Zoology Department, Lumbding College, Lumbding, Assam, India.

Corresponding author email: jashodeb@gmail.com

Article Publishing History

INTRODUCTION

Water pollution day by day has become a global matter of concern. Industrial, agricultural and domestic discharges find their ultimate recipient source as the water bodies leading to pollution in the aquatic environment, which in last few decades in researches have been reported to become a threat to aquatic ecosystem resulting in water quality damage and severe chromosomal abnormalities in aquatic organisms specially in fishes (Porto et al. 2005; Yadav et al. 2010).

Thus, Safety of aquatic environment is a major task as water resources covers two third of the globe and living organisms fully depend on it for their livelihood including humans (Krishnamurthi et al. 2003). The country administration has been continuously looking after this serious issue of cleanliness and safety of all water bodies starting from river Ganga upto the smallest stream or pond on which living organisms depends for their livelihood. Because in researches it has come to notice that people are not concerned for the cleanliness of such water bodies on which they actually depend (Ismail et al. 2014).

As a result, not only the water quality is degrading very fast but also the aquatic ecosystem is in danger including both its flora and fauna (Kohlpoth et al. 2009). Among all of the aquatic organisms, fish have been considered as an important species as it covers the majority of the aquatic fauna and it has been consumed by living organisms especially human beings for their survival (Minovski et al. 2019). So, to test the level of genotoxicity induced by industrial effluents in aquatic organisms, fish can accepted as a good test model. Simultaneously, fish have been successfully used in cytogenetic analysis, being easy to handle and adaptable with the laboratory environment, provides a relatively low-cost method (Hayashi et al. 1997; Singh et al. 2020).

Micronuclei (MN) were first described in the cytoplasm  of erythrocytes more than a century ago and were called “fragment of nuclear material” by Howell or “intraglobu- laries corpuscules” in the terminology of Jolly in the late.

laries corpuscules” in the terminology of Jolly in the latMicronuclei (MN) were first described in the cytoplasm of erythrocytes a century ago and were called ‘ fragments of nuclear material’ by Howell or ‘ Intraglobularies corpuscules ‘ in the terminology of Jolly in the late 18^th century and early 1900. Hematologisits called these structures as ‘Howell Jolly Bodies’ (Kirsch –Volders et al.., 2003)

Micronucleus assay has been successfully used as a mutagenic assay to test the induced genotoxicty in various organisms as fishes.  Among the various mutagen tests preferred for bio-monitoring contaminated environments, micronuclei assay (MN) proved relatively simple, reliable and sensitive, and has been used to evaluate the effects of mutagen compounds induced by industrial pollution and domestic discharges in aquatic ecosystem (Al-Sabti and Metcalfe 1995). The use of fish erythrocytes to test the level of toxicity leads to quick results with less chances of suffering of the test organism selected (Minissi et al. 1995; Zúñiga-González et al. 2000; Venier and Zampieron 2005; Singh et al. 2020).

Micronuclei are cytoplasmic chromatin masses that look like small nuclei as a result of lesions at the chromosomes or DNA strains, or at the level of proteins directly involved in chromosome segregation; formation of MN originating from chromosome fragments or chromosome loss events requires a miotic or meiotic division (Heddle et al. 1983). Micronucleus is composed either of small chromatin fragments which arise as a result of chromosome breaks after clastogenic action, or of whole chromosomes that do not migrate during anaphase as a result of aneugenic affects (Cavas and Ergene Gozukara 2003).

Our work was conducted on the only paper mill situated in Barak valley region, Assam from where industrial effluents after paper composition have been observed to get liberated in local river Barak without proper treatment. During exposure a corrosive smell was also found to get spread in the neighboring atmosphere which can be easily felt by any passer by fellow causing air pollution too. The exposure in river Barak was expected to cause harm not only to water body but also to aquatic life. Before exposure fish were brought to laboratory environment and acclimatized. Fish were divided into groups and exposed to three different concentrations of both Paper Mill Effluent (PME) after proper trial and observation was recorded for three consecutive days (Singh et al. 2020).

Besides the average length of the fish, body weight and survivality rate were measured in three replicates. Erythrocyte smears were obtained with heparinized syringes by puncturing the gills on previously washed microscopic slides. The fish remained unharmed and were soon returned to their natural habitat. The slides were air-dried for 24 h, fixed in a 70% methanol solution for 7 min and then dried a further 24 h. Shortly after, they were stained with Giemsa (4%) for 15 min. 3.000 intact erythrocytes were counted from each fish. Only cells that were clearly visible and isolated under a Zeiss microscope with amplification of 1000 X, were counted. Cells with more than four micronuclei were discarded so as to exclude apoptotic phenomena.

Nuclear abnormalities were manifest as changes in the normal elliptic shape of nuclei (Ferraro et al. 2004; Bolognesi et al. 2006). For a detailed description on nuclear abnormalities see previous studies (Ayllón and Garcia-Vazquez 2000; Çavas and Ergene-Gözükara 2003; Çavas et al. 2005). Micronuclei were considered as small inclusions of nuclear material inside erythrocytic cytoplasm. Criteria for identification were a round or oval shape with a flat and well-defined outline, coloration similar to that of the main nucleus and a size from 1/3 to 1/20 in relation to that of the main nucleus (Al-Sabti and Metcalfe 1995; Singh et al. 2020).

