Nerium indicum, a Botanical Pesticide Affects Ultimobranchial Gland of the Catfish Heteropneustes fossilis ManiRam Prasad, Abhishek Kumar, Sunil Kumar Srivastav, Ajai K. Srivastav Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur 273009, India
Received 23 December 2010; accepted 12 May 2011 ABSTRACT: Heteropneustes fossilis were subjected to 11.27 mg L21 (80% of 96 h LC50) and 2.81 mg L21 (20% of 96 h LC50) of Nerium indicum leaf extract for short-term and long-term, respectively. After sacrificing the fish, blood was collected on 24, 48, 72, and 96 h in short-term and after 7, 14, 21, and 28 days in long-term experiment and analyzed for plasma calcium levels. Also, ultimobranchial glands (UBG) were fixed on these intervals. Serum calcium levels of H. fossilis exhibited a decline after 48 h following exposure to Nerium indicum leaf extract. This decrease continued till the end of the experiment (96 h). Ultimobranchial cells exhibited a decrease in the cytoplasmic staining response after 72 h following the treatment. The nuclear volumes of these cells were slightly decreased. These changes were exaggerated after 96 h following the treatment. Chronically exposed fish exhibited a decline in serum calcium levels of H. fossilis on day 14. The level progressively declined till the end of the experiment. Up to day 14 following the treatment there was no change in the histological structure of UBG. A decrease in the nuclear volume of ultimobranchial cells was noticed on day 21. Moreover, the cytoplasm of these cells displayed weak staining response. The nuclear volume of these cells recorded a further decrease following 28-day treatment. Also there was noticed vacuolization and degeneration at certain places. To the best of our knowledge, the effects of any botanical pesticides on fish UBG have not been reported yet. # 2011 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2011.
Keywords: Nerium indicum; ultimobranchial gland; calcium; Heteropneustes fossilis
INTRODUCTION Organisms respond to external environment through nervous and endocrine systems (Guyton et al., 1991). Any change in the environment would initiate the primary stress response provoking modifications in hormonal levels of the organism’s body which in turn, induces secondary stress responses producing physiological and biochemical adjustment in the body of organism (Guyton et al., 1991). Because of the problem of the environmental pollution caused by continuous use of synthetic pesticides, there is renewed interest in the use of botanicals for crop protection. Botanical pesticides have been considered to cause fewer Correspondence to: A. K. Srivastav; e-mail: [email protected] Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20744
C
side effects as compared to synthetic pesticides and help keep the environment comparatively safe for future generations. Nerium is used as a rat poison, insecticide, and pesticide (Wickens, 1998; El-Shazly et al., 2000). Nerium indicum leaves contain cardiac glycosides—oleandrin and neriine (Goktas et al., 2007) and is used for the treatment of inflammation of gums, dysentery, bronchitis, asthma, and menorrhagia (Tiwari and Singh, 2005). Calcium plays a vital role in living organisms and has been implicated to control a wide variety of biological and physiological functions. Ultimobranchial gland has been implicated to control the blood calcium level by the secretion of the hypocalcemic hormone—calcitonin (CT) in nonmammalian vertebrates. There exists several reports on the impact of environmental toxicants on fish, e.g., histopathology of vital organs (Srivastava et al., 1989, 1990; Poleksic
2011 Wiley Periodicals, Inc.
1
2
PRASAD ET AL.
and Karan, 1999; Kumar et al., 2010), behavioral responses (Prasad et al., 2009; Srivastava et al., 2010), and hematological anomalies (Harikrishnan et al., 2009; Prasad et al., 2010; Kumar et al., 2011, but there exist no information regarding the impact of botanical pesticides (excluding synthetic pyrethroids) on ultimobranchial gland of fish. Hence, it was aimed in the present investigation to study the toxic effect of a botanical pesticide Nerium indicum leaf extract on the serum calcium and histological changes in the ultimobranchial gland of a freshwater catfish Heteropneustes fossilis.
