Azoxystrobin induced genotoxicity in Pethia conchonius, a freshwater fish of river Teesta, India

doi:10.21203/rs.3.rs-3241922/v1

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Azoxystrobin induced genotoxicity in Pethia conchonius, a freshwater fish of river Teesta, India

https://doi.org/10.21203/rs.3.rs-3241922/v1

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Purpose

Azoxystrobin is one of the globally used strobilurin fungicides. Indiscriminate use of azoxistrobin can contaminate the water bodies by surface run-off. The bioaccumulation of azoxistrobin may adversely affect the aquatic organisms. This study has aimed to evaluate the genotoxic effects of azoxystrobin using Micronucleus (MN) and Comet assays in the fish Pethia conchonius as an experimental organism captured from the river Teesta of the northern part of West Bengal.

Methods

The 96h LC₅₀ of azoxystrobin was estimated to be 0.514 mg/L. Three Sub-lethal concentrations (SLCs), namely SLC I = 0.025 mg/L, SLC II = 0.0514 mg/L and SLC III = 0.103 mg/L were used for the assay. The fishes were exposed to the above concentrations of azoxystrobin for 96h. The whole blood and gills were collected for MN and Comet assays, respectively at 24h interval.

Results

Nuclear abnormalities like micronuclei, notched and blebbed nuclei, and the DNA damage in terms of %Head DNA, %Tail DNA, Tail Length and Olive Tail moment were estimated by MN and Comet assays. The frequency of the nuclear abnormalities and DNA damages in the exposed groups were significantly (p˂0.05) higher than the control with increasing concentrations and the duration of exposure of azoxystrobin. The frequency of micronuclei (1.156 ± 0.011), notched (0.889 ± 0.011), blebbed (0.811 ± 0.011) nuclei and DNA damage parameters, %Head DNA (26.288 ± 1.981), %Tail DNA (73.712 ± 1.981), Tail Length (287.760 ± 11.641) and Olive Tail Moment (116.301 ± 7.636) were highest for SLC III at 96h of exposure.

Conclusion

The results strongly indicated that azoxystrobin is highly genotoxic in fish causing severe DNA damage.

Genotoxicity

Azoxystrobin

Micronuclei

Comet Assay

Pethia conchonius

Agriculture plays a crucial role in the Indian economy, employing a significant portion of the population and contributing to the country's food security. However, to meet up the food demand of the ever-increasing population, the high-yielding crop varieties were introduced in agriculture with the parallel use of agrochemicals to protect and increase productions globally including India (Abhilash and Singh 2009). The usage of pesticides to control pest infestations in agriculture has been steadily increasing during the last seven decades (Nayak and Solanki 2021). In India, insect pests are usually controlled by conventional methods, spraying a broad range of insecticides, such as cypermethrin, chlorpyrifos, imidacloprid etc. (Banerjee et al. 2014). Besides insect pests, many pathogenic fungi damage the crops by causing plant diseases, like rust, mildews and blights. The fungicides are a broad class of chemical compounds that are applied to crops to prevent fungal infections (Russel 2005). Herbicides are also in practice to maintain the growth of weeds which interfere with the growth of desired crop plants.

In India, the usage of fungicides corresponds to about 33% of the total pesticide use, compared to 51% and 16% of insecticides and herbicides, respectively (FAO 2018). After application, the pesticides drift to the air due to volatility as well as contaminate water bodies, rivers etc. by surface run-off. The fungicides accumulate in the body of the non-target organisms like fish and other vertebrates including humans through the food web (Maurya and Malik 2016; Gill and Garg, 2014). Repeated exposure to pesticides has been associated with physiological, behavioural and genetic alterations that may cause diseases leading to decreased immunity and decreased predator avoidance ultimately leading to population decline (Bony et al. 2010; Han et al. 2016; Cao et al. 2018). Fungicides are widely used to combat disease-causing fungi in agriculture as well as in aquaculture. So this is a great global health concern including India.

Among the fungicides, Azoxystrobin (AZX) is one of the highly used strobilurin fungicides worldwide. The strobilurins act to inhibit the mitochondrial respiratory system by blocking the electron transfer between cytochrome b and cytochrome c1, thereby disrupting the metabolism and preventing the growth of fungi. (Bartlett et al. 2002; Han et al. 2016). Azoxystrobin has a water solubility of 6 mg/L at 20ºC, (Bartlett et al. 2002), and a half-life of 15–28 days in aquatic environments (Tomlin 2000). Azoxystrobin can accumulate in the river by surface run-off and change the water quality that can adversely affect the non-target organisms including fish and other aquatic vertebrates inhabiting rivers or water bodies close to agricultural fields (Deb et al. 2009; Ochoa-Acuna et al. 2009).

There are several reports which show the presence of AZX along with other pesticides in various water bodies around the world (Calamari et al. 1995; Kishimba et al. 2004; Chakraborty et al. 2016; Ali et al. 2016; Mitra et al. 2019; Nag et al. 2020; Hashmi et al. 2020; and Ramírez-Morales et al. 2021). Azoxystrobin has been detected at concentrations of 0.3 µg/L in streams and above 1 µg/L in water samples from agricultural regions in Sweden (Han et al. 2016). Berenzen et al. (2005) reported the presence of azoxystrobin in a concentration of 29.7 µg/L in a stream in an agricultural environment in Germany, while in another study, the mean concentration of azoxystrobin has been estimated to be 1.43 µg/L in 20 small streams in Germany based on a spatially-explicit model (Schriever et al. 2007). Corcoran et al. (2020) reported 0.01 to 0.06 µg/L AZX in freshwater ecosystems in Argentina. The rivers near a company in Shanghai, China have been shown to be contaminated with 34 mg/L of AZX (Wang et al. 2009). In Hangzhou province (China) the residual AZX detected in the paddy field water were 0.183 mg/L, 0.035 mg/L and 0.022 mg/L after 10, 21 and 28 days of application of 506.25 g/ha of AZX, respectively (Xie and Gong 2013). Azoxystrobin residues in water and soil accumulate in the body of aquatic organisms including fish (Rossi et al. 2020) to toxic levels which are consumed by the species at the top of the food chain, thus affecting the survival and functioning of the organisms.

