Effects of Chemical Properties of on Transport through Granular Activated Carbons

A Thesis submitted to the

Division of Research and Advanced Studies

of the University of Cincinnati

In Partial Fulfillment of the

Requirements for the Degree of

MASTER OF SCIENCE

In the Department of Chemical and Environmental Engineering of the College of Engineering and Applied Sciences

2018

By

Bingran Chen

B.S., Environmental Science, Tianjin University of Science and Technology, China, 2013

Committee

Dr. Soryong Chae (Chair)

Dr. Dionysios D. Dionysiou

Dr. George A. Sorial

Abstract

Cyanobacteria are microorganisms that can multiply to produce harmful algal blooms

(HABs) under favorable conditions in the fresh waters. Many genera of cyanobacteria are identified capable of producing cyanotoxins, which are may lead to irritation, illness or even death in pets, livestock and humans. and cylindrospermosin are examples of toxins that are often detected in the fresh water.

Granular activated carbon (GAC) adsorption is considered to be one of the most successful techniques to eradicate dissolved cyanotoxins from fresh water. Numerous researches was conducted using virgin GAC that investigated the factors, which influence the adsorptions with the use of Rapid Small Scale Column Test (RSSCT). However, only a small number of studies focused on the use of reactivated GAC and assessed whether the chemical properties of cyanotoxins had an influence on the course of adsorption. This study aims to evaluate the effects of chemical properties of cyanotoxins (i.e., MC-LR, MC-RR and CYN) on adsorption by GAC and also to study the effect of physical properties (i.e., virgin vs. reactivated) of GACs on the eradication of cyanotoxins.

The eradication efficiency of three cyanotoxins (i.e. MC-LR, MC-RR, and CYN) with the two GACs (i.e. virgin GAC and reactivated GAC) was tested. The results indicated that the breakthrough was only observed from using the reactivated GAC column with CYN at the end of operation. This observation could be due to the lower molecular weight and greater hydrophilicity of the CYN compared to MC-LR and MC-RR.

In addition, as compared to the reactivated GAC, the virgin GAC exhibited a higher success in eradicating cyanotoxins, together with the total organic carbon (TOC) and dissolved organic carbon (DOC) in the substrate. The reason is that micropore allows better and greater adsorption due to its small pore size and its special structure. Hence, the virgin

GAC with more micropore demonstrated better action than the reactivated GAC.

Acknowledgements

First and foremost, I would like to devote my most sincere gratitude to my supervisor Dr.

Soryong Chae for providing this precious research opportunity. His patience, support and encouragement greatly supported me to overcome many crisis situations in all the time of working on the experiment and writing this thesis.

My deepest thanks also to Dr. George Sorial and Dr. Dionysios Dionysiou for devoting their effort and time by serving as my thesis committee. I am grateful to them for all the discussions that helped me to address the technical problems of my work.

I would also like to give many thanks to the Greater Cincinnati Water Works for this collaboration, especially to Maria Meyer, Great Cincinnati Water Works’ treatment supervisor and Dr. Ying Hong, senior engineer for their endless support on the experimental design, analysis and provision of equipment. Their guidance and encouragement enlightened me at the first glance in the real water treatment world. I would also like to express my appreciation to Kevin Reynolds for his assistance on the ELISA analysis and equipment fixation. Thanks to Dr. Toby Sanan for his assistance on the experiment samples analysis using LC/MS/MS.

I give my additional thanks to my colleague in my research group, Yootaek Oh for his help and excellent guidance on SEM as well as to all the people who delivered the water samples and picked up the samples for me; I greatly appreciate all of the group members’

contribution, kindness and friendship during my three-year research. Last but not the least, I am deeply indebted to my mother and my boyfriend. This accomplishment would not be possible without their love and support. Thank you to all the people who have supported and helped me all throughout this period.

Table of Contents

CHAPTER 1. INTRODUCTION ...... 1

CHAPTER 2. LITERATURE REVIEW ...... 5

2.1 Harmful Algal Blooms (HABs) ...... 5

2.2 Morphology and Taxonomy of cyanobacteria ...... 5

2.3 Bloom Formation of Cyanobacterial HABs ...... 6

2.4 Detected Methods of Cyanobacteria ...... 8

2.5 Cyanotoxins ...... 8 2.5.1 Microcystins (MCs) ...... 9 2.5.2 (CYN) ...... 13 2.5.3 Anatoxin-a (ANTX-a) ...... 15 2.5.4 (STXs) ...... 16

2.6 U.S. EPA Health Advisory ...... 17

2.7 Methods for Screening Cyanotoxins ...... 18

2.8 Removal of Extracellular Cyanotoxins ...... 19 2.8.1 Ultraviolet disinfection...... 19 2.8.2 Oxidants ...... 19 2.8.3 Advanced oxidation techniques ...... 22 2.8.4 Biological inactivation ...... 22 2.8.5 Membrane filtration ...... 23 2.8.6 Activated carbon (AC) ...... 25 2.8.6.1 Physical properties ...... 25 2.8.6.2 Adsorption process ...... 26 2.8.6.3 Powered activated carbon (PAC) ...... 26 2.8.6.4 Granular activated carbon (GAC) ...... 28

2.9 Research Motivation, Objectives, and Hypotheses ...... 30 2.9.1 Research motivation ...... 30 2.9.2 Research objectives ...... 31 2.9.3 Hypotheses ...... 31

CHAPTER 3. MATERIALS AND METHODS ...... 32

3.1 Materials ...... 32 3.1.1 GAC Influent ...... 32 3.1.2 Cyanotoxins...... 33 3.1.3 Granular Activated Carbon (GAC) ...... 33

3.2 Rapid Small-Scale Column Test (RSSCT) ...... 34 3.2.1 Experiment design and set up ...... 37 3.2.2 Carbon Defining ...... 38 3.2.3 Water Sample Preparation...... 39

I 3.2.4 Carbon Column Packing ...... 39 3.2.5 RSSCT Sample Collection ...... 40

3.3 Methods ...... 40 3.3.1 Sampling Procedure of RSSCT ...... 40 3.3.2 Characterization of natural organic matter ...... 41 3.3.3 Characterization of GACs ...... 42 3.3.3.1 Surface morphology ...... 42 3.3.3.2 Pore size distribution and surface area determinations ...... 43 3.3.4 Analysis of cyanotoxins ...... 44

CHAPTER 4. RESULTS AND DISCUSSION ...... 45

4.1 Characteristic of GAC influent and effluent ...... 45

4.2 Characteristic of GACs ...... 54 4.2.1 Surface morphology of virgin and reactivated GACs ...... 54 4.2.2 Pore size distributions and surface area of virgin versus reactivated GACs ...... 54

4.3 Transport of Cyanotoxins through virgin and reactivated GACs ...... 58

CHAPTER 5. CONCLUSIONS ...... 65

5.1 Summary ...... 65

5.2 Suggestions for Future Work ...... 66

II List of Figures

Figure 2. 1 Typical structure of microcystins ...... 11 Figure 2. 2 Structure of cylindrospermopsin ...... 14 Figure 2. 3 Structure of anatoxin-a ...... 16 Figure 2. 4 Structure of saxitoxins ...... 17 Figure 2. 5 Drinking water health advisories ...... 18 Figure 3. 1 Schematic diagram of water treatment process ...... 32 Figure 3. 2 RSSCT Equipment ...... 38 Figure 3. 4 LC-OCD fractions over the SEC chromatogram with OCD ...... 42 Figure 3. 5 Operation of LOC - OCD ...... 42 Figure 4. 1 Size distribution of feed solution ...... 46 Figure 4. 2 TOC removal by GACs during Run 1 (GACI only) ...... 48 Figure 4. 3 TOC removal by GACs during Run 2 (GACI + MC-LR 10 ppb) ...... 48 Figure 4. 4 TOC removal by GACs during Run 3 (GACI + MC-RR 10 ppb) ...... 49 Figure 4. 5 TOC removal by GACs during Run 4 (GACI + CYN 10 ppb) ...... 49 Figure 4. 6 TOC removal by GACs during Run 5 (GACI + MC-LR 40 ppb and CYN 10 ppb) ...... 50 Figure 4. 7 Hydrophilic DOC for Run 2 (GACI + MC-LR 10 ppb) ...... 51 Figure 4. 8 TOC removal for Run 2 (GACI + MC-LR 10 ppb) ...... 52 Figure 4. 9 Hydrophilic DOC for Run 4 ...... 53 Figure 4. 10 TOC removal for Run 4 ...... 53 Figure 4. 11 Morphology of ground and defined virgin (left) and reactivated (right) GACs. 54 Figure 4. 12 Pore size distribution for GACs before RSSCT ...... 55 Figure 4. 13 Pore size distribution for GACs before RSSCT versus after RSSCT ...... 57 Figure 4. 14 Cyanotoxins in the effluent with Run 2 (GACI + MC-LR 10 ppb) ...... 60 Figure 4. 15 Cyanotoxins in the effluent with Run 3 (GACI + MC-RR 10 ppb) ...... 60 Figure 4. 16 Cyanotoxins in the effluent with Run 4 (GACI + CYN 10 ppb) ...... 61 Figure 4. 17 Cyanotoxins in the effluent with Run 5 (GACI + MC-LR 40 ppb and CYN 10 ppb) ...... 61

III List of Table

Table 3. 1 Characteristic of GACI (1) ...... 33 Table 3. 2 Characteristics of GACI (2) ...... 33 Table 3. 3 Specifications and Typical Properties of FILTRASORB 400 ...... 34 Table 4. 1 TOC concentration of feed solutions ...... 45 Table 4. 2 DOC concentration of feed solutions...... 46 Table 4. 3 Surface area and pore volume of GACs before RSSCT ...... 55 Table 4. 4 Surface area and pore volume of GACs after RSSCT ...... 56 Table 4. 5 Surface area and pore volume of GACs before RSSCT versus after RSSCT ...... 58

IV Chapter 1. Introduction

In the summer of 2014, an outbreak of HABs in the Lake Erie caught the people’s and the government’s attention. Approximately 400,000 residents in or around the City of Toledo, Ohio faced serious drinking water concerns since the toxin was detected (Smith, King, &

Williams, 2015). By October 2015, the same issue occurred again and forced the water providers to re-examine the blooms. It was discovered that there was a toxic alga outbreak that was distributed more than 600 miles down the Ohio River through the four States (Seewer, 2015).

Although the algae toxins did not contaminate any of the municipal water supplies along the river, the government of Cincinnati still spent more than $7,700 per day to introduce chemicals to tap water in order to repel the toxins that may cause rashes, vomiting and breathing difficulty upon consumption (Seewer, 2015). Cyanotoxins has been under regulation since 2016 in Ohio.

Aside from the appearance of cyanobacterial HABs in Ohio, other states in the U.S. such as

Florida, Louisiana and North Carolina also detected cyanobacterial HABs in their area.

Furthermore, cyanobacterial HABs also affects other countries, like China, Australia and Brazil

(H. W. Paerl & Otten, 2013). These historical events heightened the people’s awareness to

“cyanotoxins” and “cyanobacteria”.

Cyanotoxins are secondary metabolites that originate from Cyanobacteria. Cyanobacteria are blue-green algae, which can be commonly found in most of the water systems. Under favorable environmental conditions, a single or several species of cyanobacteria may overgrow, which is referred to as cyanobacterial bloom (Chorus & Bartram, 1999). Some negative effects associated with cyanobacterial bloom include decline in ecosystem stability and production of

1 highly active toxic compounds. When these phenomena occur, it is named as the “harmful algal blooms”. Additionally, the incidence of Eutrophication increases the appearance and intensity of harmful cyanobacterial blooms in freshwaters, such as reservoirs, from which 50% produce cyanotoxins (Codd, 1995).

In terms of chemical structure and toxicity, cyanotoxins are diverse. Common chemical compounds in cyanotoxins comprises of cyclic peptides, alkaloids, and lipopolysaccharides

(LPSs). Microcystins (MCs) and (NODs) are examples of the common cyclic peptides, while Cylindrospermopsin (CYN) is a kind of alkaloid. On the other hand,

Hepatotoxins are cyclic peptides but are usually alkaloids (Antoniou, De La Cruz,

& Dionysiou, 2005). Based on the target organ in animals, cyanotoxins can be categorized into four: hepatotoxins, neurotoxins, cytotoxins, and skin and gastrointestinal irritants. MCs, NODs, and (CYN) all belong to hepatotoxins. Anatoxins-a (ANTX-a), antoxin-a(s) (ANTX-a(s)), saxitoxins (STXs), homoanatoxin-a, and neosaxitoxins are examples of neurotoxins. Cytotoxins include , debromoaplysiatoxins, lingbyatoxin, and lipopolysaharide endotoxin

(Falconer, 2008). Commonly, cyanotoxins exist in the cytoplasm within the cell. When the cells break down or lyse, cyanotoxins are released into the water, which poses a potential threat to the animals and humans consuming the water, especially as a potable water source. Cyanotoxins can cause illness and even death in humans (Briand, Jacquet, Bernard, & Humbert, 2003).

