The Land Snail, Eobania vermiculata , as a Bioindicator of the Heavy Metal Pollution in the Urban Areas of Sulaimani, Iraq

: Land snails are crucial consumers in the terrestrial environment and beneﬁcial signiﬁcant bioindicators to evaluate the chemical impact in the ecosystem, especially on urban lands. The present study aimed to investigate the concentration of heavy metals such as As, Cr, Ni, Pb, and Zn in urban soil and study whether Eobania vermiculata acts as a bioindicator for heavy metal contamination in an urban area. Thirty soil and snail samples in triplicate from each sampling site were taken from the urban areas of Suliamani. After a microwave-assisted digestion procedure, every sample was analyzed by inductively coupled plasma-optical emission spectrometry. Results showed that the concentration of chromium (Cr) in each snail sample was signiﬁcantly high. The maximum Cr concentration (15.87 mg kg − 1 ) was recorded in the snail sample collected from Ali Kamal Park, which was adjacent to a very crowded trafﬁc road. The As concentration in snail samples ranged from 0.08 to 1.004 mg kg − 1 , and it was below the permissible limits. However, the concentrations of heavy metals in urban soil locations were below their background measurements, except for nickel (Ni) which was above the permissible limits. The safest site in the study area was Chaviland 1, while the most contaminated site was the Ha-wary Shar Park. The snails bioaccumulated metals in their tissues in the following order, Cr > Zn > Ni, and this bioaccumulation occurred more on the main road locations, which represented potentially contaminated places due to anthropogenic activities. Moreover, there was no correlation among the heavy metals within the soil samples when compared to the similar metals in the snail samples, due to the low concentration of heavy metals in soil, excluding Ni, from where the snail samples were collected. Consequently, the land snail, E. vermiculata , is an appropriate sentinel organism for some metals, mainly for Cr, and the bioindicator monitoring with this snail should be extended to mixtures of heavy metals, since such relationships frequently occur in soil.


Introduction
Trace elements are natural parts of the earth layer and cannot be degraded. Due to their toxic nature, the regular release of these elements produces risk to both environment and human health through the food chain and other human exposure pathways [1][2][3]. One of the crucial sources of soil contamination are anthropogenic activities such as combustion of fossil fuels, smelting, and the use or production of metallic compounds, which are accountable for most of the trace element contamination [4,5]. Crowded traffic roads may emit massive pollutants such as heavy metals into the environment (soil, air, and water) and can pose severe threats to human health because of their persistency and bioaccumulation in the terrestrial environment [6][7][8]. The heavy metals may also pose threats to living organisms by entering into the food chain through biomagnification [9].
Land snails found in terrestrial environments are sensitive to pollutants and soil acidification [10]. These are available for collection in various environments including urban areas. These organisms can tolerate many contaminants, including heavy metals, through bioaccumulation [9]. Consequently, land snails have been recommended as premeditating animals for bioindication and biomonitoring heavy metals under terrestrial environments, as these can accumulate metals in their tissues [11][12][13][14].
For this reason, in the past two decades, many researchers around the world have focused on the bioaccumulation of trace elements via snail species [15]. The trace element concentration variations in snails depend on the nourishing system in the snail, the cultivation activities, and the bioavailability of metal in the natural environment. Snails can be distinguished from other groups of mollusks as they can accumulate higher concentrations of trace elements [16]. Thus, snails can provide an opportunity to evaluate trace contaminants by acting as bioindicators [17,18].
Accordingly, for monitoring contaminant transferring from diverse sources, terrestrial snails can be used [19,20]. However, land snails in some places have been eaten by humans as well as consumed by numerous animals (hedgehogs, carabid beetles, and thrushes) [21]. In the past, after France, one of the places where land snails were consumed the most frequently was Andalusia in Southern Spain, whereas, in the 1980s, Morocco and other countryside comparatively consumed more edible land snails than Spain and France [22].
Based on the above, it has been hypothesized that the land snails may serve as bioindicators of trace elements contamination in the terrestrial environment. The objective of the present study was to investigate the concentration and distribution of heavy metals (As, Cr, Ni, Pb, and Zn) in Sulaimani's urban soil and the native land snail, Eobania vermiculata, in urban areas and to estimate its potential role as a bioindicator for soil heavy metals contamination.

