Toxicological Comparison of Water, Wastewaters, and Processed

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Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Toxicological Comparison of Water, Wastewaters, and Processed Wastewaters Shengkun Dong,†,‡,⊥ Martin A. Page,¶ Nedal Massalha,‡,⊥,□ Andy Hur,¶ Kyu Hur,§,⊥ Katherine Bokenkamp,§,⊥ Elizabeth D. Wagner,§,⊥ and Michael J. Plewa*,§,⊥

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†

Guangdong Engineering Technology Research Center of Water Security Regulation and Control for Southern China, Key Laboratory of Water Cycle and Water Security in Southern China of Guangdong Higher Education Institute, Sun Yat-sen University, Guangzhou, Guangdong 510275, China ‡ Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, 205 N. Mathews Ave., Urbana, Illinois 61801, United States of America § Department of Crop Sciences, University of Illinois at Urbana-Champaign, 1101 West Peabody Dr., Urbana, Illinois 61801, United States of America ⊥ Safe Global Water Institute, University of Illinois at Urbana-Champaign, 1101 West Peabody Dr., Urbana, Illinois 61801, United States of America ¶ US Army Engineer Research and Development Center, 2902 Newmark Dr., Champaign, Illinois 61822, United States of America □ The Galilee Society Institute of Applied Research, Shefa-Amr, 20200, Israel S Supporting Information *

ABSTRACT: Drinking water utilities will increasingly rely on alternative water sources in the future, including wastewater reuse. Safety must be assured in the application of advanced oxidation processes (AOPs) and supporting treatments for wastewater effluent reuse. This study developed toxicological profiles for source and tap waters, wastewaters, and treated effluents by different processes from four military installation locations. The objective of this study was to evaluate the toxicity of extracted organics from diverse source waters and after reuse treatments. The toxicity analyses included thiol reactivity, mammalian cell cytotoxicity, and genotoxicity. Differences in toxicity between source or tap waters and effluents from wastewater treatment processes supported AOP treatment to reduce risks of potable reuse. An anoxic and aerobic activated sludge process followed by sand filtration controlled toxicity to levels similar to a municipal drinking water. An anaerobic membrane bioreactor process exceeded the toxicity levels of a typical drinking water. Two AOP processes (ultraviolet (UV) + reverse osmosis (RO) + chlorination (NaOCl) or RO + UV−H2O2 + NaOCl) significantly reduced toxicity. The integration of the wastewater systems with ultrafiltration, AOP, and RO was effective to reduce the toxicity to levels comparable to, or better than, tap water samples.

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INTRODUCTION Drinking water utilities must consider nontraditional water sources due to population growth, source water degradation and climate change.1−3 Within conventional water management frameworks, many local water sources are impacted by environmental discharges from upstream municipal wastewater treatment plants, resulting in de facto reuse.4 The application of advanced treatment technologies for the reuse of environmental discharge water represents a logical approach, as this alternate source is reliable and local.5 Advanced wastewater treatment technologies, including reverse osmosis (RO) and advanced oxidation processes (AOPs), provide alternate source waters for potable use. These management frameworks mitigate potential adverse environmental impacts by discharging reduced amounts of compromised water.1,6−8 Extended © XXXX American Chemical Society

drought was the genesis of the Colorado River municipal water district’s big spring raw water production facility in Big Spring, Texas. Since 2013, environmental discharge water was treated using microfiltration, RO, and AOPs. The treated water was blended with other source waters to feed the influent of a drinking water plant.9,10 Additional direct potable reuse (DPR) facilities are under construction or are being pilot tested. While these facilities produce clean water with fewer known contaminants than most influent sources, it is crucial to demonstrate that these advanced processes sufficiently control Received: Revised: Accepted: Published: A

February 12, 2019 June 22, 2019 June 25, 2019 June 25, 2019 DOI: 10.1021/acs.est.9b00827 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Table 1. Sample Matrix and Sample Descriptions Sample Number CERL-1 CERL-2 CERL-3 CERL-4 CERL-5 CERL-6

Sample Site Location

CERL-9 CERL-10 CERL-11 CERL-12

Fort Leonard Wood, MO Fort Riley, KS Fort Riley, KS Fort Riley, KS Fort Riley, KS Tobyhanna Army Depot, PA Tobyhanna Army Depot, PA Tobyhanna Army Depot, PA Fort Leonard Wood, MO Fort Leonard Wood, MO Moreland Hills, OH Moreland Hills, OH

CERL-13

Moreland Hills, OH

CERL-7 CERL-8

Sample Type

Sample Description

Gray water Tap water Source water Wastewater effluent Wastewater Tap water

Gray water from containerized shower systems in military field training area Treated by conventional softening, filtration, and chlorination Untreated, hard groundwater Treated in pilot scale Anaerobic Membrane Bioreactor (AnMBR) Wastewater from pump station Treated by conventional filtration and chlorination

