Potential for Pathogen Growth, Fecal Indicator Growth and Phosphorus Release
under Clam Removal Barriers in the Lake Tahoe Basin
Final Report (P056)
Mitsunori Odagiri, Alexander Schriewer, Geoff Schladow, and Stefan Wuertz
Department of Civil and Environmental Engineering
and
Tahoe Environmental Research Center
University of California, Davis
Davis, June 2012
1
Acknowledgements
This research was supported by an agreement from the USDA Forest Service Pacific Southwest
Research Station. It was conducted in part using funds provided by the Bureau of Land Management
through the sale of public lands as authorized by the Southern Nevada Public Land Management Act.
Also, it would not have been possible to accomplish this project without the student exchange
support program provided by Japan Student Service Organization (JASSO). In addition we wish to
acknowledge the assistance of Marion Wittmann, Anne Liston, Brant Allen, Katie Webb, Deborah
Hunter and John Reuter (UC Davis, TERC). We would also like to thank the Asian Clam Workgroup
(ACWG) for providing valuable inputs for experimental design. MO is indebted to Ryan Leung and
Minji Kim (UC, Davis) for assisting with the experimental set up.
2
Executive Summary
A rapid increase in the Asian clam (Corbicula fluminea) population, an invasive species considered a
major threat to the ecosystem, has been reported in Lake Tahoe since 2008. Placing rubber barriers on
top of clam beds, with ensuing anoxic conditions that will eventually suffocate clams underneath the
barriers, is one possible remedy to manage the spread of Asian clams. Although a pilot-scale
experiment conducted in Lake Tahoe showed that Asian clams were effectively killed by this
treatment, it is necessary to evaluate the water quality impacts prior to its large-scale implementation.
We examined the following four questions through microcosm experiments, which mimicked winterand summer-like conditions under the rubber barriers in a laboratory: (1) whether fecal indicator
bacteria (FIB) such as total coliforms, fecal coliforms, Escherichia coli and enterococci re-grow
under the barrier, (2) whether artificially added human pathogens (Campylobacter jejuni and
Salmonella enterica) re-grow and/or persist, (3) whether alternative fecal indicator bacteria such as
universal-, human,- dog- and bovine-associated Bacteroidales re-grow, and (4) how much nutrients
(ammonium, phosphate and dissolved organic carbon (DOC) would be released under the barrier as a
result of decaying Asian clams. The study findings revealed the following:
(1) At winter temperatures, FIB counts did not increase under the rubber barriers, whereas sporadic
increases, especially for total coliforms, were observed in some of the scenarios tested under summer
conditions.
(2) The model pathogens Campylobacter jejuni and Salmonella enterica did not increase in numbers
under the barriers at either winter or summer temperatures. Decay rate constants for these pathogens
at summer temperatures, however, were lower than those reported under ambient water conditions
elsewhere, indicating that these pathogens persisted longer under rubber barriers.
(3) Host-associated Bacteroidales DNA markers did not increase at either winter or summer
temperatures, whereas the universal-Bacteroidales DNA marker showed a slight increase at summer
temperatures.
(4) DOC release rates were the highest followed by ammonium and phosphate at both winter and
summer temperatures. Nutrient release rates at summer temperatures were one order of magnitude
higher than at winter temperatures. Release rates of ammonium and phosphate in the microcosms
at summer temperatures were 10 to 1000 times higher than release rates from sediment reported in
Lake Tahoe, suggesting that dead Asian clams were possible sources.
3
1. Introduction
The Asian clam (Corbicula fluminea) is a freshwater bivalve, and has been considered a major threat
to the ecosystem in Lake Tahoe ever since the discovery of increased Asian clam populations in
2008 (Wittmann, et al., 2009). The effects potentially caused by a rapid increase in Asian clams are
(1) water quality degradation due to excretion of nitrogen and phosphorus and the enhancement of
algal blooms, (2) aesthetic impairment due to deposition of excess shell materials, (3) promotion of
other regional exotic mussel species such as Dreissena rostriformis bugensis due to the elevation of
calcium concentration released from dead shell matters and (4) the reduction of Tahoe’s native
benthic biodiversity (Wittmann, et al., 2009).
Agencies including Tahoe Regional Planning Agency (TRPA), Tahoe Resource Conservation
District (TRCD), U.S. Fish and Wildlife Service (USFWS), Nevada Department of Wildlife
(NDOW), Nevada Division of State Lands (NDSL) and the Lahontan Regional Water Quality
Control Board (Lahontan) as well as researchers at UC Davis and University of Nevada (UNR) have
been working together as an Asian Clam Workgroup (ACWG) to develop a science-based
management plan for Asian clam population control in Lake Tahoe. Among several management
options tested in pilot-scale studies, a promising treatment is to lay rubber bottom barriers over clam
beds, which creates anoxic conditions that will suffocate clams underneath the barriers. A pilot-scale
experiment conducted by UC Davis and UNR showed that anoxic conditions were created under the
barriers within 24 h, and 100% clam mortality was achieved in 30 days at summer lake temperatures
(16 – 19°C) in Lake Tahoe in 2009. However, adverse effects of the rubber barrier installation on
water quality were also observed, such as elevated levels of FIB and phosphate under the barriers.
Given the fact that FIB levels were not dramatically changed in the water above the rubber barriers
in the pilot-scale experiment, it is not likely that the elevated FIB levels underneath barriers were the
result of recent fecal input. If the increase in FIB underneath barriers is due to their re-growth, this
will confound water quality monitoring of recent fecal contamination. Moreover, if environments
created by rubber barrier installation have a potential to grow pathogens, this treatment must be
implemented with caution. Thus, an evaluation of the water quality impacts of rubber barriers,
particularly with respect to public health aspects, is necessary prior to lake-wide implementation.
Another important aspect of this study was to include some alternative indicator bacteria, namely,
host-associated fecal Bacteroidales. Standard FIB such as total coliforms, fecal coliforms,
Escherichia coli (E. coli) and members of the genus Enterococcus (the enterococci) have been widely
used to assess fecal contamination in drinking water, recreational water and shellfish farming areas.
However, recent studies have pointed out their limitations including FIB re-growth potential outside
their hosts (Desmarais et al., 2002, Whitman et al., 2003, Ishii, et al., 2006, Yamahara, et al., 2009) and
the inability to identify sources of fecal contamination (Santo Domingo, et al., 2007). These known
shortcomings of FIB have motivated the development of alternative fecal indicators. Host-specific
4
Bacteroidales 16S rRNA gene markers are increasingly used as a potential alternative fecal indicator
in California and elsewhere (Wuertz et al., 2011). Members of the order Bacteroidales are highly
abundant bacteria in human and other animal intestines and feces (Leclerc et al., 2001). These are
strictly anaerobic bacteria (Leclerc, et al., 2001), making them particularly useful for the evaluation of
survival of fecal bacteria under clam removal barriers. In addition, host-specific sources of fecal
contamination can be identified (Wuertz et al., 2011).
