The Usefulness of physiological and biochemical stress response of soil collembola (Xenylla welchi Folsom, 1916) as a biomarker in a lead-amended garden soil
Article information
Abstract
This study investigates the physiological and biochemical stress responses of the microarthropod Xenylla welchi to different sublethal doses of lead-contaminated garden soil in microcosms, aiming to assess the impact of metallic contamination in tropical ecosystems. 24-hour LC50 for lead acetate was determined to be 2653.23 mg/kg. Chronic exposure to various sublethal concentrations (1/2, 1/4, 1/6, and 1/8 of LC50) revealed significant reductions in exuvia production, fecundity, and lifespan, particularly at higher lead concentrations. Several biochemical parameters were assessed to further understand lead-induced stress responses. A marked decrease in glutathione (GSH) levels indicated oxidative stress, while glutathione-S-transferase (GST) activity displayed temporal variations, initially increasing and then declining with prolonged exposure. Acetylcholinesterase (AChE) activity was consistently inhibited across the treatment groups, reflecting neurotoxicity. Additionally, metallothionein (MT) levels were significantly elevated after extended lead exposure, suggesting an adaptive response to metal detoxification. The cumulative responses of Xenylla welchi suggest that they could be reliable biomarkers for assessing the ecological impact of lead pollution in soil ecosystems, highlighting their potential usefulness in monitoring heavy metal contamination.
Introduction
Heavy metal soil pollution is a widespread problem that has become a major global concern, especially in urban areas due to urbanization and industrial growth [1, 2]. 13 % of total cultivated land, 40 % of lakes and rivers, and 0.24 billion hectares of the world's arable land are polluted due to heavy metals [2, 3-6]. Due to their ability to remain in the soil for a long period, heavy metals are detrimental to the health of soils and ecosystems because they can be harmful to virtually all organisms and affect many environmental processes [7-9]. The accumulation of heavy metals in different layers of soil is affected by various factors such as soil type, texture, organic matter content, and environmental conditions [10,11]. Moreover, after being released into the environment, heavy metals have the potential to travel long distances through atmospheric particles, which could increase the impact of metal pollution [1, 12].
Soil microarthropods like Collembola are important for maintaining soil ecosystem functions. They contribute significantly to energy flow and nutrient cycling and are highly sensitive indicators of changes in soil conditions among soil fauna [13, 14]. Because of its short generation time, relative sensitivity to a wide spectrum of contaminants, and ease of laboratory adaptation, it has been utilized as a standard species and genomic model organism for testing the toxicity of chemicals as well as soil quality [15, 16]. Heavy metals have been found to influence the structure of collembolan species and their behaviors significantly, given their sensitivity to pollutants [17]. As a result, the accumulation of pollutants from human activities in urban soils causes greater stress in collembola, as these pollutants can have a variety of consequences on their life-cycle properties [14, 18, 19].
Numerous studies have delved into the impact of heavy metals on Collembola; nevertheless, it is notable that most of these studies have been carried out in European soil contexts [20]. Conversely, despite the ecological significance of Collembola in tropical climates, there exists a remarkable scarcity of understanding concerning their responses to heavy metal contamination within tropical soil conditions. This glaring disparity underscores a substantial gap in our comprehension of soil ecotoxicity specifically within tropical environments, highlighting the urgent need for further research and attention in this area [21].
In this current microcosm investigation, we evaluated the physiological and biochemical stress responses of Xenylla welchi Folsom, a common tropical Collembola species, exposed to lead-treated garden soil. The study examined the effects of lead on X. welchi in terms of exuvia production, fecundity, and lifespan. Additionally, we focused on various biomarkers related to antioxidant defense and detoxifying enzymes to understand the biochemical responses of X. welchi to heavy metal pollution in tropical conditions. By focusing on the temporal dynamics (1, 3, and 7 days) of biomarker responses in X. welchi, a microarthropod native to tropical soils and relatively underexplored in the context of heavy metal stress, we address a critical gap in understanding species-specific responses to environmental contaminants. The significant changes observed in these biomarkers highlight their effectiveness in detecting lead-induced toxicity, emphasizing their potential as essential indicators for assessing metal contamination in soil environments.
