PHOTOCHEMICAL FATE OF THE ARTIFICIAL SWEETENER SUCRALOSE IN THE

ENVIRONMENT

Aleksandra M. Kirk

A Thesis Submitted to the

University of North Carolina Wilmington in Partial Fulfillment

of the Requirements for the Degree of

Master of Science

Department of Chemistry and Biochemistry

University of North Carolina Wilmington

2010

Approved by

Advisory Committee

Dr. Robert J. Kieber Jeremy B. Morgan

Ralph N. Mead

Chair

Accepted by

______________________________

Dean, Graduate School

ii

TABLE OF CONTENTS

ABSTRACT ................................................................................................................................... iii

ACKNOWLEDGMENTS ............................................................................................................. iv

LIST OF TABLES ...........................................................................................................................v

LIST OF FIGURES ....................................................................................................................... vi

INTRODUCTION ...........................................................................................................................1

METHODS ....................................................................................................................................10

Occurrence ................................................................................................................................10

Method Optimization ................................................................................................................11

Sorption .....................................................................................................................................12

Photochemistry .........................................................................................................................13

Biological Impact ......................................................................................................................15

RESULTS & DISCUSSION..........................................................................................................16

Occurrence ................................................................................................................................16

Method Optimization ................................................................................................................19

Sorption .....................................................................................................................................21

Photodegradation ......................................................................................................................23

Photocatalyzed Degradation .....................................................................................................27

Biological Impact ......................................................................................................................30

IMPLICATIONS ...........................................................................................................................32

REFERENCES ..............................................................................................................................63

APPENDIX ....................................................................................................................................68

iii

ABSTRACT

The artificial sweetener sucralose belongs to a group of compounds named Contaminants

of Emerging Concern (CECs). The persistence of sucralose in the environment coupled with

worldwide demand for its consumption necessitates expansion of the research on its fate in the

environment. This study is a continuation of preceding work by the same authors completing the

results on the first concentration data of sucralose in the United States. The sweetener has been

found in wastewater and surface waters in significant concentrations (~40 and ~3 µg/L,

respectively) but consistent with its low KOW (0.3), no sorption to various types of sediment was

observed. The assessment of its fate in the environment has been also studied. Less than 20% of

sucralose degraded during the photolysis of environmental water samples and MilliQ water.

Photocatalyzed reactions with TiO2 resulted in >90% degradation. Hydroxyl radical

photooxidation of sucralose has been suggested, and the mechanistic pathway was confirmed by

experiments conducted with the ·OH scavenger, methanol. The study has also included the

biological impact of sucralose on marine phytoplankton. There was no effect on the growth of

the four different species from the major phytoplankton groups that were monitored.

iv

ACKNOWLEDGMENTS

I would like to thank my advisory committee: Dr. Mead, Dr. Kieber, and Dr. Morgan for

always being enthusiastic about my work and for their continuous support throughout these last

few years. I am very appreciative for being given the opportunity to work in such a collaborate

environment. My special thanks goes out to Dr. Mead, for his guidance, time and effort ensuring

I got the most out of my graduate school experience. His words of encouragement, and

excitement about every step I made forward kept me going even when times were tough.

I also want to thank the whole MACRL, Ashley Myers, and Donna Glinski for being a great

group of friends. I’d also like to thank Dr. Alison Taylor for guiding me through the marine

phytoplankton experiment.

I must thank my friend Allison Stewart. Your passion for chemistry and research inspires

me every day and will never stop. Thank you for always being there for me and believing in me.

Thanks, also, to Linda Larson, for accompanying me on this journey as a great friend and fellow

graduate - coffee and noodles tasted much better in good company.

I’d like to thank my parents and in-laws, who although thousands miles away, never doubted me

and constantly reassured me of my abilities.

Finally, I would like to thank my husband, CT, for putting up with me throughout this

stressful time. It was comforting having you by my side and I can’t thank you enough for your

endless support, help, and faith you have in me.

Funding for this research was provided by UNCW Center for Marine Science Pilot Program and

NSF grant OCE-0825538. Thanks to Chemistry and Biochemistry Department for additional

funds.

v

LIST OF TABLES

Table Page

1. Examples of pharmaceuticals and other organic compounds that have been shown to

occur in the aqueous systems .............................................................................................33

2. Examples of concentrations of common artificial sweeteners found in the

Environment .......................................................................................................................34

3. Examples of sucralose concentrations in environment reported in literature ....................35

4. Concentration of sucralose found at various locations of the current research .................40

5. Method optimization experimental results.........................................................................44

6. Absorbance of different water matrices at 300 nm wavelength and spectral slopes

calculated for 240-400 nm wavelength range. ...................................................................55

vi

LIST OF FIGURES

Figure Page

1. Structure of sucrose - table sugar (a) and sucralose (b) .....................................................36

2. Hydrolysis products of sucralose .......................................................................................37

3. Microbial transformation products of sucralose: a – unsaturated aldehyde of sucralose;

b – sucralose uronic acid; C – 1,6-dichloro-l,6-dideoxy-D-fructose .................................38

4. Sucralose absorbance spectrum (a); enlarged view (b)......................................................39

5. Mass spectra of sucralose (the identifying ions at m/z 308 and 361) ................................41

6. GC chromatogram of sucralose .........................................................................................42

7. Natural sunlight vs. solar simulator comparison spectrum ................................................43

8. Extraction volume experiments (duplicates) .....................................................................45

9. Linearity of the calibration curve for the current method ..................................................46

10. Sucralose concentration in water aliquots measured during experiment on sucralose

sorption to natural, high in organic matter sediment .........................................................47

11. Sucralose concentration in water aliquots measured during experiment on sucralose

sorption to montmorillonite clay, low in organic matter sediment ....................................48

12. Effect of simulated sunlight on the degradation of sucralose in MilliQ and ASW

without the TiO2 photocatalyst ..........................................................................................49

13. Effect of humic substances on sucralose degradation under simulated sunlight in ASW

without the TiO2 photocatalyst (a) and with the TiO2 photocatalyst (b). PART I .............50

14. Effect of humic substances on sucralose degradation under simulated sunlight in ASW

without the TiO2 photocatalyst (a) and with the TiO2 photocatalyst (b) PART II ............51

15. Effect of simulated sunlight on the degradation of sucralose in different matrices

without the TiO2 photocatalyst (a) and with the TiO2 photocatalyst (b) ...........................52

16. UV-Vis absorbance spectrum of different water matrices (a); enlarged view (b) .............53

17. Effect of methanol as a hydroxyl radical scavenger on sucralose degradation in natural

waters without the TiO2 photocatalyst ...............................................................................54

vii

18. Effect of simulated sunlight on degradation of sucralose in MilliQ and ASW with

and without TiO2 photocatalyst .........................................................................................56

19. Effect of methanol as a hydroxyl radical scavenger on sucralose degradation

with TiO2 photocatalyst in ASW ........................................................................................57

20. Daily cell counts of four algal species in the experimental samples with sucralose

(Exp. 1, 2, 3) and control samples without sucralose (Contr. 1, 2, 3)................................58

21. Representation of the exponential growth four algal species in the experimental

samples with sucralose (Exp. 1, 2, 3) and control samples without sucralose

(Contr. 1, 2, 3) ....................................................................................................................60

22. Initial and final sucralose concentration in the medium of T. weiss. .................................62

INTRODUCTION

The research of water contaminants is continuously expanding and so is the list of

contaminants. Pharmaceuticals and Personal Care Products (PPCPs) belong to the major group

contributing to the problem. Prescription and over-the counter drugs, fragrances, cosmetics –

compounds that relate with human activities, wastes from pharmaceutical manufacturing or

hospitals, and agribusiness – all become a source of PPCPs in the environment. PPCPs have been

found in various aqueous systems including: surface marine and freshwater, groundwater,

wastewater and even drinking water (carbamazapine, clofibric acid). The majority of the data

collected pertains to surface freshwaters because of accessibility and sample preparation. Table 1

shows some of the drugs that were found in rivers, lakes, or ponds usually below µg/L range,

even though concentrations for some contaminants, i.e. diclofenac (Jux, 2002) or ibuprofen

(Ashton, 2004) were found to be 15 and 5 µg/L, respectively. As ideal as it would be for these

contaminants to dilute to untraceable and environmentally safe concentrations as they travel

down the rivers, more advanced analytical instrumentation allows for detections of many of

those compounds in estuarine (Thomas and Hilton, 2004) and open sea (Weigel, 2002) waters, in

low ng/L range (Table 1). Although the way that PPCPs make their way into the environment

cannot always be isolated, there is substantial evidence proving the effluents from the WWTPs

are a significant source. Table 1 lists some of the major pharmaceuticals found in the wastewater

at concentrations ranging from below 1 µg/L (carbamazepine, naproxen) up to 27 µg/L

(ibuprofen; Ashton, 2004). Although these bioactive compounds have probably been present in

the environment for decades; now, when the technology allows for their precise detection and

characterization, it’s the time to recognize their fate and an effect on the environment.

