Haemato-Biochemical and Histopathological Effects of Fungicide Pyraclostrobin on Japanese Quail (Coturnix japonica)

Use of different pesticides (insecticides, herbicides, and fungicides) is regular in crop farming which is among the major sources of environmental hazards (Abdalla et al., 2002; Zhu et al., 2022). Numerous hazards related to humans and environment are caused by the excessive use of pesticides during crop farming for achieving enhanced outputs (Chafai et al., 2022; Undugoda and Kannangara, 2022). About two hundred thousand piles of chemical pesticides are used around the year worldwide with 24% being consumed in The United States of America and 45% in Europe (Abhilash and Singh, 2009). According to Grue et al. (1991) birds that are exposed to various organophosphate (OP) and carbamate (CB) insecticides consumed less feed and display gastrointestinal problems.

Pyraclostrobin, is a common CB insecticide that is being widely used for managing critical plant diseases, delaying spore generation and mycelial development along with disrupting fungal mitochondrial activity (Bartlett et al., 2002; Li et al., 2018; Wang et al., 2021). It prevents rust, powdery mold, web blotch, downy mildew and rice blast caused by ascomycetes, oomycetes, basidiomycetes, and asexual fungal species (Gao et al., 2019). By consuming food contaminated by pesticides humans become prone to their hazardous side effects (Naraharisetti et al., 2009). Feed prepared from crops that are exposed to higher levels of pesticides can be fatal to birds and have also been reported to decrease their production performance (Harris et al., 2011).

Japanese quails (Coturnix japonica) belong to family Phasianidae and are being used as alternative research models to chickens (Farooq et al., 2022). They have a shorter life cycle, easily manageable and achieve sexual maturity around 7 to 8 weeks after hatching as reported by Nasar (2016) and Huss et al. (2008). According to Nakane and Youshimoura (2014) and Recoquillay et al. (2013) quails serve as important model species for research on genetics, behavioural norms and avian reproduction.

At different doses pyraclostrobin is toxic to terrestrial amphibians (Cusaac et al., 2017) as it decreases invertebrate pests’ ability to survive when exposed to aphids (Chirgwin et al., 2022). Inhibition of mitochondrial respiratory chain in honey bees is one of the detrimental effects of pyraclostrobin (Nicodemo et al., 2020). Pyraclostrobin, however, can kill bees as well, which might have serious negative effects on stingless bee colonies (Da Costa Domingues et al., 2020a). Pyraclostrobin fungicide residues in crops have the potential to be harmful to the environment and human health (Huang et al., 2021). In Wistar rats epoxiconazole has been reported to damage the liver and kidneys resulting in genotoxicity, oxidative stress, biochemical and histological changes. In zebrafish, pyraclostrobin aggravated DNA damage due to oxidative stress (Zhang et al., 2017; Ahmed et al., 2019; European Food Safety, 2008).

Numerous results reported in rats have suggested that the contact pesticides with aforementioned species increases the leakage of lactate dehydrogenase (LDH), alanine transaminase (ALT), urea and creatinine (Mossalam et al., 2011). Oxidative stress causes necrosis, structural alterations in various tissues and alteration of cellular macromolecules. Moreover, numerous results have revealed that extensive use of pesticides, particularly those that cause lipid peroxidation harm biological membranes (Altuntas et al., 2002). Numerous health issues in people and the surrounding environment were caused by the growing use of pesticides as a crop protection method. The objective of the current study was to confirm the hazardous risk that pyraclostrobin causes to Japanese quails.

A total number of 50 healthy quail birds were used in this study. All of the birds were kept in cages in environmentally controlled conditions. The basal diet was given twice daily along with fresh water available round-the-clock. After determining that the LD50 is 500mg/kg, several solutions were created for different groups of birds. The medication was administering orally by using 3cc syringes. Pyraclostrobin is one of the most effective fungicides. However, it can degrade via photolysis in water, it is toxic to aquatic life and if inhaled, it has a low solubility in water.

After an acclimatisation period of one week a total number of 48 quails were equally and randomly divided (n=12) into four groups: A, B, C, and D. Group A was considered as control group and was only given basal diet, whereas in Groups B, C, and D were considered as treatment groups. The body weight of the birds is measured (57±3) at the time of separation. The various dosages of Pyraclostrobin were administered orally to the various groups. After giving a first dosage of a different solution, first sampling was done. Afterwards, birds received a second dose of different solution and samples were collected. The birds were reared in semi-environmental controlled shed, and we did not notice any disease in the birds during the entire treatment period.

