Triazophos toxicity induced histological abnormalities in Heteropneustes fossilis Bloch 1794 (Siluriformes: Heteropneustidae) organs and assessment of recovery response

Abstract

Background

Agricultural pesticides have toxic effects in the aquatic ecosystem, and their persistence poses a hazard to aquatic life, as seen by fish poisoning, both acute and chronic. Triazophos, a broad-spectrum organophosphate insecticide, is used to control agricultural crops from insect pests. For a period of 10 days, Heteropneustes fossilis, a fish of great economic and therapeutic value, was exposed to various levels of triazophos toxicity (5, 10 and 15 ppm), after which they were sacrificed. For recovery tests, the treated fish were switched to clean tap water after 10 days of exposure to the toxicant, examined for another 10 days, and then sacrificed. The histological changes in the tissues of the sacrificed fishes' gill, liver, intestine, kidney, brain, and muscle (treatment and recovery) were investigated.

Results

The histology investigations revealed that the toxicant was hazardous, with histopathological changes increasing as the concentration of the toxicant increased. The gills had the most damage, with fusion of secondary lamella and epithelial hyperplasia; liver had vacuolization, pyknotic nuclei, and focal necrosis; intestine had degenerated, necrotic villi, degeneration of epithelial cells, and atropy; kidney had narrowing of the tubular lumen, pyknotic nuclei, hypertrophy, degeneration; swelling, haemorrhage, larger neuronal cells, and karyolysis were observed in the brain, whereas infiltration of leucocytes, loss of striated muscles, and an increase in intra fibril area were observed in the muscle. When compared to the treated fishes, the 10-day recovery research demonstrated tissue damage and a slower recovery pattern.

Conclusions

Triazophos caused histological changes in the gill, liver, intestine, kidney, brain and muscle of the test fish Heteropneustes fossilis. With reference to recovery response, a slow recovery was observed. Furthermore, this is the first investigation into the effects of triazophos on the recovery response in Heteropneustes fossilis.

Background

Pollution of the aquatic environment is a major international concern, and several toxicological approaches have been developed to assess the impact of aquatic contaminants on fisheries resources using physiological, biochemical, histological, and behavioural responses to contaminant exposure. Bioaccumulation, bioconcentration, and biomagnification processes enrich the aquatic food chain, extending the hazardous effects of agricultural pesticides into the aquatic ecosystem (Maurya & Malik, 2016; Ray & Shaju, 2023). Heavy metals (Abiona et al., 2019; Dane & Sisman, 2020; Garai et al., 2021; Karayakar et al., 2022; Kumar et al., 2023; Maurya & Malik, 2019; Mehmood et al., 2019; Topal & Onac, 2020) and pesticides (Amenyogbe et al., 2021; Chakraborty, 2023; Jain et al., 2021; Kumari, 2020a; Loganathan et al., 2021; Mandal & Mandal, 2023; Samuel et al., 2019a, b; Shah & Parveen, 2020; Yaseen et al., 2024) accumulation and their persistence in the aquatic environment pose a threat to aquatic life, as evidenced by acute and chronic poisoning of fish and other aquatic animals. As a result, it is critical to investigate the hazardous effects of pesticides on fish, as they are an important link in the food chain and protein source for human (Balami et al., 2019). Pesticide pollution causes an imbalance in the aquatic ecosystem (Islam et al., 2022; Shefali et al., 2021; Weiss et al., 2023). Fish have gained considerable significance as histopathological indices following exposure to toxicants (Maurya et al., 2019; Singh & Pandey, 2021). In the assessment of fish health and its target organs (gills, liver, kidney, intestine, brain, and muscle) exposed to toxicants, histopathological changes have been widely used as biomarkers (Hossain et al., 2022; Islam et al., 2020; Suchana et al., 2021). Fish health and its metabolism is negatively impacted by agricultural effluents that are purposefully or inadvertently released into adjacent aquatic bodies, thereby resulting in discernible histoarchitectural alterations in the organ tissues of the fish (Akter et al., 2020; Maurya et al., 2019; Parveen et al., 2017; Singh et al., 2019; Strzyzewska et al., 2016). For the purpose of determining the impact of toxicants and agro-pesticides on fish health and its target organs in both laboratory and field studies, histopathological assessment is a sensitive biomonitoring tool (Hossain et al., 2016; Islam et al., 2019a; Poleksic et al., 2010; Reza et al., 2020; Shah & Parveen, 2020; Sumon et al., 2019). Further, in the field of aquatic biology, histopathology is the potent gold standard and effective technique for determining the extent of toxicity, especially in terms of sublethal and chronic impacts (Reddy & Kusum, 2013), and enables the identification of long-term damage in cells, tissues, or organs as well as early warning indications of disease (Rakhi et al., 2013).

