Fertilizer is a common application used in farmland and even small gardens to help crops or plants grow, by supplying nutrients into the soil. There are many kinds of fertilizers. A common way to differentiate between fertilizers is to look at their NPK values. NPK is a measure of the number of macronutrients in a specific fertilizer. The N stands for nitrogen, the P stands for phosphorus, and the K stands for potassium. For example, if a fertilizer has an NPK value of 2:20:30, there is 2% of nitrogen in the fertilizer along with 20% of phosphorus and 30% of potassium. The other 48% left is other nutrients that do not impact the soil as much, essentially filler content. However, some scientists have also noted inherent disadvantages to using fertilizers. One such study explains how fertilizer misuse can cause massive implications for air and water pollution. Specifically, in this study, the environmental consequences of the excessive use of Nr, a synthetic nitrogen fertilizer, were observed (
Gu et al., 2015
).
Another research group looked at crop production in China, particularly the influence on the environment and how efficient the process was. The results of the study showed that most non-point source pollution (NPS)—a type of water pollution—comes from nitrogen and phosphorus residue, both common elements in fertilizers (
Liu et al., 2021
). Also, nitrogen fertilizers can have long-term effects on the soil, such as soil erosion or changes in its properties (
Daba et al., 2021
). Lastly, one study also points out that increasing the amount of nitrogen in soil could be inversely proportional to the number of microbes in the soil. The study concluded that the increase in nitrogen decreased the variability of the microbes and changed the bacteria present in the soil (
Zhou et al., 2017
). “Microbes are tiny organisms in the soil, water, and air, and play a vital role in the food web” (
National Library of Medicine, 2016
). One should also consider how the decrease in microbes would affect animals in the ecosystem.
Inorganic fertilizers especially, have been seen to damage soil long-term rather than helping. In the study by
Gupta & Hussain (2014)
, fertilizers are prone to leaching, a part of the nitrogen cycle in which nitrate goes with the water flow and potentially ends up somewhere else. Also, it was observed that chemical fertilizers can dry out the soil because of the salt commonly found inside them. These fertilizers allow for more soil erosion and create an imbalance between nitrogen and other necessary nutrients. This nitrogen accumulation can be deadly for everything near this soil (
Hashimi & Hashimi, 2020
).
Various methods have been used to limit the effect of fertilizer overuse on the environment. One group of researchers was straightforward, using techno-logical guidance and subsidies to push the experimental group of 516 households in the Heyang County of Shaanxi Province in China (
Wu & Ge, 2019
). Other scientists proposed combining synthetic fertilizer with organic manure, which they put into a Denitrification Decomposition model (DNDC) (
Lv
et al., 2020
). A DNDC model is a simulation model for N2O, CO2, and N2 emissions from agricultural soils in the U.S. (
Li et al., 1992
). The study carried out by
Lv et al. (2020)
found that using the organic matter as a replacement only slightly affected the crop yield and significantly lowered the environmental impact. Both methods described above are useful in their respective manners; however, people still haven’t taken significant steps to stop the damage the fertilizers are having on the environment.
On the other hand, one should consider the effects of fertilizer overuse on animals everywhere. Ants are important members of the environment that can be damaged because of this. In a study by
Lafleur, Hooper-Bùi, Mumma, & Geaghan (2005)
, a species of red imported fire ant was used to discover if there was a connection between them and plant growth in pine forests. Because of how ants concentrate organic matter into their ant nests, the Louisiana State scientists thought that this would help plant growth. The results showed that ants efficiently disinfected their habitat and also increased the nitrogen content in the soil.
On the other hand, another study by
Green, Pettry, & Switzer (1998)
states that the same red imported fire ants enriched the soil and could positively impact an entire landscape in about a hundred years if needed. An analysis by
Farji-Brener & Werenkraut (2017)
showed the effects of ant nests on soil fertility. They concluded that the plants with ant nests nearby had higher nutrient and calorie content than the plants not near any ant nests. Moreover, plants showed greater biomass and fitness close to ant nest soils than vice versa.
