Abstract: The toxicity of aqueous and ethanol extracts of Parkia biglobosa pods (55, 65, 75, 85 and 95 mgL ^-1) on Clarias gariepinus juveniles was investigated over a 96 h exposure period. Fish exposed to both extracts exhibited clinical signs of agitated behaviours, respiratory distress and abnormal nervous behaviours and deaths were recorded in some fish exposed to both extracts. No unusual clinical manifestations or deaths were recorded in control fish. However, signs and deaths were more in fish exposed to the ethanol extracts. The 96 h LC₅₀ values for aqueous and ethanol extracts were 296.14 and 225.48 mgL ^-1, respectively. Both extracts are of minimal toxicity and the maximum toxicant admissible concentrations for aqueous and ethanol extracts ranged between 2.96-29.61 mgL ^-1 and between 2.26-22.55 mgL ^-1, respectively. It was concluded that aqueous and ethanol extracts of P. biglobosa pods are toxic to C. gariepinus juveniles with the ethanol extract being more toxic.

INTRODUCTION

The demand for fish in Nigeria is increasing by the day because of short supply of other animal protein. This is due to the myriads of problems facing the Nigerian livestock and poultry industry (Adekunmisi et al., 2004). Fish farming or aquaculture has been described as the world ’s fastest growing food production system (Kureshy et al., 2000) and has a lso been identified as a practical and promising approach to meeting the fish demand (Olaifa et al., 2008) outside actual fishing. However, this sector is facing some challenges (Spadling et al., 1997; Adekunmisi et al., 2004; Assiah et al., 1997; Taiwo and Odunaiya, 2004) thereby making fishing the only viable alternative where both conventional and unconventional methods are employed for the sole aim of obtaining fishes from the vast water bodies for human consumption. This includes the exploitation of synthetic compounds which bio-accumulates and persist in both target and non-target animals (Arasta et al., 1996) with untold consequences for man and aquatic environment (Fafioye and Jeje, 2000).

Plant piscicides or plants that are poisonous to fishes have been recognized as effective alternatives to these harmful synthetic compounds (Dahiya et al., 2000; Fafioye, 2005). This is because they have lower toxicity against non-target animals (Chiayvareesajja et al., 1997), while being more human and environmental friendly (Marston and Hostettmann, 1985). Notable example of such plant piscicide is Parkia biglobosa (Fafioye, 2005), which is commonly found within the savannah belts of West Africa including Nigeria. Clarias gariepinus which is hardy (Hogendoorn, 1979; Olaifa et al., 2003) and indigenous to Africa (Rahman et al., 1992) is now recognized as important tropical catfish for aquaculture within the West African sub-region (Clay, 1979; Anthony, 1982) including Nigeria where it is highly valued (Olaifa et al., 2003). This study is therefore, aimed at determining the clinical manifestations and lethal concentrations of the toxicities of aqueous and ethanol extracts of P. biglobosa pods to C. gariepinus juveniles in view of the fact that there has not been any such rersearch before now.

MATERIALS AND METHODS

The dried pods of P. biglobosa were collected and later blended into fine powder. Maceration method (Bentley, 1977) was used to extract about 429 g (45.90% w/w) of the freeze-dried aqueous extract after soaking 1000 g of this fine powder with 6 L of distilled water over night prior to filtration and freeze drying. Same method was used to obtain 725 g (57.56% w/w) of the ethanol extract after soaking 1260 g of this fine powder in a separation funnel over a 48 h (h) period with 5 L of absolute ethanol prior to filtration and concentration to dryness in an evaporation dish over a 72 h period.

Juvenile C. gariepinus (25.09 ±0.52 g mean weight; mean ±SEM and 15.38 ±0.10 cm mean total lengths; mean ±SEM) were purchased and acclimatized for two weeks under laboratory conditions while being fed ad libitum daily with commercial catfish feed (Multi feed, Zelmach feed mill, Israel). Pond water was changed every other day, while acclimatization mortality was <5%. Feeding was stopped 48 h before and through out the 96 h experimental period (Adeyemo, 2005). This was to minimize interference by both the stomach contents of exposed fish and their wastes in reconstituted extracts (Olufayo, 2009).

A static bioassay (APHA, 1985) was performed for the toxicity testing after conducting a pilot study to obtain five graded concentrations (Ayotunde and Ofem, 2008) of 55, 65, 75, 85 and 95 mgL ^-1 for aqueous and ethanol extracts of P. biglobosa pods on C. gariepinus juveniles, respectively. Ten fish were randomly introduced into each of the reconstituted extracts which had been allowed to stand for 30 min (Usman et al., 2005). This is to allow for proper mixing of the reconstituted extracts. Control aquaria contained no extracts. Clinical signs and deaths were promptly monitored and recorded over the 96 h exposure period.

The Lethal Concentration (LC50) for each extract over the entire 96 h exposure period was determined by subjecting results to Probit and logit analyses (Finney, 1971). The level of toxicity of both extracts was determined based on hazard rating for synthetic agrochemicals (Louis et al., 1996). The maximum admissible toxicant concentrations for both extracts were established for C. gariepinus juveniles by multiplying the estimated 96 h LC₅₀ by a constant 0.01-0.1 (Koesoemadinata, 2000).

RESULTS

Juvenile C. gariepinus exposed to both aqueous and ethanol extracts of P. biglobosa pods exhibited similar signs of agitated behaviours, respiratory distress and abnormal nervous behaviours respectively (Table 1-3). Agitated behavious increased with increasing extracts concentrations but decreased with exposure period except for the stunned posture that persisted through out the exposure period. The exhibited signs of respiratory distress were directly proportional to increasing extracts concentrations but indirectly proportional to exposure period except for vertical positioning with exposed snouts and excessive mucus secretion whose intensity increased with exposure period.