The overall objective of this study was to evaluate impacts of the rubber barrier installation on
microbial and chemical water quality. Specific questions addressed here were (1) whether fecal
indicator bacteria (FIB) such as total coliforms, fecal coliforms, Escherichia coli and enterococci
re-grow under the barrier, (2) whether artificially added human pathogens (Campylobacter jejuni and
Salmonella enterica) re-grow and/or persist, (3) whether alternative fecal indicator bacteria such as
universal-, human,- dog- and bovine-associated Bacteroidales re-grow, and (4) how much nutrients
(ammonium, phosphate and dissolved organic carbon (DOC) would be released under the barrier as a
result of decaying Asian clams. To answer these questions, microcosms were used to mimic
environments under the rubber barriers under controlled conditions. Test scenarios included winterand summer-like conditions because water temperature could significantly affect results.
5
2. Materials and Methods
To investigate whether re-growth of FIB, human pathogens (S. enterica and C. jejuni), universal- and
host-associated Bacteroidales is possible under the anoxic and nutrient-rich environment created
underneath rubber barriers, a laboratory-based microcosm study was conducted. Winter- and
summer-like conditions were examined to evaluate seasonal differences of this treatment.
2.1. Asian clam, water sediment and green filamentous algae preparation
One crucial aspect of this study was to mimic conditions observed in Lake Tahoe as closely as possible.
Therefore, all Asian clams were collected in situ by divers in Marla bay in Lake Tahoe, while
sediments and water were collected from Round Hill Pines beach in Lake Tahoe. Furthermore,
because the physiology of Asian clams might be different in summer and winter, they were collected in
November for winter experiments and in August for summer experiments. Green filamentous algae
were collected in Putah Creek, Davis, because these algae were not found in Lake Tahoe due to
relatively lower temperature in the summer of 2011. It was confirmed by microscopic inspection that
Hydrodictyon sp. was the predominant algal species. All experiments started within 24 h after
collection of all samples listed above.
2.2. Human pathogen preparation
Salmonella enterica serovar Typhimurium (ATCC 13311) and Campylobacter jejuni (ATCC 43431)
were chosen as human pathogens in this study to evaluate the potential for pathogen re-growth after
rubber barrier installation. Primary reasons for the selection of the two pathogens are that (1)
Salmonella and Campylobacter are one of the leading bacterial agents of diarrheal diseases worldwide
(Schlossberg, 2009, Levantesi, et al., 2011), (2) both Salmonella and Campylobacter were detected in
various aquatic environments including rivers, coastal waters and estuaries (Walters et al., 2007,
Schriewer, et al., 2010, Jokinen, et al., 2011), and (3) Salmonella (facultatively anaerobic) and
Campylobacter (microaerophilic) can grow under anoxic or low DO conditions (Schlossberg, 2009). S.
enterica was incubated overnight in LB broth at 37°C and C. jejuni was incubated in sheep blood
agar at 37°C under microaerophilic conditions in GasPack® anaerobic jars (Becton Dickinson
Microbiology systems, Cockeysville, MS, USA) using CampyPack ® hydrogen + CO2 (BD, Flanklin
lakes, NJ, USA) for 48 h.
2.3. Fecal sample collection as host-associated Bacteroidales sources
As sources of human-associated Bacteroidales cells, untreated wastewater was obtained from a local
wastewater plant in the Tahoe basin (Incline Village, NV). For sources containing bovine- and
dog-associated Bacteroidales, fresh cow feces were collected from a farm at UC Davis, and fresh dog
feces were obtained in a dog park in the city of Davis, respectively. These sewage and fecal samples
were spiked into microcosms within 24 h after collection.
6
2.4. Microcosm establishment
Microcosm experiments were conducted in a constant-temperature room. Two scenarios representing
winter (6°C) and summer lake temperatures (20°C) were tested to evaluate seasonal differences in the
effects of rubber barrier installations. There were two treatments (termed “cases”) plus one control for
winter experiments and five cases plus one control for summer experiments (Table 1 and Figures 1
and 2). In all cases, 38-L rectangular aquaria (51 x 26 x 32 cm) were used to establish microcosms.
Each of the microcosms contained 32 L of lake water and sediments that were 2 cm deep. The water
volume between clams and the rubber barrier was greater than would be found in situ in the lake. This
modification was necessary to allow for a sufficient number of sampling events. One of the concerns
accompanied with this larger water volume was that it would take more time to reach anoxic
conditions than it would in situ in the lake (24 h). Keeping aerobic conditions longer could affect
growth conditions, especially for anaerobic and microaerophilic pathogens. Thus, before starting
experiments, the initial dissolved oxygen concentration in the lake water was reduced to 4 mg/L by
adding nitrogen gas to achieve an anoxic condition within 24 h. All cases but the two controls included
the actual rubber barriers (45 mil EPDM (ethylene propylene diene monomer) pond liner) to prevent
oxygen from entering the water. In the two control cases, rubber barriers were not installed, and
oxygen was continuously provided during the entire experimental period to keep the Asian clams alive.
The underlining assumption is that as long as Asian clams are alive no significant increase in FIB
occurs. Furthermore, in control cases, another tank containing 32 L of Lake Tahoe water for winter
and 64 L for summer scenarios was set next to the aquarium, and water was re-circulated between the
tank and the aquarium by a pump to dilute excreta from clams, which could be harmful for Asian
clams. Except for case F, 260 Asian clams were present in each aquarium, with a population density
equal to the 2000 individuals/m2 observed in Marla bay, Lake Tahoe, in 2008. One liter of untreated
wastewater and 15 g of suspended cow and dog feces were spiked in cases B, C, F, G, and H as
host-associated Bacteroidales sources. Two pathogens, S. enterica and C. jejuni, were spiked in cases
C, F, G, and H to mimic human pathogen sources. Green filamentous algae, in which Hydrodiction sp.
was the dominant species, were added to evaluate the effects of algae on FIB growth in summer-like
conditions in cases D, F, and G. Because no quantitative data of green filamentous algae associated
with Asian clams in Lake Tahoe were available, information from Lake Michigan was used. One
hundred and forty grams of wet algae were added per aquarium, which approximately equals 200 g dry
weight/m2.
2.5. Microbial measurement
Microbial analysis covers FIB (total coliforms, fecal coliforms, E .coli and enterococci), human
pathogens (C. jejuni and S. enterica) and universal and host-associated Bacteroidales (human, bovine
and dog). FIB were quantified using cultivation-based methods, the Colilert and Enterolert
Quanti-Tray/2000 (IDEXX Laboratories, Westbrook, ME), according to the manufacturer’s
instructions. To quantify human pathogens (C. jejuni and S. enterica) and universal- and
7
host-associated Bacteroidales, genomic DNA was extracted with PureLink™ Viral RNA⁄DNA Mini
Kit (Invitrogen, Carlsbad, CA). Amplification and quantification of target genomic DNA were
carried out by a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA) with
primer and probes shown in Table 2. Cycling conditions were as follows; 2 min at 50°C and 10 min
at 95°C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60°C.
2.6. Nutrient measurement
Nutrient analysis included ammonium nitrogen, soluble phosphorus and dissolved organic carbon
(DOC). All nutrient concentrations were measured at the analytical laboratory at UC Davis. After
water sample collection, the following pretreatments were conducted to preserve them: samples for
ammonium-nitrogen were acidified to pH < 2 using H2SO4, and analyzed within 28 days; samples for
DOC were filtered through a Whatman GF/G filter and acidified to pH < 2 using H2SO4, and analyzed
within 28 days; and samples for soluble phosphorus were filtered through a Whatman GF/G filter and
analyzed within 7 days.