2

Once the contaminants enter the environment, there are numerous pathways they are

prone to take based on their physical and chemical properties. Hydrophilicity, polarity, and

charge transfer that depends on the water pH, determine whether compounds stay dissolved in

water or adsorb onto the sediment (Giger, 2008). Usually, lipophilic compounds are more likely

to adsorb to the solid phase and can be further transported by moving particles or be deposited on

the floor of the aquatic system. In addition to the physical partitioning of the molecules, their

chemical transformations because of photolysis, hydrolysis, and biodegradation constantly take

place. This can lead to the formation of new and unknown compounds that can potentially be

more toxic than the parent compound and are exposed to the same transformations all over again

(Gomez et al., 2008). Contaminants can also enter the food chain through the consumption of

particles by fish, where in its tissue they can bioconcentrate and are being passed to the upper

trophic levels; bioconcentration of endocrine disrupting compounds (EDCs) in fish leads to its

feminization (Metcalfe et al., 2001).

Since 1977, the U.S. Food and Drug Administration (FDA) has approved numerous

artificial sweeteners that can be used in various foods. Among them are five nonnutritive

sweeteners with intense sweetening power: acesulfame-K, aspartame, neotame, saccharin, and

sucralose. The levels of these sweeteners in wastewaters and surface waters have been monitored

in multiple studies showing their relatively high concentrations, with acesulfame-K and sucralose

being the highest (Table 2, 3). Acesulfame-K was described as an ideal chemical marker of

domestic wastewater in groundwater (Buerge, 2009). However, the non-toxic and persistent

sucralose has just lately been classified as a contaminant of emerging concern (CECs,

Richardson, 2009), and its supply and demand curve is growing the fastest in the global

sweetener market (Musto et al., 2009). Sucralose, brand name Splenda ®, (1,6-dichloro-1,6-

3

dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside) was discovered in 1976

by Tate & Lyle Company and was first approved for use in Canada in 1991, in the U.S. in 1998,

and in the European Union in 2004, where it was given the additive code E955. Sucralose is a

derivative of naturally occurring disaccharide sucrose (table sugar), in which three hydroxyl

groups are replaced with three chlorine atoms (Fig. 1) in a five steps synthesis (Grice and

Goldsmith, 2000). This chlorination not only makes sucralose 600 times sweeter than sucrose,

but it also changes the conformation of the molecule making it resistant to attack from glycosidic

enzymes that break down carbohydrates (European Commission, 2000). As a result, sucralose is

poorly absorbed in the body (< 8%) and most of it is excreted in its unchanged form in feces

78.3% and in urine 14.5%; only 2% of the total dose is detected as glucoronide conjugates of

sucralose in urine (Roberts et al., 2000). It has been shown that in acidic conditions in vitro,

sucralose slowly hydrolyzes into its constituent monosaccharides, 1,6-dichloro-1,6-dideoxy-D-

fructose (1,6-DCF) and 4-chloro-4-deoxy-D-galactose (4-CG) (Fig. 2). Although the rate of the

hydrolysis is very slow, after 1 year at 25°C less than 1% hydrolysis at pH 3.0 occurs, it is

necessary to assess the safety of the hydrolysis products and the sweetener itself (Grice and

Goldsmith, 2000). Extensive pharmacokinetic studies show no evidence of hydrolysis,

dechlorination, or bioaccumulation of sucralose in the human body, indicating its effective half-

life in humans is 13 hours (Roberts et al., 2000, Griece and Goldsmith, 2000). In addition, the

results of over 113 studies on sucralose and its hydrolysis products demonstrate no toxicity and

safe human consumption (Griece and Goldsmith, 2000). Consequently, the Joint FAO/WHO

Expert Group on Food Additives (JECFA) determined Acceptable Daily Intake (ADI) of

sucralose to be 0-15 mg/kg body weight. As of 2010 sucralose is allowed for use in over 80

countries in a wide variety of foods including: tabletop sweeteners, beverages, chewing gums,

4

dry-mix products, processed foods, milk products, frozen desserts, salad dressings, and even

baked goods due to its excellent stability at high temperatures (Griece and Goldsmith, 2000).

Since it was established that sucralose is harmless to people and the sweetener has been

approved for human consumption by the FDA as well as the EU Scientific Committee on Food,

there was no environmental review conducted. Systematic examination on the environmental

effects of sucralose had not been conducted (Lubick, 2008).

It wasn’t until a study was performed by the Norwegian Pollution Control Authority in

2007 that the first results on sucralose screening in the environment were reported. Green et al.

(2007) tested influent, effluent, and sludge samples from three different Wastewater Treatment

Plants (WWTP) and found concentrations of sucralose to be 1.9 - 5.5 µg/L in the influents and

2.2 – 5.9 µg/L in the effluents. It needs to be recognized that effluent samples have higher

concentrations than influent samples, indicating that WWTPs do not eliminate sucralose during

their treatment. Concentrations in sludge were at trace levels, except one sample being 22 µg/kg

of wet weight. Sea water samples from two locations were also analyzed with highest

concentrations ranging from 18-30 ng/L. Following the Norwegian report, the Swedish

Environment Research Institute also conducted sucralose screening of native surface waters,

WWTP and biota samples (Brorström-Lunden et al., 2008; published as PART I and PART II).

Sucralose was detected in all untreated wastewater samples from two WWTPs in concentrations

between 3.5 – 7.9 µg/L. Among 29 effluent samples from 25 different WWTPs, median

concentration of sucralose was 4.9 µg/L. Just as in the Norwegian study, the effluent samples

had higher concentrations than influent waters. The highest reported removal rate was 10%, but

negative numbers were also obtained. Authors speculate that the negative removal rate may be

attributed to part of the incoming material being conjugated or complexed and transformed back

5

to parent compound during treatment. No sucralose was detected in surface waters, but effluent

receiving waters contained 400 – 900 ng/L of the sweetener. Part II of the Swedish screening

(Brorström-Lunden, 2008, PART I) reported more concentration data for sucralose in STP

influent, effluent, and sludge samples with similar or slightly lower concentrations along with

additional data from biota samples. Analysis of soft tissue from mussels (Anodonta cygnea),

previously exposed to sucralose effluent discharge for eight weeks, did not show any traces of

sucralose. No sucralose was found in the liver or muscle of perch (Perca fluviatilis), caught

downstream of STP discharge. Therefore, uptake in biota seems to be unlikely (Brorström-

Lunden, 2008, PART II).

More recent evaluation of surface waters to determine the presence of sucralose were

conducted by Loos et al. (2009) who analyzed 120 river water samples from 27 European

countries. The results show concentrations of sucralose up to 1 µg/L with clear distinction of

higher concentrations being reported in Western Europe. A recent publication from Germany

provides information on seven artificial sweeteners found in waste water, surface water, and in

soil aquifer treatment (SAT) sites, including sucralose as one of the analytes (Scheurer et al.,

2009). The study confirms the concentration of sucralose in German rivers up to 1 µg/L as

reported by Loos et al. (2009). Additionally, Scheurer et al. (2009) compared the removal rate of

sucralose in three different WWTPs. STP1 in Eggenstein-Leopoldshafen (Germany) applies

conventional mechanical and biological treatment; hydraulic retention time is 5 hours. STP2 in

Karlsruhe (Germany) uses mechanical treatment with additional phosphate precipitation,

followed by biological treatment with a denitrification/nitrification unit and additional trickling

filter; hydraulic retention time is 1 day. Both STPs remove approximately 20% of sucralose. The

solid aquifer treatment is located in Mediterranean country and includes mechanical and

6

biological treatment, after which secondary effluent is spread in percolation basins, where

infiltration through an unsaturated zone takes place; residence time in an aquifer exceeds 1.5

years. Although over 90% of sucralose is removed during the treatment, it is still present at a

concentration of 1.4 µg/L after 1.5 years in the subsurface (Scheurer et al., 2009).

The environmental lifetime of sucralose in Norwegian waters is expected to be 5 years as

reported by Green et al. (2007). This persistence is of concern because of the lack of knowledge

on how sucralose interacts with the aquatic environment.

Studies to determine whether sucralose is biodegradable were first conducted by Lappin-

Scott et al. (1987) who observed microbial transformation in soil slurries, but was not able to

isolate the organism responsible for its metabolism. Only a few years later it was reported by

Labare and Alexander (1993) that in samples spiked with sucralose, 48–60% of the sucralose is

mineralized in the soil, 1.1-4.0% in surface waters, and 23% in activated sludge. Since

mineralization did not occur in sterilized samples, degradation has to result from microbial

activity. Cometabolic process in biodegradation of sucralose was also suggested and

consequently studied by the same authors (Labare and Alexander, 1994) to evaluate this

transformation more closely and provide the evidence for its occurrence. Cultured bacteria did

not use the sucralose carbon as a source of energy and no sucralose carbon was incorporated in

their cells. They did, however, convert it into unsaturated aldehyde, 1,6-dichloro-l,6-dideoxy-D-

fructose, and the uronic acid of sucralose (Fig. 3). Although data indicates that sucralose does not

rapidly degrade, its degradation clearly depends on environmental conditions. Moreover,

generated products may accumulate. Hence, understanding of sucralose metabolism is essential

to evaluate potential environmental risks from such a wide use of the sweetener (Labare and

Alexander, 1993).

7

The occurrence and persistence of various water contaminants in the environment create

the concern about their influence on the aquatic organisms. Although sucralose is not toxic to

humans or animals, one study demonstrated sucralose inhibited 22% of sucrose transport in

sugarcane (Reinders et al., 2006). In spite of high sucralose concentrations used for this

experiment (16.5 mM), it shows that sucralose can disturb sucrose transport and if that transport

is disturbed in aquatic species, it may affect the entire ecosystem. Moreover, there are several

biological communications and orientation systems in aquatic ecosystems that are based on taste

(Giger, 2008) and the persistent existence of distinct taste, i.e. sugar like substance, may change

organisms feeding behavior (Lubick, 2008). In the interview by Lubick (2008), it was suggested

that sucralose could interfere with plant photosynthesis which could cause the problem for algae

and have the unexpected consequence of shutting down CO2 uptake.

