Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark

Egyptian Journal of Aquatic Research (2015) xxx, xxx–xxx

H O S T E D BY

National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com

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Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark Samson Eneojo Abalaka a,*, Muhammad Yakasai Fatihu a, Najume Doguwar Giginya Ibrahim a, Suleiman Folorunsho Ambali a b

b

Department of Veterinary Pathology, Ahmadu Bello University, Zaria, Nigeria Department of Veterinary Pharmacology and Toxicology, Ahmadu Bello University, Zaria, Nigeria

Received 12 July 2014; revised 28 January 2015; accepted 28 January 2015

KEYWORDS Adenium obesum; Clarias gariepinus; Toxicity; Histopathology; Gills; Skin

Abstract Histopathological effects of ethanol extract of Adenium obesum stem bark was investigated in the gills and skin of African sharptooth catfish, Clarias gariepinus over a 96-h exposure period as an endpoint of toxicity. There was a significant (p < 0.05) concentration-dependent mortality in some of the exposed fish. The median lethal concentration of the extract was 7.15 mg L 1. The extract caused some histopathological lesions in the gills and skin of the exposed fish. However, the severity but not the type of the lesions observed in the gills and skin of the exposed fish was concentration-dependent. Although the degree of tissue change (DTC) grading indicated moderate damage in the gills of the exposed fish, there were no significant (p > 0.05) differences between gills DTC of the exposed and unexposed fish. However, lesions in the skin did not affect the normal functioning of the tissue but significant (p < 0.05) differences were recorded in the DTC between the skin of the exposed and the unexposed fish. The extract was toxic to the exposed fish and therefore, A. obesum can be used as a potent organic piscicide for effective fish pond management. ª 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Aquaculture has become one of the fastest growing food production unit sub-sectors in the world (Francis et al., 2001) due to declining worldwide landings of captured fisheries * Corresponding author. Tel.: +234 8037863462. E-mail address: [email protected] (S.E. Abalaka). Peer review under responsibility of National Institute of Oceanography and Fisheries.

(Faturoti, 1999) to meet the ever increasing fish demand of the growing human population (Merino et al., 2012). However, aquaculture development is slow in Africa (Mallya, 2007) due to numerous problems (Pedini, 1997; Daramola et al., 2007) notable amongst which, is the devastating issue of unwanted fish in fish ponds (Jhingran, 1975). Unwanted aquatic animals like leeches, crayfish, snails, tadpoles, frogs and fish are known to drastically reduce aquaculture yields by competing with stocked fish for space and feed in addition to some, being predatory in nature (Jhingran, 1983). In the past, fish farmers resorted to

http://dx.doi.org/10.1016/j.ejar.2015.01.005 1687-4285 ª 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005

2 the use of synthetic chemicals to eradicate these unwanted aquatic animals from fish ponds before the stocking of the desired fish (Lennon, 1970). However, massive environmental pollution due to their non-biodegradability and therefore, residual effects in the environment has become a major drawback to this practice worldwide (Cagauan and Arce, 1992). This has shifted global focus to the use of plant piscicides instead (Fafioye, 2005) that are reportedly more environmentally friendly, as they are bio-degradable (Kela et al., 1989) with little or no residual environmental effects (Marston and Hostettmann, 1985). Adenium obesum is a globally known organic piscicide that is used to kill fish (Neuwinger, 2000; Jones, 2007; Oyen, 2008) in addition to being used worldwide for ornamental purposes (Hastuti et al., 2009). However, the plant is indigenous to Africa where it is found in places ranging from the Sahel region towards central Africa and into Natal and Swaziland as well as to south western Arabian Peninsula (Plaizier, 1980; Arbonnier, 2004). Similarly, Clarias gariepinus is indigenous to Africa (de Graaf and Janssen, 1996) where they inhabit tropical swamps, lakes, rivers and floodplains some of which are subjected to seasonal drying (Olufemi et al., 1991; Rahman et al., 1992). Although the fish is reportedly absent from the Maghreb, Upper and Lower Guinea as well as from the Cape provinces (FAO, 2011), it is also found in the Asian Minor of Israel, Syria, Southern Turkey and India (de Graaf and Janssen, 1996; Yildirim and Turan, 2010) and worldwide where it has already been introduced (Rahman et al., 1992). Besides being omnivorous in nature, C. gariepinus is known to cannibalize fish half of its size or 10% of its own body weight (de Graaf and Janssen, 1996). This is even as the fish is also known to cannibalize its own species under certain pond conditions until the fish reaches a size of between 50 and 100 g in a properly fed fish pond (Rahman et al., 1992). This is more worrisome as the fish is hardy in nature (Olaifa et al., 2003), due to the accessory breathing organs the fish possesses (Safriel and Bruton, 1984), and can even survive in burrows beneath pond beds between seasons (Van der Waal, 1998). Therefore, any piscicidal plants that can kill C. gariepinus will definitely cause similar effects on even less hardy co-inhabitant of the same aquatic environment thereby making the plant an effective tool for prompt aquaculture pond management with minimal environmental pollution, especially in some developing countries where these plants are readily available. The use of various piscicidal plants to stun and/or kill fish is well documented (Mun´oz et al., 2000; Neuwinger, 2004; Jothivel and Paul, 2008) but there is a dearth of information on the use of A. obesum, especially as it relates to its histopathological effects in exposed fish hence the need for the study. The present study evaluates histopathological changes in the gills and skin of African sharptooth catfish, C. gariepinus exposed to ethanol extract of A. obesum stem bark. This is because histopathological evaluation remains an important part of the general assessment of the adverse or toxic effects of xenobiotics in organism (Reddy and Rawat, 2013). Materials and methods Plant extraction A. obesum plants were collected from the open fields of Rurum town in Rano Local Government Area of Kano State, Nigeria