2.7. Physical parameter measurement
As for physical analysis, in situ dissolved oxygen, pH, conductivity, salinity and water temperature
were measured using the probes YSI 63 and 550A. To consider spatial distributions, all measurements
and sampling were conducted at three points (right, center and left) per aquarium, and results were
averaged. The number of dead clams in each aquarium was also counted by visual inspection.
2.8.
Decay rate and nutrient release rate calculation
A simple first-order decay model ( = × ) was used to estimate decay rate and T99
(time for 2 log reduction), where N is the number of bacteria per 100 ml or gene copies per ml at
time t, t1 is time at end of lag period, N0 is the initial concentration and k1 is the decay rate constant.
Nonlinear regression analysis and fitting of the data were performed using SigmaPlot 12.0 (Systat
Software Ink., San Jose, CA). Prior to calculating a decay rate constant, the Shapiro-Wilk test was
performed to examine the normality in the distribution of each data set using SigmaPlot 12.0. As for
nutrient release rate calculation, a linear regression equation was applied.
8
Table 1. Test conditions for the microcosms
Season
mimicked
Green
Water
Filamentous
(Fecal
Algae
Source)
✔
-
Lake Tahoe
✔
-
Lake Tahoe
Rubber
Asian
Barrier
Clams
Control
-
Case A
✔
Treatment
Temperature
Recirculation
(°C)
&Aeration
-
6
✔
-
6
-
-
6
-
6
-
Pathogen
Lake Tahoe
Case B
✔
✔
-
Winter
(W.W.a,
Cow and
Dog)
Lake Tahoe
Case C
✔
✔
-
(W.W.a,
Cow and
Dog)
✔
(Salb,
c
Campy )
Control
-
✔
-
Lake Tahoe
-
20
✔
Case D
✔
✔
✔
Lake Tahoe
-
20
-
Case E
✔
✔
-
Lake Tahoe
-
20
-
20
-
20
-
20
-
Lake Tahoe
Case F
✔
-
✔
(W.W.a,
Cow and
Dog)
Summer
Lake Tahoe
Case G
✔
✔
✔
(W.W.a,
Cow and
Dog)
Lake Tahoe
Case H
✔
✔
-
(W.W.a,
Cow and
Dog)
a
W.W., Wastewater
b
Salb, Salmonella
c
Campy, Campylobacter
✔
(Salb,
c
Campy )
✔
(Salb,
c
Campy )
✔
(Salb,
c
Campy )
9
Case A
Case B
Case C
Control
Figure 1. Visual inspection of initial conditions in microcosms
for the winter-like conditions shown in Table 1
10
Figure 2. Visual inspection of initial conditions in microcosms
for the summer-like conditions shown in Table 1
11
CAATCGGAGTTCTTCGTGATATCTA
AATCGGAGTTCCTCGTGATATCTA
FAM-TGGTGTAGCGGTGAAA-MGB
BacUni-690r1
BacUni-690r2
BacUni-656p
CGTTACCCCGCCTACTATCTAATG
FAM-TCCGGTAGACGATGGGGATGCGTT-TAMRA
BacHum-241r
BacHum-193p
GGACCGTGTCTCAGTTCCAGTG
FAM-TAGGGGTTCTGAGAGGAAGGTCCCCC-TAMRA
BacCow-205r
BacCow-257p
GGAGCGCAGACGGGTTTT
CAATCGGAGTTCTTCGTGATATCTA
AATCGGAGTTCCTCGTGATATCTA
FAM-TGGTGTAGCGGTGAAA-MGB
BacCan-545f1
BacUni-690r1
BacUni-690r2
BacUni-656p
Dog Bacteroidales
CCAACYTTCCCGWTACTC
BacCow-CF128f
Bovine Bacteroidales
TGAGTTCACATGTCCGCATGA
BacHum-160f
Human Bacteroidales
CGTTATCCGGATTTATTGGGTTTA
Oligonucleotide sequence (5'–3')a
BacUni-520f
Universal Bacteroidales
Primer/Probe
Table 2. qPCR primers and probes used in this study
(Kildare, et al., 2007)
(Kildare, et al., 2007)
(Kildare, et al., 2007)
(Bernhard and Field, 2000)
(Kildare, et al., 2007)
(Kildare, et al., 2007)
Reference
12
AGGCACGCCTAAACCTATAGCT
FAM-TCTCCTTGCTCATCTTTAGGATAAATTCTTTCACA-TAMRA
C.jejuni-r
C.jejuni-p
CTCACCAGGAGATTACAACATGG
AGCTCAGACCAAAAGTGACCATC
FAM-CACCGACGGCGAGACCGACTTT-Dark Quencher
ttr-6 (forward)
ttr-4 (reverse)
ttr-5 (probe)
Salmonella
CTGAATTTGATACCTTAAGTGCAGC
Oligonucleotide sequence (5'–3')a
C.jejuni-f
Campylobacter jejuni
Primer/Probe
Table 2. Primers and probes used (continued)
(Malorny, et al., 2004)
(Nogva, et al., 2000)
Reference
13
3. Results and Discussion
3.1. Dissolved oxygen (DO)
Winter-like conditions: DO in Cases B and C, containing untreated wastewater, decreased rapidly
reaching less than 1 mg/L within 72 h (Fig. 3, left). On the other hand, DO in Case A remained above 2
mg/L for the first 900 h (38 days). This higher DO level is explained by the fact that under low water
temperature conditions, clams and bacteria were not active enough to consume DO. Therefore, nitrogen
gas was continuously provided after 1000 h to create anoxic conditions (Fig. 3, left). DO in the Control
was relatively stable and stayed above 10 mg/L throughout the entire experiment.
Summer-like conditions: Except for the Control, DO sharply decreased within 24 h in all cases, and
remained below 1 mg/L for the entire experiment. Effects of algae and clams on DO reduction were not
observed because all cases except for the Control case showed a rapid decrease of DO. Compared to
winter-like conditions, DO decreased at a faster rate at summer temperatures, which is likely due to the
higher biological activities as well as lower DO saturation at summer temperatures. DO in the Control
mostly stayed above 6 mg/L, with continuous oxygen supply.
8
Control
Case A
Case B
Case C
16
14
12
10
8
Nitrogen gas provision
6
(Case A)
4
2
0
Dissolved Oxygen [mg/L]
Dissolved Oxygen [mg/L]
18
6
4
Control
Case D
Case E
Case F
Case G
Case H
2
0
0
500
1000
1500
Time [h]
2000
2500
0
200
400
600
800
1000 1200
Time [h]
Figure 3. Dissolved Oxygen concentration in microcosms as a function of time
at winter-like conditions (left) and summer-like conditions (right)
14
3.2. Asian clam mortality
Winter-like conditions: All Asian clams in Cases B and C were dead after 1050 h and 1150 h (44 and 48
days), respectively (Fig. 4, left and Table 3.), suggesting that they have a strong tolerance of low DO
conditions, especially under conditions of low water temperature when they can reduce their metabolic
activity. In Case A, Asian clams started dying gradually after anoxic conditions were achieved with
continuous provision of nitrogen gas. In Case A, 100% Asian clam mortality was achieved after 96 days.
