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1 STATISTICAL CORRELATION BETWEEN HEAVY METAL CONCENTRATIONS, DETERMINED BY XRF AND AAS ANALYTICAL METHODS, IN BRASSICA OLERACEA SAMPLES USED AS BIOINDICATOR. IOANA DANIELA DULAMA 1, ELENA DANIELA CHELARESCU 2, O. G. Duliu 3 1 Valahia University of Târgoviste, Multidisciplinary Research Institute for Science and Technologies, Târgoviste, , Romania 2 National Institute for Physics and Nuclear Engineering, Magurele Ilfov , Romania Corresponding authors: Elena Daniela Chelarescu, dana_chelarescu@yahoo.com 3 University of Bucharest, Faculty of Physics, Bucharest-Magurele, Romania Received January 12, 2015 Abstract.Analytical methods, Energy Dispersive X-Ray Fluorescence (EDXRF), Flame Atomic Absorption Spectrometry (FAAS) and Grafit Furnace Atomic Absorbtion Spectrometry (GFAAS), have been applied in this study of heavy metal concentrations in brassica oleracea samples used as bioindicator. The concentrations of heavy metals Cu, Fe, Mn, Ni, Zn, Cd and Pb in 36 samples of Brassica oleracea were determined. The samples were collected from agricultural area of the village Petreşti from Dâmboviţa County, which is located between Argeş and Neajlov rivers. The determined values of heavy metals concentrations showed that the plant Brassica oleracea is a very good bioaccumulator of heavy metals. The results were compared with the maximum admitted limit(mal). The aim of this work was to perform a statistical interpretation of the heavy metals concentrations in Brassica oleracea samples, to determine the relationship between the metals concentration using the model of correlation and binare regression. A linear correlations between pairs of concentrations of the elements Cu-Fe, Fe-Zn, Zn-Cu, Fe-Ni, Fe-Cd, Cu-Cd, Cu-Pb and Cd-Pb was observed. 1. INTRODUCTION To identify heavy metals in samples of the environment and determine their concentrations can be applied atomic and nuclear analytical methods of high sensitivity and precision, combined with biomonitoring technique using bioindicators. Among the most used methods of atomic and nuclear analysis of heavy metals from the environment are: Atomic Absorbtion Spectrometry in two variants, Flame Atomic Absorption Spectrometry (FAAS) and Grafit Furnace Atomic Absorbtion Spectrometry (GFAAS)[1-6]; Atomic Emission Spectrometry (AES) in two variants, Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) and Inductively Coupled Plasma with Mass Spectrometer(ICP-MS)[7,8]; Wave Dispersive X-ray Fluorescence (WDXRF), Energy Dispersive X-Ray Fluorescence (EDXRF)[9-14], Particle Induced X-Ray Emission(PIXE)[15-17] and Neutron Activation Analysis (NAA)[17-19]. Bioindicators that can be used in the study of heavy metal pollutants are collected from the flora and fauna in areas affected by pollution, which can be: vegetables, fruits, leaves of plants, mosses and lichens, etc., which have high absorption power of chemical elements in soil and atmosphere[9-13, 20,21]. Using analysis methods, EDXRF, FAAS and GFAAS were determined the concentrations of heavy metals: Cu, Fe, Mn, Ni, Zn, Cd and Pb in 36 samples of Brassica oleracea samples collected from agricultural area of the village Petreşti from Dâmboviţa County, which is located between Argeş and Neajlov rivers. Geographical location of sampling points was established by determining the geographical coordinates with GPS. Concentration values of heavy metals determined experimentally showed that the brassica oleracea vegetable plant is a bioaccumulator very good of heavy metals. The results were compared with the maximum permissible limits. We performed a statistical interpretation of the heavy metals concentrations in Brassica oleracea samples determined by the EDXRF and AAS analysis methods. For statistical interpretation we used the model of correlation and regresion in which it can describe the degree of association (the statistical relationship) between two quantitative variables normally distributed. In this model the degree of statistical relationship between two quantitative variables is expressed by "correlation coefficient" denoted by R. In this model, a measure of the strength of association of the two variables is given by the correlation coefficient values denoted by R, which are smaller in absolute value than 1 [22,23]. For values of R less than 0.25 between the two variables there is no correlation, for R (0.25, +0.50] - poor
