-------
the salmon assay in the calculation.
   Additional associations between information collected at each sampling time
and the average of the water quality measures from the same time period are
shown in Tables 4.91-  4.93. Bathers in the water was the only measure consis-
tently associated with all fecal indicators, such as  water temperature and ul-
traviolet intensity were positively correlated with Enterococcus CCE. Measures
such as wind direction, wind speed, water temperature and wave height were
also positively correlations with some fecal indicators (see Tables 4.91- 4.93).
   Associations between water quality measures and rainfall are shown in Ta-
bles 4.94. Due to the failure of the on site weather station to accurately record
data for several weeks, rainfall information was collected from an on-site rain
gauge, from the period 3:00 PM to 8:00 AM. Since this information  was not
collected during the week, except for Friday,  two day or accurate current day
lag is  unavailable for all observations, and only a one day lag (since 3:00 PM the
previous day) is reported. Precipitation was negatively correlated with Entero-
coccus delta-delta CT CCE, positively correlated with Enterococcus CFU and
was not correlated with Bacteroidales CCE.
                                    139

-------
a
--O
cr
o
m
B
1
*
c

"3
-g




0

*
i — I
CM
CM
o





, — I



*
CM



*
lO
O
CM




*
, — I
CM
oo
o


*
CO
CO
CO
0

*
CM
oo
oo




p

<— '


a
0)
1



, — i
0
0









*
, — 1
CM
CM
f^:


CM
0
0



*
oo
oo
CM




lO
1 — 1
0


CM
<=>

*
CM




oo
1 ]
o


£
0)
1



! 	 1


! 	 1
o





0
	 •
1


CM
oo
0
0



OS
OJ
0




*
OS
0
CM



i — I
0

, — 1
oo
0
0




oo
oo
'p
o


bfl
H













V
_0)
cS
^>
1
6
o
1

"3
O
ffi
O
&H


O
C5
i
ci
"3
T)

^H
O
"3
.£
cr
H
"3
O
0
1

^
"3
O
pr^
o
OH
^
O
cS
i H
,a§



























"i
^B
8
OJ
c§
s_;
-2
ffl
3cocc«s;
c
1
0
4J
H
. „
&
*s
bO
g
*s
1
_o
6
&
o








^
M
'S
OJ
>
K»
-g

>
1
e water
-^
c
"*
2
^
c5
m


i
^
to
1
m
S
S
bO
1
cS
bO
tn
72,
EH
OJ
•o
£
0
'_^
ce
•3
4^
Jltraviole
>
£




































^
0)
1
H

1
^
O
i
s
!^
<2
c
"3
c
_o
•3
•o
<

140

-------

£H
S-I
cr
Q
m
:asures.

0)
o
SH
C
"O
H
O
O
Cd
O
OH
cr
^
e
o
t-
-^
CD
SH
o

c/)
^
0)
'3
E6

1
£>
>


__j

p*

(/)
SH
0>
4^>
03
m

H
o
-o
"S
03
m


H
o
o
m







o
1 — 1
0

CM
O
1 — 1
0



CO
0
o
o

oo
oo
o
o



CO
°
o




CM
CO
1 — 1
0



^f
CM
O



OS
CO
oo
o




, — 1



H
O
"O

m



o
o
1

oo
1 — 1
CM
O



*
oo
o
CM
0

OS
OS
, — 1
o



CM
O





oo
CM
0



^f
CM
oo
o



1 — 1





OS
CO
oo
0


H
O
"O
"O

m



o
1 — 1
0

CM
i — I
0



CO
CO
CO
0

oo
0



CO
CM
oo
o




CM
oo
CM
0




i — I



CM
oo
o




CM
0


oa
^H




CO
o
0

*
, — 1
CM
CM
O



, — 1


*
IO
CM
0



*
o




*
1 — 1
CM
oo
0



*
CO
CO
CO



*
oo
0
CM
o




0
0


a
0)
^
1



1 — 1
®

, — 1




*
1 — 1
CM
CM
0

CM
lO
o



*
oo
oo
CM
(3;




i — 1
0




CM
i — I
0



*
oo
1 — 1
CM
o




CM
i — I
0


£
0)
1






1 — 1
1



CO
p
1

CM
oo
o



OS
1




*
OS
CM
0



^H
, — 1
0



0





b-
0
, — I
^


bfl
c«

H











^-^
_0)

1
o
o
2

~s
o
o
&H
•—^
c^
O
cS
i
CI3
"S
T)

C_|
O
-§
£
s
'3
cr
H
"0
O
0
1

6
o
OH
j-'
O
03
^ 1
V H
,a§























s
"3
1
tu
1
cq

j_J
0
m
§
o
^
OJ
-w
1
II.
o
^
H

'3
bO
C
'g
1
_o
6

^
O







+J
bO
'53
-^
0
i
^
^
i-T
"cS
0)
-C
o
1
"cS
m
D
^
^
t/3
fe
5
m

OJ
g
bO
"3,
s
0)
^
0
bO
C6
0
2
"

a:
2
0
1
•a
2
4^
Itraviole
^j
>
D




































^
CO
0
x;
^_l
1
o
i
s
^
Q
«
fl
"3
c
o

•3
•o
<;

141

-------
o

tp
m
a

3
cr
o
m
t/f
0)
SH
(/)
Oi
£
g

cS

0)
s
1
c
-o
c3
K-,
fa
O

r*>
8
o
£
-4*3

££l
CD
 O
1
CO
P 0
1

"g
ft o-
CD O
"§ ^
.s

^

S-H
li
^

a>

^ *
£ oo

4^
03
m


i —
o
O ^H
H



1 — '
fa
O
o
0)
H


0
i — i
	 •
^~^



CM
^





*
CO



*
oo
Q


*
CM
oo
4 — '




*
CM
oo
CM
^~^




^







*
oo
0



Bathers Wate


*
CM
0



b-
lO
^





*
, — I
CM
oo


*
CM
0


§
^





i — I




*
CM
oo
CM
0






0
0



-o
1


0
0




*
oo
oo
CM
*•—*




*
1O
°


*
oo
Q


^H






i — 1




*
CO
CM
oo
0






b-
CO
i — I
0



ft
-a


CM
oo
^".
o



CM
lO
^~i





*
LO
CM



, — I



*
oo
oo
1 — '




*
CM




*
oo
0






p



1 — '


o

^~f



*
1 — 1
CM
CM
1 — '






, — I


*
lO
CM
Q


*
0
CM
1 — '




*
, — I
CM
oo
^~^



*
CO
CO
0






CM
i — I
0



ft
0)
0)
^s


, — 1
0
0
1










^.
, — 1
CM
CM


CM
1O
0
0


*
oo
oo
CM
O




b-
1O
i — I





CM
, — I
0






oo
CM
CM
0



i


! 	 1





! 	 1
0
0





^H
CO
O
O

CM
oo
0
0


o
0





as
0
CM
O



^
0
1 — 1
o
1






oo
p
o
1



So
cS
"O
H














-— ~
+^
a
0
~3
>
'3
cr
H
"3
O
o
S
.Q
~3
o
o
DH
-S


o

0
CS
^
O
•a
T)

5~
c
OJ
5
>
c^
r^
^
D

o
CR Calibrat
OH
O"1

o
cS
10 ^
q ^
ft O



























cyj
^
1
Q
QJ
c§
o3
o
m

'/j
S
o
§
Q
QJ
g
ej

6
CD
g
H
'3
M
C
|

Q
&
"o
O
fa
O









w
'S

P
^
>

•j
-^
>
1
0
&
^
g
CO
^
0
42
Is
ffl

CD
4J
1
CO
CD
m
of
4J
M
_C

1
CO
CD
bO
c5
to
CD i — I
32 CO

JH
11
"2 "s

n C
UV: Ultraviolet
Additional infor
142

-------
Table 4.94: Spearman pairwise correlation coefficients for fecal indicator bacte-
ria and rainfall, Boqueron Beach

                                                        Rain (1-day lag)
 Entero. dCT                                               -0.149
 Entero. ddCT                                             -0.224*
 Bacter. dCT                                                0.039
 Bacter. ddCT                                             -0.031
 Entero CFU                                               0.455*
 Rain (1-day lag)                                              1
 *p<0.05
 dCT=delta CT  (qPCR Calibrator Cell Equivalents); ddCT=delta-delta CT (qPCR Calibrator Cell Equivalents)
 CFU=Colony Forming Units; Entero.=Enterococcus; Bacter.=Bacteroidales
 UV: Ultraviolet  radiation; Tide: Tide stage at sampling time; Bathers Water:  Bathers in the water; Wave ht: Wave height
 Additional information see: Table 3.1
                                       143

-------
4.2.8   Associations between water quality  and illness

Crude (unadjusted) incidence of illness among body immersion  swimmers by
tertile of indicator exposure and among non-swimmers are shown for Boqueron
Beach in Table 4.95. The crude incidence rates do not show any obvious trend
or association with exposure category with the exception of a slight increase
in respiratory illness across categories of Enterococcus CFU. Crude incidence of
illness for indicators measured by qPCR CCE calculated by the delta CT method
is also shown since generally there were considerable differences in qPCR CCE
measured by these two calculations at Boqueron Beach (Table 4.96).
Table 4.95: Number and percentage of respondents with incident illness for non-
swimmers and among body immersion swimmers by tertiles of daily average of
indicator exposures.  Boqueron Beach.  qPCR CCE determined through delta-
delta CT calculation.
GI

Enterococcus CCE
Non- Swimmer
1.8,1.98
1.98,2.15
2.15,2.67
Bacteroidales CCE
Non- Swimmer
2.58,2.95
2.95,3.18
3.18,3.38
Enterococcus CFU
Non- Swimmer
-0.0351,0.585
0.585,1.09
1.09,1.43
N

123
170
184
209

123
156
208
199

123
187
153
223
%l

4.25
4.90
4.88
4.61

4.25
4.73
5.10
4.53

4.25
4.90
4.14
5.24
URI
N

158
221
273
292

158
202
302
282

158
244
250
292
%l

5.81
6.70
7.74
6.84

5.81
6.44
7.95
6.78

5.81
6.68
7.17
7.38
Rash
N

91
156
164
241

91
138
215
208

91
177
169
215
%l

3.15
4.47
4.35
5.32

3.15
4.14
5.29
4.73

3.15
4.61
4.56
5.06
Earache
N

43
72
72
85

43
75
82
72

43
76
72
81
%l

1.47
2.05
1.90
1.86

1.47
2.24
2.00
1.62

1.47
1.97
1.93
1.89
Eye
N

111
97
152
164

111
93
157
163

111
125
131
157
%l

3.80
2.74
3.98
3.57

3.80
2.77
3.81
3.65

3.80
3.22
3.49
3.63
Diarrhea
N

84
118
118
128

84
110
131
123

84
122
95
147
%l

2.91
3.40
3.13
2.83

2.91
3.34
3.22
2.80

2.91
3.20
2.57
3.45
 1: Percentage of those within exposure category with symptom (row percentage). Number and percent not ill not shown
 CCE: logio qPCR Calibrator cell equivalents (delta-delta method). CFU: logio colony forming units
 URI: Upper respiratory illness
Enterococcus CFU (Method 1600)

The  association between culturable Enterococcus CFU exposure as measured
by EPA Method 1600 and illness are shown in Tables 4.97-4.102. During study
enrollment no single day exceeded the EPA geometric mean criteria of 35 CFU
                                   144

-------
Table 4.96: Number and percentage of respondents with incident illness for non-
swimmers and among body immersion swimmers by tertiles of daily average of
indicator exposures.  Boqueron Beach. qPCR CCE determined through delta
CT calculation.
GI

Enterococcus CCE
Non- Swimmer
1.13,1.44
1.44,1.54
1.54,1.95
Bacteroidales CCE
Non- Swimmer
1.9,2.33
2.33,2.54
2.54,2.96
Enterococcus CFU
Non- Swimmer
-0.0351,0.585
0.585,1.09
1.09,1.43
N

123
192
172
199

123
155
198
210

123
187
153
223
%

4.25
5.35
4.29
4.77

4.25
4.77
4.69
4.89

4.25
4.90
4.14
5.24
URI
N

158
236
270
280

158
205
291
290

158
244
250
292
%

5.81
6.98
7.17
7.09

5.81
6.62
7.41
7.13

5.81
6.68
7.17
7.38
Rash
N

91
166
203
192

91
142
210
209

91
177
169
215
%

3.15
4.60
5.08
4.59

3.15
4.32
4.99
4.87

3.15
4.61
4.56
5.06
Earache
N

43
87
68
74

43
75
82
72

43
76
72
81
%

1.47
2.39
1.69
1.76

1.47
2.27
1.93
1.66

1.47
1.97
1.93
1.89
Eye
N

111
122
147
144

111
94
162
157

111
125
131
157
%

3.80
3.34
3.62
3.40

3.80
2.84
3.80
3.59

3.80
3.22
3.49
3.63
Diarrhea
N

84
135
104
125

84
106
127
131

84
122
95
147
%

2.91
3.76
2.59
3.00

2.91
3.26
3.01
3.05

2.91
3.20
2.57
3.45
 CCE: (logic) qPCR Calibrator cell equivalents (delta method). CFU: (logic) colony forming units
 URI: Upper respiratory illness
per 100 ml making it not possible to compare illness incidence associated with
swimming on days with geometric means above 35 CFU per 100 ml.
   Despite the low CFU levels, there was some evidence of an association with
Enterococcus CFU and respiratory illness (AOR=1.31, p=0.06 for the daily av-
erage CFU exposure and body immersion, Table 4.99). As shown in Table 4.95,
the absolute increase in illness is modest, from about 5.8% in non-swimmers to
7.4% among swimmers exposed to greater than a geometric mean of 12 Ente-
rococcus CFU. Following adjustment for covariates, the estimated incidence of
illness  was changed as  follows:  in non-swimmers, 6.7%, swimmers  in the low-
est exposure  category,  6.5%, and swimmers above 12 CFU had an estimated
incidence of 7.8%.
   No  consistent associations between Enterococcus CFU exposure and illness
were observed among children 10 and under.  Positive but non-significant trends
were observed between  rash and respiratory illness among children (Table 4.103
and  4.104).
                                   145

-------
Table 4.97: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and GI ill-
ness. Daily, waist depth, shin depth, 8:00 AM and swimming-location indicator
averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.95
1.01
0.91
0.99
0.97

0.90
0.98
0.84
0.95
0.95
P-value Lower 95% CI

0.80
0.95
0.57
0.90
0.83

0.57
0.92
0.36
0.71
0.73

0.67
0.72
0.66
0.79
0.73

0.62
0.67
0.59
0.75
0.71
Upper 95% CI N

1.35
1.43
1.26
1.23
1.28

1.30
1.43
1.21
1.22
1.27

10802
10795
10803
10795
10802

9658
8979
8986
8986
9657
Table 4.98: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Diar-
rhea.  Daily, waist depth, shin depth, 8:00 AM and swimming-location indicator
averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.95
0.97
0.94
1.00
0.93

0.77
0.80
0.78
0.89
0.85
P-value Lower 95% CI

0.81
0.89
0.74
0.99
0.64

0.23
0.30
0.21
0.43
0.36

0.64
0.66
0.64
0.76
0.68

0.51
0.53
0.52
0.66
0.61
Upper 95% CI N

1.42
1.44
1.37
1.32
1.27

1.18
1.22
1.16
1.20
1.20

10801
10799
10802
10794
10802

8986
8986
9095
8986
8986
                                   146

-------
Table 4.99: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Respi-
ratory illness. Daily, waist depth, shin depth, 8:00 AM and swimming-location
indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.31
1.28
1.31
1.01
1.12

1.27
1.24
1.28
1.01
1.09
P-value Lower 95% CI

0.06
0.08
0.05
0.94
0.31

0.10
0.15
0.08
0.95
0.53

0.99
0.97
1.00
0.84
0.90

0.95
0.92
0.97
0.83
0.84
Upper 95% CI N

1.74
1.69
1.71
1.21
1.41

1.70
1.66
1.68
1.22
1.41

10184
10184
10184
10179
11096

9129
9129
9129
8484
8489
Table 4.100: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Rash.
Daily, waist depth, shin depth, 8:00 AM and swimming-location indicator aver-
ages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.22
1.20
1.19
1.21
1.39

1.19
1.21
1.15
1.22
1.49
P-value Lower 95% CI

0.23
0.26
0.28
0.05
0.03

0.34
0.28
0.44
0.08
0.02

0.88
0.88
0.87
1.00
1.03

0.83
0.85
0.81
0.98
1.08
Upper 95% CI N

1.68
1.64
1.63
1.46
1.87

1.71
1.72
1.63
1.51
2.05

10820
10947
10821
10821
10947

9011
9117
9011
9117
9117
                                   147

-------
Table 4.101: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Ear-
ache.  Daily, waist depth, shin depth, 8:00 AM and swimming-location indicator
averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.92
0.89
0.96
0.97
0.88

0.75
0.70
0.83
0.88
0.84
P-value Lower 95% CI

0.73
0.64
0.87
0.82
0.51

0.25
0.12
0.42
0.36
0.41

0.58
0.56
0.61
0.74
0.60

0.47
0.46
0.53
0.66
0.55
Upper 95% CI N

1.46
1.43
1.52
1.27
1.29

1.22
1.09
1.30
1.16
1.27

11872
11023
10893
10893
11878

9173
9178
9888
9758
9178
Table 4.102: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Eye
irritations.  Daily,  waist depth, shin depth, 8:00 AM and swimming-location
indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.23
1.25
1.18
1.06
1.08

1.17
1.20
1.13
1.05
1.08
P-value Lower 95% CI

0.21
0.16
0.33
0.58
0.59

0.42
0.31
0.51
0.72
0.65

0.89
0.92
0.85
0.85
0.80

0.80
0.85
0.79
0.81
0.77
Upper 95% CI N

1.69
1.71
1.64
1.33
1.47

1.70
1.70
1.63
1.34
1.52

11947
11947
11086
10955
10955

9115
9944
9224
9114
9115
                                  148

-------
Table 4.103: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Rash.
Daily, waist depth, shin depth, 8:00 AM and swimming-location indicator aver-
ages.  Boqueron Beach.  Children age 10 and under.
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.24
1.22
1.24
1.23
1.30

1.29
1.38
1.21
1.25
1.52
P-value Lower 95% CI

0.59
0.58
0.58
0.34
0.44

0.55
0.39
0.65
0.32
0.23

0.56
0.61
0.58
0.80
0.66

0.56
0.67
0.53
0.80
0.76
Upper 95% CI N

2.75
2.43
2.65
1.88
2.55

2.99
2.85
2.75
1.95
3.04

1944
2087
1944
1944
2087

1895
1895
1757
1757
1895
Table 4.104: Adjusted Odds Ratios Enterococcus CFU (Method 1600) and Res-
piratory illness. Daily, waist depth, shin depth, 8:00 AM and swimming-location
indicator averages. Boqueron Beach. Children age 10 and under.
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.19
1.05
1.29
0.87
1.04

1.15
1.01
1.31
0.85
1.06
P-value Lower 95% CI

0.50
0.87
0.36
0.45
0.85

0.67
0.97
0.37
0.40
0.82

0.72
0.57
0.75
0.61
0.67

0.60
0.51
0.72
0.60
0.62
Upper 95% CI N

1.97
1.95
2.22
1.25
1.63

2.23
2.01
2.37
1.23
1.84

1933
1810
1933
1810
1810

1632
1627
1751
1632
1630
                                   149

-------
Enterococcus qPCR Calibrator Cell Equivalents

Associations between illness and exposure to Enterococcus qPCR CCE by the
delta-delta CT method are shown in  Tables 4.105-4.110 and for the delta-CT
method in Tables 4.111-4.116.
   Overall patterns and trends with illness were inconsistent, probably reflecting
the variability in the exposure resulting from the few usable  and quantifiable
samples (see Section  4.2.6). Illness associations were not consistent across the
delta-CT  and delta-delta CT calculations.  For example, an association was
observed between skin rash and Enterococcus CE for head immersion exposure
by the delta method (Table 4.114, AOR=1.76, p=0.04), but not by the delta-
delta method (Table  4.108, AOR=1.13, p=0.72).  Marked inconsistencies were
also observed in health effects associations by sample depth and by sample time.
   Examining children separately even presented greater difficulties in interpre-
tation  as  the sample size was reduced in addition to the probable inaccuracy
in the qPCR exposure measurement.  As might be expected given these issues,
associations with Enterococcus  CCE exposure and illness among children were
inconsistent.  An inverse association was observed for diarrhea and  CCE expo-
sure among children as incidence declined with increasing levels of CCE. How-
ever, given the limitations of the exposure data, it is difficult to meaningfully
interpret these results.


Table 4.105:  Adjusted Odds Ratios Enterococcus qPCR CCE, Delta-delta CT
calculation and GI  illness.   Daily,  waist depth, shin depth,  8:00 AM and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.69
1.01
0.82
0.75
0.73

0.71
0.99
0.84
0.77
0.67
P-value Lower 95% CI

0.18
0.96
0.17
0.20
0.13

0.25
0.98
0.29
0.30
0.08

0.41
0.58
0.62
0.48
0.48

0.39
0.53
0.61
0.47
0.43
Upper 95% CI N

1.19
1.77
1.09
1.17
1.10

1.27
1.85
1.16
1.26
1.04

10934
10173
10934
10300
10934

9095
8463
9095
8574
9095
                                   150

-------
Table 4.106:  Adjusted  Odds  Ratios Enterococcus qPCR CCE, Delta-delta
CT calculation and Diarrhea.  Daily,  waist depth, shin depth, 8:00 AM and
swimming-location  indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.49
1.09
0.68
0.75
0.84

0.59
1.09
0.74
0.80
0.79
P-value Lower 95% CI

0.04
0.81
0.06
0.30
0.50

0.18
0.82
0.17
0.46
0.40

0.25
0.55
0.46
0.43
0.52

0.27
0.50
0.49
0.44
0.46
Upper 95% CI N

0.96
2.16
1.01
1.30
1.38

1.27
2.40
1.14
1.45
1.36

10933
11092
10933
10300
10802

9095
8469
9095
8574
8986
Table 4.107:  Adjusted Odds Ratios Enterococcus qPCR CCE, Delta-delta CT
calculation and Respiratory illness.  Daily, waist depth, shin depth,  8:00 AM
and swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.90
0.61
1.08
0.81
0.78

1.03
0.67
1.12
0.90
0.81
P-value Lower 95% CI

0.66
0.04
0.48
0.27
0.15

0.92
0.15
0.33
0.65
0.28

0.54
0.37
0.87
0.55
0.55

0.60
0.38
0.89
0.58
0.55
Upper 95% CI N

1.47
0.99
1.34
1.18
1.10

1.77
1.16
1.42
1.40
1.19

10184
10489
11096
9617
10310

8481
8757
9129
8019
8595
                                   151

-------
Table 4.108:  Adjusted Odds Ratios Enterococcus qPCR CCE, Delta-delta CT
calculation and Rash. Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.92
1.00
0.98
0.78
0.60

1.13
1.14
1.07
0.95
0.73
P-value Lower 95% CI

0.79
0.99
0.91
0.30
0.03

0.72
0.68
0.70
0.86
0.24

0.50
0.57
0.71
0.49
0.38

0.57
0.61
0.75
0.55
0.44
Upper 95% CI N

1.70
1.76
1.36
1.25
0.95

2.24
2.11
1.54
1.65
1.23

10815
10190
10814
10315
11793

9003
8598
9012
8498
9117
Table 4.109:  Adjusted Odds Ratios  Enterococcus  qPCR  CCE, Delta-delta
CT calculation and  Earache.  Daily, waist depth,  shin  depth,  8:00 AM and
swimming-location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.88
0.39
1.24
0.59
0.76

1.20
0.50
1.35
0.74
0.98
P-value Lower 95% CI

0.79
0.01
0.30
0.09
0.39

0.73
0.10
0.17
0.39
0.97

0.35
0.19
0.83
0.32
0.41

0.43
0.22
0.88
0.38
0.49
Upper 95% CI N

2.21
0.82
1.86
1.08
1.41

3.31
1.15
2.09
1.46
1.98

10888
11207
11028
11207
11878

9065
9339
9178
9338
9030
                                   152

-------
Table 4.110:  Adjusted Odds Ratios Enterococcus qPCR CCE, Delta-delta CT
calculation and Eye irritations.  Daily, waist depth, shin depth, 8:00 AM and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.99
1.08
0.97
1.22
0.69

1.23
1.48
1.04
1.37
0.71
P-value Lower 95% CI

0.98
0.80
0.82
0.41
0.10

0.52
0.19
0.81
0.24
0.18

0.56
0.58
0.72
0.76
0.45

0.66
0.82
0.75
0.81
0.43
Upper 95% CI N

1.75
2.02
1.30
1.98
1.07

2.31
2.65
1.45
2.33
1.18

10943
10326
10946
10450
11947

9219
8701
9111
8701
9944
Table 4.111: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation and GI illness.  Daily, waist  depth, shin depth, 8:00 AM and swimming-
location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.75
1.07
0.83
0.69
0.77

0.79
1.14
0.85
0.72
0.73
P-value Lower 95% CI

0.28
0.78
0.22
0.11
0.23

0.43
0.62
0.32
0.16
0.17

0.44
0.66
0.62
0.44
0.50

0.44
0.68
0.61
0.45
0.47
Upper 95% CI N

1.26
1.73
1.12
1.08
1.18

1.42
1.92
1.18
1.14
1.14

10934
10298
10934
10300
10803

9095
8469
9095
9238
9095
                                   153

-------
Table 4.112: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation and Diarrhea.  Daily,  waist depth, shin depth, 8:00 AM and swimming-
location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.60
1.11
0.71
0.74
0.86

0.65
1.13
0.76
0.70
0.81
P-value Lower 95% CI

0.14
0.73
0.10
0.29
0.59

0.26
0.72
0.21
0.24
0.46

0.30
0.62
0.48
0.42
0.49

0.32
0.59
0.49
0.39
0.47
Upper 95% CI N

1.18
1.99
1.07
1.30
1.50

1.36
2.17
1.17
1.27
1.42

10933
10948
10802
10300
10802

9095
8469
8986
9237
9095
Table 4.113:  Adjusted Odds Ratios Enterococcus  qPCR CCE, Delta CT cal-
culation and  Respiratory illness. Daily, waist depth, shin depth, 8:00 AM and
swimming-location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.05
0.76
1.13
1.00
0.85

1.19
0.78
1.21
1.14
0.89
P-value Lower 95% CI

0.80
0.15
0.28
0.98
0.28

0.42
0.27
0.10
0.48
0.50

0.71
0.52
0.91
0.72
0.62

0.78
0.51
0.97
0.80
0.63
Upper 95% CI N

1.56
1.11
1.40
1.41
1.15

1.82
1.21
1.52
1.63
1.25

10944
10489
11096
9605
11096

9129
8757
9129
8757
8595
                                   154

-------
Table 4.114: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcula-
tion and Rash. Daily, waist depth, shin depth, 8:00 AM and swimming-location
indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.39
1.15
1.22
1.07
0.88

1.76
1.20
1.32
1.29
1.02
P-value Lower 95% CI

0.20
0.52
0.15
0.74
0.49

0.04
0.49
0.06
0.26
0.91

0.84
0.75
0.93
0.71
0.61

1.03
0.72
0.99
0.82
0.68
Upper 95% CI N

2.31
1.77
1.59
1.63
1.27

3.01
1.99
1.77
2.02
1.53

10947
10980
10947
10195
10816

9117
8599
9117
9283
9001
Table 4.115: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation and Earache.  Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.87
0.47
1.29
0.56
0.76

1.08
0.56
1.39
0.65
0.97
P-value Lower 95% CI

0.73
0.03
0.24
0.07
0.33

0.86
0.12
0.15
0.20
0.92

0.41
0.24
0.85
0.30
0.43

0.46
0.27
0.89
0.34
0.53
Upper 95% CI N

1.88
0.92
1.95
1.04
1.33

2.56
1.16
2.17
1.26
1.76

10899
10395
11028
11204
11878

9069
8658
9178
9338
9059
                                   155

-------
Table 4.116:  Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT cal-
culation  and Eye irritations.  Daily, waist  depth, shin depth,  8:00 AM and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.24
1.20
1.08
1.36
0.88

1.39
1.35
1.13
1.47
0.88
P-value Lower 95% CI

0.40
0.47
0.59
0.13
0.49

0.27
0.27
0.38
0.10
0.56

0.75
0.73
0.82
0.92
0.60

0.78
0.79
0.85
0.93
0.57
Upper 95% CI N

2.05
1.95
1.42
2.02
1.27

2.48
2.31
1.50
2.31
1.36

11086
10450
10951
11274
11947

9224
9392
9944
9392
9939
                                   156

-------
Bacteroidales qPCR Calibrator Cell Equivalents

Associations between illness and exposure to Bacteroidales qPCR CCE by the
delta-delta CT method are shown in Tables 4.117-4.122 and for the delta-CT
method in Tables 4.111-4.128.
   As with Enterococcus CCE interpretation of the illness-Bacteroidales CCE
association was hampered by the high proportion of samples which showed in-
terference or  were not detected. Positive trends with both eye irritations  and
skin rash were observed with Bacteroidales CCE exposure by both types of CT
calculations (see Tables  4.120, 4.122, 4.126. The  interpretation of the trend
among swimmers  for eye irritations  is complicated by the finding that non-
swimmers actually experienced higher rates of eye irritations even compared to
the most highly exposed swimmer category (see Figure 4.4 and Table 4.95).  .
However, the association with skin rash was not evident for Bacteroidales CCE
calculated by the  delta-delta CT method (see Table 4.126) where statistically
significant associations were not observed.
   Positive associations between rash and  Bacteroidales CCE exposure were
observed among children 10 and under for the delta-CT calculation (AOR=2.53,
p=0.04  for body immersion  exposure,  Table 4.129), but the association  was
considerably weaker for CCE using the delta-delta CT calculation (AOR=1.24,
p=0.72 for body immersion exposure, Table 4.130).  A similar inverse association
was observed for GI illness and diarrhea for Bacteroidales CCE exposure among
children.

Table 4.117:   Adjusted Odds  Ratios Bacteroidales qPCR CCE,  Delta-delta
CT calculation and GI illness. Daily, waist depth, shin depth,  8:00 AM  and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.77
0.78
0.82
0.80
0.73

0.77
0.75
0.85
0.80
0.78
P-value Lower 95% CI

0.34
0.33
0.41
0.30
0.16

0.41
0.32
0.54
0.36
0.32

0.45
0.48
0.52
0.52
0.48

0.42
0.43
0.51
0.50
0.48
Upper 95% CI N

1.32
1.29
1.30
1.22
1.12

1.42
1.32
1.43
1.29
1.27

10934
11094
10934
11095
11611

8985
9238
8985
9238
9658
                                   157

-------
Table 4.118:  Adjusted Odds Ratios  Bacterotdales qPCR CCE,  Delta-delta
CT calculation and Diarrhea.  Daily,  waist depth, shin depth, 8:00 AM  and
swimming-location  indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.64
0.75
0.66
0.72
0.64

0.64
0.76
0.69
0.81
0.72
P-value Lower 95% CI

0.18
0.27
0.14
0.20
0.08

0.24
0.43
0.27
0.49
0.28

0.33
0.44
0.38
0.44
0.39

0.30
0.38
0.36
0.44
0.40
Upper 95% CI N

1.23
1.26
1.15
1.18
1.06

1.36
1.51
1.34
1.48
1.31

10933
11094
10933
11094
10933

9095
8470
9095
8573
9095
Table 4.119: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta-delta CT
calculation and Respiratory illness.  Daily, waist depth, shin depth,  8:00 AM
and swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.97
0.77
1.14
0.86
0.74

0.98
0.80
1.16
0.78
0.80
P-value Lower 95% CI

0.88
0.21
0.44
0.42
0.10

0.93
0.34
0.48
0.25
0.26

0.62
0.51
0.82
0.61
0.52

0.59
0.51
0.77
0.51
0.54
Upper 95% CI N

1.51
1.16
1.60
1.23
1.06

1.61
1.27
1.74
1.19
1.19

10179
10489
10944
9737
11096

8484
8634
8489
8019
9257
                                   158

-------
Table 4.120: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta-delta CT
calculation and Rash. Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.49
1.51
1.29
0.90
1.32

1.42
1.36
1.29
0.82
1.28
P-value Lower 95% CI

0.12
0.07
0.22
0.60
0.21

0.23
0.23
0.30
0.41
0.32

0.91
0.97
0.86
0.60
0.85

0.80
0.82
0.80
0.51
0.79
Upper 95% CI N

2.46
2.37
1.94
1.35
2.03

2.54
2.25
2.07
1.32
2.09

11793
11128
11793
11127
10947

9117
9283
9117
9283
9117
Table 4.121:  Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta-delta
CT calculation and  Earache.  Daily, waist depth,  shin  depth,  8:00 AM and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.73
0.58
1.03
0.67
0.76

0.99
0.87
1.19
0.81
0.91
P-value Lower 95% CI

0.39
0.11
0.92
0.14
0.37

0.99
0.71
0.60
0.45
0.77

0.36
0.30
0.56
0.39
0.42

0.46
0.42
0.61
0.46
0.47
Upper 95% CI N

1.48
1.13
1.89
1.14
1.39

2.14
1.81
2.32
1.41
1.74

10899
10395
10887
10395
10899

9030
8654
9065
8653
9060
                                   159

-------
Table 4.122: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta-delta CT
calculation and Eye irritations.  Daily, waist depth, shin depth, 8:00 AM and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.81
1.62
1.56
1.18
0.98

1.73
1.56
1.52
1.08
0.99
P-value Lower 95% CI

0.03
0.05
0.05
0.48
0.93

0.07
0.10
0.10
0.78
0.97

1.06
1.00
1.01
0.75
0.61

0.95
0.92
0.92
0.64
0.59
Upper 95% CI N

3.08
2.62
2.43
1.84
1.56

3.13
2.66
2.53
1.82
1.66

11086
10450
11086
10324
10906

9224
8701
9224
8591
9104
                                    160

-------
Table 4.123: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation and GI illness.  Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.81
0.88
0.84
0.75
0.77

0.84
0.90
0.87
0.75
0.83
P-value Lower 95% CI

0.40
0.61
0.45
0.15
0.21

0.53
0.71
0.57
0.22
0.43

0.49
0.55
0.54
0.50
0.51

0.49
0.53
0.54
0.48
0.53
Upper 95% CI N

1.33
1.42
1.31
1.11
1.16

1.44
1.54
1.41
1.19
1.31

10932
10174
10932
11092
11748

9788
8469
8985
9238
9788
                                   161

-------
Table 4.124: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta CT calcu-
lation and Diarrhea. Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.70
0.83
0.74
0.72
0.65

0.71
0.85
0.74
0.73
0.75
P-value Lower 95% CI

0.27
0.51
0.28
0.24
0.11

0.33
0.60
0.34
0.32
0.32

0.38
0.48
0.42
0.42
0.38

0.35
0.46
0.40
0.40
0.43
Upper 95% CI N

1.31
1.44
1.28
1.24
1.10

1.42
1.57
1.37
1.35
1.32

10800
11092
10933
11095
10933

8985
9113
9095
9238
9658
Table 4.125:  Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta CT cal-
culation and  Respiratory illness. Daily, waist depth, shin depth, 8:00 AM and
swimming-location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.10
0.88
1.26
1.06
0.83

1.13
0.87
1.37
1.00
0.91
P-value Lower 95% CI

0.65
0.49
0.21
0.74
0.29

0.58
0.51
0.13
1.00
0.64

0.74
0.60
0.87
0.76
0.60

0.73
0.57
0.91
0.68
0.62
Upper 95% CI N

1.63
1.27
1.81
1.46
1.16

1.75
1.32
2.04
1.48
1.34

10939
10345
10184
10339
11096

9129
8757
8489
8009
8595
                                   162

-------
Table 4.126: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta CT calcula-
tion and Rash.  Daily, waist depth, shin depth, 8:00 AM and swimming-location
indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

2.11
1.58
1.94
1.20
1.78

1.88
1.40
1.99
1.12
1.83
P-value Lower 95% CI

0.00
0.03
0.00
0.30
0.00

0.01
0.17
0.00
0.56
0.01

1.35
1.04
1.31
0.85
1.24

1.14
0.86
1.27
0.76
1.19
Upper 95% CI N

3.31
2.39
2.87
1.71
2.57

3.09
2.27
3.12
1.66
2.82

11793
11128
11793
10320
11793

9826
8603
9117
9283
9117
Table 4.127:  Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta CT cal-
culation and Earache. Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages.  Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

0.78
0.64
1.07
0.62
0.78

0.94
0.78
1.20
0.72
0.91
P-value Lower 95% CI

0.46
0.14
0.82
0.06
0.40

0.86
0.46
0.60
0.27
0.75

0.41
0.36
0.59
0.37
0.45

0.46
0.41
0.61
0.40
0.50
Upper 95% CI N

1.49
1.16
1.94
1.02
1.38

1.91
1.49
2.35
1.29
1.64

10899
10395
10892
10390
11028

9060
8648
9070
8657
9065
                                   163

-------
Table 4.128:  Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT cal-
culation  and Eye irritations.  Daily,  waist  depth,  shin depth, 8:00 AM and
swimming-location indicator averages. Boqueron Beach
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.95
1.68
1.78
1.34
1.25

1.73
1.51
1.69
1.18
1.20
P-value Lower 95% CI

0.01
0.03
0.01
0.13
0.29

0.06
0.12
0.04
0.48
0.45

1.19
1.05
1.16
0.92
0.82

0.98
0.90
1.02
0.75
0.75
Upper 95% CI N

3.22
2.69
2.72
1.95
1.88

3.04
2.55
2.82
1.86
1.92

11086
10450
11947
11274
10956

9224
8701
9224
8697
9115
Table 4.129: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta CT calcula-
tion and Rash.  Daily, waist depth, shin depth, 8:00 AM and swimming-location
indicator averages. Boqueron Beach. Children age 10 and under.
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

2.53
1.70
2.63
0.82
1.34

2.72
1.82
2.53
0.98
1.40
P-value Lower 95% CI

0.04
0.21
0.02
0.62
0.44

0.04
0.18
0.03
0.97
0.43

1.06
0.74
1.14
0.38
0.64

1.07
0.76
1.07
0.43
0.60
Upper 95% CI N

6.04
3.88
6.05
1.79
2.82

6.90
4.38
5.96
2.23
3.28

2087
1955
1944
1819
2087

1895
1782
1757
1648
1895
                                   164

-------
Table 4.130: Adjusted Odds Ratios Bacteroidales qPCR CCE, Delta-delta CT
calculation and Rash. Daily, waist depth, shin depth, 8:00 AM and swimming-
location indicator averages. Boqueron Beach. Children age 10 and under.
Exposure
Body immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
Head immersion
Daily
Waist depth
Shin depth
8:00 AM
Swimming-location
AOR

1.24
1.34
1.16
0.43
0.56

1.05
1.20
0.93
0.45
0.51
P-value Lower 95% CI

0.72
0.62
0.75
0.11
0.28

0.93
0.78
0.88
0.14
0.26

0.38
0.42
0.46
0.15
0.19

0.30
0.34
0.37
0.16
0.16
Upper 95% CI N

4.04
4.27
2.95
1.20
1.62

3.69
4.29
2.35
1.28
1.65

1944
1819
1944
1821
2087

1755
1648
1757
1650
1895
                                   165

-------
4.3    Sensitivity analyses

Several variations of the analyses were conducted to assess the stability of the
results. In the above analyses participants who reported swimming exposure in
the 1-week prior to enrollment were included (but this exposure was controlled
for in regression modeling).  Below results  are also presented for excluding re-
spondents with recent swimming exposure  and for various different approaches
to calculating qPCR CCE below the limit  of detection.  The tables below only
show results for body immersion swimming exposure and the daily indicator
average.

4.3.1    Surfside Beach

Excluding those with swimming exposure in 1-week prior to enroll-
ment

Results are shown below in Tables 4.131-4.133 for the daily averaged indicator
exposures for Enterococcus CFU and qPCR CCEs using the delta CT calcula-
tion. Sample size is greatly  reduced by  excluding those with prior swimming
exposures, but there are little differences in the effect estimates.
   For comparison with original results,  see Tables 4.33- 4.38 for Enterococcus
CFU, Tables 4.50- 4.55 for Enterococcus  qPCR CCE, and Tables 4.64- 4.69 for
B'acteroidales qPCR CCE.


Table 4.131: Adjusted Odds Ratios Enterococcus CFU  (Method 1600).  Daily
indicator averages.  Excluding those with swimming exposure in past 1-week.
Body immersion swimming exposure. Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.19
1.33
0.86
0.82
0.96
0.86
P-value Lower 95% CI
0.37 0.81
0.21
0.47
0.45
0.88
0.67
0.85
0.58
0.49
0.53
0.43
Upper 95% CI N
1.75 3815
2.09
1.28
1.37
1.72
1.73
3813
3792
3880
3884
3913
Alternate approaches for results below limit of detection for qPCR

Because even at Surfside beach there were a fairly high proportion of Ente-
rococcus qPCR CCE which were below the limit of detection (N=167), some
variation in results was expected with different approaches to handle results
below the detection limit. However, health effects associations with qPCR CCE
using the maximum likelihood approach and the regression on order statistic
approach showed no fundamental differences in interpretation as using one-half
the detection limit (Tables  4.134 and 4.135).
                                   166

-------
Table 4.132: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation. Daily indicator averages. Excluding those with swimming exposure in
past 1-week. Body immersion swimming exposure.  Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
0.98
1.18
0.76
0.50
0.81
0.92
P-value Lower 95% CI
0.91 0.71
0.38
0.15
0.01
0.44
0.72
0.82
0.52
0.29
0.47
0.59
Upper 95% CI N
1.36 3692
1.70
1.10
0.85
1.39
1.43
3690
3612
3760
3764
3787
Table 4.133: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation. Daily indicator averages. Excluding those with swimming exposure in
past 1-week. Body immersion swimming exposure.  Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
0.97
0.92
1.00
0.77
1.24
0.67
P-value Lower 95% CI
0.93
0.81
0.99
0.40
0.71
0.39
0.53
0.44
0.50
0.41
0.41
0.28
Upper 95% CI N
1.77
1.90
2.00
1.43
3.72
1.65
3690
3688
3669
3760
3758
3730
Table 4.134: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation. Maximum likelihood estimate for non-detects. Daily indicator averages.
Body immersion swimming exposure. Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.14
1.16
1.04
0.74
1.02
0.90
P-value Lower 95% CI
0.14
0.18
0.73
0.00
0.87
0.41
0.96
0.94
0.85
0.61
0.79
0.72
Upper 95% CI N
1.35
1.43
1.26
0.90
1.32
1.14
7752
7748
7618
7833
7901
7927
                                   167

-------
Table 4.135: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation. Regresson on order statistics estimate for non detects.  Daily indicator
averages. Body immersion swimming exposure.  Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.16
1.25
1.12
0.66
1.10
0.89
P-value Lower 95% CI
0.14 0.95
0.07
0.28
0.00
0.56
0.38
0.98
0.92
0.52
0.80
0.68
Upper 95% CI N
1.40 7752
1.60
1.36
0.84
1.51
1.16
7748
7725
7731
8009
7821
   Health effects associations with Bacteroidales, which had considerably fewer
non-detects results, showed essentially no differences as compared to using one-
half the detection limit (Tables  4.136 and 4.137).


Table 4.136: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation. Maximum likelihood estimate for non-detects. Daily indicator averages.
Body immersion swimming exposure. Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.16
1.09
1.14
0.68
0.99
0.95
P-value Lower 95% CI
0.49
0.75
0.59
0.12
0.98
0.88
0.76
0.65
0.70
0.41
0.46
0.50
Upper 95% CI N
1.77
1.83
1.86
1.11
2.15
1.80
7750
7746
7725
7831
7899
7817
                                    168

-------
Table 4.137: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation. Regresson on  order statistics estimate for non detects.  Daily indicator
averages. Body immersion swimming exposure. Surfside Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.22
1.14
1.18
0.74
1.00
0.91
P-value Lower 95% CI
0.31
0.62
0.49
0.20
1.00
0.76
0.83
0.68
0.73
0.46
0.47
0.49
Upper 95% CI N
1.78
1.89
1.91
1.18
2.14
1.68
7853
7746
7719
7729
7899
7817
                                   169

-------
4.3.2   Boqueron Beach

Excluding those with swimming exposure in 1-week prior to enroll-
ment

Results are shown in Tables 4.138-4.140.  The association between respiratory
illness and Enterococcus CFU was more pronounced (Table 4.138). Other as-
sociations were similar, although the same concerns regarding interpretation of
the qPCR results described previously still apply.
   For comparison with original results, see Tables 4.97- 4.102 for Enterococcus
CFU, Tables 4.111- 4.116 for Enterococcus qPCR CCE, and Tables 4.123- 4.128
for Bacteroidales qPCR CCE.
Table 4.138: Adjusted Odds Ratios Enterococcus CFU (Method 1600).  Daily
indicator averages. Excluding those with swimming exposure in past 1-week.
Body immersion swimming exposure. Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.00
0.99
1.44
1.18
1.25
1.07
P-value Lower 95% CI
0.98
0.96
0.03
0.42
0.28
0.81
0.66
0.61
1.03
0.79
0.83
0.61
Upper 95% CI N
1.50
1.60
2.02
1.74
1.89
1.89
7651
7651
7237
7672
8467
8421
Table 4.139: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation. Daily indicator averages.  Excluding those with swimming exposure in
past 1-week. Body immersion swimming exposure. Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.34
1.40
1.01
1.83
1.08
0.51
P-value Lower 95% CI
0.36 0.72
0.40
0.97
0.04
0.79
0.19
0.64
0.62
1.04
0.61
0.19
Upper 95% CI N
2.52 8335
3.09
1.65
3.20
1.90
1.39
7758
7232
8362
8467
8420
Alternate approaches for results below limit of detection for qPCR

As expected with the high proportion of results below detection, the results are
affected by the method used to handle these results, especially for Enterococcus
CCE. For Bacteroidales CCE, no fundamental differences in interpretation are
                                  170

-------
Table 4.140: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation. Daily indicator averages.  Excluding those with swimming exposure in
past 1-week. Body immersion swimming exposure. Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.18
1.26
1.07
2.13
1.38
0.61
P-value Lower 95% CI
0.51 0.72
0.44
0.75
0.00
0.14
0.22
0.71
0.71
1.35
0.90
0.28
Upper 95% CI N
1.94 7649
2.23
1.60
3.37
2.13
1.34
8204
7230
8367
8467
7828
observed and swimming-associated rash and eye irritations are still evident for
the delta-CT calculation.  However, as discussed  above, the  association  with
rash and Bacteroidales CCE is still diminished for the delta-delta CT calculation
for alternate detection limit calculations (AOR=1.38, p=0.19; and AOR=1.23,
p=0.38 for the maximum likelihood and regression on order statistics estimates).


Table 4.141: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation. Maximum likelihood estimate for non-detects. Daily indicator averages.
Body immersion swimming exposure.  Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
1.15
1.19
1.08
1.18
1.06
1.04
P-value Lower 95% CI
0.25
0.28
0.52
0.23
0.70
0.85
0.91
0.87
0.85
0.90
0.79
0.71
Upper 95% CI N
1.46
1.62
1.38
1.54
1.42
1.51
11095
11095
9733
10315
10326
10379
                                   171

-------
Table 4.142: Adjusted Odds Ratios Enterococcus qPCR CCE, Delta CT calcu-
lation. Regresson on order statistics estimate for non detects.  Daily indicator
averages. Body immersion swimming exposure.  Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
0.89
0.87
1.21
1.02
1.34
1.04
P-value Lower 95% CI
0.45 0.66
0.46
0.07
0.89
0.09
0.89
0.59
0.98
0.73
0.96
0.62
Upper 95% CI N
1.20 9666
1.27
1.50
1.43
1.88
1.74
9666
9945
9682
9928
9757
Table 4.143: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation. Maximum likelihood estimate for non-detects. Daily indicator averages.
Body immersion swimming exposure.  Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
0.88
0.83
1.03
1.89
1.94
0.78
P-value Lower 95% CI
0.63
0.58
0.90
0.00
0.01
0.46
0.52
0.42
0.69
1.21
1.17
0.40
Upper 95% CI N
1.50
1.63
1.54
2.95
3.22
1.52
10173
10174
10335
11128
10450
10263
Table 4.144: Adjusted Odds Ratios Bacterotdales qPCR CCE, Delta CT calcu-
lation. Regresson on order statistics estimate for non detects.  Daily indicator
averages. Body immersion swimming exposure.  Boqueron Beach
Illness
GI illness
Diarrhea
Respiratory Illness
Rash
Eye irritations
Earache
AOR
0.83
0.79
1.03
1.62
1.87
0.80
P-value Lower 95% CI
0.47
0.49
0.88
0.03
0.01
0.49
0.49
0.41
0.69
1.05
1.15
0.41
Upper 95% CI N
1.38
1.53
1.54
2.48
3.06
1.54
10298
10174
9608
11128
10450
10263
                                   172

-------
Chapter  5

Summary   and  discussion
5.1    Overview

This report provides a rigorous, detailed analysis and presentation of water
quality and illnesses among swimmers at studies conducted by the EPA during
the summer of 2009.   The primary goal  of this  research and  report was to
describe associations between water quality indicators and health effects at a
beach site impacted by urban runoff and a tropical beach site.  The tropical
beach site was selected with nearby discharges from sewage treatment so that
results could be compared with previous studies conducted by EPA and others
[3, 2, 54].
   Enrollment and follow up in the epidemiology study was successful. A large
number  of subjects were enrolled with more than 10,000 usable responses from
each beach site. Follow up was also very successful with more than 60% of those
initially approached completing the study at Surfside Beach and nearly 80% at
Boqueron  Beach.  Among those agreeing or eligible to be in the study, 5% or
less were lost to follow  up at both sites.
   The  overall incidence  of symptoms  appears to be consistent with what has
been previously observed,  at least for GI illness (the symptom most frequently
associated with recreational water exposure [7, 8]). At Surfside  and Boqueron
beaches  in 2009, the overall rates  of GI illness were consistent with the rates
reported in previous population based surveys and epidemiology studies. The
yearly equivalent rates  for GI illness (both swimmers and non-swimmers) were
1.56 and 2.04 per year at  Boqueron and Surfside, respectively. If swimmers are
excluded, the incidence is  lower (1.44 for Surfside and 1.41 at Boqueron). CDC's
Foodnet Survey reported  an annual rate of diarrhea of 1.4  episodes per person-
year [35].  In a review of of studies and surveys in developed countries, Roy
et.  al.  [55] found a range of rates from 0.1-3.5 per person-year. One surprise,
however, was the relatively low incidence of GI illness at Boqueron Beach, where
it was anticiapted  a higher endemic incidence may have been observed due to
the tropical setting.
                                 173

-------
   In interpreting the results, the following criteria were used to establish as-
sociations between water quality and swimming-associated illnesses.

   • The water quality (exposure) indicator must be sufficiently sensitive and
     have demonstrated accuracy and reliability.

   • The incidence of illness among swimmers should increase as  exposure to
     the concentration of the indicator increases.

        — The above association should demonstrate consistent and predictable
          patterns, such as consistency of effect across approaches to calculat-
          ing and averaging the exposure

   • The adjusted incidence  among the most highly exposed swimmers should
     generally (not necessarily always) be higher among swimmers as compared
     to non swimmers

   The last item is technically not required to establish associations with water
quality and illness, but the lack of a  difference between swimmers and non-
swimmers complicates a sensible description of risk associated with swimming
and exposure.
   Conclusions regarding relationships to health effects should hold for both ap-
proaches in calculating the Calibrator Cell Equivalents by qPCR and should be
robust to different approaches in calculating results below the limit of detection.
   Also in interpreting the results, care should be taken to not simply select a
statistically significant result as evidence of an effect or  an association. Given the
large number of analyses conducted p-values should be interpreted with caution
and meant as a guide to the relative strength of the associations presented.


5.2    General  limitations

Water quality at both beaches was relatively good compared to previous beaches
studied.  Unfortunately a low range of exposure can impact  the determination
of health  effects associations since statistical power  and  ability to detect an
association are reduced. However due to the large number of subjects enrolled,
the study likely had statistical power to observe  effects of a reasonable size.
For example, statistical simulations  of statistical  power [56] conducted using
the actual exposure data and observed incidence of GI illness indicated that for
Enterococcus qPCR CCE (delta-delta CT) there was a sufficient sample size to
observe an AOR of about 1.4 for Boqueron Beach and 1.3 for Surfside Beach.
These effect sizes are  lower than those observed at other marine sites  [57] but
slightly higher than the effect observed at freshwater beaches [3].
   As  shown in  Table 5.1 fecal indicators measured at Surfside  and Boqueron
were low compared to the other three marine beach sites studied in 2005-2007.
With the  exception of Enterococcus CFU at Goddard Beach in Rhode Island,
which had an overall  lower geometric  mean than  Boqueron  Beach,  geometric
means  for all the  indicators were lower at the 2009 beach sites.   The fecal
                                   174

-------
indicators measured by qPCR were considerably lower in 2009: less than half
the estimated CCE at the other marine beach sites. There is also a variation in
the CFU to CCE ratio across the beach sites. This could be partially explained
by the detection of non-viable organisms by qPCR.


Table 5.1: Geometric mean of fecal indicator bacteria, marine beach sites, 2005-
2009

                    Enterococcus CFU1   Enterococcus CCE2   Bactermdales CCE2
Edgewater Beach
Fairhope Beach
Goddard Beach
Surfside Beach
Boqueron Beach
8
21
4
3
6
368
258
159
55
32
2750
1791
1092
295
288
 CCE=qPCR Calibrator Cell Equivalents per 100 ml (delta-delta CT
 CFU: Colony forming units per 100 ml
   The low levels of fecal indicators were also reflected in the relatively high
number of samples where no target was detected, especially for  Enterococcus.
Even at Surfside Beach 32% of samples were not detected for Enterococcus by
qPCR. Also at Surfside, 12% of samples were not detected for Bacteroidales.
These were considerably higher than the numbers of non-detects observed at
the three  previously studied marine sites where for Enterococcus 10% of sam-
ples  were  not  detected, and for Bacteroidales 5% were not detected.  For Bo-
queron Beach, the problem with non-detected target sequences was greatly ex-
acerbated, where only a small minority of samples were actually quantifiable
(see  Section 4.2.6 for discussion).
   For some environmental measures, relying on few measures may not be prob-
lematic for establishing valid associations with health effects.  However, it has
been shown that fecal indicator bacteria can vary considerably over time, space
and  location [58, 59] and relying on few samples is likely to  result less accu-
rate  exposure measurement and classification, further reducing the ability to
determine valid associations with health effects.
   An additional issue not  seen in most previous beach studies was the low
proportion of non-swimmers.  At the three previous marine sites,  42% of re-
spondents reported body immersion swimming exposure, whereas 72% reported
body immersion exposure at Surfside and 77% at Boqueron Beach. While this
provided more statistical power to examining associations of the effects among
swimmers exposed  to varying levels of water quality, it may have reduced the
ability to accurately describe differences between swimmers and non-swimmers.
                                    175

-------
5.3    Surfside Beach

Indicator bacteria and health effects associations were originally derived by EPA
[60, 6f, 5, 6] and others [62, 54] at beaches impacted by human sewage.  These
original sites were selected for several  reasons. It was expected the indicator
bacteria and pathogen association would be more consistent at such sites  (so
long as point sources were not "small",  or experiencing outbreak conditions,  see
[5] for discussion).  Furthermore it was expected that human-feces exposures
pose a larger risk to human health [63, I].
   An issue with sites impacted by runoff or non-point source pollution is that
the fecal contamination may be from an inconsistent or variable sources which
may present  problems in understanding or deriving health effects from fecal
indicators.  It may be analogous to the  "small point source" problem described
by Cabelli  [5]:

      The rationale for the  use of guidelines and standards based on fecal
      indicator densities for indexing the health hazards in sewage polluted
      waters is that, under  average conditions of illness in the discharging
      population, there is  a reasonably constant indicator to  pathogen
      ratio  in the sewage and its receiving waters.  Thereby, an acceptable
      probability of illness caused by the pathogen can be extrapolated to
      a given indicator density, which is then recommended as a guideline
      and promulgated as a standard.  Such relationships appear to hold
      for waters receiving the discharges from relatively large municipal
      sewage  treatment  facilities.  However, as the number of individuals
      who contribute to the source of the fecal wastes becomes smaller and
      smaller, the indicator-pathogen ratio will vary more and more from
      the average upon which the guideline or standard is based. [5]

   Previous studies at runoff impacted sites in marine waters have had  mixed
results.  For example, an increased risk of illness was found among swimmers
near storm drains in Santa Monica beaches in California [14] but in Mission Bay,
California,  no associations were observed with levels of fecal indicator organisms
and illness  with the possible exception of male-specific coliphage, but this was
based on few observations [18].  Recently a randomized trial found an association
with skin symptoms at  a runoff impacted site  in Florida, but failed to find an
association with GI or respiratory symptoms [64, 65]. Enterococcus CFU at this
site during the study was higher than  at Surfside Beach with a median of 19
CFU  per 100 ml, though the sampling design was  considerably different  (see
[65]).
   Surfside Beach was selected as a "urban runoff" site and had no known point
source of human fecal contamination which affected the site.  Water quality
was generally of high quality and only one day exceeded EPA's recommended
geometric mean criterion of 35 CFU per 100  ml.  Swimmers immersing their
head had a higher incidence of skin rash, earache and GI illness compared to
non-swimmers.
                                   176

-------
   Positive but generally non-significant trends were observed between Ente-
rococcus  CFU measured by culture and GI  illness.   A slightly stronger, but
still statistically insignificant association was observed with  diarrhea. GI ill-
ness increased from 5% among non swimmers to 6% among  those exposed in
the lowest tertile to 7% among thosed exposed to the highest tertile of expo-
sure of Enterococcus CFU. Those who  were  exposed on the single day when
the geometric mean exceeded 35 CFU had a higher incidence of illness com-
pared to non-swimmers (8.6% vs. 4.7%).  Illness was also elevated compared
to other  swimmers but differences between swimmers were not stastically sig-
nificant.  Among children the incidence  of GI illness was more pronounced on
this day  (17% of those with body immersion reported illness)  but was based on
few cases of illness. No other associations were obsered with other illnesses and
Enterococcus CFU either on a continuous scale or above the criteria levels.
   Enterococcus qPCR CCE also was positively associated with both GI ill-
ness and diarrhea, but again the trends were not statistically significant.  The
strongest association was between Enterococcus qPCR CCE by the delta-delta
method and diarrhea for the daily averaged indicator values and body immer-
sion swimming exposure (AOR=1.28, p=0.08, see Table 4.45). Among children,
the trend between diarrhea and  Enterococcus CCE was not statistically signif-
icant (AOR=1.36, p=0.27 for body immersion exposure by the delta-delta CT
method). In contrast Bacteroidales exposures showed very few positive or even
borderline associations with illness.
   An unexplained inverse association was observed between Enterococcus CCE
and incidence of skin rash was observed among swimmers. Examination of nu-
merous other water quality parameters (turbidity, dissolved oxygen, conductiv-
ity, pH)  and  other risk factors for rash  could not explain this inverse associa-
tion. More puzzling was that the incidence was increased in swimmers compared
to non-swimmers, but among swimmers declined as exposures to Enterococcus
CCE increased. Since it is not entirely plausible that Enterococcus CCE would
be causally associated with a decline in rash, it may be that Enterococcus CCE
are inversely associated with other water quality factors which may cause  or
influence the risk of skin  rash.
   Respiratory symptoms were associated with Bacteroidales CCE among swim-
ming children 10 under .  The interpretation of this finding was complicated in
that the adjusted estimates affected the association considerably, which was ab-
sent from the crude and unadjusted estimates.  This is a concern because the
relatively small sample sizes among children could be producing unstable esti-
mates which are highly affected by adjustment. Because of the finding of a high
incidence of illness among the non-swimming group  and the few numbers of
children in this group, the representativeness of it as a comparison group may
be suspect for this illness outcome.  When compared against  non-swimmers,
risks are  not elevated even among the most highly exposed swimming children.
However, previous studies have also observed increased respiratory illnesses in
relation to fecal contamination [66, 14].
   The results observed  at Surfside Beach are consistent with what  would be
expected from lower illness risk  at runoff  impacted beach sites compared  to
                                   177

-------
beach sites impacted by human sewage.  However, no conclusion can be made
on the basis of these data alone due to the high quality of water observed at
Surfside Beach . .


