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Brown, Rachel R.

Burns, Eugene H. Jr.

Hinds, Terry D. Jr.

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Coliforms in experimental wetlands ♦  69Reduction of fecal coliform levels in two created wetlands at theOlentangy River Wetland Research ParkTerry D. Hinds, Jr., Rachel R. Brown, and Eugene H. Burns, Jr.*Department of Natural Sciences, Shawnee State University, Portsmouth OH 45662________________________________________*Corresponding authorEugene H. Burns, Jr., Dept. of Natural Sciences,Shawnee State University,940 Second Street, Portsmouth, Ohio 45662email: eburns@shawnee.eduphone: (740) 351-3685IntroductionThe presence of fecal coliform bacteria in aquaticenvironments is often used as an indicator of contaminationwith fecal material and other possible pollutants (Tyrrell etal., 1995; Hernandez et al., 1997; Gearheart, 1999; McMathet al., 1999; Perkins and Hunter, 2000). Fecal coliformpollution may occur in ambient water as a result of theoverflow of domestic sewage or non-point sources ofhuman or animal waste (McMath et al., 1999). River watermay be contaminated with fecal material of man or otheranimals. At the time of contamination, pathogens may beintroduced into the water (Ricca and Cooney, 1999; Tyrrellet al., 1995). Over 140 different virus types and manydifferent bacteria are excreted in animal and human fecesand urine, including Escherichia coli, Salmonella enteritis,hepatitis A and viral gastroenteritis that are pathogenic tohumans (Hernandez et al., 1997; Newman et al., 2000;Ricca and Cooney, 1999; Tyrrell et al., 1995). Thus, thepresence of fecal contamination is an indicator that apotential health risk exists for individuals exposed to thiswater (Newman et al., 2000; Ricca and Cooney, 1999;Tyrrell et al., 1995).Increasingly, constructed wetlands are being used forlowering pollutant levels in contaminated water andwastewater, including treatment for urban and agriculturalstorm water runoff that may contain chemical and otherpollutants (Carleton et al., 2001; Gerba et al., 1999;Khatiwada and Polprasert, 1999; Newman et al., 2000;Ostroumov, 1998; Perkins and Hunter, 2000; Shutes, 2001).Studies have shown that wetlands improve water qualityby reducing nutrients, chemical contaminants, andpathogenic microbes (Gerba et al., 1999; Ostroumov,1998; Lau and Chu, 2000). Previous studies at the OlentangyRiver Wetland Research Park (ORWRP) have showndecreasing levels in nitrate+nitrite, soluble-reactivephosphorus, and total phosphorus as the water passesthrough the wetlands (Mitsch et al., 2000).The purpose of this study was to examine fecal coliformlevels in two constructed wetlands at ORWRP and todetermine the ability of these basins to reduce coliforms.Samples were collected from locations throughout bothwetlands, the Olentangy River, and the swale from October2000 to June 2001 during months when the wetlands werenot frozen. Samples were subjected to the multiple tubefermentation technique using most probable number analysis(Hernandez et al., 1997; Khatiwada and Polprasert, 1999) todetermine the concentration of fecal coliforms in eachsample. The data from five months of sampling show thatthe two constructed wetlands are reducing fecal coliformlevels as water flows through them.Materials and MethodsSample CollectionSamples of 100 ml were collected from locations inwetlands 1 and 2 (Figure 1), the Olentangy River, and theentrance to the swale, using sterile 50 ml conical tubes(Becton Dickinson, Franklin Lakes, NJ). Samples weretaken monthly in October 2000, March 2001, April 2001,May 2001, and June 2001. During December 2000, January2001, and February 2001 the wetlands were frozen andsamples were unable to be collected.Figure 1. Sample locations in wetlands70  ♦  The Olentangy River Wetland Research ParkTesting for fecal coliformsSamples were subjected to presumptive, confirmed,and completed tests in the multiple tube technique asdescribed in Standard Methods for the Examination ofWater and Wastewater (APHA, 1998). Briefly, phenol redlactose fermentation broth tubes containing an invertedDurham tube for gas collection were inoculated with eachwater sample, for the presumptive test. Five tubes of 1Xlactose broth (phenol red broth base [Difco, Detroit, MI]with 5g lactose/ L) were inoculated with 0.1 ml of eachsample. Five tubes of 1X lactose broth were inoculatedwith 1.0 ml of each sample, and five tubes of 2X lactosebroth (10 g lactose/ L) were inoculated with 10 ml of eachsample. Tubes were incubated for 48 hours at 37oC.Results were recorded as positive for lactose fermentationand production of gas. In the confirmed test, each positivetube from the presumptive test was used to inoculate aneosin methylene blue agar plate (Difco) and incubated for24 hours at 37oC.  