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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
REFERENCES
Andreozzi, R., Marotta, R., Pinto, G., Pollio, A., 2002. Carbamazepine in water: persistence in
the environment, ozonation treatment and preliminary assessment on algal toxicity. Water
Research 36, 2869-2877.
Ashton, D., Hilton, M., Thomas, K.V., 2004. Investigating the environmental transport of human
pharmaceuticals to streams in the United Kingdom. Sci. Total Environ. 333, 167-184.
Arnold, W.A., McNeill, K., 2007. Transformation of pharmaceuticals in the environment:
Photolysis and other abiotic processes. Comprehensive Analytical Chemistry 50, 361-
385.
Avery, G.B., Jr., Willey, J.D., Kieber, R.J., Shank, G.C., Whitehead, R.F., 2003. Flux and
bioavailability of Cape Fear River and rainwater dissolved organic carbon to Long Bay,
southeastern United States. Global Biogeochem. Cycles 17, 1042.
Bachman, J., Patterson, H.H., 1999. Photodecomposition of the Carbamate Pestisicde
Carbofuran: Kinetics and the Influence of Dissolved Organic Matter. Environ. Sci.
Technol. 33, 874-881.
Bahnemann, D., Cunningham, J., Fox, M.A., Pelizzetti, E., Pichat, P., Serpone, N., 1994. In
Helz, G.R., Zepp, R.G., Crosby, D.G. (Eds.), Aquatic and Surface Photochemistry
(p.276). Boca Raton: Lewis Publishers.
Brorström-Lunden, E., Svenson, A., Viktor, T., Woldegiorgis, A., Remberger, M., Kaj, L., Dye,
C., Bjerke, A., Schlabach, M., 2008. Measurements of Sucralose in the Swedish
Screening Program 2007, PART I. Swedish Environmental Research Institute Ltd.
Brorström-Lunden, E., Svenson, A., Viktor, T., Woldegiorgis, A., Remberger, M., Kaj, L., Dye,
C., Bjerke, A., Schlabach, M., 2008. Measurements of Sucralose in the Swedish
Screening Program 2007, PART II. Swedish Environmental Research Institute Ltd.
Buerge, I.J., Buser, H.R., Kahle, M., Müller, M.D., Poiger, T., 2009. Ubiquitous occurrence of
the artificial sweetener acesulfame in aquatic environment: An ideal chemical marker of
domestic wastewater in groundwater. Environ. Sci. Technol. 43, 4381-4385.
Cheng, H., Selloni, A., 2010. Hydroxide Ions at the Water/Anatase TiO2 (101) Interface:
Structure and Electronic States from First Principles Molecular Dynamics. Langmuir 26,
11518-11525.
Choy, W.K., Chu, W., 2005. Destruction of o-Chloroaniline in UV/TiO2 Reaction with
Photosensitizing Additives. Ind. Eng. Chem. Res. 44, 8184-8189.
64
Cooper, W.J., Cramer, C.J., Martin, N.H., Mezyk, S.P., O’Shea, K.E., von Sonntag, C., 2009.
Free radical mechanisms for the treatment of tert-butyl ether (MTBE) via advanced
oxidation/reductive processes in aqueous solutions. Chem. Rev. 109, 1302-1345.
DeLorenzo, M.E., Fleming, J., 2008. Individual and Mixture Effects of Selected Pharmaceuticals
and Personal Care Products on the Marine Phytoplankton Species Dunaliella tertiolecta.
Arch. Environ. Contam. Toxicol. 54, 203-210.
European Commission, Health and Consumer Protection Directorate-General, 2000. Opinion of
the Scientific Committee on Food on sucralose.
Fleeger, J.W., Carman, K.R., Nisbet, R.M. 2003. Indirect effects of contaminants in aquatic
ecosysytems. Sci. Total Environ. 317, 207-233.
Frimmel, F.H., 1994. Photochemical aspects related to humic substances. Environment
International 20, 373-385.
Garbin, J.R., Milori, D.M.B.P., Simões, M.L., da Silva, W.T.L, Neto, L.M., 2007. Influence of
humic substances on the photolysis of aqueous pesticide residues. Chemosphere 66,
1692-1698.
Gao, r., Stark, J., Bahnemann, D.W., Rabani, J., 2002. Quantum yields of hydroxyl radicals in
illuminated TiO2 nanocrystallite layers. Photochem Photobiolo. A Chem. 148, 387-391.
Giger, W., 2008. Hydrophilic and amphiphilic water pollutants: using advanced analytical
methods for classic and emerging contaminants. Anal. Bioanal. Chem. 393, 37-44.