5.4    Boqueron  Beach

As described previously, interpretation of results at Boqueron Beach was seri-
ously hampered by the interference shown in the qPCR assay.  However, even
had there been no interference there was surprisingly low levels of fecal indicator
bacteria even as measured by the standard culture based method for Enterococ-
cus.
   Presently,  the exact reason for the interference is not known.  In samples
collected during dry runs prior to the start of the study, this interference was
not seen. Preliminary analyses were conducted to attempt to identify patterns
with the samples which failed the Salmon assay criterion. Birds in the water,
turbidity, density of bathers in the water, amount of debris in the water, con-
ductivity, collection time, tide stage, total algal density, and water depth were
associated with a failure of the Salmon assay criterion. In a multivariate model,
water depth, tide stage at 8:00 AM, debris and total algal density were  asso-
ciated. A formal analysis of the causes and correlates of the interference was
beyond the scope of this report, however it appears that factors associated with
floating debris in the  water could have influenced the interference.  Although
ignoring  the results of the Salmon assay was considered, there would  be con-
cern regarding the validity of the target  assay results. Some of the factors or
biological processes which could be causing the  failure would be the presence
of nucleases in the water which degrade the Salmon DNA, or humic substances
interfering with the assay.
   As a result, the health associations with qPCR CCE at Boqueron Beach are
very difficult to interpret and the attempt to draw conclusions regarding the data
reported would be questionable. Although results were presented and described,
until there is is more confidence in the exposure measure it is impossible to place
much emphasis on any associations observed. It appears the Salmon assay was
a strong  determinant in the results presented. For example,  if the delta-delta
method is used, but failure of the  ±3 CT Salmon assay criterion is  ignored
associations between Enterococcus CCE and GI illness are reversed from those
shown in the report, and are statistically significant and consistent with those
observed previously (AOR=1.22, p=0.008, data not shown). However this type
of variation across calculation approaches indicates a lack of consistency and
calls into question the confidence in the exposure measure.
   The low levels of indicator bacteria at Boqueron  Beach were unexpected.
More surprising was the  low levels seen at samples collected near the  outfalls
and discharge points for the sewage treatment plant and the mangrove swamp
(see  4.18) where it was expected that levels of fecal indicator organisms would
be high.  It may have  been that discharges from the sewage treatment plant
and various package plants were actually not impacting the beach as suspected,
                                   178

-------
despite their proximity (less than 1-mile).  For example, the bay has little wave
and tidal action and as a result there could be little circulation from the dis-
charge points to the beach. Or water could be transported away from the beach
site into the open ocean.
   Boqueron Beach had high densities of  bathers who often stayed in the wa-
ter for long periods of time.  The numbers of bathers in the water were posi-
tively associated with each of the fecal indicator measures (see Tables 4.91, 4.93
4.91). Studies have shown that bathers shed large amounts of indicator bacteria
[67] and bather load has been associated with pathogens in recreational waters
[68, 69].  Bather density was considered in the principal components analysis
of environmental measures which were then subsequently incorporated into the
regression models for health effects (see Section 3.5.4).  Although not a prin-
cipal goal of this report, models using bather  density as a direct predictor of
illness showed no associations (data not shown).  Furthermore the lack  of dif-
ferences in illnesses between swimmers and non-swimmers (with the exception
of skin rash, see Table 4.82) do not support a high risk of illness resulting from
other swimmers.  However, bather density was rather crudely categorized (see
Table 3.1)  and  may not have been adequately represented  for the purposes of
deriving health risks.
   Skin rash was the only symptom significantly elevated among swimmers com-
pared to non-swimmers.  Although associations between rash and Enterococcus
CFU among swimmers were positive, they were weak.  . There was a statistically
insignificant trend between Enterococcus CFU and respiratory illness (p=0.06).
This association improved (p=0.03) when those with recent swimming exposure
were excluded (Table 4.131). This association was present despite no overall dif-
ferences between swimmers and non-swimmers.  Two factors  can account for this
apparent contradiction:  Few numbers of non-swimmers, and few swimmers ex-
posed to poor quality water. Furthermore, the non-swimming group was quite
different from the swimming group, complicating swimmer and non-swimmer
comparisons.  This can be seen in Table 4.81 where despite higher  crude inci-
dence of respiratory illness among swimmers compared to non-swimmers (7.0%
vs. 5.8%) the adjusted risk ratio is slightly less than 1.
   Incidence of GI illness was lower than respiratory illness at Boqueron Beach.
At all previous sites studied, GI illness had the highest incidence. Furthermore,
no positive trends were observed between GI  illness and  any of the indica-
tors. One potential explanation could be  immunity in the  local  population to
pathogens causing GI illness. Furthermore, it was noted that there were cases of
the H1N1  flu in Puerto Rico at this time.  While transmission through water is
unlikely, there may have been an actual increased respiratory symptoms which
may have been exacerbated by the large  crowds on the beach.  In addition,
concern regarding H1N1 could  have resulted in an increased attention to and
reporting of respiratory symptoms.
                                   179

-------
5.5    Future work  and next  steps

Despite completion of this effort, EPA will  continue with further studies and
analyses including:

   • Evaluate the possible reasons and potential remedies for the interference
     with the PCR assay observed at Boqueron Beach. Replicate filters have
     been preserved from the  both beach sites and can be reanalyzed if the
     problem can be resolved.

   • Evaluate health effects  associations with other fecal indicator organisms
     measured by qPCR when results are available for both beach sites.

   • Evaluate additional ancillary exposures collected such as sand samples,
     composite samples and cyanbocateria samples. Compare these with health
     effects if warranted.

   • Complete testing and analyze results  of saliva samples collected at Bo-
     queron Beach. 1,209 households were enrolled in the saliva sampling pro-
     tocol and over 5,000 samples were collected.  Assays are being developed
     to detect salivary antibodies to waterborne pathogens.

   • Continue to evaluate the nature of the source of fecal contamination  at
     the two beaches
5.6    Conclusions

   • Surfside Beach

        — Enrollment, follow up and completion of health surveys was successful
          with over 11,159 completed interviews.
        — Compared with previous marine and freshwater sites [3, 2, 57], a
          high proportion of beach-goers had water exposure, immersing their
          bodies and heads in the water (73% and 58%, respectively).
        — Swimmers had  a higher incidence of GI illness, rash and  earache
          compared to non swimmers.
        — The swash impacting Surfside Beach was affected by runoff and had
          poor water quality.  The swash also had lower salinity than the beach,
          suggesting it was affected by runoff.
        — Water quality measured by Enterococcus CFU, Enterococcus CCE,
          and Bacteroidales CCE was of high quality. Only one day exceeded
          current EPA recommended criteria for Enterococcus(35 CFU per 100
          ml). This limited the ability to demonstrate associations with health
          effects.
                                  180

-------
  — Body immersion swimmers on the single day when the geometric
     mean of Enterococcus CFU exceeded 35 CFU per 100 ml had a higher
     incidence of GI illness compared to non-swimmers, but not compared
     to swimmers exposed on days when the geometric mean was below
     35 CFU per 100 ml.
  — Children  10 years of age and under who immersed their body on the
     single day when the geometric mean of Enterococcus CFU exceeded
     35 CFU per 100 ml had a higher incidence of GI illness compared to
     non-swimmers  and swimmers exposed on days when the geometric
     mean was below 35 CFU per 100 ml.
  — Upper respiratory symptoms were associated with Bacteroidales CCE
     among swimming children 10 years of age and under. There was some
     concern regarding the robustness and  stability of this finding as ad-
     justed estimates differed considerably  from unadjusted estimates.

Boqueron Beach

  — Enrollment, follow up and completion of the health survey was suc-
     cessful with 15,726 completed interviews.
  — Despite proximity to a  sewage  treatment plant discharge (less than
     1 mile from the beach), low levels of fecal indicator bacteria were
     present at the beach sites.
  — Interpretation of water  quality measures and health effects associa-
     tions using fecal indicator  bacteria measured by qPCR was compli-
     cated by  poor recovery of the salmon DNA added as an exogenous
     positive control, indicating potential interference or inhibition.
  — Interpretation of qPCR results  was further complicated by the high
     proportion of results which showed no detection for the fecal indicator
     bacteria target sequences.
  — As a result, no firm conclusions can be made regarding the associ-
     ations between health effects and water quality indicators measured
     by qPCR .
  — No single day exceeded the Enterococcus geometric mean criteria of
     35 CFU per 100 ml.


  — There was some evidence of an association between  Enterococcus
     CFU exposure and increased risk for respiratory illness among those
     without recent swimming  exposure.
                             181

-------
Chapter  6

Acknowledgements
The authors would like to acknowledge the following individuals for their sup-
port in completing these studies.

Westat, Inc. In particular: Karen Delia Torre, Kurt Patrizi, Robert Clickner,
     Sharon Jasmin-Hanif, David Bartmettler, many others.

Boqueron Beach park personnel Eddie Nieves and many others

Municipio Autonomo de Cabo Rojo Hon. Perza A Rodriguez Quinones

Puerto Rico Secretary  Department of Natural and Environmental Resources
     Marc Romaine, Javier Velez-Arocho

Puerto Rico Department of Natural and Environmental Resources Mildred
     Matos, Director of Special Projects

Puerto Rico Environmental Quality Board Angel Melendez

South Carolina Depratment of Health and Environmental Control Shannon
     Berry, Thorn Berry, Sean Torres, Ted Ambrose

Town of Surfside Beach Micki Fellner, Ed Booth

University of Puerto Rico Jose A. Norat-Ramrez, Lyzbeth A. Cordero, Lour-
     des Prieto, Gary Toranzos

US EPA field monitors and administrative and management support
     Bill Russo,  Jennifer Orme-Zavaleta, Todd Baker, Grace Robiou, Emily
     White, Mike Ray, James Kitchens, Lucas Neas, Bruce Mintz, Holly Wirick,
     Ann H. Williams, James Scott, Scott  Rhoney, Jason Mangum, Danelle
     Lobdell, Jefferson Inmon, Edward Hudgens, Carl  Axel Soderberg, Jaime
     Geliga, Lesley Vazquez-Coriano, Carlos Nunez, Jessica Montanez, Melanie
     Jardim, Joel Hansel, Doris Betancourt, Marina Evans, Christian Douglas

Computer Science Corporation Mark S. Murphy


                               182

-------
          All study participants and beaches interviewers, water sample collec-
tors and laboratory personnel.
                                    183

-------
Bibliography
 [I] USEPA. Report of the experts scientific workshop on critical reserach needs
    for the development of new or  revised recreational water quality criteria;
    2007.

 [2] Wade TJ, Calderon RL, Sams E, Beach M, Brenner KP, Williams AH, et al.
    Rapidly measured indicators of recreational water quality are predictive
    of swimming-associated gastrointestinal illness.  Environ Health Perspect.
    2006;114(l):24-28.

 [3] Wade TJ, Calderon RL, Brenner KP, Sams E, Beach M, Haugland R, et al.
    High sensitivity of children to swimming-associated gastrointestinal illness:
    results using  a  rapid assay of  recreational water quality.  Epidemiology.
    2008;19(3):375-383.

 [4] Stevenson A.  Studies of bathing water quality  and health.  Am J Public
    Health Assoc. 1953;43:529-538. Have.

 [5] Cabelli V. Health effects criteria for marine recreational waters. US Envi-
    ronmental Protection Agency; 1983. USEPA Report Number: EPA-600/1-
    80-031.

 [6] Dufour A.  Health effects criteria for  fresh recreational waters. US Envi-
    ronmental Protection Agency; 1984. EPA-600-1-84-004.

 [7] Pruss A. Review of epidemiological studies on health effects from exposure
    to recreational water. Int J Epidemiol. 1998;27(l):l-9.

 [8] Wade TJ, Pai N, Eisenberg JN, Colford JMJ. Do U.S. Environmental Pro-
    tection Agency Water Quality Guidelines for Recreational Waters Prevent
    Gastrointestinal Illness? A Systematic Review  and Meta-analysis.  Envi-
    ronmental Health Perspectives. 2003;111(8):1102-1109.

 [9] Zmirou D, Pena L, Ledrans M, Letertre A. Risks associated with the micro-
    biological quality of bodies of fresh and marine water used for recreational
    purposes: summary estimates based on published epidemiological studies.
    Arch Environ Health. 2003;58(11):703-11. 0003-9896 Journal Article Meta-
    Analysis.
                                  184

-------
[10] Yau V, Wade T, de Wilde C, Collord J. Skin-related symptoms following
    exposure to recreational water:  a systematic review and meta-analysis.
    Water Quality, Exposure and Health. 2009 May;l(2):79-103.  Available
    from: http://dx.doi.org/10.1007/sl2403-009-0012-9.

[11] von Schirnding YE, Kfir R,  Cabelli V, Franklin L, Joubert G. Morbidity
    among bathers exposed to polluted seawater. A prospective epidemiological
    study. South African Medical Journal.  1992;81(ll):543-6.

[12] Fleisher J, Kay D, Wyer M, Merrett H.  The enterovirus test in the as-
    sessment of recreational water associated gastroenteritis. Water Research.
    1996;p. 1-7.

[13] McBride GB, Salmondo CE, Bandaranayake DR,  Turner SJ, Lewis GD,
    Till DG. Health effects of marine bathing in New  Zealand. International
    Journal of Environmental Health Research. 1998;8:173-189.

[14] Haile RW, Witte JS,  Gold M, Cressey R,  McGee  C,  Millikan RC,  et al.
    The health effects of swimming in ocean  water contaminated by storm
    drain runoff.  Epidemiology.  1999;10(4):355-363.

[15] USEPA.  Method 1600:  Membrane Filter Test  for Enterococci  in Water.
    Office of Water; 1997. EPA-821-R-97-004.

[16] Haugland RA,  Siefring  SC,  Wymer LJ, Brenner KP, Dufour AP.   Com-
    parison of Enterococcus measurements in  freshwater  at two  recreational
    beaches by quantitative polymerase chain reaction and membrane filter
    culture analysis. Water  Res. 2005;39(4):559-568.

[17] Siefring S, Varma M, Atikovic E, Wymer L, Haugland RA. Improved real-
    time  PCR assays for  the detection of fecal indicator bacteria in surface
    waters with different  instrument and reagent systems. Journal of Water
    Health. 2008;6(2):225-237.

[18] Colford JM, Wade TJ, Schiff KG, Wright CC, Griffith JF, Sandhu SK, et al.
    Water quality indicators and the  risk of illness  at  beaches with nonpoint
    sources of fecal contamination. Epidemiology. 2007;18(l):27-35.

[19] Calderon R, Mood E,  Dufour A. Health effects of swimmers and nonpoint
    sources of contaminated water.  International Journal of Environmental
    Health Research. 1991;1:21-31.

[20] Kramer MH, Sorhage FE, Goldstein ST, Dalley E, Wahlquist SP, Herwaldt
    BL.  First reported outbreak in the United States of cryptosporidiosis as-
    sociated with a recreational  lake. Clin Infect Dis. 1998;26(l):27-33.

[21] Ackman D, Marks S, Mack P, Caldwell M, Root T, Birkhead G. Swimming-
    associated haemorrhagic colitis due to Escherichia coli O157:H7 infection:
    evidence of prolonged contamination of a fresh water lake. Epidemiol Infect.
                                   185

-------
[22] Soller JA, Schoen ME, Bartrand T, Ravenscroft JE, Ashbolt NJ.  Esti-
    mated human health risks from exposure to recreational waters impacted
    by human and non-human sources of faecal contamination.  Water Res.
    2010 Jun;Available from: http: //dx. doi. org/10.1016/j . watres. 2010.
    06.049.

[23] Schoen  ME,  Ashbolt  NJ.   Assessing pathogen risk  to  swimmers  at
    non-sewage  impacted recreational beaches.  Environ Sci Technol. 2010
    Apr;44(7):2286-2291.    Available  from:   http://dx.doi.org/10.1021/
    es903523q.

[24] Boehm AB,  Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA,
    et al. A sea change ahead for recreational water quality criteria.  J Wa-
    ter Health. 2009 Mar;7(l):9-20. Available  from: http://dx.doi.org/10.
    2166/wh.2009.122.

[25] Parkhurst DF, Craun  G, Soller J.   Conceptual  bases for relating illness
    risk to indicator concentrations.  In: Wymer LJ, editor. Statistical Frame-
    work for Recreational Water Quality Criteria and Monitoring. Statistics in
    Practice. Wiley; 2007. p. 19-44.

[26] Hartz A, Cuvelier M,  Nowosielski K,  Bonilla TD, Green M, Esiobu  N,
    et al. Survival potential of Escherichia coli and Enterococci in subtrop-
    ical  beach  sand:  implications  for  water  quality  managers.   J Environ
    Qual. 2008;37(3):898-905. Available from:  http://dx.doi.org/10.2134/
    jeq2007.0312.

[27] Luther K, Fujioka R.  Usefulness of monitoring tropical streams for  male-
    specific RNA coliphages. J Water Health. 2004;2(3):171-81.

[28] Fujioka RS.  Monitoring coastal marine waters for spore-forming bacteria of
    faecal and soil origin to determine point from non-point source pollution.
    Water Sci Technol. 2001;44(7): 181-188.

[29] Al-Abri SS,  Beeching NJ, Nye FJ. Traveller's diarrhoea. Lancet Infect Dis.
    2005 Jun;5(6):349-360.  Available from:  http://dx.doi.org/10.1016/
    31473-3099(05)70139-0.

[30] Payment P,  Richardson L, Siemiatycki J,  Dewar R, Edwardes M, Franco
    E. A randomized trial to evaluate the risk of gastrointestinal disease due to
    consumption of drinking water meeting current microbiological standards.
    Am J Public Health. 1991;81(6):703-8.

[31] Payment P,  Siemiatycki J, Richardson L, Gilles R, Franco E, Prevost M. A
    prospective  epidemiological study of gastrointestinal health effects due to
    the consumption of drinking water. International Journal of Environmental
    Health Research. 1997;7:5-31.
                                   186

-------
[32] Colford J, Rees J, Wade T, Khalakdina A, Hilton J, Ergas I, et al.  Partic-
    ipant blinding and gastrointestinal illness in a randomized, controlled trial
    of an in-home drinking water treatment.  Emerging Infectious  Diseases.
    2002;8(1).

[33] Colford J,  Wade T, Sandhu S, Wright C,  Lee S,  Shaw S, et al.  A ran-
    domized, controlled trial of in-home drinking water intervention to reduce
    gastrointestinal illness. Am J Epidemiol.  2005;161(5):472-82.

[34] Ferris BG.  Epidemiology Standardization Project (American Thoracic So-
    ciety). Am Rev Respir Dis. 1978;118(6 Pt 2):1-120.

[35] Herikstad H, Yang S, Van Gilder TJ, Vugia D, Hadler J, Blake P, et al. A
    population-based estimate of the burden of diarrhoeal illness in the  United
    States: FoodNet, 1996-7. Epidemiol Infect. 2002;129(1):9-17.

[36] Imhoff B, Morse D, Shiferaw B, Hawkins M, Vugia D,  Lance-Parker S,
    et al. Burden of self-reported acute diarrheal illness in FoodNet surveillance
    areas, 1998-1999. Clin Infect Dis. 2004;38 Suppl  3:8219-26.

[37] Clesceri LS,  Greenberg AE,  Eaton AD.   Standard Methods for the  Ex-
    amination of Water and Wastewater.  20th ed. American Public  Health
    Association, American Water Works Association, and Water Environment
    Federation; 1998.

[38] USEPA. Improved Enumeration Methods for the Recreational Water Qual-
    ity Indicators: Enterococci and Escherichia coli;  2000.

[39] Rinttila T, Kassinen A, Malinen E, Krogius L, Palva A. Development of an
    extensive set of 16S rDNA-targeted primers for quantification of pathogenic
    and  indigenous bacteria in faecal samples by real-time PCR.  J Appl  Mi-
    crobiol. 2004;97(6):1166-1177.

[40] Chern EC, Brenner KP, Wymer L, Haugland RA. Comparison of Fecal In-
    dicator Bacteria Densities in Marine Recreational Waters by QPCR. Water
    Quality Exposure and Health. 2009;1:203-214.

[41] Brinkman NE, Haugland RA, Wymer LJ, Byappanahalli M, Whitman  RL,
    Vesper SJ. Evaluation of a rapid, quantitative real-time PCR method for
    enumeration of pathogenic Candida cells in water. Appl Environ Microbiol.
    2003;69(3):1775-1782.

[42] Applied Biosystems. User Bulletin 2: ABI PRISM 7700 Sequence Detection
    System. Applied Biosystems Corporation; 1997.

[43] StataCorp. Stata Statistical Software: Release 10.1. College Station, Texas:
    StataCorp LP; 2008.

[44] R Development Core Team. R: A Language and Environment for Statistical
    Computing. Vienna, Austria; 2009. ISBN 3-900051-07-0.  Available from:
    http://www.R-proj ect.org.
                                   187

-------
[45] Huber P.  The behavior of maximum likelihood estimates under non-
    standard conditions. In: Proceedings of the Fifth Berkeley Symposium on
    Mathematical Statistics and Probability,  vol. I.  Berkeley, CA: University
    of California  Press; 1967. p. 221-233.

[46] White H. A heteroskedasticity-consistent  covariance matrix estimator and
    a direct test for heteroskedasticity. Econometrica. 1980;48:817-830.

[47] Rogers WH.  Regression standard errors in clustered samples. Stata Tech-
    nical Bulletin. 1993; 13:19-23.

[48] Williams RL. A note on robust variance  estimation for cluster-correlated
    data.  Biometrics. 2000;56(2):645-6.

[49] Little R, Rubin D. Statistical Analysis with Missing Data.  2nd ed. New
    York: Wiley; 2002.

[50] Greenland S,  Rothman KJ.  Modern epidemiology.  Philadelphia, PA ::
    Lippincott-Raven; 1998.

[51] USEPA. Highlights of the Child-Specific Exposure Factors Handbook (Fi-
    nal Report).   U.S.  Environmental Protection Agency,  Washington B.C.;
    2009.  EPA/600/R-08/135.

[52] USEPA. Ambient Water Quality for Bacteria-1986. Office of Water Regu-
    lations and Standards; 1986. EPA-440/5-84-002.

[53] South Carolina Department of Health and Environmental Control. Water-
    shed Water Quality Assessment: Pee  Dee River  Basin.  Bureau of Water,
    Columbia, SC. Watershed Assessment; 2007. Technical  Report No.005-07.

[54] Kay D,  Fleisher JM, Salmon RL, Jones F, Wyer MD,  Godfree AF, et al.
    Predicting likelihood of gastroenteritis from sea bathing: results from ran-
    domised exposure. Lancet. 1994;344(8927):905-9. Have.

[55] Roy SL, Scallan E, Beach MJ. The rate of acute gastrointestinal illness in
    developed countries. J Water Health.  2006;4 Suppl 2:31-69.

[56] Feiveson AH.   Power  by  simulation.   Stata  Journal.  2002;2(2):107-
    124(18).   Available  from:  http://www.stata-journal.com/article.
    html?article=st0010.

[57] Wade T,  Sams E, Brenner  K,  Haugland R,  Chern E, Beach M, et al.
    Rapidly measured indicators of recreational  water quality and swimming-
    associated illness at marine beaches:  a prospective cohort study. Envi-
    ronmental Health. 2010;9(1):66.  Available from:  http://www.ehjournal.
    net/content/9/1/66.

[58] Boehm  AB.  Enterococci concentrations  in  diverse coastal environments
    exhibit extreme variability. Environ Sci Technol.  2007;41(24):8227-32.
                                   188

-------
[59] Boehm AB, Grant SB, Kim JH, Mowbray SL, McGee CD, Clark CD, et al.
    Decadal and shorter period variability of surf zone water quality at Hunt-
    ington Beach, California. Environ Sci Technol. 2002;36(18):3885-92.

[60] Cabelli VJ, Dufour AP, Levin MA, McCabe LJ, Haberman PW. Relation-
    ship of microbial indicators to health effects at marine bathing beaches.
    Am J Public Health. 1979;69(7):690-696.

[61] Cabelli VJ, Dufour AP, McCabe  LJ, Levin MA.  Swimming-associated
    gastroenteritis and  water quality.   American  Journal of Epidemiology.
    1982;115(4):606-616.

[62] Fleisher JM, Jones F, Kay D, Stanwell-Smith R, Wyer M, Morano  R. Wa-
    ter and non-water-related risk factors  for gastroenteritis among  bathers
    exposed to sewage-contaminated marine waters. International Journal of
    Epidemiology.  1993;22(4):698-708.

[63] Pond K.  Water Recreation and Disease Plausibility of Associated Infec-
    tions:  Acute Effects, Sequelae and Mortality.  London:  IWA Publishing;
    2005.

[64] Fleisher JM, Fleming LE,  Solo-Gabriele HM,  Kish JK, Sinigalliano CD,
    Piano L,  et al.  The BEACHES Study: health effects and exposures from
    non-point source microbial contaminants in subtropical recreational ma-
    rine waters. International Journal of Epidemiology. 2010;Advance Access
    published June 3, 2010.

[65] Sinigalliano CD, Fleisher  JM,  Gidley ML,  Solo-Gabriele  HM,  Shi-
    bata  T,  Piano  LRW,  et  al.     Traditional  and  molecular   analy-
    ses for fecal  indicator  bacteria in  non-point  source  subtropical recre-
    ational  marine  waters.    Water Research.  2010;44(13):3763 -  3772.
    Available   from:    http://www.sciencedirect.com/science/article/
    B6V73-4YYRMJD-5/2/6c643faOd4bl32eOe2b8deed6d8c8d03.

[66] Fleisher JM, Kay D, Salmon RL, Jones F,  Wyer  MD, Godfree AF.  Ma-
    rine waters contaminated with domestic sewage: nonenteric illnesses asso-
    ciated with bather exposure in the United Kingdom. Am J Public  Health.
    1996;86(9):1228-34.

[67] Elmir SM,  Wright  ME, Abdelzaher  A, Solo-Gabriele HM, Fleming LE,
    Miller G, et al. Quantitative evaluation of bacteria released by bathers in
    a marine water.  Water Res. 2007;41(1):3-10.

[68] Graczyk  TK, Sunderland  D, Tamang L, Lucy FE, Breysse PN.  Bather
    density and levels  of Cryptosporidium,  Giardia,  and  pathogenic mi-
    crosporidian spores in recreational bathing water.   Parasitol Res. 2007
    Nov; 101 (6): 1729-1731.   Available  from:  http://dx.doi.org/10.1007/
    S00436-007-0734-1.
                                   189

-------
Graczyk TK, Sunderland D, Tamang L, Shields  TM, Lucy FE, Breysse
PN.  Quantitative evaluation of the impact of bather density on levels of
human-virulent microsporidian spores in recreational water. Appl Environ
Microbiol. 2007 Jul;73(13):4095-4099.  Available  from:  http://dx.doi.
org/10.1128/AEM.00365-07.
                              190

-------
Appendix A




Questionnaires
              191

-------
                                BEACH INTERVIEW
                                                     OMB Control No.: 2080-0068

                                                     Expiration Date:  xx/xx/xxxx
Site ID
Friday	Saturday	  Sunday	Monday
Date:	-	-	
Time:
1.      Have you been interviewed by the National Beaches Survey in the last 28 days?

2.      Would you be willing to participate in a study on illnesses associated with recreation at
       the beach?

         Yes (give brochure with consent form, inform about 2 follow-up calls)
         No (Terminate Interview).

2a.     Our survey  is primarily for households of one or more persons that live together at the
       same address; Do you all live at the same address?

3.      How many members in your party are at the beach today including yourself?

4.      What time did you and your household arrive at the beach today?

5.      We are interested in asking about the health of your household during the few weeks
       following your beach visit. Could you please give me your telephone number so we can
       get in touch with you in  10-12 days from now?

5a.     If "NO" Is it fro one of the following reasons? Too busy, no longer interested, will not be
       available, specify, other reason?

5b.     10-12  days from now which phone number(s) should we call?

5c.     Is this your home, vacation, or cell phone number?

5d.     Additional phone numbers?

6.      What are the best times to reach you during week days?

6a.     Can I please have your mailing  address so that we can send you your $25  Thank you
       check? We will destroy your identifying information after we mail the check.

-------
7.     Please tell me the first name of the members of your household at the beach today, their
      birth dates, gender race ethnicity, and whether they are in diapers.

8.     Will (you/all these people with you at the beach today) be living (with you) at the same
      address (es) during the next two weeks?

9.     Have any of these household members at the beach today been ill in the past 3 days with:
        Diarrhea or loose bowels
        Urinary tract infection or burning sensation
        Throwing-up or vomiting
        Sore throat or cough
        Earache, ear infection or runny ears
        Eye infection
        Rash or itchy skin
        Sunburn

10    Are there any household members NOT present at the beach today?

lOa.   Have any household members NOT present at the beach today been ill in the past 3 days
      with:
        Diarrhea or loose bowels
        Urinary tract infection or burning sensation
        Throwing-up or vomiting
        Sore throat or cough
        Earache, ear infection or runny ears
        Eye infection
        Rash or itchy skin
        Sunburn

11.    Do you or any household members at the beach today, not including anyone who stayed
      at home, suffer from any of the following chronic long-term conditions:
        Gastrointestinal problems such as Crohn's disease or irritable bowel syndrome
        Chronic respiratory diseases such as asthma or emphysema
        Allergies, other than drug allergies
        Skin problems such as psoriasis or eczema

12.    How many  times do you  usually  come to this beach each summer (Memorial Day to
      Labor Day)?

13.    How many miles did you travel to the beach today?

14.    During the past two weeks,  did you (anyone in your household at the beach today) go
      bathing or swimming anywhere - at this or some other beach, pool or lake?

14a.   Did you go bathing or swimming anywhere in the past one week (Monday  through
      Friday) at this or some other beach, pool or lake?

14b.   Did you actually get your head or face wet?

-------
14c.   During the past 2 weeks, did you get a sunburn that lasted more that 12 hours?

PART B- Exit Beach Interview

15.     Were you the person that we interview on the beach or earlier today?

16a.   Did you or anyone in your household wade, swim, or play in the water today?

16al.   Did you immerse your body, not necessarily you head, in the water today?

16a2.   Did you put your face in the water or submerge head in the water today?

16a3.   Did you get water in the mouth today?

16a4.   Did you swallow the water?

16b.   Were you in the water at the following times today? (time charts given}

16bl.   If "YES" what part of the beach did you swim in? (include all beach areas}

16b2.   If "YES" what part of the beach did you swim in most of the time?

16c.   What total time  did you stay in the water?  We are only interested in  time actually in the
       water, not the total time at the beach?

16d.   Did you engage  in any of the following water-related activities while at the beach today?
       (Dropdown list of water activities}

17.     What would you estimate your total time in  direct  sunlight was?  This does not include
       being indoors or under umbrellas, etc.?

18.     Did you engage  in any of the following activities while at the beach today?
         a.     Collecting  sea shells, rocks, feathers, etc?
         b.     Digging in sand or building sand castles?
         c.     Had their body buried in the sand?

18cl.   Did person get sand in their mouth
         After digging in sand, or building sand castles...did person eat or drink anywhere (not
         necessarily at the beach)?
         After digging in sand, or building sand castles...did person wash their hands before
         eating? Washing of hands may include the use of personal waterfree hand sanitizer?

18clb.   Was the sand the person dug in or played with dry or wet?

18d.   Did you engage  in any of the following activities while at the beach today?
       Playing with algae or seaweed

-------
18dl.   Did you get any seaweed in their mouth?

18dla. After playing with algae or seaweed...did you eat or drink anywhere (not necessarily at
       the beach)?

18dlb. After playing with algae or seaweed...  did person  wash their hands  before  eating?
       Washing of hands may include the use of personal water free hand sanitizer?

19.    Did you cut yourself today or have an open cut when you came to beach today?

20.    Did you wear sunscreen/sunblock today?

21.    What was the SPF rating of the sunscreen/sunblock you used most often today?

2la.    When you used sunscreen/sunblock today, how did you apply it? Only to  certain areas of
       my body?  All exposed skin?

22.    Did you reapply at least  once today?

23.    Did you wear a hat today?

23a.    Did the hat have a wide brim or another way to shade face, ears, and back of the neck
       from the sun?

23al.   Did you use protective equipment such as a canopy, umbrella or other type of sunshade
       today?

23b.    Did you wear protective clothing, such as a long-sleeved shirt or cover-up?

24.    During the summer, if you go out in the sun repeatedly without sunscreen or protective
       clothing, which one of these things most usually happens to your skin?
         A dark tan
         Some tanning
         No tan, maybe some freckles
         Repeated sunburns
         Other (specify)
         Never go out in the sun

25.    Did you wear insect repellant today?

26.    Did your or any member of your household consume food while at the beach today?

26a.    Was the food brought from home?

26b.    Was the food purchased  from vending machines or a vendor at the beach?

26c.    Was the food purchased  from a vendor outside the beach?

-------
27.    Did your or any member of your household consume drinks while at the beach today?

27a.   Were the drinks brought from home?
27b.   Were the drinks purchased from vending machines or a vendor at the beach?

27c.   Were the drinks purchased from a vendor outside the beach?

28.    In the past 48 hours has anyone who is at the beach today done the following...

      a.     Have you come in contact with any unknown animals?
      b.     Come in contact with someone who has complained of diarrhea, vomiting, or
             stomach illness?
      c.     Consumed raw shellfish?
      d.     Consumed rare/raw meat?
      e.     Consumed runny or raw eggs?

-------
                      Telephone Interview - follow-up (10-12 days)

Is this person (primary respondent from beach interview)?
   Yes (continue)
   No (reschedule or continue)
       Were you at the beach on (give date) with (primary respondent)?
             Yes - Continue

I'm going to ask questions about any swimming you've done and illnesses you've experienced in
the last week for the following people:

Al.    May I have your first name please?

During your beach visit where you enrolled in this study on	
A2.          Did you wear ear plugs while in the water? Ask for all household members at the
   beach.

A3.          Did you wear nose plugs while in the water?  Ask for all household members at
   the beach.

A4.          Did you wear eye goggles while in the water? Ask for all household members at
   the beach.

A5.          During the beach interview, did you have contact with an animal? Ask for all
      household members at the beach.

A6.          Between your beach visit on	date and today were you menstruating or
      pregnant? Ask for all household members at the beach.

We are now going to switch and ask you questions about activities that have occurred since the
Beach Interview.
                                        Page 6

-------
Bl.   Have you, or any of the people I just mentioned, gone bathing or swimming anywhere
      since we talked to you at the beach interview on	? Please include any bathing or
      swimming such as at a beach, waterpark, public pool, private pool, or wading pool.

B2.   Who was it that went bathing or swimming?  Ask for all household members at the
   beach.

B3a.  Did you go bathing or swimming at the beach (where the interview was taken) since the
      beach interview on this date (BEACH INTERVIEW DATE).

B3b.  Did you go  bathing or swimming at  any other beach  since the beach interview on this
      date (BEACH INTERVIEW DATE).

B3c.  Was this beach at a:
      Lake
      River
      Ocean
      Other, specify

B3d.  Did you go bathing or swimming at a waterpark?

B3e.  Did you go bathing or swimming at a public pool?

B3f.  Did you go bathing or swimming at a private pool?

B3g.  Did you go bathing or swimming in a wading pool?

B3h.  Did you go bathing or swimming any other place?

B3i.  Swim location of any other place?
                                       Page 7

-------
B4.         Did you actually get your face wet while bathing or swimming?  Ask for all
      household members at the beach.

B5.         On which days did you go bathing or swimming? Ask for all household members
      at the beach.

B6.         Have you or anyone else had a  stomachache or abdominal cramping since the
      interview at (STUDY BEACH)  ON (BEACH INTERVIEW DATE)?   Ask for all
      household members at the beach.

B6a.  Who had a stomachache  or  abdominal  cramping  since the interview at  (STUDY
      BEACH) ON (BEACH INTERVIEW DATE)?  Ask for all household members at the
      beach.

B7.         Have you or anyone else  had diarrhea or loose bowels since the interview at
      (STUDY BEACH) ON (BEACH INTERVIEW DATE)?   Ask for all household
      members at the beach.

B7a.  Who had diarrhea or loose bowels since the interview at (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)? Ask for all household members at the beach.
B8.         Have you or anyone else had nausea since the interview at (STUDY BEACH}
      ON (BEACH INTERVIEW DATE)? Ask for all household members at the beach.

B8a.  Who had nausea since the interview at I STUDY BEACH) ON (BEACH INTERVIEW
      DATE)? Ask for all household members at the beach.

B9.         Have you or anyone else  had throwing-up or vomiting since the interview at
      (STUDY BEACH) ON (BEACH INTERVIEW DATE)?   Ask for all household
      members at the beach.

B9a.  Who had throwing-up or vomiting since the interview at (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)? Ask for all household members at the beach.
                                    PageS

-------
BIO.  Have you or anyone else had urinary tract infection or burning sensation when urinating
      since the interview at I STUDY BEACH) ON (BEACH INTERVIEW DATE)? Ask for
      all household members at the beach.

BlOa. Who had urinary tract infection or burning sensation when urinating since the interview
      at (STUDY BEACH) ON (BEACH INTERVIEW DATE)?  Ask for all household
      members at the beach.

Bll.  Have you or anyone else had fever  since  the interview at  (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)?  Ask for all household members at the beach.

Blla. Who had fever  since the interview at  (STUDY BEACH) ON (BEACH INTERVIEW
      DATE)? Ask for all household members at the beach.

B12.  Have you or anyone else had headache lasting more than a few hours since the interview
      at (STUDY BEACH) ON (BEACH INTERVIEW DATE)?  Ask for all household
      members at the beach.

B12a. Who had headache lasting more than a few  hours  since  the interview at (STUDY
      BEACH) ON (BEACH INTERVIEW DATE)? Ask for all household members at the
      beach.

B13.  Have you or anyone else had sore throat since the interview at (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)?  Ask for all household members at the beach.

B13a. Who had  sore  throat  since the  interview at  (STUDY BEACH)  ON  (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

B14.  Have you or anyone else had a bad cough since the interview at (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)?  Ask for all household members at the beach.
                                     Page 9

-------
B14a. Who had a  bad  cough since the interview at  (STUDY BEACH)  ON  (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

B15.  Have you or anyone else had a cold since the interview at (STUDY BEACH)  ON
      (BEACH INTERVIEW DATE)? Ask for all household members at the beach.

B15a. Who had a cold since the interview at (STUDY BEACH) ON (BEACH INTERVIEW
      DATE)? Ask for all household members at the beach.

B16.  Have you or anyone else had a runny or stuffy nose since the interview  at (STUDY
      BEACH) ON (BEACH INTERVIEW DATE)? Ask for all household members at the
      beach.

B16a. Who had a runny or stuffy nose since the interview at (STUDY BEACH) ON (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

B17.  Have you or anyone else had an earache, ear infection, or runny ears since the interview
      at  (STUDY BEACH) ON (BEACH INTERVIEW DATE)?  Ask for all household
      members at the beach.

B17a. Who had an earache, ear infection, or  runny ears since the interview  at (STUDY
      BEACH) ON (BEACH INTERVIEW DATE)? Ask for all household members at the
      beach.

B18.  Have you or anyone else had watery eyes since the interview at (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)? Ask for all household members at the beach.

B18a. Who had watery  eyes since the  interview  at  (STUDY BEACH)  ON  (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

B19.  Have you or anyone else had an eye infection  since the interview at (STUDY BEACH)
      ON (BEACH INTERVIEW DATE)? Ask for all household members at the  beach.
                                    Page 10

-------
B19a. Who had an eye infection since the interview at (STUDY BEACH) ON (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

B20.  Have you or anyone else had an infected cut since the interview at (STUDY BEACH}
      ON (BEACH INTERVIEW DATE)? Ask for all household members at the beach.

B20a. Who had an infected cut since  the interview at (STUDY BEACH) ON (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.
B21.  Have you or anyone else had a rash or itchy skin since  the interview at (STUDY
      BEACH) ON (BEACH INTERVIEW DATE)? Ask for all household members at the
      beach.

B21a. Who had a rash  or itchy skin since the interview  at (STUDY BEACH) ON (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

B22.  Have you or anyone else had a sunburn since the  interview at (STUDY BEACH) ON
      (BEACH INTERVIEW DATE)?  Ask for all household members at the beach.

B22a. Who had  a sunburn  since  the interview at   (STUDY BEACH)  ON (BEACH
      INTERVIEW DATE)? Ask for all household members at the beach.

We will now ask about some activities people  may have done since the day of the beach
interview on the (BEACH INTERVIEW DATE)

B23a. Since the day of the beach interview, have you or anyone else come in contact with any
      animals? Ask for all household members at the beach.

B23b. Who came into contact with animals since (BEACH INTERVIEW DATE)? Ask for all
      household members at the beach.

B23c. Was this animal or any of these animals unfamiliar to you?

                                    Page 11

-------
B23d. What kind of animals were they?

B24a. Since the day of the beach interview has anyone come into contact with someone who
      has complained of diarrhea, vomiting, or stomach illness?

B24b. Who had contact with someone complaining of diarrhea, vomiting, or stomach illness
      since (BEACH INTERVIEW DATE)?

B25a. Since the day of the beach interview has anyone eaten raw shell fish,  such as oysters,
      clams, mussels, crabs?

B25b. Who has eaten  raw shell  fish, such as oysters, clams, mussels, crabs  since (BEACH
      INTERVIEW DATE)?

B26a. Since the day of the beach interview has anyone rare or raw meat?

B26b. Who has eaten rare or raw meat since (BEACH INTERVIEW DATE)?

B27a. Since the day of the beach interview has anyone eaten raw or runny eggs?

B27b. Who has eaten raw or runny eggs since (BEACH INTERVIEW DATE)?

SECTION C

This section is for all persons that experience symptoms.

Cl.   On what day did your stomachache or abdominal cramping start?

Cla.  Do you still have a stomachache or abdominal cramping?
                                      Page 12

-------
Clb.  (For persons that still have symptom) For how many days did you have a stomachache or
      abdominal cramping?

C2.   On what day did your diarrhea or loose bowels start?

C2a.  Do you still have a diarrhea or loose bowels cramping?

C2b.  For how many days did you have a diarrhea or loose bowels cramping?

C2c.  What was the maximum number of bouts or episodes of diarrhea experienced in a 24-
      hour period?  Ask for each person with symptom.

C3.   On what day did your nausea start?

C3a.  Do you still have nausea?

C3b.  For how many days did you have nausea?

C4.   On what day did your throwing-up or vomiting start?

C4a.  Do you still have throwing-up or vomiting?

C4b.  For how many days did you have throwing-up or vomiting?

C4c.  What was the maximum number of bouts or episodes of throwing-up or vomiting
      experienced in a 24-hour period? Ask for each person with symptom.

C5.   On what day did your urinary  tract infection or burning sensation start?

C5a.  Do you still have a urinary tract infection or burning sensation?

C5b.  For how many days did you have urinary tract infection or burning sensation?

                                      Page 13

-------
C6.   On what day did your fever start?

C6a.  Do you still have a fever?

C6b.  For how many days did you have a fever?

C6c.  Was your temperature taken using a thermometer?

C6d.  What is the highest temperature that you had since your beach interview on (BEACH
      INTERVIEW DATE)?

C7.   On what day did your headache start?

C7a.  Do you still have a headache?

C7b.  For how many days did you have headache?

C8.   On what day did your sore throat start?

C8a.  Do you still have a sore throat?

C8b.  (For persons that still have symptom) For how many days did you have sore throat?

C8c.  (For persons that still have symptom) Was this sore throat related to allergies?

C9.   On what day did your bad cough start?

C9a.  Do you still have a bad cough?

C9b.  For how many days did you have a bad cough?


                                       Page 14

-------
C9c.   Was this bad cough related to allergies?







CIO.   On what day did your cold start?







ClOa.  Do you still have a cold?






ClOb.  For how many days did you have a cold?







ClOc.  Was this cold related to allergies?






C11.   On what day did your runny or stuffy nose start?







Cl la.  Do you still have a runny or stuffy nose?






Cl Ib.  For how many days did you have a runny or stuffy nose?







Cl Ic.  Was this runny or stuffy nose related to allergies?






C12.   On what day did your earache, ear infection or runny ears start?







C12a.  Do you still have an earache, ear infection or runny ears?






C12b.  For how many days did you have an earache, ear infection or runny ears?







C12c.  Was this earache, ear infection or runny ears related to allergies?







C13.   On what day did your watery eyes start?






C13a.  Do you still have watery eyes?







C13b.  For how many days did you have watery eyes?









                                        Page 15

-------
C13c.  Was this watery eyes related to allergies?

C14.   On what day did your eye infection start?

C14a.  Do you still have eye infection?

C14b.  For how many days did you have eye infection?

CIS.   On what day did your cut first get infected?

C15a.  Do you still have an infected cut?

C15b.  For how many days did you have an infected cut?

C15c.  Where were you cut?  Mark all that apply

C16.   On what day did your rash, itchy skin, or skin infection start?

C16a.  Do you still have a rash, itchy skin, or skin infection?

C16b.  For how many days did you have a rash, itchy skin, or skin infection?

C16c.  Where did you have a rash, itchy skin, or skin infection?  Mark all that apply

C17.   On which parts of the body were you sunburned? Mark all that apply
       Drop down list
SECTION D
Ask only once for each person reporting symptoms
                                        Page 16

-------
Dl.    When your condition began, were you working for pay either inside or outside the home?
       Please include jobs for which you were self-employed.

D2.    During your illness, did you miss any time from work, for example because you called in
       sick or took time off to see a doctor?

D3.    How many days?

D4.    Did this illness prevent you from performing daily activities such as school, recreation, or
       vacation activities, or work around the home?
D5.    How many days?

D6.    Did this illness cause other household members to lose time at work?

D7.    How many days?

D8a.   Did you consult a healthcare provider over the phone about this illness/condition?

D8b.   Did you visit a healthcare provider?

D8c.   How many times?

D8d.   What illness did the healthcare provider say you had?

D8e.   Did you visit an emergency room?

D8f         How many times?

D8g.   Were you admitted to a hospital?

D8h.   How many days were you hospitalized?
                                       Page 17

-------
D8i.         Were you given intravenous fluids?

D9a.  Did you receive a prescription for an antibiotic or other drug for this illness/condition?

D9b.  About how much of your own or your household's money was spent altogether for these
      prescription medicines? Amount to nearest dollar.

DIOa. Did you use any over-the-counter medications, including things like  special drinks, only
      because of this illness/condition?

DIOb. About how much of your own or your household's money was spent altogether for these
      over-the-counter medications? Amount to nearest dollar.

SECTION E

El.    Before today, were you aware that people could become ill by swimming at the beach?
E2.    After today, will you change the way you use the water at the beach?


SECTION O

These are questions from beach interview to ensure data collection for important exposures. Ask
for all households.

Ql.   Did you or anyone in your household wade, swim, or play in the  water on (BEACH
      INTERVIEW DATE)?

Qla.l. Did you  immerse  your body, not  necessarily  your head in  the  water (BEACH
      INTERVIEW DATE)?

Qla.2. Did you put your  face in the water or submerge head  in the water  on (BEACH
      INTERVIEW DATE)?

                                      Page 18

-------
Qla.3. Did you get water in your mouth on (BEACH INTERVIEW DATE)?

Qla.4. Did you gag or cough after getting water in  your mouth on (BEACH INTERVIEW
      DATE)?

Qla.5. Did you swallow the water on (BEACH INTERVIEW DATE)?
                                   Page 19

-------
Appendix B

Quality Assurance Project
Plan: Survey Data
Collection
             211

-------
                           EPA Contract No. EPD-09-040
                               Work Assignment 0-01
                                    WESTAT
                                  WORK PLAN
                                       For

The National Epidemiological and Environmental Assessment of Recreational Water Study
                               for Beaches Program

                           Boqueron Beach, Puerto Rico
                                       And
                           Surfside Beach, South Carolina
                    U.S. Environmental Protection Agency (EPA)
               EPA Contracting Officer Representative: Elizabeth Sams
                                   Submitted by:
            Westat Work Assignment and Project Director: Karen Delia Torre
                                   April 27, 2009

-------
BACKGROUND

In order to meet some of the requirements of the Clean Water Action Plan, the Beach Action Plan
and the Beach Act of 2000, this beach study was initiated in 2003 to assist the Office of Water in
formulating new health and risk guidelines for recreational water.

This study is being conducted jointly by the National Exposure Research Laboratory,
Microbiological and Chemical Exposure Assessment Research Division (NERL/MCEARD), the
National Health and Environmental Research Laboratory (NHERL) and the Centers for Disease
Control and Prevention (CDC).

This information is being collected as part of a research program consistent with the Sec. 3(a)(v)(l)
of the Beaches Environmental Assessment and Coastal Health Act of 2000 and the strategic plan for
EPA's  Office of Research and Development  (ORD) and  the Office of Water entitled "Action Plan
for Beaches and Recreational Water." The Beaches Act and ORD's strategic plan has identified
research on effects of microbial pathogens in recreational waters as a high-priority research area with
particular emphasis on developing new water quality indicator guidelines for recreational waters.
This data collection  is for a series of epidemiological studies to evaluate exposure to and effects of
microbial pathogens in marine and fresh recreational waters as part of the EPA's research program
on exposure and health effects of microbial pathogens in recreational waters. The information
collected by this study program will be used to estimate the relationship between water quality
indicators  and health effects. The questionnaire health data will be compared with routinely
collected water quality measurements. The analysis will focus on determining whether any water
quality parameters are associated with increased prevalence of swimming-related health effects.

Study  Beach Site

The study period from May 15, 2009 through August 2, 2009 shall take place at Boqueron, Puerto
Rico. The study period from June 6, 2009 through September 7, 2009 shall take place at Surfside
Beach, South Carolina.  This work assignment implements field procedures for the data collection
(water  quality and human health)  at a beach study site and the follow-up telephone interviews.
Westat will travel to this site.
WORK PLAN

Westat is submitting this Work Plan detailing procedures by which support will be provided to
implement a study for the NEEAR (National Epidemiological Environmental Assessment of
Recreational) Water Study. Support will include the collection of epidemiologic data, the collection
and analysis of water and sand sample extracts, and collection of ancillary data.

TASK1
Computer Assisted Interviews:
Westat will load existing electronic beach and telephone interviews modified under EPA Contract
EP-D-04-064, Work Assignment 2-04 onto CAPI (computer-assisted personal interviews) and
CATI (computer-assisted telephone interviews) devices needed to accomplish a complete sampling
of beach goers at the designated beach area. The appropriate number of devices shall be based on
the number of expected household interviews done on individual days at the designated beach area.

-------
Westat will use Blaise® software and appropriate equipment to load the electronic questionnaires
onto tablet handheld computers and telephone interviewing computers. Westat will provide
interviewers to conduct the CAPI and CATI questionnaires by direct data entry utilizing the Blaise®
software program.  These questionnaires shall be conducted in both English and Spanish.

Implementation of Computer-Assisted Interviewer Training:
Westat will implement training programs modified in Work Assignment 2-04 for beach and
telephone interviewers. All interviewers shall undergo training using training manuals modified in
Work Assignment 2-04 as part of the training program and undergo Human Subjects Ethics
Training in accordance to the required ethics training required by the UNC biomedical Institutional
Review Board. Westat will provide documentation of training for all interviewers and produce a
report including contractor comments. For Boqueron Beach, the training programs shall be
completed by Friday, May 15,  2009 for beach interviewers and Sunday, May 24, 2009 for telephone
interviewers.  For Surfside Beach, the training programs shall be completed by Friday, June 5, 2009
for interviewers and Sunday, June 14, 2009 for telephone interviewers. EPA may be present at any
or all of the interviewing training programs and will have access to CAPI/CATI electronic devices
throughout training sessions.

Paper questionnaires shall be used  only in the event that there is a technical failure that prohibits use
of electronic CAPI or CATI devices. This study is to be almost paperless per Office of
Management and Budget's initiative to use technology and decrease the amount of paper consumed.

TASK 2
Data Collection:

Beach Interviewing:
The beach interview is to be conducted in two  parts with the  first part, Part A. containing history,
demographic information of household members and contact information for follow-up. Westat
will collect Geographic Positioning System (GPS) coordinates and transect information (See Figure
1) at the family location on the beach, concurrent with collection of Part A of questionnaire data.
Westat will ALSO COLLECT GPS COORDINATES FOR FAMILY LOCATION ON THE
BEACH FOR NONPARTICIPATING FAMILIES. The second part, PartB  (exposure date), shall
be conducted at the study station(s) located in or near the beach entrance/exit points designated by
the WACOR or COR. This data shall be included in weekly production reports and a
comprehensive database described under Task 3.

Westat will provide trained beach interviewers  (both English and Spanish bilingual) to administer
questionnaires to household units (families or individuals) beginning Friday, May 15, 2009 through
Sunday, August 2, 2009 at the  Puerto Rico beach site. Westat will provide trained interviewers for
the Surfside Beach site to administer questionnaires to household units. Spanish Bilingual
interviewers shall be scheduled each data collection day and the number of Spanish Bilingual
interviewers shall be proportionate to the mix of Spanish-speaking households that are expected to
be enrolled in the study. The questionnaires  have been reviewed by an Institutional Review Board
(IRB), approved by EPA's Human Ethics Official and have received Office of Management and
Budget (OMB) clearance under the Paper Reduction Act.

Westat will complete beach interviews for twelve (12) designated weekends and three (3) weekday
holiday from Saturday, May 15, 2009 through Sunday, August 2, 2009 at the beach site in Boqueron,

-------
Puerto Rico.  Data collection weekdays include Memorial Day and two additional Puerto Rican
holidays (July 20 and 27).

In Puerto Rico, Westat will then end beach data collection at designated beach areas Sunday, August
2, 2009 or upon technical direction from the WACOR or COR.  Westat will work all designated
non-holiday and holiday weekends and holiday weekdays except in the case of inclement weather
conditions, beach closure or technical direction by the WACOR or COR.  The status of inclement
weather conditions will be determined by the WACOR or COR.

In South Carolina, Westat will complete beach interviews for thirteen (13)  designated weekends and
two weekday  holidays  from Saturday, June 6, 2009 through Sunday, September 6. Weekday holidays
are Friday, July 3 and Monday, September 7, 2009.

Westat will set up study stations at appropriate exits at beach areas to be determined by the
WACOR or COR. Westat will provide required furniture and computer equipment to complete this
work assignment. Westat will also provide temporary "on beach" stations  for beach interviewers.
These beach stations shall display the USEPA seal and comply with park regulations concerning
location and safety.

For each of the designated data collection days at the beach site, a sampling of households will be
undertaken between 11:00 AM and 5:00 PM local time. Westat will terminate Part A Interview
collection at 5:00 PM and terminate Part B Interview collection at 6:30 PM. These  times will be
flexible depending on  beachgoer attendance at various times of day.  Westat will set up a system of
counting refusals and those who do not complete the interview processes.  Westat will require
interviewers to obtain  verbal consent from participants by distributing and discussing the consent
pamphlet.

Telephone Interviewing:
CATI telephone interviewer(s) shall implement trial interviews with at least three (3) members of the
NEEAR Water Study  project team designated by the WACOR or COR prior to Monday, June 1,
2009. This exercise allows the US EPA NEEAR Water Study project team to verify that the
telephone interviews are being properly administered to study participants.

Westat will complete telephone surveys  during a designated window of 10-12 days following the
beach interviews. Telephone interviewers shall be available beginning on Monday,  May 25, 2009
through Saturday, September 19, 2009 for beach data collection days May 15, 2009  through
September 7, 2009. The telephone surveys will collect follow-up information from all households
interviewed at the beach. This data shall be included in weekly production reports and
comprehensive database described under Task 3.

Westat will train telephone interviewers  using all data elements to be entered directly into a database
utilizing CATI instruments during the course of the telephone interview. Spanish Bilingual
interviewers shall be scheduled each data collection day and the number of Spanish Bilingual
interviewers shall be proportionate to the mix of Spanish-speaking households that enrolled in the
study and are expected to complete the telephone survey. The questionnaires have  been reviewed
by an Institutional Review Board (1KB), approved by EPA's Human Ethics Official and have
received Office of Management and Budget (OMB) clearance under the Paper Reduction Act.

-------
Field Implementation Plan:
Westat will utilize the 2007 Field Implementation Plan (FIP) describing data collection protocols for
the beach and the telephone interviews and the collection of the environmental data including
sources completed in Work Assignment 2-04 under Contract EP-D-04-064.

