Immunological responses
The study showed increased activity of SOD and CAT with increase in 2,4-D concentrations (Table1). However, ROS and lysozyme activity decreased with 2,4-D exposure (Table 1). The high level of SOD and CAT activity in the exposed fish might have occurred due to the triggering of antioxidant enzymes for defense mechanism to overcome the negative effects of 2,4 -D. While, the lysozyme activity was hindered due to the 2,4-D exposure, the activity of lysozyme decreased with increase in pesticides and its activity was significantly less compared to both positive and negative controls (Fig. 1). It is well accepted that fish phagocytes after activation are able to generate superoxide anion (O2−) and its reactive derivatives (i.e. hydrogen peroxide and hydroxyl radicals) during the period of intense oxygen consumption, called the respiratory burst. These reactive oxygen species are toxic for bacterial pathogens (Paiva and Bozza, 2014) of fish. The results show that the activity of immune parameters in serum could be used as biomarkers for monitoring environmental stressors, and indicate that the activities of specific biomarkers in C. carpio are more sensitive to pesticides.
Table 1
Immunomodulation effect of sub-lethal concentrations of 2, 4-dichlorophenoxyacetic acid in C. carpio exposed for 28 days.
Treatments | ROS | SOD | CAT |
T1 | 1.08±0.12a | 2.48±0.24a | 2.12±0.17a |
T2 | 0.81±0.10b | 3.51±0.49b | 3.82±0.45b |
T3 | 0.79±0.09b | 3.97±0.60c | 4.26±0.56c |
T4 | 0.74±0.11b | 4.21±0.61d | 5.11±0.81d |
Amongst the several immune parameters, lysozyme is one of the important defense molecules that is altered due to pesticides (Saurabh and Sahoo, 2008). Previously, a study revealed a significantly decreased lysozyme activity in plasma, liver, kidney and spleen in great sturgeon Huso huso (Khoshbavar-Rostami et al. 2006) exposed to diazonin. Similarly, it has been reported that chlorpyrifos elicited lysozyme attenuation in plasma and spleen of common carp (C. carpio) exposed acutely to 75 µg/L (Li et al. 2013). In contrast, 2.0 and 4.0 mg/L diazinon in grass carp Ctenopharyngodon idella significantly induced lysozyme activity (Soltani and Pourgholam, 2007) and chlorpyrifos (0.102 and 0.255 mg/L) in Nile tilapia Oreochromis niloticus (El-Bouhy et al. 2014). It was also reported that exposure of Nile tilapia O. niloticus to chlorpyrifos (0.102 and 0.255 mg/L) provoked an increase in the activity of lysozyme in the plasma of these organisms; however, at a lower concentration (0.051 mg/mL) the pesticide did not cause any effect on lysozyme (Zahran et al. 2018). In turn, disruption of lysozyme which has antibiotic properties originates in leucocytes that possess a broader functions (Demers and Bayne, 1997) has been frequently utilized as an indicator of non-specific immune functions, which combats primary infections in fish would deleteriously get affected due to pesticides in fish (Saurabh and Sahoo, 2008). It may be challenging to demarcate the causes for immunological responses caused by various contaminants, perhaps it is possible to consider these as suitable biomarkers for quick scrutiny of pesticides, using live fish.
Histomorphology
The current study extensively focussed on histopathology of selected tissues, since micro-architecture of the organs provides detailed information about the screeening of environmental pollutants using target tissues. The study observed a disparaging alterations in the tissues at sub-lethal levels and the responses were dose dependent, however no differences observed between control and environmental concentration (29 µg/L) of 2,4-D, hence histology pictures for environmental concentration is not provided for any of the tissues studied.
2,4-D is a synthetic auxin (a plant hormone), which is absorbed by the leaves of plants. Upon its application, plants respond with uncontrolled and unsustained growth, which ulitimately kills plant (Clark and Pazdernik, 2016). Similarly, in the current study, 2,4-D might have increased the cell numbers and formed tumours at high concentrations in fish tissues with exposure. And it is reported that nervous system is one of the targets for 2,4-D and effects were found relating to degeneration of nerve motor function, lethargic and behavioural changes (Bortolozzi et al. 2001; De Morro et al. 1993) in rats and chick embryos. In zebrafish embryos, a significant loss of nerve signals, reduced axon projections to the tectum, thin and truncated neuron axons of the motor regions were observed (DeMarco et al. 2021).
