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Genetic, enzymatic and developmental alterations observed in Caiman latirostrisexposed in ovo to pesticide formulations and mixtures in an experimentsimulating environmental exposure

Gisela L. Poletta a,b,c,n, Elisa Kleinsorge b, Adriana Paonessa b, Marta D. Mudry c,Alejandro Larriera a,d, Pablo A. Siroski a

a ‘‘Proyecto Yacare’’ - Laboratorio de Zoologıa Aplicada: Anexo Vertebrados (Facultad de Humanidades y Ciencias, Universidad Nacional del Litoral/Ministerio de Aguas, Servicios

Publicos y Medio Ambiente), CP 3000, Santa Fe, Argentinab Catedra de Toxicologıa y Bioquımica Legal, Facultad de Bioquımica y Ciencias Biologicas, Universidad Nacional del Litoral, CP 3000, Santa Fe, Argentinac Grupo de Investigacion en Biologıa Evolutiva (GIBE), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, CONICET, C1428EGA, Buenos Aires, Argentinad Catedra de Manejo de Flora y Fauna Silvestre, Facultad de Humanidades y Ciencias, Universidad Nacional del Litoral, CP 3000, Santa Fe, Argentina

a r t i c l e i n f o

Article history:

Received 21 June 2010

Received in revised form

4 November 2010

Accepted 5 December 2010Available online 24 December 2010

Keywords:

Crocodilians

Caimans

Pesticides

Field-like exposure

Genotoxicity

Enzymatic alterations

Metabolic disorders

Growth delay

a b s t r a c t

In South America, economic interests in last years have produced a constant increase in transgenic

soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was

to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman

latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental

exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were

Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed

with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated

genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to

Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of

different biological meaning is highly informative as early indicators of contamination with pesticides

and mixtures in this wildlife species.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

Current agricultural related practices affect natural ecosystemsthrough their conversion (habitat destruction) into agriculturallands and through the utilisation of agrochemicals, which subse-quent spread and runoff contaminate surrounding natural habitats(Johanson, 2004; Peruzzo et al., 2008). Agriculture expansion haslead to increased fragmentation of habitat due to deforestation anda great degradation of remnant ecosystems, with deep conse-quences for biodiversity. The use of pesticides formulated products,which are complex and variable chemical mixtures, has beenincreasing worldwide. It is estimated that pesticides reduce lossesin soybean, maize and wheat crop to a 10–15%, allowing higheryields in food production. However, only a little amount ofpesticides applied in agriculture reach target organisms directly,

while the rest disperse through the environment, affecting wildflora and fauna populations of surrounding natural areas (Donald,2004). Long-term, low level chronic exposure to chemicals mayinterfere with development and growth, haematological andphysiological parameters and genetic stability of organisms livingthere (Glusczak et al., 2006).

In Argentina, economic interests in the last years has produced aconstant increase in transgenic soybean single-cropping (glypho-sate resistant), with the corresponding rise in pesticide use,including mainly the herbicide glyphosate (GLY) and the insecti-cides endosulfan (ES) and cypermethrin (CPT) (Table 1; EXTOXNET,updated 1996). In the last season, soybean cropping rose to morethan 18 million ha cultivated and 200 million liters of pesticidesreleased to the environment, with 170 millions corresponding onlyto glyphosate-based formulations (Bolsa de cereales de BuenosAires, updated 2010; CASAFE, updated 2010). More than 50% oftransgenic soybean cultivated in the last decade has extended overareas which corresponded previously to native forest (Parueloet al., 2006).

In north-central region of Argentina, many natural environ-ments where the broad-snouted caiman (Caiman latirostris) live arein the proximity of regions with high agricultural activity, where

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journal homepage: www.elsevier.com/locate/ecoenv

Ecotoxicology and Environmental Safety

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.ecoenv.2010.12.005

n Corresponding author at: Catedra de Toxicologıa y Bioquımica Legal, Facultad de

Bioquımica y Ciencias Biologicas, Universidad Nacional del Litoral, Ciudad

Universitaria, Paraje El Pozo C.C. 242, CP 3000, Santa Fe, Argentina.

Fax: +54 342 4575221.

E-mail addresses: gpoletta@fbcb.unl.edu.ar,

gisepoletta@yahoo.com (G.L. Poletta).

Ecotoxicology and Environmental Safety 74 (2011) 852–859

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GLY, ES and CPT are extensively used as a method for pest control insoybean crops. In addition, they are extensively applied in the sameperiod of the year in which C. latirostris breeding season takes place,implying a contamination risk particularly important for develop-ing embryos and neonates (Poletta et al., 2009).

Pesticides tend to be very reactive compounds that can trigger awhole cascade of biological responses in an organism, each of whichmay, in theory, serve as a biomarker (Mitchelmore et al., 2005). Theycan form covalent bonds with different nucleophilic centres of cellularbiomolecules, and may induce reactive oxygen species (ROS) formationleading to protein, lipids, and DNA damage (Limon-Pacheco andGonsebatt, 2009). Genotoxic agents usually disrupt normal cellularprocesses and can result in direct interactions of the toxic agent withDNA, inducing structural modifications in it or the associated machin-ery (Novillo et al., 2005). Various pesticides have also been shown toinhibit or enhance many endogenous enzyme activities and metabolicparameters (Becker et al., 2009; Begun, 2007; Glusczak et al., 2006;Jiraungkoorskul et al., 2003). Enzymes are of great value for diagnosisconsidering the precocity of their variation, rather than the specificity oftheir tissular origin. All these alterations may result in malfunction ofnormal cellular physiology at individual level, with possible long-termconsequences depending on the severity of the damaged produced, andaltered genotypic diversity as well as decreased reproductive success atpopulation level (Acevedo-Whitehouse and Duffus, 2009; Mitchelmoreet al., 2005).

