Open access peer-reviewed chapter
Abstract
The objective of this chapter is to present the results of a monitoring study carried out with physiological responses (biomarkers) in Rhinella marina (giant toad) for two different years, inhabiting the low basin of Coatzacoalcos river, one of the most contaminated regions in Mexico. A decrease in delta aminolevulinic acid dehydratase (δ-ALAD) (considered inhibition) and in the condition factor (1.2–1.5 times) found in toads of the industrial zone compared with reference organisms, each year. As for the hematological parameters, the mean corpuscular hemoglobin concentrations (MCHC), in the amphibians of industrial zone of the first sampling year show a decrease, while for the second sampling year, show an increment of 1.5 times than organisms of reference site. These effects could be associated with exposure to pollutants such as heavy metals (mainly Pb), which have been registered in different studies. This study demonstrates the usefulness of giant toads as biomonitors of contaminated sites.
Keywords
- monitoring
- physiological biomarkers
- giant toads
- pollution
1. Introduction
Amphibians are vertebrates that represent the link between life in the aquatic environment and adaptation to terrestrial life. They have important characteristics; such as its ectothermic physiology, metabolism, and highly permeable skin, which makes them sensitive to disturbances in the ecosystem such as changes in water conditions, as well as the presence of pollutants and certain diseases [1, 2, 3]. This complex life cycle makes them susceptible to different routes of exposure to environmental pollutants, which is why they have been considered as bioindicators of environmental quality [4]. Currently, environmental pollution is considered one of the main factors in the worldwide decline of amphibians that has occurred since 1990 [5]. Some species of amphibians have all the characteristics of a bioindicator or biomonitor, other species can satisfy only some of the criteria and are less suitable as study animals [2, 6].
The giant toad or cane toad (
Figure 1.
Giant or cane toad (
In some of the previous research, different physiological responses have been used, from general to specific responses of pollutants from the study sites. These responses are called biomarkers, where all biochemical, physiological, histological, morphological, and behavioral measurements are quantifiable in tissue or biological fluids from different organisms, including amphibians, like the
Moreover, in Mexico, there are several types of environmental pollution scenarios that have not been assessed from the point of view of the effects to the biota living these sites. In addition to this, there are very little environmental regulations as to some types of pollutants, such as: persistent organic compounds (POPs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals. One of the sites has a higher record environmental pollution is the region of the lower Coatzacoalcos River Basin (Veracruz); region highly impacted due to industrial activities and urbanization that have contributed to the deterioration of ecosystems since the 1960s. Currently, Coatzacoalcos is considered one of the most polluted regions in Mexico [22, 23], to such a degree that, in environmental matrices such as water, soil, air, or sediment, and even in fauna, the presence of POPs, volatile organic compounds (VOCs), dioxins, heavy metals, among others, has been detected [11, 24, 25, 26, 27]. Some of these pollutants have been associated with genotoxic or enzymatic effects in aquatic and terrestrial organisms at this site [10, 11, 28, 29]. In this context, the objective of this chapter is to present and compare the results of a monitoring of physiological responses (biomarkers) in
2. Materials and methods
2.1. Study site
The lower basin of the Coatzacoalcos river is in the southeastern state of Veracruz, Mexico (18° 08′56” N; 94°24′41” W). It comprises 21 municipalities, which house petrochemical, industrial, urban complexes, and agricultural land areas. Pollution has been historic since oil exploration and refining works began at the beginning of the twentieth century up to industrialization, agricultural development, and urban growth at present, causing a rapid deterioration of the ecosystems found there.
The sampling stations were established along the basin according to the degree of contamination found. Thus, they were grouped by two zones: industrial and rural. In May 2008, the first sampling of monitoring study was carried out, while the second was carried out in February 2016 (Figure 2).
Figure 2.
Zones of sampling in low basin of Coatzacoalcos (source: Google Earth, 2016).
The industrial zone (I) was formed by the following sampling stations (red oval, Figure 2):
Ejido Cangrejera: site adjacent to the petrochemical complexes of Pajaritos and Cangrejera, where various derivatives of chlorine and ethylene compounds are produced. In addition, the presence of POPs in both environmental and biological matrices has already been evidenced in this area, as well as the effects that these could be causing in terrestrial organisms [24, 28, 29].
Estero del Pantano: site located on the banks of the Calzadas River. According to [11] the river receives discharges of sewage and industrial waters. Also, the presence of POPs and lead (Pb) has been demonstrated, as well as the effects of these compounds on terrestrial and aquatic organisms [10, 11, 30].
