Transcript Scientific Poster

Effect of Triclosan on a Freshwater Ecosystem
Lyle Rapp
Biology Department, Skyline College, San Bruno CA
Abstract
Methods
Results
Triclosan (trichloro-bisphenol) is an antimicrobial additive in over 700
household products. Studies have shown that triclosan discharged with
treated sewage persists in waterways resulting in concentrations up to 500
ppt in river. Although less than 500 ppt triclosan has no direct effect on
aquatic plants and animals, the effect on microalgae and protozoa has not
been determined. The purpose of this study was to determine the effect of
sublethal concentrations of triclosan on the productivity of a freshwater
ecosystem. Paramecium and Chlorella were grown in triclosan (5 ppm to
0.0013 ppt) in pond water. The minimum lethal concentration for
Paramecium is 1.6 ppb; Chlorella is able to grow in 5 ppm triclosan. The
effect of triclosan on an ecosystem is being determined. Triclosan (0.5 to
5000 ppb ) was added to 99 ml pond water and sediments in 150-mL
beakers. Beakers were incubated at 25°C in natural light. Population
changes for smaller organisms are being determined using an inverted
microscope. Metabolic activity is being measured with Vernier CO2 and
O2 sensors. The standard plate count will be used to count aerobic
heterotrophic bacteria in the sediments. The results of this experiment will
help determine differential sensitivities to and effect of triclosan on an
ecosystem.
Triclosan
1. Serial dilutions (5  10-4 – 5  10-10) of stock 1% Triclosan were made.
2. Spring water and sediment was collected from a natural spring located
on Skyline College campus. It contained many small ciliates and
microalgae. Paramecium (Carolina Biologicals, 13-1554) was added
for easier counting.
Ecosystems:
1. Three 150-mL beakers were prepared with 20 mL sediment and 75 mL
spring water.
2. Three 150-mL beakers were prepared with 95 mL spring water. One
150-mL beaker was prepared with 20 mL sediment and 76 mL spring
water.
3. 4 mL Paramecium was added to each beaker
4. 1mL of a triclosan dilution was added to each spring water ecosystem
and water + sediment ecosystem. Controls had no triclosan.
5. Beakers were incubated in a water bath, exposed to indirect natural
light, at room temperature (~27°C) for four weeks.
Bacteria Count
Heterotrophic plate counts were performed weekly for four weeks to count
bacteria in the sediment. Nutrient Agar plates were incubated at 35°C.
• The bacterial population declined in 500 ppm triclosan (Figure 2) .
• Paramecium and smaller ciliates were killed within 1 week in 1 ppm
triclosan (Figure 3).
• The bacterial population in the sediment decreased in the highest
concentration (Figure 4), 500 ppm, but increased in 5 ppt and 50 ppb.
• Microalgae died in the presence of triclosan (Figure 5).
• Rotifers were killed within the first week in 50 ppb beakers and decreased
by 66.66% in 5 ppt.
• The O2 and CO2 content of the water remained constant.
Hypothesis
20
0
-20
-40
-60
-80
Ciliates
Microalgae
-100
5 ppt
50 ppb
500 ppm
[Triclosan]
Control
Figure 5. Triclosan decreased the survival of ciliates and
microalgae.
8
7
Number of
Metabolism (O2 and CO2 Outputs)
O2 and CO2 Outputs were measured for three minutes once a week for four
weeks. A 20 mL pond water sample was measured using a Vernier O2 sensor
and CO2 sensor.
Percent change over 4 weeks
40
Discussion & Conclusion
6
5
5 ppm
1 ppm
8 ppt
1.6 ppt
Control
4
3
2
1
The antibacterial agent triclosan will disrupt an aquatic food chain by
killing bacteria.
The highest concentration of triclosan, 500 ppm, has a significant effect on
population of rotifers, ciliates, and bacteria. Microalgae are affected in all
concentrations. O2 and CO2 concentrations did not change over four weeks.
Perhaps due to lack of metabolic activity.
