POTENTIAL APPLICATIONS OF AMORPHOUS CALCIUM CARBONATE (ACC) IN WATER TREATMENT OPERATIONS

The increasing negative effects from anthropogenic activities has given rise for bioprospecting new and advanced materials intended for sustainable remediation and restoration efforts. There are currently efforts into exploring naturally found porous substrates to use in a variety of applications, however the cost and availability has been a limiting factor in introducing these into current practices. Many studies have explored and identified the high performance of granulated activated charcoal for water filtration but tend to disregard the environmentally harmful nuances that occur during the manufacturing process. Priority for these porous substrates focus on results, with little to no regard for environmental sustainability. In the case of this research, efforts were made to develop a low-cost, and widely applicable method for utilizing naturally occurring porous media in the form of crushed Quahog seashells for water treatment operations. Emphasis was put on using a porous media that is found naturally worldwide and simultaneously acts as a carbon sink instead of releasing carbon into the atmosphere such as is the case with activated charcoal. This study focused on exploring the versatility of different water treatment situations to show the wide range of potential applications seashells can offer in place of, or in combination with, granulated activated charcoal through chloride-based saltwater remediation and nitrogenous water scenarios. Efforts were made using bench-top water treatment system with different porous substrates, coupled with ICP-AES and ICS analytics. Results show that this method of substrate preparation is capable of producing a 99.2% pure amorphous calcium carbonate substrate (Yoon et al. 2003) (with pore sizes visible up to 2 nm in size, suggesting that Quahog shells could be a viable sorbent source for a wide range of different pollutants and analytes. Porosity values of crushed Quahog shells as a sediment has been observed between 49-67% (Pfeiffer & Rusch. 2000). To perform the analysis of CaCO3 in the water treatment study, anion quantification was conducted with an Ion Chromatography System (ICS-5000), and cation analysis was done by an Inductively Coupled Plasma Atomic Emission Spectrometer (ICPAES). Results from the water treatment section show that amorphous calcium carbonate (ACC) did not remove as much chloride-based road salts as activated charcoal did but removed more than gravel and sand. Additionally, with each column that contained shells, calcium was released into the collection containers indicating potential environmental benefits for organisms that rely on calcium for growth when shells are used in combination with road salts for deicing practices. During deicing of roadways, cations are depleted from the environment neglecting plants and organisms relying on those nutrients for growth (Kelting & Laxson, 2021.) When treating with nitrogenous waters, again activated charcoal was the top remediator, however ACC was able to remove up to 4x as much phosphate as did the gravel and sand. All results from this research will supplement the science of previous amorphous calcium carbonate water treatment studies, as well as promote the growth of aquacultures and bivalves for environmental sustainability.

practices. Many studies have explored and identified the high performance of granulated activated charcoal for water filtration but tend to disregard the environmentally harmful nuances that occur during the manufacturing process.
Priority for these porous substrates focus on results, with little to no regard for environmental sustainability. In the case of this research, efforts were made to develop a low-cost, and widely applicable method for utilizing naturally occurring porous media in the form of crushed Quahog seashells for water treatment operations. Emphasis was put on using a porous media that is found naturally worldwide and simultaneously acts as a carbon sink instead of releasing carbon into the atmosphere such as is the case with activated charcoal. This study focused on exploring the versatility of different water treatment situations to show the wide range of potential applications seashells can offer in place of, or in combination with, granulated activated charcoal through chloride-based saltwater remediation and nitrogenous water scenarios. Efforts were made using bench-top water treatment system with different porous substrates, coupled with ICP-AES and ICS analytics. Results show that this method of substrate preparation is capable of producing a 99.2% pure amorphous calcium carbonate substrate (Yoon et al. 2003) (with pore sizes visible up to 2 nm in size, suggesting that Quahog shells could be a viable sorbent source for a wide range of different pollutants and analytes. Porosity values of crushed Quahog shells as a sediment has been observed between 49-67% (Pfeiffer & Rusch. 2000). To perform the analysis of CaCO3 in the water treatment study, anion quantification was conducted with an Ion Chromatography System (ICS-5000), and cation analysis was done by an Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES). Results from the water treatment section show that amorphous calcium carbonate (ACC) did not remove as much chloride-based road salts as activated charcoal did but removed more than gravel and sand. Additionally, with each column that contained shells, calcium was released into the collection containers indicating potential environmental benefits for organisms that rely on calcium for growth when shells are used in combination with road salts for deicing practices.
During deicing of roadways, cations are depleted from the environment neglecting plants and organisms relying on those nutrients for growth  When treating with nitrogenous waters, again activated charcoal was the top remediator, however ACC was able to remove up to 4x as much phosphate as did the gravel and sand. All results from this research will supplement the science of previous amorphous calcium carbonate water treatment studies, as well as promote the growth of aquacultures and bivalves for environmental sustainability. To my family and friends back at home, thank you for your constant patience, love, and support, for I could not have gone this far without you all behind me the whole way. This thesis is dedicated to my family, who have been my foundation and inspiration for being the best I can be. I love you all.     (Brečević & Nielson 1989). Of these polymorphs, amorphous calcium carbonate (ACC) is the least understood. It is unique from the other 5 polymorphs because of its properties and the mechanisms in which it is formed; it is a biomineral , Weiner & Dove 2003, a mineral that is grown via a living organism, and is relatively stable in water compared to the other CaCO3 polymorphs. Bivalves that form ACC in their shells actively clean water they live in through filter feeding, but finding a use for the shells once the organism is deceased has not been thoroughly investigated. Additionally, shell growth acts as a carbon sink absorbing CO2 from the atmosphere as opposed to activated charcoal the introduces high concentrations of the greenhouse gas into the atmosphere, highlighting the need into further research. To better understand the potential applications of crushed seashells in water treatment, investigation into the structural morphology and element composition needs to be addressed in comparison to other types of CaCO3 and other substrates currently in practice. Amorphous calcium carbonate is abundant in waters worldwide, presenting a need for further study into the mechanisms and unique attributes that will allow for a variety of potential water treatment applications.

