Employment of different plant-based products for a range of purposes such as treatment, growth, immune-enhancement, etc. has been widely accepted owing to different phytochemicals present in them. However, their toxic effects are yet to be explored in different target and non-target species, since they also contain toxic botanicals in them it becomes an inherent part to explore such phenomenon (Okoro et al. 2019).
In the present study, the extraction yield was higher for the hydroethanolic solvent (80% ethanol) as compared to the aqueous and ethanolic solvents. The present results corroborate the findings of Senguttuvan et al. (2014) and Khan et al. (2022) as they also reported the superior ability of hydroethanolic solvent. The possible reason for the present outcome could be the cumulative effect of both polar (ethanol) and non-polar (water) solvents especially the polarity of ethanolic solvent which leads to better solvating of phyto-constituents (Vanitha et al. 2016; Akhtar et al. 2018; Nigam et al. 2021). For a plant extract, the exertion of toxic effects on fish depends on the presence of bioactive compounds, which could vary significantly depending on several factors, particularly on the type of solvent (Tung et al. 2007; Shahi and Singh 2011). For this reason, three different solvents were employed in the present study.
The study revealed presence of different toxic phytochemicals (alkaloids, saponin, tannin and rotenone) in all three P. hysterophorus extracts. In corroboration with to present findings, Nigam et al. (2021) reported alkaloids and saponin in aqueous and ethanolic extracts of parthenium. Whereas, Krishnavignesh et al. (2013), Deshpande et al (2017) and Tarekegn et al. (2021) reported the presence of alkaloids, tannin and saponin in different solvent-derived extracts including hexane, acetone, ethanol, methanol and petroleum ether. Similarly, Gupta et al. (1977) also reported saponin in the aqueous extract of gajar ghas. Moreover, present findings also revealed the superior ability of hydroethanolic solvent as compared to water and ethanolic solvent in extracting all four toxic botanicals. The reason for the hydroethanolic solvent’s superior ability could be a combined effect of both polar and non-polar solvents (Vanitha et al. 2016; Akhtar et al. 2018; Nigam et al. 2021). Moreover, reports are suggesting the better solubility of the bio-active components in organic solvents such as ethanol which could also help in explaining the present finding (De Boer et al. 2005; Salama and Marraiki 2010). Based on the quantitative phytochemical profile hydroethanolic extract was selected for further examination.
Like many other plants, P. hysterophorus extract also showed anti-bacterial activity against ten different indicator bacteria. Where the zone of inhibition varies from 17–22 mm. The present result is in agreement with the findings of earlier researchers who have also reported the anti-bacterial response of parthenium against E. coli, Bacillus subtilis, B, cereus, Enterococcus spp, Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacter aerogenes (Barsagade and Wagh 2010; Fazal et al. 2011; Khan et al. 2011; Kumar et al. 2012). Moreover, there are reports where parthenium was reported to exhibit an anti-fungal response against Aspergillus niger, Aspergillus flavus Candida albicans (Kumar et al. 2012; Malarkodi and Manoharan, 2013; Krishnaveni et al. 2015; Kaur et al. 2016; Bajwa et al. 2020; Sharif et al. 2021). The antimicrobial efficacy of plants including parthenium is largely attributed to secondary metabolites such as essential oils, flavonoids, phenolics, and tannins as well as sugars, amino acids and phenolic derivatives (Sukanya et al. 2009; Rahmat et al. 2011; Khaket et al. 2015; Veena and Shivani 2012; Malarkodi and Manoharan 2013; Shashank and Abhay 2014). Recently a non-substituted cyclopentenone moiety, parthenin was characterized which exhibited an antimicrobial response (Lalita 2018).
