New methods for detecting heavy metals in mining sites developed
By: Emelina G. Regis, Ph.D., Leo Barrameda, and Mary Ann Montaňez
Revised May 10, 2006



The methods described below are based on the findings of the study of Dela Cruz (2002) on the effects of mercury to the structure of chloroplasts. In her study, Dela Cruz was able to show through electronmicroscopy that the inner structure of chloroplasts is destroyed by mercury pollution. Likewise, Kovacs (1992) revealed that other heavy metals such as lead (Pb) and cadmium (Cd) reduce the chlorophyll content and photosynthetic efficiency of the chloroplast.

The chloroplast

Chloroplast is an organelle in the leaves of plants capable of producing food when carbon dioxide and water are absorbed by leaves and roots respectively. This process is known as photosynthesis and the end-product is a carbohydrate such as starch and oxygen, a byproduct. Inside the chloroplast, the starch grains are transported to the phloem, one of the vascular tissues of the leaves that carry food through the stem to the roots of plants. Diagrams of the leaf, leaf cross-section and chloroplast are presented in Figures 1, 1a, and 1b.

The specific site of photosynthesis is the granum (plural: grana) which is comprised of stacks of thylakoids that capture sunlight and use its energy to manufacture food such as starch in the leaves of plants. The starch is stored in the roots and accessed only for growth and respiration and other metabolic functions. The destruction of the thylakoid is similar to the destruction of a machine that does the manufacturing. Thus, no production of food (starch in this case) takes place, resulting in stunted growth or death of affected plants.

In the presence of heavy metals at concentration above the critical level for plants, mercury for instance destroys the chloroplasts (Dela Cruz, 2003). Thus, it can be predicted that the capability of the leaves to produce food is prevented when these metals are present, because no starch grain could be detected in some plants growing in metal-contaminated soil.

In mining sites, heavy metal pollution is one of the most critical problems because it releases heavy metals to the surroundings. The presence of these metals, i.e., mercury, cadmium, lead, and arsenic among others, can be detected by Bioindicators through changes in the structure and physiology of living organisms. In the case of plants, this is manifested through abortion of pollen grains (Regis and Lagunzad, 2002; Maranan and Valisto, 2004), decrease in growth rate with corresponding reduction in size (Kovacs, 1992; Regis, 1999), and changes in leaf ultrastructure (Dela Cruz, 2003) with consequent impact on physiology of photosynthesis.

Objectives of the study

Detecting heavy metals in the environment through chemical analysis is expensive. It is also problematic since collection of samples must be rigorously done following procedures that ensures no errors in the interpretation of results are committed. However, mining activities that release heavy metals into the environment happen in remote places where most people are poor and access to laboratory services are not available. Nevertheless, adjacent to these areas may be schools that have the most basic equipment, the compound microscope. Thus, the development of a cheap and simple method for detecting heavy metal pollution in the environment may be possible so that monitoring by local communities can elicit immediate response from the concerned government agencies.

In this study, the primary objective is to devise a cheap method for determining heavy metal pollution in the environment. Moreover, the study also aims to look for other possible bioindicator of heavy metal contamination in polluted sites using the abovementioned method.

The Methods

Two methods have been developed by INECAR through experimentation: a) aquatic bioindicator, Hydrilla verticillata (L.fil.) Royle (Cook et al., 1974), and b) terrestrial bioindicator using two species of grass: Cyperus kyllingia Endl. and Digitaria ciliaris (Retz.) Koeler. (FAO,

1. Hydrilla verticillata plants as bioindicator of mercury contamination*

H. verticillata plants (Figure 2) were raised in improvised aquaria using lakewater collected from Katugday Lakelet, Buhi, Camarines Sur. Prior to planting, sediments having mercury level at 12 mg/kg, taken from a contaminated area in Jose Panganiban were added to the lakewater in 8 set-ups. The same number was prepared for the control set-ups but the soil added was taken from a coconut land where no mining has been conducted. Aerators were also connected to the aquaria to provide oxygen, air circulation and prevention of natural acidification as occurring in stagnant waters. A total of 32 plants were planted in the treated set-ups. Likewise, the same number of plants was also planted in the control set-ups. Hydrilla plant was chosen for this study because its chloroplast is relatively large and can easily be seen under low power objective (LPO) of the compound microscope.

