CURRENT STATUS AND CHALLENGES FOR CONSERVATION AND SUSTAINABLE USE OF BIODIVERSITY

386 Current status and challenges for conservation and sustainable use of biodiversity Framenau, V. W. and Yoo, J. S. 2006. Nyffeler, M., Sterling, W. L. and Dean, D. A. Systematics of the new Australian wolf 1994b. Insectivorous activities of spiders in spider genus Tuberculosa (Araneae: United States field crops.J. appl. Ent. 118: Lycosidae). Invertebrate Systematics, 20(2), 113–128. 185-202. Polotow, D., Carmichael, A. and Griswold, Gertsch, W. J. 1979. American spiders (2nd C. E. 2015. Total evidence analysis of the ed.). New York, Van Nostrand phylogenetic relationships of Lycosoidea spiders (Araneae, Entelegynae). Invertebrate He,J. and Song,,D. 1996. The phylogenetic Systematics, 29(2), 124-163. relationship of Chinese wolf spiders. Zhuxingxuebao, 5(1), 3-20.Vink C.J. 2001. A Saez, N. J., Senff, S., Jensen, J. E., Er, S. Y., revision of the genus Allotrochosina Herzig, V., Rash, L. D. and King, G. F. 2010. (Lycosidae: Araneae). Invertebrate Spider-venom peptides as therapeutics. Taxonomy, 15:461-466. Toxins, 2(12), 2851-2871. Jogar K., Metspalu L. and Hiiesaar K. 2004 Vink, C. J., Mitchell, A. D. and Paterson, A. .Abundance and dynamics of wolf spiders M. 2002. A preliminary molecular analysis of (Lycosidae) in different plant communities. phylogenetic relationships of Australasian Agron. Res., 2:45-152. wolf spider genera (Araneae, Lycosidae). The Journal of Arachnology, 30(2), 227-238. Luczak, J.1979. Spiders in agrocoenoses. Polish Ecological Studies. Whitcomb, W. H. 1974. Natural populations of entomophagous arthropods and their effect Malik, T. and Barhanpurkar, S. 2018. Spider on the agroecosystem. In F. G.Maxwell& F. Silk-Properties and Uses Spider Silk- A. Harris (eds.), Proc. summer inst. on Properties and Uses. biol.control of plant insects and diseases: 150–169. Jackson, University Press of Marshall S.D. and Rypstra A.L. 1999. Mississippi. Patterns in the distribution of two wolf spiders (Araneae, Lycosidae) in two soybean Whitehouse, M.E.A. and Lawrence, L. 2001. agro-ecosystems. Environ. Entomol. Are spiders the perfect predator? Aust. 28:1052-1059. Cotton Grower, 22:30-33 Murphy N.P., Framenau V.W., Donellan Wise, D. H. 1993 Spiders in ecological webs. S.C., Harvey M.S., Park Y.C. and Austin Cambridge, Cambridge University Press. A.D. 2006. Phylogenetic reconstruction of the wolf spiders (Araneae, Lycosidae) using World Spider Catalog. 2020. World Spider sequences from 12S rRNA, 28S rRNA and Catalog. Version 20.0. [online] Available at NADH1 genes: implications for http://wsc.nmbe.ch. (Accessed on 11th classification, biogeography and the January 2020) evolution of web-building behavior. Mol. Phylogenet. Evol., 38: 583-602. Yan, Y. C. and Yan, H. M. 2007. Phylogenetic relationships of Wadicosa in Nyffeler, M. and Benz, G. 1987. Spiders in Chinese wolf spiders (Lycosidae, natural pest control: A review. J. appl. Ent., Wadicosinae). Acta Zootaxonomica Sinica, 103: 321–339 32, 996-999. Nyffeler, M., Sterling, W. L. and Dean, D. A. Young, O. P. and Edwards, G. B. 1990. 1994a. How spiders make a living. Envir.Ent. Spiders in United States field crops and their 23: 1357–1367. potential effect on crop pests. J. Arachnol., 18: 1–27. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.381-386 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 387 ISBN 978-93-5396-871-7 CHAPTER 56 MOLECULAR IDENTIFICATION AND OPTIMIZATION OF AMYLASE PRODUCING BACILLUS GINGSHENGIIS NB12 USING RESPONSE SURFACE METHODOLOGY Divya Balakrishnan, Shilpa Shaji V S and Anu Krishna K R Department of Biotechnology, Sree Narayana College, Kollam- 691001, Kerala, India. *Correspondence E-mail: [email protected] ABSTRACT Among different types of enzymes obtained from microbial sources, amylases are the most widely used in industries as well as on commercial basis. Amylase production from bacteria is economical as the enzyme production rate is higher as compared to other microorganism. The aim of the current study was to investigate the genotypic analysis of starch degrading bacteria from mangrove soil. The strain was identified as catalyse and gram positive bacteria. On the basis of phylogenetic analysis using the 16S rRNA, this strain was identified as BacillusgingshengiiSNB12.α-Amylase production by Bacillus gingshengii SNB12 was cultivated in medium containing 1% soluble starch as a carbon source. The process parameters such as incubation day, percentage of inoculum and starch were optimized for maximum amylase production using central composite design (CCD) of response surface methodology (RSM).Total 20 experiments were carried out in conical flask and a three dimensional response surface was generated to determine the effect of process variables on amylase production. Solid state fermentation (SSF) was performed using 5.0 g of Sugarcane bagasse (5g) inoculated with 1x107spores. Incubation day and percentage of starch were the most significant (p<0.05) parameter and the maximum interaction occurred between incubation day and percentage of inoculum. The results of the study indicated that amylase is maximized (274 U/ml) at optimized levels of 2, 2.5 and 3 days for percentage of inoculum, starch and incubation periods, respectively. Key words: Amylase, Phylogenetic analysis, Baciliius sps, Response surface methodology Introduction various industrial application. Amylases obtained from bacterial species are known for The starch hydrolytic amylases (α-amylase, greater stability, short growth period, high β-amylase and glucoamylase) are one of the productivity, reduced cost of production and most extensively used enzymes in present- easy manipulation of bacterial genes. day biotechnology. Amylases hydrolyze α- Increasing industrial demand for microbial 1,4glycosidic linkages of starch to yield amylases has been observed due to their dextrin and different monomeric products. specificity of reaction, mild conditions Microbial amylases are of immense value in prerequisite for the reaction, and less energy industries due to bulk enzyme production and consumption than the conventional non- easy way of genetic manipulation (Pranay et enzymatic chemical methods. al., 2019). Although amylases are produced from different sources such as Among various microbial sources Bacillus microorganisms, plants and animals sp. is ideal for industrial production of microbial amylase are most suitable for enzymes due to attributes such as short Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

388 Current status and challenges for conservation and sustainable use of biodiversity fermentation cycle, consistency, efficient 2007) which is a version with graphical user enzyme activity under stress conditions, and interface in Clustal series for multiple safe and easy handling (Souza and sequence alignment. The phylogenetic tree Magalhaes, 2014). Bacteria and fungi tend to was constructed using ClustalX with secrete amylases outside the cells to perform neighbor-joining method maximum extra cellular digestion of starch into sugars. likelihood, and presented using MEGA X Extensive application of amylase in various version (Kumar et al., 2018). The phenotypic industrial sectors such as food, starch characterization and 16S rRNA indicated that liquefaction, saccharification, brewing, bacterial cultures belongs to genera Bacillus. detergent, paper, textile and distilling The amylase production from different industries, has brought about a greater Bacillus sp. shows a great deal of variation attention for the increase in the indigenous because the production of amylase depends production of α-amylase. (Gupta et al., on the composition of medium and other 2003). Bacillus is a common bacterial source physical parameters. The present work for industrial amylase production. However, focuses on the molecular identification and different strains have different optimal optimization of bacterial strain amylase growth conditions and enzymatic production production using SSF. profile. Reportedly, Bacillus strains have been extensively used in industrials to Materials and Methods produce α-amylase which includes B. amyloliquefaciens, B.subtilis, B.licheniformi, Collection and isolation of soil sample B. stearothermophilus, and B. megaterium Sample collection Soil samples were Solid-state fermentation (SSF) uses solid randomly collected 15 cm below the soil substrates at low moisture levels and requires surface and kept in polystyrene bags from suitable agro industrial materials as solid various mangrove areas near Astamudi lake, substrate (Hamrouni et al., 2019). Various Kollam District, Kerala, India Isolation of agro industrial waste used as substrate for bacteria was performed by serial dilution in SSF which includes sunflower cake olive, which 1 g of the soil sample were weighed (Kachrimanidou et al.,2013), sugarcane and suspended into 9ml of sterile distilled bagasse (Leite et al., 2019) fruit’s peels and water taken in test tube and shaken pulps (Papadaki et al., 2019) and cereal brans vigorously with the help of a vortex machine (Socco let al., 2017). In this process, (10-1). Similarly, dilutions were carried out microorganisms grow and produce a wide up to 10-6. All the dilutions were placed on variety of products such as mushrooms Nutrient agar medium (Himedia M-001) and (Postemsky et al., 2017) microbial oil incubated at room temperature for 3days. (Tsakona et al., 2014), preservatives like fumaric acid (Papadaki et al., 2018) and Bacterial inoculums preparation enzymes (Haque et al., 2016) reducing the cost of production. 50 ml of nutrient broth (13 g/l at pH 7.4 ± 0.2) was prepared and sterilized in an Identification of the organisms was done on autoclave (NatSteel (Big), India) at 15 lbs/in the basis of 16S rDNA based molecular 2 pressure, 121°C for 20 min. Freshly grown technique. The sequence alignment was 1ml of the inoculum were aseptically carried out using ClustalX (Larkin et al., transferred to media and incubated at 37°C Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 389 overnight at 120 rpm in a incubator shaker containing 6.0 g Na2HPO4, 3.0 g KH2PO4, (Lab Tech , India). 5.0 g NH4Cl, 0.5 g NaCl, 0.15 g CaCl2, 0.25 g MgSO4 7H2O, 0.2 g Casein hydrolysate, Solid Substrate preparation and 0.10g Yeast extract, 10 g Starch per liter of Pretreament distilled water at pH 7.0. Among different agricultural residues, Enzyme production in SSF Sugarcane bagasse was used as solid substrate (support and nutrient source) for The SSF process was carried out in 250 ml SSF. Due to the water content, the residues Erlenmeyer flask using Sugarcane bagasse. were sun-dried for 2 days and then oven After proper agitation of the substrate with dried at 80°C for 24 h. The dried sugarcane 4ml of the media, it was autoclaved at 121°C bagasse was stored in air-tight container until for 15 min. Later it was allowed to cool and required. The moisture content of the solid incubated with 1ml of 24 h old bacterial substrate was estimated by drying 5 g of culture. Subsequently, incubation was carried substrate to a constant weight at 70 °C for 24 out at 37 °C for 7 days. The SSF media flask h and the dry weight was recorded. To fix the was shaken after every 24 h for uniform initial moisture content of the solid medium, mixing up of the substrate and inoculum. 5 g of substrates was soaked with 5 mL inoculums and 4mL fermentation media. Enzyme extraction After soaking, the solid substrate was again dried as described above and the percent The fermented mass was mixed with 0.1M moisture content was calculated using phosphate buffer (pH 7.0) to a volume of 100 Equation as follows mL for each flask and agitated at 180 rpm for 30 min. The slurry thus obtained was Initial moisture content (%) = squeezed through a cheese cloth, followed by centrifugation at 10,000 × g for 10 min at W Final - W Initial 100% 4°C. The supernatant was collected in vials W Initial and stored at 4°C for further analysis. Where W finalis the weight of the dried solid ������-Amylase Assay substrate after soaking and Winitialis the weight of dried solid substrate before The ������-amylase activity was estimated by soaking. From the above procedure, it was analysing reducing sugar released during found that the initial moisture content of hydrolysis of 1.0% (w/v) starch in 0.1M different solid substrate. phosphate buffer (pH 7.0) by enzyme (cell- free supernatant) incubated at 80oC for Quantitative screening for amylase 10min.The amount of reducing sugar level released in the mixture was determined by The amylase activity of the strain was the dinitrosalicylic acid (DNS) method demonstrated on starch-casein agar plate Miller. Absorbance at 550 nm was recorded containing 1% soluble starch after 4 days of by using UV-visible spectrophotometer (UV- incubation at 30°C, the plates were stained 1700 Pharmaspec Shimadzu) and activity with Gram’s iodine solution (0.2% I2 and 2% was calculated from a standard curve using KI). ������-Amylase production in solid state maltose as the standard. One unit (U) of fermentation (SSF) was carried out in 250mL enzyme activity was defined as the amount of Erlenmeyer flask using basal medium enzyme required for the liberation of 1 ������mol Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

