week 1 week 2 week 3 week 4 week 5 week 6 0%

20%

40%

60%

80%

100%

120%

13%

25%

38%44%

69%

100%

10% 10%

20%

30%

48%

80%

19% 19%

50%

75%

Fig 5. TBTCl Mortality Rate

TBTCl 1ug/LTBT 2ug/LControl

Time

Cu

mu

lati

ve F

ish

Mor

tali

ty R

ate

(%)

week 1 week 2 week 3 week 4 week 5 week 6 0%

20%

40%

60%

80%

100%

120%

0% 0%

31%

50%

69%

6%

19%

38%

63%

94%

100%

10% 10%

20%

30%

48%

80%

Fig 6. Triclosan Mortality Rate

Triclosan 20ug/LTriclosan 40ug/LControl

Time

Cu

mu

lati

ve F

ish

Mor

tali

ty R

ate

(%)

AcknowledgmentsI would first like to thank Dr. McShaffrey for helping me design this experiment and for all the feedback he has given me over the past three semesters. Also a special thanks to Dr. Spilatro and Dr. Lustofin for all their help and feedback with new ideas. In addition I would like to thank the whole senior capstone class and the rest of the Biology department for all their support.

ConclusionsThe data collected from this experiment, does in fact support my hypothesis; that exposure to triclosan and TBTCl would have similar effects on female fancy tail guppies.

However, being that triclosan has not until recently become a real interest to scientists until recently; not many studies have been done using triclosan. Therefore these results lack the needed support from outside sources. However, like stated before I feel that through using articles like the one published by Frent in 1996, I feel that these results are supported. Because through using TBTCl as a positive indicator in this experiment, I was able to compare the results of TBTCl with that of triclosan.

The effects of triclosan and tributyltin chloride on female Poecilia reticulata.

Jordan Beadling Advised by Dr. McShaffrey

Literature cited 1. Albrecht E.A, Carreno N.B, Castro-Vazquez. 1999. A Qualitative

study of Environmental Factors Influencing the Seasonal onset of Reproductive Behavior in the South American Apple-Snail Pomacea Canaliculata. Moll. Study 65: 241-250.

2. Feral C, LeGall S. 1983. The Influence of pollutant factor (tributyltin) on the neuroendocrine mechanism responsible for the occurrence of a penis in the females of Ocenebra erinacea. Molluscan Neuroendocrinology. New Holland, Amsterdam. 173-175.

3. Frent K. 1996. Ectoxicology of Organ-tin Compounds. Toxicology 26: 1-117.

4. Koplin D, Furlong E, Meyer M. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams. Environmental Science technology 36: 1201- 1211.

5. Kumiko I, Hisashi Y. 2003. Changes in dietary bioaccumulation of tributyltin chloride (TBTCl) In red sea bream (Pagrus major) with the concentration in feed. Water Research 37: 1497–1504

6. McClellan-Green P, Romano J, Rittschof D. 20006. Imposex Induction in the Mud Snail, Ilyanassa obsolete by Three Tin Compounds. Toxicology 76:581-588.

7. Oberdorster E, McClellan P. 2002. Mechanisms of imposex induction in the mud snail, Ilyanassa obsolete: TBT as a neurotoxin and aromatase inhibitor. Marine Environment Research 54 (3-5): 715-718.

8. Oehlmann J, Schulte-Oehlmann U. 2003. Endocrine disruption in invertebrates. Pure Appl. Chem 75 (11-12): 2207-2218.

9. Veldhoen N, Skirrow R.C, Osachoff H. 2006. The Bacterial agent Triclosan Modulates Thyroid Hormone- Associated gene Expression and Disrupts Postembryonic Anuran Development. Aquatic Toxicology 80: 217-227.

10. Zhou Q, Jiang G, Liu J. 2003. Acute and chronic effects of tributyltin on the Chinese rare minnow (Gobiocypris rarus). Science in China 46 (3): 1-9.