Drinking water Contaminant Candidate List (CCL) is an unregulated contaminants list published by the U.S. Environmental Protection Agency (EPA). CCL contains identified or expected contaminants to appear in public water systems in the U.S. that may pose a risk in drinking water. In 2001, four microcystin variants (RR, LR, YR, and LA), CYN, and ANTX-a were included in the U.S. priority list of freshwater algal toxins. In 2012, ANTX-a, microcystin-

2 LR (MC-LR), and CYN were listed in CCL 3 (Szlag, Sinclair, Southwell, & Westrick, 2015).

Later, in November 2016, EPA announced the Final CCL 4 on which the group of cyanotoxins includes, but is not limited to: ANTX-a, CYN, MCs, and STXs. Among these, the most widespread cyanotoxins is the MCs and the most toxic MCs is the MC-LR. Many countries such as Brazil, Canada, Australia and New Zealand have developed guidelines for cyanotoxins in drinking and recreational waters. The allowed value of MC-LR for drinking water is 1.0 g/L, a standard developed by the World Health Organization (WHO) (US EPA, 2014).

Many eradication techniques for cyanotoxins including chemical oxidation, filtration, and adsorption in drinking water treatment plants have been applied. Ultraviolet (UV) disinfection, oxidation, and advanced oxidation process (AOP) are the three major chemical removal techniques used. Among these techniques, chlorine and potassium permanganate can remove majority of the MCs and CYN, but excluding the elimination of the produced disinfection-by products (Rodriguez, Majado, Meriluoto, & Acero, 2007). UV disinfection does not introduce chemicals for disinfection; hence, it is difficult for drinking water treatment plants to identify high levels of UV. On the other hand, the biological method of eradication does not produce any harmful chemicals. However, it takes longer time to finish the reaction (Lam, Fedorak, & Prepas,

1995). Other approaches such as the use of nanofiltration and reverse osmosis membranes can achieve high removal of cyanotoxins (Merel et al., 2013). Nevertheless, the expensive cost of membrane makes it not ideal to use for most of the small drinking water treatment plants.

Usually, Activated Carbon, including GAC and powdered activated carbon (PAC), are used in drinking water treatment due to its high adsorption rate and absence of harmful chemicals production. More than 85% of the known MCs and anatoxins could be removed by PAC and

GAC (Alvarez, Rose, & Bellamy, 2010; Xagoraraki, 2007). The development of a natural

3 organic matter (NOM) layer on the AC could influence the adsorption efficiency (Ho, Lambling,

Bustamante, Duker, & Newcombe, 2011).

4 Chapter 2. Literature Review

2.1 Harmful Algal Blooms (HABs)

Algae, including macroscopic and microscopic species, are a large group of organisms.

Algal blooms are referred to as the algae’s exuberant growth under favorable environmental conditions. When an algae bloom poses a threat to the environment, the humans and the animals, this bloom is considered as HABs (Lewis & McCourt, 2004). HABs commonly occur in brackish water, like marine, estuarine and fresh water ecosystems such as lake and reservoir. Despite that within a wide range of salinity level, many algal groups form HABs, like dinoflagellates, cyanobacteria, which are the dominant source of cyanotoxins in the freshwater system (Hudnell,

2008). Cyanobacterial harmful algae blooms (CyanoHABs) exhibited the greatest health impact to the public in many areas worldwide (Lewis & McCourt, 2004). In the U.S., every coastal state was reported to be attacked by HABs (NOS, 2018).

2.2 Morphology and Taxonomy of cyanobacteria

Cyanobacteria are also referred to as the blue-green algae, which are photosynthetic bacteria.

It is very confusing to compare with the green algae for both can produce dense mats, which can impede some recreational activities, like fishing and swimming. Additionally, it may cause odor problems and oxygen depletion. However, the difference between cyanobacteria and green algae is that the former can produce toxins under favorable conditions (US EPA, 2017).

Cyanobacteria’s habitat ranges from hot springs to frozen ponds in Antarctica and can survive

5 both in the fresh water and the saline water (Whitton, 1992). They grow as single cells without nucleus, wherein they may live as single cells in colonies, or as single cells in filaments. A mucilaginous sheath like Microcystis sp. may cover the cells growing in colonies, floating mats or free-floating strands, which are another form wherein filamentous species may grow.

Numerous cyanobacterial species possess gas vacuoles, such as Anabaena flos-aquae, which allow them to float on the water surface or even in different levels below the surface of the water

(Hitzfeld, Höger, & Dietrich, 2000). This causes the cyanobacteria to converge on the surface of water and result to a pea-soup green color or blue-green “scum”. Meanwhile, other species like

Planktothrix stays in the bottom sediments and only float on the water surface in case of storms or other sediment disturbances (US EPA, 2014).

2.3 Bloom Formation of Cyanobacterial HABs

The U.S. EPA official website presents the definition of cyanobacterial HABs (cyanoHABs), as freshwater cyanobacterial blooms which produce highly potent cyanotoxins (US EPA, 2014).

Throughout the years, the incidence and intensity of cyanobacterial blooms are observed to be increasing all over the world (de Figueiredo, Azeiteiro, Esteves, Gonçalves, & Pereira, 2004).

Since cyanobacteria are primarily phototrophic microorganisms, groundwater resources are not usually affected by bloom formation, as compared to the surface water. Several factors influence cyanobacterial blooms such as environment and human interventions. Climate change, augmented external nutrient loadings by altered precipitation patterns, increased residence time, increased atmospheric carbon dioxide levels, higher salinity, and direct and indirect temperature are all anticipated to further aggravate these blooms (De Senerpont Domis et al., 2013; Hans W

6 Paerl & Huisman, 2009). In other words, water temperature, light intensity and total sunlight duration, nutrient availability (especially nitrogen and phosphorus), pH, an increase in precipitation events, water flow and water column stability may influence the formation of cyanobacterial blooms. Since, some cyanobacteria species prefer warmer water (25ºC or above), it is inevitable that global warming promotes the formation of cyanobacterial blooms (Wiedner,

Rücker, Brüggemann, & Nixdorf, 2007). Light exposure is also an important factor for most of the cyanobacteria, even though some of them can be regarded as heterotrophic or chemotrophic.

Specific species needs varying quality, intensity and duration of light. It can be observed that the pigmentation protects the cyanobacteria from photoinhibition. Photoinhibition refers to the inhibition of photosynthesis due to excessive light exposure resulting to the reduction of plant growth (Goh, Ko, Koh, Kim, & Bae, 2012). Also, it is capable of light harvesting in the visible spectrum compared to other photoplankton species (Merel et al., 2013). In addition, some species can stay in caves for a long time without visible light and can still grow immediately after light exposure (Liang, Xie, Chen, & Yu, 2011; Merel et al., 2013). Normally, cyanobacterial blooms appear at a pH level of between 6 and 9 (Liang et al., 2011). Trophic level of the aquatic system is also an important factor for bloom formation. Usually, the N/P ratio ranging from 10 to 15

(Mur, Skulberg, & Utkilen, 1999) is the ideal condition for the cyanobacterial blooms in eutrophic reservoirs (El-Shehawy, Gorokhova, Fernandez-Pinas, & del Campo, 2012). Although these favorable conditions are present in most areas of the U.S. and can promote cyanobacterial blooms in the summer, it does not mean that cyanobacterial blooms must also occur. The interrelationship of these factors fluctuates and differs from every season and every year. Some toxin-producing strains may appear in the early summer, while others may possibly occur in the late summer (US EPA, 2014).

7

2.4 Detected Methods of Cyanobacteria

The methods of detecting and monitoring the presence of cyanobacterial blooms include measuring chlorophyll a levels (Chl-a), which is a common approach; However, this method does not distinguish cyanobacteria from algae. Therefore, the combination of measuring specific cyanobacterial pigments, like phycocyanin and analysis of Chl-a may help in determining the proportion of cyanobacteria among the detected algae species (Brient et al., 2008; Merel et al.,

2013). Another common method used is microscope identification. It is more accurate to use since it can identify and convey the proportion of cyanobacteria cells. Nevertheless, it is time- consuming, and requires a high level of taxonomic expertise to be properly performed. A new technique called the polymerase chain reaction (PCR) of genes for detecting cyanobacteria has been proposed (Al-Tebrineh, Mihali, Pomati, & Neilan, 2010; Kaebernick & Neilan, 2001).

2.5 Cyanotoxins

Cyanotoxins are produced by the harmful strains of cyanobacterial algal blooms (also known as Cyano-HABs) (US EPA, 2014). Based on the damage to human organs of cyanotoxins, it can be classified into four: Hepatotoxins that induce liver injuries, Neurotoxins that alter the neuromuscular transmission, Cytotoxins, and skin and gastrointestinal irritants. Specifically,

Nodularin, Microcystins and Cylindrospermopsin are Hepatotoxins. Anatoxin-a, Anatoxin-a(S),

Saxitoxins, Neosaxitoxin, Homoanatoxin-a, and Aplusiatoxin are Neurotoxins.

Debromoaplysiatoxin, Lingbintoxin, and Lipopolysaharide endotoxin are Cytotoxins

(Kaebernick & Neilan, 2001). Structurally, cyanotoxins can be classified as cyclic peptides

8 (hepatotoxins), alkaloids (neurotoxins), and lipopolysaccharides (LPSs) (Antoniou et al., 2005).

Additionally, MC-LR, ANTX-a, and CYN are the three most important cyanotoxins and are listed on CCL3. Among the MCs, MC-LR is the most toxic.

In general, cyanotoxins appears in water in two ways: intracellular and extracellular. Most of the time, cyanotoxins are present in the cytoplasm within the cell. When the cell breaks or dies, the toxin will be released into the water or referred to as extracellular toxins. Most of the MCs are intracellular toxins, wherein 95% is intracellular toxin. On the other hand, 50% of the CYN is intracellular, while the rest is extracellular (US EPA, 2014). Generally, extracellular toxins are more difficult to be eradicated from water because it can be easily be adsorbed to clays and other organic materials present in the water system compared to intracellular toxins.

Cyanotoxins were tested in drinking water treatment plants in several countries, such as

South Korea, Italy, New Zealand and Switzerland (Dietrich & Hoeger, 2005). The commonly found cyanotoxins in the U.S. are MCs, CYN, ANTXs, and STXs (US EPA, 2014). Generally,

MCs and CYN are the most frequently detected toxins (Zamyadi et al., 2012).

2.5.1 Microcystins (MCs)

MCs are the most common toxins detected in the fresh bodies of water and are the leading cause of poisoning in animals and in humans among all the recognized cyanotoxins. Structurally,

MCs are a group of monocyclic heptapeptide hepatotoxins with a molecular weight ranging from

800 to 1100 Da (Westrick, Szlag, Southwell, & Sinclair, 2010). These cyanotoxins often produced by the five genera of cyanobacteria: Microcystis, Anabaena, Nostoc, Oscillatoria,

Anabaena and Anabaenopsis species. Currently, there are more than 60 types MCs that were

9 identified (Liu, Qian, Dai, & Feng, 2008). MCs are composed of cyclic peptides with seven amino acids connected via peptide bonds in a cyclic configuration. Specifically, these include: three D-amino acids, namely alanine (Ala), methylaspartic acid (MeAsp) and glutamic acid (Glu); two unusual amino acids, N-methyldhydroalanine (Mdha) and 3-amino-9-methoxy-2,6,8,- trimethyl-10-phenyldeca-4,6-dienoic acid (Adda); and two variable L-amino acids. The general structure of MC is presented in Figure 2.1 (Liu et al., 2008). As shown in Figure 2.1, X and Z correspond to the two variable amino acids, which are responsible for the different names of toxin molecules. For instance, when amino acid X is leucine (L is the abbreviation for leucine) and Z is arginine (short for R), this toxin is referred to as microcystin-LR (MC-LR). When the amino acids X and Z are arginine, the molecule is named MC-RR. In the structure of MC-YR, the amino acids attached are tyrosine and arginine, respectively. These two are just a few of more than one hundred variants of MCs formed by the multiple combinations of variable amino acids. Most congeners are hydrophilic. Adda is associated with toxicity due to its conjugated diene (Dawson, 1998). In environmental waters, MCs are either neutral or anionic. The cyclic structure and novel amino acids present in MCs enable it to resist heat, oxidation, and hydrolysis.