All the previous studies examined the effects of ants on the environment, and most of the studies showed that ants played a significant part in soil fertility and plant growth. However, even with this realized importance, there is little to no research on the direct impact of the increased fertilizer amount on the animals specifically. This experiment measured how different amounts of nitrogen in soil affect the survival of Pogonomyrmex occidentalis. In doing so, this can shed light on the direct impacts of fertilizer use on organisms. This study provides crucial information for maintaining ecological balance amid ongoing environmental damage. Moreover, the study helps to educate others about the potential consequences of human activity. Lastly, this information is also crucial to biodiversity conservation, to keep these ants living and well.
Pogonomyrmex occidentalis was chosen for this experiment due to its known underground foraging behavior. It is also known for its ability and willingness to communicate with other ants (
Cole, 1968
). This species of ant has also been seen to affect the nitrogen and phosphorus levels in the soil (
Green et al., 1998
).
In essence, this study examines whether there is a connection between increasing nitrogen levels in the soil and the survival of Pogonomyrmex occidentalis. The only variable that differed among the experimental groups was the amount
of nitrogenous fertilizer. It was noted how the independent variable would affect the survival of Pogonomyrmex occidentalis. The decrease in the population of Pogonomyrmex occidentalis can be attributed to nitrogen poisoning because of the added fertilizer. Based on the effects of fertilizer overuse that were described beforehand, the increase in nitrogen concentration in the soil would have a significant negative impact on the survival of the Pogonomyrmex occidentalis.
2. Materials and Methods2.1. Experimental Setup
Pogonomyrmex occidentalis ants were purchased in bulk from Insect Sales.org. Twelve plastic jars, each with a volume of 32 fluid ounces, were used in the experiment. The jars measured approximately 6.5 inches in height, with a bore radius of 1.75 inches. Each jar lid was modified with three holes to allow airflow, created using a Warrior 18V Cordless 3/4-inch drill, as shown in
Figure 1
.
The experiment included six groups: a control group and five groups treated with different concentrations of nitrogen fertilizer (1 mL, 2 mL, 5 mL, 10 mL, and 20 mL). Each group had two replicates to ensure accuracy. The experiment focused on how different nitrogen levels affected the survival of Pogonomyrmex occidentalis. All jars were stored in the Research Wing of High Technology High School, under controlled temperature (22˚C) and lighting conditions, to maintain consistency.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Figure 1. Different views of the 3.5” × 6.9” Jar.
2.2. Ant Habitat Preparation
Each jar, measuring 3.5” × 6.9”, was filled with 16 ounces of organic topsoil from Gardenpro, using an Edward Tools Garden Trowel. The soil was kept uniform across all jars, with the only difference being the nitrogen content. Since the topsoil is organic, it contains minimal additional nutrients, making it a constant factor in the experiment.
Nitrogen was added using PetraTools Liquid Nitrogen Fertilizer. A mixture of 20 mL of water and varying amounts of nitrogen fertilizer was prepared for each experimental group, as shown in
Figure 2
. For the first experimental group, 1 mL of Petra-Tools Liquid Nitrogen Fertilizer was mixed with 20 mL of water. The amounts were carefully measured using a 10 mL glass serological pipette from PYREX, ensuring accurate proportions. This mixture was poured into a jar and thoroughly combined with the topsoil.
For the second experimental group, 2 mL of PetraTools Liquid Nitrogen Fertilizer was mixed with 20 mL of water, then poured into a separate jar and blended with the topsoil. For the third experimental group, 5 mL of nitrogen fertilizer was added to 20 mL of water and mixed into another jar with topsoil. For the fourth experimental group, 10 mL of nitrogen fertilizer was combined with 20 mL of water, then mixed into a separate jar with topsoil. For the fifth experimental group, 20 mL of nitrogen fertilizer was mixed with 20 mL of water and added to another jar, which was then thoroughly mixed with the topsoil. These five experimental groups, along with the control group, were replicated to ensure accurate results. All jars were labeled accordingly for easy identification, as shown in
Figure 3
.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Figure 2. Liquid nitrogen fertilizer was measured with pipette, mixed with 20 mL water.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Figure 3. Experimental setup inside Research Lab at HTHS.