Phytoplankton plays particularly important functions in the ecosystem, e.g. producing

oxygen, providing food, and participating in nutrient cycling (Fleeger et al., 2003) and previous

studies show an effect of water contaminants, including PPCPs, on algal cells. A common

antimicrobial agent, triclosan, has been found to cause membrane disruption in marine

chlorophyte Dunaliella tertiolecta and inhibit its growth by 67% at the environmentally relevant

concentrations of 4.9 µg/L within 96 hours (DeLorenzo et al., 2008). Chronic exposure to the

contaminants that affect the individual specie’s growth rates can alter the competitive

interactions between the species and lead to a change in their abundance that will modify their

functions (Fleeger et al., 2003). In the same study, DeLorenzo et al. (2008) has tested mixtures of

PPCPs (simvastatin-clofibric acid; triclosan-fluoxetine) on the algal growth. Although no effect

was found at the environmentally relevant concentrations, the mixtures of PPCPs did appear to

have an additive toxicity effect on the marine phytoplankton and lowered their toxicity threshold

8

when compared with the effect of just the individual components of the mixtures. Therefore, the

effect of not only individual PPCPs but also their mixtures on the aquatic species that is crucial

in understanding the real time interactions of drugs with the environment. The effect of another

pharmaceutical, carbamazepine, on algal specie Ankistrodesmus braunii has been tested by

Andreozzi et al. (2002). In his study, no inhibition of growth was observed; however, after 60

days of testing carbamazepine concentration in the medium decreased by 50%. Although uptake

and accumulation of the contaminant in the algal cells and its bioaccumulation through the food

net to the consumer organisms in the upper trophic levels is a viable option (Andreozzi et al.,

2002), no significant amounts of carbamazepine were detected in A. braunii cells. It was then

stated that carbamazepine was taken up by the cells and entered the biochemical process.

Phytoplankton constitute the foundation of the food chain and accounts for half of all

photosynthetic activity on Earth, any effects that water contaminants can exhibit on it will have a

fundamental meaning in modifying their ecosystem functions.

The fate of sucralose in the environment is potentially further impacted by

photodegradation. Photochemical processes are often complicated and form multiple pathways

and multiple products that may be more toxic than the parent compound or retain the properties

of the parent compound. This has been reported for organic dye malachite green (Pérez-Estrada

et al., 2008) and for metabolite of analgesic drug dipyrone (Gomez et al., 2008). Since sucralose

does not absorb light at wavelengths that typically reach the surface of the Earth (300 – 800 nm),

Fig. 4, it will not likely undergo direct photodegradation which involves photon absorption by

sucralose molecule itself. Even though no research has been reported, it is expected indirect

photodegradation that engages photosensitizers will occur.

9

The capability of WWTP to eliminate sucralose is limited (Green et al. 2007, Brorstrom-

Lunden et al. 2008, and Scheurer et al. 2009). With the continuously discharging effluents

acting as the main source of the contaminant in receiving waters, the photodegradation process

and its products are of high significance. The purpose of this research was to study the

photodegradation rate of sucralose in different water matrices in order to establish the influence

of other water constituents on this process. The influence of potentially important physical and

chemical parameters like, salinity, dissolved organic matter, and humic substances (HS) was

observed. The conditions necessary for successful photocatalysis were examined during the

experiments to understand the mechanistic pathway of the reactions. To confirm the assumptions

of the hydroxyl radical photodegradation of sucralose, additional tests with methanol as a

hydroxyl radical scavenger, were conducted. In addition to investigating naturally occurring

photodegradation, this research also examined one of the advanced oxidation processes (AOP)

that involves UV/TiO2, called heterogeneous photocatalysis. The generation of reactive species

(mainly hydroxyl radicals ˙OH) through ultraviolet radiation in the presence of strong catalyst,

has been shown to be a successful way to degrade many organic pollutants (Choy and Chu,

2005). All photodegradation experiments with and without TiO2 catalyst were also carried out in

various matrices.

The effect of sucralose on marine phytoplankton species was examined. This research

included the study of four representative species from the major phytoplankton groups: diatoms -

Thalassiosira weissflogii, dinoflagellates – Heterocapsa triquetra, coccolithophorids – Emiliania

huxleyi, and green algae – Dunaliella tertiolecta. In addition to the growth of each species,

concentration of sucralose in the medium of T. weissflogii was measured at the beginning and at

the end of the experiment to examine uptake of sucralose by algal cells.

10

METHODS

Occurrence

Sample Collection

Water samples were collected in 1L amber glass bottles from Wilmington Southside and

Northside WWTPs and from the lower Cape Fear River - along the salinity gradient, starting in

the Wilmington area (low salinity, close to zero) and ending at the mouth of the Cape Fear River

(high salinity). Fresh water samples were collected across North Carolina and from the Potomac

River in Washington DC (Table 4). All samples were filtered through previously combusted

47mm glass microfiber filters (GF/F, 0.7 µm pore size) and stored at 4˚C until the analysis.

Extraction

For the sample preparation, 50 mL of water was passed through the C18 SPE cartridge

(0.5 g, 6 mL Hypersep). Each cartridge was conditioned with 6 mL of methanol (B&J GC2

99.99% purity, Honeywell) and 6 mL of MilliQ water obtained through Millipore system with a

resistivity of 18.2 MΩ·cm at 25°C. After the sample was extracted, the glassware and tubing

were rinsed with 5mL of water. This water was also put through the cartridge to mitigate the

analyte loss. The cartridge was then left to air dry for 30-45 min. Eight milliliters of methanol

was used as an eluting solvent. The eluent was dried over anhydrous sodium sulfate, rotovap to

~1 mL and transferred to a sample vial to be blown down to dryness under UHP nitrogen gas.

Samples were derivatized using 70 µL of MSTFA + 1% TMCS and 30 µL of pyridine, and

heated at 70˚C for 30 minutes. Once cool, samples were blown down under the nitrogen gas to

dryness at 50˚C then, dissolved in 100 µL of dichloromethane, and transferred to GC vials.

Quantification of sucralose was done using an external calibration curve prepared by

spiking the same water matrices as the tested samples with range of sucralose concentrations

11

typically from 0.5 ng/µL to 2.5 ng/µL. The created plot incorporated the analyte loss during the

sample preparation as well as the precision of sample preparation itself. If the measured

concentration of sucralose in particular sample was higher or lower than the calibration curve

range the standards were adjusted and reanalyzed along with the sample.

GC-MS Analysis

Quantitative and qualitative analysis of the analyte was done using a Varian CP-3800 GC

coupled to a Varian Saturn 2200 ion trap mass spectrometer. The GC column used was a J&W

Scientific DB-5ms with 0.25 μm phase thickness, 0.25 mm ID and was 30 m long. Instrument set

up conditions were as described by Mead et al. (2009), except for different oven temperatures:

hold at 40°C for 1 minute then ramped at 10°C/min to 200°C then to 260°C at 15°C/min then

finally to 300°C/min at 3°C/min with a hold time of 2 minutes. The adjustments in temperatures

allowed for a decreased run time while maintain the integrity of the analysis. Electron ionization

was 70 eV and the mass spectrometer operated in full scan mode from 50-600 m/z. Base ion m/z

308 and fragment ions m/z 361 and 73 were used for the identification of the TMS ether of

sucralose (Fig. 5). The total ion current was used to quantify the sucralose peak (Fig. 6)

Injection of each sample concentration was done in triplicates to estimate the instrument

precision by calculating the mean values and relative standard deviations (RSD) of the data

obtained.

Method Optimization

SPE Cartridge Selection

Four solid phase extraction cartridges with different sorbent phases were tested to

determine which has the highest recovery for sucralose extraction: diol - polar sorbent

(COHCOH, 0.2 g), cyano (CN) – polar sorbent (0.2 g), Oasis HLB - Hydrophilic-Lipophilic

12

Balance Sorbent reversed phase (0.2 g; Waters), and C18 – reversed phase nonpolar sorbent (0.5

g). For all cartridges 100 mL of MilliQ water was spiked to give a sucralose concentration of 1

ng/L. The extractions and analysis was carried out as described by Mead et al. (2009), except for

derivatization time which was 30 minutes and GC solvent, which was DCM.

Extraction Volume

To evaluate whether the amount of the sample passed through the C18 SPE cartridge

influences the analyte recovery, four different sample volumes were tested in duplicates. 200 mL

of MilliQ water was spiked with sucralose standard to obtain its final concentration of 5 µg/L.

Out of this solution, the following volumes were transferred to individual Erlenmeyer flasks: 100

mL, 50 mL, 25 mL, and 10 mL (final concentrations of these samples prepared for GC-MS

analysis were: 5, 2.5, 1.25 and 0.5 ng/L, respectively). The four samples were processed

according with the current method.