In the Control, only a few dead clams were found at the end of the experiment.
Summer-like conditions: In all Cases experiencing summer temperatures, Asian clams died faster than in
Cases exposed to winter temperatures (Fig. 4, right and Table 3.). In Cases D and G, all clams were dead
after 250 and 300 h (10 and 13 days), whereas Case E and Case H showed 100% clam mortalities at 630
and 400 h (26 and 17 days), respectively (Table 3.). Interestingly, Cases D and G, in which green
filamentous algae were provided under the rubber barriers, showed the lowest T100 values (Time (day) to
achieve 100% Asian clam mortality), suggesting that algal decay could accelerate Asian clam mortality.
It could be hypothesized that as algae were degraded, DO was consumed significantly faster on a very
local scale. This hypothesis is supported by the observation that Asian clams, when surrounded by algae,
died faster than those not surrounded. An important result here is that Case E, which mimicked the
conditions observed in Lake Tahoe in that it contained no untreated wastewater and feces, showed a
similar Asian clam mortality rate to the rate observed in the pilot-scale experiment conducted by UC
Davis and UNR in Lake Tahoe. This coherence suggests that our experimental set-up could reasonably
reproduce environmental conditions under the rubber barriers in Lake Tahoe. The Asian clam mortality
in the Control at summer temperatures is likely due to the higher accumulation rate of harmful excreta
100
Control
Case A
Case B
CaseC
80
60
40
20
0
0
500
1000
1500
Time [h]
2000
2500
Pecentage of dead Asian clams [%]
Pecentage of dead Asian clams [%]
from clams.
100
Control
Case D
Case E
Case G
Case H
80
60
40
20
0
0
200
400
600
800
1000 1200
Time [h]
Figure 4. Percentage of dead Asian clams in microcosms as a function of time
at winter-like conditions (left) and summer-like conditions (right)
15
Table 3. Time (day) to achieve 100% Asian clam mortality (T100)
Season mimicked
Winter
Summer
Case
T100 (day)
Control
Did not reach 100% clam mortality
Case A
96
Case B
44
Case C
48
Control
Did not reach 100% clam mortality
Case D
10
Case E
26
Case F
No Asian clams were provided
Case G
13
Case H
17
Figure 5. Visual inspection of dead Asian clams in microcosms at winter-like conditions
16
Figure 6. Visual inspection of dead Asian clams in microcosms at summer -like conditions
17
3.3. Fecal Indicator Bacteria (FIB)
Winter-like conditions: Although 100% clam mortality was achieved in Cases A, B and C, significant
growth of total coliforms, E. coli, fecal coliforms and enterococci was not observed at winter
temperatures, except for the slight re-growth of E. coli and fecal coliforms in Case B between 600 and 800
h (Fig. 7 (a) and (b)). In Case A and in the Control, total coliforms decayed within 600 h, whereas other
FIB were not detected at all during the whole experiment periods. Although the only difference in test
conditions between Cases B and C was whether pathogens were spiked (Case C) or not (Case B), FIB
decay patterns were different. Interestingly, Enterococcus DNA did not decrease and stably persisted in
both Cases B and C at winter temperatures, unlike enterococci enumurated with the cultivation method
(Fig. 7 (c)).
Summer-like conditions: In Case D, all FIB increased when Asian clams started dying at 100 h, and
aferwards they decayed gradually (Fig. 8 (a), right). In Case E, total coliforms increased twice before
Asian clams died at 130 h and after all Asian clams had died at 750 h. After 750 h, total coliforms
sharply decreased at first, but remained at relatively high concentration (2000 MPN/100 ml) during the
remainder of the experimental period. Other FIB were not detected throughout the experiment in Case E.
Initial FIB levels were one order of magnitude higher in Case D (with algae) than in Case E (without
algae), suggesting that a significant amount of FIB was released from algae. To further investigate the
contribution of FIB released from algae and Asian clams, FIB were measured in algae and Asian clams.
The data showed that the amount of total coliforms in algae and Asian clams were 1.6 × 10 MPN/
aquarium and 1.6 × 10 MPN/aquarium, respectively, indicating that higher initial FIB levels in
Case D were due to the release from algae. In Cases F, G and H, in which untreated wastewater and
feces were provided, FIB decreased during the entire period. In the Control, an increase in total
coliforms was observed at 400 h when 30% of Asian clams had died. Total coliforms in the Control
increased again at 1000 h, corresponding to the high Asian clam mortality rate. In Cases F, G and H,
unlike enterococci as measured by the culture method, Enterococcus DNA fluctuated and did not show
any significant decay patterns throughout the experimental period. To investigate effects of algae and
Asian clams on FIB decay rates, ANOVA was performed for FIB decay rates in the Cases F, G and H.
We found significant differences of total coliforms, E. coli and enterococci decay rates between Case G
(with algae) and H (without algae) (P value < 0.05), suggesting that algae might contribute to the faster
decay rates of clams in Case G. Significant differences in FIB decay rates between Case F (no clams
with algae) and Case G (clams with algae) were not found (P value > 0.05), except for the fecal coliform
decay rate.
To summarize, we found that FIB did not re-grow in any of the test Cases at winter temperatures,
whereas sporadic increases in FIB, especially total coliforms, were observed in Cases D, E and the
18
Control at summer temperatures. Interestingly, none of the FIB in Cases F, G and H re-grew at summer
temperatures unlike in Cases D, E and the Control. One explanation for this inconsistence is that FIB in
Cases D, E and the Control likely were more persistent compared to the FIB in Cases F, G and H.
Primary FIB sources in Cases F, G and H were untreated wastewater and animal feces, and hence these
FIB were more susceptible to environmental stress. In contrast, the primary portion of FIB in Case D, E
and Control were likely to inhabit Lake Tahoe water, algal mats and Asian clams, which implies that
these FIB had already adjusted to environments outside their hosts. In addition, it has been reported that
some FIB can originate from not only warm-blooded animals but also from temperate soils (Leclerc, et
al., 2001, Ishii, et al., 2006), and could have a higher persistence in natural environments. Cases F, G
and H were also likely to include these more persistent FIB, but we may not have detected their
re-growth due to the relatively higher number of FIB more recently released by their hosts, which then
masked the increase in more tolerant FIB.
To investigate whether FIB might persist longer than under ambient water conditions, decay rate
constants for cultivable enterococci were compared to those obtained in another study. Decay rate
constants, k1, for cultivable enterococci were 0.105 h-1 (T99 (h) = 90) in aerobic freshwater microcosms
at 22°C under dark conditions (Bae and Wuertz, 2012). This value is approximately three to seven times
higher than values obtained in our study at summer temperatures (20°C). It is, therefore, plausible that
cultivable enterococci persisted longer under anoxic and nutrient-rich conditions created by the rubber
barrier installations than they would have in the water column of Lake Tahoe.
.
