Hubbard Brook Analysis: The Effects of Increased Calcium Concentration on Tree Growth

Co-Authored with James F.

Abstract:

In our investigation, we examined the effects of elevated levels of calcium on a forest ecosystem. In the Hubbard Brook Experimental Forest Watershed One, scientists added large amounts of calcium in the form of wollastonite into the first sector, or watershed, of the forest. Our hypothesis was that this calcium, in its ionized form, precipitates out of the forest stream with ions naturally present in the water. We theorized that this precipitation could affect the pH a nearby stream by reacting with acidic or basic ions, such as HSO₄^- or OH^-, thereby pulling the ions out of solution. We also postulated that these extreme levels of calcium would be toxic to plant life, affecting leaf chemistry and potentially having adverse effects on the plants' function, possibly leading to plant death. Additionally, as a result of increased plant death and solids precipitating out of solution, we expected to see increased sediment buildup in a nearby stream. We were unable to investigate this particular hypothesis as adequate data was not recorded after calcium addition. However, we were able to examine the increased biomass of the forest floor, which we believed might correlate with the calcium addition.  For our analysis, we analyzed stream chemistry, leaf chemistry, and forest floor mass of both the control and calcium-enhanced watersheds. We found clear effects in leaf chemical levels of Ca, Mg, Mn, K, and P after calcium addition. Leaf calcium increased, leaf magnesium decreased upon addition, leaf manganese increased to compensate for increased calcium, leaf potassium decreased upon addition, and leaf phosphorous decreased upon addition.  We also noted clear increases in the chemical levels of Ca, PO₄, SiO₂, SO₄, and pH in the stream chemistry after calcium. Finally, we observed a significant increase in forest floor mass that possibly correlated with calcium addition and precipitation.

Introduction:

The purpose of this lab was to examine the effects of calcium on forest ecosystems using data gathered in the Hubbard Brook experiment.  We compared various attributes of forest watershed one before and after the addition of calcium to the system.  We also compared several attributes of forest watershed one, with the addition of calcium, and forest watershed three, the control watershed.  We theorized that calcium cations from the introduction of wollastonite, a calcium compound with the molecular formula CaSiO₃, would precipitate out of the water with other, naturally occurring anions.  We believed that this increased precipitation could result in increased sediment buildup in the river.  Additionally, we believed that the excessive levels of calcium in the environment could be detrimental to the biological function and health of the trees.  We conjectured that the trees would have increased levels of calcium, increased levels of other chemicals involved in calcium processing, and decreased levels of absorption of other necessary chemicals, owing to calcium oversaturation in the soil.  We used several sets of data to study these hypotheses.  We graphed the average thickness of soil and topsoil before and after the calcium addition, we graphed the increased and decreased presences of various substances in the trees’ leaves, and we charted the stream chemistry of the rivers in watershed one and watershed three.  We could not graph the sediment buildup owing to the fact that sediment yield data was not gathered past 1999. 

Results:

The above graph shows the average total thickness of forest floor mass before the addition of calcium to the watershed.  98 plots were surveyed eight times each.  The mean of the overall thicknesses was found to be approximately 8.0, the median thickness was found to be approximately 7.0, and the mode of the set was found to be approximately 5.4. 

The above graph shows the average thickness of the top layer (Oie) of forest floor mass before the addition of calcium to the watershed.  98 plots were surveyed eight times each.  The mean of the overall thickness was found to be approximately 3.7, the median thickness was found to be approximately 3.4, and the mode of the series was found to be approximately 3.6.

The above graph shows the average total thickness of forest floor mass after the addition of calcium to the watershed.  98 plots were surveyed eight times each.  The mean of the overall thicknesses was found to be approximately 8.2, the median thickness was found to be approximately 8.2, and the mode of the set was found to be approximately 4.8.  The mean and the median both show a significant increase form the pre-calcium addition levels, meaning that there was possibly some precipitation of solid from the calcium addition.    

