Grazing and Production by Ribbed Mussels (Geukensia demissa) in Little Pond: A Nitrogen Budget. Kimberly R. Meneo. Connecticut College

Transcription

1 1 Grazing and Production by Ribbed Mussels (Geukensia demissa) in Little Pond: A Nitrogen Budget Kimberly R. Meneo Connecticut College 13 December 2014 Adviser: Dr. Ken Foreman, Ecosystems Center, Marine Biological Laboratory, Woods Hole, MA 02543

2 2 Abstract A dramatic increase in coastal population has led to the degradation of coastal ponds and other systems. One of the biggest impacts human population has had on these systems has been an increase in nitrogen leaching. Previous scientific work has shown that shellfish have the potential to impact nitrogen fluxes at the ecosystem level. This experiment explores the filter-feeding rate of the Ribbed Mussel (Geukensia demissa), and to develop a carbon and nitrogen budget for them by observing the amount of particulate carbon and nitrogen taken up and released by the mussels in small aquaria. Additionally, the amount of carbon and nitrogen found in both the tissue of the mussel and the mussel shell was analyzed. 91% of nitrogen uptake was determined to go toward growth, with 6% being released in feces and pseudofeces. 3% of nitrogen was released in ammonia excretion. 33% of carbon went toward growth, with another 33% released in the feces and pseudofeces. 34% was released in the form of ammonia. The data found in these experiments was then used to determine if ribbed mussels could effectively remove excess nitrogen entering Little Pond, located in Falmouth, Massachusetts. Results showed that mussels do have potential in removing nitrogen from coastal systems, but that many variables impact the effectiveness of these shellfish. Key Phrases and Words: Geukensia demissa Ribbed Mussel, Little Pond Falmouth, MA, Nitrogen Uptake, shellfish, nitrogen loading

3 3 Introduction Nitrogen loading to coastal ponds has increased dramatically within recent years. Eutrophication as a result of nitrogen inputs from septic tanks, agricultural land, and combustion of fossil fuels has led to an increase in production of algal biomass and toxic algal blooms. These blooms have altered species distributions and caused decreases in species distribution and dissolved oxygen levels (Darnell and Soniat, 1981). Geukensia demissa, commonly known as the Ribbed Mussel, is a marine bivalve found along the Atlantic coast. Ribbed Mussels are filter feeders, capable of efficiently filtering large volume of water and removing the phytoplankton and particulate detritus. A portion of the ingested phytoplankton are digested, while other particulate matter is rejected in the form of pseudofeces (Dame 1996). Ribbed mussels are of particular interest, as they are known to be more efficient in filtering smaller cells such as bacteria, and can survive in temperatures up to 45 degrees Celsius and salinities ranging from 6ppt to 70ppt (Dame 1996, Puglisi). In 1976, it was discovered that bivalve filter feeders could influence nitrogen fluxes at the ecosystem level, and that cultivation of bivalve feeders could induce nitrogen fixation in marine sediments at a greater rate (Dame 1996). Therefore, a strong possibility exists that Ribbed Mussels could be used to alleviate nitrogen loading from coastal marine systems. According to a report released by the Commonwealth of Massachusetts Executive Office of Energy and Environmental Affairs, about 50% of nitrogen loading to Little Pond (a 300 hectare pond in Falmouth, MA) is caused by septic

4 4 leaching, 40% from land use, with the rest from sediments, natural background, and the atmosphere. As of 2008, the average nitrogen loading to the Little Pond embayment system was kg/day, and the concentrations of nitrogen within the system ranged from 3.18 mg/l near the head of Little Pond, to 0.49 mg/l in the lower section (Bowles 2008). Due to the top-down control of shellfish on phytoplankton concentrations, shellfish can be used to control algal blooms and to sequester nutrients and bury nitrogen in sediments through the release of feces and pseudofeces (Uline 2013). Preliminary field work showed that there is less than one ribbed mussel per square meter within Little Pond. This leaves a high potential for more Ribbed Mussels to be introduced, as a way to alleviate nitrogen loading to the system. A nitrogen budget has been developed to determine the efficiency of Ribbed Mussels, and to determine if how many mussels would be needed for effective remediation of nitrogen in Little Pond. Methods Grazing Experiments: Grazing rates were determined for 15 different mussels of varying sizes from both Little Pond and Little Sippewissett Marsh, located in Falmouth, MA and West Falmouth, MA, respectively. Grazing experiments were run for a total of 10 hours each. Each mussel was placed in a 10-liter aquarium, filled with 7 liters of 1 ųm filtered seawater. Prior to the experiments, each mussel was held in an aquarium filled with filtered seawater for a minimum of 24 hours in order for them to clear their digestive tract, so that accurate measurement of feces and pseudofeces production could be obtained. For the feeding experiments, mussels were

