Internal Assessment Biology

Topic: Effect of two heavy metal micronutrients, zinc and copper, on the frond count of
Lemna minor (duckweed) over a period of nine days

1. Background

Ever since I was little, I worked with my grandfather to take care of the garden behind our house.
He always used N-P-K (nitrogen, phosphorous and potassium) fertilizers for the plants. I always
wondered how I could make the plants grow at a faster rate. This curiosity led me to investigate the
topic which taught me that the nutritional supply of plants plays a key role in their growth and
functioning and that a variety of essential elements are needed by plants for numerous reasons, such
as for their structure. The elements can be classified into two groups: macronutrients (a large quantity
of the nutrient is required by the plant) and micronutrients (a small quantity of the nutrient is required by
the plant)1. Elements N-P-K are macronutrients2 and are the general constituents of fertilizers. I wanted
to focus on micronutrients, like zinc (Zn) and copper (Cu), as micronutrients are many a times
disregarded and are not part of several fertilizers. Thus, the degree to which micronutrients affect plant
growth became the focus of my study. On a bigger scale, I realized that this study could be applied to
boost agricultural productivity and crop yields by supplying specific micronutrients to plants in the right
amounts. Micronutrients Zn and Cu were chosen due to availability.
Zn activates enzymes for the synthesis of proteins and the formation of chlorophyll. It also helps in
the production of growth hormones, like auxins. Cu, specifically Cu2+ ions, work in the activation of
enzymes to carry out the carbohydrate and nitrogen metabolism, and is needed for synthesizing lignin
which is a polymer that forms structural elements of plants, such as cell walls3.
Different heavy metals have different effects on a plant’s growth and are required in specific
quantities, meaning an excess or deficiency of these metals can be detrimental to their growth.
Phytotoxicity – toxicity in plants – occurs when a plant absorbs excess compounds, such as chemicals
or metals, from the surroundings via its leaves and roots4. Anthropogenic activities, like pesticide use,
have led to an increase in the metal concentrations in the environment to amounts which are harmful to
a plant’s nutrition, growth and reproduction. Heavy metal pollution is not just an environmental issue,
but also a serious health concern. Metals that cannot be broken down by decomposers, are absorbed
by plants and accumulate in their body. Due to biomagnification, the concentrations of metals increase

1 Prince, Robert. “Plant Nutrients.” Plant Nutrients, www.ncagr.gov/cyber/kidswrld/plant/nutrient.htm.

2
Shah, Richa. “7 Essential Elements of Macronutrients | Plants.” Biology Discussion, 2 May 2016, www.biologydiscussion.com/plant-
physiology-2/mineral-nutrition/7-essential-elements-of-macronutrients-plants/25674.
3 “Plant Micronutrient Functions.” Ecochem, www.ecochem.com/t_micronutrients.html.
4 “Plant Phytotoxicity in the Greenhouse.” MSU Extension, Michigan State University Extension,

www.msue.anr.msu.edu/news/plant_phytotoxicity_in_the_greenhouse

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in successively higher trophic levels of the food chain, affecting the whole ecosystem5. Edible plants
contaminated with heavy metals cause crop yield losses and are injurious to human health6. On the
other hand, micronutrient deficiencies also adversely affect plants, as not enough required minerals are
available to the plant to carry out its metabolic processes7. Thus, it is imperative to monitor and
maintain optimal concentrations of metals in the water and soil to ensure that plant growth and
functioning is at its peak. This would maximize agricultural productivity and reduce health issues.
To test the effects of heavy metal micronutrients on plants I thought that bioindicators, species of
plants or animals that are used to assess the health of ecosystems, would be ideal to use as they are
more sensitive to environmental changes than other organisms8. I chose to work with aquatic plants, as
they have not been as extensively studied as land plants9. Macrophytes, like duckweed, are aquatic
bioindicators of the health of water bodies10. Lemna minor (L. minor) is a duckweed that is specifically
used for phytotoxicity studies as it has a small size, fast growth rate and accumulates metals quickly11,
making it the perfect candidate for testing the effect of heavy metal micronutrients on plant growth. I
wanted to find the concentrations of Zn and Cu at which the growth of L. minor was maximum. This can
be applied to maximizing the growth of edible aquatic plants, like watercress, to increase crop yields.