While the rural area (R) (yellow oval, Figure 2) was formed by:
Ejido Limonta: Located upstream of the Coatzacoalcos river, in the municipality of Hidalgotitlán. It presents scarce urbanization and ecosystems still well preserved. At present, concentrations of pollutants have not been reported in this sampling station.
San Carlos: Located next to the Uxpana river, upstream and that ends at the Coatzacoalcos river. It has well-preserved ecosystems. Like the previous one, no contaminant concentrations have been reported in this site.
The sampling stations of the rural area selected are characterized by semi-preserved ecosystems and minor impact by agricultural production, being susceptible areas where organophosphorus, organochlorine, or carbamate pesticides can be used to control pests or vectors.
As a reference, giant toads (seven organisms) kept in the laboratory for 1 year under conditions of feeding, humidity, and controlled temperature were selected, collected in a site outside the study area (Huasteca Potosina, San Luis Potosí) and free of exposure to pollutants.
2.2. Biological sampling
Adult male giant toads were collected per site by night transects and hand capture. In the first sampling (May 2008), 40 toads were collected, while in the second sampling (February, 2016), 30 toads were collected. The organisms were transported in containers to the laboratory (Facultad de Química-Universidad Veracruzana-Campus Coatzacoalcos). The body weight and snout-venth length (SVL) were taken. Subsequently, a blood sample (3–5 mL) was taken with heparinized syringes (following the guidelines established for amphibians and reptiles [31]). An aliquot of whole blood was stored in liquid nitrogen (−186°C) for enzymatic analysis. While with the rest the hematological parameters were analyzed. The toads were released in their respective habitat. The toads were collected under a scientific collection permit (SGPA/DGVS/09731/15) issued by the Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT).
2.3. Evaluation of physiological responses (biomarkers)
2.3.1. Delta aminolevulinic acid dehydratase
The inhibition of the activity of the enzyme δ-ALAD is a biomarker that has shown in several studies a response to a specific contaminant. This enzyme is very sensitive to the effects of increased blood lead, even at very low levels [32]. In the biosynthesis of the heme group, the prosthetic group of hemoglobin (Hb) is selectively damaged by the binding of lead, thus affecting important steps of biosynthesis, inhibiting the activity of δ-ALAD (necessary for the synthesis of the heme group), and affecting the synthesis of protoporphyrin IX and hemoglobin [33, 34].
The determination of δ-ALAD was based on the method of [35] following modifications [11]. The whole blood samples (0.05 mL) were diluted with deionized water (1:35). Then 1 mL of 10 mM aminolevulinic acid (in phosphate buffer pH 6.4) was added. The samples were incubated at 38°C for 60 min in a water bath in complete darkness. The reaction was stopped by placing 1 mL of trichloroacetic acid (10%), then the samples were centrifuged for 10 min at 2000 rpm (Thermo Scientific® Sorvall Legend X1R). The supernatant was mixed with 1 mL of the Erich’s solution. After 10 min, the absorbance was measured at 555 nm in a UV–Visible spectrophotometer (Thermo Scientific® Genesys 10S). Units of enzymatic activity were expressed as micromole per min per liter of red blood cells (RBC), which were calculated using the following formula (Eq. 1):
where Abs = absorbance of the sample,
2.3.2. Hematological parameters
The chemical and morphological parameters of the blood can provide a wide range of biomarkers; its use has increased because the sampling can be very fast and non-destructive [36]. The hematological parameters can provide evidence of pathology including anemia, dehydration, infectious processes, parasitism, or poisoning [37]. These parameters in turn may be related to pollution and its effects [38]. The volume of the cell pack or hematocrit (HT) is the percentage of the total volume of blood composed of red blood cells. The mean corpuscular hemoglobin concentration (MCHC) is the range of the weight of hemoglobin by the volume of the erythrocyte. An iron deficiency, increased immature erythrocytes (reticulocytosis) and methemoglobin can result in a decrease in MCHC values [32]. Some toxic compounds can alter the functioning of the hematological system through interference with cellular production in the bone marrow or in the synthesis of the heme group, by direct cytotoxicity to the cells, or by injuries in other tissues resulting in a loss of blood cells [32, 39].
Therefore, the hemoglobin (Hb), hematocrit (HT), and the mean corpuscular hemoglobin concentration (MCHC) were determined. The hemoglobin content (g/dL) was measured using the kit HemoCue Hb 201+ (microcuvettes and HemoCueHb 201+ Analyzer). The determination was made following the protocol of the commercial distributor [40]. The hematocrit was determined with the globular sedimentation method with the aid of the hematocrit chart (Critocaps™ tube reader). The MCHC was calculated integrating the two previous parameters following equation.
