Acute Toxicity and Stress Behaviour of Heterobranchus bidorsalis Exposed to the Detergent Nittol^® NTL

Round 1

Reviewer 1 Report

introduction need to be improve to more concise and focus

the parameters for toxicity analysis need to be added, such as abnormality or defect caused by detergent effects on the certain organs, which lead to the lethality of test fish. 

discussion need to be improve for more detailed and accurate data interpretation

Author Response

Response to Reviewers

Response to Reviewer 1

Introduction Introduction

Detergents generally contain a mixture of a wide variety of chemical substances including, water softeners, processing acids, cleaning agents, optical brighteners, perfumes and colouring agents [1, 2, 3]. These chemicals may act individually or collectively as a buildup of a more complex compound, that becomes too difficult to degrade, or cause eutrophication, in the event of trying to bring about their breakdown [4, 5, 6]. The entry point of detergent effluents from sewages, washing factories, Detergents generally contain a mixture of a wide variety of chemical substances including, water softeners, processing acids, cleaning agents, optical brighteners, perfumes and colouring agents [1, 2, 3]. These chemicals may act individually or collectively as a buildup of a more complex compound, that becomes too difficult to degrade, or cause eutrophication, in the event of trying to bring about their breakdown [4, 5, 6]. The entry point of detergent effluents from sewages, washing factories, hospitals, refineries, detergent-making industries, and agro-allied industries into the aquatic waters is a major challenge to fish farmers and scientists [7, 8, 9]. Nittol is composed of linear alkyl benzene sulphonate (LABS), sodium tripolyphosphate (SIPP), sodium carbonate, sodium sulphate, sodium per borate and sodium silicate, widely used in many homes, washing premises, and industries which channel its effluents into the receiving waters reserved for fisheries and culture of Heterobrunchus bidorsalis, an important fish in Africa [10, 11]. There is a scarcity of reports on the acute toxicity and safety level of detergents in Nigeria, and therefore the objective of this study is to determine the behaviour, acute toxicity and safety concentration of the detergent Nittol®, on exposed fingerlings of Heterobrunchus bidorsalis. It is considered to be an important culture fish in West Africa due to its fast growth, easy adaptation and hardy nature [30]. Blood parameters are important physiological indicators of animals undergoing stressful conditions such as the presence of toxicants since blood acts as a pathophysiological reflector of the whole body [32]. Haematological parameters have been recognized as valuable tools for monitoring fish health [12].

 

Parameters for toxicity Experimental fish and Nittol detergent

A total of one hundred and eighty (180) fingerlings of Heterobrunchus bidorsalis (mean weight  5.5 ± 0.3g, mean length 6.4 ±0.5cm), were obtained from Onose Farms, Ugheli, Delta State Nigeria, and transported to the Fisheries Laboratory of the Department of Animal/Fisheries Science and Management, Enugu State University of Science and Technology Enugu Lat. 7.4N; 8⁰ 7’5 and long 6⁰ 8’E. 7⁰ 6’ W. The experimental fish were maintained in four fibre-reinforced plastic (FRP) tanks, containing 600 L of de-chlorinated tap water. Aeration was provided to all tanks round the clock to maintain dissolved oxygen contents. Before the commencement of the study, the fish were acclimatized for 14 days and were fed a commercial fish diet composed of 40% crude protein. The faecal matter and other waste materials were siphoned off daily, to reduce ammonia content in the water. Nittol^® was collected from a local market and was prepared according to standard procedures Ivon et al. (2020) [12], and was dissolved in distilled water to make a stock solution for the study. Ethical clearance from the Enugu State University of Science and Technology Committee on Experimental Animal Care ESUST/CEAC/12-08-2022/015 was obtained and followed.

2.2. Acute toxicity test

The toxicity of NTL to H. bidorsalis was carried out according to the Organization for Economic and Development OECD guideline for testing chemicals No. 203 in a semi-static renewal system by using 200L capacity rectangular glass aquaria 100x40x50cm for 10 fish. Five different concentrations (0.8, 1.0, 1.2, 1.4, 1.6 ), and a control 0.00 mgL^-1 were selected and prepared in triplicates for definitive exposures after a range-finding test, and ten (10) fish were exposed to each replicate. Feed was not offered to the fish for 96 h of the test period. Dead fish were immediately removed to prevent deterioration of water quality. The exposure solution was renewed each day and was conducted under the natural photoperiod of a 12:12 light-dark cycle and the physicochemical parameters of the test water were analyzed daily, using standard methods APHA (2005) ^[13]. The test fish were sampled on hours 24, 48, 72 and 96 in each replicate, to determine the toxic effects of the detergent on the fish.  The behavioural responses in the exposed and control fish were observed and recorded daily by Little et al. 19190 [39]. The LC₅₀ was determined by Probit analysis by Finney (1971) [14]. The safe level was estimated by applying the safety application factor (AF), suggested by CCREM (1991) [15].

 

2.3 Haematological Assay

The blood sample was collected by cardiac puncture from juvenile of Clarias gariepinus into ethylene tetra-acetic acid (EDTA) bottles and were estimated for Red blood cells (RBC),  white blood cells (WBC) haemoglobin (Hb) and packed cell volume (PVC),

using the methods of Zutshi et al. (2010) [34]

 

2.4.

Discussion

4. 4. Discussion

The display of stressful responses by the test fish corroborates with the reports of other workers [16, 17]. The corroborated report of oxygen reduction in this study along with other workers [18, 19] and increased alkalinity death point, [20, 21] and temperature [22, 23], may suggest that the presence of the detergent in the aquatic environment is responsible for the significant water quality disruption since the control water maintained normal ranges. This development may have elicited insignificant opercula and tail movements to make up for the oxygen tension, due to respiratory dysfunction [24, 25 ], and an attempt to move out of the non-conducive containers, which resulted in the exhaustion of its energy [26, 27], and eventual death of the exposed fish. The median lethal value obtained for the detergent with a positive linear logarithmic probit line correlation signifies a dose-dependent mortality rate in agreement with [28] who reported that detergent has poisonous effects in all types of aquatic life if they are present in sufficient quantities. [29] Reported that most fish will die when detergent concentration approaches 15 parts per million. The median lethal dose in this study is higher than 0.9mg/L reported by [12 ] in sub-adults of C. gagiepinus. The acute toxicity of nonionic detergent oleyl-cetyl alcohol-ethylene oxide condensate (alkaline FI liquid) was reported to range from <0.1--7.9 ppm for the plankton Diaptomus forbesi,  <20.0--7,880.0 ppm for the worm Branchiura sowerbyi, and 19.9--308.0 ppm for the fish Tilapia mossambica.Chronic toxicity of detergents on fish was reported to significantly affect Feeding intake, growth rate and maturity, fecundity and egg maturity [40]

The effects of toxicants on fish can be assessed by the use of haematological indices as it has been reported to be a routine procedure in toxicological research, environmental monitoring and fish health conditions [31, 32]. Fish that inhabit a detergent-polluted environment are particularly susceptible to contaminants that can damage their haematology which causes deleterious changes in their various cellular structures and blood cells [33, 34]. The inhibition of the hybrid catfish erythrocytes and its index component PCV and haemoglobin in this investigation agrees with other workers on exposed fish to detergents [35.36], which may have resulted in haemodilution of the blood by the pollutant, erythropoietic damage, anaemic responses or as a result of reduced gill osmoregulation [37]. Increased catfish WBC observed in the present investigation agrees with other workers on exposing fish to pollutants [38] to restore the immune system of the stressed fish.