390 Current status and challenges for conservation and sustainable use of biodiversity reducing sugar as maltose per minute under Statistical analysis and modeling standard assay conditions. All experiments were carried out in triplicate and the data The data obtained from the RSM on - presented are average values. amylase production was finally subjected to the analysis of variance (ANOVA). The Optimization of incubation period, results of RSM were used to fit a second percentage of inoculums, Starch and order polynomial equation (1) as it maltose by applying RSM represented the behavior of such a system more appropriately. The characterization of three different factors for amylase production was optimized by Y= β0 + β1A + β2 B + β3 C + β1 β1 A2 + β2 β2 B2 applying the RSM. The statistical approach using the Central Composite Design (CCD) + β3 β3C2 + β1 β2 AB + β1 β3 AC + β2 β3 BC (1) developed by 'Design Expert 10.0 software Stat Ease, Inc. Minneapolis, USA, was used Where Y was response variable, β0 was to generate and analyze the 24 experimental intercept, β1, β2 and β3 were linear design. The statistical model was obtained coefficients, β1,1, β2,2 and β3,3 were with three independent variables [incubation squared coefficient, β1,2, β1,3 and β2,3 were period (A), Percentage of inoculum (B) and interaction coefficient and A, B, C, A2, B2, Starch (C). Each factor in this design was C2, AB, AC and BC were the level of studied at four different levels (Table 1). All independent. The Fisher's test value was used the variables were taken at a central coded to determine the statistical significance of the value considered as zero. The minimum and model equation and the production of maximum ranges of variables were used. The variance explained by the model was full experimental plan with respect to their specified by the multiple coefficient of values in coded form is shown in Table 2. determination, R squired (R2) value. Design After the completion of experiments, the Expert (ver, 10.0; STATEASE INC; average of amylase production was taken as Minneapolis, MN, USA) was used in this the dependent variable or response. investigation. Coded Independent variables Factor Levels Independent varibles -α Coded factor level +α 1.31 -1 +1 4.681 Incubation day 0.02 24 5.02 Percentage of inoculum 0.659 14 0.659 12 Starch Table 1. Range of the values for the response surface methodology. Effect of incubation period on enzyme loop full of bacterial strain from a slant and production was incubated at room temperature for 24 h in an orbital incubator shaker at 120 rpm. The inoculum was prepared in soluble (Orbiteck, India).Sugarcane biagasse (5 g) amylase production medium by transferring a Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 391 was taken in conical flask, moistened with 9 genomic DNA using universal primer ml of amylase medium to provide 60% (Integrated DNA Technology, India) 16S- moisture holding capacity (MHC) and the RS-F-5-CAGGCCTAACACATGCAAGTC- contents were mixed thoroughly. The bottles 3 and 16S-RS-R 5- were autoclaved at 15 lb pressure for 30 min. GGGCGGWGTGTACAAGGC-3 in a PCR After cooling at room temperature, (30°C) thermal cycler (Nubel et al., 1996).PCR these were inoculated with 1% (v/w) amplification reactions were carried out in a inoculum and incubated under static 20 µl reaction volume which contained 1X condition at room temperature for 7 days. PCR buffer, 0.2mM each dNTPs, 2.5mM Triplicate bottles were maintained for each MgCl2, 1 unit of AmpliTaq Gold DNA treatment. The contents in the bottle were polymerase enzyme, 0.1 mg/ml BSA, 4% periodically mixed by gentle tapping. At DMSO, 5pM of forward and reverse primers interval of 24 h, the enzyme was extracted and 2µl of template DNA. Amplification of twice with 25ml of 0.1M Phosphate buffer. DNA for 20 µL reaction was carried out Initially the substrate were soaked in under the following condition. The PCR Phosphate buffer for 30 min at 30°C and kept conditions were set for 35 cycles with initial in a rotatory shaker at 150 rpm. The slurry denaturation at 95°C for 5 min then final thus obtained was squeezed through a wet denaturation of 95°C for 30 sec, annealing at cheese cloth. The pooled enzyme extract 60ºC for 40 sec and final extension at 72ºC were centrifuged at 10000 rpm for 20 min in for 60 sec using Thermal cycler (GeneAmp refrigerated centrifuge and the clear PCR System 9700, Applied Biosystems). The supernatant was further stored for enzyme PCR products were detected on 0.8% agarose assay. gel. Effect of percentage of inoculum The PCR products were sent to for The influence of percentage of inoculum on sequencing. The 16S rRNA gene sequences the enzyme titer was evaluated by varying the inoculum percentage of the substrate from 1 were compared with other 16S rRNA gene to 5%. Different percentages of inoculum were incubated for 7 days at room sequences available in Gen Bank by using the temperature. BLASTN program Effect of Starch concentration (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and The effect of starch concentration on the enzyme titer was evaluated by varying the aligned with similar sequences by using starch concentration from 1 to 5% Different percentage of starch were incubated with the CLUSTX program. The phylogenetic trees sugarcane for 7 days at static condition in room temperature. were constructed by neighbor-joining Molecular identification using 16������rRNA statistical method using MEGA Gene Sequencing and Phylogenetic Analysis X(https://www.megasoftware.net/dload_win_ The total genomic DNA was extracted using gui). In order to determine the stability of NucleoSpin® Tissue Kit (Macherey-Nagel). 16S rDNA sequence were amplified from phylogenetic tree, the sequence data were sampled 1000 times for bootstrap analysis using MEGA X with 50% cut-off (Kumar et al., 2018). Results and Discussion Isolation and Screening of amylase producing Bacteria. A total of 20amylaseproducing bacterial strains were successfully isolated from Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

392 Current status and challenges for conservation and sustainable use of biodiversity different locations of mangrove soil. Among Among the isolates further screened for its the 20 strains isolated, 5 strains found to be capability to degrade starch only five isolates amylase producers based on their zone were selected based on the zone diameter formation on starch agar media (Fig1).The listed in Table 1.Among twenty strains total bacterial population (CFU/ml) was isolated, SNB12 were selected for further observed at 10-5 dilution for all the tested soil optimization and phylogenetic analysis based samples. The population was high of on the hydrolyzing zones on agar plates 2.97×107CFU/ml in the soil sample (SNB12) containing starch substrate (Fig 1). followed by the soil sample (SNB6) with 1.98×107CFU/ml. The least bacterial population was found in (SNB8) soil sample, where only1.3×107CFU/ml was observed. Screeningof amylase producing bacteria Isolates Screening test Diameter zone (mm) SNB1 + 3.6 SNB3 + 2.5 SNB7 + 1.5 SNB8 + 3.1 SNB12 + 4.2 Table 1. The hydrolyzing zone diameter of isolates Fig.1 A. Initial screening of bacterial strain in nutrient agar plate ; B. Hydrolyzing zones produced by bacterial strains on starch agar plates after flooded with iodine solution. Optimization using Response surface with the mean predicted and observed methodology responses are presented in Table 2. In the present work, experiments were planned to The Central Composite design was employed obtain a quadratic model consisting of 23 to study the effect of three independent trials. The plan includes 20 experiments and fermentation variables (incubation period, two levels of concentration for each of the Percentage of inoculum and Starch) along Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 393 component. In order to analyze the combined after the ANOVA gave the level of α- effect of these components, experiments were amylase production as a function of the initial performed at different combinations. Table 4 values of incubation period, percentage of summarizes the Central Composite inoculum and temperature. The final experimental plan along with the predicted response equation that represented a suitable and observed response for each individual model for α-amylase production was as experiment. It shows the production of alpha- below:Y=279.23-19.09A- amylase (mg/ml) corresponding to combined 10.13B+4.44C+3.40AB-0.52AC+3.95BC- effect of all three components in the specified 97.44A2-103.65XB2+4.35C2 ranges. The regression equations obtained Where Y was enzyme production, A was incubation period (h), B was percentage of inoculum (%) and C was starch (mg/ml). Std Run Incubation Percentage of Starch Amylase Predicted day inoculum (mg/ml) value 91 1.68 2.5 2.25 135.59 82 4 4 2.5 106.3 64.54 73 2 4 2.5 58.2 96.97 23 4 3 2.5 2.25 107.7 279.93 25 4 1 2 279 70.16 12 6 3 4.4 2.25 64.9 86.38 13 7 3 2.5 1.92 84 280.91 58 2 1 2.5 276.9 116.13 15 9 3 2.5 2.25 127.5 279.23 6 10 4 1 2.5 269.8 70.10 14 11 3 2.5 2.5 68.3 292.61 17 12 3 2.5 2.25 284 279.23 22 13 3 2.5 2.25 281.9 279.23 4 14 4 4 2 286.3 48.80 10 15 4.3 2.5 2.25 42.9 85.34 16 16 3 2.5 2.25 102 279.23 20 17 3 2.5 2.25 286.3 279.23 18 18 3 2.5 2.25 279.4 274.23 21 19 3 2.5 2.25 284.9 280.16 1 20 2 1 2 286.54 114.10 24 21 3 2.5 2.25 125.9 279.23 11 22 3 0.525889 2.25 297.7 113.04 19 23 3 2.5 2.25 102.8 279.23 3 24 2 4 2 273.9 79.13 86.4 Table 2. Experimental design and result of CCD of response surface methodology Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 394 The analyses of variance (partial sum of The \"Lack of Fit F-value\" of 11.68 implies squares) were shown in Table 3.The Model the Lack of Fit is significant. F-value of 159.28 implies the model is significant. There is only a 0.01% chance that There is only a0.10% chance that a \"Lack of an F-value this large could occur due to Fit F-value\" this large could occur due to noise. Values of \"Prob> F\" less than 0.0500 noise. indicate model terms are significant. In this case A, B, A2, B2 are significant model terms. The Response surface was generated by Values greater than 0.1000 indicate the plotting the response (α-amylase production) model terms are not significant. If there are on the z-axis against any two independent many insignificant model terms (not counting variables while keeping the other those required to support hierarchy),model independent variable at their zero level. reduction may improve your model. Therefore, three response surfaces were obtained by considering all the possible combinations. Source Sum of squares df Mean square F value P- value significant Model 2.250E+005 9 25003.63 159.28 <0.0001 significant A-Incubation day 4178.20 1 4178.20 26.62 0.0001 significant B- % of inoculum 1176.63 1 1176.63 7.50 0.0160 significant C- Starch 226.39 1 226.39 1.44 0.2497 not significant AB 92.48 1 92.48 0.59 0.4555 not significant AC 2.20 1 2.20 0.014 0.9073 not significant BC 124.82 1 124.82 0.80 0.3876 not significant A2 69982.59 1 69982.59 445.81 0.0001 significant B2 79181.65 1 79181.65 504.41 0.0001 significant C2 139.23 1 139.23 0.89 0.3623 Residual 2197.71 14 156.98 Lack of fit 1904.28 5 380.86 11.68 0.0010 significant Cor Total 2.272E+005 23 Std.Dev 12.53 R-squared 0.9903 Mean 182.26 Adj R-squared 0.9841 C.V% 6.76 Pred R-squared 0.9369 PRESS 14334.46 Adeq Precision 30.146 -2 Log Likelihood 176.52 BIC 208.30 AICc 213.44 Table 3. Analysis of Variance (ANOVA) of alpha amylase production depicted from CCD Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 395 An adequate precision of 30.146 for α- Effect of amylase production on amylase production was recorded. The incubation day and inoculum predicted R2 of 0.9365 was in reasonable agreement with the adjusted R2 of 0.9841. To investigate the interaction and to visualize This indicated a good agreement between the the effects of the two factors on amylase experimental and predicted value for α- activity, a graphical representation of RSM is amylase production. The model F- value of presented in Fig. 2 depicts three dimensional 159.28 and values of prob> F (< 0.05) diagram of calculated response surface from indicated that the model terms were the interaction between incubation day and significant. In analyzing the effect of percentage of inoculum while keeping the variables, normally the 3-D contour plots other variable (Starch) at ‘0’ level. The were used. response surface obtained in this study was convex in nature, which suggested that the 3-D contour plots represent the relationship optimum conditions were well defined. A of response surface function of two variables; gradual increase in α-amylase production was meanwhile another variable is maintained at observed when incubation period was zero level. The coordinates of the central increased up to third day of incubation, and point within the highest contour levels in thereafter, decline in enzyme production was these figures represent the optimum condition notified. it can be observed that the enzyme and concentrations of respective parameters. activity was increased upon the percentage of The circular shape of the curve indicates that inoculum at the range of 1 to 4%. Further no interaction occurred, whereas the elliptical increase in the percentage of inoculum and shape indicated good variation of the two prolonged incubation day has a positive variables. effect on the response until certain optimum value, whereas extremely high values of Design-Expert® Software these variables will inhibit amylase Factor Coding: Actual production. Amylase (U/dgs) Design points above predicted value Design points below predicted value 286.9 42.9 300 250 X1 = A: Aincubation day 200 X2 = B: % of inoculum Actual Factor C: Starch = 2.25 Amylase (U/dgs) 150 100 50 0 4 3.4 2.8 2.2 1.6 4 3.5 B: % of inoculum (mg/ml) 3 2A.5: Aincubation day (mg/ml) 12 Figure 2: Response surface plot showing the effect of incubation day and percentage of inoculum. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 396 Effect of amylase production on However, further increase in the incubation incubation day and starch day had no effect in the enzyme production. The concentration of starch from 2 to 2.5% In figure 3, the activity of alpha amylase resulted no increase in the enzyme increased uponDesign-Expert® Software production. Factor Coding: Actual the incubation day and reached maximum onAmylase (U/dgs) Design points above predicted value the third day. Design points below predicted value 286.9 42.9 300 250 X1 = A: Aincubation day X2 = C: Starch Actual Factor Amylase (U/dgs) 200 B: % of inoculum = 2.5 150 100 50 0 2.5 2A.5: 3 3.5 4 2.4 2.3 Aincubation day (mg/ml) C: Starch (mg/ml) 2.2 2.1 22 Figure 3: Response surface plot showing the effect of incubation day and starch Effect of amylase production on starch third day of incubation. Later on increase on and inoculum the incubation day showed a gradual decrease in enzyme production Whereas, starch When the percentage of inoculum was concentration of 2 to 2.5% showed no increase in the enzyme production. The increased, increase in amylase production details are shown in fig 4. were detectedDesign-Expert® Software and reached optimum on the Factor Coding: Actual Amylase (U/dgs) Design points above predicted value Design points below predicted value 286.9 42.9 400 300 X1 = B: % of inoculum X2 = C: Starch Amylase (U/dgs) Actual Factor A: Aincubation day = 3 200 100 0 C: St2a.r5ch2(.4mg2/.m3 l) 2.2 2.1 21 1.6 B:2%.2 2.8 3.4 4 of inoculum (mg/ml) Figure 4: Response surface plot showing the effect of percentage of inoculum and Starch Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 397 Validation of model was carried out under The amylase producing bacteria were the conditions predicted by the response recovered fromstarch agar media. The surface model. The experimental values were bacterial stain was further subjected to very close to the predicted values; hence, the molecular identification procedure which model was successfully validated. The includes extraction of total DNA, validation of the statistical model and amplification by 16S rDNA primer, finally regression equation was performed by taking the comparison of phylogenetic sequence. incubation day (third day), Percentage of inoculum (2.5%) and starch (2%) in the DNA extraction experiment. The predicted response for α- amylase production was 292.21 mg/ml, while Total genomic DNA was extracted from the actual (experimental) response was SNB12 Fig 5A. For further identification the 284.67 mg/ml, thus proving the validity. amplification of the genomic DNA was performed using 16S rDNA primer (Fig 5B). Molecular identification of keratinase Later, gene sequencing and construction of producing bacteria phylogenetic tree was done for the species identification. Fig 5. A: Genomic DNA from bacterial strain using DN A isolation kit loaded in 0.8% agarose gelB: PCR amplification of 16s rDNA fragment from bacterial sample. Sequence Analysis gene sequence of the isolate SNP1 showed high levels of sequence similarity with In this work, we have provided the molecular members of the genus Bacillus. The identification of bacteria on the basis of 16S ‘BLASTn’ tool was used for sequence rDNA analysis. The results of the forward assignment against NCBI database and and reverse primers were aligned together in highest-scoring hit from each query is taken Bioedit tool to retrieve the complete aligned for the bacterial identification. The Blast sequences. The NCBI BLAST was result predicted based on percent match given performed to confirm identity of specimens bacterial sequence belongs to Bacillus genus (Altschul, et al.,1990).The 16S rRNA gene and alignment of gene sequence with 10 sequence was compared to the Gen-bank of closely related gene sequence was performed database using the BLAST. The 16S rRNA Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