Data indicate a potential ecological danger if environmental triclosan
concentrations increase. However, it is unclear whether death of producers
and consumers is due to the death of bacteria.
0
-1
-2
0
10
20
30
Literature Cited
Time (hr)
Background
•
•
•
•
•
•
More than 700 over-the-counter products are being made with the
antimicrobial agent triclosan (Figure 1). These products include soaps,
deodorants, toothpastes, cosmetics, clothing, and children’s toys (4).
Triclosan blocks the active site of the enoyl-acyl carrier protein
reductase enzyme, an essential enzyme for fatty acid synthesis in
bacteria (3).
Most (96%) of triclosan is disposed via domestic sewage (1), and
because the half-life of triclosan is between 2 and 2000 days, the rate of
accumulation is faster than the rate of decomposition (7).
Currently triclosan levels are somewhere below 500 ppt in sewage
effluent (6).
Triclosan is a possible threat to public health because accumulation in
the aquatic food chain could select resistant bacteria (2,4).
As a broad-spectrum bacteriocide, triclosan could kill large and diverse
populations of bacteria thereby permanently disrupting food chains.
Figure 1. Triclosan (trichloro-bisphenol), an antimicrobial additive
found in over 700 household products.
Ciba Specialty Chemical. 1998. Irgasan DP 300, Irgacare MP. Toxicological and Ecological
Data. Official Registrations. Technical Brochure 2521. Basel, Switzerland.
2.
Coogan, M. A, R. E. Edziyie, T. W. La Point, and B. J. Venables. 2007. “Algal bioaccumulation
of triclocarbon, triclosan, and methyl-triclosan in a North Texas wastewater treatment plant
receiving stream.” Chemosphere 67(10): 1911-1918.
3.
Levy, C. W., A. Roujeinikovai, S. Sedelnikova, P. J. Baker, et al. 1999. “Molecular Basis of
Triclosan Activity.” Nature 398: 383-384.
4.
Levy, S. B. 2001. “Antibacterial household products: cause for concern.” Emerg Infect Dis 7 (3
Suppl):512-515.
5.
Naidenko, O., and R. Sutton. “EPA’s Decision to Support Re-registration of Triclosan.” Letter
to the U.S. Environmental Protection Agency. 19 Dec. 2008. Environmental Working Group.
<www.ewg.org/files/triclosan_EWGcomments_EPA_121908.pdf> (Downloaded 19 May
2009).
6.
Suszkiw, J. “New Test To Detect Triclosan in Water.” Agricultural Research Jan. 2009: 13.
7.
Tixier, C., H. P. Singer, S. Canonica, and S. R. Muller. 2002. “Phototransformation of triclosan
in surface waters: A relevant elimination process for this widely used biocides—Laboratory
studies, field measurements, and modeling.” Enviro. Sci Technol 36: 3482-3489.
U. S. Dept. of Health and Human Services. 1998. “Toxicological profile for chlorinated
dibenzo-p dioxins.” Public Health Service, Agency for Toxic Substances and Disease Registry;
U.S. EPA. Estimating exposure to dioxin-like compounds, Vol. II: Properties, sources,
occurrence and background exposures. Office of Research and Development. Review draft.
Washington DC.
1.00E+18
1.00E+17
CFU/g sediment
•
Figure 3. Survival rate of Paramecium over one week at
different triclosan concentrations. Error bars: S.E.
1.
Spring water and sediment, gathered from a natural spring located on
Skyline College campus, was used to examine the effect of triclosan on
a freshwater ecosystem.
Control
500 ppm
50 ppb
5 ppt
1.00E+16
1.00E+15
8.
1.00E+14
1.00E+13
1
2
3
Time (wk)
Figure 2. Aerobic heterotrophic bacteria decreased
after two weeks in 500 ppm triclosan.
Figure 4. Survival rate of bacteria is measured by a standard
plate count of a 1 g sample of pond sediment.
Acknowledgements
•Dr. Christine Case, Skyline College Professor of Biology
•Patricia Carter, Skyline College Biology Lab Technician
•Stephen Fredricks, Skyline College MESA Director