Phytoremediation of Kāneʻohe Stream and Kāneʻohe Bay using
Bacopa monnieri (ʻAe ʻAe)
By: Lilia Nakakura and Hunter Rapoza
-----------------------------------------------------------------------------------------------------------Abstract:
Sewage pollution continues to be a growing problem and threaten marine
ecosystems. Many areas in Hawaiʻi have been affected by this epidemic such as Kāneʻohe
Bay, Oʻahu, which has been a victim of several sewage spills for the past 30 years. Due to
enhancements of nutrients from sewage spills and fertilizers that flow into the bay, algae
growth has increased while water quality and clarity decreased. In addition, excessive
algae growth reduced light to coral resulting in coral bleaching, a collapse of the reef
ecosystem, and threatening biodiversity (Pastorok, 1985). Recent studies have showed that
phytoremediation as a possible strategy that can remedy eutrophication and other water
quality related problems. Phytoremediation is a type of bioremediation process that uses
plants to remove or neutralize pollutants from the environment (USDA, 2000).
Phytoremediation is natural, less costly, and an environmental friendly alternative that
hopes to remedy water pollution and eutrophication in Hawaiiʻs marine ecosystem.
In this experiment, three water samples were taken from Kāneʻohe stream and one
in Kāneʻohe Bay. Bacopa monnieri (ʻAe ʻAe) an indigenous plant to Hawaiʻi was used to
determine which study site had the most effective changes in phosphates, nitrates, algae growth
(absorbance), turbidity, pH, and salinity using phytoextraction (a type of phytoremediation). The
experiment hopes to find phytoremediation as an alternative solution that can help Kāneʻohe
Stream and Bay ecosystem back on track and help the coral reefs.
Based on the experiment, results showed that Site 3- Kāneohe Stream river mouth
worked best for phytoremediation using B. monnieri. The chlorophyll absorbance had the least
change at 0.008nm. While, the phosphate levels remained low at 0.5ppm and were normal.
The nitrate levels remained at 12.5 mg/L and normal as well. In addition, turbidity levels
had the least change at 8.0 NTU and had the clearest water. Salinity levels were 9.1 ppt and
pH levels were 4-6.5, which means that B. monnieri preferred slightly acidic medium for
growth.
--------------------------------------------------------------------------------------------------------------------Kāneʻohe Bay is located on the northeast side of
Oʻahu and is known to have lush coral reef
ecosystems. Throughout the years the pristine
coral reef ecosystem have deteriorated where
one of the major contributors is eutrophication.
Eutrophication is the process where high levels
of nitrogen and phosphates are present in the
water (Art, 1993). These pollutants are
transferred from fertilizers and sewage that
lead to high phytoplankton population and
increase algae growth in the ocean. Kāneʻohe
Figure 1: Map of Kāneʻohe Stream into Kāneʻohe Bay.
Stream is one of the main contributing rivers of Kāneʻohe Bay (Figure 1). The south sector
near Kāneʻohe Stream receives daily discharges of 12,000 m3 from the Kāneʻohe Municipal
Sewage Treatment plant. The south sector also receives considerable amounts of silted
freshwater, through streams and through the runoff from the urban and agricultural coastlands
(Hanson & Gunderson, 1975). In June 2011, 30,000 gallons of raw sewage spilled into
Kāneʻohe Bay that came from the two manholes from the Kāneʻohe Wastewater Treatment
Facility (Honolulu News & Events, 2002). In addition, heavy rains also contributed to pollution,
increase sedimentation, and decreased in water clarity. High nutrients, sedimentation, and poor
water clarity ultimately lead to devastation of the coral reefs. Corals contain symbiotic
zooxanthallae that provide nutrients and build calcium carbonate skeleton. Due to eutrophication
of Kāneʻohe Bay, a green bubble algae, Dictysosphaeria cavernosa, has killed many coral
colonies by decreasing the sunlight needed for coral’s zooxanthallae to photosynthesize. This in
turn led to coral bleaching (Pastorok & Bilyard, 1985). Studies have also shown that chlorophyll
concentration levels in Kāneʻohe Bay are currently 6.7 times higher the average of the central
and northwest sectors combined (Gulko, 1998).