Electronic Data Matching and Back-up Files:
Westat will utilize the electronic method for data matching of Part A and Part B Interviews
described in the 2007 FIP (This also included transfer of Part A data collection information for
integration in Part B questionnaires). This electronic  data matching is paperless and allows the
USEPA Field Study Monitors to ascertain whether or not households are completing Part B
Interviews that match Part A Interviews. It also allows the interviewers to see previous information
(such as, but not limited to, participant name, household member names, date of interview, and time
of arrival) collected from the participants in the Part A Interview so they will be able to coherently
collect information for Part B Interviews.

As described in the 2007 FIP, Westat will include a back-up plan for this electronic method that
duplicates data as it is collected and each individual participant record shall be stored in two separate
physical locations.  These two back-ups shall be designated as collection records only and shall not
be utilized for access. A separate log of data shall be used to complete data linkage of Part A and
Part B records.

Information Distribution:
Westat will provide electronic and written questionnaires, consent  forms, and flyers modified in
Work Assignment 2-04. Westat will provide these informational materials to potential participants
in both English and Spanish.

Incentive Distribution:
Westat will provide nonmonetary incentives to households upon completion of Beach Interview
Part A and Part B. Incentives shall be beach related, include the USEPA seal, and be distributed one
(1) per household. The USEPA seal will be provided by the WACOR or COR.

Westat will distribute $25 incentives to each household upon completion of their participation
(completion of both  beach interviews and telephone interview). Westat will not distribute more
than one check per household. Westat will distribute letters of participation status to all households
enrolled in either the beach interview or telephone interview. This letter shall indicate completion of
the study or termination of participation status.

Environmental Information:
Westat will collect the following environmental information:  air and water temperature, wind
direction, water current direction, UV radiation cloud cover and other information to be identified
by the WACOR or COR. This information shall be collected from existing sources as well as field
instrument collection.  Westat will document their sources for this  information. Westat will submit
a report evaluating the  field collection and environmental data procedures.  Existing sources of
environmental information must have approval of the WACOR or COR.

Project Final Report:

-------
Westat will provide a final work assignment report that includes overview of field procedures, field
implementation analysis, training and recommendations for improvement, and assessment of
technology.

TASK 3
Data Management:
Westat will implement a data management plan modified in Work Assignment of 2-04 t include, but
not be limited to, the following; 1) processing of beach and telephone interview data in the field
(including back-up), 2) daily count of complete and incomplete interviews, and 3) downloading of
beach data for field review by USEPA WACOR or COR.
Production Reports:
Westat will provide weekly production reports that summarize the daily completion rates to the
USEPA by the close of business on Wednesday after a data collection weekend. For weekends that
include a holiday the production report shall be due on the Friday following data collection
weekend.  On holidays not included in the weekend, the production reports are  due by the  close of
business on Wednesday of the following week. Westat will submit the production reports (beach and
phone) for the weekends for Friday, May 15, 2009 through Saturday, September 19, 2009. These
reports shall be in Excel 2000 format.

Comprehensive Database:
Westat will develop a comprehensive database that includes all data collected from beach interviews,
telephone interviews, environmental data, and water quality data. Westat will submit this database
on a weekly basis outlined in Deliverable 7. This database shall be in SAS software program
language.  Westat will submit this database with a beach identifier number for all data. Westat will
submit the database information for the data collection for Friday, May 15, 2009 through Saturday,
September 19, 2009.  The comprehensive database is due for each data collection weekend at close
of business 10 calendar days after the final collection day. Westat will provide a draft of the
comprehensive database prior to data collection weekends and obtain approval of the WACOR or
COR.  The USEPA will provide an example of a previous database. Westat will submit a report for
this comprehensive database including, but not limited to, detailed explanation of database  use,
documentation of changes made over the course of the work assignment, and detailed definitions of
variable names.

Transfer of Ownership of Project-Generated Materials:
Westat will transfer all project-generated products to the USEPA at the end of the work assignment.
Products such as, but not limited to, data, computer-assisted interviews, programs, databases,
reports and materials created under this work assignment belong to the USEPA. These products
shall be transferred electronically, when appropriate. Otherwise, products will be shipped to the
Human Studies Facility in Chapel Hill, NC.

TASK 4
Quality Assurance and Quality Control:
For all tasks except Task 5 (Sand and Water Sampling and Analysis), this project shall utilize a
project Quality Assurance Project Plan (QAPP) modified in Work Assignment 2-04. For Task  5,
this work plan will serve as the  Quality Assurance Project Plan. The QAPP shall be approved by
USEPA and implemented as written. In addition, any  data forms developed by  the contractors must
be approved by USEPA for quality assurance purposes. The QAPP will be considered draft and

-------
updated as required during the course of this work assignment. A final QAPP shall be required at
the end of the work assignment.

TASKS

Sand Sampling and Analysis:

Westat will collect 3 beach sand samples per day on Saturdays and Sundays, and on 3 holiday
weekdays, Monday May 25th, 2009 (1 day), Monday July 20th, 2009 (1 day) and Monday July 27th,
2009 (1 day) from May 15th, 2009 through August 2nd, 2009 (See Table 1), at Boqueron Beach,
Puerto Rico for microbiological analysis by two methods [the current approved membrane filter
(MF) Enterococd method (mEI Agar) and Rapid Quantitative Polymerase Chain Reaction (QPCR)].
Westat will collect 3 beach sand samples per day on Saturdays and Sundays, and on 2 holiday
weekdays, (Friday, July 3, 2009 Monday September 7, 2009) from June 6, 2009 through September 7,
2009 (See Table 2), at Surfside Beach, South Carolina for microbiological analysis. Additional
samples may need to be collected if the weather interferes with the attached schedule. The dates for
these samples will be arranged between USEPA and Westat. These samples will be taken in the
designated area of the beach associated with the water quality samples. Westat will employ a
"scoop" method to collect the samples.

Water Quality Sampling and Analysis:

Westat will collect 18 beach water samples per day on Saturdays and Sundays, and on 3 holiday
weekdays, Monday May 25th, 2009 (1 day), Monday July 20th, 2009 (1 day) and Monday July 27th,
2009 (1 day) from May 15th, 2009 through August 2nd, 2009 (See Table 1), at Boqueron Beach,
Puerto Rico for microbiological analysis by two methods [the current approved membrane filter
(MF) Enterococd method (mEI Agar) and Rapid Quantitative Polymerase Chain Reaction (QPCR)].
Additional samples may need to be collected if the weather interferes with the attached schedule.
Westat will collect 18 beach water samples per day on Saturdays and Sundays, and on 2 holiday
weekdays, (Friday, July 3, 2009 Monday September 7, 2009) from June 6, 2009 through September 7,
2009 (See Table 2), at Surfside Beach, South Carolina for microbiological analysis. The dates for
these samples will be arranged between USEPA and Westat. Westat will ensure that analysts at
laboratories  are proficient in each method and that they consult with USEPA personnel, the
technical advisors for the methods, and/or the manufacturers of the instruments to ensure proper
knowledge and use of the analytical methods. Westat will use the Global Positioning System (GPS)
to identify the location of the beach on land and the individual sample sites in the water. Westat will
transport the samples to the local analytical laboratory for analysis (MF) or processing (QPCR)
within 6 hours of collection.  Westat will ship the processed QPCR filters on dry ice to the QPCR
laboratory for analysis.  Westat will maintain a Tracking System for all samples and analyses.  Westat
will take additional measurements  (pH, turbidity, conductivity and salinity) and collect other ancillary
data (air and water temperature, cloud  cover, rainfall, wind speed and direction, current direction,
wave height, bather density in the water and on the beach, boats, animals, debris) at each sampling
visit. Westat will take photographs of the beach and the water during the sampling at least once a
day from an elevated vantage point, if possible, to aid researchers in determining the conditions at
the beach.

-------
In addition, Westat will collect additional water samples for shipment to the U.S. Geological Survey
(USGS) for chemical analyses. Westat will provide a copy of the chemical sample airbills/shipping
forms from each shipment to the WACOR or COR.

-------
Table 1. Boqueron Beach Sampling Schedule

"Weekend"
0
1


2



3


4


5


6


7


8


9


10



11



12


Totals

Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40


Date
May 6, 2009
May 15, 2009
May 16, 2009
May 17, 2009
May 22, 2009
May 23, 2009
May 24, 2009
May 25, 2009
May 29, 2009
May 30, 2009
May 31, 2009
June 5, 2009
June 6, 2009
June 7, 2009
June 12, 2009
June 13, 2009
June 14, 2009
June 19, 2009
June 20, 2009
June 21, 2009
June 26, 2009
June 27, 2009
June 28, 2009
July 3, 2009
July 4, 2009
July 5, 2009
July 10, 2009
July 11, 2009
July 12, 2009
July 17, 2009
July 18, 2009
July 19, 2009
July 20, 2009
July 24, 2009
July 25, 2009
July 26, 2009
July 27, 2009
July 31, 2009
Aug. 1, 2009
Aug. 2, 2009


Day
Wednesday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Monday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Friday
Saturday
Sunday
Monday
Friday
Saturday
Sunday
Monday
Friday
Saturday
Sunday

Water
samples
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
720
Sand
samples
o
J
3
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
120
Total
samples Notes
21 Dry Run
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21 4th of July
21
21
21
21
21
21
21
21 Luis Munoz Rivera's Birthday
21
21
21
21 Jose Celso Barbosa Birthday
21
21
21
840

-------
Table 2. Surfside Beach Sampling Schedule
Date Samples Day of the
Collected Week
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
6/1/2009 Dry Run
6/6/2009
6/7/2009
6/13/2009
6/14/2009
6/20/2009
6/21/2009
6/27/2009
6/28/2009
*7/3/2009
7/4/2009
7/5/2009
7/11/2009
7/12/2009
7/18/2009
7/19/2009
7/25/2009
7/26/2009
8/1/2009
8/2/2009
8/8/2009
8/9/2009
8/15/2009
8/16/2009
8/22/2009
8/23/2009
8/29/2009
8/30/2009
9/5/2009
9/6/2009
*9/7/2009
Mon
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Fn
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Mon
No. Water
Samples
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
No. Sand
Samples
3
3
o
J
o
J
o
J
3
3
3
3
o
J
o
J
3
3
3
o
J
o
J
o
J
o
J
3
3
3
o
J
o
J
o
J
3
3
3
o
J
o
J
o
J
o
J
No. of
Composite
Samples
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
                                        10

-------
 Sample Analysis: Westat will analyze all water samples collected at the beach by three
microbiological methods during the period of May 15, 2009 through September 7, 2009. Additional
samples, if needed, will also be analyzed by the same methods. Westat will verify 5 colonies/sample
for all samples analyzed by the MF method (Method 1600) on one day (either Saturday or Sunday)
of the first weekend. Westat and laboratories will participate in a dry run to  determine and correct
any problems in the procedures at least one week before the study begins. USEPA personnel may
attend. This dry run will consist of a full day's event of water quality sampling, water quality analysis,
and form documentation on the beach and in the laboratory as it would occur during the actual
study collection.

Data Collection and Handling: Westat will use sample collection/custody forms, approved by
USEPA. Westat will maintain sample collection/custody sheets in a binder in the laboratory where
the analysis is being performed. Westat will record and save all data from each method (counts,
estimated counts, CT values, setup values, complete run files, plots, growth curves, graphs, bit maps,
other computer files, notes, QC data, statistical parameters and analyses, etc.) and submit them to
USEPA in a hard copy and in electronic form. Westat will provide a hard copy of hand-entered
sample collection and custody sheets, data sheets, and all other forms of data for each sampling
event to the WACOR or COR (i.e., within 24 hours) during the 2-day weekend period of sampling
each week at the beach site. Westat will enter the data electronically into database
forms/spreadsheets, and the electronic file(s) will be delivered to the WACOR or COR weekly and
after the analyses have been completed at the beach site, along with all other forms of data.  Westat
will send a cover memo with the electronic data outlining the  delivery contents by method.  Westat
will maintain original copies of the sampling and data worksheets and deliver them to USEPA at the
end of the study. Additions or changes to data worksheets must be approved by USEPA.

Proficiency/Certification

There is no specific training anticipated by USEPA for the current approved membrane filter
Enterococd method (mEI Agar; see Attachments 1  and 2).  Westat will ensure that analysts are
proficient  in the above method and each of the rapid methods [Rapid Quantitative Polymerase
Chain Reaction (QPCR) and Human Specific QPCR (See Attachment 6).]. The  laboratory analysts
who will be performing the assays will be proficient in each method and will consult with USEPA
personnel, the technical advisors, and/or the manufacturers of the instruments to ensure proper
knowledge and use of the analytical methods.  Westat and laboratories will be required to process
one or two performance evaluation samples (unknowns) for all methods during the study.  General
field and lab safety protocols will also be the responsibility of the laboratory.

Safety Protocol

Westat will utilize a safety plan specific to Task 5 modified in Work Assignment 2-04 contract EP-
D-04-064.

Documents and Records

Data Deliverables

Westat will initially record membrane filter count data and lot number on field or laboratory
worksheets, then enter and save data electronically in a database (Microsoft Access) or a spreadsheet
                                             11

-------
(Lotus 123 or Excel).  Corel Word Perfect or Microsoft Word will be used for the text in reports.
The worksheets, database format, data sheets, or spreadsheets for this method and all other methods
in this study must be approved by the USEPA.  If spreadsheets are employed, a separate spreadsheet
file will be employed for each day of sample collection.  The file name will incorporate the dates or
range of dates of collection. Separate worksheets within the spreadsheet or different database forms
will be used to record the following information:

           —    Field measurement data (See below),

           —    Sample  collection time/analysis start time/incubation  start and end time (See
                 Microbiological Method Section), and

           —    Microbiological data for each sample/volume and associated quality control (QC)
                 samples.

A hard copy format of hand-entered sample collection and custody sheets, data sheets, and all other
forms of data (graphs, bit maps, CT values, setup values, plots, growth curves, notes, QC data,
statistical parameters and analyses, etc.) for each sampling event will be provided to the COR and
WACOR daily (i.e., within 24 hours) during the 2-day weekend period of sampling each week at the
beach site.  Westat will also enter the data electronically into the database  forms/spreadsheets, and,
after the analyses have been completed at the beach site, the electronic file(s) will be delivered to the
COR and WACOR along with all other forms of data (graphs, bit maps, plots, curves, setup values,
notes, QC data, statistical parameters and analysis, etc).  The delivery will  be accompanied by a brief
cover memo outlining the delivery contents by method. The memo will also indicate if there are any
unusual circumstances or known problems surrounding the deliverable, such as  QC problems in any
of the methods.

Critical data to be reported are the bacterial counts from the membrane filter tests; the complete run
files for the QPCR method, including CT and setup values,  positive and negative controls, etc; the
complete run files. All membrane filter plates will be examined and counted [If possible, plates with
up to 200 colony forming units (CPUs) are to  be considered countable, although the ideal number
of CPUs is 20-80.], and the results for all plates will be reported, including zeros and those "too-
numerous-to-count" (TNTC). An estimation  procedure for TNTC plates will be provided to the
laboratory by USEPA (Attachment 5).  The estimation data from the TNTC plates  (i.e., the five
counts from five squares on each filter) will be submitted to USEPA along with the count data for
the other samples. QC data will be reported with the sample data for all methods.

In addition to the water samples to be collected, a number of ancillary data will be collected for each
sampling visit. These are shown in Table 3, along with descriptions of their measurement. Field
data will be entered with permanent non-running ink. In addition to the items detailed in Table 3,
any items/activities  specific to the beach will be added, for example, at Edgewater Beach in Biloxi
(2005), data on the number of jet skis in and out of the water was recorded.

Any QC results associated with the collection  of ancillary data will also be reported (QC samples are
to be  specified in Methods/SOPs describing ancillary data collection methods).
                                             12

-------
Table 3. Measurements to be Recorded at/for Each Sampling Visit
Measurement
Date and Time
Air temperature
Water
temperature
Cloud Cover
Rainfall
Wind speed
Wind direction
Current
Direction
Wave height, if
applicable
Bather density
Boats
Animals /Birds
Debris
Description
Date and Time of day
Measured by thermometer at a fixed
location every visit
Measured by thermometer at a fixed
sampling location at appropriate depth for
thermometer on every visit
Sunny, Mostly Sunny (20-50% cloud
cover), Cloudy (50-70% cover) Mostly
Cloudy (70-99% cover), Overcast
Measured by rain gauge near sampling
area; collected each day at time of
sampling and any time rain is known to
have occurred at the beach since the last
measurement was taken. Current
conditions such as rain, lightning, hail, etc.
noted
Sustained speed measured by wind gauge;
gusts indicated in comments fields
Compass direction to nearest semi-
quadrant leeward measured on wind gauge
Described in relation to shoreline facing
out
Meter stick measurement at central
sampling point. This is the distance from
the low point (trough) to the high point
(peak) of the wave
Number of bathers in the water, in the
sampling area, and number of "bathers"
on beach, within outer transects to edge of
beach on land side
Number/ approximate number of boats in
the water, within approximately 500 M of
sampling area
Animals and birds potentially affecting the
water (within approximately 20M of the
sampling area in the water or laterally
within 20M of the outer transects on the
beach); also includes number of fowl or
other birds in the air near the sampling
area
Description of any debris floating in the
water or washed on shore within the
Units /Format
Mm/dd/yy;
hh:mm
°C
°C
S, MS, C, MC, O
Rain in inches;
other
observations
noted in
comments field
Miles per hour
N, NE,E, SE, S,
SW, W, or NW
Descriptive
(onshore, right,
etc.)
Meters
Categorical;
<20, 20-100,
100-200, >200
Categorical;
None, 1-5, 5-10,
10-20, 20-30,
etc., etc.
Types of
Animals,
Numbers of
Animal Types
on beach and in
water
Categorical;
"None," "Very
MQOs
V5 minutes
Vl°
Vl°
Field Person
or Team
Consensus
V 0.25 Inches
V 5 mph
Recorders
judgement
Field Person
or Team
Consensus
V0.2M
Field Person
or Team
Consensus
Field Person
or Team
Consensus
Field Person
or Team
Consensus
Field Person
or Team
                                         13

-------

bathing area
Little," "Little,"
"Lots," describe
types
Consensus
14

-------
Table 3. Measurements to be Recorded at/for Each Sampling Visit (continued)
Measurement     Description	Units/Format     MQOs
pH
Each sample measured after
microbiological analysis processing, per
"Standard Methods" (3) or equivalent.
*Equipment utilized for this measurement
must be preapproved by WACOR or COR
pH units
V 0.2 units
Turbidity
Each sample measured by nephlometer
after microbiological analysis processing,
per Standard Methods (3) or equivalent
*Equipment utilized for this measurement
must be preapproved by WACOR or COR
Nephlometric
Turbidity Units
(NTUs)
Range
dependent; see
Standard
Methods
2130B
Salinity
Each sample measured after
microbiological analysis processing, per
"Standard Methods" (3) or equivalent and
Measured on site concurrently with
temperature and air current	
Parts per
thousand
Field Person
Conductivity
Each sample measured after
microbiological analysis processing, per
"Standard Method" (3) or equivalent
microSiemens or
milliSiemens as
appropriate	
Field Person
UV Reading
Measured by UV device
Units/Format
MQOs
Geographical
Position
GPS Unit

Coordinates will be taken in 3 places for
each of the 3 transects. Total of 9
positions for each sample run (8:00 Am,
11:00 AM, 3:00 PM)	
Lat/Long
Field Person
or Team
Consensus
Swim Advisory
Flags
Flags put on beach by lifeguards or other
official to indicate if swimming is advised,
cautioned against, or unallowed for
bacteria levels, weather, or roughness of
water.

Usually Green for Safe, Yellow for
Advisory, and Red for Unsafe/Not
Allowed
Indicate if
advisory is due
to bacteria,
weather, or
roughness of
water.
Field Person
                                            15

-------
Photographic Data

To aid researchers in determining conditions at the beach that may not be readily apparent from the
ancillary data recorded, photographs of the sample locations at the beach area will be taken for the
record at least once a day during the sampling period at the beach site. While the work assignment
requires taking photographs only once a day, Westat proposes to continue taking them at every
sample collection, as in past years, because the conditions on the beach can change substantially over
the course of a day from 8:00 am to 3:00 pm. The photographs at the beach will be taken from an
elevated vantage point, if possible, on one side or the other of the study area. Photographs will be
of sufficient quality to  estimate the number of bathers on the beach and in the water.  Photos should
be labeled with the beach name,  date, target sample collection time, and actual time of photograph.
The camera will be configured to have the date (and time, if possible) displayed on the image. The
beach name, target sample time,  and actual time of photograph (if unable to configure camera to
display on image) will be part of the image name.  Submission of photographs to the WACOR or
COR will occur at the  end of the sampling period  for the beach via appropriate means, expected to
be delivery of a CD-ROM with the digital photographs.

General Laboratory Quality Control Records

Laboratories are expected to maintain records of general  laboratory quality control activities, such as
are described in this work assignment, the attachments, and some of the references (1,3,5) found at
the end of this work assignment.  Such records may become deliverables upon an amendment to the
work assignment.

Data Formats

The exact format of all data fields will be approved by USEPA prior to data collection. Formats will
be based on those specified for this project in the work assignment, in the attachments, or in the
forms recommended by the manufacturers of the instruments. Where possible,
database/spreadsheet templates will have fields preformatted.

Quality Assurance Plan and Revisions

All project personnel will receive copies of the most current version of the Quality Assurance
Project Plan (QAPP) prior to dry run.

Other Records

Various other documents and records (e.g., SOPs, reports, method validation records,  laboratory
QC, and maintenance  records) are discussed in this document in appropriate sections. The USEPA
reserves the right to request copies of any documents and records from Westats that could affect
this project. Any records that are received, and any records generated by Westat will become part of
the overall project file.
DATA GENERATION AND ACQUISTION

Sample Collection for Microbiological Analyses


                                            16

-------
Water Samples:

Three times a day, at 8:00 AM, 11:00 AM, and 3:00 PM, two water samples will be collected at the
beach along each of the three transects perpendicular to the beach shoreline, one in waist-high water
(1 ml deep) and one in shin-high water (0.3 m deep), for a total of 18 samples per day (i.e., 6 grid
locations x three times per day). See Figure la, which depicts the water sampling scheme. The
location of the transects will be at least 20 meters apart or more, if the area used by the swimmers
encompasses more than a total of 60 meters of shoreline.  The samples will be collected on
Saturdays and Sundays, and on 3 holiday weekdays, Monday May 25th, 2009 (1 day), Monday July
20th, 2009 (1 day) and Monday July 27th, 2009 (1 day) from May 15th, 2009 through August 2nd, 2009
(See Table 1), at Boqueron Beach, Puerto Rico. Westat will collect 18 beach water samples per day
on Saturdays and Sundays, and on 2 holiday weekdays, (Friday, July 3, 2009 Monday September 7,
2009) from June 6, 2009 through September 7, 2009 (See Table 2), at Surfside Beach, South Carolina
for microbiological analysis. It is intended that samples will be collected on the scheduled dates, but
other dates may be substituted if rainfall or other problems prevent swimmers from going to the
beach, prevent water sampling, or create hazardous conditions for the field personnel.  Sample
collectors will notify the WACOR or COR of adverse weather conditions or other problems and
request guidance whether to begin or continue sampling on a given day or weekend. This is
necessary because the samples must be collected when there are sufficient bathers at the beach to
allow NHEERL to conduct their concurrent epidemiological/health study.

Global Positioning System (GPS) readings of the actual water collection locations and a photo of the
sample collection sites will be taken.
Figure la. Water Sampling Locations
^^^
Left
Transect

Center
Transect

Right
Transect
                                             17

-------
Remote Chemical Analysis Samples

In addition, 3 1-liter plastic-coated glass bottles will be taken once a day, during the 11:00 AM
collection at each of the three transect locations in waist-high water and at the center transect only in
shin-high water (sampling points 2, 3, 4, and 6, as circled in Figure  Ib) each Friday, Saturday,
Sunday, and on 3 holiday weekdays (May 25th, July 20th, and July 27th) during the study for remote
chemical analysis, described in a later section (for a total of 12 samples/sampling day).  Figure Ib is a
schematic that shows the collection of water samples at 11:00 am.
Figure Ib. USGS Water Sampling Locations at 11:00 am.
Sand Samples:

Westatwill collect three sand samples per day at 8:00 AM along with the 8:00 AM water samples.
The sand samples will be collected 1 meter from the lowest water level (when the waves have
receded from the shoreline) at the same 3 transects where water samples are collected. See Figure
                                             18

-------
Ic, which is a schematic that shows the collection of the sand samples at 8:00 am (sampling points 7,
8 and 9). The sand should be wet.  If the sand is not wet at 1 meter from the water, the sand
collection location will be moved the shortest possible distance toward the water to a location where
the sand is wet. Westat will record the actual distance from the water.  Global Positioning System
(GPS) readings of the actual sand collection locations and a photo of the sample collection sites will
be taken.

The sand samples will be collected on Saturdays and Sundays, and on 3 holiday weekdays, Monday
May 25th, 2009 (1  day), Monday July 20th, 2009 (1 day) and Monday July 27th, 2009  (1 day) from May
15th, 2009 through August 2nd, 2009 at Boqueron Beach, Puerto Rico (see Table 1). Westat will
collect the sand samples Saturdays and Sundays, and on 2 holiday weekdays, (Friday, July 3, 2009
Monday September 7, 2009) from June 6, 2009 through September 7, 2009 (See Table 2), at Surfside
Beach, South Carolina for microbiological analysis. As with the water samples, it is intended that
samples will be collected on the scheduled dates, but other dates may be substituted if rainfall or
other problems prevent swimmers from going to the beach, prevent water sampling, or create
hazardous conditions for the field personnel. Sample collectors will notify the WACOR or COR of
adverse  weather conditions or other problems  and request guidance whether to  begin or continue
sampling on a given day or weekend. This is necessary because the samples must be collected when
there are sufficient bathers at the beach to allow NHEERL to conduct their concurrent
epidemiological/health study.
                                             19

-------
Figure Ic. Sand Sampling Locations at 8:00 am.

                              Water
              Waist
              depth
Composite Samples:

Westatwill collect one additional bottle at each of the 6 grid locations (see Figure la) three times a
day at SAM, 11 AM and 3PM to be used for creation of composite samples. A total of 18 bottles
will be collected per day.  In Puerto Rico, these samples will be collected on Saturdays and Sundays,
as well as 3 weekday holidays (Monday May 25th, 2009, Monday July 20, 2009, and Monday July 27,
2009) during the study period of May 15, 2009 to August 2, 2009.  In South Carolina, these samples
will be collected on Saturdays and Sundays, and on 2 holiday weekdays, (Friday, July 3, 2009 Monday
September 7, 2009) during the study period from June 6, 2009 through September 7, 2009. At the
lab these samples will be combined to create a composite sample, as described in the "Analytical
Methods" section.
Sampling Methods

Water Samples:

See Standard Methods for the Examination of Water and Wastewater. 20th edition (1998), Section
9060, for recommendations on microbiological sampling (3). Briefly, samples will be collected in
waist-high (1 m deep) and shin-high (0.3 m deep) water by serially immersing two (2) capped 1000-
mL pre-sterilized, polypropylene bottles (or four 500-mL bottles) to the appropriate sample depth,
removing the lids and allowing them to fill, re-capping lids (to prevent contamination from surface
                                             20

-------
water), raising them out of the water, removing the lids, and emptying them slightly to allow
approximately 1 inch of head space before securing the lids. Sampling devices may be employed.
Samples will be taken about 1 foot (0.3 m) under the surface of the water in waist-high water, and
shin-high samples will be taken 6 inches (0.15 m) above the bottom of the water. The samples
collected near the bottom must be taken with care so as not to introduce additional
sand/solids/debris into the samples. Sample plans may have to be altered in extreme or unusual
circumstances.  If alterations of the sample method are considered, the WACOR or COR will be
notified and guidance will be requested. Westat will utilize field protocols from Work Assignment
2-04 under contract EP-D-04-064. Such field protocols and sampling procedures will be submitted
to the WACOR or COR for approval prior to the beginning of the study.

Three 1-liter water samples will be collected aseptically, as described above, at each location on the
beach grid (Figure la) for microbial analysis:

       1.      1 liter will be used for the  membrane filter method and the ancillary measurements
              (which will be done last to prevent contamination).

       2.      1 liter will be used for the  rapid QPCR method.

       3.      1 liter will be used for the  Composite Samples

Two 1-liter plastic coated glass bottles will be used for the USGS remote chemical analysis, at the
11:00 AM sampling period, at sampling points 2, 3, 4 and 6 only.

Following collection, all samples will be placed in coolers and maintained on ice during transport
and at
1 - 4° C during the time interval before they are analyzed or shipped (samples for chemical analyses
only). No additional samples are collected for the determination of pH and turbidity. These
measurements are made from the same samples used for membrane filtration after these analyses are
completed to prevent contamination.

Any problems encountered while sampling or while taking ancillary measurements will be recorded
on data collection sheets in comment fields or on additional sheets  clearly identifying the date, time,
sample location (on grid)  and reported to  the WACOR or COR if problems may affect the analytical
results.  In the event of problems, corrective actions  taken (where possible) will be documented by
the field team leader, along with the results of such actions.
Sand Samples

Westat will collect, transport, and process the sand samples according to the protocol provided by
Kristen Brenner on April 2, 2007. The following text is taken from that protocol. Sand samples will
be collected with sterile, 2 inch x 10 inch stainless steel liners (AMS, American Falls, Idaho, or the
equivalent). The liner will be pushed into the sand at least 8 inches. The liners will be sterilized at
the lab by rinsing them with water, wrapping them in aluminum foil and heating them in the drying
oven at 170 °C overnight. The liners will remain wrapped in the aluminum foil until use. Liners
containing the sand samples will be capped at both ends, placed in zip-lock plastic bags labeled using
                                             21

-------
a simplified version of the usual alpha-numeric system (See below.), and transported to the
laboratory on ice. Samples will be stored in a refrigerator at 4 degrees C. until analyzed.

In the laboratory, sand samples will be aseptically transferred to sterile wide-mouth polypropylene
bottles (500 ml or 1- liter, depending on the quantity of the sand), also labeled using the simplified
version of the usual alpha-numeric labeling system.  For each sand sample, 75 grams of sand will be
aseptically weighed out in a sterile, pre-tared. wide-mouth 500-ml bottle (using sterile spatulas), and
300 ml of Standard Methods phosphate-buffered rinse/dilution water (3), measured with a sterile
graduated cylinder, will be added to each bottle.  Each bottle will be vigorously shaken 50 times
(Please count). Immediately after shaking, some of the contents of the bottle will be poured into
two sterile 50-ml, disposable centrifuge tubes (Corning 430829 or the equivalent) and filled to the
50-ml mark. The tubes will be centrifuged for 5  minutes at -3000 rpm (600 x g) to bring down the
sand and sediment, and the supernatant will be removed using a sterile pipette and placed in a sterile
100-ml polypropylene bottle for subsequent analysis by Method 1600 (Attachment 1) and the
Quantitative Polymerase Chain Reaction (QPCR) method (Attachment 6).

The accuracy of the 50-ml mark on the disposable tubes will be checked before the dry run by
randomly choosing 5 tubes from the package, weighing each of the 5 tubes, and recording the
weights. After 50 ml of distilled water is measured with a graduated cylinder and poured into each of
the tubes, the tubes are again weighed. The weight of the distilled water (The difference between
the two weights)  in each tube should be close to  50 grams.  Observe the position of the water
meniscus with reference  to the 50-ml mark on the tubes. In addition, 5 randomly chosen,
preweighed tubes should be filled with distilled  water so that the meniscus touches the top of the
50-ml line.  Weigh the tubes again and determine the weight of the water by difference. If the mark
is accurate, the weight of the distilled water should be close to 50 grams. Record all results, and
send a copy of the results to the WACOR and Kristen Brenner, the technical point-of-contract for
sand analyses.

During the dry run, aliquots of 10 ml and 1 ml of each undiluted sand extract and 1 ml of the 10"1 —
10~6 dilutions  of each extract in phosphate-buffered dilution water (3) will be analyzed by EPA
Method 1600 for Enterococd (Attachment 1).  The number of filtrations for the actual study will be
reduced after the normal range of concentrations in sand are determined during the dry run. Three
20-ml aliquots of each sample will be filtered, and the filters will be frozen, as described in the
QPCR Method (Attachment 6), during the dry run.  The sand extraction method described above
and the volumes  used for both tests may have to be adjusted, depending on the normal range of
concentrations of Enterococa in the extracts during the dry run. Westat will obtain EPA's approval
before changing  the protocol or volumes analyzed.

In addition, the pH of each extract will be taken  and recorded, and a 25-gram portion of each sand
sample will be dried at 100 degrees C for several  days to a week in a preweighed container. After
the samples are dry, the containers should be weighed again to determine the dry weight of the sand
samples by difference. Leftover sand samples, the bottles of the sand-buffer slurry, and extracts
should be stored in the refrigerator until all the results have been obtained with all tests (about a
week).

Sample Handling and  Custody
                                             22

-------
Westat will utilize sample collection/custody forms modified in Work Assignment 2-04 under
contract EP-D-04-064 as part of the general forms. A sample form is shown in Attachment 3.  The
distribution of each individual bottle taken at each location on the beach grid (Figure 1) must be
documented on the custody forms. A copy of each airbill for the shipment of the samples for
remote chemical analysis and a copy of the ASR custody form (Attachment 4) enclosed with each
shipment of samples must be attached to the appropriate custody forms and submitted to USEPA.
Each USGS sample must be cross-referenced with the NEEAR study locations.

Prior to sampling visits, tracking forms will be printed by a member of the site project  team. The
location, date, target collection time, field team leader, and information about all samples to be
collected during that visit will be entered on the forms, by hand (or electronically, prior to printing).
The forms will be printed on paper suitable for field work. Each cooler used to transport samples
from the site to the lab will have a copy of the appropriately completed collection/custody form(s)
in it or securely attached to it. Ideally, Westat will know in advance how many samples can fit in a
given cooler and could, therefore, prepare specific tracking sheets for each cooler prior to going to
the field.  If more than one cooler is needed, the coolers will be numbered, and cooler  numbers will
be cross-referenced on the appropriate tracking sheet. Individual bottles for the rapid  methods can
be distributed after the samples are logged in at the laboratory, and the custody forms must be
signed by each of the method analysts when portions/aliquots of the samples are removed.

Additional columns on the tracking forms include the actual collection time, the time samples arrive
at the laboratory, and their storage location. Arrival time at the laboratory can be indicated by
entering a time for the first sample on a custody sheet, and drawing a down arrow in the lab arrival
time column for the rest of the samples. The field storage location may also be  filled in this manner.

A different form (or forms) will be used to record the dates and times when analysis by QPCR and
filtering begins (MF and QPCR), the dates and time plates are placed in the water bath (or filters are
placed in the freezer for the QPCR method), the dates and time samples are removed from
incubation  (or freezer for the PCR method), and the analysis  results.  There will be spaces for
associated initials for each of the sequential steps.  The various "analysis" times will be treated on a
batch basis; i.e., a sample batch is all of the samples brought to the laboratory at the same time for
analysis, such as all 6 morning samples.

Microbiological sample containers will be labeled with water resistant sample labels. The sample
bottles will have IDs with consecutive numbers to facilitate handling in the laboratory and to
prevent errors. However, Westat will be responsible for placing the requisite additional information
onto sample bottles at the time of sampling to ensure that the samples can be clearly identified.  It is
recommended that the information (or at least alphanumeric  information, such as suggested directly
below) be added just prior to or just after sampling, as this should minimize the  chance of getting
samples in the wrong bottles.  Information to be added would include the date, scheduled and actual
time of collection, and some type of alphanumeric that identifies the  sampling location, and the
method(s) to be used.

Westat proposes to use the following sample labeling scheme for all water and sand samples.  This
scheme is the same as has been used in past years for water sampling and it was  easily modified to
accommodate the sand samples introduced this year.  Microbiological sample containers will be
labeled with water resistant sample labels using the following alphanumeric (9-character) scheme (to
avoid confusion and duplicate sample numbers):
                                            23

-------
              MMDDNSSXXB

              Where:

              MMDD is the date of the sample collection;
                    MM is the numeric month (1-12) and
                    DD is the day (01-31), e.g., 0614 for June 14,

              N is the sample point at the beach;
                    1-6 for water samples (see Figure la.)and
                    7-9 for sand samples (see Figure Ic.)

              SS is the method/bottle number, as follows:

                    01 = Membrane Filter Method 1600
                    02 = QPCR Methods
                    03 = Alternate Methods
                    4a = Chemical,  bottle a
                    4b = Chemical, bottle b
                    4c = Chemical,  bottle c
                    SI = Sand container (If in the future, more than one sand sample will be
taken                      from the same location at the same time, the second container would
be S2.)
                    Cl = Composite Sample

              XX is the planned time of day for the sample collection, as follows:

                    08 = 8:00 a.m.
                    11 = 11:00 a.m.
                    15 = 3:00 p.m.

              B designates Boqueron Beach, which is the  first initial of the beach.

Thus, for example, the water and sand  samples collected on June 23 at 8:00 am would be labeled:

                    062310108B   062310208B
                    062320108B   062320208B
                    062330108B   062330208B
                    062340108B   062340208B
                    062350108B   062350208B
                    062360108B   062360208B
                    06237S108B
                    06238S108B
                    06239S108B

Westat understands that sample containers  may be reused after proper cleaning and resterilization or
bottles, presterilized by the manufacturer, may be used. Westat will use only presterilized bottles.
                                           24

-------
Westat will obtain a copy of the manufacturer's sterilization certificate and/or record for each lot
should be obtained. In addition, the sterility of a few randomly-chosen bottles from each lot should
be tested before field use by adding sterile Trypticase Soy Broth to the bottles, incubating for 48-72
hours at 35° C, and observing the bottles for bacterial growth. Prior to leaving for the field, the
sample team leader will check to see that there are an appropriate number of sample bottles and
sample ID labels for the sampling visit (Bottles may have labels attached prior to sampling, if it is
demonstrated that this has no deleterious effects on the labels.) for the sampling visit. There should
also be extra, unlabeled sample containers, and a means to label them for back-up purposes.  Copies
of completed sample collection/custody sheets will be provided to the WACOR or COR daily along
with their associated data sheets.

As stated above, following collection, samples are to be maintained on ice during transport and at 1 -
4° C until the time of analysis.  This is the only preservation step. Microbiological analysis of water
samples will commence within six hours of collection; it is critical that sample plates from the
membrane filter methods be placed in the water bath within eight hours of sampling. In the event
of any problems or irregular occurrences, it is imperative that the WACOR or COR be called
immediately for guidance, and that the comments fields on the various data sheets be used to record
problems/corrective actions, so that the effect on data quality can be considered.  Examples of
problems that might occur include sampling difficulties, failure to ice-down samples, missed
holding/analysis times, longer than acceptable incubation times, problems with the instruments, etc.

With the rapid OPCR method, the critical step is the filtration of the water samples and storage of
the filters in the freezer within the 8 hours  after collection.  Once the filters are frozen, analysis can
be done as time allows. The times the QPCR method filters are frozen and stored, the location of
the freezer(s), and the dates and times of the analyses will be recorded.  If QPCR filters are analyzed
in another lab, they must be shipped by overnight express on dry ice. The other laboratory will
conform to the QC requirements of this document. Problems with the rapid methods, like those
with the filter methods, must be reported and guidance requested from the WACOR or COR.

Samples may be disposed of following successful microbiological processing by each  of the
microbial methods, including the counting of all plates, successful pH and turbidity measurements,
and completed  analysis of samples by all methods except for the QPCR method. However, QPCR
samples must be filtered and the filters frozen before the disposal of the samples.  Contact the
WACOR or COR about the disposition  of the samples if unusual results are obtained.

Westat will maintain a dedicated sample  record book that is used to record all sample IDs as samples
are checked into the laboratory. The record book will also  have columns for date checked in,
storage locations, and disposal dates.  Westat has the responsibility for ensuring that all sample IDs
are recorded and will initial the record book for each batch of samples received to indicate that all
expected samples were present. The USEPA may request that the record book or copies of pages
from this record book be made available for examination. Westat is also responsible  for verifying
that the arrival time at the laboratory is entered in the appropriate column on the sample collection
sheets, and will initial sample collection sheets in the appropriate space (s) to indicate such, and will
note any leaking containers or other irregularities.

Analytical Methods

       Microbiological Methods
                                             25

-------
       1.
Standard Membrane Filter Method Enterococd ("Method 1600s)
Attachment 1, "Method 1600: Membrane Filter Test Method for Enterococd in Water,"
EPA/821/R-97/004, May 1997  (4) describes the assay for Enterrococd. This method can also be
found in Attachment 2. "Improved Enumeration Methods for Recreational Water Quality
Indicators: Enterococd and Escherichia coli" EPA/821/R-97/004 (7). These attachments are detailed
enough, including descriptions of required equipment, so that the membrane filter method can be
performed.  As such, these two attachments represent the standard operating procedures (SOPs) for
the critical membrane  filter data  to be obtained from the field study.  A 1-liter sample or two 500-ml
water samples will be collected for use in performing the filtration method and the ancillary pH and
turbidity measurements, which will be performed last to avoid contamination. All collected samples
will be analyzed for Enterococd by the MF method using sample volumes of 100, 10 and 1 mL [except
for special circumstances; for example, if plates at the standard sample volumes are all TNTC, or
produce zero CPUs, then sample volumes may need to be adjusted.]  The laboratory may have to
adjust volumes using their own judgment if immediate communication with the WACOR or COR is
not possible. In the event that the laboratory must adjust  the volumes and adjust, the adjustment
and documentation of reason must be indicated in the records submitted to the USEPA. Analysis
of each sample will be initiated within 6  hours of its collection, and processing (filtration and plating)
will be completed no later than 8 hours after collection.

Specific QC requirements to be incorporated into the assays (in place of the general guidance in the
methods) can be found in the  next section of this plan. Table 3 summarizes some of the key features
of the method. Any modifications  to the method, such as  using auto-pipets or micro pipets instead
of standard glass pipets, must be approved by the WACOR or COR prior to being implemented.
Any other questions regarding the  methods should also be addressed to the WACOR or COR prior
to the start of field activity.

Table  3. Summary of the mEI Agar Method for Enterococd



Method
Enterococci
EPA 1600




Medium
mEI agar



Incubation time and
temperatures
(°Q
24 hours + 2 hours @
41 +/-0.5°C


Volumes
analyzed
(mL)
100
10
1
Detection
limits
(colonies per
plate)
1-200



Ideal 1# of
colonies per
membrane
20-60


On the sample collection/tracking sheets and final data sheets, laboratory analysts are responsible
for entering times and their initials for the following sequential steps:

           —    Analysis start time.

           —    Time at which plates being incubation in the water bath.

           —    Time at which plates are removed from the water bath for counting.
                                            26

-------
These times will be entered by hand initially, and later entered into the database electronically. The
times listed above, and initials, may be entered in a batch-wise manner. The laboratory is also
responsible for entering dilution data, count data, QC data, etc. on data sheets. Responsibility for
electronic data entry will be determined by Westat.

Samples are to be analyzed in batches. A batch will be considered to be all of the samples that were
delivered to the laboratory at the same time. The plates for each batch of samples should start their
incubation periods at the same time, and the microbiological control samples described below under
"specific filtration control tests" will accompany each analysis batch.

For the membrane filter assay, the most critical quality control requirements are as follows:

            —    Prior to any sampling/filtering,  an appropriate volume of  TSA [Tryptic Soy
                 Agar/Trypticase Soy Agar (Difco 0369-17-6, BD 4311043, Oxoid CM 0129B, or
                 the equivalent)] will be prepared, and tested as described below. These plates will
                 later by used for QC samples  during sample runs. The  recipe for TSA and the
                 contamination screening for TSA plates is described below:

                 Composition:

                             Tryptone      15 g
                             Soytone       5 g
                             NaCl          5 g
                             Agar          15 g

                 Preparation:  Add the dry ingredients listed above the 1000 mL of reagent-grade
                 distilled water, and heat to boiling to dissolve the agar completely. Autoclave at
                 121° C  (15 Ibs pressure) for 15 min. Dispense the agar into 9 x  50 mm petri
                 dishes (5 mL/plate).

                 Test for contamination: Incubate all plates for 24 - 48 hr at 35° C to check for
                 contamination. Discard any plates with growth. If > 5% of the plates show
                 contamination, discard all plates, and make new medium. Store plates in plastic
                 bags at 4°C until needed. The final pH should be 7.3 + 0.2. Records of
                 preparation and testing will be maintained, and will  be submitted to the WAM
                 upon request.

            —    Each batch of mEI agar is to be pre-tested for performance (i.e., correct enzyme
                 reaction) with known  cultures  of target (e.g.,  Enterococcus faedum or Enterocococcus
                fecalis) and  non-target  (e.g.,  Escherichia  colt or Pseudomonas species)  organisms.
                 Records of  such  tests  are  to be maintained by the  laboratory and will be
                 submitted to the COR and WACOR upon request.

            —    Specific filtration control tests, listed  below,  are  to be  performed each time  a
                 batch of samples are analyzed,  and the results recorded. Results for all filter, agar
                 or buffer controls, including counts (if any), will be  reported with  the  sample
                 results.
                                             27

-------
                 Filter Control: Place one or more membrane filters on sterile TSA plates, and
                 incubate the plates for 24 hours at 35° C. Absence of growth indicates sterility of
                 the filter(s).

                 Phosphate-Buffered Dilution Water  Controls: Filter a 50-mL volume of sterile
                 dilution water before beginning the  sample filtrations and a 50-mL volume of
                 dilution water after completing the sample  filtrations. Place the filters  on TSA
                 plates, and incubate the plates for 24 hours at 35° C. Absence of growth indicates
                 sterility of the dilution water.

                 Agar  Control: Place one or more plates of each medium, mEI and TSA, in the
                 incubator. Incubate mEI at 41° C and TSA at 35° C for 24 hours to check for
                 contamination. Absence of growth indicates sterility of the plates.

                 Optional membrane test: Test new lots of membrane filters against an acceptable
                 reference lot using the method of Brenner and Rankin (4). Although optional, this
                 test is recommended.  In lieu  of performing this test, the laboratory should
                 purchase filters from a reputable source. The USEPA has found (by the method
                 referenced)   that  Sartorious  filters  have  generally  provided   satisfactory
                 performance; however, this does not mean other filters are unacceptable.
There are no specific sample IDs for the specific filtration control samples. On the hard copy
format batch analysis sheets, their results will be reported with the following codes:

              YYZ (MEDIA), where,

              YY =  AC, PB, or MF (for Agar Control, Phosphate Buffer dilution water controls,
              or     Membrane Filter control).

              Z —   B or A, or  nothing (used only for phosphate buffer dilution water controls; B
              for    "Before filtering" control, A for "After filtering" control).

              (MEDIA) = mEI or TSA, the medium used for the control.

The methods contain other specific QC elements, such as requirements for laboratory water quality,
specifying that thermometers be NIST-traceable, calling for daily confirmation of incubator and
water bath temperatures. Such method specifications will be adhered to, and the adherence
documented. All autoclave runs will contain maximum-registering thermometers to ensure
appropriate temperatures are achieved. Additionally, at least weekly, autoclave runs will contain
spore strips or vials, which will be incubated according to the manufacturer's instructions to  check
for proper sterilizer operation. Calibration records should be maintained for laboratory balances, pH
meters, etc.

The method SOP (Attachments 1 and 2) contains procedures for verifying the correct identities of
organisms. Verification tests are required for all samples (5 colonies/sample)  from one day (either
Saturday or Sunday) of the first weekend (6 sample locations x 3 times per day x 1  day = 18 samples
                                             28

-------
total) at the beach site. Results of the verification tests will be recorded and reported to the WACOR
or COR with the other sample data in a mutually agreed upon manner.

It is expected that laboratories will follow generally accepted good microbiology laboratory practice,
such as  described in the USEPA Microbiology Methods Manual. Part IV, C (1); Section 9000 of the
20th edition of Standard Methods (3); or the QC section of the USEPA's "Manual for the
Certification of Laboratories Analyzing Drinking Water" (5). Copies of any records associated with
standard laboratory QC practices will be made available to the USEPA upon request.
       2.     Quantitative Polymerase Chain Reaction (OPCR) Method

Attachment 6 describes the procedures for the detection of Enterococd and Bacteroides in water
samples based on the collection of these organisms on membrane filters, extraction of their total
DNA, and polymerase change reaction (PCR) amplification (i.e., a process whereby the quantity of
DNA is doubled in each cycle of amplification) of a genus-specific DNA sequence using the
TaqMan™ PCR product detection system. The TaqMan™ system signals  the formation of PCR
products by a process involving the breakdown of a double-labeled fluorogenic probe that
specifically attaches to the target sequence at a site between the two PCR primer recognition
sequences. The reactions are performed in a specially-designed thermal cycling instrument that
automates the detection and quantitative measurement of the fluorescent signals produced by probe
degradation during each cycle of amplification. These signals are directly related numerically to the
quantities of PCR products produced.

Westat understands that the attachment is detailed enough, including descriptions of required
equipment, so that the method can be performed. As such, this  attachment represents the standard
operating procedure (SOP) for the critical data to be obtained from this portion of the field study. A
1-liter sample or two 500-ml water samples will be collected for use in this method. All collected
samples will be analyzed for Enterococd and Bacteroides using sample volumes of 100 mL [except for
special circumstances; for example, if this volume is found to  be impractical to filter, then sample
volumes may need to be adjusted]. Filtration of each sample will be initiated within  6 hours of its
collections. Five (5) replicate filtrations will be performed, and the filters will be transferred to
extraction tubes, as described  in the protocol (Attachment 6), and stored at -20° C for an indefinite
period. All filters will be properly labeled to identify the water sample they came from.

The local analytical lab will perform the 5 replicate filtrations and ship 3 filters to the PCR lab and 2
filters to Dr. Richard Haugland of USEPA (26 W. Martin Luther King Drive,  Mail Location 314,
Cincinnati, Ohio 45268-1314; Telephone:  513 / 569-7135), all by overnight express on dry ice on
the Monday following the weekend the samples were collected.

The PCR lab will perform the extraction to obtain DNA to be used for QPCR analyses for all
microorganisms (Enterococd and Bacteroides) as soon as possible using only one of the filters
(Attachment 6), and two filters will be stored in the freezer as backups or for other/later analyses.
At the end of this study, all remaining frozen filters will be sent  on dry ice by overnight express to
Dr. Richard Haugland of the USEPA.
                                             29

-------
Specific quality control (QC) requirements to be incorporated into these analyses are listed below, as
well as those in the method protocol.

            —    QC  requirements  for  sample  collection and filtration  are  specified  in  the
                 Microbiological Methods section.

            —    Cell  suspensions of  the calibrator  strains, Enterococcus faecalis, American Type
                 Cutlure Collection  (ATCC)  29212, Bacteroides fmgilis ATCC 25285, and reference
                 strain, Geotrichum candidum,  University of Alberta  Microfungus  Collection  and
                 Herbarium (UAMH) 7836, will be provided to the laboratory by the USEPA. The
                 cell suspensions provided must be stored by the laboratory at -70°  C, until used.
                 Preliminary QPCR analyses  must  be performed using  four tubes of these
                 suspensions prior to the start of the study, and the results (CT values and run files)
                 must be reported to  USEPA. Subsequent average  results  for  these samples on
                 each day of analysis should be within +2 CT units of the average of the initial
                 values (See paragraph below on monitoring  the performance of the  thermal
                 cycling instrument and PCR reagents).

            —    Training for the  laboratory on the highly specialized scientific PCR equipment
                 will be provided by  the government for validity of data. Westat will be responsible
                 for ensuring that the PCR technician has  documented  experience  in  QPCR
                 technology.

            —    Westat will  be required to  purchase PCR  reagents,  including  primers  and
                 fluorescently-labeled probes. Primer and probe sequences will be provided by the
                 USEPA.

            —    Westat will be  required to monitor the performance  of the thermal  cycling
                 instrument and PCR reagents based on ongoing calibrator sample analysis results.
                 (See above.) In the event of failure to meet these performance criteria, Westat will
                 be required to  prepare and  analyze a new set  of calibrator extracts, identify the
                 source of the problem (e.g., reagents or instruments), and take corrective action.

            —    Westat will be  required to provide adequate facilities and  carry out precautions
                 necessary to minimize the  likelihood of  DNA contamination. Manipulation of
                 samples and reagents will be performed in  laminar flow hoods or workstations
                 with UV light sources, and the areas will be  disinfected before and after each use
                 with 10% bleach. Disposable aerosol barrier pipette tips will be used for all liquid
                 transfers. Tubes and other disposables that are not sterilized by the  manufacturer
                 must be autoclaved before  use. All  supplies and disposables will be DNA-free.
                 Distilled water  and  other reagents must be verified to be free of target DNA in
                 negative control analyses performed with each  set of sample analyses.

            —    All pipettors used  will  be  calibrated  prior  to  commencing work and on  a
                 semiannual  basis afterwards. It  is recommended that the  pipette calibration be
                 verified weekly by weighing  several different amounts of water (in the ranges use)
                 pipetted into a properly tared container.
                                             30

-------
           —    This work assignment will also include four other combinations of reagents that
                 will be tested for each organism by PCR method.
           3. Composite Samples

Westat will create a composite sample by adding equal volumes from each of the three transects for
the different depths.  Westat will create a shin-depth composite sample by adding equal volumes
from each of the shin-depth samples. Westat will create waist-depth composite sample by adding
equal volumes from each of the waist-depth samples. Westat will create a total composite sample by
adding equal volumes from all depth samples.  There will be a total of 9 composite samples per day:
3 shin-depths (SAM, 11AM, and 3PM); 3 waist-depths (SAM, 11AM, and 3PM); and 3 all-depths
(SAM, 11AM, and 3PM).  These samples are created from parent samples (the additional 1-liter
bottle collected at each grid location).  These samples will be used to run QPCR (for Enterococd and
Bacteroides) and Method 1600 Membrane Filtration analysis.
Ancillary Measurements

Ancillary measurements listed in Table 2 will be collected by a variety of means. Some are collected
by simple observation; others involve the use of equipment, such as pH meters, wind gauges, and
rain gauges. It is noted here that WACOR or COR approval of any deviation in methods is required.

For any ancillary data collection, especially that involving specific equipment, Westat will be
responsible for documenting the exact methods used to collect the data, and to provide information
about the calibration and QC procedures for any equipment. This documentation will be provided
for approval to the USEPA WACOR or COR prior to the occurrence of any field sampling.

Appropriate field team members or lab team members are responsible for entering data on
appropriate data collection sheets. Westat may propose additional QC activities related to sampling
and analysis in their QAPP and work plan as necessary and appropriate. Any changes from the QC
specified by individual method technical point-of-contacts must be confirmed with them before
implementation.

Sample Collection and Custody for Remote Chemical Analysis

Three additional 1-liter water samples will be collected once a day, during the 11:00 AM  collection
period, in plastic-coated, ashed amber glass bottles, furnished by the U.S. Environmental Protection
Agency, at each of the three transect locations in waist-high water and at the central location in shin-
high water (sampling points 2, 3, 4 and 6 in Figure Ib) for a total of 12  1-liter samples (3 bottles at
each of 4 locations). Samples will be collected at the marine water beach by the same methods used
for collecting the water samples for microbial analysis. The bottles should be permanently labeled
with the location of sample site, the date and time of collection, and designated "For Chemical
Analysis (Unfiltered Water)." After cooling the samples on ice, the bottles will be packed in coolers
with ice along with Analytical Services Requests (ASR) (Attachment 4)  contained in double zip-lock
bags, and  shipped by overnight carrier to the U.S. Geological Survey (USGS) [Dr. Edward T.
                                             31

-------
Furlong, USFS, Denver Federal Center — Building 95, Denver, Colorado 80225-0046; Telephone
(303) 236-3941; Fax: (303) 236-3499; E-Mail efurlong@usfs.gov] for chemical analyses and
comparison of interlaboratory variation.

At the time of collection, an Analytical Services Request form (ASR), Attachment 4, will be
completed in its entirety. The ASR should be placed in a waterproof covering and shipped with the
sample to the appropriate laboratory. All sample bottles collected during a single weekend may be
sent to the USGS in one shipment (by overnight express) on the Monday following the weekend,
provided all samples are refrigerated during the entire time from collection until shipment. Coolers
and packing material to ship samples will be provided by the USEPA (Dr. Susan Glassmeyer, 26 W.
Martin Luther King Drive, Mail Location 564, Cincinnati, Ohio 45268-1564; telephone: 513 / 569-
7757), and USEPA will provide airbills with the shipping costs billed to USEPA for the field
personnel to use.

Quality Control (QC)

The most critical elements of quality control for the membrane filter method are those related to the
microbiological assays. Sampling is straightforward; Westat must ensure that the proper samples are
taken in the appropriately labeled containers. Holding time of samples will be considered critical.
Samples that have not been placed in the water bath in the membrane filter method or completely
filtered and placed in the freezer for use in  the QPCR method within eight hours of collection will
be considered to have produces invalid data. (However, all data will be collected, compiled, and
reported to USEPA). The intent of this project is to collect all of the data for subsequent evaluation
by the USEPA project team, who will ultimately determine its utility based on their collective
expertise and experience. No data will be rejected outright by the persons performing the analysis.
All data, including Too-Numerous-To-Count's (TNTC)  and zero's in the membrane filter methods,
will be reported to the USEPA. An estimation procedure for TNTC plates will be provided to the
laboratory by USEPA (Attachment 5). The estimation data from the TNTC plates (i.e., the five
counts from five squares on each filter) will all be submitted to USEPA along with the count data
for the other samples. Westat will  calibrate  and maintain the instruments according to the methods
and/or the manufacturer's recommendations. Westat will follow accepted good microbiology
laboratory practice and maintain QC records. Westat will participate in any QA audits conducted,
and will run one or more performance evaluation samples provided by the USEPA. Westat will
contact the WACOR or COR when problems occur and document corrective actions taken in a
report.

Corrective Actions

Failure to meet any QC requirements, including those associated with standard good laboratory
practice, requires that appropriate  corrective actions be taken. All QC failures, associated corrective
actions, and their effectiveness, must be documented on a corrective action form, and submitted to
the USEPA WACOR or COR as part of the weekly reports. Data associated with quality control
problems will be clearly identified  in such reports, along with an assessment as to the QC failure's
potential effect(s) on data quality. The WACOR or COR will be notified of such
problems/corrective actions as soon as possible to the time of the actual occurrence. All related
sample and ancillary data will still be reported in the standard way, with the QC problems clearly
noted on copies of the data deliverables.
                                             32

-------
Instrument/Equipment Testing, Inspection, and Maintenance

Any SOP for equipment/instrument which Westat may be required to develop or provide will
describe standard maintenance procedures for equipment. Maintenance records will be described in
the SOPs, and will be made available to USEPA upon request, including monitoring records of basic
equipment such as incubators, refrigerators, etc.

