Chlorophyll composition in sediment cores at University Lake, Bloomington Indiana: Has land use in the watershed shifted organic matter content over time? Queen, Ryan S. Advisor: Elswick, Erika R. Department of Geological Sciences, Indiana University, 1001 E. Tenth Street, Bloomington, IN 47405 Correspondence: [email protected] INTRODUCTION: University Lake was originally built in 1911. The lake and dam are located on sixteen acres of land and now function primarily as a drainage impoundment for the IU Golf Course and surrounding forest as well as an educational center for students with the creation of the IU Research and Teaching Preserve. It was hypothesized that since the creation of University Lake, nutrient loading rates have increased due to increased development and land use in the watershed, especially after the construction of the IU Golf Course in 1957-­‐1960. Sediment cores of the lake were examined by measuring organic carbon and nitrogen content as well as chlorophyll-­‐a, -­‐b and -­‐c concentrations within the cores. By using chlorophyll concentrations in the sediment as a proxy, we tracked the changes in organic carbon and other organic matter input into the lake over time. The concentrations of chlorophylls a, b, and c were calculated; each type having a specific role in nature. The most important and abundant type is chlorophyll a, which makes photosynthesis possible, participating directly in the light-­‐requiring reactions of photosynthesis. Chlorophyll a is present in every organism that performs photosynthesis. Elevated concentrations of chlorophyll a can reflect an increase in aquatic nutrient loading and is also a potential indicator of overall water quality. The second type, chlorophyll b, occurs only in green algae and plants. It is an accessory pigment and acts indirectly in photosynthesis by transferring absorbed light to chlorophyll a to enter into the light-­‐requiring photosynthetic reactions. Chlorophylls a and b are complementary to each other and absorb light more efficiently at slightly different wavelengths. The ratio of chlorophyll a to chlorophyll b in the chloroplast of the organism is 3:1. A third form, chlorophyll c, is only found in the photosynthetic members of the kingdom Chromista as well as the dinoflagellates (UCMP 2009). Organic carbon and nitrogen contents from the University Lake sediment cores were measured. Previous sedimentation and nutrient loading research has been conducted on Griffy Lake in Bloomington, Indiana (Hill, 2002). Griffy Lake was created in 1924, and is located approximately one mile downstream from University Lake. Organic carbon and nitrogen results from sediment cores from the previous Griffy study (Hill, 2002) will serve as a baseline comparison to the results obtained from University Lake. FIELD METHODS: Three sediment cores were taken from University Lake in October 2008, one near the dam, one near the center of the lake, and another towards the south end. A Wildlife Supply Co® 2427-­‐B Ogeeche Corer was used to extract sediments from the stern of a boat. Each of these cores will be referred to as Dam, Center, and South core (Fig 1). They measured roughly 50, 20, and 90 centimeters (cm) in length respectively. After the cores were obtained, they were sealed into the plastic core tube liners and stored in a cold room at 4°C until lab research was initiated. 1 Figure 1: Illustration of the University Lake watershed and locations where the South, Center, and Dam cores were extracted. University Lake is down gradient from the IU Golf Course and the IU Research & Teaching Preserve Field Laboratory. It functions as a drainage basin for the golf course (in gray) and the deciduous woods that are immediately adjacent to the lake (the area in white on the map). LAB METHODS: The Dam core was taken from the cold room, extruded from the core liner and sliced in half lengthwise. Before chemical analysis, measurements and visual descriptions were taken, sketched and recorded from the archived half. Each centimeter section of the second half of the core was placed into a separate small beaker, covered lightly with foil and placed in a drying oven. After the individual centimeter sections were dried, they were ground into powder with an agate mortar and pestle. Ten to 15 milligrams (mg) of powder from each sample was then loaded into small tin capsules for carbon and nitrogen analysis on an elemental analyzer in the IU Stable Isotope Research Facility. The Center core was short and homogenous in composition, perhaps due to a rock or cobble in the lake bottom blocking the corer from penetrating the sediment. Because the core was so short and consisted of black, organic lake bottom muck, samples from it were used to determine the methodology and optimal sample amount for carbon, nitrogen, and chlorophyll analysis. Using the chlorophyll 2 experimentation methodology listed in the following paragraph, the ideal core sample mass was calculated to be between 10 and 20 mg for an optimal absorption reading