Source water

Untreated, soft groundwater

Wastewater effluent

Effluent from centralized wastewater treatment plant with sequencing batch reactor

Gray water Recycled gray water On-site potable water Direct potable reuse water Wastewater effluent

Gray water from containerized shower and laundry systems in military field training area Treated by biofiltration, ultrafiltration, UV light, reverse osmosis, and chlorination Treated by filtration and UV light: Tangent Treated by ion exchange, ultrafiltration, reverse osmosis, UV−H2O2, and chlorination: Tangent Treated by anoxic/aerobic bioreactor and sand filtration: Tangent

insights concerning the potential for toxicity in wastewater reclamation.12,19,20 To the best of our knowledge, we present the first quantitative biological assessment of composite toxicity levels comparing advanced reuse scenarios at military bases. The objective of this study was to evaluate the modulation of toxicity of trace organics as a function of water source and treatment level at fixed Army installations. Trace organic contaminant mixtures were extracted to generate concentrated water samples (CWSs) that were analyzed for toxic characteristics. The toxicity analyses included thiol reactivity screening,17 Chinese hamster ovary (CHO) cell chronic cytotoxicity,15 and CHO cell acute genotoxicity for genomic DNA damage.15 These analytical in vitro assays were performed with the same biological platform that represented the largest quantitative comparative database for individual DBPs.15 Each assay captures a unique biologically relevant end point leading to a comprehensive measure of toxicity. Thiol reactivity detects toxicants that are reactive with biological thiols, a cellular defense against reactive oxidants. CHO cell chronic cytotoxicity captures a wide array of toxic insults by measuring the reduction in cell viability or impairments to the cell cycle as compared to untreated concurrent controls. CHO cell acute genotoxicity quantifies the level of genomic DNA damage in cell nuclei induced by toxic agents. These results provide direct comparisons of composite toxicity of trace organics from conventional and alternate water sources to evaluate DPR capability.

trace contaminants that may not be known or measurable, or potential composite effects of trace contaminants that could impair the environment or the public health.11 When implementing a new water supply technology, safety must be ensured. Unfortunately, in some cases, public perception of the potential for adverse health impacts from drinking advancedtreated wastewaters hindered the implementation of these projects.11,12 The Department of Defense (DOD) has long recognized water reuse as an opportunity for reducing logistics of expeditionary operations. Other drivers for water reuse at fixed military facilities include enhancing water security and reducing the impact on the environment. With approximately 1.9 × 1011 L of water consumed annually, the military is making progress toward the federal facility goal of a 26% reduction in water demand by 2020.4,6 Given that water reuse can drastically reduce net water demand, it is a key enabler for DOD resiliency and sustainability objectives. The U.S. Army Engineer Research and Development Center established a plan to assess direct potable reuse and other reuse frameworks for potential applications at fixed military facilities. These efforts will provide data to support health risk analyses specific to water reuse in military settings and address concerns by the general public. A key part of this effort is to incorporate toxicological approaches to augment water quality analyses and risk issues such as pathogen control. While risk assessments based on pathogens and contaminants of concern are well established, tools for assessing mixtures of known and unknown trace contaminants are not readily available. However, the integration of analytical biology to quantify mammalian cell cytotoxicity, genotoxicity, and other toxicity metrics can directly compare existing and alternate water sources.12 Such approaches have been used by the U.S. Environmental Protection Agency (EPA) and others to assess potential toxicity of water disinfection byproducts (DBPs).13−16 Employing these analytical biological assays to compare water sources could alleviate concerns of potential adverse health effects associated with direct potable reuse. Toxic agents are present in wastewaters; quantitative biological assessments of composite toxicity levels as a function of water source and treatment level is an emerging field of study.12,17,18 Quantitative comparative studies can provide

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MATERIALS AND METHODS Water Samples. Batch water samples were collected directly from process streams at four sites located in Kansas, Missouri, Ohio, and Pennsylvania, U.S.A. The test sites constituted four different design scenarios that are representative of current conditions or are under consideration for future resilient military operations. One site at Tobyhanna Army Depot in Pennsylvania served as a representative model for a conventional, centralized water supply and wastewater management framework at a military installation. The Fort Riley, Kansas site included conventional centralized water supply as well as a pilot study of a distributed low energy wastewater treatment unit with potential for future nonpotable water

