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Grey Water Sinclair 1 E scherichia coli c ontamination in Grey Water and River Water in Monteverde, Costa Rica Nicholas Sinclair University of California, Irvine Department of Biological Sciences Department of Economics EAP Tropical Biology and Conservation Spring 2007 8 June 2007 Abstract Waste water from the bathrooms (except from toilets), kitchens, and laundry machines treatment. Recently, a few grey water treatment systems have been installed in the Mon teverde area This study examines the contaminating effect of grey water discharge on a river and the effectiveness of E scherichia coli removal by grey water systems. 30 samples were taken from the following 10 water sources: 2 pa rts of Quebrada Maquina 2 parts of Quebrada Sucia, 4 untreated grey water sources, and 2 treated grey water sources. A Microlabs Chromogenic E. coli Assay was performed. I found that a ll water sources were too contaminated (considered nated) to quantify a contaminating effect of grey water on the rivers or the removal of E. coli by grey water systems. Resumen Agua s residual es de los bao s (excepto de los escusados ), de las cocinas, y de los flujos de las lavadoras (conocidas colectiv agua s gris es ) corre n sin tratamiento por las calles y los ros de Monteverde. Recientemente, algunos sistemas de tratamiento de aguas grises han sido instalados en el rea de Monteverde. Este estudio examina el efecto de contaminacin de la d escarga de aguas grises en un ro y la eficacia del retiro de Escherichia coli por los sistemas de tratamiento del agua. 30 muestras de agua fueron tomadas de las siguientes 10 fuentes de agua: 2 sitios de Quebrada Maquina, 2 sitios de Quebrada Sucia, 4 f uentes de agua s grises sin tratamiento y 2 fuentes de agua s grises tratadas Se realiz un anlisis cromatognico de E. coli de Microlabs Encontr que todas las fuentes de agua se encontraban muy con taminadas (considera n infinitamente contaminado ) c omo para cuantifica r un efecto de contaminacin de agua s gris es es en los ros o el retiro de E. coli por los sistemas de tratamiento de agu a s grises
Grey Water Sinclair 2 Introduction rey water refers to all domestic wastewater generated from bathing, washing dishes, doing laundry, and cooking (Ludwig, 2006). As it does not contain waste from toilets, people rarely associate grey water with fecal contamination. However, p ast studies have shown that grey water can contain more than 4,000 times the amount of acceptable fecal contamination of first world drinking standards (Water CASA 2004 ). This bacteria l content of grey water The majority of homes in the Monteverde Zone of Costa Rica release grey water di rectly into the environment, without treatment (Dallas, 2005). With much of this contaminated water eventually flowing back to streams, significant fecal contamination of rivers is to be expected. However, just how much the discharge of grey water affect s the rivers of the Monteverde Zone is unknown. As more research is done on the high bacterial content of grey water, many treatment options are being offered. In the Monteverde Zone, certain homeowners have installed grey water treatment systems that a grey water system is essentially a small scale sewage treatment center, with two basic steps. First, solid waste is anaerobically digested, as liquefied waste slowly flows through the tan k (Prescott et al 1999). Second, the liquefied waste enters an elongated leach pipe, where it gradually permeates the soil (known as the leach field) through holes. In the leach field, the liquefied waste still containing bacteria, is aerobi cally digeste d as the water percolates, or filters, through the soil However, little research has been done on the effectiveness of bacterial removal by installed grey water systems in the Monteverde Zone. Coliform bacteria make up approximately 10% of the intestinal microorganisms of human beings, and are readily excreted with each bowel movement (Prescott, 1999). Though fecal coliforms themselves usually do not pose any health threat, they often indicate the presence of pathogens that can cause Typhoid fever and Hep atitis A (Canter and Knox, 1988). Coliforms can survive in fresh water for extended periods of time, so they serve as good indicator organisms for water contamination (Gleeson and Gray, 1997). Specifically, Escherichia coli bacteria (a fecal coliform) is a n excellent indicator