Application

for the

Zeno Karl Schindler/EPFL Prize Environmental Sciences and/or Sustainability

Candidate:

Dr. Heather BISCHEL

Laboratory of Environmental Chemistry (LCE) Institute of Environmental Engineering (IIE) Faculty of Architecture, Civil and Environmental Engineering (ENAC)

March 2017

CONTENTS

Nomination Letter, Professor Tamar Kohn (EPFL)

List of International Referees

Research Report

Executive Summary

Background

Part I. Identification of Human and Environmental Health Hazards in Source-Separated Urine intended for Fertilizer Production1

Part II. Liquid Fertilizer Production: Evaluating the Potential for Biological Nitrification to Reduce Human Health Hazards in Source-Separated Urine2

Part III. Solid Fertilizer Production: Reducing Human Health Hazards in Source-Separated Urine through Struvite Production3

Part IV: Quantifying microbial risks of field-scale struvite production from source-separated urine using first-person videography exposure assessment4

Summary Implications

Curriculum Vitae

Publication List

Selected Additional Publications

1 Bischel, H.N., Özel Duygan, B.D., Strande, L., McArdell, C.S., Udert, K.M., Kohn, T., 2015. Pathogens and pharmaceuticals in source-separated urine in eThekwini, South Africa. Water Res. 85, 57–65. 2 Bischel, H.N., Schertenleib, A., Fumasoli, A., Udert, K.M., Kohn, T., 2015. Inactivation kinetics and mechanisms of viral and bacterial pathogen surrogates during urine nitrification. Environ. Sci. Water Res. Technol. 1, 65–76. 3 Bischel, H.N., Schindelholtz, S., Decrey, L., Bosshard, F., Buckley, C., Udert, K., Kohn, T., 2016. Bacteria inactivation during the drying of struvite fertilizers produced from stored urine. Environ. Sci. & Tech. 50.23 (2016): 13013-13023. 4 Bischel, H.N., Caduff, L., Schindelholtz, S., Kohn, T., Julian, T., 2017. Quantifying microbial risks of field-scale struvite production from source-separated urine using first-person videography exposure assessment. Manuscript in preparation.

Laboratory of Environmental Chemistry ENAC – IIE – LCE, Station 2 CH - 1015 Lausanne, Switzerland

Tél. : 0041-(0)21-693.08.91 Fax : 0041-(0)21-693.63.90 [email protected]

Lausanne, February 25, 2017

Nomination of Dr. Heather Bischel for the Zeno Karl Schindler Award

Dear members of the search committee:

It is a pleasure to provide my most enthusiastic support on behalf of Dr. Heather Bischel’s nomination for the 2017 Zeno Karl Schindler Award. I have professionally known Heather since the fall of 2012, when she joined my group as a postdoc. She had specifically sought out my group because of her wish to participate in the “VUNA” project. This project is a large collaboration with researchers from the Swiss Federal Institute of Aquatic Science and Technology (Eawag) and various institutions in Durban, South Africa. In an aggressive push to promote sanitation, over 80,000 urine-diverting dry toilets have been installed since the early 2000s in peri-urban regions of Durban. The goal of the VUNA project is to enable the recovery of nutrients from urine collected in these dry toilets, to ultimately produce a valuable fertilizer product that locally creates revenue. This project thus promotes the implementation of sanitation in areas where it is currently lacking, by offering an economic incentive for collecting urine.

What intrigued Heather about this project is that it encompasses many different aspects relevant to environmental protection and sustainability: it addresses not only public health goals (reduction of disease), but also environmental pollution issues (reduction of nutrient discharge) and societal and economic benefits (creation of revenue). However, a very important aspect of the project that was lacking, namely an assessment of the health risks of fertilizer produced from human waste. Human excreta contain pathogens, and as such are an inherently problematic matrix. Unless the pathogens can be controlled during fertilizer production, the end product will not gain consumer acceptance. Heather thus set out to assess the risks exerted by pathogens and create mitigation strategies. Specifically, she i) developed a molecular screening method for various viral and bacterial pathogens in urine; ii) applied this method in field monitoring campaigns in South Africa to inventory the pathogens present in collected urine; iii) determined if and how these pathogens can be controlled in down-stream fertilizer production methods; and iv) conducted a microbial risk assessment to determine if the presence of pathogens in urine constitutes a risk to those collecting and processing urine as well as to end users of the fertilizer product. To date, Heather has determined that – despite often being viewed as sterile - collected urine does indeed contain a wide array of both viral and bacterial pathogens. Furthermore, these pathogens are only partly inactivated during the fertilizer production methods proposed by the VUNA project. She then did an in-depth assessment of the main mechanisms leading to pathogen inactivation during fertilizer production. This allowed her to outline how minor adjustments of the treatment chain could result in significantly more hygienic outcomes.

A truly unique aspect of Heather’s project is its completeness from both a scientific and engineering point of view. The project not only asks: “Is there a problem?”, but also “How do we solve it?” and “Does this solution actually have an impact?” This holistic approach is the hallmark of a skilled engineer and methodical researcher. Consequently, Heather rapidly emerged as one of VUNA’s main drivers, and she has greatly influenced VUNA’s future direction.

Thus far, Heather’s work has resulted in three published journal articles in highly regarded journals. An additional manuscript is in preparation for a journal of equal caliber. I consider this output to be very impressive, given the

breadth of Heather’s work. She had to independently develop all tools and concepts needed for her project, ranging from molecular methods to pilot reactor operation to risk modelling. In addition, she organized and conducted several field campaigns, under often challenging circumstances with respect to both logistics and working conditions. And last but not least, Heather also managed to obtain her own funding from no fewer than three sources to support her work on this project. She obtained a US NSF postdoc fellowship, a travel award from the Rotary Foundation, and she was instrumental to the writing of a joint Eawag-EPFL proposal to support her work. Impressively, Heather managed to accomplish all this despite also having a baby and taking a maternity leave during her time at EPFL.

Heather was a candidate for the 2016 Zeno Karl Schindler Award. Since that time, she completed an important aspect of her research that brings her laboratory and field work into greater context through health risk modeling. This project required developing new skills in computer modeling, which she did independently while I was on sabbatical and through building new collaborations. She also presented her work at multiple scientific conferences and invited seminars. Heather recently accepted a position as an Assistant Professor of Environmental Engineering at the University of California, Davis, a well regarded institution known for its high-level research and its commitment to applied environmental sustainability.

I believe Heather will be a leader in the field of Environmental Engineering and Sustainability. She is not only an outstanding researcher and engineer, but also a dedicated mentor and teacher. She is furthermore a pleasant, energetic individual with great communication skills, who will serve as a great role model for young women in science and engineering. I am certain that – given her extraordinary dedication to environmental issues and sustainability and her high skill level - Heather will continue to create a positive impact on environmental research and on the training of future environmental engineers. A recognition of Heather’s work of the caliber of the Karl Zeno Schindler Award would certainly bring well-deserved attention to her research accomplished at EPFL, and will help to pave her way for a successful career as an independent researcher.

Sincerely yours,

Tamar Kohn, PhD Associate Professor Swiss Federal Institute of Technology, Lausanne (EPFL) LIST OF INTERNATIONAL REFEREES

Professor Krista Wigginton Borchardt and Glysson Water Treatment Faculty Scholar Dept. of Civil & Environmental Engineering University of Michigan

Email: [email protected] Phone: (734) 763-9661

Dr. Wigginton's research focuses on applications of environmental biotechnology in drinking water and wastewater treatment.

Professor Alexandria Boehm Associate Professor, Dept. of Civil & Environmental Engineering Stanford University

Email: [email protected] Phone: (650) 724-9128

Dr. Boehm is an expert in environmental microbiology with a focus on waterborne pathogens.

Professor Kara L. Nelson Department of Civil and Environmental Engineering University of California, Berkeley

Email: [email protected] Phone: +1 510-643-5023

Dr. Nelson is an expert in the detection, removal, and inactivation of pathogens in water and sludge, water reuse, nutrient recovery, drinking water and sanitation in developing countries.

Professor Treavor H. Boyer Associate Professor of Environmental Engineering Environmental Engineering Undergraduate Program Chair School of Sustainable Engineering and the Built Environment Arizona State University

Email: [email protected] Phone: 480-965-7447

Dr. Boyer is an expert in water sustainability and in particular urine separation and treatment as catalysis for new research directions in the water lifecycle.

Alexandria B. Boehm Dept. of Civil & Environmental Engineering Professor Y2E2 Room 189 Environmental Engineering & Science Program Stanford University (650) 724 – 9128, [email protected] Stanford, CA 94305-4020

April 11, 2017 Re: Heather Bischel’s nomination for the Zeno Karl Schindler Prize

To whom if may concern,

I enthusiastically recommend Dr. Heather Bischel for the Zeno Karl Schindler Prize. I have known Heather for over a decade, since she was a senior undergraduate at UC Berkeley. She was Stanford’s Environmental Science and Engineering MS/PhD Program’s top graduate school applicant in 2004 and I was involved in recruiting her to Stanford for her graduate degrees. I have taught Heather in three courses, worked with her in a student club as a faculty advisor, informally advised her on her PhD work and career, written a chapter with her on Oceans and Human Health for a book, and recommended her as a peer-reviewer for the California Ocean Protection Council’s research products. I have also attended a number of national and international conferences with her.

I will go into detail about Heather’s outstanding abilities below, but I want to take a moment to inform you about Heather’s most excellent quality – leadership. Since arriving at Stanford in 2004, Heather repeatedly volunteered to take leadership positions for student groups. She was president of Engineers for a Sustainable World (ESW), a national student group that leads projects to help those in developing countries with engineering projects. While she was president, the group won the Dean’s award for excellence for student groups and was awarded a large trophy. Under her leadership, the group successfully fundraised to send students to foreign countries to engage in projects. The success of the group had very little to do with their faculty advisor (me!) as I was barely involved. It is all attributable to the strength of their leadership - Heather. Her efforts for this student group were amazing, considering that at the same time, she was pursuing a PhD in environmental engineering. Besides being the president of ESW, Heather also assumed the role as student leader of a recently awarded NSF Engineering Research Center on Water at Stanford. She was in charge, with assistance from Prof. Kara Nelson (UC Berkeley) and me, of organizing student-centric education and outreach activities descriptions in the center proposal. Text she wrote was placed into the main proposal to NSF. The multi-million dollar proposal was selected for funding! Heather remained involved in the center during her post doctoral internship at Stanford which lasted just 6 months.

Heather took three classes from me and she performed at the very top of each class. Most notable was her performance in a class I team-teach with 2 lawyers (Meg Caldwell and Debbbie Sivas) “The CA coast: Science, policy, and law”. This course takes a detailed look at how science informs environmental / marine policy at the local, state, and federal level. We instill in the students the ideas that uncertainty is important and politics matter. We discuss the conflicting scientific and law doctrines that embrace and reject uncertainty, respectively, and how policies must navigate uncertainty. Heather was at the very top of the class. She mastered law readings and policy readings and was one of the most insightful when it came to in-class debates and discussions. Of course Heather’s main expertise is

Phone: 650-724-9128 • Fax: 650-725-3164 • E-mail: [email protected] • Web: www-cee.stanford.edu 1 engineering and science and her quantitative abilities and laboratory skills are excellent. She took a laboratory class from me “Environmental health microbiology” where she learned skills to enumerate pathogens in water. She also took “Coastal contaminants” from me, a class on coastal pollution that requires knowledge of physics, chemistry and biology.

I served as the co-chair of the Science Advisory Team (SAT) for the California Ocean Protection Council (OPC) and occasionally, the SAT asked to recommend peer reviewers for scientific projects commissioned by the OPC – all of which were commissioned exclusively to inform policy. Heather served as a peer-reviewer for one of these documents, so she had a glimpse first hand into science documents generated to inform policy. Her peer-review was excellent.

Heather and I co-authored a chapter “Oceans and Human Health”1. Heather is an excellent writer and it was a pleasure working with her on the chapter. She was reliable and punctual; she completed her writing sections independently and on schedule.

Scientifically, Heather is top notch. She published her PhD work in top tier environmental journals. Her PhD work was in emerging chemical contaminants. Her recent work in Prof. Kohn’s lab in Switzerland has focused more on microbiology in addition to biotechnology and chemistry. Finally, she has some experience in social science: she worked on a project that interviewed water professionals about barriers to water reuse. The results were published in ES&T.

I have reviewed Heather’s recent work on the urine separating project in South Africa and I am extremely impressed. I saw Heather present on some of her initial findings at a conference in Brazil in 2013. After being at EPFL working on the project for a short time, Heather delivered one of the best talks at the conference showing the presence of enteric pathogens in urine from the project. The audience, including me, was completely blown away with her talk. I was shocked that she was already an expert in molecular biology having just started her work at EPFL. I read through the papers she has published on the project and I am not surprised at the breadth and depth of the work. It aligns with what I already know about Heather. Her full consideration of the problem of urine separation from pathogens to chemicals, to treatment possibilities, to human health risks reminds me her similar full consideration of problems she worked on as a PhD student. I know of no other young researcher with her abilities. I am equally impressed with her ability to raise money for her research, but again, am not surprised given her success at this as a student.

I very strongly recommend Heather for this prize. Not only is she incredibly brilliant, a great oral and written communicator, she is a wonderful person who can work alone or on a team, is self-motivated, and has extremely strong leadership skills. Heather is one of the top, if not the top, young researcher in the world in our field of environmental science and engineering.

The description of the Zeno Karl Schindler Prize that I read online indicates the prize should go to someone who has completed distinguished postdoctoral work (high level research project and publications) of particular excellence performed at EPFL, in the field of

1 A. B. Boehm and H. N. Bischel. 2011. Oceans and human health. A chapter in Encyclopedia of Environmental Health edited by Jerome Nriagu. Volume 4, pages 223- 230. Burlington, Elsevier.

Phone: 650-724-9128 • Fax: 650-725-3164 • E-mail: [email protected] • Web: www-cee.stanford.edu 2 environmental sciences and/or sustainability. Heather’s work at EPFL (and elsewhere) certainly strongly fits this description. I believe she stands out well in front of her peers and those post doctoral researchers before her. The work she has done on the urine separation has had a strong impact and will continue to do so.

If you have any questions about her, please do not hesitate to call me or email me anytime.

Sincerely,

Alexandria Boehm

Phone: 650-724-9128 • Fax: 650-725-3164 • E-mail: [email protected] • Web: www-cee.stanford.edu 3

1351 Beal Ave ■ 181 EWRE ■ Ann Arbor Michigan 48109-2125 PH: 734-763-9661 ■ FAX: 734-764-4292 ■ [email protected]

April 19. 2017

To Whom It May Concern:

I am pleased to write this letter to support Dr. Heather Bischel for the Zeno Karl Schindler/EPFL Prize. I am an assistant professor of environmental engineering at the University of Michigan and have known Heather and her work since she was a graduate student at Stanford.

As her CV reveals, Heather has been extremely productive through her graduate studies and postdoctoral research. She has published in a diverse set of high-impact journals, she has demonstrated an ability to acquire competitive grant money, and has experience in teaching and mentoring students. I have no doubt she will be successful in her tenure-track position at UC Davis.

Her postdoc research focused on a topic that I am quite familiar with. Here at the University of Michigan, we have a large project on urine diversion funded by the US NSF. I believe Heather has made outstanding contributions to this topic, ranging from the mechanistic fate of pathogens in the production of fertilizer, to the risks posed by the collection and treatment of urine in developing country settings. The research has been highly collaborative and interdisciplinary, and has spanned from highly technical method development at EPFL to field-based behavioral research in developing world settings. Her three first-author manuscripts are in excellent journals.

I was excited to hear that Heather was offered the tenure-track position at UC Davis. This coveted position confirms that she is a leading young scientist in our field. I look forward to hearing about the work she initiates in her new position!

In closing, I encourage you to award Heather the Zeno Karl Schindler/EPFL Prize. Please let me know I can provide you with any more information on Heather’s qualifications

Sincerely,

Krista Rule Wigginton Assistant Professor of Environmental Engineering

UNIVERSITY OF CALIFORNIA, BERKELEY

BERKELEY • DAVIS • IRVINE • LOS ANGELES • RIVERSIDE • SAN DIEGO • SAN FRANCISCO SA NTA BAR BARA • SA NTA CRU Z

KARA L. NELSON, Professor Department of Civil & Environmental Engineering 663 Davis Hall University of California Berkeley, California 94720-1710 Tel. 510-643-5023 [email protected]

May 6, 2017

Dear Members of the Award Committee,

It is my sincere pleasure to provide this letter to support the nomination of Dr. Heather Bischel for the ZKS Foundation Prize. I have known Heather since she was an undergraduate in our CEE program at UC Berkeley. She stands out as one of the shining stars of that time. I stayed in contact with Heather when she was a PhD student and then postdoctoral researcher at Stanford; during her last year we interacted regularly through our US National Science Foundation research center called ReNUWIt. Heather demonstrated her strong leadership abilities through taking on an officer position in the Student Leadership Council. After finishing at Stanford, Dr. Bischel was a postdoctoral researcher with Prof. Tamar Kohn at EPFL; recently she started as an assistant professor at University of California, Davis. In 2015 I was on sabbatical in Zurich and had multiple opportunities to visit with her in Switzerland. My own research areas are very closely related to hers and I have thoroughly enjoyed discussing our research interests and results. From all of these interactions, I believe I am in an excellent position to judge the merits of her work.

Overall, Dr. Bischel has a very productive research track record, which is of uniformly high quality. Impressively, she has worked in at least three different research areas that are quite distinct: the fate of perfluorinated chemicals in the environment, the ecological impacts of streamflow augmentation with recycled water, and most recently the fate of pathogen indicators and pharmaceuticals in source-separated urine. The research materials submitted as part of her nomination package are related to this third area of research. I was already familiar with the first three papers as I read them when they were first published. I am very pleased to see the fourth paper, which builds on the first three by using the results on pathogen fate in urine to quantify health risks to workers collecting and producing fertilizer (struvite) from urine. To determine exposure, Dr. Bischel conducted field work using creative and careful methods, and combined the results with the laboratory results in a rigorous modeling framework. As a body of work, these four papers provide a thorough foundation for how to approach recovery of resources from urine while addressing potential human health and ecological risks due to the presence of pathogens and pharmaceuticals, respectively. I consider Dr. Bischel’s work to be a model for how high-quality research can contribute to addressing grand challenges facing humanity, in particular more sustainable management of water supplies and human waste. She approaches her work with absolute dedication, and brings her cheerful, warm personality and optimism to her work, which is a source of inspiration to all who interact with her.

In summary, I believe Dr. Bischel is a fantastic candidate for your prize, and I support her nomination enthusiastically.

Sincerely,

Kara L. Nelson

Research Report

for the

Zeno Karl Schindler/EPFL Prize Environmental Sciences and/or Sustainability

Candidate:

Dr. Heather BISCHEL

Laboratory of Environmental Chemistry (LCE) Institute of Environmental Engineering (IIE) Faculty of Architecture, Civil and Environmental Engineering (ENAC)

March 2017

EXECUTIVE SUMMARY

Regarding human waste as a valuable resource has the potential to transform waste management and the sanitation paradigm – promoting resource recovery and social enterprise, reducing discharge of harmful nutrients and other contaminants to the environment, replacing synthetic fertilizer sources, and increasing access to sanitation for improved human health. One significant obstacle towards successfully modernizing sanitation practices into ones that systematically recover and reuse waste is the need to assure human health safety in the production and application of waste-derived products. This is especially important in resource- poor settings, where an added challenge to safe waste reuse is limited financial capacity for technology implementation. Breaking down barriers to the acceptance of waste as a resource includes conducting transparent risk assessments and comprehensively addressing human health concerns. Innovative and cost- effective fertilizer product generation processes must also serve as robust waste treatment technologies. Dr. Bischel came to EPFL to work towards these goals, in collaboration with researchers and practitioners at the Swiss Federal Institute of Aquatic Science and Technology (Eawag) as well as eThekwini Water and Sanitation (EWS) and the University of Kwazulu-Natal (UKZN) in South Africa.

