Romanian Biotechnological Letters Vol. 20, Copyright © 2015 University of Bucharest

No. 1, 2015 Printed in Romania. All rights reserved ORIGINAL PAPER

Comparison of digestion methods for total content of microelements in soil samples by HG-AAS Received for publication, November 06, 2014 Accepted, December 04, 2014

STANCIU-BURILEANU MIHAELA MONICA1, VENERA MIHAELA STROE1, NINETA RIZEA1, MARIAN MUŞAT2 1 National Research-Development Institute for Soil Science, Agrochemistry, and Environment Protection, Bucharest, Bvd. Mărăşti no. 61, 011464, ROMÂNIA, [email protected] 2 University of Agronomic Sciences and Veterinary Medicine

Abstract Several digestion methods including the reverse of aqua regia and different combinations of concentrated acids (HCl, HNO3) were compared. Three acid digestion procedures were applied to certified samples to offer a proper method for determination of Zn, Cu, and Ni in soil samples. Two low pressure microwave heating programmes modified (nitric acid and Ultrapure water; nitric acid with clorhydric acid) and one procedure without pressure microwave (nitric acid with clorhydric acid and hydrogen peroxide) were tested in this study by using one standard reference material (Estuarine Sediment-1646) and ten soil samples. Short digestion time, less acid consumption, and high extraction efficiency were noted as the advantages of microwave digestion procedures. The concentration of three heavy metals (Zn, Cu, Ni) in soil samples was measured in three sampling sites of Romania, SouthEastern Romanian Plain, Central and Southern Dobrogea. The concentration of heavy metals was measured by using the atomic absorption spectrometry method and these concentration values were situated in normal limits for these chemical elements. In conclusion, to determine the total contents of heavy metals in soil samples studied, in principle any method of digestion carried out in a microwave oven can be used.

Keywords: acid digestion, heavy metals, soil

1. Introduction Soil is composed of mineral constituents, organic matter (humus), living organisms, air and water, and it regulates the natural cycles of these components [1]. Heavy metals are natural components of Earth’s crust. They cannot be degraded or destroyed. The impurification of water surfaces with heavy metals usually has disastrous effects on the environment. That’s why the determination of heavy metals concentrations at levels of ppm or ppb is strictly necessary. Industrial, economical and demographic development generates huge amounts of wastes which have different chemical composition and variable content in toxic heavy metals. A defective management of these wastes leads to environment pollution and then to its degradation [2]. The metals are classified as “heavy metals” if in their standard state they have a specific gravity of more than 5 g/cm3. There are known sixty heavy metals. Heavy metals get be accumulated in time in soils and plants and could have a negative influence on physiological activities of plants (e.g. photosynthesis, gaseous exchange, and nutrient absorption), determining Romanian Biotechnological Letters, Vol. 20, No. 1, 2015

