Special Experiment ANALYSIS OF WATER FROM BURNHAM LAKE USING MACROSCOPIC AND MICROSCOPIC PARAMETERS IN STANDARD ALLIUM TEST

Atienza, Joseph Alfonze, 1Carino, Jessa, 1Chammag, Clarisse, 1Dispo, Rabbiah Department of Biology, College of Science, University of the Philippines Baguio, Baguio City, Philippines, 26001 ABSTRACT The Allium test is a standard procedure in detecting the presence of mutagens in a given water sample. It is also used in the determination of pollution by identifying the effects of the sample to the genetic material. This can be done by the observation of onion root tips grown in the water sample. Some identified effects of the water collected from Burnham Park to the chromosomes of the onion roots were disturbed anaphase and the presence of sticky chromosomes. Macroscopically, the roots tended to present some malformations such as crochet hooks and some broken root tips which can be attributed to the toxicity present due to heavy metals and chemicals in the water. This experiment aimed to identify the level of genotoxicity of the water sample. Also, it aimed to familiarize the students with the standard Allium test procedure. INTRODUCTION Allium test is an experimental procedure that establishes the genotoxicity of ecosystems caused by chemical contaminations. This is accomplished by taking into account the cytological effects of a genotoxic substance to genetic material such as chromatin and chromosomes [1]. These effects include chromosomal aberrations that are recognized in the root tip cells of the onion or Allium cepa whenever they are exposed to contaminated water. Macroscopic observations such as root length of the onion are also employed for the analysis of this test. Allium test is important in assessing or monitoring the biological conditions of water. It is usually done to test the qualities of natural waters (e.g. lakes and rivers) drinking water, surface stagnant and flowing waters and waters from communal waste and drainages [1]. Moreover, this test is easily conducted with readily available and inexpensive equipment which is why it is commonly adapted by all laboratories. Results from this experiment can be quickly obtained and extrapolated to human cell systems [2]. This experiment aims to familiarize students with the basic procedures involved in the Allium test and also evaluate the toxicity of collected water samples by conducting microscopic and macroscopic observations of the onion bulbs. MATERIALS AND METHODS Different reagents were utilized in this experiment. Among these are the ethylenediaminetetraacetic acid (EDTA) solution and different water samples that were used as test liquids. Furthermore, reagents such as farmers solution, HCl and aceto-orcein were used for staining the samples for microscopic observations. Other materials used were test tubes, test tube racks, beakers, glass slides and droppers.
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Test Organism Small onion bulbs of equal size of about 1.5-2.0 cm in diameter were collected. Seven bulbs were utilized per test liquid. The bulbs chosen were made sure that shooting green leaves have not yet grown on them and that dried and mold-attacked bulbs were discarded. Procedure Three set-ups were prepared for this experiment, one was the positive control which was the EDTA solution and another was a negative control which contained tap water. The third one was water collected from Burnham Lake. Onion bulbs were peeled before placing them each on test tubes filled with the test liquids. Seven test tubes were employed per setup. The ring of the root primordial should be in direct contact with the water. The experiment was performed under certain conditions. Room temperature was observed and the bulbs were placed where it is not directly exposed to sunlight. Accordingly, the test liquids were also in room temperature and these were changed every day for five days. On the third day, microscopic slides of the root tips were prepared in accordance with the standard procedure for orcein staining of squashed material. Two bulbs from each of the three set-ups were utilized and observed for chromosomal aberrations. On the sixth day, macroscopic observations were accomplished according to these parameters: Root Length, Root Form and Shoot Length. All observations were recorded and photographed for documentation. RESULTS AND DISCUSSION Pollutants such as pesticides products and other chemicals contribute an inhibitory effect on the growth of the plant roots and causes chromosomal and chromatid aberrations

in the cell. Allium test is important for this by testing water samples. Its use was recommended as early as in the 1970s by the Royal Swedish Academy of Science (1973) and later by the GENE- TOX PROGRAM [3]. Compared with the other tests, this does not require preliminary processing of water samples and at the same time it is used for establishing general toxicity and genotocity. Allium test also shows an excellent correlation with tests where research on fish and mammals in vivo is carried out. Also this test is helpful in monitoring and supervising the burden of poisonous substances in the environment. Three water samples were collected from different sources one from Burnham Park, one sample with EDTA and another tap water. Onion roots were exposed to these solutions and macroscopic and microscopic testing was first done. Microscopic Effects Two of the seven samples in each setup were examined under the microscope. After preparing the root tips on the slides, chromosomal aberrations were observed. Onion root tips subjected to the distilled water eventually results to no chromosomal aberration. EDTA is primarily used as a chelating agent. It can readily bind with metal ions such as calcium and iron. In the experiment, the onion bulbs exposed to EDTA are expected to show the presence of some chromosomal aberrations in the microscopic observation of their root tips since it is considered a good positive control. Calcium, in plants, is an essential nutrient required in the formation of spindle fiber in the cell which is important to the cell division process. Because EDTA binds with calcium ions, the supply of calcium in the cell for the formation of such structures decrease, thereby resulting to disorders in the mitotic division especially during anaphase when these structures

