Hematological parameters of health status in the common toad

Bufo arenarum in agroecosystems of Santa Fe Province,

Argentina

Mariana C. Cabagna1 , Rafael C. Lajmanovich2,3 , Gabriela Stringhini1 ,

Juán C. Sanchez-Hernandez4 , Paola M. Peltzer5
1 Facultyof Biochemistry and Biological Sciences — FBCB-UNL, Paraje el Pozo s/n (3000),
Santa Fe, Argentina
2 National Council for Scientific and Technical Research (CONICET) — FBCB-UNL, Paraje el Pozo

s/n (3000), Santa Fe, Argentina

3 Corresponding author; e-mail: [email protected]
4 Laboratory of Ecotoxicology, Department of Environmental Science, University of Castilla-La

Mancha, Avda. Carlos III, 54071, Toledo, España

5 National Institute of Limnology (INALI-CONICET-UNL), José Macias 1933 (3016), Santo Tomé

(Santa Fe) Argentina

Abstract. We compared some hematological parameters and values of plasma cholinesterase activity
in adult Bufo arenarum from a control site and agricultural sites. The mean values of the plasma
cholinesterase activity did not vary among toads collected in pristine forest and agricultural sites. The
blood parameters (hematocrit, hemoglobin concentration, white blood cells, and heterophils) from
agricultural sites differed from the control site. The results suggest a high pesticide impact from
intensive cropping in mid-eastern Santa Fe Province of Argentina.

Key words: Agroecosystem; Argentina; Bufo arenarum; hematological parameters; pesticide; plasma
cholinesterase.

Introduction
Amphibians are often identified as a group of organisms that are particularly
sensitive to environmental pollutants because their double life-cycles and relatively
permeable skins provide more opportunities for exposure to pesticides than those of
other vertebrates (e.g. U.S.EPA, 2002; Mann et al., 2003). Moreover, amphibian
populations are declining worldwide and one of the many contributory factors
proposed for this phenomenon is agricultural intensification (Kiesecker et al., 2001;
Alford et al., 2001; Christin et al., 2003).
© Koninklijke Brill NV, Leiden, 2005 APPLIED HERPETOLOGY 2: 373-380
Also available online - www.brill.nl
374 Mariana C. Cabagna et al.

In Argentina a wide range of pesticides are commonly used in agricultural prac-

tices. Bullacio and Panello (1999) listed a broad group of agrochemicals such as
pyrethroids (e.g. deltamethrin, cypermethrin, alfamethrin), chlorinated (e.g. endo-
sulfan, acetochlor), organophosphorus (e.g. monocrotophos, fenitrothion) or car-
bamate (e.g. carbofuran, carbendazim) pesticides. Among these agrochemicals,
organophosphorus, carbamate and pyrethroid compounds constitute an important
group of pesticides with an intensive use in Argentina (CASAFE, 1999). There is
not a reliable database on the consumption and application rate of pesticides in
Argentina. However, studies conducted in the provinces of Entre Ríos and Santa
Fe (Argentina) during 2000-2004 revealed residues of organochlorine pesticides in
amphibian tissues (Lajmanovich et al., 2002) and high sensitivity to pyrethroids
(Izaguirre et al., 2000). In addition, malformations (Peltzer et al., 2001) and inhibi-
tion of plasma cholinesterase (Lajmanovich et al., 2004) were reported in anurans
collected in the same region. These studies indicate that investigations into the toxic
effects of pesticides in amphibian populations from the areas surrounding crop fields
in Argentina are necessary.
Biomarkers are essential to assess the environmental and health risks of exposure
to potentially toxic chemicals (NRC, 1987). In particular, changes in blood parame-
ters can be useful in some species as biochemical markers of toxicity for pesticide
exposure (Haratym-Maj, 2002). As part of a continuing study to assess the adverse
effect from pesticides on a population of anurans, the aim of this work was to es-
tablish some hematological parameters of adult Bufo arenarum from a control site
and from agricultural sites. Plasma cholinesterase (ChE) activity was also assayed.
B. arenarum was selected because it has an extensive distribution in Neotropical
regions (Cei, 1980), and it is frequently found in agricultural lands and urban terri-
tories.

