1. Rubis,Charlie 1
Research Proposal
Incorporating AllelopathicAquatic Plants for Algal Control in the Carroll Creek Linear Park in
Frederick, Maryland
Charlie Alexander Rubis
Background
Each year in Frederick Maryland, the Carroll Creek Linear Park becomes the center of
attention for many events and functions downtown as well as a site for many residence
exercising, walking to work, or eating out. This park began as a floodplain in the 1970’s and
with the population in Frederick increasing from 40,148, in the 1990 census, to 65,239, in the
2010 census, it has become the center for a changing town (Census.gov, 2015). This
development and planning to and around the Carroll Creek Linear Park will amount to about
$150 million (City of Frederick, 2011). With all of the work going in to make the area around
the creek look good, a persistent problem occurs every year within the creek; overwhelming
growth of algae in the water. There are different types of algae in the creek, including
planktonic and benthic. This research will be interested in planktonic algae. The creek may
consist of a mixture of different types of algae, ie green algae or filamentous algae. In this
research, one taxonomy won’t be of interest over another. The planktonic algae in the creek
will be looked at as a whole and not the sum of its parts.
Since 2012, the group “Color the Creek” has been adding a variety of plants and lily pads
to the creek to make it look more aesthetically pleasing. Another goal of these plantings are to
drown the light from reaching the algae in an attempt to inhibit its growth (Color the Creek,

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2015). Though this has helped some, problems still remain with the algae in the water. The
water in the Carroll Creek Linear Park is slow moving and has be shown to receive point and
non-point sources of nutrients, including from Culler Lake in Baker Park (Clark, 2014). Because
of this, the algae still have the ability to grow, even with lily pads inhibiting penetration of light
through the water. Other techniques have been employed to keep algal growth under control.
This includes using barley straw, sodium percarbonate(2Na2CO3), copper sulfate (CuSO4), and
Polyquat (Clark, 2014). All of these techniques have shown mixed results and aren’t natural.
There is a natural way to reduce and/or inhibit algae growth and its through chemicals found in
certain aquatic plants. These natural chemicals that come from certain plants inhibit growth in
a process known as allelopathy.
Allelopathy was first defined 1937 by a scientist from Austria named Hans Molischand.
This process occurs when one species has an advantageous effect over another species. These
effects can affect things such as growth and reproduction (Ferguson et al., 2003). Algae, and
phytoplankton in general, will take oxygen out of the water that other organisms may need and
can reduce the overall water quality in its ecosystem. The aquatic plants also need nutrients
that may be limited due to blooms of algae. This creates inherent competition for those
nutrients between the plants and the algae. This can lead to some plants releasing algal
inhibitory chemicals into the water, which makes the environment more favorable to them.
Some of the research on this subject involves finding out which species of plants can inhibit
which species of algae (Kong, 2006; Zuo et al., 2014). Other researchers take that idea a step
further and find out exactly what chemicals the plants are producing in order to inhibit the
algae from growing (Wu et al., 2013).

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The experiment will use two different sets of algae, each one with their own methods
and materials. The first set of algae will be directly from Carroll Creek. This type of algae will
be measured for its maximum light utilization efficiency. This simply means its photosynthetic
ability and it is a very common technique used for measuring chlorophyll fluorescence and is
commonly seen as Fv/Fm. A reduction in photosynthetic ability by the plants will drive the
Fv/Fm value further away from its maximum value of 1. The other type of algae I will use is
microcystis. This type of algae will be measured using a certain type of fluorometer and will be
measured in cells x 104/mL. Allelopathic effects will yield low cell counts per milliliter of water.
Currently, the “Color the Creek” group is not using plants specifically for their ability to
inhibit plants growth via allelopathic chemicals. This research with use plants with known
abilities to have allelopathic tendencies, and put them together with algae directly from Carroll
Creek Linear Park to see if the chosen plants have the ability to inhibit the algal growth that
occurs regularly in the creek.

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Hypotheses
H1= The treatment plants will lower the photosynthetic ability of the algae from Carroll Creek
Linear Park by their allelopathic tendencies.
Ho1: The treatments will have no effect on the photosynthetic ability of algae.
H2= The cell density of the microcystis will be lower when grown in plant media than the
control media.
Ho2= The cell density of the microcystis will not be effected by the media that it is grown
in.