Heteropneustes fossilis belongs to the genera Heteropneustes Muller (Siluriformes: Heteropneustidae), the Asian stinging catfishes (Plamoottil, 2022). They are commercially important freshwater fishes distributed throughout the South and Southeast Asian countries such as Bangladesh, India, Myanmar, Nepal, Pakistan, Sri Lanka and Thailand (Goni et al., 2020; Hossain et al., 2018; Kohinoor et al., 2012). This species lives in both freshwater and brackish waters, and mostly inhabits ponds, ditches, swamps, and marshlands, but sometimes inhabits muddy rivers and contaminated water bodies. Throughout its life cycle, it is omnivorous. Planktivorus fry eat crustacea, plants, miscellaneous matter and insects, whereas adults eat insects, detritus, and plant matter (Kohli & Goswami, 1996). The fish is elongated and heavily compressed, with maxillary barbels extending to the end of pectoral fins and mandibular pairs extending up to the base of the pelvis. The caudal fin is rounded and different from the rest of the body. Males have a narrower ventral line, whilst females do not (Rahman et al., 2019). This catfish may sting humans, and the poison produced by a gland on its pectoral fin spine has been reported to be quite unpleasant, so the name ‘stinging catfish’. It is also characterized by a long air sac, an accessory respiratory organ (air breathing organ) which enables it to exist for hours when out of water or in indefinitely oxygen-poor water and even in moist mud (Ali et al., 2014). When the oxygen concentration of the water falls below the saturation threshold, this catfish may breathe aerially by gulping in air at various intervals, hence it is a bimodal breather. This species is a key component of local commercial fisheries, as well as being farmed and sold in aquariums throughout Asia (Saikia et al., 2022). It is highly nourishing, tasty, and is widely appreciated in many parts of the Indian subcontinent due to its lack of fat, high digestibility, nutritional content, and has high economic value (Das et al., 2021; Goni et al., 2020; Singh et al., 2019). This fish has a lot of protein, iron, and calcium in its muscle, essential fatty acids, vitamins and other medicinal values (Das et al., 2021; Paul et al., 2016). It is a prized table fish in India, and this species is valued not only for its excellent taste, nutritional worth, market value, and economic relevance, but also for its medicinal benefits (Chakraborty & Nur, 2012). This type of makeup is not found in any of the other cultured fish species. As a result, physicians prescribe this fish for convalescence and fast-growing youngsters (Samad et al., 2017).

Organophosphates are systemic insecticides that are frequently used in agriculture that act as stomach and contact poisons against different types of insect pests (Shaji et al., 2021), and with high toxicity on non-target organisms and fish (Almeida et al., 2010; Diepens et al., 2014). Organophosphates damages the physiology and functioning of internal organs in fish, degrades the structural aberrations in the organs, which result in the reduction or alternation of function of the organ (Shaji et al., 2021). Triazophos, with its IUPAC name (diethoxy-[(1-phenyl-1,2,4-triazol-3-yl)oxy]-sulfanylidene-lambda5-phosphane), and molecular formula C₁₂H₁₆N₃O₃PS, is a broad spectrum organophosphate insecticide, acaricide and nematicide, to protect agricultural crops, fruits, vegetables and oilseeds (Bhandari et al., 2019; Guo et al., 2018; Hong et al., 2019; Kumari & John, 2019). This pesticide also shows a broad-spectrum insecticidal activity against a wide range of sucking insect pests and some lepidopterans (Kumari, 2020b), and toxicity effects on fishes are also reported (Bhardwaj et al., 2022; Chandra et al., 2021; Jia et al., 2018; Kumari, 2020b; Pattar & David, 2019, 2023; Singh et al., 2017, 2018; Wu et al., 2018). Changes in histoarchitectural indices serves as a biomarker for contamination in commercial fish, especially Heteropneustes fossilis, caused by the fertilizer industry (Singh & Pandey, 2021). Histopathological alterations are reported in Heteropneustes fossilis on exposure to organophosphate pesticides (Islam et al., 2019b; Khatun et al., 2016; Maurya et al., 2019; Mishra et al., 2021, 2022; Pattnaik et al., 2022; Singh & Pandey, 2021; Verma et al., 2020). Thus in the backdrop of above cited literature, histopathological changes in the gill, liver, intestine, kidney, brain and muscle upon toxicant exposure are useful tool to assess the impact of the toxicity in vital processes of fish. Hence, the present study was carried out to evaluate the histopathological changes in the gill, liver, intestine, kidney, brain and muscle of Heteropneustes fossilis in response to exposure of an organophosphate pesticide, triazophos, as well as their recovery response. Furthermore, this is the first investigation into the effects of triazophos on the recovery response in Heteropneustes fossilis.

Methods

Experimental animal

Heteropneustes fossilis adults were carefully hand netted and collected from Chengalpattu lake in Tamil Nadu, India (12.6840° N, 79.9833° E). To avoid stress and damage, the fish were immediately brought to the laboratory in wide-mouthed big plastic containers in natural water. Fish were habituated to laboratory settings for a few weeks before being kept in aquaria (30 L) with a light–dark (12:12 h) cycle and fed ad libitum with artificial pellet. The water utilized during the acclimatization and experimentation period was clear de-chlorinated ground water. The morphometrics of the fishes weighed an average of 33.0 ± 0.5 g and measured 18–20 cm in length.

Experimental design

Acclimatized fish was used for the experimental tests. They were not fed for 48 h before the start of the experiment in order to avoid any possible changes in the pesticide's toxicity in situ. Triazophos stock solution (1%) was prepared with distilled water, and thereafter from the stock solution, dosages of 5 ppm, 10 ppm and 15 ppm were arrived. For experiments, ten individuals of acclimatized fish were exposed to each of the above mentioned doses of triazophos separately for 10 days in a test tank holding 10 l of water. Controls had an equal number of acclimatized fish kept in clean tap water, and were exposed for the same amount of time. The fish's mortality was tracked at regular intervals. Fish that showed no respiratory activity or response to tactile stimulation were deemed dead and were removed as soon as possible. The treated fish were sacrificed after 10 days of exposure. For recovery tests, the treated fish were switched to clean tap water after 10 days of exposure to the toxicant, examined for another 10 days, and then sacrificed.