Researchers at Eawag have pioneered technologies to produce marketable fertilizers from urine. Urine is the primary source of nutrients in wastewater and can be collected separately from solids through urine-diverting toilets (UDTs). Thanks to progressive, forward-thinking leadership by EWS in the early 2000s, the region surrounding eThekwini (Durban) on South Africa’s northeast coast now boasts the largest network of UDTs in Africa. The source-separated urine is not yet collected for reuse at scale, it is discharged on-site, but the municipality is ripe for action on this front should the technologies prove cost-effective, efficient and safe. In 2010, Eawag began a partnership with EWS in a project called “VUNA” (Valorization of Urine Nutrients in Africa) to optimize and refine biological and chemical treatment processes to produce various urine-derived fertilizers. Fertilizer production processes were developed and implemented at a pilot scale in the first two years of the project, but regard for the human health safety of the urine and the efficacy of the treatment for human and environmental contaminants remained missing. Dr. Bischel joined the project in November 2012 to lead the assessment of the health and hygiene aspects of urine collection, processing and reuse. This research report details Dr. Bischel’s contributions towards evaluating health and hygienic hazards in the use of human urine and urine-derived products as fertilizers and to developing strategies to reduce health risks through enhanced treatment processes.

Part I of this report identifies human and ecotoxicological hazards in source-separated urine intended for fertilizer production in eThekwini, South Africa. Dr. Bischel designed and conducted a field sampling campaign to collect urine from geographically dispersed urine-collection tanks in rural townships and peri- urban settlements of eThekwini. Using a series of polymerase chain reaction (PCR) assays, Dr. Bischel detected a suite of bacterial and viral pathogens that are transmitted by the fecal oral route in all samples. Rotavirus, the cause of five percent of all deaths in children under five, was present in about a third of samples. The bacteria Shigella spp., which causes one of the most severe forms of bacterial-induced diarrhea, was found in approximately two thirds of samples. In the context of resource reuse, source-separated urine is often falsely considered “sterile” or nearly so, and low in pathogen risk. These results provided important evidence to the contrary. Collaborators at Eawag also analyzed the samples for 12 pharmaceutical compounds expected to be widely used in the region. Dr. Bischel calculated that a one-time application of urine to soils, if used directly as fertilizer for a growing season, could yield ecologically harmful concentrations of sulfamethoxazole, the most highly concentrated of the pharmaceuticals detected in the urine. To evaluate potential human health risks of sulfamethoxazole, she analyzed the urine for a sulfonamide antibiotic resistance gene, which she detected in 100% of the samples, and at high levels. While alarming, the breadth and magnitude of human and environmental health hazards Dr. Bischel and her collaborators detected in source-separated urine support the need for urine collection, and treatment for reuse, in order to reduce the discharge of these contaminants to the environment as well as their potential exposure to nearby residents. However, the fate of microbial pollutants during urine treatment and fertilizer production processes remained incomplete.

Following this field study, Dr. Bischel evaluated the potential for innovative fertilizer production technologies to inactivate or remove bacteria and viruses in urine and determined factors that govern pathogen inactivation and removal in urine recycling technologies. This work was comprised of two components: (1) evaluating pathogen and pathogen surrogate inactivation during the biological nitrification of urine to produce a stabilized liquid fertilizer, and (2) assessing the inactivation of in situ bacteria and pathogen surrogate organisms during the chemical precipitation and drying of struvite, a solid fertilizer, from urine.

Part II of this research report assesses pathogen inactivation performance and mechanisms in urine nitrification reactors. While biological wastewater treatment for nutrient and organics removal is widely applied, little is known regarding the inactivation of bacterial and viral pathogens during nitrification. Additionally, nitrification has only recently been used for the production of liquid agricultural fertilizers from urine, with pilot reactors now operating in Switzerland and South Africa. Laboratory urine nitrification reactors were built based on the pilot-scale reactors in eThekwini. Inactivation of two bacteria and three viruses was evaluated during nitrification, yielding the conclusion that biological nitrification as a fertilizer production process remains insufficient as a standalone technology for the sanitization of source-separated urine. We further utilized controlled laboratory reactors to assess the influence of physical (i.e., aeration) and biological (i.e., microbial activity) processes on inactivation as well as the role of chemical solution characteristics of nitrified urine. Importantly, a protective effect of the solution components for certain viruses in aerated nitrification reactors was identified. Downstream processing of the liquid fertilizer via distillation was recommended to ensure a microbially safe endproduct. The resulting manuscript presents the only quantitative study of pathogen inactivation during urine nitrification to date and provides comprehensive information regarding inactivation processes for nitrification systems. Because field-relevant treatment conditions were applied, the results of this work also apply broadly to the nitrification of high-ammonia concentration solutions, such as discharge from sludge dewatering facilities.

Part III of this report details an evaluation of the microbial treatment potential of struvite production from urine and recommendations to enhance the inactivation of potentially harmful bacteria in the endproduct. Growing interest has been placed in the simple process of struvite production from urine as a means for phosphorus recovery and to produce a market-attractive product (a solid product with less foul odor). However, the fate of bacteria in urine during struvite recovery and processing has not been previously described. Dr. Bischel established a field laboratory in eThekwini to monitor the in situ inactivation of heterotrophic bacteria in pilot-scale struvite reactors. In complementary laboratory studies, she evaluated bacteria inactivation mechanisms using a range of analytical techniques and target organisms, including flow cytometry as a comprehensive method to monitor the inactivation of all detectable bacteria (rather than only those that are culturable). Heterotrophs in stored urine reflected the behavior of total bacteria as detected by flow cytometry and proved to be a useful surrogate for pathogen inactivation in the field. Dr. Bischel found that while viruses and helminth (worm) ova require thorough desiccation of struvite for effective treatment, initial wet heating of the product prior to drying is required to ensure inactivation of dehydration tolerant bacteria. Such nuanced recommendations were made possible by the mechanistic understanding of inactivation developed in this study. The resulting manuscript published in Environmental Science & Technology reports this detailed and comprehensive study of bacterial inactivation during struvite production as well as recommendations to ensure the safety of the fertilizer endproduct.

Part IV of this research report reviews Dr Bischel's final contribution from her postdoctoral work at EPFL in which she sought to contextualize her laboratory and field work by developing a model of microbial health risks associated with urine collection and treatment. The unique study coupled innovative exposure analysis techniques (e.g., the use of GoPro cameras for personalized data collection) with comprehensive model development to evaluate the risks of a range of scenarios for urine reuse via struvite production. The goal of this work was to identify breaks in the waste reuse chain that may lead to unnecessary health risks and to thus point to solutions for addressing such health risks. Having completed the evaluation of treatment potential and mechanisms of the VUNA fertilizer production processes, Dr. Bischel recognized that some of the greatest risks in fertilizer production and may be those related to the exposure of municipality staff during the manual production of struvite from urine. Therefore, Dr. Bischel conducted a quantitative microbial risk assessment to compare the occupational risks of struvite production (urine treatment) to the handling of pathogen-containing urine during collection. A final field campaign was conducted to videotape the urine collection and struvite production processes and to sample hands of workers and surfaces for pathogen contamination. The video and sample data were used together with computer modeling to evaluate the risk of infection from pathogens in urine during urine collection and struvite production. Dr. Bischel found that despite lower expected risks during urine collection compared to urine treatment, exposure to pathogens and risk of infection during urine collection was actually greater than during treatment due to frequent contact with urine-contaminated surfaces. This important finding points to direct measures that can be taken to protect human health safety during urine collection and fertilizer production processes, helping to protect human health while promoting recovery of resources from waste. Quantitatively identifying risks in this way facilitates the development of control strategies to promote safety during struvite production and use. A scientific publication will be completed on the study in 2017.

The body of work presented herein describes a comprehensive evaluation of the human health risks associated with nutrient recovery from urine and proposed solutions to address these challenges, with the goal of overcoming barriers to the implementation of more sustainable sanitation technologies. In addition to the publication of multiple peer-reviewed scientific articles in high quality journals, results of this work have been communicated to the water and sanitation municipality in Durban to promote applications of the research, summarized in public brochures through the VUNA project framework to promote understanding of resource recovery opportunities, and presented at numerous invited seminars and scientific conferences to promote intellectual exchange and knowledge advancement. The work was also highlighted by the EPFL news service.

BACKGROUND

Collection of human waste and recovery of nutrients from excreta have the potential reduce pollution of water from disposal of untreated waste while generating revenue from the production of valuable fertilizers. If collected and used as fertilizer, human urine and feces could also offset more than 20% of the world’s fertilizer demand.5 A majority of the nutrients (e.g. nitrogen, phosphorus, potassium) excreted by humans is in urine; thus there is great potential for the recovery of nutrients from urine to provide significant financial and environmental benefits while promoting sanitation.

One municipality in South Africa, eThekwini Water and Sanitation (EWS) that serves the greater region of eThekwini (Durban), is leading efforts to develop such innovations. In the 2000s, EWS recognized potential benefits of collecting urine separately from solids and installed over 80,000 dry urine-diverting toilets (UDTs) in rural and peri-urban regions of eThekwini that were lacking sanitation. The “VUNA” project (Valorisation of Urine Nutrients in Africa, Figure 1) was then established in 2010 as a partnership between EWS and researchers in Switzerland to “develop a dry sanitation system, which is affordable, produces a valuable fertiliser, promotes entrepreneurship and reduces pollution of water resources.” The process engineering research team at the Swiss Federal Institute of Aquatic Science and Technology (Eawag) pioneered the nutrient recovery technologies.

Figure 1. Schematic of the “VUNA” project to promote sanitation, entrepreneurship and environmental sustainability through the recovery of nutrients in urine for use as fertilizers.

As part of the VUNA framework, two fertilizer production technologies were developed and implemented at a pilot-scale in eThekwini, namely urine nitrification and struvite precipitation. Nitrification is a biological

5 Mihelcic, J.R., Fry, L.M., Shaw, R., 2011. Global potential of phosphorus recovery from human urine and feces. Chemosphere 84, 832–839. treatment process that, when properly controlled, stabilizes the nutrients in solution so that they may be concentrated into a liquid fertilizer. Struvite is a solid fertilizer that is chemically precipitated and filtered from urine. These technologies were developed to optimize the recovery of nutrients from urine. However, evaluation of the human and environmental health hazards due to potential pathogens in the urine and urine- derived products was missing. Ensuring the safety of fertilizers produced from human urine requires comprehensive assessment of human health hazards and innovation in the fertilizer production technologies to address identified concerns. Dr. Bischel joined VUNA to lead this effort.

This research report details Dr. Bischel’s work as a Postdoctoral Research Scientist at EPFL to evaluate the human health risks of nutrient recovery from urine and reuse and to optimize fertilizer production technologies to mitigate these challenges. The ultimate goal of this work is to promote the hygienic handling of urine and to contribute to the design of fertilizer production technologies that allow for its safe enduse. Three scientific publications are presented in full followed by a brief summary of the implications of this work. PART I

IDENTIFICATION OF HUMAN AND ENVIRONMENTAL HEALTH HAZARDS IN SOURCE-SEPARATED URINE INTENDED FOR FERTILIZER PRODUCTION

Water Research 85 (2015) 57e65

Contents lists available at ScienceDirect

Water Research

journal homepage: www.elsevier.com/locate/watres

Pathogens and pharmaceuticals in source-separated urine in eThekwini, South Africa * € Heather N. Bischel a, , Birge D. Ozel Duygan b, Linda Strande b, Christa S. McArdell b, Kai M. Udert b, Tamar Kohn a a Laboratory of Environmental Chemistry, School of Architecture, Civil and Environmental Engineering (ENAC), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland b Eawag, Swiss Federal Institute of Aquatic Science and Technology, CH-8600 Dübendorf, Switzerland article info abstract

Article history: In eThekwini, South Africa, the production of agricultural fertilizers from human urine collected from Received 29 April 2015 urine-diverting dry toilets is being evaluated at a municipality scale as a way to help finance a decen- Received in revised form tralized, dry sanitation system. The present study aimed to assess a range of human and environmental 6 August 2015 health hazards in source-separated urine, which was presumed to be contaminated with feces, by Accepted 11 August 2015 evaluating the presence of human pathogens, pharmaceuticals, and an antibiotic resistance gene. Available online 14 August 2015 Composite urine samples from households enrolled in a urine collection trial were obtained from urine storage tanks installed in three regions of eThekwini. Polymerase chain reaction (PCR) assays targeted 9 Keywords: e Urine nutrient recovery viral and 10 bacterial human pathogens transmitted by the fecal oral route. The most frequently Health hazards detected viral pathogens were JC polyomavirus, rotavirus, and human adenovirus in 100%, 34% and 31% Risk of samples, respectively. Aeromonas spp. and Shigella spp. were frequently detected gram negative Sustainable sanitation bacteria, in 94% and 61% of samples, respectively. The gram positive bacterium, Clostridium perfringens, which is known to survive for extended times in urine, was found in 72% of samples. A screening of 41 trace organic compounds in the urine facilitated selection of 12 priority pharmaceuticals for further evaluation. The antibiotics sulfamethoxazole and trimethoprim, which are frequently prescribed as prophylaxis for HIV-positive patients, were detected in 95% and 85% of samples, reaching maximum concentrations of 6800 mg/L and 1280 mg/L, respectively. The antiretroviral drug emtricitabine was also detected in 40% of urine samples. A sulfonamide antibiotic resistance gene (sul1) was detected in 100% of urine samples. By coupling analysis of pathogens and pharmaceuticals in geographically dispersed samples in eThekwini, this study reveals a range of human and environmental health hazards in urine intended for fertilizer production. Collection of urine offers the benefit of sequestering contaminants from environmental release and allows for targeted treatment of potential health hazards prior to agricultural application. The efficacy of pathogen and pharmaceutical inactivation, transformation or removal during urine nutrient recovery processes is thus briefly reviewed. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction enhancing the dehydration of separately stored feces in dry toilets. Collected urine can be used directly as a fertilizer in agriculture Decentralized sanitation technologies that separate human after a period of storage or processed into concentrated powder or urine from feces at the toilet user interface have been implemented liquid products. The use or sale of urine-derived fertilizers could in low-, middle- and high-income countries, though primarily at offset the purchase of costly synthetic fertilizers or generate a pilot scales. Source-separation of urine has the advantage over source of revenue for sanitation service providers. Such a sanitation traditional toilets of sequestering nutrients primarily excreted in system would also reduce nutrient loads to treatment facilities or urine, namely nitrogen, potassium, and phosphorus, while other urine discharge locations (Larsen et al., 2013). The eThekwini Water and Sanitation (EWS) municipality in the KwaZulu-Natal province of South Africa serves the greater eThek- * Corresponding author. wini region including Durban. EWS installed over 80,000 urine E-mail address: heather.bischel@epfl.ch (H.N. Bischel). diverting dry toilets (UDDTs) in historically underserved rural and http://dx.doi.org/10.1016/j.watres.2015.08.022 0043-1354/© 2015 Elsevier Ltd. All rights reserved. 58 H.N. Bischel et al. / Water Research 85 (2015) 57e65 peri-urban areas of eThekwini, providing an ideal test case for the propagation of antibiotic resistance, especially in low-income scale-up of urine collection and use. Currently in this system, countries (Okeke et al., 2005). source-separated urine is diverted to soak pits, and feces are The objective of this study was to evaluate the presence of collected in vaults for drying. EWS would like to incorporate pathogenic organisms that are causative agents of diarrheal disease nutrient recovery from urine into the system. The VUNA (Valor- as well as pharmaceuticals in source-separated urine collected isation of Urine Nutrients in Africa) project is designing, testing and from throughout eThekwini. Based on a review of enteric patho- optimizing urine collection and nutrient recovery technologies to gens present in South Africa, polymerase chain reaction (PCR) as- produce a commercially viable fertilizer. Pilot-scale technologies says targeting 9 viral and 10 bacterial pathogens transmitted by the have been developed to produce a solid phosphate-based fertilizer fecaleoral route were selected for a screening of human pathogens (struvite) as well as a concentrated liquid fertilizer via biological in the collected urine. An initial screening of 41 TrOCs in the urine nitrification and distillation (Udert et al., 2015). Urine-derived fer- facilitated selection of 12 priority pharmaceuticals for further tilizers, and their production processes, need to be safe with respect quantitative evaluation. The presence of a sulfonamide antibiotic to environmental and public health concerns. This study thus aims resistance gene (ARG) in bacteria from urine was also evaluated. By to identify potential human and environmental health hazards in coupling analysis of pathogens and pharmaceuticals, this study source-separated urine, targeting microbial and chemical contam- seeks to identify a range of priority human and environmental inants. The ultimate goal is to promote the hygienic handling of health risk determinants in urine that is intended for fertilizer urine and to design fertilizer production technologies that allow for production and agricultural applications. its safe end use as a fertilizer. The most pressing human health concerns associated with 2. Materials and methods source-separated urine stem from the presence of human patho- gens. While enteric pathogens are primarily excreted in feces rather 2.1. Sampling than urine, the cross-contamination of source-separated urine with feces is well established (Schonning€ et al., 2002). Diseases trans- Samples were collected at four different times between 2010 mitted by the fecaleoral route are therefore relevant in the design and 2013. Samples from 2010 to 2011 were used for TrOC method of urine treatment and recycling systems. Pathogen reduction can validation and to select priority compounds for further monitoring. be partially achieved through storage, which allows inactivation by These initial urine samples were collected from two UDDTs on 10 heat or ammonia (Decrey et al., 2015; Nordin et al., 2013). The November 2010, and four composite samples consisting of urine World Health Organization (WHO) recommends storage for >6 from 29 UDDTs were obtained between 28 April and 25 May 2011. months at >20 C to achieve sufficient pathogen inactivation in The composite samples were prepared by adding urine collected urine intended for agricultural use for unprocessed food crops daily over one week to a 1500 L black polyethylene tank. At the end (WHO, 2006). While feasible in rural areas with ample space, this of the week, the tank was stirred and sampled into triplicate 50 ml storage time can be prohibitive for urine collection at scale in dense centrifuge tubes. The tank was then emptied in preparation for the urban environments due to storage volume requirements. Long subsequent composite sample. term storage can also cause odor problems and lead to loss of Samples collected in 2012 and 2013 were geographically recoverable nutrients by ammonia volatilization. These challenges distributed in the EWS service area. From December 2012 to May highlight the need for alternative urine treatment technologies. 2013, nine urine storage tanks of 1000e1500 L capacity were Urine-derived fertilizers can be produced after short-term urine temporarily installed in three distinct regions of the EWS service storage and provide a treatment benefit. However, implementation area in peri-urban Durban as part of an economic study on the of this strategy necessitates investigation of the fate of locally collection of urine using incentives (Tilley, 2015). Three urine tanks relevant pathogens in the fertilizer production processes. were installed per region, and each tank was accessible for urine While pathogens are primarily human health hazards, the pri- deposits by approximately 90e150 nearby households that were mary concerns for the presence of trace organic compounds provided with urine collection jerry cans. Tanks received deposits (TrOCs) in the environment stem from their potential ecosystem of urine carried by individuals from their homes to the tanks during health hazards (Kolpin et al., 2002). A meta-analysis of 212 phar- opening hours one day per week. Urine tanks were emptied by the maceuticals revealed that an average of 64% of the consumed mass municipality when full. The nine storage tanks were sampled on is excreted in urine (Lienert et al., 2007). The use and excretion of December 19, 2012 and subsequently on several days from April antiretroviral (ARV) drugs may be of particular interest in South 12e29, 2013 for combined TrOC and microbiological assessments. Africa where HIV prevalence is high, particularly in the KwaZulu- In December, two additional urine tanks (UT) located at the Uni- Natal province (16.9% of the population in 2012; Shisana et al., versity of KwaZulu-Natal (UKZN) were also sampled. The study 2014). South Africa has rolled out the largest ARV treatment pro- plan was approved by the UKZN Biomedical Research Ethics Com- gram in the world e in 2012, 31.2% of eligible people living with mittee (BE147/13). Specific locations of the sampling sites are human immunodeficiency virus (HIV) or Acquired Immune Defi- omitted as stipulated in the project protocol. ciency Syndrome (AIDS) had been exposed to ARV treatment (Shisana et al., 2014). While the benefits of this program are clearly 2.2. Chemical and physical urine characteristics demonstrated through the reduction of AIDS deaths in the country, antiviral pharmaceuticals were predicted to be amongst the most The urine pH, temperature, conductivity, total ammonia and hazardous therapeutic drug classes for several ecotoxicological orthophosphate concentrations were measured as described in the end-points (Sanderson et al., 2004). Commonly prescribed anti- Supporting information. bacterial compounds with high excretion rates may also be prev- alent in source-separated urine. Sulfamethoxazole and 2.3. Analytes trimethoprim, for example, are frequently prescribed together as a broad-spectrum prophylaxis against bacterial pathogens and pro- 2.3.1. Bacteria tozoa for HIV-positive patients (Zachariah et al., 2007). One po- The presence of bacterial pathogens was monitored by detection tential human health consequence of such widespread use of of bacterial DNA. Bacteria targets in this study were: Clostridium antibiotics for infectious disease therapy is the accelerated perfringens, Yersinia enterocolitica, Escherichia coli O157:H7, H.N. Bischel et al. / Water Research 85 (2015) 57e65 59 verocytotoxin producing E. coli (VTEC), Aeromonas spp. and the pH was 8.72 ± 0.10. The average temperature for all tank (A. salmonicida, A. sobria, A bivalvium, A. hydrophila), Vibrio spp. samples in April 2013 was 20.4 ± 2.0 C, ranging from 15.5 to 27.1 C (V. cholera, V parahamolyticus, and V. vulnificus), C. difficile toxin B, at the time of sampling, with pH 8.90 ± 0.09. Average ammonia Salmonella spp. (Salmonella bongori and Salmonella enterica), measured in urine tanks was 2240 ± 1180 mg/L NH4eN (range of Shigella spp. (Shigella flexneri, Shigella boydii, Shigella sonnei and 400e8550 mg/L NH4eN) and average phosphate was 240 ± 20 mg/l Shigella dysenteriae) and Campylobacter spp. (C. jejuni and C. coli). PO4eP (range of 170e290 mg/l PO4eP). Multiplex end-point PCR was conducted using two bacterial See- gene ACE Diarrheal detection kits (BÜHLMANN Laboratories AG, 3.2. Pathogen detection in source-separated urine Schonenbuch,€ Switzerland). Details of sample processing pro- cedures are available in the Supporting information. Each of the The detection frequencies of pathogenic bacteria and viruses in urine tanks were also evaluated for total viable heterotrophic the urine storage tanks during the 2012 and 2013 sampling cam- bacteria (April 2013) as an indicator of the total bacteria concen- paigns are presented in Table 2. Up to six pathogens were detected trations in source-separated urine. One ml of each of three dilutions in a single urine sample. of sampled urine (at least 100-fold dilution in phosphate buffered saline, 5 mM PO4, 10 mM NaCl, pH 7.4) from each urine tank was 3.2.1. Bacteria occurrence aliquoted on a 3M Petrifilm dryplate (3M, St. Paul, MN, USA) and The following bacteria were detected in at least two urine tank handled as per manufacturer instructions. Plates were incubated samples: C. perfringens, Y. enterocolitica, E. coli 0157:H7, for 48 h at 35 C prior to enumeration, and results are presented as verocytotoxin-producing E. coli (VTEC), Aeromonas. spp., Vibrio spp., colony forming units per ml urine sampled (CFU/ml). and Shigella spp. At least one diarrheal bacterium species was detected in each of the 18 samples (100%), and an average of three 2.3.2. Viruses bacteria species were detected in each sample. Two samples con- The presence of viruses was monitored by detection of viral RNA tained five bacteria species each, the maximum number of diar- or DNA. The viral targets were human adenovirus (HAdV), rotavirus rheal bacteria species detected in a single sample. Region 2 had the (RoV), norovirus (NoV) GI and GII, human astrovirus (HAstV), en- most total detects followed by Region 3 and Region 1 (Table 2). The teroviruses (EV), hepatitis A Virus (HAV) and JC Polyomavirus highest frequency detected bacteria were Aeromonas spp. (94%); (JCPyV). While not typically associated with fecal contamination, C. perfringens (72%); and Shigella spp. (61%). Three of the bacteria JCPyV was included herein because it has been detected consis- were not detected in any samples: C. difficile, Salmonella spp., and tently in wastewater worldwide (Bofill-Mas et al., 2000). Unlike the Campylobacter spp. Heterotrophic plate counts ranged from 1 105 other viruses evaluated, JCPyV is excreted indefinitely in the urine to 7 106 CFU/ml across all urine tanks sampled, with an average of healthy individuals following infection. As such, this virus was concentration of 2 106 CFU/ml. expected to be ubiquitously present in urine, and served as an in- ternal process control for virus extraction and detection. Bacterio- 3.2.2. Virus occurrence phage MS2 (DSMZ 13767) was used for optimization of the nucleic The following viruses were detected in at least one urine sample acid extraction procedure and PCR method. HAdV, RoV, NoV GI and from Durban: JC polyomavirus, human adenovirus, rotavirus, hep- GII, and HAstV were analyzed by viral Seegene ACE Diarrheal atitis A virus, and norovirus GII. Three to five virus species were detection kits (BÜHLMANN Laboratories AG, Schonenbuch,€ detected in each of the sampling regions, and up to three virus Switzerland), which included positive controls. EV and MS2 were species were detected in a single sample. The three most frequently analyzed by one-step reverse-transcriptase (RT)-PCR with SYBR detected viruses were JCPyV, HAdV and RoV detected in 100%, 34% Green using a Rotor-Gene 3000 thermocycler. HAV and JCPyV were and 31% of samples, respectively. In the present study, NoV GII was analyzed by RT-PCR with selective Taqman® probes. Further detected in only one sample, which was collected in April 2013. method details are available in the Supporting information. HAstV and NoV GI were not detected.