S.E. Abalaka et al. between January and April, 2011. These were authenticated by Mallam Musa Mohammed at the Herbarium of the Department of Biological Sciences of Ahmadu Bello University (A.B.U), Zaria, Nigeria where a specimen (Voucher No. 1386) has already been deposited. The bark of the stem was removed, sun-dried and pounded into powder prior to macerating 3.95 kg of it with 21 L of ethanol (96.0% vol. Sigma–Aldrich Inc., St. Louis, MO 63178, USA) in separation funnels whose mouths were plugged with cotton wools over a 72-h period. The extract was concentrated to dryness in an evaporation dish at room temperature until constant weights were obtained (Abu-Dahab and Afifi, 2007). Acute fish toxicity bioassay Adult C. gariepinus were authenticated at the Fishery Section, Department of Biological Sciences, A.B.U., Zaria, Nigeria after purchasing them from a commercial catfish farm (Fannasson Investments Limited, Kano, Nigeria). Fish were acclimatized for 21 days under natural day and night photoperiods (12/12-h) prior to the commencement of the toxicity bioassay in a rectangular plastic pond (1.54 · 1.05 · 0.75 m). Pond water was changed completely once in every 3 days. Fish were fed ad libitum twice daily with 6 mm Coppens fish feed for aquaculture (Coppens International bv., 5700 AM Helmond, Holland). Fish were observed for disease conditions and mortality as stipulated in the OECD guideline No. 203 (1992). Feeding was stopped 24-h prior to and during the 96-h exposure period so as to prevent interference with stomach contents and wastes in the fish culture water (Ayotunde and Offem, 2008). C. gariepinus adults of 265.50 ± 4.03 g mean weight and 32.85 ± 0.16 cm mean total length were exposed to 6.25 mg L 1, 7.50 mg L 1, 8.20 mg L 1, 8.80 mg L 1and 9.30 mg L 1 of the extract and a control based on the OECD guideline No. 203 (1992) in triplicate over a 96-h period. This was after performing a preliminary concentration range finding test to determine the used extract concentrations along with the control as described by Fafioye (2001). Mortality was used as an end point of toxicity. Probit analysis method (Finney, 1971) was used to determine the median lethal concentration (LC50) of the extract in the exposed fish. The temperature and pH of fish culture water were ascertained using a Hanna ‘‘Combo portable hand instrument (Hi 98129, Hanna Instrument, Mauritius) while their dissolved oxygen contents were similarly established using the modified Winkler-Azide method (Lind, 1979; APHA, 1985). Histopathological analyses The gills and skin of experimental fish were collected at the end of the 96-h exposure period. However, survivors were euthanized with 40% ethyl alcohol (Fafioye et al., 2005). The harvested tissues were fixed in 10% neutral buffered formalin before paraffin embedding, sectioning at 5 lm and haematoxylin and eosin stained (Roberts, 1978; Bancroft and Cook, 1994) for histopathological examinations under light microscopy. The degree of tissue change (DTC) method as described by Poleksic and Mitrovic-Tutundzic (1994) was modified and used to ascertain the nature and severity of the lesions in each

Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005

Histopathological evaluation of ethanol extract of Adenium obesum stem bark gill and skin of the exposed fish. Gills and skin lesions were progressively classified in three stages of tissue damage. The sum (R) of the number of lesion types within each of the stages (I, II and III) was multiplied by the stage coefficient (1, 10 and 100) to give numerical values of the DTC using the formula: DTC = (1 · R I) + (10 · R II) + (100 · R III). Lesions, which did not alter the normal functioning of the gill and skin were classified as Stage I lesions while those which were more severe and impaired the normal functioning of the gills and skin were classified as Stage II lesions. Lesions, which were very severe and induced irreparable gills and skin damage, were classified as Stage III lesions. The results were graded and interpreted as follows: 0–10 (normal gills and skin); 11–20 (slightly damaged gills and skin); 21–50 (moderately damaged gills and skin); 50–100 (severely damaged gills and skin); >100 (irreversibly damaged gills and skin). Statistical analyses Data were expressed as mean (±SEM) and subjected to student-t test, ANOVA and Tukey’s multiple comparison test for statistical significance (p < 0.05) using GraphPad software programme (GraphPad Prism, version 4.0, San Diego, California, USA, www.graphpad.com). The same software programme was used to determine differences within and between the DTC of the gills and skin of the exposed and the unexposed groups for statistical significance (p < 0.05). Results and discussion Physicochemical parameters The temperature, pH and dissolved oxygen contents of fish culture water are as shown in Table 1. The physicochemical parameters of fish culture water were all within acceptable

Table 1 Physicochemical parameters of fish culture water of Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Physicochemical parameters

Value

Temperature (C) pH Dissolved oxygen (mg L 1)

24.65 ± 0.18 7.22 ± 0.04 4.56 ± 0.63

Table 2

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limit for the growth and survival of C. gariepinus as earlier reported by Viveen et al. (1985) and Bruton (1988). Acute fish toxicity bioassay Mortality was observed in some of the exposed fish and this increased significantly (p < 0.05) with increasing extract concentration as shown in Table 2. The median lethal concentration of ethanol extract of A. obesum stem bark in the exposed C. gariepinus was therefore, established to be 7.15 mg L 1. This showed that the ethanol extract of A. obesum stem bark was toxic to the exposed C. gariepinus. The concentration-dependent nature of the observed mortality was similar to what was recorded in ticks exposed to the aqueous extract of A. obesum stem bark (Mgbojikwe, 2000). This is in addition to what was reported in land snail, Monacha cantiana exposed to benzene and methanol extracts of Adenium arabicum (Al-Sarar et al., 2012). The established LC50 value of 7.15 mg L 1 showed that the ethanol extract of A. obesum was highly toxic to the exposed C. gariepinus over the 96-h period because the lower the LC50 of a substance the more acutely toxic is that substance (Criswell et al., 2014). Although C. gariepinus has proved to be resistant to various toxicants (Datta and Kaviraj, 2002; Olaifa et al., 2004; Okomoda et al., 2010), the fish was very susceptible to the toxicity of ethanol extract of A. obesum stem bark. The plant can therefore, be used to eradicate unwanted fish from fish ponds before the stocking of desired fish species as a tool for effective pond management. This is in affirmation of the global use of the powdered roots or a decoction of the bark and the leaves of the plant to kill fish (Arbonnier, 2004; Oyen, 2008). Histopathological analyses The types and incidence of histopathological lesions in the gills and skin of the exposed fish are as shown in Table 3. However, the severity but not the type of the observed lesions in the exposed fish was concentration-dependent. Histopathological lesions were seen in the gills of the exposed fish (Figs. 1–5) with a cumulative DTC value of 23.12 ± 6.43 suggestive of moderate damage by the extract. However, the DTC in the gills of fish exposed to different extract concentrations did not differ significantly (p > 0.05) from those of the unexposed control fish (Table 4). Changes in the skin of the fish exposed to the extract (Figs. 6–8) were classified as normal based on a cumulative DTC value of 1.78 ± 0.16. However, the DTC differed significantly (p < 0.05) between the skin of the exposed and

Mortality in Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark over the 96-h period.

Extract concentration (mg L 1)

Log of concentration

Total mortality

Percent total mortality (%)

Probit value

0.00 (Control) 6.25 mg L 1 7.50 mg L 1 8.20 mg L 1 8.80 mg L 1 9.30 mg L 1

0.0000 0.7959 0.8751 0.9131 0.9445 0.9683

0.00 1.00 3.33 5.00* 5.33* 6.00*

0.00 14.28 47.57 71.43 76.14 85.71

0.0000 3.9331 4.9398 5.5651 5.7128 6.0669

 Equation of the probit regression line: Y = 6.0017 · 0.1286. 1  Estimated 96-h median lethal concentration (LC50) = 7.15 mg L . *

Statistically significant (p < 0.05) compared to the control.

Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005

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S.E. Abalaka et al. Table 3 Types and incidence of histopathological lesions in the gills and skin of Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark over the 96-h period. Stage

Degree of tissue change Gills

I

– – – – – – – –

II

– Haemorrhage (+) – Complete lamellar fusion (++) – Epithelia degeneration (+)

III

Hyperaemia (+) Epithelia proliferation (+++) Epithelia detachment (++) Lamellar oedema (++) Mucous cells proliferation (++) Mucous cells hypertrophy (++) Partial lamellar fusion (++) Epithelia cellular infiltration (+)

Skin – Mucous cells proliferation (+++) – Mucous cells hypertrophy (++) – Eroded epithelia surfaces (++)

– Epithelia necrosis (+)

(+): low incidence, (++): moderate incidence, (+++): high incidence.

Figure 1 Photomicrograph of the gill of the unexposed control Clarias gariepinus. Note the primary lamellar (arrow) and secondary lamellar (S).

Figure 2 Photomicrograph of the gill of Clarias gariepinus exposed to 6.25 mg L 1 of ethanol extract of Adenium obesum stem bark. Note lamellar necrosis (X) with mononuclear cellular infiltration (arrow) and fusion of the secondary lamellar (F).

that of the unexposed control group (Table 4). There was a significant (p < 0.05) difference between the DTC in the gills and those in the skin of C. gariepinus exposed to ethanol extract of A. obesum stem bark as shown in Table 5. There was a defensive response by the exposed fish to increase the diffusion distance between its external environment and its blood as a barrier to the entrance of the toxic extracts as evidenced by the observed gill epithelial proliferation and detachment with lamellar oedema and fusion of the primary and secondary lamellar (Mallat, 1985; Poleksic and Mitrovic-Tutundzic, 1994; Van Heerden et al., 2004). Similar lesions were reported in the gills of C. gariepinus exposed to ethanolic extract of Parkia biglobosa pods (Abalaka et al., 2010). The epithelia proliferation was also a regenerative response of stressed fish to epithelial damages (Hemalatha and Banerjee, 1997a,b; Banerjee and Chandra, 2005). However, when this is uncontrolled, it might become degenerative in nature resulting in the loss of morphological

and functional efficiency of the affected gill structures in the exposed fish (Hemalatha and Banerjee, 1997a,b) as observed in the study. This will be due to the increased interface between the blood and dissolved oxygen in the water, which impairs normal gaseous exchange (Hemalatha and Banerjee, 1997a,b). In addition to increasing the diffusion barrier to toxicants, the epithelia detachment and lamellar fusion were also defensive attempts to reduce branchial superficial area in contact with the offending toxicant (Mallat, 1985; FigueirodoFernandes et al., 2007). However, these might also impair gaseous exchange in the exposed fish by reducing the available surface area for the gaseous exchange (Susithra et al., 2007) resulting in the observed respiratory distress and mortality recorded in some of the exposed fish. The observed oedematous changes in the gill lamellar of the exposed fish might have been due to increased capillary permeability of the blood vessels of affected gills (Olurin et al., 2006), leading to epithelial detachment (Pane et al., 2004; Schwaigner

Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005

Histopathological evaluation of ethanol extract of Adenium obesum stem bark

Figure 3 Photomicrograph of the gill of Clarias gariepinus exposed to 8.20 mg L 1 of ethanol extract of Adenium obesum stem bark. Note lamellar oedema (arrow) and lamellar fusion (F).

Figure 4 Photomicrograph of the gill of Clarias gariepinus exposed to 9.30 mg L 1 of ethanol extract of Adenium obesum stem bark. Note epithelia detachment (arrows).

et al., 2004) as seen in the study. Adeogun et al. (2012) reported similar oedematous gills in C. gariepinus exposed to sublethal concentration of methanolic extract of Raphia hookeri. Mortality in some of the exposed fish could have been due to respiratory compromise caused by the observed histopathological lesions. This is because although fish have the capacity to increase its ventilation rates to compensate for low oxygen uptake (Fernandes and Mazon, 2003) by increasing the rate of water flow through the gills (Reebs, 2009), these defensive or compensatory responses could grossly affect the functional efficiency of exposed gills (Takashima and Hibiya, 1995; Bols et al., 2001; Abdel-Moneim et al., 2008). Proliferation of mucous cells (MCs) within the interlamellar spaces and on the fused surfaces of affected secondary lamellar was a protective attempt to coat absorption surfaces with mucous rather than a direct effect of toxicants (Mallat, 1985). However, such mucoid layer could coagulate thereby obstructing the gaseous exchange and osmo-regulatory

