Biological effects of environmental pollutants in American Oyster, Crassostrea virginica: a field study in Laguna de Terminos, Mexico

Int. J. Environment and Health, Vol. 1, No. 2, 2007

171

Biological effects of environmental pollutants in American Oyster, Crassostrea virginica: a field study in Laguna de Terminos, Mexico Gerardo Gold-Bouchot*, Omar Zapata-Pérez, Victor Ceja-Moreno, Gabriela RodríguezFuentes, Raúl Simá-Alvarez, Ma. Leopoldina Aguirre-Macedo and Victor M. Vidal-Martínez Departamento de Recursos del Mar, Cinvestav Unidad Mérida, Yucatán 97310, México E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] E-mail: [email protected] *Corresponding author

Luisa Da Ros and Cristina Nasci Istituto di Scienze Marine/Biologia del Mare, ISMAR-CNR, Venice, Italy E-mail: [email protected] E-mail: [email protected] Abstract: The response of Crassostrea virginica to a complex mixture of toxic contaminants was studied at four sites in Laguna de Terminos, Mexico. Contaminants assessed were heavy metals, organochlorine compounds, and hydrocarbons. Biomarkers (cholinesterase activity, neutral red retention, and metallothionein), histopathology and prevalence of Perkinsus marinus were used to evaluate the effects of contaminants. The resultant contaminant body burden was moderately as high as a whole, and biomarker levels were also moderate. Oedema in the gills and systemic haemocytosis were the only histopathological lesions observed. P. marinus was never evidenced in the oyster tissues examined. Statistical results from principal components analysis show that metallothioneins are positively correlated with copper and chromium, neutral red and oedema with chlordanes, pentachloroanisol, drins, polychlorobiphenyls and total pesticides, total hydrocarbons and polycyclic aromatic hydrocarbons, whereas cholinesterase activity is negatively correlated to Cu and Cr. The biomarkers used were sensitive indicators responding to moderate levels of pollutants. Keywords: biomarkers; cholinesterase; POPs; metallothionein; metals; neutral red; Perkinsus marinus; Terminos Lagoon.

Copyright © 2007 Inderscience Enterprises Ltd.

172

G. Gold-Bouchot et al. Reference to this paper should be made as follows: Gold-Bouchot, G., ZapataPérez, O., Ceja-Moreno, V., Rodríguez-Fuentes, G., Simá-Alvarez, R., AguirreMacedo, M.L., Vidal-Martínez, V.M., Da Ros, L. and Nasci, C. (2007) ‘Biological effects of environmental pollutants in the American Oyster, Crassostrea virginica: a field study in Laguna de Terminos, Mexico’, Int. J. Environment and Health, Vol. 1, No. 2, pp.171–184. Biographical notes: Gerardo Gold-Bouchot received his PhD from the Center for Research and Advanced Studies in 1991. He has been a Researcher at Cinvestav-Merida in the Department of Marine Resources since 1985. He studies the distribution and effects of toxic pollutants (pesticides, hydrocarbons and metals) in the coastal zone. He was the Director of Cinvestav Merida for 5 years. He also serves in several Consultative Councils of different national and international organisations. Omar Zapata-Pérez has obtained a PhD in Toxicology, and is actually a Fulltime Professor at the Center for Research and Advanced Studies (CINVESTAV). His current studies have been focused to evaluate the effects of contaminants and other human-made perturbations on important biological functions in marine organisms. His group is working with molecular, physiological and behavioural responses (biomarkers) of cells, tissues, organs and organisms to assess the exposure to polluted environments, and he is interested in the chemical–biological interactions in marine animals, including biochemical and molecular mechanisms of toxicity, bioactive marine natural products and alterations in the gene expression produced by marine pollutants. Victor Ceja-Moreno received his MSc from the Mérida Technologic Institute in 2003. He has been a research assistant at Cinvestav since 1991 in the Marine Geochemistry laboratory. He has focused on the distribution of priority pollutants such as petroleum hydrocarbons, chlorinated pesticides and trace metals in tropical aquatic environments; and their toxicological effects on aquatic organisms. Gabriela Rodríguez-Fuentes received her BSc (Environmental Engineering) degree from IPN in Mexico in 1997, MSc (Marine Biology) in 1999 from CINVESTAV-IPN in Mexico and received her PhD Degree (Marine Sciences) from CINVESTAV-IPN in Mexico in 2005. At present she holds the position of Postdoctoral Researcher at the University of California Riverside. Her research interests are in the areas of pesticide metabolism and the effect of these pollutants on cholinesterases. She has worked in the development of monitoring programmes that include the use of cholinesterases as the biomarkers of effect. Her results have been published and presented in international conferences. Ma. Leopoldina Aguirre-Macedo is a Full-time Professor at the Center for Research and Advanced Studies (CINVESTAV) in Merida Yucatan, Mexico. She has a PhD in Parasite Ecology and her research focuses on taxonomy, biology and ecology of parasites and their transmission processes in aquatic organisms. She works mainly on parasites of gasteropods and bivalves. Raúl Antonio Simá Álvarez received his MSc from the Institute of Aquaculture of the Stirling University, UK, in 1991. He has been working for about 20 years in CINVESTAV Mérida in the Marine Resources Department. He studies the diseases of aquatic organisms (fish, shrimp and molluscs), and particularly he is interested in the histopathology of aquatic organisms and their relation to exposure to pollutants.

