Biological assessment of contaminated shooting range soil using earthworm biomarkers

Environmental contamination at shooting ranges is a widely known ecological problem. The aim of the study was to evaluate the extent of contamination and the ecotoxicity of a shooting range soil assessing the physiological and biochemical effects on earthworm Eisenia fetida (Savigny). Adult E. fetida were exposed to the soils collected from different distances of the shooting range for 28 days. High concentrations of Pb (53023 mg kg−1), increased concentrations of Ni (12 mg kg−1) and Sb (600 mg kg−1), significantly higher soil organic matter content (7.2%) and density (6.14 g cm−3) were determined in the backstop berm soil. Significant weight loss (44.4%) of the adult earthworms after 28 days of exposure occurred in the most contaminated shooting range soil and significantly higher concentrations of Pb (3101 mg kg−1), Cu (51 mg kg−1), Ni (2 mg kg−1), and Sb (20 mg kg−1) were determined in the tissues of worms, and no juveniles found there. Juveniles exposed to the less contaminated soil of the shooting range (A, B and C) accumulated significant concentrations of Pb, Cu, Fe, Mn, and Zn. The antioxidant enzymatic activity (glutathione-s-transferase (GST)) decreased, and lipid peroxidation increased as indicated by an increase in malondialdehyde (MDA) level in earthworms exposed to the contaminated soil. A compensatory mechanism between the activities of glutathione reductase (GR) and GST in earthworms exposed to these soils was confirmed.


