The effects of acidity and copper toxicity on Lemna minor population growth
Ethelred II, King of England (968-1016)
Monday a.m. Biology 203
Monday, November 7, 20xx
Submitted to: Sir Percy of Kent
1

Abstract
(1) Copper and pH have both been found to negatively affect the growth rate of plants.
2
The purpose of this experiment was to determine how much pH and copper toxicity affect the growth rate of least duckweed, Lemna minor . (2) Three colonies of duckweed with five replicates each were compared. The control colony had a normal pH of 6.4 and a copper penny in each replicate; the first treatment had a pH between 4 and 5 with a copper penny in each replicate; and the second treatment had a pH between 5 and 5.5 without pennies.
(3) We found that the colonies in media with pH between 4 and 5 with a penny could not survive in these conditions. The environment was determined to be too acidic for duckweed growth. The colonies in a medium of pH between 5 and 5.5 grew well and there was no significant difference between their growth and that of the control group. The success of the second colony can be attributed to the less acidic environment and absence of copper. (4) Further studies are needed to determine how much copper and lower pH each affected the growth rates.
Introduction
Duckweed, Lemna minor , is a tiny aquatic plant that is found in many nutrient enriched, freshwater habitats such as slow-moving rivers, swamps and ditches in temperate and subtropical regions worldwide. It reproduces asexually and very quickly in ideal conditions, often forming mats on the water’s surface (Washington State Department of Ecology 20xx). For duckweed to grow at their optimal rate (with populations doubling every 2-3 days) no conditions can be limiting.
Copper is a structural catalytic component of many proteins and enzymes, which makes it an essential nutrient for plant growth. However, in higher than optimal concentrations, it has

been shown to interfere with several cellular processes including photosynthesis, respiration and
3 cell division (Charles et al. 2005). According to the Canadian Water Quality Guidelines (1987), it is unsafe for duckweed to be in an environment with a copper concentration higher than 2 µg/L at 0.12 mg/L CaCO
3
. Lemna minor has the best reproductive success in environments with pH between 6.5 and 7.5 but survives from pH 5 to 9 (Leng et al. 1995).
The present experiment was designed to test how much lowering the pH in a copper-rich environment would affect the growth rate of duckweed. We hypothesized that the duckweed colonies in the lowered-pH environment would have a slower growth rate than those colonies in the optimal pH environment. We also tested the effects that an environment with a decreased pH without copper would have on growth rate, expecting that these colonies would have an improved growth rate compared with the colonies in the more acidic environment.
Materials and Methods
To test the effects of acidity on duckweed growth rate we prepared ten, 100-mL beakers
(labeled 1-5 control and 1-5 variable) with 90 mL of the Lemna culture medium provided (for ingredients see Taylor, 20xx). In each of the beakers we put 25 healthy Lemna minor thalli along with a copper penny. We tested the pH of the growth medium, which was 6.4, with a pH meter and we the lowered the pH in the five variable beakers to 4-5. To do this, we added three to seven drops of HCl to each beaker, and tested the pH of the medium after each drop was added.
The plants were kept at a temperature around 21 o
C and under a constant 430-W high pressure sodium light source. Distilled Water was added when needed to maintain the 90 mL level in each beaker because water levels tended to drop daily due to evaporation and use by the duckweed.

4
Each day we counted and recorded the number of duckweed thalli in each beaker, and we also tested the pH in each of the populations in the reduced-pH mediums with the pH meter. If the pH in any of the beakers was higher than 5 we added HCl to return it to between 4 and 5.
By the end of the first week the duckweed in the acidic environment was not surviving.
In one beaker, however, in which we had accidentally not added a copper penny, survival was better. This population was still green and appeared to be growing, although not at a rate comparable with the control populations. We concluded from these observations that we had lowered the pH too much in this treatment, and that copper toxicity in these colonies from the penny may also have resulted in their failure to survive.
We then disposed of the five original variable duckweed populations (Treatment 1) in acidic environments and revised our experiment to include another variable group (Treatment 2).
In five new beakers we added 90 mL of the Lemna culture medium, enough drops of HCl to decrease the pH to between 5 and 5.5, and did not add a penny. We conducted the rest of our experiment the same way we had the first week, recording daily the number of duckweed thalli in each of the control and treatment beakers.
At the end of the second week we analyzed all the data we had collected over the twoweek period. To do this, I did t-tests to compare the control colony with Treatment 1 colony and
Treatment 2 colony. I also did a t-test to determine whether there was a significant difference in population growth between the treatment colonies. In addition, I did regression analysis for each colony to determine the slope of the lines of best fit and the upper and lower 95% confidence limits.