For any equipment that might affect critical data (i.e., microbiological or ancillary data), Westat will
prepare a short report for the WACOR or COR describing how the equipment was inspected and
tested upon receipt. The report will be delivered within two weeks of equipment being placed in
service.

Instrument/Equipment Calibration and Frequency

Any SOPs for instruments and equipment which Westat may be required to develop or provide will
fully describe calibration and calibration verification procedures. This should include reference to
any calibrations conducted using certified equipment and/or standards with known valid
relationships to nationally recognized performance standards. Field instruments/gauges and
laboratory measuring equipment, such as balances and volumetric measuring devices (e.g.,
micropipettes), will be professionally serviced/certified within the six months prior to the
commencement of the field/laboratory activities for this project.

Tracking and Inspection/Acceptance of Supplies and Consumables

Westat will have a system for tracking supplies, reagents, etc. prior to the start of the field season.

The membrane filter methods (Attachments 1 and 2) describe the minimum requirements for the
quality of chemicals and laboratory water. Quality control procedures for laboratory water outlined
in the USEPA drinking water certification manual (5) are recommended. Westat will at least
maintain basic records  (i.e., resistively readings, filter changes, etc.) for their laboratory water
systems.

The optional  (but recommended) filter test (2); previously described, may be employed to test new
membrane filter lots. All media prepared will be routinely tested for sterility. The goal is to have a
clear association of all microbiological data with specific lots of all materials employed in performing
analyses. All records associated with materials tracking and preparation will be made available to
USEPA upon request.

Data Management

Some elements of data management for field data and laboratory data were previously outlined.
Westat will initially hand-enter results on pre-printed forms that are  approved by USEPA.

On an approximately daily basis, completed hand-entered data sheets will be sent to the WACOR or
COR. Weekly submissions will also be submitted to the WACOR or COR.

Westat will maintain original copies of sampling and data worksheets until instructed by the
WACOR or COR on the deposition of the worksheets. Westat will maintain two  copies,  on separate
                                             33

-------
storage media, of electronic versions of data until instructed by the WACOR or COR on the
disposition of the data.

Readiness Review/Dry Runs

At least one week prior to actual sampling, sampling/analysis personnel will perform a readiness
review/dry run at the beach site. One or more USEPA representatives may attend. A checklist
modified in Work Assignment 2-04 under contract EP-D-04-064 will be utilized by Westat that
details all equipment, supplies, worksheets, logbooks, etc. required to conduct sampling, ancillary
data collection, microbiological analysis and  data recording, and reporting at the beach site. A set of
samples will be run. Data transmission will also occur as part of this  effort. Westat will observe all
activities in detail and record their observations.

Westat will be responsible for determining the results and any corrective action to be taken. After
concurrence with the USEPA, a written report on the final approach to sampling/analysis/reporting
will be provided  to the WACOR or COR.

Site Visits/Technical Systems Audits

The USEPA may, at its discretion, perform a site visit at the beach site. The site visits may include
technical systems audits (TSAs). Although any site visit or audit will likely be conducted by USEPA
personnel, the possibility of using contractor support exists. A site visit or audit will be coordinated
with Westat in advance.

Site visitors/auditors may recommend work stoppage if they observe what they deem to be critical
failings on the part of Westat. Such a recommendation will be made  to the WACOR  or COR and
the contracting officer. Work may only be stopped by the Contracting Officer until such time as
effective corrective measures are implemented, verified effective, and approved.

Following the site visit/TSA, a report will be prepared by the personnel who conducted the visit.
This report will be addressed to the WACOR or COR. Westat will be provided a copy of the report,
and will be required to respond to any corrective action recommendations. Westat will be
responsible for signing-off on the response.  A close-out memo will be issued to Westat by the
WACOR or COR following his/her approval of the response. However, USEPA does reserve the
right to revisit any identified problem areas.

Routine Surveillance

Copies of any written reports generated by Westat on routine  surveillance will be made available to
the WACOR or COR.

Data  Validation and Usability

According to USEPA Guidance for Quality  Assurance Project Plans; USEPA QA/G-5 (6),

             "the process of data verification requires confirmation  by examination or provision of
             objective evidence that the requirements of these specified QC acceptance criteria are
             met. In design and development, verification concerns  the process of examining the
                                             34

-------
             result of a given activity to determine conformance to the stated requirements for that
             activity. For example, have the data been collected according to a specified method
             and have the collected data been faithfully recorded and transmitted? Do the data
             fulfill specified data format and metadata requirements?"

Regarding validation, G-5 states,

             "The process of data validation requires confirmation by examination and provision of
             objective evidence that the particular requirements for a specific intended use have
             been fulfilled: In design and development, validation concerns the process of
             examining a product or result to determine conformance to user needs."

Based on these definitions, verification is the responsibility of Westat; however, USEPA reserves the
right to review the verification and will be responsible for validation.

Data Review, Verification, and Validation

All data will undergo several layers of review and verification, which is described in the next section.
The previously described assessments are also a key component of verification. Validation is
primarily considered part of reconciliation with project objectives. General principles guiding
acceptance/selection, verification, and validation of data are discussed immediately below.

All microbiological data will be submitted to the USEPA (i.e., data from all sample volumes or
dilutions, even if zero, uncountable or too numerous to count and all  other forms of data, described
above). The USEPA will  decide whether or not data are acceptable, and will choose which data are
included in the final data  set for the  project. The guiding principles for microbial data
acceptance/selection will be:

            —    Legible data records.

            —    Dates and times correct.

            —    Sample IDs correct.

            —    Electronic and hard-copy data concur.

            —    Results are in an appropriate format.

            —    Results reasonable (i.e., not grossly wrong).

            —    CPU counts are in the ideal range, whenever possible.

            —    Dilutions with CPU counts  outside ideal range are not grossly  incompatible with
                 those in the ideal  range.

            —    Sample holding/analysis  times were met.
                                             35

-------
            —    Associated QC sample results are acceptable.

            —    Specific acceptance criteria for the rapid methods adequate.


For ancillary data, the acceptance factors include:

            —    Legible data records.

            —    Dates and times correct.

            —    Electronic and hard-copy data concur.

            —    Results are in an appropriate format.

            —    Results reasonable (i.e., not grossly wrong).

            —    Associated QC sample results acceptable.

            —    Other factors support acceptance (or rejection).


For the remote chemical data the acceptance factors include:

            —    Legible ASR forms.

            —    Dates and times correct.

            —    Samples arrive in good condition at remote laboratory.



Verification and Validation Methods

Verification/Validation Roles and Responsibilities

Westat will inspect forms to see that all appropriate data fields have values entered, and that entries
are legible and reasonable. Westat will also ensure that all planned samples have been collected. The
verification of review will be indicated by entering their initials on the field data sheets in provided
spaces. Westat is responsible for seeing that all forms are present and that they are delivered to the
laboratory.

Westat will verify that all expected samples and field sheets are present upon arrival at the
laboratory. Westat will periodically inspect the record book and make a record of any such
inspections.
                                              36

-------
The laboratory is responsible for verifying that all microbiological (and other laboratory) data fields
are legibly filled out with apparently reasonable data. They will enter their initials and the date in
appropriate fields on the data sheets to indicate their review and acceptance. Spaces for date and
initials will be provided on all data sheets. The laboratory's initials will indicate their inspection and
acceptance of data sheets prior to their delivery to USEPA.

Transmission of deliverables is the  de facto indicator that the  data were completely reviewed and
believed to be accurate. The laboratory personnel responsible for reviewing the  data will be a person
that is different than the person who originally keyed-in the  data.

Making Corrections

On any hard copy data sheets, incorrect entries will be singly lined-out (i.e., not obliterated) and
correct results entered. If the party making the correction is  not the person who made the original
entry, then the date and  initials of the person modifying the  entry will be present next to the
correction.

Attachments

Westat will utilize the attachments referenced in the work assignment:
Attachment 1.


Attachment 2.



Attachment 3.

Attachment 4.

Attachment 5.

Attachment 6.
Total
Water
EPA Method 1600: Membrane Filter Test Method for Enterococd in
Water (www.epa.gov/microbes)

Improved Enumeration Methods for the Recreational Water Quality
Indicators: Enterococd and Escherichia cob, EPA/821/R-97/004
(www.epa.gov/microbes)

Sample Collection/Custody Form

Analytical Services Request Form for Chemical Samples

Estimation Procedure for Too-Numerous-To-Count Plates

Rapid Polymerase Chain Reaction (PCR)-Based Methods for Measuring
       Enterococod, Total Bacteroides, and Bactenoides Thetoiotaomicron in
       Samples
Attachment 7.
Method 1602
                                             37

-------
Saliva Samples:

The saliva study will be an additional effort designed to complement the basic NEEAR Water study
which relies on water samples and questionnaires. The saliva study will be conducted at the
Boqueron Beach, Puerto Rico site only.  Saliva samples will be collected from active NEEAR water
study participants using a simple sponge with a handle upon after they have completed the Part B
questionnaire at the beach site.  Active NEEAR water study participants includes persons who are
actively and successfully participating in  the current questionnaire process. Participants who are not
able to complete the questionnaires successfully will become ineligible for the saliva collection.
Participants will be mailed additional sponges to collect and return via mail to investigators  10-12
days and again six weeks  later.  The saliva sample goal for active NEEAR water study participants is
between 2500-2600 participants, each contributing a maximum of three samples.

The NEEAR Water study targets and offers enrollment to all beachgoers between 11:00 AM and
5:00 PM. Beachgoers are approached by trained interviewers between these hours and asked if they
would like to participate in a survey. Those who agree complete an initial baseline interview.  After
the Part B interview is completed, interviewers will ask participants in the  NEEAR Water study first
if they would be interested in providing saliva samples. They will briefly describe the study
emphasizing the following points: 1) three samples will be collected, today, after 10 days, and after
six weeks, and tested for germs  that cause gastrointestinal and other illness; 2) sample collection is
simple and takes about a minute (they will show the  saliva sampling kit); and 3) they will be
reimbursed $10 for each  sample and won't incur any costs  for shipping. Participants will be
provided $10 for each saliva sample received, for a maximum of
Westat will enroll active participants from the NEEAR water study survey in the saliva collection
investigation.  Adults 18 years of age and older shall be provided a consent form to review and a
contractor interviewer shall be present to answer questions about the study. Unaccompanied minors
(under 18 years of age) will not be eligible for enrollment. Parents will provide consent for children
under 7, and will provide permission for all children under the age of 18.  While the adults and
adolescents are reading and signing their consent and assent forms, the contractor interviewer shall
read the assent form to them and to any children age 7-14. Westat will store consent forms in a
locked portable filing cabinet on the beach until they can be returned to the field office at the end of
the study day. Bilingual interviewers (English/Spanish) shall be available at each site to facilitate
enrollment of Spanish speaking subjects.

Westat will ship all saliva samples to the US EPA in Chapel Hill, NC, Attn: Elizabeth Sams, 104
Mason Farm Rd., Chapel Hill, NC  27514. All samples shall be shipped with appropriate containers
and refrigeration. Westat will email the tracking numbers to sams.elizabeth(g),epa.gov. and
hudgens.edward(g),epa.gov. Westat will include all shipments with appropriate sample logs and
custodial forms. All shipment logs and custodial forms must be preapproved by the WACOR.
                                              38

-------
TASK?
Antisera:

Westat will produce coliphage antibody that will be used for the quantification and typing of
coliphage from bathing beach water. Westat will produce at least 150 ml of antisera for 7 different
coliphage antigens.  Westat will use the coliphage antigens and antisera preparation protocol by the
US EPA for producing coliphage antiserum. Westat will aliquot the antisera in 5 ml volumes in
containers suitable for freezing at very low temperatures.  Westat will deliver 200 microliter-lml of
antisera from each individual animal to the US EPA 2-3 weeks after the second injection indicated in
the protocol.
DELIVERABLES
TASK1
       Deliverable 1
       Deliverable 2
       Deliverable 3
TASK 2
       Deliverable 4
       Deliverable 5
Electronic beach questionnaires (English and Spanish) shall be loaded
to appropriate number of Beach Interviewing instruments by Friday,
May 8, 2009.  Electronic telephone interviews shall be loaded to the
appropriate number of telephone data collection computers by
Friday, May 15, 2009.

Beach Interviewer training sessions shall be completed by Friday,
May 15, 2009 in Puerto Rico and by Friday June 5 in South Carolina.
A report outlining the training schedule, verifying interviewer
completion of the program, and Westat comments shall be due June
19, 2009. EPA/CDC representatives will have access to CAPI
devices throughout training sessions.

Telephone Interviewer training sessions shall be completed by
Monday, May 25, 2009. A report outlining the training schedule,
verifying interviewer completion of the program, and contractor
comments shall be due Friday, June 19,  2009. EPA/CDC
representatives will have access to  CATI devices throughout training
sessions.
CATI telephone interviewer(s) shall implement trial interviews with at
least three (3) members of the NEEAR Water Study project team
designated by the WACOR or COR prior to Monday, May 25, 2009.

Westat will distribute $25 incentive checks and Participation Status
letters to each household within 30 days of completing the phone
interview.
                                             39

-------
TASK 3
       Deliverable 6
      Deliverable 7
      Deliverable 8
      Deliverable 9
TASK 4
       Deliverable 10
TASKS
      Deliverable 11
      Deliverable 12
Production Reports are due the close of business the Wednesday
following the weekend of data collection. The exceptions to this are
the weekends of May 22-25, 2009, July 17-20, 2009 and July 24-27,
2009 (Production Report is due the following Friday) (Production
Report is due the following Tuesday).  The final Performance
Production Reports are due September 25, 2009.

The comprehensive database is due for each data collection weekend
at close of business 10 calendar days after the final collection day.
Westat will submit this database by individual data collection days.  A
final of the comprehensive database and final database report is due
Friday, September 25, 2009.

A draft of the Final Report covering this work assignment evaluating
the questionnaires, training and recommendations for improvement is
due Friday, October 16, 2009. The Final Report, covering this work
assignment, evaluating the questionnaires, training and
recommendations for improvement is due Friday, October 30, 2009.

Transfer  of project-generated products for Tasks 1-4 (including
electronic products) are due Friday, November 20, 2009.  These
include data, computer-assisted interviews, databases, reports, and
other materials created under this work assignment.
Westat will update QAPP modified in Work Assignment
2-04 as needed and provide intermediate updates prior to next data
collection weekend (can be done electronically). A final QAPP is due
at the end of the project, February 24, 2010.
Submit the final version quality assurance project plan modified in
Work Assignment 2-04 for Task 5 and appropriate additional SOPs,
as necessary, to USEPA by the end of the project, February 24, 2010.

Submit the final version of all data and result forms to USEPA by
November 6, 2009.
      Deliverable 13
      Deliverable 14
Provide USEPA with the results of all analyses daily by facsimile,
except the PCR, for the first two weekends and weekly (i.e., after the
completion of the analyses from each weekend) thereafter. If
problems occur that result in the need to monitor the data more
closely, results shall be provided daily. PCR results shall be sent to
USEPA as soon as the analyses are completed.

Provide USEPA with a copy of the sampling records, sample custody
forms, and ancillary  data weekly.
                                            40

-------
      Deliverable 15
      Deliverable 16
      Deliverable 17
      Deliverable 18
      Deliverable 19
      Deliverable 20
TASK 6
       Deliverable 21
TASK?
      Deliverable 22
Provide USEPA with a copy of the photographs at the end of the
sampling at the beach.

Provide USEPA with a report of any problems encountered and the
actions taken as soon as possible after the occurrence.

Provide USEPA with membrane filter verification data from all of the
samples from one day (either the Saturday or Sunday) of the first
weekend.

Provide USEPA with a final report on the readiness review/dry run,
any problem encountered, and the actions taken.

Provide USEPA with a final Task 5 report within 30 days after the
completion of all analyses from all methods. This report should
include suggestions for improvement of future studies, maps of the
beach sites with the transects indicated along with the permanent
landmarks used to locate the transects, and beneficial procedures or
other observations that might be helpful in future studies.

Transfer of project-generated products for Task 5 (including
electronic products) are due Friday, January 15, 2010. These include
data, computer-assisted interviews, databases, reports and materials
created under this work assignment.
Westat will ship saliva samples to USEPA within the week of saliva
sample collection.
Westat will ship coliphage antibody by February 25, 2010.
REPORTING REQUIREMENTS
Westat will furnish a copy of the Work Plan, as well as each section of the combined monthly
technical and financial progress reports which relate to this Work Assignment directly to the Work
Assignment Contracting Officer Representative at the same time progress reports are submitted to
the Contracting Officer Representative and Contracting Officer.

Special reporting requirements include documentation of all sources and contacts so as to fully
reference the sources of all information. Three copies of each final report shall be provided to the
WACOR or COR. Deliverables shall be provided in hard copy and electronically CD ROM or via
email.  Reports shall be in Microsoft Word 2000 format or newer version, WordPerfect 9.0 or PDF
format. A complete set of all deliverables must be submitted at the end of the work assignment. A
complete checklist shall accompany this set of deliverables and the Format must be approved by the
WACOR or COR. Westat will maintain liaison with the WACOR or COR either by phone or email.
                                            41

-------
Acceptance criteria:  The quality of the data shall be judged by its internal consistency and agreement
with well established scientific knowledge, conformity with approved protocols, and completeness
both in terms of satisfying the requirements of the work assignment and in terms of adequately
characterizing the collected data in order to  ensure correct interpretation of the data users.

PERIOD OF PERFORMANCE
The period of performance of this work assignment is from the date of issue through February 24,
2010.

WORK ASSIGNMENT CONTRACTING OFFICER REPRESENTATIVE
DESIGNATION
The WACORwill be Elizabeth A. Sams, Environmental Health Scientist, US EPA/NHEERL
(MD-58C), Research Triangle Park, NC 27711.  919-843-3161 (phone) and 919-966-0655 (fax).
Sams.elizabeth(g),epa.gov

NOTICE REGARDING GUIDANCE PROVIDED UNDER THIS WORK
ASSIGNMENT
Guidance is strictly limited to technical and  analytical support. Westat will not engage in activities of
an inherently governmental nature such as the following:

       1.     Formulation of Agency policy;
       2.     Selection of Agency priorities;
       3.     Development of Agency regulations.

Should WEstast receive any instruction from an EPA staff person that Westat ascertains to fall into
any of these categories or goes beyond the scope of the contract or work assignment, Westat will
immediately contact the COR or the Contract Officer.

Westat asserts that the work under this work assignment does not contain any  real or apparent
personal or organizational conflict of interest. Westat will certify that none exist at the time the
work plan is  submitted to the EPA.

VIII.  Personnel

Westat proposes Ms. Karen Delia Torre (PL-4) as the Work Assignment Lead (WAL) and Project
Leader.  Ms. Delia Torre shall oversee all aspects of this work assignment. She has over 15 years of
experience in the environmental field,serving as program/contract director, a work assignment
manager, and senior analyst in support of EPA, including experience in managing high-visibility
heath and environmental studies.  Also, Ms. Delia Torre served as the Project Director for the
NEEAR Water Study from 2002-2008, has performed site supervision at West Beach (2003), Silver
Beach (2004), Washington Park (2004), Edgewater Beach (2005), Fairhope Beach (2007) and
Goddard Beach (2007), and  designed the CAPI data collection system  for the study.
Ms. Delia Torre holds  degrees in computer systems, medical engineering, an M.B.A. and is certified
as a Project Management Professional.

We propose Mr. Kurt Patrizi (PL-3) to serve as field director for this work assignment. Mr. Patrizi
holds degrees in Environmental Resource Management and Environmental Sciences and
Engineering and has 19 years of experience  in environmental regulation, management, policy,
                                            42

-------
analysis, training, and planning.  For over 13 years Mr. Patrizi has served as a work assignment
manager, program/contract director, and senior analyst in support of EPA.  Mr. Patrizi served as the
beach field director for the NEEAR Pilot Study during 2002 and has performed site supervision at
West Beach (2003), Silver Beach (2004), Washington Park (2004), Edgewater Beach (2005), Fairhope
Beach (2007) and Goddard Beach (2007).

We propose Dr. Robert Clickner (PL-4) to provide technical guidance for this project as necessary.
Dr. Clickner is an Associate Director at Westat and a senior statistician with 35 years of experience
in the development, implementation, and management of statistical and environmental research
projects, including four years experience  directing the Beaches water quality studies for EPA.  Dr.
Clickner has also designed, conducted and analyzed biostatistical experiments involving pesticides
and other environmental contaminants. His project management activities have included the
development and maintenance of project completion plans, quality assurance plans, schedules, and
budgets; management and coordination of multiple subcontractors, including numerous laboratories;
staff assignments; review of deliverables; and client coordination and communication. He has
developed and conducted international workshops on methodologies for human exposure
assessment field studies.

We propose Ms. Amy Kominski (PL-2) as Research Assistant to perform beach assessment tasks.
Ms. Kominski is a biologist, research assistant, with experience in collecting  epidemiologic research
data.  Prior to working at Westat, she worked at The National Institutes of Health in Bethesda,
Maryland. While at The NIH she worked in a clinical microbiology lab  and  has experience designing
and managing public health studies, including; protocol development and implementation of large
volume, multi-site research projects. Her specific experience includes infection control studies
involving antibiotic resistant bacteria, specifically Enterococd and Staphylococcus aureus. Ms Kominski is
proficient in general laboratory biochemical testing methods, quality assurance and control,
antibiotic susceptibility testing and state-of-the-art molecular assays.  Since joining Westat, Ms.
Kominski has been involved with the work done at Fairhope Beach (2007) and Goddard Beach
(2007) and has performed water sampling pilot studies and beach assessment studies.

Westat proposes Mr. David Barmettler (PL-2) to serve as site manager for the Boqueron Beach
site.  In this role, he will manage all data collection  activities performed at the site.  Mr. Barmettler
has extensive experience in supporting international public health studies and providing technical
assistance to a number of epidemiologic studies. Mr. Barmettler served EPA in the Community
Study of Common Infections  in Lawrence, MA, providing site supervision for the collection of
epidemiologic data and saliva  samples.  Mr. Barmettler has performed beach site assessments at
Goddard Beach, Huntington Beach, and  Washington Park Beach. He has coordinated travel plans,
logistic arrangements,  and equipment shipments to USAID missions in  various countries. Mr.
Barmettler holds an MPH and is fluent in Spanish.

Westat proposes Dr.  Sharon Jasim-Hanif (PL-2) as saliva study protocol developer and manager.
Having completed a Ph.D. in  Environmental Engineering and a M.Sc. in Environmental
Technology, she has extensive experience in scientific, laboratory, and field studies research. Dr.
Jasim-Hanif has provided support to many U.S. Environmental Protection Agency studies and has
served as Data Collection Supervisor for a wide range of Epidemiological field studies. For the
National Epidemiological and Environmental Assessment of Recreational Water Study, Dr. Jasim-
Hanif served as on-site Field Supervisor for the in-person interviews with beachgoers  at the
Edgewater Beach, Biloxi study site. She served as Field Supervisor for the water sample collection at
                                             43

-------
Edgewater Beach, Biloxi (MS), Washington Park, Michigan City (IN) and Silver Beach, St. Joseph,
(MI) study sites.  She is skilled in study and protocol design, fieldwork operations, data analysis and
management, literature review, and public outreach and health communication. She also has
experience interviewing, training, and supervising staff.

Westat proposes Ms. Sara Hader (PL-2) as quality assurance specialist for the environmental
sample collection.  Ms. Hader has 4 years of experience in study design, field data collection, data
analysis, and program evaluation for environmental and occupational health studies. She has
managed data and sample collection, and the packaging and shipment of environmental samples.
Ms. Hader holds a B.S. in microbiology and has served as quality assurance specialist for water and
sand sample collection at three previous NEEAR Water Study sites.

Westat proposes Ms. Naa Adjei (PL-2) as quality assurance specialist for the environmental sample
collection. Ms. Adjei has 3 years of experience in scientific study design, data collection, and data
analysis.  She has performed beach sites assessments.  Ms. Adjei holds a B.S. in neurobiology.

We propose Mr. Ron Hirschhorn (PL-3) as the systems and  data manager for the survey.  Mr.
Hirschhorn is an Associate Director with Westat who has overseen the development of many large-
scale field data collection and transmission systems. Mr. Hirschhorn holds degrees in mathematics
and has over 25 years of experience in systems analysis and design. Mr. Hirschhorn served as the
systems data manager for the NEEAR 2004 and 2005 and 2007 study beaches.

Ms. Helen Jewells (PL-2) will be the Telephone Research Center (TRC) Manager for the telephone
interviews and has  more than fifteen years of experience supervising telephone data collection staff
and conducting telephone interviews. She is responsible for training; scheduling and monitoring
interviewers; and editing, coding, and other data cleaning. She also has extensive experience with
recruiting, survey-design editing, sample reconciliation, and policies and procedures for refusal
conversion.  Ms Jewells served as the TRC interviewing manager for the 2004  and 2005 and 2007
study beaches.

We propose Ms.Stephane Ridore, Mr.  Marcus Walker, Mr. Michael Seppy, and Mr. Eddy
Mattingly (PL-1) to server as the user support specialists and to serve as interviewer trainers. Both
have over 3 years of experience  in computer applications, user support, and software testing.
                                             44

-------
Appendix C

Choosing the Best
Membrane Filter Count
             256

-------
                                        MERB SOP 041

          CHOOSING THE BEST MEMBRANE FILTER COUNT FOR THE CALCULATION
              OF THE FINAL CONCENTRATION OF MICROORGANISMS PER 100 ML
A count of 20-60 is the ideal counting range for any original Escherichi coli or Enterococcus
membrane filter colony count, regardless of the volume of sample tested. Whether a count falls
within the ideal counting range will ultimately affect the final concentration, but the ideal counting range
does not directly apply to it.  To obtain the final concentration, we look at the original counts to determine
which count to use in the calculation; then we adjust by the dilution factor to obtain the final concentration
(colonies/100 milliliters). The detailed procedure is as follows:

1.  Look at the actual colony count for the largest volume of sample tested, 100 milliliters in this case.
2.  If the count falls within the ideal counting range of 20-60, calculate the final concentration
(colonies/100 milliliters) by multiplying the count by the dilution factor. However, when the largest volume
tested is 100 milliliters, the count does not have to be adjusted (that is, the count is the final
concentration). If the largest volume is 10 milliliters or 1  milliliter, the count would have to be multiplied by
10 or 100, respectively.
3.  If the count in the largest volume of sample is  outside the ideal counting range of 20-60 on the
"low side" (that is. < 20), calculate the final concentration (colonies/100 milliliters) by multiplying that
count by the dilution factor, if any.  If the largest volume is 100 milliliters, the count js the final
concentration. If the largest volume is 10 milliliters or 1 milliliter, that count would have to be multiplied by
10 or 100, respectively.
4.  If the count in the largest volume of sample is  outside the ideal counting range of 20-60 on the
"high side" (that is. > 60), look at the actual colony count for the next (second) largest volume of
sample. In this case,  it is 10 milliliters.
5.  If the count in the second largest volume of sample falls within the ideal counting range of 20-60,
calculate the final concentration (colonies/100 milliliters) by multiplying that count by the dilution factor. In
this case, the dilution factor is 10.
6.  If the count in the second largest volume of sample is outside the ideal counting range of 20-60 on
the "low side" (that is. < 20) and the count for the largest volume of sample is > 60 but < 100, use
the count from the largest volume rather than the count from the second largest volume to calculate
the final concentration (colonies/100 milliliters) by multiplying the chosen count by the dilution factor, if
any. If the largest volume tested is 100 milliliters, the count is the final concentration.
7.  If the count in the second largest volume of sample is outside the ideal counting range of 20-60
on the "high  side" (that is. > 60), look at the count from the next (third) largest volume and repeat the
same steps as in 5-7  . Since the third largest volume is 1 milliliter, the dilution factor is 100.
8.  If the count in the third largest volume of sample falls within the ideal counting range of 20-60,
calculate the final concentration (colonies/100 milliliters) by multiplying that count by the dilution factor. In
this case, the dilution factor is 100.
9.  If the count in the third largest volume of sample is outside the ideal counting  range of 20-60 on
the "low side" (that is. < 20) and the count for the second largest volume of sample is > 60 but <
100, use the count from the second largest volume rather than the count from the third largest
volume to calculate the final concentration (colonies/100 milliliters) by multiplying the chosen count by the
dilution factor. In this case, the dilution factor would be 10.
10. If the count in the third largest volume of sample is outside the ideal counting range of 20-60 on
the "high side" (that is. > 60), look at the count from the next (fourth) largest volume,  if any, and
repeat the same steps as before .
11. If there is no additional (fourth largest) volume and the count for the third largest sample was too-
numerous-to-count, record as TNTC. greater than 100 multiplied by the dilution factor for the third
largest sample (100 in this case) or > 10.000.

-------
Appendix D

Quality Assurance Project
Plan: Water Sampling and
Testing
             258

-------
                           EPA Contract No. EPD-09-040
                           Work Assignment 0-01, Task 5
                      Quality Assurance Project Plan (QAPP)
                                  Revised Draft

                                       For

The National Epidemiological and Environmental Assessment of Recreational Water Study
                               for Beaches Program

                           Boqueron Beach, Puerto Rico

                                      And

                          Surfside Beach, South Carolina
                    U.S. Environmental Protection Agency (EPA)
               EPA Contracting Officer Representative: Elizabeth Sams
                                  Submitted by:
          Westat Work Assignment and Project Director: Karen Delia Torre
                Westat Water Quality Project Leader: Robert Clickner
                                December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                  Revised Draft
                                                                   Table of Contents
Chapter                                                                             Page

   1          INTRODUCTION AND OVERVIEW	     1

              1.1     Introduction	     1
              1.2     Background Information	     1
              1.3     Study Beach Site	     2
              1.4     Project/Task Organization	     2
              1.5     Proficiency/Certification	     4
              1.6     Safety Protocol	     4

   2          DOCUMENTS AND RECORDS	     5

              2.1     Data Deliverables	     5

   3          DATA GENERATION AND ACQUISTION	     17

              3.1     Overview   of  Sample  Collection  for  Microbiological
                     Analyses	     17
              3.2     Determination of Transects	     27
              3.3     Sampling Methods	     48

                     3.3.1    Preparation	     51
                     3.3.2    Sample Handling and Custody	     53
                     3.3.3    Labeling Procedures	     54
                     3.3.4    Water Collection at the Beach	     58
                     3.3.5    Sand Collection at the Beach	     63
                     3.3.6    Ancillary Data	     69
                     3.3.7    Afterthe Collection	     70
                     3.3.8    GPS Points and GPS ID	     71
                     3.3.9    End of Day/Weekend Procedures	     72
                     3.3.10  End  of  the  Collection  Weekend   Shipping
                             Procedures	     72

   4          ANALYTICAL METHODS	     76

              4.1     Microbiological Methods	     77

                     4.1.1    Standard Membrane Filter Method Enterococci
                             (Method  1600)	     77
                     4.1.2    Quantitative Polymerase Chain Reaction (QPCR)
                             Method	     81
WESTAT
               December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                                                 Quality Assurance Project Plan (QAPP)
                                                                   Revised Draft
                                                                    Contents (Continued)
Chapter

   5
Table

   1
   2
   4
   5

   6
   7
QUALITY CONTROL (QC)	     84

5.1     Corrective Actions	     84
5.2     Instrument/Equipment Testing, Inspection, and Maintenance	     85
5.3     Instrument/Equipment Calibration and Frequency	     85
5.4     Tracking and Inspection/Acceptance of Supplies and
       Consumables	     85
5.5     Data Management	     86
5.6     Readiness Review/Dry Runs	     86
5.7     Site Visits/Technical Systems Audits	     87
5.8     Routine Surveillance	     87

DATA VALIDATION AND USABILITY	     88

6.1     Data Review, Verification, and Validation	     88
6.2     Making Corrections	     90

PERSONNEL	     91

REFERENCES	     94


                                                                      Page

Measurements to be recorded at/for each sampling visit	     7
Measurements  recorded at/for each sampling visit and equipment
used	     9
Boqueron Beach sampling  schedule: Number of samples collected
by type of analysis	     23
Number of samples analyzed at Boqueron Beach	     24
Surfside Beach sampling schedule: Number of samples collected by
type of analysis	     25
Number of samples analyzed at Surfside Beach	     26
Summary of samples collected at each beach site	     59
Summary of the mEI Agar Method for Enterococci	     78
WESTAT
                                                                December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                   Revised Draft
                                                                        Contents (Continued)
   la          View of the initial weather station setup at Boqueron Beach, PR	      13
   Ib          Relocation of the weather station, Boqueron Beach	      14
   Ic          Westat's setup of an additional weather station	      15
   Id          Surfside Beach weather station on fishing pier	      15
   2a          Sample transects schematic	      17
   2b          Water sampling locations	      19
   2c          Water sampling locations for Cyanobacteria at 11:00 am	      21
   2d          Sand sample locations (points 7, 8 and 9) at 8:00 am	      22
   3           Schematic of Boqueron Beach	      27
   4a          Lefttransect (view from the  water)	      28
   4b          Left transect (Palm Tree)	      29
   4c          Lefttransect (Lifeguard Chair Ramp)	      30
   4d          Left transect (Lined Up)	      31
   5a          Center transect,  long view (view from water)	      32
   5b          Center transect (Palm Tree)	      33
   5c          Center transect (Handrail)	      33
   5d          Center transect (Lined-Up)	      34
   6a          Right transect (view from the water)	      35
   6b          Right transect (Front Tree)	      36
   6c          Right transect (Rear Tree)	      37
   6d          Right transect (Trees Aligned)	      38
   7           Schematic of Surfside Beach	      39
   8a          Lefttransect (view from the  water)	      40
   8b          Left transect (Edge of Building)	      41
   8c          Lefttransect (Fence Post)	      41
   8d          Left transect (Lined Up)	      42
   9a          Center transect,  (view from water)	      43
   9b          Center transect (Center of Condo)	      44
   9c          Center transect (Handrail or Walkway)	      44
   9d          Center transect (Lined-Up)	      45
   lOa         Right transect (view from the water)	      46
   lOb         Right transect (Yellow House)	      47
   lOc         Right transect (Rear House Windows)	      47
   lOd         Right transect (Structures Aligned)	      48
   11          An example of sample collection point in water	      59
   12          Start of collection	      60
   13          Submersion of bottle at  shin depth	      60
   14          Removal of excess water	      61
   15          Tightly recapping bottle	      61
   16          Collection of waist level samples	      62
   17          Continuation of waist level sample collection	      62
WESTAT
                December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                                                     Quality Assurance Project Plan (QAPP)
                                                                        Revised Draft
                                                                          Contents (Continued)
Appendices
   A
   B
Sample Flowchart	
Chain of Custody Template .
A-l
B-l
WESTAT
                                                                     December 7, 2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
                               INTRODUCTION AND OVERVIEW
1.1       Introduction

            Westat  is submitting this Quality Assurance  Project Plan (QAPP) in fulfillment of the
requirements of Work Assignment 0-01, Task 5. Under Work Assignment 0-01, Task 5,Westat conducted
and completed beach water sampling from May 15, 2009 through August 2, 2009 at Boqueron Beach,
Boqueron, Puerto Rico and from June 6, 2009 through September 7, 2009 at Surfside Beach, Surfside,
South Carolina, followed by laboratory analyses and database development.  In summary, this QAPP
seamlessly covers the work performed during this task.

1.2       Background Information

            In order to meet some of the requirements of the Clean Water Action Plan, the Beach Action
Plan and the Beach Act of 2000, this beach study was initiated in 2003 to assist the Office of Water in
formulating new health and risk guidelines for recreational water.

            This study  is being conducted jointly by the National Exposure Research Laboratory,
Microbiological and Chemical Exposure Assessment Research Division (NERL/MCEARD), the National
Health and Environmental Effects Research Laboratory (NHEERL) and the Centers for Disease Control
and Prevention (CDC).

            This information  is being collected as  part of  a research program consistent with the Sec.
3(a)(v)(l) of the  Beaches Environmental Assessment and Coastal Health Act of 2000 and the strategic
plan for EPA's Office of Research and Development (ORD) and the Office of Water entitled "Action
Plan for Beaches and Recreational Water." The Beaches Act and ORD's strategic plan has identified
research on  effects of microbial pathogens  in recreational waters as a high-priority research area with
particular emphasis on developing new water quality indicator guidelines for recreational waters.  This
data collection is for a series of epidemiological studies to evaluate exposure to and effects of microbial
pathogens in marine and fresh recreational waters as part of the EPA's research program on exposure and
health effects of microbial pathogens  in recreational waters.  The information collected by this study
program will be used to estimate the relationship between water quality indicators and health effects. The
questionnaire health data will be compared with routinely  collected water quality  measurements.  The
WESTAT                                       1                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
analysis will focus on determining whether any water quality parameters are associated with increased
prevalence of swimming-related health effects.

1.3       Study Beach Site

           The study period from took place from May 15, 2009 through August 2, 2009 at Boqueron
Beach, Boqueron, Puerto Rico  and from  June  6, 2009 through September 7, 2009 at Surfside Beach,
Surfside, South Carolina.  This  work  assignment implements field procedures for the water quality data
collection at a beach study site and the follow-up telephone interviews.  Westat traveled to these sites.

           Boqueron,  Puerto  Rico  Beach Contact:  Eddie  Nieves  Santiago,  Assistant  Park
Superintendent Boqueron Beach, PR at 787-851-1900.

           Surfside, South Carolina Beach Contact: Ed Booth, Town Manager of Surfside Beach, SC
at 843-913-6111.

           The beach sites were selected based upon specific criteria listed below:

           •   The beach was an officially designated recreational area near a large population
                center.
           •   The beach generally meets the state or local water quality standards.
           •   The beach was contaminated by a human source of pollution.
           •   The beach has a large attendance (e.g., 300-400 swimmers/day)
           •   The age  range of the swimmers was broad (i.e., includes children, teenagers, and
                adults).
           •   The swimming season was at least 90 days long.
1.4        Project/Task Organization

            This study, which was necessary to meet the requirements of the Congressionally-mandated
Beach  Act of 2000,  was administered out of the United States Environmental Protection Agency's
WESTAT                                       2                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
(USEPA) National Exposure Research Laboratory, Microbiological and Chemical Exposure Assessment
Research Division  (NERL/MCEARD),  Cincinnati, Ohio. The project was  funded by the office of
Research and Development. The water quality monitoring  study was  conducted  by Westat, and the
concurrent health study was conducted  and funded by the National Health  & Environmental Effects
Research Laboratory (NHEERL), with the assistance of Westat.

           Westat  assembled a team  consisting  of experienced  Westat environmental  researchers,
qualified locally hired staff to collect the  water samples and four laboratories to analyze the water
samples. The Westat staff are described  in the personnel section of this QAPP. It was necessary to use
four laboratories because some of the analytical methods must be performed within eight hours of the
sample  collection, which  required a laboratory close to the beaches where  sampling took place. In
addition, certain analytical procedures, for example qPCR, require special expertise and licensing, which
was possessed by only relatively few laboratories. Thus, the laboratories were:

           •    The University of Puerto Rico Microbiology Lab onsite at The Department of
                Marine Sciences, UPR,  Isla Magueyes, La Parguera, Puerto Rico, performed the
                Method  1600:  Enterococd in Water by  Membrane  Filtration using  membrane-
                Enterococcus Indoxyl-|3-D-Glucoside Agar (Method 1600 membrane filtration) (EPA
                821/R-02/022), froze,  preparatory filtration steps  of  the  Rapid  Quantitative
                Polymerase  Chain Reaction   (QPCR), pH, turbidity,  conductivity and  salinity
                analyses  on the samples collected at Boqueron, Beach. Additionally, the  UPR
                Microbiology  Lab  collected  shipped samples  for  the Cyanobateria  analyses
                performed by Green Water Laboratory, Palatka,  Florida.

           •    Environmental Systems Testing Services, Conway, South Carolina, performed
                the Method 1600: Enterococd in Water by Membrane Filtration using membrane-
                Enterococcus Indoxyl-|3-D-Glucoside Agar (Method 1600 membrane filtration) (EPA
                821/R-02/022), froze,  preparatory filtration steps  of  the  Rapid  Quantitative
                Polymerase  Chain Reaction   (QPCR), pH, turbidity,  conductivity and  salinity
                analyses on the samples collected at Surfside, Beach.

           •    Green Water Laboratory, Palatka, Florida performed analysis of water samples
                collected at Boqueron Beach, Puerto Rico for the presence of Cyanobateria.

           •    EMSL Analytical, Inc., Westmont, New Jersey, completed the qPCR analyses on
                all samples collected at both Boqueron and Surfside Beaches.
WESTAT                                       3                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
1.5        Proficiency/Certification

            There was no specific training anticipated by USEPA for the current approved membrane
filter Enterococci method (mEI Agar; see References 7  and 8).  Westat ensured that analysts were
proficient in the above method and each of the rapid methods [Rapid Quantitative Polymerase Chain
Reaction (QPCR) and Human  Specific QPCR (See Reference  9).].  The laboratory analysts  who
performed the assays were proficient in each method and consulted with USEPA personnel, the technical
advisors, and the manufacturers of the instruments to ensure proper knowledge and use of the analytical
methods.  Westat required laboratory staff to process one  or two performance evaluation  samples
(unknowns) for all methods during the study. Westat ensured that the sub-contract laboratories followed
general field and lab safety protocols throughout the study period.

1.6        Safety Protocol

            Westat utilized a safety plan specific to EPD-09-040, Work Assignment 0-01.
WESTAT                                       4                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
                          2.     DOCUMENTS AND RECORDS
2.1       Data Deliverables

            Westat used sample collection/custody forms, approved by  USEPA.  Westat maintained
sample collection/custody sheets in a binder in the laboratory where the analysis was performed. Westat's
laboratories  initially recorded membrane  filter count  data  and lot  number  on  field or laboratory
worksheets, then entered and saved data electronically in an Excel spreadsheet. Microsoft Word was used
for the text in reports.  The worksheets, database format,  data sheets, or spreadsheets for this method and
all other methods in this study were approved by the USEPA.  A separate spreadsheet file was  employed
for each day of sample collection. The file name incorporated the dates or range of dates of collection.
Separate database forms were used to record the following information:
            •   Field measurement data (See  below),
            •   Sample  collection  time/analysis start  time/incubation  start  and  end time  (See
                Microbiological Method Section), and
            •   Microbiological data for each sample/volume and  associated quality control (QC)
                samples.

            All  data from  each  of the rapid  methods (counts,  CT values,  setup values, plots, growth
curves, graphs, computer files, notes, statistical parameters and analyses, etc.) were recorded, saved, and
submitted to USEPA in a hard copy (where applicable) and in electronic form.  A copy of all computer
files was sent to USEPA electronically by E-Mail with a cover letter outlining the contents of the files.
Additions or changes to the worksheet were approved by USEPA.

            A hard copy format  of hand-entered sample collection and custody sheets, data sheets, and
all other forms of data (graphs, bit maps, CT values, setup values, plots, growth curves, notes, QC data,
statistical parameters and analyses, etc.) for each sampling event was provided to the COR and WACOR
no later than the Monday  following each 3-day  weekend  period (Friday, Saturday  and Sunday) of
sampling each week at the beach site.  Westat also  entered the data electronically into the database
forms/spreadsheets, and, after the analyses were completed at the beach site, the electronic file(s) were
delivered to  the COR  and WACOR along with all other forms of data (graphs, bit maps, plots, curves,
setup values, notes, QC data, statistical parameters and analysis, etc). The delivery was accompanied by a
brief cover memo outlining the delivery contents by method.  The memo also indicated if there were any
WESTAT                                        5                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
unusual circumstances or known problems surrounding the deliverable, such as QC problems in any of
the methods.

            Critical data that were reported included the bacterial counts from the membrane filter tests;
the complete run files for the QPCR method, including CT and  setup values,  positive and negative
controls, etc; the complete run files. All membrane filter plates were examined and counted  [If possible,
plates with up to 200 colony forming units (CPUs) were to be considered countable, although the ideal
number of CPUs is 20-80.], and the results for all plates were reported, including zeros and those "too-
numerous-to-count" (TNTC). An estimation procedure for TNTC plates were provided to the laboratory
by USEPA.  The estimation data from the TNTC plates (i.e., the five counts from five squares on each
filter) was submitted to USEPA along with the count data for the other samples.  QC data was reported
with the sample data for all methods.

            In addition to the water samples that were  collected, ancillary  data was collected for each
sampling visit.  These  are shown in Table 1. Certain ancillary data items  were  entered using specific
measuring instruments. Table 2 describes the measuring instruments,  including model, range accuracy
and calibration procedures. All field data was entered with permanent non-running ink. In addition to the
items detailed in Table 1 and 2, any items/activities specific to the beaches were added.

            Any QC  results associated with the collection of ancillary data were also reported (QC
samples were specified in Methods/SOPs describing ancillary data collection methods).
WESTAT                                         6                                  December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                    Revised Draft
Table 1. Measurements to be recorded at/for each sampling visit
Measurement
Date and Time
Air temperature
Water
temperature
Cloud Cover
Rainfall
Wind speed
Wind direction
Current
Direction
Wave height, if
applicable
Bather density
Boats
Animals/Birds
Debris
Description
Date and Time of day
Measured by thermometer at a fixed location
every visit
Measured by thermometer at a fixed
sampling location at appropriate depth for
thermometer on every visit
Sunny, Mostly Sunny (20-50% cloud cover),
Cloudy (50-70% cover) Mostly Cloudy (70-
99% cover), Overcast
Measured by rain gauge near sampling area;
collected each day at time of sampling and
any time rain is known to have occurred at
the beach since the last measurement was
taken. Current conditions such as rain,
lightning, hail, etc. noted
Sustained speed measured by wind gauge;
gusts indicated in comments fields
Compass direction to nearest semi -quadrant
leeward measured on wind gauge
Described in relation to shoreline facing out
Meter stick measurement at central sampling
point. This is the distance from the low point
(trough) to the high point (peak) of the wave
Number of bathers in the water, in the
sampling area, and number of "bathers" on
beach, within outer transects to edge of
beach on land side
Number/approximate number of boats in the
water, within approximately 500 M of
sampling area
Animals and birds potentially affecting the
water (within approximately 20M of the
sampling area in the water or laterally within
20M of the outer transects on the beach);
also includes number of fowl or other birds
in the air near the sampling area
Description of any debris floating in the
water or washed on shore within the bathing
area
Units/Format
Mm/dd/yy;
hh:mm
°C
°C
S, MS, C, MC, O
Rain in inches;
other
observations
noted in
comments field
Miles per hour
N, NE,E, SE, S,
SW,W, orNW
Descriptive
(onshore, right,
etc.)
Meters
Categorical; <20,
20-100, 100-200,
>200
Categorical;
None, 1-5, 5-10,
10-20, 20-30,
etc., etc.
Types of
Animals,
Numbers of
Animal Types on
beach and in
water
Categorical;
"None," "Very
Little," "Little,"
"Lots," describe
types
MQOs
±5 minutes
±1°
±1°
Field Person or
Team
Consensus
±0.25 Inches
± 5 mph
Recorders
judgement
Field Person or
Team
Consensus
±0.2 M
Field Person or
Team
Consensus
Field Person or
Team
Consensus
Field Person or
Team
Consensus
Field Person or
Team
Consensus
WESTAT
                 December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                    Revised Draft
Table 1. Measurements to be recorded at/for each sampling visit (continued)
Measurement
pH
Turbidity
Salinity
Conductivity
UV Reading
Geographical
Position
Swim Advisory
Flags
Description
Each sample measured after microbiological
analysis processing, per "Standard Methods"
(3) or equivalent.
* Equipment utilized for this measurement
must be preapproved by WACOR or COR
Each sample measured by nephlometer after
microbiological analysis processing, per
Standard Methods (3) or equivalent
* Equipment utilized for this measurement
must be preapproved by WACOR or COR
Each sample measured after microbiological
analysis processing, per "Standard Methods"
(3) or equivalent and Measured on site
concurrently with temperature and air current
Each sample measured after microbiological
analysis processing, per "Standard Method"
(3) or equivalent
Measured by UV device
GPS Unit
Coordinates were taken in 3 places for each
of the 3 transects. Total of 9 positions for
each sample run (8:00 Am, 11:00 AM, 3:00
PM)
Flags put on beach by lifeguards or other
official to indicate if swimming is advised,
cautioned against, or unallowed for bacteria
levels, weather, or roughness of water.
Usually Green for Safe, Yellow for
Advisory, and Red for Unsafe/Not Allowed
Units/Format
pH units
Nephlometric
Turbidity Units
(NTUs)
Parts per
thousand
microSiemens or
milliSiemens as
appropriate
Units/Format
Lat/Long
Indicate if
advisory is due to
bacteria, weather,
or roughness of
water.
MQOs
± 0.2 units
Range
dependent; see
Standard
Methods
2130B
Field Person
Field Person
MQOs
Field Person or
Team
Consensus
Field Person
WESTAT
                December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                    Revised Draft
Table 2. Measurements recorded at/for each sampling visit and equipment used
Measurement
Air temperature
Water
temperature
Rainfall
Wind speed
Salinity
Conductivity
UV Reading
Geographical
Position
MQOs
±1°
±1°
±0.25
Inches
± 5 mph
Not
specified
Not
specified
Not
specified
Not
specified
Instrument
Kestrel 4000 Pocket
Weather Tracker
Range: -29 to 70°C
Accuracy: +/-1°C
YSI Model 30/30M
Range: -5 to 95°C
Accuracy: +/-0.1°C
Cole-Parmer03319-
00
Range: 0.00 to 11. 00
in
Accuracy: +/- 0.01
in
Kestrel 4000 Pocket
Weather Tracker
Range: 0.8 to 89.0
Accuracy: 3% of
reading
YSI Model 30/30M
Range: 0 to 80 ppt
Accuracy:
+/-2%, or+/-0.1ppt
YSI Model 30/30M
Range: 0 to 49.99
mS
Accuracy: +/-0.5%
UVP, Inc. UVX
Radiometer
Range:
Oto 1999(iW/cm2
Accuracy: +/-5%
Garmin GPS 76
Accuracy: < 15 m
Calibration
Factory calibrated: Temperature response of unit
was verified in comparison with a Eutechnics
4600 Precision Thermometer or a standard Kestrel
4000 Pocket Weather Tracker calibrated weekly
with the Eutechnics 4600.
Factory calibrated. Calibration checked and
adjusted weekly in field according to
manufacturer's protocol using 50 mS/cm
calibration solution.
Rain measured in graduated cylinder calibrated
during manufacturing. No further calibration
required.
Factory calibrated: The impeller installed in the
unit was individually tested in a subsonic wind
tunnel operating at approximately 6. 1 m/s
monitored by a Gill Instruments Model 1350
ultrasonic time-of-flight anemometer. Low-speed
function of impeller further verified following
wind tunnel testing.
Factory calibrated. Calibration checked and
adjusted weekly in field according to
manufacturer's protocol using 50 mS/cm
calibration solution.
Factory calibrated. Calibration checked and
adjusted weekly in field according to
manufacturer's protocol using 50 mS/cm
calibration solution.
Calibrated according to manufacturer's
recommendations. Both the sensor and the
radiometer are calibrated every year, prior to the
start of sampling. Equipment is shipped to
manufacturer for calibration.
Factory calibrated. Time zone set for specific
beach.
WESTAT
                December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
            Photographic Data

            To aid researchers in determining conditions at the beach that may not be readily apparent
from the ancillary data recorded, photographs of the sample  locations at the beach area were taken for the
record at least once a day during the sampling period at the beach site.  While the  work assignment
required taking photographs  only once a day, Westat took them at every sample collection, as in past
years, because the conditions on the beach changed substantially over the course of the day from 8:00 am
to 3:00 pm.  Photographs were of sufficient quantity and quality to estimate the number of bathers on the
beach and in the water.

            Photos were taken with a digital camera.  They were labeled with the beach name,  date,
target sample collection time, and actual time of photograph.  The camera was configured to  have the date
(and time, if possible) displayed on the image. The beach name, target sample time, and actual time of
photograph (when unable to configure  camera to display on image) were part of  the  image name.
Submission of photographs to the WACOR or COR occurred at the end of the  sampling period for the
beach via appropriate means, expected to be delivery of a CD-ROM with the digital photographs.

            Weather Station Data

            EPA and Westat setup HOBO Weather Stations at suitable locations near  the beach sites to
automatically collect data on sequential weather parameters. At Boqueron Beach, the weather station was
initially setup and tested by EPA Athens on a handicap access ramp on the beach (Figure la). However,
EPA moved the weather station prior to the start of the study because the location on the ramp was easily
accessible to potential vandals.  EPA Athens relocated the weather station  to the top of a lifeguard  stand
located  on the beach (Figure Ib).   Shortly after the study began, Westat reported  that some of the
parameters measured by the  Boqueron weather station were not being recorded or were out of normal
range. After a series of attempts to fix the weather station, Westat set-up an additional weather station on
the opposite side of the lifeguard stand (Figure Ic).

            Prior  to setting  up another weather station,  Westat made  a  series of adjustments and
replacements in attempt to alleviate the  problematic weather station parts that were not measuring and
recording the data properly. All attempts made to correct the problems were reported to EPA and methods
were developed in consortium with EPA.
WESTAT                                        10                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
            On Thursday, June 4, 2009 Westat recognized that rain and barometric pressure data were
not being measured by the device. As a first attempt at troubleshooting the problem, Westat instructed
field staff as well as local Westat tech support staff to check all connections to and from the instrument to
verify that this was not the problem. Westat field staff reported that the cable connections were intact,
and the device was still  not measuring rain and barometric  pressure.   EPA  informed Westat that the
weather station manufacturer said that the faulty readings were likely due to bad sensors. EPA suggested
turning switching out the rain and barometric pressure sensors with replacement parts belonging to EPA
and located in the storage facility on site.

            On June  11, 2009 Westat  instructed field staff to locate the extra rain gauge and pressure
sensor and install them on the weather station. Westat field staff located the replacement parts.

            On June 25, 2009 EPA again suggested that the port that connected the sensors might have
been faulty and to  switch the ports in which the cables were connected before  installing the replacement
sensor parts.  Westat instructed field staff to attempt to fix the weather  station by testing the rain gauge
and barometric pressure sensors in different ports and to follow up with the replacement of the rain gauge
and pressure sensor if switching ports did not work.

            On the June  26, 2009 the cables were tested in all ports by Westat field staff. While all other
parameters were successfully recorded, the rain and barometric pressure data  was not recorded. Westat
staff also connected the replacement EPA rain gauge and barometric pressure  sensor and found that still
neither parameter was recorded. After testing all of the possibilities, Westat sent another replacement rain
gauge and pressure sensors as well as a replacement logger (that were not in use)  from the South Carolina
field office to Boqueron, PR. The Westat replacement rain gauge and barometric pressure sensors were
connected.

            On June 28, 2009 Westat field staff forwarded weather station data to Westat home office
which indicated that data for rain and barometric pressure was still not being measured.

            On July 9, 2009 Westat field staff examined  the weather station  again  and observed loose
connections in the data logger box. A  photograph was taken and sent  it to Westat  home office.  EPA
replied with a photo of where the cables should be connected on the instrument.  The next day, field staff
attempted to  fix the rain gauge and barometric pressure sensors by connecting  to an additional logger.
Westat staff noted that the logger was not measuring rain due to the fact that the  cable came out once the
WESTAT                                        11                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
door was closed.  The cable was replaced and the Westat logger was measuring rain and barometric
pressure data.

            On  July 11, 2009 Westat field staff checked the weather station again and found that all
parameters, with the exception of UV and battery status, were not being recorded by the EPA logger.
Westat's logger was reading rain data and barometric pressure.

            July 13, 2009 EPA  suggested a few different methods to troubleshoot the malfunctioning
weather station. Westat tried each suggestion to get the device logging properly, but was unsuccessful.

            July 15, 2009 Westat sent all remaining weather station parts from the South Carolina field
house to Boqueron, PR.  These replacement sensors were set up on the Westat logger.

            July 20, 2009 Westat reported to EPA that the additional weather station was installed and
all parameters were being collected.

            In Surfside Beach, the weather station was setup at the end of a fishing pier just south of the
sampling area (Figure Id).

            Westat collected weather parameters/units  from May 15, 2009 through August 2, 2009 at
Boqueron Beach, Boqueron, Puerto Rico and from June 6, 2009 through September 7, 2009 at Surfside
Beach, Surfside,  South Carolina.. The parameters collected include:
            •    Dew Point (F)
            •    Barometric Pressure (in Hg)
            •    Rain (in)
            •    Relative Humidity (%)
            •    Solar Radiation (W/m2)
            •    UV Voltage (in Volts)
            •    Temperature (F)
            •    Wind  Speed (mph)
            •    Gust Speed (mph)
WESTAT                                        12                                 December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                        Revised Draft
             •   Wind Direction (compass point degrees)


            Westat's weather station was set to observe conditions at 1 minute intervals and to record
data at 10 minute intervals.
            Figure la.  View of the initial weather station setup at Boqueron Beach, PR
WESTAT
13
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                     Quality Assurance Project Plan (QAPP)
                                         Revised Draft
                   Figure Ib. Relocation of the weather station, Boqueron Beach
WESTAT
14
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                         Revised Draft
                     Figure Ic. Westat's setup of an additional weather station
                     Figure Id. Surfside Beach weather station on fishing pier
WESTAT
15
December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
            General Laboratory Quality Control Records

            Laboratories were expected  to  maintain  records  of general  laboratory quality control
activities, such as are described in this QAPP and the references.  Such records may become deliverables
upon an amendment to the work assignment.

            Data Formats

            The  exact format of all data fields were  approved by USEPA prior to data collection.
Formats were based on those specified for this project in the work assignment, in the references, or in the
forms recommended by the manufacturers of the  instruments.  Where  possible,  database/spreadsheet
templates had fields preformatted.

            Quality Assurance Plan and Revisions

            All project personnel received copies of the most current version of the  Quality Assurance
Project Plan (QAPP) prior to dry run.