Introduction
Contamination of the soil with heavy metals is becoming a serious and important dimension of environmental pollution. Anthropogenic activities such as development of industry and agriculture contribute to the increased contents of heavy metals in the soil environment and potential risk to the health of organisms (Rodríguez-Seijo et al. 2017;Li et al. 2019). Shooting ranges are a potential source for contamination of soils with various heavy metals since shooting activities contribute to deposition of large amounts of heavy metals based bullets (Sorvari 2011;Sanderson et al. 2018). Contamination in the shooting range soils with heavy metals (Sb, Cu, Zn, Ni, Hg, etc.) was determined (Stauffer et al. 2017;Sanderson et al. 2018), while the concentration of Pb in the soil ranged from 10000 to 70000 mg kg −1 (Hardisonjr et al. 2004;Sanderson et al. 2018) and even up to 100000 mg kg −1 (Dinake et al. 2019). Pb is the main bullet component (>90%), while other elements are also present-Sb, Cu, As, Zn, and Ni (Sanderson et al. 2012(Sanderson et al. , 2014Dinake et al. 2019). During the shooting, bullets accumulated in the soil of the shooting ranges, especially in the backstop berm (2000-38386 mg kg −1 , Sehube et al. 2017a) or the fall-out zones (Sanderson et al. 2018).
Bioavailability of Pb depends on solubility of solid lead phases and physicochemical soil properties. Soil pH is the primary factor contributing to bioavailability of lead because it regulates solubility and soil reactions (Fayiga and Saha 2016). Soil acidity affects solubility of heavys metals in the soil solution, so the concentrations of some heavy metals (Zn, Cd, Ni, Cu) is negatively correlated with soil pH (Hou et al. 2019). Soil organic matter can immobilize heavy metals in the soil-the addition of soil amendments (organic matter, phosphates, alkalizing agents, biosolids, etc.) could reduce the solubility of metals in the soil (Kwiatkowska-Malina 2018). The solubility of heavy metals also increases in fertilized soil as nitrogen application increases their availability .
Quantification of heavy metals in the shooting range soils confirmed the potential hazard of heavy metals to receptors. Contamination of shooting range soils poses a risk of heavy metals accumulation to higher trophic levels. Due to the high level of contamination in the soils of the shooting range, the ecotoxicological risk of these sites is becoming an important field of study (Baer et al. 1995;Reid and Watson 2005;Sneddon et al. 2009;Sanderson et al. 2018;Sujetovienė and Česynaitė 2019;Dinake et al. 2019). Contamination of heavy metals results in a reduced of survival and reproduction rate of earthworms (Luo et al. 2014a;Sanderson et al. 2014), mites (Luo et al. 2015), and negatively affects all soil faunal communities (Selonen et al. 2014). Although the survival of collembola (Folsomia candida) was not sensitive to Pb contamination, the internal Pb concentration increased linearly with increasing Pb concentration in the shooting range soils (Luo et al. 2014b). The median effective earthworm concentrations determined based on the total Pb concentration in the soil ranged from 35 to 5080 mg Pb kg −1 (Lanno et al. 2019). Pb uptake was limited, and some uptake regulation occurred when Pb concentration was up to 3000 mg Pb kg −1 in soil but higher concentrations increased contaminant accumulation and organisms mortality (Davies et al. 2003). Bioaccumulation of Pb in Enchytraeus crypticus was also linearly related to the total soil Pb concentration, and the most toxic effects to these organisms were in the most contaminated and acid shooting field soil (Luo et al. 2014c).
Contamination of soil with heavy metals led to biochemical changes markers of soil fauna. In response to inorganic pollutants, changes in activity of catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), and content of malondialdehyde (MDA) have been reported in earthworms (Maity et al. 2008;Bai et al. 2020;Li et al. 2020). Glutathione metabolism is crucial as a defense response under heavy metal poisoning (Łaszczyca et al. 2004). Antioxidative enzymes-GST and GR have been previously reported as indicators of inorganic pollutants (Ojo et al. 2016). Under oxidative stress, CAT is also reported as a sensitive antioxidative enzyme (Ojo et al. 2016;Hu et al. 2016). Maity et al. (2008) reported that non-lethal Pb and Zn concentration activates GST and GR in earthworms Lampito mauritii may cause adaptive response after long-term exposure. Bai et al. (2020) also reported variations of CAT activities and MDA content in earthworms E. fetida with increasing Cd and Pb concentrations in soil (1-20 mg kg −1 and 20-500 mg kg −1 , respectively). When earthworms were exposed to low doses of Cd (1-2 mg kg −1 ), CAT activity decreased, but the stimulatory effect occurred with higher doses of Cd. As Pb levels increased, CAT activity also increased (Bai et al. 2020). MDA content increased with increasing Pb and Cd concentrations (Sinkakarimi et al. 2020;Bai et al. 2020). Although the antioxidative response describing the toxicity of individual heavy metal or even mixtures is known, there is still a lack of information describing the biochemical response of earthworm to site-specific soil contamination.
Because understanding of metal uptake by organisms is an essential aspect of safe a shooting range management, soil toxicity to earthworms was tested using contaminated soil with different physicochemical properties. In this study, we focus on the fact that an outdoor shooting range poses a threat to the physiology and biochemistry of organisms, and this could have adverse effects not only on individuals but also on ecosystems. The aim of the study was to evaluate the extent of contamination and the ecotoxicological risk in shooting range soil assessing the physiological and biochemical effects on earthworms.

Study site and sampling design
The studied shooting range is located in Alytus, southern Lithuania (54°23′48.1″N, 24°2′41.3″E). The length of the shooting range was 50 m long with two target lines at distances of 25 and 50 m. Wooden bullet traps were constructed at the end of the range. The shooting activity started in 1957. It is used seasonally (April-July) and only .22lr caliber ammunition is used in this shooting range as the range is specially designed for athletes training shooting with small-bore rifles and pistols. The total area of the range was 400 m 2 and 320 m 2 is covered by grassdominated vegetation.
Field soils were collected from the shooting field at four different distances from the firing line-5, 20, 30, and 45 m (A, B, C, D, respectively). At each distance two plots (1 × 1 m) were selected for soil sampling. At each sampling plot, five soil subsamples were collected from the corners and the center of the plot using a soil core to a depth of 10 cm. Subsamples were mixed to obtain a representative sample of each plot. Soil samples were placed in plastic bags. In the laboratory, soil was air-dried, sieved through 2-mm mesh, and homogenized. The bullets from soils were collected after sieving and then weighted. The dried samples were stored in polyethylene bags. A grassland soil near the shooting range was sampled and used as a reference soil (13 km from Alytus city, 54°25′42.7″N 24°14′03.8″ E). Soil samples were kept at 4-5°C until used in tests (within two month).

