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Introduction

India is rich in diversity of organisms and plays a prominent role in international market in ornamental fish due to its rusticity and color patterns. Despite its potential, India ranks low in the Asian market, asmost of the ornamental fish is of wild variety¹。Among the total 156 species in Tamil Nadu, 16% of them were under threat due to problem in maintaining the stock, seed production, environmental factors including contamination of water etc. Hence research in aquaculture is indeed important to maintain the potential resource of income generation through entrepreneurship in the rural areas of Tamil Nadu²。

水的质量是非常重要的生存和well-being of all the aquatic organisms. Toxicants from both point and non-point sources reach the aquatic ecosystem and pollute the environment through their receiving water bodies³。These toxicants cause adverse effects on the aquatic biota through food chains. Critical habitats are particularly vulnerable to pollution. The pollutants can directly or indirectly affect the metabolism, growth and development, and affect endocrine functions, reproduction, translation etc.,

The Endocrine Disrupting Chemicals (EDCs) affects the hormone receptors by competing with the natural hormones⁴。EDCs are widely distributed in various products such as plastic materials, pesticides, fungicides, metals, personal care products and even heavy metals⁵。When these chemicals are released into the water bodies it affects the metabolism of aquatic life. The mechanism of action is more complex which include direct actions on the receptors (mainly hormonal and/or neurotransmitter receptors), enzymes and hormone^6,7。The interference in the hormones can alter the behavioural dimorphisms particularly when exposure occurs during critical developmental periods^8,9。They are widespread throughout the aquatic ecosystems such as water and sediment disturbing the aquatic life through various routes, as runoff water from the agricultural fields, sewage/effluents of industries reaches the river and ocean.^10,11。

Among the aquatic life, fishes are mainly vulnerable to toxicants and express behavioural, structural, chemical and molecular changes¹²。Therefore, fish are considered as indicator organisms to assess the EDCs¹³which imitate the effects of natural oestrogens (xenoestrogens)^14,15,16,17。Mostly heavy metals, pesticides and insecticides acts as EDCs individually or synergistically and become toxic to the organisms. This in turn affects survival, growth and reproduction of organisms¹⁸。When the exposure level to these chemicals exceed, it can induce cancer, damage the nuclear materials and ultimately lead to lethality⁷。The lethality depends on species and concentration of toxicants, which may lead to aquatic loss and disrupts the food chain^19,20。Apart from lethality the toxicants may also affect the economy of the aquarist by affecting the behaviour, structure and health of the ornamental fishes.

因此,定期监测有助于识别ill effects of the toxicants in organisms.Therefore the present study was aimed to derive safe levels on acute and chronic concentrations of EDCs such as heavy metals (Cadmium and Arsenic) and pesticides (Chlorpyrifos and Cypermethrin) in the fresh water fish.

Materials and Methods

Experimental Fish

Freshwater fishPoecilia reticulataandPoecilia sphenopswere used to study the acute and chronic effects of the chosen chemicals. Healthy fish (1.0 ± 0.1 g) were procured from hatchery in Madurai. They were transported in the plastic bag separately with freshwater filled with oxygen to the laboratory at Madurai Kamaraj University, (9.????^?? and 78.0105^?E) in Madurai, Tamil Nadu, India. Before acclimation, fish were treated with KMnO₄solution (0.05%) for 2 min to protect from dermal infection. Healthy fish were transferred to tank in the ratio of 1 g of fish weight in 10 litre of chlorine free tape water and acclimated in the laboratory at ambient environmental conditions for 15 days. During acclimation period no mortality was observed. Local manufactured pellet fish feed was fed twice a dayad libitumand the excretory matters were frequently removed. The essential physico-chemical parameters such as temperature, pH, DO, total ammonia, phosphate, nitrate, and electrical conductivity were estimated during the test every 24 hrs, following the standard methods²¹。The photoperiod of 10 hrs light and 14 hrs dark was maintained.