2.3. Experimental Procedure
The setup consists of 12 jars, with two control and ten experimental groups. Each experimental group has a mirror of itself, to limit outliers in the dataset. To summarize the ant Habitat preparation: there are two jars with no added nitrogen content, two jars with 1 mL of added nitrogen content, two jars with 2 mL of added nitrogen content, two jars with 5 mL of added nitrogen content, two jars with 10 mL of added nitrogen content, and another two jars with 20 mL of added nitrogen content. Each of these jars has seven of the Pogonomyrmex occidentalis placed inside. The ants will be observed throughout four weeks, with the summation of ants in each jar at the end of every week. 5 mg of food will be provided two times a week through cracked corn, wheat, oats, barley, and milo. An equal amount of the food source will be provided to all jars. Next, to stimulate rain, moisten the soil by watering all of the jars with 5 mL every other day, except between the gap of Friday and Monday, which would be a two-day break. The water will be provided through the serological pipette and a Bel-Art Fast-Release Pipette Pump. The top layer of the soil should usually remain dry but everything under this layer should be kept moist.
3. Results3.1. Working Data
This study focused on the effects of increased nitrogen levels in soil on Pogonomyrmex occidentalis over four weeks. The data below (
Tables 1-8
) only refers to the number of ants that decreased over time. There are Group A and B which are replicas of each other, they have the same experimental groups. This was done to ensure that the results were accurate.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 1. Raw data table for Pogonomyrmex occidentalis alive after being exposed to control or various amounts of nitrogenous fertilizer over four weeks.
Groups
Weeks Passed
Week 0
Week 1
Week 2
Week 3
Week 4
Control A
7.0
2.0
2.0
2.0
1.0
1 mL A
7.0
4.0
2.0
0.0
0.0
2 mL A
7.0
3.0
2.0
0.0
0.0
5 mL A
7.0
0.0
0.0
0.0
0.0
10 mL A
7.0
5.0
2.0
0.0
0.0
20 mL A
7.0
0.0
0.0
0.0
0.0
Control B
7.0
3.0
3.0
2.0
1.0
1 mL B
7.0
2.0
0.0
0.0
0.0
2 mL B
7.0
2.0
2.0
0.0
0.0
5 mL B
7.0
2.0
1.0
0.0
0.0
10 mL B
7.0
4.0
2.0
0.0
0.0
20 mL B
7.0
3.0
0.0
0.0
0.0
Average
7.0
2.5
1.3
0.3
0.1
a. All values are rounded to two significant figures. This table shows the number of ants that were still alive after a certain amount of time had passed. The last row shows the average amount of Pogonomyrmex occidentalis left across all experimental groups and controls each week.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 2. Calculated data table for Pogonomyrmex occidentalis decrease over time (% of day 1) due to control or various amounts of nitrogenous fertilizer.
Groups
Weeks Passed
Week 0
Week 1
Week 2
Week 3
Week 4
Control A
0.000
71.43
71.43
71.43
87.71
1 mL A
0.000
42.86
71.43
100.0
100.0
2 mL A
0.000
57.14
71.43
100.0
100.0
5 mL A
0.000
100.0
100.0
100.0
100.0
10 mL A
0.000
28.57
71.43
100.0
100.0
20 mL A
0.000
100.0
100.0
100.0
100.0
Control B
0.000
57.14
57.14
71.43
87.71
1 mL B
0.000
71.43
100.0
100.0
100.0
2 mL B
0.000
71.43
71.43
100.0
100.0
5 mL B
0.000
71.43
87.71
100.0
100.0
10 mL B
0.000
42.86
71.43
100.0
100.0
20 mL B
0.000
57.14
100.0
100.0
100.0
Average
0.000
64.29
81.12
95.24
98.00
a. The values seen in
Table 2
were taken from
Table 1
and applied the formula for percent increase/decrease. ((xf − xi)/x1)*100 = Percent Change. All numbers are rounded to four significant figures. This shows the total decrease in the population of Pogonomyrmex occidentalis in each jar over the four weeks.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 3. Calculated data table for Pogonomyrmex occidentalis decrease over time (% of day 1) due to control or various amounts of nitrogenous fertilizer (mean of A and B).
