Assessing phytotoxicity and accumulation of trace elements to Lactuca sativa of a contaminated shooting range soil


 Shooting range soil contamination with heavy metals is a common problem around the world. Usually, lead is the main contaminant in the shooting ranges. Extreme concentrations of trace elements create a toxic living environment for various plants. The purpose of this study was to evaluate the effect on lettuce (Lactuca sativa L.) grown in contaminated shooting range soil. The results showed that physiological parameters root elongation, shoot length and fresh biomass per plant was negatively affected, especially in the most contaminated site of the shooting range. At the most contaminated shooting range site shoots accumulated higher concentrations of Ni and Zn, roots – Cu, Ni, and Zn. The roots of plants grown in the most contaminated soil accumulated significantly higher concentrations of Cu, Ni and Sb in comparison with control (p < 0.05). Accumulation of Cu, Fe, Mn, Ni, Sb and Zn in the roots of the plants grown in the most contaminated site was higher compared to shoots (p < 0.05). Bioaccumulation factor of lettuce was lower than 1, only BF of Cu and Ni in plants from most contaminated site was significantly higher compared to control. In terms of translocation factor, the TFsoil to root of Cu and Pb among all plants and Ni in plants grown in the most contaminated site above 1 shows that metals absorbed by L.sativa were accumulated in root and lower metal translocation in shoots was determined, except for Mn.


Introduction
Shooting ranges pose an environmental concern due to their contamination with trace elements (Mannine and Tanskanen 1993). The soil of shooting ranges is typically contaminated with lead (Pb), copper (Cu), antimony (Hg), arsenic (As), nickel (Ni), zinc (Zn), and silver (Ag) because these elements are Some heavy metals are essential for plant growth, but at high concentrations, they become toxic (e.g., Co, Cu, Fe, Mn, Zn), while others (e.g., Cd, Pb, Hg) are toxic even at low concentrations because they have no known physiological functions in plants (Boquete et al. 2021 objective of the study is to evaluate potential phytotoxicity and trace elements accumulation in Lactuca sativa exposed to shooting range soil.

Study site
Soil samples were collected from the shooting range located in Alytus, Lithuania (54°23'48.1"N, 24°2'41.3"E). The shooting range was opened in 1957. Since then, it has been used mostly seasonally representing site, 5 sub-samples of surface soil were collected and constituted a composite sample. Two shooting range areas were chosen: to represent a less contaminated shooting range site (5-30 m) and a more contaminated shooting range site (45 m). The reference area was selected as a grassland site in the relatively unpolluted area (54°25'42.0"N, 24°14'04.2"E) and referred to as a reference soil. Soil samples were taken from the upper layer of surface soil (10-20 cm) after removing about 2 cm of soil surface layer. Samples were mixed and homogenized and stored at 4°C until analysis.
For chemical analysis, soil was sieved to 2 mm and oven dried at 60°C for 48 h. Soil pH was measured potentiometrically in suspension of soil:water ratio of 1:5 using pH meter (inoLab 720, WTW). Total soil organic matter content was determined by loss on the ignition method. The bulk density of soil was determined by pouring air-dried soil samples in a measured cylinder.
Two replicates of samples (0.5 g of dry soil) were digested in 8 mL of HCl, 5 mL of HNO 3 , 5 mL of HBr, and 3 mL of HF in the Te on vessels using a microwave digestion system (Milestone Ethos One, Italy).
After mineralization samples were diluted with puri ed water to 50 mL. The total concentrations (mg kg − 1 ) of elements (Pb, Cu, Fe, Mn, Ni, Sb and Zn) were determined using inductively coupled plasma optical Precision of analysis was estimated by the coe cient of linear correlation and was found to be not less than 0.999 for all measured elements. At the beginning of selected elements, analysis of certi ed reference material (CRM Metals in Soil (SQC001), Sigma -Aldrich) was made, and the reproducibility was found in range ± 10 % within the certi cated values of all selected elements. During analysis every ten measurements was made QC, when the selected value exceed the established limits recalibration was performed.