2.3.3. Condition factor (CF)
Condition indices are potentially attractive biomarkers because they are simple to implement and provide information on the use of energy as well as the general health status of the organism [41]. The morphometric index most used is the condition factor (CF) expressed as the weight (g)/length (cm). Pollutants can produce rapid and marked changes in condition indices.
To calculate the CF, the snout-vent length (SVL) of the toads was taken with a vernier caliper (mm) and its body weight (BW) (g) with an electronic scale. Subsequently, these two parameters were integrated and used the following formula (Eq. (3)) to calculate the CF (%).
2.4. Statistical analysis
The statistical analysis was carried out with the GrapPhad Prism 6.0 software (for Windows, La Jolla California USA, www.graphpad.com). The results are reported in media ± standard error. A comparison analysis of means (Mann-Whitney test) was carried out to evaluate the differences between the biomarkers per years, with a level of significance of 1 and 5%. To evaluate the difference in δ-ALAD between years, zones, and the laboratory, the Kruskall-Wallis test was used. A correlation between δ-ALAD and hematologic parameters (Log-transformed) was realized with Spearman’s test.
3. Results and discussion
3.1. δ-ALAD activity
The results of the δ-ALAD activity in blood of
Figure 3.
Comparison of biological responses (biomarkers) in blood of giant toad (
The δ-ALAD activity has been shown to be a specific biomarker for evidence of lead exposure in organisms such as birds, amphibians, and mammals, both in the field and in the laboratory [42, 43, 44]. Our results show that, in 2008, toads from the industrial zone showed an inhibition of the δ-ALAD enzyme compared to those from the rural zone, which can be consistent and expected if this area is taken as a reference. This inhibition can be attributed to concentrations of lead found in the blood of giant toads, where residents of the industrial zone contained higher concentrations of this metal [11]. However, for 2016, the activity of δ-ALAD was similar for both areas. Refs. [33, 43] demonstrated in laboratory studies that exposure to lead in adult toads (
On the other hand, the δ-ALAD activity in the laboratory toads was greater for those of Coatzacoalcos; this would also confirm an enzymatic inhibition in the organisms of this region, increasing in recent years. Finally, it could be said that the δ-ALAD activity could reflect a chronic exposure to lead, because the erythrocytes that carry out the transport of hemoglobin have a half-life of between 700 and 1400 days in
3.2. Hematological parameters
The concentrations of hemoglobin and the percentage of hematocrit are presented in Figure 3. No statistical differences were found per year between zones (MW-U, p > 0.05). The levels of both parameters were similar in the toads resident in the rural area in 2008 (Hb = 9.05 ± 0.4 g/dL, HT = 34.6 ± 1.9%) and 2016 (Hb = 8.3 ± 0.4 g/dL, HT = 37.4 ± 1.6%) as well as those of the industrial zone in both years (Hb = 7.6 ± 0.3, HT = 32.3 ± 1.4; Hb = 7.9 ± 0.7 g/dL, HT = 30.7 ± 2.2%, respectively) (Figure 3B and C). However, when comparing by area in each year, a statistically significant decrease in the hemoglobin concentration of
Very few studies, in relation to pollutants and hematological parameters, have been carried out with amphibians. [51] used these parameters to evaluate the effects of agroecosystems on the health of amphibians; however, no statistically significant differences were found between the reference site and the agroecosystems. The results obtained in our study are contrary to those obtained in [52] where they showed a decrease in hematological parameters according to the presence of lead in tissues of the Egyptian toad
We consider that the hematological parameters of Coatzacoalcos toads are altered (increases or decreases) when comparing areas in both years. But not so, if they are compared per year between zones, because similar values are obtained in Hb and HT, only one alteration was found in the MCHC in the organisms of the rural area in 2016. The increase of some parameters, such as Hb, could be related to a response of organisms to a decrease in the transport of oxygen in the blood (hypoxia), derived from anemia (anemic hypoxia) [53]. Hypoxia in some vertebrate organisms (fish, amphibians) has been related to the natural and anthropogenic increase of ammonium, sulfides, or organic matter, as well as the presence of pollutants (organic or heavy metals—lead) and excess nutrients [54, 55]. In the case of MCHC, the decrease in organisms residing in industrial zones, in the sampling of 2008, can be attributed to contamination in these places, being associated with a microcytic anemia, which could have been caused by exposure to pollutants such as heavy metals (e.g. lead) [11]. But for 2016, MCHC concentrations in
3.3. Condition factor (CF)
The results of the condition factor of
The estimation of the condition factor in amphibians is important to evaluate if they are under environmental stress [56]. This biomarker is commonly used to assess the general health status of aquatic and terrestrial organisms, because it is considered a non-destructive biomarker of energy reserves [57]. This is very important given that energy reserves can be used for the maintenance, development, or reproduction of amphibians. Commonly, a greater reserve of energy in organisms gives them greater resistance without food, greater survival, and better reproductive performance compared to individuals with lower reserves [58].
