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CONTENTS

ORIGINAL PAPERS

SPATIAL VARIATIONS OF NPP IN DIFFERENT 2264 ALTITUDES AT A MEDITERRANEAN WATERSHED Cenk Donmez, Suha Berberoglu and Ahmet Cilek

ESTIMATION OF OCCUPATIONAL RISK IN HERBICIDE APPLICATION 2275 Ali Musa Bozdogan, Nigar Yarpuz-Bozdogan, Ibrahim Tobi and Bahadir Sayinci

SYNTHESIS AND CHARACTERIZATION OF ZnS NANO- 2280 CRYSTALS AND ITS PHOTOCATALYTIC ACTIVITY ON ANTIBIOTICS Yuting Wu, Liang Ni, Xinlin Liu, Mingjun Zhou, Zhi Zhu, Ziyang Lu, Changchang Ma and Pengwei Huo

EVALUATION OF PARTICLE SIZE AND INITIAL CONCENTRATION 2289 OF TOTAL SOLIDS ON BIOHYDROGEN PRODUCTION FROM FOOD WASTE Iván Moreno-Andrade and Germán Buitrón

APPLICATION OF CELLULOSE ACETATE MEMBRANES FOR 2296 REMOVAL OF TOXIC METAL IONS FROM AQUEOUS SOLUTION Nadjib Benosmane, Baya Boutemeur, Maamar Hamdi and Safouane M. Hamdi

IN VITRO ANTIOXIDANT POTENTIAL OF para-ALKOXYPHENYLCARBAMIC ACID ESTERS 2310 CONTAINING 4-(4-FLUORO-/3-TRIFLUOROMETHYLPHENYL)PIPERAZIN-1-YL MOIETY Ivan Malík, Jan Muselík, Lukáš Stanzel, Jozef Csöllei and Matej Maruniak

AN ASSESSMENT OF RUNOFF AND SEDIMENT IN SOME 2319 IRRIGATION DISTRICTS IN A SEMI-ARID REGION OF TURKEY Ali Fuat Tari, Öner Çetin, Ramazan Yolcu and Vyacheslav Bogdanets

TEMPORAL AND SPATIAL CHARACTERISTICS OF PHOSPHORUSFRACTIONS 2325 UNDER LONG-TERM WASTEWATER IRRIGATION IN TONGLIAO CITY, CHINA Yintao Lu, Fang Liu, Hong Yao and Kelin Hu

INACTIVATION OF Monopylephorus limosus BY CHLORAMINE, CHLORINE 2334 DIOXIDE, AND HYDROGEN DIOXIDE: EFFECTIVENESS AND SAFETY Kun Yao, Yao Yang, Lihong Zhao, Baiyang Chen and Xiaoshan Zhu

ADSORPTION OF LEAD METAL FROM AQUEOUS SOLUTIONS 2341 USING ACTIVATED CARBON DERIVED FROM SCRAP TIRES Ali R. Rahmani, Ghorban Asgari, Fateme Barjasteh Askari and Ameneh Eskandari Torbaghan

PREPARATION, CHARACTERIZATION OF Fe ION EXCHANGE MODIFIED TITANATE 2348 NANOTUBES AND PHOTOCATALYTIC ACTIVITY FOR OXYTETRACYCLINE Changyu Lu, Weisheng Guan, Yuexin Peng, Li Yang, Tuan K. A. Hoang and Xiao Dong Wang

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ADSORPTION CHARACTERISTICS OF PHOSPHORUS ONTO SOILS 2354 FROM DIFFERENT LAND USE TYPES IN DANJIANGKOU RESERVOIR AREA Meng Xu, Liang Zhang, Yun Du, Chao Du and Yan-Hua Zhuang

ASSESSMENT OF LIMITING FACTORS FOR POTENTIAL 2362 ENERGY PRODUCTION IN WASTE TO ENERGY PROJECTS Orhan Sevimoğlu

APPLICATIONS OF FACTOR ANALYSIS AND GEOGRAPHICAL INFORMATION 2374 SYSTEMS FOR PRECISION AGRICULTURE OVER ALLUVIAL LANDS Kadir Ersin Temizel, Hakan Arslan and Mustafa Sağlam

PHOTOSYNTHETIC ACTIVITY OF Microcystis IN FISH GUTS AND ITS 2384 IMPLICATION FOR FEASIBILITY OF BLOOM CONTROL BY FILTER-FEEDING FISHES Zhicong Wang, Zhongjie Li, Yiyong Zhou and Dunhai Li

CELLULAR LOCALIZATION OF COPPER AND ITS 2394 TOXICITY ON ROOT TIPS OF HORDEUM VULGARE Junran Wang, Qiuyue Shi, Jinhua Zou, Ze Jiang, Jiayue Wang, Hangfeng Wu, Wusheng Jiang and Donghua Liu

EVALUATION OF STABILITY AND MATURITY PARAMETERS 2406 IN WASTEWATER SLUDGE COMPOSTING WITH DIFFERENT AERATION STRATEGIES AND A MIXTURE OF GREEN PLANT WASTES AS BULKING AGENT Amir Hossein Nafez, Mahnaz Nikaeen, Bijan Bina, Akbar Hassanzadeh and Sharareh Moghim

ADSORPTION OF CARMINE ON ETHYLENEDIAMINE 2415 MODIFIED PEANUT HUSK FROM AQUEOUS SOLUTION Yinghua Song, Xi Zhang, Mei Ye, Hui Xu and Jianmin Ren

INDEX 2421

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SPATIAL VARIATIONS OF NPP IN DIFFERENT ALTITUDES AT A MEDITERRANEAN WATERSHED

Cenk Donmez1,*, Suha Berberoglu1, and Ahmet Cilek1

1 University of Cukurova, Department of Landscape Architecture, 01330 Adana, Turkey.

ABSTRACT the most important driver for global warming. Addressing to the climate change, the carbon balance is a critical issue The aim of study was to estimate the current net pri- that comprises both scientific and political importance. mary productivity (NPP) of Goksu River Basin (forest, Thus, the quantification of carbon is a major research need grasslands, bare soil, agriculture) located at the Eastern to derive management scenarios in respect to the effects of Mediterranean coast of Turkey using remote sensing and a climate change at different scales. biogeochemical model. Four elevation zones between 0 to The quantification of terrestrial carbon depends on the 2500 m were defined and spatial patterns of NPP in those net photosynthetic accumulation of carbon by plants. Ac- elevation zones were assessed to understand the impacts of cumulation of carbon by plants, also known as Net Primary topographyon local spatial patterns of productivity. The Productivity (NPP) provides the energy that drives most model results are incoorporated with available topographic biotic processes on Earth [1]. NPP is a function of standing information in watershed level. The Carnegie-Ames-Stan- biomass, an important component of the carbon cycle and ford approach (CASA) model approach was used to esti- a key indicator of ecosystem performance. NPP contributes mate annual and monthly NPP. This model uses a light-use to biological properties of Earth’s terrestrial surface by pro- efficiency (LUE) factor, which is the efficiency of conver- ducing organic matter which can be consumed by living sion of light energy into dry materials by plant, together organisms [2]. Accounting for the potential of atmospheric with remotely sensed, climate (to express the effects of air CO2 in terrestrial ecosystems depends on understanding of temperature and water stress), soil and biotic data and seasonal climate controls on NPP fluxes. Thus, the terres- ground measurements. Thus, a comprehensive spatial and trial carbon sinks can further be accounted at local and re- temporal data set including temperature, precipitation, so- gional scales. lar radiation, soil texture, percent tree cover, land cover type, and normalized difference vegetation index (NDVI) Surface temperature, precipitation, and solar radiation were used in modelling process. Percent tree cover was have been realised as the strongest controllers of annual predicted using multi-temporal LANDSAT images by ag- terrestrial NPP and its seasonal changes at different scales gregating tree cover estimates made from high resolution [1, 3, 4]. Hence, integrating of these climate forcing varia- Geo-EYE imagery in a regression tree algorithm. The re- bles into the improved techniques is essential to predict the sults indicated several interesting trends between NPP and annual and seasonal NPP fluxes. regional climate gradients. NPP was correlated strongly with solar radiation and precipitation during the growing Addressing the strong relationship between climate season suggesting that water limitation as important varia- variables and topography is known as an important factor ble controlling regional patterns of productivity. for vegetation diversity at the Mediterranean where moun- tain formations are often the dominant factor for the vegetation pattern [5, 6]. However, the impact of topography on vegetation dynamics is rather poorly understood at water- KEYWORDS: Remote sensing, NPP, spatial modelling, Mediterra- shed level, due to its complexity of the mountainous sys- nean, NASA-CASA Model. tems. Recently, remote sensing and modelling processes as improved techniques provide a better understanding and 1. INTRODUCTION quantification of the carbon sources and sinks in different land uses and land covers at different altitudes. Particu- The atmospheric concentrations of the carbon have larly, the ecosystem models based on satellite sensor data been recently increasing and its widely believed as one of have significant importance to capture variability in ecosystem processes when ground-based measurements of lo- * Corresponding author cal fluxes are not adequate.

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In previous studies, three groups of models have been grasslands and bare grounds. The climate is characterized used to estimate NPP for large areas in last decades: (i) by prevailing Mediterranean with mild and rainy winters based on satellite sensor data; such as, the Carnegie, Ames, and hot and dry summers with a mean annual precipitation of Stanford Approach (CASA) ([7, 8]; (ii) simulating carbon approximately 800 mm. Mean annual temperature is 19 Co. flux using a prescribed vegetation structure such as, the Bi- ome BioGeochemical Cycle (BIOME-BGC) model [9]; 2.2 Remote Sensing and Ancillary Data (iii) simulating both vegetation structure and carbon flux Two sets of remotely sensed images were utilized in this such as, the Dynamic Global Phytogeography (DOLY) study; i) a LANDSAT TM/ETM data set comprising five model [10]. scenes from 1999-2003, ii) Three sub-scenes of Geo-Eye The aim of this paper was to estimate the current NPP imagery representing different types of forest cover were in the Mediterranean using LANDSAT TM/ETM images used as training and testing data for percent tree cover. with a 30 m resolution and CASA model. Seasonal NPP The topographic maps in 1:25.000 scale were used to fluxes in respect to different land cover types were derived derive Digital Elevation Model (DEM). DEM was incoop- using land cover data, climate variables, soils and ground erated with the model NPP outputs to define the spatial re- truth. Spatial variations of NPP in four elevation zones lationship between elevation and NPP patterns. The forest ranging between 0 to 2500 m were examined to investigate maps were utilized for land cover mapping. influence of topography on forest land cover. This study enabled us to define the spatial composition of the NPP 2.3 Modelling Net Primary Productivity where the ground measurements are limited. It is also important to expose the variability of NPP at different alti- The CASA model was used in this study to estimate tudesto investigate the potential effects of diverse topogra- NPP in Goksu River Basin, Turkey. The model estimates phy on local NPP in a Mediterranean watershed. the monthly NPP flux as net fixation of CO2 by vegetation on the basis of light-use efficiency. Thus, NPP is calculated as a function of absorbed photosynthetically active (400 to 2. MATERIALS AND METHODS 700 nm) solar radiation (APAR), and an average light utilization efficiency (ε) [7]. Thus, the main frame of CASA 2.1 Study area model is Goksu River Basin is located in the Eastern Mediterra- NPP = APAR × ε nean region of Turkey (Figure 1). The basin covers approx- NPP=f(NDVI) × PAR × ε × g(T) × h(W) imately 10.000 km2. The land cover types of the region comprise Mediterranean evergreen needleaf forests with where APAR (in megajoules per square meter per Turkish pine (Pinus brutia) and Juniper (Juniperus excelsa), month) is a function of NDVI and downwelling photo-

FIGURE 1 - Location of the Goksu Basin, Turkey

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synthetically active solar radiation (PAR) and ε (in grams method and mapped on monthly basis. Monthly climate of C per megajoule) is a function of the maximum achiev- maps are given in Figures 2-4. able light utilization efficiency ε adjusted by functions that account for effects of temperature g(T) and water h(W) 2.5 Mapping Land Cover stress. Whereas previous versions of the CASA model [4,8] A comprehensive land cover map derived from used a normalized difference vegetation index (NDVI) to LANDSAT ETM image acquired in 2003, together with estimate FPAR, the current model version instead relies ground truth data from field surveys was used as an input upon canopy radiative transfer algorithms [11], which are to CASA model. Image classification was carried out using designed to generate improved FPAR products as inputs to maximum likelihood algorithm with supervised training. carbon flux calculations. The model was utilized to predict The classifier was provided with the spectral reflectance annual regional fluxes in terrestrial net primary production properties of each class in the form of the mean reflectance at variable degrees of C, depending on the yearly condi- for each spectral waveband and the associated covariance tions, with terrestrial net production. Several diverse da- matrix. This data set was generated from a selection of sam- tasets were used in this research. Calculation of annual ter- ple training pixels for each class using ground data. The out- restrial NPP is based on the concept of light-use efficiency, put comprised 13 land cover classes with 30 m spatial reso- modified by temperature, rainfall values and solar radiation lution initially (Figure 5). Accuracy analysis was carried out scalars. In addition, percentage of tree cover, land cover by comparing the classification map and ground truth. map of the region, soil texture and NDVI (normalized difference vegetation index) will be used to constitute this 2.6 Soil texture model. The soil texture is based on FAO soil texture classification consisted of 7 classes. The dominant soil type in a 2.4 Climate Data soil unit, the designation "coarse", "medium", "fine", or a Monthly precipitation, air temperature and solar radia- combination of these based on the relative amounts of clay, tion were used as a climate data set. These variables were silt, and sand present in the top 30 cm of soil. The regional based on 9 years (2000-2009) records of meteorological soil maps in 25.000 scale was utilized for this study and stations in and around the study region. Climate variables soil texture classes were assigned on the basis of estimated were interpolated together with DEM using co-kriging clay content according to FAO.

FIGURE 2 - Monthly precipitation maps of the study area

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FIGURE 3 - Monthly air temperature maps of the study area

FIGURE 4 - Monthly solar radiation maps of the study area

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FIGURE 5 - Land cover classification map of the study region

2.7 NDVI iv) Modelling: The relationships between tree cover Monthly NDVI images derived from 20 LANDSAT density and LANDSAT spectral values were modeled us- scenes recorded in between May 2001 and November ing RT technique. 2002. Four images were combined to comprise the study v) Accuracy Assessment and Final Map Derivation: region. The monthly composites were created and bands 4 RT model performance was measured using the correlation and 3 were used to produce NDVI. Monthly NDVI images coefficient (r) between the predicted and actual tree cover ranging between 0 and 1 were the input to CASA model. values for the set aside test samples, r can be considered a measure of the precision of prediction. Percent tree cover 2.8 Estimation of percent tree cover map derived at 30 m spatial resolution and error estimates This study utilised the Regression Tree (RT) model to obtained through validation. predict percent tree cover within a Mediterranean type forest using LANDSAT data. The methodology for deriving 2.9 Statistical analysis percent tree cover with RT consisted of five steps for this A pixel based linear regression model was constructed study [12]: between model NPP maps and DEM data. Correlation be- i) Generate reference percentage tree cover data: Dig- tween these maps represents the robust changes of NPP in ital multispectral GeoEYE images with a spatial resolution different altitudes. The linear relationship was determined of 45 cm were used to derive reference percentage tree for each elevation zone of the study region. The correlation cover data required to train the model. co-efficiency represented the relationship of local vegetation and topography, this relationship was characterized using ii) Deriving metrics from the RS data: Normalised Dif- the magnitude of estimated NPP in relation to altitude. ference Vegetation Index (NDVI) was derived as a ratio of the difference and total in reflectance between near infrared (band 4) and visible red (band 3) of the LANDSAT 3. RESULTS AND DISCUSSION standard band setting. This study showed that CASA Model and LANDSAT iii) Selecting predictor variables: The test data set were data successfully modeled the monthly NPP patterns of split into two subsets; training and testing [13]. The model land cover classes in a heterogenuos Mediterranean terrain. was fitted using the most relevant input variables selected The outputs of the model comprise monthly and total NPP using the Stepwise Linear Regression method and the maps. Monthly NPP maps are shown in Figure 6. Annual available training with the reference data derived from high total NPP map of the Goksu Basin is shown in Figure 7. resolution images, relationships between tree cover density and LANDSAT spectral values were modeled using RT Monthly NPP maps revealed that mean monthly NPP technique. A total of 20% cells were separated from the ranged from 12.9 to 144.07 gC m-2 month-1. A significant predictor variables in order to validate the model. increase was determined from March to April within spring

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FIGURE 6 - Monthly NPP maps of Goksu Basin

FIGURE 7 - Annual total NPP map of the Goksu Basin Mean annual NPP was estimated as 388 g C m-2 yr-1 for the study area.

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200 200 a) b) 150 150

100 100

50 50

NPP (gC m2y-1) 0 0

200 200

c) d) 150 150 100 100 50 NPP (gC m2y-1) 50 0 0

200 e) 150

100

FIGURE 8 - Monthly NPP results from the CASA model; a) Needleaf Evergreen Forests (NLEF), b) Broadleaf Deciduous Forests (BDF), c) grasslands, d) bare soils, e) agriculture

season in the entire basin. Slight changes in NPP were ob- Monthly NPP estimates of major land covers and land served from April to June until a dramatic decrease occured uses of Goksu Basin according to the CASA algorithm is in July. shown in Figure 9. Annual NPP of major land covers and land uses of Goksu are also shown in Table 2. Monthly NPP changes for each land cover class of The forest stands of Goksu Basin were combined to two Goksu Basin were estimated with CASA model. The NPP formation classes as NLEF and BDF. The forest NPP estima- of major land cover classes were aggregated and total an- tion using CASA model was based on those two classes that nual NPP was estimated (Table 1). Monthly NPP results comprise the main forest types of Goksu Basin. NLEF com- derived for the CASA model is shown in Figure 8. prised Juniperus excelsa, Pinus nigra, Pinus brutia, Cedrus libani and Abies cilicica. BDF comprised only Quercus sp.,. TABLE 1 - Annual net primary productivity (NPP) of major land covers and land uses of Goksu The relationship between NPP and elevation were eval- Mean NPP uated according to four general altitude zones (Figure 10). Land Cover/Land Use Classes (gC m2 yr-1) These zones were defined considering the transition bound- NLEF 742.98 aries of forest in the region. Each elevation zone represents BDF 740.80 the NPP variation in respect to its spatial distribution. Grasslands 668.92 Total annual NPP of different land cover classes was Bare soil 563.40 also estimated by incooperating the model results, land Agriculture 660.98 cover and DEM data (Table 2).

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200 NLEF BDF 150 Grasslands Bare soil 100

FIGURE 9 - Monthly NPP estimates of major land covers and land uses of Goksu Basin according to the CASA algorithm

TABLE 2 - Mean annual NPP of different altitude zones

Elevation Mean Elevation NPP NLEF BDF Grassland Agriculture Classes (m) (gC m-2 y-1) (gC m-2 y-1) (gC m-2 y-1) (gC m-2 y-1) (gC m-2 y-1) 0-500 218 765 754 755 897 758 501-1000 742 717 708 674 876 739 1001-1500 1263 708 659 659 867 704 1501-2300 1869 534 599 579 644 564

500 1800 1600

400 1400 ) -1

1200 y 300 1000 -2 200 800 600 400 Elevation (m) (m) Elevation 100 200 NPP (gC m 0 0 1 21416181101121 Number of Samples Elevation NPP

1000 1400 )

1200 -1 900 y 1000 -2 800 800 700 600 400 600 NPP (gC m Elevation (m) (m) Elevation 200 500 0 151101151 Number of Samples Elevation NPP

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1500 1600 1400 1400 1200 ) -1

1000 y

1300 -2 800 1200 600 400 1100 Elevation (m) (m) Elevation

200 NPP (gC m 1000 0 1 51 101 151 201 251 301 Number of Samples Elevation NPP

2500 1200

1000 )

2300 -1 y

800 -2 2100 600 1900 400 Elevation (m) (m) Elevation

1700 200 NPP (gC m 1500 0 1 51 101 151 201 251 301 351 401 Elevation NPP Number of Samples

FIGURE 10 - Annual NPP distribution in different altitudes in Goksu Watershed

Correlation between the spatial distribution of NPP variables at local scale. This correlation shows that the el- and elevation in Goksu Watershed is shown in Figure 11. evation is one of the main controlling factor in the vegeta- A slight decrease in forest productivity tended to occur in tion distribution and its diversity. parallel to elevation increase. NLEF NPP was estimated The Goksu Basin located in Mediterranean region is -2 -1 between 599 to 659 gC m y in the zones higher than characterized within its sparse vegetation. The complexity 1000 m. Similarly, grasslands and agriculture NPP showed of its terrain in respect to the soil types and topography a significant decrease in cooler sites in the mountainous re- greatly influence reflectance from sparse cover of the Med- gion. iterranean forests. Particularly, high reflectance from the soil causes a significant albedo effect, and hence, over- 900 ) whelms reflectance from the vegetation component, thus -1 750 y R² = 0,8129 leading to under estimation of NPP [14]. Thus, the percent -2 600 tree cover map derived using the RT method improved the 450 discrimination of vegetation cover by assisting in the ex- 300 ploration of the relationships between LANDSAT spectral 150 bands and biophysical variable of NDVI. This relationship NPP (gC m 0 assisted to calibrate the model along the entire tree cover 0 500 1000 1500 2000 2500 and other land cover types. Elevation (m) The amount of NPP within NLEF and BDF formations were lowest in winter and highest in summer. Grasslands FIGURE 11 - Correlation between the spatial distribution of NPP and elevation in Goksu Watershed that comprise seasonal herbaceous plants of the area indicated an increase of NPP amounts in spring and summer. Local NPP showed a considerable relationship with al- A dramatic decrease in NPP for the forest stands occurred titude in terms of linear changes in different elevation in July due to extreme humidity and temperature increases zones. The correlation coefficient is defined as 0.81 which in respect to micro-climatic conditions of the study region. represents a strong interaction between altitude and NPP The climate variables including precipitation, air temperature and solar radiation play a significant role in understanding the seasonal NPP changes of different land cover

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classes. Hence, integration of climate variables and spatial not exactly represent and reveal the underlying mecha- ancillary data including land cover and percent tree cover nisms of an ecosystem. In this regard, comprehensive ap- maps to the regional models may increase the potential on proaches should be applied to enable a detailed understand- NPP representation of the complex environments. Such ap- ing of the geophysical parameters controlled by topogra- proaches improve in understanding and assessing the local phy are actually the most important for local vegetation dy- and regional consequences of climate change on the namics. The future research should focus on particularly productivity of the complex and rich ecosystems such as combining functional topographic variables and plant-eco- Goksu Basin. physical parameters in micro-scale in order to improve an understanding of the relationships between topography and local vegetation dynamics. 4. CONCLUSIONS

Remotely sensed data coupled with biogeochemical ACKNOWLEDGEMENTS models may serve to better quantify and manage potential C sequestration on forest productivity [15]. However, ef- We would like to acknowledge the research project fects of topography on the spatial distribution of NPP have grants (project no: 110Y338 and 114Y273) from the Scien- been received little attention within the literature. Thus, the tific and Technological Research Council (TUBITAK) of findings of this study showed that the integration of satel- Turkey and the Scientific Research Project Administration lite images and a model algorithm can successfully be used Units of Cukurova University (project no: FYL-2015-4374). to estimate the monthly and annual dynamics of NPP within a complex semi-arid Mediterranean environment consider- The authors have declared no conflict of interest. ing the effects of altitude. LANDSAT data hold great potential for predicting NPP with CASA model, it was also enabled us to make a detailed analysis of local topographic ef- REFERENCES fects on NPP because of its spatial resolution. It can represent the spatial heterogeneity of a local environment to- [1] Potter, C., Klooster S., Steinbach M., Tan P., Kumar V., Shek- gether with altitude in the NPP modelling. CASA was the har S., Nemani R., Myneni R. (2003). Global teleconnections appropriate model for handling the variability present in of climate to terrestrial carbon flux. Journal of Geophysical Research, Vol. 108, NO. D17, 4556, complex and highly variable Mediterranean type forest doi:10.1029/2002JD002979. which has sparse coverage and high species diversity. It is promising approach for modelling NPP within various im- [2] Lobell, D.B., Hicke, J.A., Asner, G.P., Field, C.B., Tucker, C.J. (2008). Los, S.O. Satellite estimates of productivity and age resolutions in local/regional environments. light use efficiency in United States agriculture 1982–1998. In this study, local NPP patterns change and its mech- Global Change Biology, 8(8), 722-735. anism in small-scale topography was described by incoop- [3] Melillo, J.M., McGuire, A.D., Kicklighter, D.W., Moore III, erating the biogeochemical modelling results and spatial B., Vorosmarty, C.J., Schloss, A.L. (1993). Global climate change and terrestrial net primary production, Nature, 363, data. Remote sensing, GIS, and statistical and spatial anal- 234– 240. yses were used to examine the changes of NPP in various altitude zones in a Mediterranean environment. Main find- [4] Potter, C.S., Randerson, J.T., Field, C.B., Matson, P.A., Vi- tousek, P.M., Mooney, H.A., Klooster, S.A. (1993). Terrestrial ings of this study were; ecosystem production: A process model based on global satellite and surface data, Global Biogeochem. Cycles, 7, 811 – Elevation is an important factor effecting the vertical 841,. distribution of NPP in Goksu Watershed: the NPP shows a considerable distribution between the elevations of 0 m and [5] Grytnes, J. A. (2003). Species-richness patterns of vascular plants along seven altitudinal transects in Norway. – Ecogra- 500 m. NPP shows decrease in higher elevations. Highest phy 26: 291 – 300. decrease occurred between 1500 m and 2300 m. Hence, a better NPP condition occurs over a larger elevation range. [6] Sanders, N.J. and Rahbek, C. (2012). The patterns and causes of elevational diversity gradients. – Ecography 35: 1 – 3. NLEF showed a reasonable decrease of approxi- [7] Potter, C., Klooster, S., Myneni, R., Genovese, V., Tan, P.N, -2 -1 mately -230 gC m y between 500 m to 2300 m. Grass- Kumar, V. (2003) . Continental-scale comparisons of terres- lands showed the highest amount of decrease of approxi- trial carbon sinks estimated from satellite data and ecosystem mately -250 gC m-2 y-1 in higher altitudes. modeling 1982–1998. Global and Planetary Change, 39, 201– 213. Slope and aspect are expected to influence the vertical [8] Potter, C.S., Klooster, S., Steinbach, M., Tan, P., Sheikarand, distribution of NPP on higher altitudes due to their role on S., and Carvalho, C. (2004). Understanding global teleconnec- shade angles. Particularly, the shaded areas have signifi- tions of climate to regional model estimates of Amazon eco- cant importance for better vegetation growth since the system carbon fluxes. Global Change Biology, 10, 693 – 703. Goksu Watershed is located in a semi-arid region. [9] Running, S., Ramakrishna, R., Nemani, F., Heinsch, A., Mao- sheng, Z., Reeves, M., and Hashimoto, H., (2004). A Contin- However, while such an approach shows the im- uous Satellite-Derived Measure of Global Terrestrial Primary portance of topography for vegetation dynamics, it does Production. BioScience, 54, 547 – 560.

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[10] Woodward, F.I., Smith, T.M., and Emanuel, W.R. (1995). A global land primary productivity and phytogeography model. Global Biogeochemical Cycles, 9, 471 490.

[11] Knyazikhin, Y., Martonchik, J.V., Myneni, R.B., Diner, D.J., Running S.W. (1998). Synergistic algorithm for estimating vegetation canopy leaf area index and fraction of absorbed photosynthetically active radiation from MODIS and MISR data. Journal of Geophysical Research 103: 32257– 32276. [12] Donmez, C., Berberoglu, S., and Curran, P.J. (2011). Model- ling the current and future spatial distribution of Net Primary Production in a Mediterranean watershed. International Jour- nal of Applied Earth Observation and Geoinformation, Vol- ume 13: Issue 3, pp.336.-345. [13] Rokhmatuloh, Nitto, D., Al-Bilbisi, H. Tateishi. R. (2005). Percent Tree Cover Estimation Using Regression Tree Method: a case study of Africa with very-high resolution QuickBird images as training data, Geoscience and Remote Sensing Symposium IEEE 2005 International, IGARSS 05. Vol. 3, pp 2157-2160. [14] Berberoglu, S., Evrendilek, F., Ozkan C., Donmez C. (2007). Modeling Forest Productivity Using Envisat MERIS Data. Sensors, 7, 2115-2127. [15] Meydan, S.M., Evrendilek, F., Berberoglu, S., Donmez, C. (2010). Modeling above-ground litterfall in eastern Mediterra- nean conifer forests using fractional tree cover, and remotely sensed and ground data, Applied Vegetation Science, 1–26.

Received: November 10, 2014 Revised: March 31, 2015 Accepted: April 28, 2015

CORRESPONDING AUTHOR

Cenk Donmez University of Cukurova Department of Landscape Architecture 01330 Adana TURKEY

E-mail: [email protected]

FEB/ Vol 24/ No 7/ 2015 – pages 2264 - 2274

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ESTIMATION OF OCCUPATIONAL RISK IN HERBICIDE APPLICATION

Ali Musa Bozdogan1*, Nigar Yarpuz-Bozdogan2, Ibrahim Tobi3 and Bahadir Sayinci4

1Cukurova University, Agriculture Faculty, Agricultural Machinery and Technologies Engineering Dept., 01330 Adana/Turkey 2Cukurova University, Vocational School of Technical Sciences, 01350 Adana/Turkey 3Harran University, Agriculture Faculty, Agricultural Machinery Dept., Sanliurfa/Turkey 4Ataturk University, Agriculture Faculty, Agricultural Machinery Dept., Erzurum/Turkey

ABSTRACT thyl, fenoxaprop-p-ethyl, foramsulfuron, iodosulfuron methyl sodium, nicosulfuron, pinoxaden, tribenuron methyl, The aim of this study was to estimate the occupational and tritosulfuron. These a.i. consumption were 10 000 kg risk for operators and workers in herbicide application in for 2,4-D, 1 125 kg for tribenuron-methyl, 3 408 kg for di- Sanliurfa, Turkey. In this study, 2,4-D, clodinafop-propar- clofop-methyl, 360 kg for fenoxaprop-p-ethyl, 2 016 kg for gyl, dicamba, diclofop-methyl, fenoxaprop-p-ethyl, foram- clodinafop-propargyl, 243 kg for pinoxaden, 1 250 kg for sulfuron, iodosulfuron-methyl-sodium, nicosulfuron, pinox- nicosulfuron, 625 kg for tritosulfuron, 125 kg for dicamba, aden, tribenuron-methyl, and tritosulfuron were used for 225 kg for foramsulfuron, and 7.5 kg for iodosulfuron me- determining the occupational risk. In this study, it was ob- thyl sodium [3]. In Sanliurfa, total herbicide a.i. consump- tained that the highest risk was assessed in diclofop-methyl tion in cereal cultivation was 19 400 kg. as 1.913 Exceedence Factor (EF) value. The lowest risk was Pesticides used in agriculture have potential negative found in nicosulfuron due to its 0.230 EF value. In the re- effects on human health and environment. It was indicated sult of this study, it was determined that herbicide has risk that the average cost of the health treatment of the poisoned for occupational health. These risks can be minimized by worker was calculated as USD 787.97 in Brazil [5]. Pesti- using equipment such as personnel protective equipment, cide exposure on human and environment can be mini- increasing awareness of pesticide side effects on operator mized by selecting the type of tractors such as closed and and worker and advanced proper application technique and open cabin, and suitable pesticide application technology equipment, etc. such as proper anti-drift nozzles, air-assisted nozzles, pes-

ticide application methods, etc. [6-14]. Yet, these are not

KEYWORDS: Weed control, Pesticide application, Human health, enough to protect the worker and operator, some additional Environment, Cereal. precautions such as personnel protection equipment (PPE) should be taken for occupational risk during pesticide ap-

plication. Operators are persons who attend to mix, load 1. INTRODUCTION and apply pesticide in application [9, 15, 16]. Workers are persons who are occupied with thinning, pruning, and har- Pesticides are defined as chemical formulations that vesting etc. of the agricultural crop [9, 15-17]. are used for improving quality and quantity of agricultural In Turkey, generally, farmers apply overdose pesticide crops by farmers. These are mainly classified as insecti- than the recommended dose by manufacturer and do not cide, herbicide, and fungicide. Herbicides are used to con- take care of personnel protection during pesticide applica- trol weed in agricultural land. World pesticide active ingre- tions [18-20]. Demircan and Yılmaz (2005) indicated that, dients (a.i.) amount used exceeded 2 million tonnes in in apple production, farmers applied pesticides 186% more 2007. In this value, herbicide a.i. was 951 584 tonnes [1]. than the recommended dose in Isparta province, Turkey In EU-15, herbicide a.i. consumption was 70 874 tonnes in [18]. Application rate effects the pesticide exposure on hu- 2010 [2]. In Turkey, 6 056 tonnes herbicide (a.i.) was used man and environment. Application rate plays a role in the in agriculture in 2010 [2]. In Turkey, cereals are approxi- occupational pesticide exposure [19]. Yalap Tuna (2011) mately cultivated in an area of 16 million ha [3]. In San- determined that farmers were eating and drinking during liurfa, in 2013, herbicides were applied in 260 000 ha in pesticide applications. Researcher indicated that the ratio cereal cultivation [4]. In cereal cultivation areas, 11 a.i. of health problems after the applying process was 25% were used for weed control in 2013, in Sanliurfa. These a.i. amoung the farmers [20]. were 2,4-D, clodinafop-propargyl, dicamba, diclofop-me- The aim of this study was to estimate the occupational risk in herbicide applications in cereal fields, Sanliurfa, * Corresponding author Turkey.

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2,4‐D Clodinafop‐propargyl Dicamba Diclofop‐methyl

C8H6Cl2O3 C17H13ClFNO4 C16H14Cl2O4 C8H6Cl2O3 Fenoxaprop‐p‐ethyl Foramsulfuron Iodosulfuron‐methyl‐sodium Nicosulfuron

C18H16ClNO5 C15H18N6O6S C H N O S 17 20 6 7 C14H13IN5NaO6S Pinoxaden Tribenuron‐methyl Tritosulfuron

C23H32N2O4 C13H9F6N5O4S

C15H17N5O6S

FIGURE 1 - Chemical formula of a.i. [21]

2. MATERIAL AND METHODS Index (m2; default: field crops: 1), TF: Transfer factor (cm2 person-1 h-1; default: field crops: 5000), T: The duration of In this study, 11 herbicide a.i., named as 2,4-D, re-entry (h; default: 8), and P: Factor for Personal Protec- clodinafop-propargyl, dicamba, diclofop-methyl, fenoxa- tive Equipment (PPE) (default: no PPE: 1). prop-p-ethyl, foramsulfuron, iodosulfuron-methyl-sodium, Exceedence factor (EF) was determined by Equation 4 nicosulfuron, pinoxaden, tribenuron-methyl, and tritosul- [22, 24]. furon were used in cereal fields in Sanliurfa (Figure 1).  In this study, risk indices (RI) of occupational health  X TRANSFORME D  LLTRANSFORME D  EF    (4) for each a.i. were estimated via Equations 1, 2, and 3.      UL TRANSFORME D  LLTRANSFORME D 

The RI for operator was determined via Equation 1 + + + [15, 16, 22, 23]. RI , LL and UL was calculated by dividing, respectively the risk index values RI, LL and UL by UL. RI+, LL+ AR  0.292 and UL+ values were transformed with Equation 5 [22, 24]. RI operator  AOEL (1)  1  Xtransformed  log1  (5) AR: Application rate (kg a.i. ha-1); AOEL: Acceptable  X  operator exposure level (mg kg-1 body weight day-1). with The RI for worker was assessed with Equation 2 and 3 + + + [15-17, 22, 23]. X = RI , LL and UL . DE  Ab If the EF value for each a.i. is equal or lower than 0, it DE is regulate to 0 and indicates a low risk. EF value equal or RI wor ker  AOEL (2) higher than 1 is regulated to 1, and it means a high risk. An intermediate risk is found for the values between 0 and 1 AR [22, 24]. In this study, EF values of the operator and worker DE  0.01 TF T  P LAI were summed for the determination of the total risk on oc- (3) cupational health. Therefore, total EF for the occupational -1 DE: Dermal Exposure of worker (mg day ), AbDE: risk was between 0 and 2 according to POCER (The Pesti- Factor of dermal absorption (default:0.1). LAI: Leaf Area cide Occupational and Environmental Risk) indicator [22].

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3. RESULTS AND DISCUSSION cation for wheat [24]. Yarpuz-Bozdogan [25] found it as 80.07% for worker and 19.93 % for operator in herbicide EF values for worker and operator were calculated by application. Ribeiro et al. [26] notified that workers enter Eq. 1-4. These values are shown in Figure 2 presenting into greenhouse for pruning, sorting, thinning, picking etc. each a.i. with the applied doses in herbicide application. after pesticide application. Therefore, pesticide exposure on worker is higher than operator’s. In the present study, 2,4-D, diclofop-methyl, and fenoxaprop-p-ethyl have total risk on operator. As seen in Figure 2, Table 1 represents the percentage of total risk value for diclofop-methyl has the highest hazard for operator with the the occupational risk in herbicide application. EF value of 0.913. 2,4-D (EF 0.112) and fenoxaprop-p- As shown in Table 1, the theoretical total EF value was ethyl (EF 0.057) have intermediate risk. According to Fig- 2 because of the maximum 1.000 EF values of operator and ure 2, all a.i. have risk for the worker in herbicide applica- worker. The theoretical total EF value of each a.i. is 2.000 tion for cereal cultivation. EF value was calculated as 1.000 for occupational risk. The highest total occupational risk for 2,4-D, diclofop-methyl, and fenoxaprop-p-ethyl. It was in diclofop-methyl due to its 1.913 EF value (Table 1). means that they have the highest hazard for worker. Others Total occupational risk value for diclofop-methyl was cal- a.i. have intermediate risk because of the EF values be- culated as 1.000 for worker and 0.913 for operator. tween 0.000 and 1.000. Total EF value for operator was 1.082 for 2,4-D, diclo- In the present study, worker hazard in pesticide application fop-methyl, and fenoxaprop-p-ethyl a.i. If the operator had was found higher than operator hazard. As shown in Figure 1.000 EF value for each a.i., the theoretical total EF value 2, the total risk was determined as 86.69% for worker and for all a.i. would be 11.000. For this reason, in this study, 13.31% for operator. Similar results, 85.11% for worker percentage risk for operator was calculated as 9.84% of the and 14.89% for operator were founded in fungicide appli- total theoretical EF value. On the contrary, clodinafop-pro-

FIGURE 2 - EF values of the operator and worker

TABLE 1 - The applied dose, total EF value, and percentage of total risk

Active ingredient (a.i.) name Applied dose Total EF value Percentage of total risk (%) (kg a.i. ha-1) 2,4-D 0.80000 1.112 55.60 Clodinafop-propargyl 0.48000 0.958 47.90 Dicamba 0.01000 0.631 31.55 Diclofop-methyl 0.56800 1.913 95.65 Fenoxaprop-p-ethyl 0.06000 1.057 52.85 Foramsulfuron 0.04000 0.672 33.60 Iodosulfuron-methyl-sodium 0.00375 0.275 13.75 Nicosulfuron 0.05000 0.230 11.50 Pinoxaden 0.04050 0.674 33.70 Tribenuron methyl 0.00750 0.363 18.15 Tritosulfuron 0.05000 0.246 12.30

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TABLE 2 - Percentage decrease of risk for workers

EF value Active ingredient (a.i.) Percentage decrease of risk (%) no PPE with PPE

2,4-D 1.000 0.734 26.60 Clodinafop-propargyl 0.958 0.494 48.43 Dicamba 0.631 0.073 88.43 Diclofop-methyl 1.000 1.000 0.00 Fenoxaprop-p-ethyl 1.000 0.687 31.30 Foramsulfuron 0.672 0.118 82.44 Iodosulfuron-methyl-sodium 0.275 0.000 100.00 Nicosulfuron 0.230 0.000 100.00 Pinoxaden 0.674 0.121 82.05 Tribenuron methyl 0.363 0.000 100.00 Tritosulfuron 0.246 0.000 100.00

pargyl, dicamba, foramsulfuron, iodosulfuron-methyl-so- 4. CONCLUSIONS dium, nicosulfuron, pinoxaden, tribenuron-methyl and tritosulfuron have low risk for operator owing to their 0.000 In this study, all a.i. have occupational risk because of EF values. According to Table 1, the highest percentage of their 0.000 EF value. Three of eleven a.i. that named as 2,4- total risk (95.65%) was calculated in diclofop-methyl. In D, diclofop-methyl and fenoxaprop-p-ethyl have high oc- spite of that the lowest percentage of total risk (11.50%) cupational risk due to their 1.000 EF value. The percentage was determined in nicosulfuron. of a.i. with high hazard was determined as 27.27%. The highest total occupational hazard was obtained in diclofop- For all a.i., theoretical total risk is 22.000 for both methyl (EF 1.913). However, the lowest total risk for oc- worker and operator. In this study, total risk was calculated cupation was calculated in nicosulfuron (EF 0.230). Gen- as 8.131. Consequently, percentage total occupational risk erally, farmers in Turkey do not use PPE during pesticide was calculated as 36.96% for worker and operator. Yarpuz- application. If farmers do not use PPE, they can be nega- Bozdogan (2009) determined that the percentage total oc- tively affected by pesticide application. For this reason, cupational risk in herbicide application was assessed as farmers must use PPE for minimizing pesticide exposure. 31.01% in wheat cultivation in Adana, Turkey [25]. On the Four of eleven a.i. named as iodosulfuron-methyl-sodium, other hand, the percentage total occupational risk was de- nicosulfuron, tribenuron-methyl and tritosulfuron decrease termined as 50.71% in defoliant application for cotton in by 100% when PPE is used. The lesser pesticide exposure Adana, Turkey [27]. means the healthier farmers. It can be obtained from this In pesticide applications, farmers have to take precau- study that the use of PPE is important for farmers’ health tions for personnel protection. Hazards for worker and op- in herbicide applications. Public service announcement erator can be minimized by using PPE. Table 2 shows the about minimizing the pesticide exposure to operator and importance of PPE for workers. worker during application have to be broadcasted by regional media for public health. Moreover, instructions on In this study, if workers use PPE, the total value de- pesticide labels must be understood by farmers. creases up to 100% for iodosulfuron-methyl-sodium, nicosulfuron, tribenuron-methyl, and tritosulfuron (Table 2). The authors have declared no conflict of interest. On the other hand, diclofop-methyl has the highest risk for workers because of the fact that there is no decrease in its

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[6] Vercruysse, F., Drieghe, S., Steurbut, W. and Dejonckheere, [23] Vergucht, S.; De Voghel, S.; Misson, C.; Vrancken, C.; W. (1999) Exposure assessment of professional pesticide users Callebaut, K.; Steurbaut, W.; Pussemier, L.; Marot, J.; during treatment of potato fields. Pestic Sci 55, 467-473. Maraite, H. and Vanhaecke, P. (2006) Health and environmental effects of pesticide and type 18 biocides (HEEPEBI). [7] Ucar, T. and Hall, F.R. (2001) Windbreaks as a pesticide drift Available online: http://www.crphyto.be/pdf/heepebi.pdf (ac- mitigation strategy: a review. Pest Management Science 57, cessed on 11 11 2013). 663-675. [24] Bozdogan A.M. (2014) Assessment Of Total Risk On Non- [8] Colosio, C., Fustinoni, S., Birindelli, S., Bonomi, I., De Pas- Target Organisms In Fungicide Application For Agricultural chale, G., Mammone, T., Tiramani, M., Vercelli, F., Visentin, Sustainability. Sustainability 6, 1046-1058. S. and Maroni, M. (2002) Ethylenethiourea in urine as an indicator of exposure to mancozeb in vineyard workers. Toxi- [25] Yarpuz Bozdoğan N. (2009) Assessing the Environment and cology Letters, 134, 133-140. Human Health Risk of Herbicide Application in Wheat Culti- vation. International Journal of Food, Agriculture & Environ- [9] Matthews, G.A. (2006) Pesticides: Health , Safety and the En- ment 7, 775-781. vironment. Blackwell Publishing, Oxford, UK, 235 p. [26] Ribeiro, M. G., Colasso, C. G., Monteiro, P. P., Filho, W. R. P. [10] Bozdogan, A.M. and Yarpuz-Bozdogan, N. (2008) Determi- and Yonamine, M. (2012) Occupational safety and health prac- nation of dermal bystander exposure of malathion for differ- tices among flower greenhouses workers from Alto Tiete region ent application techniques. Fresenius Environmental Bulletin, (Brazil). Science of the Total Environment 416, 121-126. 12a, 2103-2108. [27] Bozdogan, A.M. and Yarpuz-Bozdogan, N. (2009) Determi- [11] Bozdogan, A.M. and Yarpuz-Bozdogan, N. (2008) Assessment nation of total risk of defoliant application in cotton on human of buffer zones to ditches of dicofol for different applied doses health and environment. Journal of Food, Agriculture & Envi- and replication numbers in pesticide applications in Adana ronment 1, 229-234. province, Turkey. Fresenius Environmental Bulletin, 3, 275- [28] Gao, B.B.; Tao, C.J.; Ye, J.M.; Ning, J.; Mei, X.D.; Jiang, 281. Z.F.; Chen, S. and She, D.M. (2014) Measurement of opera- [12] Yarpuz-Bozdogan, N. and Bozdogan, A. M. (2009) Compari- tor exposure to chlorpyrifos. Pest. Manag. Sci. 70, 636-641. son of field and model percentage drift using different types [29] Remoundou, K., Brennan, M., Sacchettini, G., Panzone, L., of hydraulic nozzles in pesticide applications. Int. J. Environ. Butler-Ellis, M. C., Capri, E., Charistou, A., Chaideftou, E., Sci. Tech. 2, 191-196. Gerritsen-Ebben, M. G., Machera, K., Spanoghe, P., Glass, R., March,s, A., Doanngoc, K., Hart, A. and Frewer, L. J. (2015) [13] Yarpuz-Bozdogan, N. and Bozdogan, A.M. (2009) Assess- Perceptions of pesticide exposure risks by operators, workers, ment of different types of nozzles on dermal bystander expo- residents and bystanders in Greece, Italy and the UK. Science sure in pesticide applications. International Journal of Food, of the Total Environment 505, 1082-1092. Agriculture & Environment 7, 678-682. [30] Yassin, M.M.; Abu Mourad, T. A. and Safi, J.M. (2002) [14] Yarpuz-Bozdogan, N. (2011) Drift of Pesticide. Editor David Knowledge, Attitude, Practice, and Toxicity Symptomps As- Pimentel Encyclopedia of Pest Management, Taylor and Fran- sociated with Pesticide Use Among Farm Workers in the Gaza cis, pp:1-4. Strip. Occup Environ Med 59, 387-394. [15] Garreyn,F.; Vagenende,B. and Steurbaut,W. (2003) “Occupa- [31] MacFarlane E., Carey, R., Keegel, T., El-Zaemay, S. and tional” Indicators - Operator, Worker and Bystander. Harmo- Fritschi, L., (2013) Dermal exposure associated with occupa- nised Environmental Indicators for Pesticide Risk, HAIR. 6th tional end use of pesticides and the role of protective measures. Framework Programme. SSPE-CT-2003-501997, 173 p. Safety and Health at Work 4, 136-141. [16] Claeys, S.; Vagenende, B.; De Smet, B.; Lelieur, L. and [32] Nuyttens, D., Braekman, P., Windey, S. and Sonck, B., (2009) Steurbaut, W. (2005) The POCER indicator: a decision tool Potential dermal pesticide exposure affected by greenhouse for non-agricultural pesticide use. Pest Management Science spray application technique. Pest Manag Sci 63, 781-790. 61, 779–786.

[17] De Schamphelerie, M.; Spanoghe, P.; Brusselman, E. and Sonck, S. (2007) Risk assessment of pesticide spray drift damage in Belgium. Crop Protection 26, 602-611. Received: December 10, 2014 Revised: February 24, 2015 [18] Demircan, V. and Yılmaz, H. (2005) The Analysis of Pesticide Accepted: March 06, 2015 Use in Apple Production in Isparta Province in terms of Econ- omy and Environmental Sensitivity Perspective. Ecoloji 14, 57: 15-25. CORRESPONDING AUTHOR [19] Baldi, I., Lebailly, P., Bouver, G., Rondeau, V., Kientz-Bou- chart, V., Canal-Raffin, M. and Garrigou, A. (2014) Levels and determinants of pesticide exposure in re-entry workers in Assoc. Prof. Dr. Ali Musa BOZDOGAN vineyards: Results of the PESTEXPO study. Environmental Cukurova University Research 132, 360-369. Agriculture Faculty, Agricultural Machinery and [20] Yalap Tuna, R. (2011) Knowledge, attitude and practice of Technologies Engineering Dept. farmers about pesticides storage conditions, safe using man- 01330 Adana ners. PhD Thesis, Erciyes University, Graduate School of TURKEY Health Sciences, Department of Public Health. (In Turkish) [21] Chemicalbook.com. (2014) Online: http://www.chemical- Phone: +90 322 338 6408 book.com. (accessed on 20/11/2014). Fax: +90 322 338 7165 [22] Vercruysse,F. and Steurbaut,W. (2002) POCER, the pesticide E-mail: [email protected] occupational and environmental risk indicator. Crop Protec- tion 21, 307-315. FEB/ Vol 24/ No 7/ 2015 – pages 2275 – 2279

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SYNTHESIS AND CHARACTERIZATION OF ZnS NANOCRYSTALS AND ITS PHOTOCATALYTIC ACTIVITY ON ANTIBIOTICS

Yuting Wu1, Liang Ni1,*, Xinlin Liu2, Mingjun Zhou1, Zhi Zhu1, Ziyang Lu3, Changchang Ma3 and Pengwei Huo1

1School of Chemistry & Chemical Engineering, Jiangsu University, Zhenjiang 212013, PR China 2School of Energy & Power Engineering, Jiangsu University, Zhenjiang 212013, PR China 3School of the Environment & Safety Engineering, Jiangsu University, Zhenjiang 212013, PR China

ABSTRACT ever, these types of nanocrystals are made from heavy metal ions (e.g., Cd(2+)), which may result in potential bio- ZnS nanocrystals were synthesized via hydrothermal logical and environmental toxicity, and this drawback seri- process and characterized by X-ray diffraction (XRD), ously hampers their practical applications [10]. ZnS nano- Transmission electron microscope (TEM), Ultraviolet–vis- crystals are one of the most important II–VI semiconductor ible spectroscopy (UV-vis) and Fourier transform infrared materials which attracted tremendous attention because of spectrometer (FT-IR). The results indicated that the ob- their outstanding properties: wide band gap energy [11], tained ZnS nanocrystals are cubic sphalerite with little par- low level of harm, stability, moderate price and easily ac- tical size and good dispersity. Under UV light irradiation cessibility. Moreover, its quantum size effect also shows and controlling the experimental conditions, the photocata- many specific photoelectric properties [12, 13]. lytic activity of ZnS nanocrystals was evaluated by degra- Techniques for preparing ZnS nanocrystals are various, dation of tetracycline (TC). Various factors have been com- such as precipitation method [14], electrodeposition method pared through a series of experiments, and the optimal reac- [15], ultrasonic-assisted method [16], microwave-assisted tion condition has been found out. Additionally, the photo- method [17] and hydrothermal method [18]. Among these catalytic activity of recycled ZnS decreased with the in- methods, hydrothermal method has the advantages of sim- creasing runs of recycling. Finally, the photocatalytic reac- ple operation, high production purity, and obtained materi- tion mechanism of ZnS nanocrystals was proposed and the als possess small crystal size, narrow size distribution, photocatalytic degradation pathway of TC was analysed. good crystallinity and high photoluminescence intensity Overall, the ZnS photocatalysis was found to be a promis- [12, 18, 19]. Recently, more and more different nanocrys- ing process for removing TC from wastewater. tals have been prepared via hydrothermal method [20-24].

Antibiotics, also known as antibacterials, are types of KEYWORDS: ZnS nanocrystals; synthesis and characterization; medications that are used for treating infections caused by tetracycline; photocatalytic degradation bacteria [25]. However, not only the extensive use of antibiotics has led to worldwide environmental pollution, but

also the development of multi-resistant bacterial strains can 1. INTRODUCTION no longer be treated by the presently known drugs [26]. Tet- racycline (TC), in virtue of its moderate price, represents a Semiconductor nanocrystals are usually composed of major proportion of the antibiotics in use currently [27], par- group II–VI or III–V elements, also termed as quantum ticularly in the field of medicines, aquaculture and veteri- dots (QDs) [1]. Because they often consist of a little number nary medicines [28]. Recently, due to its poor absorption of atoms, their particle size is very small (less than 100 nm by organisms, a significant amount of TC has been detected in three dimensions, an average of about 2~10nm). The in natural environment, causing growing concerns about its characteristic optical properties of nanocrystals include in- potential impact to the ecosystem [29]. tense fluorescence, broad excitation, narrow emission and In the present work, ZnS nanocrystals were prepared resistance to photobleaching [2]. In the past several dec- via hydrothermal synthesis method and characterized by ades, nanocrystals have received substantial attention due X-ray diffraction (XRD), Transmission electron micro- to their outstanding optical properties and biological appli- scope (TEM), Ultraviolet–visible spectroscopy (UV-vis) cations [3]. More recently, there have been intense con- and Fourier transform infrared spectrometer (FT-IR). The cerns on CdE (E = S, Se, Te) nanocrystals [4-9]. How- photodegradative effect of TC under UV light irradiation was investigated by using ZnS as the photocatalyst, and the * Corresponding author effects of solution pH, TC concentration and ZnS dosage

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were evaluated to determine the optimal reaction condi- pH adjusted by NaOH was determined by a microprocessor tion. After that, the activity of the recycled catalyst was as- pH meter (Leichi Instruments, Shanghai, China), and pho- sessed. And finally, the photocatalytic reaction mechanism todegradative experiments were carried out in a GHX-2 of ZnS nanocrystals was proposed and the photodegrada- photochemical reactor with a 500 W ultraviolet lamp. To tive process of TC was analysed. determine the initial absorbency, all samples were stirred In summary, we have found a much more simple and for 30 min in dark to ensure the adsorption equilibrium. low-cost process to synthesize ZnS nanocrystals. More im- Then the ultraviolet lamp was turned on, and we main- portantly, the as-prepared ZnS exhibited higher photodeg- tained the reactive temperature below 298 K by a flow of radative rate to TC, and showed promising prospect for the cooling water. The whole photodegradative process con- treatment of TC in future industrial application. tinued for 1 h and was conducted in 10 min interval. After that, all samples were centrifuged at 1000 rpm for 3 min and their supernatant’s UV–vis adsorption spectrum was 2. MATERIALS AND METHODS recorded by an UV–vis spectrophotometer. The adsorption detections were performed at 356nm (tetracycline), 356nm 2.1 Materials (oxytetracycline hydrochloride), 275nm (ciprofloxacin) and 190nm (danofloxacin mesylate). The degradation rate The materials used included Zn(CH COO) ·2H O and 3 2 2 was expressed as DR=(A -A)/A , where A and A are the Na S·9H O. They were purchased from Sinopharm Chem- 0 0 0 2 2 absorbance of antibiotics before and after photodegrada- ical Rengent Co. Ltd. Tetracycline, oxytetracycline hydro- tive reaction. chloride, ciprofloxacin and danofloxacin mesylate were obtained from Shanghai Shunbo Biological Engineering

Co. Ltd.. All chemicals were used as received without any 3. RESULTS AND DISCUSSION further purification and doubly deionized water was used throughout the experiments. 3.1 ZnS structural characterization

2.2 Preparation of ZnS nanocatalysts X-ray powder diffraction pattern of the synthesized ZnS nanocrystals is illustrated in Fig.1 (A). All peaks can be well Weighed 2.6341g Zn(CH COO) •2H O (0.012mol) and 3 2 2 indexed to the cubic sphalerite phase of ZnS (JCPDS No. 2.8800g Na S•9H O (0.012mol) were added separately to 2 2 05-0566) [30-32]. There are three characteristic diffraction 40 mL deionized water in the beakers. These two solutions peaks at 2θ = 28.59°, 47.78°, and 56.74°, corresponding to were mixed and stirred for 30min with continuous nitrogen the (111), (220) and (311) crystal plane of pure ZnS, re- bubbling at room temperature. Then, the mixtures were spectively. transferred to a 100 mL Teflon-lined stainless steel autoclave, followed by putting them in an electroheat blasting To further characterize ZnS nanostructures, TEM anal- baking oven with 150 °C for 10 h. After that, the products ysis was carried out. The typical TEM image of ZnS nano- were cooled to ambient temperature naturally, and the solu- crystals is shown in Fig. 1(B) and the inset figure repre- tions were centrifuged at 1000 rpm for 3 min to harvest ZnS sents its corresponding particle size distribution. Fig. 1(B) precipitate. Then the precipitates were washed with deion- exhibits that the obtained ZnS was approximately spherical ized water and ethanol several times and dried in a vacuum and homogeneous. Some of the particles were agglomer- evaporator at 60 ◦C for 48 h to obtain ZnS nanocrystals. ates. From the particle size distribution statistics, it can be seen that ZnS size distribution was in the range of about 2.3 ZnS structural Characterization 2.5–32.5 nm, and their mean diameter was 14.55 nm with The XRD pattern was obtained by a MO3XHF22 X-ray a standard deviation of 5.37. diffractometer (MAC Science, Japan) equipped with Ni-fil- UV–vis absorption spectrum is presented in Fig. 1(C). trated Cu Kα radiation (40 kV, 30 mA). The 2θ scanning an- It is similar to the image described in previous articles [33, gle range was 10–80◦ at a scanning rate of 10◦ min−1. The 34]. It is obvious that the absorption spectrum was very morphology and size of ZnS photocatalyst was studied by broad and featureless in the whole region. The band gap of TEM (JEOL IEM-200CX) microscope. UV–vis absorption materials can be estimated via the formula: Eg = 1240/λ, spectrum was obtained by dry-pressed disk samples using which Eg represents band gap energy and λ is the wave- Specord 2450 spectrometer (Shimazu, Japan), and adopted length of the absorption edge [35]. As the spectrum shows, BaSO4 was used as the reflectance sample. Fourier trans- the absorption edge wavelength of pure ZnS nanoparticles form infrared (FT-IR) spectrum was recorded by a Nicolet was about 380 nm, corresponding to a bandgap of 3.26 eV. Nexus 470 FT-IR (America thermo-electricity Company) At last, ZnS FT-IR spectrum is presented in Fig. 1(D). −1 −1 with 2 cm resolution in the range of 500–4000 cm , pellet As can be seen from the Fig. 1(D), peaks at 3420 and 1090 was prepared by mixing nanoparticles with KBr powder. cm-1 are corresponding to the O-H bond vibrations adsorbed

of -OH groups, which belong to the adsorbent H2O on the 2.4 Antibiotics photodegradative experiment ZnS surface [36]. This phenomenon indicates that Zn2+ ions 150 mL antibiotics solution was injected to quartz re- have strong bonding effect with H2O molecular on ZnS sur- actor (total volume of 200 mL) by a 100mL syringe. The face. The weak peaks located at 2900 and 2970 cm−1 are

2281 © by PSP Volume 24 – No 7. 2015 Fresenius Environmental Bulletin

A 1600 1400 1200 1000

800 600 Intensity/a.u. 400 200 0 10 20 30 40 50 60 70 80

2/ O

C D 1.4 100 1.2 90 1.0 0.8 80

0.6 70 0.4 Absorbance/a.u.

Transmitance/a.u. 60 0.2 0.0 50 200 300 400 500 600 700 800 500 1000 1500 2000 2500 3000 3500 4000 Wavelength/nm Wavelength/cm-1

FIGURE 1 - ZnS structural characterization: (A) XRD pattern, (B) TEM image, (C) UV–vis absorption spectrum, (D) FT-IR spectrum.

assigned to symmetric and asymmetric C–H stretches. pH value from 7.0 to 11.0. As can be seen in Fig. 2(B), the C–H bonds are present in monoacetate groups as inter- pH increased from 7.0 to 10.0, the TC degradation rate in- mediate products [37]. The absorption peaks at 1570 and creased from 59.66% to 89.42%. When the pH increased 1410 cm-1 represents the C=O symmetric and asymmetric from 10.0 to 11.0, the TC degradative efficiency decreased stretching vibration of the carboxylate ions (–COO−) in from 89.42% to 85.28%. -1 Zn(CH3COO)2•2H2O [38]. Moreover, 665 cm peak is The results can be explained by considering the nano- the ZnS characteristic absorption and is assigned to Zn–S crystals surface charge properties. The photodegradative stretching vibration. reaction is mainly due to the hydroxyl radical (•OH) attack

3.2 Photodegradative experiment to TC molecules. But with a low pH it is difficult to provide 3.2.1 Photodegradative performance of various antibiotics enough •OH species and they are more readily generated Fig. 2(A) illustrates the photodegradative efficiency of in higher pH condition [39]. However, the degradation rate various antibiotics. The experiments were performed on the of TC is inhibited when pH is high (> 10.0), because the initial conditions of pH=10.00, c(antibiotics)=10mg/L and •OH species will compete with TC molecules in adsorbing c(ZnS)= 400mg/L. As the investigation shows, TC and ox- on the photocatalyst surface, which may explain the varia- ytetracycline hydrochloride had higher degradation rates, tion tendency of this experiment. while ciprofloxacin and erythromycin exhibited inferior performance in the experiments. In order to make the re- 3.2.3 Effect of initial TC concentration sults more representative, we chose TC as degradative ob- The optimal pH condition has been found from the former ject in the experiments. step, but initial TC concentration is also a very important parameter in this treatment. Here we kept pH=10.0 and 3.2.2 Effects of initial solvent pH value c(ZnS)=400mg/L, changed TC concentration in the range Solvent pH is considered an important factor of antibi- of 5-20mg/L to study its influence on photodegradative ef- otics photodegradation. To study its effect, we maintained ficiency. As can be seen in Fig. 2(C), TC degradation rate c(TC)=10mg/L and c(ZnS)=400mg/L and varied the initial was increasing from 5mg/L to 10mg/L but was dropping

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from 10mg/L to 20mg/L. The result indicates that under low However, the increased of TC concentration also decreased concentration level, TC molecules on ZnS surface had not the path length of photon entering into the solution. At high reached the adsorption equilibrium, may be that ZnS pholo- concentration level, a significant amount of UV-light may catalyst mainly exhibited adsorption at low TC concentra- be absorbed by TC molecules rather than ZnS photocatalyst tion which resulted in low degradation rate. When the solu- and this may also reduce the catalytic efficiency. tion concentration increased, more TC molecules were adsorbed on ZnS surface and affected to the degradative effect.

A 1.0 B 1.0 Tetracycline pH=7.0 Oxytetracycline hydrochloride

Ciprofloxacin pH=8.0 0.8 Danofloxacin mesylate 0.8 pH=9.0 pH=10.0 pH=11.0 0.6 0.6

0.4 0.4 DR/100% DR/100% 0.2 0.2

0.0 0.0 0 102030405060 0 102030405060 Time/min Time/min

C 1.0 D 1.0 5mg/L 100mg/L 10mg/L 400mg/L 0.8 15mg/L 0.8 700mg/L 20mg/L 1000/L 1300mg/L 0.6 0.6

0.4 0.4 DR/100% 0.2 DR/100% 0.2 0.0 0 102030405060 0.0 0 102030405060 Time/min Time/min

E 1.0 Fresh ZnS Primary recycled ZnS 0.8 Secondary recycled ZnS

0.6

0.4 DR/100% 0.2

0.0 0 102030405060 Time/min

FIGURE 2 - Photodegradative experiment: (A) Photodegradative performance of various antibiotics, (B) Effect of initial pH value, (C) Effect of TC concentration, (D) Effect of ZnS dosage, (E) Activity of fresh and recycled ZnS.

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3.2.4 Effect of ZnS pholocatalyst dosage 3.3. Photodegradation mechanisms analysis For economic removal of TC effluent from wastewater, 3.3.1 Photocatalytic reaction mechanism of ZnS nanocrystals it is necessary to find out the optimal amount of photocata- ZnS nanocrystals were irradiated by the light whose lyst. In this section, we first ensured the initial pH=10.0, photon energy was larger or equal to its bandgap energy, and ZnS dosage was varied from 100 to 1300mg/L in the the electrons in valence band can be excited into conduc- 10mg/L TC solution. As presented in Fig. 2(D), with in- tion band, leading to the formation of positively charged creased ZnS amont from 100 to 400mg/L, TC degradation photogenerated holes(h+) in valence band, and negatively rate improved from 80.24% to 89.42%. However, while ZnS charged photogenerated electron (e-) in conduction band: amount exceeded 400mg/L, TC degradation rate dropped ZnS+hv→ZnS (e-,h+) (1) from 89.42% to 55.25%. As can be seen from the result, at low ZnS concentration, the number of available adsorption At this moment, photogenerated holes and electrons and catalytic sites increased with increasing ZnS dosage. have two evolve possibilities. In one case, the absorbed After optimal level, high turbidity of solution system and light energy will release as heat or photons and emitting penetration of UV light decreased thereby enhanced scat- fluorescence, all these situations can not use the absorbed tering effect and lowed the photodegradation rate. light energy. In another case, the strong oxidizing photogenerated holes will react with H2O moleculars to generate 3.2.5 Activity of recycled ZnS hydroxyl radicals(•OH), and the strong reducing photogenerated electrons will react with dissolved oxygen to pro- In practice, in order to reduce the cost of treatment and − duce oxyradicals(•O2 ). Eventually, the oxyradicals will maintaining the continuity of production, people must assess also translate into hydroxyl radicals. These reactions will the stability of photocatalyst. In the present study, we pre- separate photogenerated holes and electrons and transfer served the reaction conditions of pH = 10.00, absorbed light energy into chemical energy. The possible c(TC)=10mg/L and c(ZnS)= 400mg/L, repeated the exper- reactions are listed as follows: iment and recycled the ZnS. As the results of Fig. 2(E) sug- + + gested, photocatalytic activity of primary recycled ZnS has h + H2O→•OH + H (2) decreased to a certain extent. Furthermore, the efficiency h+ +OH−→•OH (3) of secondary recycled ZnS was unsatisfactory. The deacti- e−+O →•O − (4) vation of photocatalyst is caused by the degraded substance 2 2 − + and other impurities has been adsorbed on ZnS surface and •O2 +H →HO2• (5) also affected to its adsorption properties. 2HO2•→O2+H2O2 (6)

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FIGURE 3 - TC photodegradative process mass spectrum: (A) At the begining, (B) 30 min after UV light irradiation, (C) 60 min after UV light irradiation.