Study Area
The present research work was conducted in Sulaymaniyah, which is one of the major cities in the Kurdistan region of Iraq. The Kurdistan region is located in the north of Iraq and is famous for its mountains. The soils are calcareous. It borders Iran to the east, Syria to the west, Turkey in the north, and Saudi Arabia in the south ( Figure 1).

Sample Collection
The snail and soil samples were collected from urban areas of Sulaymaniyah during autumn and early spring 2020, as the snails remain active during these seasons. Thirty composite samples from each soil while the snail samples were taken from public parks and gardens, according to the availability of the snails in the parks. All 30 soil samples with 30 corresponding land snail samples, each containing 5 individuals, were collected from the same parks that snails are habitually available at and survive in, in all directions (north, south, east, and west) of the city. For each sampling area, 5 composite topsoil samples (01 kg) from 1-15 cm were collected from the surface around snail locations at each time to be representative of different fields, and 30 snail samples were collected in the area of the same location (Table 1, Figure 2).

Sample Collection
The snail and soil samples were collected from urban areas of Sulaymaniyah during autumn and early spring 2020, as the snails remain active during these seasons. Thirty composite samples from each soil while the snail samples were taken from public parks and gardens, according to the availability of the snails in the parks. All 30 soil samples with 30 corresponding land snail samples, each containing 5 individuals, were collected from the same parks that snails are habitually available at and survive in, in all directions (north, south, east, and west) of the city. For each sampling area, 5 composite topsoil samples (01 kg) from 1-15 cm were collected from the surface around snail locations at each time to be representative of different fields, and 30 snail samples were collected in the area of the same location (Table 1, Figure 2).
A clean sampling trowel was used to collect soil samples, which were put in polyethylene bags for transferring to the research laboratory before being air-dried for heavy metal determination concentrations. In addition, the sample snails were placed in a special plastic container and transferred on the same day to the laboratory. The size width of the E. vermiculata shell was 20-31 mm, and the shell height was around 13-22 mm.

Soil and Snail Samples Processing
The dried soil samples were gently crushed, and a proper soil grinder module was used to grind the soil, sieved by using the <2 mm sieve, and the soil was homogenized as powder.
Snails were placed in the plastic tube and transported to the laboratory. On the same day of sampling, snail samples were divided into two groups: one was submerged in ethanol (70%) for detecting the genome of the snail species. The molecular study was carried out to characterize and identify the strains of snail genetically by amplifying and sequencing the partial mitochondrial cytochrome oxides 1 gene (pmtCO1).    A clean sampling trowel was used to collect soil samples, which were put in polyethylene bags for transferring to the research laboratory before being air-dried for heavy metal determination concentrations. In addition, the sample snails were placed in a special plastic container and transferred on the same day to the laboratory. The size width of the E. vermiculata shell was 20-31 mm, and the shell height was around 13-22 mm.

Soil and Snail Samples Processing
The dried soil samples were gently crushed, and a proper soil grinder module was used to grind the soil, sieved by using the <2 mm sieve, and the soil was homogenized as powder.
Snails were placed in the plastic tube and transported to the laboratory. On the same day of sampling, snail samples were divided into two groups: one was submerged in ethanol (70%) for detecting the genome of the snail species. The molecular study was carried out to characterize and identify the strains of snail genetically by amplifying and sequencing the partial mitochondrial cytochrome oxides 1 gene (pmtCO1).
The other group of samples was starved for three days in the laboratory to empty their digestive tracts. Then, these were put in distilled water for one day, and whole soft tissues of dead snails were dissected properly and placed in an oven to dry at 105 • C for 18 h before milling with a ceramic mortar and pestle.