After 2 weeks, leaves from the 3rd upper leaf whorl of the plants were obtained and placed in a fixative (ethanol-acetic acid) which is comprised of 3 parts absolute alcohol and 1 part glacial acetic acid. After 24 hours, the fixative is replaced with 70% ethyl alcohol. This method is based on Micieta and Murin (1996).

Examination of the leaf

The sample leaves must be washed first in tap water prior to staining. Each leaf is placed on a glass slide. Then, the preparation of the leaf and subsequent examination used the method devised by Regis, the main researcher. Since chloroplast are biochemically similar to bacteria (Moore, et al., 1995), a small drop of Lugol solution to the preserved leaf (whole blade) is added. The leaf is then examined by the researchers under the compound microscope at low power objective (LPO) and at high power objective (HPO). Cells in the control set-ups showed starch grains (stained blue-black or black) inside the chloroplasts. On the other hand, cells of leaves from the treated plants did not show the starch grains; only the form of the chloroplast structure was visible (Figures 2a and 2b)

In determining the extent of contamination, Barrameda and Montaňez used a counting procedure that combines Murin’s 100 cell count (email communication in 1998) and Regis’ method of range grouping (Regis, 2004). Here, the counting of normal and affected chloroplast was done in 100 cells only as suggested by Murin. Groupings of the counted normal chloroplasts was performed to establish the extent of pollution.

2. Cyperus kyllingia and Digitaria ciliaris as bioindicators of heavy metal pollution in mining sites **

Several tests on various weeds were done between December, 2005 and January, 2006 using the method of fixation and preservation by Micieta and Murin (1996). A drop of Lugol solution was added to show the presence of starch. Lugol solution is used in this study because the chloroplast is biochemically similar to bacteria (Moore, et al., 1995) wherein cyanobacteria also responds to Lugol. Other stains applied by the researcher did not show the changes that happened in the cells. However, the minimum fixation time was reduced to 12 hours and examination of cells after preservation could be done after only 6 hours.

Prior to experimentation, several weeds were collected from various sources, i.e., weedy fields, ricefields using pesticides, solid waste dumpsite in Naga City, and coastal areas of Pasacao and Calabanga. The samples were fixed in ethanol acetic acid (3:1 v/v) and preserved in 70% ethyl alcohol (70% ethyl alcohol Casino brand can also be used). If Lugol solution is not available, iodine solution obtainable from drugstores may be used.

Each leaf sample is washed in tap water, then placed on a glass slide with its underside on top. This is because the upper epidermis is coated with waxy covering thereby obstructing the view of the inside of the leaf, while the lower epidermis has thinner coating. Then, about 1 – 2 cm of the tip of the leaf blade is removed depending upon the size of the leaf: 1 cm for small/short leaves; 2 cm for long leaves. A small drop of water is also added to the leaf section after which, about 1-2 drops of Lugol solution is added depending also on the size of the cut sample: 1drop for small/thin sample; 2 drops for large/wide sample.

Examination of the samples showed presence of starch grains in all except in those obtained from the solid waste dumpsite in Naga City where a lot of plastics are piled up. At the time of the sampling, watery effluent flowed out from a huge waste dump towards an area where few weeds grow. This was the sampling site used in collecting grasses for the initial investigation.

To validate the study, the researcher raised healthy grasses in contaminated soil taken from a gold mining area in Camarines Norte. Here, the soil used was taken from the same bundle of soil used by Barrameda and Montaňez in the first study described above. A control was also set up using soil obtained from a relatively healthy area in Pacol, Naga City. After 1 week, the 3rd leaf from each plant is removed and placed on a fixative. Harvesting of leaves was done only after 4 PM for Digitaria ciliaris (Figure 3) and after 10:30 AM for Cyperus kyllingia to ensure that starch are already produced.

