¤§ ¦    ¤  ¤§¦    ¦  ¦©  "!# $%   ¦ ¨   ¦ ¢ !¢'&( ) *+, ¨©  ¦ &( ) *%  £¥¤§¦©¨ ¦ ¤§¨ (P ty c h a dena b ib roni)

ECOLOGICALEFFECTSOFPESTICIDESCONTAMINATION ONAMPHIBIANTADPOLES(Ptychadenabibroni) OFTHE NIGERDELTAOFNIGERIA

Efeitosecológicosdacontaminaçãoporpesticidasemgirinos deanfíbios(Ptychadenabibroni)nodeltadoRio

NigernaNigéria

-.0/2143)5,63879¢3):<;¢5>=?3A@

BC D;¢:<.EBF 3)6G?D3)H IKJMLNOPRQN

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 U,VWMX>Y>Z[\X^]4_`]ba(c"[?_ Ba sudin (insec tic ide) on th e la rva l sta g es of dom ina nt frog s S,T 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"[?_ : A c ute tox ic ity ; A m ph ib ia n; S,T ; P estic ides; N ig er delta .

1 R esea rc h ers from D epa rtm ent of A nim a l a nd Environm enta l Biolog y (A EB) U niversity of Benin, Benin C ity , N ig eria . + 23 4 80 23 3 5 3 847 ,080 5 0261609. 2 R esea rc h ers from D epa rtm ent of A nim a l a nd Environm enta l Biolog y (A EB) U niversity of Benin, Benin C ity , N ig eria . + 23 4 80 23 3 5 3 847 ,080 5 0261609. C orresponding A uth or: ez em sla w @ y a h oo.c om , isq ua red27 @ y a h oo.c om

Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006 ¢¡

q'rs)t"u vw§u'x4yu z|{ vA}'u~ € { z|r>  uw ‚#ƒu œ^>žŠ‘Ÿ˜ˆ

O s efeitos ecoló gicos da contaminaç ã o por pesticidas em anfíbios no está gio larval foram avaliados usando-

se toxicidade aguda em laborató rio. O estudo comparou a toxicidade de dois pesticidas, Atrazine (herbicida) ¢ £¤ ¥¢¦>§¨\¥8©bª`©4«(¬"¨?ª e Basudin (inseticida) sobre o está gio larval de sapos ,¡ , comuns no delta do rio Niger, na Nigé ria. Para aplicaç ã o de pesticidas, a sociedade Americana de Testes e Materiais (SATM) recomenda uma

aplicaç ã o semi-está tica para posterior verificaç ã o de atividade bioló gica, sendo a CL50 medida apó s 96

horas. V alores mé dios estimados da CL50 nas 96 horas mostraram que Basudin (CL50-0,860 µg/l) era mais

tó xico que Atrazine (CL50 230,62 µg/l). V alores da CL50 diminuíram com o aumento da duraç ã o de exposiç ã o. A mortalidade e incidentes anormais ocorreram somente nas concentraç õ es maiores. A alta sensibilidade das formas larvais de 14 dias dos anfíbios, para ambos os pesticidas, demonstra a capacidade bioindicadora desses organismos aquá ticos, apontando assim para inspeç õ es regulares e monitoramento das á guas do delta do Niger. O resultado obtido é um alerta sobre o risco de uma possível diminuiç ã o da populaç ã o de

anfíbios, por contaminaç ã o de pesticidas, nas zonas ecoló gicas do delta do rio Niger, na Nigé ria.