B

DOI: 10.1021/acs.est.9b00827 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

acidified water sample was transferred onto the resin packed resin bed. The XAD resins were eluted with 400 mL of spectroscopy grade ethyl acetate, and the ethyl acetate extract was separated from the residual water. After being dried over anhydrous sodium sulfate, the extracts were concentrated to a volume of 2−3 mL using a vacuum rotary evaporator, and these were concentrated further under a gentle stream of nitrogen. The extracts were solvent exchanged into dimethyl sulfoxide (DMSO) and stored in amber vials with Teflon septa at −20 °C until use for toxicological analyses. The final concentration factor was 1 × 105. Biological and Chemical Reagents, CHO Cells. Widely used for in vitro toxicology, CHO cell line K1.AS52 (clone 11−4−8) was employed for the mammalian cell-based cytotoxicity and genotoxicity analyses.29,30 The CHO cells were maintained in Hams F12 medium containing 5% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotics (0.25 μg/mL amphotericin B, 100 μg/mL streptomycin sulfate, and 100 units/mL sodium penicillin G in 0.85% saline) at 37 °C in a mammalian cell incubator with a humidified atmosphere of 5% CO2. CHO Cell Chronic Cytotoxicity Analyses. By measuring the reduction in cell viability in comparison to that in untreated controls, cytotoxicity captures a wide array of toxic insults and adverse biological impacts. This assay measures cytotoxicity as the reduction in cell density after exposure of CHO cells to a CWS for 72 h (a chronic exposure encompassing 3−4 cell divisions) compared to that in untreated concurrent controls.15 For each experiment, a dilution series (generally 10 concentrations) was prepared by diluting the CWS into cell culture medium just prior to cell treatment. CHO cells (3 × 103 cells per well) were exposed to these treatment dilutions in 96-well microplates covered with AluminaSeal that prevented volatilization during the 72 h exposure period. After incubation in a mammalian cell incubator, the cell density per microplate well was determined by histological staining using crystal violet and absorbency at 595 nm using a microplate reader. The resulting data were saved as an Excel file. The dilution series generated from the CWS represented a range of concentration factors for the organics in the original water sample. The range in concentration factors was selected to span concentrations that induced no significant reduction in growth to concentrations that reduced cell density per microplate well. A cytotoxicity concentration−response curve for each CWS was generated from the summary data of the combined replicate experiments. The concentration factor associated with a 50% reduction in cell density compared to the negative controls (LC50) was calculated using regression analyses of the concentration−response curve. Detailed procedures for this assay and its use with water samples and individual chemical contaminants were published,15,31 and the results of this assay were used to develop risk assessment models for DBPs.32 Individual water sample cytotoxicity concentration−response curves are presented in the Supporting Information (SI) (Figures S1−S13). The lowest CWS concentration that induced a significant cytotoxic response, a summary of the mean LC50 (±SE) values, and the ANOVA statistical analyses are presented in Table S1. Single Cell Gel Electrophoresis Genotoxicity Analyses. The single cell gel electrophoresis (SCGE or comet) assay quantitatively measures genomic DNA damage such as DNA strand breaks, alkali-labile sites, incomplete excision

reuse. A third site included segregated gray water collection system at a field training area in Fort Leonard Wood, Missouri as well as an advanced treatment system that enables water recycling back into the containerized shower and laundry units. A final site included a commercial on-site DPR system (Tangent LLC, Chagrin Falls, OH) installed in a small commercial facility in Moreland Hills, OH. This system takes effluent from a conventional on-site wastewater treatment system and further purifies it using an advanced treatment train including RO, advanced oxidation, and supporting processes. From this broad range of sites, sample types included conventional source water, tap water, gray water from military shower and laundry systems, recycled gray water, wastewater, conventionally treated wastewater, and advanced-treated wastewater. The last two sample types, conventionally treated wastewater and advanced-treated wastewater, are collectively termed processed wastewater. The sample matrix is provided in Table 1. Water Sample Processing. Samples containing particulates (source water, wastewater effluent, or wastewater) were filtered through 0.2 μm microfiltration membranes. During the filtration process, the first 4 L of sample were discarded to limit potential filter adsorption effects. All samples were stored in Amber Type III soda-lime glass jugs that meet EPA performance-based specifications for semivolatile organics (Thermo Scientific 2452360). A 4 L portion of each sample was used for conventional water quality analyses, including measurement of TOC using a QbD1200 automated TOC Analyzer (Hach, Loveland, CO) according to Standard Method 5310C. An additional 20 L of each sample was delivered on ice within 24 h of collection to the University of Illinois at Urbana-Champaign for toxicity analyses. Water Sample Concentration. Within 24 h of sample receipt, the organics from each water sample were extracted over XAD resins and concentrated in spectroscopy grade ethyl acetate. Organic agents were extracted from the water samples using XAD 2/8 columns.21 We employed XAD resin extraction of organic micropollutants from water samples as recommended by the U.S. EPA.22 XAD-2 resin (Amberlite XAD-2, Millipore Sigma) isolates polyfunctional organic acids, aliphatic acids with 5 or fewer carbons, and low molecular weight solutes while XAD-8 resin (DAX-8, Millipore Sigma)23 isolates hydrophobic acid fractions, aliphatic carboxylic acids, aromatic carboxylic acids, phenols, and humic substances.24−26 We previously employed XAD resins to isolate organics from water samples for toxicological and chemical analyses, and the recovery of organics from different water types was between 64.6% and 69.5%.19,26−28 Virtually all of the cytotoxic- and genotoxic-responsive agents were recovered from water samples by XAD resins.26 Before extraction, 110 mL (wet volume) of the XAD-2 and XAD-8 resins were consecutively washed using Soxhlet extractions with spectroscopy grade solvents: methanol (400 mL), followed by ethyl acetate (400 mL), and finally methanol (400 mL), each for 24 h, respectively. A chromatography column (i.d. × length: 35 mm × 700 mm with a 1 L reservoir) was plugged with glass wool; this was followed by a 1:1 v/v mixture of XAD-2 and XAD-8 resins. The amount of resin was based on the volumetric ratio of water extract to resin and did not exceed 770:1. The packed column was rinsed with three resin volumes of deionized−distilled water, two resin volumes of 0.1 N HCl, one resin volume of 0.1 N NaOH, one resin volume of 0.1 N HCl, and two resin volumes of deionized−distilled water. Each C

