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Coliform contamination on near stream plants from polluted and pristine streams : factors that affect coliform presence ...

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Title:
Coliform contamination on near stream plants from polluted and pristine streams : factors that affect coliform presence on plants, and a potential mechanism for contamination
Translated Title:
Contaminación de coliformes fecales en plantas cercanas a las quebradas y a las fuentes de agua : factores que afectan la presencia de coliformes enpPlantas, y un mecanismo potencial para la contaminación ( )
Physical Description:
Book
Language:
English
Creator:
Jacobson, Lia
Publisher:
s.n.
Place of Publication:
Monteverde

Subjects

Subjects / Keywords:
Aguas negras
Black water
Coliform contamination
Contaminación por coliformes fecales
Quebradas
Santa Elena -- Monteverde -- San Luis
Genre:
Books / Reports / Directories   ( local )
Books / Reports / Directories   ( local )

Notes

Language:
Inglés/English.

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University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
usfldc doi - M36-00456-ML-1062
usfldc handle - m36.456-ml-1062
System ID:
SFS0001938:00001


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Contaminacin de coliformes fecales en plantas cercanas a las quebradas y a las fuentes de agua : factores que afectan la presencia de coliformes enpPlantas, y un mecanismo potencial para la contaminacin.
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Coliform contamination on near stream plants from polluted and pristine streams : factors that affect coliform presence on plants, and a potential mechanism for contamination.
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Black water
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Contaminacin por coliformes fecales
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Santa Elena -- Monteverde -- San Luis
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The State of Water in Monteverde, Costa Rica: A Resource Inventory.
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Jacobson Coliform Bacteria on Plants 1 Coliform Contamination on Near Stream Plan ts from Polluted and Pristine Streams, Factors that Affect Coliform Presence on Plants, and a Potential Mechanism for Contamination. Lia Jacobson University of California, Berkeley EAP Monteverde Tropical Bi ology and Conservation, Fall 2006 15 December, 2006 Abstract Black water contamination from human-influenced areas introduces fecal coliform bacteria into nearby water sources such as streams. Fecal co liforms, which can be pathogenic, can also be present in environments near contaminated water sources. In this study, I analyzed the presence of total coliforms, fecal coliforms and E. coli bacteria in the water and on vegetation near two polluted streams and a pristine stream in the Sant a Elena and San Luis areas. I predicted that vegetation near polluted streams would exhib it higher amounts of coliform bacteria than vegetation near a clean stream. I examined va rious environmental factors and plant distances from streams to determine any possible relationships with bacterial presence. I also tested if airborne spray from the stream introduces coliforms onto near-stream plants. From 24 November 2006 to 2 December 2006, I collected wa ter and plant samples from sites along the Quebradas Rodrguez, Cambronero and Alondra fo r bacterial analysis by incubation on 3M Petrifilm plates. I also placed open Petrifilms at various stream distances to collect potential spray, and then analyzed the films for bacterial growth. Both water and plant samples exhibited lower amounts of all three coliform types at the Quebrada Alondra than at the Quebradas Rodrguez and Cambronero. With increased distance from the stream, coliform presence on plants showed a decreasing trend. These tr ends suggest a relationship between coliform contamination on plants and near-by polluted st reams. Atmospheric temperature and percent canopy cover positively affected total coliform counts from plants, while relative humidity positively affected E. coli counts from plants. Of 50 films used to capture airborne spray, five demonstrated coliform presence from the Qu ebradas Rodrguez and Cambronero. Future studies should alter spra y collection methods and test for alte rnate contamination mechanisms. Resumen La contaminacin por aguas negr as a causa de influencia hum ana introduce coliformes fecales en las fuentes de agua cercanas. Colifomes f ecales, los cuales pueden ser patogenico pueden tambien ser encontrados cerca de fuentes de agua contaminada. En este estudio analis la presencia total de coliformes, coliformes fecales y E.coli en la vegatacin adjacented a dos quebradas contaminadas y una sin contaminacin en las areas de Santa Elena y San Luis. Yo predij un numero mayor de coliformes en la vegetacin adjacente a las fuentes de agua contaminadas y un numero mucho menor en el si tio de la fuente sin contaminacin. Examin varios factores ambientales y la cercania de las plantas a la quebrada pa ra determinar cualquier posible relacin entre el numero de coliformes presentes. Tambien examin la posibilidad de que la quebrada cree un spray por el que pueden viajar bacterias hasta las plantas adyacentes. Del 24 de Noviembre al 2 de Diciembre, colect muestras de agua y de vegetacin de la