Introduction
Amorphous calcium carbonate (ACC) is found in the shells of bivalves and mollusks found throughout the world, concentrating along the coast and in tidal estuaries. CaCO3 is found in 6 polymorphs, three anhydrous polymorphs (calcite, aragonite, and vaterite) and three hydrous polymorphs (hexahydrate, monohydrate, and amorphous calcium carbonate) (Brečević & Nielson 1989. ACC is the least stable (Zou et al. 2015) and most morphologically flexible of the six polymorphs , and Ostwald's Step Rule dictates that the least stable polymorph will be the first one to form (Myszka et al. 2019). This indicates that during biomineralization growth it is both expected and observed that ACC will be the dominant polymorph present. They are different from other calcium carbonate (CaCO3) polymorphs because they are amorphous , they do not have a defined crystalline structure visible and there is no specific pattern to how the molecules are arranged. Additionally, ACC is a biomineral grown via a living organism as opposed to the mineral and rock-like properties apparent in other polymorphs generally seen throughout the world such as limestone, marble, and chalk.
Organisms such as crabs and lobsters have a different type of biomineralization growth, primarily using calcite as their outer covering which does have a defined crystalline structure and shed their exoskeletons during each growth stage. ACC is formed in bivalves through secretions of calcium ions with carbonate taken from the water through a process called crystallization by particle attachment (CPA), wherein the structure of the shell matrix ranges from multi-ion complexes to fully formed nanocrystals (De Yoreo et al., 2015, Bots et al. 2012, Sun et al. 2018. Bivalves such as quahogs (Mercenaria mercenaria) and oysters (Magallana gigas) retain their calcium carbonate growth with them for the duration of their lives, ensuring a solid protective surface that is both strong to protect from predators, and durable in water to ensure life longevity. Current efforts into bioprospecting for sustainable sorbent substances is done through a series of systematic and organized search for useful products derived from bioresources including plants, microorganisms, animals, etc., that can then be developed further for commercialization and overall benefit society , Patricelli 2010). This has led to increased interest in the potential applications for marine organisms and their by-products . The ocean supplies a rich source of organisms that supply templates for biomimetic research (Cantaert et al. 2012), an industrial effort into copying and manufacturing the internal mechanisms of the organisms (Patricelli 2010) done to synthetically reproduce substances of similar nature (Huang et al. 1990), however this precipitated calcium carbonate (PCC) is difficult to make and does not have the same molecular structure provided by ACC derived from seashells. When alive, bivalves actively clean the water through filter feeding and absorbing heavy metals that reside in the sediments (Zuykov et al. 2013, Bozbas & Boz 2016, Setiawan et al. 2018, Tudor et al. 2006, Schenk et al. 2014), however long after bivalves are deceased their shells can often be found along beaches and waterways affected by tidal currents , indicating a possible use for them outside of the benefits already established when the organisms are alive. The objectives of this study are to use amorphous calcium carbonate as a filtration substrate as an alternative or combination to granulated activated carbon, and as an additive to normalized ground conditions while highlighting the versatility of applications ACC may offer in sustainable remediation practices. This review provides an assessment into the current known attributes and limitations of ACC, as well as the formation and mechanisms of quahog shells for the purpose of water treatment applications.

ACC formation and structure
Amorphous calcium carbonate is grown by organisms throughout the world This process is heavily influenced by the pH of the water, and the ratio of CO3 The amount of calcium carbonate in quahog shells increase incrementally as the organisms grows, and this is visible on the outer shell of the quahogs where growth lines can be seen, indicating the many different growth stages it has experienced in its lifetime. The molecular arrangement of these subsequent ACC matrices in the shell have no defined shape or pattern, and so the molecular arrangement cannot be predicted at any point in time with great accuracy. The structural process in which it is formed is by crystallization by particle attachment (CPA), adding on to the previous layers to become both thicker and longer. The microstructure of ACC is dependent on the minerology (Bots et al. 2012) and can vary between columnar prismatic, granular prismatic, fibrous, chalk and crossfoliated , Raiteri & Gale 2010, Zhang et al. 2022. This varies from anhydrous polymorphs such as vaterite, calcite, and aragonite that have a defined latticework of crystallization. Vaterite is hexagonal, calcite is trigonal, aragonite is orthorhombic, and amorphous calcium carbonate is undefined. In a pure abiogenic CaCO3 mineral, at any location in the sample the crystallized molecular structure will be the same. The resulting shape of these mineral polymorphs can be spherical, ellipsoidal, cuboidal, "flower-like", or columnar (Oral & Ercan 2018) and are consistent throughout the substance in which they are found. In temporal terms of formation, the biogenic ACC is formed much quicker than its mineral counterparts because it is physically grown by an organism, whereas the latter often must undergo many years of metamorphism or diagenesis in order to become a carbonate rock. Because of these structural differences, their physical properties and attributes also differ. The abiogenic CaCO3 are generally very water soluble and effervesce in the present of acidic condition as seen in minerals like calcite, dolomite, and other carbonate rocks. However, the biogenic ACC is stable in hydrous conditions, as exemplified by them being formed by organisms that live in most or all their lives in water. Additionally, the quahog shells do not actively effervesce in the presence of acidic conditions, but when subjugated to very low pH conditions over a period of time the structure becomes soft, malleable, and easily broken apart. This is a large reason why ocean acidification is affecting many calcium carbonate-forming organisms such as clams, crustaceans, and coral in the ocean.

