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Spectroscopic indicators for cytotoxicity of chlorinated and ozonated effluents from wastewater stabilization ponds and activated sludge Nedal Massalha, Shengkun Dong, Michael J Plewa, Mikhail Borisover, and Thanh H. Nguyen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05510 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Spectroscopic indicators for cytotoxicity of chlorinated and ozonated effluents

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from wastewater stabilization ponds and activated sludge

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Nedal Massalha,†⊥* Shengkun Dong,†⊥‡ Michael J. Plewa,§‡ Mikhail Borisover,□

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Thanh H. Nguyen†‡

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Manuscript submitted to Environmental Science & Technology

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†Department of Civil and Environmental Engineering,

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§ Department of Crop Sciences,

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‡Safe Global Water Institute,

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University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States □

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Institute of Soil, Water and Environmental Sciences, The Volcani Center, A.R.O., P.O.B. 15159, RishonLeZion7505101, Israel

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Word count = 3989 + 2100 (figures) = 6089

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*

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Nedal Massalha; e-mail: [email protected]

Author to whom correspondence should be addressed:

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ABSTRACT

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We investigated chronic mammalian cell cytotoxicity of wastewaters from four sources and their

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optical spectroscopic properties with or without chlorination or ozonation. Samples from effluents

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of activated sludge, nitrification tower, facultative waste stabilization pond, and maturation waste

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stabilization pond were either chlorinated or ozonated. The wastewater samples were analyzed for

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fluorescence excitation emission matrix, specific fluorescence index (SFI), and specific UV

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absorbance at 254 nm (SUVA). Before and after disinfection the wastewater samples were

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quantitatively analyzed for in vitro mammalian cell cytotoxicity. We found that the organic

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extracts from the ozonated samples induced lower cytotoxicity responses than those from the

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chlorinated or the non-disinfected samples. To develop correlations between SFI, SUVA, and

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cytotoxicity, we analyzed 21 independent samples. Significant linear correlations found among

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these samples suggest that under the tested conditions, cytotoxicity was preferentially influenced

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by the fluorescence and SUVA of their composite organic agents. These two spectroscopic

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parameters may be used as indicators for the potential toxicity of non-disinfected, ozonated, or

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chlorinated municipal wastewater.

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INTRODUCTION

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Wastewater reuse is an important component of sustainable wastewater management practices

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worldwide.1 Because of severe limits to fresh water resources, reuse of treated wastewater for

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irrigation has increased.2 In many regions of the USA the availability of fresh water for irrigation

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has been diminished because of climate change and droughts.3–5 Employing treated wastewater for

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agriculture irrigation reduces the demand on fresh source waters and enhances soil enrichment

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with nutrients from the effluents.6–8

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Wastewater for safe reuse demands that effluents be disinfected.9 Wastewater effluents contain

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complex organic compounds from influent organics and biodegradation byproducts formed during

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treatment processes.10 Pathogens can be inactivated by disinfecting the effluent wastewater;

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however, toxic disinfection byproducts (DBPs) are generated.11 The formation of uncharacterized

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DBPs is a public health concern

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wastewater.17–19 Chlorination is the most commonly used disinfectant for water and wastewater.20

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Ozone, an alternative disinfectant to chlorine, can inactivate even chlorine-resistant pathogens in

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drinking water 20,21 and lower the toxicity of wastewaters.22–24

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and some DBPs are more toxic than the organics in the

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Measurements of UV absorption and fluorescence of treated wastewater have been found to

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relate to the occurrence of DBPs. For example, specific absorbance at 254 nm (SUVA)25,26 and

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fluorescence index (FI)27,28 were used as surrogate measurements for dissolved organic matter

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(DOM) aromaticity and molecular weight to predict the formation of DBPs. Fluorescence of DOM

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in treated water was also linked to the formation of chlorinated DBPs.29 The three-dimensional

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fluorescence excitation emission-matrices (EEMs) were used as a basis for fingerprint information

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and on-line monitoring of DOM, with the aim of predicting DBP formation potential during water


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treatment.30–32 Several methods were used to quantitatively analyze the EEMs, for example

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conventional peak-picking, regional integration, and multivariate data analysis such as the parallel

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factor method (PARAFAC).33–35 Changes in EEMs fluorescence intensities after oxidation were

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related to DBPs formation.29,36 While the links between UV absorption and fluorescence of

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disinfected wastewater with DBP formation have been established, the potential correlations

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between mammalian cell cytotoxicity and these commonly measured properties of the wastewater

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have not been explored.

