11-ART 134-Vega Correales-Marin Vindas

Rev. Mar. Cost. ISSN 1659-455X. Vol. 6: 155-166, Diciembre 2014.

DOI: http://dx.doi.org/10.15359/revmar.6.11

Evaluation of Escherichia coli concentrations in Crassostrea gigas and seawater in two oyster growing areas in the Gulf of Nicoya, Costa Rica

Evaluación de las concentraciones de Escherichia coli en Crassostrea gigas y agua de mar en dos zonas de cultivo de ostras ubicadas en el Golfo de Nicoya, Costa Rica

Luis A. Vega Corrales1* & Carolina Marín Vindas1

1 Estación de Biología Marina Juan Bertoglia Richards, Universidad Nacional, Costa Rica. luis.vega.corrales@una.cr*

Recibido: 28 de marzo de 2014

Corregido: 10 de junio de 2014

Aceptado: 23 de junio de 2014

ABSTRACT

Costa Rica does not have references on the health quality of Crassostrea gigas growing areas, which can pose a potential public health risk. This study evaluated the temporal trends of Escherichia coli concentrations in the C. gigas oyster and in the seawater of Punta Cuchillo and Punta Morales growing areas as a basis for the classification and monitoring of bivalve mollusc production sites in the Gulf of Nicoya, Costa Rica. Monthly samples of seawater and C. gigas were collected from each site from July 2011 to June 2012, and their levels of E. coli were determined using the most probable number method. Temperature and salinity were recorded at a one meter depth. Precipitation data was provided by Instituto Meteorológico Nacional (the National Meteorological Institute). Results indicated that the evaluated areas are probably being affected by wastewater discharge. Punta Cuchillo and Punta Morales could be classified as class A or authorized areas for the production of bivalve molluscs. A significant seasonal variability of E. coli concentrations was determined in seawater and oysters from the Gulf of Nicoya, defined primarily by salinity. National legislation should be created to establish controls and implement a monitoring system ensuring the bacteriological quality of the areas used for the cultivation of bivalve molluscs in the country.

Keywords: Marine pollution, mariculture, Escherichia coli, Crassostrea gigas, Costa Rica.

RESUMEN

Costa Rica no cuenta con referencias sobre la calidad sanitaria de las zonas de cultivo de Crassostrea gigas y esto puede representar un riesgo para la salud pública. Se evaluó la tendencia temporal de las concentraciones de Escherichia coli en la ostra C. gigas y en el agua de mar de las zonas de cultivo de Punta Cuchillo y de Punta Morales como base para la clasificación y la vigilancia de los sitios de producción de moluscos bivalvos en el Golfo de Nicoya, Costa Rica. Muestras mensuales de agua de mar y de C. gigas de cada zona fueron recolectadas de julio de 2011 a junio de 2012 y se les determinó los niveles de E. coli por la técnica del número más probable. La temperatura y la salinidad fueron registradas a un metro de profundidad. Los datos de precipitación fueron suministrados por el Instituto Meteorológico Nacional. Los resultados indicaron que las áreas evaluadas podrían estar siendo afectadas por el vertido de aguas residuales. Punta Cuchillo y Punta Morales podrían clasificarse como zonas autorizadas o tipo A para la producción de moluscos bivalvos. Se determinó una variabilidad estacional significativa de las concentraciones de E. coli en agua de mar y en ostras del Golfo de Nicoya, definida principalmente por la salinidad. Se debe generar una normativa nacional que establezca controles e implemente un sistema de vigilancia que asegure la calidad bacteriológica de las áreas destinadas para el cultivo de moluscos bivalvos en el país.

Palabras claves: Contaminación marina, maricultura, Escherichia coli, Crassostrea gigas, Costa Rica.

INTRODUCTION

Marine ecosystems are vulnerable to anthropogenic pollution. According to Halpern et al. (2008), there are no marine coastal areas that have not been impacted by human influence. The discharge of wastewater in coastal areas is considered the main cause of degradation of bivalve mollusc growing areas (Oliveira et al. 2011). Evaluating bacteriological contamination in these areas is important since bivalve molluscs can accumulate bacteria potentially pathogenic to humans (Lee et al. 2008).

