Persistent Organochlorine Pollutants (POPs) and ...

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DeGarady, C. J., & Halbrook, R. S. (2003). Impacts from PCB accumulation on amphibians inhabiting streams flowing from the Paducah gaseous diffusion plant.

Water Air Soil Pollut (2013) 224:1781 DOI 10.1007/s11270-013-1781-0

Persistent Organochlorine Pollutants (POPs) and DNA Damage in Giant Toads (Rhinella marina) from an Industrial Area at Coatzacoalcos, Mexico Donaji J. Gonzalez-Mille & Guillermo Espinosa-Reyes & Norma E. Rivero-Pérez & Antonio Trejo-Acevedo & Alma D. Nava-Montes & César A. Ilizaliturri-Hernández Received: 3 May 2013 / Accepted: 14 October 2013 # Springer Science+Business Media Dordrecht 2013

Abstract Coatzacoalcos River Basin is one of the most important hydrological and ecological regions of Mexico. Persistent organic pollutants (POPs) and other contaminants have been detected in several environmental matrices in the region. So far, there have been a few efforts to evaluate exposure and effects on wildlife in the area. The purpose of this research work is to measure exposure to POPs and deoxyribonucleic acid (DNA) damage in specimens of giant toads (Rhinella marina) taken from two zones near the industrial complex by the lower Coatzacoalcos River. Total POPs levels in soil and toads' tissues are between 660.5 and 2,712.9 ng/g d.w. and 55.6 and 1,2471.9 ng/g l.w., respectively. We found differences between the body burdens of POPs from different toad tissues evaluated but it did not happen by site type. DNA damage in blood

D. J. Gonzalez-Mille : G. Espinosa-Reyes : C. A. Ilizaliturri-Hernández (*) Coordinación para la Innovación y la Aplicación de la Ciencia y Tecnología-Facultad de Medicina, Universidad Autónoma de San Luis Potosí, Sierra Leona #550, Lomas 2da Sección. CP, 78210 San Luis Potosí, SLP, México e-mail: [email protected] N. E. Rivero-Pérez : A. Trejo-Acevedo Centro Regional de Investigación en Salud Pública (CRISP), Instituto Nacional de Salud Pública, Tapachula, Chiapas, Mexico A. D. Nava-Montes Centro Nacional de Investigación y Capacitación Ambiental, Instituto Nacional de Ecología y Cambio Climático (INECC), Mexico City, Mexico

varies from 0.7 to 4.8 (olive tail moment) and 7.4 to 16.7 μm (tail length). DNA damage is found to be higher at industrial zones compared with urban zones. This study provides a data baseline about the POPs pollution status in soil and giant toads of the lower Coatzacoalcos River in Veracruz. Keywords Persistent organic pollutants . Rhinella marina . Coatzacoalcos River . Comet assay

1 Introduction Persistent organic pollutants (POPs) are stable synthetic chemical compounds resistant to degradation. Because of this, once released, they have been observed to persist in the environment for years, even decades. They are transported at a low concentration by movement of fresh and marine water. As they are semi-volatile, they are transported over long distances in the atmosphere. The result is widespread distribution of POPs across the globe, including regions where they have never been used (Buccini 2003). POPs are ubiquitous in the environment; they have high lipophilicity, tend to accumulate in fatty tissue, and bio-magnify through food chains (Walker 2009). These pollutants have been linked to human and ecological health effects, and due to these reasons they have been banned or restricted in many countries (Buccini 2003). Biomonitoring wildlife can be used to detect chemical pollution as well as to evaluate the ecosystems health by using the species as systematic models in the evaluation