The above graph shows the average thickness of the top layer (Oie) of forest floor mass after the addition of calcium to the watershed.  98 plots were surveyed eight times each.  The mean of the overall thickness was found to be approximately 4.3, the median thickness was found to be approximately 3.9, and the mode of the series was found to be approximately 3.5. The mean and the median both show a significant increase form the pre-calcium addition levels, meaning that there was possibly some precipitation of solid from the calcium addition.   

The above graph shows the effects of increased calcium concentration on leaf biochemistry.  Data was gathered on three different tree species, balsam fir (ABBA), red spruce (PIRU), and white or paper birch (BEPA).  (Hubbard Brook, Watershed 1 Temporal Canopy Leaf Chemistry)  The calcium was added in the year 2000.  (Hubbard Brook, Watershed 1 Temporal Canopy Leaf Chemistry)  After this year, there is a significant increase in the calcium concentration in parts per million, which correlates with our hypothesis. 

The above graph shows the concentration of magnesium in parts per million in leaves as a function of time (before and after the addition of calcium into the system).  As expected, for most tree species the amount of magnesium absorbed upon the addition of calcium decreased.  Two years after the addition of the calcium, the trees with the most significant absorption loss had returned to previous levels of magnesium concentration, perhaps making up for a magnesium deficit. 

The above graph shows the concentration of manganese in parts per million in leaves as a function of time (before and after the addition of calcium into the system).  Manganese is an element that is essential to calcium absorption.  (University of Maryland Medical Center, 2011) The uptake of manganese correlating with an uptake of calcium shows that the leaves have been oversaturated with calcium and need excessive amounts of other ions to process it. 

The above graph shows the concentration of potassium in parts per million in leaves as a function of time (before and after the addition of calcium into the system).  Potassium is an element that is essential to plant growth; along with calcium it is key to the growth of young and developing plants.  (Fromm, 2010)  For ABBA and PIRU species, the initial flood of calcium apparently reduced the plants’ abilities to absorb potassium.  This effect seemed also to be present in BEPA plants, though it should be noted that they made rapid recovery, and soon the potassium levels were much, much higher than in pre-calcium addition plants.

The above graph shows the concentration of phosphorous in parts per million in leaves as a function of time (before and after the addition of calcium into the system).  Phosphorous is an element that is key to sustained plant growth (Baribault, 2012).  For all species, the initial flood of calcium apparently reduced the plants’ abilities to absorb phosphorous.  However, the BEPA species quickly rebounded to levels above the initial pre-calcium levels, possibly to make up for a phosphorous deficit. 

The above graph shows the stream chemistry of watershed three, the control watershed.  This graph shows one large noticeable spike in PO₄ levels, around 400 months. 

The above graph shows the stream chemistry of watershed one, the watershed with calcium added.  This graph also shows one noticeable spike in PO₄ levels around 400 months, however its spike is larger than that of the control.  This could possibly be attributed to the calcium addition and some dissolved precipitate in the stream. 

The above two graphs show the trends of stream chemistry for watersheds three and one, respectively.  The levels of SiO₂ initially increased before leveling out in watershed one, yet in watershed 3 there was no significant trend.  This initial increase could be linked to the presence of calcium.  The levels of Ca were significantly higher in watershed one, as would be expected.  The levels of SO₄ were slightly elevated with calcium, yet both watershed three and watershed one showed a general downward trend over time, indicating that there may be some larger environmental factor affecting the levels.  The pH was elevated with calcium addition, as would be expected (calcium is a naturally basic substance).  

Discussion:

After analyzing the data from the Hubbard Brook watersheds, we found several correlations between calcium addition and changes in leaf chemistry, stream chemistry, and forest floor mass over time.  The above graphs demonstrate the correlation between elevated calcium levels and alterations in plant chemistries.

The first four graphs show an increase in forest floor mass after the addition of calcium.  This increase may be related to elevated levels of calcium leading to toxicity in some plants.  These plants would die and turn into detritus, which would increase the mass of the forest floor. 