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Jacobson Coliform Bacteria on Plants 2 Quebrada Cambronero, Rodrguez, y Alondra para enbubar bacterias y ana lisarlas en Petrifilm 3M. Tambien coloqu Petrifilm a varias distancias de la quebrad a para analisarlas despus. Las muestras de agua y vegetacin de la Qu ebrada Alondra exhibieron cantidades bajas de coliformes comparada con las otra s dos. Conform la distancia incrementaba la cantidad de coliformes disminuia mostrando un patron disminuye nte. Estos patrones sugieren una relacion entre contaminacion de las plantas en quebradas y contaminacion del agua. Presion atmosferica y porcentage cubierto del dosel afectaron positivam ente el numero de coliformes en las plantas, mientras que la humedad relative afecto positivamente la cantidad de E.coli en las plantas. Para este experimento use 50 3M Petrifilm para capturar bacterias en el aire, 5 de estos mostraron la precensia de E.coli en las quebrada Cambronero y Rodrigue z. Estudios subsecuentes deberan mejorar la metodologia de este. Streams and other water sources that run through urbanized ar eas or animal farm areas have a high potential to contain bacterial contamination, especially if run-off water is poorly treated. Black water, or water that contains hum an and animal fecal waste, is one such form of bacterial contamination. Black water contains intestinal coliform bacteria which are of public health concern not only because they signify stream pollution, but because they can have a high association with a range of viral, bacter ial and protozoan human diseases (Prescott et al 1999). Water quality assessments utilize total coliforms, fecal coliforms and Escherichia coli, a common, enteric coliform, as indicators of bacterial contamination (Prescott et al 1999). Such tests can be performed by placing collected water samples on 3M Petrifilm with media that can culture total coliforms, fecal coliforms and E. coli specifically. Not all coliforms are of intestinal origin, but Petrifilm culture s can distinguish fecal coliforms and E. coli from other coliform colonies. Coliform bacteria serve as good indicators of wate r contamination because they can survive to culturability at temperatures lower than 26 C (Prescott et al 1999). Water sources are thus suitable reservoirs for fecal coliforms to survive for several days. In addition to water source contamination, f ecal coliform bacteria can be present in environments surrounding affected water sources. This can be a result of airborne water spray from the water source by such mechanisms as ricochet off surfaces, bursting water bubbles, rain water splash and surf break (Lighthart and Frisch, 1976). Cronholm (1980) demonstrated that aerosolized bacteria from sewage treatment facilities and urban fountain spray was present in ambient air samples and on nearby foliage. Telt sch and Katzenelson (1978) determined fecal bacterial contamination from sp ray irrigation of plants with wastewater effluent on both downwind air and petri surface samples. Th eir study examined the effects of several environmental factors on the su rvivability of airborne bacter ial contamination and found that increased solar exposure had a negative correlation with airborne bacteria while greater humidity positively correlated with increased bacterial concentrations in the air. While previous studies have examined the transfer mechanisms, environmental effects and general presence of fecal coliform bacter ia in the air and on vegetation near sewage treatment facilities, urban water sources and wast ewater irrigation sources, no research has been done to examine similar bacterial presence in en vironments surrounding contaminated streams. This study analyzed the presence of to tal coliform bacteria, fecal coliform and E. coli contamination on ferns near contaminated stream sources and near a clean stream. I predicted that vegetation surrounding contaminated streams w ould exhibit higher levels of fecal coliforms and E. coli than vegetation at the clean site. I also collected and an alyzed plants at different