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of risks associated to pathways of real exposure. Wildlife species residing in polluted sites are exposed to complex mixtures of pollutants by multiple pathways which could hardly be evaluated in laboratory studies (Bernanke and Köhler 2009). Amphibians have been widely proposed as bio-indicators of environmental conditions due to the characteristics associated with their metabolism, life cycle, and ecology, while also are more vulnerable to extinction than other vertebrate groups (Sparling et al. 2010). Environmental contaminants are frequently suggested as potential primary factor or cofactor in amphibian decline (Collins and Storfer 2003); because of that, the evaluation of exposure and sublethal effects has become relevant in amphibian monitoring programs. The giant toad (Rhinella marina) is native and geographically widespread species in Mexico and Central America (Zug and Zug 1979). It is an omnivorous and opportunistic species, which indicates that toads would integrate different exposure paths due to the ingestion of a wide variety of food items and amphibious living habits. The giant toad is one of the largest amphibians in Mexico (adult body length ranges from 10 to 17 cm), with a life expectancy from 10 to 15 years in the wild. The high lipid–somatic index (2 to 10 % compared to less than 0.1 % in most anuran species after the spawning period) and the elevated hepatosomatic index along with its breeding biology make this species prone to bioaccumulation of organic pollutants and their toxicological effects. Recently, the giant toad has been used as an aquatic ecosystem biomonitor in the evaluation of infectious diseases (Zupanovic et al. 1998), organochlorine pesticides (Linzey et al. 2003), air pollution (Dohm et al. 2008), endocrine disruptors (McCoy et al. 2008), and lead pollution (Ilizaliturri-Hernández et al. 2013). The Coatzacoalcos river basin has been one of the most diverse biological areas in Mexico; since the 1970s, the Coatzacoalcos estuary has experienced a fast industrial and urban growth which combined with other productive activities such as agriculture and cattle farming have triggered a severe impact on the region's ecosystems. The Coatzacoalcos River is the fourth largest in Mexico by its annual runoff and it contributes with 18.2 % of the total flow that reaches the Gulf of Mexico. Nowadays, the Coatzacoalcos estuary houses one of the biggest and most important petrochemical industrial complexes of Mexico and Latin America (total production of petrochemical products is approximately 1.6 thousand million tons/year) and supports a population

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of 607,919 inhabitants (INEGI 2013). Environmental research at different environmental and biological compartments in the Coatzacoalcos estuary has shown the presence of heavy metals, hydrocarbon, volatile organic compounds, polybrominated compounds, dioxin, and persistent organic pollutants (Rosales-Hoz and Carranza-Edwards 1998; Bahena-Manjarrez et al. 2002; Rosales-Hoz et al. 2003; Pelallo-Martínez et al. 2011). There are a number of POPs that have been found in Coatzacoalcos. The dichlorodiphenyltrichloroethane (DDT) is an insecticide that was used to control agricultural pests and in health campaigns against within malaria endemic areas; it was abandoned in the year 2000 (Díaz-Barriga et al. 2003); presently, detectable levels of DDT and dichlorodiphenyldichloroethylene (DDE, the major degradation product of DDT) can be found in the Coatzacoalcos River. Hexachlorocyclohexane (HCH) is an insecticide with eight isomers, even though only the γisomer (lindane) has insecticidal properties. Currently, the permitted uses of lindane in Mexico are for ectoparasite control in livestock and human health control. Mirex and hexachlorobenzene (HCB) are insecticides; however, these were also used as a flame retardant in rubber, paper, plastic, and non-flammable paints. Polychlorinated biphenyls (PCBs) are industrial chemicals that had a wide variety of applications, mainly in the electrical industry in transformers and capacitors. Genotoxicity and endocrine disruption are the most worrying effects because of their ecological implications in aquatic and terrestrial ecosystems. Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms. All of the basic components of DNA (nitrogen bases and sugars) are possible targets of chemical alterations by genotoxic agents such as polycyclic aromatic hydrocarbons (PAHs), metals, and POPs. The comet assay is widely used to detect damage in vitro or in vivo caused to DNA by a broad spectrum to genotoxic agents in a wide range of ecological receptors. Recently, the comet assay has gained wide acceptance as an essential tool for genotoxicity assessment and biomonitoring studies due to sensitivity to detect low levels of DNA damage, easy and rapid implementation, flexibility, and low cost (Lee and Steinert 2003; Collins et al. 2008). Mexico has currently developed the long-term national monitoring program (PRONAME) which aims to identify and assess persistent, bioaccumulative and toxic substances (PBTs) in Mexican ecosystems and human

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populations in order to generate trustworthy information to be used for the design of public policy instruments, aimed at the reduction of the amount of these substances in the environment. The PBTs considered for monitoring environmental compartments include 12 POPs listed in the Stockholm Convention, heavy metals (Cd, Pb, Hg, As), and PAHs. The generation of information regarding the distribution and bioaccumulation of these substances as well as its effects on different groups of biota and humans is of national importance. There are a few studies proving their presence and bioaccumulation of POPs, as well as their effects on amphibian populations in Mexico. In addition to this, several genotoxic compounds (including some POPs) have been detected in the Coatzacoalcos region; therefore, the aims of this study are to (1) determine POPs levels in soil samples and different tissues of giant toad as well as (2) assess DNA damage as an integrative indicator of stress in two zones near the industrial complex (urban and industrial).