DOI: 10.1021/acs.est.9b00827 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Table 2. Toxicity Index Value Results for All Water Samples Analyzed in the Study Design Scenario

Sample Number

Conventional Centralized Supply and Wastewater Management

CERL-6 CERL-7 CERL-8

Centralized Supply and Decentralized Low Energy Wastewater Treatment

CERL-2 CERL-3 CERL-4 CERL-5 CERL-1 CERL-9 CERL-10 CERL-11 CERL-12

On-site Gray Water Recycling

On-site Direct Potable Reuse

CERL-13

Sample Type Army Tap Water Army Source Water Wastewater Effluent Army Tap Water Source Water Wastewater Effluent Wastewater Gray Water Gray Water Recycled Gray Water Tap Water Direct Potable Reuse Water Wastewater Effluent

TOCa (mg/L)

TRI ± SEb

CTI ± SEc

GTI ± SEd

1.15 1.13 2.40

1.54 ± 0.08 1.17 ± 0.10 0.99 ± 0.06

9.58 ± 0.17 8.68 ± 0.28 12.25 ± 0.34

0.95 ± 0.01 0.91 ± 0 1.17 ± 0.02

1.25 1.61 7.76 35.4 54.0 34.0 2.10 6.00 2.80

1.18 ± 0.04 0.33 ± 0.01 27.22 ± 0.13 21.60 ± 0.62 NS 17.49 ± 0.33 0.92 ± 0.002 0.81 ± 0.01 0.13 ± 0

9.60 ± 0.22 21.07 ± 0.36 260.04 ± 10.0 219.21 ± 4.29 414.18 ± 5.70 148.34 ± 1.06 5.40 ± 0.1 4.11 ± 0.01 2.26 ± 0.02

1.82 ± 0.06 4.29 ± 0.10 NS NS NS NS NS 0.47 ± 0.01 NS

14.4

3.69 ± 0.06

12.58 ± 0.07

1.35 ± 0.03

TOC, total organic carbon. bTRI, mean thiol reactivity index values ± standard error of the mean. cCTI, mean cytotoxicity index values ± standard error of the mean. dGTI, mean genotoxicity index values ± standard error of the mean; NS, no significant difference from the negative control. a

responses if the thiol pool is overwhelmed or depleted.41,42 The NAC assay employed a 96-well plate format with a working volume of 100 μL per well. The assay was divided into two parts: first, the CWS was reacted with NAC for 20 min in a volume of 50 μL, followed with the addition of 50 μL 5,5dithiobis(2-nitrobenzoic acid) (DTNB) for resolution at A412. Each experiment consisted of concurrent negative controls, positive controls, sample concentrations of the CWS, and their corresponding blanks. For the negative control, each well had 40 μL of Tris buffer, pH 8, and 10 μL of 4 mM NAC. For the positive control, each well contained 38 μL of Tris buffer, pH 8, 10 μL of 4 mM NAC, and 2 μL of 10 mM maleimide. For the treatment groups, each well had 10 μL of 4 mM NAC, a serial dilution of the CWS, and Tris buffer, pH 8. The total volume of the sample and Tris buffer together was 40 μL. Sample blanks were important because they corrected for the background A412. Each corresponding blank well typically contained the identical volume of sample and Tris buffer at pH 8. After an incubation of 20 min with NAC on a rocker platform in the dark, 50 μL of 1 mM DTNB was added to the reaction mixture to quantify the available thiol groups. Directly after the addition of DTNB, the plate was analyzed at 412 nm on a microplate reader after linear shaking of 10 s. The data were saved in an Excel spreadsheet. The A412 values for each well were blank-corrected by subtracting the A412 values of the blanks from the corresponding A412 values for each CWS treatment group. The blank-corrected negative control data were averaged; this value was divided into the individual A412 values for each treatment group ×100, and the data were expressed as the percent of the concurrent negative controls. Using these normalized data, we generated concentration− response curves. Regression analyses were used to calculate the EC50 values: the effective concentration of the CWS that induced a reduction in the NAC-thiol response by 50% compared to that in the concurrent negative controls. Individual water sample thiol reactivity concentration− response curves are presented in the SI (Figures S27−S39). The lowest CWS concentration that induced a significant thiol reactivity response, the mean EC50 value (±SE), and a summary of the ANOVA statistical test results are presented in Table S3.