IntroductionTri-organotins such as tributyltin chloride (TBTCl) have a broad range of applications with an annual world production approximating 50,000 tons/yr. These are most commonly used in marine antifouling paints, PVC stabilizers, as a biocide in agriculture and as preservatives for wood, leather, textiles and paper. Tributyltin chloride is most commonly used as an antifoulant paint additive on ships and other equipment to prevent marine growth in many different organisms [8]. Tributyltin chloride acts as a biocide against a large number of species. Unfortunately, these compounds finally end up in the marine environment as a result of leaching. Their persistence in the marine environment has lethal, immunological, carcinogenic and teratogenic effects on non-target organisms.  Triclosan is a bactericidal agent that makes up a variety of personal products such as toothpaste and soap [4]. According to a survey done by Koplin in 2000, 55% of the 139 streams surveyed across the United States had a triclosan concentration of 0.14ug/L [4]. Triclosan is a major concern, because it has the same structure as thyroid hormone [4]. Thus, one has to wonder if triclosan can potentially disrupt the normal growth and development in humans and other organisms as the thyroid hormone has been found to do [8].

It was hypothesized that exposure to triclosan and TBTCl would have a similar effect on female fancy tail guppies.

Materials and methodsDuring the experiment, the 75 fish were acclimated to laboratory conditions of a 14:10 light/dark cycle for one week. After acclimation, the fish were placed in an aquarium with a cover plate. A 5-10 cm air space above the water was maintained. Each female fish required 1.5 gallons of calcium rich water with a temperature ranging between 19-28ºC. After acclimation, the fish were divided into a control group and 2 experimental groups which received doses of either 1ug/L or 2 ug/L of TBTCl or 20ug/l or 40ug/L of triclosan every other day using a micro pipette. During this experiment the aquariums were cleaned every other day. The pH of this water was kept around 6.5-8 and the fish were fed every day.

Fig 1.

Fig 2.

Figure 1. Chemical structure for triclosan. Empirical formula is C12H7Cl3O2 .

Figure 2. Chemical structure for TBTCl. Empirical formula is [CH3(CH2)3]3SnCl.

Figure 3. Egg binding

Figure 3 shows the effects caused by both triclosan and TBTCl after only 3 weeks of exposure.

Both experimental groups showed high levels of egg binding, while the control group had 0% affected.

Figure 4 is a close up picture taken from a dissecting microscope.

Fig 5. Average survival percentage over time of both TBTCl trials compared to that of control group.

Fig 6. Average survival percentage over time of both Triclosan trials compared to that of control group.

Fig 4. Close-up of egg binding

Results

As can be seen in Figure 5 and 6 above, both TBTCl and Triclosan increased the mortality rate among female guppies. In Figure 5, both concentrations of TBTCl had a mortality rate of about 13% compared to that of the control group of only 10%. However, after comparing weeks 4 and 5, there was a greater increase in mortality rate in the experimental group than in the control group. For instance 44% mortality rate for 1ug/L TBTCl and 50% mortality rate for 2ug/L TBTCl compared to 30% mortality rate of the control; thus a 15% difference in mortality rate between the experimental group and control group. Also in week 6, there was a 100% mortality rate for TBTCl compared to only 80% in the control. However, when comparing the 2nd experimental group to the control in weeks one and two, the average mortality rate for triclosan was about 8% in week 1 and 15% in week 2 compared to 0% in the control group for the first two weeks. Also as seen in figure 5, the greatest difference in mortality rate was after week 4. In week 4, an average of 55% of the experimental group had died in both concentrations, as compared to only 30% in the control group. When comparing the data for these two graphs, both experimental groups had a higher mortality rate than the control. Also there was a 100% mortality rate in all four experimental groups after week 6, compared to the control group in which 20% of the population was still alive.

Another test showed that in both experimental treatments, no young were born and all of the female guppies were egg bound as can be seen in (Figures 3 and 4) by week 3 of the experiment. However, when analyzing the data from the control group, 14 young guppies were born in the control treatments.

Discussion

There are many scientific studies noting the different effects of TBTCl on organisms, such as a study done by Frent in 1996. Frent looked at the Ectoxicology of Organ-tin Compounds, and their effects on different organisms. Thus, through reading this article, I was able to establish a LD50 for guppies, which was around 0.2ug/L . However, when looking at his data, there was no time frame in which this LD50 was noted. As my data suggest , my concentration was much greater than that of his published findings. Thus, the reason for this significant difference was that I had only one chance to find a concentration that would have a positive effect on female guppies.