Soil pH and Organic Matter
The pH of each soil sample was measured by using around 5 g of <2 mm sieved, air-dried soil after suspension in 25 mL of distilled water and shacked at 40 rpm for half an hour. A Hanna pH 209 pH meter with a combined glass electrode (Ag/AgCl; PHE 1004) calibrated at pH 7.0 and 4.01 was used to measure soil pH.
The percentage of organic matter sample was estimated by using the loss on ignition (LOI) method. An identified weight of < 2 mm oven-dried soil in a pre-weighed ceramic crucible was placed in a muffle furnace (Gallenkamp) overnight at 550 • C, to combust organic matter. Then, for cooling purposes, each crucible and combusted soil sample was placed in a desiccator before the weighing and calculation of organic matter (%). Soil pH and organic matter were measured by using five replicates of each sample.

Soil and Snail Digestion
From every location site, three samples of soil and snail were digested by microwave acid digestion (Method 3051A, microwave-assisted acid digestion of sediments, sludge, soils, and oils). Therefore, the total number of digested samples (soil and snail) was equal to 180 samples. This microwave extraction method was designed to mimic extraction using conventional heating with nitric acid (HNO 3 ), or nitric acid and hydrochloric acid (HCl), according to EPA Method 200.2 and Method 3050. This method applies to the microwaveassisted acid extraction/dissolution of sediments, sludge, soils, and oils for the following elements: Aluminum, Arsenic, Antimony, Boron, Barium, Beryllium, Cadmium, Calcium, Chromium, Cobalt, Copper Iron, Lead, Magnesium, Manganese, Mercury, Molybdenum, Nickel, Potassium, Selenium, Silver, Sodium, Strontium, Thallium, Vanadium, and Zinc.
Each powdered sample (0.5 g) was taken into the vessel and 9 mL of HNO 3 + 1 mL H 2 O 2 were added, then digested by using Multiwave GO Plus microwave digestion (Antonpaar). All the digested samples were filtered with Whatman filter paper no. 41. Next, the snail samples were diluted to 25 mL with deionized water.
After digestion, the concentration of heavy metals in all the samples was analyzed by using an inductively coupled plasma-optical emission spectrometry (ICP-OES) instrument (iCAP™ 7600, Thermo Fisher Scientific, USA). The ICP-OES parameters/conditions were as follows: Detector type = High-performance solid-state CID86 chip, View direction = Radial, Nebulizer = V-groove, Spray chamber = Glass cyclonic, UV exposure time = 15 s, UV RF power = 1150 W, UV nebulizer gas flow = 0.5 L min −1 , VIS exposure time = 5 s, VIS RF power = 1150 W, VIS nebulizer gas flow = 0.5 L min −1 , Cool gas flow rate = 12 L min −1 , and Auxiliary gas flow rate = 0.5 L min −1 .

Quality Control
All reagents used in the present study were of analytical grade, e.g., HNO 3 and H 2 O 2 were purchased from Biochem France (analytical grade), while ICP-standard solutions, i.e., ST 1 , ST 2 , and ST 3 , having different concentrations of multi-elements standard QSC (27E) with a CRM certificate, were purchased from Chem-Lab Belgium (Supplementary Materials).

Data Analysis
The collected data were statistically analyzed regarding mean, standard deviation, and correlation, by using the Statistical Package for Social Sciences (SPSS) version 26. When testing null hypotheses, a probability level of p ≤ 0.05 was set to be statistically significant. Pearson correlation coefficients and linear regressions were calculated to detect the associations between the concentration of heavy metals in soil and snail samples. A two-way ANOVA was used to analyze the difference in heavy metals concentration under different sampling locations.