are most significantly needed. Also, calcium plays a role in cell wall rigidity where it binds with pectin to form calcium pectate molecules and finally, in membrane stability as it acts as a binder of phospholipids. Since the nuclear membrane is also composed of a lipid bilayer, it follows that decrease in calcium concentration also affects it. Chromosomal aberrations observed in the EDTA were Multipolar anaphase, laggard and binucleated. Binucleated cells (Figure 1) are commonly found at cell undergoing interphase [4]. Because of this, cells are arrested at the interphase and therefore decrease in the growth due to the inhibition of entering to the mitosis happens. Onion root tips which are exposed to the water sample taken from Burnham Park exhibited three chromosomal aberrations (Table 1). Disturbed anaphase stage with chromosomal break and laggard (Figure 2) and sticky chromosome at the anaphase stage with bridge which is shown Figure 3 were the chromosomal aberrations observed in the sample. Stickiness is induced by the effect of herbicides on chromosomal protein attributed to the improper folding of chromosome fibers or may be due to the action of herbicides on the polymerization process, resulting in the fragmentation of chromosomes and bridges forms sticky chromosome [5]. With this, probable cause of aberration may due to the presence of herbicides on the sample. The occurrence of chromosomal aberrations results in certain growth restrictions. The presence of bridges at the anaphase indicates a mutagenic event in the cell and also sticky chromosomes indicate that the chromosomes were poisoned. Macroscopic Effects For the macroscopic analysis of this test, five remaining bulbs were observed according to the following parameters: root form (twists, swellings, broken root tips, color, turgidity), root

length, and shoot length. The roots of the bulbs in the negative control (Figure 4) did not exhibit any root malformations along their length nor broken root tips. In terms of turgidity, the roots of these bulbs were the most turgid. Since these roots were placed in a hypotonic solution (distilled water), the concentration of solutes within the roots and its cells is greater compared to that of the external environment. Following osmosis, water will move into the roots making the cells and the roots charged with water and swollen an indication of the increase in turgor pressure within. The roots are then said to be in a turgid state. There were no distinct differences observed in the color of the roots from the three set-ups. The roots of the bulbs from the positive control (Figure 5) did, however, exhibit root malformations along their length and some with broken root tips. The root malformations observed were twists such as crochet hooks wherein the root tips were bent upward resembling hooks and c-tumors appearing as swellings in the root tips. The presence of crochet hooks were possibly induced by the presence of heavy metals [6] in the solution since EDTA was used and it has the ability to form strong complexes with Mn(II), Cu(II), Fe(III), Pb (II) and Co(III) [7]. The c-tumors may be caused by several compounds including metal chloride complexes formed when EDTA sequesters the calcium. Both malformations are inducible by agents that cause c-mitosis, an abortive type of nuclear division. The roots of these bulbs were also observed to be flaccid. In the hypertonic solution (EDTA), the concentration of solutes within the roots and its cells were much less as compared to the external environment where solutes and complexes were abundant. As a result, following osmosis, water moved out from the roots leaving the cell protoplasts plasmolyzed and the entire organ flaccid. The roots of the bulbs from the water sample (Figure 6) exhibited some twists (crochet hooks) as well, although only slightly bent as

compared to those of the positive control. There were also fewer broken tips and no c-tumors observed. These root malformations may be due to the presence of some heavy metals in the water sample from Burnham lake, attributed to herbicides and other dumped chemicals. In terms of turgidity, the roots from this water sample were still turgid, meaning the solution was neither hypertonic nor too hypotonic relative to the internal system of the roots. Table 2 shows the mean root lengths for each bulb from the three set-ups. Based on the results, increase in root length in the negative control was observed and the mean root length of the bulbs measured is 23.5 mm. Growth in the roots of bulbs in the positive control exhibits a degree of retardation since the mean root length of the bulbs measured is 13.2 mm. Increase in root length of the bulbs grown in the water sample was also observed and the mean root length measured is at 22.3 mm. Statistical analysis of the data, particularly t-test, showed that there is no significant difference (p>0.05) between the root lengths of the bulbs grown in the water sample and in distilled water (negative control). Furthermore, there is a significant difference (p<0.05) between the root lengths of the bulbs grown in the water sample and in the EDTA solution (positive control). This indicates that the water collected from Burnham Lake is not as polluted so as to result to the effects observed in the positive control. Table 3 shows the mean shoot lengths for each bulb from the three set-ups. No increase in shoot length was observed for the bulbs grown in the positive control and water sample. The mean shoot length measured for the bulbs in the negative control is 1.3 mm. CONCLUSION Microscopic observations by applying orcein staining to the onion root tips of the onion