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Jacobson Coliform Bacteria on Plants 3 distances from the stream and predicted that bacterial contamination would decrease with increased distance from the stream. Additionally I examined several environmental factors to determine if any play a significant role in bact erial presence in near-stream environments and predicted that greater humidity would increase coliform presence while greater sun exposure, measured inversely by percent canopy cover, would decrease coliform presence. Lastly, I attempted to determine if airborne spray from the streams is the mechanism by which bacterial contamination occurs on near stream vegetation. Materials and Methods From 24 November 2006 to 2 December 2006, I analyzed the pr esence of coliform bacterial contamination on near-s tream plants from threes site along three streams (one per stream) in the Santa Elena and San Luis area (F igure 6). The Quebrada Rodrguez runs through the town of Santa Elena and receives untreated black and gray water pollution from the urban area. The Quebrada Cambronero receives minimally treated sewage from the Monteverde Cheese Factory pig farm. I also chose a site along the Quebrada Alondra downstream from a source that is relativel y unaffected by human activity. Both the Cambronero and Alondra sites were similar in altitude and all three sites originate from a single, primary source. I collected a water sample from each stream by filling a sterile container with water while standing downstream from the collection c up. I collected stream samples at a location directly adjacent to the near-stream sites where ferns were collected for analysis and where all spray mechanism studies were performed. I collected a single water sample from each stream on 25 November 2006 and 28 November 2006 for a total of two samples per stream. In order to determine the presence and quant ity of coliform bacteria from each water source, I placed 1mL samples of collected stre am water on Total Coliform Count Petrifilm and E. coli/Coliform Petrifilm plates (3M Petrifilm). I incubated plates at 37 C for 24 hours and then counted the number of total coliform colonies, fecal coliform colonies and E. coli colonies present on each plate. To obtai n the number of bacterial colo nies present on an incubated Petrifilm plate, I utilized a 1x1-cm grid syst em present on each plate for accurate counting. Blue colonies signify E. coli growth, red colonies with associ ated gas signify fecal coliforms, and red colonies without associ ated gas signify other coliforms (C. Calvo pers. comm.). A summation of all colonies on a given plate signi fies total coliform count. Plates that demonstrated single grid squares with greater than 20 colonies were considered to have colonies too numerous to count based on AOAC Official Methods (3M Petr ifilm instruction manual). In the presentation of results, a ll plates with colonies too nume rous to count were assigned a numerical value of 2000 for statistical purposes. A gram staining procedure was performed on select colonies of apparent total coliform, fecal coliform and E. coli to verify that the colonies were indeed composed of gram negative, coliform bacteria (Prescott et al 1999; C. Calvo pers. comm.) In order to test for coliform presence on pl ants, I collected two mo rphospecies of ferns of the genera Pteris and Thelypteris for lab analysis from each site based on their common inhabitation of all three sites. I collected thr ee plant specimens at each near stream site on 25 November 2006 and 28 November 2006 for a total of six specimens per stream site. These plants were collected at distan ces of 0m, 0.5m, and 4.5m from the stream (two specimens per distance) at each site to determine if increa sed distance from the water source affects the amount of bacteria present on plant material. I chose the height of selected plant material to be within a range of 0.5m-1m from the ground. I di d this to safeguard ag ainst direct, continuous