on the spectrophotometer. The South core was the longest core and was used for chlorophyll analysis. The core was sliced in half lengthwise, measured and photographed, and half of the core was archived. Along the entire length of the remaining half, thin 1 cm notches were made to separate the core into discrete 1 cm segments. Between 10 and 20 mg of wet sample from each centimeter section of the core were placed into separate 15 milliliter (ml) centrifuge tubes, capped, labeled and covered in aluminum foil. It was essential to keep the centrifuge tubes covered with foil after extracting the samples from the core, to ensure chlorophyll within the samples was not degraded by light exposure. Six sample-­‐containing centrifuge tubes were processed at a time for spectrophotometric analysis in 4mL of 90% acetone soulution. The mixture was made by mixing 90mL of 100% reagent grade acetone with 10mL de-­‐ionized water. The mixture was then placed into centrifuge tubes with a 4mL volumetric glass pipette. The centrifuge tubes were stirred vigorously with a Vortex mixer for 60 seconds and then centrifuged at 2000 rpm for 6 minutes to separate the solid sample from the solvent to aid in minimizing turbidity. The chlorophyll-­‐containing acetone from the centrifuge tubes was then poured into quartz cuvettes and loaded into a Spectronic Instruments Genesys®5 spectrophotometer. Absorbance readings were taken for each sample at 750, 664, 647, and 630 nanometer wavelengths, blanking with 90% acetone reagent blanks before reading at each desired wavelength. Each cuvette was then acidified with 0.1mL of 0.1N HCl using an Eppendorf micropipette. After acidification the cuvettes were agitated, and left to sit for 90 seconds for acidification to take effect. Absorbencies for the acidified cuvettes were measured at 750 and 665 nanometer wavelengths, blanking again with acidified 90% acetone reagent blanks before reading at each desired wavelength. After all absorbencies were measured, they were entered into the following equations (A, B and C) to determine chlorophyll concentrations. The absorbencies in parenthesis were corrected for turbitidy by subtracting their original measured optical density (OD) values from the measured optical density readings at 750mL. The chlorophyll experiment procedure and following equations were adopted from the Standard Methods for the Examination of Water and Wastewater, 20th Edition (1998). Ca, Cb, and Cc in the following equations are chlorophyll concentrations in milligrams per Liter (mg/L), and OD 664, OD647, and OD630 are corrected optical densities with 1cm path lengths at respective wavelengths. Chlorophyll Calculations: A) Ca=11.85(OD664)-­‐1.54(OD647)-­‐0.08(OD630) B) Cb=21.03(OD647)-­‐5.43(OD664)-­‐2.66(OD630) C) Cc=24.52(OD630)-­‐7.60(OD647)-­‐1.67(OD664) RESULTS: Two of the three cores extracted yield lake stratigraphy. The two cores contain analogous organic-­‐rich layers and physical characteristics at 20cm, 36-­‐40cm, and 46cm deep, despite the difference in inlets feeding the different parts of the lake. 3 Figure 2: Physical illustrations of the Dam & South Cores. Both cores contain analogous organic-­‐rich layers and physical characteristics at 20cm, 36-­‐40cm, and 46cm deep. At a depth of 20cm in the cores there is a gradual change from solid, grey clay to a soft, black muck. From 36-­‐40cm deep, distinct color changes in both cores occur, from dark yellow clay to a grey, organic-­‐
rich band. At 46cm deep, both cores display a distinct color change; from a grainy tan band to a smooth grey, organic-­‐rich band. 4 University Lake Chlorophyll ConcentraOons Chlorophyll concentraOons (mg/L) 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0 10 Core depth (cm) 20 30 chlorophyll a 40 chlorophyll b 50 chlorophyll c 60 70 80 90 100 Figure 3: Total chlorophyll concentrations in the North Core. Chlorophyll a, b, and c concentrations remain relatively constant the entire length of the core until reaching a depth of 25 cm, where chlorophyll a concentrations drastically increase near the surface of the sediment-­‐water interface. The data show that chlorophyll values range from 0 to roughly 0.3 mg/L, and that chlorophyll concentrations decrease with increasing depth in the core. Chlorophyll a concentrations steeply rise close to the surface of the core at the lake bottom, with a maximum concentration close to 0.3 mg/L. Below 25cm, chlorophyll a, b, and c concentrations are at nearly equal concentrations. Three distinct bands occur within the core where chlorophyll concentrations exceed the expected average values for similar depths, at roughly 40, 58, and 79 centimeters deep. At these depths chlorophylls b and c are also at a higher concentration than chlorophyll a. The University Lake carbon and nitrogen results from the elemental analyzer are shown below. These graphs will also be plotted with previous Griffy Lake core data (Hill & Elswick, 2003) in the Discussion section to serve as a baseline comparison since they are similar bodies of water in a similar watershed. 