Introduction

The agriculture sector has tremendously progressed in crop production and integrated pest management1,2. Pesticides are chemicals or mixtures primarily used to control, prevent, and kill harmful pests and avoid vector-borne diseases3. Due to the rising demand for crops and vegetables, the application of pesticides in agriculture is remarkably increasing day by day4,5. Pesticides can enter the water ecosystem through surface run-off, leaching, soil erosion, or through evaporation of pesticides into the atmosphere that might precipitate as pesticide residues in the surface water6,7,8. CBSF is widely used in integrated pest management systems in rice fields to control insects9,10. This carbamate group of insecticides acts as a neurotoxicant by inhibiting synaptic transmission11,12. They are widely used as insecticides, acaricides, and nematicides13. Acetylcholine is predominantly blocked by carbamate insecticides14. This group of pesticides act on the nervous systems and are reversible inhibitors of acetylcholinesterase, the broad-spectrum activity used to control a wide variety of pests, including sucking insects, chewing insects, nematodes, and mites15. CBSF belongs to the carbamate family and is largely used against the larvae of paddy white aphids of chillies and caterpillars16. The careless use of CBSF in rural areas, as well as all over the world, leads to its entry into aquatic bodies through leaching and causes potential harm to aquatic life17. Inhibition of acetylcholinesterase disruption leads to endocrine perturbation, reproductive defects, respiratory failure, paralysis in insects, and ultimately death of the insects18,19. Being a benzofuranyl methylcarbamate, CBSF is closely connected to carbofuran’s primary metabolites20. CBSF is produced from 3-hydroxycarbofun and lastly oxidized into 3-keto carbofuran21. The activities of catalases (CATs), superoxide dismutase22and glutathione S-transferase content reflect the oxidative stress of CBSF on a particular test organism23. Recent studies have proved that the toxicity of CBSF causes DNA fragmentation in many organisms23. CBSF causes an enormous accumulation of free radicals and eventually an alteration of oxidative enzymes24. The short-term lethal (acute exposure) and long-term (sub-lethal) exposure to the carbamate insecticide CBSF causes the generation of reactive oxygen species (ROS), causing an elevation of oxidative stress25. As first line of defence against oxidative stress, organisms enhance a variety of mechanisms, including enzymatic (catalases, superoxide dismutase, glutathione peroxidases) and non-enzymatic (malondialdehyde) adaptations26.

Enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione S-transferase (GST), and other low molecular weight scavengers like glutathione (GSH) belong to part of the antioxidant defence system27. Furthermore, the GST family of multifunctional enzymes catalyses the conjugation of GSH with a variety of electrophilic metabolites that are involved in the detoxification of both reactive intermediates and oxygen radicals28. To prevent highly reactive electrophilic components from covalently attaching to tissue nucleophilic molecules and producing detrimental side effects, GST aids in the detoxification of xenobiotics29. CBSF is reported to have toxic effects on aquatic invertebrates, crustaceans, and cladoceran Daphnia magna, causing alteration of survival rate and inhibition of acetylcholine esterase (AChE) activity30. Moreover, the integrated biomarker responses (IBR) represent all oxidative biomarkers and the physiological state of the organisms, making it easier to interpret multi-biomarker responses and contamination levels31. Utilising the IBR index as a useful tool to assist scientists and executives in assessing ecological risk and as an indicator of environmental stress32.

The Tubifex tubifex is a common invertebrate species found in aquatic environments33. Tubifex tubifex is an aquatic invertebrate (Phylum: Annelida, Class: Clitellata, Order: Oligochaeta, Family: Naididae). They inhabit the uppermost layer of the freshwater sediment ecosystem and act as a “conveyor belt” deposit feeders34. Tubifex tubifex (Müller, 1774), a common benthic invertebrate with a wide range of distribution and high resistance to pollution, has been used to improve the performance of artificial wetlands due to its exceptional ability to advance the nitrogen cycle and the breakdown of sediment organic matter through a variety of pathways involving bioturbation and ingestion35.

Tubifex tubifex serves as an important bioindicator species, and due to their wide distribution and resilience to a variety of ecological factors, they are chosen as test organisms36. So, in the present investigation, Tubifex tubifex was used as an experimental organism to determine the lethal and sublethal toxicity of CBSF. One of the earliest aquatic oligochaetes to be described, Tubifex tubifex is a cosmopolitan species that is known for being extremely resistant to pollution, albeit not insensitive to it. It is frequently one of the last to leave a contaminated site and can be found in both highly and nonpolluted waters.

The main objectives of our study are to fill the knowledge gap of ecotoxicological impacts of CBSF on Tubifex tubifex regarding Toxicokinetic-Toxicodynamic (TK-TD), Species Sensitivity Distribution (SSDs), behavioural alteration, changes of oxidative biomarkers, and integrated biomarker responses (IBR) to correlate the interrelationship between the biomarker histopathological alterations and molecular docking approach to validate the molecular interaction of the toxicant with the stress regulator.

