Caffeine Effects on Reproduction, Growth, and Behavior of Small Organisms

Brianna Jones

April 26, 2022

INTRODUCTION

This experiment looks deeper into caffeine as a micropollutant and how it affects the

environment and organisms that live in it. A micropollutant is a chemical that comes from

different origins and appears in environmental sources in small amounts (Benner, et al., 2013).

Caffeine is one of the most consumed drugs by humans in the world (Reyes, Cornelius, 2018). It

can be found in substances such as coffee, energy drinks, tea, and guarana (National Library of

Medicine, 2021). Caffeine can enhance performance and decrease perception (Meeusen,

Decriox, 2018). One of the main purposes of intaking caffeine is to decrease fatigue and to

remain more “awake”. There is current research to see if caffeine can treat depression or

neurological disorders (Evans, Richards, Battisti, 2021). To be lethal in humans, the blood

concentration must reach approximately 90 μg/ml, which happens most often when more than

10 grams of caffeine is ingested (Murray, Traylor, 2021). To better understand the concept of

caffeine toxicity on larger organisms, this experiment tested the toxicity on smaller organisms.

This is important because caffeine can be found in the environment, in places such as lakes and

rivers, caused by human secretion and waste (Rodriguez del Rey, Granek, Buckley, 2011). One

of the first organisms studied in this experiment is Daphnia, and this is because of its unique

behavior and easily observable traits. They also have a short reproductive time (Stollewerk,

2010). Another key part of this experiment is the study of reproduction of yeast cells. This is due

to easy access to this organism, as well as the mechanisms behind their cell cycle (Knop, 2011).

Other organisms that will be studied in this experiment include lettuce plants, duckweed, and

darkling beetles because there is vast prior knowledge of these organisms. Previous

experiments have been done on coral to determine if caffeine exposure is linked to bleaching
(Portland State, 2011). The main research question for this study is to determine if caffeine has

toxicological effects on life of all kinds. It has an effect on smaller organisms, such as Mytilus

californianus (Rodriguez del Rey, Granek, Buckley, 2011), which leads us to hypothesize that it

will have an effect on organisms of all kinds. This study includes exposing Daphnia, lettuce

plants, duckweed, yeast cells, and darkling beetles to different concentrations of caffeine to

determine how they respond.

METHODS

Daphnia Caffeine Exposure

Five, ten fold serial dilutions were made ranging from the lowest concentration of 10-1 to

the highest concentration 10-5. The control used in the serial dilution was spring water. One 1.7

mL microcentrifuge tube was labeled for each concentration of caffeine. Six total Daphnia

magna were exposed to caffeine. The Daphnia magna were maintained in room temperature

spring water while on a 12 hour light to 12 hour dark cycle. One Daphnia magna was added per

concentration and was left to acclimate for 3 minutes. After the rest period, the Daphnia magna

were observed under 40x magnification and low light. Low light was used because it allows for

more visuals on the heart of the organism. Therefore, it is easier to count the heartbeat. The

heart rate of the Daphnia magna was observed for 15 seconds and multiplied by 4 to calculate

beats per minute. These steps were repeated twice, resulting in two trials for each

concentration. The average of the two trials per concentration were placed into a line graph

using Microsoft Excel.

Lettuce Caffeine Exposure

A solution consisting of spring water and black walnut husks was made and stored in a

sealed container in a refrigerated environment. A tube of lettuce seeds was filled with distilled
water and set to soak for 5 minutes. Filter paper was placed on the bottom of 7 petri dishes. 3

mL of distilled water was added to one petri dish to act as the control. 3 mL of the black walnut

solution was added to a second petri dish. 3 mL of each caffeine concentration was added to its

corresponding petri dish. 4 lettuce seeds were evenly placed in the petri dishes and the lids

were replaced. The dishes were placed under a grow light for a week. The number of

germinated seeds and lengths of the radicals were measured 7 days post exposure and then 14

days post exposure. 2 seedlings for each concentration were planted in soil and watered with 3

mL of its corresponding caffeine or control solution. If there were no seedlings for a

concentration, this step was not performed. The radicals of the seedlings planted in the soil

were measured after 7 days. The average radical lengths were put into a line graphing using

Microsoft Excel.