Histopathological studies

The tissues from the control and experimental groups' gill, liver, intestine, kidney, brain, and muscle were dissected out and preserved in neutral buffered formalin for 24 h before being submitted to histological procedures. The fixed tissues were subsequently preserved in 50% alcohol, dehydrated in an increasing gradient of 70%, 100% alcohol, and lastly dehydrated in isopropyl alcohol during a four-hour period. The tissues were then cleaned in xylene and imbedded in paraffin, which was then heated to 60 °C and changed every thirty minutes. Sections of 4–5 µm thickness were prepared from paraffin blocks by using a rotary microtome and sections were fixed on albuminoid slides for 24 h, deparaffinised in xylene and washed in isopropyl alcohol, 90%, 70% and 50% alcohol series and finally with water. The sections were then stained with Ehrlich’s haemotoxylin for 15 min and destained in dilute hydrochloric acid and distilled water before counterstaining with eosin. Thereafter, the stained sections were dehydrated in increasing alcohol series (50%, 70%, and 90%), cleaned in xylene, and mounted with Distyrene Plasticizer Xylene (DPX). Carl Zeiss photomicroscope at 40/100X magnifications was used for analysis of gill, liver, intestine, kidney, brain and muscle tissues of the experiment’s control, treated, and recovery group.

Statistical analysis

Probit analysis of fish mortality data was performed, and the results were converted to a probit scale. Chi-square test was performed to determine the slope function and confidential limits (lower and upper) of the regression line. With significance set at 95% confidence, all data were statistically analysed using IBM SPSS Statistics Version 27.0 (SPSS, 2021).

Results

The toxicity test convincingly demonstrated the toxicity of pesticide to the experimental fish, as major changes in its physical behaviour, viz., frequently surfacing the water, erratic swimming pattern and movement, somersaulting, and rapid and slow opercular movement were seen. The experimental fish developed clear clinical signs by structural malformations after being exposed to triazophos. The intensity of the symptoms was determined by the toxicant's nature, dose, and duration of exposure. Heteropneustes fossilis was more susceptible to the harmful effects of triazophos when the concentration was increased. There was no mortality in the controls. The LC₅₀ after 96 h was 2.303 ppm (95% confidence limit ranged from 1.892 to 2.575 ppm). The LC₅₀ values of the toxicant were observed to decrease dramatically as the exposure period was increased. Chi-square value (0.99) also revealed that it was well fit at P < 0.05 level. The intercept and slope values were 1.6 ± 0.9 and 9.1 ± 2.2, respectively. Furthermore, as the regression coefficients increased, the LC₅₀ value of triazophos decreased.

Gill

The normal anatomy of the control gill was shown by microscopic examination. The epithelial lining of the gill, interlamellar area, respiratory lamella, and supporting axis were all found to be intact. On either side of the major lamellar, each gill arch had parallel rows of elongated laterally projecting secondary gill filaments. The cartilaginous skeletal rod and blood vessels were seen in the central core of the primary gill lamella. Each secondary lamella was a flattened structure made up of two epithelial sheets divided by pillar cells. The branches of the afferent and efferent branchial arteries associated with each gill filament were linked by blood lacunae between adjacent pillar cells. Shortening of secondary lamella, necrosis, desquamation, aneurism, epithelial lifting, and hypertrophy were detected in the treated gill after exposure to a 5 ppm treatment concentration. At a concentration of 10 ppm, histological changes such as secondary lamella shortening and lifting, haematopoietic tissues, aneurism, hyperplasia, vacuolization, and epithelial lifting were observed. Gill showed subsequent lamella fusion and epithelial hyperplasia after being subjected to a 15 ppm concentration (Fig. 1). With reference to recovery period, pathological changes in the gill, such as necrosis, fusion of secondary lamellae, oedema, and hyperplasia, were found in 5 ppm concentration; aneurism, hyperplasia, vacuolization, desquamation, and necrosis were found in 10 ppm; and lifting of epithelium, hyperplasia, desquamation, and necrosis, fusion of secondary lamellae, pyknotic nuclei and epithelial hyperplasia at 15 ppm (Fig. 2).

Fig. 1

Heteropneustes fossilis gill exposed to triazophos (Treatment). SL Secondary Lamellae, EC Epithelial Cell, CC Chloride Cell, PC Pillar Cell, SSL Shortening of Secondary Lamellae, V Vacuolization, N Necrosis, D&N Desquamation and Necrosis, AN Aneurism, EL Epithelial Lifting, H Hypertrophy, LSL Lifting of Secondary Lamellae, HT Haemotopoietic Tissue, HP Hyperplasia, EL Epithelial Lifting, FSL Fusion of Secondary Lamellae, EP Epithelial Hyperplasia, D Degeneration

Fig. 2

Heteropneustes fossilis gill exposed to triazophos (Recovery). SL Secondary Lamellae, EC Epithelial Cell, CC Chloride Cell, PC Pillar Cell, N Necrosis, FSL Fusion of Secondary Lamellae, O Oedema, HP Hyperplasia, AN Aneurism, D&N Desquamation and Necrosis, V Vacuolization, LE Lifting of Epithelium, PYN Pyknotic Nuclei, EL Epithelial Hyperplasia

Liver

Large polyhedral cells grouped as a minute network of canaliculi between the liver cells defined the control liver. The nuclei were vesicular, with a big nucleolus and a bile duct distribution. Vacuolization, karyolysis, pyknotic nuclei, and hepatocyte hypertrophy were found in 5 ppm of the treated liver; haemorrhage, cloudy degeneration, binucleated hepatocyte, karyolysis, and pyknotic nuclei were found in 10 ppm of the treated liver; and vacuolization, pyknotic nuclei, and focal necrosis were found in 15 ppm (Fig. 3). In respect to recovery period, at 5 ppm, pyknotic nuclei, binucleated hepatocyte, karyolysis, vacuolization, cloudy degeneration was discovered; at 10 ppm, focal necrosis, cloudy degeneration, binucleated hepatocyte, degeneration of hepatocyte, vacuolization, necrosis, dilation of sinusoid, swelling of hepatocyte, karyolysis was observed; and at 15 ppm, pyknotic nuclei, congestion of hepatocyte, congestion of sinusoid, swelling of hepatocyte and dilation of sinusoid occurred (Fig. 4).