Therefore, they can exist in cooler, darker water bodies for several months (A. de la Cruz et al.,

2011). However, high temperature (40 ℃), acidity (pH < 1), alkalinity (pH > 9) or boiling (Tsuji et al., 1994a), chemical and biological degradation (T. & J., 1998) and sunlight (Tsuji et al., 1995) can slowly disintegrate MCs. The presence of humic acids and pigments facilitate the degradation of the toxins. The adsorption of light from the UV-Vis helps the humic acids in the formation of free radicals including hydroxyl radicals, which can degrade the toxin (Antoniou et al., 2005).

10

Figure 2. 1 Typical structure of microcystins

MC-LR (C49H74N10O12) is the identified to be the most toxic of microcystin variants. The molecular weight of MC-LR is 994, which is quite large. There are two carboxyl groups and one amino group present in MC-LR. The World Health Organization presented a standard value for drinking waters of 1.0 µg/L MC-LR. On the other hand, a 1.3 µg/L of MC-LR toxicity standard value has been set by the Australian Drinking Water Guideline (Ho et al., 2011). Since there are several species of MC in water due to ionizable groups, it becomes quite difficult to measure these species in water, hence, the n-octanol/water distribution ratio (Dow) is the only available representation of Kow of MCs. De Maaged and colleagues (de Maagd, Hendriks, Seinen, & Sijm,

1999) measured Dow of MC-LR directly by high-performance liquid chromatography (HPLC) and found that log Dow of MC-LR decreased constantly with an increase in pH from 1 to 12, which means that the hydrophilicity of MC-LR is also increasing. Liang et al. (Liang et al., 2011) confirmed the De Maaged et al. research results using liquid chromatography combined with

+ mass spectrometry (LC-MS). They found that [(COOH)2(NH2 )] is thedominant species present

- + when the pH is less than 2; when the pH is between 2 and 5.5, [(COO )(NH2 )] is the prevailing

- 2+ species in solution and ; when pH is greater than 5.5, [(COO )2(NH )] is the leading species.

The corresponsive charges at the different pH levels are positive, neutral and negative, respectively.

11 MC-RR (C49H75N13O12) has two ionizable carboxyl groups and two amino groups apart from the main molecule. The molecular weight is 1038.2. When the pH is less than 2,

+ - + [(COOH)2(NH2 )2] is the dominant species; when the pH is between 2 and 5.5, [(COO )(NH2 )2]

- 2+ is the prevailing species; when the pH is greater than 5.5, [(COO )2(NH )2] is the leading species. The corresponsive charges at the different pH levels are positive, positive and neutral, respectively.

Both MC-LR and MC-RR have two carboxyl groups, which means they have the same ability to disassociate into anion and have similar behavior in basic condition. The pKa values of the carboxyl group and amino group are 2.1 and 12.5, respectively (de Maagd et al., 1999). De

+ Maaged et al. (de Maagd et al., 1999) found that [(COOH)2(NH2 )] was much more hydrophobic

- + than [(COO )(NH2 )]. Since the two amino acids dissociated absolutely at these pH levels, the

- 2+ [(COO )2(NH )] species was not the dominant species at any pH for MC-RR. Therefore, when the pH is less than 2, MC-LR is much more hydrophobic than MC-RR. The order of

- 2+ + - hydrophilicity of species was estimated as [(COO )2(NH )] << (COOH)2(NH2 )2 < [(COO

+ - 2+ + + )(NH2 )] < [(COO )2(NH )2] = [(COOH)2(NH2 )]. Overall, (NH2 ) was more hydrophobic than

(COO-).

Numerous studies have illustrated that MCs have a bad influence on wildlife, livestock, domestic animals and humans. Handeland and Ostensvik conducted a study on roe deer and observed that it became stuporous, unresponsive, weak with fine muscle fasciculations, and exhibited liver lesions compatible with MC intoxication (Handeland & Østensvik, 2010). In another study by Matsunaga et al., it was discovered that 20 spot-billed ducks showed unnatural death in the pond with bloom in Japan. After examining the water, it was discovered that there was 161 μg/g MC-LR level in the pond (Matsunaga et al., 1999). In the fall 2011, three dogs

12 swam in the Lake Amstelmeer, The Netherlands. Then, several bad symptoms occurred on these dogs: vomiting, lethargy, difficulty breathing, signs of abdominal pain, and gastrointestinal bleeding (LÜRling, Eshetu, Faassen, Kosten, & Huszar, 2013). For humans, there were also several studies that indicated MC-LR could result in illnesses after acute exposure, such as diarrhea and vomiting (Turner, Gammie, Hollinrake, & Codd, 1990). After the chronic exposure,

Yu et al. found out that there was an incidence of primary liver cancer in the affected subjects

(Yu, Zhao, & Zi, 2001).

2.5.2 Cylindrospermopsin (CYN)

CYN has become one of the most influential and interesting compounds of toxins present in freshwaters worldwide due to the severity of health impacts it causes as well as due to the frequency of occurrence (Roegner, Brena, González‐Sapienza, & Puschner, 2014). Currently, there are three known naturally occurring CYN variants, but only two of them (7- epicylindrospermopsin and deoxycylindrospermopsin) have been characterized and isolated.

CYN was first isolated from a culture of Cylindrospermopsis raciborskii from the drinking water supply reservoir on Palm Island, Queensland, Australia (Ohtani, Moore, & Runnegar, 1992).

CYN is biosynthesized from at least five genera of cyanobacteria including Cylindrospermopsis raciborskii, Anabaen bergii, Aphanizomenon ovalisporum, Aphanizomenon flosaquae, Umezakia natans, and Raphidiopsis curvata (Falconer & Humpage, 2005).

CYN (C15H21N5O7S) is a tricyclic alkaloid that comprises of tricyclic guanidine moiety with hydroxymethyluracil (as illustrated in Figure 2.2) (Ohtani et al., 1992). The molecular weight of

CYN is approximately 415 Da. It is a highly polar hepatotoxin because it contains zwitterionic structure. The zwitterion means that the CYN contains the negatively charged sulfate group and

13 the positively charged guaido group, which makes CYN highly soluble in water (White &

Hansen, 2005) and is considered as a hydrophilic compound (Froscio, Cannon, Lau, & Humpage,

2009). CYN is stable in extreme pH, light and temperature levels. It can remain stable at temperatures ranging from 4 to 50℃ for up to 5 weeks in the darkness. It was observed that 83% of the CYN with the initial quantity of 1 mg/L concentration remains after 4 weeks at 50℃. In high purity water, CYN has a half-life greater than 10d and it cannot be degraded by different levels of pH and changes in temperature (Wormer, Cirés, Carrasco, & Quesada, 2008). However, sunlight can degrade 90% of the CYN within 3 days (Chiswell et al., 1999).

Figure 2. 2 Structure of cylindrospermopsin

CYN was identified toxic to other bacteria, plants, invertebrates and vertebrates including humans. M. Freitas et al. discovered that the high concentration (100µg/L) of CYN could influence both the yield and the nutritional condition of lettuce (Handeland & Østensvik, 2010).

In another study, Metcalf et al. assessed the effect of CYN on the brine shrimp (Artemia selina) and discovered that 20µg/L CYN was lethal and the LD50 values decreased with the time from

8.1 to 0.71 µg/ml between 24 and 72 hours, respectively (Metcalf et al., 2002). Guzmán-Guillén

14 et al. also studied the potential neurotoxicity of CYN on the tilapia fish (Oreochromis niloticus).

They were the first to confirm the neurotoxicity of CYN (L. S. Wang et al., 2009).

2.5.3 Anatoxin-a (ANTX-a)

ANTX-a is a bicyclic secondary amine and the smallest among all the cyanotoxins. Its molecular weight is 165 Da. They are mainly associated with the three cyanobacterial genera:

Anabaena, Planktothrix, and Aphanizomenon and commonly found in the U.S. (Osswald, Rellan,

Gago, & Vasconcelos, 2007), Africa (Ballot, Pflugmacher, Wiegand, Kotut, & Krienitz, 2003),

Europe (Gugger et al., 2005) and Asia (Namikoshi et al., 2003). As shown in Figure 2.3, ANTX- a has two functional groups that can be easily oxidized, the amine and α,β-unsaturated ketone.

The pKa value of the amine is 9.4, which indicates that ANTX-a is protonated and water soluble in most natural waters. Its oxidation, however, may be pH-dependent (Westrick et al., 2010).

ANTX-a can lead to paralysis by fixation on acetylcholine receptors. Finally, death can occur due to respiratory arrest. The LD50 value of mice is 375 g/kg after i.p. injection (Van

Apeldoorn, Van Egmond, Speijers, & Bakker, 2007). Even though ANTX-a could cause vomiting, convulsion and respiratory arrest, there was no record of human poisonings. Until now, there is still no official guidelines for ANTX-a in drinking water, but the formulated provisional maximum acceptable value are 6 g/L and 3.7 g/L in New Zealand and Canada, respectively.

15

Figure 2. 3 Structure of anatoxin-a

2.5.4 Saxitoxins (STXs)

STXs are also referred to as the paralytic shellfish toxin and belong to the group of neurotoxic alkaloids. Saxitoxins are mostly produced by Anabaena circinalis, Lyngbya,

Cylindrospermopsis and Aphanizomenon flos-aquae in freshwaters. However, in sea water,

STXs are produced by several species of dinoflagellates (Westrick et al., 2010). Saxitoxins are tricyclic molecules with the molecular weight ranging from 241 ~ 491 Da. Sixteen variants of

STXs were identified. As shown in Figure 2.4, STXs can be non-sulphated, single sulphated or doubly sulphated (Van Apeldoorn et al., 2007). They contain two guanidine groups with pKa values of 8.2 and 11.3. Only these two guanidine groups can undergo oxidation (Westrick et al.,

2010). Generally, STXs are stable, water-soluble and can persist more than 90 days in the freshwaters. However, high temperature can alter their structures and may be degraded into more toxic variants. The LD50 of the most toxic variant of STX was 10 g/kg for mice after i.p. injection.

16

Figure 2. 4 Structure of saxitoxins

2.6 U.S. EPA Health Advisory

Since cyanotoxins have been associated with numerous human intoxications, EPA has issued Drinking Water Health Advisories (HAs) for MCs and CYN, which is shown in Figure

2.5 (US EPA, 2014). Cyanotoxins cannot be detected in tap water at or below 0.3 micrograms per liter for MCs and 0.7 micrograms per liter for CYN. When the concentration of MCs detected in tap water is greater than 0.3 to 1.6 ppb and the concentration of CYN is greater than

0.7 to 3.0 ppb, young children and vulnerable populations were advised not to drink. When the concentration of MCs detected in tap water is greater than 1.6 ppb and the concentration of CYN detected in tap water is greater than 3.0 ppb, everyone was advised not to drink the tap water.

17

Figure 2. 5 Drinking water health advisories

2.7 Methods for Screening Cyanotoxins

Due to the high toxicity of MC-LR and the low standard set by the WHO, highly sensitive analytical methods are necessary to be developed. Reversed-phase high-pressure liquid chromatography (HPLC) with various detectors, for example, electrochemical, fluorescence, photodiode-array (DAD), and electrophoresis (CE) with UV or MS, have been developed and are currently used to detect the presence and measure the concentration of cyanotoxins. Aside from these, methods based on gas chromatography (GC) with mass spectrometer or flame ionization detectors (FID) are also utilized (Antoniou et al., 2005). Molecular test is also present in which there is a tool used to identify the presence or absence of specific cyanotoxins in a water supply, such as screening kits. Mouse, Protein Phosphatase Inhibition Assays (PPIA), and Enzyme-

Linked Immunosorbent Assays (ELISA) are rapid, sensitive, and suitable for large-scale screening. Mouse and ELISA work for both CYN and MC-LR but PPIA is only capable of screening MC-LR (US EPA, 1999). Compared to LC-MS, the sensitivity of ELISA is relatively lower. ELISA cannot distinguish MCs from . Despite its limitations, ELISA allows fast on-site detection of cyanotoxins in the absence of pretreatment (Antoniou et al., 2005).

18

2.8 Removal of Extracellular Cyanotoxins

Removal of extracellular cyanotoxins through the common drinking water treatment processes is divided into three sections: chemical inactivation, biological inactivation and physical removal. Chemical inactivation includes ultraviolet (UV) disinfection, oxidation and advanced oxidation process (AOP), while biological inactivation mainly focuses on biologically slow and rapid filtration. On the other hand, physical removal involves adsorption by granular activated carbon (GAC) or powder activated carbon (PAC), and exclusion by membrane filtration (Westrick et al., 2010).