Genotoxicity is the ability of toxic substances to damage the genetic information of cells. Among different genotoxic tests, Comet Assay or single cell gel electrophoresis (SCGE) is a rapid test for detecting different forms of DNA damage in the nucleated cells. It is widely used to assess genotoxicity in fish and other aquatic species (Mitchelmore and Chipman 1998) and environmental monitoring (Tice 1995b; Tice et al. 2000). It is employed to identify DNA damages, such as single-strand breaks, alkali labile sites, DNA cross-links (Tice 1995a), and excision repair events (Gedic et al. 1992) to establish the correlation between DNA damage and the impact of genotoxic pollutants on aquatic organisms (Lee and Steinert 2003).

The Micronucleus Assay is another simple and sensitive method that is widely used as a tool for detecting genomic alterations in animals (Bolognesi and Hayashi 2011). The micronucleus (MN) test basically involves the assessment of nuclear abnormalities (NA) (da Silva and Fontanetti 2006; Rivero-Wendt et al. 2013) such as micronuclei, blebbed, bilobed, pulverized and notched nuclei as described by Carrasco et al. (1990). The presence of micronuclei and other nuclear abnormalities in the fish erythrocytes can also be used as an important biomarker for monitoring genotoxicity in aquatic environments (Ayllon and Garcia-Vazquez 2000). Several studies tested the genotoxic impact of fungicides using the comet and MN assays in the fish blood cells (Han et al. 2016; Zhu et al. 2015; Zhang et al. 2017, 2018).

Among different aquatic organisms, fishes are highly sensitive to the contamination of water and play a pivotal role as an indicator of change in water quality (Srivastava and Singh 2013). Therefore, it can be used as a suitable experimental organism for genotoxic study. To bring forward the threats faced by fishes due to fungicides exposure, this study investigates the genotoxicological effects in the Rosy barb, Pethia conchonius, a commonly present freshwater fish in India and neighbouring countries of South Asia. This fish has been widely used in the fields of toxicology (Gill et al. 1990), genetics (Varadi et al. 1995), development biology, and behavioural study (Kirankumar et al. 2003; Bhattacharya et al. 2005). In addition, reports showed that Pethia conchonius have a high sensitivity to pesticides (Xiao et al. 2007). Being easy to maintain in the laboratory condition, with high food value and high abundance in this region, Pethia conchonius was, therefore, selected as the experimental fish for this study.

River Teesta is one of the mighty rivers in the northern part of West Bengal, India and flows through Kalimpong, Darjeeling, Jalpaiguri and Cooch Behar. Teesta is likely to be contaminated due to the indiscriminate use of different pesticides and fertilizers in agricultural fields adjacent to the banks of the river (Acharjee 2013). Our preliminary study (unpublished) has shown the presence of fungicides in the water of River Teesta. Despite the intensive use of fungicides and the associated potential ecotoxicological risks in non-target aquatic organisms, the environmental fate and effects of fungicides have received far less attention compared to insecticides and herbicides. Our laboratory has also shown the effects of insecticide (Imidacloprid) on Pethia conchonius from river Teesta (Dutta et al. 2023).

This investigation was therefore aimed to determine the genotoxic effects of the most commonly used fungicide, azoxystrobin on the fish Pethia conchonius of the river Teesta using Micronucleus and Comet Assays.

2.1 Fish Specimens

The adult Pethia conchonius (Rosy barb, Class: Actinopterygii, Order: Cypriniformes, Family: Cyprinidae) (mean length: 6.33 ± 0.51 cm and weight: 3.92 ± 0.40 g) were collected from different sites of river Teesta, Site 1: 1(26.7515°N 88.5856°E) at Gajoldoba Teesta barrage, Jalpaiguri, and Site 2: (26°50’29”N 88°36’29”E) near Odlabari, West Bengal, India). Live fish specimens were transported to the laboratory in a well-aerated condition and shifted to 20 L glass aquaria (40cm×25cm×25cm) after treatment with 0.05% potassium permanganate (KMnO₄) solution (0.05%) for 2 min for external decontamination. The fish were acclimatized for 15 days at room temperature (25 ± 2 ^oC) as per the OECD (The Organization for Economic Co-operation and Development), 2019 guidelines and fed with commercial fish food twice a day until the beginning of the experiments. The residues of the food and faecal matters were removed daily to prevent the accumulation of residual food and waste materials.

2.2 Analytical Quantification of Azoxystrobin in Test-water by LCMS/MS

The actual azoxystrobin concentrations in the exposure media were measured by liquid chromatography-tandem mass spectrometry (LCMS/MS). The water samples were extracted by AOAC Official Method 2007.01 by acetonitrile extraction and partitioning with magnesium sulphate.