Fig. 3

Heteropneustes fossilis liver exposed to triazophos (Treatment). H Hepatocyte, SN Sinusoids, V Vacuolization, K Karyolysis, PYN Pyknotic Nuclei, HH Hypertrophy of Hepatocyte, HE Haemorrhage, CD Cloudy Degeneration, BH Binucleated Hepatocyte, FN Focal Necrosis

Fig. 4

Heteropneustes fossilis liver exposed to triazophos (Recovery). H Hepatocyte, SN Sinusoids, PYN Pyknotic Nuclei, BH Binucleated Hepatocyte, K Karyolysis, V Vacuolization, CD Cloudy Degeneration, FN Focal Necrosis, DH Degeneration of Hepatocyte, N Necrosis, DS Dilation of Sinusoid, SH Swelling of Hepatocyte, CH Congestion of Hepatocyte, CS Congestion of Sinusoid

Intestine

The control intestine histological examination revealed the presence of four layers of the intestinal wall: mucosa, submucosa, muscularis, and serosa. Degeneration of epithelium, necrosis, atrophy, and infiltration of leucocytes were found in 5 ppm of treated intestine; vacuolization, infiltration of leucocytes, autolysis of mucosa were found in 10 ppm of treated intestine; and degenerated and necrotic villi, degeneration of epithelial cells, and atrophy were found in 15 ppm (Fig. 5). With regard to recovery period, degeneration of epithelial cells, oedema, autolysis of mucosa, infiltration of leucocytes were found at 5 ppm; autolysis of mucosa, erosion and necrosis of villi, vacuolization and infiltration of leucocytes were noted at 10 ppm; and autolysis of mucosa, erosion and necrosis of villi, vacuolization and infiltration of leucocytes occurred at 15 ppm (Fig. 6).

Fig. 5

Heteropneustes fossilis intestine exposed to triazophos (Treatment). VI Villi, E Epithelium, LP Lamina Propria, DE Degeneration of Epithelium, N Necrosis, AT Atrophy, INFL Infiltration of Leucocytes, V Vacuolization, ATM Autolysis of Mucosa, NV Necrotic Villi, DEC Degeneration of Epithelial Cell, DGV Degeneration of Villi

Fig. 6

Heteropneustes fossilis intestine exposed to triazophos (Recovery). VI Villi, E Epithelium, LP Lamina Propria, FV Fusion of Villi, DV Degeneration of Villi, DEC Degeneration of Epithelial Cell, O Oedema, ATM Autolysis of Mucosa, INFL Infiltration of Leucocytes, NV Necrotic Villi, EV Erosion of Villi, V Vacuolization

Kidney

A nephron with a renal capsule and a well-developed renal tubule was seen in the control kidney. A glomerulus was encircled by Bowman's capsule in the renal capsule. The glomerulus was circular, massive, and vascularized, with the Bowman's space connecting the parietal and visceral epithelium of the glomerulus to the lumen of the renal tubule. The renal tubule epithelial cells were columnar, with oval or rounded basal nuclei and brush border cells lining the inside. Histological changes in the treated kidney indicated pathogenic abnormalities, including, vacuolization, necrotic tubule, degeneration of renal epithelial cells, distortion and dilation of renal tubule, cloudy degeneration at 5 ppm; necrosis of haematopoietic tissue, dilation of glomeruli at 10 ppm; and narrowing of tubular lumen, pykonitc nuclei, hypertrophy, degeneration of renal epithelial cells, degeneration of glomerular cells, inter cytoplasmic vacuolization and necrosis at 15 ppm (Fig. 7). In recovery period, dilation of Bowman's capsule, necrosis of haematopoietic tissue, distortion of renal tubule, vacuolization, and pyknotic nuclei were found at 5 ppm; inter cytoplasmic vacuolization, cloudy degeneration, pyknotic nuclei, necrosis of haematopoietic tissue was found at 10 ppm; and hypertrophy of haematopoietic tissue, dilation of glomeruli at 15 ppm (Fig. 8).

Fig. 7

Heteropneustes fossilis kidney exposed to triazophos (Treatment). RT Renal Tubule, HT Haematopoietic Tissue, NT Necrotic Tubule, DERT Degeneration of Renal Epithelial Cell, DRT Distortion of Renal Tubule, CD Cloudy Degeneration, NHT Necrosis of Haematopoietic Tissue, DG Dilation of Glomeruli, NC Necrotic Change, NTL Narrowing of Tubular Lumen, PYN Pyknotic Nuclei, H Hypertrophy, DGC Degeneration of Glomerular Cell, ICV Inter Cytoplasmic Vacuolization, N Necrosis

Fig. 8

Heteropneustes fossilis kidney exposed to triazophos (Recovery). RT Renal Tubule, HT Haematopoietic Tissue, V Vacuolization, NHT Necrosis of Haematopoietic Tissue, DBC Dilation of Bowman’s Capsule, DRT Distortion of Renal Tubule, PYN Pyknotic Nuclei, ICV Inter Cytoplasmic Vacuolization, N Necrosis, CD Cloudy Degeneration, NHT Necrosis of Haematopoietic Tissue, HTHT Hypertrophy of Haematopoietic Tissue, DG Dilation of Glomeruli