Erythrocyte-based lytic assays have been adopted as a measure of toxicity particularly, cell membrane-targeted toxicity for various formulations and drug interactions (Zohra et al. 2014; Hussain et al. 2022; Khan et al. 2022). Additionally, the advantage of erythrocytes model is easy availability of blood cells, which resemblance with other cells and are easy to handle as per the necessity (Litman et al. 1976; Robertis and Robertis 1995). In the present study, the P. hysterophorus extract causes substantial lysis of fish red blood cells. Additionally, the haemolysis rate exhibited dose-dependent lytic potential. The possible reason for the lysis of cells could be the destruction of the lipid bilayer of plasma membrane caused by the toxic components present in the extract. Saponin present in the extract is a major compound responsible for haemolysis of blood cells because of lipid-soluble moieties present in saponin, sapogenin which destabilize the RBCs membrane (Amini et al. 2014). Additionally, other phytochemicals (rotenone, alkaloid, tannin) may also lead to cellular toxicity (Khan et al. 2022). In agreement with the present findings, Hussain et al. (2022) reported haemolysis of human RBCs upon exposure to parthenium leaf extract. The authors also reported cells treated with different extract concentrations starting from 80, 120, 160 and 200 µg were capable to cause lysis ranging from 6.09 to 76.90%. Furthermore, Bajwa et al. (2020) and Hussain et al, (2022) suggested the reason for lysis caused by parthenium extract may be because of sesquiterpene lactone, ‘parthenin’ in the extract, particularly in parthenium leaves. Parthenin content in the extract is very toxic to cells as they prevent the synthesis of important cellular enzymes and DNA and RNA (Bajwa et al. 2020). Additionally, several other botanicals present including sesquiterpene lactones (coronopilin, ambrosin, hysterone D and tetraneurin A) α-methylene-γ-lactone and some acetylated pseudoguaianolides have also associated with the cytotoxic response of the P. hysterophorus extract (Das et al. 2007; Bajwa et al. 2020). Recently Jiménez et al. (2021) also reported the haemolytic activity of ethanolic parthenium extract on human red blood cells at 200 to 800 µg ml-1 of concentration where they reported absolute haemolysis at a concentration of 800 µg ml-1.
Phyto-haemagglution activity and haemagglutination limit test in the present study also revealed the toxicity caused by the extract by causing clumping of the cells. Phytolectins in the extract are majorly responsible for the clumping of erythrocytes which ultimately hinders the normal functioning of the cells (Senthilganesh et al. 2021; Ouattar et al. 2022). A similar kind of agglutination was also reported by Abdali et al (2021) while working on Linum usitatissimum extract. Irrespective of the reason, agglutinated cells will eventually be neutralised by the organisms’ immune system which may also roots to malfunctioning of physiological functions.
The findings of the present study confirmed the toxicity of P. hysterophorus hydroethanolic extract in C. carpio. The 96-h median lethal concentration of extract was found to be 18.99 mg l-1 with Log10 (LC50) of 1.278. In alignment with the present study, Tarekegn et al. (2021) have also reported LC50 of parthenium petroleum ether extract derived from different parts leaf, stem and root with three different solvents namely hexane, acetone and petroleum ether. Where, the LC50 ranges from 10.7 ppm (for root) to 478 ppm (for leaf) in mosquito larvae, Anopheles Arabiensis. Similar larvicidal activities of parthenium extract were also reported in Culex quinquefasciatus (Bansode et al. 2016) and Aedes aegypti (Kumar et al. 2012). Additionally, present study has reported the presence of different toxic phytochemicals in extract followed by cytotoxic/ haemolytic effects. Possibly, exposure to active compounds present in the extract starts interfering with the normal physiological and metabolic processes of cells and ultimately leading to the hampered health status of fish or subsequent mortality as also suggested by Vikhar and Jadhao (2018), Cowie et al. (2020) and Tarekegn et al. (2021). Bioactive compounds present in the extract cause moderate to severe toxicity. These responses can be measured by behavioural response of the organism (Okoro et al. 2019). Behavioural expressions produced in response to toxicants were duration- and dose-dependent. As with the increasing concentration and exposure time, severe symptoms such as copious mucus production, excessive haemorrhage on the ventral body surface, etc. exhibited by the intoxicated C. carpio. In agreement with the present behavioural response Oribhabor et al (2020) and Ekpenyong et al. (2020) also reported such response in African catfish, Clarias gariepinus to Lonchocarpus cyanescens and Justicia extensa, respectively.
In determining the toxic intensity of various compounds haematological, biochemical and immunological indices play a central role as an indicator of xenobiotic compound intrusion (Nostro et al. 2000; Wu et al. 2018). Parthenium exposure in the present study at a sub-lethal concentration (T1: 0.395 mg l-1 and T2: 0.79 mg l-1) caused a significant reduction (p < 0.05) in haematological parameters including RBCs count, WBCs count, haemoglobin content and haematocrit value in comparison to extract devoid control group. In agreement with the present findings, Hussain et al. (2022) also reported a significant (p < 0.05) reduction in haematological parameters upon exposure to parthenium extract. Additionally, Hussain et al. (2022) also reported a significant reduction in differential leucocyte count (neutrophils and lymphocytes).
