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 (April – July). Only small-bore (.22 LR caliber) guns are used in this shooting range. The area of the range is about 400 m2, and about 320 m2 of it is mostly overgrown by grasses. The range consists of 6 shooting positions, and the length of the shooting area is 50 m with two target lines at 25 and 50 m.
Soil sampling and chemical analyses
Soil samples were collected according to distance from the shooting positions to the target lines at 25 m and 50 m. Soil samples were collected 5, 20, 30 and 45 m away from the shooting positions. At each 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 HNO3, 5 mL of HBr, and 3 mL of HF in the Teflon vessels using a microwave digestion system (Milestone Ethos One, Italy). After mineralization samples were diluted with purified 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 emission spectroscopy (ICP-OES, Perkin-Elmer, Optima 8000). The concentrations of selected elements were measured at wavelengths: 220.353 nm, 327.393 nm, 238.204 nm, 257.610 nm, 231.604 nm, 206.836 nm and 20.6200 nm, respectively. Calibrations of trace elements were made by analysing standard (Multi-Element Quality Control Standard, 21 Elements, Perkin Elmer) solutions in four replicates. Precision of analysis was estimated by the coefficient 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 certified reference material (CRM Metals in Soil (SQC001), Sigma – Aldrich) was made, and the reproducibility was found in range ± 10 % within the certificated 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 reflected 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 flux 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% HNO3 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 purified 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 coefficient 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 certificated 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)).
TFsoil to root = Croot/Csoil (1)
TFroot to shoot = Cshoot / Croot (2)
where Croot is the content of examined element in root, Csoil – the content of examined element in soil, and Cshoot – 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. (3)) by the method suggested by Midhat et al. (2019):
BF = Cshoot (mg kg− 1) / Csoil (mg kg− 1) (3)
where Cshoot is the content of the element in root and Csoil – the content of a tested element in soil.
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.