FECAL COLIFORM POLLUTION TESTING - BIS 293 - Coastlines and
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Figure 3, m-ColiBlue24 Broth and Colilert testing media. (Melanie Panoke, 2007) Fecal Coliform in Hick's Lake 0 100 200 300 400 500 600 Site A Site B Site C Site D Site E Site F Site G Site H Conclusion: Lowest State Standard exceeded by both tests in 3 of 8 sites. For 3 sites, Colilert passed while M-coliblue 24 failed. colonies/100mL (qty of 500 = >500) Fecal Coliform M-coliblue 24, 44.5 Fecal Coliform Colilert, 44.5 Secondary Contact Standard After Washington State Dept. of Ecology, 2002 TOTAL COLIFORM, FECAL COLIFORM, AND E. COLI Total Coliform Total Coliform = Environmetnal Contamination Fecal Coliform Fecal Coliform & E. Coli = Fecal Contamination E. Coli 0157:H7 Figure 1, After Washington State Dept. of Health, 2007. E. coli as an indicator for Fecal Coliform using Colilert (two outliers removed) R 2 = 0.5076 0 50 100 150 200 250 300 350 400 0.0 50.0 100.0 150.0 200.0 250.0 Fecal Coliform BIS 293 - Coastlines and Estuaries Prepared by: Donald Fraser [email protected] Orsolya Nance [email protected] Melanie Panoke [email protected] Christopher A. Scott [email protected] FECAL COLIFORM POLLUTION TESTING - NORTH CREEK AND HICKS LAKE Figures 2a, 2b, and 2c Site Location Maps showing Fecal Coliform testing Locations. After USGS and King County, 2007. North Creek Site A North Creek Sites B and C Hicks Lake Sample Sites BACKGROUND Fecal Coliforms are thermotolerant bacteria capable of growing at an elevated temperature of 44.5°C. This group of organisms includes species such as: Klebsiella pneumoniae and Citrobacter freundii, but are primarily represented by Escherichia coli. E.coli is known as the ‘best coliform indicator of fecal contamination’ (Hach 2007) This suggest surface waters with elevated Fecal Coliforms has the potential risk of containing harmful pathogens. (Hach Company, 2007) Poor water quality due to Fecal Coliform and E.coli contamination prevents recreational use and poses a threat to public and environmental health. High bacteria levels also lead to low dissolved oxygen levels that make sustainability hard for aquatic species (Meehan, M., & Kalenius, S. 2004, June). Washington Adminstrative Code 173-201A-200 regulates the use of fresh waterbodies when Fecal Coliform levels exceed the state standards for defined waters. Secondary Contact Recreation, waters with limited use, must meet the following criteria: ‘Fecal Coliform organism levels must not exceed a geometric mean value of 200 colonies/100mL, with not more than 10 percent of all samples (or any single sample when less than ten sample points exist) obtained for calculating the geometric mean value exceeding 400 colonies/100mL. HYPOTHESES H1: E. coli is an equivalent indicator of water quality compared to Fecal Coliform testing. H2: The Colilert test will give equivalent results to the m-ColiBlue24 Broth test for the same samples. H3: Coliform levels will be lower downstream in North Creekthan upstream due to vegetative filtering. H4: Fecal Coliform in Hicks Lake will exceed State standards. as an indicator for fecal coliform using the Colilert test at 44.5 deg. C 0 50 100 150 200 250 300 350 400 Week 1 Week 2 Week 3 Week 4 Correlations for North Creek: .97, .65, and .96 respectively. Conclusion: Good correlation between E. coli and Fecal Coliform using Colilert test Colonies/100 ml Site A: Fecal Coliform Site A: E. coli Site B: Fecal Coliform Site B: E. coli Site C: Fecal Coliform Site C: E. coli TESTING PROCEDURES Our laboratory testing was conducted at the Washington State Department of Health Public Health Laboratories in Shoreline, Washington. We performed two procedures using the Millipore m-Coliblue24® Broth and Idexx Colilert®. Each sample was run in parallel at two different temperatures, 35°C and 44.5°C, to determine Total Coliform, Fecal Coliform, and E.coli contamination levels. Idexx Colilert® Methodology: Colilert uses a ‘Defined Substrate Techology’ which allows for simultaneous detection of Total Coliforms and E.coli. Two nutrient indicators, ONPG and MUG, are used to detect metabolic activity of the enzymes •-glalactosidase and •-glucoronidase, respectfully. After adding the Colilert reagent to a 100mL sample and incubating for a 24 hour period at 35°C, detection of Total Coliforms can be observed by a color change from clear to yellow. Samples which turns yellow are subjected to long-wavelength UV light (362nm) which will fluoresce when E.coli is present. Figure 3 (bottom). Membrane Filtration Methodology using Millipore m-Coliblue24®: Millipore m-coliblue24® is a lactose based media containing inhibitors which allows for simultaneous detection of Total Coliforms and E.coli. Water samples are vacuum filtered through 47mm membrane filters with 0.45 mm porosity trapping coliforms on the filter surface. The filter is then transferred into a petridish containing an absorbent pad soaked in the m- Coliblue24 media and inverted for incubation. After a 24 hour incubation at 35°C, red and blue colonies will develop. Total Coliforms are determined by adding the total number of red and blue colonies. E.coli is determined by adding the total number of blue colonies. (Hach Company, 2007, Millipore Company, 2007) Figure 3 (top). ANALYSIS H1: E. coli is an equivalent indicator of water quality compared to Fecal Coliform testing. Conclusion: E.coli is an indicator of Fecal Coliform contamination. However, results show evidence that levels of E.coli is not directly proportional. Figure 5. H2: The Colilert test will give equivalent results to the m-ColiBlue24 Broth test for the same samples. Conclusion: Colilert and m-coliblue24 do not produce comparable results, but do provide trends of excessive coliform contamination. Colilert can produce most probable number values for a 100mL volume without the need for further dilutions. M-Coliblue24 may require additional dilutions depending on the coliform density of each sample. Confluent growth and excess debris trapped on the filter may interfere with the interpretation of results. Figure 6. H3: Coliform levels will be lower downstream in North Creek than upstream due to vegetative filtering. Conclusion: Results prove Fecal Coliforms slightly decline downstream in North Creek. However, additional testing would be required to demonstrate statistically. Figure 4. H4: Fecal Coliform in Hicks Lake will exceed State standards. Conclusion: Results prove Fecal Coliforms consistently exceed the ‘Secondary Contact Recreational Fecal Coliform Value of 200 Colonies/100mL.’ (WAC-173-201A-200, ) Figure 6. SITE INFORMATION HICKS LAKE (Figure 2c) Hicks Lake, also known as Lake Garrett, resides in Lakewood Park in Seattle, Washington. This 4-acre lake sitting on three aquifers is fed by springs, but is heavily influenced by surface run-off, neighborhood septic tanks, and the local high school athletic field. Because Hicks Lake has no natural outlets, water overflow is pumped out of the lake to nearby Salmon Creek during heavy rain events. Hicks Lake has not been used for recreational purposes since 1975 due to poor water quality. This presents concerns when contaminated lake water is pumped into Salmon Creek which feeds directly into the Puget Sound. (King County Department of Natural Resources and Parks, 2005.) NORTH CREEK (Figure 2a,b) North Creek has a 30 square mile basin running from Everett to the Sammamish River in Bothell. High Fecal Coliform contamination is the result of failing septic systems, waste from pets, livestock and wildlife. Since it is a primary salmon habitat, its contamination is a great concern for the City of Bothell (Meehan, M., & Kalenius, S. 2004). Figure 4, Comparison of Fecal Coliform and E. coli levels in North Creek samples using the Colilert method at 44.5° C. Figure 6, Fecal Coliform levels in Hicks Lake. Figure 5, Statistical corrolation between E.coli and Fecal Coliform in terms of their use as biological indicators of water quality.