References

1. Yazar, K., Johnsson, S., Lind, M. L., Boman, A., & Liden, C. (2011). Preservatives and fragrances in selected consumer‐available cosmetics and detergents. Contact Dermatitis, 64(5), 265-272.
2. Markwell, J. P., Thornber, J. P., & Skrdla, M. P. (1980). Effect of detergents on the reliability of a chemical assay for P-700. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 591(2), 391-399.
3. Garcia‐Hidalgo, E., Sottas, V., von Goetz, N., Hauri, U., Bogdal, C., & Hungerbühler, K. (2017). Occurrence and concentrations of isothiazolinones in detergents and cosmetics in Switzerland. Contact Dermatitis, 76(2), 96-106.
4. Yangxin, Y. U., Jin, Z. H. A. O., & Bayly, A. E. (2008). Development of surfactants and builders in detergent formulations. Chinese Journal of Chemical Engineering, 16(4), 517-527.
5. Scheibel, J. J. (2004). The evolution of anionic surfactant technology to meet the requirements of the laundry detergent industry. Journal of surfactants and detergents, 7(4), 319-328.
6. Smulders, E., & Rähse, W. (2002). Laundry detergents(p. 52). Weinheim: Wiley-Vch.
7. Kundu, S., Coumar, M. V., Rajendiran, S., Rao, A., & Rao, A. S. (2015). Phosphates from detergents and eutrophication of surface water ecosystem in India. Current science, 1320-1325.
8. Mousavi, S. A., & Khodadoost, F. (2019). Effects of detergents on natural ecosystems and wastewater treatment processes: a review. Environmental Science and Pollution Research, 26(26), 26439-26448.
9. Wind, T. (2007). The role of detergents in the phosphate balance of European surface waters. Official Publication of the European Water Association (EWA).
10. Adewumi, A. A., & Olaleye, V. F. (2011). Catfish culture in Nigeria: Progress, prospects and problems. African Journal of Agricultural Research, 6(6), 1281-1285.
11. Owodeinde, F. G., & Ndimele, P. E. (2011). Survival, growth and feed utilization of two clariid catfish (Clarias gariepinus, Burchell 1822 and Heterobranchus bidorsalis, Geoffroy, 1809) and their reciprocal hybrids. Journal of Applied Ichthyology, 27(5), 1249-1253.
12. Ivon, E. A., Etangetuk, N. A., Ubi, G. M., Anyanwu, C. O., Nkang, A. N., & Ekanem, A. P. (2020). Assessment of histopathological damages in African catfish (Clarias garienpinus) as Influenced by Nittol Detergent Aquatic Pollution in Nigeria. Annual Research & Review in Biology, 1-11.
13. APHA (American Public Health Association, American Waterworks Association and Water Environmental Federation). Standard Methods of Examination of Water and Wastewater. 21st ed.APHA, Washington DC 2005; pp 20001-23710.
14. Finney DJ. Probit Analysis. Cambridge University Press, London 1971; Pp123-125
15. CCREM (Canadian Council of Resources and Environmental Ministry). Canadian Water Quality Guidelines: Canadian Council of Resources and Environmental Ministry, Ottawa: Inland Waters Directorate, Environment 1991; Canada.
16. Oyoroko, E., & Ogamba, E. N. (2017). Effects of detergent containing linear alkyl benzene sulphonate on the behavioural response of Heterobranchus bidorsalis, Clarias gariepinus and Heteroclarias. Biotechnological Research, 3(3), 59-64.
17. Mekuleyi, G. O., & Faleti, B. E. (2018). Effect of detergent surfactant on some selected electrolytes and metabolites of juvenile Heterobranchus bidorsalis. Brazilian Journal of Biological Sciences, 5(10), 339-345.
18. Mohammad, N. A., Muhammed, F. B., Muhhamed, F. B., Eteng, A. O., Zainab, M. S., Bayero, U., ... & Abubakar, M. (2022). PATHOLOGIC LESIONS AND ANTIOXIDANT RESPONSE IN THE LIVER OF AFRICAN CATFISH CLARIAS GARIEPINUS (BURCHELL, 1822) JUVENILE EXPOSED TO COMMERCIAL-GRADE DETERGENT. The Bioscientist Journal, 10(3), 292-305.
19. Gabriel, U. U., Jack, I. R., Edori, O. S., & Egobueze, E. (2009). Electrolytes in selected tissues of Heterobranchus bidorsalis treated with sub-lethal levels of cypermethrin. Ethiopian Journal of Environmental Studies and Management, 2(3).
20. Uedeme-Naa, B., & Deekae, S. N. (2016). Organosomatic index and condition factors of Clarias gariepinus juvenile exposed to chronic levels of linear alkyl benzene sulphonate. Scientia, 16(3), 104-107.
21. Isyaku, B., & Solomon, J. R. (2016). Effects of detergents and the growth of the African catfish Clarias gariepinus. Tropical Journal of Zoology, 19, 198-204.
22. Biggers, W. J., Pires, A., Pechenik, J. A., Johns, E., Patel, P., Polson, T., & Polson, J. (2012). Inhibitors of nitric oxide synthase induce larval settlement and metamorphosis of the polychaete annelid Capitella teleta. Invertebrate Reproduction & Development, 56(1), 1-13.
23. Hooper, C., Day, R., Slocombe, R., Handlinger, J., & Benkendorff, K. (2007). Stress and immune responses in abalone: limitations in current knowledge and investigative methods based on other models. Fish & shellfish immunology, 22(4), 363-379.
24. Esenowo, I. K., & Ugwumba, O. A. (2010). Growth response of catfish (Clarias gariepinus) exposed to a water-soluble fraction of detergent and diesel oil. Environmental Research Journal, 4(4), 298-301.
25. Nkpondion, N. N., Ugwumba, O. A., & Esenowo, I. K. (2016). The toxicity effect of detergent on enzymatic and protein activities of African mud catfish (Clarias gariepinus). J Environ Anal Toxicol, 6(361), 2161-0525.
26. Ettah, I., Bassey, A., Ibor, O., Akaninyene, J., & Christopher, N. (2017). Toxicological and Histopathological Responses of African Clariid Mud Catfish, Clarias gariepinus (Buchell, 1822) Fingerlings Expose to Detergents (Zip and Omo). Annual Research & Review in Biology,
27. George, A., & Uedeme-Naa, B. (2020). Muscle, blood plasma and liver electrolytes of juvenile and adult freshwater catfish, Clarias gariepinus in response to treatment with detergent (Linear alkylbenzene sulfonate). Int J Fish Aquat Stud, 8(2), 285-292.
28. Sobrino-Figueroa, A. (2018). Toxic effect of commercial detergents on organisms from different trophic levels. Environmental Science and Pollution Research, 25(14), 13283-13291.
29. Bardach, J. E., Fujiya, M., & Holl, A. (1965). Detergents: effects on the chemical senses of the fish Ictalurus natalis (le Sueur). Science, 148(3677), 1605-1607.
30. Adamu, K. M. (2009). Sublethal effects of tobacco (Nicotiana tobaccum) leaf dust on enzymatic activities of Heteroclarias (a hybrid of Heterobranchus bidorsalis and Clarias gariepinus). Jordan Journal of Biological Sciences, 2(4), 151-158.