International regulations for the evaluation of the bacteriological quality of bivalve mollusc growing areas are based on the quantification of Escherichia coli as a fecal contamination indicator (Lee et al. 2008; Oliveira et al. 2011). Recent studies have shown that concentrations of E. coli in bivalve mollusc growing areas depend on, among many other factors, seasonal and climatic conditions of the area (Campos et al. 2013b; Derolez et al. 2013; Lee & Silk, 2013; Mignani et al. 2013).

Costa Rica does not have health regulations regarding mariculture, and production areas for oysters (Crassostrea gigas) do not have any references to evaluate their bacteriological quality. Oyster growing areas have been developed on a small scale in the Gulf of Nicoya. These areas are vulnerable to microbial contamination because they receive wastewater. This represents a potential risk to public health and to the development of this economic activity.

The objective of this research was to evaluate the temporal trend of E. coli concentrations in the C. gigas oyster and in the seawater in the growing areas in Punta Cuchillo and Punta Morales as a basis for the classification and monitoring of bivalve mollusc growing areas in the Gulf of Nicoya, Costa Rica.

MATERIALS AND METHODS

E. coli contamination was assessed in the oyster growing areas in Punta Cuchillo (9° 49’ 48’’ N and 84° 52’ 48’’ W) and Punta Morales (10° 4’ 84’’ N and 84° 58’ 3’’ W), located in the Gulf of Nicoya, Costa Rica (Fig. 1). A systematic random sampling plan was applied. Monthly samples of seawater and C. gigas were collected from each site from July 2011 to June 2012. With the same frequency, temperature and salinity were recorded for each site at a one meter depth using a YSI-556 multiparameter. Daily and monthly accumulated precipitation data during the sampling period was provided by Instituto Meteorológico Nacional de Costa Rica (the National Meteorological Institute of Costa Rica) and corresponded to the average existing records at the weather stations located in the study area (Puntarenas Weather Station: 09° 58’ N and 84° 49’ W, altitude = 15 masl; Paquera Weather Station: 09° 49’ N and 84° 56’ W, altitude = 15 masl) (Fig. 1).

Fig%201%20Geographic%20location%20of%20oyster%20growing%20areas.jpg

Seawater samples were collected at a one meter depth using a Niskin bottle. Between 15 and 25 oyster samples were collected directly from culture lantern nets. Samples were transported to the laboratory in ice (< 9° C) and analyzed within 8.5 h. The determination of the most probable number (MPN) of E. coli (series of five tubes and three dilutions) was performed based on Clesceri et al. (1989) and Downes & Ito (2001).

For the presumptive phase, each series of five Lauryl Tryptose Broth (BD®) tubes was inoculated with 10, 1 and 0.1 mL of seawater sample. For oyster samples 25 g of flesh and intravalvular liquid were homogenized in 225 mL of Buffered Peptone Water (BD®). Dilutions of 1:100 and 1:1000 were prepared from this mixture. From each dilution 5 Lauryl Tryptose Broth tubes were inoculated.

The confirmatory phase for both samples (seawater and oysters) was done in Brilliant Green Bile Broth (OXOID®) for total coliforms and EC Broth (OXOID®) for E. coli. E. coli was isolated using Levine Agar (EMB) (OXOID®) and identified by IMViC (OXOID®) biochemical tests and miniaturized API® 20 E (bioMérieux®) test. E. coli concentrations per type of sample were obtained from the MPN table (Clesceri et al. 1989; Downes & Ito, 2001).

The geometric mean and the 90th percentile of the E. coli concentration was calculated by sample type to classify the growing areas based on the criteria established in the United States of America (Lee et al. 2008) and the European Union (Oliveira et al. 2011) (Tables 1 and 2).

Table%201_%20Classification%20criteria%20for%20bivalve%20mollusc%20growing%20areas.jpg

Table%202_%20Classification%20criteria%20for%20bivalve%20mollusc%20growing%20areas.jpg

These regulations were chosen since both systems are important in terms of international trade and set regulations to be met by countries exporting to these regions (Lee et al. 2008).

The Pearson correlation coefficient was determined between monthly accumulated rainfall and salinity and temperature for each growing site. A Generalized Linear Model (GLM) with log link function and a Poisson error distribution was applied to evaluate the effect of seasonal variability in the E. coli concentrations in the study area.

Results were plotted and analyzed using the R programming language (R Core Team, 2013). Values below the MPN method detection limit were categorized and plotted as non-detectable.

