Carbonsulfan-induced physiological, histopathological, and ultrastructural alterations in tubifex tubifex (müller, 1774)

Abstract

The present study evaluates the toxicological effects of carbosulfan by observing the mortality, behavioural responses, and alteration of oxidative biomarkers such as CAT, SOD, and MDA. The mortality rate of tubificid worms exposed to the toxicant differed substantially (p < 0.05) from the control group at all concentrations throughout the experiment. The p-value less than 0.05 indicates that the changes in these biomarkers were statistically significant. The severity of behavioural changes included irregular movement, decreased clumping proclivity, mucus production, and a surge in autotomy. Long-term exposure to two sub-lethal doses of CBSF (0.831 mg/L and 1.632 mg/L) resulted in the disintegration of longitudinal muscles of tubificid worms, the continuity of longitudinal muscle (LM) parallel to the gut, and ruptured mucus channel (MC) from the anterior portion of the worm. In addition, scanning electron microscope images revealed the alteration of epidermal lesions, setal anomalies, and clitellar swelling due to CBSF exposure. The oxidative stress biomarkers catalase (CAT), superoxide dismutase (SOD) and malonaldehyde (MDA) increased with increasing concentrations of CBSF, i.e., 0.831 mg/L and 1.632 mg/L. The maximum elevation of CAT, SOD and MDA was observed on the 14th day. In addition, IBRv2 analysis demonstrated that tissue biomarkers were extremely sensitive to the toxicity of CBSF, and extended exposure might result in major health consequences, such as acute toxicity in tubificid worms because of increased oxidative stress biomarkers. The log-normal curve of species sensitivity distribution (SSDs) depicted the ecotoxicological risk assessment through 96 h LC_50, i.e., 8.31 mg/L. Strong interactions between CBSF and key stress biomarker enzymes were shown by molecular docking experiments, which interfered with the enzymes' activities and might have caused oxidative stress. Interactions with cytochrome c oxidase indicated disruption of the process by which cells produce energy. The findings highlight the need for more investigation to completely understand the sensitivity of tubificids to environmental pollutants and the long-term impacts of pesticide exposure on aquatic health and ecosystem stability.

Keywords: Autotomy; Behavioural changes; Benthic tubificid worm; Carbosulfan; Sub-lethal toxicity.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 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.

Fig. 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.

Fig. 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.