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May 26, 2011 - Physiological and Biochemical Responses of Nile Tilapia (Oreochromis niloticus). Exposed to Aqueous Extracts of Neem. (Azadirachta indica).

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Physiological and Biochemical Responses of Nile Tilapia (Oreochromis niloticus) Exposed to Aqueous Extracts of Neem (Azadirachta indica) Elijah Oyoo-Okoth

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, Charles C. Ngugi & Victoria Chepkirui-Boit

a

Department of Aquatic Ecology and Ecotoxicology, University of Amsterdam, Kruislaan, Amsterdam, The Netherlands b

Department of Fisheries and Aquatic Sciences, Moi University, Eldoret, Kenya Available online: 26 May 2011

To cite this article: Elijah Oyoo-Okoth, Charles C. Ngugi & Victoria Chepkirui-Boit (2011): Physiological and Biochemical Responses of Nile Tilapia (Oreochromis niloticus) Exposed to Aqueous Extracts of Neem (Azadirachta indica), Journal of Applied Aquaculture, 23:2, 177-186 To link to this article: http://dx.doi.org/10.1080/10454438.2011.581588

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Journal of Applied Aquaculture, 23:177–186, 2011 Copyright © Taylor & Francis Group, LLC ISSN: 1045-4438 print/1545-0805 online DOI: 10.1080/10454438.2011.581588

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Physiological and Biochemical Responses of Nile Tilapia (Oreochromis niloticus) Exposed to Aqueous Extracts of Neem (Azadirachta indica) ELIJAH OYOO-OKOTH1,2 , CHARLES C. NGUGI2 , and VICTORIA CHEPKIRUI-BOIT 2 1

Department of Aquatic Ecology and Ecotoxicology, University of Amsterdam, Kruislaan, Amsterdam, The Netherlands 2 Department of Fisheries and Aquatic Sciences, Moi University, Eldoret, Kenya

In this study, the physiological and biochemical response of Nile tilapia ( Oreochromis niloticus) after 96 and 24 h exposure to aqueous extracts of neem ( Azadirachta indica) in extract concentrations ranging from 0 to 32,000 mg/l was evaluated. After 96 h and 24 h exposure, the LC50 of neem extract was estimated at 3,200 and 6,800 mg/l, respectively. Plasma cortisol increased beyond pre-treatment levels at neem extract concentrations above 2,000 mg/l over 96 h and above 4,000 mg/l over 24 h. Blood glucose increased at neem extract concentrations above 1,000 and 5,000 mg/l at 24 and 96 h, respectively. Neem extract concentration had little effect on serum sodium and plasma chloride. Hematocrit was higher than the control at neem extract concentrations above 1,000 mg/l in the 96 h exposure and above 2,000 mg/l in the 24 h exposure. Plasma ammonia increased significantly at neem extract concentrations above 2,000 mg/l for both the 96 h and 24 h tests. Immediately after beginning treatment, cortisol levels increased significantly at neem extract concentrations above 2,000 mg/l in the 96 h test and 4,000 mg/l in the 24 h toxicity test. Exposure to neem extract interfered with the antioxidant defense We would like to acknowledge the financial support granted by Aquaculture and Fisheries Collaborative Research Support Program (AquaFish ACRSP). Additional funds were provided by Moi University Research Fund (MURF). Special thanks go to Professor Henk van-Maannen for the statistical analysis of the data. Address correspondence to Charles C. Ngugi, Department of Fisheries and Aquatic Sciences, Moi University, P.O Box 1125, Eldoret, Kenya. E-mail: [email protected] 177

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system of the fish by reducing liver catalase activity. Even though extracts of neem are less toxic at low concentration, concentrations exceeding 3,200 mg/l influence physiological and biochemical disturbances in fish. KEYWORDS Nile tilapia, neem extracts, lethal concentration

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INTRODUCTION Extensive research has been conducted to evaluate plants as possible pest control substances in aquaculture (Wan et al. 1996). One of the most promising is the neem tree (Azadirachta indica), whose antiviral, antibacterial, and antifungal properties are well documented (Isman et al. 1990; Harikrishnan, Rani, & Balasundaram 2003). The active ingredient in neem is azadirachtin (AZA), which has antiviral, antimicrobial, and antifungal properties (Harikshnan, Rani, & Balasundaram 2003; Krentzweiser et al. 2004). Neem extract can be useful in controlling Aeronomas, Pseudomonas, Eschiricchia coli, and molluscs. Although neem extract is of generally low toxicity to non-target organisms, tests conducted in fish have shown that it can cause mortality at higher doses (Heath 1995), particularly in warmer water. The aim of this study was to determine the physiological and biochemical response of Nile tilapia, Oreochromis niloticus, exposed to varying doses of neem extract for 96 h and 24 h in tropical warm water.