398 Current status and challenges for conservation and sustainable use of biodiversity and phylogenetic tree was constructed using identified as Bacillus qingshengii MEGA X (Fig 6.) The bacterial strain was Fig 6: Evolutionary relationships of taxa The evolutionary history was inferred using using the Maximum Composite Likelihood the Neighbor-Joining method (Saitou and method (Tamura et al., 2004). and are in the Nei, 1987). The optimal tree with the sum of units of the number of base substitutions per branch length = 0.53056807 is shown. The site. The analysis involved 33 nucleotide percentage of replicate trees in which the sequences. Codon positions included were associated taxa clustered together in the 1st+2nd+3rd+Noncoding. All ambiguous bootstrap test (500 replicates) are shown next positions were removed for each sequence to the branches (Felsenstein, 1985). The tree pair. There were a total of 1536 positions in is drawn to scale, with branch lengths in the the final dataset. Evolutionary analyses were same units as those of the evolutionary conducted in MEGA X (Kumar et al., 2014). distances used to infer the phylogenetic tree. The evolutionary distances were computed Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Molecular identification and optimization of amylase producing bacillus gingshengiis nb12 using … 399 Acknowledgements Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, The authors wish to thank the people who H., Valentin, F., Wallace, I.M., Wilm, A., helped in sampling the soil from different Lopez, R., Thompson, J.D., Gibson, T.J., mangrove areas. We also acknowledge some Higgins, D.G. 2007. Clustal W and Clustal X of the MS students and colleagues from our version 2.0. Bioinformatics, 23:2947–2948. department for their support to complete the study successfully. Leite, P., C. Silva, J. M. Salgado, and I. Belo. 2019. “Simultaneous production of References lignocellulolytic enzymes and extraction of antioxidant compounds by solid-state Felsenstein, J. 1985. Confidence limits on fermentation of agro-industrial wastes,” phylogenies: an approach using the bootstrap. Industrial Crops and Products, 137: 315–32 evolution., 39(4):783-791. Luang-In., Yotchaisarn, M., Saengha,W., Gupta,R., Gigras,P., Mohapatra, H., Udomwong, P., Deeseenthum,S., Maneewan, Goswami,V.K., and Chauhan, B. 2003. Microbial α-amylases: a biotechnological K. 2019. Isolation and Identification of perspective. Process Biochem., 38:1599– 1616. Amylase producing bacteria from soil in Hamrouni, R. and J. Molinet, L. 2019. Nasinuan Community Forest, “Production of coconut aroma in solid-state cultivation: screening and identification of MahaSarakham, Thailand. Biomed Trichoderma strains for 6-pentyl-alpha- pyrone and conidia production,” Journal of Pharmacol J., 121(3): 325-342 Chemistry, vol. Article ID 8562384, 7 pages. Miller, G.L, 1959. Use of dinitrosalicylic Kachrimanidou, V., N., Kopsahelis, A. and acid reagent for determination of reducing Chatzifragkou. 2013. “Utilisation of by- sugar. Anal Chem., 31:426-428. products from sunflower-based biodiesel production processes for the production of Nu¨bel, U., B. Engelen, A. Felske, J. Snaidr, fermentation feedstock,” Waste and Biomass A. Wieshuber, R. I. Amann, W. Ludwig, and Valorization , 4(3):529–537. H. Backhaus. 1996. Sequence heterogeneities of genes encoding 16S rRNAs in Kumar, S., Stecher, G., Michael Li, M., Paenibacilluspolymyxa detected by Knyaz, C. and Tamura, K. 2018. MEGA X: temperature gradient gel electrophoresis. J. Molecular Evolutionary Genetics Analysis Bacteriol., 178:5636–5643. across Computing Platforms. Mol Bio Evol., 35(6): 1547–1549 Papadaki,V., Kachrimanidou,S. Kumar,S., Stecher, G., Li, M., Knyaz, C and Papanikolaou,A. Philippoussis and P. Tamura, K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across Diamantopoulou. 2019 “Upgrading grape Computing Platforms. Molecular Biology and Evolution, 35( 6) ; 1547–1549. pomace through Pleurotus spp. cultivation for the production of enzymes and fruiting bodies,” Microorganisms, 7(7): 207. Postemsky, P. D., Bidegain, M. A., González-Matute,R., Figlas,ND., and. Cubitto, M. 2017. A “Pilot-scale bioconversion of rice and sunflower agro- residues into medicinal mushrooms and laccase enzymes through solid-state Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

400 Current status and challenges for conservation and sustainable use of biodiversity fermentation with Ganodermalucidum,” Bioressource Technology, ( 231): 85–93. Pranay, K., Padmadeo, S.R., Jha,V. and Prasad, B. 2019. Journal of Applied Biology and Biotechnology,7(04): 57-62. Saitou, N. and Nei, M. 1987. The Neighbor- joining method a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406-425. Souza P.M. and Magalhaes P.O. 2014. Applications of microbial α-amylase in industry—a review. Brazil J Microbiol., 41:850–61. Tamura, K., Nei, M. and Kumar, S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA), 101:11030-11035. Tsakona, S., A. Papadaki, N. Kopsahelis, V. Kachrimanidou, S. Papanikolaou, and A. A. Koutinas,“Development of a circular oriented M. A. Haque, V. Kachrimanidou, A. Koutinas, and Lin, C. S. K. 2016. “Valorization of bakery waste for biocolorant and enzyme production by Monascuspurpureus,” Journal of Biotechnology, 231:55–64. Tsakona, S., N. Kopsahelis, A. Chatzifragkou, S. Papanikolaou, I. K. Kookos, and A. A. Koutinas, 2014. “Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomycesstarkeyi,” Journal of Biotechnology, 189: 36–45. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.387-400 © Principal, Sree Narayana College, Kollam, Kerala, India

Metabolic effects of bisphenols on a fresh water fish, Oreochromis mossambicus 401 ISBN 978-93-5396-871-7 CHAPTER 57 METABOLIC EFFECTS OF BISPHENOLS ON A FRESHWATER FISH, OREOCHROMIS MOSSAMBICUS Anjali V R* and Aruna Devi C Department of Zoology, University College, Thiruvananthapuram, Kerala – 695034 *Correspondence E-mail: [email protected] ABSTRACT Bisphenol A (BPA) is the most prevalent bisphenol analogue in environment. Bisphenol A, being an endocrine disruptor and a substance of very high concern, the European Union (EU) has proposed to ban its use in consumer products and several bisphenol analogues have been introduced as a replacement in various applications. Several of the proposed BPA substitutes are chemical analogues to BPA with similar molecular structures. Bisphenol S (BPS) is an industrial chemical that has been used recently to replace the potentially toxic BPA. Potential similarities in toxicological profiles have raised concerns that the use of BPS as a substitute for BPA may result in yet another situation of a problematic chemical being distributed in consumer products. In this present study, an attempt was made to understand the role of BPS on the activities of hepatic mitochondrial and intermediary metabolism in a freshwater fish, Oreochromis mossambicus. The results revealed that sub lethal exposure of BPS affected the enzymes involved in mitochondrial metabolism such as Mitochondrial Malate dehydrogenase (MDH), Nicotinamide adenine dinucleotide (NADH) dehydrogenase, Succinate dehydrogenase (SDH) and Cytochrome C Oxidase (CCO). Changes occurred in the activities of mitochondrial enzymes which may affect the energy production and thereby Krebs cycle. It was also used to analyze the changes in the activity of enzymes involved in intermediary pathway such as Glucose 6 phosphatase (G6Pase), cytosolic Malic enzyme (cME), Isocitrate dehydrogenase (ICDH) and Lactate dehydrogenase (LDH).Significant variation(P<0.05) occurred in the activities of intermediary enzymes which may disrupt its metabolic pathways. Therefore the current study points out that BPS at environment relevant concentration impairs the activity of intermediary and mitochondrial metabolic enzymes and thereby affects the TCA cycle and electron transport system. Key words: Bisphenol S, Oreochromis mosambicus, TCA cycle, Electron transport system. Introduction the safety of BPA, several bisphenol analogues have been introduced as a Bisphenols are a group of chemicals which replacement in various applications. are used to manufacture plastics, epoxy Bisphenol S (BPS) is an industrial chemical resins and other products since the which is used as a substitute for the 1960s.Bisphenol A (BPA) is the best known potentially toxic BPA, without knowing its of all bisphenols on account of its use in possible harmful effects. The plastic products many consumer goods until its prohibition in coined as ‘BPA free’ are often made of BPS 2015. Because of concerns expressed about (Grignard et al., 2012). Bisphenol S has been Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

402 Current status and challenges for conservation and sustainable use of biodiversity used in manufacturing plastics, thermal ii.LactateDehydrogenase receipt papers, currency bills, food cartoons (LDH.,E.C.1.1.27) etc (Liao et al.,2012; Vinas et al.,2010).Experiments have proven that BPS Lactate dehydrogenase was assayed also acts as a kind of endocrine disruptors according to the method of King, (1965). (EDs)(Cao et al.,2013; Kuruto-Niwa et Read the absorbance at 550nm.The specific al.,2000) and presents a potential threat to activity of the enzyme was expressed as human health. In zebra fish (Danio rerio) IU/min/mg protein. embryos, long term exposure to different concentrations of BPS, males showed a iii.Cytosolic Malic Enzyme (cME L-Malate significant decrease in body length, weight, : NADP+ Oxidoreductase., EC.1.1.1.40) sperm count, plasma thyroid hormones and plasma testosterone. Females showed The activity of cME was determined by the decreased egg ratio and plasma thyroid method of Ochoa, (1955 a). The change in hormones (Naderi et al., 2014; Ji et al., absorbance was noted at 340nm and the 2013). The present study investigated the activity was represented as 1U/min/mg changes in the intermediary and protein. mitochondrial pathways in fresh water fish O.mossambicus induced by sub lethal iv.Cytosolic Isocitrate Dehydrogenase (ICDH)– dosages of the Bisphenol S. (IsocitrateDehydrogenaseNADP+- E.C.1.1.1.42) Materials and Methods The enzyme was assayed by the method of The animal model used in the experiment is a Ochoa, (1955 b).The change in absorbance freshwater fish, O. mossambicus (adult).The was noted at 340nm for 2min and the activity test chemical used in the experiment was represented as 1U /min/mg protein. Bisphenol S. The LC50 value of BPS was determined by probit analysis and was found Assay of mitochondrial Enzymes. to be 200mg/l BPS for adult. Based on that, three sub-lethal doses such as 150,125 and i.Succinate Dehydrogenase(SDH-Succinate 100mg/l BPS were selected for treatment and ubiquinone oxidoreductase.,E.C.1.3.5.1) the exposure period is 4, 8 and 12 days The activity of SDH was assayed according respectively. to the method of Slater and Bonner (1952).The change in OD (Optical Density) Assay of Intermediary metabolic enzymes was recorded at an interval of 15 second for 5 minute at 420nm and activity was expressed i.Glucose 6 Phosphatase (D-Glucose-6- as nmoles of succinate oxidized/min/mg protein. Phosphatase Phosphohydrolase., ii. Malate Dehydrogenase (MDHL-Malate : EC.3.1.3.9) NAD+ Oxidoreductase .,EC.1.1.1.37) The activity was assayed by the method of The activity of MDH was determined by the Swanson (1955). The absorbance was method of Mehler et al. (1948). The change measured at 640nm and the specific activity in absorbance was noted at 340nm and the of the enzyme was expressed as nano moles activity represented as 1U /min/mg protein. inorganic phosphate liberated/min/mg protein. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

Metabolic effects of bisphenols on a freshwater fish, Oreochromis mossambicus 403 iii. NADH Dehydrogenase (E.C.1.6.5.11) according to the protocol of Bradford, (1976).Data analysis was done by ANOVA. The activity of NADH dehydrogenase was The differences in means were tested by assayed according to the method of using Duncan (1995) analysis. Significant Minakami et al., (1962).The change in level used was P < 0.05. OD(Optical Density) was measured at 420nm and the activity was expressed as moles of Results NADH oxidized/min/mg protein. Activity of Intermediary Enzymes in Adult iv. Cytochrome C Oxidase (E.C.1.9.3.1 of O.mossambicus Cytochrome C Oxidase activity was assayed In the present study ,the adult fish exposed by the method of Pearl et al., (1963). The to sublethal doses (100,125 and 150 mg/l) of change in OD was recorded at 550nm for BPS after 4,8 and 12 days in a time – 5min at an interval of 15sec. The activity of dependent manner showed significant CCO was expressed as change in OD/min/mg reduction in the activity of intermediary protein. enzymes, such as G6Pase, ICDH, LDH and significant elevation in cME activity . Protein content for all enzymes was estimated using the same tissue extract 8 Activity of Glucose 6 phosphatase Activity of cytosolic Malic Enzyme b nanomoles inorganic phosphate 0.6 b d liberated/min/mg protein 6d a bc d ab 1Umin./mg protein d ab c c c 0.5 b d ac 4a 0.4 cd 2 0.3 a 0.2 0 0.1 100mg/l 125mg/l 150mg/l 0 12th day 100mg/l control 4th day 8th day control 125mg/l 150mg/l 40 a 4th day 8th day 12th day 10 Activity of ICDH dehydrogenase 30 Activity of LDH a b a a a a 20 dd cd IU/min/mg protein 8 IU/min/mg protein 10 6 b cd b cd bd c c bb 4 bc 2 0 0 100mg/l 125mg/l 150mg/l 100mg/l 125mg/l 150mg/l control 4th day 8th day 12th day control 4th day 8th day 12th day Mean values of different superscript letters (a,b,cand d) were significantly different (p<0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