Biological effects of environmental pollutants

173

Victor M. Vidal-Martínez is a Full-time Professor at the Center for Research and Advanced Studies (CINVESTAV) in Merida Yucatan, Mexico. He has a PhD in Ecology of Parasites from the University of Exeter, UK. His research is on basic parasite ecology such as the structuring processes in helminth communities of fish and its predictability, as well as the diagnoses of diseases in aquatic organisms, and the suitability of micro and macro parasites as the bioindicators of environmental impact. Luisa Da Ros is a Research Biologist at the Institute of Marine Science of the National Research Council, Italy. She has been working for about 20 years in the field of marine mollusc histopathology, and is being involved in national projects mainly regarding the monitoring of diseases and stress conditions in edible bivalves. Since 1991, she has addressed her research studies mainly to the evaluation of the well-being of marine molluscs in relation to the effects of pollutants and environmental stressors. At present, her main research interest is the deployment of histochemical and histopathological biomarkers as the early warning systems in marine environmental monitoring. Cristina Nasci served as a Senior Researcher at the Institute of Marine Science of National Research Council in Venice from 1982 to 2004, and has been involved in several national and international projects sponsored by national and international organisations (UNESCO, European Union, USA, and Mexico). She is the Project Manager at the Environmental Studies and Analysis Division of Thetis in Venice since 2004. Her main expertise is in the management of environmental quality assessment projects, in particular in the environmental quality assessment by applying biological stress indices.

1

Introduction

A biomarker is a biochemical or cellular variation that can be measured in tissue or body fluid samples of an organism which provides the evidence of exposure to and/or the effects of one or more chemical pollutants (Depledge, Aagaard and Gyorkos, 1995). Due to the fact that in nature organisms are rarely exposed to a single toxicant and the interactions between different xenobiotics might complicate the biological responses, the set-up of the suites of biomarkers focused on specific environments is largely recommended. Recent field studies in coastal marine environments have demonstrated that through the adoption of a battery of biomarkers not only diagnosis of the present pollution problems may be more effective but also early warning of damage may be successfully provided (Barhoorn and van Vuren, 2004). Among the biomarkers of exposure, the induction of metallothioneins (MTs) in response to metal exposure is well documented (Roesijadi, 1992; George and Olsson, 1994; Cosson and Amiard, 2000). George and Olsson (1994) have suggested that fish species would be better candidates for monitoring use than invertebrates; however, due to the ability of sedentary bivalves to represent the local situation and the experience gained from chemical biomonitoring (NAS, 1980; RNO, 2000), the possible use of bivalve MTs as a biomarker has given rise to a large number of studies, which have been recently reviewed (Langston, Bebianno and Burt, 1998; Cosson, 2000; Isani et al., 2000). As a result, MT analysis in the digestive gland of mussels has been recommended in the framework of the Mediterranean Action Plan (UNEP/RAMOGE, 1999).