            The next five graphs delineate the changes in leaf chemistry of calcium, magnesium, manganese, potassium, and phosphorous.  The addition of calcium in the watershed environment leads to an expected increase of calcium in the leaves themselves.  We not this effect in all of the three plant species studied.  When we analyzed the levels of magnesium, we found a general reduction in the levels of magnesium for all three plant species, noted at the year 2000 for BEPA and ABBA and approximately one year later for PIRU.  This effect is likely the result of excessive calcium saturation of the soil, which could prevent the plants from drawing other ions from the soil.  This would lower the levels of the aforementioned ions in the leaf chemistry analysis.  We subsequently observed the levels of magnesium in the leaves increasing after the initial reduction to higher levels than before.  We may attribute this phenomenon to a deficit in magnesium levels from the blockage, which would cause the plant to overcompensate to restore proper magnesium levels.  The next graph, a plot of manganese levels over time, showed an increase of manganese absorption.  We found in our research that manganese aids in calcium absorption.  Therefore, increased calcium absorption owing to the elevated calcium levels would elicit an increase in manganese absorption to allow for proper calcium processing and integration.  We have noted this general increase in the manganese absorption in all three species of plant studied after the 2000 calcium addition.  All three plant species experienced reduced levels of potassium at the 2000 calcium addition, probably owing to the introduced calcium blocking the absorption of other chemicals.  We note the BEPA plants seemed to make a rapid recovery, unlike ABBA and PIRU, which appear to recover at a slower rate.  This recovery may also be the result of a compensation for a deficiency, similarly to magnesium. Phosphorous absorption is also reduced. This may be attributed to, as noted above, extreme calcium levels blocking the absorption of other chemicals. We also note the extreme recovery of the BEPA plant, perhaps related to compensation for a previous deficiency.

            The last graphs refer to stream chemistry.  PO₄ has its own graph due to the relatively small scale of the concentrations with regards to the other chemicals in question. First, we note the clear increase in calcium levels shown by the graph due to its artificial augmentation by scientists. This calcium addition in watershed one showed a correlation to increases in SiO₂, pH, and SO₄. SiO₂ levels do not show a trend in the control, watershed three, but show a clear increase linked with the calcium addition. Therefore, the calcium addition probably caused, through an unknown set of processes, the increased SiO₂ levels. Similarly, pH was largely stable in watershed 3, but rose in watershed one in a manner tied to the calcium addition. Calcium likely is the cause of this increase, as we hypothesized. SO₄ also increased in this manner, but we note that it trended generally downward during the measurement period. This long-term downward trend is likely due to external factors, perhaps climate change, as the overall trend does not correlate with calcium levels and is present in both watersheds. However, the short-term increase of SO₄ is strongly correlated with and likely caused by calcium addition. Finally, PO₄ generally trended higher at the calcium addition at around month 400. This is increase was likely caused by calcium. Note that there is another relative increase in the PO₄ levels at 300 months. As this deviation is present in both graphs and occurs before addition of calcium, it is likely caused by a larger scale event that would affect both watersheds, such as a climatological incident.

            We do not have any known sources of error because we did not perform the data taking, although, of course, there is some extent to which there is presumable error. If we had the necessary data, we would have examined the sediment buildup as related to increased detritus from calcium toxicity (as indicated by increased forest floor mass).  Another potential project is to find exactly through what means calcium functions to increase PO₄, SiO₂, pH, and SO₄. As hypothesized in the abstract, we believed that a reduction in acidic ions such as HSO₄^- due to their precipitating out of solution by reacting with calcium ions. As for the other compounds, the processes through which they are increased could be chemical reactions or physical, i.e. calcium toxicity causing increased detritus that decomposes into some of the above chemicals.

            Overall, it seems that the introduction of calcium into a forest ecosystem results in possible toxicity to trees, precipitation of natural substances, and significant changes in leaf chemistry.  This phenomenon should be examined further before calcium is implemented in any large-scale projects in forest biomes.

Works Cited:

Baribault, Thomas W., Richard K. Kobe, and Andrew O. Finley. "Tropical Tree Growth Is Correlated with Soil Phosphorous, Potassium, and Calcium, Though Not for Legumes." Ecological Monographs 82.2 (2012): 189-203. ESA Journals. Ecological Society of America, 2012. Web. 9 Apr. 2013. <http://www.esajournals.org/doi/abs/10.1890/11-1013.1>.