Chemicals

Metals, i.e., Arsenic chloride (AsCl₃) and Cadmium chloride (CdCl₂) and commercially formulated pesticides, i.e., Chlorpyrifos 20% EC and Cypermethrin 25% EC were used for assessing the acute and chronic toxic effects. One percent stock solutions were prepared separately, for each metal and pesticide, in 1000 ml volumetric flasks. Whose concentration was 1 mg mL-1.The pesticides were dissolved in 0.5% acetone and also used in solvent control group.

Experimental Design for Acute and Chronic Toxicity Studies

Acute toxicity study was conducted to analyse the median lethal concentration (LC₅₀) and safe level of the toxicants under static renewal bioassay method after 96 hrs of exposure. Before the definitive toxicity tests, the wide range of toxicant concentrations such as 0.0001, 0.001, 0.01, 0.1, 1.0, 10 & 100 mg L^-1were used to find out the concentrations for definitive toxicity tests^21,22,23。

For acute and chronic toxicity studies, ten acclimated fish were introduced per toxicants in a separate tank. For pesticide toxicity, solvent control was maintained simultaneously. Based on the median lethal concentrations derived through definitive toxicity studies, the sub-lethal concentrations of each toxicant were selected for chronic toxicity studies by calculating 1/10^thof the LC₅₀(96h) value of each toxicant²⁴。During the chronic exposure studies the NOEC (No Observed Effect Concentration) and LOES (Lowest observed effect Concentration) were derived.

The test media were renewed every day to maintain the concentration of the toxicants. During the study period, the dead fish were removed immediately from the medium. No food was provided for test animals during the acute toxicity test. For acute toxicity testing, the mortality was observed up to 96h at an interval of 24 h.

The LC₅₀values of the toxicants were analysed at respective hours of exposure using Probit Analysis computer programme^25,26。Safe concentrations of the toxicants were also calculated following the application factor, 1/100^thof the 96h LC₅₀values^27,28。The dose (concentration)vsresponse (mortality) curves were drawn using Excel programme to find out the effect of toxicants.

To find out the chronic toxicity endpoints a 28 day exposure was conducted. The artificial pellet fish feed was givenad libitum,during chronic studies²⁹。The live animals in each media were counted every day. NOEC and LOEC were derived based on percentage survival of fishes on 28^thday of the sub-lethal tests using Dunnet analysis computer program^26,30。The statistical analysis was done using SPSS Version 23 to study the significance of the data.

Result and Discussion

Environment plays a significant role in the survival of an organism. When the fish were introduced to an induced environment, they exhibit behavioral changes to sustain in the new environment. Irrespective to the toxicants exposed both the fish species exhibited behavioral changes at higher concentration. On exposure the fish were alert and still in position. The fish exposed to metals and pesticides were found to be unsteady and showed erratic movements a sign of hyper excitability. Respiratory distresses with excess mucous secretion were observed in the toxicants exposed medium.

Their pectoral and pelvic fins were found to be hard may be due to muscle fatigue on pesticide exposure. On increasing the concentration of toxicants fish remained in a vertical position, exposing the mouth on to the surface of water indicating its effort to swallow air. The observed behavioural activity in the present study is due to increase in the level of ammonia and decrease in Na+, K+, Ca2+ in plasma which indirectly affects muscular contraction and impulse transmission³¹。

Acute Toxicity

The physicochemical parameters maintained throughout the experiments were listed in Table - 1. The toxicity endpoints are the primary criteria for the protection of fauna in the aquatic medium. Therefore, the present investigation aimed to elicit the acute and chronic endpoints of the chosen Endocrine Disrupting Chemicals such as heavy metals (Arsenic and Cadmium) and pesticides (Chlorpyrifos and Cyperimethrin) in freshwater fishviz., P. reticulataandP. sphenops.

Table 1: Water Quality Analysis.

The dose dependent increase in the mortality of metals exposed fish were shown in Fig.1. There was no mortality during the experimental period in the control and solvent control (acetone 0.5%) groups.

