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Ciencia e investigación agraria

versión On-line ISSN 0718-1620

Cienc. Inv. Agr. v.37 n.2 Santiago ago. 2010

http://dx.doi.org/10.4067/S0718-16202010000200014 

Cien. Inv. Agr . 37(2):141-149. 2010
www.rcia.uc.cl

SCIENTIFIC NOTE

 

Relations fecal coliforms/ fecal Streptococci as indicators of the origin of fecal pollution in urban and rural water bodies of Temuco, Chile

Relación coliformes fecales/estreptococos fecales, como indicador del origen de la contaminación fecal en cuerpos de agua urbano y rural de la Comuna de Temuco, Chile

 

Reinaldo Rivera, Patricio de Los Ríos, and Ángel Contreras

Escuela de Ciencias Ambientales, Facultad de Recursos Naturales, Universidad Católica de Temuco, Casilla 15-D, Temuco.

Dirección para correspondencia


Abstract

The presence of alloctone bacteria isconsidered as one of the more important biological pollutants of water bodies, existing one close relation between the fecal contamination indicators and the existence of urban and industrial zones. In the present study we utilized indicators of fecal contamination (coliforms and streptococci) and the coliform/streptococci index (FC/FE) for determine the origin of fecal pollution in two water bodies of Temuco, Northern Campus at north surrounding of Temuco, and Rucamanque source that is located in a rural zone. It utilized the multiple assays number for obtain the bacteria more probable number in 100 mi of water sample. It was done the correlations between microbiological indicators and physic-chemical variables. The results indicate that both sites have low fecal contamination. It was reported important correlation between fecal coliforms, temperature and pH, but negative correlations with dissolved oxygen. The index (FC/FE) was correlated only with dissolved oxygen. The temperature would have an important regulator role in the coliform growth, whereas the fecal streptococci have high densities at low values of temperature, conductivity and dissolved oxygen. The origin of microbiological pollution was related with the antrophogenic intervention in both studied sited, human origin in Northern Campus, and mixed origin in Rucamanque source. It was suggest the use of index as first approach for identify the origin of fecal pollution.

Key words: Fecal pollution, fecal indicator, coliforms, streptococci.


Resumen

La presencia de bacterias alóctonas es considerada uno de los contaminantes biológicos más importantes de los sistemas acuáticos, existiendo una estrecha relación entre los indicadores de contaminación fecal y la existencia de centros urbanos e industriales. Se utilizaron indicadores de contaminación fecal (coliformes y estreptococos fecales), y el índice coliformes/estreptococos fecales para determinar la fuente de contaminación fecal en dos cuerpos de agua de la comuna de Temuco, Laguna Campus Norte, situada en zona urbana y bocatoma Rucamanque, ubicada en un sitio rural. Se utilizó la técnica de tubos multiples para obtener el número más probable de bacterias en 100 mi de agua. Se efectuaron correlaciones entre los indicadores microbio lógicos y variables fisicoquímicas. Los resultados indican que ambos sitios presentaron baja contaminación fecal. Se reportan importantes correlaciones entre coliformes fecales, temperatura y pH. Los estreptococos fecales exhibieron altas correlaciones con la temperatura y pH, pero negativas con el oxígeno disuelto. El índice CF/FE se correlacionó únicamente con el oxígeno disuelto. La temperatura jugaría un rol regulador en el crecimiento de los coliformes, en cambio los FE, presentan mayores densidades a menores valores de temperatura, pH, conductividad y oxígeno disuelto. La fuente de contaminación microbio lógica se relacionó con la intervención antrópica presente en los sitios, origen humano en laguna Campus Norte y origen mixto en bocatoma Rucamanque. Se recomienda el uso del índice como primera aproximación para identificar el origen de la contaminación fecal.

Palabras claves: Contaminación fecal, indicador fecal, coliformes, estreptococos.


Introduction

Characteristics of the aquatic environment are in a large extent determined by the properties and activity of microorganisms. However, the presence of alloctone bacteria is regarded as one of the most important biological contaminants in aquatic systems, with a close relation between the increase of fecal bacteria and the closeness to urban and industrial centers (Martínez et al., 1993).