5 5 suspended over two small tins for the duration of the feeding experiments as well as for 12 hours after the termination of the feeding experiment (for a total of 22 hours).. Before beginning the experiments, 5 different species of phytoplankton cultured at the Marine Resources Center were analyzed for their concentrations. These species were Chaetoceros neogracile, Chaetoceros calcitrans, Pavlova sp., Isochrysis galbana, and Tetraselmis chui. The concentration of chlorophyll in a mixture of the stock solution (equal quantities of each species) was measured. Then, using the equation C1V1=C2V2, the quantity of phytoplankton needed for each grazing experiment was determined. A mix of equal quantities of each species of phytoplankton was then added to the 7L of filtered seawater in each experimental tank, bringing the initial concentration of phytoplankton in each experiment to 40 ug Chl a/l. This is comparable to the peak chlorophyll concentration measured within Little Pond (Howes et al ). Each tank was then set on top of a magnetic stirrer, and a stir bar was placed in the tank. When turned on at the lowest speed possible, the stir bar effectively kept the phytoplankton suspended within the water in the tank. Each grazing experiment was run for a total of 10 hours. Chlorophyll levels were measured by filtering and extracting chlorophyll in 90% acetone and measuring absorbance on a spectrophotometer before and after acidifying (Lorenzen 1967), as well as by in-vivo fluorescence (Turner Designs, 1999). The Lorenzen method was using for initial and final chlorophyll readings for each experiment, while a fluorometer was used to measure chlorophyll levels hourly. The in-vivo fluorometric measurements were then corrected to actual Chl a based upon

6 6 measurements taken from the stock solution of phytoplankton diluted to concentrations of 0, 2.5, 5, 10, 20, 40, and 200 ųg/l and analyzed using both in-vivo and spectrophotometric methods. The in-vivo measurements were then plotted against the spectrophotometric measurements to form a linear regression, from which all in-vivo measurements during the feeding experiments could be converted to actual Chl a readings. From these numbers, the filtration rate (K) of each mussel per hour was then calculated using the change in in-vivo measurements from 0 hours (T1) to 10 hours (T2), by fitting a regression to the observed data, where chlorophyll levels at Tx > Ty+1. This equation can be written as: K= (lnt1- lnt2)/δt. Therefore, the volume of chlorophyll consumed per hour per mussel is equal to the filtration rate multiplied by the volume of water in the aquarium. Data was occasionally emitted to reflect only the time that the mussels were actually filtering phytoplankton. At T 0 hours and after 10 hours, I filtered between 60 and 270 ml of water sample onto a pre-ashed 25mm GF/F filter to collect the POC/N. These samples were dried in a 60 degree Celsius oven until they could be analyzed using a Thermo Scientific N/C Analyzer. The mussels remained in the tank overnight in order to allow them to discharge any remaining feces and pseudofeces from their digestive tracts. After approximately 13 hours, feces and pseudofeces were then collected using a transfer pipette and filtered onto a pre-ashed 25 mm GF/F filter. These filters were then dried in an oven on small petri dishes. Using a CN analyzer, these filters were analyzed for carbon and nitrogen content. These measurements were then

