- Email: [email protected]
Photocatalytic transformation of acesulfame: Transformation products identification and embryotoxicity study
Accepted Manuscript Photocatalytic transformation of acesulfame: transformation products identification and embryotoxicity study Adela Jing Li, Oliver J. Schmitz, Susanne Stephan, Claudia Lenzen, Patrick Ying-Kit Yue, Kaibin Li, Huashou Li, Kelvin Sze-Yin Leung PII:
S0043-1354(15)30362-6
DOI:
10.1016/j.watres.2015.11.035
Reference:
WR 11662
To appear in:
Water Research
Received Date: 9 July 2015 Revised Date:
10 November 2015
Accepted Date: 14 November 2015
Please cite this article as: Li, A.J., Schmitz, O.J., Stephan, S., Lenzen, C., Yue, P.Y.-K., Li, K., Li, H., Leung, K.S.-Y., Photocatalytic transformation of acesulfame: transformation products identification and embryotoxicity study, Water Research (2015), doi: 10.1016/j.watres.2015.11.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Photocatalytic transformation of acesulfame: transformation products
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identification and embryotoxicity study
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Adela Jing Li1,3, Oliver J. Schmitz4, Susanne Stephan4, Claudia Lenzen4, Patrick Ying-Kit
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Yue5, Kaibin Li6, Huashou Li3, Kelvin Sze-Yin Leung*1,2
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Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong
Special Administrative Region
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Baptist University, Kowloon Tong, Hong Kong Special Administrative Region
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China Agricultural University, Guangzhou 510642, China
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Germany
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Administrative Region
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Partner State Key Laboratory of Environmental and Biological Analysis, Hong Kong
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Key Laboratory of Tropical Agro-environment, Ministry of Agriculture of China, South
Applied Analytical Chemistry, Faculty of Chemistry, University of Duisburg-Essen, Essen,
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special
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Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380,
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China
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Key Laboratory of Tropical and Subtropical Fish Breeding & Cultivation, Pearl River
*Corresponding author. Tel.: +852 34115297; fax: +852 34117348
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E-mail address: [email protected] (Kelvin S.-Y. Leung)
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Abstract
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Artificial sweeteners have been recognized as emerging contaminants due to their wide
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application, environmental persistence and ubiquitous occurrence. Among them, acesulfame
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has attracted much attention. After being discharged into the environment, acesulfame
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undergoes photolysis naturally. However, acesulfame photodegradation behavior and identity
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of its transformation products, critical to understanding acesulfame’s environmental impact,
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have not been thoroughly investigated. The present study aimed to fill this knowledge gap by
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a laboratory simulation study in examining acesulfame transformation products and pathways
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under UV-C photolysis in the presence of TiO2. Photodegradation products of acesulfame
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were isolated and analyzed using the LC-IM-QTOF-MS coupled with LC Ion Trap MS in the
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MSn mode. Our results show six new transformation products that have not been previously
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identified. The molecular structures and transformation pathways were proposed. Further
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embryotoxicity tests showed that acesulfame transformation products at the low g L-1 level
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produced significant adverse effects in tail detachment, heart rate, hatching rate and survival
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rate during fish embryo development. The identification of additional transformation
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products with proposed transformation pathways of acesulfame, the increased toxicity of
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acesulfame after photolysis, and the fact that the accumulation of acesulfame transformation
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products is increasingly likely make acesulfame contamination even more important. Water
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resource control agencies need to consider legislation regarding acesulfame and other
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artificial sweeteners, while further studies are carried out, in order to protect the safety of this
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most vital resource.
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Keywords: acesulfame, emerging contaminant, catalytic UV irradiation, transformation
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product, transformation pathway, embryotoxicity
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1. Introduction
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Artificial sweeteners (ASs) have been consumed in considerable quantities as sugar
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substitutes in low-calorie beverages, foods and personal care products since the 1950s
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(Buerge et al., 2011; Berset and Ochsenbein, 2012). They are a class of emerging
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environmental contaminants with growing scientific concern due to their potential
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undesirable impacts on ecosystems and human health (Richardson and Ternes, 2014; Sang et
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al., 2014; Tran et al., 2014). Among the ASs, acesulfame (ACE) has drawn much attention
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due to its world-wide consumption, persistence when released, and subsequent ubiquitous
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occurrence in the natural environment (Nӧdler, et al., 2013; Tran et al., 2013). Available data
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indicates that the global consumption of ACE increased from 2.5 to 4.0 metric tons from
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2001 to 2005 due to its greater sweetening power and cheaper price compared to other ASs
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(Bahndorf and Kienle, 2004; ISO, 2008; Subedi and Kannan, 2014). The ingredient is applied
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in over 4000 foods and beverages in around 90 countries (Gisel, 2009).
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Due to human excretion mostly as the parent compound and high persistence in waste
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water even after treatment (Gan et al., 2013), levels of ACE were among the highest
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measured for trace pollutants in receiving waters of surface water, groundwater and
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wastewater influent and effluent (Table S1) and even tap water (Buerge et al., 2009;
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Mawhinney et al., 2011) with up to 100% detection frequency. Once released into nature,
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ACE may undergo different kinds of transformations. Stadler et al. (2012) suggest that
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estimating ACE removal rates by monitoring only the original compound will give distorted
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figures because it fails to take into consideration transformation products (TPs). This
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viewpoint is supported by findings of several other independent studies (Gan et al., 2014;
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ACCEPTED MANUSCRIPT Sang et al., 2014; Scheurer et al., 2012; 2014). Research has subsequently shown that ACE
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can be degraded under simulated solar/UV irradiation or ozone treatment into at least ten TPs.
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The transformation ACE undergoes in the natural environment is now considered to be quite
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complex.
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Identification of the TPs structures would be extremely valuable in understanding,
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including predicting, how they might behave in the environment. Our previous study showed
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that many photo by-products were produced from ACE under prolonged UV irradiation, and
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the transformation process yielded by-products more persistent than the original ACE (Sang
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et al., 2014).
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According to several toxicological tests, ACE appears to be nontoxic to humans within
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regulated concentrations (Shankar et al., 2013; Gardner, 2014), and poses a low hazard to
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green algae Scenedesmus vacuolatus, duckweed Lemna minor and water fleas Daphnia
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magna (Stolte et al., 2013). Nevertheless, the ubiquitous occurrence of ACE raises safety
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concern and call for further research, particularly in the aspect of ACE TPs (Lange et al.,
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2012; Toth et al., 2012; Stolte et al., 2013; Richardson and Ternes, 2014). The only earlier
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relevant work by us indicated that photo-induced TPs of ACE are > 500 times more toxic
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than the mother compound in the marine bacterium Vibrio fischeri (Sang et al., 2014). The
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current study, therefore, will further evaluate the risk led by those TPs.
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The primary objectives of this study were to search for and identify the molecular
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structure of any new photo-induced ACE TP; and to evaluate the embryotoxicity of the TPs in
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mixture by embryo toxicity testing (FET) using the zebrafish Danio rerio.