Materials and Methods
Experimental Design
Test medium
Soil taken from the natural garden site is used as a test medium for the bioassay tests. The physicochemical properties of the test medium were determined as follows: soil texture using the international Piper method [22] soil pH with a Systronics model 512SE pH meter, soil organic carbon using the rapid titration method [23], and soil water holding capacity according to the methods of Lal (1977) [24] and Viji and Rajesh (2012) [25] (Table 1). The test medium was properly defaunated before being used for the bioassay [26].
Physicochemical parameters of the experimental soil
Test organism
X. welchi was collected from the soil with good organic matter and reared in polythene vials measuring 4.0 cm in diameter and 5.5 cm in height each containing 7 gm of moist soil [21]. The specimens were fed a few of Baker's yeast granules as a food source and kept in the Environmental Chamber at a fixing temperature (28⁰ C ± 0.5 °C) [27]. Moisture has been maintained by adding water occasionally [28]. The just-emerging juveniles were raised independently by moving the adults into new vials. Utilizing X. welchi stocks 12-15 days old, the toxicity tests were conducted three more times [21].
Test chemical
Fly ash dumping into the environment is currently one of the biggest issues facing the globe, particularly in developing nations [29]. While efforts to use fly ash as a fertilizer or soil amendment to improve physicochemical properties are prompted by its alkaline nature and high concentration of mineral substances, there are environmental concerns associated with its elevated levels of toxic heavy metals, such as Cu, Zn, Cd, Pb, Ni, and Cr [30-32]. Heavy metals mostly lead (Pb) and cadmium (Cd) harm several ecosystems near urban areas in various countries [6, 33-35]. For this investigation, soil tainted with coal fly ash was gathered from the Kolaghat Thermal Power Plant, situated in the Purba Medinipur region of West Bengal, India, at 22°25'28.3"N 87°51'39.4" E. Employing a randomized block design technique ensured a systematic collection process. Subsequently, collected soil samples are pooled to provide a comprehensive basis for analysis. Metallic content in the collected sample was analyzed using atomic absorption spectroscopy (AAS) (Table 2). Lead has been selected for the study as lead showed the highest concentration in the metallic composition of fly ash sample and has been used as Lead acetate trihydrate [(CH₃COO)₂ Pb.3H2O] for the present study. The metal was obtained from Merck Life Science Private Limited in Mumbai, India.
Heavy metal content of fly-ash-contaminated soil
Acute toxicological test
The current study examined the acute toxicity of lead acetate to X. welchi using standard protocols with some changes [15, 16]. The test was conducted in polythene vials (2.0 cm diameter and 2.0 cm height) filled with 2 gm of garden soil passed through a 0.25 mm mesh sieve, with the lead dosage (treatments) and (control). Different doses of lead acetate were administered into the test vials concerning the doses found in the fly ash-contaminated soil from the field. To ensure the metal solution was distributed evenly throughout the medium, the vials were kept undisturbed for about thirty minutes. To keep X. welchi from avoiding the substrate, the smaller container was employed, and the container wall was attached to ensure that X. welchi was completely exposed to the lead-contaminated soil. For each dose of lead acetate, three sets of replicates were constructed to determine the concentration causing 50 % death and the LC50. Ten X. welchi juveniles that were between the ages of 12 and 15 days were gathered and placed into each jar by timing the egg-laying of the culture animals. After exposure for 24 hours, the mortality of X. welchi was recorded [36].
Chronic toxicological test
The toxicity of lead acetate on the exuvia production, fecundity, and life span of X. welchi was assessed following the standard test procedures [16]. The following sub-lethal concentrations were employed: 331.63, 442.17, 663.25, and 1326.50 mg/kg soil to the findings of the acute toxicity study. 45 polythene vials, each measuring 4 cm in diameter and 5.5 cm in height, were filled with 7 grams of soil and had varying amounts of lead acetate added to each replicate. Three replicates in total were used in this investigation. Each vial contained ten specimens aged 12-15 days. For 28 days, the no. of exuvia was counted every 24 hours.