5 % Nitrogen-­‐ University Lake % Nitrogen 0 0.1 0.2 0.3 0.4 0.5 0.6 Core depth (cm) 0 10 20 30 40 50 60 % Carbon-­‐ University Lake % Carbon 0 1 2 3 4 5 6 7 Core depth (cm) 0 10 20 30 40 50 60 Figure 4: Nitrogen and Carbon percentages in the North Core. The graphs indicate that nitrogen concentrations mimic total carbon concentrations in University Lake, with the abundance ratio of carbon to nitrogen 10:1. The percentage of carbon mimics that of nitrogen in the University Lake South Core, with carbon being ten times more abundant than nitrogen. From a depth of 50 to 30 centimeters the percentages remain relatively constant, but a distinct break at 30 centimeters in the core shifts the nitrogen and carbon content to the left, indicating a slight decrease in total C and N percentages. From 25cm to the surface of the core there is a steady linear increase of C and N content, to maximum values of slightly more than 6% and 0.55% respectively. Physical core data reflect this trend as well, with an increase of grey, organic-­‐rich sediment starting at 25cm deep that changes to black, organic muck nearing the sediment-­‐
water interface of the each of the cores as shown in Figure 2. 6 DISCUSSION: Even though the University Lake chlorophyll data show relatively constant concentrations around 0.05mg/L the entire depth of the core, there are a few distinct areas that deviate from the normal trend. At core depths of 40, 58, and 79cm deep in the South Core there are elevated chlorophyll concentrations. 40cm down there is a bleb of black organic matter in a grey organic-­‐rich section of the core. At a depth of 58cm there is a section of gritty, not fully decomposed grey matter within a yellow clay band. At 79cm deep in the core, grey organic matter appears in yellow clay; the deepest depth in the core that organic rich sediment is identified. Each of these three depth levels coincide with darkened bands within the cores, which are possibly remnants of partially decomposed leaf or organic matter where chlorophyll concentrations still remain at elevated levels. At these depth levels, chlorophyll b and c concentrations are higher than chlorophyll a concentrations, which may be due to active decomposition of organic matter still occurring within the cores. At 25cm deep, chlorophyll a concentrations also show a sharp spike and constant increase, continuing until the surface of the core, where chlorophyll a concentrations are roughly five times greater than expected core concentrations. It may be speculated that the layer 25cm deep within the core coincides with the construction of the IU Golf Course, constructed in 1957. The continued annual use of fertilizers and organic matter on the golf course would explain the drastic increase in chlorophyll a concentrations, as fertilizer runoff would increase the aquatic algal and plant vegetation population in University Lake. Another possible conclusion to the sharp chlorophyll spike is a continued rainy season or numerous continual years of above average rainfall, which would dump an excess load of organic matter into University Lake. The excess of organic content loaded into the lake would take much longer to decompose, and would be a reasonable explanation of the elevated chlorophyll levels near the surface of the core. The percent nitrogen and organic carbon results from elemental analysis indicate that nitrogen concentrations closely mimic total carbon concentrations in University Lake, with the abundance ratio of carbon to nitrogen 10:1. It is suggested that nitrogen in the core sediments is organic nitrogen transported with organic carbon. As was the case in the chlorophyll data, there is a distinct break from a core depth of 25 to 30cm, shifting the nitrogen and carbon content to the left and indicating a decrease in carbon and nitrogen percentages. This depth range of 25 to 30cm in the core demonstrating unchanging carbon and nitrogen levels may coincide with the construction period of the golf course, which lasted 3 years. The steady linear increases in nitrogen and carbon percentages from 25cm to the surface of the core have likely been caused by increased fertilizer and organic runoff from the watershed into University Lake, inferably after construction of the IU Golf Course was complete. The sedimentation rate of University Lake needs to be established to further the understand sediment and organic matter loading into the lake. The sedimentation rate at Lake Griffy was established in 2002 to be from 7.3mm per year near the water inlet to 3.7mm per year at the Dam, and will serve as a good baseline comparison to University Lake once future sedimentation data is collected (Hill & Elswick, 2002). Graphs comparing University Lake and Griffy Lake nitrogen and carbon data are shown below. The Griffy Goose core was taken at the water inlet of Griffy Lake in very shallow water, which is also a waterfowl resting area where Geese commonly congregate. Organic content in the Goose core is higher than in the Griffy Dam core due to the aforementioned items. The Griffy Dam core will serve as a better comparison to the University Lake North core, due to the fact that both cores were taken in deep water and will have closer relative organic content values. 