Methods and materials

Experimental organisms

A full-grown, mature and annelid (Phylum: Annelida, Class: Clitellata, Order: Tubificida) was collected from an unpolluted site near the Burdwan University Campus used for bioassays with an average body size (9.0 ± 0.2) mm and a feeding rate (5–6 times/day). Before setting up the experiment, the worm was treated with KMnO4 to disinfect (concentration of 0.05% for two minutes), then protected from any kind of infection and acclimatised for 24 h before the experiment in the laboratory environment under a slow stream of water.

Results and discussion

Acute toxicity bioassays

Acute toxicity studies were carried out on freshwater benthic worms, Tubifex tubifex, to determine the toxicity of CBSF on benthic macrofauna. No test organisms died during the acclimatisation period. Table 1 shows the acute toxicity of CBSF on T. tubifex with a 95% confidence limit. No mortality was recorded in the control set during the experimental period. The mortality of the worms increased with the increasing concentrations of the toxicant. Two-way ANOVA was used for statistical evidence, and it revealed that they had a significant impact on worm mortality (P < 0.0001) and toxicant concentration (P < 0.0001). LC50 values obtained were 13.736 mg/L, 11.839 mg/L, 10.982 mg/L, and 8.314 mg/L, respectively, for 24, 48, 72 and 96 h. The Kaplan-Meier curves depicted the concentration of CBSF and duration-dependent adverse effects on worms (Fig. 1), where the chi-square value is 74.8, the degree of freedom is 10, and significance is p < 0.0001. The curve on Kaplan-Meier plots reflected that the survival rate of the worm was significantly decreased. The one-way ANOVA revealed that the concentration of toxicant and exposure period have a significant impact on the mortality of the worms. Based on 96 h LC50 values, the No Observed Effective Concentration (NOEC), Lowest Observed Effective Concentration (LOEC), and Maximum Allowed Toxicant Concentration (MATC) are 9 mg/L, 10 mg/L, and 0.8314 mg/L.

Fig. 1
figure 1

(a) The Kaplan-Meier Survival plot of Tubifex tubifex upon exposure to various exposure concentrations of CBSF (Chi-square Value 65.31, df 9, P value < 0.0001). The X-axis demonstrates Exposure time (in hours), and the Y-axis depicts the percentage survival of the test animal. (b) Determination. (c) The mean mortality of Tubifex tubifex in 24, 48, 72, and 96 h intervals is depicted by the Box and whisker plot, where the ns represent the non-significant differences (**p < 0.01, and ****p < 0.0001), data represented mean ± SD.

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General unified threshold model for survival (GUTS) analysis

In recent times, the reduced GUTS modelling is used when the internal concentration is not available, and survivorship data collected from the standard toxicity test64. Two models were predicted to assume the adverse effects of toxicants, such as the Stochastic death (SD) model and the Individual tolerance (IT) model4,65. The GUTS-SD model demonstrated the possibility of individuals dying as a result of toxicants after they attain a particular level of damage4. The GUTS-IT model demonstrated the sensitivity level of a particular chemical in an individual4. Figure 2 depicts the GUTS-SD and GUTS-IT model. Relative fit of observed and fitted values of the (a) GUTS-SD and (b) IT models at different CBSF exposure concentrations. In the present study, the GUTS-RED-SD is comparatively narrower than the GUTS-RED-IT. Further, using the simulation GUTS-RED-SD revealed that a well-fitted survival rate was observed at 0.0 mg/L for all concentrations of CBSF but overstimulated at the concentrations of 14 mg/L, 15 mg/L, 16 mg/L, 17 mg/L, and 18 mg/L. For Tubifex tubifex the Individual Tolerance level (IT) is suitably fitted at 0.0 mg/L for all concentrations of CBSF and overstimulated at the concentrations of 15 mg/L,16 mg/L, 17 mg/L, and 18 mg/L. The fitness of the model was based on the Akaike Information Criterion66 values. The lower the value of AIC denotes the better the fitness4.

In the present study, the AIC value of GUTS-RED-SD (AIC-252.35) is lower than the GUTS-RED-IT (AIC-275.32) model. The larger predicted value of GUTS-RED-IT depicts the higher sensitivity of Tubifex tubifex than the random stochastic death4. According to the GUTS-RED-SD model, the 96-hour acute toxicity of test organisms is 9.076 mg/L, which is approximately equal to our calculated LC50 value, i.e., 8.314 mg/L. The long-term acute toxicity can be measured by analysis of the GUTS-RED-SD model, the 100d LC50 is 7.844 mg/L (6.067–8.573). It is possible to gain insight into the toxicokinetic and toxicodynamic consequences of CBSF exposure in Tubifex tubifex by comparing the observed LC50 values for CBSF with predictions from the GUTS-IT and GUTS-SD models. In determining the concentration at which 50% of Tubifex tubifex would die from exposure to CBSF, the GUTS-SD model’s predicted LC50 value (5.217 mg/L) and the observed LC50 (9.076 mg/L) closely match, demonstrating the model’s accuracy and dependability. In addition, the GUTS-IT approach captures the variation in sensitivity among Tubifex tubifex individuals and offers a more accurate representation of survival dynamics by integrating individual tolerance into the model. This data is essential for evaluating CBSF’s possible ecological hazards and guaranteeing a thorough comprehension of its effects on Tubifex tubifex populations in aquatic settings.