• Qayoom, I., Balkhi, M. H., Shah, F. A., & Bhat, B. A. (2018). Toxicological evaluation and effects of organophosphate compounds on haematological profile of juvenile common carps (Cyprinus carpio var. Communis). Indian Journal of Animal Research, 52(10), 1469-1475.
• Mnkandla, S. M., Basopo, N., & Siwela, A. H. (2019). The effect of persistent heavy metal exposure on some antioxidant enzyme activities and lipid peroxidation of the freshwater snail, Lymnaea natalensis. Bulletin of environmental contamination and toxicology, 103(4), 551-558.

33. Akter, R., Pervin, M. A., Jahan, H., Rakhi, S. F., Reza, A. H. M., & Hossain, Z. (2020). Toxic effects of an organophosphate pesticide, envoy 50 SC on the histopathological, haematological, and brain acetylcholinesterase activities in stinging catfish (Heteropneustes fossilis). The Journal of Basic and Applied Zoology, 81(1), 1-14.
34. Zutshi, B., Prasad, S. G., & Nagaraja, R. (2010). Alteration in haematology of Labeo rohita under the stress of pollution from Lakes of Bangalore, Karnataka, India. Environmental monitoring and assessment, 168(1), 11-19.

 

35. Owolabi, O. D., Omojasola, P. F., Abioye, F. J., & Aina, O. P. (2019). Physiological and bacteriological profile of the fish Clarias gariepinus (Siluriformes: Clariidae) chronically exposed to different concentrations of municipal waste leachate in Nigeria. Cuadernos de Investigación UNED, 11(2), 182-197.
36. George, A. D. I., Akinrotimi, O. A., & Nwokoma, U. K. (2017). Haematological changes in African catfish (Clarias gariepinus) exposed to a mixture of atrazine and metolachlor in the laboratory. Journal of FisheriesSciences. com, 11(3), 0-0.
37. Burgos-Aceves, M. A., Lionetti, L., & Faggio, C. (2019). Multidisciplinary haematology as a prognostic device in environmental and xenobiotic stress-induced response in fish. Science of the total environment, 670, 1170-1183.

 

38. Zutshi, B., Prasad, S. G., & Nagaraja, R. (2010). Alteration in haematology of Labeo rohita under the stress of pollution from Lakes of Bangalore, Karnataka, India. Environmental monitoring and assessment, 168(1), 11-19.

 

39 Little, E. E., Archeski, R. D., Flerov, B. A., & Kozlovskaya, V. I. (1990). Behavioural indicators of sublethal toxicity in rainbow trout. Archives of Environmental Contamination and Toxicology, 19(3), 380-385.

 

40. Saxena, P., Sharma, S., Suryavathi, V., Grover, R., Soni, P., Kumar, S., & Sharma, K. P. (2005). Effect of acute and chronic toxicity of four commercial detergents on the freshwater fish Gambusia affinis Baird & Gerard. Journal of Environmental Science & Engineering, 47(2), 119-124.

 

Reviewer 2 Report

The research presented presents data on descriptive ecotoxicology, searching information on the potential effects of a contaminant released into the aquatic environment.

The serch is simple, but some complements are necessary to make its publication feasible.

METHODS 1 - Include number and date of approval by the Ethics Committee that evaluated the project; 2 - Describe which criterion was used to define the concentrations tested in the study 3 - Make it clearer in the text the volume used in each test container, containing 10 fish per concentration; 4 - Minimally describe the chemical composition of Nittol; 5 – Describe which criteria were used to evaluate the behavioral parameters of fish; 6 - In the method or in the introduction, describe biological and ecological aspects of the H. bidorsalis species, as well as its importance in the country and in the African continent;    RESULTS 7 – In Table 2, the title shown is mortality rate, but what is the number actually shown? absolute number? rate? 8 - Table 3 effectively shows the results of the experiment but obscures Table 2. What is the purpose of Table 2?   DISCUSSION 9 - Swap the presentation order of the Discussion and Conclusion topics since the discussion must come first. 10 - In the discussion, seek to present information about the susceptibility of the species H. bidorsalis to other aquatic species 11 - In the discussion comment on chronic effects of detergents, as well as the toxicity of these products to other species   CONCLUSION 12 - In conclusion, report that although the observed acute effect was in amounts greater than the limit, chronic studies are needed to ensure the safety of the aquatic ecosystem.

 

13 - Describe better what the value of 0.014 presented in the conclusion is about.

Author Response

Response to Reviewers

Response to Reviewer 2

1 Ethical clearance from the Enugu State University of Science and Technology Committee on Experimental Animal Care ESUST/CEAC/12-08-2022/015 was obtained and followed.