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2. Experimental
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2.1 Chemicals and reagents
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Chemical standard for the artificial sweetener acesulfame potassium was purchased from
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Sigma-Aldrich (≥ 99.0%, HPLC, Germany). The ACE stock solution of 400 mg L-1 was
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prepared with Milli-Q water of 18.2 ΩM cm (Millipore, Billerica, MA, USA) and stored in
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the dark at 4 ºC. Titanium (IV) oxide of 21 nm particle size was supplied by Sigma-Aldrich
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(TiO2, ≥ 99.5%, trace metals basis). For chromatographic analyses in HPLC, Milli-Q water
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and HPLC grade methanol (Tedia, OH, USA) were used to prepare the mobile phases.
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Reagents for embryotoxicity bioassay were prepared for reconstituted moderately hard
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water (MHW) using the U.S. Environmental Protection Agency recipe: 75.9 mg L-1 calcium
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sulfate dihydrate (CaSO4·2H2O, ≥ 99.0%, ISO9001:2000, China), 123.2 mg L-1 magnesium
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sulfate heptahydrate (MgSO4·7H2O, ≥ 99.0%, A.R., China), 96 mg L-1 sodium bicarbonate
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(NaHCO3, 99%, Sigma-Aldrich, China), 4 mg L-1 potassium chloride (KCl, ≥ 99.5%, A.R.,
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China). For all embryotoxicity testing, 2 times of MHW was used as test medium (Bone et al.,
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2012).
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2.2 Analysis of acesulfame transformation products
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2.2.1 TiO2-assisted photolysis experiment
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and without TiO2. The ACE (at a concentration of 400 mg/L) showed a 63.94% (n = 3)
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degradation without TiO2 while with a 99.99% (n = 3) in the presence of a catalyst.
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TiO2-assisted photodegradation was therefore adopted. Experimentally, 10 mg of ACE was
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dissolved in 25 mL of Milli-Q water. Freshly prepared TiO2 was then added to the solution at
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ACE/TiO2 mass ratio of 1:20. After the reaction under UV-C light (Sankyo G8T5, Japan;
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1630 µW/cm2) for 19 h at room temperature, the solution was filtered through 0.2 µm nylon
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membrane, and freeze-dried for use.
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2.2.2 Analytical methods
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Both ACE and its transformation products at 100 mg L-1 were analyzed by the Ion Mobility
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Q-TOF MS (Agilent 6560 IM Q-TOF) coupled with LC system (Agilent 1290 Infinity, USA)
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(LC-IM-QTOF-MS). Chromatographic separation was performed on a Phenomenex Luna 3µ
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CN column (150 × 2.0 mm, 3 µm) in the gradient elution model. The injection volume was
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1 µL. The mobile phase was composed of water and methanol, both containing 0.1% formic
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acid (98 – 100 %, Merck, Suprapur®). The flow rate was 0.2 mL/min. The gradient program
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started with 95% water for 5 min, followed by ramping to 50% water within 5 min, and then
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kept at this condition for 1 min, and then returned to the initial setup in 1 min. Q-TOF mode
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was applied with ion source of Dual AJS electrospray interface (ESI) with mass correction at
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reference masses of m/z 119.0363 and 966.0070. High resolution mass spectra (m/z 40 – 1700)
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were obtained at a rate of 2 spectra per second with electrospray ionization in the negative ion
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ACCEPTED MANUSCRIPT mode. Both positive and negative modes were initially applied to analyze samples, but only
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data from negative ion mode offered adequate information with allowance in detecting of
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potential TPs. Therefore, data derived from negative mode runs were focused for subsequent
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analyses. The dry gas of nitrogen flowed at 5 L/min under 200 ºC while sheath gas of
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nitrogen was at 12 L/min with 325 ºC, Vcap was set to 5000 V and the nozzle voltage to 500
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V.
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Specific mass of target transformation products was further fragmented by using the Ion
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Trap (Bruker amaZon speed) in the MSn mode coupled with HPLC (Thermal Scientific
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Accela). Chromatographic separation was performed under the same conditions for Q-TOF
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except with injection volume of 10 µL and m/z of 50 – 300. The fragmentation mass took
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place at energy of 2.78 V. Nitrogen was taken as dry gas with flow rate at 8.2 L/min and 350
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ºC. The nebulizer pressure was 15.0 psi.
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2.3 Embryotoxicity testing
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2.3.1 Animal care and breeding
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Adult zebrafish D. rerio were cultured in the Pearl River Fisheries Research Institute,
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Chinese Academy of Fishery Sciences, Guangzhou. In the laboratory, breeding stocks of
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males and females were maintained in separate glass aquaria filled with oxygenated tap water
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maintained at 26 ± 1 ºC and kept on a 14h:10h light:dark cycle. Fish were fed with Artemia
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franciscana nauplii. Before breeding the zebrafish in a ratio of 3:2 females: males, were kept
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in a tank with a board separating females from males, overnight. In the morning, when the
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light came on, the board was removed and the fish were allowed to breed. Embryos were
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collected after 30 minutes.
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2.3.2 Toxicity exposure
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The embryotoxicity tests of ACE and the preparation of ACE transformation products were
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carried out according to the method described by OECD (2013). Embryos of D. rerio of less
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than 32-cell blastomeres were exposed to testing compounds individually in 96-well plate
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(Thermo ScientificTM). Tests were performed with six dosage levels (100, 500, 1000, 5000,
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8500, and 10000 mg L-1). In each trial, 20 fish embryos were tested with 200 µL target
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solution, and three replicates of each dosage level were conducted. Positive and negative
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controls were performed in parallel by using 3, 4-dichloroaniline (4.0 mg L-1, 98%,
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Sigma-Aldrich) and 2 times of MHW, respectively. The criterion for positive embryotoxicity
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was more than 30% mortality after 96 h exposure, while that the criterion for negative control
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was overall survival and hatching rate of no less than 80%. The embryos were kept in an
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incubator (LRH-250, Shanghai) for 10 days with temperature maintained at 26 ± 1 ºC, pH
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8.2 - 8.3, oxygen ≥ 80% saturation and 14 h:10 h light:dark cycle. The entire test was
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repeated twice. Apical endpoints of tail-detachment, edema and coagulation after 24 h
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exposure, heart rate after 48 h exposure, hatching rate after 72 h exposure, and accumulative
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mortality were recorded.
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2.4 Statistical analysis
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data. To identify ACE TPs produced during photocatalytic transformation process,
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post-photolysis samples were compared against initial compound of ACE and the blank of
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Milli-Q water. For those reported ACE TPs, extracted m/z values were obtained based on
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previously reported m/z values. Further MSn characterization of ACE TPs was limited to the
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compounds within less than 5 ∆ppm deviation between experimental and exact m/z values.
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Data analyses in embryotoxicity tests were performed using SPSS version 19 (SPSS Inc.,
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Chicago) with mean ± standard deviation (s.d.). One-way analysis of variance (ANOVA)
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using chemical treatment as a fixed factor was used to infer the difference among chemical
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treatment groups for significance at the 5% level. If the null hypothesis was rejected by the
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ANOVA, different means were identified using Student-Newman-Keuls (SNK) test.
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3. Results and discussion
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3.1 Identification of transformation products
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UV irradiation at a wavelength 254 nm causes ACE degradation, producing a range of TPs.
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The laboratory simulated irradiation conditions including concentration of reactant,
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ACE:TiO2 ratio and reaction time were optimized through a series of experiments. It was
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found that the ACE TPs occur more easily with a higher yield at 400 mg L-1 of ACE,
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ACE:TiO2 1:20 (mass ratio), and a reaction time 19 h by UV-C irradiation, which
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corresponds to a fluence of about 1,115,000 J/m2. The TPs were concentrated and solidified
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by a freeze-drying process and then characterized by mass spectroscopic methods.