Biochemical assays
Biochemical studies were performed using natural garden soil according to OECD guidelines [16], which were identical to the previously reported chronic toxicity test. The exposure periods of 1, 3, and 7 days were chosen to capture the dynamics of biochemical responses in X. welchi to lead exposure. The 1-day period allowed for the observation of immediate stress responses, while the 3-day period provided insights into the progression of these responses. The 7-day exposure was selected to assess cumulative effects and potential recovery or adaptive responses, simulating longer-term contamination. These time points ensure a comprehensive evaluation of both acute and chronic stress, as well as recovery potential of X. welchi under heavy metal exposure.
Preparing homogenates and conducting biochemical analyses
X. welchi was cautiously taken out from each vial to extract the enzymes on days 1, 3, and 7 of the incubation periods. With three replicates for each parameter of lead and control in soil, a total of 60 vessels were made in the manner previously described. The estimations were based on the surviving specimens; each replicate comprised about 50 specimens. The current study evaluated the activities of four enzymes: reduced glutathione (GSH), glutathione-S-transferase (GST), acetylcholinesterase (AChE), and metallothionein (MT).
An important stress parameter tested in X. welchi is glutathione. According to the assay [37], 5-thio-2-nitrobenzoic acid (TNB) is produced when the sulfhydryl group of glutathione combines with DTNB (5,5ʾ -dithio-bis-2- nitrobenzoic acid). The whole-body homogenate (5 mg live weight in 2 ml of phosphate buffer, 50 mM, pH 7.0) was used for the analysis. It was centrifuged for 30 minutes at 4.0°C at 12000 rpm [21]. Using a spectrophotometer (Systronics: PC-Based Double Beam UV-VIS Spectrophotometer, Type: 2202) and DTNB (0.1M) incubation, the tissue sample's optical density (O.D.) was measured at 405 nm by comparing it to a reagent blank. Results were expressed as µ g of GSH per mg of protein [37].
The formation of glutathione and 1-chloro-2, 4-dinitrobenzene (CDNB) conjugate was utilized to test glutathioneS-transferase activity [38]. The assay mixture contained a tissue sample, 1 mM GSH, and 1 mM CDNB (substrate) in 0.1M phosphate buffer (pH 6.5). At one-minute intervals, the GS-DNB was measured at 340 nm and expressed as µ mole of DNPG generated per minute per mg of protein.
The acetylcholinesterase activity was measured using Ellman's method [39]. The rate of acetylthiocholine breakdown was measured at 412 nm by monitoring the rise in yellow color caused by thiocholine binding with 5, 5-dithiobis (2-nitrobenzoic acid). Using acetylthiocholine as the substrate, the results were represented as nmol of thiocholine created per minute per mg of protein.
Metallothionein activity was determined using a modified spectrophotometric approach [40]. Results were expressed as µ g of glutathione (GSH) per mg of protein or µ g of metallothionein (MT) per mg of protein. The standard GSH curve was created by measuring known µ g of GSH at a spectrophotometric wavelength of 412 nm, similar to the metallothionein experiment. Notably, GSH was employed as a substitute for collembola tissue in this case.
The biuret reaction of protein with alkaline cupric tartrate and the blue color produced by the amino acid tyrosine and tryptophan in the Folin-Ciocalteau reagent was used to determine the total protein content of X. welchi tissue [41]. The O.D. was determined at 660 nm with a bovine serum albumin (BSA) standard. The mg per ml value for tissue proteins was reported.
Data presentation and statistical analysis
The SPSS software and Microsoft Excel were used for statistical analysis and graphical depiction of the results. The LC50 (effect on survival) value was calculated using the probit analysis program of SPSS software version 29.0.1.0 (171). The findings about physiological and biochemical biomarkers were presented as the mean value along with its corresponding standard deviation (SD). Single-factor ANOVA was used to examine significant changes between treatments, followed by Tukey's post hoc testing at a 5 % probability level.
Results and Discussion
Acute toxicity of lead acetate and the chronic effects on exuvia Production, fecundity, and lifespan
The 24-hour LC50 value of lead acetate for X. welchi is recorded as 2653.231 mg/kg (Table 3).