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To fill this knowledge gap, the objectives of this study were to (i) examine the relationship

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between mammalian cell cytotoxicity and fluorescence indices, and mammalian cell cytotoxicity

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and UV absorbance properties, and (ii) establish correlations, if any, between mammalian cell

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cytotoxicity vs. fluorescence indices and UV absorbance properties, two commonly measured

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water quality parameters, in different municipal wastewater effluents before and after disinfection

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by chlorination or ozonation. To our knowledge, this study is the first to demonstrate that

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cytotoxicity of municipal wastewater can be inferred from commonly measured fluorescence and

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UV-absorbance spectral indicators.

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MATERIALS AND METHODS

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Sampling and Characterization of Wastewaters. Four sources of treated municipal wastewaters

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were sampled in two wastewater treatment sites in Illinois, USA: (i) the effluent of a secondary

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clarifier, which is part of an activated sludge treatment plant (ASE), (ii) the effluent of a

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nitrification tower (NIT), which was fed by a blend of trickling filter and activated sludge effluents

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(iii) the effluent of an aerated waste stabilization pond (AWSP), and (iv) the effluent of a

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maturation waste stabilization pond (MWSP). AWSP and MWSP were from the same treatment



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plant that employed passive ponds. A 50 L ASE sample was taken from the effluent of a secondary

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clarifier. A volume of 30 L was taken from the effluent of the nitrification tower. Two 50 L

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samples, one each from AWSP and MWSP, two ponds in series, were taken directly from the

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upper 30 cm of the ponds’ surface. The typical hydraulic retention time of ASE is 12 to 18 h, while

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the typical hydraulic retention times are between 4 to 8 days for AWSP and 30 to 40 days for

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MWSP. All samples were transported and stored in the dark at 4°C in 20 L polyethylene (PE)

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containers until extraction and analysis. Prior to filtration and disinfection, the source samples

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were characterized by the total chemical oxygen demand (COD) using the HACH (Loveland, CO)

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low range COD digestion vials according to USEPA Reactor Digestion Method,37 and by the

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volatile suspended solids (VSS).38 Before disinfection and concentration of the effluents, in order

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to remove coarse particles, the wastewater samples were passed through Grade 1:11 µm

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Whatman® filters pre-washed with deionized water. The filtrate was further transferred through

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1.6 µm Millipore glass fiber filters followed by 0.45 µm pre-washed nitrocellulose membranes.

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After filtration the samples were analyzed for total organic carbon (TOC) by a Shimadzu TOC

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analyzer (Columbia, MD) and ammonia nitrogen using the HACH Salicylate kit.39 The measured

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wastewater parameters are presented in the supporting information (Table 1SI).

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Disinfection Experiments. Chlorination and ozonation disinfection experiments were conducted

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with the 0.45 µm filtrate of MWSP, AWSP, ASE, and NIT samples. The filtered samples of

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MWSP, AWSP, and ASE were given the acronyms MWSP1, AWSP1, and ASE1. Chlorination

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was conducted in multiple US EPA approved 4 L amber glass bottles. A volume of 1.85 mL of

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sodium hypochlorite solution (Ricca Chemical, TX) with 27 g/L free chlorine was added to each

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4 L wastewater sample, yielding an applied chlorine concentration of 12.5 mg/L as Cl2.