Sorption

Sorption of Sucralose onto Natural Sediment Samples

Four combusted 500 mL round bottom flasks were filled with 300 mL of MilliQ water

each. All samples were spiked with 600 µL of 2.5 ng/µL sucralose/methanol standard. 10 grams

of wet sediment collected from Fishing Creek, NC was added into two experimental samples;

remaining two samples served as the controls. All flasks had stirring bars inside them and were

placed on the stirring plates for three weeks. After 24 h, and three consecutive weeks appropriate

amount of sample was taken out of each flask, centrifuged, and filtered through 0.7 µm GF/F

filter to give final volume of 50 mL per sample. Samples were analyzed for sucralose

concentration. An aliquot of the water from Fishing Creek wet sediment was centrifuged and

analyzed for the presence of sucralose.

13

Sorption of Sucralose onto Montmorillonite Clay

Water collected from Wrightsville Beach, NC was filtered through a 0.7 µm GF/F filter;

200 mL was then measured out into three combusted BOD bottles. All three samples were spiked

with 400 µL of 2.5 ng/µL sucralose/methanol standard. 0.5 g of montmorillonite clay (collected

from Wyoming) was added into two experimental samples; third sample served as a control.

Samples were swirled daily. After seven weeks, 50 mL of water was taken out from each bottle

and analyzed for sucralose concentration.

Photochemistry

Sample collection

Water samples were collected in 1L amber glass bottles, filtered through combusted GF/F

47mm glass microfiber filters (0.7 µm pore size), and stored at 4˚C (filtration doesn’t apply to

MilliQ and ASW samples). Samples were collected at Wrightsville Beach, from the Lower Cape

Fear River near Downtown, and from Northside WWTP treated effluent – all locations are in

Wilmington, NC (Table 4). Recipe for artificial seawater (ASW) was adapted from The Marine

Biological Laboratory (Appendix A). For distilled water samples we used ultrapure MilliQ water

obtained through Millipore system with a resistivity of 18.2 MΩ·cm at 25°C.

Irradiation

200 mL of previously filtered water sample was measured out into combusted 250 mL

round bottom flasks (three quartz flasks for “Light” - experimental samples and three glass flasks

for “Dark” - control samples). All samples were spiked with 65 µL of 0.12 µg/µL

sucralose/MilliQ (except effluent samples for which ambient sucralose concentration was ~40

µg/L) that yields 7.8 µg of sucralose per flask. Each “Light” sample had its equivalent “Dark”

sample wrapped in aluminum foil to serve as a control. A solar simulator reactor equipped with a

14

filter eliminating wavelengths <280 nm, which are not found in the solar spectrum, was used for

the experiments (Fig. 7). Flasks with stoppers and stirring bar inside were placed on stirring

plates and kept at a constant temperature water bath (22°C) while exposed to simulated sunlight.

Samples were radiated for 6 hours (rotated every 2 h) after which 50 mL of each sample was

measured out for sucralose analysis.

For the experiments with humic materials, appropriate amounts of Aldrich humic acid

(HA) standard solution in MilliQ were added to the flasks prior to photolysis to obtain final

concentrations of: 0.01, 0.10, and 1.0 mg HA/L.

For experiments with photocatalyst, 0.5 g of TiO2 (anatase form, Alfa Aesar) was added

to the flasks prior to photolysis and filtered out right after the photolysis through 47 mm glass

membrane filter to separate TiO2 from the solution before sample workup continued.

Sample Workup

Photolyzed samples were processed and analyzed according to the current method.

GC-FID analysis

Quantitative analysis of the analyte was done using a Hewlett-Packard HP 6890 GC

System equipped with a flame ionization detector. Instrument set up conditions were as follow: a

DB-5MS capillary column of 30 m × 0.25 mm i.d. (0.25 µm film thickness) was used and the

GC column temperature was programmed as follow: hold at 40°C for 1 minute then ramped at

10°C/min to 200°C then to 260°C at 15°C/min then finally to 300°C/min at 3°C/min with a hold

time of 2 minutes. The injection inlet was set up at 250°C with injection volume of 1 µL in

splitless mode. Helium was used as the carrier gas maintained at constant 1.2 mL/min flow. The

temperature of the flame ionization detector was 250°C. Identification of sucralose was based on

retention time of the standard.

15

Biological Impact

Culture Media

Culture medium was made up in the previously autoclaved seawater collected in the Gulf

Stream of North Carolina coast. The recipe for f/2 media that was used for growth of all

microalgae in this experiment was made up according to Guillard and Ryther, (1962) and

Guillard, (1975). After the media was made up, it was filter sterilized to remove impurities using

sterile membrane filter with 0.45 µm pore size (Sartobran 300, Sartorius Stedim Biotech GmbH).

Experimental Setup

The parent cultures of: Dunaliella tertiolecta (PLY 83), Heterocapsa triquetra, and

Emiliania huxleyi (PLY 1516) were grown in f/2 culture medium. After the cultures reached the

mid-logarithmic growth rate, they were subcultured (one culture per experiment) into six

radiation-sterilized 200 mL polystyrene bottles with filter caps (CELLSTAR Tissue Culture

Flasks, Greiner Bio-One), filled with 150 mL of f/2 medium each. The volume of each

transferred culture depended on its density and was as follows: 0.15 mL for D. tertiolecta, 1.5

mL for H. triquetra, and 0.1 mL for E. huxleyi. Three of the six bottles served as experimental

samples and were spiked with filter sterilized sucralose/MilliQ standard, to give final sucralose

concentration of 1.0 µM; three remaining bottles had no sucralose in them and served as the

controls. The microalgae were incubated for 14 days at 20 ± 2°C, 12 h light/12 h dark cycle,

under light intensity of 130 – 150 µE/m2/sec. Two milliliters of the culture from each bottle was

sampled daily, preserved with 30 µL of Lugol’s solution, and counted with a light microscope

(UNICO) in Neubauer haemocytometer.

The parent culture of Thalassiosira weissflogii (PLY 541) was also grown in f/2 culture

medium and when it reached its mid-logarithmic growth rate, 0.2 mL was subcultured into six

16

radiation-sterilized 200 mL polystyrene bottles with filter caps (CELLSTAR Tissue Culture

Flasks, Greiner Bio-One), filled with 200 mL of f/2 medium each. As in the other experiments

three of the six bottles served as experimental samples and were spiked with filter sterilized

sucralose/MilliQ standard, to give final 1.0 µM sucralose concentration; three remaining bottles

had no sucralose in them and served as the controls. Before algae were placed in the incubator,

55 mL of the culture from each bottle was measured out for the analysis of the initial

concentration of sucralose. T. weissflogii was incubated and sampled just like the other cultures.

On the last day of the experiment, 55 mL of the culture from each bottle was measured out for

the analysis of the final concentration of sucralose.

Analysis

Cell counts of the cultures obtained with the light microscope in Neubauer

haemocytometer were plotted in an Excel spreadsheet. The slopes of the logarithmic growth rate

between experimental and control samples were compared for each organism using student T-

test.

T. weissflogii samples measured out on the first and last day of the experiment were filtered

through 0.2 µm glass membrane filter to separate the cells from the solution and were worked up

according with the current method to compare initial and final concentration of sucralose in the

culture medium.

RESULTS / DISCUSSION

Occurrence of Sucralose

Lower Cape Fear River Estuary (CFR)

In the preceding study by Mead et al., (2009) sucralose was found in the waters of Cape

Fear River Estuary (~1 m below surface) in concentrations ranging from 1.94 µg/L in downtown

17

Wilmington area (station M70) to 0.38 µg/L at the mouth of the river (station M18) for the

surface waters and from 1.88 µg/L to 0.45 µg/L for deep waters (~1 m above sediment),

respectively. An additional transect collected along the CFR in February of 2009 was analyzed

as described by Mead et al. (2009) except an external calibration curve was prepared in the same

water matrices as the tested sample. Consistently, highest sucralose concentrations of 3.13 µg/L

for surface and 1.41 µg/L for deep waters were in downtown area (M70), where Northside

WWTP installed its effluent pipe, and lowest concentrations of 0.52 µg/L for surface and 0.19

µg/L for deep waters were at the mouth of the river (M18D), (Table 4). These concentrations for

the CFR are similar to concentrations reported by Loos et al. (2009) and Brorström-Lunden et al.

(2008) (Table 3). Correlation of decreasing concentrations of sucralose with increasing salinity

(R2 = 0.847) is the result of simple dilution and diffusion. Higher concentrations of sucralose in

CFR reported by Mead et al. (2009) compared with the current transect may be attributed to

lower precipitation levels in 2008 vs. 2009 (this would be consistent with higher salinity).

Seasonality, or different quantitative analysis methods used for each of the transects may also be

a factor, however, there is not enough data to support these implications.

Effluent waters

Wilmington, NC has two major wastewater treatment plants: The Northside (NS) plant

discharges ~7.4 million gallons of effluent per day (MGD) while the Southside (SS) plant

discharges ~8 MGD. Both facilities have the same physical (screening, grit removal, primary

sedimentation) and activated sludge secondary biological treatments with the exception of the

final chemical processes in which Northside uses UV disinfection and Southside uses

chlorination disinfection followed by dechlorination. Mead et al. (2009) reported sucralose

concentrations of 119 µg/L from Northside WWTP. New samples obtained from both plants in

18

July 2009 and processed using current method had sucralose levels of 40 µg/L for NSWWTP

and 46 µg/L for SSWWTP (Table 4). Based on these results and reported discharge from both

facilities, the NS plant discharges 1.13 kg and the SS plant discharges 1.30 kg of sucralose into

the CFR per day (Eq. 1 and 2). Annual sucralose discharge into the CFR is 887 kg (Eq. 3).