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Jacobson Coliform Bacteria on Plants 4 contact with stream water, and to possibly reduce the chance of plant trampling and contamination by human or animal sources (C. Ca lvo pers. comm.). I washed collected samples with 5mL of saline buffer and then transferred 1mL of the wash to Petrifilm plates. I performed incubation, counted colonies and an alyzed selected colonies by a gram stain procedure. When counting colonies, I multiplied all values by five to account for the original 5mL wash used on the plants. For every plant collection, I measured various environmental factors to determine if such factors affect amount of bacteria present. These factors included atmospheric temperature ( C), stream temperature ( C), stream water velocity (m/s), percent relative humidity and solar exposure, measured inversely by percent canopy cover. I collected atmospheric and water temperature using a standard Celsius thermometer. To calculate humidity, I collected a dry air temperature and wet bulb temperature with a standard thermometer and utilized a relative humidity calculator computer function. Wate r velocity was measured by placing a weight attached to string in the stream flow and reco rding the travel time of a 1m distance. Canopy cover was measured by taking a digital photo of the canopy over each plant collected and analyzing the percent of light pene tration with Adobe Photoshop (TM). In order to test for an airborne spray mechanism of bacterial contamination, I mounted Petrifilm plates on PVC pipes to co llect potential spray from the st reams. I placed the films at vertical distances of 0m and 0.5m from the gr ound and horizontal distances of 0m, 0.5m and 4.5m from the streams. All plates were opened to face the streams. Six plates of Total Coliform Count Petrifilms and six plates of E. Coli/Colifor m Count Petrifilms were utilized per trial at the Alondra and Cambronero sites. Two trials were performed at each site, one for 20 minutes and one for 1 hour. Due to lack of material s, one 20-minute trial was performed at the Quebrada Rodrguez site with two Petrifilm plates mounted at a vertical distance of 0.5m from the ground and a horizontal distance of 0m from the stre am. All exposed Petrifilm plates were rehydrated with 1mL of saline buffer and incubated for 24-48 hours at 37 C. Plates were then analyzed for colony growth. I analyzed my results for coliform bacteria presence in water and on plants between sites using a Kruskal-Wallis Test (JMP-IN). I al so utilized a Kruskal-Wallis Test to determine if a difference in distance from streams aff ects quantity of plant contamination. I used a multiple regression method to determine if e nvironmental factors influenced bacterial contamination on plants (JMP-IN). I perf ormed a correlation be tween water and plant contamination at a distance of 0m from the stream s to determine if a relationship exists between the quantities of bacter ial contamination found in water and on adjacent plants (Statistica). Statistical analyses were not performed for airb orne spray trials due to minimal growth of countable colonies on Petrifilm plates. Results All gram stains performed verified the presence of gram negative, coliform colonies. Water samples from the Quebrada Rodr guez and the Quebrada Cambronero had higher total coliform counts (Fig ure 1, Kruskal-Wallis=6.97, df=2, p=0.031) than the Quebrada Alondra. Quebrada Rodrguez exhibited signifi cantly higher fecal coliform counts (Figure 1, Kruskal-Wallis=6.87, df=2, p=0.032) than the Qu ebradas Cambronero and Alondra. While there was not a statistically significant difference in E. coli counts between the water sites, a trend is present in which more E. Coli colonies were counted for Quebradas Rodrguez and Cambronero than for the Quebrada Alondra (Figure 1, Kruskal-Wallis=3.6, df=2, p=0.17).