2 Materials and Methods 2.1 Study Area and Sampling Sites The lower Coatzacoalcos River area is located in the south-east of Veracruz, Mexico (18° 08′ 56″ N; 94° 24′

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41″ W); an area that is made up of urban, industrial, grazing, and agricultural areas that are immersed in wetlands. The region's wetlands are fed by the Coatzacoalcos River which is 322 km long, starting in the State of Oaxaca and going down to its mouth, draining an area of about 21,120 km2. The sampling was done in two adjacent sites to the Coatzacoalcos River in October 2006 (Fig. 1): urban zone—it is located on the banks of the Calzadas River, 7 km east of the Pajaritos Petrochemical Complex; this site was considered a reference area (minor exposure); industrial zone—it is located in favor of prevailing winds at 2 km south of the Pajaritos-Cangrejera Petrochemical Complexes. Within this area, important pollutants such as dioxins, hexachlorobenzene, and volatile compounds have been found on environmental matrices (Stringer et al. 2001). 2.2 Environmental and Biological Sampling Six sexually mature males toads were captured from both urban and industrial zones using nets in nocturnal transects within an area of 10,000 m2. Immediately after capturing toads, they were anesthetized with benzocaine. Blood samples of 1.5 mL were drawn using a heparinized needle and a syringe from cardiac puncture and then centrifuged in order to obtain plasma.

Fig. 1 Location of environmental and biological sampling points in Coatzacoalcos, Veracruz

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Dissection was performed on each of the specimens to extract the adipose, hepatic, and muscle tissues. Adipose and hepatic tissues samples were pooled to obtain the amount of samples required for analysis of contaminants. All toads were caught in October 2006 and collected with a Scientific Collector's Permit (wildlife scientific collector) issued by SEMARNAT (no. FAUT-0133) and handled in the laboratory in accordance with the Mexican normative (NOM-062ZOO-1999), for ethical and conservation purposes. Only 12 individuals were allowed to be collected. Three soil samples were taken around the toad sampling location areas. Surface soil samples (1–5 cm) were obtained with a stainless steel scoop on approximately 1 m2 surface area and stored in amber glass containers for further analysis. All samples were frozen at −20 °C until analysis. 2.3 Analysis of POPs Concentrations The following compounds were evaluated in tissue, plasma, and soil samples: α-HCH, β-HCH, γ-HCH, HCB, aldrin, dieldrin, mirex, α-chlordane, γchlordane, oxychlordane, trans-nonachlor, cisnonachlor, heptachlor epoxide, p,p′-DDT, p,p′-DDE, and PCBs (41 congeners for soil samples no. 17, 18, 28, 31, 33, 44, 49, 52, 70, 74, 82, 87, 95, 99, 101, 105, 110, 118, 128, 132, 138,149, 151, 153, 156, 158, 169, 170, 171, 177, 180, 183, 187, 191, 194, 195, 201, 205, 206, 208, 209 and only 14 congeners for tissue and plasma samples no. 28, 52, 99, 101, 105, 118, 128, 138, 153, 156, 187, 180, 183, 170). The method of extraction and quantification of POPs in tissue and plasma was carried out according to the method published by our group (Trejo-Acevedo et al. 2009; Gonzalez-Mille et al. 2010). POPs in soil were analyzed according to Espinosa-Reyes et al. (2010). The γ-HCHC13, endrin-C13, and PCB 14-C13 were used as internal standards and were added to all samples. Analysis of standard reference material EC-2 (National Water Research Institute, Canada) was conducted as quality control, with recoveries of 80 to 114 %. The detection limit for POPs was approximately 0.3 μg/L for all samples. 2.4 Comet Assay (SCGE) DNA damage was evaluated by comet assay in the blood according to that described by Singh et al.