2 correlation, for R (0.50, +0.75] - moderate correlation and for R (0.75, +1) - strong correlation [22,23]. We determined the linear correlations between pairs of concentrations of the elements Cu- Fe, Fe-Zn, Zn-Cu, Fe-Ni, Fe-Cd, Cu-Cd, Cu-Pb and Cd-Pb in samples of leaves of Brassica oleracea. 2. MATERIALS AND METHODS 2.1 Sampling and sample preparation Brassica oleracea samples were collected from six different points of Petreşti agricultural area, Dâmboviţa. Geographical location was performed using a GPS. Each of the 6 samples collected was divided into 6 other samples: outer leaves, leaves intermediate, core, spine leaf, stem and root. The resulting 36 samples were washed and oven dried at 40 C. After drying, the samples were subjected to a grinding process to be crushed and kept in sterile polypropylene vials. The method for the preparation of samples for EDXRF experiments consisted of weighing a quantity of milled material (in powder form), amount of 3-5 g of each sample placed in plastic containers provided with Mylar windows of 6 microns, containers optimally adapted to the geometry of EDXRF spectrometer. To verify the correctness of experimental measurements we used a standard reference material NIST1515-apple leaves [24] prepared by the same procedure as the samples of Brassica oleracea: dry leaf material, milled, weighed and placed in containers plastic The method for the preparation of samples for AAS measurements was as follows: an amount of approx. 0.2 g of each sample was digested with 8 ml of HNO3 (65%) and 10ml H2O2 (30%) in a microwave digestion system. Digestion was carried out at C, at a pressure of 75 MPa for 20 minutes. The solutions were filtered and placed in a glass flask containing a volume of 50 ml of deionized distilled water. To verify te accuracy of the AAS measurements, under the same conditions, was prepared a sample using the SRM-NIST1515 Apple leaves.[24]. The atomic absorption spectrometry measurements (AAS) were performed using the calibration curve. 2.2 Analytical methods The heavy metals concentrations in Brassica oleracea samples were determined using the X-Ray Fluorescence (XRF) method and Atomic Absorption Spectrometry (AAS) method. The following spectrometers were used: ElvaX Energy Dispersive X-ray spectrometer together with Avanta GBC Flame Atomic Absorption spectrometer and GBC Avanta Ultra Z Graphite Furnance Atomic Absorption spectrometer. 3. RESULTS AND DISCUSSION We verified the accuracy of the measurements using the SRM-NIST1515 Apple Leaves which contains the elements mg, Al, K, Ca, V, Mn, Fe, Ni, Cu, As, Se, Br, Rb, Sr, Mo, Cd, Pb in known concentrations. The values of concentrations of K, Ca, Mn, Cu, Fe, Zn, Cd and Pb obtained by EDXRF, FAAS and GFAAS methods are shown in Table 3.1. A good agreement between the measured values and certified values of concentrations was observed. That fact showed also the complementarity of these techniques: some elements can be determined by EDXRF technique other elements by AAS. Table 3.1 The values of concentrations of K, Ca, Mn, Cu, Fe, Zn, Cd and Pb in SRM-NIST1515 obtained by EDXRF, FAAS and GFAAS methods Concentrations Concentrations Concentrations FAAS Elements certified values (ppm): EDXRF values (ppm): and GFAAS * values (ppm): SRM-NIST1515 K 1.61± ± ±0.19 Ca 1.526± ± ±0.22 Mn 54±3 57.4± ±4.3 Cu 5.64 ± ± ±0.48 Fe 83±5 91.3± ±6.7 Zn 12.5± ± ±0.82 Cd 0.013±0.002 not detected * ±0.003 Pb 0.470±0.024 not detected 0.512*±0.029