2. Introduction

The growth of L. minor was measured by counting fronds so that the effects of different Zu and Cu
concentrations on the growth could be quantitatively compared. Measuring biomass was not opted for as
L. minor is mostly made of water, and even slight changes in drying the plant for measuring the weight
would have altered the results12.
Free metal ions could not be added to water directly, thus I thought of incorporated the metals via
salt compounds of zinc nitrate, Zn(NO3)2, and copper (II) nitrate, Cu(NO3)2, that dissociate in water into
Zn2+ and Cu2+ ions, respectively. Four concentrations of Zn(NO3)2 and Cu(NO3)2 were taken: 1.0 ppm,
2.0 ppm, 12 ppm and 20 ppm. Previous research shows that L. minor tolerates Zn at a concentration of
15 ppm, getting damaged at 18 ppm. I chose concentrations of 12 ppm and 20 ppm to test this. It also
showed that L. minor gets damaged above 0.5 ppm of Cu13. I wanted to test this concentration, but since

5 Blowes, D W. “Biomagnification.” Biomagnification - an Overview | ScienceDirect Topics, www.sciencedirect.com/topics/earth-and-

planetary-sciences/biomagnification.
6 Sethy, Sunil. “Effect of Heavy Metals on Germination of Seeds.” NCBI, 2013, www.ncbi.nlm.nih.gov/pmc/articles/PMC3783763/.
7 Sinclair, Alex H., and Anthony C. Edwards. “Micronutrient Deficiency Problems in Agricultural Crops in Europe.” SpringerLink, Springer, 1

Jan. 1970, link.springer.com/chapter/10.1007/978-1-4020-6860-7_9.

8 Holt, Emily. “Bioindicators: Using Organisms to Measure Environmental Impacts.” Nature News, Nature Publishing Group,

www.nature.com/scitable/knowledge/library/bioindicators-using-organisms-to-measure-environmental-impacts-16821310.
9 Hoagland, Robert E., et al. “Studies of the Phytotoxicity of Saponins on Weed and Crop Plants.” SpringerLink, Springer, 1 Jan. 1996,

link.springer.com/chapter/10.1007/978-1-4613-0413-5_6.
10
“Indicators: Macrophytes.” EPA, Environmental Protection Agency, 16 Aug. 2016, www.epa.gov/national-aquatic-resource-
surveys/indicators-macrophytes.
11 Cross, John W. “Phytotoxicity Test Methods.” Duckweed Biology, www.mobot.org/jwcross/duckweed/phytotox-methods.htm.
12 “Measuring Plant Growth.” Science Buddies, www.sciencebuddies.org/science-fair-projects/references/measuring-plant-growth.
13
N. Khellaf,. GROWTH RESPONSE OF THE DUCKWEED LEMNA MINOR TO HEAVY METAL POLLUTION. 2002, www.bioline.org.br/pdf?se09024.

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the least count for a milligram scale is 1 mg, I was unable to choose a smaller concentration than 1 ppm.
A study on L. minor showed that its growth decreased at a cobalt-zinc metal pair concentration of 2.00
ppm, thus I opted for 2 ppm14. I realized that different results may be obtained with the use of only Zn.
I decided to conduct my study for a period of nine days to be able to properly see any trends in my
observations. Ideally, L. minor should have been studied for a longer period of time to capture all
patterns, however due to time constraints, nine days was the longest I could take. I took observations at
intervals of 3 days (on days 3, 6 and 9) to gather sufficient data to observe overall trends in the results,
while at the same time to not have too many data points.

3. Research Question
To what extent do two heavy metal micronutrient ions, Zn2+ and Cu2+, in the water affect the frond
count (number of fronds) of Lemna minor (duckweed) over a period of nine days?

4. Hypothesis
There will be a statistically significant difference between the frond counts of L. minor on day 9
caused by the two heavy metal salt compounds considered, like Zn(NO3)2 and Cu(NO3)2, and the tap
water control. L. minor will also have the highest frond counts at a Cu concentration of 0.5 ppm and a
Zn concentration of 12 ppm.
Prior research has shown that metal micronutrients have significant effects on a plant’s growth
when they are deficient or in excess. Studies have shown that: (1) L. minor tolerates a Zn concentration
of 15 ppm, thus the growth should be maximum for 12 ppm, and (2) the plant gets damaged above a
Cu concentration of 0.5 ppm; thus, the growth should be highest for the Cu concentration of 1 ppm.

5. Variables

5.1. Independent Variable: The heavy metal salt compound - Zn(NO3)2, Cu(NO3)2 - with
concentrations of 1.0 ppm, 2.0 ppm, 12 ppm and 20 ppm

5.2. Dependent Variable: The frond count (number of fronds) of L. minor on days 3, 6 and 9

5.3. Controlled Variables

Controlled Variables Why to Control

Age, origin and number of the L. minor fronds – all Different origins and ages of plants may
fronds were taken from the same pond at the same cause differences in plant growth; hence, it
time (6 days old); 10 fronds per beaker is important to control.