In the histological investigations, tegmentum area of the brain of unexposed fish showed a well organized nerve fibres and uniformly scattered nerve granules and clearly visible erythrocytes (Fig. 2A). While, fish exposed to 2.9 mg/L 2,4-D revealed increased intensity of dark granules, and nerve fibres in bundles (Fig. 2B). However, at 29 mg/L 2,4-D, the quantity of nerve fibres decreased and the nerve granules were prominent, along with pigments. In some regions of the tegmentum, pyknosis of granules were observed along with clumping of erythrocyte (Fig. 2C). Whereas, high concentration exhibited severely intensified (dense) granules and reduced nerve fibres (Fig. 2D). It was also noticed that the cilia at the borders of the tegmentum was severely affected as the concentration of the herbicide increased. The increase in granules reflects the basic property of 2,4-D where it enhances growth of plants through increase in cell numbers, hence in fish also it might have proliferated cells leading to tumour formation. However, in zebrafish significantly increased cell death, damaged growth of motor neurons and found less teratogenic. These differences could be due to either species sensitivity and or age of the fish. The cell death observed in zebrafish embryos could be to the development of the animal where cell proliferation was inhibited (Ton et al. 2006), however the precie mechanism for such differences need to investigated.
Pineal gland, an endocrine gland that plays an imperative role in melatonin production, and also functions in photosensitivity (Dodt, 1963). A well organized pinealocytes and astrocytes were observed in pineal gland, and these cells possessed proper shapes along with uniformly distributed erythrocytes in unexposed fish (Fig. 2E). While with 2,4-D exposures, erythrocyte numbers increased with abnormal shape along with big sized blood vessels, cell necrosis and size of the pinealocytes also increased (Fig. 2F&G). Whreas at highest concentrations, vacuolization was severe and nerve fibres were disrupted disparagingly (Fig. 2H). The alterations observed would affect the melatonin secretion, followed by hypothalamic-pituitary-thyroid axis driven pigmentations, perhaps this could be used as a potential biomarker. Besides, it might affect the regulation of other physiological rhythms including translating photoperiodic stimuli into function (Walton et al. 2011; Zheng et al. 2021), since it plays an important role in regulation of circadian rhythms. It is also proven that any environmental stressor that alters the function of pineal gland would lead to jeopardy on the well-being of vertebrates including fish (Sanchez-Vazquez et al. 2019). The perturbed homeostasis of the melatonin would ultimately result in altered circadian rhythms which might hinder the fitness of the fish in terms of survival, growth and reproduction, (Zheng et al. 2021) and consequently biased sex ratio.
The tectum of the brain revealed a clearly visible mononuclear cells along with uniformly distributed nerve fibres in unexposed fish (Fig. 2I). While with increased concentrations, the fibrous dendrites, necrosis, vacuolization were initiated and the erythrocyte numbers increased (Fig. 2J&K). However, at highest concentration, multi-nucleated cells and severe vacuolization was prominent (Fig. 2L). Since tectum is a sensory and primarily visual in function (Northmore, 2011), damage in it by herbicide exposure might affect locating food and may lead to loss of prey and predator relationship. Similar alterations were also observed by Mishra and Devi (2014) in Channa sp exposed to chlorpyrifos. The alterations observed in the optic tectum reflects the motor coordination in animals (Marks and Berg, 1999; Kermer et al. 2004). The vacuolization in the tectum due to 2,4-D in the current study are similar to alterations in the retina and lens of fish contaminated by detergent (Huang and Wang, 1995). Collectively, the changes observed might influence the normal function of the brain because it plays an important role for visualization (Roy et al. 2006). The 2,4-D exposure induced cell manifestation, and may drastically affect fish behavior. The results of the study opine that 2,4-D is immunotoxic and neurotoxic and it may influence the visual and sensory functions of fish (Mishra ands Devi, 2014).
While, thalamus of the brain in unexposed fish revealed that the nerve fibres were very clearly distributed and erythrocytes were well organized with proper shapes (Fig. 2M). Whereas, fish exposed to highest concentration of 2,4-D exhibited thick blood vessels, clumping of blood cells and nerve tumours (Fig. 2N). The abnormal proliferation of cells might have led to the formation of tumor in the brain due to 2,4-D. According to our knowledge, this is the first report which identified tumor formations in fish the brain due to herbicide and it needs further studies to understand the precise mechanisms.