To predict environmental effects of chemical substances, ana-lyses use mostly data obtained from laboratory tests. However,natural ecosystems are more complex and variable than laboratorystandardised systems. Therefore, toxicity bioassays done in thelaboratory should be complemented with higher tier assessmentconducted in field-like scenarios. A major strength of field-likestudies is the incorporation of more realistic exposure regimes thatallow a better understanding of the biological effects of chemicalunder natural conditions (Graney et al., 2003). In previous studiesunder laboratory controlled conditions, we have demonstratedgenotoxic effect through the Micronucleus (MN) test and Cometassay (CA) in C. latirostris neonates after in ovo exposure to increasingconcentrations of the GLY-based formulation Roundups, applieddirectly on the eggshell (topication) (Poletta et al., 2009). In thepresent study, we aimed to model environmental conditions moreclosely through a field-like experiment simulating the exposure thata caiman nest can receive in neighbouring croplands habitats, usingpesticide practises commonly applied in agriculture. In order to

determine the effects of GLY, ES and CPT pesticide formulations onC. latirostris we analysed development, enzymatic and metabolicparameters, and genotoxic effects.

2. Materials and methods

2.1. Chemicals

Roundups Full II (66.2% glyphosate, GLY), Cypermethrin Atanors (25% cyper-

methrin, CPT) and Endosulfan Galgofans (35% endosulfan, ES) formulations were

obtained by courtesy of Establecimiento La Matuza SA, Santa Fe, Argentina.

Roundups Full II is a liquid water soluble (12 000 mg/l) herbicide, containing

glyphosate potassium salt [N-(phosphonomethyl) glycine monopotassium salt,

C3H7KNO5P] as its active ingredient (a.i.) (CAS No. 70901-12-1). CPT Atanors is a

liquid water-insoluble (0.01 mg/l) mixture of different cypermethrin isomers

(C22H19Cl2NO3, CAS No. 52315-07-8). ES Galgofans is a liquid practically water-

insoluble (0.32 mg/l) formulation, containing endosulfan as a.i. (C8H6Cl6O3S, CAS

No. 115-29-7) (EXTOXNET, updated 1996).

Dimethyl sulphoxide (DMSO) was purchased from Fluka. Low melting point

(LMP) agarose, normal melting point (NMP) agarose, ethidium bromide, the rest of

the reagents for CA and MN test and general laboratory chemicals were provided by

Sigma. RPMI-1640 medium was purchased from HyClone.

Aspartate aminotransferase (AST, EC 2.6.1.21), Alanine aminotransferase

(ALT, EC 2.6.1.21), Alkaline phosphatase (ALP, EC 3.1.3.1), Lactate dehydrogenase

(LDH, EC 1.1.1.27), Creatin kinase (CK, EC 2.7.3.2), Cholinesterase (ChE, EC 3.1.1.8),

Total Protein (TP) and Serum Albumin (ALB) commercial kits were from Wiener

Labs (Rosario, Argentina).

2.2. C. latirostris eggs

We used C. latirostris eggs coming from nests collected during ProyectoYacare

(PY) ranching activities (Larriera et al., 2008), in an area free of cropping and urban

activities in Santa Fe Province, Argentina (Natural reserve ‘‘El Fisco’’, 3011102600S;

611002700W). All eggs were harvested within 5 days after oviposition, on the same

day and maintained under the same conditions during transportation to PY facilities

in Santa Fe city. Once in the laboratory, eggs viability was determine by checking the

presence of the opaque eggshell banding (Iungman et al., 2008). Only eggs

considered viable were included in the experiments.

2.3. Experimental design and treatments

2.3.1. Experiment 1 (E1)

Three experimental groups of three artificial caiman nests each (N¼9) were

constructed separately in a field free of any contaminating activity, maintaining a

distance of approximately 100 m between each group. Vegetal mounds resembling the

nests constructed by caiman females, approximately 1 m wide and 0.60 cm high

(Larriera and Imhof, 2006), were made with vegetal material obtained from the same

non-contaminated area; based on our experience from the PY activities (www.mupcn.

Table 1Information and characteristics of glyphosate, endosulfan and cypermethrin.

Pesticide Chemical name Abbreviation Chemical class Use Persistence inenvironment

Toxicologicalclassificationn

Glyphosate N-phosphonomethyl

glycine

GLY Phosphonoglycine Systemic herbicide for non-

selective weed control in

agriculture and non-

agricultural environments.

Field half-life: from 1 to 174

days. Estimated average: 47

days in soil; from 12 days to

10 weeks in pond water.

General Use Pesticide (GUP).

Class II, moderately toxic.

Cypermethrin [(R,S)-alpha-cyano-3-

phenoxybenzyl(1RS)-

cis,trans-3-(2,2-

dichlorovinyl)-2,2-

dimethylcyclopropane-

carboxylate

CPT Pyrethroid Wide synthetic insecticide

used to control many pests,

mainly moths, in agriculture

and multiple urban

environments.

Moderately persistent in

soil, half-life from 4 days to

8 weeks. Stable in water

with a half-life from 50 to

100 days.