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(1988) with some modifications. An aliquot of 10 μL of mixture of agarose low melting point blood (30:1) was suspended in a layer of 0.5 % low melting point agarose (37 °C) and placed on a precoated slide with a layer of 0.5 % regular agarose. After, the cells were placed in a lysis solution (10 mM Tris–HCl, 2.5 M NaCl, and 0.1 M Na2EDTA, pH >13), to which 10 % dimethylsulfoxide and 1 % Triton X-100 were added just before use. The solution was chilled prior to use and the lysis duration was a maximum of 1 week at 4 °C. After cell lysis, slides were placed in an alkaline electrophoresis buffer for 5 min, followed by denaturation in alkaline solution (300 mM NaOH and 1 mM Na2EDTA, pH 13) and by electrophoresis in the same buffer for 10 min at 25 V, 300 mA. Samples were stained with ethidium bromide (0.05 mM) and examined under a fluorescence microscope (Nikon Eclipse E400). The comet image magnification was ×200. We measured the olive tail moment and tail length of 100 cells per individual with image software (Komet 4.0, Kinetic Imaging Ltd., Liverpool, UK). 2.5 Statistical Analysis The data is reported as the mean, standard error, and range. Concentrations of PCBs congeners and organochlorine compounds were added to obtain the total of these compounds (∑PCBs and ∑POPs). The concentrations in wet weight (w.w.) were standardized by lipid content (l.w.). An analysis of similarity (ANOSIM, Clarke 1993) followed by pairwise comparisons was used to investigate the differences in mixture of POPs content in tissues. ANOSIM “R” value close to +1 indicates that there are clear differences in patterns among the groups tested; a value near zero means that the distribution of patterns is as similar among the groups as within the groups. When pattern differences were identified, the similarity percentage subroutine (SIMPER) was used to identify which pollutants contributed the most to the observed differences. Nonmetric multidimensional scaling plots (Kruskal 1964) were constructed to display clustering patterns in the samples by POPs content in tissues. Multivariate analysis was performed with a nonparametric statistical software PRIMER V.6.1.15 (PRIMER-E Ltd, UK). The significant level of ANOSIM “R” value was calculated by a permutation test and was considered statistically significant at p ΣHCH > HCB > ΣDDT > Mirex. According to limits set by Screening Quick Reference Tables of National Oceanic and Atmospheric Administration (NOAA), and the Canadian Environmental Quality Guidelines (CCME), pollutants of greater concern due to their hazard coefficients

(HC = average concentration in soil/ ecological soil screening levels) are as follows: PCBs (1,656.6), δHCH (5.6), α-HCH (3.0), DDT (2.5), and HCB (1.7), which suggest risk for terrestrial organisms in the region. It must be mentioned that for some organic compounds found in site (β-HCH and Mirex), there are not levels of reference in soil. PCBs congeners found in soil samples were 52, 70, 74, 82, 87, 90, 101, 105, 110, 118, 128, 132, 138, 149, 151, 153, 156, 158, 170, and 180. Some of these congeners belong to Aroclors of more extensive use in the industry (ATSDR 2004). The Coatzacoalcos area has had a strong industrial development since the 1960s; therefore, it is possible that these oils had been used in electrical transformers and substations. Levels of PCBs and HCB are strongly associated to industrial activities. The presence of these pollutants has been observed and reported in other regions of the world where there is petrochemical activity and it is associated to production of chlorine and derivatives (ATSDR 2002a). HCB has been used as an excipient when formulating pesticides, so its presence

Table 1 Concentrations of POPs (in nanogram per gram, d.w.) in soil and tissues (in nanogram per gram, l.w.) collected in Coatzacoalcos, Veracruz Soil (n=6)

Fat body (n=6a)

Liver (n=6a)

Muscle (n=12)

Plasma (n=12)


300.5±63.4 (99.0–5.08.3)

43.1±14.1 (13.5–93.4)

39.1±21.6 (6.6–143.3)

48.0±8.3 (22.9–113.0)



33.7±9.5 (64.1–23.4)

26.4±6.5 (10.0–51.4)

3.1±2.3 (0.3–14.8)

14.7±7.0 (0.8–83.3)
























8.9±7.1 (ND–43.2)

28.7±10.4 (10.6–77.4)

9.2±5.5 (0.9–35.5)




342±107.2 (56.8–831.0)

7.7±2.3 (0.5–141)

6.0±1.6 (0.8–11.4)

3.2±0.6 (0.6–7.3)



1.2±0.7 (ND–3.8)

7.4±1.8 (3.2–15.4)

80.1±21.2 (10.6–156.5)

23.3±3.3 (0.8–36.1)






















