Effects of Nitrate, Phospate, and Hydrogen Ion Concentration on Synedra Ulna: Diatoms as Indicators of Water Composition

Part of the Young Naturalist Awards Curriculum Collection.

by Robyn, Grade 11, California - 2004 YNA Winner

The French anthropologist Claude Lévi-Strauss wrote, "The scientific mind does not so much provide the right answers as ask the right questions" (Patterson, 2003). This truism has become apparent to me through my years of performing scientific research for school science projects. Each year my projects became more sophisticated, and my love of science developed into an admiration of both the natural world and technology. I even found myself intrigued by one of the greatest naturalists and his theories. Charles Darwin and his evolutionary theory struck a chord with me. Since studying his work, I have not only developed an interest in laboratory research, but I have learned the importance of field research as well. This interest in both laboratory and field research resulted in a two-year study expedition focusing on diatoms and their possible role as indicators of water quality. I became interested in diatoms when my science teacher told me about some of the studies he had done as a part of a graduate fellowship. I was immediately interested in these amazing microscopic organisms.

In order to formulate the "right questions," my research objective was to learn as much as possible about diatoms and their environment. This would provide the foundation for a well-designed scientific investigation or research quest. My research involved reading scientific journals and books, visiting local university libraries and the California Academy of Sciences in San Francisco, and seeking advice from leading diatomists such as Sarah Spaulding. I also contacted Heal the Bay, an environmental organization that monitors water quality in southern California, and after participating in their training program, I developed a basic understanding of environmental issues and also became certified in water chemistry. 


Background Research 
While doing background research, I learned that diatoms are eukaryotic and photosynthetic organisms (Werner et al., 1977). They respond directly and sensitively to physical, chemical, and biological changes in the water (Stoermer and Smol, 1999). Many studies have linked specific diatoms to environmental conditions such as the level of nitrate in the water, the degree of pollution, and electrolyte content (Stoermer and Smol, 1999). Because the majority of the nutrients used by diatoms are derived from dissolved chemicals in the water, the presence of diatoms may be used as an indicator of the chemical environment in which they live (Patrick, 1973).

Diatoms inhabit most aquatic habitats, such as lakes, wetlands, oceans, and estuaries (Stoermer and Smol, 1999). Like most plants, diatoms need sunlight for energy, in addition to nutrients such as nitrates and phosphates and carbon dioxide (Sobehrad, 1997). They are members of the largest group of algae, the golden algae phylum. Their cell walls fit together much like a lid fits on a box (Vinyard, 1979). These walls, primarily made up of silica, are called frustules.

There are more than 12,000 species of diatoms. They live in fresh ponds, streams, rivers, or on the surface of the ocean, where they are a component of phytoplankton (Sobehrad, 1997). Though some diatoms are free-floating, some are not. Many cling to or hook onto surfaces underwater, such as aquatic plants, mollusks, crustaceans, turtles, rocks, and other surfaces (Waggoner, 2001). I spoke to Sarah Spaulding, an expert on diatoms at the California Academy of Sciences, and learned that diatoms that grow on rocks are called epilithic diatoms (Spaulding, pers. com., 2001).

Robyn collecting materials from Limekiln Creek.

Diatoms are important in aquatic ecosystems because they are part of different food webs and they oxygenate the water (Stoermer and Smol, 1999). Diatoms contribute to organic carbon fixation by photosynthesis (Waggoner, 2001). The many different species of diatoms add to the biodiversity and genetic resources in bodies of fresh and salt water.

Diatoms can be classified differently depending on the classification system used. They make up either Bacillariophyceae in the phylum Chrysophyta, or they make up the phylum Bacillariophyta in the kingdom Protista (Encarta, 1999).

