Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 57 issue 6 (june 2023) : 788-794

Short Term Effects of Antimicrobial Agent Triclosan on Oreochromis mossambicus (Peters, 1852): Biochemical and Genetic Alterations

Seenivasan Deepika1,*, Pandurengan Padmavathy1, Arasan Srinivasan1, Gopalrajan Sugumar2, Paulraj Jawahar3
1Department of Aquatic Environment Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
2Department of Fish Processing Technology, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
3Department of Fisheries Biology and Resource Management, Fisheries College and Research Institute, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Thoothukudi-628 008, Tamil Nadu, India.
Cite article:- Deepika Seenivasan, Padmavathy Pandurengan, Srinivasan Arasan, Sugumar Gopalrajan, Jawahar Paulraj (2023). Short Term Effects of Antimicrobial Agent Triclosan on Oreochromis mossambicus (Peters, 1852): Biochemical and Genetic Alterations. Indian Journal of Animal Research. 57(6): 788-794. doi: 10.18805/IJAR.B-4686.

ABSTRACT

Background: Triclosan is an antimicrobial agent which enters into the aquatic environment through wastewater discharges which causes potential health risk in human and aquatic organisms. The present study aimed to determine the toxic effects of triclosan on Oreochromis mossambicus.

Methods: The fishes were subjected to five different concentrations viz. 131, 262, 523, 1046 and 2092 µg.l-1 of triclosan for 96 h acute toxicity test. To evaluate the levels of enzymes such as acetylcholinesterase and glutathione S transferase, brain and liver tissues were collected, homogenized, extracted and stored at -20° for further analysis. The DNA damage was assessed in gill and liver tissues using single cell gel electrophoresis method.

Result: In present study, the calculated 96 h LC50 value of triclosan in O. mossambicus was 740 µg.l-1 and the fishes showed various behavioural alterations. Time and dose dependent inhibition of AChE activity in brain tissue was observed during acute toxicity test. However, the GST activity in liver tissue increased on exposure to triclosan with significant increase in concentration of toxicant. DNA damage index was higher in gill tissue compared to the liver tissue during acute exposure to TCS which could cause detrimental effects in fishes.

INTRODUCTION

In the modern world, anthropogenic pollution causes potential threat to aquatic ecosystem by interference in the normal metabolism, reproduction and development of aquatic organisms. Triclosan (TCS) (CAS registration number 3380-34-5) also known as 5-chloro-2-(2,4-dichlorophenoxy) phenol or 2,4,4'-trichloro-2'-hydroxydiphenyl ether, is a broad-spectrum antimicrobial agent in some personal care products such as toothpastes, soaps, hand washes, deodorants, kitchen and plastic wares (Fang et al., 2010; MacIsaac et al., 2014).  Its widespread use in homes and in health care centres is the reason that it has become a water micro-pollutant (Li et al., 2010; Helbing et al., 2011). Due to its high lipophilicity (log Kow = 4.8 octanol - water partition coefficient), it has serious distress about bioaccumulation in fatty tissues which raises toxicity concern in aquatic organisms (Behera et al., 2011).

Toxicity study helps to understand the effects of environmental pollutants on aquatic organisms by bioassay methods (Srivastava et al., 2016). Biomarkers can provide biologically and ecologically relevant information and considered as a valuable tool for the establishment of guidelines for effective environmental management (Tashla et al., 2018). Acetylcholinesterase (AChE) is one of the most commonly used biomarker enzymes of neurotoxicity and found at neuromuscular junctions and cholinergic synapses at the central nervous system. Main role of AChE is to regulate the nerve impulse transmission by hydrolysis of the neurotransmitter acetylcholine (Lionetto et al., 2013). Xenobiotics entering the aquatic environment can inhibit the AChE activity in brain which can lead to the behavioural alteration of fish (Ghazala et al., 2016). Some researchers have identified the reduction of AChE activity in the brain tissue due to TCS in Danio rerio, Pangasionodon hypothalamus, Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala which was found to be more intense during the early life stages (Oliveira et al., 2009; Sahu et al., 2018; Dar et al., 2020). Glutathione S-transferase (GST) is the multifunctional phase II intracellular enzyme involves in detoxification of endogenous and exogenous toxic compounds. Pollutants are known to inhibit GST activity in aquatic organisms. Elevated level of GST activity in the tissues indicates increase in the biotransformation process of toxic metabolites for excretion (Modesto and Martinez, 2010). Genotoxicity study using comet assay is a most sensitive and economical technique to detect the genetic damage at cellular level. Comet assay is a versatile and simple way of evaluating the damage caused by the pollutants by measuring the breaks in the DNA chain of animal cell (de Lapuente et al., 2015). Therefore, the above biochemical and genetic parameters were chosen for the present study to evaluate the acute toxicity of TCS in Oreochromis mossambicus which is the commercially important and candidate species for aquaculture in food security point of view.  

