higher since facilities for children. However, the recreation area (ETP-2) all of the samples contained high PIs, ranging from 1.68 to of the Children's Park demonstrated an IPI of 3.65, indicat- 6.22 for Pb, 1.12 to 4.37 for Cu, 1.25 to 3.96 for Zn and ing the presence of serious heavy metal pollution. Thus ap- from 0.35 to 4.16 for Cd, respectively. The results demon- propriate measures are badly in need for protecting chil- strated that the Pb, Cu, Zn and Cd pollution is widespread dren’s health when they are playing in the recreation area in the parks and squares of Changchun. The maximal PI for of parks. Pb was 6.22 and 22 % of soil samples were classified as According to the location, all squares investigated High PI. Thus, it was likely that many of the park and were classified into two groups: the area inside the square square soils in Changchun were highly polluted by Pb. The (L I) or the area outside the square (L II). Based on the pre- PIs of three samples for Cd were classified as High, but vious results, Cr and Ni for squares were also from natural 50% of the samples were classified as Low PI, indicating sources and less affected by the location of samples at a the absence of problematic Cd pollution of soils in Chang- regional scale. The mean values of Pb, Cu, Zn and Cd in chun parks and squares. the samples of L II were all much greater than those in the The IPIs of six elements in urban soil samples varied samples of L I (Fig. 3 b). Therefore, there were significant from 1.17 to 3.65 with an average of 1.70 (Table 4). All the correlations between the location of the samples in the parks and squares exhibited moderate to high IPIs, and 19 squares and the concentrations of Pb, Cu, Zn and Cd. % of soils investigated had high pollution levels. In partic- The squares (except for WHS ) in Changchun are not ular, the recreation area (ETP-2) of Children's Park demon- only the place for providing rest and leisure, but also as a strated an IPI of 3.65, indicating the presence of serious roundabout for relieving traffic pressure. Thus traffic is an heavy metal pollution. Thus the soil quality of Chang- important factor determining the extent of heavy metal ac- chun’s urban parks and squares has clearly been impacted, cumulation in L II of squares indicating that the squares are particularly those areas visited by large crowds of people, not suitable for leisure, expecially the area outside the such as ETP-2, SLPS-3 and ZQSS-1. squares. In the study, L II of ZQS has the highest IPI value among the squares. Station Square has a high customer traffic throughout the day, and many of the passengers take 4. DISCUSSION a rest here, so the heavy metal pollution of the square will be a threat to their health. Cultural Square, constructed in 4.1. Location of the urban soils and the extent of pollution 1996 in the centre of Changchun, is one of the largest city In this study, the assessment of the soil environment leisure squares of China. The study showed that there was quality suggests that heavy metal pollution varied greatly no obvious pollution of heavy metals in WHS, and that across the different functional areas in the parks and might be related to its relatively short time of establish- squares. Different locations of soils are associated with dif- ment. ferences in the density of human activities and traffic. As was mentioned, different areas of parks and squares Parks were mainly classified into three functional ar- has different contents of toxic elements, and the density of eas: leisure area (F I), fitness area (F II) and recreation area human activities and traffic is likely to accelerate accumu- (F III) for providing opportunities for relaxation, sports and lation of heavy metals in the soils. recreational activities. According to earlier discussion, Cr and Ni seemed to be controlled by parent material, so the Cr 4.2. Analysis of the pollutant sources and Ni in parks were less affected by human activities. The We did not find Cr and Ni pollution in the soil samples mean values of Pb, Cu and Zn in the area of F III were all of the urban parks and squares. Parent material and pedo- much greater than those in the area of F I and F II (Fig. 3 a). genic processes are major factors in the amounts and distri- The concentrations of Pb, Cu, Zn and Cd for F I were lower bution of Cr and Ni [37, 38]. In this study, the Ni concentra- than that for F II and F III. Therefore, in this study the functions in the samples were comparable with the background tional area of park was related to the amount of heavy metal values of Changchun topsoil and a little higher for Cr. accumulation found in the soil samples, particularly for Pb, The concentrations of Pb, Cu, Zn and Cd in the soil Cu, Zn and Cd. samples from most of the urban parks and squares of The recreation area is the most popular place in parks Changchun exceeded the background values of Changchun with a large quantity of visitors every year, expecially for topsoil and, indeed, serious soil pollution was found in children, for providing various kinds of recreational activ- some sites. Anthropogenic sources may contribute to this ities, such as bumper cars, roller coaster, merry-go-round elevation of Pb, Cu, Zn and Cd concentrations in the soils. and so on. As we know, children are at greater risk of ex- The content of these metals in urban soils has been readily posure to the toxic elements from contaminated soils than affected by anthropogenic factors [34, 35, 39]. Pb, Cu, Zn adults [19]. The high concentrations of metals in the F III and Cd can come from organic fertilizers [40], organic and will increase the toxicological health effects on children. inorganic fertilizers [41], and some pesticides [42], respec- For example, the Children's Park is a popular park located tively. However, the major emission source of these ele- in the Changchun and accommodates a large numbers of ments is likely to be traffic emission and industrial activi- visitors every year with more than 50 varieties of recreation ties. It had been found that Pb derived from car exhausts

1972 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

FIGURE 3 - Mean concentrations of heavy metals in the soils of parks and squares.

does not extend appreciably beyond 30 m from the road activities[34, 35] and Cd may be associated with industrial [43], but lead aerosols, industrial fumes and coal burning activities [45]. exhausts can be carried over longer distances [37], and at- In general, Pb, Cu, Zn and Cd were consistently from mospheric deposition is the main source of Pb [44], such anthropogenic sources, while Cr and Ni were from natural as industrial production and traffic activities. Although pet- sources with low concentrations. Atmospheric deposition rol with lead additives has been banned in Changchun for of heavy metals is considered to be a significant factor in several years, lead pollution may continue to affect the soil soil pollution. environment for years to come. The normal activity and deterioration of vehicles on the roads can emit heavy metals into the air, especially Cu [21, 39]. Thus, the various 5. CONCLUSION traffic densities can influence the amount of Cu emitted by local vehicles. In the present study, soil concentration of It is concluded that a significant degree of metal pollu- Zn was also found to be correlated with the level of human tion exists in some soils within the parks and squares of

1973 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

Changchun, particularly for Pb, Cu and Zn, and its concen- [7] Kleckerova, A., Docekalova, H. (2014) Dandelion Plants as a trations varied among different functional areas. All of the Biomonitor of Urban Area Contamination by Heavy Metals. International Journal of Environmental Research 8, 157-164. parks and squares had middle or high PIs of Pb, Cu and Zn, indicative of pollution from these metals, particularly the [8] Lu, S.G., Bai, S.Q. (2010) Contamination and potential mobility assessment of heavy metals in urban soils of Hangzhou, recreation area of parks and outside area of squares, such China: relationship with different land uses. Environmental as the F III of Children's Park and the L II of Station Earth Sciences 60, 1481-1490. Square. Therefore, more attention should be paid to heavy [9] Unsal, Y.E., Yilmaz, E., Soylak, M., Tuzen, M. (2013) Trace metal pollution of parks and squares in Changchun city. Cd Element Contents of Raisins, Grapes and Soil Samples from concentration in the soil samples exceeded background Incesu-Kayseri, Turkey. Fresen Environ Bull 22, 1441-1445. concentrations, but the soil pollution of Cd was low. The [10] Thornton, I., Farago, M.E., Thums, C.R., Parrish, R.R., Cr and Ni concentrations in the urban park soils samples McGill, R.A.R., Breward, N., Fortey, N.J., Simpson, P., were low and seemed less affected by human activities, Young, S.D., Tye, A.M., Crout, N.M.J., Hough, R.L., Watt, J. thus the pollution problem of Cr and Ni was not evident. (2008) Urban geochemistry: research strategies to assist risk All of the sites of parks and squares had middle or high assessment and remediation of brownfield sites in urban areas. Environ Geochem Health 30, 565-576. IPIs. Therefore, the soil quality in most of the urban parks and squares in Changchun has deteriorated. These findings [11] Wong, C.S.C., Li, X.D., Thornton, I. (2006) Urban environmen- indicate that appropriate measures should be taken to ef- tal geochemistry of trace metals. Environ Pollut 142, 1-16. fectively control heavy metal levels of park and square [12] Okorie, A., Entwistle, J., Dean, J.R. (2011) The application of soils in Changchun and thus protect human health. Urban in vitro gastrointestinal extraction to assess oral bioaccessibil- ity of potentially toxic elements from an urban recreational parks and squares are used for leisure, sporting or recrea- site. Appl Geochem 26, 789-796. tional activities and have close relations with residents and [13] Schmidt, C.W. (2010) Lead in Air Adjusting to a New Stand- tourists. So it is imperative to evaluate the potential risks ard. Environ Health Persp 118, 76-79. of pollutants in the public places. [14] Laidlaw, M.A.S., Filippelli, G.M. (2008) Resuspension of urban soils as a persistent source of lead poisoning in children: A review and new directions. Appl Geochem 23, 2021-2039. ACKNOWLEDGEMENTS [15] Siciliano, S.D., James, K., Zhang, G.Y., Schafer, A.N., Peak, J.D. (2009) Adhesion and Enrichment of Metals on Human The authors acknowledge the support of Knowledge Hands from Contaminated Soil at an Arctic Urban Brownfield. Innovative Program of The Chinese Academy of Sciences Environ Sci Technol 43, 6385-6390. (KSCX2-YW-N-077), National Natural Science Founda- [16] Li, H.B., Yu, S., Li, G.L., Deng, H., Luo, X.S. (2011) Contam- tion of China (41071056), and Strategic Priority Research ination and source differentiation of Pb in park soils along an Program of Northeast Institute of Geography and Agroe- urban-rural gradient in Shanghai. Environ Pollut 159, 3536- cology, Chinese Academy of Sciences. 3544. [17] Fong, F.T., Chee, P.S., Mahmood, A.A., Tahir, N.M. (2008) The authors have declared no conflict of interest. Possible source and pattern distribution of heavy metals content in urban soil at Kuala Terengganu town center. Malaysian J Anal Sci 12, 458-467.

REFERENCES [18] Sanchez-Camazano, M., Sanchez-Martin, M.J., Lorenzo, L.F. (1994) Lead and cadmium in soils and vegetables from urban gardens of Salamanca (Spain). Sci Total Environ 147, 163-168. [1] Imperato, M., Adamo, P., Naimo, D., Arienzo, M., Stanzione, D., Violante, P. (2003) Spatial distribution of heavy metals in [19] Granero, S., Domingo, J.L. (2002) Levels of metals in soils of urban soils of Naples city (Italy). Environ Pollut 124, 247-256. Alcala de Henares, Spain: Human health risks. Environ Int 28, 159-164. [2] Chen, T.B., Zheng, Y.M., Lei, M., Huang, Z.C., Wu, H.T., Chen, H., Fan, K.K., Yu, K., Wu, X., Tian, Q.Z. (2005) As- [20] Li, X.D., Poon, C.S., Liu, P.S. (2001) Heavy metal contami- sessment of heavy metal pollution in surface soils of urban nation of urban soils and street dusts in Hong Kong. Appl Ge- parks in Beijing, China. Chemosphere 60, 542-551. ochem 16, 1361-1368. [3] De Miguel, E., Llamas, J.F., Chacon, E., Berg, T., Larssen, S., [21] Ritter, C.J., Rinefierd, S.M. (1983) Natural background and Royset, O., Vadset, M. (1997) Origin and patterns of distribu- pollution levels of some heavy metals in soils from the area of tion of trace elements in street dust: Unleaded petrol and urban Dayton, Ohio. Environ Geol 5, 73-78. lead. Atmos Environ 31, 2733-2740. [22] Norra, S., Weber, A., Kramar, U., Stüben, D (2001) Mapping [4] Mielke, H.W., Gonzales, C.R., Smith, M.K., Mielke, P.W. of trace metals in urban soils. J Soil Sediment 1, 77-97. (1999) The urban environment and children's health: Soils as an integrator of lead, zinc, and cadmium in New Orleans, Lou- [23] Madrid, L., Diaz-Barrientos, E., Madrid, F. (2002) Distribu- isiana, USA. Environ Res 81, 117-129. tion of heavy metal contents of urban soils in parks of Seville. Chemosphere 49, 1301-1308. [5] Bullock, P., Gregory, P.J., Soils in the Urban Environment, Blackwell Scientific Publications, Oxford, UK 1991. [24] Mielke, H.W., Reagan, P.L. (1998) Soil is an important pathway of human lead exposure. Environ Health Persp 106, 217-229. [6] El Khalil, H., Schwartz, C., El Hamiani, O., Kubiniok, J., Mo- rel, J.L., Boularbah, A. (2013) Distribution of major elements [25] Szolnoki, Z., Farsang, A., Puskas, I. (2013) Cumulative im- and trace metals as indicators of technosolisation of urban and pacts of human activities on urban garden soils: Origin and ac- suburban soils. J Soil Sediment 13, 519-530. cumulation of metals. Environ Pollut 177, 106-115.

1974 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

[26] Zheng, Y.M., Yu, K., Wu, H.T., Huang, Z.C., Chen, H., Wu, [45] Charlesworth, S., Everett, M., McCarthy, R., Ordonez, A., de X., Tian, Q.Z., Fan, K.K., Chen, T.B. (2002) Lead concentra- Miguel, E. (2003) A comparative study of heavy metal con- tions of soils in Beijing urban parks and their pollution assess- centration and distribution in deposited street dusts in a large ment. Geogr Res 21, 418-424. and a small urban area: Birmingham and Coventry, West Mid- lands, UK. Environ Int 29, 563-573. [27] Guo, P. (2005) Study on Heavy Metal Contamination Mecha- nism and Countermeasure in Urban Soil of Changchun. Jilin university, Changchun. (In chinese).

[28] Wu, S.H., Zhou, S. L., Yang, D. Z., Liao, F. Q., Zhang, H. F., & Ren, K. (2008) Spatial distribution and sources of soil heavy metals in the outskirts of Yixing City, Jiangsu Province, China. Chinese Science Bulletin 53. [29] USEPA. Method 3050B (1996) Acid digestion of sediments, sludges and soils. United States Environmental Protection Agency.

[30] Meng, X.X., Li, S.Z. (1995) Study on Backgrond Value of Soil Environment of the Jilin Province. . Beijing: Science.