2.3.3. Trace organics 3.3. Pharmaceuticals in source-separated urine A method initially developed to analyze pharmaceuticals in hospital wastewater (Kovalova et al., 2012) was validated on 2010 Concentrations of pharmaceuticals from the priority list for and 2011 samples. Samples were screened for a group of 41 TrOCs samples taken from urine storage tanks in December 2012 and (see Supporting information) using liquid chromatography tandem April 2013 are shown by region in Table 2 and discussed below. Up mass spectrometry (LC-MS/MS). A group of 12 priority compounds to 10 of 12 pharmaceuticals assessed were detected in a single urine were selected for further monitoring in the 2012 and 2013 sam- sample. Results from the 2010 and 2011 screening are available in pling campaign. Concentrations are reported as averages with 95% the Supporting information. confidence intervals (CI) unless otherwise noted. Details of the analytical method as well as the process for selecting priority 3.3.1. Antibacterial detection pharmaceutical analytes are available in the Supporting The antibacterial pharmaceuticals tested in this study were: information. Methods applied for the analysis of the sulphona- sulfamethoxazole (SMX), trimethoprim (TMP), and clarithromycin. mide antibiotic resistance gene sul1 are described in the Supporting N4-acetylsulfamethoxazole (N4-AcSMX), a human metabolite of information. SMX, was also tested. SMX and its conjugate N4-AcSMX as well as TMP were frequently detected at high concentrations. SMX, N4- 3. Results AcSMX and TMP were detected in 95%, 90% and 85% of samples, respectively, with concentrations in urine reaching 6800, 3500 and 3.1. Urine tank conditions 1300 mg/L, respectively. In contrast, clarithromycin was detected in only 20% of samples and at relatively lower concentrationsP in this Characteristics of urine in the storage tanks during 2012 and study (17 ± 29 mg/L). The sum of SMX and N4-AcSMX ( SMX) 2013 sampling campaigns are presented in Table 1. The average concentrations varied widely from less than 2 mg/L to 8500 mg/L, ± m temperature for all urine tank samples in December 2012 was with an average of 2700 110 0 g/L. A weakP correlation was 25.9 ± 0.7 C, ranging from 24.8 to 34.3 C at the time of sampling, observed between TMP concentrations and SMX (R2 ¼ 0.34), 60 H.N. Bischel et al. / Water Research 85 (2015) 57e65

Table 1 Characteristics of urine in storage tanks.

a Region Sample collection Ammonia (mg/L NH4eN) Conductivity (mS/cm) Temperature ( C) pH avg. ±95% CI (range, n) avg. ±95% CI (range, n) avg. ±95% CI (range, n) avg. ±95% CI (range, n)

1 December 2012 e 30.3 ± 3.4 (25e34.3, 6) 27.6 ± 0.3 (27.2e28.1, 6) 8.65 ± 0.06 (8.58e8.75, 6) April 2013 1500 ± 530 (400e2100, 6) e 20.3 ± 0.5 (19.8e20.8, 3) 9.02 ± 0.08 (8.95e9.08, 3) 2 December 2012 e 25.7 ± 0.7 (24.8e26.8, 6) 24.5 ± 0.5 (23.7e24.9, 6) 8.88 ± 0.04 (8.81e8.9, 6) April 2013 2250 ± 640 (1600e2650, 3) e 19.2 ± 3.8 (15.5e27.1, 6) 8.91 ± 0.13 (8.68e9.08, 6) 3 December 2012 e 27.5 ± 2.3 (25.5e31.3, 6) 25.6 ± 1.0 (24.1e26.5, 6) 8.63 ± 0.24 (8.26e8.90, 6) April 2013 3650 ± 4820 (800e8550, 3) e 22.8 ± 1.6 (21.3e24.2, 3) 8.75 ± 0.15 (8.60e8.86, 3)

a In Durban, the average low temperature is 20 C in December and 18 C in April; the average high temperature is 28 C in December and 27 C in April. P such that when SMX was found in very high concentrations, TMP concentrations (30 ± 10 mg/L; max ¼ 72 mg/L). Hydrochlorothiazide, was also typically measured at elevated concentrations. a diuretic drug often prescribed to treat high blood pressure, measured on average 42 ± 18 mg/L and was found in 80% of samples. 3.3.2. Antiviral detection The beta-blocker atenolol and its metabolite atenolol acid were Among the four analyzed antivirals, only emtricitabine and ri- detected in 55% and 75% of samples, respectively, with average tonavir were detected, in 40% and 70% of samples, respectively. The concentrations of 31 ± 33 and 98 ± 110 mg/L. Atenolol acid is a concentration of emtricitabine averaged to 100 ± 97 mg/L but was human metabolite of both atenolol and metoprolol, neither of highly variable among storage tanks with a maximum concentra- which were detected in the initial 2010 and 2011 screening. tion of more than 900 mg/L. Ritonavir concentrations were typically low, with an average concentration of 1.5 ± 0.5 mg/L and a 3.4. Antibiotic resistance maximum concentration of 5 mg/L. The sul1 antibiotic resistance gene was present in 100% of bac- 3.3.3. Other pharmaceuticals terial extracts (n ¼ 18) and not detected in the two field blanks The anti-inflammatory drug diclofenac was detected consis- analyzed. A two-tailed pairwise t-test indicates that duplicate an- tently in 100% of the samples, though typically at low alyses of the same samples were not significantly different (p > 0.3).

Table 2 Detection of pathogens, pharmaceuticals, and antibiotic resistance genes in source-separated urine.

Region 1 Region 2 Region 3 UTa All samples

Dec. '12 Apr. '13 Dec. '12 Apr. '13 Dec. '12 Apr. '13 Dec. '12

Virus Detection frequency Detection frequency Detection frequency Det. freq. Freq. % (n ¼ 29) JC polyomavirus 3 6 3 6 3 6 2 100% Adenovirus 2 2 1 0 2 3 0 34% Rotavirus 3 0 3 1 0 0 2 31% Hepatitis A virus 0 0 0 1 0 1 0 7% Norovirus GII 0 0 0 1 0 0 0 3% Astrovirus 0 0 0 0 0 0 0 0% Echovirus 0 0 0 0 0 0 0 0% Enterovirus 0 0 0 0 0 0 0 0% Norovirus GI 0 0 0 0 0 0 0 0% Bacterium Detection frequency Detection frequency Detection frequency Freq. % (n ¼ 18)

Aeromonas spp. e 5 e 6 e 6 e 94% C. perfringens e 2 e 6 e 5 e 72% Shigella spp. e 2 e 5 e 4 e 61% Vibrio spp. e 2 e 2 e 0 e 22% Y. enterocolitica e 0 e 0 e 2 e 11% E.coli O157:H7 e 1 e 1 e 0 e 11% VTEC e 0 e 2 e 0 e 11% Campylobacter spp. e 0 e 0 e 0 e 0% C. difficile toxin B e 0 e 0 e 0 e 0% Salmonella spp. e 0 e 0 e 0 e 0% Pharmaceutical Concentration range (mg/L) Concentration range (mg/L) Concentration range (mg/L) Conc. range (mg/L) Freq. % >LOQ (n ¼ 20)

Diclofenac 3.2e72 20e62 1.6e45 16e44 13e56 16e44 27e30 100% Sulfamethoxazole <2e300 320e5200 800e5000 1000e6800 560e4800 1600e6400 5e12 95% N4 Acetyl-SMX <1e560 280e720 60e3500 9.6e280 8e520 34e174 <1e2 90% Trimethoprim <2e3.6 22e380 4.3e56 260e1300 <2e300 100e440 <2 85% Hydrochlorothiazide <3e19 52e94 17e120 18e64 20e134 52e94 <3 80% Atenolol acid 4.2e100 <4e100 <4e11 <4 8.0e1100 20e98 130e175 75% Ritonavir <1e2.8 <1e1.5 <1e1.7 <1e1.4 1.9e4.6 <1e1.0 2.8 70% Atenolol <1e2.9 <1e300 <1 <1 1.2e4.3 20e184 26e29 55% Emtricitabine <6 <6 <6e920 <6e240 <6e16 <6e380 26e120 40% Clarithromycin <1 <1 <1e1.1 <1 <1 4.4e300 <1 20% Atazanavir <2.5 <2.5 <2.5 <2.5 <2.5 <2.5 <2.5 0% Darunavir <1 <1 <1 <1 <1 <1 <10% Antibiotic resistance gene Detection frequency Detection frequency Detection frequency Freq. % (n ¼ 18)

sul1 e 6 e 6 e 6 e 100%

a UT refers to the two urine collection tanks at the University of KwaZuluNatal (UKZN). H.N. Bischel et al. / Water Research 85 (2015) 57e65 61

Of 36 samples tested, six were above the highest standard (106 gc/ universally detected in the samples. JCPyV already infects most reaction); one of these was excluded as an outlier because it was people asymptomatically during childhood, though its pathoge- several orders of magnitude higher in concentration than replicate nicity emerges in HIV-infected individuals (Major et al., 1992). This analyses of the sample using the same DNA extraction method consideration demonstrates the importance of considering the risk (Method A, see SI), as well as a different extraction method (Method factors of each viral target, in addition to evaluating virus preva- B, see SI). The number of sul1 genome copies detected per ml of lence, to assess its relative importance to public health. urine sample ranged from 1 108 to 4 1010. Several other viruses that are prevalent in South Africa and transmitted by the fecaleoral route were not detected in the pre- 4. Discussion sent study. These include enterovirus (Ehlers et al., 2005) and HAstV (Mans et al., 2010; Taylor et al., 1997). The prevalence of 4.1. Pathogens in source-separated urine and potential health risks these viruses could be low, as was the case for HAstV in South Af- rican patients with gastroenteritis (Taylor et al., 1997), yielding The present study is the first of its kind to assess a broad di- concentrations below the detection limit. Alternatively, limited versity of fecal pathogens in source-separated urine. The urine detection could be a result of degradation of the viral genome, an storage tanks represent a composite of urine from potentially inactivating process expected for single stranded (ss)RNA viruses in hundreds of individuals. Any urine handled downstream of this set- stored urine (Decrey et al., 2015). ssRNA viruses are less stable than up is likely to contain a mixture of pathogens originating from fecal DNA viruses and double-stranded (ds)RNA viruses, which are ex- contamination. This assumption is corroborated by our findings pected to retain their infectivity and genome integrity in urine over that demonstrate the ubiquitous presence of a wide spectrum of long periods of time (Decrey, 2015). Correspondingly, the three fecal pathogens in source-separated urine in Durban. The pathogen most frequently detected viruses in this study (JCPyV, HAdV and load and diversity will depend on the extent of fecal contamination RoV) are dsDNA or dsRNA viruses. In contrast, even ssRNA viruses of the urine, which in turn depends on the toilet user interface with high seroprevalence in South Africa, including HAV and NoV design, usage and maintenance patterns, and disposal processes, in GII (Mans et al., 2010), were detected less frequently. Their limited addition to local cultural practices. For example, whether or not detection in the present study may be indicative of genome children use the UDDTs, especially during an active gastrointestinal degradation during urine storage. infection, will influence the pathogen profile of the collected waste. Similar to viral pathogens, the bacterial pathogen targets The detection of rotavirus, a common infection in children, in- assessed in this study have each been detected in various human dicates that children likely use the UDDTs in eThekwini. and environmental matrices collected in South Africa. For example, The results presented here are based on presence of the path- Shigella spp. was commonly identified in drinking water consumed ogen DNA or RNA measured by PCR, and thus do not yield data on by individuals with HIV/AIDS in rural households (Samie et al., the infectivity of detected pathogens. However, the application of 2012) and may contribute to the chronic diarrhea commonly PCR to identify pathogens in this study is a conservative approach experienced by HIV/AIDS patients (Obi and Bessong, 2002). The for the identification of potential health hazards in urine by, for etiological agent of cholera, V. cholerae, which was also detected in example, addressing the well-known issue of viable but non- our samples, has been regarded as endemic in South Africa and is a culturable pathogens. A safe assumption for the human patho- common cause of illness and death in the country (Momba et al., gens detected is that they are infective in fresh feces and therefore 2010). While not detected in this study, Campylobacter spp. and also in freshly contaminated urine. Additionally, pathogen moni- Salmonella spp., along with other diarrhea-causing bacterial path- toring by PCR avoids exclusion of pathogens that were inactivated ogens, are known to circulate in South Africa (Obi and Bessong, at the time of sampling but may pose a health risk to individuals in 2002). These results illustrate that while country-specificpreva- contact with the material prior to inactivation. Finally, several lence studies are useful to identify a range of potential pathogen studies have evaluated the inactivation of organisms in source- hazards, the local relevance of specific pathogens varies greatly. separated urine, confirming that some bacteria and viruses may In addition to yielding an overview of the health status of the remain infective in stored urine for months (Hoglund€ et al., 1998; sample population at the time of measurement, the results indicate Vinneras et al., 2008). It is thus reasonable to assume that the potentially problematic levels of fecal contamination in terms of detected pathogens were at least in part infective, and thus health risk to humans during downstream handling and use of the constitute a potential health risk. A health-risk assessment would urine. The actual health risk to humans from this diversity of benefit from a culture-based analysis of human pathogens to assess pathogens will depend on the influent composition, pathogen the viable and culturable status of detected organisms relative to inactivation rates as well as human exposure routes during urine their total abundance at important exposure points in the urine handling and processing, which requires further evaluation. JCPyV, collection and handling chain. which is excreted in urine, may be useful as an indicator for When considering the health risks of using source-separated tracking the fate of pathogens in source-separated urine or as a urine for fertilizer, viruses are of particular relevance due to their treatment process indicator for viruses because of its consistent low infective dose and resistance to inactivation in stored urine detection. JCPyV has been proposed as a sewage indicator organism (Hoglund€ et al., 2002a). Each of the viruses detected in this study due to its high excretion titer, frequent detection in urban sewage, have been identified in various human and environmental matrices stability, and absence of animal host (Bofill-Mas et al., 2000). in South Africa. Notably, rotavirus was the most frequently detected virus in a 2008 survey of fecal specimens from patients with 4.2. Pharmaceutical excretion in urine and potential health risks gastroenteritis in the Pretoria region, followed by adenovirus and norovirus (Mans et al., 2010). Rotavirus may pose a risk in agri- Little public information is available concerning specific phar- cultural applications of urine (Hoglund€ et al., 2002b) and continues maceutical usage in South Africa, rendering estimation of con- to be a major contributor to childhood diarrheal disease in South sumption, and excreted concentrations, difficult. Therefore, average Africa, though the benefits of a vaccine implemented in 2009 are measured urine concentrations in South Africa were compared to beginning to be demonstrated (Seheri et al., 2012). In contrast to predicted urine concentrations using pharmaceutical consumption RoV, JCPyV is not expected to increase the public health concerns of data from the USA, France, Germany and Switzerland in order to urine collection and agricultural application, though it was identify outliers (Table 3). For example, low to no detection of the 62 H.N. Bischel et al. / Water Research 85 (2015) 57e65 antibacterial clarithromycin indicates limited prescription in the evaluated in this study, such as those recommended for public study region, contrary to high-income countries (Michael et al., sector ARV treatment in South Africa (WHO, 2005), may also be 2013). Similarly, concentrations of the diuretic, hydrochlorothia- present in the urine from eThekwini. zide, were relatively low in eThekwini. However, average measured One consequence of the increased prescription and consump- concentrations of SMX and TMP in urine were elevated as tion of antibiotics is the increased prevalence of antibiotic resis- compared to predicted concentrations. Concentrations of SMX in tance determinants. For example, a diversity of multidrug resistant particular were markedly high, more than an order of magnitude diarrheal bacteria, notably including Shigella spp., were isolated above the range predicted. from household water stored for use by HIV-positive patients in In addition to being widely used for the treatment of bacterial Limpopo province (Samie et al., 2012). In the present study, the infections, SMX and TMP are frequently prescribed as prophylaxis sulfonamide antibiotic resistance gene, sul1, was detected in 100% to prevent opportunistic infections in patients with compromised of urine samples. While the sul1 resistance gene is known to be immune systems (e.g., for HIV-positive individuals) (Madhi et al., widespread in the environment (e.g., (Czekalski et al., 2014)) and is 2002). In a preventative approach, patients receive such pharma- not broadly representative of other antibiotic resistance de- ceuticals regularly over an extended period of time, in contrast to terminants, it is typically detected at lower rates in urine samples targeted antibiotic treatment for acute infections. If 4e11% of all than observed in the present study. For example, in a 16-country HIV infected persons in the KwaZulu-Natal province were taking study of urine collected from patients with urinary tract infections, SMX prophylactic (see SI for estimation), this could explain the sul1 was detected in 31% of 350 uropathogenic E. coli isolates (as observed concentrations of SMX. Therefore, the surprisingly high compared to the sulfonamide resistance gene sul2 in 48% of iso- SMX concentrations detected are reasonable considering the high lates). Co-trimoxazole is a common prescription for this infection prevalence of HIV-positive people in eThekwini. Additionally, SMX (Blahna et al., 2006). Further, the concentration of sul1 gene copies and TMP prescriptions are often administered together as co- was high, from 108 to 1010 gc/ml, compared to less than 107 gc/ml in trimoxazole in a 5:1 ratio. Because N4-AcSMX is a metabolite of hospital and municipal wastewater effluents evaluated in SMX produced in the human body and deconjugates to SMX under Switzerland (Czekalski et al., 2012), while heterotrophic bacteria environmental conditions (Gobel€ et al., 2005), the sum of SMX and concentrations were similar (~106 CFU/ml) in the source-separated N4-AcSMX was expected to correlate with TMP. Correspondingly,P urine to the levels in wastewater evaluated by Czekalski et al. TMP was usually found in high concentrations when SMX also (2012). The ubiquitous detection of sul1 in the present study thus occurred in high concentrations, though this prescription ratio was encourages further evaluation of the prevalence and health risks not preserved in the samples. associated with other clinically relevant ARGs in source-separated In addition to widespread use of antibacterial pharmaceuticals urine, and especially those harbored within human pathogens. in South Africa, nationally, just over 2.0 of 6.4 million estimated Despite the potential health challenges from the spread of antibi- people living with HIV in 2012 received anti-retroviral treatment otic resistance, HIV-infected African adults receiving co- (Shisana et al., 2014). Of four ARV pharmaceuticals tested, emtri- trimoxazole currently still garner consistent benefits in survival citabine and ritonavir were detected. Target antivirals detected at even in areas where co-trimoxazole resistance is common low concentrations or not detected in storage tanks may reflect the (Zachariah et al., 2007). low consumption and/or low excretion rates of those compounds. Beyond the human health concerns associated with antibiotic Emtricitabine exhibits a urine excretion rate of 75%, whereas less resistance determinants, pharmaceutical usage and environmental than 8% of consumed atazanavir, darunavir, and ritonavir are release pose potential ecotoxicological risks. Urine discharged from excreted in urine (Table 3). Therefore, low concentrations or lack of UDDTs or urine collected and applied directly as fertilizer may detection of antivirals other than emtricitabine in the urine is not contribute to such risks. However, recommendations regarding necessarily indicative of their low consumption. The presence of environmentally safe concentrations of pharmaceuticals are feces in urine may also have contributed to the observed ritonavir, limited, especially for soil applications of human-waste derived as 41% of the consumed drug is excreted in feces. Antivirals not fertilizers (e.g., wastewater sludge, manure, or urine). Predicted no