While no influent samples were tested in this study, the high sucralose concentrations for

effluent samples from Northside (40 µg/L) and Southside WWTPs (46 µg/L) demonstrate that

sucralose is not fully eliminated. This supports results of Green et al. (2007), Brorström-Lunden

et al. (2008), and Scheurer et al. (2009) who reported low removal rate of sucralose in WWTPs.

However, results presented in this study have much higher concentrations of sucralose in effluent

waters in comparison to Green et al., 2007 (2.2 – 5.9 µg/L) and Brorström-Lunden et al., 2008

(4.9 µL), Table 3.

Fresh Waters

To complement the study by Mead et al. (2009), fresh water samples from various

locations across North Carolina were analyzed (Table. 4). Samples from Hanging Rock State

Park included the creek located at the top of the mountain, in which no sucralose was detected,

19

and one of the park’s lakes at the bottom of the mountain, where sucralose concentration was

2.06 µg/L. There is no waste water being discharged to any of the park’s waters, however, at the

bottom of the mountain visiting tourists are allowed to camp, swim in the lake, and use public

restrooms. Samples were also taken from Jordan Lake which had no detectable levels of the

sweetener but a sample from the Haw River in the Saxapahaw, NC area had concentrations of

1.35 µg/L. An additional sample from Potomac River in Washington DC area had sucralose

concentration of 1.46 µg/L (Table 4).

Including the data reported by Mead et al. (2009), the effluent water from WWTP in

Wilmington, NC has the highest sucralose concentrations of all effluent concentrations reported

to date (Table 3,4). In light of concentrations reported for other contaminants, the amount of this

chlorinated sweetener is at least an order of magnitude higher in most cases or even two orders

of magnitude for others, e.g. estrogens (Table 3). Sucralose concentrations are also higher than

reported concentrations of other artificial sweeteners (Table 2). Although occurrence itself does

not indicate sucralose is harmful to any biological systems or the environment, it is a

contaminant of emerging concern and its potential risks of persistence, bioaccumulation, toxicity,

or transformations, once it enters the environment, need to be assessed.

Method Optimization

SPE Cartridge Selection

The method described by Mead et al. (2009) (uses Oasis HLB cartridges) had the average

recoveries from CFR samples of 26 ± 10%. To improve the sucralose recovery, four different

phase sorbents of solid phase extraction (SPE) and various extraction volumes were tested.

The HLB column (Hydrophilic-Lipophilic Balance Sorbent, Waters) that was used initially had

the highest recovery (90%) in MilliQ, while the C18 column retained 2.8% of sucralose in

20

MilliQ. The diol and cyano cartridges, each consisting of relatively polar sorbent that was

expected to interact with the hydroxyl groups of sucralose resulted in 0% recovery which was

surprising (Table 5).

Sample purification was continued in CFR water by placing a C18 (0.5 g) cartridge on

top of the HLB (0.2 g) with the idea that C18 retains DOM and allows the sucralose to pass

through, to be retained by HLB. The cartridges were eluted individually yielding higher

sucralose concentration of 0.20 ng/L from the C18 sample vs. 0.14 ng/L from the HLB (Table 5).

Calculations were done using external calibration curve since CFR water was not spiked with

sucralose. Based on these results, the following experiment was repeated in MilliQ water spiked

with sucralose and recoveries were higher again for C18 than for HLB (37% and 23%,

respectively, Table 5). Subsequently, an additional experiment to compare HLB and C18

recoveries was carried out as described by Mead et al., 2009 , except the 5% methanol rinse was

omitted which resulted in 70% recovery for C18 and 62% recovery for HLB (Table 5). Although

the percent recovery for C18 was fairly low in the first experiment (7%), when placed on top of

HLB for purification purposes, it retained more sucralose than HLB in more than just one

experiment (Table 5).

Extraction Volume

In the initial method (Mead et al., 2009), 250 mL was passed through 0.2 g HLB

cartridges. It was essential to evaluate whether the amount of water sample passed through the

cartridge influences the analyte-sorbent interactions and whether it has influence on sucralose

recoveries. A C18 SPE cartridge (0.5 g, 6 mL) was used in this experiment. The results showed

linear response of the peak areas to volumes extracted for 10, 25, and 50 mL samples with

extremely high R2 values of 0.998 for the first experiment (Fig. 8, Exp. 1) and 0.997 for the

21

duplicate (Fig. 8, Exp. 2). The peak area for 100 mL sample lay below the fitted regression line

in both cases. This suggests passing more than 50 mL through the cartridge is not as efficient and

lowers the recovery that plateaus as larger volumes of samples are extracted. Extracting the 50

mL sample improved the analyte recoveries to 83% (Table 5).

A typical calibration curve for quantitative analysis of sucralose ranged from 0.5 to 2.5

ng/µL yielding R2 = 0.993 for the current method (Fig. 9). The range of the calibration curve can

be adjusted according to sample requirements indicating acceptable application of this method

for sucralose quantification at environmentally relevant concentrations. Typical RSD for all

triple GC-MS injections ranged from 1.1 – 13.5%.

Based on the results presented, the method by Mead et al. (2009) was modified to give

better performance with 83% recovery by extracting only 50 mL through the C18 column, drying

samples over sodium sulfate to get rid of any water and improve derivatization, and changing GC

solvent to DCM to make sure derivatization products are completely soluble before GC injection.

In addition, an external calibration curve, prepared in the same water matrices as the tested

sample, was used for quantitative analysis. All of these changes were incorporated into the

current method and applied during continuing research.

Sorption

Octanol-water partition coefficient (KOW) is a key parameter in determining the

environmental fate of organic compounds. It relates to water solubility, soil/sediment adsorption

coefficient, and bioconcentration factors for aquatic life. During physicochemical studies of

sucralose (Jenner and Smithson, 1989) its KOW was determined to be 0.32. This low value

indicates high hydrophilicity of the sweetener and suggests it should stay dissolved in the

aqueous phase while sediment sorption is less likely to take place. To confirm this assumption,

22

two separate sorption experiments with two different types of soils (high and low in organic

matter), were conducted. In both experiments, the sucralose concentration in water was

compared between the samples with and without the sediment. Decrease in sucralose

concentration over time in the sample with sediment compared with the sample without sediment

would suggest part of the sucralose was adsorbed onto the sediment.

Sorption of Sucralose onto Natural Sediment Samples

The predominant sorbent of organic compounds in soils is soil organic matter. The

content of the wet sediment sample from Fishing Creek was high (3-5%) in heterogeneous

organic matter creating the possibility for sucralose absorption. The concentrations of sucralose

from the experimental samples (with sediment) did not decrease but were consistently higher

than the control samples (without sediment) suggesting no sorption of sucralose to the sediment

occurred over a period of 3 weeks (Fig. 10). No sucralose was detected in the water portion of

wet sediment or in the solid phase sediment suggesting the sucralose increase in sediment flasks,

compared to the controls (Fig. 10), was not caused by the added sediment.

To make sure this outcome did not result from the ambient sucralose levels in the wet

sediment initially added to the flask, the water portion of the wet sediment and the sediment

itself were analyzed; no sucralose was detected. Therefore, varying concentrations in the samples

with sediment resulted from the experimental error.

Sorption of Sucralose onto Montmorillonite Clay

Sorption processes can be considered competitive processes which arise from site-

specific interactions of different solutes at preferred sites and (Xing, 1996). In this sorption

experiment montmorillonite clay was used because it’s mostly stripped of organic matter and has

heterogeneous adsorption potential. Results demonstrate that no sorption of sucralose to the clay

23

soil occurred over a period of 7 weeks (Fig. 11). The concentrations of sucralose from

experimental samples (with clay) did not decrease in comparison to the concentration from the

control sample (without clay).

Photodegradation

Photolysis of sucralose in MilliQ water and in artificial seawater (ASW) was conducted

in order to determine if sucralose undergoes direct photodegradation in natural waters (Fig. 12).

No significant difference in sucralose concentration between light and dark samples was

observed (t-test p = 0.28) suggesting no direct photodegradation of sucralose occurred.

An important factor in photochemical reactions occurring in environmental samples is

played by humic substances (HS), which are major organic constituents in natural waters (20 –

50% of total dissolved organic matter, DOM). Humic substances photosensitization or radical

formation enhances the rates of photodegradation of organic compounds such as microcystin-LR,

1-aminopyrene, 4-chloro-2-methylphenol, nitroaromatic compounds (Welker 2000, Zeng 2002,

Vialaton 1998, Simmons and Zepp 1986). Humic substances can also decrease the rate or

efficiency of photodegradation such as: brevetoxins, carbofuran (Khan 2010, Bachman 1999)

due to optic filter effect (Frimmel 1994), substrate and HS concentration dependence (Garbin

2007), binding of substrate to HS (Bachman 1999). The role of the chromophoric fraction of HS

can be described below, Eq. 6-8, (Zepp et al., 1985). In the present study we used Aldrich humic

acid, HA, to evaluate its effect on sucralose degradation in the controlled environment.

In the natural environment photochemical processes occur through light absorption by

CDOM forming excited triplet states 3CDOM* (Eq. 4) that can either react with the substrate

(Eq. 5) or produce highly reactive oxygen species including hydroxyl radicals (OH˙), singlet

oxygen (1O2), peroxy radicals (ROO˙), and superoxides (O2

-˙) (Eq. 6). These radicals, called

24

photochemically produced reactive intermediates (PPRIs), may further react with the substrate.