Figure 1: Percentage of mortality on exposure to Arsenic and Cadmium metals. PR –P. reticulataand PS -P. sphenops Click here to view Figure

The LC₅₀, 95% Confidence Interval and safe values ofP. reticulataandP.sphenopscalculated under acute static renewal test method were presented in Table 2. The LC₅₀values were higher during 24 hr exposure and declined on further increase in duration. The LC₅₀values ofP. reticulataandP. sphenopsexposure to metals and pesticides showed negative correlation with time. The calculated 96 h LC₅₀values forP. reticulataandP.sphenopsexposed to As were 11.387 and 10.755 mg L^-1respectively and the LC₅₀values of respective fish exposed to Cd were 24.572 and 20.036 mg L^-1respectively (Table 2). The result showed thatP. sphenopswas more susceptible to heavy metals compared withP. reticulatairrespective of the metals. The results concluded that there was a significant effect of heavy metals inP. sphenops(R² = 0.9717 and 0.9476 for As and Cd respectively) hence, there exists a strong correlation between the dose and mortality compared withP. reticulata。Overall study inferred that the highest toxicity was reported in As exposedP. sphenopsand the lowest toxicity in Cd exposedP. reticulata。On acute exposure, both fish were resistant to cadmium and sensitive to arsenic metal.

Table 2: Acute Lethal Toxicity TestUnder Static Bioassay. Click here to view Table

Similarly, in the acute toxicity test,P. reticulataandP. sphenopswere shown to be sensitivity to Cypermethrin and resistant to Chlorpyrifos. The 96 h LC₅₀values forP. reticulataandP. sphenopsexposed to Chlorpyrifos were 5.886 and 1.046 ppb and to Cypermethrin were 1.826 and 1.362 ppb respectively (Table-3; Fig 2). It was apparent thatP. sphenopswas highly sensitive to Chlorpyrifos under acute exposure. The present study on acute toxicity concluded that the pesticides were highly toxic to both fish species than heavy metals.

Figure 2: Percentage of mortality on exposure to CPF – Chlorpyrifos; CYM – Cypermethrin; PR –P. reticulataand PS -P. sphenops Click here to view Figure

The mean survival of control animals and environmental factors was substantial to EPA/COE³¹。Thus confirmed the holding facilities of test animals and other environmental factors are under acceptable levels of the toxicity testing. Under acute toxicity studies, the mortality of test fish was positively correlated with increasing acute concentrations and exposure time, coincides with earlier reports for marine organisms³²。

Among the chosen metalsP. reticulatawas more susceptible to cadmium andP. sphenops砷。因此,研究显示一个物种dependent response on exposure to heavy metals and also shown significant result to the previous investigation in freshwater fish exposed to heavy metal and pesticide under static bioassay method. The lethality depended on the test methodology, Shuhaimiet al。,³³reported less lethal concentration under static non-renewal test and Yilmaz³⁴reported higher lethal level for the same metals under static renewal test. The study confirmed that the test methods have influence on the toxic effect, due to continuous exposure to toxicants and bioaccumulation. In the present study the estimated LC₅₀values for As and Cd to test fish were higher than that of the previous studies³⁵invariable of test methods.

The calculated 96 LC₅₀values in the present study reveals that bothP. reticulataandP.sphenopswere more sensitive to As, compared toHeteropneustes fossilis(35.10 mg L^-1; Kermiuet al。,³⁶) and slightly resistant thanCatla catla³⁷(10.16 mg L^-1); and on exposure to Cd the test fish was resistance than reported inOphiocephalus striatus(0.63 mg L^-1) and sensitive thanGambusia holbrooki³⁸(37.3 mg L^-1). Similarly for cadmium metal toxicity, present study reveals that theP. reticulataandP. shenopswere sensitive thanHeteropneustes fossilis³⁹(50.41 mg L^-1) and resistant thanRasbora sumatrana³³(0.10 mg L^-1).

The lethal concentration observed in the present study on exposure to Chlorpyrifos and Cypermethrin concentrations were less when compared to the earlier reported LC₅₀value for various organisms includingP. reticulataunder different experimental setup^40,41,42,43;Danio rerio⁴⁴and rainbow trout⁴⁵。The present study also reveals thatP. shenopswas more sensitive to Chlorpyrifos and the LC₅₀values observed in the study is less compared to earlier reports.