[31] Lee, C.S., Li, X.D., Shi, W.Z., Cheung, S.C., Thornton, I. (2006) Metal contamination in urban, suburban, and country park soils of Hong Kong: A study based on GIS and multivariate statistics. Sci Total Environ 356, 45-61. [32] Moller, A., Muller, H.W., Abdullah, A., Abdelgawad, G., Utermann, J. (2005) Urban soil pollution in Damascus, Syria: concentrations and patterns of heavy metals in the soils of the Damascus Ghouta. Geoderma 124, 63-71.

[33] Yaylali-Abanuz, G. (2011) Heavy metal contamination of surface soil around Gebze industrial area, Turkey. Microchem J 99, 82-92.

[34] Artaxo, P., Oyola, P., Martinez, R. (1999) Aerosol composition and source apportionment in Santiago de Chile. Nuclear

Instruments & Methods in Physics Research Section B-Beam

Interactions with Materials and Atoms 150, 409-416. [35] Gray, C.W., McLaren, R.G., Roberts, A.H.C. (2003) Atmos- pheric accessions of heavy metals to some New Zealand pas- toral soils. Sci Total Environ 305, 105-115.

[36] Mico, C., Recatala, L., Peris, A., Sanchez, J. (2006) Assessing heavy metal sources in agricultural soils of an European Med- iterranean area by multivariate analysis. Chemosphere 65, 863-872. [37] Facchinelli, A., Sacchi, E., Mallen, L. (2001) Multivariate statistical and GIS-based approach to identify heavy metal sources in soils. Environ Pollut 114, 313-324.

[38] Rodriguez, J.A., Nanos, N., Grau, J.M., Gil, L., Lopez-Arias, M. (2008) Multiscale analysis of heavy metal contents in Spanish agricultural topsoils. Chemosphere 70, 1085-1096. Received: October 14, 2014 [39] Martin, A.C., Rivero, V.C., Marin, M.T.L. (1998) Contamina- Revised: November 20, 2014 tion by heavy metals in soils in the neighbourhood of a Accepted: November 26, 2014 scrapyard of discarded vehicles. Sci Total Environ 212, 145- 152. [40] Ni, W.Z., Ma, H.Y., He, J.X., Long, X.X. (2004) Heavy metal CORRESPONDING AUTHOR concentrations in vegetable garden soils from the suburb of Hangzhou, People's Republic of China. B Environ Contam Tox 72, 165-169. Yang Wang Northeast Institute of Geography and Agroecology [41] Csathó, P. (1994) A környezet nehézfém szennyezettsége és Chinese Academy of Sciences az agrártermelés. MTATAKI, Budapest. 4888 Shengbei Road [42] Alloway, B.J. (2005) Heavy Metals in Soils. Blackie Aca- Changchun, 130102 demic Professional. Blackie Academic Professional. P.R. CHINA [43] Smith, W.H. (1976) Lead Contamination of the Roadside Eco- system. Journal of the Air Pollution Control Association. Phone: +86-0431-85542232 [44] Dach, J., Starmans, D. (2005) Heavy metals balance in Polish E-mail: [email protected] and Dutch agronomy: Actual state and previsions for the future. Agr Ecosyst Environ 107, 309-316. FEB/ Vol 24/ No 5b/ 2015 – pages 1966 - 1975

1975 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

THE ENZYMATIC ACTIVITY AND PLASTID PIGMENT CONTENT IN MEDICAGO X VARIA T. MARTYN TREATED WITH AN EXOGENOUS GROWTH REGULATOR

Jacek Sosnowski

University of Natural Sciences and Humanities, Institute of Agronomy, ul. B. Prusa 14, 08 -110 Siedlce, Poland

ABSTRACT 50% of the applied mineral nitrogen is lost [4]. Thus, there arises a question of very low efficiency of such fertilisation The study focused on determining the effect of an ex- [5]. Moreover, studies conducted by Ilieva and Vasileva [6] ogenous growth regulator on the activity of nitrate reduc- indicate weakening of some physiological processes of lu- tase and the content of plastid pigments in lucerne. In March cerne under the influence of mineral nitrogen fertilisation. 2014, a pot experiment was conducted to grow Medicago x In this context, growth regulators based on exogenous aux- varia T. Martyn in a growth room under controlled condi- ins and cytokinins become an alternative. Numerous liter- tions (temperature 24±2/16±2ºC, photoperiod 16/8 h, light ary references [7-14] indicate positive effects of using reg- intensity of 200 µmol·m-2·s-1 achieved through the use of ulators of this type in crops of many plant species. These high-pressure sodium lamps, humidity 40%). The experi- effects include improving the plant vigour and resistance ment was carried out in 40 pots, four pots for each variant to adverse environmental conditions as well as diseases. and 3 plants in each pot. The experimental factor was an own preparation (exogenous growth regulator) protected by The aim of the study was to evaluate the effectiveness the Polish Patent Office (number of invention: P/407/790). of the growth regulator based on exogenous auxin and cy- The regulator contained synthetic auxin, cytokinin and tita- tokinin with the addition of titanium in a form of chelate nium in a form of chelate. Depending on the experimental on the nitrate reductase activity and plastid pigment content variant, spraying with the regulator was applied at the sixth in the biomass of lucerne. The experiment was to demon- true leaf stage and at the first flower bud stage. The control strate whether spraying with the regulator, applied in vari- plants were treated with distilled water. The best results were ous stages of lucerne growth and development, triggers ni- obtained after spraying plants with the regulator twice: at the trate reductase activity in lucerne leaves, stems and roots, sixth true leaf stage and at the first flower bud stage. Only in and whether it influences the content of chlorophyll a and the case of nitrate reductase activity in the roots and the ratio b, as well as carotenoids in lucerne leaves. of total chlorophyll content to carotenoids, the highest values were obtained after using the regulator once. 2. MATERIALS AND METHODS

KEYWORDS: growth regulator, synthetic auxin, synthetic cyto- In March 2014, a pot experiment was conducted to kinin, lucerne, nitrate reductase, plastid pigments grow Medicago x varia T. Martyn ‘cv. Kometa’ in a growing room at the Faculty of Natural Sciences of the Siedlce

University of Natural Sciences and Humanities. The experiment conditions were: temperature 24±2/16±2°C; photo- 1. INTRODUCTION period 16/8 hours; light intensity of 200 µmol·m-2·s-1 achieved through the use of high-pressure sodium lamps; Lucerne is considered the main plant source of protein and humidity 40%. The experiment was completely ran- in the production of protein extracts for animal feed [1]. domised, with four replications with the control subject. The question of the meaning of nitrogen fertilisation of lu- The pots were filled with 5 kg of soil each. The soil used cerne is widely discussed in the scientific literature. Ac- in the experiment was composed of loamy medium sand, cording to some authors [2], nitrogen fertilisation contrib- III soil valuation class, taken from the arable topsoil level. utes to the increase in yield of this plant, therefore it should It was marked by a very high content of assimilable phos- be implemented. On the other hand, others [3] argue that phorus and magnesium, a high content of potassium, cop- mineral nitrogen inhibits the growth and development of per and zinc, and a medium content of boron, manganese lucerne root nodules. Furthermore, it is believed that 30 to and iron (Table 1).

1976 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

TABLE 1 - Chemical composition of soil

-1 pH in KCL Humus [%] Corg [g·kg ] Dry matter [%] Humidity [%] 6.3 3.0 17.1 86 12 Content of mineral N Total content of macroelements [mg·kg -1 DM ] [g·kg -1 DM]

N-NO3 N-NH4 P K Mg 1.4 60.9 0.48 0.13 0.10 Total content of microelements [mg·kg -1 DM] B Mn Cu Zn Fe 2.3 191 8.7 22.3 1570

Lucerne seeds were sown in mid-March to a depth of - chlorophyll a content: [12.7(E 663) - 2.69(E645)] x w/v; 2-3 cm. After the seeds germinated, 3 representative plants - chlorophyll b content: [22.9(E645) - 4.68(E663)] x w/v; in each pot were left for further research. During the lu- - chlorophyll a+b content: [20.2(E645) + 8.02(E663)] x w/v; cerne growth, the factors: growth regulator and spray period were introduced (depending on the stage of growth - carotenoid content: [4.16(E440) - 0.89(E663)] x w/v; and development of plants). The applied growth regulator where: was the researcher’s own preparation protected by the E – extinction at a particular wavelength; Polish Patent Office (number of invention: P/407/790). v – amount of 80% acetone [cm3] used for extraction; The preparation contained synthetic auxin (concentration of 35 mg·dm-3) and cytokinin (35 mg·dm-3). Moreover, it w – sample weight [g]. included titanium in a form of chelate (concentration of 1 The obtained results were statistically analysed using -3 mg·dm ). The plants were sprayed until dip-off with 20 the analysis of variance and LSD0.05 (least significant dif- 3 dm of spray liquid per pot. Spraying was applied at two ferences), according to Tukey’s test. stages of lucerne growth and development: the sixth true leaf stage and at the first flower bud stage. Experimental design subjects: 3. RESULTS AND DISCUSSION . Control – spraying with distilled water at both stages The main source of nitrogen for crops are nitrate ions. of lucerne growth and development, Nitrates are absorbed by plant roots on the basis of active . AF1 – spraying with the regulator at the sixth true leaf transport, involving three types of protein transport sys- stage, and with distilled water at the first flower bud tems. Absorbed nitrates are reduced to ammonia which is stage, then incorporated into organic compounds. At the first . AF2 – spraying with distilled water at the sixth true leaf stage, the nitrate reductase enzyme reduces two-electron stage, and with the regulator at the first flower bud nitrate (V) to nitrite (III). At the second stage, nitrate re- stage, ductase transforms nitrite into ammonium ions. This reaction stage requires six electrons from the reduced ferre- . AF1,2 – spraying with the regulator at the sixth true leaf stage and the first flower bud stage. doxin. Nitrate reductase catalyses this reaction, therefore such a great role is attributed to this enzyme in the process All variants of the experiment were performed in four of forming plant metabolites [19, 20]. replications. After withering of the plants, the collection and frac- The study showed (Table 2) that the nitrate reductase - -1 tionation of biomass for roots, leaves, stems and inflores- activity in the control plants was 32.1 µmol NO2 g fresh - -1 cences was conducted. In stems, leaves and roots nitrate weight in leaves, 8.40 µmol NO2 g fresh weight in stems - -1 reductase activity was determined with the in vivo method and 4.20 µmol NO2 g fresh weight in roots, and it was according to Jaworski [15]. Moreover, plastid pigment lower than the values obtained for variants with the regu- content was marked in leaves. Chlorophyll a and b were lator. The highest nitrate reductase activity occurred in marked with the method by Arnon et al. [16] as modified leaves, and after double spraying with the growth regulator by Lichtenthaler and Welburn [17], and the content of ca- it was higher than the control subjects by 92.2%. After ap- rotenoids – using the method by Hager and Mayer- plying the regulator at the sixth true leaf stage, the nitrate - -1 Berthenrath [18]. Plant material for the marking was col- reductase activity amounted to 50.7 µmol NO2 g fresh lected from each plant at the full flowering stage (50% of weight, and after spraying lucerne only at the first flower bud open flowers). As for the pigments, the optical density of stage, the nitrate reductase activity value was 56.2 µmol - -1 the obtained supernatants was determined with the Marcel NO2 g fresh weight. This tendency was also observed in Mini spectrophotometer with wavelengths: 440, 465 and studies by other authors [3, 6, 21, 22]. In the case of stems, - -1 663 nm. Next, the results were calculated according to the the highest enzyme activity (15.0 µmol NO2 g fresh following formulas: weight) was also observed for the plants sprayed twice.

1977 © by PSP Volume 24 – No 5b. 2015 Fresenius Environmental Bulletin

TABLE 2 - Nitrate reductase activity in leaves, stems and roots of lucerne, depending on the regulator and development stage of plants

Leaves Stems Roots Objects µmol NO - g-1 µmol NO - g-1 µmol NO - g-1 2 +, -, % 2 +, -, % 2 +, -, % fresh weight fresh weight fresh weight Control 32.1 ─ 8.4 ─ 4.2 ─

AF1 50.7 + 57.9 13.4 + 59.5 5.2 + 23.8

AF2 56.2 + 75.1 11.9 + 41.7 5.0 + 19.0

AF1,2 61.7 + 92.2 15.0 + 78.6 5.1 + 21.4 LSD (P=0.05) 6.89 2.1 0.7

+, -,% – increase, decrease to the control A – regulator, F1 – sixth true leaf stage, F2 – first flower bud stage

TABLE 3 - The content of chlorophyll a and b in lucerne leaves, depending on the regulator and plant development stage

Plastid pigments [mg 100 g-1 fresh weight] Objects chl.a +, -, % chl.b +, -, % ch.a+b +, -, % Control 108.0 ─ 80.4 ─ 188.0 ─

AF1 239.0 + 121.0 108.0 + 34.3 347.0 + 84.6

AF2 201.0 + 86.1 101.0 + 25.6 302.0 + 60.6

AF1,2 284.0 +162.9 117.0 + 45.5 401.0 + 213.0 LSD (P=0.05) 33.8 14.5 43.2

+, -,% – increase, decrease to the control A – regulator, F1 – sixth true leaf stage, F2 – first flower bud stage