Concentration of Heavy Metals in Soil Samples
The descriptive statistics of the collected soil data are shown in Table 2. The pH of soils ranged from 7.94 to 8.79, showing that the soils in the study area were calcareous in nature, which have earlier been classified as sandy, silty clays, and silty loams [23]. The minimum pH value (7.94) was documented at Chwarbax Park next to Ayubia primary school, while maximum pH values (8.79) were noted at Sheikh-Muhi-Aldin Park next to Delan primary school. The pH variance represents the dissimilarity in the type of soil and underlying geology [24]. In addition, the highest soil organic matter (SOM) was documented at Woluba Park was (13.79%), whereas the Mamostayan park next to the Sheren preparatory school was recorded as the least SOM (7.18%) ( Table 2). In Iraq, there is the lack of an official guideline for standard concentrations of heavy metal in urban soils; thus, Canadian standards for heavy metal concentration in soil were used to compare the metal concentrations in urban soil ( Table 2).
The results of the descriptive statistics showed that the mean values of As, Pb, and Zn were below Canadian standards, except for Ni in all and Cr in half of the soil samples. Moreover, the concentration of Cr in the soil samples from half of the location sites was greater than Canadian standards ( Table 2). The metal concentration in Sulaimani urban soils is in the order of Ni > Cr > Zn > As > Pb. The concentration of As, Zn, and Pb in soil samples was below Canadian standards for heavy metal concentration in soil ( Table 2).
The concentrations of As ranged from 1.33 to 4.46 mg kg −1 , and these ranges were below the Canadian standard limit (70 mg kg −1 ); this might be due to the location of the sampling sites in general, which were far away from the mining sites and hazardous waste sites. Another reason might be the non-utilization of As-containing pesticides in soil parks, which have been applied in the past and are much more likely to have elevated As levels [25,26]. Earlier, a study from Kurdistan province in Iran reported high As concentrations in the soil in the cities of Qorveh (48.5 mg kg −1 ) and Bijar (107 mg kg −1 ). This elevation in As level was suggested to be due to the winding-down stages of "Tertiary-Quaternary" volcanic action, which slightly contributed to As in agricultural soils. This, in conjunction with watering arable lands with groundwater and travertine springs, enhanced severe As contamination of the agricultural soils [27]. Similar to the findings of the present research, another study in Bangladesh showed a peak level of 1.85 mg kg −1 for As, and this contamination was connected to the level of As in tube-well water, deepness, and age of the tube well [28]. A previous study reported that the level of As in urban soils from Galway (Ireland) was 8.6 mg kg −1 , and it was suggested that contamination was due to the burning of coal and peat for house heating [29]. * Each value represents mean ± SD where n = 3; OM = organic matter; NL ** = no limit given by Canadian standards. The bold values represent the concentration of metals above the permissible limit given by Canadian standards.
The Pb concentration ranged from 0.748 to 11.564 mg kg −1 , and it was below the Canadian standard limit (12 mg kg −1 ), except for one location site in Hawkary park that recorded 18.373 mg kg −1 , which was higher than the permissible limit. The mentioned area was located in a very crowded traffic road and this elevation in Pb concentration might be due to the excessive combustion of fossil fuel. It may also be due to the traffic density on the main roads and streets as well as different anthropogenic activities, or due to geologic background concentration in those areas. The results are comparable with the concentrations of Pb in the cities of Fallujah (3.82 mg kg −1 ) and Baghdad (8.34 mg kg −1 ) in Iraq [30,31]. However, the concentration of Pb in Dohuk (Iraq) was more than the current study (35.36 mg kg −1 ), which was suggested to be due to the translocation of heavy metals from polluted wastewater utilized for irrigation and the distance of farmland from the roadside [32].