2.3 Physico-chemical parameters

The physicochemical parameters of test water, like temperature, pH, total alkalinity, total hardness and dissolved oxygen were measured using the method of APHA (2017).

2.4 Acute Toxicity Bioassay

The acute toxicity test was conducted in the semi-static system in the laboratory according to Test guideline No. 203 of OECD (The Organization for Economic Co-operation and Development, guideline 2019). After acclimatization, the fishes were randomly divided into 8 glass aquaria having 20 litres of water each containing 7 (n = 7) fishes of similar size and weight. The aquaria were then exposed to eight different concentrations of AZX ranging from 0.05-5 mg/L for range finding and definitive tests. Five separate experimental concentrations of AZX i.e., 0.25mg/L, 0.6mg/L, 0.75mg/L, 1mg/L and 2mg/L were selected by a preliminary range-finding experiment. The exposure time of fish to AZX was 96 h, without adding any food. Mortality was recorded at 24, 48, 72, and 96 h of exposure, and the dead fishes were removed regularly from the experimental aquaria. All the experiments along with the control were performed with three replicates. No mortality was observed during the experimental period in the control groups. The LC₅₀ value at 96 h was estimated from the data obtained in acute toxicity bioassays, using probit analysis (Finney 1971).

2.5 Experimental Design

Based on the 96h LC₅₀ value three sub-lethal concentrations (SLC) namely, SLC I = 1/20th of LC₅₀, SLC II = 1/10th of LC₅₀ and SLC III = 1/5th of LC₅₀ were determined to evaluate the toxic effects of azoxystrobin in the fish. Twelve healthy fish (n = 12) from the stock were kept on fasting for 24 hours and introduced into each aquarium of 20 L of water capacity. The fish were exposed to sub-lethal concentrations for 96 hours. Fish kept in de-chlorinated pesticide-free water were taken as control. For each concentration, three replicates were maintained. Three (n = 3) out of twelve fishes were sampled randomly at 24 h, 48 h, 72 h and 96 h intervals for Micronucleus and Comet assays. A graphical presentation of the study has been presented in Fig. 1. No mortality was observed during this study. Animal handling and sample collection were carried out according to the protocols approved by the Institutional Animal Ethics Committee of the University of North Bengal vide ref No. IAEC/NBU/2022/31 dated 23.09.2022 (Proposal No. 13).

2.6 Micronucleus Assay

For the quantification of micronuclei and other nuclear abnormalities, three slides per fish were prepared with peripheral erythrocytes (whole blood). Blood samples were obtained from the caudal vein of the specimens by heparinized 1ml syringe and a thin smear was drawn. The slides were fixed in methanol for 20 minutes and air-dried overnight. The slides were then stained with 5% Geimsa for 15 minutes and mounted with DPX. Three slides were prepared for each fish, and a total of 3000 erythrocytes per slide i.e., a total of 9000 erythrocytes per fish were examined using a Nikon Eclipse E200 microscope, under a 100X oil immersion objective with 1000x magnification. All slides were randomly coded by another person not involved in the experiment and scored blindly by a single observer. Only the intact cell with nuclear membranes was scored. The MN test was carried out following the method of Beninca (2006). The frequency of MN and the other nuclear abnormalities (NA) were calculated as follows:

$$\text{\%}\text{M}\text{N} \text{o}\text{r} \text{N}\text{A}= \frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l}\text{s} \text{c}\text{o}\text{n}\text{t}\text{a}\text{i}\text{n}\text{i}\text{n}\text{g} \text{m}\text{i}\text{c}\text{r}\text{o}\text{n}\text{u}\text{c}\text{l}\text{e}\text{i}}{\text{T}\text{o}\text{t}\text{a}\text{l} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l}\text{s} \text{c}\text{o}\text{u}\text{n}\text{t}\text{e}\text{d}} \text{X} 100$$

The Nuclear Abnormalities (NA) were classified into two categories: blebbed nuclei (BL), and notched nuclei (NO) according to the criteria of Carrasco et al. (1990).

2.7 Comet Assay

The comet assay or single-cell gel electrophoresis (SCGE), was conducted to detect the DNA strand breaks in the Pethia conchonius according to the methods of Singh et al. (1988) with slight modifications.

Gills were dissected out and homogenized in cold homogenizing buffer (1X Hanks’ balanced salt solution (HBSS), 20mM EDTA, 10% dimethyl sulfoxide (DMSO), pH 7.0–7.5) using micro-pestle. The homogenate was then centrifuged at 3000 rpm for 5 minutes and the supernatant was collected for further steps. Fully frosted microscopic slides were coated with 50ul of 1% normal melting-point agarose and allowed to gel at 4°C for 10 min. To 80ul 0.5% low-melting agarose, 20ul supernatant was mixed. This was layered onto the slides coated with 200ul of 1% normal-melting agarose prepared in PBS and spread with a coverslip. After solidification and removal of the coverslip, the third layer of 100ul of 0.5% low-melting agarose was layered onto the slides, covered with a coverslip and allowed to gel at 4°C for 10 min. The coverslips were removed and the slides were immersed in freshly prepared 50ml cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 1% Triton X-100, 10% DMSO, pH 10.0) and kept at 4°C for 1 h. Then the slides were placed in the alkaline buffer (300 mM NaOH, 1 mM EDTA, pH ≥ 13) for 20 min to allow DNA unwinding. Electrophoresis was performed for 25 min at 18 V/m adjusted to 300 mA in the same buffer. After electrophoresis, slides were washed thrice for 5 min each in fresh neutralization buffer (0.4 M Tris-HCl, pH 7.5), dehydrated with absolute methanol for 10 min, and dried at 50ºC for 30 min in an incubator. Slides were stained with 50ul of ethidium bromide (EtBr) (10 µg/ml) and examined under a fluorescence microscope at 40x magnification. Two slides were prepared from each sample and 50 cells per slide were scored randomly, and examined under a fluorescent microscope (Nikon Eclipse E200, Nikon). The comet images were analyzed using the (Casp lab 1.2.3 b2) software. The DNA damage was quantified as %Head DNA, % Tail DNA, Tail length and Olive Tail Moment (OTM) which indicated the cellular damage (Imanikia et al. 2016).