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Preliminary experiments were done to guarantee the presence of free chlorine after 30 min reaction


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time using the US EPA DPD Method with the HACH kit (Table 2SI).40 In brief, initial free chlorine

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concentrations of 4, 8, 12.5, and 14 mg/L as Cl2 were tested in 20 mL wastewater samples for free

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residual chlorine after 30 min reaction. The bottles were sealed, kept in the dark, and stirred with

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magnetic bars for 30 min, after which the samples were measured for free chlorine and quenched

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with 1 mL of 0.1 M sodium thiosulfate (E K Industries, IL) to each 4 L wastewater sample. As

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described in the previous studies,22,23 ozonation experiments were conducted in a 10 L glass reactor

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agitated by a magnetic bar. Ozone was produced by a micro-channel plasma ozone generator (EP

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Purification Inc., IL) fed by an oxygen concentrator (Airsep, NY). The ozone-containing gas was

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bubbled through a stone diffuser into cold, deionized, distilled water (4°C) for at least 15 min. The

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aqueous ozone concentration was detected continuously with an ultraviolet spectrometer

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(Beckman Coulter Life Sciences, IN) at λ = 258 nm (ε = 3000 M−1 cm−1).41 The ozonated water

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was then quickly mixed with the wastewater samples to produce an applied ozone concentration

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of 3 mg/L. The reactor was subsequently capped and the ozonated samples were incubated for 24

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h to ensure the absence of residual ozone.

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Sample Concentration. Dissolved wastewater organics were adsorbed and concentrated by

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passing the wastewater samples through a chromatography column with a mixture of 110 mL each

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of XAD-2 (Amberlite XAD-2, Sigma-Aldrich, MO) and XAD-8 (Supelite DAX-8, Sigma-Aldrich,

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MO) resins. For 72 h, the resins were Soxhlet-cleaned with spectroscopic grade (Fisher Optima)

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methanol, followed by ethyl acetate and again with methanol. Details regarding the resins Soxhlet

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cleaning, conditioning, and the extracting of the adsorbed organics are provided in a previous

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study42,43 and in the SI. The XAD-2/XAD-8 resin mixture was packed in glass columns. The resin

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packing was conditioned by acid (1N HCl) and base (1N NaOH) before the water samples were

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passed through the columns. After passing the acidified samples (40-50 mL H2SO4 per 2L of



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sample) through the columns, the organics were eluted with Optima grade ethyl acetate (Fisher

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Scientific, PA), which was vacuum evaporated and dried with nitrogen gas. The organics were

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dissolved in ACS reagent grade dimethyl sulfoxide (DMSO) (Fisher Scientific, PA) to a

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concentration factor of 1×105 fold.

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Chinese Hamster Ovary Cells. The Chinese hamster ovary (CHO) cell line AS52 clone 11-4-2

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was maintained on glass culture plates in Ham’s F12 medium containing 5% fetal bovine serum

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(FBS), 1% antibiotics (100 U/mL sodium penicillin G, 100 µg/mL streptomycin sulfate, 0.25

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µg/mL amphotericin B in 0.85% saline), and 1% glutamine at 37°C in a humidified atmosphere of

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5% CO2.44

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CHO Cell Cytotoxicity Assay. The CHO cell chronic cytotoxicity assay measures the reduction

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in cell density as a function of the wastewater sample concentrates over a period of approximately

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three to four cell divisions (72 h), using an assay we previously developed for the analysis of

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individual chemicals as well as for complex water mixtures.15,23,45 Flat-bottom, tissue culture 96-

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well microplates were employed; 4 replicate wells were prepared for each concentration of each

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water sample extract. Eight wells were reserved for the blank control consisting of 200 µL of F12

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+ 5% FBS. The negative control consisted of 8 wells containing 100 µL of a titered CHO cell

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suspension (3×104 cells/mL) plus 100 µL F12 + 5% FBS. The wells for the remaining columns

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contained 3,000 CHO cells, F12 + 5% FBS, and a known concentration of wastewater organic

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extract (total volume per well = 200 µL). To prevent sample evaporation or cross contamination

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between wells due to evaporation, a sheet of sterile AlumnaSeal™ (RPI Corporation, IL) was

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pressed over the wells before the microplate was covered. To distribute the cells uniformly, the

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microplate was placed on a rocking platform for 10 min, and then placed in a humidified tissue

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culture incubator at 37°C, 5% CO2 for 72 h. After incubation, each well was gently aspirated, fixed 7 Environment ACS Paragon Plus

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in 100% methanol for 10 min, and stained for 10 min with a 1% crystal violet solution in 50%

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methanol. The microplate was gently rinsed in tap water, inverted, and tapped dry on paper towels.