Zn concentration ranged from 30.57 to 98.58 mg kg −1 , and it was below the Canadian standards (200 mg kg −1 ) ( Table 2). The results showed that the Zn concentration values noted in the soil samples from different locations in the present study were greater than that observed in the soil samples from the cities of Fallujah (5.50 mg kg −1 ), Haweja (51.33 mg kg −1 ), and Baghdad (33.06 mg kg −1 ) in Iraq [30,31,33]. This difference in Zn concentration might be due to soil liming with squander lime (from non-ferrous metal smelting), containing intemperate sums of overwhelming metals, which is reported as a local source of environmental contamination and potential health danger [34]. Besides, Daik Park is the third contaminated soil site, which was a great prison in Sulaimani in the past. On the other hand, the Zn concentrations in Wien (Austria), Palermo (Italy), Guangzhou (China), and Havana (Cuba) were more than the current study (137 mg kg −1 , 138 mg kg −1 , 277 mg kg −1 , and 240 mg kg −1 , respectively) [35][36][37][38].
Cr in all the soil samples ranged from 26.17 to 95.44 mg kg −1 ; more than half of the sampling sites were above Canadian standards, i.e., 64 mg kg −1 ( Table 2). Combustion of natural gas, oil, and coal as well as the chemical manufacturing industries are the two largest sources of Cr emission in the atmosphere. Therefore, most of the sites that recorded the maximum level of Cr were located on high-density traffic roads and streets while two locations were near the petrol station. These Cr samples above Canadian standards were greater than Fallujah and Baghdad in Iraq (11.59 and 0.84 mg kg −1 , correspondingly), and Wien (Austria), Palermo (Italy), Guangzhou (China), and Galway (Ireland) (36, 34, 22.4, and 33.3 mg kg −1 , respectively) [39]. However, a very high level of Cr, i.e., 310.5 mg kg −1 , was recorded in the town of Haweja in Iraq [33].
Ni concentration in all soil samples ranged from 48.38 to 95.44 mg kg −1 , and these were above the Canadian standard limit (45 mg kg −1 ). Besides, this might be due to the combustion of fossil fuels from human activities on the main road (anthropogenic source) because it is one of the major sources of Ni contamination in the soil. The other reason could be the excessive use of fertilizer and lime in the parklands [40][41][42][43]. Moreover, it may also be due to metal plating industries, industrial waste materials, and electroplating. It is released into the air by power plants and trash incinerators, settling to the ground after undergoing precipitation reactions. The highest Ni value (95.44 mg kg −1 ) was recorded in Chavy land 1, although it is the safest place in the study area, it was highly contaminated with Ni. The reason might be the geological soil background in that site location specifically, and in Sulaimani urban soil generally, or due to excessive utilizing of lime, manure, and fertilizer. The concentration of Ni recorded in the present study was higher compared to the soils in other urban cities in Iraq as Dohuk (32.24 mg kg −1 ), Haweja (152.3 mg kg −1 ), Fallujah (8.96 mg kg −1 ), and Baghdad (46.31 mg kg −1 ), as well as several other cities around the world, i.e., Wien (28 mg kg −1 ), Palermo (17.8 mg kg −1 ), Guangzhou (11.1 mg kg −1 ), and Galway (20.7 mg kg −1 ). This difference of heavy metal concentrations in dissimilar towns and cities reflects the effect of various factors, for instance, very crowded traffic roads, type of soil material itself, nature of anthropogenic activities, or maybe due to atmospheric conditions [40].
Regarding the metals correlation, the bivariate Pearson correlation was used to calculate the correlation between these heavy metals with organic matter (%) and pH (Table 3). Pearson correlation makes a sample correlation coefficient, r, which measures the strength and direction of linear associations among couples of continuous variables [44]. A significant positive correlation was found between pH with Cr (0.022) and As (0.072), whereas the organic matter (%) was significantly correlated with Zn (0.048) only. On the other hand, the correlation was not statistically significant between organic matter (%) and all metals, except Zn. Moreover, when the organic matter (%) was increased, the concentration of metals (except Zn) was decreased. In addition, among the metals, Cr was correlated significantly with other metals (Pb, As, and Ni; 0.031, 0.000, and 0.000 respectively), except Zn (0.143). However, Zn metal was significantly correlated with lead (Pb), solely. Besides, As had a significant correlation with Cr and Ni, (0.000 and 0.002, respectively). Moreover, Pb had a highly significant correlation with Zn and Cr (0.000 and 0.031, respectively).

