Introduction

The cement industry is involved in the development of the physical infrastructure of advanced industrial society but generates dust during its production. Cement dust is considered as a major source of toxic air emission [1]. Cement dust contains toxic heavy metals, such as Cobalt (Co), Iron (Fe), lead (Pb), Cadmium (Cd), Chromium (Cr), Nickel (Ni), Manganese (Mn), and arsenic (As) at different proportions. These heavy metals occupy low decay time and strong diffusivity, which in turn result in a considerable enrichment in soil and plants [2-4]. Uptake of heavy metals by two tobacco cultivars, caused a reduction on the growth and protein expression [5], alteration in morphological characters (such as chlorotic spot, stem shortening and leaf curling) and anatomical alterations (rupturing and thickening of cells) of Cenchrus ciliaris L. [6].

Accumulated HMs in soil transferred to invertebrates through the food chain via plants. This approach neglects possible differences in bioaccumulation between invertebrate species. Variability can be expected because of the variation in morphology, physiology, behavior, habitat and food preference between invertebrate species. Because of their lower metabolic rates and relatively simple enzymes system, insects may not be able to detoxify complex chemical compounds that they inhale or ingest with contaminated food. Residues of such persistent chemicals are laid down and transferred to a higher level in the food chain. Insects are usually used as bio-indicators of HMs, due to their mobility, abundance, and the variety of their physiology and morphology [7,8]. They can tolerate the accumulation of HMs in their tissues and organs, and they can survive in polluted soil because of their genetic adaptation [9,10].

Beetles are accumulators of HMs, particularly the remarkably toxic ones stored in gut [11]. Consequently, they are excellent indicators of habitat quality and are widely used in biological surveys. The selected species that belongs to Tenebrionidae; are scavengers feeding on decaying organic waste materials.

Eleodes beetles as essentially scavengers, feeding opportunistically as they wander through their habitat. Eleodes armata is one of the most conspicuous of tenebrionid beetles, not because it is particularly abundant, but because it is more frequently encountered during the daytime. The adult of E. armata is an elongate, shiny black, flightless beetle.

Because of the HMs accumulation, the biochemical stability had changed, causing physiological alterations in the beetles [12]. The detoxification (antioxidant) enzymes in insects are considered to be the defense mechanism against foreign components and have a role in preserve their normal physiological functions. Therefore, the assessment of the enzyme activities in insects exposed to HMs enables to evaluate the quantity of pollution. The exposure to heavy metals may cause stress reactions in insects: such as a reduction in antioxidant enzymes [13], an elevation in catalase (CAT) activity [14] or an increase in malondialdehyde (MDA) activity [15].

In this study, the HMs concentration in the soil, leaf litter and two species of darking beetle E. armata and E. tricostata collected from the region around the factory of cement, western region, KSA were evaluated and compared to those from the reference site. Moreover, the changes in activities of AST and ALT, as well as the protein content were evaluated in the selected two species of beetle tissues collected from the studied sites.

Materials and Methods

The study area

Two studied sites were selected; the first one was the cement site that surveyed at 0.5 km downwind from a cement factory in Al-Ahsa, about 30 Km to the south of Al- Ahsa city, in the Eastern region of Saudi Arabia (Figure 1). This area showed signs of cement dust deposition on the soil and surrounding vegetation. The second site is the reference site that was away from the first one by about 30 km.

Figure 1: Map of the studied region, Al-Ahsa city, Eastern region, Saudi Arabia.

Sampling procedure

The coleopterous insects inhabiting the selected sites were different species. Two species of beetles were selected; E. armata and E. tricostata. Soil, leaf litters and beetles’ samples were collected from the two selected sites. From Each site, ten samples of soil (each weighed about 0.5 kg, were collected from the top 10 cm by using a hand metallic soil scoop) and leaf litter were collected. Soil and leaf litter samples were transported to the laboratory in porous nylon bags. The collected beetles were separated, sorted by species and gender and maintained alive in containers in native soil and plants for 24 h to void gut contents. Beetles were kept under laboratory conditions (25±2°C, 60-70% relative humidity, and L14: D 10 photoperiod) until processing.

Chemical Analyses

For soil analysis, soil samples were air-dried at room temperature and ground to pass through a 0.2-mm sieve. Soil samples were placed in the digester for 8 h at 95°C with 10 ml of concentrated HNO₃. After soil digestion, samples were filtered and were made up to 100 ml by using distilled water. For leaf litter analysis, samples were dried at 70°C for two days and grounded. Samples were digested in concentrated sulfuric acid (H₂SO₄, 95-97%) on hotplate at approximately 270°C. Then, deionized water was added to 50 ml in a volumetric flask. 0.5 g of powdered samples of the leaf litter were carefully weighed in a 50 ml standard flask to which 5 ml of concentrated nitric acid was added. The mixture was left for 24 h and then the samples were digested on a hot plate and the temperature was gradually increased to 100°C. When all nitric oxide fumes were expelled, the temperature was raised and stabilized at 150°C until the mixture was clear. Distilled water was used to dilute the solution.

