Maribel Beltran-Cruz
Department of Biology, College of Arts and Sciences (CAS)
Krizzia Carmelle M. Bajio
Daniel Joacquin E. Bendal
Martin Neil C. Magat
Christian C. Ramos
Junbert D. Silva Jr.
B.S. Biology Program
College of Arts and Sciences (CAS)
How to Cite:
Beltran-Cruz, M., Bajio, K. C. M., Bendal, D. J. E., Magat, M. N. C., Ramos, C. C., & Silva, J. D., Jr. (2017). Cytotoxicity assessment of surface water in La Piñas–Parañaque Critical Habitat and Ecotourism Area (LPPCHEA) using Allium cepa test. NEU Knowledge Journal: A Compilation of Researches of New Era University Faculty, Staff, Students, and Administrators, 1(1), 32–43. https://doi.org/10.64303/neu-urc-kj2017-002CyAsSuWaLa
ABSTRACT
A study was conducted to assess the quality of surface waters in the Las-Piñas-Parañaque Critical Habitat and Ecotourism (LPPCHEA) area, a 175-hectare wetland ecosystem that serves as a sanctuary or haven of many endangered and threatened species of migratory birds and considered as a last mangrove frontier in Metro Manila. Water samples were collected from five sites (Pond 1, Parañaque River, North Lagoon, Las Piñas River, and South Lagoon) and evaluated including a control (tap water) using the Allium cepa test. Root number, root length (shortest and longest), and mitotic index of 120 onion bulbs (Allium cepa var. aggregatum) sample (based on 80% power, medium to large effect size, 5% significance level, and six groups ANOVA test) were assessed under controlled conditions. One-way ANOVA was used to test hypothesis of no treatment effect, with Bonferroni correction applied, and Duncan Multiple Range Test was done following significant ANOVA test.
No statistically significant difference was found for the mean number of roots per site/treatment. Statistically significant differences were found for mean shortest root, mean longest root, and mean mitotic index. Results indicated that water samples taken from Las Piñas River and South Lagoon (adjacent sites) were the most polluted.
Keywords: Cytotoxicity, surface water, Allium cepa test, mitotic index
INTRODUCTION
During the recent years, there has been an increasing awareness and concern of the ecologically-important wetland ecosystem being deteriorated around the world. Production of wastes (solid, liquid, or gaseous form) has increased exponentially as a result of rapid population growth and progressive economic activities brought about by globalization and urbanization trends. In addition, rapid population growth, progressive industrialization, and anthropogenic and biophysical factors put tremendous pressure on limited usable water resources due to increased pressure on urban hydrology (Dabgerwal, 2015).
Worse yet, surface waters and wetlands have been viewed by some people as convenient dumping sites for human and industrial and agricultural wastes (Singh et al., 2004; Abdul et al., 2014; and Alkharki, 2008). According to Abdul et al. (2014), surface waters are the most vulnerable of the degradation and pollution due to their easy accessibility for disposal of wastes.
Water pollution has been a serious problem worldwide because, among other things, polluted waters may contain hazardous substances that have mutagenic and carcinogenic effects to terrestrial and aquatic life (Tedesco et al., 2012). Many genotoxicity and mutagenicity tests for polluted water have been developed, one of which is Allium cepa test (Egito, Medeiros, & De Medeiros, 2007). The test is widely known for evaluating environmental pollutants, detecting mutagens in different environments, and evaluating many genetic endpoints.
Using the A. cepa test, Pathiratne, Hemachandra, and De Silva (2015) found that water samples collected from Kelani River in Sri Lanka statistically significantly retarded root growth and induced cytotoxicity and genotoxicity. Fiskesjo (1985) found that polluted water not only retarded root growth but also made roots less turgid or stiff, adversely affecting plant growth and development.
The Las Piñas-Parañaque Critical Habitat and Ecotourism Area (LPPCHEA) is one of the wetlands in the country which has caught environmentalists’ attention. The area is a 175-hectare wetland ecosystem and nature reserve. It consists of two islands called the Freedom and Long Island. Situated south of Manila Bay, LPPCHEA is considered as a last mangrove frontier in Metro Manila. It was recognized by the Ramsar Convention as a wetland of international importance because of the significant role it plays in the survival of threatened, restricted-range and congregatory bird species. It also serves as an important resting and refueling point for migratory birds using the East Asian Australasian Migratory Flyway. LPPCHEA hosts around 41 species of migratory birds in the area, with some coming from as far as China, Japan and Siberia (Fuentes & Bajarias, 2015). Because of its critical location in the metropolis, LPPCHEA has always been prone to the abuses of polluters for many years. This study was conducted to assess the status of surface water quality at the LPPCHEA.
METHODOLOGY
The study was done to assess the root macroscopic and microscopic properties of Allium cepa cultured in waters collected from five different water locations at LPPCHEA, namely: Pond 1, Parañaque River, North Lagoon, Las Piñas River, and South Lagoon.
Sample Size and Collection
One hundred twenty (120) Onion (Allium cepa var. aggregatum) bulbs (based on 80% power, medium to large effect size, 5% significance level, and six groups ANOVA test) were used for Allium cepa test. Twenty set-ups of Allium cepa test were used in testing water samples taken from each of the five locations in the littoral zone of LPPCHEA (Table 1).
