Transcript Document

Survival of Enterococci in Unseeded Runoff Ditch and
Sand at a Subtropical Non-point Source Beach
R.
1,2
Ramoutar ,
H.
1,2
Solo-Gabriele
Results
Methods
Abstract
To limit human contact with contaminated waters, fecal indicator bacteria (FIB) are used to
evaluate risk of illness from recreational bathing at beaches. Enterococci are one of the most
commonly used bacteria for confirmation of sewage presence in recreational water bodies.
Recent studies at a subtropical non-point source beaches have identified the intertidal zone as
a potential alternative source of FIBs; thus encouraging further experiments to obtain a better
understanding of the behavior of FIBs, particularly enterococci, within the intertidal zone.
Such experiments can aid in improved regulations and monitoring practices. This study
evaluated enterococci behavior in simulated intertidal sand and ditch environments. The
focus of the study was to determine whether there is die-off, persistence, or increases of
cultural enterococci under saturated and unsaturated conditions in the beach environment. By
utilizing a combination of experiments, the relationship of enterococci survival was
evaluated. Results exhibited a constant die off of enterococci in all simulated ditch
environments. Sand experiments showed an increase in enterococci concentration in dry
sand after wetting events, however these were not statistically significant due to the limited
number of samples (p=0.33, p=0.20 and p=0.14 respectively). All sand experiments showed
a statistically significant difference between enterococci concentration variance in dry sand
after simulated wetting (p<0.0001, p<0.0001, p=0.0005 respectively). No significant change
in enterococci die-off rates were observed in sand from both intertidal and ditch zones.
Further studies are necessary to establish correlations and statistical significance of the
change in concentration of enterococci in dry sand after wetting events.
Ocean and Rain Water Sample
Collection
Water Experiments
Sampling of FIBs in runoff water
Site
CFU/100 mL
12/5/2006
2
Ditch
Date
CFU/100mL
4000
R
3500
S+R
KL1
160,667
24,167
3000
5/22/2007
0
6/12/2007
0
5/16/2007
5/22/2007
6/17/2007
0
5/16/2007
16,000
6/6/2007
0
5/22/2007
2,500
6/6/2007
0
5/23/2007
3,000
Coral Gables Campus
12/5/2006
2
5/16/2007
6,000
RSMAS (behind
Glassel laboratory)
12/5/2006
97
5/22/2007
3,000
5/22/2007
170
RSMAS beach
Hobie beach
Hobie Beach
Date
Regrowth without Agitation
Water sample collection showed that
rainwater was virtually Enterococci free
before filtering, thus rainwater wasn’t
contributing significantly to FIBs found
after wetting events.
KL2
L
CFU/100ml
C.
1,2
Ortega-Castineiras ,
[email protected]
S+G+R
2000
1500
1000
0
0
10
20
30
40
50
60
Time (Hrs)
Sampling of FIBs in standing ditch
water and runoff water showed a
significant FIB load, particularly
Enterococci was well above regulatory
standards
Regrowth with Agitation
Sand Experiments
RSMAS
G+R
2500
500
CFU/100ml
J.
1,2
DeFrancesco ,
Jenna DeFrancesco
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
R
S+R
G+R
S+G+R
0
10
20
30
40
50
60
Tim e (Hrs)
•Rain Water Sample Collection at various sites.
•Ocean Water was collected using sterile 5L containers.
•Filtered through 0.45 µm filter for use in microcosm
experiments.
Enterococci concentrations show a steady decrease
in regrowth experiments in all water microcosm
environments which correlate to decreases in
turbidity. No significant regrowth was observed.
•FIBs in runoff water tested
•Regulatory Standards for recreational water:
U.S. (EPA) enterococci < 33 or 35 / 100 ml
Summary of Enterococci in
Sand for Dry Stage of D, I
10,000
and A
1,000
Ditch Microcosms (Undisturbed)
Ditch Microcosms (Mixed)
In a controlled environment, Enterococci concentrations
diminished as the sand from each microcosm dried.
Study Site
CFU/ g dry sand
Abstract #
R² = 0.880
Dpart 1
100
R² = 0.003
I-part
1
10
R² = 0.533
1
0.0
100.0
200.0
300.0
Time since collection (hrs)
400.0
Wetting each microcosm resulted in
pronounced regrowth in only the very
dry above sand. This was true for
both rainwater and ocean water.
W WTP
Outfall
Hobie
Beach
Sewage
Outfall
• Replicates of runoff ditch environment using:20 g sand,
10 g seaweed and 800 ml of synthetic rainwater
•Used to determine the relationship between CFU/100ml in
various ditch microcosms over time in relation to turbidity
• Microcosms were kept in the dark at a constant
temperature
• Samples were taken without mixing the microcosms
during 3 consecutive days
• Ditch microcosm experiment was repeated
•Agitation used to more closely replicate the natural
environment and obtain a more representative
enterococci count
•Used to determine the survival rate of enterococci in
various runoff ditch microcosms over time
We are here
Sand Microcosms before and after a Wetting Event
Objectives
•Evaluate the behavior of enterococci in simulated sand and ditch environments that
replicated those found at the study beach.
A series of experiments showed that regrowth was seen in the dry above sand after both rainwater and ocean water wetting
events. There was no significant regrowth found in either Ditch or Intertidal sand which may be attributed to varying moisture
content.
• .
Discussion
•Determine whether there is die-off, persistence, or increases of culturable enterococci
under saturated and unsaturated conditions.
•This study utilized a unique combination of experiments in order to simulate the different
environmental conditions and evaluate their relationship to enterococci survival.
•Overall, the study also identifies possible implications for beach regulation practice.
1University of
• Sand samples were procured via sterile containers from
each representative sampling environment
• Moisture content over time was recorded
• CFU/100ml was tracked over time to determine natural
enterococci trends in each microcosm environment
Miami, NSF NIEHS Oceans and Human Health Center, Miami, FL 33149
2Department of Civil, Arch., and Environmental Engineering, University of Miami, Coral Gables, FL 33146-0630
•Sterilized rain and ocean water were used to simulate
natural wetting events
• 3 repetitions of this sand experiment were performed
• CFU/g of sand was tracked before and after wetting to
observe behavior in each microcosm
•Explore prolonged periods of “drying” before a wetting event as well as after to follow extended
trends.
•Maintain a control throughout every experiment and repeat in triplicates to establish the
significance of the increase in the enterococci colony counts.
•Explore varying percentages of water content to determine how dry sand must be to experience
pronounced regrowth. (Between 0.2% and 4% according to data)
•*Further compare sterile ocean and rainwater in their effects on regrowth. (Rainwater showed
most regrowth at 3 hours whereas Ocean showed peak regrowth at 6 hours post wetting event)
•Determine impact of increase in enterococci CFU’s in sand on the bacterial level in the water
column.
•Repeat experiments with the addition of PCR analysis of the samples in order to establish
whether there is actual regrowth or just a strengthening of the bacteria after a wetting event.
Acknowledgements:
NSF-NIEHS Oceans and Human Health Program (P50 ES12736-01
NSF REU Program (NSF OCE 0432368),