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ACCEPTED MANUSCRIPT In literature, ten TPs were identified and reported (Gan et al., 2014; Scheurer et al.,
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2014). In our work, the chemical structures of TPs were elucidated by LC-IM-QTOF-MS
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followed by Ion Trap MSn mode. Due to the high resolution mobility separation and low
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femtogram detection limits of LC-IM-QTOF-MS, this study detected far more TPs in the
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irradiated sample. Nine out of the ten previously reported ACE TPs were found, presenting
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almost exact masses to those previous studies (Table 1). Mass differences were in the range
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of -2.1 to 2.1 ppm. The 10th TP with m/z 225.9663 that reported in Gan et al. (2014) was not
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discovered in this study.
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However, in our work, we identified six additional TPs. Most of the TPs eluted earlier
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than the parent compound ACE under the adopted HPLC conditions. This finding is
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consistent with the study by Gan et al. (2014), indicating the TPs generated upon photolysis
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were generally more polar than ACE (MacManus-Spencer et al., 2011). These additional TPs
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with extracted ion chromatograms given in Fig. S1 were structurally elucidated by
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fragmentation of Ion Trap mass spectrometry (Table 1). The MSn fragmentation patterns,
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proposed chemical structures and transformation pathways of these TPs are illustrated in Fig.
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1. The 11th TP TP 137 (C3H5O4S)- with m/z 136.9914 was identified based on its fragment
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ions appearing at m/z 120 (C3H4O3S)-, 107 (C2H3O3S)-, 96 (O4S)-, 95 (CH3O3S)-, 94
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(CH2O3S)-, 91 (C2H3O2S)-, 80 (O3S)-, 78 (CH2O2S)-, 73 (C3H5O2)-, 65 (HO2S)- and 59
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(C2H3O2)- (Fig. 1a). TP 137 was speculated to be a degradate from ACE hydrolysate by loss
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of -NCO (Fig. 2). We propose that the ACE hydrolysate was formed by addition of water
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beyond the C=C bond, which is different from the proposed pathway in Gan et al. (2014).
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Whereas, Gan et al. (2014) suggested ACE underwent hydrolysis by the addition of water at
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ACCEPTED MANUSCRIPT the C=C bond, which offered an intermediate to yield a TP with m/z 136.0074 by
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photo-rearrangement. Differences in the pathways observed, or implied, in ACE
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photo-hydration may arise from differences in the experimental designs in the irradiation
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process. Firstly, light source may be a key factor. A xenon lamp of 1 kW with UV filters to
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maintain light > 290 nm was employed as a sunlight stimulator by Gan et al. (2014), while
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UV-C lamps of 128 W at 254 nm were used in the current work. Secondly, the photo-catalytic
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process may be a factor. While in this study TiO2 was applied in the photo-catalytic process,
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and it is known to have strong photo-catalytic activity (Tang et al., 2015). Thirdly, period of
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irradiation may count. A 7-d irradiation process was used in Gan et al. (2014)’s study whereas
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19 h UV-C irradiation was adopted in this study. Finally, testing condition disparity may play
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a role. This may explain why the TP with m/z 225.9663 reported in Gan et al. (2014) was not
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found in this study.
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The 12th TP, TP 154 with m/z 153.9816 (C2H4NO5S)- was proposed to have an open ring
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structure according to fragments determined at m/z 136 (C2H2NO4S)- and 96 (H2NO3S)- (Fig.
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1b). The 13th TP 168 ((C3H6NO5S)-, m/z 167.9972) was conceived based on four fragment
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ions, namely m/z 150 (C3H4NO4S)-, 124 (C2H6NO3S)-, 96 (H2NO3S)- and 81 (HO3S)- (Fig. 1c)
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while the 14th TP 193 ((C4H5N2O5S)-, m/z 192.9925) was inferred on fragment detections at
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m/z 166 (C3H4NO5S)- and 96 (H2NO3S)- (Fig. 1d). Furthermore, it is interesting that two TPs
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of the 15th and 16th having very close in mass, namely with m/z of 229.9799 (C4H8NO6S2)-
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and 229.9976 (C4H8NO8S)-, were extracted with apparently complicated structures. Based on
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the masses detected at m/z 149 (C4H7NO3S)- or (C4H5O4S)-, 148 (C4H6NO3S)-, 122
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(C2H4NO3S)-, 106 (CNO3S)-, 84 (C4H6NO)- and 71 (C3H5NO)-, the structure of m/z 229.9799
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ACCEPTED MANUSCRIPT (TP 230a) was tentatively proposed to include a closed heterocyclic ring (Fig. 1e). However,
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a possible pathway from ACE to this intricate structure was not successfully proposed, and
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hence requires further investigation. The structure of m/z 229.9976 (TP 230b) is estimated to
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be an open ring depending on fragment ions of m/z 212 (C4H6NO7S)-, 194 (C4H4NO6S)-, 166
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(C3H4NO5S)-, 124 (CH2NO4S)-, 97 (C4HO3)-, and 96 (H2NO3S)- (Fig. 1f). Ion of m/z 96
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(H2NO3S)- was a commonly detected fragment of ACE TPs, which is also reported by Gan et
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al. (2014). The overall proposed transformation pathways of ACE upon photolysis are given
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in Fig. 2. Hydrolysis and oxidation are proposed to be the two main degradation mechanisms
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in ACE photo-induced transformation.
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For a comprehensive environmental risk assessment of any contaminant entering natural
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waters, it is essential to consider not only the parent compound but also its TPs. Concern with
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regard to TPs of emerging contaminants has been growing exponentially in recent years.
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Studies of TPs have investigated their occurrence (Nӧdler et al., 2013; Bulloch et al., 2015),
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influencing factors (Ji et al., 2013; Gan et al., 2014; Liu et al., 2015), degradation kinetics
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(Vione et al., 2013; Scheurer et al., 2014), structure (Zhou et al., 2013; Gan et al., 2014;
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Godayol et al., 2015) and mitigation (Bahnmüller et al., 2015; Postigo and Barceló, 2015).
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Nonetheless, the limited toxicological data together with the wide occurrence of these
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transformation byproducts has generated significant uncertainty with regard to their risk to
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ecological systems. Biological testing integrated with chemical analysis is unquestionably
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needed to achieve accurate characterization of the pollutants (Díaz-Cruz and Barceló, 2009;
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Stolte et al., 2013; Gan et al., 2014; Bulloch et al., 2015). In view of this point, we undertook
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an embryotoxicity study of ACE TPs in mixture.
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3.2 Embryotoxicity Stolte et al. (2013) evaluated ecotoxicity of artificial sweeteners ACE, cyclamate,
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saccharin and sucralose as well as natural sweetener stevioside to activated sewage sludge
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respiration inhibition, green algae reproduction inhibition, water flea acute immobilization
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and duckweed growth inhibition, with the No Observed Effect Concentrations (NOECs) at
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1000 mg L-1. Similar observations of relatively high NOECs were also reported in other
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persistent organic pollutants such as sucralose (Huggett and Stoddard, 2011; Soh et al., 2011),
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perfluorooctane sulfonate and perfluorooctanoic (Zhao et al., 2011). In the present
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embryotoxicity studies of zebrafish D. rerio, both ACE and ACE TPs produced statistically
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insignificant adverse effects in test concentrations up to 1000 mg L-1. ACE at ≥ 5000 mg L-1
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showed a difference in accumulative mortality but not in other apical endpoints from the
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negative control. It might be due to presence of high potassium level or ACE. However,
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treatments at rising concentrations (> 5000 mg L-1) of ACE TPs were found to induce
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significantly adverse effects on fish embryo development with regard to tail detachment (Fig.