            Other Records

            Various other documents  and  records (e.g., SOPs,  reports, method validation records,
laboratory QC, and maintenance records)  are discussed in this document in appropriate sections.  The
USEPA reserves the right to request copies of any documents and records from Westat that could affect
this project.  Any records that are received, and any records generated  by Westat became part of the
overall project file.
WESTAT                                        16                                  December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                  Quality Assurance Project Plan (QAPP)
                                     Revised Draft
                    3.     DATA GENERATION AND ACQUISTION
3.1       Overview of Sample Collection for Microbiological Analyses

           Before beginning sample  collection  at the beach  sites, the  sampling  locations  were
identified. These locations were in waist-high water (1 meter deep), shin-high water (0.3 meters deep),
and in wet-sand along pre-selected transects. The transects were lines perpendicular to the shoreline,
selected to represent the water areas frequented by the beachgoers. At both Boqueron and Surfside Beach
there were three transects, with shin-high, waist-high, and wet-sand samples along each transect, for a
total of nine sampling locations (Figure 2a).
                     transect
                                            Center
                                            transect
                      Right
                      transect
                            Beach
                            Figure 2a. Sample transects schematic
           At Boqueron Beach, Westat collected beach water samples on specified weekend (Saturday
and Sunday) sampling days during the study period of May 16 ,  2009 through August 2n , 2009,
including two weekday holidays (Mondays), May 25th, 2009 and July 27th, 2009 for microbiological
analysis by three methods [the current approved membrane filter (MF) Enterococci method (mEI Agar),
Rapid Quantitative Polymerase Chain Reaction (QPCR), and for Cyanobacteria analysis].
WESTAT
17
December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
            At Surfside Beach, Westat collected beach water samples on specified weekend (Saturday
and Sunday) sampling days during the study period of June 6, 2009 through Sept. 7, 2009, including two
weekday holidays, Friday July 3, 2009 and Monday, Sept. 7, 2009 for microbiological analysis by two
methods [the current approved  membrane  filter  (MF) Enterococci  method (mEI  Agar),  Rapid
Quantitative Polymerase Chain Reaction (QPCR)].

            Individual Water Samples

            Three times a day, at 8:00 AM, 11:00 AM, and  3:00 PM, water samples were collected at
both beach sites along each of the three transects perpendicular to the beach shoreline, one in waist-high
water (1 ml deep)  and one in shin-high water (0.3 m deep), for a total of 18 samples collected per day
(i.e., 6 grid locations x three times per day). See Figure 2b, which depicts the water sampling scheme.
Water samples were collected at the points numbered 1 through 6.  The location of the transects were at
least 20 meters apart or more, if the area used by the swimmers encompasses more than a total of 60
meters of shoreline.   It was intended that samples were collected on the scheduled dates, but other dates
may have been substituted if rainfall or  other problems prevented swimmers from going to the beach,
prevented water sampling, or created hazardous conditions for the field personnel.  Sample  collectors
notified the WACOR or COR of adverse weather conditions  or other problems and requested guidance
whether to begin or  continue sampling on  a given day or weekend.   This  was necessary  because the
samples needed to be collected when there were sufficient bathers at the beach to allow NHEERL to
conduct their concurrent epidemiological/health study.
WESTAT                                        18                                  December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                     Quality Assurance Project Plan (QAPP)
                                       Revised Draft
                               Water
            Waist
            depth
            Shin
            depth
                              Beach
                    Left
                    Transect
Center
Transect
Right
Transect
                                 Figure 2b.  Water sampling locations
            Composite Samples

            At both beach sites, additional samples were collected at the same six points and at the same
three times of day as the beach water individual samples.  These additional samples were used to develop
composite samples.  One plastic 1-liter bottle was collected at each of the six locations  on the grid in
Figure 2b for microbial analysis. At the lab these samples were combined to create a composite sample,
as described in the "Compositing Samples Protocol" and briefly here:

               •   Two 300 mL portions  from the  3 bottles collected at points 1, 3,  and  5 were
                   combined to form two shin composite samples, one for MF and one for PCR.

               •   Two 300 mL portions  from the  3 bottles collected at points 2, 4,  and  6 were
                   combined to form two waist composite samples, again, one for MF and one for PCR.

               •   150mL from each bottle collected at points 1-6 was combined to form two total
                   composite samples, one for MF and one for PCR.
WESTAT
  19
               December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
            It was intended that samples were collected on the scheduled dates, but other dates may have
been substituted if rainfall  or other problems prevented swimmers from going to the beach, prevented
water sampling, or created  hazardous conditions for the field personnel. Sample collectors notified the
WACOR or COR of adverse weather conditions or other problems and requested guidance whether to
begin or continue sampling  on a given day or weekend.  This was necessary because the samples needed
to be collected when there were sufficient bathers at  the  beach to  allow NHEERL  to conduct their
concurrent epidemiological/health study.

            Cyanobacteria Samples

            At Boqueron Beach only, Westat  collected water samples in pre-prepared  polypropylene
bottles for Cyanobacteria analysis.  Specifically, samples were collected from the waist depth location at
each of three transects  (sampling  points 2, 4,  and 6,  see Figure 2c) at the 11AM sampling  time on
weekend and holiday water sampling days.  Nine bottles total were filled.  At each of the three  sites the
following bottles were filled: two 237 ml  bottles, and one 40 ml bottle.   At the local lab, 1% Lugols
iodine was added to the 40mL bottle as a preservative. The water collectors completed documentation of
sample collection in an EPA-provided sample log.  Samples were refrigerated or kept  on ice within 30
minutes of collection and until they were shipped to Green Water Laboratories in Palatka, Florida.

            As  with  the other water samples, it was  intended that the  Cyanobacteria samples were
collected on the scheduled dates, but other dates may have been substituted if rainfall or other problems
prevented swimmers from going to the beach, prevented water sampling, or created hazardous conditions
for the field personnel. Sample collectors notified the WACOR or COR of adverse weather conditions or
other problems  and requested  guidance  whether to begin or continue sampling on a given day or
weekend.  This was necessary because the samples were collected when there were sufficient bathers at
the beach to allow NHEERL to conduct their concurrent  epidemiological/health study.
WESTAT                                        20                                 December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                      Revised Draft
                  Figure 2c. Water sampling locations for Cyanobacteria at 11:00 am.
            Sand Samples:

            Westat collected three beach sand samples per day on Saturdays, Sundays, and on weekday
holidays [Monday May 25, 2009 and Monday July 27, 2009 for Boqueron Beach and Friday July 3, 2009
and Monday September 7, 2009 for Surfside Beach] for both beach sites, for microbiological analysis by
two methods [the current approved membrane  filter (MF) Enterococci method (mEI Agar) and Rapid
Quantitative Polymerase Chain Reaction (QPCR)].  These samples were taken in the designated area of
the beach associated with the water quality samples. Westat employed a "scoop" method to collect the
samples.

            Westat collected three sand samples per day at  8:00 AM along  with the 8:00 AM water
samples.  Samples of wet sand were collected 1 meter from the lowest water level (when the waves
receded from the shoreline) at the same 3 transects where water samples were collected. See Figure 2d,
which is a schematic that shows the collection of the water and sand samples at 8:00  am.   Sand samples
were collected at points 7, 8 and 9.  When the sand was not wet at 1 meter from the water, the sand
collection location was moved the shortest possible distance toward the water to a location where the sand
WESTAT
21
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                       Revised Draft
was wet. Westat recorded the actual distance from the water.  Global Positioning System (GPS) readings
of the actual sand collection locations and a photo of the sample collection sites were taken.

            As with  the water samples,  it was  intended that  sand samples  were collected on  the
scheduled dates, but  other dates may have been  substituted if rainfall or other problems  prevented
swimmers from going to the beach, prevented water sampling, or created hazardous conditions for the
field personnel.  Sample collectors notified the WACOR or COR of adverse weather conditions or other
problems and requested guidance whether to begin or continue sampling on a given day or weekend. This
was necessary because the  samples were collected when there were sufficient bathers at the beach to
allow NHEERL to conduct their concurrent epidemic logical/health study.
                 Figure 2d.  Sand sampling locations (points 7, 8 and 9) at 8:00 am
            Table 3 details the number of samples collected and Table 4 details the number of samples
analyzed at Boqueron Beach.  Table 5 details the number of samples collected and Table 6 details the
number of samples analyzed at Surfside Beach.  Any variance in the number of samples collected is due
primarily to weather-related complications that prevented the collection of samples during the planned
collection times. These situations are documented in the following tables.
WESTAT
22
December 7, 2009

-------
National Epidemic logical and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                      Revised Draft
Table 3: Boqueron Beach sampling schedule:


Weekend
1

2


3

4

5

6

7

8

9

10


11


12

Totals


Day Date Day
1 5/16/2009 Sat
2 5/17/2009 Sun
3 5/23/2009 Sat
4 5/24/2009 Sun
5 * 5/25/2009 Mon
6 5/30/2009 Sat
7 5/31/2009 Sun
8 6/6/2009 Sat
9 6/7/2009 Sun
10 6/13/2009 Sat
11 6/14/2009 Sun
12 6/20/2009 Sat
13 6/21/2009 Sun
14 6/27/2009 Sat
15 6/28/2009 Sun
16 7/4/2009 Sat
17 7/5/2009 Sun
18 7/11/2009 Sat
19 7/12/2009 Sun
20 7/18/2009 Sat
21 7/19/2009 Sun
22 *7/20/2009 Mon
23 7/25/2009 Sat
24 7/26/2009 Sun
25 * 7/27/2009 Mon
26 8/1/2009 Sat
27 8/2/2009 Sun

Number of samples collected by type of analysis
Water Samples

MF
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
6
18
18
18
18
18
468

PCR
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
6
18
18
18
18
18
468

Composite Cyano
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
18 9
6
18 9
18 9
18 9
18 9
18 9
468 234
MF = Membrane Filtration Enteroeocci Method
PCR = Polymerase Chain Reaction Method
Composite =
Composite Samples (generated from beach water samples)

Sand
samples Total Notes
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 66
3 21 8AM Sample Collected Only
3 66
3 66
3 66
3 66
3 66
78 1,737
Cyano= Cyanobacteria
Sand Sample = Sand Samples
* Indicates a weekday sample
WESTAT
                                                                                                                                   December 7, 2009

-------
   National Epidemiological and Environmental
   Assessment of Recreational Water Study
                                Quality Assurance Project Plan (QAPP)
                                                    Revised Draft
   Table 4: Number of samples analyzed at Boqueron Beach
Weekend Date
1
2
3
4
5
6
7
8
9
10
11
12
5/16/2009
5/17/2009
5/23/2009
5/24/2009
*5/25/2009
5/30/2009
5/31/2009
6/6/2009
6/7/2009
6/13/2009
6/14/2009
6/20/2009
6/21/2009
6/27/2009
6/28/2009
7/4/2009
7/5/2009
7/11/2009
7/12/2009
7/18/2009
7/19/2009
7/20/2009
7/25/2009
7/26/2009
*7/27/2009
8/1/2009
8/2/2009
Beach Water
(separate samples were collected)
MF
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
6
18
18
18
18
18
PCR
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
6
18
18
18
18
18
Beach Composite
(collected sample was split)
MF
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
3
9
9
9
9
9
PCR
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
3
9
9
9
9
9
Sand Sample
(collected sample was split)
MF
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
PCR
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
                        474
474
237
237
81
81
Beach Water = Water Samples
Beach Composite = Composite Samples (generated from beach water samples)
Sand Sample = Sand Samples
Cyanobacteria = Remote Analysis of Cyanobacteria
MF = Membrane Filtration Enterococci Method
PCR = Polymerase Chain Reaction Method
   WESTAT
             24
                                    December 7, 2009

-------
National Epidemic logical and Environmental
Assessment of Recreational Water Study
Quality Assurance Project Plan (QAPP)
                      Revised Draft
Table 5:

Surfside Beach sampling schedule:

Weekend Day
1

2

3

4

5


6

7

8

9

10

11

12

13

14


Totals
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

Date
6/6/2009
6/7/2009
6/13/2009
6/14/2009
6/20/2009
6/21/2009
6/27/2009
6/28/2009
*7/3/2009
7/4/2009
7/5/2009
7/11/2009
7/12/2009
7/18/2009
7/19/2009
7/25/2009
7/26/2009
8/1/2009
8/2/2009
8/8/2009
8/9/2009
8/15/2009
8/16/2009
8/22/2009
8/23/2009
8/29/2009
8/30/2009
9/5/2009
9/6/2009
* 9/7/2009

Day
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Fri
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Sat
Sun
Mon
Number of samples collected by type of analysis
Water Samples

MF
-
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
12
18
18
18
18
12
18
18
18
18
18
18

PCR
-
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
12
18
18
18
18
12
18
18
18
18
18
18
510 510

Composite
-
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
12
18
18
18
18
12
18
18
18
18
18
18
510

Cyano
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
MF = Membrane Filtration Enterococci Method
PCR = Polymerase Chain Reaction Method
Composite = Composite Samples
Sand
samples
-
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
87

Total
0
57
57
57
57
57
57
57
57
57
57
57
57
57
57
57
57
57
39
57
57
57
57
39
57
57
57
57
57
57
1,617

Notes
All Collections Cancelled- Weather

















3PM Collection Cancelled- Weather




3PM Collection Cancelled- Weather







Cyano= Cyanobacteria
Sand Sample = Sand Samples
* Indicates a weekday sample
WESTAT
                                                                                                                                   December 7, 2009

-------
    National Epidemiological and Environmental
    Assessment of Recreational Water Study
                                             Quality Assurance Project Plan (QAPP)
                                                                 Revised Draft
    Table 6: Number of samples analyzed at Surfside Beach
Weekend Date
NA
1
2
3
4
5
6
1
8
9
10
11
12
13
14
6/1/2009
Dry Run
6/6/2009
6/7/2009
6/13/2009
6/14/2009
6/20/2009
6/21/2009
6/27/2009
6/28/2009
7/3/2009
7/4/2009
7/5/2009
7/11/2009
7/12/2009
7/18/2009
7/19/2009
7/25/2009
7/26/2009
8/1/2009
8/2/2009
8/8/2009
8/9/2009
8/15/2009
8/16/2009
8/22/2009
8/23/2009
8/29/2009
8/30/2009
9/5/2009
9/6/2009
* 9/7/2009
Beach Water
(separate samples were collected)
MF
18
-
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
12
18
18
18
18
12
18
18
18
18
18
18
PCR
18
-
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
12
18
18
18
18
12
18
18
18
18
18
18
Beach Composite
(collected sample was split)
MF
9
-
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
6
9
9
9
9
6
9
9
9
9
9
9
PCR
9
-
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
6
9
9
9
9
6
9
9
9
9
9
9
Sand Sample
(collected sample was split)
MF
3
-
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
PCR
3
-
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
               Total
528
528
264
264
90
90
Beach Water = Water Samples
Beach Composite = Composite Samples (generated from beach water samples)
Sand Sample = Sand Samples
MF = Membrane Filtration Enterococci Method
PCR = Polymerase Chain Reaction Method
    WESTAT
                         26
                                               December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                                                      Quality Assurance Project Plan (QAPP)
                                                                         Revised Draft
3.2
Determination of Transects
            Boqueron Beach, Puerto Rico

            Figure 3 is a schematic diagram of Boqueron Beach that identifies the transect locations and
corresponding sampling points (numbered as  1-9). The sampling area is defined as points located in the
area within the wheelchair water access structure (far left, when standing on the  shore and facing the
ocean) and the long  pier (far right, when standing on the shore facing the ocean).  Each transect was
identified by the alignment of two permanent structures.  Samples 1, 3 and 5 were the shin-depth water
sampling locations.  Samples 2,  4, and 6 were the waist-depth sampling locations.  Samples 7, 8, and 9
were the sand sampling locations.  The  left transect was the left-most sampling point when standing on
the shore and facing the water. Samples 1, 2, and 7 were taken at the left transect. The right transect was
the right-most sampling point when standing on the shore and facing the water.  Samples 5, 6, and 9 were
taken at the right transect. The center transect was in between the left and right transects. Samples 3, 4,
and 8 were taken at the center transect.
                            Figure 3:  Schematic of Boqueron Beach
WESTAT
                                  27
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                               Revised Draft
Left Transect - Samples 1, 2 and 7

The left transect was located  approximately N 18.01938 and W -67.17223.  The structures
used to identify the transect were the  furthest most left lifeguard stand (red circle)  and a
palm tree (yellow circle)  that was located behind it, as  shown  in Figure 4a, below.  The
sample point was defined by the alignment of the center of the ramp on the lifeguard stand
with the palm tree so that the palm tree was in the center of the lifeguard stand.   A close-up
view of the structures that defined the left transect can be  viewed in Figures  4b  and 4c.
Figure 4d depicts the transects  lined up. Sample  1 was  collected in shin-deep water where
these two structures appear aligned, as in Figure 4d. Sample 2 was collected in waist-deep
water along the same transect. The sand sample 7 was collected one meter from the water's
edge in wet sand along the same transect.
                      Figure 4a: Left transect (view from the water)
WESTAT
28
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                  Revised Draft
                             Figure 4b: Left transect (Palm Tree)
WESTAT
29
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                 Revised Draft
                      Figure 4c: Left transect (Lifeguard Chair Ramp)
WESTAT
30
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                  Revised Draft
                              Figure 4d: Left transect (Lined Up)
WESTAT
31
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                               Revised Draft
Center Transect - Samples 3, 4, and 8

The center transect was located approximately N 18.02076 and W -67.17220. The structures
used to identify the transect were the second or center lifeguard stand (red circle) and a palm
tree (yellow circle) that was located behind it,  as shown in Figure 5a, below. It was defined
by the alignment of the ramp handrail of the lifeguard platform with the  palm tree.  The
handrail  that was lined  up with the tree was  the  left-side rail (when sampler faced  the
lifeguard stand, and had their back to  the water).  A close-up view of the structures that
defined the center transect can  be viewed in Figures 5b and 5c.   Figure 5d depicts  the
transects lined up.  Sample 3 was collected in shin-deep water where these two structures
appear aligned, as in Figure 5d.  Sample 4 was collected in waist-deep water along the same
transect.  Sand sample 8 was collected one meter from the water's edge in wet sand along the
same transect.
                 Figure 5a:  Center transect, long view (view from water)
WESTAT
32
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                 Revised Draft
                           Figure 5b: Center transect (Palm Tree)
                            Figure 5c: Center transect (Handrail)
WESTAT
33
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                  Revised Draft
                            Figure 5d: Center transect (Lined-Up)
WESTAT
34
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                              Revised Draft
Right Transect - Samples 5, 6, and 9

The right transect was located approximately 30 feet south of N 18.02201 and W -67.17231.
The transect was identified by two  palm trees approximately 30 feet south of the lifeguard
stand (Figure 6a) that are in a line perpendicular to the shoreline.  Originally, the transect was
planned  to be in front a lifeguard stand, similar to the other transects. However,  during
training it was discovered that there were slippery rocks in the water that made collection of
the water samples hazardous. As a result the transect was moved about 30 feet to avoid the
rocks. The front tree is the second large tree to the right of the lifeguard stand, when viewed
from the shore (Figure 6b). The rear tree is several yards behind  it (Figure 6c).  Figure 6d
shows the two trees lined up. Sample 5 was collected in shin-deep water where  these two
structures appear aligned, as in Figure 6d.  Sample 6 was collected in waist-deep water along
the same transect. Sand sample 9 was collected one meter from the water's edge in wet sand
along the same transect.
                                               -30' to new transects
                     Figure 6a: Right transect (view from the water)
WESTAT
35
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                  Revised Draft
                            Figure 6b: Right transect (Front Tree)
WESTAT
36
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                               Revised Draft
           fequaretstand
                          Figure 6c: Right transect (Rear Tree)
WESTAT
37
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                              Revised Draft
                        Figure 6d: Right transect (Trees Aligned)

       Surfside Beach, South Carolina

Figure 7 is a schematic diagram of Surfside Beach that identifies the transect locations and
corresponding sampling points (numbered as 1-9). The sampling area was defined as points
located in the area within 7th Ave  N and 2nd Ave N. Each transect was identified by the
alignment of two permanent structures.   Samples 1, 3 and  5 were the shin-depth water
sampling locations. Samples 2, 4, and 6 were the waist-depth sampling locations. Samples 7,
8, and 9 were the sand sampling locations.  The left transect was the  left-most sampling
point when  standing on the shore and facing the water. Samples 1, 2, and 7 were taken at
the left transect. The right transect was the right-most sampling point when standing on the
shore and facing the water. Samples 5, 6, and 9 were  taken at the right transect. The center
WESTAT
38
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                               Revised Draft
transect was in between the left and right transects.  Samples 3, 4, and 8 were taken at the
center transect.
                                                        9 I'
                                                     t,	3= Vjr,,	„
                                                                    '•(I  II
                                                                    - --:A A.
                         Figure 7: Schematic of Surfside Beach
Left Transect - Samples 1, 2 and 7

The left transect was located approximately N 33.60884 and W - 78.96704.  The structures
used to identify  the transect were the edge of a grey condo located closest to the 6th Ave N.
entrance to of the beach (red circle) and the end of a fence located in front of the condo that
blocked the sand dunes (yellow circle), as shown in Figure 8a, below.  It was defined by the
alignment of the far left edge of the condo (when the sampler faced the building/back to the
water) ramp with the fence edge  so that the fence was directly in line with the building.   A
close-up view of the structures defining the left transect can be viewed in Figures 8b and 8c.
Figure 8d shows the transects lined up. Sample 1 was collected  in shin-deep water where
these two structures appear aligned, as in Figure 8d.  Sample 2 was collected in waist-deep
WESTAT
39
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                                Revised Draft
water along the same transect. Sand sample 7 was collected one meter from the water's edge
in wet sand along the same transect.
                       Figure 8a: Left transect (view from the water)
WESTAT
40
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                 Revised Draft
                         Figure 8b: Left transect (Edge of Building)
                                                                             "••   ' J    - ,   '
                            Figure 8c: Left transect (Fence Post)
WESTAT
41
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                               Revised Draft
                           Figure 8d: Left transect (Lined Up)

Center Transect - Samples 3, 4, and 8

The center transect was located approximately N 33.60811 and W-78.96767.  The structures
used to identify the transect were the center of a grey condo building (red  circle) and the
railing of a wooden walkway (yellow circle) that was located in front of it, as shown in Figure
9a, below.  The transect was defined by the alignment of the center of the condo building
with the right railing of the walkway (when sampler faced the building). A close-up view of
the structures that defined  the center transect can be viewed in Figures 9b and 9c. Figure
9d shows the transects lined up. Sample 3 was collected in shin-deep water where these two
structures appear aligned, as in Figure 9d. Sample 4 was collected in waist-deep water along
the same transect. Sand sample 8 was collected one meter from the water's edge in wet sand
along the same transect.
WESTAT
42
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                  Revised Draft
                        Figure 9a:  Center transect, (view from water)
WESTAT
43
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
            Quality Assurance Project Plan (QAPP)
                                 Revised Draft
                       Figure 9b: Center transect (Center of Condo)
                     Figure 9c: Center transect (Handrail of Walkway)
WESTAT
44
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
             Quality Assurance Project Plan (QAPP)
                                  Revised Draft
                            Figure 9d: Center transect (Lined-Up)
WESTAT
45
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                     Revised Draft
Right Transect - Samples 5, 6, and 9

The right transect was located approximately N 33.60763 and W -78.96815.  The structures used to
locate  the sampling location were the corner of a yellow house  (red line) and  the  second floor
windows (yellow circle) in the grey house behind the yellow house. Both structures can be viewed in
Figure lOa. The transect was defined by the alignment of the side of the yellow house so that it
blocks the 2 of the 3 windows of the grey house behind it. A close-up view of the structures defining
the center transect can be viewed in Figures lOb and lOc.  Figure lOd shows the transects lined up.
Sample 5was collected in shin-deep water where these two structures appear aligned, as in Figure
lOd.  Sample 6 was collected in waist-deep water along  the  same transect.  Sand sample 9 was
collected one meter from the water's edge in wet sand along the same transect.
                        Figure lOa: Right transect (View from the water)
WESTAT
46
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                      Revised Draft
                           Figure lOb: Right transect (Yellow House)
                        wi
                        III M ^^^Hi
                       Figure lOc: Right transect (Rear House Windows)
WESTAT
47
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                      Revised Draft
                                                     I
                         Figure lOd: Right transect (Structures Aligned)
3.3        Sampling Methods

            Water Samples:

            See Standard Methods for the Examination of Water and Wastewater. 20th edition (1998),
Section 9060, for recommendations on microbiological sampling (3).  Briefly, samples were collected in
waist-high (1 m deep) and shin-high (0.3 m deep) water by serially immersing (2) capped 1000-mL pre-
sterilized, polypropylene bottles to the appropriate sample depth, removing the lids and allowing them to
fill, raising them out of the water, and emptying them slightly to allow approximately 1 inch of head
space before replacing the lids.  Samples were taken about 1 foot (0.3 m) under the surface of the water in
waist-high water, and shin-high samples were taken 6 inches (0.15 m) above the bottom of the water.  The
samples  collected  near the  bottom were  taken  with  care  so  as  not  to introduce  additional
sand/solids/debris into the samples.  Sample plans were only altered in extreme or unusual circumstances.
When  alterations of the sample method were  considered,  the  WACOR or COR were notified and
guidance was requested.  Westat utilized field  protocols from Work Assignment 2-04  of the  previous
WESTAT
48
December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
contract.  Such field protocols  and sampling procedures were submitted to the WACOR or COR for
approval prior to the beginning of the study.

            Water samples were collected aseptically, as described above, at each location on the beach
grids (Figure 3 for Boqueron Beach and Figure 7 for Surfside Beach) for microbial analysis:

            —     1  liter  was used for the membrane filter method and the ancillary measurements
                  (which were  done last to  prevent contamination).
            —     1 liter was used for the rapid QPCR method.
            —     1 liter was used for the composite samples.
            —     Two 237 ml  bottles and  one 40 ml bottle were used for the  Cyanobacteria samples at
                  the  11:00 AM sampling  period, at sampling points 2, 4, and 6 only.  These samples
                  were only collected at the Boqueron Beach site.

            Following collection, all samples were placed in coolers and maintained on ice during
transport and at 1 - 4° C during the time interval before they were analyzed or shipped. No additional
samples were collected for the determination of pH  and turbidity. These measurements were made from
the same  samples  used  for membrane filtration  after these analyses were  completed to  prevent
contamination.

            Any  problems encountered while sampling or while taking ancillary measurements were
recorded on data collection sheets in comment fields or on additional sheets clearly having identified the
date, time, sample location (on grid)  and  reported to the WACOR or COR if problems may have
potentially affected the analytical results.  In  the event of problems, corrective actions taken (where
possible) were documented by the field team leader,  along with the results of such actions.
            Sand Samples

            Westat collected, transported,  and processed the  sand samples according to the protocol
provided by EPA on April 2, 2007.  Sand samples were collected with sterile,  2 inch x 10 inch stainless
steel liners (AMS, American Falls, Idaho, or the equivalent).  The liner was pushed into the sand at least 8
inches.  The liners were sterilized at the lab  by rinsing them with water, wrapping them in aluminum foil
or suitable bag and heating them in the drying oven  at 170°C overnight. The liners remained wrapped in
the aluminum foil until use.  Liners containing the sand samples were capped at both ends, placed in zip-
WESTAT                                        49                                 December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
lock plastic bags labeled using an alpha-numeric system (See below.), and transported to the laboratory
on ice. Samples were stored in a refrigerator at 4 degrees C. until analyzed.

            In the  laboratory,  sand  samples  were  aseptically  transferred to  sterile  wide-mouth
polypropylene bottles (500 ml or 1- liter, depending on the quantity of the sand), also labeled using the
simplified version of the usual alpha-numeric labeling system.  For each sand sample, 75 grams of sand
was aseptically weighed out in a sterile, pre-tared, wide-mouth_500-ml bottle (using sterile spatulas), and
300  ml of  Standard Methods phosphate-buffered  rinse/dilution water (3),  measured  with  a sterile
graduated cylinder, was added to each bottle.  Each bottle was vigorously shaken 50 times. Immediately
after shaking, some of the contents of the bottle were poured into two sterile 50-ml, disposable centrifuge
tubes (Corning 430829  or the equivalent) and filled to the 50-ml mark.  The tubes were centrifuged for 5
minutes at -3000 rpm (600 x g) to bring down the sand and sediment, and the supernatant was removed
using a sterile pipette  and placed in a sterile  100-ml polypropylene  bottle  for subsequent analysis by
Method 1600 (Reference 8) and the Quantitative Polymerase Chain Reaction (QPCR) method (Reference
9).

            The accuracy of the 50-ml mark on the disposable tubes was checked before the dry run by
randomly  choosing 5 tubes from the package, weighing each of the 5 tubes, and recording the weights.
After 50 ml of distilled water was measured with a graduated cylinder and poured into each of the tubes,
the tubes were again weighed.  The weight of the distilled water (The difference between the two weights)
in each tube was required to be close to  50 grams.  The position of the water meniscus was observed with
reference to the 50-ml mark on the tubes.  In addition, 5 randomly chosen, pre-weighed tubes were filled
with distilled water so that the meniscus touched the top of the 50-ml line. The tubes were weighed again
and the weight of the water was determined by difference. For the mark to be accurate, the weight of the
distilled water was required to be close to 50 grams.  All results were recorded, and a copy of the results
was sent to the WACOR and Kristen Brenner, the technical point-of-contract for sand analyses.

            During the dry run, aliquots of 10 ml and 1 ml of each undiluted sand extract and 1 ml of the
10"1  - 10"6 dilutions of each extract in phosphate-buffered dilution  water (3) was analyzed by EPA
Method 1600 for Enterococci.  The number of filtrations for the actual study was reduced after the normal
range of concentrations in sand were determined during the dry run.  Three 20-ml aliquots of each sample
was filtered, and the  filters were frozen, as described in the QPCR Method, during the dry run.  The sand
extraction method described above and the volumes  used for both tests may have  been adjusted,
WESTAT                                        50                                 December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                         Revised Draft
depending  on the normal range of concentrations  of Enterococci  in the extracts during the dry run.
Westat obtained EPA's approval before changing the protocol or analyzing volumes.

3.3.1      Preparation


            On the day prior to collection, all the EPA single-use polyethylene water collection bottles,
stainless steel sand liners, the related forms,  Cyanobacteria sample collection bottles (Boqueron Beach
only), and other materials were pre-assembled and pre-labeled for both days of the collection weekend.
These items included:


            —    Bottles and sand collection  containers for the Saturday 8:00 AM collection:  18
                  pre-labeled polyethylene bottles and 3 pre-labeled sterilized stainless steel liners per
                  beach.

            —    Bottles for the Saturday  11:00 AM collection:  18 pre-labeled polyethylene bottles
                  and 9 Cyanobacteria sample collection bottles (for Boqueron Beach only).

            —    Bottles for the Saturday 3:00 PM collection:  18 pre-labeled polyethylene bottles
                  per beach.

            —    Bottles and sand collection containers for the Sunday 8:00 AM collection:  18 pre-
                  labeled polyethylene  bottles  and 3 pre-labeled sterilized  stainless steel  liners per
                  beach.

            —    Bottles for the Sunday 11:00 AM collection:  18 pre-labeled polyethylene bottles
                  and 9 Cyanobacteria sample collection bottles (for Boqueron Beach only).

            —    Bottles for the Sunday 3:00  PM collection:  18 pre-labeled polyethylene bottles per
                  beach.

            —    6 coolers  per beach  site (one each for the 8:00, 11:00, & 3:00 EPA collections, one
                  for the sand collections, one for the Cyanobacteria collections (Boqueron Beach only),
                  and one for the icepacks)

            —    Filling the freezer with icepacks

            —    Labeling the 4-page lab transmittal sheets and ancillary data forms

            —    Checking the operation of the  Digital camera

            —    Checking the GPS device

            —    Checking the UV meter
WESTAT                                        51                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
            —     Checking the YSI  30-M Temperature/Salinity/Conductivity meter,  in  water-
                  resistant bag
            —     Checking the Handheld Weather Monitor
            —     Checking the Compass
            —     Checking the Meter Rule
            -     Checking the Rubber Mallet
            —     Checking the Water Thermometer in the waterproof pouch with a probe
            —     Checking the Sterlized end caps for sand collection
            —     Checking the Collection clipboard with wax pencil
            —     Checking the Procedures Manual
            —     Checking the EPA collection log book with paper forms
            —     Checking the 4 Storage containers (one with supplies to take to the beach, and one
                  each to hold the bottles for the Sunday 8:00, 11:00, & 3:00 collections until they can
                  be put in the coolers after the Saturday collection)

            Once all sample  containers for a collection time  period were  labeled  (see Labeling
Procedures), the set of collection containers were put into their respective coolers reserved and labeled for
each time period (8:00,  11:00, and 3:00).   The collection bottles and sand containers for the  Sunday
collections were first put into storage containers, until after the  Saturday collections were completed and
the Sunday collection bottles  and sand containers could be  transferred to their correct coolers  in
preparation for the next day. To assist in preventing the  incorrect collection of a sample in the wrong
container, the containers for each time period were separated by sample points and placed in different
mesh bags.

            All of the collection materials and  supplies such as paper towels, batteries, life vests, life
saver ring, mesh bags to hold collection bottles, scuba gloves, plastic bags to put the water collection
bottles into, paper ties to close the plastic bags, extra polyethylene (EPA), stainless steel liners (sand) and
glass  (USGS) water collection  bottles were put into a large plastic storage  container used to assist in
transporting to the beach collection site.
WESTAT                                        52                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
3.3.2         Sample Handling and Custody

            Westat utilized sample collection/custody forms modified from forms used at previous beach
sites as part of the general forms. The distribution of each individual bottle taken at each location on the
beach grid was documented on the custody forms.

            Prior to sampling visits, tracking forms  were printed by a member of the site project team.
The  location, date, target collection time, field staff, and information about all samples to be collected
during that visit was entered on the forms, by hand (or electronically, prior to printing).  The forms were
printed on laminated paper suitable  for field work. Each cooler used to transport samples from the site to
the lab had a copy of the appropriately completed collection/custody form(s) in it or securely attached to
it. Westat knew in advance how many samples could fit in a given cooler and could, therefore, prepare
specific tracking sheets for each cooler prior to going to the field. When more than one cooler were
needed, the coolers were labeled, and cooler labels were cross-referenced on the appropriate tracking
sheet.  Individual bottles for the rapid methods were distributed after the samples were  logged in at the
laboratory,  and the custody forms were signed by each of the method analysts when portions/aliquots of
the samples were removed.

            Additional columns on the tracking forms include the  actual collection time, the time
samples arrived at the laboratory, and their storage location. Arrival time at the laboratory was indicated
by entering a time for the first sample on a custody  sheet, and drawing  a down arrow in the lab arrival
time column for the rest of the samples. The field storage location was filled in this manner.

            A different form  (or forms) was used to record the dates and times when  analysis by QPCR
and filtering began (MF and  QPCR), the dates and  time plates were placed in the incubator  (or filters
were placed in the  freezer for  the QPCR method), the dates and time samples were removed  from
incubation (or freezer for the  PCR method), and the analysis results. There were spaces for associated
initials for each of the sequential steps. The various "analysis" times were treated on a batch basis; i.e., a
sample batch was all of the samples brought to the laboratory at the same time for analysis, such as all 6
morning samples.
WESTAT                                        53                                 December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
3.3.3      Labeling Procedures

            Microbiological sample containers were labeled with water resistant sample labels.  The
sample bottles had IDs with consecutive numbers to facilitate handling in the laboratory and to prevent
errors. However, Westat was  responsible for placing the requisite additional information onto  sample
bottles at the time of sampling to ensure that the samples could be clearly identified. It was recommended
that the information (or at least  alphanumeric information, such as suggested directly below) be added just
prior to or just after sampling, as this would minimize the chance of getting samples in the wrong bottles.
Information added  included the  date, scheduled and  actual  time  of collection,  and some type  of
alphanumeric that identified the sampling location, and the method(s) used.

            Westat used the following sample labeling  scheme for  all water and sand samples.  This
scheme is the similar to one that has been used in past years for sampling.  Microbiological  sample
containers will be labeled  with water resistant sample  labels  using the following alphanumeric (10-
character) scheme (to avoid  confusion and duplicate sample numbers):
               FMMDDXXNSS
               Where:
               F designates the beach area. B for Boqueron Beach and  S for Surfside Beach
               MMDD is the date of the sample collection;
                      MM is the numeric month (1-12) and
                      DD is the day (01-31), e.g., 0614 for June 14,
               XX is the planned time of day for the sample collection,  as follows:
                      08 = 8:00 am
                      11 = 11:00 am
                      15 = 3:00 pm
               N is the sample point at the beach;
                      1-6  for water samples and
                      7-9  for sand samples
                        (see Figure 2a)
               SS is the method of analysis planned for the sample/bottle number, as
       follows:
                      01 = Membrane Filter Method 1600
                      02 = QPCR Methods
                      SI = Sand container
WESTAT                                        54                                 December 7,2009

-------
National Epidemiological and Environmental                              Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                   Revised Draft
                    Cl = Beach Sample to be Composited
                    5a = Cyanobacteria, bottle a (473 mL) *
                    5b = Cyanobacteria, bottle b (237 mL) *
                    5c = Cyanobacteria, bottle c (40 mL) *

* At Boqueron Beach Site Only
       The following provides examples of the sample IDs that would be used for samples collected on
Saturday June 20. Examples of all necessary labels are given.


          Boqueron Beach, Puerto Rico
8:00 am:

Water:
MF: B062008101, B062008201, B062008301, B062008401, B062008501, B062008601

PCR: B062008102, B062008202, B062008302, B062008402, B062008502, B062008602

Sand:
B0620087S1, B0620088S1, B0620089S1

At the lab the sand samples were processed  and each split into two new "children" samples that were
analyzed by MF and PCR.

Beach Composite:
B0620081C1, B0620082C1, B0620083C1, B0620084C1, B0620085C1, B0620086C1

At the lab the composite samples were combined in this fashion:
Shin Composite = B0620081C1 + B0620083C1 + B0620085C1
Waist Composite = B0620082C1 + B0620084C1 + B0620086C1
TotalComposite = B0620081Cl+B0620082Cl+B0620083Cl+B0620084Cl+B0620085Cl+B0620086Cl

Each new child sample (8 AM shin  composite, 8 AM waist composite, and 8 AM total composite) were
split into 2 more children samples (total of 6) and analyzed for MF and PCR.


11:00 am:

Water:
MF: B062011101, B062011201, B062011301, B062011401, B062011501, B062011601

PCR: B062011102, B062011202, B062011302, B062011402, B062011502, B062011602

Composite:
B0620111C1, B0620112C1, B0620113C1, B0620114C1, B0620115C1, B0620116C1

At the lab the composite samples were combined in this fashion:
Shin Composite = B0620111C1 + B0620113C1 + B0620115C1
WESTAT                                    55                              December 7,2009

-------
National Epidemiological and Environmental                              Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                   Revised Draft
Waist Composite = B0620112C1 +B0620114C1 + B0620116C1
TotalComposite = B0620111C1+B0620112C1+B0620113C1+B0620114C1+B0620115C1+B0620116C1

Each new child sample (11 AM shin composite, 11 AM waist composite, and 11 AM total composite)
were split into 2 more children samples (total of 6) and analyzed for MF and PCR.

Cyanobacteria Water Sample:
B06201125a,  B06201125b, B06201125c,  B06201145a,  B06201145b, B06201145c,  B06201165a,
B06201165b,B06201165c
3:00 pm:

Water:
MF: B062015101, B062015201, B062015301, B062015401, B062015501, B062015601

PCR: B062015102, B062015202, B062015302, B062015402, B062015502, B062015602

Composite:
B0620151C1, B0620152C1, B0620153C1, B0620154C1, B0620155C1, B0620156C1

At the lab the composite samples were combined in this fashion:
Shin Composite = B0620151C1 + B0620153C1 + B0620155C1
Waist Composite = B0620152C1 + B0620154C1 + B0620156C1
TotalComposite = 60620151C1+B0620152C1+B0620153C1+B0620154C1+B0620155C1+B0620156C1

Each new child sample (3PM shin composite, 3PM waist composite, and 3PM total composite) were split
into 2 more children samples (total of 6) and analyzed for MF and PCR.
       Surfside Beach, South Carolina
8:00 am:

Water:
MF: S062008101, S062008201, S062008301, S062008401, S062008501, S062008601

PCR: S062008102, S062008202, S062008302, S062008402, S062008502, S062008602

Sand:
S0620087S1, S0620088S1, S0620089S1

At the  lab these samples were  processed  and each split into two new "children" samples that were
analyzed by MF and PCR.

Beach Composite:
S0620081C1, S0620082C1, S0620083C1, S0620084C1, S0620085C1, S0620086C1
WESTAT                                    56                               December 7,2009

-------
National Epidemiological and Environmental                             Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                   Revised Draft
At the lab the composite samples were combined in this fashion:
Shin Composite = S0620081C1 + S0620083C1 + S0620085C1
Waist Composite = S0620082C1 + S0620084C1 + S0620086C1
Total Composite = S0620081C1+S0620082C1+S0620083C1+S0620084C1+S0620085C1+S0620086C1

Each new child sample (8:00 am shin composite, 8:00 am waist composite, and 8:00 am total composite)
were split into 2 more children samples (total of 6) and analyzed for MF and PCR.


11:00 am:

Water:
MF: S062011101, S062011201, S062011301, S062011401, S062011501, S062011601

PCR: S062011102, S062011202, S062011302, S062011402, S062011502, S062011602

Composite:
S0620111C1, S0620112C1, S0620113C1, S0620114C1, S0620115C1, S0620116C1

At the lab the composite samples were combined in this fashion:
Shin Composite = S0620111C1 + S0620113C1 + S0620115C1
Waist Composite = S0620112C1 + S0620114C1 + S0620116C1
Total Composite = S0620111C1+S0620112C1+S0620113C1+S0620114C1+S0620115C1+S0620116C1

Each new child sample (11:00 am  shin composite, 11:00 am waist composite, and  11:00 am total
composite) were split into 2 more children samples (total of 6) and analyzed for MF and PCR.

3:00 pm:

Water:
MF: S062015101, S062015201, S062015301, S062015401, S062015501, S062015601

PCR: S062015102, S062015202, S062015302, S062015402, S062015502, S062015602

Composite:
S0620151C1, S0620152C1, S0620153C1, S0620154C1, S0620155C1, S0620156C1

At the lab the composite samples were combined in this fashion:
Shin Composite = S0620151C1 + S0620153C1 + S0620155C1
Waist Composite = S0620152C1+ S0620154C1 + S0620156C1
Total Composite = S0620151C1+S0620152C1+S0620153C1+S0620154C1+S0620155C1+S0620156C1

Each  new child  sample  (3:00  pm  shin composite, 3:00 pm waist composite,  and 3:00  pm total
composite) were split into 2 more children samples (total of 6) and analyzed for MF and PCR.
WESTAT                                    57                               December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
            Westat understood that  sample containers could be reused after  proper cleaning and
resterilization or bottles, presterilized by the manufacturer, could be used.  Westat only used presterilized
bottles.  Westat obtained a copy of the manufacturer's sterilization certificate and/or recorded for each lot
was obtained.  In addition, the sterility of a few randomly-chosen bottles from each lot was tested before
field use by adding sterile Trypticase  Soy Broth to the bottles, incubating for 48-72 hours at 35°  C, and
observing the bottles for bacterial growth.  Prior to leaving for the  field, the sample team leader checked
to see that there were an appropriate number of sample bottles and sample ID labels for the sampling visit
(Bottles had labels attached prior to sampling, as it was demonstrated that this had no deleterious  effects
on the labels.) for the sampling visit. There were extra, unlabeled sample containers, and a means to label
them for back-up purposes.  Copies of completed sample collection/custody sheets were provided to the
WACOR or COR daily along with their associated data sheets.

3.3.4      Water Collection at the Beach

            All the necessary equipment and coolers (see preparation) were brought to the beach.  Then
for each time collection period the appropriate cooler with the collection bottles was taken down on to the
beach and used to store the empty and filled water sample bottles. The sample collection started at the  1st
transect on the left-side of the beach.  For each transect, the appropriate mesh bag was taken out with the
correct pre-labeled bottles.  To enter the water, the water collectors lined themselves up  with the line-of-
site markers for the transect point, and then walked in a straight line out to the appropriate water depth to
collect the  samples. The Supervisor marked the GPS coordinates and time for each of the six collection
points while the water collectors collected the water samples. Table 3 summarizes the different samples
collected at the three sampling time periods (SAM, 11AM and 3PM) for both beach sites.
WESTAT                                        58                                  December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                                          Quality Assurance Project Plan (QAPP)
                                                             Revised Draft
Table 7: Summary of samples collected at each beach site
     Type of Sample
        Boqueron Beach
          Surfside Beach
   Sand
   microbiology
3 locations
1 time (8 am)
1 cylinder, stainless steel
MF, QPCR	
3 locations
1 time (8 am)
1 cylinder, stainless steel
MF, QPCR	
   Beach    water
   microbiology,
   individual samples
6 locations
3 times (8 am, 11 am, 3 pm)
2 bottles, 1 L plastic each
MF, QPCR	
6 locations
3 times (8 am, 11 am, 3 pm)
2 bottles, 1 L plastic each
MF, QPCR
   Beach    water
   microbiology,
   composite samples
6 locations
3 times (8 am, 11 am, 3 pm)
1 bottle, 1 L plastic
Composite (MF, QPCR)
6 locations
3 times (8 am, 11 am, 3 pm)
1 bottle, 1 L plastic
Composite (MF, QPCR)
   Cyanobacteria
   Samples
3 locations (waist deep)
1 time (11 am)
3 bottles, prepared and supplied
by GW lab,  237 mL, 237 mL, 40
mL
Refrigerate and ship to GW lab
NA
       Sample Collection Steps


       In the water, the water collectors retrieved from the mesh bag and verified each of the IDs on the
bottles before collecting a sample at each of the six sample points.  Staying in-line with the line-of-site
poles and transect points, the water collectors first pointed themselves into the direction of the current and
took the water sample pointing into the current holding the bottle in front of and away from their body.
(See Figures below for more detail).
                          Current
                                     Shore line

                    Figure 11. An example of sample collection point in water
WESTAT
                       59
                         December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                        Revised Draft
1.       The water collectors oriented and prepared themselves to collect the water samples pointing into
        the direction of the current, away from their body.
                                   Figure 12. Start of collection
2.      The collection bottle ID was checked for each bottle used at each sample point and the cap was
        slightly loosened. The cap, however, was NOT removed.
                           Figure 13.  Submersion of bottle at shin depth
WESTAT
60
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                       Revised Draft
        The bottle was submersed to a depth of approximately 6 inches for the shin level sample, and the
        cap removed to fill the bottle while holding the bottle perpendicular to the ocean bottom.  It was
        important to make sure the bottle was not very close to the ocean bottom. The start time of the
        sample collection was recorded by the field supervisor on the Water Sample Collection Log.
                               Figure 14. Removal of excess water
        Once the bottle was filled, it was recapped, and brought to the surface, to prevent surface water
        contamination. Then a small amount of water was removed, so that the  bottle was filled to the
        appropriate 1-L mark at shoulder of the collection bottle.
                               Figure 15. Tightly recapping bottle
WESTAT
61
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                        Revised Draft
        The bottle was tightly re-sealed, taking extra precautions to eliminate extended air exposure, and
        the filled collection bottles were placed into mesh bag, until they could be put back in the cooler.
                            Figure 16.  Collection of waist level samples

6.      After the shin samples were collected, the water collectors traveled out (staying in-line with the
        transect points and the line-of-site  poles) to the  1  meter collection point for the waist level
        samples.  The collection steps 1-5 were repeated.  Again, the bottle ID was verified for the
        sample point before collection, and the sample was collected into the current.
                      Figure 17. Continuation of waist level sample collection
WESTAT
62
December 7, 2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
7.     When repeating steps 1-5, field staff also made sure the bottle was only uncapped after being
       submerged to the 12 inch collection depth for waist  level samples, was recapped  before re-
       surfacing the sample (to prevent the sample from being contaminated by surface water), a portion
       of the sample was poured out so the sample level was close to the 1-L mark on the shoulder of the
       bottle, and the bottle was tightly resealed, to prevent loss of any sample collected.

8.     Then steps 1-7 were repeated for the other shin and waist level samples for the other transect
       points. NOTE: When collecting the middle transect samples, the water temperature, salinity and
       conductivity were measured by submersing the entire  probe to the collection point  depth and
       recording the temperature reading of the water during the  sample collection.  The average wave
       height was also estimated and recorded between the middle sample points.

3.3.5      Sand Collection at the Beach

            All the necessary equipment (see preparation) was brought to the beach.  For each transect
point, the appropriate  cooler with the collection containers was taken down on to the beach and used to
store the empty and filled water sand collection containers.   The sample collection  started at the 1st
transect on the left-side of the beach.  Three sand samples were collected only during the 8:00  AM water
collection time according to the following protocol.  The sand  samples were transported to the lab in a
cooler and stored in a refrigerator until analyzed.

            Saturday and Sunday 8:00 AM Sand Collection:
            1.    The sand samples were collected 1 meter from the lowest water level (when the waves
                 have receded from the shoreline) at the same 3 transects where water samples were to
                 be  collected.   The  meter stick was  laid down on the sand and the sand collection
                 sleeve was pushed  in the ground just at the top of the meter stick, so that the hole
                 created by the sleeve was not within the meter distance but the edge of the hole was a
                 meter away.  The sand should have been wet. If the sand was not wet at 1 meter from
                 the water, the sand collection location was moved the  shortest possible distance
                 toward the water to a location where the sand was wet.
            2.    The meter stick was used to record the actual distance from  the water on the sand
                 ancillary  data sheet once the sampling  location had been identified. Also anything
                 unusual or particular about the sand directly surrounding the sand sampling location
                 was recorded on the ancillary data form.
WESTAT                                        63                                 December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                       Revised Draft
            3.     A Global Positioning System (GPS) reading of the actual sand collection locations
                  was taken and the associated GPS recorded name  on the  ancillary data form was
                  recorded.

            4.     The first plastic bag labeled "7S1" from the cooler was obtained.  Then, the covered
                  sterile stainless steel sleeve was taken out.

            5.     The steel sleeve was wrapped in aluminum foil or a paper bag. If it was wrapped in
                  foil, a knife was used to remove the first two inches of aluminum foil from one end of
                  the sleeve.  The top of the aluminum foil was  removed by tracing/cutting 2 inches
                  from the top and then pulling off the end (similar to opening the top of a wine bottle).
                  The inside or the lip of the sterile steel sleeve was not touched If it was in a paper bag,
                  the bag was opened and the sleeve  removed, taking care not to touch either end.
                  The brown paper bag with the  sterilized tops was  opened.  One sterile  cap was
                  removed  while  trying not to touch any of the other caps as much as possible  or
                  touching the inside or lip of the cap itself.
WESTAT
64
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                       Revised Draft
            7.    The sterile cap was attached to the exposed end of the still covered sterile steel sleeve.
                  The rest of the aluminum foil was removed from the stainless steel sleeve and the
                  uncapped and now exposed end of the sleeve was not touched.

                  By hand, the sleeve was pushed straight down into the pre-determined sand sampling
                  location, at least 8 inches down into the sand.  See figure below.   If necessary the
                  rubber-headed mallet was used to tap the sleeve into the sand.
             10.  The sleeve was pushed into sand until only the cap showed.
WESTAT
65
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                         Revised Draft
             11.   The sleeve was pulled out of sand.
             12.   The side of the sleeve was tapped to slightly pack sand and the liner was checked that
                  it had enough sand (it should have had about 8 inches of sand).
WESTAT
66
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                     Quality Assurance Project Plan (QAPP)
                                          Revised Draft
             13.   The other end cap on was put on the sleeve.
WESTAT
67
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                        Revised Draft
            14.   The sleeve was capped and full of sand back and was placed in the same pre-labeled
                  zip-lock bag and then placed back in the cooler.
             15.   After the sand sample was collected, a picture of the sand sampling location was taken
                  that included the hole where the sand sample was removed and the water's edge. See
                  figure below.
WESTAT
68
December 7, 2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                   Quality Assurance Project Plan (QAPP)
                                      Revised Draft
            16.   The above steps 4-15 were repeated for each of the sand sampling locations, the bag
                 labeled "8S1" was removed for the second transect, and the bag labeled "9S1" was
                 removed for the third transect.

3.3.6      Ancillary Data

            Ancillary measurements listed in Table 1 were collected by a variety of means. Some were
collected by simple observation; others involved the use of equipment, such as pH meters, wind gauges,
and rain gauges.  It is  noted here that WACOR or COR approval  of any  deviation in methods was
required.

            For any ancillary data collection, especially that involving specific equipment, Westat was
responsible for documenting the exact methods used to collect the data, and to provide information about
the calibration and QC procedures for any equipment. This documentation was provided for approval to
the USEPA WACOR or COR prior to the occurrence of any field sampling.

            Appropriate field team members or lab team members were responsible for entering data on
appropriate data collection sheets. Westat may have proposed additional  QC activities related to sampling
and analysis in their QAPP and work  plan  as necessary and appropriate.  Any changes from the QC
specified  by  individual  method  technical  point-of-contacts  were  confirmed  with  them  before
WESTAT
69
December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
implementation. The ancillary data was typically taken by the field team supervisor as the water samples
were collected.  Due  to the nature  of the environment where the water collection occurred, the data
collected was put down on a clipboard using plastic-covered forms and a grease pencil.  This was to due
to the fact that the actual paper forms might get ruined from the splashing water from waves or from wind
and rain. The data was then transferred from the clipboard plastic sheet covered forms to the real forms
immediately after the sample collection.

            The data collected included air  temperature and  wind speed measured by  the handheld
weather monitor, and  wind direction using the compass.  The UV measurement was made prior to the
start of each sampling time.  Then estimates of cloud cover was made, along with an estimated count of
animals on the beach or in the water, the number of boats in the water, number of bathers at the beach or
in the water, amount of debris on the beach or in the water, rainfall measured by the rain gauge, the water
temperature, salinity and conductivity  at the middle sample points, current direction and average wave
height, and the GPS point measurements at each sample point during the collection of the  first bottle.
Additionally, approximately 5-7 digital pictures were taken of the surrounding area at each transect to
document and serve as backup for the collected ancillary data. If any problem or anything of interest was
located or occurred at the collection  site, it was recorded in comments on the collection form, and digital
images were taken, if necessary.

            The photographs were downloaded from the cameras to the field office computer and saved
as jpg images with the following file  naming convention.  The images were backed up onto CD.

            L2007MMDD  #

            Where:
            L = location or Beach (B for Boqueron Beach and S for Surfside Beach)
            M = month = 05, 06, 07, 08, or 09.
            DD = day = 01 to 31
            # = the automatic picture counter supplied by the camera software.

3.3.7      After the Collection

            After the collection,  the water sample bottles sand collection containers were  returned  to
their respective coolers.  Before transport of the samples to the water quality lab, the containers were all
WESTAT                                        70                                 December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
put through a quality control and quality assurance check to ensure all the samples were collected and the
caps on the containers were  on  tight to  prevent a loss of any water sample.  The bottles and  sand
containers were then placed into plastic bags and tied off with twist ties to prevent any separation of ID
labels and the water bottle samples during  transport to the analytical lab. The bottles and sand containers
were put back into the cooler for transport with icepacks all around to cool the samples down.  All the
ancillary data was transferred to the proper forms, double-checking to ensure that the data variable values
were correctly transcribed.  The Sample  Collection Forms  were filled  out and signed by the sample
collection team members and Lab  Transmittal forms were filled out, noting any comments about any
sample collected and initialed  by the person who actually collected the sample.  The water collector who
was transporting the samples to the lab then signed the Transmittal form denoting that they took custody
of the samples.  The field team supervisor  performed a QA/QC of these documents and then signed them
after reviewing them. The Lab Transmittal forms were then taken with the water and sand samples to the
analytical lab, where the lab would sign the transmittal form taking custody of the samples from the water
collector for filing with the study documentation and faxing to EPA.

3.3.8      GPS points and GPS ID

            The GPS points were marked as way points and stored in the GPS measuring device  used
during the water and sand sample collection. After the water and sand sample collection, the points were
retrieved and the data values for each point were recorded on the Ancillary Data forms.  To be able to
identify and store each of the marked GPS points, a unique ID scheme was developed and implemented.
At the end of each collection period, or at the end of the day, the  GPS IDs originally assigned by the  GPS
unit were changed to correspond and uniquely identify the captured GPS points for each collection period
on each of the collection days.  Listed below is the labeling scheme used to identify the collection period
and locations of stored GPS  points:

            MDDPTTL

            MDDPTTL

Where:
       MOD was the date of the sample collection;
           M was the numeric month and  DD was the day, e.g., 614 for June 14,
WESTAT                                       71                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
       P was the sample collection point at the beach (1-9), and
       TT was the planned time of day for the sample collection, using the following:

               08 = 8:00 AM
               11 = 11:00 AM
               15 = 3:00 PM
       L is the beach site location (B = Boqueron Beach and S= Surfside Beach)

3.3.9      End of Day/Weekend Procedures

            At the end of each collection weekend, several things happened.  First, all the collection
forms and logs were photocopied to be sent to EPA.  The originals were sent to Westat's main offices to
be entered into  the database maintained at the main campus. Also, the GPS IDs in the GPS unit were
changed to the  study IDs,  and noted on the Ancillary Data Forms.  If water sample collection was to
continue  on the next day, such as on Saturdays with collections scheduled to continue on Sundays, the
coolers and mesh bags were rinsed, cleaned, dried and prepared for the following day's sample collection
(putting the appropriate pre-labeled bottles into the right cooler and mesh bags).  The icepacks would also
be wiped down and returned to the freezer to be reused.  The digital pictures taken were downloaded from
the camera to the laptop at the end of  the day (or  sometimes  only at the  end of the entire collection
weekend).  Lastly, if not done already, the all of the Cyanobacteria samples (Boqueron Beach only) were
parafilmed across the cap and neck of the bottle, to prevent any  loss of a water sample during storage or
transport.

3.3.10    End of the  Collection Weekend Shipping Procedures

       Westat's subcontract laboratories were provided with  a document that detailed the required
shipping procedures.  The document highlighted the standard shipping procedures for all data, samples,
or filters sent from the local laboratory to the various analytical laboratories  during the course of the
Water Quality Study in the  summer of 2009.