Table 3 Predicted and measured pharmaceutical concentrations in 2012 and 2013 urine samples, and predicted environmental concentrations in soil following a single application of urine as a fertilizer.

c Compounds Usage Excretion Predicted concentration Measured concentration PECsoil (mg/kg) rate [%]a in urine (mg/L)b in urine (mg/L) (avg ± 95% CI; n ¼ 20) min max min max

Atazanavir Antiviral 6 1 e <2.5 0 0.3 Atenolol Beta-blocker 37 74 246 31 ± 33 0 7.5 Atenolol acid (Human) metabolite of atenolol and metoprolol NDd ND ND 98 ± 110 0 25 Clarithromycin Macrolide antibacterial 25 99 137 17 ± 29 0 5.4 Darunavir Antiviral 7.7 2 e <1 0 0.1 Diclofenac Analgesic: antiinflammaotry/antirheumatic 1 0.7 25 30 ± 10 0.2 4.7 Emtricitabine Antiviral 75 20 e 101 ± 97 0 23 Hydrochlorothiazide Diuretic 82 526 1118 42 ± 18 0.2 7.1 N4 Acetyl-sulfamethoxazole (Human) metabolite of sulfamethoxazole 50 303 468 360 ± 340 0.2 82 Ritonavir Antiviral 3.5 0.9 2.1 1.6 ± 0.5 0 0.2 Sulfamethoxazole Sulfonamide antibacterial 20 121 187 2300 ± 1000 11 390 Trimethoprim Antibacterial 60 2 120 190 ± 140 0.4 39

a Excretion rates from Swiss Compendium of Medicines by Documed: www.compendium.ch, last accessed March 10, 2015. b Predicted minimum and maximum concentrations in urine calculated based on consumption in four countries (CH, DE, FR, USA); see Supporting information Table S4. c Minimum and maximum predicted environmental concentrations in soil (PECsoil) calculated based on measured urine concentrations for a single application of urine as fertilizer for agriculture; see Supporting information. d ND ¼ Not Determined. H.N. Bischel et al. / Water Research 85 (2015) 57e65 63 effect concentrations in soil (PNECsoil) were previously estimated health effects may be experienced (ATSDR, 2004), as well as po- for SMX, TMP, and diclofenac using aquatic PNECs and compound tential detrimental environmental effects (Galloway and Cowling, physiochemical properties (Martín et al., 2012). For comparison to 2002). these values, we estimated a range of predicted environmental Besides NH3, high storage temperature is conducive to pathogen concentrations (PECsoil) and risk quotients (RQ ¼ PECsoil/PNECsoil) inactivation in urine. Measured urine tank temperatures depend on based on urine application as fertilizer in agriculture (Table 3 and the external temperature, sun exposure, tank color, and time of day Table S5). The estimated soil concentrations were based on a one- that samples were taken. Fluctuating temperatures within urine time direct urine application to achieve 150 kg N/ha (see SI for storage tanks due to diurnal heating from the sun may further calculation), assuming no degradation of the target compound in enhance inactivation (Nordin et al., 2013). Durban is characterized the soil. While the RQs for TMP and diclofenac (RQ<<1) indicated by a mild subtropical climate, with average maximum tempera- no significant environmental risk, the RQ for SMX for a range of tures of 28 or 29 C occurring between January and March and 23 or scenarios evaluated (RQ > 1) suggests a potential ecotoxicological 24 C in winters from June to August.1 Given these high tempera- risk from this compound due to its high concentration in urine and tures year-round, urine storage in Durban may achieve significant relatively low PNECsoil. The actual SMX concentration in soil, and inactivation of a range of important pathogens. Specifically, of the associated risk quotients, would depend on urine and water most frequently detected bacteria in the present study, the gram application rates, the soil characteristics, the degradation of SMX in negative bacteria Aeromonas spp. and Shigella spp. are expected to soil, and measured PNECsoil values. However, the results presented be inactivated rapidly in undiluted, stored urine with a temperature herein suggest that further detailed study on SMX ecotoxicity in above 20 C. A. hydrophila, for example, loses greater than 90% of these scenarios is warranted. infectivity per day in urine at 20 C(Hoglund€ et al., 1998). This is The breadth, high frequency and sometimes high concentra- commonly observed for other gram negative bacterial pathogens tions of pharmaceuticals detected in source-separated urine in this and fecal indicator organisms. C. perfringens, in contrast, is likely to study highlight the importance of evaluating pharmaceutical fate remain infective in stored urine for extended periods of time during urine collection and fertilizer production. Sequestering (Hoglund€ and Stenstrom,€ 1999). these compounds via urine diversion and collection prevents their Viruses in stored urine exhibit a wide range of inactivation rates release into the environment and provides an opportunity to that are typically slower than those of gram negative bacteria. As remove or transform the compounds via urine treatment. discussed above, dsDNA viruses are the most persistent in urine at urine storage temperatures expected in Durban (Decrey, 2015). The 4.3. Fate of pathogens and pharmaceuticals in treatment processes dsDNA viruses most frequently detected in this study, JCPyV and for urine-derived fertilizer production HAdV, are thus likely to remain infective in undiluted or diluted stored urine for several weeks or longer. In contrast, ssRNA viruses Due to the widespread detection of pathogenic viruses and such as HAV and NoV are expected to inactivate more rapidly than bacteria as well as high concentrations of several pharmaceuticals dsRNA or dsDNA viruses due to susceptibility of the genome to in source-separated urine in eThekwini, thorough consideration of degradation. the downstream urine treatment processes is imperative for the While the efficiency of pathogen and surrogate inactivation protection of individuals handling stored urine and for its use as an during the storage of urine under a range of conditions has been agricultural fertilizer. In eThekwini, the primary urine treatment extensively evaluated, data is lacking regarding the behavior of € and fertilizer production technologies under development for po- pharmaceuticals during urine storage. Forthcoming results (Ozel tential scale up are: (1) urine storage for pathogen inactivation; (2) Duygan et al., 2015) indicate little benefit of urine storage for the struvite fertilizer production for phosphorus recovery; and (3) transformation of pharmaceuticals detected in eThekwini, as 11 out urine nitrification for ammonia stabilization followed by distilla- of 12 compounds were very stable; only hydrochlorothiazide was tion to recover all nutrients in a concentrated liquid product (Udert degraded substantially. Nevertheless, source-separation coupled et al., 2015). In each of these technologies, processes promoting the with urine collection has the potential to sequester excreted drugs degradation, inactivation, or removal of pathogens and TrOCs may and reduce the diffuse discharge of pharmaceuticals to the occur. Below, available data from literature is interpreted in the environment. context of eThekwini to evaluate the treatment benefits of nutrient recovery processes in reducing environmental loads of human and 4.3.2. Struvite fertilizer production and drying environmental health risk determinants from the application of Struvite (MgNH4PO4$6H2O) can be precipitated from stored source-separated urine, or fertilizers produced from that urine, at urine by adding a magnesium source such as MgO, MgCl2 or bittern scale. (Etter et al., 2011). The process is completed by filtration and drying of the struvite precipitate, which can be applied directly as a 4.3.1. Urine storage fertilizer. The efficiency of pathogen indicator organism inactivation In the production of struvite, heterotrophic bacteria in urine during the storage of urine under a range of conditions has been accumulate in the solid during struvite precipitation and filtration € extensively evaluated (Hoglund et al., 2002a; Nordin et al., 2013; and are inactivated during drying with decreasing struvite moisture Vinneras et al., 2008). Uncharged ammonia (NH3), a volatile com- content (Schoger, 2011). However, concentrations of the in situ pound produced during biological urea hydrolysis, acts as an in situ bacteria in struvite may stabilize under mild drying conditions (e.g., sanitizer in stored urine (Decrey et al., 2015; Vinneras et al., 2008). 20 C, 80% relative humidity). The inactivation kinetics of patho- Measured ammonia concentrations in this study were similar to genic bacteria during struvite drying has not been considered spe- € those reported in urine storage tanks in Sweden (Hoglund et al., cifically, though achieving only partial inactivation could be 2000). To maximize its biocidal effect, NH3 losses from stored problematic for attaining high fertilizer end-product quality. urine should be minimized. Therefore, dilution of urine or the use Increased desiccation of struvite at elevated temperatures would of unsealed tanks or aeration during pumping of urine to transport facilities, which could lead to NH3 volatilization, should be limited. Ammonia losses should also be avoided to limit occupational 1 Durban Yearly Weather Summary: http://www.worldweatheronline.com/ inhalation or dermal exposure to ammonia, by which deleterious Durban-weather-averages/Kwazulu-Natal/ZA.aspx, accessed January 28, 2014. 64 H.N. Bischel et al. / Water Research 85 (2015) 57e65 likely mitigate this challenge, though struvite degrades rapidly the added benefit over struvite production of complete nutrient above 55 C due to ammonia loss (Bhuiyan et al., 2008). Thorough recovery for enhanced fertilizer product quality. dessication of struvite at temperatures above ambient conditions is also important to inactivate more resistant pathogens (e.g., hel- 4.4. Conclusion minths), which are also retained in the precipitate during filtration (Decrey et al., 2011). In contrast to bacteria, viruses present in the UDDTs provide increased access to sanitation, especially when urine are retained in struvite pore spaces but are not preferentially coupled with resource recovery and reuse, which further creates a accumulated (Decrey et al., 2011). Virus inactivation was also found financial driver for the sanitation system (Diener et al., 2014). With to increase linearly with decreasing moisture content of the struvite. a large network of dry, urine-diverting toilets in place, the oppor- Similar to viruses, pharmaceuticals in struvite were contained in tunity is ripe in eThekwini to move up the “sanitation ladder” (Mara the residual urine before struvite drying. For seven pharmaceuticals et al., 2010) by eliminating the practice of urine discharge via soak- evaluated, an average of 98% of the analytes remained in urine away pits. The presence of pathogens, pharmaceuticals and anti- following struvite precipitation (Ronteltap et al., 2007). Washing biotic resistance determinants in the source-separated urine re- struvite with water prior to drying was found to further reduce veals an important opportunity to reduce discharge of these pharmaceutical concentrations in the end-product by removing the contaminants to the environment via urine collection. However, residual urine. Such a procedure would likely reduce pathogen the implications of such human and environmental health hazards concentrations in struvite as well. also require further evaluation. In particular, the results may be With proper washing and drying procedures, the production of contextualized in eThekwini via a quantitative microbial risk struvite can thus promote the inactivation or removal of bacterial assessment of local urine collection practices, when infective and viral pathogens in fertilizers produced from source-separated pathogen concentrations are expected to be highest and opportu- urine and reduce end-product pharmaceutical concentrations. nities for accidental human-urine contact are likely greatest. These procedures facilitate the production of urine-based fertilizers Beyond urine collection, urine nutrient recovery processes must with shortened urine storage times. However, pathogens and also yield high-quality products that are protective of human and pharmaceuticals remaining in the urine after struvite precipitation environmental health and that ensure adequate fertilizer quality are not treated during this procedure and remain a potential hu- for optimal market value. The high content of such unwanted man and environmental health hazard. Longer-term field scale substances in the fertilizer source material may impact the quality, assessments as well as analysis of additional pathogens and TrOC or perceived quality, of end-products. Therefore, this study also targets are also required in order to fully assess the fate and highlights how potential environmental and human health con- transport of contaminants during struvite production. cerns could be reduced by incorporating a urine treatment step. Processes to produce urine-derived fertilizers can be designed or 4.3.3. Urine nitrification for nutrient stabilization modified to inactivate pathogens and transform or remove phar- An alternative to struvite production for urine nutrient recovery maceuticals in source-separated urine. The production of the solid is the application of biological nitrification to stabilize nutrients in fertilizer, struvite, can inactivate pathogens and remove pharma- urine (Udert et al., 2003). Recent evaluation of the inactivation of ceuticals from the end-product when implemented with proper bacterial and viral surrogates during urine nitrification indicates washing and drying procedures. Biological nitrification for urine that nitrification is insufficient as a stand-alone technology for nutrient stabilization can be coupled with the post-nitrification pathogen inactivation (Bischel et al., 2015). A relatively short steps of distillation and advanced treatment to improve the qual- nitrification reactor hydraulic retention time would likely yield ity of the liquid fertilizer in terms of pathogen and pharmaceutical only 90% or less inactivation. Viruses are likely to remain infective concentrations. through nitrification treatment. However, because volatile Further evaluation and optimization of such urine treatment ammonia is converted to nitrate during nitrification, the nitrified and fertilizer production technologies at field-scales is needed to urine product can be distilled to yield a concentrated fertilizer and a reduce occupational hazards to staff involved in urine processing pathogen treatment benefit. In a pilot reactor (Udert et al., 2015), and minimize environmental risks associated with urine manage- nitrified urine is distilled for several hours at 80 C. This is a harsher ment. Nevertheless the prevention of pathogen and pharmaceutical treatment than recommended for sanitization of compost (70 C for discharge to the environment via urine source separation and 30 min; US EPA, 2003), and can be expected to yield a microbially collection is a considerable advantage to this sanitation technology. safe product. Of the 12 pharmaceuticals studied herein, only four were found Acknowledgments € to degrade substantially during urine nitrification (Ozel Duygan et al., 2015). This is in accordance to the behavior of pharmaceuti- This study was undertaken in the framework of the Valorisation cals in municipal wastewater treatment, as many are still detected of Urine Nutrients in Africa (VUNA) project funded by the Bill & in considerable concentrations after wastewater treatment Melinda Gates Foundation (www.vuna.ch, Grant No. OPP1011603). (Michael et al., 2013). This material is also based upon work supported by the US National Nitrification of urine alone thus does not provide a significant Science Foundation International Research Fellowship Program treatment benefit for pathogens and pharmaceuticals in urine. (grant no. 1159225) and the Swiss National Science Foundation However, coupling biological nutrient stabilization with the post- (grant no. 200021_146829/1). We thank: Chris Buckley and the nitrification steps of distillation and advanced treatment can Pollution Research Group (UKZN) for laboratory access and sup- improve the quality of the liquid fertilizer by inactivating patho- port; Teddy Gounden and EWS for support of field activities; Max gens and removing pharmaceuticals, respectively. Several treat- Grau (EWS), Leanne MacGregor (VUNA) and Elizabeth Tilley (ETHZ) ment processes have been successfully tested for the removal of for assistance with sampling; Annette Remmele, Falk Dorusch and pharmaceuticals from stored urine: ozonation (Dodd et al., 2008), Birgit Beck (Eawag) for assistance with trace organics analysis; electrodialysis (Pronk et al., 2006a), and nanofiltration (Pronk et al., Sophie Campiche (Swiss Centre for Applied Ecotoxicology) for 2006b). Treatment with activated carbon was shown to be an helpful insight into ecotoxicology; Nadine Czekalski and Helmut effective process for pharmaceutical removal from nitrified urine Bürgmann (Eawag) for materials and assistance with antimicrobial € (Ozel Duygan et al., 2015). Nitrification with distillation provides resistance analysis. H.N. Bischel et al. / Water Research 85 (2015) 57e65 65

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PART II

LIQUID FERTILIZER PRODUCTION: EVALUATING THE POTENTIAL FOR BIOLOGICAL NITRIFICATION TO REDUCE HUMAN HEALTH HAZARDS IN SOURCE-SEPARATED URINE

Environmental Science Water Research & Technology

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Inactivation kinetics and mechanisms of viral and bacterial pathogen surrogates during Cite this: Environ. Sci.: Water Res. † Technol.,2015,1,65 urine nitrification

Heather N. Bischel,a Ariane Schertenleib,a Alexandra Fumasoli,b Kai M. Udertb and Tamar Kohn*a

This paper assesses the inactivation performance and mechanisms in urine nitrification reactors using bacteria and bacteriophages as surrogates for human pathogens. Two parallel continuous-flow moving bed biofilm reactors (MBBRs) were operated over a two-month period. One MBBR was used to conduct a continuous spike experiment with bacteriophage MS2. The second reactor provided the matrix for a series of batch experiments conducted to investigate the inactivation of Salmonella typhimurium, Enterococcus spp., MS2, Qβ,andΦX174 during urine nitrification. The roles of aeration, biological activity, and solution composition in inactivation were evaluated. Whereas bacteriophages ΦX174 and MS2 remained infective following urine nitrification, partial inactivation of bacteriophage Qβ was observed. Qβ inactivation was attributed primarily to aeration with a potential additive effect of biological processes, i.e.,processesthat are attributable to the presence of other microorganisms such as sorption to biomass, predation or enzy- matic activity. Tailing of Qβ inactivation to a plateau indicated a protective effect of the solution compo- nents in aerated nitrification reactors. In contrast to the bacteriophages, S. typhimurium and Enterococcus spp. were mainly affected by biological processes: they were inactivated in biologically active nitrification reactors while remaining stable in chemically equivalent filtered controls. The tested bacteria could, for example, be out-competed by other microbial communities or sorbed to biomass in the reactor. Microbial Received 8th October 2014, communities did not adapt to inactivate bacteriophage MS2 (e.g., via increased prevalence of virus preda- Accepted 3rd December 2014 tors) in the experimental time-scale evaluated, with no observed inactivation of MS2 during continuous

Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. input for 51 days in the flow-through MBBR. The compilation of these results suggests that biological nitri- DOI: 10.1039/c4ew00065j fication as a fertilizer production process remains insufficient as a stand-alone technology for the sanitiza- rsc.li/es-water tion of source-separated urine.