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3a), heart rate (Fig. 3b), hatching rate (Fig. 3c) and even lethality (Fig. 3d). Specifically, ACE
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TPs at higher levels (≥ 8500 mg L-1) had a remarkably enhanced toxicity compared with the
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same concentrations of ACE in embryo tail detachment (Fig. 3a), heart rate (Fig. 3b) and
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hatching rate (Fig. 3c). The obtained NOECs and Lowest Observed Effect Concentrations
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(LOECs) provide further evidence of the increased toxicity of ACE TPs relative to the mother
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compound (Table 2).
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ACCEPTED MANUSCRIPT The high NOECs detected for ACE and ACE TPs indicate a low hazard and risk
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potential towards zebrafish embryo development. Even so, it should be asked whether the
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current approach in assessing safety of artificial sweetener of ACE and its photo-induced TPs
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is sufficient. In fact, species sensitivity and endpoints collection are well accepted parameters
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in evaluating the potential hazards of target materials. The physiology of the amphipod
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gammarid Gammarus zaddachi and behavioral responses of the cladoceran D. magna were
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reported to be sensitive at < 5 mg L-1 sucralose (Wiklund et al., 2012). Moreover,
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environmental factors and the potential toxicity of complex mixtures with other pollutants
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should be considered when approving food additives (Schwarzenbach et al., 2006; Regulation
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(EC) No. 1333/2008). It is reported that millions of tons of micropollutants of possible
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toxicological concern at concentrations up to µg L-1 have been found ubiquitously in natural
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waters in the past 25 years (Schwarzenbach et al., 2006). Possible synergistic effects are able
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to render such mixtures potent (Pomati et al., 2006; Loos et al., 2013). Most importantly, the
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significantly elevated toxicity in ACE TPs is definitely derived from the irradiation process.
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This should be taken as a warning, since the highly persistent TPs ultimately could induce
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serious ecological consequences. Although the toxicity of ACE and its TPs to human health is
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relatively unknown at trace levels, historically continuous discharge and chronic exposure to
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these compounds may pose a threat to human wellness over a lifetime (Jones et al., 2004;
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Pomati et al., 2006; Ordóñez et al., 2012).
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4. Conclusion
ACCEPTED MANUSCRIPT By using LC-IM-QTOF-MS and Ion Trap MSn, a group of new TPs was firstly identified
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in different proposed hydrolysis and oxidative pathways under ACE irradiation. The high
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NOECs found in this developmental toxicity indicate a low hazard and risk potential towards
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zebrafish embryo, especially in view of the current environmental or estimated levels of these
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compounds. However, ACE TPs displayed an augmented toxicity relative to the mother
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compound in embryo development. The results here further emphasize that ACE should be
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paid close attention not only because of its widespread application, ubiquitous occurrence and
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persistence in the aquatic systems but also because TPs generated by photocatalytic processes
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using UV-C and TiO2 are even more toxic than the original compound. Studies on the
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environmental distribution, bioavailability, and biological effects of not only original
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compounds but also their transformation byproducts must be undertaken to ensure
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comprehensive evaluation of the risks these chemicals pose to the natural environment.
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Acknowledgements
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We thank the Hong Kong Research Grants Council (HKBU 201113) for the financial support.
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Kelvin S.–Y. Leung also thanks the grants from the Partner State Key Laboratory of
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Environmental and Biological Analysis (SKLP-14-15-P006) and Faculty of Science
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(FRG/12-13/067 and FRG/13-14/069), Hong Kong Baptist University.
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Figure Captions
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Fig. 1 – MS/MS spectra of the main transformation productions (TPs) of acesulfame and the
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detected fragmentations in (a) TP 137, (b) TP 154, (c) TP 168, (d) TP 193, (e) TP 230a and (f)
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TP 230b, by Ion Trap multiple mass spectrometry.
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Fig. 2 – Proposed acesulfame transformation pathways for the new transformation products
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under UV-C irradiation. Those masses in black represent published data whereas those in red
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are new proposed masses in present study.
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Fig. 3 – Apical endpoints of (a) tail-detachment at 24 h, (b) heart rate at 48 h, (c) hatching
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rate at 72 h and (d) accumulative mortality during 10-day exposure of zebrafish Danio rerio
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embryos to ACE and ACE TPs (mean ± 1 s.d., n = 3). Bars with different letters denote
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significantly different means (p < 0.05, Student-Newman-Keuls test).
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References
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Zhao, H., Chen, C., Zhang, X., Chen, J., Quan, X., 2011. Phytotoxicity of PFOS and PFOA to Brassica chinensis in different Chinese soils. Ecotox. Environ. Safe. 74, 1343-1347. Zhou, L., Ji, Y., Zeng, C., Zhang, Y., Wang, Z., Yang, X., 2013. Aquatic photodegradation of sunscreen agent p-aminobenzoic acid in the presence of dissolved organic matter. Water Res. 47, 153-162.
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Experimental m/z [M-H]-
Exact m/z of [M-H]-
(H2NO3S)-
2.615
95.9759
95.9761
2 3
(C3H6NO3S)(C2H2NO5S)-
2.529 2.612
136.0074 151.9660
136.0074 151.9659
4
(C2H2NO6S)-
3.638
167.9608
5
(C2H4NO6S)-
2.596
169.9768
6
(C4H6NO5S)-
2.899
179.9975
7 8 9 10 11 12 13 14 15 16
(C4H4NO6S)(C4H6NO6S)(C4H6NO7S)(C4H4NO8S)(C3H5O4S)(C2H4NO5S)(C3H6NO5S)(C4H5N2O5S)(C4H8NO6S2)(C4H8NO8S)-
2.660 3.025 2.654 N.A. 2.881 2.656 2.696 3.006 2.973 2.637
193.9769 195.9923 211.9871 N.A. 136.9914 153.9819 167.9973 192.9927 229.9804 229.9980
RT = retention time; N.A. = Not Available.
Mass error ∆ppm 2.1
Previously reported m/z 96 136 152
167.9608
0.0
168
169.9765
-1.8
170
179.9972
-1.7
180
-2.1 -1.0 -0.5 N.A. 0.0 -2.0 -0.6 -1.0 -2.2 1.74
194 196 212 226 N.A. N.A. N.A. N.A. N.A. N.A.
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Table 1–Exact mass information of acesulfame transformation products by using LC-IM-QTOF-MS.