2285 © by PSP Volume 24 – No 7. 2015 Fresenius Environmental Bulletin

OH O OH O OH OH

NH2

OH HO CH3 m/z=445.37 N(CH3)2

·OH

OH OH OH O OH OH OH OH OH OH OH NH2 NH2 + OH + OH HO CH N(CH3)2 3 m/z=120.84 N(CH3)2 m/z=318.31 m/z=162.79 m/z=274.27

-C2H5OH ·OH -CH3OH ·O H OH -H2O OH OH NH OH OH 2 + OH O OH OH m/z=162.79 m/z=96.84 m/z=148.83N(CH3)2

O m/z=246.24 OH

-C2H5OH NH2 ·OH -H O OH 2 N(CH ) m/z=162.79 3 2 OH

NH2 m/z=186.80 ·OH m/z=130.92 N(CH3)2 OH -CH3OH ·OH

NH2 m/z=120.84

m/z=112.17 -C2H5OH ·O H NHCH3

-CH3OH ·O H OH

NH2 m/z=96.84 m/z=98.93 NH2

FIGURE 4 - Photodegradative pathway of TC

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− − H2O2+•O2 →•OH + OH + O2 (7) REFERENCES − + − •O2 +H2O + H → H2O2 +OH (8) [1] Saran, A.D. and Bellare, J.R. (2010) Green engineering for − − H2O2+e →•OH+ OH (9) large-scale synthesis of water-soluble and bio-taggable CdSe and CdSe–CdS quantum dots from microemulsion by double- 3.3.2 Photodegradative process of TC capping. Colloids and Surfaces A: Physicochemical and Engi- neering Aspects 369, 165–175. In order to ensure the TC degradative pathways, the in- [2] Weilnau, J.N., Black, S.E., Chehata, V.J., Schmidt, M.P., Holt, termediates and final products were determined by mass K.L., Carl, L.M., Straka, C.J., Marsh, A.L., Patton, W.A. and spectrometry, the results taken at the beginning, 30 and 60 Lappas, C.M. (2013) ZnS nanocrystal cytotoxicity is influenced min after UV light irradiation are shown in Fig. 3(A)~(C). by capping agent chemical structure and duration of time in sus- Through the analysis, in the early stage of photodegradative pension. Journal of Applied Toxicology 33, 227–237. process, by the attacked of hydroxyl radicals, carbon–carbon [3] Yin, N.Q., Liu, L., Lei, J.M., Liu, Y.S., Gong, M.G., Wu, Y.Z., single bonds at A and C ring of TC were first broken, then Zhu, L.X. and Xu X.L. (2012) Preparation and characteriza- the m/z=274.27 and m/z=162.79 or m/z=318.31 and m/z= tion of nontoxic magnetic-luminescent nanoprobe. Chinese 120.84 substance were formed. With the increased of deg- Physics B 21, 116101. radation time, these intermediates were further splintering [4] Akshya, S., Hariharan, P.S., Vinod Kumar, V. and Anthony into small fragments. Eventually, TC was degraded thor- S.P. (2015) Surface functionalized fluorescent CdS QDs: Se- lective fluorescence switching and quenching by Cu2+ and oughly and the remains were H2O, methanol, ethanol, phe- Hg2+ at wide pH range. Spectrochimica Acta Part A: Molecu- nol, amine and some other small molecules. This process lar and Biomolecular Spectroscopy 135, 335–341. is presented Fig.4. [5] Maji, S.K., Dutta, A.K., Dutta, S., Srivastava, D.N., Paul P., Mondal, A. and Adhikary, B. (2012) Single-source precursor approach for the preparation of CdS nanoparticles and their 4. CONCLUSIONS photocatalytic and intrinsic peroxidase like activity. Applied Catalysis B: Environmental 126, 265-274.

In this research, ZnS nanocrystals was prepared by hy- [6] Kaur, G. and Tripathi S.K. (2015) Investigation of trypsin– drothermal method. The obtained ZnS was cubic sphalerite CdSe quantum dot interactions via spectroscopic methods and phase crystal, and its shape was approximately spherical. effects on enzymatic activity. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 134, 173–183. Then the optimal photodegradative conditions was discussed, while pH = 10.00, c(TC)=10mg/L and c(ZnS)= [7] Ullah, K., Jo, S.B. and Ye, S. (2015) Degradation of Organic 400mg/L, ZnS nanocrystals exhibited highest photocata- Dyes by CdSe Decorated Graphene Nanocomposite in Dark Ambiance. Fullerenes, Nanotubesa and Carbon Nanostruc- lytic activity, nearly 90% of TC was removed within 60 tures 23, 437-448. min. According to the photocatalyst recycled experiment, the photocatalytic activity of recycled ZnS was obviously [8] Song, H.L., Yang, M., Fan, X.X. and Wang, H.Y. (2014) Turn- on electrochemiluminescence sensing of Cd2+ based on CdTe declined. At last, the photocatalytic reaction mechanism of quantum dots. Spectrochimica Acta Part A: Molecular and Bi- ZnS nanocrystals for TC was discussed, the degradative omolecular Spectroscopy 133, 130–133. process of TC was analysed and its pathways were pro- [9] Mobedi, N., Marandi, M. and Zare Bidaki, H. (2014) Effect of posed. By the attacked of hydroxyl radicals, TC was de- hydrazine hydrate on the luminescence properties of MPA composed to H2O, methanol, ethanol, phenol, amine and capped CdTe nanocrystals in hot injection method. Journal of some other small molecules. Generally, this research im- Luminescence156, 235–239. plied a promising application of photodegrading TC in [10] Chen, N., He, Y., Su, Y.Y., Li, X.M., Huang, Q., Wang, H.F., wastewater treatment. Zhang, X.Z., Tai, R.Z. and Fan, C.H. (2012) The cytotoxicity of cadmium-based quantum dots. Biomaterials 33, 1238-1244. [11] Pal, S., Sharma, R., Goswami, B. and Sarkar, P. (2009) Theo- retical prediction of ring structures for ZnS quantum dots. ACKNOWLEDGMENTS Chemical Physics Letters 467, 365–368. [12] Liu, C.X., Ji, Y.Y. and Tan T.W. (2013) One-pot hydrothermal We gratefully acknowledge the financial support synthesis of water-dispersible ZnS quantum dots modified of the National Natural Science Foundation of China with mercaptoacetic acid. Journal of Alloys and Compounds 570, 23–27. (No. 21207053, 21407064), the Natural Science Founda- tion of Jiangsu Province (BK20131259, BK20130489, [13] Shahid, R., Toprak M.S. and Muhammed, M. (2012) Micro- BK20140532). wave-assisted low temperature synthesis of wurtzite ZnS quantum dots. Journal of Solid State Chemistry 187, 130–133.

The authors have declared no conflict of interest. [14] Kho, R., Torres-Mart´ınez, C.L. and Mehra, R.K. (2000) A Simple Colloidal Synthesis for Gram-Quantity Production of Water-Soluble ZnS Nanocrystal Powders. Journal of Colloid and Interface Science 227, 561–566.

[15] Monika, Kumar, R. and Chauhan, R. P. (2015) Preparation and field emission study of low-dimensional ZnS arrays and tu- bules. Journal of Experimental Nanoscience 10, 126-134.

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[16] Behboudnia, M., Habibi-Yangjeh, A. and Jafari-Tarzanag, Y. [31] Wang, L.P. and Hong, G.Y. (2000) A new preparation of zinc (2008) Preparation and characterization of monodispersed sulfide nanoparticles by solid-state method at low temperature. nanocrystalline ZnS in water-rich [EMIM]EtSO4 ionic liquid Materials Research Bulletin 35, 695–701. using ultrasonic irradiation. Journal of Crystal Growth 310, 4544-4548. [32] Wang, L., Tao, X.T., Yang, J.X., Ren, Y., Liu, Z. and Jiang, M.H. (2006) Preparation and characterization of the ZnS nan- [17] La Porta, F.A., Ferrer, M.M., Santana, Y.V.B., Raubach, ospheres with narrow size distribution. Optical Materials 28, C.W., Longo, V.M., Sambrano, J.R., Longo, E., Andrés, J., Li, 1080–1083. M. and Varela, J.A. (2013) Synthesis of wurtzite ZnS nanopar- [33] Shi, Y.H., Chen, J. and Shen, P.W. (2007) ZnS micro-spheres ticles using the microwave assisted solvothermal method. and flowers: Chemically controlled synthesis and template use Journal of Alloys and Compounds 556, 153–159. in fabricating MS(shell)/ZnS(core) and MS(M = Pb, Cu) hol- [18] Nirmala Jothi, N.S., Joshi, A.G., Jerald Vijay, R., Muthuvina- low microspheres. Journal of Alloys and Compounds 441, yagam, A. and Sagayaraj, P. (2013) Investigation on one-pot hy- 337–343. drothermal synthesis, structural and optical properties of ZnS [34] Maryam, S.A. and Masoud, S.N. (2014) Synthesis and charac- quantum dots. Materials Chemistry and Physics 138, 186-191. terization of wurtzite ZnS nanoplates through simple solvothermal method with a novel approach. Journal of Industrial [19] Liu, L.Q., Li, Y.F., Zhan, L., Liu, Y. and Huang, C.Z. (2011) and Engineering Chemistry 20, 3179–3185. One-step synthesis of fluorescent hydroxyls-coated carbon dots with hydrothermal reaction and its application to optical sensing [35] Xing, W.N., Ni, L., Huo, P.W., Lu, Z.Y., Liu, X.L., Luo, Y.Y. of metal ions. Science China Chemistry 54, 1342-1347. and Yan, Y.S. (2012) Preparation high photocatalytic activity of CdS/halloysite nanotubes (HNTs) nanocomposites with hy- [20] Hernández-Adame.L., Méndez-Blas, A., Ruiz-García, J., drothermal method. Applied Surface Science 259, 698– 704. Vega-Acosta, J.R., Medellín-Rodríguez, F.J. and Palestino, G. (2014) Synthesis, characterization, and photoluminescence [36] Maged, E.K. and Hany, E.S. (2009) Fluorescence modulation properties of Gd:Tb oxysulfide colloidal particles. Chemical and photodegradation characteristics of safranin O dye in the Engineering Journal 258, 136–145. presence of ZnS nanoparticles. Journal of Photochemistry and Photobiology A: Chemistry 205, 151–155. [21] Cao, C.Y., Luo, Z.Y., Guo, S.L., Cao, R.P., Noh, H.M., Jeong, J.H. and Xie, A. (2014) Synthesis, optical properties, and en- [37] Perez, R.C., Sandoval, O.J. , Mann, J.M. , Galvan, A.M. and 3+ 3+ ergy transfer of Ce /Tb co-doped MyGdFx (M = Li, Na, K). Delgado, G.T. (1999) Influence of annealing temperature on Spectrochimica Acta Part A: Molecular and Biomolecular the formation and characteristics of sol-gel prepared ZnO Spectroscopy 133, 457–462. films. Journal of Vacuum Science and Technology A: Vac- uum, Surfaces and Films17, 1811–1816. [22] Xu, C.J., Wang, Y.J., Chen, H.Y., Nie, D. and Liu Y.Q. (2014) Hydrothermal synthesis of silver crystals via a sodium chlo- [38] Zhao, J.J., Zhao, L. and Wang, X.P. (2008) Preparation and ride assisted route. Materials Letters 136, 175–178. characterization of ZnO/ZnS hybrid photocatalysts via microwave-hydrothermal method. Frontiers of Environmental Sci- [23] Sakoda, K. and Hirano, M. (2014) Formation of complete solid ence & Engineering in China 2, 415–420. solutions, Zn(Al Ga ) O spinel nanocrystals via hydrother- x 1-x 2 4 [39] Chow, K.L., Man, Y.B., Zheng, J.S., Liang, Y., Tam, F.Y. and mal route. Ceramics International 40, 15841–15848. Wong, M.H. (2012) Characterizing the optimal operation of [24] Zhu, X.H., Zuo, X.X., Hu, R.P., Xiao, X., Liang, Y. and Nan photocatalytic degradation of BDE-209 by nano-sized J.M. (2014) Hydrothermal synthesis of two photoluminescent TiO2.Journal of Environmental Sciences 24(9), 1670–1678. nitrogen-doped graphene quantum dots emitted green and khaki luminescence. Materials Chemistry and Physics 147, 963-967. [25] Karpecki, P., Paterno, M.R. and Comstock, T.L. (2010) Limi- tations of current antibiotics for the treatment of bacterial con- junctivitis.Optometry and Vision Science 87, 908-919.

[26] Zhu, X.D., Wang, Y.J., Sun, R.J. and Zhou, D.M. (2013) Pho- tocatalytic degradation of tetracycline in aqueous solution by Received: August 31, 2014 nanosized TiO .Chemosphere 92, 925–932. 2 Revised: January 12, 2015 [27] Maroga Mboula, V., Héquet, V., Gru, Y., Colin, R. and An- Accepted: February 13, 2015 drès, Y. (2012) Assessment of the efficiency of photocatalysis on tetracycline biodegradation. Journal of Hazardous Materi- als 209– 210, 355– 364. CORRESPONDING AUTHOR [28] Niu, J.F., Ding, S.Y., Zhang, L.W., Zhao, J.B. and Feng, C.H. (2013) Visible-light-mediated Sr-Bi2O3 photocatalysis of tet- Liang Ni racycline: Kinetics, mechanisms and toxicity assessment. School of Chemistry & Chemical Engineering Chemosphere 93, 1–8. Jiangsu University [29] Fatiha, F.S., Florence, F., Isabelle, S., Hamid, A.A., Hayet, D Zhenjiang 212013 and Abdeltif, A. (2013) Tetracycline degradation and mineral- P.R. CHINA ization by the coupling of an electro-Fenton pretreatment and a biological process. Journal of Chemical Technology and Bi- otechnology 88, 1380-1386. Phone: +86 511 8878 2621 Fax: +86 511 8879 1800 [30] Huang, Q.S., Dong, D.Q., Xu, J.P., Zhang, X.S., Zhang, H.M. and Li, L. (2010) White Emitting ZnS Nanocrystals: Synthesis E-mail: [email protected], [email protected] and Spectrum Characterization. Chinese Physics Letters 27, 057306. FEB/ Vol 24/ No 7/ 2015 – pages 2280 - 2288

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EVALUATION OF PARTICLE SIZE AND INITIAL CONCENTRATION OF TOTAL SOLIDS ON

BIOHYDROGEN PRODUCTION FROM FOOD WASTE

Iván Moreno-Andrade* and Germán Buitrón

Laboratory for Research on Advanced Processes for Water Treatment, Unidad Académica Juriquilla, Instituto de Ingeniería, Universidad Nacional Autónoma de México, Blvd. Juriquilla 3001, 76230 Querétaro, México.

ABSTRACT The H2 production yields are closely related to the fermentation pathway and end-products [1,2]. The main biochem- The influence of different parameters including parti- ical reactions for H2 production are related to the organic cle size and initial total solids on the biohydrogen produc- matter transformation into acetate and butyrate. When ace- tion from food waste was studied. The results demonstrated tic acid is the end-product of the reaction, a theoretical that the initial particle size and the concentration of initial maximum of 4 moles of hydrogen per mole of glucose can total solids (TS0) have a positive effect on hydrogen (H2) be produced (Eq. 1): production. Results showed that if no pH adjustment C H O  2H O  2CH COOH  2CO  4H (1) (buffer) or particle size selection is applied, the maximal 6 12 6 2 3 2 2 -1 volumetric hydrogen production (2.2 LH2 kg of the initial and when butyrate is the end-product, a theoretical -1 total solids added) was obtained at 90 gTS0 L . However, maximum of 2 moles of hydrogen per mole of glucose is the maximal total solids and chemical oxygen demand reproduced (Eq. 2): -1 moval percentages were obtained at 10 gTS0 L . For particles larger than 2 mm, the H2 production increases as the C6 H 12O6  CH 3CH 2CH 2COOH  2CO2  2H 2 (2) TS0 increase. In the case of particle sizes between 0.5 and If propionic acid is produced during the dark fermen- 2.0 mm, and smaller than 0.5 mm, the higher H2 production -1 -1 tation, there exist a consumption of H2 (Eq. 3), thus, reduc- was reached at 50 gTS0 L and 15 gTS0 L , respectively. Larger particles size required more time to produce the ing the H2 yields. same amount of hydrogen. The maximum hydrogen pro- C H O  2H  2CH CH COOH  2H O (3) duction rate per initial total solids was obtained with the 6 12 6 2 3 2 2 -1 smallest particle size by applying 15 gTS0 L . The princi- From the above reactions, it is clear that if the process pal volatile fatty acids generated were acetic and butyric is mainly oriented to acetic and butyric acid production acids. The values of acetic acid were similar when TS0 ap- pathway, the H2 production can be maximized. H2 produc- plied between 5 to 20 g L-1 for each different particle size. tion can be achieved in a cost-effective way by using a -1 Higher concentration of TS0 than 30 g L results in a fast wide variety of non-expensive residues such as the organic increase of acetic acid production. solid waste (OSW) as substrate [2-4].

Different initial parameters affect the hydrogen production including the feedstock source, initial pH and ini- KEYWORDS: biohydrogen; initial total solids; organic solid waste; tial total solid concentration [5-7]. In the case of methane, particle size; restaurant waste. it has been reported that particle size influences the production rate and yield [8-10]. Izumi et al. [8] showed that as result of a pre-treatment (particle size from 0.84 to 0.39 mm), 1. INTRODUCTION the total chemical oxygen demand (COD) was increased, improving the methane yield by 28%. However, it was ob- The application of the hydrolytic-acidogenic stage of served that an excessive reduction of the particle size of the the anaerobic digestion process is an appropriate alterna- substrate resulted in VFA accumulation, decreasing the me- tive to produce hydrogen (H2) and to obtain an effluent rich thane production. The reduction of the particle size gener- in volatile fatty acids, (VFA), mainly acetic, propionic and ates an increase of the surface area available to the micro- butyric acid, lactate and solvents (acetone and ethanol). organisms and thus favouring the methanogenic process [9]. For the case of hydrogen production, only few reports * Corresponding author have been published about the effect of particle size. For

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the municipal solid waste, it has been proposed that the de- lected particle sizes resulted after a mechanical pre-treat- crease of the particle size can lead to the increase of hydro- ment using a wet macerated grinder. The typical particle gen production. Yuan et al. [10] used different particles size distribution using this pre-treatment has been reported sizes (1, 5 and 10 mm) of a wheat stalk for hydrogen pro- to be around 2 mm (35%) with a substantial percentage of duction, observing differences at constant total solids particles smaller than 0.3 mm (33%) [15]. Finally, the se- value. They found that the cumulative production of hydro- lected waste was freeze at -20 °C and preserved it until the gen, acetate and butyrate decrease as the particle size in- experiments. crease. Chen et al. [11], applied different particle size (from <0.297 mm to >10 mm) of rice straw, obtaining a peak of 2.2 Hydrogen production test hydrogen production at the smaller particle size. Hydrogen production was evaluated in serum bottles of On the other hand, it has been demonstrated that the 120 mL according to Ramos et al. [6]. Anaerobic granular sludge from an upflow anaerobic sludge blanket reactor initial total solids (TS0) concentration affects the hydrogen treating brewery wastewater was used as inoculum (43.9 production [8]. High TS0 content can result in problems -1 with mass transfer between the substrate and microorgan- gTS L wet basis). A thermal pre-treatment was carried out isms, which negatively affects the production of hydrogen. at 100°C for 24 h to inhibit the bioactivity of methanogens In addition, the initial concentration of substrate (either and to select the hydrogen-producing microorganisms. After the thermal treatment, the material was broken up into a mor- solid or liquid) may result in inhibition of H2-producing tar, sieved with a # 20 mesh (850 µm) and stored in a sealed bacteria. The TS0 values applied by several authors varied from 1.3 to 120 g L-1 [6,13,14], obtaining different yields glass container at room temperature (24 ± 0.1 °C) until use, of hydrogen production. For this reason, it is necessary to according to [16]. The powder obtained was used as inoculum in each experiment in an initial concentration of 16 g of determine the effect of the particle size and different initial -1 total solids on the biohydrogen production for the feed- TS L . Boiled deoxygenated water was added to fill a total stocks used. volume of 80 mL, leaving a headspace of 40 mL. Oxygen was removed by nitrogen purging to assure an anaerobic en- The objective of this paper was to evaluate the influ- vironment. Incubation was at constant temperature of 36°C, ence of particle size and initial total solids concentration on and the content was mixed using an orbital shaking at 130 biohydrogen production for food waste from a cafeteria us- rpm. The TS0 concentration was varied from 5 to 120 gTS0 ing hydrolytic-acidogenic microorganisms in batch reac- L-1 in the experiments of set 1) and 2). In Set 1) the pH was tors operated at mesophilic conditions. not adjusted, while in Set 2), the pH were adjusted and maintained at 5.5 using a citrate buffer (sodium citrate and citric acid 1M) [6]. All the experiments were run in triplicate. An- 2. MATERIALS AND METHODS alytical analysis as TS and COD were determined according to the Standard Methods [17]. Two sets of experiments were done. Set 1) Effect of TS0 when no pH adjustment nor particle size selection is 2.3 Biogas and fermentation products analysis applied in the test, and Set 2) Effect of particle size and TS0 The production of the biogas was followed using a on the hydrogen production. pressure transducer as described by Shelton and Tiedje [18]. The pressure was monitored in regular intervals. Dur- 2.1 Food Waste Feedstock ing each pressure measurement, the bottle was allowed to The organic fraction of solid waste (fermentable mat- reach the atmospheric pressure, avoiding to work with sys- ter from food waste) was obtained from a university cafe- tems under pressure that are prone to gas leaks or inhibition teria (Universidad Nacional Autónoma de México, Ju- of hydrogen producers microorganisms. Biogas amount riquilla-campus at Querétaro). Sampling was performed was estimated from the pressure using the ideal gas law. during 15 days and refrigerated at 4 °C for preservation. In Endogenous biogas production was evaluated by tests with each collection, bones and inert material (paper and plastic) no substrate addition for the inoculum and subtracted in the were discarded; only the fermentable matter was pre- biogas calculations. The hydrogen percentage in the biogas served. The sampling of cafeteria waste showed the follow- was determined using gas chromatography (Agilent 6890 / ing fractions: residues from fruits and vegetables 69.5 TCD, column Supelco-Carboxen 1010 Plot). VFA (acetic, ±16.1 %; protein residues (meat and egg) 13.9 ± 7.3 %; propionic, butyric and iso-butyric acids) and solvents (eth- flour derived residues (including bread) 13.2 ± 8.0 % and anol and acetone) were determined by gas and liquid chro- others 3.34 ± 16.0 %. The collected raw waste was crushed matography, respectively according to Ramos et al. [6]. A in a blender, mixed and homogenized. The feedstock (after kinetic analysis of cumulative hydrogen production from blender) showed the following characteristics: moisture different particle size and initial TS was done with the ex- 82%, 206 g TS L-1; total COD 140 g L-1 and alkalinity of perimental data obtained. Each condition represents the av- -1 17.6 gCaCO3 L . For the experiments of Set 2), three dif- erage of three experiments for hydrogen production during ferent particles size were selected: a) particles larger than reaction time. The modified Gompertz equation (Eq.4) was 2 mm (named PS1), b) particles between 0.5 and 2 mm used to fit the kinetics of biohydrogen production as de- (PS2) and c) particles smaller than 0.5 mm (PS3). The se- scribed by Buitron and Carvajal [16].

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-1   2.71828* R (  t)  removal will be reach applying between 5 and 30 gTS0 L . H (t)  H *exp  exp max 1 max    For all cases, the biogas was composed mainly by CO2 (from   H max  (4) 80 to 85%) when the TS0 concentration was higher than -1 where, H(t) (mmol) represents the total amount of hy- 10 gTS0 L . The rest of the biogas composition was H2 drogen produced in time t (h); Hmax (mmol) represents the (from 12 to 20%). maximal amount of hydrogen produced during the test; Figure 2 shows the production of VFA in the experiments Rmax (mmol h-1) is the maximum hydrogen production rate done. A stable production of acetic and propionic acids was and λ (h) is the lag time before the exponential hydrogen observed until an initial TS concentration of 70 g L-1. Beyond production. this concentration, the VFA increased. There was no effect on iso-butyric and butyric acids production, where concentrations were fluctuated below 100 and 300 mg L-1, respec- 3. RESULTS AND DISCUSSION tively. It was observed that the acetone and ethanol increased as the TS0 increased. Concentrations of TS0 beyond -1 3.1 Effect of TS0 without pH adjustment 90 g L produce an exponential production of both solvents, obtaining values of 4.8, 1.5 g L-1 at TS of 100 g L-1. Lactate Figure 1 shows the hydrogen production yield (YH2), 0 the removal percentage of TS and COD applying different increased as the TS0 concentration increased. This concentration can be responsible for the decrease of hydrogen pro- TS0 concentrations. The YH2 showed a dependency between the hydrogen production and the initial total solids, duction. It has been reported that butyrate was associated -1 to H -producing pathways and a small interaction with lac- obtaining the maximum value when 90 g L was used (YH2 2 -1 tate at low concentration, this metabolic interaction be- of 2.2 LH2 kg TSadded). The removal efficiency decrease tween butyrate and lactate pathways is not well known [7]. when the TS0 increase, for the case of the COD and TS. In this sense, the optimal TS0 concentration for the H2 produc- The final pH in all the cases decreased around two -1 -1 tion was obtained at 90 gTS0 L . However, at 90 gTS0 L , units, irrespective of the TS0 concentration, i.e., the final the removal efficiency (COD and TS removal) was less pH in the all the bottles tests was 3.5 ± 0.3. Only for the -1 than 10%. A compromise between the hydrogen produc- case of 5 gTS0 L , the natural buffer capacity of the system -1 tion yield (obtaining a maximum at 90 gTS0 L ) and TS maintained the pH in 5.5. The results showed that even at and COD removal (obtaining better efficiencies at initial low pH values the hydrogen was generated, but this pro- -1 ST lower than 10 gTS0 L ) has to be found. In this aspect, duction was lower compared with those obtained when the better compromise for H2 production and COD and TS there exist an adjustment in the pH during the process [6].

FIGURE 1 - Yield of hydrogen production, TS and COD removal efficiency at different initial total solids concentrations.

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FIGURE 2 - VFA production at different TS0 during the hydrogen production.

TABLE 1 - Hydrogen and VFA production, lag time and percentage of H2 in biogas, when different particle size are applied at several TS0

-1 TS0, Hmax, mL Rmax, mL d λ, h Percentage of H2 Acetic, Propionic, Butyric, Butyric/ g L-1 in gas , % mg L-1 mg L-1 mg L-1 acetic ratio PS1 5 13 ± 3 16 ± 11 1.3 ± 1.4 27 1685 1247 2455 1.5 10 39 ± 7 31 ± 13 2.8 ± 0.3 42 1496 673 2430 1.6 15 81 ± 32 49 ± 16 3.0 ± 0.1 39 1507 532 2827 1.9 20 110± 18 72 ± 6.7 5.0 ± 1.7 45 2058 606 3425 1.7 30 170 ± 16 128 ± 18 6.7 ± 0.6 42 2506 555 6061 2.4 50 199 ± 13 177± 34 9.8 ± 2.5 39 4995 625 12938 2.6 100 195 ± 7 184± 49 10.3 ± 2.9 42 6099 404 7437 1.2 PS2 5 38 ± 4 13 ± 2 11.3 ± 1.2 33 1325 456 1603 1.2 10 56 ± 3 25 ± 3 7.7 ± 1.4 33 1590 1308 2443 1.5 15 58 ± 19 36 ± 7 3.3 + 3.1 38 1580 1135 2387 1.5 20 65 ± 10 44 ± 9 3.8 ± 0.3 30 2086 972 1513 0.7 30 75 ± 7 43 ± 3 1.2 ± 0.3 34 2851 837 1990 0.7 50 103 ± 18 54 ± 6 2.4 + 0.2 37 3136 485 3758 1.2 100 25 ± 8 33 ± 8 1.9 ± 0.1 8 4957 336 2587 0.5 PS3 5 112 ± 9 48 ± 8 9.8 ± 0.3 54 3228 330 3294 1.0 10 126 ± 5 65 ± 14 13.0 ± 3.5 32 3454 427 4426 1.3 15 154 ± 22 149 ± 17 14.3 ± 1.2 32 2822 393 6018 2.1 20 121 ± 16 131 ± 15 16.9 ± 0.2 34 3903 406 2344 0.6 30 109 ± 5 124 ± 16 17.0 ± 0.1 37 2804 353 1627 0.6 50 27 ± 2 45 ± 5 17.7 ± 0.6 9 3871 514 2750 0.7 100 8 ± 2 7 ± 3 17.5 ± 0.5 5 2209 440 1899 0.9

3.2 Effect of particle size on H2 production respectively (4.6 ± 0.3 mmol and 6.9 ± 0.1 mmol). For smaller Table 1 shows the hydrogen production for different particles, there exist higher quantity of organic matter particle size applying different TS0 concentration. In the case available than in bigger particle size. It has been observed, of PS1 (particle size >2 mm), the H2 production increased as when liquid waste is applied, that there exist a decrease in the TS0 increased. The higher H2 production was obtained at hydrogen production as the substrate concentration and the -1 TS0 of 50 g L , but this production was not significantly dif- organic loading rate increased [19,20]. -1 ferent with the condition of 100 gTS0 L (8.9 ± 0.6 and 8.7 ± 0.3 mmol H2, respectively). For PS2 and PS3 the higher The results showed that only hydrogen and CO2 were -1 -1 H2 production was obtained at 50 gTS0 L and 15 gTS0 L , present in the biogas (Table 1). No methane was detected

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in any sample. The concentration of H2 varied from 5 to particle size increased the solubility rate, increasing the ac- 54%. The higher values for hydrogen percentage in biogas cessibility of the organic matter to the microorganisms- In -1 were obtained when the TS0 were 50 gTS0 L or lower. In this sense, it was suggested that pre-treatment methods, ap- the case of PS1 the percentage of H2 was maintained stable plied to organic solid waste, make more accessible com- with percentage from 27 to 45% with an average value of plex carbohydrates resulting in an increase of biohydrogen 41.5 ± 2.3% in an initial ST from 10 to 100 g L-1. For PS2, production [7]. However, as the particle size decreased, an -1 the percentage was stable in 34.2 ± 2.9% except for the inhibitory effect was detected beyond 15 and 50 gTS0 L -1 value of 100 gTS0 L where the percentage was 8%. In for PS2 and PS3, respectively. In the case of higher TS0 -1 PS3, the hydrogen content in the biogas decreased from 54 concentrations (> 50 gTS0 L ) it is recommended to use to 5% as the TS0 increase. The percentages of H2 agree with larger particle size. the results obtained by Gomez et al. [21] and Zhu et al. [2] using food residues obtaining 25 to 45% of H2 in biogas. In a practical operation of a continuous bioreactor, it can -1 They also applied pH adjustment in order to avoid the in- be more suitable to work at concentrations below 30 gTS0 L hibition of the process and carried out at mesophilic tem- because higher values show a substrate thickness and make perature (34-35°C). difficulties in the agitation and draw of the reactor. In addition, higher levels of ST0 produce high VFA (Table 1) Figure 3 show the production of H2 obtained after 48 h concentrations that can inhibit the microorganism's activity -1 of reaction. For low concentrations of TS0 (5-15 g L ) there [21]. The principal VFA generated were acetic and butyric is an increase of H2 production in condition PS3 (6 mmol). acids in all the cases, in agreement with other studies ap- However, higher values than 30 g L-1 produced a decrease plying organic solid waste [6,22,23]. The VFAs are inter- in H2 production. Larger particles size required more time mediate products of H2 production, and a high concentra- to produce the same amount of hydrogen. When the con- tion of them causes inhibition to the microorganisms -1 centration of TS0 is between 5 to 20 g L , the best option [23,24]. The results demonstrated that when the TS0 in- is to apply a particle size smaller than 0.5 mm because the crease, the VFA production increases as well. The values production rate is higher per TS0 added. In the case of of acetic acid were similar when the TS0 concentration ap- -1 -1 15 gTS0 L the hydrogen production per TS were 0.068 ± plied was between 5 and 20 gTS0 L for each different par- -1 -1 -1 0.008, 0.078 ± 0.008 and 0.192 ± 0.009 mmol of H2 g TS d ticle size. Higher concentration than 30 gTS0 L results in for PS1, PS2 and PS3 respectively. Those hydrogen pro- a faster increase in the acetic acid production. Some studies ductions can be related to the different rate of solubility of showed that the butyric/acetic ratio (B/A) decrease as the organic matter and the influence of particle size. The small hydrogen production increase [22,25]. Low values of B/A

FIGURE 3 - Effect of different particle size and TS0 on the hydrogen production after 48 h.

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(lower than 0.8) can be obtained when pure substrates as Marie Curie Research Fellowship Programme PIRSES- sucrose or glucose are applied [12,26]. However, for the GA-2011-295170. Carlos Ramos, César Figueroa and case of organic solid waste, the values for B/A between 1.0 Mónica Rangel are acknowledged for their participation in to 5.0 has been reported [22,25]. In our study, the values test experiments. Jaime Pérez Trevilla and Gloria Moreno varied between 0.5 to 2.6. Rodríguez are acknowledged for their technical assistance.

Beyond the acetic and butyric acids as the main meta- The authors have declared no conflict of interest. bolic products, other sub-products can be obtained during the dark fermentation of food waste, e.g. lactate. The lactate can be produced due to the presence of microorgan- REFERENCES isms as lactic acid bacteria (LAB) in the food waste [27- 30]. It was demonstrated that other groups of microorgan- [1] Edwards, P.P., Kuznetsov, V.L., & David, W.I.F. and Bran- isms, including Bacteroidales strains, can produce lactate don, N.P. (2008) Hydrogen and fuel cells: towards a sustainable energy future. Energy Policy, 36, 4356-4362. as well as acetate, succinate, formate or propionate which are generated from the carbohydrates present in the food [2] Zhu, H., Parker, W., Conidi, D., Basnar, R. and Seto, P. (2011) Eliminating methanogenic activity in hydrogen reactor to im- waste [27]. However, it has been reported that the effect of prove biogas production in a two-stage anaerobic digestion LAB on H2 production depends on pH and temperature [3]. process co-digesting municipal food waste and sewage sludge. The inhibition effect on hydrogen-producing bacteria can Bioresourse Technology, 102, 7086-7092. be prevented by keeping the pH at over 5.0 [29], as the con- [3] Chu, C-F., Li, Y-Y., Xu, K-Q., Ebie, Y., Inamori, Y. and Kong, ditions that we maintain in our study. H-N. (2008) A pH- and temperature-phased two-stage process for hydrogen and methane production from food waste. Interna- Due to the use of a buffer, the average final pH values tional Journal of Hydrogen Energy, 33, 4739-4746. [4] Mohan, S.V., Mohanakrishna, G., Goud, R.K. and Sarma, P.N. were higher than 5.0 for all the particle size and TS0 tested (2009) Acidogenic fermentation of vegetable based market (5.3 ± 0.26, 5.4 ± 0.10, 5.13 ± 0.60 for PS1, PS2 and PS3, waste to harness biohydrogen with simultaneous stabilization. respectively), showing that the buffer capacity was enough Bioresource Technology, 100, 3061-3068. to avoid inhibition of the microorganism’s activity. Only [5] Fang, H.H.P., & Liu, H. (2002) Effect of pH on hydrogen pro- -1 the case PS3 with TS0 of 100 gTS0 L the final pH was 4, duction from glucose by a mixed culture. Bioresource Tech- in this case, the high VFA production decrease the pH and nology, 82, 87-93. produce a decrease on hydrogen production [24]. [6] Ramos, C., Buitrón, G., Moreno-Andrade, I. and Chamy, R. (2012) Effect of the initial total solids concentration and initial pH on the bio-hydrogen production from cafeteria food waste. 4. CONCLUSIONS International Journal of Hydrogen Energy, 37, 13288-13295. [7] Guo, X.M., Trably, E., Latrille, E., Carrere, H. and Steyer, J- P. (2013) Predictive and explicative models of fermentative The results demonstrated that there is an effect of the hydrogen production from solid organic waste: Role of butyr- initial particle size and the concentration of initial total sol- ate and lactate pathways. International Journal of Hydrogen ids on the hydrogen production. When no pH adjustment Energy, 39, 7476-7485. is applied, it was observed that the maximal hydrogen pro- [8] Izumi, K., Okishio, Y-K., Nagao, N., Niwa, C., Yamamoto, S. -1 -1 duction (2.2 LH2 kg TSadded) was obtained at 90 gTS0 L , and Toda, T. (2010) Effects of particle size on anaerobic di- but the high TS and COD removal were obtained at 10 gestion of food waste. International Biodeterioration and Bio- -1 degradation, 64, 601-608. gTS0 L . The H2 production increases as the TS0 increase when particles larger than 2 mm were used. For particle [9] Mshandete, A., Bjornsson, L., Kivaisi, A.K., Rubindamayugi, sizes between 0.5 and 2.0 mm, and smaller than 0.5 mm, M.S.T. and Mattiasson, B. (2006) Effect of particle size on bi- -1 ogas yield from sisal fiber waste. Renewable Energy, 31, the higher H2 production was reached at 50 gTS0 L and 15 2385-2392. gTS L-1, respectively. Larger particles size required more 0 [10] Yuan, X., Shi, X., Zhang, P., Wei, Y., Guo, R. and Wang L. time to produce the same amount of hydrogen. The maxi- (2011) Anaerobic biohydrogen production from wheat stalk by mal hydrogen production rate was obtained with a smaller mixed microflora: Kinetic model and particle size influence. -1 particle size applying initial 15 gTS0 L . The principal vol- Bioresource Technology, 102, 9007-9012. atile fatty acids generated were acetic and butyric. The val- [11] Chen, C-C., Chuang, Y-S., Lin, C-Y., Lay, C-H. and Sen, B. ues of acetic acid were similar when initial TS applied were (2012) Thermophilic dark fermentation of untreated rice straw -1 using mixed cultures for hydrogen production. International between 5 to 20 gTS0 L for each different particle size. -1 Journal of Hydrogen Energy, 37, 15540-15546. Higher concentration of TS0 than 30 gTS0 L results in a fast increase in the acetic acid production. [12] Maintinguer, S.I., Fernandes, B.S., Duarte, I.C.S., Saavedra, N.K., Adorno, M.A.T. and Varesche, M.B. (2008) Fermenta- tive hydrogen production by microbial consortium. Interna- tional Journal of Hydrogen Energy, 33, 4309-4317. ACKNOWLEDGMENTS [13] Fountoulakis, M.S. and Manios, T. (2009) Enhanced methane and hydrogen production from municipal solid waste and The authors gratefully acknowledge the financial sup- agro-industrial by-products con-digested with crude glycerol. port of PAPIIT (DGAPA-UNAM) project IN103315 and Bioresource Technology, 100, 3043-3047.

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[14] Ma, J., Ke, S. and Chen, Y. (2008) Mesophilic biohydrogen production from food waste. Proceedings of the 2nd Interna- tional Conference on Bioinformatics and Biomedical Engi- neering, Shanghai, 16-18 May, 2841-2844. [15] Zhang, Y. and Banks, C.J. (2013) Impact of different particle size distributions on anaerobic digestion of the organic fraction of municipal solid waste. Waste Management, 33, 297- 307. [16] Buitrón, G. and Carvajal, C. (2010) Biohydrogen production from Tequila vinasses in an anaerobic sequencing batch reactor: effect of initial substrate concentration, temperature and hydraulic retention time. Bioresourse Technology. 101, 9071- 9077. [17] APHA. (2005). Standard Methods for the Examination of Wa- th ter and Wastewater. 21 ed., APHA/AWWA/WEF, Port city press. Baltimore, Maryland.

[18] Shelton, D. and Tiedje, J. (1984) General method for determining anaerobic biodegradation potential. Applied and Envi- ronmental Microbiology, 47, 850-857.

[19] De Amorim, E.L.C., Sader, L.T. and Silva, E.L. (2012) Effect of substrate concentration on dark fermentation hydrogen production using an anaerobic fluidized bed reactor. Applied Bi- ochemistry and Biotechnology, 166, 1248-1263. [20] Shen, L., Bagley, D.M. and Lis, S.N. (2009) Effect of organic loading rate on fermentative hydrogen production from continuous stirred tank and membrane bioreactors. International Journal of Hydrogen Energy, 34, 3689-3696. [21] Gomez, X., Moran, A., Cuetos, M.J. and Sánchez, M.E. (2006) The production of hydrogen by dark fermentation of municipal solid waste and slaughterhouse waste: a two phase process. Journal of Power Sources, 157, 727-732.

[22] Zhu, H., Stadnyk, A., Bèland, M. and Seto, P. (2008) Co-production of hydrogen and methane from potato waste using a two-stage anaerobic digestion process. Bioresource Technol- ogy, 99, 5078-5084.

[23] Dong, L., Zhenhong, Y., Yongming, S., Xiaoying, K. and Yu, Z. (2009) Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture

fermentation. International Journal of Hydrogen Energy, 34, 812-820. [24] Lay, J-J., Fan, K-S., Chan, J-I. and Ku, C-H. (2003) Influence of chemical nature of organic wastes on their conversion to Received: September 02, 2014 hydrogen by heat-shock digested sludge. International Journal Revised: November 10, 2014 of Hydrogen Energy, 28, 1361-1367. Accepted: December 18, 2014 [25] Shin, H-S., Youn, J-H. and Kim, S.H. (2004) Hydrogen production from food waste in anaerobic mesophilic and thermophilic acidogenesis. International Journal of Hydrogen En- CORRESPONDING AUTHOR ergy, 29, 1355-1363. [26] Wang, J. and Wan, W. (2008) Effect of temperature on fer- Iván Moreno-Andrade mentative hydrogen production by mixed cultures. Interna- Laboratory for Research on Advanced Processes for tional Journal of Hydrogen Energy, 33, 5392-5397. Water Treatment [27] Shin, H-S., Youn, J-H. and Kim, S-H. (2004) Hydrogen pro- Unidad Académica Juriquilla duction from food waste in anaerobic mesophilic and thermo- Instituto de Ingeniería philic acidogenesis. International Journal of Hydrogen En- ergy, 29, 1355-1363. Universidad Nacional Autónoma de México Blvd. Juriquilla 3001 [28] Kim, S-H., Han, S-K. and Shin H-S. (2004) Feasibility of biohydrogen production by anaerobic co-digestion of food waste 76230 Querétaro and sewage sludge. International Journal of Hydrogen Energy, MÉXICO 29, 1607-1616. [29] Noike, T., Ko, I.B., Yokoyama S., Kohno, Y. and Li Y.Y. Phone: + 52 (442) 1926171 (2005). Continuous hydrogen production from organic waste. Fax: + 52 (442) 1926185 Water Science and Technology, 52, 145-151. E-mail: [email protected]

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APPLICATION OF CELLULOSE ACETATE MEMBRANES FOR REMOVAL OF TOXIC METAL IONS FROM AQUEOUS SOLUTION

Nadjib Benosmane1,2,*, Baya Boutemeur2,*, Maamar Hamdi2 and Safouane M. Hamdi3

1Departement of Chemistry, Faculty of Sciences, University M’Hamed Bougara de Boumerdes (UMBB), Avenue de l’indépendance 35000, Boumerdes, Algérie. 2Laboratoire de Chimie Organique Appliquée. Faculty of Chemistry, USTHB BP 32 El-Alia 16111, Alger, Algérie. 3Clinical Biochemistry Department, CHU Toulouse, University of Toulouse, UPS, Toulouse, France.

ABSTRACT Currently, due to anthropogenic activities where lead is the raw material (i.e. fuel refining and use, metallurgy, pig- In this study, we investigated the transport of heavy menting, etc.), its removal from wastewater is a problem of metals ions through a polymer inclusion membrane (PIM) great significance [2]. Due to concern for the environment containing cellulose acetate (CA) as support, calix[4]resor- and the possibility of using a very low amount of the ex- cinarenes (RC) as carriers, 2-nitrophenyloctanoate (NPOT) tractant, liquid membrane-based separation methods are and 2-nitrophenyloctylether (NPOE) as plasticizers. The considered viable alternatives to currently employed sol- effects of several transport conditions including, pH, PIM vent extraction methods [3-6]. thickness, plasticizers amount and carriers concentration Polymer inclusion membranes (PIMs) are thin, flexi- were studied. The optimal transport conditions for Pb(II), ble, and stable films, formed by casting a solution contain- Cu(II) and Zn(II) were found to be 10.8 mM of carriers, ing an extractant (carrier), a plasticiser, and a base polymer 0.13 mL/g of CA for NPOE, a pH of 5.1 and 1.5 for feeding (cellulose triacetate, CTA or poly(vinyl chloride), PVC) and receiving phases respectively. Our experiments revealed [7-15]. Selectivity, stability, and efficient transport of a facilitated counter-transport mechanism for ions transfer metal ions are the properties that make PIMs valuable tech- and showed that such PIMs can be reused for five consec- nologies for the removal of toxic ions from liquid effluents. utive cycles without appreciable deterioration. We charac- Over the last 30 years, PIMs have been widely used in the terized PIMs structure by several techniques and deter- production of chemical sensors with applications in the po- mined their water content and porosity. Applying these op- tentiometric determination of many cations and anions timized transport conditions to remove metal ions from [16]. Moreover, since the use of flammable solvents has municipal wastewater samples, we found an overall re- been eliminated from the composition of PIMs [16], many moval efficiency of 87%, 86%, 75%, 72%, and 71% for studies have presented additional applications of these Pb(II), Ni(II), Cd(II), Cu(II) and Zn(II) respectively. Of in- membranes as solid sorbents in separation and extraction terest, the residual concentrations of these heavy metal ions processes, with obvious implications for green chemistry- were below the permissible limits according to the Alge- controlled technology. rian standards for wastewaters quality. Cellulose acetate can be characterised by its extensive linearity, good flexibility, excellent durability, biodegrada- bility, non-toxicity, low cost, and displays good solubility KEYWORDS: Polymer inclusion membranes, Calix[4]resorcinarenes, Metals ions, Separation, Municipal wastewater. in organic solvents [17-23]. On the other hand, it has some disadvantages such as poor mechanical strength, low oxidation, as well as thermal and chemical resistance [24-28]. Cellulose acetate membranes have been used in many ap- 1. INTRODUCTION plications of particular interest are reverse osmosis systems, and as a neutral matrix for the incorporation of dif- Lead, copper and zinc are heavy metals of concern for ferent polymers (e.g., conducting polymers), inorganic their role in environmental pollution, as have toxic effects ions (e.g., lanthanides), and organic (e.g., pharmaceutical) in humans via inhalation, oral ingestion and/or skin con- compounds [14, 17, 18, 23, 29, 30]. tact. The presence of these metals in the human body may The calixarenes are a major class of supramolecular induce elevated blood pressure, anaemia, and gastrointes- hosts of phenolic units. One of the important properties of tinal, cardiovascular, nervous and memory diseases [1]. calixarene is its ability to recognise cationic and anionic species, as well as neutral molecules. These receptors have * Corresponding author the possibility to form interesting complexes both with metal

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cations and biological compounds by exhibiting extracta- treatment, the samples were filtered through Millipore fil- bility and selectivity. Many studies have been published ter paper (0.45 µm) and kept in Teflon® bottles, at 4 °C, in dedicated to calixarenes, particularly in the molecular in- the dark. The general characteristics of the municipal waste- clusion of biological substrates, such as amines and amino water are presented in Table 1 Concentrations of Pb(II) and acids [31-33]. Various applications of calixarenes also re- Cd(II) are high enough to be considered as a serious environ- fer to purification, chromatography, catalysis, enzyme mental problem. Therefore, they were chosen for our study. mimics, ion selective electrodes, phase transfer, transport across membranes, ion channels, and self-assembling mon- 2.2 Chemicals olayers [1, 34-41]. The aim of this work is to investigate The structures and abbreviations of the carriers used the efficiency of metal ions removal from synthetic aque- in the present study are shown in Fig. 1. The carrier, (C- ous wastewater solutions and municipal wastewater by a butyl)calix[4]resorcinarene, abbreviated as RC4, (C-oc- PIM technology. The influence of the PIM carrier concen- tyl)calix[4]resorcinarene, abbreviated as RC8, and 2-nitration, plasticizer amount, PIM thickness, pH, and phase trophenyloctanoate (NPOT) were synthesised in our la- composition were investigated for estimate their impact boratory [36,42]. Final products were purified by recrys- both on transport fluxes of the optimal membrane. tallisation and identified by FT-IR, 1H NMR, and elemental analysis.

2. MATERIALS AND METHODS Lead (II) chloride, copper (II) chloride, zinc (II) chloride, resorcinol, aldehydes, ethanol, hydrochloric acid, po- 2.1 Municipal wastewater collection tassium hydroxide, dichloromethane, cellulose acetate (CA, The triplicate samples of municipal wastewater was col- molecular weight Mw = 50.000, with an acetyl content of lected from a wastewater stream flowing over nearby location 39.7%, and refractive index =1.475), and 2-nitrophenyl-oc- of city of El Harrach (latitude deg. North 36.710455, longitude tylether (NPOE) were analytical grade reagents purchased deg. East 3.12295), 11 km east of Algiers, Algeria. Col- from Fluka. Aqueous phases were prepared by dissolving lected wastewater was brought immediately to laboratory. the different reagents in deionised water. The pH adjust- Sample was collected just before the starting of experimen- ment was done by addition of dilute hydrochloric acid or tation in order to avoid alternation in the wastewater char- potassium hydroxide to prepare the desired pH solution. acteristics mainly due to open storage of sample. Before

A B

RC4: R = C4H9

RC8: R = C8H17

FIGURE 1 - Molecular structures of calix[4]resorcinarenes. A Chemical formula and abbreviation of calix[4]resorcinarenes used as carriers. B Cartoon representation of RC8 showing the characteristic and truncated cone shape with eight hydroxyl groups on the wide rim and four hydrophobic tails on the narrow rim.

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TABLE 1 - Physical-chemical characteristics of municipal wastewater sample used for experiments.

2.3 Preparation of PIM To describe the efficiency of metal removal from the Membranes were prepared by the solution casting and source phase, the recovery factor (RF) was calculated: solvent evaporation techniques as described by Sugiura et al. RF (%) = x100 (2) [43]. The polymer matrix (CA), the plasticizers (NPOE/ NPOT), and a carrier (RC4,8) were dissolved in dichloromethane to prepare a homogenous solution. The solution Where c is metal ions concentration in the source phase was poured into a 7.6 cm diameter flat bottom glass Petri at a given time, ci is the initial metal ions concentration in dish. The organic solvent was allowed to evaporate over- the source phase. For the municipal wastewater sample. night at room temperature. The resultant translucent film The pH, electrical conductivity (EC), redox potential (Eh) was cautiously peeled off of the Petri dish and rinsed with and Total Dissolved Solids (TDS) were analyzed with a water on both sides. The resulting membrane was evalu- multifunctional pH meter (HANNA HI 2550). ated for its strength, flexibility and visually apparent ho- mogeneity. The thickness of the membrane film can be cal- 2.5 Membrane characterisation culated by micrometer To characterise the morphology and composition of This thickness could be varied by pouring more or less the PIM, several techniques were used such as FT-IR, SEM, XRS, TGA and water content measurements. FT-IR of the mixture into the casting ring, as desired. The blank −1 experiments for the transport when the ion carrier was ab- spectra were taken in the range of 650-4000 cm using an sent, i.e. with membranes containing the support (CA) and FTIR-8300 Fourier Transform InfraRed Spectrophotome- plasticizers (NPOE, NPOT) only, showed no significant ter (Shimadzu). The surface characterisation study of the flux across PIM. PIM was carried out by scanning electron microscopy (SEM) observations using a 10 kV apparatus (ESEM 2.4 Transport experiments XL30, Philips) after gold coating. X-ray analyses were recorded on a Philips model X-pert X-ray diffractometer op- A typical laboratory scale device was used for erating at 40 kV. Thermogravimetric analysis (TGA) of the transport studies. It consisted of two compartments made PIMs was done on a Setaram TG 96 thermal analysis in- of Teflon with a maximum capacity of 400 ml separated by strument from room temperature to 550ºC at a heating rate the PIM. Of interest, the film side exposed to air during of 10°C/min in a nitrogen atmosphere. solvent evaporation faced the feed compartment and the PIM area exposed to the aqueous phase was 12.56 cm². In The water content of the membranes was obtained as order to minimise the boundary layer thickness, both the follows: source and stripping compartments were provided with a Pre-weighed membranes were immersed in water for mechanical stirrer adjusted to 600 rpm [37]. The Transport 24 h and weighed after drying with blotting paper [37]. For can take place by diffusion. All transport experiments were each specimen, four samples were tested. The percent wa- carried out at 25±1°C and repeated three times. In all fig- ter content was determined according to Eq. (3) [37]: ures, the reported error bars represent the standard devia- Water content (%) = x 100 (3) tion of the data.

2.5 Analyses where m1 and m2 are the weight of the wet and dry membrane, respectively. Metal concentrations were determined by sampling at the indicated times. A 0.25 ml aliquot from both the feed and striping solutions was then analysed with an atomic ab- 3. RESULTS AND DISCUSSION sorption spectrophotometer (Varian AA 110). The mass 2 1 flux, J (µmol/m .s ), of the metal ions through the PIM was 3.1 Pb(II), Cu(II) and Zn(II) transport experiments calculated according to Eq. (1) [43]: The main objective of PIMs research is to maximise J = + n / St (1) the membrane fluxes of solutes. However, the rate of Where n represents the variation in the mole number transport of the target solutes is markedly influenced, first of metal ions in the stripping solution, during the reference by the physicochemical properties of the carrier, then by time t, and S is the active membrane surface. the chemical composition of the membrane, and finally by the chemical composition of the source and stripping solu-

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tions [44, 46]. Therefore, we investigated the impact of these 3.2 Amount of plasticizer three parameters on the facilitated transport properties of our To understand the influence of the amount of plasti- PIM system toward Pb(II), Cu(II) and Zn(II). The choice of cizer on transport flux, membranes were prepared with var- metals ions was driven by the fact that calix[4]resorcina- iable amounts of NPOE at a constant concentration of car- renes are known to form complexes with Cu(II) and Zn(II) rier the (RC4) and CA in the PIM. Fig. 2 shows the effect [47] and thus mediate the transport of Cu(II) complexes of the amount of NPOE on the transport flux of metals ions. through liquid chloroform membranes [48]. The flux was increased by increasing the amount of NPOE As seen in Table 2, the initial flow of PIM in this study until a value of 0.13 mL, further addition caused a decrease is lower than the initial flux in the previous study [36]. in the transport flux. This decrease was attributed to the in- Probably due to the difference in the nature of the support crease in the thickness and viscosity of the membrane re- (CTA/CA), the transport of metals is mainly by diffusion, sulting in lower mass transfer [49-51]. Thus, the amount of and therefore the transport rates are lower. Furthermore we NPOE was selected as 0.13 mL (4.13M) for further exper- observed that transport efficiency of PIM in this study is iments. The results (Fig. 2) show that the choice of the ap- higher than that of unplasticized PIM in previous study propriate composition of the various components of PIM [37]. The obtained data prove that the PIM with any addi- must be based not only on the interactions between the dif- tionally plasticizers is not only considerably cheaper but it ferent components, but also their effect on the extraction has enhanced metal ions transport compared with other un- and transport of the species of interest. plasticized PIMs [37].

TABLE 2 - Comparison of PIM containing RC8 for Pb(II) transport (feed phase: 1×10−2 M Pb(II), pH of feed phase 5.1, receiving phase: HCl at pH 1.5).

Support material Plasticizer Carrier Flux (μmol/m2.s1) of Pb(II) Ref Cellulose Tri Acetate (CTA) NPOE RC8 5.03 [36] Cellulose Tri Acetate (CTA) NPOT RC8 1.43 [36] Cellulose Acetate (CA) Without RC8 0.13 [37] Cellulose Acetate (CA) NPOE RC8 4.78 This study Cellulose Acetate (CA) NPOT RC8 0.98 This study

Pb(II) Cu(II) Zn(II) 3

) -1 .s -2 2

mol.m 

Flux (

0 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 Amount of NPOE (ml)

FIGURE 2 - Change of transport flux with the amount of NPOE. Transport conditions: Source phase (synthetic aqueous solution of PbCl2, -3 2 CuCl2 and ZnCl2, 10 M pH: 5.1) and stripping phase (deionized water, pH=1.5) were stirred at 600 rpm, membrane area of 12.56 cm with 10.8 mM of carrier (RC4) and [0.05-0.17] ml of NPOE, time of transport : 3 days, T =25°C.

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FIGURE 3 - Pb(II), Cu(II) and Zn(II) mass fluxes through PIMs. Transport conditions : Source phase (synthetic aqueous solution of PbCl2, -3 2 CuCl2 and ZnCl2, 10 M pH: 5.1) and stripping phase (deionized water, pH=1.5) were stirred at 600 rpm, membrane area of 12.56 cm with 10.8 mM of carrier and 0.13 ml of (NPOE/NPOT), time of transport : 5 days, T = 25°C.

3.3 Influence of carrier structure on cation flux 3.4 PIM thickness First, we wondered whether modifying the calix[4]re- In order to assess the influence of membrane thickness on sorcinarene structure (particularly the length of the alkyl metals ions transport through the membrane, transport exper- moieties, Fig. 1) would have an effect on the rate of metals iments were carried out using membranes of the same chemi- ions transport through the PIM. The collected data are de- cal composition, but with different thicknesses, i.e. 22, 28, 33, picted in Fig 3. Thus, our initial hypothesis that calix[4]re- 39 and 48 µm. As seen in Fig. 4, the metals ions flux decreased sorcinarene is able to mediate cation transfer through a CA by increasing the thickness of the membrane. This was ex- matrix was confirmed. RC8 appeared to be the most effi- plained by the appropriate formulation of Fick’s first law of cient carrier for Cu(II) with a three-fold increase in flux, diffusion illustrated in earlier studies [52,53]: followed by RC4. Rates of Zn(II) transport were below those of Cu(II), but the pattern of carrier efficiencies was J= D0 roughly the same (RC8 > RC4). We can infer from these results that metals ions fluxes depend on the alkyl chain (4) length of calix [4] resorcinarene. The influence of the Neglecting the aqueous diffusion layer, ∆x=d (d: mem- length of the alkyl chain on the transport of metal ions has brane thickness) and ∆C ≅ Ci (Ci is the initial source phase already been demonstrated for diazadibenzocrown ethers concentration). Eq. (5) can be simplified to: [16]. These results imply that the efficiency of the macrocyclic carrier based on an optimal combination of polar (5) rings and hydrophobic tails.

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On the other hand, decrease with increasing the thick- The pH of the source phase has been fixed at 5.1, tak- ness of the membrane may be due to possible interactions ing into account the maximum efficiency of experimental between the plasticizer and carrier. This can be explained biphasic extraction obtained in this pH range, from the re- in terms of the diffusion of complex through the mem- sults shown in Fig.5, when a larger difference in pH be- brane. Since the rate of mass transport is determined by tween the source and stripping phases is applied, the initial diffusion through the membrane from the source solution flux of lead (II) increases from 1.33 (at pH 3.5) to 4.68 to the stripping solution, the rate of mass transport de- µmol.m-2.s-1 (at pH 1.5). A lower pH in the stripping phase creases logically with an increase in film thickness. afforded the quantitative stripping of metals ions from the membrane phase. These observations are reasonable be- The maximum membrane transport was observed cause, according to the proton-driven mechanism dis- when the membrane thickness was 22 µm, as can be seen cussed earlier, the hydrogen ion concentration in the strip in Fig. 4. However, a negligible value was found with a phase is the only factor that influences the transport process membrane thickness of 48 µm. Therefore, the membrane [36,37]. One can suggest that the increase of the pH differ- thickness of 28 µm was used in our study. ence between the feed and the strip phases enhances the release of the metal ions in the stripping phase by a facili- 3.5 Influence of the aqueous phases composition tated protonation of carrier with two protons as expected in The facilitated transport of divalent metal ions through Eq (6): membrane systems containing acidic organic compounds 2+ + M + RC4,8 M(RC4,8)Org + 2 H (6) such as calix[4]resorcinarenes is generally ensured by a counter-transport of protons. The carrier exchanges one 3.6 Effect of the carrier concentration on initial metals ions metal ion at the source solution-membrane interface releas- flux ing two protons. The complex formed between the carrier Fig. 6 displays the variation of the entrance flux of and the metal diffuse through the membrane and one metal metals ions in the PIMs as a function of the carrier concen- ion is released at the membrane-stripping phase interface tration RC4 (the concentration is referred to the carrier con- by substitution of two protons. It is clear that the pH differ- tent in the plasticizer NPOE). When the RC4 concentration ence between source and striping aqueous solutions may increases from 1.35 mM to 10.8 mM, the initial flux in- play the role of driving force for the transfer of metal from creases by a factor Two. At lower carrier quantity the in- the source to the stripping aqueous solution [9]. terface between the donor phase and the membrane is not In order to improve the dissociation of the complexe in saturated by carrier. A further increase in the carrier con- the striping phase, the variation of the extracted metals ions centration up to about 10.8 mM resulted in only a slight M(II) amount was studied as a function of the pH of the increase in the flux. At higher RC4 concentration the or- striping phase solution, ranging from 1.5 to 3.5 by varying ganic membrane becomes saturated in M(RC4)Org complex the concentration of HNO3 (Fig. 5). A pH gradient between and initial flux remains constant, the main reason of the the source and stripping phases is the driving force for the enhancement of the flux is the increase of the kinetic of transport of metals ions through the membrane phase. So, metal-carrier association at the source membrane interface an acidic solution was needed for the stripping of Metals as the concentration of the metal ions is also increased, this ions from the PIM. plateau may be due to high viscosity in the membrane

5 Pb(II) Cu(II) Zn(II) 4

) -1

.s 3 -2

mol.m

 2

Flux ( 1

0 20 25 30 35 40 45 50

Thickness of membranes

FIGURE 4 - Influence of membrane thickness on the transport of metal ions. Transport conditions: Source phase (synthetic aqueous solution -3 of metal ions PbCl2, CuCl2 and ZnCl2, 10 M pH: 5.1) and stripping phase (deionized water, pH=1.5) were stirred at 600 rpm, membrane area of 12.56 cm2 with 10.8 mM of carrier (RC8) and 0.13 ml of NPOE, time of transport : 5 days, T = 25°C.

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Pb(II) Cu(II) Zn(II) 5

) -1

.s -2 3

mol.m

 2

Flux ( 1

0 3,5 1,5 2,5

pH FIGURE 5 - Effect of pH receiving solution on metals ions transfer through RC8 CA-NPOE membrane. Transport conditions: Source phase: -3 (synthetic aqueous solution of metal ions PbCl2, CuCl2 and ZnCl2, 10 M pH: 5.1) and stripping phase: pH = [1.5-3.5] were stirred at 600 rpm, membrane area of 12.56 cm2 with 10.8 mM of carrier (RC8) and 0.13 ml of NPOE, time of transport : 5 days. T= 25 °C.