Fromm, J. "Wood Formation of Trees in Relation to Potassium and Calcium Nutrition."Tree Physiology 9 (2010): 1140-147. PubMed. US National Library of Medicine, National Institutes of Health, 2 May 2010. Web. 9 Apr. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/20439254>.

Hubbard Brook. "Watershed 1 Temporal Leaf Chemistry." Hubbard Brook Ecosystem Study. LTER Network, n.d. Web. 9 Apr. 2013. <http://hubbardbrook.org/data/dataset.php?id=45>.

University of Maryland Medical Center. "Manganese." University of Maryland Medical Center. A.D.A.M. Inc., 2011. Web. 9 Apr. 2013. <http://www.umm.edu/altmed/articles/manganese-000314.htm>.

Data from the Hubbard Brook Ecosystem Study Data Sets

http://hubbardbrook.org/data/dataset_search.php

Oral and Sublingual Immunotherapy as Treatment for Food Allergy and Anaphylaxis

An allergic response in the human body begins upon first exposure to the allergen.  (National Institute of Allergy and Infectious Diseases, 2013)  Upon the advent of the allergen, the immune system creates one type of antibodies, known as specific immunoglobulin E, with specific affinity to that substance.  (Lerner, 2010; National Institute of Allergy and Infectious Diseases, 2013.  For a diagram of an antibody, see image 1.)  A group of immunological molecules known as interleukins promote the cloning of these IgE antibodies.  (Robinson, 2013)  During the second exposure, there is a much larger immune response and elevated production flood the bloodstream with allergen-specific IgE molecules.  (Robinson, 2013)  These antibodies move throughout the bloodstream and attach to the antigen binding cites of specialized immune cells known as mast cells and basophils. (Urry, 2011; National Institute of Allergy and Infectious Diseases, 2013; Urry, 2011) IgE levels are typically very low in the bloodstream non-allergic person, but during an allergic reaction, the body begins producing excessive amounts of the allergen specific antibody.  (de Weck, 2012; Cohen, 2012)  The IgE molecules do not remain in the bloodstream for a long period of time.  (Cohen, 2012) Instead, immediately after its proliferation, the substance binds very strongly to the membrane of mast cells in bodily tissue and blood basophils, both of which are unique types of immune system inflammatory cells.  (Tsai, 2012; de Weck, 2012; National Institute of Allergy and Infectious Diseases, 2013)  As inflammatory cells, both mast cells and blood basophils contain “inflammatory mediators,” most commonly histamine and serotonin.  (de Weck, 2012)  The strong binding of IgE molecules to the high-affinity receptors of the inflammatory cells causes cross-linking between adjacent IgE molecules.  (Tsai, 2012; Cohen, 2012)  This cross-linking triggers a series of biochemical reactions and cascades within the mast cells that eventually result in the cell’s “degranulation.” (Tsai, 2012)  When the cell becomes “degranulated” its membrane bursts and massive amount of granule-associated mediators are released.  (Parker, 2007; Tsai, 2012)  These granule-associated mediators, or inflammatory mediators, are liable for the majority of signs and symptoms associated with allergic reactions.  (Tsai, 2012)  The most common granule-associated mediator in an allergic response is the molecule histamine, which induces inflammation in various tissues, although a variety of other molecules can be produced throughout the course of the reaction. (de Weck, 2012; Cohen, 2012)  Histamine induces dilation and increased permeability of small blood vessels in various body tissues and constriction of the bronchi.  (Robinson, 2013)  These symptoms result in fluid loss and swelling of the tissues.  (Robinson, 2013)  For diagrams of the cell response to IgE, see images 2-4.