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the reductions in plant growth, dry matter accumulation and yield. In small concentrations, the traces of the heavy metals in plants or animals are not toxic. Lead, cadmium and mercury are exceptions; they are toxic even in very low concentrations [3]. Heavy metal concentration in agricultural soils can affect human beings directly, through soil ingestion or through the food web by ingestion of crops and animals. Indirectly it causes severe damage of environmental health. Atmospheric deposition of heavy metal, urban– industrial activities and agricultural practices by using agrochemical products are the main anthropic sources of heavy metals in agricultural soils [4]. Some heavy metals are important trace elements in nutrition of plants, animals or humans (e.g. Zn, Cu, Mn, Cr, Ni), while others are not known to have positive nutritional effects (e.g. Pb, Cd, As). However, all of these may cause toxic effects (some of them at a very low content level) if they occur excessively. The bioaccumulation of heavy metals over large territories and long time periods may result in the gradual damage of living organisms, which necessitates careful monitoring of the input, mobility and effects of these pollutants [5]. Plants are good environmental quality indicators and respond directly to air, soil and water quality [6, 7]. Since the plants can naturally draw the pollutants from their local environment, their chemical composition can indicate the degree of disturbances when assessed against background values obtained from unpolluted vegetation [8]. Increased introduction of foreign elements to the plant or excessive presence of some essential and trace elements can result in the toxicity of plant and hence change of colour of leaves, inhibition to the germination of seeds and growth of plants or even death of the plants [9]. Other effects of pollution can be described as inhibitory effects, by the fact that the excessive presence of some elements can result in blocking the uptake of other elements and hence depriving the plant from absorbing essential elements from the soil [10]. Organic matter and pH are the most important parameters controlling the accumulation and the availability of heavy metals in soil environment [11]. The determination of the total content of heavy metals in soils and sediments is particularly useful to collect information on the genesis of the soil and on the level of contamination [12]. Analytical instruments and techniques have been developed over the past 30 years to determine the concentrations of metals in our ecosystem; atmosphere, water, soils and sediments. In most studies, high pressure is applied to the samples by using high technology digesters [13]. Since the 1980s, microwave-assisted sample digestion techniques have become popular and are widely used. Microwave-assisted aqua regia digestion using a Teflon bomb is considered a rapid sample digestion method. This technique provides a rapid, safe, and efficient digestion and is not susceptible to losses of volatile metals [14]. In the present study, ten soil samples and standard reference material [15] were treated by micro-wave assisted digestion. The resulting solutions were analysed by HG-AAS.

2. Material and methods Reagents In this study, all chemicals used were of analytical grade (Merck, Germany). High quality water, obtained using a Milli-Q system (Purelab ELGA, Anglia), was exclusively used. Three different digestion procedures of soil samples have been analyzed. The first version used a mixture of HNO3, HCl and H2O2, in the second variant only HNO3 has been 10108

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Comparison of digestion methods for total content of microelements in soil samples by HG-AAS

used and the third digestion procedure used a mixture of HNO3 and HCl. Forwards, all procedures used are described extensively. Soil samples mineralization procedures I) Wet digestion of soil and parental material samples by HNO3, HCl şi H2O2 in microwave 1 g portion of each sample was placed into a 100 mL PTFE (polytetrafluorethylene) vessel, and 6 ml HNO3 65%, 3 ml HCl 37% and 0.25 ml H2O2 were added. After digestion the samples were cooled for 30 minutes through ventilation; they were kept in the digestion vessels at least 12 hours (overnight) and then diluted to 50 mL with ultrapure water in a volumetric flasks. The phases for the digestion procedure on microwave system for total selenium are showed in table 1. Table 1. The optimal conditions of working parameters Phase

Time (min)

Power (W)

Temp (°C)

1 2 3

15 15 15

850 850 850

150 210 210

Pre-tratation of digestion solutions: reduction of Se(VI) to Se(IV) At the end of the digestion period selenium is reduced from oxidation state +6 to +4 as follows: 3 mL concentrated HCl are added to an extract aliquot which is then heated for 30 minutes at 80°C. Samples were left to cool at room temperature and then diluted to 25 mL with ultrapure water in a volumetric flasks (Table 2) Table 2. Phase of program for pre-reduction Phase 1 2