Metabolic effects of bisphenols on a fresh water fish, Oreochromis mossambicus 404 Activity of Mitochondrial Enzymes in compared to that of control showed Adult of O.mossambicus significant reduction in the activity of mitochondrial enzymes, such as CCO, In the present study, the adult fish when MDH, NADH dehydrogenase and SDH exposed to sublethal doses (100,125 and 150 activity. mg/l) of BPS after 4,8 and 12 days 2b Activity of CCO 6 b Activity of MDH n g atom o2 consumed/min/mg1.5 a IU/min/mg protein a b ab protein d cd c a 4 a c ad c bc c d d 1 bd 2 0.5 0 0 100mg/l 125mg/l 150mg/l 100mg/l 125mg/l 150mg/l 4th day 8th day 12th day control control 4th day 8th day 12th day 0.8 Activity of NADH dehydrogenase µmol DICP reduced/min/mg protein 2.5 Activity of SDH µm mol G6Po4 converted/min/mg 0.7 a a a a aa 2 0.6 b c 0.5 d bcd b d 1.5 c bc d 0.4 1 bcd 0.3 0.5 d c b 0.2 0.1 0 125mg/l 150mg/l 0 125mg/l 150mg/l 100mg/l 100mg/l 4th day 8th day 12th day 4th day 8th day 12th day control control Mean values of different superscript letters (a,b,cand d) were significantly different (p<0.05). Discussion and dephosphorylation (Chiquoine, 1955). In adult fish inhibition of G6Pase activity may The present investigation showed a decrease be due to the damage to microsomal in the activity of glucose 6 phosphatase in membrane as this enzyme is localized adult fish. Glucose 6 phosphatase is the key exclusively in the membranes of the enzyme in glucose homeostasis, functioning endoplasmic reticulum. in both the processes of gluconeogenesis and glycogenolysis. This enzyme may be Lactate dehydrogenase activity was found to involved in the transport of glucose across be fall off significantly in adult fish. The cell membrane involving phosphorylation LDH is an anaerobic enzyme involved in the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

Metabolic effects of bisphenols on a freshwater fish, Oreochromis mossambicus 405 conversion of pyruvate to lactate in high activity of this enzyme should oxidize glycolysis. It is an important glycolytic malate very rapidly when oxygen is enzyme in biological systems and is available. inducible by oxygen stress. Therefore, the activity of several regulatory enzymes may From the present study, it can be visualized be significantly changed in order to meet the that there is a significant decrement of SDH required energy demands under toxic stress activity in the liver of fish exposed to (Mayer et al., 1989), including the activity of sublethal concentrations of BPS. Succinic lactate dehydrogenase (LDH), which helps dehydrogenase is an essential enzyme whose the continued process of glycolysis under activity takes part in oxidation of anaerobic conditions (Diamantino et al., carbohydrate through Krebs cycle. The 2001). Significant decrease in LDH activity general decrease in SDH activity was may be due to increased tissue damage and associated with the inhibition of hence LDH can be recognized as a potential mitochondrial respiratory mechanism which marker for assessing the toxicity of a prevents the transfer of electrons to chemical. molecular oxygen, resulting in the inhibition of SDH activity, and shifting the aerobic In the current investigation ICDH activity metabolism to an aerobiosis (Sweta et al., was found to decrease in O.mossambicus. 2007). The suppression of SDH activity in Nicotinamide Adenine Dinucleotide (NADP) Oreochromis mossambicus indicates dependent ICDH is structurally bounded to impairment of oxidative metabolic cycle and mitochondria and participates in Krebs cycle. hence depends on anaerobic glycolysis to This enzyme catalyses the reversible meet its energy demands. oxidation of isocitrate to oxalosuccinic acid, followed by decarboxylation, leading to the The present study observed the significant formation of α-ketoglutarate. It has been decrease in MDH activity in liver during reported that the activity of this enzyme was exposure periods of BPS . Malate increased in the muscle fibers of lead dehydrogenase converts malate to intoxicated rats(Laszcyca,1989). Ivanina et oxaloacetate and also play a significant role al. (2008) studied the combined effects of in CO2 fixation and in gluconeogenesis cadmium (Cd) and temperature on key (Lehinnger, 2008). As malate dehydrogenase mitochondrial enzymes, including NAD- and is the oxidative enzyme involved in Krebs NADP-dependent isocitrate dehydrogenases cycle, any disturbance in this enzyme activity in Crassostrea virginica. The reduced will affect the Krebs cycle. Tiwari et al.( activity of ICDH in adult liver may influence 2009) observed the decreased MDH activities energy and redox status and decrease in in tissues of Clarias batrachus on exposure activation energy of mitochondrial enzymes. to endosulfan. The decrement of MDH activity suggests that there is a shift in the Malic enzyme activity was found to be respiratory metabolism towards anaerobiosis. elevated significantly in adult when exposed to BPS. This enzyme catalyses the oxidative When experimental fish is exposed to decarboxylation of malate in the presence of sublethal doses of BPS, the activity of CCO a divalent metal ion. Assuming that malic decreased. Cytochrome C Oxidase is the enzyme is responsible for the conversion of terminal enzyme of Electron Transfer System malate to pyruvate, it could be expected that (ETS) and helps to transfer electrons to their Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

406 Current status and challenges for conservation and sustainable use of biodiversity final acceptor oxygen in ETS. Increased present work indicates that BPS can cause CCO activity in aerobically functioning considerable changes in intermediary and tissue such as liver has been demonstrated to mitochondrial metabolism and is likely to be a persistant response, which is related to induce tissue damage in O. mossambicus. the capacity of pentachlorophenols to bind The causes for these alterations appear to be very strongly to mitochondrial membranes the result of high energy demands. (Bostrom and Johansson, 1972). The decreased activity of CCO shows a clear Reference indication of decreased mitochondrial respiration and oxidative meatabolism which Bostrom, S.L. and Johansson, R.G.1972. results in reduced synthesis of ATP Effects of pentachlorophenol on enzymes production in O.mossambicus. involved in energy metabolism in the liver of the eel. Comp Biochem Physiol., 41B: 359– Activity of NADH dehydrogenase was found 369. to be declined in adult O.mossambicus in the present study. NADH dehydrogenase or Bradford, M.M. 1976. A rapid and sensitive Complex I is an electron carrier in the method for the quantitation of microgram electron transport system in the inner quantities. mitochondrial membrane. Pesticides such as pyridaben, rotenone and fenpyroximate are Chiquoine, A. and Duncan.1953. The found to decrease NADH hydrogenase distribution of glucose-6-phosphatase in the activity (Sherer et al., 2007). The liver and kidney of the mouse. J. Histochem. pyrethroids, permethrin and cyhalothrin are and Cytochem., 1: 429-435. potent inhibitors of the mitochondrial complex I (Gassner et al., 1997). Decreased Duncan, D.B.1955. Multiple range and activity on exposure indicating disturbed multiple F tests. Biometrics., 11(1):1-42. function of the mitochondrial electron transport chain leading to reduced energy Gassner, B., Wuthrich, A., Scholtysik and G., production. Solioz, M.1997. The pyrethroids, permethrin and cyhalothrin are potent inhibitors of the Thus it is evident from the current research mitochondrial complex I. J Pharmacol Exp Therap., 281 (2): 855-860. that BPS disrupted intermediate and Grignard, E., Lapenna S. and Bremer S.2012. mitochondrial metabolism in Weak estrogenic transcriptional activities of Bisphenol A and Bisphenol S. Toxicol in O.mossambicus. Vitro., 26(5):727-731. Conclusion Ivanina, A.V., Habinck, E. and Sokolova, I.M.2008. Differential sensitivity to cadmium In the present work, all of the key enzymes of of key mitochondrial enzymes in the eastern the TCA cycle were affected when exposed oyster, Crassostrea virginica Gmelin to BPS. The significant reduction in the (Bivalvia: Ostreidae). Comp. Biochem. activities of ICDH and MDH resulted in Physiol. C: Toxicol. Pharmacol., 148 (1): disruption of the TCA cycle and diminished 72–79. ATP production. Therefore, the sum of these alterations can have a significant effect on energy metabolism. In conclusion, the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

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408 Current status and challenges for conservation and sustainable use of biodiversity Tiwari., R.P.S. and Singh.,A. 2009. Changes in some biochemical parameters in the liver and muscle of Colisa fasciatusdue to toxicity of ethanolic extract of Nerium indicummill (Lal kaner) latex, Natural Product Radiance. 8(1): 48-54. Viñas, N,. Campillo, N,. Martínez-Castillo, M. and Hernández-Córdoba. 2010. Comparison of two derivatization-based methods for solid-phase microextraction? gas chromatography? mass spectrometric determination of bisphenol A, bisphenol S and biphenol migrated from food cans, Anal. Bioanal. Chem., 397:115–125. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.401-408 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxicity of azo dye tartrazine in Indian major carp, Labeo rohita 409 CHAPTER 58 ISBN 978-93-5396-871-7 HEPATOTOXICITY OF AZO DYE TARTRAZINE IN INDIAN MAJOR CARP, LABEO ROHITA Athira N* and Jaya D S Department of Environmental Sciences University of Kerala, Kariavattom Campus, Kerala, PIN- 695 581, India. *Correspondence E-mail: [email protected] ABSTRACT Wastewaters from various industries have crucial role in environmental pollution especially as they contain synthetic chemicals. The major contaminants include heavy metals, PAH, pesticides, synthetic dyes etc. Among these, synthetic dyes are in prime position in polluting water resources. Azo dyes are most extensively used among the synthetic dyes not only in textiles, but also in synthetic foods, bevarages, drugs, cosmetics, lake colours and antifungal agents. Industrial effluents containing tartrazine are not easy to treat using the conventional wastewater treatments because of its bright colour at very low concentrations and non-degradable nature. In the present study, the fish Indian major carp (Labeorohita) has been used as a model to assess the effects of azo dye tartrazine. The hepatotoxicity of azo dye tartrazine (Acid yellow 23/ FD and C Yellow No.5), which is regarded as best colouring agent in food and drug industries was studied in the fresh water fish, Labeo rohita. For the experimental study, after range finding tests, sub-lethal concentrations of 5 ppm and 10 ppm were used for 75 days chronic exposure. Fish liver forms a target organ for xenobiotics; so the biochemical composition such as total proteins, total free amino acids, total carbohydrates and total lipids were estimated. The analysis shows remarkable alterations in the liver biochemistry such as in total proteins, amino acids, carbohydrates and lipids of exposed fishes with respect to control. Histo-pathological examination of liver also showed toxicity induced changes like necrosis, vacular degeneration, chronic inflammatory cell infiltration and cell degenerations. The study revealed that chronic exposure of azo dyes like tartrazine causes hepatotoxicity in fishes. Key words: Tartrazine, Hepatotoxicity, Histopathology, Labeo rohita Introduction coal tar but now it is a petroleum derivative and the properties like low cost, stability and Aquatic ecosystems are under constant high brightness compared to natural dyes pressure due to the releasing of partially or makes synthetic dyes more popularized untreated industrial effluents. Most of the (Kobylewski and Jacobson, 2010). Azodyes, synthetic chemicals of industrial effluents are an important class among dyes having most non-degradable in nature, which can alter the deleterious effects on health of organisms as physico-chemical quality of water. Among well as in environment. Once discharged in the industrial effluents, textile, paper, leather, the natural environment, azodyes are highly food and beverage industries are more soluble in water and persist for a long time destructive due to the presence of synthetic (Bhullar and Sud, 2012). Azo compounds dyes. Originally the dyes were derived from Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

410 Current status and challenges for conservation and sustainable use of biodiversity comprise of an aromatic ring linked by an prior exposure to environmental stressors. azo bond to the second naphthalene or benzene ring. This azo group may reduce by The change in the biochemical constituents in azoreductase producing aromatic amines, some of which have been observed to be the organ reflects the physiology and overall toxic, mutagenic, and carcinogenic (Tanaka, 2008; Türkoglu, 2007). Tartrazine, most health of organisms. Proteins and amino common azo dye has extensive applications in various synthetic food products, textiles, acids forms the major building blocks of the cosmetics, pharmaceutical products, beverages, artificial lake colours like organism’s body. Carbohydrates especially aquashade and algaecides. The dye is still using without any controversy and glucose and glycogen are the major energy disagreement regarding its health effects (Mekkawy et al., 1998).So there is a chance source for the metabolism (Srivastav and of entering into the sewage and industrial effluents and to natural water bodies. There Roy, 2018). There for the assessment of these are studies about the harmful effects in rats and human beings and found that several biochemical constituents of liver can serve as allergies, reproductive abnormalities, and has immunotoxic, genotoxic and mutagenic effective in determining the toxicity induced effects (Ram and Ardern, 2001; Guendouz et al., 2013; Boussada et al., 2017). Despite of changes in fish. Histological changes provide this, there are no relevant studies regarding its specific toxic effects in environment and a rapid method to detect effects of irritants, on fishes. Tartrazine is found to be resistant to conventional wastewater treatments also especially chronic ones, in various tissues forms a matter of concern. and organs (Bernet et al., 1999). The visible For the present study, the fish Labeo rohita, commonly known as Indian major carp, has changes in the liver can be assessed by wide distribution in India and other south Asian countries was used as a model to histological examinations. Morphological assess the toxicity of tartrazine. Fishes are among the group of non-target aquatic alterations in tissues is regarded as a sign of organisms for several xenobiotics, however the adverse effects may easily reflects in manifestation of physiological and them. Fishes were always in direct contact with the water and they represent high order biochemical changes that could very well be organisms for biomarker studies. The liver of an organism has regarded as the main organ detected by microscopic examination. for detoxification (Dutta et al., 1993) that suffers serious morphological alterations in Histopathology is a very powerful subjective fish exposed to xenobiotics like pesticides (Rodrigues and Fanta, 1998). Variations in tool for the detection of such tissue level the liver biochemistry and architecture may be useful as biological markers that indicate changes in fish populations. The variation in the biochemical profile reflects the indirect destruction of tissues. The major objective of the study is to assess the changes in biochemical composition and hitstopathological alterations in the liver of Labeo rohita (Hamilton,1822) exposed to tartrazine. Materials and Methods Chemicals: Tartrazine (C.I. 19140, CAS No 1934-21-0, Mw 534,37, synonyms: E 102,Food yellow 4, Acid yellow 23, FD &C yellow No.5) is an azodye with the chemical formula 4,5-Dihydro-5-oxo-1-(4- sulfophenyl)-4-((4sulfophenyl)azo)-1-H- pyrazole-3-carboxylic acid, and its trisodium salt (95%) was purchased from TCI chemicals (Tokyo Chemical Industries, India). All the other biochemicals and chemicals used for the study are of analytical grade. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxicity of azo dye tartrazine in Indian major carp, Labeo rohita 411 Experimental Fishes: For the present study, Plummer (1988). Total carbohydrates in Indian major carp- Labeo rohita (Hamilton, tissues were estimated by anthrone method 1822) commonly known as ‘Rohu’ was (Roe, 1955). Total tissue lipids were procured from the National Fish Seed Farm, estimated by sulphophosphovanillin method Department of Fisheries, Kerala State Govt. (Floch et al., 1957). at Neyyar Dam, Thiruvananthapuram. After disinfection the fishes were acclimatized to Histological Examinations: The dissected the laboratory conditions for 21 days with liver samples of experimental fishes were cut adlibitum diet. For both acclimatization and into pieces, and fixed in neutral buffered experimental aquarium study chlorine free formalin. The tissues were subjected to water was used. The major water quality processing such as dehydration, clearing, parameters (analysed following the standard paraffin embedding and wax sectioning. Thin procedures in APHA, 2012) were remained sections (thickness between 5-7µ) were taken optimum for the survival of fish. using a microtome (Leica, UK) and stained with hematoxylin and eosin (H&E) following Experimental Design: After the staining procedure by Bucke (1972) and Bullock (1980). The microscopic acclimatization,18 healthy fishes of 15±3 cm examinations were made using an Olympus Microscope digital CH20i. body length and 27± 3 gms body weight were Results and Discussion selected for the experimental study, and were The results of the changes in the divided into test and control groups. For the morphometry (total body weight and length) of fish body, biochemical constituents of chronic experimental studies, 5 mg/L (CT1) liver and histo-pathology of liver tissue were discussed. There is a reduction in the body and 10 mg/L (CT2) concentrations of the dye weight of test fishes with respect to that of control (Table 1)and may be due to the were used and exposed the fishes for 75 days toxicity induced cell damage resulted from the destruction of building blocks of body under controlled conditions in the aquarium such as proteins, amino acids, carbohydrates and lipids. There is slight growth in terms (25L). The control group fishes were also body weight and length was observed in control fishes but not in test fishes. The maintained in same conditions like controlled avoidance behavior towards feeding and unbalanced and excess body movements as aeration and food supply. During the static adaptive mechanisms against the toxic effects also causes the reduction in body weight. The renewal-chronic assays, the important water lack of food intake may also result in the loss of reserved fats and glycogen also resulted in quality parameters were retained at every weight loss and slow growth rate of fishes. There are studies reported the stress induced renewal of test solution and the fish behavior, weight loss in fishes(Ali et al., 2003; Hussain and Ali Shah, 2016). food intake and swimming patterns etc. were also noticed. Gross observation and morphological measurements of fish body were done in the first and final day of experiment. After the exposure, the fishes were dissected and organs were separated. Determination of Biochemical composition: The fish liver tissues were washed with physiological saline and ground with homogenizing solutions for each analysis. Immediately after the sample preparation, biochemical components were determined. The total proteins were estimated using the standard procedure by Lowry et al. (1951). Free amino acids were estimated by ninhydrin method following the procedure by Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