174

G. Gold-Bouchot et al.

Similarly, many field studies have demonstrated the interest for the measurement of acetylcholinesterase (AChE) activity in invertebrates as an exposure biomarker for organophosphorus and carbamate pesticides in coastal waters and rivers (Moulton, Fleming and Purnell, 1996; Varela and Augspurger, 1996). Specifically, AChE activity is inhibited in the presence of this class of compounds (Holland, Coppage and Butler, 1967; Day and Scott, 1990). Moreover, because of the relative short life of organophosphorus pesticides in the environment, the determination of their effect on the cholinesterases (ChEs) in non-target organisms has been suggested as the most effective tool for monitoring environmental contamination and organism exposure to such xenobiotics (Fulton and Key, 2001). In contrast, the decrease of the lysosomal membrane integrity, which constitutes a very useful index of cellular damage, is considered a non-specific marker of effect, sensitive to different classes of pollutants. Lysosomes in the digestive gland of mussels and oysters are the organelles dealing primarily with nutrition, tissue repair and cellular components turnover. The stability of the lysosomal membrane is currently evaluated histochemically in the frozen sections of digestive gland, applying the lysosomal membrane stability test (Moore, 2002), as well as in vitro using the NRR assay in the lysosomes of the haemocytes (Lowe, Soverchia and Moore, 1995). The NRR quantifies the time of leakage of the dye from the lysosomes into the cytosol, which decreases in stressed mussels, reflecting the impairment/damage of the membranes due to stress conditions (Lowe and Pipe, 1994). Laguna de Terminos, in the southern Gulf of Mexico, is one of the most studied coastal ecosystems in Mexico, due to its importance as the nursery ground for several commercially important species such as shrimp and fish and because of its diversity in terms of species and habitats (Yáñez-Arancibia and Day, 1988). Because of this importance, it was declared a Protected Natural Area in 1994 by the Mexican Federal Government (INE/SEMARNAP, 1997). The continental shelf north of Laguna de Terminos is very productive for petroleum, and it has been shown that it can be a source of hydrocarbons to the Terminos Lagoon ecosystem (Noreña-Barroso et al., 1999). Moreover, the rivers that drain into the lagoon in the south are a source of agrochemicals (Gold-Bouchot, Silva-Herrera and Zapata-Pérez, 1993, 1995), and the organisms living in this lagoon system are exposed to a complex mixture of pollutants, which may complicate the interpretation of the biological effects because of possible synergistic or antagonistic interactions. This study was carried on with the aim of assessing the biological response of native American oysters, Crassostrea virginica, naturally exposed to a complex mixture of toxicants. To this end, we evaluated both micro pollutants and a suite of biomarkers in the oyster soft tissues, monitoring their histopathological condition at the same time. Moreover, as parasites have been shown to affect the responsiveness of some biomarkers, we determined the prevalence of the protozoan Perkinsus marinus as a possible confounding factor. Finally, the complexity of the biological response to mixtures of pollutants has been statistically approached by multivariate methods such as Principal Components Analysis (PCA), aiming at simplifying and visualising the correlational structure of experimental data.

Biological effects of environmental pollutants

2

175

Materials and methods

Oysters (C. virginica) were collected in November 2002 from four sites in Laguna de Terminos, a coastal lagoon in the Southern Gulf of Mexico: Boca de Atasta, Boca de Palizada, Chacahito and the centre of the lagoon (Centre), this last area being considered as the reference site (Figure 1). The sampling stations were chosen on the basis of their different pollution levels as determined from previous monitoring studies (Gold-Bouchot, Silva-Herrera and Zapata-Pérez, 1993, 1995; Gold-Bouchot, Barroso-Noreña and ZapataPerez, 1995; Noreña-Barroso et al., 1999). The concentrations of different pollutants (heavy metals, organochlorine pesticides, polychlorobiphenyls – PCBs – and hydrocarbons), were measured in the soft tissues as total body burden, and as the MTH content; neutral red assay was measured in blood cells and ChE activity in the gills. Moreover, in the same samples both light microscopy observations, to identify histopathological lesions, and cultivation of tissues, to detect Perkinsus marinus, were simultaneously carried out. Figure 1