2.8.1 Ultraviolet disinfection

Inactivation of many pathogens in drinking water relies on UV disinfection because UV energy can break their molecular bonds. The process of UV disinfection is dependent on a low to medium pressure lamp with UV doses between 10 and 40 mJ/cm2. According to literatures, the removal of CYN, MCs and anatoxin-a needs magnitude higher UV doses than the dose needed for disinfection, specifically doses ranging from 1,530 and 20,000 mJ/cm2 (Senogles et al., 2000;

Tsuji et al., 1995). Since degradation of cyanotoxins needs high doses of UV compared to disinfection, it is not readily acceptable in the drinking water treatment plants. However, the advantage of UV treatment is that this technique does not require the introduction of chemicals.

2.8.2 Oxidants

Primary oxidants added in drinking water are chlorine, chloramines, potassium permanganate and ozonation.

19 Chlorine has been playing a vital role in processing drinking water for the past 100 years.

There were also various researches that focused on the removal of cyanotoxins by chlorine. The inactivation of organic compounds is usually pH dependent since the pKa value of hypochlorous acid is 7.6 (Westrick et al., 2010). Some researches indicated that MC-LR, MC-RR and MC-YR reacted with chlorine at the same rate and had the same reduction in toxicity while other researches suggested that the reactions were different in MC-LR, MC-RR, MC-YR and MC-LA.

The order YR > RR > LR > LA indicates the order in which the different amino acids are oxidized (Acero, Rodriguez, & Meriluoto, 2005; Ho et al., 2006). In CYN, 100% removal rate can be achieved with the initial concentration of chlorine at 1.5 mg/L at pH of 7 and a temperature of 20℃. At the same time, the concentration of TTHM is 82 g/L (Acero et al.,

2005). The removal rate of ANTX-a was approximately 8% with 1.5 mg/L initial concentration of chlorine at pH 7 and a temperature of 20℃. In this case, the concentration of TTHM (chlorite, haloacetic acids and total trihalomethanes) achieved 150 g/L (Rodriguez et al., 2007). Even though there is a significant removal of cyanotoxins using chlorine, the added chlorine introduces a certain concentration of disinfection by-products.

Other oxidants, such as Chloramine and chlorine dioxide are proven not effective for treating cyanotoxins including MCs, CYN, ANTX-a, and STXs in drinking water. Several researches have proven the reaction rate constant is indeed slow. When the pH is 8, the second- order rate constant of CYN reacting with chloramine is 0.22 M/s and there is no CYN degraded.

When the pH is 7.2, the order rate constant of ANTX-a reacting with chloramine is less than 0.1

M/s and almost 0% ANTX-a is degraded (Westrick et al., 2010).

On the other hand, potassium permanganate is not very reactive with CYN but is reactive

20 with MC-LR. The apparent rate constant for MC-LR, MC-RR and MC-YR are between 350 and

420 M/s (Rodriguez et al., 2007). There was an observed 80% removal for MC-LR achieved with 1.5 mg/L of initial concentration at 20℃. The reactivity of potassium permanganate with

MC-LR does not rely on pH. However, the reactivity of potassium permanganate with CYN,

ANTX-a and STXs is pH dependent. With less than 1 mg/L initial concentration of potassium permanganate, the reactive rate constant of potassium permanganate with CYN is 0.3 M/s

(Rodríguez et al., 2006). From pH 6 to 8, the reactive rate constant of potassium permanganate with ANTX-a is constant at 25,000 M/s. It was also noted that there is no reaction between STXs and potassium permanganate at pH 8 to 10 (Rositano, Nicholson, & Pieronne, 1998). The advantage of using potassium permanganate is the restricted formation of by-products.

Meanwhile, the disadvantage is that high doses are needed to remove CYN that may lead to the formation of particulate manganese dioxide, which can cause potential problems in drinking water (Cheng, Shi, Adams, Timmons, & Ma, 2009).

Last is Ozonation, which is a powerful technique to destroy cyanotoxins and may also be used during the disinfection process. For the removal of MCs, ozone attacks the conjugated double bond and single double bond in the Adda and Mdha group, respectively. Up to 100% removal for MCs could be achieved (Xagoraraki, 2007). The reaction is also observed to be pH independent. According to Al Momani and Jarrah’s (Al Momani & Jarrah, 2010) research, 5 mg/L of MC-LR can be completely removed at pH 7 and a temperature of 20℃. Unlike the pH independent reaction with MCs, the reaction is pH dependent in CYN and ANTX-a. CYN is pH dependent from pH 4 to 10. ANTX-a is pH dependent from pH 7 to 10 (Westrick et al., 2010).

The reaction rate constant for CYN is 3.4 × 105 푀/푠 at pH 8, while the reaction rate constant is

2.5× 106 푀/푠 when pH is greater than 8. When pH is less than 8, the reaction rate constant for

21 ANTX-a is 2.8× 104 푀/푠, while the reaction rate constant increased to 8.7× 105 푀/푠 when pH is greater than 9. For STXs, ozone performs poorly (Newcombe & Nicholson, 2004).

Disadvantages of ozonation includes expensive initial cost of equipment and higher level of maintenance and operator skill since ozone is corrosive and toxic as well as the by-product formation (US EPA, 1999).

2.8.3 Advanced oxidation techniques

In the past years, advanced oxidation techniques (AOTs) have received attention for its possible use in the removal of cyanotoxins. AOTs refer to the utilization of oxidants, catalysts and radiation, or their combination to generate non-selective and highly active oxidizing radical species to detoxify organic compounds in water (A. de la Cruz et al., 2011). Common AOTs include chlorination with chlorine dioxide and hypochlorite, photochemical degradation with

Titanium dioxide (TiO2) photocatalysis under UVA and visible light radiation, Fenton and

Photo-Fenton processes, and sonolysis. Conventional water treatment processes plus AOTs can eliminate almost 100% of the cyanotoxins in water even in conditions with high cyanobacterial growth (A. de la Cruz et al., 2011). However, some limitations of AOTs exist, such as weak adsorption in sediments (Chen et al., 2008) and restricted light penetration for direct photolysis, high concentration of the oxidants, long contact times, production of carcinogenic substances and other mutagens (A. de la Cruz et al., 2011). The type of toxins, the concentration of released toxins, the contact time and the water quality all influence the efficiency of the oxidative agent

(Ho et al., 2011; Zamyadi et al., 2012).

2.8.4 Biological inactivation

22 Numerous studies had focused on the eraducation of cyanotoxins by biological degradation.

Manage et al. discovered that only few bacterial strains with degradable ability towards MCs have been isolated (Manage, Christine, & Linda, 2016). A few strains of the genus

Sphingomonas have been isolated and are capable of degrading MC-LR and MC-RR (Piccini

Ferrin, Alonso, Conde Scalone, Pernthaler, & Xavier, 2006). Harada et al. (Harada et al., 2004) reported that they have isolated another Japanese strain of Sphingomonas, 7CY, which are capable of degrading a wider range of MCs, including MC-LR, MC-RR, MC-LY, MC-LW, and

MC-LF. A microcystin-degrading bacteria strain Y2 is also capable of degrading MC-LR and

MC-LA (Ho et al., 2006). The highest reported removal percentage of MC-LR is 58% with the use of a specific strain of probiotic bacteria, Lactobacillus rhamnosus strains GG and LC-705,

Bifidobacterium longum 46, Bifidobacterium lactis 420 and Bifidobacterium lactis Bb12 (Mazur

& Pliñski, 2001). However, it is much difficult to degrade nodularin than MCs (Mazur & Pliñski,

2001). Generally, biological degradation should be used in combination with other treatment techniques, such as ozone and UV/H2O2 (Alvarez et al., 2010). Despite the usage of bacteria as a reliable, and cost-effective purification system which does not involve any harmful chemicals, the disadvantages of biological degradation cannot be neglected (Welgama, 2009). Bacteria needs longer reaction time from hours to days and, hence, not practical (Lam et al., 1995).

2.8.5 Membrane filtration

Four types of membrane filtration are classified and named based on their pore size: microfiltration (0.1 – 10 m), ultrafiltration (1 – 100 nm), nanofiltration (around 1 nm), and reverse osmosis (0.1 nm). Numerous studies have tested the potential of membrane filtration to remove microcontaminants in drinking water treatment. Due to its pore size, microfiltration membranes are not identified to be ineffective to remove extracellular cyanotoxins (Merel et al.,

23 2013). According to the study of Lee and Walker (Lee & Walker, 2006), ultrafiltration membrane is also not a reliable treatment technique for eradication of cyanotoxins. The cellulose acetate ultrafiltration membrane with 20-kDa molecular mass cutoffs did not adsorb MC-LR; while the polyethersulfone ultrafiltration membrane with 20-kDa molecular mass cutoffs can only adsorb MC-LR within the first 60 minutes. The polyethersulfone membrane with 5-kDa molecular mass cutoff can remove 8% of MC-LR, which indicates that size exclusion was the primary eliminating mechanism and that the repulsive charge interactions between the MCs and the membrane were only secondary (Lee & Walker, 2006). Nanofiltration and reverse osmosis membranes are significantly effective in the removal of extracellular cyanotoxins. More than 95% of MC-LR and MC-RR, whose initial concentrations were between 10 and 130 g/L, had been removed through the use of a reverse osmosis membrane (Neumann & Weckesser, 1998; Vuori,

Pelander, Himberg, Waris, & Niinivaara, 1997). Another type of membrane called the Trisep nanofiltration membrane with 200 Da molecular mass cutoffs achieved at least 96% removal rate for MC-LR, MC-RR, MC-YR, MC-LA and ANTX-a. However, only ANTX-a and MC-LA were identified in the permeate (Gijsbertsen-Abrahamse, Schmidt, Chorus, & Heijman, 2006). The main rejection mechanism for MCs is steric hindrance alone, whereas for ANTX-a steric hindrance and electrostatic interactions both affects the mechanism. In terms of CYN and STX, only a few studies on its removal by nanofiltration and reverse osmosis were conducted.

Despite the fact that membrane filtration is a promising technique to eradicate cyanotoxins, there are still several disadvantages of membrane filtration. It is complex and highly expensive, especially the nanofiltration and reverse osmosis. Also, successive re-mineralization of treated water is still needed due to high retention potential. Furthermore, it is too expensive for small drinking water treatment plants to pay for high-energy cost during the process (Merel et al.,

24 2013).

2.8.6 Activated carbon (AC)

AC has been widely used around the world for drinking water treatment. Common precursors of activated carbon which are used for large scale production are wood, coal and coconut. Different precursors and activated process can influence its ability to adsorb contaminants. For several types contaminant, AC can be selected. There are two types of carbon that are commonly used in the drinking water treatment: PAC and GAC. Generally, PAC is used as a temporary treatment for transient contaminants, while GAC is used in fixed bed and can reduce natural organic matter, synthetic organic compounds formed in industrialized source waters, as well as the taste and odor of compounds (Westrick et al., 2010). However, AC does not affect intracellular type of cyanotoxins and cyanobacteria. Hence, the eradication of different cyanotoxins needs different adsorbents. Many studies had shown that GAC and PAC with large amount of mesopore could effectively remove MCs and CYN (Ho, Slyman, Kaeding, &

Newcombe, 2008; Newcombe, 2002). Therefore, an AC with a larger volume of micropore is good for the removal of STXs because STXs are much smaller compared to MCs (Newcombe &

Nicholson, 2004).

2.8.6.1 Physical properties

Activated carbon is a porous material with high internal surface. Opposite to the internal surface, there is much less external surface area provided by the outside of the particle. Internal surface provides large space on which most contaminants can be adsorbed. The pores on AC can be categorized according to their sizes: Micro (< 2 nm) including primary micropores (< 0.8 nm) and secondary micropores (0.8 – 2 nm), meso (2 – 50 nm) and macro pore structure. Micropores

25 are generally described as slit-shaped, with either parallel walls or are wedge-shaped. Also, micropores are cylindrical in shape (Bansal, Donnet, & Stoeckli, 1988).

2.8.6.2 Adsorption process

The adsorption process for the removal of contaminants is highly complex. Generally, the major processes occurring during adsorption is related to diffusion. There are four steps for a molecule to be adsorbed by AC. First, a molecule must diffuse to the particle surface from the bulk liquid. Second, the molecule must pass through the liquid surface film. Third, it should pass through the pore structure of the carbon. Finally, this molecule is totally removed from the solution and adsorbed by AC. The first two steps are determined by the physical parameters of the system, such as low rates in GAC. The latter two steps rely on the AC pore size distribution and surface chemistry. Generally, the size of adsorbate should be slightly smaller than the pores which capture them, so that there would be more contact points for the compound to adhere to

(Summers & Roberts, 1988). Another important factor, which influence adsorption, is rate in which the contaminant arrives at the adsorption site. This is influenced by the structure and size of the “transport pores” and the accessibility to the internal structure through the pores on the external surface (Newcombe, 2006). AC’s surface is primarily hydrophobic (Westrick et al.,

2010).