Brain

Normal neuron cells were found in control brain tissue, and no pathogenic alterations were found. At 5 ppm, there was necrotic change, rupture and vacuolization, pyknotic nuclei, degenerative changes in nerve cells, karyolysis, and demyelination; at 10 ppm, there was binuclated cell, acentric and pyknotic nuclei, haemorrhage in neural cell; and at 15 ppm, there was swelling, haemorrhage, and enlarged neuronal cell, as well as karyolysis (Fig. 9). For recovery studies, binucleated and multinucleated cells, vacuolization, pyknotic nuclei, and karyolysis were found in 5 ppm; dissolution of cell membrane, swollen cell body, and loss of cellularity were found in 10 ppm; and profuse vacuolization, haemorrhage in neuronal cells, necrotic change, dissolution of cell membrane, and necrosis were found in 15 ppm (Fig. 10).

Fig. 9

Heteropneustes fossilis brain exposed to triazophos (Treatment). NC Neuronal Cell, R&V Rupture and Vacuolization, PYN Pyknotic Nuclei, DNC Degenerative Change in Neuronal Cell, K Karyolysis, BNC Binucleated Cell, ACN Acentric Nuclei, HNC Haemorrhage in Neuronal Cell, SNC Swelling of Neuronal Cell, ENC Enlarged Neuronal Cell

Fig. 10

Heteropneustes fossilis brain exposed to triazophos (Recovery). NC Neuronal Cell, K Karyolysis, MNC Multinucleated Cell, V Vacuolization, PYN Pyknotic Nuclei, DCM Dissolution of Cell Membrane, SCB Swelling of Cell Body, LC Loss of Cellularity, PV Profused Vacuolization, HNC Haemorrhage in Neuronal Cell, NC Necrotic Change, N Necrosis

Muscle

The myotomes of the control fish were normal, with striations in the muscle fibres. Focused necrosis and loss of striated muscles were seen in 5 ppm of the treated muscles; cellular debris in muscle fibril, vacuolization, and loss of striated muscles in 10 ppm; and infiltration of leucocytes, loss of striated muscles, and an increase in intra fibril area were seen in 15 ppm of the treated muscles (Fig. 11). Cellular distortion, dilation of muscle fibril, loss of striated muscle, muscular dystrophy were found in 5 ppm; cellular debris in myofibril area, focal necrosis was found in 10 ppm; and infiltration of leucocytes, loss of striated muscle, and cellular debris in myofibril area were found in 15 ppm in the case of recovery period (Fig. 12).

Fig. 11

Heteropneustes fossilis muscle exposed to triazophos (Treatment). MF Muscle Fibril, FN Focal Necrosis, LSM Loss of Striated Muscle, CDMF Cellular Debris in Muscle Fibril, V Vacuolization, INFL Infiltration of Leucocyte, IIA Increase in Intrafibril Area

Fig. 12

Heteropneustes fossilis muscle exposed to triazophos (Recovery). MF Muscle Fibril, LSM Loss of Striated Muscle, CD Cellular Dissolution, DMF Dilation of Muscle Fibril, MD Muscular Dystrophy, CDMA Cellular Debris in Myofibril Area, FN Focal Necrosis

Discussion

Toxicity is a typical attribute of an individual organism's response to a chemical at a specific dose over time. The current study's toxicity test convincingly demonstrated the toxicity of triazophos to Heteropneustes fossilis, as major changes in its physical behaviour were seen to avoid the toxicant/polluted water, as observed by Pattnaik et al. (2022) in Heteropneustes fossilis exposed to organophosphate pesticide. Pesticides toxic effects on fish leads to histological abnormalities in the tissue of its gill, liver, intestine, kidney, brain and muscle (Khafaga et al., 2020; Tahir et al., 2021). In the present study too, numerous histopathological alterations occurred in the gill, liver, intestine, kidney, brain and muscle tissues of Heteropneustes fossilis on exposure to triazophos. Rapid tissue changes depend on the insecticide concentrations and length of time the fish are exposed to the toxicants. Structural changes of the fish tissues in the experimented fish exposed to different concentrations of pesticides play a significant role as active response to the organisms that facilitate knowledge of the nature of toxicants (Fanta et al., 2003).

Gill

Fish gill is multifunctional (Dolenec & Kuzir, 2009). They are the primary site for oxygen uptake in fishes and are involved in respiration, maintaining acid–base balance and osmoregulation (Maurya et al., 2019; Shah & Parveen, 2020). Despite the anatomical differences across fish species gills, the cells that make up gill structure are quite similar (Wilson & Laurent, 2002). Heteropneustes fossilis gills are located near the head region on both lateral sides of the pharynx and are composed of paired gill arches which comprises epithelial, circulatory and neural tissues. The fundamental functional unit of gill tissue is represented by the long thin projections called gill filaments held apart by pillar cells, which taper at the distal end and are located lateral to the gill arch (Renu & Agarwal, 2013). Gill is the first organ that encounters toxicants and undergoes histopathological changes (Maurya & Malik, 2019; Singh & Pandey, 2021). By decreasing the total respiratory surface area, toxicity on the gills impairs respiratory function and causes respiratory failure which has a deleterious effect on fish physiology and may even be fatal (Pereira et al., 2012; Sharmin et al., 2021; Stalin, 2019; Wani et al., 2011; Yaseer & Naser, 2011). Characteristic histological changes in the gills of Heteropneustes fossilis include distortion of the gill architecture, atrophy of the gill lamellae, congestion in the blood vessels of the primary gill lamellae, curling and fusion of secondary lamellae, desquamated secondary lamellae, epithelial lifting and hyperplasia, lamellar fusion and aneurism, atrophy, hypertrophy and damage of mucus and chloride cells, vacuolization, pyknotic nuclei, apoptosis and necrosis in the epithelial cells, dilation of lamellar blood vessels, and blood spill over to the edematous space (Maurya et al., 2019; Renu & Agarwal, 2013).