193.9765 195.9921 211.9870 225.9663 136.9914 153.9816 167.9972 192.9925 229.9799 229.9976
References Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Scheurer et al., 2014 Gan et al., 2014 Gan et al., 2014 Gan et al., 2014 Gan et al., 2014 This study This study This study This study This study This study
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24-h tail detachment 24-h edema 24-h coagulation 48-h heart rate 72-h hatching rate
LOECs (mg L-1) ACE TPs ACE 10000 > 10000 > 10000 > 10000 > 10000 > 10000 8500 > 10000 10000 > 10000
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NOECs (mg L-1) ACE TPs ACE 8500 10000 10000 10000 10000 10000 5000 10000 8500 10000
Endpoints
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ACCEPTED MANUSCRIPT Highlights Acesulfame undergoes photocatalytic transformation upon UV-C irradiation; Six additional transformation products of acesulfame are identified; Hydrolysis and oxidation are proposed to be the two main degradation
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mechanisms; Transformation products in mixture produce adverse effects in fish embryo
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development.
S0043-1354(15)30362-6
DOI:
10.1016/j.watres.2015.11.035
Reference:
WR 11662
To appear in:
Water Research
Received Date: 9 July 2015 Revised Date:
10 November 2015
Accepted Date: 14 November 2015
Please cite this article as: Li, A.J., Schmitz, O.J., Stephan, S., Lenzen, C., Yue, P.Y.-K., Li, K., Li, H., Leung, K.S.-Y., Photocatalytic transformation of acesulfame: transformation products identification and embryotoxicity study, Water Research (2015), doi: 10.1016/j.watres.2015.11.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Photocatalytic transformation of acesulfame: transformation products
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identification and embryotoxicity study
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Adela Jing Li1,3, Oliver J. Schmitz4, Susanne Stephan4, Claudia Lenzen4, Patrick Ying-Kit
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Yue5, Kaibin Li6, Huashou Li3, Kelvin Sze-Yin Leung*1,2
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Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong
Special Administrative Region
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Baptist University, Kowloon Tong, Hong Kong Special Administrative Region
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China Agricultural University, Guangzhou 510642, China
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4
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Germany
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Administrative Region
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Partner State Key Laboratory of Environmental and Biological Analysis, Hong Kong
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Key Laboratory of Tropical Agro-environment, Ministry of Agriculture of China, South
Applied Analytical Chemistry, Faculty of Chemistry, University of Duisburg-Essen, Essen,
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special
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6
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Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380,
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China
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Key Laboratory of Tropical and Subtropical Fish Breeding & Cultivation, Pearl River
*Corresponding author. Tel.: +852 34115297; fax: +852 34117348
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E-mail address: [email protected] (Kelvin S.-Y. Leung)
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Abstract
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Artificial sweeteners have been recognized as emerging contaminants due to their wide
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application, environmental persistence and ubiquitous occurrence. Among them, acesulfame
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has attracted much attention. After being discharged into the environment, acesulfame
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undergoes photolysis naturally. However, acesulfame photodegradation behavior and identity
32
of its transformation products, critical to understanding acesulfame’s environmental impact,
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have not been thoroughly investigated. The present study aimed to fill this knowledge gap by
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a laboratory simulation study in examining acesulfame transformation products and pathways
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under UV-C photolysis in the presence of TiO2. Photodegradation products of acesulfame
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were isolated and analyzed using the LC-IM-QTOF-MS coupled with LC Ion Trap MS in the
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MSn mode. Our results show six new transformation products that have not been previously
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identified. The molecular structures and transformation pathways were proposed. Further
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embryotoxicity tests showed that acesulfame transformation products at the low g L-1 level
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produced significant adverse effects in tail detachment, heart rate, hatching rate and survival
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rate during fish embryo development. The identification of additional transformation
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products with proposed transformation pathways of acesulfame, the increased toxicity of
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acesulfame after photolysis, and the fact that the accumulation of acesulfame transformation
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products is increasingly likely make acesulfame contamination even more important. Water
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resource control agencies need to consider legislation regarding acesulfame and other
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artificial sweeteners, while further studies are carried out, in order to protect the safety of this
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most vital resource.
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Keywords: acesulfame, emerging contaminant, catalytic UV irradiation, transformation
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product, transformation pathway, embryotoxicity
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1. Introduction
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Artificial sweeteners (ASs) have been consumed in considerable quantities as sugar
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substitutes in low-calorie beverages, foods and personal care products since the 1950s
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(Buerge et al., 2011; Berset and Ochsenbein, 2012). They are a class of emerging
58
environmental contaminants with growing scientific concern due to their potential
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undesirable impacts on ecosystems and human health (Richardson and Ternes, 2014; Sang et
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al., 2014; Tran et al., 2014). Among the ASs, acesulfame (ACE) has drawn much attention
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due to its world-wide consumption, persistence when released, and subsequent ubiquitous
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occurrence in the natural environment (Nӧdler, et al., 2013; Tran et al., 2013). Available data
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indicates that the global consumption of ACE increased from 2.5 to 4.0 metric tons from
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2001 to 2005 due to its greater sweetening power and cheaper price compared to other ASs
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(Bahndorf and Kienle, 2004; ISO, 2008; Subedi and Kannan, 2014). The ingredient is applied
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in over 4000 foods and beverages in around 90 countries (Gisel, 2009).
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Due to human excretion mostly as the parent compound and high persistence in waste
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water even after treatment (Gan et al., 2013), levels of ACE were among the highest
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measured for trace pollutants in receiving waters of surface water, groundwater and
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wastewater influent and effluent (Table S1) and even tap water (Buerge et al., 2009;
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Mawhinney et al., 2011) with up to 100% detection frequency. Once released into nature,
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ACE may undergo different kinds of transformations. Stadler et al. (2012) suggest that
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estimating ACE removal rates by monitoring only the original compound will give distorted
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figures because it fails to take into consideration transformation products (TPs). This
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viewpoint is supported by findings of several other independent studies (Gan et al., 2014;
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can be degraded under simulated solar/UV irradiation or ozone treatment into at least ten TPs.
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The transformation ACE undergoes in the natural environment is now considered to be quite
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complex.
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Identification of the TPs structures would be extremely valuable in understanding,
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including predicting, how they might behave in the environment. Our previous study showed
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that many photo by-products were produced from ACE under prolonged UV irradiation, and
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the transformation process yielded by-products more persistent than the original ACE (Sang
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et al., 2014).
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According to several toxicological tests, ACE appears to be nontoxic to humans within
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regulated concentrations (Shankar et al., 2013; Gardner, 2014), and poses a low hazard to
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green algae Scenedesmus vacuolatus, duckweed Lemna minor and water fleas Daphnia
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magna (Stolte et al., 2013). Nevertheless, the ubiquitous occurrence of ACE raises safety
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concern and call for further research, particularly in the aspect of ACE TPs (Lange et al.,
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2012; Toth et al., 2012; Stolte et al., 2013; Richardson and Ternes, 2014). The only earlier
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relevant work by us indicated that photo-induced TPs of ACE are > 500 times more toxic
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than the mother compound in the marine bacterium Vibrio fischeri (Sang et al., 2014). The
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current study, therefore, will further evaluate the risk led by those TPs.
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The primary objectives of this study were to search for and identify the molecular
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structure of any new photo-induced ACE TP; and to evaluate the embryotoxicity of the TPs in
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mixture by embryo toxicity testing (FET) using the zebrafish Danio rerio.