14Dirilgen, N and Y. Inel, “Cobalt‐Copper and Cobalt‐Zinc Effects on Duckweed Growth and Metal Accumulation.” Journal of Environmental Science
and Health . Part A: Environmental Science and Engineering and Toxicology, vol. 29, no. 1, 1994, pp. 63–81., doi:10.1080/10934529409376022.

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 It was verified that the pH of water was 7 – the pH of
water in each beaker was measured by pH strips
every day as pH buffers were not available
Temperature – plants were kept at room temperature
(~25°C) as it was easy to maintain
Amount and intensity of light – 12 hours of daily
fluorescent light was supplied and all plants were
kept at the same distance from the light source
Time for adding the salts and measuring frond count
– the salts were added at the same time each day,
and the frond count was measured at the same time
on days 3, 6, and 9 (day 0 - start of the experiment).
To make sure the water was not acidic or
alkaline as this may affect the growth of the
plant
To make sure the temperature was constant
as it affects plant growth.
To make sure all plants received the same
amount of light (a low amount of light can
inhibit plant growth).
To make sure the data was recorded after
the same time intervals and to ensure
consistency in adding the salts, in order to
ensure the results are valid.

6. Methodology

6.1. Materials and Apparatus

• Materials
Materials Required Quantity
Lemna minor 20 g
Zinc nitrate - Zn(NO3)2 1g
Copper (II) nitrate - Cu(NO3)2 1g
Rubbing Alcohol 1 bottle
Tap Water 20 dm3
Watch/timer 1
Marker (for labelling) 1

• Apparatus
Apparatus Required Quantity
0.5L plastic beakers 37
1L glass jar 1
Magnifying glass 1
Glass stirrer 1
Lab spoon 1
Tweezers 1
Milligram scale 1

6.2. Procedure
A. Gloves were worn, and all the apparatus was wiped down with rubbing alcohol.
B. The 1L glass jar was filled with one liter of tap water.

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 C. Using the milligram scale and lab spoon, 1 mg of Zn(NO3)2 was measured and added to
the 1L glass jar to make a 1-ppm solution, and stirred with a glass stirrer.
D. This solution was poured into two 0.5L beakers, with 0.5 litres of the solution in each.
E. Steps B – D were repeated with two more beakers.
F. Steps B – E were repeated with Cu(NO3)2.
G. One 0.5L beaker was filled with 0.5 liters of tap water.
H. Using the milligram scale and lab spoon, 1 mg of Zn(NO3)2 was measured and added to
the beaker to make a 2-ppm solution, and stirred with a glass stirrer.
I. Steps G and H were repeated with three more beakers.
J. Steps G – I were repeated with Cu(NO3)2.
K. Steps G – J were repeated for concentrations of 12 ppm and 20 ppm of Zn(NO3)2 and
Cu(NO3)2. Note: 6 mg and 10 mg, respectively, of the ionic compounds were measured for
making the 12 ppm and 20 ppm concentrations.
L. The remaining five beakers were each filled with 0.5 litres of water (control).
M. Using tweezers, ten L. minor fronds were placed into every beaker.
N. Daily, the plants were supplied with 12 hours of fluorescent light – they were kept at a
distance of five feet from the light source – and the pH of each beaker was measured by
using pH strips (to verify it was still 7).
O. The fronds were counted on days 3, 6 and 9 (day 0 - start of the experiment). Note: By the
use of a magnifying glass, every visible L. minor frond was counted, including new fronds
which were budding from the larger fronds
P. Students’ t-tests (website: Statistics Kingdom) were used to analyze the findings

6.3. Experimental Design

• Experimental Setups
Each treatment consisted of tap water and a heavy metal salt, either Zn(NO3)2 or Cu(NO3)2. 4 different
concentrations of each heavy metal salt were tested – 0.5 ppm, 2 ppm, 12ppm or 20 ppm – with each
concentration having 4 replicates. Only 4 replicates (beakers) were taken for each concentration,
as the method of counting fronds was very rigorous. In all 320 L. minor fronds were added to a
total of 32 beakers, with 10 fronds in each beaker.

• Control Setups
Control setups consisted of 5 beakers that were filled with only tap water (no heavy metal was
used). In all, 50 L. minor fronds were added to a total of 5 beakers, with 10 fronds per beaker.