2.8 Statistical Analysis

Both the independent variable i.e., the concentration of azoxystrobin (AZX) and duration of exposure (24–96 h) were considered in combination to compare the responses of dependent variables. Shapiro-Wilk normality test revealed that the distribution pattern of dependent variables like %Head DNA, %Tail DNA, Tail length, OTM, Micronuclei, Blebbed and Notched nuclei percentage among the groups were significantly different from the normal distribution (p < 0.05). Therefore, Kruskal-Wallis non-parametric test followed by Dunn’s Post hoc test was performed. To compare the mean values of different concentrations of AZX exposed groups with the control groups for each duration of exposure by using the Mann-Whitney U test. Levene’s test for homogeneity of variances showed that the data were heteroscedastic (p < 0.05) for all the parameters. Therefore, all the dependent variables (DNA damage parameters and nuclear abnormalities) and the covariate (duration of exposure) were transformed into mean rank to perform non-parametric Quade’s ANCOVA considering different concentrations of AZX as fixed variables after adjusting the duration of exposure.

2.9 Chemicals and Reagents

For the present study, the technical grade azoxystrobin (23% SC) (CAS no. C22H17N3O5) with the product name ‘Amistar’, (manufactured by Syngenta India Limited, India) was procured from Kurseong Medical Store (Siliguri, West Bengal, India). Stock solutions of azoxystrobin were prepared by dissolving in distilled water. All other chemicals were of analytical grade.

3.1 Physico-chemical parameters of the test water

The temperature and the pH of the water varied from 25º±2ºC and 7.2 to 8.1, respectively. The dissolved oxygen concentration was 7.2 to 8.4 mg/L during the experimental period. The total hardness and total alkalinity ranged from 52 to 88 mg/L and 260 to 290 mg/L as CaCO₃, respectively.

3.2 Analytical Quantification of azoxystrobin

The result of the analytical quantification showed that out of 0.103 mg/L AZX, the residual of AZX concentration present was 0.086 mg/L. It indicated a degradation of 12.8% after 96 hours of exposure.

3.3 Acute Toxicity Bioassay

The 96h LC₅₀ value (with 95% confidence limits) of Azoxystrobin for Pethia conchonius was estimated to be 0.514 mg/L (0.292 to 0.687 mg/L) using SPSS by probit analysis (Finney 1971).

Based on the 96h LC₅₀ value, three sub-lethal concentrations of azoxystrobin namely SLC I (1/20th of LC₅₀ = 0.025mg/L), SLC II (1/10th of LC₅₀ = 0.0514 mg/L) and SLC III (1/5th of LC₅₀ = 0.103mg/L) were calculated for genotoxicity assays.

3.4 Micronucleus Assay

The frequency of micronuclei (MN) and other nuclear abnormalities like notched (NO) and blebbed (BL) nuclei in Pethia conchonius exposed to different concentrations of AZX as well as control groups have been shown in Table 1 and Fig. 4. The frequency (%) of MN, NO, BL were higher in the AZX-exposed groups than the control. Control groups showed no significant difference with increasing duration, whereas the AZX-exposed groups showed a significant increase in the frequency of MN and other nuclear abnormalities with increasing duration of exposure (P < 0.001). The lowest frequency of MN, NO and BL were 0.289 ± 0.011%, 0.222 ± 0.011% and 0.178 ± 0.011%, respectively for SLC I at 24 hours of exposure. Similarly, the MN, NO and BL were 0.356 ± 0.011, 0.256 ± 0.011 and 0.211 ± 0.011 for SLC II and 0.511 ± 0.011, 0.322 ± 0.011 and 0.256 ± 0.011 for SLC III, respectively at 24h of exposure. The highest percentage of MN, NO and BL were found for all the sub-lethal concentrations of AZX at 96 hours of exposure showing an increase in nuclear damage in a dose and time-dependent manner (Table 1). The frequencies of MN, NO and BL were 0.889 ± 0.011, 0.522 ± 0.011^, 0.578 ± 0.019 for SLC I, 0.956 ± 0.022, 0.644 ± 0.011, 0.678 ± 0.011 for SLC II and 1.156 ± 0.011, 0.889 ± 0.011, 0.811 ± 0.011 for SLC III, respectively at 96 h of exposure.

The mean values of each parameter within the control groups at different intervals did not differ significantly forming a single homogenous subset ‘a’ (p > 0.05) (Table 1, Fig. 4). The values belonging to identical groups of homogeneous subsets under exposure to the different concentrations were not significantly different from each other. The increasing order of homogeneous subsets for all parameters showed the lowest damage at 24 hours and highest at 96 hours indicating higher nuclear damage with increasing sub-lethal concentration and duration of exposure (Table 1).