The examination of the leaves of the sample grasses was done in the spaces between the parallel veins. The veins are not the area for determining contamination. It is the the spaces between the veins because chloroplasts with their starch grains could be seen in the mesophyll layer. This is also the area where quantitative examination and determination of the presence/absence of starch grains should be done. In this experiment, the chloroplasts of the treated grasses showed absence of starch grains whereas in the control set-up, starch grains were present.

Quantitative measurement of the extent of contamination considered the percent coverage of normal chloroplasts with black or dark gray starch grains using the microscopic field as the round grid. Percentage coverage used the following measurements: 0%, <10%, 25%, 50%, 75% and 100%. 30 grids were assessed at random but moving along the stratified layers of the leaf. This method although random, prevents the same grid from being included twice or even a number of times in the analysis.

The above method was also tried in the Lafayette Mine in Rapu-Rapu, the same results were also obtained: absence of starch grains in contaminated site and presence of starch grains in uncontaminated site (outside the path of flow of the contaminants). Figures 3a and 3b present the results.

The Researchers:

* 1. The procedure has been developed in November-December, 2005 by two Biology majors (Barrameda, Leo and Montaňez, Mary Ann) doing their thesis in consultation with one of their advisers, Dr. Emelina G. Regis who guided them through the step by step procedure. Their thesis is entitled “Hydrilla (Hydrilla verticillata) as bioindicator and phytoremediator of mercury- contaminated sediment in lake” (Barrameda and Montaňez, 2006).

** 2. The above procedure was devised and performed by Emelina G. Regis, Ph.D. between December, 2005 and February 2006 at the INECAR greenhouse, Ateneo de Naga Campus. She was assisted by Mr. Edward Kenn Ampongan, Ms. Aireen Melody Bigay, Ms Juvylin Bismonte, and Mr. Kelly Fraginal; all are INECAR staff.


Literature cited:

Barrameda, Leo and Montaňez, Mary Ann (2006) Hydrilla as indicator of mercury contamination. Undergraduate thesis for the degree of B.S. Biology, Department of Math and Sciences, Ateneo de Naga University, Naga City. Adviser: Dr. Emelina G. Regis. Co-Adviser: Engr. Irma Medrano.

Cook, CDK, B.J.Gut, E.M.Rix, J.Schneller, and M.Seitz. 1974. Water Plants of the World: a manual for the identification of the genera of freshwater macrophytes. Dr. W. Junk b.v. Publishers, The Hague. pp. 259-260

Dela Cruz, Fritzie. 2002. Effects of Bioconcentrated Mercury in Chloroplasts Ultrastructure and Chlorophyll Profile of Rice Plants from a Contaminated Gold Mining Area. Gibon, Journal of Ateneo de Naga University. Vol II No. 2 pp. 17-35

FAO. Digitaria ciliaris (Retz.) Koeler.

Kovacs, M., J. Podani, Z. Tuba, G. Turcsanyi, and L.D. Meenks. 1992. Biological Indicators in Environmental Protection. Ellis Horwood Ltd. Chichester, West Sussex. PO19 1EB, England.

Micieta, K. and G. Murin. 1996. Microspore Analysis for Genotoxicity of a Polluted Environment. Environ. And Exp. Bot. Vol 36 No. 1 pp. 21-27.

Moore, R. W.D. Clark, K.R. Stern and D. Vodopich. 1995. Botany. WmC. Brown Publisher. Kerper Boulevard, Dubuque, IA 52001 USA.

Regis, Emelina. 2005. Assessment of the Effects of Acid Mine Drainage on Mogpog River Ecosystem, Philippines, and Possible Impacts on Human Communities. A research funded by Oxfam Australia for the people of Mogpog, Marinduque.

Regis and Lagunzad, 2002. Comparison of Pollen Grain Abortiveness in Four Weed Species Treated with Mercuric Chloride. Science Diliman, Vol. 14 No.1. pp. 21-27.

Regis, Emelina. 1999. Pollen grain abortion as indicator of mercury pollution near gold mining sites in Camarines Norte. Doctoral dissertation for the degree of Ph.D. Environmental Sciences, University of the Philippines, Diliman, Quezon City.