MATERIAL AND METHODS           

For the sampling site and preparation of water samples, major objective of this current piece of study was to evaluate the acute toxicity of a paper mill effluent of Barak valley region, Assam on the behavioral responses of a regularly consumed freshwater fish Channa punctatus. Raw effluent sample was collected from the outlet discharge pipes in the local river Barak in plastic containers.  pH was measured on the very collection spot. Effluent sample was brought to laboratory and to prevent further microbial growth was stored at -20^oC. For test model, among various water borne organisms fishes have been considered to be efficient model for studying the induced level of carcinogenesis by industries and domestic affairs.

Researches have reported remarkable dose and time dependent increase in the induction of micronucleus in peripheral blood of fishes (Chaudhury et al. 2006; Ali et al. 2009; Nwani et al. 2010; Saleh and Alshehri; 2011; Pandey et al. 2014). Around 200 fishes of species Channa punctatus were used for the selected bioassays. Fish weighing 15-20 g of weight were selected and before exposure were acclimatized in the laboratory environment for 2-3 weeks. This study was conducted with prior Institutional animal ethics clearance. Fish were given proper treatment and were fed with standard fish food.

For animal exposure and micronucleus assay, after acclimatization fishes were divided into five groups, each group including 40 fishes per aquaria were exposed to three selected concentrations (10%, .25% and 50%, v/v, effluent/distilled water) of the paper mill effluent against negative (distilled water) and positive (Mitomycin C, 2mg/lit) controls for 24, 48 and 72 hrs. 5 fishes per group of treatment were selected for micronucleus assay and blood erythrocyte smears were obtained.

Three slides per sample were prepared. 3000 erythrocytes were counted and scored by a single scorer to eliminate inter-observer variation Using a light-microscope (Leica DMLS) at 1000 magnification where criteria for identification were a round or oval shape with a flat and well-defined outline, coloration similar to that of the main nucleus and a size from 1/3 to 1/20 in relation to that of the main nucleus (Al-Sabti and Matcalfe 1995).

For scoring criteria for micronuclei, following criteria for identification were used (Al-Sabti and Matcalfe 1995):-

• MN must be smaller than one third of the main nuclei,
• MN must be clearly separated from the main nuclei,
• MN must be on the same plane of focus and have same color as of the original nuclei.

For statistical analysis, the data obtained were presented as Mean±SE. Data was analyzed using One Way ANOVA and were expressed as percentage frequency for MN test. Significance at different dose levels Were studied by using Graph Pad Prism Software (Graph Pad Inc., san Diego, CA, USA).

Results AND DISCUSSION

Table 1.  Body weight variation in Channa punctatus due to Paper Mill Effluent exposure.

Dose (mg/kg)	Concentrations	Initial	24 hrs	48 hrs	72 hrs
Control	—–	17.5±0.22	17.52±0.22	17.6±0.23	17.48±0.21
Positive control	2mg/lit	17.88±0.19	17.8±0.19	17.8±0.28	17.7±0.28
Paper Mill Effluent (PME)	10%	17.1±0.31	17.08±0.30	17.02±0.30	17.0±0.31
	25%	16.90±0.16	17.0±0.16	16.75±0.23	16.67±0.24
	50%	17.7±0.11	17.66±0.12	17.42±0.16	17.50±0.18

Figure 1: Body weight Variation in Channa punctatus due to Paper Mill Effluent exposure.

Table 2. Survivality rate of Channa punctatus exposed to different concentrations of Paper Mill Effluent.

Treatment /DOSE	No. of Fish per aquarium	Concentrations	Survivality (in %)
			24 hrs	48 hrs	72 hrs
Control	50	–	100	100	100
MMC	50	2mg/L	100	80	80
Paper Mill Effluent	50	PME 10%	100	100	100
	50	PME 25%	100	80	80
	50	PME 50%	100	80	60

Figure 2: Survivality rate of Channa punctatus exposed to different concentrations of Paper Mill Effluent.

Table 3. Incidence of micronucleated PCEs in Channa punctatus induced by Paper Mill Effluent.

Dose (mg/kg)	Concentrations	Exposure Timing (In hrs)	Total PCE/n	%PCEs With MN (mean ±S.E)
Control	—	24 hrs	5001/5	0.01±0.004
		48 hrs	5002/5	0.03±0.01*
		72 hrs	4017/4	0.04±0.01*
MMC	2mg/L	24 hrs	5020/5	0.07±0.01
		48 hrs	4017/4	0.17±0.02*
		72 hrs	4018/4	0.29±0.01
Paper Mill Effluent	PME 10%	24 hrs	5019/5	0.07±0.01
		48 hrs	5022/5	0.19±0.01*
		72 hrs	5025/5	0.23±0.03
	PME 25%	24 hrs	5021/5	0.27±0.01*
		48 hrs	4017/4	0.35±0.04*
		72 hrs	4011/4	0.37±0.02***,**,*
	PME 50%	24 hrs	5022/5	0.35±0.01***,**,*
		48 hrs	4015/4	0.71±0.03*
		72 hrs	3011/3	0.79±0.02***

When compared PME with Control

 P<0.05=*, P<0.01=**, P<0.001=*** 

Figure 3: Incidence of micronucleated PCEs in Channa punctatus induced by Paper Mill Effluent 

Figure 4: Pictorial depiction of Micronucleus in Channa punctatus

Industrial discharges are recognized as one of the major resources of toxic chemicals in the environment. In the present study, the mutagenic potentiality of an effluent from a paper mill industry located in the Barak valley region of Assam, India was assessed by using an in vivo assay in fish system.