Table 1

Frequency of different nuclear abnormalities in erythrocytes of *Pethia conchonius* exposed to different concentrations of Azoxystrobin (Mean ± SE).
Exposure Duration (hours)	Concentration
Exposure Duration (hours)	Control	SLC I	SLC II	SLC III
Micronucleus%
24 h	0.089 ± 0.011^a	0.289 ± 0.011^b*	0.356 ± 0.011^bc*	0.511 ± 0.011^c*
48 h	0.100 ± 0.000^a	0.444 ± 0.011^c*	0.544 ± 0.011^cd*	0.633 ± 0.019^de*
72 h	0.078 ± 0.011^a	0.700 ± 0.019^de*	0.778 ± 0.011^ef*	0.956 ± 0.011^fg*
96 h	0.089 ± 0.011^a	0.889 ± 0.011^fg*	0.956 ± 0.022^g*	1.156 ± 0.011^h*
Notched%
24 h	0.056 ± 0.011^a	0.222 ± 0.011^ab*	0.256 ± 0.011^bc*	0.322 ± 0.011^d*
48 h	0.056 ± 0.011^a	0.289 ± 0.011^cd*	0.322 ± 0.011^d*	0.422 ± 0.011^e*
72 h	0.056 ± 0.011^a	0.378 ± 0.011^e*	0.478 ± 0.011^f*	0.611 ± 0.011^g*
96 h	0.056 ± 0.011^a	0.522 ± 0.011^f*	0.644 ± 0.011^g*	0.889 ± 0.011^h*
Blebbed%
24 h	0.044 ± 0.011^a	0.178 ± 0.011^ab*	0.211 ± 0.011^bc*	0.256 ± 0.011^c*
48 h	0.044 ± 0.011^a	0.256 ± 0.011^c*	0.311 ± 0.011^d*	0.356 ± 0.011^de*
72 h	0.044 ± 0.011^a	0.389 ± 0.011^ef*	0.422 ± 0.011^f*	0.522 ± 0.011^g*
96 h	0.044 ± 0.011^a	0.578 ± 0.011^h*	0.678 ± 0.011^i*	0.811 ± 0.011^j*
Different lowercase alphabets superscript indicates values with different homogeneous subsets significantly differ at (p < 0.05) obtained by the Kruskal-Wallis test followed by Dunn’s post-hoc analysis. Asterisk () indicates the significant pairwise difference in values of different sub-lethal concentrations with the control group within exposure durations (=p < 0.001) by the Man-Whitney U test. SLC: Sub-lethal Concentration.

Different lowercase alphabets superscript indicates values with different homogeneous subsets significantly differ at (p < 0.05) obtained from the Kruskal-Wallis test followed by Dunn’s posthoc analysis.

Asterisk (*) indicates the significant pairwise difference in values of different sub-lethal concentrations with the control group within exposure durations (* =p < 0.001) by the Mann-Whitney U test.

SLC: Sub-lethal Concentration.

3.5 Comet Assay

The fish specimens exposed to different sub-lethal concentrations of AZX exhibited significantly higher DNA damage (P < 0.001) in terms of %Head DNA, %Tail DNA, tail length and Olive Tail Moment (OTM) in the gill tissues than the control samples (Table 2, Fig. 6). No significant differences were found in the control groups with increasing duration. A significant increase in DNA damage was observed for different duration of exposure (P < 0.001) in the fish specimen exposed to AZX.

The highest %Head DNA observed at 24h of exposure for all the sub-lethal concentrations I, II and III were 94.275 ± 1.020, 88.733 ± 1.713 and 61.689 ± 1.982 respectively. There was a gradual non-linear decrease in the %Head DNA. The lowest %Head DNA observed for SLC I, SLC II and SLC III were 45.685 ± 3.469, 33.101 ± 2.950 and 26.288 ± 1.981 at 96 h of exposure, respectively, which indicated the highest DNA damage.

The lowest %Tail DNA was observed in the AZX-treated Pethia conchonius at 24h of exposure. The lowest %Tail DNA were 5.725 ± 1.020, 11.267 ± 1.713 and 38.311 ± 1.982, respectively for SLC I, SLC II and SLC III, while the highest %Tail DNA were 54.315 ± 3.469, 66.899 ± 2.950 and 73.712 ± 1.981, respectively for 9h h of exposure (Table 2).

Similar results have also been observed in the case of Tail Length and OTM. The frequency of tail length and OTM were 17.560 ± 1.205, 34.200 ± 2.707, 82.188 ± 4.110 and 1.890 ± 0.341, 6.703 ± 1.431, 21.431 ± 1.713, respectively, for SLC I, SLC II and SLC III at 24 h of exposure, while both the parameters of DNA damage were 176.780 ± 5.747, 205.340 ± 9.204, 287.760 ± 11.641 and 34.555 ± 2.228, 44.827 ± 3.070, 71.246 ± 4.523, respectively for SLC I, SLC II and SLC III at 96 h of exposure showing a gradual increase in tail length and OTM exhibiting higher DNA damage with the increasing duration of exposure (Table 2 Fig. 6)

The mean values of each parameter within the control groups at different intervals did not differ significantly forming a single homogenous subset ‘a’ (p > 0.05) (Table 2, Fig. 6) as observed in the nuclear damage parameters. The values belonging to the identical groups of homogeneous subsets under exposure to different concentrations were not significantly different from each other. The increasing order of homogeneous subsets for all parameters showed the lowest damage at 24 h and highest at 96 h indicating higher DNA damage with increasing sub-lethal concentration and duration of exposure.