Soil analysis
The soil samples were air dried, homogenized and 2 mm sieved. Soil pH was determined potentiometrically in soilwater suspension at a ratio of 1:5 (v/v) using a pH meter (inoLab 720, WTW) according to ISO guidelines (ISO 10390 2005). Total soil organic matter content was determined by loss on the ignition at 550°C for 4 h (Ben-Dor and Banin 1989). Soil bulk density was determined after collection of bullets from the soil by pouring air-dried soil in a measured cylinder and calculated as the ratio of the mass of oven-dried solids to the bulk volume of the soil (Hao et al. 2007).
Soils were extracted using a deionized water (10:1 solution/soil weight) with shaking for one hour followed by centrifugation at 4000 rpm for 10 min at 4°C. Subsequently, the colorimetric microplate assay method to determine nitrate (NO 3 --N) concentrations in the soil extracts was measured using the Griess reaction with VCl 3 as a reducing agent (Hood-Nowotny et al. 2010). An indophenol method based on Berthelot reaction was used for the determination of soil ammonium NH 4 + -N (Rhine et al. 1998). The content of dissolved inorganic P was quantified using the malachite green method (Petitou et al. 1978). Absorbance values were measured using a SPECTROstar Nano spectrophotometer (BMG Labtech, Germany). Six replicates of all treatments were made.
To determine total Pb, Cu, Fe, Ni, Sb, Mn, Zn concentrations in soil, approximately 0.5 g oven-dried soil samples were digested in 5 mL of HNO 3 (65%), 8 mL of HCl (37%), 3 mL of HF (48%), and 5 mL of H 3 BO 3 (5%) in a Teflon vessel using a microwave digestion system (Milestone Ethos One, Italy). The concentration of heavy metals was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES, Perkin-Elmer, Optima 8000). Calibration of heavy metals was made by analyzing the Quality Control Standard 21 (Perkin Elmer). Precision of analysis was estimated by the coefficient of correlation of four calibration points and was found to be 0.999 for all measured elements. For the data acquisition of the samples, a quantitative analysis mode was used. The scanning of each single sample was repeated three times to get reasonably reliable results. During measurements, care was taken to avoid memory effect and therefore a wash-out time of 1 min was used. Six replicates were used for the analytical analyses (n = 6).

Earthworm toxicity test and bioaccumulation assessment
Toxicity tests with the earthworm species Eisenia fetida were conducted in a 56-day period. These target organisms have been selected for the assessment of soil toxicity because earthworms are a representative of the soil fauna, an essential component of the soil ecosystem and important indicator of soil quality due to the direct exposure of soil contaminants and ingestion (Maity et al. 2008;Sanderson et al. 2014;Luo et al. 2014b;Bai et al. 2020). Earthworm of the strain Eisenia fetida Savigny were taken from a synchronized breeding culture kept at the laboratory of the Vytautas Magnus University. Individuals with a relatively homogeneous age structure and not different in age by more than 4 weeks were chosen for the testing. Before running exposure tests, the earthworms were left on wet filter paper for 24 h to reduce soil content into their gut. Toxicity testing was performed in plastic vessels with perforated lids. Ten mature earthworms with developed clitellum and similar in size were weighted and introduced into 500 g of dry mass of soil. The test containers were maintained in a climatic chamber at 20 ± 2°C and under controlled lightdark cycles (16/8 h light/dark) with illumination of 400 lux. Each test soil (contaminated shooting range soil of each plot and reference soil) was used in four replicates. The water content of the soil was maintained by re-weighing the test containers periodically during the testing and deficit was replenished with de-ionized water if necessary. Food was provided one day after adding the worms (about 5 g of oatmeal per container) and then once a week. After 28 days, adult worms were removed from the test containers, counted, and washed. The earthworms were placed on filter paper to empty their gut (24 h at 15°C in the dark) and then weighted. Mortality and weight were recorded for each treatment. Worms were cryopreserved by rapid freezing at -80°C until the use for biochemical analysis.
The soils were incubated for four additional weeks to allow cocoons to hatch under the same test conditions except that feeding only takes place once at the start of this phase of the test. At the end of the second 4-week period, the juveniles hatched from the cocoons in the test soil were counted. Mortality, weight change, number of juveniles was determined. At the end of the test, hatched juveniles were cryopreserved at 80°C.
For heavy metal analysis, the earthworms (adults and juveniles) were dried at 60°C to constant weight, then digested in 65% HNO 3 and 30% H 2 O 2 (1:3 v/v) using a microwave digestion system (Milestone Ethos One, Italy). The concentrations of heavy metals (Pb, Cu, Fe, Ni, Sb, Mn, and Zn) were determined using ICP-OES (Perkin-Elmer, Optima 8000). Calibrations of trace elements were also made by analyzing 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.