Calcium carbonate formation pathways, stability, and degradation
The structural mechanisms that form ACC in seashells allows for stability in hydrous conditions (Lee et al. 2018), however degradation can occur in specific parameters. The alkalinity of ACC neutralizes the acidic effects of low pH waters, but to an extent based on the available quantity. In neutral waters, the slow degradation of shells can increase the pH to slightly basic conditions which is preferential to most organisms instead of acidic conditions, as evident by the increasing ocean acidification observed today. In the ocean, the degrading shells release the elements needed by other bivalves to form their shells, causing a constant cycle of uptake and release. An important characteristic of seashells is structural stability in water; if not, the shells would easily dissolve, and the life expectancy of bivalves and mollusk would be drastically shortened. When submerged in water the shells maintain their hardness, and its solubility decreases with both rising temperatures  and rising pH.
When exposed to dry, oxygenated conditions, and UV light, degradation will occur morphing into crystalline calcite demonstrated in equation (3). This resulting calcite is brittle, easily broken down and eventually dissolved back into the environment. This can often be experienced by finding shells that have washed up on the beach and exposed to the sun for a duration of time, where the structural integrity of is severely compromised. However, the same types of shells can be found in the tidal zone of beaches, where they are often still hard and durable despite the energy of the waves crashing them against the sand.
All polymorphs of calcium carbonate are alkaline in nature and so in addition to the conditions previously mentioned, acidic waters also contribute to loss of shell structural stability, exemplified in equation (4). This is evident in coral reefs that also utilize calcium carbonate for survival when subjected to increasing levels of ocean acidification. Coral bleaching is a result of the increase in ocean pH, which is caused by the influx of CO2 being absorbed into the ocean from the atmosphere. Although bivalves sequester CO2 from the water to form the carbonate shell (Du et al. 2018), they often cannot keep up with the steadily increasing acidification largely caused by anthropogenic activities. Using hydrochloric acid (HCl) as the low pH representative, this can be visualized by the reaction sequence (equation 4).
CaCO3 + 2HCl → CaCl2 + CO2 + H2O (4) Thus, CO2 and CaCO3 are in a continuous cycle of uptake and dissolution reaction series, and so the further the scale tips towards acidic conditions, the harder it will be for calcium carbonate producing organisms to grow and survive. This can be prevented through best management practices, and efforts into both protecting and preserving not only bivalves but also the waterbodies they reside in.
New sorbent sources are needed that are sustainable, and biomimicry is a strategy made by industries to develop such sorbents based on naturally occurring materials. However, biomimetic efforts into synthesizing nanoporous materials are expensive, difficult, and time consuming, producing both unsustainable and often unreliable results (Du et al. 2018). Synthetic approaches in creating precipitated calcium carbonate (PCC) is conducted in the lab through a method similar to that found in nature, without the added benefit of sequestering CO2 from the ocean. Chemical formula-wise the compound is the same, yet the structure and morphologic composition is different. Calcium oxide (CaO) combines with water (Maciejewski & Reller 1989) to form calcium hydroxide (Ca(OH)2), that reacts with carbon dioxide (CO2) to produce precipitated calcium carbonate (PCC) and water ) (equation 5).
CaO + H2O → Ca(OH)2 + CO2 → CaCO3 + H2O This synthetic substance does not have the same structural stability in water and degrades at a faster rate than seashells in water, limiting their potential use in experimental practices. Structurally these are not amorphous and are instead similar to the spherical vateritic structure seen in abiogenic minerals.