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2. Experimental
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2.1 Chemicals and reagents
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Chemical standard for the artificial sweetener acesulfame potassium was purchased from
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Sigma-Aldrich (≥ 99.0%, HPLC, Germany). The ACE stock solution of 400 mg L-1 was
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prepared with Milli-Q water of 18.2 ΩM cm (Millipore, Billerica, MA, USA) and stored in
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the dark at 4 ºC. Titanium (IV) oxide of 21 nm particle size was supplied by Sigma-Aldrich
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(TiO2, ≥ 99.5%, trace metals basis). For chromatographic analyses in HPLC, Milli-Q water
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and HPLC grade methanol (Tedia, OH, USA) were used to prepare the mobile phases.
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Reagents for embryotoxicity bioassay were prepared for reconstituted moderately hard
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water (MHW) using the U.S. Environmental Protection Agency recipe: 75.9 mg L-1 calcium
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sulfate dihydrate (CaSO4·2H2O, ≥ 99.0%, ISO9001:2000, China), 123.2 mg L-1 magnesium
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sulfate heptahydrate (MgSO4·7H2O, ≥ 99.0%, A.R., China), 96 mg L-1 sodium bicarbonate
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(NaHCO3, 99%, Sigma-Aldrich, China), 4 mg L-1 potassium chloride (KCl, ≥ 99.5%, A.R.,
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China). For all embryotoxicity testing, 2 times of MHW was used as test medium (Bone et al.,
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2.2 Analysis of acesulfame transformation products
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2.2.1 TiO2-assisted photolysis experiment
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and without TiO2. The ACE (at a concentration of 400 mg/L) showed a 63.94% (n = 3)
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degradation without TiO2 while with a 99.99% (n = 3) in the presence of a catalyst.
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TiO2-assisted photodegradation was therefore adopted. Experimentally, 10 mg of ACE was
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dissolved in 25 mL of Milli-Q water. Freshly prepared TiO2 was then added to the solution at
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ACE/TiO2 mass ratio of 1:20. After the reaction under UV-C light (Sankyo G8T5, Japan;
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1630 µW/cm2) for 19 h at room temperature, the solution was filtered through 0.2 µm nylon
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membrane, and freeze-dried for use.
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2.2.2 Analytical methods
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Both ACE and its transformation products at 100 mg L-1 were analyzed by the Ion Mobility
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Q-TOF MS (Agilent 6560 IM Q-TOF) coupled with LC system (Agilent 1290 Infinity, USA)
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(LC-IM-QTOF-MS). Chromatographic separation was performed on a Phenomenex Luna 3µ
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CN column (150 × 2.0 mm, 3 µm) in the gradient elution model. The injection volume was
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1 µL. The mobile phase was composed of water and methanol, both containing 0.1% formic
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acid (98 – 100 %, Merck, Suprapur®). The flow rate was 0.2 mL/min. The gradient program
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started with 95% water for 5 min, followed by ramping to 50% water within 5 min, and then
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kept at this condition for 1 min, and then returned to the initial setup in 1 min. Q-TOF mode
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was applied with ion source of Dual AJS electrospray interface (ESI) with mass correction at
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reference masses of m/z 119.0363 and 966.0070. High resolution mass spectra (m/z 40 – 1700)
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were obtained at a rate of 2 spectra per second with electrospray ionization in the negative ion
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ACCEPTED MANUSCRIPT mode. Both positive and negative modes were initially applied to analyze samples, but only
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data from negative ion mode offered adequate information with allowance in detecting of
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potential TPs. Therefore, data derived from negative mode runs were focused for subsequent
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analyses. The dry gas of nitrogen flowed at 5 L/min under 200 ºC while sheath gas of
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nitrogen was at 12 L/min with 325 ºC, Vcap was set to 5000 V and the nozzle voltage to 500
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V.
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Specific mass of target transformation products was further fragmented by using the Ion
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Trap (Bruker amaZon speed) in the MSn mode coupled with HPLC (Thermal Scientific
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Accela). Chromatographic separation was performed under the same conditions for Q-TOF
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except with injection volume of 10 µL and m/z of 50 – 300. The fragmentation mass took
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place at energy of 2.78 V. Nitrogen was taken as dry gas with flow rate at 8.2 L/min and 350
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ºC. The nebulizer pressure was 15.0 psi.
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2.3 Embryotoxicity testing
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2.3.1 Animal care and breeding
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Adult zebrafish D. rerio were cultured in the Pearl River Fisheries Research Institute,
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Chinese Academy of Fishery Sciences, Guangzhou. In the laboratory, breeding stocks of
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males and females were maintained in separate glass aquaria filled with oxygenated tap water
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maintained at 26 ± 1 ºC and kept on a 14h:10h light:dark cycle. Fish were fed with Artemia
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franciscana nauplii. Before breeding the zebrafish in a ratio of 3:2 females: males, were kept
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in a tank with a board separating females from males, overnight. In the morning, when the
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light came on, the board was removed and the fish were allowed to breed. Embryos were
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collected after 30 minutes.
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2.3.2 Toxicity exposure
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The embryotoxicity tests of ACE and the preparation of ACE transformation products were
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carried out according to the method described by OECD (2013). Embryos of D. rerio of less
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than 32-cell blastomeres were exposed to testing compounds individually in 96-well plate
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(Thermo ScientificTM). Tests were performed with six dosage levels (100, 500, 1000, 5000,
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8500, and 10000 mg L-1). In each trial, 20 fish embryos were tested with 200 µL target
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solution, and three replicates of each dosage level were conducted. Positive and negative
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controls were performed in parallel by using 3, 4-dichloroaniline (4.0 mg L-1, 98%,
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Sigma-Aldrich) and 2 times of MHW, respectively. The criterion for positive embryotoxicity
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was more than 30% mortality after 96 h exposure, while that the criterion for negative control
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was overall survival and hatching rate of no less than 80%. The embryos were kept in an
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incubator (LRH-250, Shanghai) for 10 days with temperature maintained at 26 ± 1 ºC, pH
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8.2 - 8.3, oxygen ≥ 80% saturation and 14 h:10 h light:dark cycle. The entire test was
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repeated twice. Apical endpoints of tail-detachment, edema and coagulation after 24 h
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exposure, heart rate after 48 h exposure, hatching rate after 72 h exposure, and accumulative
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mortality were recorded.
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2.4 Statistical analysis
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data. To identify ACE TPs produced during photocatalytic transformation process,
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post-photolysis samples were compared against initial compound of ACE and the blank of
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Milli-Q water. For those reported ACE TPs, extracted m/z values were obtained based on
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previously reported m/z values. Further MSn characterization of ACE TPs was limited to the
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compounds within less than 5 ∆ppm deviation between experimental and exact m/z values.
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Data analyses in embryotoxicity tests were performed using SPSS version 19 (SPSS Inc.,
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Chicago) with mean ± standard deviation (s.d.). One-way analysis of variance (ANOVA)
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using chemical treatment as a fixed factor was used to infer the difference among chemical
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treatment groups for significance at the 5% level. If the null hypothesis was rejected by the
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ANOVA, different means were identified using Student-Newman-Keuls (SNK) test.
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3. Results and discussion
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3.1 Identification of transformation products
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UV irradiation at a wavelength 254 nm causes ACE degradation, producing a range of TPs.
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The laboratory simulated irradiation conditions including concentration of reactant,
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ACE:TiO2 ratio and reaction time were optimized through a series of experiments. It was
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found that the ACE TPs occur more easily with a higher yield at 400 mg L-1 of ACE,
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ACE:TiO2 1:20 (mass ratio), and a reaction time 19 h by UV-C irradiation, which
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corresponds to a fluence of about 1,115,000 J/m2. The TPs were concentrated and solidified
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by a freeze-drying process and then characterized by mass spectroscopic methods.