24 hours LC50 values (mg/kg) of lead acetate on X. welchi
Over subsequent days of lead treatment, chronic toxicity effects became evident. The number of exuvia was (22 ± 2.08) in the control group, whereas in treated vials a significant reduction (P < 0.05) of exuvia production was observed in T4 (1/2 LC50) and T3 (1/4 LC50) doses (Figure 1A). About 50 % (11 ± 2.52) and 27.36 % (16 ± 2.65) of reduction in exuvia production have been found in T4 and T3 doses concerning the control (T0) group (P < 0.05). However, for T1(1/8 LC50) and T2 (1/6 LC50) doses, no significant reduction of exuvia production has been recorded compared to the control. A substantial (P < 0.05) decline in fecundity was noted with heightened lead concentration. The control group exhibited an average fecundity rate of (59.67 ± 6.11), while discernible reductions in fecundity were observed across all treated doses (Figure 1B). The lifespan of X. welchi has also been altered with exposure to lead. The average life span of control groups was (97±1.53 days), whereas a significant reduction (P < 0.05) in life span has been observed with increased concentration of lead. For T4, T3, T2, and T1 doses there are 59.8 % (39 ± 2.08), 51.55 % (47 ± 3.21), 37.12 % (61 ± 2.52), and 25.78 % (72 ± 3.06) reduction of life-span has been recorded (Figure 1C).
Exuvia production (1A), Fecundity (1B), and life span (1C) of X. welchi exposed to sublethal doses (T1; 1/8 LC50, T2; 1/6 LC50, T3; 1/4 LC50, and T4; 1/2 LC50) of lead acetate and the control (T0); without treatment. The mean ± standard deviation is used to express the results (n = 3). Statistically significant differences between groups are indicated by different letters above the bars (p < 0.05).
This study demonstrates that lead negatively affects exuvia production, fecundity, and lifespan in the selected collembola species. Several research have corroborated this finding, highlighting the role of biological and environmental factors, such as heavy metals in the soil, regulate the reproduction of collembola [42]. The growth rate and reproduction (egg laying time) were impacted in collembola, Folsomia candida when given Cd, Pb, Cu, and Zn contaminated yeast for seven weeks [43]. Menta et al. [44] found a decrease in juvenile production in collembola at lead concentrations ranging from 500 to 1000 μg/g. The sensitivity of heavy metals like Pb and Cd on the development and reproduction of collembola was also noted by Sahana et al. [21]. Dai et al. [45] reported a substantial decrease in the survival, growth rate, and reproduction of collembola as the concentration of Pb in the soil increased. This aligns with other research findings that stated higher levels of Pb and Cd in urban soil may have a substantial negative impact on the growth, sexual development, reproduction, adult survival, and life behavior of collembola as well as the size, density, and composition of these species has also been affected [2, 5, 20, 44, 46].
Effects of lead toxicity on enzyme activities
After 1 day of treatment, the GSH concentration (μg GSH mg-1 proteins) decreased significantly (P < 0.05) for the T4 and T3 doses compared to the control (Figure 2A). For T2 and T1 doses no significant differences in glutathione content compared to control have been observed after 1 day of treatment. The inhibition persists after 3 days of treatment for T4 and T3 doses and GSH concentration was significantly lower (P < 0.05) than the control (Figure 2B). However, after 7 days, the T2, T3, and T4 groups differed significantly from the control groups (P < 0.05), and the T1 showed a non-significant difference (P > 0.05) from the control (Figure 2C).
The concentration of Reduced Glutathione (GSH) in X. welchi was measured following exposure to sublethal doses of lead acetate (T1, T2, T3, and T4) and compared to the control group (T0; no treatment) after 1 day (2A), 3 days (2B), and 7 days (2C) of exposure. Results are presented as the mean ± standard deviation (n = 3). Statistically significant differences between groups are indicated by different letters above the bars (p < 0.05)..
The activity of GST (μM DNPG/min/mg of protein) showed a significant increase (P < 0.05) in the T3 and T4 groups compared to the control after 1 day of treatment (Figure 3A). In contrast, no significant difference (P > 0.05) was observed between the T1, T2, and control groups after day 1. After 3 days of exposure, GST activity in the T1 treatment group remained insignificant to the control (Figure 3B). However, the GST activities in the T2, T3, and T4 groups were significantly lower (P < 0.05) than the control, with reductions of 9.15 %, 8.76 %, and 8.5 6%, respectively. By day 7, GST activity in all treated groups had significantly decreased (P < 0.05) relative to the control group (Figure 3C). The highest dose (T4) resulted in the greatest reduction, with a 48.92% decrease in GST activity compared to the control.