Though the cause of food allergy is unknown, the disorder’s effects on the body are well documented and well understood.  (Staff, Mayo Clinic, 2011)  The mast cells, primary proliferators of an allergic reaction, are most common in the gastrointestinal tract, respiratory tract, and skin.  (Cohen, 2012)  Therefore, it is unsurprising that these areas are the most common sites of allergic reaction in the human body.  (Cohen, 2012)  Though allergic symptoms vary widely between individuals, a handful of them are common to most people who have food allergies.  These include, itching of the mouth, swelling of the lips and the tongue, symptoms that affect the gastrointestinal tract, including vomiting, diarrhea, abdominal cramps, and abdominal pain, hives, eczema and other skin issues, constricted throat or breathing, and a drop in blood pressure.  (National Institute of Allergy and Infectious Diseases, 2013)  The inflammatory mediators released by the affected cells bring on these archetypal symptoms.  (Tsai, 2012)  If the reaction is very severe, it can trigger a response known as anaphylaxis.  This response is marked by an incredibly wide range of symptoms, most notably, though, are swelling throughout the body tissues, wheezing, weak pulse, shock, and fainting.  (National Institute of Allergy and Infectious Diseases, 2013) Such reactions are frightening, unexpected, and have the potential to be deadly.  Children and adults who live with this condition have constant anxiety about anaphylaxis and other severe reactions from ingestion and contamination.  (Fleischer, 2013)  Allergies are the single most cause of days missed from school and work, and studies have shown that food allergy has significant effects on the social activities, meal preparation, and psychological state of children impacted by the condition.  (Flesicher, 2013; Bollinger, 2010; Lerner, 2010)  Though some children eventually grow out of their allergies as they age, many do not, especially those with peanut allergy.  (Cohen, 2012; Fleicher, 2013)

Currently, there are no cures for food related allergies.  (Fleischer, 2013)  For many other types of allergy, subcutaneous shots containing progressively higher amounts of the allergen have been shown to desensitize the patient and often reduce allergy symptoms.  (Fleischer, 2013)  However, when trials of subcutaneous injections were conducted on patients with peanut allergy, many individuals had adverse reactions, and the practice was deemed unsafe.  (Fleischer, 2013)  At the moment, patients with food allergies are advised to practice strict avoidance of the dangerous food.  (Cohen, 2012)  This can be very difficult, and there is a high likelihood of possible cross-contamination of foods in cafeterias and other public dining facilities.  If an individual’s food allergies are very mild, his or her symptoms can be treated with drugs known as antihistamines.  (Staff, Mayo Clinic, 2011)  These drugs negate the more mild effects of histamine and reduce the symptoms of the condition.  More severe allergic reactions and anaphylactic reactions are treated with epinephrine injections and trips to the emergency room.  (Staff, Mayo Clinic, 2011)  Epinephrine counteracts the symptoms of high levels of histamine, increasing blood vessel diameter, reduces blood vessel permeability, and relaxes the bronchi.  (Robinson, 2013)  Yet, these treatments are only temporary.  They treat the symptoms of allergies but they do not remedy the underlying intolerance that causes the reaction. 

For years, the means by which to remedy allergic intolerance has eluded scientific researchers.  There have been no broadly available therapeutic options for allergy suffers, and there have been myriad severe and fatal anaphylactic reactions brought on by contact with food.  (Fleischer, 2013) At long last, however, there is hope for those who suffer from these allergies, which comes in the form of oral and sublingual immunotherapy.  Oral and sublingual immunotherapies, first studied more than 100 years ago, have only recently come to the forefront of scientific research.  (Nowak-Wegrzyn, 2011)  During oral and sublingual immunotherapy, patients are administered small doses of the allergic food, either mixed into other non-allergic foods or under the tongue in extract form, respectively.  (Nowak-Wegrzyn, 2011)  The amount of allergens in these doses is gradually increased over the course of many weeks, resulting in an elevated tolerance to the substance in question. (Nowak-Wegrzyn, 2011) Patients beginning treatment first establish the amount of allergen they can consume without inducing a reaction.  (Fleischer, 2013)  After the initially dosage is established, each individual begins a series of daily build-up doses, each marginally greater than the last.  (Burks, 2012)  Patients periodically test their resistance to the allergen during “food challenges” wherein an individual consumes incremental amounts of the substance in order to determine his or her tolerance. (Fleischer, 2013)  The dosages can continue as long as the individual desires, or until an adverse reaction occurs.  (Burks, 2012)  The immunotherapy trains the body, through repeated expose, to tolerate what it had once rejected.  (Fessenden, 2012)