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ACCEPTED MANUSCRIPT In literature, ten TPs were identified and reported (Gan et al., 2014; Scheurer et al.,
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2014). In our work, the chemical structures of TPs were elucidated by LC-IM-QTOF-MS
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followed by Ion Trap MSn mode. Due to the high resolution mobility separation and low
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femtogram detection limits of LC-IM-QTOF-MS, this study detected far more TPs in the
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irradiated sample. Nine out of the ten previously reported ACE TPs were found, presenting
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almost exact masses to those previous studies (Table 1). Mass differences were in the range
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of -2.1 to 2.1 ppm. The 10th TP with m/z 225.9663 that reported in Gan et al. (2014) was not
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discovered in this study.
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However, in our work, we identified six additional TPs. Most of the TPs eluted earlier
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than the parent compound ACE under the adopted HPLC conditions. This finding is
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consistent with the study by Gan et al. (2014), indicating the TPs generated upon photolysis
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were generally more polar than ACE (MacManus-Spencer et al., 2011). These additional TPs
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with extracted ion chromatograms given in Fig. S1 were structurally elucidated by
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fragmentation of Ion Trap mass spectrometry (Table 1). The MSn fragmentation patterns,
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proposed chemical structures and transformation pathways of these TPs are illustrated in Fig.
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1. The 11th TP TP 137 (C3H5O4S)- with m/z 136.9914 was identified based on its fragment
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ions appearing at m/z 120 (C3H4O3S)-, 107 (C2H3O3S)-, 96 (O4S)-, 95 (CH3O3S)-, 94
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(CH2O3S)-, 91 (C2H3O2S)-, 80 (O3S)-, 78 (CH2O2S)-, 73 (C3H5O2)-, 65 (HO2S)- and 59
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(C2H3O2)- (Fig. 1a). TP 137 was speculated to be a degradate from ACE hydrolysate by loss
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of -NCO (Fig. 2). We propose that the ACE hydrolysate was formed by addition of water
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beyond the C=C bond, which is different from the proposed pathway in Gan et al. (2014).
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Whereas, Gan et al. (2014) suggested ACE underwent hydrolysis by the addition of water at
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ACCEPTED MANUSCRIPT the C=C bond, which offered an intermediate to yield a TP with m/z 136.0074 by
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photo-rearrangement. Differences in the pathways observed, or implied, in ACE
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photo-hydration may arise from differences in the experimental designs in the irradiation
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process. Firstly, light source may be a key factor. A xenon lamp of 1 kW with UV filters to
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maintain light > 290 nm was employed as a sunlight stimulator by Gan et al. (2014), while
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UV-C lamps of 128 W at 254 nm were used in the current work. Secondly, the photo-catalytic
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process may be a factor. While in this study TiO2 was applied in the photo-catalytic process,
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and it is known to have strong photo-catalytic activity (Tang et al., 2015). Thirdly, period of
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irradiation may count. A 7-d irradiation process was used in Gan et al. (2014)’s study whereas
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19 h UV-C irradiation was adopted in this study. Finally, testing condition disparity may play
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a role. This may explain why the TP with m/z 225.9663 reported in Gan et al. (2014) was not
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found in this study.
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The 12th TP, TP 154 with m/z 153.9816 (C2H4NO5S)- was proposed to have an open ring
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structure according to fragments determined at m/z 136 (C2H2NO4S)- and 96 (H2NO3S)- (Fig.
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1b). The 13th TP 168 ((C3H6NO5S)-, m/z 167.9972) was conceived based on four fragment
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ions, namely m/z 150 (C3H4NO4S)-, 124 (C2H6NO3S)-, 96 (H2NO3S)- and 81 (HO3S)- (Fig. 1c)
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while the 14th TP 193 ((C4H5N2O5S)-, m/z 192.9925) was inferred on fragment detections at
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m/z 166 (C3H4NO5S)- and 96 (H2NO3S)- (Fig. 1d). Furthermore, it is interesting that two TPs
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of the 15th and 16th having very close in mass, namely with m/z of 229.9799 (C4H8NO6S2)-
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and 229.9976 (C4H8NO8S)-, were extracted with apparently complicated structures. Based on
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the masses detected at m/z 149 (C4H7NO3S)- or (C4H5O4S)-, 148 (C4H6NO3S)-, 122
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(C2H4NO3S)-, 106 (CNO3S)-, 84 (C4H6NO)- and 71 (C3H5NO)-, the structure of m/z 229.9799
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ACCEPTED MANUSCRIPT (TP 230a) was tentatively proposed to include a closed heterocyclic ring (Fig. 1e). However,
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a possible pathway from ACE to this intricate structure was not successfully proposed, and
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hence requires further investigation. The structure of m/z 229.9976 (TP 230b) is estimated to
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be an open ring depending on fragment ions of m/z 212 (C4H6NO7S)-, 194 (C4H4NO6S)-, 166
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(C3H4NO5S)-, 124 (CH2NO4S)-, 97 (C4HO3)-, and 96 (H2NO3S)- (Fig. 1f). Ion of m/z 96
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(H2NO3S)- was a commonly detected fragment of ACE TPs, which is also reported by Gan et
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al. (2014). The overall proposed transformation pathways of ACE upon photolysis are given
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in Fig. 2. Hydrolysis and oxidation are proposed to be the two main degradation mechanisms
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in ACE photo-induced transformation.
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For a comprehensive environmental risk assessment of any contaminant entering natural
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waters, it is essential to consider not only the parent compound but also its TPs. Concern with
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regard to TPs of emerging contaminants has been growing exponentially in recent years.
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Studies of TPs have investigated their occurrence (Nӧdler et al., 2013; Bulloch et al., 2015),
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influencing factors (Ji et al., 2013; Gan et al., 2014; Liu et al., 2015), degradation kinetics
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(Vione et al., 2013; Scheurer et al., 2014), structure (Zhou et al., 2013; Gan et al., 2014;
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Godayol et al., 2015) and mitigation (Bahnmüller et al., 2015; Postigo and Barceló, 2015).
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Nonetheless, the limited toxicological data together with the wide occurrence of these
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transformation byproducts has generated significant uncertainty with regard to their risk to
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ecological systems. Biological testing integrated with chemical analysis is unquestionably
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needed to achieve accurate characterization of the pollutants (Díaz-Cruz and Barceló, 2009;
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Stolte et al., 2013; Gan et al., 2014; Bulloch et al., 2015). In view of this point, we undertook
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an embryotoxicity study of ACE TPs in mixture.
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3.2 Embryotoxicity Stolte et al. (2013) evaluated ecotoxicity of artificial sweeteners ACE, cyclamate,
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saccharin and sucralose as well as natural sweetener stevioside to activated sewage sludge
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respiration inhibition, green algae reproduction inhibition, water flea acute immobilization
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and duckweed growth inhibition, with the No Observed Effect Concentrations (NOECs) at
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1000 mg L-1. Similar observations of relatively high NOECs were also reported in other
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persistent organic pollutants such as sucralose (Huggett and Stoddard, 2011; Soh et al., 2011),
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perfluorooctane sulfonate and perfluorooctanoic (Zhao et al., 2011). In the present
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embryotoxicity studies of zebrafish D. rerio, both ACE and ACE TPs produced statistically
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insignificant adverse effects in test concentrations up to 1000 mg L-1. ACE at ≥ 5000 mg L-1
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showed a difference in accumulative mortality but not in other apical endpoints from the
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negative control. It might be due to presence of high potassium level or ACE. However,
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treatments at rising concentrations (> 5000 mg L-1) of ACE TPs were found to induce
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significantly adverse effects on fish embryo development with regard to tail detachment (Fig.