Modern applications of calcium carbonate
The unique properties of ACC demonstrate potential uses as a sorbent source for undesired pollutants in both soil and water conditions . It is a hygroscopic material, absorbing moisture from the atmosphere similar of that to activated charcoal, and unlike other calcium carbonate polymorphs it maintains stability as the pH and temperature increase (Brečević & Nielson 1989) while submerged in water. The nanoporous characteristics of various shell types are a research topic of interest yet have not been fully investigated for Mercenaria mercenaria. The amorphous molecular structure indicates a high surface area that will allow for a variety of adsorption and absorption. Forms of calcium carbonate are present in nature and effect the environment around them in different ways, and the calcium portion of CaCO3 also has a crucial role in nature and human activities (Zuykov et al., 2013, Yoon et al., 2002, Diamadopoulos & Benedek 1984, DeJong et al., 2008.) The stoichiometric molar ratio of calcium to phosphorous is 2:1 in wastewater (Diamadopolous & Benedek 1984, Song et al. 2002, making it the limiting nutrient for phosphorous  by using seashells for any practical purpose will promote the need for bivalve growth, both naturally and through aquaculture farming, subsequently having a net effect on the removal of greenhouse gasses from the ocean and atmosphere. Bioprospecting for a sustainable porous matrix is of high interest, substituting for methods and substances currently in practice. Granular activated charcoal (GAC) is the gold standard for the filtering substrate of unwanted pollutants, however the mechanisms in which GAC is created adds to the CO2 emissions and relies on deforestation for sustenance. Carbon is taken in the form of wood from trees and "activated" through heating at a high temperature, increasing the surface area and creating microscopic pores for substances to adhere in and on to. This large surface area is efficient at removing targeted pollutants and analytes from water, but the process in which it is created is costly, labor intensive, and unsustainable for long-term ecological sustainability. Innovative studies are currently underway looking for natural nanoporous substrates such as coconut husks, sugar cane, and corn husks, but the lack of availability in efficient sorbent substrates has not been fully investigated.    Previous studies show that bivalve shells are 95-99% mineral and 0.1-5% organic material (Hare & Abelson 1965, Fakayode 2020, Li et al. 2007) and our results show that Quahog shells are 99.2% pure calcium carbonate, with weight percentages of 51.8% Oxygen, 31.0% Calcium, 16.4% Carbon, 0.5% Sodium, and 0.3% Sulfur, respectively (Figure 3). 0.5% of shell weight consisted of  The elemental distribution across the surface of granulated shell pieces was homogenous throughout the fragment, with a primary base of calcium speckled evenly with oxygen and carbon. This was created by the SEM using energy dispersive spectroscopy (EDS) which assigns color to a specific element and scans for one at a time, which then and overlapped all the layers into a single image (Figure 4). This ensures a predictably reliable natural substance capable of being implemented into future experiments, with only 0.8% of which is undesired impurities. These impurities can be attributed to free-floating elements in the water column as well as being absorbed from the sediments in which the shell resides in .

Discussion
This method is designed to provide a renewable and sustainable substrate source that is widely applicable for performing a range of water treatment studies. We found that this method was able to produce a 99.2% pure CaCO3 substance using easily available tools and chemicals with minimal cost and labor.
It is a natural organic sorbent source capable of being utilized without a need for manipulation of any sort to affect its structural integrity or porous nature and is a renewable supply source indicated by the biomineral propensity of being constantly grown by organisms. It can potentially be used for in situ operations outdoors, as it will not be adding a foreign or manmade substance to the environment that would otherwise not be present. The distribution of the element constituents in the surface topography of quahog shells are homogenous throughout, indicating a uniformity in performance expected at any point during a filtration process (Figure 4). Excess seashells were stored in a 5gallon bucket and submerged in DI water as a backup supply and still maintained their hardness even after 2 years of inactivity, implying durability and product longevity. This method is also not restricted by location as many bivalve and mollusk species around the world grow calcium carbonate as a protective defense mechanism. In terrestrial situations where access to the ocean is limited, aquacultures can be created with minimal care necessary as bivalves predominantly live a sedentary lifestyle, easily manipulated in controlled conditions. This development of methodology establishes the groundwork for using quahog shells as a viable pure sorbent media source that is otherwise deemed a waste byproduct (Claassen 2017), but the scope and efficacy of exploring different types of shells for various applications is a knowledge gap to be explored as a topic of interest for future studies. The potential applications for using crushed quahog shells in remediation practices is not well established as of yet, but instead opens up pathways into exploring various treatment methods to either replace or substitute for granulated activated charcoal. It is possible to quantify how much carbon dioxide is sequestered from the atmosphere by the weight of seashells alone, as 68.2% of quahog shells are solidified CO2 sequestered from the ocean. This is directly opposite the negative effects activated charcoal production causes to the atmosphere, as GAC requires the burning of wood collected from deforestation practices. The surface topography of quahog shells in comparison to GAC is similar down to the microscopic level and offers a larger surface area for analytes to adsorb and absorb onto, and the smaller nanopores in ACC indicate a wider range of potential analytes to be treated.
Applications of this method in preparing crushed ACC is feasible to apply into other research studies, particularly into water treatment operations that utilize a nanoporous substrate for filtration or sorption capabilities .
Pending future analyses, in situ applications of using ACC in the environment for remediation practices is also a viable option without fear of using a finite unsustainable resource. The extent and efficacy of ACC in these potential applications is not well established, therefore a wide range of experiments is suggested to find the best possible strategies for implementation with focus on comparison to the abilities of activated charcoal.   instead of salt crystals to melt ice and snow accumulating on the road. This also has drawbacks, as the efficiency of deicers decrease as the temperature drops which can cause buildup of salts that can then quickly surge into other areas as soon as the temperature rises again. 90% of the salt content is washed away with the first 20% of the meltwater . Road salt usage increase with higher latitudes in the United States, and concentrations peak during the winter seasons when ice and snow develops . Over 21 million tonnes of road salt deicers are applied annually in the United States with the majority consisting of NaCl (Jones et al. 2017). Chloride concentrations have been detected at the University of Rhode Island Kingston campus at 25-200mg/L in the winter, and 1-20mg/L for non-deicing seasons , a situation comparable to roadways around the state. The salts themselves are composed of 95% NaCl (Sodium chloride), 2.5% CaCl2 (Calcium chloride), and 2.5% MgCl2 (Magnesium chloride) (Jones et al. 2017. These chloride-based salts concentrate along urban and suburban runoff areas (Cañedo-Argüelles et al. 2013, Kaushal et al. 2005) negatively impact drinking water, groundwater  fringing vegetation , and organisms across all trophic levels . The deicers work well for melting ice on pavements, roadways, and hard surfaces, but are subject to intruding into waterways where they can both upset the nutrient balance and cause stress to organisms. Most terrestrial vegetation struggles to grow in salty soils, and many animals have specific thresholds of desired conditions where they can grow unimpeded, but this is altered with the introduction of salt into their habitat.
Additionally, the constant use of the deicers can erode pavements and concrete over time, with MgCl2 causing the worst effects out of the three . The difference between current practices and the one hypothesized is that the shells offer a large surface area for absorption and adsorption compared to gravel and sand, with the added condition of introducing calcium to the environment. The goal of this study is not to remove the salts before they melt the ice and snow, but instead to absorb and mitigate the negative effects of saltwater runoff before they are partitioned into the environment while also introducing calcium as an extra nutrient source for the ecosystem. This study will use five different columns in combinations of granulated activated charcoal (GAC), amorphous calcium carbonate (ACC in the form of quahog shells), gravel, and sand to compare the results for reducing salt concentrations both desired and optimal conditions. This study hypothesizes that using seashells in conjunction with chloride-based salt deicers will mitigate the effects of saltwater runoff, with the added effect of increasing calcium concentrations into the environment.