412 Current status and challenges for conservation and sustainable use of biodiversity Changes in Biochemical constituents of may not occur completely or partially in Liver fishes so, glycogen can be converted into glucose. Exposure of fishes with tartrazine Results of biochemical constituents in liver causes stress condition and stops feeding in are given in Table 2. There is a decrease in response cause the less availability of the concentrations of total proteins and carbohydrates. Failure in the further synthesis carbohydrates in liver tissues of tartrazine and over excretion may result in the exposed (CT1 and CT2) fishes. The proteins reduction in total carbohydrates. Similar form the major component in the tissues, due results like reduction in total carbohydrates to toxicity induced stress; proteolysis may were also recorded in the studies by Srivastav happen as reported in fish Channa punctatus et al. (2014) and by Srivastav and Roy exposed to pesticide endosulphan (Magar and (2018). sheikh, 2012). The decrease in the protein level of liver indicated that inhibition of In test fishes, the lipid concentration showed protein synthesis as a result of liver disorder significant (p<0.01) decrease in CT2 group due to the exposure of tartrazine and its fish liver and increase in CT1 group fish liver metabolites like aromatic amines. The amino compared to that of control fishes (CC).The acids are the major building blocks of decrease in lipid level indicated that organism’s body and the stress can be utilization of cholesterol to meet the energy directly affected its metabolism, synthesis requirement during stressed conditions and repair. The free amino acids were (Waghmare and Wani, 2014). The incline in increased in CT1 and decreased in liver lipid level as in CT1 may be due to structural tissues of CT2 fishes with respect to control. damage of cell organelles and cholesterol rise In CT1, the lower concentration exposed by low cholesterol catabolism because of groups; fishes may be having adaptive tartrazine toxicity.Certain heavy metals like mechanisms. The incline of free amino acids chromium can cause similar changes in may resulted from denaturation of bound cholesterol levels as reported by Meshram protein and cell disruption due to toxic and Baile (2016).The liver of experimental amines (James et al., 1979).While in CT2 group fishes forms the major target organs of group fishes exposed to higher concentration tartrazine toxicity as most of the biochemical of tartrazine the amino acid synthesis may changes were observed in the liver tissues.In inhibited due to the tartrazine induced stress test fishes, overall change in the biochemical condition. constituents like total proteins, amino acids, carbohydrates and lipids liver tissues shows The results show that there is a significant the stress induced inhibition of physiological decrease in total liver carbohydrates in test functions, metabolism and enzyme actions. fish liver (CT1 and CT2).Mainly the So the liver can be considered as a site of glycogen as carbohydrates is stored in liver action for xenobiotics like azo dye tartrazine. and muscles as a reserve for adverse conditions. In stress conditions, glycogen is Histopathological Alterations in Liver converted into glucose and completes the necessity of energy. In the present conditions Histo-pathological alterationsin liver is useful there is lack of enough oxygen, fast and markers as it is the prime target organ of irregular jurky movement induced muscular various xenobiotics and accumulation site. exercises may deplete these reserved The control fish liver (Fig.A) shows normal glycogen in the form of excess energy. architectural arrangement with normal Normally, during stress condition food intake hepatocytes (NH) with prominent nucleus, blood vessel and sinusoids. In chronic assay, Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxicity of azo dye tartrazine in Indian major carp, Labeo rohita 413 liver of test fishes CT1 (Fig.B) and CT2 In the study by Ibrahim and Mahmoud (Fig.C) shows substantial changes. CT1 fish (2005) dilation of blood vessels along with hepatocytes show vacuolar degeneration degeneration and necrosis of hepatocytes (VD), dilation of blood vessels and chronic were observed in Tilapia mossambica, inflammatory cell infiltration (IN).CT2 fish Clariasgariepinus and Mugil cephalus from hepatocytes show vacuolar degeneration Nile river as a consequence of water (VD) mild collection of blood in parenchyma pollution by industrial effluents. The (HE). In the liver, extravagated bile biochemical changes in liver profile relate to (cholestasis) was observed. Degeneration of histo-pathological changes such as hepatocytes in periportal zones implies the hepatocytes damage with significant changes influence of toxic compounds in the digestive as hyperplasia, disintegration of hepatic tract. There was rupture in endothelial lining mass, focal coagulative necrosis etc. and hepatocyte membraneswere also noted. Alterations in liver histology, such as Liver is a detoxifying organ in fish, which necrosis have been found at different levels shows alterations in histological architecture of severity depending on the contaminant, and deviation from normal biochemical and exposure time and dose or concentration physiological responses on exposure to (Oliveira Ribeiro, et al., 2002). The similar contaminants (Roy and Bhattacharya, 2006). changes were reported in fish O. niloticus The results of this study confirmed that azo exposed to red 195 dye (Ayadiet al., 2015). dye tartrazine is hepatotoxic to fishes. Table 1. Changes in the Morphometric parameters of the fishes (Chronic experiment) Fish Group Length (cms) Weight (gm) CC Initial Final Initial Final CT1 CT2 18.3±1.13 19.4±1.14 29.01±3.20 30.21±0.07 18.7±1.43 18.6±0.62 28.62±2.20 26.34±1.42 18.1±2.02 17.8±1.10 28.45±3.13 24.91±2.21 [All values are mean ± SD] Table 2. Variation in the Biochemical constituents of Fish liver Biochemical Parameters CC CT1 CT2 Total Proteins (mg/g wet wt.) 102.3 ± 0.43 86.21±0.68 59.01±1.32 Total Free Amino acids (mg/g wet wt.) 25.35±1.35 32.41±0.43 24.84±0.95 Total Carbohydrates (mg/g wet wt.) 71.23±0.25 63.44±0.94 49.02±0.43 Total Lipids (mg/g wet wt.) 96.31±0.12 107±0.38 87.04±0.32 [All values are mean± SD; n=6; p<0.01] Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

414 Current status and challenges for conservation and sustainable use of biodiversity AB C Fig. A. CC liver (H & E stain x 100); Fig. B. CT1 liver (H & E stain x 100); Fig. C. CT2 liver (H & E stain x 400). Conclusion References Hepatotoxicity of tartrazine in fresh water Ali, M., Slam, A. and Masud, S. 2003. fish Labeo rohita was studied by assessing Effect of stress induced by fasting on specific the changes in biochemical constituents and growth rate, protein growth rate and histopathology of fish liver. The reduction in condition factor of Thaila, Catla catla. Pak. J the biochemical constituents was confirmed Zool. 18:13-21. as there is a decrease in body weight of the tartrazine exposed fishes. The histological APHA 2012. Standard methods for the alterations like necrosis, vacuolar examination of water and waste water degeneration, chronic inflammatory cell analysis (22nd Edition). American Public infiltration and cell degenerations in the test Health Association, American Water Works fish liver also proved the hepatotoxicity of Association. tartrazine. Therefore the industrial effluents containing azo dyes should be chemically or Ayadi,I., Monteiro, S. M., Regaya, M., biologically treated before releasing it to the Coimbra, I., Fernandes, F., Oliveira, M.M., environment. Also there should be proper Peixotod, F. and Mnif, W. 2015. standards and guidelines for the usage of Biochemical and histological changes in the azodyes especially tartrazine in food and liver and gills of Nile tilapia Oreochromis pharmaceutical products. niloticus exposed to Red 195 dye. RSC Adv., 5: 87168. Acknowledgements Bernet, D., Schmidt, H., Meier, W., The authors thankfully acknowledge the Burkhardt-Holm, P. and Wahli, T. 1999. financial support received from Kerala State Histopathology in fish: proposal for a Council for Science, Technology and protocol to assess aquatic pollution. J. Fish Environment (KSCSTE)for this study. Dis., 22, 25-34. Gratefully acknowledge the Registrar, University of Kerala for providing the Bhullar, N. and Sud, D. 2012. Decolorization laboratory and library facilities for the study. of congo red, a carcinogenic textile dye using semiconductor mediated photocatalysis. Poll. Res., 31(1): 7-10. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxicity of azo dye tartrazine in Indian major carp, Labeo rohita 415 Boussada, M., Lamine, J. A., Bini, I., Abidi, transport : A unified theory of portal N., Lasrem, M., El-Fazaa, S. and El-Golli, N. systematic encephalopathy, Lancet. 2 : 772- 2017. Assessment of a sub-chronic 775. consumption of tartrazine (E102) on sperm and oxidative stress features in Wistar rat. Kobylewski, S. and Jacobson, M. F. 2010. Int. Food. Res. Journal., 24(4): 1473-1481. Food dyes a rainbow risk. Center for Science in the Public interest, Washington. Bucke D. 1972. Some histological techniques applicable to fish tissues, Mawdesley- Lowry, O. H, Rosenbrough N. J., Farr, A. L. Thomas. LE. Edn 10, Diseases of fish. and Randall, R. L. 1951. Protein Symposium of Zoology Society, London, measurement with the Folin phenol reagent. J Vol. 30, Academic Press, New York, 153. Biol Chem., 193: 264-275. Bullock, G.L., Conroy, D.A. and Snieszko, Magar, R. S. and Shaikh, A. 2012. Biochemical changes in proteins and amino S.F. 1980. Disease of fishes, Book 2: acids in Channa punctatus in responses to sub lethal treatment with the insecticide Bacterial Disease, Snieszko,S.F., Malathion. Trend. Lif. Sci., 1 (3): 19-23. Axelrod,H.R. (Eds.), T.H.F. Publications, Mekkawy, H. A., Ali, M. O. and El-Zawahry, A. M. 1998. Toxic effect of synthetic and Neptune, NJ. natural food dyes on renal and hepatic functions in rats. Toxicol. Let., 95(1): 155- Dutta, H. M. Adhikari, N. K. Singh, P. K. 155. and Munshi, J. S. 1993. Histo-pathological changes induced by malathion in the liver of Meshram, D. and Vidya Baile, V. 2016. a freshwater catfish, Heteropneustes fossilis Chromium induced changes in biochemical (Bloch). B. Environ. Contam.Tox., 51, 895- composition and gonado-somatic index of a 900. teleost, Oreochromis mossambicus (Peters). J. Zool. Studies., 3(5): 28-34. Folch, J., Less, M. D. and Sloane-Stanely, G. H.1957. Simple method of isolation and Oliveira Ribeiro, C.A., Schatzmann, M., purification of total lipids from animal Silva de Assis, H.C., Silva, P.H. and tissues.J. Biochem., 226: 297-308. Pelletier, E.2002. Evaluation of tributyltin subchronic effects in tropical freshwater fish Guendouz, M., Mehedi, N., Zaoui, C., Saidi, (Astyanax bimaculatus, Linnaeus, 1758). D. and Khéroua, O. 2013. Immune response Ecotoxicol. Environ. Saf. 51: 61–167. after tartrazinesubchronic ingestion in Swiss albino mice. Int. J. Pharm. Pharmaceutical Plummer, D. T. 1988. An Introduction to Sci., 5(2): 584-592. Practical Biochemistry. 3rd ed. Tata McGraw-Hill Pub. Co. Ltd. New Delhi. Hussain, M. Z. and Ali Shah, S. Q. 2016. p.360. Effect of stress conditions on condition factor and growth of farmed Rohu (Labeo rohita). Ram, F. S. and Ardern, K. D. 2001. J. Ent. Zool. St. 2016; 4(5): 258-261. Tartrazine exclusion for allergic asthma. Cochrane Database of Systematic Reviews 4: Ibrahim, S.A. and Mahmoud, S.A. 2005. CD000460. Effect of heavy metals accumulation on enzyme activity and histology in liver of Rodrigues, E. L.and Fanta, E. 1998, Liver some Nile fish in Egypt. Egypt. J. aqua. histopathology of the fish Brachydaniorerio Boil. Fish., 9(1): 203-219. James, J.H., Zipara, V. and Fischer, J.E. 1979. Hypermnonemia plasma amino acid Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