In chloroplasts there are two types of pigments: chlo- phyll pigments as compared to the control. The best results rophyll (a and b) and carotenoids. Chlorophylls are the were obtained by applying the preparation twice. The con- most important photosynthetic pigments that have the abil- tent of chlorophyll a for these objects increased by 162.9% ity to absorb the light energy and enter an “excited” state. and amounted to 284 mg 100 g-1 fresh weight, chlorophyll It determines the flow of electrons in the light-dependent b by 45.5% and amounted to 117.0 mg 100 g-1 fresh weight, stage of photosynthesis, and production of compounds nec- and the total chlorophyll content increased by 213%, giv- essary in the later stages of this process, i.e. ATP and ing 401.0 mg 100 g-1 fresh weight. It is worth noting that NADPH. In contrast, carotenoids (carotenes and their de- regardless of the type of chlorophyll pigment, the use of the rivatives containing one or more oxygen atoms – xantho- regulator at the sixth true leaf stage gave better results than phylls) act as auxiliary compounds in the process of ab- spraying the plants at the first flower bud stage. A similar sorbing the light energy at different wavelengths and trans- tendency occurred for the total chlorophyll. ferring it to the chlorophyll (light harvesting complex). These include: β- carotene, lutein, neoxanthin and violax- In the case of carotenoids, the highest increase of their anthin. Moreover, they capture free oxygen radicals, thus content (Table 4) was also obtained after double spraying protecting chlorophyll against photo-oxidation (photo-pro- with the regulator. The content of these pigments increased tective system). Here we should mention: violaxanthin, an- from 23.5 mg 100 g-1 fresh weight for the control to 41.7 mg -1 theraxanthin and zeaxanthin – VAZ [23, 24]. 100 g fresh weight for the variants AF1,2. In addition, more carotenoids in lucerne leaves were obtained after using the The study showed (Table 3) that spraying plants with regulator at the sixth true leaf stage (34.7 mg 100 g-1 fresh the regulator triggered an increase in the content of chloro- weight) than at the first flower bud stage (27.2 mg 100 g-1

TABLE 4 - The content of carotenoids in lucerne leaves and the ratio of total chlorophyll to carotenoids, depending on the regulator and plant development stage

Pigments [mg 100 g-1 fresh weight] Objects carotenoids +, -, % chl.a+b/carotenoids +, -, % Control 23.5 ─ 8.0 ─

AF1 34.7 + 47.7 10.0 + 25.0

AF2 27.2 + 15.7 11.0 + 37.5

AF1,2 41.7 + 77.7 9.0 + 12.5 LSD (P=0.05) 9.10 1.1

+, -,% – increase, decrease to the control A – regulator, F1 – sixth true leaf stage, F2 – first flower bud stage

fresh weight). It should be noted that the ratio of total chlo- [3] Kot’s, S. Ya. (2001) Physiological bases of highly efficient rophyll to carotenoids was the highest (11.0 on average) for functioning of alfalfa symbiotic systems in agrocenoses. Ph. D. thesis, NAS of Ukraine, Kyiv. the plant material treated once with the growth regulator, but only at the first flower bud stage. [4] Kostov, O. and Lynch, J.M. (1998) Composed sawdust as a carrier for Bradyrhizobium, Rhizobium and Azospirillum in crop inoculation. World Journal of Microbiology and Bio- When plants from the Fabaceae family age, chloro- chemistry, 14, 389-397. phyll and carotenoid content in tissues decreases [25]. Ac- [5] Raviv, M. M., Shlomit, Krasnovsky, A. and Ziadna H. (2004) cording to Downs et al. [26], the use of exogenous cyto- Organic matter and nitrogen conservation in manure compost for kinin may increase the content of chlorophyll in senescent organic agriculture. Compost Science and Utilization, 12, 6-10. leaf tissues, because it slows down chlorophyll degradation [6] Ilieva, A. and Vasileva V. (2013) Effect of mineral fertilization and delays the aging process. In their studies on the effects and manure on some characteristics in Alfalfa (Medicago sa- of BAP on cabbage, Costa et al. [27] found that this sub- tiva L.). World Applied Sciences Journal stance slows the degradation of total chlorophyll compared 26 (5), 630-635. to the control plants. Simultaneously, the authors demon- [7] Bai, N. R., Banu, N. R.L., Prakash, J.W. and Goldi S.J. (2007) strated a significant drop in the activity of enzymes taking Effects of Asparagopsis taxiformis extract on the growth and part in chlorophyll degradation (chlorophyllase and mag- yield of Phaseolus aureus. Journal of Basic and Applied Biol- nesium chelatase) as a result of treatment with plant hor- ogy, 1, 1, 6-11. mones. Czapla et al. [28], using two types of synthetic aux- [8] Christobel, J. G. (2008) Effect of seaweed (Sargassum wightii ins – IBA and NAA (1-naphthaleneacetic acid) and their L.) on the germination and growth of green gram (Phaseolus Au- reus L.) Journal of Basic and Applied Biology, 2, 1, 105-108. mixtures (all with a concentration of 20 mg·dm-3), observed the highest effects when using IBA. On the other [9] Thirumaran, G., Arumugam, M., Arumugam, R. and Anantha- raman, P. (2009) Effect of seaweed liquid fertilizer on growth hand, Nahar and Ikeda [29] sprayed soybean plants with and pigment concentration of Cyamopsis tetrogonolaba (L) auxin (ethyl-5-chloro-3-indazolyl acid) and found an aver- Taub. American-Eurasian Journal of Agronomy, 2, 2, 50-56. age of 23% increase in the analysed features compared to [10] Zodapea, S.T., Mukherjeea, S., Reddya, M.P. and the control. The positive effect of plant hormones on plants Chaudharya, D.R. (2009) Effect of Kappaphycus alvarezii was also reported by Nowak et al. [30], Barcley and McDa- (Doty) ex silva. extract on grain quality, yield and some yield vid [31], and Czapla et al. [28]. components of wheat (Triticum aestivum L.). International Journal of Plant Production, 3, 2, 97-101. [11] Zodape, S.T., Mukhopadhyay, S., Eswaran, K., Reddy, M.P. 4. CONCLUSIONS and Chikara, J. (2010) Enhanced yield and nutritional quality in green gram (Phaseoulus radiata L.) treated with seaweed (Kappaphycus alvarezii) extract. Journal of Scientific & In- Regardless of plant parts (leaves, stems, roots), the use dustrial Research, 69, 468-471. of the growth regulator contributed to the increase of ni- [12] El-Yazied, A., El-Gizawy, A. M., Ragab, M. I. and Hamed, E. trate reductase activity compared to the control plants. In S. (2012) Effect of seaweed extract and compost treatments on the aboveground biomass, the highest activity of this en- growth, yield and quality of snap bean. Journal of American zyme was observed after double spraying of plants with the Science, 8, 6, 1-20. regulator. Spraying plants with the regulator caused the in- [13] Sosnowski, J., Jankowski, K. and Wiśniewska-Kadżajan B. crease of the plastid pigment content in lucerne leaves. The (2013) Effect of Kelp SL on the formation of above-ground highest content of these pigments was obtained by using biomass of Festulolium baunii (K. Richt.) A. Camus, Acta Ag- the regulator at the sixth true leaf stage and the first flower robot. 66(2), 149–154, DOI: 10.5586/aa.2013.031. bud stage. The best ratio of total chlorophyll to carotenoids [14] Sosnowski, J., Jankowski, K, Wiśniewska-Kadzajan, B., Jan- was obtained for plants grown on the premises where kowska, J. and Kolczarek, R. (2014) Effect of the extract Eck- lonia maxima on selected micro- and macroelements in spraying was applied at the first flower bud stage. above-ground biomass of hybrid alfalfa. J. Elem., 2, 209-217, DOI:10.5601/jelem.2014.19.1.608. The authors have declared no conflict of interest. [15] Jaworski, E. (1971) Nitrate reductase assay in intact plant tissues. Biochemical and Biophysical Research Communications 13(6), 1274-1279. [16] Arnon, D.J., Allen, M. B. and Whatley F. (1956) Photosynthe- REFERENCES sis by isolated chloroplast. IV General concept and comparison of three photochemical reactions. Biochim. Biophys. Acta [1] Vassilev, E. (2004) Forage productivity of some Bulgarian lu- 20, 449-461, cerne cultivars in mixtures with grasses. In the proceedings of [17] Lichtenthaler, H.K. and Wellburn A.R. (1983) Determinations the 20 General Manure Meeting of the European Grassland of total carotenoids and chlorophyll a and b of leaf extracts in Federation, Luzern, Switzerland, Grassland Science in Eu- different solvents. Biochem. Soc. Trans. 11, 591-592. rope, 9, 401-403. [18] Hager, A. and Mayer-Berthenrath T. (1966) Die Isolierung [2] Tufenkci, S., Erman M., and Sonmez, F. (2006) Effect of ni- und quantitative Bestimmung der Carotenoide und Chloro- trogen application and Rhizobium inoculation on the yield and phyll von Blättern, Algen und isolierten Chloroplasten mit nutrient uptake in lucerne (Medicago sativa). Bulletin of Pure Hilfe dunnschichtchromatographischer Methoden. Planta. and Applied Sciences Botany, 25, 1. Berlin. 69, 198-217.

[19] Campbell, W. H. (1999) Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology Annu. Rev. Plant physiol. Plant Mol. Biol. 50, pp: 277-303.

[20] Kaiser, W. M., Weiner, H., Kndlbinder, A., Cyn-Bey, Tsai, Rockel, P., Sonoda, M. and Planchet E. (2002) Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction. J Exp. Bot. 53 (370), 875- 882. [21] Vasileva, V. and Ilieva A. (2007) Effect of presowing treatment of seeds with insecticides on nodulating ability, nitrate reductase activity and plastid pigments content of lucerne (Medicago sativa L.). Agronomy Research 5(1), 87-92,

[22] Vasileva, V. and Ilieva A. (2011) Chemical composition, nitrate reductase activity and plastid pigments content in lucerne under the influence of ammonium and nitrate form mineral nitrogen. Agronomy Research 9 (1–2), 357-364.

[23] Veljovic-Jovanovic, S., Bilger, W. and Heber U. (1993) Inhi- bition of photosynthesis, stimulation of zeaxanthin formation and acidification in leaves by SO2 and reversal of these effects, Planta 191, 365-376. [24] Tausz, M., Wonisch, A., Peters, J., Jiménez, M. S., Morales, D. and Grill D. (2001) Short-term changes in free-radical scav- engers and chloroplast pigments in Pinus canariensis needles as affected by mild drought stress. Journal of Plant Physiology 158, 213-219.

[25] Fu, J., Huang, B. and Zhang G. (2000): Physiological and biochemical changes during seed filling in relation to leaf senescence in soybean. Biol. Plantarum 43(4), 545-548.

[26] Downs, C. G., Somerfield, S. D. and Davey M. C. (1997) Cy- tokinin treatment senescence but not sucrose loss in harvested braccoli. Post. Biol. Technol. 11, 93-100,

[27] Costa, M. L., Civello, P. M., Chaves, A. R. and Martinez G. A. (2005) Effect of ethephon and 6-benzylaminopuryne on chlorophyll degrading enzymes and peroxidase-linked chlorophyll bleaching during post-harvest senescence of braccoli (Brassica oleracea L.) at 20oC. Postharv. Biol. Tech., 35, 191- 199. [28] Czapla, J., Nogalska, A. and Stasiulewicz L. (2003) Działanie syntetycznych auksyn na plonowanie i gospodarkę mineralną soi. Acta Sci. Pol. Agricultura 2(1), 123-131 (in Polish). [29] Nahar, B. S. and Ikeda T. (2002) Effect of different concentrations of Figaron on production and abscission of reproduc- tive organs, growth, and yield in soybean (Glycine max L.). Field Crop Res. 78, 41-50.

[30] Nowak, G. A., Klasa, A., Wierzbowska, J. and Gotkiewicz M. (1997) Plonowanie oraz zawartość makroskładników w roślin- ach bobiku w warunkach stosowania retardantów i fitohor- Received: October 23, 2014 monów. Cz. I. Plonowanie roślin. Biul. IHAR 201, 289-294 Revised: November 24, 2014 (in Polish). Accepted: December 05, 2014

[31] Barclay, G.F. and McDavid C.R. (1998) Effect of benzyloam- inopuryne on fruit set and seed development in pigeonpea (Ca- janus cajan). Scientia Hort. 72, 81-86. CORRESPONDING AUTHOR

Dr. Jacek Sosnowski Institute of Agronomy Siedlce University of Natural Sciences and Humanities 08 -110 Siedlce POLAND

E-mail: [email protected]

FEB/ Vol 24/ No 5b/ 2015 – pages 1976 – 1980

HEAVY METALS CONTENT IN AMANITA PANTHERINA IN A VICINITY OF THE THERMO-ELECTRIC POWER PLANT OSLOMEJ, REPUBLIC OF MACEDONIA

Emri Murati*, Slavcho Hristovski, Ljupcho Melovski and Mitko Karadelev

Institute of Biology, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Republic of Macedonia

ABSTRACT Heavy metals pollution is a serious problem in the Re- public of Macedonia. The heavy metals concentration in The results of the heavy metals analyses (Ni, Cu, Fe, Kichevo area is mainly a result of natural processes and of Zn, Mn, Pb and Cd) in the fruiting body of Amanita pan- the impact by Thermo-Electric Power Plant Oslomej therina (DC.) Krombh. 1846, a poisonous wild-growing (TEPP Oslomej) based on lignite combustion [5, 6]. mushroom are presented. Samples of A. pantherina and The heavy metals composition of Amanita pantherina soils were collected from Kichevo area (Republic of Mac- (DC.) Krombh. 1846 (Panther cap) and other fungi of the edonia) at different distances (~0.5, ~2.5; ~7.5 km) from genus Amanita have been studied in clean and polluted en- the Thermo-electric power plant Oslomej in 2012 and vironments [7, 8]. The potential for accumulation of Cd in 2013. The total concentration of heavy metals was ana- A. pantherina and A. muscaria has also been stressed [8, 9]. lyzed by wet digestion and atomic absorption spectrometry The aim of this work is to determine the heavy metal (Agilent 55A). concentration in soils and in the fruiting bodies of A. pan- Very high concentrations of Cd were recorded in all of therina and to assess the potential impact of the TEPP the samples indicating the bioaccumulation potential of Oslomej. Amanita pantherina for Cd. We found out that the Cd concentration in Amanita pantherina was significantly correlated to its total concentration in the soil (p<0.05; r=0.48). 2. MATERIALS AND METHODS The impact of the TEPP Oslomej was evident in the case of Cu and Zn for which a significant correlation was Study area. Kichevo valley is situated in south-west found between the distance from TEPP Oslomej and its Macedonia, surrounded by Bistra and Suva Gora moun- concentration in Amanita pantherina (p<0.05). Further- tains. Kichevo is an important industrial center in this part more, the combination of distance from TEPP Oslomej and of Macedonia, due to the iron mine in Tajmište (closed at total soil concentration of Mn gave significant multiplica- the moment), the coal mine Oslomej and the TEPP Oslomej. tive model (p<0.10) to explain the concentration of Mn in TEPP Oslomej provides for about 9% of the total electrical Amanita pantherina. energy production in the Republic of Macedonia.