The decline in erythrocyte count in extract-treated groups could be the consequence of kidney and liver injuries leading to a decline in erythropoiesis and subsequent anaemic condition as a result of phytochemical intoxication (Saha et al. 2013; Wu et al. 2018). Saha et al. (2013) reported a 20% reduction in red blood cell count and a 30% reduction in haemoglobin content after the oral treatment of methanolic parthenium extract @ 20 mg 100-1 g body weight in Wistar albino rats (Rattus norvegicus). Another possible reason may be the phytochemical-induced internal haemorrhages which were very prominent in the fish body, particularly on the ventral surface of the body during range finding assay. The decline in Hb content in the present study may be due to impaired oxygen supply resulting in a slow metabolic rate and hampered energy production because of lack of oxygen (Ahmad et al. 1995). Reduction in RBC count and haemoglobin levels can be correlated with the induction of anaemic conditions in fish (Cella and Watson 2000). Additionally, Saha et al. (2013) suggested that parthenin content induces anaemic condition either by interfering with haemoglobin synthesis or by fading RBCs survival. However, in contradiction to the present finding, Hussain et al. (2022) reported a significant increase in haemoglobin content after parthenium treatment.
Leucocytes are regarded as the major component of first-line fish defence which plays a central role in innate immune response (Menezes et al. 2006). The present study revealed a reduction in WBCs count at all sampling points. Similar to hampered RBCs levels, WBCs levels may also be correlated as a consequence of potential environmental stressor (dissolved extract), which causes an immunosuppressive condition in fish as also reported by Khedkar (2022) while studying parthenium extract effect on haematology of rohu. The possible reason could be the faulty physiological activities and subsequent vulnerable leucopoiesis. However, there are several contradictory results where reports suggested a significant rise in the leucocyte counts upon exposure to plant extract as a countering mechanism of fish’s non-specific immune response to re-establish homeostasis (Shahi and Singh 2011).
The hydroethanolic parthenium extract exposure in the present experiment also caused noteworthy changes in haematology-based indices including MCV, MCH, and MCHC. Similarly, Hussain et al. (2022) also reported deviation in haematology-based indices following exposure to leaf extract of parthenium (10, 20, 40, and 80 mg kg-1 body weight). Where a significant (p < 0.05) decline in MCV levels and an increase in MCH and MCHC were observed. The present elevation of MCV and MCH levels may be an indicator of erythrocyte swelling and resultant macrocytic anaemia upon toxicant exposure (Kavitha et al. 2012). Similar results were also reported by Suely et al. (2015) where they exposed Heteropeneustes fossils to a sub-lethal concentration of Terminalia arjuna bark extract. MCHC levels in the present study showed fluctuating levels in common carp. Fluctuation of MCHC level might be the toxic result of the extract which results in malfunctioning of normal physiological activities such as a drop in iron constitute of blood and ultimately affects MCV and MCH levels (Hodson et al. 1978). Similar fluctuations in MCHC levels were also reported by Shahi and Singh (2011) and Suely et al. (2015).
The PCV level in all the treatments upon exposure to hydroethanol-based extract showed a decreasing trend. The present finding was in collaboration with Ayotunde et al. (2004) and Anju et al. (2013) who noticed a decreasing trend in haematocrit levels in Oreochromis niloticus when exposed to aqueous extracts of Moringa oleifera seeds and in Heterobranchus longifilis upon Tephrosia vogelii extract exposure, respectively. The reduction in PCV levels may be the result of toxic components affecting erythropoiesis and causing the depletion of erythrocytes (Ahmad et al. 1995; Atamanalp and Yanik 2003). However, in contradiction Hussain et al. (2022) reported increased Hct value in experimental rabbits following parthenium leaf extract treatment.
Blood biochemical indices have exhibited significant (p < 0.05) variations in levels of blood glucose, GPT, GOT and ALP upon extract exposure in common carp. Glucose is the most important source of energy for any organism which can be affected in response to an unusual condition caused by myriads of pollutants hampering the physiological homeostasis of organisms (Manush et al. 2005). In the present study, it was observed that the glucose level followed an increasing trend from 24 h to 96 h in both treatments with a positive relation to the sub-lethal concentration of extract. Glucose serves as a vital indicator of stress caused by various irritants (Manush et al. 2005). In indirect agreement with the present finding, several studies reported the stress (gulping, copious mucus production, erratic movement, etc.) (Vikhar and Jadhao 2018; Ekpenyong et al. 2020; Oribhabor et al. 2020) caused by parthenium extract which might have rooted the increased levels of glucose in test animal. A similar type of result was also obtained by Suely et al. (2015) in H. fossilis due to exposure to T. arjuna extract. The elevation in the blood glucose found in the present study can be ascertained due to the conversion of hepatic glycogen into glucose to counter the elevated energy demand as also reported by Datta and Kaviraj (2003). However, in contradiction to the present finding, Patel et al. (2008) reported a decrease in glucose levels after the oral treatment with an aqueous extract of P. hysterophorus @ 100 mg kg-1 body weight in adult albino Wistar rats.