2.8.6.3 Powered activated carbon (PAC)

During the water treatment process, PAC can be added before coagulation, during the setting stage, during chemical addition, and prior to sand filtration. PAC is a particle form with a particle size of 10 to 100 m in diameter. Some of the benefits of PAC are convenience and rate.

It is suitable to be applied for short periods of time. When the problem occurs, PAC is applied, then, stopped when it is no longer needed. On the other hand, the disadvantage of PAC is that it

26 cannot be reused and will be disposed as waste together with the backwash water (Newcombe,

2006).

PAC can attain high removal efficiency for cyanotoxins. Up to 85% removal of extracellular MCs and about 98% removal of anatoxins can be achieved by PAC (Alvarez et al.,

2010; Xagoraraki, 2007). Ho et al. (Ho et al., 2011) studied the removal efficiency of two commercially available PACs for a mixture of cyanotoxins ( 20 g/L of CYN and 4 g/L each of

MC-RR, MC-YR, and MC-LA, and 10 g/L of MC-LR). They reported that there were no difference observed when PAC was applied in different contact times of 30, 45 and 60 min.

However, the adsorption order of microcystin variants is MC-RR > MC-YR > MC-LR > MC-LA, which is consistent with previous studies (Newcombe, 2002). In this study, they have also highlighted that the adsorption difference among microcystin variants could be attributed to the combination of hydrophobicity of the variants and the electrostatic interactions. In addition, some researchers suggested that the size and the conformation of the microcystin molecules, as well as the pore volume of the activated carbon appears to be a dominant mechanism for microcystin adsorption; while electrostatic interaction due to hydrophobic nature of microcystin molecule and low number of ionizable functional groups had minimal effect (Donati, Drikas,

Hayes, & Newcombe, 1994; Pendleton, Schumann, & Wong, 2001). They also presented that the

PAC with more mesopore volume was able to adsorb more MC-LR.

Ho et al. also discovered that the effect of water quality on the removal of MCs and CYN was negligible (Ho et al., 2011). However, other studies suggested that water quality had a strong influence on the eradication of cyanotoxins by AC since the formation of a natural organic matter (NOM) layer on the AC could conceal the original AC surface. Therefore, electrostatic

27 repulsion forces between the negatively charged solutes and the NOM negatively charged layer might decrease the solute adsorption rate (Delgado, Charles, Glucina, & Morlay, 2012). This phenomenon had been observed in the study conducted by Huang et al. (Huang, Cheng, & Cheng,

2007). Some studies even suggests that competitive adsorption is not only associated with the size of the competing compound, but is also highly dependent on the pore volume distribution

(Ho, Tanis-Plant, Kayal, Slyman, & Newcombe, 2009).

Although PAC effectively retains MC-LR, it is expensive to utilize a high and unusual amount of adsorbent for drinking water treatment plants (Lambert, Holmes, & Hrudey, 1996).

Wherein, in order to reduce the CYN concentration from 3 to below 1 g/L, a suggested dose of

29 mg/L of PAC with a contact time of 30 minutes would be required (Ho et al., 2008).

2.8.6.4 Granular activated carbon (GAC)

GAC is extensively used for removal of pesticides, tastes and orders, and industrial chemicals. Compared to PAC, the particle size is larger, usually between 0.4 and 2.5 mm. GAC is able to provide a stable barrier against unexpected contaminant from water resources. It also provide a large surface area.

There are two aims for GAC to be used in drinking water treatment. One is used as filter medium. The other one is used as an adsorber. The functions of GAC filters are mainly removing particulates, adsorbing chemicals, and biodegrading NOM. After using several years, the GAC filter has to be replaced. In contrast, the mechanism of GAC adsorber is adsorption for NOM.

The adsorption process has many advantages, including ease of operation, being suitable for batch and continuous processes, low capital cost and applicability at very low concentrations

(Mohanty, Das, & Biswas, 2006). Similar as GAC filter, GAC adsorber has to be regenerated or

28 replaced as well when total organic carbon breakthrough is high. In the aqueous adsorption onto

GAC, several variables including the GAC’s pore size distribution and internal surface area

(Rodriguez et al., 2007), the chemical properties of the GAC surface, for instance, hydrophobicity, functional groups (Tanju Karanfil, Kitis, Kilduff, & Wigton, 1999) , electrostatic interactions (Newcombe, Drikas, Assemi, & Beckett, 1997) and acid/basic characteristics, characteristics of the water (such as pH, ions in the solution), and the sizes, shapes and chemical properties of NOM can influence the adsorption (Huang et al., 2007).

GAC is high effective in removing cyanotoxins form drinking water, which has been confirmed by many water treatment studies at the laboratory and pilot plant-scale (Pantelic,

Svircev, Simeunovic, Vidovic, & Trajkovic, 2013). Up to 95% removal efficiency of MCs and anatoxin-a was achieved by GAC (Xagoraraki, 2007). According to Wang et al. (H. Wang, Ho,

Lewis, Brookes, & Newcombe, 2007), and Lambert et al. (Lambert et al., 1996) studies, removal efficiencies of MC-LR and MC-LA removed by virgin GAC were 70% ~ 80% and 40%, respectively. Removal efficiencies by preloaded carbon were not available. No studies about removal of CYN by virgin and regenerated GAC has been found.

Some studies indicate that biofilm attached on the GAC facilitates removal for MCs and anatoxin-a and increases its lifetime (Carlile, 1994; Hart & Stott, 1993). The microorganisms need several days to acclimate and consume the toxins as a food source. Thus the biodegradation only after a lag phase of several days (Carlile, 1994). Newcombe et al. (Newcombe & Nicholson,

2004) spiked MC-LA and MC-LR during a GAC pilot plant trial. The GAC was fed with treated water before chlorination and spiked the mixture of MC-LA and MC-LR at intervals. After one month, both MC-LA and MC-LR broke through and MC-LR broke through with lower level.

The authors attributed this to the level of DOC in the water (5 mg/L) providing competition with

29 for adsorption sites and reducing the adsorption for cyanotoxins. The GAC which is used in pilot trail after six months was took out and used in a laboratory scale. The inlet concentration was kept approximately 20 g/L of MC-LR and MC-LA. Both of these two variants were detected in outlet, however, no toxins was detected in the outlet after 16 days. Researchers attributed this to the biodegradation because they removed GAC and sterilized by drying using rotary evaporation at 40 ℃ after 68 days and the inlet concentration was around 30 g/L (Newcombe & Nicholson,

2004). No data are available for removal of CYN using GAC.

GAC source also influence the removal efficiency for cyanotoxins (coal, wood > peat, coconut) (Donati et al., 1994)..

2.9 Research Motivation, Objectives, and Hypotheses

2.9.1 Research motivation

Since the cyanobacterial HABs commonly occurs in freshwaters, which are the usual sources of drinking water, and cyanotoxins that are released from cyanobacteria endanger the health and even lives of human beings and animals, it is significantly necessary to determine an efficient eradication method for the dissolved cyanotoxins in water.

Although chemical treatment methods, like chlorine, UV and ozonation, are proven good at removing cyanotoxins, the disinfection by-products, high construction cost, high doses needed, and other drawbacks from these techniques have seriously prevented its application to practice.

After numerous researches in combination with the practice, GAC, as the widely used material in drinking water treatment, has turned out to be a better choice for cyanotoxins’ removal. Many researchers reported that more than 90% of extracellular cyanotoxins can been adsorbed by GAC

30 and no transformation products had been discovered during the process. However, few studies concentrated on the effects of chemical properties of cyanotoxins on adsorption of GAC and whether the physical properties of GAC, like surface area and pore structure, have an effect on the eradication of cyanotoxins. Aside from these, the virgin GAC instead of the reactivated GAC had been widely used in the previous studies. Based on these reasons, we have formulated our research objectives.

2.9.2 Research objectives

The primary aim is to determine the effects of chemical properties of cyanotoxins (i.e., MC-

LR, MC-RR and CYN) on adsorption using GAC. The second objective is to study the effect of physical properties of GACs (i.e., surface area, pore structure) on the elimination of cyanotoxins.

2.9.3 Hypotheses

Suppose that there would be no biodegradation and based on our literature review and research objectives, several hypotheses was made. The first hypothesis is that reactivated GAC has a better performance on the removal of MC-RR, MC-LR and CYN than that of the virgin

GAC since reactivated GAC has a high volume of mesopore than that of virgin GAC. In terms of the molecular weight and hydrophilicity of cyanotoxins, the smaller and the more hydrophilic it is, the more difficult it will be to be removed by GAC.

31 Chapter 3. Materials and Methods

3.1 Materials

3.1.1 GAC Influent

Sand filter effluent from the Great Cincinnati Water Works (GCWW), which is also called as the GAC Influent (GACI), was used for this study. GCWW supplies water from the Ohio

River, wherein the surface water from the Ohio River is treated at the Miller Treatment Plant. To be specific, surface water from the Ohio River initially goes into a primary sedimentation process. Lamella effluent is employed in the storage reservoirs and then reservoir settled water proceeds into the secondary sedimentation process. The next step is sand filtration. Water from the sand filtration process will go into the GAC contactor, which is referred to earlier as the GAC influent. Miller treatment plant process diagram is shown in Figure 3.1. GACI were taken on

March 7th and September 9th of 2016, and February 2nd of 2017, separately. The initial TOC concentrations were 1.4 mg/L, 1.8 mg/L and 1.7 mg/L. Other GACI chemical properties are shown in Table 3.1 and Table 3.2.

Figure 3. 1 Schematic diagram of water treatment process

32

Table 3. 1 Characteristic of GACI (1) Analyte Type Units MRL Method pH Sampling std. units N/A SM 4500-H+ TOC Sampling mg/L 0.2 SM 5310-C UV254 Sampling /cm 0.001 SM 5910-B Turbidity Sampling NTU 0.05 Turbidimeter Alkalinity Sampling mg/L as CaCO3 1 SM 2320 Calcium Hardness Sampling mg/L as CaCO3 1 SM 3500-Ca Temperature Online ℃ 0.1 Thermometer Hardness Sampling mg/L as CaCO3 1 SM 2340-C

Table 3. 2 Characteristics of GACI (2) Analyte Units 03/07/16 09/09/16 02/22/17 pH std. units 7.9 7.75 7.7 TOC mg/L 1.4 1.8 1.7 UV254 /cm 0.03 0.046 0.0352 Turbidity NTU 0.09 0.11 0.10

Alkalinity mg/L as CaCO3 53 76 55*

Total Hardness mg/L as CaCO3 NA 137 NA Calcium Hardness mg/L as CaCO3 84 91 84* Temperature ℃ 7.3 28.1 9.6 * Not measured on 02/22/16, 03/01/17 data were shown. NA: not available

3.1.2 Cyanotoxins

MC-LR, MC-RR, and CYN were tested as the model cyanotoxins for this research, which were purchased from Abraxis. Inc. Molecular weight of MC-LR (C49H74N10O12) and MC-RR

(C49H75N13O12) is 995.2 g/mol and 1038.2 g/mol, respectively. Meanwhile, the molecular weight of CYN (C15H21N5O7S) is 415.4 g/mol. The mass of each used was 0.5 mg/vial. All cyanotoxin vials were immediately stored at a temperature of -20ºC.

3.1.3 Granular Activated Carbon (GAC)

Original GAC used in GCWW is FILTRASORB 400, which can remove taste and odor compounds, organic color, TOC, and industrial organic compounds such as TCE and PCE.

33 Filtrasorb 400 is made from bituminous coal through re-agglomeration, which produces a highly durable granular carbon. There are several benefits of this type of GAC: uniform activation results with excellent adsorption properties and constant adsorption kinetics; the re-agglomerated structure removes the floating material; high mechanical strength decreases the generation of fines during backwashing; excellent reactivation performance is provided by the re-agglomerated structure with a high abrasion resistance; a greater adsorption capacity results from high density carbon. A more detailed specifications and typical properties of Filtrasorb400 are enumerated in

Table 3.3 (Calgon Carbon Corporation, 2018).