Gómez, M.J., Sirtori, C., Mezuca, M., Fernández-Alba, A.R., Agüera, A., 2008.
Photodegradation study of three dipyrone metabolites in various water systems:
Identification and toxicity of their photodegradation products. Water Research 42, 2698-
2706.
Green, N., Schlabach, M., Bakke, T., Brevik, E.M., Dye, C., Herzke, D., Huber, S., Plosz, B.,
Remberger,M., Schøyen, M., Uggerud, H., T., Vogelsang, C., 2007. Screening of selected
metals and new organic contaminants 2007. Norwegian Pollution Control Authority.
Green, S.A., Blough, N.V., 1994. Optical absorption and fluorescence properties of
Chromophoric dissolved organic matter in natural waters. Limnol. Oceanogr. 39, 1903-
1916.
Grice, H.C., Goldsmith, L.A., 2000. Sucralose-An overview of the toxicity data. Food Chem.
Toxicol. 38, S1-S36.
Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In Smith
W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals (pp. 26-60). New
York, Plenum Press.
65
Guillard, R.R.L, and Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella nana
Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8, 229-239.
Helms, J.R., Stubbins, A., Ritchie, J.D., Minor, E.C., 2008. Absorption spectral slopes and slope
ratios as indicators of molecular weight, source, and photobleaching of chromophoric
dissolved organic matter. Limnol. Oceanogr. 53, 955-969.
Helz, G.R., Zepp, R.G., Crosby, D.G., 1994. Aquatic and Surface Photochemistry (p. 451-452).
Boca Raton: Lewis Publishers.
Hernando, M.D., Petrovic, J., Fernández-Alba, A.R., Barceló, D., 2007. Removal of
pharmaceuticals by advanced treatment technologies. Comprehensive Analytical
Chemistry 50, 451-474.
Jenner, M.R., Smithson, A., 1989. Physicochemical Properties of the Sweetener Sucralose. Food
Sci. 54, 1646-1649.
Jiao, S., Zheng, S., Yin, D., Wang, L., Chen, L., 2008. Aqueous photolysis of tetracycline and
toxicity of photolytic products to luminescent bacteria. Chemosphere 73, 377-382.
Jux, U., Baginski, R.M., Hans-Günter, A., Krönke, M., Seng, P.N., 2002. Detection of
pharmaceutical contaminants of river, pond, and tap water from Cologne (Germany) and
surroundings. Int. J. Hyg. Environ. Health 205, 393-398.
Khan, U., Benabderrazik, Bourdelais, A.J., Baden, D.G., Rein, K., Gardinali, P.R., Arroyo, L.,
O’Shea, K.E., 2010. UV and solar TiO2 photocatalysis of brevetoxins (PbTxs). Toxicon
55, 1008-1016.
Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton,
H.T., 2002. Pharmaceuticals, Hormons, and Other Organic Wastewater Contaminants in
U.S. Streams, 1999-2000: A National Reconnaissance. Environ. Sci. Technol. 36, 1202-
1211.
Kosmulski, M., 2009. Compilation of PZC and IEP of sparingly soluble metal oxide and
hydroxides from literature. Advances in Colloid and Interface Science 152, 14-25.
Labare, M. P., Alexander, M., 1993. Biodegradation of sucralose, a chlorinated carbohydrate, in
samples of natural environment. Environ. Toxicol. Chem. 12, 797-804.
Labare, M.P., Alexander, M., 1994. Microbial cometabolisim of sucralsoe, a chlorinated
disaccharide, in environmental samples. J. Appl. Microbiol. 42, 173-178.
Lapin-Scott, H.M., Holt, G., Bull, A.T., 1987. Microbial transofromation of 1,6-dichloro-1,6-
dideoxy-β,D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside (TGS) by soil. J.
Appl. Microbiol. 3, 95-102.
66
Loos, R., Gawlick, B.M., Boettcher, K., Locoro, G., Contini, S., Bidoglio, G., 2009. Sucralose
screening in European surface waters using solid-phase extraction-liquid
chromatography-triple quadrupole mass spectrometry method. J. Chromatogr. A 1216,
1126-1131.
Lubick, N., 2008. Artificial sweetner persists in the environment. Environ. Sci. Technol.42,
3125-3125.
Marugán, J., Hufschmidt, D., López-Munoz, Selzer, V., Bahnemann, D., 2006. Photonic
efficiency for methanol photooxidation and hydroxyl radical generation on silica-
supported TiO2 photocatalysts. Appl. Catal. B: Environ. 62, 201-207.