Materials and Methods
Materials used in this chapter are analytical-grade NaCl, MgCl2, and CaCl2 salts purchased from Thermo Fisher Scientific and was used as the analytical standard for development of this method. 20-40 mesh size granulated activated charcoal (GAC) was purchased from Honeywell Fluka™, and DI water was obtained from a Milli-Q® Integral Water Purification System and used as the sample matrix to limit any signal interference during analysis. An 8 port MasterFlex L/S peristaltic pump was used for simultaneous pumping of standards that were held in 1 Liter amber glassware filled to no head space. An Ohaus® weighing scale was used for measuring out salts used for creating stock standards, and Quahog shells were obtained from Gardner's Wharf seafood market in Wickford, Rhode Island, where they were gathered from local waters. One inch rubber stops, tubing, anthracite sand, gravel, and 1.5 inch diameter polycarbonate columns cut into a length of 45 cm were also used to create the treatment columns for this bench-top water study. Samples were stored in a refrigerator at 37ºF and kept in dark conditions for future analysis.

Sample Preparation & Collection
Saltwater stock solutions were created in four different concentrations at 30mg/L, 60 mg/L, 120 mg/L, and 180 mg/L with replicates made for each. The Column 1 consisted of 100% GAC as the sole substrate to indicate optimal filtration scenarios. Column 2 layered in order from top to bottom was 10 cm of gravel, 20 cm of a 50-50% mixture of GAC and ACC, 10 cm of anthracite sand, and 10 cm of gravel. Column 3 was layered with 22.5 cm of sand above 22.5 cm of gravel to mimic natural ground types found along roadways, used as the control when no porous substrate is introduced. Column 4 was built similarly to column 2, with 10 cm of anthracite sand over 20 cm of ACC, 10 cm of anthracite sand, and 10 cm of gravel. Column 5 indicated a scenario where only ACC is used, with all 45 cm consisting of crushed shells.
Sample collection was performed using an 8-port Masterflex L/S peristaltic pump to ensure simultaneous and equal flow rates for each column. 1 Liter of saltwater stock solutions were pumped through each column and collected in separate 1 L amber glassware. After each sample, DI water was purged at a high flow rate through each column to mitigate any carryover effects. Two trials were conducted to confirm analyte consistency for each experiment. The samples were then stored at 37°F in an unlit fridge for future analysis.

Sample Processing Method
After completion through each column, samples were analyzed in a Thermo

Scientific iCAP 7400 DUO Inductively Coupled Plasma Atomic Emission
Spectrometer (ICP-AES) for cation analysis at Brown University. It is driven by Thermo Scientific Qtegra software and viewed under both radial and axial viewing parameters. An Ion Chromatography Spectrometer (Thermo Fisher Scientific ICS-5000) at the University of Rhode Island was run for anion analysis.
The calibration and processing method for the ICS was created through Chromeleon software also supplied by Thermo Science Fisher. Samples were diluted to a 1:10 ratio to match the detection limits for each equipment. Final solution samples were then compared to the standard stock solutions for differences in the filtration capabilities of each column.

Analyte Analysis
Samples were analyzed by their ion concentrations; three cations used for the creation of stock standards, and seven major anions. Cation focus resided on the calcium (Ca + ), magnesium (Mg 2+ ), and sodium (Na + ) concentrations that contributed to the entirety of the saltwater makeup. The seven major anions analyzed for detection were fluoride (F -), chloride (Cl -), nitrite (NO2 -), bromide (Br -), nitrate (NO3 -), phosphate (PO4 3-), and sulfate (SO4 2-), listed in the order of retention time during ICS analysis. Major emphasis was placed upon the detection of calcium, sodium, magnesium, and chloride as the main constituents predominating the saltwater samples. Two calibration standard checks and a blank DI were run after every 10 samples to ensure system stability and accuracy of results. Only select anions were to be expected based on the purity of samples and the limited number of analytes used in the preparatory steps.
The stoichiometric mass concentrations for the standard stocks were calculated and used for comparison of each sample, as concentrations of the compounds do not equate to the actual values in the saltwater samples. Table 1. Calculated values of expected stoichiometric molar mass quantities in standard stock solutions. Table 2. Actual concentration values of each standard stock solutions after ion chromatography spectroscopy (ICS) and Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES).