There is very little information on the use of the condition factor in amphibians in contaminated sites. Some researchers [58] reported a decrease in the condition factor and enzymatic alterations in semi-aquatic and terrestrial frogs resident in agricultural sites, associating it with a possible activation of compensatory or detoxification systems in amphibians in the face of environmental stress or a decrease in their prey (mosquitoes) by the application of insecticides. In other organisms, such as fish and birds, the decrease in the condition factor after exposure to heavy metals and organochlorine compounds, respectively, has been reported [59, 60]. Exposure of organisms to pollutants can increase energy requirements, decrease the metabolic or nutrient assimilation rate, and even alter digestion enzymes [41, 54, 61]. This could explain the decrease in the condition factor of the resident toads of the industrial zone in our studies (both samplings), given that, as already mentioned, a greater presence of pollutants has been demonstrated in this area. Regarding the similarity found between the toads resident in the rural area of 2016 and the laboratory ones and their increase in comparison of the organisms of the rural (2008) and industrial sites (2016), another factor that could be influencing these differences could be the scarce availability of food, caused either by natural conditions or by anthropogenic conditions.
3.4. Relationship between δ-ALAD and MCHC
The results of the correlations between the δ-ALAD enzymatic activity and the MCHC are shown in Figure 4. As observed in this figure, for the organisms of the 2008 sampling, in the rural area, there is an association between these physiological responses. This same pattern is observed considering both (total) sites in this sampling year (Figure 4A and C—2008). As previously mentioned, the relationship of the activity of this biomarker and the hematological parameters is found in the fact that the former is part of the metabolic pathway of hemoglobin, which is congruent with these results. However, when making the correlations for the toads of the 2016 sampling, only a statistically significant correlation (p < 0.05) between both biomarkers is observed for the industrial zone (Figure 4B-2016). It is important to mention that for the laboratory toads, no correlations were found between the physiological responses, besides that the values of δ-ALAD in these were found above those of Coatzacoalcos, and the hematological parameters in smaller quantity. This could support the hypothesis that organisms from this site could be producing more Hb and HT (MCHC) to defend against the effects caused by pollutants (heavy metals such as lead) found in the area, which is evident in the organisms of the rural area of the 2008 sampling. However, for the 2016 sampling, the rural area shows a decrease in the MCHC. Therefore, the hypothesis could be supported that the concentrations of lead have increased in this site and consequently the exposure, thus affecting the compensation system of the organisms in this area. In addition to confirming a chronic exposure to this pollutant.
Figure 4.
Correlations between δ-ALAD and hematologic parameters per site and per year (total) in blood of
It is important to mention that several routes and sources of exposure to lead can be found in the toads of Coatzacoalcos due to their complex life cycle. One of these can occur when the tongue of this organism catches its prey, because this organ can be in contact with the soil or sediments (which can be exposed by dredging that takes place in the area), and, as a consequence, ingest particles in which lead may be present [62]. Also, the exposure in this case could be increased by consuming preys that have been exposed to this metal and that contain it; in this case, the presence of lead has been demonstrated in insects that inhabited industrial sites [63] that could be part of the diet of the giant toad in Coatzacoalcos due to their voracious appetite [9]. Likewise, it has been demonstrated that the consumption of prey in some organisms can increase the concentrations of lead and manifest the toxic effects [64]. Therefore, this would complement the possible explanation of the decrease or increase in some physiological responses in organisms of the 2016 sampling.
Finally, it should be mentioned that the physiological responses quantified in
4. Conclusion
The δ-ALAD activity, the hematological parameters, and the condition factor can be considered as biomarkers of exposure and/or effect, non-destructive, in giant toads in monitoring studies in sites contaminated by heavy metals or other pollutants. Especially δ-ALAD, which could reflect a chronic exposure in organisms. On the other hand, the results found with the organisms of the lower basin of the Coatzacoalcos river could lead to the need to make a new monitoring with emphasis on the rural regions to affirm or discard out if there is an increase in the concentration of pollutants, especially Pb, its possible causes and, if this may be affecting other organisms and even the human settlements that are there. New studies carried out in this region should take into account this type of physiological responses in a battery of biomarkers that reflect the response to other pollutants already registered on the site.