Phytoremediation is the process of using plants to
remove pollutants from the environment such as nitrates,
phosphates, and heavy metals (USDA, 2000). The plants
used in phytoremediation act as filters or traps, where the
uptake contaminants occur in the roots. . The root
systems absorb and accumulate water and nutrients for
growth, as well as the uptake of the contaminants (Unep,
2012). There are four main types of phytoremediation
methods. 1) Phytoextraction, 2) Phytovolatization,
3) Phytostabilization, and 4) Phytodegration (Pivet,
2001). The most common type is phytoextraction. In
phytoextraction, plant roots absorb pollutants and
contaminants, but store the contaminants in aboveground
tissues and leaves. After sufficient plant growth and
accumulation, the aboveground portions of the plant are
harvested, removed, and disposed properly so pollutants
are not leached back to the environment. (Prasad and
Freitas, 2003).
Current studies and pilot programs in Hawaiʻi have
used ʻAkulikuli (Sesuvium portulacastrum) plants for
Figure 2: Phytoremediation in Ala
phytoextraction that absorb organic matter resulting in
Wai Canal, Oʻahu using ʻAkulikuli
an increase in water-clarity, decrease pollution, smell,
and balance out the habitat (Figure 2) (Leone, 2005). ʻAeʻAe (Bacopa monnieri) have also
been used in various phytoextraction experiments. B. monnieri is a fast growing wetland species
that has adapted to thrive in polluted areas that receive regular flushing of sewage waste and
industrial wastes (Hussain et. al, 2011). This plant has been recommended as an agent for
phytoremediation by Native Hawaiian plant botanist and horticulturalist Ricky Barboza
(Barboza, 2012). Barboza and Hui Kū Maoli Ola have planted several B. monnieri at the mouth
of Waimanalo River, which is also known to be one of the most polluted rivers on Oʻahu.
According to their results, the clarity of water flowing at the mouth of the river has increased and
2
the B. monnieri has thrived (Barboza, 2012). B. monnieri has also been detected as a
hyperaccumulator for toxic metals such as cadmium, chromium, lead, and mercury (McCutcheon
& Schnoor 2003). In 2010, further studies confirmed that B. monnieri has bioaccumulation
properties that detected the presence of Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn at various
levels using Atomic Absorption Spectrophotometer analyses. In addition, the same study
confirmed that xylem stem vessels and tissues of the plant contained stained deposits that
confirmed pollutants were absorbed by the plants (Hussain, 2010). In addition, further
experiments showed that B. monnieri was most effective in phytoremediation at low pH for Cd
and Hg (Hussain et. al, 2011).
In this experiment, B. monnieri (ʻAe ʻAe), an indigenous plant to Hawaiʻi, was used to
determine the effective changes of phosphates, nitrates, algae growth, turbidity, pH, and salinity
using phytoextraction of Kāneʻohe stream and Kāneʻohe Bay.
Research Question:
Which area of the Kāneʻohe Stream and Bay does ʻAe ʻAe (B. monnieri) most effective for
phytoremediation?
Hypothesis:
If ‘Ae ‘Ae (B. monnieri) were placed and grown in the heavily impacted housing area
(middle of the river), then the plant would be most effective for phytoremediation.
3
Materials and Methods:
A. Area of Study
The area chosen to study was Kāneʻohe Stream, Oʻahu. This area was chosen because it is
easily tracked from the top of the mountain all the way to the bottom, where it is released
into Kāneʻohe Bay. Four study sites and water samples were taken starting from
Ho’omaluhia gardens, second location Kāneʻohe Stream Bridge, Kāneʻohe stream mouth,
and Kāneʻohe Bay (Figure 3).
