Tillandsia usneoides: An Indicator to Air Pollution

Part of the Young Naturalist Awards Curriculum Collection.

by Caroline Grade 11, Texas - 2006 YNA Winner
Winter, 1985, Spanish moss on tree (top) Winter, 2004, the same tree, but no moss (bottom)

The breeze stirs the Spanish moss, which is draped gracefully over a sprawling oak tree. Although this is a pretty sight, it has become uncommon in the past few decades. Spanish moss is an important part of the Southern United States' ecosystem—without it, several species could become extinct. It provides a habitat for spiders, bats, snakes and birds, some of which eat insects such as mosquitoes. One species of spider (Pelegrina tillansiae) and three species of bats live  only  in Spanish moss (Adams). While looking through old pictures of Houston, I noticed that there was once more Spanish moss in the trees around the Houston area than there is today. One of my neighbors recalled, "There was a lot of Spanish moss on the trees when we moved here in 1971. Now there is no moss left in our neighborhood (Sloan)." Still, small pockets of moss can be seen thriving in certain areas, away from busy roads. What causes these pockets of Spanish moss to thrive when it has declined in other areas of Houston? And if their decline is related to air pollution, which pollutant is doing the most harm? My hypothesis was that Tillandsia usneoides is an indicator species to air pollution, that its decline is directly related to raised levels of air pollution, and that the most acidic pollutants are the most harmful.

A small mass of green, grass-like strands, Spanish moss, against a white background.
The very tip of Spanish moss as seen through a digital microscope.

Studies have been done to see which pollutants are absorbed by Spanish moss. But I was curious to discover if common pollutants in Houston affect the  life  of Spanish moss. In order to test my hypothesis, I formulated an experiment with two stages. The first stage involved taking air samples in Houston and testing for the quantity of specific pollutants using a gas chromatograph. The second stage was putting Spanish moss in an environmental study chamber (ESC), which is a closed system, and exposing it to the pollutants found in the air samples. However, I soon realized that my project had to be narrowed, as both stages would involve a lengthy amount of time and did not directly test my hypothesis. After contacting the Public Health & Environmental Services Pollution Control, I discovered that pollution monitors are located throughout the Houston area, and the levels are displayed hourly on the Internet (Van Vleck). Since I did not need to test for air pollution levels, I decided to limit my project to putting the Spanish moss in the ESC and exposing it to certain pollutants. Originally, the pollutants were the six Air Quality Index pollutants: carbon monoxide, sulfur dioxide, nitrogen dioxide, ozone, acid rain (pH 4), and particulate matter (PM 2.5). Again, this was still too broad, as accurately gathering particulate matter and obtaining ozone would be difficult. Thus, the four pollutants I chose to expose to the Spanish moss were: carbon monoxide, sulfur dioxide, nitrogen dioxide, and acid rain (pH 4). Also, I added two controls, helium and carbon dioxide. No living object can survive in a closed system with just helium. Therefore, when the Spanish moss was exposed to helium, I could see how long it took the moss to dieI also kept track of the weight difference between the live and the dead moss, which allowed me to find the percentage of weight lost in unfavorable conditions. For my second control, I kept the moss in carbon dioxide, as plants need this gas to survive. By doing this, I could find out if the moss can survive in the best conditions for the same length of time it takes the moss to die in the worst conditions. But before I could begin my experiment, I needed to learn more about Tillandsia usneoides and the four pollutants.


study of the effects of pollutants on Tillandsia usneoides
Three Erlenmeyer flasks in the Environmental Study Chamber (ESC), under plant light.
Against a white background, a close-up image of the long, thin, feathery tip of a piece of Spanish moss.
The very tip of Spanish moss as seen through a digital microscope.