Some formulations classified

as Restricted Use Pesticide

(RUP). Class II—moderately

toxic. Some formulations

toxicity Class III—slightly

toxic.

Endosulfan [6,7,8,9,10,10-hexachloro-

1,5,5a,6,9,9a-hexahydro-

6,9-methano-2,4,3-

benzadioxathiepin 3-oxide

ES Organochlorine Insecticide and acaricide

used to control a wide

variety of insects and mites

on food crops, also as wood

preservative.

Moderately persistent,

average field half-life of 50

days in soil and from 30 to

150 days in water.

Restricted Use Pesticide

(RUP). Class I, highly toxic.

Data obtained from EXTOXNET (updated 1996).

n Toxicological classification from US EPA.

G.L. Poletta et al. / Ecotoxicology and Environmental Safety 74 (2011) 852–859 853

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com.ar/yacare) the nest chamber was situated approximately at 35 cm of the nest

surface. Internal temperature was originated by sun and plant fermentation, and nest

moisture content was regulated by adding tap water when necessary (Larriera and

Imhof, 2006). Temperature inside the nests was recorded using Hobo temperature

recorders (Onset Computer Corp., Pocasset, MA, USA) since approximately 10 days

before the date stipulated for the pesticide spraying. Eggs were introduced in the nests

only when the temperature inside them were suitable and remain within a certain

range through all day (28–32 1C). In the same way, temperature was controlled while

eggs were inside the nests. A total of 81 eggs from three different clutches were equally

and randomly distributed into the three experimental groups, of 27 eggs each (9 eggs

in each of three replicates). Eggs were placed in plastic trays, using vermiculite as

substrate (Larriera et al., 2008). They were put inside the nests chamber immediately

before the moment of pesticide spraying, left in the nest for 5 days after application and

then returned to PY incubator in the same tray covered with the vegetal material of the

nest, in order to reduce any possible interference caused by external environmental

variables such as extremes temperature and predators. Conditions at PY incubator

were: 3171 1C and 90% humidity. Treatments applied to each group are presented in

Table 2. Negative control (C) nests received tap water (in the same volume that treated

groups) while those in Treatments 1 and 2 (T1 and T2, respectively) received pesticide

formulations at the following concentrations: GLY formulation: 3% (3 l/100 l water/ha;

solution prepared: 19.8 g/l), ES formulation: 0.85% (0.85 l/100 l water/ha; solution

prepared 3.03 g/l) and CPT formulation: 0.12% (0.12 l/100 l water/ha; solution pre-

pared 0.33 g/l). These concentrations correspond to those recommended in agriculture

for soybean crops and were applied in two different moment of the incubation period:

at the beginning and a month later, following exactly the same application timescale

used in agricultural practices (Table 2). Pesticide spraying in each experimental group

area was done over and around the three artificial nests constructed, with a portable

fumigation backpack, when climatic conditions were suitable: not windy and without

probability of rain during the day.

The same procedure was carried out at the second application time. Approxi-

mately two days after each pesticide application, herbicide effect on weed was

observed in the area surrounding T1 and T2 nests, but not in C nests. The plots where

the pesticides had been applied show yellowish, dry, death weed, while in the

control plot the weed remained green, without any difference observed during the

experiment (Figs. 1–3). Biological parameters analysed after exposure were

genotoxicity (MN test and CA) and developmental effects (malformations, hatching

size and post-natal growth).

2.3.2. Experiment 2 (E2)

E2 was conducted in the next C. latirostris reproductive season (12 months after E1).

Experimental design and treatments, number of eggs used and conditions of the

experiment remained exactly the same than in E1 (Table 2). Biological parameters

analysed after exposure were also the same, doing now enzymatic and biochemical

determinations as well, which had not been conducted in E1.

In both experiments (E1 and E2), eggs coming from different clutches were used

in order to control ‘‘clutch effect’’ at the moment of analysis, as it is one of the main

factors introducing variability in crocodilian studies (Verdade, 1997). Eggs were

controlled periodically during both experiments in order to identify and discard

those which became non-viable. When caimans are ready to hatch, they start to

make a characteristic sound within the eggs. At the moment these ‘‘callings’’ started,

the corresponding eggs were removed from the incubator and, if hatching did not

occur spontaneously within 24 h, they were assisted.

2.4. Analytical pesticide determination

2.4.1. Glyphosate

Glyphosate analysis was done by high-performance liquid chromatography

(HPLC) with pre-column derivatization using 9-fluorenylmethyl chloroformate

(FMOC-Cl), considering approaches developed earlier (Hogendoorn et al., 1999;

Llasera et al., 2005). Derivatization was conducted under alkaline conditions with

borate buffer. HPLC conditions were: room temperature, RP-18 5 mm (0.46 cm�

15 cm i.d.) column, injection volume 20 ml, flow volume 1.2 ml/min, fluorescence

detector (excitation 270 nm, emission 315 nm). Two different gradient elution

Fig. 1. Control experimental plot showing green weed after application of tap water

as control treatment. (For interpretation of the references to color in this figure

legend, the reader is refered to the web version of this article.)

Table 2Experimental groups and treatments applied in both experiments.

Experimentalgroup

No. of eggs/artificialnest(replica)

No. of eggs/exp. group

Application Treatmentapplied

C 9 27 11 Tap water

21 Tap water

T1 9 27 11 GLY formulation

21 GLY formulation

T2 9 27 11 GLY formulation

21 GLY form.+ES

form.+CPT form.