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Methods and Materials
Plants
I have chosen three types of allelopahtic aquatic plants to use in this experiment. All
three species of plants are native to Frederick County, Maryland. The first is Saururus cernuus L
or Lizards tail (McAvoy, 2015). These grow to about a height of 30 to 60 centimeters high. This
plant species was used in a previous experiment by a Hood College student studying allelopathy
and her results can be used comparatively with mine. The second species of plant is Pistia
stratiotes L. or water lettuce. This only grows up to about 20 centimeters high but may have
roots that grow up to 50 centimeters. It has been shown to have a strong inhibitory effect on
types of algae like Microcystis aeruginosa (Wu et al., 2013). The last species of plant for the
experiment is Eichhornia crassipes or water hyacinth. This plant can grow up to 90 centimeters
high and has been shown to inhibit growth of some types of algae, including Microcystis
aeruginosa (Kong et al., 2006). All of the plants will be bought from the Maryland Aquatic
Nursery in Jarrettsville Maryland in late June 2016.
Carroll Creek Algae Materials
The plants will be grown in the greenhouse on the campus of Hood College. Each
species of plant will be grown in their own large tub that will hold water. Inside each tub, for
the treatments and control, will be oxygen stones placed in the same position in each tub. The
stones will be placed in the tubs a week or two before the plants are added to assure the water
is of good quality for the plants. When received, the plants will have their potting soil removed
and washed of the roots in an effort to make sure the soil has no effect on the algal growth.

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Sitting on top of the water will be a floating device with nine openings. The openings will be
filled in with plastic pots that are holding the plants. Within each plastic pot will be material
from the inside of straw blankets used for grass seed germination.
A total of four large tubs will be used. The water within the large tubs will be the media
that interacts with the algae in the experiment. This includes three that will be growing plants
and one for the control, that won’t have any plants. The plastic pots in the control tub will
include the material from the straw roll and used as the baseline for the experiment. This will
be done to ensure there are no allelopathic effects coming from the straw roll material. The
same amount of nutrients will be added at the same time to each large tub. The plants will
grow for three to four weeks.
Carroll Creek Algae
To obtain the algae from Carroll Creek (Figure 1), I will be adhering 2” X 2” ceramic tiles
to cinderblocks. There will be a total of 36 tiles used for this part of the experiment. Each
treatment will have six tiles and the control will have twelve. The algae will have the ability to
grow onto the ceramic tiles. The tiles and cinderblock will be put into the creek in June 2016
and will be placed there for six weeks. After six weeks, the tiles will be taken out and processed
on campus. Tiles will be randomly placed in a treatment or control group. Each tile will be
submerged in water from one of the large tubs (treatment or control) in a plastic petri dish.
The algae will not be grown in the tubs with the plants to make sure that any inhibition of algal
photosynthetic ability will be from chemicals of the plants and not from shading from the plant
leaves. The petri dishes will consist of a bottom part that is painted black, and a lid that is clear.

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Putting the tiles and water media in their own petri dish will help make each replicate
independent from others in their own treatment. The algae will then be put into a mechanical
container that will control the temperature and the day:night light cycle. The algae will be kept
in the container for three weeks. After that time, fluorometer readings will take place every
day for two more weeks.
Carroll Creek Algae Measurements
The algae used from Carroll Creek will be measured in terms of photosynthetic ability
after being mixed with the water media. To do this, I will be using a Junior-PAM Chlorophyll
Fluorometer from WALZ (Figure 2). This device will be plugged into a computer that has the
software WinControl-3. To measure the photosynthetic ability, I will be painting a petri dish lid
black. Off centered on the lid will be a very small hole that the fiber optic cord can go through
from the PAM fluorometer. The end of the fiber optic cord will need to be just under the
water’s surface, at the same length from the tiles for each petri dish tested.
Each time a new petri dish needs to be analyzed, the blacked out lid will replace the
clear lid it initially had when it is taken out of the container. This is done to condition the algae
to a lack of light and keep as many of the photosystem 2 chlorophylls open as possible. When
they are open, most of the light energy goes towards photosynthesis. Using the black lid makes
sure little to no light from the room or the sun will be hitting the algae so that I can get a more
accurate measurement of photosynthetic ability. Using the computer, I will measure the ratio
of Fv to Fm. This will tell me the maximum light utilization efficiency. A maximum utilization
would yield a value of 1 (unit less). This directly correlates to photosynthetic ability. I will get

