Brijesh Ranjan

Environ Sci Pollut Res DOI 10.1007/s- RESEARCH ARTICLE Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation Ayan Srivastava 1 & Neeraj Verma 1 & Arup Mistri 1 & Brijesh Ranjan 1 & Ashwini Kumar Nigam 1 & Usha Kumari 2 & Swati Mittal 1 & Ajay Kumar Mittal 3 Received: 11 August 2016 / Accepted: 30 January 2017 # Springer-Verlag Berlin Heidelberg 2017 Abstract Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4 days, using 24 h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes—alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase—was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate Responsible editor: Thomas Braunbeck * Swati Mittal-Usha Kumari-Ajay Kumar Mittal-1 Skin Physiology Laboratory, Centre of Advanced Study, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221 005, India 2 Zoology Section, Mahila Mahavidyalaya, Banaras Hindu University, Varanasi, Uttar Pradesh 221 005, India 3 Present address: Department of Zoology, Banaras Hindu University, 9, Mani Nagar, Kandawa, Near Chitaipur Crossing, Varanasi 221106, Uttar Pradesh, India dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8 days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations. Keywords Labeo rohita . Skin . Histopathology . Metabolic and antioxidant enzymes . Eriochrome black T Introduction Fishes live in aquatic environment. These, in general, are highly susceptible to pollutants in water bodies inhabited by them and are considered to play an important role as Bbiological indicator^ of water quality and degree of pollution in the medium. Azo dyes are used worldwide, predominantly in dyeing industries, e.g., textile, carpet, paper, etc. These dyes are highly soluble in water and thus constitute one of the major groups of toxic compounds in effluent released into water bodies—ponds, pools, lakes, rivers, etc. Azo dyes could raise potential environmental concerns considering these are nonbiodegradable, toxic, mutagenic, and carcinogenic (Mathur et al. 2012; Ayadi et al. 2016). As a result, these dyes, in recent years, have gained more attention in the field of toxicology, environmental monitoring, and assessment (Barot and Bahadur 2015). Fish skin, in contrast to internal organs, is in direct contact with the surrounding water. It represents the principal interface between the external and the internal environments of the body and thus serves as the first line of defense to provide protection to the fish. Any change in the property of the Environ Sci Pollut Res ambient water disturbs the equilibrium and is reflected by changes in the structural organization and physiology of the skin. Alterations in the activity of metabolic and antioxidant enzymes in the skin (Hansen et al. 2006, Valavanidis et al. 2006) are considered to be sensitive biomarkers and are important parameters for testing water quality and harmful effects on fish. In literature, articles on the effect of dyes on fish are limited. Descriptions of the toxic effect of pulp mill effluents are available on the skin and gills of Carassius auratus Lidesjoo and Thulin 1994), on the skin of Anguila anguila (Pacheco and Santos 2002), and Heteropneustes fossilis (Baruah and Das 2002). Karanjkar et al. (2000) assayed mortality response in L. rohita exposed to textile wastewaters. More recently, Sharma et al. (2006) described acute and chronic toxicity of methyl red on Poecilia reticulata using indices such as mortality, reduction in red blood cell count, and morphological abnormality. Sudova et al. (2007) reviewed negative effects of malachite green on fish and its eggs. AbdelMoneim et al. (2008) evaluated 96 h LC50 of Clarias lazera exposed to wastewater, which was a combination of discharges from different processes in the industry producing dyestuffs of all groups including azo reactive dyes used in textile and paper industries, and reported the changes in hematological parameters, serum, liver function enzymes, creatinine and urea, and histopathological alterations in the gills, liver, and kidney. Nevertheless, studies on histopathological alterations and biochemical changes in the activity of metabolic and antioxidant enzymes in fish skin after exposure to azo dyes have largely been neglected. The azo dye, Eriochrome black T, is used worldwide in large quantities in carpet industries. Indiscriminate discharge of effluents of these industries in rivers, streams, lakes, and ponds is largely responsible for increasing pollution causing imbalance in the physical and chemical properties and rapid degradation of water quality. The present study was, therefore, undertaken with an aim to evaluate histopathological alterations and the changes in the activity of various metabolic and antioxidant enzymes—alkaline phosphatase (ALP, EC 3.1.3.1), acid phosphatase (ACP, EC 3.1.3.2), carboxylesterase (CBE, EC 3.1.