MATERIALS AND METHODS

Experimental fish
 
Experimental fish O. mossambicus (weight: 102.75 ± 2.63g; length: 15.73±0.68 cm) was procured from the freshwater farm facility of Fisheries College and Research Institute, Thoothukudi, Tamil Nadu, India and the study was conducted during the year 2019. The fishes were acclimatized to the laboratory condition in fibreglass reinforced plastic (FRP) rectangular tank of 500 L capacity with proper water exchange and aeration. During the acclimatization the fishes were fed with commercial fish feed. The ethical guidelines for the animal care of the institute were strictly followed during the experiment.
 
Chemicals
 
Triclosan or 2, 4, 4' - Trichloro - 2' - hydroxydiphenyl ether (CAS id: 3380-34-5) was purchased from Tokyo Chemical Industry Co., Ltd. (purity >98%) and used for the present study.  Stock solution of 200 ppm was prepared for the toxicity study using 0.01N NaOH solution as solvent for dissolving the TCS.
 
Test conditions
 
Water quality parameters of the water used for all the experiments viz. water temperature, pH, dissolved oxygen, alkalinity and hardness were analysed during the toxicity study according to standard methods of APHA (2012). Static renewal test was followed for the acute and sublethal toxicity experiments. The test solutions were totally renewed at 24 h interval during the experimental period. The tests were conducted in duplicate in plastic tanks of 50L capacity and ten fishes per tank were used for different concentrations of test chemical, solvent control (0.01N NaOH) and blank control. All experiments were conducted in indoor area with natural 12h:12h light - dark photoperiod. Before 96 h acute toxicity test, the toxicity range - finding test was conducted for 48 h by following the method of EPA (2002). Randomly selected fishes were exposed to the test concentrations in logarithmic series (i.e. 0.01, 0.1, 1.0, 10 and 100 mg.l-1) along with solvent and blank control.
 
Acute toxicity test
 
Based on the result of range - finding test, five experimental concentrations of TCS for O. mossambicus (131, 262, 523, 1046 and 2092 µg.l-1) was selected to conduct 96 h acute toxicity test and determined the median lethal concentration (LC50) of TCS. Control group (toxicant free group) of fishes were maintained as the reference group for comparison with solvent (NaOH group) and TCS exposed fishes. During the entire test period no feed was offered to the fishes. The physico chemical parameters of the test conditions were monitored daily for 96 h of the test period. Behavioural changes and fish mortality were monitored for 96 h and the dead animals were removed from the test conditions immediately to prevent contamination. The LC50 values were calculated using the mortality data by probit analysis (Finney, 1971).
 
Collection of tissues and preparation of homogenates
 
After 96 h of exposure to TCS in acute toxicity test the organs such as gill, liver and brain were collected from each treatment and control group of fishes. The collected organs were homogenised with 0.25 M sucrose buffer solution (1:10 w/v). The homogenates were centrifuged at 14,000 x g for 15 min at 4p°C to obtain supernatants which were used for further enzymatic analysis. All the supernatants were stored at -20p°C until further analysis.
 
Enzyme assay
 
The activity of acetylcholinesterase (AchE; EC 3.1.1.7) was measured in brain tissue spectrophotometrically at 405 nm at 25p°C according to the method of Ellman (1961) with some modifications described by Augustinsson (1957). The activity of AChE was expressed as µM of acetylcholine hydrolyzed min-1mg-1 of protein.

The activity of glutathione S transferase (GST; EC 2.5.1.18) was measured in liver tissue at 340 nm spectrophotometrically at 25p°C according to Habig et al., (1974). The GST activity of liver following the formation of GSH conjugate with 1-chloro-2,4-dinitrobenzene (CDNB) was expressed as n mol. CDNB conjugates formed min-1 mg-1 of protein.
 
Single cell gel electrophoresis (comet assay)
 
The alkaline single cell gel electrophoresis (SCGE) / comet assay was performed in gill and liver tissues of tilapia according to the standard procedure of Singh et al., (1988) with some modifications as described by Ali et al., (2008).
 