­®(¯%®(°±'®(²¢³©´µ¢®(°¥¶ ¢ £§¤M¥>¦>§¨\¥n©bª"©b«(¬"¨ª

: Toxicidade aguda; ,¡ ; Anfíbios; Pesticidas; Delta do Niger. „4 ^†‡ˆR‰MŠ\‹†4Œ4ˆ¢ and have vulnerable embryo and larval stage whose development coincides with pesticides application The effect of pesticides such as (6, 17). Most pesticides are applied to fields in the organophosphates and various organochlorines on beginning of the rainy season which correlates with amphibian populations are a growing concern (1, the time of the year that tadpoles of many amphibian 2, 3). Amphibian populations are declining species are developing in surface water (18) which worldwide, with 48% of rapidly declining species can alter the timing of metamorphosis. If time-to- being threatened by processes other than habitat metamorphose is delayed, tadpoles have greater destruction (4, 5). These organisms get exposed to chance of predation in the aquatic environment when pesticides by many routes but perhaps the most terrestrial emergence is postponed (19). lik ely route is agricultural runoff .W idespread Environmental contaminants that alter the pesticide use mak es it increasingly lik ely that non- time of metamorphosis could cause potential target species lik e amphibians get exposed to detrimental consequence in amphibian population pesticide contamination (6).W hile pesticides have and can affect amphibians directly or indirectly the potential to affect many aquatic taxa, the impact causing mortality (20). Examining responses of on amphibians are of particular concern because organisms to lethal concentration of chemical is a of the apparent global decline of many species (7, reliable measure of contaminant sensitivity (21). 8, 9).The list of possible causes of amphibian Typically this is accomplished using standard mean declines are numerous and pesticides have been lethal concentration (LC50) tests, allowing implicated in at least some of these declines (10). comparison of responses within and among species. Although pesticides are used on a local H owever, determining sub lethal effects (e.g. scale, they are ubiquitous and spread regionally Behavioral changes) of a chemical is equally and globally. They have been found in bodies of valuable in assessing sensitivity and is a often more frogs from areas where pesticide use has not favorable endpoint than mortality (22). occurred historically or in the past 25 years (11, The objective of this study is to compare 12, 13). Amphibians living with pesticides in their the toxicity of two widely used pesticide, Atrazine habitats exhibit physiological defects from these a herbicide and Basudin an insecticide used mainly pesticides. Recent studies provide evidence that for the control of weeds (Atrazine) and insect pests pesticide reduce hatching success, decrease size (Basudin) in most rice, yam, and vegetable farms at metamorphosis, causes physiological stress, liver in Nigeria. The study attempts to determine the and k idney degeneration, teratogenic effects, acute toxicity of these pesticides (Atrazine and

paralysis, to mention a few (14, 15, 16). Basudin) on larval stages of the dominant frog ‘’R“R”R•A–m“˜—™—š)›>–m™ Many amphibians species are vulnerable to species  Ž of Niger Delta of aquatic contamination because they experience Nigeria. The study will also serve as early warning aquatic and terrestrial stressors, play vital roles in signs of possible depletion of the amphibian comminutes, are sensitive to contaminants, complete population by pesticide contamination in the Niger their life cycles near fields where pesticides are applied Delta regional zones of Nigeria.

o>p Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006

þ þý÷§ÿ ø þ¢¡ý ÿ û÷ û¤£ý ÷§ø¦¥ÿ ü¨§ û ¥üÿ ûø¦¥ ø©¥ ü¨§ ¡ #û¤ %ûü¨¥ ÿ ü¢£¡ø ù ý ø þ¢ÿ ¢ý(û ú)ý ,ý ù©ÿ ü ø þ¢(û ú)ý %ûü

ö¥÷§ø©ù ø ú û÷§üùý (Ptychadena bibroni) ¹»ºM¼4½'¾¿4ºMÀ4Á^º¢Â^ÃÅÄƽk¼ÇÉÈRà water change because it has a minimum half-life

of 48hours in water (25).

ʢ˥̧ÌÍAÎ4ÏЧ˥ÑÒË¢ÓRÏÍAÔbÏRË¢ÕÖk×¢ÑЧÔbØÙÔm×¥Ñ?Ú ×¢Î4Îb̧ЧØÛץϧЧÜ)×¢ÏЧˢÑ

Ë¥Õ4Ïע̧ÐÏ íÙÕb×¢ÏÍ

Eggs of the amphibian species - ñ ß?àá?â?ã‘ä)å,âÅækç%ækèé¢å,ç ÝRÞ were colleted from an inlet at Ikpoba River, an inland River in southern Nigeria. Mortality was recorded at an interval of Hatching of eggs, rearing of tadpoles and testing 24 hours over a period of 4 days (96- hours). were done in the post-graduate ecotoxicological Tadpoles were taken dead when they turned upside research laboratory, at the department of Animal down and sank to the bottom of the tank or when and Environmental Biology, University of Benin, their tail showed no form of movement even when Nigeria. After hatching, emerging larvae tadpoles prodded with a glass rod (26). 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. òÉó ˢЧڥעÑÎ4Í|ÕbÍAÔ%îMË¢ÑÔ4Í Tadpoles were fed ad-libitum daily with ground maize powder. Larvae were reared on a 10: 14h Larval avoidance response was monitored light: dark cycle to mimic natural condition and in this study. Alternation in avoidance response room temperature were maintained at 30 ± 20c could increase susceptibility to predation (27, 28) through out the duration of the experiment. Larvae and proceed mortality (29). Avoidance response were subjected to concentrations of both pesticides. was assessed in-situ by gently prodding all 0, 200, 400, 600 µg/l (Atrazine) and 0, 0.1, 1, 10, 25 individual larvae and gauging their response as µg/l (Basudin). Each treatment concentration normal when larvae swims away immediately or contained 20 tadpoles each per container. Acute abnormal when there is a delayed, no response or toxicity for both pesticides was done in replicate impaired swimming ability. The avoidance response tanks. of larvae 3 hours after herbicide treatment was used

