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CONTENTS

ORIGINAL PAPERS

AIR QUALITY IN A MOUNTAINOUS CITY: A CASE STUDY IN CHONGQING, CHINA 2699 Shu-min Wang, Hui Yu, Li Song, Yun-cheng Xie and Qi-hong Zhu

BIODIVERSITY OF GRASSLAND COMMUNITIES AND SPATIAL VARIABILITY 2707 OF FUNCTIONAL GROUPS IN THE MONGOLICA FOREST-STEPPE ECOTONE Jinman Wang, Jianjun Ma, Hong Yao and Shihai Lv

ANTIBACTERIAL ACTIVITIES OF ESSENTIAL OILS, EXTRACTS AND SOME OF THEIR MAJOR 2715 COMPONENTS OF Artemisia spp. L. AGAINST SEED-BORNE PLANT PATHOGENIC BACTERIA Fatih Dadasoglu, Recep Kotan, Ahmet Cakir, Ramazan Cakmakci, Saban Kordali, Hakan Ozer, Kenan Karagoz and Neslihan Dikbas

HEAVY METAL CONCENTRATIONS IN 2725 Mytilus galloprovincialis FROM ÇANAKKALE STRAIT, NW TURKEY Serkan Özden and Sezginer Tunçer

EFFECT OF ZINC SULPHATE ON THE LEVELS OF 2732 PLASMA PARAOXONASE ACTIVITY, TOTAL OXIDANT AND HIGH DENSITY LIPOPROTEIN OF TRANSCAUCASIAN BARB (Capoeta capoeta [Guldenstaedt, 1773]) Haci Ahmet Deveci, İnan Kaya, Muhittin Yılmaz and Mahmut Karapehlivan

KINETIC AND SORPTION EQUILIBRIUM STUDIES ON PHOSPHORUS REMOVAL 2736 FROM NATURAL SWIMMING PONDS BY SELECTED REACTIVE MATERIALS Agnieszka Z. Bus and Agnieszka A. Karczmarczyk

NUTRIENT EXTRACTABILITY AND BIOAVAILABILITY OF FRESH AND 2742 COMPOSTED POULTRY LITTER AND ITS APPLICATION ON MAIZE (ZEA MAYS) CROP Faridullah, Arif Alam, Muhammad Umar, Akhtar Iqbal, Muhammad Amjad Sabir and Amir Waseem

IN VITRO WHOLE PLANT REGENERATION OF 2747 THE MEDICIAL AQUATIC PLANT-Limnophilla aromatic Mehmet Karataş and Muhammad Aasim

RESIDUE MANAGEMENT AFFECTS GREENHOUSE GAS EMISSIONS 2751 AND SOIL ORGANIC CARBON IN WHEAT-RICE ROTATION SYSTEM Jie Wu, Wenbo Wang, Xiaohua Wang, Liqun Zhu, Haishui Yang, Xinzhong Han, Jie Gao, Wei Guo and Xinmin Bian

PHYSICAL CHARACTERISATION OF NATURAL ORGANIC MATTER AND DETERMINATION 2763 OF DISINFECTION BY-PRODUCT FORMATION POTENTIALS IN ISTANBUL SURFACE WATERS Edip Avsar, Ismail Toroz and Asude Hanedar

EFFECTS OF COPPER AND LEAD ON SOME 2771 HEMATOLOGICAL PARAMETERS OF Oreochromis niloticus Nuray Çiftçi, Fahri Karayakar, Özcan Ay, Bedii Cicik and Cahit Erdem

AN EVALUATION OF THE ENVIRONMENTAL SENSITIVITY 2776 TO LAND DEGRADATION IN MURCIA REGION, SE SPAIN María José Martínez-Sánchez, Carmen Pérez-Sirvent, Mari Luz García-Lorenzo and José Molina-Ruiz

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PHYTOREMEDIATION OF SOME HEAVY METALS BY DIFFERENT TISSUES OF 2787 ROSES GROWN IN THE MAIN INTERSECTIONS IN ERZURUM CITY, TURKEY Aslihan Esringu, Elif Akpinar Kulekci, Metin Turan and Sezai Ercisli

HEAVY METALS SORPTION ON RIVER SEDIMENTS: 2792 KINETICS, EQUILIBRIA AND RELATIVE SELECTIVITIES Yuanxing Huang, Daofang Zhang, Yuanheng Li, Zhihua Xu, Shijue Yuan and Lian Wang

THE IMPACTS OF LAND USE ON MACROINVERTEBRATES 2800 COMMUNITIES IN QINGHE RIVER DRAINAGE, LIAOHE RIVER BASIN, CHINA Fayun Li, Yanjie Wang, Zhiping Fan, Xingna Lv, Yaming Hu and Lizhu Wang

TOXIC EFFECTS OF DIFFERENT LONG TERM ZINC CONCENTRATIONS 2809 ON THE ACCUMULATION IN MUSCLES, VISCERAL ORGAN AND GILLS OF JUVENILES OF Solea solea L., 1758 Oylum Gökkurt Baki, Birol Baki and Levent Bat

ADSORPTION OF COPPER(II) BY MODIFIED MAGNETITE NANOPARTICLES: 2815 ADSORPTION EFFICACY, EQUILIBRIUM, KINETIC AND REUSABILITY Ahmad Farrokhian Firouzi, Ali Akbar Babaei, Seyyedeh Maasoumeh Hosseini and Fariba Heidarizadeh

CHELATE ASSISTED PHYTOREMEDIATION OF 2824 Pb, Cd AND B BY SUNFLOWER, MAIZE AND CANOLA Ömer Vanlı and Mustafa Sait Yazgan

THE EFFECTS OF FIVE FOOD DYES ON THE LONGEVITY OF DROSOPHILA MELANOGASTER 2830 Şifa Türkoğlu, Dilek Benli and Nihan Şahin

NATURAL RADIOACTIVITY OF FERTILIZERS WIDELY USED 2837 IN THE AGRICULTURAL LANDS OF EGE REGION IN TURKEY Bihter Çolak Esetlili, Günseli Yaprak and Dilek Anaç

ENHANCEMENT OF WASTE ACTIVATED SLUDGE AEROBIC DIGESTION WITH 2843 COMBINED FENTON OXIDATION AND ELECTROCHEMICAL PRETREATMENT METHOD Haiping Yuan, Qingji Wang, Shengjuan Guo, Jie Yao, Nanwen Zhu and Yi Gong

SPINACH (Spinacia oleracea L.)RESPONSE AND ACCUMULATION OF SALTS 2850 IN SOIL UNDER SURFACE AND SUBSURFACE WASTEWATER IRRIGATION Zobia Anwar, Muhammad Irshad, Iftikhar Fareed and Faridullah

INVESTIGATION OF SOME PROPERTIES OF BACILLUS SPP. 2860 ISOLATED FROM ARCHAEOLOGICAL EXCAVATIONS SOIL Yasemin Yeşiltaş, Ferdağ Çolak and Elif Genç

INDEX 2868

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AIR QUALITY IN A MOUNTAINOUS CITY: A CASE STUDY IN CHONGQING, CHINA

Shu-min Wang1,*, Hui Yu2, Li Song1, Yun-cheng Xie1and Qi-hong Zhu1,*

1 Chongqing Key Laboratory of Environmental Material and Restoration Technology, Chongqing University of Arts and Sciences, Chongqing, 402160, China 2 Library of Chongqing University of Arts and Sciences, Chongqing, 402160, China

ABSTRACT pending on climate and topography, and on the nature of the available energy sources. Although the air quality in Air quality has become a direct threat to human health. China cities has generally improved in recent decades, air Compared to plain cities, the characteristic of temporal and pollution is still considered to be a top priority environmen- spatial distribution of air quality in mountainous cities is tal problem [1, 2]. more complex for the more intricate terrain conditions. Amongst the gaseous pollutants of atmospheric inter- Yangjiaping (YJP), Baishiyi (BSY), Chongqing Technol- est, SO2, NO2 and PM10 (particle with the aerodynamic di- ogy and Business University (CTBU) and Tongliang (TL) ameter less than 10μm) are of major concern for their con- in Chongqing (e.g., the largest mountainous city in China) tribution to global acidification and public health [3]. SO2 are selected as study areas on behalf of the central and sur- is soluble in water and can be converted into sulphuric acid rounding regions respectively. From 2002 to 2012, statisti- (H2SO4) easily within airborne water droplets, while NO2 cal analysis of SO2, PM10 and NO2 concentrations is con- can be converted into nitric acid (HNO3) with the presence ducted. Study results show that average concentrations of of humidity [4]. These acidic pollutants can be transported 3 SO2, PM10 and NO2 in YJP (e.g., 92, 126 and 52 μg/m ) are by wind over many hundreds of kilometers, and are depos- 3 higher than that in BSY (e.g., 42, 84 and 30 μg/m ), CTBU ited as acid rain then [5]. Furthermore, both SO2 and NO2 (e.g., 42, 92 and 31 μg/m3) and Tongliang (e.g., 45, 61 and are irritant gases. A great number of studies have assessed 3 31 μg/m ). Compared to secondary standard values speci- the air pollution eﬀects of nitrogen oxides on human health fied in Ambient Air Quality Standards (GB 3095-2012), [6]. Compared to NO, NO2 is the more important for air concentrations of SO2, PM10 and NO2 in surrounding cities quality since is more relevant for human health. It is an ir- are within the limited values, while they exceed the annu- ritant gas, which has both chronic and acute effects and it ally average standard value in the central city. The analysis is associated with respiratory and cardiovascular diseases of Pearson correlation indicates that concentrations of SO2, [5, 7]. PM10 is considered to be another major environmen- PM10 and NO2 are positively correlated with atmospheric tal pollutant in urban areas. It is well known that these par- pressure, but negatively with temperature and wind speed. ticulates may cause serious health problems, especially The annual concentrations of SO2, PM10 and NO2 in YJP, those associated with respiratory and cardiovascular dis- TL and CTNU Stations yearly decreased, while the ratio of eases affecting the morbidity and mortality rates [8]. NO to SO yearly increased. Meanwhile, the analysis of 2 2 Air quality investigation in many cities has been con- Multi-Pollutant Index (MPI) shows that air quality in ducted in China, such as Beijing, Shanghai, Guangzhou, Chongqing is still serious. The results of this study provide etc. [9]. However, these well monitored cities are all lo- reference for the management of air quality in mountainous cated in plain areas, and the air quality data about moun- city of Chongqing. tainous cities is still limited. Chongqing is the largest mountainous city in China, and has suffered from acid rain caused by raw coal combustion for a long time. Coal burn- KEYWORDS: ing and other industrial activities are the major source of Air quality; Mountainous city; SO2; NO2; PM10 SO2 emission, which has been conﬁrmed by many studies[10], and it has also been proved that pH of rain water can be as low as 4.5 in Chongqing [11]. Besides, spatial 1. INTRODUCTION distribution of air pollution level may be different for the complex terrain conditions and the poor air diffusion in Urban air pollution has been a world-wide problem, mountainous city. So, it is very important to analyze the air which manifests itself differently in different regions, de- quality in different regions in Chongqing. The aim of this paper is: (1) to understand the temporal and spatial distri- * Corresponding author bution patterns of SO2, NO2 and PM10 in different areas;

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and (2) to analyze influencing factors correlated with air 2.3 Evaluation method of air quality pollutant concentrations, which can be used as references Multi-Pollutant Index (MPI), formulated by Gurjar et for local environmental management. al. [13], is applicable to evaluate the integrated pollution level of several pollutants, and enables the comparison of air pollutions in different cities or urban areas. In this paper 2. MATERIALS AND METHODS we chose MPI considering the combined level of the three criteria pollutants (i.e., PM10, SO2, and NO2) in view of 2.1 Study area Ambient Air Quality Standards (GB 3095-2012)[14] and Chongqing lies in Southwest China, inland along the the World Health Organization (WHO) Guidelines for Air upper reaches of the Yangtze River. With an annual mean Quality [15] and evaluated the comprehensive air pollution temperature of about 18°C, the climate here falls into the levels in different urban functional regions. The formula is category of subtropical humid monsoon climate. The low- given as follow: est mean temperature in winter is 6°C-8°C, and the highest mean temperature in summer is above 35°C. The extreme ()ACGCGC temperature reaches 43°C as the highest and -2°C as the MPI   iii lowest. The total sunshine duration is 1000 to 1200 hr. The n annual mean rainfall is about 1100 mm, 40% to 50% in summer and only 4% to 5% in winter [12]. Four air quality Where ACiis annual mean concentration of pollutant i monitoring stations in Chongqing were selected (Table 1). (i.e., SO2, NO2 and PM10); n is total number of pollutants YJP and BSY Station are separated by Zhongliang Hill, involved in calculation; and GCi is the reference value of while TL and BSY Station are partitioned by Jinyun Hill. pollutant i obtained from the WHO standard or Ambient Meanwhile, CTBU is also determined as monitoring point Air Quality Standards (GB 3095-2012). If MPI value is reflecting air quality in Nan’an district, which is separated lower than 0, the air is clean.When MPI value is higher from Jiulongpo District by the Yangtze River. YJP Station than 0, the air is polluted.The bigger the value is, the more is about 18 Km from BSY Station, and the distance be- serious the degree of pollution would be [2]. tween TL Station and BSY Station is about 50 km.

TABLE 1 - Location anddescriptionofthe monitoring stations 3. RESULTS

Alti- Relative Station Longitude/Latitude 3.1 Air pollutant concentrations in different areas tude(m) height (m) YJP 106°30′37.2″/29°31′05.4″ 284 30 Daily average concentrations of SO2, NO2 and PM10 are summarized in Table2, and all four monitoring stations BSY 106°21′44.3″/29°29′0.2″ 320 10 had lower concentrations compared to the standard values TL 106°02′59.7″/29°50′03.5″ 292 20 limited in Ambient Air Quality Standards (GB 3095-2012) CTBU 106°34′33.03″/29°30′34.05″ 311 25 [14]. However, compared to the limited values defined by the World Health Organization (WHO)[15], the four re- 2.2 Data sources gions studied all had higher SO2 and PM10 concentration (e.g., 3 3 3 Continuous measurements of SO2, NO2, PM10 and five 92 μg/m in YJP, 42 μg/m in BSY, 45 μg/m in TL and 3 3 meteorological parameters were conducted. Specifically, 47 μg/m in CTBU for concentrations of SO2, and 126 μg/m the pollution level time-series were obtained from (i) YJP in YJP, 84 μg/m3 in BSY, 61 μg/m3 in TL and 92 μg/m3 in Station which lies in the commercial city centre, and is CTBU for concentrations of PM10, respectively). Mean- characterized by heavy traffic levels, (ii) BSY Station while, BSY, CTBU and TL Stations had similar pollutant which is located in a peripheral monitoring site with low concentrations (except of PM10), but YJP had significantly levels of traffic, (iii) TL Station lies in a residential area different pollutantdaily average concentrations, which was with moderate traffic levels and (IV) CTBU. The first loca- about 2 times and 1.7 times higher than BSY, CTBU and TL tion is selected because it reflects the maximum levels of Stations for SO2 and NO2 respectively. The lowest PM10 air pollution, whereas the second and the third monitoring daily average concentration appeared at TL Station (e.g., stations represent typical residential areas. The monitoring 61 μg/m3). YJP was the commercial central of Jiulongpo data in the first two stations were provided by Jiulongpo District, and had very heavy traffic and high population den- District Environmental Bureau and the third by Tongliang sity, which maybe the main reason of high pollutant con- Environmental Bureau. YJP Station has been established centrations. In addition, YJP was surrounded by Zhongli- for a long time, and the monitoring data continues from ang hill and Zhenwu hill, and air flow was limited, so, it 2002 to 2011. However, BSY Station just has been oper- was difficult for the diffusion of air pollutants. However, ated for a shorter period and measurement data are only TL was a small town around Chongqing City with less traf- available for one year. TL and CTBU stationsare moni- fic volume, less energy consumption and lower population tored for 3 and 4 years (e.g., from 2010 to 2012 and from density, which may lead to better air quality. Furthermore, 2009-2012), respectively. Table1 displays the location and TL was not encircled by hills, and emission pollutants were the characteristics of the stations used. easy to be transferred and diluted.

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3 TABLE 2 - Daily averageconcentrationsof SO2, PM10 and NO2 in different stations (μg/m )

Yangjiaping Baishiyi Tongliang Chongqing Technology and Business University GB2012 [14] WHO [15] (YJP) (BSY) (TL) (CTBU)

SO2 92±62 42±27 45±39 47±29 150 20 NO2 52±25 30±19 31±18 31±16 80 - PM10 126±72 84±47 61±63 92±51 150 50

3.2 Temporal distribution of pollutant concentrations 250 3.2.1 Monthly changes of pollutant concentrations YJP CTBU The mean monthly changes of SO2, NO2 and PM10 con- 200

) TL centrations for the period 2002–2012 are displayed in Fig- 3 m ures 2, respectively. The level of SO2 present in the city at- μg/

( 150 mosphere was observed to increase during the winter months 2 and decreased during summer generally. This was thought to result from the high temperature in summer. In mountain- 100 ous city, air horizontal movement was hindered by surrounding mountains, however, the high temperature in summer could lead to strong vertical convection of airflow, and dilu- 50 tion and diffusion of pollutants. The highest concentration of concentration of SO 3 SO2 throughout the study was determined as 250 µg/m in 0 February 2004. Plots of the changes of NO2 concentrations 0 20 40 60 80 100 120 140 indicated that the NO2 concentration was also higher during months the winter months than in the summer months on the whole, this phenomenon was attributed to photochemical reactions 120 by Ozcan [5]. The highest NO2 concentration was as high as YJP 3 114µg/m and also appeared in February 2004. Similar tem- CTBU

) TL poral patterns of NO2 have been reported in previous studies. 3 90 m Nguyen and Kim [6] found that concentration of NO2 in fall, 3 3 spring and summer were 18.3±5.56 μg/m , 18.1±2.53 μg/m , μg/ ( and 13.0±2.19 μg/m3 in suburban background in Korea, re- 2 60 spectively. As it also can be seen from Figure 2, during the study period the highest monthly mean concentration of 3 3 3 PM10 was 241μg/m , 160μg/m and 195μg/m in YJP, CTBU and TL, which occurred in February 2004, December 30

2010, and December 2010, respectively. Concentrations of NO of concentration PM10 were also higher in winter months, and this phenomenon was similar to other two pollutants. 0 0 20 40 60 80 100 120 140 months 3.2.2 Annual concentrations of air pollutants during study period

As seen in Figure 3, NO2 concentrations showed fluc- 250 tuations over time, rather than a steady decline throughout YJP the study period. During 2002−2012, annual NO2 concen- CTBU 3 200 ) trations at three monitoring stations ranged from 29 μg/m 3 TL to 72 μg/m3, with the minimum recorded at CTBU Station m μg/ in 2009, and the maximum at YJP Station in 2008. As ( 150 shown in Fig. 3, except in 2000, the concentrations in the 10 other years were mostly above the WHO guideline value of 3 100 40 μg/m in YJP Station [15]. Mean NO2 concentration in TL and CTBU Station was within a range between 29 μg/m3 3 and 34 μg/m , and all monitored values were lower than 50

WHO threshold value [15]. concentration of PM

The annual concentration of SO2 declined gradually 0 on the whole in all monitored stations. In YJP Station, 0 20406080100120140 annual SO2 concentration declined by 61% between 2002 months and 2010 and the annual concentration of SO2 in 2010 3 (e.g., 54 µg/m ) were lower than the value as requested in FIGURE 2 - Monthly changes of pollutant concentrations during the Ambient Air Quality Standards (GB 3095-2012). In TL and study period (μg/m3)

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3 3 80 from 55 μg/m to 160 μg/m , all significantly exceeding the YJP guideline value of 70μg/m3 given in Ambient Air Quality TL CTBU Standards (GB 3095-2012) except of TL Station. Com- 3 pared to guideline value of 20μg/m from WHO, PM10 pol- ) 3 60 lution in Chongqing was very serious. The mean concentration of the study years was 65–126 μg/m3 (e.g., 3-6 times higher than standard value in WHO) and PM10 concentrations at the three stations all showed a significant down- ward trend. On the whole, although the PM10 pollution was 40 very serious, it appeared a significant reducing trend. concentration (µg/m concentration 2 NO 3.2.3 Yearly Trends of NO2/SO2 ratio

Figure 4 presents the concentration ratio of NO2 to 20 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 SO2. The ratio NO2 to SO2 showed an increasing trend from year 2002 to 2012 in the three stations. This reflected that the NO2 concentration increased more rapidly than SO2 from 160 2002 to 2012 due to the rapid increase of motor vehicles

140 YJP and traffic exhaust and the decline of coal burning. TL

) CTBU 3 1.2 120 YJP TL 100 1.0 CTBU Linear Fit of YJP 80 Linear Fit of CTBU Linear Fit of TL 0.8 concentration (µg/m 2 60 SO

40 Ratio 0.6

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 year 0.4 180

160 YJP 0.2 TL 2000 2002 2004 2006 2008 2010 2012 2014 ) 3 CTBU year 140

g/m FIGURE 4- Annual changesof NO2to SO2ratio μ 120 3.2.4 Analysis of Pearson correlations between pollutant con- 100 centrations and meteorological parameters

80 SPSS18.0 statistical software was used to analyze the concentration ( 10 Pearson correlation coefficients between meteorological PM 60 parameters (e.g., atmospheric pressure (AP), temperature (T), wind speed (WS), and relative humidity (RH)), and 40 pollutant concentrations, and the results were shown in Ta- 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 year ble 3 and Table 4. In TL and YIP, correlation properties FIGURE 3 - Yearly changes of pollutant concentrations during the had much in common. There was a positive correlation be- 3 study period (μg/m ) tween SO2, NO2 and PM10 concentrations and atmospheric pressure. The higher the atmospheric pressure was, the CTBU Station, the annual concentrations of SO2 were all greater the SO2, NO2 and PM10 concentrations would be, within the standard values defined by Ambient Air Quality which may be attributed to the relationship between air Standards (GB 3095-2012), and were reduced by 20% and density and AP. Meanwhile, a negative correlation existed 19% during study period, respectively. Annual SO2 con- between SO2, NO2 and PM10 concentrations and air tem- centrations at three monitoring stations ranged from 39 μg/ perature and wind speed. Higher air temperature and wind m3 to 144 μg/m3, with the minimum appeared at TL Station velocity would lead to lower pollutant concentrations, in 2012, and the maximum at YJP Station in 2004. which may be because that high air temperature and wind In 2002–2012, the annual PM10 concentration at the speed were conducive to the movement of airflow and di- three uninterrupted air quality monitoring stations ranged lution of air pollutants.

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TABLE 3- Analysis of Pearson correlations between pollutant concentrations and meteorological parameters in TL Station (N=20300)

PM10 SO2 NO2 AP T WS RH ** ** ** ** ** ** PM10 1 .221 .279 .201 -.191 -.168 .057 ** ** ** ** ** SO2 1 .307 .159 -.206 -.146 -.059 ** ** ** NO2 1 .230 -.281 -.257 -.013 AP 1 -.828** -.186** .254** T 1 .226** -.427** WS 1 -.233** RH 1 “**” and “*” mean correlation was significant at the level of 0.01 and 0.05 (2-tailed), respectively

TABLE 4- Analysis of Pearson correlations between pollutant concentrations and meteorological parameters in YJP Station (N=460)

PM10 SO2 NO2 AP T WS RH PM10 1 .628** .641** .196** -.229** -.347** -.009 SO2 1 .552** .209** -.242** -.279** .031 NO2 1 .105* -.288** -.330** -.153** AP 1 -.834** -.330** .460** T 1 .311** -.561** WS 1 -.161** RH 1 “**” and “*” mean correlation was significant at the level of 0.01 and 0.05 (2-tailed), respectively

2 3.2.5 Trend of MPI Annual MPI value for each station was presented in YJP TL Fig. 5. In 2002-2012, a quick decreasing trend of PMI oc- CTBU curred due to decrease of SO2, NO2 and PM10 concentra- 1 tions (Fig.3). Meanwhile, pollution level was significantly different according to different calculation guideline. Based on standard values provided in Ambient Air Quality Standards (GB 3095-2012), there was no contamination in

0 TL and CTBU Station, however, air quality would be se- rous in all three monitored stations if calculation standard was replaced with WHO guideline. Regionally, the MPI in

MPI according to GB 3095-2012 to GB according MPI 2002–2012 was averaged 0.6 in YJP, larger than the value of -0.18 in TL and -0.04 in CTBU calculated with standard -1 value in Ambient Air Quality Standards (GB 3095-2012). 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 MPI would be averaged 2.9 in YJP, 1.0 in TL and 1.7 in year CTBU according to critical value in WHO. 4

YJP TL 4. DISCUSSION 3 CTBU 4.1 Influencing factors analysis 4.1.1 Vehicle exhaust 2 Vehicle exhaust can be the main reason of NOx pollution. Lau et al. [16] found that there is a positive correlation between traﬃc volume and concentrations of NO2 and ur- 1 MPI according to WHO ban background concentrations of NO2 could be strongly influenced by traffic volume. It had been proved that the deterioration of urban air quality is attributed to vehicle 0 emissions [16,17]. In this paper, annually number of vehi- 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 cles was analyzed according to the Statistical Yearbook of year Chongqing in Fig. 6 and we found that there was a drastic FIGURE 5 - Annual MPI value in 2002-2012 according to guideline increase in the recent ten years for the number of vehicles. values in WHO and Ambient Air Quality Standards (GB 3095-2012) 3 The quantity of motor vehicles in Chongqing increased (Guideline values for each pollutant: SO2 = 60 μg/m (GB 3095-2012), 3 3 NO2 = 40 μg/m (WHO, 2006; GB 3095-2012), PM10 = 20 μg/m from 0.5 million in 2001 to about 3.4 million in 2011 with 3 (WHO, 2006), PM10 = 70 μg/m (GB 3095-2012)) an annual growth of approximately 58%, and the concen-

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3 3 tration of NO2 rose from 40 µg/m in 2002 to 72 µg/m in fuel consumption volume. The application of coal cleaning 2007. The rapid increase in vehicles seemed good corre- technology may be quoted to explain this phenomenon. lated with the increase in NO2 concentration. Meanwhile strict standards control measures were im- 4.1.3 Other factors plemented to reduce urban air pollution caused by in- Emissions from industrial pollution sources played an creased vehicles. For example, European III standard was important role in urban air quality control. When the air- fully implemented toward the emission of exhaust pollu- flow was hampered, the point source from industrial pro- tants from vehicles in 2008 in Chongqing. So, although the duction would be more serious to urban air pollution, espe- number of vehicles increased always, concentrations of cially in mountainous cities. Many measures were imple- NO2 decreased from 2008 in YJP station (Fig.3). mented to reduce air pollution from factories. For example, moving factories out urban centre, taking desulfurization 350 and denitration technologies, and so on.

300 Number of vehicles 4.2 Air quality comparison

SO2, NO2 and PM10 concentrations in other cities in the 250 world were summarized in Table 5. The results of our comparison showed that the distinctness in the observed con- 200 centrations between cities (Table 5) seemed very large. Generally, cities in developing countries had higher PM10 150 concentrations compared to developed countries (Table 5).

100 The average PM10 concentration in Chongqing was higher than Zhengzhou, Shanghai, Guangzhou, Qinhuang- 50 dao, and Changchun, but lower than Beijing and Shenyang. Number of vehicles (10000 of vehicles) Particulate matter in atmosphere originates either from the 2000 2002 2004 2006 2008 2010 2012 direct emission (for example, road dust raised by motor ve- years hicles, power plants emissions, et al.) or from the chemical FIGURE 6 - Possession civil motor vehicles and transport vessels (10000 of vehicles) transformation of gaseous pollutants[19]. Chongqing had humid climate, and low intensity rainfall events with long 8000 duration time often occurred in winter, which may be an important reason for the high PM concentration and was electricity 10 oil similar to Chengdu. Besides, although Beijing and Shen- natural gas yang were all plain cities and had high wind speed compar- 6000 coal atively, sand storm in northwest of China would influence air quality intensively.

4000 3 TABLE 5 - SO2 and NO2concentrations in othercities (μg/m )

Cities SO2 NO2 PM10 Zhengzhou[2] 54 39 111 2000 Split[18] 22.54-54.81 48.24-56.38 -- Istanbul[5] 10.9-16.6 54.2-71.4 -- Beijing[20] 35-56 51-60 127-168 Shanghai[20] 35-72 51-67 86-103 Energy consumption (10000 tons of SCE) of tons (10000 consumption Energy 0 Guangzhou[20] 38-56 49-59 62-102 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Chengdu[20] 39-72 55-59 108-133 years Shenyang[21] 50-110 45-50 150-300 FIGURE 7 - Total Consumption of energy in major years (10000 tons Changchun[22] 18-30 28-40 97-100 of standard coal equivalent (SCE)) Qinhuangdao[23] 40 23 69 Avilés[24] 9 33 46 4.1.2 Energy use Bangkok[25] -- -- 60 Tokyo[25] -- -- 30 The energy generation pattern could have relationship Seoul[25] -- -- 61 with air pollution sources. About 90% of SO2 was resulted Jakarta[25] -- -- 100 from coal burning such as in industries and power stations Hanoi[25] -- -- 112 [18]. Coal was the main energy source in Chongqing in re- Hong Kong[25] -- -- 59 Taipei[25] -- -- 62 cent years as shown in Fig.7. Although total consumption Delhi[25] -- -- 131 of energy increased greatly in the past ten years (e.g., from Islamabad[25] -- -- 188 about 4 million in 2001 to 8 million tons of SCE in 2011), the structure of fuel consumption was not changed essen- In China, Chongqing almost had the highest average tially. However, concentrations of SO2 decreased gradually SO2 concentrations, and was close to Shenyang which on the whole (Fig.3), which was contrary to the trend of needed amount of heat by burning a great deal of coal in

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winter. Coal combustion was the main source of SO2 emis- ACKNOWLEDGEMENTS sion. In addition, if SO2 could not be transferred and diluted after being released into air, its concentration would be ac- The authors wish to thank the Environmental Monitor- cumulated and become higher. So, the poor air circulation ing Station of Jiulongpo District for their support, and the condition caused by large mountain may be a reason for the study is financially supported by the Natural Science Foun- high SO2 concentration in Chongqing. dation of Chongqing (No.cstc2014jcyjA20022), the scientific and technological research projects of Chongqing Ed- The average concentration of NO2 in Chongqing was slightly higher than other cities in China. Beside of traffic ucation Committee (No. KJ1401120) and research projects density, geographical factors were another reason leading of Chongqing University of Arts and Sciences (No. Z2013CH03). to the high NO2 concentration.

4.3 MPI comparison The authors have declared no conflict of interest. MPI had been used extensively in air quality assessment. The MPI value in Chongqing was 1.0-2.9 reflecting , the serious air pollution. Du et al. [2] found the PMI value REFERENCES in Zhengzhou was 0.17–2.23and Chan and Yao [9] found the MPI value in Tokyo, Sao Paulo, Los Angeles and New [1] Li, L. J. and Wang. Y. (2011) The characterization of NO2 pol- York was only −0.3 to −0.2 in the late 1990s, implying that lution in Beijing based on satellite and conventional observa- air pollution in Chongqing was more deteriorative. Chong- tion data. Acta Scientiae Circumstantiae, 31 (12), 2762-2768. qing is an old industrial base, and emissions from industry (in Chinese) production to air have been conducted for a long-term. Fur- [2] Du, X. L., Lu, C. H., Wang, H. R. and Ma, J. H. (2012) Trends of urban air pollution in Zhengzhou City in 1996–2008. Chin. thermore, air pollutant is very difficult to be diffused for Geogra. Sci., 22 (4), 402-413 the hindering of surrounding mountains. So, air quality in [3] Bhanarkar, A, D., Rao, P. S., Gajghate, D. G. and Nema, P. mountainous city not only had obviously spatial differ- (2005) Inventory of SO2 , PM and toxic metals emissions from ences, but also faced a more serious potential pollution. industrial sources in Greater Mumbai, India. Atmos. Environ., 39, 3851-3864 [4] Krupińska, B., Worobiec, A., Rotondo, G. G., Novakovic, V., 5. CONCLUSION Kontozova, V., Ro, C.U., Van, G. R. and De Wael, K. (2012) Assessment of the air quality (NO2, SO2, O3 and particulate matter) in the Plantin-Moretus Museum/Print Room in Ant- 1) Daily average concentrations of SO2 in the four werp, Belgium, in different seasons of the year. Microchemi- monitoring stations were within the limited values pro- cal Journal, 102, 49–53 vided by Ambient Air Quality Standards (GB 3095-2012) [5] Ozcan, H. K. (2012) Long term variations of the atmospheric (e.g., 92 μg/m3 in YJP, 42 μg/m3 in BSY, 45 μg/m3 in TL air pollutants in Istanbul City. Int. J. Environ. Res. Public and 47 in CTBU, respectively), but exceeded values spec- Health, 9, 781-790 ified by WHO. Meanwhile, the concentrations of NO2 were [6] Nguyen, H. T. and Kim, K.H. (2006) Comparison of spatio- also lower than the standard values in Ambient Air Quality temporal distribution patterns of NO2 between four diﬀerent 3 3 types of air quality monitoring stations. Chemosphere, 65, Standards (GB 3095-2012) (e.g., 52 μg/m in YJP, 30 μg/m 201–212 in BSY, 31 μg/m3 in TL and 31 μg/m3 in CTBU, respec- 3 [7] Kraft, M., Eikmann, T., Kappos, A., Kunzli, N., Rapp, R., tively). Daily mean concentrations of PM10 were 126μg/m , Schneider, K., Seitz, H., Voss, J.U. and Wichmann, H. E. 84 μg/m3, 61 μg/m3, 92 μg/m3 in YJP, BSY, TL and CTBU (2005) The German view: Effects of nitrogen dioxide on hu- Station, which 2.5, 1.6, 1.2 and 1.8 times higher than guide- man health derivation of health related short-term and long- line value given by WHO [15], respectively. term values. Int. J. Hyg. Environ. Health, 208, 305–318 [8] Godec, R., Čačković, M., Šega, K. and Beslic, I. (2012) Winter 2) In the past ten years, annual concentrations of SO2 Mass Concentrations of Carbon Species in PM10, PM2.5 and PM in Zagreb Air, Croatia. Bull Environ ContamToxicol, and PM10 decreased year by year on the whole, but NO2 con- 1 centrations fluctuated very much, which may be caused by 89,1087–1090 the growth of fuel consumption and structure, vehicles, pol- [9] Chan, C. K. and Yao, X. H. (2008) Air pollution in mega cities in China. Atmospheric Environment, 42, 1–42 lutant cleaning measures, and so on. However, the ratio of NO to SO yearly increased in YJP, TL and CTBU Stations. [10] Nguyen, H. T. and Kim, K. H. (2006) Evaluation of SO2 pol- 2 2 lution levels between four different types of air quality moni- 3) Pearson correlation coefficients analysis showed toring stations. Atmospheric Environment, 40,7066-7081 that SO2, NO2 and PM10 concentrations were positive cor- [11] Gao, S. D., Sakamoto, K., Zhao, D. W., Zhang, D.B., Dong, related with air pressure, but negative with air temperature X.H. and Hatakeyama, S. (2001) Study on atmospheric pollution, acid rain and emission control for their precursors in and wind speed. Chongqing, China.Water, Air, and Soil Pollution, 130,247- 252 4) The yearly MPI calculation indicated that MPI value [12] Wang, S. M., He, Q., Ai, H. N., Wang, Z. T. and Zhang, Q. Q. in Chongqing regions was 1.0-2.9 according to standard (2013) Pollutant concentrations and pollution loads in storm- value provided in WHO(2006), which implied that air water runoff from different landuses in Chongqing. Journal of quality in Chongqing still needed to be improved heavily. Environmental Sciences, 25 (3) ,502-510

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[13] Gurjar, B. R., Butler, T. M., Lawrence, M. G. and Lelieveld, J. (2008) Evaluation of emissions and air quality in megacities. Atmospheric Environment, 42 (7), 1593–1606.

[14] State Environmental Protection Administration of China. (2012) Ambient Air Quality Standards (GB 3095-2012) .Bei- jing: Environmental Science Press, (in Chinese)

[15] WHO. (2006) Air quality guidelines global update 2005. WHO Regional Office for Europe, Copenhagen.

[16] Lau, J., Hung, W. T., Cheung, C. S. and Yuen, D. (2008) Con- tributions of roadside vehicle emissions to general air quality in Hong Kong. Transportation Research Part D, 13, 19-26

[17] Vosniakos, F.K., Mamoukaris, A., Xipolitos, K. and Constan- tin, D.E. (2013) Vehicle emissions in the city of Leptokaria (Greece) and its contribution to the atmospheric air and noise pollution. Fresenius Environmental Bulletin, 22 (3A), 879-883

[18] Zhang, J., Ouyang, Z.Y., Miao, H. and Wang, X.K. (2011) Ambient air quality trends and driving factor analysis in Bei- jing, 1983–2007. Journal of Environmental Sciences, 23 (12), 2019–2028 [19] Ho, K. F., Lee, S. C., Cao, J. J., Chow, J.C., Watson, J.G. and Chan, C.K. (2006) Seasonal variations and mass closure analysis of particulate matter in Hong Kong. Sci. Total Environ., 355, 276–287 [20] Lin, M., Tao, J., Chan, C. Y., Cao, J.J., Zhang, Z.S., Zhu, L.H. and Zhang, R.J. Regression analyses between recent air quality and visibility changes in megacities at four haze regions in China. Aerosol and Air Quality Research, 12, 1049–1061

[21] Zhang, X.M., Chai, F.H., Wang, S.L., Hu, B.Q., Li, K. and Luo, L.B. (2006) Characteristics of air pollution in Shenyang City, China. China Environmental Science, 26 (6), 650–652. (in Chinese)

[22] Zhang, M., Lin, L. and Zhang, Z.Y. (2009) Characteristics of air pollution and its countermeasure in Changchun, China. Journal of Meteorolgy and Environment, 25 (3), 57–61. (in Chinese) [23] Zhang, B.G. and Chen, L.H. (2009) Relationship between air pollution and meteorological elements in Qinhuangdao, Hebei Province. Journal of Meteorolgy and Environment, 25 (4), 43– 47. (in Chinese) [24] Sanchez, A. S., Nieto, P. J.G., Fernandez, P.R., Diaz, J.J.D. and Iglesias-Rodriguez, F.J. (2011) Application of an SVM- Received: November 09, 2014 based regression model to the air quality study at local scale in Accepted: December 08, 2014 the Avilés urban area (Spain). Mathematical and Computer Modelling, 54, 1453–1466

[25] Colbeck, I., Nasir, Z. A. and Ali, Z. (2010) The state of ambi- CORRESPONDING AUTHOR ent air quality in Pakistan—a review. Environ. Sci. Pollut. Res., 17, 49–63 Shu-min Wang College of Material and Chemical Engineering Chongqing University of Arts and Science Chongqing 402160 P.R. CHINA E-mail:[email protected]

Qi-hong Zhu College of Material and Chemical Engineering Chongqing University of Arts and Science Chongqing 402160 P.R. CHINA E-mail: [email protected]

FEB/ Vol 24/ No 9/ 2015 – pages 2699 -2706

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BIODIVERSITY OF GRASSLAND COMMUNITIES AND SPATIAL VARIABILITY OF FUNCTIONAL GROUPS IN THE MONGOLICA FOREST-STEPPE ECOTONE

Jinman Wang1,*, Jianjun Ma2, Hong Yao2 and Shihai Lv3

1College of Land Science and Technology, China University of Geosciences, 29 Xueyuanlu, Haidian District, 100083 Beijing, People’s Republic of China 2Langfang Teacher’s College, Langfang, Hebei 065000, People’s Republic of China 3Institute of Ecology, Chinese Research Academy of Environmental Sciences, Beijing 1000l2, People’s Republic of China

ABSTRACT 1. INTRODUCTION

A sample survey was used to analyze species compo- Ecotones are bridges connecting different ecosystems sition and plant community biodiversity in a typical steppe, that also act as ecological flow channels and barriers. They a mongolica forest-steppe ecotone and the steppe of a mon- are sensitive to disturbance responses and unstable under golica forest in the pinus sylvestris var. mongolica core ecological processes [1, 2]. The community structures and area of the Hui River national nature reserve in China. The functional group diversities of plants according to the spa- changes in community characteristics along the gradients tial distribution characteristics of different scales are an im- of water, heat and altitude, and community edge effects portant issue of ecological study [3, 4]. As bridges that were also considered in this paper. The results showed that communicate the structures and functions of plant and eco- the main plants among the three plots were Gramineae, system properties, plant functional types associate the in- Compositae, Chenopodiaceae and Rosacea, which ac- dividual plant’s environment with its overall ecosystem counted for 30.0%, 20.0%, 13.3% and 10.0% of the total. structure, processes and functions [5, 6]. Therefore, the There were Artemisia frigid and Carex duriuscula in every study of community biodiversity and the spatial variability plot, which indicated that the three plots were experiencing of an ecotone’s functional groups is an effective means of different degrees of degradation. From the typical steppe to revealing the ecological environmental state, spatial varia- the Mongolica forest-steppe ecotone to the mongolica for- bility characteristics, regional resources and environmental est steppe, the cumulative dominance of every type of problems related to ecological processes. shrub, sub shrub and perennial grass decreased in order, The Hui River nature reserve is located in a transitional with a significant difference (p < 0.05). The cumulative zone from the Daxing’an Mountain forest to the Hulun- dominance of perennial forbs, however, was an increasing beier grasslands and grassy meadows to typical grasslands. trend, and the cumulative dominance of annual herbaceous It brings together forest, grassland and wetland, and com- plants in the mongolica forest-steppe ecotone was the high- bines multiple landform types including low mountain est. The biomass of shrubs, sub shrubs and perennial hills, high plains, sandy areas and valleys. It is also a rep- grasses, and the total biomass of communities, decreased resentative region for interlocking grassland areas in north- successively among the three plots, and the difference was ern China and has significantly degenerated in recent significant. The biomass of perennial forbs in the mongol- years[7]. ica forest-steppe ecotone was significantly lower than that Research on the plants in this region has mainly fo- observed in the typical steppe and the mongolica forest cused on investigating the basic characteristics of vegeta- steppe. The annual herbaceous biomass was low in every tion, analyzing floristic composition and classification and plot, but clearly the highest in the mongolica forest-steppe its transitions and other aspects[7]. Studies of the forest- ecotone. The Patrick richness index (P ), Shannon-Wiener a steppe ecotone have largely focused on the landscape level diversity index (H) and Pielou evenness index decreased at the large scale [8, 9]. On a smaller spatial scale, based successively among the three plots. on community difference, there has been less analysis of

the ecotone. The community composition and spatial vari-

KEYWORDS: Mongolica forest-steppe ecotone, plant functional ability of the diversity distribution pattern at a small scale groups, biodiversity, edge effect, Hui River national nature reserve. are important factors in maintaining community biodiversity, larger scale productivity and vegetation stability [7,

10]. Clearly, study of community biodiversity and the char- * Corresponding author acteristics of the functional groups of the forest-steppe eco-

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tone at the small-scale community level are insufficient. The reserve is located at the high-mid latitude and in Rather, research should focus on the community biodiver- the mid-temperate zone, and as such belongs to a semi-arid sity and characteristics of this ecotone’s functional groups continental monsoon climate zone. Winter is long and cold, at the community level, building on previous findings. That summer is moderate and short and the climates of spring will provide a reference and theoretical basis for the con- and autumn change greatly with an annual average temper- servation of biodiversity and sustainable use. ature of -2~-3 °C. The annual rainfall is 316 mm, the annual evaporation is 1463 mm and the area is characterized by the synchronization of high temperatures and ample pre- 2. MATERIALS AND METHODS cipitation. Main soil types include light chernozem, dark millet calcium, swamp meadow soil, saline soil, sodic soil, 2.1 Study area salt meadow soils and sandy soils. The Hui River national nature reserve is located in the The core area of the pinus sylvestris var. mongolica Ewenki Autonomous Banner at geographical coordinates forest steppe is located in the south of the reserve and is a 118°48′E～ 119°45′E and 48°10′N～48°57′N. The total geographical variant of Pinus sylvestris. There are basi- area is 3 468.48 km2 (Fig. 1). This geomorphic unit basically no shrubs in the mongolica forest, although some- cally exhibits a combination of hilly and high plain terrain. times Xeric and semi-Xeric shrubs or sub shrubs can be The majority of the eastern area consists of hilly landforms seen in this region, such as Artemisia frigid and Thymus while the natural camphor wood forest (Pinus sylvestris mongalicus, among others. At the top of the hill, Caragana var. mongolica) in the southern area belongs to the Seney microphylla can be seen under the Pinus sylvestris var. River-Honggolj-Hui River sand belt, for which the average mongolica forest and herbs are mostly distributed in the va- elevation is 700~800 m. The main landform belongs to the cant land and along the edge of the forest, mainly cover depositional landforms of the first- and second-terraces and such as Stipa baicalensis, Festuca ovina, Leymus chinen- the flood plain. The valley is low and the terrace bounda- sis, Filifolium sibiricum, Sanguisorba officinalis, Saposh- ries are clear. The western area is the Hui River valley and nikovia divaricata, Iris dichotoma and Potentilla fragari- the riparian high plain. oides, among others.

FIGURE 1 - The Hui River national nature reserve.

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2.2 Test design and vegetation sampling 2.3 Division of the community functional groups’ composition The test plot was set in the Pinus sylvestris var. mon- Based on the plant life forms of the steppe community golica core area in the south of the reserve (Fig. 1), which and the composition of the water ecotype, the functional consists of sparse forest steppe landscape. It was mainly groups were divided into four types: shrubs and sub shrubs, composed of Pinus sylvestnis var. mongolica grown as a perennial grasses, perennial forbs and annual herbaceous. plant of second-class protection in concentrated distribution that basically maintained the original state of the re- 2.4 Data analysis gion. 2.4.1 Cumulative dominance analysis Cumulative dominance is a comprehensive quantita- The influence range of the edge effect was determined tive indicator that can reflect the functional status and dis- from the research results of previous studies [11-13] and tribution of different species in the community. It was used the judgment index of the edge effect – the Jaccard index to analyze the roles that life-form functional groups play in (Cj). The Jaccard index of the typical steppe community the community, such as perennial grasses, perennial forbs outside the mongolica forest and the mongolica forest- and one or two annual herbaceous plants in different func- steppe ecotone community was 0.37, whereas that of the tional areas of the reserve. community under the Mongolica forest and the mongolica     forest-steppe ecotone community was 0.41. Both of the SDH 4  C  H  D  W / 4 (1) Jaccard indices were less than 0.5, which suggests that both where SDH 4 represents the cumulative dominance of the typical steppe outside of the mongolica forest and the     steppe under it were outside the region of the edge effect. the life-form function groups; and C , H , D and W Finally, the space settings of the three plots were deter- represent the relative coverage, relative height, relative mined (Table 1). density and relative biomass (dry weight) of the life forms, respectively. C , H  , D and W  were calculated using From July to September 2009, three plots were set in the following formulae. the Pinus sylvestris var. mongolica core area of the reserve. Relative coverage ( C ) = sum of plant coverage of They consisted of a typical steppe plot outside the mongol- any kind of life-form in the quadrat ⁄ sum of plant coverage ica forest (“outside forest” hereafter), a plot at the edge of of each species in the quadrat; the mongolica forest-steppe ecotone (“forest edge” hereafter) and a steppe plot under the mongolica forest (“inside Relative height ( H  ) = sum of plant height of any forest” hereafter). Sixteen samples (1 × 1 m) were set in the kind of life-form in the quadrat ⁄ sum of plant height of each forest edge plot, and 10 (1 × 1 m) in each of the outside and species in the quadrat; inside forest plots. The number, coverage, density, height Relative density ( D ) = sum of plant density of any and biomass of plants were measured in species. kind of life-form in the quadrat ⁄ sum of plant density of each species in the quadrat; Those factors that contribute to the changes in community biodiversity and functional groups are environmental Relative biomass (W  ) = sum of plant biomass of any factors such as elevation, moisture, light, community and kind of life-form in the quadrat ⁄ sum of plant biomass of the edge effect between communities. each species in the quadrat.

TABLE 1 - Sample description of the study area

Geographical Altitude Study area position Landform Notes (m) （、）EN The study area is far from the typical mongolica steppe, about 300m from the forest edge, and communities are rarely affected outside 119°15′3″E 697.1 high plain by mongolica. Vegetation types are typical for steppe communi- forest 48°16′33″N ties, such as agropyron mongolicum, stipa baicalensis, cleistogenes squarrosa, caragana microphylla and mesic forbs. The study area is located in the mongolica forest-steppe ecotone and the communities are widely affected by mongolica. Vegeta- aside 119°15′32″E 717.5 high plain tion types are steppe communities, such as agropyron mongol- forest 48°16′37″N icum, corispermum declinatum, artemisia frigid, cleistogenes squarrosa, leymus chinensis and mesic forbs. The study area is located in the steppe area under the mongolica inside 119°15′30″E forest. The distance from the forest edge to its inside is about 726.3 high plain 200m, and it is strongly affected by mongolica. Vegetation forest 48°16′38″N types are steppe communities, such as carex duriuscula, calamagrostis epigeios, agropyron mongolicum and mesic forbs.

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2.4.2 Diversity analysis for the edge effect [14]. When 0 ≤ Cj ≤ 0.5, it was considered

Patrick richness index (Pa): the non-affected areas of the edge effect. When 0.5 < Cj ≤ 1, it was considered the affected areas of the edge effect. P  S (2) a Based on the existence or non-existence of some spe- Shannon-wiener diversity index (H): cies between two communities, the Jaccard similarity index compares the similar degrees of communities by com- n paring their similarities. A larger Jaccard index corre- H   Pi ln Pi i 1,2,3,, n (3) sponds to a greater similarity of communities. i1 Jaccard index（Cj） : Pielou evenness index (JP): C j  c /a  b  c (5) n

where C is the Jaccard index, c is the same species JP   Pi ln Pi / ln S (4) j i1 number of two communities and a and b are the species numbers of each community. where S represents the species number, Pi=ni/N，ni represents the individual number of species(i), and N represents the total individual number of species. 3. RESULTS AND ANALYSIS We used Exce1 2000 to process data and establish charts and SAS9.0 to conduct a variance analysis of the 3.1 Spatial variability in the composition of functional groups data. In the interactions of two or more different natures of the ecosystem (or another ecosystem), the major changes 2.5 Influence range of the edge effect in some of the system components and behavior (such as In botany, similar degrees of vegetation among plots population density, productivity and diversity) can be have been compared and the borders of small-scale plant caused by some ecological factors (material, energy, infor- communities have been determined by analyzing the differ- mation and location) or differences and synergism between ent or similar degrees of samples – an approach that has system attributes [15]. Due to the differences between en- proven both mature and reliable. Edge effects mainly affect vironmental factors and the influence of the edge effect on the species composition and richness of the community. In communities, the life-form components and compositions this study, the influence range was determined by referring of the steppe community functional groups exhibited sig- to the research results of Tilman et al. and Chen et al. and nificant differences in each sample plot (Table 2). combining them with the characteristics of the study area There were 30 plants in 3 sample plots. The results in with the Jaccard index (Cj) selected as the judgment index Table 2 show that the plant composition of the Pinus syl-

TABLE 2 - The compositions of functional group life-forms in different research areas

Research objects Functional group Outside forest Aside forest Inside forest Caragana microphylla Caragana microphylla Artemisia frigida Artemisia frigida Artemisia frigida Shrub/Sub shrub Artemisia oxycephala Artemisia oxycephala Artemisia oxycephala Artemisia capillaris Thymus mongolicus Thymus mongolicus Leymus chinensis Cleistogenes squarrosa Cleistogenes squarrosa Cleistogenes squarrosa Koeleria cristata Koeleria cristata Koeleria cristata Perennial grass Agropyron mongolicum Agropyron mongolicum Agropyron mongolicum Poa sphondylodes Achnatherum splendens Poa sphondylodes Stipa baicalensis Calamagrostis epigeios Calamagrostis epigeios Festuca ovina Festuca ovina Festuca ovina Carex duriuscula Carex duriuscula Carex duriuscula Carex pediformis Serratula centauroides Serratula centauroides Potentilla chinensis Potentilla chinensis Perennial forb Potentilla acaulis Potentilla bifurca Bupleurum scorzonerifolium Allium tenuissimum Allium tenuissimum Allium anisopodium Allium anisopodium Galium verum Thalictrum squarrosum Thalictrum squarrosum Corispermum declinatum Chenopodium glaucum Saussurea japonica Annual herb Chamaerhodos erecta Carpesium abrotanoides Total 18 species 19 species 16 species

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0.7 b Outside forest 0.6 a Aside forest 0.5 b 0.4 c Inside forest

0.3 a b 0.2 a b a 0.1 c Value of cumulative dominance cumulative of Value a c 0 Ss Pg Pf Ab Life-form functional group

FIGURE 2 - The cumulative dominance value of life-form ecological types in different research areas (mean ± SE). Different letters mean significant difference (p < 0.05). Ab, Annual herb; Pf, Perennial forb; Pg, Perennial grass; Ss, Shrub, sub-shrub.

vestris var. mongolica core area in the Hui River nature re- cumulative dominance value of perennial forb increased, serve includes Gramineae (9 species), Compositae (6 spe- the cumulative dominance value of perennial forb in the cies), Chenopodiaceae (4 species) and Rosace (3 species), outside and aside forest communities exhibited no signifi- accounting for 30.0%, 20.0%, 13.3% and 10.0% of the to- cant difference (p＞0.05). The differences in the cumula- tal, respectively. tive dominance values between perennial forb in the inside forest community and that in the other two were not signif- In the life-form functional groups, there were 5 species icant. Compared with the outside and inside forests, the cu- of shrubs and sub-shrubs, with Artemisia frigid and Arte- mulative dominance value of annual herb in the aside forest misia oxycephala as mutual species. There were 9 species community was the highest, and significantly different of perennial grasses: 6 outside forest species, 7 aside forest from the other two. species and 6 inside forest species, with Cleistogenes squarrosa, Agropyron mongolicum and Koeleria cristata The dominance values of some species changed radi- as mutual species within the two sample plots. There were cally in different sample plots (Table 3). The differences in 11 perennial forb species: 6 outside forest species, 7 aside dominance values between communities led to the differ- forest species and 5 inside forest species, with Carex duri- ences in the dominance values of functional groups be- uscula as the mutual species. There were 5 annual herb spe- tween communities. cies: 2 outside forest species, 1 aside forest species and 2 in- Table 3 shows that the outside, aside and inside forest side forest species. plots create a community with Agropyron mongolicum as the dominant species. However, the outside forest commu- 3.2 Spatial variability in the dominance value of functional nity consists of Agropyron mongolicum+ Cleistogenes groups squarrosa+ Caragana microphylla; the aside forest com- Plants that have the same life-form ecological types munity consists of Agropyron mongolicum+ Corispermum within a community usually have similar functions, such declinatum+ Artemisia frigid; and the inside forest commu- that the changes in each functional group in a community nity consists of Carex duriuscula+ Calamagrostis epigeios+ can provide the response information for specific factor by Agropyron mongolicum. using the spatial information and competition relation in- An ecotone is a cross area or “tense zone” of species formation that may exist in a community. In the outside, competition with a complex condition of circumstance ca- aside and inside forest plots, the dominance values of func- pable of sustaining the different ecotypes of plant coloni- tional groups showed regular changes due to the influence zation. Annual herb plants such as Corispermum declinatum of changes in environment factors and the edge effect be- and Artemisia frigid are present in large numbers in the tween communities (Fig. 2). mongolica forest-steppe ecotone. When the transition oc- As Fig. 2 illustrates, from the outside to the aside and curring from one ecosystem to another, the ecological gra- then to the inside forest, the effect of mongolica on the dient changes significantly and many species disappear or steppes gradually strengthened. The cumulative domi- appear during these changes of circumstance. nance values of shrubs and perennial grasses in the different sample plots decreased gradually and the differences 3.3 Spatial variability in biomass of functional groups were significant (p＜0.05). Because the dominant position It is the change in the biomass of perennial grasses and of perennial grasses in the community decreased and the perennial forbs that plays the crucial role in the biomass of

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the community [16]. The biomasses of the functional community was the lowest, and it was significantly lower groups in the different research areas are shown in Fig. 3. than that in the outside and inside forest communities. The Fig. 3 reveals that the biomasses of shrubs, sub-shrubs, biomass of annual herbs in each community was very low, perennial grasses and the total biomass of the community but it was highest in the aside forest plot and significantly were gradually less from outside, to aside, to inside forest higher in the outside forest plot than it was in the inside plots and the differences were significant in different sam- forest plot. ple plots. The biomass of perennial forbs in the aside forest

TABLE 3 - Cumulative dominance values of key species in different areas

Species Outside forest Aside forest Inside forest Agropyron mongolicum 0.320 0.391 0.100 Cleistogenes squarrosa 0.152 0.030 0.063 Caragana microphylla 0.140 0.016 0.000 Carex duriuscula 0.051 0.028 0.544 Calamagrostis epigeios 0.000 0.005 0.126 Artemisia frigida 0.016 0.101 0.004 Corispermum declinatum 0.000 0.245 0.000

160 ) 2 Outside forest a 140 120 Aside forest 100 Inside forest

80 a b c 60 a 40 b b a a 20 b b b c a c 0 Biomass of functional group(g/m functional of Biomass Ss P g P f Ab T b Life-form functional group

FIGURE 3 - Biomasses of functional groups in different research areas (mean ± SE). Different letters mean significant difference (p < 0.05). Ab, Annual herb; Pf, Perennial forb; Pg, Perennial grass; Ss, Shrub, subshrub; Tb, Total biomass.

14 12 a Outside forest b x 10 Aside forest

8 c Inside forest 6

Value of inde 4 a 2 b c a b b 0 Patrick index Shannon-Wiener index Pielou index

Biodiversity index FIGURE 4 - Spatial variability of the biodiversity of functional groups in different research areas (mean ± SE). H, Shannon-Wiener index; Pa, Patrick index; JP, Pielou index.

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The change in the biomass of perennial forbs had very and Carex duriuscula in each plot. According to Dolek et little effect on perennial grasses, but a great effect on anal. and Salhab et al., the appearance of Artemisia frigid and nual herbs. The study of Watkinson et al. [17] on perennial Carex duriuscula in a steppe community marks different grass effect on the productivity of non-forb grass suggested degrees of steppe degradation [20, 21]. that removing perennial grasses creates no significant dif- The cumulative dominance values, biomasses and total ferences in the density of non-forb grass, as opposed to biomasses of shrubs and sub shrubs showed consistent vari- prompting its growth. ation that decreased gradually from the outside, to the aside, to the inside forest regions while the cumulative dominance 3.4 Spatial variability in community diversity values of perennial forbs gradually increased. Higher species The ecotone is a cause for concern among ecologists diversity increases the exploitation of plant resources and largely due to the edge effect [18] . There is high biodiver- improves the productivity of the community [22]. sity in different scales and types of biome transitional The Patrick index (P ), Shannon-Wiener index (H) and zones [19]. In this study, the species richness and biodiver- a Pielou index (JP) gradually reduced from the outside to the sity in the mongolica forest-steppe ecotone was an im- aside and then to the inside forest communities. The Patrick portant feature of the community and biodiversity of the index (P ) and the Shannon-Wiener index (H) of the mon- functional groups in the different research areas, as shown a golica forest-steppe ecotone were significantly higher than in Fig. 4. those of inside steppe community that adjoins it. This result The combined influence of the Shannon-Wiener index is consistent with the findings of other scholars [23-26]. (H), the Patrick index (P ) and the Pielou index (JP) com- a At present, human activities are changing the natural prehensively reflect the regional plant characteristics and bi- environment in a wide range of ways, and with the growth odiversity. Fig.4 shows that from the Patrick index (P ), to a of human production activities such as exploration for min- the Shannon-Wiener index (H), to the Pielou index (JP) the eral resources in grasslands forming so many mine-steppe movement was gradually less from the outside, to the aside, ecotones, those ecotones change or cut off the circulation to the inside forest communities. According to the edge ef- of energy, material and information in the original systems. fect theory, on the edges of different plant communities, A study of the impact of new ecosystem boundaries on bi- the variation and density of organisms have an aptitude to odiversity and energy, material and information flows–es- increase. The Patrick index (P ) of the outside forest com- a pecially in relation to the dynamic migration, reaction and munity and the Shannon-Wiener index (H) of the aside for- sensibility of ecotone pollutants and their scientific man- est community were significantly higher than those of the agement – would prove a fruitful direction for future re- inside forest community as the result of an inconstant mi- search. cro-environment in the edge zone that allowed species with special requirements to spread out and settle down, which helped metapopulation to exist and lead to higher species diversity. However, the Patrick index (P ) of community a ACKNOWLEDGMENTS and the Shannon-Wiener index (H) of the mongolica forest-steppe ecotone were significantly lower than those of This research was supported by Special Fund for En- the outside forest community compared with that which vironmental Protection Research in the Public Interest of was far from the mongolica forest because the Mongolica China (200809125), the Fundamental Research Funds for forest had inhabited the role of foe in relation to the plants the Central Universities China (2652012072). under the forest (such as light and chemical inhibition), which made the invasion of light-loving steppe species dif- The authors have declared no conflict of interest. ficult. Forest edge zones are usually dryer and lighter than those inside forests, which is beneficial to the existence of shade-intolerant plants [14].

REFERENCES

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[4] Alhamad, M.N., Alrababah, M.A., Bataineh, M.M., and Al- [20] Dolek, M., and Geyer, A. (2002) Conserving biodiversity on Horani, A.S. (2008) Environmental gradients and community calcareous grasslands in the Franconian Jura by grazing: a attributes underlying biodiversity patterns of semi-arid Medi- comprehensive approach. Biological Conservation 104, 351- terranean grasslands. Plant Ecology 196, 289-299. 360. [5] Diaz, S., and Cabido, M. (2001) Vive la difference: plant func- [21] Salhab, J., Wang, J., Anjum, S.A., and Chen, Y. (2010) As- tional diversity matters to ecosystem processes. Trends in sessment of the grassland degradation in the southeastern part Ecology & Evolution 16, 646-655. of the source region of the Yellow River from 1994 to 2001. Journal of Food Agriculture & Environment 8, 1367-1372. [6] Cornelissen, J.H.C., Lavorel, S., Garnier, E., Diaz, S., Buch- mann, N., Gurvich, D.E., Reich, P.B., ter Steege, H., Morgan, [22] Schlapfer, F., and Schmid, B. (1999) Ecosystem effects of bi- H.D., van der Heijden, M.G.A., Pausas, J.G., and Poorter, H. odiversity: A classification of hypotheses and exploration of (2003) A handbook of protocols for standardised and easy empirical results. Ecological Applications 9, 893-912. measurement of plant functional traits worldwide. Australian [23] Tilman, D., Wedin, D., and Knops, J. (1996) Productivity and Journal of Botany 51, 335-380. sustainability influenced by biodiversity in grassland ecosys- [7] Ma, J.J., Wang, J.M., Yao, H., Feng, Z.Y., and Li, X.F. (2013) tems. Nature 379, 718-720. Community characteristics and functional group diversity's [24] Tilman, D., Reich, P.B., and Knops, J.M.H. (2006) Biodiver- spatial variability of grassland wetland in China's Hui River sity and ecosystem stability in a decade-long grassland exper- national nature reserve. Disaster Advances 6, 94-104. iment. Nature 441, 629-632. [8] Jensen, K. (2004) Dormancy patterns, germination ecology, [25] Chalcraft, D.R. (2013) Changes in ecological stability across and seed-bank types of twenty temperate fen grassland spe- realistic biodiversity gradients depend on spatial scale. Global cies. Wetlands 24, 152-166. Ecology and Biogeography 22, 19-28. [9] Roscher, C., Schumacher, J., Baade, J., Wilcke, W., Gleixner, [26] Decasenave, J.L., Pelotto, J.P., and Protomastro, J. (1995) G., Weisser, W.W., Schmid, B., and Schulze, E.D. (2004) The Edge-Interior differences in vegetation structure and composi- role of biodiversity for element cycling and trophic interaction in a Chaco semiarid forest, Argentina. Forest Ecology and tions: an experimental approach in a grassland community. Management 72, 61-69. Basic and Applied Ecology 5, 107-121.

[10] Chalcraft, D.R., Wilsey, B.J., Bowles, C., and Willig, M.R. (2009) The relationship between productivity and multiple aspects of biodiversity in six grassland communities. Biodiver- sity and Conservation 18, 91-104.

[11] Gardner, R.H., and Engelhardt, K.A.M. (2008) Spatial processes that maintain biodiversity in plant communities. Per- spectives in Plant Ecology Evolution and Systematics 9, 211- 228.

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[13] Tallowin, J.R.B., Smith, R.E.N., Goodyear, J., and Vickery, J.A. (2005) Spatial and structural uniformity of lowland agricultural grassland in England: a context for low biodiversity. Grass and Forage Science 60, 225-236. [14] Tilman, D., and Downing, J.A. (1994) Biodiversity and stability in grasslands. Nature 367, 363-365. [15] Gaujour, E., Amiaud, B., Mignolet, C., and Plantureux, S. Received: November 13, 2014 (2012) Factors and processes affecting plant biodiversity in Revised: February 16, 2015 permanent grasslands. A review. Agronomy for Sustainable Accepted: February 23, 2015 Development 32, 133-160.

[16] Hickman, K.R., Hartnett, D.C., Cochran, R.C., and Owensby, C.E. (2004) Grazing management effects on plant species di- CORRESPONDING AUTHOR versity in tallgrass prairie. Journal of Range Management 57, 58-65. Jinman Wang [17] Watkinson, A.R., and Ormerod, S.J. (2001) Grasslands, grazing and biodiversity: editors' introduction. Journal of Applied College of Land Science and Technology of China Ecology 38, 233-237. University of Geosciences 29 Xueyuanlu [18] Weigelt, A., Schumacher, J., Roscher, C., and Schmid, B. (2008) Does biodiversity increase spatial stability in plant Beijing, Haidian District, 100083 community biomass? Ecology Letters 11, 338-347. P.R. CHINA

[19] Wardle, D.A., Bonner, K.I., Barker, G.M., Yeates, G.W., Ni- cholson, K.S., Bardgett, R.D., Watson, R.N., and Ghani, A. Phone: (+86) 010 82322135 (1999) Plant removals in perennial grassland: Vegetation dy- E-mail: [email protected] namics, decomposers, soil biodiversity, and ecosystem properties. Ecological Monographs 69, 535-568. FEB/ Vol 24/ No 9/ 2015 – pages 2707 - 2714

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ANTIBACTERIAL ACTIVITIES OF ESSENTIAL OILS, EXTRACTS AND SOME OF THEIR MAJOR COMPONENTS OF Artemisia spp. L. AGAINST SEED-BORNE PLANT PATHOGENIC BACTERIA

Fatih Dadasoglu1,6,*, Recep Kotan2, Ahmet Cakir3, Ramazan Cakmakci4, Saban Kordali2, Hakan Ozer4, Kenan Karagoz1,6 and Neslihan Dikbas5

1Department of Molecular Biology and Genetics, Faculty of Science and Letters, Agri Ibrahim Cecen University, Agri 04100, Turkey, 2Department of Plant Protection, Faculty of Agriculture, Ataturk University. Erzurum 25240, Turkey 3 Department of Chemistry, Faculty of Science and Art, 7 Aralık University, Kilis 79000, Turkey 4Department of Field Crops, Faculty of Agriculture, Ataturk University. Erzurum 25240, Turkey 5 Department of Agricultural Biotechnologie, Faculty of Agriculture, Ataturk University, Erzurum, 25240, Turkey 6 Central Research and Application Laboratory, Agri Ibrahim Cecen University, 04100 Ağrı, Turkey

ABSTRACT KEYWORDS: Antibacterial activity, essential oil, Artemisia spp., chemical composition

This study was carried out to determine antibacterial activity of essential oils, extracts and major components of three Artemisia species (A. absinthium, A. santonicum and 1. INTRODUCTION A. spicigera) against to some seed borne bacterial plant pathogens. According to our results, essential oils and some ma- Artemisia is a large, diverse genus of plants with be- jor components of Artemisia species have antibacterial ac- tween 200 and 400 species belonging to the daisy family tivities at varying rates while extracts have shown no activity Asteraceae. The genus is distributed worldwide, mainly against any of the pathogens. Disc-diffusion method was across the temperate zones of the Northern Hemisphere, used to test antimicrobial activity of the essential oils and ex- some species reaching the Arctic, but a few species can also tracts According to the results obtained, essential oil of A. san- be found on the Southern Hemisphere [1, 2]. Artemisia is rep- tonicum has antibacterial effect against to 24 of 25 bacterial resented by 23 species in the Turkish flora and among them; strains, essential oil of A. absinthium has antibacterial effect A. absinthium, A. spicigera and A. santonicum are found against 15 of 25 bacterial strains and essential oil of A. spic- growing naturally in large areas of south-eastern Anatolia re- igera has antibacterial effect against only three of 25 bacte- gion of Turkey [3]. Members of this genus, have a charac- rial strains. Additionally, constituents of the essential oils teristic scent or taste, have botanical and pharmaceutical in- were analyzed by GC–MS method. Camphor, caryophyllene terest, and are used in the liqueur-making industry. These oxide, linalool, 1,8-cineole, terpinen-4-ol, borneol and α-ter- herbs have been used worldwide in folk medicine since an- pineol were determined as predominant components. Mini- cient times [4]. There are also several reports concerning the mum-maximum inhibition zones and MIC values of linalool antimalarial, antioxidant, antibacterial, antidiabetic, and an- were 8 mm (C. violaceum RK-231) - 45 mm (X. campestris tifungal activities of different Artemisia species [5-9]. pv. vitians RK-Xcvi), 50-110 mg/ml; terpinen-4-ol 8 mm (B. In recent years, crop loss is one of the major problems pumilus RK-106) - 43 mm (X. campestris pv. vitians RK- due to plant diseases caused by plant pathogen fungi, bac- Xcvi) and MIC values 60-110 mg/ml; -terpineol 8 mm teria, viruses and insects. Microorganisms have also unfa- (P.cichorii RK-166 and X. axamopodis pv. vesicatoria RK- vorable effects on the quality, safety, and shelf life of 399) – 10 mm (P. huttiensis RK-260 and P. syringae pv. sy- foods. Nowadays, rapid and effective control of plant dis- ringae RK-204) and 60-70 mg/ml, respectively. But caryo- ease and microbial contamination in the crops is generally phyllene oxide, borneol, camphor and 1,8-cineole didn’t achieved using synthetic pesticides and sometimes, antibi- show activity against any of the pathogens. In sum, our find- otics. Control of plant bacterial diseases remains hard due ings suggest that essential oils may be valuable as potential to limited availability of commercial bactericides and pro- antibacterial agents against some plant pathogens. hibition of usage of the antibiotics in many countries. Thus, chemical control of bacterial diseases is largely dependent on the use of copper compounds. However, such control methods prevent bacterial multiplication but are not adequate against seed-borne inocula. However, these chemicals and antibiotics are associated with undesirable effects * Corresponding author and some toxic residues in the products. Furthermore,

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chemicals of this type evoke undesirable effects on the en- performed using a Thermofinnigan Trace GC/Trace vironment and leave residues toxic to mammalians [10, DSQ/A1300, (E.I. Quadrapole) equipped with a SGE- 11]. In addition, the risk of the development of resistance BPX5 MS fused silica capillary column (30 m×0.25 mm by microorganisms and the high cost-benefit ratio are other i.d., film thickness = 0.25 µm). For GC–MS detection, an disadvantages of synthetic chemicals uses [12, 13]. In ad- electron ionization system with ionization energy of 70 eV dition to microorganisms causing infectious diseases in hu- was used. Carrier gas was helium at a flow rate of 1 ml/min. mans may develop resistance to many antibiotics due to the Injector and MS transfer line temperatures were set at 220 indiscriminate use of commercial antibiotics [14, 15]. This and 290 ºC, respectively. The oven temperature was pro- problem, antibiotics are sometimes associated with adverse grammed from 50 to 150 ºC at 3 ºC /min, then held isother- effects including hypersensitivity, allergic reaction, and mal for 10 min and finally raised to 250 ºC at 10 ºC/min. immunity suppression [16]. Therefore, there has been a Diluted samples (1/100, v/v, in methylene chloride) of 1.0 µl growing interest in research concerning alternative pesti- were injected manually in the splitless mode. The relative cides and antimicrobial active compounds, including the percentage of the oil constituents was expressed as percent- plant extracts and essential oils that are relatively less dam- ages by peak area normalization. The identification of in- aging to the mammalian health and environment [11, 17, dividual compounds of essential oils was based on compar- 18]. Hence, our interest focused on the effectiveness of the ison of their relative retention times with those of authentic essential oils and extracts of Artemisia spp. samples on SGE-BPX5 capillary column, and by matching of their mass spectra of peaks with those obtained from authentic samples and/or the Wiley 7N and TRLIB libraries 2. MATERIALS AND METHODS spectra and published data [23, 24]. Authentic samples were purchased from Sigma, Fluka, Alfa or Aldrich. The 2.1 Plant Pathogenic Bacterial Strains relative percentages of major constituents of the oils are Twenty-five bacterial strains used from culture collec- presented in Table 1. Pure major components, tested for tion of Assoc. Prof. Dr. Recep Kotan (Ataturk University, antibacterial activity were purchased commercially from Agricultural Faculty, Turkey). Whole strains were tested Fluka, Merck and Sigma. formerly and they are highly virulence strains. All these strains had been determined as pathogens of different host 2.4 Antibacterial Activity Assays plants [19-22] and were stored at -80°C in 15% glycerol Antibacterial activity assays were carried out by disc and Luria Broth (LB) until use. diffusion method [25] with a minor modification using Tryptic Soy Agar (TSA, Merck, Germany) medium. The 2.2 Plant Materials, Isolation of the Essential Oil and Extrac- essential oil, extracts and pure compounds were prepared tion procedures by dissolving using 10% dimethylsulfoxide (DMSO), and The aerial parts of A. santonicum, A. spicigera and A. then were sterilized by filtration by 0.45 µm Millipore fil- absinthium were collected in Erzurum province of Turkey ters. Bacterial cultures were grown in Tryptic Soy Broth (eastern Turkey) in middle of July, at flowering stages and (TSB, Merck, Germany) and their suspension (100 µL) shaded for 7 days at room temperature. The voucher spec- containing 1×108 CFU/ml of bacteria spread by a sterile imens have been deposited in the herbarium of Ataturk swab on TSA medium. The discs (6 mm in diameter) were University, Erzurum (Turkey). The essential oils were iso- impregnated with 12.5 µl of the emulsions of the essential oils lated from the aerial parts of A. absinthium, A. spicigera prepared in 10% DMSO distilled water, and with 10.0 mg/ml and A. santonicum by hydrodistillation method using a suspensions of the extracts and pure compounds prepared in Clevenger type apparatus. The yields were based on dried 10% DMSO-distilled water. Then, they were put in the mid- materials, shaded at room temperature for 7 days and de- dle of the inoculated plates. The bacterial cultures were in- termined over (w/w). The oils were dried over anhydrous cubated at 27 ± 2 ◦C for 48 h, and then inhibition zones Na2SO4 and stored under N2 in a sealed vial until required. were measured in diameter (mm) around of the discs. Fur- The dried plant samples were powdered in a blender thermore, bactericidal and bacteriostatic activities were and then samples of 100 g extracted individually with n- also determined. The Tryptic Soy Agar samples taken from hexane, chloroform, acetone and methanol at room temper- inhibited areas around of the discs were put into nutrient ature. After filtration, the organic solvents were evaporated broth without essential oil, extracts and pure compounds under reduced pressure and temperature. For the methanol incubated at 27 ± 2 ◦C for two days. After 48 h, whether extract of the plant sample, the concentrated methanol ex- there was no bacterial growth was observed in the broth tract was individually dissolved in distilled water (60◦C) culture, it was considered as bactericidal effect or not bac- and then filtered. Thus, chlorophyll was removed from the tericidal. Oxacilin (1 µg/disc) and 10% DMSO–distilled solution. Then, this solution was lyophilized in a Labconco water were used as positive and negative controls, respec- 117 freeze-dryer at 5 m-Hg and −50◦C. tively. All the tests were made in triplicate.