There are two main types of diatoms. Pennate diatoms are usually found in shallow freshwater areas and are elongated and cigar- or pen-like in shape. Centric diatoms, the second type, are circular, triangular, or irregularly shaped, and they are primarily found in the ocean (Prescott, 1964). Synedra ulna, the diatom that my studies focused on, is an epilithic and pennate diatom. As an example of a diatom that grows best in the presence of nitrates, it can be an indicator of this water characteristic.  Synedra  are described as having narrow frustules, free-floating or sometimes in colonies (Smith, 1950). They also can be benthic, meaning they grow on different surfaces. The valves are linear and on some, slightly curved.  Synedras  live in a variety of habitats, enabling researchers to study them in various aquatic environments (Smith, 1950). As a species within the algal community,  Synedras  have specific ecological requirements and tolerances (Freedman, 1996).

Nitrates, phosphates, and hydrogen ions are three of the major chemicals present in water, and all three have effects on the health of aquatic organisms. Nitrate is the most oxidized form of nitrogen found in the natural world. It is one of the most water-soluble anions known (Campbell et al., 2001). Plants need nitrogen as a nutrient, and most plants prefer nitrate to ammonia (Campbell et al., 2001). Diatoms are forms of algae, so they also need a supply of nitrate.

Phosphates are also necessary for plants to live. Phosphates are actually one of the most important nutrients for algae because they are essential for their metabolic reactions (Campbell et al., 2001). Phosphates are formed when some or all of the hydrogen in a phosphoric acid is replaced by various metals. Phosphates also have uses as water softeners, detergents, and fertilizers (Encarta, 1999).

The term pH is an indication of the concentration of hydrogen ions within a solution. The pH scale measures the acidity or alkalinity of a solution. If a body of water is too basic or too acidic, aquatic life may be affected (Encarta, 1999). Algae grow best at a pH of 7.5 to 8.4 (Presley et al., 2001). 

The experimental setup (Red tray-pH, Blue tray-Nitrate, Green tray-Phosphate).

My early research focused on the relationship between  Synedra ulna  and the pH of the water in which it lives. Some diatoms only survive in a specific pH range. Diatoms that prefer to live in habitats with a pH above 7 are known as alkalibionte forms, while diatoms that prefer water with a pH around 7 are known as alkaliphile forms (Werner et al., 1977). The acidophile forms of diatoms prefer a pH below 7, and those diatoms that prefer pHs of 5 and below are the acidobionte forms. Some diatoms will live in a wide range of pH levels, both above and below 7, and these are called indifferent forms (Werner et al., 1977). The tests I conducted during the first year of my research on samples taken directly from Limekiln Creek, in Northridge, California, established that Synedra ulna  is an example of an alkalibionte diatom. 

Robyn recording measurements and observations.

From my research, I learned that water temperature is another factor that affects aquatic organisms. Changes in water temperature impact both their chemical and biological characteristics, including the capacity of aquatic plants to perform photosynthesis and the ability of organisms to resist parasites, pollution, and disease (Myers et al., 2001). Aquatic organisms can only exist in narrow temperature ranges, which vary among species. If the temperature of a river were to increase a few degrees, some plants and native fish could die (Hart, 1999). Thermal pollution, or the introduction of warm water into a cooler body of water, is often the result of runoff from power plants and urban areas. Thermal pollution starts a domino effect that harms aquatic life. Specifically, warmer water is less capable of holding dissolved oxygen. A reduced level of dissolved oxygen leads to weakened aquatic organisms that are more susceptible to disease, parasites, and pollution (Myers et al., 2001). 

Water pollution is a significant problem facing us today. When there are harmful pollutants in the water, we are directly affected by it, and so is the surrounding aquatic life (Hart, 1999). Water in our environment can be classified as either eutrophic or oligotrophic. Eutrophic water is water which is rich in plant growth and mineral nutrients (Vinyard, 1979); and eutrophication is the process of enrichment. Oligotrophic water does not contain many mineral nutrients or much active plant growth (Vinyard, 1979).

I designed my experiment to investigate whether Synedra ulna  could be used as an indicator of the concentrations of nitrate, phosphates, and hydrogen ions in water. I hypothesized that diatoms such as Synedra ulna could be used as effective indices of these characteristics, and hence could be useful in assessing the quality of the water in my community.