Pb(II) 3,0 Cu(II) Zn(II)

2,5

) 2,0

-1 .s

-2 1,5

mol.m  1,0

Flux (

0,5

0,0 024681012141618 Carrier concentration (RC4).103M

FIGURE 6 - Metal ions initial flux vs. calix[4]resorcinarene (RC4) concentration. Transport conditions: Source phase: synthetic aqueous so- -2 lution of metal ions PbCl2, CuCl2 and ZnCl2, 10 M at pH= 5.1. Stripping phase: HCl pH =1.5. Were stirred at 600 rpm, membrane area of 12.56 cm2 with [1.35-17.55] mM of carrier (RC4) and 0.13 ml of NPOE, Time of transport: 3 day, T = 25 °C.

which limits the diffusion of carrier-cation complexes in remove specific pollutants from polluted water. Municipal the PIM [7,53]. The course of the curve can be interpreted wastewater collected from the city of El Harrach. The con- as evidence for a carrier-mediated transport mechanism centration of metals ions was measured for the three sam- due to the fact that a percolation threshold was not ob- ples, and the Zn(II), Ni(II) and Cu(II) concentrations were served in Fig. 6. In these experiments, the diffusion of the found to be lower than the Permissible limit of Algerian complex across the membrane was the rate-controlling step Standards. in the transport process [36]. Concentrations of heavy metals in the municipal 3.7 Environmental applications of PIM to remove toxic metals wastewater, before and after the process of treatment and ions from municipal wastewater their removal degrees, are shown in Table 3. After five It is very important to use real water samples in order days the concentrations of heavy metals in the municipal to investigate the applicability of a certain type of PIMs to wastewater were reduced below the permissible limits for

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wastewater in Algeria. The uptake of Pb(II) from sample PIM membrane as; 3468, 2910, 1717, 1614, and 1536 cm-1 by the PIMs was high (87%). The percentage of metal ions for OH, CH (Aliph) C=O(carbonyl), CH (Aromatic), and removal varied from 71% to 87%. (C=C Aromatic) (Table 4). The most part of the bands are due to the support (CA) and carriers. The additional main 3.8 Membrane characterisation bands at 1536 cm−1 were observed for the PIM containing 3.8.1 SEM and FTIR characterisation RC4, which correspond to the (C=C)Ar groups of RC4. Fur- thermore, the bands shape of the C=O stretch at 1746 cm-1 The surface morphology of prepared PIMs was evalu- was greatly influenced by the inclusion of calix [4] resorcin- ated by using SEM. The SEM micrographs suggest that the arene. This was most likely due to hydrogen bonds between before addition of carrier onto membrane the structure or the polymer carbonyl moieties and OH groups on the car- morphology of the membrane was microporous as reported rier. According to the results of the FTIR spectra, there was by the suppliers (Fig. 7a). But after the addition of carrier no formation of covalent bonds between the constituents of onto the membrane the morphology of membrane was en- the membrane, and there were only weak interactions be- tirely changed. The pores of membrane were cov- tween the constituents, i.e. van der Waals and hydrogen ered/blocked by carrier (Fig. 7b and c). This suggests that, bonds [54]. Consequently, the analysis and comparison of after the evaporation of dichloromethane, the plasticizer the obtained spectra revealed that all the membrane con- and the carriers diffuse homogeneously through the mem- stituents remained as pure components inside the membrane and filled the pores. brane [55,56]. The significant characteristics for FTIR spectrum of the blank membrane (Table 4) shows bands at 2932 and 3.9 X-ray Diffraction 2880 cm−1 attributed to C–H bonds. The bending peaks of The modified surface morphology of the PIM led us to C=O (carbonyl) and C-O were observed at 1746 and 1061 evaluate the degree of crystallinity by X-ray diffraction. cm−1, and the wide band detected in the 3600–3400 cm−1 Fig. 8 depicts the X-ray diffractograms of the CA and RC4 region was attributed to O–H bond stretching modes. The membranes. Two unresolved bands of diffuse diffraction FT-IR bands of carrier RC4 and RC8 were observed at were observed between 10° and 20° for the CA membrane 3468, 2910 and 1536 cm-1 and 3481, 2988 and 1527 cm-1 which confirmed an amorphous structure. When RC4 was for OH, C-H (Aliph) and CH (Aromatic) respectively (Ta- added to CA, a minor modification was observed (a slight ble 4). While after the addition of carrier a small shifting of increase of the broad peak around 20°). Similar results bands were observed due to the vibrational coupling of were obtained with RC8. These findings allow us to ignore bands. The significant bands were interpreted which con- carrier crystallisation within the membrane and suggest an forms the impregnation of carrier RC4 and RC8 onto the amorphous state of the prepared PIMs [8].

TABLE 3 - Metals ions removal from municipal wastewater sample using PIMs

SD: Standard Deviation, sample volume treated: 400 mL Transport conditions : feed phase (municipal wastewater adjusting the pH at 5.1) and receiving phase (deionized water, pH=1.5) were stirred at 600 rpm, membrane area of 12.56 cm2 with 10.8 mM of carrier (RC8) and 0.13 ml of NPOE, Time of process: 5 Days, T = 25°C. * Algerian standards of water for human consumption, Official journal of the algerian republic, N° 18, 2011.

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(a) (b)

(c)

FIGURE 7 - Surface view of membranes. (a) CA ; (b) CA-RC4-NPOE ; (c) CA-RC8-NPOE.

TABLE 4 - FTIR characterization: absorption peak values and the corresponding moieties in CA membrane and PIMs.

Membrane Peak value (cm-1) Corresponding moieties CA 3400-3600 O-H 2932 C-H ( CH3) 2880 C-H ( CH2) 1746 C=O 1253 C-O 1194 asym( C-O-C ) CA-NPOE Same bands and 1372 1497 1554 C-N

C=C (NPOE) NO2 (NPOE) CA-NPOT Same bands and 1370 1495 1531

C-N C=C (NPOT) NO2 (NPOT) CA-NPOE-RC4 same bands and (C=C)Ar (RC4) 1527 CA-NPOT-RC4 same bands and (C=C)Ar (RC4) 1536

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3.10 Thermal analysis (TGA) of PIMs exist two aggregates: polymer CA and aggregates of the Fig. 9 shows the TGA curves of the CA, CA-NPOE carrier solubilised in plasticizer, in theory the thermogram and CA-NPOT membranes at a heating rate of 10 °C/min of the system (support-carrier+plastifiant) gives two losses in N2. The thermal degradation of the CA membrane con- of mass. This weight loss was mainly due to the evapora- sisted of a series of degradation reactions, such as dehydration of bonding water on the CA membrane. Thermal py- tion at 100°C. rolysis of cellulose acetate skeleton was found at ≈330°C.

In order to investigate the heat resistance of CA+plas- In the case of the NPOE-CA membrane, weight loss ticizer+carriers membranes, and then we then used thermo- started at around 206°C, which was due in part to the gravimetric analyses (TGA) to link specific temperature NPOE, since the boiling temperature of NPOE is 198°C. and mass changes to the degradation of a specific com- At around 370°C, CA polymer degradation started. The pound. It should be noted that normally according to the PIM containing NPOT-CA showed a first weight loss at results of the FTIR: there are not new covalent bands be- 215°C which was due in part to the NPOT, since the boil- tween the carrier and polymer, thus in the membrane there ing temperature of NPOT is 200°C. In this case, CA polymer degradation started at 357°C [36].

FIGURE 8 - X ray diffractogramms of RC(4,8), CA membrane alone and PIMs.

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FIGURE 9 - Thermogrammes of CA membrane alone, CA-NPOE membrane and CA-NPOT membrane

TABLE 5 - Chemical and physical characteristics of PIMs

Membrane Quantity (g) of carrier Weight (mg/cm2) Thickness (μm) Water content (%)

CA 0 4.30 18.83 37.37 CA-NPOE 0 5.36 23.60 13.98 CA-NPOT 0 5.29 20.43 10.86 CA-NPOE-RC4 0.015 5.81 25.17 11.90 CA-NPOT-RC4 0.015 5.67 21.40 09.56 CA-NPOE-RC8 0.013 5.73 28.91 10.78 CA-NPOT-RC8 0.013 5.43 23.43 09.10

FIGURE 10 - Fluxes in consecutive transport experiments performed on the PIM with RC8 and (NPOE/NPOT). Transport conditions: Source -3 phase (synthetic aqueous solution of PbCl2: 10 M, pH: 5.1) and stripping phase (deionized water, pH=1.5) were stirred at 600 rpm, membrane area of 12.56 cm2 with 10.8 mM of carrier (RC8) and 0.13 ml of (NPOE/NPOT), T = 25 °C.

3.11 Water content This phenomenon can be attributed to the enhanced hydro- Water content is used to investigate the hydrophilicity phobic character of the PIM surface. A possible explana- of the material surface, as shown in Table 5. When the ali- tion of these results is the adsorption and/or inclusion pro- phatic chain length of calix[4]resorcinarene was increased cess of the carrier molecules at the interface between air in the PIM at a constant carrier concentration, a continuous and the surface of the membrane. As the hydrophobic part decrease in the water content of the PIM was observed. of the molecular skeleton is oriented toward the air inter-

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face, it is also possible that a hydrophobic PIM could re- [2] Salazar-Alvarez, G. Bautista-Flores, A.N. de San Miguel, pulse water molecules inside the membrane matrix. This E.R. Muhammed, M. and Gyves, J. (2005) Transport characterisation of a PIM system used for the extraction of Pb(II) means that embedding calix[4]resorcinarene with long ali- using D2EHPA as carrier, J. Memb. Sci, 250, 247-257. phatic chains into the CA matrix could create a membrane [3] Alguacil, F. J. Coedo, A. G. Dorado, M. T. and Padilla, I. with greater hydrophobic content [37]. (2001) Phosphine oxide mediate transport: modeling of mass transfer in supported liquid membrane transport of gold (III) 3.12 Stability of the PIM using Cyanex923, Chem. Eng. Sci, 56,3115-3122. The stability of the PIM was evaluated under the same [4] Boyadzhiev, L. Lazarova, Z. and Noble, R.D. Stern (Eds.), conditions where the feed and strip phases were renewed S.A., (1995) Membrane Separations Technology: Principles every 24 h without changing the membrane. During the and Applications, Elsevier Science B.V,. pp. 28-300. transport of Pb(II) ions through the PIM carried out for 24 [5] Kocherginsky, N.M. Yang, Q. and Seelam, L. (2007) Recent h (first cycle), no decrease in transport efficiency was ob- advances in supported liquid membrane technology, Sep. Pur. Tech, 53, 171-177. served. However, when further transport experiments were carried out using the same membrane, it was observed that [6] Mohapatra, P.K., and Manchanda, V.K. (2003) Liquid membrane based separations of actinides and fission products, the PIM was stable during the first five cycles (Fig. 10). Ind. J. Chem., 42(A): 2925-2939. From the sixth cycle on, a remarkable decrease was observed, particularly after seven cycles, by approximately [7] Bhattacharyya, A., Mohapatra, P.K., Hassan, P.A. and Manchanda, V.K. (2011) Studies on the selective Am3+ 20%, as shown in Fig. 10. The decrease in the stability of transport, irradiation stability and surface morphology of the membrane may have been caused by the partitioning of polymer inclusion membranes containing Cyanex-301 as the carrier between the membrane and the aqueous solution carrier extractant, J. Hazard. Mat., 192: 116-123. [55]. Moreover, the stability of the PIM was influenced by [8] Cherif, A.Y., Arous, O., Amara, M., Omeiri, S., Kerdjoudj, H., the properties of plasticiser used in the PIM [36,57]. and Trari, M. (2012) Synthesis of modified polymer inclusion membranes for photo-electrodeposition of cadmium using po- larized electrodes, J. Hazard. Mat., 227-228: 386-393.

[9] Gherasim, Cristina-Veronica I. , Bourceanu, G., Olariu, R.I. and Arsene, C. (2011) A novel polymer inclusion membrane 4. CONCLUSIONS applied in chromium (VI) separation from aqueous solutions, J. Hazard. Mat., 197:244-253. Homogeneous, transparent, and mechanically stable [10] Lamb, J. D,; West, J. N., Shaha, D. P. and Johnson, J. C. PIMs with CA as base polymer, NPOE/NPOT as plasti- (2010) An evaluation of polymer inclusion membrane per- ciser and calix[4]resorcinarene as carrier were prepared, formance in facilitated transport with sequential membrane and tested for their ability to extract Pb(II), Zn(II) and reconstitution, J. Memb. Sci., 365:256-259. Cu(II) from their acidic aqueous solution. The optimal [11] Panja, S., Mohapatra, P.K., Tripathi, S.C. and Manchanda, transport conditions were obtained at pH 5.1 in the feed V.K. (2011) Facilitated transport of uranium(VI) across sup- phase and pH 1.5 in the receiving phase. The experiments ported liquid membranes containing T2EHDGA as the carrier extractant. J. Hazard. Mat., 188: 281-287. demonstrated that the mechanism of metal ions transport by the PIM system occurred by facilitated counter- [12] St John, A. M., Cattrall, R. W. and Kolev, S. D. (2012) Transport and separation of uranium(VI) by a polymer inclu- transport mechanism. Also, the optimal content of PIM was sion membrane based on di-(2-ethylhexyl) phosphoric acid. found to be 0.13 ml of plasticiser (NPOE) per 1.0 g of CA J. Memb. Sci., 409- 410:242-250. with 10.8 mM of calix[4]resorcinarene. The membrane has [13] Raut, D.R., Kandwal, P., Rebello, G. and Mohapatra, P.K. been reused for five consecutive cycles without apprecia- (2012) Evaluation of polymer inclusion membranes contain- ble deterioration in performance, which showed the good ing calix[4]-bis-2,3-naptho-crown-6 for Cs recovery from stability. This work provides an opportunity to explore the acidic feeds: Transport behavior, morphology and modeling efficiency of a PIMs system (with CA as base polymer, studies. J. Memb. Sci., 407-408: 17-26. NPOE/NPOT as plasticiser and calix[4]resorcinarene as [14] Radzyminska-Lenarcik, E. and Ulewicz, M. (2012) Selec- carrier) in treatment of municipal wastewater. Although, tive Transport of Cu(II) across a Polymer Inclusion Mem- brane with 1-Alkylimidazole from Nitrate Solutions, Sep. results clearly indicates the efficacy of PIMs system in Sci. Tech., 47: 1113–1118. wastewater treatment but further detailed studies are still [15] Zawierucha, I., Kozlowski, C. and Malina, G. (2013) Re- required to answer few key issues of this system. moval of toxic metal ions from landfill leachate by comple- mentary sorption and transport across polymer inclusion membranes. Waste Management., 33 :2129-2136. The authors have declared no conflict of interest. [16] Nghiem, L.D., Mornane, P., Potter, I.D., Perera, J.M. and Cattrall, R.W.; Kolev, S.D. (2006) Extraction and transport of metal ions and small organic compounds using polymer inclusion membranes (PIMs ). J. Memb. Sci., 281:7-41. REFERENCES [17] Crowley, M.M., Schroeder, B., Fredersdorf, A., Obara, S., Tal- arico, M., Kucera, S. and McGinity, J.W. (2004) Physico- [1] Sgarlata, C. Arena, G. Longo, E. Zhang, D. Yang, Y. R.A. chemical properties and mechanism of drug release from ethyl and Bartsch, R.A. (2008) Heavy metal separation with pol- cellulose matrix tablets prepared by direct compression and ymer inclusion membranes. J. Memb. Sci, 323,444-451. hot-melt extrusion. Int. J. Pharm., 269:509-522.

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Preparation characterization and applicability of cellulose acetate–polyurethane blend membrane in separation tech- [44] Harrick, N.J. (1971) Determination of refractive index and niques. Colloids Surfaces A: Physicochemical and Engineer- film thickness from interference frings. Applied optics., 10 ing Aspect., 370: 120-128. :2344-2349. [29] Regel-Rosocka, M., Nowak, L. and Wisniewski, M. (2012) [45] Zhang, P., Yang, L.C., Li, L., Qu, Q.T., Wu, Y.P. and Removal of zinc(II) and iron ions from chloride solutions with Shimizu, M. (2010) Effects of preparation conditions on po- phosphonium ionic liquids. Sep. Puri. Tech., 97: 158–163 rous polymer membranes by microwave assisted efferves- cent disintegrable reaction and their electrochemical proper- [30] Valente, A.J.M., Polishchuk, A.Y., Burrows, H.D. and ties. J. Memb. Sci., 362: 113-118. Lobo, V.M.M. 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Tech., 27: 269-276. bit-[5]uriluril, p-tert-butylcalix[4]arene and p-tertbutyl- calix[6]arene in aqueous solutions using a spectrophotomet- [48] De San Miguel, E.R., Aguilar, J.C. and De Gyves, J. (2008) ric method. Mat. Sci. Eng C., 14: 35-39. Structural effects on metal ion migration across polymer inclusion membranes: Dependence of transport profiles on na- [32] Danil de Namor, F., Cleverley, R.M. and Zapata-Ormachea, ture of active plasticizer. J. Memb. Sci., 307 :105-116. M.L. (1998) Thermodynamics of calixarene chemistry,. Chem. Rev., 98:2495-2525. [49] Arous, O., Kerdjoudj, H. and Seta, P. (2004) Comparison of carrier-facilitated silver (i) and copper(ii) ions transport mech- [33] Ikeda, A. and Shinkai, S. (1997) Novel cavity design using anisms in a supported liquid membrane and in a plasticized calix[n]arene skeletons: Toward molecular recognition and cellulose triacetate membrane. J. Memb. Sci., 241 :177-185. metal binding. Chem. Rev., 97 1713-1734. [50] Gherrou, A., Kerdjoudj, H., Molnari, R. and Seta, P. (2005) [34] Antipin, I., Stoikov, I.I., Pinkhassik, E.M., Fitseva, N.A., Preparation and characterization of polymeric plasticized Stibor, I. and Konovalov, A.I. (1997) Calix[4]arene based membranes (PPM) embedding a crown ether carrier applica- a-aminophosphonates: Novel carriers for zwitterionic amino tion to copper ions transport. Mat. Sci. Eng C., 25(4) :436- acids transport. Tetra. Lett., 33:5865-5868. 443.

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[51] Pośpiech,B. (2012) Separation of Silver(I) and Copper(II) from Aqueous Solutions by Transport through Polymer In- clusion Membranes with Cyanex 471X. Sep. Sci. Tech., 47: 1413-1419. [52] Konczyk, J., Kozlowski, C. and Walkowiak, W. (2010) Re- moval of chromium(III) from acidic aqueous solution by polymer inclusion membranes with D2EHPA and Aliquat 336. Desalin., 263:211-216. [53] Mohapatra, P.K., Pathak, P.N., Kelkar, A. and Manchanda, V.K. (2004) Novel polymer inclusion membrane containing a macrocyclic ionophore for selective removal of strontium from nuclear waste solution. N. J. Chem., 28 :1004-1009. [54] Kebiche-Senhadji, O., Mansouri, L., Tingry, S., Seta, P. and Benamor, M. (2008) Facilitated Cd(II) transport across CTA polymer inclusion membrane using anion (Aliquat336) and cation (DE2HPA) metal carriers. J. Memb. Sci., 310:438- 445. [55] Bayou, N., Arous, O., Amara, M. and Kerdjoudj, H. (2010) Elaboration and characterization of a plasticized cellulose triacetate membrane containing trioctylphosphine oxyde (TOPO): application to the transport of uranium and molybdenum ions. Comp. Rend. Chim., 13 :1370-1376. [56] Kebiche-Senhadji, O., Tingry, S., Seta, P. and Benamor, M. (2010) Selective extraction of Cr(VI) over metallic species by polymer inclusion membrane (PIM) using anion (Aliquat 336) as carrier. Desalin., 258: 59-65. [57] Scindia, Y.M., Pandey, A.K. and Reddy, A.V.R. (2005) Cou- pled-diffusion transport of Cr(VI) across anion-exchange membranes prepared by physical and chemical immobilization methods. J. Memb. Sci., 249:143-152.

Received: September 08, 2014 Revised: February 02, 2015 Accepted: February 19, 2015

CORRESPONDING AUTHORS

Nadjib Benosmane Departement of Chemistry Faculty of Sciences University M’Hame Bougara de Boumerdes (UMBB), Avenue de l’indépendance 35000, Boumerdes ALGERIA and Laboratoire de Chimie Organique Appliquée Faculty of Chemistry USTHB BP 32 El-Alia 16111 Alger ALGERIA E-mail: [email protected]

Baya Boutemeur Laboratoire de Chimie Organique Appliquée Faculty of Chemistry USTHB BP 32 El-Alia 16111, Alger ALGERIA E-mail: [email protected]

FEB/ Vol 24/ No 7/ 2015 – pages 2296 - 2309

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IN VITRO ANTIOXIDANT POTENTIAL OF para-ALKOXY- PHENYLCARBAMIC ACID ESTERS CONTAINING 4-(4-FLUORO- /3-TRIFLUOROMETHYLPHENYL)PIPERAZIN-1-YL MOIETY

Ivan Malík1,*, Jan Muselík2, Lukáš Stanzel1, Jozef Csöllei3 and Matej Maruniak1

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic 2Department of Pharmaceutics, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic 3Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

ABSTRACT antioxidant properties of concerned compound and the impacts on cardiac remodeling could be also responsible for its Antioxidant properties of certain β-adrenoceptor block- final beneficial outcome [3]. The potency of carvedilol to in- ing agents (β-ABAs) may provide their significant benefit in hibit reactive oxygen-derived free radicals might also be rel- the therapy of cardiovascular diseases. Considering men- evant in heart failure therapy [4-6]. tioned, the potential of original β-ABAs, para-alkoxy- As shown in Figure 1, chemical structure of carvedilol phenylcarbamic acid esters 6i–6l and 8f–8i with incorpo- consists of some fundamental parts: (i) lipophilic carbazole rated 4-(4-fluoro- or 3-trifluoromethylphenyl)piperazin-1- moiety (the substituent R) which is responsible for antioxi- -yl fragment, to reduce stable 2,2-diphenyl-1-picrylhydra- dant properties, (ii) etheric oxygen directly connected to zyl radical (DPPH•) applying UV/VIS spectrophotometry 2-hydroxypropane-1,3-diyl chain which is essential for the and to scavenge peroxynitrite ions (ONOO–) using HPLC β-blockade and (iii) basic fragment (guaiacoxyethylamino method, has been in vitro investigated. It has been observed group) which primarily invokes blocking ability on α-adre- that para-position of attached alkoxy group has appeared noceptors resulting in moderate vasodilator properties to be most suitable for antioxidative efficiency. The in- of considered molecule [7, 8]. crease in lipophilicity in homological series 6i–6l and 8f– During past two decades, in vitro and in vivo antioxidant 8i could be regarded as important but it has been questiona- potency of various third-generation non-selective β-blockers ble whether principal factor in terms of capabilities of these with both β - and β -adrenoceptor as well as α -adrenergic esters being effective reductants of DPPH• and the ONOO– 1 2 1 receptor blocking activities have been investigated [9, 10]. scavengers as well. From entire evaluated set, para-meth- These compounds (Figure 1), namely ferulidilol which was oxy substituted molecule 6i has been able to reduce refer- derived from ferulic acid and isoeugenodilol which was de- ence DPPH• most markedly. The in vitro experiments have rived from isoeugenol, isoform of eugenol, have contained also revealed the most pronounced ONOO– scavenging po- lipophilic vanilloid base. The modification of lipophilic and tential of para-propoxy substituted compound 8h. salt forming compartments and isosteric replacement of polar etheric bridge by carbamoyloxy moiety has led to the

KEYWORDS: β-adrenoceptor antagonists, antioxidant efficiency, class of presently investigated para-alkoxyphenylcarbamic positional isomerism acid derivatives 6i–8i (Table 1).

Concerning legislative complexity which has been connected with preparation and realization of pharmacological 1. INTRODUCTION experiments focused on the ability of inspected derivatives to influence the functions of cardiovascular system, it has In clinical practice, β-adrenoceptor blocking agents (β- been possible to investigate relatively narrow group of the -ABAs) have shown several beneficial cardiovascular con- compounds. Preliminary pharmacological evaluation has in- tributions to patients with hypertension, angina pectoris dicated the ability of some molecules from the set 6 to pro- or myocardial infarction [1]. It has been reported that part tect heart against extrasystoles, fibrillations and heart failure of carvedilol´s effects, a lipophilic third-generation non-se- exceeding the effect of propaphenone [11]. Following phar- lective vasodilating β-ABA [2], might be related to the re- macological screening of the compound 6j [12], it has been duction of myocardial oxygen demand and cardiac workout suggested that an incorporation of carbamoyloxy group has as a result of β-adrenoceptor blockade. In addition, notable not significantly influenced its β-adrenolytic activity compared to original aryloxyaminopropanols. Moreover, its * Corresponding author ability to in vitro inhibit contractibility of aortal strips and

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to decrease a blood pressure in normotensive and hyperten- Other compounds, which have been used in carried in sive rats has enabled to consider 6j as a proper representa- vitro experiments, were purchased: tyrosine (BDH Chemi- tive of potential antihypertensives [12]. Fundamental prop- cals, United Kingdom), 3-nitrotyrosine (Sigma-Aldrich, Ger- erty of β-ABAs to antagonize the influence of β-sympatho- many), carvedilol (Sigma-Aldrich, Germany) and Trolox, mimetics has been in vitro evaluated for the compound chemically (±)-6-hydroxy-2,5,7,8-tetramethylchromane- 6j [12] and 6k [11], respectively. The experiments have -2-carboxylic acid (Fluka Chemie, Switzerland). All these been based on the molecules´ characteristics to specifically chemicals were of an analytical grade. block positively chronotropic effect of isoprenaline. The estimated value of pA2 for 6j was 7.9 and for 6k was 9.2 2.2 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Reduction which indicated their notable anti-isoprenaline Assay (β-adrenolytic) efficiency [11, 12]. Free radical reduction ability of tested compounds was Outlined backgrounds have motivated current research determined with the DPPH assay by following the proce- to explore whether any of above mentioned compounds 6i– dure described in research articles of Malík et al. [17] 8i could be also antioxidants under in vitro conditions. and Masteiková et al. [18]. The solution (200 µL) of particular compound dissolved in ethylene glycol monomethyl –1 ether (c=0.1 mg·mL ) was made up to 2.0 mL with meth- –1 2. MATERIAL AND METHODS anolic solution of DPPH (c=0.1 mmol·L ), one of a few stable organic nitrogen radicals with strong visible absorption 2.1 Synthesis and physicochemical parameters determination [19]. After 5 min, the absorbance value was measured at the of the compounds under the study wavelength of 517 nm using the UV/VIS spectrophotometer Preparation of currently evaluated compounds 6i–6l HP 8453 (Hewlett Packard, USA). Decrease in the absorb- and 8f–8i (Table 1), chemically 1-[3-(4-alkoxyphenylcar- ance of respected DPPH solution has indicated increase in bamoyloxy)-2-hydroxypropyl]-4-(4-fluoro-/3-trifluoro- DPPH radical (DPPH•) scavenging effect [19, 20]. methylphenyl)piperazinium chlorides (where alkoxy= Reduction of DPPH• was calculated relatively to the methoxy to butoxy group), their spectral characteristics measured absorbance of control as means ± standard devi- and elemental analyses data have already been pub- ation (SD) of three parallel measurements according to the lished [13, 14]. equation given below: Determination of some physicochemical parameters of  A  these molecules, i.e. solubility profile, dissociation con- %DPPH 1 sample  100     stant pKa and lipophilicity descriptors (the log Pexps esti-  Acontrol  mated by shake-flask method in the octan-1-ol/buffer medium with pH=7.3, the log k´s from RP-HPLC, the RMs where: %DPPH – the percentage of the DPPH reduc- from RP-TLC), with corresponding readouts can be found tion; Asample – the absorbance at 517 nm; Acontrol – the ab- in research papers [13-16]. sorbance at 517 nm in control measurement.

TABLE 1 – The potential of evaluated compounds 6i–8i to reduce DPPH radicals (%DPPH) and to protect tyrosine from peroxynitrite mediated nitration (%INH).

OH H H N O N N . Cl

O R2 R1

1 2 a Entry R R log Pexp %DPPH %INH

6i OCH3 4´-F 3.42 7.7 ± 0.2 -8.5 ± 0.3 6j OC2H5 4´-F 3.28 5.3 ± 0.3 -10.4 ± 0.3 6k OC3H7 4´-F 3.12 7.1 ± 0.2 -11.7 ± 0.4 6l OC4H9 4´-F 3.54 7.2 ± 0.2 -10.0 ± 0.2

8f OCH3 3´-CF3 3.60 1.2 ± 0.2 2.4 ± 0.3

8g OC2H5 3´-CF3 3.71 0.9 ± 0.1 -0.1 ± 0.2 8h OC3H7 3´-CF3 3.92 1.0 ± 0.1 3.6 ± 0.2 8i OC4H9 3´-CF3 3.98 1.0 ± 0.1 -1.3 ± 0.3 Carvedilol – – – 15.4 ± 0.2 0.8± 0.5 Trolox – – – 94.7 ± 0.3 51.3 ± 0.2 a The log Pexp readouts of investigated compounds have been adopted from the article [15]

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FIGURE 1 – Chemical structure of some non-selective α-/β-adrenoceptor blocking agents which have shown antioxidant properties.

2.3 Peroxynitrite Scavenging Potential column temperature was set to 25 °C, mobile phase con- 2.3.1 Preparation of Solutions sisted of 90 % HCOOH (c=40 mmol·L–1) and 10 % –1 Peroxynitrite solution was prepared by applying previ- CH3CN (v/v) at a flow rate of 1 mL·min . Chromatograms ously published method [21]. Aliquots of H2O2 (c=0.6 were detected at the wavelength of 276 ± 20 nm against –1 –1 mol·L ) dissolved in HClO4 (c=0.5 mol·L ) and NaNO2 reference wavelength of 600 ± 100 nm. (c=0.5 mol·L–1) in water (10 mL each) were pre-cooled to - The activity of inspected compounds (including refer- 2 °C and rapidly mixed. The reaction was quenched imme- ence drugs) was calculated relative to the measured 3-ni- diately by an addition of 5 mL cold NaOH (c=3.5 mol·L–1). trotyrosine peak area of the control as means ± standard Unreacted H2O2 was removed by a treatment with an excess deviation (SD) of three parallel measurements according of MnO2 and the solution was filtered. The concentration of following equation: ONOO ions was determined spectrophotometrically in al- 1 1  AUCsample  kaline solution using 302=1670 L·mol ·cm . The stock so- %INH  1 100   lutions were stored frozen at -80 °C for several months with-  AUCcontrol  out noticeable decomposition. where: %INH – the percentage of inhibition of tyrosine The volume of 8 μL of prepared peroxynitrite solution nitration; AUCsample – the area under curve of 3-nitrotyro- (c=15 mmol·L–1) in NaOH (c=50 mmol·L–1) was drawn and sine peak; AUCcontrol – the area under curve of 3-nitrotyro- rapidly mixed in the injector of HPLC autosampler with sine peak in control measurement. 42 μL of tyrosine solution (c=1.0 mmol·L–1) in 80 mmol·L–1 The percentage of inhibition of tyrosine nitration was KH PO –Na HPO buffer (pH=6.0) containing 0.25 mmol· 2 4 2 4 compared to that of the Trolox standard. L–1 of tested compound and ethylene glycol monomethyl ether (in a 1:1 ratio with water solution).

2.3.2 Peroxynitrite Scavenging Assay Using HPLC System 3. RESULTS AND DISCUSSION

The reaction mixture was directly injected into the In this study, both in vitro antioxidant tests, i.e. the HPLC system HP 1100 with autosampler, quaternary pump DPPH radical reduction assay and the peroxynitrite scav- and diode-array detector (Agilent Technologies, USA). The enging evaluation, were used to examine, compare and ex- separation was carried out with Supelcosil ABZ + Plus col- plain (i) differences in the potency of para-alkoxyphenyl- umn (250 × 4.6 mm, 5 μm particle size; Supelco, USA),

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carbamic acid esters 6i–8i (Table 1) and their previously in- loss (ET-PT) with the proton being initially transferred into vestigated positional ortho- and meta-alkoxy isomers [17]. a solvent molecule to which is phenol hydrogen-bonded. (ii) Furthermore, the compounds of the series 6 and 8 have In terms of stereochemistry, currently (6i–8i) and pre- been varying in hydrogen bonding ability, electrostatic and viously (6a–8e) in vitro evaluated compounds have been steric effects due to different fluorine-containing substitu- racemates. They have primarily differed in position of ent which has been directly bonded to the aromatic ring alkoxy side chain attached to lipophilic aromatic ring. In of 4-(phenyl)piperazin-1-yl moiety. For completeness, pre- addition, all the molecules within the series of 6 (6a–6f and vious paper [17] has focused, among others, on the factors 6i–6l) have contained 4-(4-fluorophenyl)piperazin-1-yl stated in paragraph (ii) so they will be indicated in the cur- fragment compared to the compounds from the set 8 (8a–8e rent research only briefly. and 8f–8i) bearing more lipophilic 4-(3-trifluoro- Reaction mechanism of DPPH with potential antioxi- methylphenyl)piperazin-1-yl moiety. The idea that antiox- dants, particularly with phenols and polyphenols, is still a idant properties of ortho-/meta-/para-alkoxyphenylcar- subject of controversy. Taking into the consideration chem- bamic acid esters could be modified by a suitable selection ical reactions involved, hydrogen atom abstractions from of lipophilic fragment R (Figure 1), has been inspired by phenols are now recognized to proceed by four different the research of Feuerstein et al. [7]. mechanisms [22-25]: hydrogen-atom transfer (HAT), pro- In terms of the ability to reduce the DPPH radicals, it ton-coupled electron-transfer (PCET), sequential proton- has been reported [17] that ortho-alkoxy substituted com- loss electron transfer (SPLET), and electron-transfer proton- pounds 6a–6c (within general chemical structure in Table 1,

FIGURE 2 – The formation of virtual five-membered ring (A) for the compounds 6a–6c in contrast to the activating electronic effect of ortho- -alkoxy moiety serving for the stabilization of radical (B).

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methoxy, ethoxy or propoxy substituent R1 was attached to which has contained identical lipophilic part as the sub- the position 2 of phenyl ring) have been considered more stance 6a, has shown that MAMM had an almost planar effective (%DPPH=2.0 ± 0.1 – 7.6 ± 0.2) than meta-alkoxy structure and intramolecular N–H ··· O hydrogen bond has substituted (the R1 substituent attached to the position 3) formed a planar five-membered ring which has helped to derivatives 6d–6f (%DPPH=0.4 ± 0.1 – 1.3 ± 0.1) flatten the molecule. Following calculated data, the dis- As current research has revealed, para-alkoxyphenylcar- tance between hydrogen of NH-group and oxygen of bamic acid esters 6i–6l (the R1 substituent attached to 4 posi- 2-methoxy moiety has been 2.18 Å. Planar structure has tion of phenyl ring) have been regarded as slightly more effi- been favorable for conjugation between donor and acceptor cient than the compounds 6a–6c. Furthermore, all currently parts of the derivative [29]. According to Pauling’s princi- and previously inspected substances containing 4-(3-tri- ple [30], the strength of that hydrogen bond has increased fluoromethylphenyl)piperazin-1-yl group have shown the with the increase in the electronegativity of the acceptor %DPPHs not higher than 1.4 ± 0.2 [17]. (oxygen) atom. Following the values of the bond lengths, trigonal nitrogen atom has shown stronger conjugation Based on resonance theory [26], the linearity of these with double bond than with the aromatic ring [29]. aromatic compounds with the substituent attached to para- -position has made resonance (mesomeric) effect at phenyl Another compound which contained very similar lipo- ring which has affected their electron distribution and lipophilic fragment to the derivative 6a, N-(4-amino-2-meth- hydrophilic properties as well. para-Alkoxy fragments have oxyphenyl)acetamide, has been the product of second step primarily acted through the resonance as electron-donating in the synthesis of N-(4-amino-2-methoxyphenyl)me- groups which have been able to enhance the basicity of ni- thanesulfonamide, the side chain of an anticancer drug. trogen atom. Given substituents could distribute negative Following its hydrogen-bonding geometry data, the discharge towards amino moiety as a part of carbamate group fa- tance between oxygen of 2-methoxy group and hydrogen cilitating its protonation. Nevertheless, described elec- of anilide moiety has been lower than 2.50 Å (it has been tron-donating resonance effect has been countered by 2.19 Å, in fact) indicating the formation of five-membered the electron-withdrawing inductive one of these alkoxy quasicycle [31]. substituents. Anyway, for para-position, positive me- When considering the compound 6a the most effective someric effect has dominated [27]. reductant of DPPH• within the 6a–6c set, it could be sug- Similarly, in the series of ortho-alkoxy substituted com- gested an additional improvement of such ability. Theoret- pounds 6a–6c [17], the introduction of alkoxy group has led ically, the breakage of IHB could be achieved by (i) so- to the increase in electron density of phenyl ring due to called secondary substituent effect, i.e. by the introduction above mentioned activating positive mesomeric effect. On of another meta-alkoxy side chain (manning para-position the other hand, it has been suggested [17] that intramolecular towards original ortho-methoxy substituent) into the chem- hydrogen bond (IHB) has been formed between N–H group ical structure of the molecule 6a. Firstly, the electrons of carbamoyloxy fragment and oxygen atom of ortho-alkoxy would flow from alkoxy to methoxy by ortho-, para-reso- side chain. The consequence of that process was the creation nance [32]. Consequently, electron density of original or- of virtual five-membered ring, as illustrated in Figure 2 (part tho-methoxy group would be enhanced by strong electron- A). All these intramolecular interactions have supported the -donating nature of attached alkoxyl and methoxy moiety forming of regions of negative electrostatic potentials, serv- might have more electron density than it could accommo- ing thus as electron-donating site. It should be further clari- date. That electron excess would be handed to ortho-posi- fied that hydrogen bond has opposed the activating elec- tion of methoxy group. (ii) The incorporation of electron- tronic influence of ortho-alkoxy moiety (stabilization of the -donating substituents into para-position (e.g. methyl, radical), as drawn in Figure 2 (part B), and has slightly de- methoxyl) would lead to the increase in electron density on creased the reactivity of ortho-alkoxy positional isomers. the „aniline“ nitrogen. In the structure of such ortho-/para- Pankratov and Shalabay [28] have studied electronic -substituted molecule, the radical electron would be stabi- structure of the series of organic molecules including the lized on para-position to increase antioxidant capability of given derivative. derivatives of aniline 2-XC6H4NH2 (where X=CHO, COOH, NO, NO2, OH, OCH3, SH, SCH3, F, Cl or Br) by In a series of meta-alkoxy substituted substances 6d– means of ab initio Hartree-Fock (HF) method with the 6f [17], assuming the existence of nitrogen radical on car- 6-311G(d,p) basis set. That theory level HF/6-311G(d,p) bamoyloxy moiety, strong electron-withdrawing influence has been sufficient for the overview of tendency in electron of the radical has caused that meta-alkoxy fragment has density redistribution on the IHB formation in that series. acted as relatively weaker electron-donating group. Their research has proven the IHB establishment to lead On the other hand, positive mesomeric effect of para- to a local electron redistribution in quasicycle and primar- -alkoxy chain (with no hydrogen bond) and inferential in- ily to the electron density transfer between direct IHB par- crease in the electron density on aromatic ring has slightly ticipants of binding – from „mobile“ hydrogen atom to- favored the compounds 6i–6l in terms of their potential to ward the proton-acceptor atom [28]. interact with DPPH• (Table). Resonance (valence-bond) X-Ray crystallographic investigation of [(2-methoxy- structures proposal of the radicals for considered deriva- anilino)methylene]malononitrile (MAMM), a molecule tives 6i–6l has been shown in Figure 3.

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FIGURE 3 – Resonance (valence-bond) structures proposal of the radicals for investigated compounds 6i–6l.

Quantitative and qualitative spin density distribution and to scavenge ONOO– ions as well. The most efficient within the set of 6i–6l will be the subject of further research methoxy derivative 6i (%DPPH=7.7 ± 0.2) has not been employing computational ab initio methods. regarded as the most lipophilic one [15]. In addition, given From structural viewpoint, the presence of another molecule was slightly less lipophilic, log Pexp=3.42 (Ta- phenyl ring within basic part of investigated derivatives ble), than the compound 6a, log Pexp=3.61 [34], which has 6i–8i has established the possibility for DPPH• to interact shown comparable potential to reduce DPPH• expressed by with this moiety. By contrast, strong electron-withdrawing the readout of %DPPH=7.6 ± 0.2 [17]. inductive effect, which has been invoked by 3-trifluorome- Following previous pharmacological evaluation of the thyl group, has meant the decrease in electron density compounds 6k, its capability of being moderate DPPH re- on the aromate. Following mentioned, DPPH• as an oxi- ductant (%DPPH=7.1 ± 0.2) could contribute to notable dant has been able to abstract electron from aromatic ring β-adrenolytic efficiency which given molecule has shown of 4-(3-trifluoromethylphenyl)piperazin-1-yl more heavily previously [11]. and that was the reason why the compounds 8f–8i have shown lower %DPPH data compared to the series of 6i–6l For comparison, Oettl et al. [35] have published that (Table 1). carvedilol has acted both as metal chelator and in vitro radical scavenger. However, that β-ABA has been considered The research of Butler et al. [33] has pointed out that selective in a reacting with different types of radicals – it carvedilol´s lipophilic carbazole group (the R substituent in has provided no reaction with nitrogen-centered DPPH• Figure 1) has played dominant role in bilayer perturbation. and it has not been regarded as an electron-donating radical The combination of specific chemical structure of carve- scavenger. Current research has indicated that carvedilol dilol and its high lipophilicity has appeared as required for has shown the value of %DPPH=15.4 ± 0.2 (Table 1) and carvedilol´s antioxidant potential. Current investigations has been considered doubly more efficient than the com- have indicated that the increase in lipophilicity (presuming pound 6k. its certain „requisite“ degree) could be important, but it has been not clear if principal factor in terms of the abilities of Following current and previously published experi- these alkoxyphenylcarbamic acid esters to reduce DPPH• mental observations, it could be hypothesized that the re-

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placement of para-alkoxyphenyl moiety by the 2,6-dime- to be a potent scavenger of reactive oxygen radicals and thylphenyl or 2,4,6-trimethylphenyl one would lead to the singlet oxygen quencher as well [40, 41]. Considering compounds with more promising antioxidative potential. given substance to be an antiradical drug, it was also wor- The introduction of methyl groups would mean the in- thy note that lidocaine could not donate its hydrogen atom crease in electron density on the aromate which has been (anilide group has been supposed to be a donor) to the attributed to hyperconjugation effect. It has taken place DPPH. Atom of nitrogen in lidocaine radical has been at- through the interaction of σ electrons of carbon-hydrogen tached to phenyl ring directly (Figure 4) so aromate could bond with π-electrons of aromatic system. Such hypercon- stabilize a single electron. Meanwhile, two methyl groups jugation occurs through hydrogen atom bonded to α-car- have also supported the stabilization of thus formed N-cen- bon which is directly attached to the aromate [36]. Conse- tered radical. Following mentioned, concerned LA has scav- quently, methyl groups have shown electron-donating (ac- enged radicals preferentially by reducing them rather than tivating) properties. The selection of electron-donating sub- donating its hydrogen atom radicals [42]. Analogously with stituents, however, should be made with care. The electronic the research of Ohkatsu and Nishiyama [43], the presence of nature and steric hindrance have been believed to be im- α-hydrogen of ortho-methyl group could be used for regen- portant, in order to explain the function of ortho-substitu- eration of 2,6-dimethylphenylcarbamoyl moiety. The au- ents. For example, the bonding of tert-butyl group into the thors have also suggested that electron-withdrawing group ortho-position would lead to slightly complicated situation. on α-carbon of ortho-substituent (i.e. ortho-chloromethyl) The antioxidant capability of thus substituted derivative could enhance effectiveness of thus derivatives. could be negatively influenced by expected inhibition Turning an attention to the inspection of peroxynitrite of free rotation of N–H group due to steric hindrance. scavenging potential, current research has shown that addi- Three methyl groups are included in the chemical struction of almost all evaluated compounds, except of 8f and 8h, ture of non-selective β-blocker metipranolol which has been has led to catalysis of peroxynitrite mediated nitration pro- also used to reduce intraocular pressure in the treatment cess. That fact has been indicated by negative values of of glaucoma [37]. Given compound and its active metabolite %INH (Table 1). The highest ability to inhibit the nitration desacetylmetipranolol have shown the ability to inhibit both of tyrosine has been assigned to the molecule 8h (%INH=3.6 iron/ascorbate and sodium nitroprusside-induced lipid pe- ± 0.2). That derivative has been considered more efficient roxidation in rat brain homogenates [38]. Described struc- than carvedilol (%INH=0.8 ± 0.7). The %INH readouts of tural arrangement could influence the lipid peroxidation. ortho-/meta-alkoxy substituted derivatives 6a–6f and 8a– For completeness, to be the best of authors´ knowledge, no 8e [17], respectively, have been, however, lower compared research describing metipranolol´s potential to interact to the value of Trolox (%INH=51.3 ± 0.2). with DPPH• has been reported in literature yet. In contrast, it has been reported that carvedilol treat- In addition, phenyl ring substituted by methyl groups in ment has significantly uppressed inducible NO synthase the positions 2 and 6 is also incorporated into chemical struc- expression (and subsequent ONOO– formation), decreased ture of well-known potent local anaesthetic (LA) agent, li- nitrotyrosine formation, improved endothelial function, docaine [39]. and reduced vascular cell death in animal models [44]. How- Another structural similarity of given LA with the mol- ever, peroxynitrite released by inflammatory cells might ecules included in currently tested set is in the nature of affect the potency of lidocaine through the inhibition nitrogen-containing moiety. Carbamate bond (NHCOO) of of its membrane-fluidizing effect. That interaction might 6i–8i is isosterically replaced by the anilide (NHCO) one, be related to the failure of local anaesthetic in inflammed as drawn in Figure 4. Lidocaine has been previously found tissues [45, 46].

FIGURE 4 – Chemical structure of lidocaine and its N-centered radical which is stabilized by hyperconjugation effect of both methyl groups.

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4. CONCLUSIONS [7] Feuerstein, G.Z., Bril, A. and Ruffolo, Jr., R.R. (1997) Protective effects of carvedilol in the myocardium. Am. J. Cardiol. 80, 41-45. Major novel findings of current study has been that the [8] Yoshikawa, T., Port, J.D., Asano, K., Chidiak, P., Bouvier, M., Dutcher, D., Roden, R.L., Minobe, W., Trammel, K.D. and Bris- potency of evaluated compounds 6i–8i has been closely tow, M.R. (1996) Cardiac adrenergic receptor effects of carvedilol. connected with the position of alkoxy side chain at- Eur. Heart J. 17 (Suppl. B), B8-B16. tached to phenylcarbamoyloxy fragment. In regard to posi- [9] Huang, Y.-Ch., Yeh, J.-L., Wu, B.-N., Lo, Y.-Ch., Liang, J.-Ch., tional isomerism and electronic effects, it seemed that Lin, Y.-T., Sheu, S.-H. and Chen, I.-J. (1999) Ferulidilol: A vaso- dilatory and antioxidant adrenoceptor and calcium entry blocker, para-alkoxy substitution as well as the selection of the sub- with ancillary β -agonist activity. Drug Dev. Res. 47, 77-89. stituents with electron-donating and suitable steric proper- 2 [10] Yeh, J.-L., Yang, T.-H., Liang, J.-Ch., Huang, Y.-Ch., Lo, Y.-Ch., ties has contributed most positively to the reduction effect Wu, J.-R., Lin, Y.-T. and Chen, I.-J. (2000) Isoeugenodilol: A vas- of investigated compounds towards DPPH•. It has been orelaxant α/β-adrenoceptor blocker with antioxidant activity. Drug provided that bonding of short alkoxy group has been more Dev. Res. 51, 29-42. favorable than the presence of prolonged side chain. These [11] Račanská, E., Tumová, I. and Foltánová, T. (2005) 2-, And 4-fluor- structural features have notably influenced such effective- ophenylpiperazine derivatives of alkoxyphenylcarbamic acid and their potential antidysrhythmic effect. Farm. Obzor 74, 247-250. ness together with balanced lipohydrophilic properties. Current experimental observations have also shown that [12] Racanska, E., Balaz, M. and Malik, I. (2007) Effect of a newly synthesized fluorophenylpiperazine derivative on basic cardiovas- the introduction of strongly electron-withdrawing substitu- cular functions. Biomed. Pap. 151 (Suppl. 1), 74-75. ent, i.e. 3-trifluoromethyl group, to 4-(substituted phe- [13] Malík, I., Sedlárová, E., Csöllei, J., Račanská, E., Čižmárik, J. and nyl)piperazin-1-yl has provided slightly better prospect to Kurfürst, P. (2004) Synthesis, physico-chemical properties and bi- scavenge ONOO– ions than the incorporation of 4-fluoro ological activity of 1-(4-fluorophenyl)-4-[3-(2-, 3- and 4-alkyloxy- phenylcarbamoyloxy)-2-hydroxypropyl]piperazinium chlorides. substituent. Sci. Pharm. 72, 283-291.

[14] Malík, I., Sedlárová, E., Csöllei, J., Andriamainty, F., Kurfürst, P. and Vančo, J. (2006) Synthesis, spectral description, and lipo- ACKNOWLEDGEMENTS philicity parameters determination of phenylcarbamic acid derivatives with integrated N-phenylpiperazine moiety in the structure. The authors thank the anonymous reviewers for their Chem. Pap. 60, 62-67. valuable comments and helpful revision suggestions. [15] Malík, I., Sedlárová, E., Čižmárik, J., Andriamainty, F. and Csöl- lei, J. (2005) Study of physicochemical properties of 4-alkoxyphenylcarbamic acid derivatives with various substituted N-phe- The authors have declared no conflict of interest. nylpiperazin-1-yl moiety in the basic part of the molecule. Farm. Obzor 74, 211-215. [16] Sedlárová, E., Malík, I., Andriamainty, F., Kečkéšová, S. and Csöllei, J. (2007) Study of lipophilicity of phenylcarbamic acid derivatives with substituted N-phenylpiperazine moiety. Farm. Ob- REFERENCES zor 76, 86-90. [17] Malík, I., Muselík, J., Sedlárová, E., Csöllei, J. and Stanzel, Ľ. [1] Mak, I.T. and Weglicki, W.B. (2004) Potent antioxidant properties (2014) In vitro antioxidant properties of basic ortho-/meta-alkox- of 4-hydroxyl-propranolol. J. Pharmacol. Exp. Ther. 308, 85-90. yphenylcarbamic acids esters bearing 4-(4-fluoro-/3-trifluoro- [2] Bristow, M.R., Larrabee, P., Minobe, W., Roden, R., Skerl, L., methylphenyl)piperazin-1-yl fragment. Fresen. Environ. Bull. 23, Klein, J., Handwerger, D., Port, J.D. and Müller-Beckmann, B. 2361-2368. (1992) Receptor pharmacology of carvedilol in the human heart. J. [18] Masteiková, R., Muselík, J., Bernatonienė, J., Majienė, D., Cardiovasc. Pharmacol. 19 (Suppl. 1), S68-S80. Savickas, A., Malinauskas, F., Bernatonienė, R., Pečiūra, R., Cha- lupová, Z. and Dvořáčková, K. (2008) Antioxidant activity of tinc- [3] Yue, T.-L., Cheng, H.Y., Lysko, P.G., McKenna, P.J., Feuerstein, tures prepared from hawthorn fruits and motherwort herb. Čes. R., Gu, J.L., Lysko, K.A., Davies, L.L. and Feuerstein, G.Z. (1992) slov. Farm. 57, 35-38. Carvedilol, a new vasodilator and beta adrenoreceptor antagonist, is an antioxidant and free radical scavenger. J. Pharmacol. Exp. [19] Kaurinovic, B., Popovic, M., Vlaisavljevic, S., Zlinska, J. and Ther. 263, 92-98. Trivic, S. (2011) In vitro effect of Marrubium peregrinum L. (La- miaceae) leaves extracts. Fresen. Environ. Bull. 12, 3152-3157. [4] Chin, B.S.P., Langford, N.J., Nuttall, S.L., Gibbs, Ch.R., Blann, A.D. and Lip, G.Y.H. (2003) Anti-oxidative properties of [20] Mosquera, O.M., Correa, Y.M., Buitrago, D.C. and Niño, J. (2007) beta-blockers and angiotensin-converting enzyme inhibitors in Antioxidant activity of twenty five plants from Colombian biodi- congestive heart failure. Eur. J. Heart Fail. 5, 171-174. versity. Mem. Inst. Oswaldo Cruz 102, 631-634. [5] Gilbert, E.M., Abraham, W.T., Olsen, S., Hattler, B., White, M., [21] Vančo, J., Švajlenová, O., Račanská, E., Muselík, J. and Valen- Mealy, P., Larrabee, P. and Bristow, M.R. (1996) Comparative he- tová, J. (2004) Antiradical activity of different copper(II) Schiff modynamic, left ventricular functional, and antiadrenergic effects base complexes and their effect on alloxan-induced diabetes. J. of chronic treatment with metoprolol versus carvedilol in the fail- Trace Elem. Med. Biol. 18, 155-161. ing heart. Circulation 94, 2817-2825. [22] Litwinienko, G. and Ingold, K.U. (2007) Solvent effects on the [6] Rouleau, J.L., Roecker, E.B., Tendera, M., Mohacsi, P., Krum, H., rates and mechanisms of reaction of phenols with free radicals. Katus, H.A., Fowler, M.B., Coats, A.J., Castaigne, A., Scherhag, Acc. Chem. Res. 40, 222-230. A., Holcslaw, T.L. and Packer, M.; Carvedilol Prospective Ran- [23] Tishchenko, O., Truhlar, D.G., Ceulemans, A. and Nguyen, M.T. domized Cumulative Survival Study Group. (2004) Influence of (2008) A unified perspective on the hydrogen atom transfer and pretreatment systolic blood pressure on the effect of carvedilol in proton-coupled electron transfer mechanisms in terms of topo- patients with severe chronic heart failure: the Carvedilol Prospec- graphic features of the ground and excited potential energy sur- tive Randomized Cumulative Survival (COPERNICUS) study. J. faces as exemplified by the reaction between phenol and radicals. Am. Coll. Cardiol. 43, 1423-1429. J. Am. Chem. Soc. 130, 7000-7010.

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[24] Litwinienko, G. and Ingold, K.U. (2005) Abnormal solvent effects [44] Ma, X.-L., Gao, F., Nelson, A.H., Lopez, B.L., Christopher, T.A., on hydrogen atom abstraction. 3. Novel kinetics in sequential pro- Yue, T.-L. and Barone, F.C. (2001) Oxidative inactivation of nitric ton loss electron transfer chemistry. J. Org. Chem. 70, 8982-8990. oxide and endothelial dysfunction in stroke-prone spontaneous hy- pertensive rats. J. Pharmacol. Exp. Ther. 298, 879-885. [25] Galian, R.E., Litwinienko, G., Pérez-Prieto, J. and Ingold, K.U. (2007) Kinetic solvent effects on the reaction of an aromatic ke- [45] Ueno, T., Mizogami, M., Takakura, K. and Tsuchiya, H. (2008) tone π,π* triplet with phenol. Rate-retarding and rate-accelerating Membrane effect of lidocaine is inhibited by interaction with per- effects of hydrogen-bond acceptor solvents. J. Am. Chem. Soc. oxynitrite. J. Anesth. 22, 96-99. 129, 9280-9281. [46] Ueno, T., Mizogami, M., Takakura, K. and Tsuchiya, H. (2008) [26] Carey, F.A. and Sundberg, R.J. (2007) Advanced Organic Chem- Peroxynitrite affects lidocaine by acting on membrane-constituing istry, Part A: Structure and Mechanisms. 5th Ed. Springer, New lipids. J. Anesth. 22, 475-478. York, 1199 pp.

[27] Dewick, P.M. (2006) Essentials of Organic Chemistry: For Stu- dents of Pharmacy, Medicinal Chemistry and Biological Chemis- try. 2nd Ed. John Wiley and Sons, Chichester, 710 pp. [28] Pankratov, A.N. and Shalabay, A.V. (2007) Electronic structure of planar-quasicycled organic molecules with intramolecular hydrogen bond. J. Serb. Chem. Soc. 72, 265-273. [29] Nesterov, V.N., Viltchinskaia, E.A. and Nesterova, S.V. (2003) [(2-Methoxyanilino)methylene]malononitrile. Acta Cryst. E59, o625-o627. [30] Pauling, L. (1960) The Nature of the Chemical Bond. An Introduc- tion to Modern Structural Chemistry. 3rd Ed. Cornell University Press, Ithaca, New York, 664 pp. [31] Robin, M., Galy, J.-P., Kenz, A. and Pierrot, M. (2002) N-(4-Amino-2-methoxyphenyl)acetamide. Acta Cryst. E58, o644- o645. [32] Kajiyama, T. and Ohkatsu, Y. (2002) Effect of meta-substituents of phenolic antioxidants – proposal of secondary substituent effect. Polym. Degrad. Stabil. 75, 535-542.

[33] Butler, S., Wang, R., Wunder, S.L., Cheng, H.-W. and Randal, C.S. (2006) Perturbing effects of carvedilol on a model membrane system: Role of lipophilicity and chemical structure. Biophys. Chem. 119, 307-315.

[34] Malík, I., Sedlárová, E., Čižmárik, J., Andriamainty, F. and Csöl- lei, J. (2005) A study of physicochemical properties of 2-, 3-, 4-alkoxyphenylcarbamic acid derivatives with a substituted N-phenylpiperazine moiety in the basic part. Čes. slov. Farm. 54, 235-239. [35] Oettl, K., Greilberger, J., Zangger, K., Haslinger, E., Reibnegger, G. and Jürgens, G. (2001) Radical-scavenging and iron-chelating properties of carvedilol, an antihypertensive drug with antioxidative activity. Biochem. Pharmacol. 62, 241-248. [36] Pahari, A.K. and Chauhan, B.S. (2006) Engineering Chemistry. Received: September 27, 2014 Laxmi Publications, New Delphi, 583 pp. Revised: January 22, 2015 [37] Battershill, P.E. and Sorkin, E.M. (1988) Ocular metipranolol. Accepted: February 11, 2015 Drugs 36, 601-615. [38] Melena, J. and Osborne, N.N. (2003) Metipranolol attenuates lipid peroxidation in rat brain: A comparative study with other antiglau- coma drugs. Graefe´s Arch. Clin. Exp. Ophthalmol. 241, 827-833. CORRESPONDING AUTHOR [39] Gordh, T., Gordh, T.E. and Lindqvist, K. (2010) Lidocaine: The origin of a modern local anesthetic. Anesthesiology 113, 1433- Ivan Malík 1437. Department of Pharmaceutical Chemistry [40] Das, K.C. and Misra, H.P. (1992) Lidocaine: a hydroxyl radical Faculty of Pharmacy scavenger and singlet oxygen quencher. Mol. Cell. Biochem. 115, 179-185. Comenius University Odbojárov 10 [41] Lee, J.M., Suh, J.K., Jeong, J.S., Cho, S.Y. and Kim, D.W. (2010) Antioxidant effect of lidocaine and procaine on reactive oxygen Bratislava, 832 32 species-induced endothelial dysfunction in the rabbit abdominal SLOVAK REPUBLIC aorta. Korean J. Anesthesiol. 59, 104-110. [42] Tang, Y.-Z., Liu, Z.-Q. and Wu, D. (2009) Lidocaine: An inhibitor Phone: (+421)-2-50117-226 in the free-radical-induced hemolysis of erythrocytes. J. Biochem. Fax: (+421)-2-50117-100 Mol. Toxicol. 23, 81-86. E-mail: [email protected] [43] Ohkatsu, Y. and Nishiyama, T. (2001) Phenolic antioxidants-effect of ortho-substituents. Polym. Degrad. Stabil. 67, 313-318. FEB/ Vol 24/ No 7/ 2015 – pages 2310 - 2318

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AN ASSESSMENT OF RUNOFF AND SEDIMENT IN SOME IRRIGATION DISTRICTS IN A SEMI-ARID REGION OF TURKEY

Ali Fuat Tari1, Öner Çetin2,*, Ramazan Yolcu3 and Vyacheslav Bogdanets4

1Harran University, Agricultural Faculty, Dept. of Agricultural Structures and Irrigation, Sanliurfa, Turkey 2Dicle University, Agricultural Faculty, Dept. of Agricultural Structures and Irrigation, Diyarbakir, Turkey 3Regional Directorate of State Hydraulic Works, Diyarbakır, Turkey 4National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine

ABSTRACT agement is often viewed as the main cause of accelerated erosion rates. Soil erosion is, thus, one of the main envi- This study was carried out to assess runoff and mass ronmental problems in irrigated areas. sediment (soil losses) from 3 irrigation districts in the south- There are four basic methods of applying irrigation eastern Anatolia Region of Turkey in 2005 and 2006. The water: surface (or flood), sprinkler, trickle, and subsurface. total irrigating area was 3582 ha, 10044 ha, and 4758 ha for The choice of a specific irrigation method is usually based Çinar-Göksu, Devegecidi, and Kralkızı Districts, respec- on crop type, land slope, soil texture, cost, and local cus- tively. The numbers of samples in the irrigation districts for toms. Furrow irrigation, however, can be a major contrib- measurement of runoff and sediment ranged from 2 to utor to soil loss. Furrow irrigation results in greater loss 9 depending on the exit of these districts. A volumetric cup because furrow irrigation uses the soil as the transmission was used for discharge of runoff based on time. To estimate line and distributes the water along the irrigation furrow. sediment in the runoff, water samples were collected using More efficient irrigation water use not only saves money a 1-liter bottle. The total soil loss was estimated using the but can also reduce irrigation-induced erosion and reduce amount of soil mass lost with runoff per second and per leaching potential. There are several ways of improving ir- liter. Runoff and mass sediment losses from these irrigation rigation efficiency, including the use of pressurized irriga- districts ranged from 28.0–42.4% and 98.4–4503.6 tons for tion systems such as drip, sprinkler, and gated pipe and one irrigation season, respectively. The reasons for excess surge irrigation [1]. runoff and mass sediment might be attributed to excessive flooding of lands and inappropriate irrigation methods. Roughly 75 billion tons of fertile topsoil is lost world- These runoff and soil losses are not acceptable for sustain- wide from agricultural systems every year [2]. Erosion re- able irrigation and environmental pollution, and these sults in the degradation of a soil’s productivity in a number losses were affected by size of irrigation districts, land of ways: it reduces the efficiency of plant nutrient use, dam- slopes, crop pattern, irrigation methods, amount of irriga- ages seedlings, decreases plants’ rooting depth, reduces the tion water applied, and climatic conditions. soil’s water-holding capacity, decreases its permeability, increases runoff, and reduces its infiltration rate.

Irrigation-induced erosion begins when water is first KEY WORDS: applied to the soil surface where the land slope is sufficient irrigation, surface irrigation, runoff, sediment, erosion that the moving water has enough shear force energy to de-

tach soil particles from the soil mass and transport them as suspended sediment or bed load. Some of the first irrigation 1. INTRODUCTION was done in this way by wild flooding [3]. Most high-value crops are planted in rows, and to sur- Irrigation is a basic and inevitable factor in increasing face irrigate them, small ditches are made parallel to these and securing the agricultural production in arid and semi- crop rows; thus, furrow irrigation has become a common arid regions. Irrigation is, thus, essential to profitable crop method. production. Irrigation and drainage projects have very im- In general, soil erosion is a three-step process. It begins portant impacts on both agriculture and human life. with the detachment of soil particles, continues with the On the other hand, soil erosion is acknowledged as a transport of those particles, and ends with the deposition of major environmental problem, threatening sustainable live- soil particles in a new location. Bare soils (soils that lack a lihoods around the world. Inappropriate land use and man- cover of living or dead plant biomass) are highly susceptible to erosion, even on flat land. There are three main types of * Corresponding author water- induced soil erosion: sheet, rill, and gull [2].