Glutathione-S-transferase (GST) concentration of X. welchi exposed to sublethal doses (T1, T2, T3, and T4) of lead acetate and the control (T0; without treatment) after 1 (3A), 3 (3B) and 7 (3C) days of exposure. The mean ± standard deviation is used to express the results (n = 3). Statistically significant differences between groups are indicated by different letters above the bars (p < 0.05).
The activity of AChE (nmol/min/mg of protein) was reduced significantly after 1 day of lead exposure across all treated concentrations (P < 0.05) (Figure 4A). This inhibition remained evident after 3 days for all treated concentrations when compared to the control group (P < 0.05) (Figure 4B). After 7 days of exposure, compared to the control groups, no significant differences were found in the T1 and T2 treatments but for T3 and T4 treatments, a significant (P < 0.05) decrease in AChE activities has been noted (Figure 4C). For the T3 and T4 treatment, AChE activity was significantly reduced throughout the exposure period in comparison to the controls (P < 0.05).
Acetylcholinesterase (AChE) activity of X. welchi exposed to sub-lethal doses (T1, T2, T3, and T4) of lead acetate and the control (T0; without treatment) after 1 (4A), 3 (4B) and 7 (4C) days of exposure. The mean ± standard deviation is used to express the results (n = 3). Statistically significant differences between groups are indicated by different letters above the bars (p < 0.05).
For MT, after 1 day of treatment, the treated groups showed no significant difference (P > 0.05) in MT activity with the control (Figure 5A). After 3 days, the T4 group was found to have a significant increase (P < 0.05) in MT concentration compared to the control. The rest of the treated doses showed no significant change in MT activity with the control group. (Figure 5B). However, after 7 days, except the T1, all other treated groups have shown a significant increase of MT concentration concerning the control group (P < 0.05) (Figure 5C).
Metallothionein (MT) activity of X. welchi exposed to sub-lethal doses (T1, T2, T3, and T4) of lead acetate and the control (T0; without treatment) after 1 (5A), 3 (5B) and 7 (5C) days of exposure. The mean ± standard deviation is used to express the results (n = 3). Statistically significant differences between groups are indicated by different letters above the bars (p < 0.05).
Exposure to xenobiotics can increase reactive oxygen species (ROS) in cells and alter the presence of antioxidant defense enzymes, reducing an organism's ability to withstand increased oxidative stress and causing reproductive organ malformations and decreased fertility [47]. Antioxidants such as GSH and GST protect cells by minimizing the damaging effects of reactive oxygen species (ROS). In this study, the glutathione content decreased in the tissues of X. welchi exposed to lead acetate for 1, 3, and 7 days of treatment. Furthermore, the enzyme activities in X. welchi were shown to be concentration-dependent, with the highest applied concentration corresponding to the lowest enzyme activity. Studies on glutathione levels have also been conducted in various invertebrate species. Glutathione levels in Enchytraeus albidus were significantly lowered by exposure to Cd and Cu at doses of 100 mg/kg and 750 mg/kg, respectively [48,49]. Another study stated that in Eutyphoeus waltoni, GSH levels increased dramatically with increasing Cd exposure dose, whereas in Eisenia fetida, acute exposure to Cd led to declines in glutathione levels [50]. The decline in GSH content may stem from heavy metals directly hindering antioxidant functions or from the overall deterioration of an organism's health [51]. Conversely, the rise in content could be viewed as an organism's adaptive response to counter oxidative stress-induced damage [52, 31, 5]. The reduction in GSH levels and enzyme activity observed in X. welchi due to exposure to lead acetate in this study suggests a major weakening of the antioxidant defense in this organism, which may increase susceptibility to oxidative stress and related problems.