organism, because it is always found in mammalian waste and rarely occurs in natural fresh water (Pepper, et al. 1995). Therefore, by testing for the presence of E. coli in freshwater samples, one can efficiently asses the presence of mammalian feces. The purpose of this study was two fold to examine the contaminating effects of gray water runoff on Monteverde rivers and the effectiveness of bacteria removal by grey water systems in the Monteverde Zone. With these two objectives, I formulated two major predictions. First, I hypothesize d that I would find an increase in fecal contamination (indicated by increased E. c oli presence) as rivers flow past more grey water sources Second, I predict ed that I would find a decrease in bacteri al contamination levels in grey water samples that had been treated. Methods and Materials Experimental Location and Sample Collection I collected two water samples from Quebrada Maquina and two water samples from Quebrada Sucia, both in the Monteve rde Zone of Costa Rica, Central America. For each river, I
Grey Water Sinclair 3 took one water sample 1 Km upstream and one water sample 1 Km downstream from the first major human disturbance I also collected two untreated grey water samples between the two aforementioned upp er and lower river samples To find untreated grey water samples, I visually surveyed the area for two days and found grey water outlets of at least four houses. Once I found four untreated grey water outlets, I observed what time of day the largest amount s of effluent were discharged. River samples were taken during midday (before rain) and untreated grey water samples were taken in the morning To test grey water treatment systems, I collected wate r samples from the system outlet of two single famil y hous es in the Monteverde Zone (Fig 1) The first grey water system (System A) discharged grey water above ground, so samples were collected directly from the end of the outlet pipe. However, the second grey water system ( System B ) discharged grey water approxi mately 2 feet underground so I collected samples both directly from the treatment outlet and after the grey water had traveled through 1 m of organic soil. All sample locations were replicated a total of three times, although System B was collected twice at the end of the outlet pipe and once af ter 1 m of organic soil. Thus, I collected total of 30 samples of ten different types of water (Fig 2). For each sample, I collected 50 mL of water in a sterile urine flask and tested for E. coli within the subseque nt 18 hours E. coli Analysis For all 30 samples, I followed the standard procedure from Microlabs Chromogenic Assay for E. coli (1998). For each sample, I filled f ive sterile test tubes with 5 mL of the given water sample and 1 mL of Microlabs Chromoge nic Lac+ broth After an 18 hour incubation period at 37 Celsius, I analyzed the tube with a black light. If the tube fluoresced under black light, it was counted as a positive. After analyzing all five tubes of each sample, the positives were summed. Tab le 1 was used to correla te number of positive tubes to M ost Probable N umber of E. coli per 100 mL of sample water (hereafter referred to as MPN) As Table 1 shows, this nitely will refer to a MPN greater than 23). Results Of the 30 water samples tested, 27 contained a MPN greater than 23 E. coli coliforms per 100 mL of water. Only three samples had an MPN less than 23: two r eplicates of treated grey water from System A (MPN < 1.1 ) and one replicate of upper Quebrada Maquina (MPN = 9.2) (Table 2) As only three of the water samples have a quantifiable MPN, descriptive statistics were not possible. Therefore, n o statistical ana lyses were performed. Discussion With regards to my first objective, I cannot quantify the extent to which grey water sources contribute to river contamination However, water is flowing directly into the rivers one can assume that it must cause an increase in river contamination The most unexplainable result is the E. coli content of the upper regions of Quebrada Maquina and Quebrada Sucia. Although Quebrada M aquina had one replicate with a MPN of 9.1, that is still nine times the standard of acceptable drinking water in most first world countries