416 Current status and challenges for conservation and sustainable use of biodiversity after acute exposure to sublethal levels of the Srivastav, A.K., Srivastava, S.J. and Roy, D. organophosphate dimethoat 500. Rev. Bras. 2014. Microcystin – LRinduced changes in Zool., 15: 441-450. the carbohydrate metabolism the freshwater fish, Cyprinus carpio (Linn.). J. Exp. Zoo. Roe, J. H.1955. The determination of sugar India., 17(2):581-583. in blood and in spinal fluid with anthrone reagent.J.Biol. Chem.212: 335-343. Tanaka, T., Takahashi, O., Oishi, S. and Ogata, A. 2008. Effects of tartrazine on Roy, S. and Bhattacharya, S. 2006. Arsenic- exploratory behaviour in a three-generation induced histopatology and synthesis of stress toxicity study in mice. Repro. Toxicol., 26(2): proteins in liver and Kidney of Channa 156-163. punctatus. Ecotoxicol. Environ. Saf., 65, 218- 229. Türkoğlu, S. 2007. Genotoxicity of five food preservatives tested on root tips of Allium Srivastav, A.K., and Roy, D. 2018. Acute cepa L. Mut. Res., 626(1-2): 4-14. toxicity of malachite green (Triarylmethane dye) and pyceze (Bronopol) on carbohydrate Waghmare, S.Y. and Wani, G.P. 2014. metabolism in the freshwater fish Cholesterol alteration in a fresh water fish, Heteropneustes fossilis (Bloch.). Int. J. Fish. Labeo rohita after exposure to an insecticide Aqua. St., 6(1): 27-30. polo. Int.J. Curr. Microbiol. App. Sci., 3(11): 221-225. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.409-416 © Principal, Sree Narayana College, Kollam, Kerala, India

ISBN 978-93-5396-871-7 CHAPTER 59 EFFECT OF 4 –NONYLPHENOL ON MITOCHONDRIAL AND INTERMEDIARY METABOLISM IN A FRESHWATER FISH, LABEO ROHITA Remya V S *and Aruna Devi C Department of Zoology, University College, Thiruvananthapuram, Kerala *Correspondence E-mail: [email protected] ABSTRACT Nonylphenol ethoxylate (NPE) is one of the most dangerous chemical that are recorded in aquatic environments. 4-Nonylphenol (4-NP) is a breakdown product of NPE and attains much concern because of its persistence and toxicity to the aquatic organisms. The present study aimed to evaluate the effect of sub lethal concentrations of 4-NP on major carp, Labeo rohita in the mitochondrial and intermediary metabolism in the hepatic tissue. Exposure to 4-NP shows significant changes in the activities of mitochondrial enzymes like nicotinamide adenine dinucleotide (NADH) dehydrogenase , cytochrome C oxidase (CCO), malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and intermediary enzymes like lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), glucose 6 phosphatase (G6Pase) and cytosolic malic enzyme (cME) when compared to the control (p<0.05). Therefore the current study indicates that 4-NP at environment concentration impairs the activity of intermediary and mitochondrial metabolic enzymes and thereby affects the Tricarboxilic acid(TCA) cycle and electron transport system. The alteration of these parameters can be effectively used to monitor the impact of 4- NP in aquatic system. Key words: 4 -Nonylphenol, TCA cycle, Electron transport system Introduction reduced growth etc.,( Madsen et al.,1997, Ashfield et al.,1998, Chaube et al., 2013). In 4-Nonylphenol is a known endocrine- addition to the tissue specific estrogenic disrupting chemical, due to its ability to effects, 4-NP has non estrogenic adverse mimic or partially mimic naturally occurring effects on liver and kidney function through estrogen.4-Nonylphenol (4-NP) is widely activation of other metabolism related gene used in the production and formulation of distinct from estrogen- responsive gene. This many commercially sold products such as study aimed to investigate the effects of 4-NP industrial and commercial detergent, on some mitochondrial and intermediary pesticides, spermicides, paints, wetting enzymes in a fish, Labeo rohita (L.rohita). agents, textiles, plastics, paper products etc.4- Nonylphenol causes adverse effect on Materials and Methods aquatic animals, especially fishes (Madigou et al., 2001; Marin et al., 2008).4- The animal model, freshwater teleost fish, L. Nonylphenol causes a number of deleterious rohita used in the experiment were collected effects in fishes including reduced from local suppliers and brought to the fertilization, embryonic and larval toxicity, laboratory. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

418 Current status and challenges for conservation and sustainable use of biodiversity Experimental setup  Cytochrome C oxidase ; Pearl et al.,1963 The laboratory acclimated fish were divided  Malate Dehydrogenase ; Mehler,1948 into 4 groups of ten each in separate glass tanks. LC50 dose of 4-NP was determined by  Succinate dehydrogenase ; Slater and probit analysis as 3.14µl/l. Based on that Bonner(1952). three sub lethal doses such as1.5µl/l ,2µl/l and 2.5µl/l of 4-NP were selected for Results exposure Results of mitochondrial enzymes (NADH Analytical method of intermediary dehydrogenase, CCO, MDH and SDH) and enzymes intermediary enzyme (LDH, ICDH, G6Pase, and cME) are shown in figure 1,2,3,4,5,6,7  Lactate dehydrogenase ; King , 1965 and 8. Administration of 4-NP for 7, 14 and 21 days significantly decreased the activity of  Isocitrate dehydrogenase; Ochoa ,1955 b CCO and MDH and significant increment in time dependent manner in NADH  Glucose 6 phosphatase; Swanson,1955 dehydrogenase and SDH activity. The activity of intermediary enzymes like LDH,  Malic enzyme; Ochoa ,1955 a ICDH, G6Pase and cME when exposed to 4- NP for 7,14 and 21 days shows significant Analytical method of mitochondrial reduction when compared to that of control enzymes  NADH Dehydrogenase ; Minakami et al.,1962 Figure 1. Activity of NADH dehydrogenase Figure 2. Activity of CCO µm mol F6Po4 converted/min/mg1c bc cd 0.5 a b ab protein0.9 d b a c a 0.45 d n g atom o2 consumed/min/ mg protein0.8a c 2.5µl/l 0.4 d 2.5µl/l 0.7 b 2µl/l 0.6 d 0.35 b 2µl/l 0.5 a 0.3 0.4 0.25 c 0.3 0.2 d 0.2 0.15 0.1 0.1 0 0.05 1.5µl/l 0 1.5µl/l control 7th day 14th day 21st day control 7th day 14th day 21st day Figure 3. Activity of MDH Figure 4. Activity of SDH Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

Effect of 4 –nonylphenol on miotochondrial and intermediary metabolism in a freshwater fish.. 419 IU/min/mg protein 0.6 a ab a µmol DICP reduced /min/mg 12 bcd d 0.5 protien c c b 10 a b 0.4 d c 0.3 b cd 2µl/l a 2µl/l 8d 7th day 14th day c 7th day 14th day 2.5µl/l 6b 2.5µl/l 0.2 d 21st day 4a 21st day 0.1 2 0 0 1.5µl/l 1.5µl/l control control Mean values of different superscript letters (a, b, c, d) were significantly different (p<0.05) Figure 5. Activity of LDH Figure 6. Activity of ICDH 1.2 ab 1.6 cd 1 ab cd a b cd 1.4 2.5µl/l 1.2 a 0.8 21st day IU/min/ mg protein a a IU/min/mg protein 1b b c cd bcd 0.6 0.8 d 0.6 2µl/l 2.5µl/l 0.4 14th day 21st day 0.4 0.2 0.2 0 2µl/l 0 1.5µl/l 1.5µl/l control 7th day 14th day control 7th day Mean values of different superscript letters (a, b, c, d) were significantly different (p<0.05) Figure 7. Activity of G6Pase Figure 8. Activity of cME 2 ab a 0.3 a a a b 0.25 c bc d 1.8 a c c 0.2 b b d d cd nanomols inorganic phosphate 1.6 IU/min/mg protein 0.15 d liberated/min/mg protein 2µl/l 2.5µl/l 1.4 bc 14th day 21st day 1.2 1 0.8 0.1 0.6 d 0.4 0.05 0.2 0 0 1.5µl/l 1.5µl/l 2µl/l 2.5µl/l control 7th day control 7th day 14th day 21st day Mean values of different superscript letters (a, b, c, d) were significantly different (p<0.05) Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

420 Current status and challenges for conservation and sustainable use of biodiversity Discussion Malate dehydrogenase is an enzyme that reversibly catalyses the oxidation of malate The present study was focused to evaluate the to oxaloacetate. In this present study, 4-NP effect of 4-NP on some metabolic enzymes decreased the MDH activity which indicates such as mitochondrial and intermediary that it inhibits aerobic as well as anaerobic enzymes in hepatocytes of Labeo rohita. metabolism in exposed fish. Several studies are done related to the decreased activities of The current investigation shows the TCA cycle enzymes to the changes in the increasing activity of NADH dehydrogenase integrity of mitochondria. Srinivasamoorthy, in hepatic tissues of fish, L.rohita on 1983 investigated decreased MDH activity in exposure to sublethal concentrations of 4-NP tissue of muscle (Lamellidens marginalis) during 7, 14 and 21 days when compared exposed to deltamethrin. with their respective controls. NADH dehydrogenase is the first enzyme complex in In the present investigation, it can be the respiratory chain for generation of ATP. visualized that there is a rapid increase of 4-Nonylphenol has the ability to alter the SDH activity in liver tissues of fish L. rohita activity of NADH dehydrogenase which on exposure to sublethal concentrations such cause liver dysfunction. This occurs because, as 1.5µl/l ,2µl/l and 2.5µl/l of 4-NP after 7 4-NP can increase the activity of NADH ,14 and 21 days when compared with their dehydrogenase, which leads to the imbalance respective controls. Succinate dehydrogenase of mitochondrial electron transfer activities is an important enzyme of citric acid cycle and ATP synthesis. Pesticides such as and catalyzes the reversible oxidation of pyridaben, rotenone and fenpyroximate succinate to fumarate. Succinate decreases the activity of NADH dehydrogenase is a primary enzyme in the dehydrogenase (Sherer et al., 2007). oxidative catabolism of sugars and is used According to Gassner et al., 1997, effectively as a marker of mitochondrial pyrethroids, permethrin and cyhalothrin are activity. In fish, mitochondrial respiratory potent inhibitors of NADH dehydrogenase. enzymes such as lactate dehyrogenase (LDH) and succinic dehydrogenase (SDH) are From the present study, the activity of involved in cellular respiration through Cytochrome C Oxidase decreases by after glucose metabolism which yields ATP. exposure of 4-NP. Cytochrome C Oxidase Several authors (Rao, et al., 2003; carries electrons from cytochrome C to Shivakumar et al., 2004; Vutukuru., et al., molecular oxygen, reducing it to water 2005; Vineet Kumar, et al., 2008) have (H2O), in the final step of the respiratory reported that the disturbance in oxidative chain (Tyler, 1992). The decreased activity of metabolism led to alteration in the whole cytochrome oxidase, therefore it shows a animal oxygen consumption in different clear indication of decreased mitochondrial species of fish. respiration and oxidative metabolism in L. rohita. In the current investigation the activity of LDH is decreased. LDH catalyses the Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

Effect of 4 –nonylphenol on miotochondrial and intermediary metabolism in a freshwater fish.. 421 reversible conversion of lactate to pyruvate production, ICDH has been extensively and vice versa. Thus, the significant changes studied with respect to its isoenzymes, in enzyme activity of LDH indicate damage regulation, kinetics and its presence in other to any or all organs producing this enzyme species including prokaryotes. during liver or kidney injuries (Latner, 1975). On the view of Usharani and Ramamurthi The activity of the G6Pase in the liver on (1987) LDH activity was increased in exposure to sub lethal doses showed a freshwater fish exposed to sub-lethal significant reduction when compared to the concentrations of cadmium. LDH is generally control. Glucose 6 phosphatase is associated with cellular metabolic activity responsible for contributing free glucose to and so, its inhibition may be due to ion the blood from liver glycogen pool and from imbalance or plasma membrane damage other precursors of glucose 6 phosphate and (Sastry and Gupta, 1980) and may also due to catalysis a critical step in gluconeogenesis formation of enzyme inhibitor complex and plays an important role in regulation of (Singh and Sharma, 1998; Rajanna et al., glucose homeostasis (Nordlie et al., 1993). 1999). Decrease in LDH activities was observed after exposure to endosulfan and The activity of the cytosolic malic enzyme in fenvalerate on freshwater fish Clarias the liver shows significant decrease during batrachus, which indicates decrease in sublethal exposure of 4-NP. Malic enzyme aerobic and anaerobic capacity of fish (Rao, catalyses the reversible decarboxylation of 1999). Changes in the enzyme activity may malate to form pyruvate in the presence of provide direct and indirect evidence of the NADP coenzyme. Malic enzyme activity in cellular damage and can indicate the toxic crustacean and fish muscles is much higher mechanism. Here decrease in activity than that observed in most terrestrial species indicates decrease in aerobic and anaerobic (Skorkowski, 1988).Inhibition of ME activity capacity of fish. by 4-NP, and in consequence the decreasing formation of NADPH, could interfere with In the present study, 4-NP exposure the cellular mechanism against detoxification significantly reduced ICDH activity after 7, and oxidative stress. 14 and 21 days of exposure. Isocitrate dehydrogenase catalyses the reversible According to the present study, 4- oxidation of isocitrate to oxalosuccinic acid, Nonylphenol performs non-estrogenic followed by decarboxylation, leading to the adverse effects in liver tissue of L. rohita in formation of alpha keto glutarate. It is found addition to tissue-specific estrogenic effects. only in the mitochondria and this enzyme Based on the study, 4-NP negatively affect appears to participate in the TCA cycle. The the intermediary enzymes such as LDH, reduced activity of ICDH indicated that, 4- ICDH, G6Pase and cME. However, 4-NP NP exposure in fish may influence energy significantly increased the activity of some and redox status. On the view point of mitochondrial enzymes like NADH Benderdour et al., (2004), because of its role dehydrogenase and SDH. But in the case of in intermediary metabolism and energy CCO and MDH, 4 –NP inhibited their activity significantly. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