Table 2

The %Head DNA, %Tail DNA, Tail Length and OTM in both control and AZX exposed *Pethia conchonius* for different sub-lethal concentrations and duration (Mean ± SE).
Exposure Duration (hours)	Concentration
Exposure Duration (hours)	Control	SLC I	SLC II	SLC III
% Head DNA
24 h	99.659 ± 0.119^a	94.275 ± 1.020^b*	88.733 ± 1.713^c*	61.689 ± 1.982^f*
48 h	99.239 ± 0.275^a	78.669 ± 2.264^d*	70.786 ± 2.072^e*	57.095 ± 2.903^f*
72 h	99.678 ± 0.092^a	59.459 ± 2.281^f*	50.241 ± 2.595^g*	44.381 ± 2.598^g*
96 h	99.342 ± 0.233^a	45.685 ± 3.469^g*	33.101 ± 2.950^h*	26.288 ± 1.981^h*
% Tail DNA
24 h	0.341 ± 0.119^a	5.725 ± 1.020^b*	11.267 ± 1.713^c*	38.311 ± 1.982^f*
48 h	0.761 ± 0.275^a	21.331 ± 2.264^d*	29.214 ± 2.072^e*	42.905 ± 2.903^f*
72 h	0.322 ± 0.092^a	40.541 ± 2.281^f*	49.759 ± 2.595^g*	55.619 ± 2.598^g*
96 h	0.658 ± 0.233^a	54.315 ± 3.469^g*	66.899 ± 2.950^h*	73.712 ± 1.981^h*
Tail Length
24 h	3.306 ± 0.110^a	17.560 ± 1.205^b*	34.200 ± 2.707^c*	82.188 ± 4.110^f*
48 h	3.440 ± 0.172^a	59.240 ± 2.509^d*	73.520 ± 3.552^e*	148.857 ± 6.592^h*
72 h	3.440 ± 0.125^a	123.780 ± 3.596^g*	134.680 ± 4.323^gh*	186.920 ± 8.900^i*
96 h	3.574 ± 0.117^a	176.780 ± 5.747^i*	205.340 ± 9.204^i*	287.760 ± 11.641^j*
OTM
24 h	0.033 ± 0.014^a	1.890 ± 0.341^b*	6.703 ± 1.431^c*	21.431 ± 1.713^e*
48 h	0.079 ± 0.030^a	11.448 ± 1.290^d*	19.514 ± 2.055^e*	46.336 ± 4.109^g*
72 h	0.056 ± 0.018^a	34.555 ± 2.228^f*	44.827 ± 3.070^g*	71.246 ± 4.523^h*
96 h	0.011 ± 0.003^a	50.707 ± 4.374^g*	75.127 ± 5.846^h*	116.301 ± 7.636^i*
Different lowercase alphabets superscript indicates values with different homogeneous subsets significantly differ at (p < 0.05) obtained by the Kruskal-Wallis test followed by Dunn’s post-hoc analysis. Asterisk () indicates the significant pairwise difference in values of different sub-lethal concentrations with the control group within exposure durations (=p < 0.001) by the Man-Whitney U test SLC: Sub-lethal Concentration.

Asterisk (*) indicates significant pairwise difference in values of different sub-lethal concentrations with the control group within exposure durations (* =p < 0.001) by the Mann-Whitney U test.

SLC: Sub-lethal Concentration.

3.6 Quade’s ANCOVA

According to Quade’s ANCOVA analyses, significant differences were found in the DNA damage parameters (% Head DNA, % Tail DNA, tail length, OTM) and nuclear abnormalities (%MN, %NO, %BL) among different sub-lethal concentrations of AZX adjusted with the duration of exposure. The Partial Eta squares (R²) (% variance) represent the % variation of DNA damage parameters and nuclear abnormalities (effect size) that can be explained by the effects of different sub-lethal concentrations of AZX.

Table 3

Quade’s ANCOVA analysis of DNA damage parameters and nuclear abnormalities adjusted with the duration of exposure.
Independent variable	F value	df	P value	R²	Adjusted R²
DNA damage parameters
% head DNA	629.754	3	< 0.001	0.705	0.704
% tail DNA	629.783	3	< 0.001	0.705	0.704
tail length (L tail)	1073.414	3	< 0.001	0.803	0.802
OTM	801.560	3	< 0.001	0.753	0.752
Nuclear Abnormalities
% Micronuclei	87.610	3	< 0.001	0.857	0.847
% Notched nuclei	89.584	3	< 0.001	0.859	0.850
% Blebbed nuclei	76.871	3	< 0.001	0.848	0.829

df: degree of freedom.

A large number of studies indicate that fungicides are highly toxic not only to fish but also to the other organisms which constitute the major component of food chain (Taxvig et al. 2008; Capriglione et al. 2011; Schmidt et al. 2016; De la Paz et al. 2017). Long-term exposure to fungicides induces physiological, behavioural, haematological, and histopathological alterations, biochemical changes, immune suppression, hormone disruption, reproductive abnormalities and cancers (Pinho et al. 2013; Gao et al. 2014; Du et al. 2018; Souders II et al. 2018; Cao et al. 2018; Li et al. 2019; Kumar et al. 2020). AZX is one of the widely used fungicides for the prevention of fungal infections in agricultural plants. It affects not only target organisms but also the non-target organisms including human beings, a higher level consumer in food chain. After application, fungicides can accumulate in the water body by surface run-off and can cause genotoxic effects on aquatic organisms by DNA damage interacting with DNA (Li et al. 2018; Wang et al. 2018; Jiang et al. 2018, 2019a, 2019b). Comet and MN assays are widely used techniques in genotoxicity-studies to detect the level of genomic damages. These assays alongside other physiological, biochemical, and histopathological assessments can provide a more comprehensive understanding of the overall toxicological impact of fungicides on fish.