P. reticulatais also more sensitive to chlorpyrifos than other organic chemicals including pyrethroid⁴⁶。Similarly, sensitive than other freshwater species i.e.,Oreochromis niloticus⁴⁷(12.6 µg L^-1);Heterobranchus bidorsalis⁴⁸(36 mg L^-1) andCyprinus carpio⁴⁹(3.31 µg L^-1). Hence the present study concluded that there exists a species dependent response to the toxicant.

Chronic Toxicity

In the present study the NOEC, LOEC and chronic value for Arsenic exposedP. reticulataandP. sphenopswere similar i.e., 0.07, 0.140 and 99.0 µg L^-1respectively. Nevertheless, there was variation in the value for Cadmium exposedP. reticulata(0.15, 0.31 and 216.0 µg L^-1) andP. sphenops(0.13, 0.25 and 180.0 µg L^-1) (Table 3). The pesticide exposed fish showed very less chronic endpoints (NOEC and LOEC) than that of metal exposed fish. The NOEC and LOEC value ofP. reticulataandP.sphenopsexposed to Chlorpyrifos were 0.038 and 0.007 ppb respectively and the Chronic value ranged between 0.53 and 0.010 ppb. At the same time the chronic value for cypermethrin was noted to be lower forP. reticulata。

Table 3: Chronic Toxicity Assay.

Similar to acute exposure, the fish reared in control media (freshwater control and solvent control) showed 100% survival during the period of chronic studies. The survival rate was observed to be 85% forP. reticulataon exposure to higher sub-lethal concentration of cadmium and chlorpyrifos. In the lowest sub-lethal concentrations of metals and pesticides, the fish survival was between 97.5% and 100%, hence these concentrations were found to be suitable for survival test^50,51,52。

Acute and Chronic Safe Concentrations

Analogous to the acute safe level, the chronic safe values were also derived using the NOEC and LOEC values obtained through survival studies. The chronic values for Arsenic toP. reticulataandP. sphenopswere same i.e., 99 µg L^-1while the chronic values of Cd for respective fish were 216 and 180 µg L^-1。The chronic value for Cd exposed P。reticulatawas slightly higher. `Likewise, the chronic values of chlorpyrifos to respective fish were 0.053 and 0.010 µg L^-1and for cypermethrin were 0.016 and 0.010 µg L^-1。

No mortality in the control and solvent control groups were noticed in the present study ie., 100% survival rate observed in chronic test. Only 32.5% mortality was observed that too in the higher concentration of pesticide on the third week of exposure.

Conclusion

Maintenance of water quality is essential for survival, growth and physiology of aquatic organisms. The release of various pollutants including endocrine disruptive chemicals affects the quality of aquatic medium and leads to impairment of life in the aquatic environment. Therefore, the strict acute and chronic safe level criteria should be adopted to maintain water quality for the protection of most natural bio resources. Thus the present study on acute and chronic values of the chosen EDCs reveals that it was in good agreement with the criteria developed by USEPA²⁶under safe levels to protect the freshwater bodies. The data obtained in the present study can be used to assess and monitor the organic or inorganic contaminates in the aquarium fish culture. This in turn helps to improve the economy of the aquarist and growth of entrepreneurship in the ornamental fish trade of the world market.