Water impact Recently, nitrification has been applied at pilot scales for the production of agricultural fertilizers from urine. While little is known regarding the inactivation of bacterial and viral pathogens during nitrification, such information is instrumental in evaluating the hygiene and safety of the nitrification end product. This study quantitatively assesses pathogen surrogate inactivation performance and mechanisms in urine nitrification reactors using two bacteria (Salmonella typhimurium and Enterococcus spp.) and three indicator viruses (MS2, ΦX174, and Qβ). Further, the influence of physical (i.e., aeration) and biological (i.e., microbial activity) processes on inactivation as well as the role of chemical solution characteristics of nitrified urine was assessed. Results indicate that biological nitrification is insufficient as a stand-alone technology for the sanitization of source-separated urine.

a Laboratory of Environmental Chemistry, School of Architecture, Civil and Introduction Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. E-mail: [email protected] Nutrients excreted in urine have long been used as a fertilizer b Eawag: Swiss Federal Institute of Aquatic Science and Technology, for agricultural applications of sewage sludge1 and waste- 8600 Dübendorf, Switzerland water2,3 or more directly through the application of source- † Electronic supplementary information (ESI) available: Contains results from separated urine.4,5 Urine contains the major fraction of nutri- the monitoring of physiochemical parameters in continuous flow MBBRs as – – well as inactivation rate constants calculated for supplementary batch tests for ents found in human excreta: 80 90% of the nitrogen, 55 4,6,7 ΦX174 and Qβ. See DOI: 10.1039/c4ew00065j 67% of the phosphorus and 50–80% of the potassium.

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Harvested urine not only provides value as an alternative fer- biomass recirculation.16 MBBRs are currently used for urine tilizer, but its use could also reduce pollution from unsafe nitrification in Durban and Dübendorf.13 The use of several excreta disposal, lower the costs of wastewater treatment, and MBBRs in this study allowed evaluation of several bacterio- lessen the ecological burden of fertilizer production and sur- phages and bacteria under field-relevant as well as varied plus use of chemical fertilizers.6,8 experimental conditions. Due primarily to the fecal contamination of source- Inactivation was evaluated using gram negative and gram separated urine,9 handling of urine and direct application as positive bacteria (Salmonella typhimurium and Enterococcus fertilizer in agriculture can pose microbial health risks.10 spp., respectively) as well as three bacteriophages (MS2, Qβ, Storage of urine for 6 months at 20 °C is recommended prior and ΦX174) that served as surrogates for human enteric to handling and usage in order to reduce or eliminate the viruses. The Salmonella genus consists of rod-shaped flagel- risks.11 However, in the scale-up of urine nutrient recovery lated facultative anaerobes of the family Enterobacteriaceae. such storage time requirements are prohibitively long. Alter- S. typhimurium, one of thousands of non-typhoidal serotypes native urine treatment and nutrient stabilization technologies of the medically important species S. enterica, causes most – are now available to produce marketable fertilizers.12,13 A cases of non-typhoidal Salmonella across Africa.17 19 Entero- promising nutrient recovery process that yields a chemically coccus spp. is a common fecal indicator bacteria, selected stable solution from stored urine is nitrification.14 In config- because of its slightly longer persistence in urine relative to urations where nitrification is applied in combination with gram negative indicator organisms.20 evaporation, a concentrated solution that preserves nearly all The inclusion of virus surrogates is particularly important nutrients in the urine can be produced.15 Pilot reactors for in this study as viruses are expected to persist much longer combined nitrification/distillation systems are operated at than gram negative and non-spore forming gram-positive eThekwini Water and Sanitation in Durban, South Africa and bacteria in stored urine10,21 and are thus anticipated to be at the Swiss Federal Institute of Aquatic Science and Technol- infective in the influent of field urine nitrification reactors. ogy (Eawag) in Dübendorf, Switzerland as part of the The selection of several bacteriophages also facilitates an Valorisation of Urine Nutrients in Africa (VUNA) project.13 evaluation of the effect of urine nitrification on viruses with a β Nitrification of urine, i.e., the oxidation of ammonia (NH3) range of characteristics. MS2 and Q are positive-sense single- − to nitrate IJNO3 ), prevents ammonia volatilization, enhancing stranded RNA bacteriophages that infect Escherichia coli.MS2 the recovery of nitrogen in urine. Two groups of nitrifying and Qβ are structurally similar, small in size (21–29 nm bacteria are involved in this process: ammonia oxidizing bac- diameter), and frequently used as models for enteric viruses.22 teria (AOB) and nitrite oxidizing bacteria (NOB). Nitrification With an isoelectric point of 3.9 and 5.3, respectively,23 MS2

typically oxidizes half of the NH3 content of urine. In addi- and Qβ are expected to be negatively charged in urine or tion, it reduces the pH of urine from 9 to around 6, which nitrified urine. ΦX174 is a single-stranded DNA bacterio-

causes the remaining NH3 to become protonated to the non- phage with a diameter of 27 nm and an isoelectric point of + 14 23 volatile NH4 (pKa = 9.25). Concomitantly to nitrification, 6.6. Because of its low hydrophobicity and high stability Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. heterotrophic bacteria consume biologically degradable organic against many environmental stressors,24 including stored substances so that malodor is removed as well. urine,20 ΦX174 has been suggested as a model for more con- Refinement of urine nitrification processes presents sev- servative virus inactivation. eral challenges. At a technical level, nitrifying bacteria are Using the test organisms described above and varied sensitive to environmental conditions and the intermediate continuous-flow and batch reactor conditions, the specific product nitrite.15 From a public health perspective, the path- objectives of this study were to (1) assess the inactivation of ogen inactivation or removal efficacy during urine nitrifica- gram negative and gram positive bacteria during urine nitrifi- tion is unknown. Such information is instrumental in cation, (2) similarly evaluate the inactivation of several bacte- assessing the hygiene and safety of the nitrification end prod- riophages as surrogates for human viruses, (3) elucidate the uct (fertilizer) and to minimize health risks to reactor opera- roles of physical and biological processes as well as the tors. The present study aimed to establish and operate chemical solution composition of urine nitrification reactors bench-top urine nitrification reactors to evaluate the inactiva- in the inactivation of test organisms. Results are anticipated tion of viral and bacterial pathogen surrogates during this to inform the understanding of pathogen inactivation during nutrient recovery process. Inactivation kinetics for five organ- urine nitrification and more broadly for other applications of isms were established and compared, and the main modes of nitrification for wastewater treatment. inactivation were identified. Two types of nitrification reactors were operated in this Materials and methods study: two 6.5 L continuous flow moving bed biofilm reactors (MBBR) and several smaller-volume batch MBBRs. In MBBRs, Materials floating plastic carriers are used to provide surfaces for bio- Approximately 25 L of nitrified urine and 10 L of Kaldnes® films. The biofilm prevents washing out of slow growing bac- carriers with active biofilm were obtained from a 120 L urine teria such as nitrifiers and thereby allows high volumetric nitrification reactor operating at Eawag and were stored at conversion rates without the need for membrane filtration or 4 °C until use in the laboratory reactors. Urine (100 L) to feed

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reactors was collected from the men's NoMix storage tank at Eawag and stored at 4 °C. Autoclaved phosphate-buffered 2− saline (PBS; 5 mM PO4 , 10 mM NaCl, pH 7.5) was used for storage of bacteriophage stocks; PBS was adjusted to pH 6.1 using HCl for batch reactor experiments.

Bacteriophage Bacteriophages MS2 (DSMZ 13767), ΦX174 (DSMZ 4497), and Qβ (DSMZ 13768) and their respective bacterial hosts E. coli (DSMZ 5695 for MS2 and Qβ and DSMZ 13127 for ΦX174) were used. Bacteriophage stocks were prepared and enumer- ated by the double-layer agar method as described previously.25 The stock was conserved in PBS at 4 °C, and the volume of stock used to spike the batch reactors was <1% of the reactor volume. Phage concentrations are reported as plaque forming units (pfu) per mL. A PBS blank and an E. coli host blank were plated at each time point. Bacteria An attenuated derivative of Salmonella typhimurium (strain Fig. 1 Continuous flow MBBR set-up with a urine influent container SL1344) was generously provided by the Microbiology Depart- (1), a nitrified urine effluent tank (2), a urine nitrification MBBR (3), a pH transmitter (4), a peristaltic pump (5), a DO transmitter (6), pH and DO ment of Eawag. Isolates were grown to log phase in LB broth probes (7), a data logger (not shown), and an aeration device (9).The −1 with 100 μgmL ampicillin and stored in aliquots with 15% pH transmitter records the instantaneous pH value of the MBBR and glycerol at −80 °C. S. typhimurium was grown overnight from activates the peristaltic pump to inject stored urine into the reactor storage in ampicillin-containing lysogeny broth immediately when required to increase the reactor pH. prior to use in experiments. Bacteria were pelleted and resuspended in PBS prior to reactor spiking (spike volume was <1% of reactor volume). The spread-plate method with as determined by the specific surface area of the Kaldnes® − 100 μL sample on ampicillin containing agar was used for (460 m2 m 3)carriers. enumeration and concentrations are reported as colony Stored urine (9.0 ± 0.1) was the sole input to the reactor, forming units (cfu) per mL. Spike concentrations were serving as the ammonia source for nitrifying bacteria, the selected to be more than four orders of magnitude above low organic input for heterotrophic bacteria, and to balance the background concentrations of ampicillin resistant colonies decrease in pH that occurs with ammonia nitrification. To Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. detected in unspiked nitrified urine. Enterococcus spp. colo- control the urine input, a proportional-integral-derivative nies were isolated from wastewater treatment plant influent (PID) controller was configured for each reactor using a (Vidy, Lausanne) on Bile Esculin Agar (Sigma-Aldrich, St. Louis, Liquisys M CPM 223 regulator (Endress+Hauser AG, Reinach, MO, USA) and subsequently grown in azide glucose broth Switzerland) that regulated the inflow based on the pH in the (Sigma-Aldrich). Aliquots of log-phase growth Enterococcus reactor. pH was measured with a pH ISFET combination − ° spp. were stored with 15% glycerol at 80 C. Enterococcus electrode (Endress+Hauser). The PID parameters (Kp, tmin)of spp. from stored aliquots were grown overnight in azide glucose the regulator were empirically determined to yield a rapid broth immediately prior to use in batch experiments. 100 μLof response to pH changes and to maintain the pH target of sample and 50 mL of PBS were filtered using the membrane 6 to 6.1 (pH set-point, pH = 6.1; proportional gain, Kp = 3;

filtration EPA method 1600 (ref. 26) for enumeration. A PBS minimal length of response, tmin = 0.1 s). The minimal length blank was plated at each time point. of response allowed at least one drop of urine input in the reactor per urine input event. The reactor was aerated Continuous flow MBBRs with humidified air through a 125 mm Hobby Flexi Diffuser Two bench-top 6.5 L MBBRs made of PVC were operated in (Saint Vincent Group, Dubai, UAE). PVC tubing was used to parallel (Fig. 1). Material from reactor 1 served as the matrix deliver the urine input and moistened air within the reactor. for batch MBBR experiments described below, and reactor 2 To reduce evaporation and prevent escape of foam, the top of was continuously spiked with MS2 to evaluate potential the reactors was plugged. Effluent from the reactor surface phage inactivation during nitrification. Reactors were seeded was continuously captured into a separate storage container. with the contents of an active urine nitrification reactor (i.e., nitrified urine and Kaldnes® K1 carriers with biofilm from Monitoring of physiochemical parameters in continuous Eawag) such that nitrifying organisms were in place at initia- flow MBBRs tion. The filling ratio of the reactor was 50%, yielding a total The pH and dissolved oxygen (DO) in the reactors were approximate biofilm carrier surface area of 1.63 m2 per reactor, monitored and recorded once per minute using an Ecograph

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−1 T RSG35 data logger (Endress+Hauser). The DO probes (e.g., in pfu mL ), Qin is the flow rate of the influent (L per

(Mettler-Toledo, Greifensee, Switzerland) were configured to day), Qout is the flow rate discharging from the reactor (L per separate COM223F regulators (Endress+Hauser). pH and DO day), and k is the first-order inactivation rate constant (per probes were calibrated weekly, according to the manufac- day). To model the concentration of MS2 in the reactor with turer's instructions. The weights of influent and effluent time, the average concentration between two MS2 spike refer- tanks were recorded daily to determine the flow rate. When ence samples, along with the flow rate of the MS2 spike and nitrified urine and biofilm carriers were removed from the the urine input, was used to determine the input concentra-

reactor for batch experiments, the same volume of material tion, Cin. The time between each sample measurement was was replaced using the nitrified urine stock stored at 4 °C the model input time step. The generalized mass balance

and acclimated to room temperature. Temperature was (eqn (1) with Qin = Qout) can be solved under steady state con-

recorded daily from the pH transmitter. Nitrite was moni- ditions to determine the removal efficiency, C/Cin, for a con- tored approximately once per week using Nitrite Test strips tinuously mixed reactor with known hydraulic retention time (Merck Millipore, Darmstadt, Germany) to avoid nitrite accu- (HRT = V/Q) and constant inflow of a microorganism that fol- mulation in the reactors. Hach-Lange cuvette tests were used lows first-order removal kinetics, yielding:

to monitor ammonium (LCK 303), nitrite (LCK 342), total 1 nitrogen (LCK 338) and chemical oxygen demand (COD; LCK C  V  1 k  (2) 614) in reactor influent and effluent. For chemical analysis, Cin  Q  18 mL samples were taken and filtered through 0.45 μm cellulose nitrate filters (Albet LabScience, Dassel, Germany), Batch and semi-batch MBBRs discarding the first half of the filtrate prior to analysis according to the manufacturer's instructions. Samples were To test the inactivation of bacteriophages and bacteria under diluted with Milli-Q water to within the appropriate measure- varying experimental conditions, a series of 500 mL semi- ment range and measured with a DR 3900 spectrophotometer batch and batch MBBRs were established in washed and (Hach Lange GMBH, Düsseldorf, Germany). The volume, autoclaved 1 L Pyrex glass bottles (Fisher Scientific, Reinach, weight, date and time of samples were recorded. Measure- Switzerland). Batch reactors refer to reactors with neither an ments of the input stored urine (Table 2) were consistent influent flow nor an outflow, while semi-batch hereafter with other analysis of urine from the NoMix men's storage refers to reactors that received influent for the duration of tank at Eawag.15,27 Unless otherwise noted, results are the experiment but did not have an outflow. When biofilm presented as average values with standard deviations (SD). carriers were added to the reactors, a filling ratio of 50% was used. Reactors were covered with parafilm to reduce loss by Continuous MS2 input to continuous flow MBBR evaporation. Five types of reactors (Table 1) were evaluated for all target organisms, with Qβ and ΦX174 tested together Reactor 2 was amended continuously with MS2 for 51 days. The in one series and MS2 tested with bacteria in a separate initial MS2 concentration was forced to steady-state by first Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. series. The main reactor conditions and parameters studied injecting a high concentration pulse followed by a reduced were given as follows: actively nitrifying urine reactors vs. fil- concentration continuous spike delivered via a syringe pump tered nitrified urine controls (to evaluate the effect of biologi- and a headspace-free syringe. This was achieved by first − − cal activity), aerated reactors vs. non-aerated reactors (effect adding 1010 pfu mL 1 MS2 at 0.001 mL min 1 for 1 hour − of aeration), and urine reactors vs. PBS reactors (effect of followed by a continuous input of 107 pfu mL 1 MS2 at − matrix composition). 0.001 mL min 1. The syringe was refilled every 2–5days.To The baseline data set was obtained from replicate actively monitor the inflow concentration, MS2 concentrations were nitrifying semi-batches that were seeded with the contents of measured in a side-by-side reference solution of the spike the continuous flow reactor 1. Reactors were aerated and solution kept at the same room temperature as the syringe received urine input over 8 days. The influent flow rate was used to amend reactor 2. The reference solution MS2 concen- empirically determined to maintain a pH of 6.1 to 6.2. Reac- tration showed agreement with the syringe MS2 concentra- tor material for the ΦX174 and Qβ semi-batches was removed tions and allowed more frequent input concentration mea- from reactor 1 on day 40 when the reactor 1 nitrification rate surements (data not shown). − was 0.479 gN m 2 per day (day 39). Material for the MS2 and The concentration of a microorganism with first order bacteria semi-batches was obtained on day 70; the nitrifica- removal kinetics in a continuous flow reactor can be calcu- − tion rate on day 65 was 0.267 gN m 2 per day. The reactor lated assuming a perfectly mixed system of constant total − nitrification rate per surface area (r ,gNm2 per day) was reactor volume, V (6.5 L), according to the rate of change n calculated as: mass balance equation: NHQQ NH  dC Q Q 4,in in 4,out out in out rn  (3) Cin CkC (1) dt V V Atot

where C is the microorganism concentration in the reactor, where NH4,in is the ammonia concentration in the influent −1 Cin is the concentration of the microorganism in the influent (gN L ), NH4,out is the ammonia concentration in the

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Table 1 Semi-batch and batch reactors

Reactor type Description Composition Nitrified urine MBBR semi-batch Biologically active system: conducted in duplicate Reactor content: 500 mL of nitrified urine; for all test organisms 250 mL of active biofilm carriers Reactor input: stored urine Aeration: yes Filtered nitrified urine semi-batch Filtered control: to test the role of solution Reactor content: 500 mL of 0.45 μm-filtered composition relative to biological activity nitrified urine; 250 mL of clean biofilm carriers Reactor input: 0.45 μm-filtered nitrified urine Aeration: yes Nitrified urine batch Aeration control: to test the role of aeration when Reactor content: 500 mL of nitrified urine compared with nitrified urine MBBR semi-batch Reactor input: none Aeration: none Phosphate-buffered saline (PBS) Clean aerated matrix control: to compare inactivation Reactor content: 500 mL of PBS; 250 mL of aerated batch in aerated nitrified urine matrix to a clean PBS matrix clean biofilm carriers and to evaluate the role of aeration in a clean matrix Reactor input: none Aeration: yes PBS batch Clean matrix control: to determine baseline inactivation Reactor content: 500 mL of PBS at the experimental temperature, to compare inactivation Reactor input: none in non-aerated nitrified urine matrix to a clean PBS Aeration: none matrix, and to evaluate the role of aeration in a clean matrix

−1 effluent (gN L ), and Atot is the total biofilm carrier surface Data analysis 2 area (m ). First-order inactivation rate constants (k, per day) were deter- To disentangle the effect of biological activity from that of mined from a least-square linear regression of data from solution composition on inactivation, filter-sterilized controls each batch and semi-batch experimental condition according with aeration were established for comparison with actively to the equation: nitrifying reactors. These biologically inactive semi-batch reactors contained new biofilm carriers (cleaned with bleach C ln kt (4) and rinsed several times with sterile water) and were filled C0 with 0.45 μm-filtered nitrified urine from the continuous flow MBBR. The same filtered nitrified urine also served as the where C0 is the initial concentration of the target organism. influent at the rate established for the urine inflow to the Without an outflow, the volumes of semi-batches increased Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. active nitrification semi-batch. slightly during the experiment. For batch tests, evaporation To assess the physical role of gas bubbles on inactivation reduced the reactor volume slightly over the course of the (i.e., aeration), a nitrified urine control without inflow, bio- experiment. Therefore, concentrations and inactivation rates film carriers or aeration was tested for comparison with the were corrected for the changes in volume due to inflow and actively nitrifying, aerated semi-batch. Some biological activ- evaporation. Specifically, semi-batch and batch reactor ity was expected to be sustained in these reactors, although volumes were recorded initially and at the conclusion of the at lower rates than the aerated nitrifying semi-batch. inactivation tests. A linear rate of evaporation or inflow was Finally, PBS batch controls either with clean biofilm car- assumed between the initial and final measured volumes. riers and aeration or without both were tested to compare This rate was then used to correct the inactivation rates and inactivation in nitrified urine to that in a simple matrix (rep- concentrations of microorganisms for changes in the reactor resented by PBS). volume. For Qβ, first-order inactivation rate constants were In the tests containing nitrifying bacteria, pH and nitrite calculated for the first 4 days of the test only, prior to signifi- concentrations were measured daily to verify the batch reac- cant onset of tailing of the inactivation curve towards a pla- tor stability. For the remaining reactors, the pH and tempera- teau. Reaction rates are reported only when the slope (k) was ture were measured at the beginning of the experiment, and different from zero within 95% confidence. pH stability was verified at the end of the experiment. For ΦX174 and Qβ, additional PBS batch tests without biofilm Results and discussion carriers were conducted with and without aeration to verify observations. For Qβ, additional replicate nitrified urine The operation stability of laboratory urine nitrification reac- batch tests without urine input or biofilm carriers were tors established in this study is first reported for comparison conducted with and without aeration. Material for these tests with field nitrification activities, followed by a discussion of was obtained from reactor 1 when the nitrification rate was the inactivation of pathogen surrogates observed in batch, − 0.218 gN m 2 per day. semi-batch and continuous reactors.

