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The Effects of Atrazine on Tail Regeneration in Salamanders
Carlena K. Johnson
Department of Biology
Hartwick College
Oneonta, NY
This thesis is submitted in partial satisfaction of the requirements for the degree of Bachelor
of Arts or Bachelor of Sciences from the Department of Biology, Hartwick College.
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Thesis Advisor
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Date
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Chair, Biology Department
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Date
The Effects of Atrazine on Tail Regeneration in Salamanders
Carlena Johnson
Dr. Stanley K. Sessions
ABSTRACT
Atrazine, a widely used herbicide, is thought to be contributing to amphibian
declines. In addition, it may be working as an endocrine disruptor in amphibians. To better
understand if atrazine affects cell proliferation I studied the effects of atrazine on dividing
cells in regenerating tails of the Red-backed Salamander (Plethodon cinereus). It was found
that there was a significant difference between the percentages of dividing cells in the
experimental salamanders, but not between any of the experimental groups and the control.
Specifically, the tissues appear to show a dose-dependent effect in which an increase in
cellular growth was seen only at the lowest concentration. This result is consistent with
atrazine acting as an endocrine disruptor. Dead cells were also observed in most of the
regenerating tails, but were not quantified in this study. Further research should be
conducted into the effects of atrazine on both cellular growth and cell death during
amphibian growth and development.
INTRODUCTION
Over the past decade the issue of declining amphibian populations has been in the
forefront of both scientific and popular literature. Amphibians are important environmental
indicators because they have thin moist skin, which is used as a respiratory surface, and
because they lay their eggs in the water. In this way, they may be exposed to chemicals and
other pollutants very easily. Also, the basic development and the immune system of
amphibians are similar to that of humans and other vertebrates.
Many hypotheses have been suggested as to why amphibian populations are
declining. Some of these include: chemical pollution, UV radiation, diseases, parasite, global
climate change, over-collecting and general habitat destruction. It is very likely however that
it is a combination of some or all of these, or something else that has not been thought of yet.
The herbicide atrazine is the most common contaminant of ground, surface, and
drinking water (Hayes, 2002). About 27 million kilograms of atrazine are used each year in
the US, mostly in corn growing regions (Withgott, 2002). The EPA has set a maximum limit
of 3 ppb atrazine in drinking water (U.S. Environmental Protection Agency (U.S. EPA),
2006). It is therefore apparent that contamination of ground water exists, but the
consequences of this contamination have been disputed (Coady et al., 2004; Filipov et al.,
2005; Hayes, 2004). One study shows that atrazine affects the immune systems of juvenile
mice by adversely affecting lymphocyte distribution as well as decreasing cellularity within
the spleen and thymus (Filipov et al., 2005). It also shows that some effects of the herbicide
remain well after exposure to it has ended. A study that looked at the effects of atrazine on
embryos and larvae concludes that atrazine does not have any adverse effects on hatchability,
post hatch mortality, or swimming speed and concludes that “direct toxicity of atrazine is
probably not a significant factor in recent amphibian declines” (Allran and Karasov, 2001).
Another study looked at the effects of atrazine on growth and gonad development (Coady et
al. 2004). Coady et al. (2004) conclude that there is no difference in weight or length of the
exposed vs. control individuals and also says that there is no significant difference in the
gonadal development of the frogs, even though multiple testes, size irregularity, and
“rudimentary hermaphroditism”, including intersex gonads and testicular oocytes, were
observed. Other recent studies have shown that even low doses of atrazine, such as those
found in agricultural runoff, can cause adverse effects on male African clawed frogs
(Xenopus laevis), such as hermaphroditism and the demasculization of the larynges of
exposed males (Hayes et al. 2003). Hayes et al. (2002), as well as others, have concluded
that atrazine works as an endocrine disruptor resulting in the effects cited by these articles.
Rohr et al. (2006) investigated possible persistent effects of atrazine after exposure to the
herbicide was stopped on the salamander Ambystoma barbouri. They found that there was a
significant difference in survival between the three experimental groups (4, 40, and 400 ppb)
and the control and also cite a non-linear pattern in their results, which is characteristic of an
endocrine disruptor. A study by Storrs & Keisecker (2004) also showed a non-linear rate of
survivorship during a study into the effects of long-term exposure to low levels of atrazine.