Plant toxicity study
The contaminated shooting range soil was used to evaluate the toxicity of heavy metals in the soils.
Lettuce (Lactuca sativa L.) has been chosen for this study as a reference species to characterize plant response in pot experiments. The selection of this species was based on its relevance to phytotoxic investigations, as it is known as a bioindicator species of heavy metals. It is also considered an accumulator of heavy metals (Cd, Pb, Zn). It is proved that physiological changes of L. sativa re ected the quality and characteristics of the environment (Moreira et al. 2020). This plant is amenable to testing in the laboratory and grows relatively fast. Research shows that it can survive extreme conditions in shooting range soils heavily contaminated with heavy metals. We consider it suitable to evaluate potential shooting range soil toxicity to plants. The phytotoxicity test was carried out according to the OECD guidelines for the testing of chemicals (OECD/OCDE 208 2006). 200 g of each homogenized and sieved (2 mm) soil was placed in pots, and 9 lettuce seeds were evenly sown into the soil. The test soil was hydrated, and distilled water was added daily to maintain 50% water holding capacity of the soil. The plants were grown for 21 days in a climate chamber where the average temperature was 20 ± 2°C and relative humidity was 60%. An average photon ux density was 180-200 µmol m − 2 . Three replicates (pots) of all treatments were made.
Seed germination was observed after 7 days. The germination rate (%) was calculated as the number of seeds sprouted divided by the total number of seeds and multiplied by 100. At the end of the experiment, seedlings were harvested, and the plant fresh weight was assessed. The plant height and root length were measured. Subsequently, shoots and roots were separated and dried at 60°C in an oven until a constant weight was obtained. Three sub-samples of soil and plant were used for the analytical analyses (n = 3).
For heavy metal analysis, dry material was homogenized (Retsch HM400, Germany). Two replicates of homogenized material of samples were followed by acid digestion in 65% HNO 3 and 30% HF (v/v = 8/2) using a high-pressure microwave digestion system (Milestone ETHOS One, Italy). Samples were diluted to 45 mL with puri ed water. After that, the concentration of elements (Pb, Cu, Fe, Mn, Ni, Sb and Zn) were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES, Perkin-Elmer, Optima 8000). Selected elements were measured at the same wavelength as soil samples. Calibrations of trace elements were also made by analysing standard (Multi-Element Quality Control Standard, 21 Elements, Perkin Elmer) solutions in four replicates. Precision of analysis was also estimated by the coe cient of linear correlation and was found to be not less than 0.999 for all measured elements. At the beginning of selected elements, analysis of the plant reference material (BCR-129, hay powder) was made. The reproducibility was found in range ± 10 % within the certi cated value of Zn and the other elements selected in CRM: Cu, Fe, Mn. During analysis every ten measurements also was made QC test, when the selected value exceed the established limits recalibration was performed. These quality control measurements ensured the reliability of the results.
Translocation factor (TF) was calculated to evaluate the ability to translocate elements from soil to root and from root to shoot. TF was calculated by the method suggested by Sun et al. (2017) from soil to root (Eq. (1)) and from root to shoot (Eq. (2)). TF soil to root = C root /C soil (1) TF root to shoot = C shoot / C root (2) where C root is the content of examined element in root, C soil -the content of examined element in soil, and C shoot -the content of examined element in shoot.
In terms of bioaccumulation of heavy metals, we evaluated bioaccumulation factor (BF). Plants with BF above 1 are reported as hyperaccumulators (Yazdi et al. 2019). BF was calculated (Eq. where C shoot is the content of the element in root and C soil -the content of a tested element in soil.

Statistical analyses
To analyze the effects of the study area, data were grouped in three units according to the lead contamination: 1) reference soil; 2) soil from less contaminated areas (5-30 m) were combined into one forming a medium contaminated study plot, and 3) the heavily contaminated area (45 m) of the shooting range. Relationships between concentrations of trace elements and plant parameters, as well as differences between TFs and BFs of heavy metals were assessed using the Mann-Whitney U test (p < 0.05). Spearman correlation was used to identify the relationship between heavy metal concentration in soil and in the tissue of plants (p < 0.05). The statistical analysis was condicted by using IBM SPSS Statistics 25.