RESULTS

The presence of E. coli was determined in 42% of the seawater and oyster samples collected in the Punta Cuchillo growing area. In the Punta Morales growing area this bacterium was found more frequently in the seawater samples (83%) than in the oyster samples (25%). The geometric mean and the 90th percentile of the E. coli concentration in the seawater and oysters for the two growing areas did not exceed the limit of the best sanitary quality classification category. Therefore, Punta Cuchillo and Punta Morales could be classified, taking into consideration only the bacteriological criteria established in the United States of America (Table 1) and in the European Union (Table 2), as class A or authorized areas for the production of bivalve molluscs (Fig. 2).

Fig%202%20Escherichia%20coli%20concentrations%20in%20seawater%20samples.jpg

Daily precipitation in the study area, according to data provided by Instituto Meteorológico Nacional de Costa Rica (IMN) (the National Meteorological Institute of Costa Rica), confirms that the country’s Pacific Regime has a well defined dry season (December to March) and rainy season (May to October). April and November are considered transition months (IMN, 2014). Salinity showed minimum values in the month of October for the two growing sites. During the dry season, this parameter increased in both areas due to the decreased precipitation in the Gulf of Nicoya. The minimum temperature was recorded in October and the maximum in April (Punta Morales) or May (Punta Cuchillo) (Fig. 3).

Fig%203%20Daily%20precipitation%20mm%20and%20salinity%20PSU%20and%20temperature.jpg

Monthly accumulated precipitation and salinity presented high negative correlation for both study areas. The Pearson correlation coefficient was -0.92 (95% CI: -0.98, -0.74) in Punta Cuchillo and -0.89 (95% CI: -0.97, -0.65) in Punta Morales. The recorded temperature in the growing areas did not show a clear tendency determining the seasonal variability in the Gulf of Nicoya (Punta Cuchillo: Pearson’s r = -0.34, P > 0.05; Punta Morales: Pearson’s r = -0.36, P > 0.05).

The GLM applied between salinity and E. coli concentration was significant for both types of samples. Coefficients estimated by the model indicated a negative trend between recorded E. coli concentrations and salinity (Table 3). As salinity increased in the growing areas E. coli concentration decreased in both types of samples. The GLM determined, for the study period, a significant seasonal variability in the E. coli concentration in seawater and oysters in the Gulf of Nicoya. During the dry season E. coli was not detected in the oyster samples from either growing area. Only the seawater samples from Punta Morales showed E. coli during this period (Fig. 4).

Table%203_%20Coefficients%20estimated%20using%20the%20Generalized.jpg

Fig%204_%20Seasonal%20variability%20of%20the%20Escherichia%20col.jpg

DISCUSSION

Total coliforms have been used to assess sanitary quality of coastal areas. However, E. coli is considered a more specific indicator of fecal contamination, mainly in tropical coastal areas where the values of total coliforms may include bacteria that do not constitute a health risk (Lee et al. 2008). E. coli is also used for the bacteriological monitoring and classification of the bivalve mollusc growing areas (Almeida & Soares, 2012; Bettencourt et al. 2013).

Previous studies reported the presence of fecal coliforms or E. coli in samples of seawater (Acuña et al. 1998; García et al. 2006) and mollusc (Vega et al. 2013) from the Gulf of Nicoya; however, there are no references on the bacteriological classification of growing sites or their seasonal variability. E. coli concentrations in seawater and oyster samples analyzed during the study period allowed to classify, on a preliminary basis, Punta Cuchillo and Punta Morales growing areas as authorized (category A) areas for the cultivation of C. gigas in Costa Rica. This classification category establishes that produced molluscs do not require any post harvest treatment and can be directly used for human consumption (Lee et al. 2008; Oliveira et al. 2011).

The salinity variation recorded in this study reinforces the observations by Peterson (1960) and Voorhis et al. (1983), who established that this parameter is a seasonality indicator in the Gulf of Nicoya. Salinity in Punta Cuchillo and Punta Morales growing areas increased during the dry season. Increased precipitation and surface runoff during the rainy season reduce salinity and, usually, increase concentrations of organic matter and microorganisms in coastal environments (Barrera-Escorcia et al. 1999). Rain is, therefore, the parameter most commonly associated with the maximum levels of organisms indicating fecal contamination in coastal areas (Campos et al. 2013b).