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The topographical structure of Turkey is generally Yearly average precipitation and evaporation are 475 mm sloping, high land, and mountainous. In Turkey nearly 63% and 897 mm, respectively. In summers, the rainfall ranges of the land has slopes steeper than 15% on average, even from 0.6 mm through 7.9 mm. In the irrigated areas, the in the coastal areas. The percentage of the land with high soils have, in general, clay texture and flat lands. There are slopes is very high and 48.0% of the soil is on a slope of no significant drainage problems. Irrigation water quality 20%. High slopes are associated with soil erosion, which is reasonable and the salinity level is 0.45 dS m-1. is one of the major problems in Turkey. About 73% of cul- Irrigation reservoir, Devegecidi Dam, and the irriga- tivated land and 68% of prime agricultural land are prone tion scheme were built in 1971. The irrigation scheme was to erosion in Turkey. Areas with moderate or high rates of operated and managed by State Hydraulic Works (DSI), precipitation erosion, caused largely by water soil erosion, governmental sector, between 1971 and 1994. The govern- are the biggest problems for Turkey. In Turkey, 59% of mental organizations have been mandated to transfer oper- land is exposed to severe and very severe soil erosion. In ation and maintenance irrigation systems to the Water User these lands soil fertility is decreased by about 50% and they Organizations (WUOs) of Devegecidi. The main canal cannot be used economically [4]. flow is 9.3 m3 sec.-1 It has a gross command area of 10,044 ha. GAP, the Southeastern Anatolia Project, in Turkey is The average land holding in the irrigation district is 2.5 ha. one of the world’s largest and most ambitious regional de- Water to the scheme is delivered by means of the main con- velopment projects, which includes a giant water resources veyed canal (9.3 m3 sec.-1, max capacity). The irrigation development plan. It includes developing irrigation infra- comprises 47 km of main canals, 13 km of secondary, and structure to increase the income in the region through di- 126 km of tertiary canals [7]. versified crop patterns including main field and horticultural crops and also secondary crops that require intensive Kralkızı dam and P-II irrigation district: This dam farm workers, alleviating poverty to some extent. Totally, was constructed for electricity production and irrigation in 1.82 million hectares of land will be irrigated by GAP [5]. 1998. There are different components of this irrigation dis- Although there are many agronomical and economic bene- trict, and P-II is one of these districts. The main irrigation fits, such as this big-scale irrigation project, there will also resource of the P-II District is provided by means of pump- be some negative impacts on the environment, such as ero- ing systems from the Kralkizi Dam and open canal systems sion. Soil erosion is the main threat to the sustainability of are used to convey irrigation water into the irrigation dis- agriculture in the Mediterranean region [6]. trict. The total irrigation area is about 4,758 ha. Most of the farmers use surface irrigation including furrow irrigation. In this article, the impacts of soil erosion in irrigated lands in the GAP region, soil losses, and runoff from some 2.2 Runoff and mass sediment measurements irrigation districts are presented and discussed. A volumetric cup was used for discharge of runoff based on time. The amount of irrigation water discharge, 2. MATERIALs AND METHODS which was released to the main canals in the irrigation districts, was measured using an orifice system. To estimate 2.1 Irrigation districts sediment in the runoff, water was collected using a 1-liter bottle. The number of samples in the irrigation districts This study was carried out in three irrigation districts ranged from 2 to 9 depending on the exit of these irrigation in the southeastern Anatolia Region of Turkey, Diyarbakır. districts, and all samples represented for each irrigation These were Çınar-Goksu, Devegeçidi, and P-II. The char- district. acteristics of irrigation districts are given below. The quantity of sediment transported from a small land Çinar-Göksu reservoir and district: This reservoir area can be, thus, measured by trapping the water and sed- was constructed in 1997 and it has been operated only for iment runoff in a catch pit, which is a tank constructed from irrigation. The total operation area for irrigation is 3582 hec- plastic. tares (ha). The total capacity of the reservoir is 56.5x106 m3 The runoff samples collected were put into an oven, and the active capacity (operation capacity) is 46.5x106 m3. thus water in the runoff samples was evaporated and resi- The capacity of the main conveying canal is 3,226 m3 sec-1. due in the samples was weighed; thus mass sediment was Irrigation methods used in the district were mainly surface calculated for each different discharge of runoff. irrigation methods and furrow irrigation. The irrigation period and total runoff during the irriga- Devegeçidi reservoir and district: The main water tion season were considered when measuring the total resource for the reservoir is Devegecidi Creek. The water- amount of sediment deposited in runoff water. Thus, the shed of the dam is originated by the surrounding lands, mass of sediment collected from the irrigated area repre- which have altitudes between 580 and 730 m. The slope of sented the covered area. the lands ranges from 0.5% to 10%. The irrigated areas are Runoff was calculated as discharge of water in exits of dominated by storage reservoirs (dams) supplying only the irrigation districts. The total soil loss (mass sediment) was surface waters conveyed through large open channels. estimated using the amount of soil mass lost with runoff

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per second and per liter. Thus, total discharge of runoff and leased to the irrigation district. In this district, the samples total irrigation period were considered for that. The total were collected at 10 different outlets of the irrigation dis- irrigation period was approximately 100 days. Thus, to es- trict in 2005. The total runoff from all irrigated areas was timate soil loss, the equation 1 given below was used. 3,014 L sec.-1. Thus the runoff ratio was 42.4%. To estimate the amount of mass sediment from the district area, SL = S x R x IP x 3,600 x 24 (1) samples were also taken from 10 different sampling points (Table 2, Fig. 1). In 2006, the amount of irrigation water of Where SL stands for soil losses (in grams); S, sedi- 9,009 L sec.-1 from the reservoir was released to the irriga- ment, in grams per liter per second (g L-1 sec.-1); R, dis- tion district. Runoff was measured at 5 different places. charge of total runoff (L sec.-1); IP, irrigation period (100 The total runoff from all irrigated areas was 2,818 L sec.-1. days); 3,600 (seconds); 24 (hours) Thus the runoff ratio was 31.3%. To estimate the amount of mass sediment from the district area, samples were taken from 5 different sampling points. Excessive runoff and ero- 3. RESULTS AND DISCUSSION sion from the irrigation district area was attributed to irrigation methods used (flood irrigation), irrigation through 3.1 Runoff variations the slope, greater length of furrows, greater water discharge In the Çınar-Göksu irrigation district, runoff water than infiltration rate of the soil, frequently of irrigation samples were collected in 2006. There was no data in 2005. (shorter irrigation intervals), and tenant farmers in the irri- Runoff discharge of 2 sampling points ranged from 79 to gation district area. 578 L sec.-1 (Table 1). These runoffs varied considerably In the P-II irrigation district, the amount of irrigation depending on the size of the irrigation area represented by water of 6,075 L sec.-1 from the reservoir was given to the the outlet of the runoff, crop pattern, land slope, and irriga- irrigation district. Runoff was measured at a total of 5 points tion methods. The amount of irrigation water of 2,333 L of the district in 2005. The total runoff from all irrigated areas sec.-1 from the reservoir was given to the irrigation district. was 1,494 L sec.-1. Thus, runoff ratio was 24.6% (Table 3, The total runoff from all irrigated areas was 657 L sec.-1. Fig. 1). To estimate the amount of mass sediment from the Thus, the runoff ratio was 28.2% (Table 1, Fig. 1). district area, samples were taken from 5 different sampling points. To pump this amount of water costs approximately In the Devegeçidi irrigation district, the amount of ir- $600,000. Considering the amount of runoff, approximately rigation water of 7,112 L sec.-1 from the reservoir was re- $150,000 is wasted for one irrigation season. In 2006, the

TABLE 1 - Measurement results of runoff and sediment in Cınar-Goksu irrigation district in 2006.

Sampling points Runoff discharge Mass sediment in runoff Mass sediment loss per unit time (L sec.-1) (g L-1) (g sec.-1) 1 79 0,105 8,295 2 578 0,045 26,01 Total 657 Total 34.31 Totally active irrigation area: 1394 ha.

TABLE 2 - Measurement results of runoff and sediment in Degecidi Irrigation District.

Years 2005 2006 Runoff Mass sediment Mass sediment Runoff Mass sediment Mass sediment discharge in runoff loss per unit time discharge in runoff loss per unit time Sampling points (L sec.-1) (g L-1) (g sec.-1) Sampling points (L sec.-1) (g L-1) (g sec.-1) 1 36,6 0,047 1,720 1 2112 0,155 327,360 2 39 0,009 0,351 2 105 0,217 22,785 3 165 0,027 4,455 3 39 0,64 24,960 4 191 0,027 5,157 4 61 0,325 19,825 5 72 0,009 0,648 5 401 0,315 126,315 6 200 0,003 0,600 Total 2818 Total 521.300 7 138 0,002 0,276 8 1706 0,006 10,236 9 318 0,006 1,908 10 148 0,025 3,700 Total 3014 Total 29.05 Totally active irrigation area : 5800 ha Totally active irrigation area : 6400 ha

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TABLE 3 - Measurement results of runoff and sediment in PII irrigation district.

Years 2005 2006 Runoff Mass sediment in Mass sediment Runoff Mass sediment in Mass sediment discharge runoff lost per unit time discharge runoff lost per unit time Sampling points (L sec.-1) (g L-1) (g sec.-1) Sampling points (L sec.-1) (g L-1) (g sec.-1) 1 90 0,016 1,440 1 239 0,384 91,776 2 207 0,014 2,898 2 55 0,59 32,450 3 587 0,008 4,696 3 141 0,748 105,468 4 27 0,001 0,027 4 88 0,632 55,616 5 583 0,004 2,332 5 231 0,163 37,653 Total 1494 Total 11.390 6 5 0,21 1,050 7 51 0,041 2,091 8 1.549 0,033 51,117 9 154 0,036 5,544 Total 2513 Total 382.770 Totally active irrigation area :5021 ha Totally active irrigation area : 4582 ha

FIGURE 1 – Runoff according to the studied years in the irrigation districts.

amount of irrigation water of 7,230 L sec.-1 from the reser- When water reaches the lowest part, or end, of the voir was given to the irrigation district. The total runoff from field, “tailwater” runoff begins unless the water is shut off all irrigated areas was 2,513 L sec.-1. Thus the runoff ratio or the end of the field is blocked with berms to keep the was 34.8%. (Table 3, Fig. 1). To estimate the amount of water in the field. Allowing runoff is a common part of mass sediment from the district area, samples were taken normal surface irrigation to ensure that sufficient water is from 9 different sampling points. To pump this amount of applied at the lowest end of the field. With border irrigation, water costs approximately $650,000. Considering the this often means that the water must be stopped before it amount of runoff, approximately $253,000 is wasted for one advances to the end of the field, since water will continue irrigation season depending on energy prices. to advance even after it is turned off. Water will not ad- Runoff or tailwater (24.6–42.4%) in all irrigation dis- vance far after a furrow irrigation system is shut off; crops tricts was significantly higher. Schwankl et al. [8] calcu- at the end of the row may be under irrigated water if this lated the tailwater volume to be 15 to 25 % of the water technique is used with furrow irrigation [11]. applied to an irrigation set. On the other hand, cotton-cultivated areas ranged from 3.2 Sediment measurements 50 to 98% in all irrigation districts [9]. Cotton is one of the To estimate the amount of mass sediment from the dis- crops that consume much more irrigation water compared trict area, samples were taken from 2 different sampling to other field crops [10]. Cotton is irrigated almost entirely points in the Çınar-Göksu irrigation district. The irrigation by surface irrigation. All these reasons might cause the use period was approximately 100 days in this irrigation district. of more irrigation water in the irrigation districts. Considering this irrigation period, the total amount of soil

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FIGURE 2 – Amount of soil losses according to the studied years in the irrigation districts.

losses with erosion was calculated as 296.4 tons year (y)-1 Most of fields in irrigation district areas used surface (Fig. 2). In addition, sediment losses from this irrigation dis- irrigation systems such as furrow irrigation or border (or trict on irrigated cropland were 212.6 tons per hectare dur- flood) irrigation. Over-irrigation, in which more water is ing one irrigation season. Considering the irrigation period, applied than can be stored in the crop root zone, can lead 100 days, the total amount of soil losses with erosion was to greater tail-water runoff problems. Thus, under surface calculated to be 251.0 and 4503.6 tons y-1 in 2005 and 2006 irrigation, water is introduced into furrows or border in the Devegeçidi irrigation district, respectively (Fig. 2). checks at the top of the fields. As the water flows Soil loss in 2006 was higher compared to that in 2005. One downslope, some of the water infiltrates the soil. Water is of the reasons might be the higher use of irrigation water in applied in excess of that which infiltrates and the excess 2006 because discharge of irrigation water released into the water flows, or advances, across the field. irrigation scheme was 9,009 L sec.-1, while it was 7,112 L sec.-1 in 2005. Furrow irrigation erosion redistributes topsoil by eroding upper ends of fields and depositing sediment on Sediment losses from the fields on irrigated cropland downslope portions, causing a several-fold topsoil depth ranged from 43.3 to 703.7 tons per ha from the fields dur- difference on individual fields. Loss of topsoil due to ero- ing one irrigation season. Considering the results that sed- sion causes the loss of growing areas for crop production. iment losses from furrow erosion on irrigated cropland ranged from 0.5 to 142 metric tons per hectare fields during Sediment in irrigation return flows arises primarily one irrigation season [12], these soil losses were not ac- from furrow erosion and subsequent surface runoff. The ceptable for sustainable irrigation and environmental pol- sediment concentration in irrigation return flows varies lution. widely with time during the irrigation season. Concentra- tions in the Hansen drain varied from 280 to 14500 ppm, Surface runoff accounted for 50 % or more of the water while concentrations in other drains were as low as 10 ppm. applied to 13 fields. These results indicate that furrow The seasonal soil loss from fields into drains on a 65,350 ha- streams were considerably larger than needed. Our obser- irrigated tract was 4.0 tons ha-1 [13]. vations also indicated that few farmers reduced furrow stream inflow after setting the water at the beginning of the When water flows over cultivated land, erosion may irrigation. This practice conserves labor but increases ero- occur. When surface runoff from eroding fields enters a sion. Furrow streams can be reduced manually or by using surface river or stream, it contains sediment. Sediment automatic cutback systems. Furrow erosion varied consid- losses from furrow erosion on irrigated cropland ranged erably during the irrigation season. from 0.5 to 142 metric tons per hectare during one irrigation season [12]. Another reason could be considered for this. The flow and/or amount of water coming into the reservoir in 2006 Furrow erosion can be reduced by: (a) reducing furrow was 192x106 m3, while it was 101x106 m3 in 2005 because stream size when water reaches the furrow ends, (b) avoid- the rainfall in 2005 and 2006 was 408 and 566 mm, respec- ing irrigation of row crops on slopes that are too steep, (c) tively. Thus a flood occurred in 2006 and some of the water keeping the tail-water ditch shallow and the water in it in the reservoir was released by means of the spillway. moving slowly, (d) installing tail-water control systems, Some areas and canals were exposed to the accumulation and (e) using alternate-furrow irrigation [12]. of sediment.

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4. CONCLUSION [4] Haktanır, K., Karaca, A. and Omar, S.M. (2004). The pro- spects of the impact of desertification on Turkey, Lebanon, Syria and Iraq. In: Marquina, A. (Ed.) Environmental Chal- The most important problem in the irrigation districts lenges in the Mediterranean 2000-2050. Kluwer Academic is environmental damage by soil erosion and soil lost. Run- Publishers, Netherland, Chapter 9, 139-154 off and mass sediment losses from these irrigation districts [5] GAP-BKİ (2014). What is GAP? (Available: ranged from 28–42.4 % and 98.4–4503.6 tons for one irri- http://www.gap.gov.tr, accessed on: September 14, 2014) gation season, respectively. These runoff and soil losses are [6] Boulal, H., Gomez-Macpherson, H., Gomez, J.A. and Mateos, not acceptable for sustainable irrigation and environmental L. (2011). Effect of soil management and traffic on soil erosion pollution. in irrigated annual crops. Soil and Tillage Research 115-116, 62-79 Sustainable irrigation is at risk due to excessive flood- [7] Üzen, N., Yolcu, R. and Cetin, Ö. (2013). Evaluation of De- ing of lands with inappropriate irrigation methods. Exces- vegecidi irrigation scheme on the irrigation management in sive irrigation and soil erosion occurred since the amount Southeastern Anatolia Region of Turkey. AgroLife Scientific of irrigation water applied to the lands of the farmers was Journal 2 (1), 151-156 not measured. Some technically improper practices in- [8] Schwankl, L.J., Prichard, T.L. and Hanson, B.R. (2007). clude: inappropriate irrigation methods, sides of cultivation Tailwater return systems. University of California, Division of and furrow parallel to the land slope, longer furrow lengths, Agriculture and Natural Resources. Publication 8225 CA, USA use of higher discharges than infiltration rate for the lands, and tenant irrigation instead of property owner irrigation in [9] DSI (2007). Reports on irrigation districts. X. Regional Direc- the irrigation schemes. torate of State Hydraulic Works, Diyarbakir, Turkey (in Turk- ish). The goal of every irrigator should be to apply the right [10] Çetin, Ö. and Bilgel, L. (2002) Effects of different irrigation amount of water as uniformly as possible to meet the crop methods on shedding and yield of cotton. Agricultural Water needs. To do this, irrigators need to take into account how Management 54 (1), 1-15 much water is applied during irrigation and where the wa- [11] Schwankl, L.J., Prichard, T.L. and Hanson, B.R. (2007). ter goes. Factors affecting furrow erosion are the slope Causes and management of runoff from surface irrigation in orchards. University of California, Division of Agriculture and along the furrow, stream size, residue, surface roughness, Natural Resources. Publication 8214, CA, USA and cropping sequence. [12] Berg, R.D. and Carter, D.L. (1980). Furrow erosion and sedi- Tail-water return systems, in which tail-water runoff is ment losses on irrigated cropland. Journal of Soil and Water Conservation 35 (6), 267- 70 collected in a pond and reused, are common in surface-irrigated row and field crops. However, they are not as com- [13] Carter, D.L. (1976). Guidelines for sediment control in irrigation return flow. Journal of Environmental Quality 5 (2), 119- mon in these irrigation districts areas. In addition, there is 124. no reason the farmers should be used because farmers do not pay water prices based on the volumetric unit for the amount of irrigation water applied. In fact, a tail-water return system collects the runoff water in a storage pond until it can be reused for irrigation on another section of the field or on other land being irrigated. Reusing the collected water maintains high irrigation efficiency and makes room in the pond for additional runoff. Received: October 06, 2014 Revised: January 09, 2015 Accepted: February 20, 2015 The authors have declared no conflict of interest.

CORRESPONDING AUTHOR

REFERENCES Öner Çetin Dicle University [1] Anonymous. (2013). Erosion control for irrigated land. ES-6. (Available: http://www.cals.ncsu.edu, accessed on: October Agricultural Faculty 12, 2013) Dept. of Agricultural Structures and Irrigation Diyarbakir [2] O’Geen, A.T. and Schwankl, L.J. (1986). Understanding soil erosion in irrigated agriculture. University of California, Divi- TURKEY sion of Agriculture and Natural Resources. Publication 8196 CA, USA Phone: +90 412 248 85 09 [3] Carter, D.L. (1990). Soil erosion on irrigated lands. In: Stew- Fax: +90 412 248 81 53 art, B.A. and Nielsen, D.R. (Eds.) Irrigation of Agricultural E-mail: [email protected] Crops. Agronomy Monograph No. 30, ASA-CSSA-SSSA, 677 South Segoe Road, Madison, WI 53711, 1143-1171. FEB/ Vol 24/ No 7/ 2015 – pages 2319 - 2324

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TEMPORAL AND SPATIAL CHARACTERISTICS OF PHOSPHORUSFRACTIONS UNDER LONG-TERM WASTEWATER IRRIGATION IN TONGLIAO CITY, CHINA

Yintao Lu1,2,*, Fang Liu1,2, Hong Yao1,2, and Kelin Hu3 1 School of Civil Engineering, Beijing Jiaotong University, Beijing 100044, China 2 Beijing Key Laboratory of Aqueous Typical Pollutants Control and Water Quality Safeguard, Beijing 10044, China 3College of Resources and Environmental Sciences, China Agricultural University, Beijing 100083, China

ABSTRACT for reclaimed wastewaters is irrigation of agricultural, for- ested, and urban landscapes [2-5]. Effluent reuse allows eu- The irrigation reuse of municipal and industrial efflu- trophication-promoting nutrients to enhance plant growth ents conserves freshwater resources and avoids direct nu- and reduce water-quality effects. Phosphorus (P) is a plant trient discharges to surface waters. The sustainability of ag- nutrient present in treated sewage effluents, which rarely ricultural reuse programs, however, may be challenged by affects the daily operation ofmunicipal wastewater effluent changes in phosphorus (P) availability. To understand the irrigation systems [6].P is not typically considered as a lim- characteristics of P fractions, 130 soil samples were col- iting parameter for effluent irrigation because only a small lected around an irrigation drain in May 2009, at the begin- amount of P is typically added to soil by irrigation with ning of the growing season. In the study area, total phos- treated sewage effluents [7-9]. phorus (TP) varied from 302.32 to 716.30 mg/kg, residual However, soluble P can be easily fixed in soils, result- P varied from18.21 to 126.08 mg/kg, and Olsen P varied ing in low P availability for plant utilization [10]. P frac- from 0.71 to 90.04 mg/kg. The relative abundance detected tionation studies considering fertilizer applications of sev- for nearly every P fraction followed the order Ca-P>resid- eral years or longer have been conducted to understand P ual P>NaOH-Po≈NaOH-Pi>KCl-P. Although NaOH-Pi and availability in soil [11].Calcium-bound phosphorus (Ca-P) most of the Ca-P fractions contributed markedly to P avail- is the dominant inorganic form of P [12]. The availability ability, NaOH-Po and Ca-P contributed more to residual P of P fractions to plants depends on time-varying soil when different phosphate sources were added to the soil. changes[13]. Despite an increase in the total soil P over the However, the available P in the studied soils represented life of a system, most fields have inadequate plant-availa- only 5.5% of the TP, which was inadequate for the growth ble P [14]. After 70 years of irrigating citrus crops with and development of crops. The influence of wastewater ir- wastewater from Cairo and Alexandria (Egypt), the surface rigation differs significantly at different depths. Wastewater soil contained only 10-11 mg/kg Olsen P [15]. Unfortu- irrigation is favorable to the transformation of stable P nately, few studies have considered the variation of P frac- (NaOH-Po and Ca-P) into available P (NaOH-Pi), and tions in wastewater-irrigated areas. cancause an increase in residual P in the shallow layer (0- 60 cm), as well as an increase in TP in the middle layer (60- In northern China, P-deficient soils are widespread. 110 cm). However, wastewater and groundwater irrigation Although wastewater has beenwidely used as an important is beneficial to the increase in soil P concentration in the irrigation source in China, relevant studies evaluating the deep layer (110-170 cm). These effects may depend on soil effects ofreclaimed wastewater irrigation on soil P are properties, water cycles and biological processes, among stilllacking. This study aimed to investigate the proportions other factors. and relationships of soil P fractions in wastewater-irrigated and groundwater-irrigated areas, and to study the transport and conversion of soil P with respect to spatial and vertical KEYWORDS: Phosphorus transport,soil phosphorus, wastewater variationsin soil P fractions. irrigation, groundwater irrigation

1. INTRODUCTION 2. MATERIALS AND METHODS

2.1 Site description Wastewater effluentsare increasingly being viewed as potential water supply sources [1]. The major reuse option The experimental area is located in the northeastern region of China, downstream of the Xiliao river catchment (43°38′11″–43°45′50″ N; 122°19′50″–122°31′52″ E). The * Corresponding author site lies in the semi–arid grassland area of the North Tem-

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perate Zone and belongs to the continental monsoon cli- tection threshold. All parameters were within the limits es- mate, with a mean annual temperature of 5 °C. This region tablished by the GB20922–2007 standard and in accordance is arid with an annual water deficit of 350 mm, due to po- with agricultural water quality standards. The COD, BOD5, tential evaporation exceeding precipitation. The dry season TN and TP of irrigation wastewater after 1985 were greater spans from November to April, and the wet season spans than those of groundwater; thus, long-term wastewater irri- from June to September. The dominant vegetation species gation can enhance these concentrations in soil. is corn. 2.2 Sampling and analysis methods TABLE 1 - Water quality indices of wastewater and groundwater 2.2.1 Soil collection Chemical parameter USW TSW Groundwater Field soil samples were collected in Tongliao, China, pH 7-8 7-8 7-8 in May 2009 (Fig. 1), at the beginning of the growing sea- COD (mg/L) 568.90 95.55 - son. In total, 130 soil samples were collected around the BOD (mg/L) 193.50 20.96 - irrigation drain and in the farming area. Most of the soil 5 samples were taken at 50-m intervals along a 250-m tran- TN (mg/L) 113.71 59.15 1.04 sect that was perpendicular to the direction of the irrigation TP (mg/L) 6.60 0.04 0.07 drain and was approx. 400 m downstream of the heads of *“USW” refers to the untreated sewage from municipal and industrial the furrows. Side-by-side undistributed soil cores were ob- wastewater and can reflect the irrigation wastewater level from 1985 to 2007; “TSW” represents treated sewage, which can reflect the irrigation tained at soil depths of 0-20 cm at the 130 sample sites. An wastewater level since 2007. adequate amount of bulk soil was obtained and sub-divided for the different types of measurements performed. Six The wastewater treatment plant in Tongliao City treats sampling lines were selected (Fig. 1). Lines 1 and 2 were approximately 70,000 m3·d–1 of sewage. Most of the located 50 m from the drain on either side, and lines 3 and treated effluent flows into the Xiliaoriver, and a smaller 4 were located 200 m from the drain on either side. Lines amount is used for the irrigation of suburban farms. Farm- 5 and 6 were perpendicular to the drain. Three sampling ers used a primary sedimentation pond to treat effluents sites (denoted S1, S2, and S3, as indicated in Fig. 1) were without any biological treatment from 1985 to 2007; after selected from among the sampling points in the southeast- 2007, farmers used a secondary sedimentation pond after ern section of the irrigation drain, and they represent the the effluent had been subjected to an anaerobic-anoxic- 3 irrigation water types (wastewater, wastewater and ground- oxic process at wastewater treatment plants. There are no water, and groundwater, respectively). The soil cores were detailed data regarding the use of wastewater for irrigation collected at 9 different depths from the top of the soil: 0-20, from 1985 to 2007. However, the main quality parameter 20-40, 40-60, 60-80, 80-100, 100-110, 110-130, 130-150, and levels of TSW (the treated sewage) and groundwater were 150-170 cm. After extraction, soil samples were preserved in just below those of USW (the untreated sewage; Table 1). ice-packed coolers until they were transferred to the labora- All heavy metals in the treated sewage were below the de- tory and stored in a 4 °C cold room until analysis.

FIGURE 1 - Location of the study site (square-labelled with numbers are sites for study of the vertical distribution of soil properties).

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2.2.2 Soil sample tests NaOH-Pi and NaOH-Po were the least abundant P Samples were analyzed for various phosphate charac- fractions in the soils (Fig. 2). NaOH-Pi constituted between teristics using a UV-VIS spectrophotometer (Shimadzu 2.3 and 4.8% of the TP and averaged 3.6% for all the soils UV1800). TP was measured by Mo-Sb colorimetry after at the 6 sites, whereas NaOH-Pi constituted between 2.2 HClO4–H2SO4 digestion. Olsen P was determined by shak- and 3.9% of the TP and averaged 3.6% for all the soils at ing 100 ml of a 0.5 M NaHCO3 solution at pH 8.5 with 5 g the 6ix sites. However, the percentages of NaOH-Pi and soil for 30 min. The P content of the samples was deter- NaOH-Po were similar for all the soils at the 6 sites. The mined using the manual colorimetric procedure of Murphy availability of soil P to plants depends on the replenishment and Riley [16]. of soil KCl-P from other P fractions [18]. NaOH-Pi and NaOH-Po are believed to be held to Fe and Al complexes Soil P fractions were measured sequentially using a by chemisorption but may be released due to desorption modified version of the Hedley procedure [17] as follows: and may be used by plants when KCl-P is exhausted [19]. (1) KCl-P (exchanged P): add fresh soil samples (0.5 g In this study, KCl-P concentrations were below the limit of oven-dry equivalent) to 25 ml of 1 mol/L KCl prepared ac- detection; thus, the KCl-P fraction was negligible. The cording to Tessien (1993) into a 100-ml capped plastic cen- NaOH-Pi fractions constituted a greater proportion of the trifuge tube (4000 rpm), shaken for 1 h, and centrifuged for available P at the study sites. 8 min at 25 C; (2) NaOH-Pi and NaOH-Po: resuspended P accumulation largely occurred in the form of cal- residue from (1) in 40 ml of a 0.1 mol/L NaOH solution in cium-bound phosphorus (Ca-P), constituting between 60.6 a 100-ml capped plastic centrifuge tube (4000 rpm), shaken and 93.3% of the TP (representing75.8% of TP on average) for 17 h and centrifuged for 10 min at 25 C; NaOH-Po is (Fig. 2), which suggests that a high proportion of P occurs the difference between NaOH-TP and NaOH–Pi; (3) Ca-P: in a stable inorganic form. Many previous studies have resuspended residue from (2) in 40 ml of a 0.25 mol/L shown that Ca-P is the main P fraction in calcareous soil H2SO4 solution, shaken for 1 h and centrifuged for 8 min; [20, 21]. Our results support this conclusion. Diazet al. [22] (4) Residual P: digested residue from (3) in concentrated indicated that P, adsorbed and precipitated with Ca, repre- H2SO4 and H2O2; for (2) to (4): the supernatant was filtered sents a more stable fraction than the KCl-P and NaOH-P through a 0.20-µmmicropore filter, and the inorganic P fractions [22]. Thus, this fraction is unlikely to contribute concentration in the filtrate was colorimetrically deter- to eutrophication [23], and leaching. mined using the molybdate blue method [16]; for (2): the Residual P constituted between 1.4 and 33.0% of the inorganic P and TP concentrations in the extracts were de- TP and averaged 17.0% for all the soils at the 6 sites. Re- termined after digesting the extracts in concentrated H SO 2 4 sidual P as a percentage of TP shows a trend opposite to and H O , respectively, and the organic P concentration in 2 2 that exhibited by Ca-P.Turneret al.[24] indicated that the the extracts was calculated as the difference between the residual P pool is a stable chemical fraction, and is com- total P concentration and the inorganic P concentration. posed of lignin and organo-metallic complexes [24]. Crop plants can only uptake available P, but other fractions of P, 2.3 Statistical analysis such as NaOH-Po and Ca-P, are depleted due to crop Statistical analyses and corresponding graphs were growth [25]. Pheavet al. [26] indicated that soil reduction performed and generated, respectively, using SPSS 10.0 mobilizes P from all pools, including residual P. software. Geostatistics software (GS +) was used to assess The relative abundances detected for nearly every P the spatial structure of TP, residual P and Olsen P. GS + fraction were determined to follow the order Ca-P>residual features a number of models that can be fitted to estimate P>NaOH-Po≈NaOH-Pi>KCl-P. Other scholars have ob- semi-variograms by using a nonlinear square procedure. served this phenomenon as well.It was reported that in Is- The spherical, exponential, and linear models featured in fahan Province of central Iran, a P fraction order of residual the program were used in this study. The selection of the P>NaOH-P>KCl-P was found [27], and the order residual best model was based on the most favorable weighted re- P>NaOH-Pi>NaOH-Po was also found in Palampur[28]. sidual mean square value and was visually fitted to the data Shen and Jiang [29] indicated that the proportions of Ca - with short lags. Contour maps were developed using the 2 P, Ca -P and Ca -P were approx. 1-2:10:70 [29]. Thus, we software program Surfer. 8 10 can calculate that the concentration of Ca -P, which is an 2 available P fraction, constitutes only 2% that of TP. Ac- cording to Fig. 2, NaOH-Pi constitutes only 3.5% of TP. 3. RESULTS AND DISCUSSION These results indicate that the available P sources in this soil represent only 5.5% of TP (approx. 25 mg/kg), which 3.1 Statistical analysesof P fractions and changes in ratio is lower than the concentration required to support plant In general, the utilization rate of soil P is low (10-25% growth and development (30-50 mg/kg) [30]. The soil has of the annual applied P). Thus, large amounts of insoluble limited bio-availability of P in the study area; therefore,we and more stable P (residual P) can be accumulated in soils should take appropriate measures (such as the application with regular P application. The soil P fractions (as percents of N and P fertilizer) to increase the available P and to en- of TP) for the studied soils at 6 sites are presented in Fig. 2. sure the normal growth of plants.

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FIGURE 2 -Soil P fractions as percentages of total P for the studied soils at six sites.

TABLE 2 - Pearson correlation coefficients of TP, Olsen P and P frac- (Table 2). The NaOH-Pi and Ca-P fractions contributed tions markedly to P availability when different phosphate TP Olsen NaOH‐ NaOH‐Po Ca‐P Residual P sources were added to the soil, confirming the findings re- P Pi ported in [13]. In all treatments, the organic P fraction TP 1.000 0.667** 0.774** ‐0.486** 0.956*** 0.573** (NaOH-Po) and Olsen P correlated negatively (P<0.01). ** ** ** * Olsen P 1.000 0.571 ‐0.569 0.653 0.365 Organic P not only constituted a low percentage of TP but NaOH‐Pi 1.000 ‐0.115NS 0.775** 0.312NS NaOH‐Po 1.000 ‐0.465** ‐0.359* was also not an available P source in these soils. Ca‐P 1.000 0.347* Residual P 1.000 Residual P and 2 P fractions (NaOH-Po and Ca-P) n=39. NS: not significant at the P≤0.05 level. *P=0.05; **P=0.01; ***P=0.001 were correlated (P<0.05); this finding suggests that NaOH- Po and Ca-P contribute more to residual P. Conversely, the The Ca-P fraction and TP were significantly (P<0.001) NaOH-Pi fraction did not correlate significantly with resid- positively correlated, with a Pearson correlation coefficient ual P (P≥0.05), which suggests that the NaOH-Pi fraction of 0.956; these results emphasize that Ca-P is the main cannot be converted to residual P and can be used by plants. fraction contributing to TP. At a calcareous soil site, simi- In addition, Olsen P was positively correlated with residual lar results were reported [31]. The NaOH-Pi and Ca-P frac- P (P<0.05), which demonstrates that Olsen P and residual tions and Olsen P were positively correlated (P<0.01); P can be converted into one another. these 2 fractions were correlated with P removal by plants

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These results suggest that there is a dynamic equilib- groundwater-irrigated areas. Some studies have reported a rium process between the P fractions in the soil P cycle, relationship between soil TP and wastewater irrigation and that all P fractions can affect and restrict one another. time. Plots receiving effluents for 0, 6, 19, and 38 years in The Olsen P concentration depends on the distribution and Lubbock, Texas, showed ammonium acetate-extractable P transformation direction of the other P fractions: the varia- levels of 21.3, 24.2, 107, and 202 mg/kg, respectively. tion of any P fraction can cause an increase or decrease in Rusanet al. [7] reported Olsen P levels of 10, 15, 40, and Olsen P. 45 mg/kg in different plots after 0, 2, 5, and 10 years of P fractionation varies with soil depth, and significant effluent irrigation, respectively. Our results and those re- differences were observed between S1 (wastewater irriga- ported in the aforementioned studies suggest that waste- tion), S2 (groundwater and wastewater irrigation), and S3 water irrigation can increase TP and Olsen P in soil. Fig. 4 (groundwater irrigation); see Fig. 3. These differences were shows that the residual P distribution was irregularly scat- mainly evident in the concentrations of Ca-P and residual P. tered, suggesting that the residual P concentration in top- In profile S1, the percentage of Ca-P increased with soil soil is unrelated to wastewater irrigation. Residual P is af- depth, and that of residual P decreased with depth. In profile fected by many factors, including soil physical and chemi- S3, the Ca-P percentage was the highest at a soil depth of cal properties, and the P content in soil inputs and the P 100-110 cm. Because S2 is irrigated by wastewater and uptake of plants. groundwater, the variation is complex, and no significant The vertical distribution trends observed at sites S1, S2 trends were apparent. When comparing the 3 sampling sites, and S3 were different, but the trends of TP, NaOH-Pi, NaOH- it can be concluded that wastewater irrigation can cause an Poand Ca-P were all similar at any given site (Fig. 5). In pro- increase in residual P in topsoil, and can lead to P accumu- file S1, the TP, Ca-P and NaOH-Po concentrations were the lation in the soil environment. In wastewater-irrigated ar- highest at a soil depth of 40-110 cm, the NaOH-Pi concen- eas, residual P was gradually converted to Ca-P with in- tration showed the highest value at a depth of 60-80 cm, and creasing depth. residual P decreased with soil depth. In profile S2, the TP and Ca-P concentrations increased with soil depth; the Spatial variation of soil P and effect of wastewater irrigation NaOH-Po and NaOH-Pi concentrations showed peak val- The spatial variations in TP and Olsen P were similar, ues at 110-130 cm and 130-150 cm, respectively. In profile and these parameters were affected by wastewater irriga- S3, TP, NaOH-Pi, NaOH-Po and Ca-P all showed 2 peak tion (Fig. 4). Other factors (including soil properties, geog- values at depths of 40-60 cm and >130 cm. The residual P raphy, climate, and farming practices) could affect the spa- concentration showed the same trend in profiles S2 and S3: tial distribution patterns of TP, Olsen P and residual P. peaks were observed at depths of 40-60 and 110-130 cm, respectively. However, the largest concentrations of resid- In the study area, TP varied from 302.32 to 716.30 ual P in profiles S1 (136.02 mg/kg) and S2 (122.03 mg/ kg) mg/kg, residual P varied from 18.21 to 126.08 mg/kg, and were significantly higher than the residual P concentration Olsen P varied from 0.71 to 90.04 mg/kg. The concentra- in profile S3 (84.19 mg/kg). tions of TP and Olsen P in the northern wastewater-irrigated areas were higher than those in the more southern

FIGURE 3 -Soil P fractions as percentages of total P at different depths at sites S1, S2 and S3 (S1 is irrigated by wastewater; S2 is irrigated by wastewater and groundwater; S3 is irrigated by groundwater).

2329 © by PSP Volume 24 – No7. 2015 Fresenius Environmental Bulletin

FIGURE 4 -Horizontal distributions of soil properties (including (a) TP, (b) Olsen P, and (c) residual P) in irrigated areas (yellow line represents wastewater irrigation drain, green line is the boundary between wastewater irrigation zones and groundwater-irrigated areas).

TABLE 3 - Mean values of soil P at different soil depths at sites S1, S2 and S3 (mg/kg)

TP NaOH-Pi NaOH-Po Ca-P Residual P Shallow layer S1 366.14 12.42 11.85 228.9 112.97 S2 214.43 8.58 7.63 157.69 40.53 S3 328.56 11.57 14.23 243.79 58.98 Middle layer S1 437.77 15.72 16.2 346.36 59.49 S2 351.21 15.15 13.01 306.79 16.24 S3 305.65 10.78 10.36 252.39 32.12 Deep layer S1 313.65 11.44 10.51 270.53 21.15 S2 446.33 14.39 13.90 376.37 64.51 S3 356.61 12.68 13.12 284.00 46.81 *Shallow layer represents a soil depth of 0-60 cm; middle layer represents a soil depth of 60-110 cm; and deep layer represents a soil depth of 110-170 cm.

To analyze the effects of long-term wastewater irriga- increase in TP and residual P in topsoil, as shown in Fig. 3. tion, the vertical profile can be divided into 3 sections: a These results also underline the fact that, compared with shallow layer (0-60 cm), middle layer (60-110 cm) and that observed in groundwater-irrigated areas, the transfor- deep layer (110-170 cm), as shown in Table 3. In the shal- mation efficiency of P fractions into stable P (NaOH-Po) low layer, TP, the NaOH-Pi and residual P contents follow was lower, whereas the transformation efficiency of P frac- the order S1>S3>S2, whereas NaOH-Po and Ca-P follow tions into available P (NaOH-Pi) was greater under the the order S3>S1>S2. Wastewater irrigation can cause an long-term wastewater irrigation conditions. In the middle

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layer, all P fractions, except for the residual P, followed the vals, and quantities [32]. Previous studies have reported order S1>S2>S3. These results indicate that wastewater ir- that dry/wet cycles may result in the chemical breakdown rigation can cause an increase in TP in the middle layer. In of organic matter, thereby increasing P availability [33]. In the deep layer, all forms of P followed the order S2>S3>S1, irrigated areas, the dry/wet cycles are more striking in clay suggesting that wastewater and groundwater irrigation is soils, which might enhance the mineralization of soil or- beneficial to the increase in soil P content. ganic matter, thereby increasing P availability. Moreover, microbiological processes might play an essential role in The observed P distribution depends on soil properties, the transformation between inorganic P fractions [34]. Rel- water cycles, biological processes, etc. In the study area, a ative to those observed in wastewater-irrigated areas, large increase in clay content was observed at a depth of NaOH-Pi fractions in groundwater-irrigated areas accumu- 50-90 cm. Clay has a high adsorption capacity and high late more easily in soil. The P contained in the NaOH-Pi moisture content, and thus, it bcan induce an increase in P fraction may be unstable under fluctuating redox condi- content. In addition,the phenomena illustrated in Fig. 5 and tions [35]; thus, there may be considerable movement of P Table 3 may be a consequence of the integrated effect of into and out of this fraction depending on the environmen- different soil moisture regimes and dry/wet cycles induced tal conditions of these soils. by treatments with different irrigation frequencies, inter-

FIGURE 5 - Spatial distributions of TP, NaOH-Pi, NaOH-Po, Ca-P and residual P - (a) TP; (b) residual P; (c) NaOH-Pi; (d) NaOH-Po; (e) Ca-P.

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4. CONCLUSIONS [5] Blum, J., Melfi, A.J., Montes, C.R. and Gomes, T.M. (2013) Nitrogen and phosphorus leaching in a tropical Brazilian soil cropped with sugarcane and irrigated with treated sewage ef- There is a dynamic equilibrium process among all P fluent. Agricultural Water Management 117, 115-122. fractions in the soil P system, and all P fractions can affect and restrict one another. The relative abundance detected [6] Angelakis, A.N.,Marecos, M.H.F., Do Monte,Bontoux, L. and Asano, T. (1999) The status of wastewater reuse practice in the for nearly every P fraction followed the order Ca-P>resid- Mediterranean basin: Need for guidelines.Water Research33, ual P>NaOH-Po≈NaOH-Pi>KCl-P. NaOH-Pi and a part of 2201–2217. the Ca-P fraction contributed markedly to P availability [7] Rusan, M.J.M., Hinnawi, S. and Rousan, L. (2007) Long term when different phosphate sources were added to soil. effect of wastewater irrigation of forage crops on soil and plant Wastewater irrigation has various effects on P. In the shal- quality parameters. Desalination 215, 143-152. low layer, the transformation efficiency of P fractions into [8] Jaiswal, D. and Elliott, H.A.(2011) Long-Term Phosphorus stable P (NaOH-Po) was lower than that in the groundwa- Fertility in Wastewater-Irrigated Cropland. Journal of Envi- ter-irrigated area, whereas the transformation efficiency of ronment Qualit 40 (1), 214-223. P fractions into available P (NaOH-Pi) was greater under [9] Guo, H.A. andJaiswal, D.(2012) Phosphorus management for long-term wastewater irrigation conditions. In the middle sustainable agricultural irrigation of reclaimed water. Journal layer, wastewater irrigation can cause an increase in TP, of Environmental Engineering138, 367-374. and in the deep layer, wastewater and groundwater irriga- [10] Park, M., Singvilay, O., Shin, W., Kim, E., Chung, J. andSa, tion is beneficial to the down-transformation of P.These re- T.(2004) Effect of long-term compost and fertilizer applica- sults may depend on soil properties, water cycles, and bio- tion on soil phosphorus status under paddy cropping sys- logical processes, among other factors. tem.Communications in Soil Science and Plant Analysis 35 (11–12), 1635-1644. [11] Scherer, H.W. and Sharma, S.P.(2002) Phosphorus fractions and phosphorus delivery potential of a luvisol derived from lo- ACKNOWLEDGEMENTS ess amended with organic material. Biology and Fertility of Soils 35, 414-419. This study was supported by the National Natural Sci- [12] Shen, J., Li, R., Zhang, F., Fan, J., Tang, C. andRengel, Z. ence Foundation of China (No. 41103007) and the Appli- (2004) Crop yields, soil fertility, and phosphorus fractions in response to long-term fertilization under the rice monoculture cation and Development Fund from the Ministry of Sci- system on a calcareous soil. Field Crops Research6, 225-238. ences and Technology of the Inner Mongolia Autonomous Region (No. 2009058). The authors are grateful to the State [13] Guo, F., Yost, R.S., Hue, N.V., Evensen, C.I. and Silva, J.A. (2000) Changes inphosphorus fractions in soils under inten- Key Laboratory of Environmental Aquatic Chemistry, the sive plant growth. Soil Science Society of America Journal 64, Research Center for Eco-Environmental Sciences, and the 1681–1689. Chinese Academy of Sciences for their assistance in soil [14] Hu, C., Zhang, T.C., Kendrick, D., Huang, Y.H. andSuram- sampling and analysis. We also thank all supporters from palli, R.(2006) Muskegon wastewater land treatment system: the Tongliao Environmental Protection Agency and all re- Fate and transport of phosphorus in soils and life expectancy viewers and editors for their valuable comments and sug- of the system. Engineering in Life Science 6 (1), 17-25. gestions during the review process. [15] Waly, T.M., Elnaim, E.M., Omran, M.S. and El Nashar, B.M.B.(1987) Effect of sewage water on chemical properties The authors have declared no conflict of interest. and heavy metals content of ElGabal El Asfar sandy soils. Bi- ological wastes 22 (4), 275-284. [16] Murphy, J. and Riley, J.P. (1962) A modified single solution method for the determination of phosphate in natural waters. REFERENCES AnalyticaChimicaActa 27, 31-36. [17] Huguenin-Elie, O., Kirk, G.J.D. andFrossard, E.(2003) Phos- [1] O’Connor, G.A., Elliott, H.A.,and Bastian, R.K. (2008) De- phorus uptake by rice from soil that is flooded, drained or graded water reuse: An overview. Journal of Environmental flooded then drained. European Journal of Soil Science 54(1), Quality 37, S157-S168. 77-90.

[2] Hamilton, A., Stagnitti, F., Xiong, S.Z., Kreidl, S., Benke, K., [18] Zhang, T.Q., MacKenzie, A.F., Liang, B.C. and Drury, C.F. and Maher, P. (2007) Wastewater irrigation: The state of (2004) Soil test phosphorus and phosphorus fractions with play.Vadose Zone Journal6, 823-840. long-term phosphorus addition and depletion. Soil Science So- ciety of America Journal 68, 519-528. [3] Egwu, G.N. andAgbenin, J.O. (2012) Lead enrichment, adsorption and speciation in urban garden soils under long-term [19] Babatunde, A.O. and Zhao, Y.Q. (2009) Forms, patterns and wastewater irrigation in northern Nigeria.Environmental Earth extractability of phosphorus retained in alum sludge used as Sciences 69, 1861-1870. substrate in laboratory-scale constructed wetland systems. Chemical Engineering Journal 152 (1), 8-13. [4] Guo, G.X., Deng, H., Qiao, M., Yao, H.Y. and Zhu, Y.G. (2013) Effect of long-term wastewater irrigation on potential [20] Jalali, M. and SajadiTabar, S. (2011) Chemical fractionation denitrification and denitrifying communities in soils at the wa- of phosphorus in calcareous soils of Hamadan, western Iran tershed scale. Environmental science & technology 47, 3105- under different land use. Journal of Plant Nutrition and Soil 3113. Science 174, 523-531.

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[21] Yang, J.C., Wang, Z.G., Zhou, J., Jiang, H.M., Zhang, J.F., Pan, P., Han, Z., Lu, C., Li, L.L. and Ge, C.L. (2012) Inorganic phosphorus fractionation and its translocation dynamics in a low-P soil. Journal of Environmental Radioactivity 112, 64- 69. [22] Diaz, O.A., Daroub, S.H., Stuck, J.D., Clark, M.W., Lang, T.A. and Reddy, K.R. (2006) Sediment inventory and phosphorus fractions for Water Conservation Area canals in the Ev- erglades. Soil Science Society of America Journal 70, 863- 871. [23] Wright, A.L. (2009) Soil phosphorus stocks and distribution in chemical fractions for long-term sugarcane, pasture, turfgrass, and forest systems in Florid. Nutrient Cycling in Agroecosys- tems 83, 223-231.

[24] Turner, B.L., Cade-Menun, B.J., Condron, L.M. and Newman, S. (2005) Extraction of soil organic phosphorus. Talanta 66, 294-306. [25] Saleque, M.A. and Kirk, G.J.D. (1995) Root-induced solubili- zation of phosphate in the rhizosphere of lowland rice. New Phytologist 129, 325-336. [26] Pheav, S., Bell, R.W., White, P.F. and Kirk, G.J.D. (2002) Phosphate sorption–desorption behaviour, and phosphorus release characteristics of three contrasting lowland rice soils of Cambodia. Cambodian Journal of Agriculture 6, 39-54. [27] Mohsen, J. and Narges, H.M. (2013) Soil phosphorus forms and their variations in selected paddy soils of Iran. Environ- mental Monitoring and Assessment 185, 8557-8565. [28] Verma, S., Subehia, S.K. and Sharma, S.P. (2005) Phosphorus fractions in an acid soil continuously fertilized with mineral and organic fertilizers. Biology and Fertility of Soils 41, 295- 300.

[29] Shen, R. and Jiang, B. (1992) Distribution and availability of various forms of inorganic-P in calcareous soils. ActaPedolog- icaSinica 29 (1), 81-86 (in Chinese)

[30] Chu, Q., Wang, X.X., Yang, Y., Chen, F.J., Zhang, F.S. and Feng, G. (2013) Mycorrhizal responsiveness of maize (Zea mays L.) genotypes as related to releasing date and available P content in soil. Mycorrhiza 23, 497-505.

[31] Li, B., Ma, Q. and Guo, B. (2008) Forms and bioavailability of phosphorus in sand samples from Wulanbuhe Desert. Jour- nal of Ecology and Rural Environment 24 (3), 86-88, 93(in Chinese). [32] Wang, Y. and Zhang, Y. (2012) Soil inorganic phosphorus fractionation and availability under greenhouse subsurface irrigation. Communications in Soil Science and Plant Analysis 43, 519-532. [33] Magid, J., Kjærgaard, C., Gorissen, A. and Kuikman, P.J. Received: October 06, 2014 (1999) Drying and rewetting of a loamy sand soil did not in- Accepted: January 08, 2015 crease the turnover of native organic matter, but retarded the decomposition of added 14C-labelled plant material. Soil Bi- ology and Biochemistry 31, 595-602. CORRESPONDING AUTHOR [34] Nguyen, B.T. andMarschner, P.(2005) Effect of drying and rewetting on phosphorus transformations in red brown soils with Yintao Lu different soil organic matter content. Soil Biology and Bio- chemistry 37, 1573-1576. School of Civil Engineering Beijing Jiaotong University [35] Moore, P.A. and Reddy, K.R. (1994) Role of Eh and pH on phosphorus geochemistry in sediments of Lake Okeechobee, Beijing 100044 Florida. Journal of Environmental Quality 23, 955-964. P.R. CHINA

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INACTIVATION OF Monopylephorus limosus BY CHLORAMINE, CHLORINE DIOXIDE, AND HYDROGEN DIOXIDE: EFFECTIVENESS AND SAFETY

Kun Yao1,2,3, Yao Yang3, Lihong Zhao2, Baiyang Chen4 and Xiaoshan Zhu2,*

1School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, China 2Shenzhen Public Platform for Screening and Application of Marine Microbial Resources, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China 3College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China 4Harbin Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China

ABSTRACT been found in the tap water of Shenzhen, Guangzhou, Wujiang, Tianjin, and Shanghai cities in China, and also oc- The occurrence of redworms in water supplies raises cur in Essex of Britain and Lowell, Tacoma and Colcord of concerns among consumers regarding the safety of their the United States [1-10]. The presence of red worms in wa- drinking water. In this study, the inactivation effects of ter supplies raises concerns among consumers regarding three disinfectants, chloramines, chlorine dioxide (ClO2), the safety of their drinking water. Thus, inactivation of red and hydrogen peroxide (H2O2), on the redworm (Mono- worms in the water system while pose minimal risk to con- pylephorus limosus) in tap water were comparatively eval- sumers is desired. This paper investigates the killing effect uated. After inactivation, a Photobacterium phosphoreum of chloramine, chlorine dioxide, and hydrogen dioxide on acute toxicity experiment and a zebrafish embryonic devel- redworm (Monopylephorus limosus) eggs, larvae, and opment toxicity experiment were conducted to evaluate the adults found in the tap water of Tianjin, China. Because safety of the disinfected water. The results show that chlo- disinfection byproducts in drinking water may have certain ramines with 4 mg/L residual chlorine has relatively good toxic effects on the human body [2], the chemical safety of effect in killing redworm, and the disinfected water main- the disinfected water was evaluated using a Photobacte- tains low health risks; in contrast, the tap water is high toxic rium phosphoreum acute toxicity test and a zebrafish em- after killing redworm with even a low concentration (0.005%) bryonic development test. The purpose is to identify an ef- of H2O2, suggesting that H2O2 is probably not a good disin- fective, safe, and economical disinfectant that can improve fectant for controlling redworm in water supply; ClO2 at 6- the quality of urban water supply and ensure its chemical 8 mg/L level can inactive redworm effectively while the safety. risk of disinfected water to human health remains low. Based on the overall results of disinfection effectiveness, side-effect chemical safety, and practicality, chloramines 2. MATERIALS AND METHODS and ClO2 appear to be useful in reducing redworm risks in water supplies. 2.1 Experimental organisms

All the experiments using animals in this study were approved by the Animal Welfare and Ethics Committee of KEYWORDS: Tsinghua University (Shenzhen), China (No. 2012-XSZ- Redworm; inactivation; tap water; safety F66).

2.1.1 Red Worms 1. INTRODUCTION The red worms used in this experiment were M. limosus of phylum Annelida, class Oligochaeta, family Tubifi- “Red worm” is a group of benthic organisms that in- cidae, and genus Monopylephorus. M. limosus were col- clude various types of annelids (such as tubificids) and in- lected from the tap water of three consumers in Tianjin and sects (such as chironomid larvae) [1]. Red worms are com- cultivated in the environmental science laboratory of Nan- monly detected in water sources and secondary tanks. They kai University [8, 9]. M. limosus eggs (Figures 1A) and can grow and distribute in water supply systems. They have non-hatching larvae (Figures 1B) were picked from the culture dish using tweezers. Healthy eggs whose internal de- * Corresponding author velopment could be observed were selected by microscopy

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(Leica Company, Germany). M. limosus larvae 3 mm to H2O2 inactivation solution: H2O2 solution with an orig- 5 mm long (Figure 1C) were obtained from newly hatched inal mass fraction of 30% was dissolved in water to create eggs. The adults selected were grown to sexually mature inactivation solutions with different mass percentages. zooids with clitellum from 30 mm to 50 mm in length (aver- ClO2 inactivation solution: ClO2 release agent was age 37 mm) and weighing from 7 mg to 14 mg (Figure 1D). mixed with water at the same volume, according to the The samples were aspirated using a dropper and moved into product manual, to prepare inactivation solutions with dif- a culture disk filled with water from a breeding cylinder. ferent concentrations.

2.3.2 Inactivation of M. limosus adults Chloramine inactivation: The initial residual chlorine concentrations for the chloramine inactivation solutions prepared were 1.0, 2.0, 3.0, 4.0, and 8.0 mg/L. For each concentration, 20 red worms were used, and each was placed in a 100 mL beaker with 40 mL of inactivation solution. In control treatment, individual worms were placed in 100 mL beakers with 40 mL of water. Each beaker was wrapped with double layer aluminum foil to have dark conditions, in order to simulate the actual environment in tap water pipelines. Before testing, organic matter on the body surface of the red worms was removed as much as possible FIGURE 1 - The redworm at different stages. A. the redworm eggs by washing with water to prevent it from consuming the at the stage of granules; B. the redworm non-hatching larvae in egg available chlorine in the solution. The experimental and cocoon; C. the redworm larvae hatched within 24h; D. the mature redworm control beakers were maintained at 20 ± 1 °C. Red worms are very resilient at 20 °C [12], thus the experiments were 2.1.2 Luminescent bacteria Photobacterium phosphoreum conducted at this temperature to get the best inactivation. The variations in residual chlorine values were recorded at P. phosphoreum T3 spp. freeze-dried powder was purchased from the Institute of Soil Science, Chinese Acad- 0.5 h, 1.0 h, 1.5 h, and 2.0 h intervals. The mortality of red emy of Sciences. worms was detected after 2 hours treating. Red worms were considered dead when their somites were unable to 2.1.3 Zebrafish (Danio rerio) Embryos move independently [13]. Adult zebrafish were purchased from a market and fed H2O2 inactivation: The mass percentages of H2O2 in in an aquarium (2:1 female to male ratio). The water in the the inactivation solution were 0.005%, 0.006%, 0.0075%, aquarium was fully aerated and the water temperature was 0.01%, and 0.05%. Treatments and controls were set up in maintained at 26 °C ± 1 °C. The fish were fed twice daily the same manner as for chloramine inactivation. Experi- with frozen chironomid larvae disinfected using infrared mental beakers were wrapped with double layer aluminum radiation. The light/dark cycle was 14 h: 10 h. Fish eggs foil to simulate the dark condition in tap water pipelines, were collected, and fertilized eggs (embryos) were selected and to prevent volatilization of H2O2. The mortality rate of using a stereo microscope (Leica Company, Germany) af- red worms was measured after 2 hours. ter a month of cultivation. ClO2 inactivation: The initial ClO2 concentrations in the inactivation solution were 2.0, 4.0, 5.0, 6.0, and 8.0 mg/L, 2.2 Chemicals respectively. The procedure is the same as that for H2O2 Chloramine (chlorine water + ammonia water), 30% inactivation. hydrogen dioxide (H2O2, analytically pure), and chlorine dioxide (ClO2) release agent (liquid dual product, Tianjin 2.3.3 Inactivation of M. limosus eggs and non-hatching larvae Lvyuan Environmental Resources Technology Develop- Chloramine inactivation: Chloramine inactivation so- ment Co., Ltd.) were used as the disinfectants. Other rea- lutions, with initial residual chlorine values of 2.0, 3.0, and gents were all analytically pure, and the water used for the 4.0 mg/L, were prepared. Non-hatching larvae in egg co- experiments was aerated tap water. coons (Figure 1B) were used for the experiment. Prior to the experiment, organic matter was removed as much as 2.3 Inactivation Tests possible from the surface of the egg cocoon by washing 2.3.1 Preparation of inactivation solution with water. For each treatment and control, 10 egg cocoons Chloramine inactivation solution: Liquid chlorine was were placed in a 100 mL beaker with 40 mL of inactivation added into aerated water, followed by ammonia addition solution or water. Each beaker was wrapped with alumi- and 10 minutes shaking to prepare a chloramine inactiva- num foil to simulate the environment in tap water pipelines. tion solution with different residual chlorine values. The Beakers were maintained at 20 ± 1 °C, and the inactivation residual chlorine was measured using Ortho-toluidine vis- solution was changed every day. The results were observed ual colorimetry [11,12]. using microscopic examination, and recorded. The experi-

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ment was continued until the larva in all egg cocoons either T(%) ＝ [Bioluminescence in disinfected water (mv)/ Bio- hatched out or died (approximately 5 to 10 days). Mortali- luminescence in control (mv)]×100% (1) ties of the egg cocoons were subsequently calculated. Non- hatching larvae within a cocoon were considered dead To assess the safety of disinfected water, in this study, when they did not move within 15-30 s of observation after mercuric chloride (HgCl2) was used as the reference sub- gentle agitation of the test solution. The experiments were stance for toxicity, and the T (%) under different HgCl2 conducted with 3 replicates. concentrations was measured to obtain a standard curves.

H2O2 inactivation: Inactivation solutions of approxi- The linear regression equation for HgCl2 concentration (C) mately 6%, 3%, 1.5%, 1%, 0.5%, 0.1%, and 0.05% were versus T (%) based on the standard curve is as follows: 2 prepared. The red worm eggs at the stage of granule (Fig- T (%) ＝ 105.23 - 420.59 Cmercuric chloride (r ＝ 0.9901) (2) ure 1A) were used for this experiment. For each treatment, 10 eggs were placed into a 50 mL volumetric flask with Based on the standard curve and the linear regression 50 mL of inactivation solution or water. The volumetric equation, a toxicity concentration (equivalent to the mer- flask was used because the small neck of each flask can curic chloride concentrations (mg/L)) was adopted to rep- help to prevent volatilization of H2O2. The neck of each resent and classify the biological toxicity of the disinfected flask was tightly plugged and the flask wrapped in alumi- water (Table 1) [11, 12, 15, 16]. num foil to create inactivation conditions without illumina- tion. Flasks were maintained at 20 ± 1 °C. The experiment TABLE 1 - The toxicity classification standard of Photobacterium was continued for 0.5 h. After that, the damages and mor- phosphoreum toxicity test method tality of redworm eggs were observed using microscopic Relative Equivalent to the mercuric Toxicity examination, and recorded. luminosity (T) chloride concentrations (C) classification (%) (mg/L) 2.3.4 Inactivation of M. limosus hatching larvae T > 70 C < 0.07 low Chloramine inactivation solutions with initial residual 50 < T ≤ 70 0.07 ≤ C < 0.09 moderate chlorine values of 1.0, 1.3, 1.5, and 2.0 mg/L were prepared 30 < T ≤ 50 0.09 ≤ C < 0.12 serious with water. For each treatment, five larvae were placed into 0 < T ≤ 30 0.12 ≤ C < 0.16 high a 100 mL beaker with 40 mL inactivation solution or water. T ≤ 0 C > 0.16 very high Each treatment was replicated 10 times. The experimental conditions were the same as those described above. The The zebrafish embryonic development toxicity exper- mortality rate and variations in residual chlorine values iment was designed based on the operating instructions of were measured after 2 hours. All larvae that survived after the Organization of International Economy Cooperation and 2 hours were kept in their experimental conditions for one Development [14]. Intact zebrafish embryos at the blastula more day to observe additional mortality. The death criteria stage were immersed in the disinfected water or aerated tap for the larvae were the same as those for the adults. water (as control). Three parallel samples were used for each concentration. The tested embryos were then placed 2.4 Safety of tap water after disinfection of M. limosus in an illuminated incubator at 26 °C ± 1 °C for hatching 2.4.1 Tap water after disinfection of M. limosus (disinfected with a 14 h: 10 h light/dark cycle. The developmental water) stages of the embryos and fish larvae were observed at 2- For the safety assessment, tap water after 2-hour inac- 4, 12, 24, 36, 48, 60, 72, 84, and 96 h after contamination tivation by chloramine (2, 4, and 8 mg/L), ClO2 (4, 8, and using an inverted microscope (Leica Company, Germany). 16 mg/L), and H2O2 (0.005%, 0.01%, and 0.02% w/w), The end points that indicate toxicity, namely, the survival were used. A photobacterium phosphoreum acute toxicity rate of embryos or fish larvae, the embryo hatching rate, experiment and a zebrafish embryonic development tox- heartbeat, and pericardial edema were observed. Deformi- icity experiment were conducted directly to evaluate the ties among the embryos and fish larvae in the control and safety of the disinfected water. the experimental treatments were documented with a cam- era mounted on a stereo microscope. 2.4.2 Safety tests A P. phosphoreum acute toxicity experiment (GB/ T15441—1995) [11] and a zebrafish embryonic develop- 3. RESULTS AND DISCUSSION ment toxicity experiment [14] were used to measure the biological toxicity of the disinfected water. 3.1 Inactivation effects of different disinfectants on M. limosus adults P. phosphoreum acute toxicity was measured using the DXY-2 Biotoxicity Analyzer (Institute of Soil Science, Chi- 3.1.1 Inactivation effect of chloramine nese Academy of Sciences, Nanjing, China) by quantifying The inactivation effects of chloramine solution with the decrease in light emission from the bacteria as a result of different residual chlorine concentrations on the red worm exposure to the disinfected water for 15 min. The relative adults were shown in Table 2. After 2 h incubation, the in- luminosity T (%) was calculated based on equation (1): activation rate of chloramine increased with increasing

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concentration of residual chlorine, showing a dose-de- with increasing ClO2 concentration. For 5, 6, and 8 mg/L pended inactivation pattern. The non-inactivation concen- ClO2, the inactivation rate reached or exceeded 40%, 90%, tration was found to be 1.0 mg/L, whereas at concentration and 100%, respectively. Similar inactivation effects of of 8.0 mg/L the inactivation rate was high to 100%. More- ClO2 were reported on the other kind of red worm, such as over, a persistent inactivation effect was found for chlora- Chironomus kiiensis [17]. ClO2 with low concentration of mine with a function of exposure time. For example, the 2.5 mg/L could kill most of the C. kiiensis after treating for inactivation rate in 2.0 mg/L treatment was 25% after 2 h 24 hour. exposure while these surviving adults died with continuing exposure. 3.2 Inactivation effect of the disinfectants on M. limosus eggs and larvae TABLE 2 - The initial concentrations of disinfections and their red- 3.2.1 Inactivation effect of chloramine worm inactivation effects in 2 h The inactivation effects of chloramine solution with Disinfectants Initial concentration Inactivation effects different residual chlorine concentrations on the redworm (%) eggs and larvae were measured (Table 3). The mortality of chloramines 1 mg/L 0±0 eggs and non-hatching larvae increased with increasing 2 mg/L 25±5 concentration of residual chlorine. At low concentrations 3 mg/L 45±10 (< 2.0 mg/L), chloramine did not exert any inhibition on 4 mg/L 75±15 the development of larva in cocoons. At concentration of 8 mg/L 100±0 2.0 and 3.0 mg/L, only 33.3% of the eggs and non-hatching chlorine dioxide 2 mg/L 0±0 larvae were killed. However, at concentration of 4.0 mg/L, (ClO ) 2 chloramine solution effectively inhibited the development 4 mg/L 0±0 of the eggs causing completely death. Newly hatched lar- 5 mg/L 45±5 vae were used to further investigate the inactivation effect 6 mg/L 90±10 of chloramine solution at low concentrations (Table 3). The 8 mg/L 100±0 results indicate that the final 2-hour inactivation rate of hydrogen perox- 0.005 % 60±15 ide (H2O2) chloramine at 1.0 mg/L for larva reached 55%. The inacti- 0.006 % 85±5 vation rate would be higher up to 85% with longer expo- 0.0075 % 90±5 sure of 24 hours. Moreover, the mortality rate at 2.0 mg/L 0.01 % 100±0 treatment reached 100 % only after 2 hours of exposure. 0.05 % 100±0 These results again suggested that the inactivation effects of chloramine are related to the dose used and the exposure 3.1.2 Inactivation effect of H2O2 time. It is obvious that red worm larvae are more sensitive

The 2-hour inactivation effects of different H2O2 concen- than eggs in this experiment, which may reflect the weak trations on adult red worms were also shown in Table 2. Sim- penetrability of chloramine into egg cocoon. ilar to chloramine, the inactivation rates of H2O2 increased with increasing concentration. It was interesting to find, 3.2.2 Inactivation effect of H2O2 even at low concentration of 0.005%, the 2-hour inactiva- Inactivation experiments were conducted on red worm tion rate was high to 60 %. The 100% inactivation rate was eggs using H2O2 solutions at 6%, 3%, 1.5%, 1%, 0.5%, found when the mass percentage was increased to 0.01%. 0.1%, and 0.05% (w/w). Eggs were killed effectively within half an hour at 0.05%. The eggs in cocoons immersed in 3.1.3 Inactivation effect of ClO 2 H2O2 solution shrank by varying degrees and appeared de- As shown in Table 2, ClO2 ≤ 4 mg/L did not kill the formed (Figure 2). H2O2 might kill the red worm eggs red worms; however, the inactivation rate increased rapidly through the formation of highly oxidative free hydroxyl

TABLE 3 - The inactivation effects of chloramines solution to redworm Non-hatching (in egg cocoons) and hatching larvae

chloramines solution 24 h mortality of Non-hatching larvae in 2 h mortality of hatching larvae 24 h mortality of hatching (mg/L) egg cocoons (%) (%) larvae (%) 1.0 nd 55±5 85±10 1.3 nd 70±10 100±0

1.5 nd 90±10 100±0

2.0 33.3±10.2 100±0 100±0 3.0 33.3±10.2 nd nd 4.0 100±0 nd nd Note: nd means no detection

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TABLE 4 - The safety assessment of the disinfected waters based on P. phosphoreum toxicity assay

Disinfectants Initial Relative luminosity (T) Equivalent to the mercuric Toxicity concentration (%) chloride concentrations (C) (mg/L) classification chloramines 2 mg/L 89.87±5.07 0.0365 low 4 mg/L 88.24±5.58 0.0404 low 8 mg/L 75.23±2.66 0.0713 moderate

ClO2 4 mg/L 86.47±5.82 0.0446 low 8 mg/L 78.92±6.45 0.0626 low 16 mg/L 72.07±9.31 0.0788 moderate

H2O2 0.005 % 21.08±3.66 0.2001 very high 0.01 % 7.91±1.67 0.2314 very high 0.02 % 2.22±0.22 0.2449 very high

radicals and active derivatives [1,18]. It could damage the 3.3.2 Safety of H2O2 disinfected water permeability of biomembranes and cytomembranes via The safety of H2O2 disinfected water was also tested ionization by disrupting fatty acid chains. Once the mem- by P. phosphoreum toxicity assay (Table 4). The results brane structure is destroyed, its osmotic pressure and per- showed that the toxicity of H2O2 disinfected water is high, meability change, thereby killing the red worm eggs [19]. and it remains high even after 72 hours of aeration. At low H2O2 concentration of 0.005%, the disinfected water was highly toxic equaling to a very high toxic mercuric chloride with concentration of 0.2001 mg/L. Further testing using a zebrafish embryonic development assay showed that the all zebrafish embryos died after 36 hours of exposure to 0.02% H2O2 disinfected water. At 0.01% and 0.005% H2O2, the hatching of embryos were prolonged to different extents; and the hatched larvae exhibited physical deformities (the FIGURE 2 - The redworm eggs before (A) and after (B) immersing pericardium and abdominal were severely edematous) and in H2O2 solution (0.05%, w/w) for half an hour. died within 24 h (Figure 3). The high toxicity of the H2O2 disinfected water may be related to the hydroxyl radical (-OH) 3.3 Safety of Tap Water after Killing the Red Worms with Dis- infectant [23], because H2O2 dissociates in solution. Unlike the other disinfectants, H2O2 has strong inactivation effects on both 3.3.1 Safety of the chloramine disinfected water adults and eggs. However, H2O2 disinfected water is highly After aeration for 36 hours, the safety of chloramine toxic (Figure 3), and thus poses a higher health risk to hu- disinfected water was tested by a P. phosphoreum toxicity mans. assay. The results showed that chloramine solutions with higher residual chlorine resulted in higher toxicity in disinfected water (Table 4). The relative luminosity of P. phosphoreum was 75.2 % at 8 mg/L, which is equivalent to a moderately toxic mercuric chloride with concentration of 0.0713 mg/L. However, at lower concentration of 4 mg/L, the disinfected water was only slightly toxic after aeration. Further test using zebrafish embryonic development toxicity assay showed that the malformation rate and mortality of zebrafish embryos exposed to high concentration of 8 mg/L were not significantly (t-test, P > 0.05) different from those of the control. Moreover, during the whole disinfection, only limited amount of disinfection by-products (such as DBPs) were released, and free chloramine was FIGURE 3 - Effects of H2O2 disinfected water on zebrafish embryos continuously emitted [20]. Thus, the toxicity of the tap wa- and larvae. A: control embryo at 36 hpf (hour post-fertilization); B: ter disinfected by chloramine solution should be low. Heng dead embryo at 36 hpf; C: control larva, developing normally at 96 et al. (2003) [21] indicated that the health risk of chlora- hpf; D: larva with pericardium and abdominal edema at 96 hpf. mine for disinfection is lower than that using liquid chlorine. She and Xiao (2001) also showed that drinking water with 3.3.3 Safety of ClO2 disinfected water residual chlorine concentrations up to 5 mg/L for 12 weeks The toxicity of ClO2 disinfected water after 36 hours produces no adverse symptoms [22]. of aeration was measured and the results were shown in

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Table 4. The disinfected water from high concentration [3] Bay, E.C. (1993) Chironomid (Diptera: Chironomidae) larval (8 mg/L) ClO treatment had low toxicity (Figure 3), equiv- occurrence and transport in a unicipal water system. J. Am. 2 Mosq. Control. Assoc. 9, 275-284. alent to 0.0626 mg/L mercuric chloride. Further checking using a zebrafish teratogenicity test revealed that the 8 mg/L [4] Berg, M.B. (1995) Infestation of enclosed water supplies by Chironomids (Diptera: Chironomidae): Two case studies. Chi- ClO2 disinfected water was not teratogenic to zebrafish, ronomids from genes to ecosystems. Australia：CSIRO pub- again suggesting that the toxicity of ClO2 disinfected water lications, East Melbourne. is low. Moreover, considering the toxicity of ClO2 could be [5] Cui, F., Sun, X., Zhang, J., Xu, F., Liu, L. (2005) A review on minimized by aeration in the water plant and consumption preventions of chironomus larva pollution in water treatment in the pipe network. Therefore, although the concentration system. Environ. Pollut. Control. 27, 235-238. of 6 mg/L or 8 mg/L may be slightly high in the redworm [6] Dong, J. (2002) Causes and preventive measures of blood- inactivation, their disinfected water still remains low toxic. worm pollution in drinking water. Jiangsu. J. Prev. Med. 13, Nevertheless, during the actually redworm inactivation op- 47-48. eration, the ClO2 dosage should be optimized to maximize [7] Lu, J. (2001) Growth pattern and the control strategies of chi- water quality and inactivation effect while minimizing the ronomid (Tendipes gr. thammi) larvae in tap water. Chin. Wa- ter. Wastewater. 17, 53-54. health risks. [8] Chen, X., Zhu, L., Wang, Q., Yang, Y., Zhao, W., He, W., Han, H., Zhang, X. (2005a) Pilot study on control of redworm in drinking water. Water. Wastewater Eng. 31, 7-10. 4. CONCLUSION [9] Chen. X., Zhu, L., Wang, Q., Yang, Y., Zhao, W., He, W., Han, H., Zhang, X. (2005b) Pilot study on growth and repro- The inactivation effects of chloramine, ClO2 and H2O2 duction of Red Worm. Water. Wastewater. Eng. 31, 38-40. on M. limosus eggs, larvae, and adults were compared and [10] Eaton, K. (2013) Red worms found in drinking water supply the safeties of the resulting tap water were evaluated. The for Oklahoma town. NBC News. key finding are summarized as following: (1) 4 mg/L of http://www.nbcnews.com/news/other/red-worms-found- chloramine has an excellent effect on killing M. limosus drinking-water-supply-oklahoma-town-f8C11028887. adults, eggs, and larvae with very low chemical toxicity [11] MEPPRC, Ministry of Environmental Protection of the Peo- identified for the resulting tap water. (2) Although H O ple’s Republic of China. (1995) GB/T-15441. Water quality— 2 2 Determination of the acute toxicity—Luminescent bacteria has a significant effect on killing both adults and eggs of test. redworms, the resulting water is too toxic thus may pose a http://kjs.mep.gov.cn/hjbhbz/bzwb/shjbh/sjcgfffbz/199508/t1 relatively high risk to human health. (3) ClO2 at a range 9950801_67352.htm from 6 mg/L to 8 mg/L is very effective in killing M. limo- [12] SAPRC, Standardization Administration of the People’s Re- sus and poses low toxicity for the resulting water. These public of China. (2006) GB5750.11. Standard examination results suggested that chloramines and ClO but not H O methods for drinking water – disnifectants parameters. 2 2 2 http://www.moh.gov.cn/zwgkzt/pgw/201212/33655.shtml probably should be good disinfectants for controlling redworm in tap water. The findings in this paper may be help- [13] Song, ZH., Chen, TY. (1998) Toxicity of Tributyltin to Chi- ronomus Larvae. Environ. Sci. 19, 87-91. ful for water treatment plants to better control the redworm pollution in water distribution systems. [14] OECD. (1998) OECD212. Fish, short term toxicity test on embryo and sac-fry stages. OECD Guideline for Testing of Chemicals.