Results
The results of soil analyses show that the pH of the studied shooting range and control soil was close to neutral. The soil from 45 m distance had a signi cantly lower pH (p < 0.05; Table 1). Soil organic matter content and density did not show any signi cant differences compared to control (p > 0.05). Ammonium content in the 5-30 m area was signi cantly higher compared to control. Signi cantly lower phosphorus content was observed in the 45 m area of shooting range soil compared to control (p < 0.05). Physiological parameters of L. sativa exposed to shooting range soil show that seed germination rate was no different between shooting range soil and control ( Table 2). Plants grown in contaminated shooting range soil had signi cantly lower root length than the reference. The lowest roots had plants exposed to soil from the 45 m area (p<0.05). Although seed germination in plants grown in the most contaminated site was similar to the control, after a few weeks of the experiment, differences in grow were visible -the growth of the plant in the most contaminated site was retarded. Analyses of shoot length and fresh biomass clearly showed the plants exposed to the most contaminated site had signi cantly lower shoot length (p<0.05) and fresh biomass per plant than the reference soil (p < 0.05).
Lettuce exposed to contaminated shooting range soil accumulated high concentrations of lead (Fig. 1). Signi cantly higher Pb concentrations were observed in shoots of plants grown in the contaminated soil compared to the reference soil, with the highest concentration being 3963 mg kg -1 . The roots of plants grown in the soils from the shooting range accumulated signi cantly higher concentrations of lead compared to the reference soil. Eight times more lead was accumulated in the plants grown in soil from the 5-30 m area, and 687 times more lead was accumulated in plants grown in soil from the 45 m area compared to the reference soil (Fig. 2). The roots accumulated signi cantly more lead compared to shoots (p<0.05). The roots of plants grown in the most contaminated site accumulated 10 times more lead than shoots of the same site. The statistically signi cant correlations between total Pb concentration in soil and Pb concentration in shoots (r=0.97, p<0.05) and roots (r=0.91, p<0.05) were determined.
No signi cant differences were determined among all heavy metal concentrations in the shoots of lettuce grown in the soil from the 5-30 m site of the range compared to reference shoots (p > 0.05; Fig. 2). In shoots of lettuce grown in the most contaminated site of the shooting range, signi cantly higher Cu, Fe, Mn, Ni, Sb, and Zn concentrations were found compared to control shoots. In shoots of plants grown in the most contaminated study site, signi cantly higher concentration of Cu, Fe, Mn, Sb, and Zn were observed than shoots of plants grown in soil from the 5-30 m area (p<0.05). The roots of lettuce grown in the soil from the 5-30 m area of the range accumulated signi cantly higher concentrations of Cu, Fe, and Ni (p < 0.05) compared to the control (Fig. 2). The roots of L. sativa grown in the most contaminated range soil accumulated signi cantly higher concentrations of Cu, Fe, Ni, and Sb compared to the control (p < 0.05). The roots of plants grown in the 5-30 m area soil accumulated signi cantly higher concentrations of Cu, Fe, Mn, and Zn than shoots of the same plants (p < 0.05, Fig. 2). Among plants grown in the soil from the most contaminated site, signi cantly higher Cu, Fe, Mn, Ni, Sb, and Zn concentrations were observed compared to shoots of plants grown in the soil from the same area. A signi cant correlation was determined between total Ni concentration in soil and Ni concentration in shoot (r = 0.97, p < 0.05) and root (r = 0.99, p < 0.05).
The BF values in lettuce of all sites were lower than 1 ( Table 3). BF of Cu in plants grown in the most contaminated shooting range soil was 0.98. It was signi cantly higher compared to control (p<0.05). Signi cantly higher than control, but still relatively low BFs of Fe, Mn, Ni, Pb, Zn were also calculated in lettuce from the 45 m area of the shooting range (p<0.05). BF values of Pb were very low (0.02 at 5-30 m and 0.04 at 45 m), which could be because of extremely high soil Pb concentrations.
The TF of soil to root of Cu, Fe, Ni, Pb, Zn was signi cantly higher than TF of root to soil in lettuce grown in the 5-30 m area of the range (Fig. 3). The TF of soil to root of Cu, Fe, Ni, and Pb was signi cantly higher compared to TF of root to shoot in lettuces grown in shooting range soil in the 45 m area. TF soil to root of Cu (5-30 and 45 m), Ni (45 m), Pb (5-30 and 45 m), and Zn (5-30 m) were above 1. A strong positive correlation between total Ni concentration in soil and bioconcentration factor of Ni (r = 0.89, p < 0.05) and TF root to shoot (r = 0.97, p < 0.05) was determined. On the contrary, a strong negative correlation was determined between total Ni concentration in soil and TF shoot to root (r = -0.96, p < 0.05). The TF of Pb from roots to shoots signi cantly correlated with total soil Pb concentration (r = 0.90, p < 0.05; Fig. 3).