422 Current status and challenges for conservation and sustainable use of biodiversity Conclusion Gassner, B., Wuthrich, A., Scholtysik, G. and Solioz, M. 1997. The pyrethroids, Exposure of L.rohita to sub lethal permethrin and cyhalothrin are potent concentrations of 4-NP significantly inhibitors of the mitochondrial complex I. J diminish, the intermediary enzymes such as Pharmacol Exp Therap., 281 (2): 855-860. LDH, ICDH, G6Pase and cME. On the other hand, it also affected the mitochondrial King, J. 1965. The dehydrogenases or enzymes such as NADH dehydrogenase oxidoreductases lactate dehydrogenase. In CCO, MDH and SDH. The present study Practical clinic Enzymol London Van reports presence of 4-NP even at very low Nostrand D Company Ltd :83-93. concentrations in the aquatic environments may cause harmful effects on aquatic Latner, A. L. 1975. Contarow and Tumer organisms. The parameters studied in this Clinical Biochemistry, 7th ed. Philadelphia, study could be used as potential biomarkers PA: W.W. Saunders Co. in assessing toxic effects of 4-NP. Madsen, S. S., Mathiesen, A. B. and References Korsgaard, B. 1997. Effects of 17-estradiol and 4-nonylphenol on smoltification and Ashfield, L. A., Pottinger, T. G. and vitellogenesis in Atlantic salmon (Salmo Sumpter, J. P. 1998. Exposure of female salar). Fish Physiol Biochem., 17:303-12. juvenile rainbow trout to alkylphenolic compounds results in modifications to growth Madigou, T., Le Goff. P., Salbert, G., and ovosomaticindex. Environ Toxicol Chem., 17: 679-86. Cravedi, J. P., Segner. H., Pakdel,. F. and Benderdour. M., Charron.G., Comk. B., Valotaire, Y. 2001. Effects of nonylphenol on Ayoub. R., Beaundry. D., Foisy. S., De Blois. D. and Des Rosier. C. 2004. Decreased estrogen receptor conformation, cardiac mitochondrial NADP+ isocitric dehydrogenase activity and expression:a transcriptional activity and sexual reversion marker of oxidative stress in hypertrophy development. Am. J. Physiol.Heart in rainbow trout (Oncorhynchus mykiss). Circ.Physiol., 287:2122-2133. Aqua. Toxicol., 53: 173-186. Marin, M.G., Rigato, S., Ricciardi, F. and Matozzo, V. 2008. Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastodermaglaucum, Marine Pollut. Bull, 57: 552-558. Chaube, R., Gautam, G. J. and Joy, K. P. Mehler, A. H., Kornberg. A., Grisolia. S. and 2013. Teratogenic effects of 4-nonylphenol Ochao. 1948. Chemistry and methods of on early embryonic and larval development enzyme: Desmolases. J. Biochem., 174: 961- of the catfish Heteropneustes fossilis. Arch 977. Environ Contam Toxicol., 64: 554-61. Minakami, S., Ringleo, R.C. and Finney, D. 1971. Probit analysis. Cambridge University Press, Cambridge. Singer,T.P.1962. Respiratory chain linked dihydrodiphosphopyridine nucleotide hydrogenase assay of enzyme in soluble Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

Effect of 4 –nonylphenol on miotochondrial and intermediary metabolism in a freshwater fish.. 423 particulate and insoluble preparation. J. Biol., Greenamyre, J.T. 2007. Mechanism of 237: 569-579. toxicity of pesticides acting at complex I: Relevance to environmental etiologies of Nordlie. R.C., Bode, A.M and Foster, J. D. Parkinson's disease. J Neurochem., 1993. Recent advances in hepatic glucose-6- 100(6):1469-79. phosphatase regulation and function. Proc Soc Exp Bio Med., 203:274-285. Shivakumar, R. and David, M. 2004. Toxicity of endosulfan to the freshwater fish, Ochoa, S. 1955a. Malic Enzyme Methods. Cyprinis carpio. Ind. J. of Ecology, 31: 27- Enzymol., 66: 323-325. 29. Ochoa, S. 1955b. Isocitrate dehydrogenase Singh. R.K.. and Sharma.B. 1998. methods. Enzymol., 66: 232-236. Carbofuran-induced biochemical changes in Clarias batrachus. Pest Sci, 53: 285-290. Peal, W., Cascarano. J and Zweifach. B W. 1963. Micro determination of cytochrome Skorkowski, E. F. 1988. Mitochondrial malic oxidase in rat tissues by the oxidation on N- enzyme from crustacean and fish muscle, phenyl p-phenylene diamine or ascorbic acid. Comp. Biochem. Phys. B, 90 (1): 19–24. J hostochem cytochem.,11:102-104. Slater. E.C. and Bonner, W. D. 1952. Effect Rajanna. B., Chetty. C S., McBride. V. and of fluoride on succinate oxidase system. Rajanna. S. 1999. Effects of lead on K+-para- Analytical Biochem:384-399. nitrophenyl phosphatase activity and protection by thiol reagents. Biochem. Int., SPSS.,1998. SPSS for Windows. SPSS Inc, 20: 1011-1018. Headquarters. Chicago. Rao, J. V., Rai. C H S., Kavitha. P., Rao. R Srinivasamoorthy, K. 1983. Modulatio of N., and Mahavendra. S. S.2003. Toxicity of Carbohydrate and Associated Metabolism in chlorpyrifos to the fish, Oreochromis the Selcted Tissue of Marine Prawn Penaeus mossambicus. Bull. Environ. Contam. indicus During Induced Methyl Parathion Toxicol., 70: 285-292. Stress, Tirupathi, India: Ph.D Thesis. S. V. University. Rao, K. R. S. 1999. Pesticide Impact on Fish Metabolism, New Delhi, India: Discovery Swanson, M. A. 1955. Glucose-6- publishing House 111-112. phosphatase from liver. Meth. Enzymol., 2: 541-543. Sastry, K..V and Gupta, P.K. 1980. Alterations in the activities of a few Tyler, D. D. 1992. The mitochondrion in dehydrogenases in the digestive system of 2 health and desease, New York, VCH teleost fishes exposed to lead nitrate. publishers: 318-323. Ecotoxicol. Environ. Safe, 4: 232-239. Usha Rani, A and Ramamurthy, P. 1987. Sherer, T.B., Richardson, J.R., Testa, Effect of sublethal concentration of cadmium C.M., Seo, B.B., Panov, A.V., Yagi, T., on oxidative metabolism in the fresh water Matsuno-Yagi, A., Miller, G.W. and Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

424 Current status and challenges for conservation and sustainable use of biodiversity teleost, Tilapia mossambica, Indian Journal comparative Animal Physiology, 5(2): 71-74. Vineet Kumar, K., Patel. and David, M. 2008. Behaviour and respiratory dysfunction as an index of malathion toxicity in freshwater fish, Labeo rohita. Turkish J. of Fisheries and Aquatic Sciences. 8: 233-237. Vutukuru, S.S. 2005. Acute effects hexavalent chromium on survival, O2 consumption, hematological parameters and some biochemical profiles of the Indian major carp, Labeo rohita. Ind. J. Environ. Res. Pub. Health, 2: 456-462. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.417-424 © Principal, Sree Narayana College, Kollam, Kerala, India

Biomarkers as tools to characterize the contaminated ecosystem 425 ISBN 978-93-5396-871-7 CHAPTER 60 BIOMARKERS AS TOOLS TO CHARACTERIZE THE CONTAMINATED ECOSYSTEM Sulekha B T* and Anna Mercy T V** P G and Research Department of Zoology, Sree Narayana College, Kollam, Kerala ** KUFOS, Panangad, Kochi, Kerala *Correspondence E-mail: [email protected] ABSTRACT Pesticides are one of the dangerous pollutants which cause great harm to the animals present in the aquatic environment including fishes. Assessing the extent of pesticide induced pathogenesis in the kidney cells of Anabas testudineus, a fresh water food fish inhabiting the paddy fields of Kuttanad. As the concentration of Phosphamidon increased, the damage was also increased. Shrinkage of renal tubules may be due to the reaction of pesticides on the wall of the renal tubules or due to the osmotic imbalance and it gradually diminishes the excretory surface area. Later it causes the reduction of excretion and gradually leads to the death of the fish itself. The melano-macrophage centers are also the indicator of stressful environmental conditions. This show how toxic is this pesticide, Phosphamidon to the fish species. It may affect the human health as it is one of the common edible species of the inhabitants of Kuttanad. Hence, its use should be reduced and/ or regulated. Key words: Pesticides, Biomarkers, Anabas testudineus, Phosphamidon Introduction variety of agricultural pests. Pesticides reach water either through direct application or Pesticides are one of the dangerous pollutants indirectly from agricultural fields, spray drift, which cause great harm to the animals rainwater, sewage and effluents from present in the aquatic environment including industries manufacturing pesticides or using fishes. The world over, human health is at them in their processes. Pesticides are risk. While on one hand people are dying due becoming potential dangers for the to scarcity of food, on the other they are rejuvenation of fish wealth of the country. slowly being poisoned by the chemicals used for growing food (Shobha Nagnur, 2012). Kuttanad, the rice bowl of Kerala, is a region where there is overdose application of Agricultural pesticides are nowadays being pesticide during the punja cultivation periods. extensively used for the control of a wide A survey conducted by a volunteer group Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.425-430 © Principal, Sree Narayana College, Kollam, Kerala, India

426 Current status and challenges for conservation and sustainable use of biodiversity makes an observation regarding reports on Results the reduction in the fish population and massive deaths due to ulceration in fish in In the laboratory conditions the mean Kuttanad appear very frequently in the local temperature, pH and DO values for Anabas media (Indiradevi, 2007). The Kerala testudineus ranged from 27.00 to 28.71oC, Agricultural University restricts the use of 7.30 to 7.60 and 6.10 to 7.10 mg.l-1 certain chemicals for rice in the area (methyl respectively. parathion and BHC). Interestingly, the most commonly used chemicals in the region were The kidney of A. testudineus maintained in methyl parathion, followed by the control exhibited intact vascularised monocrotophos and phosphamidon. The dose glomeruli, renal tubules and hematopoietic of the spray fluid used in the study area was cells(Fig.1). 0.5ppm and 1.0ppm found to be much higher than the phosphamidon treated A. testudineus showed recommended level in all cases. almost similar structures to the control fishes. Whereas 2.0ppm treated A. testudineus The present study is aimed at assessing the exhibited moderately shrunken renal tubules extent of pesticide induced pathogenesis in (SR) and few regions of melano-macrophage the kidney cells of Anabas testudineus, a centres (ML) (Fig.2). But fishes in 5.0ppm fresh water food fish inhabiting the paddy showed perceptible variation from the other fields of Kuttanad. lower concentrations. Most of the renal tubules were considerably shrunken (SR) and Materials and methods also exhibited severe shrinkage of glomeruli (SG)(Fig 3). A. testudineusin 10.00ppm 48 hr LC50 value of Phosphamidon for revealed severe shrinkage of glomerulus Anabas testudineus was initially determined (SG) and degeneration of renal tubules by acute bioassay. Juveniles of Anabas (DR)(Fig. 4). It frequently exhibited the testudineus(Length 67.5 3.0 mm and weight accumulation of darkly stained melano- 330  80mg) were exposed to sublethal macrophage centers (ML)and shrunken renal concentrations of phosphamidon for a period tubules(Fig. 5). of 30 days. Maximum and minimum sublethal concentrations were chosen based The histological conditions of fishes on Konar (1969) and Sprague (1973). The collected from Kuttanad paddy fields showed concentrations were 0.5ppm, 1.0ppm, the toxicity of the environment. They showed 2.0ppm, 5.0ppm and 10.0ppm in addition to degenerated renal tubules (DR) (Fig. 6), the control group. After 30 days, the kidney melano-macrophage centers (ML) (Fig. 7), was dissected out and prepared for the shrunken renal tubules (SR) and shrunken histological analysis. Simultaneously Anabas glomerulus(Fig. 8). testudineuscollected from the paddy fields of Kuttanad were also subjected to histological analysis. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.425-430 © Principal, Sree Narayana College, Kollam, Kerala, India

Biomarkers as tools to characterize the contaminated ecosystem 427 Fig.1 - Control Fig. 2 - 2.0ppm Fig. 3 - 5.0ppm Fig. 4. 10.0ppm Fig. 5 -10.0ppm Fig. 6 -Kuttanad Fig. 7 –Kuttanad Fig. 8 -Kuttanad (DR - Degenerated renal tubule, ML - Melano-macrophage, SG - Shrunken glomerulus, SR - Shrunken renal tubules, BC - hematopoietic cells, Kuttanad - Sections of kidney of fishes collected from Kuttanad) Discussion environmental conditions due to pesticides, as reported by Agius (1985), Wolke et al. In the case of kidney, lesions are non-specific (1985b), Blazer et al. (1987) and Bucke and hence it is not possible to attribute the (1991). Many studies have suggested that the occurrence of a given type of lesion to a general functions of these aggregates are the particular pollutant but an attempt is made centralization of destruction, detoxification here to discuss the pathological changes in or recycling of endogenous and exogenous relation to concentrations between chemical materials (Vogelbein et al., 1987; Wolke, pollution and specific kidney conditions in 1992). Macrophage centres ingested with the fishes under study. processed material in them can also act as germicidal centres for the induction of In the present study, the melano-macrophage immune reactions (Lamers and De Haas, centres were observed in higher 1985) and they have an important role in the concentrations. Here the melano-macrophage initial responses of fish to foreign material, centres are the clear indicator of stressful Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.425-430 © Principal, Sree Narayana College, Kollam, Kerala, India

428 Current status and challenges for conservation and sustainable use of biodiversity (Wishkovsky et al., 1989). According to concentrations. This degeneration of renal Anderson (1990) phagocytic cells are tubules probably occurred because of the lack involved in the initial steps of the fishes' of oxygen as a result of acute anemia as defensive system and play a fundamental role suggested by Taveekijakran et al. (1996). in resistance to diseases. Thus the activity of Different renal and glomerular changes were macrophages is one of the first indicators for reported by Ayoola and Ajani (2008) in the presence of a stressor. fishes due to cypermethrin exposure. The frequently observed shrinkage and Histopathological changes in the glomerulus degeneration of renal tubules in the kidney of have been reported in many disease processes A. Testudineus collected from the paddy but no systematic study has so far been fields and polders of Kuttanad also revealed carried out. In the present study the the impact of uncontrollable use of pesticides glomerular shrinkage was observed in 5.0 in the paddy fields (Sulekha and Anna and 10.0 ppm treated fishes. This glomerular Mercy, 2015a & b). shrinkage resulted in an increase in Bowman's space. The shrinkage of Conclusion glomerulus might be due to the pesticide reaction on the walls of the capillaries and it As the concentration of Phosphamidon might diminish the surface area of the increased, the damage was also increased. glomeruli. Later, it may lead to the reduction Shrinkage of renal tubules may be due to the of excretion and finally causes the death of reaction of pesticides on the wall of the the fish. Similar pathological symptoms renal tubules or due to the osmotic imbalance were also observed by workers in response to and it gradually diminishes the excretory sublethal exposure to the pesticides like surface area. Later it causes the reduction of lindane (Colisa fasciatus - Verma et al., excretion and gradually leads to the death of 1975), monocrotophos (Puntius conchonius - the fish itself. The melano-macrophage Kumar and Pant, 1985; Sulekha & Anna centres are also the indicator of stressful Mercy, 2015b), malathion (Leponius environmental conditions. This show how macrochirues - Dutta and Marclino, 1990) toxic is this pesticide, Phosphamidon to the and phosphamidon (Sulekha and Anna fish species. It may affect the human health Mercy. 2015a). as it is one of the common edible species of the inhabitants of Kuttanad. Hence, its use The shrinkage of renal tubules may be due to should be reduced and/or regulated.The the reaction of pesticides on it or due to the histopathological changes give an early osmotic imbalance and it gradually warning of the damage caused in the fish at diminishes the excretory surface area. Later the histological level before their mortality. it causes the reduction of excretion and Hence preventive measures can be taken to gradually leads to the death of the fish itself. protect the fishery resources. Pandey et al. (1997) reported this condition as oedema. In the present study, degenerated renal tubules were observed in higher Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.425-430 © Principal, Sree Narayana College, Kollam, Kerala, India