19
500
80
400
60
300
40
200
20
100
0
0
500
1000
1500
2000
0
2500
700
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
600
500
80
400
60
300
40
200
20
100
0
0
500
1000
Time [h]
1500
2000
0
2500
Percentage of dead Asian clams [%]
600
Fecal indicator bacteria [MPN/100 ml]
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
Percentage of dead Asian clams [%]
Fecal indicator bacteria [MPN/100 ml]
700
Time [h]
Figure 7. (a) Fecal Indicator Bacteria (FIB) in Control (left) and Case A (right)
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
107
106
80
105
60
104
40
103
20
102
101
0
200
400
600
0
800 1000 1200 1400 1600
108
100
107
80
106
60
105
104
40
103
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
102
20
101
0
200
400
Time [h]
600
0
800 1000 1200 1400 1600
Percentage of dead Asian clams [%]
100
Fecal indicator bacteria [MPN/100 ml]
108
Percentage of dead Asian clams [%]
Fecal indicator bacteria [MPN/100 ml]
at winter-like conditions as a function of time
Time [h]
Figure 7. (b) Fecal Indicator Bacteria (FIB) in Case B (left) and Case C (right)
107
80
106
105
60
104
40
103
102
Enterococcus
Asian clams
101
0
200
400
600
20
0
800 1000 1200 1400 1600
108
100
107
80
106
105
60
104
40
103
102
Enterococcus
Asian clams
101
0
200
400
Time [h]
600
20
0
800 1000 1200 1400 1600
Persentage of dead Asian clams [%]
100
Enterococcus [gene copies/ml]
108
Persentage of dead Asian clams [%]
Enterococcus [gene copies/ml]
under winter-like conditions as a function of time
Time [h]
Figure 7. (c) Enterococcus 23 S rRNA gene as measured by DNA in Case B (left) and Case C (right)
at winter-like conditions as a function of time
20
80
2000
60
1500
40
1000
20
500
0
0
200
400
600
800
1000
0
1200
106
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
105
104
80
60
103
40
102
20
101
100
0
200
400
600
800
1000
0
1200
Percentage of dead Asian clams [%]
2500
Fecal indicator bacteria [MPN/100 ml]
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
Percentage of dead Asian clams [%]
Fecal indicator bacteria [MPN/100 ml]
3000
Time [h]
Time [h]
Figure 8. (a) Fecal Indicator Bacteria (FIB) in Control (left) and Case D (right)
8000
80
6000
60
4000
40
2000
20
0
0
200
400
600
800
1000
0
1200
Fecal indicator bacteria [MPN/100 ml]
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
Percentage of dead Asian clams [%]
10000
107
Total coliform
E.coli
Fecal coliform
enterococci
106
105
104
103
102
0
200
Time [h]
400
600
800
1000
1200
Time [h]
Figure 8. (b) Fecal Indicator Bacteria (FIB) in Case E (left) and Case F (right)
at summer-like conditions as a function of time
Enterococcus [genescopies/ml]
Fecal indicator bacteria [MPN/100 ml]
at summer-like conditions as a function of time
108
107
106
105
104
103
102
101
0
200
400
600
800
1000
1200
Time [h]
Figure 8. (c) Enterococcus 23 S rRNA gene as measured by DNA in Case F
at summer-like conditions as a function of time
21
80
105
60
104
40
103
20
102
0
200
400
600
800
1000
0
1200
107
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
106
80
105
60
104
40
103
20
102
0
200
400
Time [h]
600
800
1000
0
1200
Percentage of dead Asian clams [%]
106
Fecal indicator bacteria [MPN/100 ml]
100
Total coliform
E.coli
Fecal coliform
enterococci
Asian clam
Percentage of dead Asian clams [%]
Fecal indicator bacteria [MPN/100 ml]
107
Time [h]
Figure 8. (d) Fecal Indicator Bacteria (FIB) in Case G (left) and Case H (right)
107
80
106
105
60
104
40
103
102
Enterococcus
Asian clams
101
0
200
400
600
Time [h]
800
1000
20
0
1200
108
100
107
80
106
105
60
104
40
103
102
Enterococcus
Asian clams
101
0
200
400
600
800
1000
20
0
1200
Persentage of dead Asian clams [%]
100
Enterococcus [gene copies/ml]
108
Persentage of dead Asian clams [%]
Enterococcus [gene copies/ml]
at summer-like conditions as a function of time
Time [h]
Figure 8. (e) Enterococcus 23 S rRNA gene as measured by DNA in Case G (left) and
Case H (right) at summer-like conditions as a function of time
22
Table 4 (a). Kinetic parameters of fecal indicator bacteria at winter temperatures (6 °C)e
Season
k1 (h-1)
S.E.a
t1 (h)
R2
T99b (h)
Total coliforms
0.010
0.002
0
1.00
475
E .coli
N.D.c
Fecal coliforms
N.D.c
enterococci
N.D.c
Total coliforms
N.A.d
E. coli
N.D.c
Fecal coliforms
N.D.c
enterococci
N.D.c
Total coliforms
N.A.d
E. coli
N.A.d
Fecal coliforms
N.A.d
enterococci
0.007
0.001
118
0.99
776
Total coliforms
N.A.d
E. coli
N.A.d
Fecal coliforms
0.009
0.000
96
1.00
625
Case
mimicked
Control
Case A
Target FIB
Winter
Case B
Case C
enterococci
N.A.
d
a
S.E., standard error
b
T99, Time for two log reduction
c
N.D., not detected
d
N.A., not applicable due to the violation of normality assumption for the data set
e
The model is as follows;
=
×
where N is the number of bacteria per 100 ml at time t, t1 is time at
end of lag period, N0 is the initial concentration and k1 is the decay rate constant
23
Table 4 (b). Kinetic parameters of fecal indicator bacteria at summer temperatures (20 °C)e
Season
mimicked
Case
Control
Case D
Case E
k1 (h-1)
Target FIB
S.E.a
t1 (h)
R2
T99b (h)
Total coliforms
N.A.d
E. coli
N.D.c
Fecal coliforms
N.D.c
Enterococci
N.D.c
Total coliforms
0.015
0.002
154
0.96
457
E. coli
0.005
0.001
98
0.86
1038
Fecal coliforms
0.006
0.001
130
0.94
924
Enterococci
0.026
0.000
98
1.00
274
Total coliforms
N.A.d
E. coli
N.D.c
Fecal coliforms
N.D.c
Enterococci
N.D.c
Total coliforms
0.017
0.001
0
1.00
265
E. coli
0.028
0.003
0
1.00
164
Fecal coliforms
0.012
0.001
0
1.00
374
Enterococci
0.019
0.001
0
1.00
240
Total coliforms
0.020
0.002
0
1.00
230
E. coli
0.025
0.002
0
1.00
188
Fecal coliforms
0.002
0.000
98
0.97
2191
Enterococci
0.023
0.001
0
1.00
200
Total coliform
0.006
0.001
0
0.93
781
E. coli
0.004
0.001
0
0.98
1071
Fecal coliform
0.003
0.000
98
0.96
1804
enterococci
0.015
0.002
0
0.98
318
Summer
Case F
Case G
Case H
a
b
S.E., standard error, T99, Time for two log reduction
c
N.D., not detected, d N.A., not applicable due to their re-growth
e
The model is as follows;
=
×
where N is the number of bacteria per 100 ml at time t, t1 is time at
end of lag period, N0 is the initial concentration and k1 is the decay rate constant
24
3.4. Human pathogens (Campylobacter jejuni and Salmonella tenrerica)
Winter-like conditions: At winter-like conditions, neither Campylobacter nor Salmonella DNA markers
increased significantly before and after 100% Asian clam mortality was achieved (Fig. 9). To calculate a
decay rate, a first-order decay model ( = × ) was applied to the Salmonella data set,
showing exponential decay with a rate constant k1 of 0.003 h-1 (Table 5.). On the other hand,
Campylobacter data set was not normally distributed, and hence the model was not applied.