4.1 Acute Toxicity Bioassay

The acute toxicity bioassay or the LC₅₀ value offers a quantitative measure of toxicity allowing researchers to compare the relative toxicity of different substances. It helps in the safety evaluation of chemicals and assesses the potential impact of these chemicals on the ecosystem. By measuring the sensitivity of various organisms to a specific substance researchers can evaluate the potential ecological risks and design appropriate strategies for environmental protection (Arome and Chinedu 2013). Results from acute toxicity tests serve as a guide in dose selection for long-term toxicity studies as well as other studies that involve the use of animals (Maheshwari & Shaikh 2016).

In the present study, the LC₅₀ value of AZX, estimated to be 0.514 mg/L at 96 hours in Pethia conchonius is within the range as shown by other workers. Several studies had been conducted on the acute toxicity of AZX in fish. Liu et al. (2013) determined the 48h LC₅₀ value of azoxystrobin in the grass carp (Ctenopharyngodon idella) juveniles to be 0.549 mg/L. Jiang et al. (2018) reported the 96h LC₅₀ values of azoxystrobin in the developmental stages of zebrafish as 0.777 mg/L, 0.810 mg/L, 0.987 mg/L and 1.08 mg/L in larval, embryonic, juvenile and adult forms, respectively while, Mu et al. (2017) have demonstrated the 96h acute toxicity of azoxystrobin in the three developmental phases of zebrafish were 0.39 mg/L, 0.61 mg/L and 1.37 mg/L, respectively in the larvae, embryos and adults, which are lower than the findings of Jiang et al. (2018) except adult stage.

The effect of AZX has also been studied in non-chordates. The 96 h LC₅₀ value of 0.31 mg/L was reported in Daphnia magna (Ochoa-Acuna et al. 2009). Ali et al. (2021) determined the mean LC₅₀ value at 96 h of AZX exposure in Lymnea luteola, which was 0.79 mg/L in a static system. Interestingly a very high 96 h LC₅₀ value of azoxystrobin as 1656 mg/L was reported in the crayfish (Astacus leptodactylus) (Uckun and Oz 2021). The differences in the LC₅₀ value of azoxystrobin in different organisms as cited above varies according to the application period and the life stage of the organisms and also with other factors.

4.2 Micronucleus Assay

The present study showed the induction of MN and other nuclear abnormalities in peripheral erythrocytes of Pethia conchonius when exposed to different sub-lethal concentrations of the fungicide, azoxystrobin (AZX). The lowest MN, NO and BL frequencies were 0.289 ± 0.011, 0.178 ± 0.011 and 0.144 ± 0.011, respectively for SLC I at 24h of exposure while, the highest MN frequency of 1.156 ± 0.011 was found at 96 h of AZX exposure for SLC III. Similar results were also found for the NO (0.889 ± 0.011) and BL (0.881 ± 0.011) nuclei showing a concentration and time-dependent increase in nuclear damage (Table 1). The frequency of MN, NO and BL is in descending order from MN > NO > BL for almost all the sub-lethal concentrations and exposure duration. Compared to the control the percentage of MN increased by 10–13 fold for SLC I to SLC III, respectively. Similarly, the percentages of NO and BL increased by 9–15 and 13–18 fold, respectively. Han et al. (2016) reported similar results showing an increase in nuclear damage with increasing dose and duration of exposure of AZX in the liver tissue of Danio rerio at all concentrations tested. Crupkin et al. (2021) reported increased MN and Notched nucleus in the erythrocytes of both the juvenile and adult Australoheros facetus at 50µg/L concentration of AZX. In one study, Shahi and Singh (2014) showed a higher MN induction after exposure to the fungicide Mancozeb (dithiocarbamate) on the fish Clarias batracus. Shrivastava and Singh (2015) reported significantly higher frequencies of MN in the fish Clarias batracus exposed to Propiconazole fungicide (1.11 and 2.23 mg/L) than the control fish throughout all treatment periods (p < 0.05). Several other studies have also shown that the occurrence rate of micronucleated erythrocytes in different fish species varies from 0 to 1.75% (Bolognesi et al. 2006; Xia et al. 2016; Iturburu et al. 2017).

Comparatively higher MN, NO, and BL induction at SLC III at 96 h of exposure in the present study may be due to the high production of reactive oxygen species (ROS) and oxidative stress (Shen and Liu 2006). It has also been suggested that abnormal cell division due to the blocking of cytokinesis and the dysfunction of the mitotic spindle or breaks in DNA strands can cause nuclear abnormalities that may lead to a genetic imbalance in RBCs (Rodilla 1993; Alimba et al. 2017).

According to Kirsch-Volders et al. (2006), MN assay is a multi-endpoint test of genotoxic responses to clastogens. The frequency of micronuclei (MN) is extensively used as a biomarker of genomic stability (Norappa and Falak 2003). Formation of these abnormalities in fish erythrocytes indicates that AZX has high genotoxic potency and it is very harmful to the aquatic ecosystem.

4.3 Comet Assay

The comet assay is a widely used rapid technique in genetic toxicology and environmental monitoring. It can be used on a small number of cells and is compatible with different tissues, making it a versatile tool for a wide range of applications, including biomonitoring, genotoxicity testing, and mechanistic studies (Tice et al. 2000; Wood et al. 2015).

