Abstract: Acute toxicity of water extract from dry Euphorbia heterophylla (Linnaeus) plant stem on Barbus occidentalis (Boulenger, 1920) fingerlings was conducted using static bioassay tests over a period of 96 h. The range finding test was used to determine the lethal concentration of the botanical on B. occidentalis and was found to induce varying behavioral response in the fish. The 96 h median lethal concentration, LC50 of 1.81 g L-1 and safety level of 0.18 g L-1 with lower and upper 95% confidence limits being 1.51 and 2.18 g L-1, respectively were determined for B. occidentalis fingerlings exposed to water extract from dry E. heterophylla plant stem. A concentration dependent relationship was established for the effect of the toxicants on the test organisms. Percentage survival of the test organisms followed a regular pattern increasing with decreasing concentration. Prior to death fish were observed to swim actively at the bottom, came up to the surface of the water to gasp for air, there was erratic swimming behaviour, spiral uncoordinated movement and darkened in skin.
INTRODUCTION
The usefulness of some of plants as Piscicidal has been reported by Reed et al. (1969) and Adewole (2002) in replacement for obnoxious chemicals. The position and importance of some of this piscicidal plants is unique, whereby most fish farmers and fishermen indiscriminately use various kinds and parts of these plants extract due to their narcotic, pesticidal and molluscidal properties in other to induce fish for easy catch and clean up the aquatic systems off some pests and Molluscs. Earlier research have been carried out on the responses of fish to some plants toxicants (Omoregie and Okpanachi, 1992; Omoregie and Ufodike, 1994; Ufodike and Omoregie, 1994; Ayuba and Ofojekwu, 2002; Wade et al., 2002) but the piscicidal effect of E. heterophylla (L.) on fish has not been given much attention despite its economical and ornamental importance and wide usage by most fishermen in the south-west geographical region of Nigeria (Adewole, 2002). Toxic activity of leaf extracts of Polygonum hydropiper (L.) and Pogostemon parviflorus (Benth) were tested and reported at LD50 was 758.58 mg kg-1 in male albino mice by Rahman et al. (2005). Akinwande et al. (2007) also reported that the 96 h LC50; 81.28 mg L-1 while the threshold value was 21.13 mg L-1 for Heteroclarias exposed to mesocarp of Neem plant.
This plant belongs to the family Euphorbiaceae which embraces about 7,000 species distributed all over the temperate and tropical world (Bradley, 1979) and produces milky irritating juice which contains some bioactive ingredients like diterpene together with aleuritolic acid, oleanolic acid and betulin diacetate sesquiterpene-coumarin and a quinoid-type diterpenoid which negate physiological activities in fish (Madureira et al., 2004a, b). The test organisms, Barbus occidentalis is an ornamental fish species of great economical value and belongs to the family Cyprinidae. It is a common freshwater species, slivery black in colour and often tinged with yellow or pink and hardy (Reed et al., 1969). The choice of the species was based on its ecological and economic significance.
The primary objective of this research is to establish the piscicidal effect of the indiscriminate used E. heterophylla, in harvesting fish and to determine the 96 h median lethal concentration, LC50 of the plant extract to the test organism.
MATERIALS AND METHODS
Fingerlings of B. occidentalis with a mean weight and total length of 3.1 g and 6.2 cm, respectively, were obtained from fish farm of the Department of Wildlife and Fisheries Management, University of Ibadan. They were transported to the Fisheries and Hydrobiology Post Graduate Research Laboratory, Department of Zoology, University of Ibadan in plastic bucket containing cool water, in the morning before sunrise to prevent the fish from being stressed. The fishes were acclimatized in the laboratory by being held in tanks (30x20x15 cm) containing clean dechlorinated water for seven days. The water in each tank was replaced thrice a week.
The fishes were fed with crushed pellets twice daily. The left over feed and feaces were siphoned off promptly and dead fish were promptly removed to avoid contamination. The percentage of death recorded during acclimatization was less than 5% as such the fishes were accepted as being adapted to the laboratory conditions. Feeding was discontinued 24 h prior to the commencement of the bioassay test (Ruparelia et al., 1990).