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27), succinate dehydrogenase (SDH, EC 1.3.5.1), catalase (CAT, EC 1.11.1.6), and peroxidase (POX, EC 1.11.1.7)—in the skin of a freshwater teleost, L. rohita, exposed to sublethal concentration of the azo dye, Eriochrome black T. Experiments were also set to assess the restoration in the structural organization and the activity of enzymes on transfer of fish to freshwater after cessation of Eriochrome black T exposure. L. rohita (common name in Hindi, Rohu; Family, Cyprinidae; Order, Cypriniformes; Taxonomic Serial Number 163681; retrieved from Integrated Taxonomy Information System, 2008) is considered a valuable source of food and is cultured extensively in India. Knowledge of changes in the structural organization and activity of enzymes in relation to the toxicity of Eriochrome black T in fish could provide useful guidelines for its safe use. Materials and methods Collection and maintenance of fish Live specimens of L. rohita (mean ± SD; standard length, LS, 95 mm ± 10 mm; weight 22 ± 2 g; N = 168) were collected from local ponds at Varanasi, India and were maintained in the laboratory at controlled room temperature (25 ± 2 °C) in glass aquaria containing continuously aerated water. Fish were acclimatized with the laboratory conditions for 15 days prior to the commencement of the experiments. Fish were fed once daily with commercially available fish food, Tokyu® pellets, containing 46 % protein, 6 % fat, 5 % fiber, minerals, and vitamins (M.C.T. Aquarium, Import and Export, Changwat Nakhon Pathom, Thailand). Water quality characteristics were determined following APHA, AWWA, and WPCF (1985). Water quality parameters (mean ± SD) used for acclimation, in controls, and for the preparation of test solution were temperature, 25 ± 2 °C; dissolved oxygen, 7.06 ± 0.2309 mg/L; pH, 7.77 ± 0.0754; alkalinity, 249.32 ± 2.3094 mg/L; methyl orange as HCO3−; and hardness, 186.00 ± 3.4641 mg/L as CaCO3. Preparation of test solution and exposure Eriochrome black T (molecular formula C20H12N3O7SNa) (Qualigens Fine Chemicals, Part of Thermo Fisher Scientific India Pvt. Ltd., Mumbai, India, Cat. No. 39952), a mono azo dye, was used to determine its effect on fish skin histopathology and enzyme activity. The experiments were conducted to understand the short-term toxic effect of Eriochrome black T on the skin of the fish Labeo rohita. The 96-h LC50 value of Eriochrome black T for the fish was 8.89 mg/L at 0 % trimming and 8.88 mg/L at 10 % trimming (present authors, unpublished data). In this study, sublethal concentration (0.89 mg/L, i.e., 10 % of 96 h LC50 value) of the azo dye Eriochrome black T was selected randomly based on the survivability response of the fish following Altinok and Capkin (2007). Test solution was prepared by diluting the stock solution (5 g/L) of Eriochrome black T with water. Fish were exposed to the sublethal test concentrations of the dye, using 24 h renewal bioassay method (APHA, AWWA, WPCF 1985). Fish were divided into three groups: (1) fish without any treatment (control fish), (2) fish exposed to Eriochrome black T (experimental fish), and (3) fish exposed to Eriochrome black T for 4 days and then transferred to freshwater for recovery (recovery fish). Fish of the three groups were cold anesthetized following Mittal and Whitear (1978) at different intervals. Fish of the first and second groups (control fish and experimental fish) were cold Environ Sci Pollut Res anesthetized at intervals 1, 2, 3, and 4 days and those of the third group (recovery fish) at intervals 2 days of recovery (2dr), 4dr, 6dr, and 8dr. Histopathology Fish of the three groups, i.e., control, experimental, and recovery, were cold anesthetized at different intervals. Skin pieces (5 mm) from the dorsal side of the head near the snout were then excised, rinsed in physiological saline, and were fixed in aqueous Bouin’s fluid and Carnoy’s fluid following Bancroft and Gamble (2002) for histochemical and/or histopathological investigations. The fish were euthanized after sampling. Fixed tissues were dehydrated in an ethanol series of ascending concentrations, cleared in cedar wood oil, and then were embedded in paraffin wax (melting point 58–60 °C) (E-Merck, India). Serial sections of embedded tissues were cut at a thickness of 6 μm using a Leica Rotary Microtome (Model RM 2125RT, Leica Mikrosysteme Vertrieb GmbH-DSA, Germany). The tissue sections were mounted on ethanolcleaned glass slides and were kept in an oven at 37 °C overnight to dry. The tissue sections were de-paraffinized in xylene and hydrated in an ethanol series of descending concentrations. Tissue sections were then stained with Ehrlich’s hematoxylin and eosin (H/E) for histological organization of the tissue or with Alcian Blue at pH 2.5 followed by Periodic acid Schiff (AB 2.5/PAS) for localization of mucopolysaccharides in cells following Bancroft and Gamble (2002). Stained sections were dehydrated in an ethanol series of ascending concentrations, cleared in xylene, and mounted in distrene dibutyl phthalate xylene (DPX, Merck, Merck Specialities Pvt. Ltd., Mumbai, India). Observations were made using a Leitz BLaborlux S^ microscope (Ernst Leitz GmbH, Wetzlar, Germany). Results were recorded using a digital camera system Leica DFC 290 (Leica Microsystems Ltd. Germany) on an Intel® Pentium® D computer (Model dx2280 MT, HP Compaq, USA). Activity of LDH, CAT, and POX was determined by the methods following Worthington Enzyme Manual (2011a, 2011b, 2011c). The activity of SDH was determined by the method following Padh (1992). Specific activity of each of these enzymes was