Biochemical assays
Prior to freezing, earthworms were washed with deionized water and placed on filter paper to clear their gastrointestinal tracts for 24 h. Three frozen worms from each replicate were used for analysis of the catalase (CAT), glutathione reductase (GR), and glutathione S-transferase (GST) activities. Tissues (0.15-0.20 g) were homogenized in a 1.5 ml elution extraction buffer containing 200 ml potassium phosphate buffer (pH 7.8), 61.72 mg dichloro-diphenyl-trichloroethane + 5.8 mg ethylenediaminetetraacetic acid, 1 g polyethylene glycol 4000, 2 g polyvinylpolypyrrolidone. The homogenate was centrifuged at 13000 rpm (4°C) to obtain the supernatant for the assay of CAT, GR, and GST. Six replicates of all treatments were made (n = 6).
GR reaction was based on nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidation when GR reduces glutathione disulfide. NADPH concentration was estimated at 340 nm (Bailly et al. 1996).
Catalase activity was determined by measuring the rate of dissociation of hydrogen peroxide at 240 nm (Bailly et al. 1996;Claiborne 1985).
GST activity was determined using the method described by Habig et al. (1974) with 1-chloro-2,4dinitro-benzene as substrate for the measurement at 340 nm spectrophotometrically.
Total protein contents were determined according to the Bradford method (1976). Homogenate (with potassium phosphate buffer) was centrifuged at 13000 rpm for 15 min at 4°C. Bradford reagent was diluted with water (1:5). Absorption was measured at 660 nm. The enzyme activities were expressed as nmol min −1 mg −1 of protein.

Statistical analysis
Bioaccumulation factor (BAF) was calculated as the ratio dividing of the element concentrations in the tissue earthworm and by the total concentrations of the element in the soil. Log-transformed metal concentrations were used to normalize their distribution. Significant differences between the parameters were assessed using the nonparametric Mann-Whitney U test (p < 0.05). Spearman's rank-order correlation was used to identify the relationship between physicochemical properties of soils and toxicity data (p < 0.05). Multiple regressions were used to determine the relationship between E. fetida bioassay endpoints (mortality, weight loss, Pb accumulated) and soil physicochemical properties. The statistical analysis was carried out using Statistica 10 software.

Soil physical and chemical characteristics
The amounts of sieved bullets were significantly higher in the shooting range soils in comparison with the reference soil (p < 0.05, Table 1). More than half of the soil mass (55.4%) contained bullets at site D (at 45 m from the fire line). Site D is strongly influenced by direct impact of bullets and ricochet because this site is located at the end of the range. The shooting range soils were more acid and denser, lower in nitrate, phosphorus, and organic matter content compared to the reference soil.
Total Pb concentrations in the shooting field soils ranged from 217 to 53023 mg kg −1 ( Table 1). The shooting range soils contained elevated Pb concentrations compared to the background (15 mg kg −1 ) and maximum (100 mg kg −1 ) lead concentrations in Lithuanian soils (HN 60:2004). Significantly higher level of total Pb was observed in all sites of the shooting range compared to reference (p < 0.05), and the highest levels of Pb were observed at site D (53022 mg kg −1 ). Heavy contamination in Sb was also characteristic of site D (599 mg kg −1 ) while its concentration was below the detection level in other study sites. Ni concentrations were significantly higher in all sites than the reference (p < 0.05) except site C. At the most contaminated shooting range site (site D), the soil was contaminated with Pb and Sb. The soil bulk density was positively correlated with the mass of the sieved bullets (r = 0.75, p < 0.05) indicating the direct impact of the bullets on the soil. Most of the sieved bullets were presented at the end of the shooting range (near the target lines). Bullets that hit the ground disturb the soil layer, which contributes to changes in soil properties, in this case soil density.