Grey Water Sinclair 4 (equivalent to approximately 9.1 mg of feces for every 1000 L of water) (Ludwig 2006). A similar result was found in a 1998 study of the Rio Guacimal, where extrem ely high E. coli contamination was found at the headwater of that river ( Hansen and Silvia 1998). The high fecal contamination could not be explained then, and it seems unexplainable still. However, g iven the aforementioned trend that E. coli rarely occurs in clean fresh water, the contamination must be coming from some external source. It is possible that the contamination is from wild mammals (monkeys, sloths, agoutis, etc.) living in the forests around the rivers While fecal coliforms can live in freshw ater for days traveling considerable distances it seems unlikely that there would be enough animal s to give such a high reading. This is an area of research that should be developed further in the future. While grey water treatment systems may be somew hat effective at reducing the bacteria count, treatment is not reducing the bacteria count to a level below 23 MPN This notion is also important to the difference between the grey water from the outlet pipe of System B and the grey water of System B after it had filtered through on e meter of organic soil. Again, although this study does not show a difference, it has been shown that Monteverde organic soil is effective at removing E. coli (Escalmado 2006). Therefore, a method that will test higher values of MPN should be utilized in future experiments of grey water treatment efficiency. Also, it is important to note that t he two grey water treatment samples that had zero contamination (System A) were taken after a family was doing large amounts of laundry. L aundry detergents and bleach ar e excellent at killing bacteria, and the zero contamination therefore, is most likely a product of the detergent, not the treatment system. However, overuse of detergents and bleach is not an acceptable alternative to proper treatment, because these substances will also kill naturally occurring microorganisms and alter the natural micro environment. As more contaminated grey water flows into the local rivers, various environmental threats arise. Since fecal coliforms are facultatively anaerobic, i ncreased fecal contamination will lower the amount of dissolved oxygen in a river and hinder the ability of fish and other organisms to thrive. Also, the increase in coliform contamination in a river can greatly affect the pH of t he water. Fecal coliforms use respiration to produce ATP and therefore produce carbon dioxide. As fecal contamination increases, then, carbon dioxide levels increase, causing the pH of the river to decrease This decrease in pH and dissolved oxygen can dec rease both the abundance and species richness of fish and macroinvertebrates (Prescott et. al 1999). For example, nine days after a large amount of sewage run off entered a river in Australia, 5000 fish from 18 species were found dead. After numerous wate r tests, biologists found coliform concentrations were several magnitudes higher than normal and dissolved oxygen levels were greatly reduced. According to Townsend, low levels of dissolved oxygen were responsible for this massive fish kill ( 1992 ). Alt hough tank treatment of grey water is a move in the right direction, it might not be the most practical and beneficial removal process for domestic waste water. It is now proposed that instead of treating grey water in a tank system, it should be recycled as water for home gardening (Ludwig 2006). Grey water is comprised of organic matter, which can greatly increase plant growth in home gardens. While the CO 2 producing bacteria, phosphorus, nitrogen, and potassium will negatively affect rivers, they are ess ential to the growth of plants. Also, the top layers of organic soil are excellent at filtering the bacterial components of grey water before it is ever released to any natural water source (Escalmado, 2006) Therefore, the re use of grey water in gardens has three distinct benefits: (1) decreases the amount of water that must be taken from