About 15 beetle samples were collected from each site and were anesthetized in ethanol (95%). After cutting the head, elytra, wings, and legs, the beetles dissected in Petri dishes containing drops of 1% Ringer's saline solution. The separated soft tissues were grounded and the prepared whole-body tissue samples subjected to digestion by adding 5-mL of supra-pure grade concentrated HNO₃ in the beaker and slightly heated up at 80°C to dehydration in the oven. After cooling, 5-mL of concentrated sulphuric acid (H₂SO₄) were added, and the mixture was heated for one hour then allowed to cool down. Then, 2-mL of 30% hydrogen peroxidase (H₂O₂) solution was added to the beaker contents and reheated. The final treatment reiterated until the mixture was clear [16].

The determination of HMs; Mg, Cu, Zn, Fe, Pb, Cd and Ni concentration of all collected samples were performed by using Atomic Absorption and Emission Spectrometry model Shimadzu-AA7000. Concentrations of heavy metals were determined according to Allen et al. [17]. All data are presented as concentration per unit wet weight of the sample (as mg/kg). All analyses were carried out on triplicate samples.

Biochemical analysis

Some biochemical parameters; enzyme levels of AST and ALT, and Protein content were determined in the soft tissues of males and females of the two selected beetle species, collected from the inspected sites. Levels of enzymes AST and ALT were assayed according to the method outlined by Nasr, et al. [15]. Protein concentration that assessed according to Crissman and Steinkamp [18], was measured in µg/100 mg tissue.

Statistical Analysis

Statistical analysis was performed using the program SPSS. Statistical analysis was carried out using an analysis of variance (ANOVA) according to Sokal and Rohlf [19] to analyze the significant differences in the HMs concentrations between the studied sites and the Pearson Correlation test to evaluate the relations between HMs concentrations in soil, leaf litter and beetles.

Results and Discussion

Bioaccumulation of heavy metals

Cement dust that originates from raw materials, contains HMs and deposited into soil. HMs are toxic to living organisms and could accumulated to a different extent in different organ/tissue [20]. In the cement site, the lowest concentration of HMs in soil samples was recorded for Cd (2.30±0.03 mg/kg), while the highest concentration was recorded for Pb (42.43±0.31 mg/kg) (Figure 2). This can be attributed by the fact that cement dust and its products incorporated a high quantity of lead [21]. Concentrations of Cd in soil from the cement site was three times higher than in soil from reference site. The concentration of HMs in soil collected from the two inspected sites were in order of Pb > Mg > Fe > Zn > Cu > Ni > Cd. Statistical analysis showed that the concentrations of Mg, Cu, Zn, Fe, Pb and Cd in soil collected from cement site were obviously significantly higher than those recorded in soil collected from the reference site. The concentration of Ni was insignificantly differed in soil collected from the studied sites (Table 1).

Table 1: One-way analysis of variance of HMs concentrations in soil and leaf litters collected from the two studied sites.

Figure 2: Heavy metals concentrations (mg/kg) in the soil collected from the two studied sites.

The concentrations of HMs in leaf litter have the same order recorded for soil, Pb > Mg > Fe > Zn > Cu > Ni > Cd (Figure 3). Based on the one way of ANOVA, there were significant differences between the concentrations of HMs in leaf litter collected from the reference site and those collected from the cement site (Table 1). Only, Ni concentration showed insignificant difference (p >0.05) between the two selected sites. The HMs’ concentrations in leaf litter were slightly higher than those recorded for soil. The concentrations of Mg, Fe and Pb are the highest concentrations of HMs, there were insignificant difference in their concentrations in soil and leaf litter (t = 1.14, p > 0.30; t = 1.99, p > 0.20; t = 1.29, p >0.30, respectively).

Figure 3: Heavy metals concentrations (mg/kg) in leaf litter collected from the two studied sites.

Cement dust reduces the absorption of mineral substances from the soil, resulting in changes in the plant physiology and morphology [22] as well as a reduction on the growth and protein expression [5]. In our study, the HMs’ concentrations were significantly higher in leaf litter collected from the cement site than those collected from the reference site; these data confirm those obtained by some previous studies [6,23]. The present recorded concentration of Mg and Fe was higher in leaf litter collected from the cement site, these obtained results confirmed those obtained by Raajasubramanian et al. [24].