Toxicity test and bioaccumulation of heavy metals
After 4 weeks, exposure and mortality of E. fetida were lower than 10%, even in site D where soil is heavily contaminated with Pb (Fig. 1A), but significantly lower weight of earthworms (E. fetida) exposed to the contaminated soils of the shooting range was observed (p < 0.05; Fig. 1B). The weight loss ranged from 5.8 to 6.6% at sites A and B to about 44.4% at site D. At site D, no juvenile was produced. No significant differences of juvenile production were observed between reference and sites A, B and C (Fig. 1C).
Pb concentration in earthworm tissues was significantly higher in all soil samples of the shooting range than the reference (p < 0.05) with the highest bioaccumulation (3101 mg kg −1 ) at site D ( Fig. 2A). Adult earthworms exposed to the soil from the end of the range (D site) accumulated significantly higher levels of Cu (51 mg kg −1 ), Fe (954 mg kg −1 ), Sb (599 mg kg 1 ), and Ni (3 mg kg −1 ) compared with the reference soil (p < 0.05, Fig. 2B, D, F, G). The concentrations of Pb, Cu, Fe, Mn, Sb, and Zn in juvenile tissues were significantly higher in all soil samples than the reference (Fig. 3).
The total bioaccumulation factor (BAFs) of Pb in exposed earthworms ranged from 0.06 to 0.33 and sampling plots were arranged in the following order: B > C > A > D ( Table 2). The BAFs was slightly higher in juveniles than in adults (0.07-0.40) and were in the following order: C > B > A (no juveniles were present in site D). BAF of the site D was relatively low because of the extremely high total Pb concentration in the soil.

Relationship between E. fetida bioassay endpoints and soil properties
Weight loss of E. fetida was significantly positively correlated with total Pb in the soil (Table 3). Pb accumulation in earthworms was significantly positively correlated with total Pb concentration, while other soil properties had negligible effects on the accumulation of Pb in the exposed organisms. Higher mortality was positively related with total Mn, and Zn concentrations in soil and negatively with Fe and Cu (Table 3).

Biomarker responses of earthworm E. fetida to contaminated soil
MDA concentration in all test samples of E. fetida was significantly higher than the reference (p < 0.05; Fig. 4), but no significant correlation between MDA content and physicochemical properties was observed (Table 4). CAT activities showed no significant differences (Fig. 4).
Significantly higher activities of GR were determined in the earthworms exposed to soil from site D compared to reference (p < 0.05; Fig. 4). Significant positive correlations were determined between GR activities and soil properties Values are mean ± SE, n = 6. The same letters above the columns indicate no significant differences between the treatments at p > 0.05 using a Mann Whitney U test  (Table 4). A positive linear correlation was observed between total Pb concentration, total Zn concentration and GR activities in earthworms (r = 0.57, p < 0.05; r = 0.60, p < 0.05, respectively). Negative relationship between bioaccumulation of Pb and GR activities in earthworms was also observed (r = −0.79, p < 0.05). fetida after a 4-week exposure to soils from the shooting range (A, B, C, D-plots at different distances from the fire line in the shooting range (5, 20, 30, and 45 m, respectively)) and reference (R) soil. Values are mean ± SE, n = 6. The same letters above the columns indicate no significant differences between the treatments at p > 0.05 using a Mann Whitney U test No significant tendency in GST (Fig. 4D) activities in adult earthworms at the end of the test was determined. In site D, where soil was heavily contaminated with Pb, GST activity tended to be even lower than the other shooting range sites. Interestingly, negative correlation between GST activity of E. fetida and total Pb Zn in soil, organic matter  Fig. 3 Tissue trace element concentrations in juveniles of E. fetida after a 4-week exposure to soils from the shooting range (A, B, C, D-plots at different distances from the fire line in the shooting range (5, 20, 30, and 45 m, respectively)) and reference (R) soil. Values are mean ± SE, n = 6. The same letters above the columns indicate no significant differences between the treatments at p > 0.05 using a Mann Whitney U test content, weight loss of E. fetida was determined (p < 0.05). Positive linear correlation between GR activity and bioaccumulation of Pb was observed. Also, negative linear correlation between GR activity and GST activity in earthworms was determined, meaning that with the increase of GR activity, GST decreased (r = −0.51, p < 0.05), indicating a compensatory mechanism between the activity of GR and GST.