2 &3 The toxicity of NTL to H. bidorsalis was carried out according to the Organization for Economic and Development OECD guideline for testing chemicals No. 203 in a semi-static renewal system by using 200L capacity rectangular glass aquaria 100x40x50cm for 10 fish. Five different concentrations (0.8, 1.0, 1.2, 1.4, 1.6 ), and a control 0.00 mgL^-1 were selected and prepared in triplicates for definitive exposures after a range-finding test, and ten (10) fish were exposed to each replicate. Feed was not offered to the fish for 96 h of the test period. Dead fish were immediately removed to prevent deterioration of water quality. The exposure solution was renewed each day and was conducted under the natural photoperiod of a 12:12 light-dark cycle and the physicochemical parameters of the test water were analyzed daily, using standard methods APHA (2005) ^[13].

 

4 Nittol is composed of linear alkyl benzene sulphonate (LABS), sodium tripolyphosphate (SIPP), sodium carbonate, sodium sulphate, sodium per borate and sodium silicate

5 Materials and Methods

2.1. Experimental fish and Nittol detergent

A total of one hundred and eighty (180) fingerlings of Heterobrunchus bidorsalis (mean weight  5.5 ± 0.3g, mean length 6.4 ±0.5cm), were obtained from Onose Farms, Ugheli, Delta State Nigeria, and transported to the Fisheries Laboratory of the Department of Animal/Fisheries Science and Management, Enugu State University of Science and Technology Enugu Lat. 7.4N; 8⁰ 7’5 and long 6⁰ 8’E. 7⁰ 6’ W. The experimental fish were maintained in four fibre-reinforced plastic (FRP) tanks, containing 600 L of de-chlorinated tap water. Aeration was provided to all tanks round the clock to maintain dissolved oxygen contents. Before the commencement of the study, the fish were acclimatized for 14 days and were fed a commercial fish diet composed of 40% crude protein. The faecal matter and other waste materials were siphoned off daily, to reduce ammonia content in the water. Nittol^® was collected from a local market and was prepared according to standard procedures Ivon et al. (2020) [12], and was dissolved in distilled water to make a stock solution for the study. Ethical clearance from the Enugu State University of Science and Technology Committee on Experimental Animal Care ESUST/CEAC/12-08-2022/015 was obtained and followed.

2.2. Acute toxicity test

The toxicity of NTL to H. bidorsalis was carried out according to the Organization for Economic and Development OECD guideline for testing chemicals No. 203 in a semi-static renewal system by using 200L capacity rectangular glass aquaria 100x40x50cm for 10 fish. Five different concentrations (0.8, 1.0, 1.2, 1.4, 1.6 ), and a control 0.00 mgL^-1 were selected and prepared in triplicates for definitive exposures after a range-finding test, and ten (10) fish were exposed to each replicate. Feed was not offered to the fish for 96 h of the test period. Dead fish were immediately removed to prevent deterioration of water quality. The exposure solution was renewed each day and was conducted under the natural photoperiod of a 12:12 light-dark cycle and the physicochemical parameters of the test water were analyzed daily, using standard methods APHA (2005) ^[13]. The test fish were sampled on hours 24, 48, 72 and 96 in each replicate, to determine the toxic effects of the detergent on the fish.  The behavioural responses in the exposed and control fish were observed and recorded daily by Little et al. 19190 [39]. The LC₅₀ was determined by Probit analysis by Finney (1971) [14]. The safe level was estimated by applying the safety application factor (AF), suggested by CCREM (1991) [15].

 

2.3 Haematological Assay

The blood sample was collected by cardiac puncture from juvenile of Clarias gariepinus into ethylene tetra-acetic acid (EDTA) bottles and were estimated for Red blood cells (RBC),  white blood cells (WBC) haemoglobin (Hb) and packed cell volume (PVC),

using the methods of Zutshi et al. (2010) [34]

 

6 It is considered to be an important culture fish in West Africa due to its fast growth, easy adaptation and hardy nature [30]. Blood parameters are important physiological indicators of animals undergoing stressful conditions such as the presence of toxicants since blood acts as a pathophysiological reflector of the whole body [32]. Haematological parameters have been recognized as valuable tools for monitoring fish health [12].

 

7, 8 table 2 has been removed andreplaced by table 3

9,10 and 11    4. Discussion

The display of stressful responses by the test fish corroborates with the reports of other workers [16, 17]. The corroborated report of oxygen reduction in this study along with other workers [18, 19] and increased alkalinity death point, [20, 21] and temperature [22, 23], may suggest that the presence of the detergent in the aquatic environment is responsible for the significant water quality disruption since the control water maintained normal ranges. This development may have elicited insignificant opercula and tail movements to make up for the oxygen tension, due to respiratory dysfunction [24, 25 ], and an attempt to move out of the non-conducive containers, which resulted in the exhaustion of its energy [26, 27], and eventual death of the exposed fish. The median lethal value obtained for the detergent with a positive linear logarithmic probit line correlation signifies a dose-dependent mortality rate in agreement with [28] who reported that detergent has poisonous effects in all types of aquatic life if they are present in sufficient quantities. [29] Reported that most fish will die when detergent concentration approaches 15 parts per million. The median lethal dose in this study is higher than 0.9mg/L reported by [12 ] in sub-adults of C. gagiepinus. The acute toxicity of nonionic detergent oleyl-cetyl alcohol-ethylene oxide condensate (alkaline FI liquid) was reported to range from <0.1--7.9 ppm for the plankton Diaptomus forbesi,  <20.0--7,880.0 ppm for the worm Branchiura sowerbyi, and 19.9--308.0 ppm for the fish Tilapia mossambica.Chronic toxicity of detergents on fish was reported to significantly affect Feeding intake, growth rate and maturity, fecundity and egg maturity [40]

The effects of toxicants on fish can be assessed by the use of haematological indices as it has been reported to be a routine procedure in toxicological research, environmental monitoring and fish health conditions [31, 32]. Fish that inhabit a detergent-polluted environment are particularly susceptible to contaminants that can damage their haematology which causes deleterious changes in their various cellular structures and blood cells [33, 34]. The inhibition of the hybrid catfish erythrocytes and its index component PCV and haemoglobin in this investigation agrees with other workers on exposed fish to detergents [35.36], which may have resulted in haemodilution of the blood by the pollutant, erythropoietic damage, anaemic responses or as a result of reduced gill osmoregulation [37]. Increased catfish WBC observed in the present investigation agrees with other workers on exposing fish to pollutants [38] to restore the immune system of the stressed fish.

 

12             5. Conclusion

The 96 h LC₅₀ value of 1.41 mg/L of Nittol detergent on Heterobrunchus bidorsalis fingerlings caused behavioural responses, mortality and significant effects on the haematology of the test fish. Therefore, its value greater than the safety amount of 0.014 mg/L should be disallowed into receiving culture water for Heterobranchus bidorsalis fingerlings. Although the observed acute effect was in amounts greater than the limit, chronic studies are needed to ensure the safety of aquatic ecosystems.