Summer-like conditions: Neither Campylobacter nor Salmonella DNA markers increased significantly
in Cases F, G and H at summer-like conditions (Fig. 10). Different decay patterns were observed
depending on pathogens and cases. As for Campylobacter, Case G showed exponential decay patterns
with a rate constant, k1, of 0.003 h-1 (Table 5.). Campylobacter in Case H also decayed with a rate
constant k1 of 0.002 h-1, but the data fluctuated more, resulting in lower R2 value (R2 = 0.62). Salmonella
decay patterns were different from Campylobacter in that Salmonella DNA decreased at the beginning
of this experiment, and were relatively stable or slightly increased from 200 to 800 h in Cases G and H.
After 800 h, Salmonella DNA decayed again. These decay patterns were poorly expressed by the
first-order decay model (R2 = 0.57 and R2 = 0.35, respectively), whereas the decay pattern of Case F
fitted well with a first-order decay model, suggesting that nutrients released from dead Asian clams may
contribute to the different decay patterns observed in Cases G and H.
In conclusion, we found that neither Campylobacter nor Salmonella re-grew under the rubber barriers at
winter- and summer-like conditions, although Campylobacter (microaerophilic) and Salmonella
(facultatively anaerobic) have an ability to grow under anoxic or low DO conditions (Schlossberg, 2009).
The reason why these two pathogens did not re-grow is likely due to their growth temperature ranges. C.
jejuni have a relatively high minimum growth temperature around 30°C (Hazeleger, et al., 1998), whereas
S. enterica growth rates are significantly decreased below 20°C (Balamurugan and Dugan, 2010),
implying that test temperature conditions (6°C and 20°C) typically observed in winter and summer in
Lake Tahoe do not allow these pathogens to grow significantly under the rubber barriers.
In implementing this treatment, however, another important question is whether these two pathogens
could survive longer than under ambient conditions in the lake, which could eventually pose higher
public health risks due to the installation of the treatment. A study has reported that decay rate constant
values for k1 for Campylobacter and Salmonella DNA were 0.064 h-1 and 0.043 h-1, respectively, in
aerobic freshwater microcosms at 22°C under dark conditions (Bae and Wuertz, 2012). These values are
larger than those obtained in this study at summer temperatures (20°C). Primary differences between the
reference and this study are DO (aerobic and anoxic) and nutrient conditions (ambient and nutrient-rich)
as well as experimental set-up (flowing and non-flowing). In this study, anoxic condition may inhibit
25
predation activities of higher organisms. Furthermore, the periods when Salmonella were stable
correspond to the periods when DOC levels increased in Cases G and H (Fig. 14 (e) and (f)). This
correspondence supports the idea that nutrient rich conditions could help Salmonella persist longer. In
addition, aerobic conditions are harmful for Campylobacter, as they are microaerophilic bacteria. These
factors could lead to longer persistence of two pathogens’ DNA in this study. Finally, it should be noted
that these results are based on presence of their DNA alone, which does not yield any information on
infectivity or viability of cells.
26
100
107
80
106
60
105
40
104
20
C.jejuni
S.enterica
Asian clams
103
102
0
200
400
600
0
800 1000 1200 1400 1600
Percentage of dead Asian clams [%]
Campylobacter and Salmonella [gene copies/ml]
108
Time [h]
Figure 9. Campylobacter and Salmonella as measured by DNA
in Case C at winter-like conditions as a function of time
Figure 10. (a) Campylobacter and Salmonella as measured by DNA
in Case F (left) and Case G (right) at summer-like conditions as a function of time
Figure 10. (b) Campylobacter and Salmonella as measured by DNA
in Case H at summer-like conditions as a function of time
27
Table 5. Kinetic parameters of Campylobacter and Salmonellad
Season
mimicked
Winter
Case C
Case F
Summer
Target pathogen
k1 (h-1)
Campylobacter jejuni
N.A.c
Salmonella enterica
0.003
Case
Case G
Case H
S.E.a
t1 (h)
R2
T99b (h)
0.001
96
0.94
1631
c
Campylobacter jejuni
N.A.
Salmonella enterica
0.030
0.007
50
0.95
206
Campylobacter jejuni
0.003
0.000
122
0.99
1518
Salmonella enterica
0.002
0.001
60
0.57
2618
Campylobacter jejuni
0.002
0.001
60
0.62
2769
Salmonella enterica
0.003
0.002
60
0.35
1831
a
S.E., standard error
b
T99, Time for two log reduction
c
N.A., not applicable due to the violation of normality assumption for the data set
d
The model is as follows;
=
×
where N is the number of gene copies per ml at time t, t1 is time at
end of lag period, N0 is the initial concentration and k1 is the decay rate constant
28
3.5. Universal and host-associated Bacteroidales
Winter-like conditions: At winter-like conditions, all universal-, human-, dog- and bovine-associated
Bacteroidales DNA did not increase in Case B and C throughout the experiment period (Fig. 11),
although 100% Asian clams were achieved in both cases. The first-order decay model (
)
=
×
2
could reproduce their decay patterns well and provided high R values in Case B (Table 6.),
whereas, except for the dog marker, the data set for Case C was not normally distributed (P < 0.05), and
hence the model was not applied. Decay rate constants k1 for universal and host-associated Bacteroidales
in Case B were comparable (Table 6).
Summer-like conditions: At summer-like conditions, human-, and dog-associated Bacteroidales DNA
decayed and did not increase during the entire period. On the other hand, bovine-associated
Bacteroidales DNA slightly increased from 400 to 800 h in Cases F, G and H. Universal-Bacteroidales
DNA showed different patterns compared to host-associated Bacteroidales DNA, in that
universal-Bacteroidales DNA was more stable and increased in Case G and H after 100% clam
mortality was achieved (Fig. 12). Because universal-Bacteroidales DNA unexpectedly survived longer,
cloning was conducted to confirm that our universal-Bacteroidales assay detected Bacteroidales DNA
fragments. In total, 11 clones amplified with this universal-Bacteroidales primer set were taken from
Cases F, G and H at 500 h, and their sequence results showed that all clones were closely related to
uncultivated Bacteroidales with 97 to 100% similarity, indicating that the assay properly detected
Bacteroidales. Decay rate constants k1 for most of host-associated Bacteroidales DNA were one order
higher at summer-like conditions than constants in Case B at winter-like conditions (Table 6.). To
investigate the effects of algae and Asian clams on human-associated Bacteroidales decay rates,
one-way ANOVA was performed. The results showed that the decay rate constants of Case F (no clams
with algae), G (clams with algae) and H (clams without algae) were not statistically different (P = 0.947),
demonstrating that these effects on human-associated Bacteroidales DNA were not significant under
these conditions.