MATERIALS AND METHODS Adult freshwater teleost Heteropneustes fossilis (both sexes body weight 32–44 g) were collected locally. Healthy fish showing no external signs of injury and disease were selected for experiments and were acclimatized to laboratory conditions (under natural photoperiod 11:20–12:18 and temperature 26.748C 6 2.118C; pH 7.26 6 0.09; hardness 167.97 6 5.69 mg L21 as CaCO3 dissolved oxygen 7.85 6 0.36 mg L21) for 15 days in dechlorinated tap water. In the present study, Nerium indicum leaf extract was used. The 96 h LC50 value of Nerium indicum leaf extract (14.0 mg L21 for the fish H. fossilis) was reported by Prasad et al. (2009). In short-term exposure the fish were subjected to 11.27 mg L21 of Nerium indicum leaf extract (80% of 96 h LC50 value). In long-term exposure the fish were subjected to 2.81 mg L21 (20% of 96 h LC50 value) of Nerium indicum leaf extract. Simultaneously, a control group was also run for comparison by using the tap water containing ethanol. Fish were kept in groups of 10 in 40-L media. Nerium indicum leaf extract was weighed and stock solution (50 mg mL21) was prepared in 100% ethanol. Six fish were sacrificed on each time intervals from control and experimental (Nerium indicum) groups after 24, 48, 72, and 96 h in short-term exposure and after 7, 14, 21, and 28 days in long-term experiment. Blood samples were collected by sectioning of the caudal peduncle of fish. The sera were separated by centrifugation at 3500 rpm and analyzed for calcium levels (calcium kit, RFCL Limited India, for details see Prasad et al., 2010). After the collection of blood samples, the area adjoining the heart along with the esophagus were removed and fixed in aqueous Bouin’s fluid. Tissues, thus fixed were routinely processed in graded series of alcohols, cleared in xylene, and then embedded in paraffin wax. Serial sections were cut at 6 lm and stained with hematoxylin-eosin (HE). Nuclear indices (maximal length and maximal width) of ultimobranchial cells that were determined (50 nuclei were measured per specimen; thus 300 nuclei were measured from six specimens) were taken with the aid of ocular micrometer and then the nuclear volume was calculated as volume 5 4/3 p ab2, where ‘‘a’’ is the major semiaxis and ‘‘b’’ is the minor semiaxis.
Environmental Toxicology DOI 10.1002/tox
Fig. 1. Serum calcium levels (mg/100 mL) of short-term Nerium indicum leaf extract-treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
All samples were estimated in duplicate. All data were presented as the mean 6 S.E. of six specimens and student’s t test was used for the determination of statistical significance. In all studies, the experimental group was compared to its specific time control group. Two-way analysis of variance (ANOVA) was used for multiple group comparisons.
RESULTS In short-term experiment the serum calcium levels of H. fossilis exhibited a decline after 48 h following exposure to Nerium indicum leaf extract. This decrease continued till the end of the experiment (96 h) (Fig. 1). Analysis of
Fig. 2. Serum calcium levels (mg/100 mL) of long-term Nerium indicum leaf extract-treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
Nerium indicum AFFECTS FISH ULTIMOBRANCHIAL GLAND
Fig. 3. Ultimobranchial gland of control H. fossilis exhibiting follicles and cell cords. HE 3 500.
3
Fig. 5. Nuclear volume (lm3) of ultimobranchial cells of short-term Nerium indicum leaf extract treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
variance indicated that the level of serum calcium were significantly different between groups (between intervals F 5 9.38, P \ 0.0001 between treatment F 5 55.78, P \ 0.0001). In long-term experiment, Nerium indicum leaf extract provoked a decline in the serum calcium levels of H. fossilis on day 14. The level progressively declined till the close of the experiment (28 days) (Fig. 2). Analysis of variance indicated that the level of serum calcium were significantly different between groups (between intervals F 5 16.46, P \ 0.0001 between treatment F 5 113.7, P \ 0.0001). The histological structure of UBG of control fish usually consisted of solid parenchyma which was composed of cell cords and small follicles (Fig. 3). All the cells were alike
and their cell boundaries were indistinct. When stained with HE, the cytoplasm of these cells was noticed slightly eosinophilic. Up to 48 h following Nerium indicum treatment no histological change was noticed in the ultimobranchial gland of fish. Ultimobranchial cells exhibited a decrease in the cytoplasmic staining response after 72 h following the treatment (Fig. 4). The nuclear volume of these cells was slightly decreased though not significantly (Fig. 5). These changes were exaggerated after 96 h following treatment with Nerium indicum leaf extract (Fig. 5). Analysis of variance indicated that the nuclear volume of ultimobranchial cells was not significantly different between groups
Fig. 4. Ultimobranchial gland of 72 h Nerium indicum leaf extract treated H. fossilis showing decreased staining response of the cytoplasm. HE 3 500.