Tillandsia usneoides, commonly called Spanish moss, is a relative of the pineapple (order Bromeliales, family Bromeliaceae, genus Tillandsia (air plant), and species usneoides) (Spanish moss). In fact, it is an epiphyte, a plant that gains all of its moisture and nutrients from the air (Arny). Thus, it is not parasitic and rarely harms trees. Since I was unable to find much information about the anatomy of Tillandsia usneoides, I also used information on Tillandsia recurvata L. (ball moss). These two plants only differ by shape; thus, they were easy to compare. The leaves of Tillandsia usneoides are awl-shaped and pointed. Looking through a Motic Images 2000 digital microscope, I observed that the thin trichomes (scales) that cover the whole plant grow larger towards the base of the leaf, where it merges with the stem. Researching further, I found that these trichomes play an important role in the absorption of moisture and nutrients from the air. The trachomas act as pumps, and draw moisture and dissolved minerals into the plants through the stomata (Arny). This indicates that whatever is present in the air—including pollutants—will be absorbed by the plants. And if the trichomes are harmed, the plant, or a section of the plant, might soon die from lack of nutrients and moisture. The stomata are found just beneath the wings of the trachomas. Perhaps the pollutants that I exposed to the Spanish moss would damage the trichomes. I would find all this out in my experiment.

A pollutant is defined as a chemical that is in the wrong place at the wrong time (Wellburn). I exposed the Spanish moss to carbon monoxide (CO), sulfur dioxide (SO2), nitrogen dioxide (NO 2 ), and acid rain (pH 4). Carbon monoxide is formed when incomplete combustion occurs, or when something is burned in limited oxygen. This pollutant is mainly caused by emissions from humans, and studies on CO in regard to plants are limited (Wellburn). However, CO does not linger long, because plants oxidize it to CO2. However, I was still curious to see how CO affected Spanish moss, as it is a unique plant. According to Mansfield, NO2 is often less toxic than other pollutants. However, when NO 2  combines with water, it forms nitric acid: NO2 + H2 + O = HNO3. Sulfur dioxide is a more potent pollutant. When SO2 combines with oxygen and water, sulfuric acid is formed: 2SO2 + O2 + 2H2 + O = 2H2SO4. Plants mainly absorb these three pollutants during the day through their stomata when they are open. Plants that have a thicker waxy cuticle are more resistant to sulfur dioxide; Spanish moss does not have this protective barrier. Acid rain is composed of two-thirds sulfuric acid and one-third nitric acid (Gordon). Acid rain is considered any rain that is below pH 5; normal rain is pH 5.5 (Acid Rain).

Gas Chromatograph (GC) used at school.

After completing my background research, I began my experiment. Helium was the first control. Helium is lighter that air and easily escapes from containers; thus, it was not practical to pump helium into the large ESC. Therefore, I decided to use 250ml Erlenmeyer flasks. I observed the Spanish moss through the digital microscope and then weighed out three trial groups of moss. Next, I placed the moss into three labeled flasks. Two capillary wires were put through the stopper holes: one wire came from the helium tank and one wire was attached to the gas chromatograph (GC). Clay was used to seal the holes around the wires, and the inverted flasks were placed in a stand. After opening the helium valves, the pressure of the helium was turned to 40 PSI, which was kept constant for all three flasks. After letting the helium enter the flask for 30 minutes, the pressure increased and the stopper had to be taped to prevent it from popping off. I took an air sample from the air in the room and ran it through the GC. Then, I ran the air from the flask into the GC. The amount of air in the room was compared to the amount of air left in the flask. The amount of air in the flask was lower than the air in the room due to the fact that the helium had pushed out most of the air. The GC employed used helium as a detector gas; therefore, the helium in the flask did not show on the graph, as the helium from the flask cannot be distinguished from the detector helium. Helium was kept flowing until there was almost pure helium in the flasks, which took about an hour per flask. The flasks were sealed and placed inverted into the ESC to prevent the helium from escaping. I placed a plant light on top and a timer was set from 9 a.m. to 6 p.m. A sheet was placed over the ESC to exclude all other light; the ESC was kept at a constant 20 C. to 22 C. The light and temperature was kept constant for all six tests. After 10 days, the moss was withered and dead—it was clearly dead. I was amazed at the difference between the live and the dead moss under the digital microscope.