Discussion
In this study, basic soil analyses re ected site-speci c shooting range contamination with heavy metals and changes in physicochemical soil properties. The changes in soil properties such as pH and organic matter can change heavy metal solubility. For example, some heavy metals have a strong attraction to soil organic matter, which means that the organic matter should be highly e cient in the sorption of heavy metals. Also, the binding of heavy metals with organic matter is a strongly pH-dependent process (Lewińska and Karczewska 2019). Soil pH at the most contaminated shooting range soil was lower compared to control and this is in accordance with the nding when pH values of the full-bore military shooting ranges soil ranged from 5. . During our study, the plant was capable of surviving at higher Pb concentrations, but adverse effects on growth were visible. In general, lead as a non-essential element negatively affects root, plant growth, inhibits photosynthesis, enzymatic activities and, at very high concentrations, leads to cell death (Hadi and Aziz 2015). Total concentrations of Cu, Fe, Mn, and Ni do not pose an environmental risk, as concentrations do not exceed limit concentrations (HN 60:2004). However, the total concentration of Sb exceeded the concentration limit (10 mg kg − 1 ) (HN 60:2004). Pollution with Sb was reported in shooting range areas. It is known that Sb is more mobile compared to Pb. Therefore, shooting ranges can have larger long-term environmental problem related to groundwater pollution (Shtangeeva et al. 2011). The suppression of shoot and root biomass under an increase of antimony concentration could result from plant Sb accumulation. Under our circumstances, both shoot and root systems of plant grown in the most contaminated soil accumulated signi cantly higher concentration of antimony and growth of these plants was inhibited. Studies show that environmental stressors, such as heavy metals, can disturb plant growth and development (Yazdi et al. 2019). This study revealed that above-ground and below-ground plant growth was negatively affected by heavily contaminated (Pb, Sb) shooting range soil. Numerous studies show that the reduction of root length in the presence of heavy metals appears as the reason for metal interference with the process of cell division, which causes chromosomal aberration and abnormal mitosis (Yazdi et al. 2019).
Accumulation of heavy metals in the tissues of exposed plants depends on metal supply and plant species. Plants can absorb heavy metals through passive transport by water mass ow or through active transport by the plasma membrane of the root cell (Midhat et al. 2019). During this study lead was the main contaminant in soil. In the most contaminated site of the shooting range, roots of L. sativa accumulated almost 10 times more Pb compared to shoots. Pb content was also higher in roots compared to leaves of the Brassica campestris seedlings (Zhang et al. 2020). In the case of Ni, Zhao et al. (2019) reported higher concentration in roots than shoots. Our results agree in the most Nicontaminated site; Ni concentration in L. sativa roots were signi cantly higher. The tendency of higher heavy metal accumulation in roots than shoots were also reported in other studies (Zhao et al. 2019;Steliga and Kluk 2020). The roots are the main organ of the plant for stabilization and collection of nutrients (Steliga and Kluk 2020). Translocation from roots to shoot might be limited by precipitation of heavy metals on the root membrane. This barrier could control metal mobility from root to shoot (Shtangeeva et al. 2011). In terms of BF, results concerning BF' of Cu and Ni in plants from the most contaminated site stand out. In the 5-30 m area of the shooting range, BFs were very low. However, in L. sativa, exposed to the most contaminated BF can be put together in the following sequence: Sb < Fe < Pb < Mn < Zn < Ni < Cu, but only BF of Cu, Fe, and Ni were signi cantly higher. Very low Pb bioaccumulation could be explained by low concentration of soluble Pb in soil; as the pH and organic matter values of studied soil do not drastically change the solubility of Pb (Ashworth and Alloway 2008; Lewińska and Karczewska 2019). Similar results reported (Dradrach et al. 2020) with bioaccumulation of As in Festuca rubra L. values of TF con rm better translocation from "soil to root" system than "root to shoot" at translocation of Cu, Fe, Ni and Pb. TF soil to root of Cu and Pb (in all test samples) and Ni (45 m) > 1 shows that metals absorbed by Lactuca sativa are accumulated in its root tissue rst and poorer metal translocation in shoots was visible, except for Mn.

Conclusions
The present study demonstrated physicochemical properties and phytotoxicity of the shooting range soil.
The shooting range soil was site-speci c with high contamination in Pb and Sb. A high concentration of antimony, which is more mobile in the soil than lead, in the most contaminated site of the range revealed that pollution of shooting range still needs better understanding and approaches for remediation of these sites must be considered to avoid its hazardous leaching into groundwater. Our results showed that in the most contaminated site, the inhibition of tested plant growth was observed. Translocation of heavy metals from root to shoot was suppressed, and to our knowledge, this could be caused by very extreme heavy metal concentrations when heavy metals on the root membrane form a barrier that controls metal mobility from root to shoot.