Fig. 6. Nuclear volume (lm3) of ultimobranchial cells of long-term Nerium indicum leaf extract treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
Environmental Toxicology DOI 10.1002/tox
4
PRASAD ET AL.
DISCUSSION
Fig. 7. Ultimobranchial gland of 21 days Nerium indicum leaf extract treated H. fossilis showing decreased staining response of the cytoplasm. HE 3 500.
(between intervals F 5 0.80, P \ 0.499 between treatment F 5 1.36, P \ 0.250). Up to day 14 following Nerium indicum leaf extract treatment there was no change in the histological structure of UBG. A decrease in the nuclear volume of UB cells was noticed on day 21 following treatment (Fig. 6). Moreover, the cytoplasm of these cells displayed weak staining response (Fig. 7). The nuclear volume of these cells recorded a further decrease following 28-day treatment (Fig. 6). Also there was noticed vacuolization and degeneration at certain places (Fig. 8). Analysis of variance indicated that the nuclear volume of UB cells are significantly different between groups (between intervals F 5 2.41, P \ 0.081 between treatment F 5 11.83, P \ 0.001).
Fig. 8. Ultimobranchial gland of 28 days Nerium indicum leaf extract treated H. fossilis exhibiting degeneration (arrows) and vacuolization. HE 3 500.
Environmental Toxicology DOI 10.1002/tox
UBG exhibited inactivity in Nerium indicum leaf extracttreated fish which was expressed by decreased staining response and nuclear volume of UB cells. In UBG of treated fish vacuolization and degeneration were also noticed. Inactivity of UBG could be explained on the basis of prolonged hypocalcemia caused by Nerium indicum leaf extract exposure. Earlier to the present study no report existed in literature regarding the activity of UBG of fish after exposure to Nerium indicum leaf extract. The observed inactivity of UBG in treated fish derive support from similar observations reported by other investigators after exposure of the fish to different toxicants—deltamethrin (Srivastav et al., 2002), metacid (Mishra et al., 2004), cypermethrin (Mishra et al., 2005), and cadmium (Rai et al., 2009). The present study is also in conformity with the earlier reports in which hypoactivity/inactivity of the UBG was noticed in response to calcitonin-induced hypocalcemia in the fish—Anguilla anguilla (Peignoux-Deville et al., 1975), Gasterosteus aculeatus (Wendelaar Bonga, 1980), Clarias batrachus (Srivastav et al., 1989), Amphipnous cuchia (Tiwari, 1993) and Heteropneustes fossilis (Srivastav et al., 2009). Prolonged hypocalcemia rendered continuous disuse of the UBG which caused degeneration and vacuolization in the gland. It is concluded that Nerium indicum leaf extract can affect the activity of the ultimobranchial gland and may pose a threat to the physiology of the fish.
REFERENCES El-Shazly MM, El-Zayat EM, Hermersdorfer H. 2000. Insecticidal activity, mammalian cytotoxicity and mutagenecity of an ethanolic extract from Nerium oleander (Apocynaceae). Appl Biol 136:153–157. Goktas O, Mammadov R, Duru ME, Ozen E, Colak AM. 2007. Application of extracts from the poisonous plant, Nerium oleander L. as a wood preservative. Afr J Biotech 6:2000–2003. Guyton AC. 1991. Text Book of Medical Physiology, 8th ed. W.B. Saunders and Company, Philadelphia. Harikrishnan R, Balasundaram C, Kim M, Kim J, Heo M. 2009. Effective administration route of azadirachtin and its impact on haematological and biochemical parameters in goldfish (Carassius auratus) infected with Aeromonas hydrophila. Bull Vet Inst Pulawy 53:613–619. Kumar A, Prasad M, Srivastava K, Tripathi S, Srivastav Ajai K. 2010. Branchial histopathological study of catfish Heteropneustes fossilis following exposure to purified neem extract, azadirachtin. World J Zool 4:239–243. Kumar A, Prasad M, Mishra D, Srivastav SK, Srivastav Ajai K. 2011. Effects of Euphorbia tirucalli latex on blood calcium and phosphate of the freshwater air-breathing catfish Heteropneustes fossilis. Toxicol Environ Chem 93:585–592.