for statistical analysis (30).

êɽ>Á¼4¿4ë¿4ÃM½kÁ

ôAÏעϧÐÔ4ϧÐÎ4×¥ÌM×¥Ñ?×¥Ì íkÔ4ЧÔ

The pesticides used for the 96-hour acute toxicity test were the Organochlorine, Atrazine The susceptibility of the tadpoles to both (Atraforce, 80% Top Atrazine) and the pesticides was determined using the probit method Organophosphate, Basudin. Both of these pesticides of analysis (31) for median lethal concentration are commonly used on farms in Nigeria for the control LC50 at 96 hours. Computations of confidence of weeds (Atrazine) and soil insects (Basudin). interval of mortality rate were also obtained from the probit analyses used to determine the LC50

(Probit software).The two-factor ANOVA (analysis ÐË¢×¥Ô4Ô4×#í|îMÕbË¢Îbï¢Í)Ú¢ðÕ4Í ì of variance) in Microsoft Excel was used to test the variables at P < 0.05 level of significance. Multiple Stock solutions of the required bar graphs were also used in this study for the concentrations were prepared for both pesticides. pictorial representation of assessment endpoint. Serial dilutions of 200, 400 and 600 µg/l for Atazine

(20). and 0.1, 1, 10, 25µg/l for Basudin (23) were Í)Ô4ð?ÌÏ§Ô made. The semi-static renewal bioassay procedure õ started with a range finding test (24). This was used to determine the range of concentrations to be The results of the acute toxicity of tadpoles tested and approximate the range that would exposed to varying concentrations of Atrazine and produce the desired LC50 effect for the different Basudin pesticides are presented in Tables 1, 2 life stages. A new stock solution for Atrazine was and 5 and further illustrated in F igures 1 and 2. made up every 3 days immediately before each

Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006 ·¢¸

vw¢xEy z {}|bz~DWz €Y¦{W‚z¨ƒ¤„ †¦€Yw„ ‡¦z| ˆD‰¤z

 ! #"%$&(')+*-,/.10324)654742 89.6:;24)6<1=;.654'>?'@6=;.A>B'!.6:6H;2¤03)1IJ7¤*!'

=;'>B2474FB74

TG CU%$&(')+*-,/.10324)654742 89.6:;24)6<1=;.A54'>!'E@6=V.6>B'!.A:YXT)6>BZT<67¤*

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\+]1^`_bac]+d e6_¨a4f+gEhb^3iYikj1l6]1m4n3a4i

No mortality or morphological The test organisms exposed to varying Atrazine changes were observed in the controls for the concentrations at 96- hrs recorded mortality in all the

96-hour acute toxicity test. Tadpoles in the control concentrations. The mean % mortality for the 2 weeks qsctBur experiment appeared active and healthy old tadpoles of oWpqr exposed to Atrazine were throughout the test period. The proportion of 43%, 80% and 90% for 200, 400, 600 µg/l concentrations abnormal avoidance response in the control was respectively. This indicated that mortality increased with less than 10%. increased concentrations (Table 1 and 3; Figure 3).

 Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006

¾ ¾½·b¿ À3¸¦¾¢ÁB½ À†¿ »¤· »¤Â¢½ À3·b¸¦Ã}¿ ¼¨Ä » Ã}¼¢¿ »¤¸¦ÃG¸©ÃG¼¨Ä Á ÅD»¤Æ[»¤¼¨ÃG¿ ¼¢ÂÁB¸¦¹¦½ À ¸¦¾+¿ Å+½Ç » ºE½ ÈÉJ½ ¹©¿ ¼ ¸¦¾¢Ç » ºE½ È[»¤¼

¶1·b¸©¹¦¸¦º†»¤·b¼¨¹½ (Ptychadena bibroni)