2.3 GC-MS Analyses 2.5 Determination of Minimal Inhibition Concentration (MIC). The oil composition was analyzed by gas chromatog- Minimum inhibition concentrations (MICs) of the es- raphy–mass spectrometry (GC–MS). GC–MS analysis was sential oils were tested by using a two-fold serial dilution

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method [26]. Two fold serial dilutions of the liquid sub- nuciferol butanoate (8.24%), nuciferol propionate (5.13%), stances, essential oil was prepared by diluting 10% DMSO caryophyllene oxide (4.28%) (E)-sabinene hydrate (2.87%), to achieve a decreasing concentration ranging from 500 µl/ cis-sesquisabinene hydrate (2.67%), -terpineol (2.38%) ml to 3.125 µl/ml. However, solutions of the solid sub- and geranyl isobutyrate (2.32%) as major components. Es- stances, the extracts and fractions were prepared by diluting sential oils of A. santonicum and A. spicigera are more sim- 10% DMSO at concentrations ranging from 50 to 110 mg/ ilar to each other in terms of chemical composition. These ml. Using 100 µl of suspension containing 1×108 CFU/ml essential oils contain camphor (18.18 % and 34.85), 1,8-cin- was measured spectrophotometrically at 600 nm of bacte- eole (7.53% and 9.48%), cubenol (4.21% and 0.21%), bor- ria spread on TSA plates. The blank discs (Oxoid) were neol (4.02% and 5.10%), terpinen-4-ol (3.47% and 1.24%), impregnated with 12.5 µl of the solutions tested. Then, they -terpineol (4.07% and 1.64%), -selinene (2.38% and were put in the middle of inoculated TSA plates. The bac- 0.49%) and bornyl acetate (2.17% and 1.00%) as major com- terial cultures were incubated at 27 ± 2 ºC for 48 h. The ponents, respectively. lowest concentration of the essential oils, extracts and fractions showing a clear zone of inhibition were considered as 3.2 Antibacterial test results of the main components in the the MIC. 10% DMSO was used as negative control. All the essential oil tests were carried out in triplicate. Table 2 represent the antibacterial activities of major components; namely linalool, terpinen-4-ol, -terpineol, caryophyllene oxide, borneol, camphor and 1,8-cineole, 3. RESULTS obtained from essential oils linalool, terpinen-4-ol and - terpineol has shown varying diameters of inhibition zones 3.1 The chemical composition of essential oils obtained by hydrodistillation method of aerial parts of plants against pathogens, -terpineol being the weakest. Caryo- phyllene oxide, borneol, camphor and 1,8-cineole didn’t The hydrodistillation essential oil composition of Turk- show activity against any of the pathogens. ish three Artemisia species and the relative amounts of the components are shown in Table 1. This table has shown that Minimum-maximum inhibition zones and MIC values the chemical composition of the three types of essential oil of linalool were 8 mm (C. violaceum RK-231) - 45 mm (X. has differed from each other. In particular, the essential oil campestris pv. vitians RK-Xcvi), 50-110 mg/ml; terpinen-4- of A. absinthium is different than other types of essential oils. ol 8 mm (B. pumilus RK-106) - 43 mm (X. campestris pv. Essential oil of this type contains, chamazulene (17.77%), vitians RK-Xcvi) and MIC values 60-110 mg/ml; -terpin-

TABLE 1 - Chemical composition of the essential oils of test plants

RIb Components A. absinthium (%) A. santonicum (%) A. spicigera (%) Identification methods 1042 1,8-Cineole 1.48 7.53 9.48 GC, MS, RI 1106 Linalool 0.23 0.57 0.40 GC, MS, RI 1153 Camphor 1.41 18.18 34.85 GC, MS, RI 1172 Borneol 0.61 4.02 5.10 GC, MS, RI 1178 Terpinen-4-ol 1.80 3.47 1.24 GC, MS, RI 1190 -Terpineol 2.38 4.07 1.64 GC, MS, RI 1579 Caryophyllene oxide 4.28 1.66 1.76 GC, MS, RI 930 -Thujene - 0.10 - GC, MS, RI 938 -Pinene - 0.59 - GC, MS, RI 957 Camphene - 1.00 0.10 GC, MS, RI 978 5-Methyl-3-hexen-2-one - - 0.27 MS 979 Sabinene - 0.10 - GC, MS, RI 983 β-Pinene - 0.19 - GC, MS, RI 988 3-Octanone - 0.11 - GC, MS, RI 994 β-Myrcene 0.19 0.54 0.10 GC, MS, RI 1012 -Phellandrene - - 0.33 GC, MS, RI 1023 -Terpinene - 0.34 0.16 GC, MS, RI 1034 p-Cymene 0.61 0.39 0.49 GC, MS, RI 1037 Limonene 0.10 0.18 0.69 GC, MS, RI 1055 (E)-β-Ocimene 0.11 - - GC, MS, RI 1067 -Terpinene - 0.45 - GC, MS, RI 1071 Artemisia ketone - - 0.10 GC, MS, RI 1079 (Z)-Sabinene hydrate - 0.98 - GC, MS, RI 1080 (E)-Arbuscolene - - 0.31 MS, 1081 cis-Linalol oxide (furanoid) 0.41 - - GC,MS, RI 1084 Artemisia alcohol - - 0.10 MS, RI 1084 Camphenilone - - 0.35 MS, RI 1088 Fenchone 0.12 - - GC, MS, RI 1090 trans-Linalol oxide (furanoid) 0.27 - - MS, RI 1091 Terpinolene - 0.13 - GC, MS, RI 1095 p-Cymenene 0.10 - - MS, RI 1099 -Pinene oxide - - 0.10 MS, RI 1103 Pentyl butyrate - 0.13 - MS, RI 1113 Isopentylisovalerate - 0.26 - MS, RI

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1114 cis-Thujone 0.16 - - GC, MS, RI 1117 (E)-Sabinene hydrate 2.87 - 0.73 GC, MS, RI 1122 trans-Vertocitral C - 1.15 - MS, RI 1125 trans-Thujone 0.16 - - GC, MS, RI 1130 cis-p-Menth-2-en-1-ol 0.24 0.26 1.68 GC, MS, RI 1134 -Campholenal 0.10 0.10 - GC, MS, RI 1134 Terpinen-1-ol - - 0.30 MS, RI 1141 Nopinone - - 0.10 MS, RI 1143 Isocyclocitral - 0.51 - MS, RI 1145 trans-Pinocarveol 0.72 - 1.19 GC, MS, RI 1147 trans-p-Menth-2-en-1-ol - - 1.41 GC, MS, RI 1147 trans-Sabinol - 0.44 - MS, RI 1150 trans-Verbenol 0.29 0.27 - GC, MS, RI 1158 Neo-3-Thujanol 0.10 - - MS, RI 1162 Sabinaketone 1.19 - 0.30 MS, RI 1163 Isoborneol - 0.11 - GC, MS, RI 1164 cis-Chrysanthenol - 2.03 1.33 MS, RI 1170 δ-Terpineol 0.10 - - MS, RI 1185 Isomenthol 0.16 - 0.16 GC, MS, RI 1185 p-Cymen-8-ol 1.66 0.15 1.02 GC, MS, RI 1190 Myrtenol eser - 0.55 GC, MS, RI 1199 (E)-4-Decenal - - 0.10 MS, RI 1200 Verbanol - - 0.86 GC, MS, RI 1201 D-Verbenone 0.10 0.10 0.33 GC, MS, RI 1203 Isodihydrocarveol - 0.12 - GC, MS, RI 1210 trans-Pulegol - 0.25 - MS, RI 1211 trans -Carveol 0.12 - 0.23 GC, MS, RI 1212 cis-Sabinene hydrate acetate - - 0.26 MS, RI 1217 Nerol 0.49 0.10 - GC, MS, RI 1218 Isobornyl formate 0.13 - 0.27 MS, RI 1242 Cuminaldehyde 0.89 - 0.49 GC, MS, RI 1243 trans-Chrysanthenyl acetate 0.10 0.76 0.12 MS, RI 1254 Piperitone 0.13 - 2.56 GC, MS, RI 1263 cis-Chrysanthenyl acetate 0.10 1.26 - MS, RI 1264 Geranial 0.11 0.10 - GC, MS, RI 1267 Nonanoic acid 0.10 - 0.13 GC, MS, RI 1273 Neoisopulegol acetate - - 0.16 MS, RI 1275 Isopulegol acetate - - 0.18 MS, RI 1278 Bornyl acetate 0.33 2.17 1.00 GC, MS, RI 1285 Lavandulyl acetate - 0.73 - MS, RI 1289 Thymol 0.31 0.27 0.58 GC, MS, RI 1287 (E)-Anethole 0.26 - 0.50 GC, MS, RI 1293 Phenyl 2-methylpropionate - 0.10 - MS, RI 1296 Carvacrol 0.48 0.12 0.32 GC, MS, RI 1322 2,4,6-trimethyl acetophenone 0.20 - - MS 1327 Methyl decanoate - - 0.11 MS, RI 1333 Isodihydrocarvyl acetate - - 0.11 MS, RI 1337 trans-Carveol acetate 0.36 0.14 0.22 GC, MS, RI 1346 -Terpineol acetate - 0.10 0.31 GC, MS, RI 1349 -Longipinene - 0.10 - GC, MS, RI 1357 Eugenol 0.27 0.80 0.10 GC, MS, RI 1367 -Yılangene 0.10 - 0.10 GC, MS, RI 1373 -Copaene 0.18 0.10 0.40 GC, MS, RI 1377 Isobornyl propionate - 0.12 - MS 1383 β-Bourbonene 0.14 - 0.10 GC, MS, RI 1404 (Z)-Isoeugenol 0.10 0.44 0.57 MS, RI 1405 β-Isocomene 0.19 - - MS, RI 1406 Isoaryophyllene 0.20 - 0.26 GC, MS, RI 1412 Phenyl hexanal* - 0.10 - MS 1413 -Cedrene 0.77 - - GC, MS, RI 1420 cis-threo-Davanofuran - - 0.10 MS, RI 1419 -Caryophyllene 1.09 1.15 0.39 GC, MS, RI 1431 p-Cymen-7-ol acetate - - 0.14 MS, RI 1433 -Gurjunene 0.10 - - GC, MS, RI 1442 Aromadendrene 0.10 - - GC, MS, RI 1453 (Z)-β-Farnesene - 0.10 0.10 MS, RI 1460 -Humulene 0.11 0.13 eser GC, MS, RI 1463 -Patchulene - 0.10 - MS, RI 1467 Linaly isovalerate 1.36 - - MS,RI 1470 Cyclamen aldehyde 0.10 - - MS, RI 1474 -Gurjunene - 0.13 - GC, MS, RI 1476 β-Chamigrene 0.10 0.43 0.20 MS, RI 1478 -Muurolene 0.16 0.11 - GC, MS, RI 1484 -Cyclogeraniol acetate - - 0.10 MS, RI 1486 Germacrene-D - 1.30 - GC, MS, RI 1486 (E)-β-Ionene - - 0.10 MS, RI 1488 β-Selinene 1.97 - - MS, RI 1493 Neryl isobutyrate 0.81 - - MS, RI 1499 -Selinene - 2.38 0.49 MS, RI 1500 Benzyl tiglate - - 0.10 MS, RI 1504 Neryl butyrate 0.53 - - MS, RI 1507 (E,E)--Farnesene - 0.10 0.15 GC, MS, RI

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1508 (Z)--Bisabolene 0.22 - 0.44 MS, RI 1513 -Cadinene - - 0.10 GC, MS, RI 1514 Cubebol 0.12 - 0.90 MS, RI 1515 Geranyl isobutyrate 2.32 0.10 - MS, RI 1516 Dehydro-ar--himachalene 0.10 - - MS, RI 1521 (Z)-Nerolidol 0.19 - - MS, RI 1522 cis-Calamenene - 0.11 0.22 MS, RI 1523 Artedauglasia oxide-A - - 0.55 MS, RI 1539 cis-Sesquisabinenehydrate 2.67 - - MS, RI 1541 -Calacorene 0.26 0.10 0.27 MS, RI 1546 Elemol - 0.10 - MS, RI 1551 Germacrene-B - 0.10 - MS, RI 1555 Geranyl butyrate 1.69 - - MS, RI 1555 (E)-Nerolidol - 0.13 0.28 GC, MS, RI 1574 Spathulenol 1.75 1.31 3.70 GC, MS, RI 1585 2-Phenylethyl tiglate - - 0.33 MS, RI 1593 Globulol - - 0.20 MS, RI 1590 Gleenol 0.17 0.10 0.21 MS, RI 1593 Davanone 0.10 0.10 - MS, RI 1595 Viridiflorol 0.30 - 0.26 MS, RI 1604 (Z)-Sesquilavandulol 0.38 0.44 0.83 MS, RI 1632 (E)-Sesquilavandulol - - 0.33 MS, RI 1633 β-Acorenol 0.24 - - MS, RI 1634 epi--Cadinol 0.40 - - MS, RI 1642 Cubenol 0.13 4.21 0.21 MS, RI 1650 (Z)-Methyl jasmonate - - 0.39 MS, RI 1651 Vulgarone-B 0.19 - - MS, RI 1654 Cedr-8(15)-en-9--ol 0.39 0.46 0.50 MS, RI 1656 Longipinocarvone - - 0.28 MS, RI 1658 -Eudesmol 1.07 7.19 0.60 GC, MS, RI 1659 -Cadinol 0.21 - - GC, MS, RI 1660 7-epi--Eudesmol 1.32 - 0.10 MS, RI 1682 Epi--Bisabolol - 0.10 0.22 MS, RI 1690 -Bisabolol 0.34 1.02 0.25 GC, MS, RI 1695 (Z)--trans-Bergamatol - 0.10 0.11 MS, RI 1700 n-Heptadecane - - 0.10 GC, MS, RI 1731 (E,Z)-Farnesal - 0.29 - MS, RI 1736 14-Hydroxy--humulene - 0.16 - MS, RI 1745 Chamazulene 17.77 0.32 - GC, MS, RI 1756 (E,E)-Farnesol - 0.42 - GC, MS, RI 1760 -Bisabolol oxide A - 0.25 - MS, RI 1784 Benzyl benzoate - 0.65 - MS, RI 1790 (Z)-Lanceol 0.45 0.10 - MS, RI 1794 Guaiazulene 0.89 - - GC, MS, RI 1796 (E)--Atlantone 0.15 - - MS, RI 1797 14-Hydroxy--muurolene - - 0.10 MS 1810 (Z,E)-Farnesyl acetate - 2.53 - GC, MS, RI 1838 (E,E)-Farnesyl acetate - 0.10 - GC, MS, RI 1844 (Z,Z)-Farnesyl acetone 1.20 0.10 0.12 MS, RI 1867 Diisobuthyl phthalate 1.33 0.26 0.15 MS, RI 1885 (Z)-Nuciferol acetate 0.29 - - MS, RI 1898 (Z)-Lanceol acetate 0.51 - - MS, RI 1902 Farnesyl propionate* 0.59 0.10 - MS 1914 Nuciferol propionate* 5.13 2.05 - MS 1916 Lanceol propionate* 0.90 1.28 - MS 1900 n-Nonadecane 0.6 - - GC, MS, RI 1930 Cedrane-8,13-diol 0.20 - - MS 1930 Nuciferol butanoate* 8.24 0.53 - MS 1932 Lanceol butanoate* 0.17 0.30 - MS 1940 Cembrene 0.27 - - MS 1949 Lanceol pentanoate* 1.22 - - MS 1951 Phytol* 0.61 0.10 0.18 GC, MS, RI 1963 Ethyl hexadecanoate 0.36 - - MS 1964 Nuciferol hexanoate* 0.70 - - MS Grouped components (%) Monoterpene hydrocarbons 1.11 3.62 1.38 Oxygenated monoterpenes 23.58 50.34 70.11 Sesquiterpene hydrocarbons 6.42 7.26 2.93 Oxygenated sesquiterpenes 35.61 26.39 12.29 Diterpenler 0.61 0.10 0.18 Aromatic monoterpenes 22.83 3.82 3.91 Others 3.30 0.96 1.47 Total identified (%) 93.46 92.49 92.27 tr, traces (less than 0.07%). bRetention index relative to n-alkanes on SGE-BPX5 capillary column; GC, identification was based on retention times of authentic compounds on SGE-BPX5capillary column; MS, identification was based on computer matching of the mass spectra of peaks with Wiley 7N and TRLIB libraries and published data [23]. RI, tentatively identified based on comparison of retention index of the com-pounds compared with published data [23].

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TABLE 2 - Antibacterial activities of the major components.

Caryophyllene Terpinen-4-ol Linalool -Terpineol 1,8-Cineole Borneol Camphor oxide Strains IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC

A. piechaudii RK-155 11* 90.0 12* 90.0 – – – – – – – – – – B. pumilus RK-106 8* 90.0 10* 90.0 – – – – – – – – – – C. violaceum RK-231 11* 70.0 8* 110.0 – – – – – – – – – – C. michiganensis subsp. michiganensis 9* 90.0 10* 110.0 – – – – – – – – – – RK-Cmm E. intermedius RK-90 17* 80.0 14 90.0 – – – – – – – – – – E. amylovora RK-228 20* 70.0 13* 80.0 – – – – – – – – – – E. caratovora subsp. atroceptica RK-462 10* 90.0 18* 80.0 – – – – – – – – – – E. chrysanthemi RK-421 15* 80.0 14* 90.0 – – – – – – – – – – E. rhapontici RK-208 15* 70.0 13* 90.0 – – – – – – – – – – Flavobacter sp. RK-299 11* 90.0 29 90.0 – – – – – – – – – – P. agglomerans RK-84 12* 90.0 13* 80.0 – – – – – – – – – – P. aeruginosa RK-168 17* 70.0 – – – – – – – – – – – – P. cichorii RK-166 16 70.0 – – 8 70.0 – – – – – – – – P. huttiensis RK-260 27* 60.0 25* 80.0 10 60.0 – – – – – – – – P. putida RK-249 9* 110.0 – – – – – – – – – – – – P. syringae pv. syringae RK-204 31* 60.0 26* 80.0 10 60.0 – – – – – – – – P. syringae pv. tomato RK-Pst1 26 80.0 34* 90.0 – – – – – – – – – – X. axamopodis pv. malvacearum RK-401 21* 70.0 33* 80.0 – – – – – – – – – – X. axamopodis pv. vesicatoria RK-399 25* 70.0 23* 60.0 8 70.0 – – – – – – – – X. campestris pv. campestris RK-Xcc 20 70.0 24 80.0 – – – – – – – – – – X. campestris pv. raphani RK-Xcr 22* 70.0 14 80.0 – – – – – – – – – – X. campestris pv. vesicatoria RK-Xcv1 36* 70.0 21* 80.0 – – – – – – – – – – X. campestris pv. vesicatoria RK-Xcv 761 22* 70.0 21* 60.0 – – – – – – – – – – X. campestris pv. vitians RK-Xcvi 43* 60.0 45* 50.0 – – – – – – – – – – X. campestris pv. zinia RK-Xcz 9* 110.0 10* 110.0 – – – – – – – – – – X. malvacearum RK-397 22 70.0 21 80.0 – – – – – – – – – – X. pelargonii RK-406 21 70.0 33 80.0 – – – – – – – – – –

IZ, inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 1.25 mg of the extracts and 12.5 μL of the essential oil; MIC, minimal inhibitory concentration as μLmL−1 for the essential oil and in mg mL−1 for the extracts. * Bactericidal effect was observed; – , not active.

eol 8 mm (P.cichorii RK-166 and X. axamopodis pv. vesi- dishes while the extracts have no antibacterial effects. The catoria RK-399) – 10 mm (P. huttiensis RK-260 and P. sy- highest inhibition zone with 15 mm was observed against the ringae pv. syringae RK-204) and 60-70 mg/ml, respec- strains of X. axonopodis pv. Pelargonii RK-Xa-pel. Also, tively. the MIC value against this bacterial strain was 250 μl/ml. Considering the test results of main components of es- The results regarding the essential oil and extracts sential oils, antibacterial effect is thought to arise from the of A. santonicum have been shown in the Table 4. Accord- substances such as linalool and terpinen-4-ol. It is believed ing to these results, 24 of 25 bacterial strains were inhibited that these substances are used for disinfection against path- by essential oil of A. santonicum while the extracts have no ogens, which is known as seed borne such as X. campestris antibacterial effects. Antibacterial activity defined against pv. campestris, X. campestris pv. vesicatoria and X. cam- some strains is also important in terms of having a bacteri- pestris pv. vitians. cidal effect. The highest inhibition zone with 29 mm was observed against the strains of X. axonopodis pv. cam- 3.3 Antibacterial test results of the essential oils and extracts pestris RK-Xa-cam. Also, the MIC value against this bac- In the present study, antibacterial activities of hydro- terial strain was 125 μl/ml. distillated essential oils, and the extracts isolated from the The results regarding the essential oil and extracts of A. aerial parts of the plant species studied with n-hexane, spicigera have been shown in the Table 5. According to CHCl3, acetone and methanol were tested against 25 plant these results, A. spicigera essential oil has antibacterial ef- pathogenic bacterial strains (Tables 3-5). fect against to 3 of 25 bacterial strains while the extracts have The results regarding the essential oil and extracts no antibacterial effects. The highest inhibition zone with of A. absinthium have been shown in the Table 3. Accord- 16 mm was observed against the strains of P. syringae pv. ing to these results, essential oil of A. absinthium show in- syringae RK-204. Also, the MIC value against this bacterial hibition zone against 15 of 25 bacterial strains in petri strain was 500 μl/ml.

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TABLE 3 - Antibacterial activities of the essential oil and extracts of Artemisia absinthium

Extracts PC NC Essential oil Hexane Chloroform Aceton Methanol OX 10% Strains DMS IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC IZ IZ A. piechaudii RK-155 – – – – – – – – – – – – B. pumilus RK-106 – – – – – – – – – – 11 – C. violaceum RK-231 – – – – – – – – – – – – C. michiganensis subsp. michiganensis Cmm – – – – – – – – – – 11 – E. intermedius RK-90 – – – – – – – – – – – – E. amylovora RK-228 – – – – – – – – – – – – E. caratovora subsp. atroceptica RK-462 7 500 – – – – – – – – – – E. chrysanthemi RK-421 9 500 – – – – – – – – – – E. rhapontici RK-208 – – – – – – – – – – – – Flavobacter sp. RK-299 11 500 – – – – – – – – – – P. agglomerans RK-84 – – – – – – – – – – – – P. aeruginosa RK-168 7 500 – – – – – – – – – – P. cichorii RK-166 – 250 – – – – – – – – 7 – P. huttiensis RK-260 7 250 – – – – – – – – – – P. putida RK-249 – – – – – – – – – – 7 – P. syringae pv. syringae RK-204 11 250 – – – – – – – – – – P. syringae pv. tomato RK-Ps-tom 7 500 – – – – – – – – – – X. axonopodis pv. malvacearum RK-Xa-mal 13 500 – – – – – – – – – – X. axonopodis pv. vesicatoria Xcv110c 9 500 – – – – – – – – – – X. axonopodis pv. campestris RK-Xa-cam 9 500 – – – – – – – – 7 – X. campestris pv. raphani RK-Xc-rap 8 500 – – – – – – – – 7 – X. axonopodis pv. vesicatoria RK-Xcv761 7 250 – – – – – – – – 8 – X. axonopodis pv. vitians Xa-vit 7 500 – – – – – – – – – – X. campestris pv. zinniae Xc-zin 10 250 – – – – – – – – – – X. axonopodis pv. pelargonii RK-Xa-pel 15 250 – – – – – – – – 7 – IZ, inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 1.25 mg of the extracts and 12.5 μL of the essential oil; MIC, minimal inhibitory concentration as μLmL−1 for the essential oil and in mg mL−1 for the extracts; PC, positive control (OX: Oxacilin), NC, Negative control, – , not active, .

TABLE 4 - Antibacterial activities of the essential oil and extracts of Artemisia santonicum

Extracts PC NC Essential oil Hexane Chloroform Aceton Methanol OX 10% Strains DMS IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC IZ IZ

A. piechaudii RK-155 10 250 – – – – – – – – – – B. pumilus RK-106 11 250 – – – – – – – – 11 – C. violaceum RK-231 9 125 – – – – – – – – – – C. michiganensis subsp. michiganensis Cmm – – – – – – – – – – 11 – E. intermedius RK-90 9 250 – – – – – – – – – – E. amylovora RK-228 14* 250 – – – – – – – – – – E. caratovora subsp. atroceptica RK-462 15* 250 – – – – – – – – – – E. chrysanthemi RK-421 9 125 – – – – – – – – – – E. rhapontici RK-208 16 125 – – – – – – – – – – Flavobacter sp. RK-299 20* 125 – – – – – – – – – – P. agglomerans RK-84 10 500 – – – – – – – – – – P. aeruginosa RK-168 7 – – – – – – – – – – – P. cichorii RK-166 7 – – – – – – – – – 7 – P. huttiensis RK-260 12 125 – – – – – – – – – – P. putida RK-249 11 250 – – – – – – – – 7 – P. syringae pv. syringae RK-204 28 125 – – – – – – – – – – P. syringae pv. tomato RK-Ps-tom 11 250 – – – – – – – – – – X. axonopodis pv. malvacearum RK-Xa-mal 27* 125 – – – – – – – – – – X. axonopodis pv. vesicatoria Xcv110c 15* 125 – – – – – – – – – – X. axonopodis pv. campestris RK-Xa-cam 29* 125 – – – – – – – – 7 – X. campestris pv. raphani RK-Xc-rap 14 125 – – – – – – – – 7 – X. axonopodis pv. vesicatoria RK-Xcv761 17* 125 – – – – – – – – 8 – X. axonopodis pv. vitians Xa-vit 15 125 – – – – – – – – – – X. campestris pv. zinniae Xc-zin 11 125 – – – – – – – – – – X. axonopodis pv. pelargonii RK-Xa-pel 15 125 – – – – – – – – 7 – IZ, inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 1.25 mg of the extracts and 12.5 μL of the essential oil; MIC, minimal inhibitory concentration as μLmL−1 for the essential oil and in mg mL−1 for the extracts. * Bactericidal effect was observed; PC, positive control (OX: Oxacilin), NC, Negative control, – , not active.

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TABLE 5 - Antibacterial activities of the essential oil and extracts of Artemisia spicigera

Extracts PC NC Essential oil 10% Strains Hexane Chloroform Aceton Methanol OX DMS IZ MIC IZ MIC IZ MIC IZ MIC IZ MIC IZ IZ

A. piechaudii RK-155 – – – – – – – – – – – – B. pumilus RK-106 – – – – – – – – – – 11 – C. violaceum RK-231 – – – – – – – – – – – – C. michiganensis subsp. michiganensis Cmm – – – – – – – – – – 11 – E. intermedius RK-90 – – – – – – – – – – – – E. amylovora RK-228 12 500 – – – – – – – – – – E. caratovora subsp. atroceptica RK-462 – – – – – – – – – – – – E. chrysanthemi RK-421 – – – – – – – – – – – – E. rhapontici RK-208 – – – – – – – – – – – – Flavobacter sp. RK-299 – – – – – – – – – – – – P. agglomerans RK-84 – – – – – – – – – – – – P. aeruginosa RK-168 – – – – – – – – – – – – P. cichorii RK-166 – – – – – – – – – – 7 – P. huttiensis RK-260 – – – – – – – – – – – – P. putida RK-249 – – – – – – – – – – 7 – P. syringae pv. syringae RK-204 16 500 – – – – – – – – – – P. syringae pv. tomato RK-Ps-tom – – – – – – – – – – – – X. axonopodis pv. malvacearum RK-Xa-mal – – – – – – – – – – – – X. axonopodis pv. vesicatoria Xcv110c – – – – – – – – – – – – X. axonopodis pv. campestris RK-Xa-cam – – – – – – – – – – 7 – X. campestris pv. raphani RK-Xc-rap – – – – – – – – – – 7 – X. axonopodis pv. vesicatoria RK-Xcv761 10 500 – – – – – – – – 8 – X. axonopodis pv. vitians Xa-vit – – – – – – – – – – – – X. campestris pv. zinniae Xc-zin – – – – – – – – – – – – X. axonopodis pv. pelargonii RK-Xa-pel – – – – – – – – – – 7 –

IZ, inhibition zone in diameter (mm) around the discs (6 mm) impregnated with 1.25 mg of the extracts and 12.5 μL of the essential oil; MIC, minimal inhibitory concentration as μLmL−1 for the essential oil and in mg mL−1 for the extracts; PC, positive control (OX: Oxacilin), NC, Negative control, – , not active.

4. DISCUSSION AND CONCLUSIONS tected in the oils of A. santonicum was reported to exhibit antibacterial activity [33-35]. These reports are compatible Our results showed that essential oils (12.5 μL disc−1) with our results in the present study. of A. absinthium, A. santonicum and A. spicigera exhibited Although there are numerous reports on the analyses a broad spectrum of potent antibacterial activity against of essential oils from Artemisia species in the literature, some tested bacterial strains producing 7–29 mm inhibition some Artemisia oils were tested against only a limited zones depending on the bacterial strains tested. However number of bacteria [6, 8]. On the other hand, using essen- the extracts have no antibacterial effects. The results pre- tial oils of the Artemisia species against seed-borne bacte- sented in Tables 3, 4 and 5 show that the MIC values of the rial pathogens is not enough work available. Therefore, this oils vary with the bacterial strains tested, ranging from study performed has a great importance. 125.0 to 500.0 μLmL−1. Furthermore, A. santonicum oil had bactericidal activity against 7 bacterial strains tested In conclusion, the development of natural antimicrobi- (Table 2), whereas A. absinthium and A. spicigera oils als will help to decrease the negative effects (residues, re- didn’t show any bactericidal activity against all of the path- sistance, and environmental pollution) of synthetic drugs. ogen strains (Table 1,3). Numerous reports indicated that a In this respect, natural antimicrobials may be also effective, high percentage of essential oil having antimicrobial activ- selective, biodegradable, and less toxic to environment. In ity [27-32]. The oil of A. absinthium showed the weak an- addition, the risk of the development of resistance by mi- tibacterial activity at a broader spectrum. However, the an- croorganisms and the high cost-benefit ratio are other dis- tibacterial effect of A. spicigera was found to be low as advantages of synthetic chemicals uses [13]. In addition to compared to the essential oil of A. santonicum and A. ab- microorganisms causing infectious diseases in humans sinthium with regard to low inhibition zones and high MIC may develop resistance to many antibiotics due to the in- values. However, A. santonicum were active against most discriminate use of commercial antibiotics [14]. This prob- of the bacterial strains. Previously, we have reported that lem, antibiotics are sometimes associated with adverse ef- the essential oils of A. santonicum species have chemical fects including hypersensitivity, allergic reaction, and im- compositions and major components such as terpinen-4-ol, munity suppression [36]. Thus, this study matters to high- camphor, 1,8- cineole and borneol [8]. The wide antibacte- light the successful usage an environment-friendly, natural, rial spectra of A. santonicum oil may also be attributed to risk free for health of humans and other livings product their relatively high content of oxygenated monoterpenes against some seed-borne pathogens in substitution for the [8]. Recently, oxygenated monoterpenes such as camphor, chemical pesticides that are intensely used and harmful for 1,8- cineole, terpinen-4-ol, and borneol, which were de- environment, natural balance and human health. In view of

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the present results, it is concluded that these essential oils [16] Cakir, A., Kordali, S., Kilic, H. and Kaya, E. (2005). Antifun- can be used as antimicrobial agents for control of seed- gal properties of essential oil and crude extracts of Hypericum linarioides Bosse. Biochem. Syst. Ecol. 33, 245-256. borne bacterial plant pathogens. However, the safety and toxicity of these compounds will need to be addressed. [17] Misra, G. and Pavlostathis, S. G. (1997). Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems. Appl. Microbiol. Biotechnol., 47, 572-577.

[18] Costa, T. R., Fernandes-Orionaldo, F. L., Santos, S. C., Ol- ACKNOWLEDGEMENTS iveria, C. M. A., Liao, L. M., Ferri, P. H., Paulo, J. R., Ferreira, H. D., Sales, B. H. N. and Silva, M. R. R. (2000). Antifungal The research is financed by Scientific and Technolog- activity of volatile constituents of Eugenia dysenterica leaf oil. ical Research Council of Turkey (TUBITAK) (project TO- J. Ethnopharmacol., 72, 111-117. VAGT-107 O 525). [19] Sahin, F. and Kotan, R., (1999). First observation of Xan- thomonas campestris pv. vesicatoria race T2P7 isolated from The authors have declared no conflict of interest. pepper in the Philippines. Plant Disease 83: 590. [20] Kotan, R., and Sahin, F. (2002). First record of bacterial canker, caused by Pseudomonas syringae pv. syringae, on apricot trees in Turkey. Plant Pathol 51:798–798.

REFERENCES [21] Sahin, F., Uslu, H., Kotan, R. and Donmez, M.F. (2002). Bac- terial canker, caused by Clavibacter michiganensis subsp. michiganensis, on tomatoes in Eastern Anatolia region of Tur- [1] Bremer, K. (1994). Asteraceae: cladistics and classification. key. Plant Pathol 51:399. Timber Press: Portland. [22] Kotan, R., Sahin, F. and Ala, A., (2005). Identification and [2] Ling, Y.R. (1994) The genera Artemisia L. and Seriphidium pathogenicity of bacteria isolated from pome fruits trees in (Bess.) Poljak in the world. Compositae Newslett., 25: 39-45. eastern Anatolia region of Turkey. Journal of Plant Diseases [3] Baytop T. (1999). Turkiye’de Bitkiler ile Tedavi: Gecmişte ve and Protection 113: 8-13. Bugun. Nobel Tıp Kitabevi. İstanbul. [23] Adams, R.P., 2007. Identification of Essential Oil Compo- [4] Der, H. S. (2002). Wermut - Artemisia absinthium L. nents by Gas Chromatogra-phy/Mass Spectrometry, 4th Edi- Zeitschrift Phytother 23:187-194. tion. Allured Publishing Corp, Carol Stream, IL,USA, pp. 803. [24] Jennings, W.; Shibamoto, J. 1980. In Qualitative Analysis of [5] Anonymous (1997). ESCOP. Absinthii herba. In: ESCOP (European Scientific Cooperative on Phytotherapy) Mono- FlaVor and Fragrance Volatiles by Capillary Gas Chroma- tography; Academic Press: New York. graphs on the Medicinal Uses of Plant Drugs. Fascicule I; pp. 1-5. [25] Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. and Yolke, R. H. (1995). Manual of Clinical Microbiology; ASM: [6] Juteau, F., Jerkovic, I., Masotti, V., Milos, M., Mastelic, J., Washington, DC, Vol. 6. Bessiere, J. M., and Viano, J. (2003). Composition and antimicrobial activity of the essential oil of Artemisia absinthium [26] Kotan, R., Cakir, A., Dadasoglu, F., Aydin, T., Cakmakci, R., from Croatia and France. Planta Med. 69, 158-161. Ozer, H., Kordali, S., Mete, E. and Dikbas, N. (2010). Anti- bacterial activities of essential oils and extracts of Turkish [7] Sayyah, M., Nadjafnia, L. and Kamalinejad, M. (2004). Anti- Achillea, Satureja and Thymus species against plant patho- convulsant activity and chemical composition of Artemisia genic bacteria. Journal of the Science of Food and Agriculture dracunculus L. essential oil. J. Ethnopharmacol. 94, 283-287. 90: 145-160. [8] Kordali, S., Cakir, A., Mavi, A., Kilic, H., and Yildirim, A. [27] Basim, H., Yegen, O. and Zelker, W. (2000). Antibacterial ef- (2005). Screening of chemical composition and antifungal and fect of essential oils of Thymbra spicata L. var spicata on some antioxidant activities of the essential oils from three Turkish plant pathogenic bacteria. Journal of Plant Disease and Pro- Artemisia species. J. Agric. Food Chem, 53, 1408-1416. tection 107: 279-284. [9] Zang, H., Bai, X and Wu, B. (2012). Evaluation of antimicro- [28] Sokovic M, Van Griensven LJLD 2006. Antimicrobial activity bial activities of extracts of endophytic fungi from Artemisia of essential oils and their components against the three major annua. Bangladesh J Pharmacol; 7: 120-123. pathogens of the cultivated button mushroom, Agaricus bispo- [10] Barnard, M., Padgitt, M. and Uri, N. D. (1997). Pesticide use rus. Eur J Plant Pathol 116: 211-224. and its measurement. Int. Pest Control, 39, 161-164. [29] Kordali, S., Cakir, A., Ozer, H., Cakmakci, R., Kesdek, M., [11] Isman, M. B. (2000). Plant essential oils for pest and disease and Mete, E. (2008). Antifungal, phytotoxic and insecticidal management. Crop Prot, 19, 603-608. properties of essential oil isolated from Turkish Origanum acutidens and its three components, carvacrol, thymol and [12] Brent, K. J. and Hollomon, D. W. (1998). Fungicide Re- pcymene. Bioresource Technology 99: 8788-8795. sistance: The Assestment of Risk, FRAC; Monograph 2; Global Crop Protection Federation: Brussels, Belgium, pp 1- [30] Dadasoglu, F., Aydin, T., Kotan, R., Cakir, A., Ozer, H., Kor- 48. dali, S., Cakmakci, R., Dikbas, N. and Mete, E. 2011. Antibac- terial Activities of Extracts And Essential Oils of Three Origa- [13] Roy, N. K. and Dureja, P. (1998). New ecofriendly pesticides num Species Against Plant Pathogenic Bacteria And Their Po- for integrated pest management. Pest World, 3:16-21. tential Use as Seed Disinfectants. Journal of Plant Pathology, 93:2, 271-282. [14] Service, R. F. (1995). Antibiotics that resist resistance. Sci- ence, 270:724-727. [31] Kotan, R., Dadasoglu, F., Karagoz, K., Cakir, A., Ozer, H., Kordali, S, Cakmakci, R. and Dikbas, N. 2013. Antibacterial [15] Mukherjee, P. K., Saritha, G. S. and Suresh, B. (2002). Anti- activity of the essential oil and extracts of Satureja hortensis microbial potential of two different Hypericum species availa- against plant pathogenic bacteria and their potential use as ble in India. Phytother. Res, 16, 692-695. seed disinfectants. Scientia Horticulturae, 153, 34-41.

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[32] Kotan, R., Cakir, A., Ozer, H., Kordali, S., Cakmakci, R., Da- dasoglu, F., Dikbas, N., Aydin, T. and Kazaz, C. 2014. Anti- bacterial effects of Origanum onites against phytopathogenic bacteria: Possible use of the extracts from protection of disease caused by some phytopathogenic bacteria. Scientia Horticul- turae, 172, 210-220. [33] Pattnaik, S., Subramanyam, V. R., Bapaji, M. and Kole, C. R. (1997). Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios, 89, 39-46. [34] Tabanca, N., Kirimer, N., Demirci, B., Demirci, F. and Baser, K. H. (2001). Composition and antimicrobial activity of the essential oils of Micromeria cristata subsp. phrygia and the enantiomeric distribution of borneol. J. Agric. Food Chem., 49, 4300- 4303.

[35] Setzer, W. N., Vogler, B., Schmidt, J. M., Leahy, J. G. and Rives, R. 2004. Antimicrobial activity of Artemisia dauglasiana leaf essential oil. Fitoterapia 75, 192-200.

[36] Dey, S. K., Banarjee, D., Chattapadhyay, S. and Karmakar, K. B. (2010). Antımıcrobıal Actıvıtıes Of Some Medıcınal Plants Of West Bengal. International Journal Of Pharma And Bio Sciences. 1:3, 1-10.

Received: November 18, 2014 Accepted: February 19, 2015

CORRESPONDING AUTHOR

Fatih Dadasoglu Department of Molecular Biology and Genetics Faculty of Science and Letters Agri İbrahim Cecen University Agri 04100 TURKEY

Phone: +90 472 2154073 Fax: +90 472 215 6554 E-mail: [email protected]

FEB/ Vol 24/ No 9/ 2015 – pages 2715 - 2724

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HEAVY METAL CONCENTRATIONS IN Mytilus galloprovincialis FROM ÇANAKKALE STRAIT, NW TURKEY

Serkan Özden1,* and Sezginer Tunçer2

1Department of Environmental Engineering, Engineering Faculty, Çanakkale Onsekiz Mart University, 17100, Çanakkale, Turkey 2Faculty of Marine Science and Technology, Çanakkale Onsekiz Mart University, 17100, Çanakkale, Turkey

ABSTRACT waste materials and irregular industrial progress. Also marine productions (fish, mussel and marine vegetations) Mytilus galloprovincialis, a bioindicator, was collected have been consumed by humans as a cheap food material from the Çanakkale Strait to investigate heavy metal con- along these industrial sea side cities [2, 3]. Therefore, toxic centrations related to contamination. Pb, Cu, Zn and Fe heavy metals are directly transferring from marine environ- heavy metal concentrations were determined in the mussel ment to human through the food chain. From these marine at six selected stations, namely Hamzakoy (Station 1), Ge- foods, mussel (M. galloprovincialis) is known well as a bi- libolu Tersanesi (2), Kilya Koyu (3), Eceabat Çam Burnu oindicator to conducted potentially toxic heavy metals [1- (4), Kilitbahir (5) and Büyük Kemikli Burnu (6). These mus- 4]. These metals in mussels and their tissues have shown in sel samples were collected six times in 2009, 2010, 2012- several monitoring studies [5-18] along the north and west 2013 during winter, spring, summer and autumn seasons. seas, costs, bays and straits of the industrial cities in Turkey Zinc (19.15 µg/g) is at the highest level in spring 2010 and last 20 years. Unfortunately, among these regions, Çanak- Pb (0.05 µg/g) has the lowest in winter 2010. While maxi- kale Strait (Dardanelles) in eastern costs of the Gelibolu mum values of the heavy metal concentrations are observed Peninsula (Fig. 1) presented by no actual and few data in at Station 2, the minimum values are from Station 6. All sta- restricted monitoring periods [11-17] to understand of pol- tions are associated with mean seasonal values: Station 2 ˃ lution level. Station 3 ˃ Station 4 ˃ Station 5 ˃ Station 1 ˃ Station 6. The current study has been conducted to possible con- There were significant differences between heavy metal tamination levels by mussels seasonally sampled during re- concentrations among the first four seasons (p˂0.05). Val- cent six different periods between 2009 and 2013 years, ues determined as seasonal averages (Fe ˃ Zn ˃ Cu ˃ Pb) and six different stations along the eastern coasts, bays and have been discussed, based on Anonymous, FAO, WHO/ estuaries of the Çanakkale Strait in Gelibolu Peninsula an- FAO, EPA, TME data and previous studies in the region, alyzed for toxic heavy metals (Pb, Cu, Zn and Fe). within the limits of the northern Aegean and southern Mar- mara Sea. In conclusion, it is suggested that these heavy metal levels do not pose an environmental risk in Çanak- 2. MATERIALS AND METHODS kale Strait. The biological samples used in the course of this study

were M. galloprovincialis a species in the Mytilidae family KEYWORDS: Contamination, heavy metals, M. galloprovincialis, in the class Bivalvia. This species, called the Mediterranean Çanakkale Strait, Turkey mussel, has a wide distribution area from the Mediterra-

nean to the Black Sea [2, 3]. As mussels are organisms that filter feed on organic material and phytoplankton in water, 1. INTRODUCTION they are a very good biological indicator species used to research marine pollution. In addition during the water fil- Heavy metals, an environmentally polluting material, tration procedure they may filter toxic material [8]. increase in water and sediment layers as a result of transport Sampling (12 to 15 number mussel sample from each by rivers, erosion, rain and flood waters in aqueous environ- station) occurred at six different stations (Hamzakoy (Sta- ments [1]. In addition human activities may cause an in- tion 1), Gelibolu Tersanesi (Station 2), Kilya Koyu (Station crease in heavy metals in marine environments [1, 2]. 3), Eceabat Çam Burnu (Station 4), Kilitbahir (Station 5) Moreover, fast developed industrial cities along the costs, and Büyük Kemikli Burnu (Station 6)), along the coast of bays and straits in Mediterranean region have environmen- Çanakkale Strait between June 2009 and May 2010 to in- tal pollution problems by the human activities, domestic clude all four seasons and also twice in October 2012 and February 2013 for a total of six times (Fig. 1-2, Table 1). * Corresponding author During biological sampling weather conditions, seasonal

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FIGURE 1 - M. galloprovincialis sample stations from Çanakkale Strait.

and monthly average temperatures formed parallel values. oven set to 105 ºC before being weighed again to calculate The topic of the research, M. galloprovincialis biological dry weight and dry weight percentage (Table 1). The heavy samples, was collected on the coast by hand using waders metal contents of the prepared samples were determined in and gloves with a steel spatula. Biological samples col- COMU Central Laboratory using an AAS (Table 2). lected at all stations were put in clean polyethylene bags The statistical analysis of data obtained in this study and labeled, brought to the laboratory preserving the cold from M. galloprovincialis were seasonally compared using chain and stored at -25 ºC [9] until analysis. the Friedman Test [19]. To compare the mussels seasonally Mussel samples, brought to laboratory temperatures, the first four samplings were compared with due regard for were well washed, first with tap water and then distilled wa- the characteristics of the seasons (summer 2009, fall 2009, ter. Later each mussel sample was measured for length (the winter 2010 and spring 2010). Later samples taken in the longest length of the mussel shell) and weighed (Table 1). same two seasons (fall 2009 and fall 2012) were compared. After data recording the mussels were separated from the Again for a later comparison in the same two seasons (win- shell using a steel-tipped scalpel and forceps, and left on ter 2010 and winter 2013) the Friedman test was used. In blotting paper until a constant weight was obtained (until situations where the difference was significant to deter- true weight was reached). Samples were transferred to mine which season caused significance the “multiple com- flasks of known weight and weighed on a sensitive scale. parison test” was used. Depending on the sample weight HNO3:HClO4 (ratio 5:1) was added [3] and left for two hours for extraction. All mineralization processes were conducted on a heat-con- 3. RESULTS AND DISCUSSION trolled hot plate for 48-96 hours. Samples with organic dis- solution completed were filtered through filter paper and When the sampling periods and stations of M. gallo- brought to a fixed volume with distilled water. The samples provincialis are considered during the summer of 2009 the were stored in polyethylene containers at +5 ºC until anal- longest average mussels were samples from Gelibolu Ter- ysis. The same samples were placed in glass petri dishes of sanesi, Kilya Koyu and Büyük Kemikli Burnu with a known weight and weighed and were left overnight in an length of 4.9 cm, while the shortest were 3.8 cm from Ham-

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zakoy station. The largest mussels weighed 19.66 g in sum- with an average of 37.5%. The lowest values were from sam- mer 2009 from Büyük Kemikli Burnu with the smallest ples from Kilitbahir with an average of 28.48% (Table 1). In from Hamzakoy station weighing 9.05 g (Table 1). Accord- general the results of the measurements show that the length ing to the dry weight percentages of M. Galloprovincialis, of the mussels did not change through the seasons but the the highest values were in samples from Gelibolu Tersanesi weight increased slightly in fall and spring months with

TABLE 1 - Data from the sample stations, average length (cm/±0.02), weight (g/±0.03) and average percent dry weight (%/±0.03) of Mytilus galloprovincialis during each period.

Stations Hamzakoy Gelibolu T.. Kilya Koyu E. Çam Burnu Kilitbahir B. Kemikli B. (Station 1) (Station 2) (Station 3) (Station 4) (Station 5) (Station 6) Coordinates 40º 24’ 48’’ N 40º 19’ 13’’ N 40º 12’ 09’’ N 40º 11’ 34’’ N 40º 08’ 51’’ N 40º 18’ 55’’ N 26º 40’ 46’’ E 26º 34’ 54’’ E 26º 21’ 30’’ E 26º 21’ 36’’ E 26º 22’ 51’’ E 26º 14’ 18’’ E Period/Habitat Sandy, Slimy Slimy Sandy Stony, Rocky Gravelly, Rocky Stony, Rocky Summer’09 3.80 cm 4.90 4.90 4.40 4.30 4.10 9.05 g 15.43 18.63 17.93 15.10 12.00 42.40 % 45.40 37.80 35.20 34.30 39.40 Autumn’09 4.00 4.80 4.80 4.20 4.30 4.90 10.85 16.03 15.80 11.22 12.82 19.66 33.50 37.40 36.50 36.20 32.60 35.30 Winter’10 4.30 4.40 4.20 4.30 4.20 4.60 12.11 13.47 11.95 11.52 12.58 14.01 34.50 38.20 24.70 26.30 23.50 26.80 Spring’10 4.40 4.70 4.80 4.30 4.40 4.80 13.73 16.76 17.86 14.73 14.05 16.83 42.50 44.60 37.20 36.50 30.20 38.80 Autumn’12 4.30 4.70 4.30 4.30 4.50 4.60 14.55 16.27 14.36 15.02 14.93 16.02 28.40 32.80 31.30 30.60 27.50 29.40 Winter’13 4.40 4.60 4.20 4.20 4.30 4.40 15.84 17.02 13.75 13.07 13.59 16.04 23.10 26.40 24.20 25.60 22.80 24.80

TABLE 2 - Pb, Cu, Zn and Fe concentrations in Mytilus galloprovincialis (µg/g dry weight/±0.03).

Metal Station/ Hamzakoy Gelibolu T. Kilya Koyu E. Çam B. Kilitbahir B.Kemikli B. Period (Station 1) (Station 2) (Station 3) (Station 4) (Station 5) (Station 6) Pb Summer’09 0.26 0.47 0.36 0.23 0.28 0.12 Autumn’09 0.13 0.36 0.15 0.17 0.18 0.10 Winter’10 0.19 0.27 0.18 0.11 0.14 0.05 Spring’10 0.35 0.56 0.38 0.27 0.28 0.12 Autumn’12 0.14 0.24 0.12 0.15 0.11 0.10 Winter’13 0.22 0.28 0.13 0.17 0.16 0.09 Average 0.22 0.36 0.22 0.18 0.19 0.10 Cu Summer’09 0.41 0.48 0.77 0.47 0.50 0.37 Autumn’09 0.39 0.40 1.00 0.56 0.43 0.24 Winter’10 0.20 0.23 0.52 0.36 0.37 0.18 Spring’10 0.42 1.52 1.30 0.53 0.54 0.46 Autumn’12 0.29 0.80 1.13 0.42 0.33 0.22 Winter’13 0.25 0.97 1.24 0.38 0.27 0.19 Average 0.33 0.73 0.99 0.45 0.41 0.28 Zn Summer’09 8.88 18.8 12.90 13.05 11.20 6.00 Autumn’09 8.64 16.83 11.23 12.88 10.00 3.27 Winter’10 7.32 11.23 9.40 10.00 6.16 2.28 Spring’10 9.86 19.15 15.64 13.62 12.05 5.64 Autumn’12 8.55 16.83 9.72 11.39 7.94 3.00 Winter’13 6.24 15.30 8.87 10.43 7.17 2.78 Average 8.25 16.36 11.30 11.89 9.09 3.83 Fe Summer’09 9.40 11.30 9.90 10.00 10.20 5.63 Autumn’09 7.45 9.23 6.35 11.60 11.37 5.30 Winter’10 7.20 7.63 5.17 9.37 7.12 4.20 Spring’10 11.22 17.06 12.68 11.03 10.97 6.82 Autumn’12 6.31 8.77 6.45 6.85 7.92 4.12 Winter’13 5.11 9.57 6.28 7.02 7.14 2.23 Average 7.78 9.32 7.80 9.30 9.12 4.72

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FIGURE 2 - Pb, Cu, Zn and Fe concentrations in M. galloprovincialis (µg/g dry weight).

slight decreases in winter and summer months. In addition > Station 5 > Station 1 > Station 6 (Fig. 2, Table 2). Ac- this study determined there was no correlation between cording to the results of the research when the Zn data from heavy metal pollution values and length-weight changes, M. Galloprovincialis are considered the highest heavy however the fact that the largest and heaviest forms were metal concentrations were found in Gelibolu Tersanesi, Ki- found at Büyük Kemikli Burnu which had the lowest heavy lya Koyu, Eceabat Çam Burnu and Kilitbahir stations. In metal contents is interesting. In a similar study by Prota- spring 2010 Gelibolu Tersanesi had the highest Zn concen- sowicki et al. [20] of samples collected from the Baltic Sea, tration of 19.15 µg/g dry weight, while the lowest concen- no clear relationship was identified between heavy metal tration was from Büyük Kemikli Burnu in winter 2010 with pollution values and length-weight changes. 2.28 µg/g dry weight. When seasonal averages are consid-

According to the results of the research when the Pb ered Zn concentrations were found in the order; Station 2 data from M. Galloprovincialis are considered, the highest > Station 4 > Station 3 > Station 5 > Station 1 > Station 6 heavy metal concentrations were found in Gelibolu Ter- (Fig. 2, Table 2). The Fe data from M. Galloprovincialis sane, Kilya Koyu, Kilitbahir and Hamzakoy stations. In indicates that the areas with highest heavy metal concen- spring 2010 Gelibolu Tersanesi had the highest Pb concentration are Gelibolu Tersanesi, Kilya Koyu, and Hamzakoy tration of 0.56 µg/g dry weight, while the lowest concen- stations. The highest value was from Gelibolu Tersanesi in tration was from Büyük Kemikli Burnu in winter 2010 with spring 2010 with a Fe concentration of 17.06 µg/g dry 0.045 µg/g dry weight. When seasonal averages are con- weight with the lowest value from Büyük Kemikli Burnu in sidered Pb concentrations were found in the order; Station winter 2013 with a concentration of 2.23 µg/g dry weight. 2 > Station 3 > Station 1> Station 5 > Station 4 > Station 6 When the seasonal averages are considered the Fe concen- (Fig. 2, Table 2). The Cu data from M. Galloprovincialis trations were in the order; Station 2 > Station 4 > Station 5 > indicates that the areas with highest heavy metal concentra- Station 3 > Station 1 > Station 6 (Fig. 2, Table 2). According tion are Gelibolu Tersanesi, Kilya Koyu, Eceabat Çam Burnu to the heavy metal analysis results considering the seasonal and Kilitbahir stations. The highest value was from Gelibolu averages the metals were found in M. Galloprovincialis in Tersanesi in spring 2010 with a Cu concentration of 1.52 µg/g the order Fe ˃ Zn ˃ Cu ˃ Pb. The seasonal averages of heavy dry weight, with the lowest value from Büyük Kemikli Burnu metal concentrations were in the order Station 2 > Station 3 in winter 2010 with a concentration of 0.18 µg/g dry weight. > Station 4 > Station 5 > Station 1 > Station 6. When the seasonal averages are considered the Cu concen- Statistical analysis results from the mussels, compared trations were in the order Station 3 > Station 2 > Station 4 with respect to the four seasons, indicates that for Pb, Cu,

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Zn and Fe p˂0.05. This shows that the difference in heavy metals Co, Cu, Fe, Ni, Pb and Zn and that especially Pb and metals in mussels during the seasons is significant. Accord- Zn were above acceptable limits. Similarly in a study of ing to the statistical analysis results for mussels, compari- Kepez Port, Çanakkale, the Cd and Pb concentration levels son of characteristics in fall 2009 and 2012 (same season) were high in seawater at the port [16]. The authors stated that showed Pb p˃0.05, Cu p˃0.05, Zn p˂0.05 and Fe p˂0.05 rivers linked to the Kepez delta carried Cd and Pb and that which indicates the difference in heavy metals in the sea- this might be related to mining. Çayır et al. [17] researched sons is significant. Statistical analysis results from the mus- the metal concentrations and effect on human health of M. sels compared with respect to winter 2010 and 2013 (same galloprovincialis species from the Çanakkale Straits. Sam- season) indicates that for Pb, Cu, Zn and Fe p>0.05. This pling was during 2007, 2008 and 2009 from six stations. The shows that the difference in heavy metals in mussels during concentrations of the elements Cd, Cr, Cu, Pb, Zn, Fe and Ni the seasons is not significant. Uysal et al. [5] analyzed Cu, were 1.59, 6.04, 12.01, 6.03, 319.6, 402.79 and 3.52 mg/kg, Zn, Pb, Fe, Cd and Hg levels in some mussels and fish be- respectively. These values did not reach the recommended tween 1984-1988 from the coast of the Aegean Sea and risk values for human health. Küçüksezgin et al. [18] studied found that heavy metal changes were proportionally higher heavy metal levels from the whole of the eastern Aegean in mussel species (Fe>Zn>Cu>Cd>Pb>Hg) than in pelagic coasts and bays and stated that, apart from Zn, there was no fish species but determined that the heavy metal concentra- risk to public health. In a study investigating the bacterial tion was not observed at levels dangerous to public health. loading of mussels collected from the Çanakkale Strait, Topçuoğlu et al. [10] along the northern strip of the Sea of Şener et al. [4] found more bacteria in mussels from the Marmara in sedimentary and biological samples from Med- Anatolian coastline hunted in the summer months, com- iterranean mussels identified metals from highest to lowest pared to those from the European coastline hunted in the as Ni, Mn, Cu, Pb, Cr, Cd, and Co. In the south coast of the winter. They noted that to prevent risks to human health Çanakkale Strait, Özden [11] found heavy metal concentra- attention should be paid to thermal processing and storage tions in M. galloprovincialis, Patella vulgata, Monodonta conditions before consumption. turbinata and the algae Ulva lactuca, Cystoseira barbata, and Enteromorpha sp. from 9 stations were in the order Çanakkale Strait, the northern Aegean and southwest- Fe> Zn> Cu> Pb> Cd. In the study and in our study span- ern Sea of Marmara in M. galloprovincialis in marine en- ning 2009-2013, the heavy metal concentrations have sim- vironments, coasts and bays polluted by terrestrial inputs ilar patterns. Karafistan and Ormancı [12] researched the heavy metal concentrations have reached certain levels, metal contents (Pb, Cu, Cd, Ni, Al and Zn) from M. gallo- however in many places they are observed at levels close provincialis from the south coast of the Çanakkale Strait to the limits. In our study the heavy metal concentrations and found Cu, Cd and Zn levels occasionally exceeded in M. galloprovincialis were high in Gelibolu Tersanesi, food index values. They emphasized that the metal con- Kilitbahir and Eceabat Çam Burnu. The most important tents of Mediterranean mussels (p<0.05) had rates of reason for this was both the increased rate of ship mainte- Pb>Cd>Cu and when the frequency of consumption is con- nance, construction and repair during spring and summer sidered, this mussel species may occasionally be unsafe. months, and the intensity of traffic on the straits and port They stated regional mining activity and other industrial activity. Another reason for the intense pollution at these wastes were sources of the pollution. Lök et al. [13] in a stations is explained by the fact that they are open to expo- study of heavy metal concentration of M. galloprovincialis sure by polluting factors. These three stations should be species from Çanakkale Strait attempted to determine lev- considered for pollution studies and require monitoring els for As, Cd, Cr, Cu, Hg, Ni, Pb and Zn metals. These over longer periods. Similarly Akbaş and Güneyli bays, metals were observed at levels above accepted Turkish close to streams entering the Gulf of Saros and Çanakkale Standard Institute limits. In M. galloprovincialis samples Straits from the Gelibolu Peninsula, should be monitored. from the Sea of Marmara and Çanakkale Strait, Mol and In addition as there is no mining activity close to our study Alakavuk [14] observed Cu, Cd and Hg contents were area, it is thought that Pb inputs have stayed at low levels. within safe limits while Pb and Zn contents were high. In However the cause of high Zn, Cu and Fe may be the samples of Mytilus galloprovincialis collected from maintenance, construction and repair of ships, together Yalova on the Sea of Marmara, Culha et al. [15] identified with shipping traffic [21] on the straits. The comparison no significant seasonal difference (p>0.05) in the heavy station (Station 6) with lowest concentrations of heavy

TABLE 3 - Pb, Cu, Zn and Fe concentrations acceptable upper limits about pollution in official sources in M. galloprovincialis (dry weight).

Anonymous FAO WHO/FAO 1999, EPA TME This study Heavy Metals 1995 1983 2004 (PTWI) 2012 2012 (max. level) [22] [23] [24, 25] [26] [27] Pb 1.0 mg/kg 0.5 mg/kg 2 mg/kg 5 mg/L 0.1 mg/L 0.47 µg/g Cu 20 mg/kg 30.0 mg/kg 2 mg/kg 0.2 mg/L 0.01 mg/L 1.52 µg/g Zn 50 mg/kg 30.0 mg/kg 7 mg/kg 2.0 mg/L 0.1 mg/L 19.15 µg/g Fe - - 7 mg/kg 5 mg/L - 17.06 µg/g

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TABLE 4 - Comparison of Pb, Cu, Zn and Fe heavy metal concentrations determined in mussels (M. galloprovincialis) in mainly Çanakkale Strait (1); also Soutwestern Marmara Sea (2) and Northern Aegean Sea (3) stations (dry weight).

H.Metals Pb Cu Zn Fe Method References Regions 1,3 6.49 µg/g 3.20 µg/g 42.46 µg/g 94.97 µg/g AAS Uysal et al., 1989 [5] 1,2 5.2 µg/g g-1 9.5 µg/g g-1 319.95 µg/g g-1 415 µg/g g-1 AAS Topçuoğlu et al., 2004 [10] 1 0.39 µg/g 0.90 µg/g 16.50 µg/g 11.75 µg/g ICP-AES Özden, 2005 [11] 1 18.474 µg 0.661 µg 65.612 µg - AAS Lök et al., 2010 [13] 1 82.515 µg/g g-1 2.752 µg/g g-1 34.122 µg/g g-1 - ICP-AES Karafistan and Ormancı, 2010 [12] 2 0.56 mg kg 2.36 mg kg 97.13 mg kg - ICP-MS Mol and Alakavuk, 2011 [14] 2 2.92 mg kg 5.54 mg kg 106.23 mg kg 156.72 mg kg ICP-AES Culha et al., 2011 [15] 1 6.03 mg/kg 12.01 mg/kg 319.6 mg/kg 402.79 mg/kg ICP-AES Çayır et al., 2012 [17] 1,3 1.55 mg/kg-1 5.92 mg/kg-1 142.3 mg/kg-1 - AAS Küçüksezgin et al., 2013 [18] 1 0.47 µg/g 1.52 µg/g 19.15 µg/g 17.06 µg/g AAS This Study

metals was identified as Büyük Kemikli Burnu. This station [4] Şener, A., Demir, N., Çakıcı, N., Çakıcı, H., Kaya, H. and Ba- is spatially a headland that is naturally cleaned by marine kar, C. (2013). Çanakkale Boğazından avlanan kara midyelerinin (M. galloprovincialis) mikrobiyolojik incele- currents. This headland has no terrestrial inputs. mesi. Nobel Med., 9, 2, 69-73.

When the results of our study of M. Galloprovincialis [5] Uysal, H., Yaramaz, Ö. Tunçer S. and Parlak, H. (1989). Ege Denizi kıyılarında pollusyon durumu, organizma ve ekosistem are compared with previous studies findings in and around üzerindeki etkileriyle ilgili araştırmalar. Ege Üniv. Su Ürünleri of the study area (Table 3), the data are in accordance with Dergisi, 6, 21-24. and are below the highest levels for heavy metals deter- [6] Egemen, Ö., Alparslan, M. and Sunlu, U. (1997). Çanak- mined by data from the Anonymous [22], FAO [23], kale’de (Karacaören ve Kepez) toplanan midyelerde (M. Gal- WHO/FAO [24, 25], EPA [26] and TME [27] (Table 4). In loprovincialis, Lamarck) bazı ağır metal düzeylerinin araştırıl- light of data obtained from M. galloprovincialis from Ça- ması. Ege Üniversitesi Su Ür. Der., 14, 1-2, 189-196. nakkale Strait around Gelibolu Peninsula in the four sea- [7] Balkıs, N., Aksu, A. and Hiçsönmez, H. (2013). Pollution sons from June 2009 to May 2010 and in fall 2012 and win- Monitoring Using Mytilus Galloprovincialis and Fishes: A ter 2013 in this study, we conclude that pollution in the Case Study on the Southern Black Sea Shelf. Asian Journal of study period did not reach dangerous levels on a regional Chemistry, 25, 1, 450-454. scale. [8] Bat, L., Gündoğdu, A. Öztürk, M. and Öztürk, M. (1999). Cop- per, Zinc, Lead and Cadmium concentrations in the Mediter- ranean mussel M. Galloprovincialis (L. 1718) from the Sinop coast of the Black Sea. Turkish. J. Zool., 23, 321-326. ACKNOWLEDGMENTS [9] Tüzen, M., (2003). Determination of Heavy Metals in Fish Samples of the Middle Black Sea (Turkey) by Graphite Fur- nace Atomic Absorbsion Spectometry. Food Chemistry, 80, This study forms part of a ÇOMU Arts and Science 119-123. Institute doctoral thesis. It was also supported by COMU- BAP project number 2009/145. Catherine Yiğit is thanked [10] Topçuoğlu, S., Kırbaşoğlu, Ç. and Yılmaz, Z. (2004). Heavy metal levels in biota and sediments in the northern coast of the for her contribution to the English of the manuscript. Marmara Sea. Environ. Monit. Assess., 96, 183-189.

The authors have declared no conflict of interest. [11] Özden, S. (2005). Çanakkale Boğazı ve çevresindeki bazı midye ve alg türlerinin ağır metal düzeylerinin belirlenmesi. ÇOMU Fen Bilimleri Ensititüsü, Yüksek Lisans Tezi, 32 s., Çanakkale.

REFERENCES [12] Karafistan, A. and Ormancı, H.B. (2010). Metal concentrations in M. Galloproviancialis from Southern Dardanelles, Turkey. ESAIJ, 5, 3, 201-204. [1] Egemen Ö. (2000). Çevre ve Su Kirliliği. Kitap, Ege Üniver- sitesi Su Ürünleri Fakültesi Yayını No: 42, 120 sayfa, İzmir. [13] Lök, A., Çolakoğlu, S., Acarlı, S., Serdar, S., Küçükdermenci, A., Yiğitkurt, S., Kırtık, A. and Güler, M. (2010). Heavy metal [2] Başçınar S.N., 2009. Bentik Canlılar ve Biyoindikatör Tür. concentrations in the Mediterranean Mussels (M. galloprovin- Yunus Araştırma Bülteni, Su Ürünleri Araştırma Enstitüsü cialis) collected from the Dardanelles. Int. Mer. Medit., 39, Yayını (2): 5-8. Trabzon. 278-285.

[3] Atabeyoğlu, K. and Atamanalp, M. (2010). Yumuşakçalarda [14] Mol, S. and Alakavuk Üçok, D. (2011). Heavy metals in mus- (Mollusk) yapılan ağır metal çalışmaları. Atatürk Üniv. Vet. sels (M. galloproviancialis) from Marmara Sea, Turkey. Bio. Bil. Derg., 5, 1, 35-42. Trace Elem. Res., 141, 184-191.

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[15] Culha, S.T., Koçbaş, F., Gündoğdu, A., Baki, B., Culha, M. and Topçuoğlu, S. (2011). The seasonal distribution of heavy metals in Mussel sample from Yalova in the Marmara Sea, 2008-2009. Enviromental Mon. Assess., 183, 1-2, 525-529.

[16] Yılmaz, S. and Sadıkoğlu, M. (2011). Study of heavy metal pollution in seawater of Kepez Harbor of Çanakkale (Turkey). Environ. Monit. Assess., 173, 899-904.

[17] Çayır, A., Coşkun, M. and Coşkun, M. (2012). Evaluation of metal concentrations in mussel M. galloprovincialis in Darda- nelles Strait, Turkey in regard of safe human consumption. Bull. Environ. Contam. Toxi., 89, 91–95. [18] Küçüksezgin, F., Pazı, I., Gier-Yücel, G., Akçalı, B. and Gal- gani, F. (2013). Monitoring of heavy metal and organic compound levels along the eastern Aegean cost with transplanted mussels. Chemosphere, 93, 1511-1518. [19] Mendeş, M. (2012). Uygulamalı bilimler için istatistik ve araştırma yöntemleri. Kriter Yayınları, Kitap, s. 457, İstanbul. [20] Protasowicki, M., Dural, M. and Jaremek, J. (2008). Trace metals in shelle of Blue Mussels from the Poland Coast of Bal- tic Sea. Environ. Monit. Assess., 141, 329-337. [21] Çiner, F. and İnan, H. (1996). Gemi taşımacılığından kaynak- lanan deniz kirlenmesi. Yerleşim ve Çevre Sorunları: Çanak- kale İli Sempozyumu, 1-10, Çanakkale. [22] Anonymous (1995). Resmi Gazete Sayı: 22223, sayfa: 1361. [23] FAO (1983). Compilation of legal limits for hazardous substances in fish and fishery products, FAO Fishery Circular, 464, 5–100. [24] WHO/FAO (1999). Summary Report of the 53rd meeting of the joint FAO/WHO expert committee on food additives. JEFCA, Roma. [25] WHO/FAO (2004). Summary of evaluations performed by the joint FAO/WHO Expert Committee on Food Additives (JECFA 1956–2003). ILSI Press, International Life Sciences Institute, Washington, DC. [26] EPA (2012). Environmental Protection Agency national recommended water quality criteria. http://water.epa.gov. [27] TME (T.C. Çevre ve Şehircilik Bakanlığı Su Kirliliği Kontrolü Yönetmeliği) (2012). Resmi Gazete (31.12.2004 Resmi Gazete Sayısı: 25687; Güncelleme Tarihi 2012) 3. Bölüm, Madde 11, 51 sayfa.

Received: November 24, 2014 Accepted: December 17, 2014

CORRESPONDING AUTHOR

Serkan Özden Department of Environmental Engineering Engineering Faculty Çanakkale Onsekiz Mart University 17100 Çanakkale TURKEY

E-mail: [email protected]

FEB/ Vol 24/ No 9/ 2015 – pages 2725 - 2731

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EFFECT OF ZINC SULPHATE ON THE LEVELS OF PLASMA PARAOXONASE ACTIVITY, TOTAL OXIDANT AND HIGH DENSITY LIPOPROTEIN OF TRANSCAUCASIAN BARB (Capoeta capoeta [Guldenstaedt, 1773])

Haci Ahmet Deveci1, İnan Kaya2, Muhittin Yılmaz2,* and Mahmut Karapehlivan3

1Islahiye Vocational School, Gaziantep University, 27800 Gaziantep, Turkey 2Department of Bioengineering, Faculty of Engineering and Architecture, Sinop University, 57000 Sinop, Turkey 3Biochemistry, Faculty of Veterinary Medicine, Kafkas University, 36100 Kars, Turkey

ABSTRACT The importance of Zn+2 for living organisms originate from being an integral part of cofactor and metalloenzyms Zinc and its compounds have different effects in chem- for activity of enzymes related to specific Zn+2 as carbonic ical or toxicological terms on many living species in the anhydrase, alkaline phosphatase and alcohol dehydrogen- aquatic areas. In this study, total plasma paraoxonase ac- ase [5]. In case of Zn+2 deficiency or high Zn+2 amount in tivity (PON1), total oxidant (TOS) and high density lipo- aquatic environment, Zn+2 has been taken by gill cells at a protein (HDL) levels in response to different doses of zinc large rate [6, 7]. Reactive oxygen species (ROS) originat- sulphate (ZnSO4) in Capoeta capoeta were investigated. ing from environmental polluters cause harmful effects on The fishes were kept in tanks for 15 days for adaptation. oxidant/antioxidant balance [8]. Analysis of total oxidant Three groups of fish (control, 1st and 2nd), each containing status (TOS) for the organism has been reported to be more nine fishes, were placed in separate tanks containing no 5 useful, rather than to determine oxidants parameters [9]. and 10 mg/L ZnSO4, respectively for 10 days. At the end Paraoxonase (PON1, EC 3.1.8.1) related to HDL is rec- of the study, blood samples were taken, and plasma PON1, orded to have an important protective effect as antioxidant TOS and HDL levels were analyzed. An increase in plasma defense system element due to its LDL oxidation reduction TOS level was found along with increasing ZnSO4 amounts [10]. Changes on HDL also affect PON1 activity linked to compared to the control group, and a decrease in PON1 and HDL lipids by means of hydrophobic N-terminal region HDL levels was observed. In conclusion, it was determined [11]. Capoeta capoeta from Cyprinidae family, which is that levels of PON1, TOS and HDL were altered depending spread in North East Anatolian Region as an aquatic living, on ZnSO4 doses applied in Capoeta capoeta. has economic importance in terms of aquatic nourishment

[12, 13]. Our study aims to investigate plasma PON1, TOS and HDL levels of Capoeta capoeta in an aquatic environ- KEYWORDS: freshwater fish, Capoeta capoeta, zinc, paraoxonase activity, total ment, in which ZnSO4 is applied. oxidant status, high density lipoprotein

2. MATERIALS and METHODS 1. INTRODUCTION In this study, a total of 27 Capoeta capoeta [Gulden- Recently, heavy metal pollution in environment has staedt, 1773] between weights 200-250 g was used. quickly increased because of household, industrial and ag- Fishes captured from Kars Creek were taken into tanks in ricultural wastes [1, 2]. It has been reported that a lot of laboratory and waited for the purpose of adaptation to en- heavy metals act as environmental polluters and constitute vironment conditions for 10 days. Among the three an important risk for aquatic organisms in terms of genetic, groups with nine fishes each, first group was in tank with physiological, biochemical and behavioral parameters [3]. normal water, second and third group fishes were in water Although a lot of polluters disappear by breaking down environment including 5 and 10 mg/L ZnSO4 for 10 days. biologically, biological metals can neither be constituted Afterwards, blood samples were collected intracardially into nor disappeared [4]. tubes with heparine and centrifuged in +4ºC and 3000 rpm for 10 minutes. Plasma samples obtained from centrifuga- tion were kept at -20ºC until analyses. * Corresponding author

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In the measurement of PON1 activity, paraoxon was sue of proteins required for the transporting of Zn+2 in the used as substrate. Absorbance of the colour, which emerges blood [21]. In the present study, the opinion that decreasing as the result of the hydrolysis of paraoxon at 37°C, was de- PON1 enzyme activity levels in the groups with ZnSO4 termined at 412 nm [14]. Plasma HDL concentration was may be associated with Ca+2 channel and aminoacids measured using spectrophometric method by autoanalyzer linked Zn+2 in gills is reached. (Huma Star 600, Germany) with commercially available It may be variances in terms of sensitivity to metals in kit. The level of TOS was measured by using commercial species due to changes in the cellular mechanism of anti- diagnostic kit (Gaziantep, Turkey), which is used by Erel oxidant defense system and differences encountered in dis- [9] at auto analyzer (Aeroset®, Abbott®, Illinois, USA). tribution and accumulation in tissue of metals [21,22]. In the present study, reducing PON1 levels directly and HDL 2.1 Statistical Analysis levels indirectly as an antioxidant molecule in the groups Results were expressed as median (X)±standard decli- treated with ZnSO4 indicated that antioxidants in Capoeta nation (SD). Importance level of difference between group capoeta are highly sensitive to metals. In a study performed averages was determined by using statistic packet program on Channa punctatus, it is stated that liver and kidney are (IBM SPSS version 20 for Windows) with variance analy- target tissues with regard to Zn+2 toxicity [23]. Also, in a sis and Tukey polycomparison test. study applied on Scyliorhinus canicula, it was reported that metal accumulation is maximum in the liver tissue among tissues in medium containing of sublethal Zn+2 concentra- 3. RESULTS AND DISCUSSION tions [24]. In the light of these studies and our findings, it comes to mind that the important difficulties encountered The levels of plasma PON1, TOS and HDL in blood during production in liver of PON1 and other antioxidant samples taken after 10 days from control group fishes, enzymes may be due to metal accumulation. whose environment did not include ZnSO4 but was added 5 It is pointed out that acute effects of heavy metals and 10 mg/L ZnSO4 ,are shown in Table 1. According to our change the osmoregulatory system, function of metalo- findings, plasma PON1 and HDL levels were determined to tiyonin and binding abilities of metals associated with inhi- be lower when plasma TOS level was a significant increase bition of ATPase and consequently loss of electrolyte cause in other two groups compared to the first group. oxidation in cell membranes [25-27]. NADPH oxidases, It is reported that acute effects occurred as a result of which are a group of enzymes depending on plasma mem- high concentrations of Zn+2 in fishes cause the condition brane, produce reactive oxygen radical from oxygen using of hypoxia, depending on structural disorder in gills and an NADPH as the electron donor [28]. Zinc is an inhibitor for increase in mortality eventually [15]. In a study, which an- these oxidase enzymes and present in structure of superoxide alyzed the metal accumulation in the gill, muscle and liver dismutase (SOD) which has antioxidant property [29]. It is tissue of Cyprinus carpio was recorded that Zn+2 as heavy also stated that Zn+2 is an OH radical scavenger and induces metal was rather tend to accumulation in tissues compared synthesis metalotiyonin, which is a very rich protein in terms other heavy metals [16]. Zinc is taken by an apical Ca+2 of cysteine [29]. Proteins such as metalotiyonin, which have channel in mitochondria-rich ion carrier cells especially in a property of binding Zn+2 and are from important antioxi- gills of freshwater fishes [17, 18]. In addition, Ca+2 is dants, may reduce TOS [30]. Although the antioxidant de- claimed to be directly related to the absorption of Zn+2, and fense system supported by low zinc concentrations, high is required for stability and activity of PON1 enzyme, de- zinc concentrations may cause oxidative damage [31]. It was pending on HDL in the plasma [19, 20]. In the present reported that high Zn+2 concentrations in environment study, it was found that decrease of plasma PON1 activity caused changes on the level of lipid peroxidation related to of fishes held for 10 days in tanks containing 5 and 10 mg/L oxidative stress in Capoeta capoeta [13]. In the present ZnSO4 compared to the control group. It is suggested that study, it was determined that TOS levels including lipid pe- these reductions in PON1 enzyme activity might be origi- roxidation increased in ZnSO4 treated (5 and 10 mg/L for 10 nated from absorption relation between Ca+2 and Zn+2 cat- days) groups and that high Zn+2 concentration could be toxic ions. In a study, it was recorded that protein levels of Cy- in oxidative terms to fish in aquatic environment. The con- prinus carpio living in the Zn+2 added mixture decreased centrations of Zn+2 used in this study concluded that the ox- in muscle tissue corresponds to increased gills and liver. idative stress may create the pathogenesis of a lot of disor- Accordingly, it is reported to be provided from muscle tis- der, which is rather important.