Once again, it was confirmed that the giant toad (
Acknowledgments
This work was supported by Fondo de Apoyo a la Investigación (C17-FAI-06-27-27) by Universidad Autónoma de San Luis Potosí and Secretaría de Educación Pública - Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT-Ciencia Básica-178778). We are also grateful to the Catedras CONACyT-UASLP project (No. 553). Special thanks to Universidad Veracruzana-Campus Coatzacoalcos for the facilities granted for sampling and obtaining samples of the giant toads.
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this chapter.
References
- 1.
Duellman EW, Trueb L. Biology of Amphibians. 1st ed. USA: The Johns Hopkins University Press; 1994. pp. 1-47 - 2.
Samaniego-Herrera A, Peralta-García A, Aguirre-Muñoz A. Vertebrados de las islas del Pacífico de Baja California-Guía de Campo. 1st ed. Mexico: Grupo de Ecología y Conservación de Islas, A.C.; 2007 - 3.
Wells KD. The Ecology and Behavior of Amphibians. Chicago, USA: University of Chicago Press; 2007 - 4.
Sparling WD, Linder G, Bishop AC, Krest KS. Ecotoxicology of Amphibians and Reptiles. SETAC Technical Publications Series. 2nd ed. Florida, USA: CRC Press; 2010. pp. 13-167 - 5.
Alford R. Declines and the global status of amphibians. In: Sparling WD, Linder G, Bishop AC, Krest KS, editors. Ecotoxicology of Amphibians and Reptiles. 2nd ed. USA: CRC Press; 2010. pp. 13-45 - 6.
Venturino A, Rosenbaum E, Caballero de Castro A, Anguiano OL, Gauna L, Fonovich de Schroeder T, Pechen de D’Angelo AM. Biomarkers of effect in toads and frogs. Biomarkers: Biochemical Indicators of Exposure, Response, and Susceptibility to Chemicals. 2004; 8 (3-4):167-186 - 7.
Zug GR, Zug PB. The marine toad, Bufo marinus : A natural history Resumé of native populations. Smithsonian Contributions to Zoology. 1979;284 (284):1-58 - 8.
Feder EM, Burggren WW. Environmental Physiology of the Amphibians. USA: The University of Chicago Press; 1992 - 9.
Pizzatto L, Shine R. The behavioral ecology of cannibalism in cane toads ( Bufo marinus ). Behavioral Ecology and Sociobiology. 2008;63 (1):123-133 - 10.
González-Mille DJ, Espinosa-Reyes G, Rivero-Pérez NE, Trejo-Acevedo A, Nava-Montes AD, Ilizaliturri-Hernández CA. Persistent organochlorine pollutants (POPs) and DNA damage in giant toads ( Rhinella marina ) from an industrial area at Coatzacoalcos, Mexico. Water, Air, and Soil Pollution. 2013;224 (11):1-14 - 11.
Ilizaliturri-Hernández CA, González-Mille DJ, Mejía-Saavedra J, Espinosa-Reyes G, Torres-Dosal A, Pérez-Maldonado I. Blood lead levels, δ-ALAD inhibition, and hemoglobin content in blood of giant toad ( Rhinella marina ) to asses lead exposure in three areas surrounding an industrial complex in Coatzacoalcos, Veracruz, Mexico. Environmental Monitoring and Assessment. 2013;185 (2):1685-1698 - 12.
Solís F, Ibáñez R, Hammerson G, Hedges B, Diesmos A, Matsui M, et al. Rhinella marina . The IUCN Red List of Threatened Species. 2009 - 13.
SEMARNAT. Norma Oficial Mexicana NOM-059-SEMARNAT-2001: Protección ambiental-Especies nativas de México de Flora y Fauna Silvestres-Categorías en Riesgo y Especificaciones para su Inclusión, Exclusión o Cambio-Lista de Especies en Riesgo, Diario Oficial de la Federación 06-03-2002, México - 14.
Dohm MR, Mautz WJ, Doratt RE, Stevens JR. Ozone exposure affects feeding and locomotor behavior of adult Bufo marinus . Environmental Toxicology and Chemistry. 2008;27 (5):1209-1216 - 15.
Zupanovic Z, Musso C, Lopez G, Louriero CL, Hyatt AD, Hengstberger S, Robinson AJ. Isolation and characterization of iridoviruses from the giant toad Bufo marinus in Venezuela. Diseases of Aquatic Organisms. 1998;33 (1):1-9 - 16.