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6.4. Instrumental Uncertainty
Instrument Least Count Uncertainty Purpose
Milligram Scale 0.001 g ± 0.001 g To measure the heavy metal salts
Plastic Beaker (500 cm3) 10 cm3 ± 5.0 cm3 To measure the tap water for
preparing metal concentrations

7. Safety, Ethical and Environmental Issues

Safety Issues – The heavy metals salts may have caused irritation in case of contact with the skin,
thus gloves were worn while handling the salts for protection.

Ethical Issues – There were no ethical issues in this experiment.

Environmental Issues – The heavy metal salts and tap water used in the experimental setups were
placed in a sanitary drain (drain disposal)15. The unused salts were returned to the school laboratory.

8. Results and Analysis

8.1. Processed Data Tables

Table 1: Average frond count of L. minor on days 3, 6, and 9 caused by Zn(NO3)2 treatment and the
tap water control

Average frond count of L. minor caused by Zinc Nitrate Zn(NO3)2 and standard deviation
Concentrations of Zinc Day 0 (start of Day 3 Day 6 Day 9
Nitrate (ppm) the experiment)
Control (tap water) 10.0 ± 0.0 17.3 ± 1.1 24.3 ± 1.3 34.8 ± 1.3
1 ppm 10.0 ± 0.0 17.3 ± 1.1 36.3 ± 1.8 46.3 ± 4.0
2 ppm 10.0 ± 0.0 20.3 ± 4.4 29.5 ± 3.4 39.5 ± 3.8
12 ppm 10.0 ± 0.0 16.8 ± 2.9 26.3 ± 2.5 34.3 ± 3.6
20 ppm 10.0 ± 0.0 13.3 ± 1.3 20.8 ± 2.2 27.3 ± 1.5

Table 2: Average frond count of L. minor on Days 3, 6, and 9 caused by Cu(NO3)2 treatment and the
tap water control

Average frond count of L. minor caused by Copper (II) Nitrate Cu(NO3)2 and standard deviation
Concentrations of Day 0 (start of Day 3 Day 6 Day 9
Copper (II) Nitrate (ppm) the experiment)
Control (tap water) 10.0 ± 0.0 17.3 ± 1.1 24.3 ± 1.3 34.8 ± 1.30
1 ppm 10.0 ± 0.0 21.7 ± 0.5 31.3 ± 0.5 41.0 ± 2.2
2 ppm 10.0 ± 0.0 19.5 ± 2.5 31.3 ± 4.0 42.0 ± 3.9
12 ppm 10.0 ± 0.0 23.3 ± 0.8 29.5 ± 2.3 38.3 ± 2.2
20 ppm 10.0 ± 0.0 14.3 ± 1.5 20.0 ± 3.9 25.0 ± 4.7

15
“ VII. Chemicals: Managing, Handling and Disposing.” School Improvement in Maryland,
mdk12.msde.maryland.gov/instruction/curriculum/science/safety/chemicals.html.

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8.2. Graphical Analysis

Figure 1: A comparison of the average frond counts of L. minor on days 0, 3, 6, and 9 caused by
Zn(NO3)2 treatment and the tap water control

The Average Frond Count of Lemna minor caused by Four Different

Concentrations of Zinc Nitrate (1 ppm, 2 ppm, 12 ppm, 20 ppm) and the
Control (tap water) on Days 0, 3, 6 and 9
60
*Error bars show
Average Number of L. minor Fronds

standard deviation
50

40

30

20

10

0
0 (start of experiment) 3 6 9
Number of Days

1 ppm 2 ppm 12 ppm 20 ppm Control

As seen, the highest average frond count of L. minor, on day 9, was caused by the Zn(NO3)2
concentration of 1 ppm, while the lowest frond count was caused by the concentration of 20 ppm.
Zn(NO3)2 concentrations of 1 ppm and 2 ppm caused a relatively higher frond count, on day 9, in
comparison to the tap water control, while the concentrations of 12 ppm and 20 ppm had a relatively
lower frond count than the control. Graphically, the Zn(NO3)2 concentration of 12 ppm shows a similar
frond count of L. minor on day 9 to that of the control. Interestingly, on day 3, the Zn(NO3)2
concentration of 2 ppm caused a higher frond count than the concentration of 1 ppm, however, on day
9, the concentration of 1 ppm resulted in the highest average frond count than all of the other Zn(NO3)2
concentrations and the control. Similarly, on day 6, the 12-ppm concentration caused a relatively higher
L. minor frond count than that of the control, however on day 9, the average frond count caused by the
control surpassed the frond count of the 12-ppm concentration. Overall, the Zn(NO3)2 concentrations
and the tap water control in order of decreasing average frond count on day 9 were: 1 ppm, 2 ppm, tap
water control control, 12 ppm, and 20 ppm.