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50 μL of DMSO/methanol (3:1 v/v) was added to each well for 10 min before the microplate was

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analyzed in a microplate reader for absorbance at 595 nm. The blank-corrected absorbance value

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of the negative controls (cells with medium only) was set at 100%. The absorbance for each

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treatment group was converted into a percentage of the negative controls. A concentration-

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response curve was generated for each wastewater sample extract, and a regression analysis was

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conducted for each curve. The LC50 values were calculated from each regression analysis, where

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the LC50 represents the concentration factor that induced a 50% reduction in cell density as

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compared to the concurrent negative controls. A DMSO control demonstrated that no significant

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cytotoxicity was induced throughout the solvent concentrations used in these experiments.

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Spectroscopic Analysis. The fluorescence EEMs were measured at room temperature with a

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Shimadzu RF-5301PC spectrofluorometer employing a quartz cuvette with 4 × 1.0 cm windows.

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The slit widths for excitation and emission were 5 nm. Excitation wavelengths varied from 250 to

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400 nm with increments of 10 nm, and emission wavelengths varied between 280 to 550 nm with

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1 nm increments. Samples for EEM analysis were diluted with fluorescence-free Milli-Q deionized

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water to ensure that solution absorbance did not exceed 0.1 cm-1 at wavelengths ≥ 250 nm, thus

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minimizing the inner filter effect.46 The pH of the filtered samples was within the 8.3 ± 0.2 range.

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The dilution Milli-Q water was used as the blank for fluorescence EEM to be subtracted from the

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sample EEM. No fluorescence was detected in Milli-Q water after passing through pre-washed

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filters.

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For quantitative analysis of the EEM plots, a peak picking algorithm-based approach was used

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to determine the maximum peak intensities within pre-defined regions of interest.26,27 Four specific 8 Environment ACS Paragon Plus


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regions of interest were chosen in excitation emission wavelength boundaries as defined

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previously:47 humic acid (HA)-like at excitation wavelengths of 280 to 370 nm and emission

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wavelengths of 400 to 480nm; fulvic acid (FA)-like at excitation wavelengths of 250 to 260 and

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emission wavelengths of 400 to 460 nm; tryptophan (protein)-like (TP) at excitation wavelengths

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of 260 to 280 nm and emission wavelengths of 330 to 380 nm; tyrosine (protein)- like (TY) at

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excitation wavelengths of 260 to 280 nm and emission wavelengths of 290 to 330 nm. Determined

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EEMs of fluorescence were multiplied by a dilution factor used for sample preparation. The

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fluorescence index (FI) was taken as the ratio of emission intensities at 470 nm and 520 nm for an

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excitation at 370 nm.27 The specific fluorescence index (SFI) was calculated as the ratio of FI to

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the corresponding dissolved organic carbon (DOC). Absorbance at 254 nm (Abs254) was obtained

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in a 1.0 cm quartz cuvette using an ultraviolet-visible spectrometer (Shimadzu UV-2450). The

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absorbance value (cm−1) was expressed as the ratio of optical density to optical length (1.0 cm).

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SUVA, determined by the ratio of UV254 to DOC, is an indicator of the presence of aromatic

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components in organic matter.26 The values of SUVA were reported in liter per milligram of C per

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meter (Abs254/DOC × 100).

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Statistics. The LC50 values were converted into cytotoxicity index values CTI = LC50-1 × 103 for

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the ANOVA test statistic to determine significant differences amongst the toxicity of the

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wastewater samples. The statistical power was maintained at ≥ 0.8 at α = 0.05. The values of the

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DOC-normalized cytotoxicity index (NCTI) were calculated by dividing the mean CTI values by

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the corresponding DOC of the wastewater sample. Possible correlations between the NCTI and

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spectroscopic properties were evaluated using the Pearson Product-Moment test (OriginPro, MA).