The study presented a well organised primary and secondary lamellae along with uniformly distributed chondrocytes in control group (Fig. 3A). Whereas, fish exposed to 2.9 and 29 mg/L 2,4-D exhibited a minute telangiectasia, increased erythrocytes, necrosis, leukocyte infiltration, fused secondary lamellae, vasodilatations and decreased chondrocyte size (Fig. 3B&C). At highest concentration, numerous aneurysms, congregated erythrocytes in aneurysms, and reduced chondrocytes in primary lamellae were prominent (Fig. 3D). Similar observations were also made in P. vivipara exposed to 2,4-D (Vigario and Saboia-Morais, 2014). The histopathological alterations observed for pesticide exposures in the earlier investigations have revealed that the gills are the primary routes for entry of several contaminants, and possess large suface area having direct contact with water (Simonato et al. 2008; Abdel-Moneim et al. 2012; Baskar, 2014). The numerous aneurysms recorded at the higher concentrations of 2,4-D exposure could be due to the rupture of pillar cells associated with increased blood flow (Camargo and Martinez, 2007; Paulino et al. 2014) which might have caused by the stress that is induced by the pesticide exposure. The alterations observed in gills might be because of polarity of 2,4-D and it can contact and interact easily with the epithelial cells (Vigario and Saboia-Morais, 2014). As per the current and earlier studies, histopathology of gills is one of the excellent biomarkers for environmental impact assessment and provide relevent information on the health status (Strzyżewska-Worotyńska et al. 2017).
Concurrently, the study recorded a uniformly distributed proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) in unexposed fish (Fig. 4A). Whereas with 2,4-D exposures, lumen in PCT was noticeable, and damage of hematopoietic tissue was evident (Fig. 4B). With increased concentration (29 mg/L), Bowman’s space increased, minute tumour nodules and melanomacrophages (MMCs) were prominent (Fig. 4C). At highest concentration, fluid accumulations (exudates) were commonly found, severe damage of kidney tissue through the formation of tumour was more prominent along with huge vacuolisations (Fig. 4D). Similar alterations were also observed in Clarias gariepinus exposed to cypermethrin (Velmurugan et al. 2009) and Piaractus mesopotamicus exposed to trichlorofon (Mataqueiro et al. 2009). The yellowish to black MMCs observed in hematopoetic tissues of kidney and liver indicated the development of chronic inflamatory lesions (Agius and Roberts, 2003) or stress (Peters and Schwarzer, 1985) are a consequences of 2,4-D exposure. These alterations implicate the deterioration of the renal tubules, glomuruli and tumour formation, and the intensity of damage was increased with increased concentrations and similar changes were also observed in rats exposed to malathion (Al-Attar, 2010). The disparaging alterations observed in kidney in various investigations reveal that these changes might take place to perform the sequestration of the pesticides for their degradation to reduce the toxicity of the contaminants (Agius and Roberts, 2003), by compromising their micro-architecture.
Liver, an organ for metabolism, detoxification and accumulation of toxic substances. In the current investigation, uniformly distributed hepatocytes and Kupffer cells were observed in unexposed fish (Fig. 5A). However, at environmental concentration, slight vacuolization was initiated to appear and with a few anucleated hepatocytes (figure not shown). While at sub-lethal concentrations, yellowish to black MMCs were noticed (not shown in figure), pancreas appeared thickening and vacuolization was more prominent with pyknosis of hepatocytes (Fig. 5C&D). These responses are in agreement with the Vigario and Saboia-Morais (2014) in P. vivipara exposed to 2, 4-D. In both studies the responses were dose dependent and severity of effects increased with increase in 2,4-D concentration. The vacuolization observed at higher concentration indicate the toxic effect of 2,4-D and this might lead to tumour formation through spongiosis (Hinton and Lauren, 1990; Ortiz et al. 2003). The disaapearance of nuclei or micronuclei observed in the exposed fish might be an indication of genotoxicity of 2,4-D, and perhaps this would be adpoted as genotoxic assessment tool as it is a simple and short term assay which helps monitoring the water quality (Grisolia, 2002).
In summary, the study showed 2,4-D as an immunotoxic, and perturbed multiple tissues investigated. Further studies are warranted to understand its precise neurotoxic effects. The histo-architecture witnessed that 2,4-D can cause tumours in multiple tissues through cell proliferation. However, future studies are warranted to know the molecular insights of herbicides impact either in fish or any other aquatic animals.