Body weight and Survival rate variation in exposed groups: In current investigation, variation in body weight was measured in fishes exposed to different concentrations of PME. Minute variation in body weight of fishes were recorded in exposed groups compared to negative and positive control. Whereas Remarkable mortality rate was recorded in exposed sets where dose dependent and time dependent death was noted.

Micronuclei Formation: Micronuclei are chromatin masses in the form of small nuclei which appear within the cytoplasm and close to the main nucleus in inter­phase cells. They are originated spontaneously or as consequence of clastogenic and/or aneugenic effects, which generate acentric chromosomal fragments and/or lagging chromosomes during the mitotic anaphase (Fenech 2003). Due the nucleated nature of erythrocytes in fish, the practicality of MN test has gain high relevance in bio-monitoring of aquatic environments, also including assessment of water quality (Xing et al. 2012; Chen et al. 2016).  

Originally the micronucleus test was developed for application in mammals, it was subsequently modified and used in fish (Schmid 1976; Houk 2007). Later, the analysis of MN was increasingly used for assessing environmental genotoxicity in fish (Al-Shabti and Metcalfe 1995; Ayllon and Garcia-Vazquez 2000; Jha 2000; Oost 2003). The micronuclei assay is one of the best biomarkers that clearly correlate with pollution load, as it has been shown in a number of studies (Belfiore et al. 2001; Kirch and Sofuni 2003; Cavas and Ergene-Gozukara 2005; Singh et al. 2020).

Several studies throughout years have demonstrated increases in micronuclei frequency in species of marine fish in polluted areas, and their use as genotoxicity markers in accordance with the present study, and also in the laboratory (Al-Sabti and Metcalfe 1995; Hayashi et al. 1998; Ateeq et al. 2002; Teles et al. 2003; Buschini et al. 2004; Çavas et al. 2005).

Many researchers have demonstrated the success of using micronucleus test in the evaluation of environmental quality, by using several freshwater fish (Hayashi et al. 1998). Group of researchers (Lopes-Poleza 2004) evaluated the genotoxic effect methylmercury (CH3Hg+) in Hoplias malabaricus, using chromosomal aberrations (anterior kidney), micronuclei, and DNA damage by the comet assay in erythrocytes (Claxton et al. 2008; Singh et al. 2020).

Just like previous, our study also confirmed the usefulness of the erythrocyte micronucleus as a powerful monitoring tool for detecting genotoxic agents in a coastal environment (Nunes et al. 2015; Singh et al. 2019). Significant increase in the frequency of micronucleated erythrocytes has been observed after exposing Channa sp. to various concentrations of industrial effluents.  Industrial effluents have already been reported to have genotoxic effects on fishes which arise in the form of micronucleus (Serrano and Montero 2001; Araújo et al. 2006). Present investigation confirmed the importance of erythrocyte micronucleus assay as an effective tool for detecting genotoxic agents.

In present investigation, selected paper mill effluent has been observed to induce MN in the blood erythrocytes of fish. The frequency of micronuclei was significantly higher than in the negative control and Mitomycin C. There was a significant difference between frequency of miconuclei among the negative and positive control. PME lead to the formation of micronucleus were compared to 10% concentration, 25% and 50% concentration showed more micronucleus formation in both PME and DS (Singh et al. 2019).

Frequency of micronucleated PCEs increased from 24hrs to 48hrs and from 48hrs to 72hrs of time interval. (p<0.05, p<0.001) designating the time dependent increase of genotoxicity. With time the chances of toxicity increase leading to any kind of chromosomal abnormality which may ultimately lead to death of the aquatic organisms in long run. Same happened in our case too (Singh et al. 2020). 

CONCLUSION

The findings of the present study suggests that the risk factor graph of pollution of water bodies due to direct industrial exposure is consistently affecting the aquatic ecosystem and human life. Based on previous research reports, current research was conducted using Micronucleus assay as an effective and verified model to monitor the level of induced toxicity which was found up to the mark, as it came out with remarkable results. Cachar paper mill effluent has been visualized to have genotoxic potentials via the tested toxicity parameter as Micronucleus assay.

Thus, we hope that we will try to get better details about the mutagenicity caused by applying other parameters and our piece of work will motivate other researchers to work on this issue from other aspects which will attract the attention of government for protection of such water resources from water pollution and make public aware about it , so that such exposures can be prevented and measures will be undertaken to aware people against direct use of untreated water from these polluted water bodies.

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