In this study, while GST activity significantly rose in the T3 and T4 groups after 1 day, it significantly declined after 3 days and further dropped in all treated groups by 7 days compared to the control. This finding aligns with a prior study that stated that the activity of GST was reduced in another collembola, Cyphoderus javanus after seven days of exposure to soil treated with Pb and Cd [21]. A study by Kovaĉević et al. [53] revealed that during the initial days of azoxystrobin treatment, GST activity in F. candida increased at certain concentrations. As the treatment continued, GST activity significantly decreased over time. Numerous investigations have revealed a decrease in the activity of antioxidant enzymes in insects exposed to pollutants [54-56]. According to Zhang et al. [57] at one or more sampling intervals, GST activity was suppressed in an earthworm E. fetida after exposure to the neonicotinoid insecticide imidaclothiz. When exposed to wastewater from olive mills, E. albidus and E. fetida have similarly been shown to have decreased GST activity [58, 59] as have several earthworm species exposed to pesticides [60, 61]. One of the main roles of the GST enzymes is to catalyze the conjugation of xenobiotics with glutathione, which leads to its detoxification. Metals can disrupt the function of GST, potentially leading to harmful toxic effects on the organism. Several studies have shown that metals decrease GST activity, reinforcing the idea that GST is vital for defending cells against oxidative stress, performing roles of both GST and glutathione peroxidase. As a consequence, metal-induced inhibition of GST interferes with the intracellular detoxification of organic xenobiotics metabolite which may lead to unpredictable toxicological consequences for organisms [62].
In various sub-lethal dosages of lead treatment, AChE activity of X. welchi was significantly impacted. Acetylcholine (ACh) is essential for nerve transmission, and exposure to heavy metals and other xenobiotic compounds can interfere with it, which can affect the neurological system of collembola. The metal prevents the breakdown of ACh in the synaptic cleft by binding to the active site of the cholinesterase (ChE) enzyme. This induces ACh accumulation in the synaptic cleft, which promotes overstimulation of neuronal cells and neurotoxicity in collembola [27]. Corroborating to our results, Chakravorty et al. [63] found that the AChE activity in C. javanus has been suppressed by organophosphate and carbamate group of insecticide, methyl parathion, and carbaryl. In F. candida, significant suppression of AChE activity was seen at all tested azoxystrobin doses and time points [53]. In fly ash-treated soil the AChE of collembola is compromised within 7 days of exposure to heavy metals [64].
The present study found that after 7 days of treatment, the T2, T3, and T4 groups displayed a significant increase in MT concentration compared to the control group. This result corroborates the finding of Maria et al. [65] who reported an increase in the MT levels in collembola F. candida after 6 days of exposure to heavy metals Cu and Cd. In a prior study, Maity et al. [66] documented the induction of MT in earthworm tissue following Pb exposure. Metallothioneins are tiny cysteine-rich proteins that scavenge ROS and maintain metal homeostasis [67]. Elevated MT levels may increase ROS scavenging, shielding cells from oxidative damage, and the sustained increase in MT levels indicates that the treatment had a cumulative effect over time. It may be associated with adaptive responses or cellular processes initiated by the treatment.
Conclusions
This study shows that lead exposure has a considerable impact on the physiological and biochemical parameters of X. welchi, with negative impacts on exuvia production, fecundity, lifespan, enzyme activities, and acetylcholinesterase levels. The concentration-dependent decreases in GSH and GST activity and higher MT levels highlight the complicated metabolic reactions to heavy metal exposure. These findings highlight the importance of multi-biomarker analysis in environmental health evaluation. Biomarkers, as accurate indicators of biological responses to environmental stresses, are critical for assessing the degree and severity of pollutant exposure. Future studies should explore the long-term effects of sub-lethal lead exposure, interactions among multiple heavy metals, potential genetic adaptations in X. welchi, and the broader ecological impacts on soil-dwelling organisms to improve our understanding of environmental toxicology and inform mitigation strategies.
Notes
Acknowledgement
The authors are grateful for the facilities provided by the Principal, Raja Narendra Lal Khan Women's College (Autonomous), Natural and Applied Sciences Research Centre, Paschim Medinipur, West Bengal, India. PS is grateful for Junior Research Fellowship from the University Grant Commission (UGC) to conduct the research work, and PPC is grateful to DST-CURIE for providing infrastructure assistance.
Conflict of interest
The authors declare that they have no competing interests.
CRediT author statement
PS: Writing – Original draft preparation, Data curation, Formal analysis; PPC: Project administration, Supervision; BD: Supervision