Materials and Methods

The study area was situated in the mid-eastern area of Santa Fe Province (Argentina)
(fig. 1). Because intensive agriculture was introduced to this area in the 20th century,
the remaining forest is restricted primarily to riparian woodlands surrounded by
croplands and pastures.
24 adult male B. arenarum were collected by hand from four sampling sites
(n = 6 for each site); one control site, located in a pristine forest (PRB: Paraná River
Boundary, 31◦ 42 S 60◦ 30 W), and three agricultural sites: Monte Vera (site A),
Monte Vera (site B) and Angel Gallardo, situated in suburban woodlands (fig. 1).
These three areas differed in the degree of human disturbance, successional stages
and crop. Monte Vera (site A) (MVA, 31◦ 32 S 60◦ 41 W), Monte Vera (site B)
(MVB, 31◦ 30 S 60◦ 40 W) and Angel Gallardo (AG, 31◦ 33 S 60◦ 40 W) were often
used for intensive agricultural production with transgenic soybean. Vegetables such
as lettuce, tomato, and spinach were also cultivated.
Health status in the common toad Bufo arenarum 375

Figure 1. Locations of the sampling sites. PRB = Paraná River Boundary; MVA = Monte Vera
(site A); MVB = Monte Vera (site B); AG = Angel Gallardo.
The toads were captured in January 2004, with a mean air temperature of 25◦ C
and rainfall of 1000 mm. Animals were transported to the laboratory within 1 h, in
a covered bucket with water, to avoid stress. Head-cloaca length and body weight
were recorded. Duplicate samples of blood (ca. 500 µL) were taken from animals
anaesthetized with 30% ethyl alcohol by cardiac puncture using a heparinized small
needle (20 mm length) following location of the heart via palpation. The toads were
released after external examination of general body condition, at the same sites
where they had been captured. Plasma ChE activity was determined colorimetri-
cally by the method of Ellman et al. (1961) with modifications for optimal assay
conditions (Lajmanovich et al., 2004). The plasma was separated by centrifugation
at 4500 r.p.m. for 10 min. Variations in optical density were measured at 405 nm for
2 min at 25◦ C. Slides were prepared and stained with the May Grunwald-Giemsa
method (Dacie and Lewis, 1991). It is important to note that amphibian blood con-
tains nucleated erythrocytes, leucocytes and thrombocytes. The red blood cells, ery-
throcytes, typically are nucleated and elliptical. The white cells are made up of
agranular leucocytes (lymphocytes and monocytes) and granular leucocytes (ba-
sophiles, heterophils, and eosinophils) (Duellman and Trueb, 1986).
Hematocrit (Hct) was determined by centrifugation of whole blood at 12000 × g
for 5 min. in heparinized capillary tubes. Hemoglobin (Hb) concentration was mea-
376 Mariana C. Cabagna et al.

sured by the cyanmethemoglobin method (reading at 540 nm); the red blood cell
(RBC) and white blood cell (WBC) counts were determined in a Neubauer cham-
ber after diluting the samples in Natt and Herrick solution (Fudge, 2000). Mean cell
hemoglobin (MCH), mean cell volume (MCV) and mean cell hemoglobin concen-
trations (MCHC) were calculated using standard methods for amphibian blood (Peri
et al., 1998). The data were analyzed statistically using the non-parametric Kruskal-
Wallis ANOVA; pairwise comparisons between samples from the four sampling
sites were tested by the Dunn test for post-hoc multiple comparisons. A level of
probability below 0.05 was considered to be significant.