Nerium indicum AFFECTS FISH ULTIMOBRANCHIAL GLAND
Mishra D, Srivastav SK, Srivastav Ajai K. 2004. Plasma calcium and inorganic phosphate levels of a teleost Heteropneustes fossilis exposed to metacid-50. Malaysian Appl Biol 33:19–25. Mishra D, Srivastav SK, Srivastav Ajai K. 2005. Effects of the insecticide cypermehrin on plasma calcium and ultimobranchial gland of the teleost Heteropneustes fossilis. Ecotox Environ Safe 60:193–197. Peignoux-Deville J, Lopez E, Lallier F, Bagot EM, Milet C. 1975. Responses of ultimobranchial body in eels (Anguilla anguilla L.) maintained in seawater and experimentally matured to injections of synthetic salmon calcitonin. Cell Tissue Res 164:73–83. Poleksic V, Karan V. 1999. Effects of trifluralin on carp: Biochemical and histological evaluation. Ecotox Environ Safe 43:213–221. Prasad M, Kumar A, Mishra D, Srivastav SK, Suzuki N, Srivastav Ajai K. 2009. Acute toxicities of diethyl ether and ethanol extracted Nerium indicum leaf to the fish, Heteropneustes fossilis. Nij J Nat Prod Med 13:53–57. Prasad M, Kumar A, Mishra D, Srivastav SK, Srivastav Ajai K. 2010. Alterations in blood electrolytes of a freshwater catfish, Heteropneustes fossilis in response to treatment with a botanical pesticide, Nerium indicum leaf extract. Fish Physiol Biochem. DOI: 10.1007/s10695-010-9452-1. Rai R, Mishra D, Srivastav SK, Srivastav Ajai K. 2009. Ultimobranchial gland of a freshwater teleost, Heteropneustes fossilis in response to cadmium treatment. Environ Toxicol 24:589–593. Srivastav Ajai K, Srivastava SK, Mishra D, Srivastav S, Srivastav SK. 2002. Ultimobranchial gland of freshwater catfish, Heteropneustes fossilis in response to deltamethrin treatment. Bull Environ Contam Toxicol 68:584–591.
5
Srivastav Ajai K, Singh S, Mishra D, Srivastav SK. 2009. Ultimobranchial gland of freshwater catfish, Heteropneustes fossilis in response to calcitonin administration. Pesquisa Veterina´ria Brasileira 29:963–968. Srivastav SP, Swarup K, Singh S, Srivastav Ajai K. 1989. Effect of calcitonin administration on ultimobranchial gland, Stannius corpuscles and prolactin cells in the male catfish, Clarias batrachus. Arch Biol (Bruxelles) 100:385–392. Srivastava Anoop K, Mishra D, Srivastava S, Srivastav SK, Srivastav Ajai K. 2010. Acute toxicity and behavioral responses of Heteropneustes fossilis to an organophosphate insecticide, dimethoate. Int J Pharma Biosci 1:359–363. Srivastava SK, Tiwari PR, Srivastav Ajai K. 1989. Chlorpyrifos induced histological changes in the gill of freshwater catfish Heteropneustes fossilis. Bolm Fisiol Anim 13:23–28. Srivastava SK, Tiwari PR, Srivastav Ajai K. 1990. Effects of chlorpyrifos on the kidney of freshwater catfish Heteropneustes fossilis. Bull Environ Contam Toxicol 45:748–751. Tiwari PR. 1993. Endocrinal Regulation of Calcium in Teleost. Ph.D. Thesis, University of Gorakhpur, Gorakhpur, India. Tiwari S, Singh A. 2005. Possibility of using latex extracts of Nerium indicum plant for control of predatory fish Channa punctatus. Asian Fish Soc 18:161–173. Wendelaar Bonga SE. 1980. Effect of synthetic salmon calcitonin and low ambient calcium on plasma calcium, ultimobranchial cells, Stannius bodies and prolactin cells in the teleost Gasterosteus aculeatus. Gen Comp Endocrinol 40:99–108. Wickens GE. 1998. Ecophysiology of Economic Plants in Arid and Semi-Arid Lands. Springer-Verlag Berlin and Heidelberg GmbH & Co. KG p 243.