MATERIALS AND METHODS This study was conducted at Moi University, Eldoret, Kenya (0◦ 34 13.8 N; 35◦ 18 49.8 E), from June to October 2008. Moi University is situated in the highlands, west of the Rift Valley at an altitude of 2180 m asl with an average day and night temperature of 23.6◦ C and 14.2◦ C, respectively. Nile tilapia juveniles (mean weight = 31.4 ± 2.1 g) were obtained from Moi University growout ponds. A total of 500 test animals were acclimated for 6 days in glass aquaria each measuring 60 × 75 × 50 cm and containing 100 L of dechlorinated tap water in a recirculating water system aerated with diffusers. The stocking density of 3 juveniles/L was intended to reduce any chance of stress. The water flow through each aquarium was maintained at 0.4 L/min to ensure a renewal rate of at least once every 5 h. Water was filtered and treated with a germicidal UV lamp. Water temperature was maintained at 24.5 ± 0.5◦ C using thermostat-controlled heaters. Dissolved oxygen (DO) was monitored each morning and ranged from 6-8 mg/L. Fish were fed commercial pellets twice a day at 4% body weight. Twelve hours before the experiment, feeding of the fish was stopped.

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Leaves of the neem tree, Azadirachta indica, used in this experiment were obtained from the Mt. Kenya forest and finely chopped and weighed. Thirty-six grams of chopped neem leaves was dissolved in 1 L of water and allowed to sit for 24 h at room temperature (approx. 20◦ C). This mixture was then filtered, and an extract used immediately for experimental work in different dilutions making up concentration of 0, 500, 1,000 2,000, 4,000, 8,000, 16,000, and 36,000 mg of extract per liter. Short-term (24 h), long-term (96 h), and static toxicity tests were run to determine the median lethal concentration (LC50 ) of aqueous neem extract to O. niloticus following OECD (1992) guidelines. Fish were distributed in eight groups of 20 each in 100-L glass aquaria. Each of the seven concentrations of neem extract prepared above, plus one pure water control, was administered for 24 h or 96 h. The tests were conducted in triplicate and lethal concentration to death TC50 recorded every 6 hours. After 24 h and 96 h of exposure, ten fish from each treatment were anaesthetized using benzocaine (0.1 g/L) and blood sampled from the caudal vein using a 1 ml heparinized syringe. At least 5 ml of blood was obtained from the vein of all fish groups. Immediately after blood sampling, fish were sacrificed through cervical sectioning and their livers was removed. The livers were quickly frozen at −80◦ C, while gills and kidneys were fixed in Bouin’s solution. To determine hematocrit, blood was centrifuged for 5 minutes at 5,000 rpm in glass capillaries using a micro-hematocrit centrifuge (LC5, Luguimac SRL, Argentina). Blood glucose was measured according to King and Garner (1947). Heparinized blood was centrifuged at 3,000 rpm for 10 min and plasma frozen at -96◦ C in liquid nitrogen for cortisol analysis (Radioimunoassay with a Coat-to-Count Kit, Diagnostic Products Corp., Los Angeles, Calif.), chloride (Labtest Kits), and osmolarity in a freezing microsomometer (Osmont 030, Genotec, Germany). Blood without anticlotting agent was centrifuged as described earlier and the serum frozen at −96◦ C for analysis of serum sodium by flame photometry. Enzyme activity was measured spectrophotometrically at 25◦ C. Samples of frozen liver were weighed and homogenized in 0.1 M KPO4 buffer (1:10 w/v) and centrifuged (14,000 rpm) for 20 min at 40◦ C (Joan BR-4i, France). The supernatant was then separated for catalase (CAT) and glutathione-S-transferase (GTS) assay. CAT activity was determined by measuring the rate of decomposition of hydrogen peroxide at 240 nm as described by Beutler (1975). The GST activity was measured by enzymatic conjugation of reduced glutathione (GSH) with 1-chloro-2,4-dinitrobenzane at 340 nm (Keen, Habig, & Jakoby 1976). LC50 over 24 h and 96 h were estimated using a logistic regression model for dose-response treatment. Since mortality under dose-response treatments is a binary variable, it follows that the binomial distribution (mortality and non-mortality) data in the current study consisted of both dead fish and