The microbiological indicators frequently used in quality evaluations of continental waters correspond to fecal coliform bacteria, Escherichia coli, fecal streptococci and spores of sulfite-reductor anaerobes (Wyer et al., 1995; Gesche et al., 2003). Unlike pathogenic bacteria, indicator bacteria may be easily detected and quanti-fied by simple and economic methods (Murgel, 1984), allowing the sanitary classification of water for different uses, the determination of criteria and compliance of quality standards, the identification of contaminant discharges, the control of processes of water treatment, and epidemiológica! studies among others (EPA, 1997; Gesche et al., 2003; Horan, 2003). However, there is no universal indicator group of fecal contamination; therefore, the appropriate bacterial group must be selected for the specific situation under study (Suárez, 2002).

In Chile, the microbiological indicators of contamination for surface waters correspond to the coliform group (e.g., INN, 1978; Ministerio Secretaría General de la Presidencia, 2001; CONAMA 2004; INN, 2005), which are conformed by bacillary bacteria, facultative aerobes and anaerobes, negative Gram, non-spore-forming and lactose fermenters, corresponding approximately to 10% of human and animal intestinal microorganisms (Mariñelarena and Mariazzi, 1995; Romero, 1999; Prescott et al., 2004). The presence of these bacteria is considered as an index of fecal contamination and therefore contamination with pathogenic organisms (Sankararamakrishnan and Guo, 2005; Bastholm et al., 2008).

In regard to the determination of the animal or human origin of the contamination, it is proposed the use of fecal streptococci, bacterial group comprising several species of the genera Enterococcus and Streptococcus (Suárez, 2002; Horan, 2003). Fecal streptococci include species found in feces from humans and hot-blooded animals (Sinton et al., 1993). They present survival rates similar to enteric pathogens and they do not proliferate in the environment (Horan, 2003). Fecal streptococci have been used by sanitary authorities in different countries to evaluate the quality of their water resources, along with fecal coliforms to differentiate the fecal contamination of human and animal origin (Marín, 2003; Sankararamakrishnan and Guo, 2005). However, factors like the differences in mortality ranges in the environment between these two bacterial groups, variable survival of streptococclospecies and methods for determination questioned their use for these purposes (Sinton et al., 1993; Marín, 2003). Nevertheless, the fecal coliform /fecal streptococci relation to differentiate contamination has been used essentially in tropical zones, and the application in témplate zones is still unknown (Chagas et al., 2006).

The objective of the present study was to evaluate the source of microbiological contamination through the relation FC/FE in two bodies of water, one located in an urban zone and the other located in a rural zone of the commune of Temuco, Region of La Araucanía.

Materials and methods

Sites under study

The first site corresponded to the urban La-goon Northern Campus, located in a campus of Catholic University of Temuco (Table 1). The origins of the lagoon date back to the late nineties where it was the extension of a small wetland. The second site corresponded to the water intake of the Rucamanque land, located at 12 km North West from the city of Temuco, Region of La Araucanía, Chile (Table 1). The intake is surrounded by native relict forests in the Central Valley (Ramírez et al., 1988). The hillsides of the Rucamanque land are drainedby the Chivilcán creek, where an intake was built in 1931 to provide water to the city of Temuco (Magofke, 1985).


The criteria applied for the sites selectionconsisted on their anthropic intervention, as well as their artificial origin. The Lagoon Northern Campus is located inside the city and highly influenced by anthropic activities from urban zones, unlike the intake in the Rucamanque land, which is located in a natural environment away from urban centres; therefore, a minimal alteration is expected. The main morphometric characteristics of both sites are detailed in Table 1.

Physical-chemical variables

The water temperature, pH, electric conductiv-ty and oxygen dissolved inboth bodies of water were measured in situ by digital instruments. The samples were taken in fall and summer as they are periods where the lowest and highest environmental temperatures are present in the sites under study. The samples collection for physical-chemical variables was made in the deepest zone (Sosnovsky and Quirós, 2006).

Microbiological analysis

The concentration of total coliform bacteria (TC), fecal coliforms (FC) and fecal streptococci (FS) were quantified through the fermentation technique in multiple tubes (APHA, 1992; Astorga et al., 1998), with three dilutions and three replications per site. The samples were collected in 1-liter-sterile flasks, which were transponed to the Microbiology Laboratory of Catholic University of Temuco in a thermal container for analysis.

An aliquot was inoculated for the TC and FC quantification with a nichrome loop from each sample in tubes with lauryl sulfate broth, and incubated for 48 hours at 35 ± Io C. The positive tubes were inoculated in tubes of brilliant green bile broth (BGB) selective for coliform bacteria. The formation of turbidity and gas in 48 hours, at 35 ±1° C confirmed the presence of TC. At the same time, FC bacteria were confirmed, as an aliquot of positive lauryl sulfate broth was inoculated in tubes with EC broth (Escherichia coli). The production of turbidity and gas in 48 hours of incubation at 44.5 °C confirmed the presence of this group (Astorga et al., 1998). The cultivation medium manifesting gas production as a consequence of lactose fermentation was considered positive (Gesche et al., 2003).