TABLE 1 - The levels of PON1, TOS and HDL in blood samples of fishes in life environments with/without ZnSO4

Parameters (n=9) Control ZnSO4 (5 mg/L) ZnSO4 (10 mg/L) P PON1 (U/L) 58.57±4.32a 44.71±8.04b 38.65±6.52b <0.001 a ab b TOS (μmol H2O2 eq/L) 10.81±0.87 12.04±0.76 12.33±1.09 <0.05 HDL (mg/dl) 71.42±3.74a 57.01±3.91b 51.14±5.36b <0.001 a, b Values with different letter indicates significant differences

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As a result, the environmental conditions, which have [13] Yılmaz, M., Karapehlivan, M. and Kaya, I. (2012) Effects of high Zn+2 concentrations, were found to cause significant zinc sulphate on transcaucasian barb, (Capoeta capoeta [Gul- denstaedt, 1773]) plasma nitric oxide, malondialdehyde and changes on the levels of plasma PON1 activity, TOS and total sialic acid levels. Journal of the Faculty of Veterinary HDL in Capoeta capoeta. In addition, it was concluded Medicine, University of Kafkas, 18, 61-64. that more detailed studies on the protective mechanisms [14] Eckerson, H.W., Romson, J., Wyte, C.M. and La Du, B.N. +2 against the Zn toxicity were needed. (1983) The human serum paraoxonase polymorphism: Identi- fication of phenotypes by their response to salts. American The authors have declared no conflict of interest. Journal of Human Genetics, 35, 214-227. [15] Skidmore, J.F. (1964). Toxicity of zinc compounds to aquatic animals, with special reference to fish. Quarterly Review of Biology, 39, 227-247. REFERENCES [16] Benzer S, Arslan H, Uzel N, Gül A, Yılmaz, M. (2013). Con- centrations of metals in water, sediment and tissues of Cypri- [1] Park, S.W., Lee, J.Y., Yang, J.S., Kim, K.J. and Baek, K. nus carpio L., 1758 from Mogan Lake (Turkey). Iranian Jour- (2009) Electrokinetic remediation of contaminated soil with nal of Fisheries Science, 12, 44-55. waste-lubricant oils and zinc. Journal of Hazardous Materials, [17] Glover, C.N., Bury, N.R. and Hogstrand, C. (2004) Intestinal 169, 1168-1172. zinc uptake in freshwater rainbow trout: evidence for apical pathways associated with potassium efflux and modified by [2] Coruh, S. and Ergun, O.N. (2010) Use of fly ash, phosphogyp- calcium. Biochimica et Biophysica Acta, 1663, 214-221. sum and red mud as a liner material for the disposal of hazardous zinc leach residue waste. Journal of Hazardous Materials, [18] Zhang, L. and Wang, W. (2006). Alteration of dissolved cad- 173, 468-473. mium and zinc uptake kinetics by metal pre-exposure in the black sea bream (Acanthopagrus schlegeli). Environmental [3] Scott, G.R., and Sloman, K.A. (2004) The effects of environ- Toxicology and Chemistry, 25, 1312-1321. mental pollutants on complex fish behavior: integrating behavioral and physiological indicators of toxicity. Aquatic Toxicol- [19] Babacan, F., Işık, B. and Bingöl, B. (2011). Changes in serum ogy, 68, 369-392. paraoxonase activity, calcium and lipid profiles in pre-eclamp- sia, a preliminary study. Journal of Turkish Society of Obstet- [4] Wepener, V., Van Vuren, J.H.J. and Du Preez, H.H. (2001) ric and Gynecology, 8, 169-174. Uptake and distribution of a copper, iron and zinc mixture in gill, liver and plasma of a freshwater teleost, Tilapia spar- [20] Cicik, B. (2003) The effects of copper-zinc interaction on the manii. Water SA, 27, 99-108. accumulation of metals in liver, gill and muscle tissues of common carp (Cyprinus carpio L.). Ekoloji, 12, 32-36. [5] Adhikari, S. and Ayyappan, S. (2004) Behavioural role of zinc on primarymproductivity, plankton and growth of a freshwater [21] Grosell, M., Nielsen, C. and Bianchini, A. (2002) Sodium turn- teleost, Labeo rohita (Hamilton). Aquaculture, 231, 327-336. over rate determines sensitivity to acute copper and silver exposure in freshwater animals. Comparative Biochemistry and [6] Galvez, F., Nebb, N., Hogstrand C. and Wood, C.M. (1998) Physiology Part C, 133, 287-303. Zinc binding to the gills of rainbow trout: the effect of long- term exposure to sublethal zinc. Journal of Fish Biology, 52, [22] Garcia-Santos, S., Fontainhas-Fernandes, A. and Wilson, J.M. 1089-1104. (2006) Cadmium tolerance in the nile tilapia (Oreochromis niloticus) following acute exposure: assessment of some ion- [7] Giguere, A., Campbell, P.G.C., Hare, L. and Rasmussen, J.B. oregulatory parameters. Environment Toxicology, 21, 33-46. (2004) Influence of lake chemistry and fish age on cadmium, [23] Murugan, S.S., Karuppasamy, R., Poongodi, K. and Puvanes- copper and zinc concentrations in various organs of indige- wari, S. (2008) Bioaccumulation pattern of zinc in freshwater nous yellow perch (Perca flavescens). Canadian Journal of fish Channa punctatus (Bloch) after chronic exposure. Turkish Fisheries and Aquatic Sciences, 61, 1702-1716. Journal of Fisheries and Aquatic Sciences, 8, 55-59. [8] Parvez, S. and Raisuddin, S. (2005) Protein carbonyls: novel [24] Sanpera, C., Vallribera, M. and Crespo, S. (1983) Zn, Cu and biomarkers of exposure to oxidative stress-inducing pesticides Mn levels in the liver dogfish exposed to Zn. Bulletin of Envi- in freshwater fish Channa punctata (Bloch). Environmental ronmental Contamination and Toxicology, 31, 415-417. Toxicology and Pharmacology, 20, 112–117. [25] Dautremepuits, C., Parıs-Palacıos, S., Betoulle, S. and Vernet, [9] Erel, Ö. (2005). A new automated colorimetric method for G. (2004) Modulation in hepatic and head kidney parameters measuring total oxidant status. Clinical Chemistry, 38,1103- of carp (Cyprinus carpio L.) induced by copper and chitosan. 1111, Comparative Biochemistry and Physiology Part C, 137, 325– 333. [10] Cao, H., Girard-Globa, A., Berthezene, F. and Moulin, P. (1999) Paraoxonase protection of LDL against peroxidation is [26] Atlı, G., and Canlı, M. (2007) Enzymatic responses to metal independent of its esterase activity towards paraoxon and is exposures in a freshwater fish Oreochromis niloticus. Com- unaffected by the Q to R genetic polymorphism. Journal of Li- parative Biochemistry and Physiology Part C, 145: 282-287. pid Research, 40, 133-139. [27] Pandey, S., Parvez, S., Ansari, R.A., Ali, M., Kaur, M., Hayat, [11] Mackness, M.I., Mackness, B., Durrington, P.N., Connelly, F., Ahmad, F. and Raisuddin, S. (2008) Effects of exposure to P.W. and Hegele, R.A. (1996) Paraoxonase: biochemistry, ge- multiple trace metals on biochemical, histological and ultra- netics and relationship to plasma lipoproteins. Current Opin- structural features of gills of a freshwater fish, Channa punc- ion in Lipidology, 7, 69-76. tata Bloch. Chemico-Biological Interactions, 174, 183-192. [12] Karakuş., S. and Gey., H. (2006) A preliminary study of heavy [28] Pinton, R., Cakmak, I. and Marschner, H. (1994) Zinc defi- metals in transcaucasian barb (Capoeta capoeta capoeta Gul- ciency enhanced NAD(P)H-dependent superoxide radical pro- denstaedt, 1772) from the Kars Creek, Turkey. Revue de duction in plasma membrane vesicles isolated from roots of Medecine Veterinaire, 157, 551-556. bean plants. Journal of Experimental Botany, 45, 45-50.

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[29] Prasad, A.S., 2002. Zinc in health and Diaease. Kuwait Medi- cal Journal, 34, 91-93. [30] Chung, M.J., Kang, A.Y., Lee, K.M., Oh, E., Jun, H.J., Kim, S.Y., Auh, J.H., Moon, T.W., Lee, S.J. and Park, K.H. (2006) Water-soluble genistin isoflavone up-regulate antioxidant metallothionein expression and scavenge free radicals. Journal of Agricultural and Food Chemistry, 54, 3819-3826. [31] Davis, C.D., Milne, D.B., Nielsen, F.H. (2000) Changes in dietary zinc and copper affect zinc-status indicators of postmen- opausal women, notably, extracellular superoxide dismutase and amyloid precursor proteins. American Journal of Clinical Nutrition, 71,781-788.

Received: November 25, 2014 Revised: December 11, 2014 Accepted: February 13, 2015

CORRESPONDING AUTHOR

Muhittin Yılmaz Department of Bioengineering Faculty of Engineering and Architecture Sinop University 57000 Sinop TURKEY

E-mail: [email protected]

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KINETIC AND SORPTION EQUILIBRIUM STUDIES ON PHOSPHORUS REMOVAL FROM NATURAL SWIMMING PONDS BY SELECTED REACTIVE MATERIALS

Agnieszka Z. Bus* and Agnieszka A. Karczmarczyk

Faculty of Civil and Environmental Engineering, Warsaw University of Life Sciences – SGGW, Nowoursynowska Street 166, 02-787 Warsaw, Poland

ABSTRACT and nature based methods of water purification. Instead of chemicals, natural ponds use macrophytes (e.g. Iris The aim of the study is to select the best P-reactive ma- pseudacorus L., Schoenoplectus lacustris L., Palla, Jun- terial for implementation in the natural swimming ponds cus L., Nymphaea L., Acorus calamus L.) planted in the based on the kinetic and sorption equilibrium studies. substrate, zooplankton and extended water treatment on re- Seven different (natural and man-made) reactive materials active materials. (A, B, C, D, E, F, G) were chosen to these tests. The kinetic Reactive materials are referred as adsorbents or sorption of tested reactive materials ranged from 0.147 sorbents and specially interact with targeted chemical spe- mg/g to 2.066 mg/g. Three materials (A, B, G) character- cies such as phosphorus [6]. The P-sorption mainly de- ized by the best sorption properties were selected to further pends on content of Ca, Al or Fe fractions [7, 8]. Reactive sorption equilibrium studies. The obtained experimental materials used in swimming ponds are designed to remove data for that materials were fitted to the Freundlich and excess of phosphorus from water. Even a small surplus of Langmuir adsorption isotherms. Two of the materials (A phosphorus may leads to water quality deterioration, alga and B) are characterized by good adjustment to the Lang- grows, water toxicity and in consequence to the water eu- muir isotherm (0.9855 and 0.8615) while the third one (G) trophication [9-10]. To secure maintenance the phosphorus to the Freundlich isotherm (0.8747). Maximum sorption concentration should be limited below 0.01 mg/L [2]. capacities (Smax) calculated on the base on the Langmuir isotherm for the A and B materials are 46.98 mg/g and The aim of the study is to select and asses the best re- 13.87 mg/g, respectively. active materials for the control of the P content in the natural swimming ponds.

KEYWORDS Freundlich isotherm, kinetic models, Langmuir isotherm, natural swimming pond, phosphorus 2. MATERIALS AND METHODS

2.1 Adsorbents

1. INTRODUCTION In this study seven different reactive materials were used as adsorbents (A-G). All of them were selected and Natural swimming ponds are artificially created water supplied by local company specialized in construction of bodies that are separated from surface water and ground- natural swimming pools and ponds. water and have no chemical disinfection or sterilization Materials A-D are made of a silica-calcite sedimentary system [1]. Swimming ponds are natural friendly alterna- rock opoka, thermally treated. Material A and B were ex- tive of traditional swimming pools. Water treatment is car- tracted in eastern Poland, and were heated at temperature ried out through mechanical techniques, biological filters of 950°C and 800°C, respectively. Material C and D are and plants growing in the system [2]. The swimming ponds extracted in south-east part of Poland, both heated at tem- are generally divided into two zones: deep swimming zone perature of 900°C. After heating process, the COCO3 (one and regeneration zone (the filter) which is usually shallow of the main components of opoka) is transformed to the what favors quick water heating with the planted zone to more reactive CaO form. Such process is aimed to enhance achieve effective water and sediment treatment [3-5]. The the P sorption capacity. The material varied only in used main difference between traditional swimming pools and fraction (Table 1). Materials E and F are a sedimentary rock swimming ponds is that swimming ponds use ecological formed from ooids (ooloite), spherical grains composed of concentric layers, used material come from Germany (E) * Corresponding author and Poland (F).

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TABLE 1 - Characteristic of reactive materials

Adsorbents Grain size [mm] Bulk density Porosity [%] pH* [-] EC* [µS/cm] [g cm-3] A 1-5 0.62 67.9 11.9 1000 B 1-5 0.68 68.1 10.8 551 C 5-12 0.69 59.2 9.4 72 D 1-5 0.64 63.5 9.2 208 E 1-3 0.97 57.4 9.0 110 F 5-12 1.29 50.7 9.5 530 G 1-3 4.12 70.1 8.8 135 *Results taken after 1 h mixing (400 rpm) 1g of reactive material with 100 mL distilled water: pH=8.6; EC=0.00.

Material G is a commercial product (FerroSorp®) used to log ∙log (3) remove phosphate from water and wastewater [11]. where: aF – constant which express the adsorbent ca- Used materials were sieved with a screens of mesh di- pacity (the larger value, the higher the capacity), bF – het- ameter from 1 to 12 mm. Bulk density was determined by erogeneity factor, qs – sorption, Cc – solute concentration 3 strew method using 100 cm volume cylinder. Total poros- at equilibrium. ity was calculated as a ratio of volume of the sample’s free space to the total sample’s volume. The pH and electrolytic The Langmuir isotherm describes adsorption on ho- conductivity (EC) were marked by LF-340 (WTW) and mogenous surfaces while the Freundlich isotherm assumes SensoDirect Con110 (Lovibond), respectively. The physi- surface which is heterogeneous. cal and chemical properties of used materials are presented in Table 1. 3. RESULTS AND DISCUSSION 2.2 Adsorbates 3.1 Kinetic tests Varying concentrations of the artificial P solution prepared of KH2PO4 were used in all batch tests for assessing The sorption capacity sequence of tested reactive ma- P sorption. The triplicate samples of material were mixed terials after 300 minutes is as follow: A (2.066 mg/g) > B in Erlenmeyer glass flask, each contained 1 g of material (1.985 mg/g) > G (1.952mg/g) > D (0.636 mg/g) > C and 100 mL of the various P solution added. The kinetic (0.377 mg/g) > F (0.156 mg/g) > E (0.147 mg/g). tests were performed at various contact times (5-300 min) In Figure 1 the calculated P reduction of the three best and the constant P solution concentration of 2.0 mg P- tested materials is shown versus varying contact time. The PO4/L which simulates the natural swimming pond condi- fastest P removal was observed for A, B and G reactive tions. The sorption equilibrium tests were performed at materials. The A material needs 15 minutes to remove various P solution concentrations (2.066-864.200 mg P- 100% P from adsorbate. The B and G materials need 300 PO4/L) and a constant time (1 h). The P equilibrium con- minutes to remove 96% and 88% of P, respectively. Other centrations were measured by flow injection analyses using tested materials are characterized by low P sorption (up to FIAstar 5000. The P-removal ratio R [%] was calculated 35% after 300 min.). based on the equitation: % ∙ 100% (1) 3.2 Sorption equilibrium tests Based on the result of the kinetic tests, three materials where C0 and Cc are the initial and equilibrium P concentration (mg/L). (A, B, G) were selected for the detailed sorption tests. The experimental data obtained for those materials were fitted 2.3 Sorption equilibrium models to the Langmuir and Freundlich adsorption isotherms (Ta- ble 2). The A and B reactive materials better fitted to the Description of the sorption process between solid Langmuir than to the Freundlich isotherm model. This ad- phase and solution was made based on mathematical equa- sorption model is used to explain monomolecular adsorp- tions given by Langmuir [13]: tion on the surface of an adsorbent. In case of G reactive material the data fitted better to Freundlich isotherm ∙ (2) model. This can be explained by the removal P sorption mechanism of the G reactive material which is based on Fe where: KL – constant parameter reflects the solute ad- content of the material [11,14]. sorptivity, aL – constant parameter related to the energy of adsorption, KL/aL ratio is defined as a adsorption capacity, On the base of the Langmuir isotherm maximum sorp- qs – sorption, Cs – solute concentration at equilibrium, and tion capacity Smax were calculated (Table 3) for A, B and G Freundlich equation [13]: reactive materials. The highest Smax value is obtained for A

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material and it is a result of fast P removal in time and prob- from the table all of tested materials (A, B, G) are charac- ably high content of Ca [15]. The lowest value of Smax in terized of good sorption properties. On the other hand, all case of G material with simultaneous high calculated sorp- of compered batch tests differ from each other the condition may be a consequence of better fitting to the Freun- tions under which the tests were performed. This may sig- dlich than to the Langmuir isotherm model (Figure 2). nificantly influence on the final sorption properties. Too However, the equilibrium adsorption capacities in case of long contact time of the batch test may not able to be used A and G materials are on the same level. in field conditions and give the overestimated sorption results. Also the material to solution ratio may significantly In case of the A material, the high value on the axis influence on the equilibrium constant. The smaller ratios 1/Cs is the result of a very rapid and fast sorption of phos- may lead to higher concentrations of P sorbed to the mate- phorus by the material (figure 2). At the lowest initial con- rial, that is, a higher percentage of P removal, but not nec- centration (2.066 mg/L), the solute concentration at equi- essarily a higher sorption capacity [6]. The fine fraction of librium (Cs) is 0.001 mg /L. In the same initial concentra- the material gives a good sorption result but in field condition materials B and G have not as fast as A material sorption tion can lead to a rapid clogging of the filter. In a batch test properties. The Cs concentration is 0.273 and 0.710 mg /L for the material should be used in the same form as is expected the A and G material respectively. to be used in field conditions [16]. A comparison of the phosphorous sorption efficiency of different reactive materials is shown in Table 4. As seen

FIGURE 1 - The phosphorus ion reduction [%] in time for A, B and G reactive materials.

TABLE 2 - Values of the Langmuir and Freundlich isotherms approximation constants calculated for the reactive materials used in the study

The Langmuir isotherm The Freundlich isotherm Adsorbent 2 2 KL [L/g] aL [L/mg] R aF [L/g] bF [-] R A 0.0046 0.2161 0.9855 0.3060 0.3449 0.9593 B 4.4122 0.318 0.8600 0.4227 0.5355 0.6748 G 1.2321 0.6731 0.7559 0.5962 0.6873 0.8747

TABLE 3 - Values of apparent P-sorption capacity obtained from the Langmuir isotherm and calculated of the equilibrium tests.

A B G

Maximum sorption capacity Smax [mg/g] 46.98 13.87 1.83 Equilibrium adsorption capacities [mg/g] 20.85 10.06 19.15

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FIGURE 2 - The Langmuir (on the left) and Freundlich (on the right) isotherm approximation for materials A, B and G.

TABLE 4 - Comparison of various reactive materials and their phosphorous removal efficiency based on batch tests.

Fraction Concentrations Contact time Material/solution ratio Sorption Adsorbent Reference [mm] [mg/L] [h] [g/mL] [mg/g] Filtralite P® 0.5-4.0 0-480 24 1/30 0.00213 [17] Shell sand 3.0-7.0 0-480 24 1/30 0.00924 Limestone 2.8-12.7 2-40 24 1/25 0.1 [16] Zeolite 2.8-12.7 2-40 24 1/25 0.03 Blast furnace slag 0.01-15 0-1000 6 1/50 46.5 [18] Steal furnace slag 0.01-15 0-1000 6 1/50 33.3 A 1-5 2-864 1 1/100 20.85 B 1-5 2-864 1 1/100 10.06 This study G 1-3 2-864 1 1/100 19.15

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3.3 Practical recommendations to provide suitable habitat for plants and at the same time The idea of natural swimming ponds is taken from nat- prevent the water from eutrophication (low P concentra- ural ponds and lakes. The extensive swimming ponds are tion). The reference value for the pH ranges from 6 to 9. the first and the oldest type of swimming pond, constructed The values for conductivity and phosphorous should not without using mineral filters to purify the water. The re- exceed 1000 µS/cm and 0.01 mg/L, respectively. The con- moval of nutrients is performed by plants and microorgan- ductivity of all tested materials complies with the require- isms. Such process is used, for example, in constructed ments of natural swimming ponds. From all tested material, wetlands. The disadvantage of such system is an aerial pro- the pH value is compiled only with G material. portion of swimming zone (30%) to cleaning zone (70%). Using of the reactive materials as the support of the natural treatment process is much more effective [19]. In that case 4. CONCLUSIONS (intensive ponds) the ratio of recreation zone to cleaning zone is 40% to 60%. Implementation of reactive materials into regeneration The materials used in natural swimming ponds should zone of natural swimming ponds increases the share of the be available locally which significantly decreases the con- recreation area and contributes to reduction of P concentra- struction costs of ponds and pools. Thus why, apart from tion, alga grows and in the consequence to water eutrophi- commercial products for phosphorus control there is a need cation. The use of reactive materials in natural swimming for alternative materials which are available in the local ponds in the form of extended filter for water treatment is scale. It this study it has been found that some of calcifer- indispensable step to remove phosphorus from water and ous bed rocks widely available in Europe can be even more to protect it against undesirable alga grow. The important effective than commercial products. However, in case of issue of the stage of the selection of reactive material, apart the reactive materials of natural origin there is a risk of in- of its P-sorption properties, is the availability. Searching homogeneous composition which will influence on P-sorp- for and using local materials rather than imported products tion properties. results in decreasing the overall price of system and reduce the environmental impact associated with transport. Provided that the ratio between the cleaning zone and recreation zone is 2:3, the average depth of the cleaning The authors have declared no conflict of interest. zone (filled by reactive material) and recreation zone is 0.3 m and 2.0 m, respectively, the amount of material use in the cleaning zone (including the porosity of the material) was calculated. Assuming that the water in the volume of REFERENCES swimming zone should by filtered through cleaning zone within 24 h, the number of water exchanges was calculated [1] Casanovas-Massana, A., Blanch, A. R. (2013) Characteriza- with the retention time in cleaning zone and it can be esti- tion of microbial populations associated with natural swim- mated for 98 minutes (A and B materials) and 101 minutes ming pools. International Journal of Hygiene and Environ- (G material). Based on the results of kinetic test, after this mental Health 216, 132– 137 time the P-PO4 removal can reach 100%, 60% and 56% for [2] Recommendations for Planning, Construction, Servicing and A, B and G materials, respectively. For the rest of tested Operating of Outdoor Swimming Pools with Biological Water Purification (Swimming and Bathing Ponds) (FLL 2011) reactive materials the estimated retention time range from 73 to 91 minutes with the significantly lower removal of P- [3] Radic, D., Gujanicic, V., Petricvic, J., Raicevic, V., Lalevic, B., PO (from 3 to 35%). Rudic, Z., Bozic, M. (2013) Macrophytes as remediation tech- 4 nology in improving Ludas lake sediment. Fresenius Environ- According to the guidelines for natural swimming mental Bulletin 22, 6, 1787-1791 ponds [2] the concentration of phosphorus in swimming [4] Dhote, S. (2007) Role of Macrophytes in improving water zone should not exceed 0.01mg/L and this condition could quality of an aquatic eco-system. J. Appl. Sci. Environ. Man- be fulfilled only in case of implementation of A material. age. December, 11 (4), 133 - 135 However, the negative effect of using this material is strong [5] Dhote, S., Dixit, S. (2009) Water quality improvement through alkaline pH (11.90). Using G material for filling up the fil- macrophytes—a review. Environ Monit Assess 152, 149–153 ter will not affect the pH but the contact time necessary for [6] Cucarella, V., Renman, G. (2009) Phosphorus Sorption efficient P removal is much longer (300 minutes) than for Capacity of Filter Materials Used for On-Site Wastewater A material. Treatment Determined in Batch Experiments - A Comparative Study. J. Environ. Qual. 38, 381-392 Filters used in natural swimming ponds have to fulfill a number of requirements [2]. In multi-layer filter, the suitable [7] Klimeski, A., Chardon, W.J., Turtola, E., Uusitalo, R. (2012) diameter for the main filter body is 1-5 mm, and 2-11 mm Potential and limitations of phosphate retention media on water protection: A process-based review of laboratory and field- for the plants matrix. Permeability of the filter should be at scale tests. Agric. Food Sci. 21, 206-223 the level of k ≥10-3 m/s. Based on physical properties ma- [8] Karczmarczyk, A., Bus A. (2014) Testing of reactive materials terials such as C and D can be used as an addition to sub- for phosphorus removal from water and wastewater – compar- strate for plants implementation. The chemical values of ative study. Ann. Warsaw Univ. of Life Sci. – SGGW, Land water in natural swimming pond after pre-treatment have Reclam. 46 (1), 57-67

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[9] Correll, D. L. (1998) The Role of Phosphorus in the Eutrophi- cation of Receiving Waters: A Review. J. Environ. Qual. 27 (2), 261-266 [10] Fondu, L., De Bo, I., Van Hulle, S.W.H. (2014) Phosphate adsorption capacity testing of natural and industrial substrates in view of application in swimming and fish pond water treatment system. Desalination and Water Treatment, DOI: 10.1080/19443994.2014.899930

[11] Adsorbents based on iron hydroxide for the remediation and

restoration of lakes http://www.ferrosorp.de/english/produkte/ferrosorpgw/index.html (accessed on 19.10.2014)

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[14] Sibrell, P.L., Montgomery, G.M., Ritenour, K.L., Tucker, T.W. (2009) Removal of phosphorus from agricultural wastewaters using adsorption media prepared from acid mine drainage sludge. Water Research, 43, 8, 2240–2250 [15] Bus, A., Karczmarczyk, A. (2014) Properties of lime-siliceous rock opoka as reactive material to remove phosphorus from

water and wastewater. Infrastructure and Ecology of Rural Ar- eas, II, 1, 227-238 [16] Drizo, A., Frost, C.A., Grace J., Smith K.A. (1999) Physico- chemical screening of phosphate-removing substrates for use in constructed wetland system. Water Research, 33,17, 3595- 3602 [17] Ádám, K., Krogstad, T., Vråle, L., Søvik, A.K., Janssen, P.D. (2007) phosphorus retention in the filter materials shellsand and Filtralite P®-Bath and column experiment with synthetic P solution and secondary wastewater. Ecological Engineering, 29, 200-208

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moval from aqueous solution by blast furnace slag and steal furnace slag. Journal of Zhejiang University SCIENCE A, 9, 125-132

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Received: December 01, 2014 Revised: March 13, 2015 Accepted: June 02, 2015

CORRESPONDING AUTHOR

Agnieszka Bus Warsaw University of Life Sciences – SGGW Faculty of Civil and Environmental Engineering Department of Environmental Improvement Nowoursynowska 166 Warsaw, 02-776 POLAND

E-mail: [email protected]

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NUTRIENT EXTRACTABILITY AND BIOAVAILABILITY OF FRESH AND COMPOSTED POULTRY LITTER AND ITS APPLICATION ON MAIZE (ZEA MAYS) CROP

Faridullah1, Arif Alam2, Muhammad Umar3, Akhtar Iqbal1, Muhammad Amjad Sabir3 and Amir Waseem4,*

1Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad-campus, Abbottabad-22060, Pakistan 2 Department of Developmental Studies, COMSATS Institute of Information Technology, Abbottabad-campus, Abbottabad-22060, Pakistan 3Department of Earth Sciences, COMSATS Institute of Information Technology, Abbottabad-campus, Abbottabad-22060, Pakistan. 4Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.

ABSTRACT The environmental impacts of the industrial waste of poultry have gained worldwide importance. Increasing in- Organic farming is the best option for producing qual- terest in the recycling of waste has also raised concerns ity and healthy agriculture products on cheaper costs. The over the possible contamination of soil and water. The in- present study was conducted to assess the influence of tensive use of chemical fertilizer inputs has aggravated the composted poultry litter (CPL) and fresh poultry litter global environmental system since nutrients are usually (FPL) on the extractability and bioavailability of nutrients subjected to different losses in the soils. The form in which for application in Maize crop. The mean levels for the re- fertilizer is applied may influence the nutrient dynamics in lease of these metals were obtained in the order of Cu>Zn> the soil plant system. Mn>Ni irrespective of the manure type. Other examined Composting is considered to be a sustainable and en- nutrients recorded in the order of P> Ca>K>Mg from poul- vironmental friendly alternative to manage and recycle or- try waste after 100 days of composting process as well as ganic solid wastes, with the aim of obtaining a quality or- fresh manure. The concentrations of these nutrients were ganic product, known as compost, to be used as organic found apparently higher in composted material as com- amendment in agriculture. Composting poultry litter may pared to fresh one. The release of water soluble phosphorus provide a beneficial alternative method for handling litter obtained was higher in compost amended soil as compared due to immobilization of nutrients and a reduction of litter to fresh one. Total phosphorus was also higher in compost volume [2]. Among the plants nutrients, phosphorus is im- amended soil compare to fresh manure. The concentration portant and is often supplied to the field crops in organic of nutrients in Maize tissue varied in the order of P > Ni> forms such as manures or bio solids. Organic waste appli- Mn> Cu> Zn in compost manure amended soil. The effi- cation for the reduced P losses and enhanced environmen- ciency of manure used is highly dependent upon soil type tal quality is also an important consideration when recy- and source of manure added. The applications of CPL to cling farm wastes as soil amendment. As a result of the re- fields could reduce both synthetic fertilizer inputs and im- peated applications of the manure in areas with intensive prove soil qualities. livestock farming, soils are often enriched with P at levels,

which exceed those removed in the harvested crops [3,4]. Heavy metal contamination in soil is a major concern be- KEYWORDS: cause of their toxicity and threat to human life and the en- Heavy metal, Poultry litter, composting, biodiversity vironment. Toxic heavy metals entering the ecosystem may lead to geo-accumulation, bio-accumulation and bio- magnifications. Environmental contamination and expo- 1. INTRODUCTION sure to heavy metals such as mercury, cadmium and lead is a serious growing problem throughout the world especially Organic fertilizers such as manure have been used in the third world [5,6]. Human exposure to heavy metals has agriculture for thousands of years; ancient farmers did not risen dramatically in the last 50 years as a result of an ex- understand the chemistry involved, but they did recognize ponential increase in the use of heavy metals in industrial the benefit of providing their crops with organic material. processes and products [7]. When composted manure is ap- The use of manure in agricultural land is beneficial to the plied to soils, it is necessary to further investigate the dy- physical, chemical and biological properties of the soils [1]. namics of heavy metals during composting. Although the basic composition of animal wastes has been documented * Corresponding author with respect to major constituents and nutrients, such as C,

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N, P, K and organic matter, and some reports include in- evaluate the concentration of P, Ni, Cu, Zn and Mn in formation on minor and trace elements, detailed studies of Maize plant tissues. Quantity of water applied to pots was trace element composition are lacking. Thus it is important manipulated according to the requirement of each treat- to have information on the nutritional and toxic element ment. Pots were reshuffled after every 48 h to avoid micro- status of manure amendments. Therefore, this investigation climatic effects. Plants were harvested, rinsed with distilled was aimed to compare the extractability and bio availabil- water and oven dried at 65 oC for 48 hours to determine ity of heavy metals and other plant nutrients to maize crop plant dry matter. Thereafter, plant samples were ground following composted poultry litter application. sieved via 0.5 mm sieve and digested by a strong acid mixture of HNO3 and HClO4 to measure the content of above mentioned heavy metals and nutrients in shoot by atomic 2. MATERIALS AND METHODS absorption spectrophotometer. Data were statistically analyzed and results were expressed on oven-dry basis. The Two types of poultry litter (PL), fresh (FPL) and com- overall differences among treatments were tested using posted (CPL), were collected from different poultry farm analysis of variance. A probability level of <0.05 was con- in Abbottabad, KPK. Litters were air dried, crushed, and sidered significant and means were compared by least sig- sieved (< 0.5 mm) to ensure homogeneity and digested in nificant difference (LSD-test). a mixture of duplicate acids (HNO3 and HClO4).

Total elements (P, K, Ca, Mg, Ni, Cu, Zn and Mn) in 3. RESULTS AND DISCUSSION the samples were determined with a mixture of HClO4- HNO . Sample weighing 0.25 g was digested with 5 mL 3 The application of various manure composts to agri- concentrated HClO by gradual heating it over a hot plate 4 cultural soils is associated with several benefits. However, for 1 h. After drying 20% HNO was added to the sample 3 certain composts may contain high concentrations of trace and it was heated again for 1h. The solution was diluted to elements that may be detrimental to plants and environ- 50 mL with deionized water and passed through a 0.22 μm ment. When rates of manure application are greater than filter. The P was determined on a spectrophotometer using crop demand, crop nutrient imbalance and soil or water the phosphomolybdate blue method. Absorbance was de- pollution often occur. An experiment was conducted to as- termined at a wavelength of 710nm. Ca, Mg and K were sess the influence of composted and fresh poultry litter on extracted with water to determined water soluble Cations. the extractability and bioavailability of P, K, Ca, Mg and The soil samples weighing 3g were measured in soil– water some heavy metals in the fresh and composted poultry ma- (1:5; w:v) suspensions placed in a 100 mL centrifuge tube nure. The mean levels for the release of these metals were and shaken mechanically for 1 h and filtered into a 100 mL obtained in the order of Cu>Zn >Mn> Ni irrespective of volumetric flask. The material was shaken for 2 h at room the manure types. The other nutrients were found in the or- temperature. After each end-to-end shaking the tubes, were der of P >Ca >K > Mg from poultry waste. The concentra- centrifuged at 10000 rpm for 10 min., and the supernatants tions of these nutrients were found apparently higher in were filtered by a 0.2 µm filter. The contents were deter- composted material as compared to fresh one. The slow re- mined using atomic absorption spectrophotometer (Model lease of nutrients from composted poultry litter (CPL) may Z-2300 Hitachi corp., Japan). lessen adverse environmental effects from leaching of N in The experiment was conducted in the facility of COM- runoff from farmlands [8]. Increased soil organic matter SATS Institute of Information Technology, Abbottabad and cation exchange capacity from CPL applications may under green house condition. Two manures fresh and com- improve nutrient retention in soils. Thus, applications of posted poultry litter, chemical fertilizer i.e, KHPO4 @ 100 CPL to fields could reduce both synthetic fertilizer inputs kg ha-1 and control treatment (without any amendment) and improve soil qualities. The concentration of nutrients were mixed with sandy soil in the pots. Four Maize (Zea varied in the order of P>Ca> K>Mg both in compost and mays.) plants in each pot were grown for eight weeks to fresh amended soil (Table 1). There has recently been a

TABLE 1 - Chemical properties of fresh and composted poultry Litter used in this study

H2SO4+HNO3Digested Water Soluble (mg kg-1) (mg kg-1) Treatment Sample P(gkg-1) K Ca Mg P K Ca Mg Fresh S1 2.3 721 1234 409.8 834 257 431 183 S2 2.2 718 1227 412.1 829 249 439 177 S3 2.2 714 1232 415.4 833 243 442 171 S4 2.3 728 1239 419.3 841 251 429 169 Compost S1 2.4 812 1378 437.7 893 293 472 194 S2 2.3 817 1368 439.9 889 308 482 197 S3 2.4 821 1375 433.2 888 301 477 191 S4 2.4 816 1371 435.6 897 298 488 201 S1 to S4 denotes Samples collected from different sites

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TABLE 2 - Physicochemical properties of amendments used in this study (mg kg-1).

Treatment Sample Ni Cu Zn Mn pH (1:5) Fresh S1 53.8 99.8 129 99.4 7.9 S2 57.9 93.2 133 96.4 7.7 S3 54.7 97.4 123 97.3 7.8 S4 51.3 95.5 126 95.8 7.6 Compost S1 57.8 129.2 127 107.1 7.8 S2 59.1 133.1 129 109.5 7.7 S3 55.9 127.8 123 103.4 7.9 S4 53.8 135.8 126 106.8 7.8

renewed interest in using livestock manure to improve soil available and thus have potential to contribute to the envi- fertility and crop production. The manure may be applied ronmental contamination [12]. Table 1 shows total phos- fresh or composted prior to application in fields; however, phorus in g kg-1, the higher phosphorus was observed in composted manure is preferred because of its reduced vol- compost as compared to fresh with (2.35 and 2.24g kg-1). ume and ease of handling due to smaller particle size that In our previous research higher percentage of P was re- facilitates more uniform application [9]. leased from PLA as compared to PL samples [13]. Signif- icance differences were observed between fresh and com- The determination of the nutrient level contained in post amended soil. The pH was slightly higher for poultry any manure is essential for its safe and balanced use as soil fresh manure as compare to composted manure. pH of the amendment. Appropriate management of various manures FPL and CPL in our case are 7.75 and 7.8 respectively, the and organic wastes is one of the best means of improving ag- higher pH values may stabilize the phosphorous in ionic ricultural soils. Generally the order of acid digested nutrients form by making salt which will prevent volatilization of in poultry litter was found as P > Ca> K > Mg (Table 1). phosphates. The data of water soluble K, Ca and Mg, also showed that water soluble cations were higher in composted material as The concentrations of metals were significantly dif- compared to fresh poultry manure. There was a progressive fered between compost and fresh poultry litter. The amount increase in the concentrations of K, Ca and Mg during of total Cu, Zn, Mn and Ni were increased in composted as composting. These increases could be due to the concen- compared to fresh manure (Table 2). The concentration of tration effect as compost volume decreased with time ac- metals varied considerably in the order of Cu> Zn>Mn> Ni companied by losses of the more volatile elements such as in compost amended soils (Ni 56.7mg kg-1), (Cu 131.4 mg carbon and nitrogen under aerobic conditions. It has been kg-1), (Zn 125.5 mg kg-1), and (Mn 106.7 mg kg-1), whereas reported that cattle manure contains relatively higher levels contents observed in fresh amendment was as (Ni 54.4 mg of K as compared to Ca and Mg, [9] and the major concern kg-1), (Cu 96.5 mg kg-1), (Zn 127.8 mg kg-1) and (Mn 97.2 of soil pollution with available heavy metals by the use of mg kg-1), respectively. The ashing of chicken and duck litter manures can be minimized through composting under aer- at various temperatures had a substantial impact on the obic conditions. Phosphorus plays key role both as a struc- chemical forms of the trace elements [14]. The total con- tural element of biologically important compounds and as centrations of trace elements increased significantly with catalyst for several biological reactions. Therefore, it is degree of burning. Water soluble metals readily release to quite necessary to determine the water extractable phos- the environment. The available pool of metal is also the phorus (WEP) contents of manure. We have compared most labile form in the soil environment and greater leach- fresh and composted poultry litter for water extractable P ing potential than the other forms [15]. (WEP), total P (TP) and other nutrient contents and the results show that the composted poultry litter is rich in WEP 3.1 Pot Experiment in compared to FPL. The WEP contents of FPL and CPL It is important to compare different waste material in are 0.83 g kg-1 and 0.89 g kg-1, respectively (Table 1). It order to enhance an efficient fertilizer management system was shown that WEP contents in CPL were not signifi- for infertile soils. This study was basically aimed to evalu- cantly different from FPL after composting. There may be ate the influence of fresh and composted poultry litters on multiple explanations for this difference; WEP contents of the growth of Maize crop (Zea mays) plants and changes in FPL are non-volatile and therefore on composting the rel- their mineral composition at an early growth stage. Regard- ative ratio of WEP increases. This implies that the phos- less of the types of material, the application of both fresh phorus is present primarily in non-labile form such as non and composted poultry litters significantly increased plant labile phospholipids or non degradeable fulvic and humic growth as compared to the control. Significant Zn, Ni,Cu, acids. P fractionation studies also revealed that water solu- Mn concentration was found in plant shoots grown in com- ble P was reduced significantly whereas other P fractions posted and fresh poultry manure as compared to control were increased with incineration of PL [10,11]. The meas- (Table 3). The application of poultry manure had a substan- urement of water soluble P in the manure is important be- tial impact on elemental concentrations of plants as com- cause it include forms of P that can immediately be bio- pared to unamended control. Organic matters are rich source

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TABLE 3 - Nutrient concentration in Maize shoot (mg kg-1).

Source P (g/kg) Ni Cu Zn Mn Compost 0.155 14.4 11.45 8.4 7.3 Fresh 0.125 13.45 8.15 7.55 9.6 Chemical Fertilizer 0.45 3.35 2.35 3.25 4.2 Control 0.13 13 9.6 6.35 4.4

of macro (K, Ca and Mg) and micro nutrients (Cu, Ni, Cu, bio availability of plant nutrients to maize crop following Mn etc). However, balanced manure should contain composted poultry litter application. enough quantities of minerals for normal growth of plants. Excess quantities can cause damage to human being via food chain and on the other hand, deficient quantities ren- 4. CONCLUSIONS der incomplete plant growth. Composting is also known to decrease the bioavailability of heavy metals to plants and The overall goal of this study was to determine nutri- reduces their toxicity [15]. Therefore it is quite essential to ents and some heavy metals in poultry litter and its effect determine the amount of these nutrients in poultry litter on the maize crop in fresh and composted poultry litter (FPL and CPL) and also their uptake by plants. Mineral amended soil. Extractability of plant nutrients was found values of poultry manure and uptake by plants has been highly dependent upon the type of PL applied. Nutrient studied, albeit less extensively. Similarly the P concentra- concentrations were higher in Composted litter as com- tion in maize shoot was found maximum in compost pared to fresh. Application of composted PL to the soils amended soil with 0.155g kg-1 where as lowest was ob- produced higher maize (Zea mays) biomass yield as com- served in control with (0.12g kg-1). Through composting, pared to fresh PL chemical fertilizer and control. The con- active organic matters in fresh waste are converted into centrations of other nutrients were also higher in com- source of nutrients for plant growth and soil conditioner for posted manure. The general increase in nutrient content improving soil physical properties. Significant changes can and uptake of maize shoot in amended soils indicated that occur in its elemental composition that may influence its compost could result in a greater pool of plant available use as soil amendment. The presence of organic and inor- nutrients. Poultry compost litter exhibited less water solu- ganic contaminants in compost, pose a grave danger to the ble P fraction, which may reduce the risk of P transfer from environment. Literatures on the effect of compost use on soil to the surface water. The total P was higher in compost the release of elements in the soil environment show that it poultry litter than fresh poultry litter. For the sustainable varies according to the soil types, plant species and com- soil fertility management, it is important to understand the post quality. For the sustainable nutrient management of impacts of poultry litter on the chemical forms of elements crops, it is important to understand the effects of compost- in soils. Therefore, this investigation was aimed to compare ing on the chemical forms of elements in waste. The total the extractability and bio availability of heavy metal and concentration of elements in manures may not provide the other plant nutrients to maize crop following composted best indication of their bioavailability. In a pot experiment, poultry litter application. we have evaluated the influence of fresh and composted poultry litters on the growth of maize crop (zea mays) The authors have declared no conflict of interest. plants along with mineral uptake at early stages of growth. Regardless of the type of material, application of both fresh and composted poultry litters significantly enhanced the REFERENCES plant growth compared to the control. Nickel, Copper, Manganese and zinc contents in Zea Mays shoots are com- [1] Little, I.P. (1997). The fate of nitrogen fertilizers added to red gradational soils of Batlow, New South Wales and its implica- parable for plants grown with CPL and chemical fertilizer tions with respect to soil acidity. Australian Journal of Soil Re- which indicates that CPL is the best alternative of chemical search, 35, 863-880. fertilizer for these elements; however plants grown with [2] Preusch, P.L., Alder, P.R., Sikora, L.J., and Tworkoski, T.J. FPL comparatively showed less amounts of these elements (2002). Nitrogen and phosphorus availability in composted (Table 3). On the other hand, Ni and Cu contents of zea and uncom posted poultry litter. Journal of Environ Quality, mays shoots were remarkably higher for CPL, compared to 31, 2051–2057. other treatments. The environmental impacts of the indus- [3] Koopmans, G.F., Van der Zeeuw, M.E., Chardon, W.J., and trial waste of poultry have gained worldwide importance. Dolfing, J. (2001). Selective extractive of labile phosphorus using dialysis membrane tubes filled with hydrous iron hy- Research reports on the comparison of composted and droxide. Soil Science, 166, 475-483. fresh organic wastes on various crops are sufficient, but the [4] Sims, J.T., Edwards, A.C., Schoumans, O.F., and Simard, R.R. nutrients release and their bio availability to maize crop (2000). Integrating soil phosphorus testing into environmen- from composted poultry litter are scanty. Therefore, this tally based agricultural practices. Journal of Environmental investigation was aimed to compare the extractability and Quality, 29, 60-71.

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[5] Hassan, N.U., Mahmood, Q., Waseem, A., Irshad, M., Farid- ullah, and Pervez, A. (2013). Assessment of heavy metals in wheat plants irrigated with contaminated wastewater. Polish Journal of Environmental Studies, 23, 115–123. [6] Waseem, A., Arshad, J. Iqbal, F., Sajjad, A., Mehmood, Z., and Murtaza, G. (2014). Pollution status of Pakistan: A retro- spective review on heavy metal contamination of water, soil and vegetables. BioMed Research International article ID 813206, vol.2014, 29. [7] Ullah, H., Noreen, S., Rehman, F.A., Waseem, A., Zubair, S., Adnan, M., Ahmad, I. (2014) Comparative study of heavy metals content in cosmetics products of different countries marketed in Khyber Pakhtunkhwa, Pakistan. Arabian Journal of Chemistry, doi:10.1016/j.arabjc.2013.09.021.

[8] Chang, C., and Janzen, H.H. (1996). Long-term fate of nitrogen from annual feedlot manure applications. Journal of Envi- ronmental Quality, 25, 785-790. [9] Eneji, A.E., Yamamoto, S., Honna, T., and Ishiguro, A. (2001). Physicochemical changes in livestock feces during composting. Communication in Soil Science and Plant Analy- sis, 32, 477–489. [10] Faridullah, Yamamoto, S., Irshad, M., Uchiyama, T., and Honna, T. (2008). Phosphorus fractionation in chicken and duck litter burned at different temperatures. Soil Science 73, 287-295. [11] Faridullah, Pervaiz, A., Hafeez, S., Alam, A., Ma, J., Shah, S.H., and Waseem, A. (2014). Evaluating nutrient elements and heavy metals in fresh and composted manure of different livestock. Fresenius Environmental Bulletin. 23, 1773-1777.

[12] Canet, R., Pomares, F., and Tarazona F (1997). Chemical extractability and availability of heavy metals after seven years application of organic wastes to a citrus soil. Soil Use Man- agement, 13, 117-121.

[13] Faridullah, Irshad, M., Yamamoto, S., Ahmad, Z., Endo, T., and Honna T (2009a). Extractability and Bioavaiability of Phosphorus from Soil Amended with Poultry Litter and Poul- try Litter Ash. Journal of Food Agriculture and Environment, 7, 292-297. [14] Faridullah, Irshad, M., Yamamoto, S., Eneji, A.E., Uchiyama, T., and Honna T, (2009b). Recycling of Chicken and Duck Litter Ash as a Nutrient Source for Japanese Mustard Spinach. Journal of Plant Nutrition, 32, 1082 -1091.

[15] Bolan, N.S., Adriano, D.C., and Mahimairaja, S. (2004). Dis- tribution and bioavailability of trace elements in livestock and poultry manure by-products. Critical Reviews in Environmen- tal Science and Technology, 34, 291-338. Received: December 05, 2014 Revised: February 24, 2015 Accepted: March 04, 2015

CORRESPONDING AUTHOR

Amir Waseem Department of Chemistry Quaid-i-Azam University Islamabad, 45320 PAKISTAN

Phone: (+92)-5190642021 Fax: (+92)-90642241 E-mail: [email protected]

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IN VITRO WHOLE PLANT REGENERATION OF THE MEDICIAL AQUATIC PLANT-Limnophilla aromatic

Mehmet Karataş1,* and Muhammad Aasim2

1Department of Biotechnology, Faculty of Science, Necmettin Erbakan University, Konya, Turkey 2Department of Biology, Kamil Ozdag Faculty of Science, Karamanoglu Mehmetbey University, Yunus Emre Campus, 70200, Karaman, Turkey

ABSTRACT ponent. Besides that, perillaldehyde and ketone, cis-4- caranone are other compounds found in the oil [7]. Anti- Linmophila aromatica is an important aquatic or semi- tumor promoting [8, 9] and antimutagenic ingredients [10] aquatic medicinal herb of South East Asia used for curing are other important characters of this plant. Besides that, different diseases and disorders. Shoot tip explants were anti-poisoning and pain killer activity, removal of mucus surface-sterilized with 16% H2O2 (v/v) and inoculated on from the respiratory tract (as expectorant), curing fever and MS medium having 0.25-2.0 mg/L BA+0 and 0.25 mg/L treatment of many diseases, such as intestinal worms, also NAA. 100% shoot regeneration frequency was scored on make this plant important. In addition, it is commonly used all culture mediums. Shoots per explants ranged 19.61- for dysentery, elephantiasis, indigestion and menstrual 44.22 and 14.28-30.72 on MS medium with 0.25-2.0 mg/L problems [11]. BA or 0.25-2.0 mg/L BA+0.25 mg/L NAA. Increased BA L. aromatica is a very important medicinal plant but concentration with or without resulted in a decreased num- no effort has been made to propagate this plant under in ber of shoots per explant. Shoot length was scored between vitro conditions, which is a very useful tool for obtaining 1.02-2.60 cm and 1.74-4.23 cm, respectively, for MS me- or altering the metabolites. Keeping in view, the present dium with 0.25-2.0 BA or 0.25-2.0 mg/L BA+0.25 mg/L study was designed to develop an efficient and reliable in NAA. In vitro regenerated shoots were rooted 100% on MS vitro shoot regeneration protocol for L. aromatica. medium with 0.25-1.0 mg/L NAA. Rooted plantlets were acclimatized in the aquarium provided wih tap water and oxygen. 2. MATERIAL AND METHODS

L. aromatica plants purchased from local aquatic plant KEYWORDS: traders of Karaman province of Turkey were surface-steri- In vitro, medicinal, shoot regeneration, shoot tip lized with H2O2 at a rate of 16% (v/v) for 10 min. Thereaf- ter, plants were rinsed with water thrice for 5 min, each with sterilized distilled water in order to remove traces. Af- 1. INTRODUCTION ter sterilizatrion process, twigs (Fig. 1a) were cut off into small pieces of 1-2 cm length with 1-2 nodes for 15 days Rice paddy herb (Linmophila aromatica) is an on MS [12] medium to get contamination-free explants. aquatic or semi-aquatic perennial ornamental and me- Shoot tip (Fig. 1b) and leaf were used as explants in this dicinal plant of Scrophulariaceae family. It is a tropical study and cultured on MS medium containing 0.25-2.0 plant that is also cultivated in South East Asian countries mg/L BA alone, or with 0.25 mg/L NAA (Table 1) in Ma- [1] as spice and medicinal herb [2]. The plant has a unique genta GA7 vessels. Mediums were incubated in a culture combined flavour of lemon and cumin, and is used for room at 16-h light photoperiod under white light emitting making soups. The stem is submersed with emergent and diodes (LEDs). in amphibious form. The flowers are single and stalkless with different colors like pink, white, purple, or ranged Regenerated shoots obtained after 8 weeks of culture from blue to lavender colour. The fruit is a capsule which were isolated and transferred onto a rooting medium pro- contains approximately 150 seeds [3]. vided with 0.25-1.0 mg/L NAA in Magenta GA7 vessels The plant contains several flavonoids like nevadensin, for 4 weeks. Rooted plantlets were cleaned under tap water nevadensin-7-O-b glycopyranoside, gardenin B, and other to remove the gel adhering to roots, and acclimatized in the flavones [4], with antioxidant activity [5, 6]. The leaves aquariums containing tap water. All culture media (regen- contain 0.1% essential oil [3] with limonene as main com- eration and rooting) used in the study were provided with 30 g/L (w/v) sucrose and 0.65% (w/v) agar at 5.8 pH. Au- toclaving of all culture media was performed at 118 kPa * Corresponding author atmospheric pressure and 120 °C for 21 min.

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FIGURE 1 - Multiple shoot regeneration from shoot tip explant of L. aromatica: (a) sterilized twig, (b) shoot explant, (c, d) multiple shoot induction after 4 weeks, and (e, f) after 8 weeks.

TABLE 1 - In vitro shoot regeneration from shoot tip explant of L. aromatica on different concentrations of BA-NAA.

BAP NAA Frequency of shoot regeneration Shoots Shoot (mg/L) (mg/L) (%) per explant length (cm) 0.25 - 100ns 20.89f 2.60c 0.50 - 100 28.28d 2.16e 1.00 - 100 44.22a 1.65h 1.50 - 100 31.17b 1.21i 2.00 - 100 19.61g 1.02j 0.25 0.25 100 14.28h 4.23a 0.50 0.25 100 21.17f 3.76b 1.00 0.25 100 25.44e 2.44d 1.50 0.25 100 28.22d 2.09f 2.00 0.25 100 30.72c 1.74g Values within a column followed by different letters are significantly different at 0.01 level of significance using DMRT.

The experiments were run in triplicate for both shoot 3. RESULTS proliferation and rooting experiments. Data pertaining frequency of shoot regeneration, shoots/explant and shoot In this study, sterilization, shoot regeneration potential length were analyzed using statistical analysis by One Way and acclimatization of aquatic Limnophilla plants were in- ANOVA. SPSS17 for Windows by IBM was employed for vestigated on different BA-NAA media as plant growth statistical analysis, and Duncan’s multiple range test regulators in the culture medium. 16 % H2O2 proved to be (DMRT) was computed for post-hoc tests. All data were sufficient for gaining contamination-free leaf and shoot tip transformed into percentages ande subjected to arcsine explants. Leaf explants did not show any sign of callus or transformation [13]. shoot induction and become necrotic within 15 days whereas, shoot buds initiation started from the basal part of shoot tip explant within 15 days with clear multiple shoot formation after 4 weeks of culture (Figs. 1c, d). After 8 weeks of culture, data regarding frequency of shoot regeneration,

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shoots per explants and shoot length were taken under and was reported as one of the most potent explants for shoot aseptic conditions. The data were subjected to statistical regeneration of other aquatic plants [15, 17-19]. Shoot tip analysis which showed statistically insignificant effects of explants induced shoot buds from the basal part of the ex- BA-NAA concentrations on shoot regeneration frequency plant that has also been reported previously in other aquatic and was recorded as 100%. On the other hand, shoots per plants like C. demersum [15]. Results further revealed the explants and shoot length were statistically (p≤0.05) af- greater response of shoot tip explants to PGRs which re- fected by BA-NAA. sulted in 100% shoot regeneration. Karataş et al. [14] achieved 100 % shoot regeneration of Bacopa using 1st, 2nd Shoots per explant responded clearly to the BA or and 3rd internodes and leaf explants cultured on the medium BA+NAA concentrations in the culture medium. Shoots containing BA-NAA concentrations. per explants on MS medium with 0.25-2.0 mg/L BA ranged between 19.61-44.22 shoots with maximum of Shoots per explants also showed importance of PGRs 44.22 shoots on the medium having 1.0 mg/L BA. Whereas type and concentration in the culture medium. BA alone minimum shoots per explants (19.61) were scored on the induced more shoots than compared to BA-NAA which medium containing 2.0 mg/L BA. Contrarily, provision of confirms the previous findings of Aasim et al. [20], but 0.25 mg/L NAA with all BA concentrations resulted in a contrarily to the findings of Manik et al. [19]. On the other decreased number of shoots per explants compared to BA hand, increased concentration of BA singly or in combina- used singly that ranged between 14.28-30.72 with maxi- tion with NAA showed a decreased pattern and resulted in mum shoots at 2.0 mg/L BA+0.25 mg/L NAA. a relatively less number of shoots. The results are in line with the previous results of Sharma et al. [21] and Karataş Shoot length also clearly showed the importance of and Aasim [16] in B. monieri. Similarly, increased BA con- plant growth regulators that ranged between 1.02-2.60 cm centration inhibiting the shoot length are in line with the on the medium containing 0.25-2.0 mg/L BA. Maximum previous findings of Karataş et al. [15] and Karataş and longer shoots (2.60 cm) were obtained from the medium Aasim [16]. However, Vijayakumar et al. [22] achieved with 0.25 mg/L BA, and increased BA concentration in the longer shoots with increased BA concentration of B. mon- culture medium resulted in shorter shoots. On the other ieri. Results further showed that NAA with all combina- hand, shoot length on the medium having 0.25-2.0 mg/L tions of BA also inhibited the shoot length which was more BA+0.25 mg/L NAA was 1.74-4.23 cm. In general, adding prominent with increased BA concentration. These results 0.25 mg/L NAA promoted the mean shoot length on all BA show contradiction to the findings of Manik et al. [19], who concentrations compared to BA used singly. Likewise BA reported positive bearings of NAA on shoot length. Simi- used singly, increased BA in the presence of NAA, and also larly, Karataş et al. [23] declared positive effects of IBA resulted in decreased shoot length. with KIN or TDZ on shoot length of Hygrophila poly- For rooting of in vitro regenerated shoots, shoots with sperma. more than 1 cm length were incubated on rooting medium Rooting of in vitro regenerated aquatic plants is im- provided with 0.25-1.0 mg/L NAA. Rooting started within portant for successful acclimatization in the aquariums. 8-10 days, and 100 % rooting was scored after 4 weeks of Different concentrations of NAA proved to be excellent for culture, irrespective of concentrations. Besides that, multiple 100% rooting confirming the higher rooting frequency of shoot regeneration was also observed in the rooting medium other aquatic plants [16]. Besides that, multiple shoot in- which was more prominent on higher NAA concentration. duction in the rooting medium was also recorded which is Rooted plantlets were, thereafter, acclimatized in the aquar- an unknown phenomenon and was reported in other plants iums having tap water and provided with oxygen. Plants sur- like cowpea [24] and chickpea [20]. However, there is no vived and continued their growth in the aquariums. report available on multiple shoot induction of aquatic

plants in the rooting medium. Last step for successful re-

generation protocol of in vitro regenerated aquatic plant is 4. DISCUSSION the acclimatization in the aquariums. Rooted plantlets adapted well in the aquariums and did not show any sign of necrosis. In vitro shoot regeneration protocol for aquatic plants in- Recent studies on other aquatic plants showed successful cludes sterilization, shoot regeneration, rooting and acclimati- acclimatization of in vitro regenerated plantlets like coon- zation in the aquariums. Each step has immense importance tail [15], dwarf hygro [23], Alternatharia sessilis [25], Ve- but sterilization of aquatic plants using twigs is a prerequisite ronica anagallis aquatica [26], Cryptocoryne beckettii, C. for obtaining contamination-free explants. H2O2 is the most wendtii [27] and Roundleaf toothcup [28]. preferable sterilizing agent for aquatic plants. A lot of previous studies on aquatic plants also showed the successful use of H O for surface sterilization of aquatic plants at different 2 2 5. CONCLUSION concentrations and timings [14-16]. In this study, we checked 2 different explants, but leaf The present study is the first report on successful explants did not show any response to PGRs in the culture whole plant regeneration of an important medicinal aquatic medium. Shoot tip explant showed great response to PGRs plant under in vitro conditions. This protocol can be em-

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ployed for further studies like secondary metabolite pro- [17] Kane, M.E., Philman, N.L., Bartuska, C.A. and Mcconnel, D.B. (1993). Growth regulator effects on in vitro regeneration of Cras- duction, followed by their activities. sula helmsii. Journal of Aquatic Plant Management, 31, 59-64.

[18] Kanchanapoom, K., Chunui, P. and Kanchanapoom, K. (2012). The authors have declared no conflict of interest. Micropropagation of Anubias barteri var. Nana from shoot tip culture and the analysis of ploidy stability. Notulae Botanca Horti Cluj Agrobotanica, 40(2), 148-151. [19] Manik, S.R., Yatoo, G.M., Ahmad Z. and Nathar, V.N. (2012). REFERENCES Direct organogenesis of Mentha piperata L. from shoot tip, nodal and sucker explants. Journal of Agricultural Technology, 8(2), 663-669. [1] Gorai, D., Jash, S.K., Singh, R.K. and Gangopadhyay, A. (2014). Chemical and Pharmacological Aspects of Limnophila aromatica [20] Aasim, M., Day, S., Rezaei, F. and Hajyzadeh, M. (2013). Multiple (Scrophulariaceae): An Overview. AJPCT, 2(3), 348-356. shoot regeneration of plumular apices of chickpea. Turkish Journal of Agriculture and Forestry, 37(1), 33-39. [2] Vo, C.V. (1999). Dictionary of Medicinal Plants in Vietnam. Med- icine Publishing House, pp. 58 [21] Sharma, S., Kamal, B., Rathi, N. Chauhan, S., Jadon, V., Vats, N., Gehlot, A. and Arya, S. (2010). In vitro rapid and massmultiplica- [3] Brahmachari, G. (2008). Limnophila (Scrophulariaceae): Chemi- tion of highly valuable medicinal plant Bacopa monnieri (L.) cal and Pharmaceutical Aspects. The Open Natural Product Jour- Wettst. African Journal of Biotechnology, 9(49) 8318–8322. nal, 1, 34-43. [22] Vijayakumar, M., Vijayakumar, R. and Stephen, R. (2010). In [4] Bui, M. L., Grayer, R. J., Veitch, N. C., Kite, G. C., Tran, H., Ngu- vitropropagation of Bacopa monnieri L., A multipurpose plant. In- yen, Q.C. K., (2004). Uncommon 8- oxygenated flavonoids from dian Journal of Science and Technology, 3(7), 781-786. Limnophila aromatica (Scrophulariaceae). Biochemical Systemat- [23] Karataş, M., Aasim, M., Çınar, A. and Dogan, M. (2013). Adventi- ics and Ecology, 32, 943-947. tous shoot regeneration from leaf explant of dwarf hygro (Hy- [5] Kukongviriyapan, U., Luangaram, S., Kukongviriyapan, V. and grophila polysperma (Roxb.) T. Anderson). TheScientific- Pakdeechote, P. (2003). Alleviation of oxidative damage after Lim- WorldJournal, http://dx.doi.org/10.1155/2013/680425. nophila aromatica treatment in phenylhydrazine induced anemic [24] Aasim, M., Khawar, K.M. and Özcan, S. (2008). In vitro micro rats. Proceeding of the sixth JSPS-NRCT joint seminar: Recent ad- propagation from shoot meristems of Turkish Cowpea (Vigna un- vances in natural medicine research, December 2-4, 2003, Bang- guiculata L.) cultivar Akkiz. Bangladesh Journal of Botany, 37(2), kok, Thailand: 202. 149-154. [6] Sribusarakum, A, Bunyapraphatsara, N., Vajragupta, O. and [25] Gnanaraj, W.E., Marimuthu, J., Subramanian, K.M. and Nallyan, Watanabe H. (2004). Antioxidant activity of Limnophila aro- S. (2011). Micropropagation of Alternanthera sessilis (L.) using matica Merr. Thai Journal of Phytopharmacy, 11(2), 11-17. shoot tip and nodal segments. Iranian Journal of Biotechnology, 9(3), 206-212. [7] Katzer G. (2014). Rice Paddy Herb (Limnophila aromatica [Lomk.] Merril). (http://gernot-katzers-spice- [26] Shahzad, A., Parveen, S. and Fatema, M. (2011). Development of pages.com/engl/Limn_aro.html) a regeneration system via nodal segment culture in Veronicaa- nagallis-aquatica L.-An amphibious medicinal plant. Journal of [8] Murakami, A., Jiwajinda, S., Koshimizu, K. and Ohigashi, H. Plant Interaction, 6, 61-68. (1995). Screening for in vitro anti-tumor promoting activities of edible plants from Thailand. Cancer Letters, 95(1/2), 139-46. [27] Stanly, C., Bhatt, A. and Keng, C.L. (2011). An efficient in vitro- plantlet regeneration of Cryptocoryne wendtii and Cryptocoryne [9] Murakami, A., Nakamura, Y., Ohigashi, H. and Koshimizu, K. becketti through shoot tip culture. Acta Physiologia Plantarum, (1997). Cancer chemo preventive potentials of edible Thai plants 33(2), 619-624. and some of their active constituents. School of Biology- Oriented Science and Technology Kinki University, 1, 1-23. [28] Karatas, M., Aasim, M.and Çiftçioğlu, M. (2014). Adventitious shoot regeneration of Roundleaf toothcup-Rotala rotundifolia [10] Nakahara, K., Roy, M.K., Alzoreky, N.S., Thalang, V.N. and [(Buch-Ham. ex Roxb) Koehne]. Journal of Animal and Plant Sci- Trakoontivakorn G. (2002). Inventoryvof indigenous plants and ences, 24(3): 838-842. minor crops in Thailand based on bioactivities. 9th JIRCAS Inter- national Symposium 2002-“Value-addition to Agricultural Prod- ucts”, 135-9. [11] Bhuiyan, N.I., Akter, F., Chowdhury, J.U. and Begum, J. (2010). Chemical constituents of essential oils from aerial parts of Aden- Received: December 08, 2014 osma capitatum and Limnophila aromatica, Bangladesh Journal of Revised: February 23, 2015 Pharmacology, 5(1), 13-16. Accepted: February 24, 2015 [12] Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473-497. CORRESPONDING AUTHOR [13] Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods. The Iowa State University Press, Iowa, USA. Mehmet Karataş [14] Karatas, M., Aasim, M, Dogan, M. and Khawar, K.M. (2013). Ad- Department of Biotechnology ventitious shoot regeneration of the medicinal aquatic plant water Faculty of Science hyssop (Bacopa monnıeri L. PENNELL) using different internodes. Archives of Biological Sciences Belgrade, 65(1), 297-303. Necmettin Erbakan University Konya [15] Karataş, M., Aasim, M. and Dogan, M. (2014). Multiple shoot regeneration of Ceratophyllum demersum L. on agar solidified and TURKEY liquid mediums. Fresenius Environmental Bulletin, 23(1), 3-9. [16] Karataş, M. and Aasim, m. (2014). Efficient adventitious shoot re- Phone: +90 338 226 2151-3827 generation of medicinal aquatic plant water hyssop (Bacopa mon- E-mail: [email protected] nieri L. Pennell). Pakistan Journal of Agricultural Sciences, 51(3), 665-670. FEB/ Vol 24/ No 9/ 2015 – pages 2747 - 2750

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RESIDUE MANAGEMENT AFFECTS GREENHOUSE GAS EMISSIONS AND SOIL ORGANIC CARBON IN WHEAT-RICE ROTATION SYSTEM

Jie Wu, Wenbo Wang, Xiaohua Wang, Liqun Zhu, Haishui Yang, Xinzhong Han, Jie Gao, Wei Guo and Xinmin Bian*

College of Agriculture, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT 1. INTRODUCTION

Straw returning is an important factor in taking full ad- Carbon dioxide (CO2), methane (CH4) and nitrous ox- vantage of the resources in the agricultural ecosystem. In ide (N2O) are potent greenhouse gases (GHGs), the pres- this study, a ﬁeld study was conducted to compare the ence of which in the atmosphere is leading to global warm- greenhouse gas (GHG) emissions and soil organic carbon ing. The global atmospheric concentrations of CO2, CH4 (SOC) under different rice (R) and wheat (W) straw return- and N2O have increased from a pre-industrial value of ap- ing modes (RDI, rice straw returned in wheat season and proximately 250 ppmv, 0.7 ppmv and 255 ppbv, respec- WDI, wheat straw returned in rice season) and ditch bury- tively, to concentrations of CO2, CH4 and N2O of 367 ppmv, ing with different depths, viz., 20 cm (RD20 and WD20), 1.76 ppmv and 312 ppbv, respectively, in the last century 40 cm (RD40 and WD40) and 60 cm (RD60 and WD60) in a [1]. Agriculture is a major emission source, which accounts wheat-rice rotation system. Ditch burying treatments sig- for 13.5% of the total GHG emission [1]. Soil is the largest nificantly decreased GHG emissions compared with RDI, active carbon pool in terrestrial ecosystems, storing more while RD40 and RD60 had no significant difference in the carbon than either the atmosphere or vegetation [2]. Mean- total global warming potential (GWP) compared to RCK while, agricultural soil organic carbon (SOC) is diminish- (no rice straw returning). The SOC and the carbon pool ing due to long-term intensive cropping [2]. Agricultural management index (CPMI) with RDI (WDI) were signifi- soil can provide a large potential for carbon sequestration cantly higher than those with the other four treatments in to mitigate the increase of atmospheric CO2 [3]. Therefore, the initial stage after straw returning. However, the differ- improving agricultural practices is a recommended strat- ence in SOC and CPMI between RDI (WDI) and the other egy to mitigate GHG emissions and increase soil carbon four treatments exhibited a decreasing trend. In addition, storage [1]. ditch burying treatments exhibited no significant differ- There are 13 million hectares of rice-wheat cropping ence in the crop yield in relation to CK. This result suggests in China [4], which produces increasing amounts of fresh that in eastern China, straw ditch burying should be a better rice and wheat residue. Large amounts of crop residue are approach to reducing GHG emissions and improving soil simply burnt to save time and labor; however, this practice carbon quality without affecting the crop yield. results in significant environmental pollution [3]. Retain- ing crop residues in the field is an important measure for comprehensive utilization and recycling of resources in an KEYWORDS: agricultural ecosystem. Residue retention is a key factor af- Straw; Ditch burying; Carbon pool management index fecting GHG production, depending on the straw amount, type and returning mode [5]. Straw retention and incorporation can increase CO2 emissions from soil [6; 7] by changing the soil microbial activity and the decomposition processes ABBREVIATIONS that transform straw-derived C into soil organic matter and CO2 [8]. Previous studies on the effect of straw incorpora- CPMI, carbon pool management index; GHG, green- tion on CH4 emissions were primarily applied to the rice house gas; GWP, global warming potential; ROC, oxidiz- fields; the results indicated that residue retention and incor- able organic carbon; SOC, soil organic carbon poration can increase CH4 emissions in most conditions in China [9-11]. However, the effects of straw incorporation on N2O emissions are inconsistent. A previous study demon- * Corresponding author strated that the application of wheat straw increased N2O

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emissions in the following maizerotation by 58% [12]. main cropping in the region is a rice-wheat rotation under Mixing wheat or alfalfa residues with soil resulted in a subtropical monsoon climate. It has a mean annual tem- greater generation of N2O than the soil without straw in- perature of 15.3°C and a precipitation of 1117.1 mm, corporation [13]. However, N2O emissions decreased after which is concentrated from June to September. The soil at straw incorporation in rice paddies [9; 11] and in rice-wheat the experimental site was a slightly alkaline sandy loam rotation fields [10]. The inconsistency in the observed (pH = 7.9), with a total organic C of 0.77%, total N 0.09%, changes in N2O emissions may be due to the C/N ratio of the available P 2.78% and available K at 9.61%. straw, the mechanical treatment of the straw, and the distribution of the straw in the soil, as well as soil structure, com- 2.2. Treatments and field work paction, level of waterlogging and fertiliser [13]. In addition, Rice straw (R) and wheat straw (W) were returned to the previous studies mostly examined the GHG emissions different experiment fields on October 29th, 2010 and June during a single season; as a result, little is known regarding 9th, 2011, respectively. Under each case, five treatments the further effect in the following seasons. were set in a randomized block design with three replicates. Previous studies indicated that straw retention and in- Residue was applied on plots of 3 × 2 m2 at the same rate corporation to cropland increased soil carbon storage by of 10 t ha-1 before crop cultivation. After residue retention, 12% [14]. Straw incorporation could improve the accumula- shallow rotary tillage was applied with the depth of 5 cm. tion of carbon in the soil surface to maintain and even im- The five treatments were as follows: (1) RCK and WCK, prove the long-term productivity of soil in Southwest Eng- no residue returning applied (the previous crop harvested land [15]. In addition, straw incorporation significantly in- to ground level and all residue removed); (2) RDI and WDI, creased the carbon pool management index (CPMI), which residue incorporated into the topsoil (0-20 cm); (3) RD20 indicated the increase in soil organic carbon (SOC) [16; 17]. and WD20, residue without chopping was buried in a ditch in However, conventional straw incorporation also aggravates the middle of the plot and covered with soil to the field level; off-site transport of nutrients and decline of tilling quality the ditch was 3 m in length, 0.02 m in width and 0.02 m in [18; 19]. depth; (4) RD40 and WD40, residue was buried in a ditch of 3 m in length, 0.02 m in width and 0.04 m in depth; (5) RD Burying straw in ditches was first reported by Han 60 and WD , residue was buried in a ditch of 3 m in length, (1992) [20]; its impacts on crop yield and soil physical and 60 0.02 m in width and 0.06 m in depth. The gas fluxes from chemical properties were recently studied [21; 22]. Com- treatments (3), (4), and (5) included both the ditch and the pared with conventional straw returning, burying straw treat- non-ditch areas. Another three rice straw returning treatments improved the soil carbon storage of the 0- to 20-cm ments were set to collect the gas data from the ditch for the soil layer [23], which favored the crop growth and yield [21]. narrow of the ditch area. The residue was applied at the Several studies also reported the effect of burying wheat same rate of 10 t ha-1. The three treatments were as follows: straw on CH and N O emissions from rice fields [24-26]. 4 2 (1) RDP , 3 m in length, 2 m in width and 0.02 m in depth Compared with incorporating straw, ditch burying (includ- 20 for the ditch area of treatment RD ; (2) RDP , 3 m in ing ditch area and non-ditch area with a ratio of 1:4) almost 20 40 length, 2 m in width and 0.04 m in depth for the ditch area emitted the same amount of CH and N O. However, the im- 4 2 of treatment RD ; (3) RDP , 3 m in length, 2 m in width pact of rice straw burying in ditch on GHG emissions from 40 60 and 0.06 m in depth for the ditch area of treatment RD . In the following wheat rotation and the later rice season re- 60 addition, the micro region of 1 × 1 m2 without plants for mains unknown. each treatment was set up to eliminate the influence of In this study, we compared greenhouse gas emissions plants respiration. and SOC under the conditions of all residue removal, resi- In the wheat season of 2010, wheat seeds (cv. Yangmai due incorporation, and ditch burying of all residue in a rd wheat-rice rotation farming system. The objectives of this 158) were sown on November 3 and the resulting wheat was harvested on June 7th, 2011. The fertilization was ap- study were to: (a) investigate the characteristics of CO2, plied according to the local farming regime. Diammonium CH4 and N2O emissions from wheat-rice rotation field un- phosphate of 150 kg·hm-2 was applied as the basal fertilizer. der residue returning; (b) to investigate the dynamic -2 changes of crop yield and SOC from wheat-rice rotation Urea of 465 kg·hm was applied, with 55% as the basal fertilizer and 45% as the top dressing at the booting stage. Min- system after rice/wheat straw returning; and (c) to deter- -2 mine which straw retention method is more beneficial to eral compound fertilizerof 150 kg·hm (N: P2O5: K2O = the environment. 15%: 15%: 15%) was applied as the return green fertilizer. In the rice season of 2011, rice seedlings (cv. Nangeng 46) were transplanted on June 26th, with harvested on October 2. MATERIALS AND METHODS 27th. Urea of 492 kg·hm-2 was applied, with 20% as basal fertilizer, 50% at tillering and 30% at the panicle stage. Di- 2.1. Site description ammonium phosphate of 82.5 kg·hm-2 was applied at tiller- -2 A field experiment was conducted in Chongming Island ing. Mineral compound fertilizerof 150 kg·hm (N: P2O5: in Shanghai, China (31° 31' N, 121° 54' E) commencing in K2O= 15%: 15%: 15%) was applied at the panicle stage. The the wheat season of 2010 to the wheat season of 2012. The rice field was flooded for 36 days before a one-week mid-

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season drainage, followed by intermittent irrigation until the of the cropping seasons. The wheat and rice yields were crop was harvested. The crop cultivation, fertilization re- recorded at harvest. gimes and irrigation methods were same for the remainder

0~20cm

Straw DI

10~20cm 10~20cm

D20 Straw

FIGURE 1 - Schematic diagrams of different straw returning modes. CK, no straw returning; DI, straw evenly incorporated into the topsoil; D20, straw buried in ditch, taking 20 cm for example

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2.3. GHG emissions monitoring of the soil mineral N (ammonium and nitrate) at the main The investigation of GHG emissions were performed growing stages during the wheat growth season and the from November, 2010 to October, 2011 in the rice straw mature stage of the rice season in 2010-2011. Fresh soil retention treatments. The static chamber technique was was extracted with 0.01 M CaCl2 in a 1:10 ratio of soil to adopted for sampling CO2, CH4 and N2O. To collect the extractant. The concentrations of ammonium and nitrate in gas sampling, the chamber (0.5 m × 0.5 m × 0.5 m, with the extract were analyzed using continuous flow analytical the addition of a hollow chamber (0.5 m × 0.5 m × 0.5 m) system [29]. Surface soil samples (0-20 cm) were collected during the later stage of crops) was placed over the crop for the analysis of the soil organic carbon and the oxidiza- (Fig. 1). The chamber was equipped with a circulating fan ble organic carbon at the mature stage of the crop season. to ensure complete gas mixing and was wrapped with a The soil was air-dried and then was ground to pass a 100- layer of sponge and aluminum foil to minimize air temper- mesh sieve. The organic carbon of the sieved soil samples ature changes inside the chamber during the period of sam- were analyzed using dichromate oxidation titration [29]. pling. Aluminum ﬂux collars (0.5 m × 0.5 m × 0.2 m) sup- The oxidizable organic carbon was measured using the porting the chambers were permanently installed in all the KMnO4 oxidation method [30]. plots before the measurements. During the wheat-growing To monitor the soil characteristics of D , D and D , season, gas samples were collected once approximately 20 40 60 soil samples in the ditch area and non-ditch area were col- every 10 days between 14:00 and 16:00 hours, except from lected in each plot. Therefore, the soil nutrient contents of November to February due to the cold weather. During the D , D and D were calculated as follows: C = rice-growing season, gas samples were collected once 20 40 60 D (C +9C )/10. The carbon pool management index every two weeks between 8:00 and 10:00 hours. Gas sam- ditch non-ditch (CPMI) was calculated as follows [30]: ples were drawn from the chambers through a three-way stopcock using an airtight syringe with volume of 50 ml at CPMI= CPI×AI×100 (1) 0, 5, 10 and 15 min after closure, and immediately transferred into 50ml vacuum glass container. Where CPI is the carbon pool index, and AI is the activity index of the carbon pool. CPI and AI were calculated The mixing ratios of CO2, CH4 and N2O were analyzed as follows: using a modified gas chromatograph (Agilent 4890D) equipped with a flame ionization detector (FID) and an CPI=SOCt/SOCc (2) electron capture detector (ECD) [27]. N2 was used as the carrier gas and an Ar-Ch4 gas mixture as the make-up gas AI=At/Ac (3) for ECD analysis of CO , CH and N O. N O was separated 2 4 2 2 A=AOC/NAOC=AOC/ (SOC- AOC) (4) by two stainless steel columns, which were packed with 80-

100 mesh Porapack Q. N2O was detected by ECD, while SOCt represents the soil organic carbon of the treated CO2 and CH4 was detected by FID. The oven temperature soils; SOCc represents the soil organic carbon of the control was controlled at 55 ºC, and the temperatures of ECD and soils. At is the carbon pool activity of the treated soils; Ac FID were set at 330 ºC and 200 ºC, respectively. Each sam- is the carbon pool activity of the control soils. The meas- pling was subdivided four times in 5-min intervals, and urement of AOC (active organic carbon) has not been in- then the fluxes were determined by the slope of the four vestigated to date, and ROC (oxidizable organic carbon) is samples. The sample sets were rejected unless their linear thought to be the best representation of AOC [31]. NAOC regression value was greater than 0.90. All of the flux rates refers to non-active organic carbon. were adjusted for the air temperature, the air pressure, and the area and volume of the chamber [28]. The average 2.5. Statistical analyses GHG fluxes were calculated using the results of triplicate All data were examined using a one-way analysis of plots. The seasonal accumulation amounts of GHG emis- variance (ANOVA) test. The statistical analysis was per- sions were calculated from the averaged emissions of each formed using SPSS 20.0 (IBM SPSS Statistics, Chicago, IL, two adjacent intervals of measurements. USA). Correlation analyses between CO2 flux and soil tem- The gas fluxes from RD20, RD40 and RD60 included the perature for different rice straw returning treatments were ditch and the non-ditch areas. The flux from the ditch area also applied. (Fditch) was measured in the plots of RDP20, RDP40 and RDP60, and the flux from the non-ditch area (Fnon-ditch) was measured in the plots of RD20, RD40 and RD60. Therefore, the 3. RESULTS fluxes of the treatments of RD20, RD40 and RD60 (FD) were calculated as follows: FD= (Fditch+9Fnon-ditch)/10, where the 3.1. GHG fluxes and yield-scaled GWP ditch accounted only for 1/10 of the ditch treatments. During the wheat growth season, we found the CO2 fluxes under all treatments varied similarly in their temporal 2.4. Soil mineral N and organic carbon contents trends but differently in their amplitudes (Fig. 2a). The CO2 Fresh surface soil samples (0-20 cm) at all rice straw re- fluxes were generally higher in the early stage of the wheat- turning plots and RCK plots were collected for the analysis growing season from sowing and then decreased in winter;

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in spring, the CO2 fluxes increased steadily with the in- though the average temperature during the rice growth sea- creases in temperature. There was a linear positive correla- son was higher than that of the wheat growth season, the tion between the CO2 fluxes and the soil temperature at the CO2 fluxes were relatively lower than those of the wheat sea- 2 soil depth of 5 cm (Fig. 3a, R = 0.76, P < 0.001). Further- son (Fig. 2a). The correlation analysis between the CO2 more, an obvious increase in CO2 production with RDI was fluxes and the soil temperature at the soil depth of 5 cm in- observed compared with the other four treatments when air dicated that the CO2 fluxes increased with temperature to a temperature was more than 20°C in the late reproductive certain extent and then decreased with temperature (Fig. 3b, 2 stage. The total CO2 emissions during the wheat season of R =0.70). A significant difference was observed between the ditch-burying treatments RD20, RD40 and RD60 exhibited RDI and the other four treatments in the total CO2 emis- slight differences in relation to RCK and RDI (Table 1). Al- sions during the rice season (P<0.05), while no significant

) 500 -1 (a) RCK RD RD

·h I 20 -2 400 RD40 RD60 300

200 fluxes (mg·m

2 100 CO 0

) 50 -1 (b) RCK RD RD

·h I 20 -2 40 RD RD 40 60 30 20 fluxes (mg· m

4 10

CH 0

) 0.10 -1 (c) RCK RD RD ·h I 20 -2 0.08 RD RD 40 60 0.06

0.04 fluxes (mg·m

O 0.02 2 N 0.00 Nov 1 Jan 1 Mar 1 May 1 Jul 1 Sep 1 Nov 1 Date FIGURE 2 - Seasonal patterns of CO2, CH4 and N2O emissions from the wheat-rice rotation field. RCK, no rice straw returning; RDI, rice residue incorporated into the topsoil (0-20 cm); RD20, RD40, RD60, rice residue was buried in a depth of 0.02 m, 0.04 m and 0.06 m, respectively.