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Figure 5 Photomicrograph of the gill of Clarias gariepinus exposed to 9.30 mg L 1 of ethanol extract of Adenium obesum stem bark. Note lamellar haemorrhage (arrows).

capacity of the affected gills with untold respiratory consequences (Tamse et al., 1995). This is because although periodic regeneration of new MCs, almost exclusively in gill filaments, normally re-establishes the much desired protective mucoidal coating that allows for the regeneration and/or multiplication of the gill epithelia cells, the recorded mortality in some of the exposed fish might have however, been due to asphyxiation caused by the altered morphology and physiology of the affected gills in the exposed fish as earlier reported by Banerjee, (2007). This is because although the front-line protective effect offered by the mucous coating against the continuous absorption of the toxic xenobiotic is quite effective at the initial stage of the exposure, it usually does not last long as this eventually collapses (Banerjee, 2007). Similar mucous cell proliferation was reported in Heteropneustes fossilis exposed to Azadirachta indica (Kumar et al., 2010). The erosion and consequent alterations in the chemical compositions and thickness of the mucous layer and the epithelia linings after interaction with xenobiotics resulted in the adhesion or fusion of the secondary lamellar to the primary lamellar (Daoust et al., 1984) as observed in the study. The observed lamellar haemorrhage was due to damage to pillar cells with the subsequent loss of their supportive properties resulting in red blood cells leakages into the epithelial layer, especially as the noxious extract was not immediately removed from the affected gill surfaces (Camargo and Martinez, 2007). This could have affected the free flow of blood with subsequent impairment of gaseous and/or ionic exchange at the very expense of the exposed fish. The observed lamellar mononuclear cellular infiltrations were inflammatory responses to tissue damage (necrosis) in affected gills. Similar cellular infiltrations were reported in the gills in C. gariepinus exposed to Malathion (Sharipudin et al., 2012). Unlike what was obtained in the study, vascular changes are usually more prominent in affected gills than the epithelial changes and it has been reported that although recovery from these vascular changes might be possible, it is usually more difficult compared to recoveries from epithelia changes (Poleksic and MitrovicTutundzic, 1994). Although stage III alterations were seen in the gills of the exposed fish and none in the gills of the

Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005

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S.E. Abalaka et al. Table 4 Degree of tissue change in the gills and skin of Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark over the 96-h period. Tissue

Gills Skin *

Degree of tissue change per extract concentration 0.00 (Control)

6.25 (mg L 1)

7.50 (mg L 1)

8.20 (mg L 1)

8.80 (mg L 1)

9.30 (mg L 1)

0.71 (±0.29) 0.67 (±0.21)

23.57 (±15.39) 1.71 (±0.36)

23.57 (±13.03) 2.17 (±0.40)*

15.25 (±7.12) 1.33 (±0.21)

35.50 (±26.03) 2.14 (±0.40)*

17.00 (±1.68) 1.50 (±0.34)

Statistically significant (p < 0.05) compared to the control.

Figure 6 Photomicrograph of the skin of the unexposed control Clarias gariepinus. Note the dermis (D), epidermal layer (double arrow), mucous cell (X) and epithelia surface (arrow).

Figure 8 Photomicrograph of the skin of Clarias gariepinus exposed to 8.80 mg L 1 of ethanol extract of Adenium obesum stem bark. Note the dermis (D), proliferated and hypertrophied mucous cells (X), eroded epithelia surface (arrow) and elaborated mucous (S).

Table 5 Degree of tissue change between the gills and skin of Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark over the 96-h period. Histopathological lesions

Gill

Skin

Degree of tissue change

23.12 ± 6.43

1.78 ± 0.16*

*

Figure 7 Photomicrograph of the skin of Clarias gariepinus exposed to 6.25 mg L 1 of ethanol extract of Adenium obesum stem bark. Note thickened epidermal layer (double arrow) with proliferated and hypertrophied mucous cells (X) and eroded epithelia surface (arrow).

unexposed fish, the non-significant (p > 0.05) differences between the DTC in the gills of the exposed and the unexposed fish are not fully understood thereby requiring further investigation(s).