Table 3. 3 Specifications and Typical Properties of FILTRASORB 400 Specifications1 FILTRASORB 400 Iodine Number, mg/g 1000 (min) Moisture by Weight 2% (max) Effective Size 0.55-0.75 mm Uniformity Coefficient 1.9 (max) Abrasion Number 75 (min) Typical Properties2 FILTRASORB 400 Apparent Density (tamped) 0.54 g/cc Water Extractables <1% Non-Wettable <1% 1 Calgon Carbon test method 2 For general information only, not to be used as purchase specifications

Spent GAC can be reactivated on-site by multi-hearth furnaces in the Miller plant. These furnaces can reactivate approximately 40,000 pounds of GAC per day. Spent GAC in Cincinnati refers to the carbon which cannot produce an effluent with less than 1.0 mg/L of TOC (Moore et al., 2001). The virgin and reactivated GACs for this study are carbons, which are processed by ground and carbon defining.

3.2 Rapid Small-Scale Column Test (RSSCT)

34 One of the best methods to predict GAC performance is with the use of rapid small-scale column test (RSSCT). It is a scaled-down version of a pilot- or full scale GAC column, which is designed by Frick (Frick, 1982) and Crittenden et al. (Crittenden et al., 1991). RSSCT imitates the design of a full-scale GAC adsorption process, which involves arduous and expensive pilot- plant studies. However, procedures have been developed and applied over the last twenty years.

In order to reduce the size of a full-scale adsorber to a small column, a mass transfer model is utilized in the RSSCT method. Similarly, selecting the particle size, empty bed contact time

(EBCT) and hydraulic loading can ensure large-scale adsorbers’ operational parameters. Three primary benefits of using RSSCT should not be neglected. The first one is that an RSSCT needs lesser time than the tests conducted in pilot scales. Second, it is easy to process and explain the model since it does not need extensive isotherm or kinetic studies, which are necessary in predictive mathematical models. Lastly, only a small amount of water is required for the test, which makes it more convenient to be transported to laboratories for further evaluation

(Crittenden et al., 1991). A RSSCT should have an identical breakthrough curves similar to full- scale adsorber, theoretically. Nevertheless, in reality, there are differences due to the water quality, the RSSCT scaling assumptions and the biological processes present (Westerhoff,

Highfield, Badruzzaman, & Yoon, 2005).

Three types of mechanisms, including external mass transfer resistance or film transfer, the internal mass transfer resistances of pore and surface diffusion, and axial mixing resulting from dispersion, result in the distribution in the mass transfer zone and breakthrough curve. Those mechanisms define the selection of the hydraulic loading and EBCT of the RSSCT. The internal mass transfer resistance is more essential than the external mass transfer resistance for large molecules (> 300 MW) if the distribution of breakthrough curve is evident, while they are

35 equally important for small molecules (100-200 MW). Generally, dispersion can be negligible if the product of the Reynolds number and the Schmidt number is in the mechanical dispersion region of 200,000 to 200. The Reynolds number refers to the dimensionless ratio of the inertial forces over the viscous forces in a fluid, while the Schmidt number pertains to the dimensionless ratio of the diffusion of momentum over the diffusion of mass. The product of these two numbers can determine the minimum Reynolds number of the RSSCT.

The EBCT of the RSSCT, known as the EBCTSC, originates from the intraparticle mass transfer resistances. Before calculating the scaling equations, three conditions must first be satisfied. The first one is that boundary conditions for the full-scale and small-scale processes must be at the same dimensionless coordinate values in the dimensionless differential equations.

Second, regardless if its full-scale or small-scale, the parameters in equations must be identical.

Third, when reducing the size of the process, there should be no charge occuring in the mechanism. The proper scaling between the small- and large- column EBCTs can be calculated from Eqs. (1). In Equation (1), EBCTSC and EBCTLC pertains to the EBCTs of the small and large columns wherein: dp,SC and dp,LC are the adsorbent particle size for the small and the large

GAC; tSC and tLC represent the corresponding elapsed time in the small- and large- column tests, respectively. X is the dependence of the intraparticle diffusion coefficient on a particle size.

There are two kinds of diffusivities considered in the scaling, surface diffusivity and pore diffusivities. For surface diffusivity, X is defined in Eqs. (2), wherein Ds,SC and Ds,LC are the surface diffusivities of the GAC in the RSSCT and large-scale column, respectively. For pore diffusivity, X is defined from Eqs. (3), in which Dp,SC and Dp,LC are the surface diffusivities of the GAC in the RSSCT and large-scale column, respectively. If the intraparticle diffusivities is dependent on particle size, i.e. X is 0, RSSCT can be designed using the Eqs. (1) and Eqs. (4).

36 This is referred to as constant diffusivity. Eqs. (4) means that if the Reynolds number for the small- and large-GAC particles are set equal, an equal amount of dispersion related to the adsorber length and spreading in the mass transfer zone resulting from the external mass transfer can be calculated. VSC and VLC are hydraulic loadings in the RSSCT and pilot columns. On the contrary, there is proportional diffusivity. Specifically, intraparticle diffusivity is proportional to the particle radius (X is 1) and the surface diffusion is dominant. RSSCT design uses Eqs. (1) and Eqs. (2). When internal mass transfer is limiting, proportional diffusivity has been applied on

GAC systems. For GAC systems, intraparticle surface diffusion is more dominant than internal pore diffusion.

2−푋 EBCT d t SC = [ p,SC ] = SC ------Eqs. (1) EBCTLC dp,LC tLC

푋 D d s,SC = [ p,SC ] ------Eqs. (2) Ds,LC dp,LC

푋 D d p,SC = [ p,SC ] ------Eqs. (3) Dp,LC dp,LC

V d SC = p,SC ------Eqs. (4) VLC dp,LC

3.2.1 Experiment design and set up

The methodology is adopted from GCWW (Metz, 1995). In order to compare the removal efficiency on CYN, MC-RR and MC-LR by two types of GACs, feed solutions with different concentration was made. Five runs were designed in this research. Run 1 was the control test

(GACI). GAC influent spiked with MC-LR stock solution results in MC-LR 10 ppb was the Run

37 2. Run 3 represents the GAC influent with MC-RR 10 ppb. The feed solution in the Run 4 was

GAC influent spiked with CYN 10 ppb. In the Run 5, both GAC columns were preloaded with

10 days GACI and then, added and mixed with 40 ppb MC-LR and 40 ppb CYN for each column. The equipment used in this experiment were pressure gauge, dampener, feed solution brown bottle, carbon column rack and fraction collector. For the comparison of the removal efficiency of virgin and reactivated GACs, parallel equipment was set (Figure 3.2). The feed solution went through the pump, dampener, and pressure gauge. After passing through the column, the effluent was gathered by the fraction collector.

Figure 3.2 RSSCT Equipment

3.2.2 Carbon Defining

The carbon sample evaluated was riffled down to a 5 g sample. The 5g sample was ground and sieved to a 230(63 m) x 325(m) mesh size. This was accomplished by serial grinding until the entire amount of the carbon passed through the 230-mesh sieve. After sieving, the ground

38 carbon was slurred in 50 mL portions of boiled double distilled water. The slurry was sonicated for 30 seconds and the water was decanted. This process was repeated several times to effectively remove the fines. Further decantation without sonication was also performed to wash the carbon further until no fines were observed and the carbon slurry could settle quickly in about 30 seconds. The process could take up several hours. This was done to reduce the head loss buildup in the RSSCT. The carbon was then dried in an oven at 95°C and stored in a sealed organic free vial until used. The final GACs with around 54 m of diameter was used in the

RSSCT (Metz, 1995).

3.2.3 Water Sample Preparation

Before each RSSCT experiment, the water samples were placed to room temperature overnight and placed in a stir bar. Then, it was degassed for 1 hour (for each 4 L brown bottle).

After, the bottle was capped. Degas water on the same day for RSSCT experiment to avoid air getting back to water during storage (Metz, 1995).

3.2.4 Carbon Column Packing

Before packing carbon column, there was a need to prepare carbon. Exactly, 0.25 g of the carbon was weighed in a small beaker and then, approximately 10 mL of boiled double distilled water was added into the carbon. The next step was vacuum. The slurry was vacuumed in a desiccator for about 45 mins to remove the trapped air. The RSSCT setup was assembled by adding a small wet plug of glass wool into a glass column (0.38 cm internal diameter and 12-15 cm length). Then, the glass wool was pushed to the bottom of the column to provide a flat surface for the carbon. A few drops of boiled double distilled water were added. Slurred carbon was added drop-wise to the column and allowed to settle for an hour to give a bed height of

39 around 4.1 cm. The column was allowed to sit vertically in a clear vial (e.g, a 60 ml HAA vial) filled with boiled double distilled water until all carbons inside settle down. Another small wet plug of glass wool was added to the top of the column and pushed down to the GAC bed to get rid of the remaining air bubbles. A stainless-steel nut with a Teflon front and back ferrule was attached to the top and bottom of the column, and tightened. The pump effluent collection system was then assembled by connecting it to the column. Make sure there was no air gap present on the top of the carbon column. The influent end was connected to a bottle that contains the degassed water. Make sure the influent filter was attached (Metz, 1995).

3.2.5 RSSCT Sample Collection

There were three loadings for each run. There were 600 tubes utilized in total. Thirty effluent samples were collected for each run. Each effluent sample was from the three tubes, which contains 72 mL in total. The pump flow was set to ~ 0.57 ml/min. The packed carbon column height was fixed on 4.1 cm. The collected sample from feed solution was 70 mL. The operation was directly sucking 70 mL from the brown bottle using eppendorf. All samples were immediately placed into a 4 °C refrigerator after collection.

3.3 Methods

3.3.1 Sampling Procedure of RSSCT

Each effluent sample was separated into three parts: 50 ml for TOC, 2.5 ml for ELISA, 1 mL for LC/MS/MS (only available for MC-LR and MC-RR). Vials for TOC that were already added H3PO4 as preservative were provided by GCWW.

40 3.3.2 Characterization of natural organic matter

TOC was analyzed by Teledyne Tekmar TOC Fusion at GCWW. These analyses were performed with U.S. EPA Method 415.3, Rev.1.2: Determination of total organic carbon and specific UV absorbance at 254 nm in source water and drinking water (Potter, 2005). The limit of detection ranged from 0.2 ppb to 4,000 ppm. All samples were filtered (0.45 µm) before analysis.

Size distribution of dissolved organic carbon (DOC) was analyzed using Liquid

Chromatography – Organic Carbon Detection (LC-OCD) at Busan Drinking Water Treatment,

South Korea. The detection limit ranged from less than 1 ppb to 50 ppb. Size exclusion chromatography in combination with organic carbon detection is an established method to separate the pool of NOM into major fractions of different sizes to qualify these on basis of organic carbon (Huber, Balz, Abert, & Pronk, 2011). LC-OCD is able to separate five size fractions: Biopolymers ( > 20,000 Da), Humics ( ~ 1000 Da), Building Blocks ( 300 ~ 500 Da),

Low Material Weight (LMW) Acids ( < 350 Da) and LMW Neutrals (Figure 3.3) (Simon et al.,

2013). The operation of the system is shown in Figure 3.4 (DOC-Labor, 1998). Mobile phase was precleaned by UV-oxidation in the DOCOX-reactor and pumped to an autosampler and then to the chromatographic column. The liquid entered the UV-detector and then the organic carbon detector. The first detector after chromatographic separation was non-destructive, with a fixed wavelength UV of 254 nm. At the inlet of OCD, the solution was acidified to convert the carbonates to carbonic acid.

41

Figure 3. 3 LC-OCD fractions over the SEC chromatogram with OCD

Figure 3. 4 Operation of LOC - OCD

3.3.3 Characterization of GACs

3.3.3.1 Surface morphology

To obtain the morphology of ground and defined virgin and reactivated GACs, scanning electron microscope (SEM) was used to examine GAC samples of the study at the University of

Cincinnati. The instrument used was Philips XL-30 FEG Environmental Scanning Electron

Microscope (ESEM). First, the sample was dried in an oven at 60 ℃ for 3 hours. The second step

42 was loading the sample to the SEM holder. Finally, turn on the ESEM.

3.3.3.2 Pore size distribution and surface area determinations

Surface area and pore size distribution samples were determined by nitrogen adsorption isotherms from relative pressure of 10-6 to 0.99 at 77K with Micromeritics ASAP 2060. Pore size distribution was determined for virgin GAC and reactivated GAC before RSSCT and after

RSSCT 10 days. Carbon sample weighed about 0.1 g was dried in an oven at 110°C for 24h and then put the sample into sample tube for degassing at 110°C, 5×10-3 Torr, or 7×10-4 kPa for 24h, which was the aim for removing any moisture and weakly volatiles that might interfere in the analysis. Then, the sample tube was placed on the machine and the analysis process was started.

The process involved two stages: dose amount check and pore size distribution measurement.