Helium Control Trial 1 Trial 2 Trial 3 Average
Before 3.1038g 3.2572g 3.2670g 3.2093g
After 2.1682g 2.1607g 2.2441g 2.1910g
%Weight Loss 30.166% 33.664% 31.310% 31.713%
Moss before and after carbon dioxide exposure.

From these results, I decided to expose the Spanish moss to the four pollutants individually for 10 days, since it took that many days for it to die completely. Using the difference in weights, I was able to get a quantitative measurement to scientifically define "dead" Spanish moss. If the Spanish moss lost 31.713 percent of its weight after being exposed to a pollutant, it was considered 100 percent dead in this experiment. This information was used to analyze the results of the other four pollutants.

For the second control, dry ice was used to obtain carbon dioxide. Again, I looked at the moss under the digital microscope and weighed it using the same analytical balance. I put approximately 15g of dry ice in each flask. After half of each cube had sublimated, I put the stopper in each flask. Pressure built up beneath each stopper as the dry ice continued to sublime and the stoppers popped off. This showed that the CO2 had filled the flask and the air was being pushed out, since CO2 is heavier than air. I kept the moss in the carbon dioxide for 10 days. Then, its weight change was recorded and the moss was inspected under the digital microscope. This time, the moss was a healthy light green color. 

CO 2  Control Trial 1 Trial 2 Trial 3 Average
Before 3.6634g 3.5374g 3.5822g 3.5943g
After 3.6977g 3.5276g 3.5222g 3.5825g
% Weight Loss -0.936% 0.277% 1.675% 0.338%

From these results, I found that the Spanish moss could live and even grow, as seen from the negative weight loss, in the ESC for a period of 10 days.

Each time the samples of Spanish moss were collected, I collected the moss off of the same branch of the same live oak tree in order to make sure that the moss was as similar as possible. Looking at the pollution results from January 2004 to October 2004, I was able to get an average amount of pollutants in the Houston air for carbon monoxide, nitrogen dioxide, and sulfur dioxide. I obtained these results from the monitor nearest to where the samples of Spanish moss were collected: (Air Quality Index and Lang C408).

Carbon monoxide Nitrogen dioxide Sulfur dioxide
1.2 ppm 38.59 ppb 7.5 ppb

For the acid rain, I collected rainwater in a sterile container and measured the pH. I found the average pH of Houston's water to be 4.5.

In order to obtain carbon monoxide, nitrogen dioxide, sulfur dioxide, and acid rain, I synthesized them in my school's laboratory with teacher supervision. Carbon monoxide was made by combining eight drops of concentrated sulfuric acid with eight drops of formic acid (H 2 SO 4  HCOOH → CO + H2SO4:H2O; the sulfuric acid dehydrates the formic acid). I used the thermal method—the two chemicals were heated in a test tube and the waste was collected in one syringe and the carbon monoxide in the other syringe. A clamp was used to prevent mixing of carbon monoxide and the waste product (dilute sulfuric acid). Sulfur dioxide was made with sodium bisulfate and concentrated hydrochloric acid (H2SO4 + NaHSO4  → SO2 + NaCl + H2O). The reaction did not require heating but was collected in a similar manner to the carbon monoxide. Nitrogen dioxide was made by reacting copper with nitric acid (4HNO3 + Cu → 2NO2 + 2H2O + Cu(NO3)2). The reaction did not require heat as a catalyst, and the two chemicals were mixed in an Erlenmeyer flask and allowed to react. After the air was pushed out and the nitrogen dioxide produced, a syringe was placed over the opening and the nitrogen dioxide was collected. A wet piece of litmus paper was held over the opening of the flask; if it turned red I knew that I had collected pure nitrogen dioxide. Unlike the other two pollutants above, the nitrogen dioxide had to be made each day since it was so corrosive and could not be kept in the plastic syringe for long. The acid rain was made by mixing three liters of water with 250ml of sulfuric acid and 125ml of nitric acid. Small amounts of sodium hydroxide, a base, were added to the solution until pH 4.5 was reached. While synthesizing these "pollutants" in the hood, I wore goggles, an apron, and gloves and kept MSDS sheets on hand for all chemicals.