Yeast Cell Caffeine Exposure

3 control conical tubes were created by adding 7.5 mL of YDP solution. 3, 5 mM caffeine

concentration tubes were created by adding 375 ul of 100 mM stock caffeine concentration and

7.125 mL of YDP media. 3, 50 mM caffeine concentration tubes were created by adding 3,750

ul of 100 mM stock caffeine solution and 3.75 mL of YDP media. Using a micropipette, 2.5 mL of

S. cerevisiae cells were added to all 9 tubes. One of each tube per concentration was examined

at 0 hours, 1 hour, and 1.5 hours. The tubes to be examined at 1 hour and 1.5 hour were placed

in the incubator at 30C. The samples were analyzed using a haemocytometer using a 4x4 set.

The pictures were uploaded to Fiji and each individual yeast cell was counted in both squares.

This was repeated for all 9 tubes - one of each caffeine concentration and a control at each time

point. The average cell concentration was put into a line graph using Microsoft Excel.
Duckweed Caffeine Exposure

4 conditions were made using differing volumes of caffeine solution and Hoagland’s

media. The control was Hoagland’s media (100 mL). 1 mg/L concentration was a solution of 0.1

mL caffeine solution and 99.9 mL Hoagland’s media. 10 mg/L concentration was a solution of 1

mL caffeine solution and 99 mL of Hoagland’s media. 100 mg/L concentration was a solution of

10 mL and 90 mL of Hoagland’s media. 50 mL of each condition was added to 2 jars. 8 green

fronds of duckweed were added to each jar. The jars were placed under a grow light. Using Fiji,

the number of adult fronds and emerging fronds were counted, as well as the length of 3 fronds

in mm. The number of fronds and length measurement was conducted on both jars for each

condition at 0 days, 7 days, and 21 days. The average frond length for each condition were put

into a line graph using Microsoft Excel.

Darkling Beetle Caffeine Exposure

Enclosures for the Tenebrio molitor were created by adding 200 g of dried oats to a

plastic storage container. At random, caffeine was added to certain enclosures. 4 adult and 8

larvae (mealworms) were weighed and added to each enclosure. After 14 days, the living adults

were counted and weighed again, as well as the living larvae (mealworms). An antipredator test

was performed on 1 of the living larvae in each enclosure. This was done by placing the larvae

in a plastic cup, placing it on a balance, and hitting the top of the cup with a plastic ruler to a

pressure of 1 g. The time for the larvae to begin moving again was recorded. This was repeated

for all 4 enclosures. A choice test was performed on 1 adult from each enclosure. An adult was

placed under a cup in a plastic container for a 30 second rest period. On one side of the cup,

there were distilled water crystals and on the other side, caffeine crystals. Once the cup was

removed, the time for the adult to choose a crystal was recorded, as well as which one was

chosen. Once the tests were completed on all 4 enclosures, the adults and larvae were placed

back in their home enclosure.

RESULTS

Daphnia Caffeine Exposure

Daphnia magna were exposed to 5 different concentrations of caffeine ranging from 0.1

to 0.00001 g/L and viewed under a microscope using low light and magnification. The number

of heartbeats post exposure counted and calculated as beats per minute (BPM). The average

heart rate of the Daphnia magna in the control condition of spring water was 192 bpm. The

average heart rate in beats per minute at the highest concentration of caffeine was 226. In

comparison to the highest caffeine concentration (0.1 g/L), there was not a significant change in

the organism’s heart rate. As shown by Fig. 1, the average beats per minute for the intermediate

caffeine concentrations did not have a significant increase or decrease. However, an

unexpected average heart rate of 175 bpm was calculated for the 10-3 concentration. A one-way

ANOVA test was performed to determine the statistical significance. The p value for the data

shown is 0.727, which is not significant on a p>.05 scale.