Fig. 2
figure 2

Comparison of the parameters estimated by the General Unified Threshold Model for Survival (GUTS). Observed vs. Predicted survival plot of CBSF for the calibration of (a) GUTS-RED-SD and (b) GUTS-RED-IT models, (c) Comparison between GUTS-SD (Stochastic Death) and GUTS-IT (Individual Tolerance) based on AIC values (Akaike Information Criterion), (d) Prediction of lethal concentration on long-term exposure, created in OpenGUTS® software.

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Altered physiological biomarkers

The present study is also designed to observe the ethological response of Tubifex tubifex, thus this worm serves as a bioindicator of the toxicity of CBSF in the environment (Fig. 3). According to the current study, there is no mucus secretion, and high clumping tendencies were observed in the control group and healthy worms. Similar results were observed when Tubifex tubifex was exposed to the fungicide propiconazole67. The mucus secretion of the worms increased with increasing concentrations of the toxicant. This is due to the avoidance reaction of worms from toxicants. Mucus secretion is an evolutionary response against toxicants68. Mucus secretion rises steadily throughout the 96-hour toxicological exposure period and quickly from 13 mg/L to 18 mg/L. Secretion of mucus against the toxicant represents the physiological resistance against the hazardous materials69. The toxic action of the worm due to the precipitation of mucus-toxicant on the epidermal layer of the worm inhibits the exchange of respiratory gases70. Similar effects were observed when tubificid worms aromatic acetic acid and benzoic acid68. The clumping tendency of the worm gradually decreases as the concentration of toxicants during 24 h to 96 h (Fig. 3). As the exposure concentration rises, the clumping or aggregation tendency falls. The clumping characteristics gradually deteriorate in the current experiment. Aggregation properties in the current experiment decrease time-dependently from 12 mg/L to 18 mg/L. During the experimental period, the control worms were active and had no erratic movement or wrinkling, whereas the treated worms displayed this abnormal behaviour, including enhanced erratic movement and wrinkling activity. Among all cases, the treated worms of higher concentration exhibit the maximum alteration of behaviours at 96 h, toxicological end-points, than the control group. When worms were kept in a control group, there was no mortality or autotomy seen. Increasing the concentrations of CBSF, on the other hand, resulted in a substantial (p < 0.05) increase in the percentage of autotomy (Fig. 2). The maximum percentage of autotomy was found at or above concentrations of 15 mg/L. The shedding of the caudal portion or autotomy increases with the increasing concentration from 14 mg/L.

Fig. 3
figure 3

A heatmap for the changes in the behavioural pattern and autotomy of T. tubifex due to concentration, along with time-dependent exposure to CBSF. The higher intensity of colour indicates the behavioural alteration is highly correlated to the exposure concentration of CBSF.

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Species sensitivity distribution analysis

The species sensitivity distributions are widely used to determine the ecological risk assessments of certain pollutants or groups of pollutants to a system or community based on the Potentially Affected Fraction (PAF) and combined potentially affected factor as a quantitative indicator of the environment49,71,72. The SSD analysis is generally performed to determine the safe level of the toxicants in the environment73. In the present study, SSDs were used to assess the toxic impact of the CBSF and safe level estimation on Tubifex tubifex (Fig. 4). The SSDs distribution curve was employed in this study to evaluate overall ecological risk, with a particular emphasis on the benthic tubificid worm. Environmental contaminant threshold concentrations must be determined by environmental risk evaluators to safeguard the diversity of species and the essential components of natural ecosystems74. The 96-hour median lethal concentration in the current study, 8.314 mg/L, is between the lower log-normal curve’s Oncorhynchus mykiss, Channa punctatus, and Danio rerio, indicating a severe impact of CBSF on those organisms. In contrast, the upper part of the curve, which includes Gara mullya, Tubifex tubifex, and Labeo rohita, indicates a low stressor intensity of CBSF.

The sensitivity of different species to environmental contaminants varies significantly, and this variance may be characterised by creating a Species Sensitivity Distribution. The stressor sensitivity curve demonstrates the propensities of affecting species following the order of Oncorhynchus mykiss > Oryzias latipes > Channa punctatus > Clarius batrachus > Danio rerio > Gara mullya > Tubifex tubifex > Duttaphrynus melanostictus.

Fig. 4
figure 4

The species sensitivity distribution curve depicts the proportion of species and Stressors intensity (LC50 96 h), the red line indicates the 95% confidence intervals, and the blue line central tendency.

