This study was conducted with the objective of examining the acute and sublethal toxicity of Prosopis africana pods methanolic extract on the biomarkers: haematological test, biochemical parameters and histopathological indices of Clarias gariepinus juveniles using static and renewal bioassay methods. Five hundred Fish were collected and acclimatized for two weeks out of two hundred and ten experimental fish were exposed to acute concentrations (0.00, 0.80, 1.60 and 2.4 g/l) for 96 hours and chronic (sublethal) concentrations (0.00, 0.08 and 0.16g/i) for 21 days. Phytochemical screening of Prosopis africana pods methanolic extract reveals the presence of alkaloids-prosopine and saponins which indicated the toxicity of the plant. Mortality of fish occurred in the acute exposure and preceded by pronounced behavioural changes such as erratic swimming, dart, respiratory distress and excessive mucous secretions. The Lethal Concentration (LC50) was 1.6 g/l for 96 hour exposure. At the end of the acute and chronic exposures, haematological characteristics [Red Blood Cell (RBC), White Blood Cells (WBC), Haemoglobin (Hb), the haematocrit (Ht) level, Mean Cell Volume (MCV), Packed Cell Volume (PCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin Concentration (MHCH), Platelet (PLT) and Lymphocites (LYM%)], biochemical characteristics [Alaninetransaminase (ALT), Aspertate aminotransferase (AST), Lactate dehydrogenase (LDH), Malondialdehyde (MDA)- as an index of lipid peroxidation, catalase (CAT), glutathione peroxidase (GSH-Px) and Superoxide dismutase (SOD), high-density lipoproteins (HDL), low-density lipoproteins (LDL),urea, total cholesterol(TChol), triglycerides (Trig), glycogen (Gly), Albumin, Total protein, Creatinine, Globulin, Glucose] as well as the histological profiles were estimated in blood, gill, and liver. The changes of haematological, biochemical and histological constituents from the control which was all time and dosage dependent were noted. The results are statistically analyzed and most values are found to be significant (p<0.05). Some liver enzymes (AST, ALT) in the blood serum are decreased, while others (CAT, SOD, GPx) increased. The histological parameters reveal clubbing, odema, necrosis of the primary and secondary lamella of the gill and degenerative changes in livers of fish exposed to Prosopis africana extract compared to the control. It is therefore concluded that Prosopisafricana pods extract is toxic with deleterious effects on the haematological, biochemical and histological profiles of Clarias gariepinus juveniles. And the use of prosopisafricana for fish harvesting should be discouraged by way of legislation as applications may directly or indirectly cause hazards to aquatic organisms, as well as humans via food chain.
INTRODUCTION AND LITERATURE REVIEW
Africa and Nigeria in particular with some of the world longest rivers, swamps and lakes has been richly blessed with inland waters and not surprisingly the indigenous people have a successful tradition of fish harvesting or subsistence fishing, a practice which, of course,is universal and includes harvesting the ocean. Moreover, demand is increasing for fish and fish protein -partly because fish protein is considered healthier than other animal proteins (Fafioye, 2001).
Fish farming has taken its place in the frontline of food production worldwide (Francis et al., 2001). This is due to high feed conversion ratio of fish, high utilization of both agricultural and animal wastes leading to high productivity and high returns and provision of the much needed employment opportunities (Katina, 2000). However, factors such as the presence of pests, predators and competitors of cultivated fishes affect aquaculture production negatively (Chiavareesajja & Chiavareesajja, 1997). At stocking these undesirable fishes may be introduced into aquaculture through water supplies or along with desired fishes (Cagauan et al., 2004) and their control is vital to the success or productivity of the fish farm (Jothivel and Paul, 2008).
Some measures such as complete pond drainage, sun drying for cracked pond bottoms and the use of screening as a standard method of preventing the entry of unwanted fishes into ponds are often undertaken but they are inadequate (Bardach et al., 1972; Jhingran, 1983; Cagauan et al., 2004). These have led to the rampant use of synthetic compounds by farmers but for their undesirable toxic consequences on man and the environment (Terry, 1987; Fafioye and Jeje, 2000). However, piscicidal plants are now seen as effective alternatives to these toxic synthetic compounds (Dahiya et al., 2000) because they are bio-degradable (Kela et al., 1989) and are less harmful to man and the environment than synthetic compounds (Ahmed and Grainge, 1986).
The use of plant poisons to catch fish is still used in many places in the world today. Though this easy and simple method of fishing is forbidden by law, most indigenous cultures across the Africa used poisonous plants to catch fish (BioinformaticsCentre, 1998).