In the present study, the observed DNA damage in the Pethia conchonius at different sub-lethal concentrations (0.103mg/L, 0.0514mg/L, and 0.025mg/L) was significantly higher than the controls. The % Head DNA decreased significantly with increasing dose and duration of exposure (P < 0.001), whereas the increasing % Tail DNA, Tail Length and OTM showed increased DNA damage in a similar manner indicating the genotoxic nature of AZX in fish. Compared to the control the % Head DNA decreases in an ascending order from 45–74% for each sub-lethal concentration at 96 hours, respectively (Table 2). The observed increase of % Tail DNA, tail length and OTM corresponds to the increased DNA damage as found in %Head DNA in Pethia conchonius. The induction of DNA damage as observed in the present study is in accordance with the findings of Crupkin et al. (2021), who showed DNA damage at 0.05 mg/L AZX for acute exposure to Australoheros facetus. Han et al. (2016) detected DNA damage in terms of OTM in liver cells of zebrafish both males and females exposed to 10 µg/L AZX at day 7 of exposure, whereas Bony et al. (2010) observed increased %Tail DNA in liver and sperm cell of Danio rerio by Comet Assay when exposed for 7 days at 0.48 µg/L AZX. In one survey, Bony et al. (2008) examined the genotoxic pressure of AZX in fish and documented increased level of DNA damage in terms of %Tail DNA by comet assay in early life stages of brown trout (Salmo trutta fario) collected from a river contaminated with AZX. A 3–5-fold increase in the DNA damage in European topminnow (Phoxinus phoxinus) exposed to an environmentally realistic concentration of a mixture of diuron and AZX in the laboratory was reported by Bony et al. (2008). Ali et al. (2021) showed a dose and time-dependent increase in DNA damage in terms of the percentage of tail DNA and olive tail moment in the digestive gland cells of the snail Lymnea luteola.

The DNA damage detected in this study may have resulted from DNA single-strand breaks, double strands break, DNA adduct formations, DNA–DNA and DNA–protein cross-links (Mitchelmore and Chipman 1998) due to the interaction of toxic substances or their metabolites with DNA (Fairbairn et al. 1995). Genotoxic compounds may cause strand breaks directly through apoptosis or necrosis, or through the interaction with oxygen radicals as a consequence of excision repair enzymes (Eastman and Barry 1992; Speit and Hartmann 1995; Lee and Steinert 2003).

Significantly higher %Head DNA, %Tail DNA, tail length, OTM and the induction of MN and other nuclear abnormalities in the AZX exposed groups than the control indicated the high genotoxic nature of AZX. The frequency of micronuclei and other nuclear abnormalities and DNA damage showed a dose and time-dependent pattern. The results highlight the significance of utilizing the micronucleus (MN) and Comet assay as effective biomarkers for assessing exposure to clastogenic pollutants in aquatic environments. Therefore, the observed high DNA damage indicates that AZX is a potential genotoxic fungicide in the fish Pethia conchonius raising significant concerns about its potential hazards to aquatic organisms and its application in agricultural practices.

Statements & Declarations

Funding

This work was partially supported by the Council of Scientific Research-Human Resource Development Group (CSIR-HRDG Vide File No. 09/285(0095)/2019-EMR-I) as fellowship to the scholar.

Conflict of Interests

The authors declare that there is no conflict of interest regarding the publication of this paper.

Ethical permission

The present investigation has been approved by the Institutional Animal Ethics Committee of the University of North Bengal vide ref No. IAEC/NBU/2022/30 dated 23.09.2022 (Proposal No. 13).

Author’s contribution

The original research was conducted by Arpita Ray and Debojit Dutta. Statistical analysis was conducted by Arpita Ray and Bappaditya Ghosh. The manuscript was written by Arpita Ray. Min Bahadur guided the research, edited and reviewed the manuscripts as supervisor of the Genetics and Molecular Biology Laboratory, Department of Zoology, University of North Bengal. The authors have no financial or non-financial interests to disclose.

Data Availability Statement

The data that supports the findings are available from the corresponding author upon reasonable request.

Acknowledgement: Authors are thankful to the Head, Department of Zoology, University of North Bengal for providing the Departmental Central Instrument Facility under the support of the Fund for Improvement of Science and Technology Infrastructure Program (FIST), Department of Science and Technology, New Delhi, India, and the Council of Scientific and Industrial Research, New Delhi, for providing fellowship to first Author. The authors are also grateful to the people who supported this research.

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Azoxystrobin induced genotoxicity in Pethia conchonius, a freshwater fish of river Teesta, India

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Abstract

Purpose

Methods

Results

Conclusion

Figures

1 Introduction

2 Materials and Methods

2.1 Fish Specimens

2.2 Analytical Quantification of Azoxystrobin in Test-water by LCMS/MS

2.3 Physico-chemical parameters

2.4 Acute Toxicity Bioassay

2.5 Experimental Design

2.6 Micronucleus Assay

2.7 Comet Assay

2.8 Statistical Analysis

2.9 Chemicals and Reagents

3 Results

3.1 Physico-chemical parameters of the test water

3.2 Analytical Quantification of azoxystrobin

3.3 Acute Toxicity Bioassay

3.4 Micronucleus Assay

3.5 Comet Assay

3.6 Quade’s ANCOVA

4 Discussion

4.1 Acute Toxicity Bioassay

4.2 Micronucleus Assay

4.3 Comet Assay

5 Conclusion

Declarations

References

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