ECOLOGICAL EFFECTS OF PESTICIDES CONTAMINATION ON AMPHIBIAN TADPOLES (Ptychadena bibroni) OF THE NIGER DELTA OF NIGERIA

Ecolog ic a l effec ts of pestic ides c onta m ina tion on a m ph ib ia n la rva l sta g es w ere a ssessed using a c ute tox ic ity in th e la b ora tory . T h e study c om pa red th e tox ic ity of tw o w idely used pestic ides, A tra z ine (h erb ic ide) a nd Ba sudin (insec tic ide) on th e la rva l sta g es of dom ina nt frog s S,T U,V WMX>Y>Z [\X^]4_`]ba(c"[?_ of N ig er D elta of N ig eria . T h e A m eric a n soc iety of T esting a nd M a teria l (A S T M ) rec om m ended sem i-sta tic renew a l b ioa ssa y w a s em ploy ed, a nd L C 5 0 w a s m ea sured a t 96 h ours. M ea n estim a ted 96 h ours L C 5 0 va lues sh ow ed th a t Ba sudin (L c 5 0 -0.860 μg /l) w a s m ore tox ic th a n A tra z ine (L C 5 0 23 0.62 μg /l). D erived L C 5 0 va lues dec rea sed w ith inc rea sed in ex posure dura tion. S ub sta ntia l m orta lity a nd inc idenc es of a b norm a l a voida nc e response oc c urred m ore a t h ig h er c onc entra tions. T h e h ig h sensitivity of 14 da y s old test org a nism to b oth pestic ides provides a ra tiona l for reg ula tory surveilla nc e a nd m onitoring of th e w a ters of th e N ig er D elta ec olog ic a l z one. T h e result ob ta ined is a n indic a tion of ea rly w a rning sig ns of possib le depletion of th e a m ph ib ia n popula tion from pestic ides c onta m ina tion in th e N ig er D elta ec olog ic a l Z ones of N ig eria . dRe(f gZ [mXn]4_`]ba(c"[?_ ; P estic ides; N ig er delta .