I spent a lot of time considering the different ways in which I could test my hypothesis. I consulted teachers and experts in the field for feedback on my ideas. I decided to create three different environments to determine how Synedra ulna would be affected by ecological changes in its environment. I altered the pH, nitrate, and phosphate levels of the stream water used in my experiments. I then made observations of diatoms in each environment, focusing on Synedra ulna.

On the first day of my expedition, I created the baseline environments with materials I collected from Limekiln Creek. Creek water was sampled from a site whose coordinates (34°17.554 N, 118°33.350 W) I determined by global positioning and that I knew from my earlier study contained Synedra ulna. This site was my favorite from the earlier tests I had conducted at three locations along the creek. I felt a special connection with it, perhaps because of the small waterfalls that trickled lightly over the rocks, or the peaceful and serene atmosphere. It embodied unadulterated nature, which is so rare in today's world. I distributed rocks, sediment, and water from this site into six 3.5-liter glass containers. Substrate, rocks, and 2.5 liters of stream water were distributed evenly and carefully into each container. The containers were labeled: 1) Nitrate Control, 2) Nitrate Treatment Sample, 3) pH Control, 4) pH Treatment Sample, 5) Phosphate Control, 6) Phosphate Treatment Sample.

The ratio of Synedra ulna per 100 diatoms in all six containers was then determined following a specific procedure. I took a sample from each artificial environment using a centrifuge tube. I scraped the rock with the open tube multiple times, and each time I poured out the excess water. I then added ethyl alcohol (70 percent) to each sample (2 parts alcohol to 1 part creek water) in order to preserve the diatoms and stop diatom motility. This step was crucial so that I could accurately determine the ratio of Synedra ulna per 100 diatoms. I also measured and recorded the water temperature, pH, nitrate content, and phosphate content in each container, using HACH water-testing equipment. 

Robyn conducts chemical testing.

I tested the effect of reducing the pH of the water on the ratio of Synedra ulna per 100 diatoms by adding an acid to the pH Treatment environment. I measured the pH in both containers by using an Oakton pH meter (pH Testr 1). One of my favorite processes was calibrating the pH meter prior to use, with HACH buffer powder pillows of 4.01, 7.00, and 10.01 and de-ionized water. After extensive research, I decided to use a hydrochloric acid standard solution of 0.1N to alter the pH of the water. I added this standard solution to the pH Treatment sample container, and I monitored the pH until it had been lowered to pH 6.6 (this value indicated that the water's pH was below neutral).

I then measured the water's nitrate and phosphate content. I performed these measurements at both the start and the completion of the six-day test cycle in order to see if the concentration of these chemicals was affected by the change in pH in the samples. I thought this would produce interesting data regarding the interaction of various water conditions. I was looking for evidence that the levels of nitrate and phosphate in the stream water might be altered, directly or indirectly, by a change in the water's pH. I measured the nitrate and phosphate content by using HACH water reagent testing kits (Model NI-11, Ca. No. 1468-03 and Model P0-19, Cat No. 2248-00). The tests I decided to use were color disk and reagent pouch kits. I added the reagent to the tube of creek water. I put a separate tube of creek water, without chemicals, into the color disk apparatus. With both samples in place, I rotated the color disk until the colors of both samples matched, and then I calculated and recorded the result.

I altered the nitrate environment in the Nitrate Treatment sample by adding a nitrate standard solution of 15.0 ppm (parts per million) to the nitrate treatment sample until the nitrate solution was 50 to 75 percent greater than the baseline control value. I believed this increase would be large enough to affect the abundance of Synedra ulna. I altered the phosphate environment in the Phosphate Treatment sample by adding a phosphate solution of 10.00.1 mg/L to the sample, raising the phosphate content 100 percent from the baseline control.

After observing the natural conditions of the creek, I decided it was necessary to use Aqua Culture aquarium air pumps in all six bowls to simulate creek flow and aeration. I used one pump with a T-connector for two containers. I also placed the containers outdoors in natural conditions to replicate the weather conditions of the stream from which the samples were taken. 