Environmental Toxicology DOI 10.1002/tox
Received 23 December 2010; accepted 12 May 2011 ABSTRACT: Heteropneustes fossilis were subjected to 11.27 mg L21 (80% of 96 h LC50) and 2.81 mg L21 (20% of 96 h LC50) of Nerium indicum leaf extract for short-term and long-term, respectively. After sacrificing the fish, blood was collected on 24, 48, 72, and 96 h in short-term and after 7, 14, 21, and 28 days in long-term experiment and analyzed for plasma calcium levels. Also, ultimobranchial glands (UBG) were fixed on these intervals. Serum calcium levels of H. fossilis exhibited a decline after 48 h following exposure to Nerium indicum leaf extract. This decrease continued till the end of the experiment (96 h). Ultimobranchial cells exhibited a decrease in the cytoplasmic staining response after 72 h following the treatment. The nuclear volumes of these cells were slightly decreased. These changes were exaggerated after 96 h following the treatment. Chronically exposed fish exhibited a decline in serum calcium levels of H. fossilis on day 14. The level progressively declined till the end of the experiment. Up to day 14 following the treatment there was no change in the histological structure of UBG. A decrease in the nuclear volume of ultimobranchial cells was noticed on day 21. Moreover, the cytoplasm of these cells displayed weak staining response. The nuclear volume of these cells recorded a further decrease following 28-day treatment. Also there was noticed vacuolization and degeneration at certain places. To the best of our knowledge, the effects of any botanical pesticides on fish UBG have not been reported yet. # 2011 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2011.
Keywords: Nerium indicum; ultimobranchial gland; calcium; Heteropneustes fossilis
INTRODUCTION Organisms respond to external environment through nervous and endocrine systems (Guyton et al., 1991). Any change in the environment would initiate the primary stress response provoking modifications in hormonal levels of the organism’s body which in turn, induces secondary stress responses producing physiological and biochemical adjustment in the body of organism (Guyton et al., 1991). Because of the problem of the environmental pollution caused by continuous use of synthetic pesticides, there is renewed interest in the use of botanicals for crop protection. Botanical pesticides have been considered to cause fewer Correspondence to: A. K. Srivastav; e-mail: [email protected] Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20744
C
side effects as compared to synthetic pesticides and help keep the environment comparatively safe for future generations. Nerium is used as a rat poison, insecticide, and pesticide (Wickens, 1998; El-Shazly et al., 2000). Nerium indicum leaves contain cardiac glycosides—oleandrin and neriine (Goktas et al., 2007) and is used for the treatment of inflammation of gums, dysentery, bronchitis, asthma, and menorrhagia (Tiwari and Singh, 2005). Calcium plays a vital role in living organisms and has been implicated to control a wide variety of biological and physiological functions. Ultimobranchial gland has been implicated to control the blood calcium level by the secretion of the hypocalcemic hormone—calcitonin (CT) in nonmammalian vertebrates. There exists several reports on the impact of environmental toxicants on fish, e.g., histopathology of vital organs (Srivastava et al., 1989, 1990; Poleksic
2011 Wiley Periodicals, Inc.
1
2
PRASAD ET AL.
and Karan, 1999; Kumar et al., 2010), behavioral responses (Prasad et al., 2009; Srivastava et al., 2010), and hematological anomalies (Harikrishnan et al., 2009; Prasad et al., 2010; Kumar et al., 2011, but there exist no information regarding the impact of botanical pesticides (excluding synthetic pyrethroids) on ultimobranchial gland of fish. Hence, it was aimed in the present investigation to study the toxic effect of a botanical pesticide Nerium indicum leaf extract on the serum calcium and histological changes in the ultimobranchial gland of a freshwater catfish Heteropneustes fossilis.