Positive colonies produce a dark centeror green metallic sheen. Positive colonies from theconfirmed test were then subjected to the completed test.The positive colonies were inoculated into 1X lactosefermentation broth and streaked on nutrient agar plates(Difco). Fermentation tubes and plates were incubated for24 hours at 37°C. Tubes were recorded as positive if theyshowed lactose fermentation and gas production. Colonieson nutrient agar were subjected to Gram stains and recordedas positive if they showed Gram negative rods (Khatiwadaand Polprasert, 1999). Positive results at the end of thecompleted test were used for most probable number analysisto determine the fecal coliform concentration per 100 mlin each wetland sample (Khatiwada and Polprasert, 1999;Hernandez et al., 1997).ResultsTo determine the effects of water flow through thewetlands on fecal coliform concentration, samples werecollected each month from October 2000 to June 2001,except during months when the wetlands were frozen. Thesampling pattern was designed to distribute samples acrosseach wetland and provide adequate space for reduction offecal coliforms between each water sample (see Figure 1).Sampling in different locations within each wetlandprovided data to analyze the reduction of fecal coliformlevels throughout each wetland. In addition, one samplewas collected in the Olentangy River and the swale. Fecalcoliform levels recorded each month at the inlet and outletof wetlands 1 and 2, the river, and the swale are shown inTable 1.The maximum coliform concentration in the river wasTable 1: Reduction of fecal coliforms in each wetland during five months sampled.________________________________________________________________________________________Month Inlet1 Outlet2 Number Reduced3   %Reduction4________________________________________________________________________________________October 2000   Wetland 1 22     2 20        90.9   Wetland 2 33     9 24        72.7River to Swale5 50     2 48        96.0March 2001   Wetland 1 27     15 12        44.4   Wetland 2 23      9 14        60.9   River to Swale 33     13 20        60.6April 2001   Wetland 1 49      7 42         85.7   Wetland 2 30     16 14         46.7   River to Swale 47      5 42         89.4May 2001   Wetland 1 37     4 33         89.2   Wetland 2 44     6 38         86.4   River to Swale 37    18 19         51.3June 2001   Wetland 1 40     4 36         90.0   Wetland 2 33     2 31         93.9   River to Swale 60     4 56         93.3__________________________________________________________________________________________1 Fecal coliforms / 100ml in samples collected at the inlet to the wetland or in the river or swale.2Fecal coliforms/ 100ml in samples collected at the outlet of each wetland or in the river or swale.3Difference in fecal coliform concentration between inlet and outlet.4Percent reduction in fecal coliform concentration from inlet to outlet calculated as (number reduced/ inletconcentration) x 100.5Fecal coliform levels for the river sample are reported as inlet values and fecal coliform levels at the entrance to theswale are reported as outlet values.Coliforms in experimental wetlands ♦  7160 coliforms/100ml, in June 2001, whereas, the maximumcoliform concentration found in either wetland basin was49 coliforms/100ml, found in wetland 1 in April 2001.With the exception of wetland 1 in June 2001 and wetland2 in May 2001 the data consistently show a slight reductionin coliforms between the river sample and the sample takenat the inlet to the wetland basin.Table 1 shows that coliform concentration varied frommonth to month, but always decreased from inlet to outletof the wetland. Wetland 1 showed fecal coliform reductionbetween 44% and 91% with an average reduction of 80%± 18%. Wetland 2 reduced fecal coliform levels an averageof 72 ± 17% with monthly reduction varying between 47%and 94%. Figure 2 shows average fecal coliform concentration ineach sample site in wetland 1 and wetland 2 over the fivemonth sampling period. These data show a steady decreasein fecal