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Figure 2: A comparison of the average frond counts of L. minor on days 0, 3, 6, and 9 caused by
Cu(NO3)2 treatment and the tap water control

The Average Frond Count of Lemna minor caused by 4 Different

Concentrations of Copper (II) Nitrate (1 ppm, 2 ppm, 12 ppm, 20 ppm) and
the Control (tap water) on Days 0, 3, 6 and 9
60
*Error bars show
standard deviation
Average Number of L. minor Fronds

50

40

30

20

10

0
0 (start of experiment) 3 6 9
Number of Days

1 ppm 2 ppm 12 ppm 20 ppm Control

As seen, the highest average frond count of L. minor, on day 9, was caused by the Cu(NO3)2
concentration of 2 ppm, while the lowest frond count was caused by the concentration of 20 ppm.
Cu(NO3)2 concentrations of 1 ppm, 2 ppm and 12 ppm caused a relatively higher frond count, on day
9, in comparison to the tap water control, while the concentration 20 ppm had a relatively lower frond
count than the control. On day 3, the Cu(NO3)2 concentration of 12 ppm caused the highest frond
count than all of the other concentrations and the tap water control, however, on day 9, it resulted in a
relatively lower frond count than both Cu(NO3)2 concentrations of 1 ppm and 2 ppm. Remarkably, the
concentrations of 1 ppm, 2 ppm and 12 ppm all resulted in similar frond counts of L. minor on day 6
graphically. Overall, the Cu(NO3)2 concentrations and the tap water control in order of decreasing
average frond count on day 9 are: 2 ppm, 1 ppm, 12 ppm, tap water control and 20 ppm.

8.3. Statistical Analysis

Student’s t – test16
All of the Students’ t-tests (Welch’s test – 2 sample unequal variance) were two-tailed, had degrees of
freedom = 7, and used a 95% level of significance (α = 0.05)

16
“Statistics Calculators.” Shapiro-Wilk Calculator, www.statskingdom.com/.

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Null Hypothesis: There will be no significant difference between average frond count of L. minor on
day 9 of the two groups (treatments) under consideration: treatments were the concentrations (1 ppm,
2 ppm, 12 ppm or 20 ppm) of either zinc nitrate or copper (II) nitrate, and the tap water control

Table 3: Students’ t-test results of the effects of the four different concentrations of Zn(NO3)2 and the
tap water control on the average frond count of L. minor on day 9
Comparison 1 Comparison 2 Comparison 3 Comparison 4
Groups Compared - Control 1 ppm Control 2 ppm Control 12 ppm Control 20 ppm
Zinc Concentration
and Tap Water Control
Sample Size 5 4 5 4 5 4 5 4
Mean 34.8 46.3 34.8 39.5 34.8 34.3 34.8 27.3
Standard Deviation ±1.30 ±4.02 ±1.30 ±3.77 ±1.30 ±3.56 ±1.30 ±1.48
P - value 0.00534177 0.0757731 0.802657 0.000207792
Statistical
Significance YES NO NO YES

The average L. minor frond counts caused by Zn(NO3)2 concentrations of 1 ppm and 20 ppm on
day 9 differed significantly from that of the tap water control. While, the concentration of 1 ppm
showed a higher frond count than that of the control, 20 ppm resulted in a lower frond count
compared to the control. On the contrary, the average frond counts caused by the 2 ppm and 12 ppm
did not show significantly different results from that of the tap water control on day 9. The 2-ppm
concentration caused a relatively larger frond count than the control, while 12 ppm caused a lower
frond count. Overall, the Zn(NO3)2 concentration of 1 ppm resulted in the highest frond count than all
the other Zn concentrations and the tap water control, while 20 ppm resulted in the lowest.

Table 4: Students’ t-test results of the effects of the four different concentrations of Cu(NO3)2 and the
tap water control on the average frond count of L. minor on day 9
Comparison 1 Comparison 2 Comparison 3 Comparison 4
Groups Compared – Control 1 ppm Control 2 ppm Control 12 ppm Control 20 ppm
Cu(NO3)2 concentration
and Tap Water Control
Sample Size 5 4 5 4 5 4 5 4
Mean 34.8 41.0 34.8 42.0 34.8 38.3 34.8 25.0
Standard Deviation ±1.30 ±2.16 ±1.30 ±3.94 ±1.30 ±2.17 ±1.30 ±4.74
P - value 0.00391704 0.0247756 0.0361045 0.0277180
Statistical Significance YES YES YES YES

The average L. minor frond counts caused by all Cu(NO3)2 concentrations – 1 ppm, 2 ppm, 12
ppm, 20 ppm – on day 9 significantly differed from the average frond count caused by the tap water
control. Cu(NO3)2 concentrations of 1 ppm, 2 ppm, and 12 ppm showcased a relatively higher frond

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count compared to the control, whereas 20 ppm had a lower frond count than that of the control.
Overall, the Cu(NO3)2 concentration of 2 ppm resulted in the highest frond count than all the other
concentrations and the tap water control, while 20 ppm resulted in the lowest frond count.