Figure 1. This graph shows the average heart rate of Daphnia magna once exposed to differing
caffeine concentrations. The p value shows that the data is not statistically significant on a p>.05
scale. The heart rate decreased as the concentration of caffeine increased.
Lettuce Caffeine Exposure

4 lettuce seeds were set to germinate on petri dishes containing caffeine concentrations

ranging from 0.1 to 0.00001 g/L, as well as two control dishes - one with spring water and the

other with a black walnut solution. After 7 days the number and size ( in cm) of germinated

seeds were recorded, and done again after 14 days. After being planted and watered with

caffeine solution until day 21, the length of each radicle was measured. The concentration with

the highest number of seeds germinated the 0.001g/L as it gave an average of 0.5 cm.

Compared to the control and the rest of the caffeine concentration, there was no major increase

or decrease. The seed germination data stayed relatively similar, regardless of the caffeine it

was exposed to. However, when looking at the radicle length data, it is obvious that the lowest

concentration of caffeine yielded the lowest length with an average of less than 0.5 cm. Again,

the third caffeine concentration gave the highest length, with an average of almost 2.5 cm. The

other concentrations and control gave relatively similar data. Finally, the spring water control

gave the highest seedling height after being planted with an average of over 2.5 cm. Although

the caffeine concentrations studied gave similar numbers, the 0.001 g/L gave the lowest

seedling height with an average of a little over 1.5 cm. A one-way ANOVA test was performed to

determine the statistical significance of the data. The statistics of the p value for the lettuce seed

germination data is 0.937615, which shows that the data is not significant at p<.05. The data

was also not statistically significant in the radicle lengths, due to a p value of 0.052171, using

p<.05. Finally, and consistently, the data for the average seedling high was also not significant.

The p value for this was 0.559033 at p>.05.

Figure 2. This shows the average number of seed germination over a 7 day period. The data
was not statistically significant on a p>.05 scale. Caffeine tube 3 showed the highest level of
seed germination, compared to the other conditions and the control.

Figure 3. This graph shows the average radicle length following a 14 day period. The data was
not significant on a p>.05 scale. Caffeine tube 3 showed the longest radicle length (cm) after 21
days.
Figure 4. This graph shows the average height of the seedlings following a 21 day period. The
data was not significant on a p>.05 scale. Plants watered with spring water yielded the highest
length of seedlings.

Yeast Caffeine Exposure

S. cerevisiae cells were added to tubes filled with 375 ul of caffeine and 7.125 mL of

YDP media, 3,750 ul of caffeine and 3.75 mL of YDP media, and just 7.5 mL of YDP media.

The number of S. cerevisiae cells in the tube were counted at 0 hours, 1 hour, and 1.5 hours

after they were placed in the refrigerator. The average cells in the control at the zero time point

was 939 cells. As the time passed increased, the number of cells stayed relatively constant with

776 cells at 1 hour and 807 cells at 1.5 hours. There was no major decrease in cell number but

the amount fluctuated as time passed. In the 5mM concentration, the average cell count at 0

hours was 864 cells. After an hour passed, the cell count decreased to 827 cells then increased

to 908 cells after 1.5 hours. The 50mM caffeine concentration averaged 815 cells at 0 hours,

which then decreased to 548 cells after 1 hour. After sitting for 1.5 hours, the average cell

concentration decreased to 442 cells. Comparing the controls to the 1.5 hour time points, it is

obvious that the number of S. cerevisiae cells decreases the longer it is placed in a caffeine
solution. A one-way ANOVA test was to be conducted to determine statistical significance, but

due to shared data issues, this could not be done.

Figure 5. This graph shows the average cell concentration of yeast cells over a 2 hour time
period. Statistical significance cannot be determined due to shared data issues.

