1. Presence of Microbial Indicators in Reid Park Wetlands Jepson Sutton Scott Stine SWES 574

2. Purpose Determine the effect of water flow rate on microbial indicator concentration –Stagnant Pond, Turbulent Pond, and Waterfall Determine the effect of aquatic plants on microbial indicator concentrations –Open water vs. Planted area Presence/Effect of chlorination and pH Compare presence of coliforms and enterococci

3. Experimental Problems It was determined that the ponds contained a shading dye that eliminated all plant growth.

4. Methods Collect samples from three varied areas of constructed wetlands at Reid Park –Located at 22nd Street and Country Club Use Colilert and Enterolert (IDEXX) to determine indicator concentrations

5. Methods Make dilutions and add Colilert/Enterolert reagents

6. Methods Pour into Quantitray (IDEXX) and seal using sealer

7. Methods Count number of positive wells Enumerate using MPN table

8. Total Coliforms Includes Escherichia, Citrobacter, Enterobacter, and Klebsiella Used as a standard for assessing fecal contamination of recreational and drinking water. Problems in accuracy due to ability to replicate in environment. Colilert positive=Yellow well

9. Fecal Coliforms Includes Escherichia and Klebsiella Definitive indication of fecal contamination Unable to replicate in the environment Colilert can enumerate E. coli concentrations Colilert positive=fluoresce under UV lamp

10. Colilert

11. Colilert (IDEXX) Defined substrate technology (DST) simultaneous detection of total coliforms and E. coli Contains specific indicator nutrients: ortho- nitrophenyl-beta-D-galactopyranoside (ONPG) and 4-methyl-umbellifery-beta-D- glucuronide(MUG)

12. Colilert (IDEXX) Total coliforms react with ONPG to form yellow color. E. coli react with MUG to form fluorescent color Can detect low levels of total coliforms and E. coli (1CFU/100ml)

13. Total Coliforms

14. E. coli

15. Fecal Streptococci Indicators Fecal Streptococci have advantages over coliform indicators: –they rarely multiply in water –they are more resistant to environmental stress and chlorination –they generally persist longer in the environment

16. Enterococci Members of Lancefield Group D of Streptoccocus (fecal streptococci subgroup) “Enterococci” - normal colonist of the human large intestine Streptococcus often given as genus of this group in water industry

17. Enterococci Includes Enterococcus species faecalis, faecium, avium, and durans E. faecalis: – infects elderly patients undergoing surgery urinary tract, wounds, blood, appendix, intestinal structures –emerging nosocomal opportunist due to: rising incidence of multi-drug-resistant strains ease of person to person transfer

18. Enterococci Have been suggested as indicators of: –presence of enteric viruses in environment biosolids and seawater –risk of gastroenteritis for recreational bathers

19. Enterolert (IDEXX)

20. Uses a nutrient-indicator to detect enterococci –fluoresces when metabolized by enterococci Compared to standard membrane filtration (MF) method: –50% fewer false positives –95% fewer false negatives

21. Results

22. pH / Chlorine

23. FC/FS Ratio >4.0 = contamination of human origin <0.7 = contamination of animal origin Pond 1: FC/FS = 5 Pond 2 : FC/FS = 15 Waterfall : FC/FS = 0.8

24. Discussion Probable pathogen removal factors –temperature, natural die-off, sedimentation sedimentation most significant Studies have shown slower flow rates allow more settling of microorganisms –Pond 1 would be expected to have lowest numbers

25. Discussion The highest amount of contamination in all categories occurred in Pond 2 –Area where ducks gather Total coliforms were present in the largest amounts in all samples –Total coliforms broadest of groups assayed Presence of microorganisms could vary over time

26. Discussion Pond 1 and Pond 2 have similar distributions in all categories Waterfall does not follow the trend –Total coliforms higher compared to other samples Why? –Turbulence –Cycling of water

27. Discussion FC/FS Ratio suggests: –Pond 1 and Pond 2 contamination is of human origin –Waterfall contamination is predominantly of animal origin

28. Discussion Values used to calculate ratio are not completely representative of FC and FS –E. coli is not only member of FC group –Enterococci is only a subgroup of FS Ratio only valid for recent (24 hrs) fecal pollution Validity of ratio has been questioned