Table 1:	Agitated behaviours
Experimental group (Exp. group), Frequent Surface to Bottom Movements (FSBM), Aqueous extract (A), Ethanol extract (B), None (-), Weak (+), Moderate (++) and Strong (+++)

Table 2:	Respiratory distress
Experimental group (Exp. group), Vertical Posture with Exposed Snouts (VPES), Excessive Mucus Secretion (EMS), Aqueous extract (A), Ethanol extract (B), None (-), Weak (+), Moderate (++) and Strong (+++)

Table 3:	Abnormal nervous behaviour
Experimental group (Exp. group), Sluggish and Swirling Movements (SSM), Different Postures (DP), Aqueous extract (A), ethanol extract (B), None (-), Weak (+), Moderate (++) and Strong (+++)

The exhibited abnormal nervous behaviours increased with both increasing extracts concentrations and exposure period except for sudden darts whose intensity decreased with exposure period. Even though clinical manifestations were similar in fish exposed to both extracts, signs and death were more in C. gariepinus juveniles exposed to the ethanol extracts of P. bigloboss pods than in fish exposed to their aqueous counterparts while there were no deaths or any form of unusual clinical manifestations in control fish. The probit and logit analyses for determining the 96 h LC₅₀ for both extracts were as presented in Fig. 1 and 2 and the 96 h LC₅₀ values for aqueous and ethanol extracts of P. biglobosa pods were 296.14 and 225.48 mgL ^-1, respectively.

Fig. 1:	Log of concentrations of aqueous extracts of P. biglobosa pods and its probit value in exposed C. gariepinus juvenile (96 h)

Fig. 2:	Log of concentrations of ethanol extracts of P. biglobosa pods and its probit value in exposed C. gariepinus juvenile (96 h)

The maximum toxicant admissible concentrations ranged between 2.96-29.61 mgL ^-1 for the aqueous extract and between 2.26-22.55 mgL ^-1 for the ethanol extract, respectively.

DISCUSSION

The exhibited agitated behaviours were fish flight response from making contact with reconstituted extracts so as to prevent the absorption of offending extracts. The findings agree with reports of Omoniyi et al. (2002) and Usman et al. (2005). The observed respiratory distress may have been due to both decreasing dissolved oxygen content of reconstituted extracts with decreasing capacity and/or ability of exposed fish to oxygenate their blood via respiration. The decreasing dissolved oxygen content of reconstituted extracts with exposure period may be due to the nature of phytochemical constituents of aqueous and ethanol extracts of P. biglobosa pods coupled with their subsequent continuous oxidative bio-degradation over time. However, the extent of such oxygen depletion due to the continuous oxidative bio-degradation of both extracts may depend on such extract concentrations. The decreasing capacity and/or ability to respire may be due to pathological alterations in the gills and skin (the primary and secondary respiratory structures) of exposed fish where the extent of such decreasing incapacitation could be directly proportional to the level of pathological compromise of these vital respiratory structures. Excessive mucus secretions are natural defense mechanisms by exposed fish to coat their body surfaces in order to prevent and/or reduce the absorption of offending toxicants (Cagauan et al., 2004). However, such excessive mucus secretions are reported to reduce respiratory activity in fishes (Konar, 1975) which together with decreasing oxygen content of reconstituted extracts results in the creation of hypoxic states in exposed fishes (Usman et al., 2005) leading to subsequent respiratory distress and deaths in exposed fishes (Omitoyin et al., 1999). The findings of exhibited excessive mucus secretions in exposed fish agree with the report of Jothivel and Paul (2008). Abnormal nervous behaviours are associated with the impacts of the toxicants on fishes (Fafioye, 2005). This may be due to nervous system involvement or failure (Ufodike and Omoregie, 1994; Oti and Ukpabi, 2000) or may be due to biochemical body derangement including hepatic compromise (Fadina et al., 1991).

The findings agree with the reports of Omitoyin et al. (1999), Omoniyi et al. (2002) and Ayotunde and Ofem (2008). The observed increasing state of inactivity with increasing extracts concentrations and exposure period are considered normal in both acute and chronic toxicity testing (Kulakkattolickal and Kramer, 1997).

The estimated 96 h LC₅₀ values were due to the toxicities of both extracts on exposed fish. These values were much higher than 96 h LC₅₀ values of 0.36 and 13.18 mg L ^-1 reported by Omitoyin et al. (2006) and Usman et al. (2005), respectively meaning that the pollutants used by these researchers were more toxic to C. gariepinus juveniles than aqueous and ethanol extracts of P. biglobosa pods. Even though, the ethanol extract of P. biglobosa pods was more toxic compared to their aqueous counterparts from their estimated 96 h LC₅₀ values and exhibited clinical signs, both extracts of P. bigloobsa pods are of minimal toxicity to exposed C. gariepinus juveniles based on the hazard ratings for synthetic agrochemicals (Louis et al., 1996). The findings of ethanol extract being more toxic to xposed C. gariepinus juveniles agree with the report of Fafioye et al. (2004). The estimated maximum toxicant admissible concentrations for aqueous and ethanol extracts of P. biglobosa pods were concentration ranges which will not have any adverse effect on exposed fishes or the physicochemical constituents of their immediate external environment (Lesnikov, 1979).

CONCLUSION

In this study, we observed clinical signs and deaths of exposed C. gariepinus juveniles including the established 96 h LC₅₀ values for both extracts implies that the aqueous and ethanol extracts of P. biglobosa pods are toxic and therefore, can be used as effective piscicide in place of synthetic compounds to harvest fishes from our abundant water bodies.