Time (min) 10 30

Power (W) 450 450

Temp (°C) 80 80

II) For determination of total content element, about 0,25 g finely ground soil sample was weighed with a precision of 0.1mg and digested in a microwave digestion system Ultraclave III (MLS, Leutkirch, Germany) in quartz tubes with 2 mL nitric acid and 2mL Ultrapure water. The tubes were transferred to a teflon support and covered with teflon caps, support settling into the microwave. After closing the system, it was applied a pressure of argon for 4x 106Pa, and the mixture was heated to 250ºC for 30 minutes, following a program of temperature: (a) from room temperature to 80ºC in 5', (2)-150ºC in 15', (3) 150-250ºC in 15', (4) 250ºC for 30' (total time about including ventilation (60'): 2:05h). After mineralization, samples were diluted with Ultrapure water volume of 10 mL polypropylene tubes (Greiner Bio-One, Frickenhausen, Germany). Total element calibration curve is obtained from standard solution of single element: Zn, Cu, Ni in 2%HNO3-3μg/mL 1000 ±100mL, ICC, Lot 0CF092. III) For determination of total content element, about 0,25 g finely ground soil sample was weighed with a precision of 0.1mg and digested in a microwave digestion system Ultraclave III (MLS, Leutkirch, Germany) in quartz tubes with 2 mL nitric acid and 2mL Romanian Biotechnological Letters, Vol. 20, No. 1, 2015

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chlorhydric acid. The tubes were transferred to a teflon support and covered with teflon caps, support settling into the microwave. After closing the system, it was applied a pressure of argon for 4x 106Pa, and the mixture was heated to 250ºC for 30 minutes, following a program of temperature: (a) from room temperature to 80ºC in 5', (2)-150ºC in 15', (3) 150-250ºC in 15', (4) 250ºC for 30' (total time about including ventilation (60'): 2:05h). After mineralization, samples were diluted with Ultrapure water volume of 10 mL polypropylene tubes (Greiner Bio-One, Frickenhausen, Germany). Total element calibration curve is obtained from standard solution of single element: Zn, Cu, Ni in 2%HNO3-3μg/mL 1000 ±100mL, ICC, Lot 0CF092.

3. Results and discussion After digestion of certified reference material with different mixtures of strong acids by microwave and dosage by atomic absorbtion spectrometry, the following observations have been highlighted: - the three digestion procedures performed in the microwave mineralization indicates values of heavy metals (Zn, Cu, Ni) very close between them, values obtained by atomic absorbtion spectrometry; but total contents of Zn obtained by the three methods have almost double values than its concentration in the certified reference material, and the total contents of other trace elements (Cu, Ni) also obtained through the same procedures of digestion situates around concentrations being in certified reference material (Figure 1).

T o tal co n ten t elem en t (m g /kg )

140 Total Zn

120

Total Cu

100

Total Ni

80 60 40 20 0 MRC certified values

MRC values obtained (I)

MRC values obtained (II)

MRC values obtained (III)

Figure 1. Total content of Zn, Cu and Ni in certified reference material resulting from different digestion procedures (I – digestion by nitric acid, hydrochloric acid and hydrogen peroxide; II – digestion with Ultrapure water MilliQ and nitric acid; III – digestion by nitric acid and hydrochloric acid)

- from the digestion procedures, the digestion procedure which use a mixture of nitric acid with ultrapure water in a microwave oven under argon atmosphere according to a specific program gives good results on the total content of heavy metals

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Comparison of digestion methods for total content of microelements in soil samples by HG-AAS

250

Recovery (%)

200 150

Total Zn content Total Cu content

100

Total Ni content 50 0

1

2

3

Procedure digestion

Figure 2. Recovery of microelements content obtained after digestion procedures (1 – digestion by nitric acid, hydrochloric acid and hydrogen peroxide; 2 – digestion with Ultrapure water MilliQ and nitric acid; 3 – digestion by nitric acid and hydrochloric acid)

For determination of heavy metals contents in soil samples for analyse a total of 10 samples analyzed in triplicate have been taken, soil samples being collected from the upper horizon (0-20 cm), South-Eastern Romanian Plain, Central and Southern Dobrogea. In tables 3, 4 and 5 total content of heavy metals obtained by the three digestion procedures performed in the microwave mineralization are presented. Table 3. Total content of zinc (mg·kg-1) in soil samples digested by the three digestion procedures Areas

South-Eastern Romanian Plain

Central Dobrogea Southern Dobrogea

I

Location N Orezu P1 Orezu P1 Orezu Deschidere P1 E Bueşti basis 5-6 cm SE Slobozia V Sudiţi S Ţăndărei N Râmnicu de Jos S Râmnicu de Jos Pietreni loess