Field collection of wild mushrooms. The samples KEYWORDS: Amanita pantherina, mushroom, heavy metals, were collected from Kichevo area at various distances from Thermo-Electric Power Plant. TEPP Oslomej in the period of 2012 and 2013 (exclusively from spring to autumn). In total, 19 samples of A. pantherina from 11 localites at different distances (~0.5, ~3 1. INTRODUCTION and ~7.5 km) and orientations (north, west, east and south) from TEPP Oslomej were collected (Tab. 1). All of the Fungi in general are known to be potent accumulators samples were collected in Italian and Turkey oak forests of heavy metals from soils and thus were used as indicators (Quercetum frainetto-cerris Horvat, 1954). Soils from for heavy metal pollution [1-4]. Compared to green plants, these localities at depth of 5-15 cm were also collected (in mushrooms can build up large concentrations of some heavy two replicates). Dried samples were kept in the Macedo- metals, particularly Cd, Hg, Cu and Pb. This suggests that nian Collection of Fungi (MCF). mushrooms possess a very effective mechanism that enables them readily to take up heavy metals from soil [4]. Analytical methods. All samples of A. pantherina were ground and wet digested with HNO3 and H2O2. Sam- * Corresponding author ple of 0.5 g was measured on analytical balance (0.0001 g)

and transferred into digestion flask. Immediately, 8ml of 3. RESULTS AND DISCUSSION HNO3 were added followed by the addition of 4 ml of H2O2 (1 ml in one-hour intervals). The samples were digested on The concentration of heavy metals in soils of 11 sampling temperature of 160˚C during 24 hours. localities around TEPP Oslomej is presented in Table 1. Con- The concentration of heavy metals in soils was deter- centrations of 19 elements were studies in a extensive study mined by wet digestion of approximately 1 g dry soil sam- based on 52 soil samples from Kichevo valley [6]. The val- ple in 10 ml of digestion solution HNO3:HCLO4:H2SO4= ues for Mn (Table 1) are lower than the ones reported [6]; 10:2:1 [10]. all of the other elements are comparable between these two studies. All of the analyzed heavy metals were determined by flame atomic absorption spectrometry on Agilent 55A. The Analyses of the heavy metals concentrations in the concentration of heavy metals was calculated and corrected fruiting body of A. pantherina showed considerable varia- by subtracting the blanks. tions between localities, especially for Fe (Figure 1).

TABLE 1 - Total (T) and available (A) concentration (mg·kg-1) of heavy metals in soils of 11 sampling localities around TEPP Oslomej.

Ni Cu Zn Fe Mn Pb Cd Locality

T A T A T A T A T A T A T A Distance Orientation Zhubrino 0.5-0.8 N 4.6 0.032 4.2 0.15 11.3 0.05 4726.9 6.3 72.2 1.7 17.6 0.06 0.17 0.003 Oslomej - W 0.5 W 10.3 0.102 7.7 0.16 24.1 0.07 8780.7 4.4 142.0 2.9 19.8 0.14 0.05 0.004 Oslomej - S 0.5 S 8.9 0.052 5.5 0.09 19.2 0.08 4691.6 4.3 105.9 5.1 18.1 0.21 0.05 0.005 Gorica 3.25 N 8.7 0.021 5.1 0.08 17.0 0.04 9399.0 3.2 171.3 1.8 17.3 0.05 0.05 0.002 Stragomishte 3-3.5 W 15.5 0.044 23.7 0.14 37.0 0.03 7590.7 3.4 110.8 3.8 16.8 0.23 0.05 0.002 Crvivci 3 S 19.1 0.100 13.3 0.13 39.9 0.10 6315.6 3.1 102.7 5.4 16.9 0.10 0.10 0.006 Zadel-Srbica 2.5-3.0 E 16.1 0.029 20.1 0.12 32.7 0.05 9455.2 2.6 160.7 0.9 39.2 0.02 0.12 0.003 Jagol-Dolenci 7-7.5 N 9.5 0.015 5.1 0.05 15.6 0.02 4792.3 2.0 111.4 5.7 17.4 0.05 0.05 0.001 Zajas 7-7.5 W 34.2 0.104 21.5 0.12 43.2 0.18 4085.9 3.8 101.3 1.1 17.2 0.12 0.07 0.002 Krushino 7-7.5 S 9.3 0.041 10.1 0.17 25.6 0.15 9376.4 3.1 177.4 4.3 19.3 0.06 0.05 0.003 Novo Selo 7-7.5 E 3.3 0.053 3.0 0.09 10.1 0.07 3690.7 4.9 54.5 2.5 16.8 0.14 0.07 0.003

500 mg·kg-1 392,0 400

300

200

100 67,6 28,1 7,3 16,1 6,2 4,1 0 Ni Cu Zn Fe Mn Pb Cd

FIGURE 1 - Concentration of analyzed heavy metals in the fruiting body of Amanita pantherina (vertical bars represent standard errors).

The concentration of Cd was high (4.1 mg·kg-1 in av- As in case of Cu, the concentration of Mn in A. pan- erage) with a maximum of 10.05 mg·kg-1. Even higher Cd therina was dependent on both the soil concentration and concentrations of 12.3±3.58 mg·kg-1 have been reported in distance from TEPP Oslomej (p<0.1). four different areas in Hungary [8]. Similar results have been published for A. muscaria [9]. In other edible mushroom 12 species in Kichevo area lower values were found [11]. )

-1 10 The concentration of Ni was also high (7.3 mg·kg-1 in average) compared to one study in Hugary where average 8 value of 1.92 mg·kg-1 for Ni in A. pantherina was reported [8]. High values for Ni in Kichevo are probably due to the Cd (mg·kg 6 high concentration of Ni in the soils (Table 1). The concentration of Ni and Fe in soils is high and predominantly a 4 result of the natural lithologic composition [6]. This is further supported by very high value of Fe in A. pantherina 2 (Figure 1), especially compared to the results of other studies [8, 10]. 0 0,00 0,10 0,20 0,30 0,40 The concentrations of Zn and Cu in A. pantherina from -1 Kichevo area (Figure 1) are lower than the values reported Cd in soils (mg·kg ) in other studies [8, 10]. FIGURE 3 - Correlation between concentration of Cd in soils and in the fruiting body of Amanita pantherina. We found significant correlation between the distance from TEPP Oslomej (Kichevo, Macedonia) and concentration of Cu in the fruiting body of A. pantherina (Figure 2; 4. CONCLUSIONS p<0.05, r=0.76). This provides evidence of the pollution impact of TEPP Oslomej with Cu although its soil concen- The study on the impact of Thermo-Electric Power tration was regarded as mainly dependent on natural lithol- Plant Oslomej in Kichevo area (Republic of Macedonia) on ogy [6]. However, high values of Cu (enrichment factor > the heavy metals concentration in Amanita pantherina 2) were reported in the lignite from Oslomej coal mine [5]. showed that: The correlation in the case of other heavy metals was not  The concentration of Cd is very high as a result of statistically significant. Multiple regression analyses natural potential of this species for accumulation of showed that the concentration of Cu in A. pantherina is Cd. However, the accumulation of Cd was dependent both dependent on the soil concentration and distance from on the soil concentration of Cd; TEPP Oslomej (p<0.05).  The concentration of Ni and Fe was also high and this 120 is probably a result of the high soil concentration (nat- )

-1 ural lithology); 100  The impact of the pollution of TEPP Oslomej was ev- 80 ident in the case of Cu and Zn, although the natural soil concentration of Cu is an important factor. 60 The authors have declared no conflict of interest.

Cu and Zn Zn (mg·kg Cu and 40

20 REFERENCES 0 [1] Kalač P., Nižnanská M., Bevilaqua D. and Stašková I. (1996) 02468 Concentrations of mercury, copper, cadmium and lead in fruiting bodies of edible mushrooms in the vicinity of a mercury Distance from TEPP Oslomej (km) smelter and a copper smelter. Science of the Total Environ- FIGURE 2 - Distance from TEPP Oslomej and concentration of Cu ment, 177 (1), 251-258. (dashed line) and Zn (full line) in the fruiting body of Amanita pantherina. [2] Svoboda L., Havlíčková B. and Kalač P. (2006) Contents of cadmium, mercury and lead in edible mushrooms growing in a his- torical silver-mining area. Food chemistry, 96 (4), 580-585. Significant correlation was found between the concentration of Cd in soils and in the fruiting body of A. pan- [3] Stihi C., Radulescu C., Busuioc G., Popescu V., Ghebolanu A. and Ene A. (2011) Studies on Accumulation of heavy metals therina (Figure 3; p<0.05, r=0.48). Such analyses for other from substrate to edible wild mushrooms. Rom. Journ. Phys., heavy metals were not statistically significant. 56 (1-2), 257-264.

[4] Radulescu C., Stihi C., Busuioc G., Popescu I. V., Gheboianu A. I. and Cimpoca V. G. (2010) Evaluation of essential elements and heavy metal levels in fruiting bodies of wild mushrooms and their substrate by EDXRF spectrometry and FAA spectrometry. Romanian Biotechnological Letters, 15 (4), 5444-5456. [5] Yossifova M., Lerouge C., Deschamps Y., Serafimovski T. and Tasev G. (2009). Inorganic chemical chracterization of the Bitola, Oslomej, and Berovo coals and their waste products from burning, FYROM. Geolines 22, 95-103. [6] Stafilov T., Šajn R., Sulejmani F. and Bačeva K. (2011) Geo- chemical atlas of Kičevo and the environs. Faculty of Natural Sciences and Mathematics, Skopje, 70 pp. [7] Allen R. O. and Steinnes E. (1978) Concentrations of some potentially toxic metals and other trace elements in wild mushrooms from Norway. Chemosphere, 7 (4), 371-378. [8] Vetter J. (2005) Mineral composition of basidiomes of Ama- nita species. Mycol. Res. 109, 6, 746-500. [9] Drewnowska M., Lipka, K., Jarzynska, G., Danisiewicz- Czuprynska, D. and Falandysz, J. (2013). Investigation on metallic elements in fungus Amanita muscaria (fly agaric) and the forest soils from the Mazurian lakes district of Poland. Fresenius Environmental Bulletin, 22 (2), 455-460. [10] Allen S. E. (ed.) (1989). Chemical analysis of ecological materials. Second edition. Blackwell scientific publications. Ox- ford-London-Edinburgh-Boston-Melbourne, 368 pp. [11] Murati E., Hristovski S., Melovski Lj. and Karadelev M. (2014). Heavy metals content in some wild edible mushrooms in Kichevo area, Republic of Macedonia. Proceedings Book: Essays on Ecosystem and Environmental Research. ICE 2014 Conference, May 23-26, 2014, Tirana, Albania, p. 527-532.

Received: October 28, 2014 Accepted: November 20, 2014

CORRESPONDING AUTHOR

Emri Murati Institute of Biology Faculty of Natural Sciences and Mathematics Ss. Cyril and Methodius University Skopje REPUBLIC OF MACEDONIA

E-mail: [email protected]

FEB/ Vol 24/ No 5b/ 2015 – pages 1981 - 1984

HEMATOLOGICAL PARAMETERS IN NILE TILAPIA (Oreochromis niloticus) EXPOSED TO SUB-LETHAL CONCENTRATIONS OF CADMIUM AND ZINC

Özgür Fırat1,* and Ferit Kargin2 1; University of Adiyaman, Faculty of Science and Letters, Department of Biology, 02040 Adiyaman, Turkey 2; University of Cukurova, Faculty of Science and Letters, Department of Biology, 01330 Adana, Turkey

ABSTRACT Most studies of the effects of metals on the hematology of fish have focused on a single metal [3-5]. Few studies The aim of the present study was to assess the hemato- have investigated the effects of metal mixtures, despite the logical alterations induced by Zn and Cd individually or in fact that fish are normally affected by a combination of combination in Nile tilapia (Oreochromis niloticus). Fish metals in their natural environments [6, 7]. Nile tilapia were exposed to 0.5 and 5.0 mg/L Zn, 0.1 and 1.0 mg/L Cd, (Oreochromis niloticus) is a tropical freshwater fish and cul- and 0.5 mg/L Zn + 0.1 mg/L Cd and 5.0 mg/L Zn + 1.0 mg/L tured extensively for its economic importance [8]. Due to its Cd mixtures for 7 and 28 days. Significant changes in all he- easy handling, culture and maintenance in the laboratory, matological parameters were found to be either dose or time and because it responds promptly to environmental altera- dependent. The exposure of O. niloticus to metals resulted tions, it is also a well-established model for toxicological re- in decreases in red blood cell, hemoglobin, hematocrit, search [9-11]. As hematological parameters are effective and and white blood cell, which shows the anemic state of the sensitive indicators to monitor physiological and pathologi- fish and the weakening of the immune system. Our work cal changes induced by environmental stressors, alterations showed that hematological parameters are sensitive indicators in the hematology in response to metal exposures may pro- to monitor physiological and pathological changes induced vide important information about the general physiology by metals. and health status of the organism as well as water quality.