Mead, R.N., Morgan, J.B., Avery, G.B., Kieber, R.J., Kirk, A.M., Skrabal, S.A., Willey, J.D.,
2009. Occurrence of the artificial sweetener sucralose in coastal and marine waters of the
Unitated States. Marine Chemistry 116, 13-17.
Metcalfe, C.D., Metcalfe, T.L., Kiparissis, Y., Koenig, B.G., Khan, C., Hughes, R.J., Croley,
T.R., March, R.E., Potter, T., 2001. Estrogenic potency of chemicals detected in sewage
treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias
latipes). Environ. Toxicol. Chem. 20, 297-308.
Musto, C.J., Lim, S.H., Suslick, K.S., 2009. Colorimetric Detection and Identification of Natural
and Artificial Sweeteners. Anal. Chem. 81, 6526-6533.
Pérez-Estrada, L.A., Agüera, A., Hernando, M.D., Malato, S., Fernández-Alba, A.R., 2008.
Photodegradation of malachite green under natural sunlight irradiation: Kinetic and
toxicity of the transformation products. Chemosphere 70, 2068-2075.
Richardson, S.D., 2009. Water Analysis: Emerginng Contaminants and Current Issues. Anal.
Chem. 81, 4645-4677.
Reinders, A., Sivitz, A.B., His, A., Grof, C.P.L., Perroux, J.M., Ward, J.M., 2006. Sugarcane
ShSUT1: analysis of sucrose transport activity and inhibition by sucralose. Plant Cell
Environ. 29, 1871-1880.
Roberts, A., Renwick, A.G., Sims, J., Snodin, D.J., 2000. Sucralose metabolism and
pharmokinetics in man. Food Chem. Toxicol. 38, S31-S41.
Schreurer, M., Brauch, H.J., Lange, F.T., 2009. Analysis and occurrence of seven artificial
sweeteners in German waste water and surface water and in soil aquifer treatment (SAT).
Anal. Bioanal. Chem. 394, 1585-1594.
Simmons, M.S., Zepp, R.G., 1986. Influence of humic substances on photolysis of nitroaromatic
compounds in aqueous systems. Water Research 20, 899-904.
67
Smith, D.L., 1977. Guide to Marine Coastal Plankton and Marine Invertebrate Larvae.
Dubuque, Iowa: Kendall/Hunt.
Stackelberg, P.E., Furlong, E.T., Meyer, M.T., Zaugg, S.D., Henderson, A.K., Reissman, D.B.,
2004. Persistence of pharmaceutical compounds and other organic wastewater
contaminants in a conventional drinking-water-treatment plant. Sci. Total Environ. 329,
99-113.
Ternes, T.A., 1998. Occurrence of drugs in German sewage treatment plants and rivers. Wat.
Res. 32, 3245-3260.
Thomas, K.V., Hilton, M.J., 2004. The occurrence of selected human pharmaceutical compounds
in UK estuaries. Marine Pollution Vuletin 49, 436-444.
Vialaton, D., Richard, C., Baglio, D., Paya-perez, A., 1998. Phototransformation of 4-chloro-2-
methylphenol in water: influence of humic substances on the reaction. Photochem.
Photobiolo. A: Chem. 119, 39-45.
Wang, C., Rabani, J., Bahnemann, D.W., Dohrmann, J.K., 2002. Photonic efficiency and
quantum yield of formaldehyde formation from methnol in the presence of various TiO2
photocatalysts. Photochem. Photobiolo. A: Chem. 148, 169-176.
Weigel, S., Kuhlmann, J., Hühnerfuss, H., 2002. Drugs and personal care products as ubiquitous
pollutants: occurrence and distribution of clofibric acid, caffeine and DEET in the North
Sea. Sci. Total Environ. 2002, 131-141.
Welker, M., Steinberg, C., 2000. Rates of Humimc Substance Photosensitized Degradation of
Microcystin-LR in Natural Waters. Environ. Sci. Technol. 34, 3415-3419.
Xing, B., Pignatello, J.J., Gigliotti, B., 1996. Competitive Sorption between Atrazine and Other
Organic Compounds in Soils and Model Sorbents. Environ. Sci. Technol. 30, 2432-2440.
Zeng, K., Hwang, H.M., Yu, H., 2002. Effect of dissolved humic substances on the
photochemical degradation rate of 1-aminopyrene and atrazine. Int. J. Mol. Sci. 3 (10),
1048-1057.
Zepp, R.G., Schlotzhauer, P.F., Sink, R.M., 1985. Photosentisized Transformations Involving
Electronic Energy Transfer in Natural Waters: Role of Humic Substances. Environ. Sci.
Technol. 19, 74-81.
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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