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surviving fish. To determine the efficacy range of the chemical dosages, a logistic regression model, which can be used to identify non-linear responses to the range of concentrations, was fitted to the data. The logit model:   logit θ (x) = log



 θ (x) = β0 + β 1 x 1 + β 2 x 2 + . . . + β i x i 1 − θ (x)

is a general logistic model, which takes the form:  Downloaded by [UVA Univeriteitsbibliotheek] at 07:06 30 September 2011

log

 ρ = βo + β 1 C + β 2 C 2 + β 3 C 3 1−ρ

in dose response treatments (Agresti 1990), where rdenotes the probability of survival, b0 is an intercept, b1 is the coefficient of concentration C, and b2 is the coefficient of quadratic response in C, while b3 is the coefficient of cubic response in C. We fitted the model using GENSTAT (GenStat Release 4.24DE) statistical software program. Model fit was based on residual likelihood ratio chi-square statistic. For each parameter analyzed, differences among groups exposed to different neem extract concentrations were tested by one-way ANOVA and their means compared using Tukey’s HSD test. All analysed results were declared significant at P < 0.05.

RESULTS During the toxicity tests, dissolved oxygen, pH, and temperature remained at 5.5 ± 0.52 mg O2 /L, 7.2 ± 0.22, and 21.3 ± 0.32◦ C, respectively. However, conductivity increased with increasing concentration of neem extract as shown in Table 1. The 24 h LC50 neem extract for O. niloticus was estimated at 6,800 mg/l, with a confidence interval ranging from 5,400 to 8,200 mg/l, while the TABLE 1 Values of Dissolved Oxygen (DO), pH, Temperature, and Conductivity of the Water, at the Beginning and at the End of the Toxicity Tests with Different Neem Leaf Extract Concentrations (0, 500, 1,000 2,000, 4,000, 8,000, 16,000, and 36,000 mg/l) Neem concentration 0 (control) 500 1000 2000 4000 8000 16000 36000

DO (mg/L)

pH

Temperature (◦ C)

Conductivity

6.2–5.5 6.1–5.4 6.2–5.7 6.6–5.6 6.3–5.5 6.0–5.5 6.2–6.0 5.4–5.9

7.1–7.3 7.6–7.3 7.6–7.1 7.2–7.1 7.3–7.2 7.4–7.1 7.3–6.9 7.3–6.9

21.1–21.5 21.5–21.6 21.5–20.7 21.7–21.3 21.4–21.6 21.3–21.3 21.5–21.6 21.4–21.6

152–164 424.0–426.0 626.0–661.0 926–761.0 1065.1–854.2 1242.3–1129.1 1471.2–1322.1 1492.1–1326.2

Initial (t = 0) and final (t = 24 h) measurements are shown for each group.

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Response of Nile Tilapia Exposed to Neem Extract 100.0 90.0 24 h

80.0

96 h

70.0

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Survival

60.0 50.0 40.0 30.0 20.0 10.0 0.0 0

4000

8000

12000 16000 20000 Concentration (ppm)

24000

28000

32000

FIGURE 1 Survival of O. niloticus under chronic (96 h) and acute (24 h) exposure to extracts of A. indica.