Œ+VŽJ#‘%’“(”•+–-—/˜1™3š4•6›4œ4š 9˜6ž;š4•6Ÿ1 ;˜6›4”¡?”¢6 ;˜A¡B”ŸCš4˜CŸAœ4ž4ž4”E™3”E–GšY£B˜1–!£B”–!š¤™3•AšDœ4˜1–!¡!˜6ž6;š¤™}•+¤Jœ¤–G”

;”¡Bš4œ4£Bœ4Ÿ6”L¥ ¦6§V’ ¨;˜A©™3¡ ª«¥¦ N¬L­6®[­6¯1ª

Œ+VŽJ#Ê%’“(”•+–-—/˜1™3š4•6›4œ4š 9˜6ž;š4•6Ÿ1 ;˜6›4”¡?”¢6 ;˜A¡B”ŸCš4˜CŸAœDž4ž4”™3”E–!š;£B˜1–!£B”E–Gš¤™3•6š4œ4˜1–!¡!˜6ž;Ž•A¡B©ŸAœ¤–

;”¡Bš4œ4£Bœ4Ÿ6”L¥ ¦6§V’ ¨;˜A©™3¡ ª Œ+VŽJ#°%’Y±#;²1–!˜+™B—9•6›;•6Ÿ;³˜6œ4Ÿ6•1–!£B”´™3” ;˜+–G¡B”µž4˜+™!;š¤™3•1¤Jœ¤–!”

Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006 Š+‹

ôõ¢öE÷ ø ù}úbøûDüWø ýYþ¦ùWÿø¡ £¢ ¤†þ¦ýYõ¥¢ ¦¦øú §©¨¤ø

The concentrations also had varying Í`Î1Ï4Ð3Ñ+ÒbÓÕÔkÖ1×6Ø1Ï4Ð`ÙcÔ degrees of behavioral alternations in the surviving tadpoles as observed in the avoidance response. Mean % mortality increased also with The avoidance response was also concentration increased concentrations for Basudin pesticide dependent. In the highest treatment concentration, exposure (Table 2 and 4; figure 4). The mean % many tadpoles displaying abnormal avoidance mortality were 35%, 58%, 68%, 75% for 0.1, 1.0, responses at approximately 3 hours post-treatment 10.0 and 25µg/l concentrations respectively. perished in subsequent days. Avoidance response was similar to Atrazine and

was also concentration dependent.

Ú+ÛVÜÝJÞ#ßáà;âCÛVã+äGå+æBç/èAéYèAê;ëEåAì4ê6è1ä!íBî´æ3îï ðVå+äGïBîµñ4å+æòÜèAïBóêAì¤ä

¥ !Þ%à â(îè+ä-ÛVã+ä!å1æBç/è6é6Û ëå6ì4ê6è1ä!íBî´æ3îï ð;å1ä!ïBîµñ4å+æ!Û¤æ}èJì¤äGî¦ð ! £!"#

ËÌ Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006

‡ ‡s†s~€ˆ ‰>h‡|Š6† ‰Mˆ „£~ „£‹|† ‰>~€hŒJˆ ¡@„ ŒJ |ˆ „£hŒW‚ŒW ¡@ŠcŽ©„£„£ ¡ŒWˆ |‹sŠ6hh† ‰ h‡ˆ Ž†’‘„ ƒ2† “1”p† ‚ˆ @h‡|‘„ ƒ2† “„£

}B~€‚hhƒM„£~€ ¡1† (Ptychadena bibroni)

D

Q¥R SUT1'WVYX[Z\](¥,BAG^ A)7B=6_`,&*&^a027!/-C&,BA)46(b=>(¥EcF97AWE6(3<-7=ed,!E65C!/£AgfhFi j!k£j!lm

')(+*-,&.-/102(3,!465.-(3.-789/-46/-. :;7&<,&.£=>,?@/£A)(3,BA)C

more toxic than Atrazine (230.621µg/l) to

D ,!E65C!/£AGF(+E6.-/-46/-C&(¥E

tadpoles of HJI K LMK N6O>PL . Derived LC50 values for 24,48,72 hours followed a similar trend as On the basis of the derived 96-hrs described for 96 - hrs values of the test

LC50, Basudin (0.860µg/l) was found to be compounds (Table 5).

n^9d opVl[Z+\](+,AGopq!l&jrM,!*-5(¥E)7!<@^9.£=>,?p/£AW(3,AWCd,!E65C!/sAt(28&F97&E65u=>(3,&..£v(3(2AWC7&<w&x9Z v7!5 =>E yb/-Ez465E6E6/-7BA