Most fish poisons, also called icthyotoxins or piscicides, occur in several related plant species. A variety of chemicals found in these plants will stun fish when it passes through the gills or in some cases ingested. The fish then floats to the surface for easy capture (Campbell and Paul, 1991)
The active ingredient is released by mashing the appropriate plant parts, which are then introduced to the water environment. Poisoning was generally done in stagnant pools or slow-flowing streams and rivers that allow the pounded bark, leaf, seed, root or fruit, to concentrate its power without being washed away or diluted by a strong current. Sometimes streams would be partly dammed/ blocked to slow down the water flow. Gathering the fish was usually done by hand, but baskets, spears and nets were sometimes employed. Although primarily used in fresh water areas, Australian Aborigines and Californian Indians also used this technique in saltwater environments for octopus and low-tide shellfish fishing as well as for catching fish trapped in inter-tidal pools (Fallon and Enig, 1999).
According to St. Onge (2002) the two primary chemicals that occur in most plants used for stunning fish are saponin and rotenone. Saponins normally break down in the digestive system and must enter the bloodstream to be toxic, but fish take in saponins directly into their bloodstream through their gills. The toxin acts on the respiratory organs of the fish without affecting their edibility. Saponins also cause the breakdown of red blood cells that help the toxin to spread quickly. Even though the effects of the poison are powerful, they are not usually fatal. Fish that are washed away into untainted water revive, and can return to their pre-toxic condition. Because of this, the fishermen would have to gather the stunned fish quickly as they float to the surface.
According to Armstrong (2001) saponins are one of a group of glucosides found in many plant species with known foaming properties when mixed with water. Saponins lower the surface tension of water allowing the formation of small stable bubbles. The amount of foam created by a crushed plant sample, shaken with water in a jar, is a good indication of the amount of saponins present. Saponins have been used in modern times in the manufacture of fire extinguisher foam, toothpaste, shampoos, liquid soaps, and cosmetics and to increase the foaming of beer and soft drinks (Seigler, 2002).
Plant families that contain significant saponins are: Amaryllidaceae, Convolvulaceae, Dioscoreaceae, Lamiaceae, Lecythidaceae, Liliaceae, Loganiaceae, Meliaeae, Menispermacea, Papilionaceae, Solanaceae, Sapindaceae, Sapotaceae, Scrophulariaceae, Solanaceae and Verbenaceae. Plants containing rotenones are the second most utilized as a fish poison. Rotenone is an alkaloid toxin, in a group called flavonoids and stuns fish by impairing their oxygen consumption. The plant is toxic only to cold-blooded creatures and is found almost exclusively among the family of legumes (Papilionaceae, Mimosaceae, Cesalpiniaceae). Rotenone is also used today as an insecticide. In Africa, leaves and sap of pencil tree, milk bush, (Euphorbia tirucalli) and parquatina nigrecensare utilized as fish poisons while the dry fruits (pods) of ‘Guele’, Ironwood, Prosopis africana and Parkia biglobosaare normally used (Houerou, 2002).
The toxicity of piscicidal plants to fishes have been reported (Chiavareesajja et al., 1997; Usman et al., 2005). Akinbulumo et al.(2008) observed that as many as one percent of all plant species may be toxic to humans and animals, their effects ranging from skin irritation to hallucinations, bone marrow destruction, paralysis, vomiting, and heart failure. In particular, these effects have been found to affect fish organs and tissues. According to Fafioye et al. (2004), the higher the concentrations of P. biglobosa and R. vinifera, the more severe the degree of damages to fish gill and liver. Poisonous plants occur in all habitats and many cultivated gardens. Common varieties include star-of-Bethlehem, belladonna, poison ivy, poison oak, yew, oleander, wisteria, and poison hemlock (Akinbulumo et al., 2008).
Plants are natural biocides (Burkill, 1985) and due to recent wide use their contamination of natural waters has become inevitable in Nigeria. Piscicidal plants like Blighia sapida, Kigelia africana, Tetrapleura tetraptera, Raphia vinifera, Parkia biglobosa and Tephrosia vogelii are frequently in use by the fisher folks because they are highly potent (Fafioye, 2001). However, the presence of these botanicals in high concentrations may have adverse effects on aquatic organisms.