Results and Discussion

Mean (± SD) length and body mass of male toads were 96 ± 13 mm and 90.3 ±
3.5 g, respectively. The mean values of the plasma ChE activity varied from
139.2 µmol seg−1 L−1 for the toads collected from the pristine forest (PRB) to
166.4 µmol seg−1 L−1 for individuals captured from an agriculture site (MVB)
(table 1).
In amphibian and reptile populations exposure to anti-cholinesterase pesticides
generally is evaluated by comparison of enzyme activity from animals collected
in non-polluted areas with those from agricultural areas (Sparling et al., 2001;
Sánchez-Hernández, 2003). In our study the means of ChE activity did not show
significant differences (P > 0.05). Several authors have documented differences
in the sensitivity to the anti-pesticides among enzymes of the same tissue from
different species (Johnson and Wallace, 1987; Monserrat and Bianchini, 1998;
Pan and Dutta, 1998) and suggested that species-related differences in enzyme
susceptibility can primarily be due to dissimilar enzyme amounts and inhibitor
affinity degree to cholinesterase receptors.
The blood parameters of toads from agricultural sites did not differ significantly
from those of the control (table 1), with the exceptions of Hct, Hb, WBC and
Table 1. Mean values (± SD, n = 6) of some hematological parameters and plasma cholinesterase
activity in specimens of Bufo arenarum sampled in control site (PRB) and agricultural sites (MVA,
MVB and AG) in Argentina (fig. 1).
Parameter Sampling site
PRB MVA MVB AG
RBC (×1012 /L) 0.57 ± 0.11 0.46 ± 0.13 0.55 ± 0.06 0.55 ± 0.15
MCHC 19.95 ± 0.63 19.35 ± 1.71 22.40 ± 3.90 21.39 ± 2.47
MCH (pg) 143 ± 15.0 125 ± 15.6 181 ± 23.1 160 ± 46.0
MCV (fl) 718 ± 77 655 ± 125 650 ± 82 636 ± 101
Eosinophils (%) 17 ± 8.2 16 ± 9.8 12 ± 4.3 10 ± 10.0
Lymphocytes (%) 62 ± 7.4 71 ± 7.1 59 ± 11.9 64 ± 27.7
Monocytes (%) 1.0 ± 0.51 1.0 ± 0.89 1.0 ± 0.07 2.0 ± 0.5
ChE activity (µmol seg−1 L−1 ) 139 ± 52 146 ± 72 166 ± 47 141 ± 39
Health status in the common toad Bufo arenarum 377

Figure 2. Mean (± SD) values of hematocrit (Hct), hemoglobin (Hb), white blood cells
counts (WBC), and heterophils in common toad (Bufo arenarum) collected from control (PRB) and
agricultural (MVA, MVB and AG) sites in Argentina. ∗ P < 0.05, ∗∗ P < 0.01.

heterophils in MVA site (fig. 2). Blood parameters of control toads were similar
to those from another study of B. arenarum (Peri et al., 1998). Lymphocytes were
the dominant cell among the various types of leucocytes. We did not identify
basophils. Our investigation showed in MVA samples a normochromic/normocytic
anemia (Hct 29.16%, control 41; Hb 5.65 g dL−1 , control 8.19). Low values
of Hct (anemia) may indicate bacterial infections and gastrointestinal disorders,
including parasitism and hemorrhages (Dein, 1986). Elevated leucocyte number
378 Mariana C. Cabagna et al.

(leucocytosis) can be symptomatic of stress syndrome and inflammatory/infective

and neoplastic processes (Ots et al., 1998). In this context, U.S. EPA (1982) and
WHO (1996) suggested that pesticide exposure can cause leucocytosis. Finally, no
abnormalities were observed in the erythrocytes and leucocytes.
Notably, the MVA site heterophils (%) were lower than in the other sites
(P < 0.05). Recent studies have shown that pesticide exposure causes measurable
alterations in the immune function of amphibians (e.g. Carey et al., 1999; Gilbertson
et al., 2003). Other studies on humans working in the production of liquid pesticides,
reported a significant decrease in the number of neutrophils (Klucinski et al., 1996).
Neutropenia/Heteropenia is a serious disorder because it makes the body vulnerable
to bacterial and fungal infections and is often the result of exposure to pesticides
(Berkow, 1992).
In nature, wildlife is often exposed to mixtures of chemicals and their degradation
products (Ensenbach and Nagel, 1995). The interactions between various combina-
tions of pesticides can affect hematological parameters (Dolara et al., 1994). In-
deed, another possible explanation for variation in the blood parameters of toads at
the sites studied is contamination as a consequence of prolonged use of pyrethroids
(Desi et al., 1985; Garg et al., 1997; Haratym-Maj, 2002). Although these results are
preliminary, the differences in hemopoietic and leucocyte levels in the toads from
sampling site MVA may possibly be due to a high pesticide impact from an intensive
cropping in mid-eastern Santa Fe Province of Argentina. It is important to note that
toads collected from MVA had been in contact with clothes and tools that farmers
utilize to fumigate. Clearly, the interaction of human-induced habitat modifications
and disease dynamics in agroecosystems deserves further study.