Acknowledgment

The authors thank the Madurai Kamaraj University for providing necessary facility for the present study and Dr.C.M. Ramakritinan for his valuable suggestion in preparing the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

References

1. http://www.dof.gov.in/sites/default/files/2020-07/Ornamnetal_fisheries_development
2. Mogalekar H. S., Canciyal J. Freshwater fish fauna of Tamil Nadu, India.Proceedings of the International Academy of Ecology and Environmental Sciences。2018; 8 (4):213 – 230.
3. Nagarani N., Anand M., Kumaraguru A.K. Environmental monitoring using biomarkers in relevance to heavy metal pollution in coastal areas of the Gulf of Mannar.Indian J Exp Biol。2020; 58:794-802.
4. Mary C Reiley. Science, policy, and trends of metals risk assessment at EPA: How understanding metals bioavailability has changed metals risk assessment at US EPA.Aquatic Toxicology.2007; 84:292–298. https://doi.org/10.1016/j.aquatox.2007.05.014.
   CrossRef
5. Segner H. Zebra fish (Danio rerio) as a model organism for investigating endocrine disruption.Comp Biochem Phys C。2009; 149:187–195.
   CrossRef
6. Gore A.C., Patisaul H.B. Neuroendocrine Disruption: Historical Roots, Current Progress, Questions for the Future.Front Neuroendocrinol。2010; 31(4): 395–399.
   CrossRef
7. Janaki Devi V., Nagarani N., Yokesh Babu M., Kumaraguru A.K., Ramakritinan C.M. A study of proteotoxicity and genotoxicity induced by the pesticide and fungicide on marine invertebrate(Donaxfaba).Chemosphere,2013;90(3):1158-66.
   CrossRef
8. Adeel M., Song X., Wang Y., Francis D., Yang Y.Environmental impact of estrogens on human, animal and plant life: A critical review.Environ Int.2017; 99:107-119.
   CrossRef
9. Sona S., Eva R., Alzbeta B.M. Impact of endocrine disrupting chemicals on onset and development of female reproductive disorders and hormone-related cancer,Reprod Biol。2016; 16(4):243-254.
   CrossRef
10. Dan Liu., Wu S., Xu H., Zhang Q., Zhang S., Shi L., Yao C., Liu Y., Cheng J. Distribution and bioaccumulation of endocrine disrupting chemicals in water, sediment and fishes in a shallow Chinese freshwater lake: Implications for ecological and human health risks.Ecotoxicol Environ Saf.2017; 140: 222-229.
    CrossRef
11. Zhang C., Li Y., Wang, Niu L., Cai W. Occurrence of endocrine disrupting compounds in aqueous environment and their bacterial degradation: a review.Crit. Rev. Environ. Sci. Technol。2016; 46: 1–59.
    CrossRef
12. Ayobahan S.U., Eilebrecht S., Baumann L., Teigeler M., Hollert H., Kalkhof S., Eilebrecht E., Schäfers C. Detection of biomarkers to differentiate endocrine disruption from hepatotoxicity in zebrafish (Danio rerio) using proteomics,Chemosphere。2020; 240:124970.
    CrossRef
13. Jin Y., Chen R., Liu W., Fu Z. Effect of endocrine disrupting chemicals on the transcription of genes related to the innate immune system in the early developmental stage of zebrafish (Danio rerio).Fish Shellfish Immunol。2010; 28: 854–861.
    CrossRef
14. Falconer I.R., Heather F.C., Michael R.M., Geetha R. Endocrine Distrupting Compounds: A review of their challenge to sustainable and safe water supply and water reuse. Inc.Environ Toxicol.2006; 21: 181–191.
    CrossRef
15. Kolpin D.W., Furlong E.T., Meyer M.T., Thurman E.M., Zaugg S.D., Barber L.B. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999–2000: a national reconnaissance.环境科学工艺, 2002; 36:1 202–11.
    CrossRef