Analyte Removal
Results in the chloride-based road salts filtration indicates consistent results for the remediation capabilities of every column, with column 1 (100% GAC) having a slightly higher quantity of salts removed, and column 3 (half sand & half gravel) interacting and removing the least for all analytes evaluated. This is to be expected for GAC, as activated charcoal is proven to be one of the best for filtrating a wide variety of substances. This data is backed by the comparison between column 2 and 4. They are virtually the same in every aspect, except that column 2 used a 50-50 mix of ACC and GAC instead of just shells. The difference in the column substrate is the reason for slightly lower sodium and chloride than columns 3,4, & 5 that contained no GAC. Therefore, we can conclude that the majority of the filtration can be attributed to the mechanisms of GAC in the column. Column 3 (gravel and sand) mimicked natural untouched ground scenarios and yielded slightly higher concentrations in each trial run, indicating that any additional amount of ACC or GAC introduced to a system will be more effective than a natural untouched setting. Columns 2 (50-50 GAC/ACC mix), 4 (gravel, shell, & sand), and 5 (100% ACC) were similar in sodium and chloride removal rates, with each containing seashells of various quantities in the column. Since 95% of the standard saltwater stock were of NaCl, the sodium and chloride concentrations consisted of the majority of the analytes, and removal rates for both were proportional to each other, excluding the anomaly in column 1 at 30 mg/L.  The effects of filtration on magnesium for all columns was negligible, as so little was used in the stock standards that the combined effects of human error and machine accuracy yielded no conclusive correlation. Since the concentration values were so small, the error margins did not fall below 55%. The highest concentration detected was 1.3 mg/L in column 3 using the 180 mg/L stock standard sample. The effects of these substrates on The analysis of calcium in the ion chromatography yielded different results from the magnesium despite using the same amount of each in preparing stock standards. This is highly due to the excess calcium present in seashells, that when dried turn into calcite and easily eroded by the flow of water. Columns 2, 4, and 5 that used seashells as a substrate had a noticeable increase in calcium that was not originally present in the saltwater stock samples, in one case at a magnitude as high as 50x more than columns 1 & 3 in the 30 mg/L concentration, and no smaller than 5x as was the case in the 120mg/L concentration. For the columns that contained seashells, the lowest quantity of calcium did not fall below 8.3 mg/L at any point, as was the case in column 4 at 60 mg/L concentrations, quadrupling both its shell-less counterparts and the standard stock sample. Unanimously for all 4 standard stock concentrations prepared, the largest amount of excess calcium came from column 4, which used a 50-50 mix of GAC and ACC as its primary filtration matrix. For the shell-less columns 1 & 3, no calcium concentration came higher than 4.5 mg/L, which arose from column 3 (only sand and gravel) at the 180 mg/L concentration solution. Therefore, we can determine that for any column that uses ACC as a filtration substrate, calcium is observed in higher quantities relative to the amount in the initial water system.
This implies that during in situ operations calcium will be released into the environment post de-icing practices, however the ecological effects of this immediate influx of calcium on the environment have yet to be analyzed in detail.
The pH of the filtered waters were analyzed with a handheld Apera® Instruments handheld pH meter. The average of pH of each column was quantified, and for columns 2, 4, & 5 that contained ACC, the pH was higher by a range of 0.165 -0.571. Column 4 averaged out to having the highest pH of 8.04, whereas column 3 containing only gravel and sand had a pH of 7.46. Even in small quantities, enough of the seashells dissolved into the water to raise the pH by at least half a value. Although these values may seem insignificant, the pH scale is measured on a logarithmic scale, meaning the change from neutral to basic conditions can have a large impact. Even though this particular study was a small sample size conducted in a bench-top water treatment method, this trend can be assumed to continue with the increased quantity of seashells being utilized. Increasing the pH in water bodies is an unintended benefit not originally planned as a point of focus in this experiment, however the documented advantages of introducing a neutralizing/alkaline agent into the environment has been well established combatting ocean acidification and fortifying the habitat parameters of organisms that prefer slightly basic conditions over acidic settings. Saltwater Column pH 30 mg/L 60 mg/L 120 mg/L 180 mg/L larger scale operations. As expected, GAC filtered slightly more Na + and Clthan any other column, however the realistic applications of adding GAC to chloride based road salts during freeze events is counterproductive to the goal of melting the ice and snow, and the added cost of implementing activated charcoal is expensive. All columns that contained seashells only slightly removed some NaCl, but was consistent in the performance of removing more than Column 3 (gravel and sand). These results show that using seashells as an additive substrate in deicing maneuvers will not hinder the melting of ice and snow, and will remove a portion of saltwater melt before it reaches runoff pathways. In a small scale setting the data appears insignificant, however when applied in vast quantities over a large area the accumulative amounts of salt removed is beneficial for the ecosystem. There is no cleanup afterwards because even though the salt is absorbed into the shell granules, it will slowly dissipate into the environment instead of shocking the system in quick, large doses. The added benefit of introducing calcium as a nutrient byproduct can potentially offset the negative impacts briny melt waters have on waterways and soils, but the extent of these tradeoffs is a knowledge gap to be investigated in future studies (APHA 1999).
In conclusion, using the columns for a controlled experimental setup in conducting saltwater remediation was an efficient analytical method that yielded consistent results.