Therefore, in this study, the effects of single and combined Zn and Cd on RBC, WBC, Hb, and Hct of O. niloticus have KEYWORDS: been investigated to elucidate the cause-effect relationship Oreochromis niloticus, Zn, Cd, hematological parameters between metals and hematological responses. Also, Zn or Cd levels in blood of fish exposed to metals were measured. 1. INTRODUCTION

Increased discharge of both essential and non-essential 2. MATERIALS AND METHODS metals into natural aquatic ecosystems can expose aquatic organisms to unnaturally high levels of these metals [1]. 2.1 Test Animal and Metal Treatment Fish live in very intimate contact with their environment, and are therefore very susceptible to physical and chemical O. niloticus (80.5 ± 0.9 g, 17.1 ± 0.8 cm, as mean ± changes [2]. S.E.) obtained from Cukurova University Fish Culture Farm, were transferred to the laboratory. Fish were acclima- Hematological indices are very important parameters tized to laboratory conditions in glass tanks for one month for the evaluation of fish physiological status under metallic before exposure. The laboratory was illuminated for 12 h stress [3]. A number of hematological indices such as red with fluorescent lamps (daylight 65/80 W). Experimental blood cell (RBC), hemoglobin (Hb), hematocrit (Hct) and so tanks contained 120 L of dechlorinated and gently aerated on, are used to assess the functional status of the oxygen car- tap water: temperature 21.1 ± 0.2 °C, pH 8.1 ± 0.1, dis- rying capacity of the bloodstream and have been used as an solved oxygen 7.4 ± 0.1 mg/L, alkalinity 203 ± 6.1 mg/L indicator of metal pollution in the aquatic environment [4]. CaCO and total hardness 349 ± 2.2 mg/L CaCO . In fish exposed to metals, several hematological studies have 3 3 been reported anemia and white blood cell (WBC) counts Fish were divided into seven groups each containing changes due to a response of the major hematopoietic organs. 10 fish. Group I was held in tap water as control and other groups were exposed to 0.5 and 5.0 mg/L Zn (ZnCl2); 0.1 * Corresponding author and 1.0 mg/L Cd (CdCl2·H2O), or 0.5 mg/L Zn + 0.1 mg/L

Cd and 5.0 mg/L Zn + 1.0 mg/L Cd for 7 and 28 days. The and osmoregulatory dysfunction or to an increase in the Zn and Cd concentrations were selected based on 96-h rate of erythrocyte destruction in hematopoietic organs due LC50 values (60 mg/L for Zn and 16 mg/L for Cd) of O. to heavy metal toxicity. The our results are in agreement niloticus [12]. Throughout the experiments, control and ex- with the findings of Maheswaran et al. [5] who found that perimental fish were fed daily with a commercial fish food RBC, Hb and Hct decreased in Clarias batrachus follow- (Pinar Yem, Turkey), at approximately 3% of their body ing mercuric chloride exposure. Previous studies [15-17] weight. The test media were changed every two days to reported that the hematology disturbances induced by met- minimize decreases in the metal concentrations. als may be due to were associated with an the inhibition of iron absorption, defective iron metabolism shortening the 2.2 Preparation of Samples and Biochemical Determinations life span in erythrocytes, the decline in activity of ALA-D After each test period blood samples were taken from catalyzed the metabolic synthesis of the heme group and the caudal vein of each fish that is the best place to obtain the osmotic changes resulting in hemodilution or hemocon- fish blood [13] into tubes containing 0.072 mL of 7.5% centration. K3EDTA (Vacuette EDTA, Greiner Bio-One GmbH, The count of RBC is quite a stable index and the fish Kremsmünster, Austria) as anticlotting agent. Some of the body tries to maintain this count within the limits of phys- whole blood was used immediately to determine the levels iological standards, using various physiological mecha- of hematological parameters. The RBC, WBC, Hb and Hct nisms of compensation, especially under stress [3]. The de- were analyzed using a Beckman Coulter LH 750 hematol- cline in RBC count of O. niloticus is attributed to either a ogy analyzer (Beckman Coulter Inc, Miami, FL). direct action of metals on these cells causing hemolysis or Metal levels in remaining whole blood after hemato- impaired erythropoiesis due to an indirect effect of metals logical analysis were measured according to Ince and Kunc on hematopoietic centers (kidney/spleen). It was observed [14]. Blood digested with H2O2 (30%) and concentrated ni- that the exposure to the heavy metals resulted in a decrease o tric acid (1:1 v/v) at 100 C for one hour. After digestion in RBC count of fish [5, 18]. the volume was made up to 2.5 mL with redistilled water, Zn and Cd concentrations of the tissue were measured by The use of immune system parameters to assess alter- atomic absorption spectrophotometer (Perkin Elmer AS ations in fishes experiencing heavy metal exposure and in- 3100, Waltham, USA). terest in defense mechanisms stem from the need to de- velop healthy management tools to support a rapidly grow- 2.3 Data Analysis ing aquaculture industry [19]. WBC, the major cells com- Statistical analysis was carried out using One-way ponent involved in immune responses, can be used as indi- analysis of variance (ANOVA) followed by Student New- cator of health status in fish. Alterations in WBC count of man-Keul’s Test using SPSS 10.0 statistical software fish may probably be attributed to the activity of the spleen, (SPSS Inc., Chicago, IL, USA). Differences were consid- which sequesters and store blood cells under resting condi- ered significant if P < 0.05. tions and releases them into circulating blood during contraction associated with various states of stress [20], and to the gradual repairment of damaged hemopoietic tissues [21]. The decreased number of WBC of O. niloticus may 3. RESULTS AND DISCUSSION be the result of bioconcentration of the metals in the spleen and kidney, which shows the weakening of the immune The individual and combined effects of metals, except system. The reduction in WBC was observed in Tilapia in their lower concentrations at 7 days, caused a decline in zilli [22] and Cyprinus carpio [23] following Cd exposure. RBC and WBC counts at both exposure periods (Figure 1A and B). Although no significant change in Hb content of Hb concentrations reflect the supply of an organism fish in response to single and combined Zn and Cd expo- with oxygen and the organism itself tries to maintain them sure was observed at 7 days, it decreased at the end of the as much stable as possible [24]. The decline in Hb content exposure period in all tested concentrations of metals ex- of O. niloticus may be due to increased rate of destruction cept in lower concentration of Zn (Figure 1C). There was a or reduction in the rate of formation RBC. This premise is decline in Hct value of fish exposed to concentrations of supported by reported low of RBC in the fish exposed to Cd and Zn+Cd combination for 28 days, while O. niloticus metals. Maheswaran et al. [5] suggested that the reduction exposed to Zn showed an increase in its value at 7 days in Hb content following metal exposure may be attributed followed by a return to control levels at the end of the ex- to a decreased rate of production of RBCs or an increased posure period (Figure 1D). loss of these cells. In addition, the lower Hb levels of treated fish in the present study may be a result of the dis- The results of our study showed that effects of heavy ruption of the iron synthesizing machinery. Shah [25] re- metals caused the alterations in hematology of O. niloticus ported that Hb decreased due to a direct action of metal on and those significant changes in all hematological parame- the pathway of hemoglobin synthesis. ters were found to be either dose or time dependent. A reduction in hematological values observed in the present Measurement of Hct is a commonly used assay for study may be attributed to erythropoiesis, hemosynthesis monitoring hemolysis [26]. The Hct depends on the varia-

2 a 60 a a a a

# # a a a a a b b b # 50 1,6 b b b b b # # b b # /µL) # /µL) 40 # 6 b 3 c c b b b b 1,2 b (10 (10

30 count

count

0,8 20 WBC RBC 0,4 10

0 0 728 728 Exposure time (in days) Exposure time (in days)

A B 10 a 40 a a b a a a b a a a

b b # 8 b a # a a a a 30 a a a # # c c b b 6 b b 20

4 Hct value (%) Hb content (g/dL) Hb content

10 2

0 0 728 728 Exposure time (in days) Exposure time (in days)

C D

FIGURE 1 - RBC (A), WBC (B), Hb (C) and Hct (D) levels of O. niloticus exposed to metals for 7 and 28 days. Data are expressed as mean±standard error (N=5). Different letters indicate significant differences among groups at the same time (P<0.05). # shows significant differences between time for the same exposure group (P<0.05).

tion in plasma volume, the rate of erythrocyte production- count, Hb content and Hct value. Vutukuru [3] suggested and destruction, dehydration, toxins, and direct blood loss that anemia in fish is an early manifestation of acute and as a result of injury [27,28]. A reduction in Hct of O. nilot- chronic intoxication of metal. The anemia may be probably icus may be a result of the hemadilution or the decline in due to the inhibition of erythropoiesis and hemosynthesis count of blood cells. Similarly Kori-Siakpere and Ubogu and to an increase in the rate of erythrocyte destruction in [29] concluded that the decrease in Hct value following hemopoietic organs. metal exposure in fish, Heteroclarias sp (Clariidae) might be an indication of hemodilution. Zn level in the blood of all Zn and Zn + Cd groups increased compared to control group during 7 and 28 days Our results indicated that metal exposure of O. nilot- (Table 1). Zn level of fish exposed to Zn only was much icus induced an anemia following reducing of the RBC higher when compared with Zn + Cd group. Significant el-

TABLE 1 - Zn level (µg/mL) in blood tissue of O. niloticus under Zn and Zn+Cd exposures

Period Metal Concentrations (mg/L) 7 days 28 days

0.0 77.4±1.4 ax 80.9±1.9 ax 0.5 Zn 100.3±0.9 bx 138.3±3.1 by 5.0 Zn 148.9±1.2 cx 211.3±1.6 cy

0.0 77.4±1.4 ax 80.9±1.9 ax 0.5 Zn+0.1 Cd 88.3±2.2 bx 110.8±0.3 by 5.0 Zn+1.0 Cd 110.7±2.3 cx 142.5±2.3 cy Values are expressed as mean±standard error ((N=5). Letters a, b and c show the differences between groups at the same time and letters x and y show differences between time for the same group (P<0.05).

TABLE 2 - Cd level (µg/mL) in blood tissue of O. niloticus under Cd and Zn+Cd exposures

Period Metal Concentrations (mg/L) 7 days 28 days

0.0 N.D. N.D.

0.1 Cd 0.102±0.002 ax 0.198±0.029 ay 1.0 Cd 0.212±0.001 bx 0.357±0.037 by

0.0 N.D. N.D. 0.5 Zn+0.1 Cd 0.090±0.003 ax 0.125±0.002 ay 5.0 Zn+1.0 Cd 0.188±0.002 bx 0.285±0.001 by Values are expressed as mean±standard error (N=5). Letters aand b show the differences between groups at the same time and letters x and y show differences between time for the same group (P<0.05). N.D.: Not Determined

evations in blood Cd levels occurred in all concentrations anemic state of the fish and the weakening of the immune tested Cd and Zn + Cd mixtures (Table 2). In both exposure system. The present study confirmed that hematological concentrations, Cd levels of fish exposed to Zn + Cd com- parameters are sensitive indicators to monitor physiologi- binations for 7 and 28 days were lower when compared cal and pathological changes induced by metals. with Cd group. Zn and Cd level in the blood of O. niloticus increased with increasing exposure periods in all concentrations tested. ACKNOWLEDGEMENT In the present study, there was increasing level of the metals in the whole blood with increasing concentrations This study was supported by a grant FEF 2006 D16 of Zn, Cd individually or in combination in the exposure from Cukurova University. medium, and with increasing duration of exposure. Differ- ent authors have demonstrated that metal levels increased The authors have declared no conflict of interest. in blood of fish exposed to heavy metals [30,31]. We observed that when fish were exposed to the mixtures of Zn and Cd, concentrations of these metals in their blood were REFERENCES lower than in fish exposed to individual metals. It is possible that one metal blocks or even antagonizes the gill epi- [1] Van Dyk, J.C., Pieterse, G.M., van Vuren, J.H.J. (2007) His- tological changes in the liver of Oreochromis mossambicus thelium absorption of the other and thereby limits the dis- (Cichlidae) after exposure to cadmium and zinc. Ecotoxol. En- tribution of the metal in blood. viron. Safe., 66: 432-440.

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[15] Tort, L. and Torres, P. (1988) The effects of sublethal concentrations of cadmium on hematological parameters in the dog Received: October 31, 2014 fish, Scyliorhinus canicula. J. Fish Biol., 32: 277-282. Revised: December 01, 2014 [16] Liu, J., Liu, Y., Habeebu, S.S., Klaassen, C.D. (1999) Metal- Accepted: December 05, 2014 lotionein-null mice are highly susceptible to the hematotoxic and immunotoxic effects of chronic DcCl2 exposure. Toxicol. Appl. Pharmacol., 159(2): 98-108. CORRESPONDING AUTHOR [17] Adhikari, S., Sarkar, B., Chatterjee, A., Mahapatra, C.T., Ay- yappan, S. (2004) Effects of cypermethrin and carbofuran on Özgür Fırat certain hematological parameters and prediction of their recovery in a freshwater teleost; Labeo rohita (Hamilton). Eco- University of Adiyaman toxol. Environ. Safe., 58: 220-226. Faculty of Science and Letters [18] Bhagwant, S. and Bhikajee, M. (2000) Induction of hypo- Department of Biology chromic macrocytic anaemia in Oreochromis hybrid (Cichli- 02040 Adiyaman dae) exposed to 100 mg/L (sublethal dose) of aluminium. J. TURKEY Sci. Tech., 5: 9-20. [19] Jones, S.R.M. (2001) The occurrence and mechanisms of in- Phone: +90 416 2231771 nate immunity against parasites in fish. Dev. Comp. Immunol., Fax: +90 416 2231774 25: 841-852. E-mail: [email protected] [20] Yamamoto, K.I. (1988) Contraction of spleen in exercised freshwater teleost. Comp. Biochem. Physiol., 89A: 65-66. FEB/ Vol 24/ No 5b/ 2015 – pages 1985 - 1989

ASSESSMENT OF HEAVY METALS CONTAMINATION IN FEN RIVER CHANNELS

Yu Qi1, Jun Ma2 and Guisheng Fan3,*

1College of Environmental Science and Engineering, Taiyuan University of Technology, Taiyuan, 030024, China 2Key Lab of Advanced Transducers and Intelligent Control System, Ministry of Education, Taiyuan University of Technology, Taiyuan, 030024, China 3College of Water Resources Science and Engineering, Taiyuan University of Technology, Taiyuan, 030024, China

ABSTRACT suspended forms and sedimentary phases [5]. Large quantities of the heavy metals are deposited in the sediment be- The Fen River is an important tributary of the Yellow cause of adsorption, hydrolysis and co-precipitation pro- River. And the water body was affected by various human cess [6-9]. It is estimated that between 30 and 98% of the activities. To fully understand the heavy metal pollution in heavy metals entering into rivers can be transported in a the river, water samples in the riverbed soil of the typical sediment-associated form [10]. Metalliferous sediments section along the Fen River were collected and the concen- should be deposited on the channel banks, bed or adjacent trations of Cr, Cd and Pb in all samples were determined. floodplain, which have the potential to serve as future Based on data analysis, this paper evaluated the state of mi- sources of pollution. gration and characteristics of accumulation, and analyzed The Fen River is the second largest tributary of the the distribution and the content variation in the riverbed Yellow River in china, which flows from the north to the soil. According to the research results, the maximum Pd, south through Taiyuan city, and it has many confluent Cr and Cd contents in soil around the Fen River respec- streams. Heavy metal was used widely in production and tively reach to 21.2, 8.0 and 2.0 mg/kg. Compared to the people’s life, and formed several kinds of heavy metal pol- background value of the heavy metal contents in soil (Pb lution sources, which polluted the Fen River for a long =14.7 mg/kg, Cr=55.3 mg/kg, Cd=0.1 mg/kg) in Shanxi time. Therefore, investigating the transformation and dis- Province, the Cd pollution is very serious. On basis of the tribution mechanisms of the heavy metal in sediment be- distribution of Cd, therefore, a feasible renovating plan is comes necessary. proposed. The aim of this study is to assess the extent of heavy

metal pollution resulting from industrial discharges in the KEYWORDS: Fen River, Shanxi Province. In order to evaluate the extent Fen River; Heavy metal; Riverbed soil of heavy metal contamination in the sediments of the Fen River, sediment samples were taken at three sampling lo-

cations and the metals (Cr, Cd and Pb) concentrations were 1. INTRODUCTION determined.