expressed in terms of nanomoles of product released per minute per milligram protein. Measurement and statistical analysis Area and density of the mucous goblet cells were measured in tissue sections stained with AB 2.5/PAS and those of club cells were made in the tissues sections stained with H/E. Leica Qwin3, an advanced image processing and analysis software (Leica Microsystems, Germany), was used for the measurement of area of cells. A combination of a stage micrometer (object micrometer) with scale graduated in units of 1/100 (1 division = 0.01 mm) and an eyepiece graticule (ocular micrometer) with square grid (Carl Zeiss, Jena, Germany) was used for the measurement of density of mucous goblet cells and club cells. All results were expressed throughout as mean ± SD (n = 3). Samples of ten randomly selected sites of skin were analyzed for each estimation. Estimations were based on the data obtained from three fishes. Each experiment was repeated three times to determine the reproducibility. Statistical differences were analyzed between data obtained from control fish and experimental fish and from control fish and recovery fish at different intervals using one-way ANOVA followed by Dunnett’s post hoc test. All statistical analyses were carried out through Statistical Package for the Social Sciences (SPSS) for Windows (standard version 11.5) software. P < 0.05 was accepted as the level of statistical significance. Results Histopathology Enzyme activity in fish epidermis Group 1 fish (control) For biochemical estimation of enzyme activity, skin pieces of fish of the three groups, i.e., control, experimental, and recovery, were excised at different intervals, homogenized in 0.1 M phosphate buffer (pH 7.1), and centrifuged at 10,000×g for 10 min at 4 °C. Corresponding fresh supernatants were used to assay the activities of ALP, ACP, CAT, LDH, CBE, and POX. The pellet was processed for determining the activity of SDH. Absorbance of solutions for enzyme activity was measured using Genesys 10 UV–Visible Spectrophotometer (Thermo Fisher Scientific, NY, USA). Assay of ALP and ACP activity was made by the method following Korndat and Braunbeck (2001). CBE activity was determined by the method following Thompson (1999). In control fish, the epidermis is stratified and is composed mainly of the epithelial cells. In general, these cells appeared vertically flattened or wedge shaped in the superficial layer, polygonal or vertically elongated in the middle layer, and columnar in the basal layer. The basal layer epithelial cells lie on a thin non-cellular basement membrane. In between these cells, small rounded lymphocytes were discernible. Interspersed between the epithelial cells were the gland cells—the mucous goblet cells and the club cells (Fig. 1a). In general, the mucous goblet cells were located mainly in the outer layers of the epidermis. In H/E-stained preparations, secretory contents of the mucous goblet cells were moderate to strongly basophilic; Environ Sci Pollut Res the nuclei of these cells were basal, flattened, or crescent shaped and strongly basophilic (Fig. 1a). Contents of mucous goblet cells stained bluish purple with the method AB 2.5/PAS for mucopolysaccharides (Fig. 1b). The club cells were voluminous and, in general, did not open on the outer surface of the epidermis (Fig. 1a). With H/E, the contents of the club cells were slightly eosinophilic. Each club cell had a single, central, healthy appearing moderately basophilic nucleus. The contents of the club cells stained light pink with the method AB 2.5/PAS (Fig. 1b). Group 2 fish (experimental fish exposed to the azo dye, Eriochrome black T) In fish exposed to the dye, epithelial cells in the superficial, middle, and basal layer of the epidermis at 1–4 days, compared to those in controls, in general, appeared swollen, hypertrophied, and loosely arranged (Fig. 1c). Further, intercellular spaces between the epithelial cells were more distinct. Mucous goblet cells at 1 day, in general, secreted profusely on the surface of the skin (Fig. 1c, d). At 2 days, a large number of mucous goblet cells were observed in the outer layers, and newly differentiated mucous goblet cells were discernible in deeper layers of the epidermis (Fig. 1d). Subsequently at 3 and 4 days, these cells showed a decline in their density. Nevertheless, it remained significantly high compared to those of the controls (Fig. 2). The mucous goblet cells did not show any significant change in their area throughout the experiment (Fig. 3). Club cells showed gradual degenerative changes characterized by vacuolization and shrinkage of their contents at 1–4 days. These cells often showed confluence with each other with simultaneous disappearance of the cell membrane and releasing their contents in the intercellular spaces (Fig. 1e, f). Density of the club cells showed a significant decline at 1–4 days (Fig. 2). An insignificant increase in the density of club cells was, however, observed at 2 days (Fig. 2). The area of club cells showed a gradual decrease and was significantly low at 3–4 days (Fig. 3). Group 3 fish(fish for recovery after exposure to the azo dye, Eriochrome black T) In the fishes, on their transfer to freshwater for recovery, epithelial