Specimen of the fresh stem of E heterophylla was collected from a cassava farm near Oba reservoir in University of Ibadan. The stems were oven dried between 40 and 50°C and crushed into powdered form using mortal and pestle. Five hundred grams of fine particulate powders were dissolved in 2 L distilled water of warm temperature 23.3±0.5°C for 24 h. The extract was filtered using Whatmann’s filter paper No. 1 using a vacuum pump. The filtrate was freeze-dried and store in the refrigerator for use. A range finding test was conducted to determine the concentration to be used in the definitive tests. Test concentration in the range finding tests were selected at intervals based on logarithm ratio 0.00.1, 0.01 and 0.1 (Parish, 1985). The fishes were disturbed randomly at the rate of ten per tank using hand net. Observations were made on the behavior of the test organism at 1, 2, 4, 8, 16, 24, 48, 72 and 96 h. Dead specimens were removed immediately death was confirmed. After the range finding test, some concentration were chosen for the definitive tests. A stock solution of the freeze extract was made and delivered into the experimental aquaria as 0.0 mg L-1 (control), 1.2, 1.8, 2.4, 3.0 and 3.6 g L-1 concentrations. Eighteen aquaria of dimension 0. 20x0.15x0.10 m were used. The fishes were disturbed randomly at the rate of ten per tank to each of the five concentrations in triplicates using hand net. Each tank contained 5 L of water for each test concentration. The toxicant solutions and test water were renewed after 48 h in each bioassay. Water characteristics were monitored after 24 h using methods described by APHA (1980). Test aquaria were examined for fish mortality on a daily basis. Fish were confirmed dead when they showed no response to touch by glass rod. Dead specimens were removed immediately and recorded. The 96 h LC50 for each extract was determined as summary of percentage mortality data as a probit analysis. The lower and upper confidence of limits of the LC50 was determined as described by UNEP (1991). Observations were made on the behavioral response of the test organism at 24, 48, 72 and 96 h. Results were subjected to statistical analysis using SPSS10 Windows 2000 to test for the significant difference (p<0.05) between the various concentrations of E. heterophylla.
RESULTS
The water quality parameters within the treatment tanks as shown in Table 1 did not vary significantly (p>0.05) to what is obtained from the control. The mean water quality values for temperature were 24.12±0.27°C, dissolved oxygen was 6.36±0.14 mg L-1, pH was 6.95±0.03 and free carbon dioxide is 4.84±0.01 mg L-1. At concentration of 3.6, 3.0 and 2.4 g L-1 fishes were observed to swim actively at the bottom, came up to the surface of the water to gasp for air, there was erratic swimming behavior, spiral uncoordinated movement.
In the definitive test, at concentration 3.6 g L-1, 100% mortality was recorded at the 96 h exposure time. While at concentration of 3.0 and 2.4 g L-1, 87 and 63% mortality were recorded, respectively. The toxicity of the plant varied with concentration and time of exposure of test organisms. In the control group no mortality was recorded (Table 2). The 96 h LC50 was determined using probit values and regression analysis 1.81 g L-1 (Fig. 1) with a safety concentration of 0.18 g L-1 for the exposed organism. At any concentration of the ichthyotoxic plant, the regression equation Y = 1.4083+11.816 log conc. and R = 0.9120 will produced a projected mortality.
A golden brown colouration which persisted until 48 h was observed immediately the dried plant was introduced during the preparation of the stock solution. After this time duration, the intensity of the colouration decreased and precipitates of the plant were observed to settle at the bottom of the experimental tank during the bioassay. At the 96 h there was production of foul odour and some foam on the surface of the experimental solution. The intensity of the color was observed to decrease with deceased in the concentration of the test solution. Dark patches and fading off of the golden silvery colouration of the skin were observed as the fish died.
Ninety six hour LC16 and LC84 were also using probit method as 1.60 and 2.21 g L-1, respectively. 2.21 remain the MATC. The operculum ventilation rate/minute increase with increase in concentration of the test solution and the exposure time. Ninety six hour had the lowest operculum ventilation rate (Fig. 2). The ventilation rate of the fish during the exposure period of 96 h gave the highest correlation value R = 0.9934, p<0.05 between the experimental treatments of concentrations 2.4 and 3.0 g L-1 while the lowest correlation value of R = –0.2471, p>0.05, p>0.01 was recorded between the concentrations 1.2 and 3.6 g L-1 (Table 3).