Heavy metals pollution had become a major concern worldwide because of their toxicity, abundance and persis- 2. MATERIALS AND METHODS tence, and subsequent accumulation in aquatic habitats. 2.1 Study area Heavy metal residues in contaminated habitats may accumulate in microorganisms, aquatic flora and fauna, and The Fen River has been an important river throughout then affect human health through food chain [1-3]. Cad- history and is one of the main water sources of the Taiyuan mium (Cd), lead (Pb) and Chromium (Cr) are classified as city in Shanxi province. The Fen River drains the center of the priority pollutants, because these metals are not required Shanxi Province, China. It rises in the Guancen Mountains for metabolic activity and can be toxic even at quite low con- of Ningwu County in northeast Shanxi, flows southeast centrations [4]. into the basin of Taiyuan, and then south through the central valley of Shanxi before turning west to join the Yellow Heavy metals discharged into the aquatic environment River west of Hejin. The Fen and the Wei Rivers are the two by natural or anthropogenic sources are usually distributed largest tributaries of the Yellow River. The river is 694 km in several ways as follows: water-soluble species, colloids, long and drains an area of 39,417 km2, 25.3% of the area of Shanxi Province. The Fen River is the longest river in * Corresponding author Shanxi province, northern China. It is also the second

FIGURE 1 - Fen River location

FIGURE 2 - The schematic diagram of typical cross section soil profile stratified and sampling position distribution

longest tributary of the Yellow River (Fig.1). The Fen nels. Thus, heavy metal and nutrient concentrations in wa- River in Taiyuan city is throughout from north to south, the ter column and sediments at the sites upstream and down- length of Fen River in Taiyuan City is 100 kilometer and stream were compared. occupies one seventh of the entire Fen River. Site-1 is located in the midstream of the Fen River (Qingxu County, Taiyuan City, Shanxi Province). Site-2 is 2.2 Sampling sites located at the downstream of the river channel (Yaodu Dis- Previous investigations indicated that the surface water trict, Linfen City, Shanxi Province). Site-3 is situated at the quality stood at inferior class V in midstream and down- section Yellow river estuary (Wanrong County, Yuncheng stream of the Fen River [11]. The situation has improved City, Shanxi Province). These settings provide us an ideal to some extent because of the efforts to curb the basin-wide place for studying the transformation and distribution mech- environmental pollution by the local governments and rel- anisms of the heavy metal in sediment of the Fen River. evant sectors [12]. It was also necessary to discuss the relevant environmental variations and analyze the spatial dis- 2.3 Sampling collection and chemical analysis parities of Pb, Cd, Cr in river channel along the river. The The typical cross section soil profile stratified and sediment samples were collected from three sites along the sampling position distribution are shown in Fig. 2. Typi- Fen River. The sampling sites were chosen to assess the cally, at each sampling site, five stations were set up along heavy metals distribution and migration in Fen River chan- the traverses to channel at intervals of 30 m. Seven sedi-

ment samples were collected at each station along the ver- 2.4 Study equipments tical profile to the underground water level at intervals of Fig. 3 is the self-made soil water infiltration device for 40 cm. The sediment samples were taken by using a cus- the soil column experiments. The device consists of a water tom-made Luoyang spade and they were stored according supply system (Markon barrel) and a seepage soil column to the GB-standard [13]. After sampling, the sediment sam- which is composed of water supply room, seepage soil col- ples were dried at room temperature and crushed to a particle umn and water output room. size less than 0.15 mm. Then they were immediately transported to the laboratory for further analysis. The heavy metal content was determined with an atomic adsorption spectro- 3. RESULTS AND DISCUSSION photometer (TAS-986, China). Total nitrogen (TN) was determined by the semi-micro Kjeldahl method [14] and soil 3.1. TN and TP concentrations total phosphorus (TP) was determined colorimetrically [15]. The TN and TP concentrations in sediments of the Fen Samples went through wet digestion with H2SO4+HClO4 River are shown in Table 1. As shown in Table 1, the TN [16]. concentrations in the sediment ranged from 0.864-0 mg/kg, while TP concentrations varied from 11.207-2.146 g/kg. The data shows that the soil nutrient element content of Fen River is in the medium level, which indicates that the nutrient content has certain influence on the soil environment of this region. In addition, the TN and TP content in soil is lower than the background value of soil element in the Shanxi Province, and is lower than the national soil quality specification, which indicate that the soil environment of the Fen River basin is less eutrophication.

3.2. Traverses distribution of heavy metals in sediment To investigate the traverses distribution of heavy metals in the Fen River, Site 1 Five sediment samples of site 1 was chosen as the present research subjects. According to

FIGURE 3 - The schematic diagram of the test equipment analytical results of measured data, as shown in Fig. 4, the

TABLE 1 - TN and TP contents of the sediment in Fen River

No. Distance(m) Depth(cm) Qingxu site Wangrong site Yaodu site TN TP TN TP TN TP Sation 1 0 0-40 0.045 9.455 0.728 7.313 0.325 9.87 40-80 0.287 6.095 0.169 5.663 0.125 9.553 80-120 0 8.793 0.684 6.120 0.11 7.754 120-160 0.133 5.546 0.062 6.263 0.03 9.07 160-200 0.759 8.751 0.253 6.518 0.798 9.47 Sation 2 30 0-40 0.141 9.445 0.033 11.07 0.303 3.924 40-80 0.094 7.59 0.016 11.20 0.34 5.033 80-120 0.574 10.479 0.013 9.745 0.67 7.109 120-160 0.049 2.146 0.03 9.82 0.484 8.328 160-200 0.549 9.48 0.034 10.32 0.473 6.656 Sation 3 60 0-40 0.72 10.427 0.033 8.733 0.401 7.46 40-80 0.488 3.789 0.016 7.84 0.651 11.207 80-120 0.162 10.705 0.013 8.125 0 10.988 120-160 0.108 8.936 0.03 6.13 0.109 10.184 160-200 0.101 10.824 0.034 6.283 0.556 8.683 Sation 4 90 0-40 0.864 7.45 0 6.883 0.162 11.15 40-80 0.074 9.528 0.096 6.643 0.145 10.291 80-120 0.058 9.155 0.058 6.264 0 9.218 120-160 0.179 6.284 0 5.958 0.164 10.249 160-200 0.484 10.181 0.119 7.123 0 10.524 Sation 5 120 0-40 0.304 9.622 0.853 10.506 0.043 4.78 40-80 0.661 9.634 0.728 10.394 0.068 9.008 80-120 0 8.359 0.674 8.665 0.027 9.835 120-160 0.666 7.646 0.478 9.93 0.037 10.126 160-200 0.097 4.277 0.304 6.96 0.01 6.55

14 Pb Cr 12 Cd 10

Concentration (mg/g) Concentration 4

0 20 40 60 80 100 120 140 160 180 200 220 Depth (cm) Qingxu Site

FIGURE 4 - Traverses distribution of heavy metals in Fen River

concentration of heavy metals (Pb, Cr and Cd) in sediment 18 decreased as distance away from the fen river main channel. Adopting the heavy metal contents in soil of Shanxi 15 Province are as the background value (Pb =14.7 mg/kg, Cr= Pb 55.3 mg/kg, Cd=0.1 mg/kg) [17]. As compared to the back- 12 ground value of Pb, the soil was affected by the heavy metal Cr Cd pollution of Fen River in the 100 m range from the channel. 9 Data shows that the Cr content in the sediment at different sampling points was lower than the soil background 6 value of Shanxi Province, which indicated that the Cr con- Concentration (mg/kg) tent has a tiny effect to the soil. The results showed that the 3 concentration of Cd in the sediment are higher than the background value of Shanxi Province, indicated that the Cd con- 0-40 40-80 80-120 120-160 160-200 tent has an potential ecological risk to the ecology system. Depth (cm)

Yaodu Site 3.3 Vertical distribution of heavy metals in sediment The dates of the station 1 in site 1 were collected to FIGURE 5 - Vertical distribution of heavy metals in Fen River investigate the vertical distribution of heavy metals in the The sediment can be divided into three layers: the soft sediment. As shown in Fig. 5, the change in depth of the black ooze layer, the brown mud layer and the sandy layer Pb, Cr and Cd concentrations suggests that the deposition according to the sampling observation. The black ooze rate decrease with distance from the channel. layer is obviously in a flow state with a peculiar smell, the brown layer contain more water but not flow with smelly. The third layer is sandy layer and is wet with little smelly. 18 Combined with the characters of initial station sectional 16 soil layer, the black ooze layer is formed from the sedimen-

14 tation and accumulation of mineral, sediment and contaminant that carried by the river ford. The soil layer is rather 12 Pb rough, the particles are bigger, the soil pore space is a little 10 Cr bit bigger. In the saturated state, the pores are filled with Cd 8 water and the average pore velocity of soil water move- ment is larger, and the convection velocity of the soil so- 6 lutes is faster, and the exchange between the pollutants and

Concentration (mg/g) 4 the soil surface layer is very quick. The contaminants in

2 river water directly affects the contaminant contents of black ooze layers. Due to the long-term contamination of 0 0-40 40-80 80-120 120-160 160-200 the Fen River, the lead content level is the highest in this Depth (cm) layer. The heavy metal Pb, Cr and Cd in the brown soil Wanrong Site layer is mainly come from the Pb, Cr and Cd vertical migration in the black ooze layer and the accumulation of Pb, Cr and Cd infiltration with the river water, so, in this layer,

TABLE 2 - Heavy metals contents of the sediment in Fen River

Yaodu Site Wanrong Site Qingxu site Distance Depth NO. Heave Metals Heave Metals Heave Metals (m) (cm) Pb Cr Cd Pb Cr Cd Pb Cr Cd 0-40 18.4 5.6 1.4 17.8 8.2 2.0 19.4 9.2 1.4 40-80 17.6 3.2 1.2 17.2 7.6 1.6 16.8 7.4 1.2 Station 1 0 80-120 15.8 2.8 1.2 16.2 7.2 1.4 16.0 5.2 1.0 120-160 15.0 2.4 1.0 15.6 6.8 1.2 15.4 3.8 1.0 160-200 14.8 1.9 0.8 15.4 6.7 1.2 15.3 3.2 0.8 0-40 21.2 8.0 1.4 18.2 7.2 1.2 22.8 9.6 1.6 40-80 19.2 6.0 1.2 16 7.0 1.0 21.4 7.2 1.2 Station 2 30 80-120 16.4 5.4 1.0 14.2 6.6 1.0 19.2 5.4 1.0 120-160 18.8 5.0 1.0 13.2 6.2 0.8 18.4 5.4 0.8 160-200 18.6 4.6 0.8 11.8 6.2 0.8 17.6 4.6 0.8 0-40 16.8 4.6 1.6 16.4 6.2 2.0 21.8 10.6 1.4 40-80 16.4 4.2 1.1 14.2 5.4 1.4 20.8 8.4 1.2 Station 3 60 80-120 16.0 3.8 0.8 12.8 5.4 1.2 19.8 7.2 1.0 120-160 15.8 3.6 0.6 12.6 5.0 1.2 18.8 6.8 1.0 160-200 15.6 3.4 0.4 12.6 4.8 0.8 16.4 6.6 0.8 0-40 16.2 5.4 1.2 17.0 4.6 1.0 23.2 4.0 1.6 40-80 14.8 5.0 1.0 15.2 3.6 0.8 21.8 3.8 1.4 Station 4 90 80-120 14.6 4.4 0.8 14.2 3.4 0.5 21 3.6 1.0 120-160 14.4 4.2 0.6 13.6 3.1 0.3 20.6 3.4 0.8 160-200 14.2 4.2 0.6 13.4 3.0 0.2 20.4 3.4 0.8 0-40 19.4 4.8 1.2 12.2 5.8 1.4 19.2 5.2 1.4 40-80 18.0 4.0 0.8 12.0 5.4 1.2 16.0 4.8 1.0 Station 5 150 80-120 14.6 4.2 0.8 11.6 5.2 1.2 14.8 4.4 0.8 120-160 14.4 4.0 0.6 11.4 4.6 1.0 14.6 4.2 0.6 160-200 14.2 4.0 0.6 11.0 4.2 1.0 14.3 4.0 0.5

the content of heavy metals take second place. The sandy heavy metals gradually reduces, resulting in the moisture layer locates the lowest in this soil picking depth, the pol- content of the soil is reduced, which changes from satu- lutant transport path is long, so pollution level is the mini- rated to unsaturated soil. Consequently, the main influence mum, and the concentration of heavy metals is the least. factors for the heavy metals concentrations are the molecular diffusion and atmospheric precipitation. Therefore, the 3.4 The distribution of heavy metals along Fen River pollution of the heavy metals at the point of 30m from the As shown in Table 2, the highest heavy metal concen- riverbed and water boundary is the most serious area; es- trations were recorded at site 3 (Qingxu County). This may pecially the concentration of Cd exceeds the standard of be due to the industrial and domestic sewerage discharged Class III (1.0 mg/kg) of the soil environment quality from the Taiyuan city and the sewerage carried from Jin- (GB15618-1995, National Standard of the People’s Repub- zhong city which emptied into the Fen River. lic of China). As the soil of the Fen River is weakly alkaline, and polluted heavily by cadmium which exists in re- The heavy metal content in site 2 (Yaodu County) is fractory state, it is suggested to be taken the following relatively low though located in the downstream of Fen measures for prevention: River. It can be attributed to the input of the surface runoff from the Linghuo mountain gorges. The soil quality in the 1. We should increase the intensity of environmental Wanrong county area was obviously worse compared to monitoring, control the industrial waste water and emis- the Yaodu area. It can be attributed to the lower water ve- sions strictly, and be reasonable mining and smelting [18]. locity and sewage collected from the Yuncheng and Linfen 2. To plant cruciferous fixing blue food or willow area. The selected sample sections were representation for within the scope of the Fen River along [19]. other area can reveal the diffusion and distribution of heavy metals in sed- be changed into non-agricultural construction land based iment along the Fen River. on needs. As is shown in Table 2, the heavy metals concentrations along the Fen River reach maximum at 30m in the boundary of riverbed. The possible reasons are as follows: 4. CONCLUSIONS in the range of 0-30m, the concentrations of the heavy metals in the soil are affected by the Fen River’s water convec- Sediment samples were collected from three sites in tion and molecular diffusion. Beyond 30m, the influence of the Fen River system and tested for nutrient (TN and TP) the Fen River water convection on the concentrations of the and heavy metal contamination (Pb, Cr and Cd). The re-

