FROGLOG - Amphibian Specialist Group

Population and Health of common toads across ag landsContinued from previous pageMid-west region of Entre Ríos Province.It presents the biggest expansionof agricultural lands and fragmentationof the landscape. Tensoybean fields and two control siteswere sampled for larvae and adultsduring breeding events (coincidentwith period of soybean cultivation).During the day, surveys were conductedto detect anuran egg massesand tadpoles, while night surveysincluded active searches to detectnon-calling anurans and listeningfor calling male anurans, followingthe guide by Heyer et al. (1994).Individual adults were transportedin buckets to Ecotoxicology Lab ofFBCB-UNL for laboratory analysis.Each individual was anesthetized usingthe guide by ASIH et al. (2001),sexed, measured for snout-ventlength (SVL; to nearest mm) with acaliper and mass (g) with a balance.Adult malformations were analyzedfollowing the descriptionsdetailed in the field guide to malformationsof frogs and toads (Meteyer,2000). To take representativesamples and comparable data weused adult of three bufonid speciesfor this parameter. Samples of blood(ca. 300-500 µL) were extracted inanesthetized adult animals by cardiacpuncture using a small heparinizedneedle, previously locating the heartvia palpation. The anurans then werereleased in the same sites in whichthey were captured. Plasma BChEactivity was determined colorimetricallyby the method of Ellman etal. (1961) with some modifications(Sánchez-Hernández & Moreno Sánchez,2002). Hematocrit, hemoglobinconcentration, mean of cell hemoglobin,mean cell volume, meancell hemoglobin concentrations,red blood cell, and white blood cellwere determined for each toad speciesfollowing the protocols of Cabagnaet al. (2005).For micronuclei (MN) testswe conducted field experimentswith Scinax nasicus (Anura, Hylidae)tadpoles caged in agriculturalponds, because this species is notgregarius and is larger than Chaunustadpoles. Three replicates ofeach in situ chamber (ten tadpolesof similar Gosner developmentstage (st. 37) were located in eachpond under plant shade). Samplesof blood was extracted in anesthetizedtadpoles by cardiac puncture.Two peripheral blood smearsfor each tadpole were prepared onclean slides, fixed and stained bythe May-Grunwald-Giemsa method.The MN frequency was determinedin 1,000 erythrocytes fromeach tadpole using 1,000 X magnification.Other alterations of theerythrocytes were also registered.Relative abundance.The most common toad speciesobserved was Chaunus fernandezae,recorded in 65% of the total pondssampled surrounded by or withinsoybean croplands, followed by C.schneideri (21%) and C. arenarum(14%). Tadpoles and eggs in jellystrand were recorded for the threetoad species in reference sites, butevidence of reproduction was onlyobserved for C. fernandezae in pondssampled surrounded by sor withinsoybean croplands. The proportionof males and females of C. fernandezaedid not differed signifcantlyamong pond samples (agriculturalsites and controls). The proportionof sexes of C. arenarum and C. schneiderivaried significantly amongagricultural ponds and referenccesites, being biased towards malesin the former.We found upper eye-lid (n = 1),hindlimb (n = 3), and forelimb (n= 3) malformations in toad specimens(total specimens review n =124) collected across agriculturalsites of the Mid-west Entre RíosProvince. Moreover, similar malformationswere recorded in othernative species (Leptodactylus ocellatus,L. chaquensis, L. gracilis, Physalaemusbiligonigerus) that commonlyoccur in agroecosystemsalongside the three study species.Cholinesterase analysis and haematologicalanalysis:The mean values of plasma BChEactivity varied from 1 µmol min-1ml-1 (C. schneideri at agriculturalsites) to 17.31 µmol min-1ml-1(C. fernandezae at references sites).BChE activity was statisticallylower in the amphibians collectedfrom the agricultural sites thanthose from the reference site (KStest for the three species P < 0.05).Moreover, the differences in theactivity of BChE were significantamong the three study species (P< 0.05). In addition, the increasein plasma BChE was inverselyproportional to the body weight ofthe three anurans (r = - 0.85; P 0.01), in all caseslower values in agricultural siteswere recorded for each haematologicalparameter.The erythrocystes of caged tadpoles in natural sites were oblongovalshaped with centric nuclei.5