Terminos Lagoon, showing the sampling stations

Hydrocarbon and chlorinated organic compound concentrations (pesticides and PCBs) were determined according to the procedures described in Sericano, Atlas and Wade (1990). Briefly, freeze-dried tissue was extracted with hexane and methylene chloride. Extracts were purified and fractionated to facilitate the analysis in an alumina : silica column. The aromatic fractions, containing both the PAHs and PCBs, were further purified to remove lipids by size exclusion chromatography. Organic compounds were determined by gas chromatography using a Hewlett Packard 5890 Series II gas chromatograph equipped with a 30 m × 0.25 mm HP-5 column. Hydrocarbons were quantified with a flame ionisation detector, and chlorinated pesticides and PCBs with an electron capture detector. Heavy metals were analysed by digesting the soft tissues with fuming nitric acid and hydrogen peroxide, and measuring the concentrations in a Perkin Elmer Spectrum Emission Plasma 400 Inductively Coupled Plasma (ICP) spectrometer. Quality assurance of the analytical procedures included the addition of internal standards and the analysis of a procedural blank and a spiked blank for each set of

176

G. Gold-Bouchot et al.

samples. Recovery of internal standards was between 88 and 107%. Reproducibility of duplicate samples was between 10 and 15%. Certified reference materials were analysed in parallel with the samples. For metals, the Community Bureau of Reference reference material No. 279 (Sea Lettuce, Ulva lactuca) was used. For organic pollutants, the National Institute of Standards and Technology reference material No. 1946 (Lake Superior fish tissue) was used. Concentrations obtained were within the certified range for all analytes. ChE activity was measured individually in the gills of five organisms per station, by a modification of the method of Ellman et al. (1961) using acetylthiocholine iodide (AChI) as substrate, and following the formation of 5-thio-2-nitrobenzoic acid at 412 nm with a spectrophotometer. Protein content was determined according to Lowry et al. (1951), using Bovine Serum Albumin (BSA), fraction V, as standard. For the evaluation of MTH content, samples were prepared as described in Viarengo et al. (1997). This method involves the evaluation of the MT concentration in a partially purified metalloprotein-containing fraction obtained by acidic ethanol/chloroform fractionation of tissue homogenate. The procedure includes the precautions for obtaining a complete MT precipitation and avoiding sulphydryl oxidation and contamination by soluble low molecular weight thiols and enzymatic protein degradation, which can occur during sample preparation. NRR times were individually evaluated in the haemocytes of 15 oysters from each site according to the methods of Lowe, Soverchia and Moore (1995) and Ringwood, Conners and Hoguet (1998). Briefly, 5 Pl of haemolymph syringed from the posterior muscle were left on glass slide where haemocytes are able to adhere. Following the addition of neutral red, haemocytes were then examined systematically under a light microscope every 15 min, the end point of the test being the evidence of dye loss from the lysosomes into the cytosol in at least 50% of the examined cells. Immediately after haemolymph withdrawal, oysters were dissected. A small portion of gills and digestive gland were incubated in thioglycolate medium for 7 days in the dark at room temperature to detect the parasite Perkinsus marinus (Ray, 1952). The tissues of each oyster left over were fixed in Davidson’s solution for subsequent histological processing. Following paraffin embedding, tissues were sectioned at 5 Pm and the resulting section slides stained with haematoxilin and eosin (Howard and Smith, 1983). Microscopic observations were carried out, aiming at both the qualitative evaluation of the tissues and the quantitative assessment of the gill oedema (Gold-Bouchot et al., 1995). To visualise the correlational structure of the data, a PCA was done on the Spearman non-parametric correlation matrix. In this way, data do not have to be standardised. XlStat version 7.06 (Addinsoft) was used.

3

Results

The highest concentrations of organochlorine pesticides in oysters are related to 6Drins and 6DDTs with values of 84.48 and 45 ng g1, respectively, and were found in the organisms collected in Atasta; however, 6HCHs tended to be more widely present in all sampling stations (Table 1). In general, total organochlorine pesticides and 6PCBs presented their highest values at Atasta, and lowest at Chacahito. Endosulphan and Mirex were not detected in any of the sampling stations.

Biological effects of environmental pollutants Table 1

Chlorinated pesticides and PCBs concentrations (on a dry weight basis) in tissues of the American oyster, Crassostrea virginica, from Laguna de Terminos, Mexico

Sample

ȈTCBs (ng g1)

PC ANISOL (ng g1)

Atasta