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Oxidative stress enzyme biomarkers assessment

Tubificid worms were exposed to two sublethal concentrations of CBSF (0.831 mg/L and 1.662 mg/L, respectively) for 14 days and showed substantial changes in antioxidant enzyme activities (Fig. 5a-c). In the present study, the elevation of CAT was observed to be significantly, on the initial day, nearly 32.29% (****p < 0.0001), and 52.86% (****p < 0.0001) on exposure to 0.831 mg/L (T1) and 1.632 mg/L (T2) compared to the control group. The maximum level of detoxifying enzyme CAT observed at the 14th day of exposure subsequently increased by 79.28% (****p < 0.0001). and 83.45% (****p < 0.0001) on exposure to 0.831 mg/L (T1) and 1.632 mg/L (T2), respectively, compared with the control.

The activity of superoxide dismutase (SODs) increased significantly on exposure of two-sublethal doses 0.831 mg/L (T1) and 1.632 mg/L (T2). SODs activity increased by 254.902% and 514.216%, respectively, on the first day (1d) of exposure in contrast to the control (****p < 0.0001). The highest activity of SODs noticed at 7th day, consequent increase of 852.247% and approximately 1220.79% for the exposure of 0.831 mg/L (T1) and 1.632 mg/L (T2) respectively, compared with control (****p < 0.0001) (Fig. 5b). In thiobarbituric acid (TBARS), a lipid peroxidation product created when singlet oxygen reacts with unsaturated fatty acids, is regarded as a practical and trustworthy marker of oxidative damage in cells and tissues. In the present study, a remarkable level of cellular damage was observed on exposure to CBSF. On the initial day (1d), the level of malonaldehyde (MDA) increases by 399.35% (****p < 0.0001) and 732.90% (**p < 0.001) at exposure of 0.831 mg/L (T1) and 1.632 mg/L (T2), respectively, compared with the control. On the 7th day of exposure, the elevation of LPO was observed to be approximately 643.91% (***p < 0.001) and 939.15% (****p < 0.0001) in contact with 0.909 mg/L (T1), compared with the control group. The highest amount of TBARS was formed on the 14th day, approximately 852.24% (****p < 0.0001), and 1220.78% (***p < 0.001) on exposure to 0.831 mg/L and 1.632 mg/L, compared with the control group (Fig. 5c).

This rise in MDA could be linked to the interaction of CBSF with the polyunsaturated fatty acids, with cellular antioxidant deficiency, such as reduced GST activity, as reported earlier66. This increase in MDA levels changes the cell membrane permeability, allowing toxicants to enter the cell and eventually cause DNA deterioration and apoptosis75. The production of CATs and SODs is the first line of defence against Reactive Oxygen Species76. To combat the reactive oxygen species, the level of superoxide dismutase (SOD) level elevated, the similar observation reported on tubificid worm exposed to the difenoconazole for chronic period77. Subsequently, H2O2, another reactive species, is catalysed by the enzyme catalase; the elevation of CAT activity indicated higher H2O2 production in the system, which is generally scavenged O2-, H2O2, and other reactive species to mitigate the toxic effects. The elevation of CAT activity reported in Eisenia fetida when exposed to the imidacloprid-contaminated soil78. Data are shown as mean ± SEM (n = 3), and the Duncan multiple range test was performed.

Fig. 5
figure 5

Effects of different sublethal concentrations of CBSF on (a) CAT, (b) SOD, and (c)MDA level in Tubifex tubifex at different exposure periods. Where ns represents the non-significant differences and the * denotes the level of significance, (**p < 0.01, ***p < 0.001, and ****p < 0.0001). Data are shown as mean ± SEM (n = 3), and the Duncan multiple range test was performed.

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Docking analysis of the stress enzymes

We performed a thorough molecular docking investigation with important stress indicator enzymes from Tubifex tubifex, which include catalase, superoxide dismutase, and cytochrome c oxidase, in conjunction with CBSF. This investigation attempted to identify potential interactions among these enzymes with CBSF (Fig. 6A, 6 C, and 6E). The molecular docking investigation found that CBSF has binding affinities of -6.5, -5.4, and − 7.3 kcal/mol for catalase, superoxide dismutase, and cytochrome c oxidase from Tubifex tubifex, respectively. Notably, the catalase enzyme from Tubifex tubifex showed many alkyl and pi-alkyl interactions involving CBSF, particularly with amino acid residues like Met24, Arg15, and Tyr27. CBSF also exhibited π-stacked interactions with the Tyr27 residue of the catalase enzyme (Fig. 6B). Cytochrome c oxidase from Tubifex tubifex, an additional important enzyme in our research, is involved in the mitochondrial electron transport chain, aiding oxidative phosphorylation as well as catalysing the reduction of oxygen to water. The molecular docking investigation of cytochrome c oxidase and fluorene found two forms of intermolecular interactions: alkyl and pi-alkyl, unlike catalase. CBSF interacts with cytochrome c oxidase residues Ile59, Ile208, Ala63, Phe231, and His69 via alkyl, pi-alkyl, as well as π-stacking interactions with Trp56 and Phe212 (Fig. 6F). An investigation of the possible interactions between superoxide dismutase and CBSF also revealed alkyl and pi-alkyl connections with Phe58, Phe6, Val78, Leu65, and Phe84 (Fig. 6D).