Brodkin et al. (2007) found that atrazine may also work as an immune disruptor in
amphibians. It is obvious that there is dispute over the consequences of atrazine on
amphibians, but it is clear that this issue deserves continued study to help clarify any possible
effects on amphibian populations.
Previous senior research theses (Wilkes, 2004 & Owens, 2005) have shown that
atrazine has a negative effect on growth and development, as well as the immune system. To
better understand how atrazine may cause these negative effects I choose to investigate the
mechanism by which atrazine works. I chose to study the effects of atrazine on tail
regeneration in salamanders to see if atrazine would affect cell proliferation in a rapidly
growing tissue.
MATERIALS AND METHODS
A total of 18 Red-backed salamanders (Plethodon cinereus) were caught from the
area surrounding Strawberry field (Hartwick College, Oneonta NY). The salamanders were
brought back to the lab and acclimated for one week. Before being introduced to the
treatment, about half of each salamander’s tail was removed with a clean blade. The
salamanders were then randomly assigned one of four treatment groups (0 ppb control, 1ppb
atrazine, 10 ppb atrazine, or 100 ppb atrazine). The atrazine treatment solutions were made
from a series of dilutions from a “stock” atrazine solution. The stock solution included
ethanol to dissolve the atrazine itself and aged tap water. The control solution was made
with aged tap water and the same amount of ethanol as the 100 ppb treatment. Each
salamander was kept in a Petri dish with filter paper and 10mls of the assigned treatment
solution. Three salamanders were included in the 0 ppb control group and five salamanders
were in each of the three experimental atrazine groups. The salamanders were kept at 15º C.
New treatment solutions were made and replaced in each Petri dish every three days for four
weeks. In addition, each salamander was fed a few fruit flies every third day. At the end of
the exposure period, each salamander was injected with 0.3% colchecine twenty-five hours
prior to harvesting the tail regenerate. The tails were then placed in 3:1 ethanol: acetic acid
fixative. They were then embedded in wax, following standard histological procedure, and
sectioned at 10µm. Slides were then stained with hematoxylin-eosin. The number of mitotic
cells/total number of cells for at least 5 sections of each tail were counted and totaled to
determine a proportion of mitotic cells for each salamander tail.
RESULTS
Over the four week period during which tail regeneration occurred, the average length
of the regeneration bud for the control, 1 ppb, 10 ppb, and 100 ppb groups, respectively were:
0.4cm, 0.37cm, 0.39cm, and 0.36cm. There is no statistical significance between any of the
groups regarding regeneration bud length. In addition, no adverse effects on the salamanders
were noted during the one month exposure period (such as loss of appetite or death). There
was no correlation between the amount of growth of the regeneration bud and the
concentration of atrazine. In addition, there is no correlation between the amount of growth
and the proportion of dividing cells. For each tail, 5-9 sections were used to record the
proportion of mitotic cells (Fig. 1). Only one tail (in the 10 ppb treatment group) used five
sections, while most tails contained six sections from which data was recorded. The number
of mitotic cells per section were counted (Fig. 2) and the total for each section were added
together to give a total number of mitotic cells for a given tail. In the same manner, the total
number of cells per section were counted and added together for a total number of cells for a
given tail. The proportion of mitotic cells was calculated from this data (Table 1).
Figure 1. This shows two cells undergoing mitosis (arrows). Hematoxylin
and eosin staining of 10 micrometer paraffin section.
Mean Proportion of mitotic cells
6.000
5.000
4.000
3.000
2.000
1.000
0.000
Control
1 ppb
10 ppb
100 ppb
Atrazine Concentration
Error bars: 95% CI
Figure 2. This shows the average proportion of mitotic cells for each
treatment group. Note that there is a significant difference between the
experimental groups 1 ppb & 10 ppb.
Table 1. This shows each individual used in the four treatment groups. Each individual is
separated into the total number of mitotic cells, total number of cells in the tail and the
proportion of mitotic cells found for each tail. The total mitotic cells and the total cells were
found by adding the numbers for each section of a given tail.