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Continuous flow MBBR performance (approximately 2 L of nitrified urine and 1 L of biofilm car- The pH, DO, temperature and nitrification rates of the two riers) was removed for batch experiments and replaced by continuous-flow urine nitrification reactors are presented stored material. As the nitrification rate decreased in the in Fig. S1 (reactor 1) and S2 (reactor 2).† Average measured reactors, a lower urine input flow rate was required to main- physical and chemical parameters are shown in Table 2. tain the set pH. Therefore the input and output flow rates Approximately half of the total ammonia in urine was oxi- decreased over time in both reactors, and changes were more † dized under optimum reactor conditions, indicating active pronounced in reactor 1 than those in reactor 2 (Fig. S3 ). biological nitrification. This is consistent with the literature: a maximum of 50% of ammonia in urine was converted to Bacteria and bacteriophage inactivation in semi-batch and nitrate in either a MBBR, a continuous flow stirred reactor, or a batch reactors sequencing batch reactor.14 Heterotrophic bacteria degraded Inactivation of bacteriophages MS2, ΦX174 and Qβ as well as approximately 90% of the COD in the influent. The average pH the bacteria Enterococcus spp. and S. typhimurium was tested in the reactors was stable at 6.05 (SD < 0.01) throughout the over 7 to 10 days in semi-batch MBBRs containing either operating and experimental period, except for short periods active nitrifying biofilm carriers or filtered nitrified urine of electronic malfunction and during the first several days of with clean biofilm carriers (Table 1, Fig. 2). Enterococcus spp. operation when urine input was limited to prevent nitrite and S. typhimurium underwent 4–5 log inactivation over 7 accumulation in the reactor. pH is an important parameter days in the active biological batch, compared to no inactiva- influencing AOB and NOB activity.14 DO was also relatively tion in the filtered control. While ΦX174 and MS2 were not stable throughout operation. or only minimally inactivated in either the active nitrification The average nitrification rate in continuous flow reactors system or the filtered nitrified urine, infective Qβ concentra- − over the course of operation was 0.56 ± 0.14 gN m 2 per day in tions decreased by 3–4 logs in both reactor types. − reactor 1 and 0.49 ± 0.10 gN m 2 per day in reactor 2, lower Inactivation of target organisms was also tested in aerated − than the reported maximum nitrification rate of 1.7 gN m 2 batch reactors containing only PBS and clean biofilm car- per day in a 2.8 L reactor.14 Over time the nitrification rate riers, without any input (Fig. 3). Only Qβ was inactivated with also decreased. The lower nitrification rate may be explained k > 0.5 per day in repeat aerated PBS batch tests. For an by temperature: the average temperature in both MBBRs unknown reason, the concentration of ΦX174 decreased sub- was approximately 5 °C lower than the reported temperature stantially in one aerated PBS control. In 10 other batch or of operating reactors in the literature.15 Temperature is an semi-batch tests for ΦX174, including duplicate aerated PBS important factor influencing the growth of nitrifiers; a 5 °C control batches (Table S1†), inactivation of ΦX174 was mini- decrease in temperature could reduce the biomass activity of mal. This outlier was therefore excluded from the subsequent AOB by 25%.28 However, temperature measurements were rate calculation and discussion. taken only once per day, limiting a detailed analysis of this Finally, inactivation of all test organisms was monitored effect. Furthermore, the salt concentrations were higher than in nitrified urine and PBS batch reactors without aeration Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. in the reactor from which the inoculum originated. High salt (Fig. 3). In biologically active nitrified urine held without aer- concentrations are known to inhibit nitrifying bacteria.29 ation, Enterococcus spp. and S. typhimurium exhibited inacti- The greatest decrease in nitrification rate appeared in reactor vation similar to aerated semi-batches. The bacteria were 1, where a substantial fraction of the reactor material stable or exhibited comparably low inactivation rates in PBS

Table 2 Average physical and chemical parameters of continuous-flow MBBR influent and reactor contents (effluent)a

Reactor 1 Reactor 2 Parameter Influent (Avg. ± SD) Reactor content (Avg. ± SD) Influent (Avg. ± SD) Reactor content (Avg. ± SD)

−1 b NO2 [mg L ]NM 1.37 ± 0.43 NM 1.27 ± 0.30 −1 NH4,tot [mg L ] 3760 ± 180 1980 ± 130 3570 ± 300 1850 ± 250 −1 c Ntot [mg L ] 3970 ± 440 4270 ± 330 3780 ± 170 4250 ± 150 − COD [mg L 1] 3870 ± 61 419 ± 72 3890 ± 260 401 ± 70 −1 COD/N [mg O2 mg N] 1.04 ± 0.12 0.09 ± 0.01 1.06 ± 0.08 0.09 ± 0.01 pH [−] 9.04 ± 0.09 6.05 ± < 0.01 9.04 ± 0.1 6.05 ± < 0.01 Temperature [°C] NM 19.1 ± 1.8 NM 20.2 ± 1.3 −2 d rn [gN m per day] N/A 0.56 ± 0.14 N/A 0.50 ± 0.10 HRT [day] N/A 20.7 ± 8.2 N/A 17 ± 3.7 Losses [vol%] N/A 1.1 ± 0.9 N/A 1.6 ± 0.9

a Averages for pH, temperature, nitrification rate (rn), hydraulic retention time (HRT) and volume losses for the reactor contents are calculated from the data presented in Fig. S1–S3. The average (Avg.) and standard deviation (SD) are based on n = 8 measurements for the influent and other reactor content measurements. b NM = not measured; nitrite concentrations were below detection in the initial tests of the urine influent c and are usually negligible in stored urine. Higher apparent Ntot in the effluent can be explained in part by water loss in addition to measurement accuracy. d N/A = not applicable.

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slowly inactivated in PBS without aeration. MS2 and ΦX174 were stable in nitrified urine or only slowly inactivated in PBS without aeration. First-order inactivation rate constants (k) were calculated for all semi-batch and batch systems (Tables 3 and S1†). Reproducibility between replicate active nitrification semi- batch reactors was high; for each of the three bacteriophages and two bacteria, there was no significant difference within 95% confidence limits between first-order inactivation rates measured in replicate reactors. The inactivation curve observed for Qβ in aerated nitrified urine reactors consisted of an initial exponential decrease and a secondary plateau. The first-order rate constants (k) were therefore calculated for the initial decrease that occurred in the first 4 days of the experiment. A comparison of the inactivation kinetics observed in the different reactors allowed us to identify the main modes of bacteria and virus inactivation during nitrification. Specifi- cally, we could assess (1) the physical role of the air–water interface, (2) the role of a biologically active bacterial commu- nity, and (3) the role of chemical solution conditions. These mechanisms are discussed below, and a recapitulation of the primary observed modes of inactivation is presented in Table 4.

Inactivation at the air–water interface Aeration of the MBBRs is essential in order to maintain dissolved oxygen levels, as well as to provide mixing of the solu- tion. Previous studies have found that viruses can become – inactivated at the air–water interface.30 32 In a study of bacte- riophages MS2 and ΦX174 at the air–water interface (AWI), Thompson et al.30 propose that loss of infectivity occurs when hydrophobic regions of the virus capsid partition out of solu- Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. tion into the gas phase via reconfiguration of the capsid pro- teins. More precisely, inactivation was shown to occur at the triple-phase-boundary at the interface of air, liquid and solid, and inactivation is influenced by both the hydrophobicity of the solid phase30,31 and the surface properties of the virus.32 In the present study, aerated reactors in glass bottles also contained polyethylene biofilm carriers, PVC tubing and air diffusers, providing several surface characteristics at which the liquid–air–solid interface may be formed. Aeration, there- fore, may lead to inactivation by increasing the air–water– solid interface compared to non-aerated reactors. This assumption was evaluated by comparing inactivation in aerated and non-aerated nitrified urine semi-batches or PBS control batches (Fig. 3). Little to no difference in

Fig. 2 The fraction of surviving organisms IJC/C0) over time during Enterococcus spp. or S. typhimurium inactivation was observed replicate semi-batch urine nitrification studies (solid lines, solid circles) between aerated and non-aerated batches of similar solution or filtered nitrified urine semi-batches (dashed lines, open squares). Both and biological conditions, indicating that the physical pres- types of semi-batches were aerated. C0 is the initial spiked concentra- − − − ence of air bubbles did not affect bacteria viability. Similarly, tions of MS2 (106 pfu mL 1), ΦX174 (106 pfu mL 1), Qβ (106 pfu mL 1), − − Φ S. typhimurium (108 cfu mL 1), or Enterococcus spp. (105 cfu mL 1). little to no difference in the infectivity of X174 and MS2 was observed between aerated and unaerated nitrified urine or PBS reactors. without aeration. Qβ also exhibited modest inactivation in In contrast, aeration did cause inactivation of Qβ in both nitrified urine without aeration and was relatively stable or nitrified urine and PBS (with or without biofilm carriers

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Fig. 3 Inactivation of target organisms in aerated or unaerated batch PBS reactors (left column) and aerated or unaerated batch nitrified urine reactors (right column). Results from aerated reactors are shown with solid black lines and solid circles. Unaerated reactor results are shown with dashed lines and open squares. Additional tests for ΦX174 and Qβ in aerated PBS without biofilm carriers or aerated nitrified urine without biofilm carriers are shown with solid grey lines and solid triangles.

present). In PBS, 4- to 5-log inactivation was observed over 6 leading to a 3- to 4-log decrease in infective Qβ over the days in reactors with aeration, compared to 1-log inactivation course of 4 days. Interestingly, the inactivation rate slowed in PBS without aeration. In nitrified urine, significant inacti- down after approximately 4 days in the aerated reactors that vation was observed both in the presence and absence of aer- contained nitrified urine (either unfiltered or filtered), lead- ation (Fig. 3 and Table S1†). Inactivation of Qβ can therefore ing to a tailing inactivation curve. This feature was not not be explained by aeration alone. However, aeration caused observed in any other Qβ experiments. A tailing inactivation the initial inactivation to proceed at a markedly faster rate, curve has been observed previously in phage disinfection

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Table 3 First-order inactivation rate constants [per day] in batch and semi-batch reactors, determined based on n time points, and standard error (SE)a

PBS batch, PBS batch, Biologically active nitrified Filtered nitrified urine Nitrified urine aerated with not aerated, urine semi-batches semi-batch, aerated semi-batch, not biofilm carriers without biofilm [per day]b [per day] aerated [per day] [per day] carriers [per day] MS2 NSc 0.17 ± 0.04 NS NS 0.13 ± 0.04 (n =6;R2 = 0.79) (n =6;R2 = 0.69) Qβd 1.77 (1.46–2.04) 1.88 ± 0.32 1.50 ± 0.07 1.82 ± 0.24 0.39 ± 0.09 (n =6,5,3,3; (n =6;R2 = 0.90) (n =6;R2 = 0.99) (n =8;R2 = 0.91) (n =7;R2 = 0.81) R2 = 0.62, 0.74, 0.97, 0.996) ΦX174 NS NS NS 0.38 ± 0.01 0.39 ± 0.01 (n =3;R2 = 0.999) (n =7;R2 = 0.99) S. typhimurium 1.42 (1.31–1.52) NS 1.50 ± 0.30 NS NS (n =5,6;R2 = 0.83, 0.85) (n =5;R2 = 0.89) Enterococcus spp. 0.92 (0.91–0.92) NS 0.71 ± 0.15 0.35 ± 0.09 0.27 ± 0.02 (n =5,6;R2 = 0.91, 0.83) (n =6;R2 = 0.85) (n =6;R2 = 0.79) (n =6;R2 = 0.98)

a Standard error of regression for the slope coefficient (k) determined from the log-transformed culturable fraction versus time, based on n data points. b Average of semi-batch results reported with range and R2 for each replicate experiment. c NS = not significantly different from zero at a 95% confidence level. d Calculated k for the first 4 days of the biologically active nitrified urine semi-batches and the filtered nitrified urine semi-batch.

Table 4 Recapitulation of the observed modes of inactivation for each target organism

Mode of inactivation MS2 ΦX174 Qβ S. typhimurium Enterococcus spp. (1) Physical effect of the air–water interface No No Yesa No No (2) Presence of biologically active community No No Possible Yes Yes (3) Chemical matrix effects No No No No No

a A protective effect of the chemical matrix was observed for Qβ during aeration, leading to tailing of the inactivation curve. This effect could also be relevant for MS2 and ΦX174 but could not be observed due to the overall lack of inactivation of these two bacteriophages.

kinetics and, for disinfection of MS2 by ClO2, was recently activated sludge wastewater treatment, viruses and bacteria attributed to deposition of protective material on the phage can be adsorbed on sludge flocs.35,36 Pathogens may also be exterior.33 Therefore, while the aeration of nitrified urine is out-competed by active biological communities. For example, Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. initially the primary cause of Qβ inactivation, it may also the regrowth of S. typhimurium has been suppressed by indig- facilitate the creation and deposition of protective material enous microflora in biologically active compost relative to for Qβ or other viruses, protecting them from complete sterilized compost.37 Predation of viruses or bacteria by pro- inactivation. tozoa or other sludge microbes as well as enzymatic activity – To rationalize why Qβ was susceptible to aeration whereas can also inactivate pathogens.38 40 A comparison of actively MS2 and ΦX174 were not, their surface properties must be nitrifying semi-batches with semi-batches containing filtered considered. From the literature, it is known that viruses nitrified urine (i.e., no bacteria larger than 0.45 μm) facili- containing hydrophobic regions on the capsid are more sen- tated evaluation of the role of microbial activity in the inacti- sitive to AWI inactivation.31,32 While highly similar in struc- vation of target organisms during nitrification. ture to MS2, Qβ is more hydrophobic than MS2,34 a charac- The concentrations of Enterococcus spp. and S. typhimurium teristic likely contributing to its susceptibility to inactivation were stable in the absence of bacteria (i.e., in filtered nitrified at the triple-phase boundary. In the context of urine nitrifica- urine). On the contrary, inactivation was observed in nitrifica- tion, aeration may thus contribute to the inactivation of tion batches, reaching 3-log reduction for Enterococcus spp. viruses with hydrophobic capsids, although protection from and 5-log inactivation for S. typhimurium over 6 days (Fig. 2). inactivation may limit the extent of inactivation. In contrast, Biological activity therefore had an effect on the survival of more hydrophilic viruses and bacteria appear to be resistant bacteria. Because of the similarities between temperature, to AWI inactivation. aeration, pH and other solution conditions between the two systems, the resulting difference in inactivation is likely attributable to biological processes relevant within the Role of biological treatment in the inactivation of experimental time frame, such as competition for nutrients pathogen surrogates with the indigenous organisms, sorption to Kaldnes® bio- In biological treatment systems, several physiochemical and films and predation. Inactivation of both S. typhimurium biological processes can lead to pathogen inactivation. In and Enterococcus spp. was similar between aerated and

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non-aerated (unfiltered) nitrified urine, despite the expecta- tion of reduced biological activity in the non-aerated batch. This indicates that adsorption to biomass or enzymatic activity, rather than competition in growth, may have played important roles. Additionally, because the HRT of field nitri- fication reactors is expected to be shorter than the duration of batch studies conducted, competition is expected to be less important. Further study is required to evaluate the relative contribution of different biological processes to inactivation. Concentrations of infective MS2, Qβ and ΦX174 followed the same evolution in both aerated biologically active and filtered systems (Fig. 2). This result indicates that the biologi- cal activity in the nitrification reactors did not cause bacterio- Fig. 4 MS2 was spiked continuously in a continuous flow MBBR for 51 phage inactivation, and inactivation of Qβ was largely attrib- days. A tracer was modeled in the reactor using the measured MS2 utable to aeration. However, inactivation of Qβ was also input concentrations and reactor flow rates. observed in non-aerated nitrified urine held without biofilm carriers (Fig. 3), indicating an additional inactivating effect of biological activity on Qβ. This finding was surprising because biological activity was expected to be reduced in the absence response to new substrates is highly variable. This effect of aeration. Qβ inactivation reached 4–5 logs in 8 days for could be further evaluated after months or even several years the unaerated system with a starting nitrification rate of of exposing an operating nitrification reactor to different − 0.5 gN m 2 per day, while inactivation reached only 2–3 logs (pathogen surrogate) substrates. in 7 days in the unaerated system with a starting nitrification − rate of 0.2 gN m 2 per day, suggesting that higher microbial activity may lead to more inactivation. This corresponded to Solution chemistry matrix effects an inactivation rate constant in the batch with higher micro- In stored urine, the three key parameters governing pathogen

bial activity (Table 3) that was approximately twice that of the inactivation are free ammonia (NH3) activity, pH and temper- † 9,20,21,27,41 lower microbial activity batch (Table S1 ). Therefore, while ature. NH3 is a known biocide for most organisms, aeration appears to be a primary mode of inactivation for Qβ, as is high pH.42 The survival time of bacteria and inactivation in unaerated nitrified urine batch controls is viruses in urine declines with increasing temperatures.20,21 likely due to biological processes of suspended microbial Gram-negative bacteria are generally more rapidly inactivated communities not attached to biofilm carriers or to degrada- in stored urine than gram-positive bacteria, and viruses are

tion by proteolytic enzymes present in the unaerated nitrified typically more persistent than both. In this study, NH3 con- Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. urine batch. centrations were reduced by microbial oxidation and pH To evaluate the ability of the biological community to was lowered from that of the influent, yielding less detrimen- adapt to inactivate persistent bacteriophage, MS2 was contin- tal conditions for the test microorganisms following nitrifica- uously spiked into the continuous MBBR over 51 days. Mea- tion than during urine storage at high pH. sured MS2 concentrations in the continuous-flow MBBR mir- To evaluate the role of the bulk nitrified urine solution rored the expected concentration of a modeled conservative composition on target organism inactivation, aerated batches tracer added with equivalent influent concentrations and no containing PBS were compared to aerated nitrified urine and degradation (k = 0 in eqn (1), Fig. 4). The difference between filtered nitrified urine. In the chemically complex solutions measured MS2 and modeled tracer concentrations was less (i.e., nitrified urine and filtered nitrified urine, Table 3), the than 0.5 log over the course of the experiment, indicating inactivation of bacteriophage was either comparable to or that little to no MS2 was lost due to adsorption to the reactor less pronounced than in the buffer (Tables 3 and S1†). As or to inactivation. This is consistent with little to no inactiva- was observed for Qβ, the solution could also provide a protec- tion of MS2 observed in all batch and semi-batch reactors. tive coating for the other phage, but this was not further eval- Because inactivation of MS2 was not enhanced through time, uated due to the lack of overall inactivation of MS2 and the biological community facilitating nitrification and ΦX174. organic degradation in the MBBR did not adapt to alter MS2 The concentrations of Enterococcus spp. and S. typhimurium infectivity within the experimental time scale. It was postu- decreased substantially in active nitrification reactors but lated that the microbial community could adapt with MS2 as were unchanged in filtered nitrified urine and relatively a continuously added substrate. Bacteria, protozoa or other stable in PBS. This suggests that the sole mode of inactivation organisms can engulf viruses or release virucidal agents, so during nitrification was biological processes, and there was the long-term input of MS2 could favor the growth of these no additional effect of the matrix composition. Further, the organisms and lead to increased MS2 inactivation. However, solution did not provide protection for bacteria as observed the time-scale over which microbial communities change in for Qβ.