The concentration of pesticide (Pyraclostrobin) exposed to different groups of birds is as follows; after acclimatisation period, the Group A was administered distilled water with no pyraclostrobin for one week. Group B was given oral solution of comprising of 15 mg of pyraclostrobin diluted in 5 ml of distilled water. Group C was administered 30 mg of pyraclostrobin dissolved in 5 ml of distilled water (Akbel et al., 2018). Whereas, Group D was given 50 mg of pyraclostrobin diluted in 5 ml distilled water. The 2nd dose of pyraclostrobin was administered orally @ 15, 30, 50 mg/kg of body weight to group B, C, and D, respectively after one week of first dose. The Chemical formula of pyraclostrobin is given in Figure 1.

After the pesticide exposure, on 7 and 15-days birds from control and treatment groups were randomly selected. The samples of blood were taken from four birds of each group. Haematological tests were conducted on blood sample for determining of WBC, RBC counts, hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) according to previous study (Tchoupou-Tchoupou et al., 2022; Ahmed et al., 2023; Sindi et al., 2022) by using commercial haematology analyser.

Serum was harvested for determining serum biochemical profile that included aspartate aminotransferase (AST) and alanine aminotransferase (ALT) which were determined spectrophotometrically using commercially available kit according to previous study (Naseem et al., 2022; Nadeem et al., 2023; Vo et al., 2022).

On 7th and 15th day of experiment four quails from each group were slaughtered and visceral organs were obtained that included liver, heart, spleen, gizzard, proventricular, lungs, and kidneys. Immediately after slaughter small parts of kidney, lungs, liver, spleen, gut, heart, and spleen were fixed in 10% neutral alkaline formaldehyde (pH 7.2). Tissues were then prepared for embedding in paraffin wax. According to the routine histological protocol 4-5µm thick sections were cut and stained in the haematoxylin and eosin (H and E) (Saleemi et al., 2023; Kazmi et al., 2023). Using a photomicroscope, three sections from each specimen were examined at 100x.

No clinical symptoms were noticed in the birds of control group. However, multiple changes especially behavioural changes with minimal fluctuation were observed in treatment groups in dose and time dependent manner. Open mouth breathing, ruffled feathers, tremors, drabness, wet droppings, torticollis, salivation and decreased crowing was observed. Every bird was severely impacted compared to control group. In addition to this it was also observed that the birds of treatment group significantly less feed intake compared to the birds of control group.

Results revealed that there was variation in the selected haematological parameters after administration of different concentration of pyraclostrobin. The quantity of WBCs increased in the treatment group compared to the control group. However, in comparison to the birds of control group it was discovered that the birds of the treatment groups had lower values for RBCs, HB, HCT, MCV, and MCH. Details in variations of haematological parameters in quails after administration of different concentrations of pyraclostrobin are given in Table I.

The quails in control group remained healthy and active. But after administration of different concentration of pyraclostrobin the value of AST and ALT altered. The value of AST increased in treatment groups compared to control. The high value of AST shows that liver was infected after administration of pyraclostrobin. The value of ALT also increased in treatment groups compared to control. Variations in biochemical parameters of quails after administration of different concentration of pyraclostrobin are given in Table I.

Microanatomical features of lung, liver, kidney and spleen of birds in control group did not show any abnormalities throughout the course of the investigation. However, the birds administered different amounts of pyraclostrobin had mild to moderate physical abnormalities in lungs, liver, kidneys and spleen. At the 7th day minor cytoplasmic vacuolation in the livers of group B quail that had received dosages of (15 mg/kg body weight) were observed. Whereas, on the 15th day of experiment the birds of groups C and D which were administered dosages of (30 and 50 mg/kg), exhibited infiltration of leukocytes and nuclear condensation.

Table I. Effect of pyraclostrobin administered as 15, 30 and 50 mg/Kg body wt on haematological parameters and aminotransferases of Japanese quails.

Different superscript inf the same row indicates significant difference. Hb, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin and MCHC, mean corpuscular hemoglobin concentration; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

However, excessive dose of pyraclostrobin, caused liver congestion, pyknosis, karyolysis, fatty alteration, edema, karyorrhexis, atrophy of hepatocytes and haemorrhages as shown in Figure 2.

Several histopathological alterations were observed in kidneys. Examination on the 7th day of treatment groups revealed congestion, increase in urinary spaces, and necrosis of renal tubular epithelial cells in the kidneys of birds of group B that were administered dosage of (15 mg/kg). Renal tubule degradation, enlargement of Bowman’s gap, infiltration of inflammatory cells and necrosis of renal epithelial cells was seen in tissues sections from the kidneys of quail from groups C and D that were administered dosages of (30 and 50 mg/kg). In certain birds’ renal tubular necrosis and occasional kidney haemorrhages were also seen during trial at higher dosage as shown in Figure 3.

Spleen of birds in control group had normal histological features. However, with dosages of (30 and 50 mg/kg) birds of group C and D presented mild histopathological abnormalities in splenic parts that included deterioration of lymphoid tissues, deterioration of white and red pulp along with edema as shown in Figure 4.