Statistical analysis
 
Differences in the test concentration and control were subjected to one way analysis of variance (ANOVA) followed by Turkey’s multiple range test using statistical package SPSS 22.0. Difference in the mean range test and influence of TCS concentration in fishes were determined at 5% probability level. The results were presented as mean ± standard error.

RESULTS AND DISCUSSION

Determination of LC50 and behavioural responses
 
Triclosan has been classified as a class III drug (compounds with low permeability and high solubility) by FDA due to their broad spectrum antimicrobial activity (Dhillon et al., 2015). Toxicity studies conducted on various organisms proved that TCS is acutely and chronically toxic to all the organisms especially to the aquatic organism through wastewaters from different sources. Earlier studies observed the median lethal concentration (LC50) of TCS on different fishes as 4400 µg.l-1 in Onchorynchus mykiss (Adolfssonerici et al., 2002), 370 and 1700 µg.l-1 in Oryzias latipes  (Orvos et al., 2002 and Nassef et al., 2009), 340 µg.l-1 in Danio rerio (Oliviera et al., 2009), 1480 µg.l-1 in Xiphophorus helleri (Liang et al., 2013), 2810 µg.l-1 in Oreochromis niloticus (Vijitha et al., 2017), 1458 µg.l-1  in  Pangasianodon hypophthalmus (Sahu et al., 2018), 1767 µg.l-1 in Anabas testudineus (Priyatha and Chitra, 2018), 360 µg.l-1 in Gibelion catla and 390 µg.l-1 in Labeo rohita (Hemalatha et al., 2019a and 2019b). In the present study, the LC50 value of TCS on O. mossambicus was calculated based on the 96 h mortality data of fishes exposed to TCS. The data were plotted as graph and there was a positive correlation (P<0.05) between the mortality of fishes and concentration of TCS (Fig 1). The calculated 96 h LC50 value at 95% confidence limits for TCS for O. mossambicus was 740 µg.l-1. During the exposure period the fishes showed behavioural irregularities such as mucous formation, surfacing, erratic movement, air gulping, hanging at the top and discoloration of skin.

Fig 1: Mortality of Oreochromis mossambicus on exposure to different concentration of TCS. C1 – Control; C2 – Solvent Control; T1 – 0.131 ppm; T2 – 0.262 ppm; T3 – 0.523 ppm; T4 – 1.046 ppm; T5 – 2.092 ppm.


 
Activity of acetylcholinesterase enzyme
 
Acetylcholinesterase (AChE) activity is widely being used as a biomarker to study the neurotoxic effects of wide range of toxicants in the aquatic environment. Inhibition of AChE activity results in build up of acetylcholine which causes prolonged excitatory post synaptic potential which negatively impacts the overall behaviour and nervous system of fishes (Banaee et al., 2011). In the present study, the mean AChE activity of brain tissue of tilapia exposed to control medium was 0.345±0.01 μM.min-1.mg.protein-1. In tilapia, on exposure to 96 h acute toxicity of TCS the AChE activity of the brain got significantly reduced to 0.266±0.03, 0.209±0.02, 0.175±0.01 and 0.088±0.01 μM.min-1.mg.protein-1 at 131, 262, 523 and 1046 µg.l-1 respectively (Fig 2). AChE activity in the brain tissue of tilapia was significantly (p<0.05) reduced on exposure to different concentrations of TCS. There was no significant difference in enzyme activity between the control and solvent control group of tilapia in the acute toxicity test during the study period. The degree of reduction of enzyme activity was found to be increase with increase in concentration of TCS during the exposure. The reduction of AChE activity in tilapia on exposure to acute concentrations of TCS is shown in Fig 2. Highest amount of inhibition of AChE was observed at 1046 µg.l-1 of TCS in tilapia. Similar observation of reduction in enzyme activity in brain tissue was observed in zebra fishes by Oliviera et al., (2009) and Pullaguri et al., (2020).
 

Fig 2: Mean acetylcholinesterase activity in O. mossambicus exposed to acute concentration of TCS for 96 h. [Superscripts indicate significant difference (p<0.05) between control and different concentrations within the same exposure period. Error bars denote standard error (n=3)]. C1 - Control; C2 - Solvent Control; T1 - 0.131 ppm; T2 - 0.262 ppm; T3 - 0.523 ppm; T4 - 1.046 ppm.