References
Alford, R.A., Dixon, P.M., Pechmann, J.H. (2001): Global amphibian population declines. Nature
414: 449-500.
Berkow, R. (1992): The Merck Manual of Diagnosis and Therapy. Rahway, NJ: Merck Research
Laboratories.
Bullacio, L.G., Panello, M.S. (1999): Evaluación de medidas de seguridad en el manejo de fitosanitar-
ios para cultivos extensivos en dos localidades de la República Argentina. Acta Toxicol. Argent.
7: 32-35.
Carey, C., Cohen, N., Rollins-Smith, L. (1999): Amphibian declines: an immunological perspective.
Dev. Comp. Immunol. 23: 459-472.
CASAFE (1999): Cámara de Sanidad Agropecuaria y Fertilizantes de la República Argentina. Buenos
Aires, Guía de Productos Fitosanitarios para la República Argentina.
Cei, J.M. (1980): Amphibians of Argentina. Firenze, Monitore Zoologico Italiano.
Christin, M.S., Gendron, A.D., Brousseau, P., Manard, L., Marcogliese, D.J., Ruby, D.S., Fournier, M.
(2003): Effects of agricultural pesticides on the immune system of Rana pipiens and on its
resistance to parasitic infection. Environ. Toxicol. Chem. 22: 1127-1133.
Dacie, J.V., Lewis, S.M. (1991): Practical Hematology. New York, Churchill Livingstone.
Dein, J. (1986): Haematology. London, Saunders.
Desi, I., Varga, L., Dobronyi, I., Szklenarik, G. (1985): Immunotoxicological investigation of the
effect of a pesticide; cypermethrin. Archiv. Toxicol. 8: 305-309.
Health status in the common toad Bufo arenarum 379