Though oral and sublingual immunotherapy procedures are still in trial phases, the results have been promising.  (Fleischer, 2013)  Sublingual immunotherapy treatment has been shown to raise tolerance in patients allergic to kiwi, hazelnut, peach, milk, and, most recently, peanut.  (Fleischer, 2013)  In individuals allergic to peanut, the most deadly known food allergy, sublingual immunotherapy raised tolerance from less than two grams of peanut protein to over ten grams in some cases.  (Fleischer, 2013)  Tolerance increased greatly when therapy was continued over many more weeks.  (Fleischer, 2013)  A similar study of oral immunotherapy conducted with egg protein was even more successful, with 70% of participants able to consume a cumulative dose of five grams of powder at ten months of treatment.  (Burks, 2012)  Oral and sublingual immunotherapy show great promise as food allergy treatment.  Measurements of the immune components of the subjects also revealed encouraging results.  In studies of the peanut specific antibody levels in immunotherapy patients, trial participants were found to have a decreased range of allergen-specific IgE molecules and greater levels of polyclonal allergen-specific IgG₄ serum.  (Vickery, 2012; Burks, 2013; Fessenden, 2012)  IgG₄ is another antibody, the serum of which is essential to the promotion of IgE in allergic reactions.  (Vickery, 2012)  There was also a decrease in peanut-specific interleukin production; interleukins, which promote the propagation of IgE molecules, are key to a strong allergic response.  (Blumchen, 2010; Robinson, 2013)  All of these biochemical signs correlate with a reduction or possible discontinuation of allergic response.  Furthermore, the treatment was found to be incredibly safe.  (Hofmann, 2009)  Very few individuals had adverse reactions, and those reactions were always in the hospital during build up days, where they could be safely treated.  (Hofmann, 2009) Unlike subcutaneous immunotherapy, which was discontinued because it was too dangerous for many patients, oral and sublingual immunotherapy treatments seem to be safe, even for those with serious peanut allergies.  (Blumchen, 2010; Hoffman, 2009)

Food allergy is a serious medical condition that affects approximately 20% of Americans today.  (Lerner, 2010)  For many years, individuals with the condition have lived in fear of anaphylactic shock and severe reaction.  (National Institute of Allergy and Infectious Diseases, 2013) A cure, other than symptomatic treatment, has long evaded researches and medical practitioners.  (Burks, 2012)  Recently, however, oral and sublingual immunotherapy have risen to prominence.  (Nowak-Wegryzn, 2011)  By slowly increasing consumption of allergen, these techniques increase body tolerance to the offending substance.  (Fleischer, 2013)  The treatment results in decreased diversity of allergen specific IgE and increased levels of allergen specific IgG₄, both of which are good indicators of decreased allergic response.  (Vickery, 2012)  The treatment was found to be both safe and very effective.  (Hoffman, 2013; Fleischer, 2013)  It seems that the future advancements for allergy suffers lie in the very substances to which they are allergic.  As oral and sublingual immunotherapy gain prevalence and become widely used, one day we may see a world without food allergy. 

Appendix

Image 1

Image from the Biology 570 Immunology PowerPoint

Image 2

This image gives a basic overview of the IgE propagation, binding, and subsequent histamine release in allergic tissue

Lewis, Ricki. An Allergic Reaction - Overview. McGraw-Hill Companies, Inc, Digital Image. Nutri-Living, nd. Web. 25 Feb. 2013. <http://dft.ba/-allergicreaction>.

Image 3

This image shows the specific IgE molecules on an immune effector cell, and their cross-linking in the presence of an allergen.

Nature Reviews. Allergen Activation and Cross-Linking of IgE molecules. Digital image. Nature. Nature Reviews, n.d. Web. 25 Feb. 2013. <http://www.nature.com/nrd/journal/v3/n7_supp/images/nrd1408-f1.jpg>.

Image 4

An image displaying the various paths of mast-cell activation.  Note the presence of IgE and allergens, as well as the granule-associated mediators leading to inflammation of the tissue.

Mast Cell Activation. Digital image. AccessScience. McGraw-Hill Education, 2012. Web. 25 Feb. 2013. <http://www.accessscience.com/content/Mast%20cells/900114>.

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