C: control; T1: treatment 1; T2: treatment 2; 11: applied at 5th day of incubation

period; 21: applied at 35th day of incubation period (a month later than 11).

Fig. 2. T1 experimental plot showing herbicide effect. It can be seen as the yellowish,

dry, death weed area. (For interpretation of the references to color in this figure

legend, the reader is refered to the web version of this article.)

G.L. Poletta et al. / Ecotoxicology and Environmental Safety 74 (2011) 852–859854

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programs were used for separation, using acetonitrile (A) and a NaH2PO4 (B) (pH 6.4)

water solution, as it is shown in Table 3.

2.4.2. Cypermethrin

Cypermethrin determination was conducted by Gas chromatographic method

(GC) following the AOAC Official Method 985.03 (AOAC Official Methods of Analysis,

1995). Sample was dissolved in CH2CL2 containing dicyclohexyl phthalate, and

1.0 ml is injected into capillary GC in split mode, with flame ionisation detection.

Peak areas are measured for each cypermethin isomer and dicyclohexyl phthalate

and compared with those from standard injection.

2.4.3. Endosulfan

Endosulfan was determined by GC following AOAC Official Method 983.08

(AOAC Official Methods of Analysis, 1995). Sample is extracted with toluene and

a- and b-endosulfan isomers are determined separately by flame ionisation GC,

using di (2-ethylhexyl) phthalate as internal standard.

2.5. Developmental parameters

Immediately after hatching, caimans were weighed (OHAUSs Compact scale

CS200, precision 0.1 g) and measured in total length (TL) and snout-vent length

(SVL) (tape measure, precision 0.5 cm), and then individually identified by two

numbered webbing tags in the hindlegs (National Band and Tag Co.s, Newport, KY).

After that, they were maintained in rearing pools under common controlled

conditions used in PY facilities. Animals of all experimental groups were maintained

in the same pool at a density of 12 caiman/m2, feeding ad libitum 5 times/week with

a mixture of 60% minced chicken head and 40% dry pellets. At 3 and 12 months of age

they were measured and weighed again in order to evaluate the effect of treatments

on subsequent animals growth during the first year of life. Sex of the animals was

determined when they were captured at 12 months of age by examination of their

genitalia. In younger animals it is not accurate to differentiate between males and

females due to similarity of size and shape of sexual organs.

2.6. Blood collection

All animals had been treated following the Ethical Reference Framework for

Biomedics Researches: ethical principles for research with laboratory, farm and wild

animals (CONICET, 2005), using non-harmful techniques of blood collection and

minimising stress and suffering by suitable management methods. At hatching,

whole blood samples (0.5 ml) of all animals were obtained from the spinal vein

(Zippel et al., 2003) with heparinised disposable syringes, in order to perform

genotoxic techniques (MN and CA) on erythrocytes (Poletta et al., 2008).

In E2, when animals were capture for size measure at 3 and 12 months old, they

were bleed again (1 ml) without anticoagulant to conduct biochemical and

enzymatic determinations. These samples were immediately centrifuged at 450 g

approximately for 20 min; serum aliquots were storage at 4–10 1C (ALT, AST, LDH,

CK, ChE, TP and ALB) or �20 1C (ALP) and processed within 24 h.

2.7. Micronucleus (MN) test

Micronuclei are formed by condensation of acentric chromosomal fragments

or by whole chromosomes that are left behind during anaphase movements

(lagging chromosomes). The presence of micronuclei can therefore be taken as an

indicator of the previous existence of chromosomal aberrations (Schmid, 1975).

The MN test was applied on erythrocytes of C. latirostris (Poletta et al., 2008). Two

smears were prepared on clean glass slides from each animal, coded for ‘blind’

analysis, fixed with methanol for 10 min, and stained with Acridine Orange (AO)

supravital stain at the moment of microscopic analysis. MN frequency (MNF) was

scored using a fluorescent microscope (Olympus CX 40) equipped with a U-RFLT

50 excitation filter. For each individual 1000 erythrocytes were analysed in two

replicated slides and MNF (No. of cells containing MN/1000 cells analysed) was

recorded.

2.8. Comet assay (CA)

CA can detect DNA single strand breaks, double strand breaks, alkali labile sites,

DNA adducts, and oxidative damage (Singh et al., 1988). This assay has provided the

opportunity to study DNA damage, repair and cell death (programmed cell death

including apoptosis) in different cell types of natural biota, without prior knowledge

of karyotype and cell turnover rate. This is particularly important when cytogenetic

and molecular assays are either not available or difficult to adopt (Jha, 2008). Cell

viability was determined before the application of the CA by fluorescent DNA-

binding dyes (100 mg/ml AO and 100 mg/ml ethidium bromide—EB). A total of 100

cells were counted per sample under a fluorescent microscope (Olympus CX 40) and

the percentage of viable cells was determined (Poletta et al., 2009). The CA was

performed as described by Singh et al. (1988) with modifications required by

C. latirostris erythrocytes, determined in previous studies (Poletta et al., 2008). In

brief, blood samples were diluted 1:19 (v/v) with RPMI-1640 medium, 1.5 mL of each

diluted sample (4.0�103 erythrocytes, approximately) was added to 100 mL of LMP

agarose and slides prepared following standard procedure. Slides were immersed in

lysis buffer for 24–48 h, incubated in freshly made alkaline buffer (pH 13),

electrophoresed at 300 mA and 25 V (0.90 V/cm) and then neutralised (pH 7.5).