repair sites, and interstrand cross-links in the nuclei of cells.33−35 CHO cells (4 × 104) were treated in individual wells of a 96-well microplate with a series of concentrations of each CWS for 4 h at 37 °C. For each experiment, a concurrent negative control, a concurrent positive control of 3.8 mM ethylmethanesulfonate, and nine concentrations of a specific CWS were conducted. After treatment, the cells were removed from the microplate wells using a trypsin-EDTA solution. An aliquot of the cell suspension was used to determine the acute cytotoxicity by employing trypan blue vital dye.36 SCGE data were not used if the acute cytotoxicity exceeded 30%. The remainder of the cell suspension was incorporated into agarose microgels; the cell membranes were lysed in situ. The microgels were electrophoresed and stained with a fluorescent DNA binding dye to resolve the migration of damaged DNA streaming from the nucleus. The microgels were analyzed with a Zeiss fluorescence microscope with an excitation filter of 546/10 nm and a barrier filter of 590 nm. A computerized image analysis system (Comet Assay IV; Instem PLC, Staffordshire, U.K.) was applied to measure a number of specific SCGE parameters of the nuclei per microgel. The fluorescent intensity of the DNA that migrated away from the nucleus (%Tail DNA) was the primary metric of DNA damage that was used for the concentration−response curves.37 The digitalized data were automatically transferred to an Excel spreadsheet for subsequent statistical analysis. A regression analysis of the SCGE concentration−response curve was conducted to obtain the concentration factor that induced a 50% Tail DNA value. The details of SCGE analyses for individual DBPs or CWSs have been published.15,38 Individual water sample genotoxicity concentration−response curves are presented in the SI (Figures S14−S26). For each CWS, the lowest concentration that induced a significant genotoxic response, the mean 50% Tail DNA values (±SE), and a summary of the ANOVA statistical test results are presented in Table S2. N-Acetylcysteine Thiol Reactivity Analyses. We demonstrated that N-acetylcysteine (NAC) thiol reactivity was a predictor of adverse biological effects including CHO cell toxicity.17,39,40 The cysteine thiol is a major reductant against reactive toxicants, which can induce adverse biological D

DOI: 10.1021/acs.est.9b00827 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Statistical Analyses. Statistical analyses were conducted on the data for each toxicological end point assay (Tables S1− S3). After a concentration−response curve from combined replicate experiments was generated, a test for significance using a one-way analysis of variance (ANOVA) test was conducted. If a significant F value of P ≤ 0.05 was obtained, a Holm−Sidak multiple comparison versus the control group analysis was conducted with the power (1−β) ≥ 0.8 at α = 0.05 to identify the lowest concentration factor that was significantly different from the negative control.43,44 After nonlinear regression analyses of the three data sets, LC50 values were determined for CHO cell cytotoxicity, 50% Tail DNA values for CHO cell genotoxicity, and EC50 values for NACthiol reactivity. A bootstrap statistic was conducted for each assay data set, and mean toxicity index values (±SE) were calculated.45,46 When conducting direct comparisons of the biological and thiol reactivity responses, we used index values such that the larger the value, the more toxic or reactive the sample. The cytotoxicity index (CTI) value is the LC50−1 × 103; the genotoxicity index (GTI) value is the 50% Tail DNA−1 × 103; the thiol reactivity index (TRI) value is the EC50−1 × 103. Using these index values, an ANOVA test was conducted to identify significant differences among specific CWS groups.17

Figure 1. Comparison of the average toxicity index values of thiol reactivity (TRI), cytotoxicity (CTI), and genotoxicity (GTI) from concentrated water samples from Ft. Riley. CERL-3 was a source water sample; CERL-2 was tap water. CERL-5 was a wastewater sample, and CERL-4 was an AnMBR-treated wastewater sample. The error bars represent standard error of the mean (n = 8) generated by bootstrap statistics. NS indicates no significant difference from the negative control; the color indicates the specific toxicity index category.