Activity of glutathione S transferase
 
Glutathione S transferase is involved in detoxification of xenobiotics at cellular level and their activity in different organs acts as a potential biomarker in toxicity tests (Sturve et al., 2008). In the present study, the observed increase in concentration of GST enzyme in liver tissue can be attributed to the detoxification process of toxic metabolites and it is an indicator of defence mechanism (Modesto and Martinez, 2010; Sahu et al., 2018). The mean GST activity in liver tissue of tilapia at control medium during acute toxicity test was 2.637±0.13 n mol. min-1 mg.protein-1. The GST activity was induced on exposure to TCS in fishes with significant increase in activity with increase in concentration of TCS. The GST activity on exposure to 96 h acute toxicity test significantly increased to 3.022±0.06, 3.346±0.10, 3.860±0.10 and 3.551±0.10 n mol. min-1 mg.protein-1 at 131, 262, 523 and 1046 µg.l-1 respectively (p<0.05) (Fig 3). Similarly, an elevation in GST activity due to TCS toxicity were reported in zebra fish by Oliviera et al., (2009), in sword tail fish by Liang et al., (2013) and also in mussels and zooplankton (Canesi et al., 2007; Binelli et al., 2011; Peng et al., 2013; Han et al., 2016). GST inhibition at the early larval stages of Solea senegalensis observed by Araujo et al., (2019) proves that TCS inflicts more damage to fishes at their early life stages.

Fig 3: Mean glutathione S transferase activity in O. mossambicus exposed to acute concentration of TCS for 96 h. [Superscripts indicate significant difference (p<0.05) between control and different concentrations within the same exposure period. Error bars denote standard error (n=3)]. C1 – Control; C2 – Solvent Control; T1 – 0.131 ppm; T2 – 0.262 ppm; T3 – 0.523 ppm; T4 – 1.046 ppm


 
DNA damage in gill and liver of tilapia exposed to acute and sublethal toxicity of TCS
 
The integrity of DNA damage in blood cells and different tissues have been proved as potential biomarker for environmental toxicology. In the present investigation, significant dose dependent DNA damage intensity was recorded in gill and liver tissues of tilapia and rohu. The difference in extent of DNA damage in various tissues could be attributed to number of alkali – labile sites and variability in DNA of different tissues (Paul et al., 2019) because different cells have different levels of DNA single strand breaks due to variation in antioxidant concentration, metabolic and repair activity (Lee and Steinert, 2003). Effect of TCS on the index of DNA damage (DI) on gill and liver tissues of tilapia during acute toxicity test is presented in Fig 4. DNA damage was not observed in the control group of fishes in both gill and liver tissues of tilapia during 96 h acute toxicity test. In gill tissue, the mean DI values were observed as 37.564 ± 1.92, 49.275±2.34, 61.005±1.47 and 98.014±1.69 at 96 h acute toxic concentrations of 131, 262, 523 and 1046 µg.l-1 respectively. In liver, the mean DI values at the concentrations of 131, 262, 523 and 1046 µg.l-1 were 28.789±1.86, 38.877±1.39, 46.226±1.32 and 50.705±1.58 respectively. The DNA damage increased with increased in concentration of TCS in gill and liver tissue. The DI value was found to be higher in gill than the liver tissue.  The TCS induced genotoxicity study on rainbow trout by Capkin et al., (2017) in red blood cells showed significant DNA damage. In addition to fishes, studies by Silva et al., (2015) and Xu et al., (2015) revealed the TCS induced DNA damage in live food organisms such as Daphnia magna and Artemia salina. So, the present investigation proved that TCS can affect the target tissue of fishes and induces DNA strand breakage.

Fig 4: Mean DNA Damage Index (DI) in gill and liver of O. mossambicus exposed to acute concentration of TCS for 96 h. [Superscripts indicate significant difference (p<0.05) between control and different concentrations within the same exposure period. Error bars denote standard error (n=3)]. C1 – Control; C2 – Solvent Control; T1 – 0.131 ppm; T2 – 0.262 ppm; T3 – 0.523 ppm; T4 – 1.046 ppm.

CONCLUSION

The toxicity result of the investigation shows that it has the potential to affect the tilapia at the median lethal concentration of 740 µg.l-1 respectively. It also has the potential to affect the fishes biochemically and genetically. The present detailed investigation on enzyme activity and DNA damage clearly showed that the TCS could alter the normal metabolic processes of the aquatic organisms. So, this preliminary study of TCS determination in fishes would gain interest in regular monitoring to obtain the contamination profile of emerging pollutants of personal care products. So, it is necessary to study in detail about the polluting agents of personal care products, their fate and transportation in the aquatic environment. 

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