MATERIALS AND METHODS Adult freshwater teleost Heteropneustes fossilis (both sexes body weight 32–44 g) were collected locally. Healthy fish showing no external signs of injury and disease were selected for experiments and were acclimatized to laboratory conditions (under natural photoperiod 11:20–12:18 and temperature 26.748C 6 2.118C; pH 7.26 6 0.09; hardness 167.97 6 5.69 mg L21 as CaCO3 dissolved oxygen 7.85 6 0.36 mg L21) for 15 days in dechlorinated tap water. In the present study, Nerium indicum leaf extract was used. The 96 h LC50 value of Nerium indicum leaf extract (14.0 mg L21 for the fish H. fossilis) was reported by Prasad et al. (2009). In short-term exposure the fish were subjected to 11.27 mg L21 of Nerium indicum leaf extract (80% of 96 h LC50 value). In long-term exposure the fish were subjected to 2.81 mg L21 (20% of 96 h LC50 value) of Nerium indicum leaf extract. Simultaneously, a control group was also run for comparison by using the tap water containing ethanol. Fish were kept in groups of 10 in 40-L media. Nerium indicum leaf extract was weighed and stock solution (50 mg mL21) was prepared in 100% ethanol. Six fish were sacrificed on each time intervals from control and experimental (Nerium indicum) groups after 24, 48, 72, and 96 h in short-term exposure and after 7, 14, 21, and 28 days in long-term experiment. Blood samples were collected by sectioning of the caudal peduncle of fish. The sera were separated by centrifugation at 3500 rpm and analyzed for calcium levels (calcium kit, RFCL Limited India, for details see Prasad et al., 2010). After the collection of blood samples, the area adjoining the heart along with the esophagus were removed and fixed in aqueous Bouin’s fluid. Tissues, thus fixed were routinely processed in graded series of alcohols, cleared in xylene, and then embedded in paraffin wax. Serial sections were cut at 6 lm and stained with hematoxylin-eosin (HE). Nuclear indices (maximal length and maximal width) of ultimobranchial cells that were determined (50 nuclei were measured per specimen; thus 300 nuclei were measured from six specimens) were taken with the aid of ocular micrometer and then the nuclear volume was calculated as volume 5 4/3 p ab2, where ‘‘a’’ is the major semiaxis and ‘‘b’’ is the minor semiaxis.
Environmental Toxicology DOI 10.1002/tox
Fig. 1. Serum calcium levels (mg/100 mL) of short-term Nerium indicum leaf extract-treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
All samples were estimated in duplicate. All data were presented as the mean 6 S.E. of six specimens and student’s t test was used for the determination of statistical significance. In all studies, the experimental group was compared to its specific time control group. Two-way analysis of variance (ANOVA) was used for multiple group comparisons.
RESULTS In short-term experiment the serum calcium levels of H. fossilis exhibited a decline after 48 h following exposure to Nerium indicum leaf extract. This decrease continued till the end of the experiment (96 h) (Fig. 1). Analysis of
Fig. 2. Serum calcium levels (mg/100 mL) of long-term Nerium indicum leaf extract-treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
Nerium indicum AFFECTS FISH ULTIMOBRANCHIAL GLAND
Fig. 3. Ultimobranchial gland of control H. fossilis exhibiting follicles and cell cords. HE 3 500.
3
Fig. 5. Nuclear volume (lm3) of ultimobranchial cells of short-term Nerium indicum leaf extract treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
variance indicated that the level of serum calcium were significantly different between groups (between intervals F 5 9.38, P \ 0.0001 between treatment F 5 55.78, P \ 0.0001). In long-term experiment, Nerium indicum leaf extract provoked a decline in the serum calcium levels of H. fossilis on day 14. The level progressively declined till the close of the experiment (28 days) (Fig. 2). Analysis of variance indicated that the level of serum calcium were significantly different between groups (between intervals F 5 16.46, P \ 0.0001 between treatment F 5 113.7, P \ 0.0001). The histological structure of UBG of control fish usually consisted of solid parenchyma which was composed of cell cords and small follicles (Fig. 3). All the cells were alike
and their cell boundaries were indistinct. When stained with HE, the cytoplasm of these cells was noticed slightly eosinophilic. Up to 48 h following Nerium indicum treatment no histological change was noticed in the ultimobranchial gland of fish. Ultimobranchial cells exhibited a decrease in the cytoplasmic staining response after 72 h following the treatment (Fig. 4). The nuclear volume of these cells was slightly decreased though not significantly (Fig. 5). These changes were exaggerated after 96 h following treatment with Nerium indicum leaf extract (Fig. 5). Analysis of variance indicated that the nuclear volume of ultimobranchial cells was not significantly different between groups
Fig. 4. Ultimobranchial gland of 72 h Nerium indicum leaf extract treated H. fossilis showing decreased staining response of the cytoplasm. HE 3 500.