9. Discussion

On day 9, Zn(NO3)2 concentrations of 1 ppm and 20 ppm caused a significantly different

average frond count from that of the control (see Table 7), while all of the Cu(NO3)2 concentrations (1
ppm, 2 ppm, 12 ppm and 20 ppm) showed significantly different results in comparison to the control
(see Table 8). As a whole, this supports the first part of experimental hypothesis because a
significant difference existed between the frond counts of L. minor on day 9 between the
different concentrations of the two heavy metals salts, Zn(NO3)2 and Cu(NO3)2, and the tap
water control. The second part of the hypothesis that the highest frond counts will be
observed at a Zn concentration of 12 ppm and Cu concentration of 0.5 ppm was not supported
as the highest frond counts were caused by a Zn concentration of 1 ppm and Cu
concentration of 2 ppm. While the highest frond count was observed at a Zn(NO3)2 concentration of
1 ppm, the optimal concentration of Zn, at which the growth of L. minor is maximum, could not be
assumed because a concentration lower than 1 ppm could not be taken due to limited resources. As
the Cu(NO3)2 concentration of 2 ppm resulted in the highest frond count, it can be assumed that the
range for the optimal concentration of Cu in the environment, at which the growth of L. minor is
maximum, is between 1 ppm to 12 ppm, as both of these concentrations resulted in a lower frond
count than for the 2 ppm concentration. Further research needs to be done to find a smaller range for
the optimal concentrations of Zn and Cu required by L. minor.

The findings also showed that, the 20-ppm concentrations of both heavy metal salts showed a
significantly different and lower frond count than that of the tap water control. This reflects the fact
that micronutrients, like Zn and Cu, are only needed by L. minor in small amounts in order for it to
have optimal growth. If an excessive amount of these micronutrients is present in the surroundings, it
can cause phytotoxicity in plants, inhibiting their growth. This shows the extent to which water and
soils polluted with heavy metals can have detrimental effects on a plant’s growth and health.

On day 9, the Zn(NO3)2 concentrations of 2 ppm and 12 ppm did not show significantly different
results from that of the tap water control (see Table 7), thus the null hypothesis was accepted. This
suggests that the differences seen in the frond counts between the tap water control, and the Zn
concentration of 2 ppm or 12 ppm, can only be attributed to random chance. However, the 2-ppm
concentration caused a relatively higher frond count than that of the control, while the 12-ppm
concentration resulted in a relatively lower frond count compared to the control.

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10. Conclusion

This study was aimed to see the effect of two heavy metal micronutrient ions (Zn2+ and Cu2+) on
the frond count (number of fronds) of Lemna minor (duckweed) over a period of nine days. As a
whole, the first part of the experimental hypothesis was accepted: most of the Zn(NO3)2 and Cu(NO3)2
concentrations showed significantly different results from that of the tap water control on day 9,
however, the Zn(NO3)2 concentrations of 2 ppm and 12 ppm did not cause significantly different frond
counts from the control. The second part of the experimental hypothesis, that the highest frond count
would be observed at a Zn concentration of 12 ppm and Cu concentration of 0.5 ppm was rejected.
Overall, it was seen that L. minor needs a Zn concentration of 1 ppm and Cu concentration of
2 ppm in its surroundings (water) in order to have maximal growth in terms of frond count.
The lowest frond counts were observed at 20 ppm concentrations of both heavy metal salts
compared to all the other concentrations and the tap water control, showcasing how heavy metal
pollution can have harmful effects on a plant’s growth. The study also showed that deficiency or
absence of the micronutrients, such as in the tap water control treatment, does not lead to maximum
plant growth, showcasing that a certain concentration of the metal is required by L. minor and is
crucial for the plant to have optimum growth.
Hence, it was concluded that different Zn and Cu concentrations significantly differ in
their effects on the growth of L. minor, and that the plant showcases optimal growth at only
certain concentrations of the two heavy metals. Beyond the optimal concentration range of the
essential metal, either deficiency or pollution, the growth of the L. minor is inhibited. Thus, it is crucial
to maintain optimal concentrations of essential micronutrients needed by certain plants to maximize
their growth, and thus maximize agricultural outputs and productivity.