 

 

 

 

 

 

Find Sample of the revised paper

Abstract: The acute toxicity of the detergent Nittol^® 0.8, 1.0, 1.2, 1.4, 1.6, and 0.0 mg NTL/L of clean water on Heterobrunchus bidorsalis, 5.5 ± 0.3g, 6.4 ±0.5cm were investigated, using semi-static bioassay, for 96 hours in 50 L capacity plastic test bowls. The fingerlings of the same broodstock and age were collected from Onose Farms Limited, Ughelli, Delta State to the University Research Laboratory, Enugu Lat. 7.4N; 8⁰ 7’5 and long 6⁰ 8’E. 7⁰ 6’ W. The test fish were acclimatized for 14 days, and fed at 3% body weight once daily, on a 40% CP commercial diet. Feeding was suspended 24h before and during the range finding and acute tests. The whole set-up was replicated thrice and no death was recorded during the acclimatization period and in the control. A total of 180 fingerlings were used, and 10 fingerlings were assigned to each replicate. The test set-up was monitored daily for water quality parameters, opercular ventilation, tail fin beat frequency, and mortality. Dose and time-dependent behavioural patterns exhibited by the test fish, during the exposure periods include rapid swimming, air gulping, loss of balance and a period of convulsion before death. Significant elevation in pH and temperature, reduction of DO compared to the control (p < 0.05) in the water quality, and dose-dependent early elevation of the tail and fin movements declined towards the end of the experiment. The 96 h LC₅₀ was determined to be 1.41 mg/ L, indicating that the detergent NTL is toxic to the test fish. The haematological parameters were significantly (P<0.05) reduced in the treated ranges of RBC 5.20±0.07 – 8.00±0.02 x 10⁶ mm³ ,  HB 7.53±0.50-10.72±0.14 g/dl, PCV 13.20±0.8.50 – 18.00±0.43 % below their elevated respective controls of 10.50±0.01 x 10 ⁶ mm³, 11.00±0.01 g/dl  and 23.48±0.2.6 % . The white blood cells WBC recorded a significant (P<0.05) increase in ranges of 23.72±0.14 – 51.80±1.9 x 10³ mm³ above its control value of 11.00±0.01 x 10³ mm³Therefore, its value is greater than a safe amount of 0.014 mg/L should be disallowed into receiving culture waters for Heterobrunchus bidorsalis fingerlings.

Keywords: acute toxicity; Nittol detergent; behavior; Heterobranchus bidorsalis; exposure

 

1. Introduction

Detergents generally contain a mixture of a wide variety of chemical substances including, water softeners, processing acids, cleaning agents, optical brighteners, perfumes and colouring agents [1, 2, 3]. These chemicals may act individually or collectively as a buildup of a more complex compound, that becomes too difficult to degrade, or cause eutrophication, in the event of trying to bring about their breakdown [4, 5, 6]. The entry point of detergent effluents from sewages, washing factories, hospitals, refineries, detergent-making industries, and agro-allied industries into the aquatic waters is a major challenge to fish farmers and scientists [7, 8, 9]. Nittol is composed of linear alkyl benzene sulphonate (LABS), sodium tripolyphosphate (SIPP), sodium carbonate, sodium sulphate, sodium per borate and sodium silicate, widely used in many homes, washing premises, and industries which channel its effluents into the receiving waters reserved for fisheries and culture of Heterobrunchus bidorsalis, an important fish in Africa [10, 11]. There is a scarcity of reports on the acute toxicity and safety level of detergents in Nigeria, and therefore the objective of this study is to determine the behaviour, acute toxicity and safety concentration of the detergent Nittol®, on exposed fingerlings of Heterobrunchus bidorsalis. It is considered to be an important culture fish in West Africa due to its fast growth, easy adaptation and hardy nature [30]. Blood parameters are important physiological indicators of animals undergoing stressful conditions such as the presence of toxicants since blood acts as a pathophysiological reflector of the whole body [32]. Haematological parameters have been recognized as valuable tools for monitoring fish health [12].

 

2. Materials and Methods

2.1. Experimental fish and Nittol detergent

A total of one hundred and eighty (180) fingerlings of Heterobrunchus bidorsalis (mean weight  5.5 ± 0.3g, mean length 6.4 ±0.5cm), were obtained from Onose Farms, Ugheli, Delta State Nigeria, and transported to the Fisheries Laboratory of the Department of Animal/Fisheries Science and Management, Enugu State University of Science and Technology Enugu Lat. 7.4N; 8⁰ 7’5 and long 6⁰ 8’E. 7⁰ 6’ W. The experimental fish were maintained in four fibre-reinforced plastic (FRP) tanks, containing 600 L of de-chlorinated tap water. Aeration was provided to all tanks round the clock to maintain dissolved oxygen contents. Before the commencement of the study, the fish were acclimatized for 14 days and were fed a commercial fish diet composed of 40% crude protein. The faecal matter and other waste materials were siphoned off daily, to reduce ammonia content in the water. Nittol^® was collected from a local market and was prepared according to standard procedures Ivon et al. (2020) [12], and was dissolved in distilled water to make a stock solution for the study. Ethical clearance from the Enugu State University of Science and Technology Committee on Experimental Animal Care ESUST/CEAC/12-08-2022/015 was obtained and followed.

2.2. Acute toxicity test

The toxicity of NTL to H. bidorsalis was carried out according to the Organization for Economic and Development OECD guideline for testing chemicals No. 203 in a semi-static renewal system by using 200L capacity rectangular glass aquaria 100x40x50cm for 10 fish. Five different concentrations (0.8, 1.0, 1.2, 1.4, 1.6 ), and a control 0.00 mgL^-1 were selected and prepared in triplicates for definitive exposures after a range-finding test, and ten (10) fish were exposed to each replicate. Feed was not offered to the fish for 96 h of the test period. Dead fish were immediately removed to prevent deterioration of water quality. The exposure solution was renewed each day and was conducted under the natural photoperiod of a 12:12 light-dark cycle and the physicochemical parameters of the test water were analyzed daily, using standard methods APHA (2005) ^[13]. The test fish were sampled on hours 24, 48, 72 and 96 in each replicate, to determine the toxic effects of the detergent on the fish.  The behavioural responses in the exposed and control fish were observed and recorded daily by Little et al. 19190 [39]. The LC₅₀ was determined by Probit analysis by Finney (1971) [14]. The safe level was estimated by applying the safety application factor (AF), suggested by CCREM (1991) [15].