Duckweed Caffeine Exposure

Duckweed fronds were placed into 3 different solutions using Hoagland’s media and

stock caffeine solution (1 mg/L, 10 mg/L, and 100 mg/L). The control was entirely Hoagland’s

media. The number and length of the fronds in millimeters were recorded at 0 days, 7 days, and

21 days. The average frond length at 0 days in the control solution was 1.681 cm, which

increased to 2.551 cm after 21 days. The 1 mg/L concentration started with an average frond

length of 1.765 cm and increased to 2.302 cm after 21 days. Both the 10 mg/L concentration

and the 100 mg/L concentration started with an average frond length of about 1.7 cm which

increased to around 2.7 cm after 21 days in both. In all of the concentrations, including the

control, the number of duckweed fronds in the jars increased significantly. The 0 day jars all

began with approximately 8 fronds for each concentration. After 21 days the control averaged

71 fronds, 1 mg/L averaged 52 fronds, 10 mg/L averaged 39 fronds, and 100 mg/L averaged 57

fronds. A one-way ANOVA test was conducted to determine statistical significance of the data.
Data between the control and 10 mg/L concentrations was significant with p values of .018231

and .000651. 1 mg/L and 100 mg/L yielded opposite results because their data was not

significant, with p values of .208326 and .068623. These were done on a p>.05 scale. The

statistical significance of average frond count will be determined at a later time due to shared

data issues. The statistical significance of the data from the 7 day time point of each condition

shows that it was not significant, with a p value of .370259.

Figure 6. This graph shows the average duckweed length over a 21 day period. Most of the
concentrations resulted in increased length after 21 days, other than 1 mg/L. Statistical
significance shows that the data from the control as well as the 10 mg/L were significant, but the
data from the 1 mg/L and 100 mg/L were not. The blue and yellow asterisk indicate that the data
for the control and the 10 mg/L concentration of caffeine are significant at the 7 day period.
Figure 7. This graph shows the average duckweed cell count after a 21 day period. The dats is
significant at 21 days for the 1 mg/L caffeine concentration, as well as the 100 mg/L caffeine
concentration. Statistical significance could not be calculated for 10 mg/L due to shared data
issues.

Tenebrio molitor Caffeine Exposure

Adults and larvae Tenebrio molitor were exposed to either caffeine or distilled water

crystals for 21 days. An antipredator test and a choice test was conducted on the organisms at

day 7 and day 21. The data collected was the time it took the larvae to begin moving after a

stimulus and the time/choice an adult took when given the choice between caffeine or distilled

water. After gathering the final data, it was determined that the both conditions, caffeine and

control, caused the Tenebrio molitor to lose mass the longer they were placed inside the

enclosures. The average beginning mass of the control Tenebrio molitor at day 0 was 0.127,

however by day 21, that average decreased to -0.05. This means in 21 days, the subjects lost

0.05 grams. The average mass at day 21 for the caffeine Tenebrio molitor was also negative,

which indicates that the subjects lost mass again, however this group lost 0.037 grams.

According to a T test of two independent samples, the data is not statistically significant. The p
value was .385967. On a p>.05 scale, the data was not significantly different from each other.

The behavioral tests showed that it took the larvae Tenebrio molitor a significant amount of time

to recover post stimulus. As for the adults, when given the choice between caffeine and distilled

water, most adults chose the caffeine, in a relatively fast time frame.

Figure 8. This graph shows the average larval mass of Tenebrio molitor after 21 days of either
caffeine or distilled water exposure. Both of the conditions lost mass. According to a p value of
.385967, there was no statistically significant data difference.

CONCLUSIONS

To understand how different concentrations of caffeine affected the heart rate of Daphnia

magna, 5 concentrations were created in which one organism was placed in each solution. After

looking closely at how the Daphnia magna responded to caffeine, it can be concluded that

caffeine, at any concentration, does not give a significant effect on the heart rate (bpm). None of

the data yielded a statistical significance, meaning that the caffeine did not cause a major

change in any of the Daphnia magna's heart rate. A source of error could be that the Daphnia

magna were not left to rest long enough in the respective caffeine concentration before the heart

rate was recorded. This experiment was important to the bigger picture because it is known, and
mentioned in the introduction, that caffeine can raise an individual’s heart rate. Therefore, if the

heart rate of the Daphnia magna was raised, it would be a step in the process of determining

how caffeine affects organisms that are larger. Future directions for this specific experiment

include letting the organism acclimate longer to the caffeine concentration before examining and

also finding a more precise and organized way to measure the heart rate.