11. Evaluation
11.1. Strengths
This study allowed the investigation of how two different heavy metal micronutrients, Zn and Cu,
effect the frond count of L. minor over a period of nine days. A statistical analysis was done (Students
t-test) with 95% level of significance, thus ensuring the accuracy and significance of the findings, by
ruling out random errors with a confidence level of 95%.
As a laboratory experiment, the study had a high degree of control over confounding variables,
such as a constant temperature (~25°C) and pH (~7) of water, meaning that the change seen in the
frond count of the L. minor (dependent variable) was only due to the manipulation of the different
concentrations of the two heavy metal salts (independent variable), and not due to any other factors.
Thus, a causal relationship could be established between the different concentrations of either
Zn(NO3)2 or Cu(NO3)2 and the fron count of L. minor on day 9.

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11.2. Limitations
Being an artificial setup, however, may reduced the external validity of the study, meaning that the
same results may not be applicable in a real-life setting. This study could be replicated by adding heavy
metals in a small freshwater pond which already has L. minor growing in it to see the effects of the
metals on the plant in its natural habitat. A large number of replicates could not be taken (n=4) as the
method of counting the fronds was rigorous. This may have reduced the reliability (consistency) of the
results. At least five replicates should have been taken. As only the L. minor species was used, the
results cannot be generalized to all aquatic plants. A variety of plant species, such as watercress and
hydrilla, should be tested to increase the generalizability of the findings. Another limitation is that, due
to time constraints, the growth of L. minor could only be observed for nine days. This period may be
insufficient to see all trends of the growth of L. minor at different concentrations of Cu and Zn, so the
plants should be observed for a longer period of time. Lastly, heavy metal salts, Zn(NO3)2 and
Cu(NO3)2, were used to incorporate metal ions in water. Nitrogen (N) is essential for plants and
stimulates their growth as it is a main component of chlorophyll and amino acids17. This means that the
dissociated nitrate ions, NO32-, may have affected the growth of L. minor, and interfered with the
results. Zn2+ and Cu2+ ions should be added directly to the water to get credible results.

12. Application and Extension

In a larger biological context, this study can have possible applications in detecting effluent
toxicity of a facility’s wastewater, which can have harmful effects on the survival and reproduction of
aquatic organisms. WET (Whole Effluent Toxicity) tests are done by exposing organisms to the
wastewater and testing its effects on them in to ensure that the facility is not discharging pollutants in
toxic amounts18. L. minor can be used as a bioindicator for heavy metal pollution. By knowing optimal
concentrations of certain heavy metals at which the growth of L. minor is maximum, a reduction in the
plant’s growth could be used potentially indicate toxicity of a heavy metal.
An extension to this experiment can be testing the effect of other heavy metal micronutrients,
such as nickel and manganese, on the frond growth of L. minor, to examine the toxic effects of these
elements on plants. Another extension could be to investigate the effect of the heavy metals on the
growth of other macrophytes, such as water hyacinth or hydrilla, in order to investigate trends and to
be able to increase the generalizability of the results to other species of plants.

13. Bibliography
1. Prince, Robert. “Plant Nutrients.” Plant Nutrients, www.ncagr.gov/cyber/kidswrld/plant/nutrient.htm

17 “Nitrogen.” Fertilizers And Soil Acidity | Mosaic Crop Nutrition, www.cropnutrition.com/efu-nitrogen.

18
“Whole Effluent Toxicity Methods.” EPA, Environmental Protection Agency, 19 Apr. 2018, www.epa.gov/cwa-
methods/whole-effluent-toxicity-methods.