7 7 compared to the carbon and nitrogen concentrations found at hours 0 and 10 of the feeding experiments. The carbon and nitrogen concentrations of the feces and pseudofeces were subtracted from the total carbon and nitrogen consumed by the mussel to account for the amount of carbon and nitrogen consumed that contributed toward mussel growth and shell composition. Respiration and Excretion: In order to estimate respiration and excretion of the mussels, oxygen uptake and ammonia production were measured. After removing the feces and pseudofeces from the tanks, each mussel was transferred to a small, cylindrical, sealed chamber with a volume of about 1.6 liters. Care was taken to exclude air bubbles when sealing the chamber. Oxygen consumption was measured and water samples were taken for analysis of ammonium, both at hours 0, 3, 5, 7, and 10. Water removed for ammonium samples was then replaced with filtered seawater. Oxygen was measured using a WTW oxygen probe, and the ammonium samples were filtered through pre-ashed 25 mm GF/F filters, acidified, and stored in a fridge. Ammonium samples were then analyzed using protocol developed by Strickland and Parsons (1972) and modified based upon the phenol-hypochlorite method of Solarzano (1969.) Size and Age Assessment: Twenty mussels, ranging in length from mm to 33.3 mm, were selected for size and age determination. The exterior of each shell was cleaned by removing attached organisms and detrital material. Mussels were placed in filtered seawater for 24 hours prior to tissue extraction to remove any material from their digestive

8 8 tracts. Using an Neiko 0-150mm digital caliper, mussel length (anterior to posterior), width (ventral to dorsal), and thickness were measured. Mussels were then microwaved for a maximum of 30 seconds to open their shells. Mussel tissue was removed from the shell, and using an Ohaus Adventurer Balance, the wet weight of both the mussel meat and the wet weight of the shell were recorded. Both were then placed in an oven and dried for 1 week at 60 degrees Celsius. Both the shell and the tissue were then measured again for dry weights. Tissue and shells were then broken up and ground into powder using a Wigglebug for CN analysis in order to estimate the mass of carbon and nitrogen biomass for each mussel. The periostracom taken from one valve of each mussel shell was removed by placing it into 5% sodium hypochlorite for 24 hours in order to more easily visualize the growth rings and estimate mussel age (Brousseau 1984). Results Results from chl a measurements using the Lorenzen method for the feeding experiments were inconclusive, but in vivo chlorophyll was converted into actual chlorophyll using a linear regression (Figure 1). Overall, the average grazing rate per mussel was ųg of phytoplankton per hour. Mussel grazing rates were then compared with the length and total volume of the mussel, respectively (Figure 2, Figure 3). In both cases, as mussel length and volume increased, the grazing rate of mussels increased as well. These trends showed a strong correlation, with R 2 values of 0.78 and 0.77 respectively. This data allows for an estimation of feeding rate to be calculated for any mussel with a known length or volume (Table 3).

9 9 Measurements of ammonium excretion and respiration were taken for each of the grazing experiment mussels. Oxygen uptake rates were strongly linear, whereas ammonium excretion rates were less so (Figures 4 and 5, respectively). Interestingly, rate of oxygen uptake was most closely correlated to mussel thickness (Figure 6). Oxygen uptake was translated to amount of carbon sequestered by assuming a molar ratio of 1:1 drawn from the chemical equation for respiration. Data acquired from particulate organic carbon and nitrogen during the feeding experiments as well as from the oxygen uptake and ammonium release was compiled to form both a carbon and nitrogen budget for each individual mussel and then amassed to create an average budget (Figures 7 and 8). Consumption was calculated by subtracting the final particulate carbon and nitrogen from the initial particulate carbon and nitrogen found in the phytoplankton. Pseudofeces and feces egestion was directly analyzed for carbon and nitrogen contents, along with respiration and oxygen uptake (as described above). The average nitrogen budget for a ribbed mussel contributes about 91% to growth, while 6% is excreted in feces and pseudofeces, and 3% is excreted in the form of ammonium. The average carbon budget for a ribbed mussel contributes about 33% to growth, while 33% is excreted in feces and pseudofeces, and 34% is respired. There was little correlation between mussel size and carbon and nitrogen consumption. Data for individual mussels can be found in Tables 1 and 2. Results from the carbon and nitrogen composition of mussels showed mussel shells to be an average of 12.61% carbon and 0.553% nitrogen. Mussel tissue was composed of an average of 38.4% carbon and 8.65% nitrogen. Tissue dry weight,