These findings in Tubifex tubifex have significant toxicological significance in comprehending the causes of CBSF-induced toxicity in aquatic species. The molecular docking investigation (-6.5, -5.4, and − 7.3 kcal/mol for catalase, superoxide dismutase, and cytochrome c oxidase) found high binding affinity scores for CBSF, indicating potential interaction with these enzymes. These interactions can alter enzyme activity and cause oxidative stress, a common toxicological reaction to environmental contaminants79. The alkyl, pi-alkyl contacts and π-stacking interactions among CBSF and amino acid residues provide insights into the molecular-level mechanisms of interaction, which may disrupt active sites, catalytic activities, and enzyme functions. Disruption of antioxidant enzymes, like catalase and superoxide dismutase, might hinder the body’s ability to detoxify reactive oxygen species (ROS) produced during CBSF-exposure68. Cytochrome c oxidase, an enzyme in the mitochondrial electron transport chain responsible for oxidative phosphorylation, may be affected by its capacity to effectively reduce oxygen to water through interactions with CBSF residues80. This could disrupt cellular energy production and contribute to oxidative stress. These interactions can disrupt Tubifex tubifex’s antioxidant defence system, leading to increased oxidative stress, cellular damage, lipid peroxidation, and cell death81.

Fig. 6
figure 6

A. CBSF bound with Catalase. C. CBSF bound with SOD. E. CBSF is bound to Cytochrome c oxidase. (B, D, F). 2D Diagrams of Ligand-Protein Interaction.

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Fig. 7
figure 7

The star plot for the integrated biomarker response (IBR) index for the tubificid worm, exposed to the CBSF for the chronic study period: 3 days (a) 1d exposure period, (b) 7d exposure period, (c) 14d exposure period, (d) Individual IBRv2 values for two sub-lethal doses (T1 and T2) for 1d, 7d and 14d multi-biomarker study.

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Fig. 8
figure 8

Longitudinal section (H&E) of T. tubifex. a-b: control- normal appearance of the glandular cells of the epidermis (GC), epidermis (Ep) and fully stretched continuity of longitudinal muscle (LM), mucus channel (MC) from the anterior portion of worm; c-d: treated with 1.662 mg/L CBSF- disintegrated longitudinal muscles (DL) and extensive obliteration of longitudinal muscles and mucus channels in segments and hypertrophied remnants of longitudinal muscles (HLM).

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Integrated biomarker response (IBRv2) determination

This study examined the sub-chronic toxicity that caused oxidative stress in a time-dependent manner by calculating the integrated biomarker responses of the tubificid worm after exposure to CBSF. The star plot used in this study illustrates the cumulative effect of oxidative stress biomarkers, such as CAT, SOD, and MDA. The IBRv2 index increased over the exposure period as the exposure duration increased. Superoxide dismutases (SOD) and malaonaldehyde (MDA) had the highest standardized value in the star-plot on the first day (1d), while the cumulative IBRv2 value was 1.394 (Fig. 7a). The highest standardized values were displayed by SOD and catalase (CAT) on the seventh day of exposure, and the overall IBRv2 was 1.983 (Fig. 7b). CAT and SOD activity reached their maximum standardized value on the fourteenth day of exposure, and the total IBRv2 was 2.799 (Fig. 7c). The individual IBRv2 values for the two sub-lethal exposure concentrations—0.831 mg/L and 1.632 mg/L—against the exposure duration were also displayed in the bar plot (Fig. 7d). In this study, we assessed the toxicity of CBSF in tubificid worms using three biomarkers: CAT, SOD, and MDA. Furthermore, the scores of the three biomarkers shown in the star plots showed a clear trend of change. In the current experiment, the 14-day exposure period had the highest IBRv2 (IBRv2 = 2.799), followed by 7d (IBRv2 = 1.983) and 1d (IBRv2 = 1.394), respectively. This suggests that CBSF may have more hazardous qualities or be a more intense stressor for tubificid worms. Similar results were observed when Apis mellifera was exposed to different contaminants, indicating that EMS has the highest toxicity82. When Eisenia fetida was exposed to perfluorooctanoic acid (PFOA), which was evaluated for toxicity using an integrated biomarker approach, similar patterns were observed in another result83.

Histopathological alterations

Histological sections taken longitudinally through the body show an outer layer of epithelial cells, or the epidermis, which appears to be continuous with the integumental surface (Fig. 8). In the control, the glandular cells of the worm’s GC and Ep, as well as the longitudinal muscle (LM) running parallel to the gut and the mucus channel (MC) from the front part of the worm, appeared normal (Fig. 8a-b). Mucus channels (MC) in segments, hypertrophied remnants of longitudinal muscles (HLM), and extensive disintegrated longitudinal muscles (DL) were observed in the group treated with 1.662 mg/L CBSF (Fig. 8c-d). T. tubifex photomicrographic cross-section (Fig. 8c-d) of worms treated with 1.662 mg/L CBSF at the sub-acute level showed a gross depletion of longitudinal muscles and a preponderance of epidermal cell thinning and degeneration77.