Statistically significant (p < 0.05) between the gills and the skin.

The observed proliferated mucous cells within the gills and skin of the affected fish were for the continuous elaboration of mucous, which helps to clean up these respiratory surfaces in facilitating the removal of trapped toxicants from them (Chandra and Banerjee, 2004). This is because C. gariepinus also respires through the skin on its dorsum (Bruton, 1988) even as the skin of C. striatus and Clarias batrachus (L.), which are its close relatives, reportedly acts as an accessory respiratory organ (Banerjee and Mittal, 1976). The skin and the gills, which constitute the external boundary tissue of the fish, are normally not keratinized and covered by a layer of slimy mucous (El-Sayyad et al., 2010) but the amount of the mucous secreted is usually much more when these tissues are challenged as observed in the study. However, the protective

Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005

Histopathological evaluation of ethanol extract of Adenium obesum stem bark roles of the elaborated mucous may not last too long due to the rapid exhaustion of the mucous cells with the extensive loss and the altered nature of the excreted mucous following prolonged exposure, which might have caused the erosion of the superficial cells of the skin of the exposed fish (Chandra and Banerjee, 2004; Singh and Banerjee, 2008) as seen in the study. Chandra and Banerjee (2003) reported similar tear and wear leading to sloughing off of skin surfaces that resulted in haemorrhage in the skin of C. batrachus exposed to the air. The continuous exposure of these respiratory surfaces to the toxicant within the reconstituted fish culture water necessitated the observed spontaneous reactions as was earlier reported in Labeo rohito exposed to hexachlorocyclohexane (Das and Mukherjee, 2000). Conclusion Ethanol extract of A. obesum stem bark was toxic to the exposed C. gariepinus as evidenced by the recorded mortality in some of the exposed fish, including the observed histopathological lesions in the gills and skin of the exposed fish. Therefore, the plant is a readily available potent organic piscicide that can be meaningfully exploited by fish farmers in place of the environmentally harmful synthetic piscicides to eradicate unwanted fish from ponds prior to the stocking of desired fish species for maximum aquaculture yields. Acknowledgements The contributions of Mr. S.M. Meslam, Mr. A. Umar and Mr. J.D. Yaro to the work are highly appreciated. References Abalaka, S.E., Fatihu, M.Y., Ibrahim, N.D.G., Kazeem, H.M., 2010. Histopathologic changes in the gills and skin of adult Clarias gariepinus exposed to ethanolic extract of Parkia biglobosa pods. Basic Appl. Pathol. 3, 109–114. Abdel-Moneim, M.A., Shabana, N.M., Khadre, S.E.M., AbdelKader, H.H., 2008. Physiological and histopathological effects in catfish (Clarias lazera) exposed to Dyestuff and chemical wastewater. Int. J. Zool. Res. 4 (4), 189–202. Abu-Dahab, R., Afifi, F., 2007. Anti-proliferative activity of selected medical plants of Jordan against a breast adenocarcinoma cell line (MCF7). Sci. Pharm. 75, 121–136. Adeogun, A.O., Alaka, O.O., Taiwo, V.O., Fagade, S.O., 2012. Some pathological effects of sub-lethal concentration of the methanolic extract of Raphia hookeri on Clarias gariepinus. Afr. J. Biomed. Res. 15, 105–115. Al-Sarar, A., Husseini, H., Abobakar, Y., Bayoumi, A., 2012. Molluscicidal activity of methomyl and cardenolide extracts from Calotropis procera and Adenium arabicum against the land snail, Manacha cantiana. Molecules 17, 5310–5318. APHA, 1985. Standard Method for the Examination of Water and Waste Water, 15th ed. American Public Health Association, Washington, DC, USA, pp. 413–426. Arbonnier, M., 2004. Trees, shrubs and lianas of West African dry zones. CIRAD, MARGRAF Publishers, GMBH, MNHN, p. 161. Ayotunde, E.O., Offem, B.O., 2008. Acute and chronic toxicity of pawpaw (Carica papaya) seed powder to adult Nile tilapia (Oreochromis niloticus Linna 1757). Afr. J. Biotechnol. 7 (3), 2265–2274.

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Please cite this article in press as: Abalaka, S.E. et al., Gills and skin histopathological evaluation in African sharptooth catfish, Clarias gariepinus exposed to ethanol extract of Adenium obesum stem bark. Egyptian Journal of Aquatic Research (2015), http://dx.doi.org/10.1016/j.ejar.2015.01.005