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RESULTS AND DISCUSSION Table 2 summarizes the results by sample number and type for NAC-thiol reactivity, CHO cell cytotoxicity, and CHO cell genotoxicity. Detailed concentration−response curves and statistical parameters are provided in the SI. The comparisons of the water samples were divided into groups depending upon the military design scenario: centralized supply and decentralized low energy wastewater treatment, conventional centralized supply and wastewater management, on-site gray water recycling, and on-site DPR. Centralized Supply and Decentralized Low Energy Wastewater Treatment. This study leveraged an ongoing Army decentralized low energy wastewater treatment demonstration. Water samples from Fort Riley, KS represented two distinct categories (Figure 1). The centralized tap and source water samples (CERL-2, CERL-3) provided benchmark data to compare with the decentralized low-energy wastewater treatment system. The pilot wastewater related samples were CERL-4 and CERL-5. The source water was statistically more cytotoxic (P ≤ 0.01) than its corresponding tap water despite similar TOC values (Table 2). Compared to other source waters that we analyzed, the Fort Riley source water was substantially more toxic than what would be expected for groundwater. The genotoxicity analyses complemented the cytotoxicity data in that the source water was significantly more genotoxic than the tap water (P ≤ 0.001). As illustrated in Figure 1, the Ft. Riley untreated wastewater (CERL-5) was 10.4× more cytotoxic (P ≤ 0.001) and 66× more thiol reactive (P ≤ 0.001) than the source groundwater (CERL-3). The untreated or treated wastewaters did not induce a genotoxic response. Anaerobic membrane bioreactor (AnMBR) is a processing system that combines biological anaerobic treatment with physical separation membranes that returns particulate matter and biomass. The AnMBR treatment (CERL-4) did not reduce the toxicity of the untreated wastewater sample (CERL-5). Although the TOC of the AnMBR-treated wastewater was lower than that of the untreated wastewater by 78% (Table 2), the AnMBR-treated

wastewater was 26% more thiol reactive (P ≤ 0.001) and 19% more cytotoxic (P ≤ 0.001) than untreated wastewater (Figure 1). Both wastewater samples were 18.3× and 10.4× more toxic than the tap or source water samples. The AnMBR-treated wastewater also contained relatively high concentrations of micropollutants (data not shown). These data suggest that biological processes during anaerobic wastewater treatment, although beneficial regarding the reduction of TOC and biochemical oxygen demand, may produce nitrogenous metabolites and organic transformation intermediates that may add to the toxic burden of the processed wastewater. This finding suggests that AnMBR was good in removing organic matter but may release other organics with higher toxicity, though further studies with more diverse feed wastewaters may be needed. Therefore, AnMBR may need to be followed by other treatment processes in order to deal with effluent toxicity characteristics. Conventional Centralized Supply and Wastewater Management. The Tobyhanna Army Depot samples included a ground source water (CERL-7), tap water (CERL-6), and wastewater effluent (CERL-8). We determined any significant differences in thiol reactivity, CHO cell cytotoxicity, and CHO cell genotoxicity as a progression from source water to tap water to wastewater effluent (Table 2). For thiol reactivity, there was no significant difference between the source water and the wastewater effluent (P = 0.132). However, a significant difference in thiol reactivity was observed between source water and tap water, and between tap water and wastewater, (P ≤ 0.007 and P ≤ 0.001, respectively). The tap water was more cytotoxic than the source water (P ≤ 0.024) while the wastewater effluent was significantly more E