Figure 3: Study Sites
Site 1: Hoʻomaluhia Garden
Site 2: Kāneʻohe Stream,
Housing Area
Site 3: Kāneʻohe Stream,
River Mouth
Site 4: Kāneʻohe Stream,
River Mouth
4
B. Plant Used
The plant used in the experiment was
ʻAeʻAe (B. monnieri)(Figure 4), an
indigenous plant to Hawaiʻi, which were
obtained from Hui Kū Maoli Ola in
Kāneʻohe, Oʻahu. The plant roots were
removed from the soil and washed
thoroughly with tap water.
Figure 4: ʻAe ʻAe (B. monnieri)
C. Experimental Design
Water samples were taken from four study sites in Kāneʻohe Stream using a twogallon water jug that were labeled with each location at the same day and same time. Five
galloon (5 gallon) fish tanks were used and filled with 4700mL of water from each location
using a graduated cylinder. The fifth fish tank served as the control and was filled with
4700mL of distilled water. One hundred grams (100 grams) of ‘Ae ‘Ae (B. monnieri) (with
roots in tact) were placed in each fish tank. The roots were submerged in the water, but the
leaves were above the water (Figure 5). The experiment was placed in room temperature
environment with adequate amounts of sunlight. Water quality tests and absorbance of
chlorophyll were taken starting at day zero every other day for 10 days.
Figure 5: Experimental set-up of ʻAe ʻAe (B. monnieri)
5
D. Water Quality Testing
Various water quality tests were taken using Logger Pro Vernier Software for
Turbidity, Salinity, and pH. Aquarium test kits were used for Phosphates and Nitrate water
quality tests.
E. Absorbance
A colorimeter (logger pro) was used at 480 nanometers to determine the
absorbance amount of chlorophyll A for each water sample (Figure 6).
Figure 6: Vernier Colorimeter
6
Results
Absorbance (nm)
Nanometers (NM) Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
Control
Distilled Water
.002
.002
0.001 0.001 0.001 0.001
Site 1
Top of River
Site 2
Housing Area
.002
.002
0.001 0.001 0.030 0.036
.034
.034
0.033 0.035 0.044 0.045
Site 3
Mouth of River
.031
.032
0.032 0.001 0.002 0.039
Site 4
Bay Water
.040
.043
0.051 0.050 0.058 0.114
Summary: Absorbance increased for Site 1(top of the river) a total 0.034nm. Site 2
(absorbance increased a total of 0.014nm. Site 3 (river mouth) absorbance-increased
0.008nm. Site 4 (Kāne’ohe Bay) absorbance increased a total 0.071nm. Site 4 had greatest
absorbance change, showing that the amount of algae and chlorophyll increased the most
in Site 4.
7
Phosphate (PPM)
PPM
Control
Distilled Water
Site 1
Top of River
Site 2
Housing Area
Site 3
Mouth of River
Site 4
Bay Water
Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
0
0
0
0
0
0
0
0.2
0.2
0.1
0.1
0.1
0
0.2
0.1
0.1
0.2
0.2
0
0.1
0.2
0.5
0.5
0.5
0.1
0.1
1.0
1.0
2.0
2.0
Summary: Phosphate levels for Site 1 (top of river) & Site 2 (housing area) were in the
normal range. However in Site 3, the phosphate levels began to slightly increase, but still
normal. In Site 4 (Kāneʻohe Bay), the phosphate level increased from 0.1 ppm to 2.0 ppm,
where levels are harmful for marine life and correlates to increase of algae growth.
8
Nitrate (Mg/L)
Mg/L
Control
Distilled Water
Site 1
Top of River
Site 2
Housing Area
Site 3
Mouth of River
Site 4
Bay Water
Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
Summary: The nitrate levels did not change overtime in all sites. There is possibility of
experimental error or expiration of the nitrate test kit. A logger pro is needed in the future.
9
pH
Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
Control
Distilled Water
Site 1
Top of River
Site 2
Housing Area
Site 3
Mouth of River
Site 4 Bay Water
6.76
6.76
6.65
6.5
6
6
6.8
6
6.0
4
4.5
5
7.0
8.0
8.5
5.5
5.5
6
7.8
5.0
6.2
5
6.5
6.5
8.4
8.0
6.5
7.5
9
9
Summary: The pH of Site 1 (top of river) became more acidic over time. Site 2 (housing
area) became acidic over time as well. Site 3 (river mouth) had the most fluctuations of pH.