In summary, except for universal-Bacteroidales DNA at summer temperatures, universal- and
host-associated Bacteroidales did not re-grow under the rubber barrier. Host-associated Bacteroidales
represent uncultivated Bacteroidales mainly inhabiting their host guts (Kildare, et al., 2007), and hence
it is expected that they require relatively high optimum growth temperatures, which could be one reason
why host-associated Bacteroidales could not re-grow under the rubber barriers even though anoxic
conditions were achieved. However, the universal-Bacteroidales assay could detect a wider variety of
Bacteroidales, which might include Bacteroidales with lower optimum growth temperatures.
29
Although host-associated Bacteroidales DNA did not increase, decay rate constants for
human-associated Bacteroidales were compared to values obtained in other studies to investigate
whether the DNA markers might persist longer than under ambient water conditions in the freshwater
environment. For example, decay rate constants k1 for human-associated Bacteroidales DNA were 0.059
h-1 in aerobic freshwater microcosms at 22°C under dark conditions (Bae and Wuertz, 2012). This value
is twice as large as the values obtained in this study at summer temperatures (20°C), which demonstrates
that human-associated Bacteroidales could persist longer under anoxic nutrient-rich conditions created
by the rubber barrier installations.
30
108
107
60
106
40
105
20
104
103
0
200
400
600
0
800 1000 1200 1400 1600
Time [h]
Universal Bacteroidales
Human Bacteroidales
Dog Bacteroidales
Bovine Bacteroidales
Asian clams
1010
109
100
80
108
107
60
106
40
105
20
104
103
0
200
400
600
0
800 1000 1200 1400 1600
Percentage of dead Asian clams [%]
80
Bacteroidales [gene copies/ml]
109
100
Percentage of dead Asian clams [%]
Bacteroidales [gene copies/ml]
Universal Bacteroidales
Human Bacteroidales
Dog Bacteroidales
Bovine Bacteroidales
Asian clams
1010
Time [h]
Figure 11. Universal and host-associated Bacteroidales as measured by DNA
in Case B (left) and Case C (right) at winter-like conditions as a function of time
Figure 12. (a) Universal and host-associated Bacteroidales as measured by DNA
in Case F (left) and Case G (right) at summer-like conditions as a function of time
Figure 12. (b) Universal and host-associated Bacteroidales as measured by DNA
in Case H at summer-like conditions as a function of time
31
Table 6. Kinetic parameters of universal and host-associated Bacteroidalese
Season
mimicked
Target Bacteoridales
k1 (h-1)
S.E.a
t1 (h)
R2
Universal Bacteroidales
0.004
0.001
98
0.97
1343
Human Bacteroidales
0.005
0.000
8
0.99
1009
Dog Bacteroidales
0.005
0.001
8
0.94
894
Bovine Bacteroidales
0.004
0.002
8
0.85
1287
Universal Bacteroidales
N.A.(a)c
Human Bacteroidales
N.A.(a)c
Dog Bacteroidales
0.004
0.001
34
0.97
1157
Bovine Bacteroidales
N.A.(a)c
Case
Case B
T99b (h)
Winter
Case C
Universal Bacteroidales
N.A.(b)
Human Bacteroidales
0.029
Case F
Summer
200
d
0.008
50
0.94
209
0.011
50
0.85
224
c
Dog Bacteroidales
N.A.(a)
Bovine Bacteroidales
0.027
Universal Bacteroidales
N.A.(b)d
Human Bacteroidales
0.025
0.008
60
0.92
241
Dog Bacteroidales
0.004
0.001
60
0.81
1211
Bovine Bacteroidales
0.019
0.009
60
0.67
305
Universal Bacteroidales
N.A.(b)d
Human Bacteroidales
0.028
0.006
60
0.96
226
Dog Bacteroidales
0.018
0.009
60
0.68
320
Bovine Bacteroidales
0.002
0.001
60
0.42
2938
Case G
Case H
a
S.E., standard error
b
T99, Time for two log reduction
c
N.A.(a), not applicable due to the violation of normality assumption for the data set
d
N.A.(b), not applicable due to their re-growth
e
The model is as follows;
=
×
where N is the number of gene copies per ml at time t, t1 is time at
end of lag period, N0 is the initial concentration and k1 is the decay rate constant
32
3.6. Nutrient release
Winter-like conditions: Except for the Control in which only a few Asian clams were dead, ammonium
and DOC concentrations sharply increased as Asian clams started dying (Fig. 13), whereas the increase
of phosphate concentration was less significant compared to ammonium and DOC. Nutrient release rates
were calculated with a linear regression model, using the data which showed linear increases of nutrients
when Asian clams were dying. DOC release rates were largest, followed by ammonium and phosphate
in all Cases (Table 7 (a).).
Summer-like conditions: Similarly, ammonium and DOC concentrations increased more significantly
than phosphate as Asian clams died at summer temperatures (Fig. 14). In Case F in which no Asian
clams were provided, increases in ammonium and DOC were not as drastic as in Cases G and H,
suggesting that dead Asian clams were primary nutrient sources in addition to decomposing organic
substances derived from untreated wastewater. The effect of algae was not clear because the differences
between Cases G (with algae) and H (without algae) were not significant. Interestingly, DOC levels in
Cases D, G and H decreased at 800 h. This implies that DOC was rapidly released from dead Asian
clams, while simultaneously DOC was being consumed by anaerobic bacteria. Nutrient release rates at
summer temperatures were one order of magnitude higher than at winter temperatures (Table 7 (b).).
It has been reported that ammonium and phosphate release rates from sediments in Lake Tahoe under
anoxic conditions are 0.49 and 0.22 mg/m2/day (Coats, et al., 2010). Release rates obtained in this study
were 10 to 1000 times higher at both winter and summer temperatures, suggesting that decomposition of
dead Asian clams could produce significant amounts of nutrients. Among three nutrients measured in
this study, release rates of phosphate were the smallest, and those of DOC were the largest in all Cases.
However, DOC concentrations eventually decreased at summer temperatures likely due to biological
uptake. In conclusion, the rubber barrier treatment could affect water quality especially by increasing
DOC and ammonium levels. Further investigation is required to estimate how much nutrient
concentrations can be increased by decomposition of dead Asian clams after rubber barriers are removed,
taking overlying water dilution factors into account.