16. Metcalfe C.D., Tracy L.M., Yiannis K., Brenda G.K., Colin K., Richard J.H., Timothy R.C., Raymond E.M., Thomas P. Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka(Oryzias latipes).Environ Toxicol Chem。2001; 20(2): 297–308.
    CrossRef
17. Piquer I.F., Silvia B., Fabiana P., Stefania S.D., Vincenzo D.M., Hamid R.H., Francesca M., Oliana C. Effects of BPA on zebrafish gonads: Focus on the endocannabinoid system,Environ Pollut.2020; 264:114710.
    CrossRef
18. Balasubramani A., Pandian T. J. Endosulfan suppresses growth and reproduction in zebrafish,Curr. Sci.2008; 883-890.
19. Fu J., Hu X., Tao X., Yu H., Zhang X. Risk and toxicity assessments of heavy metals in sediments and fishes from the Yangtze River and Taihu Lake, China.Chemosphere, 2013; 93 (9):1887-1895.
    CrossRef
20. Afshan S., Ali S., Ameen U.S., Farid M., Bharwana S.A., Hannan F, Ahmad R. Effect of different heavy metal pollution on fish.Res. J. Chem. Environ. Sci.2014; 2 (1): 74-79.
21. APHA/AWWA/WEF, 1998. Standard Methods for the Examination of Water and Wastewater. 20th Ed. American Public Health Association, Washington DC, p. 1220.
22. Sprague J.B. The ABCs of pollutant bioassay using fish. In: Cairns J, Dickson DL (eds) Biological methods for assessment of water quality.ASTM Special Technical。1973; 528: 6–30.
23. OECD. 1993. OECD guidelines for testing of chemicals. OECD, Paris.
24. Palanikumar L., Kumaraguru A.K., Ramakritinan C.M., Anand M. Biochemical response of anthracene and benzo[a] pyrene in milkfishChanos chanos. Ecotox Environ Saf。2012; 75:187–197.
    CrossRef
25. Finney, D.J. (1971) Probit Analysis. 3rd Edition, Cambridge University Press, Cambridge.
26. USEPA. 1994. US EPA toxicity data analysis software. EMSL, Cincinnati
27. Kameswara R. The Comparative Toxicities of Organophosphorus and Carbamate Pesticides.Mahasagar。1974; 7(1&2).
28. Ramakritinan C.M., Rathishri C., Kumaraguru A.K. Acute toxicity of metals: Cu, Pb, Cd, Hg and Zn on Marine Mollusc,Cerithidiac ingulataandModiolus phillipinarum H. Indian J Geo- Mar Sci。2012; 41(2): 141-145.
29. Gundersen D.T., Kristanto S.W., Curtis L.R., A1-Yakoob S.N., Metwally M.M., A1-Ajmi D. Subacute Toxicity of the Water-Soluble Fractions of Kuwait Crude Oil and Partially Combusted Crude Oil onMenidia beryllinaandPalaemonetes pugio.ArchEnviron Con Tox。1996; 31: 1-8.
    CrossRef
30. USEPA (1985) Water quality standards. Environmental Protection Agency, Taipei, Taiwan, no. 547327 Meade, J. W. (1989)Aquaculture management。Van Nostrand Reinhold, New York.
31. Mohan S. Behavioural responses in effect to chemical stress in fish: A review.Int. J. Fish. Aquat.2019; 7(1): 01-05.
32. USEPA and USACE.Evaluation of dredged material proposed for ocean disposal, Testing manual,(Washington, DC, EPA-503/8-91/001), 1991.
33. Shuhaimi-Othman M., Nadzifah Y., Nur-Amalina R., Umirah N.S. Deriving freshwater quality criteria for copper, cadmium, aluminum and manganese for protection of aquatic life in Malaysia.Chemosphere.2013;90: 2631–2636.
    CrossRef
34. Yilmaz M., Ali Gül., Erba?l K. Acute toxicity of alpha-cypermethrin to guppy (Poecilia reticulata, Pallas, 1859),Chemosphere.2004; 56(4): 381-385.
    CrossRef
35. Zakaria N.A., Kutty A.A., Mahazar M.A., Zainal Abidin M. Arsenic acute toxicity assessment on select freshwater organism species in Malaysia.AIMS Environ. Sci.2016; 3(4): 804-814.
    CrossRef