Although CDOM is a principal photosensitizer in natural waters, iron (III) organic complexes,

nitrate, and nitrite also produce hydroxyl radicals (Zepp et al., 1985, Arnold and McNeill, 2007).

CDOM + hv → 1CDOM* →

3CDOM* (Eq. 4) (Eq. 3)

3CDOM* + substrate → CDOM + photoproducts (Eq. 5)

3CDOM* + O2 → CDOM + PPRIs (Eq. 6) (Eq. 5)

To evaluate the influence of environmental dissolved organic matter on sucralose degradation,

photolysis experiments were conducted with different concentrations of humic acid in artificial

seawater. There was a <20% decrease in sucralose concentration in the “experimental” samples

with varied concentrations of HA exposed to light compared to controls (Fig. 13a).

Photochemical degradation increased from 14.8 to 15.7 to 18.7% as the concentration of humic

acid increased from 0.01 to 0.1 to 1.0 mg/L, respectively (Fig. 14a). Although increase in

degradation is not large from sample to sample, it suggests its correlation to the concentration of

humic acid: more HA, higher degradation. Similar observations were made by Jiao et al. (2008)

who reported increasing photooxidation rate in tetracyclines (widely prescribed antibiotics in

agriculture) with increasing concentrations of HA (0, 3.75, 7.5 mg HA/L). Based on the results

from degradation of sucralose in MilliQ and ASW where a decrease of 7 and 3%, respectively,

was observed (Fig. 12), it appears that the degradation of sucralose is enhanced (up to 15.7%) by

the reactive species formed by the HA.

The natural waters included samples with low DOM concentrations (~200 µM DOC,

Avery et al., 2003) collected from Wrightsville Beach (WB), samples with high DOM

concentrations (~800 µM DOC, Avery et al., 2003) collected from Cape Fear River (CFR), and

effluent samples from Northside WWTP in Wilmington, NC. The results showed 16%, 9%, and

25

3% sucralose degradation in WB, CFR, and effluent, respectively (Fig. 15a). In accordance with

the humic acid experiment, higher amount of DOM should have resulted in higher sucralose

degradation; the results were opposite: samples higher in DOM from CFR degraded 9% vs

samples lower in DOM from WB, which degraded 16%. It appears that low amounts of DOM

have small but proportional to concentration photocatalytic effect (Fig. 13a, 14a). At higher

DOM concentrations, however, degradation efficiency goes down implying that another process

that competes with sucralose degradation takes place simultaneously. The same relationship was

described by Garbin et al. (2007) and Jiao et al. (2008). In both studies HS was able to

photogenerate hydroxyl radicals that increase degradation of certain pesticides or antibiotics; but

at higher concentrations HS also react with these hydroxyl radicals leading to a decreased rate of

the photolysis.

The UV-Vis absorption spectra of chromophoric dissolved organic matter (portion of the

DOM responsible for absorption of UV and visible light) provide quantitative information about

the abundance of CDOM (Fig. 16). The absorbance (A) at selected wavelength (λ) that typically

reach the surface of the Earth (300 – 800 nm) represents the organic compounds that actively

absorb visible light. According to Beer-Lambert Law (A = εcl for a given wavelength) the

absorbance is linear with the concentration of the sample. Therefore, if molar absorptivity (ε) and

the path length (l) stay constant, Aλ will yield the relative amount of CDOM present in the

sample. The absorbance A300 for WB, CFR, and effluent water was: 0.006, 0.534, and 0.120,

respectively, (Table 6). The high organic matter content in CFR and effluent water can compete

with the sucralose for photons or it can increase the water opacity resulting in lower percent

degradation in these samples compared with optically more clear samples from WB.

26

Since CDOM can form hydroxyl radicals as one of the PPRIs (Eq. 6), the ·OH mediated

degradation was a possible pathway for indirect degradation of sucralose. The additional

experiments with methanol, a well known hydroxyl radical scavenger, were conducted. As

described in the literature (Marugán et al., 2006; Wang et al., 2002; Gao et al., 2002)

photogenerated ˙OH (Eq. 6) reacts with methanol molecule by H atom abstraction (Eq. 7) which

in the presence of oxygen forms formaldehyde (Eq. 8).

˙OH + CH3OH → H2O + ˙CH2OH (Eq. 7)

˙CH2OH + O2 → ˙O2CH2OH → HCHO + ˙O2- (Eq. 8)

Photolysis samples of sucralose solutions prepared in natural waters of Wrightsville

Beach and Cape Fear River were spiked with methanol and compared to corresponding controls

without methanol (Fig. 17). There was a decrease in degradation from 16% in samples without

methanol to 0% in samples with methanol. Samples from CFR had a 9% degradation in samples

without methanol and 1.4% in samples with methanol suggesting hydroxyl radical scavenger

degradation of sucralose in natural waters.

According to the results from all the photolysis experiments conducted without the

photocatalyst (Fig. 12, 13a, 15a), sucralose degradation does not exceed 20% of its initial

concentration. Even though the nature and the amount of organic matter present in the water may

play an important role in the process, degradation occurs in organic free matrices as well. If only

20% of the sucralose in the environment photodegrades, its high concentrations in µg/L range

(Mead et al., 2009), implicate accumulation of this contaminant in the surface waters over time

accompanied by its high input from WWTPs to the surface waters (Eq. 3).

27

Photocatalyzed Degradation

Titanium dioxide (TiO2) is a naturally occurring oxide of titanium found in minerals

like rutile, anatase, or brookite (these differ in their crystal structures and stability). It is

chemically and biologically inert, environmentally safe, inexpensive, and widely available (Khan

et al., 2010). The electronic structure of TiO2 as a semiconductor is characterized by a filled

valence band and an empty conduction band. During irradiation of TiO2 with energy of light

≤385 nm, that matches or exceeds the bandgap energy (3.2 eV), an electron, eCB-, from the

valence band VB is promoted to the conduction band CB, leaving a hole, hVB+, behind (Eq. 9).

The generated electron-hole pairs can react with electron donors and electron acceptors adsorbed

at the surface of the semiconductor or they can recombine and dissipate the input energy as heat.

In aqueous solutions valence-band hole usually reacts with hydroxyl anions forming hydroxyl

radicals (Eq. 10) that further react with organic molecules (Eq. 11). Excited-state conduction-

band electron is scavenged by molecular oxygen, producing superoxide anion radicals (Eq. 12),

(Helz, 1994, Gao et al., 2002, Wang et al., 2002).

TiO2 + hv → hVB+ + eCB

- (Eq. 9)

hVB+

+ ¯OH → ˙OH (Eq. 10)

˙OH + substrate → substrate ˙ (Eq. 11)

eCB- + O2 →

˙O2

¯ (Eq. 12)

When sucralose solutions of MilliQ and ASW were photolyzed in the presence of TiO2

photocatalyst, degradation increased dramatically to 96% and 95%, respectively (Fig. 18). When

0.098 mM sucralose solutions were photolyzed with and without methanol (10 mM)

accompanied by TiO2, 95.1% sucralose degradation for the sample without methanol while no

degradation for 10 mM methanol solution was observed (Fig. 19). This suggests the presence of

28

hydroxyl radical scavenger dramatically reduces/prevents sucralose degradation. This is

consistent with Cooper et al., 2009 who found that the ether linkages, which are present in

sucralose structure, are particularly reactive towards hydroxyl radicals.

Photolysis experiment with humic substances in artificial seawater with the addition of

TiO2 showed enhanced photodegradation compared to photolysis of TiO2 with no HS (Fig. 13).

The sucralose breakdown was 94.1%, 98.3%, and 99.7% for 0.01, 0.1, and 1.0 mg HA/L,

respectively (Fig. 14b). As in the previous experiment with humic acid (Fig. 13a), increase in

degradation from sample to sample is not large but proportional to low HA concentrations. The

effect of low humic acid concentrations in both experiments, with and without TiO2 is consistent,

although the mechanisms in those two cases would expect to be different and probably more

complex for TiO2 samples.

Photolysis experiment with dissolved organic matter in natural waters was also repeated in

the presence of TiO2. Sucralose degradation was 14.9%, 81%, and 100% for Wrightsville Beach,

Cape Fear River, and effluent, respectively (Fig. 15b). The higher amount of DOM in CFR and

effluent water (Table 6) could have resulted in higher number of produced hydroxyl radicals

explaining more successful degradation in these waters compared to WB water. This is similar to

what was observed for the enhanced photodegradation in the presence of HS and TiO2. Too high

DOM concentrations could potentially retard photodegradation, which could explain why

degradation in CFR sample was lower when compared with effluent.