The recorded concentrations of HMs in E. armata and E. tricostata collected from the two studied sites were obviously showed that beetle accumulated HMs in the same order recorded for the soil and leaf litter (Pb > Mg > Fe > Zn > Cu > Ni > Cd) (Figure 4). However, studies on the leaf beetles, Oulema gallaeciana and Lachnaia paradoxa showed that the mean HMs levels in beetles decreased in the subsequent order Fe > Zn > Cu > Mg > Pb > Ni > Cd [23,25].

Figure 4: Heavy metals concentrations (mg/kg) in (a) E. armata and (b) E. tricostata collected from the two studied sites.

The statistical analysis showed that there were significant differences between the concentration of all HMs in E. armata and E. tricostata collected from cement site and those from the reference site (Table 2). These results concord with those results obtained by Walker et al. [26]; Naccarato, et al. [27] and Nasr et al. [15]. The concentrations of Mg, Fe and Pb are the highest concentrations of HMs, there were significant difference in their concentrations between the two selected species of beetles (t = 5.37, p < 0.033; t = 11.31, p < 0.0001; t = 66.40, p < 0.0002, respectively) collected from the cement site. The reported data showed different capabilities of the beetle species involved in accumulating HMs.

Table 2: One-way analysis of variance of HMs concentrations in E. armata and E. tricostata collected from the two studied sites.

Additionally, the present results cleared that the two selected beetle species accumulated Cd and Ni less than Cu and Zn, these results similar to those recorded by Jelaska et al. [28] and Nasr et al. [11]. While, beetles accumulated high concentration of Pb, as recorded in Enchytraeus crypticus in highly contaminated habitats [29].

It is obvious that the concentrations of HMs were accumulated by transferring from soil to leaf litter and concentrated in beetle tissues (Figure 5). There was significant correlation of the concentration of HMs in soil, leaf litter and beetles (for Mg, Cu, Zn, Fe, Pb, Cd, and Ni the correlation coefficient was 0.963, 0.8471, 0.933, 0.9245, 0.949, 0.986 and 0.941, respectively). This biomagnification of metals has been reported at previous studies [28,30]. This increase of Pb and Cd concentration transferring from soil to plant to insect was observed in respect that Cd and Pb are non-essential elements [31]. It had reported that, metal associated with heat-stable, low molecular weight metal binding proteins, is highly available to predators, therefore, some complex mechanisms might exist in the higher tropic level of the food chain to tolerate the contaminated environment [29] (Table 3).

Table 3: Statistical analysis of the activities of AST, ALT and protein content (µg/100 mg) in body of males and females (a) E. armata and (b) E. tricostata sampled from the inspected sites.

Figure 5: Heavy metal concentrations in soil, leaf litter, E. armata and E. tricostata collected from cement site.

Biochemical assessment

The levels of AST and ALT of males were lower than females of the two studied species of beetles collected from the two selected sites. While, protein content of males was higher than females of the two studied species of beetles collected from the cement site (Figure 6). The statistical analyses showed that the activities of transminases (AST, ALT) and protein content were decreased significantly (p < 0.05) in both sexes of the two studied species of beetles collected from the two selected sites. The accumulation of HMs in beetles inhibits the tissue enzyme activities, specially transaminases, and resulted in tissue damage. The reduction in enzyme activity could be explained due to the interaction of HMs with enzyme SH group. It has been shown in various studies that toxic contaminants cause a change in protein content of the organisms studied [32]. Ortel found that feeding on diet contaminated with Cd, Cu, Pb and Zn decreased total haemolymph protein in Lymantria dispar [33]. Also, a reduction in the CAT activity of male B. polychresta stressed with HMs could be due to decreased SOD activities at polluted sites [34].

Figure 6: The activities of AST, ALT and protein content (µg/100 mg) in body of males and females (a) E. armata and (b) E. tricostata sampled from the inspected sites.

Conclusion

The heavy metals in the cement dust are present at different levels in the soil, plants and insects in the area around the industrial cement factory, Eastern region of Saudi Arabia. The data recorded in this study for HMs concentrations accumulated by transferring from soil to plant to insect. There was an accumulation of HMs from soil to leaf litter to beetles tissues. Moreover, the activities of transaminases; AST and ALT) as well as the protein content of both sexes were significantly lower in beetles’ tissues collected from the cement site than the reference one.

Acknowledgements

The authors acknowledge the Deanship of Scientific Research at King Faisal University, Saudi Arabia, for the financial support under the annual research project (Grant No.2099).

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