The Effects of Atrazine on Tail Regeneration in ... - Hartwick College

<strong>The</strong> <strong>Effects</strong> <strong>of</strong> <strong>Atrazine</strong> on Tail Regeneration in Salamanders
Carlena K. Johnson
Department <strong>of</strong> Biology
Hartwick College
Oneonta, NY
This thesis is submitted in partial satisfaction <strong>of</strong> the requirements for the degree <strong>of</strong> Bachelor
<strong>of</strong> Arts or Bachelor <strong>of</strong> Sciences from the Department <strong>of</strong> Biology, Hartwick College.
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<strong>The</strong>sis Advisor Date
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Chair, Biology Department Date
<strong>The</strong> <strong>Effects</strong> <strong>of</strong> <strong>Atrazine</strong> on Tail Regeneration in Salamanders

Carlena Johnson
Dr. Stanley K. Sessions
ABSTRACT
<strong>Atrazine</strong>, 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 <strong>of</strong> atrazine on dividing
cells in regenerating tails <strong>of</strong> the Red-backed Salamander (Plethodon cinereus). It was found
that there was a significant difference between the percentages <strong>of</strong> dividing cells in the
experimental salamanders, but not between any <strong>of</strong> 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 <strong>of</strong> the
regenerating tails, but were not quantified in this study. Further research should be
conducted into the effects <strong>of</strong> atrazine on both cellular growth and cell death during
amphibian growth and development.
INTRODUCTION
Over the past decade the issue <strong>of</strong> declining amphibian populations has been in the
forefront <strong>of</strong> 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 <strong>of</strong>
amphibians are similar to that <strong>of</strong> humans and other vertebrates.
Many hypotheses have been suggested as to why amphibian populations are
declining. Some <strong>of</strong> 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 <strong>of</strong> some or all <strong>of</strong> these, or something else that has not been thought <strong>of</strong> yet.
<strong>The</strong> herbicide atrazine is the most common contaminant <strong>of</strong> ground, surface, and
drinking water (Hayes, 2002). About 27 million kilograms <strong>of</strong> atrazine are used each year in
the US, mostly in corn growing regions (Withgott, 2002). <strong>The</strong> EPA has set a maximum limit
<strong>of</strong> 3 ppb atrazine in drinking water (U.S. Environmental Protection Agency (U.S. EPA),
2006). It is therefore apparent that contamination <strong>of</strong> ground water exists, but the
consequences <strong>of</strong> this contamination have been disputed (Coady et al., 2004; Filipov et al.,
2005; Hayes, 2004). One study shows that atrazine affects the immune systems <strong>of</strong> 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 <strong>of</strong> the herbicide
remain well after exposure to it has ended. A study that looked at the effects <strong>of</strong> 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 <strong>of</strong> atrazine is
probably not a significant factor in recent amphibian declines” (Allran and Karasov, 2001).
Another study looked at the effects <strong>of</strong> atrazine on growth and gonad development (Coady et
al. 2004). Coady et al. (2004) conclude that there is no difference in weight or length <strong>of</strong> the
exposed vs. control individuals and also says that there is no significant difference in the
gonadal development <strong>of</strong> 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 <strong>of</strong> atrazine, such as those
found in agricultural run<strong>of</strong>f, can cause adverse effects on male African clawed frogs
(Xenopus laevis), such as hermaphroditism and the demasculization <strong>of</strong> the larynges <strong>of</strong>
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 <strong>of</strong> atrazine after exposure to the
herbicide was stopped on the salamander Ambystoma barbouri. <strong>The</strong>y 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 <strong>of</strong> an
endocrine disruptor. A study by Storrs & Keisecker (2004) also showed a non-linear rate <strong>of</strong>
survivorship during a study into the effects <strong>of</strong> long-term exposure to low levels <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> atrazine on tail
regeneration in salamanders to see if atrazine would affect cell proliferation in a rapidly
growing tissue.
MATERIALS AND METHODS
A total <strong>of</strong> 18 Red-backed salamanders (Plethodon cinereus) were caught from the
area surrounding Strawberry field (Hartwick College, Oneonta NY). <strong>The</strong> salamanders were
brought back to the lab and acclimated for one week. Before being introduced to the
treatment, about half <strong>of</strong> each salamander’s tail was removed with a clean blade. <strong>The</strong>
salamanders were then randomly assigned one <strong>of</strong> four treatment groups (0 ppb control, 1ppb
atrazine, 10 ppb atrazine, or 100 ppb atrazine). <strong>The</strong> atrazine treatment solutions were made
from a series <strong>of</strong> dilutions from a “stock” atrazine solution. <strong>The</strong> stock solution included
ethanol to dissolve the atrazine itself and aged tap water. <strong>The</strong> control solution was made
with aged tap water and the same amount <strong>of</strong> ethanol as the 100 ppb treatment. Each
salamander was kept in a Petri dish with filter paper and 10mls <strong>of</strong> the assigned treatment
solution. Three salamanders were included in the 0 ppb control group and five salamanders
were in each <strong>of</strong> the three experimental atrazine groups. <strong>The</strong> 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 <strong>of</strong>
the exposure period, each salamander was injected with 0.3% colchecine twenty-five hours
prior to harvesting the tail regenerate. <strong>The</strong> tails were then placed in 3:1 ethanol: acetic acid
fixative. <strong>The</strong>y were then embedded in wax, following standard histological procedure, and
sectioned at 10µm. Slides were then stained with hematoxylin-eosin. <strong>The</strong> number <strong>of</strong> mitotic
cells/total number <strong>of</strong> cells for at least 5 sections <strong>of</strong> each tail were counted and totaled to
determine a proportion <strong>of</strong> mitotic cells for each salamander tail.