 

2.3 Haematological Assay

The blood sample was collected by cardiac puncture from juvenile of Clarias gariepinus into ethylene tetra-acetic acid (EDTA) bottles and were estimated for Red blood cells (RBC),  white blood cells (WBC) haemoglobin (Hb) and packed cell volume (PVC),

using the methods of Zutshi et al. (2010) [34]

 

2.4. Statistical analysis

Data were expressed as a mean ± standard error and were analyzed using the statistical package SPSS 20.0 computer program (SPSS Inc. Chicago, Illinois, USA). Differences in the test concentrations and control were subjected to a one-way analysis of variance (ANOVA), followed by Duncan range tests to determine the significant mean differences.

3. Results

The behavioural changes of the test fish exposed to the acute doses of NTL are shown in Table 1, while Tables 2 represent the mortality rate and cumulative probit mortality, and Table 3 represents mean water quality. Figure 1 represents the logarithmic probit line for 96 h LC₅₀ during the acute exposure of NTL on the experimental fish and Figure 2 indicates the mean opercular ventilation OVR and Tail fin beat frequency TBF of exposed fish to NTL. Table 4 represents the haematological parameters of the experimental fish exposed to the detergent.

Table 1. The behaviour of Heterobranchus bidorsalis exposed to acute doses of NTL.

Behavioural parameters	Period (hours)
	Concentration mg/L	24	48	72	96
Rapid swimming	1.60	++++	++++	+++	++
	1.40	+++	++	++	+
	1.20	-	++	+	+
	1.00	-	-	+	+
	0.80	-	-	-	+
	0.00	-	-	-	-
Air gulping	1.60	++	++	++++	++++
	1.40	+	+	++	+++
	1.20	-	+	+	++
	1.00	-	-	+	++
	0.80	-	-	-	+
	0.00	-	-	-	-
Loss of balance	1.60	++	++	++++	++++
	1.40	+	+	++	+++
	1.20	-	+	+	++
	1.00	-	-	+	++
	0.80	-	-	-	+
	0.00	-	-	-	-
Period of convulsion	1.60	++	++	++++	++++
	1.40	+	+	++	+++
	1.20	-	+	+	++
	1.00	-	-	+	++
	0.80	-	-	-	+
	0.00	-	-	-	-

Key: -none, + mild, ++ moderate, +++strong, ++++ very strong.

Table 2. Cumulative and probit mortality of experimental fish exposed to acute doses of the detergent NTL.

Concentration mg/L	log concentration	No exposed fish	Replicate1	Replicate2	Replicate3	Cumulative mortality	% mortality	Probit mortality
0	0	30	0	0	0	0	0	0
0.8	-0.09	30	1	2	2	5	16.67	4.01
1	0	30	2	2	3	7	23.33	4.23
1.2	0.07	30	3	4	4	10	33.33	4.56
1.4	0.14	30	4	5	4	13	43.33	4.82
1.6	0.2	30	6	7	7	20	66.66	5.41

 

Figure 1. Logarithmic probit line to determine the 96 h LC 50 of detergent NTL exposed on the fish.

Table 3. Mean water quality parameters.

Parameters	Toxicant concentration (MgL-1)
	1.6	1.4	1.2	1.0	0.8	0.0
Temp. (0C)	27.50±0.50b	27.50±0.35	26.75±0.32	26.50±0.54	26.75±0.48	26.38±0.50a
DO(mgL-1)	4.99±0.06a	5.98±0.05b	5.94±0.05b	6.99±0.09c	6.96±0.07c	8.03±0.07d
pH	9.88±0.18c	9.70±0.15c	9.60±0.15c	7.48±0.13b	7.43±0.17b	6.19±0.32a

Figure 2. Mean OVR/TBF of exposed fish on acute doses of NTL

Dose and time-dependent behavioural patterns exhibited by the test fish during the exposure periods include rapid swimming, air gulping, loss of balance, and a period of convulsion before death (table 1). Significant elevation in pH and temperature and reduction of DO compared to the control (p < 0.05) in the water quality (table 4), and insignificant early elevation of the OVR and TBF movements (p > 0.05), declined towards the end of the experiment (figure 2). The 96 h LC₅₀ determined to be 1.41 mg/ L (tables 2 and 3; figure 1) indicated that the detergent NTL was toxic to the test fish.

 

Table 4: Mean Blood parameters ± SEM of Hetrobranchus bidorsalis exposed to acute concentrations of Nittol for 96 h

Concentration of Nittol (mg/L)

	1.6	1.4	1.2	1.0	0.8	0.00
RBC 106/mm6	5.2±0.07e	6.4±0.08d	7.2±0.06c	7.4±0.05c	8.0±0.2b	10.5±0.1a
WBC103/mm3	51.80±1.9b	50.15±1.4c	45.58±0.75c	38.30±0.16a	23.72±0.14a	11.00±0.1a
Haemoglobin (g/dl	7.53±0.5e	8.24±40.31d	8.95±0.25c	9.32±0.16c	10.72±0.14b	11.00±0.1a
PCV(%)	13.2±8.5d	15.4±1.2c	16.5±1.01c	17.1±1.01b	18.0±0.44b	23.48±2.6a

 

Differences in letter superscript indicate significant differences at P<0.05

 

Haematology

 

The haematological parameters were significantly (P<0.05) reduced in the treated ranges of RBC 5.20±0.07 – 8.00±0.02 x 10⁶ mm³ ,  HB 7.53±0.50-10.72±0.14 g/dl, PCV 13.20±0.8.50 – 18.00±0.43 % below their elevated respective controls of 10.50±0.01 x 10 ⁶ mm³, 11.00±0.01 g/dl  and 23.48±0.2.6 % . The white blood cells WBC recorded a significant (P<0.05) increase in ranges of 23.72±0.14 – 51.80±1.9 x 10³ mm³ above its control value of 11.00±0.01 x 10³ mm³ (Table 5).