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3a), heart rate (Fig. 3b), hatching rate (Fig. 3c) and even lethality (Fig. 3d). Specifically, ACE
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TPs at higher levels (≥ 8500 mg L-1) had a remarkably enhanced toxicity compared with the
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same concentrations of ACE in embryo tail detachment (Fig. 3a), heart rate (Fig. 3b) and
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hatching rate (Fig. 3c). The obtained NOECs and Lowest Observed Effect Concentrations
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(LOECs) provide further evidence of the increased toxicity of ACE TPs relative to the mother
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compound (Table 2).
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ACCEPTED MANUSCRIPT The high NOECs detected for ACE and ACE TPs indicate a low hazard and risk
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potential towards zebrafish embryo development. Even so, it should be asked whether the
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current approach in assessing safety of artificial sweetener of ACE and its photo-induced TPs
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is sufficient. In fact, species sensitivity and endpoints collection are well accepted parameters
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in evaluating the potential hazards of target materials. The physiology of the amphipod
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gammarid Gammarus zaddachi and behavioral responses of the cladoceran D. magna were
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reported to be sensitive at < 5 mg L-1 sucralose (Wiklund et al., 2012). Moreover,
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environmental factors and the potential toxicity of complex mixtures with other pollutants
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should be considered when approving food additives (Schwarzenbach et al., 2006; Regulation
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(EC) No. 1333/2008). It is reported that millions of tons of micropollutants of possible
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toxicological concern at concentrations up to µg L-1 have been found ubiquitously in natural
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waters in the past 25 years (Schwarzenbach et al., 2006). Possible synergistic effects are able
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to render such mixtures potent (Pomati et al., 2006; Loos et al., 2013). Most importantly, the
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significantly elevated toxicity in ACE TPs is definitely derived from the irradiation process.
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This should be taken as a warning, since the highly persistent TPs ultimately could induce
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serious ecological consequences. Although the toxicity of ACE and its TPs to human health is
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relatively unknown at trace levels, historically continuous discharge and chronic exposure to
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these compounds may pose a threat to human wellness over a lifetime (Jones et al., 2004;
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Pomati et al., 2006; Ordóñez et al., 2012).
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4. Conclusion
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in different proposed hydrolysis and oxidative pathways under ACE irradiation. The high
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NOECs found in this developmental toxicity indicate a low hazard and risk potential towards
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zebrafish embryo, especially in view of the current environmental or estimated levels of these
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compounds. However, ACE TPs displayed an augmented toxicity relative to the mother
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compound in embryo development. The results here further emphasize that ACE should be
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paid close attention not only because of its widespread application, ubiquitous occurrence and
315
persistence in the aquatic systems but also because TPs generated by photocatalytic processes
316
using UV-C and TiO2 are even more toxic than the original compound. Studies on the
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environmental distribution, bioavailability, and biological effects of not only original
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compounds but also their transformation byproducts must be undertaken to ensure
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comprehensive evaluation of the risks these chemicals pose to the natural environment.
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Acknowledgements
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We thank the Hong Kong Research Grants Council (HKBU 201113) for the financial support.
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Kelvin S.–Y. Leung also thanks the grants from the Partner State Key Laboratory of
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Environmental and Biological Analysis (SKLP-14-15-P006) and Faculty of Science
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(FRG/12-13/067 and FRG/13-14/069), Hong Kong Baptist University.
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Figure Captions
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Fig. 1 – MS/MS spectra of the main transformation productions (TPs) of acesulfame and the
330
detected fragmentations in (a) TP 137, (b) TP 154, (c) TP 168, (d) TP 193, (e) TP 230a and (f)
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TP 230b, by Ion Trap multiple mass spectrometry.
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Fig. 2 – Proposed acesulfame transformation pathways for the new transformation products
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under UV-C irradiation. Those masses in black represent published data whereas those in red
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are new proposed masses in present study.
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Fig. 3 – Apical endpoints of (a) tail-detachment at 24 h, (b) heart rate at 48 h, (c) hatching
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rate at 72 h and (d) accumulative mortality during 10-day exposure of zebrafish Danio rerio
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embryos to ACE and ACE TPs (mean ± 1 s.d., n = 3). Bars with different letters denote
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significantly different means (p < 0.05, Student-Newman-Keuls test).
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Experimental m/z [M-H]-
Exact m/z of [M-H]-
(H2NO3S)-
2.615
95.9759
95.9761
2 3
(C3H6NO3S)(C2H2NO5S)-
2.529 2.612
136.0074 151.9660
136.0074 151.9659
4
(C2H2NO6S)-
3.638
167.9608
5
(C2H4NO6S)-
2.596
169.9768
6
(C4H6NO5S)-
2.899
179.9975
7 8 9 10 11 12 13 14 15 16
(C4H4NO6S)(C4H6NO6S)(C4H6NO7S)(C4H4NO8S)(C3H5O4S)(C2H4NO5S)(C3H6NO5S)(C4H5N2O5S)(C4H8NO6S2)(C4H8NO8S)-
2.660 3.025 2.654 N.A. 2.881 2.656 2.696 3.006 2.973 2.637
193.9769 195.9923 211.9871 N.A. 136.9914 153.9819 167.9973 192.9927 229.9804 229.9980
RT = retention time; N.A. = Not Available.
Mass error ∆ppm 2.1
Previously reported m/z 96 136 152
167.9608
0.0
168
169.9765
-1.8
170
179.9972
-1.7
180
-2.1 -1.0 -0.5 N.A. 0.0 -2.0 -0.6 -1.0 -2.2 1.74
194 196 212 226 N.A. N.A. N.A. N.A. N.A. N.A.
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Table 1–Exact mass information of acesulfame transformation products by using LC-IM-QTOF-MS.
193.9765 195.9921 211.9870 225.9663 136.9914 153.9816 167.9972 192.9925 229.9799 229.9976
References Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Scheurer et al., 2012 Gan et al., 2014 Scheurer et al., 2014 Gan et al., 2014 Gan et al., 2014 Gan et al., 2014 Gan et al., 2014 This study This study This study This study This study This study
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24-h tail detachment 24-h edema 24-h coagulation 48-h heart rate 72-h hatching rate
LOECs (mg L-1) ACE TPs ACE 10000 > 10000 > 10000 > 10000 > 10000 > 10000 8500 > 10000 10000 > 10000
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NOECs (mg L-1) ACE TPs ACE 8500 10000 10000 10000 10000 10000 5000 10000 8500 10000
Endpoints
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ACCEPTED MANUSCRIPT Highlights Acesulfame undergoes photocatalytic transformation upon UV-C irradiation; Six additional transformation products of acesulfame are identified; Hydrolysis and oxidation are proposed to be the two main degradation
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mechanisms; Transformation products in mixture produce adverse effects in fish embryo
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development.