FE quantification was made by the procedure described by Fernández et al. (2007). The samples were inoculated in tubes with glucose azide broth and incubated at 37 ± 1 °C during 48 hours. The tubes presenting turbidity were transferred to tubes with broth of bromocresol purple cultivation. At 48 hours, the tubes presenting turbidity and change from purple to green yellow were considered positive.

The calculation of concentration TC, FC and FE bacteria was determined based on tables of most probable numbers (NMP), expressing the results as NMP/100 mi of water (Mariñelarena and Mariazzi, 1995; Astorga et al., 1998).

Source of microbiological contamination

The determination of the source of fecal contamination was determined by the relation fecal coliform /fecal streptococci (FC/FE) established by Geldreich and Kenner (1969). It indi-¿cates human origin if the relation FC/FE > 4.0, it indicates animal origin if the relation FC/FE < 0.7. The interval < 4.0 and > 0.7 was considered as a mixed contamination.

Statistical analysis

The microbiological variables TC, FC, FE and the relation FC/FE measured in both sites were compared by the non parametric Mann-Whitn-ney test. The microbiological indicators were correlated with the physical-chemical parameters from both periods by the Spearman non parametric correlation coefficient (Zar, 1999). The analyses were made by the XLSTAT software, version 7.5.2. (www.addinsoft.com).

Results

The average and standard error of the microbiological and physical-chemical variables of the sites under study are presented in Table 2, as well as the correlations existing among them.


The microbiological analysis carried out for the summer and fall seasons in both sites (Table 2) is not an indication of a tendency of the TC and FC bacteria in the studied periods. The concentration of TC bacteria, in both the Rucamanque intake and the Lagoon Northern Campus was remarkably higher than the FC concentration, especially for fall. In regard to the FE, the high-est concentrations were recorded in the Ruca-manque intake, in both summer and fall, coinciding with lower values of temperature, conductivity, pH and dissolved oxygen compared to the higher values in the Lagoon Northern Campus in both periods.

The analysis of correlation on the variables quantified in the Lagoon Northern Campus showed high relations between FC and pH. The indicator group conformed by FE showed significant and positive correlations with temperature, but negatively with the DO. The analysis of correlation in the Rucamanque intake revealed that the TC were positively and significantly correlated with the temperature and the pH (Table 2), but negatively although significantly with the dissolved oxygen. In regard to the FC quantified inthe intake, a significant correlation with the temperature was observed. Likewise, the FE showed a high correlation with the pH. The relation FC/FE revealed a correlation with the DO, although negatively (Table 2).

In regard to the source of microbiological contamination (Table 3), it was observed that the sites under study presented a defined pattern inboth sam-pling periods, corresponding to human origin in the Lagoon Northern Campus (FC/FE> 4.0) and mixed contamination in the Rucamanque intake (FC/FE> 0.7 and <4.0), and the source of contamination for this site was not identified.


The comparative analysis (Table 4) revealed that the TC and FE bacteria present significant differences in the sites under study (p< 0.05 and p<0.004 respectively). However, the FC bacteria and the relation FC/FE might indicate a similar contribution from fecal bacteria in both the Rucamanque intake and the Lagoon Northern Campus, due to the absence of significant differences (p< 0.581 and p< 0.055 respectively).


Discussion

The pristine site versus contaminated site criterion fixed a priori was not corroborated by the indicators of FC microbiological contamination and the relation FC/FE, due to the absence of significant differences, which indicates similar contributions of fecal matter in the bodies of water. However, the FE density, was higher in the Rucamanque intake, supported by a significant difference Z= -2.892 p< 0.004), which generates the differentiating element inthe bodies of water. Nevertheless, the presence of FE could be related to contributions of fecal matter distant from the site where the samples were collected, situation described by Marín (2003), which does not necessarily involve recent fecal contamination in the intake.

It is noteworthy to remark the significant correlation between TC and the water temperature in the Rucamanque intake, which is coherent to the proposals by Atlas and Bartha (2002) on the close relation between bacteria and temperature. Similarly, a high correlation between the FE and temperature was recorded in the Lagoon Northern Campus, which may be explained by the FE physiology as they present higher resistance to the environmental stress (Godfree et al., 1997; Barrera et al., 1998). Even though there was a high water temperature, conductivity and pH during both sam-pling periods in the lagoon, these conditions might not represent extreme conditions for these microorganisms.