I determined the ratio of Synedra ulna per 100 diatoms by making a wet mount slide. This was one of the most trying tasks of my procedure. Not only did it take a lot of time, but the procedure I developed to count the diatoms took me several trials to perfect. My final procedure for this task was to use a pipette to put three drops of the sample onto a slide and then place a cover slip over it. I then examined the wet mount slide in a consistent sequence at 400x magnification. I counted the number of Synedra ulna per 100 diatoms. I counted two slides per sample, for a total of 200 diatoms for each of the six containers. I then determined and recorded the average of the two slides. I followed this procedure for all six centrifuge tubes.

Six days after the establishment of the artificial environments, I took diatom samples from all the containers being used, and I repeated my original protocol to determine the ratio of Synedra ulna per 100 diatoms. I also repeated my original protocol for measuring the temperature, pH, nitrate, and phosphate levels of all six bowls. I performed four separate and independent trials during an eight-week period.


Discussion and Conclusions 
The hypothesis that diatoms such as Synedra ulna could be used as an index of water characteristics impacting water quality could not be supported by my data. The water data and the Synedra ulna counts from the four trials were inconsistent. The data did not specifically or consistently demonstrate a relationship between levels of nitrate, phosphate, and hydrogen ions and the rate of Synedra ulna reproduction as determined by the ratio of Synedra ulna to other species of diatoms.

Even though I was unable to answer my original question about Synedra ulna and its interaction with nitrate, phosphate, and pH, I was able to make three observations about Synedra ulna and other water organisms from my research. First, Synedra ulna was shown to be a very durable organism. It survived and reproduced at varying concentrations of hydrogen ions, nitrate, and phosphate. It is significant that it survived both low and high concentrations of each of these agents.

Second, I observed unexpected changes in the pH Treatment sample. The pH of the treatment sample had been lowered to 6.6, but six days later its pH was 8.7, higher than the baseline pH of the control sample. Perhaps diatoms or other organisms such as bacteria in the water used the hydrogen ions. Alternatively, perhaps the bacteria were able to change the pH level from acidic to alkaline so that the water was a desirable place to live. Bacteria's ability to form a biofilm is an example of this phenomenon. In a biofilm, different species of bacteria work together and secrete a mucus-like layer to make a community where they can carry out specific tasks and regulate certain characteristics in their environment, such as pH (Greenberg, 2001). Based on the observations and results from my previous research, I had expected that the presence of Synedra ulna could be used as an indicator that a body of water has a pH of 7 or more. Synedra ulna grows best in water with a high nitrate content, which in turn has a pH above 7. Therefore, Synedra ulna may be an indicator that the water is more basic than acidic. However, my data did not support or disprove this hypothesis.

Finally, I found that an increase in the concentration of nitrate in the water may inhibit the reproductive rate of Synedra ulna. In my control samples, the Synedra ulna count per 100 diatoms nearly doubled at the end of each testing series. This did not occur in the Nitrate Treatment samples, where there was little change in the ratio of Synedra ulna compared to other diatoms. The relative change in the ratio of  Synedra ulna to other diatoms in the treatment samples remained unchanged throughout the experiment.

I tested for nitrate because diatomists had previously demonstrated that Synedra ulna grows best in high-nitrate-content water (Werner et al., 1977). Nitrate was also tested because it is affected by pH. For example, a reduction in pH will lead to an increase in ammonia and, in turn, a decline in nitrate. I also wanted to explore whether there were other negative or positive effects caused by nitrate's presence.

Though aquatic plants need nitrate, there are some negative effects from this form of nitrogen when there is an excessive amount of it in the water. A certain level of nitrate stimulates the growth of plankton and weeds, which are food for fish. In turn, the fish population grows. But if there is too much nitrate in the water and too much plankton growth, the level of dissolved oxygen in the water will decline and the fish will die (Presley et al., 2001). Monitoring nitrate levels to maintain this balance is important to preserve the ecosystem. Nitrates are widespread contaminants of ground and surface waters around the world (Campbell et al., 2001).