500 150 )

(a) ) (b) -1 2 -1

·h RCK y = 49.10 + 11.90x y = -896.52 + 75.66x - 1.43x ·h -2

RD -2 400 I r2 = 0.76 120 r2 = 0.70 P = 0.076 RD 20 P < 0.001 RD 300 40 90 RD 60

200 60

100 (mg·m season rice of fluxes 30 fluxes of (mg·m wheat season 2 2 CO CO 0 0 10 20 30 20 25 30 35 Temperature (oC) Temperature (oC) FIGURE 3 - Correlation analyses between CO2 flux and soil temperature for different rice straw returning treatments. RCK, no rice straw returning; RDI, rice residue incorporated into the topsoil (0-20 cm); RD20, RD40, RD60, rice residue was buried in a depth of 0.02 m, 0.04 m and 0.06 m, respectively.

2755 © by PSP Volume 24 – No 9. 2015 Fresenius Environmental Bulletin

TABLE 1 - Effects of different rice straw returning modes on total CO2, CH4 and N2O emissions during the wheat and rice seasons of 2010- 2011 in the wheat-rice rotation field.

Treat- 2010 Wheat season (kg ha-1) 2011 Rice season (kg ha-1) 2010-2011 Wheat-Rice rotation (kg ha-1) ments CO2 N2O CO2 CH4 N2O CO2 CH4 N2O RCK 6191.76±322.67b 0.72±0.02c 1905.33±93.20bc 176.01±42.90b 0.84±0.03a 8097.09±288.52b 176.01±42.90b 1.56±0.05b RDI 7351.85±486.13a 1.10±0.09a 2410.33±117.64a 301.91±30.17a 0.90±0.07a 9762.18±466.73a 301.91±30.17a 2.00±0.13a RD20 6314.05±285.67ab 0.84±0.03bc 1981.91±71.33b 276.03±40.06a 0.87±0.03a 8295.96±259.71b 276.03±40.06a 1.71±0.05b RD40 7050.02±255.67ab 0.83±0.01bc 1899.32±72.91c 187.89±36.65b 0.88±0.03a 8949.34±238.64ab 187.89±36.65b 1.72±0.04b RD60 6547.08±290.97ab 0.87±0.01b 1974.17±72.34bc 202.28±34.82b 0.85±0.03a 8521.25±263.04b 202.28±34.82b 1.72±0.03b Values are means ± standard deviation of three replicates. Different small letters in the same column refer to significant difference between treatments at P < 0.05 level. RCK: no rice straw returning; RDI: rice straw incorporation in wheat season; RD20:rice straw ditch burying with the depth of 0.02 m; RD40:rice straw ditch burying with the depth of 0.04 m; RD60:rice straw ditch burying with the depth of 0.06 m.

25000 CO (a) 2

CH4 ) 20000 N O -1 2 eq ha 2 15000

10000 GWP CO (kg

5000 1500

) (b) -1 1200 eq t 2

900

600

300 Yield-scaled GWP (kg CO 0 RCK RD RD RD RD I 20 40 60 Treatments FIGURE 4 - Overall GWP of GHGs (a) and yield-scaled GWP (b) for different rice straw returning treatments. The error bar in (a) was the standard error of overall GWP of CO2, CH4 and N2O emissions. RCK, no rice straw returning; RDI, rice residue incorporated into the topsoil (0-20 cm); RD20, RD40, RD60, rice residue was buried in a depth of 0.02 m, 0.04 m and 0.06 m, respectively.

difference was observed between RCK and the ditch-bury- Table 1). No significant difference was observed among ing treatments (Table 1). RD20, RD40 and RD60 in the total N2O emissions. During the rice growing season, the peak fluxes of N O emission A very similar seasonal trend of the N2O fluxes in all 2 treatments was also observed during the wheat-growing appeared when the field was dry. In addition, there was no significant difference between all these treatments in the season (Fig. 2c). The peak fluxes of N2O emission appeared in the wheat seedling stage, and then the fluxes were main- total N2O emissions during the rice season (Table 1). -2 -1 tained at a low level between 0.01 and 0.02 mg m h . The The CH4 fluxes varied over a large range, from 0.10 -2 -1 -2 -1 seasonal pattern of the N2O fluxes was not interpreted from mg m h to 25.87 mg m h , during the rice growing sea- the variation of temperature. The N2O fluxes from RDI son, with a peak flux in the tillering stage (Fig. 2b). After were much higher than those from the other four treat- the one-week-long mid-season drainage, the CH4 fluxes -2 -1 ments, while the total N2O emission was approximately maintained a low level between 0.10 and 6.95 mg m h . 35% lower for RCK than that for RDI(P<0.05) (Fig. 2c, No significant difference was observed between RDI and

2756 © by PSP Volume 24 – No 9. 2015 Fresenius Environmental Bulletin

RD20 in the total CH4 emissions during the entire rice sea- (Fig. 4a). Compared to RCK, RDI and RD20 was 37% and son. Compared with RDI, treatments RCK, RD40 and RD60 20% higher in the total GWP, respectively (P<0.05). CO2 were approximately 42%, 38% and 33% lower in the total and CH4 were the main GHGs from the wheat-rice rotation CH4 emissions, respectively(P<0.05) (Table 1). fields, which contributed 55 to 65% and 31 to 42% to the overall GWP under these treatments, respectively. The The total GWP of the CO2, CH4 and N2O emissions during the wheat and rice growing seasons was 12610.73, yield-scaled GWP calculated by GWP per unit crop yield varied between 763.96 and 960.16 with the lowest value in 17303.03, 15154.01, 13782.36 and 13686.61 kg CO2 eq -1 RCK and the highest value in RD (Fig. 4b). hm for RCK, RDI, RD20, RD40,and RD60, respectively I

TABLE 2 - Effects of different rice straw returning modes on soil mineral nitrogen during the wheat and rice seasons of 2010-2011 in the wheat-rice rotation field.

+ -1 - -1 NH4 -N(mg kg ) NO3 -N(mg kg ) 2011 Rice 2011 Rice 2010 Wheat season 2010 Wheat season Treat- season season ments over- over- jointing booting mature jointing booting mature wintering filling stage mature stage wintering filling stage mature stage stage stage stage stage stage stage stage stage RCK 5.95±0.07a 4.26±0.15a 2.84±0.29a 3.80±0.23b 2.76±0.34b 4.44±0.22b 49.57±1.84a 45.51±5.01a 22.08±3.64a 23.64±3.11a 15.06±0.84b 12.68±0.45b RDI 5.50±0.31a 4.17±0.04a 2.46±0.18a 4.51±0.26ab 3.96±0.45ab 5.39±0.19a 31.35±2.48b 34.86±1.28b 24.28±1.11a 30.24±2.41a 19.16±0.31a 13.94±0.70a RD20 5.93±0.07a 4.26±0.15a 2.83±0.24a 3.83±0.19b 2.82±0.28b 4.75±0.22b 49.07±1.97a 45.25±4.49a 21.82±3.53a 23.60±3.10a 15.46±0.69b 13.80±0.35ab RD40 5.90±0.05a 4.25±0.14a 2.80±0.28a 3.81±0.20b 2.79±0.32b 4.66±0.19b 48.50±2.02a 44.78±4.52a 21.75±3.33a 23.46±3.14a 15.34±0.75b 13.07±0.40ab RD60 5.91±0.06a 4.25±0.13a 2.78±0.27a 3.81±0.21b 2.79±0.34b 4.48±0.19b 48.33±1.63a 44.58±4.45a 21.32±3.44a 23.23±2.89a 15.21±0.69b 12.72±0.27ab Values are means ± standard deviation of three replicates. Different small letters in the same column refer to significant difference between treatments at P < 0.05 level. RCK: no rice straw returning; RDI: rice straw incorporation in wheat season; RD20: rice straw ditch burying with the depth of 0.02 m; RD40: rice straw ditch burying with the depth of 0.04 m; RD60: rice straw ditch burying with the depth of 0.06 m.

25 RCK RD RD (a) I 20

) RD RD

-1 20 40 60

5 Soil orrganicSoil carbon (g kg 0 0.5Year 1Year 1.5Year 2Year 2.5Year 25 WCK WD WD (b) I 20

) WD WD 40 60

-1 20

5 Soil organic carbon (g kg 0 0.5Year 1Year 1.5Year 2Year Time of straw returning

FIGURE 5 - Soil organic carbon (SOC) for different rice and wheat straw returning treatments. RCK, no rice straw returning; RDI, rice residue incorporated into the topsoil (0-20 cm); RD20, RD40, RD60, rice residue was buried in a depth of 0.02 m, 0.04 m and 0.06, respectively. WCK, no wheat straw returning; RDI, wheat residue incorporated into the topsoil (0-20 cm); WD20, WD40, WD60, wheat residue was buried in a depth of 0.02 m, 0.04 m and 0.06 m, respectively.

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6 RCK RD RD (a) I 20 RD40 RD60

) 4 -1

2 Soil ROC(g·kg Soil

0 0.5Year 1Year 1.5Year 2Year 2.5Year 6 WCK WD WD (b) I 20 WD WD 40 60

) 4 -1

2 Soil ROC(g·kgSoil

0 0.5Year 1Year 1.5Year 2Year Time of straw returning FIGURE 6 - Oxidizable organic carbon (ROC) for different rice and wheat straw returning treatments. RCK, no rice straw returning; RDI, rice residue incorporated into the topsoil (0-20 cm); RD20, RD40, RD60, rice residue was buried in a depth of 0.02 m, 0.04 m and 0.06 m, respectively. WCK, no wheat straw returning; RDI, wheat residue incorporated into the topsoil (0-20 cm); WD20, WD40, WD60, wheat residue was buried in a depth of 0.02 m, 0.04 m and 0.06 m, respectively.

3.2. Soil mineral nitrogen treatments in SOC over two-and-a-half years. Addition- - + The peaks of the soil NO3 -N and NH4 -N contents ap- ally, no significant difference was observed between WDI peared at the over-wintering stage during the wheat growth and the other four treatments in the SOC, except for the - season of 2010. The soil NO3 -N content with RDI was sig- difference at half-a-year after the wheat straw returning nificantly lower at the over-wintering stage and the joining (Fig. 5b). stage, while the content was significantly higher at the ma- The ROC for RDI was significantly higher than that for ture stage than that of the other four treatments during the the other four treatments within one-and-a-half years after wheat season (P<0.05) (Table 2). There was no significant rice straw returning (P<0.05) (Fig. 6a). The peak of ROC + difference in the soil NH4 -N content among the different with RDI appeared at one-year after straw returning, while treatments during the entire wheat season. However, the the other four treatments exhibited an increasing trend. + soil NH4 -N content was significantly higher with RDI at Compared with the other four treatments, the ROC was sig- the rice mature stage than that of the other four treatments nificantly higher with WDI, except for two years after + (P<0.05) (Table 2). The soil NH4 -N contents for different wheat straw returning (P<0.05) (Fig. 6b). In addition, treatments at the rice mature stage exhibited higher contents WD20 exhibited a significant difference in ROC with WCK than those at the wheat mature stage (P<0.01) (Table 2). and WD60 two years after the straw returning.

The CPMI for RDI was significantly higher than that 3.3. SOC, ROC, CPMI and crop yield for the other four treatments in half-a-year and one-and-a- The SOC for RDI was significantly higher than that for half years after the rice straw returning (P<0.05) (Table 3). the other four treatments in a half a year and one-year after The CPMI for RDI was higher in the early period after- the rice straw returning (P<0.05) (Fig. 5a). However, the straw returning and then decreased after a peak at one year difference between RDI and the other four treatments ex- after straw returning, while the CPMI for RD20, RD40 and hibited a decreasing trend. In addition, there was no signif- RD60 exhibited an increasing trend. Compared with the icant difference between RCK and the three ditch burying other four treatments, the CPMI was significantly higher

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TABLE 3 - Effects of different rice and wheat straw returning modes on crop yield and carbon pool management index (CPMI) of top soil (0- 20cm) during 2010-2012 in the wheat-rice rotation field.

Straw species Treatments Yields (t ha-1) CPMI 2010 Wheat 2011 Rice 2011 Wheat 2012 Rice 2012 Wheat 0.5 Year 1Year 1.5 Year 2 Year 2.5 Year RCK 7.69±0.72a 9.02±1.17b 7.32±0.61a 9.23±0.47a 7.59±0.54a 100.00±0.00b 100.00±0.00a 100.00±0.00b 100.00±0.00a 100.00±0.00a RDI 7.63±0.57a 10.45±1.05a 7.37±0.46a 9.23±0.33a 7.59±0.56a 118.10±6.26a 126.33±18.06a 110.62±3.76a 105.90±8.81a 102.65±5.72a Rice straw RD20 7.77±0.63a 9.12±1.14b 7.40±0.54a 9.41±0.48a 7.68±0.49a 100.10±0.88b 103.69±0.76a 103.74±0.69b 104.47±0.53a 104.83±1.35a RD40 7.69±0.65a 9.12±1.14b 7.33±0.56a 9.31±0.47a 7.62±0.49a 99.53±0.49b 100.87±1.70a 101.78±1.46b 101.73±0.37a 102.18±1.15a RD60 7.68±0.56a 9.05±1.11b 7.33±0.52a 9.26±0.47a 7.60±0.46a 100.03±1.46b 101.27±1.95a 101.32±1.13b 101.14±0.50a 101.38±0.86a 2011 Rice 2011 Wheat 2012 Rice 2012 Wheat 0.5 Year 1Year 1.5 Year 2 Year WCK 9.09±0.44b 7.47±0.18a 9.25±0.28a 7.65±0.21a 100.00±0.00b 100.00±0.00b 100.00±0.00b 100.00±0.00b WDI 10.45±1.05a 7.54±0.98a 9.19±0.19a 7.63±0.14a 126.00±8.89a 131.18±9.67a 115.55±6.79a 103.09±2.06ab Wheat straw WD20 9.22±0.4b 7.61±0.14a 9.44±0.21a 7.71±0.18a 101.67±1.34b 102.98±0.15b 104.06±0.40b 104.27±1.03a WD40 9.13±0.38b 7.47±0.25a 9.25±0.23a 7.67±0.19a 99.57±0.67b 100.44±2.36b 101.80±0.81b 102.08±1.14ab WD60 9.10±0.36b 7.43±0.15a 9.27±0.26a 7.64±0.18a 99.26±0.45b 99.51±0.18b 100.04±0.51b 100.38±0.23b Values are means ± standard deviation of three replicates. Different small letters in the same column refer to significant difference between treatments at P < 0.05 level. RCK: no rice straw returning; RDI: rice straw incorporation in wheat season; RD20: rice straw ditch burying with the depth of 0.02 m; RD40: rice straw ditch burying with the depth of 0.04 m; RD60: rice straw ditch burying with the depth of 0.06 m. WCK: no wheat straw returning; WDI: wheat straw incorporation in rice season; WD20: wheat straw ditch burying with the depth of 0.02 m; WD40: wheat straw ditch burying with the depth of 0.04 m; WD60: wheat straw ditch burying with the depth of 0.06 m.

with WDI, except at two years after the wheat straw return- treatments during the wheat season (P<0.05), while no sig- ing (P<0.05) (Table 3). WD20 exhibited a significant dif- nificant difference was found between the five treatments ference in the CPMI with WCK and WD60 two years after during the following rice season (Table 1). The result is in straw returning. good agreement with the results of a previous study that There was no significant difference in the crop yield indicated that mixing residues with soil generally gener- among all the treatments of R or W, except the rice yield in ated more N2O than the soil without straw [13]. However, several studies reported that straw incorporation reduces 2011 was significantly higher for RDI or WDI than that for the other treatments (P<0.05) (Table 3). N2O emissions compared with the control [10; 11]. This inconsistency might be attributed to the soil type, agricultural management type, and straw type. The N2O produced 4. DISCUSSION by denitrification was likely to be further reduced to N2, leading to a decrease in N2O emission [26]. Furthermore, - N2O emission are closely correlated with soil N (NO3 -N The emissions of CO2, CH4 and N2O from the wheat- + rice rotation field were investigated under different rice and NH4 -N), which provided a N source for N2O emission straw returning modes in eastern China. The accumulated [33]. During the initial stage of the wheat growing season, N might be immobilized in the microbial biomass because CO2 emissions during the rice growth season in this study were higher than that in the paddy field in southern China of the large C/N ratio of the rice straw [34]. The microbial [6], which might be attributed to the difference in the N biomass N is subsequently released into the soil when the fertilizer application rate, climate factors, and soil proper- crop requires it for growth, which led to the relatively higher soil N for RDI during the later period of crop growth ties. In this study, lower levels of CO2 emissions from the wheat-rice rotation field were found under the treatments (Table 2). In a previous study, under conventional irrigation, the incorporation of wheat straw in rice season was of RCK, RD20, RD40 and RD60 in relation to RDI (Table 1), and no significant difference was found between RCK and observed to reduce the N2O emissions in the following the other three ditch burying treatments. The relatively wheat season [35]. However, the impact of rice straw returning during the wheat season on N2O emissions from the lower CO2 emission under the ditch burying treatments was mostly caused by the straw returning mode, where the ditch following rice season was slight in this study. This result area only occupied 1/10 of the entire plot. Previous studies might be affected by the different C/N ratios of straw and the initial conditions for straw decomposition. reported that straw returning can increase the CO2 emissions from the soil [7] due to the improved decomposition There is no doubt that straw returning could increase processes, which transform straw-derived C into soil or- CH4 emissions from agricultural soils [36; 37]. The previ- ganic matter and CO2 [8]. Therefore, the relatively lower ous studies of Ma et al. (2010) and Zhang et al. (2009) SOC in the wheat-rice rotation field with ditch burying found that the application of rice residue during the wheat treatments can reduce the CO2 emission more effectively season increased CH4 by 40% and 41% from the following than RDI (Fig. 5a). This result might be attributed to the rice season in China, respectively [38; 39]. In the present limited soil- residue contact of the ditch burying treatments study, compared with RDI, the treatments of RCK, [13]. The seasonal CO2 emissions were significantly af- RD40,and RD60 decreased the total CH4 emissions by 42%, fected by the soil temperature, which was consistent with 38% and 33% during the entire rice season, respectively the results of a previous study [32]. (P<0.05) (Table 1). This result might due to the greater rate Field observations indicated the total N2O emissions of decomposition of straw for RDI, which offered the pre- from RDI were much higher than that from the other four dominant source of methanogenic substrates [11]. How-

2759 © by PSP Volume 24 – No 9. 2015 Fresenius Environmental Bulletin

ever, no significant difference was observed between RDI ence in CPMI compared with those of WCK and WD60 two and RD20 in the total CH4 emissions. CH4 emission from years after straw returning. This result suggested that straw the non-ditch area was much lower than that from RDI, but ditch burying might be an effective way to improve soil it was counter-balanced by high CH4 emission from the quality. ditch area. Although the soil-residue contact was limited In the present study, the yield of wheat ranged from for RD20, the high temperature and moisture in the topsoil 7.33 to 7.77 t ha−1, while the rice yield ranged from 9.02 to from summer paddies provided advantages for straw de- −1 10.45 t ha (Table 3). In addition, the RDI and WDI had composition. The treatments of RD40 and RD60 avoided higher grain yield in the rice season of 2011. A previous such situations due to their deep burial. Although the total study also observed that the combined application of straw GWP of CO2, CH4 and N2O emissions during the wheat incorporation and an inorganic fertilizer was more effec- and rice growing seasons decreased with the depth, no sig- tive in obtaining a higher grain yield [42]. This result may nificant difference was observed among these three ditch be related to the availability of nutrients from straw decom- burying treatments. Further study for long-term effects is position. still required. In the present study, the SOC for RDI and WDI were found to be significantly higher than the SOC for the other 5. CONCLUSION four treatments within one year after straw returning (Fig. 5). The result is in good agreement with previous study that in- Straw returning modes significantly affected the GHG dicated that conventional incorporation of straw to emissions and the SOC in a wheat-rice rotation field in east- cropland could increase soil carbon [14]. It was also re- ern China. Ditch burying treatments (RD20, RD40 and RD60) ported that rotary tillage with residues incorporated leads significantly reduced the GHG emissions in a short time to higher SOC stock [18]. RDI and WDI could provide compared with RDI. In addition, ditch burying treatments large quantities of organic carbon inputs to cropland soils exhibited no significant difference in the crop yield with CK in a short time for rapid decomposition, thereby promoting but exhibited a slowly increasing trend in the SOC and the soil microbial biomass and its activity, and subsequently CPMI. Thus, straw ditch burying should be a better approach increasing the SOC [40]. to reducing GHG emissions and improving the soil carbon ROC, as the representation of active organic carbon quality without crop yield reduction in eastern China. from the soil, can reflect the activity level of soil carbon. ROC plays an important role in soil carbon transformation and is closely related to soil productivity [30]. In this study, the ROC ranged from 21% to 36% of the total SOC, and ACKNOWLEDGEMENTS these values were larger than those reported by Xu et al. [17]. This difference might be due to the different straw This work was financially supported by the National amounts and the returning modes. The ROC for RDI and Science & Technology Pillar Program of the Ministry of WDI was significantly higher than those for the other four Science and Technology of China during the Eleventh treatments within one and a half years after straw returning Five-Year Plan Period (No. 2010BAK69B17-3). (Fig. 6). The increase in ROC might be ascribed to the priming effect of the applied inorganic N on the fresh or- The authors have declared no conflict of interest. ganic material in the soil, which stimulated the microbial activity that results in the decomposition of the soil organic matter [41]. Furthermore, the ROC for WD20 exhibited a significant difference with the ROC for WCK and WD60 REFERENCES two years after straw returning. The results indicated that conventional corporation of straw only worked in rela- [1] IPCC (2007) Climate change 2007: The physical science basis. Cambridge University press, Cambridge, United Kingdom and tively short period, and its beneficial effect on ROC would New York. be weakened after complete decomposition of the straw. [2] Schmidt, M.W.I., Torn, M.S., Abiven, S., Dittmar, T., Gug- In the present study, CPMI was observed to significantly genberger, G., and Janssens, I.A., et al. (2011) Persistence of increase under straw incorporation compared with other treat- soil organic matter as an ecosystem property. Natutre ments within one and a half years after rice straw returning. 478(7367), 49-56. The result is in good agreement with the results of previous [3] Powlson, D.S., Whitmore, A.P., and Goulding, K.W.T. (2011) studies [17; 42]. 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[31] Lefroy, R.D.B., and Blair, G.J. (1993) Changes in soil organic matter with cropping as measured by organic carbon fractions [14] Lu, F. (2014) How can straw incorporation management im- 13 pact on soil carbon storage? A meta-analysis Mitigation and and C natural istope abundance. Plant soil. 1(155-156), 399- Adaptation Strategies for Global Change. Mitig. Adapt. 402. Strateg. Glob. Change. 1573-1596. [32] Song, C., and Zhang, J. (2009) Effects of soil moisture, tem- [15] Hazarika1, S., Parkinson, R., Bol, R., Dixon, L., Russell, P., perature, and nitrogen fertilization on soil respiration and ni- and Donovan, S., et al. (2009) Effect of tillage system and trous oxide emission during maize growth period in northeast straw management on organic matter dynamics. Agron. Sus- China. ActaAgr. Scand. B-S. P. 59(2), 97-106. tain. Dev. 29 (4), 525-533. [33] Mu, Z.J., Kimura, S.D., Toma, Y., and Hatano, R. (2007) Ni- [16] Blair, N., Faulkner, R.D., Till, A.R., and Poulton, P.R. (2006) trous oxide fluxes from upland soils in central Hokkaido, Ja- Long-term management impactions on soil C, N and physical pan. J. Environ. Sci. 20(11), 1312-1322. fertility. PartI: broadbalk experiment. Soil Till. Res. 91, 30-38. [34] Paul, B.K., Lubbers, I.M., and van Groenigen, J.W. (2012) [17] Xu, M.G., Lou, Y.L., Sun, X.L., Wang, W., Baniyamuddin, Residue incorporation depth is a controlling factor of earth- and M., Zhao, K. (2011) Soil organic carbon active fractions worm-induced nitrous oxide emissions. Global Change Biol. as early indicators for total carbon change under straw incor- 18(3), 1141-1151. poration. Biol. Fert. Soils. 47(7), 745-752. [35] Zou, J.W., Huan, G.Y., Zong, L.G., Jiang, J.Y., Zheng, X.H., [18] Shadrack, B.D., Chen, Z., Lal, R., Zhang, H.L., Chen, F. and Wang, Y.S. (2003) Effects of water regime and straw ap- (2014) Changes in soil organic carbon and nitrogen as affected plication in paddy rice season on N2O emission from following by tillage and residue management under wheat-maize crop- wheat growing season. Sci. Agr. Sin. 36 (4), 409-414. ping system in the North China Plain. Soil Till. Res. 144, 110- 118. [36] Lu, F., Wang, X.K., Han, B., Ouyang, Z.Y., and Zheng, H. (2010) Straw return to rice paddy: Soil carbon sequestration [19] Li, S.K., Wang, K.R., Feng, J.K., Xie, R.Z., and Gao, S.J. and increased methane emission. Chin. J. Appl. Ecol. 21(1), (2006) Factors affecting seeding emergence in winter wheat 99-108. under different tillage patterns with maize stalk mulching returned to the field. Acta. Agron. Sin. 32(3), 463-465. [37] Wassmann, R., Neue, H.U., Lantin, R.S., Makarim, K., Chareonsilp, N., and Buendia, L.V., et al. (2000) Characteri- [20] Han, C.G., Qiu, B.T., Qian, H.B., Xue, A.H., and Yan, G.Z. zation of methane emissions from rice fields in Asia. II. Dif- (1992) Research and application of technique of burying straw ferences among irrigation, rainfed, and deepwater rice. Nutr. in ditches. Soils Fert. 6,5–8. Cycl. Agroecosys. 58(1), 13-22.

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[38] Ma, E.D., Ma, J., Xu, H., Cai, Z.C., and Yagi, K. (2010) Ef- fects of rice straw returning methods in wheat-growing season on CH4 emissions from following rice-growing season. Ecol. Environ. Sci. 19(3), 729-732.

[39] Zhang, G.B., Ma, E.D., Zhang, X.Y., Ma, J., Xu, H., Cai, and Z.C. (2009) Effects of rice straw incorporation and land management in winter on methane emission during rice-growing season. J. Agro-Environ. Sci. 28(12), 2501-2505. [40] Lal, R., Follett, F., Stewart, B.A., and Kimble J.M. (2007) Soil carbon sequestration to mitigate climate change and advance food security. Soil Sci. 172(12), 943-956. [41] Brar, B.S., Singh, K., Dheri, G.S., and Kumar, B. (2013) Car- bon sequestration and soil carbon pools in a rice–wheat cropping system: Effect of long-term use of inorganic fertilizers and organic manure Soil Till. Res. 128, 30-36. [42] Bhattacharyyaa, P., Roya, K.S., Neogia, S., Adhyaa, T.K., Raoa, K.S., and Mannab, M.C. (2012) Effects of rice straw and nitrogen fertilization on greenhouse gas emissions and carbon storage in tropical flooded soil planted with rice. Soil Till. Res. 124, 119-130.

Received: December 11, 2014 Revised: March 13, 2015 Accepted: June 02, 2015

CORRESPONDING AUTHOR

Xinmin Bian College of Agriculture Nanjing Agricultural University Nanjing, 210095 P.R. CHINA

Phone: (+86) 25 8439 5018 Fax: (+86) 25 8439 5018 E-mail: [email protected]

FEB/ Vol 24/ No 9/ 2015 – pages 2751 - 2762

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PHYSICAL CHARACTERISATION OF NATURAL ORGANIC MATTER AND DETERMINATION OF DISINFECTION BY-PRODUCT FORMATION POTENTIALS IN ISTANBUL SURFACE WATERS

Edip Avsar1,*, Ismail Toroz2 and Asude Hanedar3

1Bitlis Eren University, Engineering and Architecture Faculty, Environmental Engineering Dept, Rahva Campus, Bitlis- Turkey. 2Istanbul Technical University, Civil Engineering Faculty, Environmental Engineering Dept, Ayazaga Campus, Istanbul- Turkey. 3Namik Kemal University, Engineering Faculty, Environmental Engineering Dept, Tekirdag-Turkey

ABSTRACT 1. INTRODUCTION

Natural organic matter (NOM) is a complex mixture of Natural organic matter (NOM) originates from the phys- various organic molecules mainly originating from aquatic ical chemical and biological natural and anthropogenic ac- organisms, soil and terrestrial vegetation which enters sur- tivities in the water source and the watershed that surrounds face and ground waters. Through the different reaction re- the water source. Dissolved organic matter (DOM) can be activity of the NOM components with disinfectants, isola- defined operationally as the portion of the NOM which can tion and fractionation of NOM into more homogenous pass through the 0.45 µm membrane filter. components is the better way of determining the DBP for- DOM has several adverse effects on water sources mation potential of the waters. Within the aim and scope of such as; this paper, raw water samples were taken from Buyuk- cekmece (BC) and Omerli (OM) surface water sources  Some aesthetic problems including color, taste, odor; which supply drinking water to Istanbul, in eight different  DOM can transport in water via binding organic and months between February 2010 and February 2011. At first inorganic pollutants; water quality parameters were analysed. Then, the physical  Reacts with disinfectants and produces unexpected dis- structure of the waters’ dissolved organic matter (DOM) infection by-products (DBPs) content, seasonal variations of this structure, reactivity of  As a carbon source for microbial reactions causing ac- DOM by means of DBP production and the effects of the cumulation in the water body, seasonal variations on this reactivity were investigated. In  Contributes to the formation of photochemical pro- terms of specific reactivity of the OM and BC physical cesses. fractions, <1kDa fraction was determined as the most reac- Through these adverse effects, the chemical structure tive fraction for STHMFP and SHAAFP per mg DOM. and physical properties of DOM should be investigated in >5kDa fraction was also determined as the most reactive terms of improvement of drinking water treatment quality. part of the DOM in terms of SAOXFP for both waters. However, these structures and properties are unstable and Both of the waters SUVA values were increased due to the vary depending on the water supply location, surrounding increase of DOM molecular weight but there was no simi- activities and seasons. For this reason, in order to improve lar trend in the formation of by-products. The main reason the quality of drinking water treatment, it is necessary to know for this situation is the lower SUVA and DOM values of the DOM’s composition and seasonal changes on DBPs for- the waters investigated. In this context, it can be said that mation reactivity [1-4]. OM and BC waters have special characteristics. For the better explaining DOM’s complex structure and investigating its behaviour in terms of water treatment, fractionation of DOM is one of the useful tool. DOM is the complex mixture of macro molecular humic matter, low KEYWORDS: Natural organic matter (NOM), physical characterisation, chlorination, disinfection by-products (DBPs) molecular weight hydrophilic acids, proteins, lipids, carboxylic acids, amino acids, carbohydrates and hydrocarbons. The importance of DOM has been increased by means of drinking water treatment while discovering the chlorine organic substances interactions can produce potential car- * Corresponding author cinogenic compounds such as THMs [5-11].

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DOM include precursor compounds in terms of DBP 2.2 DOM Isolation and Fractionation Procedure formation and can be fractionated as hydrophilic (HPI) and Isolation and fractionation procedure was applied ac- hydrophobic (HPO) fractions. HPI fractions include humic cording to Avsar et al. [21]. Samples filtered from 0.45 and acids; HPI fractions include low molecular weight carbo- 0.22 µm pore sized membrane filters respectively for the hydrates, proteins and amino acid. Humic matter consti- pretreatment. Then DOMs were fractionated to molecular tutes approximately 40-80% of the DOM, including both weight bigger than 5 kDa (>5kDa), molecular weight be- humic and fulvic acids and this site is the most important tween 5kDa and 1kDa (5-1kDa) and molecular weight part of DOM by means of DBPs formation. DOMs can be smaller than 1 kDa (<1kDa), fractions by means of Pall also fractionated by the use of UF membranes to molecular Omega PES (Polyethersulphone) 5kDa (product code: size fractions. DOM’s removal efficiency by means of a con- OM005076, 76 mm) and 1kDa (product code: OM001076, ventional treatment plant consisting of coagulation, sedi- 76mm) ultrafiltration membranes. mentation, and filtration is limited. Therefore, enhanced co- Before the usage of membranes, cleaning procedure agulation, granular activated carbon, membrane filtration occurred. Membranes were washed with 0.1 N NaOH for systems or advance oxidation processes are required in terms 1 hour then washed with distilled water for 1 hour. Then of effective DOM removal according to DOM’s specific the first 5kDa membrane was placed next to the Amicon molecular weight, size and fractions [2, 11-13]. 8400 stirred cell and the sample passed through the mem- UF is an appropriate method and has the lowest price for brane by means of ultrapure nitrogen gas pressure. DOM the determination of molecular size distribution of NOM. bigger than 5kDa size were collected on the membrane sur- Different molecular weight cutoffs, such as 500; 1,000; face. This fraction was dissolved by means of flushing 3,000; 10,000; 30,000 and 100,000 Daltons, are used for ul- membrane 3 sample volumes of distilled water. Filtrate trafiltration and fractionation of NOMs. Many researchers was passed through 1kDa membrane. DOM between 5kDa have used the technique in their different studies [14-17]. As and 1kDa size were collected on the membrane surface. a result, they have seen that the molecular weight of fulvic This fraction was dissolved by means of flushing mem- acid rarely surpasses 10 kDa. The major part of fulvic acid brane 3 sample volumes of distilled water too. Filtrate of fraction is smaller than 1 kDa or between 5 kDa and 10 kDa the 1kDa membrane contained DOMs smaller than 1kDa. [18]. In addition, humic acid fraction is monitored between General process diagramme is given in Figure 1. 10 kDa and 300 kDa [19]. The aim of this paper is to investigate the DOMs isolated from the OM and BC Lakes (at Istanbul/Turkey) molecular size distribution, the effects of seasonal changes on this distribution, and to determine the relationship between DOM fractions and THMs, HAAs and AOX formation potentials. According to Wei et al. [4], the chemical and physical properties of DOM are of the greatest significance in the water treatment process selection and understanding these properties is the key to better drinking water treatment.

2. MATERIAL AND METHODS

2.1 Water Quality Istanbul is the most populated metropolitan city, located in north-west Turkey with a population of approximately 13,200,000. Approximately 2,200,000 m3 water is treated by the Istanbul Metropolitan Municipality every day. The OM and BC Water Dams are the two big surface water sources of Istanbul providing approximately 55% of FIGURE 1 - Physical Fractionation Process Diagramme the city’s drinking water requirements. In this study, water samples were taken from OM and BC surface waters in After fractionation DOC and UV measurements were eight different months by seasonal basis between February conducted in terms of characterisation of phases. DOC con- 2010 and February 2011. In terms of water quality; pH, centrations were measured according to Standard Methods chloride, bromide, sulphate, hardness, alkalinity, conduc- 5310 D [20]. Measurements were conducted using TOC An- tivity, salinity, total dissolved solid (TDS), dissolved or- alyser (Shimadzu TOC Vcph). According to DOC results, ganic carbon (DOC) and UV254 parameters were monitored organic matter mass balances were calculated for determin- during the sampling period according to Standard Methods ing the DOM recoveries of all water samples. Average DOM [20]. recovery was 101.3% for OM and 104.6 for BC. According

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to Kitis et al. [22], the positive errors in the recovery were parameter concentrations (except UV254 and SUVA254) indicated as analytical errors, including the very low-level than OM Raw water. This difference is the result of sea DOC measurements required for some fractions. water intrusion from the Marmara Sea into BC. However, UV254 results indicate that the aromaticity of DOM is 2.3 Chlorination Procedure higher than BC. This result differs from previous studies Chlorination procedure was applied according to [23, 29]. For OM, the aromaticity of the water has in- Standard Methods 5710 B [20]. 5 mg/mL NaOCl dosing creased year by year. But in terms of BC aromaticity in- solution was prepared for chlorination procedure. Before creased at first time then decreased and in due course OM chlorination all of the samples were maintained to pH=7 by UV254 value exceeded the BC value. The DOC, UV and means of 6N and 1N NaOH solutions. After that all of the SUVA results for the same water sources from earlier stud- fractions were chlorinated to six times their DOC concen- ies are given in Table 2. trations by means of the dosing solution. After chlorination, all of the samples were placed in the incubator and TABLE 2 - Water quality characteristics of two surface water sources from earlier studies (average values for the sampling period) stored in dark conditions at 25 0C. Literature 2.4 DBPs Measurement Procedure Water Ates et al. (2007) [29] Uyak et al. (2008) [23] Source DOC* UV254 SUVA DOC* UV254 SUVA After a seven day reaction period, samples were taken OM 3.90 0.066 1.732 4.08 0.072 1.785 from incubator, dechlorinisated with 0.1 mL sodium sul- BC 3.90 0.075 1.95 4.55 0.100 2.33 phide solution (100 g/L) and then THMFP and HAA9FP *DOC measurements were conducted according to SM 5310B measurements were conducted via GC-µECD (Agilent 6890) equipped with DB 1 (30m x 0.32mm I.D.x 1 μm, It was noted that DOC levels of OM had not varied for J&W Science) column according to Uyak et al. [23], Bay- high intervals but the UV254 and SUVA254 values of OM tak et al. [24] and Avsar [25]. The procedure is based on increased year by year. For BC there was no regular trend EPA 551.1 [26] for THM4 and EPA 552.3 [27] for HAA9. by means of DOC and UV. This varied characteristic of BC Sample injection was carried on in split/splitless mode. He- is important by means of water treatment. According to lium was used as carrier and nitrogen as make up gas. Min- these results, DBPFP of OM was expected to be higher imum reporting levels (MRLs) are 0.1μg/L for THMs and than BC, which differs from the previous studies [23, 29]. HAAs. AOXFP measurement was conducted according to Physical fraction distributions of OM and BC waters ISO 9562:2004 [28] and Avsar [23] via Behr Cl10 AOX are given in Figure 2a and 2b. The total DOC distribution Analyser. Oxygen and Argon gases were used as process of OM water varied between 2.109 and 2.672 mg/L and for gases and the measurement interval of the method varied BC water it varied between 2.126 and 3.250 mg/L. <1kDa between 10-300 μg pure Cl-. fraction was determined as the dominant phase for both of the waters in the sampling period.

3. RESULTS AND DISCUSSION In terms of sampling period average values; <1kDa fraction is approximately 47% of DOC in OM water. When Average water quality characteristics of the two sur- the other physical fractions are analysed, it can be seen that face waters for the sampling period are given in Table 1. 5-1kDa fraction (approximately 33%) is more dominant with respect to >5kDa fraction (approximately 20%) and TABLE 1 - Water quality characteristics of two surface water sources that both fractions exhibit instability characteristics in the (average values for the sampling period) monitoring period.

Parameters Unit OM BC In BC water, similar to OM water, <1kDa fraction is pH - 7.73 8.23 the dominant fraction (approximately 51%) of the mass Cl- mg/L 18.2 37 distribution and varies seasonally. In addition, 5-1kDa Br- mg/L 0.04 0.10 2- fraction (approximately 24%) is more dominant with re- SO4 mg/L 22 55 Hardness mgCaCO3/L 98 200 spect to >5kDa fraction (approximately 22%) and both the TKN-N mgN/L 0.7 0.7 fractions varied in the monitoring period. As a result, the NH3-N mgN/L 0.14 0.24 seasonal changes in the fractionation occasionally caused Alkalinity mgCaCO3/L 78 161 changes in the dominant physical organic matter fraction. Conductivity µS/cm 238 505 % 0.011 0.023 Salinity UV254 results of OM and BC waters physical fractions TDS mg/L 116 249 DOC mg/L 3.595 3.765 are given in Figure 2c and 2d. When the effects of fractions -1 UV254 cm 0.093 0.083 in BC and OM waters are examined in terms of UV254 ab- SUVA254 L/mg.m 2.587 2.205 sorbance, it was seen that the effects have shown an alteration in the monitoring period and <1kDa fraction had the According to Table 1, BC Raw water pH, chloride, supreme effect on UV254 absorbance. The average effects bromide, sulphate, hardness, alkalinity, conductivity, salin- of <1kDa fraction in terms of UV254 are 69.1% in OM wa- ity and total dissolved solid (TDS) had higher water quality ter and 69.9% in BC water. As a consequence, the main

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a b

c d

e f OM BC

FIGURE 2 - OM and BC waters physical fractions DOC (a-b), UV254 (c-d) and SUVA254 (e-f) distribution

effect on aromatic structure originated from micro mole- was approximately 20% and the effect of >5kDa was ap- cules. When we look at the other phases in OM water, the proximately 10.2%. In terms of total UV254 value, the results effect of >5kDa fraction to the absorbance value (16%) is show that absorbance values decrease generally with precip- bigger than 5-1kDa fraction effect (14.9%). In comparison itation decreasing and rises in temperature had little effect on to DOC in OM water, despite DOC mass in >5kDa fraction decreasing in organic matter in several months (February- being very low, obtained absorbance value in 254 nm for March 2010 and May-June 2010 interval). the >5kDa fraction is higher than 5-1kDa fraction because Because varying absorbance values in the water of the humic structure. In addition, total UV254 value had a sources (BC and OM) show the changing of water reactiv- variable increase/decrease tendency. It was thought that the ity, seasonal water analysis becomes a very important sub- rising of UV254 values in the spring were caused by in- ject in terms of drinking water obtained from these water creases in water-borne organic matter due to increases in sources. temperature, precipitation and organic matter in the soil. SUVA254 concentrations of OM and BC waters physi- Especially precipitation and soil originated organic mat- cal fractions are given in Figure 2e and 2f. When the OM ter had the most effect on decreasing and increasing organic fractions sampling period averages are analysed in terms of matter. The reactivity of water needs to be monitored sea- SUVA parameters, the maximum effect on SUVA coming sonally because of varying precipitation and temperature. In from >5kDa fraction is 41.5%. In addition, the effect of BC water, as distinct from OM, 5-1kDa fraction had a <1kDa fraction on SUVA is 31.4% and the effect of 5- stronger effect on UV254 absorbance values in comparison 1kDa fraction is 27.1% during the monitoring period. Ac- with >5kDa fraction. The effect of 5-1kDa fraction on UV254 cording to literature, increases in SUVA with molecular

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weight indicate that molecular size is related to aromatic fraction and STHMFP increases in parallel with decreasing structure [22]. In terms of the SUVA tendency in the mon- molecular size. The other fractions respectively 5-1kDa itoring period the situation in OM water is unsuited to lit- (approximately OM: 29.9%; BC: 27.9%) and >5kDa (ap- erature, SUVA values of 5-1kDa fraction is smaller than proximately OM: 19.3%; BC: 16.2%) had effects on the SUVA values of <1kDa. During the monitoring period to- STHMFP. In terms of total reactivity of fractions, maxi- tal SUVA variation has parallels with variation of SUVA mum reactivity in OM and BC respectively were measured values effected by >5kDa fraction. as 985 µg/mg in February 2010 and 434 µg/mg in Decem- In BC water sampling period averages, SUVA values ber. The minimum reactivity in OM and BC respectively increase with molecular size as stated in literature. The were measured as 200 µg/mg in August and 243 µg/mg in maximum effect on SUVA is 36% originated by >5kDa, March. approximately 32.6% of effect is coming from 5-1kDa Specific HAA9FP of raw waters and fractions are fraction and finally 31.4% of SUVA is coming from <1kDa given in Figure 4a and 4b. Physical fractions and percent- fraction. Consequently, during the monitoring period the age SHAA9FP distributions are given in Figure 4c and 4d. average values indicate that increases in molecular size and In terms of raw waters, the highest results were obtained in aromatic structure tend to raise SUVA values. summer for OM and in spring and summer for BC. For raw Specific THMFP of raw waters and fractions were cal- waters and fractions thrichloroacetic acid (TCAA) and di- culated by means of dividing raw water or fraction THMFP chloroacetic acid (DCAA) were the dominant species. to its own SUVA254 value and are given in Figure 3a and About fractions, similar to the STHMOP, <1kDa were 3b. DOM fractions and percentage STHMFP distributions the most active site of the DOM and had higher SHAA9FP are given in figure 3c and 3d. In terms of raw waters, high- (sampling period average is 228 µg/mg) than >5kDa est results were obtained in the winter period. (181 µg/mg) and 5-1kDa (145 µg/mg) phases for OM. For About fractions, <1kDa was the most active site of BC, <1kDa fraction was the most active site of DOM and had DOM and had higher STHMFP (sampling period average higher SHAA9FP (151 µg/mg) than 5-1kDa (116 µg/mg) and is 222.46 for OM and 188.19 µg/mg for BC) than 5-1kDa >5kDa (92 µg/mg) phases. According to our results, (OM: 156.78 µg/mg and BC: 107.04 µg/mg) and >5kDa SHAA9FP of both waters and fractions was higher than (OM: 81.38 µg/mg and BC: 56.30 µg/mg) phases. Chloro- their STHMFP. form (TCM) and bromodichloromethane (BDCM) were In terms of SHAA9FP, similar to the STHMFP, the most the dominant species of THMs. effective fraction is <1kDa (approximately OM 45.5%; BC: In both waters the main effect on STHMFP (approxi- 44.3%). The effect of >5kDa fraction is 30% and the ef- mately OM: 50.7%; BC: 55.9%) originated from <1kDa fect of 5-1kDa fraction is 24.5% on SHAA9FP. As for the

a c

b d

FIGURE 3 - OM and BC waters and fractions STHMFP results (a-b) and distributions (c-d)

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c a

b d

FIGURE 4 - OM and BC waters and fractions SHAA9FP results (a-b) and distributions (c-d)

a c

b d

FIGURE 5 - OM and BC waters and fractions SAOXFP results (a-b) and distributions (c-d)

other phase in BC water, active fractions were respec- tion reactivity. In terms of total reactivity, SHAA9FP was tively 5-1kDa and >5kDa fractions with 29.8% and 25.9% approximately 1.2 times higher than STHMFP. of SHAA9FP reactivity. In comparing THM and HAA reactivities in BC water, In comparison with the STHMFP results in OM, rising in reactivity with decreasing in molecular size has SHAA9FP for 5-1kDa fraction was lower than >5kDa frac- shown in terms of both parameters. For both THM and

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HAAs chlorinated species were more prevalent than bro- of specific reactivity of the BC physical fractions, <1kDa minated species because Turkish water sources have low fraction was determined as the most reactive fraction for bromine levels as mentioned by Ates et al. [29]. STHMFP and SHAAFP. >5kDa fraction was also determined as the most reactive part of the DOM in terms of Specific AOXFP of raw waters and fractions are given SAOXFP for both waters. in Figure 5a and 5b. DOM fractions percentage SAOXFP distributions are given in Figure 5c and 5d. As for OM and Both of the waters SUVA values were increased due BC fractions, different from SHAA9FP and STHMOP, to the increase of DOM molecular weight but there was no >5kDa was the most active site for DOM (sampling period similar trend in the formation of by-products. The main average OM: 2947 µg/mg; BC: 3976 µg/mg) and had reason for this situation is the lower SUVA and DOM val- higher SAOXFP than 5-1kDa (sampling period average ues of the waters investigated. In this context, it can be said OM: 1836 µg/mg; BC: 2643 µg/mg) and <1kDa (sampling that OM and BC waters have special characteristics. period average OM: 823 µg/mg; BC: 828 µg/mg) fractions. The authors have declared no conflict of interest. The effects of >5kDa fractions of OM and BC waters were 52.6% and 53.4% respectively on SAOXFP. Alt- hough the dominant fraction was <1kDa fraction in terms of organic matter, >5kDa fraction was the most active frac- REFERENCES tion in terms of reactivity. Specific reactivity in both waters increases with molecular weight. According to our results, [1] Leenheer, J.A. and Croue, J.P. (2003). Characterizing aquatic 5-1kDa fraction was the second fraction for effect on SAO- dissolved organic matter Environmental Science & Technol- XFP in both waters (average OM: 32.8%; BC: 35.5). ogy, 37, 1, 18A-26A. <1kDa fractions of both waters had minimum effects on [2] Kanokkantapong, V., Marhaba, T.F., Pavasant, P. and SAOXFP (OM: 14.7%; BC: 11.1%). Pinyaphol, B.P. (2006). Characterization of haloacetic acid precursors in source water, Journal of Environmental Man- There were no strong correlations between SUVA254 agement, 80, 214-221. and STHMFP formations and SUVA and SHAA9FP were [3] Lu, J., Zhang, T., Ma, J. and Chen, Z. (2009). Evaluation of found in both waters. Also, there was no strong relationship disinfection by-products formation during chlorination and between THMFP and HAAFP either. This result is similar chloramination of dissolved natural organic matter fractions with literature [29, 30]. isolated from a filtered river water, Journal of Hazardous Ma- terials, 162, 140–145.

[4] Wei, Q., Feng, C. H., Wang, D., Shi, B., Zhang, L. and Wei, 4. CONCLUSION Q., Tang, H. (2008). Seasonal variations of chemical and physical characteristics of dissolved organic matter and trihalomethane precursors in a reservoir: a case study, Journal of Haz- Physical fraction distribution and DBP analysis results ardous Materials, 150, 257–264. indicate that; <1kDa fraction is the dominant fraction for [5] Bellar, T.A., Lichtenberg, J.J. and Kroner, R.C. (1974). The both OM and BC waters in terms of DOM mass basis. In occurrence of organohalides in chlorinated drinking waters. terms of physical fractions contribution to the UV254 ab- Journal AWWA 66(12), 703-706. sorbance, it was determined that the most important contri- [6] Rook, J.J. (1974) Formation of haloforms during chlorination bution came from <1kDa fraction. This result reflected that of natural water. Water Treatment and Examination, 23(2), an important part of the aromaticity was constituted by mi- 234-243. cro molecules. DBPFP analysis of the chlorinated physical [7] Chen, C., Zhanga, X., Zhu, L., Liua, J., He, W. and Han, H., fractions showed that according to measured by-products, (2008). Disinfection by-products and their precursors in a wa- the physical fraction responsible for the formation of this ter treatment plant in North China: Seasonal changes and frac- product was changed. In terms of OM physical fractions; tion analysis, Science of The Total Environment, 397, 140–147 <1kDa fraction is responsible from the highest THMFP, [8] Colman, J., Rice, G.E., Wright, J.M., Hunter, H.S., Teuschler, HAAFP and AOXFP results, In terms of BC physical frac- L.K., Lipscomb, J.C., Hertzberg, R.C., Simmons, J.E., Fran- tions; <1kDa fraction is responsible from the highest sen, M., Osier, M. and Narotsky, M.G., (2011). Identification of developmentally toxic drinking water disinfection byprod- THMFP, HAAFP and AOXFP results. ucts and evaluation of data relevant to mode of action, Toxi- cology and Applied Pharmacology, 254, 100-126. According to the sum of physical fractions DBPFP results, dominant THM species is the TCM for both of the wa- [9] Hebert, A., Forestier, D., Lenes, D., Benanou, D., Jacob, S., ters and the brominated THM species percentage is higher Arfi, C., Lambolez, L. and Levi, Y. (2010). Innovative method for prioritizing emerging disinfection by-products (DBPs) in in BC than OM. Dominant HAA species is the MCAA for drinking water on the basis of their potential impact on public OM and DCAA for BC and looks like the THM results bro- health, Water Research, 44, 3147-3165. minated HAA species percentage is higher in BC than OM. [10] Kitis, M. (2001). Probing chlorine reactivity of dissolved or-

ganic matter for disinfection by-product (DBP) formation: Re- In terms of specific reactivity of the OM physical frac- lation with specific ultraviolet absorbance (SUVA) and devel- tions, <1kDa fraction was determined as the most reactive opment of the DBP reactivity profile, PhD Thesis, The Grad- fraction for STHMFP and SHAAFP per mg DOM. In terms uate Scholl of Clemson University, Clemson, South Carolina.

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[11] Zhao, Z., Gu, J., Fan, X. and Li, H. (2006). Molecular size dis- with Electron-Capture Detection Revision 1.0 National Expo- tribution of dissolved organic matter in water of the Pearl sure Research Laboratory Office of Research and Develop- River and trihalomethane formation characteristics with chlo- ment U.S. Environmental Protection Agency Cincinnati, Ohio rine and chlorine dioxide treatments, Journal of Hazardous 45268. Materials, 134, 60–66. [27] Domino M. M., Pepich B.V., Munch D.J., Fair P.S. and Xie Y. [12] Kim, H.C., and Yu, M.J. (2007). Characterization of aquatic (2003). Method 552.3 Determination of Haloacetic Acids and humic substances to DBP’s formation in advanced treatment Dalapon in Drinking Water By Liquid-Liquid Microextrac- processes for conventionally treated water, Journal of Hazard- tion, Derivatization, and Gas Chromatography with Electron ous Materials, 143, 486-493. Capture Detection Revision 1.0 Technical Support Center Of- fice of Ground Water and Drinking Water U.S. Environmental [13] Leenheer, J. A. (2009). Systematic Approaches to Comprehen- Protection Agency Cincinnati, Ohio 45268. sive Analyses of Natural Organic Matter, Annals of Environ- mental Science, 3, 1-130 [28] ISO 9562: (2004). Water quality — Determination of adsorb- able organically bound halogens (AOX). [14] Newcombe, G., Drikas, M., and Hayes, R. (1997). Influence of characterizes natural organic material on activated carbon [29] Ates, N., Kaplan, S., Sahinkaya, E., Yetis, U., Dilek, F.B. and adsorption: II effect on pore volume distribution an adsorption Kitis, M. (2007). Occurrence of disinfection by-products in of 2-Methylisoborneol, Water Research, 31, (5), 1065-1073. low DOC surface waters in Turkey, Journal of Hazardous Ma- terials, 142, 526–534. [15] Koechling, M.T. (1998). Assessment and modeling of chlorine reactions with natural organic matter: impact of source water [30] Aydin, E., Yaman, B.F., Ates-Genceli, E., Topuz, E., Erdim, quality and reaction condutions, PhD Thesis, University of E., Gurel, M., Ipek, M. and Pehlivanoglu-Mantas, E. (2012). Cincinnati, Ohio. Occurrence of THM and NDMA precursors in a watershed: Effect of seasons and anthropogenic pollution, Journal of Haz- [16] Cai, Y. (1999). Size distribution measurements of dissolved ardous Materials, 221-222, 86-91. organic carbon in natural waters using ultrafiltration technique, Water Research, 33, 13, 3056-3060. [17] Lin, C.W., Huang, F.J., and Hao, O.J., (1999). Ultrafiltration processes for removing humic substances: effect of molecular weight fractions and PAC treatment, Water Research, 31, (5), 1252-1264.

[18] Rasid, M.A. (1985). Geochemistry of Marine Humic Com- pounds, Springer Verlag, New York, NY, USA.

[19] Alberts. J.J. and Schindler, J.E. (1976). Elemental infrared spectrophotometeric and electron spin resonance investigation of non-chemically isolated humic material, Geochimica et. Cosmochimica Acta, 40: 369. [20] American Public Health Association (APHA), American Wa- ter Works Association (AWWA), and Water Environment Federation (WEF). 1998. Standard Methods for the Examina- tion of Water and Wastewater 20th Edition. United Book Press, Inc., Baltimore, Maryland. [21] Avsar, E., Toroz, I., Hanedar, A., and Yılmaz, M. (2014) Chemical Characterization of Natural Organic Matter and De- termination of Disinfection By-Product Formation Potentials in Surface Waters of Istanbul (Omerli and Buyukcekmece Wa- ter Dam), Turkey, Fresenius Environmental Bulletin, 23 (2a), 494 – 501. [22] Kitiş, M, Karanfil, T., Wigton, A. and Kilduff, J.E. (2002). Probing reactivity of dissolved organic matter for disinfection Received: December 17, 2014 by-product formation using XAD-8 resin adsorption and ultra- Accepted: March 09, 2015 filtration fraction, Water Research, 36, 3834-3848. [23] Uyak, V., Ozdemir, K. and Toroz, I. (2008). Seasonal variations of disinfection by-product precursors profile and their re- CORRESPONDING AUTHOR moval through surface water treatment plants, Science of the Total Environment, 390, 417-424. Edip Avsar [24] Baytak, D., Sofuoglu, A., Inal, F. and Sofuoglu, C.S. (2008). Seasonal variation in drinking water concentrations of disin- Bitlis Eren University fection by-products in Izmir and associated human health Engineering and Architecture Faculty risks, Science of The Total Environment, 407, 286-296. Environmental Engineering Department [25] Avsar, E. (2013) Physical and chemical characterization of Rahva Campus natural organic matter in Istanbul surface waters and determi- Bitlis nation of disinfection by product formation potentials, PhD TURKEY Thesis, Istanbul Technical University Graduate School of Sci- ence Engineering and Technology (In Turkish) Phone/Fax: +904342220000-3201/+904342229145 [26] Munch D.J. and Hautman D.P. (1995). Method 551.1Determi- nation Of Chlorination Disinfection Byproducts, Chlorinated E-mail: [email protected] Solvents, And Halogenated Pesticides/Herbicides in Drinking Water By Liquid-Liquid Extraction And Gas Chromatography FEB/ Vol 24/ No 9/ 2015 – pages 2763 - 2770

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EFFECTS OF COPPER AND LEAD ON SOME HEMATOLOGICAL PARAMETERS OF Oreochromis niloticus

Nuray Çiftçi1,*, Fahri Karayakar2, Özcan Ay2, Bedii Cicik2 and Cahit Erdem3

1 University of Mersin, Vocational School of Silifke, Aquaculture Programme, Yenimahalle, Kayraktepe Mevkii, Silifke, Mersin, Turkey 2 University of Mersin, Faculty of Aquaculture, Yenişehir Kampüsü, C Blok, Kat 2, 33169 Mersin, Turkey 3 University of Çukurova, Faculty of Art and Sciences, Biology Department, 01330 Balcalı, Adana, Turkey

ABSTRACT elevated aquatic pollution results in mortality in flora and fauna which, in turn, cause permanent changes in structural Effects of sub-lethal concentrations of copper and lead components of the environment [4]. Prolonged exposure to on hematocrit and mean cell volume (MCV) levels and on low levels of metals results in tissue accumulation in erythrocyte numbers, erythrocyte and erythrocyte nucleus aquatic organisms, and is transferred to higher trophic lev- areas were determined after exposing Oreochromis nilot- els through the food chain [5]. icus to 4 ppm Cu and 0.2 ppm Pb over 7, 15 and 30 days. Copper is present as a structural component of living Micro-hematocrit methods were adopted in determin- organisms at low concentrations. It acts as a prosthetic ing hematocrit levels and microscopic methods were used group in various proteins and as a cofactor in enzymes, and to determine MCV, erythrocyte numbers, erythrocyte and also has functions in the formation of connection and bone erythrocyte nucleus areas. tissues, impulse transmission, and the antioxidant defence Hematocrit and MCV levels and erythrocyte and nu- system. Erythrocuprein, a copper-containing metallothi- cleus areas increased significantly (P<0.05), whereas the onein, is required for hemoglobin synthesis [6]. erythrocyte numbers decreased under the effect of both Copper was used in making various kitchen and orna- metals at the exposure periods tested, except at 30 days of mental materials over long periods [7]. Today, it is widely copper exposure. used in electric, electronic, construction and chemistry in- There was an increase in MCV levels under the effect dustries, algaecide, detergent, dye, insecticide and artificial of lead and an increase in erythrocyte and nucleus areas agricultural fertilizer production and in strengthening of under the effect of both metals with increasing exposure cement and reinforced concrete beams. Both production periods. Prolonged exposure periods decreased other he- and usage of wastes of these activities constitute the main matological parameters studied. source of copper in aquatic environments. Hematological parameters studied were higher in fish Lead is a toxic heavy metal which has no biological exposed to lead compared to copper during the experi- function in animals. It is used in accumulator production, ments, except the erythrocyte area. isolation of underground cables, and in steel constructions as It was concluded that both copper and lead caused sig- a corrosion preventer; its tetraethyl and tetramethyl com- nificant changes in hematological parameters of O. nilot- plexes are used in fuels as octane adjuster, and in nuclear icus which, in turn, affected the physiological conditions of energy plants against radioactive emittance [8]. Studies car- fish. ried out with various fish species showed that lead has a neu- rotoxic effect, since it passes the blood-brain barrier, it causes changes in hematologic parameters, and causes struc- KEYWORDS: tural deformations in various tissues including bones [9, 10]. O. niloticus, copper, lead, hematological parameters Heavy metals up-taken through gills and the digestive system are firstly bound to carrier proteins in the blood 1. INTRODUCTION stream; hence, their first effects can be observed on hematological parameters. Hematological parameters, such as Heavy metals are natural constituents of the earth crust. hematocrit and hemoglobin levels and erythrocyte num- Their levels in aquatic environments, however, increase bers, not only reflect the oxygen carrying capacity to vari- mainly due to increasing agricultural and industrial activities ous tissues but also allow to determine the changes in organ causing environmental and health problems [1-3]. Acute and organ systems [11].

Studies carried out with various fish species have * Corresponding author shown that heavy metals can cause polycythemia, anemia,

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morphological changes in erythrocytes, and inclusions in Three fish were removed from each aquarium at the nucleus or cytoplasm [12-14]. end of experimental periods and were anaesthetized using Experimental animal O. niloticus is an economically anesthetic ethylene glycol monophenyl ether (= phenoxi- important fish species that is widely consumed in the equa- ethanol, C8H10O2). After washing the fish with tap water to torial region as a protein source. The reproduction of the remove metal residues, they were dried with Whatman dry- species is easy, it is resistant to diseases and is used for ing paper and prepared for sampling. plantation of inland water resources. Predetermination of Blood samples to be used in determining the blood pa- heavy metal toxicity, in aquatic organisms, especially in rameters were obtained by cutting the caudal peduncle ver- economically important species, is important as far as envi- tically. Among the hematological parameters to be deter- ronmental and human health is concerned. Hence, the pre- mined, heparinized capillary hematocrit pipettes were used sent study was undertaken to find out the effects of 4.0 ppm in determining hematocrit levels according to Wedemayer Cu and 0.2 ppm Pb, which are 10% of the LC50 level of each and Yasutake [15]; erythrocyte numbers were determined metal, on hematocrit and MCV levels, erythrocyte number, using EDTA (ethylene-diamine tetraacetic acid)-contain- erythrocyte and nucleus areas of O. niloticus after exposing ing tubes, and spread slides were used to determine eryth- the animals to these metals over 7, 15 and 30 days. rocyte and erythrocyte areas. Erythrocyte numbers of blood samples were determined by counting on thoma slides using a light micro- 2. MATERIALS AND METHODS scope (Leica, Model CME) [15]. Erythrocyte numbers and MCV levels were determined according to the formulas Experimental material O. niloticus was obtained from given below: the implementation unit of Aquaculture Faculty of Mersin University. The total length and weight of the fish used in experiments were 18.58±0.91 cm and 110.27±3.29 g, re- 100 spectively. Fish were placed in 6 glass aquaria, 40x100x 40 cm in size, in the basic sciences laboratory under 24± 1 °C constant temperature conditions, and 12-h light and 10 12-h dark illumination period was applied. Experiments were started after two weeks of adaptation period. Four glass aquaria of the same size were used in the Erythrocyte and nucleus areas were determined by mor- experiments. Experimental solutions were prepared from phometric measurements of dyed spread slides under microscope. Giemsa method was adopted in preparing dyed water-soluble compounds of copper (CuSO4) and lead spread slides [16]. Erythrocyte and nucleus areas were cal- ((Pb(NO3)2). Tri-sodium citrate was used in the preparation of stock solutions in order to prevent precipitation of metal culated according to the following formulas after measuring salts. 120 L of 4.0 ppm Cu and 0.2 ppm Pb, which are the the length and width of at least 150 erythrocyte and their nucleus under a microscope (Nicon, H550-L) [17]. 10% of LC50 levels of each metal, were added in the first two aquaria. The same amount of metal-free tap water was added to the third and the fourth aquaria, and were used as controls. 2 Experiments were run in triplicate, being 3 fish in each replicate. Hence, 9 fish were placed in each aquarium taking the 1, 7 and 15 days of experimental periods into account. Some physical and chemical characteristics of ex- 2 perimental aquaria are given in Table 1. Data were analyzed by a series of variance analysis and TABLE 1 - Some physical and chemical parameters of experimental water. Student Newman Keuls test (SNK) using SPSS statistical package. Arcsine transformation was applied on hemato- Temperature (°C) 24.0 ± 1.0 crit levels, since they were expressed in percentages. pH 8.62 ± 0.16 Dissolved oxygen (mg L-1) 5.29 ± 0.7 -1 Total Hardness (mg L CaCO3) 227 ± 0.48 -1 Alkalinity (mg L CaCO3) 332 ± 0.50 3. RESULTS

Fish were fed once a day with ready fish feed (Pınar, No mortality was observed during the 30 days of ex- Bream feed, Pellet No:2) at amounts of 2% of total bio- posure to the metal concentrations tested. The same behav- mass. Central aeration system was used to aerate experi- ioral and morphological changes were observed at the be- mental tanks. Experimental waters were changed once in ginning of experiments as indicated by Cicik [18], except two days to prevent possible alterations in experimental so- for darkening of the skin in fish exposed to Cu continued lutions due to evaporation, precipitation and adsorption. throughout the experiments.

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Hematocrit levels increased compared to control in Toxicants, such as heavy metals, may directly cause fish exposed to metals up to 7 days of exposure which then structural changes, or they may cause stress which, in turn, decreased on the 30 days of exposure, compared with the results in behavioral changes in fish. Same behavioral 7th day (P<0.05) (Table 2). The decrease in hematocrit lev- changes were observed in O. niloticus under the effect of els was higher in fish exposed to Pb than to Cu (P<0.05). Cu [21] and Pb [22], at the beginning of experiments as Erythrocyte numbers decreased compared to control with mentioned in previous studies. increasing exposure periods under the effect of both metals Hematologic parameters, such as hematocrit and he- (P<0.05). The decrease in erythrocyte numbers was higher in moglobin levels, MCV, erythrocyte numbers, erythrocyte fish exposed to copper than to Pb (P<0.05) (Table 2). and erythrocyte nucleus areas, leucocyte and thrombocyte Exposure to copper increased MCV compared to con- numbers are normally kept at given levels by homeostatic trol, except on 30 days of exposure (P<0.05). There was mechanisms. They, however, instantly change under the also an exposure period-dependent increase in MCV in fish effect of chemical toxicants, infection or other stress fac- exposed to lead (P<0.05). Lead had a greater effect on tors [19, 23]. MCV, compared to copper (P<0.05) (Table 2). Studies carried out with various fish species, hypoxic Erythrocyte and erythrocyte nucleus areas increased conditions, and metal effects demonstrate increase of mu- significantly with increasing exposure periods under the ef- cus secretion which covers gill surface area and increase fect of both metals (P<0.05). Copper had a more pro- oxygen diffusion distance. The resulting inhaling difficul- nounced effect on erythrocyte and nucleus areas than lead ties increase adrenalin secretion and cause contraction in (P<0.05), except on day 30 (P>0.05) (Table 2). spleen and increase in erythropoiesis [12]. Sub-lethal concentrations of copper increase hematocrit and hemoglobin levels and erythrocyte numbers in Ichtalurus nebulosus 4. DISCUSSION AND CONCLUSION [24], Scyliorhinus canicula [25], Oreochromis mosambi- cus [26], Heteropneustes fossilis [27], Oncorhynchus Effects of heavy metals on fish mortality depend on the mykiss [28], Clarias gariepinus [29] and Prochilodus metal concentration, exposure period, environmental fac- scrofa [30]. This increase was reported to be a result of tors, species, developmental stage and sex. Mortality in- hemo-concentration due to osmoregulation failure. The increases rapidly over a given concentration [19, 20]. No crease in hematocrit levels, MCV, erythrocyte and erythro- mortality was observed in O. niloticus exposed to 4.0 ppm cyte nucleus areas in O. niloticus exposed to Cu and Pb Cu and 0.2 ppm Pb over 7, 15 and 30 days which might be might be either hemo-concentration or stimulation of due to tested concentrations of these metals not being lethal erythropoiesis by feedback mechanisms. to this species at exposure periods tested, or detoxification The loss of membrane-selective permeability under the mechanisms might be stimulated against metal toxicity. effect of metals results in hemo-dilution and osmotic hemo-

TABLE 2 - Effects of copper and lead on some hematological parameters of O. niloticus.

Exposure Period (Days) Metal 0 (Control) 7 15 30

X  Sx * X  Sx * X  Sx * X  Sx * Hematocrit Cu 33,52±0,31 a 38,94±0,29 b 35,97±0,30 c 24,73±0,38 d (%) Pb 31,25±0,31 a 41,27±0,29 b 40,69±0,29 b 39,52±0,30 c

Erythrocyte num- Cu 2,58±0,002 a 1,27±0,01 b 1,14±0,02 b 0,80±0,13 c bers (cell/mm3) Pb 2,60±0,05 a 1,86±0,03 b 1,60±0,03 c 1,31±0,05 d

MCV Cu 166,24±2,78 a 331,06±3,33 b 292,44±6,73 b 78,29±1,71 c (fl)** Pb 159,24±2,78 a 234,92±0,18 b 285,61±3,92 c 294,02±2,94 d

Erythrocyte Area Cu 0,87±0,01 a 1,13±0,015 b 1,45±0,015 c 1,45±0,007 c (µm3) Pb 0,90±0,009 a 0,94±0,009 b 1,23±0,015 c 1,68±0,003 d

Erythrocyte Nu- Cu 0,13±0,006 a 0,17±0,003 b 0,18±0,007 b 0,25±0,003 c cleus Area (µm3) Pb 0,15±0,007 a 0,20±0,006 b 0,23±0,006 c 0,26±0,006 d *SNK; Letters a, b, c and d show differences between exposure periods. Data shown with different letters are significant at the P<0.05 level. X  Sx : mean± standard error; **fl; femtoliter (10-15 L)

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lysis of erythrocytes [31]. Acute effect of 3.2 mg L-1 Cu [5] Prasuna Solomon, G., Pavani, V. and Nageswara, R.A. (2012) caused hemolysis and anemia in Clarias lazera [32]. Sub- Food Chain Bioaccumulation of Lead in Chrissia halyi (Fer- guson 1969) Using Cladophora as Feed. European Journal of lethal concentrations of Pb decreased erythrocyte numbers, Experimental Biology, 2(1):83-87. hemoglobin and hematocrit levels in O. mykiss [33], Bar- bus conchonius [34] and Oreochromis aureus [35], which [6] Cicik, B. (2003) Bakır + Çinko Etkileşiminin Sazan (Cyprinus carpio)’nın Karaciğer ve Kas Dokularındaki Metal Birikimi was related to hemolysis of erythrocytes under the effect of Üzerine Etkileri. Ekoloji Çevre Dergisi, 48, 32-36. metals [35]. The tested concentrations of Cu and Pb de- [7] Eisler, R. (2000) Handbook of Chemical Risk Assessment: creased erythrocyte numbers and hematocrit levels in O. Health Hazards to Humans, Plants, and Animals, Metals, Vol- niloticus at the exposure periods tested. This might be ei- ume 1., Lewis Publishers, Boca Raton London New York ther osmotic hemolysis depending upon deterioration of Washington, D.C., 95p. the erythrocyte membrane under the effect of metals, or [8] Sorensen, E. M. (1991) Metal Poisining in Fish, CRC Press, functional disturbances caused by accumulation of metals Boca Raton, FL., 243 pp. in hematopoietic tissues, together with other tissues. [9] Seymore, T. (1995) Manganese, Lead and Strontium Bioaccu- Uptake, tissue accumulation and excretion rates, bio- mulation in the Tissues of the Yellow Fish, Barbus marequen- logical functions and toxic effects of heavy metals differ sis from the Lower Olifants River, Eastern Transvaal Water, between aquatic organisms [19]. Acute effects of mercury South Africa, pp 241-244. increased hematocrit and hemoglobin levels and erythro- [10] Jezierska, B. and Witeska, M. (2001) Metal Toxicity to Fish. cyte numbers in Tinca tinca [36], whereas sub-lethal levels Wydawnictwo Akademii Podlaskiej, Siedlce, 318. of copper decreased erythrocyte numbers in Salmo gaird- [11] Witeska, M., Kondera, E. and Belniak, N. (2013) Hematolog- neri [37]. Lead caused more changes in blood parameters ical and Hematopoietic Changes Induced by Formaldehyde of O. niloticus than copper. Since copper is an essential el- and Malachite Green in Common Carp (Cyprinus carpio L.). ement, it might be less toxic than lead which has no bio- Zoology and Ecology, DOI: 10.1080/21658005.2013.821790. logical function, or lead might stimulate erythropoiesis by [12] Vosyliene, M. Z. (1999) The Effect of Heavy Metals on He- inhibiting ALA-D (delta-aminolevulinic acid dehydratase) matological Indices. Acta Zoologica Litvanica Hydrobiologia, enzyme activity which plays a role in heme synthesis. 9: 76-82. Witeska et al. [38] reported that lead caused morpholog- [13] Clauss, T. M., Dove, A. D. M. and Arnold, J. E., 2008. Hema- tologic Diorders of Fish. Vet. Clin. Exot. Anim., 11, 445-462. ical changes in erythrocyte nucleus, structural deformations and spreading in chromatin material. They concluded that [14] Orun, I. and Talas, Z.S. (2008) Antioxidative Role of Sodium +2 +3 lead might cause distortion in erythrocyte and erythrocyte Selenite against The Toxic Effect of Heavy Metals (Cd , Cr ) on some Biochemical and Hematological Parameters in Blood nucleus membrane permeability or RNA synthesis. of Rainbow Trout (Oncorhynchus mykiss Walbaum, 1792). In conclusion, hematocrit and MCV levels, erythrocyte Fresenius Environmental Bulletin, 17 (9A), 1242-1246. numbers, erythrocyte and erythrocyte nucleus areas [15] Wedemeyer, G.A. and Yasutake, W.T. (1977) Clinical Meth- changed significantly in O. niloticus exposed to 4 ppm Cu ods for The Assessment of The Effects of Environmental and 0.2 ppm Pb over 7, 15 and 30 days. These changes in Stress on Fish Health, United States Technical Papers. Fish Wild Services, 89: 1-18. blood parameters under the effect of Cu and Pb might be due to the influence of metals on osmoregulation, and on [16] Grimstone, A.V. and Skaer, R.J. (1972) A Guide Book to Mi- membrane permeability or stimulation of feedback mecha- croscopical Methods. Cambridge Uni. Press, 138 pp. nisms under the effect of metals. Hence, changes in hema- [17] Gregory, T. R. (16th October 2003) Fish Erythrocyte Sizes. tologic parameters under the effect of metals reflect the Animal Genome Size Database. Retrieval: http://www.ge- th physiological conditions of organisms. nomesize.com/cellsize/fish.htm [02 February 2009]. [18] Cicik, B., Ay, Ö. and Karayakar, F. (2004) Effects of Lead and The authors have declared no conflict of interest. Cadmium Interaction on the Metal Accumulation in Tissue and Organs of Nile Tilapia (Oreochromis niloticus). Bull. En- viron. Contam. Toxicol., 72 (1), 141-148. REFERENCES [19] Heath, A.G. (1995) Water Pollution and Fish Physiology. De- partment of Biology Virginia Polytechnic Institute and State [1] Puel, D., Zsuerger, N. and Breittmayer, J.P. (1987) Statistical Uniandrsity Blacksburg, Virginia, 4: 67-76. Assessment of a Sampling Pattern for Evaluation of Changes in Hg and Zn Concentration in Patella coerulea. Bull. Envi- [20] Mzimela H,M., Wepener, V. and Cyrus, D.P. (2010) The Sub- lethal Effects of Copper and Lead on the Haematology and ron. Contam. Toxicol., 38: 700–6. Acid-base Balance of the Groovy Mullet, Liza dumerili. Afri- [2] Javed, M. and Hayat, S. (1999) Heavy Metal Toxicity of River can Journal of Aquatic Science, 27:1, 39-46. Ravi Aquatic Ecosystem. Pakistan J. Agric. Sci., 36: 1–9. [21] Sağlamtimur, B., Cicik, B. and Erdem, C. (2003) Farklı Ortam [3] Orun, I. Talas, Z.S. and Alkan, A. (2011) Modulating Effect Derişimleri Etkisinde Bakır, Bakır + Kadmiyum Karışımının of Selenium on Gills of Fish Exposed to Heavy Metals. Frese- Oreochromis niloticus (L.)’un Solungaç, Karaciğer, Böbrek ve nius Environmental Bulletin, 20(1): 104-108. Kas Dokularındaki Bakır Birikimi Üzerine Etkileri. Turkish J. Vet. Ani. Sci., 27, 813-820. [4] Papagiannis, I., Kagolou, I., Palelogos, E., Karayannis, M. and Kalfakakou, V. (2002) Heavy Metals in Lake Pamvotis (NW. [22] Doaa, M.M. and Hanan, H.A. (2013) Histological Changes in Greece) Ecosystems. Fresenius Environmental Bulletin, Selected Organs of Oreochromis niloticus Exposed to Doses 11(9b):659-664. of Lead Acetate. J. Life Sci. Biomed., 3(3):256-263.