Dolara, P., Torricelli, F., Antonelli, N. (1994): Cytogenetic effects on human lymphocytes of a mixture
of fifteen pesticides commonly used in Italy. Mutat. Res. 325: 47-51.
Duellman, W.E., Trueb, L. (1986): Biology of Amphibians. Baltimore, The Johns Hopkins University
Press.
Ellman, L., Courtey, K.D., Andreas, V., Featherstone, R.M. (1961): A new rapid colorimetric
determination of cholinesterase activity. Biochem. Pharmacol. 7: 88-95.
Ensenbach, U., Nagel, R. (1995): Toxicity of complex chemical mixtures: acute and long-term effects
on different life stages of zebrafish (Brachydanio rerio). Ecotoxicol. Environ. Safety 30: 151-157.
Fudge, A. (2000): Laboratory Medicine: Avian and Exotic Pets. New York, W.B. Saunders Company.
Garg, S.K., Ayub Shah, M.A., Garg, K.M., Farooqui, M.M., Sabir, M. (1997): Biochemical and
physiological alterations following short term exposure to fluvalinate — a synthetic pyrethroid.
Ind. J. Pharmacol. 29: 250-254.
Gilbertson, M.K., Haffner, G., Rouillard, G.D., Albert, A., Dixon, B. (2003): Immunosuppression in
the northern leopard frog (Rana pipiens) induced by pesticide exposure. Environ. Toxicol. Chem.
22: 101-110.
Haratym-Maj, A. (2002): Hematological alterations after pyrethroid poisoning in mice. Ann. Agric.
Environ. Med. 9: 199-206.
Izaguirre, M.F., Lajmanovich, R.C., Peltzer, P.M., Peralta Soler A., Casco, V.H. (2000): Cypermethrin-
induced apoptosis in the telencephalon of Physalaemus biligonigerus tadpoles (Anura: Lepto-
dactylidae). Bull. Environ. Contam. Toxicol. 65: 501-507.
Johnson, J.A., Wallace, K.B. (1987): Species-related differences in the inhibition of brain acetyl-
cholinesterase by paraoxon and malaoxon. Toxicol. App. Pharmacol. 88: 234-241.
Kiesecker, J.M., Blaustein, A.R., Belden, L. (2001): Complex causes of amphibian population
declines. Nature 410: 681-684.
Klucinski, P., Hrycek, A., Stasiura-Zielinska, H., Kossmann, S., Tustanowski, J., Friedek, D.,
Kaminska-Kolodziej, B. (1996): Humural and cellular immunity rates in chemical plant workers
employed in the production of liquid pesticides. Int. J. Occup. Med. Environ. Health. 9: 103-110.
Lajmanovich, R.C., Lorenzatti, E., De la Sierra, P., Marino, F., Peltzer, P.M. (2002): First registrations
of organochlorines pesticides residues in amphibians of the Mesopotamic region, Argentina.
Froglog 54: 4-5.
Lajmanovich, R.C., Sánchez-Hernández, J.C., Stringhini, G., Peltzer, P.M. (2004): Levels of serum
cholinesterase activity in the rococo toad (Bufo paracnemis) in agrosystems of Argentina. Bull.
Environ. Contam. Toxicol. 72: 548-591.
Mann, R.M., Bidwell, J.R., Tyler, M.J. (2003): Toxicity of herbicide formulations to frogs and the
implications for product registration: a case study from Western Australia. Appl. Herpetol. 1: 13-
22.
Monserrat, J.M., Bianchini, A. (1998): Main kinetic characteristics of thoracic ganglia cholinesterase
of Chasmagnathus granulata (Decapoda, Grapsidae). Comp. Biochem. Physiol. 120: 193-199.
NRC (1987): National Research Council committee on biological markers. Environ. Health. Perspect.
74: 3-9.
Ots, I., Murumägi, A., Hõrak, P. (1998): Haematological health state indices of reproducing great tits:
methodology and sources of natural variation. Funct. Ecol. 12: 700-707.
Pan, G., Dutta, H.M. (1998): The inhibition of brain acetylcholinesterase activity of juvenile
largemouth bass Micropterus salmoides by sublethal concentrations of Diazinon. Environ. Res.
79: 133-137.
Peltzer, P.M., Ponzza Ma.L., Lajmanovich, R.C. (2001): Caso de malformación en Leptodactylus
mystacinus (Anura: Leptodactylidae). Nat. Neotrop. 32: 173-176.
Peri, S.I., Fink, N.E., Salibián, A. (1998): Hematological parameters in Bufo arenarum injected with
sublethal dose of lead acetate. Biom. Environ. Sciences 11: 70-74.
Sánchez-Hernández, J.C. (2003): Evaluating reptile exposure to cholinesterase-inhibiting agrochemi-
cals by serum butyrylcholinesterase activity. Environ. Toxicol. Chem. 22: 296-301.
380 Mariana C. Cabagna et al.

Sparling, D.W., Fellers, G.M., McConnell, L.L. (2001): Pesticides and amphibian population declines
in California, USA. Environ. Toxicol. Chem. 20: 1591-1595.
U.S. EPA (1982): Recognition and Management of Pesticide Poisonings. 3rd ed, EPA-540/9-80-005.
Washington, DC, U.S. Environmental Protection Agency.
U.S. EPA (2002): Methods for Evaluating Wetland Condition: Using Amphibians in Bioassessments
of Wetlands. EPA-822-R-02-022. Washington, DC, Office of Water, U.S. Environmental Protection
Agency.
WHO (1996): Environmental Health Criteria 180: Principles and Methods for Assessing Direct
Immunotoxicity Associated with Exposure to Chemicals. Geneva, World Health Organisation.

Accepted: February 4, 2005.