Water samples were collected (during normal operating hours) 15 to 30 cm below the surface water or halfway to the bottom of the reservoir. The area for water sampling were not frequented by bathers at the time of sampling (Centre d’expertise en analyse environnementale du Québec, 2009).
Selection of the Biological Test Material
Onion (Allium cepa var. aggregatum) bulbs used in the experiment was selected according to size and quality. They were bought from Farmer’s Market, Cubao, Quezon City. The onion bulbs were identified and certified as Allium cepa var. aggregatum by the Department of Botany, National Museum, Manila. Only bulbs without shoot and root outgrowth and fungus infection were used.
Onion Preparation
Onion (Allium cepa var. aggregatum) bulbs of the same size were stripped and scraped to prime outer scale. During cleansing, the onion bulbs were put in distilled water at the constant room temperature to protect the root primordia from dehydration (Ukaegbu & Odeigah, 2009).
Allium cepa test set-ups were kept away from direct contact of sunlight. The onion bulbs were placed in the experimental water containers. Initially, root growth was started in tap water. When the roots reached 1-2 cm long, different water samples were successively applied and performed at the certain time intervals: 24 hrs., 48 hrs., and 72 hrs. In each 24-hour interval, sample of water solution was taken. Subsequently, after 72 hours, the onion bulbs were removed from experimental water containers (Firbas & Amon, 2013) and macroscopic morphological structures of tissue were further investigated.
Fixation of Onion Root Tips
Onion root tips per sample sites were harvested and cut into about 0.5 to 1.5 mm. Fixation was done at 12:00 noon in a 3:1 acetic alcohol (3 parts ethanol: 1 part glacial acetic acid) in order to stabilize the structures and prevent chemical and structural changes whilst the material was undergoing staining and mounting. Afterwards, root tips were stored in frigo for 24 hours.
Squash Preparation for Microscopic Examination
After fixation, root tips were hydrolyzed in 1N HCL at 60oC for one minute. Afterwards, they were transferred to the watch glass containing aceto-orcein and 1N HCL (9:1). They were heated interminably for 5 to 10 minutes. Root tips were cut and placed on a glass slide. Then, 45% glacial acetic acid was added. Root tips were macerated using a flat-ended glass rod. Before the cover slip was placed, a drop of 0.2% aceto-orcein was added. Large cell clumps were macerated by tapping the slip with the end of a pencil. The slide were blotted and passed through a low flame for a second using an alcohol lamp (Salvador, 2007).
The slides were observed and scanned at low power magnification and selected cells were analyzed at higher power magnification using Optika Microscope B-380 Series. After the observation, the sides of the cover slip were sealed with nail polish (semi-permanent mount).
Macroscopic and Microscopic Parameters
After three days of cultivation, onion bulbs were assessed on the following root parameters:
Root number or count, root length (shortest and longest), and cell mitotic index (number of dividing cell/total number of cells). Mitotic index indicates cell proliferation or reproductive capability. For mitotic index, onion tips were scored on 5000 cells (500 cells per slide).
Statistical Analysis
One-way ANOVA was used to test the hypothesis that there is no treatment effect across the six treatment groups in terms of root number, shortest root, longest root, and mitotic index. Duncan Multiple Range Test (DMRT) was performed following significant ANOVA test.
RESULTS AND DISCUSSION
Root Number
No statistically significant difference was found for the mean number of roots per site/treatment, F(5,114)=0.75, p=0.588(Table 2).
Root Length
Shortest Root. The shortest roots of bulbs grown from the six treatments differed statistically significantly,F(5,114)=11.97, p<0.001 (Table 3). DMRT indicated that bulbs cultured in water sample from North Lagoon had the least mean shortest roots (1.40 mm) (Table 4). This mean value was not statistically significantly different from those of Las Piñas River (2.35 mm) and South Lagoon (3.10 mm). This finding indicates that the waters in the North Lagoon, Las Piñas, and South Lagoon areas contain pollutants which have the most adverse effect on the development of plant root system.
Longest Root. Means of longest roots of bulb cultured from the six water samples are statistically significantly different, F(5,114)=130.19, p<0.001 (Table 5). DMRT shows that the mean of longest roots from water collected from Parañaque River (49.50 mm) was the greatest. However, this mean value was not statistically significantly different from that of Pond 1 (47.10 mm) and the control (48.25 mm) (Table 6). Although, this finding may suggest that waters in the Parañaque River and Pond 1 have the least adverse effect on plant root system, root tips showed tendency of malformation such as bending and swelling.
Mitotic Index
ANOVA test revealed that waters from the five test sites including the control induced statistically significant differential mean mitotic index, F(5,114)=37.03, p<0.001 (Table 7). Apparently, mean mitotic index of bulbs grown in tap water (control) (7.01) was statistically significantly greater than those grown in the five water samples from LPPCHEA; Las Piñas River showed the least (4.16).
Because decrease in the mitotic index signifies abnormality in mitotic or cell division, waters in the five test sites seem to have a detrimental effect on cell reproduction and growth. Mean mitotic indexes for South Lagoon, Pond 1, and North lagoon were found not statistically significantly different (Tabl0e 8).
CONCLUSION
Allium cepa test on shortest root, longest root, and mitotic index indicates that surface water samples from Las Piñas River and South Lagoon (adjacent sites) are the most polluted.
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