Linzey D, Burroughs J, Hudson L, Marini M, Robertson J, Bacon J, Nagarkatti M, Nagarkatti P. Role of environmental pollutants on immune functions, parasitic infections and limb malformations in marine toads and whistling frogs from Bermuda. International Journal of Environmental Health Research. 2003; 13 (2):125-148 - 17.
McCoy KA, Bortnick LJ, Campbell CM, Hamlin HJ, Guillette LJ, St. Mary CM. Agriculture alters gonadal form and function in the toad Bufo marinus. Environmental Health Perspectives. 2008; 116 (11):1526-1532 - 18.
Walker CH. Organic Pollutants: An Ecotoxicological Perspective. USA: CRC Press/Taylor & Francis; 2009 - 19.
Roméo M, Giambérini L. History of biomarkers. In: Amiard-Triquet C, Amiard JC, Rainbow SP, editors. Ecological Biomarkers. USA: CRC Press/Taylor & Francis; 2013. pp. 14-44 - 20.
Sibley PK, Hanson ML. Ecological impacts of organic chemicals on freshwater ecosystems. In: Sánchez-Bayo F, Van den Brink P, Mann MR, editors. Ecological Impacts of Toxic Chemicals. Bentham e Books; 2011. pp. 138-164 - 21.
Linder G, Lehman C, Bidwell J. Ecotoxicology of amphibians and reptiles in a nutshell. In: Sparling WD, Linder G, Bishop AC, Krest KS, editors. Ecotoxicology of Amphibians and Reptiles. 2nd ed. USA: CRC Press; 2010. pp. 69-103 - 22.
Vázquez-Botello A, Páez FE. Problema Crucial: La Contaminación. 1st ed. Mexico: Centro de Ecodesarrollo; 1987 - 23.
Bozada-Robles L, Bejarano-González F. Los Contaminantes Orgánicos Persistentes en el Istmo Mexicano. 1st ed. México: Red de Acción sobre Plaguicidas y Alternativas en México (RAPAM); 2006 - 24.
Stringer R, Labunska I, Bridgen K. Organochlorine and Heavy Metal Contaminants in the Environment around the Complejo Petroquimicos Paharitos, Coatzacoalcos, Mexico. Exeter, UK: Greenpeace Research Laboratories, Department of Biological Sciences, University of Exeter; 2001 - 25.
Blake A. The Next Generation of POPS: PBDEs and Lindane. “Keep the Promise, Eliminate POPs Campaign” Campaing and Community Monitoring Working Group of the International POPs Elimination Network (IPEN) Report; 2005 - 26.
Riojas-Rodríguez H, Baltazar-Reyes MC, Meneses F. Volatile organic compound presence in the environmental samples near a petrochemical complex in Mexico. Epidemiology. 2008; 19 (1):219 - 27.
Espinosa-Reyes G, Ilizaliturri-Hernández C, González-Mille D, Mejía-Saavedra J, Nava AD, Cuevas M, Cilia-López G. Contaminantes orgánicos persistentes en la cuenca baja del río Coatzacoalcos, Veracruz. In: Botello AV, Rendón von Osten J, Benítez JA, Gold-Bouchot G, editors. Golfo de México. Contaminación e impacto ambiental: diagnóstico y tendencias. 2da. edición. Mexico: UAC, UNAM-ICMYL, CINVESTAV-Unidad Mérida; 2013. pp. 309-322 - 28.
Espinosa-Reyes, G, Ilizaliturri-Hernández CA, González-Mille DJ, Costilla R, DíazBarriga F, Cuevas MdC, Martínez MA, Mejía-Saavedra J. DNA damage in earthworms ( Eisenia spp.) as an indicator of environmental stress in the industrial zone of Coatzacoalcos, Veracruz, Mexico. Journal of Environmental Science and Health. Part A, Toxic/Hazardous Substances & Environmental Engineering. 2010;45 (1):59-55 - 29.
González-Mille DJ, Ilizaliturri-Hernández CA, Espinosa-Reyes G, Costilla-Salazar R, Díaz-Barriga F, Ize-Lema I, Mejía-Saavedra J. Exposure to persistent organic pollutants (POPs) and DNA damage as an indicator of environmental stress in fish of different feeding habits of Coatzacoalcos, Veracruz, Mexico. Ecotoxicology. 2010; 19 (7):1238-1248 - 30.
Pelallo-Martínez NA, Ilizaliturri-Hernández CA, Espinosa-Reyes G, Carrizales-Yáñez L, González-Mille DJ. Assessment of exposure to lead in humans and turtles living in an industrial site in Coatzacoalcos Veracruz, Mexico. Bulletin of Environmental Contamination and Toxicology. 2011; 86 (6):642-645 - 31.