bulbs showed that the water sample collected from Burnham Park can be considered polluted with genotoxic substances that can cause disturbances in the mitotic division of cells such as sticky chromosomes and chromosomal breaks. Observation of such in the slides and macroscopic observations of twists or crochet hooks and broken root tips of the roots were attributed to the hypothesis made that the water in Burnham Lake is contaminated with herbicides that commonly cause such aberrations and root malformations but not to the same degree as those observed in the EDTA solution. The objectives of the experiment were attained in that the researchers were able to observe the presence of chromosomal aberrations in the water sample which concludes that Burnham Lake is polluted with genotoxic substances. In the analysis of the onion root tips, it is important to remember to take note of as much data as possible to be able to qualify the data for more statistic parameters especially in the macroscopic parameters such as root length, turgidity, root color, and the presence of hooks or twists. CONFLICT OF INTEREST There are no conflicts of interest. CONTRIBUTION AUTHOR OF INDIVIDUAL

Everyone from the group participated and contributed in the experiment as well as in the construction of the laboratory report. Alfonze authored the abstract, conclusion, conflict of interest, contribution of individual author and he was also in charge of the compilation of the report. Jessa and Rabbiah authored the results and discussion part of the paper while Clarisses parts included the introduction and materials and methods.

REFERENCES [1] Peter Firbas, 2011. Use of genotoxicity ALLIUM anaphasetelophasegenotoxicity assay with common onion (Allium cepa L.) grown in water samples. 25.6 28.6. Private Laboratory of Plant Cytogenetics.

[7]

Holleman, A. F. and Wiberg, E. 2001. Inorganic Chemistry. San Diego: Academic Press.

APPENDIX Table 1. Chromosomal aberrations observed in the negative control, positive control, and water sample. Water Samples Distilled water EDTA Water sample from Burhnham Park Aberrations observed None Multipolar anaphase, Laggard, Bipolar Disturbed anaphase stage with chromosomal break and laggard, sticky chromosome at the anaphase stage with bridge

[2]

Kendler, B. S &Koritz, H. G. 1990. Using the Allium Test to Detect Environmental Pollutants. 52: 372-375. The American Biology Teacher. https://www.google.com.ph/url?sa=t&r t=j&q=&esrc=s&source=web&cd=3& ed=0CDcQFjAC&url=http%3A%2F% Fwww.flaska.si%2Ffiles%2Foriginal% F756.pdf&ei=CTIGU9y6OM UiQeMnICgDg&usg=AFQjCNHYjaX rx4kRzlopIGJEFIHl0n yA&sig2=Yb2LMj8lwgQ4dYYOrVV fA. Retrieved on February 25,2014. Iwalokun, B.A..et,al .2011. Analyses of Cytotoxic and Genotoxic Potentials of Loranthus micranthus using the Allium cepa Test. Current Research Journal of Biological Sciences, 3(5). Sharma, Sonia and Vig, Adarsh Pal. 2012. Genotoxicity of Atrazine, Avenoxan, Diuran and Quizalofop-P ethyl Herbicides iusing the Allium cepa Root Chromosomal Aberration Assay. Terrestrial and Aquatic Environmental Toxicology: Global Science Books. Odeigah, P.G.C, J. Makinwa, B. Lawal and R. Oyeniyi. 1997. Genotoxicity screening of leachates from solid industrial waste evaluated Allium test. ATLA. 25:311-321.

[3]

[4]

[5]

Figure 1. Binucleated cell observed on the onion root tip subjected on the EDTA solution.

[6]

Figure 2. Disturbed anaphase stage with chromosomal break and laggard observed at the onion root tip subjected on the water sample from Burnham Park.

Figure 5. Macroscopic effects on Allium cepa cultivated in the positive control, EDTA solution.

Figure 3. Sticky chromosome at the anaphase stage with bridge observed at the onion root tip subjected on the water sample from Burnham Park.

Figure 6. Macroscopic effects on Allium cepa cultivated in the water sample from Burnham Park. Table 2. Root length of Allium cepa after cultivation in three different set-ups. Negative Control 22 mm 35 mm 25 mm 21 mm 14.5 mm 23.5 mm 2.3 0.1698 Positive Control 11.5 mm 12 mm 12 mm 19 mm 11.5 mm 13.2 mm 1.0 0.0027 Water Sample 33.5 mm 31 mm 25 mm 22 mm 0 mm 22.3 mm 4.0

Figure 4. Macroscopic effects on Allium cepa cultivated in the negative control, distilled water.

Bulb 1 Bulb 2 Bulb 3 Bulb 4 Bulb 5 Mean root length SE p-values from t-test
(+/- vs water sample)

Table 3. Shoot length of Allium cepa after cultivation in three different set-ups. Negative Control 1 mm 2 mm 2.5 mm 1 mm 0 mm 1.3 mm 0.3 Positive Control 0 mm 0 mm 0 mm 0 mm 0 mm 0 mm Water Sample 0 mm 0 mm 0 mm 0 mm 0 mm 0 mm

Bulb 1 Bulb 2 Bulb 3 Bulb 4 Bulb 5 Mean shoot length SE