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For each correlation, a Pearson Product-Moment correlation coefficient (r) was evaluated to assess

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possible correlation. All tests were performed at a 95% confidence level (α = 0.05).


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Additional samples for correlations: To expand the samples size for the correlations of NCTI

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values vs. SFI and SUVA, nine samples of municipal wastewater were used together with the 12

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samples from the four different sources to establish the correlations between cytotoxicity and

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SUVA or fluorescence indices. These samples were taken from different locations and sampling

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seasons that included early spring, early summer, peak summer, and late fall, encompassing a wide

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range of water qualities and temperatures. The nine additional samples included four chlorinated

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and ozonated unfiltered effluents from a facultative waste stabilization pond, and a maturation

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waste stabilization pond (named chlorinated MWSP2, ozonated MWSP2, chlorinated AWSP2,

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and ozonated AWSP2), two activated sludge samples taken at different times (named ASE2 and

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ASE3), and one ozonated sample of ASE3, and two non-disinfected and chlorinated municipal

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activated sludge effluents in Israel (named IS and chlorinated IS). All 21 samples were processed

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using the same protocols.

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RESULTS AND DISCUSSION

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Fluorescence Characteristics of the Studied Wastewater before and after Chlorination or

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Ozonation. Fluorescence EEMs of the source samples are presented in Figure 1a, 1d,1g, and 1j.

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The four key fluorescence peaks indicating the presence of the fluorophores in HA, FA, TP, and

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TY were resolved. For source wastewaters, despite having the highest DOC, MWSP1 had lower

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fluorescence peaks for all organic fractions than those of AWSP1, ASE1, or NIT (Figure 1a, 1d,

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1g, 1j and Table 3SI). Comparing non-disinfected wastewater samples from AWSP1 with those

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from ASE1, the fluorescence peaks of HA, FA, and TP were lower by 53, 17, and 33%,

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respectively, and higher by 26% in TY (Figure 1d, 1g, and Table 3SI). The fluorescence peaks of

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HA and FA in the non-disinfected ASE1 and NIT were very close. TP and TY in ASE1 and NIT

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were also slightly different (Figure 1g, 1j, and Table 3SI). For the MWSP1 sample, chlorination 10 Environment ACS Paragon Plus


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reduced the fluorescence peaks of HA and FA by 24 and 18%, respectively, and increased TP and

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TY by 22 and 16%, respectively (Figure 1a, 1b, and Table 4SI). In contrast, ozonation lowered the

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concentrations of all fluorophores, as compared to those in the non-disinfected MWSP1, by 42,

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37, 69, and 77% for HA, FA, TP, and TY, respectively (Figure 1a, 1c, and Table 4SI). For both

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chlorinated and ozonated AWSP1 samples, the fluorescence peaks in the FA region decreased by

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28 and 34% after chlorination and ozonation as compared to the non-disinfected AWSP1 sample,

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respectively (Figure 1d, 1e, 1f, and Table 4SI). Chlorination reduced the fluorescence peaks in the

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regions of TP, TY, and HA of the AWSP sample by 29, 54, and 30%, respectively, while ozonation

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reduced these fluorescence peaks by 38, 63, and 39%, respectively (Figure 1d, 1e, 1f, and Table

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4SI). Comparing non-disinfected ASE1 sample to chlorinated ASE1 sample, the latter showed

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about 36% reduction of the fluorescence peak in the regions of TP and TY, and about 56% for the

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HA, while ozonation lowered the fluorescence peaks of TP and TY by about 59% and by about

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47% for FA fluorescence peak (Figure 1g, 1h, 1i, and Table 4SI). For the NIT sample, chlorination

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reduced the fluorescence peaks of HA, FA, and TP by 32, 18, and 27%, respectively, and increased

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TY by 17% (Figure 1j, 1h, and Table 4SI). In contrast, ozonation lowered the concentrations of all

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fluorophores, as compared to those in the non-disinfected NIT, by 79, 65, 54, and 67% for HA,