5
Results
The first step in my analysis involved taking the mean number of thalli (N t
) per beaker in each population for each day of the study. I then calculated the standard deviation to analyze the data’s variance, and the standard error of the data to determine how closely our sample means estimated the true population means. This I did instead of using error bars to reduce crowding on the graph. There was one day that I did not record the population numbers, which is why there are missing values on day six of the study.
Table 1: This table includes the means, standard deviations and standard errors for the control, treatment 1 and treatment 2 populations for each day of the study. Please note that recording day does not mean the day of the study.
Control Treatment 1 Treatment 2
______________________________________________________________________________
Recording Day
N t
SD SE N t
SD SE N t
SD SE
______________________________________________________________________________
1 25 0 0 25 0 0 25 0 0
2 27.4 0.8944 0.3651 26.2 1.6432 0.6708 29.6 2.6077 0.9856
3 28.4 1.1402 0.4655 25.8 1.3229 0.5401 31.4 3.2094 1.213
4 35 3.937 1.6263 27 3.5355 1.4434 33.8 6.8337 2.5829
5 45 7.4833 3.055 25 7 2.8577 36.6 9.3167 3.5214
6 40.2 10.3779 3.9225
7 49.2 10.2811 4.1972 24.75 5.1235 2.0917 43.8 12.3774 4.6781
______________________________________________________________________________
I then plotted the mean number of thalli (N t
) against time for all three populations to visualize the growth of the three duckweed colonies (Figure 1).

To compare growth rates, I took the Ln of the means (Ln(N t
)) of the three colonies and plotted them against time (Figure 2).
6

I then did regression analysis to find the slope of these lines, and from the confidence intervals determine if they were significantly different. the growth rate of Treatment 1 is
7 significantly different from the control colony, as Treatment 1 has a negative slope of -0.0046
(day
-1
) and the control colony has a positive growth rate of 0.1249. We therefore accept our first hypothesis that lowering the pH would decrease the growth rate of duckweed.
The slope (r-value) of the Treatment 1 colony is 0.0874, which is within the 95% confidence interval of 0.07924 of the control colony, which indicates that these slopes are not significantly different. Evidently, the slopes of Treatment 1 and Treatment 2 are very different.
This supported our second hypothesis since it showed that Treatment 2 colony’s growth rate was much better than first treatment colony’s growth rate, and was even statistically no different than the control colony’s growth rate.
Table 2: regression equations, the r-value (slope) and r
2
for the control populations, the lowered pH with penny populations (Treatment 1) and the lowered pH without penny populations
(Treatment 2).
__________________________________________________________________________
Control Treatment 1 Treatment 2
__________________________________________________________________________
Regression y = 0.1249x+
Equation 3.0642
y = -0.004x+
3.2599
y = 0.0874x+
3.1713
Slope (r) 0.1249 -0.0046 0.0874
Lower 95% 0.07924 -0.6523 0.07445
Upper 95% 0.1706 0.4209 0.1004
_________________________________________________________________________
I then did three t-tests with 95% significance levels. In the first t-test, I compared the control colony with the first treatment colony. For this test the t-value was 2.28, which was greater than the critical t-value, which was 1.81. This result indicates that the two groups were growing at entirely different rates. In the second t-test I compared the control and Treatment 2

populations, in which our calculated t-value was 0.138 and the critical t-value was 1.796. This
8 showed that these colonies did not have significantly different growth rates. The third t-test compared the first and second treatment colonies. For this comparison the calculated t-value was3.5527 and critical t-value was 1.796, indicating that there is a significant difference in growth rates between these two colonies, additionally supporting our second hypothesis.
Discussion
Our finding that the Treatment 1 colony in the acidic environment of pH 4-5 did not survive, agrees with previous studies showing that duckweed cannot survive in a low pH. The duckweed in the beaker in which we forgot to put the copper penny, however, was still green and appeared to be growing slightly better than the other populations. This indicated that it was perhaps a combination of acidity and copper toxicity in the other four beakers that was killing the duckweed.Charles et al. (2005), growth rate of duckweed was inhibited by 50% in acopper concentration of 16 ± 1µg/L.
Treatment 2 colony grew well in the environment with pH between 5 and 5.5 without the copper. This could be due to both the less acidic environment and the absence of copper. In future studies, I would suggest treatments with different copper concentrations and varying pH levels between 4 and 5.5 to determine how much each variable inhibits the growth of the duckweed.
Literature Cited
Caicedo, J.R., Van der Steen, N.P. Arce, O. and Gijzen, H.J. 2000. Effect of total ammonia nitrogen concentration and pH on growth rates of duckweed. Water Research, 34 : 3829-3835.

Charles, A.L., Markich, S.J. and Ralph, P. 2005. Toxicity of uranium and copper individually, and in combination, to a tropical freshwater macrophyte ( Lemna aequinoctialis ). Chemosphere,
Leng, R.A., Stambolie, J.H. and Bell, R. 1995. Duckweed- A potential high-protein feed resource for domestic animals and fish. Livestock Research for Rural Development. 7 : 11-33
Nixdorf, B., Fyson, A. and Krubeck, H. 2001. Review: Plant life in extremely acidic waters.
Environmental and Experimental Botany, 46 : 203-211.
Taylor, B.R. 20xx. Introductory ecology: Laboratory manual 20xx. St. Francis Xavier
University, Antigonish, NS, Canada.
Washington State Department of Ecology. Free Floating Plants. http://www.ecuy.wa.gov/programs/wq/plantgs/plantid2/descriptions/lemmin.html
. (6 November
20xx)
9