96 h LC50 of neem extract for O. niloticus was estimated at 3,200 mg/l, with a confidence interval ranging from 2,700 to 4,200 mg/l (Figure 1). Blood physiological parameters for O. niloticus at different neem extract concentrations are shown in Figure 2. Blood glucose was significantly higher (P < 0.05) in fish exposed to neem extract concentrations above 1,000 mg/l and 500 mg/l in 96 h and 24 h toxicity tests, respectively, compared to the control. Plasma cortisol was significantly elevated (P < 0.05) in neem extract concentrations above 2,000 mg/l in the 96 h test and 4,000 mg/l in the 24 h toxicity test. Fish exposed to neem extract concentrations above 2,000 ml/l exhibited slightly elevated serum sodium levels, but these were not significantly different from the control (P > 0.05). Neem extract concentration above 4,000 mg/l raised plasma chloride above the control in the 96 h toxicity test but not in the 24 h toxicity test. Plasma ammonia increased significantly in neem extract concentration above 2000 mg/l for both the 96 h and 24 h tests. Immediately after beginning treatment, cortisol levels increased significantly at neem extract concentrations above 2,000 mg/l in the 96 h test and 4,000 mg/l in the 24 h toxicity test. Blood glucose level was significantly higher in fish exposed to neem extract concentrations above 8,000 mg/l and 5,000 mg/l in the 96 h and 24 h toxicity tests, respectively. Hematocrit was higher than the control at neem extract

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a) Plasma cortisol

250.0

Concentration (mg dl–1)

Concentration (ng ml–1)

300.0

200.0 150.0 100.0 50.0

c) Serum Na

60.0 40.0 20.0

140.0 Concentration (ng ml–1)

Concentration (M Eq l–1)

80.0

0.0

0.0 160.0 120.0

80.0 40.0 0.0

d) Plasma chloride

120.0

100.0

80.0 f) Hematocrit

35.0 8.0

e) Plasma ammonia Concentration (m mol–1)

Concentration (m mol–1)

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b) Blood glucose

6.0 4.0 2.0 0.0

24 h 96 h

28.0 21.0 14.0 7.0 0.0

0

4000

8000 12000 16000 20000 24000 28000 32000

0

4000 8000 12000 16000 20000 24000 28000 32000

Concentration (ppm)

FIGURE 2 Plasma concentrations of cortisol, blood glucose, serum sodium (Na+ ) and chloride (Cl− ), ammonia, and hematocrit of O. niloticus exposed for 24 h to different neem leaf extract concentrations. Concentration 0 corresponds to control group (CTR). Bars represent means, and vertical lines represent the SE (n = 10).

concentrations above 1,000 mg/l in the 96 h exposure and above 2,000 mg/l in the 24 h exposure. As shown in Figure 3, hepatic catalase (CAT) activity in fish livers exposed to neem extract was significantly lower (P < 0.05) than in the control at extract concentrations above 1,000 mg/l and 4,000 mg/l for 96 h and 24 h tests, respectively. The glutathione-S-transferase (GST) activity was, however, significantly higher (P < 0.05) in fish exposed to more than 2,000 and 8,000 mg/l of neem extract for 96 h and 24 h, respectively.

DISCUSSION The LC50 of neem leaf extract used for O. niloticus was estimated at 6,800 and 3,200 mg/l, respectively for 24 h and 96 h exposure. Though LC50 values need be interpreted with caution, given what is often a lack of

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Response of Nile Tilapia Exposed to Neem Extract Glutathione-S-transferase * * 24 h 96 h

180.0

Catalase

70.0

60.0

150.0

*

*

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Mmol.min–1mg protein–1

* 50.0

*

*

120.0 40.0 90.0 30.0 *

60.0

*

20.0

*

*

*

*

10.0

*

30.0 *

*

*

* 0.0

0.0 0

500

1000 2000 4000 8000 16000 32000

0

500 1000 2000 4000 8000 16000 32000

Concentration (ppm)

FIGURE 3 Catalase and glutathione-S-transferase liver activities of O. niloticus exposed for 24 h and 96 h to different neem leaf extract concentrations. Concentration 0 corresponds to control group (CTR). Bars represent means and vertical lines the SEM (n = 10). ∗ Indicates a significant difference from CTR (P ≤ 0.05).