In general, changes in the cells of the alimentary canal in exposed groups are toxic-related and concentration-dependent. The structure of epithelial cells, particularly in the digestive system, was also altered by exposure to various concentrations of pesticides.

Scanning electron micrograph study

The setae of untreated T. tubifex showed no obvious ultrastructural alterations (Fig. 9a). The scanning electron microscopic picture of the skin revealed complete setae in untreated worms (Fig. 9a). Setal damage was observed in worms subjected to 1.662 mg/L CBSF concentrations (Fig. 9b-d). Structural abnormalities in setae were also observed in worms exposed to high concentrations of CBSF. CBSF at high concentrations caused setae to fold laterally (Fig. 9b-d). Setal structural abnormalities were also observed in worms subjected to a high dose of CBSF (Fig. 9b-d).

There were no apparent morphological changes in the cuticle or epidermis of untreated T. tubifex. However, worms exposed to 1.662 mg/L CBSF concentrations acquired wrinkled skin with ulcers. (Fig. 9e). Anomalies such as segment loss, constriction of the body, discolouration, and swellings were noticed in worms exposed to 1.662 mg/L CBSF concentrations. Clitellar oedema was seen in worms treated with CBSF. Clitellar oedema was observed due to damage to the tissue in the clitellar region which led to fluid accumulation and inflammation, and a similar result was observed when Eudrilus eugeniae was exposed to the agrochemicals61. Electron microscopic pictures revealed ruptured skin and muscle under 1.662 mg/L CBSF concentrations (Fig. 9e).

Fig. 9
figure 9figure 9figure 9

Scanning electron micrographs (5.00 kV, WD- 11.8 mm, 21660× magnification) showing alterations in the structure of T. tubifex (a) control, (b-e) treated with 1.662 mg/L CBSF.

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Conclusion

Our research on the toxicity of CBSF to tubificid worms may serve as a foundation for future researchers who wish to list the environmental stressors that aquatic vertebrates and invertebrates experience as a result of environmental or man-made changes to the physical characteristics of the aquatic system. The results obtained from our study shows that the behavioural and stress enzyme biomarker responses elicited by our annelid model T. tubifex, can act as a potent biomonitoring of environmental CBSF contamination in natural water bodies and support ecological health and risk assessment. In addition, the toxic effects of CBSF and its metabolites are well documented in human cell lines, causing a partial loss of spindle microtubules and significantly increasing activities of caspase-3 observed as well as programmed cell death. In the present study, the toxicological effects of exposure to CBSF on Tubifex tubifex, an essential element of aquatic ecosystems, were thoroughly investigated in this study. The research revealed concentration-dependent CBSF-toxicity and delayed response through a variety of analyses, including survival assessment, behavioural responses, antioxidant enzyme reactions, histopathological examinations, molecular docking studies, and scanning microscopy image analysis. These findings have important implications for the organism’s sediment-dwelling habit, which is known to be a sink for hydrophobic contaminants like carbamates. The study would therefore be the first to document the impact of stress related to CBSF on tubificid oxidative status, along with histopathological alterations, morphological changes, and molecular docking studies to ascertain protein affinities.

Table 1 Acute lethal concentration values (LC10,30,50,60,70,80,90) with 95% confidence limit of Carbosulfan to T. tubifex at 24, 48, 72, and 96 h exposure period.
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Ethical approval

Current regulatory studies do not require any ethical approval for invertebrates like T. tubifex.

Test chemicals

Carbosulfan (25% EC; trade name: Marshal), a broad-spectrum insecticide, was collected from the local market of Burdwan. CBSF (2,2-dimethyl-3H-1-benzofuran-7-yl) N-(dibutyl amino) sulfanyl-N-methylcarbamate, (molecular formula C20H32N23S, molecular weight: 380.5, topological tolar surface area: 67.3 Å2 ) was used as a test chemical.

Experimental water

After 72 h of acclimatisation in the laboratory, the experiment was performed in pollution-free dechlorinated water with the following physicochemical parameters as specified by the American Public Health Association37 (pH 7.3 ± 0.4, temperature 29.0 ± 0.5 °C, free CO2 18.4 ± 0.6 mg/L, DO 5.9 ± 0.8).

Acute toxicity bioassay and projection of survival rate

After the acclimatisation in the laboratory condition, static replacement bioassays were conducted in a 250 ml Borosil glass beaker, each containing 200 ml of water for the experiment in the laboratory condition to determine the acute sensitivity, and ten worms were placed in each beaker. And no mortality was observed. Different concentrations of test chemicals were prepared by diluting the distilled water and preparing the test concentration. The final concentration of the acute experiment was determined by the 96-hour preliminary range-finding experiment. To determine the acute toxicity of CBSF, various concentrations were used, i.e., 9 mg/L, 10 mg/L, 11 mg/L, 12 mg/L, 13 mg/L, 14 mg/L, 15 mg/L, 16 mg/L, 17 mg/L, and 18 mg/L. Soft tweezers were used to periodically assess the worms’ viability; if there was no movement, they were deemed to be dead and responded within 10 s to reflex action38,39. The number of dead worms was counted at intervals of 24 h to prevent the biological decay of the organism. In the present investigation, the acute toxicity and behavioural response were recorded at an interval of 24 h40. To confirm the mortality of the tubificid worms, they were observed under a light microscope.