Discussion
The implications of this study prove that using seashells in de-icing practices can add Calcium to the environment, mitigating the harm salt has in plants and aquatic areas. Future efforts should be evaluated in looking to implementing crushed shells as an additive to the normal sand/salt mix currently used for both traction control and environmental effects. GAC would be best used as a cleanup action after deicing, however the realistic application of that is not desirable due to the cost and harm in higher CO2 atmospheric levels from making GAC. Using seashells is a natural substance that can be left in the environment permanently with no need for cleanup efforts afterwards. Additionally, the extra Calcium will have positive impacts on plant growth, which was seen in the columns months after the experiment was finished. The extent and limitations of plant growth after deicing is a subject for future experiments.
One of the most important factors in considering substrates for water treatment is the porosity, which is the air spaces between the grains instead of the nanopores that are on the individual grains themselves. In order of decreasing porosity, the values are as follows; column 1=67.8% , column 5=42.8%, column 3= 42.8%, column 4=35.7%, and column 2=28.5%. This shows that the higher the rate of homogeneity in the column, the higher the porosity.
Columns 4 & 2 are a mix of stratified gravel, sand, shell/1:1 GAC:ACC mix, with another layer of sand and gravel. The difference in grain sizes may limit the permeability of the water to pass through the column, leaving air pockets that could otherwise be filled with water. Columns 1 & 5 are 100% GAC and ACC, respectively, whereas column 3 is half sand-half gravel. This does not relate to the nanoporosity that characterizes individual grains but is a factor to consider when doing permeability studies. In conclusion, using amorphous calcium carbonate in the form of discarded quahog shells for the purpose of water treatment offers a range of potential applications needing further investigation for remediation practices, while also emphasizing the need to both protect and preserve bivalve communities that benefit ecosystems and societies alike.