[15] Gu, Z., Xie, S., Wu, L., et al. (1983) The bio-toxicity determi- ACKNOWLEDGEMENTS nation of contaminated water using photobioluminometer. En- viron. Sci. 4, 30-33. This project was supported by the National Natural [16] Qiao, H., Gu, Z. (1993) Use of luminescent bacteria test for Science Foundation of China (21107058, 41373089), the quantitatively determining the toxicity of contaminated water. Shenzhen Key Laboratory for Coastal Ocean Dynamic and Environ. Her. (5), 11-13. Environment (ZDSY20130402163735964), the Scientific [17] He W. (2005) The control methods for Chironomus Kiiensis, Research Foundation for the Returned Overseas Chinese and the molecular classification of Chironomus Species in municipal water supply system. Thesis for master degree. China Scholars，State Education Ministry of China. Agricultural University, Beijing, China.

[18] Ye, J., Li, B. (2001) The killing experiments of Chironomid The authors have declared no conflict of interest. larvae as well as several associated issues. The 8th annual meet- ing of water supply committee, Water Industry Branch, China Civil Engineering Society. Chengdu, China. [19] Ge, Y., Huan, P. (1999) Hydrogen peroxide disinfection and REFERENCES its application. Sh. J. Prev. Med. (11), 157-158. [20] Zhang, X., Chen, C., He, W., et al. (2004) Control of disinfec- [1] Zhou, L., Zhang, J., Lei, P., Liang, M. (2003) The advances on tion byproducts in safe-chlorine-disinfection process. China the bloodworm control in water purification process. Water. Water. Wastewater. 20, 13-16. Wastewater. Eng. 29, 25-28. [21] Heng, Z., Li, C., Jiang, W., et al. (2003) Effect of chloramine [2] Michael, K. (1997) New strategies for the control of the par- disinfection on the formation of drinking water disinfection thenogenetic chironomid. J. Am. Mosq. Control. Assoc. 13, by-products and their muta-genicity. J. Environ. Heal. 20, 189-192. 134-136.

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[22] She, L., Xiao, G. (2001) The toxicological character of Cl2 and chlorine dioxide used in drinking water disinfection. Fujian. Chem. Industry. (1), 15-17.

[23] Liu, X., Tang, Y., Xu, S., et al. (2003) Study on treatment of pre-oxidation hanjiang river water with hydrogen peroxide in pilot plant. J. Hunan. Ur. Construc. Col. 12, 23-25.

Received: October 12, 2014 Revised: December 11, 2014 Accepted: January 08, 2015

CORRESPONDING AUTHOR

Xiaoshan Zhu Shenzhen Public Platform for Screening and Application of Marine Microbial Resources Graduate School at Shenzhen Tsinghua University Shenzhen 518055 P.R. CHINA

Phone: +86-755-26036381 Fax: +86-755-26036381. E-mail: [email protected]

FEB/ Vol 24/ No 7/ 2015 – pages 2334 - 2340

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ADSORPTION OF LEAD METAL FROM AQUEOUS SOLUTIONS USING ACTIVATED CARBON DERIVED FROM SCRAP TIRES

Ali Reza Rahmani1, Ghorban Asgari2, Fateme Barjasteh Askari3,* and Ameneh Eskandari Torbaghan4

1 Environmental Health Engineering Department, Faculty of Health and Research Center for Health Sciences, Hamadan University of Medical Sciences, Hamadan, Iran 2 Department of Environmental Health Engineering, Faculty of Health, Hamadan University of Medical Sciences, Hamadan, Iran 3 Department of Environmental Health Engineering, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran 4 Department of Environmental Health Engineering, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran

ABSTRACT taining large quantities of heavy metals like cadmium, lead and copper [1]. Lead is one of those heavy metals which is Heavy metals in water-bodies are one of the serious en- harmful to human health causing different signs, such as vironmental problems in many communities. In this study, anemia, headache etc. [2]. Different techniques, such as the potential of activated carbon derived from scrap tires chemical precipitation, coagulation and flocculation, are was investigated for adsorption of lead metal cations (Pb2+) used to remove highly concentrated and heavy metal-con- from aqueous solutions. Activated carbon was prepared taining wastewaters [1, 3]. However, most of them are not from scrap vehicle tires using a thermo-chemical activation practical enough due to the low concentration of heavy method. Scanning electron microscopy (SEM), BET and metals; therefore, these methods should be used along with BJH analyses were used to characterize the prepared acti- supplementary techniques [1]. vated carbon. Batch adsorption experiments were carried Ion exchange and membrane filtration technologies out to determine the effects of initial concentration of the can reduce heavy metal concentrations to acceptable levels ions, pH, contact time and adsorbent dosage. Adsorption of environmental regulations, but high costs and opera- isotherms of the metal ions on the adsorbent were meas- tional problems are the drawbacks of these methods [3, 4]. ured and correlated with the Langmuir and Freundlich iso- During the recent decades, the use of activated carbon in therm models. The predominant component of prepared water and wastewater treatment industries has been consid- activated carbon is carbon (76.481%) with 185.046 m2/g ered [5, 6]. Activated carbon had successfully been able to surface area and an average pore diameter of 52 nm. The remove a wide range of pollutants from the environment. results showed that there was a decrease in removal of Pb2+ It can also remove most heavy metals [5]. In general, find- with increasing contact time, adsorbent dosage and pH. Ex- ing economic ways of water and wastewater treatment have perimental adsorption data were best fitted by Freundlich been a controversial issue among experts. model. In view of the findings, it can be claimed that the production of the activated carbon from scrap tires can pro- Activated carbon can be produced from raw materials vide a green and cost-effective method for heavy metals having high amounts of carbon and small parts of inorganic removal. matters [7]. The most prevailing ones in this case are wood, some plants, piceous coal, brown coal, lignin, coconut shell, fruit shells like nut shells, almond shells, rice husks etc. [8,

KEYWORDS: 9]. Activated carbon, adsorption, scrap tire, thermo-chemical Just in Iran, about 250000 tons of scrap tires are produced annually (6.3 kg per capita per year) [10]. The methods used to recycle scrap tires are not efficient enough [10]. 1. INTRODUCTION Accumulated masses in the environment cause environmental problems and lead to an increase in flies and rodent Industrialization has increased the environmental pol- numbers; on the other hand, illegally piled scrap tires can lution through producing huge amounts of wastes. Heavy set fires [11]. The positive aspects of using scrap tires to metals are classified as the most important environmental make new products are cheapness and abundance. One of pollutants due to their high toxicity to humans and ecosys- the products that can be obtained from the scrap tires is ac- tems. Some human activities, such as mining, extraction tivated carbon. A fixed amount of carbon (about 25% of industries and metal melting, discharge wastewaters con- mass) and very low levels of volatile ash (>5%) of rubber are the advantages of the derived carbon [7, 11]. Pyro-oil * Corresponding author and pyro-gas produced beside the pyrolysis of scrap tires

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to produce activated carbon, can be used as a source of re- 2.3. Adsorption experiments newable energy [7, 8]. The adsorption tests were performed in a batch system, Production of scrap tire-derived carbon could have and the effective parameters on the adsorption process in- commercial value in air quality and wastewater treatment cluding the contact time (30-180 min), pH of solution (2, industries, and can be used to store gas, to separate and 4, 6 and 8), adsorbent dosage (0.1-0.6 g), and initial Pb2+ treat various pollutants [12]. As a whole, economical prof- concentration (10-100 mg/L) were studied in different itability and eco-friendly properties are the two main ad- stages. The speed of stirring was 200 rpm, the tempera- vantages of producing activated carbon from scrap tires [9]. ture was 25±1 °C, and the size of adsorbent particles was In this study, the ability of prepared activated carbon 100 mesh. The volume of test solutions was 50 ml and the 2+ in removal of Pb2+ as a heavy metal from aqueous solutions adsorbent particles were separated after the Pb removing was investigated. process by centrifugation. The tests were performed by making the other variables constant. To study the isotherm model fitting, Langmuir and Freundlich models were used.

2. MATERIALS AND METHODS In order to determine pHZPC, the equilibrium technique in a batch system was applied [14]. The percent of removed This experiment involves three separate but sequential Pb2+ by activated carbon was calculated from Eq. 1: steps: activated carbon production, characterization of ac- 2+ tivated carbon, and adsorption experiments. Activated car- Removed Pb (%) = [(Ca-C)/Ca]×100 (1) bon was produced by a thermo/chemical method through Where, Ca is the initial Pb2+ concentration before ad- the pyrolysis process. Scrap tire samples were pyrolysed dition of the adsorbent and C is Pb2+ concentration after by a muffle furnace (model Exiton). All the experiments adsorption, with produced activated carbon, and with Eq. were carried out based on the standard methods mentioned 2, the carbon yield after pyrolysing was calculated. in reference [13]. The unknown concentrations of Pb2+ solutions were measured by atomic absorption (Analyst 700, Carbon yield% = Wac/Wti (2) Perkin Elmer). NaOH or H2SO4 solution (1N) was used to In the equation, Wti is the weight of used scrap tire for adjust the pH during the experiments (pH-meter, model pyrolysing and Wac is final produced activated carbon. PP-50, Sartorius). All the chemicals used in this study were prepared from Merck and Aldrich companies. Distilled water was prepared by a Fater Electronic water distiller model 3. RESULTS AND DISCUSSION 2104 (Tehran, Iran). For all batch experiments, glassware and bottles were washed and rinsed with HNO3 before use, 3.1. The structural characteristics of the prepared activated and then by distilled water. A 301 Sigma centrifuge was carbon used for sample separation. Based on the conducted analyses, carbon is the main element (76%) of the structure of the processed activated 2.1. Adsorbent preparation carbon (Table 1). The result of this section are consistent Scrap tire samples were crushed into 0.2-0.3 cm pieces with the findings of other investigators. Teng et al. (2000) and put into KOH solution for 3 h to become activated [7], Galvagno et al. (2002) [8] and Mahramanlioglu et al. (mass ratio for KOH to tire was 4:1). Next, the mixture was (2006) [15] reported on carbon as the main compound of heated up to 110 °C in an oven for 24 h. Then, the tires scrap tire-derived activated carbon. As shown in Table 1, were pyrolysed (oven temperature raised to 700 °C during the specific surface area of prepared activated carbon was 2 h). The pyrolysis products were washed with 250 ml HCl determined 185 m2 g-1 (Table 1). solution (0.5 N) at 85 °C for 30 min. In the next step, the products were washed with water until the pH of water and TABLE 1 - Characteristics of the prepared activated carbon carbon mixture reached above 6.9. At last, the final product BET(m2gr-1) 185.046 was dried at 110 °C for 24 h. The produced samples were BJH(m2gr-1) 146.4 sieved by standard ASTM sieves into 100 mesh (149 µm) Total pore volume (cm3 gr-1) 0.58 particles and kept for the next usages. pHzpc 6.87 Particle size (µm) <149 2.2. Characterization of prepared activated carbon Average pore size (nm) 52.461 C 76.481 A scanning electron microscope (SEM) of Majlesi O 10.136 Components (wt.%) University of Material Engineering (Kv 20.0, take off an- Al 7.29 gle 25.0º and elapsed lifetime 10.0) was used to show the K 6.093 morphologic properties. Specific surface area, total pore volume and average pore size were measured by BET (Bar- In the preparation process, the production efficiency of ret, Brunner and Emerett) isotherm using Belsorp software activated carbon from scrap tires was 32 and 36.5%. A (BEL, Japan, Inc.). The distribution size of the pores was SEM micrograph of the produced activated carbon is determined by BJH (Barrett, Joyner Halenda) method. shown in Fig. 1. Based on the SEM micrograph, processed

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activated carbon has a non-crystalline structure with a wide At pH values above pHzpc, the carbon surface has more neg- range of pore sizes. (Fig. 1). ative charge and lead as a cation attracts more to the adorbent surface whereas at pHs below pHzpc, lead cation adsorption decreased. In the present study, the highest removal efficiency was obtained at pH 8. That is to say, in acidic condi- + tions, H3O ions which compete with metal cations are adsorbed on active sites of the adsorbent. When saturating these sites, the adsorption of metal cations decreased [18]. Mehrasbi et al. (2008) [19] investigated the removal of heavy metals from an aqueous solution by adsorption on almond shells; their results show that the adsorption values decreased when lowering pH value, and optimum pH was determined to be 5-6 [19]. In this study, almond shells were separately treated with acidic solution (0.4 mol/L HNO3) and basic solution (0.4 mol/L NaOH). Kannan and Rengasamy (2005) [17] studied the adsorption of nickel(II) ions on different carbons. In this study, it was reported that the increase in pH increased the removal percentage of Ni(II) FIGURE 1 - SEM micrograph of scrap tire derived carbon ions which is in accordance with our results [17]. Meena et al. [12] investigated the efficiency of heavy metal removal 3.2. The effect of pH on adsorption process by granular activated carbon; the results show that at pH 4, bivalence cations like mercury, manganese and cadmium Adsorption experiments were conducted at different have the highest removal efficiencies, and up to pH 7, Cd pH values while other parameters were constant. Experi- removal efficiency decreased. But for other 3 bivalence cat- ments showed that metal removal efficiency increased with ions (Pb, Ni, Zn), pH 8 was determined as the optimum pH increasing pH (Fig. 2). Alkaline pH was found as the opti- [12]. The results of this research are consistent with those of mum pH. Meanwhile, the blank solution was used during the present study. Increase in adsorption efficiency at pHs the rest of the experiment. Regardless of the different val- >6 is due to the reaction of metal hydroxide species with the ues of adsorption efficiencies, adsorption was observed at micropores of activated carbon. The differences in results of all pHs and metal ions were removed from the solution. different pH values can be because of the various effects of The process can highly be used to remove heavy metals pH on adsorbents used in the different studies [2, 19, 20]. from effluents at different pH values (Fig. 2). Moreover, in the mentioned studies, the concentration of the The pH value is one of the most effective parameters metal cations are different from examined concentrations in in adsorption process. Hydrogen ions compete with cations this study (more or less than 10 ppm), and this can be another (adsorbate fraction) in adsorption process [16]. The pH can reason for these differences. At higher initial concentration, also affect adsorbent surface charge. According to other the decrease of adsorption rate resulted in equilibrium time studies, the pH range for adsorption on different activated reduction. Hossein Zadeh et al. (2010) [20] reported that the carbons is between 4 and 8 [16-18]; therefore, this range most efficient acidic dye removal on scrap tire-derived acti- was determined for the experiments in this study. As can vated carbon was observed in acidic conditions [20]. In gen- be seen from Fig. 2, the removal efficiency of lead cations eral, according to the obtained results and also to parallel under acidic conditions was lowest, particularly at pH 2. studies, it is concluded that in adsorption process, the pH plays a significant role in pollutants removal. That is to say, 100 the effect of pH on adsorption differs with the types of both adsorbent and pollutant. 80 60 3.3. The effect of contact time on adsorption process 2+ 40 The effect of contact time on removal of Pb by produced activated carbon indicated that with increasing the 20 contact time, the adsorption rate was increased (Fig. 3). The metal cations adsorption on activated carbon reached

Adsorption efficiency % efficiency Adsorption 0 an equilibrium after 3 h, and after that, the amount of ad- 2468 sorption remained constant. These results show that the in- pH itial adsorption on scrap tire-derived activated carbon is a FIGURE 2 - Effect of solution pH on Pb2+ adsorption: time=60 min., rapid process which is related to activated carbon structure Pb2+=10 mg/L, activated carbon 0.1 g (activated carbon pores are classified in the meso range). The blanks, which were used to show the effects of pH on The pHzpc is a parameter that affects the adsorption removal efficiency in different contact times, removed the efficiency, and it was determined to be 6.87 in this study. pollutants to some extent but it was negligible (Fig. 3).

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100 nificant and can be due to the either increase of adsorption activated sites or general increase in specific surface area 80 of the adsorbent. These results are consistent with the findings of Meena et al. (2010) [12]. Conversely, in studies 60 with herbal biomass or other living organisms as adsor- 40 bents, the removal efficiency decreased with increasing ad- Ex perimental samples sorbent dose, and the results were not the same as in our 20 study. This decrease can be because of a rise in bioadsor- Adsorption efficiency,% Blank samples bent concentration which prevents metal cations from ad- 0 sorbing into the cells. In addition, as the amount of bio- 0 30 60 90 120 150 180 sorbent increases, the density of cellular compounds in- Contact time, min creases, and leads to adsorption sites reduction [22], while

2+ for activated carbon it i not like that. Kim et al. (2005) [21] FIGURE 3 - Effect of contact time on removal of Pb : activated car- 2+ 2+ bon 0.1 g, pH=8, Pb2+ =10 mg/L reported that the adsorption of Pb and Cu increased with increasing the bioadsorbent dose while for Cd this was Determining optimum contact time is required to use quite opposite [21]. In Elaigwu et al. (2010) [23], the in- an adsorbent for pollutant removal. Thus, the adsorption crease in activated carbon dose enhanced the Pb2+ removal efficiency was investigated at different times (30, 45, 60, efficiency. 90, 120 and 180 min). As can be seen in Fig. 3, for lead, the greatest amount of adsorption happened up to 30 min, 100 and after that, amount increased slightly with a constant trend; this is also mentioned in the study carried out by 80 Meena et al. (2010) [12]. The reason is that, at first, more 60 adsorption sites are available and a higher driving force transferred the solute to the adsorption sites more easily. 40 Experimental samples But after a while, the number of active sites occupied by 20 adsorbate were diminished and, consequently, adsorption Blank samples 0 rates decreased [12]. This can be related to the tiny struc- % efficiency, Adsorption 0.1 0.2 0.3 0.4 0.5 0.6 ture of pores on the surface of adsorbents (Fig. 1). Since the adsorption is an equilibrium reaction, the adsorption of Adsorbent dose, gr metal cations on the activated carbon continued to reach an 2+ equilibrium. If the active adsorption sites are more acces- FIGURE 4 - Effect of adsorbent dose on removal of Pb : time=180 min., Pb2+ =10 mg/L, pH=8 sible to the soluble metal cations or other adsorbate components, less time will be required for the maximum ad- 3.5. The effect of initial concentration of the metallic solution sorption, and otherwise, it will take more time [20]. Meena Lead removal under various initial concentrations did et al. (2010) [12] investigated the removal of heavy metals not show a clear trend but the highest removal efficiency on granular activated carbon, the equilibrium time was ob- (%99.166) was observed in a 60 ppm Pb solution. (Fig. 5). tained to be 48 h while it was determined to be 3 h in this study. In Hossein Zadeh et al. (2010) [20], adsorption of acidic dyes on activated carbon derived from scrap tire was 100 studied, and the equilibrium time was measured to be 2 h; 80 in the study of Kim et al. (2005) [21], the equilibrium time With activated carbon Without activated carbon for Pb and Cu adsorption on brewery biomass was meas- 60 ured to be 60 min which is inconsistent with our findings. Uzun et al. (2000) [16] found that the equilibrium time for 40 adsorption of various metal cations on activated carbon 20 was 130-150 min which was similar to the results of our study. The difference in our findings is a result of structural .Adsorption efficiency, % 0 properties of the used adsorbents. 10 20 40 60 80 100 Pb concentration, mg/L 3.4. The effect of adsorbent dose on adsorption process FIGURE 5 - Effect of initial concentration on removal of Pb2+: Lead adsorption was investigated by using different time=180 min., activated carbon 0.1 g, pH=8 amounts of the produced activated carbon (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 g). As shown in Fig. 4, removal efficiency The results showed that the adsorption capacity of the increased with increasing adsorbent dose. The Pb removal sorbent increased by raising the initial concentration of efficiency increased from 68.3 to 88.1% (Fig. 4). metal ions, which may be due to an increase in metal ions Increasing the adsorbent dose (activated carbon) raised driving force. Under concentrations higher than the opti- the lead metal cation removal efficiency. This rise is sig- mum level, the adsorption sites get saturated which reduces

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more adsorption capacity and removal efficiency [12]. The results showed that the adsorption isotherms of Pb2+ Meena et al. (2010) [12] results conform to our findings on activated carbon were better fitted by the Freund-lich whereas in Kim et al. (2005) [21] experiments, the results model (0.982

Freundlich Langmuir 2 1 y = 0,115x + 0,2789 1,6 R² = 0,9607 0,8 0,6 1,2 qe 0,8 0,4 y = 0,533x + 0,3541 Ce/qe Log R² = 0,9827 0,2 0,4

0 0 -1-0,500,511,5 -0,2 0510 Ce Log Ce

FIGURE 6 - Sorption isotherms with fitted models for Pb2+ metal sorption: pH=8, activated carbon=0.6 gr

TABLE 2 - Isotherm parameters for Pb2+ adsorption on the prepared activated carbon

2 Isotherm model R K N qm (mg/g)

Freundlich 0.982 2.259 1.876 -

Langmuir 0.960 3.197×10-2 - 8.695

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4. CONCLUSION [6] Sciban M., Radetic B., Kevresan Z. and Klasnja M. (2007). Adsorption of heavy metals from electroplating wastewater by wood sawdust. Bioresource Technology, 98, 402-409. The results showed that there was a decrease in removal of Pb2+ with increasing contact time, adsorbent dos- [7] Teng H., Lin Y-C and Hsu L-Y (2000). Production of Acti- vated Carbons from Pyrolysis of Waste Tires Impregnated age and pH. The optimum pH was found to be 8 and the with Potassium Hydroxide. Air & Waste Management Asso- adsorption data were best fitted to Freundlich model. ciation, 50(11), 1940-6. Activated carbon has been used under different aspects [8] Galvagno S., Casu S., Casabianca T., Calabrese A. and Cor- of environmental issues to remove various pollutants but nacchia G. (2002). Pyrolysis process for the treatment of scrap the production of commercial activated carbon is costly. tyres: preliminary experimental results, Waste Management, Moreover, the management of solid wastes, especially non- 22(8), 917-23. biodegradable wastes such as scrap tires, is one of the most [9] Ioannidou O. and Zabaniotou A. (2007). Agricultural residues common and important environmental issues. Therefore, as precursors for activated carbon production—A review, Re- newable and Sustainable Energy Reviews, 11(9), 1966-2005. preparing activated carbon from scrap tires can be an economic solution to solve the accumulation problems of high [10] Brady T.A., Rostam-Abadi M. & Rood M.J. (1996). Applica- volumes of used tires. On the other hand, there is a wide tions for activated carbons from waste tires: Natural Gas Stor- age and Air Pollution Control, Journal of Separation and Puri- range of pollutants in industrial effluents which are dis- fication, 10(2), 97-102. charged into the environment. The best way ever to remove [11] Ko D.C.K., Mui E.L.K., Lau K.S.T. and McKay G. (2004). Pro- these pollutants, in terms of low costs, simple design, ease duction of activated carbons from waste tire - process design and of operation and insensitivity, is adsorption. Thus, with re- economical analysis, Waste Management, 24(9), 875-88. gard to environmental and economical features, the scrap [12] Meena A.K., Chitra R., Kiran and Mishra G.K. (2010). Re- tire derived activated carbon can be profitable although fur- moval of heavy metal ions from aqueous solutions using ther research should be done. chemically (Na2S) treated granular activated carbon as an adsorbent, Journal of Scientific & Industerial Research, 69(6), 449-53.

[13] Glesceria L.A., Greenberg E. and Eaton A.D. (1998). Stand- ards method for the Examination of Water and Wastewater. ACKNOWLEDGMENT 20th edition. This study was carried out with technical and financial [14] Hameed B.H., Tan I.A.W. and Ahmad A.L. (2008). Adsorp- tion isotherm, kinetic modeling and mechanism of 2,4,6-tri- support of the Vice Chancellor for Research and the Re- chlorophenol on coconut husk-based activated carbon, Chem- search Center for Health Sciences of Hamadan University ical Engineering Journal, 144, 235–244. of Medical Sciences. The authors of the manuscript do not [15] Mahramanlioglu M., Bicer I.O., Misirli T., Caliskan E., Gül S. have any relation with the commercial identities mentioned and Misirli C. (2006) The removal of anionic naphtalene de- in the manuscript. rivatives by the adsorbents produced from used tires, Fresenius Environmental Bulletin, 15(9b), 1150-55. The authors have declared no conflict of interest. [16] Uzun I. and Guzel F. (2000). Adsorption of some heavy metal ions from aqueous solution by activated carbon and comparison of percent adsorption results of activated carbon with those of some other adsorbents, Turkish Journal of Chemistry 24, 291-7. REFERENCES [17] Kannan N., Rengasamy G. (2005). Studies on the removal of nickel(II) ions by adsorption using various carbons - a com- [1] Lagoa R. and Rodrigues J.R. (2007). Evaluation of dry proto- parative study, Fresenius Environmental Bulletin, 14(6), 435- nated calcium alginate beads for biosorption applications and 43. studies of lead uptake. Applied Biochemistry and Biotechnol- ogy, 143(2), 115–28. [18] Taty-Costodes V.C., Fauduet H., Porte C. (2003). Removal of Cd(II) and Pb(II) ions, from aqueous solutions, by adsorption [2] Khalir W., Hanafiah M., So’ad S. and Ngah W. (2011). Ad- onto sawdust of Pinus sylvestris, Hazardous Materials, 105, sorption behavior of Pb (II) onto xanthated rubber (Hevea 121-142. brasiliensis) leaf powder. Polish Journal of Chemistry, 13(4), 84-88. [19] Mehrasbi M. R., Farahmandkia Z., Taghibeigloo B. and Taromi A. (2009). Adsorption of lead and cadmium from [3] Fu F. and Wang Q. (2011). Removal of heavy metal ions from aqueous solution by using almond shells, Water, Air, and Soil wastewaters: A review. Journal of Environmental Manage- Pollution, 199(1-4), 343-351. ment, 92(3), 407–418. [20] Hoseinzadeh E., Rahmani A.R., Asgari Gh., McKay G. and [4] Kurniawan T.A., Chan Y.S. G., Lo W-H and Babel S. (2006). Dehghanian A.R. (2012). Adsorption of acid black 1 using ac- Physico–chemical treatment techniques for wastewater laden tivated carbon prepared from scrap tires: kinetic and equilib- with heavy metals. Chemical Engineering Journal, 118(1-2), rium studies, Journal of Scientific & Industrial Research, 71, 83-98. 682-689. [5] Amuda O.S., Giwa A.A. and Bello I.A. (2007). Removal of [21] Kim T-Y., Park S-K., Cho S-Y., Kim H-B., Kang Y., Kim S- heavy metal from industrial wastewater using modiﬁed acti- D., Kim S-J. (2005). Adsorption of heavy metals by brewery vated coconut shell carbon. Biochemical Engineering Journal, biomass, Korean Journal of Chemical Engingeering, 22(1), 36, 174–181. 91-8.

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[22] Vasudevan P., Padmavathy V., Dhingra S.C. (2003). Kinetics of biosorption of cadmium on bakers yeast, Bioresource Tech- nology, 89(3), 281-287.

[23] Elaigwu S.E., Usman L.A., Awolola G.V., Adebayo G.B., Ajayi R.M.K. (2009). Adsorption of Pb(II) from aqueous solution by activated carbon prepared from cow dung, Advances in Natural and Applied Sciences, 3(3), 442-46. [24] Seki K, Saito N, Aoyama M. (1997). Removal of heavy metal ions from solutions by coniferous barks. Wood Science Tech- nology. 31(6), 441-7. [25] Özdemir M., Şahin Ö. and Güler E. (2004). Removal of Pb(II) ions from water by sunflower seed peel, Fresenius Environ- mental Bulletin, 13(6), 524-531.

Received: October 16, 2014 Revised: December 11, 2014 Accepted: March 04, 2015

CORRESPONDING AUTHOR

Fateme Barjasteh Askari Torbat Heydariyeh University of Medical Sciences Department of Environmental Health Engineering Torbat Heydariyeh IRAN

E-mail: [email protected]

FEB/ Vol 24/ No 7/ 2015 – pages 2341 - 2347

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PREPARATION, CHARACTERIZATION OF Fe ION EXCHANGE MODIFIED TITANATE NANOTUBES AND PHOTOCATALYTIC ACTIVITY FOR OXYTETRACYCLINE

Changyu Lu1,2, Weisheng Guan1,*, Yuexin Peng1, Li Yang1, Tuan K.A. Hoang2 and Xiao Dong Wang2,3

1School of Environmental Science and Engineering, Chang’an University, Xi’an, 710054, China 2Department of Chemical Engineering, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo Ontario N2L 3G1, Canada 3 State Key Laboratory of Coal Conversion, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan, 030001, China

ABSTRACT the low efficiency and high cost. Recently, the photocatalytic oxidation has been established to be one of the most To enhance the photo degradation efficiency for oxy- prospective technologies for environment remediation and tetracycline (OTC), Fe-TiNTs were prepared via a simple provided a promising proposal for the transformation and and fast hydrothermal process using TiO2 powder as pre- degradation of OTC [5]. cursors. A wide range of techniques, such as X-ray diffrac- TiO , due to its nature of chemical stability, environ- tion (XRD), X-ray photoelectron spectroscopy (XPS), trans- 2 mental-friendliness, and high activity, has been widely mission electron microscopy (TEM), Brunauer-Emmett- used as a highly-efficient photo-catalyst [6, 7]. Titanate Teller (BET) and Ultraviolet-visible spectroscopy (UV- nanotubes (TiNTs) with a high specific surface area, ion- vis) were applied to characterize as-obtained Fe-TiNTs. changeable potentiality, and photocatalytic ability have Moreover, we have studied the photocatalytic properties of been considered for an extensive number of applications the Fe-TiNTs in the degradation of OTC. The results of our [8, 9]. Therefore, preparation of TiNTs by simple hydro- investigation and analysis have shown that regularly uni- thermal method has caught the attention of the scientific form nanotubes were fabricated by the hydrothermal treat- community. Unfortunately, as is well known, TiO , due to ment, and they had homogeneous tubular structures and 2 its wide band gap (Eg = 3.2 eV), can only respond to ultra- open ends. UV-vis spectra of Fe-TiNTs demonstrated its violet light (less than 387 nm), which means a low utiliza- absorption edge had remarkable red shift to the visible light tion rate of solar energy [10]. Meanwhile, the low photo region. Fe-TiNTs exhibited higher photocatalytic activities efficiency of TiNTs caused by their wide band gap and than that of TiNTs and TiO powder when they were eval- 2 quick recombination of electron-hole pairs limit the practi- uated to degrade OTC under visible light irradiation. cal use of TiNTs as an efficient photocatalyst [11]. Metal ion [12-15] modified TiNTs will definitely change their

KEYWORD: properties, however, as a result of the metal ion modifica- titanate nanotubes; photocatalysis; ion exchange; oxytetracycline. tion the structure of the nanotubes can be strongly influenced as well. Recently, many researchers have been re-

ported for the intercalation of alkaline ions [16] and some 1. INTRODUCTION transition-metal ions [17] between the layers in the multi- layered nanotubes by ion-exchange method. Some groups Antibiotics have become a special group of pharma- incorporated Fe, Ni into TiNTs via a simple hydrothermal ceuticals used to control and treat infection diseases in hu- procedure and investigated their electronic, optical, and man and veterinary medicine [1]. The resulting environmen- magnetic properties [18, 19]. tal contamination by the use of antibiotics in a wide range of Herein, we for the first time report the photocatalytic human activities has been receiving special attention in re- degradation of OTC on Fe-TiNTs under visible-light. Fe- cent years. Oxytetracycline (OTC) is a famous broad-spec- TiNTs photocatalyst is synthesised via a fast and conven- trum antibacterial agent widely used for treating bacterial in- ient method under a simple and mild reaction condition. fection [2]. And it has been found in a wide range of envi- Furthermore, Fe-TiNTs was characterized by XRD, XPS, ronmental samples including surface water, groundwater, TEM, BET and UV-vis. Moreover, we have also studied wastewater and drinking water [3, 4]. Unfortunately, the the properties of photoabsorption and photocatalytic deg- conventional degradation processes are often constrained by radation of the as-prepared Fe-TiNTs. Then, our study is necessary and significant for Fe-TiNTs from the value of * Corresponding author practical applications.

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2. MATERIALS AND METHODS 3. RESULTS AND DISCUSSION

All of the reagents were of analytical grade and were To investigate the crystalline structures of composite used without further purification and the water used was photocatalysts, XRD studies of TiO2 power, TiNTs and Fe- been deionized. TiNTs were successfully prepared via the TiNTs were conducted. Fig.1 exhibits the corresponding hydrothermal synthesis as follow: Firstly, 1 g of TiO2 was XRD patterns of as-obtained samples. As shown in Fig. 1, added to 60 ml of 8 M NaOH solution, and the mixture was the diffraction peak of TiNTs and Fe-TiNTs both are at 2θ hydrothermally reacted at 150 °C for 15 h under stirring in = 24.5°, 28.6°, 48.3° which can be confidently correspond a Teflon-lined stainless steel autoclave. The resulting white to the (110), (130), (200) reflection of the H2Ti3O7. Other- precipitate was washed with 3% HNO3 and finally with wise, Fe-TiNTs has extra peak at 25.3°which is correspond water again to obtain a neutral salt-free product. The nano- to the (101) reflection. XRD results displayed that the TiO2 tubes were dried at 80 °C. We prepared the Fe-TiNTs in have been transformed to TiNTs completely and no anatase the same procedures as the TiO2 nanotubes except instead or rutile was found in the obtained nanotubes. However, we of washing with 3% HNO3 solution, without roasting at a high can’t find diffraction peak about Fe in the XRD results. XPS temperature, we washed the precipitate with 3% Fe(NO3)3 so- measurement was applied to measure the chemical state of lution. the elements in Fe-TiNTs samples. As shown in Fig. 2, the Ti 2p3/2 peaks of the Fe-TiNTs appear at 458.53 eV [20]. 2.1 Characterization and measurements. The O 1s has a binding energy of 530.48 eV [21]. In addi- The products were characterised by X-ray diffraction tion, the peaks of 709.57 eV and 711.55 eV [22, 23] are measurements performed on an X-ray diffractometer response to the Fe2p3/2 and indirectly indicates that the Fe (XRD, Bruker D8 Advance diffractometer) with Cu-Ka ra- exists in the sample. diation in the range of 10-80° at a scanning rate of 7°min−1. The typical TEM images of TiNTs and Fe-TiNTs are The morphology of the products was observed by a trans- presented in Fig. 2. Fig. 2(a) demonstrates the similar micro- mission electron microscope (TEM, JEM-2100). The UV- structure of TiNTs with an outer tube diameter of 10 nm, vis diffused reflectance spectra of the samples were ob- inner diameter of 5 nm and a wall thickness of about 2-3 nm. tained from a UV-vis spectrophotometer (UV2550, Shi- The smooth nanotubes are hollow and the two terminals are madzu, Japan), BaSO4 was used as a reflectance standard. opening. In Fig. 2(b), the surface of Fe-TiNTs is still The investigation of the surface of the sample was by an smooth and the tube did not change much after Fe ion ex- X-ray photoelectron spectrometer (XPS, PHI-5300). The change treatment which suggested the Fe did not accumu- Brunauer-Emmett-Teller (BET) surface areas were deter- late on the cover of the nanotube [24]. Photo-generated mined from nitrogen adsorption-desorption isotherms at charge carries can be quickly transferred to the surface of 77 K (ASAP 2020, Micromeritics, America). nanotubes, because walls of these nanotubes are rather thin.

2.2 Photocatalytic degradation of antibiotic. BET results, summarized in Table 1, the surface areas and bore diameter of Fe-TiNTs, TiNTs, and TiO2 powder The photocatalytic properties of the powders were car- were 174.02, 176.29 and 43.78 m2/g, 5.76, 5.82 and 3.76 Å, ried out at 298 K in our home-made instruments. The pho- respectively. The surface area was significant increased after tochemical reactor contains 0.1 g Fe-TiNTs and 100 mL of hydrothermal treatment, which was strongly related to the 20 mg/L OTC aqueous solution. To exclude the influence morphological change from nanoparticles to elongated of physical adsorption, the reactor was kept into darkness nanostructures with hollow tubes. Moreover, the large spe- for 30 min to reach the adsorption equilibrium. The photo- cific surface area of nanotubes also provides more effective chemical reactor was irradiated with a 300 W xenon lamp reaction sites for photocatalysis [25]. which was located with a distance of 8 cm at one side of the containing solution. UV lights with wavelengths less To further understand Fe-TiNTs, we have studied the than 420 nm were removed by a UV-cutoff filter. The sam- UV-vis diffused reflection spectra. As shown in the Fig. 3, pling analysis was conducted in 15 min interval. The pho- the absorption cut-off wavelength for TiO2 is determined tocatalytic degradation ratio (DR) was calculated by the to be about 400 nm, which means TiO2 only absorb ultra- following formula OTC absorption concentration was de- violet (UV) light and transmit visible light. The absorption termined using the TU-1800 UV-vis spectrophotometer spectrum of TiNTs presented in Fig. 3 shows the similar (Shanghai AoXi technology Instrument CO., Ltd.) by re- curve in UV light, but the adsorption is weaker than TiO2. cording the variations of the absorption band maximum at This phenomena belongs to the blue shift in the UV-vis λ = 267nm (OTC) .The degradation rate (DR) was calcu- wavelength which be attributed to the reduce of the particle lated by this formula (1): size after the synthesis of nanotube by hydrothermally reacted [26]. In Fig. 3, the absorption edge of the Fe-TiNTs

DR = [(1–Ai/A0)] ×100%. (1) displays an obvious shift to the visible light region as compared to TiNTs. That may be attributed that Fe exchanged Where A0 is the initial absorbency of the OTC antibi- Ti and formed impurity levels near or above the valence otic waste water solution which reached absorbency bal- band resulting in a decrease in band gap energy. Another ance and Ai is the absorbency of reaction solution. reason underlying this phenomenon that Fe 3d electron en-

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FIGURE 1 - XRD pattern (a) of the typically as-synthesized TiO2, TiNTs and Fe-TiNTs sample. XPS spectra of Ti peaks (b), Fe peaks (c) and O peaks (d) of Fe-TiNTs

FIGURE 2 - TEM images of TiNTs (a) and Co-TiNTs (b).

TABLE 1 - Physicochemical properties of the prepared samples

Average value of Average value of Samples BET surface area (m2/g) BET bore diameter (Å)

TiO2 43.78 3.76 TiNTs 176.29 5.82 Fe-TiNTs 174.02 5.76

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FIGURE 3 – UV-Vis diffuse reflection absorption spectra of TiO2, TiNTs and Co-TiNTs.

FIGURE 4 - The adsorption-desorption (a) and photocatalytic degradation (b) on tetracycline hydrochloride of two samples: TiO2, TiNTs and Fe-TiNTs.

ergy levels are higher than that of Ti 3d, leading to the lev- gap without changing its valence band edge, which results in els emerging in the band gap region between the O 2p and the absorption of visible light for metal modified TiO2 [28]. Ti 3d dominant bands. From this interesting result it can be TOC analysis is an effective method which can be used considered that the Fe-TiNT material is a hopeful candidate to further demonstrate mineralisation of organic pollutants, for solar energy applications [27] especially the properties of the photocatalysts. Figure 4b To demonstrate the potential application of Fe-TiNTs showed the degradation of the organic carbon content with in photocatalytic degradation of OTC and investigate influ- Fe-TiNTs, TiNTs and TiO2 photocatalysts on the degrada- ences of morphology on the photocatalytic activity, we tion of OTC under visible irradiation. TOC contents of dif- evaluated the photocatalytic degradation of OTC on Fe- ferent samples decreased in the same order as that of the TiNTs in relation to TiNTs and TiO2 under visible light. As photocatalytic degradation curves (Figure 4a) could also be shown in Fig. 4a, the degradation rates of Fe-TiNTs, TiNTs found out from Figure 4b. However, the removal rate of and TiO2 are 74.89%, 32.41% and 17.84%, respectively. It TOC is lower than that of the DR. This result is reasonable can be concluded that Fe-TiNTs shows the highest visible due to the degradation data were detected after centrifuga- light photocatalytic activity. Some reasons are proposed to tion. TOC removal and the degradation curves with similar account for the high photocatalytic activity of the Fe- trends indicate that photodegradation experiments have TiNTs: as previously reported, metal ions can introduce successfully eliminated the effect of physical adsorption new energy levels of the transition metal ions into the band and the photocatalytic activity of different photocatalysts

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was be correctly evaluated. Moreover, widely environmen- [5] Zhao C., Zhou Y., Ridder D. J., Zhai J., Wei Y. M. and Deng tal applications, such as the mineralization, would be im- H. P. (2014) Advantages of TiO2/5A composite catalyst for photocatalytic degradation of antibiotic oxytetracycline in plemented by Fe-TiNTs, since OTC the reduced TOC con- aqueous solution: Comparison between TiO2 and TiO2/5A tents suggests. composite system. Chem. Eng. J., 248,280-289. [6] Fan W. Q., Bai H. Y., Zhang G. H., Yan Y. S., Liu C. B. and Shi W. D. (2014) Titanium dioxide macroporous materials 4. CONCLUSION dopedwith iron: synthesis and photo-catalytic properties. Cryst. Eng. Comm., 16, 116-122. In summary, Fe-TiNTs were successfully synthesized [7] Lu C. Y., Guan W. S., Zhang G. H., Ye L. J. and Zhang X. via a fast and convenient method under a simple and mild (2013) TiO2/Fe2O3/CNTs magnetic photocatalyst: a fast and convenient synthesis and visible-light-driven photocatalytic reaction condition. The obtained nanotubes are hollow and degradation of tetracycline, Micro. Nano. Lett., 8, 749-752. the two terminals are opening with an outer tube diameter of 10 nm, inner diameter of 5 nm and a wall thickness of [8] Chen X., Mao S. S. (2007) Titanium dioxide nanomaterials: synthesis, properties, modifications and applications, Chem. about 2-3 nm. In addition, we have also studied the mor- Rev., 107, 2891–2959. phology and photocatalytic degradation of the as-prepared [9] Ou H. H., Liao C. H., Liou Y. H., Hong J. H. and Lo S. L. Fe-TiNTs through the different characterization testing (2008) Photocatalytic oxidation of Aqueous Ammonia over technology. Moreover, the photocatalysis of the compo- Microwave-Induced Titanate Nanotubes. Environ. Sci. Tech- sites are better than that of TiO2 for the change of structure nol.,42, 4507-4512. and properties. [10] Wang Q., Chen C. C., Ma W. H., Zhu H. Y. and Zhao J. C. (2009) Pivotal role of fluorine in tuning band structure and visible-light photocatalytic activity of nitrogen-doped TiO2. ACKNOWLEDGEMENTS Chem.-Eur. J., 15, 4765-4769. [11] An H. Q., Li J. X., Zhou J., Li K. R., Zhu B. L. and Huang W. This work was supported financially by the National P. (2010) Iron-coated TiO2 nanotubes and their photocatalytic Natural Science Foundation of China (No. 21407012 and performance. J. Mater. Chem., 20, 603-610. 41472220), the Doctor Postgraduate Technical Project of [12] Jum S. J., Dong H. K., Sun H. C., Ji W. J., Hyun G. K. and Chang’an University (No. 2014G5290009), China Schol- Lee, J. S. (2012) In-situ synthesis, local structure photoelectron chemical property of Fe-intercalated titanate nanotube. arship Council (No.201406560003, NO.201506560022) , Int. J. Hydrogen. Energy., 37, 11081-11089. Research and Development Project of Science and Tech- [13] Wang L. S., Xiao M. W., Huang X. J., Wu Y. D. (2009) Syn- nology for Shaanxi Province (No. 2013JQ2019 and No. thesis, characterization, and photocatalytic activities of titan- 2015KJXX-25), the Fundamental Research Funds for the ate nanotubes surface-decorated by zinc oxide nanoparticles. Central Universities (2014G3292007, 2014G2290017 and J. Hazard. Mater.,161, 49-54. 310829153507), Project funded by Postdoctoral Science [14] Dong H. K., Jum S. J., Nam H. G., Min S. K., Jin W. L., Sun Foundation (No. 2014M552400), Foundation of State Key H. C., Dong W. S., Kim S. and Kyung S. L. (2009) Structural Laboratory of Coal Conversion, China (J14-15-603), Na- characterization and effect of dehydration on the Ni-doped ti- tional Training Projects of the University Students’ Inno- tanate nanotubes, Catal. Today., 146, 230-233. vation and Entrepreneurship program (No. 201510710071 [15] Rónavári A., Buchholcz B., Kukovecz Á. and Kónya Z. (2013) and No.201510710077). Structure and stability of pristine and Bi and/or Sb decorated titanate nanotubes, J. Mol. Struct., 104, 104-108. The authors have declared no conflict of interest. [16] Ma R., Sasaki T., Bando Y. (2005) Alkali metal cation intercalation properties of titanate nanotubes. Chem. Commun., 7, 948-950.

REFERENCES [17] Sun X., Li Y. (2003) Synthesis and characterization of ionex- changeable titanate nanotubes. Chem- Eur. J, 92,229-2238. [1] Pereira J. H., Queirós D. B., Reis A. C., Nunes O. C., Borges [18] Han W. Q., Wen W., Ding Y., Liu Z. X., Mathew M. M., M. T., Boaventura R. A. and Vilar V. J. (2014) Process en- Lewis L., Hanson J. and Gang O. (2007) Fe-doped trititanate hancement at near neutral pH of a homogeneous photo-Fenton nanotubes: formation, optical and magnetic properties, and reaction using ferricarboxylate complexes :Application to ox- catalytic applications, J. Phys. Chem. C, 111, 14339-14342. ytetracycline degradation. Chem. Eng. J., 253, 217-228. [19] Jiang J., Gao Q., Chen Z., Hu J. and Wu C. (2006) Syntheses, [2] Verlicchi P., Galletti A., Petrovic M., Barceló D. (2010) Hos- characterization and properties of novel nanostructures con- pital effluents as a source of emerging pollutants: an overview sisting of Ni-titanate and Ni-titania. Mater. Lett., 60, 3803- of micropollutants and sustainable treatment options. J. Hy- 3908. drol., 389, 416-428. [20] Gonçalves J. E., Castro S. C., Ramos A. Y., Alves M. C. and [3] Jones O. A. H., Voulvoulis N., Lester J. N. (2002) Aquatic en- Gushikem Y. (2001) X-ray absorption and XPS study of tita- vironmental assessment of the top 25 English prescription nium mixed oxides synthesized by the sol–gel method, J. Elec- pharmaceuticals. Water. Res., 36, 5013-5022. tron. Spectrosc., 114-116, 307-311. [4] Brown K. D., Kulis J., Thomson B., Chapman T. H. and [21] Leinen D., Fernández A., Espinós J. P., Holgado J. P. and Gon- Mawhinney D. B. (2006) Occurrence of antibiotics in hospital, zález-Elipe A. R.. (1993) An XPS study of the mixing effects residential, and dairy effluent, municipal wastewater, and the induced by ion bombardment in composite oxides. Appl. Surf. Rio Grande in New Mexico. Sci. Total. Environ., 366 ,772-783. Sci., 68, 453-459.

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[22] Bajnóczi É. G., Balázs N., Mogyorósi K., Srankó D. F., Pap Z., Ambrus Z., Canton S. E., Norén K., Kuzmann E., Vértes A., Homonnay Z., Oszkó A., Pálinkó I. and Sipos P. (2011) The influence of the local structure of Fe(III) on the photocatalytic activity of doped TiO2 photocatalysts-An EXAFS, XPS and Mössbauer spectroscopic study. Appl. Catal. B-Environ., 103, 232-239. [23] Sathe A., Peck M. A., Balasanthiran C., Langell M. A., Roux R. M. and Hoefelmeyer J. D. (2014) X-ray photoelectron spectroscopy of transition metal ions attached to the surface of rod- shape anatase TiO2 nanocrystals, Inorg. Chim. Acta., in press, DOI: 10.1016/j.ica..08.011. [24] Xiong L., Yang Y., Mai J. X., Sun W. L., Zhang C. Y., We D. P., Chen Q. and Ni J. R. (2010) Adsorption behavior of methylene blue onto titanate nanotubes. Chem. Eng. J., 156, 313- 320.

[25] Liang H. C., Li X. Z. (2009) Effects of structure of anodic TiO2 nanotube arrays on photocatalytic activity for the degradation of 2, 3-dichlorophenol in aqueous solution, J. Hazard. Mater, 162, 1415-1422. [26] Yao B. D., Chan Y. F., Zhang X. Y. and Zhang W. F. (2003) Formation mechanism of TiO2 nanotubes. Appl. Phys. Lett., 82, 281-283. [27] Ohsaki Y., Masaki N., Kitamura T., Wada Y., Okamoto T., Sekino T., Niihara K. and Yanagida S. (2005) Dye-sensitized TiO2 nanotube solar cellsfabrication and electronic characterization. Phys. Chem. Chem. Phys., 7, 4157-4163. [28] Litter M. I. (1999) Heterogeneous photocatalysis-Transition metal ions in photocatalytic systems, Appl. Catal. B-Environ., 23, 89-114.

Received: November 02, 2014 Revised: December 08, 2014 Accepted: January 22, 2015

CORRESPONDING AUTHOR

Weisheng Guan School of Environmental Science and Engineering Chang'an University Yanta Road 126 Xi'an, 710054 P. R. CHINA

Phone: +86 02982334561 E-mail: [email protected]

FEB/ Vol 24/ No 7/ 2015 – pages 2348 - 2353

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ADSORPTION CHARACTERISTICS OF PHOSPHORUS ONTO SOILS FROM DIFFERENT LAND USE TYPES IN DANJIANGKOU RESERVOIR AREA

Meng Xu1,2, Liang Zhang1,*, Yun Du1, Chao Du1,2, and Yan-Hua Zhuang1

1.Institute of Geodesy and Geophysics, Chinese Academy of Sciences, Wuhan 430077, China 2.University of Chinese Academy of Sciences, Beijing 100049, China

ABSTRACT industry though, the excess P entering into water body may cause eutrophication among other environmental problems Characteristics of phosphorus adsorption onto soils [1]. Agricultural runoff is considered to be the largest have been studied in many researches to reveal the adsorp- source of phosphate causing eutrophication because of the tion capacities and to explore the mechanism of phospho- overuse of soluble phosphate as a fertilizer [2]. The sedi- rus behavior in water-sediment interface. In this study, the ment-water interface such as riparian zone, lakeside zone land use types in Danjiangkou reservoir riparian area was or water-level-fluctuate zone are always considered to be investigated in April 2013 before the soon-coming impound- buffer zones when receiving runoff containing P, since ment of Danjiangkou reservoir for the first time. The inves- they act as both sinks and sources of P [3,4]. These buffer tigation was implemented between 160m and 170m water- zones between the pollutant source areas and the receiving lines, and samples of soil in five different land uses waters were considered to have potential use in improving (cropland, forest land, abandon cropland, grass land, orange water quality decades ago [5,6] and their effectiveness of orchard) were taken in the elevations of 160m, 165m and removing P has been confirmed in many later researches 170m. After the field work, the analysis of chemical com- [7]. The study of the buffer zones functioning has per- ponents of the soil and the experiments of phosphorus ad- suaded researchers to explore the mechanism of P behavior sorption capacity of these samples were conducted in labor- in these sediment-water interfaces. As an important ap- atory. The results showed that: (a) the traditional Langmuir proach to reveal the various biogeochemical processes oc- and Freundlich equations can be well fitted to experiment curring in sediment-water interfaces, the study of adsorp- data, the Qmax obtained ranged from 141.89~209.96 mg/kg tion of P has always been concerned. The primary re- with an average value of 191.61mg/kg; (b) The values of searches of P adsorption during 1950s to 1960s focused on EPC0 ranged from 0.05~0.59 mg/L, which were higher phosphatic fertilizers in lakes and sediments [8], later ex- than the liquid phosphate concentrations. This indicated periments confirmed that P was an important limiting fac- these samples had a trend of releasing P as a source; (c) the tor in eutrophication [9,10]. adsorption capacity and isotherm parameters were influ- To study the adsorption characteristics of P, adsorption enced by sediment components, especially by the content capacity was explored by many experiments as an essential of Fe and Al, while surface area and sample elevation point to predict long-term P availability [11-14]. To reveal showed no obvious effect in the present system. the P adsorption behavior and response in nature, environmental parameters which may affect the adsorption were

always concerned [15-17] and many of the researches fo- KEYWORDS: water-level fluctuations; South-North Water Diver- cused on the effect of land use types [18,19]. Moreover, sion Project; cropland; sediment; isotherm there were researchers found the traditional adsorption models failed in some way to explain the mechanism, and then tried to provide new theories and models [20].

1. INTRODUCTION The Danjiangkou reservoir is an important drinking water source protection area and also part of the middle route for China’s South-to-North Water Diversion Project. As an essential element for plants and crops, phospho- The project has changed some of the land use types and the rus (P) is also one of the most important components of water-level-fluctuated zone in this area would be operated non-point source pollution. The worldwide application of out-of-season due to the impoundment and future dis- phosphatic fertilizers brings great prosperity in agricultural charge. The consequent variations of water levels and the changing of hydrological regime in this area would make * Corresponding author water quality protection a new challenge in the future. The

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study of potential pollutant and assessment of future risks year. The average annual precipitation was 830mm and would be necessary and appreciated. To explore the P con- 70–80 % occurs between April and October. The mean an- tent and retention in this special background, this study fo- nual temperature ranged from 12~16 ºC. According to the cused on P adsorption in typical land uses of Danjiangou. statistic data in 2010, the land area in Danjiangkou city was Batch experiments of adsorption and analysis of soil com- 3121km2 including 204.2km2 cultivated land and the forest ponents were conducted to: (a) investigate the P sorption cover was 39.9%; the water quality of Danjiangkou reser- isotherm of soils from different land uses and (b) explore voir held steady in class II national water quality standard the relationship between p adsorption and soil characters in [21]. The official website of south-to north water diversion this area. announced the water transfer would be launched in late Oc- tober of 2014, right after the flood season. The protection of water quality in Danjiangkou reservoir is becoming well 2. MATERIALSAND METHODS concerned as the water transfer project has changed both the hydrological and the environmental conditions. 2.1 Study site This study was conducted in Danjiangkou reservoir 2.2 Land use investigation method area(32°11'34''~32°52'40''N，109°23'7''~111°57'55''E) The land use investigation of Danjiangkou reservoir (Fig. 1). This area belongs to the subtropical monsoon of was based on the USGS land use classification system. The the north subtropical zone. The distribution of precipitation image data used in this study was from HJ-1 with a spatial is declining from south to north, shaped by both the topog- resolution of 30m. Geometric correction and atmospheric raphy and the monsoon. The annual precipitation fluctu- correction was conducted before classification. Supporting ated from 500mm in low flow year to 1500mm in high flow data also included DEM, slope and social references of

FIGURE 1 -Map of study site and sampling locations

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Danjiangkou. Land use information was extracted and flasks with 50mL 0.02M KCl solutions containing 0, 1, 2, 4, practiced by classification and regression tree (CART). 6, 8 and 110 mg/L P in the form of KH2PO4. Then the sus- pensions were placed in the shaker for 3 hours to achieve the 2.3 Sampling and soil analysis equilibrium. Subsequently, the solutions were centrifuged at Seven surface soil samples (0~20cm) were taken from 8000r/min for 5mins and then filtered through a 0.45μm Danjiangkou reservoir including 5 different land use types membrane. (Fig. 1 & Table 1). Samples were taken specifically be- The P concentrations in these filtrates were analyzed tween the elevations from 160m to 170m because this area by molybdenum blue colorimetry using a visible spectro- would be the new-born water-level-fluctuated zone since photometer (UNIC, 7200). These experimental conditions the dam of Danjiangkou reservoir has been heightened for were chosen and optimized after several preliminary exper- China’s South-to-North Water Diversion Project. Soil iments. components were analyzed by X-ray Fluorescence Spec- trometer (Bruker AXS, S4 Pioneer) using standardless quan- 2.5 Statistic method titative analysis method [22]. Surface area were analyzed by Model fitting and curve plotting was performed by us- BET method [23]. ing the non-linear trial-and-error regression method.