In the present study, the gills of Heteropneustes fossilis displayed significant histological alterations like epithelial lifting, hypertrophy, epithelial hyperplasia, lifting and fusion of secondary lamellae. Lifting of epithelia functions as a defense mechanism because separating the epithelia of the secondary lamellae increases the distance across which water-borne irritants pass into the circulation. Lamellae have a clubbed look due to lamellar hyperplasia, which occurs when cells generated from primary lamellae migrate to the distal end. This causes clubbing of secondary lamellae, which is the aggregation of cells along the leading edge of secondary lamellae (Samuel et al., 2008). The fish tried to limit the available surface area to the water sample by atrophy or dystrophy, bending, clubbing, and fusing of the secondary lamellae. Vascular changes in exposed fish gills could be an attempt by the fish to boost oxygen intake and delivery to the internal organs by supplying more blood to the gills. According to Chang et al. (2020), hyperplasia causes lamellar fusion, which lessens the amount of toxicant that comes into contact with blood and lessens its effects on fish. Edema with lamellar fusion caused by the lifting of the lamellar epithelium, acts as a defensive mechanism (Pribadi et al., 2017). The extent of gill damage is determined by the toxicant's concentration and the length of exposure. Islam et al. (2019b) reported lamellar fusion in Heteropneustes fossilis at 20 ppm, and distortion of its gill filaments and gill arches at 25 ppm. Further, Pattnaik et al. (2022) reported that organophosphate insecticides cause mild hyperplasia of epithelial cells, and secondary lamellae curling and fusing at low doses; sloughing of the epithelium layer from secondary lamellae at medium concentrations; and complete loss of gill architecture, deformation of primary and secondary lamellae, rupture of lamellae, desquamation, hyperplasia and severe necrosis at high concentrations in Heteropneustes fossilis.

Liver

Fish liver is an essential organ for detoxification site, and a trustworthy biomarker of health because of its many functions, which include regulating blood circulation, detoxification, biotransformation, glycogen storage, glucose release into the bloodstream, red blood cell breakdown, and synthesis of various blood plasma components (Hossain et al., 2016; Maurya et al., 2019; Sharmin et al., 2021; Suchana et al., 2021). The most common cell type, hepatocytes, carry out most of the body's essential functions, such as metabolism, detoxification, storage, and excretion of xenobiotics, as well as the conversion of glucose to glycogen, lipid regulation, and amino acid deamination (Kobayashi et al., 2019; Liu et al., 2017). Liver is the site of parenchymal injury since it is the primary organ targeted by xenobiotics and insecticides (Montaser et al., 2010). The liver is particularly susceptible to toxicants due to its extensive blood supply and role in metabolism, hence its histochemical alteration affect metabolism of carbohydrates, lipids, and particularly proteins (Karami-Mohajeri & Abdollahi, 2010). The most frequent negative consequences of pesticide exposure on the liver include necrosis, cellular deformity linked to nuclear hypertrophy and vacuolization, sinusoidal enlargement, and congestion of sinusoidal spaces caused by WBC infiltration (Verma et al., 2020). Hepatocyte lysis causes necrosis, central vein congestion, blood vessel disruption leading in a haemorrhagic region, cirrhosis, nuclear pyknosis in most hepatic cells, and finally death (Loganathan et al., 2006; Samuel et al., 2019b). Liver becomes congested along the hepatic arteries and bile duct due to acute and widespread necrosis of liver cells (Pandey & Dubey, 2015). Liver dysfunction stemming from pesticides cause structural damage to hepatocytes, leading to vacuolated hepatocytes and necrosis (Weber et al., 2020). Hepatic lipid accumulation (Tanaka et al., 2002; Xu et al., 2009), imbalances in the assimilation index (Hadi & Alwan, 2012) interpreted as pathological response also contribute to hepatocyte vacuolization.

Significant histopathological changes, viz., pyknotic nuclei, hepatocyte hypertrophy, haemorrhage, sinusoid vacuolization, and necrosis were observed in Heteropneustes fossilis liver in the present study. According to Kaur and Mishra (2019) and Özaslan et al. (2018), the liver of fish exposed to pesticides experiences notable structural alterations and responds differently to the type and intensity of the toxicant. In response to exposure to organophosphate pesticides, Barbhuiya and Dey (2014) observed that Heteropneustes fossilis had central venous congestion, hepatocyte degradation, cytoplasmic vacuolization, vacuoles in sinusoids, and hepatocytes with pyknotic nuclei after 21 days. On exposure to 5, 10 and 20 ppm concentrations of an organophosphate pesticide for 25 days, Heteropneustes fossilis showed cytoplasmic degeneration, pyknosis in liver tissues, vacuoles in hepatic cells, and rupture in hepatic blood vessels (Islam et al., 2019b). These findings corroborate the results of the current experiment. According to Pandey and Dubey (2015), hepatocytes exposed to organochlorine pesticides display dissociation and a granular appearance, along with varying degrees of hepatic damage inside and surrounding the hepatic parenchyma. Begum et al. (2013) reported in Heteropneustes fossilis that after 15 days of exposure to 7 ppm arsenic concentration, the hepatocytes underwent primary degeneration, and at 20 ppm, the hepatocytes lost their polygonal shape and became irregular, with areas containing eosinophilic cytoplasm.