 

 

 

 

 

 

 

4. 4. Discussion

The display of stressful responses by the test fish corroborates with the reports of other workers [16, 17]. The corroborated report of oxygen reduction in this study along with other workers [18, 19] and increased alkalinity death point, [20, 21] and temperature [22, 23], may suggest that the presence of the detergent in the aquatic environment is responsible for the significant water quality disruption since the control water maintained normal ranges. This development may have elicited insignificant opercula and tail movements to make up for the oxygen tension, due to respiratory dysfunction [24, 25 ], and an attempt to move out of the non-conducive containers, which resulted in the exhaustion of its energy [26, 27], and eventual death of the exposed fish. The median lethal value obtained for the detergent with a positive linear logarithmic probit line correlation signifies a dose-dependent mortality rate in agreement with [28] who reported that detergent has poisonous effects in all types of aquatic life if they are present in sufficient quantities. [29] Reported that most fish will die when detergent concentration approaches 15 parts per million. The median lethal dose in this study is higher than 0.9mg/L reported by [12 ] in sub-adults of C. gagiepinus. The acute toxicity of nonionic detergent oleyl-cetyl alcohol-ethylene oxide condensate (alkaline FI liquid) was reported to range from <0.1--7.9 ppm for the plankton Diaptomus forbesi,  <20.0--7,880.0 ppm for the worm Branchiura sowerbyi, and 19.9--308.0 ppm for the fish Tilapia mossambica.Chronic toxicity of detergents on fish was reported to significantly affect Feeding intake, growth rate and maturity, fecundity and egg maturity [40]

The effects of toxicants on fish can be assessed by the use of haematological indices as it has been reported to be a routine procedure in toxicological research, environmental monitoring and fish health conditions [31, 32]. Fish that inhabit a detergent-polluted environment are particularly susceptible to contaminants that can damage their haematology which causes deleterious changes in their various cellular structures and blood cells [33, 34]. The inhibition of the hybrid catfish erythrocytes and its index component PCV and haemoglobin in this investigation agrees with other workers on exposed fish to detergents [35.36], which may have resulted in haemodilution of the blood by the pollutant, erythropoietic damage, anaemic responses or as a result of reduced gill osmoregulation [37]. Increased catfish WBC observed in the present investigation agrees with other workers on exposing fish to pollutants [38] to restore the immune system of the stressed fish.

5. 5. Conclusion

The 96 h LC₅₀ value of 1.41 mg/L of Nittol detergent on Heterobrunchus bidorsalis fingerlings caused behavioural responses, mortality and significant effects on the haematology of the test fish. Therefore, its value greater than the safety amount of 0.014 mg/L should be disallowed into receiving culture water for Heterobranchus bidorsalis fingerlings. Although the observed acute effect was in amounts greater than the limit, chronic studies are needed to ensure the safety of aquatic ecosystems.

 

References

1. Yazar, K., Johnsson, S., Lind, M. L., Boman, A., & Liden, C. (2011). Preservatives and fragrances in selected consumer‐available cosmetics and detergents. Contact Dermatitis, 64(5), 265-272.
2. Markwell, J. P., Thornber, J. P., & Skrdla, M. P. (1980). Effect of detergents on the reliability of a chemical assay for P-700. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 591(2), 391-399.
3. Garcia‐Hidalgo, E., Sottas, V., von Goetz, N., Hauri, U., Bogdal, C., & Hungerbühler, K. (2017). Occurrence and concentrations of isothiazolinones in detergents and cosmetics in Switzerland. Contact Dermatitis, 76(2), 96-106.
4. Yangxin, Y. U., Jin, Z. H. A. O., & Bayly, A. E. (2008). Development of surfactants and builders in detergent formulations. Chinese Journal of Chemical Engineering, 16(4), 517-527.
5. Scheibel, J. J. (2004). The evolution of anionic surfactant technology to meet the requirements of the laundry detergent industry. Journal of surfactants and detergents, 7(4), 319-328.
6. Smulders, E., & Rähse, W. (2002). Laundry detergents(p. 52). Weinheim: Wiley-Vch.
7. Kundu, S., Coumar, M. V., Rajendiran, S., Rao, A., & Rao, A. S. (2015). Phosphates from detergents and eutrophication of surface water ecosystem in India. Current science, 1320-1325.
8. Mousavi, S. A., & Khodadoost, F. (2019). Effects of detergents on natural ecosystems and wastewater treatment processes: a review. Environmental Science and Pollution Research, 26(26), 26439-26448.
9. Wind, T. (2007). The role of detergents in the phosphate balance of European surface waters. Official Publication of the European Water Association (EWA).
10. Adewumi, A. A., & Olaleye, V. F. (2011). Catfish culture in Nigeria: Progress, prospects and problems. African Journal of Agricultural Research, 6(6), 1281-1285.
11. Owodeinde, F. G., & Ndimele, P. E. (2011). Survival, growth and feed utilization of two clariid catfish (Clarias gariepinus, Burchell 1822 and Heterobranchus bidorsalis, Geoffroy, 1809) and their reciprocal hybrids. Journal of Applied Ichthyology, 27(5), 1249-1253.
12. Ivon, E. A., Etangetuk, N. A., Ubi, G. M., Anyanwu, C. O., Nkang, A. N., & Ekanem, A. P. (2020). Assessment of histopathological damages in African catfish (Clarias garienpinus) as Influenced by Nittol Detergent Aquatic Pollution in Nigeria. Annual Research & Review in Biology, 1-11.
13. APHA (American Public Health Association, American Waterworks Association and Water Environmental Federation). Standard Methods of Examination of Water and Wastewater. 21st ed.APHA, Washington DC 2005; pp 20001-23710.
14. Finney DJ. Probit Analysis. Cambridge University Press, London 1971; Pp123-125
15. CCREM (Canadian Council of Resources and Environmental Ministry). Canadian Water Quality Guidelines: Canadian Council of Resources and Environmental Ministry, Ottawa: Inland Waters Directorate, Environment 1991; Canada.
16. Oyoroko, E., & Ogamba, E. N. (2017). Effects of detergent containing linear alkyl benzene sulphonate on the behavioural response of Heterobranchus bidorsalis, Clarias gariepinus and Heteroclarias. Biotechnological Research, 3(3), 59-64.
17. Mekuleyi, G. O., & Faleti, B. E. (2018). Effect of detergent surfactant on some selected electrolytes and metabolites of juvenile Heterobranchus bidorsalis. Brazilian Journal of Biological Sciences, 5(10), 339-345.
18. Mohammad, N. A., Muhammed, F. B., Muhhamed, F. B., Eteng, A. O., Zainab, M. S., Bayero, U., ... & Abubakar, M. (2022). PATHOLOGIC LESIONS AND ANTIOXIDANT RESPONSE IN THE LIVER OF AFRICAN CATFISH CLARIAS GARIEPINUS (BURCHELL, 1822) JUVENILE EXPOSED TO COMMERCIAL-GRADE DETERGENT. The Bioscientist Journal, 10(3), 292-305.
19. Gabriel, U. U., Jack, I. R., Edori, O. S., & Egobueze, E. (2009). Electrolytes in selected tissues of Heterobranchus bidorsalis treated with sub-lethal levels of cypermethrin. Ethiopian Journal of Environmental Studies and Management, 2(3).
20. Uedeme-Naa, B., & Deekae, S. N. (2016). Organosomatic index and condition factors of Clarias gariepinus juvenile exposed to chronic levels of linear alkyl benzene sulphonate. Scientia, 16(3), 104-107.
21. Isyaku, B., & Solomon, J. R. (2016). Effects of detergents and the growth of the African catfish Clarias gariepinus. Tropical Journal of Zoology, 19, 198-204.
22. Biggers, W. J., Pires, A., Pechenik, J. A., Johns, E., Patel, P., Polson, T., & Polson, J. (2012). Inhibitors of nitric oxide synthase induce larval settlement and metamorphosis of the polychaete annelid Capitella teleta. Invertebrate Reproduction & Development, 56(1), 1-13.
23. Hooper, C., Day, R., Slocombe, R., Handlinger, J., & Benkendorff, K. (2007). Stress and immune responses in abalone: limitations in current knowledge and investigative methods based on other models. Fish & shellfish immunology, 22(4), 363-379.
24. Esenowo, I. K., & Ugwumba, O. A. (2010). Growth response of catfish (Clarias gariepinus) exposed to a water-soluble fraction of detergent and diesel oil. Environmental Research Journal, 4(4), 298-301.
25. Nkpondion, N. N., Ugwumba, O. A., & Esenowo, I. K. (2016). The toxicity effect of detergent on enzymatic and protein activities of African mud catfish (Clarias gariepinus). J Environ Anal Toxicol, 6(361), 2161-0525.
26. Ettah, I., Bassey, A., Ibor, O., Akaninyene, J., & Christopher, N. (2017). Toxicological and Histopathological Responses of African Clariid Mud Catfish, Clarias gariepinus (Buchell, 1822) Fingerlings Expose to Detergents (Zip and Omo). Annual Research & Review in Biology,
27. George, A., & Uedeme-Naa, B. (2020). Muscle, blood plasma and liver electrolytes of juvenile and adult freshwater catfish, Clarias gariepinus in response to treatment with detergent (Linear alkylbenzene sulfonate). Int J Fish Aquat Stud, 8(2), 285-292.
28. Sobrino-Figueroa, A. (2018). Toxic effect of commercial detergents on organisms from different trophic levels. Environmental Science and Pollution Research, 25(14), 13283-13291.
29. Bardach, J. E., Fujiya, M., & Holl, A. (1965). Detergents: effects on the chemical senses of the fish Ictalurus natalis (le Sueur). Science, 148(3677), 1605-1607.
30. Adamu, K. M. (2009). Sublethal effects of tobacco (Nicotiana tobaccum) leaf dust on enzymatic activities of Heteroclarias (a hybrid of Heterobranchus bidorsalis and Clarias gariepinus). Jordan Journal of Biological Sciences, 2(4), 151-158.