Transcript of FECAL COLIFORM POLLUTION TESTING - BIS 293 - Coastlines and
Figure 3, m-ColiBlue24 Brothand Colilert testing media.(Melanie Panoke, 2007)
Fecal Coliform in Hick's Lake
0
100
200
300
400
500
600
Site A Site B Site C Site D Site E Site F Site G Site H
Conclusion: Lowest State Standard exceeded by both testsin 3 of 8 sites. For 3 sites, Colilert passed while M-coliblue
24 failed.
colo
nies
/100
mL
(qty
of 5
00 =
>50
0)
Fecal Coliform M-coliblue 24, 44.5
Fecal Coliform Colilert, 44.5
Secondary Contact Standard
After Washington StateDept. of Ecology, 2002
TOTAL COLIFORM, FECALCOLIFORM, AND E. COLI
Total ColiformTotal Coliform = EnvironmetnalContamination
Fecal ColiformFecal Coliform & E. Coli =Fecal Contamination
E. Coli 0157:H7
Figure 1, After Washington StateDept. of Health, 2007.
E. coli as an indicator for Fecal Coliform usingColilert (two outliers removed)
R2 = 0.5076
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50
100
150200
250
300
350
400
0.0 50.0 100.0 150.0 200.0 250.0Fe
cal C
olifo
rm
BIS 293 -Coastlines and Estuaries
Prepared by:
Donald [email protected] [email protected] [email protected] A. [email protected]
FECAL COLIFORM POLLUTION TESTING -NORTH CREEK AND HICKS LAKE
Figures 2a, 2b, and 2c Site Location Maps showingFecal Coliform testing Locations. After USGS and King County, 2007.
North Creek Site A North Creek Sites B and C Hicks Lake Sample Sites
BACKGROUNDFecal Coliforms are thermotolerant bacteria capable of growing at an elevatedtemperature of 44.5°C. This group of organisms includes species such as:Klebsiella pneumoniae and Citrobacter freundii, but are primarily representedby Escherichia coli. E.coli is known as the ‘best coliform indicator of fecalcontamination’ (Hach 2007) This suggest surface waters with elevated FecalColiforms has the potential risk of containing harmful pathogens. (HachCompany, 2007)
Poor water quality due to Fecal Coliform and E.coli contamination preventsrecreational use and poses a threat to public and environmental health. Highbacteria levels also lead to low dissolved oxygen levels that make sustainabilityhard for aquatic species (Meehan, M., & Kalenius, S. 2004, June).
Washington Adminstrative Code 173-201A-200 regulates the use of freshwaterbodies when Fecal Coliform levels exceed the state standards for definedwaters. Secondary Contact Recreation, waters with limited use, must meetthe following criteria: ‘Fecal Coliform organism levels must not exceed ageometric mean value of 200 colonies/100mL, with not more than 10 percentof all samples (or any single sample when less than ten sample points exist)obtained for calculating the geometric mean value exceeding 400colonies/100mL.
HYPOTHESESH1: E. coli is an equivalent indicator of water quality
compared to Fecal Coliform testing.
H2: The Colilert test will give equivalent results tothe m-ColiBlue24 Broth test for the samesamples.
H3: Coliform levels will be lower downstream inNorth Creekthan upstream due to vegetativefiltering.
H4: Fecal Coliform in Hicks Lake will exceed Statestandards.
as an indicator for fecal coliformusing the Colilert test at 44.5 deg. C
0
50
100
150
200
250
300
350
400
Week 1 Week 2 Week 3 Week 4Correlations for North Creek: .97, .65, and .96 respectively.
Conclusion: Good correlation between E. coli and Fecal Coliform usingColilert test
Col
onie
s/10
0 m
l
Site A: Fecal Coliform
Site A: E. coli
Site B: Fecal Coliform
Site B: E. coli
Site C: Fecal Coliform
Site C: E. coli
TESTING PROCEDURESOur laboratory testing was conducted at the Washington State Department of Health PublicHealth Laboratories in Shoreline, Washington. We performed two procedures using theMillipore m-Coliblue24® Broth and Idexx Colilert®. Each sample was run in parallel at twodifferent temperatures, 35°C and 44.5°C, to determine Total Coliform, Fecal Coliform, andE.coli contamination levels.
