Essay: Thesis Proposal about Shell – Free Essay Example

Nitrogen isotope records in Unionid mussels: assessing fidelity to stream conditions and post-mortem shell degradation

Statement of Purpose

Several recent studies have demonstrated that nitrogen isotope measurements (15N) in the organic fraction of mollusk shells can give insight into nutrient flux and some anthropogenic impacts on habitats. For example, Oczkowski et. al. (2016) used Native American shell middens to measure 15N values in clam shells, showing insight into how nitrogen dynamics have changed over thousands of years in coastal ecosystems, and providing historical information regarding nitrogen reduction. Carmichael et. al. (2008) measured differences in 15N between shell material and soft tissue, a method that could be applied to refine modern and historical assessments and discern natural from anthropogenic influences on coastal ecosystems. Black et. al. (2017) demonstrated the potential utility of shell 15N proxies, suggesting that detailed records of anthropogenic nitrogen can be created from shell-midden deposits. The baseline collected from the Black et al. study is not only valuable in addressing modern pollution concerns but also in assessing the extent of ancient human habitation, land use, agricultural practice, and related activities. However, key questions remain regarding quantifying the relationship between the environment, soft tissue, and the growing shell, and understanding how isotope records change after death. Additionally, few shell 15N studies have been conducted on freshwater taxa to assess for anthropogenic impacts on N loading in rivers. To further examine these concerns, samples of freshwater Unionid mussels were collected from two separate locations in Alabama: the Sipsey River in western Alabama and Bogue Chitto Creek in the Black Belt Prairie. The Sipsey River collection location has suffered the comparatively little anthropogenic impact, while the Bogue Chitto Creek area is downstream from areas of greater human population density and intense agricultural activity. Comparisons in shell 15N records between these two regions may yield insight into the sensitivity of these proxy methods of assessing anthropogenic N inputs. Furthermore, ancient shells from archaeological sites and museum collections from both regions may be analyzed to assess for 15N changes over time. Additionally, some shells collected from the Bogue Chitto site appear to have lived for several decades, permitting the first time-series 15N analysis performed on a freshwater mussel shell. Comment by Andrus, C. Fred: Good job bringing new citations in, but why only these two? Is there a way you could list some other relevant ones with these?

To address concerns of diagenesis, a subset of the collected shells from the Sipsey River was tagged, placed in mesh bags, and affixed to the river bottom near the collection site for over two years to simulate pre-depositional exposure. 15N data were measured in local seston, benthic organic matter, and bulk soft tissue of these shells soon after collection. After retrieval, comparisons of shell and periostracum 15N data will be made between those shells placed in the river and the others retained in a dry and stable laboratory climate. The resulting data permits an assessment of the fidelity of the shell isotope record to that of its habitat, and the impact of changes to the shell isotope profiles soon after death that may contribute to a better interpretation of nitrogen isotope records in ancient shells.

Introduction

This study will focus on two locations: The Sipsey River in western Alabama, and Bogue Chitto Creek, a tributary to the Alabama River in central Alabama. The Sipsey River is a tributary of the Tombigbee River and drains an area of 2044 km2 (McGregor et. al 1992, McCullagh 2002). The headwaters originate in Marion County Alabama at the confluence of New River and Little New Rivers and flows to the south and southwest for over 180 km through west-central Alabama to join the Tombigbee River in Greene County, Alabama. The Sipsey River is free-flowing (McCullagh 2002) and is not in a location downstream from any major cities. Bogue Chitto Creek, however, is downstream from two major cities (Birmingham to the north, Montgomery to the east), and is in an area that is primarily agricultural. Bogue Chitto Creek is designated a critical habitat for several species of crayfish and mussels. Comment by Andrus, C. Fred: The map does n make it look like it is downstream from Montgomery really, but maybe I just can’t understand the river flow patterns. Comment by Dawson, Jamekia A: For future figures, I can label the rivers. The Alabama flows from east to west and then makes a gradual turn south towards the Mobile delta.