II

III -1

54.6±0.1 51.3±0.8 60.5±0.2 47.9±0.2 49.0±0.2 55.7±0.1 42.7±0.1 61.1±0.1 65.8±0.2 60.6±0.1

mg·kg 52.4±1.2 52.4±1.1 57.4±0.8 48.2±1.3 46±1.2 48.2±1.5 38.4±1.1 54.4±0.4 48.1±0.9 47.9±1.6

49.5±0.1 50.3±0.3 54.3±2 45.5±0.7 46.4±5.1 45.5±0.3 37.6±0.4 52.1±0.4 47.4±0.8 45.5±0.3

Table 4. Total content of Cu (mg·kg-1) in soil samples digested by the three digestion procedures Areas

South-Eastern Romanian Plain

Central Dobrogea Southern Dobrogea

N Orezu P1

21.7±0.2

II mg·kg-1 21±0.2

Orezu P1 Orezu Deschidere P1 E Bueşti basis 5-6 cm SE Slobozia V Sudiţi S Ţăndărei N Râmnicu de Jos S Râmnicu de Jos Pietreni loess

22.2±0.3 25.7±0.2 20.3±0.2 19.0±0.2 19.7±0.1 15.7±0.2 19.8±0.3 18.6±0.1 18.3±0.2

21±0.1 22.2±0.4 19.4±0.2 17.4±0.3 18.1±0.2 13.9±0.01 18.5±0.5 17.3±0.2 16.7±0.2

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I

III 19.0±0.1 19.2±0.2 21.0±0.3 17.1±0.6 16.9±0.5 17.1±0.1 13.3±0.3 17.8±0.5 16.1±0.3 15.1±0.1

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STANCIU-BURILEANU MIHAELA MONICA, VENERA MIHAELA STROE, NINETA RIZEA, MARIAN MUŞAT Table 5. Total content of Ni (mg·kg-1) in soil samples digested by the three digestion procedures Areas

South-Eastern Romanian Plain

Central Dobrogea Southern Dobrogea

Location N Orezu P1 Orezul P1 Orezul Deschidere P1 E Bueşti basis 5-6 cm SE Slobozia V Sudiţi S Ţăndărei N Râmnicu de Jos S Râmnicu de Jos Pietreni loess

I 29.5±0.1 30.7±0.2 32.4±0.1 27.2±0.2 29.6±0.1 28.9±0.1 23.5±0.1 29.9±0.1 29.4±0.1

II mg·kg-1 28.4±0.7 25.8±1.2 28±3 26±3 26±1 25±0.3 21±1.2 28±1 26±1

III 23.9±0.5 24.7±1.6 24.2±1.3 23.0±0.1 22.7±1.2 22.1±0.4 18.0±1.4 23.7±0.2 21.3±1.7

29.5±0.1

26±0.5

22.4±1.7

4. Conclusions This study tested two programs of microwave digestion modified by low-pressure (ultrapure water and nitric acid; hydrochloric acid and nitric acid) and a digestion procedure without involving any pressure (hydrochloric acid, nitric acid and hydrogen peroxide) using a standard reference material (Estuarine Sediment-1646) and ten soil samples. The three digestion procedures performed in the microwave mineralization indicates values of heavy metals (Zn, Cu, Ni) very close between them; but total contents of Zn obtained by the three methods have almost double values than its concentration in the certified reference material, and the total contents of other trace elements (Cu, Ni) also obtained through the same procedures of digestion situates around concentrations being in certified reference material. From the digestion procedures, the digestion procedure that use a mixture of nitric acid with ultrapure water in a microwave oven under argon atmosphere according to a specific program gives good results on the total content of heavy metals. In conclusion, to determine the total contents of heavy metals in soil samples studied, in principle any method of digestion carried out in a microwave oven can be used.

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