TABLE 1 - Brief description of sampling sites 3. RESULTS AND DISCUSSION Sample Land use Elevation S1 cropland 160m 3.1 Land use distribution S2 cropland 165m S3 cropland 170m To study the adsorption characteristics of soils from wa- S4 forest land 165m ter level fluctuation area, the land use distribution was inves- S5 abandoned cropland 165m tigated between elevations of 152m (current water level in S6 grass 165m 2013) to 170m (highest water level in the future). The study 2 2 S7 orange orchard 165m area was 1405.83km in total, including 504.03km water areas. The land use of the study area was varied. Fig. 2 showed 2.4 Adsorption experiment the land use distribution percentage of this area and the water area was taken off in this histogram. Cropland ac- Batch adsorption experiments were performed in an counted for more than 60% of the total land areas. Respec- orbit water bath shaker (LSHZ-300A) under 25 ºC. To in- tive distribution of bushes, orchard and bare land were hibit the microbial activity, all the samples were heated at nearly 10%. As the heightening of the dam, basin clearing 170 ºC for 2h [24]. has been conducted in this area all along. Field investiga- Equilibrium isotherms were conducted as followed. tion of the study area in April, 2013 found most cropland For each sample, 2g soil samples (air-dried, ground to pass had been abandoned. The clearing of orchards and forest a 100-mesh sieve) were placed in a series of conical glass land was also under going by then.

wetland

grass construction land forest land

bare land

orchard

bushes

cropland

0 10203040506070 Land use areas (%)

FIGURE 2 -Distribution of different land use areas

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3.2 Soil components and characteristics some other researches [25,28]. However the retention of P The general physicochemical properties of sediments due to Fe and Al was not permanent and these phosphorus were presented in Table 2. can be released under certain environmental conditions [29]. The results in our study implied there may be some TABLE 2- Composition and BET surface area of samples other environment factors which promoted the releasing of phosphorus in some sites. BET surface Sample Fe (%) Al (%) P (%) C (%) area(m2/g) Another factor affecting the phosphorus retention is S1 3.931 8.15 0.0605 1.37 31.2732 the surface area. Concentrations of BET surface areas of these sediments ranged from 1.9m2/g~39.7 m2/g, sediment S2 4.405 9.288 0.0873 0.79 37.6955 of forest land had a much smaller surface area than others. S3 3.875 7.846 0.0674 1.83 39.7162 Phosphorus content seemed irrelevant to BET surface area S4 2.436 8.872 0.0867 2.15 1.9339 based on our data. S5 4.999 10.56 0.0777 0.506 20.0681 Moreover, rare earth element Y was also detected in these samples except for other ordinary elements in soil. S6 4.967 10.22 0.0677 0.559 32.4675 There were articles studied how REEs (rare earth elements) S7 4.951 10.25 0.0533 0.166 37.8823 would affect the behavior of phosphorus and some correlations between some REEs and phosphorus in runoff was Generally, carbon concentration in sediments was detected [30], while the relationship between element Y highly concerned due to its influence on sediments reten- and phosphorus was not found. tion capacity of P. The total C concentration showed the 3.3 Equilibrium isotherm study lowest value of 0.166% in orange orchard sediment and the highest of 2.15% in forest land sediment. P concentration Langmuir model and Freundlich model were widely in these sediments taken from elevation of 165m were in applied to study the isotherms of phosphorus adsorption on decreasing order as cropland＞forestland＞abandoned soils. The Langmuir model was generally expressed as: cropland＞grass＞orange orchard. The sediment (from or- Q C ange orchard) which obtained the lowest value of total C max eq Q Qe  (1) concentration also showed the lowest value of phosphorus K L  Ceq concentration, while the highest values of C and P concentrations were not synchronized. Familiar research in Three- Where Qe (mg/kg) is the amount of adsorbate of adsor- gorge reservoir observed synchronization in the highest bent, Ceq (mg/L) is the equilibrium concentration of ad- values of organic matter and TP content among several sorbate in solution, Qmax (mg/kg) is the theoretical maxi- samples, and sample with lowest content of organic matter mum of adsorption capacity and KL (mg/L)is a Langmuir showed a second highest TP content [25]. This indicated constant related to adsorption rate. that P content in sediments was relevant to the existence of The Freundlich equation was expressed as: carbon. However the P retention in sediments was determined by many other complex factors and the relationship 1 n between carbon and phosphorus cannot be an easy correl- Qe  K F Ceq Q KC (2) ativity. The content of Fe and Al has been considered as a main factor that enhances phosphorus retention because of KF and 1/n are purely empirical parameters which the high specific surface [26]. The average content of Fe stand for the Freundlich capacity factor and the Freundlich and Al in these samples were 4.22±0.92 % and 9.31±1.08% intensity respectively[31].Qe (mg/kg) is calculated by ex- respectively, which were higher than the soil background perimental data as: values of china [27]. Concentrations of metal elements like Fe, Ni, and Ti were correlated with each other (p＜0.05), ‐ (Cadd  Ceq )V Q Q  (3) and metal elements including Fe, Ni, Zn, Ti, and Al were cor- e w related with total C concentration (p＜0.05). According to Ta- ble1, Fe+Al content of sediments from different land use at Where Cadd is the initially added phosphorus concen- the same elevation showed a decreasing order as abandoned tration in solution of batch experiments (mg/L); V is solu- cropland＞orange orchard＞grass＞cropland＞forest land. tion volume (L); w is dry weight of added sediment (kg); And the phosphorus concentration of these sediments was Experiments data and fitting results of 7 samples were sequenced as cropland＞forestland＞abandoned cropland＞ illustrated in Fig.3 and Table 3. Table 3 shows that adsorp- grass＞orangeorchard. In our study, lower phosphorus tion data of S1~S5 and S7 were well fitted to both Lang- concentration was observed in the sediments group (taken muir and Freundlich equations with R2 ranging from0.95 from abandoned cropland, orange orchard and grass) with to 0.99. In the contrast, adsorption data of S6 can be de- higher contents of Fe+Al. Sediments with higher content scribed fairly by both equations with R2 of 0.92 and 0.88 of Fe or Al were observed to obtain more phosphorus in for Langmuir and Freundlich equations respectively. The

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Langmuir equation gave a Qmax of these samples ranged Based on the theory of modified isotherm and taking from 141.89 to 211.33mg/kg. NAP into account, the modified Freundlich equation [32] can be described as: TABLE 3 - Summary of the fitting results of the traditional Langmuir and Freundlich equations (9) Langmuir equation Freundlich equation Q Sample max K R2 1/n K R2 (mg/kg) L F (10) S1 206.97 1.26 0.98 0.51 83.28 0.95 S2 209.96 1.02 0.98 0.49 94.56 0.99 S3 156.76 2.21 0.99 0.52 48.31 0.99 (11) S4 141.89 7.03 0.99 0.69 18.97 0.97 S5 211.33 0.99 0.99 0.49 95.89 0.98 S6 208.12 1.02 0.92 0.49 92.67 0.88 (12) S7 206.24 0.29 0.98 0.38 145.79 0.98

However, the study of exchangeable P on natural sed- With the experiment data, modified Langmuir and iments indicated that native adsorbed exchangeable phos- Freundlich equations can be applied to describe the rela- phorous (NAP) of sediment also takes part in the process tionship between concentration of adsorbate and the of adsorption [32] and should be taking into the isotherms. amount of adsorption using non-linear trial-and-error regression method [33]. Taking NAP into account, the modified Langmuir ad- The intersection of y-axes and the regression curves sorption equation could be written as [29]: refers to NAP value and the x-intercept of the model indi-

cates EPC0 value. These two parameters are highlighted (4) because they play important roles in the retention and release of phosphorus in sediment-water interface. NAP

Where NAP stands for the amount of total native ex- stands for native adsorbed exchangeable phosphorous, and EPC0 indicates the critical point of adsorption and desorp- changeable phosphorus (mg/kg) In the case of Cadd  0 , tion. When actual phosphorus concentration in water were C  C 0 , the Eq. (4) can be described as: higher than EPC0, the sediment would absorb phosphorus eq eq from water, otherwise the phosphorus obtained in sediments would release. Table 4 shows that for sample S2 to S5, adsorption data were well fitted to both adsorption equations, with all the (5) R2 value over 0.95. Although the R2 of both Langmuir and

Substituted NAP in equation (4) by equation (5) to ob- TABLE 4- Summary of the fitting results of the modified Langmuir tain the modified Langmuir isotherm equation [29]: and Freundlich equations

Modified Langmuir Modified Freundlich Fitting results 2 2 (6) Sample Qmax KL R 1/n KF R S1 337.22 0.84 0.84 0.019 3048.56 0.94 Zero-equilibrium P concentration, which is usually S2 215.02 0.82 0.98 0.483 91.89 0.98 known as EPC0 refers that there is no net adsorption or re- S3 157.71 1.98 0.99 0.628 37.27 0.98 lease and phosphorus on sediments and in water phase is in S4 130.26 5.16 0.99 0.885 11.80 0.95 S5 216.86 0.78 0.98 0.459 97.53 0.98 dynamic equilibrium. When Ceq  Cadd , EPC0 can be S6 501.40 0.13 0.91 0.003 18410.87 0.96 obtained by equation (6) as: S7 277.26 0.29 0.89 0.002 18410.87 0.96 Calculated parameters

NAP EPC0 KP NAP EPC0 KP (7) S1 109.91 0.41 0.27 2997.59 0.42 7.16 S2 12.81 0.05 0.25 22.13 0.05 0.42 The partitioning coefficients (KP) can be calculated by: S3 5.35 0.07 0.08 7.14 0.07 0.10 S4 9.27 0.40 0.02 7.38 0.59 0.01 S5 14.52 0.06 0.26 26.11 0.06 0.46 S6 328.79 0.26 1.28 18339.58 0.27 67.54 (8) S7 50.66 0.06 0.79 18289.98 0.06 285.89

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Freundlich equations were over 0.9 when data of S1, group. This result indicates the varied adsorption capacity S6 and S7 were fitted, the inapplicability in these 3 cases of samples is relevant to soil components. Resemblance of was obvious due to the unreasonably high NAP. The mod- components contributes to similarity in adsorption capacity ified Freundlich equation may lack some scientific founda- and isotherms. tion and appear unsuitable in our cases. Although the surface area was usually considered to be The results of EPC0 ranged from 0.05~0.59 mg/L, S4 responsible for adsorption capacity of absorbent [21,34], from forest land showed the highest EPC0 and S2 from the data of BET surface area showed no correlations with cropland obtained the lowest EPC0. The values of EPC0 by the parameters of adsorption. However, the soil sample S4 modified Langmuir model and modified Freundlich model with the lowest adsorption capacity also has a much were nearly the same, while the NAP values showed some smaller BET surface area than other samples. BET surface abnormal values. The abnormal NAP values might indicate area may not influence phosphorus adsorption in an obvi- the inapplicability of modified Freundlich model when de- ous way though, significant decrease in surface area would scribing adsorption on complex natural sediments such as affect the adsorption capacity. soils. The adsorption capacity can be related to many factors 3.4 Effect of land uses and soil characters especially when complicate adsorbent such as soil involved. The metal content was considered to be the main S1 to S7 present different phosphorus adsorption iso- factor that determines soil adsorption capacity, because the therms in Fig.3. The Qmax obtained from Langmuir equa- iron/aluminum hydroxides in soil has high specific surface tion ranged from 141.89~209.96 mg/kg with an average [26]. Table 5 illustrates the correlations between adsorp- value of 191.61mg/kg. According to different land use, the tion parameters and Fe, Al contents in this study. order of adsorption capacity was abandoned cropland＞ cropland＞grass＞orange orchard＞forest land. In the case The parameters obtained from both equations were generally correlated with Fe content in soil, indicating Fe of cropland, the adsorption capacity of samples taken at content to be an important factor of phosphorus adsorption 160m and 165m were nearly the same, while the sample at in this case. The contribution of Al content to phosphorus 170m showed an obvious decrease in Q . S1, S2, S5 and max adsorption was present in affecting NAP value. Samples S6 shared a similar Langmuir isotherm with a Qmax about with more Al resulted in a higher NAP. Generally, samples 209mg/kg, while the adsorption capacity of S3 and S4 were with higher content of Fe and Al can adsorb more phospho- relatively lower. Cluster analysis based on sample compo- rus from natural environment to obtain a higher NAP, and nents data showed that S2, S5 and S6 can be classified to the adsorption capacity of soil will increase with content of one group, while S1, S3 and S4 were classified to the other Fe and Al as well.

200

S 150 1 S 2 S 3 100 S 4 S P adsorbed (mg/kg) P adsorbed 5

0 0246810 Equilibrium P Concentration (mg/L)

FIGURE 3 -Adsorption isotherms fitted by traditional Langmuir isotherm

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TABLE 5- Correlations between isotherm parameters and hydrox- REFERENCES ides in sediments [1] Smith, V. H. (2003). Eutrophication of freshwater and coastal Fe Al marine ecosystems a global problem. Environmental Science Modified Langmuir * & Pollution Research, 10(2), 126-139. KL -0.976 -0.480 [2] Butusov, M. and Jernelöv, A. (2013) Eutrophication. In: Bu- Qmax 0.947 0.667 tusov, M. Phosphorus: An Element that could have been called NAP 0.478 0.945 Lucifer. Springer New York, New York, Heidelberg, Dor- drecht, London, 57-68. EPC -0.918 -0.189 [3] Weissteiner, C. J., Bouraoui, F., and Aloe, A. (2013). Reduc- K 0.891 0.733 P tion of nitrogen and phosphorus loads to european rivers by Modified Freundlich KF 0.902 0.772 riparian buffer zones.Knowledge & Management of Aquatic Ecosystems, 483(408), 1-7. n -0.907 -0.744 [4] Li, H., Sun ,A.H., Hou, J., Chen, X., Bai, Y.Q., Cao, X.Y., NAP 0.839 0.859 Song, C.L. and Zhou, Y.Y. (2014) Phosphorus fractions, sorp- EPC -0.921 -0.193 tion characteristics, and its release in sediments of a Chinese eutrophic river (Nanfei river). Fresenius Environmental Bulle-

KP 0.896 0.765 tin, 23, 1222-1231. * indicates significance at 0.05 level [5] Muscutt, A. D., Harris, G. L., Bailey, S. W., and Davies, D. B. (1993). Buffer zones to improve water quality: a review of their potential use in uk agriculture. Agriculture Ecosystems & 4. CONCLUSIONS Environment, 45(1-2), 59–77. [6] Norris, V. (1993). The use of buffer zones to protect water Sediments taken from five typical land use type in quality: a review. Water Resources Management, 7(4), 257- Danjiangkou reservoir area were studied. The adsorption 272. isotherms were both fitted to traditional Langmuir and [7] Uusi-Kämppä, J. (2005) Phosphorus purification in buffer Freundlich equations except for S6. Qmax obtained by tra- zones in cold climates. Ecological Engineering, 24, 491–502. ditional isotherm equations of these samples ranged from [8] Hepher, B. (1958) On the dynamics of phosphorus added to 141.89 to 211.33mg/kg. Taking NAP into account, the fish ponds in Israel. Limnol. Oceanogr, 3, 84-100. modified Langmuir and Freundlich equations were also used to describe the adsorption isotherm. The modified [9] Schindler, D.W. (1974) Eutrophication and recovery in experimental lakes: implications for lake management. Science, equations gave information of NAP and EPC0, while also 184, 897-899. showed inapplicability in some cases (S1, S6 and S7). The adsorption capacity was influenced by sediments compo- [10] Schindler, D.W. (1977) Evolution of phosphorus limitation in lakes. Science. 195, 260-262. nents. Samples from different land use types with different resemblance of components varied in adsorption capacity, [11] Holford, I. and Mattingly, G. (1976) Phosphate adsorption and while the effect by BET surface area and elevation of sam- availability plant of phosphate. Plant Soil, 44, 377-389. ples were not obvious. The parameters obtained by modi- [12] Richardson, A.E. and Simpson, R.J. (2011) Soil microorgan- fied Langmuir and Freundlich equations were generally isms mediating phosphorus availability update on microbial correlated with Fe content in sediments and the content of phosphorus. Plant Physiology, 156, 989-996. Al content resulted in affecting NAP value. Generally, the [13] Vohla, C., Kõiv, M., Bavor, H.J., Chazarenc, F. and Mander, existence of higher content of Fe and Al refers to higher Ü. (2011) Filter materials for phosphorus removal from NAP and lower EPC0, which enhance the probability to ad- wastewater in treatment wetlands—a review. Ecological Engi- sorb phosphorus from natural environment rather than de- neering, 37, 70-89. sorption. [14] Fondu, L., De Bo, I. and 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 systems. Desalination & Water Treatment, 54, 2461- 2467. ACKNOWLEDGEMENTS [15] Pratt, C., Parsons, S.A., Soares, A. and Martin, B.D. (2012) Biologically and chemically mediated adsorption and precipi- The authors thank the support by the National Natural tation of phosphorus from wastewater. Current Opinion in Bi- Science Foundation of China (41471433 and 41001333), otechnology, 23(6), 890–896. the National Key Technology R&D Program of China [16] Weng, L., Van Riemsdijk, W.H. and Hiemstra, T. (2012) Fac- (2012BAC06B03) and the Central Public-interest Scien- tors controlling phosphate interaction with iron oxides. Journal tific Institution Basal Research Fund (2014-37). We also of Environmental Quality, 41(3), 628-635. thank Ms Y. Hu for remote sensing image data collection and analysis. [17] Devau, N., Hinsinger, P., Le Cadre, E., Colomb, B. and Gérard, F. (2011) Fertilization and pH effects on processes and mechanisms controlling dissolved inorganic phosphorus in soils. Geo- chimica Et Cosmochimica Acta, 75(10), 2980–2996. The authors have declared no conflict of interest.

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[18] Majumdar, B., Venkatesh, M. and Saha, R. (2013) Long term effect of land use systems on phosphorus adsorption behaviour under acidic alfisol of Meghalaya, India. Agrochimica, 55(4), 233-247.

[19] Alt, F., Oelmann, Y., Herold, N., Schrumpf, M. and Wilcke, W. (2011) Phosphorus partitioning in grassland and forest soils of Germany as related to land-use type, management intensity, and land use-related pH. Journal of Plant Nutrition & Soil Science, 174(2), 195–209. [20] McGechan, M. and Lewis, D. (2002) Sorption of phosphorus by soil, part 1: Principles, equations and models. Biosystems engineering, 82, 1-24. [21] Li, D. (2011) National economic statistic information of Dan- jiangkou city, 2010. In: Luo, T.Y. National economic statistic information of Danjiangkou city. Statistics Bureau of Dan- jiangkou city, Danjiangkou city, 16-18.

[22] Arai, T. (2007) Quantitative Analysis. In: Beckhoff, B., Kan- ngieber, B., Langhoff, N., Wedell, R., Wolff, H.H. Handbook of practical X-ray fluorescence analysis. Springer, Berlin, Hei- delberg, 36-39.

[23] Brunauer, S., Emmett, P.H. and Teller, E. (1938) Adsorption of gases in multimolecular layers. J. Am. Chem. Soc, 60, 309- 319. [24] Labeda, D., Balkwill, D. and Casida Jr, L. (1975) Soil sterili- zation effects on in situ indigenous microbial cells in soil. Ca- nadian Journal of Microbiology, 21(3), 263-269. [25] Wang, Y., Shen, Z., Niu, J. and Liu, R. (2009) Adsorption of phosphorus on sediments from the Three-Gorges Reservoir (China) and the relation with sediment compositions. Journal of Hazardous Materials,162(1), 92-8. [26] Brinkman, A.G. (1993) A double-layer model for ion adsorption onto metal oxides, applied to experimental data and to natural sediments of Lake Veluwe, The Netherlands. Hydrobio- logia, 253, 31-45.

[27] Zhang, N.M. and Wang, S.Q. (2005) Capacity and speciation of phosphorus in soils. In: Chen, H. Environmental Soil Sci- ence. Science Press, Beijing, 164-170.

[28] Lai, D.Y.F. and Lam, K.C. (2009) Phosphorus sorption by sediments in a subtropical constructed wetland receiving storm- water runoff. Ecological Engineering, 35(5), 735-743.

[29] Zhou, A., Tang, H. and Wang, D. (2005) Phosphorus adsorption on natural sediments: Modeling and effects of pH and sediment composition. Water Research, 39(7), 1245-1254. [30] Wang, L., Liang, T., Kleinman, P.J.A. and Cao, H. (2011) An Received: November 03, 2014 experimental study on using rare earth elements to trace phos- Revised: December 11, 2014 phorous losses from nonpoint sources. Chemosphere, 85(6), Accepted: December 18, 2014 1075-1079.

[31] Achife, E. C., and Ibemesi, J. A. (1989). Applicability of the freundlich and Langmuir adsorption isotherms in the bleaching of rubber and melon seed oils. Journal of Oil & Fat Indus- CORRESPONDING AUTHOR tries, 66(2), 247-252. Liang Zhang [32] Aminot, A. and Andrieux, F. (1996) Concept and determination of exchangeable phosphate in aquatic sediments. Water Re- Institute of Geodesy and Geophysics search, 30(11), 2805-2811. Chinese Academy of Sciences [33] Zhang, L., Hong, S., He, J., Gan, F. and Ho, Y.S. (2011) Ad- Wuhan, Hubei Province sorption characteristic studies of phosphorus onto laterite. De- P.R. CHINA salination & Water Treatment, 25(1), 98-105. [34] Xiang, Q., Xie, H. Q., Henderson, H., Kay, A. F., Fontes, M. P. Phone: +86 27 68881362 F., and Weed, S. B. (1996). Phosphate adsorption by clays Fax: +86 27 68881362 from brazilian oxisols: relationships with specific surface area E-mail: [email protected] and mineralogy.Geoderma, 72(1-2), 37–51. FEB/ Vol 24/ No 7/ 2015 – pages 2354 - 2361

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ASSESSMENT OF LIMITING FACTORS FOR POTENTIAL ENERGY PRODUCTION IN WASTE TO ENERGY PROJECTS

Orhan Sevimoğlu

Istanbul Metropolitan Municipality, Şehzadebaşı Caddesi, 34134 Fatih / Istanbul /Turkey

ABSTRACT in the energy market [6]. There are 76 sanitary landfill sites that are used by 1010 municipalities with 16 operating The popularity of landfill gas to energy projects in- WTE plants which are forecasted to grow in number in re- creased worldwide as well as in Turkey in the last two dec- spect to the new subventions given by the government. ades due to the importance of renewable energy sources These projects are mostly designed based on generally ac- and the green house gas emission control in environmental cepted rules and regulations of especially United States protection. Many Waste-to-Energy (WTE) projects focus (US) and European techniques. The goals of WTE projects on methods that employ landfill gas (LFG) from landfills from an environmental regulations standpoint are the re- to produce electric power. Most of the projects refer to gas duction of greenhouse gas emissions to the atmosphere prognosis models such as USEPA-LandGEM, GasSim, from landfills [7] and the climate change impact [8], all the EPER, and IPCC to determine the installation power ca- while utilizing methane of LFG in energy production pacity. However, problems related to landfill, power gen- [9,10]. In order to evaluate the acceptability of these pro- eration and energy delivery have limiting factors for land- jects, a detailed gas prognosis is needed to check the feasi- fill gas and energy production. In this study, the limiting bility for an accurate investment [11]. Provided that the parameters with their assessments are discussed for prognosis of LFG recovery is done incorrectly, the conse- Odayeri and Kömürcüoda Sanitary Landfills, Istanbul, quence will be failure in terms of cost-benefit analysis of Turkey. To overcome these issues, several improvements the project [12]. One of the methods for a high-quality as- were done such as top surface cover application to both sessment is performing a pump trial on the landfill site [13] landfill sites, buffer gas tank installation to minimize the to get an actual LFG flow rate from the landfill site. An- fluctuation of LFG quality and reduction of leachate water other assessment method can be surface emission measure- level via pumping. Accordingly, an increase in the recov- ment [14, 15] by which an analogy can be made to flow ery of LFG has been accomplished. The recovery LFG rate rate to evaluate the real potential. On the other hand, math- to theoretical extractable LFG rate ratios reached to 75 % ematical models are mostly used for the evaluation of the- and 60% in 2012 for Odayeri and Kömürcüoda landfills, oretical LFG potential. The LandGEM (USEPA), GasSim, respectively. EPER, IPCC LFG estimation models are widely used in many applications of LFG emission. The models require waste character and empirical parameters to calculate LFG

KEYWORDS: emission. However, the potential of actual gas production waste-to-energy, landfill gas, limitations, energy, recovery depends on many factors, especially landfill site conditions such as the level of anaerobic activity, solid waste contents, rainfall, surface covering. Besides, the recovery of LFG de-

pends on the design quality of the extraction system and 1. INTRODUCTION the performance of the operation as well as management of the collection system. In many applications, the theoretical Municipal solid waste (MSW) is generally disposed in landfill gas potential is calculated by the accepted models, a sanitary landfill site due to environmental concerns and and then WTE projects are designed based on the estima- public health [1]. Stored MSW produces LFG including tion of LFG recovery for each year of the project life. The methane, carbon dioxide, and trace gases under anaerobic considered recovery rate of LFG is in the wide range of conditions. Energy recovery utilizing LFG has been ac- 90% to 10%. In many cases, the extracted landfill gas vol- cepted as a widely used application for several decades ume was lower than the estimated recovery LFG volume around the world [2, 3] as well as in Turkey [4, 5]. The first which is misleading for inventors [16]. power plant utilizing LFG in Turkey was built in Bursa There are several reasons for low extraction rates of province at Demirtas landﬁll in 1997. Since then electricity landfill gas such as poor sanitary landfill site management, production from renewable energy sources in Turkey has failure to comply with waste storage techniques, lack of ac- enormously increased due to updates and new regulations curate characterization of waste and absence of complete

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anaerobic conditions [17]. Inadequate plant design and sys- tribution in the metropolitan area of Istanbul is 63% in Eu- tem operations also limit gas extraction resulting from ropean and 37% in Asian side. Approximately 9500 tons of leachate accumulation in lateral gas collection pipes, parti- waste was disposed in Odayeri Landfill, and 5000 tons of cles and sludge clogging the vertical wells, scale formation waste was disposed in Kömürcüoda Landfill on daily basis of chemicals in the pipes, deformation and cracking of gas during 2009. The estimated collected recyclable waste was extraction pipes, and waste sliding with the well. about 350 tons per day [20]. The municipal solid waste characterized by researchers is shown in Table 1 indicating This study focuses on the major causes of decrement that about 48-50% of the disposed waste contains food in LFG recovery rates from landfills when compared with waste. All the stored waste including organic materials has the estimated theoretical LFG potential. The encountered the high potential to produce LFG under anaerobic condi- problems in the acquisition process of Odayeri and tion after being stored for about 6 months [21]. While Kömürcüoda landfill sites in Istanbul were studied to elu- MSW has been stored in the landfill sites in Odayeri and cidate these problems. The limitation parameters in energy Kömürcüoda since 1995, the produced LFG was emitted to production were investigated as well. atmosphere through the ventilation wells that contributed

to GHG in the atmosphere as well as causing loss of profit

until WTE projects were implemented in both landfill sites 2. MATERIALS AND METHODS in 2009.

2.1 The profile of the landfill sites 2.2. Theoretical LFG versus recovery LFG There are two major sanitary landfill sites in the City The environmental impact of the greenhouse gas emis- of Istanbul: Odayeri (Europe) landfill site (41° 13' 02.9"N; sion from the landfill sites is considered by the climate 28° 51' 11.3"E) with total area of 115 ha and 32 million change initiatives due to global warming issue. Mathemat- tons of waste stored and Kömürcüoda (Asia) landfill site ical models and software programs to predict greenhouse (41° 10' 30.6"N; 29° 22' 16.2"E) with total area of 89 ha gas emission from the sites were developed by United and 15 million tons of waste stored since the established States Environmental Protection Agency (USEPA), Inter- date of 1995 to early 2009. Odayeri and Kömürcüoda Land- governmental Panel on Climate Change (IPCC) and other fills are located outside the city center with 25 and 55 km private organizations. These models are primarily used away, respectively. to estimate the amount of theoretical LFG emission from Municipal waste production rate for each person in Is- landfill sites. Once energy recovery proposals became tanbul depends on daily habits, economical poverty, and more popular in the utilization of LFG, the WTE projects social status [18]. In Istanbul, the waste production per cap- expanded around the world especially in the developed ita is about 0.9-1.1 kg/person/day [19]. The population dis- countries to produce renewable energy as well as reducing

TABLE 1 - Istanbul municipal solid waste composition

Componentsa Kömürcüodab (%) Odayerib (%) Averageb (%) Istanbulc (%)

Paper 6.27 11.27 8.77 8.4 Cardboard 3.49 5.56 4.53 Colorful glass 2.05 3.44 2.74 2.3 Colorless glass 3.01 3.15 3.08 2.3 PET bottles 0.99 2.05 1.52 Plastic bags 8.42 10.55 9.48 Plastic 3.21 3.56 3.39 11 Sack and similar 0.25 0.45 0.35 Metals (iron) 0.89 0.87 0.88 2.3 Aluminum 0.45 0.92 0.68 Other metals 0.13 0.00 0.07 Organics 56.31 44.13 50.22 48 Diapers 3.72 4.08 3.90 3.2 Wood 0.50 0.53 0.51 Electrical Equipments 0.11 0.20 0.15 Batteries 0.02 0.00 0.01 Textile 4.77 5.80 5.28 2.9 Tetra-pak 0.66 0.61 0.64 Other combustibles 2.62 1.58 2.10 6.3 Ash 1.51 1.17 1.34 13.2 Stone, rock, etc. 0.64 0.09 0.36 Total 100 100 100 100 a Composition is as w/w b [21] Kanat (2010) c [32] Arikan and Toroz (1999)

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greenhouse gas (GHG) emissions. Although these models the waste stored in both landfill sites is 50.22% organic as are used for calculating GHG emissions from landfill sites reported in Table 1 which is considered as midlevel fast to report to climate change departments, WTE project de- decaying. signers also use them to calculate LFG flow rate for the Each of the organic waste categories is assigned a power plant capacity. The rate of theoretical LFG varies in unique k and L pair that is used to calculate LFG genera- calculation due to the range of parameters used for the 0 tion. The Model’s LFG generation calculations also in- models. The estimation of LFG emission rate for Odayeri clude an adjustment to account for aerobic waste decay and Kömürcüoda Landfills were calculated using the known as the methane correction factor (MCF), and a fire USEPA Land GEM model. The expectation is that results constant (F). LFG recovery is estimated by the Model by should meet the recovery LFG rate. multiplying projected LFG generation with the estimated

2.3 Theoretical LFG estimation of the landfill sites collection efficiency. The theoretical LFG were calculated using the parameters established in Table 3. LandGEM is based on a first-order decomposition rate equation for quantifying emissions from the decomposition of waste in MSW landfills [22]. This software provides an TABLE 2- Disposed waste amount in both landfill sites uncomplicated approach to estimating landfill gas emis- Odayeri Kömürücüoda sions. Model defaults require empirical data determined by Year Metric Tons Metric Tons investigating the landfill directly. The first order decompo- 1995 524,013 228,546 1996 1,112,443 628,622 sition rate equation used to estimate the landfill gas emis- 1997 1,521,541 779,389 sion and the LFG generation for a given year from cumu- 1998 1,775,983 885,427 lative waste disposed up to that year for the Odayeri and 1999 2,220,225 965,168 Kömürcüoda Landfills is given below: 2000 2,282,265 1,037,624 2001 2,205,211 1,013,196 2002 2,281,219 1,032,549 n 1 2003 2,452,301 1,099,974 M i  ktij QLFG  2kL0  (e )(MCF)(F) 2004 2,848,277 1,314,152 tj1 0.1  10  2005 3,145,032 1,459,226 2006 3,469,871 1,620,796 2007 3,469,871 1,639,000 QLFG = annual LFG generation in the year of the cal- 2008 2,970,000 1,642,500 culation (m3/year) Total 32,278,252 15,346,169 i = 1 year time increment n = (year of the calculation) - (initial year of waste ac- TABLE 3 - Used parameters in the model ceptance) j = 0.1 year time increment Unit Odayeri Kömürcüoda -1 k 1/year 0.065 0.065 k = methane generation rate (year ) Lo m3/ton 90 90 3 Lo = potential methane generation capacity (m /Mg) MCF % 50 50 F 1 1 Mi = mass of waste accepted in the year (Mg) th tij = age of the j section of waste mass Mi accepted in the ith year (decimal years, e.g., 3.2 years) Fig. 1 shows the theoretical (potential), estimated re- MCF = methane correction factor covery (extractable) and recovered (actual extraction) LFG F = fire adjustment factor. for Odayeri and Kömürcüoda Landfill Sites. Recovered LFG is lower than the expected recovery (extractable) Table 2 shows the amount of disposed waste in tons value (60%) used in the gas prognosis calculation. The year for both landfill sites. Total LFG generation is equal to of the calculation used in the model is 35 years. twice the amount of the calculated methane generation. The calculated total amount of theoretical extractable The model landfill characteristics and the model parame- LFG volumes for Odayeri and Kömürcüoda Landfills are ters were set based on the literature values and site specifi- 1,929 x 106 m3 and 918 x 106 m3 in 35 years, respectively. cations. All of the used parameters are summarized in Ta- ble 3. 2.4. Utilization of landfill gas The four organic waste categories grouped according It is very important to utilize LFG as soon as the waste to waste decay rates which has an impact on the estimation is disposed to landfill. In Istanbul, unfortunately, the LFG of LFG in calculations can be listed as: 1. Very fast decay- utilization was achieved 14 years after the first waste was ing waste (food waste, other organics), 2. Medium fast de- disposed to both landfill sites. Hence, as much as 850 mil- caying waste (e.g. garden waste and green waste), 3. Me- lion m3 and 400 million m3 theoretical extractable LFG es- dium slow decaying waste (e.g. paper, cardboard and tex- caped to the atmosphere causing energy loss and emissions tiles), 4. Slow decaying waste (e.g. wood, rubber, leather, of GHG to atmosphere which was about 8 million tons and bones and straw). LFG generation rate from composite 3.7 million tons equivalent waste is estimated based on these categories. For instance,

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FIGURE 1 - Theoretical and practical LFG rates in Odayeri and Kömürcüoda landfills.

CO2 from Odayeri and Kömürcüoda Landfills, respec- tracted LFG is directed to the gas engines. The selected gas tively. After evaluation and inspection of the utilization of engines are suitable for electricity generation in both sites LFG, the WTE projects were implemented in both landfill based on LFG quality. Each gas engine has a capacity of sites. The recovered LFG from both landfill sites are used 1.4 MW. The installed energy production capacity in 2010 for electricity production via gas engines. The vertical gas was 18 MW and 7 MW at Odayeri and Kömürcüoda energy extraction wells are placed in the waste body and the ex- recovery facilities, respectively. The produced electricity is

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transferred to the national grit with a high power transmis- landfill top covering, and landfill body stability. Major lim- sion line of 34.5 kV. itation factors seen in both landfill sites are summarized in Table 4.

3. RESULTS AND DISCUSSION 3.1.1 Disposed waste character in landfill gas production The characterization of MSW plays an important role There are three main areas of the limitations of WTE in the estimation of LFG production [19]. The variety of projects, categorized based on landfill site, energy conver- biodegradable organic materials in waste composition sion and distribution. Fig. 2 gives a schematic description causes variations in LFG generation rate. Fast biodegrada- of these limitations. ble organic waste (e.g. food waste) generates biogas once the anaerobic condition is reached [23, 24]. Food waste starts

3.1 Limitations on LFG production from landfill sites decomposing in aerobic conditions by the time its being The LFG production capacity is limited by waste char- placed in the garbage until pre-cover or final cover of the acter, compacting and waste height, leachate in the waste, waste. The aerobic process will occur especially during the

Limitations of WTE Project

Landfill Site Energy Conversion Energy Distribution

Waste Character Gas Extraction and Gas Transmission Line Collection

Compacting and Height Gas Quality National Grit

Leachate in Landfill Power Generation System

Landfill Top Final Cover

Landfill body stability

FIGURE 2 - Limitations of WTE projects

TABLE 4 - Major limitation factors seen in both landfill sites.

Limitation factors Assessments Waste Characterization About 50% waste fast degradable organic waste disposed. Stored waste depends on the years 13 years old waste. Fresh wastes produce more LFG than old waste. Waste compacting Midlevel compacting for both landfill sites. Waste cell height Changeable in 5-7 m. Landfill daily coverage Midlevel Landfill segment coverage Midlevel Landfill site final cover The final cover was finalized in 2010 for both landfill sites.. Landfill site leachate collection system Active bottom leachate collection system.. Landfill site rainwater drain system No surface water collection system on the surface for both landfill sites. Air intrusion While vacuum to landfill site.

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disposal process due to light compaction and delayed soil stored in the landfill site, leachate production rate begins to covering, downwards into a depth of two meters or even increase. In addition, if rain water penetrates into the non- more [25]. 90 % of the degradable carbon will be destroyed covered landfill site, it increases the leachate flow rate as during one year in aerobic landfills [26]. A first order well. Unfortunately, both of the landfill sites top surface model can be used to calculate the degradation of the or- final covers were finalized in 2010. The average leacheate ganic matter, eg. in the first 3 months nearly 50 % will be flow rate at Odayeri and Kömürcüoda landfill sites were degraded [27, 28]. Therefore, it is very important to con- about 2100 m³/day and 1000 m³/day, respectively in 2008. sider decomposed organic waste in aerobic conditions for The organic matters dissolve during rainwater infiltration the right prognosis and to estimate the influence of daily through the waste and leave the waste with leachate which covering. Early waste decomposing in aerobic conditions causes a decrease in the LFG generation potential due to causes potential LFG loss as well. To avoid immediate de- the lower organic content of the waste [34]. The leachate composition the organic matter needs to be collected regu- accumulation in the waste body observed in both landfill larly from the daily top soil cover. sites is over 40 meters high, attributable to a 13 year period of rainfall on the open landfill site. The high waste height 3.1.2 Compacting and height of disposed waste. causes high pressure on the bottom layer causing it to be Compacting of solid waste is an important factor in bi- more compact. ogas generation. Fewer voids in the waste volume increase The LFG collection wells are the most important part the LFG production [29]. Compactors were used in both of a WTE plant. High leachate level in the waste body and landfill sites, however, high quality compaction could not high waste body height have a potential on limiting the for- be achieved due to field problems (rainfall, compactor fail- mation of LFG as well as extraction [35], which in fact ure) and high waste load in a short period of time. Another were observed in Odayeri and Kömürcüoda landfill sites. significant factor is covering the waste with soil as soon as Once the LFG extraction wells were placed in the waste possible. When the waste remains open to atmosphere in body, leachate increased by capillary effect by causing it to the landfill site for a long time the biodegradable organic fill the holes in the perforated wells. The remaining water waste degrades aerobically that causes a loss in LFG pro- free perforated wells available for gas extraction during the duction capacity. There is a high waste load in Odayeri and vacuum application in Odayeri and Kömürcüoda landfill Kömürcüoda landfills; consequently, considerably less sites are 16% and 40% percent, respectively. Hence, high daily coverage over the waste. The old waste is usually vacuum should be applied to the wells to extract LFG from covered by fresh waste daily. Once the lot is filled with the mass of waste. In order to decrease level of leachate in waste, the top is covered with soil. the wells, it is discharged by the help of a pump, which The height of the waste in the landfill is another im- increased the amount of water free perforated wells used in portant limitation parameter in the utilization of LFG. The gas extraction. This additional step brought increased gas extraction wells can be drilled up to 40 meters. The workload for the maintenance of the wells as well as con- height of the waste has reached up to 90 meters in both struction and operating costs, in both landfill sites. Table 5 landfills, thus it is not feasible to extract LFG below the represents the values of the wells. In order to decrease extraction wells. leachate in the wells, the submerge pumps were placed in all the gas extraction wells. The height of the available wa- 3.1.3. Leachate in landfill ter free perforated wells increased from 16% to about 30 % in Odayeri and from 40% to about 45% Kömürcüoda, re- The moisture in the waste encourages bacterial growth spectively. and transports nutrients and bacteria to all areas within a landfill [30]. The moisture content of 40% or higher, based 3.1.4. Top cover impermeability on wet weight of waste promotes maximum gas production, especially in a capped landfill [31]. The disposed Top cover is an important aspect of gas extraction perfor- waste in both Odayeri and Kömürcüoda landfill sites con- mance needed for blocking air intrusion into waste body [36]. tains about 50%-70% water [19, 32, 33]. Once the waste is Anaerobic reactions should be sustained during gas extrac-

TABLE 5 - The values of wells

Odayeri Kömürcüoda Average Well depth (m) 35 23,2 Average perforated well height (m) 29,4 18,8 Average water depth height (m) 24,7 10,7 Total pipe length in the wells (m) 4685,5 2760 Total perforated pipe length (m) 3933,5 2403 Total free slotted pipe length (m) 626,4 1423 Closed perforated pipe length with leachate (m) 84% 40%

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tion period. Oxygen in the air kills methanogenic bacteria of leachate. Remaining motionless for a long time in the that stop methane production in the landfill. Odayeri and well environment, the leachate goes through various phys- Kömürcüoda landfills were finally covered with soil after ical, chemical and biological processes and solidifies. At the LFG extraction wells were placed in 2010. The maxi- the end of this process, the already high rate of solids in- mum allowed oxygen level in the extracted LFG is 2% in creases even further and leads to the clogging of well cas- both landfill site based on operation experience. The me- ing [40] as well as the pumps resulting in their unavailabil- thane ratios in the LFG in both sites are kept in about 45- ity from time to time. 50% for optimum energy production. The fluctuation ratios There are several types of scale formation in the land- of the methane were rarely changing ascribable to the air in- fill gas extraction wells in Odayeri and Kömürcüoda Land- trusion and landfill site problems. Higher fluctuation ratios fills with the first one being the chemicals accumulation on have negative impact on the gas engine. Therefore, surface the surface of imperforated part of the extraction pipe re- top cover should be sealed with natural soil and clay, ge- sembling a hard rock. The microbiological activity pro- omembrane is also used in some advanced applications [37]. duces CO2, H2O, NOx, H2S, SOx, which are carried through the flow of the pipe. These compounds condensate on the 3.1.5. Landfill body stability surface of the pipe over 50oC. Although this initial deposit Municipal solid waste is stored in the landfill site with tries to corrode the polymers of the pipe, salicylic acids, the slope of one-third ratio. The disposed waste, based on calcium salts, and alkaline salts that are carried with land- a certain time period, settles down because of the decom- fill gas neutralize the radicals and stop the corrosion. Nev- posing of fast degradable waste [29], seen in both Odayeri ertheless these salts accumulate and the deposition occurs and Kömürcüoda landfill sites. The accumulation of leach- on the surface of the inner pipe between 1 cm to 2 cm, re- ate in the waste body decreases stability especially in the sulting in the reduced diameter, 19% to 36%, of the 20 cm Kömürcüoda landfill that cause the sliding of the waste. extraction pipe (Fig 4a). The unstable landfill site area is inefficient in terms of land- Another type of formation is the sludge, silt and chemi- fill gas intake [38]. Gas extraction wells were clogged or cal salts accumulated in the gas extraction wells carried via cracked (Fig. 3) due to the waste body settlement that re- leachate [41]. The salt compounds accumulated in the wells quired new extraction well installations with additional in- due to chemical equilibrium cause the precipitation inside vestment costs [39]. the pipe (Fig. 4b). The accumulated sludge in the extraction pipe depends on the length of the pipe; especially around the 3.2 Energy conversion from landfill gas leachate level between 2 m to 6 m. Sludge resists removal 3.2.1. Deposit formation in wells from the pipe via any drain cleaners making it unusable. Ex- The most important problem encountered in vertical traction well replacements were placed to the sites which wells that are used to collect gas is the excessive amounts brought in additional construction and operation costs.

FIGURE 3 - Cracked and perforated pipes of LFG extraction wells

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FIGURE 4 - (a) Scale formed in the inner LFG extraction pipe and (b) clogged LFG extraction pipe.

3.2.2. Effects of landfill gas quality on energy production and RUK are located in Bruckberg and Longuich, Ger- CH4 and CO2 are the main components of LFG that many, respectively. Both laboratories use standard meas- also contain trace amounts of compounds such as hydrogen urement techniques and procedures published by German sulphide, mercaptans, halogenated hydrocarbons, NOx, authority for specific chemical analyses. and siloxanes [42]. Landfill gas components were analyzed to determine the parameters mentioned. The landfill gas There are various methods to remove trace compounds samples from both landfills were collected in the metal from LFG, to prevent gas engine damage or failure, avail- canister (by Agrolab) and Tedlar bag (by RUK). After col- able commercially such as adsorption, absorption, deep lection, samples were shipped to the laboratory immedi- chilling, biological removing, catalytic processes and ately to be analyzed by GC/MS. The compounds were membranes [43]. The components of LFG withdrawn from identified by conventional simple comparison of retention the field must meet the requirements of gas engine to run times using selective detector (FID, ECD, WLD) as well as at full performance. Additional requirements include sus- by mass spectra. The samples were sent to different labor- taining parameters such as gas pressure, methane ratio, atories to be analyzed as mentioned in Table 6. Agrolab temperature, relative humidity, heating value fluctuation

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and contaminants. Landfill gas quality in both landfill sites However, after improvements to the system, the me- are given in Table 6. Earlier on, the methane ratios were thane ratios increased to over 50%. Methane and other between 45-50% in both landfill sites. compound concentrations were detected in variation dur-

TABLE 6 - Quality of landfill gas in Kömürcüoda and Odayeri landfill sites

a Laboratory ID 1212 b,c,d Sampling date parameter Sep‐09 Apr‐11 Aug‐09 Apr‐11 Sampling Site Kömürcüoda LF Odayeri LF Before Gas Before Gas Before Gas f e Compounds Name Units DL Main Pipe Engine Engine Engine Method Oxygen % (v/v) 0.5 7,7 0,6 3,4 0,9 GC/MS Nitrogen % (v/v) 0,5 35 7,7 14,9 4 GC/MS Carbon dioxide % (v/v) 0,02 23,1 37 35,1 40 GC/MS Methane % (v/v) 0,2 35,4 54,6 46,5 55,1 GC/MS hydrogen % (v/v) 0,01 0,01 42 0,01 NM GC/WLD Hydrogen sulfide mg/m³ 0,3 NR 122 260 406 GC/FPD calorific value Kcal/m³ 1000 11,8 19,61 15,6 17,6 DIN 51900 Trichlorofluoromethane (R11) mg/m³ 0,2 <0,2 0,1 <0,2 NM A, GC/MS Dichlorodifluoromethane (R12) mg/m³ 0,5 2,2 0,4 1,3 0,4 B, GC/MS dichlorofluoromethane (R21) mg/m³ 0,5 <0,5 0,3 <0,5 0,2 B, GC/MS Chlorodifluoromethane (R22) mg/m³ 0,5 5,4 0,4 7,6 0,5 B, GC/MS 1,1,2‐Trichlorotrifluoroethane (R113) mg/m³ 0,2 <0,2 <0,1 <0,2 <0,1 B, GC/MS Vinyl chloride mg/m³ 0,3 4,1 2,7 2,8 4,6 B, GC/MS Dichloromethane mg/m³ 0,1 0,3 <0,1 <0,1 <0,1 B, GC/MS 1,1‐Dichloroethane mg/m³ 0,1 <0,1 0,1 <0,1 <0,1 B, GC/MS 1,2‐Dichloroethane mg/m³ 0,1 <0,1 <0,1 <0,1 0,1 B, GC/MS 1,1‐Dichloroethene mg/m³ 0,1 <0,1 <0,1 2,4 0,3 B, GC/MS cis‐Dichloroethene mg/m³ 0,1 8,4 10,5 NR 26,9 B, GC/MS trans‐Dichloroethene mg/m³ 0,1 <0,1 <0,1 0,2 0,2 B, GC/MS Trichloromethane mg/m³ 0,1 <0,1 <0,1 <0,1 <0,1 B, GC/MS 1,1,1‐Trichloroethane mg/m³ 0,1 <0,1 <0,1 <0,1 <0,1 B, GC/MS Trichloroethene mg/m³ 0,1 2 0,7 5,4 2 B, GC/MS Tetrachloromethane mg/m³ 0,1 <0,1 <0,1 <0,1 <0,1 B, GC/MS Tetrachloroethene mg/m³ 0,1 0,6 0,6 1,5 2,1 B, GC/MS sum fluorine mg/m³ 0,1 3,1 2,9 3,8 4,9 B, GC/MS sum chlorine mg/m³ 0,1 14 13 25,0 24,9 B, GC/MS Benzene mg/m³ 0,2 2,6 3,5 4,4 5,1 B, GC/MS Toluene mg/m³ 0,2 150 204,9 350 518 B, GC/MS Ethylbenzene mg/m³ 0,1 22 38,2 23,0 36 B, GC/MS m,p‐Xylene mg/m³ 0,2 39 62,4 37 59,1 B, GC/MS o‐Xylene mg/m³ 0,2 10 16,3 9,9 15,3 B, GC/MS Cumene mg/m³ 0,1 1,2 3,4 2,3 4,6 B, GC/MS Styrene mg/m³ 0,1 1,1 1,3 0,9 2,1 B, GC/MS Mesitylene mg/m³ 0,1 2 5,3 2,8 6,3 B, GC/MS 1,2,3‐Trimethylbenzene mg/m³ 0,1 4,7 11,1 1,7 19 B, GC/MS 1,2,4‐Trimethylbenzene mg/m³ 0,1 3,8 15,2 7,0 20,7 B, GC/MS a 1: Agrolab 2: RUK b ND : not dedected. c NR : not reported. d NM : not measured. e A is analog VDI 3865 and B: VDI 3865, Bl.4 f DL: Detection limit

TABLE 7 - Siloxanes commonly measured in LFG

Odayeri Komurcuoda Siloxanes Chemical Formula Average (mg/m³) Average (mg/m³)

Decamethyltetrasiloxan (L4) C10H30Si4O3 <0.5 <0.5 Dekamethylcyclopentasiloxan (D5) C10H30O5Si5 3.18 ± 0.61 2,6 Dodecamethylcyclohexasiloxan (D6) C12H36O6Si6 <1.0 <1.0 Hexamethyldisiloxane (L2) C6H18Si2O 1.95 ± 0,40 0,6 Hexamethyl-(cyclo)-trisiloxane (D3) C12H18O3Si3 <1.0 <1.0 Octamethylcyclotetrasiloxan (D4) C8H24O4Si4 5.93 ± 0,88 4,3 Octamethyl-trisiloxane (L3) C8H24Si3O2 <0.5 <0.5

ing the application which is not a desired condition. There- faults from time to time resulting in the interruption of en- fore, the landfill gas parameters were stabilized by placing ergy delivery to the national grit as well. The failure period buffer tanks before the gas engines. Constructing an inter- is at least one hour or more. For instance, a delay of one hour mediate buffer storage tank to ensure that stable and homo- in energy production in Odayeri and Kömürcüoda cause loss geneous gas is being sent to the gas engine is a suitable of profit as a result of unproduced 18000 kwh and 7000 kwh method to avoid any failure. The variation of major com- energy, respectively. During the failure period, burning of ponents in LFG impacts engine performance and reduces LFG in the burner instead of being emitted to atmosphere is energy production rate [44]. Unused LFG is stored in a the most appropriate method in order to optimize LFG ex- buffer tank to be used in energy production at a later time. traction from the landfill. If the withdrawal of LFG from the Optimal compensation of fluctuations increases gas engine field is halted, it may result in burst of piping systems that availability and components lifetime. Two 16000 m3 and was previously experienced in both landfill sites. one 16000 m3 buffer tanks were installed in both Odayeri and Kömürcüoda WTE plants, respectively. 3.4 Considerations on landfill gas potential for future applications 3.2.3. Limitations on power generation systems Any landfill may have a potential in producing energy The gas engines are affected by the compounds in the by utilizing LFG. However, only the correctly identified landfill gas during energy production. Especially the silox- parameters used in a selected prognosis model lead to the anes in landfill gas hamper to the engine in energy produc- ideal estimation of production of gas. Furthermore, accu- tion [45]. Siloxanes contain silicon atoms attached to rate gas emission estimation helps make appropriate in- VOCs [43]. Common siloxane derivatives in LFG of both vestments. Even if all investments are in a position to meet sites are shown in Table 7. Siloxanes are especially prob- the operating costs, it is also important to collect LFG in lematic in landfill gas to energy projects since they result such a way that environmental damage thru the release of in deposit formation in gas engines [46] and effect engine LFG to the atmosphere is minimized thus reducing green- performance [47]. When LFG containing siloxanes are house gas emissions. burned in the combustion chamber, siloxanes are converted into silicates (SiO2 or SiO3) [48] causing accumulation at various points of the combustion system. Siloxane in the 4. CONCLUSIONS presence of gas is usually deposited as a layer of white powder that causes mild stratification in the internal parts Generating electricity from landfill gas is a widely of a combustion engine [46].The deposit formation reused method worldwide. The benefits of generating elec- sulted in untimely engine cleaning, failure of gas engines, tricity from LFG can be two fold. Firstly, it helps avoid the high engine lube oil consumption, low lube oil filter life, contribution of greenhouse gases to the atmosphere formed valve damages for gas engines in both Odayeri and in landfills and secondly, LFG is utilized as a fuel in dif- Kömürcüoda landfills. Gas engine failure during energy ferent revenue-generating energy systems. production or early maintenance period results in loss of A successful WTE project reaches its targets by fol- energy production. The longer maintenance period or fail- lowing these steps: 1) A mathematical gas prognosis is ure time, the higher the loss of energy production. done to see if the landfill is suitable for a WTE project. 2) Landfill site is inspected with static tests to see the influ- 3.3 Limitations on power distribution ence on gas production such as bad compaction, waste A major aspect of a WTE project is delivering the elec- height, steep slopes, high water table, shallow areas and tricity to the end users continuously. However, short cir- leachate accumulation. 3) Site pumping test is applied in cuits caused by birds perching on the energy voltage line the field to see if real LFG is carried out over a certain pe- that carry the energy to the national grit were seen in both riod of time using a full scale collection system for at least Odayeri and Kömürcüoda landfills. The produced energy three months. not being able to be delivered to the national energy grit Mathematical or experimental LFG gas prognosis is causes loss of profit. The local transformer system has used for construction of energy conversion facilities in the

feasibility phase. The installation capacity of a facility is [5] Gokcol, C., Dursun, B., Alboyaci, B. and Sunan E., (2009) Im- determined by calculating recovered LFG, utilizing a re- portance of biomass energy as alternative to other sources in Turkey. Energy Policy, 37, 424-431. covery constant. The recovered LFG rate is used for calculating installation capacity of the energy conversion facil- [6] Dolgen, D., Sarptas, H., Alpaslan, N. and Kucukgul, O., (2005) Energy potential of municipal solid wastes. Energy ity. In many applications, field LFG extraction rate is usu- Sources, 27, 1483-1492. ally lower than the estimated recovery LFG rate. The ab- [7] Lombardi, L., Carnevale, E. and Corti, A., (2006) Greenhouse sence of accurate estimations in WTE projects prevents effect reduction and energy recovery from waste landfill. En- them from being income-generating projects. Even when ergy, 31, 3208-3219. the recovered LFG is estimated accurately, operational [8] Chun, S.K. and Bae, Y.S. (2012) An impact analysis of landfill problems are often encountered such as engine damage and for waste disposal on climate change: Case study of Sudokwon clogging of the wells. Problems related to landfill site, landfill Site 2nd landfill in Korea. Korean Journal of Chemical power generation and energy delivery are limiting factors Engineering, 29, 1549-1555. for landfill gas and energy production. Some of these prob- [9] Papageorgiou, A., Barton, J.R. and Karagiannidis, A. (2009) As- lems occur naturally and they can be reduced by taking sessment of the greenhouse effect impact of technologies used necessary measures. for energy recovery from municipal waste: A case for England. Journal of Environmental Management, 90, 2999-3012. LFG production and collection systems as well as en- [10] Kumar, A. and Sharma, M.P. (2014) Estimation of GHG emis- ergy production and distribution systems of a WTE project sion and energy recovery potential from MSW landfill sites. should be checked regularly for it to continuously produce Sustainable Energy Technologies and Assessments, 5, 50-61. high-performance energy and be revenue-generating. As [11] White, A.L. and Zack, M. (1989) Avoided-cost pricing of elec- was the case in Odayeri and Kömürcüoda WTE applica- tricity from waste-to-energy plants the regulatory economics tions, parameters were established and collected regularly of a negative cost fuel, Energy Policy, 17, 370-381. and analyzed for optimum energy production. The esti- [12] Niskanen, A., Värri, H., Havukainen, J., Uusitalo, V. and mated amount of LFG to be recovered in both landfills was Horttanainen, M. (2013) Enhancing landfill gas recovery. determined by taking 60% of the value of the prognosis Journal of Cleaner Production, 55, 67-71. model. 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[36] Rajesh, S., Gourc, J.P. and Viswanadham, B.V.S. (2014) Eval- uation of gas permeability and mechanical behaviour of soil barriers of landfill cap covers through laboratory tests. Applied Clay Science, 97–98, 200-214. Received: January 01, 2015 [37] Simon, F.G. and Müller, W.W. (2004) Standard and alternative Revised: February 23, 2015 landfill capping design in Germany. Environmental Science & Accepted: March 11, 2015 Policy, 7, 277-290. [38] Elagroudy, S.A., Abdel-Razik, M.H., Warith, M.A. and Ghobrial, F.H. (2008) Waste settlement in bioreactor landfill CORRESPONDING AUTHOR models. Waste Management, 28, 2366-2374. [39] Yi, F., Xu, Y., Tian, Y. and Jiang, F. (2014) The influence of Orhan Sevimoğlu landfill settlement on the efficiency of landfill gas collection. Advances in Science and Technology of Water Resources, 34, Istanbul Metropolitan Municipality 30-33. Şehzadebaşı Caddesi 34134 Fatih / Istanbul [40] Beaven, R.P., Hudson, A.P., Knox, K., Powrie, W. and Robin- son, J.P. (2013) Clogging of landfill tyre and aggregate drain- TURKEY age layers by methanogenic leachate and implications for practice. Waste Management, 33, 431-444. Phone: +90 (212) 455 33 00 [41] Van Gulck, J.F. and Rowe, R.K. (2004) Inﬂuence of landﬁll E-mail: [email protected] leachate suspended solids on clog (biorock) formation. Waste Management, 24, 723–738. FEB/ Vol 24/ No 7/ 2015 – pages 2362 - 2373

APPLICATIONS OF FACTOR ANALYSIS AND GEOGRAPHICAL INFORMATION SYSTEMS FOR PRECISION AGRICULTURE OVER ALLUVIAL LANDS

Kadir Ersin Temizel1,*, Hakan Arslan1 and Mustafa Sağlam2

1Ondokuz Mayis University, Faculty of Agriculture, Department of Agricultural Structures and Irrigation, 55139 Samsun-Turkey 2Ondokuz Mayis University, Faculty of Agriculture, Department of Soil Science and Plant Nutrition, 55139 Samsun-Turkey

ABSTRACT farmers have started to be aware of the advantages brought by the implementations carried out in agriculture through In this study, soil samples were taken from 32 different considering the spatial differences. Knowledge about the locations from two different soil depths (0-30 and 30-60 factors exhibiting spatial and temporal variations plays a cm) and 16 physical and chemical soil properties were as- significant role for optimum profit, sustainability and envi- sessed through factor analysis (FA) and geographical in- ronmental protection. Knowledge and technology-based formation systems (GIS). FA revealed 5 factors for the sur- precision agriculture are used to analyze and manage such face soil depth (0-30 cm) explaining 79.58% of total vari- factors and recently has a wide range of application in ag- ation in data set. These factors were entitled as factor 1 ricultural practices [1]. Precise agricultural implementa- “soil water holding capacity”, factor 2 “microelement tions are basically composed of accurate analysis of present availability”, factor 3 “organic matter”, factor 4 “soil reac- conditions and performance of implementations in accord- tion” and factor 5 “soil salinity”. FA for the subsurface ance with the present needs. Precision agriculture inte- depth (30-60 cm) revealed 4 factors explaining 75.14% of grates developing technologies into agricultural production total variation in data set. These factors were entitled as and covers the practices aiming maximum gain and taking factor 1 “nutrient availability–soil reaction relationship”, the environmental protection principles into consideration factor 2 “soil water holding capacity”, factor 3 “organic together with various low-cost inputs [2]. matter” and factor 4 “soil salinity”. A total of nine spatial distribution maps were prepared for these factors by using Multivariate statistic is a general term defining the sta- the factor scores obtained from FA for both soil depths. tistical methods allowing the analysis of two or more fac- Significant similarities were observed in both factor com- tors together [3]. In multivariate statistical analysis, there ponents and spatial distribution patterns of both soil depths. are multiple interrelated variables in a system. The appli- It was concluded that FA with various soil properties used cations of multivariate analytical methods such as cluster as multiple variables might reveal significant hidden infor- analysis (CA), FA and principle component analysis mation about soil properties and yield highly valuable out- (PCA) to environmental data have tremendously increased comes for the management and planning of precise agricul- and yielded meaningful information. FA is a multivariate tural practices. statistical technique used to get less and unrelated variables by using multiple interrelated variables. CA and PCA have been widely used in soil surveys [4-6]. FA and GIS have also together been used in several studies investigating soil KEYWORDS: Precision agriculture, factor analysis, geographical characteristics [7, 8] Shan et al. [9] carried out a study to information systems, soil properties, alluvial lands find out the heavy metal source of soils and used multivariate statistical techniques and GIS together. Soil samples were taken from 149 different points in that study and 11 different soil parameters were determined. Researchers de- 1. INTRODUCTION termined anthropogenic factor as factor 1, lithogetic factor as factor 2 and prepared spatial distribution map of each Precision agriculture represents the approaches allow- factor. ing the implementation of environment-friendly methods and techniques in agricultural production activities. Paral- The present study was conducted to present the bene- lel to developments in global positioning systems (GPS), fits and advantages brought by combined use of FA and GIS in planning and management of precision agriculture * Corresponding author implementations.

2. MATERIALS AND METHOD Global positioning (GPS) device was used to locate the soil sampling points. The distance between soil sampling 2.1 Study area and sampling points was 70 m. Disturbed soil samples were taken from The study area is located within the Black Sea Coastal 0-30 cm and 30-60 cm soil depths. Since horticultural crops Region of the Samsun province (between 41º30’- 41º45’ are grown over the research site of the present study and latitude and 35º30’-36º15’ longitude) in the northern Tur- rooting depths of most of these crops are within the initial key (Figure 1). Soils are formed over alluvial deposits of 60 cm soil layer, soil samples were taken from these Kizilirmak River at various elevations and classified as depths. Samples were air dried at laboratory and sieved Typical Ustifluvents, Chromic Haplusterts and Vertic Hap- through 2 mm sieve to make them ready for analyses. Soil lustepts [10]. Bafra plain has a mild climate with annual texture was determined by Bouyoucos hydrometer method average temperature of 14.4 °C and annual precipitation of [11]; lime content was determined with Scheibler calcime- 802.6 mm. The main rock of Bafra Plain and close sur- ter [12]; organic matter content was determined in accord- roundings are composed of Mesozoic Tertiary and quater- ance with Smith-Weldon method [13]; soil pH [14] and nary lands. Quaternary lands are composed of alluvial and electrical conductivity [15] were determined in 1:2 soil- terrace depots. The alluvium over the flood plains along the water mixture; Kjeldahl method was used to determine the Kızılırmak crossing the plain in North-south direction and total nitrogen [16]; available phosphorus was determined other streams are silty and gravelly and he gravels are by Olsen method [17]; extractable potassium (Kext) was de- mostly composed of volcanic rocks and lime stones. The termined in accordance with Soil Survey Staff [18]; DTPA- soils of Bafra Plain may be broadly characterized as deep, extracted solution was used to determine available iron, alluvial delta and colluvial terrace-alluvial fans. These are copper, zinc and manganese in an atomic adsorption device young soils extremely variable, ranging from sandly loans [19]. The analyses on entire soil characteristics were per- to heavy clays. Particularly paddy and tomato, pepper, formed in three replications and the data set was used melon, watermelon and eggplant are grown in Bafra Plain. through taking the arithmetical mean of these replications. Over these lands, generally surface gravity irrigation methods and randomly sprinkler and drip irrigation method are 2.2 Principle component and spatial analysis methods used in irrigations. Since Bafra Plain is close to sea, there Soil samples were taken from 32 different locations is a drainage problem in some sections of the plain. and 16 different physical and chemical analyses were per-

FIGURE 1 - Location of Study Area and Distribution of Sampling Points

formed on these samples. Analyses results were assessed water properties [25-26]. To explore the spatial distribution through FA and GIS. FA is a multivariate statistical tech- of FA results, ordinary kriging method was used with PC nique and uses multiple interrelated variables to derive un- software ArcGIS 10.0. related new variables. It includes dimension reduction and dependence elimination phases and brings together p number of interrelated variables of a case and tries to find less 3. RESULTS AND DISCUSSION unrelated new (common) variables [20]. While finding the number of principle components, the ones with an eigen 3.1 Definition of descriptive statistics value over 1 are selected. This method has been utilized in Descriptive statistics for surface (0-30 cm) and subsur- assessment of soil data in several studies [21, 22]. In FA, face (30-60 cm) soil depths are provided in Table 1 and resultant factors are assessed as: f > 0.75 strong, 0.50< f assessment of soil characteristics with regard to threshold <0.75 medium and 0.30 < f <0.50 weak [23]. In FA of the values of these parameters in plant production are provided present study, varimax rotation was employed to reduce the in Table 2. number of factors through maximizing the variations of factor loads. Statistical analyses were performed by using Considering the average values, soil texture of surface SPSS 21 software. and subsurface depths was classified as clay (Table 1). For unsampled locations, kriging is considered as an While field capacity (FC) of the surface depth varied be- optimal method of spatial prediction that provides a supe- tween 25.26-39.18%, permanent wilting points (PWP) var- rior liner unbiased estimator for spatially varying quantities ied between 11.85-22.60%. EC values of the surface depth [24]. Various researchers have used Geographic Infor- varied between 0.23-0.51 dSm-1 and a salinity problem was mation System (GIS) to investigate a wide range of soil and not observed in surface depth considering the threshold

TABLE 1 - Descriptive statistics for soil samples of surface (0-30 cm ) and subsurface (30-60 cm) soil depths.