Fig. 6. Nuclear volume (lm3) of ultimobranchial cells of long-term Nerium indicum leaf extract treated H. fossilis. Values are mean 6 SE of six specimens. Asterisk indicates significant differences (P \ 0.05) from control group.
Environmental Toxicology DOI 10.1002/tox
4
PRASAD ET AL.
DISCUSSION
Fig. 7. Ultimobranchial gland of 21 days Nerium indicum leaf extract treated H. fossilis showing decreased staining response of the cytoplasm. HE 3 500.
(between intervals F 5 0.80, P \ 0.499 between treatment F 5 1.36, P \ 0.250). Up to day 14 following Nerium indicum leaf extract treatment there was no change in the histological structure of UBG. A decrease in the nuclear volume of UB cells was noticed on day 21 following treatment (Fig. 6). Moreover, the cytoplasm of these cells displayed weak staining response (Fig. 7). The nuclear volume of these cells recorded a further decrease following 28-day treatment (Fig. 6). Also there was noticed vacuolization and degeneration at certain places (Fig. 8). Analysis of variance indicated that the nuclear volume of UB cells are significantly different between groups (between intervals F 5 2.41, P \ 0.081 between treatment F 5 11.83, P \ 0.001).
Fig. 8. Ultimobranchial gland of 28 days Nerium indicum leaf extract treated H. fossilis exhibiting degeneration (arrows) and vacuolization. HE 3 500.
Environmental Toxicology DOI 10.1002/tox
UBG exhibited inactivity in Nerium indicum leaf extracttreated fish which was expressed by decreased staining response and nuclear volume of UB cells. In UBG of treated fish vacuolization and degeneration were also noticed. Inactivity of UBG could be explained on the basis of prolonged hypocalcemia caused by Nerium indicum leaf extract exposure. Earlier to the present study no report existed in literature regarding the activity of UBG of fish after exposure to Nerium indicum leaf extract. The observed inactivity of UBG in treated fish derive support from similar observations reported by other investigators after exposure of the fish to different toxicants—deltamethrin (Srivastav et al., 2002), metacid (Mishra et al., 2004), cypermethrin (Mishra et al., 2005), and cadmium (Rai et al., 2009). The present study is also in conformity with the earlier reports in which hypoactivity/inactivity of the UBG was noticed in response to calcitonin-induced hypocalcemia in the fish—Anguilla anguilla (Peignoux-Deville et al., 1975), Gasterosteus aculeatus (Wendelaar Bonga, 1980), Clarias batrachus (Srivastav et al., 1989), Amphipnous cuchia (Tiwari, 1993) and Heteropneustes fossilis (Srivastav et al., 2009). Prolonged hypocalcemia rendered continuous disuse of the UBG which caused degeneration and vacuolization in the gland. It is concluded that Nerium indicum leaf extract can affect the activity of the ultimobranchial gland and may pose a threat to the physiology of the fish.
REFERENCES El-Shazly MM, El-Zayat EM, Hermersdorfer H. 2000. Insecticidal activity, mammalian cytotoxicity and mutagenecity of an ethanolic extract from Nerium oleander (Apocynaceae). Appl Biol 136:153–157. Goktas O, Mammadov R, Duru ME, Ozen E, Colak AM. 2007. Application of extracts from the poisonous plant, Nerium oleander L. as a wood preservative. Afr J Biotech 6:2000–2003. Guyton AC. 1991. Text Book of Medical Physiology, 8th ed. W.B. Saunders and Company, Philadelphia. Harikrishnan R, Balasundaram C, Kim M, Kim J, Heo M. 2009. Effective administration route of azadirachtin and its impact on haematological and biochemical parameters in goldfish (Carassius auratus) infected with Aeromonas hydrophila. Bull Vet Inst Pulawy 53:613–619. Kumar A, Prasad M, Srivastava K, Tripathi S, Srivastav Ajai K. 2010. Branchial histopathological study of catfish Heteropneustes fossilis following exposure to purified neem extract, azadirachtin. World J Zool 4:239–243. Kumar A, Prasad M, Mishra D, Srivastav SK, Srivastav Ajai K. 2011. Effects of Euphorbia tirucalli latex on blood calcium and phosphate of the freshwater air-breathing catfish Heteropneustes fossilis. Toxicol Environ Chem 93:585–592.