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While the temperature was not specifically controlled in example, although energy intensive, provides the production the continuous or batch MBBRs (laboratory temperature, benefit of concentrating the liquid nitrified urine fertilizer ~20 °C), batch controls in PBS conducted at the same temper- and producing a clean water by-product, while also confer- ature as urine batch tests indicated little additional inactiva- ring a pathogen treatment benefit. tion effect of temperature for the duration of the experiment. Phage and bacteria remained relatively stable in unaerated Acknowledgements PBS (Fig. 3). In summary, the nitrified urine solution compo- sition and experimental temperature had little biocidal effect We thank our funding sources: the US National Science Foun- on bacteria or bacteriophage and, in the case of Qβ, may dation International Research Fellowship Program (grant no. instead contribute to protection of the virus from complete 1159225), the Swiss National Science Foundation (grant no. inactivation during aeration. 200021_146829/1), and the Bill and Melinda Gates Foundation. This study was part of the research project VUNA – Promoting sanitation and nutrient recovery through urine separation. Implications for urine nitrification applications While results of batch and semi-batch MBBRs cannot be References extrapolated directly to the continuous flow MBBRs because several parameters were different (e.g., aeration rate, reactor 1 Sewage sludge: Land utilization and the Environment, dimensions and material), they permit evaluation of several ed. C. E. Clapp, W. E. Larson and R. H. Dowdy, inactivation mechanisms for the bacteria and bacteriophage American Society of Agronomy, Inc., Madison, WI, 1994. and can inform further research with continuous flow MBBRs 2 H. N. Bischel, G. L. Simon, T. M. Frisby and R. G. Luthy, (Table 4). Bacteriophages ΦX174 and MS2 were more resis- Environ. Sci. Technol., 2012, 46, 180–188. tant to inactivation during urine nitrification than Qβ or 3 Water reuse for irrigation: agriculture, landscapes, and turf tested bacteria. The presence of active nitrification relative to grass, ed. V. Lazarova and A. Bahri, CRC Press, 2004. controls inactivated the tested bacteria but did not directly 4 T. Karak and P. Bhattacharyya, Resour., Conserv. Recycl., affect bacteriophages. Conversely, bacteriophages may be 2011, 55, 400–408. protected by macromolecules or particles generated during 5 M. Johansson, H. Jönsson, C. Höglund, A. Richert Stintzing aeration in nitrified urine, as observed for Qβ. This protective and L. Rodhe, Final Rep. R&D Proj. Source-Separated Hum. or tailing effect was evident for Qβ only and not for the tested Urin. – a Futur. source Fertil. Agric. Stock. Reg., 2001. bacteria. Qβ was sensitive to aeration in batch reactors, while 6 H. Heinonen-Tanski and C. van Wijk-Sijbesma, Bioresour. MS2, ΦX174 and bacteria were not. Technol., 2005, 96, 403–411. In further development of nitrification for the production 7 T. A. Larsen and W. Gujer, Water Sci. Technol., 1996, 34, of fertilizers from source-separated urine, it is anticipated 87–94. that nitrification will provide inactivation capacity for bacte- 8 K. M. Udert and M. Wächter, Water Res., 2011, 46, 453–464. Published on 04 December 2014. Downloaded 30/01/2015 11:30:08. rial pathogens but viruses may remain infective following 9 C. Schönning, R. Leeming and T. A. Stenström, Water Res., treatment. For example, field-scale nitrification reactors 2002, 36, 1965–1972. established in the VUNA project have a HRT of 3 to 6 days. 10 C. Höglund, T. A. Stenström and N. Ashbolt, Waste Manage. Under these conditions, assuming steady state of the reactor Res., 2002, 20, 150–161. has been reached (eqn (2)), and applying first-order inactiva- 11 WHO, Guidelines for the safe use of wastewater, excreta and tion rates presented in Table 3, S. typhimurium and Enterococ- greywater. Volume 4 excreta and greywater use in Agriculture, cus spp. are expected to undergo 0.7 to 1-log and 0.6 to 0.8- World Health Organization, 2006, vol. 4. log inactivation, respectively. Bacteriophage Qβ could reach 12 M. Maurer, W. Pronk and T. A. Larsen, Water Res., 2006, 40, 0.8 to 1.1-log removal if no protective effect of the matrix is 3151–3166. assumed, while no treatment benefit is expected for MS2 or 13 K. M. Udert, C. A. Buckley, M. Wächter, C. S. McArdell, ΦX174. The persistence of these viruses raises concern for T. Kohn, L. Strande, H. Zöllig, A. Hug, A. Oberson and the treatment capacity of urine nitrification for human B. Etter, in WISA Biennial Conference, Mbombela, viruses and therefore its ability to improve the hygiene of Mpumalanga, South Africa, 2014. urine fertilizer production. 14 K. M. Udert, C. Fux, M. Münster, T. A. Larsen, H. Siegrist Nitrification of urine removes a significant amount of the and W. Gujer, Water Sci. Technol., 2003, 48, 119–130. biocidal effect afforded by ammonia in stored urine. The 15 K. M. Udert and M. Wächter, Water Res., 2012, 46, 453–464. inactivation of bacteria and viruses could be enhanced via 16 E. Morgenroth, in Biological Wastewater Treatment, longer storage of urine prior to nitrification, but stabilization Principles, Modelling and Design, ed. M. Henze, M. C. M. van of the urine for nutrient recovery remains important. Addi- Loosdrecht and G. A. Ekama, WA Publishing, London, UK, tionally, because some viruses as well as spore-forming bacte- 2008. ria are known to persist in stored urine, even with extended 17 E. L. Hohmann, Food Saf., 2001, 32, 263–269. storage times, downstream treatment of nitrified urine would 18 M. Mcclelland, K. E. Sanderson, J. Spieth, S. W. Clifton, be necessary to inactivate such pathogens. Distillation for P. Latreille, L. Courtney, S. Porwollik, J. Ali, M. Dante, F. Du,

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SOLID FERTILIZER PRODUCTION: REDUCING HUMAN HEALTH HAZARDS IN SOURCE-SEPARATED URINE THROUGH STRUVITE PRODUCTION

Article

pubs.acs.org/est

Bacteria Inactivation during the Drying of Struvite Fertilizers Produced from Stored Urine † † ∥ † ∥ † ‡ Heather N. Bischel, Simon Schindelholz, , Manfred Schoger, , Loïc Decrey, Christopher A. Buckley, § † Kai M. Udert, and Tamar Kohn*, † Laboratory of Environmental Chemistry, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fedéralé de Lausanne (EPFL), CH-1015 Lausanne, Switzerland ‡ Pollution Research Group, Faculty of Engineering, University of KwaZulu-Natal, Durban 4041, South Africa § Eawag: Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland

*S Supporting Information

ABSTRACT: Human urine can be processed into market-attractive fertilizers like struvite; however, concerns regarding the microbial safety of such products remain. The present study evaluated the inactivation of in situ heterotrophs, total bacteria as observed by flow cytometry, and inoculated Enterococcus spp. and Salmonella typhimurium during the drying of struvite under controlled temperature (from 5 to 35 °C) and relative humidity (approximately 40 and 80%) as well as dynamic field conditions. Bacteria accumulated in the struvite cake during struvite filtration. Despite the use of sublethal temperatures, all bacteria types were subsequently inactivated to some degree during struvite drying, and the inactivation typically increased with increasing drying temperature for a given relative humidity. Heterotrophic bacteria inactivation mirrored the trend in total bacteria during struvite drying. A linear relationship was observed between inactivation and sample moisture content. However, bacteria survivor curves were typically nonlinear when struvite was dried at low relative humidity, indicating bacterial persistence. Weibull model survivor curve fits indicated that a shift in the mechanism of inactivation may occur with changing humidity. For increased efficiency of bacterial inactivation during the production of struvite, initial heating under moist conditions is recommended followed by desiccation.

■ INTRODUCTION more than 10% of the total global P demand.10 Urine can be · collected and stored separately from solids through urine- Struvite (MgNH4PO4 6H2O) has long been known in wastewater treatment as a scale deposit that causes maintenance diverting toilets, from which point it can be used directly as a ° 11 problems and reduces treatment efficiency.1 More recently, fertilizer following a minimum of 6 months storage at 20 C. centralized wastewater treatment plants are recognizing the Alternatively, urine can be processed into more manageable, potential of struvite as a phosphorus-rich fertilizer, simulta- market-attractive products (e.g., with less foul odor) like neously removing scale throughout the treatment works while struvite. The high pH and high P levels in stored urine require ff 2 only the addition of a magnesium source for spontaneous o setting some treatment costs from its sale. Recycling of P in 12 this way is important since modern agriculture depends on the precipitation of struvite. nutrient, but it is mined from finite global reserves.3 Struvite’s Struvite precipitation has received much attention due to its high P content, low solubility in water and convenience as a process simplicity, but the hygienic quality of the end-product fi solid, dense and odorless fertilizer contribute to its market generated in eld settings has not been thoroughly evaluated. potential as a slow-release fertilizer in agriculture.4,5 While Source-separated urine collected in Durban, for example, struvite production from wastewater or dry sanitation systems contains a diversity of pathogens and high concentrations of 13 has not yet proven economically self-sustaining,2,6 revenue can locally prescribed pharmaceuticals. When struvite is precipi- be generated alongside meeting waste discharge requirements tated from urine, pharmaceuticals largely remain in the effluent by producing increasingly valuable fertilizers, especially as the of struvite reactors, yielding low concentrations in the struvite market price of P continues to rise. Many researchers have focused on struvite production from Received: July 15, 2016 5,6 7 human urine, for example in Nepal, Vietnam, and Durban, Revised: October 26, 2016 South Africa.8,9 Human urine is the main source of P in Accepted: November 8, 2016 wastewater. The P in human urine, if collected, could offset Published: November 8, 2016

© 2016 American Chemical Society 13013 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article product.14,15 Viruses are retained in residual urine in the pump.16 For pilot-scale experiments, four struvite batches were struvite after filtration, and human virus surrogates ΦX174 were produced from reactors built and maintained by EWS.9 Urine shown to inactivate in struvite under mild temperature drying storage tanks were mixed with a peristaltic pump prior to being conditions due to reduction in the product moisture content.16 pumped into the reactor. For a single struvite batch, up to 320 Larger sized helminth ova accumulate in struvite during L of urine was dosed with industrial grade magnesium sulfate fi · ltration and require elevated temperatures and reduced heptahydrate (MgSO4 7H2O, Strathmore Mine, Malelane, moisture contents in the struvite to achieve inactivation.16 South Africa), filtered sequentially through cotton filter bags The fate of bacteria in urine during struvite recovery and (∼100 μm pore size), and air-dried following previously processing has not been previously described. described procedures.9 Here, we evaluate the fate of in situ bacteria in urine as well Controlled Struvite Drying. Struvite cakes were dried by as surrogates for human pathogens (Salmonella typhimurium evaporation under a combination of various controlled and Enterococcus spp.) during the production of struvite from temperature and relative humidity conditions. The temper- laboratory reactors and a pilot-scale reactor operated in atures were 5 °C, 20 °C, or 35 °C, and the low and high relative Durban. Test conditions (5−35 °C, 40 and 80% relative humidity levels were approximately 40% or 80%, respectively. humidity) were selected to reflect struvite production in the Relative humidity was maintained using 150 mm × 150 mm field without additional energy input to create harsher drying desiccators (Nalgene, Thermo Fisher Scientific, Waltham, MA) conditions and corresponded to conditions previously used to containing 50 mL LiCl or NaBr saturated salt solutions (88 g/ study virus and helminth inactivation.16 Results from isothermal mL and 95 g/mL, respectively) and a water-soaked tissue for drying conditions conducted in the laboratory were also low and high relative humidity conditions, respectively. The compared to drying struvite under fluctuating relative humidity relative humidity stabilized after about 6 h. The temperature and temperature in the field. These studies together were used and relative humidity were observed using an Irox Piccolo to develop recommendations for enhanced struvite production indoor thermometer/hygrometer (OS Technology AG/SA, procedures and to increase the safety of using urine-derived Gümligen/Bern, Switzerland) placed inside the desiccator for fertilizers. each experiment. Controlled drying experiments were conducted in duplicate, ■ MATERIALS AND METHODS such that two struvite cakes were dried in the same desiccator Inactivation of bacteria in struvite dried under a range of with the same temperature and humidity conditions. The ff θ controlled and uncontrolled (ambient) conditions was e ective moisture content of struvite cakes ( g) is reported as quantified for heterotrophic bacteria, the Gram-negative − Salmonella typhimurium, and Gram-positive Enterococcus spp. MMww dw θg(%) = via plate counts as well as for total bacteria via flow cytometry Mdw (FC). This range of analytical techniques was selected to provide information on different end-product quality targets where Mww and Mdw are the wet weight (ww) and dry weight and insight into inactivation processes. The experimental (dw) masses of struvite, respectively. The dw was measured as approach also included the production of struvite from urine the stable weight of struvite when dried at reference laboratory collected from different sources, and drying experiments were conditions (20 ± 3 °C and 30 ± 5% RH). conducted in the laboratory and the field. Ambient Struvite Drying. A total of nine struvite cakes were Urine Sources and Characterization. Source-separated dried outside at EWS facilities in Durban, South Africa. Four of urine was obtained from the men’s urine storage tank at the these struvite cakes were produced from the pilot-scale reactors Swiss Federal Institute of Aquatic Science and Technology with cloth filters and dried under cover to prevent exposure to (Eawag, Switzerland) to produce struvite at a laboratory-scale direct sunlight, four were produced at laboratory-scale with and evaluate the inactivation of spiked Enterococcus spp. and S. nylon filters and dried under cover, and one was produced at a typhimurium as well as in situ heterotrophic bacteria under laboratory-scale and dried under natural sunlight. The temper- controlled struvite drying conditions. Additionally, source- ature and relative humidity were monitored with a HOBO Pro separated urine was obtained from mixed-gender urine storage v2 external temperature/relative humidity data logger (Onset tanks maintained by eThekwini Water and Sanitation (EWS) Computer Corporation, Bourne, MA) during the drying of and from household storage tanks and a community ablution pilot-scale struvite cakes and with a TFD 128 temperature/ block in eThekwini Municipality (the municipality in the relative humidity data logger (Conrad Electronic, Berlin, Durban region) to evaluate variability in the inactivation of in Germany) for the laboratory-scale struvite cakes. situ heterotrophs in urine or struvite produced at a laboratory- Bacteria Enumeration by Plate Count. Urine and struvite scale. Struvite was also produced from pilot-scale reactors at samples were enumerated for heterotrophic bacteria, Salmonella EWS using urine obtained from the EWS mixed-gender urine typhimurium, and Enterococcus spp. Details regarding the source storage tanks; these bags of struvite were dried under ambient and enumeration of the organisms are described in the (uncontrolled) conditions. A subset of urine samples were Supporting Information. Concentrations are reported as colony characterized for ammonium, phosphate, conductivity, chemical forming units (cfu) per mL urine or cfu per g dw struvite, oxygen demand and pH as described in the Supporting unless otherwise stated. Information (Table S1). Bacteria Enumeration by Flow Cytometry. Flow cytometry Struvite Production. For laboratory-scale experiments, with appropriate cell staining procedures provides a culture- each struvite cake was produced in sterile reactors as previously independent method of evaluating the inactivation of all · described from 1 L of urine by adding 1.5 g MgCl2 6H2O bacteria, including viable and culturable bacteria, viable but (Acros Organics, Geel, Belgium), gently stirring for 10 min, and nonculturable bacteria and nonviable bacteria. An FC viability filtering through a nylon filter (pore diameter ≅ 18−240 μm) assay was conducted on a subset of struvite suspensions to cut to fit the filtration unit (45 mm diameter) using a manual provide insight into the mechanisms of bacterial inactivation

13014 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article

Figure 1. Accumulation of bacteria during struvite formation. The concentration expected in struvite (CFU/g dw) was calculated from the urine influent concentration (CFU/g) and the initial sample moisture content in the struvite sample. Error bars here are 95% confidence intervals. and to evaluate the robustness of the simple plating procedures inactivation. HPC and total bacteria were never observed below to reflect overall trends in bacterial inactivation. Total cell the detection limit. The slope of the linear regression was counts (total bacteria), total intact cell counts (live bacteria), considered significantly different from zero if p < 0.05 by and membrane-compromised cell counts (dead bacteria) were ANOVA. measured in urine and dissolved struvite samples using a fl CyFlow CL ow cytometer (Sysmex Partec GmbH, Görlitz, ■ RESULTS AND DISCUSSION Germany), applying a live/dead staining procedure (e.g., Figure S1−S3). The SYBR Green and propidium iodide (PI) staining Viable Bacteria Content in Stored Urine Is Highly procedure was adapted from Berney et al.,17 as described in the Variable. Heterotrophic plate count (HPC) bacteria were × 2 Supporting Information. detected in all urine samples tested, ranging from 4 10 to 3 × 8 ff Data Analysis. The Weibull model in the form presented by 10 CFU/mL in urine collected from di erent sources and at ff Mafart et al.18 was selected to model survivor curves and was di erent times. The range of in situ heterotrophic bacteria fl applied to log-transformed data using Microsoft Excel and the concentrations re ects the variability of contamination of the add-on tool Ginafit.19 This model allows for a dispersion of urine from fecal matter or other sources as well as variability in resistance toward inactivation described by the Weibull the storage times and temperatures of the urine before sampled. distribution.20 The concentration of surviving organisms (C, Flow cytometry results confirm the presence of a large number CFU/g struvite or counts/g struvite) is modeled as a function of live bacteria in stored urine (Figure S1). The approximate of time (t) by the equation: residence time of urine in the sampled tank was between 37 and 47 days.21 As expected, HPC detected only a small fraction ⎛ t ⎞p of the total bacteria present in the urine. In side-by-side log (CC )=− log ( ) ⎜⎟ 10 10 0 ⎝ δ ⎠ measurements, concentrations of heterotrophic bacteria in urine ranged from 4 × 102 to 2 × 104 CFU/ml (n = 8), while 7 where C is the initial concentration, δ is the time for first total intact cell counts (live bacteria) ranged from 1 × 10 to 7 0 8 decimal (90%) reduction, and p is a shape parameter. The × 10 counts/mL. 22 Weibull model has the advantage of describing nonlinear While Gram-negative bacteria inactivate rapidly in urine, survival curves that have a shoulder (p > 1) or tail (p <1) our data indicate that some in situ HPC bacteria are expected without substantial additional empirical complexity. Because to persist and remain detectable. Bacterial communities in the δ and p parameters in the Weibull model are source-separated urine from two locations in the United States autocorrelated, the model is sometimes applied with a fixed p were dominated by Clostridiales and Lactobacillales after 80 days 23 value, usually the average p from several survivor curves of storage. Spore forming Gram-positive bacteria such as evaluated at different temperatures. The special case of p =1 Clostridium perfringens are known to resist inactivation in stored simplifies to the classic log−linear inactivation equation, and δ urine.24 Therefore, source-separated urine should not be becomes D, the time for decimal reduction that is independent considered sterile, even after long-term storage. A reduction of elapsed experimental time. Weibull model parameters were in the live bacteria content during urine processing to fertilizer determined without a fixed p for each experimental condition is thus desirable. using the average bacterial concentrations in two side-by-side Bacteria Accumulate in Struvite during Filtration. An struvite cakes. In addition, an average p was calculated for accumulation of heterotrophic bacteria and total bacteria in struvite dried in field conditions (p = 0.38), and the model was struvite was observed for struvite produced from either the rerun with this fixed value on those curves. nylon or the cloth filters used for laboratory and pilot-scale Nondetect data were excluded from the fits, except for the production, respectively (Figure 1). An expected concentration first data point below the detection limit when available, which of bacteria in the struvite was calculated based on the residual was set to the limit of quantitation (see Supporting urine present in the struvite, determined from the effective Information) as a conservative estimate of the extent of moisture content of struvite samples taken immediately after