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[23] Witeska, M. Kondera, E. Lipionoga, J. and Jastrzebka, A. (2010) Changes in Oxygen Consumption Rate and Red Blood Parameters in Common Carp Cyprinus carpio L. After Acute Copper and Cadmium Exposures. Fresenius Environmental Bulletin, 19(1): 115-122. [24] Christensen G.M., McKim, J.M, Brungs, W.A. and Hunt, E.P. (1972) Changes in the Blood of the Brown Bullhead (Ictalurus nebulosus Lesuer) Following Short and Long Term Exposure to Copper. Toxicol. App. Pharmacol. 23:417-427.

[25] Tort, L., Torres, P. and Flos, R. (1987) Effects on Dogfish Haematology and Liver Composition after Acute Copper Ex- posure. Comp. Biochem. Physiol., 87(C), 349–353. [26] Cyriac, P.J. Antony, A. and Nambiasan, P.N.K. (1989) Hemo- globin and Hematocrit Values in the Fish Oreochromis mos- sambicus (Peters) After Short Term Exposure to Copper and Mercury. Bull. Environ. Contam. Toxicol., 43: 315-320. [27] Singh, H.S. and Reddy, T.V. (1990) Effect of Copper Sulfate on Haematology, Blood Chemistry and Hepato-Somatic Index of and Indian Catfish, Heteropneustes fossilis (Bloch), and its Recovery. Ecotoxicol. Environ. Safe., 20: 30-35. [28] Svobodova, Z., Vykusova, B. and Machova, J. (1994) The Ef- fects of Pollutants on Selected Haematological and Biochemi- cal Parameters in Fish, In: R. Müller and R. Lloyd (eds.), Sub- lethal and Chronic Effects of Pollutants on Freshwater Fish. Fishing New Boks, London. [29] Van Vuren, J.H.J., Van Der Merve, M. and Du Prezz, H.H. (1994) The Effects of Copper on the Blood Chemistry of Clarias gariepinus (Clariidae). Ecotoxicology and Environ- mental Safety., 29:187-199. [30] Mazon, A.F., Monteiro, E.A.S., Pinheiro, G.H.D. and Fer- nandes, M.N. (2002) Hematological and Physiological Changes Induced by Short-Term Exposure to Copper in the Freshwater Fish Prochilodus scrofa. Braz. Jç Biol., 62(4A): 621-631. [31] Witeska, I. (2004) The Effect of Toxic Chemicals on Blood Cell Morphology in Fish. Fresenius Environmental Bulletin, 13(12A): 1379-1384. [32] El Domiaty, N.A. (1987) Stress Response of Juvenile Clarias lazera Elicited by Copper. Comp. Biochem. and Physiol. C88(2): 259-262. [33] Johansson-Sjöbeck, M.L. and Larsson, A. (1979) Effects of Inorganic Lead on Delta-Amunolevulinic Acid Dehydratase Activity and Hematological Variables in the Rainbow Trout, Salmo gairdneri. Arch. Environ. Contam. Toxicol., 8: 419- 431. [34] Tewari, H., Gill, T.S. and Pant, J. (1987) Impact of Chronic Lead Poisoning on the Hematological and Biochemical Pro- Received: December 18, 2014 files of a Fish, Barbus conchonius (Ham.). Bull. Environ. Con- Revised: February 17, 2015; March 23, 2015 tam. Toxicol., 38: 748-752. Accepted: March 25, 2015 [35] Allen, P. (1983) Accumulation Profiles of Cadmium and Their Modification by Interaction with Lead and Mercury in the Ed- ible Tissues of Oreochromis aureus., Fresenius Environmental CORRESPONDING AUTHOR Bulletin, 2: 745-751. [36] Shah, S.L. and Altindag, A. (2004) Hematological Parameters Nuray Çiftçi of Tench (Tinca tinca L.) after Acute and Chronic Exposure to University of Mersin Lethal and Sublethal Mercury Treatments. Bull. Environ. Con- tam. Toxicol., 73: 911-918. Vocational School of Silifke Aquaculture Programme [37] Dick, P.T. and Dixon, D.G. (1985) Changes in Circulating Blood Cell Levels of Rainbow Trout, Salmo gairdneri Rich- Yenimahalle, Kayraktepe Mevkii ardson, Following Acute and Chronic Exposure to Copper. J. Silifke, Mersin Fish Biol., 26, 475–481. TURKEY [38] Witeska, M., Kondera, E., Szymanska, M. and Ostrysz., M. (2010) Hematological Changes in Common Carp (Cyprinus E-mail: [email protected] carpio L.) after Short-Term Lead (Pb) Exposure, Polish. J. of Environ. Stud., 19(4): 825-831. FEB/ Vol 24/ No 9/ 2015 – pages 2771 - 2775

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AN EVALUATION OF THE ENVIRONMENTAL SENSITIVITY TO LAND DEGRADATION IN MURCIA REGION, SE SPAIN

María José Martínez-Sánchez1, Carmen Pérez-Sirvent1, Mari Luz García-Lorenzo2,* and José Molina-Ruiz3

1 Department of Agricultural Chemistry, Geology and Pedology. Faculty of Chemistry, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain 2 Department Petrology and Geochemistry, Faculty of Geology. University Complutense of Madrid, 28040 Madrid, Spain 3 Department of Geography. Faculty of Geography. University of Murcia, Campus de la Merced, 30001 Murcia, Spain

ABSTRACT radation on the basis of their environmental characteristics. An environmentally sensitive area to degradation (ESA) An assessment of the sensitivity to land degradation could be considered as a spatially delimited area in which was carried out in the Murcia Region (SE Spain) by means some key aspects related to its sustainability are unbal- of a modelling approach developed by the European Com- anced, and not sustainable for a particular environment [3]. mission funded MEDALUS project (Mediterranean Deser- Then, determination of environmental sensitive areas has a tification and Land Use). These areas are identified accord- great importance in the identification of desertification risk ing to ESA index (Environmental Sensitive Areas index) areas, and to define adequate mitigation measures [4]. that incorporates data on environmental quality (climate, vegetation, soil) together with management data. The ob- The European Commission funded MEDALUS pro- tained results were integrated into a Geographic Infor- ject (Mediterranean Desertification and Land Use) [5, 6] mation System (GIS), and maps of environmental sensitiv- and in its frame, a set of desertification indicators were es- ity to degradation were created. The proposed methodol- tablished in order to identify the sensibility to land degra- ogy allows to identify different degrees of vulnerability to dation and desertification. These indicators provide a pre- land degradation, and also to analyse specific factors af- cise diagnosis of the present state of the environment to fecting desertification. Results showed that the study area formulate effective recovery strategies. This approach in- is mostly located in the critical desertification class. Based cludes parameters which can be easily found in existing on the results, almost all the land is included in high-critical soil, vegetation, and climate reports [7]. class (C3), showing that the whole region is at high deser- This model has been tested in most of the Mediterra- tification risk. nean basin countries including south-eastern Spain [8],

Iyzad Khast plain of Iran [9], Algeria [10], Lebanon [11], Extremadura region in Spain [12], in Italy [4, 13], Portugal KEYWORDS: Land degradation, desertification risk, MEDALUS [14], and Greece [15], showing good results. project, sensitivity areas, Mediterranean environments, mapping. Geographical information system (GIS) methodology

is a useful tool for the identification of the areas prone to 1. INTRODUCTION desertification at a regional scale because the use of a GIS facilitates the establishment of standardised procedures to Desertification is a major environmental, social and integrate alphanumeric and cartographic data with re- economic problem to many countries in all parts of the motely sensed information [3]. world. The United Nations Environmental Programme has The main aim of this study was to identify places with defined desertification as ‘land degradation in arid, semi- different environmental sensitivity to land degradation in arid and dry sub-humid areas resulting from various fac- Murcia Region (SE Spain), by means of one of the most tors, including climatic variations and human activities” pertinent assessing and mapping methods of desertifica- [1]. There are many factors that can contribute to desertifi- tion-sensitivity in the Mediterranean regions. This method cation and land degradation; these factors include soil, veg- was carried out by a modelling approach developed in the etation, climate, demographic and human activities [2]. European Commission (MEDALUS project, Mediterra- nean Desertification and Land Use) which identifies envi- In this context, the concept of environmental sensitiv- ronmental-sensitive areas on the basis of an index (ESA in- ity was defined in order to identify areas prone to land deg- dex) in which environmental quality (climate, vegetation, soil) as well as anthropogenic factors (management) are in- * Corresponding author corporated and mapped.

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2. MATERIALS AND METHODS four areas were selected, being all of them affected by agricultural activities. These areas were Upper Segura River 2.1 Study area Valley, Middle Segura River Valley, Campo de Cartagena The Region of Murcia is located in the southeast of the and Guadalentín Valley (Fig. 1). In the selected zones, the Iberian Peninsula, with a surface of 11 317 km2, and char- main land-uses are represented, corresponding to 4 groups: acterised by a semi-arid climate. Average temperature is drip irrigation, inundation irrigation, unirrigated soils and 18.5 ºC and precipitation ranges from 150 to 400 mm, with soils with natural vegetation, which correspond to aban- an average value of 325 mm. Murcia Region is a typical doned agricultural areas. In order to study the desertifica- Mediterranean area prone to land degradation and deserti- tion risk in Murcia Region, 207 soil samples were studied fication due to both unfavourable bio-physical conditions in the selected areas. Sampling sites were disposed in a and adverse human activities. Such factors include erratic 3 km square grid, and sampling density was 1 sampling site precipitation, which frequently occur between the end of per km2. Soil samples were collected from the surface soil summer and autumn high summer temperature and fre- (0-25cm). Five subplots (25x 25cm) for soil sampling were quent seasonal droughts, poor and erodible soils, rugged selected at the corners and the centre of the plot (1x1m). topography, extensive human-induced deforestation and Soil samples were collected with a shovel in each subplot, forest losses due to frequent wildfires, land abandonment then mixed and homogenized, and a subsample (about and deterioration, unsustainable exploitation of water re- 2 kg) was taken. sources, salinization and exhaustion of aquifers, uncon- trolled urbanization, concentration of economic activities The selected areas have been previously studied be- in coastal areas, tourism pressure, etc. The most intense ag- cause they were included in DESERTNET project [17]. ricultural areas of the Murcia Region are located close to The DESERTNET project was managed in the framework the most important watercourses of Segura River and Gua- of the Community Initiative Programme (CIP) Interreg III dalentín River [16]. Other very important agricultural areas B-Western Mediterranean. The main aim of the DESERT- are placed in Campo De Cartagena zone. For this study, NET project was to advance the study of the desertification

FIGURE 1 - Land use in the four established areas

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processes in the Mediterranean regions through carrying SQI = (texture × parent material × rock × fragment out pilot studies, exchanging experiences and developing depth × slope × drainage) 1/6 models of evaluation. The Region of Murcia (SE, Spain) All selected variables were determined in the frame- participated in this project, since it is sensitive to desertifi- work of DESERNET and DESERTNET II projects. Tex- cation processes due to its climatic characteristics and tak- tural analysis was performed after dispersion of the fine ing into account the intense agricultural use. soil, and by combining extraction by Robinson pipette and sieving. The parent rock material was obtained according 2.2 Applied methodology to the geological map. The other soil variables (rock frag- The MEDALUS model was used for calculating the ments, depth, slope and drainage) were determined in situ ESA index and to determine the situation and tendency of when sediment samples were collected. desertification in Murcia Region. The ESA index is composed by 4 quality indices, calculated from some individual 2.2.2 Climate quality indicator (CQI) variables: soil quality, climate quality, vegetation quality The CQI index could be determined as: and management quality, and human factors [18]. Desertifi- CQI = (rainfall × aridity × aspect) 1/3 cation indicators are combined into four quality indices, the Soil Quality Index (SQI), the Climate Quality Index (CQI), Climate quality was assessed by using parameters that the Vegetation Quality Index (VQI), and the Management influence water availability to the plants, such as amount Quality Index (MQI), briefly explained in this section. of rainfall, air temperature, aridity, as well as aspect. The following data on climate were required for the 2.2.1 Soil quality indicator (SQI) assessment of climate quality and obtained from the Re- Soil quality indicators for mapping environmental sen- gional Meteorological Centre: sitive areas could be associated to: (a) water availability, - Temperature-mean monthly air temperature (ºC) and (b) erosion resistance. These qualities can be evaluated - Precipitation-mean monthly precipitation amount (mm) by using simple soil properties, such as soil depth, soil tex- - Frost-mean monthly number of days with minimum ture, drainage, parent material, slope grade or stoniness temperature < 0 ºC (Table 1). The SQI (Soil Quality Index) is estimated as:

TABLE 1 - Classes and assigned weighted indices for the various parameters used for assessment of soil quality.

Parameter Class Texture Index Good L, SCL, SL, LS, CL 1.0 Moderate SC, SiL SiCL 1.2 Soil texture Poor Si, C, SiC 1.6 Very poor S 2.0 Parameter Class Parent material Index Good Shale, schist, basic, ultra basic, conglomerates, unconsolidated 1.0 Limestone, marble, granite, Parent material Moderate 1.7 rhyolite, ignibrite, gneiss, siltstone, sandstone Poor Marl*, pyroclastics 2.0 Parameter Class Rock fragment cover (%) Index Very stony >60 1.0 Rock fragments Stony 20-60 1.3 Bare to slightly stony <20 2.0 Parameter Class Slope (%) Index Very gentle to flat <6 1.0 Gentle 6-18 1.2 Slope Steep 18-35 1.5 Very steep >35 2.0 Parameter Class Soil depth (cm) Index Deep >75 1.0 Moderate 75-30 2.0 Soil depth Shallow 15-30 3.0 Very shallow <15 4.0 Parameter Description Index Well drained 1.0 Drainage Imperfectly drained 1.2 Poorly drained 2.0 Soil quality index (SQI) Class Description Range 1 High quality <1.13 2 Moderate quality 1.13-1.45 3 Low quality >1.45 * For perennial vegetation, marls are removed to class 1.

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- Potential evapo-transpiration-mean monthly potential evapotranspiration (mm). 2 Annual precipitation is classified in three classes considering the annual precipitation of 280 mm as a crucial where, ti is the mean air temperature for month i (ºC), value for soil erosion and plant growth (Table 2). Pi is the total precipitation for month i (mm), and ki represents the proportion of the month during which 2ti - Pi >0. TABLE 2 - Classes and assigned weighted indices for the various parameters used for assessment of climate quality. Slope aspect is considered herein for climate quality assessment by distinguishing two classes: one with NW Parameter Class Rainfall (mm) Index and NE aspects and class two with SE and SW aspects. 1 >650 1.0 This variable was determined in situ when sediment sam- Rainfall 2 280-650 2.0 ples were collected. 2 <280 4.0

Parameter Class BGI* range Index 2.2.3 Vegetation quality indicators 1 <50 1.0 2 50-75 1.1 Vegetation quality is assessed in terms of fire risk and 3 75-100 1.2 Aridity ability to recover, erosion protection offered to the soil, 4 100-125 1.4 drought resistance and percentage of plant cover (Table 3). 5 125-150 1.8 6 >150 2.0 The in the Mediterranean region existing dominant types Parameter Class Slope aspect Index of vegetation are grouped into 4 categories according to the 1 NE, NW 1.0 fire risk (Table 3). Also 4 categories are used for classifying Slope aspect 2 SE, SW 2.0 the vegetation according to the protection to the soil from Climate quality index Class Description Range erosion (Table 3). Five categories are used for classification (CQI) of vegetation, with respect to drought resistance. Finally, 1 High quality <1.15 plant cover is distinguished into 3 classes. All these variables Moderate qual- 1.15- 2 ity 1.81 were determined during the sampling collection in the 3 Low quality >1.81 framework of DESERNET project. *BGI: Bagnouls-Gaussen aridity Index The VQI (Vegetation Quality Index) is assessed as fol- The concept of Bagnouls-Gaussen bioclimatic aridity lows: index was used for determining the aridity index and is de- VQI = (fire risk× erosion protection × drought re- fined as: sistance × vegetation cover) ¼

TABLE 3 - Classes and assigned weighted indices for the various parameters used for assessment of vegetation quality.

Parameter Class Description Type of vegetation Index Bare land, perennial agricultural crops, 1 Low 1.0 annual agricultural crops (maize, tobacco, sunflower) Annual agricultural crops (cereals, grasslands), Fire risk 2 Moderate 1.3 deciduous oak, (mixed), mixed Mediterranean, macchia/evergreen forests 3 High Mediterranean macchia 1.6 4 Very high Pine forests 2.0 Parameter Class Decription Vegetation types Index 1 Very high Mixed Mediterranean macchia/evergreen forests 1.0 Mediterranean macchia, pine forests, 2 High 1.3 Permanent grasslands, evergreen perennial crops. Erosion protection Moderate Deciduous forests 1.6 3 Low Deciduous perennial agricultural crops (almonds, orchards) 1.8 4 Very low Annual agricultural crops (cereals), annual grasslands, vines, 2.0 Parameter Class Description Types of vegetation Index Mixed Mediterranean macchia/evergreen forests, 1 Very high 1.0 Mediterranean macchia 2 High Conifers, deciduous, olives 1.2 Drought resistance 3 Moderate Perennial agricultural trees (vines, almonds, orchard) 1.4 4 Low Perennial grasslands 1.7 5 Very low Annual agricultural crops, annual grasslands 2.0 Parameter Class Description Plant cover (%) Index 1 High >40 1.0 Plant cover 2 Low 10-40 1.8 3 Very low <10 2.0 Vegetation quality index (VQI) Class Description Range 1 High quality <1.2 2 Moderate quality 1.2-1.4 3 Low quality >1.4

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2.2.4 Management quality and human factors ESAI= (SQI × CQI × VQI × MQI)1/4 In addition to the physical indicators, when ESA are de- The ESAI index identifies 4 main classes of areas fined the human-induced stress, has also to be assessed. De- threatened by land degradation (“critical”, “fragile”, “po- pending on the particular type of management, land re- tentially affected” and “non-affected”) that could be further sources are subject to a given degree of stress. Moreover, the differentiated into subclasses that describe the sensitivity existence of environmental policies which apply to a certain to desertification risk as C3-critical > C2-critical > C1-crit- area, moderate the anticipated impacts of a given land-use ical > F3-fragile > F2-fragile > F1-fragile > potential > type compared to the situation where no such policies are in non-affected [18]. effect (Table 4). The MQI is assessed as the product of land- The ranges of ESAI for each type of ESA are shown in use intensity and the enforcement of policy for environmen- Table 5. tal protection using the following algorithm: MQI = (land use intensity x policy enforcement)1/2 2.4 Geographic Information System (GIS) methodology

The integration, management and processing of the 2.3 Matching the results data and maps were performed by means of ArcGIS, v.9 The values of the quality indices are obtained as geo- software. GIS methodology allows representing indicators metric mean of the scores assigned to each single indicator. and environmental sensitive areas in the studied area. In Then, the Environmental Sensitive Areas Index (ESAI) is our study, map intervals were established according to the determined as follows: ESAI categories.

TABLE 4 - Classes and assigned weighted indices for the various parameters used for assessment of management quality.

Parameter Description Index Low land use intensity (LLUI) 1.0 Cropland Medium land use intensity (MLUI) 1.5 High land use intensity (HLUI) 2.0 Parameter Description Stocking rate Index Low ASR< SSR 1.0 Pasture Moderate ASR=SSR to 1.5*SSR 1.5 High ASR>1.5*SSR 2.0 Parameter Class Management characteristics Index Low A/S = 0 1.0 Natural areas Moderate A/S < 1 1.2 High A/S = 1 or greater 2.0 Parameter Class Erosion control measurements Index Low Adequate 1.0 Mining areas Moderate Moderate 1.5 High Low 2.0 Parameter Class A/P visitors ratio Index Low <1 1.0 Recreation areas Moderate 1-2.5 1.5 High <2.5 2.0 Parameter Class Degree of enforcement Index High Complete: >75% of the area under protection 1.0 Policy Moderate Partial: 25-75% of the area under protection 1.5 Low Incomplete: <25% of the area under protection 2.0 Management quality index (MQI) Class Description Range 1 High quality <1.25 2 Moderate quality 1.25-1.50 3 Low quality >1.50

TABLE 5 - Types of ESAs and corresponding ranges of indices. 2.5 Map Validation

Type Subtype Range of ESAI In order to validate the model and the maps resulting C3 >1.53 from the classification made over the ESA index, the final Critical C2 1.42-1.53 ESA maps were compared with the obtained results for the C1 1.38-1.41 salinization rate index, determined in the framework of F3 1.33-1.37 DESERTNET project. The indicator was defined as the Fragile F2 1.27-1.32 F1 1.23-1.26 electrical conductivity temporal variation in the soil satu- Potential P 1.17-1.22 ration extract for each soil sample. This indicator was cal- Non affected N <1.17 culated comparing the salinization state in 2 periods of time, thus providing a measure of the temporal evolution

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of the salinization degradation of soils. To determine the mainly due to its texture. Samples with moderate soil qual- salinization rate indicator, electrical conductivity values ity were also developed on marl parent material, but they were employed, obtaining the variations between sampling present a loamy texture, a deeper soil depth and a well periods, and next, dividing them between the years elapsed drainage. Climate quality index results ranged from mod- between sampling periods. After the electrical conductivity erate (8%) to low quality (92%). Samples with moderate increase/decrease was determined, the results were reclas- climate quality index correspond to slopes with a NW or sified in 5 intervals for GIS representation. NE aspect. Vegetation quality index values ranged from high quality (1.16) to low quality (1.80), presenting 6% of the territory high quality, 1% moderate and the rest, low 3. RESULTS vegetation quality. With regards to this index, high and moderate quality values correspond to samples with Medi- 3.1 Application of the proposed methodology to Murcia region terranean macchia or perennial agricultural trees, and with Soil quality index values ranged from 1.17 to 1.61 and more than 40% of plant cover. Similar results were ob- an average value of 1.41 (moderate quality). Results show tained for management quality index in this area, 93% of that 49% of the territory showed low soil quality and 51% samples showed low quality, and 7% moderate quality, moderate quality. Samples with low soil quality index cor- ranging from 1.41 to 1.73 (Fig. 2). All samples in this area respond to soils with a clayey texture, developed on a marl showed similar management policies; then, differences in parent material, shallow soil depth and poorly drained, management quality index are due to land-use intensity.

FIGURE 2 - SQI, CQI, VQI and MQI results in Campo de Cartagena

FIGURE 3 - SQI, CQI, VQI and MQI results in Guadalentín River Valley

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In Guadalentín Valley (Fig. 3), soil quality index val- trees, like almonds or orchards, increases drought re- ues ranged from 1.11 (high quality) to 1.61 (low quality), sistance and erosion protection. Results showed that plant with an average value of 1.39. The obtained results sug- cover percentage was higher than 40% and vegetation qual- gested that 34% of collected samples showed low soil quality index was moderate in this zone. All samples in this area ity, 63% moderate quality and 3% high soil quality. Sam- showed similar management policies; then, differences in ples with high soil quality index value correspond to Cal- management quality index are due to moderate and low cisols [19] with a loamy texture, deep depth and well land-use intensity. drained. All samples in this area showed low climate quality because the area showed scarce rainfall, high aridity in- In Upper Segura River Valley (Fig. 4), obtained results dex, and slopes with a SE or SW aspect in all cases. Vege- for soil quality index suggested that 59% of samples in this tation quality index results ranged from 1.16 (high quality) area show low quality and the rest, 41% moderate quality. to 1.80 (low quality), with an average value of 1.47. Man- Soils with moderate soil quality in this area correspond to agement quality index is comprised in the interval (1.41- Fluvisols [19] with a loamy texture, well drained, with 1.73). For both indexes, 91% of samples showed low qual- scarce presence of rock fragments (<20%), a very gentle ity and 9% moderate quality. Even, if vegetation showed slope, and a moderate soil depth. However, soils with low similar fire risk, the presence of perennial agricultural soil quality were Regosols [19] with a clayey texture, im-

FIGURE 4 - SQI, CQI, VQI and MQI results in Upper Segura River Valley

FIGURE 5 - SQI, CQI, VQI and MQI results in Middle Segura River Valley

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FIGURE 6 - ESAI results in the study zones

perfectly drained with a shallow soil depth. All samples in class (C2) and 92% to high critical class (C3); in Guada- this area showed moderate climate quality because in this lentín Valley, 14% belongs to medium critical class (C2) area annual rainfall is between 280-650 mm, slightly and the rest to high critical class (C3); in Middle Segura higher than annual rainfall in the other areas (values rang- River Valley, 56% belongs to medium critical class (C2) ing from 1.34 to 1.69). Vegetation and management quality and 44% to high critical class, and in Upper Segura River indices showed similar results, 91% of samples with low Valley 9% belongs to high fragile class (F3), 50% belongs quality and 9% with moderate quality. Almost all samples to medium critical class (C2), and 41% to high critical class showed low management quality, because they are sub- (C3). jected to medium land-use intensity. In Fig. 7, the maps showed the distribution of sub-clas- In Middle Segura River Valley (Fig. 5), soil quality in- ses of sensitivity to degradation in the 4 study areas. The dex results are comprised in the interval (1.41-1.73), 59% high-critical class (C3) dominates in Campo de Cartagena of samples showed low quality, and 41% moderate quality. and Guadalentín valley while medium-critical class domi- In this area, soils with low quality correspond to Fluvisols nates in Upper and Middle Segura River Valley. The criti- [19] with a clayey texture, developed on marl parent mate- cal areas (C1, C2 and C3) are very sensitive to degradation rial and a moderate soil depth. On the other hand, soils with under any change to the delicate balance of climate and moderate soil quality correspond to Calcisols [19], with land-use. loamy or sandy texture, well drained, stony (20-60% rock fragments), and with a deep soil depth. When climate qual- In order to validate the obtained results, the ESAI re- ity index results are evaluated, 89% of samples showed low sults were compared with other indicators studied in these quality and 11% moderate quality. Samples with low qual- areas [17]. The salinization rate was defined as the electri- ity are located in slopes with SE or SW aspects, and annual cal conductivity temporal variation in the soil saturation rainfall in this area is lower than 280 mm. Vegetation qual- extract for each soil sample. This indicator was calculated ity index in this area noticed that 41% of samples have low comparing the salinization state in 2 periods of time, thus quality and 59% moderate quality, ranging from 1.16 to providing a measure of the temporal evolution of the sali- 1.68. Moderate vegetation quality was noticed in samples nization degradation of soils. To determine the salinization with high plant cover of perennial crops or Mediterranean rate indicator, electrical conductivity values were em- macchia, which increases drought resistance and erosion ployed, obtaining the variations between sampling periods, protection. Finally, management quality index in this area and next, dividing them between the years elapsed between suggested that 59% of samples have low quality and 41% sampling periods. After the electrical conductivity in- moderate quality; differences between them are due to land crease/decrease was determined, the results were reclassi- use intensity. fied in 5 intervals for GIS representation. Fig. 6 shows ESAI results for the study zones. Except Guadalentín River Valley and Campo de Cartagena are of Upper Segura River Valley, where 9% of land is classi- the most sensitive areas to salinization and showed the fied as fragile, all the studied territory is classified as criti- highest values for salinization rate indicator, being some cal. In Campo de Cartagena, 8% belongs to medium critical points affected by a serious or very serious salinization risk

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(Fig. 8). In these areas, an intensive agricultural activity is agree with the ESAI results and suggested that both Campo carried out and salinization problems are mainly due to the de Cartagena and Guadalentín River Valley are most sen- poor quality of the irrigation water. The salinization results sitive to degradation and desertification processes.

FIGURE 7 - Map of eight classes of environmental sensitivity in study zones

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FIGURE 8 - Salinization rate indicator in the studied areas

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4. CONCLUSIONS [12] Lavado Contador, J.F., Schnabel, S., Gómez Gutierrez, A. and Pu- lido Fernández, M. (2009). Mapping sensitivity to land degradation in Extremadura, SW Spain. Land Degradation & Develop- In this work, desertification risk of Murcia region was ment 20, 129-144. assessed in 4 areas considering a set of indices involving [13] Coscarelli, R., Minervino, I. and Sorriso-Valvo, M. (2005). Meth- both the natural ecosystem and the human social system. ods for the characterization of areas sensitive to desertification: An The results suggested that Murcia Region showed an ele- application to the Calabrian territory. Italy. IAHS-AISH Publica- vated desertification risk according to the applied method- tion 299, 23-30. ology. The presented methodology could be used for the [14] Roxo, M.J., Mourão, J.M., Rodrigues, L. and Casimiro, P.C. (1999). The Alentejo region (Mértola municipality, Portugal). In definition of priorities in the adoption of strategies to miti- C. Kosmas, M.J. Kirkby, N. Geeson (eds.), Manual on key indica- gate desertification not only in Murcia Region but also in tors of desertification and mapping environmentally sensitive ar- other semi-arid areas of the Mediterranean region. The eas to desertification. Medalus project- Mediterranean desertification and land use (pp.80-87). Luxembourg: European Commission MEDALUS approach is conceptually very simple and easy Publication EUR 18882. to implement as a GIS-based method from local to regional [15] Kosmas, C., Gerontidis, St., Detsis, V., Zafiriou, Th. and Marathi- or national scale. anou, M. (1999a). The Lesvos island (Greece). In C. Kosmas, M.J. Kirkby, N. Geeson (eds.), Manual on key indicators of desertifica- The authors have declared no conflict of interest. tion and mapping environmentally sensitive areas to desertification. Medalus project- Mediterranean desertification and land use (pp.66-74). Luxembourg: European Commission Publication EUR 18882. REFERENCES [16] Pérez-Sirvent, C., Martínez-Sánchez, M.J., Vidal, J. and Sánchez, A. (2003). The role of low-quality irrigation water in the desertifi- [1] UNEP (United Nations Environment Programme) (1994). United cation of semi-arid zones in Murcia, SE Spain. Geoderma 113, Nations Convention to Combat Desertification in those countries 109-125. experiencing serious drought and/or desertification, particularly in [17] Martínez-Sánchez, M.J., Pérez-Sirvent, C., Molina-Ruiz, J., Africa. Text with annexes. Geneva: United Nations Environment Tudela, M.L. and García-Lorenzo, M.L. (2011). Monitoring sali- Programme for the Convention to Combat Desertification (CCD), nization processes in soils by using a chemical degradation indica- Interim Secretariat for the CCD. tor. Journal of Geochemical Exploration 109, 1-7. [2] Pavari, S.Y., Pahlavanravi, A., Nia, A.R.A., Dehvari, A. and Par- [18] Kosmas, C., Ferrara, A., Briasouli, H. and Imeson, A. (1999b). vari, D. (2011). Application of methodology for mapping environ- Methodology for mapping environmentally sensitive areas (ESAs) mentally sensitive areas (ESAs) to desertification in dry bed of to desertification. In C. Kosmas, M.J. Kirkby, N. Geeson (eds.), Hamoun Wetland (Iran). International Journal of Natural Re- Manual on key indicators of desertification and mapping environ- sources and Marine Sciences 1, 65-80. mentally sensitive areas to desertification. Medalus project- Med- [3] Basso, F., Bove, E., Dumontet, A., Ferrara, A., Pisante, M., iterranean desertification and land use (pp.31-47). Luxembourg: Quaranta, G. and Taberner, M. (2000). Evaluating environmental European Commission Publication EUR 18882. sensitivity at the basin scale through the use of geographic information systems and remotely sensed data: An example covering [19] IUSS Working Group WRB (2006). World reference base for soil the Agri basin (Southern Italy). Catena 40, 19-35. resources 2006. World Soil Resources Reports No. 103. FAO, Rome. [4] Ladisa, G., Todorovic, M. and Trisorio Liuzzi, G. (2012). A GIS- bases approach for desertification risk assessment in Apulia Re- gion, SE Italy. Physics and Chemistry of the Earth 49, 103-113. [5] European Commission. Mediterranean Desertification and LandUse (MEDALUS) (1999). MEDALUS Office, Landen. [6] Brandt J.C. and Thornes J.B. (1996). Mediterranean Desertifica- tion and Land Use. John Wiley & Sons, London. [7] Kosmas, C., Kairis, Or., Karavitis, Ch., Ritsema, C., Salvati, L., Acikalin, S., et al. (2012). Evaluation and Selection of Indicators Received: December 18, 2014 for Land Degradation and Desertification Monitoring: Methodo- Accepted: February 28, 2015 logical Approach. Environmental Management, doi 10.1007/s00267-013-0109-6.

[8] Hooke, J.M., Brookes, C.J., Duane, W. and Mant, J.M. (2005). A simulation model of morphological, vegetation and sediment CORRESPONDING AUTHOR changes in ephemeral streams. Earth Surface Processes and Land- forms 30, 845-866. Mari Luz García-Lorenzo [9] Farajzadeh, M. and Egbal, M.N. (2007). Evaluation of MEDALUS Department Petrology and Geochemistry model for desertification hazard zonation using GIS; study area: Faculty of Geology Iyzad Khast plain, Iran. Pakistan Journal of Biological Sciences 10, 2622-2630. University Complutense of Madrid 28040 Madrid [10] Benabderrahmane M.C. and Chenchouni, H. (2010). Assessing environmental sensitivity areas to desertification in Eastern Algeria SPAIN using Mediterranean Desertification and Land Use “MEDALUS” Model. International Journal of Sustainable water & Environmen- Phone: +34 913944912 tal systems 1, 5-10 Fax: +34 91544 2535 [11] Darwish, T., Zdruli, P., Saliba, R., Awad, M., Shaban, A. and Faour, G. (2012). Vulnerability to desertification in Lebanon based E-mail: [email protected] on geo-information and socioeconomic conditions. Journal of En- vironmental Science and Engineering 1, 851-864. FEB/ Vol 24/ No 9/ 2015 – pages 2776 - 2786

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PHYTOREMEDIATION OF SOME HEAVY METALS BY DIFFERENT TISSUES OF ROSES GROWN IN THE MAIN INTERSECTIONS IN ERZURUM CITY, TURKEY

Aslihan Esringu1, Elif Akpinar Kulekci2, Metin Turan3 and Sezai Ercisli4,*

1Ataturk University Narman Vocational School, 25530 Narman-Erzurum, Turkey 2Ataturk University Faculty of Architecture and Design, Department of Landscape Architecture 25240 Erzurum, Turkey 3Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and Bioengineering, 34755 Kayisdagi, Istanbul, Turkey 4Ataturk University Agricultural Faculty, Department of Horticulture, 25240 Erzurum, Turkey

ABSTRACT Heavy metals are conventionally defined as elements with metallic properties and an atomic number >20. The In this study, we assessed the level of some heavy met- most common heavy metal contaminants are Cd, Cr, Cu, als such as lead (Pb), nickel (Ni), cadmium (Cd), and zinc Hg, Pb, and Zn. Metals are natural components in soil [4]. (Zn) in both soils and leaf and roots of roses (Rosa gallica) Some of these metals are micronutrients necessary for plant planted at six of the busiest intersections that have heavy growth, such as Zn, Cu, Mn, Ni, and Co, while others have vehicle traffics in Erzurum city center. The results showed unknown biological function, such as Cd, Pb, and Hg [5]. that the accumulation of heavy metals (Pb, Ni, Cd, and Zn) Metal pollution has harmful effect on biological systems in the rose leaves were significantly higher than in roots. and does not undergo biodegradation. Toxic heavy metals The distribution of the heavy metals in the soils decreased such as Pb, Co, Cd can be differentiated from other pollu- in the order Pb>Zn>Ni>Cd. This observation indicated that tants, since they cannot be biodegraded but can be accumu- the emissions of heavy metals from the vehicles on traffic- lated in living organisms, thus causing various diseases and congested intersections might be deposited on the leaves disorders even in relatively lower concentrations [6]. rather than in the roots of roses. Hence, roses may be an Heavy metals, with soil residence times of thousands of important plant for aesthetic use and also for phytoremedi- years, pose numerous health dangers to higher organisms. ation purposes to clean up soils and environment contami- They are also known to have effect on plant growth, ground nated with heavy metals and the possibility of using the cover and have a negative impact on soil microflora [7]. It plant in phytomining. is well known that heavy metals cannot be chemically degraded and need to be physically removed or be transformed into nontoxic compounds [5].

KEYWORDS: There are various ways and means to mitigate the ur- Roses, phytoremediation, plant, heavy metals ban environmental pollution. The use of plant species for cleaning polluted soils has gained increasing attention since the last decade, as it is an emerging cheap technology. Many studies have been conducted in this field in the last 1. INTRODUCTION two decades. Numerous plant species have been identified and tested for their traits in the uptake and accumulation of In biology, “heavy” refers to a series of metals and also heavy metals such as lead, cadmium, chromium, arsenic metalloids that can be toxic to both plants and animals even from soil in different tissues [1-3, 8]. at very low concentrations. Therefore the term “heavy metals” in general refers to potentially phytotoxic elements Planting of trees and shrubs for abatement of pollution [1,2]. Heavy metals are accepted as major pollutants that and improvement of environment is an effective and well are accumulated in the environment. The accumulation of recognized way throughout the world. Proper planning and heavy metals in the soil has become a worldwide serious planting scheme depending upon the magnitude and type environmental problem due to rapid industrialization and of pollution, selection of pollution-tolerant and dust scav- random urbanization. Over exploitation of open spaces, enging trees and shrubs should be done for bioremediation ever-increasing number of automobiles and demographic of urban environmental pollution. pressure has further aggravated the problem [3]. The rose has been used for recreational purposes in urban areas for a long time [9]. The genus is also well known for its compatibility with industrial and urban environments * Corresponding author

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pollution. It shows a good tolerance against air pollution, Plant samples were taken from each location, then dust, UV light and noise pollution [10]. Roses like damask dried in the open air for 48 hours and allowed to plants fix the sulfur dioxide (SO2) in their tissues. Roses are com- samples was dry oven at 68 °C. Pb, Cd, Ni and Zn content patible in aluminum factories and absorb the fluoride proof dried plant samples were determined after wet digestion duced by the factory [10]. of dried and ground sub-samples in microwave (Bergof The City of Erzurum is a relatively densely populated Speedwave Microwave Digestion Equipment MWS-2) area and 90% percent of all the job opportunities are local- [11]. Tissue Pb, Cd, Ni and Zn were determined by an In- ized in the aforementioned areas, resulting in a huge daily ductively Couple Plasma spectrophotometer (Perkin- movement of people to and from work with high vehicular Elmer, Optima 2100 DV, ICP/OES, Shelton, CT 06484- traffic on the major roads in the city center. Over the past 4794, USA) [12]. twenty years the number of vehicles in the city has increased and the city has been put under increased toxic Soil samples were taken from 10-20 cm depth and stress due to increased emission of metals, such as lead. were also sampled from root area and both samples were mixed. We sampled 5 soil samples per location. The soil The aim of the study was firstly, to determine the accu- samples were air-dried, crushed, and passed through a mulation of heavy metals (Pb, Cd, Ni and Zn) in soil at six 2-mm sieve prior to chemical analysis. Electrical conduc- major vehicle traffic congestion sites and secondly, to deter- tivity (EC) was measured in saturation extracts [13]. Plant- mine if roses accumulate Pb, Cd, Ni and Zn after being ex- available P was determined by using the sodium bicar- posed to the contaminated soil from the sampling sites. bonate method [14]. Soil pH was determined in 1:2 ex-

tracts, and calcium carbonate concentrations were deter-

mined [15]. Soil organic matter was determined using the 2. MATERIALS AND METHODS Smith-Weldon method [16]. Micro elements in the soils were determined by Diethylene Triamine Pentaacetic Acid 2.1. Sampling area (DTPA) extraction methods [16]. The soil characteristics The sampling area is Erzurum city, a city that has of sampling locations are given in Table 1. around 500.000 inhabitants. The city is located in eastern Anatolia of Turkey. The sample locations are 5 main road conjunctions (intersections) in the city center including 3. RESULTS AND DISCUSSION Havuzbasi, Gez, Sanayi, Migros and Havaalani kavsagi. The average heavy metal content in soil of 6 main in- 2.2 Preparation and analysis of samples tersections that has higher vehicle traffic congestion in Er- For each intersection, a total of ten Rosa gallica plants zurum city is shown in Table 2. There were statistically im- were sampled in June 2013. We rooted out 10 whole plants portant differences (p≤0.01) among soils of six intersec- per sampling location and 10 roots and 40 leaves per locations in terms of all four heavy metals (Pb, Cd, Ni and Zn) tion are used in the study. content (Table 2).

TABLE 1 - Soil characteristics of sampling locations

Location code Location pH CaCO3 Organic Matter EC P (%) (%) (µS/cm) (mg kg-1) A1 Havuzbasi 7.21 5.62 7.98 351 75 A2 Gez 7.31 1.85 4.92 406 72 A3 Sanayi 6.87 1.46 6.93 461 142 A4 Migros 7.16 2.66 2.03 481 87 A5 Universite 7.11 0.97 0.64 272 12 A6 Havaalani 7.18 1.14 5.43 360 85

TABLE 2 - Pb, Cd, Ni and Zn content (mg kg-1) in soils of six main intersections in Erzurum city center

Location code Location Pb Cd Ni Zn

A1 Havuzbasi 0.83 b *** 0.039 b *** 0.25 a *** 0.22 d *** A2 Gez 0.83b 0.041a 0.03c 0.39b A3 Sanayi 0.74d 0.036c 0.14b 0.44a A4 Migros 0.79c 0.034d 0.11d 0.36c A5 Universite 0.91a 0.031e 0.07e 0.08f A6 Havaalani 0.71d 0.029f 0.06f 0.23d

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According to the results, Pb was the dominant heavy to human health. In fact, exposure to lead adversely affects metal. It accumulated in all sampled soils and the concen- hemoglobin synthesis (causing anemia), the kidneys, the tration varied from 0.71 (A6) to 0.91 mg kg-1 (A5). Zn var- neurological system, as well as the cardiovascular system ied from 0.08 (A5) to 0.44 mg kg-1 (A3) and Ni varied from [21, 22]. Mean ratios of the heavy metal concentrations in the 0.03 (A2) to 0.25 mg kg-1 (A1), respectively (Table 2). The leaves and roots in rose plants found at six intersections are overall, Cd content was the lowest and varied from 0.029 shown in Figures 1-4. The heavy metal accumulation caused (A6) to 0.041 mg kg-1 (A2), respectively (Table 2). A pre- by exhaust gases generated by vehicles around six main in- vious study [17] also indicated that the major causes of en- tersections in Erzurum city was highly affected by plant tis- vironmental pollution arise from vehicular emissions con- sue (leaf vs. root) and also the location (Figures 1-4). taining unoxidized traces of tetra ethyl lead (TEL) and its oxidized form of lead. Sutherland and Tack [18] showed Soil can be contaminated with Pb from several other that cars are a source of several pollutants to the environ- sources such as industrial sites, from leaded fuels, old lead ment, particularly lead, and Harper et al. [19] found that plumbing pipes, or even old orchard sites in production older vehicles are associated with higher levels of lead pol- where lead arsenate is used. Lead accumulates in the upper lutant emissions. Nabulo et al. [20] reported that contami- 20 cm of the soil and is highly immobile. Contamination is nation of the roadside environment is a result of the use of long-term. Without remedial action, high soil lead levels leaded petrol. Lead exposure is a health hazard, recognized will never return to normal [23]. worldwide. There is no knowledge of lead being beneficial

Leaf Root Leaf Root

) 0,08

1 2 ) ‐ 1 a ‐ a a kg

kg b 1,6 0,06 c (mg

(mg b d 1,2 a b f e f d b c c 0,04 content g

d content 0,8

Pb c e Cd d d e 0,4 f 0,02

0 0 A1 A2 A3 A4 A5 A6 A1 A2 A3 A4 A5 A6

Sample locations Sample locations

FIGURE 1 - Pb content in leaf and root of rose plants sampled FIGURE 2- Cd content in leaf and root of rose plants sampled from 6 locations from 6 locations

Leaf Root Leaf Root

8 )

1 a ‐ 50 kg )

b 1 a ‐ b 6 d kg 40 c

(mg

f e a (mg g a d 4 30 b c e f

e d f content c b 20 d 2 Zn e content f Ni 10 0 A1 A2 A3 A4 A5 A6 0 Sample locations A1 A2 A3 A4 A5 A6 Sample locations

FIGURE 3 - Ni content in leaf and root of rose plants sampled FIGURE 4 - Zn content in leaf and root of rose plants sampled from 6 locations. sampled from 6 locations

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For all locations, leaves of roses accumulated more [3] Tangahu, B.V., Abdullah, S.R.S.A., Basri, H., Idris, M., heavy metals (Pb, Cd, Ni and Zn) rather than roots (Figure Anuar, N. and Mukhlisin, M. (2011). Review on heavy metals (As, Pb, and Hg) uptake by plants through Phytoremediation. 1-4). In general plants accumulate heavy metals in aerial International Journal of Chemical Engineering 939161: 31. parts via phytoextraction. Phytoextraction is the uptake/ab- [4] Lasat, M.M. (2000). Phytoextraction of metals from contami- sorption and translocation of contaminants by plant roots nated soil: a review of plant/soil/metal interaction and assess- into the above ground portions of the plants (shoots, leaves) ment of pertinent agronomic issues Journal of Hazardous Sub- that can be harvested and burned gaining energy and recy- stance Research 2 (5):1-25. cling the metal from the ash [24, 25]. [5] Gaur, A. and Adholeya, A. (2004). Prospects of arbuscular mycorrhizal fungi in phytoremediation of heavy metal con- There were statistical significant differences (p≤0.01) taminated soils Current Science, vol. 86, no. 4, pp. 528–534, 2004. among locations in terms of Pb, Cd and Zn content in both leaf and roots (Figure 1, 2 and 4). However there were no [6] Pehlivan, E., Ozkan, A.M. and Parlayici, S. (2009). Adsorp- tion of Cu2+ and Pb2+ ion on dolomite powder. Journal of statistical differences among locations in terms of Ni con- Hazardous Materials 167 (1–3): 1044–1049. tent (Figure 3). [7] Roy, S. Labelle, P. Mehta et al., “Phytoremediation of heavy metal and PAH-contaminated brownfield sites,” Plant and The distribution of the heavy metals in the leaves of Soil, vol. 272, no. 1-2, pp. 277–290, 2005. roses decreased in the order Zn>Ni>Pb>Cd. Our results [8] Yang, X.E., Li, T.Q., Yang, J.C., He, Z.L., Lu, L.L. and Meng, demonstrated that the heavy metal content in plant tissue F.H. (2006). Zinc compartmentation in root, transport into xy- was higher than in soil indicating heavy metal accumula- lem, and absorption into leaf cells in the hyperaccumulating tion capacity of rose leaves. species of Sedum alfredii Hance. Planta 224 (1), 185-195. [9] Ercisli, S. (2004). Rose germplasm resources of Turkey. Ge- Zn concentrations were 18.9-42.9 mg kg-1in leaves of netic Resources and Crop Evolution 52:787-795. roses in the studied areas. Zinc is an essential element in all [10] Janfaza, S. and Janfaza, E. (2012). The study of medicinal plants usage through urban green space. Annals of Biological organisms and plays an important role in the biosynthesis Research 3 (4), 1934-1937. of enzymes, auxins and some proteins [26, 27, 28]. A crit- -1 [11] Mertens, D. (2005a). AOAC Official Method 922.02. Plants ical toxic level of Zn in the leaves is about 100 mg kg in Preparation of Laboratuary Sample. Official Methods of Anal- dry plant matter [3]. Zinc levels can be enhanced in auto- ysis, 18th edn. Horwitz, W., and G.W. Latimer, (Eds). Chapter mobile exhaust, also may be elevated near roadways due to 3, pp1-2, AOAC-International Suite 500, 481. N F Avenue, tire wear. According to the results, Zn level in rose plants Gaitherburg, Maryland 20877-2417, USA. is not harmful level as a major threat to the human and en- [12] Mertens, D. (2005b). AOAC Official Method 975.03. Metal in vironment. Plants and Pet Foods. Official Methods of Analysis, 18th edn. Horwitz, W., and G.W. Latimer, (Eds). Chapter 3, pp 3-4, AOAC-International Suite 500, 481. N F Avenue, Gaither- burg, Maryland 20877-2417, USA. 4. CONCLUSION [13] Rhoades, J.D. (1996). Salinity: Electrical conductivity and total dissolved solids, pp, 417-436. In: Methods of soil analysis. In conclusion, the present study provides data on Part III. Chemical Methods (Bartels, J.M., and J.M. Bigham heavy metal (Pb, Cd, Ni, Zn) distribution in the soil and eds.) 2nd Ed. ASA SSSA Publisher Agronomy. No: 5 Madi- son, Wisconsin, USA. leaves and roots of roses planted in six main intersections in Erzurum city center. High levels of Pb, Ni, Cr, and Zn [14] Olsen, S.R., Cole, C.V., Watanabe, F.S. and Dean. L.A. (1954). Estimation of Available Phosphorus in Soils by Ex- contents were obtained for the plant tissue rather than soils traction with Sodium Bicarbonate. USDA, Circ 939, Washing- indicating phytoremediation capacity of roses. We there- ton, DC. fore suggest that plantation of roses on main intersections [15] McLean, E.O. (1982). Soil pH and lime requirement, pp,199- in city centers both for recreational purposes and also in 224. Methods of soil analysis. Part II. Chemical and microbi- order to prevent excessive accumulation of these metals in ological properties In: (Page, A.L., R.H. Miller and D.R. nd the soils. Keeney eds). 2 Ed., ASA SSSA Publisher, Agronomy. No: 9 Madison, Wisconsin, USA.

The authors have declared no conflict of interest. [16] Lindsay, W.L. and Norvell, W.A. (1978). Development of a DTPA soil test for zinc, iron, manganese and copper. Soil Sci- ence Society of American Journal 42, 42-428.

[17] Ren, H.M., Wang, J.D. and Zhang, X.L. (2006). Assessment of soil lead exposure in children in Shenyang, China. Environ- REFERENCES mental Pollution 144 (1), 327-335. [18] Sutherland, R.A. and Tack, F M. (2000). Metal phase associa- [1] Hassan, M. and Mansoor, S. (2014). Oxidative stress and anti- tion in soils from an urban watershed, Honolulu, Hawaii. Sci- oxidant defense mechanism in mung bean seedlings after lead ence of Total Environment 256 (2-3), 103-113. and cadmium treatments.Turkish Journal of Agriculture and Forestry 38 (5), 55-61. [19] Harper, C.C., Mathee, A., von Schirnding, Y., De Rosa, C.T. and Falk, H. (2003). The health impact of environmental pol- [2] Turan, M. and Ercisli, S. (2007). Evaluation of phytoremedia- lutants: a special focus on lead exposure in South Africa. In- tion potential of Rose species, Asian Journal of Chemistry 19 ternational Journal Hygiene and Environmental Health 206, (6), 4895-4902. 315-322.

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[20] Nabulo, G., Oryem-Origa, H. and Diamond, M. (2006). As- sessment of lead, cadmium, and zink contamination of roadside soils, surface films, and vegetables in Kampala City, Uganda. Environmental Research 101(1), 42-52. [21] EPA (United States Environmental Protection Agency). (1998) Reducing health risks worldwide. EPA's Int Lead Risk Reduc Pro, 3-4. EPA 160-K-98-Q01. [22] ILO (Encyclopaedia of Occupational Health and Safety). (1998) Fourth addition, Volume III, Metals: Chern Prop and Tox, Lead 63, 21. International Labor Office, Geneva. [23] Traunfeld, J.H. and Clement, D.L. (2001). Lead in Garden Soils. Home and Garden. Maryland Cooperative Extention, University of Maryland. http://www.hgic.umd .edu/ media/documents/hg18.pdf [24] Erakhrumen, A. and Agbontalor, A. (2007). Review Phytore- mediation: an environmentally sound technology for pollution prevention, control and remediation in developing countries. Educational Research and Review 2 (7):151–156. [25] Erdei, L., Mezosi, G., Mecs, I., Vass, I., Foglein, F. and Bulik, L. (2005). Phytoremediation as a program for decontamination of heavy-metal polluted environment. Acta Biologica Szege- diensis 49 (1-2): 75-76. [26] Guderian, R. (1977). Air pollution, phytotoxicity of acidic gases and its significance in air pollution control. Springer Verlag, Berlin. [27] Onder, S. and Dursun, S. (2006). Air borne heavy metal pollution of Cedrus libani (A.Rich) in the city centre of Konya (Tur- key). Atmospheric Environmental 40, 1122-1133. [28] Glass DJ (2000) Economical potential of phytoremediation. In: Raskin I, Ensley BD (eds), Phytoremediation of toxic metals: Using plants to clean up the environment, Wiley, New York, 15-31.

Received: August 06, 2014 Accepted: May 29, 2015

CORRESPONDING AUTHOR

Sezai Ercisli Ataturk University Agricultural Faculty Department of Horticulture 25240 Erzurum TURKEY

Phone: +90 442 2312599 E-mail:[email protected]

FEB/ Vol 24/ No 9/ 2015 – pages 2787 - 2791

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HEAVY METALS SORPTION ON RIVER SEDIMENTS: KINETICS, EQUILIBRIA AND RELATIVE SELECTIVITIES

Yuanxing Huang, Daofang Zhang*, Yuanheng Li, Zhihua Xu, Shijue Yuan and Lian Wang

School of Environment and Architecture, University of Shanghai for Science and Technology, Shanghai, P. R. China, 200093

ABSTRACT ment (GB 18668-2002) [5]. Lead (Pb) toxicity stems from its ability to mimic biologically essential metals such as The sorption of heavy metals by river sediment is en- iron, calcium and zinc, which can be toxic to aquatic or- vironmentally significant in that it affects the migration and ganisms even in trace amounts [6]. It was once commonly transformation of heavy metals. Heavy metals (Cr, Cd, Cu, used in gasoline, nowadays most of the lead is used for bat- Ni, Pb and Zn) sorption experiments were conducted to de- teries [1]. Chromium (Cr) is moderately toxic to aquatic fine kinetics parameters and selectivity sequence for eight organisms. Large volumes of trivalent and hexavalent toxic sediments collected from a typical East China river. The Cr compounds are used and generated from industrial pro- relationships between sorption and metal concentrations cesses such as mining, plating, tanning or corrosion inhibi- were fitted with Langmuir and Freundlich models respec- tion, and discharged into the aquatic environment. Water- tively. The results indicated that all six heavy metals borne Cr exists essentially as hydroxy-complexes and achieved sorption equilibrium on the river sediments in ﬁnally accumulates into the sediment [7, 8]. about 30 min, and the empirically determined sorption ki- In natural rivers, heavy metals exist mainly in the sed- netics fitted a pseudo-second order reaction model well. iments, water body and organisms. Their distribution Freundlich isotherm described the sorption process better changes dynamically through complicated biogeochemical than Langmuir model. The selectivity sequences of the six exchanges among different compartments under the influ- studied heavy metals by eight sediments were established ences of anthropogenic activities as well as natural im- on the basis of distribution coefficients Kd, which sug- pacts, and pose a risk to water quality and human health gested that Pb, Cu and Zn had higher affinity to the inves- [9]. The ecological risk of heavy metals in aquatic systems tigated sediments than Cd, Cr and Ni. is generally determined by their mobilisation more than by

their accumulation process [10]. For those reasons, it is of KEYWORDS: River sediments; Heavy metals; Sorption; Kinetics; great importance to study the transportation of heavy met- Selectivity sequences als, among which the sorption of heavy metals on river sediments was one of the essential topics.

1. INTRODUCTION Sediments in aquatic systems are products of soil erosion and rock weathering. They are the most important re- Due to rapid development, lots of environmental issues pository for heavy metals that enter the aquatic environ- have occurred, especially the pollution caused by heavy ments in that heavy metals are transported predominantly metals. In the past few decades, the heavy metals contami- in association with particulate matter, and furthermore sed- nation of rivers has been attributed to the effluents from in- iments also act as a sink of various other contaminants [11- dustries such as electronics, battery, petrochemical, electro- 14]. The sorption of heavy metals by sediments are highly plating, steel and iron, and dumping of rubbish [1]. Heavy influenced by pH, redox potential, sediment constituents metals are considered as not naturally decompo sable. They and fraction, particle size, temperature, heavy metal speci- are often toxic, persistent and have the characteristics of ation, ionic strength, mixing, and time, etc. [15, 16]. The bioaccumulation, thus increased their threat to water secu- threat of heavy metals pollution usually includes more than rity [2]. one metal, so the study on sorption of more than one heavy Heavy metal cadmium (Cd) is an important toxicant of metal by river sediments is desirable to examine this pro- major environmental concern, whose levels in air, water cess [17, 18]. and soils have increased several-fold in many parts of the In this paper, the following works were systematically world as a result of human activities [3]. The maximum conducted: (1) determining the sorption kinetics and equi- levels of Cd established by the Ministry of Environmental libria of sediments collected from a typical East China river Protection of China are 1.0 mg Kg-1 for class III soil (GB and its tributaries for heavy metals chromium (Cr), cad- 15168-1995) [4] and 5.0 mg Kg-1 for class III marine sedi- mium (Cd), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn); (2) examining the selectivity sequence of the 6 heavy * Corresponding author metals on the river sediments.

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2. MATERIALS AND METHODS 2.2 Sorption experiments The tests of heavy metals sorption on sediments were 2.1 Sediments sampling and characterization performed using synthetic single metal ion solutions of This study was conducted with sediments taken from CuSO4, Pb(NO3)2, Cr(NO3)3, Ni(NO3)2, Zn(NO3)2 and a typical East China river and its tributaries. Eight surface Cd(NO3)2, respectively. The kinetics experiments were sediments (depth 0-10 cm) were collected at eight different performed under 25°C with 1 g sediment (disinfected un- sites: S1 through S8 in the study area of 120.69°E, 30.61°N der 120°C for 20 min before use) suspended in 200 mL to 121.98°E, 31.63°N (Fig.1). From each site a total of sorption solution containing 100 mg L-1 each of above 2 Kg sediments were collected by a Peterson sampler (A17, metal ions, which had an initial pH of 7 (adjusted with 1/40, China). Each of the sediment was placed in polyeth- NaOH). The contact time was from 5 min to 24 h. Sorption ene bottle, sealed and labelled, then kept in ice box during isotherms were obtained from solutions containing each of transportation, and stored under -20°C in the laboratory. Be- above metal ions of 10-100 mg L-1 buffered at pH 7. An fore use, the sediment was dried at 50°C for 48 h, homoge- amount of 0.5 g disinfected sediment was suspended in 50 nized and passed through a 40 mesh sieve. The organic mat- mL of sorption solution, equilibrated under the temperature ter was quantified by measuring the total volatile solid of 25°C on a shaker for 24 h. After sorption the mixture (TVS) under 600°C. The particle size distribution was ana- was centrifuged at 4000 rpm for 10 min and filtered by a lyzed using a Laser Size Analyzer (Microtrac S3500, USA). 0.45 μm membrane. The residual heavy metals concentra- The water parameters of temperature, pH, total dissolved tions in the filtrate were quantified by an Atomic Absorp- solid (TDS) and dissolved oxygen (DO) were measured in tion Spectrophotometer (AAS, TAS-990, PERSEE). All situ with a portable water analyzer (WTW, Multi 3410, Ger- tests were performed in duplicate. many). To measure concentrations of the six heavy metals in From the mass balance, the quantity of adsorbed met- the sediments, 0.1 g of each sediments were digested with als was determined by the following equation (Eq.1): 5 mL nitric acid, hydrofluoric acid and hydrogen peroxide q = (C – C )V/m (1) (volume ratio 3:1:1) in a TOPEX (PreeKem) closed-vessel t i t microwave digestion system, the operation program used −1 were recommended by the manufacturer as follows: 160 Where qt (mmol g ) is the amount of metals sorbed at −1 for 3 min under the pressure of 15 atm, 190 for 4 min time t; Ci (mmol L ) is the initial concentration of the −1 under the pressure of 18 atm, and 210 for 15 min under metal ions, Ct (mmol L ) is the metal ions concentration at the pressure of 22 atm. After digestion, the digests were di- time t; m (g) is the adsorbent or sediment mass and V (L) luted to a final volume of 100 mL and centrifuged for is the solution volume. 20 min under 4000 rpm. The heavy metals concentrations Sorption isotherms were fitted using the Langmuir in the centrifuged solutions were quantified by an Induc- (Eq.2) and Freundlich (Eq.3) models: tively Coupled Plasma Mass Spectrometry (ICP-MS, Nex- qe = qmKLCe/(1+KLCe) (2) ION300, Perkin Elmer, United States). Where qe is the mass of metals adsorbed per unit mass −1 All the equipments used were thoroughly cleaned and of adsorbent at equilibrium (mmol g ), qm is the empiri- left in 10% nitric acid for 24 h to prevent contamination. cally derived maximum sorption capacity to form a mono- −1 Before use it was rinsed three times by deionized water layer on adsorbent surface (mmol g ), KL is the empirically -1 supplied by Milli-Q Advantage system and dried in the derived Langmuir constant (L mmol ), Ce is the equilib- fume hood. rium concentration of metal ions (mmol L−1). 1/n qe = Kf Ce (3) 1/n (1- Where Kf is the Freundlich capacity factor (L mmol 1/n)g-1), 1/n is Freundlich intensity parameter[19]. -1 The sorption distribution coefficients Kd (Lg ) for the studied metals in different sediments were calculated from Eq.4:

Kd = qe/Ce (4)

3. RESULTS AND DISCUSSION

3.1 Characterization of sediments The sediments in the studied river and its tributaries are mostly the products of soil erosion. They have similar characteristics with the ambient soil, and often exhibit some properties of various sources such as anthropogenic FIGURE 1 - Location of sampling sites inputs and geological matrix [14]. The geochemical prop-

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erties such as redox potential, mineral and chemical com- 3.2 Kinetics of sorption ponents, particle size, and organic matter content of the Sorption kinetics for the 6 studied metals was investi- sediments could reveal the condition of the surrounding gated to find the time needed to reach equilibrium between water environment. solid and aqueous phase. It indicated that the metal ions Table 1 shows the chemical properties of the studied transferred from water to sediments quite quickly. Nearly river sediments and overlying water. Among the eight sam- all metals achieved sorption equilibrium in about 30 min pling sites, S1 and S2 are located in a typical urban resi- under the given condition. After 200 min sorption, the met- dential area mainly receiving domestic sewage; S3 and S7 als concentrations in the aqueous phase began to decrease are located in a suburb residential area; S4 is in an an- slowly. At 1440 min most of the metal concentrations cient town dependent on tourism; S5 is located in the up- reached a constant value, and the rest process will continue stream and is one of the drinking water sources; S6 and S8 very slowly for around 5 days, as shown in Fig. 2. This are in an agricultural area. means the process could be divided into 2 steps: rapid sorption stage and slow sorption stage. Previous researches The overlying water in S5, S6 and S8 had higher DO suggested the metal sorption kinetics could be fitted by a value indicating relatively clean water quality. Most of the non linear pseudo-second order reaction model, the pH values fell in near-neutral range, except that of S5, pseudo-second order reaction model assumed sorptions as which exhibited a basic pH of 9.7. The contents of organic the rate limiting process [23, 24], as Eq.5: matter in the 8 sediments ranged from 29.4 to 57.9 mg g-1. 2 The particle sizes of the 8 sediments varied with the aver- t/qt = 1/(k qe ) + t/qe (5) -1 -1 age particle size Dav ranging from 9.35 to 36.97 μm, and where k (g mmol min ) is the rate constant of sorp- particle size distribution showed that the river sediments tion. comprised mainly of silt (60%, D: 5-50 μm) and clay (30%, The calculation results were summarized in Table 2, D＜5 μm), with a small amount of sand (6%, D: 50-500 which showed that the correlation coefficients for the μm), according to the logarithmic particle size method pro- pseudo-second order reaction model were greater than 0.99 posed by Udden and Wentworth [20, 21]. in most of the cases. Moreover, the experimental and theo- The background values of Cr, Cd, Cu, Ni, Pb and Zn retical qe values were in excellent agreement. in the sediments were 0.085 mg g-1, 0.260 μg g-1, 0.032 mg In summary, the sediment of S2 exhibited the highest g-1, 0.032 mg g-1, 0.023 mg g-1 and 0.078 mg g-1, respec- sorption capacity for all metals, while the sediment of S5 tively, according to China Stream Sediment Reference Ma- had the lowest affinity for most of the metals. However, the terial [22]. The actual values varied with the location and differences of sorption capacity of all the sediments were community function of different areas, in the studied area, not remarkable for their organic matter contents and parti- the content of Cr ranged from 0.03 to 0.29 mg g-1, the Cd cle sizes, which had important impact on their sorption ca- content was from 0.20 to 1.02 μg g-1, Cu was from 0.01 to pacity, were similar; thus the results might be representa- 0.22 mg g-1, Ni was from 0.02 to 0.09 mg g-1, Pb was from tive for the river sediments in the studied region. Interpre- 0.02 to 0.09 mg g-1and Zn was from 0.03 to 0.52 mg g-1, tations of the results also revealed that Zn and Cu were which were close to the background values and had very sorbed in the greatest amounts, followed by Cr, then Cd low values that could be negligible for the performed sorp- and Ni, while Pb was sorbed in the smallest amounts in tion experiments. most of the sediments.

TABLE 1 - Selected characteristics of the studied river sediments

S1 S2 S3 S4 S5 S6 S7 S8 TDS of water (mg L-1) 0.63 0.97 0.47 0.62 0.45 0.42 0.59 0.42 DO in water (mg L-1) 0.9 0.3 0.5 0.5 6.1 5.9 0.4 4.9 Water pH 7.4 8.2 7.8 7.5 9.7 7.3 7.6 7.3 Organic matter in sediment (mg g-1) 57.9 44.3 29.4 31.5 39.0 53.9 34.7 44.7 Particle size of sediment (μm)

D5 1.04 1.08 1.10 1.13 0.91 0.68 0.96 4.96

D50 17.63 16.38 24.85 22.80 16.58 6.33 12.39 15.92

D95 71.00 66.07 110.6 82.05 81.32 28.83 45.17 63.41

Dav 25.22 23.03 36.97 29.05 29.88 9.35 16.66 22.14

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.25 .25 S2 S1 .20 .20 ) ) -1 -1 .15 .15

.10 .10 q (mmolg q (mmol g

.05 .05 Cr Cd Cu Ni Pb Zn 0.00 0.00 0 200 400 600 800 1000 1200 1400 1600 0 200 400 600 800 1000 1200 1400 1600 Time (min) Time (min)

.25 .25 S3 S4 .20 .20 ) ) -1 .15 -1 .15

.10 .10 q (mmolg q (mmol g

.05 .05

0.00 0.00 0 200 400 600 800 1000 1200 1400 1600 0 200 400 600 800 1000 1200 1400 1600 Time (min) Time (min)

.25 .25 S5 S6 .20 .20 ) )

-1 .15 -1 .15

.10 .10 q (mmol g q (mmol g

.05 .05

0.00 0.00 0 200 400 600 800 1000 1200 1400 1600 0 200 400 600 800 1000 1200 1400 1600 Time (min) Time (min)

.30 .25 S7 S8 .25 .20

) .20 ) -1 -1 .15 .15 .10 .10 q (mmol g q (mmol g

.05 .05

0.00 0.00 0 200 400 600 800 1000 1200 1400 1600 0 200 400 600 800 1000 1200 1400 1600 Time (min) Time (min)

FIGURE 2 - Sorption kinetics for Cr, Cd, Cu, Ni, Pb and Zn onto the studied river sediments (the legend is common for all plots)

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TABLE 2 - The rate constants and qe of pseudo-second order reaction equations for the sorption of 6 heavy metals

2 2 Metal Sediment k qe R Exp. qe Sediment k qe R Exp. qe Cr S1 0.51 0.16 0.999 0.16 S5 0.46 0.07 0.996 0.07 S2 0.35 0.19 0.998 0.19 S6 0.34 0.09 0.998 0.09 S3 2.09 0.17 1.000 0.17 S7 0.14 0.15 0.990 0.15 S4 0.43 0.10 0.998 0.10 S8 0.22 0.08 0.996 0.08

Cd S1 1.39 0.09 0.999 0.09 S5 13.39 0.08 1.000 0.08 S2 0.85 0.12 0.999 0.12 S6 1.26 0.09 0.999 0.09 S3 0.88 0.12 0.999 0.12 S7 5.71 0.08 0.999 0.08 S4 3.64 0.08 0.999 0.08 S8 2.18 0.08 0.999 0.08

Cu S1 1.03 0.18 0.999 0.18 S5 3.77 0.17 1.000 0.17 S2 0.22 0.22 0.996 0.23 S6 20.54 0.22 1.000 0.22 S3 0.23 0.21 0.996 0.21 S7 0.15 0.25 0.995 0.25 S4 2.39 0.18 1.000 0.18 S8 0.90 0.19 0.999 0.19

Ni S1 0.12 0.08 0.974 0.08 S5 1.25 0.06 0.999 0.07 S2 0.11 0.13 0.978 0.13 S6 0.49 0.10 0.998 0.10 S3 0.16 0.10 0.978 0.10 S7 0.54 0.10 0.998 0.10 S4 0.53 0.08 0.996 0.08 S8 0.59 0.09 0.999 0.09

Pb S1 0.37 0.07 0.995 0.07 S5 1837 0.04 0.999 0.04 S2 0.69 0.08 0.999 0.08 S6 1.37 0.06 0.999 0.06 S3 0.40 0.07 0.996 0.07 S7 1.72 0.07 0.999 0.07 S4 0.95 0.05 0.998 0.05 S8 0.31 0.07 0.990 0.07

Zn S1 1.20 0.19 0.999 0.19 S5 0.76 0.18 0.999 0.18 S2 0.81 0.19 0.999 0.19 S6 0.44 0.22 0.999 0.22 S3 0.65 0.20 0.999 0.20 S7 0.46 0.23 0.999 0.23 S4 0.57 0.19 0.999 0.19 S8 0.40 0.19 0.998 0.20

3.3 Sorption equilibria .18 2D Graph 1 Sorption equilibrium isotherms were generated with .16 S1 sediment chosen to be the model sediment, and the equi- ) -1 .14 librium data were fitted using both the Langmuir and Freundlich models. .12 .10 Because of the heterogeneity of the sediments, most of the isotherms curves in Fig. 3 were irregular. Another reason .08 .06 Cr was that the experiments were performed at pH of 7 to sim- Cd ulate the natural environment. Usually under this condition, Cu .04 Ni not all metals were fully soluble. Part of the metals was re- g (mmol sorbed Metals Pb moved through the mechanism of precipitation or surface .02 Zn precipitation, which is a function of pH and the quantities of 0.00 metals and anions. Moreover, a few amounts of precipitates 0.00 .05 .10 .15 .20 .25 .30 might be adsorbed by sediments in the form of particle, thus -1 Equilibrium concentration (mmol L ) led to non-ideal results. The sorption equilibrium isotherms were similar upward sloping curves and indicated a multi- FIGURE 3 - Sorption isotherms of six studied heavy metals layer adsorption. Actually, the retention mechanism of metal ions on surface of soils or sediments is often complex. Three Table 3 lists the binding capacity and binding affinity important processes including adsorption, surface precipita- of the two models. As shown, the experimental data fitted tion and fixation are generally applied to explain the transfer the Freundlich model more closely than the Langmuir of metal ions from aqueous phase to the contiguous solid model. Langmuir model is an empirical model that as- phase. The organic matter and hydrous oxide minerals exist- sumes a fixed number of accessible sites available on the ing in the sediments often possess surface carboxylic and hy- adsorbent surface, and the number of adsorbed and de- droxyl groups which could exchange or bind with metal ions sorbed molecules on unit surface in unit time is equal. in surrounding solution. The products of these processes are However, departure from the assumptions, which is very usually a ternary metal complex composed of three compo- common for natural sediments, can have a canceling effect, nents – metal, ligand and reactive surface [25-27]. so the interaction between the studied metals and sediment

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surface sites could not be described by a single parameter. water temperature, which always changes with the seasons. The source of deviations between experimental data and Although some literatures suggest an increase in loading calculated results mainly stems from the imperfect crystal capacities of metals with increasing temperature [29, 30], lattice as well as different nature and position of charges on there are also some literatures which imply that the influ- the sediment surface [26]. ence of temperature on metal sorption is not evident, or at least, temperature is not the dominant factor to impact the 3.4 Sorption selectivity sequence sorption of metals [31, 32]. The parameters controlling heavy metal sorption and The present study examined the sorption behaviour of their distribution between sediment and water include pH, the six metals on the river sediments by comparing their organic matter content, sediment minerals, metal specia- distribution coefficients Kd. Many literatures have deter- tion, particle size and temperature, etc. [26, 28]. Generally mined the distribution of metals between solid matrix and for natural water bodies in a relatively small geographic metal ion solution by using Kd. It is assumed that the sorp- area, the environmental condition such as pH, oxidation re- tion or desorption equilibrium was achieved during a cer- duction potential and mineral constituents does not fluctu- tain period of contact time and calculated for the purpose ate wildly, as in the case of this work. The exception is the of ranking metals according to their extent of sorption. From

TABLE 3 - Langmuir and Freundlich correlations for the sorption process

Metal Langmuir Freundlich 2 2 Equation KL qm R Equation Kf n R Cr y=0.71x-0.51 0.31 1.41 0.884 Cd y=6.54x+5.84 105.8 0.15 0.875 Cu y=2.11x+3.32 103.2 0.47 0.978 Ni y=2.88x+1.38 2.08 0.35 0.56 y=0.78x-0.47 0.34 1.28 0.986 0 Pb y=3.62x+7.47 107.4 0.28 0.940 Zn y=3.25x+0.45 7.21 0.31 0.67 y=0.80x-0.07 0.85 1.25 0.987 2

-1 TABLE 4 - The sorption distribution coefficients (Kd, Lg ) for the 6 heavy metals

Metal Sediment Kd sorption Sediment Kd sorption Cr S1 0.14 S5 0.05 S2 0.19 S6 0.06 S3 0.15 S7 0.13 S4 0.07 S8 0.05

Cd S1 0.22 S5 0.14 S2 0.43 S6 0.19 S3 0.39 S7 0.16 S4 0.15 S8 0.15

Cu S1 0.28 S5 0.25 S2 0.54 S6 0.46 S3 0.43 S7 0.79 S4 0.26 S8 0.30

Ni S1 0.06 S5 0.05 S2 0.13 S6 0.08 S3 0.08 S7 0.08 S4 0.06 S8 0.07

Pb S1 0.64 S5 0.15 S2 1.25 S6 0.40 S3 0.62 S7 0.71 S4 0.19 S8 0.46

Zn S1 0.35 S5 0.31 S2 0.36 S6 0.52 S3 0.37 S7 0.59 S4 0.33 S8 0.36 Initial concentration of the studied heavy metals in aqueous phase: 100 mg L-1.

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TABLE 5 - The sorption sequences of the 6 studied heavy metals by 8 sediments and sediment ranking according to Kd

Selectivity sequences S1 Pb＞Zn＞Cu＞Cd＞Cr＞Ni S5 Zn＞Cu＞Pb＞Cd＞CrNi S2 Pb＞Cu＞Cd＞Zn＞Cr＞Ni S6 Zn＞Cu＞Pb＞Cd＞Ni＞Cr S3 Pb＞Cu＞Cd＞Zn＞Cr＞Ni S7 Cu＞Pb＞Zn＞Cd＞Cr＞Ni S4 Zn＞Cu＞Pb＞Cd＞Cr＞Ni S8 Pb＞Zn＞Cu＞Cd＞Ni＞Cr Sediment ranking Cr S2＞S3＞S1＞S7＞S4＞S6＞S8S5 Cd S2＞S3＞S1＞S6＞S7＞S8S4＞S5 Cu S7＞S2＞S6＞S3＞S8＞S1＞S4＞S5 Ni S2＞S3S6S7＞S8＞S1S4＞S5 Pb S2＞S7＞S1＞S3＞S8＞S6＞S4＞S5 Zn S7＞S6＞S3＞S2S8＞S1＞S4＞S5

Kd values it is also possible to estimate the mobility and ACKNOWLEDGEMENTS fate of competing metals in the sediments. High Kd values indicated stronger metal sorption or retention by sedi- The authors would like to thank the Shanghai Science ments, while low values signified the important proportion and Technology Development Fund for Environmental of metal desorption from the sediments and a high amount Protection (No.2012-03), the Innovation Program of of metal remains in the solution [28, 33, 34]. Shanghai Municipal Education Commission (Grant number 15ZZ075) and the Hujiang Foundation of China Table 4 shows the Kd sorption for the 6 metals in 8 sed- (B14003) for funding support. iments. Table 5 shows the sorption selectivity sequences of the 6 studied metals by the 8 sediments and sediment rank- The authors have declared no conflict of interest. ing. It is indicated that in most of the cases, Pb, Cu and Zn had the highest sorption affinity to the studied sediments, followed by Cd, while Cr and Ni had the least sorption affinity to the studied sediments with little variation. This REFERENCES means that under competitive condition, the preferentially sorbed metals would be Pb, Cu and Zn. The selectivity of [1] Ismail, Z., Salim, K., Othman, S.Z., Ramli, A.H., Shirazi, heavy metals was related to their relevant properties such as S.M., Karim, R. and Khoo, S.Y. (2013) Determining and comparing the levels of heavy metal concentrations in two selected first hydrolysis constants of the metal cations, the atomic urban river water. Measurement 46, 4135-4144 weight and ionic radius [34, 35]. The sediment ranking was [2] Xu, L., Wang, T., Ni, K., Liu, S., Wang, P., Xie, S., Meng, J., established based on the Kd value. It can be seen that among Zheng, X. and Lu, Y. (2013) Metals contamination along the the 8 sediments, S2 had the highest sorptive capacities for watershed and estuarine areas of southern Bohai Sea, China. Cr, Cd, Ni and Pb, and the least sorbent sediment was S5. Marine Pollution Bulletin 74, 453-463 This might be ascribed to many reasons, such as sediment [3] Galunin, E., Ferreti, J., Zapelini, I., Vieira, I., Ricardo Teixeira constituents and mineral composition, or different intrinsic Tarley, C., Abrão, T. and Santos, M.J. (2014) Cadmium mo- mechanisms in sediments sorption properties towards differ- bility in sediments and soils from a coal mining area on Tibagi ent metals. This deserves further exploration. River watershed: Environmental risk assessment. Journal of Hazardous Materials 265, 280-287.