Types of Samples
During this time period, the subcontract laboratory was responsible for shipping the following items to
the respective locations:
WESTAT                                       72                                December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
    1.  Sand Sample Extracts (300 g of sand was removed from each sand sample prior to processing)
       Shipped to: Emylee Prevette
                    University of North Carolina Env. Sci and Engineering
                    Room 1108
                    135 Dauer Drive Route 1A
                    Chapel Hill, NC 27599
       Electronic COCs were  sent to Elizabeth Sams.
    2.  Water Sample PCR Filters (total of 7 filters per sample)
       Shipped to: EMSL Analytical in Westmont, NJ (3 filters) c/o Charlie Li
                   USEPA in Cincinnati, OH (2 filters) c/o Rich Haugland
                   USEPA in Athens, GA (2 filters) c/o Marirosa Molina

    3.  Sand Sample PCR Filters
       Shipped to: EMSL Analytical in Westmont, NJ (3 filters) c/o Charlie Li

    4.  Transmittal Sheets, Pictures & MF /Turbidity/Ancillary Data Result Sheets
       Shipped to: Westat in Rockville, MD c/o Robert Clickner

    5.  * Cyanobacteria Samples *
       Shipped to: Greenwater Labs in Palatka, FL c/o Amanda Foss

       * Only for the Puerto Rico Beach Site *

Samples, Filters and Data were shipped  out on Mondays following a sample collection weekend via
FedEx overnight priority delivery  to  their respective  locations.  On weekends where  there was a
Monday holiday,  shipments went out on Tuesdays.   Integrity of the shipment was maintained by
following the protocol  specifications regarding the method for  shipping  and  maintaining the
appropriate temperatures.


Westat's subcontract laboratories were provided with a flowchart that depicted the  process for each
sample from collection through processing or analysis and onto  shipping/transport. See Appendix
A, Flowchart of Samples  Collected.
Labeling

All child samples were labeled with the waterproof polyester labels supplied by Westat. These labels
were made of the same material as the labels supplied for the collection of the samples.  The child
samples included the composited samples and the QPCR filters.
WESTAT                                      73                                December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
Chain of Custody

All shipments were  accompanied with a detailed chain of custody (COG) that indicated every item
that was included in the shipment, number of duplicates, sample id, sampling  location, collection
time, freeze time, etc.

            A Chain of Custody template (See Appendix B) was provided by Westat for the study
dates.  There were separate Chain of Custody for each recipient; the intended recipient was indicated
in the  Chain of Custody heading. The information that was pre-filled consisted of the sample ID,
sampling date, nominal collection time, sample location, sample type.

            At the laboratory, lab technicians were responsible for completing the following fields
on the Chain of Custody:
            •   Actual number of bottles/filters with that ID;
            •   Actual date processed/analyzed;
            •   Shipping date; and,
            •   Any relevant comments.

            As a form of quality control, someone other than they person who packed the shipping
container would double check the shipment and corresponding Chain of Custody to verify that it
was correct.

In addition to the packed Chain  of Custody, the subcontract laboratories sent out electronic versions
as well.

Packing and Shipping Bottles and Filters

            When packing the  shipments,  the  subcontract laboratory was provided with the
following checklist to ensure a safe shipment and to help maintain the integrity of the sample.
            •   Ensure all bottles/filters are properly labeled.
            •   Seal the bottle caps and necks with parafilm or electric tape.
WESTAT                                       74                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
                Double-bag each bottle in sealed zip-lock bags. This is not needed for the QPCR
                filters, since they were packed in compartmentalized shippers.
                Individually wrap each bottle in bubble wrap or other protective container/packing
                material. This was not needed for the QPCR filters.
                Place plenty of ice or ice packs or dry ice (as appropriate) on the bottom and sides
                of the cooler or shipping container.
                Add the samples and additional ice/packs/dry ice and bubble wrap.
                Lay  more  ice/packs/dry ice  and bubble  wrap  on top  of the  samples before
                shipping.
                Add the printed COG.
Sample-Specific Procedures and Shipping Requirements

            The  following  paragraphs describe the  specific shipping  requirements  that were
followed for the different type of items that were shipped throughout this study.

            Data  Sheets and Lab Transmittal Forms:   The  local lab shipped copies of all
laboratory transmittal forms and data sheets (MF results, ancillary data, pH and turbidity reading) to
Westat on the Monday or Tuesday after all analyses were complete.

            Sand  Samples:   Sand samples collected during the SAM sample collection time on
Saturdays, Sundays and Holidays during the study period were taken to the local lab for analysis via
MF and processing for PCR.  Prior to processing the sand,  300g of each sample was ascetically
weighed out and transferred to a separate plastic container.  These sand samples (created from the
parent sand samples) were stored until  in the lab refrigerator, until shipped on Monday. During the
Monday shipment the samples were sent on ice packs to the USEPA Lab in Chapel Hill, NC.

            PCR Filters:  PCR filters were frozen and stored at temperatures of at least -20° C until
shipped on  Monday.  Filters were sent frozen and on dry ice to EMSL Laboratories in Westmont,
NJ, USEPA Lab in Athens, GA, and the USEPA Lab  in Cincinnati, OH.   Filter blanks were sent
along with the samples, one  blank  filter for every 6 samples was  shipped, in  accordance with the
June 5, 2009 email from Kris Brenner.  Filter blanks were included on the chain of custody forms.
WESTAT                                      75                                December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
                              4.     ANALYTICAL METHODS
            Following collection, samples were maintained on ice during transport and at 1 - 4° C until
the time of analysis.  This was the only preservation step.  Microbiological analysis of water and sand
samples commenced  within six hours of collection. Further, it was critical that sample  plates from the
membrane  filter methods be placed  in  the  incubator within eight hours of sampling.   This was
accomplished for all samples. In the event of any problems or irregular occurrences, it was imperative that
the WACOR or COR be called immediately for guidance, and that the comments fields on the various
data sheets was used to record problems/corrective actions, so that the effect on data quality could be
considered.  Examples of problems that could occur included sampling difficulties, failure to ice-down
samples,  missed holding/analysis times, longer than acceptable incubation times, problems with  the
instruments, etc.

            With the rapid QPCR method, the critical step was the filtration of the water samples and
storage of the filters  in the freezer within the 8  hours after collection. Once the  filters were frozen,
analysis could be done as the time allowed. Again, this was accomplished for all samples. The times the
QPCR method filters  were frozen and stored, the location of the freezer(s), and the dates and times  of the
analyses were recorded.  If QPCR filters were analyzed in another lab, they were shipped by overnight
express on dry ice. The other laboratory conformed to the QC requirements of this document. Problems
with the rapid methods, like those with the filter methods, were reported and guidance requested from the
WACOR or COR.

            Samples were disposed of following successful microbiological processing by each of the
microbial methods, including the counting of all plates, successful pH, conductivity, salinity and turbidity
measurements, and completed analysis of samples by  all methods except for the QPCR method.
However, QPCR samples were filtered and the filters frozen before  the disposal of the samples.  The
WACOR or COR was contacted about the disposition of the samples if unusual results were obtained.

            Westat maintained a dedicated sample record book that was used to record all sample IDs as
samples were checked into the laboratory.  The record book also had columns for date checked in, storage
locations, and disposal dates. Westat was responsible for ensuring that all sample IDs were recorded and
initialed the record book for each batch of samples received to indicate that all expected samples were
present. The USEPA could request that the record book or copies of pages from this record book be made
WESTAT                                       76                                 December 7,2009

-------
National Epidemiological and Environmental                               Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
available for examination. Westat was also responsible for verifying that the arrival time at the laboratory
was entered in the appropriate column on the sample collection sheets, and initialed sample collection
sheets in the appropriate space(s) to indicate such, and noted any leaking containers or other irregularities.

4.1        Microbiological Methods

4.1.1      Standard Membrane Filter Method  Enterococci (Method  1600)

            Reference 8, EPA/821/R-97/004, describes the membrane filtration assay for Enterrococci.
This method can also be found in Reference 7:  "Improved Enumeration Methods for Recreational Water
Quality Indicators: Enterococci and Escherichia coli" EPA/821/R-97/004. These references are detailed
enough, including  descriptions  of required equipment,  so that the membrane  filter  method  can be
performed.   As such, these two  references represent the standard operating procedures (SOPs) for the
critical membrane filter data to be obtained from the field study. A 1-liter sample was collected for use in
performing the filtration method  and the ancillary pH, conductivity, salinity and turbidity measurements,
which was performed last to avoid contamination.  All collected samples were analyzed for Enterococci
by the MF method using sample volumes of 100,  10 and  1 mL .[except for special circumstances; for
example, if plates at the standard sample volumes were all  TNTC, or produced zero CPUs, then sample
volumes needed to be adjusted.]  In  the event that the laboratory needed to adjust the  volumes, the
adjustment and documentation of reason was indicated in the records submitted to the USEPA. Analysis
of each sample was initiated within 6  hours of its collection, and processing (filtration and plating) was
completed no later than 8 hours after collection.

            Specific  QC requirements  to  be  incorporated into the assays (in  place of the  general
guidance in the  methods) can be found in the next section of this plan. Table 8 summarizes some of the
key features of the method. Any modifications  to the method, such as using auto-pipets or micro pipets
instead of standard glass pipets, was approved by the WACOR or COR prior to being implemented. Any
other questions regarding the methods were also addressed  to the WACOR or COR prior to the start of
field activity.
WESTAT                                       77                                 December 7,2009

-------
National Epidemiological and Environmental
Assessment of Recreational Water Study
                    Quality Assurance Project Plan (QAPP)
                                        Revised Draft
Table 8. Summary of the mEI Agar Method for Enterococci



Method
Enterococci
EPA 1600




Medium
mEI agar



Incubation time and
temperatures
(°Q
24 hours ± 2 hours @
41+/-0.5°C


Volumes
analyzed
(mL)
100
10
1
Detection
limits
(colonies per
plate)
1-200



Ideal 1# of
colonies per
membrane
20-60


            On the sample collection/tracking sheets and final data sheets, laboratory analysts were
responsible for entering times and their initials for the following sequential steps:

            —    Analysis start time.
            —    Time at which plates being incubation in the water bath.
            —    Time at which plates are removed from the water bath for counting.

            These times were entered by hand initially, and later entered into the database electronically.
The  times listed above, and initials, were entered in a batch-wise manner. The laboratory was also
responsible  for  entering dilution  data, count data, QC  data,  etc.  on data sheets.  Responsibility for
electronic data entry was determined by Westat.

            Samples were analyzed in batches. A batch was considered to be all  of the samples that were
delivered to the laboratory at the same time. The plates for each batch of samples started their incubation
periods at the same time, and the microbiological control samples  described below under  "specific
filtration control tests" accompanied each analysis batch.
WESTAT
78
December 7, 2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                         Revised Draft
            For the membrane filter assay, the most critical quality control requirements are as follows:

            —     Prior  to any sampling/filtering,  an  appropriate volume  of TSA  [Tryptic  Soy
                  Agar/Trypticase  Soy Agar (Difco 0369-17-6, BD 4311043, Oxoid CM 0129B, or the
                  equivalent)] was prepared, and tested as described below. These plates were later used
                  for QC samples during  sample runs.  The recipe for TSA and  the  contamination
                  screening for TSA plates is described below:

                  Composition:

                              Tryptone       15 g
                              Soytone        5 g
                              NaCl           5 g
                              Agar           15 g

                  Preparation:   Add the dry ingredients listed above to  the 1000 mL of reagent-grade
                  distilled water, and heat to boiling to dissolve the agar completely. Autoclave at 121°
                  C (15 Ibs pressure) for 15 min. Dispense the  agar into 9 x 50 mm petri dishes (5
                  mL/plate).

                  Test for contamination: Incubate all plates  for 24 -  48 hr at 35° C to check for
                  contamination.  Discard any plates  with growth.  If >  5% of the plates  show
                  contamination, discard all plates, and make new medium. Store plates  in plastic bags
                  at 4°C until needed. The final pH was  7.3 ± 0.2.  Records of preparation and testing
                  were maintained, and were submitted to the WAM upon request.

            —     Each batch of mEI agar was pre-tested for performance (i.e., correct enzyme reaction)
                  with known cultures of target (e.g., Enterococcus faecium or Enterocococcus fecalis)
                  and non-target (e.g., Escherichia coli or Pseudomonas  species) organisms. Records of
                  such tests were  maintained by the laboratory and was submitted to the COR and
                  WACOR upon request.

            —     Specific filtration control  tests, listed below, were performed each time a batch of
                  samples was  analyzed, and the results  recorded. Results for all filter, agar or buffer
                  controls, including counts (if any), were reported with the sample results.

            —     Filter Control: Place one or more membrane filters on sterile TSA plates, and incubate
                  the plates for 24 hours at 35° C. Absence of growth indicates sterility of the filter(s).

            —     Phosphate-Buffered Dilution  Water Controls: Filter a 50-mL  volume of sterile
                  dilution water before beginning the sample filtrations and a 50-mL volume of dilution
                  water was filtered after completing the sample filtrations. Place the filters on TSA
                  plates, and incubate the plates for 24  hours at 35° C. Absence of growth indicates
                  sterility of the dilution water.
WESTAT                                       79                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
                  Agar Control: Place one or more plates of each medium, mEI and TSA, in the
                  incubator. Incubate mEI at  41° C and the TSA at 35° C for 24  hours to check for
                  contamination. Absence of growth indicated sterility of the plates.
                  Optional  membrane test: Test new lots of membrane filters against an acceptable
                  reference lot using the method of Brenner and Rankin (4). Although optional, this test
                  was recommended. In lieu  of performing this  test, the laboratory purchased filters
                  from a reputable source. The  USEPA has found  (by the method referenced) that
                  Sartorious filters have generally provided satisfactory performance; however, this
                  does not mean other filters were unacceptable.
            There were no specific sample IDs for the specific filtration control samples. On the hard
copy format batch analysis sheets, their results were reported with the following codes:
               YYZ (MEDIA), where,
               YY =  AC, PB, or MF (for Agar Control, Phosphate Buffer dilution water controls, or
                      Membrane Filter control).
               Z =    B or A, or nothing (used only for phosphate buffer dilution water controls; B for
                      "Before filtering" control, A for "After filtering" control).
               (MEDIA) = mEI or TSA, the medium used for the control.

            The methods contain other specific QC elements, such as requirements for laboratory water
quality, specifying that thermometers be NIST-traceable, calling for daily  confirmation of incubator and
water bath temperatures. Such method specifications were adhered to, and the adherence documented. All
autoclave runs  contained maximum-registering thermometers to ensure appropriate  temperatures were
achieved. Additionally, at  least weekly,  autoclave runs  contained spore strips  or  vials,  which were
incubated according to the manufacturer's instructions to check for proper sterilizer operation. Calibration
records were maintained for laboratory balances, pH meters, etc.

            The method SOP contained procedures for verifying the  correct identities of organisms.
Verification tests were performed for all samples  (5 colonies/sample) from one day  (either Saturday or
Sunday)  of the  first weekend (6 sample locations  x 3 times per day x 1 day =18 samples total) at the
beach site.  Results of the verification tests were recorded and reported to the WACOR or COR with the
other sample data in a mutually agreed upon manner.

            It  was expected that laboratories would  follow generally  accepted good  microbiology
laboratory  practice, such as described in the USEPA Microbiology Methods Manual,  Part IV, C (1);
WESTAT                                        80                                 December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
Section 9000 of the 20th edition of Standard Methods (3); or the QC section of the USEPA's "Manual for
the Certification of Laboratories Analyzing Drinking Water" (5). Copies of any records associated with
standard laboratory QC practices were made available to the USEPA upon request.

4.1.2      Quantitative Polymerase Chain  Reaction (QPCR) Method

            Reference 9 describes  the  procedures for the detection  of total  Enterococci  and total
Bacteroides in water samples based on the collection of these organisms on membrane filters, extraction
of their total DNA, and polymerase change reaction (PCR) amplification (i.e., a process whereby the
quantity of DNA is doubled in each cycle of amplification) of a genus-specific DNA sequence using the
TaqMan™ PCR product detection system. The TaqMan™ system signals the formation of PCR products
by a process involving the breakdown of a double-labeled fluorogenic probe that specifically attaches to
the target sequence  at a site between the two PCR primer recognition sequences. The reactions were
performed in  a  specially-designed thermal  cycling  instrument that automated  the  detection  and
quantitative measurement of the fluorescent signals produced by probe degradation during each cycle of
amplification. These signals were directly related numerically to the quantities of PCR products produced.

            The protocol was detailed enough, including descriptions of required equipment, so that the
method could be performed. As such, this reference represents the standard operating procedure (SOP) for
the critical data obtained from this portion of the field study. A 1-liter sample was collected for use in this
method. All  collected  samples were analyzed for total Enterococci and total Bacteroides using sample
volumes  of  100 mL [except for special circumstances; for example, if this volume  was found to be
impractical to  filter, then sample volumes may  have been adjusted].  Filtration of each sample was
initiated within 6  hours of its collections. Seven (7) replicate filtrations were performed, and the filters
were transferred to extraction tubes, as described  in the protocol and stored at -20° C for an indefinite
period. All filters were properly labeled to identify the water sample they came from.

            The local lab performed the 7 replicate filtrations and shipped 3  filters to the PCR lab, 2
filters to  Dr. Richard Haugland of USEPA, and 2 filters to Dr. Mariosa Molina of USEPA. All silters
were sent by overnight  express on dry  ice on the  Monday following the weekend the  samples were
collected.

            The PCR lab performed the  extraction to obtain DNA to be used for QPCR analyses for all
microorganisms (total Enterococci and  total  Bacteroides) as soon  as  possible  using only one  of the
WESTAT                                       81                                 December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
filters, and two filters were stored in the freezer as backups or for other/later analyses. At the end of this
study, all remaining frozen filters were sent on dry ice by overnight express to Dr. Richard Haugland of
the USEPA.


            Specific quality control  (QC) requirements that were incorporated  into these analyses are
listed below, as well as those in the method protocol.

            —     QC  requirements  for  sample  collection  and  filtration  are   specified  in  the
                  Microbiological Methods section.

            —     Cell  suspensions  of the calibrator  strains, Enterococcus faecalis, American Type
                  Cutlure  Collection (ATCC) 29212, Bacteroides fragilis ATCC 25285, and reference
                  strain,  Geotrichum  candidum, University of Alberta Microfungus  Collection and
                  Herbarium (UAMH) 7836, were provided to the laboratory by the USEPA. The cell
                  suspensions provided were stored by the laboratory at -20° C,  or preferably at -70° C,
                  until used. Preliminary QPCR analyses  were performed  using four  tubes of these
                  suspensions prior  to the start  of the study, and the results (CT values and run files)
                  were reported to USEPA. Subsequent average results for these samples on each day of
                  analysis were within +2 CT units of the average of the  initial values (See paragraph
                  below on monitoring the performance of the thermal cycling instrument and PCR
                  reagents).

            —     Training for the laboratory on the highly specialized scientific PCR equipment was
                  provided by the government for validity of data. Westat was responsible for ensuring
                  that the PCR technician had documented experience in QPCR technology.

            —     Westat purchased  PCR reagents, including primers and fluorescently-labeled probes.
                  Primer and probe sequences were provided by the USEPA.

            —     Thermal cycling instrumentation  (SMART Cycler TD System, Cepheid,  Sunnyvale,
                  California) was provided by the USEPA. Westat monitored  the performance of the
                  thermal  cycling instrument and PCR  reagents based on ongoing calibrator sample
                  analysis results. (See above.) In the event of failure to meet these performance criteria,
                  Westat prepared and analyzed a new set of calibrator extracts, identify the source of
                  the problem (e.g.,  reagents or instruments), and take corrective action.

            —     Westat provided adequate facilities and carry out precautions necessary to minimize
                  the  likelihood of  DNA contamination. Manipulation of samples  and reagents was
                  performed in laminar flow hoods or workstations with UV light sources, and the areas
                  were disinfected  before and  after each use with  10% bleach. Disposable aerosol
                  barrier pipette tips were used for all liquid transfers. Tubes and other disposables that
                  were not sterilized by the manufacturer were autoclaved before use. All supplies and
                  disposables were DNA-free. Distilled water and other reagents were verified to be free
                  of target DNA in negative control  analyses performed  with each  set of sample
                  analyses.
WESTAT                                        82                                 December 7,2009

-------
National Epidemiological and Environmental                                  Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                         Revised Draft
                  All pipettors used were calibrated prior to commencing work and on a semiannual
                  basis afterwards. It was recommended that the pipette calibration be verified weekly
                  by weighing several  different amounts of water (in the ranges  use) pipetted into a
                  properly tared container.

                  This work assignment  also included four other combinations of reagents that were
                  tested for each organism by PCR method.
WESTAT                                        83                                  December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
                             5.     QUALITY CONTROL (QC)
            The most critical elements of quality control for the membrane filter method were those
related to the microbiological assays. Sampling was straightforward; Westat was required to ensure that
the proper  samples  were taken  in the appropriately labeled containers. Holding time of samples  was
considered critical. Samples that had not been placed in the water bath in the membrane filter method or
completely filtered and placed in the freezer for use in the QPCR method within eight hours of collection
was considered to have produced invalid data.  (However, all data was collected, compiled, and reported
to USEPA). The intent of this project was to collect all of the data for subsequent evaluation by the
USEPA project team,  who  ultimately determine its utility  based on their collective  expertise  and
experience. No data was rejected outright by the persons performing the analysis. All data, including Too-
Numerous-To-Count's (TNTC) and zero's in the membrane filter methods, was reported to the USEPA.
An estimation procedure for TNTC plates was provided to the laboratory by USEPA. The estimation data
from the TNTC plates (i.e., the five counts from five squares on each filter) were all submitted to USEPA
along with the count data for  the other samples.  Westat calibrated and  maintained the  instruments
according to the methods and/or the manufacturer's  recommendations.  Westat followed accepted good
microbiology laboratory practice and  maintained QC records. Westat participated in all QA  audits
conducted,  and ran one or  more performance  evaluation samples provided by  the USEPA. Westat
contacted the WACOR or  COR when problems  occurred and documented corrective actions taken in a
report.

5.1       Corrective Actions

            Failure  to  meet any QC requirements, including those  associated  with standard good
laboratory practice,  requires that appropriate corrective actions be taken.  All QC failures, associated
corrective actions, and their effectiveness, were documented on a corrective action form, and submitted to
the USEPA WACOR or COR as part of the weekly reports. Data associated with quality control problems
was clearly identified in such reports, along with an assessment as to the QC failure's potential effect(s)
on  data quality. The WACOR  or COR was notified of such problems/corrective actions as soon as
possible to the time of the actual  occurrence. All related sample and ancillary data was still reported in the
standard way, with the QC problems clearly noted on copies of the data deliverables.
WESTAT                                       84                                 December 7,2009

-------
National Epidemiological and Environmental                              Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                   Revised Draft
5.2       Instrument/Equipment Testing, Inspection, and
           Maintenance

           Any  SOP for equipment/instrument which Westat was required to develop or  provide
described standard maintenance procedures for equipment. Maintenance records were described in the
SOPs, and were made available to USEPA upon request, including monitoring records of basic equipment
such as incubators, refrigerators, etc.

           For any equipment that might have affected critical data (i.e., microbiological or ancillary
data),  Westat prepared a short report for the WACOR or COR describing how the equipment was
inspected and tested upon receipt. The report was delivered within two weeks of equipment being placed
in service.

5.3       Instrument/Equipment Calibration and Frequency

           Any  SOPs for instruments and equipment which Westat may  be required to develop or
provide would fully describe calibration and calibration verification procedures. This included reference
to any calibrations conducted using certified equipment and/or standards with known valid relationships
to nationally recognized performance standards. Field instruments/gauges  and  laboratory measuring
equipment, such as balances and volumetric measuring devices (e.g., micropipettes), were professionally
serviced/certified within the six months prior to the commencement of the field/laboratory activities for
this project.

5.4       Tracking  and   Inspection/Acceptance   of  Supplies   and
           Consumables

           Westat had a system for tracking supplies, reagents, etc., and submitted its procedures to the
WACOR or COR for approval prior to the start of the field season.

           The membrane filter methods describe  the minimum  requirements for the quality  of
chemicals and laboratory water. Quality control procedures for laboratory water outlined in the  USEPA
drinking  water certification manual  (5) were  recommended. Westat maintained basic records (i.e.,
resistively readings, filter changes, etc.) for their laboratory water systems.
WESTAT                                    85                              December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                       Revised Draft
            The optional (but recommended) filter test (2); previously described, may be employed to
test new membrane filter lots. All media prepared was routinely tested for sterility.

            The goal was to have a clear association of all microbiological data with specific lots of all
materials  employed  in  performing  analyses.  All  records  associated  with  materials tracking and
preparation were made available to USEPA upon request.

5.5        Data Management

            Some  elements  of data management for field data and laboratory data were previously
outlined. Westat initially hand-entered results on pre-printed forms that were approved by USEPA.

            On  an approximately daily basis, completed hand-entered  data sheets were sent to  the
WACOR or COR. Weekly submissions were also submitted to the WACOR or COR.

            Westat maintained original  copies of sampling and data worksheets  until instructed by the
WACOR or COR on the deposition of the worksheets. Westat maintained two copies, on separate storage
media, of electronic versions of data until instructed by the WACOR or COR on the disposition of the
data.

5.6        Readiness Review/Dry Runs

            At least one  week  prior to actual  sampling,  sampling/analysis personnel performed a
readiness review/dry run at the beach site. USEPA representatives attended. A  checklist modified in Work
Assignment 2-04,  under the previous contract, was utilized by Westat that detailed all equipment,
supplies,  worksheets,  logbooks, etc.  required  to  conduct  sampling,  ancillary  data  collection,
microbiological analysis and data recording, and reporting at the beach site.  A set of samples was run.
Data transmission also occurred as part of this effort. Westat observed all activities in detail and recorded
their observations.

            Westat was responsible for  determining the results and  any corrective action that needed to
be  taken.  After  concurrence  with the  USEPA,  a   written  report  on the  final  approach  to
sampling/analysis/reporting was provided to the WACOR or COR.
WESTAT                                       86                                December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                     Revised Draft
5.7       Site Visits/Technical Systems Audits

           The USEPA performed a site visit at the beach site. The site visit included a technical
systems audit (TSAs). The site visit or audit was coordinated with Westat in advance.

           Site visitors/auditors may recommend work stoppage if they observe what they deem to be
critical failings  on the part of Westat.  Work may  be  stopped until such time as effective corrective
measures were implemented, verified effective, and approved.

           Following the  site visit/TSA, a report was prepared by the  personnel who conducted the
visit. This report was addressed to the WACOR or COR. Westat was provided a copy of the report, and
was  required to  respond  to  any corrective action  recommendations.  Westat  was responsible for
signing-off on the response. A  close-out memo was issued to Westat by the WACOR or COR following
his/her approval of the response. However, USEPA reserved the right to  revisit any identified problem
areas.

5.8       Routine Surveillance

           Copies  of  any written reports generated by Westat on  routine  surveillance was made
available to the WACOR or COR.
WESTAT                                      87                                December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
                       6.     DATA VALIDATION AND USABILITY
            According to USEPA Guidance for Quality Assurance Project Plans; USEPA QA/G-5

(6),

             "the process of data verification requires confirmation by examination or provision of
             objective evidence that the requirements of these specified QC acceptance criteria are met.
             In design and development, verification concerns the process of examining the result of a
             given activity to determine conformance to the stated requirements for that activity. For
             example, have the data been collected according to a specified method and have the
             collected data been faithfully recorded and transmitted? Do the data fulfill specified data
             format and metadata requirements?"

            Regarding validation, G-5 states,

             "The process of data validation requires confirmation by examination and provision of
             objective evidence that the particular requirements for a specific intended use have been
             fulfilled: In design and development, validation concerns the process of examining a
             product or result to determine conformance to user needs."

            Based on these definitions,  verification is the responsibility of Westat; however, USEPA
reserves the right to review the verification and will be responsible for validation.
6.1       Data Review, Verification, and Validation


            All data were subjected to several layers of review and verification, which is described in the
next section. The previously described assessments were also a key component of verification. Validation
was  primarily considered  part  of reconciliation with project  objectives. General principles guiding
acceptance/selection, verification, and validation of data are discussed immediately below.

            All microbiological data was submitted to the USEPA (i.e., data from all sample volumes or
dilutions, even if zero, uncountable  or too numerous to count and all other forms of data, described
above). The USEPA would decide whether or not data are acceptable, and chose which data were to be
included  in  the  final data  set for  the  project.  The guiding  principles   for  microbial   data
acceptance/selection were:

            —     Legible data records.
WESTAT                                       88                                 December 7,2009

-------
National Epidemiological and Environmental                                  Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                         Revised Draft
            —    Dates and times correct.

            —    Sample IDs correct.

            —    Electronic and hard-copy data concur.

            —    Results are in an appropriate format.

            —    Results reasonable (i.e., not grossly wrong).

            —    CPU counts are in the ideal range, whenever possible.

            —    Dilutions with CPU counts outside ideal range are not grossly incompatible with those
                  in the ideal range.

            —    Sample holding/analysis times were met.

            —    Associated QC sample results are acceptable.

            —    Specific acceptance criteria for the rapid methods adequate.

            For ancillary data, the acceptance factors included:

            —    Legible data records.

            —    Dates and times correct.

            —    Electronic and hard-copy data concur.

            —    Results are in an appropriate format.

            —    Results reasonable (i.e., not grossly wrong).

            —    Associated QC sample results acceptable.

            —    Other factors support acceptance (or rejection).

            For the remote chemical data the acceptance factors included:

            —    Legible ASR forms.

            —    Dates and times correct.

            —    Samples arrive in good condition at remote laboratory.
WESTAT                                        89                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
            Verification and Validation Methods

            Westat inspected forms to see that all appropriate data fields had values entered, and that
entries were legible and reasonable. Westat also ensured that all planned samples had been collected. The
verification of review was indicated by entering their initials on the field data sheets in provided spaces.
Westat  was responsible for  seeing that all forms were present and that they were delivered to the
laboratory.

            Westat verified that all expected samples and field sheets were present upon arrival at the
laboratory. Westat periodically inspected the record book and made a record of any such inspections.

            The laboratory was responsible for verifying that all microbiological (and other laboratory)
data fields were legibly filled out with apparently reasonable data. The verifier entered their initials and
the date in appropriate fields on the data sheets to indicate their review and acceptance. Spaces for date
and  initials  were provided on all data sheets. The laboratory's initials  indicated their  inspection and
acceptance of data sheets prior to their delivery to USEPA.

            Transmission  of deliverables  was  the  de facto  indicator that the data were completely
reviewed and believed to be accurate. The laboratory personnel responsible for reviewing the data was a
person that was different than the person who originally keyed-in the data.

6.2       Making Corrections

            On any hard copy data sheets, incorrect entries were singly lined-out (i.e., not obliterated)
and correct results entered. If the party making the correction was not the person who made the original
entry, then the date and initials of the person modifying the entry was present next to the correction.
WESTAT                                        90                                  December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                        Revised Draft
                                     7.     PERSONNEL
            Westat proposed the following personnel for this work assignment.  Dr. Robert Clickner was
the Water  Quality  Project Director.  Karen Delia Torre was  the Westat Project Leader, and Work
Assignment Leader. In addition to Dr. Clickner, Amy Kominski,  Sara Hader, Naa Adjei, and Rebecca
Birch served as study support. All of the staff worked on the Beaches projects in previous years.

            Robert Clickner,  Ph.D.: Dr.  Clickner is an  Associate Director at Westat and a senior
statistician  with over 35 years of experience in the development, implementation, and management of
statistical and environmental research projects, including two years  experience  directing the Beaches
water quality studies for EPA.   Dr. Clickner has also  designed, conducted and  analyzed biostatistical
experiments  involving pesticides  and  other  environmental contaminants.  His project management
activities have included the development and maintenance of project completion plans, quality assurance
plans,  schedules, and budgets;  management and coordination of multiple subcontractors, including
numerous  laboratories;   staff assignments;  review  of deliverables;  and  client coordination and
communication. He has developed and conducted international workshops on methodologies for human
exposure assessment field studies.

            Karen Delia Torre, MS, MBA, PMP: Karen Delia  Torre  is a Senior Study Director and
systems manager with more than 15 years of experience in managing multiple research efforts in  support
of Federal  government  initiatives,  including  epidemiologic studies.  She has managed large  studies
involving the collection of environmental data, questionnaire data, and biological measurements. She has
designed and  supervised the development  of paperless  data collection and transmission systems for
environmental and health studies. Ms.  Delia Torre has managed field studies using hand-held devices to
capture survey data, performed reliability testing of computer equipment in field conditions, and reviewed
new technologies  for application to  field  studies  and other data collection  efforts  as they  become
commercially  available.  She  supervises  a  staff  of  data  collection  specialists,  systems  analysts,
programmers, web  developers,  database developers, survey designers,  and subject-area specialists to
produce systems to track environmental and biological specimens, collect data via the Internet, collect
data  using hand-held  computers,  administer  computer-based  surveys,  and  produce  searchable
environmental and medical literature databases. Ms. Delia Torre holds a master's degree in biomedical
engineering. She has  performed research on diagnostic  imaging  procedures, computerized diagnostic
WESTAT                                        91                                 December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                         Revised Draft
systems, and medical history data and image transmission techniques.  She also holds an M.B.A. in
Information Systems, specializing in database design and Internet applications.

            Rebecca Jeffries, MPH: Rebecca Jeffries is an epidemiologist with 4 years of experience in
study design, field  data collection,  data analysis,  and  program evaluation  for environmental  and
occupational health studies. She has managed studies, data and sample collection, and the packaging and
shipment of environmental samples.  She has also designed data collection forms and study protocols,
pilot tested survey instruments, supervised listers in preparing for sample selection, developed databases,
analyzed data, and developed a manual of operating procedures. In addition, Ms. Jeffries has experience
teaching at the high school and college levels, including instruction of non-native English speakers and
international teaching experience in rural Kenya.

            Ms.  Amy  Kominski, BS:  Amy Kominski  is an assistant  study  manager and research
assistant at Westat.   Ms.  Kominski  is  a biologist,  research assistant,  with experience in collecting
epidemiologic research data.  Prior to working at Westat, she worked at The National Institutes of Health
in Bethesda, Maryland.  While at The NIH she worked in a clinical microbiology lab and has experience
designing and managing public health studies, including;  protocol development and implementation of
large volume, multi-site research projects.  Her specific experience  includes infection control  studies
involving  antibiotic  resistant bacteria,  specifically  Enterococci  and Staphylococcus  aureus.     Ms
Kominski is proficient in general laboratory biochemical testing methods, quality assurance and control,
antibiotic susceptibility testing and state-of-the-art molecular assays.  Since joining Westat, Ms. Kominski
has been involved with the work done  at Fairhope Beach (2007) and Goddard Beach  (2007) and has
performed water sampling pilot studies and beach assessment studies.

            Sara Hader,  BA:   Sara Hader  is an assistant study manager and  research assistant in
Westat's Health  Studies Sector.   After graduating  with a bachelor's degree  in microbiology,  she
performed medical literature research for defense medical malpractice cases in trial law.  Since joining
Westat, Ms. Hader has  supported studies for the Centers for Disease Control  and Prevention and the
National Cancer Institute. Ms. Hader has experience in coordinating forms and records request, receipt,
and processing.   She  works with project directors, operations staff, programmers, and support staff in
managing  data   collection  and  tracking  systems  on   several  environmental,  occupational,  and
epidemiologic studies.
WESTAT                                        92                                  December 7,2009

-------
National Epidemiological and Environmental                                 Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                         Revised Draft
            Ms. Naa  Adjei  BS.  Naa  Adjei has  experience  as  quality assurance  specialist  for
environmental sample collection. Ms. Adjei has 3  years  of experience in scientific study design,  site
selection for field study, data collection, and data analysis. Naa is experienced in developing databases
for data entry and  reporting. She has performed beach site assessments and has worked as a quality
assurance specialist to ensure that data collection and analysis adhere to strict protocols.  Ms. Adjei holds
a B.S. in neurobiology and physiology.

            Field  Staff.   The field staff collected the water  and sand samples, processed them  and
delivered them to the local laboratory, and performed related tasks. They worked under the supervision
of a Westat person  as well as the supervision of the subcontract laboratory.  These staff had some post-
secondary  education  in biology,  environmental science,  or a  related discipline. Westat provided  the
necessary project-specific training.
WESTAT                                        93                                   December 7,2009

-------
National Epidemiological and Environmental                                Quality Assurance Project Plan (QAPP)
Assessment of Recreational Water Study                                                      Revised Draft
                                   8.     REFERENCES
    1.  Bordner, R., J. Winter, and P. Scarpino (eds). 1978. Microbiological methods for monitoring the
       environment: water  and wastes.  EPA/600/8-78/017, Environmental  Monitoring and Support
       Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH.

    2.  Brenner, K. P., and  C. C. Rankin. 1990. New screening test to determine the acceptability of
       0.45-[Lm membrane  filters for analysis of water. Appl. Environ. Microbiol. 56:54-64.

    3.  Clesceri, L. S.,  A. E. Greenberg, and A.  D.  Eaton (eds.). 1998. Standard Methods for the
       Examination  of Water  and  Wastewater, 20th edition. American Public Health Association,
       American Water Works Association and Water Environment Federation. Washington, DC.

    4.  Messer,  J.  W. and A. P. Dufour. 1998. A rapid, specific  membrane filtration  procedure for
       enumeration of enterococci in recreational water. Appl. Environ. Microbiol. 64:678680.

    5.  U.S.  Environmental  Protection Agency. 1992.  Manual  for the  certification of laboratories
       analyzing  drinking   water:   Criteria  and  procedures,  quality   assurance,   3rd   edition.
       EPA/814B/92/002, Office of Ground Water and  Drinking Water,  Technical Support Division,
       U.S. Environmental Protection Agency, Cincinnati, OH.

    6.  U.S.  Environmental  Protection Agency.  1998. EPA Guidance for Quality Assurance Project
       Plans; EPA QA/G-5 (EPA/600/R-98/018),  U.S.  Environmental Protection Agency,  Office  of
       Research and Development, Washington, DC.

    7.  U.S.  Environmental  Protection  Agency.  2000.  Improved enumeration  methods  for  the
       recreational water quality indicators: Enterococci and Escherichia coll. EPA/821/R97/004, Office
       of Science and Technology, U.S. Environmental Protection Agency, Washington, DC.

    8.  U.S.  Environmental  Protection Agency.   2002.   Method 1600: Enterococci  in  Water by
       Membrane  Filtration  Using  membrane-Enterococcus  Indoxyl-fi-D-Glucoside  Agar  (mEI).
       EPA/82 l/R-02/022, Office of Water, U.S. Environmental Protection Agency, Washington, DC.

    9.  Rapid Polymerase Chain Reaction  (PCR)-Based Methods for Measuring Total  Enterocococi
       and Total Bacteroides, in Water Samples  [revised March 2009]
WESTAT                                       94                                 December 7,2009

-------
Appendix E




Wade et al.  2008
              358

-------
                                              ORIGINAL  ARTICLE
        High  Sensitivity  of  Children to  Swimming-Associated
                                     Gastrointestinal  Illness
           Results Using  a Rapid Assay  of Recreational Water Quality
     Timothy J. Wade,a Rebecca L.  Calderon,a Kristen P. Brenner,h Elizabeth Sams,a Michael Beach,1
                          Richard Haugland,b Larry Wymer,b and Alfred P.  Dufour0
 Background: Culture-based methods of monitoring fecal pollution
 in recreational waters require 24 to 48 hours to obtain results. This
 delay leads to potentially inaccurate management decisions regard-
 ing beach safety. We evaluated the quantitative polymerase chain
 reaction (QPCR) as a faster method to  assess recreational water
 quality and predict swimming-associated illnesses.
 Methods: We enrolled visitors at 4 freshwater Great Lakes beaches,
 and contacted them 10 to 12 days later to ask about health symptoms
 experienced since the visit. Water at the beaches was polluted by
 point sources that carried treated sewage. We tested water samples
 daily for Enterococcus using QPCR and membrane filtration (EPA
 Method 1600).
 Results: We completed 21,015 interviews and tested 1359 water
 samples. Enterococcus QPCR cell equivalents (CEs) were positively
 associated with swimming-associated gastrointestinal (GI) illness
 (adjusted odds ratio per 1 Iog10 QPCR CE =1.26;  95% confidence
 interval =  1.06-1.51). The association between  GI illness and
 QPCR  CE was stronger among children  aged 10 years and below
 (1.69; 1.24-2.30). Nonenteric illnesses were not consistently asso-
 ciated with  Enterococcus QPCR CE  exposure, although rash and
 earache occurred more frequently  among swimmers. Enterococcus
 QPCR  CE exposure was more strongly associated with GI illness
 than Enterococcus measured by membrane filtration.
 Conclusions: Measurement of the indicator bacteria Enterococci in
 recreational  water using a rapid QPCR method predicted swimming-
 associated GI  illness at  freshwater beaches polluted by sewage
 Submitted 1 May 2007; accepted 25 October 2007; posted 25 March 2008.
 From the aUS Environmental Protection  Agency,  National Health  and
    Environmental Effects Research Laboratory, Chapel Hill, NC; bUS
    Environmental Protection Agency, National Exposure Research Labora-
    tory, Cincinnati, Ohio; and "Centers for Disease Control and Prevention,
    Atlanta, GA.
 Supported by US Environmental  Protection Agency  and Westat Corp.
    (contact 68-D-02-062). This document has been subjected to review by
    the National Health and Environmental Effects Research Laboratory and
    approved for publication. Approval does not signify that  the contents
    reflect the views of the Environmental Protection Agency.
 F3 Supplemental material for this article is available with the online version
    of the journal  at www.epidem.com; click on "Article Plus."
 Correspondence: Timothy J. Wade, US EPA Human Studies Division, MD
    58 C, RTF, NC 27711. E-mail: wade.tim@epa.gov.

 Copyright © 2008 by Lippincott Williams & Wilkins
 ISSN: 1044-3983/08/1903-0375
 DOI: 10.1097/EDE.Ob013e318169cc87
 Epidemiology •  Volume 19, Number 3, May 2008
discharge. Children at 10 years or younger were at greater risk for GI
illness following exposure.

(Epidemiology 2008;19: 375-383)
  It would be useful to monitor recreational waters continu-
  ously for human pathogens as a way to prevent swimming-
associated infections.  However, there  is considerable  diffi-
culty and expense associated with testing for the vast number
of potentially pathogenic microorganisms.  Instead, fecal in-
dicator bacteria such as Escherichia coli or Enterococcus are
used to assess  the microbial safety of recreational waters.
These indicator bacteria are generally not harmful but can be
a marker for the presence of sewage and human feces, and the
health  risks  that result from such exposures. Because  cur-
rently used methods require 24 to 48 hours to obtain results,
monitoring does not immediately detect changes in exposure,
leading to delays in notifying beach-goers of possible risks.
     A faster  method of measuring water quality could
improve protection of public  health by reducing the time
between exposure measurement and management decisions,
potentially providing  same-day results before most beach-
goers enter the water. We previously  reported that a faster
method of measuring fecal indicator bacteria using quantita-
tive polymerase chain reaction (QPCR) showed promise in its
ability to predict swimming-associated gastrointestinal (GI)
illness.1 After sample collection and  transport, the QPCR
method can  be performed in 3 hours  or less. With further
improvements this  may be shortened to 2 hours or less.2
     We  expand  on  our  previous analysis to  include  2
additional freshwater beaches, an assessment of nonenteric
illnesses (upper respiratory illness [URI], rash, eye irritations,
and earaches), and separate analyses by age.

                     METHODS
Study Design
     We conducted a prospective study of visitors to fresh-
water Great  Lake beaches on Lake Michigan and Lake Erie
during the summers of 2003 and 2004. The data  collection

                                                   375
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction  of this         is prohibited.

-------
 Wade et al
          Epidemiology • Volume 19, Number 3, May 2008
 methods have been described previously.1 In brief,  we at-
 tempted to enroll all beach-goers between 11:00 AM and 5:00
 PM  during summer weekends  and holidays. We  excluded
 unaccompanied minors (below  18 years) or those who could
 not speak English or Spanish. We interviewed volunteers as
 they were leaving  the beach to ascertain information about
 swimming and other activities. Ten to 12  days later,  one of
 the adults in the household was interviewed by telephone
 about health symptoms experienced by participating  house-
 hold members. All  subjects provided oral consent. The study
 procedures were approved by the Institutional Review Board
 for the Centers for Disease Control and Prevention.

 Beach Descriptions
       Human-derived pollution sources generally cause the
 most health concern,3 and beaches with such pollution were
 the focus  of these studies. In 2003, we conducted studies at
 West Beach (on Lake Michigan in Indiana Dunes National
 Seashore in Indiana) and Huntington Beach (on  Lake Erie
 near Cleveland, OH). In 2004, we studied 2 additional Lake
 Michigan Beaches: Silver Beach,  near St. Joseph,  Michigan,
 and Washington Park Beach in Michigan City, Indiana. The
 range  of fecal  indicator-bacteria concentration  at these
 beaches is related to  contamination by effluent from sewage
 treatment plants. Water quality  at each beach was influenced
 by  point-source tributaries  that received  combined  treated
 sewage treatment discharges from communities with popula-
 tions of at least 38,000 and with flow rates of over  10 million
 gallons per day (see  Appendix A, available with  the online
 version of this article, for additional details). These sewage
 plants provided secondary treatment as well  as disinfection
 with chlorine or ultraviolet radiation during the summer.

 Water Sample Collection and  Sample Analysis
       Water samples were tested for  fecal indicator bacte-
 ria Enterococcus and Bacteroides using QPCR. Because of
 problems in the sensitivity of the Bacteroides QPCR assay,
 the data in 2004 were insufficient to assess this indicator in
 relation to health effects.  Samples  were also tested for
 Enterococcus using  EPA Method 1600,4 one of the cul-
 ture-based methods  currently recommended by the EPA
 for recreational freshwater monitoring.5
       We  collected water samples at 8:00 AM, 11:00 AM, and
 3:00 PM, along 3 transects perpendicular to the shoreline—
 one  sample in waist-high  water  (1m deep) and  one  in
 shin-high water (0.3 m deep). Transects were located  at least
 60 m apart to encompass the swimming area. Because rock
 jetties  at  Huntington Beach prevented free circulation  of
 water, we collected 4 additional  samples  at  each sampling
 time to better characterize the water quality. Following col-
 lection, samples were placed in coolers and maintained on ice
 at 1 to 4°C.  Analyses of samples  by  Method 1600 for
 Enterococcus were performed by local laboratories within 6
 hours of collection. Samples were filtered for QPCR analysis
 376
within 6 hours of collection. To ensure consistency across the
4 beaches, the filters were frozen and sent on dry ice by
overnight  express  for analysis by  EMSL Analytical,  Inc.
Laboratory (Westmont, NJ).
     The  QPCR method used in this study has been previ-
ously described.1'6 In brief, organisms in water samples were
collected by membrane filters, total DNA was extracted, and
polymerase chain reaction (PCR) amplification of a genus-
specific  DNA sequence of Enterococcus was carried out
using the TaqMan PCR product detection system. The reac-
tions were performed in a thermal cycling instrument (Smart-
Cycler System, Cepheid, Sunnyvale, CA) that automated the
detection and quantitative measurement of the fluorescent
signals produced by probe degradation during each cycle of
amplification. Ratios of the target sequences in a test sample
were compared with a calibrator sample using  an arithmetic
formula, referred to as  the Comparative Cycle Threshold
Method.7 These ratios were converted to measurements of
calibrator cell equivalents in test samples through the use of
calibrator samples processed in the same manner as the test
samples and containing a known quantity of the target organ-
ism cells.  Results  are reported  in  QPCR cell equivalents
(QPCR CE) per 100 mL of original sample.
     At each sampling time we recorded environmental
conditions, including air and water temperature, cloud cover,
rainfall,  wind speed and direction, wave height, beach pop-
ulation density, boats, animals  (number and type), and debris.

Health Assessments
     We assessed  5 endpoints, defined a priori, and similar
to those previously studied.8"13
1. "Gastrointestinal illness" (GI  illness) was defined as any
   of the following: diarrhea (three or more loose stools in a
   24-hour period); vomiting; nausea  and  stomach ache;
   nausea  or  stomach ache, and interference  with regular
   activities (missed time  from  work or school, or missed
   other regular activities as a result of the illness).
2. "Upper respiratory illness"  (URI) was defined as any 2 of
   the following: sore throat, cough, runny nose,  cold, or
   fever.
3. "Rash" was defined as a rash or itchy skin.
4. "Eye ailments"  were defined  as either eye infection or
   watery  eye.
5. "Earache"  was defined as earache, ear infection, or runny
   ears.
     Consistent with some other studies,8'10'11 URI and GI
illness were not restricted to persons with fever, since infec-
tions can produce these  illnesses without fever (eg, E.  coli
0157:H7 and  norovirus infections). We were also concerned
about the accuracy  of self-reported low-grade fever.
     People who were ill within 3  days before their beach
visit were excluded for the outcome with which they had been
afflicted.  We examined various definitions of GI illness

                       © 2008 Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this          is prohibited.

-------
 Epidemiology •  Volume 19, Number 3, May 2008
Rapid Water Quality Measures and Children's Sensitivity to Illness
 including diarrhea (three or more loose stools in a 24-hour
 period) alone and GI illness with complications (defined as
 missing regular activities,  using medications, or visiting a
 health provider as a result of a GI symptom).

 Definition of Swimming
       "Swimmers" were those who reported immersing their
 body to their  waist  or  higher. In our  previous analysis,
 immersion to the waist showed a pattern of risk similar to
 head immersion.1 Nonswimmers were defined as those who
 reported no contact with water. Those entering the water not
 up to their waist were classified as "waders."

 Statistical Analysis
       Because QPCR CE were highly skewed, raw data were
 log-transformed (base 10).  The arithmetic mean of the log-
 transformed values was used to summarize water quality at a
 given day, time, or location. We previously used a maximum-
 likelihood method to impute results for samples  below  the
 limit of detection.1 However, QPCR CE from 2004 beaches
 were not as well approximated by a log-normal distribution,
 making imputation based on this exact distributional assump-
 tion questionable. Furthermore, we  had concern that partial
 inhibition may have  been responsible for some nondetected
 results, making the imputed  detection  limits incorrect. We
 therefore excluded samples below the limit of detection from
 the calculation of averages. However, this choice of method
 for dealing with the limit of detection did not affect the results
 (see Appendix B, available with the online version  of this
 article). We focused  the analyses primarily on 2 summary
 measures: the daily average of all samples and the average of
 the 8:00 AM samples.  The daily average represented average
 water quality at a beach on a particular day. The 8:00 AM
 average was used to determine if morning water quality was
 predictive  of  illness  among swimmers exposed later that
 day—an important consideration for assessing the utility of a
 faster method such as QPCR for water quality evaluation.
 Analyses using  depth-specific  averages were  also con-
 ducted and results are shown in Appendix B. Analysis of
 variance  models were used to  explore the  relationship
 between Iog10 QPCR CE with beach, collection date, time,
 and  sample depth.
       To account for correlated environmental measurements
 as potential confounders of the swimming and health effects
 relationships, we  used principal-components  analysis  to pro-
 duce summary components. The 5 principal components that
 accounted for  the majority of the  variability  (54%) were
 included in health effects regression models. These 5 com-
 ponents were beach-goer density, temperature (water and air),
 rainfall, wind direction and debris, and wind speed and wave
 height. To avoid data loss when one or more of the environ-
 mental observations were missing (18 of 85  days), principal
 components were imputed using best-subset  regression.14

 © 2008 Lippincott  Williams & Wilkins
       We used generalized linear-regression models to eval-
 uate the association between water quality and health effects.
 Logistic regression models were used to describe the strength
 of the association between the QPCR CE  measures and
 incidence of  illness among  swimmers.  Models using an
 identity link and a binomial error structure (linear model)
 were  used to directly estimate the attributable risk15 (swim-
 mer risk minus nonswimmer risk),  which we refer  to as
 "swimming-associated  illness." Although the linear and lo-
 gistic models produced  similar results, the  linear models
 allowed direct estimation of the attributable risk, which is often
 considered a more meaningful and direct statement of risk.5
 Nonswimmers were  included in models and  were assigned
 water quality exposures of zero. Indicators for "swimming" and
 "beach" were included in all models. Log-linear models were
 used to estimate the adjusted cumulative incidence ratio associ-
 ated with swimming (without regard to water quality).15 Robust
 estimates of variance were used to account for the nonindepen-
 dence of observations within household.16"18
       Covariates strongly associated with swimming, water
 quality or illness, or  those considered by investigators to be
 potential confounding factors were considered for inclusion
 in regression models. These factors included age, sex, race,
 contact with animals, contact  with other persons with diar-
 rhea,  number of other visits to the beach, any other chronic
 illnesses  (GI, skin, asthma), digging in sand,  and the first 5
 principal components of the  environmental/meteorological
 factors (described above). An indicator was also created for a
 festival that took place at Silver Beach, drawing 17,000 visitors
 to an area adjacent  to the beach. For URI,  rash, and eye
 outcomes, use of insect repellent and sun block  were also
 considered. For each analysis, the set of covariates was reduced
 through a change-in-estimate procedure.19 A criterion of a 5%
 change was  used, although this was occasionally relaxed to
 obtain a parsimonious model. The selection procedure generally
 reduced the numbers of covariates to 7 or fewer.
       To evaluate heterogeneity in the indicator/illness rela-
 tionship  across the beaches,  we  graphically examined the
 relationship  at each  beach and conducted likelihood ratio
 tests.  These tests compared models  with  interaction terms
 between  beach and water quality (which allowed slopes to
 differ across beaches) with restricted models constrained to a
 single slope across the  4 beaches.
       We conducted separate analysis for the age categories 0
 to 10 years,  11 to 54 years, and 55 years and older. The age
 groups were selected  a  priori based on  sample  size and
 investigators' judgment.
                        RESULTS
       A total of 21,015 interviews from 10,093 household
 groups were completed (Appendix B). Respondents at the 4
 beaches differed by age, race, miles traveled to the beach and
 proportion of swimmers (Appendix B). Respondents were

                                                     377
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this          is  prohibited.

-------
  Wade et al
                     Epidemiology • Volume 19, Number 3, May 2008
 85% white and 56% female, with a median age of 27 years.
 Swimmers were younger than nonswimmers (median age 19
 and 35 years, respectively) but were equally likely to report
 rash, sore throat, vomiting, and eye irritations in the 3 days
 prior  to the beach visit,  chronic respiratory illness  (eg,
 asthma), and chronic skin problems (Table 1). Slightly fewer
 swimmers compared with nonswimmers reported chronic GI
 conditions (2% vs. 3%), GI symptoms (other than vomiting)
 in the 3  days prior to the beach visit (2% vs.
chronic
 allergies (18% vs. 21%), and consumption of red or raw meat
 prior to or immediately after the beach visit (8% vs.  10%).
 There  were more female beach-goers  than male in all 3
 water-use groups, with the  largest discrepancy among the
 waders (63% vs. 37%) and the smallest among the swimmers
 (52% vs. 48%). Most were white. The percentages of other
 races were similar across water-use groups, except that the
 percentage of Hispanic/Latino respondents was highest among
the swimmers. More swimmers than nonswimmers reported
using sunblock (61% vs. 40%), insect repellant (3% vs. 2%), and
having had contact with animals (79% vs. 75%).

Water Quality
     Enterococcus  QPCR CE differed by beach (Table  2)
and  sample depth. Median QPCR CEs at shin depth were
higher than waist depth  (93  and 65  QPCR CE/100  mL,
respectively). Collection time was not an important factor in
the variability of QPCR CE, although QPCR CE levels
measured at 3:00 PM were slightly higher than at 8:00 AM
(median QPCR CE/100 mL was 74, 78, and  80 at 8:00 AM,
11:00 AM, and 3:00  PM, respectively).
     Twenty-five of 78  days (32%) exceeded the current
geometric mean  guideline value of 33  colony forming units
(CFU)/100 mL Enterococcus  measured by Method  1600.5
                  TABLE 1.   Characteristics of Nonswimmers, Waders, and Swimmers
                                                           Nonswimmers
                                                            (n = 6888)
                                                              No. (%)
                           Waders
                          (n = 3597)
                           No. (%)
                              Swimmers
                             (n = 10,436)
                              No. (%)
                  Age (yrs)
                    0-4                                       365 (5)          303 (9)          975 (10)
                    5-10                                      242 (4)          231 (7)         2156 (21)
                    11-19                                      815(12)         360(10)        2007(20)
                    20-54                                     4574(68)        2304(66)        4599(45)
                    55+                                       776(11)         319(9)          386(4)
                  Sex
                    Male                                     2786(40)        1332(37)        5049(48)
                    Female                                    4098(60)        2260(63)        5366(52)
                  Race
                    White                                     5848(86)        3143(89)        8617(84)
                    Black                                      231 (3)          102 (3)          260 (3)
                    Asian                                      122(2)           64(2)          118(1)
                    American Indian                               21 (<1)          16(1)          27 (<1)
                    Hispanic/Latino                              554(8)          197(6)         1138(11)
                    Multiethnic/other                              51 (1)           26 (1)          129 (1)
                  Conditions in the 3 d prior to the beach visit
                    Vomiting                                    59 (1)           41 (1)          95 (1)
                    Other GI symptoms                           178 (3)           79 (2)          179 (2)
                    Sore throat                                  397 (6)          201 (6)          605 (6)
                    Rash                                      155(2)           74(2)          227(2)
                    Eye irritations                                 35(1)           16 (<1)        47 (<1)
                    Earache                                     84 (1)           35 (1)          147 (1)
                  History of allergies                             1467(21)         781(22)        1879(18)
                  History of chronic GI illness                       208 (3)          106 (3)          218 (2)
                  Any history of chronic GI illness, asthma, or          1980 (29)        1061 (30)        2707 (26)
                      allergies
                  Contact with animals 48 h prior to or after           5178(75)        2821(78)        8221(79)
                      beach visit, or between beach visit and
                      phone interview
                  Consumption of raw meat 48 h prior to beach          701(10)         333(9)          881(8)
                      visit or between beach visit and phone
                      interview


 378                                                                               © 2008 Lippincott Williams & Wilkins

Copyright © Lippincott Williams & Wilkins.  Unauthorized reproduction of this         is prohibited.