Freshwater ecosystems have been modified and impacted extensively by anthropogenic factors such as the construction of levees and dams, which has disconnected rivers from their backwaters and floodplains, and dredging for navigation that has altered natural river channels (Fritts et. al 2017). In addition to physical anthropogenic impacts, nitrogen fertilizer runoff and human and animal waste input to rivers have been linked to nutrient enrichment, hydrologic alteration, habitat degradation, loss, and declines in biotic integrity in streams. While nutrients, such as nitrogen and phosphorous, are critical for healthy and diverse aquatic environments, an excess of nutrients can have undesirable effects on water quality (Harned et. al. 2004), potentially affecting organisms that live in these ecosystems. This runoff from agricultural use is expected to increase with population growth (Atkinson et. al. 2014).

Freshwater mussels furnish an abundance of information about past and current conditions in aquatic ecosystems (Fritts et. al. 2017). They are large, typically long-lived, primary consumers that perhaps can provide temporal assimilation of nitrogen inputs into watersheds through their filter feeding (Atkinson et. al. 2014). Filter feeding transfers organic materials and nutrients from the water column to the surrounding benthic area (Howard and Cuffey 2006, Vaughn et. al. 2007, Atkinson et. al. 2014), while also ingesting and assimilating a wide range of suspended fine particulate organic matter (FPOM) originating from both aquatic and terrestrial sources (Atkinson et. al. 2014). Other bivalves, such as oysters, have been used as proxies for estuarine nitrogen because they approximate 15N in suspended particulate organic matter (Darrow et. al. 2017). Regarding ancient shells, preserved shell middens have a similar potential to preserve valuable information about organic matter sources dating back centuries (Darrow et. al. 2017). The 15N relationship between the ancient and modern shells in both the Sipsey River and Bogue Chitto Creek may depict changes in land use over centuries.

Proposed Research

I propose to analyze suites of live-collected mussels for shell 15N from the two aforementioned watersheds (Figure 1). Dr. Carla Atkinson has extensive collections of mussels Pleurobema decisum from the Sipsey River and has measured soft-tissue and seston 15N. I propose to measure the whole shell and time-series shell 15N from subsets of these mussel collections. I will conduct a similar analysis of shells from Bogue Chitto Creek (some collected by Dr. Alex Huyrn, UA Biological Sciences, others I will collect). The resulting data will be compared to assess differences in nitrogen inputs between the environments. Shells from pre-industrial periods may also be compared at one or both locales (depending on availability) to assess for potential changes in N inputs over time, similar to Payne, (2016); Black et al., (2017), and Darrow et al, (2017). My hypothesis is that the shells from the Sipsey River will contain more negative (lighter) 15N values than the shells collected from Bogue Chitto Creek, based on factors of higher agricultural land use and being downstream from two major cities. This is due primarily to differences in 15N values between potential sources of N (Figure 2). Similarly, pre-industrial age shells would contain more negative 15N values than modern shells from anthropogenically-impacted rivers. Some limiting factors to these hypotheses include the short residence time of nitrogen in river systems, as rivers flush nitrogen out quickly and the process of diagenesis in these environments alters the 15N values in shells over time. Additionally, impacts from synthetic fertilizers will not likely be evident in shell 15N values because the N source of such fertilizers is air, which has similar 15N values to many other natural N sources. [image: ] Comment by Andrus, C. Fred: Isotope measurements are ratios, not concentrations. All samples have about the same concentration of isotopes overall, but the ratio of heavy to light will vary.

Research Questions

1. Is freshwater mussel shell 15N useful as a paleoenvironmental proxy for observing changing waste N input over time?

Several studies have been done in Europe on the effects of 15N bivalves and in coastal areas of North America on the effects of 15N in bivalves. In a study of scallops in France, Gillkin et. al. (2017) concluded that bivalve carbonate-bound organic matter (CBOM) can be used similarly to soft tissue 15N values and also track various biological events. In regard to 15N effects on coastal ecosystems, Kovacs et. al. (2010) determined that 15N values in oyster shells may be useful to enable anthropogenic source tracing and refine food web reconstructions in areas with remnant shells where oysters or other bivalves have declined or already lost. In these and other studies (e.g. Payne, 2016; Black et al 2017) all of the study’s species were from marine or estuarine habitats. To date, this approach has not been tested in a river, though Fritts et al., (2017) detected changes in N input to the Illinois River by measuring 15N from ancient and modern shell periostracum. Comment by Andrus, C. Fred: What year?