[4] China, M.o.E.P.o.t.P.s.R.o. 1995 Environmental quality stand- 4. CONCLUSIONS ard for soils, GB15168-1995, Beijing. [5] General Administration of Quality Supervision, All the 6 investigated heavy metals achieved sorption I.a.Q.o.t.P.s.R.o.C. 2002 Marine sediment quality, GB 18668- 2002, Beijing. equilibrium on the studied river sediments in about 30 min, and the following slow sorption continued for the rest of [6] Marasinghe Wadige, C.P.M., Taylor, A.M., Maher, W.A., Ubrihien, R.P. and Krikowa, F. (2014) Effects of lead-spiked the time. The sorption kinetics fitted the pseudo-second or- sediments on freshwater bivalve, Hyridella australis: linking der reaction model well with good correlation coefficients. organism metal exposure-dose-response. Aquatic Toxicology Because of the heterogeneity of the studied river sedi- 149, 83-93. ments, most of the sorption isotherm curves were irregular. [7] Dong, C.-D., Chen, C.-W. and Chen, C.-F. (2013) Distribution The sorption process fitted the Freundlich isotherm better and contamination status of chromium in surface sediments of than the Langmuir model. By comparing the distribution northern Kaohsiung Harbor, Taiwan. Journal of Environmen- tal Sciences 25, 1450-1457. coefficients Kd sorption, it was concluded that the studied river sediments had higher sorptive capacity for Pb, Cu and [8] Pazos-Capeáns, P., Barciela-Alonso, M.C., Herbello-Her- Zn, while Cr and Ni had the least sorption affinity to the melo, P. and Bermejo-Barrera, P. (2010) Estuarine increase of studied sediments. chromium surface sediments: Distribution, transport and time evolution. Microchemical Journal 96, 362-370.

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[9] Zhang, M., Cui, L., Sheng, L. and Wang, Y. (2009) Distribu- [27] Temminghoff, E.J.M., Van der Zee, S.E.A.T.M. and de Haan, tion and enrichment of heavy metals among sediments, water F.A.M. (1997) Copper Mobility in a Copper-Contaminated body and plants in Hengshuihu Wetland of Northern China. Sandy Soil as Affected by pH and Solid and Dissolved Organic Ecological Engineering 35, 563-569. Matter. Environmental Science & Technology 31, 1109-1115. [10] Helios Rybicka, E., Calmano, W. and Breeger, A. (1995) [28] Cerqueira, B., Covelo, E.F., Andrade, L. and Vega, F.A. Heavy metals sorption/desorption on competing clay minerals; (2011) The influence of soil properties on the individual and an experimental study. Applied Clay Science 9, 369-381. competitive sorption and desorption of Cu and Cd. Geoderma 162, 20-26. [11] Gao, X. and Li, P. (2012) Concentration and fractionation of trace metals in surface sediments of intertidal Bohai Bay, [29] Green-Ruiz, C., Rodriguez-Tirado, V. and Gomez-Gil, B. China. Marine Pollution Bulletin 64, 1529-1536. (2008) Cadmium and zinc removal from aqueous solutions by Bacillus jeotgali: pH, salinity and temperature effects. Biore- [12] Ridgway, J. and Shimmield, G. (2002) Estuaries as Reposito- source Technology 99, 3864-3870. ries of Historical Contamination and their Impact on Shelf Seas. Estuarine, Coastal and Shelf Science 55, 903-928. [30] Sirola, K., Laatikainen, M. and Paatero, E. (2010) Effect of temperature on sorption of metals by silica-supported 2-(ami- [13] Yu, G.B., Liu, Y., Yu, S., Wu, S.C., Leung, A.O.W., Luo, nomethyl)pyridine. Part II: Sorption dynamics. Reactive and X.S., Xu, B., Li, H.B. and Wong, M.H. (2011) Inconsistency Functional Polymers 70, 56-62. and comprehensiveness of risk assessments for heavy metals in urban surface sediments. Chemosphere 85, 1080-1087. [31] Aston, J.E., Apel, W.A., Lee, B.D. and Peyton, B.M. (2010) Effects of cell condition, pH, and temperature on lead, zinc, [14] Yuan, X., Zhang, L., Li, J., Wang, C. and Ji, J. (2014) Sedi- and copper sorption to Acidithiobacillus caldus strain BC13. ment properties and heavy metal pollution assessment in the Journal of Hazardous Materials 184, 34-41. river, estuary and lake environments of a fluvial plain, China. CATENA 119, 52-60. [32] Biesuz, R., Pesavento, M., Gonzalo, A. and Valiente, M. (1998) Sorption of proton and heavy metal ions on a [15] Jho, E.H., Lee, S.B., Kim, Y.J. and Nam, K. (2011) Facilitated macroporous chelating resin with an iminodiacetate active desorption and stabilization of sediment-bound Pb and Cd in group as a function of temperature. Talanta 47, 127-136. the presence of birnessite and apatite. Journal of Hazardous Materials 188, 206-211. [33] Chaturvedi, P.K., Seth, C.S. and Misra, V. (2007) Selectivity sequences and sorption capacities of phosphatic clay and hu- [16] Murakami, M., Nakajima, F. and Furumai, H. (2008) The sorp- mus rich soil towards the heavy metals present in zinc mine tion of heavy metal species by sediments in soakaways receiv- tailing. Journal of Hazardous Materials 147, 698-705. ing urban road runoff. Chemosphere 70, 2099-2109. [34] Usman, A.R.A. (2008) The relative adsorption selectivities of [17] Chiang, Y.W., Ghyselbrecht, K., Santos, R.M., Martens, J.A., Pb, Cu, Zn, Cd and Ni by soils developed on shale in New Swennen, R., Cappuyns, V. and Meesschaert, B. (2012) Ad- sorption of multi-heavy metals onto water treatment residuals: Valley, Egypt. Geoderma 144, 334-343. Sorption capacities and applications. Chemical Engineering [35] Vega, F.A., Covelo, E.F. and Andrade, M.L. (2006) Competi- Journal 200–202, 405-415. tive sorption and desorption of heavy metals in mine soils: In- [18] Covelo, E.F., Vega, F.A. and Andrade, M.L. (2007) Heavy fluence of mine soil characteristics. Journal of Colloid and In- terface Science 298, 582-592. metal sorption and desorption capacity of soils containing endogenous contaminants. Journal of Hazardous Materials 143, 419-430. [19] George Tchobanoglous, F.L.B., H. David Stensel 2003 Wastewater Engineering, Treatment and Reuse, McGraw-Hill.

[20] Wentworth, C.K. (1922) A scale of grade and class terms for clastic sediments. Journal of Geography 30, 377-392.

[21] Udden, J.A. (1914) Mechanical composition of clastic sediments. Geological Society of America Bulletin 25, 655-744. Received: September 01, 2014 [22] Zhang, W., Feng, H., Chang, J., Qu, J., Xie, H. and Yu, L. Revised: December 10, 2014 (2009) Heavy metal contamination in surface sediments of Accepted: January 23, 2015 Yangtze River intertidal zone: An assessment from different indexes. Environmental Pollution 157, 1533-1543. [23] Božić, D., Gorgievski, M., Stanković, V., Štrbac, N., Šerbula, CORRESPONDING AUTHOR S. and Petrović, N. (2013) Adsorption of heavy metal ions by beech sawdust – Kinetics, mechanism and equilibrium of the process. Ecological Engineering 58, 202-206. Daofang Zhang School of Environment and Architecture [24] Kwon, J.-S., Yun, S.-T., Lee, J.-H., Kim, S.-O. and Jo, H.Y. University of Shanghai for Science and Technology (2010) Removal of divalent heavy metals (Cd, Cu, Pb, and Zn) and arsenic(III) from aqueous solutions using scoria: Kinetics No. 516 Jungong Road and equilibria of sorption. Journal of Hazardous Materials 174, Shanghai, 200093 307-313. P. R. CHINA [25] Benjamin, M.M. and Leckie, J.O. (1981) Conceptual model for metal-ligand-surface interactions during adsorption. Envi- Phone: 86-21-55275979 ronmental Science & Technology 15, 1050-1057. Fax: 86-21-55275979 [26] Bradl, H.B. (2004) Adsorption of heavy metal ions on soils E-mail: [email protected] and soils constituents. Journal of Colloid and Interface Science 277, 1-18. FEB/ Vol 24/ No 9/ 2015 – pages 2792 - 2799

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THE IMPACTS OF LAND USE ON MACROINVERTEBRATES COMMUNITIES IN QINGHE RIVER DRAINAGE, LIAOHE RIVER BASIN, CHINA

Fayun Li1,2,*, Yanjie Wang1, Zhiping Fan1, Xingna Lv3, Yaming Hu3 and Lizhu Wang4

1Institute of Eco-environmental Sciences, Liaoning Shihua University, Fushun, 113001 China 2School of Environmental Sciences, Liaoning University, Shenyang, 110036 China 3Tieling Academy of Environmental Sciences, Tieling, 112000 China 4Institute for Fisheries Research, Michigan Department of Natural Resources, University of Michigan, 1105 N. University, Ann Arbor, MI 48109, USA

ABSTRACT been extensively used for biomonitoring programs of freshwater systems. Macroinvertebrate assemblage structure can As part of the basin-wide restoration effort of Liaohe provide consistent and complementary information with River, we studied macroinvertebrate communities and rel- other aquatic organisms (e.g., fish) on environmental quality. evant physic-chemical measures at 18 locations within the Furthermore, their characteristics are known in relation to four major land-use types across the Qinghe River drain- multiple environmental factors such as water quality, hydro- age. A total of 36 macroinvertebrates taxa belonging to three logical regimes, and geomorphological alterations [5, 6]. phyla and three classes were found. The dominant macroinvertebrate assemblages and densities of benthic fauna were The impact of different land-use types on river health assessment has been considered as one of the most promi- distinct among the four groups. Significant variation in the environmental parameters was observed among different nent environmental issues in many regions [7]. Variation groups, except pH, COD , and SS. Using a combination in land uses could change habitat of river ecosystem and Mn water quality, then affect benthic macroinvertebrate com- of No. taxa, Diversity, Richness, Evenness, No. EPT, and EPT%, we found that most of these biological indices re- munities [8]. The effects of land desertification have been vealed significant differences (P<0.05) between four investigated on benthic fauna of rivers and their relationship was linked to environmental conservation [9]. land-use groups. NMDS and BIOENV analyses implied that macroinvertebrate assemblage characteristics were Macroinvertebrate species are sensitive to disturbance strongly linked with the health conditions of the river and could be used to represent functional relationships with segments and their associated physicochemical measures. important environmental selective forces [10, 11]. Multi-

variate approaches based on statistical relationships between macroinvertebrate composition and selected envi- KEYWORDS: Qinghe River, macroinvertebrates, land use, environmental features could be used to discern patterns in the ronmental variables, biological indices. assemblage composition and to provide a statistical objective method for grouping sites with similar macroinvertebrates communities. Wang et al. [12] established a benthic 1. INTRODUCTION index of biotic integrity (B-IBI) based on biological integrity for China's fluvial ecosystem. Along with its rapid eco- Increasing impact of stresses on ecosystems by anthro- nomic growth during the past decades, China is facing un- pogenic activities has forced the development of monitor- precedented environmental issues. Such issues are most se- ing programs and bioassessment techniques to find a uni- vere in the Liaoning Province due to its National importance versal indicator that are capable of evaluating river quality for having heavy industry centers as well as its rapid popu- and can account for a variety of effects in ecosystems [1, lation growth. Therefore, it is critically important to under- 2]. Macroinvertebrate communities play a key role in the stand the environmental risk resulted from economic de- freshwater ecosystem function and process, and are one of velopment so that the environmental impacts could be min- the most important aquatic groups selected by the Water imized and the economic development could be sustained. Framework Directive in European Commission [3] for Based on previous studies of benthic macroinvertebrates evaluating the biological integrity of the ecological status and the actual status of rivers in Liaoning province in North process [4]. Recently, macroinvertebrate assemblages have Eastern China, we studied benthic macroinvertebrates in the Qinghe River drainage of Laiohe River basin, where * Corresponding author water quality has been heavily impacted by anthropogenic

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activities. The objectives of this study were to describe the discharges, alteration of hydrological and thermal regimes, macroinvertebrate characteristics, identify their linkages and direct water uses by industrial, urban, and agricultural with the physicochemical measures, and assess stream seg- activities. Intensive agriculture, urbanization, and industri- ment health conditions in the Qinghe River system. Our alization in its watershed maintain a constant increasing goal was to contribute a subset of bioassessment data to the stress on this river. The watersheds associated with the basin-wide efforts of Liaohe River, into which the Qinghe sampling sites were relatively similar in geomorphology River flows. Furthermore, we wanted to evaluate the suit- and climate, but primarily differed in land uses and associ- ability of using macroinvertebrate communities in order to ated variation in stream water quality conditions. assess the river health to provide management with monitoring and assessment tools to sustain economic growth. 2.2 Sampling methods Eighteen river sites, that were selected based on acces- sibility, longitudinal distribution, and representation of dif- 2. MATERIALS AND METHODS ferent land uses, were sampled for macroinvertebrates and physicochemical measures in October, 2009 (Fig. 1). Five 2.1 Study area sites (W1-W5) were located upstream of Qinghe river and Qinghe River drainage is located in the North East of were classified as woodland. Four sites (C1-C4) were situ- Liaoning Province, a tributary of Liaohe River basin in ated in the upper part of the Qinghe reservoir, where the North Eastern China, with an average annual precipitation land-use type was dominated by both woodland and farm- o and temperature of 692 mm and 6.5 C, respectively. The land. Six sites (F1-F6) were located around Qinghe reser- Qinghe River drainage region is characterized by its temper- voir, where the major land utilization was agricultural. The ate monsoon climate. The maximum average precipitation remaining three sites were situated in the lower part of the and river discharge are in August, and the dry period extends basin. The sampling sites and land use types are described from November to March (autumn/winter seasons). The in Table 1. We compared the variation of macroinverte- 2 Qinghe River drains a watershed area of 5674.28 km with a brate community structures to evaluate their differences in total river length of 217 km. Water quality in the river has the four land-use types. been heavily impacted by industrial and urban pollutants’

FIGURE 1 - Sampling sites of Qinghe River in the Liaoning Province, northeastern China.

TABLE 1 - Sampling sites and four major land-use types in Qinghe River.

Sampling sites Group code Land use 1 W1, W2, W3, W4, W5 groupI Woodland 2 C1, C2, C3, C4 groupII Combination of woodland and farmland 3 F1, F2, F3, F4, F5, F6 groupIII Farmland 4 U1, U2, U3 groupIV Urban

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Macroinvertebrate samples were collected using a suber primarily Leptophlebiidae and Caenidae), Trichoptera sampler (size of 0.3×0.4 m; pore diameter of 0.425 mm) at (32%, mainly Hydropsychidae, Rhyacophilidae, and Poly- four locations on each of the 18 sites. Macroinvertebrate centropopidae), Plecoptera (22%, Nemouridae and Per- samples were collected by placing the net on rocky river lidae). At the Combination of woodland and farmland substrates and disturbing an area of 0.12 m2 immediately group, the assemblage was still dominated by Ephemerop- upstream of the net to dislodge and wash the macroinver- tera (28%) and Trichoptera (23%), but these two numbers tebrates into the net. Individual rocks in the sampled area were reduced and instead the percentage of Diptera was in- were picked up and the attached bugs were removed. The creased. The farmland macroinvertebrate communities four samples from different locations were combined and were less dominated by a single order (Diptera, Coleoptera, all of the collected aquatic organisms were preserved in and Basommatophora made up 65% of it). At the urban 70% ethanol. group, the percent of Oligochaeta (Tubificidae and Naidi- Taxonomic analysis of macroinvertebrates was carried dae) and Hirudinea clearly increased and the proportion of out in the laboratory using several identification keys and EPT contribution decreased. methods [13-15]. Identifications were made to the lowest Non-metric Multidimensional Scaling (NMDS) ordina- taxonomic group possible, and most taxa were identified to tions were used to evaluate the differences in macroinverte- the level of genus or species. Water quality samples were brate community structures. We ran a multiple response per- collected before macroinvertebrate sampling at the sa- mutation procedure to check out significant differences in meriver locations. For those samples, were measured dis- macroinvertebrate community composition among the four solved oxygen (DO), permanganate index (CODMn), dichro- land-use groups. In the NMDS analysis, the two-dimen- mate index (CODCr), biological oxygen demand (BOD5), sional ordinations produced reliable and acceptable final ammonia-nitrogen (NH3-N), suspended solids (SS), Con- stress values. The NMDS ordination plot highlighted four ductivity, total phosphorous (TP), total nitrogen (TN), and major, well-discriminated groups of macroinvertebrate as- nitrate (NO3-N) in our laboratory. semblages delineating contrasting land-use types on the basis of Bray-Curtis similarity (Fig. 2). NMDS axis 1 indicated 2.3 Data analysis a major gradient dominated by Ephemeroptera (e.g. Hepta- Based on the macroinvertebrate data, we calculated the geniidae, Baetidae), Trichoptera (e.g. Hydropsychidae, No. of taxa, Shannon–Wiener Diversity [16], Margalef Limnephilidae), Plecoptera (e.g. Nemouridae, Perlidae) to Richness [17], Pielou’s Evenness [18], No. EPT (Ephem- the negative side of the axis which was associated with sam- eroptera, Plecoptera and Tricoptera), % EPT, % intolerant pling sites of woodland group. Axis 2 was dominated by taxa and % tolerant taxa. One-way ANOVA of variance Diptera (e.g. Chironomidae, Tipulidae), Coleoptera (e.g. and post hot test of Least Significant Difference (LSD) Haliplidae, Dytiscidae), Basommatophora (e.g. Lymnae- were used to test for significant differences in macroinver- idae, Planorbidae) at the negative side of the axis. Macroin- tebrate community’s descriptive parameters and environ- vertebrate communities from groups II and III were graded mental factors between sampling sites. Macroinvertebrate to the left and right bottom of the diagram at the negative community similarities were described by non-metric direction of NMDS axis 2, respectively. The positive side of multi-dimensional scaling (NMDS) [19]. The data was axis 2 was mainly represented by Oligochaeta (e.g. Tubifici- log(x+1) transformed and tested. To assess the relationship dae, Naididae), Hirudinea (e.g. Erpobdellidae, Glossiphoni- between macroinvertebrate community structures and en- idae) which were associated with urban sites. vironmental factors, BIOENV program was carried out using Primer5.0 package. The density of benthic macroinvertebrate community was calculated as the number of individuals per square meter according to the sampling surface area. Benthic ma- 3. RESULTS croinvertebrate community density ranged from 369 to 1082 ind·m−2, where the highest and lowest densities were 3.1 Spatial variation in macroinvertebrate assemblage compo- observed in combination of woodland and farmland groups sition and environmental parameters (Fig. 3), and farmland group, respectively. The results in- We collected 36 benthic macroinvertebrate taxa that be- dicated significant variations (F=11.496, p<0.001) in the longed to three phyla (Annelida, Mollusca, Arthropoda) and mean densities of the four different land use groups. three classes (Hirudinea, Gastropoda, Insecta) from Qinghe Among the groups, the highest mean density of all the ben- River sampling sites. Overall, macroinvertebrate communi- thic organisms was recorded for group II (885 ind·m−2), ties were predominated by aquatic insects (accounted for followed by group IV (805 ind·m−2), group I(635 ind·m−2), 78.8%). Diptera accounted for 39% of macroinvertebrate and group III (458 ind·m−2). The differences on the mean community, with Ephemeroptera (26%) and Trichoptera density between groups were significant (on average (22%) being the next most abundant orders. The macroin- p<0.01), with the exception of group IV vs I and IV vs II. vertebrate families appeared to be strongly associated with The difference of mean density between group I and III was different land-use types (Table 2). At the woodland group, significant at the 5% level. The results did not indicate sig- the major macroinvertebrate taxa recorded were Ephemer- nificant variations in density across group I and IV, and optera (comprised of 35% macroinvertebrate communities, group II and IV.

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TABLE 2 - Percentage of the most common macroinvertebrate families at four major land-use types.

Sampling sites Macroinvertebrate Combination of woodland order and family Woodland (%) Farmland (%) Urban (%) and farmland (%)

Diptera 17 22 29 32 Chironomidae Simuliidae 39 35 26 0 Tipulidae 36 41 19 4 Tabanidae 12 23 31 34

Trichoptera Hydropsychidae 41 32 21 6 Rhyacophilidae 49 34 17 0 Limnephilidae Polycentropopidae 33 42 21 4 55 21 24 0 Ephemeroptera Baetidae 19 24 39 18 Heptageniidae 37 33 20 10 Leptophlebiidae 46 38 15 1 Caenidae 62 34 4 0 Plecoptera Nemouridae 58 42 0 0 Perlidae 63 36 1 0 Coleoptera Elmidae 24 31 28 17 Haliplidae 18 36 24 22 Dytiscidae 21 38 36 5 Hemiptera Corixoidea 19 25 34 22 Basommatophora Lymnaeidae 17 26 30 27 Planorbidae 21 28 32 19 Hirudinea Erpobdellidae 5 17 38 40 Glossiphoniidae 7 22 29 42 Oligochaeta Tubificidae 2 10 23 65 Naididae 9 19 34 38

1300 U1 U3 1200 Axis 2 U2

1100

1000

900 2 800

700 W1 600

W4 ind/m Density 500 W2 W3 Axis 1 400 W5 300

200

100

C3 0 C1 Woodland Combination Farmland Urban C2 F2 F3 C4 FIGURE 3 - The densities (bars indicated standard deviation) of ben- F6 F5 thic macroinvertebrate communities for the four different land-use F4 F1 types in the Qinghe River drainage. FIGURE 2 - Non-metric multidimensional scaling (NMDS) ordination plot of community composition (Bray–Curtis similarity on pres- A range of environmental variables was recorded within ence/absence data) for all sampling sites. each of the four groups (Table 3). Except for pH, CODMn,

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and SS, significant variation was observed in the environ- ness decreased across the four groups ranging from an aver- mental parameters among different groups (ANOVA results age of 24 taxa at group I to nine taxa at group IV. With Shan- of LSD test, P<0.05). Physiochemical data provided a clear non-Wiener diversity index, we incorporated both the Mar- trend of increased or decreased from the upper catchment galef Richness as well as the Pielou’s Evenness of the num- groups and those in the middle and lower catchments. The bers of individuals in each taxon. Higher values of the three LSD test results of NH3-N and TN revealed significant dif- indices were registered in the woodland group, while lower ference at the 1% value within each pair of the four groups. values were recorded in the urban group. The highest mean Mean NH3-N and TN increased from group I and IV. The value of No. EPT was found at group I (13.8), followed by DO and conductivity were significantly varied among the group II (11.25), group III (6.83), and group IV (1.33). The zones considered (p<0.05). The mean values of conductiv- mean percent EPT taxa was higher in the woodland group, ity were 207-218 µs·cm−1 within the forward three groups, while it was successively decreased for other groups. The whereas it reached 356 µs·cm−1 in the urban group. The percent of intolerant and tolerant taxa was employed for LSD test indicated that group IV differed significantly benthic fauna in order to determine characteristics of ma- from the other three groups. The DO showed a higher level croinvertebrate assemblages. in combination of woodland and farmland groups (mean of 8.52 mg/l), followed by the single woodland group (mean There were significant differences between the three of 7.89 mg/l), reaching its lowest level in the urban group pairs of groups (I-III, I-IV, II-IV) in the total of eight biolog- (mean of 6.7 mg/l). There is significant difference of DO ical metrics at the 1% level based on the ANOVA and LSD between group II and group IV by LSD test. Mean values test (Table 4). Significant differences in No. taxa were found of CODCr (12.89-24.55), NO3-N (0.99-1.86), and TP (0.05- between group I and II, and group III and IV (P<0.05), while 0.32) were 2-3 folds higher at the downstream groups as no significant differences were recorded for No. taxa be- compared to those of the upper basin groups. The distinc- tween group II and III (P>0.05). Group I, II, and III had tion was outlined by LSD test, each pair of the four groups significantly higher values as compared to those of the revealed significant difference (p<0.05). Mean values of group II (p<0.01), III and IV (P<0.05), respectively. The BOD5 ranged from 3.31 to 5.19 at all groups, where higher richness and evenness differences were observed and out- values were registered in the urban group, while lower val- lined by a multiple comparison test (P<0.01) between farm- ues were observed near woodland. Group I and II differed land and urban groups. The richness and evenness did not significantly from the other two groups. significantly vary from group I to II, and from group II to III (ANOVA results of LSD test, P>0.05). The number and 3.2 The variation of biological indices in spatial distribution percent of EPT taxa at groups I and II were significantly The macroinvertebrate community structures were higher (p<0.01) when compared with those of the farmland evaluated by six structural metrics (No. taxa, Diversity, and urban site values. No. EPT and %EPT significantly dif- Richness, Evenness, No. EPT, and %EPT), where two tol- fered between land uses, being higher in the woodland erance level measured metrics (% Intolerant taxa, % Toler- group. Significant variations in intolerant taxa levels were ant taxa) were also incorporated in each group. The benthic found among the four land-use types (p<0.01), except only macroinvertebrate fauna exhibited diversities in biological one pair (groups III and IV, p<0.05). A strong difference was indices across the study area. The boxplots revealed the detected in the percent of tolerant taxa within different land- variation trend of different biological indices within the four use groups (p<0.01), except for that between groups II and major land-use types (Fig. 4). Macroinvertebrate taxon rich- III.

TABLE 3 - ANOVA results showing mean values with standard deviation, F-ratio (F) and significance level (p<0.05) of environmental variables measured in different land uses.

Sites Environmental Combination of wood- variables Woodland Farmland Urban F p land and farmland pH 7.56 (0.16) 7.58 (0.15) 7.45 (0.59) 7.28 (0.08) .462 ns DO 7.89 (1.18) 8.52 (0.26) 7.14 (1.04) 6.70 (0.70) 2.876 *

CODMn 2.61 (0.70) 2.10 (0.80) 2.66 (0.88) 2.71 (0.43) .549 ns

CODCr 12.89 (1.61) 18.72 (0.98) 22.03 (3.11) 24.55 (3.17) 3.072 *

BOD5 3.31 (0.53) 3.76 (0.21) 4.14 (0.55) 5.19 (0.29) .345 *

NH3-N 0.09 (0.01) 0.11 (0.02) 0.12 (0.04) 0.55 (0.19) 25.984 ** SS 0.22 (0.06) 0.24 (0.02) 0.24 (0.10) 0.18 (0.30) .181 ns Conductivity 218.24 (46.64) 207.85 (53.18) 216.38 (46.78) 356.67 (86.31) 5.492 * TP 0.05 (0.03) 0.17 (0.02) 0.28 (0.05) 0.32 (0.06) 1.420 * TN 1.12 (0.28) 1.23 (0.29) 1.32 (0.22) 2.73 (0.92) 10.720 **

NO3-N 0.99 (0.37) 1.22 (0.40) 1.48 (0.49) 1.86 (0.20) 3.585 *

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40 3.0

2.5

2.0

20 1.5 Diversity NO.taxa 1.0

0.5

0 0.0 Woodland Combination Farmland Urban Woodland Combination Farmland Urban

1.0 3.5

3.0 0.8

2.5

0.6 2.0 Evenness

0.4 1.5 Richness

1.0 0.2

0.5 0.0 Woodland Combination Farmland Urban Woodland Combination Farmland Urban

20 35

15 25

10 EPT% 15 NO.EPT

10 5

0 0 Woodland Combination Farmland Urban Woodland Combination Farmland Urban

30 70 65

25 60 55 50 20 45 40 15 35

Tolerant taxa% 30

Intolerant taxa% 10 25 20 5 15 10 0 5 Woodland Combination Farmland Urban Woodland Combination Farmland Urban

FIGURE 4 - Box-Whisker plots (Whisker: maximum, minimum; Box: 0.25 quartile, median and 0.75 quartile) for comparisons of macroinvertebrate community indices at woodland, combination of woodland and farmland, farmland and urban groups in the Qinghe River drainage.

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TABLE 4 - Mean difference and significance level of biological indices between four land-use types.

Intolerant Tolerant Groups No. taxa Diversity Richness Evenness No. EPT EPT% taxa% taxa% I--II 6.00* 0.57 ** 0.39 0.14 2.55 * 9.72* 7.41** -6.74** I--III 9.17** 1.10 ** 0.58 ** 0.23 ** 6.97 ** 13.12** 13.24** -9.89** I--IV 15.00** 1.44 ** 1.37 ** 0.45 ** 12.47** 19.45** 17.80** -33.28** II--III 3.17 0.52 * 0.19 0.09 4.42 * 3.40* 5.83** -3.15 II--IV 9.00** 0.86 * * 0.99 ** 0.31 ** 9.92 ** 9.73 ** 10.40** -26.54** III--IV 5.83* 0.34* 0.80** 0.23** 5.50** 6.33** 4.57* -23.39**

Table 5. BIOENV analysis of environmental variables to macroinvertebrate communities. Correlation Number of variables Environmental variables Significance coefficients (r) DO 0.523 * Conductivity 0.436 * Single variables CODCr 0.418 * TN 0.397 * NH3-N 0.362 * DO, Conductivity 0.624 * Double variables DO, CODCr 0.605 * The best explanatory combination of variables DO, Conductivity, CODCr, TN, NH3-N 0.826 * Asterisks indicated correlation significance level, *P<0.05

3.3 Relationships between macroinvertebrate community and taxa found in the samples in groups I and II [8]. These ma- environmental parameters croinvertebrate taxa were regarded as sensitive organisms, We used BIOENV analysis to evaluate the associa- which adapted to clean environment and stone substrate tions between macroinvertebrate assemblage and environ- [21, 22]. Analysis of the benthic macroinvertebrate assem- mental variables. Correlations of environmental variables blages was consistent with the pollution trends of different with macroinvertebrate community and significance levels land-use types. Homologous CODCr, NH3-N, and conduc- are presented in Table 5. Among all the environmental var- tivity were higher in the urban group, meanwhile farmland iables, the best single explanatory variable was DO group had higher values of TP and TN [23]. Macroinverte- (r=0.523), followed by Conductivity, CODCr, TN, NH3-N, brate taxa were dominated in farmland by Tabanidae, Dy- the correlation coefficients of the four environmental vari- tiscidae, and Chironomidae, which were tolerant to nitrog- ables were 0.436, 0.418, 0.397,0.362, respectively. The enous compounds from agricultural activities, especially double environmental variables which best explained ma- from fertilizer of cropland [24]. In woodland group, there croinvertebrate communities were DO & Conductivity was a high concentration of DO and a low concentration of (0.624), followed by DO & CODCr (0.605). The best ex- nitrogen, phosphorus, and organic pollution. The analysis planatory combination of environmental variables included of benthic fauna assessed potential features of certain con- DO, Conductivity, CODCr, TN, NH3-N, which explained tamination rather than contaminants themselves [25]. the difference of macroinvertebrate communities in the study area. The ANOVA result revealed that land-use types had a significant impact on the macroinvertebrate biological indices of Qinghe river. The differences were supported by 4. DISCUSSION NMDS ordination, which indicated distinct differences in benthic fauna structures in the four major land-use types. The percent of Chironomidae, Hirudinea (e.g. Erpob- Macroinvertebrates with high tolerance values were abun- dellidae), and Oligochaeta (e.g.Tubificidae) were espe- dant in the farmland and were dramatically increased in the cially elevated in urban group (groups III and IV) relative urban group [26]. Lower macroinvertebrate community di- to that of the former two groups, which was consistent with versity and taxonomic richness typically revealed that ur- other studies [20]. These macroinvertebrate organisms have ban group was negatively affected by the pollutants [27]. short life cycles and doughty adaptability, enabling them to However, the averaged transformed values for EPT taxa survive and establish communities in severe habitat. Exam- and percent of EPT were significantly improved in the ination of the raw data for farmland and urban groups re- woodland. Although the woodland group had a low ma- vealed low percent and density of Ephemeroptera (e.g. croinvertebrate density as compared with groups II and IV, Heptageniidae, Leptophlebiidae, Caenidae), Plecoptera it exhibited a high No. EPT and % EPT, which was con- (e.g. Perlidae, Nemouridae), and Trichoptera (e.g. Rhya- sistent with characteristics of a rare anthropogenic disturb- cophilidae, Polycentropopidae) taxa, the two dominant ance [28, 29]. Sensitivity to living habitat would help in

2806 © by PSP Volume 24 – No 9. 2015 Fresenius Environmental Bulletin

explaining the variation in EPT taxa and percentage in the Key Lab of Urban Water Resource and Environment (HIT, four groups. No.HC200902), Natural Science Foundation of Liaoning Province (No.20092013), Hundred Talents Program of BIOENV analysis suggested that macroinvertebrate Liaoning Province (No.2008921082), and the National assemblages were strongly affected by environmental con- Key Project of Sciences and Technology R & D Program ditions, which increased the variability in different groups (No.2006BAB0203). The authors are grateful to Professor [30, 31]. According to the analysis of environmental vari- Yang Mingxian for his valuable comments. ables, sites in the woodland group were characterized by high concentrations of DO in comparison with other The authors have declared no conflict of interest. groups. Group I had a higher taxa and high abundance for each taxon macroinvertebrates that were associated with oxygen content. 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[13] Annon. (1982) Freshwater Biology (the first volume: tax- [31] Matthew S.B., Musa C.M. and Jenny A.D. (2013) Macroinver- ology). Beijing: Agriculture Press. tebrates as unreliable indicators of human disturbance in tem- porary depression wetlands of the south-western Cape, South [14] Morse J.C., Yang L.F. and Tian L.X. (1994) Aquatic insects Africa. Hydrobiologia, 720: 19-37. of China useful for monitoring water quality. Nanjing: Hohai University Press. [32] Kaller M. and Kelso W. (2007) Association of macroinverte- [15] State Administration of China for Standardization. GB/T brate assemblages with dissolved oxygen concentration and wood surface area in selected subtropical streams of the south- 12763.6-2007 (2008) Specifications for oceanographic survey- eastern USA. Aquat Ecol, 41, 95–110. Part 6: Marine biological survey. Beijing: China Standard Press. [16] Shannon C.E. and Weaver W. (1949) The mathematical theory [33] Benson E.R., Wipfli M.S., Clapcott J.E. and Hughes N.F. of communication. Urbana IL: University of Illinois Press. (2013) Relationships between ecosystem metabolism, benthic macroinvertebrate densities, and environmental variables in a [17] Margalef R. (1968) Perspective in ecological theory. Chicago: sub-arctic Alaskan river. Hydrobiologia, 701:189–207. University Chicago Press. [34] Masson S., Desrosiers M., Pinel-Alloul B. and Mantel L. [18] Pielou E.C. (1975) Ecological diversity. New York: Wiley In- (2010) Relating macroinvertebrate community structure to enters. vironmental characteristics and sediment contamination at the [19] McCune B., Grace J.B. and Urban D.L. (2002) Analysis of scale of the St.Lawrence River. Hydrobiologia, 647:35–50. Ecological Communities. MjM Software Design, Gleneden [35] Johnson R.C., Jin H., Carreiro M. and Jack, J.D. (2013) Ma- 711 Beach, Oregon. croinvertebrate community structure, secondary production [20] Mesa L. (2010) Effect of spates and land use on macroinver- and trophic-level dynamics in urban streams affected by non- tebrate community in Neotropical Andean streams. Hydrobio- point-source pollution. Freshwater Biol, 58: 843-857. logia, 641:85–95.

[21] Vale M., Cabral H. and Andrade F. (2010) Distribution and structure of the upper sublittoral macrobenthic communities of Tróia sand beaches (Setúbal, Portugal) and their relationship with environmental factors. J. Environ. Monit., 12, 964–972. [22] Holmes K.L., Goebel P.C., Williams L.R. and Schecengost M. (2011) Environmental influences on macroinvertebrate assemblages in headwater streams of northeastern Ohio. J. Freshwa- ter Ecol, 26(3): 409-422. [23] Zhang Y.X., Dudgeon D., Cheng D.S., Thoe W., Fok L., Wang Z.Y. and Lee J.H.W. (2010) Impacts of land use and water quality on macroinvertebrate communities in the Pearl River drainage basin, China. Hydrobiologia, 652:71–88. [24] Pan B.Z., Wang Z.Y., Xu M.Z. and Xing L.H. (2012) Relation between stream habitat conditions and macroinvertebrate assemblages in three Chinese rivers. Quatern Int, 282:178-183. [25] Wittman J., Weckwerth A., Weiss C., Heyer S., Seibert J., Kuennen B., Ingels C., Seigley L., Larsen K. and Enos-Berlage J. (2013) Evaluation of land use and water quality in an agricultural watershed in the USA indicates multiple sources of bacterial impairment. Environ Monit Assess, 185:10395– 10420.

[26] Rasmussen J.J., Baattrup-Pedersen A., Larsen S.E. and Kron- vang B. (2011) Local physical habitat quality cloud the effect of predicted pesticide runoff from agricultural land in Danish streams. J. Environ. Monit., 13, 943-950. [27] Evans-White M.A., Dodds W.K., Huggins D.G. and Baker D.S. (2009). Thresholds in macroinvertebrate biodiversity and Received: September 01, 2014 stoichiometry across water-quality gradients in Central Plains Revised: December 31, 2014 (USA) streams. J. N. Am. Benthol. Soc., 28, 855–868. Accepted: January 30, 2015 [28] McDermond-Spies N., Broman D., Brantner A. and Larsen K. (2014) Family-level benthic macroinvertebrate communities indicate a successful relocation and restoration of a northeast CORRESPONDING AUTHOR Iowa stream. Ecol. Restor. 32 (2): 161-170. [29] Zhang Y., Zhao R., Kong W.J., Geng S.W., Bentsen C.N. and Fayun Li Qu X.D. (2013) Relationships between macroinvertebrate Institute of Eco-environmental Sciences communities and land use types within different riparian widths in three headwater streams of Taizi River, China. J. Liaoning Shihua University Freshwater Ecol, 28(3): 307-328. Fushun, 113001 P.R. CHINA [30] Robertson D.M., Graczyk D.J., Garrison P.J., Wang L.Z., Laliberte G. and Bannerman R. (2006) Nutrient concentrations and their relations to the biotic integrity of wadeable streams E-mail: [email protected] in Wisconsin. Professional Paper 1722, U.S. Department of the Interior, U.S. Geological Survey, Reston, VA. FEB/ Vol 24/ No 9/ 2015 – pages 2800 - 2808

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TOXIC EFFECTS OF DIFFERENT LONG TERM ZINC CONCENTRATIONS ON THE ACCUMULATION IN MUSCLES, VISCERAL ORGAN AND GILLS OF JUVENILES OF Solea solea L., 1758

Oylum Gökkurt Baki1,*, Birol Baki2 and Levent Bat3

1Sinop University, Vocational School of Environmental Health Program, 57000 Sinop, Turkey 2Sinop University, Fisheries Faculty, Department of Aquaculture 57000 Sinop, Turkey 3Sinop University, Fisheries Faculty, Department of Hydrobiology 57000 Sinop, Turkey

ABSTRACT Status of the Seas (GES) to the Marine Environment Policy Marine Strategy Framework Directive (MSFD) acknowl- The present study focused on the uptake and toxicity of edges that biota especially fish are important matrices for Zn in Solea solea. Laboratory studies were performed where the monitoring of certain pollutants with significant poten- metal content, were measured as a response to exposure to tial for bioaccumulation [1]. nominal concentrations of 0.01, 0.1 and 0.5 mg/L of zinc The accumulation of heavy metals, by living organ- salts (ZnCl2) for 60 days. The levels of Zn accumulated in isms is often a good integrative indicator of exposure, and edible tissues and gills of S. solea correlated well to exposure has been extensively used to assess contamination levels of concentrations and the greater the uptake. Significant accu- heavy metals in polluted ecosystems [2]. These chemicals mulations were observed in muscle Zn levels at the 0.5 mg/L often enter marine environment from industry, agriculture, treatment in comparison to controls (p<0.05) with maximum settlements, atmospheric etc. The Black Sea ecosystem has average of 3.99±0.2 mg Zn/g wet weight. Also, there were been seriously damaged as a result of these loads arise from significant increases in gill Zn concentrations at two exper- rivers, notably the Danube, Dnieper, Dniester, Don, South- imental groups compared to the controls (p<0.05) with ern Bug, Chorokh, Rioni, Yeşilırmak, Kızılırmak, Sakarya maximum averages of 82.59±6.6 and 84.48±7.2 mg Zn/g in the six Black Sea countries but also from eleven non- gill wet wt. at 0.1 and 0.5 mg/L treatments, respectively. coastal countries that belong to the Black Sea basin contrib- No significant changes were observed in visceral organ Zn ute to the loads of contaminants entering the Black Sea [3- levels after 60 days of ZnCl2 exposures. Mortality in- 5]. However the toxicity of many chemicals is relatively creased with increase in concentration of Zn on S. solea unknown, particularly to marine fish. Essential heavy met- and time of exposure. als are naturally considered as component elements of ma-

rine ecosystems and they play important roles in survival of organisms. However, if the concentrations of these met- KEYWORDS: als exceed to certain limit, it can produce cumulative dele- Solea solea, zinc, toxicity, accumulation terious effects in a wide variety of fish, because it is dis-

rupted ecology balance and caused a loss of the ecosystem. Fish may accumulate large amounts of heavy metals from 1. INTRODUCTION contaminated water. Therefore various fish species have been studied to assess the health status of marine ecosys- The pollution of marine environment with chemicals tems to monitor heavy metal pollution that could be bio- has become one of the most critical environmental prob- magnified in the food chain. lems of the world. As a result of the pollutants transport The common flatfish sole is widely distributed in the from industrial areas into the environment and their chem- shallow marine and estuarine benthic habitats of European ical persistence, many marine ecosystems are faced with waters, the Mediterranean and the Black Sea and has a pref- alarming high levels of chemicals including heavy metals. erence for relatively shallow water with sand or mud cover- The assessment of the chemical status of marine ecosys- ing the bottom. Imsland et al. [6] emphasized that S. solea tems is based on compliance with Good Environmental appears as credible candidate for marine culture. The studies on heavy metal accumulation in fishes are generally in- * Corresponding author tensified on demersal types [7, 8]. However, there is no

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available information concerning the long-term toxicity of to about 7 to prevent adsorption of the metals on the wall Zn on S. solea. Common flatfish sole has an economic value of the container. Several experimental plastic containers for the Black Sea. with 10-l aquaria of dimensions 35 x 25 x 15 cm were Zinc was chosen for the present study because of its cleaned and washed by running tap water, then by clean sea toxicity to fish was well documented [9, 10]. The objec- water. The aquaria were filled with a known volume of sea tives of the present study were to determine the 96-h and water before adding the test solutions. In the experiment different concentrations of zinc solutions (0 as control, 0.01, 0.1 60-d LC50 values of Zn, and to study their accumulations -1 in muscles, visceral organ and gills of juveniles of the com- and 0.5 mg L ) were used. No zinc was added into the con- mon flatfish sole exposed to various concentrations of zinc. trol groups. A total of 96 healthy and active juveniles were divided randomly into 4 groups, making 8 individuals in each group with 3 replicates. In one hour any juveniles that 2 MATERIALS AND METHOD were showed abnormal behaviour were removed and replaced. During the course of the experiments juveniles fed 2.1 Fish collection regularly same as acclimatization period and the test solutions were renewed weekly. This was possible because an Live juveniles of common flatfish sole (mean weight abundant supply of very clean seawater was available. The of 2.72±0.03 g and mean length of 5.84±0.14 cm) are ob- photoperiod was 8:16 h light: dark. Observations for mor- tained with shore trawl in the Province of Sinop, Akliman tality were made daily and dead fish were removed and rec- district, at a distance of 5-10 m to coast, at 1-2 m depth orded. The test was terminated after 60 days. Temperature, (Fig. 1). After collection they were transported to the wet dissolved oxygen, salinity and pH were measured in all ex- lab of Fisheries Faculty in aerated tank. periments and all replicates twice daily. Treatments were

2.2 Experimental procedure exposed to the same ecologic factors to reduce any potential physiological stress on the juveniles of S. solea encoun- Fish were kept in a holding tank (100 cm x 50 cm wide tered during bioavailability of compounds. The test organ- and 40 cm deep) filled with clean seawater as stock. They isms were weighed for each group at the beginning and at were acclimatized for 30 days and up to 1% of their the end of experiment for determination of a growth end- weights were fed with commercial dry feed pellets with point. 55% crude protein, 14% raw oil twice daily (both morning and evening). The quality of the analysis was assessed by ensuring that blanks were acceptable and also by the inclusion of a Certified Reference Material (CRM) in each analysis. Dog- fish muscle and liver (DORM 2 and DOLT 4) as reference materials from National Research Council of Canada was used as the CRM in the present study. These standards were treated and analysed under the same conditions as the samples and recoveries of the metals ranged from 96 to 104%.

2.3 Analysis of zinc End of the experiment, S. solea samples are washed in distilled deionized water and then dissected into three parts. First one included muscle tissue, second one included gills and third one included liver, kidney and digestive system as visceral organs. Each of these tissue samples was homogenized and weighed. Samples were put Erlen- meyer flasks then placed in a vented drying oven at 1050C for 24 hours to dry constant weight. Each sample was weighed again for a dry weight measurement. Then 10 mil- lilitres of HNO3 per g of dry wt. were added to each Erlen- FIGURE 1 - Location of Sinop and sampling point of the S. solea ju- meyer flask covered with watch glass and the solution was veniles. evaporated to dryness on a hot plate. The Erlenmeyer flasks were cooled room temperature overnight and they were re- The seawater used for the experiment was taken from placed to the hot plate at 600C until samples’ coloured va- Akliman of Sinop coast through a biological filter to avoid pours lose the colour in fume hood gradually. After allow- water containing suspended particles were put into a tank ing the samples to cool 1 mL HNO3 was added to bring the continually aerated in order to maintain the dissolved oxy- volume to 25 mL for muscles and to 10 mL for gills and gen levels above 60% of the air saturation value. Stock so- visceral organs with distilled water. Then they were kept at o lutions of 1000 mg Zn/1 were prepared freshly using ZnCl2 +4 C until analysis. The tissues of the fish were prepared of pure grade. The pH of the stock solutions was adjusted for analysis according to the method described by Bernhard

[11] and UNEP [12]. Zn concentrations in the samples perature, dissolved oxygen, salinity and pH values ob- were determined by using (Inductively Coupled Plasma– tained during the course of the experiments are given in Mass Spectrometer) ICP-MS mass spectrometer. The the- Table 1. Mortalities in controls were lower than 10%, indi- ory and application of Spectrochemical Analysis have been cating that a good water quality was maintained. described in detail by Lajunen [13]. The difference between groups in temperature and salinity values was not significant (p>0.05), while the dif- 2.4 Statistical analyses ference between control group and other groups in terms The statistical methods described by Finney [14] were of dissolved oxygen amount and pH values was signifi- used to calculate the LC50 for Zn. The numbers surviving cant (p<0.05). Moore and Ramamoorthy [17] pointed out in the control group were compared with those recorded in that Zn is relatively rare in nature but has a long history the sea water containing zinc, by one-way analysis of var- of use it and extraction from ores. Zn is classified as a iance (ANOVA). If the results of ANOVA were found to borderline element and reflected in its ability to form be significant, Tukey’s multiple significant difference tests bonds with oxygen. Pagenkopf [18] studied the toxicity using the software program (IBM SPSS 21) were carried of heavy metals including copper, cadmium, lead and zinc out [15]. Differences were regarded as significant when to fishes and concluded that an increase in water hardness p<0.05 level. All data prior to statistical analyses were extends to reduce the heavy metals toxicity and naturally pressed as µg/g dry weight. occurring materials often reduce the concentrations of toxic species. For determination of the changes in weights of the 3. RESULTS AND DISCUSSION fishes in the beginning and end of the study, mean weights of fishes used in each concentration is measured. In the pre- The experiments were conducted for 60 days between sent study the control and the test juveniles were fed exactly December 25, 2012 and February 22, 2013. The effect of the same amounts of food to avoid the problem of weight metal toxicity in fishes varies depending on environmental loss. In the beginning of the experiment, mean weight and conditions such as dissolved oxygen, hardness, tempera- length of juveniles was 2.72±0.03 g and 5.84±0.14 cm in ture, pH, salinity and existence of other metals in waters in each group, respectively. Mean weight and length of juve- which fishes live. Accordingly, environmental conditions niles at the end of bioassay is given in Table 2. There was affect as increase or decrease in tolerance of metal concen- not a substantial change in their body weights and lengths tration in water to metal tolerance [16]. Mean water tem- at the end of the test period (p>0.05).

TABLE 1 - Mean water temperature, dissolved oxygen, salinity and pH values determined in experimental plastic containers.

Groups Parameters Control 0.01 ppm 0.1 ppm 0.5 ppm Temperature (°C) 15.47±0.11a 15.36±0.11a 15.33±0.11a 15.48±0.10a Dissolved oxygen (mg L-1) 7.49±0.11a 7.93±0.10b 7.71±0.12b 7.68±0.11b Salinity (‰) 17.68±0.05a 17.72±0.06a 17.65±0.06a 17.68±0.05a pH 8.21±0.02a 8.31±0.01b 8,29±0,01b 8.32±0.01b Each value refers to mean ± standard error. The difference between the values marked with different letters in same line (p<0.05).

TABLE 2 - Mean weights of juveniles of S. solea at the end of bioassay (g)

Group

Control 0.01 ppm 0.1 ppm 0.5 ppm Weight of fishes 3.02±0.04a 2.99±0.02a 2.92±0.03a 2.89±0.04a Lengths of fishes 6.14±0.12a 6.32±0.16a 6.21±0.07a 5.99±0.09a Each value refers to mean ± standard error. The difference between the values marked with different letters in same line (p<0.05).

TABLE 3 - Analysis results of zinc concentrations determined in muscle, visceral organ and gills tissues of juvenile S. solea for each zinc concentrations and control group (µg/g dry wt.)

Group

Control 0.01 ppm 0.1 ppm 0.5 ppm Muscle 2.87±0.1ax 3.18±0.3abx 3.59±0.3abx 3.99±0.2bx Visceral organ 30.51±3.1ay 22.6±2.4ay 44.44±5.7by 32.23±4.8ay Gill 31.51±2.6ay 43.22±1.8az 82.59±6.6bz 84.48±7.2bz Each value refers to mean ± standard error. The difference between the values expressed with different exponential letters in same line (a, b) and same column (x, y, z) (p<0.05).

TABLE 4 - ANOVA test comparisons and Tukey post-hoc multiple comparison test results conducted with Zn concentrations determined in muscle, visceral organ and gills tissues of juvenile S. solea

df F Sig. Post Hoc Tests (between groups) Control 0.01 0.1 0.5 Control - ns ns * 0.01 ns - ns ns Muscle 3 3.979 0.053 0.1 ns ns - ns 0.5 * ns ns - Control - ns * ns 0.01 ns - * ns Visceral organ 3 1.590 0.266 0.1 * * - * 0.5 ns ns * - Control - ns * * 0.01 ns - * * Gill 3 35.755 0.000 0.1 * * - ns 0.5 * * ns - (*p<0.05; ns: non-significant).

Zinc concentrations in muscle, gills and visceral or- groups (p<0.05). Highly significant (p<0.05) differences gans of juveniles of S. solea within 60 days period are were observed in gill of 0.1 and 0.5 ppm groups compared given in Table 3. with control group. However, no significant difference was found in gill of 0.01 ppm group compared to the control There were variations in the accumulation of ZnCl2 in the muscles, visceral organ and gills of juveniles of S. solea group (Table 4). Gündoğdu et al. [10] showed that there (p<0.05). However there were no significant differences was no mortality of Onchorhyncus mykiss at all zinc con- Zn concentrations in muscle were compared with the concentrations within the 30-days period, demonstrating that trol group except the highest concentration in test organism they were not lethal for rainbow trout but the accumulation exposed 0.5 ppm. Accumulation occurred in the visceral of zinc in the tissue and organs of the rainbow trout in- organs but with lower concentrations recorded in 0.01 ppm creased with increasing metal concentrations in the me- group. Highest Zn level was recorded in the visceral organs dium. of juveniles of S. solea at 0.1 ppm group. The Zn concen- The overall mean survival in all the controls was more trations in visceral organs of control and 0.5 ppm groups than 90%, demonstrating that the holding facilities, water were not significant. Pattern of accumulation results ob- and handling techniques were acceptable for conducting tained from this study showed that ZnCl2 was accumulated bioassays. Survival of juveniles of S. solea decreased with in the gills at a fast rate than those in muscle and visceral increasing zinc concentrations (Fig. 2), but juveniles was organ of the juveniles of S. solea. Accumulation of Zn in less affected by zinc of 0.5 ppm at the beginning. However, gills of juveniles of S. solea was higher than those in mus- mortality of fish increased after 21 day, this becoming sig- cle and visceral organ particularly at 0.1 and 0.5 ppm nificant at following days. The reason for that may be in- groups (Table 3). This is due to the major role they play in terpreted as organisms’ being exposure to high concentra- gaseous exchange in fish, which also makes them a target tion performing more resistant attitude mechanism and try- site for active uptake of Zn that is present in the surrounding to protect themselves from the effects of toxicants ing water. ANOVA test and Tukey multiple comparison found in the environment by living more stable. It is known test results of Zn concentrations obtained among tissues are that organisms have such kind of attitude mechanism given in Table 4. against toxicants. Murugan et al. [19] reported lower concentration of Zn Healthy and active juveniles of similar size were taken in the gill of freshwater fish Channa puncatus at the end of from plastic stock tank and randomly assigned to bioassay the 45-day of exposure period as compared to levels found containers. Each treatment was three replicates, giving a at 30 days, and attributed this trend to induction of regula- total of 8 juveniles exposed per treatment. For LC50 calcu- tory process and biochemical defence mechanisms. Zn is lation, plastic containers were examined twice daily for pe- an essential element which can be regulated by fish over riods of 96 hours and 60 days and any dead juveniles of S. wide range concentrations [20]. solea removed. The LC50 (lethal concentration) with 95% According to the one-way analysis of variance, no sig- confidence limits was calculated by probit analysis [14]. nificant difference in Zn concentrations were recorded in Acute toxicity (96-h) of Zn for juveniles of the common fish muscle of 0.01 and 0.1 ppm groups compared with flatfish was found to be 7.09 ppm. The upper and lower control group. Zn concentrations in fish muscle of 0.5 ppm 95% confidence limits were found to be 8.51 and 5.67 ppm, group is significantly higher (p<0.05) compared to the con- respectively. Similarly, Hemalatha and Banerjee [9] ob- trol group. The accumulation of 0.1 ppm group in the vis- tained acute toxicity values of 7.5 mg/l of zinc chloride ceral organs of fish is significantly higher between all other against catfish (Heteropneustes fossilis).

100

90 Control 80 0.01 ppm

70 0.1 ppm 0.5 ppm 60

40 Survival (%)

0 0 5 10 15 20 25 30 35 40 45 50 55 60 Days

FIGURE 2 - Percent survival of juveniles of S. solea placed in sea water containing zinc (each concentration included 3 replicates).

100 90 80 70 (%)

60 50 40 Mortality 30 20 10 0 Control 0.01 ppm 0.1 ppm 0.5 ppm Concentration

FIGURE 3 - Mortality (%) of S. solea after 60 days placed in sea water containing zinc solution (each treatment included 3 replicates).

Mortality of S. solea after 60 days of exposure to zinc a focusing on the zinc and other essential elements that can contaminated sea water was given in Fig.3. The 60-day produce a hazardous influences on fish. LC50 was 0.031, ranging from 0.024 to 0.039 ppm. It is evident from the results that even low concentrations of Zn In the present study different behavioural responses of are toxic to juveniles of S. solea. However, there is no Zn salts to juveniles of S. solea were also observed available data concerning the long-term toxicity of Zn on throughout the bioassay period. The control group behaved S. solea. The variation in the LC50 values for the same in natural manner i.e., they were active with their well-co- metal may be due to species, chemical structure of metal ordinated movements. When the Zn concentrations in- compound and the conditions of the bioassays (water tem- creased gradually they slowly became lethargic, hyper ex- perature, salinity, oxygen content and pH). It can be con- cited, restless and secreted excess mucus all over the body. cluded that, although zinc is an essential metal for various Mucus secretion in fish forms a barrier between body and physiological processes, it showed a toxic effect to juve- toxic media thereby probably reduces contact of toxicant niles of S. solea. This result should be impelled us to make so as to minimize its irritating effect, or to eliminate it

through epidermal mucus. At the 0.1 and 0.5 ppm groups [9] Hemalatha, S. and Banerjee, T.K. (2003). Estimation of acute whitening in derma colour, deformation in dermis and fins toxicity of zinc chloride by histopathological analysis of the epidermal linings of operculum of the catfish Heteropneustes and scale off in fish were observed. Moreover, juveniles fossilis (Bloch). Toxicology International, 10 (1), 11-22. started to take baits as of 8th day of the bioassay. However, [10] Gündoğdu, A., Yardım, Ö., Bat., L. and Çulha, S. T. (2009). alterations to the tissues described in the present study may Accumulation of zinc in liver and muscle tissues of Rainbow be as a result of exposure to Zn salts since Zn is the only trout (Onchorhyncus mykiss Walbaum 1792). Fresenius Envi- contaminant used. Further proof of this is that no form of ronmental Bulletin,18 (1), 40-44. damage was observed in the control juveniles of S. solea. [11] Bernhard, M. (1976). Manual of möethods in Aquatic Envi- ronmental Research. Part 3. Sampling and Analysis of Biolog- Chemicals affect aquatic organisms including fish in ical Material, FAO Fish. Tech. Pap. FIRI/T, Rome, No: 158, case of food chain. In conclusion the common flatfish sole 123 pp. is one of the ecologically important fish species in the [12] UNEP (1984). Determination of Total Cd, Zn, Pb and Cu in Black Sea and juveniles may be useful as an indicator of Selected Marine Organisms by flameless AAS. Reference metal toxicity. Methods for Marine Pollution Studies, 11 Rev 1. [13] Lajunen, L.H.J. (1992). Spectrochemical Analysis by Atomic Absorption and Emission. The Royal Society of Chemistry, Cambridge. ACKNOWLEDGEMENTS [14] Finney, D.J. (1971). Probit Analysis (3rd Edition). Cambridge The authors wish to special thanks Prof. Dr. Osman University Press London, 256 pages. Nuri Ergun and Assoc. Prof. Dr. Semra Çoruh from On- [15] Zar, J.H. (1984). Biostatistical analysis. Second edition. Pren- dokuz Mayis University Engineering Faculty Environmen- tice Hall, Int., New Jersey. tal Engineering Department for their advice and contribu- [16] Witeska, M. and Jezierska, B. (2003). The effects of environ- tion to the emergence of this research. mental factors on metal toxicity to fish (Review). Fresenius Environmental Bulletin, 12: 824-829. The authors have declared no conflict of interest. [17] Moore, J.W. and Ramamoorthy, S. (1984). Heavy metals in natural waters, applied monitoring and impact assessment. Springer-Verlag, New York. [18] Pagenkopf, G. K. (1986). Metal ion speciation and toxicity in REFERENCES aquatic systems. In: H. Sigel. (Ed.) Concepts on metal ion toxicity, pp. 101-118. [1] Official Journal of the European Union (25.6.2008). DIREC- [19] Murugan, S.S., Karuppasamy, R., Poongodi, K., and Pu- TIVES DIRECTIVE 2008/56/EC OF THE EUROPEAN vansewari, S. (2008). Bioaccumulation pattern of zinc in fresh- PARLIAMENT AND OF THE COUNCIL of 17 June 2008 water fish Channa punctatus after chronic exposure. Turkish establishing a framework for community action in the field of Journal of Fisheries and Aquatic Sciences, 8:55-59. marine environmental policy (Marine Strategy Framework Di- rective). L 164: 19-40. [20] Spry, D.J., Hodson, P.V. and Wood, C.M. (1988). Relative contributions of dietary and waterborne zinc in the rainbow [2] Phillips, D.J.H. and P.S. Rainbow (1994). Biomonitoring of trout, Salmo gairdneri. Can. J. Fish. Aquat. Sci., 45: 32−41. trace aquatic contaminants. Environmental Management Se- ries, Chapman & Hall, London. [3] Bakan, G. and Büyükgüngör, H. (2000). The Black Sea. Ma- rine Pollution Bulletin, 41(1-6), 24-43.

[4] Altas, L. and Büyükgüngör, H. (2007). Heavy metal pollution in the Black Sea shore and offshore of Turkey. Environmental Geology, 52(3), 469-476. [5] Bat, L., Gökkurt, O., Sezgin, M., Üstün, F. and Sahin, F. Received: October 02, 2014 (2009). Evaluation of the Black Sea Land Based Sources of Revised: January 13, 2015 Pollution the Coastal Region of Turkey. The Open Marine Bi- Accepted: January 29, 2015 ology Journal, 3, 112-124. [6] Imsland, A.K., Foss, A., Conceiçâo, L.E.C., Dinis, M.T., Del- bare, D., Schram, E., Kamstra, A., Rema, P. and White, P. CORRESPONDING AUTHOR (2003). A review of the culture potential of Solea solea and S. senegalensis. Reviews in Fish Biology and Fisheries, 13, 379- 407. Oylum Gökkurt Baki Sinop University [7] Usero, J., Izquierdo, C., Morillo, J., Gracia, I. (2003). Heavy Vocational School of Environmental Health Program metals in fish (Solea vulgaris, Anguilla Anguilla and Liza au- rata) from salt marshes on the southern Atlantic coast of 57000 Sinop Spain. Environmental International, 29, 949-956. TURKEY [8] Vasconcelos, R. P., Reis-Santos, P., Anabela, M., Ruano, M., Costa, M. and Cabral, H. N. (2011). Trace metals (Cu, Zn, Cd E-mail: [email protected] and Pb) in juvenil fish from estuarine nurseries along the Por- tuguese coast. Scientia Marina, 75 (1), 155-162. FEB/ Vol 24/ No 9/ 2015 – pages 2809 - 2814

ADSORPTION OF COPPER(II) BY MODIFIED MAGNETITE NANOPARTICLES: ADSORPTION EFFICACY, EQUILIBRIUM, KINETIC AND REUSABILITY

Ahmad Farrokhian Firouzi1, Ali Akbar Babaei2,3,*, SeyyedehMaasoumeh Hosseini1and Fariba Heidarizadeh4

1Department of Soil Science, Faculty of Agriculture, ShahidChamran University, Ahvaz, Iran. 2Environmental Technologies Research Center, Ahvaz Jundishapur University of medical Sciences, Ahvaz, Iran. 3Department of Environmental Health Engineering, School of Public Health, Ahvaz Jundishapur University of medical Sciences, Ahvaz, Iran. 4Department of Chemistry, School of Science, ShahidChamran University, Ahvaz, Iran.

ABSTRACT electrodialysis, ion exchange, adsorption, biological treatment and photocatalysis[4, 5]. Among different physico- In the present study, sodium dodecyl sulphate–coated chemical processes, adsorption onto solid substrate materi- Fe3O4 (SDS–Mag) nanoparticles were prepared, character- als has been used as one of the most suitable process owing ized and applied to remove Cu(II) ions from aqueous solu- to its economical feasibility and environmental friendly be- tions. Scanning Electron Microscopy measurements demon- havior for the removal of heavy metals from water and strated that the particle size was about 40-60 nm. The results wastewater but low cost alternatives or cost-effective ad- indicated that Cu(II) adsorption increased with increasing sorbents are still needed. Several low cost adsorbents have solution pH up to 5.0. Kinetic studies revealed that Cu(II) been used for the removal of heavy metals including clays uptake was fast with 90% or more of the adsorption occur- [6], agricultural waste biomass [7], metal oxides [8], mi- ring within first 15 min of contact time. The adsorption of croorganisms [9], sewage sludge [10] and fly ash [11]. Cu(II) ions was relatively fast and the Avrami fractional The magnetite is among the most effective adsorbent order and pseudo–second–order kinetic models showed to remove contaminants from the aqueous medium due to satisfactory fit with the experimental data. The equilibrium high specific surface area, high reactivity and catalytic po- data were well fitted by the Liu isotherm model, with a tential [12], which is recently investigated to remove heavy maximum sorption capacity of 110.9 mg g–1. Desorption metal ions from aqueous solutions [4, 13]. Magnetite nano- studies using an acidic eluent showed maximum recovery particles separated easily by a strong magnetic field, which of adsorbent and sorption capacity was dropped only 9% is helpful in terms of recycling and reuse [4]. Since the ag- after five cycles adsorption/desorption experiments. glomeration reduces the specific surface area and reduction

potential of magnetite nanoparticles, therefore, methods such as application of a surfactants, silica particles, poly- KEY WORDS:Adsorption, copper ion, modified magnetite, nano- mers, zeolites and organic ingredients have been developed particles, desorption. to solve the agglomeration problem [14]. In many studies, surfactants have been used as stabilizers and as a coating agent in the surface modiﬁcation of 1. INTRODUCTION iron oxide particles. Emulsifying properties of sodium dodecyl sulfate (SDS), an anionic surfactant, lead our interest Among the commonly encountered metals of concern to study the feasibility of synthesizing magnetite nanopar- copper is hazardous and has been included in the Priority ticles modified with sodium dodecyl sulfate (SDS–Mag) Pollutants List by United States Environmental Protection and to evaluate the modified magnetite nanoparticles as ad- Agency (USEPA) [1]. Copper has been reported to cause sorbent for the adsorption of Cu(II) from aqueous solu- neurotoxicity, jaundice, and liver toxicity [2]. High dose of tions. Moreover, reusability of the prepared adsorbent was copper (Cu) concentrations can lead to weakness, lethargy, examined. anorexia and damage to the gastrointestinal tract [3]. There- fore, removal of copper from water and wastewater by an appropriate treatment becomes necessary. Conventional 2. MATERIALS AND METHODS methods for heavy metal removal from water and wastewater include chemical precipitation, membrane processes, 2.1. Chemicals and materials All chemicals were of analytical reagent grade or the * Corresponding author highest purity available from Merck Company (Germany).

Double distilled water (DDW) was used throughout the Jena, Vario 6, Germany). All the adsorption experiments study. Copper nitrate (Cu(NO3)2) was used for the prepara- repeated triplicates and the mean and standard deviation tion of stock solutions of Cu(II). Analytical grade reagents (SD) of the values were used for compute the final results. such as ferrous chloride tetra-hydrate (FeCl ,4H O, 99%) 2 2 The adsorption capacity (q ) and the removal effi- and sodium hydroxide (NaOH) were used for the synthesis e ciency (R) of Cu(II) were obtained via the following equa- of magnetite. Sodium dodecyl sulfate (NaC H SO ) was 12 25 4 tions: used as magnetite modifier. All working solutions were prepared by diluting the stock solution with distilled water (Co  Ce )V to the needed concentrations. qe  (2) m 2.2. Adsorbent preparation and characterization C  C The modified Si et al. (2004) method was used for R  o e 100 (3) synthesizing SDS–Mag nanoparticles [15]. Briefly, at first, Co –1 50 mL of an aqueous solution of FeCl2·4H2O (5.0 g L as Fe) –1 where qe (mg g ) is the amount of Cu(II) ions ad- was added dropwise to a 0.625% (w/v) SDS (NaC12H25SO4) under continuous shaking. The mixture was shaken for 30 min sorbed onto the unit amount of the adsorbent, Co and Ce –1 to allow the formation of a homogeneous SDS–Fe2+ solution. (mg L ) are the initial and equilibrium Cu(II) ion concen- Then the pH of the solution was increased slowly to 12 by trations, respectively, V (L) is the volume of the solution, adding of 0.5 M NaOH solution. The SDS–Mag was pre- and m (g) is the adsorbent mass in dry form. pared via controlled oxidation of a Fe2+ ion solution ac- After optimization of pH, the adsorption kinetic of cording to the following equation: Cu(II) onto SDS–Mag was investigated using contact time of 0-120 min. The equilibrium isotherms were also inves- 6SDS–Fe2+ + O + 12OH–→2SDS–Mag + 6H O (1) 2 2 tigated using different adsorbent mass over a range of 0.1 The reaction mixture was subsequently aged for 1 h to 5.0 g L–1 under optimal conditions obtained (optimum with constant shaking [15]. After the reaction, excess SDS pH and contact time). was removed by washing with double distilled water. The To conduct the desorption study, appropriate amount resultant solid magnetite materials were then dried in a vac- ° of SDS–Mag nanoparticles was set into the 150 mL Erlen- uum oven at 40 C for 12 h. All of the prepared samples meyer flasks containing Cu2+ solutions (50 mL, 50 mg L–1). were kept on a vacuum desiccator before being used for They were then shaken using a horizontal shaker for approx- adsorption experiments. imately 2 h. In the next step, the Cu2+–loaded SDS–Mag X-ray diffraction (XRD X’pert Philips, HOLAND) was separated magnetically and cleansed sequentially via analysis was performed to identify the structure and the both distilled and deionized water several times in order to composition of freshly synthesized SDS–Mag. BRUKER, eliminate the concentration of unattached metal ions. The Vertex 70, Germany infrared spectrometer was used for the concentration of copper ions was measured using an atomic Fourier transform infrared (FTIR) spectroscopy analysis. absorption spectrometry instrument and the removal effi- The surface morphologies of SDS–Mag nanoparticles were ciency of Cu2+ ions was calculated according to Eq. (3). In performed using a Philips XL-20 scanning electron micro- the desorption experiments, the Cu2+–loaded SDS–Mag scope (SEM) (Philips Co., The Netherlands). were mixed in 50 mL of HNO3solutions (pH~1) and stirred at 150 rpm. At appropriate time intervals, the aliquots were 2.3. Adsorption experiments withdrawn from the solutions. The aliquots were analyzed In the present study adsorbent efficiency for the removal using flame atomic absorption spectrometer to determine the Cu(II) ion concentration. Then, separated SDS–Mag of Cu(II) was evaluated through investigation of various pa- ° rameters. The adsorption experiments was performed at rinsed with Milli-Q water and was dried at 80 C followed room temperature (25±1 °C) using 100 mL amber glass bot- by five sequential sorption/desorption cycles. The desorp- tles containing 50 mL of Cu(II) solution and varying adsor- tion efficiency (DE) was determined using the following bent dosages. Stock solution of Cu(II) (1000 mg L-1) was Eq. (4): prepared by Cu(NO3)2 and all concentration range of Cu(II) C prepared from stock solution varied between 10 to 50 mg L- % DR  des 100 (4) 1 . The initial pH of solution was adjusted in the range of 2 to Cads 6 by adding 0.1M HCl and 0.1 M NaOH. A known amount of SDS–Mag nanoparticles was added to 50 mL of the cor- where % DR is the desorption ratio, Cads is the amount 2+ responding Cu(II) solution over a period of desired time on of Cu sorbed on the adsorbent, and Cdes is the amount of 2+ a shaker at 150 rpm. At appropriate time intervals, the ali- Cu desorbed from the adsorbent. quots were withdrawn from the solutions and SDS–Mag particles was separated magnetically. The residual Cu(II) 2.4. Adsorption isotherm and kinetic models concentration in the supernatant solutions was determined To determine the parameters of the isotherm and ki- by using flame atomic absorption spectrometer (Analytic netic models, the Cu(II) adsorption data were fitted to the

non-linear kinetic and isotherm models using MATLAB® n n 2 2 7.11.0 (R2010b), with successive interactions calculated qi,exp  qi,exp  qi,exp  qi,calc by the Levenberg-Marquardt algorithm. Since the un- 2 i11i (4) R  n wanted falsification of error distribution occurs due to data 2 transformation to a linear form, therefore, the nonlinear  qi,exp  qi,exp method is superior to the linear one in order to determine i1 the parameters of the isotherm and kinetic models [16]. 2 2  n 1  R 1 1 R .  (5) Determination of adsorption isotherm and related pa- adj  n  p  rameters are a basic requirement for the design of adsorp-   tion systems. To remove contaminant such as heavy metals n 2 from water and wastewater, it is necessary to know the re- SSE  q  q (6) moval rate for the design and the quantitative evaluation of  i,calc i,exp i1 adsorbent. In addition, the kinetics describes the adsorbate i uptake rate which control the residence time of adsorbate n uptake at the adsorbent–solution interface. Therefore, it is 2 qi,exp  qi,calc important to be able to predict the metallic ion uptake re-  RMSE  i1 (7) moval rate from aqueous solutions in order to design an n appropriate adsorption unit. In this study, three widely–used adsorption isotherm where qi,calc is each value of q predicted by the fitted models (Langmuir, Freundlich, and Liu) and five general model, qi,exp is each value of q measured experimentally, q adsorption kinetic models (pseudo–first–order equation of i,calc is the average of qi,calc, q i,exp is the average of q meas- Lagergren, pseudo–second–order equation of Ho, Elovich, ured experimentally, n is the number of experiments per- fractional power function, and Avrami fractional order) formed and p is the number of parameters of the fitted model. were used to describe the adsorption equilibrium and the adsorption kinetics of Cu(II) onto SDS–Mag nanoparticles, respectively [17]. 3. RESULTS AND DISCUSSION

2.5. Models ﬁtness 3.1. Characterization of SDS–Mag To select of the most suitable kinetic and isotherm Fig. 1 shows the X–ray diffraction pattern of the SDS– model, it is necessary to evaluate their validity. Here, the Mag nanoparticles. This pattern indicates that the diffraction validity of the kinetic and isotherm models were assessed peaks (088-0315 ICSD) are in agreement with the standard by such criteria as the determination coefficient (R2), the pattern for crystalline magnetite with spinel structure with 2 adjusted determination coefficient (R adj), the sum squared PDF2 card of Fe3O4. The characteristics and purity of the error (SSE), and the root mean square error (RMSE). These SDS–Mag nanoparticles were evident because XRD peaks criteria describe the goodness of fit between the experi- of the nanocomposite matched well with standard Fe3O4 and mental and predicted data. The best model was chosen based other crystalline phases were not detected (Fig. 1). on the lowest RMSE and SSE, as well as with R2adj and R2 The FTIR spectrum of SDS–Mag nanoparticles shows as close as possible to one. R2, R2 adj, SSE and RMSE are Fe–O groups with wave numbers around 575 cm–1, which given below (RMSE) [17]: confirms the metal–oxygen stretching and indicates the

FIGURE 1 - X–ray diffractographof SDS–Mag nanoparticles.

FIGURE 2 - FTIR spectra of the prepared SDS–Mag nanoparticles.

FIGURE 3 - SEM image of the SDS–Mag nanoparticles.

presence of Fe in SDS–Mag nanoparticles (see Fig. 2). The 3.2. Cu(II) adsorption bands around 3400 cm-1 indicated the presence of stretch- 3.2.1. Effect of solution pH ing O-H vibrations. Other peaks at 1628, 1481 and 1020 The aqueous solution's pH plays a key role in the ad- cm-1 were due to C=O, (CH2)n and C-O, respectively, sorption processes of heavy metals. The effect of pH on the which is the characteristic absorption peak of SDS. The adsorption of Cu(II) by SDS–Mag nanoparticles is shown FT–IR studies show that the required bonds have been in Fig. 4. With increasing pH from 2 to 5, the removal ef- formed in sample structures and SDS was coated onto ficiency and sorption capacity by SDS–Mag increased Fe O nanoparticles. 3 4 from 22.1% to 98.5% and 2.3 to 9.9 mg g–1, respectively. The surface morphologies of SDS–Mag were observed However, the removal efficiency and sorption capacity re- using a scanning electron microscope (SEM). As shown in main almost constant at higher pH values. At pH 2 and 3, Fig. 3, SDS–Mag nanoparticles prepared are in the range competition between H+ and Cu2+ ions could thus explain of 40–60 nm (average 50 nm) in diameter. As evident in the weak adsorption in acid medium. This pH dependency the ﬁgure, the produced SDS-Mag nanoparticles has a spher- has previously been attributed to the form surface complexes ical shape and porous internal structure and the aggregation between the functional groups (≡FeOH) and the Cu(II) with of nanoparticles was decreased, as well. the possible reaction given as follows [18]:

2+ (2-q-s) sH2O + q≡ FeOH+ rCu ↔ (≡ FeOH)qCur(OH)s Mag was rapid, reaching equilibrium in a relatively short + (s+q)H+ (8) period of time. The maximum adsorption occurred at the ﬁrst 30 min, and then the sorption rate reached a constant The high removal efficiency of Cu(II) at pH values value, which is probably due to plentiful sorption sites and higher than 5 may be attributable to the precipitation of large surface area available for the adsorption of Cu(II) on Cu(OH) rather than the adsorption of Cu(II) by SDS– 2 SDS–Mag. This may as well be attributed to the external Mag. Therefore, the optimum pH for Cu(II) ion sorption surface adsorption. Thus, 30 min was determined as the op- is found in the pH 5.0, which is in accordance with pre- timal contact time for Cu(II) adsorption onto SDS–Mag. In vious reports [19]. the subsequent experiments, the contact time of 60 min was

3.2.2. Effect of contact time and adsorption kinetics chosen to ensure reaching equilibrium. This indicates that a significant part of the adsorption sites of the adsorbent The adsorption efficiency and sorption capacity of exist in the exterior of the SDS–Mag nanoparticles and are SDS–Mag nanocomposites was determined by varying the easily accessible by Cu(II) species, thus resulting in a rapid contact time in the range of 2–120 min. As it is evident approach to equilibrium. from Fig. 5 the initial adsorption rate of Cu(II) onto SDS–

100 20 90 18

80 R (%) 16 70 14 q (mg/g) 60 12 50 10 40 8 30 6 Removal efficiency(%) 20 4 capacitySorption (mg/g) 10 2 0 0 1234567 pH

FIGURE4 -The effect of pH on the Cu(II) adsorption by SDS–Mag (contact time 60 min, adsorbent dose 1.0 g L–1, Cu2+ 10 mg L–1).

40 100 35

80 30

25 60 20

40 15 Removal efficiency Adsorption capacity Removal of Cu (II) (II) (%) Cu of Removal

10 (mg/g) capacity Adsorption 20 5

0 0 -5 25 55 85 115

Contact time (min) FIGURE 5 - Effect of contact time on the Cu(II) ions adsorption onto SDS–Mag (adsorbent dose 2.0 g L–1, pH 5.0±0.1, Cu2+ 50 mg L–1).