Various plants are reputed for their medicinal and antimicrobial values (Nascimento et al., 2000; Adamu et al. 2005), including their pesticidal, acaricidal and trypanocidal properties (Jhingran, 1975; Mgbojikwe and Okoye, 1998; Atawodi, 2005). Yet, some are known potent arrow and fish poisons (Geidam et al., 2007; Kamalkishor and Kulkarni, 2009) depending upon the type and the concentrations of their bio-active constituents. This is because plants contain structurally diverse biological substances with varying properties (Istvan, 2000)
Prosopis africana is a flowering plant species in the genus Prosopis found in Africa. Its common names include African mesquite, iron tree, “gele” (Malinke) (traditional ‘djembe’ wood) or somb tree. Other species of the genus Prosopis are Prosopis juliflora, Prosopis alpataco, Prosopis tamarugo, P. chilensis, P. dendans, P. farcta
Seeds of P. africana are used in Nigeria to prepare “daddawa”, “kpaye” or “okpeye”, fermented products are used as food condiments. The Fabaceae or Leguminosae, commonly known as the legume, pea, or bean family, are a large and economically important family of flowering plants. It includes trees, shrubs, and perennial or annualherbaceous plants, which are easily recognized by their fruit (legume) and their compound, stipulated leaves. Many legumes have characteristics of flowers and fruits. The Fabaceae family includes a number of important agricultural and food plants, including Glycine max (soybean), Phaseolus (beans), Pisum sativum (pea), Cicer arietinum(chickpeas), Medicago sativa (alfalfa), Arachis hypogaea (peanut), Ceratonia siliqua (carob), and Glycyrrhiza glabra (liquorice). A number of species are also weedy pests in different parts of the world, including: Cytisus scoparius (broom), Robinia pseudoacacia(black locust), Ulex europaeus (gorse), Pueraria lobata (Lupinus species kudzu), a number of and Parkia biglobosa.
Prosopis africana is common in the middle belt region (Kwara, Niger, Kogi, Benue, Nasarawa and Plateau states) of Nigeria and they are generally called “okpeye” or “kiriya” in Hausa language. The plant is of immense importance and uses; every part of the plant being effectively utilized. Phytochemical constituents of Prosopis africana consist of prosopine, harman, tyramine, and prosopinine which are alkaloids and can intercalate with DNA. The poisonous principles in Prosopis africana are alkaloids that affect the nervous system and induce trembling, loss of coordination, and paralysis of respiration.
Also, Prosopis spp have caffeic acid derivatives with antibiotic activities against viruses, bacteria and fungi (Harzallah-Shhiri et al. 2005, Sirmah et al., 2009).
Leaf extractives analyzed by gas chromatography-mass spectrometer reveals the presence of physiologically relevant fatty acids such as hexadecanoic acid, octadecanoic acids, glucopyranose, hydroquinone, glucopyranosides and galactose sugars (Aqeel et al., 1989). In particular it has been reported that the leaves are employed in the treatment of headache and toothaches as well as other various ailments. Its leaves and bark are combined to treat rheumatism, remedies for skin diseases, fever and eye washes are obtained from the bark. The roots are a diuretic and are used to treat gonorrheoa, tooth and stomach aches, dysentery and bronchitis. In Mali, the leaves, bark, twigs and roots are used to treat and relieve bronchitis, dermatitis, tooth decay, dysentery, malaria and stomach cramps (Ajiboye, 2009). Dry pods are used as fish poison and for treating wounds and tooth decay (Abbiw, 1990).
The seeds are fermented and used as food condiment; towards the end of the dry season the leaves and fodder become essential commodities (Ajiboye, 2009). Prosopis africana is used to control erosion, the bark is used to make beehives, the leaves and shoots are palatable to livestock and suitable for shades in homesteads in dry areas. It has the potential to fix atmospheric nitrogen; provides useful mulch for soil improvement, suitable as avenue tree, its pod ashes are a source of potash for soap making, it has a potential for parkland agro forestry systems and for improved agro forestry technologies (Ajiboye, 2009).
Biological activities of Prosopis africana have been reported where an oral median lethal dose of the methanolic extract of the plant at 3.808g/kg in mice and >5g/kg in rat; and the study result support the traditional claim of the use of Prosopis africana for the analgesic and anti-inflammatory activities (Ayanwuyi et al., 2010). Abbiw (1990) reports that dry pods of Prosopis africana are used in fish poisoning.