Finally, they were dehydrated in ethanol and left to dry (Poletta et al., 2008). During

all procedures, the preparations were kept in dark to prevent additional DNA

damage. All samples were coded for ‘blind’ analysis, stained with EB (2 mg/ml) and

comet images of 100 randomly selected cells (50 cells from each of two replicated

slides) were scored from each animal under the fluorescent microscope Olympus CX 40.

Cells were visually classified into five classes according to tail size and intensity

(from undamaged, class 0, to maximally damaged, class 4), resulting in a single DNA

damage score (damage index, DI¼n1+2 � n2+3 �n3+4 �n4) for each animal, where:

n1, n2, n3 and n4 are the number of cells in each class of damage, respectively

(Poletta et al., 2008).

2.9. Laboratory techniques

Enzymatic and metabolic determinations were carried out following

conventional techniques as previously applied in this species (Barboza et al.,

2008; Coppo et al., 2005) and in other crocodiles (Barnett et al., 1999; Millan et al.,

1997). Enzymatic determinations of AST, ALT, LDH and CK were conducted by

UV method and read at 340 nm while ALP and ChE by kinetic method and read

at 405 nm. They were all measured at 37 1C using a Spectrophotometer

(Jenways Geneva) and all data expressed as U/l. TP and ALB determinations

were done by colorimetric method using the analyser Targa B 53000s and data

expressed as g%.

2.10. Statistical analysis

Statistical analysis was performed using the software SPSS 14.0 for Windows

(2005). Variables were tested for normality with Kolmogorov–Smirnov test and

homogeneity of variances between groups was verified by Levene test. Data from

MN test, CA, enzymatic and metabolic parameters were analysed by one-way

ANOVA followed by the Tukey’s test or Kruskal–Wallis followed by the Mann

Whitney U-test, depending on data homogeneity and normality assumption;

differences between sex were analysed by T-test. Data from TL, SVL and weight

were analysed by general lineal model: repeated measures. Linear regressions

were applied for the relation between genotoxic and morphological endpoints.

A difference of po0.05 was considered statistically significant.

Table 3Gradient elution programs used in HPLC glyphosate determination.

Time (min) % (A) % (B)

0 8 92

5 8 92

10 30 70

15 40 60

25 40 60

30 10 90

A: acetonitrile; B: NaH2PO4 water solution (pH 6.4). Constant flow volume of

1.2 ml/min.

Fig. 3. T2 experimental plot showing herbicide effect. It can be seen as the yellowish,

dry, death weed area. (For interpretation of the references to color in this figure

legend, the reader is refered to the web version of this article.)

G.L. Poletta et al. / Ecotoxicology and Environmental Safety 74 (2011) 852–859 855

Author's personal copy

3. Results

Analytical pesticide determinations done in the aqueous solu-tions prepared for spraying the nest areas give a concentration of17.25 g/l of GLY, 0.26 g/l of CPT and 2.47 g/l for ES.

From the total number of C. latirostris eggs exposed, eightresulted non-viable in E1 (C: n¼2; T1: n¼3; T2: n¼3) and six inE2 (C: n¼1; T1: n¼2; T2: n¼3) and all of them were discardedduring the experiments. The period of time went by from treatmentto hatching was between 78 and 84 days in E1 and between 76 and80 days in E2.

No difference was found between replicates in any treatments(p40.05) for any of the parameters evaluated so, all results areinformed as mean values7SE per experimental group.

Under the experimental conditions set, no external malforma-tions were observed in any of the caimans of the different groups.Cell viability conducted as a requirement for genotoxic techniqueswere found to be more than 95% for all the samples (data notshown). In both experiments, results obtained from the CA and theMN test as genotoxic endpoints, demonstrated that both T1 and T2

induced an increase in DNA damage. In E1, the DI and MNF of T1 andT2 were significantly higher compared to the control (po0.05).However, there were no differences between T1 and T2 neither inthe CA (p¼0.370) nor in the MN test (p¼0.097). Similar resultswere obtained in E2 (po0.001), but in contrast to E1, the CA shown asignificant difference between T1 and T2 (p¼0.05) remainingwithout difference in the MN test (p¼0.932) (Table 4). Nodifference between sexes (p40.05) or clutch effect (p40.05) wereobserved neither for CA nor for MN test in any of the experimentscarried out.

Concerning developmental parameters, data obtained in E1

demonstrated that none of the treatments applied produced anyeffect on TL, SVL or weight of the animals at birth, or in subsequentgrowth evaluated after 3 or 12 months (p40.05). In E2, however,animals from T1 presented a significantly lower TL and SVLcompared to control group at birth (po0.05), while the samewas observed for both T1 and T2 at 3 months (po0.05), but not at 12months (p40.05). No effect was observed on weight of the animals(Table 5). We found clutch effect for TL, SVL, and weight at threemeasures made (birth, 3 and 12 months), but no relations wereobserved between DI or MNF and size of the animals at birth(p40.05 in all performed analysis). Sex of the animals determinedat 12 months of age shown a ratio of 60% females and 40% males inE1 and 58% females and 42% males in E2. No differences wereobserved in the sex ratio between treatments in any of theexperiments (p40.05).