Kinetics describes solute uptake rates and defines the bounds using non–linear equations of Pseudo–first–order residence time of the adsorbate at the solid–liquid interface (PFO) of Lagergren, Pseudo–second–order (PSO) of Ho, as well. Additionally, valuable insights into the reaction Elovich, fractional power function (FPF), and Avrami frac- pathways and the adsorption mechanisms can be achieved tional order (AFO) kinetic models. The kinetic parameters via kinetic studies. acquired from non–linear fitting results are presented in Ta- ble 1. The higher values of the adjusted determination coef- As shown in Fig. 5, Cu(II) uptake seemed to occur in ficient (R2–adj> 0.99) and the lower values of the sum two steps. The first step involved extremely fast metal up- squared error (SSE<10) and the root mean square error take within the first 15 min of contact followed by the sub- (RMSE <1.0) revealed that the Avrami fractional order and sequent removal of Cu(II), which continued for a relatively the pseudo–second–order kinetic models were the most ap- short period of time until adsorption equilibrium was ob- propriate to represent the Cu(II) adsorption onto SDS–Mag tained. The fitting plots using five kinetic equations are nanoparticles indicating that the chemical reaction seems shown in Fig. 6, which were simulated with 99% confidence significant in the rate–controlling step and no involvement

20 PFO PSO 15 FPF q (mg/g) 10 AFO Elovich 5 q-exp.

0 -5 15 35 55 75 95 115

time (min) FIGURE 6 - Non-linear adsorption kinetics curves of Cu(II) by SDS–Mag adsorbent (pH 5.0±0.1, adsorbent dose 2.0 g L–1, Cu2+ 50 mg L–1).

TABLE 1 - Kinetic parameters for Cu(II) adsorption using SDS–Mag adsorbent (Conditions: pH 5.0±0.1, adsorbent dose 2.0 g L–1, Cu2+ 50 mg L–1).

2 Adsorption kinetic models Parameters (unit) Values R adj RMSE SSE –1 Pseudo–ﬁrst order (Lagergern) kf (min ) 0.305 0.98 1.291 10.01 –1 qt  qe (1 exp(k f t)) qe (mg g ) 23.9 –1 –1 2 kS (g mg min ) 0.016 0.995 0.6266 2.355 ksqe t Pseudo–second order (Ho) qt  –1 qe (mg g ) 26.4 1 qekst Elovich α (mg g–1 min–1) 36.19 0.96 1.817 19.8 1 q  ( ) ln(1  t) –1 t  β (g mg ) 0.226 –1 Avrami fractional order kAV (min ) 0.261 0.999 0.3433 0.589 –1 n qe (mg g ) 24.9 q  q 1exp[(k t)] AV  t e AV nAV (–) 0.704 Fractional power function a (mg g–1 min–b) 11.45 0.92 2.548 38.94 b qt  at b (–) 0.216

of a mass transfer in solution [1, 2]. Also, the calculated Liuusing non–linear equations (see Fig. 7). The parameters equilibrium adsorption capacity (qcal) for the Avrami frac- obtained from the studied isotherms are given in Table 2. tional order and pseudo–second–order kinetic models (24.9 and 26.4 mg g–1, respectively) were well approximated to the The high adjusted correlation coefficient (R2–adj) and –1 experimental value (qexp) (24.9 mg g ), indicating better the low RMSE and SSE values indicated that the experi- suitability of these kinetic models to describe adsorption ki- mental data fitted satisfactorily the Liu isotherm model, netics of Cu(II) onto SDS–Mag. Nonetheless, the highest which means that the q fit by the isotherm model was close R2–adj and lowest RMSE and SSE values for the Avrami to the q measured experimentally (Table 2). Based on the fractional order kinetic model in Table 1 suggests this model Liu model assumptions, all reactive sites onto the surfaces can be used to represent the kinetic uptake of Cu(II) onto of the adsorbent have no similar energy. Hence, the adsor- −2 SDS–Mag. The value of kAV was found to be 26.1×10 bent would have several reactive sites that tend to be occu- −1 −1 2+ min at 50 mg L Cu concentration. The values of kAV, pied with the adsorbate molecules [20]. The Langmuir and shown in Table 1, indicate that SDS–Mag nanoparticle can the Freundlich isotherm models were not suitably fitted, be used for the Cu(II) removal from aqueous solution. presenting sum squared error (SSE) values ranging from 3.8 to 15.3–fold higher than the SSE value obtained by the 3.2.2. Adsorption isotherms Liu isotherm model. The maximum amounts of Cu(II) up- The adsorption isotherms are usually considered as take were 110.9 mg g–1 for SDS–Mag. This value indicates feasible tools to understand the adsorption process. In this that SDS–Mag is a very good adsorbent for heavy metal work, experimental data were fitted to threewidely used removal from aqueous solutions. Additionally, the adsorp- isotherm models, namely, Langmuir, Freundlich, and tion capacities of Cu(II) ions removal by means of various

40 Lang. Freun. 30 Liu q (mg/g) q 20 q-exp

0 0 5 10 15 20 25 30 35 40 45

Ce (mg/L) FIGURE 7 - Non-linear ﬁt of experimental data obtained using Langmuir, Freundlich and Liu isotherm models (contact time 60 min, pH 5.0±0.1, Cu2+ 50 mg L–1, adsorbent dose 0.1-5.0 g L–1).

TABLE 2 - Isotherm parameters for Cu(II) adsorption using SDS–Mag adsorbent (Conditions: contact time 60 min, pH 5.0±0.1, adsorbent dose 0.1–5.0g L–1, Cu2+ 50 mg L–1).

2 Adsorption isotherm models Parameters (unit) Values R adj RMSE SSE –1 Langmuir qm (mg g ) 71.4 0.958 5.97 249.8 K q C L m e –1 qe  KL (L mg ) 0.796 1 K LCe –1 –1 –n Freundlich KF (mg g )(mg L ) 31.4 0.989 2.97 61.9 1 n n (–) 4.32 qe  KFCe –1 Liu qm (mg g ) 110.9 0.997 1.65 16.3 –1 q (K C )nL Kg (L mg ) 0.116 m g e qe  n L nL(–) 0.411 1 (K gCe )

adsorbents are presented in Table 3, which apparently con- adsorption/desorption experiments show that the SDS–Mag firms the higher potential of SDS–Mag to remove the adsorbent can be reused with a slight decrease in the ad- Cu(II) from aqueous solutions. sorption capacity. The maximum recovery percentage was about 97%. In addition, the SDS–Mag exhibit good recy- TABLE 3 - Comparison of maximum adsorption capacity (qm) for clability and only 9.0% of the sorption capacity is lost after Cu(II) with various adsorbents. five consecutive cycles of adsorption/desorption (Fig. 8b). Adsorbent q max (mg g–1) References Numerous studies have reported large elution efficiency Modiﬁed litchi pericarp 23.70 [2] figures for several heavy metals, especially Cu2+, by means Nano–Fe3O4 8.90 [18] of acidic solutions as an eluant [23, 24]. The findings of the Alginate–immobilized bentonite 131.6 [1] clay desorption survey confirmed that no considerable loss EDTA functionalized Fe3O4 46.27 [19] could be observed in the performance of SDS–Mag nano- magnetite nanorods 79.10 [21] particles over at least ﬁve cycles of adsorption/desorption, SDS–Mag 110.9 Present work which proved the reusability of SDS–Mag nanoparticles in adsorbing heavy metals from contaminated waters. 3.3. Desorption studies and reusability Desorption experiments can be considered as an appropriate tool to recover the adsorbed metal ions from the 4. CONCLUSIONS adsorbent surfaces. Furthermore, it will enhance the reusability of the adsorbent to adsorb more metal ions and will Aiming at the removal of Cu(II) adsorptive form aque- also improve the sorption process [22]. ous solutions, SDS–Mag nanoparticles were successfully synthesized, characterized and applied in a batch system. (a) 100 Considering the batch experiments, the sorption process of Cu(II) onto the surfaces of SDS–Mag seems to be a pH de- 80 Cycle 1 pendent process where the maximum amount of Cu(II) removal efficiency was obtained at pH 5.0. Moreover, kinetic Cycle 2 60 studies of the adsorption of Cu(II) onto the surfaces of Cycle 3 SDS–Mag by means of various general kinetic models were investigated. The sorption seems to be governed by 40 Cycle 4 chemical forces rather than physical electrostatic interac- Desorbed(%) Cu Cycle 5 tions, possibly best described by the Avrami fractional or- 20 Mean der and pseudo–second–order kinetic models, where the rate limiting step is assumed to be the chemical sorption 0 between the adsorbate and the adsorbent. The results of 0 20406080100120equilibrium studies, in addition, show that the obtained Desorption time (min) data of Cu(II) adsorption were well described by Liu model with a high coefficient of correlation (R2=0.997) and the 112 (b) sorption capacity of 110.9 mg g–1. Desorption of Cu2+ ions 109 and feasible recovery of SDS–Mag nanoparticles was achieved by using an acidic eluent with efficiencies of 106 greater than 95 %. Ultimately, the results clearly indicated

103 that the SDS–Mag nanoparticles could be easily synthesized as an environmentally friendly and robust adsorbent, (mg/g) e

q 100 and could be a very attractive candidate for the removal of toxic metals from water and wastewater. 97 94

91 ACKNOWLEDGEMENTS 12345 Adsorption-Desorption Cycle The financial support of the Environmental Technolo- gies Research Center (ETRC) and the Research and Tech- FIGURE 8 - Desorption studies of SDS–Mag nanoparticles (a) Re- lease curve of Cu2+ ions; (b) Reusability of adsorbent. nology Deputy of Ahvaz Jundishapur University of Medi- cal Sciences (grant No: ETRC9004) for this study are Desorption of Cu ions from the SDS–Mag adsorbent is thankfully acknowledged. most effectively achieved by dilute nitric acid solution (pH~1). The results indicate a rapid desorption behavior of The authors have declared no conflict of interest. copper, with approximately 78.3 (±3.8)% of the desorption being completed within the first 2 min (Fig. 8a). Consecutive

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[10] Gutiérrez-Segura, E., Solache-Ríos, M., Colín-Cruz, A. and Fall, C. (2014) Comparison of Cadmium Adsorption by Inor- ganic Adsorbents in Column Systems. Water, Air, & Soil Pol- lution 225, 1-13. [11] Shyam, R., Puri, J. K., Kaur, H., Amutha, R. and Kapila, A. (2013) Single and binary adsorption of heavy metals on fly ash samples from aqueous solution. Journal of Molecular Liquids Received: October 18, 2014 178, 31-36. Revised: December 30, 2014 [12] Giraldo, L., Erto, A. and Moreno-Piraján, J. (2013) Magnetite Accepted: March 09, 2015 nanoparticles for removal of heavy metals from aqueous solutions: synthesis and characterization. Adsorption 19, 465-474. [13] Babaei, A. A., Bahrami, M., Farrokhian Firouzi, A., Rama- CORRESPONDING AUTHOR zanpour Esfahani, A., Alidokht, L. (2014) Adsorption of cadmium onto modified nanosized magnetite: kinetic modeling, Ali Akbar Babaei isotherm studies, and process optimization. Desalination and Water Treatment, 1-13, doi: 10.1080/19443994.2014.972986. Environmental Technologies Research Center Ahvaz Jundishapur University of Medical Sciences [14] Yuan, P., Liu, D., Fan, M., Yang, D., Zhu, R., Ge, F., Zhu, J. Ahvaz and He, H. (2010) Removal of hexavalent chromium [Cr(VI)] from aqueous solutions by the diatomite-supported/unsup- IRAN ported magnetite nanoparticles. Journal of Hazardous Materi- als 173, 614-621. Phone: +98 611 3738269 [15] Si, S., Kotal, A., Mandal, T. K., Giri, S., Nakamura, H. and Fax: +98 611 3738282 Kohara, T. (2004) Size-Controlled Synthesis of Magnetite Na- E-mail: [email protected] noparticles in the Presence of Polyelectrolytes. Chemistry of Materials 16, 3489-3496. FEB/ Vol 24/ No 9/ 2015 – pages 2815 - 2823

CHELATE ASSISTED PHYTOREMEDIATION OF Pb, Cd AND B BY SUNFLOWER, MAIZE AND CANOLA

Ömer Vanlı and Mustafa Sait Yazgan*

Istanbul Technical University, Environmental Engineering Department, 34469, Istanbul, Turkey

ABSTRACT paints and pigments, plastic stabilizers, electroplating, incineration of cadmium-containing plastics and phosphate Phytoremediation is a relatively recent technology fertilizers [8, 9]. Combustion of leaded petrol, battery man- with research conducted mostly during the last twenty ufacture, herbicides and insecticides are among the major years. Research in this field is shifted towards the selection sources of Lead [7, 10]. Cd has carcinogenic, mutagenic, of more effective plant species and chelating agents to en- and teratogenic effects and it interferes with calcium regu- hance element uptake. In this study, EDTA enhanced phy- lation in biological systems and causes renal failure and toremediation performance of Pb, Cd and B from soil by chronic anemia [8, 11]. Pb causes poisoning problems in sunflower, maize and canola crops was studied. The trans- children such as impaired development, reduced intelli- fer processes of elements from root to stem and leaves were gence, loss of short-term memory, learning disabilities and also discussed. Although canola has the maximum accu- coordination problems and renal failure [1, 7, 12, 13]. mulation capacity, all crops showed a high uptake perfor- On the other hand Boron (B) is an element which nat- mance. Addition of chelate to the soil considerably in- urally occurs in soils. It is an essential trace element re- creased Cd and Pb uptake till 5 mmol chelate dosages and quired for normal growth of crops. Boron requirement of then it started to decrease. This trend was described by sec- crops varies and the range between deficient and toxic con- ond order polynomial equation (y=ax2+bx+c). Maximum centrations is smaller than other nutrient elements [14-16]. and minimum accumulated elements were B and Pb, re- However, many species are quite sensitive to elevated B spectively. All crops had an efficient transfer of the ele- levels in their tissues, showing severe toxicity symptoms at ments from roots to top organs and they are evaluated as tissue levels of about 50 mg/kg. Such levels can be found good collectors according to Baker Theory. Although, ac- in tissues when the available soil Boron exceeds 3 mg/kg cumulation of high heavy metal concentration by edible [17]. Toxic effects are more obvious in dry seasons, when plants, such as maize and sunflower, is a major risk for roots penetrate deeper into the soil [18]. public health, the results are positive from a phytoremedi- Use of phytoremediation for removal of heavy metals ation perspective. and other pollutants is a cost-effective, efficient, in situ applicable and solar-driven remediation strategy [4, 19-31]. Plants generally handle the contaminants without affecting KEYWORDS: Accumulation, bioremediation, chelates, heavy metals, soil topsoil, thus conserving its utility and fertility. They may improve soil fertility with inputs of organic matter. They have a great ability to uptake pollutants from the soil and accomplish their detoxification by various mechanisms [1, 1. INTRODUCTION 32- 34]. Phytoremediation technology is a relatively recent tech- Heavy metals are considered to be a significant environ- nology with research studies conducted mostly during the mental problem in the world. Industrial, agricultural and nat- last two decades. It is suitable for application at very large ural processes are the major sources for these elements in the field sites where other remediation methods are not cost ef- environment. Heavy metal pollution is becoming more and fective or practicable. Research shifted towards the plant more serious with the rapid increase in industrialization species chelating agents (such as EDTA) that could be used and disturbance of natural biogeochemical cycles [1, 2]. to enhance their metal uptake. While the enhanced mobility Cadmium (Cd) and lead (Pb) are two of the mostly ob- of chelated metal complexes in the soil solution increases the served heavy metals in the environment. Like other heavy metal availability to the plant, it also increases the risk of metals, they originate from natural and anthropogenic inadvertently spreading the contamination [35-39]. sources. [3-7]. The anthropogenic sources of cadmium are In the literature, there are many phytoremediation studies focusing on removing heavy metals by using crops. * Corresponding author However, there are limited works focusing on the use of

edible plants in phytoremediation studies. Use of edible soil by dissolving it in the irrigation water. EDTA concen- plants such as sunflower (Helianthus annus) and maize trations were chosen as 0, 2.5, 5 and 10 mmol/kg soil by (Zea mays) are critical for both remediation performance taking into consideration the literature data. and public health consideration. On the other hand, number of studies on boron removal from soil is limited, since Bo- 2.2 Methods ron is not toxic to the human health. Experiments were conducted in pot environment and The aim of this paper is to determine the EDTA en- in open air conditions with a temperature range of 19 and hanced phytoremediation performance of sunflower, maize 30 0C. A total number of 72 sunflower, maize and canola and canola crops in order to uptake the elements of Pb, Cd crops were used with four chelate doses in randomized de- and B from soil. Elements of concern are the ones that are sign with two replicates. Pb, Cd and B added soil samples abundant Turkey's soil. Pb is present in regions with dense (each of 3 kg) were put into pots and were seeded with sun- traffic load and Cd is found in agricultural land where flower, maize and canola. The crops were periodically irri- phosphorous fertilizers are applied. Natural B on the other gated and no fertilizer was applied. hand is abundant everywhere. Besides, variations in the el- During the experiments, canola was affected by high ement uptake of the crops are investigated by adding che- B concentration in the soil and that it started to dry. Thus, late at varying amounts. For the phytoremediation purpose; B dosage was dropped to 25 mg and all experiments were sunflower, maize and canola crops are used and the uptake repeated. of the elements and their transfer from root to stem and Nine weeks after seeding, EDTA was added to soil leaves are discussed. samples and crops were harvested after one week. EDTA

was added just immediately after the vegetation period of

the crops was completed. This is the stage when the genera- 2. MATERIALS AND METHODS tive organs start developing. The growth periods of sunflower, maize and canola are known as 12, 18 and 19 weeks, 2.1 Materials respectively. Crops were then cut with a steel scissor just Phytoextraction of Pb, Cd and B elements was investi- 2 cm above the soil. The roots were separated from stem gated through the crops of maize (Zea mays) from Gra- and leaves and all the samples were left drying after wash- mineae family, sunflower (Helianthus annus) from Com- ing them with deionized water. positae family and canola (Brassica napus L.) from Cru- Elemental analyses were conducted on the different or- ciferae family. gans of the crops (root, stem and leaves) in order to deter- Soil samples used in the experiments were gathered mine the element concentrations. For this purpose, ele- from the top 20 cm of the sampling area which is located ments in the crop samples were extracted and the obtained at the Asian side of Istanbul. Table 1 shows the quality solution was analyzed by Atomic Absorption Spectrometry analysis of the soil. (AAS) for Pb and Cd and by Inductively Coupled Plasma (ICP) for B. Cd and Pb concentrations were analyzed by TABLE 1 - Soil Quality Analysis using Perkin-Elmer (Norwalk, CT) Optima 3000 DV In- Parameter Value ductively Coupled Plasma-Optical Emission Spectrometer Saturation with water (%) 35 (ICP-OES). All ICP measurements were carried out in the pH 6,48 axial mode. A 999 ± 0.2 µg/mL standard solution from In- Total Salinity (%) 0,02 organic Ventures including pure heavy metal, water and CaCO (%) none 3 5% HNO3 (v/v) was used as an internal standard for cali- Organic Matter (%) 2,4 bration as recommended by the ICP manufacturer and cor- Sand (%) 53 relation coefficient was 0.9998. All samples and ICP stand- Clay (%) 22 ards were acidified by using 0.5% trace metal grade HNO . Silt (%) 25 3 Soil Structure Sandy clay loam Boron concentration was determined by atomic absorption

Phosphorous P2O5 (kg/da) 31,2 spectrometry on 0.5 g samples that were overnight predi-

Potassium K2O (kg/da) 38,6 gested in 5 ml concentrated HNO3 and then digested add- 0 Pb (mg/kg) <1 ing 1ml HClO4 at 220 C in an aluminum block digester. Cd (mg/kg) <1 B (mg/kg) 5,5 3. RESULTS Pb, Cd and B elements were added to soil samples in the form of Pb(NO3)2, CdCl2 and H3BO3, respectively. The total 3.1 Element uptake performance of sunflower amounts in the soil were 250 mg/kg for Pb and 400 mg/kg Pb accumulation of sunflower under various chelate for Cd. Since the soil had initial B concentration of 5.5 mg/ concentrations is given in Figure 1. According to the figure, kg, 19.5 mg/kg of B were added to get 25 mg/kg. Pb uptake linearly increased with increase in chelate concen- EDTA with molecular weight of 372.24 gram and sol- tration. The highest uptake is achieved 97 mg at 10 mmol/kg ubility of 100gr/lt was used as chelate. It was mixed with chelate doses. For Cd, uptake of sunflower increase is de-

tected till addition of chelate up to 5 mmol; however, a de- In general, Cd uptake is obtained more than Pb uptake crease is observed beyond this amount. The highest uptake in sunflower samples. Chelate addition has affected the up- is achieved in 5 mmol/kg chelate doses (i.e. 139 mg). On take performances both for Pb and Cd, however no consid- the other hand, uptake of B decreases with chelate addition erable change has occurred in the uptake of B. and then it slightly increase with an increase in the chelate concentrations. In the case of no chelate addition, a high B 3.2 Element uptake performance of maize uptake is observed. The highest accumulation is detected Pb uptake by maize under variable chelate dosages is under chelate addition of 10 mmol/kg soil (i.e., 701 mg). given in Figure 2. Pb uptake increased as chelate doses in- The minimum accumulation on the other hand was realized creased. However, this increase ceased after addition of as 645 mg where chelate addition was 2.5 mmol. 5 mmol. Similarly, up to 5 mmol, Cd uptake by maize in-

FIGURE 1 - Element uptake diagram of sunflower

FIGURE 2 - Element uptake diagram of maize

FIGURE 3 - Element uptake diagram of canola

creased as chelate doses increased. After that value, Cd uptake decreased. In case of B, higher ratios of uptake oc- TABLE 2 - Statistical analysis of uptake performances curred even without chelate addition. 2 a b c r 3.3 Element uptake performance of canola Sunflower Pb -2,32 32,93 21,80 0,99 Element uptake by canola under variable chelate dos- Cd -1,14 17,15 26,32 0,95 ages is given in Figure 3. According to the figure, Pb and Maize Pb -2,76 32,83 36,95 0,95 Cd uptakes increased in parallel to an increase in the che- Cd -2,99 38,93 25,59 0,99 late doses. The maximum Cd and Pb accumulation oc- Canola Pb -2,25 35,37 57,85 0,98 curred at 10 mmol addition of chelate with a value of 364 Cd -3,14 49,48 162,85 0,94 mg and 215 mg, respectively. In case of B, it is seen that B uptake at higher ratios occur even without the addition of The highest leaf accumulation of Pb, Cd and B oc- chelate. Generally speaking, B uptake was greater than Cd curred with canola. Regarding root zone accumulation, uptake by canola. Among all the metals Pb has the lowest maize had a higher capacity among the other crops. Highly uptake. developed canopy root structure of maize is probably the reason for this. Similarly, lower accumulation at the root zone occurred with sunflower. This situation is due the dif- 4. DISCUSSON ferent root type of sunflower as it has a tap type of root. According to Baker Theory that is used for the assess- The highest uptake performance for all elements is re- ment of element uptake values, crops are divided into good alized by canola. Sunflower and maize showed similar up- collectors and root accumulators. A good collector crop is take performance for Pb and Cd, although canola showed defined as a one that accumulates high amount of elements higher Cd performance. Higher amounts of B were accu- in its above surface parts. Thus, good collector crops are mulated in three crops even without chelate addition. classified as those whose element accumulation in the In general Pb and Cd uptake by all plants increased above surface parts over the accumulation in the roots is with chelate application till 5.0 mmol/kg soil dosages then greater than one. When this ratio becomes less than one; it slowed down and decreased in most of the application. this type of plants is called as root collectors. Good collec- This trend was described by second order polynomial equa- tors are used in the phytoextraction method whereas the tion (y=ax2+bx+c). Table 2 gives the constants and corre- root collectors are favored in the phytostabilization method lation coefficients (r2) for each application. [40]. However, in case of B uptake, chelate addition didn’t The findings of the experimental work indicate that the affect the uptake ratios. It was considered that lower per- amount of element resent in the above surface parts of the formances obtained by addition of high chelate concentra- crop is statistically higher than that of accumulation in the tions were due to a decrease in the pH of the soil. root zones (Table 3). This means that selected crops

showed a successful element uptake performance and tions, to minimize the crop losses, and to prevent the soil transfer of elements to the above surface parts of the crops. of experimental conditions. This period is accepted as short Thus, the crops used in the experiments are evaluated as since their normal growth period is around 14-15 weeks. good accumulators based on Baker Theory. The crop uptake ratios would be higher if the total vegetation period is completed. Moreover, in order to increase the TABLE 3 - Phytoremediation performance of three crops according amount of elements in the crops, it is important to raise the to Baker Theory above surface parts of the crops and this can be realized by Element in stem and leaves/element some agricultural practices such as applying nutrients. Element in root (mean) Addition of chelate is a commonly applied practice Pb 196 CANOLA with the aim of increasing the performance of phytoreme- Cd 354 diation. On the other hand, addition of chelate causes an B 145 extra cost and pollution load to soil; thus, it must be added Pb 10 MAIZE at a minimum level. To fulfill this purpose, it is important Cd 6 to select convenient crops that will lead to maximum pol- B 33 lutant removal with minimum or no chelate consumption. Pb 68 SUNFLOWER The aim of accumulating an element in a crop is to con- Cd 42 vert the elements adsorbed on soil to a more transportable B 545 and controllable form. Thus, phytoremediation method

should not be considered as final disposal and removal 5. CONCLUSIONS method. As a final disposal it can be either disposed as hazardous waste safely in specialized dumps or if economically In this study, determining the EDTA enhanced phy- feasible, processed for biorecovery of precious and semipre- toremediation performance of elements Pb, Cd and B from cious metals (a practice known as phytomining). soil by various edible plants is aimed. As expected, the Although phytoremediation is a promising approach maximum accumulation of all elements was observed with for remediation of heavy metal-contaminated soils, it also canola. This condition validated the high uptake perfor- suffers from some limitations such as long time required mance of canola, a crop widely used in phytoremediation for clean-up, risk of food chain contamination or phyto- process and known as a hyper accumulator. toxic effect in heavily polluted soils. Although the chelate addition to the soil considerably As a conclusion, phytoremediation is considered as a increased the Cd and Pb uptake, it didn’t increase the B method that bears a significant capacity regarding the re- uptake. This is because chelate increased the amount of moval of pollutants from soil. It is applicable to sites with metals dissolving in to the soil mixture. B is present in soil low to moderate levels of metal contamination and ex- mostly in the form of borate salt; which therefore was not pected to be a commercially viable technology for phytom- affected by chelate addition. It is known that higher amount ining of heavy metals in future. of B dissolve in the soil mixture when the water content of soil is kept high. The authors have declared no conflict of interest. When the crops are investigated, it can be stated that maximum accumulation are detected for B whereas the minimum values are observed for Pb. All crops used in the experimental work uptook the elements with success and REFERENCES performed an efficient transfer of the elements to top or- [1] Ali, H., Khan, E. and Sajad, M.E. (2013) Phytoremediation of gans and hence they are evaluated as good collectors ac- heavy metals - Concepts and applications - Review, Chemosphere cording to Baker Theory. This result is positive from a phy- 91, 869–881. toremediation point of view. However from public health [2] Khan, S., Hesham, A.E.L., Qiao, M., Rehman, S. and He, J. Z. point of view, collecting of high amount of heavy metals (2010) Effects of Cd and Pb on soil microbial community structure by edible plants such as maize and sunflower will consti- and activities, Environ. Sci. Pollut. Res. 17, 288–296. tute a significant health risk. [3] Modaihsh, A., Al-Swailem, M. and Mahjoub, M. (2004) Heavy When the uptake periods of crops are considered; all metal contents of commercial inorganic fertilizer used in the King- dom of Saudi Arabia, Agri. Mar. Sci. 9, 21–25. the periods were long except for B. However, it should be noted that element concentrations used in this study was [4] Chehregani, A. and Malayeri, B.E. (2007) Removal of heavy metals by native accumulator plants, Int. J. Agri. Biol. 9, 462–465. comparatively high. In the case where lower element concentrations are used, time scales would be much shorter. [5] Fulekar, M., Singh, A. and Bhaduri, A.M. (2009) Genetic engineering strategies for enhancing phytoremediation of heavy met- Besides, the crops used in the experiments were harvested als, Afr. J. Biotechnol. 8, 529–535. after 10 weeks of growth without waiting the completion [6] Sabiha, J., Mehmood, T., Tufai, M. and Irfan, N. (2009) Heavy of normal vegetation periods. Such an approach was cho- metal pollution from phosphate rock used for the production of fer- sen in order to make the calculations regarding concentra- tilizer in Pakistan, Microchem. J. 91, 94–99.

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THE EFFECTS OF FIVE FOOD DYES ON THE LONGEVITY OF DROSOPHILA MELANOGASTER

Şifa Türkoğlu*, Dilek Benli and Nihan Şahin

Cumhuriyet University, Faculty of Science, Department of Biology, 58140, Sivas, Turkey

ABSTRACT and synthetic dyes have been eliminated from both the national and international lists of permitted food colors be- In this study, the effects of Sunset Yellow, Ponceau cause of their mutagenic or carcinogenic properties [8-10]. 4R, Allura Red, Brilliant Blue FCF, and Brown HT on the Sunset Yellow (E110), Ponceau 4R (E124), Allura Red longevity of Drosophila melanogaster was investigated. (E129), Brillant Blue FCF (E133), and Brown HT (E155) The effects of different concentrations of these food dyes are dyes used asfood additives in several countries, includ- (0.5 mg-1 mL, 1 mg-1 mL, 1.5 mg-1 mL, and 2 mg-1 mL) ing Turkey. Sunset Yellow is a synthetic yellow azo dye were separately administered one by one to female and manufactured from aromatic hydrocarbons, which are de- male populations of Drosophila melanogaster for applica- rivatives of petroleum. It is used in orange sodas, marzipan, tion groups. In all of application groups of each population Swiss rolls, apricot jam, citrus marmalade, lemon curd, the longevity decreased, depending on the concentrations packet soups, margarine, energy drinks, snack chips, phar- of food dyes. It was found that the difference between the maceutical pills, and even prescription medicines (espe- groups was significantly important (p<0.05). We observed cially children’s medicines). Ponceau 4R is a synthetic col- that Brilliant Blue FCF caused the biggest decreased in life orant that may be used as a food-coloring additive. It is a span amoung the other food colouring dyes followed by red azo dye, which is usually synthesized from aromatic Ponceau 4R, Sunset yellow, Brown HT and Allura Red petroleum-derivative hydrocarbons, and can be used in a during this study. wide variety of food products. Agarwal et al. [11] showed that Ponceau 4R is clastogenic. Allura Red, which is added to several food products, including orange soda, gelatins,

KEYWORDS: Drosophila melanogaster, Sunset Yellow, Ponceau puddings, confectionary, beverages, and condiments, is a 4R, Brilliant Blue FCF, Allura Red, Brown HT, longevity widely used synthetic azo dye. Brilliant Blue FCF (Blue 1) is a colorant for foods and other substances. It is a synthetic dye produced from aromatic petroleum derivatives. Bril- liant Blue FCF is often usedin ice cream, packaged soups, 1. INTRODUCTION blue raspberry-flavored products, sweets, and drinks, as well as in hygiene and cosmetic products such as soaps, Food additives are substances that are added to food to shampoos, and mouthwashes. Brown HT, also called modify its visual appearance, taste, texture, processing, or Chocolate Brown HT, is a brown synthetic coal tar diazo shelf life. In most countries, the use of food additives is dye. Its E number is 155 when used as a food dye substitute regulated by law. Only specific additives are permitted, and for cocoa or caramel, and it imparts brown coloration to these may be added to only selective food items in an products such as chocolate cakes and milk. amount within the predetermined maximum concentration Drosophila melanogaster is commonly used as an in limit. Natural as well as synthetic food colorants are widely vivo model organism in toxicology testing as it offers the used by food manufacturers to attract consumers. In fact, following advantages: 1. It is easy to maintain under labor- the total amount of global colorant production is estimated atory conditions. 2. It has a short generation time that al- to be 80,000,000 tons per year [1]. Consequently, thesafety lows for quick genotoxic evaluation. 3. Its metabolic activ- and acceptable daily intake of food dyes are continually ity can lead to activation of promutagens and procarcino- evaluated by the US Food and Drug Organization (FDA) gens, which is analogous to that of the liver in mammals and World Health Association (WHA) [2]. The European [12-13]. 4. About 75% of known human disease genes have food Safety Authority (EFSA) has allocated an acceptable a recognizable corresponding match in the genome of Dro- daily intake (ADI) of Sunset Yellow, Ponceau 4R, Allura sophila, and 50% of fly protein sequences have mamma- Red, Brillant Blue FCF and Brown HT at 1, 0.7, 7, 6 and lian homologues [14]. The aim of the present study was to 1.5 mg/kg bw/day, respectively [3-7]. Numerous natural investigate the possible toxic effects of five food dyes (Sunset Yellow, Ponceau 4R, Allura Red, Brilliant Blue, * Corresponding author and Brown HT) on the longevity of D. melanogaster. As

these food dyes are widely used worldwide as well as in mean longevity values of the control and applications Turkey, it is necessary to gather more data on mutagenicity groups. The significance level was set at p˂0.05. in order to assess the potential hazards of these food dyes for human health. 3. RESULTS AND DISCUSSION

2. MATERIALS AND METHODS In the present study, the changes in the longevity of D. melanogaster, which were exposed to food dyes at differ- Flies belonging to the Oregon R wild-type strain of D. ent treatment doses, were investigated. The lethal concen- -1 melanogaster were used in the experiments. The stock of tration 50 (LC50) value for these dyes was 6–10 mg mL. this fly had been maintained over several years in the la- The concentrations below the LC50 value, namely, 0.5, 1, boratory of the Department of Biology, Cumhuriyet Uni- 1.5, and 2 mg-1 mL, were tested in the experiments. Tables versity, Sivas, and is highly inbred with little genetic vari- 1 through 5 and Figures 1 through 4 show the effect of dif- ation. The flies were maintained at a constant temperature ferent concentrations of Sunset Yellow, Ponceau 4R, Al- of 25°C ± 1°C on a standard medium composed of maize lura Red, Brilliant Blue FCF, and Brown HT on longevity. flour, agar, sucrose, dried yeast, and propionic acid (Stand- In this study, we determined that these food dyes decreased ard Drosophila Medium; SDM). The flies were kept in the the maximum mean life-span of the female and male D. dark, except during the transfer onto fresh medium (usually melanogaster populations in comparison with the control twice a week). The humidity of the experimental chamber groups not exposed to the dyes. The maximum life-span of was 40–60%, and the female flies used in this experiment this fly was 54–73 days for the female flies and 55–70 days were virgins. for the male flies in the control groups, whereas it was 54, 51, In this study, the effects of five food dyes (Sunset Yel- 47, and 40 days for female flies and 56, 49, 46, and 39 days -1 low, Ponceau 4R, Allura Red, Brilliant Blue, and Brown for male flies, respectively, for the lowest (0.5 mg mL) and HT) on longevity were studied separately in female and highest (2 mg mL) application groups among the adult pop- male populations of D. melanogaster. Different concentra- ulations of D. melanogaster exposed to Sunset Yellow. -1 tions of food dyes were added to the culture medium and The difference between the control and 0.5 mg mL appli- in each culture medium 20 females and 20 males were cation groups was not statistically significant (p˃0.05). On mated. After 24 h the numbers of dead and living individ- the other hand, the difference between the control and the uals were counted and the death rate was calculated. Ac- other application groups as well as the difference between -1 the 0.5, 1, 1.5, and 2 mg-1 mL application groups was sta- cordingly, the LC50 concentration was between 6 mg mL and 10 mg-1 mL. For the experiments 4 concentrations be- tistically significant (Table 1). -1 low the LC50 value were used (0.5, 1, 1.5, and 2 mg mL). Table 2 shows the effect of different concentrations of The food dyes were dissolved in distilled water. Control Ponceau 4R on longevity. The life-span of the control sub- groups were exposed to the distilled water. Each experi- jects was 60 and 61 days for female and male flies, respec- ment was repeated three times. To obtain same-aged flies, tively. Comparison of the maximum life-span of the con- adult individuals were mated in culture vials containing trol and application groups showed that the result for the only SDM and used to prepare prestocks. On an average, male control was highest. However, for Ponceau 4R, there 100 individuals were collected from among the same-aged was no significant difference between the maximum life- female and male flies that were not mated and obtained span of the female flies from the control group and the fe- from pupa. Then, the individuals gathered were put into male flies from the 0.5 and 1 mg-1 mL application groups. empty culture vials and starved for 2 h before application. In addition, there was no significant differencebetween the For the application groups, small plastic vials containing mean life-span of 0.5, 1, and 1.5 mg-1 mL application fe- 20 g SDM and 5 mL of the respective test solutionswere male flies. For Allura Red, the maximum life-span for the used; 100 larvae were embedded in the medium in these lowest and highest application doses was observed to be vials. The larvae were fed different concentrations of the 54, 52, 50, and 50 days for female flies and 55, 54, 56, and five test dyes (0.5, 1, 1.5, and 2 mg-1 mL). Feeding was 52 days for male flies. The difference between the 1.5 and terminated with the pupation of the surviving larvae. The 2 mg-1 mL application doses for female flies and 0.5 and experiments forboth the control and application groups 1 mg-1 mL application doses for male flies was not statisti- were initiated at the same time. All vials were incubated in cally significant. In addition, in male flies, the difference appropriate thermal cabins. During the experiments, the between the control and 1.5 mg-1 mL group was not signif- feed material was refreshed twice a week. The number of icant. Table 4 shows the effect of different concentrations individuals was controlled both at the beginning and at the of Brilliant Blue FCF on the longevity. In the control end of each application day; dead individuals were rec- group, the maximum life-span for female flies was 67 days, orded and then removed from the culture vials. The appli- while that for the male flies was 63 days. However, the cation was performed until the last individual died. The maximum life-span decreased to 46 days for female flies data obtained were analyzed by using SPSS (version 15.0). and 36 days for male flies in the application groups. The One-way analysis of variance (One-way ANOVA) and results suggest that longevity decreased with increase in the Least Significant Difference (LSD) test were used for the concentrations of Brilliant Blue. The difference between

the control and Brilliant Blue-applied groups was statisti- life-spans of the female and male flies in the control and cally significant. The maximum life-span for the lowest application groups were compared, it was observed that the (0.5 mg-1 mL) and highest (2 mg-1 mL) application groups longevity of the flies in the application groups was shorter among the adult populations of Drosophila subjected to thanthat in the control group. As shown in Tables 1-5, ex- Brown HT were observed to be 73, 70, 67, 61, and 53 days cept for some of the application groups (in female flies: for female flies and 70, 68, 60, 58, and 51 days for male control–0.5 mg-1 mL for Sunset Yellow and Allura Red, in flies, respectively. The difference between the control and male flies: control–0.5 mg-1 mL for Sunset Yellow), the the application groups was statistically significant for the difference between the groups in longevity was statistically female flies. However, there was no difference between the significant (p<0.05). Figures 1-4 show the maximum and maximum life-span of male flies in the control group and 0.5 mean life-span curves of female and male D. melanogaster and 1 mg-1 mL application groups. In addition, in male flies, in media treated with different concentrations of food dyes the difference among the results for 0.5, 1, 1.5, and 2 mg-1 during their adult stage of life. mL application groups was not significant. When the mean

TABLE 1 - Effects of Sunset Yellow on maximum life span and mean life span of D. melanogaster.

Doses Number of flies Max. lifespan±S.E. Mean lifespan±S.E. (mg-1 mL ) ♀ ♂ (in days) (in days) ♀ * ♂* ♀ * ♂ * Control 100 100 54 a 55 a 41.27±1.92 a 40.21±0.17 a 0.5 100 100 54 a 56 a 40.13±1.68 ab 39.56±1.13 a 1 100 100 51 b 49 b 38.51±0.97 b 34.28±1.21 b 1.5 100 100 47 c 46 c 35.20±1.28 c 32.54±1.82 c 2 100 100 40 d 39 d 30.91±1.13 d 29.99±1.33 d S.E.: Standard Error, Max.: Maximum, * Means with the same letters do not significantly differ at 0.05 level.

TABLE 2 - Effects of Ponceau 4R on maximum life span and mean life span of D. melanogaster.

Doses Number of flies Max. lifespan±S.E. Mean lifespan±S.E. (mg-1 mL) ♀ ♂ (in days) (in days) ♀ * ♂ * ♀ * ♂ * Control 100 100 60 a 61 a 57.23±1.19 a 58.27±3.20 a 0.5 100 100 57 ab 59 b 52.28±0.92 b 55.33±1.15 b 1 100 100 56 ab 57 c 53.97±1.83 b 54.27±2.18 b 1.5 100 100 50 b 49 d 49.19±1.72 c 50.91±1.19 c 2 100 100 41 c 39 e 39.21±1.10 d 41.41±1.00 d S.E.: Standard Error, Max.: Maximum, * Means with the same letters do not significantly differ at 0.05 level.

TABLE 3 - Effects of Allura Red on maximum life span and mean life span of D. melanogaster.

Doses Number of flies Max. lifespan±S. E. Mean lifespan±S. E. (mg-1 mL) ♀ ♂ (in days) (in days) ♀ * ♂ * ♀ * ♂ * Control 100 100 55 a 57 a 61.13±0.66 a 59.83±1.53 a 0.5 100 100 54 a 55 b 59.82±0.09 ab 58.21±1.26 b 1 100 100 52 b 54 b 58.28±0.75 b 56.56±0.26 c 1.5 100 100 50 c 56 a 56.13±1.18 c 54.10±1.92 d 2 100 100 50 c 52 c 55.92±2.73 c 54.90±1.74 d S.E.: Standard Error, Max.: Maximum, * Means with the same letters do not significantly differ at 0.05 level.

TABLE 4. Effects of Brilliant Blue FCF on maximum life span and mean life span of D. melanogaster.

Doses Number of flies Max. lifespan±S.E. Mean lifespan±S.E. (mg-1 mL) ♀ ♂ (in days) (in days) ♀ * ♂ * ♀ * ♂ * Control 100 100 67 a 63 a 51.22±1.26 a 56.74±1.43 a 0.5 100 100 61 b 57 b 48.97±0.95b 52.21±1.48 b 1 100 100 56 c 55 c 46.27±0.89 c 47.33±0.93 c 1.5 100 100 47 d 41 d 43.04±1.28 d 41.11±1.21 d 2 100 100 46 d 36 e 39.61±1.64 e 36.85±2.56 e S.E.: Standard Error, Max.: Maximum, * Means with the same letters do not significantly differ at 0.05 level.

TABLE 5 - Effects of Brown HT on maximum life span and mean life span of D. melanogaster.

Doses Number of flies Max. lifespan±S.E. Mean lifespan±S.E. (mg-1 mL) ♀ ♂ (in days) (in days) ♀ * ♂ * ♀ * ♂ * Control 100 100 73 a 70 a 67.24±0.54 a 66.361.18 a 0.5 100 100 70 b 68 ab 64.41±0.59 b 62.471.72 b 1 100 100 67 c 60 abc 56.28±0.72 c 59.570.65 c 1.5 100 100 61 d 58 bc 56.39±0.66 c 43.251.75 d 2 100 100 53 e 51 c 44.13±0.05 d 44.291.69 d S.E.: Standart Error, Max.: Maximum * Means with the same letters do not significantly differ at 0.05 level.

FIGURE 1 - The maximum life span curves of female individuals the D. melanogaster living medium applied with different concentrations of food dyes during adult stages.

FIGURE 2 - The maximum life span curves of male individuals the D. melanogaster living medium applied with different concentrations of food dyes during adult stages.

FIGURE 3 - The mean life span curves of female individuals the D. melanogaster living medium applied with different concentrations of food dyes during adult stages.

FIGURE 4 - The mean life span curves of male individuals the D. melanogaster living medium applied with different concentrations of food dyes during adult stages.

Julie Miller Jones, a professor of food and nutrition at Tanaka [20] studied the neurobehavioral effects, and the College of St. Catherine in St. Paul and the author of McCann et al. [18] reported that exposure to a mixture con- Food Safety [15] states that the amount of food additives taining Ponceau 4R gave rise to increased hyperactivity in consumed in a year by the average American is equivalent 3-year-old children. However, a long–term toxicity study to the body weight. The commonly used food dyes pre- in rats exposed in utero showed no carcinogenicity [21]. pared from petroleum and its derivatives pose a “rainbow Overdose of Allura Red has been shown to affect the re- of risks.” These risks include hyperactivity in children, can- productive functions when tested at the experimental level cer (in animal studies), and allergic reactions. Sunset Yellow [22]. Hyperactivity in children has been reported as a result itself may be responsible for causing an allergic reaction in of ingestion of Allura Red in high concentrations [23]. Bril- people with aspirin intolerance [16], resulting in various liant Blue FCF has previously been banned in Austria, Bel- symptoms, including gastric upset, diarrhea, vomiting, urti- gium, Denmark, France, Germany, Greece, and Spain, alt- caria, angioedema, and migraines [17]. Food coloring used hough it is certified as a safe food additive in the European to impart color to foods has been linked to hyperactivity in Union and is currently legal for use as a food additive in young children by McCann et al. [18]. Ponceau 4R is con- most countries. In fact, Maloney et al. [24] reported this sidered to be carcinogenic in some countries, including the food additive to be a carcinogenic agent. In addition, there USA, Norway, and Finland, and is currently listed as a is strong evidence that it causes reproductive and neurolog- banned substance by the US FDA (Food and Drug Admin- ical disorders [25], severe allergies [26], and anaphylactic istration Compliance Program Guidance Manual). Moreo- reactions [27], including rashes, swelling, and difficulty in ver, Tsuda et al. [19] analyzed the magnitude of DNA mi- breathing in humans. The genotoxicity data on Brown HT gration by using an in vivo comet assay (genotoxicity), are limited to bacterial tests. In a genotoxicity screening

test, 25 food dyes currently in use in foods were evaluated In a recent study, the toxic effects of three different for their mutagenic effect in bacterial assay systems. food dyes on D. melanogaster were investigated. Uysal et Brown HT did not show any mutagenic effects [28]. In ad- al. [40] reported that food dyes exert toxic effects in the dition, several bacterial assays, including the Ames’ muta- order of Carmine>Turmeric>Annatto. genicity test, revealed no evidence of genotoxicity, as reported in the BIBRA [29] toxicity profile on Brown HT. In this study, the mean life-span of D. melanogaster 4. CONCLUSIONS was found to be the maximum in the control group. In addition, the female individuals were found to have greater In conclusion, the number of studies on the action or longevity than the male individuals in all application mechanism of these food additives on the life-span of D. groups. Our results suggested that Brilliant Blue FCF melanogaster is in sufficient. We believe that the results of caused the greates decrease in life-span among the food this study may provide a basis for further studies in this coloring dyes tested, followed by Ponceau 4R, Sunset Yel- subject. As food additives form an inevitable part of our low, Brown HT, and Allura Red. Our results are in agree- daily life, it should be kept in mind that they may pose se- ment with those of studies that investigated the effects of rious health risks, particularly in children, necessitating different test materials on the life-span and survival rate of stringent regulations for their use. D. melanogaster. Sarıkaya et al. [30] studied the effects of sodium nitrate, potassium nitrite, and potassium nitrate on The authors have declared no conflict of interest. the life-span of D. melanogaster. They found that the life- span of the groups subjected to these substances was shorter than that of the control groups. In another study, Sarıkaya et al. 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(2002) Food dye use in enteral feedings: a review and a call for a moratorium. Nutrition in Clinical Practice 17 (3): 169-81. Received: October 27, 2014 [26] Mortelmans, K., Haworth, S., Lawlor, T., Speck, W., Tainer, B., Revised: January 29, 2015 and Zeiger, E. (1986) Salmonella mutagenicity tests. II. Results Accepted: March 04, 2015 from the testing of 270 chemicals. Environmental Mutagenesis 8 (Suppl 7): 1–119. [27] Metheny, N.A., Dahms, T.E. and Stewart, B.J. (2002) Efficacy of CORRESPONDING AUTHOR dye-stained formula in detecting pulmonary aspiration. Chest 122: 276-281. Şifa Türkoğlu [28] Haveland-Smith, R.B. and Combes, R.D. (1980) Screening of food dyes for genotoxic activity. Food and Cosmetics Toxicology 18: Cumhuriyet University 215-221. Faculty of Science [29] BIBRA, (1989) The British Industrial Biological Research Asso- Department of Biology ciation: working group. Toxicity Profiles, Brown HT was only 58140, Sivas shown at BIBRA symposium, (as referred to by TemaNord, 2002). TURKEY [30] Sarıkaya, R., Çakır, Ş. and Solak, K. (2006) The effects of food preservatives on the longevity of Drosophila melanogaster (mwh x flr). Kastamonu Eğitim Dergisi 14(1): 173-184. Phone: 90 346 2191010 Fax: 90 346 2191186 [31] Sarıkaya, R., Selvi, M., Akaya, N., Acar, M. and Erkoç, F. (2010) Effects of food dyes in different concentrations on percentage of E-mail: [email protected] survival in Drosophila melanogaster (mwh x flr). Süleyman De- mirel Üniversitesi Fen Dergisi 5(1): 38-46. FEB/ Vol 24/ No 9/ 2015 – pages 2830 - 2836

NATURAL RADIOACTIVITY OF FERTILIZERS WIDELY USED IN THE AGRICULTURAL LANDS OF EGE REGION IN TURKEY

Bihter Çolak Esetlili1,*, Günseli Yaprak2 and Dilek Anaç1

1Department of Soil Science and Plant Nutrition, Faculty of Agriculture, Ege University, 35100, Bornova, Izmir, Turkey 2 Institute of Nuclear Science, Ege University, 35100 Bornova, Izmir, Turkey

ABSTRACT products are strictly inspected in order to be certified. There is also organic farming; however, in the country generally This is a preliminary study to determine the natural ra- conventional farming system prevails and not much control dionuclides of the commercial chemical fertilizers and rock exits in this regard. phosphates which are widely used in the agricultural lands The rapidly rising world population is increasing the of Ege region in the west of Turkey. Mainly the fundamen- demand for food. Due to the limited extent of arable agri- tal raw materials of all phosphorous (P) containing fertiliz- cultural land, it is essential to sustain the soil health and ers are examined. fertility by environmentally friendly techniques. Large Gamma spectrometric measurements of 226Ra, 232Th quantities of commercial chemical fertilizers are being and 40K activities were made on sixty-eight straight and used to replenish the plant nutrients depleted from the soil. compound commercial fertilizers, as well as on the nine For instance, the use of phosphorous (P) fertilizers is a very phosphate rocks. The activity concentrations of 226Ra in the common practice since it is a primary plant nutrient [1]. phosphate rocks were found to be in the range of 326 and However, P fertilizers also contain radioactive elements, in 788 Bq kg-1 while those of 232Th and 40K were found to be some cases at toxic levels. Their extensive application much lower, ranging from 50 Bq kg-1 to levels so low that threatens the natural balance by accumulating in the soil, they were undetectable. Normal super phosphate (NSP) accessing into the food chain or leaching into water and triple super phosphate (TSP) exhibited relatively sources. Therefore, it is important to prevent the misuse of higher 226Ra activities than the rest of studied P based fer- P fertilizers as well as other agrochemicals. It is also im- tilizers. 40K activities in potassium sulfate and nitrate ferti- portant to obtain accurate data on the chemical and radio- lizers w up to about 104 Bq kg-1 . In particular, the activity logical impacts of the agricultural activities [2-5]. concentrations of the 226Ra in NP and the majority of NPK Rock phosphate is the main source of P for fertilizers fertilizers, which are the most widely used types in the cul- [6]. The activity concentrations of the natural radionuclides tivated soils of the Ege region, appeared much lower, and in the rock phosphates vary noticeably depending on their in some cases, were undetectable. These results are of great geological origin. According to the recent UNSCEAR Re- significance in terms of sustainable agriculture involving ports [6-8] sedimentary origin P, which covers 85% of cur- the use of phosphate-based fertilizers. rent demand, contains relatively high concentrations of 238 226 U and its decay products like Ra [6]. It is widely doc- KEYWORDS: Natural radionuclides, chemical fertilizers, rock umented [3,9,10] that the continuous long-term application phosphate, agricultural land, Ege region of the P and/or K fertilizers to soils could eventually increase the activity concentrations of natural radionuclides

in the soil, leading to a considerable rise in the number of 1. INTRODUCTION people exposed to higher gamma radiation levels. Turkey has 77.9 million ha of land in total, 26% of The first detailed reports on the activity concentrations which is covered by forests, 16% by meadow and pastures, of the natural radionuclides in the cultivated soils of the and 35% by agricultural lands [1]. In the country, the vari- three basins in the Ege Region of western Anatolia were ation in climate allows the production of a wide range of from a team in the Nuclear Science Institute [12-14]. These agricultural crops. Some part of the agricultural land is authors claimed that phosphate and/or potassium contain- managed under the Good Agricultural Practices (GAP) ing fertilizers could lead to a slight increase in the natural farming system where synthetic pesticides and fertilizers radioactivity concentration in the soils (Table 1). can be used only according to the related regulations dif- On the basis of the recently reported data [16], the con- fering from the conventional farming. Therefore, GAP sumption of chemical fertilizers in Turkey has reached 5 million tons, 65 % of which is N fertilizers, 32 % P ferti-

* Corresponding author lizers and the remaining 3% is K (Table 2) [16-18].

TABLE 1 - Activity concentrations of natural radionuclides of the soils in Ege Region [12-14] Activity concentrations (Bq kg-1) Basin Radionuclide N* Average Standard deviation Range 226Ra 130 36 9 19-72 Gediz 232Th 31 8 10-65 40K 608 144 168-965 226Ra 105 34 7 17-76 Küçük Menderes 232Th 32 6 17-51 40K 585 158 296-1305 226Ra 43 29 21 7-84 Büyük Menderes 232Th 22 12 10-48 40K 464 186 100-864 N*: Number of samples.

TABLE 2 - Consumption of chemical fertilizers in Turkey (thousand tonnes)

Fertilizers 1985-1989 1990-1995 1996-2000 2001-2005 2006-2010 2011 2012 2013 Ammonium Sulphate 440.5 342.7 310.6 305.8 391.6 413.1 467.7 499.1 Ammonium Nitrate (%26 N) 1584 1401 1228 933.1 896.0 733.2 883.2 798.6 Ammonium Nitrate (%33 N) 1.75 132.2 401.1 752.7 883.6 842.1 995.9 1040.1 Urea 517.7 650.0 838.6 781.3 807.6 760.3 872.4 1100.9 Normal Super Phosphate 8.4 0.004 - 10.9 10.3 7.1 3.0 1.95 Triple Super Phosphate 263.9 128.2 60.6 39.4 31.2 29.2 24.2 26.2 Di-Ammonium Phosphate 431.9 658.9 633.7 501.8 475.0 386.5 460.5 615.7 Potassium Sulphate 20.4 16.3 16.9 17.1 19.6 19.7 15.7 13.7 Compounds 1171 1395.6 1627 1509 1463 1575.2 1617.3 1717.3 Total 4451 4727 5116 4852 4978 4766 5340 5814

. All of the above reported scientific background estab- cay products before undergoing gamma spectroscopy meas- lished the objective of this study. For a sustainable agricul- urements. The period is also long enough to ensure equilib- tural productivity and food safety, the natural radionuclides rium between 220Rn and its decay products (approximately of the chemical fertilizers widely used in Ege region of four days) in the 232Th series. Turkey and their raw material-rock phosphate- was aimed to be examined . 2.2 Gamma-spectrometric measurements Gamma spectrometry measurements of 226Ra, 232Th and 40K activities were carried out in a total of 77 samples, 2. MATERIALS and METHODS including the raw materials of the P fertilizers using a Can- berra 76x76 mm, NaI(Tl) scintillation detector connected 2.1 Sample collection and preparation to a PC-based 8K multichannel analyzer, which uses only In Turkey, there are seven major fertilizer companies 2048 channel to avoid poor counting statistics per channel, which are responsible for the production of most common and associated software. The detector was shielded by standard fertilizers. For this study, 68 fertilizers and 9 rock 100 mm thick lead bricks internally lined with 1.5 mm cop- phosphates were randomly collected from these companies per foil to reduce the background counting rate. in the years 2012 and 2013. The studied samples were tri- The gamma spectroscopic analysis using NaI(Tl) for ple superphosphate, diammonium phosphate, mono ammo- U, Th and K assays both in the laboratory and field are well nium phosphate, normal super phosphate from the standard described in the relevant publications [19, 20, 21]. straight P fertilizers and NP/NPK compounds; urea, ammonium nitrate, potassium sulphate, potassium nitrate In this method, the calculation of the activity concentration is based on the solution of three linear equations, from the P free fertilizers and FeSO4.7H2O, Fe-EDTA and Fe-EDDHA from the iron fertilizers. involving K1, K2 and K3 sensitivity factors, and ,  and  stripping ratios as gamma spectrometer calibration param- For gamma analysis, the samples were dried at room eters, which are related to the counting rates in selected temperature, further oven dried at 65 °C, crushed to pass three energy peaks or windows, corresponding to the con- through a 2mm sieve, powdered and well homogenized. tents of Th, U and K in the sample. Accordingly, equations The densities of the samples ranged from 0.8 to1.7 gcm-3 for determination of eTh, eU and K activity concentrations (dry density). Approximately 100 g of each sample was in a sample are as follows [22]; placed in a 100 cc PVC can, sealed and stored at least 30 days to allow for a secular equilibrium between 226Ra and its de- eTh(Bq/kg)= C(Th)/K1 (1)

-1 eU(Bq/kg) =[ C(U)- αC(Th) ]/ K2 (2) was estimated to be 6, 6 and 18 Bq kg , respectively, for the counting time of 30.000 second and a sample weight of 100 g. K(Bq/kg) =C(K)- C(U) -  C(Th)  -  C (Th) / K3 (3) The background spectra were used to correct the net peak In the above equations, the term “e”, i.e. equivalent, areas for the related radionuclides. Activity concentrations indicates that series equilibrium conditions are assumed. of 226Ra, 232Th and 40K were systematically determined The constants K1, K2 and K3 are the count rates per unit 232 238 40 with one sigma error and expressed relative to dry weight. activity concentration of Th, U and K and are thus Relative uncertainties of the activity concentrations were the spectrometric sensitivities for these radioelements. The usually lower than 30 %. fractions ,  and  are referred to as the stripping ratios of the gamma spectrometer. The contribution from Th in the U window and in the K window are represented by the count 3. RESULTS AND DISCUSSION rates αC(Th) and C(Th), while the count rate C(U) represents the contribution from U in the K window. Without Rock phosphate is the starting raw material for all of compensation for these count rates, it is not possible to de- the P fertilizers which are chemically processed from the termine U and K concentrations spectrometrically [22-26]. rocks. Results showed that the activity concentrations of The U and Th standards in radioactive equilibrium pur- natural radionuclides in the P based fertilizers exceed the chased from IAEA were prepared by the Nuclear Science world wide average values in soils [29-33]. The investiga- Institute to ensure the same geometry as the samples. In a tions on the radionuclide contents of the input raw materi- similar process, the K standard was prepared from pure po- als (rock phosphate) and output products as final P fertiliz- tassium chloride (KCl), purchased from Merck. These stand- ers are of great significance as they can contribute to the ard materials were used to find the stripping ratios and sen- identification of an unpolluted/polluted agricultural envi- sitivity factors of the spectrometric system. The activities of ronment. 232 238 40 Th, U and K standards are 2442, 1458 and 16210 Bq It is known that the 70% of the world rock phosphate -1 kg respectively. It is simple to determine these stripping reserves is located in sedimentary marine deposits of the factors by making measurements on calibration sources con- Upper Cretaceous and Eocene ages of the Mediterranean taining only one of K, U and Th [22]. phosphogenic province. These deposits were laid down in In the conventional NaI(Tl) -ray spectrometry method, the ancient Tethys Sea in the Mesozoic and Tertiary ages, the activity concentration of 214Bi is used for measuring the which occupied an area covering Morocco, Spain, Sahara, activity concentrations of 238U. On the other hand, 214Bi ac- Algeria, Tunisia, Egypt, Israel, Jordan, Syria, Saudi Ara- tivity should be considered only as a measure of the 226Ra bia, Turkey and Iraq [34]. 238 content of the sample rather than U itself due to the radio- Since the phosphate deposits in Turkey itself are gen- 238 active disequilibrium between U and the long-lived erally low grade, almost all of the studied rock phosphates 234 230 226 daughter products, mainly U and Th, prior to Ra. Ac- are imported from other countries. Accordingly, the results 214 cordingly, a measurement of Bi activity provides a relia- of the gamma spectroscopic analyses of 226Ra, 232Th and 226 226 ble measurement of the Ra activity. The Ra was evalu- 40K in the phosphate rocks from countries in the Mediter- 214 ated, in all cases, from the 1764.49 keV peak of Bi, while ranean region, including Turkey, are presented in Table 3, 232 the Th activity was determined from the 2614.53 keV while the other measurements for different P fertilizers, in- 208 40 peak of Tl, and the K activity was determined from the cluding NP and NPK samples, are presented in Table 4. 40 K peak at 1460.75 keV. The measurements of P free compound fertilizers are given The minimum detectable activity (MDA) based on Cur- in the Table 5. rie [27] for 226Ra, 232Th and 40K of the gamma spectrometer

TABLE 3 - Activity concentrations of 226Ra, 232Th and 40K in the phosphate rocks

Activity concentrations (Bq kg-1 ) Phosphate rock 226Ra 232Th 40K Turkey 620±17 32±6

Table 3 shows that the activity concentrations of 226Ra (NSP). This value is much higher than those reported in in the phosphate rocks are in the range of 326 and 788 Bq Saudi Arabia (75 Bq kg-1) and Finland (54 Bq kg-1) but very kg-1 while those for 232Th and 40K are much lower, ranging similar to the values reported in USA (780 Bq kg-1) and from a maximum of 50 Bq kg-1 to undetectable. As ex- Germany (520 Bq kg-1) [37, 38]. The 226Ra activity concen- pected, the activity concentrations of 232Th and 40K in these trations in TSP varied in the range of

TABLE 4 - Activity concentrations of 226Ra, 232Th and 40K in the P based chemical fertilizers

Activity concentrations (Bq kg-1 ) Fertilizer No. of samples 226Ra 232Th 40K Mean (Range) Mean (Range) Mean (Range) NSP 4 494±19 (352-788) (

TABLE 5 - Activity concentrations of 226Ra, 232Th and 40K in the P-free chemical fertilizers

Activity concentrations (Bq kg-1 ) Fertilizers No. of samples 226Ra 226Ra 40K Mean (Range) Mean (Range) Mean (Range) Ammonium Sulphate 2

fertilizers, the activity concentrations of 40K in the samples [4] Dora, O.Ö., Candan, O., Dürr, S. and Oberhansli, R. (1995) reached 94 Bq kg-1. In these fertilizers, 40K might be de- New evidence on the geotectonic evolution of the Menderes Massif. International Earth Sciences Colloquium On The rived from phosphate ores [45]. Generally, the NPK ferti- Eagean Region, Izmir-Turkey, V.1, 53-72. lizers exhibit proportional changes in 40K value based on 226 232 [5] Kocman, A. (1993) Climatical conditions through the Turkish their K content, while the levels for Ra and Th are al- Aegean Subregion plains from the standpoint of the human most all below the instrument detection limits. and environmental effects. Published By Ege University, Fac- ulty of Arts, No, 73 (Turkish With English Abstract), 143. Table 5 shows that natural activity concentrations were found to be lower than the instrument detection limits for [6] Ahmed, N.K. and El-Arabı, A.M. (2005) Natural radioactivity in farm soil and phosphate fertilizer and its environmental im- ammonium sulphate, ammonium nitrate, urea, potassium plications in Qena Governorate, Upper Egypt” Journal Envi- sulphate, and potassium nitrate, and also iron fertilizers. On ronmental Radioactivity, Vol. 84, No. 1, pp. 51-64. the other hand, the activity concentrations of up to approx- 4 -1 40 [7] Lovborg, L. (1971) Assesment of uranium by gamma ray spec- imately 10 Bq kg for K in potassium sulfate and potas- trometry. Proceeding of a Nato-Sponsored Advanced Study sium nitrate were detected. Although some fertilizers do Institude On Methods Of Prospecting For Uranium Minerals, not contain K, very low levels of 40K were found, which London. may be attributed to the filling materials (clay, sand, etc.) [8] Killeen, P.G. (1979) Gamma ray spectrometric methods in used in the production of fertilizer. uranium exploration—application and interpretation; in Geo- physics and Geochemistry in the Search for Metallic Ores; Hood, P.J., ed., Geol. Surv. Can. Econ. Geol. Report 31:163- 229.

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[34] Aydin, I., Aydin, F., Saydut, A., Bakirdere, A.G. and CORRESPONDING AUTHOR Hamamci, C. (2010) Hazardous metal geochemistry of sedimentary phosphate rock used for fertilizer (Mazıdag, SE Ana- tolia, Turkey) Microchemical Journal 96, 247–251. Bihter Çolak Esetlili Department of Soil Science and Plant Nutrition [35] Hussein, E. M. (1994) Radioactivity of phosphate ore, superphosphate and phosphogypsum in Abu-Zaabal phosphate Faculty of Agriculture plant, Egypt. Journal of Health Physics, 67, 280–283. Ege University 35100, Bornova, Izmir [36] Ahmed, N. K. and El-Arabi, A. M. (2005) Natural radioactivity in from soil and phosphate fertilizer and its environmental TURKEY implication in Qena govemorate, Upper Egypt .Jourmal of En- vironmetal Radioactivity, 84:51-64. E-mail: [email protected] [37] Gulmond, R. J and Windham, S. T. (1975) Radionuclides in [email protected] the Environment. Technical Note No. ORP/CSD-75-3 (Wash- ington, D.C.: USEPA) 228–229. FEB/ Vol 24/ No 9/ 2015 – pages 2837 - 2842

ENHANCEMENT OF WASTE ACTIVATED SLUDGE AEROBIC DIGESTION WITH COMBINED FENTON OXIDATION AND ELECTROCHEMICAL PRETREATMENT METHOD

Haiping Yuan1, Qingji Wang2, Shengjuan Guo2, Jie Yao3, Nanwen Zhu1,* and Yi Gong1

1School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China 2 Daqing Oilfield Engineering Co., Ltd, CNPC, Daqing, Heilongjiang, 163712, P. R. China 3 Shanghai Municipal Sewerage Co., LTD., Shanghai 200233, China

ABSTRACT tion and odor problems of sludge should be reduced prior to the ultimate disposal [1-3]. The treatment and disposal The effects of hybrid electrochemical (EC)-Fenton of WAS is a problem of growing importance, representing pretreatment method on the efficiency of aerobic sludge di- up to 50% of the current operating costs of wastewater gestion were studied. The effects of H2O2 dosage, initial treatment plants (WWTP)[4].The most common treatment 2+ pH, reaction time, and [H2O2]/[Fe ] were investigated by of sludge was stabilization including biological processes Fenton oxidation alone or by EC-Fenton combined pre- of anaerobic digestion and aerobic digestion as well as treatment. Based on both the removal of VSS and the sol- physical and chemical methods. Aerobic digestion is a very ubilization of WAS, the proper Fenton reaction conditions popular alternative for small or medium-sized WWTPs [5]. were recommended as follows: H2O2 dosage = 1.0 g/L,ini- Furthermore, compared with anaerobic digestion, the ad- 2+ tial pH = 3, reaction time = 60 min, and [H2O2]/[Fe ]= vantages of rapid biomass degradation, short sludge reten- 10.0. The sludge SCOD concentration of EC-Fenton com- tion time (SRT) is obtained in aerobic sludge digestion. bined pretreatment was observed to be synergistically en- However, sludge reduction and stabilization through the hanced from 104 mg/L (raw) to 624 mg/L, higher than that microbial function appears to be of low efficiency in full- of Fenton oxidation pretreatment alone. The highest VSS scale aerobic wastewater treatment processes. Conventional removal efficiency could reach up to 13.3% after EC-Fen- aerobic digestion still requires large digestion tanks due to ton combined pretreatment. Furthermore, the batch aerobic its relatively long retention time (15-30 days) [6]. digestion system led to exceeding 38% VSS degradation after 12-day digestion, 12 days earlier than the control WAS is known to be very difficult to digest due to rate without pretreatment. EC-Fenton pretreatment was shown limiting cell lysis which has been reported in many re- to offer a promising alternative method of enhancing aero- searches [7]. Thus sludge requires pretreatment to speed up bic sludge digestion. the low rate of biodegradation and to enhance the digesti- bility. Recently, various new pretreatment methods have focused specifically on the disintegration of WAS by me-

KEYWORDS: Waste activated sludge; Aerobic digestion; Stabili- chanical, thermal hydrolysis, ozonation, acidification or al- zation; Fenton oxidation; Electrochemical pretreatment kaline hydrolysis, freezing and thawing, ultrasound and biological approaches [8-13].

Due to the fast development of electrode materials, 1. INTRODUCTION electrochemical (EC) technology has been introduced as a promising method for effective destruction of organic com- Along with a rapid industrial growth of urban area, pounds [14], especially in the pollution control of various many developing countries, especially China, are facing wastewaters [15]. However, there have been little reports waste activated sludge (WAS) disposal problem due to the about the utilization of EC in the treatment of sludge until continual increase in the production of excess sludge. The now. Complete mineralization or partial degradation of or- Environmental Protection Agency (EPA) has enunciated ganic pollutants depends on the anode materials during the that WAS requires prior treatment of organic residues in EC process. It was reported that the use of Ti/RuO anode order to acquire ‘class A’ sludge property before final dis- 2 could produce a series of EC steps which converted high posal. Therefore, the organic content, pathogen contamina- biopolymer substances to low-molecular-weight products [16]. The low-molecular-weight products then can be eas- * Corresponding author ily degraded by the subsequent biological treatment [17].

Recently, the feasibility of Advanced Oxidation Pro- TABLE 1 - Characteristics of waste activated sludge used in this cesses (AOPs) in excess sludge reduction has been investi- study gated intensively [18, 19]. Many researches showed that Parameters Values AOPs have great potential to reduce the production of ex- pH 6.69-7.07 cess sludge. AOPs are based on the generation of hydroxyl Water content (%) 99.2-98.2 radicals (·OH) to degrade organic matters. Among the Conductivity (μS/cm) 794-1,148 Chemical oxygen demand (COD)(mg/L) 17,462-18,990 AOPs, the low cost Fenton’s oxidation process is an effec- Soluble chemical oxygen demand 36-52 tive method to remove organic pollution [20]. (SCOD)(mg/L) In the past few years, many experiments have been car- Total solid (TS) (g/L) 8.0-18.2 Volatile solid (VS) (g/L) 5.5-12.7 ried out to treat excess sludge in combined processes. Fen- Suspended solid (SS) (g/L) 7.6-17.0 ton oxidation process is one of the commonly used ad- Volatile suspended solid (VSS) (g/L) 5.4-12.6 vanced oxidation techniques. Fenton's reagent is a mixture Organic content (VSS/SS) (%) 0.68-0.74 of H2O2 and ferrous iron. The ferrous iron initiates and cat- 2.2. Pretreatment processes alyzes the decomposition of H2O2, resulting in the generation of highly reactive hydroxyl (·OH) radicals [21]. How- Batch pretreatment experiments were carried out to deter- ever, the performance of H2O2 in the disintegration of ex- mine the parameters affecting WAS disintegration. Samples cess sludge has not yet been investigated. Therefore, the of excess sludge (300 mL) were batch-treated in a 500 mL main objective of this research was to achieve sludge re- beaker, which were initially adjusted to the required pH value duction by combining Fenton oxidation and EC pretreat- by sulfuric acid (H2SO4) and ferrous sulfate (FeSO4·7H2O) ment followed with aerobic sludge digestion process. was then added into the sludge samples, subsequently Fen- Herein, in order to enhance solid reduction in aerobic ton reaction was initiated after adding H2O2(30%v/v). After sludge digester, the optimum condition for Fenton oxida- Fenton oxidation, EC pretreatment was carried out in the tion and the effect of combined Fenton oxidation and EC same beaker equipped with two RuO2/Ti mesh electrodes pretreatment on the efficiency of the aerobic sludge diges- (with a total effective area of 70 cm2) (Fig. 1a). The distance tion process were investigated in this study. between the two electrodes was set to 2 cm in all experiments. The voltage (15V) and treating time (30 min) were maintained at constant values according to the previous 2. MATERIALS AND METHODS study [22].

2.1. Sewage sludge 2.3. Aerobic digestion reactor set up In this study, excess sludge was obtained from the sec- Aerobic sludge digestion experiments were carried out ondary sedimentation tank of a municipal wastewater treat- in plexiglass cylinders with an effective volume of 5 L each ment plant in Shanghai, China. The plant treats 50,000 m3/d (Fig. 1b). D0 digester filled with sludge untreated was set of wastewater with the anaerobic-anoxic-aerobic process. as control, D1 digester was filled with EC pretreated The sludge samples were thickened to meet the require- sludge, D2 digester was filled with Fenton oxidation pre- ment of solid concentrations and stored at 4 prior to use. treated sludge, and D3 digester was filled with Fenton-EC The characteristics of the sludge were shown in Table 1. combined pretreated sludge. After adjusting pH of the pre-

(b)

(a)

(1) Electrochemical cell; (2) anode; (3) cathode; (4) DC stabilized power supply; (5) flow control; (6) aerator pipe; (7) sludge

FIGURE 1 - Experimental device of electrochemical treatment (a) and aerobic digestion (b)

treated sludge to 7.0 approximately, inoculations were per- initial concentration at the Fe(II) to H2O2 ratio. While the formed with 10% (V/V) of fresh activated wastewater SCOD concentration rose to 614mg/L, increasing by about sludge at a solid concentration of about 20 g/L. The di- 5 times compared to the initial Fe (II) to H2O2 concentra- gesters were aerated by an air compressor (AIRPUMP, X- tion when treated by the combination of Fenton and EC 6500) to maintain a uniform oxygen concentration of 2 mg pretreatment. The predominant radical species is ·OH [24]. O2/L as to keep good mixture. All the digesters were op- These ·OH are responsible for the degradation of a number erated at room temperature (252℃) for 24 days. Samples of organic substances via oxidation. Thus, sludge disinte- were taken from the biological reactors at regular intervals, gration is the result of oxidizing effect of ·OH. The ·OH filtered and analyzed. radicals generated during the Fenton oxidation have an oxidizing potential of 2.8 V and are capable of oxidizing a 2.4. Analytical methods wide range of organic compounds including sludge cells Soluble COD (SCOD), Total phosphorous (TP), Total which resulted in the increase of SCOD and decrease of nitrogen (TN), Suspended Solids (SS) and Volatile Sus- VSS [25]. In all likelihood, more ·OH radicals were gener- pended Solids (VSS) were analyzed using standard meth- ated at high concentration ratios, but ·OH radicals were ods [23]. In order to remove suspended particles, samples also scavenged by reacting with excess Fe (II) as shown in were filtered through a 0.45 µm membrane filter (What- Eq. (1) [26]. Furthermore, excess ·OH radicals may react man, Germany) to obtain soluble nutrient samples. pH val- with themselves, as shown in Eq. (2) [27]. ues were measured with a pH meter (pHS-3C, Leici Co., Fe2++ ·OH Fe3++ OH- (1) Ltd., Shanghai, China). Dissolved oxygen (DO) concentrations were measured using a DO meter (MI-65 Martini In- HO· + HO· H2O2 (2) struments). 2+ Therefore, extremely high [H2O2]/[Fe ] ratio is not beneficial to the removal of VSS and solubilization of 3. RESULTS AND DISCUSSION sludge. Taking both VSS removal and sludge solubilization into consideration, the Fe (II) to H2O2 concentration 3.1. Optimizaion of Fenton oxidation pretreatment conditions ratio of 10 is recommended.