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Environmental Science & Technology cytotoxic than either the source water or the tap water (P ≤ 0.001 and P ≤ 0.001, respectively). The disinfected tap water was slightly more genotoxic than the source water (P ≤ 0.014), and the wastewater effluent was more genotoxic than the tap water (P ≤ 0.001) or the source water (P ≤ 0.001). These comparative data demonstrated that the assays employed were able to identify significant differences within a water process stream. They also provided a general baseline for a conventional system against which other design scenarios could be compared. On-Site Gray Water Recycling. On-site treatment of gray water, such that it can be recycled into the shower and laundry systems, is an important capability that is currently in development for military operations and training areas. In addition to the removal of pathogens, advanced treatment to remove trace chemicals should be applied, since chlorination of waters with high organic content may generate toxic DBPs.14,18 Sample CERL-1 was gray water from showers only, whereas sample CERL-9 contained gray water from both shower and laundry systems. Sample CERL-10 was collected downstream of the treatment system, which applies a series of biological filtration, ultrafiltration, and RO prior to residual disinfection with free chlorine (Table 1). No significant genotoxic response was detected in the on-site gray water or the advanced-treated recycled gray water. This result may be due to the lack of genotoxic agents or the high cytotoxicity of the samples. An ANOVA all pairwise comparison of the CTI values of the CERL-1, CERL-9, and CERL-10 samples demonstrated that they were statistically different from each other (P ≤ 0.001) (Figure 2). At Ft. Leonard Wood, both types of gray waters (CERL-1 and CERL-9) were at least 1.9 fold more cytotoxic than some of the most toxic chlorinated municipal secondary effluents from a previous study.12 The low CTI value of CERL10 indicated that the treatment train of biological filtration (BF) + ultrafiltration (UF) + UV radiation (UV) + reverse osmosis (RO) + HOCl reduced or removed approximately 96.4% of the extractable cytotoxic agents from the wastewater. The reduced cytotoxicity of CERL-10 was in concert with its reduced thiol reactivity (TRI = 0.92) as compared to its corresponding gray water sample (CERL-9 TRI = 17.49) (P ≤ 0.001). The removal of thiol-reactive agents by AOP treatment, and the corresponding reduction in cytotoxicity, is important because biological thiols are major cellular reductants that protect against oxidative stress and are involved in disease prevention.41 On-site Direct Potable Reuse. The samples from the DPR demonstration site included treated tap water (CERL11), effluent from an on-site wastewater treatment system (CERL-13), and advanced-treated water that was further subjected to RO and AOP purification (CERL-12) (Table 1). The on-site wastewater effluent (CERL-13) included conventional biological treatment followed by sand filtration. Even with these treatments, CERL-13 was cytotoxic and genotoxic and induced thiol reactivity to a significantly greater extent than the associated tap water sample (CERL-13 versus CERL11) (Figure 3). However, further processing of the water with advanced treatment, including RO and AOP (CERL-12), significantly reduced all toxicity end points of the feedwater (partially treated wastewater) to a level that was significantly lower than that of the tap water. Of importance were the facts that the AOP treated wastewater expressed no genotoxicity and the water quality defined by these toxicity characteristics was superior to a disinfected tap water.47 This observation was

Figure 2. Comparison of the average toxicity index values of thiolreactivity (TRI), cytotoxicity (CTI), and genotoxicity (GTI) from concentrated water samples from Ft. Leonard Wood. CERL-1 was gray water from showers, CERL-9 gray water was from showers and laundry systems, and CERL-10 was gray water after AOP treatment. The error bars represent standard error of the mean (n = 8) generated by bootstrap statistics. NS indicates no significant difference from the negative control; the color indicates the specific toxicity index category.

compared to CTI values derived from CHO cell cytotoxicity LC50 values from 11 European disinfected drinking waters.38 The CTI values for the European water samples ranged from 1.7 to 12.6, while the CTI value of the Tangent tap water was 4.11 (CERL-11) and that of the Tangent AOP-treated wastewater was 2.26 (CERL-12). The anaerobic + aerobic activated sludge process followed by sand filtration (CERL-13, CTI = 12.58) produced toxicity levels close to a municipal drinking water, although they were approaching the upper boundary. The DPR process eliminated the genotoxicity observed in the feed wastewater sample (CERL-13) and was similar to disinfected drinking waters from 11 cities.38 We speculate that AOP (UV−H2O2) significantly lowered the aromaticity, a characteristic significantly correlated to toxicity in a previous study.48 Toxicity Characteristics of Water Types. Toxicity assays that were previously employed for drinking water DBPs and wastewater evaluation were also effective in analyzing a variety of water and wastewater reuse processes. A comparison of the toxicity of 11 municipal disinfected drinking waters with the toxicity of the tap and source waters of the present study is presented in Figure 4. The horizontal blue line in Figure 4 represents the mean CTI value (7.3) for the municipal drinking waters;38 the dotted blue lines represent ±1 standard deviation of the mean. Note that this comparison is to various municipal tap (finished) waters, whereas the data collected in this study also includes untreated source waters. Only the Ft. Riley source water sample (CERL-3) deviated from this trend and expressed a high level of cytotoxicity. This example may F

DOI: 10.1021/acs.est.9b00827 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

Figure 4. Comparison of the average toxicity index values of thiolreactivity (TRI), cytotoxicity (CTI), and genotoxicity (GTI) from the XAD 2/8 organic extracts of the source and tap waters at Ft. Riley (CERL-3, CERL-2), Tobyhanna Army Depot (CERL-7, CERL-6), and Tangent Engineering (CERL-11) (Table 1). The error bars represent standard error of a minimum of 8 replicates. The solid blue horizontal line represents the average cytotoxicity index value of 11 municipal disinfected drinking (tap) waters;38 the dotted blue lines represent ±1 standard deviation. The solid red horizontal line represents the average genotoxicity index value of 11 municipal disinfected drinking waters; the dotted red lines represent ±1 standard deviation.