Intestine

The second most important portion of a fish alimentary canal is the intestine, which is responsible for absorption of food. It is exposed to a variety of hazardous substances either directly through the ingestion of contaminated food or indirectly through the blood and/or lymph (Maurya et al., 2019). Mucosa, submucosa (lamina propria), muscularis, and thin serosa are the four histological layers that make up the intestine of Heteropneustes fossilis (Samanta et al., 2016; Vishwakarma et al., 2022). Mucous cells sustain the intestinal mucosa. Simple long finger shaped villi held by absorptive columnar epithelial cells make up the mucosa. Loose connective tissues compose the submucosa, and the serosa layer has a dense network of blood vessels, and a thin top plate of brush borders embraces the villi. Histopathology of the intestine indicate raising of columnar epithelium of villi and hyperplasia as responses representing defense mechanisms, with villi rupture, loss of structural integrity of mucosal folds, and degeneration and necrosis of the submucosa in the intestine (Samuel et al., 2019a). The same was observed in the present study with degenerated epithelium, autolysis of mucosa, degenerated and necrotic villi. Begum et al. (2013) reported that Heteropneustes fossilis when subjected to 7 ppm of arsenic concentration for 15 days had degenerated villi, and at 20 ppm, damaged serosa was detected, leading to mucosal fusion and edema. Further Islam et al. (2019b) reported that Heteropneustes fossilis when subjected to organophosphate pesticide, showed swelling, disintegrating sub-mucosa, mildly damaged serosa, and fused or ruptured villi at concentrations ranging from 20 to 25 ppm.

Kidney

As the primary organ engaged in fish fluid and ionic homeostasis, the kidney is recognized as the basic excretory and detoxifying organ (Rakhi et al., 2013). They function as the majority of teleosts primary haematopoietic organ, and are in charge of maintaining stable internal homeostasis, osmoregulation, electrolyte and water balance, and acid–base balance, all of which are crucial for controlling the amount and composition of extracellular fluid (Hyodo et al., 2014; Kobayashi et al., 2019). Fish kidney is composed of two parts, viz., head and body. Avascular lymphoid mass predominates in the head kidney of Heteropneustes fossilis, which lacks functional features like glomeruli, but the kidney located in the posterior trunk is functional and has a large number of uriniferous tubule (Verma et al., 2020). Fish kidney histopathological changes are a well-known bioindicator for assessing the effects of toxins (Ramesh et al., 2018). Toxicants often target the kidneys, which might impair the organ's ability to maintain homeostasis and impact the fish physiological condition (Maurya et al., 2019; Verma et al., 2020). Pesticides primarily damage the kidney's renal tubules, resulting in renal lesions, dilatation of Bowman's space, and swelling of the tubules (Singh, 2012). In the present study, alterations like distortion of renal tubule, haematopoietic tissue, dilation and degeneration of glomeruli and necrosis were seen in the kidney of Heteropneustes fossilis. Islam et al. (2019b) found necrosis of the kidney's tubular and hematopoietic cells, bleeding of the renal tubules, and necrotic alterations of the glomerulus in Heteropneustes fossilis when subjected to organophosphate insecticide at concentrations ranging from 20 to 25 ppm.

Brain

The brain serves as a relay station, controlling all functions and movements in the fish body, and it regulates several physiological processes, particularly when pesticides are involved in their mode of action in the nervous system (Rao et al., 2005). The telencephalon is the most peculiar part of the teleost’s brain comprising the cerebral hemisphere, olfactory lobe and olfactory bulbs. The telencephalon aids in the processing of visual inputs and is involved in feeding, defense, schooling, aggressive and reproductive behavior (Berntssen et al., 2003), as well as the expression of learning and memory (Salas, 2006). According to Nordgreen et al. (2007) and Rodríguez et al. (2002), the telencephalon in fish is most likely comparable to the hippocampus and amygdala in mammals. Toxicants reduce the action of the acetylcholinesterase enzyme, which is present in the brain and regulates physiological and behavioural responses in fish (Modesto & Martinez, 2010). Degenerative changes in neuronal cells were observed with histological abnormalities like pyknotic nuclei, karyolysis, swelling and haemorrhage in neuronal cells in the treated fish's brain in the present study. Mishra et al. (2021, 2022) reported that acute toxic effects and degeneration of neuronal cells in the optic tectum and cerebellar region of the fish brain alters the locomotory behavior of Heteropneustes fossilis on exposure to organophosphate pesticides.

Muscle

Fish muscles are frequently contaminated with chemicals, pesticides, and harmful heavy metals (Dutta et al., 2017). Fish muscles are active and high in protein, and they generate mechanical tissue that aids in the fish's navigation through water. They do not participate in any metabolic activity (Tasneem & Yasmeen, 2020). The majority of the energy used by the body comes from muscular action. The metabolism of fats and carbohydrates produces ATP, which is used as energy by all muscle cells. Myotomes in fish muscles are typical, with muscle bundles evenly spaced. Skin muscle is the primary site of exposure, and pollutants affected the epidermis abruptly (Rakhi et al., 2013). The skin and muscle tissue come into direct contact with toxins dissolved in water as gills, which can cause severe thickening and separation of muscle bundles, haemolysis, necrosis, lesions with decreased compactness, pronounced intramuscular oedema, and degeneration in muscle bundles with inflammatory cell aggregations, among other histopathological changes (Srivastava, 2019). According to Prasad and Shil (1993), the primary purpose of the fish skin is to shield it from numerous dangers it encounters when interacting with its surroundings and to preserve its inside environment (milieu intérieur). Pollutant levels in fish muscle are influenced by factors such as fish length, species, trophic level, habitat type, feeding habitat, and fish movement patterns and time spent in contaminated areas (Bonito et al., 2016). Muscle damage in response to organophosphate pesticides is revealed by elevated heat-shock protein expression, which serves as defense mechanism (Karami-Mohajeri & Abdollahi, 2010).