Soil properties Unit Min. Max Mean S.D. CV (%) Skewness Kurtosis Transformation Surface depth (0-30 cm) Sand % 9.27 24.12 15.23 4.15 27.24 0.49 2.12 Normal Clay % 33.88 58.10 48.73 6.51 13.36 -0.76 2.85 Lognormal Silt % 27.98 43.95 36.04 3.53 9.78 -0.19 3.20 Normal FC % 25.26 39.18 32.55 3.37 10.34 -0.28 2.28 Normal PWP % 11.85 22.60 17.41 2.60 14.94 -0.34 2.62 Normal EC dS m-1 0.232 0.511 0.344 0.073 21.35 0.56 2.33 Lognormal pH 7.44 8.12 7.91 0.16 2.00 -1.25 4.70 Lognormal OM % 0.52 2.55 1.73 0.50 28.62 -0.59 2.84 Lognormal

CaCO3 % 3.88 8.54 6.53 1.20 18.37 -0.17 2.12 Normal Ntotal % 0.03 0.13 0.09 0.02 28.62 -0.59 2.85 Lognormal -1 Feav mg kg 14.89 32.76 22.19 4.46 20.08 0.14 2.35 Normal -1 Cuav mg kg 3.77 6.50 4.79 0.64 13.34 0.49 3.03 Normal -1 Znav mg kg 0.31 1.16 0.60 0.26 42.82 0.80 2.35 Lognormal -1 Mnav mg kg 9.52 21.23 14.18 3.24 22.87 0.66 2.47 Lognormal -1 Kext cmol(+) kg 0.37 1.09 0.71 0.14 19.88 -0.04 3.98 Normal -1 Pav mg kg 7.57 46.40 16.80 7.53 44.81 2.18 9.06 Lognormal Subsurface depth (30-60 cm) Sand % 8.63 25.87 17.24 5.08 29.48 0.23 1.76 Normal Clay % 31.32 55.78 44.58 6.36 14.26 -0.17 2.66 Normal Silt % 30.94 51.41 38.184.79 12.54 0.77 3.12 Lognormal FC % 25.40 40.00 33.75 4.63 13.73 -0.21 1.91 Normal PWP % 11.97 23.60 18.47 2.93 15.88 -0.30 2.45 Normal EC dS m-1 0206 0.541 0.350 0.087 24.96 0.52 2.41 Lognormal pH 7.57 8.18 7.97 0.12 1.51 -1.05 5.27 Lognormal OM % 0.30 2.06 1.48 0.42 28.16 -0.98 3.35 Lognormal

CaCO3 % 4.66 8.93 6.93 1.10 15.93 -0.01 2.11 Normal Ntotal % 0.02 0.10 0.07 0.02 28.16 -0.97 3.35 Lognormal -1 Feav mg kg 17.87 61.06 25.32 7.78 30.71 3.07 15.05 Lognormal -1 Cuav mg kg 4.25 7.29 5.40 0.69 12.83 0.57 3.12 Lognormal -1 Znav mg kg 0.34 1.25 0.65 0.27 42.16 1.09 2.92 Lognormal -1 Mnav mg kg 12.25 22.21 16.90 2.53 14.99 -0.01 2.36 Normal -1 Kext cmol(+) kg 0.34 1.19 0.70 0.16 23.03 0.36 4.51 Normal -1 Pav mg kg 6.25 47.88 15.90 7.64 48.02 2.55 11.09 Lognormal

FC: Field Capacity; PWP: Permanent Wilting Point; OM: Organic Matter; Ntotal: Total Nitrogen; Feav: Available Iron; Cuav: Available Copper; Znav: Available Zinc; Mnav: Available Manganese; Kext: Extractable Potassium; Pav: Available Phosphorus. S.D: Standart Deviation

TABLE 2 - Threshold values for soil properties

Soil properties Units Very Low Low Moderate High Very High References OM % 0-1 1-2 2-3 3-4 >4 Ülgen and Yurtsever [27] CaCO3 % 0-1 1-5 5-15 15-25 >25 Ülgen and Yurtsever [27] EC dS m-1 0-2.0 2-4 4-8 8-16 16 < Ülgen and Yurtsever [27]

Ntotal % <0.045 0.045-0.09 0.09-0.17 0.17-0.32 >0.32 FAO [37] -1 Fe av mg kg < 0.2 0,2-4,5 >4,5 Lindsay and Norvell [38] -1 Zn av mg kg 0.2 0.2-0.7 0.7-2.4 2.4-8.0 >8.0 FAO [37] -1 Mn av mg kg <4 4-14 14-50 50-170 >170 FAO [37] 1 Kext cmol(+) kg- <0.13 0.13-0.28 0.28-0.74 0.74-2.56 >2.56 FAO [37] -1 P av mg kg <2.5 2.5-8.0 8.0-25 25-80 >80 FAO [37] -1 Cu av mg kg <0.2 >0.2 Follet [39]

values for soil salinity. Soil pH values of the surface depth classified as limey and medium limey with regard to varied between 7.44-8.12 and soils in general had slightly CaCO3 contents; they were classified in low, medium and alkaline reaction [27]. Organic matter (OM) content of the high classes with regard to Pav content; in medium and high surface depth varied between 0.52-2.55% and total nitro- classes with regard to Kext contents. On the other hand, with gen (Ntotal) content varied between 0.03-0.13%. Consider- regard to micro element threshold values, entire micro nu- ing the threshold values, both OM and Ntotal values varied trient values of the subsurface depth soils were classified among very low, low and medium levels. With regard to within the same classes with the soils of the surface depth. maximum and minimum values, soils of the surface depth With regard to CV values of the subsurface depth soils, were considered as limey and medium limey; available clay, silt, FC, pH, Cu av and Mn av contents exhibited slight phosphorus (Pav) levels were at low, medium and high lev- variation; sand, PWP, EC, OM, CaCO3, Ntotal, Feav and Kext els; extractable potassium (Kext) levels were medium and contents exhibited moderate variation; Zn av and P av con- high. With regard to maximum and minimum values of mi- tents exhibited high variation. cro nutrients, soils of surface depth were not deficient in available iron (Feav) and copper (Cuav); zinc (Znav) and 3.2 Factor Analysis manganese (Mn ) levels were low and medium. With re- av Before the factor analysis, suitability of soil character- gard to coefficient of variation (CV) of soil characteristics istics for normal distribution was tested by Kolmogorow- of the surface depth, clay, silt, FC, PWP, pH and Cu con- av Simirnow test and transformations were applied to non- tents exhibited slight variation; sand, EC, OM, CaCO , N 3 to- normally distributed data sets. The data sets subjected to , Fe , Mn and K contents exhibited moderate variation; tal av av transformations and applied transformation methods are Zn and P contents exhibited high variation. According av av provided in Table 1. to classification of CV by Wilding [28], an attribute with a CV ≤ 15% is deemed slightly variable, between 16 and Correlation coefficients between the variables of the 35% moderately variable, and CV > 36% highly variable. data set should be evaluated and Kaiser-Meyer Olkin Various other researchers also reported slight, moderate and (KMO) should be performed to assess the availability of high CV values of soil characteristics [29-32]. In addition, data set for factor analysis. In this study, correlations be- Tsegaye and Hill [33], Aimrun et al. [34], and Mousavifard tween soil characteristics were determined and KMO test et al. [35] reported a lower variance of soil pH compared to was performed to assess the availability of data sets of both other soil chemical attributes. The pH values are a log scale soil layers for factor analysis. The correlation between the of proton concentrations in the soil solution and there would variables should be over 0.30 to perform factor analysis.S be much higher variability if soil acidity was expressed in uitability of the data set about soil properties of surface and terms of proton concentrations directly [36]. subsurface depths soils for FA was assessed based on Kai- ser-Meyer-Olkin (KMO) value. While Sharma [40] classi- While FC of the lower level varied between 25.40- fied the suitability of the data set with a KMO value of 0.9 40.00%, PWP varied between 11.97- 23.60%. Soil pH and for FA as perfect; the values between 0.8-0.5 were classi- EC values of the lower level were similar to values of sur- fied as very good, good, medium and weak; a value below face depth and subsurface depth soils were also had slight 0.5 was classified as not suitable for FA. KMO value of the alkaline reaction and they were unsaline. OM and N total present study was above 0.5 and such a value indicated that content of soils decreased with increasing soil depths but data set on soil properties of both soil depths was suitable the values were classified in similar levels with the surface for FA. depth based on threshold values. Considering the maximum and minimum values for OM and Ntotal, soils of the At the end of FA on surface soil depth, 5 factors had subsurface depth distributed within very low, low and mean eigen value ≥1 and these factors represented 79.58% of dium levels. While the soils of the subsurface depth were total variation in data set (Table 3).

TABLE 3 - Rotated factor patterns of four factors after varimax rotation (0-30 cm)

Parameters PC1 PC 2 PC 3 PC 4 PC 5 Clay 0.888 0.044 0.238 -0.153 -0.165 PWP 0.879 0.042 0.042 0.240 0.033 FC 0.866 0.079 0.174 0.195 0.241 Sand -0.800 -0.074 -0.314 0.228 -0.223

Kext 0.724 0.325 0.259 -0.147 -0.267 Silt -0.700 0.007 -0.071 0.014 0.568

Feav 0.150 0.852 -0.068 0.042 0.209

Mnav -0.167 0.823 -0.059 0.010 -0.169

CaCO3 -0.168 -0.670 -0.181 0.304 -0.020

Znav -0.048 0.613 -0.062 -0.524 0.169

Cuav 0.327 0.573 0.276 0.016 0.081 OM 0.300 0.040 0.921 0.070 -0.083

Ntotal 0.315 0.062 0.916 0.077 -0.076 pH 0.088 -0.037 0.057 0.938 -0.095

Pav 0.278 0.420 -0.377 -0.479 -0.006 EC 0.058 0.116 -0.096 -0.149 0.917 Eigen value 5.468 3.183 1.532 1.349 1.20 Total variance (%) 34.176 19.896 9.574 8.433 7.503 Cumulative variance (%) 34.176 54.073 63.646 72.08 79.582 Bold and italic values indicate strong (> 0.75) and moderate (0.75 – 0.50) loadings, respectively

Factor 1 was able to explain 34.18% of total variation positively represented; CaCO3 was medium negatively in data set. In this factor, clay, PWP and FC were strong represented; Znav and Cuav were medium positively repre- positively represented, sand was strong negatively represented. Considering the soil properties assigned as factor sented, Kext and silt were respectively medium positively components, the factor was entitled as “microelement and negatively represented. Considering the soil properties availability”. The negative relationship between microele- assigned as variable in this factor, it was observed that gen- ments and CaCO3 contents indicated increasing microele- erally physical soil properties related to water holding in ment availability with decreasing CaCO3 contents. Feav, soil were assigned as the variables. Clay content with pos- Cuav, Znav and Mnav are the essential micro nutrients for itive impacts on water holding capacity had positive factor plant growth and development. [41] reported that availabil- loading, but sand and silt contents with negative impacts ity of micro nutrients were especially sensitive against var- on soil water holding capacity had negative factor loadings. iations around the soils and indicated the soil properties Thus, considering the factor components and relationships like OM, pH, CaCO3, clay, silt and sand content as the sig- among them, this factor was entitled as “soil water holding nificant factors effecting micro nutrient contents of the capacity”. The spatial distribution of the scores for factor 1 soils. The spatial distribution of the scores for factor 2 (mi- (soil water holding capacity) are shown in Figure 2. For croelement availability) are shown in Figure 2. The spatial each factor, higher values were indicated by darker colors distribution map of the factor indicated heterogeneity in and lower values were indicated by lighter colors. microelement availability over the study area. Microele- Spatial distribution map of the factor indicated high ment availability was found to be high over the southern water holding capacities of the soils over the southern, east- sections of the study area and low over the western and ern and south-eastern sections of the study area, on the northern sections of the study area. Therefore during the other hand indicated low water holding capacities of the precise agricultural practices to be implemented over the soils over the northern and north-westerns sections of the study area, it was recommended for the areas with low mi- study area. Therefore during the precise agricultural prac- croelement availability levels that foliar micro nutrient tices to be implemented over the study area, it was recom- treatments might improve the availability of microele- mended for the areas with low water holding capacities that ments. either more frequent irrigations should be applied or Factor 3 was composed of OM and Ntotal components measures should be taken to improve water holding capac- and was able to explain 9.57% of total variation in data set. ities of the soils through improving organic matter con- Thus, this factor was entitled as “organic matter”. Spatial tents. distribution map of the factor indicated low OM levels over Factor 2 was able to explain 19.90% of the total varia- the south-western and north-western sections of the study tion in data set. In this factor, Feav and Mnav were strong area and high OM levels over the south-eastern and north-

FIGURE 2 - Spatial distribution maps of the factors (0-30cm)

eastern sections of the study area (Figure 3). Therefore dur- level with clay and OM content and increased Feav levels ing the precise agricultural practices to be implemented with sand content. The distribution map of factor 2 sup- over the study area, it was recommended for the areas with ports the earlier recommendation of “foliar application of low OM contents together with low water holding capaci- micro elements” to be made to improve the availability of ties that plant production activities to improve OM contents microelements according to distribution map of factor 2. might also improve water holding capacities. Factor 5 including soil EC was able to explain 7.50% Soil pH was assigned as the variable in factor 4 and it of total variation in data set. Considering the soil property was able to explain 8.43% of total variation in data set. The assigned as variable, the factor was entitled as “soil salin- factor can be defined as “soil reaction”. The spatial distri- ity”. Spatial distribution map of the factor indicated low bution map of factor 4 indicated high pH levels over the EC values over northern and north-eastern sections of the north, north-eastern and north-western sections of the study area and high EC values over southern and south- study area and low pH levels over the south, south-western western sections of the study area which may create a sa- and south-eastern sections of the study area (Figure 3). linity risk over these sections. Maximum and minimum EC When the spatial distribution map of factor 4 was assessed values of surface and subsurface soil depths did not indi- together with the spatial distribution map of factor 2 (mi- cate a salinity problem for the study area. However, inten- croelement availability), it was observed that maps re- sive agricultural practices over the study area, seasonal vealed significant information about the relationships be- changes in irrigation water quality, evaporation from soil tween microelement availability and pH values. The north, surface and possible drainage problems because of inten- north-eastern and north-western sections of the study area sive irrigations may eventually result in a salinity problem. with high pH values had lower microelement availability Although currently a salinity problem does not exist over levels and the southern sections with low pH values had the study area, a leaching water requirement should be cal- increased microelement availability levels. Similar to find- culated and added to irrigation water to reduce the soil sa- ings of the present study, Chhabra et al. [42] reported de- linity during the implementation of precise agricultural creased Feav and Mnav levels with soil pH, increased Cuav practices.

TABLE 4 - Rotated factor patterns of four factors after varimax rotation (30-60 cm)

Parameters PC1 PC 2 PC 3 PC 4

Znav 0.811 0.110 0.109 -0.040

Feav 0.750 0.141 0.546 -0.007

Cuav 0.719 0.288 0.189 0.155

Pav 0.717 0.134 0.418 -0.134

CaCO3 -0.706 -0.130 0.193 -0.039 pH -0.615 0.364 0.481 -0.008 FC 0.106 0.920 -0.175 0.177 PWP -0.010 0.903 0.031 0.090

Kext 0.455 0.707 -0.141 -0.230 Silt -0.292 -0.664 0.235 0.097

Ntotal -0.057 0.333 -0.860 -0.029 OM -0.057 0.333 -0.860 -0.029

Mnav 0.297 0.067 0.550 -0.389 Sand 0.151 -0.135 0.121 -0.868 EC 0.365 -0.211 0.317 0.694 Clay 0.099 0.608 -0.274 0.621 Eigen value 4.556 3.94 1.851 1.675 Total variance (%) 28.472 24.627 11.57 10.471 Cumulative variance (%) 28.472 53.099 64.669 75.14 Bold and italic values indicate strong (> 0.75) and moderate (0.75 – 0.50) loadings, respectively

At the end of FA on subsurface soil depth, 4 factors the vertical variations in soil properties should be taken had an eigen value ≥1 and these factors represented 75.14% into consideration since nutrient availability–soil reaction of total variation in data set (Table 4). factor had the highest value in subsurface depth soils of the southern, south-western and south-eastern sections of the Factor 1 explained 28.47% of total variation in data set. study area with the lower soil reaction values in surface In this factor, Zn and Fe were strong positively repre- av av depth. Besides the horizontal variations, vertical variations sented, Cuav and Pav were medium positively represented, in pH should also be investigated to benefit from the micro CaCO3 and pH were medium negatively represented. Con- nutrients at uppermost level in fields over which precise sidering the soil properties assigned as variable, the factor agricultural practices are to be implemented. Considering was entitled as “nutrient availability–soil reaction relation- the variations in pH values with the depths in detail, it was ship”. The spatial distribution of the scores for factors is observed that increasing pH values were observed with in- shown in Figures 3. The spatial distribution map of factor creasing soil depths. Two factors were considered to be ef- 1 indicated higher values for nutrient availability levels fective to have increasing pH values. The first one is the over the southern, south-western and south-eastern sec- leaching of cations to lower depths in alkaline soils through tions of the study area and lower values over the northern, the irrigations. The second is the increasing water tables north-western and north-eastern sections of the study area. because of intensive irrigations over alluvial soils. There- Opposite signs of factor loadings of nutrients and pH- fore, water table rises should be prevented and more effi- CaCO3 indicated increasing nutrient availability with de- cient irrigation management should be practiced to prevent creasing pH and CaCO3 contents and vice versa. When the the increases in pH levels of lower soil depths of the sites spatial map of factor 4 representing the variations in soil over which precise agricultural practices are to be imple- reactions of surface depth and map of factor 1 representing mented. the relationships between nutrient availability and soil re- In factor 2 of the subsurface depth, FC and PWP were action were assessed together, it was recommended while strong positively represented, K was medium positively deciding about precise agricultural practices on nutrient represented and silt was medium negatively represented. availability that not only the horizontal variations but also The factor was able to explain 24.63% of the total variation

FIGURE 3 - Spatial distribution maps of the factors (30-60cm)

in data set. Considering the soil properties assigned as var- In factor 3 of the subsurface depth, OM and Ntotal were iables in this factor, it was observed that the factor gener- strong negatively represented and Mnav was medium posi- ally included the information about water holding capacity tively represented. The factor was able to explain 11.57% of soils. Thus, as it was in surface depth, the factor was also of total variation in soil properties. Such a negative rela- entitled as “soil water holding capacity” in subsurface tionship for OM, Ntotal and Mnav has not been explained in depth. When the spatial distribution maps on soil water previous literatures. Thus, considering the soil properties holding capacity factors of surface and subsurface depths assigned as variable, a proper title was not assigned to this are assessed together, it was observed that same regions factor. However, spatial distribution map of the factor in- had the minimum and maximum values. Thus in precise dicated that lower factor values over the southern, northern, agricultural practices to be implemented over the study eastern and south-eastern sections of the study area were area, decisions on soil water holding capacities should in- actually high in soils since they were represented by nega- clude both the horizontal and vertical variations, so the rec- tive factor loadings. Combined assessments of spatial maps ommendations made for the soil water holding capacity of for factor 3 of surface depth in which OM and Ntotal were the surface depth are also valid for the subsurface depth. represented by positive factor loadings and factor 3 of sub-

surface depth in which OM and Ntotal were represented by and vertical variations in soil characteristics. It was proved negative factor loadings revealed that low and high OM in this study that combined use of FA and GIS might reveal and Ntotal values were observed in surface and subsurface successful outcomes for the implementation of precise ag- depths of the similar sections of the study area. Although ricultural activities. Thus, these methods may also be used Mnav was assigned as a variable in factor 3 of the subsur- in larger-scale studies toward the planning of precise agri- face depth, OM and Ntotal were determinants of the factor cultural practices. Considering current international trends, based on factor loadings of determinant factors. The factor it is evident that sustainable development is only possible which was not assigned with any titles initially may then through the performance of right implementations at right be assessed as “organic matter” factor. Since the soil prop- time in right places. erties assigned to factor 3 of both soil depths and their distributions in spatial distribution maps were similar to each The authors have declared no conflict of interest. other, precision agricultural practices should take both horizontal and vertical variations in OM contents into consideration. REFERENCES

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PHOTOSYNTHETIC ACTIVITY OF Microcystis IN FISH GUTS AND ITS IMPLICATION FOR FEASIBILITY OF BLOOM CONTROL BY FILTER-FEEDING FISHES

Zhicong Wang, Zhongjie Li, Yiyong Zhou and Dunhai Li*

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P.R. China

ABSTRACT 1. INTRODUCTION

In a background of increasingly frequent and intensive Eutrophication is an increasingly serious problem in Microcystis blooms occurred in eutrophic lakes, many freshwater ecosystems, and can be accompanied by fre- countries, particularly China, have used filter-feeding fish quent cyanobacterial blooms, which, particularly for spe- to control algal blooms according to non-traditional bio- cies producing the toxin microcystin, may be extremely manipulation theory. However, there is little information harmful to aquatic ecosystems and human health [1-3]. concerning the potential photosynthetic activity of cyano- Several methods for bloom control are currently available bacteria in the guts of planktivorous fishes. To make a sci- and employ engineering [4, 5], physical [6, 7], chemical [8, entific assessment of this algal control technology, we 9] or biological methods [10]. Some researchers observed evaluated the effects of digestion by two fish species, silver that water blooms were dissipated in lakes stocked with fil- carp and bighead carp, on the growth potential of Micro- ter-feeding fish, which led to the conclusion that grazing cystis blooms in terms of photosynthetic activity, meta- pressure by planktivorous fishes is a key factor in eliminat- bolic activity, up-floating velocity, morphological size, and ing water blooms from lakes [11]. Previous studies also toxin production. The results showed that: 1) the potential suggested that cyanobacterial blooms consumed by plank- photosynthetic activity of Microcystis dropped signifi- tivorous fish, could result in compositional changes of the cantly in fish foreguts after ingestion, but gradually in- phytoplankton community [12]. Also some researchers have creased and recovered in the midgut and hindgut; 2) diges- reported the change of photosynthesis in Microcystis colo- tion by planktivorous fish could significantly decrease the nies after gut passage through crucian carp and koi carp colonial size and up-floating velocity of Microcystis; 3) se- [13]. But this research studied the algae outside the guts of lective digestion by bighead carp caused a sharp rise in cel- fish. Furthermore, the fishes studied are not planktivorous lular microcystin levels; and 4) Microcystis retained a high fishes. Therefore, we think it is valuable and interesting to growth potential after digestion by filter-feeding fish. These investigate the photosynthetic activity, growth potential, results indicate that planktivorous fish could be used for morphological changes and microcystin content of Micro- controlling blooms due to the degree of digestion and long cystis in the guts of planktivorous fishes. retention of Microcystis in fish guts, but the controlling effect was limited because the potential photosynthesis ac- The silver carp (Hypophthalmichthys molitrix) and the tivity of algae gradually recovered along the gut sections bighead carp (Aristichthysnobilis) are the main species for and the digested algae could grow normally after subse- freshwater fishery and aquaculture in China [14]. Aided by quent release into lakes. In addition to being less effective a filter-feeding organ comprised of gill rakers, a gill rak- at controlling algal blooms than silver carp, the digestion ernetwork, palatal rugae and a gill raker tube [15], most process in bighead carp might confer an advantage to toxic bloom-forming cyanobacterial colonies can be consumed Microcystis species. by silver carp and bighead carp. Algae sized 8-100 μm can be filter-fed by silver carp, while those sized 17-3,000μm can be consumed by bighead carp [16]. However, most co- KEYWORDS: Microcystis bloom; filter-feeding fish; microcystin; lonial Microcystis cannot be completely digested by plank- photosynthetic activity. tivorous fishes due to their cell wall composition that includes cellulose and pectic substances. Therefore, the digestion of Microcystis colonies in the gut is sometimes referred to be as “pseudo-digestion” because only the algal colony morphology is changed after digestion, and thus in- * Corresponding author tact algal cells are excreted back to the lake water driven by continuous consumption and/or intestinal peristalsis.

The chlorophyll fluorescence parameters which showed 104 to 106 algal cell/ mL for measurement of chlorophyll the potential photosynthetic activity of higher plants and al- fluorescence parameters. Five representative gut sections gae were used as important indicators for algal cell vitality were chosen to assess the composition change of different- and the survival state of Microcystis in fish guts. Therefore, sized Microcystis colonies, and these gut sections were re- our study focused on the effects of planktivorous fishes on numbered as I (numbers 1-3), II (numbers 8-10), III (num- the bloom-forming genus Microcystis in terms of (1) changes bers 15-17 for silver carp and numbers 16-18 for bighead in colony size, photosynthetic activity and metabolic activity carp), IV (numbers 21-23 for silver carp and numbers 23- in various fish gut sections; (2) gut sections in which algal 25 for bighead carp) and V (numbers 28-30 for silver carp cells are digested; (3) the effects of digestion on Micro- and numbers 31-33 for bighead carp). Microcystis in the cystis community structures and microcystin content; and above-mentioned sections I, III and V (representing the (4) whether Microcystis that has been digested and ex- foregut, midgut and hindgut, respectively) were collected creted back into lake water can serve as a source for subse- for measurement and analysis of the up-floating velocity, quent blooms. Our analysis could reveal the mechanism colony composition, autofluorescence, biochemical com- and efficiency of bloom removal promoted by planktivo- position and microcystin content. rous fishes. 2.2 Up-floating or sinking velocity of Microcystis colonies The average floating velocities of Microcystis colonies 2. MATERIALS AND METHODS were measured as described previously [18], and the velocities were used to evaluate colony buoyancy. 2.1 Gut content analysis From September 1-6, 2011, 30 silver carp and 30 big- 2.3 Microcystis colony size determination head carp were randomly collected from the large shallow Microcystis colony morphology was investigated based eutrophic Lake Taihu (China), having the specific location on previous studies [9]. After dilution with 5 mL of distilled N31°14′10″, E119°57′1″. The basic characteristics of the water, 0.1 ml sample was assayed for phytoplankton number sampled fish are shown in Table 1. The open fishing season and type using a light microscope. By measuring the longi- for silver carp and bighead carp as designated by the Taihu tudinal axis length of each Microcystis colony, colonies Basin Authority (Ministry of Water Resources, contact num- were divided into eight types based on the single colony ber: +86 021-65425171) is September 1st to January 30th size: 3–5, 5–10, 10–20, 20–40, 40–100, 100–300, 300–900 of the subsequent year. Since the experiments were carried and >900 μm. To evaluate the effect of filter-feeding on out during the open fishing season, no specific permissions algal colony size, the total number of Microcystis cells in were required. The fishes used were collected with nets, and different gut sections was normalized to 1×108 cell/mL, then carefully dissected so that the guts could be sampled such that all above-mentioned algal colonies could be com- according to the steps described in the next paragraph. Be- pared between any two sections where the total cell number cause these two kinds of filter-feeding fish are not protected was the same. species and are common food sources, no approval was needed from an Institutional Animal Care and Use Commit- 2.4 Chlorophyll fluorescence tee (IACUC) or equivalent animal ethics committee. The chlorophyll fluorescence parameters of the num- The foregut contents were collected from the proxi- bered gut contents were measured with PAM2100 (Pulse- mate end of the intestine to the middle of the first loop [17]. Amplitude-Modulation Chlorophyll Fluorescence 2100; The hindgut contents were collected from the middle of the WALZ, Germany) [18]. The Fv/Fm was obtained from meas- last loop to the anus, and the mid-gut was between the fore- urements taken with a light less than 0.15 μmol photons· gut and hindgut. The entire gut was sampled and divided m-2·s-1 after incubation in the dark for 10 min. Maximum into 30 equal portions for silver carp or 33 equal portions photochemical efficiency of PSII (Fv/Fm), actual photochem- for bighead carp, and sequentially numbered 1 to 30 or 1 to ical efficiency of PSII (ÖPSII), photochemical quenching (qP) 33 from foregut to hindgut. The digestive residues in each and non-photochemical quenching (qN) were automatically gut portion were immediately removed and suspended in measured under an active light (AL) with an intensity of 5 ml distilled water before further dilution with water to 256 μmol photons·m-2·s-1 following adaptation for 5 min.

TABLE 1 -Basic characteristics of sampled planktivorous fishes

Fish Silver carp (n=30) Bighead carp (n=30) Body length (cm) 39.88 ± 3.47 (36.0-43.5)* 39.80 ± 2.26 (37.5-45.0) Total length (cm) 46.75 ± 4.43 (42.0-51.0) 46.95 ± 2.47 (44.5-53.0) Body weight (kg) 1.58 ± 0.31 (2.5-3.8) 1.70 ± 0.22 (2.8-4.3) *The data in parentheses are the lower and upper limits of the measurements.

2.5 Fluorescence microscopy nificantat 95% confidence if p< 0.05 for a given F statisti- Microcystis sampled from fish guts were diluted with cal test value. distilled water and immediately placed in a refrigerator at 4 °C and analyzed within 24 hours. The morphological and fluorescence features of Microcystis colonies were ob- 3. RESULTS served using a fluorescence microscope (Nikon ECLIPSE E600, Japan), and images were recorded using a micro- 3.1 Up-floating velocity of Microcystis in different gut sections scopic photograph system (Pixera, USA). After being filter-fed and digested by planktivorous fishes, the up-floating velocities of Microcystis colonies 2.6 Re-culture of Microcystis sampled from fish gut significantly decreased (Table 2). Using the foregut as the Microcystis collected from various gut sections was re- standard value, in both silver carp and bighead carp the Mi- cultured in conical flasks filled with BG11 medium [19] to crocystis colony buoyancy in hindguts showed a larger de- evaluate its photosynthetic activity and growth potential crease than the midgut (p< 0.01). under suitable simulated conditions. The initial Microcystis 3.2 Microcystis colony size changes in different gut sections biomass was set to 2.0×105 cell/L, then cultured under a 12:12 LD (Light: Dark) cycle at 30 ± 2 °C with a light in- The distribution of Microcystis colonies in different tensity of 60 μmol photons s-1 m-2 provided by cool white gut sections of silver carp and bighead carp is shown in fluorescent tubes. Cell growth was measured based on Fig.1. The numbers of small colonies (single cells and 5- 25μm) were significantly higher (p< 0.05) in the gut sec- changes in cell concentration and Fv/Fm. tions as compared to that found in lake waters (Fig. 1a, b). 2.7 Microcystin extraction and quantification Silver carp showed smaller colonies, including single cells and 5-25 μm colonies that were mostly located in the hind- To evaluate changes in the microcystin content after gut while large colonies (25-900 μm) were distributed in filter feeding by fish, approximately 4 g wet weight of Mi- the foregut and midgut. In contrast to silver carp, the dis- crocystis was divided into two equal parts, with one part tribution pattern of different-sized Microcystis in gut sec- used to measure the dry weight after freeze-drying to a con- tions from bighead carp showed no significant difference stant weight, and the other used to determined microcystin. in the distribution pattern of small colonies (single cells Microcystin in the wet algal sample was extracted and meas- and 5-25 μm) among all gut sections, while compared with ured according to methods reported by Xiao et al. [20]. the foregut and midgut, large colonies (25-900 μm) in-

2.8 Carbohydrate and protein content creased significantly in the hindgut, especially near the clo- acal pore. Soluble carbohydrate content was measured according to the phenol-sulfuric acid method reported by Kochert 3.3 Chlorophyll fluorescence parameters in rapid light curves [21], and soluble protein was determined by the method of (RLCs) Coomassie Brilliant Blue G-250 proposed by Bradford Fv/Fm values of Microcystis in all gut sections from [22], with bovine serum albumin used as a standard. planktivorous fishes were lower than that in lake water (0.44-0.46). For silver carp, the photosynthetic activity of 2.9 Statistical analysis Microcystis from the foregut to hindgut showed a signifi- The analysis of variance (ANOVA) was used to exam- cant increase as suggested by rETRmax (r = 0.460, p < ine whether there were significant differences among each 0.01), ΦPSII (r = 0.378, p < 0.05) and qP (r= 0.350, p> physiological or ecological parameter of Microcystis sam- 0.05) (Fig. 2a-f). Fv/Fm increased in the foregut and pled from different gut sections. The parameters include reached relatively stable values. The declining non-photo- the up-floating velocity, photosynthetic activity, biochem- chemical quenching (qN), which is related to excess exci- ical component content and intracellular microcystin con- tation, suggested that there was a certain degree of recovery tent of Microcystis. Results of all tests were considered sig- for photosynthetic reaction centers.

TABLE 2 - Changes in Microcystis floating velocity in different fish gut sections

Silver carp Bighead carp Fish Foregut Midgut Hindgut Foregut Midgut Hindgut

Up-floating rate (cm/min) 1.02±0.11 0.57±0.08 0.33±0.07 0.96±0.06 0.52±0.09 0.38±0.04

Buoyancy decrease (%) — 44.11*** 67.65*** — 45.83*** 60.42***

Note: Bars represent SD of means, n = 9. *, **, and *** indicate values that differ significantly from the others at P <0.05, P <0.01 and P <0.001, respectively.

FIGURE 1 - The distribution of various sized Microcystis colonies in different gut sections of silver carp (a) or bighead carp (b). Columns represent means, and error bars are not shown, n = 9.*, **, and *** indicate a significant quantity difference of various colonies between the foregut and other gut sections or in lake water at p<0.05, p <0.01 and p<0.001, respectively.

FIGURE 2 - Microcystis chlorophyll fluorescence parameters (a) rETRmax, (b) Ik, (c)Fv/Fm, (d) ΦPSII, (e) qP and (f) qN plotted against specific gut-section number (i) in different gut sections of silver carp. k values denote the slope of the regression line of photosynthetic parameters versus specific gut section number. The correlation coefficient (r) and significant level (p) are presented in each figure.

FIGURE 3 - Microcystis chlorophyll fluorescence parameters (a) rETRmax, (b) Ik, (c)Fv/Fm, (d) ΦPSII, (e) qP and (f) qN plotted against specific gut-section number (i) in different gut sections of bighead carp. k values are as described for Fig. 2. The correlation coefficient (r) and significant level (p) are presented in each figure.

FIGURE 4 - Changes in cell density (a, c) and Fv/Fm (b, d) in Microcystis re-cultures sampled from different silver carp (a, b) and bighead carp (c, d) gut sections.

The photosynthetic activity of the digestive residues and fluorescence microscopy (Fig.5 and 6). In the foregut from bighead carp also showed an increase along the intes- of silver carp, Microcystis colonies showed strong auto- tinal tract that was similar to that of silver carp (Fig. 3a-f). fluorescence, which weakened along with the decrease in The increasing degree indicated by k, especially the Fv/Fm colonial size observed in the midgut and hindgut (Fig.5). (k = 0.003), was more apparent for bighead carp compared Some M. flos-aquae colonies showed pigment bleaching and to silver carp (k = 0.00002). However, qN was relatively autofluorescence loss while M. aeruginosa and M. wesen- stable in all gut sections from both types of fish. bergii still had a relatively strong autofluorescence, which indicated that some Microcystis species, such as M. flos- 3.4 Re-culture of Microcystis sampled from gut aquae, were more vulnerable to digestion and diminished The results of re-culture of Microcystis sampled from metabolic activity. different gut sections of silver carp and bighead carp are In contrast, for bighead carp there was no obvious min- shown in Fig. 4. For silver carp, there were obvious differ- iaturization of Microcystis colonies in the midgut and hind- ences in the growth curves and Fv/Fm changes in the foregut as compared to the foregut, and the outer edge of the col- gut, midgut and hindgut, while no apparent differences onies was clear and their autofluorescence relatively strong were observed for bighead carp. Comparing the midgut and (Fig. 6). These results suggested that the digestion effect of hindgut of silver carp, Microcystis from the foregut showed bighead carp on Microcystis colonies was weaker than sil- a significantly higher specific growth rate (μ) (p< 0.01, and ver carp. μ are 0.317, 0.176 and 0.237 d-1 in hindgut, midgut and foregut, respectively) and cell concentration (ρ) (p< 0.01, 3.6 Biochemical components and microcystin content of Micro- and ρ are 7.480, 6.380 and 2.627 in hindgut, midgut and cystis in different gut sections foregut, respectively). Also for silver carp, after re-cultur- Significant differences in microcystin (MC-RR/ MC- ing for 5 days, the Microcystis photosynthetic activity LR) content between the foregut and other gut sections (Fv/Fm) from the midgut was restored to the same level as from silver carp and bighead carp were observed (Fig. 7). that in the foregut. However, the Fv/Fm recovery of Micro- Compared to the foregut in silver carp, the MC-RR and cystis from the hindgut was very slow and stable at 80% of MC-LR in the midgut were significantly (p< 0.05) de- the maximal value (0.42) after 12 days of re-culture. For creased by 23.6% and 28.0%, respectively, and in the hind- bighead carp, digestion appeared to have no effects on gut they were further (p< 0.005) decreased by 51.6% and these parameters as observed by re-culturing (Fig. 4 c, d). 53.6%, respectively. However, for bighead carp, the MC- RR and MC-LR in the midgut were respectively increased 3.5 Microcystis autofluorescence in different gut sections by 129.9% (p< 0.05) and 145.3% (p< 0.01), and further re- The morphology of Microcystis colonies sampled from spectively increased by 228.0% and 219.2% over that in different gut sections was observed by light microscopy the foregut.

FIGURE 5 - Light and fluorescence microscopy of various Microcystis (M. flos-aquae, M. aeruginosa and M. wesenbergii) colonies in different gut sections from silver carp. Panels (a), (b) and (c) show light-microscopic photographs of the foregut, midgut and hindgut, respectively, while panels (d), (e) and (f) are fluorescence-microscopic photographs of the foregut, midgut and hindgut, respectively.

FIGURE 6 - Light microscopy and fluorescence microscopy of various Microcystis (M. flos-aquae, M. aeruginosa and M. wesenbergii) colonies in different gut sections from bighead carp. Panels (a), (b) and (c) show light-microscopic photographs of the foregut, midgut and hindgut, respectively, while panels (d), (e) and (f) are fluorescence-microscopic photographs of the foregut, midgut and hindgut, respectively.

FIGURE 7 - Microcystin contents (a) and physiological components (b) of Microcystis in different gut sections. Bars represent SD of means, n = 9. *, **, *** indicate a significant difference between the foregut and midgut or between the foregut and hindgut at P <0.05, P <0.01 and P <0.001, respectively.

The algal biochemical composition is shown in Fig. 7b. 4. DISCUSSION AND CONCLUSION In silver carp the hindgut showed a significant decrease (p< 0.05) in soluble protein and carbohydrate contents. For big- Because results from reports on the capacity of silver head carp, the carbohydrate contents in the midgut and the carp and bighead carp to control cyanobacteria blooms hindgut were significantly lower (p< 0.05) than that in the have been contradictory, we evaluated several morpholog- foregut, and protein content decreased, albeit to a non-sig- ical, ecological, and photosynthetic indictors to determine nificant level (p> 0.05). how these fish species affect Microcystis blooms (Table 3). Our results suggested that planktivorous filter-feeding fish do indeed have some effects on Microcystis colonies. The photosynthetic activities of algae in fish guts were lower

than those growing in lakes, which could have resulted The nutrient composition of algal residues in different from the decomposition and digestion related to gastroin- gut sections of planktivorous fishes was also analyzed. In testinal microorganisms and digestive enzymes, respec- silver carp the carbohydrate and soluble protein contents tively. were significantly lower in the hindgut than those in the foregut, while there were no significant differences between the The quantity of small-sized Microcystis in the guts of foregut and midgut of the bighead carp (Fig. 7b). This result planktivorous fish was significantly larger compared to suggested that some algal nutrients could be digested and ab- that in lake water. The decreasing Microcystis colony size sorbed by planktivorous fishes, with digestion and absorp- in the foregut can be mainly attributed to the filtering effect tion mainly occurring in the hindgut section for silver carp, of the gill rakers [23] or grinding by the pharyngeal teeth which might be attributed to decreased enzyme activity [24], and the increase in small-sized colonies in the midgut caused by the relatively low pH present in the silver carp and hindgut might be caused by the digestion process [25]. foregut [33]. And this decline of enzyme activity along the Potential photosynthetic activities of Microcystis colonies guts might be the main reason for the slowest recovery of in silver carp gut were analyzed by chlorophyll fluores- Microcystis growth in the foregut. However, for algae in var- cence [26], and the results showed that the digestion pro- ious gut sections of bighead carp, photosynthetic activities cess had almost no effect on the activity of M. aeru- and nutrient contents of the Microcystis residues showed no ginosaand M. wesenbergii while significantly decreasing significant changes. These digestion differences between sil- the activity of M. flos-aquaeas indicated by the observed ver carp and bighead carp might be attributed to the gut decrease in fluorescence intensity and bleached pigment. length (the ratio of gut length and body length was 5.33 and The reason why M. flos-aquae might be more prone to lose 3.93 measured for silver carp and bighead carp, respectively, activity during the digestion process could be because there as reported by Ni & Jiang [23]) and differences in digestive is a higher proportion of bound water as suggested by an enzymes between these two fishes [34]. In addition, these apparently higher level of transparency in the gelatinous two fish species have different community structures of in- envelope of algal colonies as compared to other Micro- testinal microorganisms [35], which might also contribute to cystis species. the above-mentioned digestion differences.

TABLE 3 – Comparison of the efficiency of using filter-feeding fish to control cyanobacterial bloom from the present study and previous studies

Fish species used Algal species Lake name Algal control Main monitoring indicators Reference for algal control monitored or number effect Bacillariophyta Lake Victoria Not effective for Tilapia esculenta Cyanophyta Algal biomass in all fish gut sections [27] (Kenya, Africa) cyanobacteria Chlorophyta Cyanobacteria Silver carp Not effective for Chlorophyta 17 lakes (China) Algal biomass in all fish gut sections [23] Bighead carp cyanobacteria Euglenophyta Microcystis Silver carp Lake Donghu (Cyanophyta) Algal biomass in all fish gut sections Effective [28] Bighead carp (China) Euglena Not effective Silver carp Microcystis Lake Donghu Algal biomass in water column under high algal [25] Bighead carp (Cyanophyta) (China) density Silver carp Microcystis Lake Donghu Algal species and biomass in water Significant [11] Bighead carp (Cyanophyta) (China) column efficiency Silver carp Microcystis Man-made eutrophic Algal count and chlorophyll-a in the Only effective for Bighead carp [29] (Cyanophyta) lake (India) gut content or aquaria silver carp Tilapia esculenta Algal biomass All species in phyto- Villerest Reservoir Silver carp Algal size Not effective [30] plankton community (France) Fish biomass Effective in All species in phyto- Saidenbach Reser- Algal size Silver carp trophical lake [31] plankton community voir (Germany) Algal biomass with bloom Effective for Mi- Enclosures in Silver carp All species in phyto- Algal species and biomass in enclo- crocystis but not Yuqiao reservoir [32] Bighead carp plankton community sures for total algal bio- (China) mass Silver carp Microcystis Algal consumption in gut sections of Only effective Taihu Lake (China) [12] Bighead carp (Cyanophyta) fish for silver carp Algal colony buoyancy Algal colony size Silver carp Microcystis Not significant Present Taihu Lake (China) Algal photosynthesis Bighead carp (Cyanophyta) effective study Re-culture of Microcystis in gut Algal autofluorescence

Chlorophyll fluorescence parameters are widely stud- Although they had no significant removal effects on ied and applied as indicators of photosynthetic activity blooms, the filter-feeding of planktivorous fishes could [36]. The measurements here showed that Microcystis pho- postpone the occurrence of Microcystis blooms for two rea- tosynthesis gradually increased from the foregut to the sons: 1) algae cannot grow well in fish guts due to the lack hindgut in both kinds of planktivorous fish studied here, of light and nutrients as well as exposure to digestive stress; which would appear to contradict the conclusion that some and 2) the up-floating velocity of residual Microcystis de- algae lost metabolic activity in the hindgut of silver carp creased, such that light becomes a limiting factor for ex- (Fig. 5c, f). This contradiction could have two explana- creted Microcystis colonies since they are suspended in the tions. On the one hand, chlorophyll fluorescence measure- water column rather than floating on the water surface. ments are based on the average photosynthetic activity of Therefore, we conclude that, to a certain extent, the filter- single intact algal cells containing chlorophyll [18, 37], and feeding by planktivorous fishes, and in particular silver the average photosynthesis increased due to some Micro- carp, could alter the morphology (i.e. decrease colony cystis species such as M. flos-aquae being bleached and in- size), decrease the up-floating velocity, and inactivate pho- activated after digestion, which results in complete loss of tosynthesis by Microcystis colonies. These changes could autofluorescence while other Microcystis species main- decrease the frequency and intensity of Microcystis tained a high level of photosynthesis. On the other hand, blooms, which may explain the disappearance of blooms in photosynthetic activity might slightly increase due to the eutrophic lakes such as Lake Donghu [11, 24] after stock- decreased digestive enzyme content in the hindgut that is ing with some planktivorous fishes. related to the re-absorption process, which is consistent with previous research [33, 34, 38]. Therefore, some recovery of photosynthetic activity along the foregut to hindgut ACKNOWLEDGEMENTS should be expected. Microcystin contents in various gut sections were de- We thank Mr. Fangtian Cui for his assistance in exper- termined, and the results showed that the microcystin con- imental material collection. This study was jointly sup- tent per gram dry weight decreased significantly in the ported by the National Science Foundation of China midgut and hindgut of silver carp, but increased signifi- (41230748; 31300391), the State Key Laboratory of Fresh- cantly in the midgut and hindgut of bighead carp after di- water Ecology and Biotechnology (2011FBZ15), and the gestion, as compared with those in the foregut. However, Major Science and Technology Program for Water Pollu- some previous reports indicated that filter-feeding by tion Control and Treatment (2012ZX07103-004-02). planktivorous fish led to a decrease in the microcystin content [17]. 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CELLULAR LOCALIZATION OF COPPER AND ITS TOXICITY ON ROOT TIPS OF HORDEUM VULGARE

Junran Wang, Qiuyue Shi, Jinhua Zou, Ze Jiang, Jiayue Wang, Hangfeng Wu, Wusheng Jiang and Donghua Liu*

Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China

ABSTRACT The nucleolus is a nuclear structure, responsible for ri- bosome biogenesis and transcription [8]. It can be selec- Copper (Cu) localization and cell damage in Hordeum tively stained by silver as it contains a set of acidic, non- vulgare root tips under Cu stress by means of electron mi- histone proteins. It has been reported that nucleoproteins, croscopy with energy dispersive X-ray spectrum analysis detected by silver staining and described as “silver-stained (EDXA), propidium iodide (PI), Carbol Fuchsin and silver particulate material”, is extruded into the cytoplasm in the staining and indirect immunofluorescent microscopy were root tip cells of plants exposed to Cu [9–10]. However, lim- investigated. The results indicated that the mitotic index ited information is available about the nature of these sil- (MI) in the root tips decreased significantly by increasing ver-stained particles and the effects of Cu on nucleolus and the concentration of Cu. Cu accumulated in the meristem nucleoproteins, especially what types of nucleoproteins are and disturbed cell division, inducing C-mitosis, anaphase scattered in the nuclei or extruded into the cytoplasm under bridges (two types) and chromosome stickiness, which re- Cu stress. sulted in the inhibition of root growth. Cu had toxic effects Cu toxicity in plants has been known for a long time on nucleoli in root tip cells. It could induce some particles [2, 10–13]. The comprehensive evaluation of toxic effects containing argyrophilic proteins scattered in the nuclei and of Cu on microlocalisation, and cell division, especially on extruded from nucleoli into cytoplasm. Using indirect im- nucleoli in the root tip cells of H. vulgare has not been re- munofluorescent microscopy, we found that the two nucle- ported before. The objective of this study was to try to pro- olar proteins, nucleolin and fibrillarin, were translocated vide direct evidence of Cu toxic effects on H. vulgare root from nucleolus to nucleoplasm and cytoplasm. Data from tips under Cu stress from Cu localization and cell damage EDXA showed that the mature, elongation and meristem (cell mitosis and nucleoli) aspects by means of electron mi- zones in the root tips could absorb Cu ions. The Cu level in croscopy with EDXA, PI, Carbol Fuchsin and silver stain- meristem zone in the root tips was higher, suggesting that ing and indirect immunofluorescent microscopy. it could be considered as a primary target of Cu toxicity.

There was a clear correlation between Cu content and cell damage in root tips of H. vulgare. The degree of cell dam- 2. MATERIALS AND METHODS age increased with prolonged period of treatment. 2.1 Culture condition and copper treatment

KEYWORDS: Hordeum vulgare; copper (Cu); Cd-localization; nu- Healthy seeds and equally-sized seeds of H. vulgare cleoprotein; cell division; cell damage were collected and used for the present investigation. Be- fore starting the experiments, they were soaked in distilled water for 24 h, and then germinated in moistened gauze in 1. INTRODUCTION the dark at 23 for 12 h. The seedlings were grown in plastic containers at 23 for 24 h, producing roots reach- Plants are very sensitive to Cu both the deficiency and ing about 0.6 cm length. Fifteen seedlings each treatment the excess [1]. High Cu levels in soil can be phytotoxic, were selected and grew in a growth chamber under con- causing deleterious effects both morphologically and phys- trolled climatic conditions: 15 h light / 9 h dark at 23-25 ºC iologically [2–5]. Root tip systems of various plants have diurnal cycle, 80% relative humidity. They were treated been used for the assessment. This is because the root tips with the different concentrations of Cu solutions (1, 10 and are often the first to be exposed to chemicals spread in na- 100 μM) for 24, 48 and 72 h. The test liquids were renewed ture in soil and water [6]. H. vulgare is well known as an regularly every 24 h. Cu was provided as copper sulfate excellent model plant and a useful biomarker for the detec- (CuSO4 · 5H2O) manufactured by Sigma. Cu stock solution of heavy metal pollution in the laboratories [7]. tion was prepared in distilled water. The solutions were aerated by pump, which connected to the containers with * Corresponding author pump lines. Control seedlings were grown in distilled wa-

ter alone. The length of roots were observed, measured and with 4% (w/v) paraformaldehyde in phosphate-buffered recorded at the end of each time interval (24 h). All treat- saline (PBS, pH 7.0) for 1.5 h in darkness at room temper- ments were done in three replicates. ature and then they were washed with the same buffer. The methods are described in more detail [17]. 2.2 Carbol Fuchsin-staining and silver-staining Twenty root tips in each treatment group were cut 2.6 Statistical analysis every 24 h, respectively. They were fixed in 95% ethanol Each treatment was replicated 3 times for statistical va- and acetic acid (3:2) for 1 h and hydrolyzed in 1 M hydro- lidity. SPSS computer software was used for statistical chloric acid, 95% ethanol and 99.8% acetic acid (5:3:2) for analyses (SPSS Japan Inc., Shibuya, Tokyo, Japan) and 5 min at 60 °C. Then the tips were squashed in Carbol SigmaPlot 8.0 software was used for mapping. Any differ- Fuchsin solution [14] for counting the cell mitosis and ob- ences between treatments were determined using one-way serving the variation of chromosome. For the observation analysis of variance (ANOVA) and t-test was used to de- of changes in nucleolus, the root tips were squashed in 45% termine the significance at P < 0.05. acetic acid and stained with silver nitrate after the slide was dry [9]. 3. RESULTS In order to obtain mitotic index (MI), approximately 3,000 cells (about 1,000 cells each slide) were observed 3.1 Cellular localization of Cu each treatment group at the end of each time interval. The evidence from EDXA revealed cellular localiza- 2.3 Propidium iodide staining tion of Cu in the root tips, as well as Wt% of Cu in localization sites in H. vulgare exposed to 100 μM Cu for 48 h To visualize the damaged root tip cells of H. vulgare (Fig. 1). The levels of Cu on the surfaces of the different induced by Cu, the intact root tips from the seedlings ex- regions, mature zone, elongation and meristem zone, were posed to 100 μM Cu for 1, 3, 6, 12, 24 and 48 h were 1.54, 3.79, and 1.80 Wt%, respectively (Fig. 1A–C), which stained with PI (PI, P4170; Sigma, Sigma-Aldrich, Buchs, exhibited that elongation zone was one of Cu main accu- Switzerland), according to Koyama et al. [15] as modified mulation and poisoning sites. In transverse section of the from Jones and Senft [16]. PI provides red fluorescence of mature zone, the Cu content in epidermal cells of the root nuclei in damaged cells, as PI has very low penetrability tip was the lowest (1.2 Wt%) when compared with cortical across intact membranes. The fluorescence density was cells (2.13 Wt%) and vascular cylinder cells in the center calculated using “Analyse and Measure” function of the of roots (1.54 Wt%) (Fig. 1D–F). EDXA spectra revealed Image J software (NIH, Bethesda, MD, USA) to analyze that Cu was found in all cortical cells after Cu stress. Dis- distribution in the intact root tips of H. vulgare seedlings tribution of Cu in these cells was detected in both cyto- under Cu stress. plasm and cell walls. The vascular tissues were also storage sites of Cu, where they were mainly located in the cell 2.4 Sample preparation for scanning electron microscope walls. Data from transverse section of the meristem zone Elemental distribution and composition of experi- (Fig. 2 A–D) showed a low Cu accumulation in the proto- mental plants was determined from samples of freeze-dried derm cells (0.8 Wt%) and high contents in internal cell of root materials. Ten root samples of 1 cm length were cut meristem such as, ground meristem (5.41 Wt%) and pro- from the root tips of H. vulgare exposed to 100 μM Cu for cambium (4.02 Wt%). From a transverse section point of 48 h and rapidly frozen in liquid nitrogen and lyophilized. view, the Cu level in meristem zone was high in compari- Cross-sections of the roots (about 1 mm) were coated by son with mature zone (Fig. 1D–F; Fig. 2A–D). gold using the sputter/coater (EMITECH K550X, Quorum Group, England). The energy dispersive X-ray microana- 3.2 Effects of Cu on cell damage lytical studies were carried out using scanning electron mi- To visualize damaged root tip cells induced by Cu, the croscopy (FEI Nova NANOSEM 230, Oregon, USA) pro- root tip cells of H. vulgare exposed to 100 µM Cu for 0, 1, vided with EDXA (Genesis Apollo 10, EDAX, USA). 3, 6, 12, 24 and 48 h were stained with PI. Red fluorescence EDAX spectra were collected by 20 KV and X-ray detector is an indicator of cell damage, as PI enters cells. The toxic equipped with a super ultra thin window. The collection effects of Cu on cell damage in root tips of H. vulgare var- time of spectra was 30-40s. The Cu composition in the ied with the different treatment times (Fig. 3). Weak fluo- roots was showed by wt% (weight percent), which means rescence was observed in control roots (Fig. 3A), suggest- that concentration percent on the basis of weight (or mass) ing there were some cell damages in root tips during the of a particular element relative to the total elements weight plant growth period, although the root tip cells without Cu (or mass). treatment. The low level of fluorescence intensity was observed in the cells after 1 h Cu treatment, indicating that Cu 2.5 Indirect immunofluorescent microscopy could induce the cell damages as early as 1 h Cu exposure For the visualization of nucleolin and fibrillarin, meri- (Fig. 3B). The fluorescence intensity was more pronounced stematic zones of root tips from control and seedlings of H. with prolonged exposure (Fig. 3B-G). Data from the fluo- vulgare treated with Cu 100 μM 48 h were cut and fixed rescence density analysis by Image J software could con-

FIGURE 1 - EDXA spectrum taken from the site of the SEM micrographs, showing Cu localization in different zones of root tip cells in H. vulgare exposed to 100 µM Cu for 48 h. A–C. Showing Cu localization on the surfaces of elongation, meristem and mature zones. A. Mature zone. B. Elongation zone. C. Meristem zone. D–F. Showing transverse section of the mature zone. D. Epidermal cells. E. Cortical cell wall. F. Vessel cells. Red “*”site of the analysis.

FIGURE 2 - EDXA spectrum taken from the site of the SEM micrographs, Cu localization in meristem zone in root tip cells of H. vulgare exposed to 100 µM Cu for 48 h. A. Showing transverse section of meristem zone. B. Protoderm cells. C. Ground meristem. D. Procambium. Red “*”site of the analysis.

FIGURE 3 - Micrographs of roots from H. vulgare exposed to 100 µM Cu using PI. Red fluorescence due to PI entering cells is an indicator of cell damage, showing status of cell death of the roots exposed to 100 μM Cu for 0, 1, 3, 6, 12, 24 and 48 h, respectively. Scale bar=200 µm

g chromosome bridges were noticed. First, anaphase bridges 25 100 μM involved one or more chromosomes (Fig. 5B-D). Second, sticky bridges were observed only after 48 h treatment with 20 f 10 to 100 µM Cu (Fig. 5E-F). Chromosome stickiness e arises from improper folding of the chromosome fiber into single chromatids and chromosomes become attached to d 15 each other by subchromatid bridges. The frequency of cells with chromosome stickiness progressively increased with

10 c increasing Cu concentration and prolonged duration of b treatment. Two types of chromosome stickiness were observed. First, sticky chromosomes at anaphases were no- 5 ticed in the root tips exposed to higher concentrations of Fluorescence density (rel) a Cu (mainly at above 10 µM) (Fig. 5G). Second, the sticky

0 chromosomes at metaphases (Fig. 5H) in root tips exposed 0 1 3 6 12 24 48 to 10 and 100 µM Cu. Besides, chromosome fragments were also noted in some root tip cells. Treatment time (h) FIGURE 4 - PI fluorescence density in the intact roots exposed to 3.5 Effects of Cu on nucleoli 100 μM Cu for 0, 1, 3, 6, 12, 24 and 48 h treatment time. Normally, the diploid nucleus of H. vulgare contains one or two nucleoli (Fig. 6A). The effects of Cu on nucleoli firm the findings (Fig. 4). The cell damage increased sig- varied with the different concentrations of Cu solutions used. nificantly with prolonged treatment time (P < 0.05). Three phenomena were observed after Cu treatments. Firstly, nucleoli in the root tips exposed to 1 µM Cu for 48 h 3.3 Effects of Cu on mitotic index or 10 µM Cu for 24 h were irregular (Fig. 6B). Big swollen The MI values for each group were given in Table 1. nucleolus occupied large areas of the nucleus was also The MI rate decreased significantly (P < 0.05) with in- noted (Fig. 6C-D). Secondly, some tiny silver-stained parti- creasing concentrations of Cu and prolonged treatment cles were observed together with the main nucleolus/nucle- time when compared with control. This fited well with the oli in the nucleus of some root-tip cells exposed to 10 µM below mentioned effects of Cu on root growth. The MI Cu for 48 h or 100 µM Cu for 24 h (Fig. 6C-D), and the could be correlated with the rate of root growth, suggesting amount of the particulates increased progressively (Fig. that the inhibition of root growth resulted from inhibition 6E-F). Thirdly, nucleoli were aggregated into irregular of the cell division. shapes and nearly was occupied the whole nucleus after increasing Cu concentration and prolonged treatment time. TABLE 1 - Effects of Cu on mitotic index (%) in the root tip cells of Some silver-stained particles were leached out from the nu- H. vulgare cleus to the cytoplasm after 10 µM Cu for 72 h or 100 µM Cu for 48 h (Fig. 6G-I). Treatment Treatment (μM） Time (h) Control 1 10 100 3.6 Effects of Cu on nucleoproteins 24 23.81±0.63a 20.28±0.34b 9.07±1.35c 7.52±1.97d In order to investigate, in more detail, the localization of 48 19.92±0.47a 16.02±0.92b 8.71±0.36c 6.77±0.33d nucleoproeins in the root tip cells after Cu stress, immunofluorescence localizations of nucleolin and fibrillarin were 72 14.29±0.47a 13.22±0.87a 7.33±0.22b 5.05±0.04c carried out in the present investigation. The antibodies used could produce positive reactions with two nucleolar proteins 3.4 Effects of Cu on chromosome morphology above. Obvious toxic effects on the two nucleoproteins in The mitotic anomalies in root tips of H. vulgare ex- the root tip cells of H. vulgare treated with 100 µM Cu in posed to different Cu concentrations were shown in Fig. 5. comparison with control cells. Nucleolin was marked with The mitotic preparation from root tips of Cu treated seeds FITC and produced green fluorescent signal under confo- exhibited several chromosomal abnormalities such as C- cal microscopy. Nucleolin in control cells of H. vulgare mitosis, chromosome bridges and chromosome stickiness. was localized in the nucleolus (Fig. 7A1–A4). On treat- C-mitosis was observed in the root tip cells of all treated ment with 100 μM Cu for 24 h, it migrated from the nucle- groups after exposure to Cu. The severely condensed chro- olus to the nucleoplasm (Fig. 7B1–B4), and on the way mosomes were randomly scattered in the cell (Fig. 5A). Cu from nucleoplasm to cytoplasm (48 h) (Fig. 7C1–C4). Pro- had C-mitotic effects as its main effects at low dose (1 µM) longing the treatment time (72 h), the nucleolin were scat- for 24 h treatment. Chromosome bridges at anaphase were tered in the cytoplasm (Fig. 7D1–D4). Fibrillarin was also due either to breaks in chromosomes or chromatids (often observed using the green fluorescent signal. The intracel- resulting in fragments) or to chromosome stickiness (dis- lular distributions of fibrillarin were very similar to nucle- turbing the normal cell division). In the roots treated with olin using confocal laser scanning microscopy. Fibrillarin Cu studied, anaphase bridges were observed. Two types of is normally present only in the nucleolus in untreated con-

FIGURE 5 - The effects of Cu on chromosomal morphology in root tip cells of H. vulgare. A. C-metaphase (1 μM Cu, 24 h). B–D. Chromosome bridges (10 μM Cu, 24 h). E–F. Sticky chromosome bridges (10 μM Cu, 24 and 48 h). G. Sticky chromosome at anaphase (10 μM Cu, 72 h). H. Sticky chromosome at metaphase (100 μM Cu, 48 h). Scale bar=10 μm.

FIGURE 6 - Effects of Cu on nucleoli in root tip cells of H. vulgare. A. Control cell. B. Showing irregular nucleoli. B. Showing swollen nucleolus occupied large areas of the nucleus. C–D. Showing big swollen nucleolus, which occupied large areas of the nucleus. E–F. Showing silver- stained particles together with the nucleolus scattered in the nucleus. G-I. Showing silver-stained materials extruded from the nucleus into the cytoplasm, the particulates aggregated into irregular shapes and nearly occupied the whole nucleus. Scale bar=10 μm.

FIGURE 7 - Simultaneous detection of nucleolin after incubation with primary anti-nucleolin antibody and secondary antibody conjugated with FITC (green), and of DNA after incubation with DAPI (blue) in the same single optical section using confocal microscopy. (A1,B1,C1,D1) Nucleolin detection; (A2,B2,C2,D2) DNA detection; (A3,B3,C3,D3) Merged image of “nucleolin detection” and “DNA detection”; (A4,B4,C4,D4) bright field image. (A1–A4) Nucleolin in nucleolus of control cells; (B1–B4) The migration of nucleolin from the nucleolus to the nucleoplasm in cells treated with 100 μM Cu for 24 h; (C1–C4) Nucleolin on the way from nucleoplasm to cytoplasm in the cytoplasm of cells treated with 100 μM Cu for 48 h. (D1–D4). Nucleolin scatted in the cytoplasm. Scale bar=10 μm.

trol cells of H. vulgare (Fig. 8A1–A4). In comparison with 3.7 Effects of Cu on growth control cells, it was transferred from nucleolus to nucleo- Effects of Cu on root growth of H. vulgare varied with plasm (Fig. 8B1–B3). Fig. 8C1–C3 it was showed on the the different concentrations of Cu used (Fig. 9A). The root way from nucleoplasm to cytoplasm. Finally fibrillarin was growth per day decreased with increasing Cu concentration scattered in the cytoplasm in some cells (Fig. 8D1–D3). and prolonged treatment time. Cu inhibited the root growth significantly (P < 0.05) at all concentrations (1 to 100 µM Cu) during the entire treatment (72 h) in comparison with

control. At 10 and 100 µM Cu, the root length was strongly The effects of Cu on the morphology of the roots of H. inhibited after 24 h of treatment and there was no any root vulgare also varied with the different Cu concentrations growth at 10 and 100 μM Cu during the whole period of (Fig. 9B). At 1 μM Cu, the morphology of the roots was treatment. more or less the same as control during the whole treatment (72 h). At 10 to 100 μM Cu, the root tips were browning, twisting, stunted and reduction of lateral roots.

FIGURE 8 - Simultaneous detection of fibrillarin after incubation with primary anti-fibrillarin antibody and secondary antibody conjugated with FITC (green), and of DNA after incubation with DAPI (blue) in the same single optical section using confocal microscopy. (A1,B1,C1,D1) Fibrillarin detection; (A2,B2,C2,D2) DNA detection; (A3,B3,C3,D3) Merged image of “fibrillarin detection” and “DNA detection”; (A4,B4,C4,D4) bright field image. (A1–A4) Fibrillarin in the nucleolus of control cells; (B1–B4) Migration of fibrillarin from the nucleolus to the nucleoplasm. (C1–C4) Fibrillarin on the way from nucleoplasm to cytoplasm. (D1–D4) Fibrillarin scattered in the cytoplasm. Scale bar=10 μm.

Control 6 A 1 ¦ÌM 10 ¦Ì¦¬ 100 ¦ÌM a 5 a 4 b 3 a b 2

Root length (cm) b c 1 a aaa c d c d d

0 02448 72 Time (h)

FIGURE 9 - Effects of Cu on root and seedling growth of H. vulgare. A. Root length. B. Seedling growth. Vertical bars denote SE. Values with different letters differ significantly from each other (n = 10, P < 0.05).