Ec olog ic a l effec ts of pestic ides c onta m ina tion on a m ph ib ia n la rva l sta g es w ere a ssessed using a c ute tox ic ity in th e la b ora tory .T h e study c om pa red th e tox ic ity of tw o w idely used pestic ides, A tra z ine (h erb ic ide) a nd Ba sudin (insec tic ide) on th e la rva l sta g es of dom ina nt frog s D D w b b R @ 4 c of N ig er D elta of N ig eria .
T h e A m eric a n soc iety of T esting a nd M a teria l (A S T M ) rec om m ended sem i-sta tic renew a l b ioa ssa y w a s em ploy ed, a nd L C 5 0 w a s m ea sured a t 96 h ours.M ea n estim a ted 96 h ours L C 5 0 va lues sh ow ed th a t Ba sudin (L c 5 0 -0.860 µg /l) w a s m ore tox ic th a n A tra z ine (L C 5 0 23 0.62 µg /l).D erived L C 5 0 va lues dec rea sed w ith inc rea sed in ex posure dura tion.S ub sta ntia l m orta lity a nd inc idenc es of a b norm a l a voida nc e response oc c urred m ore a t h ig h er c onc entra tions.T h e h ig h sensitivity of 14 da y s old test org a nism to b oth pestic ides provides a ra tiona l for reg ula tory surveilla nc e a nd m onitoring of th e w a ters of th e N ig er D elta ec olog ic a l z one.T h e result ob ta ined is a n indic a tion of ea rly w a rning sig ns of possib le depletion of th e a m ph ib ia n popula tion from pestic ides c onta m ina tion in th e N ig er D elta ec olog ic a l Z ones of N ig eria .
d e @f ¢g `h ei kj el : A c ute tox ic ity ; A m ph ib ia n; D 1 1 ¢ b m n R @ 4 c ; P estic ides; N ig er delta .The effect of pesticides such as organophosphates and various organochlorines on amphibian populations are a growing concern (1,2,3).Amphibian populations are declining worldwide, with 48% of rapidly declining species being threatened by processes other than habitat destruction (4,5).These organisms get exposed to pesticides by many routes but perhaps the most lik ely route is agricultural runoff .W idespread pesticide use mak es it increasingly lik ely that nontarget species lik e amphibians get exposed to pesticide contamination (6).W hile pesticides have the potential to affect many aquatic taxa, the impact on amphibians are of particular concern because of the apparent global decline of many species (7,8,9).The list of possible causes of amphibian declines are numerous and pesticides have been implicated in at least some of these declines (10).
Although pesticides are used on a local scale, they are ubiquitous and spread regionally and globally.They have been found in bodies of frogs from areas where pesticide use has not occurred historically or in the past 25 years (11,12,13).Amphibians living with pesticides in their habitats exhibit physiological defects from these pesticides.Recent studies provide evidence that pesticide reduce hatching success, decrease size at metamorphosis, causes physiological stress, liver and k idney degeneration, teratogenic effects, paralysis, to mention a few (14,15,16).
Many amphibians species are vulnerable to aquatic contamination because they experience aquatic and terrestrial stressors, play vital roles in comminutes, are sensitive to contaminants, complete their life cycles near fields where pesticides are applied and have vulnerable embryo and larval stage whose development coincides with pesticides application (6,17).Most pesticides are applied to fields in the beginning of the rainy season which correlates with the time of the year that tadpoles of many amphibian species are developing in surface water (18) which can alter the timing of metamorphosis.If time-tometamorphose is delayed, tadpoles have greater chance of predation in the aquatic environment when terrestrial emergence is postponed (19).
Environmental contaminants that alter the time of metamorphosis could cause potential detrimental consequence in amphibian population and can affect amphibians directly or indirectly causing mortality (20).Examining responses of organisms to lethal concentration of chemical is a reliable measure of contaminant sensitivity (21).Typically this is accomplished using standard mean lethal concentration (LC 50 ) tests, allowing comparison of responses within and among species.H owever, determining sub lethal effects (e.g.Behavioral changes) of a chemical is equally valuable in assessing sensitivity and is a often more favorable endpoint than mortality (22).
The objective of this study is to compare the toxicity of two widely used pesticide, Atrazine a herbicide and Basudin an insecticide used mainly for the control of weeds (Atrazine) and insect pests (Basudin) in most rice, yam, and vegetable farms in Nigeria.The study attempts to determine the acute toxicity of these pesticides (Atrazine and Basudin) on larval stages of the dominant frog species 1 e m ) # A b m of Niger Delta of Nigeria.The study will also serve as early warning signs of possible depletion of the amphibian population by pesticide contamination in the Niger Delta regional zones of Nigeria.
Eggs of the amphibian species -Ý Þ ß cà &á câ cã ä Aå Dâ Åae kç 7ae kè %é ¢å Dç were colleted from an inlet at Ikpoba River, an inland River in southern Nigeria.Hatching of eggs, rearing of tadpoles and testing were done in the post-graduate ecotoxicological research laboratory, at the department of Animal and Environmental Biology, University of Benin, Nigeria.After hatching, emerging larvae tadpoles were distributed into six (22 x 22cm) plastic tanks each containing 1 litre of dechlorinated tap water and were allowed to acclimatize for seven days.Tadpoles were fed ad-libitum daily with ground maize powder.Larvae were reared on a 10: 14h light: dark cycle to mimic natural condition and room temperature were maintained at 30 ± 2 0 c through out the duration of the experiment.Larvae were subjected to concentrations of both pesticides.0, 200, 400, 600 µg/l (Atrazine) and 0, 0.1, 1, 10, 25 µg/l (Basudin).Each treatment concentration contained 20 tadpoles each per container.Acute toxicity for both pesticides was done in replicate tanks.

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The pesticides used for the 96-hour acute toxicity test were the Organochlorine, Atrazine (Atraforce, 80% Top Atrazine) and the Organophosphate, Basudin.Both of these pesticides are commonly used on farms in Nigeria for the control of weeds (Atrazine) and soil insects (Basudin).

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Stock solutions of the required concentrations were prepared for both pesticides.Serial dilutions of 200, 400 and 600 µg/l for Atazine (20).and 0.1, 1, 10, 25µg/l for Basudin (23) were made.The semi-static renewal bioassay procedure started with a range finding test (24).This was used to determine the range of concentrations to be tested and approximate the range that would produce the desired LC 50 effect for the different life stages.A new stock solution for Atrazine was made up every 3 days immediately before each water change because it has a minimum half-life of 48hours in water (25).

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Mortality was recorded at an interval of 24 hours over a period of 4 days (96-hours).Tadpoles were taken dead when they turned upside down and sank to the bottom of the tank or when their tail showed no form of movement even when prodded with a glass rod (26).
ò Éó Ë ¢Ð §Ú ¥× ¢Ñ #Î RÍ |Õ Í eÔ 7î wË ¢Ñ #Ô RÍ Larval avoidance response was monitored in this study.Alternation in avoidance response could increase susceptibility to predation (27,28) and proceed mortality (29).Avoidance response was assessed in-situ by gently prodding all individual larvae and gauging their response as normal when larvae swims away immediately or abnormal when there is a delayed, no response or impaired swimming ability.The avoidance response of larvae 3 hours after herbicide treatment was used for statistical analysis (30).