Appendices
The overlying theme of this study has been to highlight potential applications of amorphous calcium carbonate via crushed quahog seashells for a variety of implementational practices. In doing so, emphasis is stressed upon maintaining and improving bivalve populations for the impacts they have on the environment. Drawing attention to the functional use of discarded seashells will give further precedence into both the protection and preservation of aquacultural communities. Supplemental research into different treatment methods furthermore establishes prominence amorphous calcium carbonate has to offer as evident by these additional experiments.
Eutrophication events have been a topic of concern for numerous communities that suffer an influx of nutrients that cannot be remediated in an efficient manner. These events are not solely subjugated to oceanic waters but are commonly seen in freshwater and source waters used for drinking water.
Inputting crushed seashells into the parameters that define these contaminated waters is a knowledge gap to be investigated further but is investigated as additional evidence into the potential applications of amorphous calcium carbonate.
Experiments were conducted using replicates of the columns established in chapter 3, however instead of chloride-based road salts, nitrogenous spiked waters were used to mimic high eutrophicate events. Concentrations were run at 800 mg/L, 400 mg/L, 200 mg/L, 100 mg/L, and 50 mg/L to mimic different high eutrophicate scenarios, and the collected water samples were analyzed for cation and anion quantities.   The results show that GAC has the best filtration capability out of all the substrates and columns evaluated and is backed up by previous studies.
However, when comparing columns using seashells in a either a portion or all of the matrix, filtration capabilities far exceeded those of gravel and sand that is normally seen in average ground type conditions. The major attribute of applying seashells in water treatment operations was the ability to reduce the concentrations of phosphate, a notoriously difficult analyte to remove from the environment and one that has the tendency to dwell in sediments for extended periods of time. Additionally, phosphate is a key nutrient that contributes to eutrophicating events supporting algal growth, evident in harmful algal bloom (HABs) that diminish the habitats of organisms in aquatic ecosystem. Throughout every concentration range of nitrogenous waters treated, column 3 containing increasing concentration. The resulting data further indicates that inputting seashells into high nutrient contained waters will either limit or prevent the formation of HABs, or restrict the impacts from anthropogenic fertilizer runoff.
Furthermore, in waters already contaminated by high eutrophic conditions, the addition of crushed ACC will absorb phosphates, while also contributing to the capabilities established in previous chapters discussed in this paper.
A separate subset of this study is to investigate the potential for ACC to be used as a substrate for dispersive micro solid phase extraction (D-μ-SPE). This differs from commonly used method of liquid-liquid phase extraction (LLE) which uses a liquid solely as a binding agent, and solid phase extraction (using just a solid with no additional liquid for sorption); focus is stressed upon suspending the target analyte in a liquid that will then be sorbed by the solid at the same time, and furthermore desorbed for further analysis. The term "micro" refers to the sorption capabilities of removing miniscule quantities in the range of ppmppb, and could potentially be used in last step efforts of clean up methods, or for removing small quantities of harmful substances. This novel method has primary focus on the adsorption and absorption capabilities of a nanoporous material (commonly GAC), for a high percentage of removal and subsequent remediation practices. SPE is used for removal of pollutants in mass quantities, and LLE uses specific liquids targeted for specific analyte pollutants but does not cover a wide range of analytes. Furthermore, in-situ applications of LLE is difficult to control and fully collect, limiting its' use to laboratory and highly controlled settings. This proposed method of D-μ-SPE using ACC offers a solution to both these issues using a sustainable sorbent source with a high sorption rate uncommonly seen in modern remedial practices.
As determined previously, using an easily obtainable sorbent source for both remediation and laboratory needs is a high topic of focus for both industrial and scientific purposes. In this experiment the pollutant of focus is polycyclic aromatic hydrocarbons (PAHs), a ubiquitous group of known carcinogens in nature commonly seen as a byproduct of anthropogenic activity and is present as thousands of distinct types, however only 16 of which are currently regulated by the U.S EPA. Those 16 are present throughout the world and are concentrated in both waters and soils around urban environments, with minimal concentrations in the range of ppb needed to cause lifelong detrimental effects to biogenic organisms. Remediation of these PAHs from the environment is costly and time consuming, and collecting the entirety of these plumes represents a muchneeded focus of study. D-μ-SPE studies using ACC is currently a topic of novel research focus with sorption capabilities for remediating pollutants such as PAHs and oil/gas leaks.
The method used is adapted from  that used waste avian eggshells as a substrate source for uptake of PAHs, however this study used naturally found Quahog shells as the nanoporous sorbent source. First 50 mg of pure ACC derived from seashells (as explained in chapter 1) were collected and rinsed with 5 ml of dichloromethane (DCM) and ultrasonicated for 5 minutes to coat the entirety of the surface area. The shell was then placed in a 10 ml glass vial filled with 5 ml of DI water and spiked with either 25 μl or 35 μl of a 16-PAH (100 mg/l) standard mixture. The hydrophobic PAH/DCM solution is concentrated at the bottom of the vial with the ACC shell fragment and is ultrasonicated for 30 minutes for analyte sorption into the pores and crevices. Afterwards, it is taken out and centrifuged at 3000 rpm for 2 minutes and then the aqueous solution is discarded and the shell dabbed dry with lint free tissue. The PAH/DCM has been fully sorbed into the shell at this point, so now desorption of the pollutant from the solid is needed for analyte analysis. This is conducted by placing the shell into a separate 10 ml glass vial and filled with 2 ml of DCM, and ultrasonicated again for 15 minutes. Ideally all of the PAH will then be desorbed from the shell and present in the 2 ml of DCM, which will then be microsyringed out and injected into a 2 ml amber glass vial. The resulting 2 ml was then sent out to Brown University for gas chromatography mass spectrometry (GC-MS) analysis where concentrations were evaluated for each of the 16 PAHs used in the standard mixture. From start to finish (excluding analysis), this method took 52 minutes and used less than 10 ml of DCM, using commonly accessible equipment found in an average laboratory setting.
Results from previous D-μ-SPE experiments using ACC as a substrate yielded 95% or more sorption capability of all PAHs tested. The methodology of this experiment using Quahog derived ACC seashells has never been tested before, yielding interesting results that are subject to major discussion in future experiments. Figure 18. Trial 1 of a 16 PAH-mixture standard sorbed and desorbed with DCM from ACC and spiked afterwards with 500 μl of a 16 PAH-mixture standard for GC-MS analysis.  Figure 19. Trial 2 of a 16 PAH-mixture standard sorbed and desorbed with DCM from ACC and spiked afterwards with 500 μl of a16 PAH-mixture standard for GC-MS analysis.
In both trials (Figures 18 & 19) the results were consistent across each PAH in the 16 PAH-mixture standard that were tested. However, the difference in the sorption capabilities of ACC for each PAH is subject for debate and urged for future experimentation. The reasoning for the variations and possible causes of those difference may be due to a single or combination of factors, many of which may stem from the amorphous surface morphology and varying pore sized of ACC derived from the seashells, while also the molecular weights of the PAHs evaluated: 1) The pore sizes of the ACC from seashells are so small that they are able to absorb low molecular weight PAHs but are reluctant to desorb them after the sorption process has occurred.
2) The high molecular weight PAHs are able to adsorb onto the high surface area of ACC from Quahog shells (less absorption into pores), and thus easier to be removed during the desorption process.
3) Human and/or machine error; detection limits of the GC-MS during analysis, residual concentrations sticking to the glass vial, not completely microsyringing out the entire analyte mixture. 4) Volatilization of the PAH/DCM mixture during the D-μ-SPE process, with low molecular weight PAHs evaporated into the gas phase more so than the high molecular weight PAHs.
Human and machine error is a factor that is a possible contribution to all scientific evaluations (3), however the other three scenarios may have a high influence based on the physical properties of the substances used. Traube's law dictates that the solubility of organic molecules decreases with increasing molecular weight (White 2013), and so DCM may not fully solubilize all of the PAHs fully. During the ultrasonification step, the vibrations naturally cause the water bath in which the glass vials are soaking in to heat up, possibly causing an increase in the rate of volatilization of the DCM/PAH mixture turning it from the liquid phase into the gas phase (4). In terms of molecular weight, the PAHs on the x-axis of Figures 18 & 19 are listed in order of increasing molecular weight from left to right, which is a possible reasoning for the sorption capabilities/differences in pore sizes (1 & 2). As established from the SEM images, the ACC has crevices and pore sizes that are detectable in the low nanometer range which could possibly trap the low molecular weight PAHs (absorb into the pores) and not release them; or the large molecular PAHs are too large to fit into those pores and crevices, and are therefore only subjected to adsorption processes (being attached to only the surface) of the ACC which are then easier to be removed by desorption during the second round of ultrasonication. These causes may either individually or collectively contribute to the differences in the detected PAH concentrations, and so it is implored for future studies to investigate further into establishing the scope and capabilities of using ACC in D-μ-SPE practices for removing PAHs.