KJLsKJN2O+P{L are available for comparison with our results. The results of the experiment demonstrated Until recently, the adverse effects of that pesticides can have adverse direct (mortality) pesticides on non-target organisms have not been and indirect (physiological) effects on tadpoles. seriously considered in Nigeria, and toxicological Examination of our results indicates that studies with amphibians are relatively limited in both pesticides – Atrazine and Basudin varied

number (32). Consequently, limited data on the greatly in their effects on survival and behaviour HBI!K¥L£K+N|OPpL toxicity of pesticides to amphibian larvae of HBI of . The highest mortality was found at

Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006 $&%

ŒÆ|Ç2ÈcÉ ÊJˀɒ̩ÍzÉ Î³ÏhÊzÐ’É¡Ñ£Ò ÓMÏhÎ³Æ¥Ò ÔhÉsË Õ©Ö£É ¹@º+»)¼½©¾9¿À©º+» the highest concentrations, suggesting dose – dependent survival and concentration graded response. Basudin significantly induced mortality recording This study has unequivocally demons- complete mortality at the highest treatment trated that exposure of amphibian tadpoles to concentrations within 96 – hours while Atrazine had pesticides (Atrazine and Basudin) resulted in

the least adverse lethal effects. Mortality rates were concentration dependent mortality and abnormal š œ‚©™!ž

comparable to those reported for —>˜J™!˜›š œ and avoidance response. Atrazine (Herbicide) and

˜¥¢£œ‚¤ ˜¥™&ž —>˜J™!˜ Ÿ’¡ exposed to similar concentrations of Basudin (Insecticide) usage in the Nigerian Niger Basudin (23). Atrazine affected larval survival in this Delta is a common practice that often lead to the study but mortality was not as significant as for Basudin. contamination of water bodies with pesticides Atrazine did not affect larval survival in gray tree frogs through storm water runoffs. A sustainable (33) Northern leopard frogs (34) and American toads biomonitoring program for pesticide

(35). However, significant lower survival was reported contamination of the aquatic environment is ¦¢£˜3©B˜Jª«©B¦u¬1ª«œ

in streamside salamanda (¥@¢£¦¨§Jž¡¤ ) and advocative for the Niger Delta ecological zone.

°¬1¯‚¦U˜¥¢£|ª«œ Ÿ2˜¥™9¬1ž

spring peepers (­>ž¡B¬B®¥˜JŸ’ª«œ‚žŸ’ªz¬1Ÿ’œ‚¯‚©ª , This program will enhance the protection of the

˜¥¢²œ‚¤ ˜¥™!ž —>˜¥™!˜³ž §¥¡s´€˜¥¤ œ Ÿ’˜ —>˜¥™ ˜±Ÿ’¡ , ) at low concentrations observed amphibian decline in this sensitive of 3ppb and 4ppb respectively (36, 37). At higher ecological region of Nigeria.

concentrations such as 200 – 600µg/l, Atrazine ˜JB´€œ‚ž

produced mortality on µ±©™!¦¨š¬1ž ¡ as reported by

»)¼ ¿

Á)Â¥Ã-’Ä>  Freeman and Rayburn (20) and is consistent with our study.

The less sensitivity of ­+¶·©Jœs©>ª’¦+™¸œ to 1. Bishop CA. The effects of pesticides on Atrazine may be due to species differences in amphibians and the implications for susceptibility. (38, 35). Larval death occurred most determining the causes of decline in amphibian rapidly in the highest concentration of Basudin population. In: Bishop CA, Pettit K E, editors. indicating that short – term exposure to this Decline in Canadian amphibian population: concentration would be more detrimental to this designing a natural monitoring strategy. Ottawa amphibian species. The mortality of larvae could on: Canadian wildlife Service Occasional paper; be explained by bioconcentration of these 1992. agrochemicals. Each of the two focal compounds 2. Hall RD, Henry PFP. Review Assessing effects in our study has been reported to bio concentrate of pesticides on amphibians and reptiles: Status in amphibian tadpoles (34, 39, 40). This is an issue and needs Herpetol J. 1992; 2:65-71 of serious ecological consequence. Larval mortality occurred more during exposure to high 3. Phillips K . Tracking the vanishing frog: an concentrations of Atrazine and Basudin than the ecological mystery. New Y ork: St. Martin’s; 1994. controls, suggesting that death may have been 244 p. due to the presence of the pesticides. Pesticide exposure induced abnormal avoidance response, 4. Colborn T, Clement C. Chemically induced skittishness and possible nervous system alternations in sexual and functional malfunction. Motor disorders in animals exposed development: the wildlife human connection. to Atrazine and most organophosphate Princeton NS: Princeton Scientific; 1992. 403 p. compounds like Basudin have been linked to their 5. Stuart SN, Chnason JS, Cox NA, Y oung BE, toxic effects in the Central Nervous System (35, Rodrigues ASL, Fischman DL. Status and trends 41). Most organophosphates like Basudin have of amphibian declines and extinctions been found to concentrate in tissues of frogs with worldwide. Science. 2004; 306:1783-1786. depressed cholinesterase activity (42) and induce hyper activity in frogs followed by paralysis (35). 6. Blaustein AR, K iesecker JM, Complexity in conservation: lessons from the decline of amphibian population. Ecol Lett. 2002; 5:597- 608.