Another approach to explain the difference in the percent degradation of sucralose, in

addition to the amount of DOM present in the water, is the nature of the DOM itself. The

absorption coefficients calculated from the UV-Vis absorption spectra of CDOM, can be fitted

into Equation 13 producing spectral slope, S (nm-1

) (Helms et al., 2008). Spectral slope provides

29

details regarding the average characteristics of CDOM (chemistry, source, diagenesis) by

implementing the general trend: S increases with decreasing molecular weight (MW) and

decreasing aromaticity (Green and Blough, 1994). Spectral slopes from WB, CFR, and effluent

water were: 0.0188, 0.0118 and 0.0132 nm-1

, respectively (Table 6). Highest S value for organic-

poor coastal WB water, corresponding to the lowest molecular weight and lowest degree of

aromaticity, represents aquatic source of DOM (Helms et al.,2008), e.g. in situ production by

phytoplankton (Avery et al., 2003). Estuarine waters with terrestrial source of DOM (e.g. runoff)

have higher molecular weights based on their low spectral slopes (Helms et al., 2008),

representing organic-rich waters. Effluent waters with spectral slopes in between WB and CFR

had an organic content of higher MW than the WB water but lower MW compared with CFR,

which may be linked to the wastewater treatment biodegradation processes that eliminate

complex organic matter.

aλ = aλref e-S(λ – λref)

(Eq. 13)

An additional factor influencing the rate of TiO2 photocatalytic degradation is the

presence of positive and negative ions. The point of zero charge, pHPZC, for anatase TiO2 was

approximately 5.3 -5.6 (Kosmulski 2009). All water matrices used in the experiment had

pH~7.0; therefore, at the pH greater than PZC, an excess of negative charge was present on the

TiO2 surface (Cheng and Selloni, 2010). In seawater samples, this negative charge can influence

the behavior of ions in multiple ways. It could lower the concentration of anions around the TiO2

surface by electrostatic repulsion, increasing the concentration of cations in that area. Reaction of

cations with OH-, that could have potentially formed hydroxyl radicals, can decrease the rate of

degradation (Khan et al., 2010). Another possibility is that the anions compete with OH- for

adsorption on TiO2 surface lowering the chances of hydroxyl radical formation (Khan et al.,

30

2010). Finally, hydrogen carbonate (HCO3-) present in natural waters has a detrimental effect on

photodegradation rates of water contaminants because it efficiently scavenges ˙OH radicals

(Bahnemann et al., 1994). Although sucralose degradation in artificial seawater has been shown

to be successful in this research (>90%, Fig. 13b, 15b, 18), degradation in Wrightsville Beach

water resulted in only 15% decrease in sucralose concentration (Fig. 15b). This can potentially

be explained by the fact that salinity of ASW was 32‰ compared with 39‰ in WB (Table 4).

Therefore, higher concentration of ions in WB water could have resulted in more interference

with hydroxyl radical formation or its scavenging.

Successful degradation (100%) of sucralose was achieved in effluent water (Fig. 15b).

This suggests an effective way of treating the contaminant at its source is incorporating TiO2/UV

photocatalysis by WWTP. Although employing TiO2/UV system by most plants has technical

drawbacks, i.e. energy efficiency (Hernando et al., 2007), the demand for more successful

methods for removal of water contaminants is increasing and this advanced oxidation process

could prove to be valuable.

Biological Impact

By examining different types of phytoplankton, the study meant to establish better

understanding of sucralose’s biological impact on the species of a potential risk. It has been

suggested that sucralose can potentially interfere with algae photosynthesis and may shut down

their CO2 uptake (Lubick, 2008). Since the sweetener was found in marine coastal waters,

diatoms and dinoflagellates species were of significant importance to this experiment. Diatoms

are the major contributors to primary production in coastal waters. Although they are widespread

in the oceans, they are most abundant in colder, nutrient-rich, nearshore waters. Their walls are

built of silica and pectin and they float in the water or attach to surfaces as single cells or chains.

31

Dinoflagellates are responsible for the formation of red tides. They are second to diatoms

contributors in primary production and are mostly found in nutrient-poor waters offshore. Most

are unicellular and usually equipped with two flagella enabling them to move. Coccolithophorids

are most abundant in open ocean waters and their single cells are covered with calcium carbonate

platelets called coccoliths (Smith, 1977). Emiliania huxleyi is the most abundant specie of

coccolithophorids and can potentially be found in the Gulf Stream where low sucralose levels

were detected (Mead et al., 2009). Green algae were selected because they are most closely

related to the higher plants, for which sucralose concentrations were shown to inhibit sucrose

transport (Reinders et al., 2006). The representative specie D. tertiolecta used in this study is a

marine unicellular flagellate although green algae are also commonly found in the freshwaters.

Daily cell counts of species’ batch cultures were recorded over a two week period and plotted to

represent algal growth curves (Fig. 20). The statistical analysis of the exponential growth slopes

from the logarithmic graphs (Fig. 21) indicated no difference between experimental (exposed to

sucralose) and control (no sucralose exposure) samples of all four species. The concentration of

sucralose in the T. weiss. medium at the end of the experiment was unchanged compared to the

concentration at the beginning of the experiment (Fig. 22), suggesting no sucralose uptake by

algal cells.

Although results from both experiments did not show any type of sucralose effect on

marine phytoplankton, this does not mean an effect does not exist. Additional research that

includes other species, longer experimentation times, or tests with a mixture of contaminants,

that can concomitantly with sucralose be present in the environment, may result in different data.

On the contrary, sucralose was designed to have no biological effects in humans and therefore it

may not cause any biological effects in non-target organisms.

32

IMPLICATIONS

The results of this study are important because they demonstrate that sucralose is present

in relatively high concentrations in treated waters, surface fresh and marine waters, with no

noticeable sorption of the sweetener onto the sediment. The fact that sucralose had no effect on

the growth of selected marine phytoplankton species, shows that occurrence itself does not

necessarily indicate that sucralose is harmful to any biological systems; however, potential risks

of its transformations are still unclear. The results show that sucralose doesn’t undergo direct

photodegradation, but it does photodegrade (15-20%) in the presence of the humic substances

into unknown possibly chlorinated products. This is of significant importance since humic

substances are the major constituents of natural waters and products formed during

photochemical processes can potentially be more toxic than the parent compound or at least

retain its properties. Therefore, isolation and characterization of sucralose photoproducts should

be an integral part of further research on sucralose’s effect on environmental ecosystems.

The present study also reveals that sucralose can be degraded via hydroxyl radicals

mediated process during heterogeneous photocatalysis involving UV/TiO2. The UV/TiO2 system

is one of the advanced oxidation processes used in removal of organic water contaminants and its

90 – 100% efficiency in the breakdown of sucralose makes it a viable option for wastewater

treatment plants to eliminate this contaminant of emerging concern before it enters the

environment, where its persistence and bioaccumulation can bring unforeseen effects.

33

Table 1. Examples of pharmaceuticals and other organic compounds that have been shown to

occur in the aqueous systems.

Substance

Concentration

(µg/L) Sample Type Reference

carbamazepine 0.258 drinking water Stackelberg et al. 2004

up to 6.3 wastewater Ternes, 1998

caffeine 0.119 drinking water Stackelberg et al. 2004

up to 0.16 open sea Weigel et al. 2002

clofibric acid up to 0.001 open sea Weigel et al. 2002

bisphenol A 0.42 drinking water Stackelberg et al. 2004

diclofenac 0.0011 - 15.033 freshawater Jux et al. 2002

estrogens (E1, E2, E3,

EE2) 0.009 - 0.073 freshawater Kolpin et al. 2002

ibuprofen <0.002 - 5.044 freshawater Ashton et al. 2004

<0.008 - 0.698 estuarines Thomas and Hilton, 2002

0.05 -7.11 wastewater Andreozzi et al. 2003

naproxen 0.8 -2.6 wastewater Andreozzi et al. 2003

trimethoprim < 0.004 - 0.569 estuarines Thomas and Hilton, 2002

34

Table 2. Examples of concentrations of common artificial sweeteners found in the

environment.

Concentration (µg/L) Sample Matrix References

acesulfame 20 wastewater Scheurer et al., 2009

14 -46 wastewater Buerge et al., 2009

2 surface freshwater Scheurer et al., 2009

2.8 surface freshwater Buerge et al., 2009

saccharin 2.8 wastewater Scheurer et al., 2009

0.27 - 3.2 wastewater Buerge et al., 2009

0.050 - 0.15 surface freshwater Scheurer et al., 2009

0.18 surface freshwater Buerge et al., 2009

cyclamate 2.8 wastewater Scheurer et al., 2009

0.11 - 0.82 wastewater Buerge et al., 2009

0.050 - 0.15 surface freshwater Scheurer et al., 2009

0.13 surface freshwater Buerge et al., 2009

35

Table 3. Examples of sucralose concentrations in environment reported in literature.

Concentration (µg/L) Sample Matrix References

2.2 - 5.9 wastewater Green et al. 2007

2 - 8.8 wastewater Buerge et al. 2009

0.6 surface freshwater Buerge et al. 2009

4.9 surface freshwater Brorström-Lunden et al. 2008

up to 1.0 surface freshwater Loose et al. 2009

0.4 - 0.9 effluent receiving waters Brorström-Lunden et al. 2008

0.018 - 0.030 seawater Green et al. 2007

36

Figure 1. Structure of sucrose - table sugar (A) and sucralose (B).

37

Figure 2. Hydrolysis products of sucralose

38

Figure 3. Microbial transformation products of sucralose: A – unsaturated aldehyde of

sucralose; B – sucralose uronic acid; C – 1,6-dichloro-l,6-dideoxy-D-fructose.

39

Figure 4. Sucralose absorbance spectrum (a); enlarged view (b)

(a)

(b)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

150 250 350 450 550 650 750

Abso

rban

ce

Wavelength (nm)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

180 190 200 210 220 230 240 250

Abso

rban

ce

Wavelength (nm)

λ max = 193 @ A = 1.20

40

Table 4. Concentration of sucralose found at various locations of the current research.