Results from enzymatic determinations conducted at 3 monthsshown an increase in the level of CK, AST and ALT in exposed groupscompared to control group. There was a significant increment ofALT level in both T1 and T2 (po0.05) while CK and AST levels weresignificantly increased only in T2 (po0.05). No alterations werefound in LDH, ALP or ChE of exposed groups compared to control,and no differences were observed between T1 and T2 in any of theenzymatic parameters evaluated (p40.05) (Table 6). Metabolicparameters indicated a significant decrease in TP and TP/ALBrelation in T2 compared to control (po0.05) but no alteration inALB level was observed (p40.05) (Table 6). At 12 months afterhatching, enzymatic or metabolic alterations were no longerobserved in exposed animals compared to controls (p40.05 inall performed analysis). The level of CK, AST, ALT and ChE at 12months of age show a significant increase compared to those at 3months of age (po0.05 in all performed analysis). No differenceswere observed between sexes for any of the enzymes or metabolicparameters evaluated at 3 or 12 months of age (p40.05 in allperformed analysis).

Table 4Micronucleus frequency and damage index observed in C. latirostris hatchlings in

different experimental groups.

Experimental group MNF DI

E1

C 2.6170.43 114.0073.97

T1 5.5770.62n 167.88711.84nn

T2 7.5370.85nn 175.7577.88nn

E2

C 1.0870.19 173.8777.61

T1 4.4670.26nn 256.776.98nn

T2 4.2770.40nn 280.877.17nn

All values are expressed as mean7SE. E1: Experiment 1; E2: Experiment 2;

C: control; T1: treatment 1; T2: treatment 2; MNF: micronucleus frequency;

DI: damage index.

n o0.05 compared to control.nn o 0.001 compared to control.

Table 5Growth data observed in hatchlings, 3 and 12 month old C. latirostris from different

groups of Experiment 2.

Experimental group

C T1 T2

TL (cm)At birth 24.4370.13 24.0370.19n 24.1770.17

3 month 31.7170.55 30.5070.51n 30.8470.59n

12 month 63.6671.73 63.0671.63 63.8171.92

SVL (cm)At birth 11.8470.58 11.5770.76n 11.6870.63

3 month 15.9070.68 14.3470.23n 14.5370.27n

12 month 30.3570.84 30.9170.84 31.5370.87

Weight (g)At birth 48.5070.88 47.2471.02 47.5571.03

3 month 115.6378.39 104.9576.75 113.3577.67

12 month 920.54774.98 934.3777.21 929.18794.21

C: control; T1: treatment 1; T2: treatment 2; TL: total length; SVL: snout-vent length.

n o0.05 compared to control.

Table 6Enzymatic and metabolic parameters observed in 3 months old C. latirostris from

Experiment 2.

Experimental group

C T1 T2

Enzymatic parametersCK (U/L) 215.52728.48 318.40744.93 703.07291.50n

ALT (U/L) 13.6673.07 29.0373.15 n 26.0772.89 n

AST (U/L) 49.3376.58 50.2977.52 64.3878.35 n

LDH (U/L) 850.267102.51 712.677114.61 951.32782.58

ALP (U/L) 85.23711.45 108.09716.09 118.88715.02

ChE (U/L) 660.6729.64 842.87146.91 874.757175.12

Metabolic parametersTP (g%) 3.6770.97 3.3970.46 3.3770.47 n

ALB (g%) 1.8470.5 1.8570.62 1.9170.67

TP/ALB relation 1.9970.11 1.8470.65 1.7770.44 n

All values are expressed as mean7SE. C: control; T1: treatment 1; T2: treatment 2;

CK: Creatin kinase; ALT: Alanine aminotranferase; AST: Aspartate aminotrasferase;

LDH: Lactate dehydrogenase; ALP: Alkaline phosphatase; ChE: Cholinesterase;

TP: Total Protein; ALB: Serum Albumin.

n Statistically significant compared to control.

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4. Discussion

Analytical proof of exposure pesticide concentrations demon-strated that for the three formulations used, the concentrationsdetected in the solutions prepared for spraying the nest areas werea slight lower than those used in agriculture and calculated fromthe percentage of a.i. informed in the commercial labels. This couldbe explained considering that pesticide formulations often containless a.i. than what is said. In the present study, we tried to simulatean environmental exposure that a caiman nest could receive insurrounding agricultural habitats. Even when direct pesticide sprayover caiman nests is not a common situation, it is a fact thatneighbouring croplands habitats receive continuous exposure tolow concentration of pesticides that could have cumulative dele-terious effects on animals living there, especially considering thosewith particular sensitivities (Freeman and Rayburn, 2004). Underthese field-like exposure conditions, we demonstrated genotoxicalterations, growth delay as well as enzymatic and metabolicdisorders in caimans exposed in ovo to GLY formulation aloneand also in combination with ES and CPT formulations, as they arecommonly applied in agricultural practices related to soybeancrops.