3 We performed, in the same experimental conditions as for the standard sample, the experiments of EDXRF and AAS, for different components of Brassica oleracea samples: outer leaves, inner leaves, core, spine of leaves, stems and root. By EDXRF were determined concentrations of Cu, Fe, Mn and Zn. By FAAS was determined the concentration of Ni and by GFAAS were determined the concentrations of Cd and Pb. The average values of heavy metals concentrations determined in the aerial parts Brassica oleracea samples are given in Table 3.2. Aerial parts of Brassica oleracea are denoted as follows: A (outer leaves), B (inner leaves), C (core), D (spine leaves) and E (strain). Table 3.2. The average concentrations of heavy metals (ppm) determined from aerial parts of Brassica oleracea samples Concentrations (ppm) Heavy metal A B Minimum Maximum Ratio Outer leaf Inner leaf C Core D Spine leaves E Stem value value max/min Cu 9.443± 12.88± 14.21± 8.654± 10.35± 8.654± 14.21± Fe 224.7± ± ± ± 165.1± 161.3± ± Mn 87.86± 62.14± 63.55± 49.71± 35.53± 35.53± 87.86± Ni 2.534± 2.305± 2.281± 1.822± 1.719± 1.719± 2.534± Zn 24.83± 40.66± 62.10± 29.81± 12.07± 12.07± 62.10± Cd 1.067± 0.892± 0.727± 0.627± 0.455± 0.455± 1.067± Pb 1.923± 1.263± 1.013± 0.945± 0.682± 0.682± 1.923± Maximum admitted limit (MAL, ppm); Vegetables / Vegetable leaves: Cu(5.0), Zn(15), Cd( ), Pb(0.5). Ratio max/mal From Table 3.2 we observe a higher concentration of Cu and Zn in the core of Brassica oleracea and a higher concentration of Fe, Mn, Ni, Cd and Pb in the outer leaf. Maximum admitted limit it is exceeded, approximately of 1.7 times for Cu, 4 times for Zn, 5 times for Cd and 4 times for Pb. By using PAST software [23], we realized the distribution diagrams of average values of heavy metals concentrations in aerial parts of Brassica oleracea samples. It is known that the PAST software is used to analyze the data as graphs, statistics, analysis of environmental data, etc. The distribuition diagrams of the average values of concentration of Cu, Fe, Mn, Ni, Zn, Cd and Pb from aerial parts of Brassica oleracea samples are shown in the figures 3.3a, b, c, d, e, f, g. a. b. Fig. 3.1a,b. Diagrams of Cu and Fe concentrations in the aerial parts of Brassica oleracea samples
4 c. d. Fig. 3.1.c,d. Diagrams of Mn and Ni concentrations in the aerial parts of Brassica oleracea samples e. f. Fig. 3.1.e,f. Diagrams of Zn and Cd concentrations in the aerial parts of Brassica oleracea samples g. Fig. 3.1g. Diagram of Pb concentrations in the aerial parts of Brassica oleracea samples From the distribution diagrams (fig.3.1) it follows that the outer leaves of Brassica oleracea contain heavy metals in a higher concentrations than inner leaves, core, spine leaves and strain. The results show that the Brassica oleracea plant, by aerial parts, could be a bioaccumulator of heavy metals in atmosphere [25]. Heavy metal concentrations, determined by EDXRF and ASA methods, from samples of Brassica oleracea were interpreted in correlation and regression statistical model [23,24]. The mean values of concentrations (in ppm) of heavy metals from the aerial parts are given in table 3.3. Table 3.3.The mean concentrations (ppm) of heavy metals determined in brassica oleracea samples (leaf outer, leaf inner, core, spine leaf, strain),determined by EDXRF and GAAS + methods. Proba Cu Fe Mn Ni + Zn Cd + Pb + M ± ± ± ± ± ± ± M ± ± ± ± ± ± ± M ± 177.8± 41.69± 2.076± 22.48± 0.828± 1.116± 0.48 M ± ± ± ± ± ± ± 0.053