Water Source Collection Site Date

collected

Sample

Depth Latitude Longitude Salinity

(‰)

Concentration

(µg/L)

WWTP Effluent Northside 1/16/2008 34 15.5 N -77 55.2 W 0 119

7/21/2009 34 15.5 N -77 55.2 W 46

Southside 7/21/2009 34 09.6 N -77 56.5 W 40

LCFR HB 2/13/2009 1 m 34 17.1 N -77 57.2 W 2.0 2.07

M70 2/13/2009 1 m 34 14.4 N -77 57.4 W 3.13

M70 2/13/2009 15 m 1.42

M61 2/13/2009 1 m 34 11.6 N -77 57.4 W 7.5 0.94

M54 2/13/2009 1 m 34 08.3 N -77 56.7 W 13.0 0.45

M54 2/13/2009 15 m 1.47

M42 2/13/2009 1 m 34 05.4 N -77 56.0 W 17.5 0.27

M23 2/13/2009 1 m 24.5 0.14

M18 2/13/2009 1 m 33 54.7 N -78 01.0 W 28.0 0.52

M18 2/13/2009 15 m 0.19

Fresh Waters NC

Hanging Rock State Park

Creek

3/10/2009 1 m 36 23.5 N -80 16.1 W N.A.

Hanging Rock State Park

Lake

3/10/2009 1 m 36 23.3 N -80 16.1 W 2.06

Jordan Lake 3/11/2009 1 m 35 44.0 N -79 01.1 W N.A.

Haw River, Saxapahaw 3/11/2009 1 m 35 56.5 N -79 19.2 W 1.35

Potomac River,

Washington DC

8/16/2010 0.5 m 38 86.6 N -77 03.8 W 1.46

41

Figure 5. Mass spectra of sucralose (the identifying ions at m/z 308 and 361).

42

Figure 6. GC chromatogram of sucralose.

43

Figure 7. Natural sunlight vs solar simulator comparison spectrum.

0

50

100

150

200

250

280 380 480 580 680 780

wavelength (nm)

solar simulator

sun full spectrum

Inte

nsit

y (m

W/c

m2)

44

Table 5. Method optimization experimental results.

Individual extractions Phase % recovery

MilliQ HLB 90

C18 2.8

Diol 0

Cyano 0

HLB 62a

C18 70a

C18 83ab

C18 on top of HLB but eluted separately

MilliQ HLB 23

C18 37

CFR HLB 0. 14 ng/uL

C18 0.20 ng/uL

a no 5% MeOH rinse

b 50 mL sample volume

45

Figure 8. Extraction volume experiments (duplicates).

y = 60754x + 170413R² = 0.9975

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

7.00E+06

0 20 40 60 80 100 120

Peak

Are

a

Volume Extracted (mL)

Experiment 1

y = 47201x - 11712R² = 0.9973

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

0 20 40 60 80 100 120

Peak

Are

a

Volume Extracted (mL)

Experiment 2

46

Figure 9. Linearity of the calibration curve for the current method.

y = 189712x - 18749R² = 0.993

0.00E+00

1.00E+05

2.00E+05

3.00E+05

4.00E+05

5.00E+05

0 0.5 1 1.5 2 2.5 3

Pe

ak A

rea

Sucralose (ng/µL)

47

Figure 10. Sucralose concentration in water aliquots measured during experiment on sucralose

sorption to natural, high in organic matter sediment.

0

0.5

1

1.5

2

2.5

24 h week 1 week 2 week 3

Sucr

alose

conce

ntr

atio

n (

ng/µ

L)

no sediment

sediment

48

Figure 11. Sucralose concentration in water aliquots measured during experiment on sucralose

sorption to montmorillonite clay, low in organic matter sediment.

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

3.50E+06

4.00E+06

No clay Clay 1 Clay 2

Pea

k A

rea

49

Figure 12. Effect of simulated sunlight on the degradation of sucralose in MilliQ and ASW

without the TiO2 photocatalyst.

0

20

40

60

80

100

MilliQ ASW

Ram

ainin

g o

f S

ucr

alose

(%

)

Dark

Light

50

Figure 13. Effect of humic substances on sucralose degradation under simulated sunlight in ASW

without the TiO2 photocatalyst (a) and with the TiO2 photocatalyst (b). PART I

(a)

(b)

0

20

40

60

80

100

0.01 mg HA/L 0.1 mg HA/L 1.0 mg HA/L

Rem

ainin

g S

ucr

alo

se (

%)

Dark

Light

0

20

40

60

80

100

0.01 mg HA/L 0.1 mg HA/L 1.0 mg HA/L

Rem

ainin

g S

ucr

alose

(%

)

Dark

Light

51

Figure 14. Effect of humic substances on sucralose degradation under simulated sunlight in

ASW without the TiO2 photocatalyst (a) and with the TiO2 photocatalyst (b). PART II

(a)

(b)

0

5

10

15

20

0 0.2 0.4 0.6 0.8 1

% d

egra

dat

ion

[Humic Acid]

60

70

80

90

100

0 0.2 0.4 0.6 0.8 1

% d

egra

dat

ion

[Humic Acid]

52

Figure 15. Effect of simulated sunlight on the degradation of sucralose in different matrices

without the TiO2 photocatalyst (a) and with the TiO2 photocatalyst (b).

(a)

(b)

0

20

40

60

80

100

MilliQ ASW WB CFR Effluent

Rem

ainin

g o

f S

ucr

alo

se (

%)

Dark

Light

0

20

40

60

80

100

MilliQ ASW WB CFR Effluent

Rem

ainin

g o

f S

ucr

alo

se (

%)

Dark

Light

53

Figure 16. UV-Vis absorbance spectrum of different water matrices (a); enlarged view (b).

(a)

(b)

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

200 300 400 500 600 700

Abso

rban

ce

Wavelength (nm)

ASW Effluent WB CFR

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

240 290 340 390

Abso

rban

ce

Wavelength (nm)

ASW Effluent WB CFR

54

Figure 17. Effect of methanol as a hydroxyl radical scavenger on sucralose degradation in natural

waters without the TiO2 photocatalyst.

0

20

40

60

80

100

WB without

methanol spike

WB with

methanol spike

CFRW without

methanol spike

CFRW with

methanol spike

Rem

ainin

g o

f S

ucr

alose

(%

)

55

Table 6. Absorbance of different water matrices at 300 nm wavelength and spectral slopes

calculated for 240-400 nm wavelength range.

A300 S (nm-1

)

WB 0.006 0.0188

CFR 0.534 0.0118

Effluent 0.12 0.0132

56

Figure 18. Effect of simulated sunlight on degradation of sucralose in MilliQ and ASW with

and without TiO2 photocatalyst.

0

20

40

60

80

100

MilliQ ASW MilliQ ASW

Rem

ainin

g o

f S

ucr

alose

(%

)

without TiO2 with TiO2

Dark

Light

57

Figure 19. Effect of methanol as a hydroxyl radical scavenger on sucralose degradation with

TiO2 photocatalyst in ASW.

0

20

40

60

80

100

sample spiked with

sucralose/MQ standard

sample spiked with

Sucralose/MeOH standard

(10mM MeOH)

Rem

ainin

g o

f S

ucr

alose

(%

)

Dark

Light

58

Figure 20. Daily cell counts of four algal species in the experimental samples with sucralose

(Exp. 1, 2, 3) and control samples without sucralose (Contr. 1, 2, 3).

0.00E+00

5.00E+04

1.00E+05

1.50E+05

2.00E+05

2.50E+05

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Num

ber

of

cell

s/m

L

Days

Culture growth of Thalasiossira weissflogii

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

0 2 4 6 8 10 12 14

Num

ber

of

cell

s/m

L

Days

Culture growth of Heterocapsa triquetra

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

59

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

0 2 4 6 8 10 12 14

Num

ber

of

cell

s/m

L

Days

Culture growth of Emiliania huxleyi

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

3.00E+06

0 2 4 6 8 10 12 14

Num

ber

of

cell

s/m

L

Days

Culture growth of Dunaliella tertiolecta

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

60

Figure 21. Representation of the exponential growth four algal species in the experimental

samples with sucralose (Exp. 1, 2, 3) and control samples without sucralose (Contr. 1, 2, 3).

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

log (

# o

f ce

lls/

mL

)

Days

Thalasiossira weissflogii

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 2 4 6 8 10 12 14

log (

# o

f ce

lls/

mL

)

Days

Heterocapsa triquetra

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

61

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0 2 4 6 8 10 12 14

log (

# o

f ce

lls/

mL

)

Days

Emiliania huxleyi

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 2 4 6 8 10 12 14

log (

# o

f ce

lls/

mL

)

Days

Dunaliella tertiolecta

Exp 1 Exp 2 Exp 3 Contr. 1 Contr. 2 Contr. 3

62

Figure 22. Initial and final sucralose concentration in the medium of T. weiss.

0.00E+00

2.00E+06

4.00E+06

6.00E+06

8.00E+06

1.00E+07

Initial Final

Pea

k A

rea

63

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APPENIDX

Appendix A. Artificial seawater (ASW) recipe was adapted from The Biological Bulletin

Compendia “Physiological Solutions”, published by The Marine Biological Laboratory (MBL).

“MBL” Formula ASW #1

Ingredients Formula Weight (FW)

Grams/Liter

NaCl 58.44 24.72

KCl 74.56 0.67

CaCl2 (anhydrous) 110.99 1.03

MgCl2 (anhydrous) 95.22 2.18

MgSO4.7H2O 246.50 6.29

NaHCO3 84.01 0.18