Biomarkers as tools to characterize the contaminated ecosystem 429 References and sublethal concentrations of monocrotophos. Bull. Environ. Contam. Agius, C. 1985. The melano-macrophage Toxicol., 35(2): 228-233. centers of fish: a review. In: Fish Immunology (M.J. Manning and M.F. Tatner. Lamers, C.H.J. and De Haas, M.J.H. 1985. eds.) pp. 85-105. Academic Press, London. Antigen localization in the lymphoid organ of carp(Cyprinus carpio). Cell tissue Res., 242: Anderson, D.P. 1990. Immunological 491-498. indicators: effects of environmental stress on immune protection and disease outbreaks. Pandey, A.K., George, K.C. and Mohamed, Am. Fish. Soc. Symp., 8: 38-50. M.P. 1997. Histopathological alterations in the gill and kidney of an estuarine marine Ayoola, S.O. and Ajani, E.K., 2008. mullet, Liza parcia (Hamilton Buchanan) Histopathological effects of cypermethrin on caused by sublethal exposure to lead (Pb). juvenile African catfish (Clarias gariepinus). Indian J. Fish, 44(2): 171-180. World Journal of Biological Research, vol. 1, pp. 1-14. Shobha Nagnur, Vijaya Hosamani and Anitha Shapur. 2012. Training on organic Blazer, V.S., Wolke, R.E., Brown, J. and farming practices for women –An impact Powell, C.A. 1987. Piscine macrophage study. Karnataka J. Agric. Sci.,25 (2) : (253- aggregate parameters as health monitors: 255). effect of age, sex, relative weight, season and site quality in largemouth bass (Micropterus Sprague, J.B. 1973. The ABC's pollutant salmoides). Aquat.Toxicol., 10: 199-215. bioassay using fish. In: Biological methods for the assessment of water quality. (J. Bucke, D. 1991. Current approaches to the Cairns, Jr. and K.L. Dickson eds.). pp.6-30 study of pollution-related diseases in fish. ASTM STP 528, American Society for Bull. Eur. Assoc. Fish pathol., 11: 46-53. testing and Materials, Philadelphia. Dutta, H.M. and Marclino, J., 1990. Effects Sulekha B. T. and T. V. Anna Mercy. of malathion on kidney and skin of bluegill fish, Lepomius macrochirus. J. Freshwat. 2015a.Phosphamidon Induced Biol., 2(2): 77-88 Histopathological Changes in the Kidney of Indira Devi, P. 2007. Health damages due to pesticide use- a study, LEISA India. 9(3): 28. Freshwater Fish, Etroplus maculatus. Holistic Konar, S.K., 1969. Two organophosphorus Thought. Vol. XIV No.2. 5-12. insecticides DDVP and phosphamidon as selective toxicants. Transactions of the Sulekha B. T. and T. V. Anna Mercy. 2015b. American Fisheries Society, 98: 430- 437. Histopathological Effects of Pesticide on the Kumar, S. and Pant. S.C. 1985. Renal pathology in fish (Punchius conchonius Kidney of Etroplus maculatus in the Paddy Ham.) following exposure to acutely lethal Fields of Kuttanad. Proceedings. Solid Waste Management:Emerging Trends and Challenges. St Stephens College, Pathanapuram. Taveekijakran, P., Miyazaki, T., Matsumolo, M. and Arai, S. 1996. Studies on Vitamin K

430 Current status and challenges for conservation and sustainable use of biodiversity deficiency in amago salmon. Oncorhynchus rhodurus (Jordan and McGregor). J. Fish Dis., 19: 209–214. Verma, S.K. Gupta, S.P. and Tyagi, M.P. 1975. Studies on the toxicity of lindane on Colisa fasciatus (Part I: TLM measurement and histopathological changes in certain tissues). Gegenbaues Morph. Jahrb. Leipzig, 121(1): 38-54 Vogelbein, W.K., Fournie, J.W. and Overstreet, R.M. 1987. Sequential development and morphology of experimentally induced hepatic melano- macrophage centers in Rivulus marmoratus. J. Fish. Biol., 31 (Suppl. A), 145-153. Wishkovsky, A., Mathews, E.S. and Weeks, B.A. 1989. Effect of tributyltin on the chemiluminesence response of phagocytes from three species of estuarine fish. Arch. Environ. Contam. Toxicol., 18: 826-831. Wolke, R.E., George, C.J. and Blazer, V.S. 1985b. Pigmented macrophage accumulations (MMC; PMB): Possible monitors of fish health. In: Paracitology and Pathology of Marine Organisms of the World Oceans (W.J. Hargis, Jr. eds.). NOAA Tech. Rep. NMFS, 25: 93-97. Wolke, R.E. 1992. Piscine macrophage aggregates: a review. Annu. Rev. Fish Dis., pp. 91-108. Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.425-430 © Principal, Sree Narayana College, Kollam, Kerala, India

431 Current status and challenges for conservation and sustainable use of biodiversity ISBN 978-93-5396-871-7 CHAPTER 61 HEPATOTOXIC EFFECTS OF 4-NONYLPHENOL ON OXIDATIVE STRESS ANDANTIOXIDANT RESPONSES IN THE INDIAN MAJOR CARP, LABEO ROHITA Reshmi S* and Aruna Devi C Department of Zoology, University College, Thiruvananthapuram-695034, Kerala * Correspondence E-mail: [email protected] ABSTRACT 4-Nonylphenol (4-NP) is a well-known endocrine-disrupting chemical (ECD) present in the aquatic environment, but little is known about its oxidative stress effects. An emerging literature suggests that early life exposure to 4-NP, may increase the risk of metabolic syndrome. Due to its hydrophobicity and long half-life, 4-NP can easily accumulate in living organisms, including humans, where it displays a seriesof toxic effects. In the present study, the oxidative stress and antioxidant parameters of Labeo rohita after exposure to various sublethal concentrations of 4-NP for 5, 10 and 15 days, respectively was examined. The Glutathione content (GSH) and the activity of Glutathione Peroxidase (GPx) were significantly inhibited, whereas, the activity of Superoxide Dismutase (SOD), Catalase (CAT), Glutathione-S- Transferase (GST) and Glutathione Reductase (GR) and the Lipid Peroxidation products(LPO) such as Malondialdehyde (MDA) and Conjugated Diene (CD) were significantly elevated, indicating the occurrence of oxidative stress. Exposure to the chemical was found to enhance the production of hydroxyl radicals and lipid peroxidation in a concentration-dependent manner. The resultsdemonstrated that 4-NP in aquatic systems can affect antoioxidant responses. Key words: Endocrine-disrupting chemical, Oxidative stress, Antioxidant responses. Introduction manufacturing, textile processing, pesticide emulsifiers, pulp and paper production, and Nonylphenol ethoxylates (NPEs) represents personal care products. Many studies are the most critical metabolite of alkylphenols focused on the effect of 4-NP on the (APs) and alkylphenol ethoxylates (APEs), reproductive impairments including non-ionic surfactants widely used in the developmental and physiological formulation of domestic and industrial abnormalities related to estrogenecity but products. Quite recently, 4-NP has been recent studies reported that 4-NP alters described as priority hazardous compound antioxidant enzymes in fish and mammals. 4- (EC, 2002). Moreover, 4-NP is a lipophilic Nonylphenol has the potentiality to bring compound and may consequently be taken up about all these alterations in mammalian and accumulated by aquatic organisms. species and a few aquatic species, but the These non-ionic surfactants are used in oxidative potential of 4-NP has not been cleaning products, plastic and elastomer much reported in sub-mammalian species, Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

432 Current status and challenges for conservation and sustainable use of biodiversity including fish. Notwithstanding their chemicals U.S.A. ecological importance, the phenomenon of oxidative stress in fishes to 4-NP still needs Statistics in-depth studies. Hence, in this regard it was thought worthwhile to investigate the All the statistical analysis was carried out oxidative potential of 4-NP, if any, in L. using the software SPSS 22.0 package for rohita. Windows. Data analysis was done by one- way analysis of variance (ANOVA). The Materials and methods difference in means were analysed by using Duncan multiple range tests (1955). Experimental Design Significant level was p˂0.05. The association of variables was studied by Pearson The experimental model Labeo rohita were correlation test. collected from local suppliers. The fish were divided into 10 groups, weighing 30±5 g and Results acclimatized in the laboratory. After this, the fish were exposed to various sub-lethal doses Total Protein Level (1.0µl/l, 1.5µl/l and 2.0µl/l) of 4- Nonylphenol (4-NP) for a periodof 5, 10 and A significant increase was recorded in protein 15 days in aquarium tanks. After stipulated level after 10 and 15 days of exposure to period of exposure, fish were sacrificed and 1.0µl/l4-NP but the activity was depleted at 5 liver was excised and frozen for enzyme assay days of exposure. However, with 1.5 and at -200 C. The lab acclimatized fish kept in 2.0µl/l 4-NP, the protein activity remained dechlorinatedtap water served as control. significantly elevated after 5, 10 and 15 days of exposure compared to control. Biochemical Analysis Activities of antioxidant enzymes The activity of Superoxide dismutase (SOD) A. Superoxide Dismutase (EC.1.15.1.1), Glutathione peroxidase (GPx) (EC.1.11.1.9), Catalase (CAT) (EC.1.11.1.6), There was a significant increase in the SOD Glutathione content (GSH), Glutathione-S- activity in liver tissue after 5 and 15 days of Transferase (GST) (EC.2.5.1.18), Glutathione exposure to 1.0µl/l 4-NP but the activity reductase (GR) (EC.1.6.4.2) and protein were decreased after 10 days of exposure. determined using UV- Visible However, with 1.5µl/l and 2.0µl/l 4-NP, SOD spectrophotometer (Perkin Elmer). Protein activity increased significantly from 5 days was estimated according to the protocol of and the elevation was maintained till 10 and Bradford, (1976), SOD was estimated 15 days with respect to control. according to the protocol of Kakkar et al., (1984), GPx according to Lawrence et al., B. Catalase (1976) as modified by Agergaard and Jenson (1982), CAT activity was measured Catalase activity did not change significantly according to Maehly et al., (1954), GSH after 5 days of exposure to 1.0µl/l 4-NP. according to Benke et al., (1974), G-S-T However significant elevation was observed according to Habig et al., (1974) and GR after 10 and 15 days of exposure to 1.0µl/l 4- according to David et al., (1983) and lipid NP. Catalase activity showed a significant peroxidation products; MDA according to increase in the liver tissue of fish after 10 and Nichans et al., (1968) and CD according to 15 days of exposure to 1.5µl/l 4-NPbut there Recknagel et al., (1966). Chemicals were of was no notable difference after 5 days of analytical grade and purchased from Sigma exposure to 1.5µl/l 4-NP. Likewise, exposure to 2.5µl/l 4-NP significantly increased CAT Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxic effects of 4-nonylphenol on oxidative stress 433 activity in the fish after 5, 10 and 15 days. significant change in GPx activity after 5 days of exposure. Similarly, fish exposed to C. Glutathione-S-Transferase 2.0µl/l 4-NP showed significant decrease in GPx activity after 5, 10 and 15 days as The activity of GST measured with CDNB as compared to that of control. substrate increased significantly in hepatic tissue of fish exposed to 1.0µl/l, 1.5µl/l and F. Glutathione Content 2.0µl/l 4-NP for 5,10 and 15 days at all exposure in a dose- and time-dependent Fish exposed to 1.0µl/l, 1.5µl/l and 2.0µl/l 4- manner. NP had significantly depleted hepatic concentration of GSH after 5, 10 and 15 days D. Glutathione Reductase in comparison with the control group. There is a significant increase in the activity Levels of Lipid Peroxidation Products of GR to 1.0µl/l 4-NP after 5, 10 and 15 days of exposure to 4-NP in a time- and dose- A. Malondialdehyde Content dependent manner. These elevated levels were similarly maintained throughout 10 and The hepatic concentration of MDA, which 15 days of exposure to 1.5µl/l and 2.0µl/l 4- reflect the degree of lipid peroxidation is NP. illustrated a significant elevation in the concentration of MDA in fish exposed to E. Glutathione Peroxidase 1.0µl/l, 1.5µl/l and 2.0µl/l 4-NP after 5, 10 and 15 days of exposure to 4-NP. One-Way ANOVA tests showed that there was significant decrease in GPx activity after B. Conjugated Diene Content 5, 10days and 15 days of exposure to 1.0µl/l 4-NP. Glutathione peroxidase activity Conjugated diene content increased significantly reduced after 10 and 15 days significantly in a dose- and time-dependent exposed to 1.5µl/l but there was no manner in1.0µl/l, 1.5µl/l and 2.0µl/l 4-NP after 5, 10 and 15 days of exposure d cd a ac b Fig. 1: The effect of sub-lethal doses of 4-NP (1.0µl/l, 1.5µl/l, 2.0µl/l) on the level of Total Protein (A) and activities of SOD (B) and CAT (C), GPx (D) and GST (E), in the liver of L. rohita with respect to control. Each column is mean ± SEM for six fish. Mean values of different superscript letters (a, b, c, d) were significantly different (p˂0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

434 Current status and challenges for conservation and sustainable use of biodiversity d d c b c b Fig. 2: The effect of sub-lethal doses of 4-NP (1.0µl/l, 1.5µl/l, 2.0µl/l) on the activity of GR (A) and level of GSH (B), MDA (C) and CD (D), in the liver of L. rohita with respect to control. Each column is mean ± SEM for six fish. Mean values of different superscript letters (a, b, c, d) were significantly different (p˂0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India

Hepatotoxic effects of 4-nonylphenol on oxidative stress 435 Fig. 3: The effect of sub-lethal doses of 4-NP (1.0μl/l, 1.5μl/l, 2.0μl/l) on the activity of GPx (A) and level of GSH (B), in the liver of L. rohita with respect to control. Each column is mean ± SEM for six fish. Mean values of different superscript letters (a, b, c, d) were significantly different (p˂0.05). Current Status and Challenges for Conservation and Sustainable use of Biodiversity|2020 | pp.431-440 © Principal, Sree Narayana College, Kollam, Kerala, India