2+ 3.1.2. Effect of [H2O2]/[Fe ] on the sludge oxidation pretreatment The VSS removal efficiency and sludge solubilization were very common used to measure the effect of Fenton oxidation on sludge, and the sludge solubilization was measured in terms of the SCOD concentration. The effects 2+ of [H2O2]/[Fe ] (from 1.0 to 50.0) on the VSS removal efficiency and SCOD concentration were investigated with the H2O2 dosage, reaction time and initial pH of 1.0 g/L, 60 min and 3, respectively. As was illustrated in Fig. 2, the rate of VSS removal increased considerably when the 2+ [H2O2]/[Fe ] increased from 2.5 to 10.0. However, the continuous increasing of the concentration ratio, even to 50.0, brought a sustainable decline in VSS removal efficiencies, to the values nearly the same as that treated by the 2+ [H2O2]/[Fe ] of 2.5. When the sludge pretreated by Fenton 2+ FIGURE 2 - Influence of the [H2O2]/[Fe ] ratio on the sludge reduc- oxidation alone, the VSS removal efficiencies could reach tion and SCOD concentration (Note: error bars represent standard as high as 7.3%, while according to the treatment of the deviation) combination of EC and Fenton oxidation method, it could reach even as high as 12.9%, increased by 76.7% at the 3.1.3. Effect of H2O2 dosage on the sludge oxidation treatment 2+ [H2O2]/[Fe ] ratio of 10.0. The effects of H2O2 dosage (from 0 to 2.0 g/L) on the 2+ The increase of [H2O2]/[Fe ] ratio can also improve VSS removal efficiency and SCOD concentration were in- 2+ 2+ the solubilization of sludge after the [H2O2]/[Fe ] ratio ex- vestigated with the [H2O2]/ [Fe ], reaction time and initial ceeding 10.0, but it showed a slight increase of SCOD pH of 10.0, 60 min and 3, respectively, as were shown in 2+ when the [H2O2]/[Fe ] ratio exceeding 30.0 and somewhat Fig. 3. The removal efficiencies of VSS increased markedly 2+ slowed down when the [H2O2]/[Fe ] ratio reached 50.0. along with the increase of H2O2 dosage until to 1.0 g/L, and However, the SCOD concentration of sludge decreased then rose steadily when the H2O2 dosage further increased 2+ when the [H2O2]/[Fe ] ratio was less than 10.0 (Fig. 2). In to 2 g/L. The highest VSS removal efficiency of sludge addition, the SCOD concentration of sludge treated by Fen- could be obtained at about 7% when treated by Fenton ox- 2+ ton oxidation alone was 414 mg/L when the [H2O2]/[Fe ] idation alone, while 12.5% when followed by the treatment ratio was 30.0, increasing by about 3 times compared to the of EC method with the 2.0 g/L H2O2 dosage (Fig. 3). The

2+ 3+ - VSS removal efficiencies increased from 5.3% to 11.4% Fe + H2O2 Fe +·OH + OH (3) when the H O dosage increased from 0 to 1.0 g/L followed 2 2 2H2O2 O2+2H2O (4) by the EC treatment at the conditions mentioned above. The formation of ·OH can rupture sludge microbial cell In a word, taking the sludge reduction and sludge solu- walls and release intracellular material, leading to the de- bilization as well as sludge treatment cost into consideration, crease of VSS [28]. As a result, the H2O2 concentration of the H2O2 dosage of 1.0 g/L is recommended for the pre-treat- 1.0 g/L was chosen as the optimal dosage taking the cost ment of sludge using Fenton-EC oxidation treatment. of H2O2 dosage and VSS removal efficiency into account. 3.1.4. Effect of initial pH and reaction time on the sludge oxidation treatment Batch experiments were carried out to investigate the effects of initial pH on the process of sludge reduction by Fenton and EC combined oxidation. The effects of pH (from 2 to 7) on the VSS removal efficiency and SCOD 2+ concentration were investigated with the [H2O2]/[Fe ], H2O2 dosage, and reaction time of 10.0, 1.0 g/L and 60 min, respectively. As was illustrated in Fig. 4a, the removal efficiencies of VSS decreased with the increase of pH by both the treatment of Fenton oxidation alone and the combination of Fenton and EC oxidation, because pH can influence the generation of ·OH radical. The removal efficiencies of VSS increased notably with the decline of pH from 7.0 to 2.0, (Fig. 4a), from 0.9% to 5.9% with the treatment of Fenton oxidation alone and from 3.2% to 12.3% FIGURE 3 - Effect of H2O2 dosage on the sludge reduction and solu- with the treatment of Fenton-EC oxidation, respectively. It bilization could be concluded that the VSS removal efficiency under the application of Fenton-EC oxidation were about 2 times Solubilization of WAS during Fenton oxidation pro- greater than that under the pretreatment of Fenton oxida- cess was evaluated based on a variety of sludge character- tion alone when treated at the pH of 2. istics, such as SCOD concentrations used in this study. As The rise of pH can cause the formation of an inactive was shown in Fig. 3, similar changes of SCOD concentra- hydroperoxide anion due to H O dissociation [30]. When tions could be observed with the increase of H O dosage 2 2 2 2 pH was greater than 4, the concentration of free iron spe- by the different pretreatments. The SCOD concentration cies decreased due to formation of complexes that could treated by Fenton oxidation alone was 325 mg/L and 585 not react with H O easily [31]. H O became unstable and mg/L treated by Fenton and EC combined oxidation, with 2 2 2 2 decomposed to oxygen when pH was high. It also lost its the addition of 2 g/L H O dosage, respectively (Fig. 3). 2 2 oxidation ability, as presented in Eq. (4). The SCOD concentration observed during the oxidation pretreatment at H2O2 dosage of 2.0 mg/L when treated by The decrease of pH caused the increase of SCOD (Fig. Fenton oxidation alone was 1.8 times greater than that 4a). When pH decreased from 7.0 to 2.0, the SCOD concen- treated at H2O2 dosage of 1.0 g/L by Fenton- EC oxidation tration increased slightly compared with VSS removal effi- pretreatment. With the improvement of the sludge’s solu- ciencies. It increased from about 250 mg/L to about 350 mg/L bilization during Fenton oxidation, organic substances were with the treatment of Fenton oxidation alone, while from transferred from the solid to the aqueous phase, resulting in 400 mg/L to 624 mg/L with the combined treatment of an increase in soluble protein and carbohydrate concentra- Fenton and EC oxidation. Extreme acidic conditions may tions in the aqueous phase [29]. However, the continuous lead to serious lysis or disintegration of sludge cells, and oxidation with excess oxidant may also make the released thus cause the significant decrease of VSS and increase of organic material mineralized, resulting in the decrease of SCOD. Furthermore, such low pH does not facilitate the SCOD. As it could be seen from Fig. 3, the SCOD concen- subsequent sludge biological treatment, such as aerobic di- tration decreased a little when the H2O2 dosage was more gestion. Therefore, considering both VSS removal effi- than 0.3 g/L, while after increasing more than 0.6 g/L, the ciency and subsequent sludge solubilization, the proper pH SCOD concentration increased all the way. value should be set around 3. It is revealed that dissolved iron concentration would become rather low, because fer- The reacting system of Fenton oxidation applicable in ric iron would precipitate as Fe(OH) when pH> 4, which this study is promised to be effective. As shown in Eq. (3), 3 would lower its potential ability to catalyze H O [32]. more OH radicals were produced at increased dosages of 2 2 H2O2, resulting in higher VSS removal efficiency. How- The effects of the reaction time (from 0 to 240 min) on ever, due to the self-decomposition of H2O2 shown in Eq. the VSS removal efficiency and SCOD concentration were 2+ (4), extremely high dosages of H2O2 did not improve re- investigated with the [H2O2]/[Fe ], H2O2 dosage, and ini- moval efficiency [26]. tial pH of 10.0, 1.0 g/L and 3, respectively. The VSS re-

moval efficiencies increased at longer reaction times (Fig. was slightly broken up and was not destroyed effectively 4b). The degradation of organic matters in sludge was fast [33], resulting in the insignificant release of intracellular during the first 45 min but did not vary significantly at re- and extracellular organic compounds. However, when the action times exceeding 60 min. The VSS removal efficien- reaction time further increased from 120 to 240 min, the cies of sludge treated by Fenton oxidation alone and Fen- SCOD concentration decreased slightly (Fig. 4b). This ton-EC oxidation were 6.9% and 13.3% with the reaction means reaction time of < 60 min is needed for effective time of 60 min, respectively. While the VSS removal effi- solubilization of WAS and VSS degradation. ciencies could reach as high as 9.2% and 19.0%, respec- The extension of reaction time may increase the con- tively, after reacting for 240 min. sumption of the Fenton reagent, taking both the removal of VSS and sludge treatment cost into consideration, the reaction time of 60 min is recommended. (a) As a result of the above experimental findings, Fenton- EC oxidation can be considered as an effective sludge pretreatment method. Under the following recommended conditions: H2O2 dosage = 1.0 g/L, initial pH = 3, reaction time = 60 min, and Fe(II) to H2O2 concentration ratio= 10.0, the removal efficiencies of VSS could reach as high as 7.3%, and the SCOD concentration increased from 104 to 414 mg/L; While EC oxidation treated after Fenton reaction as following conditions: the distance between electrodes was set to 2 cm, the voltage was 15V and the EC time was 30 min, the removal efficiencies of VSS could reach 13.3%, and the SCOD concentration could increase to 624 mg/L.

3.2. Effect of different pretreatments on the efficiency of aerobic sludge digestion Batch experiments were conducted to evaluate the ef- (b) fects of different pretreated WAS on the efficiency of aerobic digestion. Fig. 5 showed the comparison of aerobic digestion efficiency of control and the other three pretreated sludges by EC, Fenton and EC+ Fenton pretreatments. Pretreated sludge showed greater removal efficiency of SCOD from the very beginning of the aerobic sludge digestion (Fig. 5a). 37.5%, 33.3% and 29.6% reduction in SCOD concentration were observed only after four days of aerobic digestion period, while that of the control run increased from 130 mg/L to 250 mg/L. Moreover, the SCOD concentrations of the pretreated sludges were reduced from 550-650 mg/L to about 300 mg/L after 20 days of the digestion period. This fast degradation of SCOD concentration is due to the presence of high soluble organic content FIGURE 4 Effect of pH (a) and reaction time (b) on sludge reduction in pretreated sludge, which is readily biodegradable. An and solubilization overall SCOD removal of about 50% was observed after 20 days of digestion period with the different pretreat- Reaction time was mainly influenced by the rate of for- ments, and there was no obvious differences between them mation of ·OH and its reaction rate with organics. At the (p <0.05) (Fig. 5a). However, the SCOD concentration of beginning of the Fenton reaction, the concentration of ·OH the control run maintained about 250 mg/L during the 20 was very high, which allowed considerable contact with days digestion period, and decreased below 200 mg/L after organic matter. As ·OH was consumed, the degradation 24days of digestion period. rates of organic matter began to slow down. Fig. 5b showed the trend of VSS removal during the The SCOD concentration of sludge also increased with aerobic digestion of pretreated and control sludge. The di- the increase of reaction time (Fig. 4b), indicating that the gesters fed with pretreated sludge all achieved over 38% organic part of the solids was released continuously into removal efficiency of VSS after 20 days digestion as com- the water phase during Fenton oxidation. When the reac- pared to 36% removal efficiency of VSS in the control dition time exceeded 60 min, the solubilization rate increased gester. Nevertheless, the VSS removal efficiency of the slightly. Fenton reaction treatment did not present obvious control reactor reached over 38% after 24 days digestion effect on microbes of sludge, and hence, the floc structure period. Furthermore, the digester fed with Fenton + EC

pretreated sludge achieved stabilization after only 12 days Fenton reaction. Under these conditions, the removal effi- digestion period compared to that fed with EC and Fenton ciency of VSS could reach as high as 13.3%, and the SCOD pretreated sludge alone reaching stabilization after 18 days concentration could increase from 104 mg/L to 624 mg/L. and 16 days digestion period, respectively. Thus, the di- Sludge pretreatment with Fenton + EC oxidation could en- gester fed with the EC+ Fenton pretreated sludge achieved hance sludge stabilization synergistically and attained stabilization 12 days earlier than the control run. This showed greater sludge solubilization than the additive value of Fen- that the level of sludge degradability is significantly higher ton oxidation alone. Reduction of 12 days stabilization pe- and more rapid in the case of the pretreated sludge, espe- riod can be achieved after the aerobic digestion of the Fenton cially the EC+ Fenton pretreatment, than control sludge. + EC pretreated sludge in comparison with the control reactor. The results showed that Fenton-EC oxidation could be useful for the improvement of aerobic sludge digestion.

(a)

ACKNOWLEDGMENTS

This study was financially supported by the National Hi-Tech Research and Development Program of China (863) (No. 2011AA060906), the National Natural Science Foundation of China (No. 51208295) and the Key project of Science and Technology Commission of Shanghai Mu- nicipality (No. 12231202101).

The authors have declared no conflict of interest

REFERENCES

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SPINACH (Spinacia oleracea L.) RESPONSE AND ACCUMULATION OF SALTS IN SOIL UNDER SURFACE AND SUBSURFACE WASTEWATER IRRIGATION

Zobia Anwar1,*, Muhammad Irshad1, Iftikhar Fareed2 and Faridullah1

1Department of Environmental Sciences, COMSATS Institute of Information Technology Abbottabad Pakistan 2Department of Natural Resources Engineering and Management, University of Kurdistan, Hewler, Iraq

ABSTRACT 1. INTRODUCTION

Demand for higher agricultural yield has enhanced the There is a shortage of water resources for agriculture use of wastewater for irrigation. This study was carried out in the arid regions of the world[1]. The increasing need for to compare surface- and subsurface irrigation on the growth, water in the arid areas of the world has resulted in the emer- water use efficiency (WUE), nutrient uptake of spinach (Spi- gence of wastewater application for agriculture and land- nacia oleracea L.) and accumulation of soluble salts in the scape. The environmental effects of irrigation and drainage soil. Results showed that crop response was significantly are widespread and significant. The use of non-conven- affected with the wastewater irrigation. Subsurface waste- tional water resources and opportunities for achieving food water irrigation increased plant growth than surface irriga- security in water-scarce countries are presented by Qadir et tion. Higher biomass was produced with the application of al. [2]. sewage and municipal wastewater. The lower biomass was Pakistan is an agrarian country of Southeast Asia lo- noticed in the industrial wastewater. Higher application of cated in the severe water-scarce zone and increased indus- wastewater significantly reduced WUE. An enhanced WUE trialization and urbanization have resulted in discharge of was achieved with subsurface irrigation. Higher concentra- effluents of toxic nature into the waterways, thus polluting tions of nutrients in the plants were achieved under 100% and rendering the water bodies unfit for consumption for both surface and subsurface irrigation than 50% irrigation. agricultural sector [3-5]. In the absence of fresh water for Surface irrigation enhanced concentrations of elements in agricultural purposes, the use of wastewater for agriculture plants. Calcium and Mg concentrations in plants were ob- could be a viable option. Kahlown et al. [6] reported the tained as sewage water > industrial waste water > municipal situation in Pakistan, where the country with a population wastewater > fresh water. Heavy metals concentrations in of more than 150 million, cannot meet its need for food, if plants remained identical to other elements under both irri- adequate water is not available for crop production. A sub- gation systems. Higher accumulation of salts occurred in surface irrigation system can reduce the need for costly nd the 2 layer (30-60 cm) of the soil column under 100% sur- wastewater treatment, protect against environmental con- face irrigation. Wastewater differed for soil Ca in the order tamination and enhance efficient water use for many crops. of sewerage water > industrial wastewater > municipal This system provides better control of theapplication rate wastewater > fresh water. Potassium and Mg concentrations and distributes effluent uniformly, therebyminimizing rd were higher in the 3 layer (60-100 cm) of soil under sub- groundwater contamination risks. Agricultural irrigation surface irrigation. Wastewater differed for K concentration with industrial wastewater is a common practice in arid and in soil: sewage water > municipal wastewater > industrial semiarid regions and it is used as a readily available and wastewater > fresh water. Higher EC was seen in the middle inexpensive option to fresh water. layer of columns where greater accumulations of cations oc- Subsurface drip irrigation (SDI) is an effective method curred. Irrigation with 100% PE increased EC more than for wastewater application because it ensures desired soil 50% PE irrigation water. saturation [7] and the wastewater is not exposed on the sur-

face to other organisms including humans. The availability

KEYWORDS: of water supply and the demand for higher agricultural surface irrigation, sub-surface irrigation, wastewater, spinach yield have raised the interest for sub-surface irrigation. growth,salts concentrations, heavy metal uptake Safe use of poor quality water and enhanced productivity of water in agriculture enhanced the use of sub-surface irrigation. Several studies have been conducted to evaluate * Corresponding author the effects of irrigation methods using wastewater on

crops. Studies on the comparison of surface and subsurface each soil column. There were 2 irrigation methods at 2 lev- irrigation on the nutrients uptake and spinach growth as af- els. Plants were harvested, rinsed with distilled water and fected by subsurface irrigation are deficient. Therefore, this then oven dried at 65o C for 48 h to determine dry biomass. study investigated the spinach response and accumulation Thereafter, plant samples were ground, sieved by 0.5mm of salts in the soil under surface and subsurface wastewater sieve and then digested in a mixture of HNO3 and HClO4 irrigation systems. acids. Shoot concentrations of Ca, Mg, K and Na were determined to assess the irrigation systems for the accumulation of cations in the spinach plant and soils by atomic ab- 2. MATERIALS AND METHODS sorption spectrophotometer (AAS). Water use efficiency (WUE) was calculated by dividing the plant dry plant bio- Municipal wastewater was collected from Abbottabad mass (kg) by the total amount of water applied (M3) as re- city, Pakistan. Nutrients elements (Ca, Mg, K and Na) and ported by Sinclair et al. [8]. various trace elements (Cu, Fe, Zn and Mn) were determined in the filtered wastewaters by atomic absorption Some selected properties of the post harvest soil were spectrophotometer (AAS). Electrical conductivity (EC) also measured. Electrical conductivity (EC) and pH of soil and pH were determined by EC meter and pH meter, re- was determined in 1: 5 soil water suspensions after shaking spectively. A sandy soil was filled up in PVC pipes (1m for an hour by a pH meter and EC meter in CIIT laboratory. height and 0.2m diameter). Wastewater was applied based Water soluble Ca, Mg, Na, K and trace elements were deter- on 100% and 50% daily pan evaporation either by surface mined in the soil sampled in 3 layers (0-30; 30-60 and 60- or subsurface method. Three spinach plants were grown on 100 cm) from each treated column in soil water suspension.

140 120 100 Ca 80 Mg 60 K 40 Na 20 0 Fresh water Industrial Municipal Sewage water water water

14 12 10 Zn 8 Cu Fe 6 Mn 4 2 0 Fresh water Industrial Municipal Sewage water water water

FIGURE 1 - Chemical characteristics of wastewaters used

TABLE 1 - Electrical conductivity, pH and turbidity of wastewater irrigation

Parameters Freshwater Industrial wastewater Municipal wastewater Sewage water EC (dS m-1) 0.2 1.9 1.8 2.1 pH 6.7 8.3 8.2 7.8 Turbidity (NTU) 1.2 21.5 32.6 40.7

2.1 Pot experiment was obtained with the application of sewage and municipal Four types of wastewaters (municipal wastewater, waters (Table 6). The lower yield was noted in pots irri- sewerage water, industrial wastewater and freshwater, gated with industrial wastewater as compared to fresh wa- sampled from different locations of Abbottabad city were ter. The reduced biomass was observed with industrial compared for spinach growth in sandy soil. Municipal wastewater applications. This might be due to the presence wastewater was collected from a drain in front of Army Burn of higher contents of non-essential elements that may have Hall College (boys) Abbottabad. Sewerage wastewater was impaired the growth of plants. The higher biomass under collected from sewerage drain. Industrial wastewater was sewage and municipal waters could be attributed to the collected from Pakistan Ordinance Factory, Havelian. Four higher fertility status of these waters. Tamoutsidis et al. [9] kg soil was filled in small plastic pot. Some chemical prop- reported that treated urban wastewater effectively in- erties of the water are given in Figure 1 and Table 1. Three creased the yield of cultivated forage crop species, proba- spinach plants were grown. Surface irrigation system was bly due to the nutritive value of the wastewater. Oron et al. applied at the rate of 100% daily pan evaporation. After 8 [10] reported a higher corn yield with subsurface drip irri- weeks plants were harvested, rinsed with distilled water, gation (SDI). Phene et al. [11] demonstrated significant oven dried at 65o C for 48 h to determine plant dry biomass. yield increases in tomatoes with the use of high frequency The material was ground, sieved and digested in a mixture SDI and precise fertility management. Hutmacher et al. of HNO3 and HClO4. The digest was analyzed for the [12] also reported yield increases in alfalfa production us- above mentioned elements by AAS. Post harvest soil was ing SDI systems buried at a depth of 0.7 m. also sampled and analyzed for the above nutrients elements, EC and pH. Data were statistically analyzed by Stat- 3.2 Water use efficiency view software, and results were expressed on oven-dry ba- Water use efficiency (WUE) of spinach was signifi- sis. The overall differences among treatments were tested cantly affected by the wastewater application through sur- using analysis of variance. Mean separations was done us- face and subsurface irrigation method (Figure 2). The effi- ing LSD at P < 0.05. ciency of water use represents a given level of plant biomass per unit of water consumed physiologically by the plants. With the increased application of wastewater, evapo- 3. RESULTS AND DISCUSSION transpiration was increased and WUE was significantly decreased.Regardless to the irrigation methods, 100% irriga- 3.1 Plant growth tion based on pan evaporation (PE) significantly reduced The study demonstrated that the growth and WUE of WUE as compared to 50% PE. The WUE was increased in spinach (Spinacia oleracea L.) and its mineral composi- subsurface treatment as compared to the surface irrigation. tions were profoundly affected by the application of The higher WUE could be attributed to the higher plant bi- wastewater irrigation based on both 50% and 100% pan omass and low ET under the effect of subsurface irrigation evaporation. Municipal wastewater was used for irrigation.. method.The improved crop WUE under subsurface irriga- Subsurface wastewater irrigation (50% and 100%) signifi- tion could confirm the ameliorative effects of this method cantly increased the plant growth as compared to the surface under deficit water conditions.Marked reduction in the irrigation with wastewater (Figure 2). The higher dry bio- WUE of spinach under 100% water application could be mass was obtained with the application of 100% subsurface attributed to the higher loss of water through evaporation wastewater irrigation. Plant biomass obtained from soil ir- and transpiration processes.Fischer [13] reported the yield rigated with surface wastewater with 50% and 100% pan of a crop under deficit water as a function of crop evapo- evaporation were 20.3g and 26.7g, respectively. The gen- transpiration (ET), WUE and moisture-use pattern.Arafa et eral increase in the growth of spinach by wastewater irri- al. [14] concluded that subsurface drip irrigation was effi- gation through subsurface method indicated that polluted cient for intensive field crop production. water with harmful or toxic material can be used successfully. There is a general consensus that wastewater is an 3.3 Plant analysis inexpensive source of fertilizer for crop plants and could 3.3.1 Calcium prove to be a management option for suitable crop produc- Concentrations of cations in plant tissue were the function. This experiment indicated that the utilization of tion of irrigation methods. A significant effect on Ca con- wastewater via subsurface could be more efficient and pro- centration in plants was observed with wastewater appli- ductive than surface irrigation. The higher biomass yield cation (Figure 3). Higher Ca concentration was found in

FIGURE 2 -Spinach biomass and water use efficiency as affected by surfaceandsubsurfaceirrigation

FIGURE 3 - Ca and Mg concentrations in spinach as affected by surface and subsurface irrigation

plants irrigated with100% surface and subsurface. Surface The water with higher concentration of respective metal irrigation enhanced Ca concentration than subsurface irri- also increased the respective concentration in soil and gation. This could be due to the accumulation of Ca in the plants. surface soil layers under surface irrigation. Calcium concentration was higher in plants irrigated with wastewater than fresh water irrespective of the type of water applied. Among wastewaters, Ca concentration (mg g-1) was observed in plants in the order of sewage water > industrial wastewater > municipal wastewater > fresh water. This was because of higher Ca concentration in the sewage wastewater (96.4 mg L-1) and industrial wastewater (88.5 mg L-1)and greater bioavailability of Ca from soil applied with these waters.

3.3.2 Magnesium Methods of irrigation significantly affected the concentration of Mg in plants (Figure 3). Higher concentration of Mg was observed in plants irrigated with 100% surface -1 irrigation (2.2 mg g ) and 100% subsurface irrigation water (1.8 mg g-1). Magnesium concentration in plants was FIGURE 4 - K concentration in spinachasaffectedbysurfaceandsub- obtained as 1.2 and 1.0 mg g-1 in plants irrigated with both surfaceirrigation surface ad subsurface irrigation systems, respectively. Sur- face method accumulated more Mg in plants than subsurface method. Surface irrigation accumulated more Mg in soil than subsurface irrigation. When wastewaters were compared for Mg concentration in spinach shoots, higher Mg level was found with sewage and industrial wastewaters than municipal wastewater. A substantial Mg increase in plants was observed in pots irrigated with wastewater. These differences in plant Mg could be attributed to the concentration differences of Mg among waters. Sewage water exhibited a greater pool of plant nutrients than other wastewaters.

3.3.3 Potassium and sodium Based on the plant analysis, significant K and Na concentrations were observed under surface wastewater irrigation (50% and 100%) than subsurface irrigation (Figures 4 and 5). Higher K and Na concentrations were found in plants under 100% surface irrigation and lower concentrations were found in 50% subsurface irrigation. Greater wastewater applications significantly varied concentrations of elements in plants. This could be due to higher uptake and availability of these elements due to surface irrigation. Higher magnitudes of K and Na concentrations were achieved in pots of sewage water and municipal wastewater, respectively. This showed that these wastewaters are the potential sources of nutrients for plants as compared to industrial wastewater. Similar to the above elements, wastewaters significantly enhanced K and Na concentrations as compared to the freshwater.

3.3.4 Heavy metals Cumulative heavy metals concentration followed a similar pattern as noticed for the above cations in plants FIGURE 5 - Heavy metalconcentrations in spinachasaffect- either irrigated with surface or subsurface water (Figure 5). edbysurfaceandsubsurfaceirrigation

TABLE 2 -Accumulation of elements (mg kg-1) in post-harvest soil irrigated with surface and subsurface wastewater

Surface irrigation Subsurface irrigation Parameters 50% 100% 50% 100% Ca

030 142.7 184.8 135.8 167.7

3060 172.3 196.3 146.7 173.0

60100 159.5 176.5 170.4 191.1

030 115.2 135.9 95.5 119.1

3060 119.6 129.5 117.2 124.9

60100 117.4 125.1 121.3 131.2

030 112.2 133.9 101.5 141.1

3060 131.6 143.5 128.2 135.9

60100 127.4 137.1 149.3 145.2

3.4 Soil analysis identical and highly dependent on irrigation methods and 3.4.1 Cations quantity of wastewater applied. The amount of K and Mg was higher in the middle layer of soil under surface irriga- Based on the analysis of variance, the effect of irrigation whereas the concentrations of these elements were tion methods significantly varied Ca concentration among higher in the lower zones of soil column under subsurface soils layers (Table 2). The highest Ca concentration (196.3 irrigation. The cations level reduced with the movement of mg kg-1) was recorded in the 2nd layer (30-60 cm) under wastewater towards the soil surface. This indicated that 100% surface irrigation. This could be due to the presence while moving upward the wastewater was filtered by sand. of salts in wastewater (municipal water) that contained The concentrations of chemical constituents in soil layers (96.4 mg Ca L-1). Overall Ca concentration was higher in have been influenced by water movement patterns, chemi- the 1st and 2nd layer in the soil column under surface irriga- cal concentrations in irrigation water and plant uptake [15]. tion (both 50% and 100% pan evaporation) while in the 3rd Moreover from the surface soil, nutrients were uptaken by layer of the soil contained higher Ca concentration under the spinach plants. Across all soil layers, elemental con- subsurface irrigation system as compared to the surface ir- centrations were ordered as Ca > K > Mg > Na in the post- rigation. This might be due to the greater residence time of harvest soil under both irrigation systems. Heidarpour et al. wastewater above or below the soil column. This differ- [15] reported greater Na and Mg in the 1st layer of soil (0– ence could also be attributed to the plant uptake, reaction 15 cm) with subsurface irrigation than with surface irriga- of Ca or movement of Ca ions among layers under both tion. The EC, Ca and Mg in the 2nd and 3rd soil layers irri- irrigations. Irrespective of the methods, water application gated with wastewater were less in surface irrigation than at the rate of 100% added more Ca in soils as compared to subsurface irrigation. The amount of K in the 1st and 2nd the irrigation at 50% pan evaporation. layers of soil irrigated with wastewater was significantly Calcium accumulation also differed among the types greater than those irrigated with groundwater. of wastewaters. Wastewaters were differed for soil Ca in the order of sewage water > industrial wastewater > mu- For K concentration, wastewaters differed as sewage nicipal wastewater > fresh water. The higher level of Ca in water > municipal wastewater > industrial wastewater > sandy soil was directly related to the Ca concentration in fresh water (Table 6). Magnesium differed among the respective irrigation water. wastewaters in the order of sewage water > industrial water Potassium and Mg concentrations were found higher > municipal water > fresh water. Soils applied with in the soil in the 3rd layer (60-100 cm) of the soil (149.3 mg wastewater differed for cations concentrations in a similar kg-1) under subsurface irrigation with both 100% and 50% pattern of cations found in a wastewater. Waly et al.[16] re- pan evaporation rate (Table 2). Surface irrigation enhanced ported an increase in the K content of soils with sewage wa- the level of K and Mg in the zone of 30-60 cm. The accu- ter. Heidarpour et al. [15] related K concentrations in soils mulation of these cations among soil layers were found layers to its concentrations to the applied water. Cations

were varied in the sewage water and municipal wastewater 3.4.2 Heavy metals in soil in the order of K > Ca > Mg > Na. The industrial wastewater Heavy metals determined in the wastewater applied in was varied for elements as Ca > Mg > K > Na. the form of surface and subsurface irrigation, were found The accumulation of Na in the soil was higher under as Fe > Mn > Zn (Tables 3 and 4). The concentrations of sub-surface irrigation (Table 2). The higher accumulation heavy metals in soils layers followed a similar pattern as occurred in the lower zone of soil than the surface soil un- noticed for other elements. Surface irrigation significantly der subsurface irrigation. Under surface irrigation, the Na increased the magnitude of heavy metals in middle layer of was higher in the middle layer of soil. This might be due column whereas the sub-surface water accumulated these to the leaching of the water soluble Na from the surface soil metals in the 3rd layer of soil columns. For instance, Mn layer under surface irrigation [15]. Subsurface irrigation was higher (20.6 mg kg-1) in the 2nd layer (30-60 cm) under initially increased the concentration of Na in the 3rd layer. 100% surface irrigation. Zinc was comparatively less af- Sodium was differed among soil layers as 60-100 > 30-60 fected among soil layers as compared to Mn and Fe. Over- > 0-30. all 100% surface irrigation significantly affected heavy In the pots, all types of wastewaters (i. e., industrial, metals in soil layers than the subsurface irrigation.This in- municipal and sewage) gave enhanced Na concentration dicated that subsurface wastewater irrigation (50% and in soil as compared to the freshwater (Table 6). Higher 100%) method was efficient as compared to the surface ir- Na in soil indicated higher concentration of this element rigation by reducing heavy metal contamination and also in the wastewater. Among wastewaters, sewage water ap- reduced the uptake of heavy metals by plants. This has plied soil gave a Na level of 45.4 mg kg-1 followed by been supported by the higher biomass yield in the subsur- municipal wastewater (32.3 mg kg-1) and industrial face irrigation. Najafi [17]revealed that drip irrigation and wastewater (29.0 mg kg-1). Concentration patterns of Na subsurface drip irrigation could decrease the level of pol- in the soils were highly associated with the Na level of lution in soil as compared to surface irrigation. Oron et al. wastewater. The lower concentration of Na was observed [10]showed a reduced soil contamination when using sub- in the soil irrigated with freshwater (23.5 mg kg-1). surface drip irrigation.

TABLE 3 -Accumulation of elements (mg kg-1) in post-harvest soil irrigated with surface and subsurface wastewater

Surface irrigation Subsurface irrigation Parameters 50% 100% 50% 100% Na 030 17.2 43.9 20.5 34.1 3060 23.8 36.5 26.2 39.9 60100 21.6 26.1 34.3 42.2 Zn 030 4.61 5.60 4.54 5.40 3060 4.62 5.65 4.87 5.67 60100 3.40 5.45 4.41 6.43 Mn 030 9.54 19.57 8.50 9.50 3060 12.56 20.61 10.68 10.56 60100 8.63 19.56 12.49 19.45

TABLE 4 - Accumulation of elements (mg kg-1) in post-harvest soil irrigated with surface and subsurface wastewater

Surface irrigation Subsurface irrigation Parameters 50% 100% 50% 100% Fe 030 7.2 15.4 7.7 12.2 3060 8.8 17.3 7.6 15.6 60100 6.4 13.4 9.1 16.5

Soil moisture (%) 030 13 18 13 17 3060 15 21 18 15 60100 10 14 23 20

Zinc and Fe was higher in sewage water and municipal provides better control of the application rate and distrib- wastewater (Table 6). Industrial wastewater contained uteseffluents uniformly and minimizes contamination risks lower amount of these elements. Manganese and Cu could of groundwater. Sustained efforts are needed to evaluate not differ among wastewaters. The cummulative concen- the safe use of waste water in soil plant system. Turbidity trations of trace elements in soils were achieved according (NTU) was higher in the order of sewerage water > munic- to the elemental concentrations present in the respective ipal water > industrial water > fresh water. wastewater. Malla et al. [18] reported the application of sewage water improved the physico-chemical properties 3.4.3Soil moisture and nutrient status of the soil. Abide-Koupai et al. [19] re- Soil moisture content was higher (23 %) in the 3rd soil ported the accumulation of Pb, Mn, Ni and Co in soil with layer (60-100 cm) under both 100% and 50% subsurface wastewater than groundwater application and also reported irrigation (Table 4). Less water has been moved upward to the accumulation of decreased metals with soil depth and the surface soils. Surface irrigation had lowest moisture irrigation systems were not differed for extractable heavy level in the 3rd followed by the 2nd soil layer. The inferior metals. Galavi et al. [20] showed that wastewater irrigation water content in the 1st soil layer could be attributed to the led to the significant increase in the Zn, Fe and Mo in soil higher evapo-transpiration rate over the soil surface. and Cu and Fe in sorghum plants as compared to control treatment. Arora et al.[21]also found higher concentrations 3.4.4 Electrical conductivity of heavy metals in radish, spinach, turnip, brinjal, cauli- The electrical conductivity (EC) of soils depended on flower and carrot grown with wastewater as compared to the amount of water and the method of irrigation used dur- those with clean water. Findings of Malla et al. [18] also ing the experiment (Table 5). Soil EC is typically used to indicated a higher concentration of metallic cation in soils indicate soluble salt concentration in soil. Therefore the EC and vegetables due to the application of sewage water irri- values among soil layers have been attributed to the salts gation. movement and their distribution in soil under the influence of water movement [22]. Higher EC was recorded in the There is a risk of contamination associated with the middle layers of soil columns where greater accumulations continuous application of waste water. Therefore the irri- of cations were obtained as discussed above under surface gation of wastewater may carefully be rationalized espe- irrigation. Subsurface irrigation apparently enhanced the cially for food crops. Reuse criteria can be relaxed some- EC values in the lower part of the soil column. Irrigation what when using a drip irrigation and primarily subsurface with 100% pan evaporation increased EC more than 50% drip irrigation because the soil acts as a complementary irrigation water. Our results were agreed well with the re- biofilter and there is no contact between the effluent and sults reported by Mohammad and Mazahreh [23]. Wastewater workers or the plants parts above the soil [10]. This system has been reported as a main source of salts in soils [24].

TABLE 5 - Electrical conductivity and pH of post-harvest soil irrigated with surface and subsurface wastewater

Surface irrigation Subsurface irrigation Parameters 50% 100% 50% 100% EC ( dS m-1) 030 0.56 0.87 0.49 0.66 3060 0.67 0.93 0.45 0.82 60100 0.54 0.80 0.63 0.97 pH 030 7.74 8.12 7.63 8.01 3060 7.89 8.23 7.94 8.13 60100 7.87 7.95 7.97 8.16

TABLE 6 - Plant growth and accumulation of elements in soil irrigated with surface and subsurface wastewater

Parameters Freshwater Industrial water Municipal water Sewerage water Plant biomass (g) 29.3 18.4 23.3 31.8 Ca (mg kg-1) 47 189 170 201 Mg (mg kg-1) 34 167 156 183 K (mg kg-1) 84 147 187 203 Na (mg kg-1) 23.5 29.0 32.3 45.4 Zn (mg kg-1) 1.2 12.9 19.7 23.6 Cu (mg kg-1) 0.2 4.7 6.4 7.5 Fe (mg kg-1) 4.5 56.6 76.8 86.7 Mn (mg kg-1) 3.7 45.6 51.6 67.8 EC (dS m-1) 0.4 0.7 1.1 1.3 pH 7.1 7.8 7.9 8.0

Different types of wastewaters (industrial, municipal The authors have declared no conflict of interest. and sewage) differed EC values of soils depending on water used for irrigation. Electrical conductivity values (dS m-1) of soils were noticed as 1.3, 1.1, 0.7 and 0.4 in sewage water, municipal water, industrial water, and fresh water irriga- REFERENCES tion. These values were directly related to the EC values of the respective soils after irrigation.Sial et al. [26].reported [1] Haytham, E., Irshad,M. El-Serfy, A.M., Honna, T., Hassan, a raised level of pH, EC, TDS, SAR, RSC with 100% sew- A.K.S., Mohamed, T., Mahmoud, E. and Yamamoto. S. (2004) The effect of water quality on grain yield and nutrient uptake age wastewater irrigation than with 100% canal water irri- of rice (Oryza sativa L.). Acta Agronomica Hungarica, 52(2), gation. 141-148.

[2] Qadir, M., Sharma, B.R., Bruggeman, A., Choukr-Allah, R. 3.4.5 pH and Karajeh, F. (2007) Non-conventional water resources and Soil pH is the measure acidity or basicity of soils. The opportunities for water augmentation to achieve food security pH values under surface and subsurface wastewater irriga- in water scarce countries. Agricultural Water Management, 87 tion (50% and 100%) were slightly alkaline. The highest (1), 2–22. pH value was observed in the 2nd layer (30-60cm) 8.23 un- [3] Sheikh, K.H. and Irshad M. (1980) Wastewater effluents from der 100% surface irrigation (Table 5). Overall there was an a tannery: their effects on soil and vegetation in Pakistan. En- increase in the pH in the 2nd and 3rd layer of the soil (7.94 vironmental Conservation, 7(4): 319-324. and 7.97, respectively) under subsurface irrigation as com- [4] Wahid, A., Ahmad, S.S. and Nasir, M.G.A. (1999) Water pol- pared to surface irrigation. Surface application of water lution and its impact on fauna and flora of a polluted stream of produced slightly greater pH value as compared to the sub- Lahore. Acta Cient, 9(2), 65-74. surface regardless to the soil layers. This could be due to [5] Wahid, A., Nasir, M.G.A. and Ahmad, S.S. (2000) Effects of the accumulation of basic salts present in municipal water. water pollution on growth and yield of soybean. Acta Cient,10, Most of the soil layers were found statistically not different 51-58. for pH under both methods of irrigations. [6] Kahlown, M.A., Raoof, A., Zubair, M. and Kempera. W.D. Highest soil pH was achieved by industrial water and (2007) Water use efficiency and economic feasibility of growing rice and wheat with sprinkler irrigation in the Indus Basin municipal water when compared with freshwater. The soil of Pakistan. Agricultural Water Management, 87(3), 292-298. applied with these waters exhibited greater pH values as compared to normal water irrigation. Soil pH influences [7] Watson, J.T. and McEntyre, C.L. (2004) Peer Reviewed Guidelines for Wastewater Subsurface Drip Distribution (Pub- the availability and plant uptake of micronutrients includ- lication No. 701P0104). In On-Site Wastewater Treatment X ing heavy metals. Wastewater has been reported to have a Conference Proceedings (pp. 068-072). Sacramento, Califor- direct impact on soil pH [25]. Heidarpour et al. [15] re- nia, USA ported no significant effect on soil pH due to wastewater [8] Sinclair, T.R., Bingham, G.E., Lomon, E.R., and Allen, J.R. application. (1984) Water use efficiency of field-grown maize during moisture stress. Plant Physiology, 56, 245-249. [9] Tamoutsidis, E., Lazaridou, M., Papadopoulos, I., Spanos, T., 4. CONCLUSIONS Papathanasiou, F., Tamoutsidou, M., Mitlianga, P. and Vasil- iou, G. (2009) The effect of treated urban wastewater on soil properties, plant tissue composition and biomass productivity It is concluded that the growth and water use efficiency in berseem clover and corn. Journal of Food, Agriculture and of spinach and its mineral compositions were affected by Environment, 7(3&4), 782-786. the methods of wastewater irrigation. Subsurface wastewater irrigation at the rate of 50% and 100% pan evapora- [10] Oron, G., Campos, C., Gillerman, L. and Salgot, M. (1999) Wastewater treatment, renovation and reuse for agricultural ir- tion (PE) significantly increased plant growth as compared rigation in small communities. Agricultural Water Manage- to the surface irrigation. Higher biomass was obtained with ment, 38, 223-234. the application of sewage and municipal waters. The lower [11] Phene, C.J., Davis, K.R. and McCormick, R.L. (1987) Ad- plant biomass was found with industrial wastewater. vantages of subsurface drip irrigation for processing tomatoes. Higher WUE was achieved with subsurface irrigation. Sur- Acta Horticulturae, 200, 101-113. face irrigation enhanced elemental concentrations in plants [12] Hutmacher, R.B., Mead, R.M. and Shouse, P. (1996) Subsur- as compared to the subsurface irrigation. Calcium and Mg face drip: improving alfalfa irrigation in the west. Irrigation concentrations in the plants were obtained as sewage water Journal, 45, 48-52. > industrial wastewater > municipal wastewater > fresh [13] Fischer, R.A. (1979) Growth and water limitation to dry land water. Irrigation with 100% PE increased EC more than wheat yield in Australia: A physiological framework. Journal 50% PE irrigation water. Surface water produced slightly of the Australian Institute of Agricultural Science, 45, 83-94. greater pH value than sub-surface irrigation. The research [14] Arafa, Y.E., Wasif E.A. and Mehawed, H.E. (2009) Maximiz- information on the meaningful utilization of wastewaters ing water use efficiency in wheat yields based on Drip irriga- for the crop productivity would address issues of waste- tion systems. Australian Journal of Basic and Applied Sci- water disposal. ences, 3 (2), 790-796.

[15] Heidarpour, M., Mostfazadeh-Fard, B., Abedi Koupai, J. and Malekian, R. (2007) The effect of treated wastewater on soil chemical properties using subsurface and surface irrigation methods. Agricultural Water Management, 90, 87-94.

[16] Waly, T.M., Abd Elnaim, E.M., Omran, M.S. and El Nashar, B.M. (1987) Effect of sewage water on chemical properties and heavy metals content of El Gabal El Asfar sandy soil. Bi- ological Wastes, 22(4), 264-275. [17] Najafi, P. (2006) Effects of using subsurface drip irrigation and treated municipal waste water in irrigation of tomato. Pa- kistan Journal of Biological Sciences, 9(14), 2672-2676. [18] Malla, R., Tanaka, Y., Mori, K. and Totawat, K.L. (2007) Short term effect of sewage irrigation on chemical buildup in soil and vegetables. The Agric. Engg. Int. The CIGR J.Manu- script LW 07 006. Vol IX. Aug. 2007. Pp 14. [19] Abide-Koupai, Afyoni, M., Mosavi, F., Mostafazade, B. and Bagheri, M. (2003) Impact of surface and sprinkle irrigation by treated wastewater on soil salinity. Water and Wastewater, 45: 2-12.

[20] Galavi, M., Jalali, A., Mousavi, S.R. and Galavi, H. (2009) Ef- fect of treated municipal wastewater on forage yield, quantitative and qualitative properties of sorghum (S. bicolor Speed feed). Asian Journal of Plant Sciences, 8, 489-494. [21] Arora, M., Bala, K., Rani, S., Rani, A., Kaur, B. and Mittal, N. (2008) Heavy metal accumulation in vegetables irrigated with different water sources. Food Chemistry,111, 811-815. [22] Nakayama, F.S. and Bucks, D.A. (1986) Trickle Irrigation for Crop Production: Design, Operation and Management. Else- vier, New York, NY. [23] Mohammad, M.J. and Mazahreh. N. (2003) Changes in soil fertility parameter in response to irrigation of forage crops with secondary treated wastewater. Communications in Soil Science and Plant Analysis, 34, 1281-1294.

[24] Hussain, G. and Alsaati. A.J. (1999) Wastewater quality and its reuse in agriculture in Saudi Arabia. Desalination, 123, 241-251. [25] Sial, J.K., Bibi, S. and Qureshi, A.S. (2005) Environmental impacts of sewage water irrigationon groundwater quality. Pakistan Journal of Water Resources, 9(1), 49-53. [26] Rabia, N., Irshad, M. and Faridullah (2011) Assessing cadmium and lead in vegetables and soils irrigated with wastewater at Haripur, Pakistan. Minerva Biotechnologica (accepted).

Received: October 31, 2014 Revised: January 29, 2015 Accepted: February 16, 2015

CORRESPONDING AUTHOR

Zobia Anwar Department of Environmental Sciences COMSATS Institute of Information Technology Abbottabad PAKISTAN

E-mail: [email protected]

FEB/ Vol 24/ No 9/ 2015 – pages 2850 - 2859

INVESTIGATION OF SOME PROPERTIES OF BACILLUS SPP. ISOLATED FROM ARCHAEOLOGICAL EXCAVATIONS SOIL

Yasemin Yeşiltaş1, Ferdağ Çolak1,* and Elif Genç2

1 Department of Biology, Faculty of Arts & Science, University of Dumlupinar, Kütahya, Turkey 2Department of Archaeology, Faculty of Science and Letters, Çukurova University, Adana, Turkey

ABSTRACT 1. INTRODUCTION

In this study, soil samples were taken from the Kuriki The Kuriki Mound is located near the village of Mound, the silo, the furnace ruins, and graves within the Oymataş, 14 kilometers south of the city of Batman, on the excavation area, 14 km south of the city of Batman. VITEK left bank of the Batman River. The mound is located at the biochemical identification tests determined that there were confluence of the River Tigris and the Batman River, 8 isolates of Brevibacillus borstelensis, 7 isolates of Bacil- within the rivers’ terrace lands, which are suitable for agri- lus cereus, 6 isolates of B. subtilis, 1 isolate of Paenibacil- cultural production. Two main areas of occupation have lus macerans, 1 isolate of B. popillia, 1 isolate of B. poly- been distinguished and named Kuriki Mound 1 and Kuriki myxa isolates, 2 isolates of B. coagulans, and 1 isolate of Mound 2, as shown in Figure 1. The first season of archae- B. larvae. The identified strains were subjected to heavy ological excavation at Kuriki Mound was conducted in metals and antibiotic resistance profiling, antimicrobial 2009, as part of the ILISU and hydroelectric power plant and enzyme activity, and PHB formations were investi- (HES) Projects. The settlement contained four levels, which gated. The highest heavy metal resistance in terms of Min- were subdivided into twelve phases, dating back about 5000 imal Inhibitory Concentration (MIC) value was observed years. The first occupation documented at the site dates back against the manganese (16Mm), in comparison to the ref- to the end of the 4th/beginning of the 3rd millennia. After a erence strain (B. subtilis NRRL B-209). The highest level long period of abandonment, Kuriki was re-occupied during of antibiotic resistance was observed against cefotaxime, the 1st millennium BC, and the latest occupation occurred while the lowest antibiotic resistance was exhibited against during the late 1st millennium AD [1-4]. . gentamycin. Bacillus spp., antimicrobial activity against Spores of various Bacillus species are formed in spor- Gram-negative/positive bacteria, yeast, mold and clinical ulation, a process that is generally induced by reduced lev- isolates was determined using the agar well diffusion tech- els of nutrients in the environment. The formation of a nique. The tested isolates were devoid of any antimicrobial spore generates a cell type that can survive for extended activity against Gram negative bacteria. The bacilli ob- periods with little or no nutrients, yet is poised to return to tained in the course of our study showed activity against life if nutrients become available. As spores may have to the isolates B. cereus, S. epidermidis and B. subtilis. Clini- survive for long periods without nutrients, they are meta- cal isolates showed maximum antimicrobial activity bolically dormant, contain little or no high energy com- against Acinetobacter spp., while against Vancomycine re- pounds such as ATP and NADH, exhibit no detectable me- sistant Enterecocci (VRE) they showed no antimicrobial tabolism of endogenous or exogenous compounds and lit- activity. The bacillus Bacillus isolates showed antifungal tle, if any, enzyme activity in the spore core, the analogue activity against the previously mentioned yeasts (Saccho- of the protoplast of a growing cell [5]. romyces cerevisia, S. baulardi and Rhodotorula. rubra). 10% of the Bacillus isolates showed amylase and protease Heavy metals as natural components of the earth’s crust activity. Poly-ß hydroxybutyrate (PHB) was observed in are increasingly found in microbial habitat due to several only 4 out of the 27 Bacillus isolates. natural and anthropogenic processes. However, microbes have evolved mechanisms to tolerate the presence of heavy metals either by efflux, complexation or reduction of metal ions or to use them as terminal electron acceptors in anaero- KEYWORDS: Antimicrobial activity, archaeology, Bacillus spp, en- bic respiration [6, 7]. It has been shown that a correlation zymes, heavy metal, PHB. exists between metal tolerance and antibiotic resistance in bacteria because of the likelihood that resistance genes to both (antibiotics and heavy metals) may be closely located * Corresponding author on the same plasmid in bacteria and the presence of the organisms that possess specific mechanisms of resistance to

FIGURE 1 - Kuriki mound location map.

heavy metals increases destruction or transformation of toxic favored by environmental stresses, such as nitrogen, phos- substances in the natural environment. Consequently, the phate or oxygen limitation. PHB and other PHAs are syn- range of genes carried on these plasmids (frequently asso- thesized and deposited intracellularly in the form of gran- ciated with these heavy metal resistant determinants) was ules and might amount up to 90% of the cellular dry shown to extend far beyond those coding for antibiotic re- weight. Accumulation of intracellular storage polymers has sistance [8]. been considered a strategy used by bacteria to increase sur- Heavy metals are stable and persistent environmental vival in a changing environment [10]. contaminants since they cannot be degraded or destroyed. Of all the known PHAs, poly- 3-hydroxybutyrate, Therefore, they tend to accumulate in soils and sediments. PHB, is the most commonly and widely produced homo- While some heavy metals are required in trace amounts as polymer by many bacteria. Current applications of PHB- nutrients, they become strongly inhibitory for microorgan- based polymers or composites include the packaging in- isms at relatively low concentrations. Toxicity occurs dustry, medicine, pharmacy, agriculture, food industry, through the displacement of essential metals from their na- raw material for enantiomerically pure chemicals and the tive binding sites or through ligand interactions. Toxicity paint industry [11]. results from alterations in the conformational structure of The Bacillus species that produce antibiotics are B. nucleic acids and proteins and interference with oxidative subtilis, B. polymyxa, B. brevis, B. licheniformis, B. circu- phosphorylation and osmotic balance. To survive under lans and B. cereus. The majority of studied antibiotics pro- metal-stressed conditions, bacteria have evolved several duced by Bacillus strains are polypeptides of low molecular types of adaptation mechanisms to tolerate the uptake of weight that are synthesized by ribosomal or nonribosomal heavy metal ions. These mechanisms include the efflux of mechanisms [12]. Polypeptide antibiotics produced by Ba- metal ions outside the cell, accumulation and complexation cillus that are used in medical treatments are bacitracin, of the metal ions inside the cell, and the reduction of the gramicidins, polymyxin, and tyrotricidin [13]. B. cereus and heavy metal ions to a less toxic state. Bacteria have adapted some closely related species from the genus Bacillus have to heavy metals through a variety of chromosomal-, trans- several features including the production of various biologi- poson-, and plasmid-mediated resistance systems [9]. cally active metabolites, i.e. antibiotics, proteinases and PHB is well known as a carbon and energy reserve pro- bacteriocins. It is well-known that most, if not all, bacterial duced by a variety of microorganisms and its synthesis is species are capable of producing a heterogeneous array of

molecules in the course of their growth in vitro (and pre- CrCl36H2O, PbCl2, MnCl2.4H2O, Cu(CH3COO)2.H2O and sumably also in their natural habitats) that may be inhibi- CdCl2.4H2O. Stock solutions of the metal salts were pre- tory to other bacteria [14]. pared in sterile distilled water. The solutions were then Therefore, we have designed this study with the aim of sterilized by filtration with syringe type filters (with a pore isolating endospore forming bacilli from Kuriki mound. diameter of 0.22 μm) and were added to the NA plates in Further, our aim was to characterize the isolated bacteria various concentrations which were then spot-inoculated 8 for their antibiotic and heavy metal resistance pattern as with approximately 10 CFU/mL. The density of this cul- well as enzyme activities and PHB formation. ture was adjusted to 0.5 McFarland (at 625nm, 0.08-0.1 absorbance). The plates were incubated at 37°C for 24 hours. The lowest concentration of metal inhibiting the growth of 2. MATERIALS AND METHODS the microorganisms was considered as the MIC of the metal against the strain tested. The isolates were consid- 2.1 Isolation and Identification of Bacillus strains ered resistant if the MIC values exceeded that of the B. sub- Soil samples were taken from excavated soil at three tilis NRRL B-209 strain, which was used as the control. different locations (silo, furnace ruins, and graves from the soil), at Kuriki mound. An impressive monumental build- 2.3 Sensitivity to the Antibiotics ing of the Level II, dating to the end of the 1st millennium Bacillus isolates were cultured in Nutrient Broth (NB) BC/beginning of the 1st millennium AD, was excavated on medium and incubated at 37°C for 24 hours, and a suspen- the top of the mound. This building was centered on a long sion containing 108CFU/mL for each isolate was prepared. corridor overlooked by two pairs of three rooms. In order 100μL of each suspension was spread on the plates con- to establish a stratigraphic sequence of the settlement, a taining Mueller-Hinton Agar (MHA) medium (15 mL). deep sounding was dug. The main soil was reached by dig- Antibiotic discs, namely, amikacin (AK 30µg), gentamicin ging as far as five meters down from the corridor, and here (CN 30µg), tetracycline (TE 30µg), cefotaxime (CTX the remains of a silo were found and containing a huge 30µg), vancomycin (VA 30µg), oflaxacin (OFX 5µg), amount of carbonized lentils and wheat. A cist tomb made sulbactam (SAM 30µg), chloramphenicol (C 30µg), oxa- by slab stones containing a 5-6 month [1-4]. cillin (OX 1µg), ampicilin (AMP 10µg), clindamicin (DA Ten grams of soil sample was serially diluted in sterile 10µg), streptomicin (S 10µg), meticilin (Met 10µg), kana- water and dilutions were surface-plated on Nutrient agar. mycine (K 30µg), imipenem (IMP 10µg), erythromycin (E The soils were pasteurized at 80ºC for 10 minutes to select 15µg (Bioanalyse LTD, Turkey)), were placed on MHA the Gram positive spore forming bacteria, Bacillus spp. Af- plates and the diameters of inhibition zones formed follow- ter spreading 100 μL of each suspension on Nutrient Agar ing 24-hour incubation at 37°C were measured [18]. Inter- (NA), the sample was aerobically incubated for 2 days at pretation of the response of Bacillus to these antibiotics 37°C. After the incubation periods, colonies were selected was presented as resistant, intermediate or sensitive, based for isolation. The purified isolate was maintained on agar on the size of the inhibition zones for individual antibiotics medium, and also stored as glycerol (20%, v/v) stocks at - according to the instructions given in the manufacturer’s 18° C. manual. The morphological and some biochemical properties of the isolate were determined by carrying out Gram stain, 2.4 Determination of antimicrobial activity endospore stain, catalase test, anaerobic growth, voges The antimicrobial activity of Bacillus strains isolated prouskauer test, hydrolysis of casein, gelatin, and starch, from soil was tested against 27 microbial genera including growth at different pH, temperature, and NaCl concentra- 14 bacteria (Bacillus cereus ATCC 7064, Staphylococcus. tions in accordance with Bergey’s Manual of Systematic epidermidis ATCC 12228, S. aureus ATCC 25923, B. sub- Bacteriology [15]. The bacteria were identified using a VI- tilis NRRL B-200, MRSA (clinic isolate), Enterococcus TEK Compact 1 System based on the differences of carbon fecalis ATCC 29112, Enterobacter aerogenes ATCC source utilization. 13048, Pseudomonas aeroginosa ATCC 27853, Proteus vulgaris NRRL B-123, Escherichia coli NRRL 3704, Aer- 2.2 Determination of Minimum Inhibitory Concentrations (MIC) omonas hydrophilia NRRL 406, VRE (clinic isolate), Aci- of heavy metals netobacter spp. (clinic isolate), Moraxella catarrhalis The Minimal Inhibitory Concentration (MIC) of the (clinic isolate), 3 yeasts (Saccharomyces cerevisa (wild), metals for the isolate was determined by the plate dilution S. boulardii (wild), and Rhodotorula rubra DSM 70403), method, as adopted by Alam and Malik [16] and Alam et and 2 mold species (Aspergillus flavus NRRL 1957, and A. al. [17]. The MIC for two bacterial isolates in terms of fumigatus NRRL 163). The Bacillus strains were grown in seven heavy metals was determined using NA containing 300mL Erlenmeyer flasks containing 50mL SG (pepton Ni2+, Zn2+, Co2+, Fe 3+, Cr6+, Pb2+, Mn2+, Cu2+ and Cd +2 at 20.0g, glycerol 20.0mL, tap water 1000mL) medium, with concentrations ranging from 0.0625mM/mL to 16mM/mL. a rotary shaker at 160rpm at 37°C. The SG medium con- The metals were added as Ni(CH3COO)2.4H2O, sisted of 2% glycerol and 2% peptone in tap-water, pH 7.5 Zn(CH3COO)2.H2O, Co(CH3COO)2.4H2O, FeSO4.7H2O, [19]. After allowing maximum biomass formation for

3 days in SG medium, the cultures were centrifuged. The The Bacillus was isolated from aquatic environments [22], obtained supernatant was passed through 0.22µm Milli- various parts of insects [23] and grain textures [24]. pore filter. All supernatants were maintained at +4°C until The results of the Gram stain, endospore stain and cat- they could be used for the agar well diffusion method. The alase tests, as well as the morphological properties of the determination of the inhibitory effect of the extracts from isolates, revealed that all of them belonged to the Bacillus isolates on test microorganisms was carried out according genus. Identification of the 27 isolates was achieved ac- to the agar well diffusion method. All bacteria were cul- cording to biochemical tests and the VITEK identification tured on NB medium and incubated at 37°C for 24 hours. system. MHA medium (15mL) was poured into each sterile Petri- dish. A suspension (100μL) containing 108CFU/mL for According to the biochemical properties, 27 isolates bacteria, 107CFU/mL for yeasts, or 105 spores/mL for were identified as these being Brevibacillus borstelensis (8 molds was spread on the plates of MHA or Sabouraud dex- isolates), B. cereus (7 isolates), B. subtilis (6 isolates), B. trose agar medium respectively. Suspensions (100μL) of macerans (1 isolate), B. popillia (1 isolate), B. polymyxa (1 the target strain cultured for 24 hours were spread on the isolate), B. coagulans (2 isolates), B. larvae (1 isolate). Ten plates, and wells of 10mm diameter were punched in the Bacillus cereus strains were isolated from different soil agar with a sterile steel borer. The extracts (100μL) were samples. Bacillus cereus is found frequently as a sapro- poured into the wells and the plates were incubated at 37°C phyte in soil, water, vegetation, and air. The colonization for 24 hours for bacterial strains, 48 hours for yeasts and at of different ecological niches is enabled by its extremely room temperature for 72 hours for fungi. good adaptability and resistance to various influences [25]. The microbial level of resistance or tolerance of each 2.5 Screening for amylase-producing strains concentration of heavy metal was depicted by the level of Bacterial isolates were screened for amylolytic proper- growth on the agar. The microbial load decreased with an ties using a starch hydrolysis test on starch agar plate. The increase in the concentration. In the present study, re- microbial isolates were streaked as a line on the starch agar sistance to nine heavy metals Ni2+, Zn2+, Co2+, Fe 3+, Cr6+, plate and plates were incubated at 37oC for 24 hours. The Pb2+, Mn2+, Cu2+ and Cd +2 was investigated in all the iso- plates were flooded with a 1% prepared iodine solution at lates. The MICs of the isolates ranged from 0.0625mM/mL the end of incubation. A clear zone of hydrolysis surround- to 16mM/mL. Among all the isolates, resistance to heavy ing the growth indicates a positive result while the presence metals was determined in comparison to the reference of a blue color around the growth indicates a negative result strain (B. subtilis NRRL B-209), (Table 1). Which [20]. achieved the following results: Mn2+ [A2 (B. cereus), A4, A7 (B. borstelensis), C1 (B. subtilis), C4 (B. cereus), D4 2.6 Screening of protease-producing strains (B. polymyxa, E3 (B. larvae)] 11.17% and Cd +2 [A2, A3, Bacterial isolates were screened for extracellular pro- A5 (B. cereus), A11 (B. borstelensis] 8.51%. The Bacillus tease production by streaking onto skim milk agar plates. isolates were not found to be resistant to other heavy met- The plates were incubated at 37oC for 24 hours. Protease als. Çolak et al. [26] reported that isolated Paenibacillus production was demonstrated by the clearing of opaque polymyxa was found to be tolerant to different concentra- milk proteins in the area surrounding the colony [20]. tions of heavy metals, as evidenced by its MICs ranging from 25µg/mL to 1600µg/mL. In assessing the range of 2.7 PHB producing Bacillus Detection MICs obtained, a maximum MIC (1600µg/mL) was ob- Microscopic techniques were used to detect PHB-pro- served for Mn and Cu, with a minimum for Cd (25µg/mL). ducing strains. The strain Sudan Black was used to identify However, the control strain showed a maximum MIC of PHB under bright field microscopes. Sudan black was 1600µg/mL for Mn and a minimum of 25µg/mL for Cd and placed on heat-fixed samples and prepared as 0.3g Sudan Cu. A heavy metal resistance pattern of Cu=Mn > Black B dissolved in 70mL 95% ethanol, bringing it to Pb>Ni=Zn>Hg was observed in P. polymyxa. A metal-re- 100mL with distilled water. Samples were stained with Su- sistant microbial community was likely to be dependent on dan Black solution for 10 minutes, dried with filter paper the presence of the metal in the growth medium [27]. In and clarified using xylene drops, dried again with filter pa- this study, the soil sample used for the isolation of bacteria per and counterstained with 0.5% aqueous safranine for 5 contained serpentine soil. Çolak et al. [28] reported that seconds [21]. they isolated B. cereus and B. pumilus from a serpentine soil and found that both isolates were resistant to copper and lead metals. Retaining suitable concentrations of es- 3. RESULTS AND DISCUSSION sential metals such as copper, while rejecting toxic metals like lead and cadmium was probably one of the toughes A total of 27 isolates were obtained from the Kuriki challenges of living cells. The first response to toxic metal Mound, approximately 1km south-west of the village of contamination is a large reduction in microbial activity. Oymataş at the confluence of the Tigris River (Fig 1). Soil This is confirmed by the fact that habitats that have had samples were taken from different locations; these being high levels of metal contamination for years still have mi- the silo, the furnace ruins and graves within the excavation. crobial populations and activities that are smaller than the

TABLE 1 - Heavy metals resistance profiles of isolated bacteria

Heavy metals n Heavy metal concentrations mM /mL of medium Resistant isolates

0.0625 0.125 0.25 0.5 1 2 4 8 16 n % Ni2+ 27 - - - 3 24 -* - - - 0 0.0 Zn2+ 27 1 3 5 18 - - -* - - 0 0.0 Co2+ 27 1 3 5 18 - -* - - - 0 0.0 Fe 3+ 27 - - - 3 7 17 -* - - 0 0.0 Cr6+ 27 - - - 8 5 14 -* - - 0 0.0 Pb2+ 27 - - 1 7 1 2 16 -* - 0 0.0 Mn2+ 27 ------8 12* 7 19 11.17 Cu2+ 27 2 7 18 *- - - - 0 0.0 Cd +2 27 4 *- 23 ------23 8.51 * Heavy metals reference strains MIC values

microbial populations in uncontaminated habitats. and 2 mold species (A. flavus, A. fumigatus). The microorgan- Konopka et al. (1999) argued that resistance mechanisms isms were tested and the results can be found in Table 3. do not offer protection at extremely high levels of free metal ions and with a lethal toxic effect [8]. TABLE 2 - Antibiotic resistance profiles of isolated bacteria Also there is the use of different types of microorgan- Antibiotics Resistance pattern isms such as algae, fungi and bacteria that remove metals R(%) MS (%) S(%) from solution. It would necessarily be of immense benefit AK (30 µg) 2 (7.40) - 25 (92.59) exploiting microorganisms for this purpose, but the at- CN (30µg) - - 27 (100) tendant health implications that may result when these or- TE (30µg) 3 (11.11) 1 (3.70) 23 (85.18) ganisms develop resistant genes invariably becomes a CTX (30µg) 14 (51.85) 1 (3.70) 12 (44.44) source of concern for disease treatment and management [8]. VA (30µg) 4 (14.81) 5 (18.51) 18 (66.66) OFX (5 µg) 1 (3.70) - 26 (96.29) The highest level of antibiotic resistance was observed SAM (30µg) 4 (14.81) 2 (7.40) 21 (77.77) against cefotaxime while the low level of antibiotic re- C (30µg) 4 (14.81) 1 (3.70) 22 (81.48) sistance was exhibited against gentamycin clindamycin OX (1 µg) 9 (33.33) 3 (11.11) 15 (55.55) (3.7%), ofloxacine (3.7%), and none of the isolated bacte- AMP (10µg) 6 (22.22) 1 (3.70) 20 (74.07) ria were resistant to gentamisine (Table 2). Ombui et al. DA (10µg) 2 (7.40) 2 (7.40) 23 (85.18) [29] reported that all B. cereus isolates were resistant to S (10µg) 5 (18.51) 1 (3.70) 21 (77.77) ampicillin but susceptible to streptomycin, and that re- MET (10µg) 7 (25.92) 1 (3.70) 19 (70.37) sistance to gentamicin was about 7%. Jensen et al. [30] in- K (30 µg) 2 (28.57) - 25 (92.59) vestigated the antimicrobial resistance among B. cereus IPM (10µg) 1 (3.70) - 26 (96.29) group isolates from Danish agricultural soil, and recorded E (15µg) 3 (11.11) - 2488.88 the resistance to bacitracin and erythromycin. In this study, R: Resistant (≤14), MS: Moderately Sensitive (15-18), S: Sensitive all the isolates were resistant to ampicillin and cephotax- (≥19mm) ime, but susceptible to kanamycin. However, a differing degree of susceptibility and resistance was observed for Due to the fact that Bacillus species have produced another antibiotics tested. It was also reported that since most tibiotics, in the form of soluble protein, have been found Bacillus species populate the same ecosystems as Strepto- cheaper and more effective in studies conducted so far, myces and other antibiotic producers, they might have ac- therefore these microorganisms are preferred for commer- quired resistance to antibiotics produced under natural con- cial production. It was reported that members ofthe species ditions. Since many bacteria exhibit production of second- Bacillus generally produced polypeptide-type bacteriocins, ary metabolites mainly in the late exponential or in the sta- and that these antibiotics generally affect Gram-positive tionary phase, we investigated whether this is also the case bacteria. 27 Bacillus isolates were found most effective for the production of antimicrobial agent [31]. The antimi- against B. cereus S. epidermidis and B. subtilis. Bacillus crobial activity of Bacillus strains isolated from the soil isolates were found exhibiting antibacterial activity against was tested against 27 microbial genera, including 14 bac- B. cereus with zone size ranging from 13-30mm. The Ba- teria (B. cereus, S. epidermidis, S. aureus, B. subtilis, cillus isolates showed no activity against S. aureus strains. MRSA (clinic isolate), E. fecalis, E. aerogenes, P. aer- Bacillus isolates were also found non-inhibitory against oginosa, P. vulgaris, E. coli, A. hydrophilia, VRE (clinic non-clinical Gram-negative isolates such as E. fecalis, E. isolate), Acinetobacter spp. (clinic isolate), M. catarhalis aerogenes, P. aeroginosa, P. vulgaris, E. coli and A. hy- (clinic isolate), 3 yeasts (S. cerevisa, S. boulardii, R. rubra), drophila. Oscariz et al. [32] identified and isolated a bac-

TABLE 3 - Identification and some of the characteristic of isolated bacteria

Origin Isolates Identification Antimicrobial activity (zone size in mm ) Amylase/ protease activity PHB Silo A1 B. borstelensis - -/- - A2 B. cereus B. cereus (13), S. epidermidis (30), -/- + B. subtilis (20), Acinetobacter spp. (23), M. catarrhalis (22), S. cerevisia (15), R. rubra (15) A3 B. cereus B. cereus (20) 9/0 + A4 B. borstelensis B. cereus (25) 11/0 + A5 B. cereus Acinetobacter (19) -/- + A6 B. subtilis B. cereus (18), S. epidermidis (15), -/- + Acinetobacter (16), S. cerevisia (12), S. boulardi (16) A7 B.borstelensis B. cereus (20) -/- + A11 B. borstelensis B. cereus (16) S. epidermidis (16) -/- + MRSA (12), Acinetobacter spp (20) M. catarrhalis (20) B1 B. subtilis B. cereus (14) Acinetobacter spp (13) -/- + B3 B. subtilis B. cereus (16), S. epidermidis (14) B. subtilis -/- + (15), Acinetobacter spp.(16) C1 B. borstelensis Acinetobacter spp.(15) S. boulardi (17) -/- + C2 B. subtilis B. cereus (20) -/20 - C4 B. cereus B. cereus (20) -/10 + C5 B. borstelensis - -/- + Furnace ruins D1 P. macerans 12/- + D3 B. popilliae -/- + D4 B. polymyxa -/9 - D5 B. cereus 14/15 + D7 B. subtilis B. cereus (21) 14/17 - D8 B. coagulans B. cereus (30) -/- + D9 B. cereus - -/- + D10 B. coagulans B. cereus (21) -/- + D11 B. borstelensis B. cereus (26) -/- + D13 B. cereus B. cereus (12) Acinetobacter spp (15) 15/15 + D14 B. borstelensis B. cereus (14) Acinetobacter spp (12) -/- + Graves from the soil E2 B. subtilis B. cereus (12) -/- + E3 B.larvae B. cereus (20) 15/- + Note: (+) indicates positive (-) indicates negative

teriocin-producing strain of Bacillus cereus from a soil when many Gram positive pathogens have developed re- sample. Wherein the compound, cerein 7, was found active sistance mechanisms to almost all known antibiotics. In ad- against most Gram-positive bacteria. However, the same dition, there is a need to develop an antifungal agent with compound was reported ineffective Gram-negative bacte- fewer side effects, since serious fungal infections, such as ria. Güven et al. [25] reported that, had moderate activity invasive aspergillosis, have increased dramatically in re- against diverse Gram-positive bacteria and certain cent years. The A2, A3, D5, D13 (B. cereus), A4 (B. bor- Gramnegative bacteria. Bacillus isolates were found to dis- stelensis), D1 (P. macerans), D7 (B. subtilis), and E3 (B. play antibacterial activity against MRSA, Acinetobacter larvae) isolates showed amylase activity, with D13 and E3 spp., and M. catarrhalis Bacillus isolates were also found demonstrating the highest amylase activity with a zone di- to exhibit antibacterial activity against B. cereus with zones ameter of 15mm. The isolates indicating protease activity sizes ranging between 13-30 mm, however, they showed were found to be A2, C4, D5, D13 (B. cereus), C2, D7 (B. no activity against the VRE strain. Three yeasts (S. cere- subtilis) and D4 (B.polymyxa). The Bacillus isolate with visiae, S. boulardii, R. rubra) and 2 fungi (A. fumigatus, A. the highest protease activity was determined to be C2with flavus) showed antifungal activity against the species eval- a zone diameter of 20mm. Of the four Bacilli isolated from uated. Yeast strains showed antifungal activity, but other the fish gut (B1, B2, B3 and B4), only one isolate (Bacillus fungi strains were not inhibitory. The Bacillus species have sp. B1) showed activity for protease, lipase, amylase and a wide range of antibacterial activities and are also used as cellulase enzymes [20]. The poly-β-hydroxybutyrate anti-fungals. These results provide us with novel tools for (PHB) is a compound accumulated as a cellular energy and antimicrobial therapy, which is particularly urgent at a time carbon reserve by a large variety of bacteria that include

the genera Alcaligenes, Pseudomonas, Rhizobium and Ba- cess is needed. The physiological characteristics of the or- cillus. It is deposited intracellularly in amorphous state in ganism are important for the bioprocess to produce poly- inclusions in the cytoplasm and inclusion levels depend on mer. This strain might be employed in the industrial pro- the nutritional (carbon source, C/N ratio, etc.) and the en- duction of PHA. This study aimed to shed light on the ex- vironmental conditions (pH, oxygen, etc.) during growt cavations made in Turkey. [33]. Considering the poly-β-hydroxybutyrate PHB presence of isolates, PHB presence is not found in all of the isolates other than A1 (B. borstelensis), C2, D7 (B. subtilis) and D4 (B. polymyxa) strains (Table 3). Gram positive bac- ACKNOWLEDGEMENT teria reporthave not been reported to accumulate large amountsof polyhydroxyalkonate and hence have not been This work has been dedicated/ conducted with the head considered as potent candidates for industrial production. of the Batman Museum Directorate, under the auspices of A number of Bacillus spp. has been reported to accumulate Ministry of Culture and Tourism, General Directorate for 9-67%dry cell weight PHB. Cultural Heritage and Museums and supported by the Gen- eral Directorate of State Hydraulic Works (DSI).

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

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[25] Guven, K., Ilhan, S., Mutlu, M. and Colak, F., (2008). Diver- sity, characterization and antimicrobial activities of Bacillus cereus strains isolated from soil. Fresen. Environ. Bull., 17 Received: January 12, 2015 (3), 303-310. Revised: March 20, 2015 Accepted: April 15, 2015 [26] Çolak, F., Olgun, A., Atar, N. and Yazıcıoğlu D. (2013) Heavy metal resistances and biosorptive behaviors of Paenibacillus polymyxa Batch and column studies. Journal of Industrial and Engineering Chemistry, 19, 3, 863–869. CORRESPONDING AUTHOR [27] Hassen N., Saidi M., Cherif A. Boudabous (1998), Resistance of environmental bacteria to heavy metals. Bioresour. Tech- Dr. Ferdağ ÇOLAK nol. 64, 7–15. Department of Biology [28] Çolak F., Atar N., Yazıcıoğlu D., Olgun A. (2011) Biosorption Faculty of Science and Art of lead from aqueous solutions by Bacillus strains possessing Dumlupınar University heavy-metal resistance. Chem. Eng. J. 173, 422-428. Kütahya [29] Ombui, J.N., Mathenge, J.M., Kimotho, A.M., Macharia, J.K., TURKEY and Nduhiu, G. (1996). Frequency of antimicrobial resistance and plasmid profiles of Bacillus cereus strains isolated from Phone: +90 274 2652031/3152 milk. East Afr. Med. J. 73, 380-384. Fax: +90 274 2652014 [30] Jensen, G.B., Hansen, B.M., Eilenberg, J. and Mahillon, J. Email: [email protected] (2003). Minireview: The hidden lifestyles of Bacillus cereus and relatives. Environ. Microbiol.,5, 631-640. FEB/ Vol 24/ No 9/ 2015 – pages 2860 - 2867

SUBJECT INDEX

A H accumulation 2809 heavy metals 2792 accumulation 2824 heavy metals 2824 adsorption 2815 heavy metal uptake 2850 aerobic digestion 2843 hematological parameters 2771 agricultural land 2837 high density lipoprotein 2732 air quality 2699 Hui River national nature reserve 2707 Allura Red 2830 antibacterial activity 2715 I Artemisia spp. 2715 in vitro 2747

B K biodiversity 2707 kinetic models 2736 biodiversity 2742 kinetics 2792 biological indices 2800 bioremediation 2824 L Brilliant Blue FCF 2830 land degradation 2776 Brown HT 2830 land use 2800 Langmuir isotherm 2736 C lead 2771 Çanakkale Strait 2725 longevity 2830 Capoeta capoeta 2732 carbon pool management index 2751 M chelates 2824 M. galloprovincialis 2725 chemical composition 2715 macroinvertebrates 2800 chemical fertilizers 2837 mapping 2776 chlorination 2763 MEDALUS project 2776 composting 2742 medicinal 2747 contamination 2725 Mediterranean environments 2776 copper 2771 modified magnetite 2815 copper ion 2815 Mongolica forest-steppe ecotone 2707 mountainous city 2699 D desertification risk 2776 N desorption 2815 nanoparticles 2815 disinfection by-products (DBPs) 2763 natural organic matter (NOM) 2763 ditch burying 2751 natural radionuclides 2837 Drosophila melanogaster 2830 natural swimming pond 2736 NO2 2699 E edge effect 2707 O Ege region 2837 O. niloticus 2771 electrochemical pretreatment 2843 environmental variables 2800 P essential oil 2715 paraoxonase activity 2732 phosphorus 2736 F physical characterisation 2763 Fenton oxidation 2843 phytoremediation 2787 freshwater fish 2732 plant functional groups 2707 Freundlich isotherm 2736 plant 2787 PM10 2699 H Ponceau 4R 2830 poultry litter 2742 heavy metal 2742 heavy metals 2725 Q heavy metals 2787 Qinghe River 2800

AUTHOR INDEX

R A River sediments 2792 Aasim, Muhammad 2747 rock phosphate 2837 Alam, Arif 2742 Roses 2787 Anaç, Dilek 2837 Anwar, Zobia 2850 S Avsar, Edip 2763 salts concentrations 2850 Ay, Özcan 2771 selectivity sequences 2792 sensitivity areas 2776 B shoot regeneration 2747 Babaei, Ali Akbar 2815 shoot tip 2747 Baki, Birol 2809 SO2 2699 Baki, Oylum Gökkurt 2809 soil 2824 Bat, Levent 2809 Solea solea 2809 Benli, Dilek 2830 sorption 2792 Bian, Xinmin 2751 spinach growth 2850 Bus, Agnieszka Z. 2736 stabilization 2843 straw 2751 C sub-surface irrigation 2850 Cakir, Ahmet 2715 Sunset Yellow 2830 Cakmakci, Ramazan 2715 surface irrigation 2850 Cicik, Bedii 2771 Çiftçi, Nuray 2771 T Çolak, Ferdağ 2860 total oxidant status 2732 toxicity 2809 D Turkey 2725 Dadasoglu, Fatih 2715 Deveci, Haci Ahmet 2732 W Dikbas, Neslihan 2715 waste activated sludge 2843 wastewater 2850 E Ercisli, Sezai 2787 Z Erdem, Cahit 2771 zinc 2732 Esetlili, Bihter Çolak 2837 zinc 2809 Esringu, Aslihan 2787

Fan, Zhiping 2800 Fareed, Iftikhar 2850 Faridullah 2742

Faridullah 2850 Firouzi, Ahmad Farrokhian 2815

G Gao, Jie 2751 García-Lorenzo, Mari Luz 2776 Genç, Elif 2860 Gong, Yi 2843 Guo, Shengjuan 2843 Guo, Wei 2751

H Han, Xinzhong 2751 Hanedar, Asude 2763 Heidarizadeh, Fariba 2815 Hosseini, Seyyedeh Maasoumeh 2815

H W Hu, Yaming 2800 Wang, Jinman 2707 Huang, Yuanxing 2792 Wang, Lian 2792 Wang, Lizhu 2800 I Wang, Qingji 2843 Iqbal, Akhtar 2742 Wang, Shu-min 2699 Irshad, Muhammad 2850 Wang, Wenbo 2751 Wang, Xiaohua 2751 K Wang, Yanjie 2800 Karagoz, Kenan 2715 Waseem, Amir 2742 Karapehlivan, Mahmut 2732 Wu, Jie 2751 Karataş, Mehmet 2747 Karayakar, Fahri 2771 X Karczmarczyk, Agnieszka A. 2736 Xie, Yun-cheng 2699 Kaya, İnan 2732 Xu, Zhihua 2792 Kordali, Saban 2715 Kotan, Recep 2715 Y Kulekci, Elif Akpinar 2787 Yang, Haishui 2751 Yao, Hong 2707 L Yao, Jie 2843 Li, Fayun 2800 Yaprak, Günseli 2837 Li, Yuanheng 2792 Yazgan, Mustafa Sait 2824 Lv, Shihai 2707 Yeşiltaş, Yasemin 2860 Lv, Xingna 2800 Yılmaz, Muhittin 2732 Yu, Hui 2699 M Yuan, Haiping 2843 Ma, Jianjun 2707 Yuan, Shijue 2792 Martínez-Sánchez, María José 2776 Molina-Ruiz, José 2776 Z Zhang, Daofang 2792 O Zhu, Liqun 2751 Özden, Serkan 2725 Zhu, Nanwen 2843 Ozer, Hakan 2715 Zhu, Qi-hong 2699

P Pérez-Sirvent, Carmen 2776

Sabir, Muhammad Amjad 2742

Şahin, Nihan 2830

Song, Li 2699

Toroz, Ismail 2763 Tunçer, Sezginer 2725 Turan, Metin 2787 Türkoğlu, Şifa 2830

U Umar, Muhammad 2742

V Vanlı, Ömer 2824

2870