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Jacobson Coliform Bacteria on Plants 5 Fern samples collected from the Quebradas Rodrguez and Cambronero sites demonstrated higher total coliform colony count than those from the Quebradas Alondra site (Figure 2, Kruskal-Wallis=8.23, df=2, p=0.01). Ther e was no statistically significant difference in fecal coliform count (Kruskal-Wallis=2.77, df=2, p=0.25) or E. coli count ( KruskalWallis=3.63, df=2, p=0.16) over the three sites. However, Figure 2 demonstrates a trend in which greater amounts of fecal coliform colonies and E. Coli colonies were cultured from plants at the Quebradas Rodrguez and Cambrone ro sites than from the Quebrada Alondra site. No significant correlations exist between water contamination and plant contamination at 0m for total coliform, fecal coliform or E coli counts (Figure 5; r=0.57, p=0.08, N=12; r=0.30, p= 0.39, N=12; r=-0.21, p=0.74, N=6). Howe ver similar trends in water and plant samples suggest less contamination at the Qu ebrada Alondra site for both analyses when compared to the Quebradas Rodrguez and Cambronero (Figures 1 and 2). While there was no significant difference in total coliform, fecal coliform or E. coli quantity on plants at three diffe rent distances from the stream (Kruskal-Wallis=1.68, df=2, p=0.43; Kruskal-Wallis=0.51, df=2, p=0.78; Kruskal-Wallis=2.47, df=2, p=0.29), a visible trend demonstrates decreased colony counts for all three coliform types from plants at increased distances from the streams (Figure 3). Of the environmental factors measured, increased atmospheric temperature and percent canopy cover positively affected the am ount of total coliform bacterial counts from ferns (Table 1, Appendix 1). Water temperature, percent relative humidity and water velocity did not significantly affect to tal coliform count (Table 1, A ppendix 1). No significant relationships were found between fecal coliform counts and environmental factors measured (Table 2, Appendix 1). For E coli counts, percent relative hu midity was significant and demonstrated a positive relationship (Table 3, Figure 4, Appendix 1). Of 50 films used to test air borne spray, five demonstrated growth of bacterial colonies after incubation. Less than five colonies of f ecal coliforms and total coliforms were counted on both the films placed at the Quebrada Rodrguez si te. Of the plates set out at the Cambronero site, two plates placed at 0.5m from the stream developed one colony of fecal coliforms. One film placed 0m from the stream developed a colony of E. coli. All three films developed 5 or less colonies of total coliform. No films set out at the Alondra site demonstrated coliform growth after incubation. Discussion Water sample comparisons between the th ree stream sites de monstrate that the Quebrada Alondra is less contaminated by to tal coliforms and fecal coliforms than the Rodrguez and Cambronero Quebradas a nd appears to be less contaminated by E. coli as well (Figure 1). These results are consistent with th e natural history of the Monteverde area, as the Rodrguez and Cambronero Quebradas are exposed to human and animal fecal contamination from the densely populated Santa Elena area and Monteverde Cheese Factory pig farm respectively, while the Quebrada Alondra is relatively unaffected by human influence. Similar to water sampling results, total coli form counts from ferns at the three nearstream sites demonstrate significantly more c ontamination of plants at the Rodrguez and Cambronero Quebrada sites than at the Alondra Qu ebrada site. Trends for fecal coliform and E. coli colony counts suggest the same pattern (Figure 2). Trends for all three coliform types suggest that with increased distance from the streams, plants exhibited decreasing amounts of bacterial contaminati on (Figure 3). While

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Jacobson Coliform Bacteria on Plants 6 these comparisons were not significant, the tren ds suggest that plant contamination may be resultant of stream contamination. An increas e in plant sample size from three distances may provide more significant results. While no statistically significant correlation between water and plant samples was presented for relative amounts of bacterial contamination, comparative trends suggest a potential relationship between water and subsequent plant co ntamination when comparing polluted stream sites to a pristine stream site (F igures 1 and 2). An increase in sample size and a decrease in standard deviations may demons trate more a significant correlation in future studies. Analyses of environmental factors suggest a positive relations hip between percent canopy cover and total coliform count. As pe rcent canopy cover inversely measures solar exposure, a negative relationship between solar exposur e and total coliforms can be inferred. This result supports the documente d effects of sunlight exposure on coliform bacteria: solar exposure increases the die-out rate of colif orms in natural environments (Prescott et al. 1999). Relative humidity also positively affected tota l coliform presence on plants. This result is consistent with past research demonstrating increased airborne coliform b acteria with increased relative humidity (Teltsch and Katzelson 1978). This study also demonstrated a positive relationship between increased atmospheric temperature and E. coli bacteria on plants. Coliform bacteria however, survives better at lower temperatures (Prescott et al. 1999). This inconsistency may be due to relatively low temperature fluctuations in the data (Appendix 1). From my obser vations at thr ee sites over two days, atmospheric temperature ranged from 18 C-19.5 C. Five of the 50 Petrifilm plates set out to collect stream spray and associated bacterial contamination presented a small number of total coliform, fecal coliform and E. coli colonies. These five films were set out at the polluted Rodrguez and Cambronero Quebrada sites, and were placed at either 0m or 0.5m from the streams. No films co llected from the Alondra site or far from the streams (4.5m) demonstrated growth. These results could suggest contamination by airborne spray. The minimal number of Petrifilms to exhi bit growth may be explained by a few factors. During stream spray collection, many of the Petrifilm plates used were exposed to excessive moisture or became discolored with pr olonged exposure to sunlight. This may have negatively affected several of the Petrifilms ability to culture coliform bacteria (3M Petrifilm instruction manual). Additionally Teltsch and Katzenelson (1978) demonstrated the presence of airborne bacteria downwind from an irrigation source by setting out Petri materials in collection sieves for 48 hours. Perhaps future studies co uld utilize prolonged, prot ected exposure of Petri materials or alter the means of collection to dete rmine if airborne contamination from streams is a mechanism for coliform presence on near-stream plants. It should also be mentioned that insects were noted to land on films during trials. This could be another possible mechanism for fecal bacterial transfer on plants (Geldreich et al ., 1968). Animals or people could also introduce bact erial contamination either from the streams or from an alternate source. Further studies should be done to determine the mechanism of bacterial transfer onto near-stream pl ants from polluted streams. Water contamination by black water and consequent fecal coliforms is a continuing problem for water sources near human-influenced areas. This study adequately demonstrates that fecal coliforms are present in streams in the Monteverde area. Coupled with the threat of water pollution by coliform bacter ia, is the potential for asso ciated pathogenic disease.