The applied GLM estimated a significant negative trend between salinity and E. coli concentrations in seawater and oysters samples. In this way, precipitation in the Gulf of Nicoya seems to define a seasonal pattern in the variability of salinity and the registered E. coli contamination. Results of this study are consistent with other research projects. The inverse relationship between salinity and fecal coliform bacteria in water has been demonstrated in laboratory tests (Šolić & Krstulović, 1992) and in coastal marine environments (Barrera-Escorcia & Namihira-Santillán, 2004; Mignani et al. 2013). Anacleto et al. (2013) indicated that E. coli concentrations in seawater and sediments of the Targus Estuary (Portugal) were significantly higher during the rainy season. The same seasonal trend was found in the Thau Lagoon in France (Derolez et al. 2013).

Levels of thermotolerant coliforms in bivalve molluscs may differ with fecal contamination in the surrounding water (Martínez & Oliviera, 2010) and may change from one time period to another (Campos et al. 2013a; Soegianto & Supriyanto, 2008). Derolez et al. (2013) noted an inverse relationship between E. coli concentrations in seawater and in C. gigas oysters. In contrast, Anacleto et al. (2013) found a positive correlation between E. coli concentrations in water and in the Venerupis pullastra clam. The present study showed that E. coli concentrations in seawater and in C. gigas showed the same trend as Derolez et al. (2013). During the dry season, Punta Morales growing area presented higher levels of E. coli in the seawater than in oysters, possibly because the oyster farm is located at the mouth of Estero Morales. This could suggest that Punta Morales growing area receives direct contamination from the surrounding towns.

E. coli accumulation percentages among bivalve molluscs species in the same area may also show differences. Younger & Reese (2013) concluded that the percentage of accumulation between Cerastoderma edule, Tapes philippinarum and Mytilus spp. were equivalent in general terms, and that each of these species showed greater levels of accumulation than C. gigas and Ostrea edulis. In a controlled field study, E. coli accumulation was significantly higher in Pecten maximus than in Mytilus spp. and C. gigas (Lee & Silk, 2013). Programs assessing the microbiological quality of bivalve molluscs growing areas should monitor all cultivated species or select an indicator species that presents levels of pollution accumulation equivalent to or higher than the species they represent (Younger & Reese, 2013).

It is concluded that Punta Cuchillo and Punta Morales C. gigas growing areas are possibly being affected by wastewater discharge. This contamination is attributed to the coastal towns nearby and the river systems flowing into the Gulf of Nicoya from major urban, industrial and agricultural areas of the country. However, the bacteriological quality of seawater and oysters determined in this study suggests that these sites may be suitable for growing C. gigas in Costa Rica.

National legislation should be created to establish controls and implement a monitoring system ensuring the bacteriological quality of the areas used for cultivation of bivalve molluscs in Costa Rica. It is also important to supervise the treatment of wastewater to reduce the vulnerability of this estuarine system.

Depuration and proper handling of harvested molluscs is recommended to decrease potential dangers that may arise from the E. coli levels detected and sporadic contamination of the evaluated areas.

These provisions, in addition to protecting the consumer and public health in general, promote the mariculture development in the country.

ACKNOWLEDGMENTS

To Asociación de Mujeres de Punta Morales (the Punta Morales Women’s Association) and the Peralta family for providing the oyster samples. Instituto Meteorológico Nacional de Costa Rica (the National Meteorological Institute of Costa Rica) for providing the precipitation data. This research was funded by Ley de Pesca y Acuicultura (Fisheries and Aquaculture Law) of the Government of Costa Rica as part of the project entitled “Composición nutricional y efecto de las alteraciones bioquímicas, microbiológicas y ambientales en la calidad y la frescura de algunas especies de interés comercial capturadas en el Golfo de Nicoya” (Nutrient Composition and Effect of Environmental, Microbiological, and Biochemical Alterations in the Quality and Freshness of Some Commercial Species Caught in the Gulf of Nicoya) and by the Consejo Nacional de Rectores (National Provost Council) as part of the project entitled “Incremento en la competitividad de las PYMES del Pacífico Central mediante un plan de fortalecimiento interuniversitario regional” (Increasing Competitiveness of SMEs in the Central Pacific by an Inter-University Regional Strengthening Plan). The authors wish to thank the anonymous evaluators who made valuable contributions in the development of the paper.

Evaluation of Escherichia coli concentrations in Crassostrea gigas and seawater in two oyster growing areas in the Gulf of Nicoya, Costa Rica por Revista Ciencias Marinas y Costeras se distribuye bajo una Creative Commons Reconocimiento-NoComercial-SinObraDerivada 3.0 Costa Rica License.
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