The bacterial group formed by the FC presented a higher density in the Lagoon Northern Campus for the summer season, which is related to the high environmental temperature (c.a 30 °C, Ramírez, 2007), which is similar to the records by Fernández et al. (2007) for the El Sauce lagoon in the Argentinean pampas, and by Rivera et al. (2004) for the Cautín river in the zone bordering the city of Temuco. According to these last authors, the FC concentrations found in ritral zones of the Cautín river would be similar to the reports for the Rucamanque intake, which shows their relatively pristine character.

In regard to the TC, a higher concentration was recorded in both sites in comparison to the FC, which is expected because the TC refer to the total of coliform bacteria from human, animal or edaphic origin (Romero, 1999). The FC did not present significara differences, which indicates that both bodies of water might receive similar bacterial contributions from the mentioned origins.

In Chile, only the TC and FC are used as parameters of microbiological evaluation, which must not exceed a value of 1000 NMP/lOOml for irrigation or recreational purposes with direct contact (INN, 1978). This allows indicating that, based on the current regulation; the Rucamanque intake and the Lagoon Northern Campus would be adequate for the mentioned uses. In regard to the FE, Chile has regulations for determining these bacteria (INN, 2002) although unlike Central American countries, the FE have been regarded as indicators of fecal contamination, used along FC as differen-tiators of fecal contamination (Barrera et al., 1998; Suárez, 2002).

The determination of the source of fecal contamination allowed showing that the pattern of mixed contamination found in the Rucamanque intake considered as natural site, may be related to the existence of cattle and poultry existing inthe land. The low density of fecal streptococci and FC/FE coefficients in the interval 0.7 and 4.0 might indicate that there are not any importara contributions of fecal matter by home or wild animals. On the contrary, the Lagoon Northern Campus presented a microbial load of human origin, due to the relation FC/FE that remarkably exceeded the value established by Geldreich and Kenner (1969) as fecal human contamination, which is expected due to the contributions to the lagoon tributary on the path for the urban zone of Temuco, as it was described by Habit et al. (2005) for the Quilque river in the city of Los Angeles and by Martínez et al. (1993) for the Bío Bío river.

The use of the relation between the FC compared to the FE as a differentiating index of fecal contamination was common until recent years (Howell et al., 1996; Sankararamakrishnan and Guo, 2005). Nevertheless, the relation FC/FE has involved questioning, due to the different indexes of survival in the species of fecal streptococci (Marín, 2003; Horan, 2003), as well as by the methods of FE count. This information was considered by standardizing the methodology for FC and FE quantification, using only the technique of multiple tubes to avoid errors in the bacterial count. The fact that the validity of the relation FC/FE has been questioned mainly in tropical áreas deserves special attention (Suárez, 2002). However, its use has not been rejected in températe áreas (Chagas et al., 2006) as made in this study.

If both antecedents are considered, the use of the relation FC/FE could represent the first approach in regard to the origin of the fecal contamination in the Central-Southof the country, which has shown an increased on cattle activities in recent years (Alfaro and Salazar, 2005), where the aquatic systems were exposed to a higher contribution of waters contaminated with fecal matter of animal origin. Nevertheless, it becomes necessary to analyze the different genera and species composing the group of fecal enterococcus for a correct determination of the fecal origin of the contamination, by biochemical and/or serological tests which demand higher resources (e.g., financing, infrastructure) unlike the relation FC/FE, which may be made in laboratories with less equipment.

Based on the results obtained, we may concoide that: 1) the bacterial load indicates that both sites under study present a low fecal contamination, which might be adequate for irrigation or recreational use with direct contact, according to the current legislation. 2) The bacterial load was positive and significantly correlated to the temperature, and negatively with the dissolved oxygen, therefore, the temperature would regúlate the bacterial load in both sites. 3) The higher FE concentration was recorded at lower values in the physical-chemical variables, which indicates a higher environmental tolerance unlike the coliform. 4) The relation FC/FE indicated fecal contamination from human origin for urban environment and of mixed type in natural environment, with a relation between the load of fecal bacteria and the surroundings of both bodies of water.

Acknowlegement

The authors thank the School of Environmental Sciences of Catholic University of Temuco for the technical and financial facilities for the development of this work.

 

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Received July 07,2009. Accepted August 19,2009.

Corresponding author: reijavier@gmail.com

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