| Table 1: | Mean±SE of water quality parameters during the acute bioassay exposure of B. occidentalis to E. heterophylla dried stem water extract |
| Table 2: | Mortality rate of B. occidentalis fingerlings exposed to acute concentrations of E. heterophylla (L.) dried stem water extract |
| Fig. 1: | Porbit mortality of Barbas occidentalis exposed to water extracts of dried Euphorbia heterophylla plant stem |
| Fig. 2: | Operculum ventilation rates of B. occidentalis exposed to various acute concentrations of water extract of dried stem, E. heteropohylla plant |
| Table 3: | Correlation matrices (r) of the ventilation rate |
DISCUSSION
The ichthyotoxic potential and phytotoxic properties of plants extracts have been reported by Reed et al. (1969), Ufodike and Omorege (1994) and Adewole (2002). Various forms of abnormal behaviors were observed in B. occidentalis when exposed to different concentrations of E. heterohpylla stem water extracts. These include erratic swimming, occasional darting of fish up and down the water column, aggregation of fishes below the water surface gasping for breath and change in colour from slivery black to pale colour. Some of these behavioural responses have been reported by Cruz- Lacerda (1993), Chlayvareesalla et al. (1997), Ayuba and Ofojekwu (2002), Wade et al. (2002) and Oti (2003) for Clarias and Puntius gonionotus, exposed to Maesa ramenticae, Chanos Chanos (milk fish) and Oreochromis mossombicus exposed to rotenone, Clarias gariepinus exposed to Datura innoxia root extract, O. niloticus when exposed different concentration of cassava effluent under laboratory conditions and Heteroclarias exposed to water extract of bark of Thevetia peruviana, respectively. The behavioural response of the test organism was observed to be dose dependent reducing with decreasing concentration. Chin and Chen (1987) also observed similar response in Penaeus monodon exposed to different concentrations of ammonia. Adeogun (1994) reported similar response on O. niloticus and Clarias gariepinus due to the effect of Raphia hookeri. Loss of balance and direction in the test organisms, uncordnated spiral movement and occasional jumping exhibited to a nervous reaction of the test organism to the toxicant have been earlier been reported by Adeogun (1994).
Emission of strong foul odour from the test solution by the end of the 96 h test may be attributed to oxygen depletion. The stressful breathing behavior exhibited by the fish may be as result of respiratory impairment due to effect of toxicant on the gills. The inability of the gills surface to actively carry out gaseous exchange might be responsible for the recorded mortalities which significantly different (p>0.05) and directly proportional to the exposure concentration and period (Fig. 2). The increase in mortality reported in this study as a result of increase in the concentrations of the plant extract was due to the impairment of normal metabolism by the inhibitory parents in the extract which could produce digitalis-like action that my result into arrhythmia, loss of normal heart beat rhythm, leading to serious disorder and severe cases may be fatal resulting to death (Oti, 2003).
Changes in the skin colouration of the fish observed in each experimental tanks was similar to the observation made by Oti (2003) when Heteroclarias fingerlings were exposed to Thevetia peruviana bark water extract. This may be as a result of the dispersion response of the melanin pigments in the chromatophores which move towards the periphery by pituitary hormone intervention, Melanocyte Stimulating Hormone (MSH) (Oti, 2003; Oti and Ukpabi, 2005). It has been stated earlier by Novales (1959) that MSH is the most important pigment movement determinant factor within the chromatophores. E. heterophylla like others toxicants contain some chemicals which affects dispersion and as well induces melanocyte stimulating hormones, since dark patches and other changes in colourations were only observed on fish in the treatments group and not dependent on the variation of the concentration of the extract (toxicants).
Based on the 96 h LC50 (1.81 g L-1) and an empirical applicable factor of 0.1 (Sprague, 1971), the safety level was 0.18 g L-1 water extract of E. heterophylla concentrations on B. occidentalis was lower to safety level of 0.24 g L-1 reported for the fresh plant exposed to the same fish under the same experimental condition (Dan-Ologe et al., 2006). The threshold concentration that produces statistically significant deleterious effect as seen in Probit mortality (Table 2 and Fig. 1) is commonly expressed as the maximum acceptable toxicant concentration (Wickins, 1976). The indiscriminate utilization of this plant extract by fish farmers as ichthyotoxin should be discourage since a lower concentration will even lead to higher mortality of fish apart from causing some obvious physiological disorderliness. The identified bioactive ingredients by bioactive ingredients (Madureira et al., 2004a, b) from the milky juice of this plant may not only cause physiological stress but have adverse effect on the ecosystem.