First, the sample tube was evacuated (1×10-5 Torr, or 1.3×10-6 kPa) for 1 to 3 h and then submerged into a liquid nitrogen bath. The second step was that the sample was dosed with incremental amount of gaseous nitrogen that was 99.999% pure (supplied by Wright Brothers

Industries) until the pseudo-equilibrium conditions had been reached. The instrument repeated this process until an adsorption isotherm covering the relative pressure from 10-6 to 0.99 (Moore et al., 2001). Pore size distribution was determined by Density Functional Theory (DFT) software (N2-DFT model). The pore volume distributions were computed for 77 pore sizes, over the range of 4 to 2166 Å. The micropore volume was calculated by Dubinin-Radushkevich equation with relative pressure range of 10-5 to 10-1. The total pore volume was calculated from the adsorbed volume of gas near the saturation point (P/P0 = 0.98). Surface area was calculated from Brunauer-Emmett- Teller (BET) equation. BET surface area of microporous activated carbons was calculated by relative pressure from 0.01 to 0.1 (T. Karanfil et al., 2007).

43 3.3.4 Analysis of cyanotoxins

In this research, Enzyme-Linked Immunosorbent Assay (ELISA) and Liquid

Chromatography/Tandem Mass Spectrometry (LC/MS/MS) were used as analysis tools to measure the concentration of MC-LR, MC-RR and CYN. For ELISA, U.S. EPA Method 546:

Determination of Total Microcystins and Nodularins in Drinking Water and Ambient Water by

Adda Enzyme-Linked Immunosorbent Assay (US EPA, 2016) was utilized for measuring MC-

LR, MC-RR and CYN. The detection limit for MC-LR and MC-RR was between 0.3 ppb to 5 ppb. The detection limit for CYN ranged from 0.05 ppb to 2 ppb. The method for LC/MS/MS was the U.S. EPA Method 544: A Case Study in USEPA Drinking Water Method Development

(Shoemaker, 2016) and the detection limit was 0.05 ppb for MC-LR and MC-RR. Measurement for CYN by LC/MS/MS was not available.

44 Chapter 4. Results and Discussion

4.1 Characteristic of GAC influent and effluent

TOC concentration of feed solution for each run was measured at room temperature

( 23℃ ~ 25 ℃ ). The results were shown in Table 4.1, in which C0 represents the TOC concentration of feed solution. According to Table 4.1, TOC concentration ranges from 1.4 ppm to 1.85 ppm. The averaged initial TOC concentration measured in each run were close to the

TOC concentrations measured at GCWW.

Table 4. 1 TOC concentration of feed solutions Run 1 Run 2 Run 3 Run 4 Run 5 GACI with GACI with GACI GACI with MC-LR Control Test MC-LR 10 MC-RR 10 with CYN 40 ppb and CYN 40 (GACI only) ppb ppb 10 ppb ppb CO, ppm 1.85 1.73 1.4 1.84 1.68

Duration, 10 days with GACI only days 18 after that with MC-LR and CYN Temp., ℃ 23 ~ 25

Meanwhile, the DOC concentrations are shown in Table 4.2. Hydrophobic DOC and hydrophilic DOC were reported. Hydrophilic DOC is dominant in the dissolved DOC, specifically more than 90% for each feed solution and the concentration is around 1.7 ppm. Even though the hydrophilic DOC has slightly increased in feed solution with cyanotoxins, the values are all around 1.7 ppm. This indicates that the influence of cyanotoxins on DOC concentration can be neglected. Hydrophobic DOC concentrations for each feed solution are all lower than 0.2

45 ppm (less than 10%).

Table 4. 2 DOC concentration of feed solutions Hydrophilic DOC (ppm) Hydrophobic DOC (ppm) GACI 1.66 (91%) 0.16 (9%) GACI + 10 ppb MC-LR 1.71 (91%) 0.18 (9%) GACI + 10 ppb MC-RR 1.68 (90%) 0.18 (10%) GACI + 10 ppb CYN 1.7 (92%) 0.16 (8%)

Size distributions of DOC for each feed solution (Figure 4.1) are clearly displayed. The size distributions of DOC in each feed solution are similar. Biopolymers (>>20000 Da), humic substance (~1000 Da), building blocks (300~500 Da), low molecular weight (LMW) neutrals

(<350 Da) were all measured. Among them, humic substance is the dominant fraction (around

50%).

Figure 4. 1 Size distribution of feed solution (a. GACI, collected in January 2017, TOC = 1.87 mg/L; b. GACI + MC-LR 10 ppb; c. GACI + MC- RR 10 ppb; d. GACI + CYN 10 ppb)

46 The change of TOC during each run was measured (Figure 4.2 ~ Figure 4.6). The bottom horizontal axis stands for time (day) for field scale; the top horizontal axis stands for time (day) for RSSCT. The unit of vertical axis is dimensionless, which is easy to compare with the removal efficiency of TOC by GACs. C/C0 in which C represents TOC concentration of GACI at time t;

C0 represents TOC concentration of feed solution averaged for each run. Generally, for each run, the TOC concentrations in the effluent through virgin GAC column are obviously lower than those through reactivated GAC column at any time, which means that virgin GAC is marginally effective for the removal of TOC than reactivated GAC. Also, some abnormally high points occurred initially within the virgin GAC column. This may be due to some defined carbon particles that went through the column. Furthermore, the largest removal efficiency among all

RSSCT tests is 90% in the 40 ppb MC-LR and 40 ppb CYN run. All the highest removal efficiency during each run occurred around 44.29 large scale run days, that equates to 2.39 days in RSSCT, except for the control test (11.34 large run days). On the other hand, all the lowest removal efficiencies occurred near 311.11 large-scale run days (about 16.8 days in RSSCT) except for mixed 40 ppb, which occurred near 248.46 large-scale run days (13.42 days in RSSCT.

This confirmed that the removal of TOC in each run were stable. In the later period of RSSCT, porosity was not the only factor important in the removal of TOC, for other factors could work like biological growth (B.C. Moore et al., 2001).

47

Figure 4. 2 TOC removal by GACs during Run 1 (GACI only)

Figure 4. 3 TOC removal by GACs during Run 2 (GACI + MC-LR 10 ppb)

48

Figure 4. 4 TOC removal by GACs during Run 3 (GACI + MC-RR 10 ppb)

Figure 4. 5 TOC removal by GACs during Run 4 (GACI + CYN 10 ppb)

49

Figure 4. 6 TOC removal by GACs during Run 5 (GACI + MC-LR 40 ppb and CYN 10 ppb)

The size distribution of effluent solution for Run 2 (GACI + MC-LR 10 ppb) and Run 4

(GACI + CYN 10 ppb) were also examined (Figure 4.7, Figure 4.8). The blue columns stand for the concentration of feed solution in different size part. The orange columns stand for the concentration of solution in different size part through virgin GAC column and grey columns represent the concentration of solution in different size part through reactivated GAC column.

50

Figure 4. 7 Hydrophilic DOC for Run 2 (GACI + MC-LR 10 ppb)

As seen in Figure 4.7, the concentration of hydrophilic DOC though GAC columns is dramatically decreased, from 1.713 ppm to around 1.2 ppm. The virgin GAC performed better than reactivated GAC since the concentration of hydrophilic DOC in effluent solution through virgin GAC column is 1.229 ppm, which is lower than 1.278 ppm through the reactivated GAC column. The specific removal rates in different size fraction are shown in Figure 4.8. The blue columns stand for removal rate of DOC in different size fraction through virgin GAC column, while the blue ones stand for those that went through the reactivated GAC column. There was a

28.3% removal efficiency achieved by virgin GAC column while there was a 25.4% removal efficiency achieved by the reactivated GAC column. This demonstrates that virgin GAC is more efficient than reactivated GAC for removal of TOC in the solution. Among different size fractions of hydrophilic DOC, building blocks (300 ~ 500 Da) made the most contribution, 15.4% by virgin GAC and 13% by reactivated GAC. The biopolymers (>> 20000 Da) contributes the least for removal efficiency, which is less than 1%.

51

Figure 4. 8 TOC removal for Run 2 (GACI + MC-LR 10 ppb)

In order to confirm the stability of DOC removal by GAC columns, DOC concentration in different size fraction for Run 4 (GACI + CYN 10 ppb) was examined as well. According to

Figure 4.9, the hydrophilic DOC of the effluent decreased a lot after RSSCT, which is similar as for Run 2. The final hydrophilic DOC concentration in the effluent through virgin GAC column was 1.216 ppm while the hydrophilic DOC concentration in the effluent through reactivated

GAC column was 1.242 ppm, which means virgin GAC marginally performed better than reactivated GAC and confirmed the removal of TOC was dependent of cyanotoxins. The removal rate for hydrophilic DOC through virgin GAC column reached 28.8% compared to 27.2% through the reactivated column (Figure 4.10).

52

Figure 4. 9 Hydrophilic DOC for Run 4

Figure 4. 10 TOC removal for Run 4

According to the results form TOC and DOC, the performance of removal efficiency of

TOC and DOC by virgin GAC was better than that achieved by reactivated GAC. Approximately diameter of NOM fraction with molecular weight between 500 and 3000 Da (Newcombe et al.,

1997) is 11Å. The molecular weight of 90% NOM in out feed solution are all around 1000 Da, which means that their diameters are all around 11 Å. Considering that the diameter of micropore

53 is less than 20 Å, most fraction of NOM in the feed solution can be captured by micropore. The volume of micropore used in virgin GAC was more than that of the reactivated GAC, so it was very possible that virgin GAC adsorbed more NOM than reactivated GAC. Thus, the removal efficiency of NOM by virgin GAC was higher than that achieved by the reactivated GAC.

4.2 Characteristic of GACs

4.2.1 Surface morphology of virgin and reactivated GACs

Before RSSCT, morphology of ground and defined virgin and reactivated GAC were examined (Figure 4.11). From Figure 4.7, typical pores cannot be seen in the 2500 magnitude.

This implied GACs do not contain many macropore (> 50 nm).

Figure 4. 11 Morphology of ground and defined virgin (left) and reactivated (right) GACs

4.2.2 Pore size distributions and surface area of virgin versus reactivated GACs

Pore size distributions of GACs had been examined before RSSCT and after RSSCT in order to further understand how physical pore structure of GACs influence the adsorption of

NOM and cyanotoxins. The trend of cumulative pore volume of GACs before RSSCT is

54 displayed in Figure 4.12. As seen in the Figure 4.12, the reactivated GAC had more pore volume than the virgin one. The virgin GAC contained a volume of micropores (< 20 Å ) that was roughly twice the amount present in reactivated one. After 100 Å, the cumulative pore volume of reactivated GAC was greater than that of virgin GAC because the reactivated GAC pores had been widened through the multiple reactivation cycles. Specific pore volume and surface area are shown in Table 4.3. The micropore (52%) is dominant in the virgin GAC; while the mesopore (51.78%) is dominant in reactivated one. The micropore BET surface area of virgin

GAC was greater than that of reactivated GAC. The micropore volume of virgin one which was

0.26 cm3/g is greater than 0.15 cm3/g in the reactivated one as well.

Figure 4. 12 Pore size distribution for GACs before RSSCT

Table 4. 3 Surface area and pore volume of GACs before RSSCT Virgin Reactivated Micropore BET surface 837.31 603.47 area (m2/g) Total pore volume (cm3/g) 0.5 0.56 Micropore volume (cm3/g) 0.26 (52%) 0.15 (26.78%) Mesopore volume (cm3/g) 0.12 (24%) 0.29 (51.78%) Macropore volume (cm3/g) 0.12 (24%) 0.12 (21.43%)

55

Pore size distribution of GACs after RSSCT was also checked. Total pore volume of virgin

GAC was greater than that of reactivated one (Table 4.4). Micropore (53.33%) instead of mesopore (26.67%) became dominant in virgin one; while mesopore was still dominant part in reactivated one. Micropore BET surface area, the values of micropore volume and total pore volume in virgin one was all greater than those of reactivated one.

Table 4. 4 Surface area and pore volume of GACs after RSSCT Virgin Reactivated Micropore BET surface 250 100.68 area (m2/g) Total pore volume (cm3/g) 0.15 0.11 Micropore volume (cm3/g) 0.08 (53.33%) 0.02 (18.18%) Mesopore volume (cm3/g) 0.04 (26.67%) 0.07 (63.64%) Macropore volume (cm3/g) 0.03 (20%) 0.02 (18.18%)

Comparison of pore size distribution of virgin and reactivated GACs between before

RSSCT and after RSSCT is clearly shown in Figure 4.13 in which the green triangle represents virgin GAC while the yellow square represents reactivated GAC; the green line stands for the trend of cumulative pore volume of GACs before RSSCT whereas the yellow line stands for the trend of cumulative pore volume of GACs after RSSCT. For micropore (< 20 Å), the decreased volume in virgin one, which is the triangle area enclosed by the two triangles lines, was more than compared to the the triangle area enclosed by the two squared lines in the reactivated GAC.