sults show that the heavy metals content decrease with the [11] Hou, J.H., Feng, M.Q., Xing, X.P., Hou, Z.H. (2012) Research increase of horizontal distance from the main channel in on the pollution diffusion regularity near sewage outlet areas in Yuncheng reach of the Fen River. Advanced materials Re- the horizontal direction along the transverse section. search, 383-390, 2430-2436. The characteristic of vertical distribution of heavy [12] Local legislation to support transjurisdictional water pollution metals (Pb, Cr and Cd) suggests that the deposition rate de- management. Final Report, 2003, Environment Protection and crease with distance from the channel, and the correlation Natural Resources Conservation Committee, National Peo- analysis between the characteristics of soil and the content ple’s Congress, PRC. TA3588-PRC. of heavy metals were studied. This study indicates that the [13] Zhang, B., Fang, F., Guo, J.S., Chen, Y.P., Li, Z., Guo, S.S. riverbed sediment was polluted with nutrient and heavy (2012) Phosphorus fraction and phosphate sorption-release metals. The results demonstrate the pollution has an eco- characteristics relevant to the soil composition of water-level- fluctuating zone of three gorges reservoir. Ecological Engi- logical risk and need of the development of an efficient neering, 40, 153-159. strategy to reduce local pollution and contamination. [14] Guebel, D.V., Nudel, B.C., Giulietti, A.M. (1991) A simple and rapid micro-kjeldahl method for total nitrogen analysis. Biotechnology techniques, 6, 427-430. ACKNOWLEDGEMENTS [15] Wang, B., Liu, G. B., Xue, S., Zhu, B.B. (2011) Changes in soil physico-chemical and microbiological properties during natural succession on abandoned farmland in the Loess Plat- This work was supported by the Program of River Man- eau. Environ Earth Sci, 62, 915-925. agement Service Station of Shanxi Province (2010-11). [16] Parkinson J A, Allen S E. (1975). A wet oxidation procedure suitable for determination of nitrogen and mineral nutrients in The authors have declared no conflict of interest. biological material. Communications in Soil Science and Plant Analysis, 6, 1-11. [17] Yang, G. Y., He, Q. S. and Yang, Z. J. (2011) Research on ecological risk and control of Cr pollution of Fenhe River in Taiyuan section. Communications in Computer and Infor- REFERENCES mation Science, 158, 206–213.

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GLOBAL ENVIRONMENTAL INPUT–OUTPUT RESEARCH TRENDS DURING 1900–2013: A BIBLIOMETRIC ANALYSIS

Jingqing Zhou, Zhen Wang*, Beibei Niu and Song Hong

School of Resource and Environmental Sciences, Wuhan University, Wuhan 430079, China

ABSTRACT used in many aspects of resource and environment, such as environment, ecology, geoscience, and agriculture. To This article applied a bibliometric analysis on the in- some extent, these studies have transformed environmental put–output analysis of environmental issues in the SCIE impact assessment from a traditionally production-oriented and the SSCI databases during 1900–2013. The results way to a consumption-oriented way, which has profoundly showed that the number of articles increased annually, influenced policies regarding world trade, energy, green- reaching a total of 3,532. Environmental Science & Ecol- house gas emissions, etc. Despite the growing application ogy and Energy Policy were the most productive Web of of EIO, existing studies are dedicated to specific fields, Science category and scientific journal, respectively. The such as the relationship of embedded energy and carbon USA, the UK, and China were ranked as the three most ac- emission [4, 5], environmental impact generated by trade tive countries in this field and served as the center of three [6], and the method’s principles, application fields, and ad- geographical research aggregation areas: North America, vantages and disadvantages [3]. These studies have inves- Western Europe, and East Asia. Other researchers in this tigated past and current research qualitatively. There is no field were mostly from developed countries located in holistic review on the past EIO studies. these areas. Developed European countries played an im- Bibliometrics is a technique for the analysis of litera- portant role in international cooperation, which could sig- ture that uses mathematical and statistical methods to quan- nificantly raise the impact factor of articles published by titatively summarize research trends in a particular area [7- authors in developing countries. Keyword analysis indi- 9]. Conventional bibliometrics has focused on changes in cated that the hot spots in this field were climate change, the volume of published scientific papers and the publica- international trade, energy consumption, and CO emis- 2 tion outputs of different countries, institutions, authors, re- sions. China became a hot study region of environmental search fields, and journals [10, 11]. Recent bibliometric input–output in the last 5 years. A co-word analysis re- studies have focused on cooperation between countries, in- vealed that the transfer of energy and environmental pres- stitutions, and authors and on the role of citations in char- sure in the process of international trade have attracted in- acterizing the influence of a paper [12, 13]. Co-word anal- creasing attention. ysis is also widely employed in bibliometrics [14-16].

The objective of this study was to quantitatively deter- KEYWORDS: Environmental input–output; Bibliometric; Research mine the research status of EIO, as a supplement to previ- trend; International collaboration; Research hotspot ous reviews, and to inform subsequent research. In this paper, we present a bibliometric analysis of published EIO

1. INTRODUCTION studies during 1900–2013.

Input–output analysis is a practical method of eco- 2. MATERIALS AND METHODS nomic analysis, first introduced by Leontief [1, 2]. It is used to study the interdependent relationships between sectors in an economic system, and to systematically analyze We collected literature data from the Science Citation the internal complex transactions among various indus- Index-Expanded (SCIE) and the Social Science Citation tries. Environmental input–output (EIO) is formed by add- Index (SSCI) databases covering 1900–2013. The search ing environmental factors (including energy, environmen- term “input–output” or “input and output” was used to tal pollution) to the conventional input–output tables based identify all papers containing either of these keywords in on the proportional relationship between pollutant output their title, keywords, or abstract. To restrict our study to the and material input in one industrial sector [3]. EIO has been field of resource and environment according the EIO concept mentioned in the Introduction, we refined the subject categories to fifteen relevant Web of Science categories * Corresponding author and they were environment, energy & resources (Environ-

mental Sciences, Environmental Studies, Energy Fuels, discussions (4), book chapters (2), and one software re- Engineering Environmental, Water Resources, Soil Sci- view. In common with other bibliometric studies, only ar- ence, Meteorology Atmospheric Sciences), ecology (Ecol- ticles were used for further study, as they reflected the fo- ogy), geoscience (Geography, Oceanography, Geosci- cus of the research. The first EIO paper was published in ences Multidisciplinary), agriculture (Agronomy, Agricul- 1940, but the field of study did not emerge until the 1980s. ture Multidisciplinary, Agricultural Engineering, Agricul- Its prominence has increased dramatically in the past two tural Economics Policy), respectively. Then we retrieved decades. We focused on research published during 1994– the relevant scientific papers on EIO published during 2013 as the number of articles increased from 51 in 1994 1900–2013. The information extracted included publica- to 303 in 2013, with a 9.8% average annual growth rate. The tion type, publication year, author name and affiliation, standardized number of articles on EIO, which is defined as subject category, citations, journal name, keywords, etc. the ratio of the number of EIO articles to the total number of We then built a bibliometric dataset containing the extracted articles in the SCIE and SSCI databases, increased from information for subsequent analysis. First, some of the data 0.00558% in 1994 to 0.0166% in 2013, which indicated a were preprocessed to avoid confusion. For the authors’ ad- general trend of increasing interest in EIO research. dresses, England, North Ireland, Scotland, and Wales were replaced by the United Kingdom (UK). We treated publica- 3.2 Subject categories and journal distribution tions from Mainland China, Hong Kong, Macau, and Tai- The annual growth in the number of articles in the five wan separately. Characteristics of publication outputs, the most productive Web of Science categories (see FIGURE 1) distribution of subject categories and journals, and a con- indicated a growing annual trend. Environmental Science ventional analysis of countries and keywords were com- & Ecology was the most productive category, with a total piled using Microsoft Excel 2010. Collaborative networks of 1,470 (56%) articles, increasing from 30 in 1994 to 168 between countries and co-word analysis were processed in in 2013, with an average annual growth rate of 3.0% in the Ucinet 6. The addresses of authors were geocoded by first decade and 11.5% in the second decade. The largest in- CiteSpace [17], and Arcgis10.0 was used to map the crease found in this category reflected increasing concerns global geographic distribution of authors. around sustainability and climate change, which involves a complex interaction between the economy and the environment that can be investigated using EIO models. Engineer- 3. RESULTS AND DISCUSSION ing was the second most productive category, with 693 publications, and 67.0% of these appeared simultaneously with 3.1 Characteristic of publication outputs Environmental Science & Ecology. Articles pertain to En- A total of 3,532 EIO papers were identified in the ergy & fuels displayed rapid growth after 2008. Prior to SCIE and SSCI, among which there were 13 different types 2008, the number of articles in this category was small and of document. Articles (3,009) accounted for the largest basically unchanged. A dramatic rise in energy prices began share, with approximately 85% of the total productions. from 2008, and efforts to reduce carbon dioxide emissions Proceeding papers (263) had the next largest share, com- may explain the increasing interest in energy input–output prising 7.4% of the total, followed by reviews (95; 2.7%), analysis. The numbers of articles in the Agriculture and book reviews (69; 2.0%), meeting abstracts (32), notes Business & Economics were 397 and 353 respectively, and (19), editorial materials (18), letters (14), corrections (6), both exhibited small but steady annual growths.

FIGURE 1 - The growth of the five most productive subject categories

EIO articles were published in 519 journals during followed by the International Journal of Life Cycle Assess- 1994-2013. Table1 showed the 10 most active journals. ment (5.0%) and Annals of Regional Science (4.7%). These 10 (1.9%) out of the 519 journals published 744 (28.5%) out of the 2613 articles. The 5-year impact factor 3.3 Geographic distribution and international collaboration (IF-5) of the journals was obtained from the 2012 Journal The affiliations of authors were used to conduct a col- Cited Report (JCR) and was used to evaluate the academic laboration analysis. There were 2,383 (79.2%) articles af- influence of each journal [18-20]. We also used the average filiated with independent countries/territories, and 588 ar- citations per article in the journal (ACA) to characterize the ticles involving international collaboration. No address in- influence of the articles, and we calculated the ratio of EIO formation was given for the authors of 38 articles. Geo- articles in a journal to the total journal publications coded CiteSpace analysis, shown in FIGURE 2, demon- (TA/TJP) to characterize the journal’s coverage of EIO. As strated that the authors were clustered in North America, seen in TABLE 1, Energy Policy published 147 (5.6%) Western Europe, and East Asia. The USA contributed the EIO articles, being followed by Ecological Economics most publications in the North America region. The cluster (140). Environmental Science & Technology was the third in Western Europe was dominated by authors from the UK, most productive journal, publishing 86 articles, but its IF- the Netherlands, Spain, Germany, and France. Eastern 5 was ranked the first place, which suggested that this jour- China and Japan were the main locations for the authors of nal had the largest influence on EIO research. The Journal EIO-related publications in Asia. These clusters of authors of Industrial Ecology gave the largest coverage to EIO were consistent with the fact that these regions are developed studies, as evidenced by its largest TA/TJP ratio (10.4%), economies that have a large number of academic institutions.

TABLE 1 - The 10 most active journals in environmental input-output research

Journal IF-5 TA TA(%) TC ACA TA/TJP(%) Energy Policy 3.382 147 5.6 2375 16.2 2.1 Ecological Economics 3.732 140 5.4 3005 21.5 3.5 Environmental Science & Technology 5.865 86 3.3 2169 25.2 0.4 Energy 4.107 76 2.9 899 11.8 1.4 Journal of Industrial Ecology 3.424 70 2.7 953 13.6 10.4 International Journal of Life Cycle Assessment 3.550 55 2.1 704 12.8 5.0 Journal of Cleaner Production 3.587 46 1.8 567 12.3 1.8 Annals of Regional Science 1.155 45 1.7 295 6.6 4.7 Ecological Modelling 2.399 43 1.6 796 18.5 0.8 Water Resources Research 3.448 36 1.4 1828 50.8 0.5 TA: total published articles in corresponding journal; TA(%): the percent of articles in corresponding journal out of total published articles; TC: total citations during 1994-2013; ACA: average of citations per article in corresponding journal during 1994-2013; TJP: total journal publications during 1994-2013; TA/TJP(%): the ratio of EIO articles to total publications in the journal.

