A R T I C L E  I N F O		ABSTRACT
ORIGINAL ARTICLE		Introduction: This study aimed to assess a good protocol for the contamination indexes, concentration, spatial analysis, and source identification of toxic metals in top soils. Materials and Methods: In the first step, samples were taken from top soil (30 cm) and the metals were extracted and detected with ICP-AES. In the second step, Enrichment Factor, Geoaccumulation Index, and Contamination Factor of metals were calculated to determine soil contamination degree. Furthermore, the principal component analysis and correlation between metals were conducted for source identification. Results: Spatial analysis, as an important section of the present protocol, was performed using Arc GIS, kriging, and Moran's I models. As results of Moran's I model showed, distribution pattern for Fe, As, Cd, Cu, Ni, Pb, and Zn were random (z-scores ranged from -1.17 to 1.09), indicatingthat these elements could be emitted from different potential sources. In Moran's model, spatial autocorrelation of each pollutant could be measured based on its value and location. Conclusion: The finding of this protocol can be used for extraction of contamination indexes, concentration, spatial analysis, and source identification of toxic metals in top soils.
Article History: Received: 22 September 2020 Accepted: 20 November 2020
*Corresponding Author: Amir Mohammadi Email: mohammadiurm@gmail.com Tel: +98 9177094695
Keywords: Environmental Pollution, Metals, Heavy, Spatial Analysis, Moran's model.

Introduction
The soil is part of environment in earth, that is largely  exposed to heavy metal aggregation ¹. Soil pollution with heavy metals (HM) possibly results in contamination of rural products and causes other unhealthy impacts The soil pollution by HM is associated to nature and texture of soil, content of clay fraction, and its physicochemical quality ². In the cities and villages residential regions, HM in soils can penetrate the human tissues through direct skin contact, ingestion, and breath. Consequently, they may  gather in various body tissues including the kidneys, bones, and liver ³. The main toxic heavy metal included Pb, As, Hg, Cd, Cu, and Cr. The Cu had a harmful effect on the liver and kidney role, lung cancer, and chief nervous system. Long-term contact of lead can harm the nervous system creating symptoms, for example insomnia, memory loss, and headache .The Cr affect gastrointestinal disorders and sometime may lead to death. The high dose contact to Arsenic can be damaging to the skin as well as the respiratory and cardiovascular systems .The Cd accumulation in human tissues can harm the nervous system and resulted to renal ^3-6. In several studies soil pollution was reported by high values of heavy metals; for example, in northern Colombia high values of Pb, Cd and Hg were reported ⁷. In Tabriz, a industrial City of Iran, contamination with Cd, Cu, Cr, and Pb were mentioned ⁸. In some cities of China, high levels of Cr, , Pb, As, Hg, and Cd ⁹ were found. In Zwierzyniec of Poland, contamination with lead and copper was indiacted.¹ In Van region in Eastern Turkey, contamination with Cd, As, Pb, Cr, and Hg was represented ¹⁰. In Gebze town of Kocaeli City in Turkey, contamination with Cd, Pb, Cu, and Co was reported¹¹. This study aimed to found a protocol for the contamination indexes, concentration, spatial analysis, and source identification of the toxic metals in top soils.

Materials and Methods
Study area and sampling points
The study was conducted around Lake Urmia in North-west of Iran. The region of study has about 4000 km² area¹². Also the control station selected in prevented land in south of Urmia city. To select the study location, GIS was used and 32 zones were selected. Furthermore, one control point was selected from unpolluted soil in Urmia (Figure 1). The methods applied to select the study area and sampling points were performed and explained in previous studies ^{13, 14}.

Figure 1: Geographic location of present protocol study (“S: sampling point)”
 

Soil sampling and chemical analysis
To conduct the study, 97 topsoil (0-30 cm) samples were collected in triplicate. Sampling was done in non-rainy days.  During the sampling period, the average temperature was 16 °C. The dry weight of each sample was about 1 kg, which was collected from three subsamples in approximately 5 meters within each sampling zone to prepare the composite ¹⁴.  Samples were packed into bags of polyethylene and moved to the soil lab. Soil samples were dried for 24 hours at 60 °C before analysis; later, they were cleaned and selected using a sieve with size100 mesh (0.15 mm) ^{7, 14} Extraction of HM was done using the USEPA 1996 Technique 3050B. First 0.5 gram of each prepared soil sample was warmed (2 hour at 50 ºC) and poured into the solution with 70 % HNO₃ (9 ml) and HCL (3 ml), and 40 % HF (1 mL). This mixture was mineralized to digested using a Teflon high-pressure digestion vial (Applied Plastics Technology, Inc., Bristol, USA) in 170 °C for 4 h. In next step, digested solution were cooled and then were added H₂O₂ (2 mL - 30%) and concentrated HNO₃ (3 mL) into it ¹⁴. This extraction liquid using Milli-Q water was weakened to 50 mL, and finally, by a micro-porous membrane with the pore size of (0.45 μm) filtered into bottles (PET). Also, a blank digest solution used for quality control.
In last section, HM of soil sample were detected using US EPA Method 200.7 by inductively coupled plasma-atomic emission spectrometry (ICP-AES, Model: Arcous, German),
Indexes of soil pollution
Enrichment factor (EFs)
EF was used to determine the probable pollutant origin, the impacts of unnaturally actions on the grade of soil pollution and health risk. For each metal, EF was calculated separately by formula. (2) based on the formula reccomended by Taylor ¹⁵.