cells at 2dr were observed less hypertrophied compared to those exposed to Eriochrome black T for 1– 4 days. Subsequently, these cells gradually become similar to those of the controls by 8dr (Fig. 1g, h). At 6dr, juvenile club cells, small in dimension, could be observed in the deeper layer of epidermis (Fig. 1g). Density and area of the mucous goblet cells and the club cells started Fig. 1 Photomicrographs of cross sections of the skin of the fish Labeo rohita: a, b control fish, c–f fish exposed to 0.89 mg/L of Eriochrome black T, and g, h fish transferred to freshwater for recovery. a Histological organization of the stratified epidermis showing epithelial cells (dotted arrow), mucous goblet cells (arrows), and club cells (arrowheads). Note rounded lymphocytes (barred arrows) between basal layer epithelial cells (H/E). b Mucous goblet cells (arrows) stained bluish purple (AB2.5/ PAS). c Hypertrophied epithelial cells (open arrows). Note the presence of intercellular spaces between the epithelial cells (arrow) (1 day, H/E). d A large number of mucous goblet cells (arrows) in the outer layers as well as in the deeper layers are discernable (2 days, AB2.5/PAS). e The club cells reduced in dimension and showed confluence with each other (arrowheads). Note the shrinkage of their contents (2 days, H/E). f Vacuolated club cells (arrowheads) devoid of secretory contents (4 days, H/E). g Juvenile club cells (arrowheads) are discernible in the deeper layers of the epidermis (6dr, H/E). h Compare with a. Epidermis appears similar to that of control. Note the epithelial cells (dotted arrows), mucous goblet cells (arrows), and club cells (arrowheads) (8dr, H/E). Scale bars = 50 μm recovering from 2dr. Subsequently, these gradually recovered and became similar to those of control by 8dr (Figs. 2 and 3). Enzyme activity in fish epidermis Group 1 fish (control) Specific activity of the enzymes, ALP, ACP, CBE, LDH, SDH, CAT, and POX in the skin of L. rohita under control conditions, is summarized in Table 1. It was observed that the enzyme CBE exhibited high activity Environ Sci Pollut Res Fig. 2 Comparison of density of mucous goblet cells (MGC) and club cells (CC) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. d, day; dr, day of recovery; asterisk on bars indicates significant difference from control (p < 0.05) (87.08 ± 1.26 nmol min−1 mg−1 protein (p < 0.05)). Activity of the other enzymes, compared to that of CBE, was much lower and was in the descending order: ACP, ALP, SDH, LDH, CAT, and POX (p < 0.05). Activity of POX was lowest (0.07 ± 0.003 (p < 0.05)) (Table 1). Group 2 fish (experimental fish exposed to the azo dye, Eriochrome black T) On exposure to the sublethal concentration of Eriochrome black T (0.89 mg/L), activity of ALP and SDH declined significantly at 1 day (Figs. 4 and 5), CBE, CAT, POX at 2 days (Figs. 6, 7, and 8), and ACP at 3 days (Fig. 9). Activity of all the enzymes further decreased gradually and was found to be lowest at 4 days (Figs. 4, 5, 6, 7, 8, and 9). Nevertheless, LDH showed an increase in activity at 1 and 2 days, which increased significantly at 3 days and was highest at 4 days (Fig. 10) (p < 0.05). Fig. 3 Comparison of area of mucous goblet cells (MGC) and club cells (CC) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Fig. 2 Group 3 fish (fish for recovery after exposure to the azo dye, Eriochrome black T) In the fishes on their transfer to freshwater for recovery, the activity of ALP and POX compared to those of the controls remained significantly low up to 2dr. Subsequently, the activity recovered and became similar to those of the controls by 8dr (Figs. 4 and 8). Activities of SDH, CBE, CAT, and ACP, however, remained significantly low as compared to control up to 8dr (Figs. 5, 6, 7, and 9). Activity of LDH on the contrary showed a gradual decline and became similar to control by 8dr (Fig. 10) (p < 0.05). Discussion The present study showed marked histopathological changes in the structural organization of the epidermis of the fish, Environ Sci Pollut Res Table 1 Specific activity (nanomoles min−1 mg−1 of protein) of different enzymes in skin of Labeo rohita in control condition, i.e., without exposure of Eriochrome black T Enzyme ALP ACP CBE LDH SDH CAT POX Specific activity 3.05 ± 0.14 7.54 ± 0.37 87.08 ± 1.26 0.27 ± 0.01 0.4 ± 0.01 0.09 ± 0.002 0.07 ± 0.003 Values = mean ± SD; N = 24 ALP alkaline phosphatase, ACP acid phosphatase, CBE carboxylesterase, LDH lactate dehydrogenase, SDH succinate dehydrogenase, CAT catalase, POX peroxidase L. rohita, exposed to the azo dye Eriochrome black T for different durations. Epidermis, being the outermost layer of the skin, is highly vulnerable to environmental hazards. Epithelial cells in the superficial, middle, and basal layers appeared swollen and hypertrophied as a response to stress caused by the dye. It could be suggested that the change is an attempt to protect the epidermis from the harmful impact of the dye, e.g., disruption and tissue damage. A significant increase in the density of mucous goblet cells, during 1–2 days of exposure of the fish to the dye, results in profuse secretion of mucus at the outer surface of the epidermis. This could be considered to cope up