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The susceptibility of the tadpoles to both pesticides was determined using the probit method of analysis (31) for median lethal concentration LC50 at 96 hours.Computations of confidence interval of mortality rate were also obtained from the probit analyses used to determine the LC50 (Probit software).The two-factor ANOVA (analysis of variance) in Microsoft Excel was used to test the variables at P < 0.05 level of significance.Multiple bar graphs were also used in this study for the pictorial representation of assessment endpoint.

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The results of the acute toxicity of tadpoles exposed to varying concentrations of Atrazine and Basudin pesticides are presented in Tables 1, 2 and 5 and further illustrated in F igures 1 and 2.
Estud.Biol., v. 28, n. 64, p. 91-100, jul./set.2006 No mortality or morphological changes were observed in the controls for the 96-hour acute toxicity test.Tadpoles in the control experiment appeared active and healthy throughout the test period.The proportion of abnormal avoidance response in the control was less than 10%.

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The test organisms exposed to varying Atrazine concentrations at 96-hrs recorded mortality in all the concentrations.The mean % mortality for the 2 weeks old tadpoles of o p 'q r q s t fu 'r exposed to Atrazine were 43%, 80% and 90% for 200, 400, 600 µg/l concentrations respectively.This indicated that mortality increased with increased concentrations (Table 1 and 3; Figure 3).Estud.Biol., v. 28, n. 64, p. 91-100, jul./set.2006 Estud.Biol., v. 28, n. 64, p. 91-100, jul./set.2006 The concentrations also had varying degrees of behavioral alternations in the surviving tadpoles as observed in the avoidance response.The avoidance response was also concentration dependent.In the highest treatment concentration, many tadpoles displaying abnormal avoidance responses at approximately 3 hours post-treatment perished in subsequent days.
¥ I9 % @ ¥D fe q h e AU fa T D 8B 8 H PU 3G Eg 8D fe AR T@ a b@ ¥i p WU %e i T@ QÈU %a ed D 3i TS g 3G £e gf hp )i 1j 3k £j 3l %m n % Wd o p l C @ CD %e qo pq 3l 8j )r wD 3B ES @ ¥i AU 3d WF £a bD %c pG £e @ QD %e g !d D 3i TS g 3G se t@ PV 8p WU 8i TS ua b@ QD 8F #F £v #@ Q@ Pe g !U 8#w 8x W v #U 3S a bi y G Ei zR TS i Ti TG EU fe Until recently, the adverse effects of pesticides on non-target organisms have not been seriously considered in Nigeria, and toxicological studies with amphibians are relatively limited in number (32).Consequently, limited data on the toxicity of pesticides to amphibian larvae of r fs u tv su tx Py C {v are available for comparison with our results.The results of the experiment demonstrated that pesticides can have adverse direct (mortality) and indirect (physiological) effects on tadpoles.
The less sensitivity of C ¶ •© t s© bª ¦ C ¸ to Atrazine may be due to species differences in susceptibility.(38,35).Larval death occurred most rapidly in the highest concentration of Basudin indicating that short -term exposure to this concentration would be more detrimental to this amphibian species.The mortality of larvae could be explained by bioconcentration of these agrochemicals.Each of the two focal compounds in our study has been reported to bio concentrate in amphibian tadpoles (34,39,40).This is an issue of serious ecological consequence.Larval mortality occurred more during exposure to high concentrations of Atrazine and Basudin than the controls, suggesting that death may have been due to the presence of the pesticides.Pesticide exposure induced abnormal avoidance response, skittishness and possible nervous system malfunction.Motor disorders in animals exposed to Atrazine and most organophosphate compounds like Basudin have been linked to their toxic effects in the Central Nervous System (35,41).Most organophosphates like Basudin have been found to concentrate in tissues of frogs with depressed cholinesterase activity (42) and induce hyper activity in frogs followed by paralysis (35).

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This study has unequivocally demonstrated that exposure of amphibian tadpoles to pesticides (Atrazine and Basudin) resulted in concentration dependent mortality and abnormal avoidance response.Atrazine (Herbicide) and Basudin (Insecticide) usage in the Nigerian Niger Delta is a common practice that often lead to the contamination of water bodies with pesticides through storm water runoffs.A sustainable biomonitoring program for pesticide contamination of the aquatic environment is advocative for the Niger Delta ecological zone.This program will enhance the protection of the observed amphibian decline in this sensitive ecological region of Nigeria.

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