Figure 3. Comparison of the average toxicity index values of thiolreactivity (TRI), cytotoxicity (CTI), and genotoxicity (GTI) from concentrated water samples from Tangent Engineering. CERL-11 was a tap water internal control, CERL-13 was the on-site wastewater effluent after biological treatment and nutrient removal, and CERL-12 was the wastewater effluent after AOP treatment. The error bars represent standard error of the mean (n = 8) generated by bootstrap statistics. NS indicates no significant difference from the negative control; the color indicates the specific toxicity index category.

Compared to a typical municipal drinking water, these AOPbased DPR processes abated the toxicity. With the exception of one source water, the cytotoxicity and genotoxicity of the source and tap waters at these Army installations were similar to municipal drinking waters. We conducted a Pearson Product Moment Correlation test to identify relationships among total organic carbon (TOC), cytotoxicity (CTI), genotoxicity (GTI) and thiol reactivity (TRI) measurements of the water samples (Figure S40, Table S4). TOC was significantly correlated with TRI and CTI, r = 0.70; P ≤ 0.05 and r = 0.84; P ≤ 0.001, respectively. TOC and GTI were not correlated. TRI and CTI were highly and significantly correlated (r = 0.99; P ≤ 0.001) which agrees with our previous work.17 Finally, for this study, CTI and GTI values were highly and significantly correlated (r = 0.91; P ≤ 0.004). Thus, these toxicity metrics integrate well with standard measurements of water samples. Identifying individual causative chemicals for toxicity was beyond the scope of the study. These toxicity studies demonstrated the efficacy of this approach to evaluate the potential adverse health impacts of water and wastewater reuse systems and provide valuable guidance for engineering designs. Safe water is a commodity that cannot be compromised. As the world turns to increasingly engineered solutions that alter conventional frameworks, it is prudent to fully explore all potential risks. The results of this study support the need for advanced treatment technologies that convert wastewaters into a reliable and safe source for potable supply.

indicate that low levels of contamination in source waters with toxicity potential can be mitigated by drinking water treatment processes (CERL-3 vs CERL-2). The horizontal red line in Figure 4 represents the mean GTI value (0.684) for the municipal drinking waters;38 the dotted red lines represent ±1 standard deviation of the mean. The genotoxicity of installation source and tap waters were similar to that observed with municipal drinking waters. However, the source water (CERL-3) and the resulting tap water from Ft. Riley (CERL-2) expressed increased levels of genotoxicity of 6.3× and 2.7×, respectively. The goals of this work were to determine if advanced treatment technologies reduced toxicity in processed water and compare the toxicity of potable reuse waters versus conventional source and drinking waters. In grouping the water samples by type (Figure S40), trends became apparent for some but not all of the index values. Wastewaters were more toxic than source or tap waters, as expected. However, the smaller differences between source or tap waters and effluent from wastewater treatment processes supported the case for advanced treatment to reduce risks of potable reuse activities. Among biological-based wastewater treatment processes, an anaerobic + aerobic activated sludge process followed by sand filtration controlled toxicity to levels similar to a municipal drinking water; whereas an AnMBR process effluent exceeded the toxicity levels of a typical drinking water. Two advanced treatment configurations (BF + UF + UV + RO + NaOCl and water softening + UF + RO + UV−H2O2 + NaOCl) significantly reduced nearly all of the toxicological end points. G

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.9b00827.

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Detailed data on individual water samples and statistical analyses (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Shengkun Dong: 0000-0002-0264-0621 Nedal Massalha: 0000-0003-4265-9869 Elizabeth D. Wagner: 0000-0002-3198-2727 Michael J. Plewa: 0000-0001-8307-1629 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge funding from the Army Environmental Quality Technology program of the Assistant Secretary of the Army for Installations, Energy and Environment, via grant number CESU W9132T-16-2-0005 (M.J.P.) administered through the U.S. Army Engineer Research and Development Center. Vaadia-BARD Postdoctoral Fellowship Award No. FI552-2016 is acknowledged for supporting N.M. Chris Otto (Fort Riley), Amalia O’Brien (Tobyhanna Army Depot), and Adam Arnold (Tangent LLC) helped to coordinate site sampling. This work was conducted at the U.S. Army Construction Engineering Research Laboratory (CERL), Champaign, IL and at the University of Illinois at UrbanaChampaign, Urbana, IL.

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DOI: 10.1021/acs.est.9b00827 Environ. Sci. Technol. XXXX, XXX, XXX−XXX