coliform concentration as water moves away fromthe inlet through the wetland. The difference in averagefecal coliform reduction between the two wetlands is notstatistically significant (t-test, p= 0.2801). These datademonstrate that both wetlands are reducing fecal coliformconcentration in a similar manner.Interestingly, the data show differences in fecal coliformreduction between summer (June 2001) and winter (March2001) months. The samples taken in March 2001 were onlyreduced in fecal coliform levels by 44% in wetland 1 and61% in wetland 2. Samples taken in June 2001 werereduced 90% in wetland 1 and 94% in wetland 2. The fecalcoliform reduction in wetland 1 in March 2001 was 51%less than in June 2001. Wetland 2 reduced fecal coliformconcentration 35% less in March 2001 than in June 2001.DiscussionFecal coliforms have long been used as indicators ofpollution in water (Gearheart, 1999; Hernandez et al., 1997;McMath et al., 1999; Perkins and Hunter, 2000; Tyrrell etal., 1995) due to the potential for introduction of pathogensand other pollutants along with these bacteria  (Ricca andCooney, 1999; Tyrrell et al., 1995). This study examinedthe concentration of fecal coliforms in two created wetlandsat the Olentangy River Wetlands Research Park. During themonths sampled, both wetlands consistently showed fecalcoliform reduction ranging from 44 to 96% with averagereductions of 80 ± 18% and 72% ± 17% for wetland 1 andwetland 2, respectively. The data did not show a significantdifference in fecal coliform reduction between the twowetlands. The level of reduction in fecal coliforms observedis similar to that reported by Perkins and Hunter (Perkinsand Hunter, 2000) and others (Lau and Chu, 2000; Newmannet al., 2000; Perkins and Hunter, 2000) in constructedwetlands in other areas. Similar reductions in fecal coliformshave also been reported in natural wetland areas (Lau andChu, 2000).Reduction in fecal coliforms by wetlands may involveseveral factors including amount of plant coverage, hydraulicretention time, and settling of microorganisms (Perkins andHunter, 2000; Khatiwada and Polprasert, 1999; Shutes,2001). Seasonal changes in any of these factors may accountfor the variability in reduction from month to month.Interestingly, this study showed substantial differences infecal coliform reduction potential observed in winter monthsas compared to summer months. In fact, fecal coliformreduction was 1.5 to two times greater in June as comparedto March. Further studies will be needed to determine thecause of these differences but Newman et al. (2000) andothers (Khatiwada and Polprasert, 1999) have suggestedthat differences in reduction potential between winter andsummer months may be due to differences in plant coverage,temperature, and retention time.Increased plant coverage in summer months may lead togreater filtration through plant material providing moreentrapment of bacteria and more time for sedimentation.Gerba et al. (1999) suggested that microorganism removalas water flows through constructed wetlands may beprimarily due to sedimentation. Preliminary studies of totalbacterial burden in the wetlands suggest that sedimentationof bacteria is occurring in the ORWRP (data not shown).Further studies will be necessary to confirm these preliminaryobservations and to relate bacterial sedimentation to coliformreduction.This study also showed a decrease in fecal coliforms aswater traveled from the river through the pumping systeminto the wetlands, although this is unlikely due tosedimentation. With the exception of wetland 1 in June2001 and wetland 2 in May 2001, the data show a 10% toA B C D E01020304050BSample Number1 2 3 4 501020304050ASample NumberFigure 2. Fecal coliform levels in the wetlands. A) meanfecal coliforms/ 100 ml  ± standard deviation in eachsample site in wetland 1 for the five months sampled.  