The pH decreased from neutral in Day 0 to acidic in Day 2-6, but in Day 6-8 was slightly
increased to slightly acidic and neutral. Site 4 (Kāneʻohe Bay) also fluctuated but the pH
was mostly basic over the 10-day period.
10
Salinity (PPT)
PPT
Control
Distilled Water
Site 1
Top of River
Site 2
Housing Area
Site 3
Mouth of River
Site 4
Bay Water
Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
0
0
0
0
0
0.1
0
0
0
0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
7.3
7.3
8.5
8.6
9.1
9.9
31.1
31.1
25.1
25
26.5
30.6
Summary: In Site 1 (top of river) and Site 2 (housing area), there were no salinity changes.
The salinity in Site 3 (river mouth) increased over time and Site 4 (Kāneʻohe Bay) salinity
decreased on Day 4 - 8, but on Day 10, it increased to 30.6 ppt.
11
Turbidity (NTU)
NTU
Control
Distilled Water
Site 1
Top of River
Site 2
Housing Area
Site 3
Mouth of River
Site 4 Bay Water
Day 0 Day 2 Day 4 Day 6 Day 8
Day 10
24.2
24.2
24.2
32.6
30.3
22.9
32.3
24.8
15.2
32.3
30.1
44.9
24.2
24.6
31.0
49.5
61
51.2
28.2
35.4
20.9
31.5
45.3
36.2
24.3
38.9
31.5
98
110.1 246.1
Summary: Site 1 (top of river) had a total change of 12.6 NTU. The total change of Site 2
(housing area) was 27.0 NTU. In Site 3 (river mouth), the difference in turbidity was 8.0
NTU, which was the least amount of change. Site 3 (river mouth) also had the clearest
water. Site 4 (Kāneʻohe Bay) had a change of 221.8 NTU, which was the greatest change in
turbidity. The water clarity was poor, murky and the plants did not survive in Site 4
(Kāneʻohe Bay).
12
Discussion
In this experiment phytoextraction (a type of phytoremediation) using Bacopa
monnieri (ʻAe ʻAe) was used to determine the effective changes of algae growth (absorbance)
phosphates, nitrates, turbidity, salinity, and pH of Kāneʻohe stream and Kāneʻohe Bay.
Results showed that Site 4 (Kāneʻohe Bay) absorbance increased a total of 0.071 nm, thus
having the most chlorophyll change in 10 days. On the other hand, Site 3 (River Mouth) had the
least increase at 0.008nm, thus having the least chlorophyll changes.
For phosphate, Site 1 (Top of River) & Site 2 (Housing Area) were in the normal
range. However, in Site 3 (River Mouth) the phosphate levels slightly increased, but still
normal. In Site 4 (Kāneʻohe Bay), phosphate levels increased from 0.1 ppm to 2.0 ppm
leading to dangerous levels for aquatic life. The high increase of phosphate directly
correlated to the increase of chlorophyll or absorbance because high phosphates increase
algae growth.
In the nitrate test, all four sites had the same level at 12.5 mg/L. This could be due to
experimental error or expiration of test kits. In the pH levels, B. monnieri was also found to work
best in slightly acidic conditions of ph 4-6.5, which referred to Site 1 (Top of River), Site 2
(Housing Area), and Site 3 (River Mouth). B. monnieri was not able to grow and thrive at basic
pH, which was Site 4 (Kāneʻohe Bay). Site 3 (River mouth) showed the clearest water, where
the turbidity levels total change was of 8.0 NTU. However in Site 4 (Kāneʻohe Bay), the
turbidity total change was 221.8 NTU and showed the greatest change. In Site 4 (Kāneʻohe
Bay), the water was murky, poor, and plants did not survive. Turbidity correlated to
absorbance of chlorophyll as well because the higher the turbidity the more likely algae
growth increased.