5 M ± 171.9± 58.69± 1.892± 29.31± 0.853± 1.272± M ± ± ± ± ± ± ± A.M.L Mi (i=1,2,3,4,5,6)- Aerial parts of brassica oleracea samples: mean concentrations A linear correlation between pairs of concentrations of the elements Cu-Fe, Fe-Zn, Zn-Cu, Fe-Ni, Fe-Cd, Cu-Cd, Cu-Pb and Cd-Pb in samples of Brassica oleracea was observed. For linear correlation graphs we used the mean concentrations (in ppm) of heavy metals from the aerial parts given in table 3.3. Linear correlation graphs were made with the Excel software and are presented in Figures 3.2a, b, c, d, e, f, g, h. a. b. Fig.3.2a,b. Linear correlation between the pairs of mean concentrations (ppm), Fe-Cu and Fe- Zn from aerial parts of rassica oleracea samples Fe (m g/ kg) y = x R 2 = Ni (mg/ kg) y = x R 2 = c. d. Fig.3.2c,d.Linear correlation between the pairs of mean concentrations, Ni-Fe and Cu-Zn from aerial parts of rassica oleracea samples Zn (mg/ kg) Cu (mg/ kg) e. f. Fig.3.2e,f. Linear correlation between the pairs of mean concentrations, Fe-Cd and Cu-Cd from aerial parts of rassica oleracea samples
6 g. h. Fig.3.2g,h. Linear correlation between the pairs of mean concentrations, Cu-Pb and Cd-Pb from aerial parts of rassica oleracea samples From figures 3.2 it can see an increasing linear dependence between mean concentrations of heavy metals in aerial parts of Brassica oleracea samples for pairs: Cu-Fe, Fe-Zn, Zn-Cu, Fe-Ni, Fe-Cd, Cu-Cd, Cu-Pb and Cd-Pb. The values of square correlation coefficient, [22,23], for each pair of mean concentrations (Fig.3.2 and Table 3.2) show that there is a moderate correlation between the pairs concentrations Cu-Fe, Fe-Cd, Cu-Cd and Cu-Pb, and a strong correlation between the pairs concentrations Fe-Zn, Zn- Cu, Fe- Ni and Cd- Pb. This demonstrates that the absorption process by Brassica oleracea (as bioindicator) of a heavy metal from soil by plant root and from the atmosphere occurs in correlation with other heavy metal. Considering the average concentrations of heavy metals (Table 3.2) from the aerial parts of Brassica oleracea, which significantly exceed the maximum admitted limit (MAL) and from correlation coefficient values (Table 3.4) we obtained that the absorbtion process of heavy metals by Brassica oleracea plant is a correlative complex process. Table 3.4. The correlation coefficient values obtained by Figure 3.2 Pairs heavy metals R 2 R Correlation Cu-Fe moderate Fe-Zn strong Zn-Cu strong Fe-Ni strong Fe-Cd moderate Cu-Cd moderate Cu-Pb moderate Cd-Pb strong
7 4. CONCLUSIONS This study shows that the brassica oleracea plant can be used as suitable bioindicator for environmental pollution with heavy metals in specially for Cu, Fe, Mn, Ni, Zn, Cd and Pb. From statistical analysis it's obtained that the absorption process by Brassica oleracea, as bioindicator, of an heavy metal from soil by plant root and from the atmosphere occurs in correlation with other heavy metal. The results show that the maximum concentrations of heavy metals Fe, Mn, Ni, Cd and Pb, determined by EDXRF and AAS methods, are in outer leaves; this means that these heavy metals, partially, were absorbed by outer leaves of brassica oleracea from the atmosphere of the geographical zone. Considering that the mean concentrations of heavy metals (Table 3.2) from the aerial parts of Brassica oleracea, which significantly exceed the admitted maximum limit (AML), and from the correlation coefficient values (Table 3.4), it follows that the absorption of heavy metals by Brassica oleracea plant is a correlative complex process. Hence it can conclude that Brassica oleracea samples collected from different geographical zones can be used as bioindicator of the heavy metals pollution. REFERENCES 1. M.B.SPERLING, B. WELZ, Atomic Absorbtion Spectroscopy, Weinheim, Wiley-VCH.,ISBN J.A.C. Broekaert (1998), Analytical Atomic Spectrometry with Flames and Plasmas, 3rd Edition, Wiley-VCH, Weinheim, Germany. 3.S. Apostol, C. Stihi, Spectroscopic techniques and methods used in environmental monitoring. Ed. Bibliotheca, Târgovişte, J. Sardans, F. Montes, J. Peñuelas, Determination of As, Cd, Cu, Hg and Pb in biological samples by modern electrothermal atomic absorption spectrometry, Spectrochimica Acta Part B., 65, (2010). 