Treatment
Control
1 ppb
10 ppb
100 ppb
Total Mitotic cells
171
224
252
257
135
310
295
392
153
200
155
186
213
163
246
349
213
Total cells
4878
7961
6319
4974
3411
6894
6670
12164
6268
5941
5909
12317
8055
6095
8152
8313
6779
Proportion of mitotic cells
3.506
2.814
3.988
5.167
3.958
4.497
4.423
3.223
2.441
3.366
2.623
1.51
2.644
2.674
3.018
4.194
3.142
One tail from the 10 ppb experimental group was not included in data analysis due to
poor histology and a resultant inaccuracy in counting cells. This reduced the number of tails
in the 10 ppb group to four, as compared to the 1 ppb and 100 ppb experimental groups
which contain five tails each. Overall, it is apparent that the proportion of mitotic cells is
largest at the 1 ppb concentration of atrazine, while it is smallest at the 10 ppb concentration
of atrazine (Fig. 2, Table 2). A one-way ANOVA was preformed on the data and the results
were found to be significant (F3,13 = 5.192, P = 0.014). Afterwards, post hoc tests (LSD,
SNK, bonferroni, and tukey) were preformed on the data to find which treatments were
significantly different. It was found that there was a statistically significant difference
between the 1 ppb and 10 ppb treatment groups only. There was no difference between any
of the experimental treatment groups and the control.
Table 2. This table shows the average proportion of mitotic cells
for each treatment group. Note that there is a significant difference
between the experimental groups 1 ppb & 10 ppb.
Treatment
Mean Proportion of Mitotic
Cells
Control
3.44 ± 0.59
1 ppb
4.25 ± 0.72
10 ppb
2.49 ± 0.76
100 ppb
3.14 ± 0.63
DISCUSSION
Overall, atrazine appears to have little or no effect on cell proliferation in
regenerating salamander tails. There may be a small dose dependent effect (as seen by the
significance between the 1 ppb and 10 ppb experimental groups), which is characteristic of
an endocrine disruptor. This is consistent with other research that concludes that atrazine
works as an endocrine disruptor (Hayes et al. 2002, Sessions et al. unpublished). It is also
important to note the high degree of variance within the control. Due to the variance within
the control, a possible pattern may have been masked. Further steps should be taken to better
understand if atrazine has an effect on cell proliferation before it is ruled out as the
mechanism by which the herbicide is functioning. Other protocols, such as the use of BrdU
to label mitotic cells, may prove to be a better measure of mitotic activity, though
preliminary tests show that background staining may be an issue that would need to be
resolved to get an accurate count of cells. I also propose that similar studies should be
conducted on frogs to rule out the possibility of a species-specific reaction to salamanders
that may not be applicable to amphibians as a whole (though Rohr et al., 2006, showed that
atrazine does have a negative effect on salamanders exposed to the chemical before
metamorphosis).
In addition, it was noted that many of the tail sections contained dead or dying cells.
The proportion of dying cells was not quantified at this time, but the possible interrelated
effects of atrazine on cell proliferation and dying cells should be looked at in future research.
Studies should be done to determine the extent to which atrazine may simply be causing too
much cell death and the possible effect that cell death may have on increasing the rate of
mitosis, especially at low levels.
It is important to consider the mechanism by which atrazine may be causing the
adverse effects that many studies have found on amphibians because by better understanding
how this herbicide stunts growth and development and the immune system we may be able to
better understand the complex mixture of issues that may be contributing to amphibian
declines. For example, a reduction in the ability of the immune system to function properly
may make it easier for parasites/fungi/diseases to infect amphibians, thus causing a higher
death rate in a population exposed to atrazine than one that was not exposed to the chemical.
Only by better understanding how individual causes may be negatively affecting amphibian
populations can we begin to understand the entire scope of these effects, not only on
amphibians, but also so that we may apply this understanding to other species that are
declining.
ACKNOWLEDGEMENTS
I would like to thank Megan Irland and Nancy Johnson for giving me advice and
assisting me with my research, Dr. Mary Allen for her assistance with statistics, and Dr. Stan
Sessions for all of his help with my senior research project. I would like to dedicate my
research to my grandmother, Betty I. Laitsch, who’s never ending dedication to animals and
the environment has inspired me to continue my career in scientific research.
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