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2. “Plant Micronutrient Functions.” Ecochem, www.ecochem.com/t_micronutrients.html.
3. “Plant Phytotoxicity in the Greenhouse.” MSU Extension, Michigan State University
Extension, www.msue.anr.msu.edu/news/plant_phytotoxicity_in_the_greenhouse
4. Sethy, Sunil. “Effect of Heavy Metals on Germination of Seeds.” NCBI, 2013,
www.ncbi.nlm.nih.gov/pmc/articles/PMC3783763/.
5. Sinclair, Alex H., and Anthony C. Edwards. “Micronutrient Deficiency Problems in
6. Agricultural Crops in Europe.” SpringerLink, Springer, 1 Jan. 1970,
www.link.springer.com/chapter/10.1007/978-1-4020-6860-7_9.
7. “What Is Bioremediation?” Conserve Energy Future, 25 Dec. 2016,
www.conserve-energy-future.com/what-is-bioremediation.php.
8. Holt, Emily. “Bioindicators: Using Organisms to Measure Environmental
Impacts.” Nature News, Nature Publishing Group,
www.nature.com/scitable/knowledge/library/bioindicators-using-organisms-to-measure-
environmental-impacts-16821310.
9. “Indicators: Macrophytes.” EPA, Environmental Protection Agency, 16 Aug. 2016,
www.epa.gov/national-aquatic-resource-surveys/indicators-macrophytes.
10. Cross, John W. “Phytotoxicity Test Methods.” Duckweed Biology,
www.mobot.org/jwcross/duckweed/phytotox-methods.htm.
11. Hoagland, Robert E., et al. “Studies of the Phytotoxicity of Saponins on Weed and
Crop Plants.” SpringerLink, Springer, 1 Jan. 1996, www.link.springer.com/chapter/10.1007/978-1-
4613-0413-5_6.
12. Cross, John W. “Phytotoxicity Test Methods.” Duckweed Biology,
https://www.mobot.org/jwcross/duckweed/duckweed-measuring-growth.htm
13. N. Khellaf, and M. Zerdaoui. GROWTH RESPONSE OF THE DUCKWEED LEMNA
MINOR TO HEAVY METAL POLLUTION. 2002, www.bioline.org.br/pdf?se09024.
14. Dirilgen, N and Y. Inel, “Cobalt‐Copper and Cobalt‐Zinc Effects on Duckweed Growth and Metal
Accumulation.” Journal of Environmental Science and Health . Part A: Environmental Science and
Engineering and Toxicology, vol. 29, no. 1, 1994, pp. 63–81., doi:10.1080/10934529409376022.
15. “VII. Chemicals: Managing, Handling and Disposing.” School Improvement in Maryland,
www.mdk12.msde.maryland.gov/instruction/curriculum/science/safety/chemicals.html
16. “Statistics Calculators.” Shapiro-Wilk Calculator, www.statskingdom.com/.
17. “Nitrogen.” Fertilizers And Soil Acidity | Mosaic Crop Nutrition, www.cropnutrition.com/efu-nitrogen.
18. “Whole Effluent Toxicity Methods.” EPA, Environmental Protection Agency, 19 Apr. 2018,
www.epa.gov/cwa-methods/whole-effluent-toxicity-methods.

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 14. Appendices
14.1. Zinc Raw Table

Day Zn(NO3)2 - Concentration (ppm)

1 ppm 2 ppm
Replicates (increase in number of Average Replicates (increase in number of Average
fronds) fronds)
1 2 3 4 1 2 3 4
0 (start of 10 10 10 10 10.00 10 10 10 10 10.00
experiment)
3 17 16 17 19 17.25 26 19 14 22 20.25
6 34 36 36 39 36.25 33 30 24 31 29.50
9 40 46 48 51 46.25 42 41 33 42 39.50

Day Zn(NO3)2 - Concentration (ppm)

12 ppm 20 ppm
Replicates (increase in number of Average Replicates (increase in number of Average
fronds) fronds)
1 2 3 4 1 2 3 4

0 (start of 10 10 10 10 10.00 10 10 10 10 10.00

experiment)
3 16 17 21 13 16.75 15 12 14 12 13.25
6 28 28 27 22 26.25 24 18 21 20 20.75
9 37 38 33 29 34.25 29 25 28 27 27.25

14.2. Copper Raw Table

Day Cu(NO3)2 - Concentration (ppm)

1 ppm 2 ppm
Replicates (increase in number of Average Replicates (increase in number of Average
fronds) fronds)
1 2 3 4 1 2 3 4

0 (start of 10 10 10 10 10.00 10 10 10 10 10.00

experiment)
3 22 22 12 21 21.67 16 23 19 20 19.50
6 31 31 13 32 31.33 25 36 31 33 31.25
9 43 42 15 38 41.00 36 46 45 41 42.00

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 Day Cu(NO3)2 - Concentration (ppm)
12 ppm 20 ppm
Replicates (increase in number of Average Replicates (increase in number of Average
fronds) fronds)
1 2 3 4 1 2 3 4
0 (start of 10 10 10 10 10.00 10 10 10 10 10.00
experiment)
3 24 22 23 24 23.25 16 12 15 14 14.25
6 33 28 27 30 29.50 25 14 20 21 20.00
9 38 35 39 41 38.25 28 17 26 29 25.00

14.3. Control Raw Table

Day Tap Water - Control

Replicates (increase in number of fronds) Average
1 2 3 4
0 (start of 10 10 10 10 10.00
experiment)
3 17 19 17 16 17.25
6 25 26 23 23 24.25
9 36 36 34 33 34.75

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