The histological abnormalities in the muscle of Heteropneustes fossilis in this study included loss of striated muscle, muscle fibre degradation and necrosis. The histopathological changes identified in fish muscles in this study are consistent with prior research by Begum et al. (2013) who reported atrophy, vacuolar degeneration, and degeneration in the muscle bundles of Heteropneustes fossilis, along with isolated regions of necrosis. After 15 days of exposure to arsenic concentration of 7 ppm, the muscle tissue showed dystrophic changes with marked thickening and separation of muscle bundles. At 20 ppm, the muscle tissues showed severe intramuscular edema along with dystrophic changes with marked thickening and vacuolar degeneration of muscle bundles. It is true that, similar to gills, dissolved toxins and pollutants can get into intimate contact with muscle tissue. Histopathological alterations in the fish muscle show a high level of pesticide accumulation, which affects contractile ability and can lead to muscle fibre disfunction (Lakshmaiah, 2016). Fatma (2009) observed vacuolar degeneration of muscle bundles, necrosis, and atrophy of muscle bundles. The current findings on muscle histology are also consistent with those of Dhevakrishnan and Zaman (2012), Kaur et al. (2018), and Patnaik et al. (2011) who reported numerous pathological changes in muscle tissue including disorganization of muscle bundles, dystrophy, severe intramuscular edema, and muscle bundle dissociation and necrosis.

Recovery studies

The elimination of toxicant during recovery phase in fish is responsible for the improvement in its histological architecture. According to Narra et al. (2017), recovery studies are now a vital tool for enhancing the quality of fish life exposed to pesticides. Recovery studies have been described in the organs of fish such as Clarias batrachus (Begum, 2004), Danio rerio (Sancho et al., 2009), Cyprinus carpio (Blahova et al., 2014; Mikulikova et al., 2013), Carassius gibelio (Velcheva et al., 2013), Anabas testudineus (Jayakumar et al., 2014), Barbonymus gonio (Moniruzzaman et al., 2017), Barbonymus gonionotus (Islam et al., 2019c), Oreochromis niloticus (De Mello et al., 2021; Hasan et al., 2022; Meng et al., 2021), Trachinotus ovatus (Liu et al., 2021), and Heteropneustes fossilis (Loganathan et al., 2021). Hasan et al. (2022) reported that in most cases of recovery, the abnormalities becomes moderate where damages were severe, and mild where the damages were moderate. Gaber (2007) and Velcheva et al. (2013) reported that the beginning of recovery process is slow, long-lasting difficult process, and it requires a long period of time. This statement corroborates the slow recovery response observed in the present study. In gill, the recovery response was reduced, revealing necrosis, secondary lamellae fusion, hyperplasia, and desquamation. In liver, the recovery response was lower, with swollen and congested hepatocytes, pyknotic nuclei, karyolysis, vacuolization, necrosis, and sinusoid dilation. In intestine, the recovery pattern was less severe, with fusion, degeneration, erosion, and necrosis of the villi, autolysis of the mucosa, and infiltration of leucocytes. In kidney, the recovery response was weaker, with dilation of Bowman's capsule and glomeruli, deformed renal tubule, and necrosis and hypertrophy of haemotopeotic tissue. In brain, the recovery response was even lower, as it revealed disintegration of cell membrane, bleeding in neural cells, loss of cellularity, and profuse vacuolization. In muscle, the recovery response was lower, as it showed loss of striated muscles, cellular debris in the myofibril area, dilatation of muscle fibril, and muscular dystrophy.

Recovery assessment is frequently included in lower-tier toxicity assessments, and the possibility for recovery is influenced by the length of time between doses and the pesticide's unique mechanism of action. Once a contaminant has harmed an organism's system, the rate of recovery is influenced by the contaminant's persistence as well as the ecology, physiology, and biochemistry of the organism's system, as well as its closeness to the system. The dissipation half-life of the pesticide, the initial exposure concentration, and the dangerous concentration are all factors that can be used to forecast how quickly an organism's system will recover. This is especially important for fish that are only in a region of a water body for a brief time and may move away from a hazardous system. Furthermore, a variety of factors influence an organism's recovery after a large perturbation, one of which is the fish's life history, which varies greatly in terms of reproductive and dispersion characteristics (Boxall et al., 2002). Therefore, from a practical perspective, research on recovery process is useful.

Conclusions

Agrobased organophosphate pesticides create a high risk to fish species and their long-term exposure causes incalculable abnormalities and reduces the quality of fish life. The present study conclude triazophos to be toxic to fish as the pesticide affected Heteropneustes fossilis vital organs, viz., gill, liver, intestine, kidney, brain and muscle, via histopathological investigations as sensitive bio-indicators in fishes confirming pesticide toxicity. Additionally, recovery response studies responsible for the improvement in the histological architecture showed a slow recovery response. Therefore, from a practical perspective, research on recovery studies is also useful in determining the health of the fish after recovery. Furthermore, as this is the first investigation into the effects of triazophos on the recovery response in Heteropneustes fossilis more research is needed to determine the link between the treated and recovered responses.

Declaration