•+– Estud. Biol., v. 28, n. 64, p. 91-100, jul./set. 2006

ø øs÷sñ€ù ú>òhø|û6÷ úMù õ£ñ õ£ü|÷ ú>ñ€òhýJù ö¡þ@õ ýJö|ù õ£òhýWò‚ýWö¡þ@ûcÿ©õ¡ õ£ö¡ýWù ö|üsû6òhóh÷ ú òhøù ÿ÷£¢õ ô2÷ ¤¦¥p÷ ó‚ù ö@òhø§¢õ ô2÷ ¤õ£ö ðBñ€ò‚óhòhôMõ£ñ€ö¡ó1÷ (Ptychadena bibroni) 7. Blaustan AR, Wake DB. Declining amphibian 18. Rayburn AL, Norcott CA. Flow cytometric populations: a global phenomenon? Trends in monitoring of aquatic ecosystems subjected to Ecology and Evolution. 1990; 5:203-204. periodic flooding Illinous G roundwater Consorhum Research on Agricultural Chemicals 8. Alford RA, Richards SJ. G lobal amphibian declines: in Illinois of G roundwater: Status and future a problem in applied ecology. Annual Review of Directions viii. Proceeding of Eight Annual Ecology and Systematics. 1999; 30:133-165. conference; 1998 April 1-2; USA: Makanda, Il; 9. Holihan JE, Findlay CS, Schmidt BR, Meyers 1998. p. 70-82. A.H, Kuzmin SL. Q uantitative evidence for 19. Werner EE. Amphibian metamorphosis: G rowth global amphibian population declines. rate, predation risk, and the optimal size at Nature. 2001; 404:752-755. transformation. Am Nat. 1986; 128:319-341. 10. McConnell LL, Le Noir JS, Datta S, Seiber JN.

20. Freeman JL, Rayburn AL. 2004 .Developmental Ûàá

Wet deposition of current – use pesticides in Ý Úpß

impact of Atrazine on metamorphing Þ â

the Sierra Nevada mountain range. California, ÝJã-Üsá²äUá

Ù revealed by nuclear analysis and USA: Environmental Toxicology and morphology. Environmental Toxicology and Chemistry. 1998; 17:1908-1916. Chemistry. 1986; 7 :1648-1653. 11. Datta S, Hansen L, McConnell L, Baker J, Le 21. Bridges CM. Tadpoles swimming performance Nior J, Seiber JN. Pesticides contaminants in and activity affected by exposure to Sublethal fish and tadpoles from the Kaweah River levels of carbaryl. Arch Environ contain Toxicol. basin, California. Environ Contam Toxicol. 1997; 16:1935-1939. 1998; 60:829-836. 22. Little EE, Archeski RD, Flerov BA, Kozlovskaye 12. Russell RW, Hecnar SJ Haffner G D. VI. Behavioural indicators of sublethal toxicity Organochlorine pesticides – residues in Ontario in rainbow trout. Arch Environ. contain. Toxicol. spring peepers. Environ Toxicol Chem. 1995; 1990; 19:380-385. 14:815-817. 23. Harris ML, Bishop CA, Strugers J, Ropley B, 13. Russell RW, G illan KA, Haffer G D.

Bogart JP. The functional integrity of

Û{Ü¡Ý á

Polychlorinated biphenyls and chlorinated Ü Ú

Northern leopard frog ( ØpÙ&Ú¸Ùæå ) and á

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