Identifying the primary sources of fecal contamination along the beaches and rivers of Trinidad

The aim of this study was to identify the main sources of fecal pollution at popular beaches and rivers in the island of Trinidad. Escherichia coli enumeration and microbial source tracking (MST) were used to identify the primary sources of fecal bacteria contamination at the sites. Nineteen sites exceeded USEPA water quality standards for safe recreational use. Highest levels of fecal contamination were recorded on the central and west coasts of the island and included Brickfield River (4,839 MPN 100 ml ), Orange Valley Bay (2,406.6 MPN 100 ml ) and Chaguaramas Bay (1,921.2 MPN 100 ml ). MST detected human (HF183) fecal pollution at ∼63%, birds at ∼67%, chicken at ∼36% and cattle (BacCow) at ∼34% of the sites. MST is a useful and rapid method for identifying major sources of fecal pollution in rivers and beaches. In Trinidad water bodies, the main sources of fecal pollution were humans and birds. The large number of sites with elevated levels of fecal pollution detected is particularly alarming and represents a serious public health risk. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/wh.2020.047 ://iwaponline.com/jwh/article-pdf/18/2/229/709069/jwh0180229.pdf Ronell S. H. Bridgemohan Dave S. Bachoon (corresponding author) Yingfan Wang Christine Mutiti Department of Biological and Environmental Sciences, Georgia College and State University, Milledgeville, GA 31061, USA E-mail: dave.bachoon@gcsu.edu Puran Bridgemohan Waterloo Research Campus, The University of Trinidad and Tobago, Waterloo Estates, Carapichaima, Trinidad and Tobago Adesh Ramsubhag Department of Life Sciences, University of the West Indies, St. Augustine, Trinidad and Tobago This article has been made Open Access thanks to the generous support of a global network of libraries as part of the Knowledge Unlatched Select initiative.


INTRODUCTION
In the Caribbean, fecal contamination of freshwater and marine environments is a growing threat to human health, tourism and the food industry (Shuval ; Walker et al. IMA ). As such, contamination of the island's marine and freshwater environments with high levels of fecal pollution, and possibly pathogenic bacteria from human and non-human sources, can present a public health concern. However, in Trinidad, routine monitoring of the level of fecal pollution in rivers and beaches is not conducted and watershed management plans are lacking.
In water quality management systems, regulatory agencies generally focus on fecal indicator bacteria (FIB) and identifying the source of the FIB to adequately assess . Although no standard for E. coli has been adopted for marine waters, the geometric mean standard for freshwater should be <126 CFU 100 ml À1 and the statistical threshold value (STV), which is a value that approximates the 90th percentile of the water quality distribution and should not be exceeded by more than 10 percent of the samples taken, fluctuates from 320 to 410 CFU 100 ml À1 The aim of this study was to conduct an island-wide survey of the level and sources of fecal contamination in Trinidad. Thirty-five sampling sites, including 23 marine and 12 freshwater locations were surveyed and for each location, physiochemical parameters were recorded, E. coli levels were determined, and MST assays for human, cattle and birds were conducted. Information obtained will be useful for public health risk assessment and for guiding the development of the most appropriate mitigation steps to improve water quality on the island.

Microbial source tracking
Quantitative PCR assays were conducted on a CFX 9600 (Bio RAD) Real-Time PCR system for the detection of human and animal fecal bacteria (Table 1). All primers used in this study were based on sensitivity and selectivity cited previously (Table 1), optimized to avoid nonspecific cross reaction against the appropriate non-target fecal DNA from chicken, cattle, goat and human (with local samples), and increased specificity. PCR assays were conducted with 1 μl of sample DNA (approximately 10 ng/μl) and a ten-fold diluted DNA sample with annealing temperatures and primer/probe sequences for each marker gene as listed in Table 1. Standard curves for each quantitative PCR (qPCR) (controls and samples) were linear and had coefficients of determination (r 2 ) of 0.99. Negative controls without DNA, which were run with each reaction, always exceeded the cycle threshold at a mean C q of 39.58. Each hydrolysis probe was labeled at the 5 0 end with the reporter dye 6-FAM (6-carboxy-fluorescein) and at the 3 0 end with the quencher dye TAMRA (6-carboxytetramethyl-rhodamine).

Physiochemical parameters
The observed temperature, pH, dissolved oxygen ( (Figure 1, Table 2). Overall, the majority of the 19 sites that had exceedingly high E. coli levels were on the western and central regions of the island (Figure 1).

MST:
The PCR assay CP1 for Clostridium tetani of the avian (chicken and other birds) origin indicated that birds were the most common source (65.52%) of fecal pollution to water systems on the island. The chicken-specific marker (CP29F/R) suggested that over half of the sites (32.38%) impacted by bird fecal pollution contained chicken fecal bacteria ( Table 2). The highest concentration of fecal pollution from avian sources was detected along the western coast of Trinidad. Human fecal Bacteroidales (HF-183) pollution was detected at 63.8% of the sites and was more predominant along the western region of the island. Similar trends were found for cattle BacCow at (34.48%) sites (Table 2). In addition, over 20% of the sampling areas along the island were impacted by human, avian and ruminant fecal pollution, and at 12% (7 sites), the MST assays used did not detect any human or animal sources of fecal pollution. In spite of these reports, researchers and regulatory agencies in the tropics continue to use FIB concentrations for monitoring or detecting fecal pollution. Previous research in Trinidad has indicated varying levels of marine and freshwater quality with many popular beaches,      A positive (þ) detection was based on if any sample or its replicate had a threshold above the limit of detection determined by the standard curve of the assay.

DISCUSSION
extensive or seasonal sampling regime of fecal pollution on the island.
In marine and freshwater systems, human fecal pollution is considered to carry a higher public health risk than contamination originating from non-human fecal sources (Holman et al. ; Bradshaw et al. ).
The HF-183 PCR assay for human fecal pollution is widely accepted as a reliable and sensitive PCR assay for Cuevas and Toco Bay) in the northeastern region of the island were contaminated with avian fecal bacteria; however, the levels of E. coli at these beaches were relatively low (<400 MPN ml À1 ).
Along with human and birds, cattle/ruminants were expected to be a major source of fecal pollution in Trinidad because many people on the island rare domesticated goats, sheep and cattle. In addition, it is common practice to use cattle manure fertilizer for crop farming. The BacCow assay indicated that at least 34% of the sites contained fecal bacteria of cattle origin, and in general the incidence of cattle fecal pollution was concentrated in the central and western sites on the island. Symonds et al. () found that the BacCow had high sensitivity (88%) at detecting cattle fecal pollution in Costa Rician samples but is prone to cross-reaction. Surprisingly, cattle fecal pollution was detected at Maracus River, Las Cuevas and Chaguaramas even though there were no noticeable cattle farms in these areas. Therefore, the detection of cattle fecal pollution at these sites could be attributed to run-off of cattle manure that is widely used in food-crop production in the island. It should be noted that the cattle fecal bacteria marker (BacCow) used in this study has been reported to crossreact with fecal bacteria from other ruminant sources, including deer (Boehm et al. ). However, deer was not present or common at any of the sampling areas used in this study.