Behavioural toxicity

During the bioassays, all physiological responses are recorded by physical observation. Using a semi-quantitative scoring method and a 96-hour exposure period, the behavioural changes, such as mucous production, erratic movement, and low-level clumping tendency, caused by exposure to different doses of CBSF were analysed every 24 hours39,41,42. The proportion of autotomy (loss of caudal or posterior segments) during 96 h exposure to organic acids was calculated following the earlier standard method43. According to the loss of caudal segments of worms (autotomy) was also counted in intervals of 24 h. The behavioural responses of tubificid worms (CT = Clumping tendency, EM = Erratic Movement, MS = Mucus Secretion, AT = Autotomy are represented as ‘-’ absent, 1: mild, 2,3: moderate, 4,5: high) at the different exposure concentrations of CBSF and exposure period were observed and represented using Likert scale in a heat map44. Three different observers verified the autotomy, clumping tendency, wrinkling effect, and mucus secretion through visual inspection45.

Survival rate analysis

The General Unified Threshold model (GUTS) is depicted as a mathematical model, primarily applied to environmental toxicology questions, developed due to different doses applied to the organisms46. The General Unified Threshold (GUTS) modelling was used to derive a set of relevant toxicokinetic and toxicodynamic parameters relating to the organism’s response to exposure over time47.The Toxicokinetic-Toxicodynamic (TK-TD) models demonstrated the uptake of internal concentration of chemicals as well as their elimination rate, known as toxicokinetics, and internal damage leading to the death of individuals, known as toxicodynamics48. The General Unified Threshold model (GUTS) equation is widely used for ecological risk assessment purposes and individual tolerance (IT) and stochastic death (SD) to predict carbosulfan (CBSF)-induced toxicity. The validation of the results was performed by juxtaposing the LC50 values of 96 h in different intervals.

Species sensitivity distribution analysis

Through the use of protective benchmark concentrations (like the predicted-no-effect concentration [PNEC]) and environmental quality standards, SSDs are related to contemporary worldwide practices in chemical safety assessment, environmental quality assessment, and management49,50. Ecological assessments, like life cycle impact assessments, are linked to the latter SSDs and are currently utilised globally to estimate the probable consequences of chemical pollution51. Considering data on compound-specific ecotoxicity, an SSD is constructed by fitting only a few statistical models (such as log-normal)51. Many policies and jurisdictions are required for creating SSD plots, i.e., for the minimum number of data points necessary for achieving the minimum study quality. To create the SSD plots, the list of aquatic organisms was gathered, and the LC50 value of particular chemicals was determined. The aquatic organisms were listed according to the concentration of particular chemicals, and the rank was converted into proportion by using the equation below:

$${\rm The\: proportions\: of\: species\: affected\: = (rank- 0.5)/number\: of\: species}$$

The bell-shaped log-normal plot draws the concentration of particular chemicals along the X-axis and the proportion of species along the Y-axis52. Probit values, an inverse cumulative distribution function of the normal distribution with a mean of 5 and a standard deviation of 1, were created by transforming proportions to these values. Probit values were used instead of proportions in addition. All probit values throughout the study had to be non-negative; hence, a mean of 5 was chosen to ensure this. Regression analysis was used to determine the central tendency from log10 concentrations (on the x-axis) and probit (on the y-axis).

Experimental paradigm for chronic bioassay

The worms underwent sublethal doses of CBSF at two concentrations, which corresponded to 10% and 20% of the 96-hour LC50 (i.e., 0.831 mg/L and 1.632 mg/L) during 14 days, and the corresponding concentrations of CBSF on median lethal concentration LC50 of 96 h were 0.831 mg/L (T1) and 1.662 mg/L (T2), respectively (median lethal concentration of 96 h toxicological endpoints). Using the selection criteria, the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC) that might be present in aquatic environments or linked to environmentally significant concentrations were determined. Approximately 106 tubificid worms were added to each aquarium for the chronic bioassay. A Venus Aqua Ap-208 aerator was used to aerate 5 L of unchlorinated water for each aquarium-housed worm, which weighed about 20 g. The test medium was then replenished every two days with corresponding toxicants at a concentration of 10% of the original quantity53. For every treatment, control groups were kept in triplicate to ensure accuracy.