Discussion
Our study showed that Pb concentrations in the entire soil of the shooting range and Sb concentration in the berm were much higher than the background levels in Lithuanian soils (Pb = 15 mg kg −1 ; Sb = 1.0-1.5 mg kg −1 ) (HN 60-2004(HN 60- 2004. Such contamination in the berm soil is directly related to the accumulation of bullets and shots (Cao et al. 2003) where the highest mass of the sieved bullet was found. Also shooting range soil had higher density especially this was characteristic for the soil at the end of the shooting range (site D) which was associated with the highest mass of the sieved bullets. Therefore, as the distribution of metal contaminants at shooting ranges is predominantly associated with spent bullets (Sanderson et al. 2010), This study demonstrated significant differences in the shooting range soil properties such as lower organic matter content and higher acidity. Such changes in soil may contribute to the transformation of Pb into different weathering products (soluble ion complexes), such as PbO, PbCO 3 , Pb 3 (CO 3 ) 2 (OH) 2 , PbSO 4 , etc. (Fayiga and Saha 2016;Sehube et al. 2017;Sanderson et al. 2019) while higher pH experienced the highest accumulation of total Pb in the shooting range soils (Cao et al. 2003). The results of this study were consistent with other studies showing a total lead concentration of 10068-70350 mg kg −1 in soils of different shooting ranges (Fayiga and Saha 2016). Other studies showed that there was very high levels of Pb contamination in shooting ranges-up to 81000 mg kg −1 (in stop berm soil) and even up to 21000 mg kg −1 (Sanderson et al. 2010). In the target ranges, as in the case of our study, the highest pollution was in the fallout zone, closest to the D site (Sanderson et al. 2018). Cao et al. (2003) agreed that Pb concentration increased with increasing distance from the firing line. These assumptions confirmed that the main problem of shooting range contamination is high Pb levels, especially at the end of the range, near the target lines.
As earthworms are an important component of the soil biota, their response to the contaminated soils is important for the ecological assessment of heavy metals in the shooting range soils. Although survival of E. fetida was not reduced in the shooting range soil, the growth of the earthworms was a sensitive indicator of the studied soil toxicity to earthworms after 4 weeks of exposure. Significant weight loss of the adults (44%) and lower juveniles' numbers were observed in the heavily Pb and Sb contaminated berm soil. Although Luo et al. (2014b) observed that no significant weight loss of earthworms occurred in soil where Pb concentrations were up to 656 mg kg −1 , our study results showed significant weight loss of the earthworms at total Pb concentrations of 216-443 mg Pb kg −1 in the soil. The results highlighted those site-specific conditions could modify the effects of soil contamination on the survival of earthworms. Severe weight loss and lowered reproduction rate are key components that could lead to a decrease in a whole population of these essential organisms (Uwizeyimana et al. 2017).
When exposed to contaminated shooting range soil, adult and juvenile earthworms accumulated Pb in and the uptake Table 3 Multiple-regression equations and coefficient of determination (R 2 ) of relationships between physicochemical properties of soil and toxicity to earthworm E. fetida