10 10 mass of nitrogen, and mass of carbon all increased exponentially with length (Figure 9). The equations derived from these charts can be extrapolated to determine these measurements for any mussel with a known length (Table 3) See Table 4 for a complete list of mussel size and percent composition of carbon and nitrogen. Discussion The data collected and studied in this experiment can be extrapolated and used to estimate the total dry mass as well as the carbon and nitrogen mass of any Ribbed Mussel of a known length. Additionally, knowing the length of any mussel, approximate age can be determined. Age can then be compared to either total dry mass or carbon or nitrogen mass. Lastly, filtration rate versus the volume of the mussel showed a positive correlation, and knowing the volume of any mussel, the rate at which it will filter phytoplankton can be estimated. These equations may be of particular use in similar studies, or for expanding upon the results found in this experiment. Although budgets for both carbon and nitrogen were calculated, it is possible that the nitrogen budget does not accurately portray the typical budget. Studies completed previously have shown that nitrogen uptake is greatest in the fall season, when this study was completed (Jordan and Valiela). However, it should be noted that fall is also the time of year when most nitrogen would be entering the system, due to the increase in summer residents in the area surrounding Little Pond, and due to the delay in time it takes for nitrogen removal from lawn and septic systems to reach the pond.

11 11 It is likely that additional carbon and nitrogen is used to development of byssal threads and other bodily functions, which was not accounted for in my experiments. Therefore, a smaller amount of both carbon and nitrogen is actually being used for mussel tissue growth than what was reported in these experiments. Ammonia excretion, in particular, likely plays a larger role than was found through these experiments. The chamber the mussels were placed in was fairly large, so ammonia concentrations were so low that it was hard to distinguish any measureable changes over a period of ten hours. Using a smaller chamber would have provided greater changes in ammonia concentrations. Using information discovered from these experiments, it is then possible to estimate the number of mussels needed to filter the excess nitrogen coming into Little Pond. As stated earlier, the nitrogen loading to Little Pond is approximately kg/day, and the area of little pond is 300 hectares, or 3 million meters squared. From these experiments, the average uptake rate per mussel was determined to be about 1.06mg of nitrogen every ten hours. It was also determined that about 94% of nitrogen that was taken up is retained in each mussel. Using this information, it is possible to calculate the number of mussels needed to filter that much nitrogen (Appendix I). Approximately 10 million mussels would be needed in order to do this, which works out to be about 3.3 mussels per square meter. Based upon this calculation, mussel addition to Little Pond may be a way to remediate nitrogen loading. However, there are many other variables that come into play. Firstly, it is unknown if all of the excess nitrogen coming into Little Pond is in a form that can be filtered by the mussels. Also, these mussels must be placed over

12 12 an area where they can access the nitrogen coming into the system. It is also important to note that Little Pond is not a closed system, so some of the excess nitrogen surely leaches out into the ocean. Lastly, unlike other types of shellfish, ribbed mussels are not typically used as food source. Because of this, the mussels taking up the nitrogen are unlikely to be removed from the pond, and the nitrogen will remain within the system. Although ribbed mussels seem to be an important sink for excess nitrogen, more research must be done in order to determine if they could effectively help to restore impacted systems, giving the rate of filtration per hour. After completing this calculation, an exponential decay curve was fitted to the filtration rates observed in the feeding experiments by taking the natural logs and finding the slope of the line. Multiplying that number by the volume of the aquaria (7L), the volume of water filtered per hour is given. Based upon these values, it was determined that the mussels used for the grazing experiments filtered at an average rate of L/day. Multiplying that by the volume of Little Pond, about 5.3 million mussels would be needed to filter all of the water in the pond, which is a little more of half of the mussels needed to filter all of the nitrogen entering the system. However, assuming the mussels only filter during high tide, they would filter approximately 18.6 L/day. In this case, it would require 10.5 million mussels to filter all of the water: only about 3.5 mussels per square meter.