5. L. Barbes, A.Barbulescu, C.Radulescu, C,Stihi, E.D.Chelarescu; Determination of heavy metals in leaves and bark of Populus Nigra L by Atomic Absorbtion Spectrometry; Romanian Reports in Physics, Vol. 66, No.3, P , C. Radulescu, C. Stihi, I. V. Popescu, I. Ionita, I. D. Dulama, A. Chilian, O. R. Bancuta, E. D. Chelarescu, D. Let, Assessment of heavy metals level in some perennial medicinal plants by flame Atomic Absorption Spectrometry, Romanian Report in Physics, 65(1), 246, L. Yongsik, Atomic Emission Spectroscopy, 2004; ICP/AES; 8. C. STIHI, I.V. POPESCU, A. BANCUTA, V. STIHI, GH. VLAICU, Inductively coupled plasma (ICP) and total dissolved solids (TDS) measurements of surface waters from Ialomita river, Rom. Journ. Phys., 50, Nos. 9 10, pp , G. State, I.V. Popescu, C. Radulescu, C. Macris, C. Stihi, A. Gheboianu, I. Dulama, O. Niţescu,Comparative Studies of Metal Air Pollution by Atomic Spectrometry Techniques and Biomonitoring with Moss and Lichens, Bulletin of Environmental Contamination and Toxicology, 89, 3, (2012). 10. G. State, I.V. Popescu, A. Gheboianu, C. Radulescu, I. Dulama, I. Bancuta, R. Stirbescu, Identification of air pollution elements in lichens used as bioindicators by the XRF and AAS methods, Romanian Journal of Physics, 56, (2011). 11. I. Dulama, I.V. Popescu, C. Stihi, C. Radulescu, G.V. Cimpoca, L.G. Toma, R. Stirbescu, O. Nitescu, Studies on accumulation of heavy metals in Acacia leaf by EDXRF, Romanian Reports in Physics, 64, (2012).EDXRF 12. C. RADULESCU, C. STIHI, G. BUSUIOC, A. GHEBOIANU, I.V. POPESCU, Studies Concerning Heavy Metals Bioaccumulation of Wild Edible Mushrooms from Industrial Area by Using Spectrometric Techniques, Bulletin of Environmental Contamination and Toxicology, 84(5), pp , C.RADULESCU, C. STIHI, G. BUSUIOC, I.V. POPESCU, A. GHEBOIANU, G.V. CIMPOCA, Evaluation of essential elements and heavy metal levels in fruiting bodies of wild mushrooms and their substrate by EDXRF spectrometry and FAA spectrometry, Romanian Biotechnological Letters, 15(4), pp , M. Ataabadi, M. Hoodaji, P. Najafi, F. Adib, Evaluation of airborne heavy metal contamination by plants growing under industrial emissions, Environmental Engineering and Management Journal, 9, 7, (2010). 15. S. A. E. Johansson, J. L. Campbell, K. G. Malmqvist, Particle Induced X-Ray Emission Spectrometry (PIXE), Volume 133, John Wiley&Sons, Inc., S. A. E. Johansson, J. L. Campbell, PIXE: A Novel Technique for Elementals Analysis, Campbell s First Book on PIXE, John Wiley&Sons, Inc., A. Ene, Metode radiometrice de analiză multielementală, Ed. Cartea Universitară, Bucureşti, I.V.POPESCU, M. FRONTASYEVA, C. STIHI, G.V. CIMPOCA, C. RADULESCU, A. GHEBOIANU, C.OROS, G. VLAICU, M. PETRE I. BANCUTA, I. DULAMA, Nuclear and nuclear
8 related analytical methods applied in environmental research, Romanian Journal of Physics, 55(7-8), pp , I.V. Popescu, M. Frontasyeva, C. Stihi, G.V. Cimpoca, C. Radulescu, G. State, A. Gheboianu, C. Oros, O. Cilicov, I. Bancuta, I. Dulama, Atomic and nuclear methods applied in the study of heavy polluting elements, Romanian Reports in Physics, 63, (2011). 20. C.STIHI, C. RADULESCU, G. BUSUIOC G, I.V.POPESCU, A. GHEBOIANU, A. ENE, Studies on accumulation of heavy metals from substrate to edible wild mushrooms, Romanian Journal of Physics, 56, Nos.1-2, pp , C. Radulescu, C. Stihi, I.V. Popescu, I.D. Dulama, E.D.Chelarescu, A. Chilian, Heavy Metal Accumulation and Translocation in different parts of Brassica Oleracea L., Romanian Journal of Physics, 58(9-10), pp , T. Colton, Statistics in Medicine, Little Brown and Company, New York, NY O. Hammer, PAST - PAleontological STatistics, Reference manual, University of Oslo, Certificate of Analysis,Standard Reference Materials 1515, Apple leaves, National Institute of Standards and Technology, USA. 25. Ordinul Nr.975 din 16 decembrie 1998 privind aprobarea normelor igienico-sanitare pentru alimente, Ministerul sanatatii.
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