2. How are the signatures in the stream environment related to signatures in the shell?

As mentioned previously, freshwater mussels have a vital ecological role through their filter feeding (Atkinson et. al. 2014). These activities transfer organic materials and nutrients from the water column to their environment (Howard and Cuffey 2006, Vaughn et. al. 2007, and Atkinson et. al. 2011, 2014), while also assimilating a variety of particulates from aquatic and terrestrial sources. Despite differences in diet, caused by particle size preferences (Leff et. al. 1990, Galbraith et. al. 2009, Atkinson et. al. 2011 and 2014), different species of unionid mussels tend to have similar isotopic signatures within the same site, allowing for cross-species comparisons (Christian et. al. 2009, Atkinson et. al. 2011 and 2014). In filter-feeding activities, 15N ratios are correlated within the soft tissue, periostracum, and shell (e.g. O’Donnell et al., 2003). Comment by Andrus, C. Fred: Here you should add some general statements about the known relationship between food, soft tissue, periosteum, and shell d15N. Dos this relationship remains true in freshwater mussels?

3. What factors may limit the usefulness of shell 15N record in mussels?

Unlike coastal systems where nitrogen typically has a higher residence time, dynamic river systems quickly flush out nitrogen downstream. As a consequence, the impacts of waste input may be less intense than what was seen in prior similar studies in estuaries with longer residence times (e.g. Black et al. 2017). Freshwater mussel shells are sometimes less robust than marine shells, especially in an archaeological context (Andrus, 2012), thus preservation issues may impact the applicability of ancient shells to 15N analysis.

Methodology

Periostracum Preparation

The methods used to prepare the periostracum for analysis have been adopted from the preparations of Fritts et. al. (2017) and Black et. al. (2017). The shells are initially cleaned using DI water and a soft bottle brush followed by approximately five minutes of ultrasonic cleaning. The shells are then air-dried in stable laboratory conditions. In the removal of the periostracum, a small steel blade was used to separate the periostracum from the shell layer. For preliminary analyses, one sample of periostracum was obtained from approximately 20 shells from the Sipsey River. The samples ranged in weight from 1.6-4.3 mg. Each sample was sealed in 5x9mm size tin foil cups. If time-sequence data are required, periostracum can be sampled across ontogeny using similar methods, whereby each sample represents a discrete time interval of growth.

Shell Preparation

Bulk shell preparation methodology has been adopted by Black et. al (2017). Upon removal of the periostracum by abrasion, a hand-held variable speed drill is used to obtain powder across the transect of the shell perpendicular to growth lines, approximately 1 mm deep. Care must be taken to ensure sampling was from the upper layer of the shell. The samples will range in weight from 35-40 mg (Figure 3). Each sample will be sealed in 5x9mm size tin foil cups. If time-sequence data are required, transects will be milled parallel to growth lines in a series of trenches spanning all parts of the shell large enough to meet the minim sample size requirements.

Soft Tissue Analysis

Soft tissue analysis methods are similar to those listed in Atkinson et. al. (2010). The length (in mm) and wet mass (g), including the shell, were recorded. After dissecting the gut of the sample, all body tissue samples were collected from sacrificed individuals and dried at 45C, and ground separately. Total carbon and nitrogen isotope signatures were analyzed for the tissue samples in the Alabama Stable Isotope Laboratory (ASIL). The carbon data collected are not relevant to this study.

EA-IRMS Analysis

These methods were adopted by Black et. al. (2017). To obtain comparable amounts of nitrogen (mass spectrometer peak area in millivolts (mv)) between the soft tissue, periostracum, shell layer, and ancient samples, varying masses of each component were used. The samples were analyzed in the Alabama Stable Isotope Laboratory (ASIL), located in the Department of Geological Sciences at the University of Alabama, using a Costech ECS 4010 elemental analyzer (EA) coupled via a continuous flow of ultrapure helium to a Thermo Delta V isotope ratio mass spectrometer. The resulting 15N data were reported relative to AIR. The EA was fitted with a carbon trap (glass trap (Costech part no. 071121)) filled with CO2 absorbent (Costech part no. 021020) to remove CO2 and operated combustion temperature of 1020℃. Two standards were analyzed to calibrate %N measurements: B2151 High Organic Content Sediment 5gm (0.62%) and Acetanilide 135.17gm (10.36%). IAEA-N-2 Ammonium Sulfate (20.3 ± 0.2 ‰) and B2151 High Organic Content Sediment (4.42‰) were used as the isotope standards. δ15N data are reported in parts per mil (‰) vs. air. Precision (1σ) was better than 0.09‰ based on analysis of multiple standards over a range of isotopic values.