Other observations deserve comment as well. Phosphate was tested for its impact, both negative and positive, on the reproduction rate of Synedra ulna. Phosphates are important for the metabolisms of both plants and animals, but like many other compounds and elements, phosphates in excessive amounts have harmful effects. Awareness has been raised about the harmful environmental effects of detergents (Encarta, 1999). Now, phosphates are considered to be water pollutants, because if too much wastewater containing phosphates is dumped into a body of water, algae may grow in excess (because phosphates are its primary nutrient) and cause eutrophication.

The excessive growth in algae over time may choke a lake or river, and affect dissolved oxygen levels and aquatic life (Encarta, 1999). Since some diatoms can live in water that is mildly polluted, they may be useful as markers for pollution levels. But if the water becomes critically polluted, then a whole species of diatoms may become extinct. The only diatoms able to survive would be those diatoms adapted to critically polluted water. An example of a diatom that can tolerate critically polluted waters is Rhoicosphernia curvata (Cox, 1996). On the other hand, Gyrosigma acuminatum would not be able to survive in those conditions (Cox, 1996). Because the counts of Synedra ulna in the Phosphate Treatment samples were inconsistent, no specific relationship to phosphate content could be made by the present study.

This graph displays the consistent increase in the ratio of Synedra ulna after each six day period in the controls. There was no consistent increase or decrease in the Synedra ulna count in the treatment samples.

I wondered why my results were inconsistent even though I performed my experiment carefully. The results may have been influenced by variables that I was unaware of, or that I was unable to control. First, there are other organisms in the creek water besides the diatoms; these organisms consume the same nutrients as the diatoms, and may have had an effect as well on the concentrations of nitrate, phosphate, and hydrogen ions in the water. Although I did not observe any grazers that consume diatoms in the samples, my observations do not rule out the possible presence of microscopic diatom consumers. Possible grazers are animal larvae such as flattened mayfly larvae (Leptophlebiidae) and beetle larvae (Elmidae) (Winterbourn and Townsend, 1991).

The weather was also an uncontrollable variable, as the bowls were kept outside to replicate the conditions at the source, Limekiln Creek. Temperature, wind, and the amount of sunlight may have affected the metabolic and reproductive activity of the organisms in the bowls. During the last two trials the weather was unseasonably cold, so this may have also affected the abundance of Synedra ulna

The time in between trial measurements may have also been a factor. Because of the amount of time required to perform all of the testing and obtain all the measurements, each trial lasted six days and only four trials could be performed. All trials were conducted over an eight-week period. 

Another factor that could have affected my results was the existing concentration of nitrate in the source water at Limekiln Creek. After correspondence with mentors, I found that even the "dirtiest" site tested by Heal the Bay, an organization that monitors water quality in southern California, had a nitrate concentration of less than 10mg/L. However, the water samples I collected and distributed into the six bowls showed nitrate levels consistently above 10mg/L, and the concentration of nitrate in the treatment samples was greatly increased over that baseline measurement. The baseline nitrate levels in the creek may have been too high for me to get conclusive results.

An alternate reason for my inconsistent results is the optimum nutrient ratio theory. This theory suggests that some nutrients work in pairs such that if the concentration of one nutrient is increased, the concentration of the "partner" also needs to be increased to allow the ratio for optimum growth to remain the same (Spaulding, pers. com, 2002). It is possible that nitrate and phosphate interact in this manner. If so, when I increased the concentration of nitrate in the Nitrate Treatment sample, I should also have increased the concentration of phosphate.