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FA, TP, and TY, respectively (Figure 1j, 1i, and Table 4SI).The reduction in wastewater

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fluorescence after chlorination is in agreement with the observation reported in the previous

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studies.29,48–50 Possible reasons for the increase in the intensity of fluorescence peak of TP and TY

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for chlorinated MWSP1, and TY for chlorinated NIT (Table 4SI), after chlorination may include

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both formation of fluorescent DBPs and a selective degradation of naturally present quenchers of

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organic matter fluorescence. Consistent with previous observations,51–53 ozonation reduced the

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fluorescence intensities of major fluorophores, especially protein-like substances in all samples.


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The observed changes in the EEMs of fluorescence caused by chlorination or ozonation suggest

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breakdown or alteration of the organics in the treated wastewaters and perhaps the generation of

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DBPs.53,54

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CHO Cell Cytotoxicity of Concentrated Effluents. As shown in Figure 2, for non-disinfected

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samples, ASE1 and AWSP1 have lower cytotoxicity than NIT (P < 0.05, Table 5SI). These three

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samples were more cytotoxic than MWSP1 samples (P < 0.05, Figure 2, 3, and Table 5SI). The

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obtained higher cytotoxicity of NIT, compared to the other two secondary effluents AWSP1 and

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ASE1, is likely because the NIT sample was taken from a nitrification tower fed by a blend of

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activated sludge and trickling filter effluents. The measured higher cytotoxicity of NIT, compared

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to the other two secondary effluents AWSP1 and ASE1, is likely due to the fact that the NIT

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sample was taken from a nitrification tower fed by a blend of activated sludge and trickling filter

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effluents (Table 1SI). The significant 1.74-fold lower cytotoxicity of MWSP1 compared to

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AWSP1 (P < 0.05, Table 5SI) indicates that the maturation treatment process reduced the available

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cytotoxic materials in the wastewater (Figure 2a, 2b, and 3). For disinfected wastewaters,

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chlorinated samples generated at least 1.1-fold higher cytotoxicity than the non-disinfected

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controls for ASE1, AWSP1, and NIT, while chlorination of MWSP1 did not change the

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cytotoxicity (P > 0.05, Figure 3 and Table 5SI). Consistent with previous studies,23,31 ozonated

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samples were at least 1.6-fold less cytotoxic than the chlorinated samples (Figure 3), and were

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significantly less cytotoxic than the non-disinfected controls (P < 0.05, Table 5SI). AWSP1 that

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expressed the highest reduction in cytotoxicity after ozonation (2.4-fold decrease) showed the

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lowest increase in cytotoxicity after chlorination (1.1-fold increase).



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MWSP1, the wastewater possessing the lowest cytotoxicity but the highest DOC among the

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non-disinfected samples, produced the least cytotoxic chlorinated or ozonated wastewaters

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compared to the disinfected AWSP1, ASE1, and NIT samples (Figure 3).

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The increase in toxicity after chlorination is in agreement with a previous study,55 likely due to

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the reactions between chlorine and the organics in wastewaters that produced a number of

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halogenated aliphatic or aromatic compounds. Ozonation not only efficiently removes wastewater

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contaminants but also produces byproducts such as aldehydes, ketones, and organic acids56–59 that

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are in general less toxic than the precursors in wastewater or the halogenated DBPs from

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chlorination. Our observation that ozonationled to a lower toxicity compared to chlorination is

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likely a result of the increased degradation of toxic organic compounds in the studied wastewaters

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by ozone and increased formation of toxic halogenated DBPs with chlorination. These different

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trends observed for chlorination and ozonation suggest that between the two alternative

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disinfectants, ozone is a better candidate to minimize cytotoxicity.

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Correlation Analyses between CHO Cell Cytotoxicity and Spectroscopic Indicators. Linear

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and positive correlations were found among the DOC-normalized cytotoxicity index (NCTI)

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values of the non-disinfected, chlorinated, or ozonated samples and SFI values (Figure 4a, 4b, 4c,

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and Table 6SI). For a given SFI value, the NCTI ascending rank order was as follows: ozonated

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