Sample size requirements

To determine the suitable range of sample sizes, a whole mussel shell collected from Lake Tuscaloosa was ground to a fine powder in an iron mortar and pestle, then sub-samples were removed at sizes ranging from 25 mg to 100 mg. The data produced from the EA showed δ15N values ranging from 4.8-5.3‰. Based on the results, there is sufficient δ15N to analyze the shells. Sample sizes should not be a negative factor in obtaining results in ancient shells that have lost a great deal of organic matter. If necessary, these samples can be made several times larger, yet still be small enough to fit in the EA, thus analysis of ancient samples is likely possible. [image: ]

References

1. Andrus C. F. T. (2012). Isotope sclerochronology in southeastern US archaeology to estimate season of capture. in Reitz, E. J, Quitmyer, I. R., and Thomas, D. H. eds. Seasonality and Human Mobility along the Georgia Bight. American Museum of Natural History Anthropological Papers. v. 97. p. 123-133.
2. Atkinson, C. L., Opsahl, S. P., Covich, A. P., Golladay, S. W. & Conner, L. M. (2010). Stable isotopic signatures, tissue stoichiometry, and nutrient cycling (C and N) of native and invasive freshwater bivalves. J. North Am. Benthol. Soc. v. 29. p. 496–505.
3. Atkinson, C. L First, M. R Covich, A. P Opsahl, S. Pand Golladay, S. W. (2011). Suspended material availability and filtration–bio deposition processes performed by native and invasive bivalve species in streams. Hydrobiologia 667: 191– 204.
4. Atkinson, C. L., Christian, A. D., Spooner, D. E., & Vaughn, C. C. (2014). Long-lived organisms provide an integrative footprint of agricultural land use. Ecological Applications. http://doi.org/10.1890/13-0607.1
5. Black, H. D., Andrus, C. F. T., Lambert, W. J., Rick, T. C., & Gillikin, D. P. (2017). δ15N values in Crassostrea virginica shells provide early direct evidence for nitrogen loading to the Chesapeake Bay. Scientific Reports. http://doi.org/10.1038/srep44241
6. Darrow, E. S., Carmichael, R. H., Andrus, C. F. T., & Jackson, H. E. (2017). From middens to modern estuaries, oyster shells sequester source-specific nitrogen. Geochimica et Cosmochimica Acta. http://doi.org/10.1016/j.gca.2016.12.023
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10. McCullagh, W.H., Williams, J.D., McGregor, S.W., Pierson, J.M., & Lyndeard, C. (2002). The unidoid (Bibalbia) fauna of the Sipsey River in northwestern Alabama, is an aquatic hotspot. Retrieved from https://www.researchgate.net/publication/290278845
11. McGregor, S.W. and O’Neil P.E. (1992). The biology and water quality monitoring of the Sipsey River and Lubbub and Bear Creeks, Alabama. Geological Survey of Alabama Circular 169. p. 1-44.
12. Oczkowski, A., Gumbley, T., Carter, B., Carmichael, R., & Humphries, A. (2016). Establishing an Anthropogenic Nitrogen Baseline Using Native American Shell Middens. Frontiers in Marine Science. http://doi.org/10.3389/fmars.2016.00079.
13. O’Donnell, T. H., Macko, S. A., Chou, J., Davis-Hartten, K. L. & Wehmiller, J. F. (2003) Analysis of δ13C, δ15N, and δ34S in organic matter from the biominerals of modern and fossil Mercenaria spp. Organic Geochemistry. 34, 165–183.
14. Payne, T.N., Andrus, C.F.T., Zierden, M.A., Tobin, T.S., and Dimova, N. T. (2016). 15n as a potential proxy for anthropogenic nitrogen loading in Charleston harbor, South Carolina. Electronic Thesis. p. 1-38.
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16. Wilson, W. A., Fritts, A. K., Fritts, M. W., Unrine, J. M., & Casper, A. F. (2018). Freshwater mussel (Unionidae) shells document the decline of trace element pollution in the regional watersheds of Chicago (Illinois, USA). Hydrobiologia. http://doi.org/10.1007/s10750-018-3582-3