Where, the nth metal concentration (mg/kg) is Cn and Iron concentration is C_Fe (reference metal used for standardization in our study, mg/ kg) in the soil of experienced environment and the reference environment.
In this Eq, normaliization of a HM was used against a reference HM to recognize accumulation of HM in soil. The greatest conventional reference HM were applied in previous studies comprising Al and Fe ^{10, 16}.  Gradation of metal pollution may be categorized  into 7 groups as the rate of EF in soil: no enrichment (EF < 1); minor enrichment (1 ≤ EF < 3); moderate enrichment (3 ≤  EF < 5); moderately severe enrichment (5 ≤ EF < 10); severe enrichment (10 ≤ EF < 25); very severe enrichment (25 ≤  EF < 50); extremely severe Enrichment (EF > 50) ⁷.

Index of geoaccumulation (I_geo)
The geoaccumulation (I_geo) index estimates the levels of HM contamination in n cities and villages soils. The I_geo was founded the including  between the current and pre-industrial HM value ¹⁷. This index is measured by Eq. (3):

Here, C_n are the concentrations of HM evaluated in the soil samples of environment and B_n  is related soil sample of the control area. The coefficient 1.5 use to evaluate usual fluctuations in contents of a given substantial in the environment and to discriminate very slight manmade influences. The l_geo consists  of 7 groups  ⁹: uncontaminated (I_geo ≤ 0); uncontaminated to moderately contaminated (0 <  l_geo ≤ 1); moderately contaminated (1 < I_geo ≤ 2); moderately to heavily contaminated (2 < l_geo ≤ 3); heavily contaminated (3 < I_geo ≤ 4); heavily to extremely contaminated (4 < I_geo ≤ 5), and extremely contaminated (5 ≤ I_geo).
Contamination degree (C_deg)
The another factors to identify soil contamination in heavy metals are the contamination factor (CF) and contamination degree (CD)  ¹⁸. The contamination factor is computed by Eq. (4) regarding value of each HM in the sampling soil point and background soil:
 
The CF is separated into 4 groupings ¹⁰: Low contamination (CF < 1); Moderate contamination (1 < CF ≤ 3); moderate to strong contamination (3 < CF ≤ 6) and very strong contamination (CF > 6).
Additionally, the C_deg is extracted using Eq (5) that considers the sigma of CF for heavy metal pollutants:

The C_deg is divided into 4 cataloging ¹⁰: Low degree of contamination (C_deg < 8); Moderate degree of contamination (8 < C_deg ≤ 16); moderate to a strong degree of contamination (16 < C_deg ≤ 32); and a very strong degree of contamination (C_deg > 32).
Spatial distribution of metal elements and C_deg
C_deg maps were drawn using ArcGIS 10.1 to show the spatial distribution in assessing contamination values of the heavy metals in around Urmia Lake. To create raster layers in independent form for C_deg, the Kriging interpolation technique was applied. Afterwards, the raster computer function was recycled to overlay each layer to create distribution map pollutants averages (Figure 2).

Figure 2: Spatial distribution map of soil samples' contamination with heavy metals
 

The regions with high and low contaminated of HM were highlighted by rane of high and low color . Based on the findings, red region, showing heavy metal concentrations was wider than the green region with low soil contamination.
Spatial autocorrelation
Global Moran's Index (Moran's I) was applied to evaluated the local variation distribution pattern (which can be as a disperse, random or clustered) of metal elements in soil samples. In this model, spatial autocorrelation of each pollutant can be measured based on its value and location. In Moran's I, the null hypothesis showed random distribution of  values across the study area ^{19, 20}. In the spatial autocorrelation model, in addition to Morans I, z-score and P-value can be measured to evaluate significance of the mentioned Index ²¹. The Global Moran's I can be calculated as follows ^{20, 22, 23}:

Where, (S₀) and (n)  are the sum of all spatial weights and the total number of features, respectively; wij is the spatial weight between feature i and j; and zi represents the deviation of an attribute for feature i from its mean (xi – X) ^{23, 24}:

Finally, the ZI-score for the statistic can be calculated as follows:
  
 Where, E[I] and V[I] are equal to -1/(n-1) and E[I2] – E[I]2, respectively ²¹.
In this model, the z-scores lower than -1.65 indicated dispersed emission pattern, z-scores from -1.65 to 1.65 represent random emission, and z-scores higher than 1.65 indicated clustered emission pattern ^{21, 25}. According to the results of Moran's I model in figure 3, distribution patterns for Fe, As, Cd, Cu, Ni, Pb, and Zn were random (z-score ranged between -1.17 to 1.09), indicating that these elements could be emitted from different potential sources.