Groups
Weeks Passed
Week 0
Week 1
Week 2
Week 3
Week 4
Control A
0.000
64.29
64.29
71.43
87.71
1 mL A
0.000
57.15
85.72
100.0
100.0
2 mL A
0.000
64.29
71.43
100.0
100.0
5 mL A
0.000
85.72
93.86
100.0
100.0
10 mL A
0.000
35.72
71.43
100.0
100.0
20 mL A
0.000
78.57
100.0
100.0
100.0
a. The formula for the calculated data table above is from averaging groups A and B. (a + b/2) The data for the table was obtained from
Table 2
.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 4. Summative data table for Pogonomyrmex occidentalis decrease over time (% of day 1) by varying amounts of nitrogen (Group A).
Groups
Groups (A)
Control A
1 mL A
2 mL A
5 mL A
10 mL A
20 mL A
Mean
75.50
78.57
82.14
100.0
75.00
100.0
SD
8.140
27.35
21.43
0.000
33.76
0.000
Var
66.26
748.2
459.2
0.000
1140
0.000
n
4.000
4.000
4.000
4.000
4.000
4.000
a. All numbers are rounded to four significant figures. This is the summary of the decrease in the population of Pogonomyrmex occidentalis over time (% of Day 1) by Varying Amounts of Nitrogen for Group A.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 5. Summative data table for Pogonomyrmex occidentalis decrease over time (% of day 1) by varying amounts of nitrogen (Group B).
Groups
Groups (B)
Control A
1 mL A
2 mL A
5 mL A
10 mL A
20 mL A
Mean
75.50
78.57
82.14
100.0
75.00
84.67
SD
8.140
27.35
21.43
0.000
33.76
15.5
Var
66.26
748.2
459.2
0.000
1140
239
n
4.000
4.000
4.000
4.000
4.000
4.000
a. All numbers are rounded to three significant figures. This is the summary of the decrease in the population of Pogonomyrmex occidentalis over time (% of Day 1) by Varying Amounts of Nitrogen for Group B.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 6. Statistical table describing results of ANOVA test for Pogonomyrmex occidentalis decrease, week three.
P-Value
Alpha
0.002872821965
0.05
a. An ANOVA test was conducted to compare the twelve data sets, to week Three. The p-value for week Three is 0.002872821965.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 7. T-test results for Pogonomyrmex occidentalis decrease, week three (Group A).
T-test Comparison
P-Value
Control – 1 mL
0.008153917065
Control – 2 mL
0.005185066967
Control – 5 mL
0.009187730962
Control – 10 mL
0.009759577837
Control – 20 mL
0.009187730962
a. A two-tailed T-test was used for this Statistical Data Table. Two-tailed T-test can show if the control and the experimental group are different.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Table 8. T-test results for Pogonomyrmex occidentalis decrease, week three (Group B).
T-test Comparison
P-Value
Control – 1 mL
0.004108037084
Control – 2 mL
0.001920721572
Control – 5 mL
0.002017648658
Control – 10 mL
0.003357581280
Control – 20 mL
0.001114389600
a. A two-tailed T-test was used for this Statistical Data Table. Two-tailed T-test can show if the control and the experimental group are different.
3.2. Presentation of Results
The graphs and images (
Figures 4-5
) below show the data in more comprehensive matter, showing the negative correlation between increasing nitrogen fertilizer and ant population over time. They also illustrate the other unexpected variables in the experiment, suggesting that the correlation is not objectively clear.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Figure 4. Graph showing the average of the experimental groups and the controls with one standard deviation. Mean values were taken from <xref ref-type="table" rid="table3">
Table 3
</xref> and the standard deviation values were taken from <xref ref-type="table" rid="table5">
Table 5
</xref>.
<xref ref-type="bibr" rid="scirp.145510-"></xref>Figure 5. Control A (shown above) and others were found to have plants growing inside of them.