with the adversity in the environment and serve to protect the skin from the toxic impact of the azo dye. Fish skin mucus has fairly been recognized as a novel protective covering over the body surface. It remains in intimate contact with surrounding aquatic environment and acts as a barrier against mechanical, physical, and chemical stressors and pathogenic attacks (Shephard 1994; Nigam et al. 2014a). Further, in L. rohita, appearance of new differentiated mucous goblet cells in large numbers at 1– 4 days exposure of the fish to the dye, compared to those in controls, could be to provide sustenance of excessive secretion of mucus at the surface, to back up and continue protection against harmful effect of the dye in the surrounding medium. Bonga (1997) reported that, in gills, Fig. 4 Comparison of specific activity of alkaline phosphatase (ALP) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Cont., control; Exp., experimental. Rest of the abbreviations are the same as in Fig. 2 the hypersecretion of mucus is often followed by its depletion and the differentiation of new cells as part of a compensatory response to a variety of stressors. In L. rohita, significant decline in the density of club cells in the fish exposed to the dye could be considered as a result of degenerative changes in these cells, with the simultaneous confluent of the contents of the adjacent club cells and release of their contents into the intercellular spaces. Mittal and Garg (1994) reported similar changes in the club cells in the epidermis of Clarias batrachus exposed to sodium dodecyl sulfate and suggested that the contents of club cells released into the intercellular spaces might serve to plug the intercellular channels, preventing indiscriminate entry of foreign matter that might be initiated due to the disruption of the superficial layer of the epidermis. Disappearance of club cells following vacuolization and attenuation, in response to wound healing in L. rohita, has also been reported by Kumari et al. (2016). Statistically significant increase in the number of club cells in the fishes on their transfer to freshwater for recovery might be associated with differentiation of juvenile club cells. These cells appeared in the deeper layers in order to replenish degenerated club cells. Similar observations were also reported during effect of an anionic detergent in the epidermis of C. batrachus (Mittal and Garg 1994). The present study reports a significant decline in the activities of ALP and ACP in the skin of L. rohita exposed to Eriochrome black T. Recently, Kaur and Kaur (2015) also Environ Sci Pollut Res Fig. 5 Comparison of specific activity of succinate dehydrogenase (SDH) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Figs. 2 and 4 reported a decline in the activity of ALP and ACP in the gills of L. rohita exposed to another azo dye Acid Black. The inhibition of ALP may be due to a breakdown of the membrane transport system and an inhibitory effect on cell growth and proliferation (Lakshmi et al. 1991). While the inhibition of ACP may be due to accumulation of organic mercury compound in the lysosomes, it acts as a labilizing agent and ruptures the lysosomal membrane (Lakshmi et al. 1991). In addition, Gill et al. (1990) stated that the decrease in the activity of ACP could be attributed to the structural damage to the cellular machinery concerned with enzyme production. Significant decline in CBE activity of the skin of L. rohita on exposure to the azo dye could be as a result of instantaneous binding of the enzyme to the dye. Xing et al. (2010) also reported inhibition of CBE activity in Cyprinus carpio exposed to chlorpyrifos and atrazine. de Lima et al. (2013) showed inhibition of CBE activity in metal-treated Danio rerio. Solé and Sanchez-Hernandez (2015) reported a Fig. 6 Comparison of specific activity of carboxylesterase (CBE) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Figs. 2 and 4 decrease in CBE activity in several aquatic organisms on exposure to pharmaceuticals and personal care products. CBEs are serine hydrolases that catalyze the hydrolysis of esters, amides, thioesters, and carbamates (Laizure et al. 2013). These enzymes lack substrate specificity, so they are involved in many diverse physiological and toxicological processes (Wheelock and Nakagawa 2010). The natural substrate for most CBEs is not known; therefore, their physiological function has not been well understood. More recently, Nigam et al. (2014a) reported that these enzymes are important for the metabolism of endogenous and exogenous compounds. A significant increase in the activity of LDH in the skin of L. rohita on exposure to the azo dye corroborates with Rani et al. (2000) who also reported increased LDH activity in Tilapia mossambica following exposure to arsenic and Kamalaveni et al. (2003) who reported increased LDH activity in the liver of C. carpio exposed to group II pyrethroids (deltamethrin, cypermethrin, fenvalerate, and fluvalinate). More Environ Sci Pollut Res Fig. 7 Comparison of specific activity of catalase (CAT) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Figs. 2 and 4 recently, Manjunatha et al. (2015) reported a significant rise in LDH activity in Oreochromis