Idexx Colilert® Methodology:Colilert uses a ‘Defined Substrate Techology’ which allows for simultaneous detection of TotalColiforms and E.coli. Two nutrient indicators, ONPG and MUG, are used to detect metabolicactivity of the enzymes •-glalactosidase and •-glucoronidase, respectfully. After adding theColilert reagent to a 100mL sample and incubating for a 24 hour period at 35°C, detectionof Total Coliforms can be observed by a color change from clear to yellow. Samples whichturns yellow are subjected to long-wavelength UV light (362nm) which will fluoresce whenE.coli is present. Figure 3 (bottom).
Membrane Filtration Methodology using Millipore m-Coliblue24®:Millipore m-coliblue24® is a lactose based media containing inhibitors which allows forsimultaneous detection of Total Coliforms and E.coli. Water samples are vacuum filteredthrough 47mm membrane filters with 0.45 mm porosity trapping coliforms on the filter surface.The filter is then transferred into a petridish containing an absorbent pad soaked in the m-Coliblue24 media and inverted for incubation. After a 24 hour incubation at 35°C, red andblue colonies will develop. Total Coliforms are determined by adding the total number of redand blue colonies. E.coli is determined by adding the total number of blue colonies. (HachCompany, 2007, Millipore Company, 2007) Figure 3 (top).
ANALYSISH1: E. coli is an equivalent indicator of water quality compared
to Fecal Coliform testing.Conclusion: E.coli is an indicator of Fecal Coliform contamination. However, results show evidencethat levels of E.coli is not directly proportional. Figure 5.
H2: The Colilert test will give equivalent results to the m-ColiBlue24 Broth test for the same samples.Conclusion: Colilert and m-coliblue24 do not produce comparable results, but do provide trendsof excessive coliform contamination. Colilert can produce most probable number values for a 100mLvolume without the need for further dilutions. M-Coliblue24 may require additional dilutions dependingon the coliform density of each sample. Confluent growth and excess debris trapped on the filtermay interfere with the interpretation of results. Figure 6.
H3: Coliform levels will be lower downstream in North Creek than upstream due to vegetative filtering.Conclusion: Results prove Fecal Coliforms slightly decline downstream in North Creek. However,additional testing would be required to demonstrate statistically. Figure 4.
H4: Fecal Coliform in Hicks Lake will exceed State standards.Conclusion: Results prove Fecal Coliforms consistently exceed the ‘Secondary Contact RecreationalFecal Coliform Value of 200 Colonies/100mL.’ (WAC-173-201A-200, ) Figure 6.
SITE INFORMATIONHICKS LAKE (Figure 2c)Hicks Lake, also known as Lake Garrett, resides in Lakewood Park in Seattle,Washington. This 4-acre lake sitting on three aquifers is fed by springs, butis heavily influenced by surface run-off, neighborhood septic tanks, and thelocal high school athletic field. Because Hicks Lake has no natural outlets,water overflow is pumped out of the lake to nearby Salmon Creek during heavyrain events. Hicks Lake has not been used for recreational purposes since1975 due to poor water quality. This presents concerns when contaminatedlake water is pumped into Salmon Creek which feeds directly into the PugetSound. (King County Department of Natural Resources and Parks, 2005.)
NORTH CREEK (Figure 2a,b)North Creek has a 30 square mile basin running from Everett to the SammamishRiver in Bothell. High Fecal Coliform contamination is the result of failing septicsystems, waste from pets, livestock and wildlife. Since it is a primary salmonhabitat, its contamination is a great concern for the City of Bothell (Meehan,M., & Kalenius, S. 2004).
Figure 4, Comparison of Fecal Coliform and E. colilevels in North Creek samples using the Colilert methodat 44.5° C.
Figure 6, Fecal Coliform levels in Hicks Lake.Figure 5, Statistical corrolation between E.coli andFecal Coliform in terms of their use as biological indicatorsof water quality.