Herpetological Animal Care and Use Committee (HACC). Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research. 2nd ed. USA; 2010 - 32.
Fairbrother A. Clinical biochemistry. In: Fossi MC, Leonzio C, editors. Nondestructive Biomarkers in Vertebrates. USA: Lewis Publishers; 1994. pp. 63-91 - 33.
Arrieta MA, Bruzzone L, Apartín C, Rosenberg CE, Fink NE, Salibián A. Biosensors of inorganic lead exposure and effect in an adult amphibian. Archives of Environmental Contamination and Toxicology. 2004; 46 (2):224-230 - 34.
Tokar JE, Boyd AW, Freedman HJ, Waalkes PM. Toxic Effects of Metals. In: Klassen DC, editor. (Ed.), Casarett & Doull’s Toxicology. The Basic Science of Poisons. 8th ed. USA: McGraw Hill Education; 2013. p. 981-1030 - 35.
Berlin BA, Schaller KH. European standardized method for the determination of δ-aminolevulinic acid dehydratase activity in blood. Journal of Clinical Chemistry and Clinical Biochemistry. 1974; 12 (8):389-390 - 36.
Hopkins WA, Rowe C. Interdisciplinary and hierarchical approaches for studying the effects of metals and metalloids on amphibians. In: Sparling WD, Linder G, Bishop AC, Krest KS, editors. Ecotoxicology of Amphibians and Reptiles. 2nd ed. USA: CRC Press; 2010. pp. 325-336 - 37.
Fossi MC, Leonzio C. Nondestructive Biomarkers in Vertebrates. USA: Lewis Publishers; 1994 - 38.
Blaxhall PC. The haematological assessment of the health of freshwater fish: A review of selected literature. Journal of Fish Biology. 1972; 4 (4):593-604 - 39.
Lajmanovich R, Cabagna M, Peltzer P, Stringhini G, Sanchez-Hernandez J. Hematological parameters of health status in the common toad Bufo arenarum in agroecosystems of Santa Fe Province, Argentina. Applied Herpetology. 2005;2 (4):373-380 - 40.
Hemocue America. HemoCue® Hb 201+ System Instructions. Hemocue America, California, USA; 2003 - 41.
Linder G, Palmer B, Little E, Rowe C, Henry P. Physiological ecology of amphibians and reptiles. In: Sparling WD, Linder G, Bishop AC, y Krest KS, editors. Ecotoxicology of Amphibians and Reptiles, 2nd ed. USA: CRC Press; 2010, p. 105-166 - 42.
Stansley W, Roscoe DE. The uptake and effects of lead in small mammals and frogs at a trap and skeet range. Archives of Environmental Contamination and Toxicology. 1996; 30 (2):220-226 - 43.
Arrieta MA, Perí SI, Apartín C, Rosenberg CE, Fink NE, Salibián A. Blood lead concentration and δ-aminolevulinic acid dehydratase activity in adult Bufo arenarum . Archives of Physiology and Biochemistry. 2000;108 (3):275-280 - 44.
Espín S, Martínez-López E, Jiménez P, María-Mojica P, García-Fernández AJ. Delta-aminolevulinic acid dehydratase (δALAD) activity in four free-living bird species exposed to different levels of lead under natural conditions. Environmental Research. 2015; 137 :185-198 - 45.
Gómez-Ramírez P, Martínez-López E, María-Mojica P, León-Ortega M, García-Fernández AJ. Blood lead levels and δ-ALAD inhibition in nestlings of Eurasian eagle owl ( Bubo bubo ) to assess lead exposure associated to an abandoned mining area. Ecotoxicology. 2010;20 (1):131-138 - 46.
Martínez-López E, Sousa AR, María-Mojica P, Gómez-Ramírez P, Guilhermino L, García-Fernández AJ. Blood δ-ALAD, lead and cadmium concentrations in spur-thighed tortoises ( Testudo graeca ) from southeastern Spain and northern Africa. Ecotoxicology. 2010;19 (4):670-677 - 47.
Rosales-Hoz L, Cundy AB, Bahena-Manjarrez JL. Heavy metals in sediment cores from a tropical estuary affected by anthropogenic discharges: Coatzacoalcos estuary, Mexico. Estuarine, Coastal and Shelf Science. 2003; 58 (1):117-126 - 48.