36. Kermi T, Sunkam N.R, Bidyadhar d .屁股的效果nic (As) and lead (Pb) on glycogen content and on the activities of selected enzymes involved in carbohydrate metabolism in freshwater catfish,Heteropneustes fossilis. Int Aquat Res。2019; 11:253–266.
    CrossRef
37. Kousar S., Javed M. Heavy metals toxicity and bioaccumulation patterns in the body organs of four fresh water fish species.Pak. Vet. J.2014; 34(2): 161?164.
38. Bais U.E., Lokhande M.V. Toxicity evaluation of cadmium chloride in fresh water fishOphiocephalus striatus。Int J Fish Aquat Stud.2017; 5(1): 519-521
39. Singh A., Jain D.K., Puneet Kumar. Determination of LC₅₀of cadmium chloride inHeteropneustes fossilis,GERF Bulletin of Biosciences。2010; 1(1): 21-24.
40. Sharbidre A.A., Vimal M., Priyanka P. Effect of methyl parathion and chlorpyrifos on certain biomarkers in various tissues of guppy fish,Poecilia reticulata. Pestic Biochem Phys.2011; 101: 132–141.
    CrossRef
41. Abdolreza J., Fardin, S., Hedayati, A. Mortality Response of Silver Carp (Hypophthalmicthys molitrix), Gold Fish (Carassius auratus) and Roach (Rutilus rutilus) to Acute Exposure of Copper Sulphate.World Journal of Fish and Marine Sciences。2012; 4 (4): 418-421.
42. Shen M.F., Anupama K., Shu-Yan Ding., Sonia G. Comparative study on the toxicity of pyrethroids, α-cypermethrin and deltamethrin toCeriodaphnia dubia。Ecotox Environ Safe.2012; 78: 9-13.
    CrossRef
43. Restrepo D.E., Jaramillo-Colorado B.E., Duarte-Jaramillo L. Effects of chlorpyrifos on the crustaceanLitopenaeus vannamei. PLoS ONE,2020; 15(4): e0231310.
    CrossRef
44. Ahmad S., Vineeta Y., Kaneez Z. Determination of LC50 of Chlorpyrifos with special reference to Behavioral and Morphological changes in zebra fish,Danio rerio。IJRAR2019; 6(1): 655-668.
45. Topal A., Atamanalp M., Oruc E., Demir Y., Beydemir S., Isik A. In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout.Arh. Hig. RadaToksikol。2014; 65: 377-385.
    CrossRef
46. Mustafa C. Assessment of acute toxicity of cypermethrin alone and synergized with piperonylbutoxide to the male guppies, (Poecilia reticulataPeters, 1859).Fresenius Environmental Bulletin。2017; 26(12): 7458-7462.
47. Yaji A.J., Auta J., Oniye S.J., Adakole J.A., Usman J.I. Effects of cypermethrin on behaviour and biochemical indices of fresh water fishOreochromis niloticus。Elec. J. Env.Agricult. Food Chem.2011; 10(2): 1927-1934.
48. Olufayo M.O., Alade O.H. Acute toxicity and histological changes in gills, liver and kidney of catfish,Heterobranchus bidorsalisexposed to cypermethrin concentration.African Journal of Agricultural Research.2012; 7(31): 4453-4459.
    CrossRef
49. Neelima P., GovindaRao K., GopalaRao N., Chandra SekharaRao J. Acute toxicity of cypermethrin (25% EC) on nucleic acids (DNA and RNA) inCyprinus carpio(linn.).Int J. Recent Sci. Res.2015; 6(7):5219-5224.
50. ASTM, Standard guide for conducting 10-day static sediment toxicity tests with marine and estuarine amphipods, (ASTM E 1367-90, American Society for Testing and Materials, Philadelphia, PA) 1990, pp. 1-24.
51. Palanikumar L., Kumaraguru A.K., Ramakritinan C.M., Anand M. Toxicity, biochemical and clastogenic response of chlorpyrifos and carbendazim in milkfishChanos chanos。Int. J. Environ. Sci. Technol。2014; 11:765–774.
    CrossRef
52. Shahbaa K., AL-Taee., Karam H., Hana Kh.Ismail. Review on Some Heavy Metals Toxicity on Freshwater Fishes,J. Appl.Vet. Sci.2020; 5(3): 78 - 86. DOI: https://dx.doi.org/10.21608/javs.2020.100157.
    CrossRef