Concentration of Heavy Metals in Snail Samples
Thirty samples of land snail species (Eobonia vermiculata) in urban parks in Sulaimani had been analyzed, and the maximum and the minimum mean concentration results of the heavy metals in snails were as follows: 0.99 to 15.87, 0.044 and 10.92, 70.74 and 203.63, 0.32 to 2.25, and 0.08 to 1.00 mg kg −1 for Cr, Ni, Zn, Pb, and As, respectively ( Table 4). The order of metal concentration in Sulaimani urban snails was in the following order, i.e., Cr > Ni > Zn > Pb > As. There is no standard guideline regarding the permissible limits of metals in snails and these are regarded as a beneficial source of food, therefore, the recommended maximum permitted limit value for heavy metals by the Codex Alimentary Commission of the WHO was used [45].   per billion) for Pb, As, Cr, Ni, and Zn, respectively; NL ** = no limit given by the WHO. The bold values represent the concentration of metals above the permissible limit given by the WHO.
The As concentrations of this current research in all urban land snails examined in the city of Sulaimani were between 0.08 and 1.00 mg kg −1 , which was below the permissible limit [46]. In the state of Akwa Ibom, Nigeria, the concentration of As ranged from 0.21 to 0.81 mg kg −1 , which was similar to the current study [46]. In Can Gio, Vietnam, the higher As concentrations were reported in four snail species (Chicoreus capucinus, Littoraria melanostoma, Cerithidea obtusa, and Nerita balteata) were 10.9, 3.64, 4.32, and 2.83 mg kg −1 , respectively, and these were higher than the permissible level and dissimilar to the present study findings [47].
The concentration level of Cr in all urban land snail-sampling areas in Sulaimani ranged from 0.99 to 15.87 mg kg −1 , and these were greater than the recommended maximum permitted limit value for heavy metals by the Codex Alimentary Commission [45]. The higher value might be due to road dust from catalytic converter erosion, asbestos brakes, topsoil, and rocks in the sample location sites. Although, the Malaysian Food Act 1983 and Regulation 1985 as well as WHO/FAO 2004 did not set any limit for Cr in bivalves [48]. In comparison to the present study, a lower concentration of Cr in snails (Achatina achatina) in Nigeria was reported by Nwoko et al. [49], i.e., 1.00 mg kg −1 . Another study from the Benue River reported Cr concentration as 92.9 mg kg −1 in Coptodon zillii and 88.5 mg kg −1 in Clarias gariepinus, which was much higher than the current study [46].
Pb is one of the toxic metals, which has adverse health effects (cardiovascular, nervous, urinary tract, and reproductive system) in humans. As previously reported, liver and kidney damage is one of the Pb effects on human organ health [49]. The maximum permissible limit for Pb is 1.5 mg kg −1 , as indicated by the Food and Agriculture Organization/World Health Organization [45]. In the present study, the concentration of Pb in land snail (Eobonia vermiculata) analyzed in the city of Sulaimani ranged from 0.32 to 2.25 mg kg −1 , with around a quarter of the samples above the maximum permissible limit.
Seven out of thirty location sites in Sulaimani recorded the higher Pb concentration, which was higher than the maximum permissible limit recommended for heavy metals as stated by the Codex Alimentarius Commission [45]. Principal Pb contamination sources may include the gaseous emissions from vehicles and unprocessed wastes from manufacturing plants, which pollute crop plants via the soil due to the flow of wastes into the irrigation channels [55].
The concentrations of nickel (Ni) in all the land snails samples inspected in the city of Sulaimani ranged from 0.07 to 10.92 mg kg −1 , which was greater than the permissible limits, i.e., 0.1 mg per liter [45]. The Ni values in half of the snail samples were above the permissible limit. This might be due to the excessive concentration of Ni in all soil sites that the snails are exposed to. Research conducted in Can Gio, Vietnam reported Ni concentration in four snail species as C. capucinus (2.07), L. melanosome (3.34), C. obtuse (6.15), and N. articulate (3.58 mg kg −1 ), respectively [47]. In another research study conducted at Ancona (Italy), Ni concentration in the snail H. aspersa from five urban sites ranged from 0.39 to 2.64 mg kg −1 [13]. Another study reported Ni concentration tested in snail (Nerita lineata) between 3.37 and 28.67 mg kg −1 [17]. The maximum concentration of Ni, i.e., 28.67 mg kg −1 , in the mentioned study was higher than the present study. Hence, these maximum concentrations of some heavy metals in snail samples are crucial evidence that snails are significant bioindicators especially for Cr, Zn, and Ni, while not important bioindicators for As and Pb metals in urban soil in Sulaimani.
Overall, Table 5 showed the relationship between heavy metals in soil and snail, which had no associations among the heavy metals in the soil and the corresponding metals in the snail. For that reason, the bioaccumulation of heavy metal in gastropods tissues was based on the species of metal, the environmental habitat, and the physiological situation [56]. Table 5. Pearson correlation and R-squared (R 2 ) among the same element in soil and snail; the bold numbers are R-squared (R 2 ) of each metal in both soil and snail samples, and the other numbers are Pearson correlation within the metals in each soil and snail samples.