7 Nitrogen: Griffy & University Lake Core Comparison % Nitrogen 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 Core Depth (cm) 10 Griffy Goose 20 Griffy Dam 30 University Lake 40 50 60 70 Figure 5: Nitrogen percentages remain relatively constant until 25cm deep within the cores, where nitrogen percentages in the University Lake core drastically increase near the surface of the core, nearly tripling that of Griffy core percentages. Carbon: Griffy & University Lake Core Comparison % Carbon 0 2 4 6 8 0 Core depth (cm) 10 Griffy Goose 20 Griffy Dam 30 University Lake 40 50 60 70 Figure 6: Carbon percentages in each core are relatively equal showing a gradual increase of carbon with decreasing depth. At 25cm deep University Lake carbon percentages drastically increase nearing the surface, tripling that of Griffy carbon percentages. In comparing the Griffy Lake and University Lake core data above, some interesting results are found. When looking at carbon and nitrogen core percentages, the data suggest that the rate of nutrient loading in Griffy Lake has remained fairly constant over the years. But for University Lake the data indicate substantial nutrient loading. From about 20cm deep to the surface of the University Lake core, 8 carbon and nitrogen percentages drastically increase, with a steadily increasing linear curve. Carbon and nitrogen concentrations at the surface of the University Lake core are roughly 3 times larger than in the Griffy Lake cores, demonstrating that substantial nutrient loading has occurred in recent years at University Lake, while Griffy Lake has remained relatively stable. The most interesting data occur from 25 to 30cm deep within the cores. At this depth range, chlorophyll, nitrogen, and carbon levels in the Griffy and University Lake cores are almost equal. But starting at 25cm in the University Lake core, values start deviating considerably from the Griffy cores. It can be inferred that this deviation has arisen due to substantially higher nutrient loading in the University Lake watershed, mainly due to the construction of the IU Golf Course or recent years of above-­‐average rainfall. For anyone interested in furthering research at University Lake or expanding upon the above research, there are a few suggested recommendations. The University Lake sediment cores have yet to be dated, and this can be done using gamma counting to determine Cs-­‐137 and Pb-­‐210 levels at different depths within the cores. The depth producing the highest Cs-­‐137 count can be assumed to correspond to the year 1964, when thermonuclear testing was at a maximum. Pb-­‐210 radiometric dating could also be used, as it corresponds to an exponential decay curve, with an equation describing the decay to be calculated. With age data on the cores, sedimentation rates could be determined, as well as a dated analysis on how land use in the watershed through time has affected University Lake. The bulk density of University Lake also still needs to be determined. With a bulk density calculated, chlorophyll a concentrations could be refigured according to separate equations not used in this report, located in Standard Methods (1998). Griffy Lake core data conducted in 2002 was used as a corollary to the University Lake cores. But no chlorophyll research has been done at Griffy Lake as of yet. To better understand Griffy Lake and corroborate Griffy data to University Lake data, new cores should be taken at Griffy Lake with chlorophyll research being conducted. ACKNOWLEDGEMENTS: I would first like to thank Dr. Erika Elswick for her continued guidance, support, and funding throughout the duration of my research. The majority of lab analysis and sample preparation was performed in her Analytical Geochemistry Laboratory. I would also like to thank Dr. Peter Sauer and the Stable Isotope Research Facility for assistance with elemental and isotopic analysis on the sediment cores. Thank you to Dr. Chris Craft’s lab, in particular John Marton, for use of their spectrophotometer. Finally, I would like to thank the IU Research and Teaching Preserve for the research grant received to assist in funding this project. REFERENCES: American Public Health Association, American Waterworks Association and the Water Environment Federation. Standard Methods for the Examination of Water and Wastewater, 20th Edition. 1998, p.10.18 -­‐ 10.25. Hill, Megan, “An Investigation of carbon, nitrogen, and sediment loading at Griffy Reservoir, Bloomington, IN.” Department of Geological Sciences, BSES, Indiana University, 2002. University of California Museum of Paleontology. “Photosynthetic Pigments”. Regents of the University of California, copyright 1994-­‐2009. http://www.ucmp.berkeley.edu/glossary/gloss3/pigments.html. 9