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this by hitting “Fo/Fm” on the software which will give a strong beam of light to the algae in the
blacked out petri dish.
The WinControl-3 software will be able to give me a graph of Fv/Fm over time. This
procedure will be repeated multiple times for each tile and all of the data from photosynthetic
ability for the plant treatments will be combined based on plant species for that day (ie: 10
PAM readings for tiles with lizard tail water averaged with a standard deviation). This process
will be repeated over a minimum of two weeks, and may go longer if deemed necessary, to see
if there is a trend in photosynthetic ability of the algae.
Microcystis Algae
In the second part of this experiment, I will be testing the same water media from the
tubs, used for the Carroll Creek algae, but on microcystis. The strain of microcystis is currently
being processed on campus and is from Lake Erie. This side experiment will follow a known
protocol used by the former Hood College student as mentioned above. For this experiment,
the water media from the tubs will be mixed with the microcystis algae on a welled plate. Here,
I am going to measure fluorescence of the algae, which will relate to algal growth. The well
plates with plant water will be compared to plates with the control water, as that will be the
baseline. The plates will be measured for their pigment concentration using a BMG Labtech
FLUOstar Omega Plate Reader Flourometer. This will give measures of fluorescence. The units
will be in cells x 104/mL. I will use this along with a hemocytometer and microscope to get a
measurement of cell density. Measurements will be taken on a regular basis. Through time, I
will look for trends in cell density of the microcystis.

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Statistical Methods
For both experiments being run, I will be using a one-way ANOVA to compare the
treatments to the control and to each other. Comparing the treatments to the control using
SPSS will let me know if the plants effected the health of the algae (photosynthetic ability)
significantly or not. I will be using SPSS on the computers on the Hood College campus.

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Anticipated Results
Carroll Creek Algae
For my main experiment, I believe that the plants will have some effect on the health of
the algae in their ability to do photosynthesis. I think each species of plant will yield different
results. Since I will be using the actual algae from the creek, the results will be generalizable to
the creek. The algae in the creek may be a mixture of different types of phytoplankton. If there
ends up being a low species richness or diversity, one (or two of the) plant(s) may be more
effective than the others in inhibiting the algae. I will need to use enough tiles and repeat the
steps over multiple days to give my results more statistical power and lower my chance for a
beta-error.
For the microcystis side experiment, I anticipate that the water lettuce and water
hyacinth will have a significant effect on the algal growth as their cases for allelopathy have
been well documented. In general, I also think the treatments with plant media will be yield
different results for cell density of the algae than those samples with the treatment water.

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References
Census.gov. 2015. State and County Quick facts: Frederick (city), Marylan
http://quickfacts.census.gov/qfd/states/24/2430325.html?cssp=SERP. Retrieved
November 5, 2015.
City of Frederick. 2011. Economic Development. Carroll Creek Park Overview.
http://cityoffrederick.com/DocumentCenter/Home/View/1100. Retrieved November 5,
2015.
Clark, J. 2014. Chapter 3: Algaecides. Algae in Carroll Creek: Green Infrastructure an
Community Greening Approaches. University of Maryland College of Agriculture and
Natural Resources. Pp: 16-21.
Color the Creek. 2015. “Color the Creek”. Retrieved on 13 Jun 16 from
http://www.coloronthecreek.com/color-on-the-creek.html.
Ferguson, J. J., & Rathinasabapathi, B. (2003). Allelopathy: How plants suppress other plants.
University of Florida Cooperative Extension Service, Institute of Food and Agricultural
Sciences, EDIS.
Kong, C. H., Wang, P., Zhang, C. X., Zhang, M. X., & Hu, F. (2006). Herbicidal pote
allelochemicals from Lantana camara against Eichhornia crassipes and the alga
Microcystis aeruginosa. Weed research, 46(4), 290-295.
Wu, X., Wu, H., Chen, J., & Ye, J. (2013). Effects of allelochemical extracted from water lettuce
(Pistia stratiotes Linn.) on the growth, microcystin production and release of Microcystis
aeruginosa. Environmental Science and Pollution Research, 20(11), 8192-8201.
Zuo, S P; Wan, K; Ma, S M; Ye, L T. 2014. Combined allelopathic potential of aquatic plants
species to control Algae. Allelopathy Journal. 34.2: 315-323

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Figures
Figure 1. Location in Carroll Creek Linear Park that the algae will be collected from on the tiles.
Source: Google Maps
Figure 2. Image of Junior-PAM Fluorometer that will be used in the experiment
Source: http://www.walz.com/images/junior-pam/intro/junior-pam_2l.jpg