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Jacobson Coliform Bacteria on Plants 7 Presence of fecal contamination in areas su rrounding polluted streams could increase this potential exposure for nearby human and animal communities. While this study demonstrated evident trends to support the re lationships between coliform contamination found in water and in near-by environments, further studies with increased sample size could show improved statistical significance of such trends. Acknowledgements I would like to thank Ramsa Chaves for her endless guidance, patience, and friendship throughout my project. I would also like to th ank Ruth Salas for her dedication and impeccable attention to detail. Many thanks to Dr. Carl os Alfonso Calvo for his enthusiasm for teaching and his insight into the world of fecal coliforms and Petrifilm, and Erick McAdam for the drives to San Luis and his vast knowledge about relative humidity. Literature Cited Cronholm, L. S. 1980. Potential health hazards from microbial aerosols in densely populated urban regions. Applied and Environmental Microbiology. 39: 6-12. Geldreich, E. E., B. A. Kenner and P. W. Kabler. 1964. Occurrence of Coliforms, Fecal Coliforms, and Streptococci on Vegetation and Insects. Applied Environmental Microbiology 12: 63-69. Lighthart, B. and A. S. Frisch. 1976. Estimation of viable airborne microbes downwind from a point source. Applied and Environmental Microbiology. 31: 700-704. Prescott, L. M., J.P. Harley, and D. A. Klein. 1999. Microbiology. WCB/McGraw-Hill, Boston. Teltsch, B. and E. Katzenelson. 1978. Airborne enteric bacteria and viruses from spray irrigation with wastewater. Applied and Environmental Microbiology. 35: 290-296.

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Jacobson Coliform Bacteria on Plants 8 -100 -50 0 50 100 150 200 250 300 350 400 450 500 Alondra Cambronero Rodriguez SiteColonies/mL tc fc ec EC FC Figure 1: Colonies of total coliform (t c: N=12), fecal coliform (fc: N=12) and E. Coli (ec: N=6) cultured from 1mL water samples at three sites. -500 0 500 1000 1500 2000 2500 AlondraCambronero TC FC EC Supermerc ado SitesColonies/5mL EC EC FC Rodriguez Figure 2: Colonies of total coliform (t c: N=12), fecal coliform (fc: N=12) and E. Coli (ec: N=6) cultured from 5mL saline wash of fern samples at three sites.