4. DISCUSSION to metal interference with cell division, including induce- ment of chromosomal aberrations and abnormal mitosis Cu is an essential nutrient for plants and plays an irre- [24–26]. Chromosomal aberrations are changes in chromo- placeable role in the function of a large number of enzymes some structure resulting from a break or exchange of chro- catalyzing oxidative reactions in a variety of metabolic mosomal material. Based on the results of the present in- pathways [18]. However, it can induce severe damage to vestigation, it is quite clear that Cu is capable of inducing plant growth and development when absorbed in large such cytological abnormalities as c-mitosis, bridges, stick- amounts [19]. The root is the most sensitive and accessible iness, fragments, etc. The frequency of abnormalities in- part of the plant to Cu stress. Inhibition of root growth is creases, in most cases, as the concentration of the metal in- typical symptoms of Cu toxicity. Barley seeds grown in creases. The results from the present investigation are in different concentrations of Cu showed a significant de- agreement with the findings by Agarwal et al. [27], Jiang crease of root growth and mitotic index and higher number et al. [28] and Liu et al. [29], but with some differences. 1) of chromosomal abnormalities. The MI, characterized by Chromosome bridges exhibiting stickiness were observed the total number of dividing cells in cell cycle, has been in the treatment with 10 Cu µM for 48 h. 2) Anaphase con- used as a parameter to assess the cytotoxicity of heavy met- figuration with chromosome bridges exhibiting disintegra- als in monitoring environmental pollution [20]. In the pre- tion was found only after 72 h of treatment with 100 µM sent study, we determined that the MI in root tip of barley Cu. The chromosomal bridge aberrations and chromosome decreased significantly by increasing the concentration of stickiness appeared in the root tips exposed to higher con- Cu (see Table 1). The mitotic cells decreased in the root centration of Cu (100 µM) are lethal and probably lead to tips of H. vulgare, indicating that Cu was accumulated in cell death. C-mitosis more often observed in low concen- the meristem where it disturbed cell division. Thus, it is tration of Cu, in which the chromosomes spread irregularly clearly that the inhibition of the root growth resulted from over the cell, may be due to disturbance of the spindle ap- inhibiting the cell division of root tips. This fits well with paratus. This kind of chromosomal aberration reflects the above mentioned effects of Cu on root growth. slight or moderate cytological toxicity. Chromosome aberrations have been used as a measure The nucleolus is well known as the site of transcription of reproductive success and as a method for the detection of ribosomal genes and further transcript process [30], of possible genetic damage by environmental agents (such which also contains a set of acidic, nonhistone proteins that as herbicides, insecticides, fungicides and heavy metals in bind silver ions and are selectively visualized by silver plant for many years [21]. Plant chromosomes are easy to methods. It is well known that silver impregnation is re- analyze in terms of size, morphology and number, and regarded as a specific stain of the nucleolus. To better under- spond to treatments with toxins at an early stage [22]. stand the nucleolar cycle and the nucleolar organization, Chromosomal aberrations and proportion of mitotic phases the silver staining technique has been widely used in cyto- can be accepted as indicators of genetic damage induced logical studies in both animals and plants. The results in by stress conditions and the anaphase–telophase chromo- this work showed that Cu could induce toxic effects on nu- some aberration assay has been shown to be highly reliable cleoli, causing some particulate silver-stained material in genotoxicity testing [23]. Several authors reported that containing the argyrophilic proteins scattered in the nuclei the inhibition of root elongation caused by Cu may be due and some particles extruded from the nucleus into the cy-

toplasm. The results here are similar to those under Cu EDXA following freezing of the plant sample has been stress described by Liu et al. [31] for the effects Cu on the employed systematically in biological sciences to localize nucleolus of Allium cepa root tip cells, Jiang et al [25] for ions in cellular compartments, to monitor ion distribution Cu on Helianthus annuus, Jiang et al. [28] for Cu on Zea changes during physiological processes, to determine the mays and Liu et al. [29] for Cu on Allium sativum, but with behavior of toxic ions incorporated by cells, and thus can a few differences. (1) There are not so many nucleolar par- provide information on mechanisms of metal toxicity and ticles containing the argyrophilic proteins scattered in the tolerance [43–45]. EDXA was used to determine Cu local- nuclei and large amount of the materials extruded from the ization in root tips of H. vulgare exposed to Cu in this nucleus into the cytoplasm in the present study, when com- study. The results here can be summarized as follows. 1) pared with the findings of Liu et al. [29, 31]. (2) Cu has The three zones, mature, elongation and meristem zone can weak toxic effect on nucleolus in root tip cells of H. annuus absorb Cu ions. 2) In mature zone, root hairs are found only and Z. mays [25, 28], where the nucleolar material is only in the region of maturation of the root, and they are out- scattered in the nucleus, and not extruded from the nucleus growths at a tip of the plants roots. They have a large sur- into the cytoplasm when compared with the results here. face area, which makes absorbing both water and minerals Thus, the toxicity of Cu on the nucleoli varied with differ- more efficient using osmosis. Once Cu enters the roots, it ent species plants. The phenomenon Cu induced in the pre- easily penetrates the root through the cortical tissue and is sent investigation is in line with the previous findings, in- translocated to the above-ground tissues. This can ex- dicating that besides Cu some other metal ions can affect plained why the level of Cu in mature zone is low when toxic effects on nucleolus in the same way, such as alumi- compared to meristem zone. 3) From a transverse section num (Al) [32], cadmium (Cd) [17] and lead (Pb) [20, 33]. point of view, the Cu level in meristem zone is high in com- Some reports indicated that the extrusion of nucleolar mate- parison with mature zone (Fig. 1D–F; Fig. 2A–D), suggest- rial containing argyrophilic proteins from the nucleus into ing that root tip meristem of plants is a primary target of the cytoplasm may be correlated with damage to the nuclear Cu accumulation and toxicity. Meristem cells are small and pore complex (NPC) proteins, causing the NPC to lose se- have thin walls without differentiation. These cells have lectivity [34]. However, the mechanism needs to be studied not yet differentiated and have poor transport capacity up. further. Although there are some reports concerning Cu toxic Thus, excess Cu can easily induce damage of cell construc- effects on nucleolus in plant root tips, they hardly indicated tion. Evidence here shows Cu toxic effects on meristem what kinds of proteins involved in those reports. cells under Cu stress, causing obviously toxic characteristics, such as low MI, chromosome aberrations, damage of The nucleolus has long been known playing important nucleolus and production of large damaged cells. The re- roles in the regulation of many fundamental cellular pro- sults also confirmed indirectly the previous results reported cesses [35–36]. It is well known that the nucleolus contains by Liu et al. [26] where they found that excess Cu could several kinds of proteins. Nucleolin and fibrillarin are ma- cause adverse effects on garlic meristem cells after 1 h treat- jor nucleolar proteins conserved in all eukaryotic organ- ment with Cu, increasing dictyosome vesicles in number, isms [37]. They are multifunctional proteins involved in forming of cytoplasmic vesicles containing electron dense different cellular aspects like chromatin organization and granules, altering mitochondrial shape, disrupting nuclear stability, DNA and RNA metabolism, assembly of ribo- membranes ect. Due to Cu toxicity on meristem, the cell nucleoprotein complexes, cytokinesis, cell proliferation division was affected, leading to the mitotic cells decreased and stress response [37, 38]. Evidence obtained using in- and interphase cells increased. direct immunofluorescent microscopy in the present investigation revealed that the two nucleolar proteins, nu- PI, an intercalating agent and a fluorescent molecule cleolin and fibrillarin were localized in nucleoli in control with a small molecular mass of 668.4 Da that can be used groups. However, under Cu stress the two nucleolar pro- to stain DNA, has been considered as a alternative agent to teins presented abnormal nucleoplasm and cytoplasmic study cell membrane damage [46]. The amount of PI enter- location in the nucleoli of H. vulgare root tips, which con- ing into cells reflects the degree of cell membrane damage, firmed the observation with the silver impregnation stud- and morphological changes in the cells may be related to ies here. Recent investigations indicated that heavy met- damage to the integrity of the cell membrane [47]. Here, als such as Cd and Pb could induce nucleolar proteins, we can confirm that Cu has toxic effect on the cell mem- nucleophosmin, nucleolin, and fibrillarin migrated from brane damage in the root tip cells of H. vulgare using PI nucleolus to nucleoplasm and cytoplasm in root tip cells staining. The cell damages were observed mainly in the of Vicia faba [17] and Allium cepa [20]. Qin et al. [39, meristem and elongation zone of root tip exposed to Cu for 40] and Zhang et al. [41] also reported that Al had the 1 h (Fig. 3). The data from PI staining here are in good same toxic effects on nucleolus in root tip cells of V. faba agreement with the evidence obtained from EDXA in the and H. vulgare. These observations are in good agreement roots of H. vulgare under Cu stress, indicating that there is with the findings here, suggesting that nucleolus is a cen- a clear correlation between Cu content and cell damage in ter to sense stress and can coordinate the stress response. root tips of H. vulgare. At the cellular level, Cu toxic ef- Thus nucleolar morphology, composition and function fects at excess Cu concentrations is oxidative stress caused will change upon stress [42]. by the increased concentration of reactive oxygen species

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EVALUATION OF STABILITY AND MATURITY PARAMETERS IN WASTEWATER SLUDGE COMPOSTING WITH DIFFERENT AERATION STRATEGIES AND A MIXTURE OF GREEN PLANT WASTES AS BULKING AGENT

Amir Hossein Nafez1,*, Mahnaz Nikaeen1, Bijan Bina2, Akbar Hassanzadeh3 and Sharareh Moghim4

1Department of Environmental Health Engineering, School of Public Health, Kermanshah University of Medical Sciences, Kermanshah, Iran 2Department of Environmental Health Engineering and Environment Research Center, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran 3Department of Statistic and Epidemiology, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran 4Department of Medical Microbiology and Virology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

ABSTRACT and high efficiency processes for recycling of this waste as an alternative to dumping and incineration [4]. WS is a The aim of this study was to investigate the influence good candidate for composting and subsequently for agri- of bulking agent volumetric ratio on the stability and ma- cultural purposes since its pH is neutral to slightly alkaline turity parameters in composting process of anaerobically and its organic matter (OM) can vary from 50% to 70% of digested sludge (ADS) with different aeration strategies. the total solids content and rich in both phosphorus and ni- Two main influencing factors including aeration method trogen [4-6]. Composting of WS is a biological process (aerated static pile and windrow process) and proportion of which converts OM to stabilized humic substances through green plant wastes (GPW) and ADS (1:1, 2:1 and 3:1, GPW: mineralization with a significant reduction (40-50%) in vol- ADS) were investigated through the evaluation of physico- ume [4, 7]. In composting process pathogens can be killed chemical and biological parameters during the 83 days of by heat generated in the thermophilic phase [7]. Further- composting. Heavy metals concentration, germination in- more, final product of composting process could act as a dex (GI), and microbiological indicators were also deter- soil conditioner or fertilizer. mined in the final products. The results showed that wind- However, the high heavy metal content of WS com- row method was superior to the aerated static piles, since post is a major limitation for land application of this end the former enhances the composting process by favoring product. WS also has a compact structure because of its high organic matter (OM) stabilization. Based on these results it moisture content even if a dewatering treatment is used [1- appears that windrow method with 2:1 mixture of GPW: 2, 5]. Many studies have verified that WS alone generates ADS was best for ADS composting due to the highest OM poor quality compost [1, 4, 8]. Due to the high moisture degradation ability and maximum GI in the final product. content and low C/N ratio of WS they need to be mixed

with some materials as bulking agents which absorb the

moisture and provide sufficient porosity and air permeabil-

KEYWORDS: Compost, Green plant wastes, Wastewater sludge, ity for composting of WS [4, 5, 9]. A long list of waste Static pile composting, Windrow composting materials (e.g. municipal refuses, agricultural residues, animal manures and forest wastes) have been proposed as bulking agents in several studies [1, 2, 10, 11], but the most widely used materials are fibrous carbonaceous and ligno- 1. INTRODUCTION cellulosic materials with low moisture content such as wood chips, straw and sawdust. These lignocellulosic materials Wastewater sludge (WS) is a by-product and insoluble have also the ability to uptake heavy metals and reduce residue remaining following wastewater treatment. Inap- heavy metals availability. propriate management of WS could lead to major public However, the type of bulking agent and the volume ra- health and environmental concerns due to high amounts of tio of WS to bulking agent could strongly influence the pathogens and heavy metals [1-3]. With the increased pro- property of composting process and mineralization [5]. For duction of WS, there is a great need to develop low cost instance, Chazirakis et al. [12] investigated the effects of different mixture ratios of sewage sludge, organic fraction * Corresponding author of municipal solid waste (OFMSW) and yard trimmings

(YT) on the performance of windrow composting process. plant (Isfahan, Iran). The green plant wastes (a mixture of Their results showed that the 3:1:2 mixture of OFMSW: grass clippings, tree leaves, foliage and small twigs) were SS: YT was the most beneficial in composting. Using a used as a bulking agent in this study. Physico-chemical forced-aeration static pile composting process, Luo et al. characteristics of the feedstocks and initial characteristics [13] reported that increasing volumetric mixing ratio of of the mixture ratios (1:1, 2:1 and 3:1, GPW: ADS) are bulking agent to sewage sludge in compost piles improved shown in Table 1. the temperature rising rate and shortened the composting cycles. However, due to the feedstock differences as well 2.1.2. Pilot specifications as the extensive diversity of composting situation (e.g. fa- Composting experiments were carried out at the uncov- cility-scale, aeration schemes, temperature, pH and mois- ered composting platform. In order to survey the conditions ture content), the results of these studies are often incom- of ADS composting, 3 piles were prepared by blending the parable. Therefore, composting process and compost prod- GPW with ADS at different ratios (v/v: based on the raw uct quality should be evaluated in different areas. Compost matter) in two trials including aerated static pile (ASP) and quality is closely related to its stability and maturity. Sev- windrow (W) process. GPW and ADS mixed thoroughly eral physico-chemical and biological parameters such as with a front-end loader to obtain GPW: ADS ratios of 1:1 temperature, OM reduction, nitrogen compounds evolu- (ASP1, W1), 2:1 (ASP2, W2) and 3:1 (ASP3, W3). A pile tion, C/N ratio, specific oxygen uptake rate, and germina- containing ADS without any addition (ASP0, W0) was also tion index (GI) have been proposed for evaluating compost used as a control in both trials. The approximate dimen- stability and maturity [1, 3-5, 14]. sions of the piles were as follows: width: 2.5 m; height: The green plant wastes (GPW) such as grass and leaves 1.25 m; length: 3–4 m and they were trapezoidal in shape. are lignocellulosic materials which could be used as readily In the ASP, a perforated plastic pipe was laid under the available and low-cost bulking agents in composting of composting piles and blowers were employed to supply air WS. However, up to now, a small number of researches at a rate of about 0.5 L/kgOM.min throughout the compost- have been considered the application of GPW for composting process. For the windrows, mechanical turning used for ing of WS [15, 16]. Furthermore, in many studies, com- aeration and the piles were turned every 7-10 days during posting process of WS was performed in windrows or re- the composting process with a front-end loader. actors, and little information is available about the aerated static pile composting of WS [11, 13, 17]. The objective of 2.2. Physicochemical analysis and respirometric activity this study was to evaluate the feasibility of composting of Samples were collected weekly from the piles for ana- dewatered anaerobically digested sewage sludge (ADS) for lyzing of physico-chemical parameters and respirometric sustainable management of these organic wastes in a semi- activity. Each composting sample consisted of three sub- arid area. Therefore, this study was performed to: 1) com- samples collected from different points of each pile to en- pare the performance of two aeration methods for compost- sure representative sampling. For measurement of temper- ing of ADS, 2) determine the influence of using different ature, rod thermometer was inserted into the middle of each ratios of GPW as a bulking agent on the performance of the pile at different locations and from different directions. pH composting process through evaluation of common stabil- of compost samples was measured in a 1:5 slurry (w: v) ity and maturity indices, and 3) evaluate the quality of final using a pH meter (Eutech pH1500, Singapore). The mois- products. ture content was determined by oven-drying of compost

samples at 70±5°C for 24 h [18]. The organic matter con-

tent was measured as volatile solids (VS) at 550°C for 2 h 2. MATERIALS AND METHODS in a muffle furnace and the carbon content (%C) was calculated from the ash content, according to the following 2.1. Experiment establishment formula [19, 20]: 2.1.1. Composting materials The dewatered anaerobically digested sewage sludge %C = (100-%Ash) / 1.8 was obtained from the South Isfahan wastewater treatment

TABLE 1 - Physicochemical parameters for ADS and GPW, and initial characteristics of the mixture ratios (1:1, 2:1 and 3:1, GPW: ADS)

Parameter ADS GPW 1:1 mixture 2:1 mixture 3:1 mixture Moisture content (%) 77.8±3.1 51.5±5.2 64.7 ± 4.2 60.3±4.7 58.0±3.8 7.2±77.4 4.5± 70.6 9.1±63.8 15.4±85.3 3.9±52.8 (٭OM content (%DW pH 7.6±0.4 - 7.6±0.2 7.6±0.3 7.5±0.2 Organic carbon (%DW) 29.3±7.9 47.4±6.8 35.4±5.9 39.2±4.2 43.0±4.1 Total nitrogen (%DW) 2.50±0.29 0.50±0.07 2.14±0.18 1.40±0.25 1.04±0.13 C/N ratio 11.7±0.6 94.8±8.5 16.6±5.3 28.0±2.6 41.3±3.9 dry weight٭

Total Kjeldahl nitrogen (TKN), ammonium nitrogen windrow and static pile, respectively. This can be ex- ( -N) and nitrates nitrogen ( -N) contents were as- plained by higher porosity and improved moisture of these sayed as described by the US Department of Agriculture piles as a consequence of the higher proportion of bulking and US Composting Council (2001) [18], and their concen- agent [5]. The control piles, without bulking agent, did not trations determined by DR5000 Spectrophotometer (Hach show any significant variation in temperature. Moreover, Co. USA). Respirometric activity was determined by spe- the temperature of 1:1 piles not only increased slowly but cific oxygen uptake rate (SOUR) of the compost samples. also decreased rapidly which could be explained by an in- The maximum rate of oxygen uptake was measured using hibition effect on microorganism’s activity caused by high a dissolved oxygen sensor (5740sc Galvanic Membrane, moisture content. Faster increase of the temperature and Hach Company, USA) in an aqueous suspension of 3-8 g longer periods of thermophilic phase for the mixtures with compost (wet weight) in 500 ml distilled water, at 30°C. higher ratios of GPW reflect the greater content of easily SOUR was determined from the slope of oxygen level de- biodegradable C-fractions in 2:1 and 3:1 mixtures and also crease according to the method described by Lasaridi and indicates high capability of GPW for preserving heat. Stentiford (1998) [14]. These results confirm the hypothesis that the self-heating ability is directly associated to the feedstocks characteris- 2.3. Final compost quality assessment tics [4, 12]. The duration of thermophilic phase also de- To assess the final compost quality, analysis of micro- pends on the chemical composition of feedstocks [4]. The bial indicators, heavy metals content and cress seed germi- temperature of 2:1 and 3:1 mixtures exceeded 55°C on day nation were performed on samples were taken from each 13-19 and lasted for more than 15 days which be enough to pile at the end of the composting process (day 83). Micro- ensure sanitization of composted materials [1]. bial analyses including fecal coliforms and Salmonella were carried out according to Standard methods [21], and Comparison of Fig. 1a and 1b shows that the tempera- results expressed on a dry mass basis. In order to determine ture of the W piles not only increased faster, but also lasted the available fraction of heavy metals including Cd, Cu, Pb, longer time in comparison to the static piles. Therefore, as Cr, Ni, and Zn in final compost, the water-soluble fraction well as the feedstock composition, the aeration method also of heavy metals was digested and determined by flame has a considerable effect on duration of thermophilic phase atomic absorption spectrometry (Varian SpectrAA220FS, in the composting process. By days 47–54, the temperature Australia). The seed germination bioassay was evaluated in piles decreased gradually, reaching a second mesophilic in the final samples according to the method of Tiquia et phase and maturation process took place during the last al. [22]. stage of composting. At the end of process, after 83 days of composting, the temperature remained at 31°C in all of the mixtures. 3. RESULTS AND DISCUSSION 3.2. Mineralization of OM 3.1. Temperature evolution Reduction of OM is mainly caused by microbial activ- Graphs in Fig. 1a and 1b show the temperature varia- ity which significantly influences the composting process tions of the composting piles as a function of the waste and quality of the final product [5, 24]. In both trials, the mixtures and aeration methods. Temperature follows a typ- OM values decreased mainly in the initial stages of the ical temperature profile of organic waste composting for all composting process, which is related to the thermophilic mixtures [1, 23]. However, variation in mixing ratios caused phase and mineralization process. The decrease of OM different thermophilic temperatures. Throughout the experi- content in ASP was 12.9%, 44.2%, and 59.2% for 1:1, 2:1, mental period, the ambient temperature ranged from 24 to and 3:1 mixtures, respectively (Fig. 1c). While the OM 39°C. Initially the temperature of the compost mixtures content reduction for W mixtures was 18.2%, 65.3%, and was in the mesophilic phase and near to the ambient tem- 54.4% for the 1:1, 2:1, and 3:1 piles, respectively (Fig. 1d). perature. Temperature increased to 40–50°C in 2 weeks The 1:1 mixtures showed a lower mineralization rate than (start of the thermophilic phase), reached between 55 and the other piles. This can be explained by the greater activity 65°C in 4-5 weeks (except for ADS: GPW, 1:1) as a con- of microorganisms in 2:1 and 3:1 piles, which used a large sequence of the vigorous biodegradation of the easily bio- quantity of easily biodegradable compounds in these mix- degradable OM. As shown in Fig. 1a and 1b, after the start tures. The remaining OM content at the end of the process of composting, the temperatures increased in both trials; was 55.6%, 39.4%, 31.6%, 52.2%, 24.5% and 30.3% for however, the rate of rise, as indicated by the gradient of the ASP1, ASP2, ASP3, W1, W2 and W3 mixtures, respec- curves, was much faster in the windrow method than ASP. tively, which indicated a good stability condition in 2:1 and As have been reported, due to the lack of heat exchange in 3:1 mixtures for both trials. Tognetti et al (2007) reported the static piles, a longer time is needed for OM stabilization that OM content in the products of co-composting munici- in ASP process [1, 15]. pal organic waste with biosolids (at ratios of 1:1, 2:1, and 3:1 municipal organic waste /biosolids) ranged between As shown in Fig. 1a and 1b, higher temperatures (66 39% and 45%. The remained OM content in the final com- and 63°C) were achieved in the 2:1 and 3:1 mixtures in post of Rihani et al. [1] study also was higher than our com-

(a)

(b)

(c)

(d)

FIGURE 1 - Temperature (a and b) and OM content evolution (c and d) during composting of ADS and GPW. (ASP0, ASP1, ASP2, and ASP3: aerated static piles of control, 1:1, 2:1, and 3:1 mixtures, respectively. W0, W1, W2, and W3: windrows of control, 1:1, 2:1, and 3:1 mixtures, respectively).

posts (49.7%-58.3%), which may be due to the type and along with the transformation of organic nitrogen to am- proportion of the bulking agent, stabilization degree of WS monia were led to increase in pH [1, 12]. Thereafter, and composting duration. The results suggest that the deg- through ammonia volatilization and its oxidation to nitrates radation of OM under W method was greater than that un- by the action of nitrobacteria [1], the ammonia content and der ASP, signifying a lower microbial activity in the latter thus the pH gradually decreased in both trials and reached and consistent with the higher temperatures observed in the to neutrality at the end of the composting process (values W piles during the thermophilic phase. The reason might between 7.5 and 8). These results are consistent with the be the content of oxygen with a higher value in windrow results of Rihani et al. [1] and Tognetti et al. [24]. They system for aerobic microorganisms, whereas the activities reported that the decrease of pH at the final stage of com- of aerobic microorganisms were limited by lack of enough posting indicates the production of humic matter which oxygen during static composting. By increasing distance acts as buffer. The composts obtained from 2:1 and 3:1 from the aeration pipes in the static piles, a smaller amount mixtures showed pH values that were suitable from an ag- of oxygen reaches to the microbial population and there- ricultural point of view, and were within the range of val- fore due to the lack of turning, the outer layers have insuf- ues currently found for this type of waste [1, 5, 12]. The ficient oxygen for microbial activity. Moreover, there was trend of this parameter during composting was similar in less moisture content within the W piles due to the turning, all the composting piles regardless of the proportion of which improved the aeration and microbial activity. bulking agent or the aeration method. Therefore it can be concluded that bulking agent ratio did not have a signifi- 3.3. Other physico-chemical parameters cant influence on pH, which confirmed results of Ponsa et To assess the effectiveness of composting process, the al. [4], but in contrary to the findings of Yanez et al. [27]. C/N ratio is frequently used, as it may show the compost Compare with other organic wastes, ADS is character- stability [1, 4, 12]. Apparently, the characteristics of feed- ized by its high nitrogen content, and the nitrogen occur- stock have a significant effect on C and N profiles [4, 24]. ring mostly in the organic form [5]. The quantity and form The addition of GPW as bulking agents increases the C/N of nitrogen present in compost is important in determining ratio in the optimal range (between 25:1 and 35:1) for well- the product quality [1, 12, 28]. The addition of bulking organized composting. According to Table 2, the C/N ratio agent had a diluting effect on total kjeldahl nitrogen (TKN) presents a descending trend for both ASP and W compost. content, since the value for 3:1 mixtures (1.04%), with At the beginning of process, the C/N ratio of 1:1, 2:1, and lower amounts of ADS, was lower than in the 1:1 mixtures 3:1 mixtures was 16.6, 28, and 41.3 respectively, and at the (2.14%), which is consistent with the Yanez et al.(2009) end of composting period reached about 11.7, 10.5, and results [4]. TKN in the products was 2.65%, 2.09%, and 10.8 in ASP and 10.6, 12.2, and 10 in W piles, which is 1.62% for 1:1, 2:1, and 3:1 ASP, respectively, and 2.73%, similar to that obtained by Rihani et al. [1] and Tognetti et 1.12%, and 1.97% for the 1:1, 2:1, and 3:1 W, respectively al. [24]. Researchers have suggested different ideal C/N ra- which have a good compliance with the findings of tios from lower than 12 to 25 in final product; however, the Tognetti et al. [24]. A decrease in TKN may be expected optimum value is often related to the initial feedstock. For due to the mineralization of nitrogen and transformation to example, Wong et al. [24] and Tognetti et al. [25] reported ammonia and afterward nitrates [12]. However, this trend that compost may be considered mature when the C/N ratio was not observed and the TKN concentration during com- is lower than 17 and 20, respectively. At the end of process, posting remained constant or even increased in some piles once the readily available carbon compounds were de- by the end of process. The TKN increase generally was clined, the C/N ratio steadily decreased to less than 15 in observed in the piles in which the OM reduction had been all mixtures, which is qualified as finest compost which can particularly observed, which is compatible with the results be used in agricultural land [12, 25]. of Banegas et al. [5] and Chazirakis et al. [12]. Banegas et Moisture content of initial mixtures, in both aeration al. [5] and Rihani et al. [1] reported that because of the OM methods, varied depending on the bulking agent ratios and mineralization and consequent decrease of weight in the was in a range of 60–70%, which seems to be appropriate mass being composted, there is an increase in total nitrogen for composting [26]. At the thermophilic phase of com- concentration in terms of dry weight. posting the moisture was gradually decreased, principally The nitrogen is mostly metabolized to ammonium due to the evaporation of water as a result of biological heat rd (ammonification) during composting [1]. Tognetti et al., production [12], and at the final day (83 day) of the pro- [24] reported that during the composting process, the am- cess reached 32.7-37.6% and 20.7-26.3% in ASP and W monium content decreased and nitrate concentration in- piles, respectively which indicates that no intensive biolog- creased. High initial ammonium contents were observed in ical process can take place in such low moisture contents the primary stages of composting process. The solubiliza- [1]. tion of the ammonia led to the formation of ammonium and In the initial stage of composting the pH decreased an increase in the pH values in the composting mixtures. slightly due to the formation of acids resulted from the de- However, ammonium concentration decreased from 1131, composition of OM. Afterward, intense activity of proteo- 624, and 596 mg/kg at first day to 320, 143, and 146 mg/kg lytic bacteria and degradation of short chain fatty acids in 1:1, 2:1, and 3:1 ASP, respectively and from 1219, 766,

and 485 mg/kg to 286, 166, and 204 mg/kg in 1:1, 2:1, and time are shown in Fig. 2. In our study the SOUR of raw -1 -1 3:1 W, respectively at the final day of composting. The de- ADS was between 10.8-13.5 mg O2 g VS h . According crease in ammonium content was mainly due to the stimu- to Gomez et al.(2006), the SOUR of raw WS may vary in -1 -1 lation of nitrification and immobilization by microorgan- the range of 10 to 20 (or more) mg O2 g VS h , but an isms and partly caused by volatilization of gaseous ammo- effectively processed compost should display a value of -1 -1 nia, as a result of slight alkaline pH of the compost. The less than 3 mg O2 g VS h following two months of pro- highest initial concentrations of ammonium corresponded cessing [29]. An increase in the respiration rate was ob- to the 1:1 treatments, but all treatments reached similarly served in the early stages of composting. The highest low values at the end of composting process. The 2:1 mix- SOUR values were recorded during the thermophilic de- ture in W method showed the highest ammonium deple- composition phase due to the higher microbial activity. Af- tion. Apparently, the concentrations of this parameter were terward, SOUR decreased in 2:1 and 3:1 mixtures, as did below the recommended value (< 400 mg/kg) for the stable temperature and OM content, indicating a decrease in bio- compost [24], which shows a stabilized compost product in degradable OM. At the latest stage of composting process all mixtures at the final stage. (days 54-83), a slightly increase of SOUR was observed for The concentration of nitrate was very low at the begin- both trials, which may be attributed to the degradation of ning of the composting process and increased after 83 days of slowly biodegradable compounds [27]. Comparing the composting from 69, 64, and 111 to 467, 268, and 376 mg/kg piles, it seems that higher SOUR values were achieved in in 1:1, 2:1, and 3:1 ASP, respectively and from 83, 86, and the thermophilic phase for W (Fig. 2). 58 to 545, 508, and 444 mg/kg in 1:1, 2:1, and 3:1 W, re- However, during composting of 1:1 and control mix- spectively at the end of process. An increase in the nitrate tures the SOUR did not change significantly in the thermo- concentration was observed, but this increase in 2:1 and 3:1 philic phase, and only a slight increase in SOUR occurred piles for both trials, was low compared to the ammonium after 30-40 days of composting (data not shown). In addi- depletion. The 1:1 piles yielded higher nitrate content in tion, final SOUR values in control and 1:1 mixtures were the final composts than the 2:1 and 3:1 mixtures, which higher than those established in the standards for mature probably was the result of the higher amount of ADS in the -1 -1 composts (0.5–1.5 mg O2 g VS h ) which confirms the 1:1 piles with higher amount of organic nitrogen. Compar- low biological activity in these piles and unfinished coming the piles, nitrification process was favored in W mix- posting process [2, 18]. In the 1:1 mixtures, with higher tures. The degree of stability and maturity of the compost proportion of ADS, microbial activity may hindered due to is also related to the NH /NO ratio (nitrification index), the inhibitory effect of reducing compounds as a conse- which ratios lower than 0.5 is indicative of mature compost quence of anaerobic digestion [5]. These results suggested [1]. NH /NO ratio decreased from 5.35-9.75 and 8.36- that biological activity in 2:1 and 3:1 mixtures were higher, 8.92 in the initial mixtures to 0.39-2.72 and 0.33-1.92 for since SOUR is usually considered as an indicator of the ASP and W, respectively (Table 2). According to the microbial activity of the compost. Furthermore, at the final NH /NO ratio the 2:1 W and 3:1 ASP piles with ratio of stage of composting, the mixtures in windrows showed 0.33 and 0.39 have the greatest maturity, respectively. lower SOUR values than the static piles (Fig. 2), which can be explained by the higher microbial activity in the wind- 3.4. Respiratory activity rows as a consequence of better supplying of oxygen and The respiration activity in terms of SOUR is widely faster stabilization in the windrows. Although the 3:1 and -1 used to monitor microbial activity and OM decomposition 2:1 mixtures had a similar final SOUR close to 1 mg O2 g -1 -1 -1 during composting [14, 27]. The changes in SOUR over VS h , only W, with a SOUR of 1.2-1.3 mg O2 g VS h ,

TABLE 2 - Evolution of C/N ratio and / ratio during composting process

Days C/N ratio NH /NO ratio ASP 2:1 ASP 3:1 W 2:1 W 3:1 ASP 2:1 ASP 3:1 W 2:1 W 3:1 1 28 41.3 28 41.3 9.75 5.35 8.92 8.36 8 24.5 31.7 23.9 36.2 6.07 4.44 7.80 5.62 13 23.4 24.6 18.9 26.5 4.19 3.28 5.81 5.23 19 18.4 22.1 16.6 23.3 4.12 1.91 4.32 1.97 26 17.1 19.8 12.3 19.4 2.21 1.68 2.18 1.82 33 16.2 20.2 10.8 18.3 1.42 1.59 1.91 1.43 41 14.1 19.1 11.7 15.8 1.37 1.43 1.66 1.30 47 13.6 15.6 11.3 16.7 1.32 1.04 1.42 0.97 54 13.6 13.8 14.5 15.5 0.81 0.67 1.30 0.91 68 12.1 11 12.9 12.9 0.63 0.67 0.70 0.69 83 10.5 10.8 12.2 8.5 0.53 0.39 0.33 0.46

FIGURE 2 - SOUR variations for ASP and W mixtures with 2:1 and 3:1 proportions of GPW: ADS. (ASP2 and ASP3: aerated static piles of 2:1 and 3:1 mixtures, respectively. W2 and W3: windrows of 2:1 and 3:1 mixtures, respectively).

TABLE 3 - Microbial indicators, Heavy metals and GI in the final compost products (after 83 days of composting)

Aerated static piles Windrows Control 1:1 2:1 3:1 Control 1:1 2:1 3:1 Total coliforms a 21.43 23.47 39.8 94.9 3.67 244.9 3.64 ND Fecal coliforms a 3.06 3.06 3.06 3.67 3.06 3.06 ND ND Salmonella b ND ND ND ND ND ND ND ND Zn (mg/kgDW c) 1280 720 690 490 1240 720 470 430 Cu (mg/kgDW) 365.7 280.6 10 164.8 319.2 131.3 104.2 89.4 Ni (mg/kgDW) 108.2 87.5 73.7 62.7 163 113.7 81 61.3 Pb (mg/kgDW) 114.8 102.1 98.3 80.8 160.4 88.3 79.5 72.2 Cd (mg/kgDW ) 4.6 1.9 1.8 1.2 6.1 1.76 1.35 1.28 Cr (mg/kgDW) 110.3 84.3 71.1 63.1 103.4 40.7 32.1 31 G I (%) 0 20 80 80 0 30 100 90 a (MPN/gDW), b(MPN/4gDW), c dry weight, ND = not detected

had a value lower than the limit established to qualify the cress seeds were not germinated in control samples due to compost as stable product [18]. However, the effectiveness the phytotoxic effect of inhibiting substances such as am- of this parameter for the evaluation of stability in compost monia and other toxic substances derived from the anaero- is controversial, since different trends have been found de- bic digestion. In the 2:1 and 3:1 mixtures this toxic effect pending on the aeration method and starting feedstocks [3, would be diluted by the higher proportion of bulking agent. 24]. SOUR evolution in the mixtures was also dependent The results revealed that germination percentage in 3:1 and on the GPW: ADS ratio and non-similar trends were found 2:1 piles was over 50%. It should be noted that this perfor different mixtures, which confirms the results of Yanez centage in the W mixtures was higher than that obtained in et al. [4]. ASP (Table 3). The 2:1 mixture in windrows showed the highest germination index value, presumably due to the ap- 3.5. Quality assessment of the composted sludge propriate mineralization.

Seed germination bioassay is commonly used as a One of the major concerns in land application of WS compost maturity test. The seed germination test helps us compost is the presence of high concentrations of heavy to establish the efficiency of the composting process for re- metals, which can penetrate the soil and may affect soil moval of phytotoxic substances. The results of phytotoxi- properties, plant growth and human health [1, 8, 30]. As city analysis are presented in Table 3. No inhibitory effect described by several studies, the most important heavy on seed germination being observed in 2:1 and 3:1 mix- metals which are present in WS are including lead (Pb), tures at different aeration methods. Results showed that the zinc (Zn), cadmium (Cd), chromium (Cr), copper (Cu), and

nickel (Ni) [1, 8]. Concentration of these heavy metals in dian, Mr. Rabierad, Mr. Amini, and Ms. Javadi, from Isfa- the end-product of composting piles has been evaluated as han Water and Wastewater Co., for technical support and shown in Table 3. Heavy metals concentration of compost Mr. Farrokhzadeh, Ms. Vahid Dastjerdy, and Ms. Hatam- products were found in levels lower than the international zadeh for their assistance in supplying laboratory facilities limits approved by USEPA (1995) [31], due to the fact that of this research and valuable advice. the main load of the treated wastewater was municipal sewage. According to these limits, all composts could be clas- The authors have declared no conflict of interest. sified as type A compost. Some studies [8, 30] confirmed that the concentration of heavy metals in municipal WS compost is in the acceptable limits. Heavy metals are much REFERENCES less soluble at high pH levels of compost, making them less bioavailable in soil [8]. 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[20] Zeng, G., Yu, M., Chen, Y., Huang, D., Zhang, J., Huang, H., Jiang, R. and Yu, Z. (2010) Effects of inoculation with Phan- erochaete chrysosporium at various time points on enzyme activities during agricultural waste composting. Bioresource Technol. 101: 222-227. [21] American Public Health Association (APHA). (2012) Stand- ard Methods for the Examination of Water and Wastewater, nd 22 ed, Washington DC. [22] Tiquia, S., Tam, N. and Hodgkiss, I. (1996) Effects of composting on phytotoxicity of spent pig-manure sawdust litter. Environ. Pollut. 93: 249-256.

[23] Lu, L.A., Kumar, M., Tsai, J.C. and Lin, J.G. (2008) High-rate composting of barley dregs with sewage sludge in a pilot scale bioreactor. Bioresource Technol. 99: 2210-2217.

[24] Tognetti, C., Mazzarino, M. and Laos, F. (2007) Cocompost- ing biosolids and municipal organic waste: effects of process management on stabilization and quality. Biol. Fert. Soils 43: 387-397.

[25] Wong, J., Mak, K., Chan, N., Lam, A., Fang, M., Zhou, L., Wu, Q.T. and Liao, X.D. (2001) Co-composting of soybean residues and leaves in Hong Kong. Bioresour. Technol. 76: 99- 106. [26] Malinska, K. and Zabochnicka-Swiatek M. (2013) Selection of bulking agents for composting of sewage sludge. Environ. Prot. Eng. 39: 91-103. Received: October 30, 2014 Revised: December 29, 2014 [27] Ponsa, S., Pagans, E. and Sanchez, A. (2009) Composting of Accepted: January 19, 2015 dewatered wastewater sludge with various ratios of pruning waste used as a bulking agent and monitored by respirometer. Biosyst. Eng. 102: 433-443. CORRESPONDING AUTHOR [28] De Guardia, A., Petiot, C., Rogeau, D. and Druilhe, C. (2008) Influence of aeration rate on nitrogen dynamics during composting. Waste manage. 28: 575-587. Amir Hossein Nafez [29] Gomez, R.B., Lima, F.V. and Ferrer, A.S. (2006) The use of Department of Environmental Health Engineering respiration indices in the composting process: a review. Waste School of Public Health Manage. Res. 24: 37-47. Kermanshah University of Medical Sciences [30] Zorpas, A.A., Inglezakis, V.J. and Loizidou, M. (2008) Heavy Kermanshah metals fractionation before, during and after composting of IRAN sewage sludge with natural zeolite. Waste manage. 28: 2054- 2060. Phone: +98 833 826 4447 [31] USEPA (1995) guide to the biosolids risk assessment for the Fax: +98 833 826 3048 EPA Part 503 Rule EPA: B32-B-93-005. United States Envi- E-mail: [email protected] ronmental Protection Agency Office of Wastewater Manage- ment, Washington DC. FEB/ Vol 24/ No 7/ 2015 – pages 2406 – 2414

ADSORPTION OF CARMINE ON ETHYLENEDIAMINE MODIFIED PEANUT HUSK FROM AQUEOUS SOLUTION

Yinghua Song*, Xi Zhang, Mei Ye, Hui Xu and Jianmin Ren

Department of Chemistry and Chemical Engineering; Chongqing Key Lab of Catalysis & Functional Organic Molecules, Chongqing Technology and Business University, Chongqing 400067, China

ABSTRACT degraded because of their complex aromatic species and xenobiotic properties. Among the most useful dyes, there A new adsorbent was synthesized from peanut husk, an is carmine that is used as an additive in pharmaceutical tab- agricultural by-product, in which epichlorohydrin was used lets and capsules, in confectionery items and etc. It has as a cross-linking agent and ethylenediamine as a modifica- been shown that carmine may cause increasing risk of can- tion agent. Factors affecting the adsorption behavior of this cer and many other diseases due to its carcinogenic and adsorbent for carmine in aqueous solution, namely, pH value, mutagenic characteristics. adsorption time, temperature and initial carmine concen- There are many physical and chemical processes avail- tration were evaluated through experiments in a batch sys- able to remove dyes, such as advanced oxidation, com- tem. It was concluded that the maximal adsorption capacity bined chemical and biochemical process, adsorption mem- of this adsorbent for carmine was 95.2 mg / g at 303 K, brane filtration, aerobic and anaerobic digestion. Nowa- which was improved 17.6 times as much as that of the un- days activated carbon works as adsorbent to remove dyes modified peanut husk. The Langmuir isotherm model can in most commercial systems because of its high adsorption provide a better description for the adsorption equilibrium ability. But the processing costs are still expensive. So peo- when it was compared with the Freundlich equation under ple try to develop cheaper and effective adsorbents to re- the conditions of the present study. In order to examine the move dyes and find an alternative method from different controlling mechanisms of the process, kinetic equations of starting materials such as bagasse pith [3], rice husk [4], the mass transfer and chemical reaction, the Largergren first clay [5], sawdust [6], hen feather [7], polar leaf [8], papaya order model, the pseudo-second order model and the intra- seed [9], and etc. particle diffusion model were used to correlate the experi- Peanut husk, an agricultural by-product available in mental data. It was found that the intra-particle diffusion is large quantity in China, is often burned and discarded di- the significant controlling step under the experimental con- rectly, which produced waste gas and dust. Fortunately, a ditions but it was not the unique one and it accompanied possible solution to utilize peanut husk as an adsorbent has with chemical reactions. It showed that the peanut husk been found to remove contaminants in waste water. Peanut modified with ethylenediamine had good performance for husk was introduced directly as a low-cost adsorbent for removal of azo-dye and can be used as a highly efficient the efficient removal of Sunset Yellow dye in aqueous so- biomass adsorbent to treat dyes-containing wastewater. lution [10]. However, raw peanut husk cannot be used as a good natural adsorbent due to two major limitations. Firstly, on its contact with water, there is a leaching of yel- KEYWORDS: peanut husk; ethylenediamine; modification; adsorption; carmine lowish color into solutions; secondly, on its prolonged contact with water, peanut husk tends to scatter. Researchers have studied the removal of heavy metals [11], phenol [12]

1. INTRODUCTION and dyes [13, 14] by partially carbonized peanut husk or by activated carbon from peanut husk. The purpose of this work was to produce a new adsorbent from peanut husk Synthetic dyes are widely used in various industries, chemically modified with ethylenediamine and the adsorp- such as paper, textiles, leather, plastics, printing and cos- tion of carmine on it was investigated. The experiments metics. The discharged colored wastewater not only causes were done in the same batch system to evaluate the adsorp- serious environmental issues, but also hazards human be- tion capacity of the adsorbent, and the adsorption of car- ings’ health due to their toxicity and carcinogenicity [1, 2]. mine was studied on account of the initial carmine concen- Such effluent must be treated before discharged into the tration, pH, adsorption time and temperature. The equilib- environment. But many synthetic dyes are difficult to be rium of adsorption was modeled with the Langmuir and the

Freundlich isotherms. The kinetic parameters and intra- * Corresponding author particle diffusion were also determined for this process.

2. MATERIALS AND METHODS The adsorption capacity q (mg·g-1) was calculated as follow: 2.1. Preparation of Ethylenediamine Modified Peanut Husk v(c  c ) (EMPH) q  0 t (1) The peanut husk used in this study was purchased from m a farmers’ market in Chongqing, Rep. of China. The peanut where c0 and ct are the initial concentration and the husk was first washed copiously with distilled water to re- concentration at t moment (mg·L-1); v is the volume of car- move dust and impurities and dried at 60 in an air circu- mine solution used (L); and m is the mass of the dry EMPH lating oven. In order to receive uniform modification and used(g). reproducible results, the peanut husk was sieved into 40- 60 meshes before modification. 2.0 g (dry weight) of 2.4. Adsorption isotherms the raw peanut husk was mixed with a solution of NaOH The Langmuir and the Freundlich isotherms are the (45 mL, 1 mol·L-1) and epichlorohydrin (30 mL), then the most widely used isotherms since they can be applied to a mixture reacted for 2 h at (45 ± 1) . The biomass residue wide range of adsorbate concentrations. The linear form of was rinsed with distilled water and oven-dried. the Langmuir equation is generally accepted in the follow- Then 2.0 g (dry weight) of the epichlorohydrin crossing variation: linked peanut husk was heated at 55 with 100 mL of so- c 1 c dium carbonate solution (0.1 mol·L-1) and 2.5 mL of ethylene- e   e (2) diamine for 90 minutes. The peanut husk (EMPH) modified qe K Lqmax qmax with ethylenediamine was washed thoroughly with distilled where ce is the equilibrium concentration of dye solu- water, filtered and then oven-dried and stored in a desiccator. -1 tion(mg·L ), qe is the equilibrium capacity of dye on the -1 The general reaction scheme can be expressed as [15, 16]: adsorbent (mg·g ), qmax is the maximum monolayer ad- -1 O O sorption capacity of the adsorbent (mg·g ), and KL is the -1 Cell NaOH Cell Langmuir adsorption constant (L·mg ). OH + Cl CH2 CH CH2 O CH2 CH CH2 OH The Freundlich isotherm equation is a semi-empirical

NH2CH2CH2NH2 , NaCO3 Cell O CH CH CH one employed to describe heterogeneous system: 2 2 NHCH2CH2NH2 1 ln q  ln k  lnC (3) 2.2. Chemicals e F n e Stock solution was prepared by dissolving 1.0 g of car- where k (L·mg-1)and n (dimensionless) are the Freund- mine dye in 1000 mL of twice-distilled water. The test so- F lich adsorption isotherm constants, indicating the capacity lutions were prepared by diluting the stock solution to the and intensity of the adsorption, respectively. desired concentration. All reagents used in this study were of analytical reagent grade and used without further purifi- 2.5. Adsorption Kinetics cation. The initial pH was adjusted to the pre-determined value (2.00 - 12.00) ± 0.10 using NaOH or HCl solutions In order to examine the controlling mechanism of this prior to the addition of adsorbent. process, pseudo-first order, pseudo-second order and intraparticle diffusion kinetic equations were used to correlate the 2.3. Batch adsorption studies experimental data. The pseudo- first order kinetic model was Batch adsorption experiments were conducted in suggested by Lagergren [17] for the adsorption of solid/liq- 100mL conical flasks containing 0.1 g of EMPH and car- uid systems and its integrated form is given below: mine solutions (30 mL) of the desired concentration at con- ln(q - q ) = lnq - k t (4) stant temperature. The flasks were oscillated on a shaker at e t e 1 -1 constant 150 rpm shaking rate for given time. Samples where qt(mg·g ) is the adsorption capacity at time t -1 -1 were taken out from mixture and centrifuged and the su- (min ) and k1 (min ) is the rate constant of the pseudo-first pernatant liquid was analyzed spectrophotometrically at adsorption. 508 nm to determine the residual dye concentration in the The kinetic data were further analyzed with Ho’s system. All the experiments were carried out twice in par- pseudo-second order kinetics model [18] expressed as: allel and average values were calculated further. For kinetic studies, a series of flasks for each initial concentration t t 1   (5) were prepared, each data point at pre-determined time in- q q k q 2 terval was obtained from an individual flask and therefore, t e 2 e no correction was necessary due to withdrawal of sampling The adsorption process follows generally three consec- volume. For isotherms studies, a series of solution contain- utive steps of external diffusion, intraparticle diffusion and ing 30 mL of carmine in the range of 250 - 1000 mg·L-1 actual adsorption. One or more of these steps control the were prepared. EMPH (0.1 g) was added to each solution adsorption kinetics altogether or individually. The kinetic and then the mixtures were agitated at temperature of 293, data were also be analyzed by intraparticle diffusion kinet- 303 and 313K respectively for 24 h. ics model [19] to determine the rate controlling step:

q  k t1/ 2  C (6) ture 303K. According to the results (Fig. 2), the removal of t p carmine was faster in initial 150 minutes and then became -1/2 -1 where kp (mg·min ·g ) is the intraparticle diffusion slower and finally reached equilibrium at approximately -1 -1 -1 rate constant and C (mg·g ) is a constant, k2 (g·mg ·min ) 1200 minutes. During initial stages, a large number of va- is the rate constant of the pseudo-second order adsorption. cant sites were available on the surface of EMPH and the For elucidation of the adsorption rate controlling adsorption of carmine was very quick. Then the adsorption mechanism, the Wünwald-Wagner intraparticle diffusion process became slower due to gradual occupancy of these model was also proposed to give the intraparticle diffusion sites. With increasing time, the occupation of the remain- coefficient, D(cm2·s-1) [20]: ing vacant sites was more difficult due to increased repul- sive forces between dye molecules and bulk solution. q 6 4 2 D lg(1 t )  lg( )  t (7) 2 2 100 qe  2.303d where d (cm) is the mean particle diameter. 80

-1

60 □ 563.3 mg·L-1 3. RESULTS AND DISCUSSION -1 / mg.g

t ■410.7 mg·L

q 40 ○254.1 mg·L-1 3.1. Effect of initial pH of solution The pH value of the solution was an important factor 20 that must be considered in the adsorption process. The initial pH of the working solutions was adjusted to between 0 pH 2.0-12.0 by addition of diluting HCl or NaOH solution. 300 600 900 1200 The effect of pH on the adsorption of carmine was shown t / min in Fig. 1. FIGURE 2 - Effect of contact time on the adsorption of carmine (T = As shown in Fig. 1, a sharp decrease in the adsorption 303K, pH = 2.0, rpm = 150) capacity occurs as the pH of the initial solution was increased from 2.0 up to 4.0, beyond which the adsorption Fig. 2 also shows that the equilibrium capacity of EMPH capacity remains almost constant. For the subsequent stud- increased from 71.48 to 86.15 mg·g-1 as increasing initial con- ies, pH 2.0 was selected as an optimal pH value. centration of carmine because the initial carmine concentra- The higher adsorption capacity of carmine at low pH tion provides an important driving force to overcome all mass may be due to the enhanced protonation of -NH2 at the sur- transfer resistance. The increase of loading capacity of EMPH face of the EMPH. It contributed to the preferential adsorp- with increasing carmine concentration may also be derived tion of the negative dye ions to positive active sites and fa- from the higher interaction between carmine and EMPH. cilitates the diffusion process in the working solution. As increasing pH, protonation reduces and electrostatic repul- 3.3. Adsorption isotherm parameters sive force becomes dominant, which inhibits the diffusion The experimental adsorption isotherms of EMPH at and adsorption of carmine on EMPH. different temperatures are depicted in Fig.3. According to Fig.3, the adsorption of carmine onto EMPH was enhanced 100 by increasing the temperature from 293 K to 313 K at different initial dye concentration. The Langmuir and the 80 Freundlich adsorption isotherms were used to evaluate the adsorption equilibrium data. The Langmuir plot of c / q 60 e e -1 against ce yields straight lines (Fig.4). The isotherm con- 40 stants and correlative coefficients were tabulated in Table 1.

/ mg.g 2 e The correlative coefficient R values in Table 1 confirm q 20 that the adsorption equilibrium data fitted very well with the Langmuir model under the studied conditions. This indicates uniform adsorption and strong dye-adsorbent inter- 02468101214 actions over the surface of the adsorbent. The maximum pH monolayer capacity of EMPH is determined as 87.7, 95.2 -1 FIGURE 1 - Effect of pH on the adsorption of carmine (T = 303K, c0 and 101.0 mg·g for 293, 303 and 313 K, respectively, = 396.3 mg·L-1, contact time = 24 h, rpm = 150) which suggests an endothermic process. The values of Freundlich constant, kf, increased with increasing tempera- 3.2. Effect of contact time ture and showed easy uptake of carmine by EMPH. All n Removal of carmine by EMPH was performed with values were found to be greater than 1, which indicated a three different initial carmine concentrations at tempera- favorable adsorption condition during the process.

100 10

80 8

60 6 -1 -1 .

□ 313K L / g □ 313K -1

40 e 4

/ mg.g ■ 303K ■ 303K e .q e q ○ 293K c ○ 293K 20 2

0 0 200 400 600 800 200 400 600 800 -1 c / mg. L -1 e c / mg. L e FIGURE 3 - Adsorption isotherms of Carmine on EMPH at different FIGURE 4 - Linear fit of the Langmuir isotherm temperatures (pH = 2.0, contact time = 24 h, rpm = 150)

TABLE 1 - Langmuir and Freundlich isotherm constants of carmine on EMPH.

Langmuir constants Freundlich constants T / K -1 -1 2 2 qmax / mg · g KL / L· mg R n kf R 293 87.7 15.0 0.9989 5.2 86.4 0.9623 303 95.2 16.3 0.9964 5.3 99.0 0.9880 313 101.0 24.8 0.9980 7.9 101.3 0.9687 303* 5.4 1.0 0.9991 3.3 6.75 0.7968 (303*——Isotherms regression data of the unmodified peanut husk at 303 K)

Table 1 also showed that the maximal adsorption ca- 20 pacity of EMPH for carmine was 95.2 mg · g-1 at 303 K, which was improved 17.6 times as much as that of the un- 15 modified peanut husk (5.4 mg · g-1).

3.4. Kinetic parameters of adsorption t 10 □ 563.3 mg·L-1 -1 In order to investigate the adsorption process of car- q t / ■410.7 mg·L ○254.1 mg·L-1 mine onto EMPH, the pseudo first-order, the pseudo sec- 5 ond-order and the intraparticle diffusion model were used to correlate the experimental data of Fig.2. The values of k ,q and correlative coefficients of the 0 1 eq 500 1000 1500 2000 pseudo first-order model were listed in table 2. Low correl- t / min ative coefficients and a large difference of equilibrium adsorption capacity (qe) between the experiment and calcula- FIGURE 5 - Pseudo-second order adsorption kinetics tion reveal a poor fit to the experimental data, which indicate that the film mass transfer can be ignored under the 100 present experiment conditions. The rate constant k2, the qe value and correlative coef- 80 ficient R2 of the pseudo-second order kinetics were calculated from the linear plots of t/qt against t (Fig.5) and the 60 results were given in table 2. The correlative coefficients -1 are all greater than 0.99 and the calculated qe values are 40 / mg.g also very close to the experimental data, which indicated t q that the pseudo-second order model can provide a perfect 20 fit to the experimental data. The adsorption of carmine onto EMPH can be considered as a chemical reaction between 0 carmine and EMPH based on electron exchange or charge 0 5 10 15 20 25 30 35 40 sharing [21]. 1/2 1/2 t / min The possibility of intraparticle diffusion cannot be over- FIGURE 6 - Intra-particle diffusion model plots at different initial looked because of the long adsorption equilibrium time in concentrations

TABLE 2 - Statistical results of the application of the kinetic models

Model Initial carmine concentration / mg·L-1 254.1 410.7 563.3 -3 -1 First order kinetic k1 (10 ) Rate constant, min 3.9 3.7 2.8 -1 qe,cal Equilibrium capacity, mg·g 39.3 35.7 29.3 2 R Correlation coefficient 0.9536 0.8589 0.9252 -3 -1 -1 Second order kinetic k2 (10 ) Rate constant, g·mg ·min 3.3 3.6 4.2 -1 qe,cal Equilibrium capacity, mg·g 73.5 82.6 88.5 R2 Correlation coefficient 0.9987 0.9992 0.9996 -1/2 -1 Intraparticle diffu- kp Rate constant, mg·min ·g 26.6 36.8 49.3 sion C Intercept 2.0 2.0 1.8 R2 Correlation coefficient 0.9588 0.9706 0.9684 Wünwald-Wagner Effective diffusion coefficient, D (10-10) 5.2 4.1 3.4 intraparticle diffusion cm2·s-1 Intercept -0.5877 -0.7016 -0.7202 R2 Correlation coefficient 0.9568 0.9561 0.9557 q Experimental data of the e,exp 71.8 81.0 87.4 Equilibrium capacity, mg·g-1

the present study. For obvious illustration of this, intra- intra-particle diffusion, accompanying with chemical reac- particle diffusion model was proposed to describe the ad- tions. The results of the study indicated EMPH can be used sorption process (Fig.6). The time dependence of qt in as an effective adsorbent for dye removal. Fig.6 could presented two straight lines which could be well fitted linearly. The multi-linearity suggested that the intraparticle diffusion was dominant in carmine adsorption. The qt in the first portion showed a rapid increase with ACKNOWLEDGEMENT time, which was attributed to the rapid external diffusion of dyes to the surface of EMPH. The second portion corre- This work was supported by the Project Foundation of sponded to the intraparticle diffusion effect. The values of kp, Chongqing Municipal Education Committee (KJ110724), C and correlation coefficients were also shown in Table 2. It and the Project Foundation of Environmental Pollution is found that correlation coefficients for the intraparticle dif- Control Technology Innovative Team. fusion model were all greater than 0.95. However, the linear plots did not pass through the origin of coordinates, The authors have declared no conflict of interest. which indicated that the intraparticle diffusion was not the sole rate-controlling step. A similar finding was observed for the adsorption of methylene blue by polar leaf and the adsorption of sunset yellow by peanut husk [8,10]. REFERENCES

Table 2 also listed the calculated results of Wünwald- [1] Sharma P. and Kaur H. (2011) Sugarcane bagasse for the re- Wagner intraparticle diffusion model. The values of the in- moval of erythrosin B and methylene blue from aqueous waste. Appl. Water Sci. 1, 135- 145. ternal diffusion coefficient fall well within the magnitudes reported in literature [20], which suggested that the adsorp- [2] Attallah M.F., Ahmed I.M. and Hamed, M.M. (2013) Treat- ment of industrial wastewater containing Congo Red and tion of carmine should be governed by intraparticle diffu- Naphthol Green B using low-cost adsorbent. Environ Sci Pol- sion mechanism. lut Res. 20, 1106- 1116.

[3] Mitter E. K., Santos G. C., Almeida E. J. R., Morao L. G., Ro- drigues H. D. P. and Corso C. R. (2012) Analysis of acid aliz- 4. CONCLUSIONS arin violet N dye removal using sugarcane bagasse as adsorbent. Water Air Soil Pollut. 223,765- 770. The adsorption of carmine dye onto EMPH as a novel [4] Shamik C. and Papita D. S. (2013) Artificial neural network adsorbent was carried out in batch mode. The effects of dif- (ANN) modeling of adsorption of methylene blue by NaOH- ferent operating parameters such as pH, contact time, ini- modified rice husk in a fixed-bed column system.Environ Sci tial carmine concentration were evaluated. The results of Pollut Res. 20, 1050- 1058. the present study concluded 2.0 of the optimal pH, 24 h to [5] Tahir S. S. and Rauf N. (2006) Removal of a cationic dye from reach adsorption equilibrium, and increasing adsorption aqueous solutions by adsorption onto bentonite clay. Chemo- sphere. 63, 1842- 1848. capacity with increasing temperature. Based on the correlative coefficients, the Langmuir isotherm model can pro- [6] Ansari R., Seyghali B., Mohammad-khah A., and Zanjanchi vide a better description when it was compared with the M. A. (2012) Highly efficient adsorption of anionic dyes from aqueous solutions using sawdust modified by cationic surfac- Freundlich equation. Kinetics reveals that the adsorption tant of cetyltrimethylammonium bromide. J Surfact Deterg. process of carmine on EMPH tends to be controlled by the 15, 557- 565.

[7] Mittal A., Thakur V. and Gajbe V. (2013) Adsorptive removal of toxic azo dye Amido Black 10B by hen feather.Environ Sci Pollut Res. 20, 260- 269. [8] Han X., Niu X. and Ma X. (2012) Adsorption characteristics of methylene blue on polar leaf in batch mode:Equilibrium,kinetics and thermodynamics. Korean J Chem Eng. 29, 494- 502. [9] Weber C. T., Foletto E.L. and Meili L. (2013) Removal of tan- nery dye from aqueous solution using papaya seed as an efficient natural biosorbent. Water Air Soil Pollut. 224, 1427- 1437. [10] Song Y., Liu Y., Chen S., Xu H. and Liao Y. (2014) Sunset yellow adsorption by peanut husk in batch mode. Fresenius Environmental Bulletin. 23, 1074- 1079.

[11] Ricordel S., Taha S., Cisse I. and Dorange G. (2004) Heavy metal removal by adsorption onto peanut husks carbon:characterization,kinetic study and modeling. SepPurif Technol. 24, 389- 401.

[12] Gonzo E.E. and Gonzo L.F. (2005) Kinetics of phenol removal from aqueous solution by adsorption onto peanut shell acid activated carbon. Adsorp Sci Technol. 23, 289- 302. [13] Gong R., Ding Y., Li M., Yang C., Liu H. and Sun Y. (2005) Utilization of powdered peanut hull as biosorbent for removal of anionic dyes from aqueous solution. Dyes Pigments. 64, 187- 192. [14] Ozer D., Dursun G. and Ozer A. (2007) Methylene blue adsorption from aqueous solution by dehydrated peanut hull. J Hazard Mater. 144, 171- 179. [15] Li B., Li L. and Sun X. (2011) Preparation of Ethylenediamine modified peanutshell and its adsorption behavior for anionic dyes.J South—Central Univ for Nationalities (Nat. Sci. Edi- tion). 30, 20- 24. (in Chinese) [16] Zou W., Li K., Gao S. and Bai H. (2013) Study on Congo Red adsorption with ethylenediamine modified sawdust. J Zheng- zhou Univ (Engineering Science). 34, 28- 31,36. (in Chinese)

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Handlingar. 24, 1- 39. [18] Ho Y.S.and McKay G. (1999) Pseudo-second order model for sorption processes. Process Biochem, 34, 451- 465.

[19] Weber W.J. and Morriss J.C. (1963) Kinetics of adsorption on carbon from solution. J Sanit Eng Div. 89, 31- 60. Received: October 24, 2014 [20] Kalavathy M.H., Karthikeyan T., Rajgopal S. and Miranda L. Revised: December 15, 2014 R. (2005) Kinetic and isotherm studies of Cu(II) adsorption Accepted: January 09, 2015 onto H3PO4-activated rubber wood sawdust. J Colloid and In- terface Science. 292, 354- 362.

[21] Wang G., Li L., Li Y. and Xue Q. (2010) Study on the prepa- CORRESPONDING AUTHOR ration of bamboo activated carbon and its phenol adsorption properties. J Chem Eng of Chinese Univ. 4, 700- 704. (in Chi- nese) Dr. Yinghua Song Department of Chemistry and Chemical Engineering College of Environmental and Biological Engineering Chongqing Technology and Business University Chongqing 400067 P.R. CHINA

Phone: +86-023-62769785(office) Fax: +86-023-62769785 E-mail: [email protected]

FEB/ Vol 24/ No 7/ 2015 – pages 2415 – 2420

SUBJECT INDEX

A M activated carbon 2341 metals ions 2296 adsorption 2341 microcystin 2384 adsorption 2415 Microcystis bloom 2384 alluvial lands 2374 modification 2415 antioxidant efficiency 2310 municipal wastewater 2296

B N β-adrenoceptor antagonists 2310 NASA-CASA Model 2264 biohydrogen 2289 NPP 2264 nucleoprotein 2394 C Calix[4]resorcinarenes 2296 O carmine 2415 organic solid waste 2289 Cd-localization 2394 oxytetracycline 2348 cell damage 2394 cell division 2394 P cereal 2275 compost 2406 particle size 2289 copper (Cu) 2394 peanut husk 2415 cropland 2354 pesticide application 2275 phosphorus transport 2325 photocatalysis 2348 E photocatalytic degradation 2280 energy 2362 photosynthetic activity 2384 environment 2275 polymer inclusion membranes 2296 erosion 2319 positional isomerism 2310 ethylenediamine 2415 precision agriculture 2374

F R factor analysis 2374 recovery 2362 filter-feeding fish 2384 redworm 2334 remote sensing 2264 G restaurant waste 2289 geographical information systems 2374 runoff 2319 green plant wastes 2406 groundwater irrigation 2325 S

safety 2334 H scrap tire 2341 Hordeum vulgare 2394 sediment 2319 human health 2275 sediment 2354 separation 2296 I soil phosphorus 2325 inactivation 2334 soil properties 2374 initial total solids 2289 South-North Water Diversion Project 2354 ion exchange 2348 spatial modelling 2264 irrigation 2319 static pile composting 2406 isotherm 2354 surface irrigation 2319 synthesis and characterization 2280 L landfill gas 2362 T limitations 2362 tap water 2334 tetracycline 2280 M thermo-chemical 2341 Mediterranean 2264 titanate nanotubes 2348

AUTHOR INDEX

W A waste-to-energy 2362 Arslan, Hakan 2374 wastewater irrigation 2325 Asgari, Ghorban 2341 wastewater sludge 2406 Askari, Fateme Barjasteh 2341 water-level fluctuations 2354 weed control 2275 B windrow composting 2406 Benosmane, Nadjib 2296 Berberoglu, Suha 2264 Z Bina, Bijan 2406 ZnS nanocrystals 2280 Bogdanets, Vyacheslav 2319 Boutemeur, Baya 2296 Bozdogan, Ali Musa 2275 Buitrón, Germán 2289

C Çetin, Öner 2319 Chen, Baiyang 2334 Cilek, Ahmet 2264 Csöllei, Jozef 2310

D Donmez, Cenk 2264 Du, Chao 2354 Du, Yun 2354

G Guan, Weisheng 2348

Hamdi, Maamar 2296 Hamdi, Safouane M. 2296 Hassanzadeh, Akbar 2406

Hoang, Tuan K. A. 2348 Hu, Kelin 2325 Huo, Pengwei 2280

Jiang, Wusheng 2394 Jiang, Ze 2394

L Li, Dunhai 2384 Li, Zhongjie 2384 Liu, Donghua 2394 Liu, Fang 2325 Liu, Xinlin 2280 Lu, Changyu 2348 Lu, Yintao 2325 Lu, Ziyang 2280

M Ma, Changchang 2280 Malík, Ivan 2310 Maruniak, Matej 2310 Moghim, Sharareh 2406

M Z Moreno-Andrade, Iván 2289 Zhao, Lihong 2334 Muselík, Jan 2310 Zhou, Mingjun 2280 Zhou, Yiyong 2384 N Zhu, Xiaoshan 2334 Nafez, Amir Hossein 2406 Zhu, Zhi 2280 Ni, Liang 2280 Zhuang, Yan-Hua 2354 Nikaeen, Mahnaz 2406 Zou, Jinhua 2394

P Peng, Yuexin 2348

R Rahmani, Ali R. 2341 Ren, Jianmin 2415

S Sağlam, Mustafa 2374 Sayinci, Bahadir 2275 Sevimoğlu, Orhan 2362 Shi, Qiuyue 2394 Song, Yinghua 2415 Stanzel, Lukáš 2310

T Tari, Ali Fuat 2319 Temizel, Kadir Ersin 2374 Tobi, Ibrahim 2275 Torbaghan, Ameneh Eskandari 2341

W Wang, Jiayue 2394 Wang, Junran 2394 Wang, Xiao Dong 2348 Wang, Zhicong 2384 Wu, Hangfeng 2394 Wu, Yuting 2280

X Xu, Hui 2415 Xu, Meng 2354

Y Yang, Li 2348 Yang, Yao 2334 Yao, Hong 2325 Yao, Kun 2334 Yarpuz-Bozdogan, Nigar 2275 Ye, Mei 2415 Yolcu, Ramazan 2319

Z Zhang, Liang 2354 Zhang, Xi 2415

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