  
Winter, 1985, Spanish moss on tree (top) (Click to view) Winter, 2004, the same tree, but no moss (bottom) (Click to view)
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.
Close up of Spanish moss
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.
Three Erlenmeyer flasks in the Environmental Study Chamber (ESC), under plant light.
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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 (SO 2), 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 CO 2. However, I was still curious to see how CO affected Spanish moss, as it is a unique plant. According to Mansfield, NO 2 is often less toxic than other pollutants. However, when NO 2 combines with water, it forms nitric acid: NO 2 + H 2O = HNO 3. Sulfur dioxide is a more potent pollutant. When SO2 combines with oxygen and water, sulfuric acid is formed: 2SO 2 + O 2 + 2H 2O = 2H 2SO 4. 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.
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