Alkaline phosphatase (ALP) is a marker enzyme which plays an essential role in membrane transport as well as is a significant bio-indicator of stress in fish. ALP level was found to be significantly increased in fish from 24 h to 96 h in all the treatments. And the probable reason could be an acceleration of transphosphorylation activity as a result of the toxic effect of phytochemicals present in the hydroethanolic extract as also reported by Sharma (1990). Similarly, Hussain et al. (2022) reported a significant (P < 0.05) increase in serum ALP level with the increasing concentration (10, 20, 40 and 80 mg kg-1 body weight) of parthenium extract. These variations in serum ALP levels are indicators of liver (chief detoxification organ) damage, as this enzyme is a recognized marker associated with hepatocyte damage (necrosis and cytosol drip) caused by the accumulation of toxic compounds (Cho et al. 2006; Ozer et al. 2008; da Silva et al. 2020).
Glutamate-oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) are other vital enzymes which play a central role in carbohydrate and protein metabolism and liver function (Nemcsok and Benedeczky 1990). In the present study, GPT and GOT levels increased significantly (p < 0.05). In corroboration to the present finding, Hussain et al. (2022) also reported a significant increase in GPT level with the increasing extract concentration. A similar type of finding was reported by Jessa et al. (2015) due to exposure to the extract of Derris elliptica stem extract on Clarius gariepinus. Gabriel et al. (2009) also suggested elevation may be the consequence of a disturbed Kreb cycle and also indicates hepatic cell damage caused by the toxic components present in the plant extracts.
Serum immunological parameters such as lysozyme activity, NBT, total protein, albumin and globulin levels have recorded significant (p < 0.05) decline in the levels following extract exposure. Lysozyme is a major immune factor which aids as an indicator of innate immunity (Tort et al. 2003). Lysozyme activity in the present study has shown a decreasing trend with an increasing concentration of crude extract. However, contradictory results were obtained by Huet and Wu (2004) in Jian carp while working on Astragalus Root (Radix astragalin seu Hedysari) and Chinese Angelica Root (Angelicae Sinensis) extract and observed the positive influence in the lysozyme level due to exposure of herbal extract. NBT assay helps to determine production of free oxygen radicals by phagocytes especially macrophages or monocytes (Dalmo et al. 1997). Respiratory burst activity is considered a vital indicator of innate immunity, which is a measure of increased oxidation level against foreign compounds. Similar to lysozyme activity, respiratory burst response also revealed a significant decrease in the levels following extract exposure at all the sampling hours. The reduction in the overall immune response of fish observed in the present study, may be due to reduced levels of leucocytes, which serves as a critical compromising factor of the immune system (Misra et al. 2006a, b) as also reported in the present study as a consequence of extract exposure.
Protein could be an alternative source of energy for carbohydrates in fish under certain conditions, which may result in hypoproteinaemia (Martinez et al. 2004). The present study revealed a marked decline in serum protein levels upon extract exposure in C. carpio at every sampling point. The present findings may be the result of protein utilization to meet the energy demand under the stressed condition created by the experimental condition as also reported by Tiwari and Singh (2003). Omoniyi et al. (2002) also reported significant hypoproteinaemia possibly due to cellular injuries or necrosis with subsequent impairment of protein synthesis while working on the effect of Tobacco leaf extract in C. gariepinus. Similar results were also observed by Adamu et al. (2009) under the influence of tobacco leaf dust on Catfish (C. gariepinus) and by Shahi and Singh (2011) while working on euphorbious plants on C. punctatus. However, on contrary, there are several reports suggesting hyperproteinaemia in fish exposed to plant extracts may be because of water loss from plasma, elevated de novo synthesis or malfunctioning of blood protein mobilization (Al-Attar 2005; Abalaka et al. 2011). Similarly, albumin and globulin content also depicted declining levels in a dose-dependent manner. The decreasing levels of different protein constituents in serum can be negatively correlated with the disturbed homeostasis caused by external stress stimuli, gajr ghas hydroethanolic extract.