B)mean fecal coliforms/ 100 ml  ± standard deviation ineach sample site in wetland 2 for the five monthssampled.72  ♦  The Olentangy River Wetland Research Park56% decrease in fecal coliform concentration between theriver and the wetland inlet. These data suggest that bacteriamay be accumulating in the pipes and pumps. Otherresearchers have noted differences in nitrate+nitrites andphosphorus between river samples and samples taken at theinlet to the wetlands (Mitsch, 2000). These differences inchemical concentrations may be related to bacterial metabolicprocesses in the piping machinery. Biofilm formation inpipes is a common occurrence (Batista et al., 2000; Jenkinsonand Lappin-Scott, 2001; MacDonald et al., 2000) that mayeventually lead to pipe damage (Batista et al., 2000;MacDonald et al., 2000). Further research will be necessaryto determine the presence and extent of biofilms in theORWRP pump and piping system.Because this study only measured fecal coliform levelsfor five months, observations are continuing. However,these preliminary data suggest that biofilm may be formingin the pumping machinery, and the wetlands at ORWRPmay be reducing fecal coliforms in a similar manner asobserved at other constructed wetlands and in natural wetlandareas.AcknowledgmentsThe authors thank Dr. Robert Deal, Dr. Bill Mitsch, JudyGardner, and Hui Suk Jones. This research was supportedby the Department of Natural Sciences, Shawnee StateUniversity and a grant from the Ohio Board of RegentsIncentive for Performance Fund Research ChallengeProgram.ReferencesAPHA. 1998. Standard Methods for the Examination ofWater and Wastewater, 20th ed.. American PublicHealth Association, Washington, DC.Batista, J. F., R. F. C. Pereira, J. M. Lopes, M. F. M.Carvalho, M. J. Feio, and M. A. M. Reis. 2000. In situcorrosion control in industrial water systems.Biodegradation 11: 441-448.Carleton, J. N., T. J. Grizzard, A. N. Godrej, and H. E.Post. 2001. Factors affecting the performance ofstormwater treatment wetlands. Water Research 35:1552-1562.Gearheart, R. A. 1999. The use of free surfaceconstructed wetland as an alternative processtreatment train to meet unrestricted water reclamationstandards. Water. Science Technology 40: 375-382.Gerba, C. P., J. A. Thurston, J. A. Falabi, P. M. Watt, andM. M. Karpiscak. 1999. Optimization of artificialwetland design for removal of indicatormicroorganisms and pathogenic protozoa. WaterScience Technology 40: 363-368.Hernandez, F., R. Monge, C. Jimenez, L. Taylor. 1997.Rotavirus and Hepatitis A virus in market lettuce(Latuca sativa) in Costa Rica. International Journal ofFood Microbiology 37: 221-223.Jenkinson, H. F. and H. M. Lappin-Scott. 2001. Biofilmsadhere to stay. TRENDS in Microbiology 9: 9-10.Khatiwada, N. R. and C. Polprasert. 1999. Kinetics offecal coliform removal in constructed wetlands. WaterScience Technology 40: 109-116.Lau, S. S. S. and L. M. Chu. 2000. Nutrient and faecalcontamination and retention in wetland enclosures(gei wais) in the Mai Po Marshes, Hong Kong.Hydrobiologia 431: 81-92.MacDonald, R., M. Santa, and V. S. Brozel. 2000. Theresponse of bacterial biofilm community in astimulated industrial cooling water system totreatment with an anionic dispersant. Journal ofApplied Microbiology 89: 225-235.McMath, S. M., C. Sumpter, D. M. Holt, A. Delanoue,and A. H. L. Chamberlain. 1999. The fate ofenvironmental coliforms in a model water distributionsystem. Letters in Applied Microbiology. 28: 93-97.Mitsch, W. J., V. Bouchard, L. Zhang, and M. Hunter.2000. Biogeochemical and nutrient removal patternsof created riparian wetlands: Sixth-year results. In:Mitsch, W. J. and V. Bouchard (ed.), Olentangy RiverWetland Research Park at The Ohio State University,Annual Report 2000. Columbus, OH, pp. 83-90.Newman, J. M., J. C. Clausen, and J. A. Neafsey. 2000.Seasonal performance of a wetland constructed toprocess dairy milkhouse wastewater in Connecticut.Ecological Engineering. 14: 181-198.Ostroumov, S. A. 1998. Biological Filtering andEcological Machinery for Self-Purification andBioremediation in Aquatic Ecosystems: Towards aHolistic View. Biology Forum. 91: 221-232.Perkins, J., C. Hunter. 2000. Removal of enteric bacteriain a surface flow constructed wetland in Yorkshire,England. Water Research 34: 1941-1947.Ricca, D. M. and J. J. Cooney. 1999. Coliphages andIndicator Bacteria in Boston Harbor, Massachusetts.John Wiley & Sons, Inc. 404-408.Shutes, R.B.E. 2001. Artificial wetlands and waterquality improvement. Environment International. 26:441-447.Tyrrell, S. A., S. R. Rippey, and W. D. Watkins. 1995.Inactivation of bacterial and viral indicators insecondary sewage effluents, using chlorine and ozone.Water Research 29: 2483-2490.

Reduction of fecal coliform levels in two created wetlands at the Olentangy River Wetland Research Park

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Reduction of fecal coliform levels in two created wetlands at the Olentangy River Wetland Research Park

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