In both experiments carried out genotoxic effects were inducedby GLY formulation alone (T1) as well as by the mixture of GLY, ESand CPT tested (T2), showing a higher increase in DI (CA) for themixture of pesticides. Similarly to our previous work in 10 monthsold caimans (Poletta et al., 2008), we found no differences betweensexes neither for the MN test nor for the CA. In other study,genotoxicity of the same GLY formulation (Roundups) has beenobserved in erythrocytes of caimans exposed in ovo under labora-tory controlled conditions, showing a clearly dose-dependenteffect in DI and MNF as well as reproducibility in separateexperiments (Poletta et al., 2009). MNF found in the presentstudy for T1 are similar to those shown in the previous work bycaimans exposed topically to 500 and 750 mg/egg while the groupexposed to the mixtures of pesticides (T2) shown MNF similar tothose exposed topically to the higher Roundups concentration(1750 mg/egg). In the case of the Comet assay, DI found in E2 arehigher than those observed in E1 in all groups, including thenegative control. Anyway, we can say that DI observed in caimansexposed by pesticide spraying on the nest are comparable with, oreven higher, than that of the group exposed topically to the higherconcentration of Roundup (1750 mg/egg). These results could meanthat, even inside the nest, eggs would receive pesticides anddamage would be produced at a level similar to that reportedafter topical exposure, demonstrating that risk exist for caimansliving in agricultural areas. Red-eared sliders (Trachemys scripta

elegans) exposed in ovo to another GLY formulation (Glypros)presented lower hatching success and dose-dependent geneticdamage measured by flow cytometry (Sparling et al., 2006). Themixture of the herbicides Roundups (GLY) and Hexarons (Hex-axinone 13.2%+Diuron 46.8%) significantly increase the MNF inerythrocytes of Astianax sp, while no one of them had any effect byseparate (Rossi, 2008), indicating a possible synergic effect. CPT hasbeen shown to induce a significant increase in DI and MNF inerythrocytes of the fish Prochilodus lineatus (Poletta et al., 2005;Simoniello et al., 2009) and a dose-dependent increase in DNAdamage (CA) in multiple organs and tissues of mouse (Patel et al.,2006). ES has increased DNA damage (CA) in gill and kidney of thefish Channa punctatus (Pandey et al., 2006) and the MNF inerythrocytes of Hyla pulchella tadpoles (Lajmanovich et al.,2005). The mechanisms by which pesticides induce geneticdamage vary greatly with their chemical nature. One of them isthe production of reactive oxygen species (ROS), which could leadto single-strand breaks and mutations (Zegura et al., 2004). Studieshave demonstrated generation of ROS induced by GLY in the

goldfish Carassius auratus (Lushchak et al., 2009), by ES in rainbowtrout, Oncorhynchus mykiss (Dorval and Hontela, 2003), and by CPTin different experimental systems (Giray et al., 2001).

In our study, enzymatic and metabolic alterations wereobserved three months after hatching of caimans exposed to bothpesticides treatments (T1 and T2). Animals in T1 show an increaseonly in one enzymatic parameter, ALT, while the animals in T2

presented increased ALT, AST and CK, as well as a reduction inmetabolic parameters TP and the relation TP/ALB. AST and ALT areoriginated mainly in liver, and also in skeletal muscle, heart, kidneyand nervous tissues. They are released as a result of destruction ofcells or changes in membrane permeability. CK comes mainly fromheart, skeletal muscle and brain, and an increase in serum level isindicative of cellular lession. Total serum proteins and lipid contenthave also been used as additional endpoints of overall health of theorganism (Angel and Angel, 2000). These results agree with those ofJiraungkoorskul et al. (2003) who reported a significant increase inAST, ALT and ALP in the Nile tilapia (Oreochromis niloticus) aftersubchronic-sublethal exposure to GLY formulation. Glusczak et al.(2006) found that the same formulation decreased plasma protein,brain Acetylcolinesterase (AChE), and some haematological para-meters in piava fish (Leporinus obtusidens). Similar effects wereobserved on AchE of the sunfish (Lepomis macrochirus) exposed toES (Dutta and Arends, 2003). Begun (2007) reported CPT-inducedreduction in proteins as well as increased ALT and AST in muscleand kidney of the fish Clarias batrachus. Proteins of the kidney werelower in the catfish Rhamdia quelen exposed to pesticides instreams near agricultural fields (Becker et al., 2009). On the otherhand, and in agreement with our study, Cabagna et al. (2005) foundno alterations in plasma ChE activity in adult Bufo arenarum fromagricultural sites in mid-eastern Santa Fe Province (Argentina), buthaematological parameters differed significantly from animals ofcontrol sites. It has to be noted that this is the same region whereC. latirostris is naturally distributed. Our results shown an increasein the level of CK, AST, ALT and ChE at 12 months of age compared tothose at 3 months. Similarly, Coppo et al. (2005) reported that CK,AST, LDH and ChE increased gradually with growth of sub-adultscaimans. On the contrary, Barboza et al. (2008) observed nodifference between 3 different age range groups of sub-adultsC. latirostris for any of the enzymes analysed, except ChE. Evenwhen biochemical parameters appear to show no defined patternin relation to age, a gradual increase may be expected with growth,as a result of increase in muscular volume (Coppo et al., 2005). Inrelation to sex, we found no differences between males and femalesin any of the parameters analysed. The same was observed byBarboza et al. (2008) in sub-adults C. latirostris while Coppo et al.(2005) reported that CK and LDH were higher in males than infemales sub-adults (1–5 years old) C. latirostris and C. yacare undercaptivity, but as they analysed data from both species together, wedo not know if the effect correspond to one of the two species orto both.