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Jacobson Coliform Bacteria on Plants 9 -1000 -500 0 500 1000 1500 2000 2500 00 54 5 Distance (meters)Colonies/5mL tc fc ec Figure 3: Colonies of total coliform (t c: N=12), fecal coliform (fc: N=12) and E. Coli (ec: N=6) cultured from saline wash of fern samples at three distances from streams. Table 1: Regression values for the effects of environmental factors on total coliform colony counts from fern samples at three near stream sampling sites. Data collected over two days at each site. N=30 Rsquare=0.0000 Parameter NDF SS F Ratio Prob>F Temperature C 1 4717953 9,554 0,0045 Water Temperature C 1 17681,33 0,027 0,8713 % Relative Humidity 1 1853219 3,090 0,0897 Water Velocity (m/s) 1 283414,8 0,435 0,5151 % Canopy Cover 1 5317307 11,256 0,0023 FC EC Table 2: Regression values for the effects of environm ental factors on fecal colifor m colony counts from fern samples at three near stream sampling sites. Data collected over two days at each site. N=30 Rsquare=0.0000 Parameter nDF SS "F Ratio" "Prob>F" Temperature C 1 217667.8 1.127 0.2974 Water Temperature C 1 21505.87 0.107 0.7455 % Relative Humidity 1 142816.7 0.73 0.4003 Water Velocity (m/s) 1 6654.017 0.033 0.8568 % Canopy Cover 1 26876.34 0.134 0.7166 Table 3: Regression values for the effects of environmental factors on E. coli colony counts from fern samples at three near stream sampling sites. Data collected over two days at each site. N=15 Rsquare=0.0000 Parameter NDF SS "F Ratio" "Prob>F" Temperature C 1 10800 0,036 0,8528 Water Temperature C 1 734410 2,987 0,1076 % Relative Humidity 1 1596008 8,888 0,0106 Water Velocity (m/s) 1 865790,3 3,673 0,0776

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Jacobson Coliform Bacteria on Plants 10 % Canopy Cover 1 139620,9 0,479 0,5011 a) 0 500 1000 1500 2000 2500 5060708090100 % Canopy CoverTC Colonies/5mL b) Figure 4: Total coliform count s according to a) % Canopy Cover and b) Atmospheric Temperature. 0 500 1000 1500 2000 2500 17.5 18 18.5 19 19.5 20 Atmospheric Temperature (C)TC Colonies/5mL -500 0 500 1000 1500 2000 2500 80828486889092 % Relative Humidity Colonies/5mL

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Jacobson Coliform Bacteria on Plants 11 Figure 5: E. coli colony counts on ferns according to %Relat ive Humidity at three near stream sampling sites. Appendix 1: Multiple regression data for total coliforms (tc), fecal coliforms (fc) and E. coli (ec) on ferns and environmental factors. TC EC FC Temperat ure (C) Water Temperature (C) % Relative Humidity Water Velocity (m/s) % Canopy Cover 164 0 0 23 19 83 0.24 77.8 600 150 23 19 83 0.24 77.8 2000 30 0 23 19 83 0.24 91.5 2000 40 23 19 83 0.24 91.5 2000 0 5 23 19 83 0.24 91.6 2000 10 23 19 83 0.24 91.6 185 5 0 23 19 83 0.24 61.2 210 10 23 19 83 0.24 61.2 415 0 65 23 19 83 0.24 85.2 660 10 23 19 83 0.24 85.2 60 0 10 23 19 83 0.24 55.9 100 15 23 19 83 0.24 55.9 2000 135 0 21 19 86 0.2 79.3 2000 0 21 19 86 0.2 79.3 2000 0 5 21 19 86 0.2 94.3 2000 0 21 19 86 0.2 94.3 2000 205 0 21 19 86 0.2 95.6 2000 125 21 19 86 0.2 95.6 2000 2000 0 22 19.5 91 0.28 77.6 2000 2000 22 19.5 91 0.28 77.6 1290 730 0 22 19.5 91 0.28 73.5 1125 585 22 19.5 91 0.28 73.5 2000 0 0 22 19.5 91 0.28 80.8 2000 0 22 19.5 91 0.28 80.8 2000 10 1120 20.5 18 81 0.17 93.6 940 940 20.5 18 81 0.17 93.6 2000 45 35 20.5 18 81 0.17 93.6 2000 115 20.5 18 81 0.17 93.6 95 0 0 20.5 18 81 0.17 91.6 2000 0 20.5 18 81 0.17 91.6

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Jacobson Coliform Bacteria on Plants 12 Figure 6: Correlation of plant samples versus water samples for total coliforms (TC), fecal coliforms (FC) and E. coli (EC).

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Jacobson Coliform Bacteria on Plants 13 Figure 7: Topographical map of the Monterverd e Area. Three near-stream sites are shown.