13 13 Acknowledgements Thank you to the Marine Biological Laboratory and especially the Ecosystems Center for providing me with the proper funding and expertise to complete this project. I would especially like to acknowledge Rich McHorney, Fiona Jevon, Tyler Messerschmidt, Nick Barrett, and Ken Foreman for their guidance and never-ending willingness to help me. Lastly, I would like to thank Scott Lindell and Dave Bailey at the Marine Ecosystems Center for allowing me to use their phytoplankton.

14 14 Literature Cited: Bowles, Ian A., Laurie Burt, and Glen Haas. Little Pond Embayment System Total Maximum Daily Loads for Total Nitrogen Mass.gov. Brousseau, Diane J. Age and Growth Rate Determinations for the Atlantic Ribbed Mussel, Geukensia demissa Dillwyn (Bivalvia: Mytilidae). Estuaries 7:3: Dame, Richard F Ecology of Marine Bivalves: An Ecosystem Approach. CRC Press Inc., Boca Raton, FL, USA. Darnell, Rezneat M. and T.M. Soniat Nutrient Enrichment and Estuarine Health. Pages in B.J. Neilson and L.E. Cronin, editors. Estuaries and Nutrients. Humana Press, Clifton, New Jersey, USA. Howes, B.L., et al. Linked Watershed-Embayment Model to Determine Critical Nitrogen Loading Thresholds for Little Pond System, Falmouth, MA. Boston: SMAST/DEP Massachusetts Estuaries Project, Massachusetts Department of Environmental Protection, Lorenzen, C.J Determination of Chlorophyll and Pheo-pigments: Spectrophotometric Equations. Limnology and Oceanography 12: Jordan, Thomas E. and Ivan Valiela A nitrogen budget of the ribbed mussel, Geukensia demissa, and its significance on nitrogen flow in a New England salt marsh. Limnology and Oceanography. 27: Puglisi, Melany P. Geukensia demissa. Smithsonian Marine Station, 2008.

15 15 Solarzano, L Determination of Ammonium in Natural Wates by Phenol Hypochlorite Method. Limnology and Oceanography. 14: Strickland, J.D.H. and T.R. Parsons A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada. Uline, Nicholas M Powerhouse Plankton Removers: Can shellfish remediate nutrient loading to the coastal pond ecosystems? Gettysburg College, Gettysburg, PA, USA.

16 Spectrophotometer Readings- Lorenzen (ug/l) y = x R² = Fluorometer Readings- in vivo (ug/l) Figure 1: Recorded chlorophyll levels from the dilutions of phytoplankton mixture used in the feeding experiments. Dilutions were made at concentration of 0, 2.5, 5, 10, 20, 40, and 200 ųg/l. Results from the Lorenzen method measured spectrophotometrically were then compared with in vivo results measured fluorometrically in order to create a linear regression. Using this regression, in vivo chlorophyll measurements can be converted to actual chl a concentrations.

17 Filtration Rate: L filtered/mussel/hr Filtration Rate: L filtered/mussel/hr y = x R² = Mussel Length (mm) Figure 2: Graph depicting the correlation between mussel length and filtration rate of that mussel. As mussel length increases, so does the rate at which they are able to filter phytoplankton y = 4E-06x R² = Mussel Volume (mm 3 ) Figure 3: Graph depicting the correlation between mussel volume and filtration rate of that mussel. As mussel volume increases, so does the rate at which they are able to filter phytoplankton.

18 Oxygen (mg/l) y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = Time (Hours) Figure 4: Oxygen consumption in milligrams per liter of seawater over time (in hours) for individual mussels used in feeding experiments. y = x R² =

19 Ammonium (umol/l) y = 7E-05x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = y = x R² = Time (Hours) Figure 5: Raw absorbance values of ammonium excretion in micromoles of seawater per liter over time (in hours) for individual mussels used in feeding experiments.

20 Mussel Thickness (mm) y = 0.007x R² = O2 Uptake: mg O2 consumed/mussel/hr Figure 6: Rate of O2 uptake as compared to mussel thickness. As the rate of oxygen consumption increased, so did the measured thickness of the mussel.