4. Summary of Results and Conclusions4.1. Conclusions
This study specifically tested whether increasing nitrogen levels in the soil had a significant negative effect on the survival of Pogonomyrmex occidentalis. The results showed a negative relationship between increasing nitrogen levels and the survival of the ants. Within one week of being introduced into the experimental conditions, most ants in the higher nitrogen concentration groups (such as 20 mL A, 10 mL A and B, and 5 mL) exhibited immediate mortality as shown in
Table 1
. While it was expected that the nitrogen fertilizer would harm Pogonomyrmex occidentalis, the rapid and extensive mortality was unexpected.
As shown in
Table 4
,
Table 7
, and
Table 8
, a significant impact can be observed on the ant populations due to nitrogen levels. However, while the differences were statistically significant, the high mortality rates in the experimental groups made it difficult to assess the precise relationship between nitrogen levels and ant survival. As shown in
Table 6
, there was insufficient variability among the twelve experimental groups by Week 3, likely due to the high levels of mortality.
Table 2
and
Table 3
show the percentage of the ant population that died throughout the study. By the end of Week 4, all ants in the experimental groups were dead, with only a few remaining in the control groups.
Figure 4
illustrates the increasing mortality over the four weeks, with most experimental groups reaching 100% mortality by the end. These results suggest a strong negative effect of nitrogen levels on the survival of Pogonomyrmex occidentalis, though the high mortality rates likely resulted from the fertilizer doses being too high.
Several inconsistencies were noted during the study. The most significant unforeseen variable was the growth of plants in the jars (see
Figure 5
). Even by Week 1, plants began to grow in both the control and experimental jars. It was later determined that the food provided by InsectSales.org, which included seeds and honey, was likely the source of the plant growth. It is important to note that there were no light sources, but the seeds appeared resilient, germinating despite the lack of light. Interestingly, most plants grew in the control groups A and B.
Another inconsistency was the experimental habitat itself, which was somewhat unrealistic. The ants were confined to small jars with limited space to burrow—only about 3 inches deep—and a barren environment. These factors may have contributed to the high mortality. Given the large number of deaths, it is likely that the fertilizer doses were too high for the ants to survive.
4.2. Future Studies and Research Impact
Future research could entail the effects of supplementing organic matter with artificial fertilizer on the survival of Pogonomyrmex occidentalis. This could help determine if organic fertilizer is a better environmental option than synthetic fertilizers. In the future, lower concentrations of fertilizer should be utilized to assess more subtle effects on ant survival. This would help identify more precise effects of nitrogen exposure to ants or other organisms.
Other studies that could stem from this research include using a different ant species such as the red imported fire ant (Solenopsis invicta). This experiment would examine how increasing fertilizer levels affect the survival of Solenopsis invicta and thus would get closer to finding out if the negative effects of fertilizer are universal to all species of ants.
Additionally, another study could be the effects of phosphorus fertilizer on the survival of Pogonomyrmex occidentali, branching the type of fertilizer beyond just nitrogen-based fertilizer. Since nitrogen is relatively inert, with phosphorus reactivity, the study could be even more significant than the effects observed in this experiment.
The potential impacts of this study could be from many industries such as farming, agriculture, and even animal conversation. With the correct principles of what fertilizer to use, farmers could now use healthier alternatives and possibly reduce the damage artificial fertilizers have done to the environment. With agriculture, farming practices improved and more organisms, the industry could benefit from more plant growth and produce. The importance of ants could raise important questions about how human practices are affecting organisms and what can be done to solve this.
Acknowledgements
Thank you to Dr. Dina Ellsworth for direction through this research project and for helping when my organisms did not come as expected. Thank you to Mr. Craig Queenan for answering all my questions. Thank you to Mrs. Agnostak for helping with the figures and format. This work was also supported by the Monmouth County Board of Commissioners, for their support of MCVSD, the MCVSD Board of Education and Administration, including Dr. Charles Ford and Mr. Sean Meehan, the HTHS Faculty and Administrators, for their support of the research program, and the PFA for financially supporting the experiment. Special thanks to the Singh family for funding the experiment and helping with the setup as well.
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