Lettuce was used in this study to see if caffeine affects the rate in which it grows, as well

as caffeine’s toxicity. The major conclusion drawn from this experiment was that intermediate

concentrations of caffeine do expedite the growth of lettuce seeds while they are in the

germination phase. However, once the seeds are planted in soil, caffeine does not give the

same effect on growth that caffeine does. Although the caffeine did not kill the lettuce seeds, it

also was not the ideal hydration source once they were placed in the soil. Sources of error for

this experiment include not using the correct amount of caffeine solution due to micropipetting

issues as well as shared data issues. Again, this relates to the big picture because it helps

prove that caffeine has stimulatory effects. Future directions regarding the lettuce in this

experiment would be to keep them planted for longer than 21 days while still hydrating them

with the caffeine source. This could show toxicity of the caffeine to the lettuce, or in turn, show

that after the lettuce is accustomed to the new soil, it can also acclimate to the caffeine

concentration.

Understanding how caffeine can affect the reproductive rate of S. cerevisiae (yeast cell)

required them to be placed in these concentrations for extended periods of time. The data

collected shows that as the concentration of caffeine increases, the number of cells that occupy

the space decreases. Caffeine concentration and amount of cells are inversely correlated.

However, in the control concentration and the 5 mM concentration, the cell count decreased and

then increased again as more time passed. This could be due to the cells acclimating to the

caffeine concentration before reproduction increased again. The original experiment

recommended that the cells be counted at 0 hours, 1 hour, and 2 hours. However, due to
outstanding circumstances, the cells were counted at 1.5 hours, which could be a source of

error. This is important to the bigger world because it shows that caffeine is a stimulant. In the

introduction, it is mentioned that caffeine has stimulatory effects on certain subjects. Therefore,

the increase in cell concentration as the concentration and time increases shows that caffeine

does in fact show stimulatory effects.

The experiment to understand the effect of time and caffeine on the length and

reproduction of duckweed fronds had to be conducted over a long period of time (21 days) and

using many increasing concentrations of caffeine. After 21 days, all of the concentrations

yielded an increased amount of fronds. Statistical significance cannot be determined at this time

due to shared data issues. However, from day 0 to day 21, all concentration jars showed an

increase in frond count. Frond length also increased in almost all the jars. Around day 7, the 10

mg/L jar came to a plateau in length at around 2 cm. However, the rest of the fronds in each

concentration continued to grow to higher lengths. The data of the frond length was both

statistically significant and insignificant. Data from the control and the 10 mg/L concentration

showed a significant difference in length from day 0 to day 21. However, 1 mg/L and 100 mg/L

showed no significant difference in length. Some sources of error in this experiment could be

due to insufficient number of starting fronds, errors when counting and measuring on Fiji, and

also issues with sharing data as some data was initially lost or needed to be done twice for

correctness. Future directions for this experiment would be to move the duckweed from caffeine

concentrations, place them in distilled water for a rest period, and then place them back into

concentrations of caffeine to see if they would still be functional.

To further prove that caffeine has stimulatory effects, Tenebrio molitor adults and larvae

were tested, regarding their antipredator response and their choice when it came to caffeine or

distilled water. As shown by the results, both the caffeine and control caused the larvae to lose a

small amount of mass. This was not the initial hypothesis when the experiment was created.

None of the data was significant for these two groups because the difference in mass lost was
not a large change. This experiment contained a few sources of error. On day 7, it was apparent

that there was a significant amount of cannibalism in the enclosures, as less and less larvae

and beetles were found. Also, the enclosures contained adults and larvae of different ages.

Meaning, the organisms could have been ending or beginning any one of the life stages,

resulting in skewed results when searching for and weighing the organisms. This was important

to the bigger picture because it showed that caffeine can have stimulatory effects, as well as

addictive effects if an individual is exposed long enough. Future directions include moving the

Tenebrio molitor that were exposed to caffeine into an enclosure full of distilled water. This

would help to see if the adults or larvae would die from lack of caffeine, showcasing withdrawal

symptoms.

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