Pb in Soil
As The difference noted in the values of heavy metals concentration in the current study and earlier studies might be due to the difference in the snail species investigated, the body size of the animal, the main road or street distance where the snails were collected, environmental emissions, and the nature of the studied places [13,52].
In general, few data, comparatively, are available on relationships between extractable soil concentrations and concentrations in wild animal tissues [57][58][59]. The current study result was similar to a study in the Banat area of Romania, which collected soil and Roman snail (Helix pomatia) samples from eight locations, concluding that all snails originated in habitats with low heavy metal accumulation in soil. In addition, it was determined there was a lack of association between the concentration of heavy metals in snail tissues with the same metal value in soil and nettle leaves, except for Cu levels in soil [9]. This differential accumulation of heavy metals in the body of snail was dependent on the origin of the area under study and the snail body part investigated. The results of another study conducted at Metaleurop Nord, Nord-Pas-de-Calais, France, showed that the species impact was the greatest significant variable explaining the soft tissue concentrations of snails [60]. A similar study result in the Netherlands, which investigated three species of earthworms in a moderately contaminated Dutch floodplain, found that the soil concentrations were non-significantly correlated with worm tissue concentrations [61].
Regarding, the molecular characterization and identification of the snail species, the snail samples were morphologically different. All types of snail samples were subjected to molecular characterization through polymerase chain reaction and found positive as visualized on 1% agarose gel electrophoresis. After blasting sequences in NCBI, the sequence analysis and phylogenetic tree indicated the common land snail in Sulaimani/Iraq was Eobania vermiculata. The sequences were deposited in Genebank under the following accession numbers: Gnus = Eobania with some genetic variation (MZ486014, MZ486015, MZ486016, MZ486017, MZ486018, and MZ486019).
As the concentration of heavy metals (except Ni) in the soil samples was lower than Canadian standards for soil guidelines, it might be the reason for the lack of correlation between the heavy metal accumulations in both soil and snail samples.

Conclusions
The concentration of Ni in the soil samples and half of the Cr samples was greater than the permissible limits as given by Canadian standards of soil quality, while the other metals (As, Pb and Zn) were lower than Canadian standards. This might be due to the combustion of fossil fuels from human activities on the very crowded traffic road (Anthropogenic source) and to excessive use of fertilizer and lime in the parklands. It may also be due to the type of soil material itself (natural source) and atmospheric condition. The snails bioaccumulated certain metals (Cr > Zn > Ni), and this bioaccumulation occurred more on the main road locations, which represent potentially contaminated places due to anthropogenic activities. Therefore, the results of the present study suggest that the terrestrial snail (Eobonia vermiculata) can be a good bioindicator for Cr. Moreover, no correlations among the heavy metals within the soil samples were recorded when compared to the similar metals in the snail samples. It was mainly due to the low concentration of heavy metals, except for Cr and Ni, in the soil samples from where the snail samples were collected.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.