-------
 Epidemiology  •  Volume 19, Number 3, May 2008
Rapid Water Quality Measures and Children's Sensitivity to Illness
                  TABLE 2.  Entemcoccus QPCR CE by Beach (QPCR CE/100 ml)

                                                                          25th       75th
                                        No.    Mean (SD)   Median   Min   Percentile   Percentile    Max
All beaches
West Beach
Huntington Beach
Silver Beach
Washington Park
Beach
1359
320
339
352
348

770 (10,800)
572 (1280)
450 (1300)
553 (6260)
1480a (20,300)

76.4
134
127
56.8
60.0

0.050
0.080
0.050
0.14
0.080

27.5
41.7
32.8
15.1
23.1

267
486
327
143
165

376,000
15,800
14,800
117,000
376,000

aMean influenced by outlying maximum value.
 Twenty-two percent (333 of 1482)  of  individual  samples
 exceeded the single sample maximum of 61 CPU/100 mL.5

 Health Effects
       The incidence of new GI illness  was 7.3% (1497 of
 20,414) during the 10 to 12 day follow-up period. GI illness
 incidence was highest among children younger than 5 years
 (9.0%) and lowest among those aged 55 and older (4.9%).
 The adjusted risk of GI illness was  1.44 times higher in swim-
 mers than nonswimmers (95% CI = 1.27-1.64; Table 3). The
 risks among children aged 10 and  younger, and children and
 adults aged 11 to 54, were similar  to the pooled risk. Among
 those aged 55 and older, swimmers  reported 2.3 times as many
 illnesses as nonswimmers of the same age (1.33-3.99; Table 4).
 Children aged 5 and younger showed a similar pattern of risk as
 those aged 10 years and younger, but with the exception of GI
 illness (1.67 [CI = 1.03-2.69]), small sample sizes prohibited
 making conclusions about this age group.
       Approximately 5.7% of respondents reported URL In-
 cidence was highest in children younger than 5 (10.6%) and
 lowest in those aged 55 and older (2.5%). The crude  inci-
 dence of URI was higher among  swimmers than nonswim-
 mers, but after adjustment there was  little difference in risk
 TABLE 3.   Illness Incidence and Adjusted Cumulative
 Incidence  Ratios  (aCIR) Comparing Swimmers With
 Nonswimmers (Excluding Waders)
Incidence

Illness
GI
URI
Rash
Eye ailments
Earache
Nonswimmers
No. (%)
397 (6.0)
321 (5.0)
144(2.1)
219 (3.2)
78 (1.2)
Swimmers
No. (%)
849 (8.3)
589 (6.0)
305 (3.0)
280 (2.7)
190 (1.9)

aCIR (95% CI)
1.44(1.27-1.64)
1.06 (0.90-1.24)
1.38(1.12-1.72)
1.00 (0.81-1.24)
1.63(1.23-2.17)
    Numbers are those reporting new symptoms, among those without baseline symp-
 toms. For GI illness, subjects reporting vomiting or other GI symptoms in the past 3 d
 shown in Table 1 were excluded. Fourteen nonswimmers and 15 swimmers reported
 both vomiting and other GI symptoms at baseline. aCIR estimated from log-linear
 regression model.
 © 2008 Lippincott Williams & Wilkins
 (1.06 [0.90-1.24]; Table3].  Age was a strong  confounder
 because young respondents were both more likely both to
 swim and report URI. Among children  aged 10 years  and
 younger, URI risk was not elevated among swimmers (0.95
 [0.66-1.38]; Table 4).
       Approximately 2.7% of all respondents reported rash,
 with the  highest incidence in children younger than 5 years
 (4.1%), and the lowest in those aged 55 and older (2.1%).
 Swimmers reported more rash than nonswimmers (1.38 [CI =
 1.12-1.72]; Table 3).  Rashes occurred more frequently on the
 upper and lower back (26%) of swimmers reporting rash than of
 nonswimmers reporting rash (12%).
       The incidence  of eye irritations  and infections was
 2.9%; these were reported with equal frequency by swimmers
 and nonswimmers (1.00 [0.81-1.24]; Table 3).

 Relationships Between Water  Quality and
 Health
       The incidence of GI illness was consistently associated
 with Entemcoccus QPCR CE exposure (Table 5, Figs. 1  and
 2). Among all subjects, a 1 Iog10 increase in the daily QPCR
 CE average resulted in a 1.26 increase in the risk (odds) of GI
 illness (95% CI = 1.06-1.51). The relationship was stronger
 among children, with a similar association for those aged 10
 years and younger (1.69 [1.24-2.30]), 5 and younger (1.67
 [1.08-2.57]), and 2  and younger  (1.65 [0.81-3.36]). The
 association between  the  8:00 AM Entemcoccus QPCR CE
 average and GI illness was nearly identical to that of the daily
 average. As illustrated in Figure 1,  1000  swimmers exposed
 to 100 Entemcoccus QPCR CE would experience an average
 of 34 more episodes of GI illness than nonswimmers. One
 thousand swimming  children aged 10 and younger would
 experience an  average of 49 more  episodes than nonswim-
 ming children (Fig. 2). The associations between Entemcoc-
 cus QPCR CE and GI illness were  positive at each of the 4
 beaches, and tests for heterogeneity indicated no difference in
 these relationships across the 4 beaches  among all subjects
 (P = 0.84), or among children aged  10  and younger (P =
 0.65). Crude rates of GI illness  and numbers exposed are
 presented in Appendix C (available with the online version of
 this article).
                                                    379
Copyright ©  Lippincott Williams & Wilkins. Unauthorized reproduction of this         is prohibited.

-------
 Wade et al
          Epidemiology •  Volume 19, Number 3, May 2008
        TABLE 4.  Adjusted Cumulative Incidence Ratios (aCIR) Comparing Swimmers With Nonswimmers by Age
        and Beach


Age (yrs)
<10
11-54
55 +
Beach
West Beach
Huntington Beach
Washington Park
Silver Beach
aCIR estimated from loj
GI Illness
aCIR (95% CI)

1.42(0.99-2.11)
1.40(1.22-1.61)
2.30 (1.33-3.99)

1.90 (1.23-2.92)
1.39(1.03-1.86)
1.32 (0.99-1.74)
1.43 (1.18-1.74)
'-linear regression model.
URI
aCIR (95% CI)

0.95 (0.66-1.38)
1.12(0.93-1.35)
0.89 (0.38-2.06)

1.29 (0.74-2.24)
1.08 (0.70-1.68)
0.98 (0.64-1.50)
1.03 (0.83-1.29)

Rash
aCIR (95% CI)

1.38 (0.81-2.36)
1.40 (1.10-1.79)
0.86(0.31-2.38)

2.31 (1.23-4.32)
0.81 (0.44-1.51)
1.33 (0.85-2.08)
1.39 (1.02-1.88)

Eye
aCIR (95% CI)

1.65 (0.78-3.52)
1.01 (0.80-1.27)
0.63 (0.30-1.33)

1.32 (0.77-2.27)
0.63 (0.36-1.10)
1.41 (0.87-2.27)
0.94(0.70-1.27)

Earache
aCIR (95% CI)

1.56(0.78-3.12)
1.77 (1.28-2.45)
0.62 (0.09-4.47)

1.83 (0.71-4.71)
1.97 (0.78-4.98)
1.32 (0.71-2.46)
1.60 (1.05-2.45)

 TABLE 5.  Adjusted Odds Ratios (aOR) for Illness Associated With a 1-log Increase in Entemcoccus QPCR CE Exposure (Daily
 Average and 8:00 AM Average)


Age
(yrs)
All

slO

11-54

55 +




Average
Daily
8:00 AM
Daily
8:00 AM
Daily
8:00 AM
Daily
8:00 AM


GI Illness
aOR (95% CI)
1.26(1.06-1.51)
1.29(1.10-1.52)
1.69 (1.24-2.30)
1.67 (1.25-2.22)
1.13 (0.93-1.39)
1.16 (0.96-1.39)
1.21 (0.47-3.09)
1.17 (0.53-2.54)


Diarrhea
aOR (95% CI)
1.31 (1.06-1.61)
1.36(1.13-1.63)
2.02 (1.39-2.93)
1.98 (1.36-2.88)
1.17 (0.92-1.49)
1.21 (0.97-1.51)
0.68 (0.23-2.02)
1.02 (0.43-2.43)
GI Symptom
With
Complications
aOR (95% CI)
1.27 (1.04-1.56)
1.19(0.98-1.46)
1.60 (1.04-2.45)
1.56(1.05-2.31)
1.19(0.95-1.50)
1.09 (0.86-1.37)
1.62 (0.54-4.83)
1.63 (0.54-4.83)


URI
aOR (95% CI)
0.87 (0.69-1.09)
0.95(0.76-1.17)
0.83 (0.54-1.27)
1.05 (0.71-1.54)
0.91 (0.72-1.15)
0.93 (0.74-1.16)
0.42 (0.05-3.28)
0.34(0.05-2.19)


Rash
aOR (95% CI)
1.21 (0.92-1.58)
1.03 (0.80-1.32)
1.58 (0.90-2.76)
1.18 (0.68-2.06)
1.17 (0.87-1.59)
1.02 (0.77-1.35)
0.82(0.07-10.13)
0.55 (0.03-9.57)


Eye
aOR (95% CI)
0.80 (0.59-1.07)
0.91 (0.70-1.18)
0.80 (0.45-1.42)
1.14(0.67-1.93)
0.73 (0.54-0.99)
0.89 (0.68-1.15)
0.40 (0.06-2.47)
0.46 (0.06-3.38)


Earache
aOR (95% CI)
1.01 (0.69-1.48)
0.96 (0.69-1.33)
0.73 (0.41-1.28)
0.61 (0.34-1.09)
1.19(0.77-1.84)
1.16(0.80-1.68)
NA
NA
    aOR estimated from logistic regression model.
    NA indicates not applicable (only 8 subjects aged 55 and older reported earache).
      As time spent in the water increased beyond 1.5 hours,
 the association between Entemcoccus QPCR CE and GI
 illness  also increased. Among subjects exposed at least 2
 hours,  the risk of GI illness associated with Entemcoccus
 QPCR CE exposure increased (1.89 [1.07-3.35]). Children
 aged 5 to 10  years spent the most time  in the water, an
 average of 1.5 hours compared  with  1.2 hours for those
 younger than 5 years, 1.2 hours for those aged 11 to 20, and
 less than an hour for those older than 20.
      Other illnesses did not show strong or consistent asso-
 ciations with Entemcoccus QPCR CE. For example, rash was
 positively  associated overall  with Entemcoccus QPCR CE
 exposure among all subjects (aOR =  1.21 [CI = 0.92-1.58];
 Table 5) and particularly among children (1.58 [0.90-2.76];
 Table 5). However, there was  significant (P = 0.02) variation
 in the association across the 4 beaches, with strong positive
 associations at 2 of the beaches (at Silver Beach, aOR =1.78
 [CI = 1.08-2.94] and at Washington Park Beach aOR = 1.60
 [0.59-4.31]. At the other 2 beaches there was no evidence of
 380
an association (Huntington Beach 0.87 [0.27-2.85], and West
Beach 0.92 [0.57-1.48]). The data were too sparse to reliably
assess heterogeneity on the association of beach-specific rash
with Entemcoccus QPCR CE among children.

Enterococcus Measured by Method 1600 and GI
Illness
     Swimmers  exposed above the guideline  value of 33
CFU/100 mL had higher risks than nonswimmers or swim-
mers exposed below this value (Table 6). As with QPCR CE,
the risks associated with Enterococcus  CPU exposure were
more pronounced among children aged  10  and younger.
     Enterococcus QPCR CE levels were a stronger predic-
tor of GI illness than the CPU measure. Among all subjects,
a quartile increase in Enterococcus QPCR CE was associated
with a  1.44 (95% CI  =  1.10-1.90) increase in the odds of
illness  for all subjects,  whereas a quartile increase Entem-
coccus CPU was associated with only a  1.04 (0.90-1.21)
increase. Among children, a quartile increase in Enterococcus

                       © 2008 Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins.  Unauthorized reproduction of this         is prohibited.

-------
 Epidemiology • Volume 19, Number 3, May 2008
Rapid Water Quality Measures and Children's Sensitivity to Illness
  •- 40
  e>
  I
  "cS
  "o 20
  o
  v>
  cc
  O)
  £  0
  E
                 10             100             1,000
           Enterococcus Geometric Mean (daily average, QPCR CE/100ml)
 FIGURE 1. Swimming-associated Gl illness  rate (rate in swim-
 mers minus rate  in nonswimmers)  among all subjects as a
 function of daily average Enterococcus QPCR Cell Equivalent
 exposure. Swimming-associated  illness rate estimated  from
 linear  regression  model, adjusting  for factors described in
 TableS. Swimming-associated Gl illness = -0.0091816 + log
 10  Enterococcus QPCR CE  X 0.0213998. Solid line indicates
 rate; dashed line indicates 95% confidence interval.
    160

  o" 140

  § 1,0
                 10              100            1,000
           Enterococcus Geometric Mean (daily average, QPCR CE/100ml)
 FIGURE 2. Swimming-associated Gl illness rate (rate in swim-
 mers minus rate in  nonswimmers) among children aged  10
 years and below as  a function of daily average Enterococcus
 QPCR Cell Equivalent exposure.  Swimming-associated illness
 rate estimated  from linear regression  model, adjusting  for
 factors described  in Table 5.  Swimming-associated illness =
 -0.04821  + log 10 Enterococcus QPCR CE X 0.0486077.
 Solid line indicates rate; dashed line indicates 95% confidence
 interval.
 QPCR CE was associated with a 2.27 increase in the odds of
 illness (1.34-1.68) compared with a 1.21 (95% CI =  0.94-
 1.55) increase for a quartile increase in Enterococcus CPU. hi
 models including both Enterococcus measurements, a quar-

 © 2008 Lippincott Williams & Wilkins
                                                             tile increase  in  the  daily QPCR average  and illness was
                                                             strengthened  (1.56 [1.14-2.12]), while the relationship be-
                                                             tween a quartile increase in CPU and Gl illness was weak-
                                                             ened (0.95 [0.79-1.13]).
                      DISCUSSION
       A molecular method for rapid measurement of water
 quality (Enterococcus QPCR CE) was consistently associated
 with swimming-associated Gl illness at 4 freshwater beaches.
 Furthermore, the Enterococcus QPCR CE showed that chil-
 dren up to age 10 years were especially susceptible to Gl
 illness following swimming  exposure. While a  sensitivity
 among children to illness following recreational water expo-
 sure has often been hypothesized,3'20"23 this is the first study
 to demonstrate this  sensitivity as a function of microbial
 water quality. At least one  previous study has  observed
 higher rates of swimming-associated  illness among children,
 but the  authors did  not attribute the increased illnesses to
 measures of water quality.24 Children may be more likely to
 swallow water,25 transfer water to their mouth after exposure,
 or, as we observed, spend a longer time in water, resulting in
 a greater likelihood of contact with pathogens. Children are at
 increased  susceptibility to infection  and illness caused by
 several enteric pathogens.26'27 Such susceptibility may be due
 to differences in immune system function, hygiene, and other
 physiological and behavioral  differences.27
       We saw no evidence of increased susceptibility among
 those aged 55 and  older, but  our  ability  to make valid
 conclusions among this group was limited because they swam
 infrequently  and  reported the lowest incidence  of  illness.
 Swimmers in this age group did have  a higher overall risk for
 Gl illness compared  with nonswimmers, but the relative risk
 may have been skewed by the low incidence of Gl illness
 among nonswimmers.
       Some of the health endpoints  were nonspecific, and
 may have  been affected  by recall  bias. Broad  endpoints
 accounted  for the diverse range of symptoms potentially
 associated  with recreational water exposure  but such broad
 symptoms may obscure more  specific effects of water quality
 and  swimming exposure.  The association between Entero-
 coccus QPCR CE and Gl illness, however,  was  robust to
 different definitions (diarrhea, Gl illness with complications).
 While swimmers may have been more likely to recall illness
 than nonswimmers,  it is unlikely such a recall bias would
 occur among swimmers at varying levels of water quality. As
 with our previous analysis, a more  stringent definition of
 swimming with head immersion did not substantially alter the
 results (data not shown).
       Numerous studies  have considered associations be-
 tween fecal indicator bacteria and symptoms  of illness. The
 majority of these studies have  observed some association
 with Gl illness.22'28'29 Associations between  fecal indicator
                                                     381
Copyright © Lippincott            & Wilkins,                                   of this         is prohibited.

-------
  Wade et al
           Epidemiology  •  Volume 19,  Number 3, May 2008
            TABLE 6.  Adjusted Cumulative Incidence Ratios (aCIR) for Gastrointestinal Illness and Exposure to
            Current  Freshwater Enterococcus Method 1600 Guideline Value (33 CFU/100 ml)
                                                                 Reference Group
Nonswlmmers

Nonswimmers
Swimmers
<33 CFU/100 mL
>33 CFU/100 mL
All Subjects
aCIR (95% CI)
1.00a
1.43 (1.24-1.64)
1.61 (1.36-1.91)
Children Aged 10
and Younger
aCIR (95% CI)
1.00a
1.30 (0.89-1.91)
1.72 (1.13-2.60)
Swimmers
Children Aged 10
All Subjects and Younger
aCIR (95% CI) aCIR (95% CI)
NA NA
1.00a 1.00a
1.13 (0.96-1.32) 1.32 (1.00-1.73)
aReference category.
 bacteria and  nongastrointestinal  (nonenteric) health condi-
 tions appear to be less consistent. Several studies9"11'13 ob-
 served  associations  with respiratory  illness,  although not
 ajj 8,12,30-33 Sylvia,- inconsistencies have been observed for
 skin, ear, and eye ailments.8"10'12'30'31'34 Earaches and ear
 infections  are often associated with  swimming  and water
 exposure, but associations with specific  indicator organisms
 have been inconsistent.8"10'30'35
       Enterococcus QPCR CE was more strongly associated
 with illness than the currently recommended  culture-based
 method of measuring Enterococcus. The QPCR measure may
 be a truer representation of fecal contamination,  because it
 measures all  Enterococcus  associated with feces,  not just
 viable  cells.  The molecular  measurement of Enterococcus
 DNA provides a stable,  conservative  means of quantifying
 the  level of  fecal contamination, which is not  subject to
 die-off but may mirror the dilution and dispersion of fecal
 material. Studies have demonstrated that pathogenic micro-
 organisms (especially viruses and certain protists) are capable
 of surviving  the  sewage treatment process.  Levels of such
 pathogens  in treated effluent are often poorly correlated with
 indicator bacteria measured by cultural methods.36 Whereas
 fecal indicator bacteria are often nondetectable  by culture
 methods following sewage treatment, these same bacteria can
 be detected by QPCR.37 A recent study found human adeno-
 viruses at  both Silver Beach and Washington Park Beach,
 with municipal discharges as the  likely source.38
       The water quality at the beaches we studied was  influ-
 enced by human sources of pollution. We do not know  if the
 relationships we observed between Enterococcus QPCR CE and
 GI illness can be extended to marine beaches, or to recreational
 waters affected by different sources of fecal contamination. Our
 failure  to observe consistent associations between nonenteric
 illness and fecal indicator bacteria suggests a continuing need to
 investigate the causes of excess nonenteric illnesses commonly
 observed among swimmers.

 382
               ACKNOWLEDGMENTS
      We gratefully acknowledge the work and cooperation
of the following: Karen Delia Torre, Kurt Patrizi,  Robert
Clickner, Richard Whitman, Justin Telech, Joel Hansel, Ann
Williams, Mark Murphy,  Westat, Inc., the NEEAR field team,
National Park Service-Indiana Dunes National Lakeshore,
USGS-Lake Michigan Ecological Research  Station,  Cleve-
land  Metroparks-Bradley  Woods/Huntington Reservation,
Cuyahoga County Board of Health,  Berrien County Parks
and Recreation Commission, Michigan City Parks and Rec-
reation,  La Porte County Health Department, and the U.S.
Army  Corps  of Engineers. A special  acknowledgment  is
reserved for all of the families and individuals who took the
time to participate in the NEEAR Water Studies.

                      REFERENCES
 1. Wade TJ, Calderon RL, Sams E,  et al. Rapidly measured indicators of
   recreational water quality are predictive of swimming-associated gastro-
   intestinal illness. Environ Health  Perspect. 2006;! 14:24-28.
 2. Siefring S, Atikovic MVE, Wymer L, et al. Improved real-time PCR
   assays for the detection of fecal indicator bacteria in surface waters with
   different instrument and reagent systems. / Water Health. 2008;6:225-
   237.
 3. Pond K. Water Recreation and Disease Plausibility of Associated Infec-
   tions:  Acute Effects, Sequelae and Mortality. London: IWA Publishing;
   2005.
 4. USEPA. Method 1600: Membrane Filter Test for Enterococci in Water.
   Washington DC: Office of Water; 1997.
 5. USEPA. Ambient Water  Quality for Bacteria-1986. Cincinnati, OH:
   Office of Water Regulations and  Standards; 1986, p. 18.
 6. Haugland RA, Siefring SC, Wymer LJ, et al. Comparison of Enterococ-
   cus measurements in freshwater at two recreational beaches by quanti-
   tative  polymerase chain reaction  and membrane filter culture analysis.
    Water Res. 2005;39:559-568.
 7. Applied Biosystems. User Bulletin #2:  ABI  PRISM 7700 Sequence
   Detection System. Foster City, CA: Applied Biosystems Corporation;
    1997.
 8. Colford JM Jr, Wade TJ, Schiff KC, et al. Water quality indicators and
   the risk of illness at beaches with nonpoint sources of fecal contamina-
   tion. Epidemiology. 2007; 18:27-35.
 9. Fleisher JM, Kay D, Salmon RL, et al. Marine waters contaminated with
   domestic sewage: nonenteric illnesses associated with bather exposure in
   the United Kingdom. Am JPublic Health. 1996;86:1228-1234.
10. Haile  RW, Witte JS, Gold M, et al. The health effects of swimming in

                         © 2008 Lippincott Williams & Wilkins
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this          is prohibited.

-------
  Epidemiology  •  Volume 19,  Number  3, May 2008
Rapid Water Quality Measures and Children's Sensitivity to Illness
     ocean water contaminated by storm drain runoff. Epidemiology. 1999;
     10:355-363.
  11. McBride GB, Salmondo CE, Bandaranayake DR, et al. Health effects of
     marine bathing in New Zealand. IntJEnviron Health Res. 1998;8:173-189.
  12. Prieto MD, Lopez B, Juanes JA, et al. Recreation risks in coastal waters:
     health risks associated with bathing in sea water. JEpidemiol Commun
     Health.  2001;55:442-447.
  13. von Schirnding YE, Kfir R, Cabelli V, et al. Morbidity among bathers
     exposed to polluted  seawater. A prospective epidemiological study.
     SAfrMedJ.  1992;81:543-546.
  14. Little R, Rubin D. Statistical Analysis with Missing Data. 2nd ed. New
     York: Wiley; 2002.
  15. Jewell NP. Statistics for Epidemiology. Texts in Statistical Science. Vol.
     58. Boca Raton: Chapman & Hall/CRC; 2004.
  16. Huber P. The Behavior of Maximum  Likelihood Estimates Under Non-
     Standard Conditions.  Berkeley,  CA: University of  California Press;
     1967, pp. 221-233.
  17. Rogers  W. Regression standard errors in clustered samples. Stata Tech
     Bull. 1993;13:19-23.
  18. White H. A heteroskedasticity-consistent covariance matrix estimator and a
     direct test for heteroskedasticity. Econometrica.  1980;48:817-830.
  19. Greenland  S, Rothman KJ. Modern epidemiology.  Philadelphia, PA:
     Lippincott-Raven, 1998.
  20. Alexander LM, Heaven A, Tennant A, et al. Symptomatology of chil-
     dren in  contact with sea water contaminated with sewage. J Epidemiol
     Commun Health. 1992;46:340-344.
  21. Dorfman M. Testing the Waters: A Guide to Water Quality at Vacation
     Beaches. Natural Resources Defense  Council; 2005.
  22. Wade TJ, Pai N, Eisenberg JN, et al. Do U.S. Environmental Protection
     Agency  Water Quality Guidelines  for Recreational Waters  prevent
     gastrointestinal illness? A  systematic review and meta-analysis. Environ
     Health Perspect. 2003;111:1102-1109.
  23. World Health Organization. Bathing Water Quality and Human Health:
     Faecal  Pollution.  Outcome of an Expert Consultation.  Farnham, UK:
     World Health Organization; 2001, p. 58.
  24. Cheung WH,  Chang KC, Hung RP, et al. Health effects of beach water
     pollution in Hong Kong. Epidemiol Infect. 1990;105:139-162.
  25. Dufour AP, Evans O, Behymer TD, et al. Water ingestion during swimming
     activities in a pool: a pilot  study. / Water Health. 2006;4:425-430.
 26. Koehler KM, Lasky T, Fein SB, et al. Population-based  incidence  of
     infection with selected bacterial enteric pathogens in children  younger than
     five years of age, 1996-1998. Pediatr Infect Dis J. 2006;25:129-134.
 27. Nwachuku  N, Gerba CP.  Health  risks of enteric viral infections  in
     children. Rev Environ Contam Toxicol. 2006;186:l-56.
 28. Pruss A. Review of epidemiological studies on  health  effects from
     exposure to recreational water. Int JEpidemiol.  1998;27:l-9.
 29. Zmirou D,  Pena L, Ledrans M, et al. Risks associated with the micro-
     biological  quality of bodies of fresh and marine water used for recre-
     ational purposes: summary estimates based on published epidemiologi-
     cal studies. Arch Environ Health. 2003;58:703-711.
 30. Ferley JP, Zmirou D,  Balducci F, et al. Epidemiological  significance of
     microbiological pollution criteria for river recreational waters. Int J
     Epidemiol.  1989;18:198-205.
 31. Kueh CSW, Tarn TY, Lee T, et al. Epidemiological study of swimming-
     associated illnesses relating to bathing-beach water quality.  Water Sci
     Technol. 1995;31:l-4.
 32. Lee JV, Dawson SR, Ward  S, et al. Bacteriophages  are a better indicator
     of illness rates than bacteria amongst users of a white water course fed
     by a lowland river. Water Sci Technol. 1997;35:165-170.
 33. Seyfried PL, Tobin RS,  Brown NE,  et al.  A prospective  study  of
     swimming-related illness. II. Morbidity and the microbiological quality
     of water. Am J Public Health. 1985;75:1071-1075.
 34. Marino F, Morinigo M,  Martinez-Manzanares  E, et  al.  Microb-
     iological-epidemiological  study   of selected  marine  beaches  in
     Malaga (Spain). Water Sci Technol.  1995;31:5-9.
 35. Corbett SJ, Rubin GL, Curry GK, et al. The health  effects of swimming
     at Sydney beaches. The Sydney Beach  Users Study Advisory Group.
     Am J Public Health. 1993;83:1701-1706.
 36. Harwood VJ, Levine AD, Scott TM, et al. Validity of the  indicator
     organism paradigm for pathogen reduction in reclaimed water and public
     health protection. App! Environ Microbiol. 2005;71:3163-3170.
 37. He JW, Jiang S. Quantification of enterococci and  human adenoviruses
     in environmental samples by real-time PCR. Appl Environ Microbiol.
     2005;71:2250-2255.
 38. Xagoraraki I, Kuo  DH-W, Wong K,  et  al. Occurrence of human
     adenoviruses in two great lake recreational beaches.  Appl  Environ
     Microbiol 2007;73:7874-7881.
  © 2008 Lippincott Williams & Wilkins                                                                                               383

Copyright © Lippincott Williams & Wilkins. Unauthorized  reproduction of this            is prohibited.

-------
Appendix F

Report on  additional
monitoring for Urban
Runoff Sites
             368

-------
                   DRAFT Final Report

Monitoring of Marine Beaches Impacted by Urban Runoff


                        Prepared for:
    United States Environmental Protection Agency, Office of Water
             Standards and Health Protection Division
                    Contract: EP-C-06-033
                    Work Assignment: 2-17

            James Kitchen, Work Assignment Manager
                      ORD/NERL/ERD
                    960 College Station Rd.
                     Athens, GA 30605
                    Phone: 706-355-8043
                E-mail: kitchens.james@epa.gov

     Samantha Fontenelle, Alternate Work Assignment Manager
                  OW/OST/SHPD MC-4305T
                  1200 Pennsylvania Ave., NW
                    Washington, DC 20460
                    Phone: 202-566-2083
              E-mail: fontenelle.samantha@epa.gov
                        Prepared by:
                       GLEC
               Great Lakes Environmental Center
                     739 Hastings Street
                    Traverse City, Ml 49686
                    Phone:231-941-2230

           Dennis McCauley, Work Assignment Leader
                 E-mail: dmccauley@glec.com

         Jamie Saxton, Alternate Work Assignment Leader
                   E-mail: jsaxton@glec.com
                      February 27, 2009

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


                               TABLE OF CONTENTS

List of Figures                                                                   3

List of Tables                                                                    5

1.0 Introduction                                                                  6

2.0 Materials and Methods	7

2.1  Sample Locations	7
       2.2 Sampling Procedures	7
              2.2.1 South Carolina	7
                    2.2.1.1 Baseline Sampling	7
                    2.2.1.2 Rain Event Sampling	8
                    2.2.1.3 Duplicate and Field Blank Sample Collection               8
              2.2.2 Florida	8
                    2.2.2.1 Baseline Sampling	8
                    2.2.2.2 Rain Event Sampling	9
                    2.2.2.3 Duplicate and Field Blank Sample Collection	9
       2.3 Sample Collection	9
       2.4 Collection of Ancillary Data	10
       2.5 Sample Filtration and Processing                                         10
              2.5.1 qPCR Samples	10
              2.5.2 Dissolved Organic Carbon Samples                              11
              2.5.3 Bacteriological Samples                                         11
                    2.5.3.1 Enterococci Samples                                   12
                    2.5.3.2 Pseudomonas Samples                                12

3.0 Results and Discussion                                                        14
       3.1 Verification Tests and Laboratory Audits                                   14
       3.2 Manipulation of Laboratory Data                                          15
       3.3 Pseudomonas Data                                                     15
              3.3.1 Baseline Data	16
              3.3.2 Rain Event Data	16
       3.4 Enterococci Data                                                       17
              3.4.1 Baseline Data	17
              3.4.2 Rain Event Data	18
       3.5 Field Blanks and Duplicates	18
       3.6 Sources of Urban Runoff	19

4.0 Literature Cited	20

Figures	21

Tables	48

APPENDIX A: QUALITY ASSURANCE PROJECT PLAN

APPENDIX B: SAMPLING AND ANALYSIS PLAN

APPENDIX C: FLORIDA AND SOUTH CAROLINA MICROBIOLOGICAL DATA

APPENDIX D: SOUTH CAROLINA MICROBIOLOGICAL DATA (ALTERED BY GLEC)
                                                                          Page 2 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
                                   LIST OF FIGURES

Figure 1.       Locations of beach and ditch sampling locations at Canes Patch Swash in Myrtle
               Beach, South Carolina.

Figure 2.       Locations of beach and ditch sampling locations at Surfside Swash in Surfside,
               South Carolina.

Figure 3.       Locations of beach and ditch sampling locations at Withers Swash in Myrtle
               Beach, South Carolina.

Figure 4.       Locations of beach sampling locations at Florida Shores in Daytona Beach,
               Florida.

Figure 5.       Locations of beach sampling locations at Silver Beach in Daytona Beach, Florida.

Figure 6.       Digital picture of Canes Patch Swash at conjunction with the Atlantic Ocean
               looking north in Myrtle Beach, South Carolina.

Figure 7.       Digital picture of Canes Patch Swash Transect 1 (CPS-DT1) looking upstream,
               Myrtle Beach, South Carolina.

Figure 8.       Digital picture of Canes Patch Swash Transect 2 (CPS-DT2) looking
               downstream, Myrtle Beach, South Carolina.

Figure 9.       Digital picture of Canes Patch Swash Transect 3 (CPS-DT3) looking
               downstream, Myrtle Beach, South Carolina.

Figure 10.      Digital picture of Surfside Swash at conjunction with the Atlantic Ocean looking
               north in Myrtle Beach, South Carolina.

Figure 11.      Digital picture of Surfside Swash Transect 1 (SS-DT1) looking downstream,
               Myrtle Beach, South Carolina.

Figure 12.      Digital picture of Surfside Swash Transect 2 (SS-DT2) looking upstream, Myrtle
               Beach, South Carolina.

Figure 13.      Digital picture of Surfside Swash Transect 3 (SS-DT3) looking downstream,
               Myrtle Beach, South Carolina.

Figure 14.      Digital picture of Withers Swash at conjunction with the Atlantic Ocean looking
               south in Myrtle Beach, South Carolina.

Figure 15.      Digital picture of Withers Swash Transect 1 (WS-DT1) looking upstream, Myrtle
               Beach, South Carolina.

Figure 16.      Digital picture of Withers Swash Transect 2 (WS-DT2) looking upstream, Myrtle
               Beach, South Carolina.

Figure 17.      Digital picture of Withers Swash Transect 3 (WS-DT3) looking upstream, Myrtle
               Beach, South Carolina.

Figure 18.      Digital picture of Florida Shores Beach looking south in Daytona Beach, Florida.

Figure 19.      Digital picture of Silver Beach looking south in Daytona Beach, Florida.
                                                                              Page 3 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
                                   LIST OF FIGURES

Figure 20.      Box and whisker plot depicting baseline Pseudomonas concentrations at each of
               eight sites sampled in the vicinity of Canes Patch Swash, Myrtle Beach, South
               Carolina.

Figure 21.      Box and whisker plot depicting baseline Pseudomonas concentrations at each of
               eight sites sampled in the vicinity of Withers Swash, Myrtle Beach, South
               Carolina.

Figure 22.      Box and whisker plot depicting baseline Pseudomonas concentrations at each of
               eight sites sampled in the vicinity of Surfside Swash, Surfside, South Carolina.

Figure 23.      Box and whisker plot depicting baseline Pseudomonas concentrations at each of
               six sites sampled in the vicinity of Myrtle Beach, South Carolina.

Figure 24.      Box and whisker plot depicting baseline Enterococci concentrations at each of
               eight sites sampled in the vicinity of Canes Patch Swash, Myrtle Beach, South
               Carolina.

Figure 25.      Box and whisker plot depicting baseline Enterococci concentrations at each of
               eight sites sampled in the vicinity of Withers Swash, Myrtle Beach, South
               Carolina.

Figure 26.      Box and whisker plot depicting baseline Enterococci concentrations at each of
               eight sites sampled in the vicinity of Surfside Swash, Surfside, South Carolina.

Figure 27.      Box and whisker plot depicting baseline Enterococci concentrations at each of six
               sites sampled in the vicinity of Daytona Beach, Florida.

Figure 28.      Box and whisker plot depicting baseline Enterococci concentrations at each of
               eight sites sampled in the vicinity of Daytona Beach,  Florida and Myrtle Beach,
               South Carolina.

Figure 29.      Locations of outfalls in the vicinity of sites sampled at Canes Patch Swash in
               Myrtle Beach, South Carolina.

Figure 30.      Locations of outfalls in the vicinity of sites sampled at Withers Swash  in Myrtle
               Beach, South Carolina.
                                                                               Page 4 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
                                   LIST OF TABLES

Table 1.       Summary of deviations from the Quality Assurance Project Plan and the
              Sampling and Analysis Plan.

Table 2.       GPS coordinates for sites sampled in Myrtle Beach, South Carolina and Daytona
              Beach, Florida.

Table 3.       Ancillary measurements recorded during each sampling visit.

Table 4.       Concentration  of Enterococci and Pseudomonas in samples collected at five
              beach and three ditch transects in the vicinity of Canes Patch Swash, Myrtle
              Beach, South Carolina.

Table 5.       Concentration  of Enterococci and Pseudomonas in samples collected at five
              beach and three ditch transects in the vicinity of Withers Swash, Myrtle Beach,
              South Carolina.

Table 6.       Concentration  of Enterococci and Pseudomonas in samples collected at five
              beach and three ditch transects in the vicinity of Surfside Swash, Surfside, South
              Carolina.

Table 7.       Mean Enterococci and Pseudomonas concentrations in baseline and rain event
              samples collected in the vicinity of Silver Beach and Florida Shores, Florida and
              Canes Patch Swash, Withers Swash and Surfside Swash, South Carolina.

Table 8.       Concentration  of Enterococci and Pseudomonas in samples collected at three
              beach transects in the vicinity of Silver Beach, Daytona Beach, Florida.

Table 9.       Concentration  of Enterococci and Pseudomonas in samples collected at three
              beach transects in the vicinity of Florida Shores, Daytona Beach, Florida.

Table 10.      Concentrations of Enterococci and Pseudomonas in field blank samples
              collected in Myrtle Beach, South Carolina and Daytona Beach, Florida.

Table 11.      Concentrations of Enterococci and Pseudomonas in investigative and field
              duplicate samples collected in Myrtle Beach, South Carolina and Daytona Beach,
              Florida.
                                                                              Page 5 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


1.0 Introduction

The United States Environmental Protection Agency's (EPA's) recreational water criteria are
based on epidemiology studies of publicly owned treatment works (POTW) impacted waters. The
Critical Path  Science Plan (CPSP), an effort by the EPA Office of Water (OW) and the EPA Office
of Research  and Development (ORD) to assess recreational criteria, includes the investigation of
non-POTW impacted  recreational waters to determine whether potential health risks are different
than those in recreational waters associated with POTWs. To assist with this investigation, EPA's
Standards and Health Protection Division (SHPD) contracted Great Lakes Environmental Center
(GLEC) to provide support for a study designed to monitor recreational waters impacted primarily
by urban/suburban runoff, which may carry a variety of pollutants including bacterial pathogens
and indicators of fecal contamination.

The primary  objective of this study was to perform preliminary microbial monitoring (i.e.,
enumeration of enterococci and Pseudomonas aeruginosa, and qPCR analysis)  in  support of
EPA's search fora marine, non-POTW impacted beach affected by urban runoff. Urban runoff is
defined as storm water from rain, snowmelt or irrigation that flows over the land surface and is not
absorbed into the ground, instead flowing into streams or other surface waters or land
depressions, including the possible discharges of storm water or storm water runoff. The marine
waters selected for this study are not known by EPA to be impacted by: (1) discharges from
POTWs or combined sewer overflows (CSO) or (2) identified discharges of untreated human
waste from sanitary sewer systems. Therefore, the study was designed to collect data that will
allow for the  determination of a relationship between human illness and fecal indicators that
originate from urban runoff in the absence of POTW and CSO discharges and untreated human
wastes.
                                                                              Page 6 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


2.0 Materials and Methods

The following section briefly outlines the methods by which samples were collected, processed
and analyzed for this monitoring effort. Extensive details concerning the collection protocols and
analytical methods utilized for this project are provided in the project's Quality Assurance Project
Plan (QAPP) and Sampling and Analysis Plan (SAP) (Appendix A and Appendix B, respectively).

Occasionally there were instances when the procedures outlined in the QAPP and SAP were not
followed in entirety. Deviations from the QAPP and SAP are explained throughout the text of this
report and are summarized in Table 1.

2.1 Sample Locations

Through rigorous selection criteria based on Westat and Molina (2008), five marine beaches were
identified by  the EPA for monitoring. Three of these beaches are located in South Carolina and
two are located in  Florida. The South Carolina beaches include: Canes Patch Swash in Myrtle
Beach, Surfside Swash in Surfside and Withers Swash  in Myrtle Beach. All of these beaches are
located in Horry County. The Florida beaches include: Silver Beach  and Florida Shores. Both of
these beaches are located in Volusia County, north of Daytona Beach. Maps of the sampling
locations at each of the five beaches monitored during this study are provided in Figures 1
through 5. Digital pictures collected by GLEC at each beach and swash (where appropriate, see
Sampling Procedures below) are provided in Figures 6 through 9 (Canes Patch Swash), Figures
10 through 13 (Surfside Swash), Figures 14 through 17 (Withers Swash),  Figure 18 (Florida
Shores) and  Figure 19 (Silver Beach). Global positioning system (GPS) coordinates for each
sampling location are provided in Table 2.

2.2 Sampling Procedures

2.2.1 South  Carolina

2.2.1.1 Baseline Sampling

At each of the three South Carolina beaches (Figures 1  through 3), the sampling area was
divided into five transects, each located perpendicular to the shoreline, with approximately  100 m
between each transect. The sampling area was selected by identifying the main ditch or storm
drain affecting the bathing zone. The middle transect was delineated at the main ditch or storm
drain. Samples were collected from this transect, and also from two  transects to the right (north)
of the drain and from two transects to the left (south) of the drain. Therefore, a total of five
transects, each located 100 m apart, were sampled at each of the three South Carolina beaches.
Three water  samples were collected from one location per transect at waist deep (approximately
1.0 m deep, 0.3  m below the surface) in 500 ml, pre-sterilized polycarbonate bottles and then
composited into a  2 L pre-sterilized polycarbonate bottle. This  composite sample was used for
enterococci,  Pseudomonas and qPCR sample analyses.

In addition to sample collection at each of the five beach transects, sampling was also conducted
in the ditch or storm drain stream used to delineate the  beach transects (Figures 1 through 3).
The open ditch or stream was divided into three segments, each between 100 and 300  m apart.
Three water  samples were collected per segment at a depth of 0.3 m, if water depth allowed for
such an interval, in 500 ml, pre-sterilized polycarbonate bottles. If water depth was inadequate to
collect a sample from a 0.3 m depth, samples were collected to avoid re-suspension of bottom
sediments to the greatest degree possible.

With two exceptions, baseline sampling at each of the three South Carolina beaches was
conducted by GLEC three times per week (Sundays, Tuesdays and  Thursdays), over a period of
five weeks from  December 16, 2008 through January 18, 2009. Because this sampling effort
coincided with the Christmas and New Year holidays, sampling originally scheduled to be
                                                                              Page 7 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


completed on these days was moved to the Friday immediately after the holidays. GLEC
attempted to collect the samples within a two hour time frame during the morning hours (9:00 to
11:00 AM) at each beach and ditch/storm drain. This was accomplished for most sampling dates.
However, there were instances when GLEC was unable to complete sample collection within the
two hour window because of weather, beach conditions and/or access (see Table 1). Excluding
day one of the sampling effort, the longest window during which all samples were collected on a
given day in  South Carolina was approximately 2.5 hours (December 23, 2008).

On Tuesdays only, one additional water sample was collected for dissolved organic carbon
(DOC) analysis from each of the five beach transects and three storm drain/ditch locations (for
each of the three South Carolina beaches). This sample was collected in an ashed (at > 400°C
for two hours) 250 ml glass bottle, at the same location and depth as the composite samples
used for enterococci, Pseudomonas and qPCR analyses.

2.2.1.2 Rain Event Sampling

When there was an appreciable rain event (> 0.25 inches of rain) that was not captured during
the scheduled baseline sampling days, additional sampling was conducted within two to four
hours after accumulation of 0.25 inches or more of rainfall. In  South Carolina, rain event samples
were collected on January 13 and January 29,  2009. For each of these two rain events, GLEC
followed the  sampling procedures, and sampled at the same locations, as those outlined  above.

2.2.1.3 Duplicate and  Field Blank Sample Collection

For enterococci, Pseudomonas  and qPCR samples collected  during the baseline monitoring
period, field duplicates were collected at a rate of > 10%. These samples were equally divided
across the three beaches and ditch/storm drain locations. Duplicate samples were collected in the
same manner in which investigative samples were collected. Field blank samples for enterococci,
Pseudomonas and qPCR collected during the baseline monitoring period were collected  at a rate
of > 5%. These samples were also equally divided across the three beaches and ditch/storm
drain locations. Field blanks for the bacterial  analyses were collected by filling three of the 500
ml, pre-sterilized polycarbonate sample bottles with  sterile phosphate buffer and compositing
them into a 2 L pre-sterilized polycarbonate bottle. The field blanks were then handled in  the
same manner as investigative samples.

For DOC, field duplicate samples were collected in the same manner in which investigative
samples were collected: every Tuesday, in conjunction with collection of the investigative DOC
samples. One DOC field duplicate sample was collected at each beach or ditch/storm drain
location every Tuesday. Field blank samples for DOC were collected every Tuesday in
conjunction with the collection of the investigative DOC samples by filling a 250 ml ashed glass
bottle with 250 ml of laboratory de-ionized (Dl) water. The field blank samples were then
processed in the same manner as the investigative DOC samples.

Rain event field blank and field duplicate samples were collected at a rate of one blank and one
duplicate for each rain  event at each beach or dith/storm drain location.

2.2.2 Florida

2.2.2.1 Baseline Sampling

At each of the two Florida beaches (Figures 4 and 5), the sampling area was divided into three
transects, with approximately 200 m between each transect. Each transect was situated
perpendicular to the shoreline. Three water samples were collected from one location per
transect at waist deep (approximately 1.0 m deep, 0.3 m below the surface) in 500 ml, pre-
sterilized polycarbonate bottles. The three samples were then composited into a single 2 L pre-
sterilized polycarbonate bottle.
                                                                             Page 8 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


With three exceptions, baseline sampling at each of the two Florida beaches was conducted
three times per week (Sundays, Tuesdays and Thursdays), over a period of five weeks from
December 16, 2008 through January 18, 2009. Because this sampling effort coincided with the
Christmas and New Year holidays, sampling originally scheduled to be completed on these  days
was moved to the Friday immediately after the holidays. In addition, samples were not collected
on December 28 (Sunday) because of a flight cancellation for the GLEC field crew leader (Table
1). Therefore, this sample was collected the next day (December 29, 2008). With few exceptions,
GLEC collected the samples within a two hour time frame during the morning hours (9:00 to
11:00 AM) at each beach.

On  Tuesdays only, one additional water sample was collected for DOC analysis from the three
transects at each of the two beach locations. This sample was collected  in an ashed (at > 400°C
for two hours) 250 ml glass bottle at each transect, at the same location and depth as the
composite samples used  for enterococci, Pseudomonas and qPCR analyses.

2.2.2.2 Rain Event Sampling

When there was an appreciable rain event (> 0.25 inches of rain) that was not captured during
the  baseline sampling period, additional sampling was conducted within  two to four hours after
accumulation of 0.25 inches of rainfall.  In Florida, samples were collected during one rain event
on January 30, 2009. During this rain event GLEC followed the sampling procedures, and
sampled at the same locations, as those outlined above.

2.2.2.3 Duplicate and Field Blank Sample Collection

For enterococci, Pseudomonas and qPCR samples collected during the  baseline sampling
period, field duplicates were collected at a rate of > 10%. These samples were equally divided
across the two beaches. Duplicate samples were collected in the same manner in which
investigative samples were collected. Field blank samples for enterococci, Pseudomonas and
qPCR collected during the baseline monitoring period were collected at a rate of > 5%.  These
samples were also equally divided across the  two beaches. Field blanks for the bacterial
analyses were collected by filling three  of the 500 ml, pre-sterilized polycarbonate sample bottles
with sterile phosphate buffer and compositing  them into a 2 L pre-sterilized polycarbonate bottle.
The field blanks were then handled in the same manner as investigative samples.

For DOC, field duplicate samples were  collected in the same manner in which investigative
samples were collected: every Tuesday, in conjunction with collection of the investigative DOC
samples. One DOC field duplicate sample was collected at each beach every Tuesday. Field
blank samples for DOC were collected  every Tuesday in conjunction with the collection of the
investigative DOC samples by filling a 250 ml ashed glass bottle with 250 ml of Dl water. The
field blank samples were then processed in the same manner as the investigative DOC samples.

Rain event field blank and field duplicate samples were collected  at a rate of one blank and  one
duplicate for the rain event at each beach location.

2.3  Sample Collection

Samples were collected at each location using the methods outlined in Section 9060 of APHA et
al. (1998). A brief summary of this method is outlined below. Extensive details concerning the
methods by which samples were collected are also provided in the QAPP and SAP (Appendix A
and B,  respectively).

Using aseptic techniques (i.e. latex or nitrile gloves and, where necessary, shoulder length
polyethylene gloves, and  waders), three water samples were collected from one location per
transect at waist deep (approximately 1.0 m) in 500 ml, pre-sterilized polycarbonate bottles. To
collect these samples, the un-capped, face down 500 ml sample bottle was lowered to a depth of
                                                                             Page 9 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


approximately 0.3 m below the water surface, taking care to avoid surface scum, vegetation and
substrates. The mouth of the container was pointed away from the sampler. The bottle was then
righted, with  the opening facing away from the body, and raised through the water column,
allowing the  bottle to fill completely. This process was repeated three times (once per bottle).
Bottles were capped immediately after sample collection.

The three water samples were then composited into a 2 L, pre-sterilized polycarbonate bottle,
and a reducing agent (three tablets of sodium thiosulfate (Na2S2O3)) was added to prevent the
continuation  of bactericidal action and to reduce any strong oxidants that may have been present
in the sample and interfered with the analysis. Samples were placed in a cooler immediately after
collection and maintained  at < 4°C on wet ice.

A 500 ml portion of each of the composited water samples was removed from the 2 L
polycarbonate bottle and transferred to a 500 ml pre-sterilized polycarbonate bottle and stored
on wet ice for later filtration (within 6 hours) for qPCR analysis. There were three individual filters
processed for each qPCR sample collected in South Carolina and five individual filters were
processed for each qPCR sample collected in Florida. Each of these filters was maintained in a
cooler on dry ice. The remaining portion of the composite sample (1,000 ml) was delivered to the
local analytical laboratory on wet ice where it was filtered for enumeration of culturable
enterococci and Pseudomonas aeruginosa.

2.4 Collection of Ancillary Data

Site-specific ancillary data were collected during sampling visits at  each of the five beaches and
each of the nine ditch/storm drain locations. The parameters that were measured in the field are
shown in Table 3, along with descriptions of their measurement. The ancillary data collected for
this effort are provided on  the accompanying  project DVD and are not discussed directly in this
report.

2.5 Sample  Filtration and Processing

2.5.1 qPCR Samples

With few exceptions, filtration of the composite water samples forqPCR analysis was performed
by GLEC within six hours of sample collection. However, there were instances (particularly in
South Carolina) when, because of the exceptional level of effort required for sample filtration, the
field crew exceeded the six hour filtration threshold. Instances when the six hour threshold was
exceeded are highlighted in the final data files (see project DVD) and Table 1.

In addition to occasionally exceeding the recommended holding time, no qPCR filters were
collected in South Carolina on December 16, 2008 because of the  extraordinary time  required to
collect and filter the samples (filtration of the first of six batches of samples on this date was not
completed until approximately six hours after the recommended holding time). Because of the
holding time  exceedance forqPCR filters on December 16, 2008 (the first day of sample
collection), GLEC reduced the filtration effort  by only collecting two filters per sample on
December 18 and 21, 2008. Per EPA's guidance, three qPCR filters were collected per sample in
South Carolina beginning on December 23, 2008 (and continuing for the remainder of the study).
However, only one qPCR filter was collected  per sample during the rain event on January 29,
2009 in South Carolina because of the concern of exceeding the holding time for all samples if
three filters were collected per sample. Exceptions to the QAPP in  regards to the number of
qPCR filters  collected in South Carolina are summarized in Table 1.

Specific details regarding the procedure by which qPCR samples were filtered and preserved are
provided in the project's SAP (Appendix B). Briefly, five 100 ml aliquots of the composited water
sample were filtered using separate 47 mm, 0.4 urn polycarbonate filters. These filters were then
placed in microcentrifuge tubes and stored on dry ice (approximately -80°C) until shipment. In
                                                                             Page 10 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


Florida two of the five qPCR filters for each sample were shipped on dry ice to NRMRL/WSWRD,
Cincinnati, OH, and the other three filters were shipped to NERL/ERD, Athens, GA (see full
addresses below). In South Carolina one of the three qPCR filters for each sample was shipped
on dry ice to NRMRL/WSWRD, Cincinnati, OH, and the other two filters were shipped to
NERL/ERD, Athens, GA. The filters were shipped to each laboratory after the first two weeks of
sample collection, and then after the last three weeks of sample collection. Additional samples
(rain events) were shipped separately to the EPA laboratories. All qPCR sample filters were
shipped packed in dry ice (approximately -80°C) via overnight courier.

    1.  Marirosa  Molina
       USEPA
       NERL/ERD
       960 College Station Rd
       Athens, GA 30606
       706-355-8113 voice
       706-355-8104 fax
       Molina.Marirosa@epa.gov

    2.  Cathy Kelty
       USEPA
       NRMRL/WSWRD
       Microbial Contaminants Control Branch
       26 West Martin Luther King Drive
       MS 387
       Cincinnati, Ohio 45268
       (513) 569-7080 voice
       (513) 569-7328 fax
       Kelty.catherine@epa.gov

In addition to investigative samples, negative controls for the rinse procedure (i.e. equipment
blanks) using PCR grade water were collected twice in both South Carolina and  Florida for each
laboratory (i.e. two blanks per state per EPA lab for a total of eight equipment blanks). In addition,
a suspension with a known concentration of Enterococcus faecaliswas filtered to provide a
positive control. The positive control samples were provided to each EPA laboratory receiving
qPCR samples at a rate of two positive controls per state (total of eight samples).

The analytical results forqPCR are available from EPA and are not discussed directly in this
report.

2.5.2 Dissolved Organic Carbon Samples

The DOC samples were kept on wet ice or in a refrigerator and stored at approximately 4 °C until
sample filtration. With several exceptions, each DOC sample was filtered within 24 hours of
sample collection (see project DVD and Table 1). The method by which samples were filtered is
provided in the project's SAP (Appendix B). Once filtered, the DOC samples were shipped to
NERL/ERD, Athens, GA (see address,  above) after the first two weeks of sample collection, and
after the last three weeks of sample collection. The samples were shipped packed in wet ice via
overnight courier.

The analytical results for DOC are available from EPA and are not discussed directly in this
report.

2.5.3 Bacteriological Samples

The remaining portion of the 2 L composite samples (after the removal of the 500 mL sample for
qPCR processing) was delivered to local analytical laboratories on wet ice (approximately 4°C)
                                                                            Page 11 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


where they were filtered for enumeration of cultivable enterococci and Pseudomonas aeruginosa.
With few exceptions, they were processed within six hours of sample collection (see project DVD
and Table 1). In Florida, laboratory analyses were completed by the Volusia County
Environmental  Health Laboratory. In South Carolina, laboratory analyses were completed by
Environmental  Systems Testing Services.  Contact information for these laboratories is provided
below.

    1.  Volusia County Environmental Health Laboratory
       1250 Indian Lake Road
       Daytona Beach, FL 32124-3518
       Contact: Jack Towle
       Phone:386-248-1781
       Fax:386-248-1785
       Email: jack_towle@doh.state.fl.us

    2.  Environmental Systems Testing Services
       Post Office Box 1615
       Conway, SC 29528-1615
       Contact: Kellah Webster
       Phone: 843-347-7688
       Fax: 843-347-6739
       Email:  kellahwebster@msn.com

2.5.3.1 Enterococci Samples

Enterococci enumeration followed EPA Method 1600 on mEI agar plates (EPA 2006; Haugland et
al. 2005). For the culturable enterococci analyses, volumes of 100 mL, 50 mL and 10 mL of the
water sample will be filtered, if necessary.  These dilutions were adjusted for each beach
depending on historical or previous  samples. Colony counts from the 100 mL sample volumes
were reported unless they exceeded 150,  in which case counts from one or the other of the
smaller volumes were used after multiplying by an appropriate  correction factor to express the
enterococci counts in CFU/100 mL.

As described in the method, verification  tests on the identities of five colonies  per sample were
performed for all water samples collected during the first day of the study at each beach site. This
verification step was completed at GLEC by a GLEC microbiologist. GLEC coordinated the
shipment of processed (counted and marked) filters with the laboratories to avoid weekend
delivery (for temperature maintenance concerns) to GLEC.

Each new batch of mEI agar was tested for positive performance using pure cultures of
Enterococcus faecalis, and for negative  performance using a pure culture of a non-target
organism, e.g. E. coli. The sterility of the filters and  phosphate-buffered water used for rinsing the
filtration apparatus was also tested with  each batch of samples arriving together at the laboratory.

2.5.3.2 Pseudomonas Samples

For enumeration of Pseudomonas aeruginosa, the laboratories followed ASTM Method D5246-92
(2004). A volume of 100 to 200 mL was  filtered, with appropriate dilutions when bacterial
concentrations  were high. Colony counts were reported on a per 100 mL basis.

As described in the method, verification  tests on the identities often colonies per sample were
performed in skim  milk agar, and were performed for all water samples collected on one day
during the first week of the study at each beach site. This verification was completed at GLEC by
a GLEC microbiologist. GLEC coordinated the shipment of processed (counted and marked)
filters with the laboratories to avoid weekend delivery (for temperature maintenance concerns) to
GLEC.
                                                                            Page 12 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff                          27 Feb 2009
Each new batch of agar was tested for positive performance using pure cultures of Pseudomonas
aeruginosa, and for negative performance using a pure culture of non-target organisms, e.g. £.
feacalis. The laboratories reported results from plates producing between 20 and 80 colonies,
and not more than 150 colonies total per plate.
                                                                               Page 13 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


3.0 Results and Discussion

3.1 Verifications Tests and Laboratory Audits

The Volusia County Environmental Health Laboratory and Environmental Systems Testing
Services provided final microbiological plates to GLEC for verification of the procedures for
isolating enterococcus and Pseudomonas aeruginosa. Plates from early sample events were sent
on ice via FedEx Priority Overnight service from the laboratories to GLEC. Colonies which were
counted for data were identified on the plates and subjected to verification procedures as outlined
in  Method 1600 for enterococcus and Method D5246-92 for Pseudomonas (see QAPP in
Appendix A). Eight plates were selected at random from each laboratory for evaluation of
enterococcus. In addition, eight plates were selected at random for evaluation of Pseudomonas
from the South Carolina laboratory, but due to lack of growth, only a single plate was available
from the Florida laboratory.

Verification results for enterococcus from both the Florida and South Carolina laboratories
indicated that all selected counted colonies were correctly verified as enterococcus using the
multi-stage process outlined in the Method.  Isolation of enterococci in water using mEI media is
quite good, with fairly low false positive and false negative results in  a variety of environmental
water samples.

Verification results for Pseudomonas from the Florida laboratory for the single plate containing
colony growth were verified as Pseudomonas aeruginosa. Seven of the eight plates selected at
random for the South Carolina laboratory were  also positively identified tor Pseudomonas
aeruginosa. One plate contained colonies thought to be Pseudomonas, but did not produce the
correct clearing of the medium or fluorescent green-yellow pigment as described in the Method
verification procedure. These colonies had a more "yellow to white" color than the "pink to brown"
color specified  in the Method. The laboratory was provided with this information to correct future
plate counts.

The Florida and South Carolina laboratories were also subjected  to audits in order to ensure that
the data generated by each laboratory were of the quality necessary to meet the goals of the
study.