niloticus exposed to potassium cyanide. According to Puvanshwari et al. (2006), increased LDH activity may reflect energy demand for anaerobic burst swimming. Increased activity of LDH is a characteristic feature of a shift from aerobic to anaerobic metabolism leading to an elevated rate of pyruvate conversion into lactate, resulting in lactic acidosis (Abdel-Hameid 2009). Since SDH is a primary enzyme in the oxidative catabolism of carbohydrates, a significant decrease in the activity of SDH reported in the present study indicates depression of cellular metabolism resulting in a shift to anaerobic metabolic pathway to meet the increased energy demand under stress. Chen et al. (2012) also reported a decrease in SDH activity in Pelteobagrus fulvidraco on exposure to copper (Cu). Inhibition of SDH activity could be due to binding of Cu or Fig. 8 Comparison of specific activity of peroxidase (POX) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Figs. 2 and 4 its metabolites with the enzyme molecules and/or by blocking the enzyme synthesis leading to impairment of aerobic metabolism (Rajeswari and Reddy 1989). Antioxidant defense mechanism in fish helps to maintain health and prevent oxidative lesions. Catalase is a scavanger of the reactive oxygen species acting on hydrogen peroxide (Vinay et al. 2013). Peroxidases are large family of enzymes found in all aerobic cells and function to decompose toxic hydrogen peroxide into water and oxygen gas (Petersen and Anderson 2005). The present investigation shows a significant decrease in the activity of CAT in the skin of L. rohita exposed to the azo dye. This corroborates with Ayadi et al. (2015) who also reported a decrease in the level of CAT in the liver of O. niloticus exposed to Red 195, a reactive azo dye. They however reported that, in the gills of the fish exposed to the Environ Sci Pollut Res Fig. 9 Comparison of specific activity of acid phosphatase (ACP) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Figs. 2 and 4 dye, the activity of CAT initially decreases and then after 7 days increases. An increase in CAT activity in different organs of O. niloticus following exposure to tea seed cake was reported by El-Murr et al. (2014). Paulino et al. (2012) assessed the impact of environmentally realistic atrazine concentrations on the gills of Prochilodus lineatus. They reported that acute (48 h) and sub-chronic (14 days) exposure to atrazine at 2 or 25 mg/L did not change the activity of CAT. However, sub-chronic (14 days) exposure to 10 mg/L increased the CAT activity. Sayeed et al. (2003) also demonstrated a decrease of 45 % in hepatic catalase activity in freshwater fish Channa punctatus exposed to the insecticide deltamethrin. According to Zhang et al. (2003), severe oxidative stress may suppress antioxidant defense enzyme activities, due to oxidative damage and a loss of compensatory mechanisms. Crestani et al. (2007) reported a decrease in CAT activity in Rhamdia quelen on exposure to sublethal concentration of an Fig. 10 Comparison of specific activity of lactate dehydrogenase (LDH) (mean ± SD; n = 15) in the skin of the fish Labeo rohita of control fish, fish exposed to Eriochrome black T, and fish for recovery after exposure, at different intervals. Rest of the abbreviations are the same as in Figs. 2 and 4 herbicide clomazone. Very recently, Melo et al. (2015) reported inhibition of CAT activity in the embryos of Danio rerio exposed to rotenone. Recovery of L. rohita exposed to azo dye for 4 days and then transferred to freshwater for 8 days suggests that changes in altered environment were to overcome the period of stress. Gradual restoration of the morphology of epithelial cells, density and area of mucous goblet cells and club cells, and general recovery of the activity of different enzymes similar to that of control in the skin of L. rohita indicate that the epidermis has the potential to recover from the toxic effect of the dye. Crestani et al. (2007) in R. quelen reported a recovery response in CAT activity after 96 h of transfer of fish into herbicide-free water. Nigam et al. (2014a, 2014b) reported a gradual recovery of cholinesterase and carboxylesterase activity, respectively, in the skin mucus of Cirrhinus mrigala, L. rohita, and Catla catla exposed to the sublethal Environ Sci Pollut Res concentrations (5 and 15 mg/L) of Nuvan for 4 days and then transferred to freshwater for recovery. In conclusion, exposure to sublethal concentration of the azo dye—Eriochrome black T—induced significant alterations in histopathology and enzyme activities in the epidermis of L. rohita. This study could thus be useful in providing useful guidelines for the release of these dyes, in effluents of various textile and carpet industries in the concentrations, which are not threatening to fish. This will prove to be a great asset in protecting fish population from harmful effects of the dye and may benefit the fish famers in increasing the production of fish being consumed as food by human beings. Further this study will undoubtedly provide more relevant insight of threat posed to aquatic wildlife, human health, and local ecosystems. Acknowledgements Mr. Ayan Srivastava was supported by Banaras Hindu University