Percentage of Weight Loss
Percentage of Weight Loss
Percentage of Weight Loss

I exposed three trials of Spanish moss to one chemical at a time for 10 days. Each trial was observed under the microscope before and after exposure and weighed using the same analytical balance. A damp paper towel was placed at the back of the ESC to keep the humidity constant—around 85 percent. The air pump was kept running constantly in order to circulate the air and pollutants in the closed system. The light and temperature was kept the same as with the controls. I used a three-milliliter syringe to inject the pollutant into the ESC through a stoppered hole daily at 7:30 a.m., including the weekends. I mixed the acid rain solution in a spray bottle and thoroughly sprayed the moss daily. The averages of the data are below:

  Before After % Weight Loss % Dead
Helium 3.2097g 2.1910g 31.71% 100%
Carbon dioxide 3.5943g 3.5825g 0.34% 1.07%
Carbon monoxide 3.7756g 3.3297g 11.79% 37.18%
Sulfur dioxide 3.6988g 3.1689g 14.30% 45.09%
Nitrogen dioxide 3.9122g 3.3371g 14.69% 46.33%
Acid rain 4.0744g 3.1220g 23.39% 71.48%

Moss after acid rain exposure
Digital microscope images of live Spanish moss (top) and moss after being exposed to acid rain for 10 days (bottom). The acid rain was the most harmful pollutant. The trichomes are almost gone and the width is significantly decreased. (Click to enlarge)

After all four tests were done, I was surprised with the results. Using the helium control as a standard for 100 percent dead Spanish moss, I was able to get a qualitative number for how "dead" the moss was after being exposed to the four pollutants. Acid rain killed the moss 71 percent.  Moss after acid rain exposure (Click to view) Acid rain levels are frequently higher in cities than in the country, perhaps explaining why the Spanish moss is disappearing. Also, the Spanish moss that remains in Houston is found on the lower branches of the larger, more densely leafed trees. Perhaps the tree leaves protect the Spanish moss from acid rain; thus it is able to survive in certain protected microclimates.

Carbon monoxide killed the moss 37 percent,  which is worse than I expected. However, it did the least amount of damage; thus the Spanish moss must have oxidized some of the CO to CO2. Sulfur dioxide and nitrogen dioxide had similar results, possibly because both create a weak acid when mixed with water from the air. From these results, my hypothesis can be accepted: Tillandsia usneoides is an indicator species to air pollution in that its decline is directly related to raised levels of air pollution, and the most acidic pollutant (acid rain) is doing the most harm.

Moss after carbon monoxide exposure
Digital microscope images of live Spanish moss (top) and moss after being exposed to carbon monoxide for 10 days (bottom). After exposure trichomes are still present, but shorter and there is a slight overall decrease in width.(Click to enlarge)
Moss after sulfur dioxide exposure
Digital microscope images of live Spanish moss (top) and moss after being exposed to sulfur dioxide for 10 days (bottom). After exposure trichomes are shorter and the width is decreased even more than the CO results.(Click to enlarge)
Moss after nitrogen dioxide exposure
Digital microscope images of live Spanish moss (top) and moss after being exposed to nitrogen dioxide for 10 days (bottom). After exposure trichomes are considerable shorter and width is also decreased significantly.(Click to enlarge)
Tree with Spanish moss
Tree with Spanish moss

After performing this experiment, I have new understanding of the problems that air pollution can create. It is fascinating to know that an indicator plant to air pollution is living in my city. Even though Houston is at attainment levels for the four pollutants that I exposed to Spanish moss, those levels clearly can still be unhealthy. When the maximum "safe" pollution levels are decided, the main thought of safety is for humans. Although we are able to adapt to certain pollutants, more delicate plants and wildlife may be hurt. Thus, over time, humans may be gradually harmed. We must start preserving our environment before it is too late. By experimenting and seeing how air pollution impacts plant life, I now want to act to encourage others to do so as well. There is no better time than the present.



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Wellburn, Alan.  Air Pollution and Acid Rain: The Biological Impact . Library of Congress, 1988.