detailed information on methods and water chemistry, inter alia, compared to other synthetic insecticides used in aquaculture, such as carbamates and organophosphates, neem-based products are certainly less toxic to fish. For example the 96 h LC50 of carbofuran and malathion for the silver salmon, Onchorhynchus kisutch, were 0.5 and 0.2 mg/L, respectively, compared to the neem products, Margosan-O and Pherotech, and neem extract of 38, 81, and 13 mg/L, respectively (Wan et al. 1996). Observed increased glucose levels could be attributed to a hyperglycemic response to satisfy the raised energy demands arising from the chemical stress. Hyperglycemia was also found in O. niloticus acutely exposed to lead for 6, 12, and 24 h as reported by Martinez et al. (2004). According to Heath (1995) high doses of some pesticides cause immediate hyperglycemia followed by hypoglycemia shortly before fish die. The absence of a hyperglycemic response above the 24 h LC50 thus represents exhaustion eventually leading to death (Wendelaar Bonga 1997). A variety of toxicants can interfere with fish osmoregulation, and the usual method used to detect the effects of compounds on osmotic and ionic regulation has been to measure the concentrations of individual ions and total osmolarity in fish plasma (Heath 1995; Barcarolli & Martinez 2004). In this study, neem extract concentration above 4,000 mg/l increased plasma sodium and chloride above the control groups in 96 h toxicity test, but no significant impact was discerned for the 24 h exposure in all the neem extract

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concentrations. The changes in the plasma chlorides are a possible indicator of osmoregulatory disturbances in fish at longer exposure times. However, it appears that the exposure a neem extract concentration of 4,000 mg/l for 24 h did not interfere with the osmoregulation processes of O. niloticus. There was increased accumulation of plasma ammonia with exposure to neem extract for 96 h. The high NH3 after treatment suggested increased physiological activity of the fish during osmoregulation. This could be associated with possible stress caused by increased physiological activity in fish such as increased oxygen consumption and increased heart rate (El-Shafey 1998). However, the exact cause of the increased plasma ammonia needs to be further investigated. In this study, neem extract in the water led to significant changes in the hematocrit, which is in agreement with Harikrishnan, Rani, and Balasundaram (2003), who observed increases in the hematocrit, hemoglobin (Hb) content, and number of erythrocytes in carp infected with A. hydrophila and treated for 20 or 30 days with neem extract (10,000 mg/l). Many toxic agents used in aquaculture have been reported to promote hematological changes of this nature (Carvalho & Fernandes 2006). Many environmental pollutants, including pesticides, are capable of inducing oxidative stress in fish (Dorval, Leblond, & Hontela 2003; Pandey et al. 2003; Sayeed et al. 2003; Monteiro et al. 2006). In the current study, fish exposed to neem showed significant reduction in hepatic CAT activity, which is likely to affect the capacity of liver cells to defend themselves and respond to contaminant-induced oxidative stress. A lower level of CAT activity might be due to an increased production of the superoxide radical (O2− ), as an excess of this anion is known to inhibit CAT activity (Bainy et al. 1996). Reduced hepatic CAT activity was also found in carp (Cyprinus carpio) exposed for four days to 300 mg/L of chitosan, a natural polymer extracted from the exoskeleton of crustaceans that has insecticidal properties (Dautremepuits et al. 2004). This results in the formation of highly reactive compounds such as free radicals or oxyradicals (O2 and H2 O2 ) that frequently react with cellular macromolecules, leading potentially to enzyme inactivation, lipid peroxidation, DNA damage, and even cell death (Van der Oost, Beyer, &. Vermeulen 2003). Catalase is the primary cellular enzymatic defense against H2 O2 , converting it into H2 O and O2 , and is critical for the process of scavenging free radicals (Dorval, Leblond, & Hontela 2003). Glutathione-S-transferases (GST) are a group of enzymes that catalyze the conjugation of reduced glutathione (GSH) with a variety of electrophilic metabolites, and are involved in the detoxification of both reactive intermediates and oxygen radicals (Van der Oost, Beyer, & Vermeulen 2003). It has been demonstrated that the activity of these enzymes may be enhanced in fish exposed to polycyclic and polychlorinated hydrocarbons (Zhang, Andersson, & Förlin 1990) and organophosphorus insecticides (Monteiro

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et al. 2006). Even low-level organic contamination can lead to increased hepatic GST activity in fish (Machala et al. 1997). Many plant extracts and other chemicals are being promoted for use as probiotics in aquaculture. Results from this study indicate the need to assess the functional and morphological responses in fish exposed to such plant biocides. The neem aqueous extract, although comparatively less toxic to O. niloticus than other pesticides, does promote functional and morphological changes in fish, thus requiring further work to develop criteria for its safe use in aquaculture.

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