• Qayoom, I., Balkhi, M. H., Shah, F. A., & Bhat, B. A. (2018). Toxicological evaluation and effects of organophosphate compounds on haematological profile of juvenile common carps (Cyprinus carpio var. Communis). Indian Journal of Animal Research, 52(10), 1469-1475.
• Mnkandla, S. M., Basopo, N., & Siwela, A. H. (2019). The effect of persistent heavy metal exposure on some antioxidant enzyme activities and lipid peroxidation of the freshwater snail, Lymnaea natalensis. Bulletin of environmental contamination and toxicology, 103(4), 551-558.

33. Akter, R., Pervin, M. A., Jahan, H., Rakhi, S. F., Reza, A. H. M., & Hossain, Z. (2020). Toxic effects of an organophosphate pesticide, envoy 50 SC on the histopathological, haematological, and brain acetylcholinesterase activities in stinging catfish (Heteropneustes fossilis). The Journal of Basic and Applied Zoology, 81(1), 1-14.
34. Zutshi, B., Prasad, S. G., & Nagaraja, R. (2010). Alteration in haematology of Labeo rohita under the stress of pollution from Lakes of Bangalore, Karnataka, India. Environmental monitoring and assessment, 168(1), 11-19.

 

35. Owolabi, O. D., Omojasola, P. F., Abioye, F. J., & Aina, O. P. (2019). Physiological and bacteriological profile of the fish Clarias gariepinus (Siluriformes: Clariidae) chronically exposed to different concentrations of municipal waste leachate in Nigeria. Cuadernos de Investigación UNED, 11(2), 182-197.
36. George, A. D. I., Akinrotimi, O. A., & Nwokoma, U. K. (2017). Haematological changes in African catfish (Clarias gariepinus) exposed to a mixture of atrazine and metolachlor in the laboratory. Journal of FisheriesSciences. com, 11(3), 0-0.
37. Burgos-Aceves, M. A., Lionetti, L., & Faggio, C. (2019). Multidisciplinary haematology as a prognostic device in environmental and xenobiotic stress-induced response in fish. Science of the total environment, 670, 1170-1183.

 

38. Zutshi, B., Prasad, S. G., & Nagaraja, R. (2010). Alteration in haematology of Labeo rohita under the stress of pollution from Lakes of Bangalore, Karnataka, India. Environmental monitoring and assessment, 168(1), 11-19.

 

39 Little, E. E., Archeski, R. D., Flerov, B. A., & Kozlovskaya, V. I. (1990). Behavioural indicators of sublethal toxicity in rainbow trout. Archives of Environmental Contamination and Toxicology, 19(3), 380-385.

 

40. Saxena, P., Sharma, S., Suryavathi, V., Grover, R., Soni, P., Kumar, S., & Sharma, K. P. (2005). Effect of acute and chronic toxicity of four commercial detergents on the freshwater fish Gambusia affinis Baird & Gerard. Journal of Environmental Science & Engineering, 47(2), 119-124.

 

Round 2

Reviewer 1 Report

"Fish that inhabit a detergent pollutedvenvironments are particularly susceptible to contaminants that can damage their haematology which causes deleterious changes in their various cellular structures and blood cells"

picture of blood cells is recommended to be presented as support for the said statement

Author Response

RESPONSE TO REVIEWER 1

Find herewith the attached copy of the response to reviewer 1

Author Response File: Author Response.docx

Reviewer 2 Report

The comments sent were answered and included in the text. 

I suggest that adjustments be made to the final version, as well as to the order of citations, especially in the Introduction, since the numbering has been changed.

Author Response

RESPONSE TO REVIEWER 2

Find herewith the attached copy of the response to Reviewer 2

Author Response File: Author Response.docx