Grey Water Sinclair 5 rivers in Monteverde, (2) increases plant yields for home g ardens, and (3) removes fecal bacteria decreasing groundwater and river contamination. However, grey water r ecycling should be performed with caution. Soil percolation is effective at removing fecal bacteria for two reasons. First, the porous soil acts as a physical filter to trap the bacteria as the water flows through. Second, naturally occurring m icroorganism s digest the trapped fecal bacteria (Prescott, et al. 1999) As more water percolates through the soil, it will rise to the surface, forming springs and streams People rely on these springs and streams for drinking water, because they are usually relative ly clean. However, when soil becomes highly saturated from rainfall, percolation will occur more quickly and with much lowered efficiency. Therefore, if we are adding contaminated grey water to the soil during heavy rains, the grey water will not stay in t he lower layers of soil long enough to be properly digested (Canter and Knox, 1988) Because the Monteverde area receives heavy rainfall during parts of the year, grey water recycling may only be a viable treatment option during the drier seasons. Ackn owledgments your knowledge of everything, and your help with my ringwo rm. Ramsa, thank you for always giving your opinion, especially when we were re designing my sample collection. I would like to thank Carlos Alfonso Calvo, without him this project never would have happened. I would like to thank the Fennell Arguedas famil y for housing and feeding me, and not getting angry when I would come home covered in mud and highly contaminated water. I would not have known which streams were which without the help of Erick McAdam. Finally, I would like to thank all the EAP students a nd staff (especially Federico, my favorite vegan) for cheering me up when it References Canter, L.W. and R.C. Knox. 1988. Septic tank systems e ffect on ground water q uality Lewis Publishers, Inc. : Chelsea, MI. Dall as, S.C. B. Scheffe, and G. Ho. 2005. Reedbeds for greywater t reatment case study in Santa Elena Monteverde, Costa Rica, Central America. Ecological Engineering. 23: 55 61 Esclamado, J.S. 2006. Determining the filtration quality of different soil types using turbidity, bacterial content, and drainage ability for filter greywater. EAP Instituto Monteverde, Fall. Gleeson, C. and Gray, N.F. 1997. Coliform index and waterborne d isease Taylor and Francis Publishers USA Oasis Design 2006. 28 May 2007 http://www.oasisdesign.net/greywater/index.htm Oasis Design 2006. 28 May 2007 http://www.oasisdesign.net/water/quality/coliform.htm Microlabs Standard Procedure: Choromogenic E.coli Assay Pepper, I.L., C.P. Gerba, J.W. Brendecke. 1995. Environmental Microbiology: Laboratory Manual Academic Press USA
Grey Water Sinclair 6 Prescott, L.M., J.P. Harley, and D.A. Klein. 1999. Microbiology WCB/McGraw Hill, Boston. Silvia, H.M. and L.A. Hansen. 1998. Biotic indicators of water quality in the Rio Guacimal: from the headwaters to the estuary. EAP Instituto Monteverde, Fall. Townsend, S.A ., K.T. Bowland, T.J. Wrigley. 1992. Factors contributing to a fish kill in the Australian wet/dry tropics. Water Research. 26(8): 1039 44. Water CASA. Residential Grey Water Study. 2004. 27 May 2007 http://www.watercasa.org/research/residential/resindex.htm
Grey Water Sinclair 7 Tables and Figures Figure 1. Typical grey water treatment system. Depending on replicate and site, treatment samples were taken fro m the end of the leach pipe and/or 1 meter of soil after the end of the leach pipe.
Grey Water Sinclair 8 Figure 2. A map of the Monteverde area, displaying where the ten types of sample water were collected. Table 1. Table used to quantify the c orrelation between numbe r of fluorescent tubes (positive tubes) and MPN of E. coli per 100 ml of H 2 O. # of Positive Tubes MPN per 100 mL 0 <1.1 1 2.2 2 5.1 3 9.2 4 16.1 5 <23 (Infinite)
Grey Water Sinclair 9 Table 2. Overall results for the most probable number of E. coli cells per 100 mL of water sample, for all 30 water samples. An Replicate One Replicate Two Replicate Three Quebrada Sucia Upper Infinite Infinite Infinite Untreated A Infinite Infinite Infinite Untrea ted B Infinite Infinite Infinite Quebrada Sucia Lower Infinite Infinite Infinite Quebrada Maquina Upper 9.2 MPN Infinite Infinite Untreated C Infinite Infinite Infinite Untreated D Infinite Infinite Infinite Quebrada Maquina Lower Infin ite Infinite Infinite System A Infinite <1.1 MPN <1.1 MPN System B Infinite Infinite Infinite (after 1 meter of soil filtration)
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Contaminacin de Escherichia coli en aguas grises y agua de los ros en Monteverde, Costa Rica.
Escherichia coli contamination in grey water and river water in Monteverde, Costa Rica.
g El 8 de junio 2007/June 8, 2007.
Books / Reports / Directories
Scanned by Monteverde Institute.
The State of Water in Monteverde, Costa Rica: A Resource Inventory.