Rosales-Hoz L, Carranza-Edwards A. Estudio Geoquímico de Metales en el estuario del Río Coatzacoalcos. In Botello AV, Rendón von Osten J, Benítez JA, y Gold-Bouchot G. (Eds.), Golfo de México. Contaminación e impacto ambiental: diagnóstico y tendencias. 2nd ed. UAC, UNAM-ICMYL, CINVESTAV-Unidad Mérida, Mexico; 2005. p. 389-406 - 49.
Ruelas-Inzunza J, Gárate-Viera Y, Páez-Osuna F. Lead in clams and fish of dietary importance from Coatzacoalcos estuary (Gulf of Mexico), an industrialized tropical region. Bulletin of Environmental Contamination and Toxicology. 2007; 79 (5):508-513 - 50.
Altland PD, Brace KC. Red cell life span in the turtle and toad. American Journal of Physiology. 1962; 203 (6):1188-1190 - 51.
Cabagna MC, Lajmanovich RC, Peltzer PM. Induction of micronuclei in tadpoles of Odontophrynus americanus (Amphibia: Leptodactylidae) by the pyrethroid insecticide cypermethrin. Toxicological & Environmental Chemistry. 2006;88 (4):37-41 - 52.
Said EMR, Saber AS, Osman GMA. Haemotoxic and genotoxic potential of lead on the Egyptian toad Amietophrynus regularis . International Journal of Ecotoxicology and Ecobiology. 2016;1 (3):94-102 - 53.
Cazenave J, Wunderlin DA, Hued AC, Bistoni MDLÁ. Haematological parameters in a neotropical fish, Corydoras paleatus (Jenyns, 1842) (Pisces, Callichthyidae), captured from pristine and polluted water. Hydrobiologia. 2005; 537 (1-3):25-33 - 54.
Rice TMR, Blackstone BJB, Nixdorf WLN, Taylor DHT. Exposure to lead induces hypoxia-like responses in bullfrog larvae ( Rana catesbeiana ). Environmental Toxicology and Chemistry. 1999;18 (10):2283-2288 - 55.
Wu RSS. Hypoxia: From molecular responses to ecosystem responses. Marine Pollution Bulletin. 2002; 45 (1-12):35-45 - 56.
Bӑncilӑ IR, Hartel T, Plӑiaşu R, Smets J, Cogӑlniceanu. Comparing three body condition indices in amphibians: a case study of yellow-bellied toad Bombina variegata , Amphibia-Reptilia. 2010;31 (4):558-562 - 57.
Amiard-Triquet C, Cossu-Leguille C, Mouneyrac C. Biomarkers of defense, tolerance, and ecological consequences. In: Amiard-Triquet C, Amiard JC, Rainbow SP, editors. Ecological Biomarkers. USA: CRC Press/Taylor & Francis; 2013. pp. 45-74 - 58.
Brodeur JC, Suarez RP, Natale GS, Ronco AE, Elena Zaccagnini M. Reduced body condition and enzymatic alterations in frogs inhabiting intensive crop production areas. Ecotoxicology and Environmental Safety. 2011; 74 (5):1370-1380 - 59.
Bervoets L, Blust R. Metal concentrations in water, sediment and gudgeon ( Gobio gobio ) from a pollution gradient: Relationship with fish condition factor. Environmental Pollution. 2003;126 (1):9-19 - 60.
Jaspers VLB, Covaci A, Voorspoels S, Dauwe T, Eens M, Schepens P. Brominated flame retardants and organochlorine pollutants in aquatic and terrestrial predatory birds of Belgium: Levels, patterns, tissue distribution and condition factors. Environmental Pollution. 2006; 139 (2):340-352 - 61.
Dedourge-Geffard O, Palais F, Geffard A, Amiard-Triquet C. Origin of energy metabolism impairments. In: Amiard-Triquet C, Amiard JC, Rainbow SP, editors. Ecological Biomarkers: Indicators of Ecotoxicological Effects. USA: CRC Press/Taylor & Francis; 2013 - 62.
Gans C, Gorniak GC. Functional morphology of lingual protrusion in marine toads ( Bufo marinus ). The American Journal of Anatomy. 1982;163 (3):195-222 - 63.
Hsu MJ, Selvaraj K, Agoramoorthy G. Taiwan’s industrial heavy metal pollution threatens terrestrial biota. Environmental Pollution. 2006; 143 (2):327-334 - 64.
Reinecke AJ, Reinecke SA, Musilbono DE, Chapman A. The transfer of lead (Pb) from earthworms to shrews ( Myosorex varius ). Archives of Environmental Contamination and Toxicology. 2000;39 (3):392-397