33
80
Asian clams
0.3
60
0.2
40
0.1
20
0.0
0
500
1000
1500
2000
0
2500
20
100
Dissolved Organic Carbon
Asian clams
80
15
60
10
40
5
20
0
0
500
Time [h]
1000
1500
2000
0
2500
Percentage of dead Asian clams [%]
PO4-P
0.4
Dissolved Organic Carbon [mg/L]
100
NH4-N
Percentage of dead Asian clams [%]
NH4-N and PO4-P [mg/L]
0.5
Time [h]
Figure 13. (a) Nutrient concentration in Control at winter-like conditions as a function of time
PO4-P
8
80
Asian clams
6
60
4
40
2
20
0
0
500
1000
1500
2000
0
2500
20
100
Dissolved Organic Carbon
Asian clams
80
15
60
10
40
5
20
0
0
500
Time [h]
1000
1500
2000
0
2500
Percentage of dead Asian clams [%]
100
NH4-N
Dissolved Organic Carbon [mg/L]
NH4-N and PO4-P [mg/L]
10
Percentage of dead Asian clams [%]
(left: ammonium and phosphate, right: Dissolved Organic Carbon)
Time [h]
Figure 13. (b) Nutrient concentration in Case A at winter-like conditions as a function of time
PO4-P
80
Asian clams
30
60
20
40
10
20
0
0
200
400
600
0
800 1000 1200 1400 1600
Time [h]
120
100
Dissolved Organic Carbon
Asian clams
100
80
80
60
60
40
40
20
20
0
200
400
600
0
800 1000 1200 1400 1600
Percentage of dead Asian clams [%]
100
NH4-N
Dissolved Organic Carbon [mg/L]
NH4-N and PO4-P [mg/L]
40
Percentage of dead Asian clams [%]
(left: ammonium and phosphate, right: Dissolved Organic Carbon)
Time [h]
Figure 13. (c) Nutrient concentration in Case B at winter-like conditions as a function of time
(left: ammonium and phosphate, right: Dissolved Organic Carbon)
34
80
60
20
40
10
20
0
0
200
400
600
0
800 1000 1200 1400 1600
Time [h]
120
100
Dissolved Organic Carbon
Asian clams
100
80
80
60
60
40
40
20
20
0
0
200
400
600
0
800 1000 1200 1400 1600
Percentage of dead Asian clams [%]
PO4-P
Asian clams
30
Dissolved Organic Carbon [mg/L]
100
NH4-N
Percentage of dead Asian clams [%]
NH4-N and PO4-P [mg/L]
40
Time [h]
Figure 13. (d) Nutrient concentration in Case C at winter-like conditions as a function of time
(left: ammonium and phosphate, right: Dissolved Organic Carbon)
35
Figure 14. (a) Nutrient concentration in Control at summer-like conditions
as a function of time (left: ammonium and phosphate, right: Dissolved Organic Carbon)
Figure 14. (b) Nutrient concentration in Case D at summer-like conditions
as a function of time (left: ammonium and phosphate, right: Dissolved Organic Carbon)
Figure 14. (c) Nutrient concentration in Case E at summer-like conditions
as a function of time (left: ammonium and phosphate, right: Dissolved Organic Carbon)
36
Figure 14. (d) Nutrient concentration in Case F at summer-like conditions
as a function of time (left: ammonium and phosphate, right: Dissolved Organic Carbon)
Figure 14. (e) Nutrient concentration in Case G at summer-like conditions
as a function of time (left: ammonium and phosphate, right: Dissolved Organic Carbon)
Figure 14. (f) Nutrient concentration in Case H at summer-like conditions
as a function of time (left: ammonium and phosphate, right: Dissolved Organic Carbon)
37
Table 7. (a) Nutrient release rates at winter temperatures (6°C)
Season
mimicked
Case
Control
Case A
Nutrient
Release rate
(mg/m2/day)
S.E.a
R2
NH4-N
N.D.b
PO4-P
N.D.b
DOC
N.D.b
NH4-N
76.2
22.8
0.92
PO4-P
3.6
1.8
0.82
DOC
149.4
60.0
0.86
NH4-N
252.6
25.8
0.98
PO4-P
7.8
1.2
0.98
DOC
551.4
41.4
0.98
NH4-N
214.2
19.2
0.98
PO4-P
12
2.4
0.91
DOC
483
35.4
0.98
Winter
Case B
Case C
a
S.E., standard error
b
N.D., not determined because most of data points were below detection limits
38
Table 7. (b) Nutrient release rates at summer temperatures (20 °C)
Season
mimicked
Case
Control
Case D
Case E
Nutrient
Release rate
(mg/m2/day)
S.E.a
R2
NH4-N
60.0
3.0
1.00
PO4-P
N.D.b
-
-
DOC
136.8
28.8
0.92
NH4-N
583.8
127.2
0.95
PO4-P
19.2
8.4
0.83
DOC
2485.2
331.8
0.98
NH4-N
538.2
60.0
0.99
PO4-P
4.2
3.0
0.66
DOC
822
168.0
0.96
NH4-N
70.8
7.2
0.98
PO4-P
25.8
16.8
0.70
DOC
702.0
295.2
0.85
NH4-N
664.8
51.0
0.99
PO4-P
24.6
15.6
0.56
DOC
2561.4
216.6
0.99
NH4-N
669.6
215.4
0.83
PO4-P
46.2
16.2
0.80
DOC
2571.0
752.4
0.85
Summer
Case F
Case G
Case H
a
S.E., standard error
b
N.D., not determined because most data points were below detection limits
39
4. Conclusions
The study aimed to evaluate impacts of rubber barrier installations on water quality in Lake Tahoe. A
microcosm study was performed, mimicking environmental conditions under the rubber barriers in the
laboratory. We found the following results;
FIB did not increase under the rubber barriers at winter temperatures in any of the Cases studies,
whereas sporadic increases in FIB, especially total coliforms, were observed in some Cases at
summer temperatures.
The model pathogens Campylobacter jejuni and Salmonella enterica did not significantly increase
in numbers under the barriers at either winter or summer temperatures as measured by DNA. The
pathogen decay rate constants at summer temperatures, however, were lower than those reported
under ambient water conditions elsewhere, indicating that these pathogens persisted longer under
rubber barriers.
Host-associated Bacteroidales DNA did not increase at either winter or summer temperatures,
whereas universal-Bacteroidales DNA showed a slight increase at summer temperatures.
Dissolved Organic Carbon (DOC) release rates were the highest followed by ammonium and
phosphate at both winter and summer temperatures. Nutrient release rates at summer temperatures
were one order of magnitude higher than at winter temperatures. Release rates of ammonium and
phosphate estimated at summer temperatures were 10 to 1000 times higher than release rates from
sediment reported in Lake Tahoe, suggesting that dead Asian clams were possible sources.
According to the major results above, a large-scale implementation of the rubber barrier treatment
during summer might lead to an increase in FIB, especially total coliforms, under the barriers, which
might confound routine monitoring of recent fecal contamination. Moreover, the barrier treatment might
contribute to longer survival times of pathogens such as microaerophilic Campylobacter jejuni and
facultatively anaerobic Salmonella enterica. This finding needs to be further investigated because results
are based on DNA measurements and provide no information on infectivity or viability. As for nutrient
release rates, phosphate release might not be considered as the primary concern, but ammonium and
DOC could be of concern in implementing this treatment as they can be massively released from dead
Asian clams. In conclusion, considering the fact that no FIB increase was observed and lower nutrient
release rates were measured at winter-like conditions, installation of rubber barriers during winter could
minimize the impacts on water quality, but this could also lead to a longer lead time before achieving
100% Asian clam mortality.
40
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