FIGURE 2 - Global geographic distribution of authors and the article output of different countries

The top 20 most active countries/territories were revealed no significant difference between single-country listed in TABLE 2. Among these, 12 came from Europe,5 publications and collaborative publications (p = 0.1294, > from Asia, 2 from North America, and 1 from Oceania. 0.05) for the top 10 countries. However, significant growth Most of them were developed countries, except China, In- in the ACA, from 10.19 to 12.93 (p < 0.01), was found in dia, and Iran. Norway ranked first in the average citations publications based on international collaboration compared per article (ACA, 24.8), although articles authored in Nor- with single-country publications for the next 10 countries. way (73) ranked just thirteenth, and the Netherlands ranked It indicated that international collaboration was a feasible second in average citations. In contrast, although China had way for these countries to improve their influence in this the third highest publication record, its average number of field. citations was only 7.6, which was lower than most listed countries. The top 10 countries in respect to ACA were all The number of internationally co-authored articles has developed countries and had average ACAs of 20.46 and grown annually, as shown in FIGURE 3. However, the per- 18.28 for single-country publications and internationally centage of internationally co-authored articles declined in co-authored publications, respectively. Statistical testing 1998 and again in 2007–2008. This may be linked to the two

TABLE 2 - The 20 most productive countries/territories

Single-country Internationally-collaborated Country/territory TA TA(%) SA SA(%) TC ACA CA CA(%) TC ACA USA 897 30 645 72 13039 20.2 252 28 4226 16.8 UK 279 9.3 174 62 2574 14.8 105 38 1917 18.3 China 271 9.0 167 62 1276 7.6 104 38 887 8.5 Netherlands 175 5.8 99 57 2616 26.4 76 43 1498 19.7 Australia 160 5.3 93 58 1489 16 67 42 1377 20.6 Japan 149 5.0 102 68 907 8.9 47 32 403 8.6 Spain 139 4.6 98 71 905 9.2 41 29 500 12.2 Germany 137 4.6 82 60 932 11.4 55 40 907 16.5 Canada 130 4.3 74 57 907 12.3 56 43 715 12.8 France 107 3.6 59 55 1151 19.5 48 45 909 18.9 Italy 101 3.4 68 67 713 10.5 33 33 494 15.0 India 96 3.2 78 81 760 9.7 18 19 221 12.3 Norway 73 2.4 36 49 1116 31 37 51 694 18.8 Turkey 73 2.4 64 88 1137 17.8 9 12 97 10.8 Taiwan 70 2.3 59 84 674 11.4 11 16 139 12.6 Iran 67 2.2 56 84 348 6.2 11 16 175 15.9 Sweden 60 2.0 35 58 636 18.2 25 42 493 19.7 Switzerland 48 1.6 19 40 421 22.2 29 60 466 16.1 Austria 47 1.6 19 40 351 18.5 28 60 648 23.1 Finland 46 1.5 34 74 501 14.7 12 26 179 14.9 SA: single-country articles; SA(%): percentage of single-country articles out of TA; ACA: average of citations per article; CA: internationally-collaborated articles; CA(%): percentage of internationally-collaborated articles

FIGURE 3 - Annual growth of single-country and internationally collaborative papers

global economic crises in these periods. The lack of avail- with China (49), the UK (36), the Netherlands (26), and Aus- able funds hindered international collaboration, and con- tralia (20). Therefore, the USA held a central position in the cerns about energy use and CO2 emissions spurred single- collaboration network. The USA’s largest partner, China, country research on EIO, which enabled resource and en- cooperated less with other countries/territories, such as Ja- ergy flows in various economic sectors to be tracked. Over pan (18), the UK (11), and Canada (11). FIGURE 4 (b) the whole period, the proportion of collaborative articles showed the average citation of each country in collaborative still increased, from 14% in 1994 to 23% in 2013. publications. The Netherlands, France, Austria, Switzerland, Norway, Italy, and Germany had important roles in high- International collaboration networks of the 20 most quality collaborative articles. For example, the ACA of arti- productive countries/territories were present in FIGURE 4. cles involving collaborative work by the USA with the UK, The size of nodes in FIGURE 4 (a) and (b) represented the the Netherlands, France, Norway, and Austria were 21.69, amount and the ACA of internationally co-authored articles 27.61, 21.07, 28.55, and 34.67, respectively, higher than that for each country/territory, respectively. The thickness of for the USA’s single-country articles (20.2). A similar im- lines in FIGURE 4 (a) and (b) represented the amount and provement in the ACA was also found between China and the ACA of two connected countries/territories. FIGURE 4 these European countries. (a) showed that in terms of participating in international collaboration, the USA published a larger share of articles than 3.4 Hot spots did other countries/territories, followed by the UK, China, Author keywords reflect the focus of a paper’s con- the Netherlands, and Australia. The USA cooperated closely tents [21, 22]. They can be used to track hot spots and re-

(a)

(b) FIGURE 4 - Collaborative networks of the 20 most active countries/territories in (a) number of articles and (b) average citations

search trends. There were 6,452 keywords in the 3,009 ar- analysis as a method of environmental life cycle assess- ticles. However, 5,319 keywords appeared only once, and ment, called economic input–output life cycle assessment 947 keywords appeared two to five times. The 20 most fre- (EIO-LCA), was mainly used to analyze the environmental quent keywords were separated into four periods of five- impact of a product or service chain [23, 24], e.g., the cal- years duration each (1994–1998, 1999–2003, 2004–2008, culation of greenhouse gas emissions that were implicit in and 2009–2013) to analyze their percentages and frequen- international trade [25]. Another hot spot was neural net- cies in each period (see TABLE 3). A co-word analysis of works, including the terms “artificial neural network” and these 20 frequently used keywords was also conducted, as “neural network”. Neural network model can optimize in- depicted in FIGURE 5. put–output systems. There were also some frequent keywords regarding specific research methods used in input– Considering the methodology associated with EIO, output models, including “sensitivity analysis”, “structural the hottest spot was “life cycle assessment”. Input–output decomposition analysis”, and “ecological footprint”.

TABLE 3 - The 20 most frequently used author keywords

1994-1998 1999-2003 2004-2008 2009-2013 Author keywords TA % A A(%) A A(%) A A(%) A A(%) input-output 398 15 22 7.03 42 11.08 119 17.42 215 17.37 life cycle assessment 92 3.5 0 0 9 2.37 22 3.22 61 4.93 artificial neural network 68 2.6 3 0.96 10 2.64 22 3.22 33 2.67 industrial ecology↑ 57 2.2 0 0 0 0 10 1.46 47 3.80 model 48 1.8 3 0.96 5 1.32 16 2.34 24 1.94 China↑ 47 1.8 0 0 1 0.26 8 1.17 38 3.07 CO2 emissions↑ 47 1.8 2 0.64 1 0.26 13 1.90 31 2.50 neural network 45 1.7 0 0 7 1.85 19 2.78 19 1.53 energy↑ 39 1.5 3 0.96 4 1.06 9 1.32 23 1.86 nitrogen 38 1.5 3 0.96 8 2.11 16 2.34 11 0.89 sustainability 32 1.2 8 2.56 5 1.32 2 0.29 17 1.37 international trade↑ 32 1.2 0 0 3 0.79 8 1.17 21 1.70 ecological footprint↑ 31 1.2 1 0.32 3 0.79 9 1.32 18 1.45 sensitivity analysis 29 1.1 0 0 2 0.53 11 1.61 16 1.29 phosphorus 29 1.1 4 1.28 7 1.85 8 1.17 10 0.81 climate change↑ 27 1.0 0 0 3 0.79 6 0.88 18 1.45 agriculture 26 1.0 1 0.32 3 0.79 9 1.32 13 1.05 energy efficiency↑ 25 0.96 0 0 1 0.26 1 0.15 23 1.86 energy consumption 23 0.88 2 0.64 0 0 3 0.44 18 1.45 structural decomposition analysis 22 0.84 1 0.32 1 0.26 2 0.29 18 1.45 TA: total articles; %: the percentage of articles; A/A(%): articles /percentage in the study period; ↑: the upward trend in rank

FIGURE 5 - Collaborative networks of the 20 most frequently used author keywords

Sensitivity analysis studied the impact of inputs and mental science & ecology, engineering, energy & fuels, ag- outputs data changes on effectiveness of decision making riculture, and business & economics were the top five most unites in the multi-inputs and multi-outputs system[26, 27]. productive categories. Energy Policy contained the most Structural decomposition analysis, in the respect of input- EIO related articles, whereas Environmental Science & output, was mainly used in studies of energy utilization Technology had the highest influence in this field. structures and/or CO2 emissions change in economic sys- On the national scale, 103 countries/territories con- tems [28-31]. Ecological footprint analysis was used to cal- ducted EIO research. The authors were clustered in North culate the ecological footprint of specific environmental America, Western Europe, and East Asia. Developed coun- impacts caused by industries on the regional-scale [32], na- tries, especially the USA and Western European countries, tional-scale [33] or international-scale [34, 35] from the were very active and influential in this field. The propor- perspective of land use and energy consumption [35]. tion of articles with international collaboration increased in the past two decades. International collaboration clearly With regard to the application of an EIO model, “in- enhanced the influence of an article, and it should be en- dustrial ecology” ranked the hottest (57 times total), having couraged in countries such as China and India. The USA emerged in recent decades. Inter-industry interactions and was the leading country in terms of both single-country re- the interaction between industries and the environment search and international collaboration. The developed Eu- were the two major foci of EIO studies [36, 37]. The key- ropean countries had an important role in international col- word “China” did not emerge in EIO articles until 2003. laboration, which significantly improved the average cita- However, it is the only country listed in the top 20 key- tions per article. words, growing from a single article in 1999–2003 to 38 articles in 2009–2013. Due to its size, rapid economic The EIO model was usually combined with life cycle growth, and increasing environmental pressure, China has assessment, neural network analysis, and structural decom- struggled to mitigate its energy consumption and to reduce position analysis. It was widely applied in the areas of cli- environmental impacts during the economic transition pro- mate change, international trade, energy consumption, and cess [38, 39]. These efforts could be seen in FIGURE 5, CO2 emissions. China became a hot study region in the last 5 years. The transfer of energy and environmental pressure where “China” was linked with “CO2 emission”, “international trade”, “climate change”, and “energy consump- in the process of international trade was a topical hotspot. tion”. The percentage composition and occurrences of these linked keywords increased in the last two decades and became hot spots in EIO research. Energy had a central ACKNOWLEDGEMENT position in the application of EIO, including structural changes in industrial energy efficiency [31], the relation- This work was supported by National Natural Science ship between energy consumption and international trade Foundation of China (Grant No. 41101567), and the Youth [40], and the embodied carbon from energy consumption Chen-guang Project of Science and Technology of the City [41, 42]. Due to the close relationship between energy and of Wuhan (Grant No. 2014070404010199). We also thank the financial award from State Scholarship Fund of China CO2 emissions, CO2 emissions [43] and the related topic of climate change [44] also became popular in recent years. Scholarship Council (File No. 201308420277) to support The high pairwise frequency of appearance of these key- the research at UCSB. words was shown in FIGURE 5, indicating that studies of the relationship between energy consumption and carbon The authors have declared no conflict of interest. emissions, and the transfer of energy and environmental pressure in the process of international trade [45] were the research hotspots. REFERENCES

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Received: October 14, 2014 Revised: November 10, 2014 Accepted: November 18, 2014

CORRESPONDING AUTHOR

Zhen Wang Room 225 School of Resource and Environmental Sciences Wuhan University Wuhan 430079 P.R. CHINA

Phone: +86 17771465253 E-mail: [email protected]

FEB/ Vol 24/ No 5b/ 2015 – pages 1996 - 2004

SUBJECT INDEX

A M active carbon nanoparticles (MAC-NPs) 1947 mercury 1958 Amanita pantherina 1981 mushroom 1981 antioxidant parameters 1958 Apolaccase (ApoLac) 1947 N nitrate reductase 1976 B Basic red 9 (BR9) 1947 O bibliometric 1996 Oreochromis niloticus 1958 Oreochromis niloticus 1985 C oxidative stress 1958 cancer proliferation 1920 Cd 1940 P Cd 1985 plastid pigments 1976 chorotype 1932 pollution index 1966 Chrysomelidae 1932 corrosion 1926 R cyprodinil 1920 research hotspot 1996 research trend 1996 D riverbed soil 1990 decolorization 1947 Drosophila melanogaster 1920 S selenium 1958 E SEM-EDS 1909 electrode material 1940 soil 1940 electrokinetic remediation 1940 sorption/retention capacity 1909 environmental input–output 1996 source identification 1966 synthetic auxin 1976 F synthetic cytokinin 1976 fauna 1932 Fen River 1990 T Fenton 1947 Thermo-Electric Power Plant 1981 fludioxonil 1920 trace elements 1909 Tunisia 1909 G Turkey 1932 genotoxic effect 1920 growth regulator 1976 U urban park and square 1966 H W heavy metal 1966 heavy metal 1990 wastewater 1947 heavy metal cations 1926 weak magnetic field 1926 heavy metals 1981 wing spot test 1920 Helophoridae 1932 hematological parameters 1985 Z Hydrophilidae 1932 Zero valent iron 1926 hysteresis 1909 Zn 1985

I International collaboration 1996

L lucerne 1976

AUTHOR INDEX

A Q Andrade, María Luisa 1909 Qi, Yu 1990 Aslan, Baran 1932 Qiao, Junlian 1926 Aslan, Ebru G. 1932 S B Sahraoui, Hamdi 1909 Bayram, Fatma 1932 Savaş, Burhan 1920 Sosnowski, Jacek 1976 C Cai, Zongping 1940 T Celebi, Neslihan 1947 Tarhouni, Jamila 1909 Chen, Dongrui 1940 Topcuoğlu, Ş. Fatih 1920 Çoğun, Hikmet Y. 1958 Covelo, Emma F. 1909 V Van Doren, Jeremiah 1940 F Vega, Flora Alonso 1909 Fan, Guisheng 1990 Fang, Zhanqiang 1940 W Fırat, Özge 1958 Wang, Chao 1926 Fırat, Özgür 1958 Wang, Lei 1926 Fırat, Özgür 1985 Wang, Yang 1966 Firidin, Beran 1958 Wang, Zhen 1996 Firidin, Gülbin 1958

X G Gungor, Azize Alayli 1947 Xiao, Zhongjin 1926 Xing, Lidan 1940 H Y Hachicha, Mohamed 1909 Hong, Song 1996 Yılmaz, Ayçin 1932 Hristovski, Slavcho 1981 Yüzereroğlu, Tüzin A. 1958

K Z Karadelev, Mitko 1981 Zhou, Jingqing 1996 Karadeniz, Asuman 1920 Zou, Mingying 1966 Kargın, Ferit 1958 Kargin, Ferit 1985 Kaya, Bülent 1920

L Li, Weishan 1940 Liu, Jingshuang 1966 Liu, Qiang 1966

M Ma, Jun 1990 Melovski, Ljupcho 1981 Murati, Emri 1981

N Nadaroglu, Hayrunnisa 1947 Niu, Beibei 1996

O Oliveira, Luis Felipe Silva 1909

2006