In our present contribution, developmental analysis demon-strated that animals exposed to GLY formulation and to the mixtureof pesticides presented a lower size at birth and during first monthsof life compared to control group. These results show detrimentaleffect on post-natal caiman growth after in ovo exposure topesticides. Less hatchling relative weight in C. latirostris was alsoreported by a study made on ES effects (Beldomenico et al., 2007).Different GLY formulations induced less weight at birth in T. scripta

elegans after in ovo exposure (Sparling et al., 2006) and a decrease ingrowth embryos of different frog species (Edginton et al., 2004).Chronic sublethal exposure to contaminants has been shown toresult in elevated Standard Metabolic Rate (SMR) in reptiles(Mitchelmore et al., 2005). Given no compensatory increase infeeding or assimilation, individuals having SMRs elevated abovenormal would be expected to experience fitness costs associated

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with reduced growth. Environmental factors that lead to a period ofreduced growth can be particularly important during the juvenileperiod, when animals grow at a maximum rate, attaining a size atwhich certain predators can be avoided. Therefore, growth reduc-tions at this moment will result in a longer period of risk of size-dependent mortality (Mitchelmore et al., 2005).

We found clutch effect for both length and weight at threedifferent measures made (birth, 3 and 12 month). This demon-strated that animals coming from different clutches grew atdifferent rates during their first year of life, as it was extensivelyreported in previous studies with this species (Pina et al., 2005;Schulte and Chabreck, 1990; Verdade, 1997). Results indicated norelation between genotoxic endpoints and weight or length of theanimals at birth. Previous works done in laboratory controlledconditions with C. latirostris exposed to Roundups shown similarresults (Poletta et al., 2009).

Alterations found in enzymatic and metabolic systems as well asin growth of caimans at birth and three months after hatching wereno longer observed at 12 months old. Considering this, we mayassume that once a certain time has passed from pesticide exposureand in the absence of a new exposure event, enzymatic andmetabolic systems can recover from damage, possibly returningto normal levels. This may allow exposed animals to reach a similarsize than control ones at a year of life approximately. However, innatural environments this situation could be worse by repeatedpesticide exposures and unfavourable conditions produced by coldthat, in ectotherms like caimans, implies lack of food intake andhence, poor nutritional state on animals. Immediately after hatch-ing, caimans have to face extremely low temperatures typical ofwinter, without almost any previous nutritional contribution(Larriera et al., 2008). Caimans can be exposed to pesticides asembryos and neonates (December–March), because the moment ofmaximal pesticide application coincides with incubation periodand hatching. Later, these animals can receive new successiveexposures at 8–12 months old, moment in which extensivefumigations start again, mainly in relation to first and secondsoybean cropping (November–March) (Paruelo et al., 2006). Underthese conditions, it is difficult to ensure that animals can recover atshort term from genetic damage, enzymatic and metabolic dis-orders, and growth delay, as we observed in this field-like study. Itis important to highlight that recent studies made in the pampasicregion of Argentina revealed GLY residues in surface water afterspraying, as a consequence of drift and runoff (Peruzzo et al., 2008).Moreover, it is known that GLY is extensively metabolised by someplants but remains totally intact in others (EXTOXNET (updated1996)). This could be relevant at the moment that caiman femalesstart to construct the nest, as they normally use vegetal materialavailable in surrounding areas.

In the present work, all parameters analysed indicated a highertoxicity for the mixture of pesticides than for GLY formulationalone, as it has been widely reported in the literature when toxicityof mixtures and single compounds were compared (Hayes et al.,2006; Rossi, 2008). It has to be noted that application conditions setin T2 attempted to simulated quite closely agricultural pesticidepractices commonly used nowadays in relation to soybean crops,where successive sprayings that combine mainly GLY, ES and CPTformulations are made. The use of the present field-like studyextends and supports the information derived from tests donepreviously with GLY formulation Roundups in laboratory condi-tions (Poletta et al., 2009), allowing a better understanding of thebiological effects of chemical in current environmental situations.These results indicate that genotoxic, enzymatic, and growthparameters may be good early indicators of contamination withpesticide single compounds and mixtures in C. latirostris. Evalua-tions made through different biological endpoints could be highlyvaluable at population and community levels of wildlife species.

5. Conclusions

Field-like exposure to the single formulation GLY and to themixture of GLY, ES and CPT formulations in conditions commonlyapplied in agricultural practices, induced genotoxic alterations,growth delay as well as enzymatic and metabolic disorders incaimans exposed in ovo. All parameters analysed indicated a highertoxicity for the mixture of pesticides than for GLY formulation alone.In natural environments this situation could be worse by repeatedpesticide exposures and unfavourable conditions produced bylack of food intake and poor nutritional state on animals duringwinter. Genotoxic, enzymatic and metabolic biomarkers as well asgrowth parameters demonstrated to be good early indicatorsof contamination with pesticide single compounds and mixturesin C. latirostris.

Acknowledgments

This work was supported by Proyecto Yacare and YacaresSantafesinos (Gob. Sta. Fe/MUPCN), Universidad Nacional delLitoral, Consejo Nacional de Investigaciones Cientıficas y Tecnicas– CONICET (PIP 5012 to MDM), and Universidad de Buenos Aires(UBACyT X154 to MDM). We would like to thank other members ofProyecto Yacare (www.mupcn.com.ar/yacare) and Bioq. Ana ClaraLorenzi for their help in this study. We are especially grateful toCarlos Mastandrea (Cat. Toxicologıa y Bioquımica Legal, Fac.Bioquımica y Ciencias Biologicas, UNL), Horacio Beldomenico,Ma. Rosa Repetti and Silvia R. Garcıa (Area Plaguicidas, LaboratorioCentral, Fac. Ingenierıa Quımica, UNL) for analytical pesticidedeterminations. We also thank to Establecimiento La Matuza SA(Santa Fe, Argentina) for providing pesticides used.

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