Oxidative biomarker analysis

To measure the oxidative stress of Tubifex tubifex, 20 g of wet worms were kept in a 10 L glass aquarium, each containing 5 L of unchlorinated tap water. For the assessments of the toxicity of CBSF, the Tubificid worms are exposed to two sub-lethal concentrations, i.e., 0.8314 mg/L and 1.66 mg/L. During the intervals of 1, 4, and 7 days, 1 g of CBSF-exposed worms were collected from the beaker and homogenised in phosphate buffer (0.1 M, pH 7.6). Then the homogenate was centrifuged at 11,180 × g at 4 °C for 10 min. For measuring the enzyme activity, the supernatant was collected, and the pellet portion was discarded. After a week of storage at -20 °C, the resultant supernatant was utilised to measure the enzymatic activity. Standard protocols were applied to measure the activity of different oxidative stress biomarkers, i.e., CAT, SOD, GSH, and MDA. According to the methods of Bradford54 the protein content was analysed. The catalase activity was measured by the residual H2O2 spectrophotometrically at 240 nm55. The superoxide dismutase activity was detected by the photoreduction inhibition method of nitroblue tetrazolium (NBT)56. The level of malondialdehyde (MDA) was measured by the formation of thiobarbituric acid reactive substance (TBARS)57. The activity of CAT and SOD is expressed in the unit of nmol TBARS/min/mg protein. The enzyme activities were determined by using an ultraviolet-visible spectrophotometer (Cecil 150149 Aquarius CE 7400).

Molecular docking analysis

Firstly, we intended to track down the three-dimensional (3D) crystal structures of three crucial stress indicator enzymes, catalase, superoxide dismutase, and cytochrome c oxidase, of Tubifex tubifex from the Protein Data Bank (PDB)58. However, we found no experimentally determined 3D structures of the proteins. Concerning this, we utilised the Swiss Model for homology modelling, in which AlphaFold59a database known for generating 3D protein structures with exceptional precision, was considered for the 3D models of these proteins. We then performed a molecular docking analysis using these three 3D structural models of enzymes with CBSF. This experiment was conducted using PyRx software, where the molecular docking program was carried out using Vina Wizard60. This investigation used a conventional approach to investigate potential intermolecular interactions among the aforementioned enzymes and CBSF, providing insights into the macromolecular complex formation and associated mechanisms.

Histological procedure

Haematoxylin and eosin (H&E) were used to stain longitudinal and transverse sections of tubificid worms treated with CBSF, and microscopic images were captured. The tubificid worms were exposed to two chronic doses (10% of the LC50, noted as T1, and 20% of the LC50, noted as T2), then sacrificed, and dehydrated using a graded alcohol treatment. The dehydrated tissues were cleaned in xylene and embedded in a paraffin block. Then the tissue-embedded block was sectioned at 5 μm in a microtome machine. The tissues were arranged on a grease-free slide and stained by standard methods with H&E, mounted in DPX, and covered by a glass coverslip. The lesion of treated tissues was captured and examined under an Olympus Microscope (Olympus, BH-2, Japan).

Analysis of ultrastructural alteration: through scanning electron microscopy (SEM)

The CBSF-treated tubificid worms of 1d, 4d, and 7d intervals were sacrificed to visualize morphological alterations. The tubificid worms were fixed in 2.5% glutaraldehyde, 0.1 M sodium cacodylate, followed by 0.05% calcium chloride overnight at 4 °C and rinsed in distilled water. The worms were dehydrated in a series of acetone concentrations (50%, 70%, 90%, and 100%), followed by critical point drying (BAL-TEC, CPD030), and finally coated with metal61.

Calculation of IBRv2

The IBRv2 index was computed using three oxidative stress parameters by Sanchez et al.62. Initially, each parameter, or biomarker, in each group underwent the following log transformation:

$$\:Yi\hspace{0.17em}=\hspace{0.17em}log\:(Xi/X0)$$
(1)

in which X0 symbolizes the mean value of this biomarker in the control, and Xi indicates the mean value of each biomarker in a single treatment.

Second, this biomarker’s standardized value was determined to be:

$$\:Zi\:=\:(Yi\hspace{0.17em}-\hspace{0.17em}M)/S$$
(2)

where M and S stand for the overall mean and standard deviation, respectively, for every biomarker across all treatments, including control.

Third, for each biomarker in each treatment group, a deviation index (Ai) is obtained:

$$\:Ai\hspace{0.17em}=\hspace{0.17em}Zi\:-\:Z0$$
(3)

The standardized value of the control group is denoted by Z0.

After that, the IBRv2 was calculated for each treatment calculated

$$\:IBRv2\hspace{0.17em}=\hspace{0.17em}\:\left|Ai\right|$$
(4)

The standardized value of the control group is denoted by Z0.

Statistical analysis

The median lethal concentration (LC50) with a 95% confidence limit in the interval of 24 h was determined by the standard method of PROBIT analysis performed by IBM SPSS version 16 63. Survival curves were analysed using Kaplan-Meier survival analysis. GraphPad Prism 8 was used to statistically analyse the collected data. The one-way ANOVA was performed to analyse the time and concentration dependence elevation of the oxidative stress biomarker (CAT, SOD and MDA), followed by a Duncan multiple range test performed to compare the mean value. All the data are represented as mean ± standard deviation (where n = 3), and p < 0.05 was the accepted level of statistical significance.