Overall, Site 3 (River Mouth) had the best results for phytoremediation of B.
monnieri, thus the hypothesis was not supported. Site 4 -Kāneʻohe Bay was not effective for
the B. monnieri to grow because Site 4 has a high salinity for ocean environments and basic pH,
which are components to ocean conditions. Based on background research, B. monnieri was
most effective in estuary ecosystems that require fresh or brackish environment and acidic
pH, which Site 3 provided.
Suggestions for future research include 1) to redo nitrate test using logger pro for
accuracy, 2) test various river mouth water samples in Oʻahu to reconfirm effectiveness of
B. monnieri, with repeated trials and to also ensure that the increase of absorbance is really due
to growth of algae not microorganisms on the water (cyanobacteria, diatoms, dinoflagellates,
etc), 3) detect leaf and stem samples of B. monnieri for signs of pollutants, and 4) test
B. monnieri for phytoextraction of heavy pollutant chemicals, and 5) determine proper and safe
ways of plant disposal if used in future trials. With such future research, it is the hope that this
environmental friendly and natural alternative of removing pollutants in Hawaiiʻs water
ecosystems can be effective and ensure coral reef ecosystems of Kāneʻohe Bay can be enjoyed
for future generations.
13
References
Art. "Eutrophication Definition Page." USGS Toxic Substances Hydrology
Program. 1993. Web. 26 Jan. 2012.
<http://toxics.usgs.gov/definitions/eutrophication.html>.
Barboza, Rick. "Phytoremediation with AeʻAe." Personal interview. 12 Jan.
2012.
Hanson, Roger B., and K. Gunderson. Influence of Sewage Discharge on Nitrogen
Fixation and Nitrogen Flux from Coral Reefs in Kaneohe Bay, Hawaii. 6th ed. Vol. 31.
American Society for Microbiology, 1976. Print. Honolulu News & Events. "KANEOHE BAY
SEWAGE SPILL, City and County of
Honolulu." City and County of Honolulu, Official Web Site for The City and County of
Honolulu. July 2002. Web. 25 Jan. 2012.
<http://www1.honolulu.gov/refs/csd/publiccom/honnews02/kbayspill2.htm>.
Hussain, K. Journal of Stress Physiology & Biochemistry. 3rd ed. Vol. 6.
2010. 91-101. Print.
Hussain, Abdussalam, Ratheesh Chandra, and Nabeesa Salim. Journal of Stress
Physiology & Biochemistry. 4th ed. Vol. 7. 2011. Print.
Leone, Diana. "Honolulu Star-Bulletin News /2005/05/08." Hawaii Archives –
Honolulu Star-Bulletin Archives - Starbulletin.com - Archives.starbulletin.com. 2005. Web. 25
Jan. 2012. <http://archives.starbulletin.com/2005/05/08/news/story3.html>.
McCutcheon & Schnoor 2003, Phytoremediation. New Jersey, John Wiley & Sons,
page 898.
Pastorok, Robert A., and Gordon R. Bilyard. Effects of Sewage Pollution on Coral-reef
Communities. 175-189 ed. Vol. 21. 1985. Print.
Pivetz, Bruce E. Phytoremediation of Contaminated Soil and Ground Water at
Hazardous Waste Sites. 2001. Print.
Prasad, Majeti, and Helena Freitas. "Metal Hyperaccumulation in Plants." Electric
Journal of Biotechnology. Nov. 2003. Web. 2012.
<http://www.ejbiotechnology.info/content/vol6/issue3/full/6/>.
UNEP. "What Is Phytoremediation." International Environmental Technology
Centre (IETC) – Homepage. Web. 25 Jan. 2012.
<http://www.unep.or.jp/ietc/publications/freshwater/fms2/1.asp>.
USDA. "Phytoremediation: Using Plants To Clean Up Soils." ARS: Home. June 2000.
Web. 25 Jan. 2012. <http://www.ars.usda.gov/is/ar/archive/jun00/soil0600.htm>.
14