For mesopore (20 ~ 500 Å), the decreased volume in virgin one was less than in the reactivated

GAC.

56

Figure 4. 13 Pore size distribution for GACs before RSSCT versus after RSSCT

Specific values of pore volume and surface area of GACs before RSSCT and after RSSCT are available in Table 4.5. For virgin GAC, the micropore volume decreased the most compared to mesopore volume and macropore volume. The micropore volume was decreased from 0.26 cm3/g to 0.08 cm3/g. The decreased micropore volume was 0.18 cm3/g; while the mesopore volume was decreased from 0.12 cm3/g to 0.04 cm3/g. The decreased mesopore volume was 0.08 cm3/g. For reactivated GAC, the mesopore volume decreased most compared to micropore volume and macropore volume. The micropore volume was decreased from 0.15 cm3/g to 0.02 cm3/g. The decreased mesopore volume was 0.13 cm3/g; while the mesopore volume was decreased from 0.29 cm3/g to 0.07 cm3/g. The decreased mesopore volume was 0.22 cm3/g. This indicates that micropore were occupied more than meso- and macropore of virgin GAC; while mesopore contributes more than the other two pores of reactivated GAC. Compared with virgin and reactivated GACs, the decreased amount of micropore BET surface area in virgin one was more than that of reactivated one. The micropore BET surface area of virgin GAC was decreased from 837.31 m2/g to 250 m2/g. The decreased micropore BET surface area was 587 m2/g;

57 whereas the micropore BET surface area of reactivated GAC was decreased from 603.47 m2/g to

100.68 m2/g. The decreased micropore BET surface area was 503 m2/g. Also, the decreased amount of micropore volume of virgin GAC (0.18 cm3/g) was more than that of reactivated one

(0.13 cm3/g).

Table 4. 5 Surface area and pore volume of GACs before RSSCT versus after RSSCT

Virgin GAC Reactivated GAC

Before use After use Before use After use Micropore BET surface area 837.31 250 603.5 100.7 (m2/g) Total Pore 0.5 0.15 0.56 0.11 volume (cm3/g) Micropore 0.26 0.08 0.15 0.02 volume (cm3/g) Mesopore 0.12 0.04 0.29 0.07 volume (cm3/g) Macropore 0.12 0.03 0.12 0.02 volume (cm3/g)

4.3 Transport of Cyanotoxins through virgin and reactivated GACs

During the transport, the cyanotoxin concentrations in the effluent were examined. In order to assure that the concentration of cyanotoxins in feed solution was kept stable, the cyanotoxins concentration in feed solution was measured as well. In Figure 4.14 ~ Figure 4.17, the yellow square stands for the concentration of cyanotoxins in effluent with each run through reactivated

GAC column; the green triangle represents the concentration of cyanotoxins in effluent with each run through virgin GAC column; the orange dash line and circle represent the concentration of cyanotoxins in feed solution. The left vertical axis stands for the concentration of cyanotoxins

58 in the effluent; the right vertical axis represents the concentration of cyanotoxins measured in the feed solution during the RSSCT.

The concentration of different cyanotoxin measured in feed solution were basically equivalent to their initial spiked concentration during each run, which suggested that the RSSCT were carried out using a constant influent concentration. For Run 2 (GACI + MC-LR 10 ppb) and Run 3 (GACI + MC-RR 10 ppb), no breakthrough in the effluent was detected for 300 days with two GACs. For Run 4 (GACI + CYN 10 ppb), breakthrough in the effluent through the reactivated GAC column were detected at the end of the operation (about 280 days). The detected concentrations were 0.056 g/L (280 days) and 0.059 g/L (300 days). During the Run

5 (GACI + MC-LR 40 ppb and CYN 40 ppb), there was no cyanotoxins added in the feed solution for both columns for the first 10 small scale run days and 40 ppb MC-LR and 40 ppb

CYN were added into the feed solution on 11th small scale run days (about 180 large scale run days). Only breakthrough with CYN occurred at the end of operation from reactivated GAC column. The detected concentration is 0.065 g/L (280 days). Consequently, the breakthroughs only occurred in Run 4 (GACI + CYN 10 ppb) and Run 5 (GACI + MC-LR 40 ppb and CYN 40 ppb) and both breakthroughs occurred from reactivated GAC.

59

Figure 4. 14 Cyanotoxins in the effluent with Run 2 (GACI + MC-LR 10 ppb)

Figure 4. 15 Cyanotoxins in the effluent with Run 3 (GACI + MC-RR 10 ppb)

60

Figure 4. 16 Cyanotoxins in the effluent with Run 4 (GACI + CYN 10 ppb)

Figure 4. 17 Cyanotoxins in the effluent with Run 5 (GACI + MC-LR 40 ppb and CYN 10 ppb)

For pH range (6 ~ 8.5), the dominant functional groups for MC-LR and MC-RR are [(COO-

2+ - 2+ )2(NH )] and [(COO )2(NH )2], respectively (Liang et al., 2011). The negative charged groups resulted from the dissociated carboxyl groups (COO-) on D-glutamamate and D-ery-thro-β −

61 methylaspartic acid. The positive charge is from the variable amino acid, arginine which has a basic amino group (NH2+) (Liang et al., 2011). Therefore, MC-LR is negative charge; while MC-

RR is neutral. The charge of CYN for pH ranging 6 to 8 is zero (Newcombe, 2006).This characteristic might affect the adsorption result. Also, according to log Kow reported by UKWIR

(UKWIR, 1997), the Kow for MC-RR is lower than that of MC-LR, which indicates that MC-LR is more hydrophobic than MC-RR. CYN is a zwitterion due to the negatively charged sulfate group and the positively charged guanido group, and is highly water soluble (White & Hansen,

2005). CYN is much hydrophilic than MC-RR and MC-LR (Westrick et al., 2010). Based on the above information and the molecular size of MC-LR (995 Da), MC-RR (1038 Da), and CYN

(415 Da), the expected result was that the removal for MC-LR would be much easier than MC-

RR and CYN. Therefore, in our experiment, there were no breakthroughs for MC-LR and MC-

RR with GACs, excluding CYN. CYN is the most hydrophilic and with the smaller size compared with MC-LR and MC-RR. Besides, the surface diffusion coefficient (Ds) for CYN is bigger (~ 10-9 cm2/s) than that for MC-LR (~ 10-11 cm2/s), which suggests that CYN diffuses faster than MC-LR (Ho et al., 2008) to the particle surface. This improves the adsorbed possibility of CYN, but the adsorption process is complicated and it is not logical to expect the result based on just one factor. It is difficult to expect the adsorb order for MC-LR and MC-RR since there is no breakthrough for these two MCs analogue. According to their hydrophilicity, we can speculate that removal for MC-LR might be easier than MC-RR. However, many studies showed removal result by PAC were opposite to the expectation (Donati et al., 1994; Ho et al.,

2011; Newcombe, Cook, Brooke, Ho, & Slyman, 2003). Ho et al. (Ho et al., 2011) removed a mixture of cyanotoxins (20 g/L of CYN and 4 g/L each of MC-RR, MC-YR and MC-LA, and

10 g/L MC-LR) by PAC under 30, 45 and 60 minutes, and discovered that the ease of removal

62 was in this order: MC-RR > MC-YR > MC-LR > MC-LA. They attributed this result to attractive or repulsive forces between the MCs molecule and the AC’s surface. In addition, molecular size and conformation of these molecules might also influence the adsorption. It is very likely that the initial concentrations in the spiking material resulted in the difference in the adsorption of the MCs variants. The difference in adsorbability of MC analogue is still under investigation (Cook & Newcombe, 2002).

By examining the pore size distribution for GACs, virgin GAC owns more internal surface area and micropore than reactivated GAC, and less mesopore than reactivated GAC. Generally, the shape of micropore is considered as slit-shaped, with either parallel walls, or, when the crystallites are at an angle, wedge-shaped, whereas there is no such structure for mesopores and macropores which are more cylindrical in shape (Newcombe, 2006). The special shape of micropore may contribute to the removal of cyanotoxins. Also, there is a sharp pore volume decrease ranging from 10 Å to 20 Å for virgin GAC. This pore range belongs to secondary micropores (0.8 nm – 2 nm) (Newcombe, 2006). It is believed that most cyanotoxins were adsorbed on the secondary micropores. Since the molecular weight of CYN is only 415 Da, which is much smaller than that of MC-LR and MC-RR (around 1000 Da), CYN could be captured by small pores of GAC instead of the bigger ones. Considering that the micropore (< 2 nm) is smaller than mesopore (2 ~ 50 nm) and there are more micropore occupied in virgin GAC than that of reactivated GAC during the RSSCT according to pore size distribution, it is very likely that CYN were captured by micropore instead of mesopore. Thus, partial CYN cannot be adsorbed by reactivated GAC and the breakthrough occurred from reactivated GAC. It is difficult for CYN to be adsorbed on the GAC surface whose surface is hydrophobic. Considering the small molecular weight of CYN and micropores shape as well as more micropore used for

63 virgin GAC, all these factors may explain why there was only breakthrough with CYN from reactivated carbon.

Another possibility for only CYN breakthrough is because NOM competed with the adsorption process. By examining the different fractions of DOC removal using LC-OCD, the most fraction of removal for DOC is building blocking within 300 ~ 500. This corresponds to the molecular size of CYN (415 Da). Given that NOM is also absorbed by micropore, CYN broke out at the end of the operation when there is not enough micropores for reactivated GAC.

64 Chapter 5. Conclusions

5.1 Summary

For the purpose of exploring the effect of chemical properties of cyanotoxins (MC-LR, MC-

RR and CYN) on removal efficiency and physical properties of GACs (i.e., virgin and reactivated) on removal of cyanotoxins, five RSSCT operations were conducted. To be specific, the concentration of TOC and different size fraction of DOC, pore size distribution and surface area of GACs, as well as transport of cyanotoxins through virgin and reactivated GACs were all analyzed.

The removal efficiency for MC-LR and MC-RR was higher compared to CYN removal by

GACs. In terms of chemical property of cyanotoxins, CYN carried with zwitterion is more hydrophilic than MC-LR and MC-RR, and more soluble in the water. Therefore, compared with

MCs, CYN is inclined to stay in water and a slightly difficult to be removed. Also, the molecular size might influence the adsorption. The molecular weight of CYN (415 Da) is much smaller than that of MC-LR and MC-RR (~ 1000 Da). On the other hand, secondary micropore has more advantages in adsorption due to its smaller pore diameter (0.8 ~ 2 nm) and the slit-shaped structure compared with mesopore (2 ~ 50nm) with cylindrical structure.

Both chemical and physical properties of cyanotoxins influenced the adsorption. However, considering that there was no breakthrough with the virgin GAC, which owns more micropore, we concluded that the pore size distribution of GAC was the dominant factor for the adsorption of CYN, if there was no biodegradation. However, this breakthrough resulted from the NOM

65 competition for adsorption sites, considering that the most decreased fraction of DOC was 300 ~

500 Da corresponding to the 415 Da size of CYN. Nevertheless, virgin GAC also adsorbed more fraction of DOC (300 ~ 500 Da) and no breakthroughs occurred on it. This again demonstrated that the effect of micropore volume of GAC on the removal for MC-LR, MC-RR and CYN was dominant.

5.2 Suggestions for Future Work

In this research, only 10 ppb and 40 ppb was tested for the three types of cyanotoxins and there is no breakthrough in MC-RR and MC-LR. All operations were once tested and no repeat.

For further exploration on the limit concentrations GAC can hold, higher concentration in feed solution is necessary. Also, repeating tests are necessary in order to get a stable experiment result.

Furthermore, only three types of cyanotoxins, namely MC-RR, MC-LR and CYN, were tested in the research, but other important cyanotoxins were not included, such as (STX),

Anatoxin-a and Nodularin as well as other MCs variants, like MC-LA, MC-LW and MC-LY. In the future work, more types of cyanotoxins will be tested using GAC. For ground and defined

GACs, accurate density and size cannot be known because of the absence of specific measurement, which may influence the experiment results. Therefore, density and size of ground and defined carbon will be accurately measured. Additionally, the influence of biodegradation was not considered during the RSSCT. It was possible that some bacteria grew during the test that consumed some of the cyanotoxins. The following work should conduct biodegradation test to determine whether microorganisms degrade some of the cyanotoxins during the RSSCT.

66

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