21 mg Carbon consumed mg Nitrogen consumed Mussel Growth Respiration Feces+Pseudofeces Mussel Figure 7: Fate of both carbon and nitrogen consumed by each mussel used for feeding experiments. Budgets are totaled by subtracting the mass of C or N found in feces and pseudofeces as well as the mass found in either respiration or excretion from the total amount of phytoplankton consumed in the grazing experiments. The mass left was assumed to be the N or C used to contribute toward mussel growth.

22 (6%) Nitrogen (3%) (91%) Feces+Pseudofeces NH4 Excretion Growth Carbon Feces+Pseudofeces Respiration Growth 0.99mg (33%) 0.98mg (33%) 1.04mg (34%) Figure 8: Average fate of carbon and nitrogen consumed by all mussels used for feeding experiments. Data averaged from that shown in Figure 7. Data is shown in terms of average mass as well as average percentages of sum total.

23 Carbon Mass (g) Nitrogen Mass (g) Total Dry Mass (g) y = x R² = Length (mm) y = x R² = Length (mm) y = x R² = Length (mm) Figure 9: Relationships between total dry mass, nitrogen mass, and carbon mass (all in grams) as compared to mussel length (in millimeters). All graphs show an exponential relationship between the x and y values.

24 24 Mussel Length (mm) Mean C Consumption (mg/10hr) C Feces+Pseud o (mg/10hr.) Respiration (mg C/ 10 hr) Assimilation (Consumptio n- Feces+Pseud o) Net Growth (mg C /10hr) % Assimilation Efficiency (Assimilation /Consumptio n) % Net Growth Efficiency (Growth/Assi milation) Table 1: Total mass of carbon consumed for all mussels used in feeding experiments. Includes the mass of biodeposits, mass of carbon lost through respiration, assimilation, assimilation efficiency, and net growth efficiency. Average values for all experiments are given in the far right column.

25 25 Mussel Length (mm) Mean N Consumption (mg/10hr) N Feces+Pseud o (mg/10hr.) *Excretion (ug N/ 10 hr) Assimilation (Consumptio n- Feces+Pseud o) Net Growth (mg N /10hr) % Assimilation Efficiency (Assimilation /Consumptio n) % Net Growth Efficiency (Growth/Assi milation) * Excretion shown in micrograms not milligrams due to the small amount of ammonium produced. Table 2: Total mass of carbon consumed for all mussels used in feeding experiments. Includes the mass of biodeposits, mass of carbon lost through respiration, assimilation, assimilation efficiency, and net growth efficiency. Average values for all experiments are given in the far right column.

26 26 Mussel Width (mm) Thickness (mm) Length (mm) Initial chl a (ug/l) Final chl a (ug/l) R 2 value Number of points used Rate of Filtration Estimated Weight (g) Table 3: Table depicting the mussel width, thickness, and length used from feeding experiments as well as an estimated weight of each mussel, computed using the equation taken from Figure 9. In addition, initial and final chlorophyll concentrations are given, as well as the rate of filtration of that mussel during that experiment, as well as the number of data points used to calculate the filtration rate and the R 2 value of that correlating line.

27 27 Width Thickness Length DW Tissue DW Shell Tissue % N Mussel (mm) (mm) (mm) (g) (g) Shell % C Shell % N Tissue % C Table 4: Table depicting the number of mussels used to determine the percent concentrations of carbon and nitrogen in both mussel shell and tissue. Dry weights of both the shell and tissue were also recorded, as well as the length, width, and thickness of each mussel.

28 28 APPENDIX I: Calculation of number of mussels needed to remediate nitrogen loading to Little Pond - N loading to Little Pond: kg/day - Area of Little Pond: 3,000,000m 2-94% N retention per mussel (Figure 8) mg N/10hrs average uptake (Table 2) ( 1.06mgN 10hours ) hours (24 10hours 1day ) = 2.54mgN/day ( 2.54mgN 1kg ) ( day 1,000,000mg ) = kgn mussel/day 22.76kgN mussel day = 9,895,653mussels kgn mussel day 9,895,653mussels 3,000,000m 2 = 3. 3mussels m 2