The African catfish, Clarias gariepinus is an ecologically important and commercially valued fish for the Nigerian fishing industry (Ita, 1980). These mudfish are frequently and widely cultured in ponds and they also occur freely in Nigerian natural fresh waters. The demand for this fish species by almost 75% of Nigerian population has necessitated the cropping of it in large number using poisons. Based on the piscicidal properties of Prosopis africana pods, there is the need to evaluate its potential as catfish poison. Therefore this study will present a much greater information on the acute and chronic toxicity of the methanolic extract of Prosopis africana and its effects on the haematology, biochemistry and histopathology of C. gariepinus juveniles.
Lyon et al. (2006) reported that the latex part of Parquetina nigrescens (African Parquetina) is toxic and even used in making fish poison.
Franket al. (2016) reported a work on the ichthyotoxic effect of Phyllantus niruri on Clarias gariepinus where the fishes exposed to the plants extract showed a range of changes from lamellae inflammation and distortion in 100 and 150mgdm-3. Serious gill shrinkage and lamellae erosion was observed in the highest concentration of 200mgdm-3. It was noted that the observed mortality rate of the fingerlings is concentration and time dependent as it is directly proportional to the level of the extract in the aquarium water and the exposure time. After four days (96h), the LC50 occurred at 200mgdm-3. The observed behavioral changes include weakness, erratic swimming, moribund behavior, loss of balance and finally death.
The ichthyotoxic activity of Ageratum conyzoides against the African catfish, Clarias gariepinuswas well documented. It was observed that ichthyotoxic constituents of Ageratum conyzoides are more soluble in ethanol than in water. The affected fishes started with moribund behavior, erratic swimming raising their head up apparently to get oxygen and depigmentation before their death. The death of the fishes must have occurred as a result of the plant fish poison as well as the effect of the plant material which lowers the level of dissolved oxygen and affects other water parameters.The LC50 for the ethanol and aqueous extracts were observed at 5.3 and 8mgdm-3 respectively (Frank and Ogie, 2016).
Abalaka and Auta (2010) observed that a96 hour LC50 values of 105.83mg/L and 135.64mg/L for both aqueous and ethanol extracts of P. biglobosa pods were much higher than 0.19mg/L and 4.2mg/L reported by Ayotundeet al. (2008) and Wadeet al. (2002). This means that the aqueous and ethanol extractsof P. biglobosa pods were less toxic to C. gariepinus than the extracts of Carica papaya and Manihot esculenta that were used on Oreochromis niloticus adults respectively by both authors.
A paradox in metabolism is that, while the vast majority of complex life on Earth requires oxygen for its existence, oxygen is a highly reactive molecule that damages living organisms by producing reactive oxygen species- ROS- H2O2, O2−&-OH) (Davies, 1995). Consequently, organisms contain a complex network of antioxidant metabolites and enzymes that work together to prevent oxidative damage to cellular components such as DNA, proteins and lipids(Vertuani et al., 2004). In general, antioxidant systems either prevent these reactive species from being formed, or remove them before they can damage vital components of the cell. However, ROS also have useful cellular functions, such as redox signaling. Thus, the function of antioxidant systems is not to remove oxidants entirely, but instead to keep them at an optimum level (Rhee, 2006). The ROS produced in cells are chemically reactive chemical species containing oxygen. Examples include hydrogen peroxides (H2O2), superoxide anion (O2−), free radicals such as hydroxyl radical (·OH), and singlet oxygen. The hydroxyl radical is particularly unstable and will react rapidly and non-specifically with most biological molecules. This species is produced from hydrogen peroxide in metal-catalyzed redox reactions such as the Fenton reaction. These oxidants can damage cells by starting chemical chain reactions such as lipid peroxidation, or by oxidizing DNA or proteins. Damage to DNA can cause mutations and possibly cancer, if not reversed by DNA repair mechanisms, while damage to proteins causes enzyme inhibition, denaturation and protein degradation (Hirst., 2008). However, during times of environmental stress (e.g. Prosopis africana exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. Thus important biomarkers and bio-indicators such as haematological data, biochemical or enzymatic activities and alterations in tissue structures are usually employed to measure the effects of toxicants on aquatic organisms. The use of haematological technique in fish culture is growing in importance for toxicological research and environmental monitoring of fish health condition. Haematological indices are important parameters to evaluate the general physiological status of fishes and may be considered as stress indicators for estimation of the response reactions of the fish to various environmental conditions (Docan et al., 2010). It may be considered useful in assessing the health of fish subjected to changing environmental conditions (Blaxhall, 1972 and Nair et al, 1984) and have proven useful in monitoring stress responses as bio-indicators (Bridges et al, 1976; Soivio and Oikari, 1976).