Pesticides and herbicides are extensively used in agro-production to safeguard cereals and vegetable crops. Herbicides are harmful to plants in a variety of ways (Hussain et al., 2012). The present investigation reveals an unreported relationship between pyraclostrobin levels and macro and micro anatomical alterations caused by it in the selected visceral organs.

The lymphoid organs of developing avian species have a high degree of differentiation, making them more vulnerable to poisoning (Joshi et al., 2012; Khan et al., 2023). Pyraclostrobin (C19H18ClN3O4) has been reported to negatively affect humans. Pyraclostrobin is a fungicide which is extremely hazardous to living organisms but still being used extensively worldwide (Zemheri-Navruz et al., 2023; Wang et al., 2021). The current level of information regarding its detrimental effects to fish and aquatic species have been documented (Kiran et al., 2022).

The current study reveals that WBC increased in treatment groups compared to control group. The RBCs, Hb, HCT, MCV, and MCH values were found to be lower in treatment groups compared to the control group. Regarding haematological parameters the MCH concentration and MCV considerably dropped when the current study was compared to the prior study. From these results, it can be concluded that the quails of treatment groups had anaemia (macrocytic hypochromic). The findings of the current study are comparable to those of Kumar et al. (2010) who suggested anaemia in broilers treated with triazophos. The drop in haemoglobin content is due to decreased feed intake.

The values of AST and ALT in treatment groups were significantly higher than those of control groups. The elevated level of AST and ALT show that liver had undergone significant alterations. The impact of pyraclostrobin on avian blood biochemical changes has not been previously documented in literature to the best of our knowledge.

In all treatment groups significant abnormalities in liver as congestion, the kidney as inflammation and spleen as oedema were observed. Pyraclostrobin has been linked to negative impacts in non-target species like honeybees (Batista et al., 2020; Degrandi-Hoffman et al., 2015) and Danio rerio (Zhang et al., 2017). Our results reveal that C. Japonica birds were susceptible to every parameter examined following exposure to pyraclostrobin, during the experiment. In accordance with recent literature, other sublethal studies have also shown adverse effects of pyraclostrobin on different organs such as the midgut, mandibular and hypopharyngeal glands (da Costa Domingues et al., 2020b; Zaluski et al., 2017). Adverse effects of pyraclostrobin have also been documented on the midgut of Brazilian native stingless bee Melipona scutellaris (da Costa Domingues et al., 2020a, b) and on the fat body of neotropical solitary bee Tetrapedia diversipes (Rondeau and raine, 2022).

According to the results of the current study, pyraclostrobin administration at different dosages significantly increased liver weight at higher dosages (30 and 50 mg/kg). However, after 15 days of the birds being exposed to larger dosages of the fungicide pyraclostrobin (30 and 50 mg/kg) the kidney weight was dramatically decreased. it has been reported recently that liver exhibits a variety of histological changes that include oedema, pyknosis, fatty changes, congestion and hepatocyte atrophy. According to Ambali et al. (2007) malnutrition and condensed chromatin are the primary causes of liver cell atrophy and congestion. These conclusions concurred with those given by Grote et al. (2006). The effects on liver weight in quails of treatment group is a sign of either increased substrate availability that induces pesticide metabolism or minimal liver-toxic impacts of the insecticide exposure on birds.

In the current experiment, degeneration, leukocyte infiltration, haemorrhages, inflammatory cells, necrosis and edema were seen in kidney and spleen. These necrotic alterations result from necrotic cells releasing more IL-1 and IL-33 along with producing more cytoplasmic peptides and neuropeptides (Hussain and Mahmood, 2013). This systemic toxicity is the cause of observed histological lesions. Birds and mammals have also exhibited same abnormalities in the liver, kidneys, and spleen (Mossalam et al., 2011; Mahmoud et al., 2012). The buildup of organophosphorus and its pathophysiological effects in the visceral organs of birds is another cause of histological changes (Zhu et al., 2022; Mahmoud et al., 2012). The results of this study have significant relevance for identifying hazardous environmental effects of fungicide and an indicator for its accurate ecological effects related to stress.

It is concluded that exposing with pyraclostrobin to C. japonica birds has lethal effect which alters the histological features of liver, kidney, and spleen. Pyraclostrobin exposure negatively impacts haemato-biochemical profiles that may lead to anemia, hematopoietic stress and immune-suppression. This study warns about further investigations related to genotoxicity and to adopt precautionary measures to control pesticides usage in the field.

The authors extend their appreciation to the Researchers supporting project number (RSP2023R349) King Saud University, Riyadh, Saudi Arabia.

The study received no external funding.

The authors have declared no conflict of interest.