An initial site visit for each laboratory was conducted by GLEC research staff in order to assess
general laboratory operations and to confirm that each microbiology laboratory was clean and
prepared, and all necessary equipment was present and calibrated. Observations were made of
sample receipt and sample tracking using customary chain-of-custody reports. In addition to the
onsite visit, GLEC participated in several pre-implementation phone calls with each laboratory to
provide technical support for the laboratory for the selected methods. During the project, GLEC
was also in contact with the laboratories to answer questions, and to provide additional technical
support as required.

GLEC required that each laboratory send Enterococci and Pseudomonas plates from the final
project sample event for an assessment and quality control (QC)  check of colony counts. Plates
were counted by the laboratories, sealed and sent on ice via FedEx Priority Overnight. Upon
receipt, GLEC staff independently assessed colony numbers for 10 and 6 randomly selected
plates from the South Carolina and Florida laboratories, respectively. Colony numbers were
assessed for both enterococcus and Pseudomonas. Colony counts were within ± 5% for the
Florida laboratory for both bacteria assessed. The South Carolina laboratory was ± 10% for 8 of
10 samples of enterococcus, and 7 of 10 for Pseudomonas. Colony numbers for both bacterial
types were much higher in South Carolina than Florida (see results below).

At the conclusion of the project, GLEC solicited additional laboratory documentation for quality
assurance (QA)/QC data, equipment calibration logs, laboratory supplies and consumables to
verify this documentation was in place and up-to-date. No deficiencies for either lab were noted.
                                                                              Page 14 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


Data sheets for the verification and plate counting audits are provided on the project
DVD.

3.2 Manipulation of Laboratory Data

The enterococci and Pseudomonas concentration data reported by the Florida and South
Carolina microbiological laboratories are provided in Appendix C. After reviewing these data,
GLEC determined that there were inconsistencies in the  method by which the South Carolina
laboratory determined plate counts for the samples. On occasion the laboratory ceased counting
colonies of Pseudomonas or enterococci bacteria once 60 colonies were identified in a particular
dilution. In these instances the laboratory reported values as >60 CFU/100 ml (for a 100 ml
sample), or another value appropriate for the dilution  factor (>240, >600 or >6,000 CFU/100 ml
for 25, 10 and 1 ml dilutions, respectively). However, there were also instances when the
laboratory counted  more than 60 colonies on a plate for any of a number of dilutions. For
example, the laboratory may have counted 120 colonies  in 25 ml of sample. In this case the
South Carolina laboratory may have reported a value of 480 CFU/100 ml.

In  addition to the inconsistencies reported above, the South Carolina microbiological laboratory
also (on occasion) reported values for all dilutions, regardless of which data were most
appropriate (based  on the method) for reporting purposes.

To address these inconsistencies, GLEC altered some of the South Carolina laboratory data
based on the following rules:

1)  If a direct count was reported by the laboratory (i.e., no > sign associated with the data), this
value was used without alteration by GLEC.

2)  If two direct counts were reported by the laboratory for two separate dilutions of the same
sample, GLEC used the count between 20 and 60 for a particular dilution to determine the
reported value. If colony counts for a particular dilution were not between 20 and 60, the dilution
volume with a count closest to the 20 to 60 range was used to determine the value reported by
GLEC.

3)  When >60 colonies were reported for all dilutions for the same sample, the smallest dilution
was used to determine the reported value.

The GLEC altered data (with original data as reported by the South Carolina  laboratory) are
provided in Appendix D; these altered data were used in this report. Please note that no data
from the Florida microbiological laboratory were altered by GLEC; these data  are provided in
Appendix C.

Because the inclusion of > or < symbols during the calculation of data means, figure generation,
etc. is problematic,  GLEC chose to remove the > symbol from data, where appropriate. For
example, data with  a reported value of >60 were assigned a value of 60 to allow for the
generation of data means, figures,  etc. in this report.  In addition, values less than the reporting
limit (e.x.  <2) were assigned a value of 0 for the generation of data means, figures, etc. in this
report.

3.3 Pseudomonas Data

Tables 4 through 6  present Pseudomonas concentration data for beaches sampled in South
Carolina and Tables 7 and 8 present Pseudomonas concentration data for beaches sampled in
Florida.
                                                                             Page 15 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


3.3.1 Baseline Data

In South Carolina, concentrations of Pseudomonas bacteria collected during the baseline
monitoring period ranged between <2 and >600 CFU/100 ml at sites associated with Canes
Patch Swash (Table 4), between <2 and 144 CFU/100 ml at sites associated with Withers
Swash (Table 5) and between <2 and 176 CFU/100 ml at sites associated with Surfside Swash
(Table 6). Mean concentrations at each of the three sampling areas, averaged across the entire
baseline sampling period, ranged from 17 to 35 CFU/100 ml (Table 7).

Across the entire  baseline sampling period, Pseudomonas concentrations at the South Carolina
beach transects ranged between  1.7 (Surfside Swash BT-3) and 14 CFU/100 ml (Canes Patch
Swash BT-2) (Table 7). When averaging across beach transects, concentrations of
Pseudomonas bacteria collected during the baseline period were most elevated at Canes Patch
Swash (11 CFU/100 ml) and least at Surfside Swash (3.7 CFU/100ml_).

Across the entire  baseline sampling period, Pseudomonas concentrations at the South Carolina
ditch/storm drain sampling locations ranged  between 18 (Canes  Patch Swash  DT-3) and 123
CFU/100 ml (Canes Patch Swash DT-2) (Table 7). When averaging across ditch/storm drain
sampling locations in South Carolina, concentrations of Pseudomonas bacteria collected during
the baseline period were most elevated at Canes Patch Swash (74 CFU/100 ml) and least at
Withers Swash (43 CFU/100ml_). The relatively high Pseudomonas bacteria counts associated
with the ditch/storm drain at Canes Patch Swash corresponded to elevated concentrations of
Pseudomonas bacteria observed at the beach transects (Table 4).

Overall, mean concentrations of Pseudomonas bacteria in South Carolina were more elevated
and variable at the ditch/storm drain sampling locations than at associated beach locations
(Figures 20 through 23 and Table 7).

In Florida,  Pseudomonas bacteria collected during the baseline monitoring period were only
measured  above the method detection limit in two samples collected at Silver Beach (Table 8)
and two samples  collected at Florida Shores (Table 9). These four samples had reported
Pseudomonas concentrations of 1 CFU/100 ml. Three of these four samples were collected on
December 18, 2008.

3.3.2 Rain Event Data

In South Carolina, concentrations of Pseudomonas bacteria collected during the rain events
(January 13 and 29, 2009) ranged between <2 and 168 CFU/100 ml at sites associated with
Canes Patch Swash (Table 4), between 4 and >120 CFU/100 ml at sites associated with Withers
Swash (Table 5) and between <2 and >120 CFU/100 ml at sites associated with Surfside Swash
(Table 6). Mean concentrations at each of the three sampling areas, averaged across the two rain
events, ranged from 41 to 60 CFU/100 ml (Table 7). With the exception of the ditch/storm drain
sampling locations at Canes  Patch Swash, concentrations of Pseudomonas bacteria in South
Carolina were always more elevated during rain events than during the baseline monitoring
period (Tables 4 through 7).

When considering the two rain events in South Carolina, Pseudomonas concentrations at the
beach transects ranged between  5.0 (Surfside Swash BT-2 and BT-3) and 66 CFU/100 ml
(Withers Swash BT-3) (Table 7). When averaging across beach transects, concentrations  of
Pseudomonas bacteria collected during the rain events were most elevated at  Canes Patch
Swash (52 CFU/100 ml) and least at Surfside Swash (17 CFU/100ml_). This pattern was also
observed for Pseudomonas concentrations observed at the beach transects during the baseline
monitoring period.

Across the two rain  events, Pseudomonas concentrations at the  South Carolina ditch/storm drain
sampling locations ranged between <2 (Canes Patch Swash DT-2) and 168 CFU/100 ml (Canes
                                                                           Page 16 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


Patch Swash DT-1) (Table 7). When averaging across ditch/storm drain sampling locations in
South Carolina, concentrations of Pseudomonas bacteria collected during the rain events were
most elevated at Withers Swash (87 CFU/100 ml) and least at Canes Patch Swash (72
CFU/100ml_). Contrary to the pattern observed during the baseline monitoring period, the
relatively high Pseudomonas bacteria counts associated with the ditch/storm drains during the
rain events did not necessarily translate to elevated concentrations of Pseudomonas bacteria
observed at the beach transects (Table 7).

In Florida, Pseudomonas bacteria collected during the rain event (January 30, 2009)  were never
measured above the method detection limit.

3.4 Enterococci Data

Tables 4 through 6 present enterococci concentration data for beaches sampled in South
Carolina and Tables 7 and 8 present enterococci concentration data for beaches sampled in
Florida.

3.4.1 Baseline Data

In South Carolina, concentrations of enterococci bacteria collected during the baseline monitoring
period  ranged between <2 and 876 CFU/100 ml at sites associated with Canes Patch Swash
(Table  4), between <4 and 2,760 CFU/100 ml at sites associated with Withers Swash (Table 5)
and between <4 and 2,470 CFU/100  ml at sites associated  with Surfside Swash (Table 6). Mean
concentrations at each of the three sampling areas, averaged across the entire baseline sampling
period, ranged  from 86 to 231 CFU/100 ml (Table 7).

Across the entire baseline sampling period, enterococci concentrations at the South Carolina
beach transects ranged between 23 (Surfside Swash BT-1)  and 177 CFU/100 ml (Canes Patch
Swash BT-5) (Table 7). When averaging across beach transects,  concentrations of enterococci
bacteria collected during  the baseline period were most elevated at Canes Patch Swash (163
CFU/100 ml) and least at Surfside Swash (25 CFU/100ml_).

Across the entire baseline sampling period, enterococci concentrations at the South Carolina
ditch/storm drain sampling locations ranged between 19 (Canes Patch Swash DT-3)  and 992
CFU/100 ml (Withers Swash DT-2) (Table 7). When  averaging across ditch/storm drain sampling
locations in South Carolina, concentrations of enterococci bacteria collected during the baseline
period were most elevated at Withers Swash (654 CFU/100  ml) and least at Canes Patch Swash
(189 CFU/100ml_). Elevated enterococci bacteria counts associated with the ditch/storm drains
did not necessarily translate to elevated concentrations of enterococci bacteria observed at the
associated beach transects (Table 7).

Overall, mean concentrations of enterococci bacteria in South Carolina were more elevated and
variable at the ditch/storm drain sampling locations than  at associated beach transects (Figures
24 through 26 and Table  7).

In Florida, concentrations of enterococci bacteria collected during  the baseline monitoring period
ranged between <1 and 47 CFU/100 ml at sites associated  with Silver Beach (Table 8) and
between <1 and 108 CFU/100 ml at sites associated with Florida Shores (Table 9). Mean
concentrations at each of the two sampling areas, averaged across the entire baseline sampling
period, were 12 and 20 CFU/100 ml for Silver Beach and Florida  Shores, respectively (Table 7).
Concentrations of enterococci measured during the baseline monitoring period were also more
variable at Florida Shores than at Silver Beach (Figure 27). Across the entire baseline sampling
period, enterococci concentrations at the Florida beach transects  ranged between 10 (Silver
Beach  transect 2) and 26 CFU/100 ml (Florida Shores transect 3) (Table 7).
                                                                             Page 17 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


Enterococci concentrations measured during the baseline monitoring period in South Carolina
were more elevated than those measured during the same time period in Florida (Tables 4
through 6, Tables 8 and 9 and Figure 28). Mean concentrations of enterococci bacteria, averaged
across the entire baseline sampling period for each of the five beach locations, never exceeded
20 CFU/100 ml in Florida and were never less than 86 CFU/100ml_ in South Carolina (Table 7).

3.4.2 Rain Event Data

In South Carolina, concentrations of enterococci bacteria collected during the rain events
(January 13 and 29, 2009) ranged between <4 and 1,120 CFU/100 ml at sites associated with
Canes Patch Swash (Table 4), between <4 and 2,740 CFU/100 ml at sites associated with
Withers Swash (Table 5) and between <4 and >2,000 CFU/100 ml at sites associated with
Surfside Swash (Table 6). Mean  concentrations at each of the three sampling areas, averaged
across the two rain events, ranged from 279 to 816 CFU/100 ml (Table 7).

When considering the two rain events in South Carolina, enterococci concentrations at the beach
transects ranged between 112 (Canes Patch Swash BT-4) and 446 CFU/100 ml (Withers Swash
BT-4) (Table 7). When averaging across beach  transects, concentrations of enterococci bacteria
collected during the rain events were  most elevated at Withers Swash (415 CFU/100 ml) and
least at Canes Patch  Swash (129 CFU/1 OOmL). This pattern was different than that observed for
enterococci concentrations at the beach transects during the baseline monitoring period; Canes
Patch Swash had the most elevated enterococci concentration during the rain events (when
averaged across beach transects).

Across the two rain events, enterococci concentrations at the South Carolina ditch/storm drain
sampling locations ranged between 64 (Canes Patch Swash  DT-3) and 1,750 CFU/100 ml
(Withers Swash DT-2) (Table 7).  When averaging across ditch/storm drain  sampling locations in
South Carolina, concentrations of enterococci bacteria collected during the rain events were most
elevated at Withers Swash (1,485 CFU/100 ml) and least at Canes Patch  Swash (530
CFU/1 OOmL). The relatively high  enterococci bacteria counts associated with the ditch/storm
drains during the rain events translated directly to elevated concentrations  of enterococci bacteria
observed at the beach transects  (Table 7).

With the exception of the beach sampling locations at Canes Patch Swash, mean concentrations
of enterococci bacteria in South Carolina were always more  elevated during rain events than
during the baseline monitoring period (Tables 4  through 7).

In Florida, concentrations of enterococci bacteria collected during the one rain event (January 30,
2009) ranged between 3 and 13  CFU/100 ml at sites associated with Silver Beach (Table 8) and
between 21 and 24 CFU/100 ml at sites associated with Florida Shores (Table 9). Mean
concentrations at each of the two sampling areas, averaged across the entire baseline sampling
period, were 8.3 and 22 CFU/100 ml for Silver Beach and Florida Shores,  respectively (Table 7).
During the one rain event, enterococci concentrations at the Florida beach  transects ranged
between 3 (Silver Beach transect 2) and 24 CFU/100 ml (Florida Shores transect 1)  (Table 7).

Enterococci concentrations measured during the rain events  in South Carolina were more
elevated than those measured during the rain event in Florida (Tables 4 through 6, Tables 8 and
9). Mean concentrations of enterococci bacteria, averaged across the rain  events and sites
associated with the five beaches, never exceeded 22 CFU/100 ml in Florida and were never less
than 279 CFU/1 OOmL in South Carolina (Table 7).

3.5 Field Blanks and Duplicates

In general there were relatively few (if any) Pseudomonas or enterococci bacteria detected in field
blank samples collected as part of the quality assurance/quality control QA/QC program (Table
10). Concentrations of enterococci bacteria were always less than the method detection limit with
                                                                             Page 18 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


three exceptions: two field blanks collected on January 29 (Canes Patch Swash BT1 and Withers
Swash BT5) had concentrations of 36 and 4 CFU/100 ml, respectively. These samples were
associated with the second rain event in South Carolina. One additional enterococci field blank
sample collected on January 15, 2009 at Wither Swash (BT3) also had a measurable
concentration of 24 CFU/100 ml. When reviewing the Pseudomonas field  blank data, there was
only one sample that was measured above the method detection limit. This sample was collected
on December 28, 2008 at Withers Swash (BT3) (measured concentration of 4 CFU/100 ml).

Duplicate samples, collected as part of the QA/QC program,  showed considerable agreement
with investigative samples (Table 11). With few exceptions, the relative percent difference
between the investigative sample and the field duplicate were within acceptable limits as defined
in the project's QAPP (Appendix A).

3.6 Sources of Urban Runoff

As depicted in Figures 29 and 30, there are a considerable number of storm water outfalls in
Myrtle Beach, South Carolina in the vicinity of the sampling sites that were monitored by GLEC.
(Please note that there were no data  available for outfalls in the vicinity of Surfside Swash in
Surfside, South Carolina. However, we  can reasonably assume that a similar configuration exists
in this area). Each of these outfalls has  the capacity to carry storm water runoff from rain,
snowmelt or irrigation and may potentially affect the data that were collected for this project
(particularly those data collected during the two rain events).

Volusia County, Florida, which includes the city of Daytona Beach, has no direct storm sewer
outfalls to the ocean; storm drains empty directly into the Halifax River (Towle 2009). The outlet of
this river is located approximately eight  miles south of the closer of the two Florida beaches
monitored during this study (Florida Shores).  However, there are sites at the beach by which
stormwater could directly enter the ocean; these sites are primarily associated with
condominium/apartment complexes, parking lots or beach approaches (streets that allow cars
direct access to the beach) rather than from an outfall (Winters 2009).
                                                                             Page 19 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff	27 Feb 2009


4.0 Literature Cited

ASTM. 2004. Standard Test Method for Isolation and Enumeration of Pseudomonas aeruginosa
from Water. ASTM Method D5246-92.

American Public Health Association (APHA) American Water Works Association and Water
Environment Foundation. 1998. Standard Methods for the Examination of Water and Wastewater,
20th Edition. Section 9060: Sample Collection. American Public Health Association, Washington,
DC.

Haugland, R. A., S. C. Siefring, L. J. Wymer, K. P. Brenner, and A. P. Dufour. 2005. Comparison
of Enterococcus measurements in freshwater at two recreational beaches by quantitative
polymerase chain reaction and membrane filter culture analysis. Water Research 39: 559-568.

Molina, Marirosa. 2008. Procedure for Selection on Non-POTW Impacted Marine Beaches
Affected by Urban Runoff. November 14, 2008.

Towle, Jack. 2009. Email to Jamie Saxton.  February 3. jack_towle@doh.state.fl.us.

U.S. Environmental Protection Agency (EPA). 2006. Method 1600: Enterococci in Water by
Membrane Filtration Using membrane-Enterococcus Indoxyl-B-D-Glucoside Agar (mEI). EPA-
821-R-06-009. Office of Water, Environmental  Protection Agency, Washington, DC.

Westat. Technical Support for EPA's Search for Potential Non-POTW Impacted Beaches Affected
by Runoff from Non-point Sources. Revision 1. Prepared for USEPA  under Contract EP-D-04-
064.

Winters, Jennifer. 2009. Email to Jamie Saxton. February 4. jwinters@co.volusia.fl.us.
                                                                           Page 20 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff                          27 Feb 2009
                                     FIGURES
                                                                             Page 21 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 1. Locations of beach and ditch sampling locations at Canes Patch Swash in Myrtle
Beach, South Carolina.
                                                                             Page 22 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 2. Locations of beach and ditch sampling locations at Surfside Swash in Surfside,
South Carolina.
                                                                              Page 23 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 3. Locations of beach and ditch sampling locations at Withers Swash in Myrtle
Beach, South Carolina.
                                                                             Page 24 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 4. Locations of beach sampling locations at Florida Shores in Daytona Beach,
Florida.
                                                                              Page 25 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 5. Locations of beach sampling locations at Silver Beach in Daytona Beach, Florida.
                                                                              Page 26 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 6. Digital picture of Canes Patch Swash at conjunction with the Atlantic Ocean
looking north in Myrtle Beach, South Carolina.
                                                                              Page 27 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 7. Digital picture of Canes Patch Swash Transect 1 (CPS-DT1) looking upstream,
Myrtle Beach, South Carolina.
                                                                             Page 28 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 8. Digital picture of Canes Patch Swash Transect 2 (CPS-DT2) looking downstream,
Myrtle Beach, South Carolina.
                                                                            Page 29 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 9. Digital picture of Canes Patch Swash Transect 3 (CPS-DT3) looking downstream,
Myrtle Beach, South Carolina.
                                                                            Page 30 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 10. Digital picture of Surfside Swash at conjunction with the Atlantic Ocean looking
north in Myrtle Beach, South Carolina.
                                                                              Page 31 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 11. Digital picture of Surfside Swash Transect 1 (SS-DT1) looking downstream,
Myrtle Beach, South Carolina.
                                                                             Page 32 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 12. Digital picture of Surfside Swash Transect 2 (SS-DT2) looking upstream, Myrtle
Beach, South Carolina.
                                                                             Page 33 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 13. Digital picture of Surfside Swash Transect 3 (SS-DT3) looking downstream,
Myrtle Beach, South Carolina.
                                                                             Page 34 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 14. Digital picture of Withers Swash at conjunction with the Atlantic Ocean looking
south in Myrtle Beach, South Carolina.
                                                                              Page 35 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 15. Digital picture of Withers Swash Transect 1 (WS-DT1) looking upstream, Myrtle
Beach, South Carolina.
                                                                             Page 36 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 16. Digital picture of Withers Swash Transect 2 (WS-DT2) looking upstream, Myrtle
Beach, South Carolina.
                                                                             Page 37 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 17. Digital picture of Withers Swash Transect 3 (WS-DT3) looking upstream, Myrtle
Beach, South Carolina.
                                                                             Page 38 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 18. Digital picture of Florida Shores Beach looking south in Daytona Beach, Florida.
                                                                               Page 39 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 19. Digital picture of Silver Beach looking south in Daytona Beach, Florida.
                                                                                Page 40 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
                                                        27 Feb 2009
Figure 20. Box and whisker plot* depicting baseline Pseudomonas concentrations at each
of eight sites sampled in the vicinity of Canes Patch Swash, Myrtle Beach, South Carolina.
     600
   IT 500-
   E
   o
   ° 400 H

   o
   In 300 -
   ro
   O
   § 200 H
     100-
                                       I
            BT1
BT2
BT3
BT4     BT5

   Site ID
                 DT1
        DT2
        DT3
* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
Figure 21. Box and whisker plot* depicting baseline Pseudomonas concentrations at each
of eight sites sampled in the vicinity of Withers Swash, Myrtle Beach, South Carolina.
     160
   o
   o
     140 -
     120 -
   5 100 H
   LL.
   o
   ^  80-I
   n:

   I  60-I
   I/)
   Q_
      40 -
      20 -
            BT1
BT2
BT3
BT4     BT5

   Site ID
DT1
DT2
                                  DT3
* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
                                                                            Page 41 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
                                                        27 Feb 2009
Figure 22. Box and whisker plot* depicting baseline Pseudomonas concentrations at each
of eight sites sampled in the vicinity of Surfside Swash, Surfside, South Carolina.
     180

     160 -

   E 140 -
   o
   ° 120 -

   O 100 -
   in
   JS  80 -
   o
   §  60 H

   S  40 -
   Q.
      20 -

       0
            BT1
BT2
BT3
BT4     BT5

   Site ID
DT1
DT2
DT3
* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
Figure 23. Box and whisker plot* depicting baseline Pseudomonas concentrations at each
of six sites sampled in the vicinity of Myrtle Beach, South Carolina.
j 500-
E
o
? 400-
LL.
0
7 300-
ra
c
o
§ 200-
•o
0)
If mn -
^ i \j\j





























•











1

1 •
pb 1 b±d
                CPS-BT   CPS-DT  WS-BT   WS-DT   SS-BT    SS-DT

                                        Site ID

* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
                                                                            Page 42 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
                                                       27 Feb 2009
Figure 24. Box and whisker plot* depicting baseline Enterococci concentrations at each of
eight sites sampled in the vicinity of Canes Patch Swash, Myrtle Beach, South Carolina.
     900
     800 -

   ^ 700 -

   o 600 -

   2 500 -
   O,

   •g 400 -

   § 300 -
   k.

   c 200 -
   LLJ

     100 -

       0
             BT1      BT2      BT3      BT4      BT5      DT1      DT2     DT3

                                         Site ID

* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
Figure 25. Box and whisker plot* depicting baseline Enterococci concentrations at each of
eight sites sampled in the vicinity of Withers Swash, Myrtle Beach, South Carolina.
     3,000
   _ 2,500 -
   o 2,000 -
   ii  1,500 -
   'o
   o
oco
   a  l,ooo^
   I
   £   500 -I
              BT1
BT2
BT3
BT4     BT5

   Site ID
DT1
DT2
DT3
* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
                                                                             Page 43 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
                      27 Feb 2009
Figure 26. Box and whisker plot* depicting baseline Enterococci concentrations at each of
eight sites sampled in the vicinity of Surfside Swash, Surfside, South Carolina.
     2,500
     2,000 -
   o
   o
      1,500 -
   O
      1,000 -
       500 -
BT1
                                                L
BT2
BT3
BT4     BT5

   Site ID
DT1
                                                                DT2
DT3
* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
Figure 27. Box and whisker plot* depicting baseline Enterococci concentrations at each of
six sites sampled in the vicinity of Daytona Beach, Florida.
_ 100 -
E
o 80-
u.
^ 60-
o
o
o
8 40-
1
iS 20-
0-















»

I






FS-1














*





















I

__l , 	 ,
* i— H


FS-2 FS-3 SB-1 SB-2 SB-3


Site ID
* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
                                                                             Page 44 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 28. Box and whisker plot* depicting baseline Enterococci concentrations at each of
eight sites sampled in the vicinity of Daytona Beach, Florida and Myrtle Beach, South
Carolina.
     3,000
   _ 2,500 -
   § 2,000 -
   £  1,500 -
   |
   §  1,000 -
   I

   £   500 4
                                                                 1
              FS       SB    CPS-BT  CPS-DT  WS-BT   WS-DT   SS-BT    SS-DT

                                          Site ID

* Each box exhibits the inner quartiles, the whiskers represent the outer quartiles, the median is
represented by a solid line and the mean is presented as a red diamond.
                                                                             Page 45 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Figure 29. Locations of outfalls in the vicinity of sites sampled at Canes Patch Swash in
Myrtle Beach, South Carolina.
                                                                        Legend
                                                                          •   Sampling localions
                                                                          ffl   Outfall
                                                                          — Slormwater Pipes and Culverts
                                                                          — Surface Water Channels
                                                                             Surface Water Ponds
                                                                                      Page 46 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
                 27 Feb 2009
Figure 30. Locations of outfalls in the vicinity of sites sampled at Withers Swash in Myrtle
Beach, South Carolina.
 Avx/Golf Course
 Drainage Basin
Legend
 •   Sampling locations
 9   Outfall
  — Stormwaler Pipes and Culverts
 — Surface Water Channels
     Surface Water Ponds

                                             0  125 250    500    750   1,000
                                                                                         Page 47 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff                        27 Feb 2009
                                     TABLES
                                                                           Page 48 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 1. Summary of deviations from the Quality Assurance Project Plan and the Sampling
and Analysis Plan.
SOUTH CAROLINA
Sample Date Exception
12/16/2008
12/18/2008
12/21/2008
12/23/2008
12/26/2008
12/28/2008
12/30/2008
1/2/2009
1/4/2009
1/6/2009
1/8/2009
1/11/2009
1/13/2009
1/15/2009
1/18/2009
1/29/2009
qPCR, Sample
Collection
qPCR
qPCR
qPCR, Sample
Collection
qPCR
qPCR
qPCR
qPCR
qPCR, Sample
Collection
qPCR
qPCR
qPCR
qPCR, Rain
Event
qPCR
qPCR, Ancillary
Data
qPCR
Notes 1
all qPCR filters discarded due
to gross overages in holding
times
two qPCR filters for each site
two qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
three qPCR filters for each site
one qPCR filterfor each site
Notes 2
sample collection window
greater than two hours (last
sample was collected at 4:20
PM)
qPCR holding time slightly
exceeded

DT#3 not filtered for qPCR
across all beaches due to time
constraints




CPS-BT#1 andCPS-BT#2
samples not collected due to
beach re nourishment
construction denying access



field blank samples associated
with rain event not collected
because weather data were not
available until after the
sampling event (i.e., rain event
not confirmed until after
sample collection)

pH data not recorded for CPS
due to meter ma If unction
qPCR holding time exceeded
Notes 3



sample collection window
greater than two hours (last
sample was collected at 1 1 :30
AM)












                                                                             Page 49 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 1 (cont'd). Summary of deviations from the Quality Assurance Project Plan and the
Sampling and Analysis Plan.
FLORIDA
Sample Date Exception
12/16/2008
12/21/2008
12/23/2008
12/29/2008
12/30/2008
1/2/2009
1/11/2009
1/13/2009
1/15/2009
1/18/2009
1/30/2009
qPCR, Ancillary
Data
qPCR, Ancillary
Data
Sample
Collection,
DOC, Ancillary
Data
Sample
Collection,
qPCR, Ancillary
Data
Sample
Collection,
qPCR, DOC,
Ancillary Data
qPCR
Ancillary Data
Sample
Collection
qPCR
Sample
Collection
qPCR, DOC
Notes 1
used 2 20 ml aliquots of
sterile phospate buffer to
rinse all qPCR filters
FS-1 filter 5: only 80 ml
sample filtered and only one
10 ml phosphate buffer
rinse was filtered for qPCR
no DOC field blanks
collected at FS or SB due to
lack of clean (baked) glass
bottles
samples collected on 12/29
instead of 12/28 as originally
planned due to travel issues
returning from Christmas
holiday
FS-2, FS-3 and Duplicate
qPCR filter 5 slightly over
holding time
used 2 20-ml aliquots of
sterile phospate buffer for
each qPCR filter blank
no photographs collected at
FS
field blanks not collected due
to lack of sterile phosphate
buffer (were instead
collected on 1/15)
all qPCR filters were
accidentally allowed to thaw
for over 24 hours due to lack
of adequate dry ice
SB-3 sample collected
outside of 2 hour sampling
window (11:07 AM)
FS duplicate qPCR sample
exceeded holding time by 5
minutes
Notes 2
FS field blank for qPCR
exceeded holding time by
two hours; SB duplicate and
field blank exceeded holding
time by 48 minutes
unsure whether conductivity
measurement was
temperature corrected (i.e.
conductivity or specific
conductance)
no initial rinse completed for
FS-1, FS-2 or FS-3 DOC
samples
sample collection window
greater than two hours;
sample collection occurred
between 9:56 AM and 12:07
PM
no FS DOC field blank
collected due to lack of clean
(baked) glass bottles


DOC field blanks collected
outside of 2 hour sampling
window (11:45 AM)


DOC filtration completed on
2/01 due to lack of personnel
availability
Notes 3
unsure whether conductivity
measurement was
temperature corrected (i.e.,
conductivity or specific
conductance)

unsure whether conductivity
measurement recorded was
temperature corrected (i.e.,
conductivity or specific
conductance)
FS-1 and FS-2 filter 5 slightly
over holding time
DOC filtration completed on
12/31 due to lack of clean
(baked) glassware






Notes 4



unsure whether conductivity
measurement recorded was
temperature corrected (i.e.,
conductivity or specific
conductance)
unsure whether conductivity
measurement recorded was
temperature corrected (i.e.,
conductivity or specific
Conductance)






                                                                             Page 50 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 2. GPS coordinates for sites sampled in Myrtle Beach, South Carolina and Daytona
Beach, Florida.
State
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
South Carolina
Florida
Florida
Florida
Florida
Florida
Florida
Sampling Location
Canes Patch Beach
Canes Patch Beach
Canes Patch Beach
Canes Patch Beach
Canes Patch Beach
Canes Patch Swash
Canes Patch Swash
Canes Patch Swash
Withers Swash Beach
Withers Swash Beach
Withers Swash Beach
Withers Swash Beach
Withers Swash Beach
Withers Swash
Withers Swash
Withers Swash
Surfside Beach
Surfside Beach
Surfside Beach
Surfside Beach
Surfside Beach
Surfside Swash
Surfside Swash
Surfside Swash
Florida Shores Beach
Florida Shores Beach
Florida Shores Beach
Silver Beach
Silver Beach
Silver Beach
Site ID
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
FS-1
FS-2
FS-3
SB-1
SB-2
SB-3
Latitude (N)
33° 44.269'
33° 44.237'
33° 44.216'
33° 44.1 73'
33° 44. 136'
33° 44.293'
33° 44.486'
33° 44.51 9'
33° 40.873'
33° 40.837'
33° 40.801'
33° 40.766'
33° 40.731'
33° 40.822'
33° 41 .036'
33° 41 .330'
33° 36.850'
33° 36.814'
33° 36.770'
33° 36.682'
33° 36.682'
33° 36.843'
33° 36.974'
33° 36.998'
29° 11. 011'
29° 10.907'
29° 10.81 7'
29° 12.854'
29° 12.754'
29° 12.654'
Longitude (W)
78° 49.235'
78° 49.282'
78° 49.305'
78° 49.391'
78° 49.437'
78° 49.384'
78° 49.504'
78° 49.695'
78° 53.343'
78° 53.394'
78° 53.441'
78° 53.492'
78° 53.539'
78° 53.452'
78° 53.569'
78° 53.684'
78° 57.765'
78° 57.808'
78° 57.849'
78° 57.888'
78° 57.927'
78° 57.921'
78° 58.244'
78° 58.535'
80° 59.056'
80° 59.003'
80° 58.965'
80° 59.986'
80° 59.935'
80° 59.890'
                                                                             Page 51 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 3. Ancillary measurements recorded during each sampling visit.
Measurement
Date and Time
Air temperature
Water
temperature
Cloud Cover
Rainfall
Wind speed
(local)
Wind direction
Current
Direction
Wave height
(avg)
Boats
Animals/Birds
Description
Date and Time of day
Measurement taken from nearby weather
station* each sampling day
Measured by YSI thermometer at sampling
location on center transect (1m deep, 0.3m
below surface) on every visit
Evaluated by approximate areal coverage:
Sunny (<20% cloud cover), Mostly Sunny
(20-50% cover), Cloudy (50-70% cover)
Mostly Cloudy (70-99% cover), Overcast
(1 00% cover)
Measurements taken from nearby weather
station each sampling day for rainfall since
last sample collection; current conditions
such as rain, lightning, hail, etc. should also
be noted
Measurements taken from nearby weather
station* each sampling day
Compass direction to nearest semi-quadrant
leeward measured on wind gauge
Described in relation to shoreline facing out
Meter stick measurement at central sampling
point
Approximate number of Sailboats, Rowboats,
and Powerboats/Jet skis in the water, within
500 m of sampling area
Animals and birds potentially affecting the
water (within approximately 20 m of sampling
area in water or laterally within 20 m of outer
transects on beach), also includes number of
fowl or other birds in the air near the
sampling area
Units/Format
mm/dd/yy; hh:mm
Quantitative: °C
Quantitative: °C
Categorical: S,
MS, C, MC, 0
Quantitative: rain
in inches
Descriptive:
current conditions
Quantitative:
miles per hour
Categorical: N,
NE, E, SE, S,
SW, W, or NW
Descriptive: e.g.
onshore, right,
etc.
Quantitative:
meters
Categorical:
S, R, P; None, 1-
5,5-10, 10-20,
20-30, etc.
Descriptive: types
of animals
Quantitative:
numbers of each
type on beach
and in water
MQOs
5 minutes
+/-1°
+/-1°
Field Person or
Team
Consensus
+/- 0.25 Inches
5 mph
Weatherstation
Field Person or
Team
Consensus
0.2m
Field Person or
Team
Consensus
Field Person or
Team
Consensus
                                                                              Page 52 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 3. Ancillary measurements recorded during each sampling visit.
Measurement
Debris
PH
Turbidity
Salinity
Conductivity
Presence of
SSOs , CSOs,
leakage of
sanitary sewers,
and location of
storm sewers
Geographical
Position
Beach Facilities
Description
Approximate amount of Woody debris, Plant
matter, and Trash/Litter within bathing area;
evaluated on areal coverage basis in 10x40m
plot around center transect (20m parallel to
shore on either side of transect, 5m onshore,
and 5m out from shoreline)
Measured by YSI pH meter at sampling
location on center transect (1m deep, 0.3m
below surface) on every visit
Measured by turbidity meter from water
sample collected in 500 ml Dl-rinsed
Nalgene bottle at sampling location on center
transect (1m deep, 0.3m below surface) on
every visit
Measured by YSI salinity meter at sampling
location on center transect (1m deep, 0.3m
below surface) on every visit
Measured by YSI conductivity meter at
sampling location on center transect (1m
deep, 0.3 m below surface) on every visit
Information regarding possible local SSOs,
leakage of sanitary sewers that could have
affected the beach, presence of municipal
storm sewers, quality of the sanitary sewer
system, recent malfunctions, accidental
bypasses, etc. determined through interviews
with local managers
Coordinates taken using handheld GPS unit
in 3 places for each transect
Facilities at the beach or accessible to beach
goers such as: public restrooms, camping,
picnic areas, food stands, city parks, etc.
Units/Format
Categorical; W,
P, T; 0=Absent
(0%), 1=Sparse
(<10%),
2=Moderate (10-
40%), 3=Heavy
(40-75%), and
4=Very Heavy
(>75%)
Quantitative: pH
units
Quantitative:
Nephlometric
Turbidity Units
(NTUs)
Quantitative:
parts per
thousand
Quantitative:
microSiemens or
milliSiemens, as
appropriate
Descriptive:
location of SSO
and other
sources of
sanitary sewer
contamination
Quantitative:
Lat/long,
ddd°mm'ss.s"
Descriptive:
description and
location of
facilities relative
to the beach (i.e.,
on beach,
walking distance,
adjacent to
beach, but not on
beach, etc)
MQOs
Field Person or
Team
Consensus
0.2 units
Range
dependent; see
Standard
Methods 21 SOB
1 part per
thousand
Range
dependent
Field or team
contact person
0.1 seconds
Field Person or
Team
Consensus
                                                                              Page 53 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 4. Concentration of Enterococci and Pseudomonas in samples collected at five
beach and three ditch transects in the vicinity of Canes Patch Swash, Myrtle Beach, South
Carolina.
Sample ID
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT1-D
CPS-DT2-FLDB
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT5-D
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT4-FLDB
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-DT1-D
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
sample
Date
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
bnterocci
(CFU/100mL)
16
8
8
8
16
96
156
24
8
<4
32
20
12
16
8
310
136
20
200
204
192
256
260
312
228
24
256
16
8
4
8
16
160
76
16
<4
12
20
12
40
80
300
>240
32
272
<4
20
4
<4
4
Pseudomonas
(CFU/100mL)
16
120
<4
72
56
100
600
24
28
<4
20
8
10
10
16
590
>600
14
8
2
8
16
44
56
14
20
24
20
2
<2
<2
8
122
206
44
<2
4
2
<2
<2
<2
6
18
18
28
2
<2
<2
<2
<2
Sample ID
CPS-DT1
CPS-DT2
CPS-DT3
CPS-DT2-D
CPS-DT2-FLDB
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT1-D
CPS-BT1-FLDB
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT4-D
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT3-D
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT4-D
CPS-BT4-FLDB
CPS-BT1
bam pie
Date
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
11-Jan-09
bnterocci
(CFU/100mL)
228
96
8
68
<4
24
4
12
8
8
168
132
40
28
20
12
16
12
28
52
8
4
<4
64
96
96
272
220
<4
44
536
384
484
480
524
350
320
8
412
116
228
430
320
370
290
300
40
320
<4
824
Pseudomonas
(CFU/100mL)
2
20
18
54
<2
<2
<2
<2
<2
<2
34
18
12
4
2
<2
<2
<2
12
18
8
4
<2
6
22
16
38
18
8
8
16
12
26
6
4
50
34
24
22
18
14
<2
<2
6
44
32
14
6
<2
10
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                            Page 54 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 4 (cont'd). Concentration of Enterococci and Pseudomonas in samples collected at
five beach and three ditch transects in the vicinity of Canes Patch Swash, Myrtle Beach,
South Carolina.
Sample ID
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-BT5-D
CPS-BT1*
CPS-BT2*
CPS-BT3*
CPS-BT4*
CPS-BT5*
CPS-DT1*
CPS-DT2*
CPS-DT3*
CPS-DT1-D*
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-DT2-D
CPS-DT2-FLDB
CPS-BT1
CPS-BT2
CPS-BT3
CPS-BT4
CPS-BT5
CPS-DT1
CPS-DT2
CPS-DT3
CPS-DT3-D
CPS-BT1*
CPS-BT2*
CPS-BT3*
CPS-BT4*
CPS-BT5*
CPS-DT1*
CPS-DT2*
CPS-DT3*
CPS-BT1-D*
CPS-BT1-FLDB*
sample
Date
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
tnterocci
(CFU/100mL)
688
852
768
732
860
690
44
876
184
244
224
184
230
470
1110
56
420
200
208
164
108
232
248
176
4
116
<4
80
104
140
124
120
680
540
<4
<4
88
56
36
40
<4
350
1120
72
60
36
Pseudomonas
(CFU/100mL)
4
20
10
14
36
76
22
8
124
106
108
124
50
168
62
78
138
2
2
6
4
2
14
50
8
68
<2
4
16
20
4
6
16
22
14
10
<2
<2
6
2
2
30
<2
92
4
<2
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                            Page 55 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 5. Concentration of Enterococci and Pseudomonas in samples collected at five
beach and three ditch transects in the vicinity of Withers Swash, Myrtle Beach, South
Carolina.
Sample ID
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT5-D
WS-BT4-FLDB
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-DT3-D
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-DT3-FLDB
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT2-D
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
sample
Date
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-OS
21-Dec-OS
21-Dec-OS
21-Dec-OS
21-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
bnterocci
(CFU/100mL)
<4
88
80
112
40
160
184
2000
84
<4
8
24
4
16
<4
16
640
>600
28
32
32
24
24
>240
>240
>240
>240
28
48
20
40
20
108
>240
>240
<4
160
128
200
284
188
>240
532
>240
160
24
20
28
84
32
Pseudomonas
(CFU/100mL)
<4
12
20
32
52
36
40
80
<4
<4
<2
<2
8
10
6
6
84
24
<2
<2
<2
12
2
40
>120
28
24
4
8
4
8
6
144
>120
82
<2
2
<2
<2
6
<2
40
18
32
<2
<2
<2
8
4
<2
Sample ID
WS-DT1
WS-DT2
WS-DT3
WS-BT3-D
WS-BT3-FLDB
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT5-D
WS-BT5-FLDB
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-DT1-D
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-DT2-D
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-DT3-D
sample
Date
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
bnterocci
(CFU/100mL)
460
310
1020
12
<4
32
28
<4
4
32
460
1530
800
16
24
28
4
12
64
96
680
20
<4
16
28
16
12
32
272
1180
310
320
<4
8
8
12
8
340
340
>600
520
72
36
36
32
60
500
910
1190
920
Pseudomonas
(CFU/100mL)
4
16
28
2
4
<2
4
2
<2
10
42
66
100
<2
<2
<2
<2
<2
<2
<2
20
<2
<2
4
2
4
<2
8
12
28
32
14
2
6
4
2
<2
46
24
22
68
36
2
6
4
14
36
90
66
50
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 56 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 5 (cont'd). Concentration of Enterococci and Pseudomonas in samples collected at
five beach and three ditch transects in the vicinity of Withers Swash, Myrtle Beach, South
Carolina.
Sample ID
WS-DT3-FLDB
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT1-D
WS-BT1*
WS-BT2*
WS-BT3*
WS-BT4*
WS-BT5*
WS-DT1*
WS-DT2*
WS-DT3*
WS-BT2-D*
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT3-D
WS-BT3-FLDB
WS-BT1
WS-BT2
WS-BT3
WS-BT4
WS-BT5
WS-DT1
WS-DT2
WS-DT3
WS-BT3-D
WS-BT1*
WS-BT2*
WS-BT3*
WS-BT4*
WS-BT5*
WS-DT1*
WS-DT2*
WS-DT3*
WS-BT4-D*
WS-BT4-FLDB*
sample
Date
8-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
1 3-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
1 8-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
tnterocci
(CFU/100mL)
<4
290
450
570
280
310
380
240
1210
300
>800
>800
>800
>800
>800
1820
2740
>2000
>800
28
60
28
28
24
24
92
2760
28
24
80
92
56
28
28
1800
>2000
>2000
80
<4
8
12
92
40
400
760
1190
92
<4
Pseudomonas
(CFU/100mL)
<2
38
12
18
6
36
14
46
50
48
34
16
116
34
18
28
94
66
46
<2
4
2
10
<2
<2
36
6
<2
<2
4
4
10
8
16
56
38
40
14
4
8
16
40
36
>120
116
98
30
<2
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 57 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 6. Concentration of Enterococci and Pseudomonas in samples collected at five
beach and three ditch transects in the vicinity of Surfside Swash, Surfside, South Carolina.
Sample ID
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-DT1-D
SS-DT1-FLDB
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-DT2-D
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT2-FLDB
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT3-D
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
Sample
Date
1 6-Dec-OS
1 6-Dec-OS
16-Dec-08
1 6-Dec-OS
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
1 6-Dec-OS
16-Dec-08
1 8-Dec-OS
1 8-Dec-OS
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-OS
23-Dec-OS
23-Dec-OS
23-Dec-OS
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
Enterocci
(CFU/100mL)
4
10
4
10
12
96
400
1300
350
<4
8
8
8
<4
<4
56
160
>600
12
28
32
24
16
28
304
>240
296
20
8
28
8
8
40
>240
>240
<4
8
4
<4
8
12
4
92
>240
4
12
20
12
28
32
Pseudomonas
(CFU/100mL)
<4
<4
<4
<4
<4
48
160
<4
176
<4
6
10
<2
6
2
34
130
22
<2
2
<2
<2
14
22
16
14
>120
4
4
2
10
4
36
>120
28
<2
<2
2
2
2
<2
28
>120
144
2
<2
<2
4
6
6
Sample ID
SS-DT1
SS-DT2
SS-DT3
SS-BT4-D
SS-BT4-FLDB
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-DT1-D
SS-DT1-FLDB
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-DT2-D
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-DT3-D
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT1-D
Sample
Date
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
28-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
2-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
4-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
Enterocci
(CFU/100mL)
40
50
810
20
<4
4
4
4
8
28
20
40
>600
16
8
16
8
8
12
36
710
12
<4
12
<4
<4
8
8
88
36
1550
16
16
12
28
8
28
24
132
1940
2070
8
<4
12
<4
12
44
316
>600
<4
Pseudomonas
(CFU/100mL)
20
132
88
<2
<2
2
<2
<2
<2
2
12
50
74
<2
<2
<2
6
<2
12
60
62
18
<2
<2
<2
<2
<2
<2
34
>120
84
>120
10
2
4
4
14
14
92
106
82
2
4
4
<2
2
18
124
60
<2
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 58 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 6 (cont'd). Concentration of Enterococci and Pseudomonas in samples collected at
five beach and three ditch transects in the vicinity of Surfside Swash, Surfside, South
Carolina.
Sample ID
SS-BT1-FLDB
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT2-D
SS-BT1*
SS-BT2*
SS-BT3*
SS-BT4*
SS-BT5*
SS-DT1*
SS-DT2*
SS-DT3*
SS-BT3-D*
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT4-D
SS-BT4-FLDB
SS-BT1
SS-BT2
SS-BT3
SS-BT4
SS-BT5
SS-DT1
SS-DT2
SS-DT3
SS-BT4-D
SS-BT1*
SS-BT2*
SS-BT3*
SS-BT4*
SS-BT5*
SS-DT1*
SS-DT2*
SS-DT3*
SS-BT5-D*
SS-BT5-FLDB*
Sample
Date
8-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
29-Jan-09
Enterocci
(CFU/100mL)
<4
156
160
168
168
136
140
330
290
220
568
692
680
608
560
104
450
>2000
552
44
56
36
40
24
96
236
2470
40
<4
8
16
28
28
20
4
88
1230
28
4
<4
4
4
10
100
690
1210
<4
4
Pseudomonas
(CFU/100mL)
<2
20
24
4
4
20
10
102
40
8
96
6
6
36
10
36
112
56
6
<2
2
<2
4
6
10
60
68
2
<2
4
2
4
12
12
12
42
112
6
<2
4
4
4
2
46
>120
>120
4
<2
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 59 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 7. Mean Enterococci and Pseudomonas concentrations in baseline and rain event
samples collected in the vicinity of Silver Beach and Florida Shores, Florida and Canes
Patch Swash, Withers Swash and Surfside Swash, South Carolina.




Site ID
FS-1
FS-2
FS-3
FS*
SB-1
SB-2
SB-3
SB*
CPS-BT-1
CPS-BT-2
CPS-BT-3
CPS-BT-4
CPS-BT-5
CPS-DT-1
CPS-DT-2
CPS-DT-3
CPS-BT*
CPS-DT*
CPS*
WS-BT-1
WS-BT-2
WS-BT-3
WS-BT-4
WS-BT-5
WS-DT-1
WS-DT-2
WS-DT-3
WS-BT*
WS-DT*
WS*
SS-BT-1
SS-BT-2
SS-BT-3
SS-BT-4
SS-BT-5
SS-DT-1
SS-DT-2
SS-DT-3
SS-BT*
SS-DT*
SS*
Baseline Measurements
Mean
Enterococcus
Concentration
(CFU/100ml_)
16
18
26
20
14
10
12
12
160
147
171
161
177
307
240
19
163
189
173
56
76
79
69
58
362
610
992
67
654
231
23
24
27
25
25
49
176
916
25
380
86
Mean
Pseudomonas
Concentration
(CFU/100ml_)
0.07
0
0
0.02
0
0.07
0.07
0.04
10
14
6.9
10
12
80
123
18
11
74
35
6.4
3.9
6.1
7.3
11
34
52
44
6.9
43
17
3.4
3.7
1.7
3.9
5.9
22
95
64
3.7
60
21
Rain Event Measurements
Mean
Enterococcus
Concentration
(CFU/100ml_)
24
21
22
22
13
3.0
9.0
8.3
136
150
130
112
115
410
1,115
64
129
530
279
400
404
406
446
420
1,110
1,750
1,595
415
1,485
816
286
346
342
306
285
102
570
1,605
313
759
480
Mean
Pseudomonas
Concentration
(CFU/100ml_)
0
0
0
0
0
0
0
0
62
53
57
63
26
99
31
85
52
72
60
19
12
66
37
27
74
105
82
32
87
53
48
5.0
5.0
20
6.0
41
116
88
17
82
41
 = Includes all transects in this category.
                                                                           Page 60 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 8. Concentration of Enterococci and Pseudomonas in samples collected at three
beach transects in the vicinity of Silver Beach, Daytona Beach, Florida.
Sample ID
SB-1
SB-2
SB-3
SB-1-D
SB-1-FLDB
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-2-D
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-3-D
SB-2-FLDB
SB-1
SB-2
SB-3
sample
Date
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
29-Dec-08
29-Dec-08
29-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
2-Jan-09
2-Jan-09
2-Jan-09
bnterocci
(CFU/100mL)
4
16
4
5
<1
20
7
7
21
6
6
1
<1
<1
<1
15
8
16
16
5
12
5
9
2
2
<1
18
13
16
Pseudomonas
(CFU/100mL)
<1
<1
<1
<1
<1
<1
1
1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
Sample ID
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-2-D
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-1
SB-2
SB-3
SB-2-D
SB-3-FLDB
SB-1
SB-2
SB-3
SB-1*
SB-2*
SB-3*
SB-2-D*
SB-2-FLDB*
sample
Date
4-Jan-09
4-Jan-09
4-Jan-09
6-Jan-09
6-Jan-09
6-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
8-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
1 3- Jan-09
1 3- Jan-09
1 3- Jan-09
1 5- Jan-09
1 5- Jan-09
1 5- Jan-09
1 5- Jan-09
1 5- Jan-09
1 8- Jan-09
1 8- Jan-09
1 8- Jan-09
30-Jan-09
30-Jan-09
30-Jan-09
30-Jan-09
30-Jan-09
bnterocci
(CFU/100mL)
19
15
20
39
29
22
2
5
6
2
6
6
5
32
29
47
4
5
4
5
<1
<1
<1
5
13
3
9
6
<1
Pseudomonas
(CFU/100mL)
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 61 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 9. Concentration of Enterococci and Pseudomonas in samples collected at three
beach transects in the vicinity of Florida Shores, Daytona Beach, Florida.
Sample ID
FS-1
FS-2
FS-3
FS-1-D
FS-1-FLDB
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-2-D
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-3-D
FS-2-FLDB
FS-1
FS-2
FS-3
sample
Date
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
16-Dec-08
18-Dec-08
18-Dec-08
18-Dec-08
21-Dec-08
21-Dec-08
21-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
23-Dec-08
26-Dec-08
26-Dec-08
26-Dec-08
29-Dec-08
29-Dec-08
29-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
30-Dec-08
2-Jan-09
2-Jan-09
2-Jan-09
bnterocci
(CFU/100mL)
10
17
70
11
<1
21
22
14
3
4
5
21
4
8
3
9
12
11
10
4
3
16
22
13
12
<1
15
20
19
Pseudomonas
(CFU/100mL)
<1
<1
<1
<1
<1
1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
1
<1
<1
<1
<1
Sample ID
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-1-D
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-1
FS-2
FS-3
FS-2-D
FS-3-FLDB
FS-1
FS-2
FS-3
FS-1*
FS-2*
FS-3*
FS-2-D*
FS-2-FLDB*
sample
Date
04-Jan-09
04-Jan-09
04-Jan-09
06-Jan-09
06-Jan-09
06-Jan-09
08-Jan-09
08-Jan-09
08-Jan-09
08-Jan-09
11-Jan-09
11-Jan-09
11-Jan-09
13-Jan-09
13-Jan-09
13-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
15-Jan-09
18-Jan-09
18-Jan-09
18-Jan-09
30-Jan-09
30-Jan-09
30-Jan-09
30-Jan-09
30-Jan-09
bnterocci
(CFU/100mL)
5
16
7
4
9
3
28
30
92
28
20
9
3
72
92
108
7
10
28
10
<1
4
2
8
24
21
22
28
<1
Pseudomonas
(CFU/100mL)
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 62 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 10. Concentrations of Enterococci and Pseudomonas in field blank samples
collected in Myrtle Beach, South Carolina and Daytona Beach, Florida.
Sample ID
CPS-BT4-20081223-FLDB
CPS-DT2-20081228-FLDB
CPS-BT1 -200901 02-FLDB
CPS-BT4-200901 08-FLDB
CPS-DT2-20090115-FLDB
C PS-BT1 -200901 29-FLDB
CPS-DT2-20081216-FLDB
WS-BT4-20081 216-FLDB
WS-DT3-20081 223-FLDB
WS-BT3-20081 228-FLDB
WS-BT5-200901 02-FLDB
WS-DT3-20090108-FLDB
WS-BT3-20090115-FLDB
WS-BT4-200901 29-FLDB
SS-DT1-20081 216-FLDB
SS-BT2-20081 223-FLDB
SS-BT4-20081 228-FLDB
SS-DT1-200901 02-FLDB
SS-BT1 -200901 08-FLDB
SS-BT4-20090115-FLDB
SS-BT5-200901 29-FLDB
FS-1-20081216-FLDB
FS-2-20081230-FLDB
FS-3-20090115-FLDB
FS-2-20090130-FLDB
SB-1-20081 216-FLDB
SB-2-20081230-FLDB
SB-3-20090115-FLDB
SB-2-20090130-FLDB
Enterocci
(CFU/100mL)
<4
<4
<4
<4
<4
36*
<4
<4
<4
<4
<4
<4
24
<4
<4
<4
<4
<4
<4
<4
4
<1
<1
<1
<1
<1
<1
<1
<1
Pseudomonas
(CFU/100mL)
<2
<2
<2
<2
<2
<2*
<4
<4
<2
4
<2
<2
<2
<2
<4
<2
<2
<2
<2
<2
<2
<1
<1
<1
<1
<1
<1
<1
<1
* Represents data collected during a rain event (defined as > 0.25 inches of rain in the 12 hour
period prior to sampling).
                                                                             Page 63 of 64

-------
DRAFT Final Report
SHPD WA 2-17 Monitoring of Marine Beaches Impacted by Urban Runoff
27 Feb 2009
Table 11. Concentrations of Enterococci and Pseudomonas in investigative and field
duplicate samples collected in Myrtle Beach, South Carolina and Daytona Beach, Florida.
Sample ID
CPS-BT1-20081216
CPS-BT5-20081221
CPS-DT 1-20081 226
CPS-DT2-20081228
CPS-BT 1-200901 02
CPS-BT4-20090104
CPS-BT3-20090106
CPS-BT4-20090108
CPS-BT5-20090111
CPS-DT 1-200901 13
CPS-DT2-20090115
CPS-DT3-20090118
CPS-BT 1-200901 29
WS-BT5-20081216
WS-DT3-20081221
WS-BT2-20081226
WS-BT3-20081228
WS-BT5-20090102
WS-DT1-20090104
WS-DT2-20090106
WS-DT3-20090108
WS-BT1-20090111
WS-BT2-20090113
WS-BT3-20090115
WS-BT3-20090118
WS-BT4-20090129
SS-DT1-20081216
SS-DT2-20081221
SS-BT3-20081226
SS-BT4-20081228
SS-DT1-20090102
SS-DT2-20090104
SS-DT3-20090106
SS-BT1-20090108
SS-BT2-20090111
SS-BT3-20090113
SS-BT4-20090115
SS-BT4-20090118
SS-BT 5-20090129
FS-1-20081216-P
FS-2-20081223-P
FS-3-20081230-P
FS-1 -200901 08-P
FS-2-20090115-P
FS-2-20090130-P
SB-1-20081216-P
SB-2-20081223-P
SB-3-20081230-P
SB-1-20090108-P
SB-2-20090115-P
SB-2-20090130-P
Enterocci -
Investigative
(CFU/100mL)
16
260
300
96
28
96
484
320
732
470
176
<4
88
40
>240
128
28
12
272
340
1190
290
>800
28
56
92
96
304
<4
28
12
36
1940
8
160
680
40
28
10
10
4
13
28
10
21
4
<1
<2
<2
5
3
Enterocci -
Duplicate
(CFU/100mL)
8
256
272
68
4
44
412
320
876
420
116
<4
60
84
>240
160
12
20
320
520
920
300
>800
28
80
92
350
296
4
20
12
16
2070
<4
220
552
40
28
<4
11
3
12
28
10
28
5
<1
<2
<2
5
6
Pseudomonas -
Investigative
(CFU/100mL)
16
44
6
20
4
22
26
<2
14
168
50
14
<2
52
28
<2
8
<2
12
24
66
38
16
2
10
40
48
16
2
6
12
>120
106
2
24
6
4
12
2
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
Pseudomonas -
Duplicate
(CFU/100mL)
28
24
28
54
4
8
22
6
8
138
68
10
4
<4
24
<2
2
<2
14
68
50
48
46
<2
14
30
176
>120
2
<2
18
>120
82
<2
8
6
2
6
4
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
                                                                            Page 64 of 64

-------