In designing an improved experimental procedure, I would consider the following changes. First, I would include other diatom species in addition to Synedra ulna to see if I could find similarities in how the other species react to the changes in the environment. Second, I would also raise the pH (in addition to lowering it) to determine whether the raised pH would return to its baseline range. I would raise the other water conditions that were tested to different levels as well. Third, I would try different time intervals, shorter and longer, between collecting and testing, to see how that affects the diatoms' rate of reproduction. The intervals for this experimental procedure were determined by school schedules and by the time required to perform the chemical tests and microscopy. Finally, I may need to develop a more consistent method of evenly distributing the collected stream diatoms, and especially Synedra ulna, into each bowl.

In summary, my experiment demonstrated that artificial aquatic environments can be constructed that allow for the individual chemical contents to be altered in order to determine their effects on diatoms. However, my experiment was not conclusive as to the specific effects of changed chemical levels on the ratio of Synedra ulna in a diatom-rich environment.

Though my data was inconclusive as to the specific effects of changed chemical levels on the ratio of Synedra ulna, I found myself fulfilled after the completion of my experimentation. At the end of my quest I found I had more questions than answers, but according to Lévi-Strauss, raising questions is as important in scientific inquiry as finding solutions. When I consider my research, I am reminded of Charles Darwin, who wrote of his experiences on the voyage of the Beagle as the most important event in his life. He felt it had provided the first real training and education of his mind. Darwin said, "I worked to the utmost during the voyage from the mere pleasure of investigation, and from my strong desire to add a few facts to the great mass of facts in natural science" (Howard, p. 4). Perhaps someday, through persistent curiosity, questioning, and investigation, I will be able to make a noteworthy contribution to the field of science. My research has motivated me to continue working toward my goal of becoming a scientist. Being involved in the field of science is in itself a lifelong expedition in pursuit of the right questions.



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Personal Interviews

Abramson, Mark. Heal the Bay, Stream Team Director. Personal communications 8 January 2002 and 4 February 2002.

Campbell, Craig. Sierra Canyon Science Department. Personal communications 9, 13 July 2001; 27 September 2001; 15, 20, 28 October 2001; 1, 2 December 2001, and 28 January 2002.

Spaulding, Sarah. California Academy of Science, Diatomist. Personal communications 23, 30 January 2001; 14, 15, 18 February 2001; 29 September 2001; 18 October 2001; 26 November 2001; 17

December 2001; 28 January 2002; and 5 February 2002.


Web Sites

Campbell, Wilbur H., et al.  Nitrate: Health Risks to Consumers. Retrieved from the World Wide Web on 9 December 2001. http://www.nitrate.com/nitrate1.htm

Charles, Donald and Pat Kociolek.  Freshwater Diatoms: Indicators of Ecosystem Change. Retrieved from the World Wide Web on 2 December 2001. http://biology.usgs.gov/st/noframe/m2097.htm "Diatom."  Encarta Encyclopedia 2000 . Microsoft, 1999.

EPA United States Environmental Protection Agency. "Periphyton as Indicators." Retrieved from the World Wide Web on 27 November 2001. http://www.epa.gov/bioiweb1/html

Heal the Bay. "Stream Team." Retrieved from the World Wide Web on 21 September 2001. http://www.healthebay.org/streamteamhome.asp

Myers, Dr. Robert, et al.  Water Quality Assessment. Retrieved from the World Wide Web on 11 November 2001. http://www.coft.edu/ete/modules/waterq3/Wqassess1.html "pH."  Encarta Encyclopedia 2000 . Microsoft, 1999.

"Phosphoric Acid."  Encarta Encyclopedia 2000. Microsoft, 1999.

Presley, Richard, et al.  Important Water Quality Factors. Retrieved from the World Wide Web on 7 December 2001. http://www.hach.com/h2ou/h2wtrqual.htm

Susan Sobehrad.  Diatoms: Living Snowflakes. Retrieved from the World Wide Web on 6 January 2001. http://bellnetweb.brc.tamus.edu/diatoms.htm.

"Temperature."  Encarta Encyclopedia 2000. Microsoft, 1999. Waggoner, Ben. Diatoms: Life History and Ecology. Retrieved from the World Wide Web on 12 January 2001. http://www.ucmp.berkeley.edu/chromista/diatoms/diatomlh.html