Results of Assessments of the Effects of Holding Times On E. coli Concentrations In Environmental Samples

Transcription

1 Results of Assessments of the Effects of Holding Times On E. coli Concentrations In Environmental Samples Jon Standridge Wisconsin State Laboratory of Hygiene Mary Ann Feige USEPA Misty Pope, Yildiz Chambers, Ken Miller DynCorp/CSC Biology Studies Group

2 Background E. coli Excellent indicator of fecal contamination Greatly expanded use improvements in test protocols new regulations

3 Background Little or no data available on sample storage Forced USEPA and Standard Methods to recommend stringent sample storage guidelines Icing 6 hours transit + 2 hours in lab

4 Background Relaxed sample transit and storage guidelines would expand the utility of using E. coli as a monitoring tool. A decision to relax the guidelines must be based on data

5 Background USEPA initiated studies in three phases 1. A variety of contaminated waters stored at controlled temperatures, tested with 2 common methods 2. A variety of waters stored under field icing conditions, tested with 2 common methods 3. A variety of waters from geographically diverse areas, stored under field icing conditions and tested with 4 methods

6 Phase 1-3 Standard Protocols Preliminary E. coli levels determined Low level samples were spiked wild environmental isolate used Time 0 was defined as when the spiking occurred and/or when the samples were placed at holding temperature Always less than 3 hours from collection Temperatures monitored with ibuttons All analyses done in triplicate

7 ibutton Digital Thermometer

8 Phase 1 Four Wisconsin surface waters pristine to polluted Four controlled storage temperatures 4, 10, 20 and 35 degrees C Analysis at 0, 8, 24, 30 and 48 hours Two common methods Colilert Quantitray 2000 TM mfc agar with nutrient agar MUG

9 Colilert Quantitray 2000 and Quantitray

10 mfc NAMUG Test mfc NAMUG

11 Positive NAMUG Rxn

12 Statistical Analysis Dunnett s Test tests pair-wise differences in means identifies data points where you can be 95% confident that the concentration has changed significantly from time zero

13 L.Winnebago Spiked and Tested with Colilert 300 E. coli/100 ml deg. C 10 deg. C 20 deg. C 35 deg. C time 8 hrs 24 hrs 30 hrs 48 hrs time in hours

14 L.Winnebago Spiked and Tested with mfc/namug 250 E. coli/100 ml deg. C 10 deg. C 20 deg. C 35 deg. C 0 0 time 8 hrs 24 hrs 30 hrs 48 hrs time in hours

15 Rainbow Lake Spiked and Tested with Colilert E. coli/100 ml time 8 hrs 24 hrs 30 hrs 48 hrs time in hours 4 deg. C 10 deg. C 20 deg. C 35 deg. C

16 Phase 1 - Incidence of Changes from Time Zero using Colilert 8 Hr 24 Hr 30 Hr 48 Hr 4 deg. C /4 10 deg. C 1/4 0 1/4 2/4 20 deg. C 0 1/4 3/4 4/4 35 deg. C 0 2/4 3/4 4/4

17 Phase 1 - Incidence of Changes from Time Zero using mfc/na-mug 8 Hr 24 Hr 30 Hr 48 Hr 4 deg. C 0 1/4 1/4 1/4 10 deg. C 1/4 1/4 1/4 3/4 20 deg. C 1/4 2/4 3/4 4/4 35 deg. C 1/4 3/4 3/4 3/4

18 Observations From Phase 1 Significant E. coli die-off occurs when samples are stored at 20 and 35 degrees Acceptable storage length may be impacted by method

19 Phase 2 Examine a variety of waters Field icing conditions wet ice Utek Ice packs 2 commonly-used methods Colilert mfc-namug

20 Bethel - Spiked E. coli/100 ml time 8 hrs 24 hrs 30 hrs 48 hrs time in hours Wet ice Colilert wet ice mfcnamug Utek Colilert Utek mfcnamug

21 Williamsburg - Spiked 2500 E. coli/100 ml Wet ice Colilert wet ice mfcnamug Utek Colilert 0 0 time 8 hrs 24 hrs 30 hrs 48 hrs time in hours Utek mfcnamug

22 Phase 2 - Incidence of Changes from Time Zero using Colilert 8 Hr 24 Hr 30 Hr 48 Hr Wet ice 0/7 0/7 2/7 2/7 Utek 1/7 0/7 1/7 1/7

23 Phase 2 - Incidence of Changes from Time Zero using mfc/na-mug 8 Hr 24 Hr 30 Hr 48 Hr Wet ice 0 2/7 3/7 3/7 Utek 1/7 2/7 2/7 4/7

24 Observation From Phase 2 Samples may freeze with both Utek ice packs and wet ice. Chilling samples appears to allow longer storage times

25 Phase 3 Examine a variety of geographically diverse waters Field icing conditions wet ice Utek Ice packs A variety of commonly-used methods Colilert mtec mfc-namug mendo agar- NAMUG

26 mtec Test

27 Phase 3 Colilert 120 Mean percent recovery WV IN TX IN 0 AZ AZ 0 time 8 hrs 24 hrs 30 hrs 48 hrs time in hours

28 Phase 3 mtec 300 Mean percent recovery MO NV 0 0 time 8 hrs 24 hrs 30 hrs 48 hrs time in hours

29 Phase 3 mfc-namug (NJ) 120 Mean percent recovery time 8 hrs 24 hrs 30 hrs 48 hrs time in hours

30 Phase 3 mendo-namug 250 Mean percent recovery time 8 hrs 24 hrs 30 hrs 48 hrs time in hours OR NY WI del. Inc. WI WI del Inc. WI

31 Summary: Incidence of changes from Time Zero for Chilled Samples held 24 Hours Enzyme based minimal media tests (Colilert ) 1 out of 35 (3%) Membrane based tests (MTEC, MFC and mendo with nutrient agar MUG) 7 out of 29 (24%)

32 Summary Comprehensive study 24 sites 11 laboratories 4 commonly-used methods

33 Conclusions Water samples should be tested as soon after collection as possible Samples must be stored at less than 10 O C and not allowed to freeze Sample storage prior to testing may be method dependant

34 Conclusions Most raw water samples being tested for E. coli can be chilled and held beyond 8 hours and still generate valid data.

35 Results of Assessments of the Effects of Holding Times On E. coli Concentrations In Environmental Samples Jon Standridge Wisconsin State Laboratory of Hygiene Mary Ann Feige USEPA Misty Pope, Yildiz Chambers, Ken Miller DynCorp/CSC Biology Studies Group <