Fellowship (Scheme No. 5012) sponsored by the University Grants Commission, Government of India. Neeraj Verma was supported as Junior Research Fellow and then as Senior Research Fellow under the Centre of Advanced Study scheme (Award No. 14740) sponsored by the University Grant Commission, Government of India. Arup Mistri was supported as Junior Research Fellow and then as Senior Research Fellow under Rajiv Gandhi National fellowship (RGNF-201314-SC-WES-36992) scheme, University Grant Commission, Government of India. Brijesh Ranjan was supported as Project Fellow under the project P01/651 sponsored by the University Grant Commission, Government of India. We hereby declare that the experiments comply with the current laws of the country (India) in which they were performed. Author contributions Ayan Srivastava and Neeraj Verma designed the experiments and wrote the manuscript. Arup Mistri, Brijesh Ranjan, Ashwini Kumar Nigam, and Usha Kumari assisted in execution of experiments and data interpretation. Swati Mittal and Ajay Kumar Mittal were involved in critical reading, editing of manuscript, and data analysis. All authors discussed the results and approved the final version of the manuscript. Compliance with ethical standards Conflict of interest The authors declare that there are no financial or others conflicts of interest associated to this work. References Abdel-Hameid NAH (2009) A protective effect of calcium carbonate against arsenic toxicity of the Nile catfish, Clarias gariepinus. Turkish J Fisheries Aquatic Sci 9:191–200 Abdel-Moneim AM, Abou Shabana NM, Khadre SEM, Abdel Kader HH (2008) Physiological and histopathological effects in catfish Clarias lazera exposed to dyestuff and chemical wastewater. Int J Zool Res 4(4):189–202 Altinok I, Capkin E (2007) Histopathology of rainbow trout exposed to sublethal concentrations of methiocarb or endosulfan. Toxicol Pathol 35:405–410 American Public Health Association (APHA), American Water Works Association (AWWA) and Water Pollution Control Federation (WPCF) (1985) Standard methods for the examination of water and wastewater, 16th edn. American Public Health Association, Washington Ayadi I, Monteiro SM, Regaya I, Coimbra A, Fernandes F, Oliveira MM, Peixoto F, Mnif W (2015) Biochemical and histological changes in the liver and gill of Nile tilapia Oreochromis niloticus exposed to Red 195 Dye. RSC Adv 5:- Ayadi I, Souissi Y, Jlassi I, Peixoto F, Mnif W (2016) Chemical synonyms, molecular structure and toxicological risk assessment of synthetic textile dyes: a critical review. J Develop Drugs 5:151. doi:10. 4172/- Bancroft JD, Gamble M (2002) Theory and practice of histological techniques, 5th edn. Churchill Livingstone, London Barot J, Bahadur A (2015) Toxic impacts of C.I. Acid Orange 7 on behavioural, haematological and some biochemical parameters of Labeo rohita fingerlings. IJSRES 3(8):- Baruah BK, Das M (2002) Study of the effect of paper mill effluent on the morphology of fish Heteropneustes fossilis (Bloch). J Nature Conservator 14(2):367–369 Bonga SEW (1997) The stress response in fish. Physiol Rev 77(3):591–625 Chen QL, Luo Z, Zheng JL, Li XD, Liu CX, Zhao YH, Gong Y (2012) Protective effects of calcium on copper toxicity in Pelteobagrus fulvidraco: copper accumulation, enzymatic activities, histology. Ecotox Environ Saf 76:126–134 CrestaniM,MenezesC,GlusczakL,MironDS,SpanevelloR,SilveiraA, Goncalves FF, Zanella R, Loro VL (2007) Effect of clomazone herbicide on biochemical and histological aspects of silver catfish (Rhamdia quelen) and recovery pattern. Chemosphere 67:- de Lima D, Roque GM, de Almeida EA (2013) In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio). Mar Environ Res 91:45–51 El-Murr A, Ali HA, Eldeen NAMN (2014) Molecular, biochemical and histological effects of tea seed cake on different organs of Oreochromis niloticus. Glob Vet 13(5):711–719 Gill ST, Tewari H, Pande J (1990) Use of the fish enzyme system in monitoring water quality: effects of mercury on tissue enzymes. Com Biochem Physiol C 97:287–292 Hansen BA, Romma S, Garmo OA, Olsvik PA, Andersen RA (2006) Antioxidative stress proteins and their gene expression in brown trout (Salmo trutta) from three rivers with different heavy metal levels. Comp Biochem Physiol C 143:263–274 Kamalaveni K, Gopal V, Sampson U, Aruna D (2003) Recycling and utilization of metabolic wastes for energy production is an index of biochemical adaptation of fish under environmental pollution stress. Environ Monit Assess 86:255–264 Karanjkar DM, Muley DV, Manjare SA (2000) Acute toxicity of industrial effluents to Labeo rohita. Proc Acad Environ Biol 9(Sppl):30 Kaur S, Kaur A (2015) Variability in antioxidant/detoxification enzymes of Labeo rohita exposed to an azo dye, acid black (AB). Comp Biochem Physiol C 167:108–116 Korndat J, Braunbeck T (2001) Alterations of selected metabolic enzymes in fish following long-term exposure to contaminated streams. J Aquat Ecosys Stres Recov 8:299–318 Kumari U, Verma N, Nigam AK, Mittal S, Mittal AK (2016) Woundhealing potential of curcumin in the carp, Labeo rohita. Aquac Res 2016:1–17. doi:10.1111/are.13077 Laizure SC, Herring V, Hu Z, Witbrodt K, Parker RB (2013) The role of human carboxylesterases in drug metabolism: have we overlooked their importance. Pharmacotherapy 33:210–222 Lakshmi R, Kundu R, Thomas E, Mansuri AP (1991) Mercuric chloride induced inhibition of acid and alkaline phosphatase activity in the kidney of mudskipper, Boleophthalmus dentatus. Actahydrochim Hydrobiol 3:341–344 Lidesjoo E, Thulin J (1994) Histopathology of skin and gills of fish in pulp mill effluents. Dis Aquat Org 18:81–93 Environ Sci Pollut Res Manjunatha B, Tirado JO, Selvanayagam M (2015) Sub-lethal toxicity of potassium cyanide on Nile tilapia (Oreochromis niloticus): biochemical response. Int J Pharm Pharm Sci 7(3):379–382 Mathur N, Bhatnagar P, Sharma P (2012) Review of the mutagenicity of textile dye products. UJERT 2:1–18 Melo KM, Oliveira R, Grisolia CK, Domingues I, Pieczarka JC, Filho JS, Nagamachi CY (2015) Short-term exposure to low doses of rotenone induces developmental, biochemical, behavioral, and histological changes in fish. Environ Sci Pollut Res 22:- Mittal AK, Garg TK (1994) Effect of an anionic detergent—sodium dodecylsulphate exposure on club cells in the epidermis of Clarias batrachus. J Fish Biol 44:857–875 Mittal AK, Whitear M (1978) A note on cold anaesthesia of poikilotherms. J Fish Biol 13:519–520 Nigam AK, Kumari U, Mittal S, Mittal AK (2014a) Characterization of carboxylesterase in skin mucus of Cirrhinus mrigala and its assessment as biomarker of organophosphate exposure. Fish Physiol Biochem 40:635–644 Nigam AK, Srivastava N, Rai AK, Kumar U, Mittal AK, Mittal S (2014b) The first evidence of cholinesterase in skin mucus carps and its applicability as biomarker of organophosphate exposure. Environ Toxicol 29:788–795 Pacheco M, Santos MA (2002) Biotransformation, genotoxic, and histopathological effects of environmental contaminants in European eel (Anguilla anguilla L.). Ecotoxicol Environ Saf 53:331–347 Padh H (1992) Organelle isolation and marker enzyme assay. Tested studies for laboratory teaching. In: C.A. Goldman (Ed). Proceedings of the 13th Workshop/Conference of the Association for Biology Laboratory Education (ABLE) 13:129–146 Paulino MG, Souza NES, Fernandes MN (2012) Subchronic exposure to atrazine induces biochemical and histopathological changes in the gills of a Neotropical freshwater fish, Prochilodus lineatus. Ecotoxicol Environ Saf 80:6–13 Petersen CE, Anderson BA (2005) Investigations in the Biology 1151 Laboratory. Stipes Publishing L.L.C, Champaign, p 45 Puvanshwari N, Muthukrishnan J, Gunasekaran P (2006) Toxicity assessment and microbial degradation of azo dyes. IJEB 44:618–626 Rajeswari K, Reddy J (1989) Impact of thiodon on the metabolic pathway of the fish Tilapia mossambica. Environ Ecol 7:863–866 Rani AS, Sudharsan R, Reddy TN, Reddy PUM, Raju TN (2000) Alternations in the levels of dehydrogenases in a freshwater fish, Tilapia mossambica (Peters) exposed to arsenic toxicity. Indian J Environ Health 42:130–133 Sayeed I, Parvez S, Pandey S, Bin-Hafeez B, Rizwanul H, Raisuddin S (2003) Oxidative stress biomarkers of exposure to deltamethrin in freshwater fish, Channa punctatus Bloch. Ecotoxicol Environ Saf 56:295–301 Sharma S, Sharma S, Sharma KP (2006) Identification of a sensitive index during fish bioassay of an azo dye methyl red (untreated and treated). J Environ Biol 27(3):551–555 Shephard KL (1994) Functions for fish mucus. Rev Fish Biol Fisher 4: 401–429 Solé M, Sanchez-Hernandez JC (2015) An in vitro screening with emerging contaminants reveals inhibition of carboxylesterase activity in aquatic organisms. Aquat Toxicol 169:215–222 Sudova E, Machova J, Svobodova Z, Vesely T (2007) Negative effects of malachite green and possibilities of its replacement in the treatment of fish eggs and fish: a review. Vet Med 52(12):527–539 Thompson HM (1999) Esterases as markers of exposure to organophosphates and carbamates. Ecotoxicology 8:369–384 Valavanidis A, Vlahogianni T, Dassenakis M, Scoullos M (2006) Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol Environ Saf 64:178–189 Vinay TN, Chang-Su P, Heung-Yun K, Sung-Ju J (2013) Toxicity and dose determination of quillaja saponin, aluminum hydroxide and squalene in olive flounder (Paralichthyso livaceus). Vet Immunol Immunopatho 25 l-261 Wheelock CE, Nakagawa Y (2010) Carboxylesterases from function to the field: an overview of carboxylesterase biochemistry, structure– activity relationship. J Pestic Sci 35:215–217 Worthington Enzyme Manual. Worthington K, Worthington V (2011a) Worthington Biochemical Corporation. July, 2014 (http://www. worthington-biochem.com/CTL/default.html) Worthington Enzyme Manual. Worthington K, Worthington V (2011b) Worthington Biochemical Corporation. July, 2014 (http://www. worthington-biochem.com/HPO/default.html) Worthington Enzyme Manual. Worthington K, Worthington V (2011c) Worthington Biochemical Corporation. July, 2014 (http://www. worthington-biochem.com/LDH/default.html) Xing H, Wanga J, Li J, Fan Z, Wang M, Xu S (2010) Effects of atrazine and chlorpyrifos on acetylcholinesterase and carboxylesterase in brain and muscle of common carp. Environ Toxicol Pharmacol 30:26–30 Zhang J, Shen H, Wang X, Wu J, Xue Y (2003) Effects of chronic exposure of 2,4-dichlophenol on the antioxidant system in liver of freshwater fish Carassius auratus. Chemosphere 55:167–174