RESULTS
Over the four week period during which tail regeneration occurred, the average length
<strong>of</strong> 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. <strong>The</strong>re is no statistical significance between any <strong>of</strong> 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 <strong>of</strong> appetite or death). <strong>The</strong>re
was no correlation between the amount <strong>of</strong> growth <strong>of</strong> the regeneration bud and the
concentration <strong>of</strong> atrazine. In addition, there is no correlation between the amount <strong>of</strong> growth
and the proportion <strong>of</strong> dividing cells. For each tail, 5-9 sections were used to record the
proportion <strong>of</strong> 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. <strong>The</strong> number
<strong>of</strong> mitotic cells per section were counted (Fig. 2) and the total for each section were added
together to give a total number <strong>of</strong> mitotic cells for a given tail. In the same manner, the total
number <strong>of</strong> cells per section were counted and added together for a total number <strong>of</strong> cells for a
given tail. <strong>The</strong> proportion <strong>of</strong> mitotic cells was calculated from this data (Table 1).
Figure 1. This shows two cells undergoing mitosis (arrows). Hematoxylin
and eosin staining <strong>of</strong> 10 micrometer paraffin section.

6.000
Mean Proportion <strong>of</strong> mitotic cells
5.000
4.000
3.000
2.000
1.000
0.000
Control
1 ppb
10 ppb
<strong>Atrazine</strong> Concentration
Error bars: 95% CI
100 ppb
Figure 2. This shows the average proportion <strong>of</strong> 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 <strong>of</strong> mitotic cells, total number <strong>of</strong> cells in the tail and the
proportion <strong>of</strong> mitotic cells found for each tail. <strong>The</strong> total mitotic cells and the total cells were
found by adding the numbers for each section <strong>of</strong> a given tail.
Treatment Total Mitotic cells Total cells Proportion <strong>of</strong> mitotic cells
Control 171 4878 3.506
224 7961 2.814
252 6319 3.988
1 ppb 257 4974 5.167
135 3411 3.958
310 6894 4.497
295 6670 4.423
392 12164 3.223
10 ppb 153 6268 2.441
200 5941 3.366
155 5909 2.623
186 12317 1.51
100 ppb 213 8055 2.644
163 6095 2.674
246 8152 3.018
349 8313 4.194
213 6779 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 <strong>of</strong> 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 <strong>of</strong> mitotic cells is
largest at the 1 ppb concentration <strong>of</strong> atrazine, while it is smallest at the 10 ppb concentration
<strong>of</strong> atrazine (Fig. 2, Table 2). A one-way ANOVA was preformed on the data and the results

were found to be significant (F 3,13 = 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. <strong>The</strong>re was no difference between any
<strong>of</strong> the experimental treatment groups and the control.
Table 2. This table shows the average proportion <strong>of</strong> mitotic cells
for each treatment group. Note that there is a significant difference
between the experimental groups 1 ppb & 10 ppb.
Treatment
Mean Proportion <strong>of</strong> 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. <strong>The</strong>re may be a small dose dependent effect (as seen by the
significance between the 1 ppb and 10 ppb experimental groups), which is characteristic <strong>of</strong>
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 <strong>of</strong> 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 <strong>of</strong> BrdU
to label mitotic cells, may prove to be a better measure <strong>of</strong> mitotic activity, though
preliminary tests show that background staining may be an issue that would need to be
resolved to get an accurate count <strong>of</strong> cells. I also propose that similar studies should be
conducted on frogs to rule out the possibility <strong>of</strong> 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 <strong>of</strong> the tail sections contained dead or dying cells.
<strong>The</strong> proportion <strong>of</strong> dying cells was not quantified at this time, but the possible interrelated
effects <strong>of</strong> 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 <strong>of</strong>
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 <strong>of</strong> issues that may be contributing to amphibian
declines. For example, a reduction in the ability <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> 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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