Nerium indicum AFFECTS FISH ULTIMOBRANCHIAL GLAND
Mishra D, Srivastav SK, Srivastav Ajai K. 2004. Plasma calcium and inorganic phosphate levels of a teleost Heteropneustes fossilis exposed to metacid-50. Malaysian Appl Biol 33:19–25. Mishra D, Srivastav SK, Srivastav Ajai K. 2005. Effects of the insecticide cypermehrin on plasma calcium and ultimobranchial gland of the teleost Heteropneustes fossilis. Ecotox Environ Safe 60:193–197. Peignoux-Deville J, Lopez E, Lallier F, Bagot EM, Milet C. 1975. Responses of ultimobranchial body in eels (Anguilla anguilla L.) maintained in seawater and experimentally matured to injections of synthetic salmon calcitonin. Cell Tissue Res 164:73–83. Poleksic V, Karan V. 1999. Effects of trifluralin on carp: Biochemical and histological evaluation. Ecotox Environ Safe 43:213–221. Prasad M, Kumar A, Mishra D, Srivastav SK, Suzuki N, Srivastav Ajai K. 2009. Acute toxicities of diethyl ether and ethanol extracted Nerium indicum leaf to the fish, Heteropneustes fossilis. Nij J Nat Prod Med 13:53–57. Prasad M, Kumar A, Mishra D, Srivastav SK, Srivastav Ajai K. 2010. Alterations in blood electrolytes of a freshwater catfish, Heteropneustes fossilis in response to treatment with a botanical pesticide, Nerium indicum leaf extract. Fish Physiol Biochem. DOI: 10.1007/s10695-010-9452-1. Rai R, Mishra D, Srivastav SK, Srivastav Ajai K. 2009. Ultimobranchial gland of a freshwater teleost, Heteropneustes fossilis in response to cadmium treatment. Environ Toxicol 24:589–593. Srivastav Ajai K, Srivastava SK, Mishra D, Srivastav S, Srivastav SK. 2002. Ultimobranchial gland of freshwater catfish, Heteropneustes fossilis in response to deltamethrin treatment. Bull Environ Contam Toxicol 68:584–591.
5
Srivastav Ajai K, Singh S, Mishra D, Srivastav SK. 2009. Ultimobranchial gland of freshwater catfish, Heteropneustes fossilis in response to calcitonin administration. Pesquisa Veterina´ria Brasileira 29:963–968. Srivastav SP, Swarup K, Singh S, Srivastav Ajai K. 1989. Effect of calcitonin administration on ultimobranchial gland, Stannius corpuscles and prolactin cells in the male catfish, Clarias batrachus. Arch Biol (Bruxelles) 100:385–392. Srivastava Anoop K, Mishra D, Srivastava S, Srivastav SK, Srivastav Ajai K. 2010. Acute toxicity and behavioral responses of Heteropneustes fossilis to an organophosphate insecticide, dimethoate. Int J Pharma Biosci 1:359–363. Srivastava SK, Tiwari PR, Srivastav Ajai K. 1989. Chlorpyrifos induced histological changes in the gill of freshwater catfish Heteropneustes fossilis. Bolm Fisiol Anim 13:23–28. Srivastava SK, Tiwari PR, Srivastav Ajai K. 1990. Effects of chlorpyrifos on the kidney of freshwater catfish Heteropneustes fossilis. Bull Environ Contam Toxicol 45:748–751. Tiwari PR. 1993. Endocrinal Regulation of Calcium in Teleost. Ph.D. Thesis, University of Gorakhpur, Gorakhpur, India. Tiwari S, Singh A. 2005. Possibility of using latex extracts of Nerium indicum plant for control of predatory fish Channa punctatus. Asian Fish Soc 18:161–173. Wendelaar Bonga SE. 1980. Effect of synthetic salmon calcitonin and low ambient calcium on plasma calcium, ultimobranchial cells, Stannius bodies and prolactin cells in the teleost Gasterosteus aculeatus. Gen Comp Endocrinol 40:99–108. Wickens GE. 1998. Ecophysiology of Economic Plants in Arid and Semi-Arid Lands. Springer-Verlag Berlin and Heidelberg GmbH & Co. KG p 243.
Environmental Toxicology DOI 10.1002/tox