Values represented the mean, standard error and (range) N.D. non detected a

Pooled samples\


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added to mirex in the zone may be related to farming activities, mainly upriver of the Coatzacoalcos' river (Gonzalez-Mille et al. 2010). DDT was used in Mexico in order to maximize agricultural production and control diseases transmitted by vectors (ATSDR 2002b). Even though DDT was prohibited in the beginning of the 1990s because of its effects in the environment, DDT residual stills exist in the environment because of the characteristics of persistence in this compound. The DDT/DDE relationship obtained from the average of samples of soil is greater than 1, which suggests that recent DDT is being incorporated in the environment either by environmental means or by the alterations of natural reservoir such as sediment resuspension or soil removal; therefore, the main cause is continuously dredged to avoid its obstruction and allow the traffic of commercial vessels. The presence of DDT in Coatzacoalcos may be related to vector control campaigns carried in the zone to control malaria. It is also important to considerer the possible recent use of pesticide, which may enter the country in a clandestine manner. HCH concentrations were present in the following proportions, α-HCH (77 %), β-HCH (9 %), and γ-HCH (14 %), which could correspond to the formulation of HCH technical grade [α-HCH, 60–70 %; β-HCH, 5– 12 %; and γ-HCH, 10–15 %] (Willett et al. 1998; Yim et al. 2005). HCH technical grade is mainly used to control ectoparasites in cattle, so it is possible that concentrations registered in soil may be due to contributions from municipalities with greater cattle activity located in the upper Coatzacoalcos River. Fig. 2 MDS of the distribution of contaminants in giant toad (R. marina) by tissue (Stress 2D: 0.09). FB, fat bodies; M, muscle; L, liver; P, plasma

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3.2 Concentration of POPs in Giant Toad Tissues POPs concentrations in different giant toad tissues are shown in Table 1. Seven organochlorine pesticides and 9 polychlorinated biphenyls were detected in the 15 and 14 analyzed, respectively. All captured organisms showed detectable levels of DDE, lindane, and PCBs. With the exception of plasma, the same pattern of contaminant presence was found (∑DDT > ∑HCH > ∑PCBs, HCB, and mirex) in the adipose, liver, and muscular tissue. Figure 2 shows the samples sorting diagram in relation to POPs body loads by kind of tissue. The diagram shows a clear pooling of samples, which suggests significant differences among tissues (ANOSIM global R=0.621, pβHCH>γ-HCH, except the liver tissue that presented this pattern α-HCH > γ-HCH > β-HCH. According to literature, general distribution patterns in vertebrates (mammals, birds, and fish) is β-HCH>α-HCH >γHCH; this distribution pattern is mainly determined by the compound persistence, route of exposure, species metabolism, and trophic position (Willett et al. 1998). Data found in tissue belong to the pattern observed in soil, which suggests the exposure of these organisms to polluted soil with technical-grade lindane used with the livestock in the region. One possible explanation to

interpret the presence of the isomer γ-HCH in plasma samples is the chronic exposure to isomer by different routes. γ-HCH is more volatile in comparison to other isomers, which implies an important transport by air (Walker 2009). The giant toad has been used as a biomonitor of atmospheric pollutants because of its capacity of pulmonary and cutaneous respiration more developed than other amphibians that may represent a greater exposure to these kinds of pollutants. Because of the above, there may exist other important exposure routes towards HCHs besides soil that have not been explored in toads and that may contribute in a relevant manner to body load and its distribution in the tissues. Differences in proportions of isomers may indicate different sources, routes, and times of exposure, and it is still unknown in a clear way for many species the influence of processes such as intake, distribution, metabolism, and storage in the differences of distribution of isomers in tissues (Willett et al. 1998). The main levels of γ-HCH detected in the study are comparable with those observed in other studies conducted with adult amphibians in the wild (N.D.– 10.0 mg/kg w.w, Table 3). PCBs congeners present in the tissue samples were 52,101, 105, 118, 138, 153, 156, 170, and 180. These congeners (except 52) belong in a greater proportion (>30 %) to Aroclor 1254, one of the best-selling commercial mixes in the world, which suggests that the source of these compounds may be related to the use of these oils at sub-electrical stations located in the in the region's industrial zones. Congeners detected are characterized by being some of the most persistent in the environment and by being absorbed in a greater proportion in organisms. Congeners presence pattern

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Table 3 Concentration of organochlorine pesticides and polychlorinated biphenyls (in milligram per kilogram w.w unless specified) in amphibians Specie





Chaumus arenarum


ND–4.5 ND–5.6


Jofré et al. (2008)


Jofré et al. (2008)


Jofré et al. (2008)


Jofre et al. (2008)

Hypsiboas cordobae

Leptodaclylus mystacinus

Melanophyniscus Stelzneri






1.1–1.7 3.9–5.4






ND–6.0 3.5–6.9






10.6 6.9





Mixed species




DeGarady and Halbrook (2003)

Mixed species




Phaneuf et al. (1995)

Necurus maculosus



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