Equation Statistics
Weight  was the highest than that of other elements. Earthworms serve as bioindicator of metals showed clear relationship between metal concentration in worm tissues and surrounding soils (Suthar et al. 2008). These findings are consistent with the results obtained by Luo et al. (2014a) where significant increase in bioaccumulation of Pb in worms Eisenia andrei occurred when soil Pb concentration was higher than 88 mg kg −1 . Beside bioaccumulation of Pb, adult earthworms exposed to the soil from the berm (D site) also accumulated significant amounts of Cu, Fe, Ni and Sb, while juveniles-Cu, Fe, Zn, and Mn. Previous studies also demonstrated that the higher availability of heavy metals in the shooting range soils resulted in the accumulation of other elements such as Cu and Zn, but much lower level compared to Pb (Sanderson et al. 2014;Wang et al. 2018). Bioaccumulation of essential metals such as Fe, Cu, Zn, and Mn could also cause toxic effects under the high concentrations. Non-essential metals such as Ni and Pb are toxic even in small amounts (Gasic and Korban 2006). At excess concentrations, these metals could become detrimental to living organisms, and in the case of the shooting range we studied, high levels of pollution and bioaccumulation indicate that this poses a significant risk to the health of living organisms, including a health risk to its participants. Therefore, although lead is a major pollutant in the shooting ranges, other contaminants may raise important ecotoxicity concerns.  Fig. 4 Lipid peroxidation (MDA) and activities of antioxidant enzyme (CAT, GR, GST) in earthworm E. fetida exposed to the shooting range (A, B, C, D-plots at different distances from the fire line of the shooting range (5, 20, 30, and 45 m, respectively)) and reference (R) soil. Values are mean ± SE, n = 6. The same letters above the columns indicate no significant differences between the treatments at p > 0.05 using a Mann Whitney U test MDA content of earthworms was used to as an indicator of lipid peroxidation to assess oxidative stress status (Bai et al. 2020). In our study, MDA content in E. fetida increased significantly after 28 days of the contaminated shooting range soil exposure. Despite that there was no relation between MDA content and total heavy metal concentrations, higher content of contaminants in the treated soil induced MDA increase in tested organisms as has been found in previous studies (Hu et al. 2016;Sinkakarimi et al. 2020;Li et al. 2020). Toxic effects of heavy metals, including Pb, on earthworms result in the formation of oxygen metabolites-reactive oxygen species (ROS) that could react with cellular components resulting to peroxidation of lipid membranes and increase in MDA content (Saint-Denis et al. 2001).
Antioxidant enzymes play an vital role in lowering the ROS levels and helping to avoid oxidative stress (Elavarthi and Martin 2010). Such enzymes as CAT and GR help organisms to adapt to stressful conditions and the inhibit of specific enzymes by the elimination of ROS. In the present study, the activity of CAT of in earthworms exposed to contaminated soils were not affected. This could be associated with high cellular stress under which CAT activity can decrease with exposure time (Chao et al. 2016). With increasing total Pb concentration, the activity of GR in earthworms E. fetida increased only at the D site, where the highest Pb and Sb contamination was found. A positive relationship was determined between GR and heavy metal concentrations in soil were determined, especially with total Pb and Zn concentrations. Because glutathione plays a protective role in cellular tissue against heavy metal toxicity, high concentrations of heavy metals had a significant effect on the activity of GR (Maity et al. 2008). At the same time, the activity of GST was the lowest where Pb concentration in soil was the highest. The study by Saint-Denis et al. (2001) also showed that GST activity decreased significantly in earthworms E. fetida andrei exposed to a Pb-contaminated soil. GST is a metabolic stress enzyme and is important as biomarker in earthworms. The negative relationship found between GR and GST indicated a compensatory mechanism between these enzymes. Łaszczyca et al. (2004) reported that the highest GR activity but the lowest activity of GST was found in the earthworms taken directly from the contaminated site. This type of mechanism resulted from the induced changes between reduced and oxidized glutathione, leading to increased consumption of this peptide for the synthesis of heavy metal-binding proteins (Łaszczyca et al. 2004). Because antioxidant enzymes in the earthworms are involved in the adaptation for survival and neutralization of free radicals and reactive oxygen species that cause cell and tissue damages, these enzymes could protect cells (Jeyanthi et al. 2016). The results of this study confirm the importance of using different biochemical indicators to monitor and assess environmental pollution.

Conclusions
A shooting range soil was contaminated with Pb and Sb along with higher soil density. The backstop berm soil had the highest total Pb and Sb concentrations, negatively affecting the survival and reproduction of the exposed earthworm E. fetida. Concentrations of Pb in E. fetida linearly increased with increasing concentration of Pb in the soils. Toxic effects of heavy metals on earthworms resulted in the formation of ROS that reacted with cellular components and resulting to peroxidation of lipid membranes as MDA content increased. GR activity increased significantly, and GST activity decreased in earthworms exposed to the contaminated soils. Toxicity tests coul help to indicate the ecological risk of the contaminated soils.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.