13015 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article

Figure 2. Inactivation of bacteria (Enterococcus spp., S. typhimurium, and heterotrophic bacteria) during the drying of struvite with controlled temperature and relative humidity (a). Linear regression when plotting the relative concentration of bacteria (log10 C/C0) in struvite against sample θ moisture content (log10 g), shown with regression standard error in gray (b). Heterotrophic bacteria inactivation was evaluated in repeat controlled drying experiments for struvite produced from different urine sources, with emphasis on the extreme conditions (low humidity/35 °C and high humidity/5 °C). Concentrations of bacteria are reported as CFU/g struvite (dry weight); the moisture content was calculated on a dry weight basis. Results were marked with “°” if at least one of the samples had no measurable residual moisture; results were marked with “*” if at least one of the concentrations was below the limit of detection. production, and the measured concentration of bacteria in the concentration following struvite production is required to source urine. The measured concentrations of bacteria in the achieve a net benefit in microbial quality as compared to that struvite were greater than these expected concentrations by up required for urine (on an equivalent mass basis). The to 2 orders of magnitude, suggesting additional mechanisms of inactivation of bacteria during struvite drying is subsequently bacterial retention besides the residual moisture. This was true evaluated, first in controlled conditions followed by field for HPC (ratios of measured/expected concentrations ranged settings. from 31 to 250, n = 16), total intact cells detected by FC (ratios Inactivation of in Situ Heterotrophic Bacteria during from 30 to 340, n = 4), and total cells (ratios from 20 to 350, n Controlled Drying of Struvite. The inactivation curves for = 4) in struvite produced using nylon filters. Struvite produced HPC bacteria during the drying of struvite under controlled using cloth filter bags at the pilot-scale also retained temperature and relative humidity are displayed in Figure 2a heterotrophic bacteria, with the ratios of measured to expected (lowest panels). In some struvite batches, up to approximately concentration ranging from 10 to 61 (n = 4). The nylon and 3-log inactivation was achieved in less than 100 h drying. cloth filters used for drying have similar pore sizes, so the However, the extent of inactivation ranged widely for different reduced accumulation of bacteria in the field setting relative to drying conditions and often did not reach this level, even after the laboratory may be due to the reactor configuration and field almost 200 h drying. Struvite was also produced from urine that struvite production protocol, in which elevated pressure is originated from different sources, either eThekwini (South manually applied to increase urine flow rates through the filter. Africa) or Eawag (Switzerland), to evaluate the variability of in The observed accumulation of bacteria in struvite from urine situ heterotrophic bacteria inactivation under repeat controlled indicates that at least two-log reduction in live bacteria drying experiments with emphasis on the extreme conditions

13016 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article

Table 1. Weibull Model Parameters for Inactivation of Heterotrophic Plate Count (HPC) Bacteria Sourced from Eawag, Switzerland or Durban, South Africa as well as Enterococcus spp., S. typhimurium, Total Intact Cells (TIC) and HPC Measured alongside TIC in Struvite Dried at Controlled Temperature and Relative Humidity

a 2 δ b c d drying condition bacteria RMSE R (h) p log10(C0) t4D (d) 35 °C/low RH HPC (Eawag) 0.91 0.76 8 ± 26 0.33 ± 0.30 6.6 ± 0.9 23 0.50e 0.94 2 ± 4 0.41 ± 0.19 7.4 ± 0.5 3 HPC (eThekwini) 0.11 0.98 10 ± 7 0.20 ± 0.04 6.5 ± 0.1 415 0.43 0.92 2 ± 4 0.24 ± 0.10 7.9 ± 0.4 24 0.23 0.97 2 ± 3 0.25 ± 0.06 7.7 ± 0.2 25 Enterococcus spp. 0.41 0.94 6 ± 5 0.50 ± 0.12 6.0 ± 0.4 4 S. typhimurium 0.43 0.96 4 ± 2 0.85 ± 0.21 6.3 ± 0.4 1 TIC 0.23 0.95 18 ± 6 0.84 ± 0.25 10.6 ± 0.2 4 HPC (alongside TIC) 0.27 0.94 33 ± 5 2.4 ± 0.9 6.1 ± 0.1 2

35 °C/high RH HPC (Eawag) 0.87e 0.88 13 ± 20 0.64 ± 0.41 7.9 ± 0.8 5 Enterococcus spp. 0.03 1.00 44 ± 1 1.11 ± 0.03 6.01 ± 0.02 6 S. typhimurium 0.19 0.99 14 ± 3 2.0 ± 0.8 4.8 ± 0.1 1

20 °C/low RH Enterococcus spp. 0.41 0.84 20 ± 28 0.35 ± 0.15 6.0 ± 0.4 47 S. typhimurium 0.72 0.89 4 ± 6 0.41 ± 0.16 6.0 ± 0.7 4 TIC 0.26 0.83 82 ± 55 0.46 ± 0.23 10.8 ± 0.2 68 HPC (alongside TIC) 0.25 0.80 119 ± 65 0.53 ± 0.30 6.4 ± 0.2 66

20 °C/high RH Enterococcus spp. 0.14 0.99 123 ± 10 1.5 ± 0.1 5.9 ± 0.1 13 S. typhimurium 0.38 0.97 32 ± 12 1.1 ± 0.3 5.8 ± 0.3 5

5−6 °C/low RH HPC (Eawag) 0.17e 0.98 15 ± 8 0.41 ± 0.09 7.5 ± 0.2 18 Enterococcus spp. 0.13 0.93 325 ± 59 0.63 ± 0.15 6.1 ± 0.1 123 S. typhimurium 0.35 0.85 114 ± 70 0.56 ± 0.21 6.0 ± 0.2 57

5−6 °C/High RH HPC (Eawag) 0.22 0.94 144 ± 31 1.1 ± 0.3 7.4 ± 0.2 22 HPC (eThekwini) 0.11 0.90 265 ± 33 2.3 ± 1.1 6.4 ± 0.1 20 0.29 0.95 23 ± 16 0.50 ± 0.15 7.9 ± 0.3 15 0.36 0.93 36 ± 26 0.62 ± 0.25 7.9 ± 0.3 14 Enterococcus spp. 0.08 0.99 275 ± 11 1.7 ± 0.1 5.92 ± 0.03 25 S. typhimurium 0.20 0.98 170 ± 21 1.3 ± 0.1 5.9 ± 0.1 21 TIC 0.07 0.54 784 ± 1096 1.2 ± 1.2 10.62 ± 0.04 101 HPC (alongside TIC) 0.07 0.85 217 ± 17 6.3 ± 3.7 6.34 ± 0.03 11 a bδ fi c d RMSE, Root mean sum of squared errors. , time for rst decimal reduction. p, Weibull model shape parameter. t4D, time for 4-decimal (99.99%) reduction projected from model fit. eFive observations used for model fit. A minimum of at least six observations is preferable for Weibull model parametrization. ° ° (low humidity/35 C and high humidity/5 C). Heterotrophic ionic strength and the concentration of NH3, which is a known bacteria from different sources exhibited variable susceptibility biocide. Decrey et al.16 performed a simulation of these to inactivation (Figure 2, Table 1 and Table S2). At a given parameters as a function of moisture reduction in struvite (at 35 ° relative humidity and within the same urine batch, inactivation C, Low RH). The NH3 concentration declines rapidly with was generally more efficient at higher temperatures. Conversely, moisture, and the pH approaches neutral. Therefore, after the effect of relative humidity on inactivation at a given about 20% moisture loss, these two parameters are not temperature was less clear. At the low temperature condition, expected to contribute to inactivation. The ionic strength increased relative humidity increased the time for 1-log does increase at low moisture (after about 80% moisture loss). reduction, but this effect was not observed during higher Such low moisture conditions were achieved rapidly in most temperature storage. Elsewhere it has been observed that struvite drying tests (Figure S4); thus ionic strength may heterotrophs are more sensitive to heating in wet soil than dry promote inactivation in our samples. soils.25 In all cases, HPC bacteria were not reduced below the Changes in conditions at the cellular level during desiccation, detection limit over the time period monitored. including increased ionic strength, changes in cellular compart- Decrey et al.16 found that the reduced moisture content of ment volumes, increased viscosity, the crowding of macro- struvite during drying was the primary cause of inactivation of molecules, damage to external layers, and changes in bacteriophage and helminth ova. Reduction in moisture content physiological processes, contribute to inactivation.27 Reduced is also known to inactivate bacteria, such as during the drying of stability of DNA under such altered conditions is also known as wastewater sludge.26 Inactivation may stem from changes in a major contributor to cell death during dehydration.28 In our porewater solution conditions and from cellular changes during data, a linear relationship was observed independently of the desiccation. In the porewater, parameters of interest are the pH, drying condition between heterotrophic bacteria inactivation

13017 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article C C ff θ θ Table 2. Linear Regression Results for Inactivation (Log10 C/C0 or / 0) versus E ective Moisture Content (Log10 g or g) θ a θ b log10 C/C0 vs Log10 g C/C0 vs g bacteria group drying condition R2 slope ± SEc intercept ± SE R2 slope ± SE intercept ± SE heterotrophic bacteria field drying in cloth bag filters 0.28 0.60 ± 0.14 −0.39 ± 0.11 0.11 0.82 ± 0.33 0.22 ± 0.30 heterotrophic bacteria field drying on nylon filters 0.68 0.71 ± 0.07 −0.30 ± 0.10 0.11 0.60 ± 0.23 0.33 ± 0.26 heterotrophic bacteria controlled drying on nylon filters 0.50 0.65 ± 0.05 −0.27 ± 0.08 0.34 0.48 ± 0.05 0.20 ± 0.05 Enterococcus spp. controlled drying on nylon filters 0.45 0.66 ± 0.08 −0.14 ± 0.08 0.77 0.73 ± 0.04 0.12 ± 0.04 S. typhimurium controlled drying on nylon filters 0.53 1.04 ± 0.11 −0.47 ± 0.11 0.74 0.65 ± 0.04 −0.003 ± 0.03 a θ b θ c Excludes data where g =0. Includes data where g =0. SE, standard error

Figure 3. Concentrations (log10 C) of in situ heterotrophic bacteria (CFU/g struvite) monitored side-by-side with total intact cells (counts/g struvite) by flow cytometry in struvite dried at different temperature and relative humidity conditions (a). Representative FL1 (green) vs FL3 (red) fluorescence emission dot plots from struvite drying experiments conducted at 35 °C and low relative humidity (∼40%) (1000-fold dilution of struvite sample) demonstrate the transition of cells from intact (right side gated region) to membrane compromised (left side gated region) with time (b). Each dot on the plot represents a detection event (256 channels per axis), and the color gradient represents the z-axis with a single event shown in blue and greater than 16 events shown as black. ff θ (log10 C/Co) and the e ective moisture content (log10 g, relative humidity (Figure 2a), thereby limiting the utility of this Figure 2b). The correlations of residual moisture content with proxy at low water contents. bacteria inactivation, however, were often weak (Table 2). Inactivation of Human Pathogen Surrogate Bacteria Therefore, the reduction in sample moisture content could be during Controlled Drying of Struvite. While HPC have used as a rudimentary proxy for estimating the log reduction of been used historically to provide an indication of the overall bacteria during struvite drying over a range of drying microbial status of an environment, HPC bacteria include temperatures and relative humidity. However, concentrations mostly nonpathogenic organisms. Evaluation of fecal indicator of in situ heterotrophic bacteria in struvite may stabilize during bacteria and Salmonella were conducted to assess the drying, as indicated by the tailing inactivation curves at low agreement of the findings using HPC with human health

13018 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article relevant bacteria. The inactivation of inoculated Enterococcus Nonlinear Inactivation Kinetics and Bacterial Persis- spp. and S. typhimurium was monitored during struvite drying tence during Drying at Low Relative Humidity. At low under controlled temperature and relative humidity for relative humidity, heterotrophic bacteria in urine from different comparison to results for heterotrophic bacteria (Figure 2a). sources as well as Enterococcus spp. and S. typhimurium tended In a study of thermal resistance to eight nonspore forming to be inactivated rapidly initially but stabilized with time. Such bacteria in mild heating conditions, Enterococcus faecalis and tailing of the inactivation curve was typically not observed in Salmonella typhimurium represented extreme responses (most the higher relative humidity drying condition in the time frame resistant and least resistant, respectively).29 In the present monitored, though inactivation was generally slower at higher study, both bacteria were partly inactivated during struvite relative humidity. The tailing feature observed at lower relative drying, and the inactivation typically increased with increasing humidity implies the need for a nonlinear inactivation model, drying temperature (from 5 to 35 °C) for a given relative such as the Weibull model, to predict the time to achieve humidity, with a more pronounced increase with temperature desired inactivation (e.g., the time for 4-log reduction). Weibull for S. typhimurium relative to Enterococcus spp. model parameters determined for all test organisms are listed in fi As observed for heterotrophic bacteria, the varied behaviors Table 1, and corresponding rst-order inactivation rate fi observed for Enterococcus spp. and S. typhimurium dried under constants (from the application a Weibull model with xed p different conditions do approximate into a linear relationship = 1) for each are listed in Table S2. fi when inactivation (log C/C ) is plotted against the effective For heterotrophic bacteria, the time for rst log reduction 10 o δ struvite moisture content (Figure 2b). Therefore, the moisture ( ) in struvite was 1 day or less when dried at low humidity and ° δ content of the struvite cake can be a useful parameter to 35 C, but ranged from 1 to 11 days when struvite was dried evaluate the extent of microbial inactivation. Gram positive at high humidity and low temperature. Conversely, extrap- Enterococcus spp. responded to drying at similar rates to olation of the Weibull model predicted a longer time on heterotrophic bacteria, while Gram-negative S. typhimurium was average for 4-log reduction at low humidity/high temperature inactivated more efficiently than both, resulting in a steeper compared to the high humidity/low temperature conditions. inactivation vs moisture content relationship (Table 2). This This is attributable to the occurrence of tailing (p < 1) at low humidity and shouldering (p > 1) at high humidity. Notably, suggests that results from HPC provide a good indicator of fi treatment processes efficiency for Enterococcus spp. and a applying a simple rst-order inactivation model would either conservative indicator for S. typhimurium. under- (low humidity) or overpredict (high relative humidity) Inactivation of the Total Bacterial Population. FC the required 4-log inactivation times, highlighting the need for a more complex inactivation model. Similar results were observed measurements of in situ total bacterial population were taken for both Enterococcus spp. and S. typhimurium in which the alongside HPC measurements for a series of controlled struvite average p was greater than one when struvite was dried under drying conditions (Figure 3) to evaluate the ability of the high relative humidity but less than one when dried at low cultivation-dependent method to reflect total bacteria behavior relative humidity. Consistent with the previous discussion, this during struvite drying and to explore the mechanism of indicates that it is beneficial to dry struvite at higher inactivation. The ease of use and comprehensive detection temperature but not reduced relative humidity. Further, the capabilities of FC render this method favorable for rapid and relatively consistent change in curve shape with relative extensive data collection. humidity may be indicative of a shift in the primary inactivation Similar to HPC bacteria, total cell counts and total intact cells mechanism under different drying conditions. from FC analysis were consistently detected above the Nonlinear microbial inactivation curves are commonly detection limit throughout the drying experiments. Total intact observed during mild heat treatment, though the causes of cells declined over time during drying (Figure 3), indicating this behavior, whether physical, chemical or biochemical, are loss of the cytoplasmic membrane integrity of detected cells, still debated.31 For example, the appearance of a shoulder, or a while total cell counts remained stable throughout the lag-time before inactivation, has been attributed to an experiments (Figure S5). A strong correlation between HPC accumulation of sublethal injury prior to inactivation, repair and total intact cells in the same samples was observed (Figure of cellular damage and aggregation of cells.32,33 Tailing of heat- S6) independently of drying condition, suggesting that HPC driven inactivation curves has been proposed as a consequence does serve as a useful proxy for following the progression of of the decrease in the probability of a collision between a water overall bacterial inactivation, as characterized by membrane molecule that carries sufficient thermal energy to be lethal and a integrity, during struvite drying. The strong correlation microbial particle as microorganisms are inactivated.34 In a observed may be a result of a relatively homogeneous starting study of bacterial inactivation in soils, evaporation of moisture 23 bacterial population in the stored urine. Storage of urine prior during heating in low relative humidity cooled the wetted to use for production of struvite is known to rapidly inactivate inoculum relative to external temperatures and reduced some bacteria, while those remaining must be tolerant of the inactivation.35 This could explain tailing observed in the high ammonia levels and elevated pH typical of stored urine present study at low relative humidity. An alternative − + − 30 (2.6 3.3 g/L N-NH4 and 8.5 9.4, respectively). explanation for tailing is the presence of a resistant Despite the benefits of using FC, our cultivation-independent subpopulation. Removing water from cells causes damage to assessment of viability just mirrored the inactivation trends as cellular components, leading to death in most organisms. For captured via HPC under a range of conditions. The Gram- example, DNA double strand breaks are high under desiccation positive bacteria Enterococcus spp. also followed a progression conditions in Escherichia coli.36,37 However, organisms that similar to heterotrophic bacteria during drying. Therefore, our experience frequent droughts, such as in soils, are known to results suggest that HPC continue to be a useful method for exhibit mechanisms for protection against desiccation or to assessing struvite end-product quality, especially in low- repair ensuing damage.27 Desiccation resistant microorganisms resource field settings. such as the pathogen Staphylococcus aureus tend to be Gram-

13019 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article

Figure 4. Replicate struvite cakes produced from cloth filters (a) or nylon filters (b) and dried at ambient conditions, as outlined in Table S3. θ θ Relative concentrations of heterotrophic bacteria in replicate struvite cakes (log C/C0, top) and relative moisture content ( g / g0, middle) were monitored with time. Error bars represent the range of results for duplicate samples taken at the same time from different locations in the struvite cake. Relative heterotrophic bacteria concentrations from individual samples are shown separately with their corresponding moisture content to evaluate the relationship between these variables (bottom). positive and small.27,28,38 However, Gram-negative Salmonella scavenge reactive oxygen species, during slow dehydration can has also been shown to survive in low moisture content influence bacterial survival.37,41 However, high relative humidity conditions26 due at least in part to the water-retaining capacity can also lead to more efficient inactivation of dehydration 39 of expressed lipopolysaccharides. This is consistent with tolerant bacteria, as shown for Deinococcus radiodurans cells.36,37 observed tailing of the S. typhimurium survivor curves at low With regards to struvite, initial retention of moisture during relative humidity in the present study. thermal treatment may aid in achieving inactivation objectives. Dynamic processes during struvite drying, such as rates of Because struvite cannot be dried at high temperatures heating and dehydration or changes in struvite porewater ° fl (approximately >55 C) without some reduction of fertilizer chemical composition with time, may also in uence inactivation quality due to loss of ammonia,42 low levels of moisture are kinetics. Struvite porewater ionic strength increases rapidly in likely present in the final product in any case. For long-term laboratory-produced struvite when the moisture content is 16 storage of sludge, maintaining a moisture content of 15% was reduced by more than approximately 80%. Drying struvite at ff recommended to encourage inactivation of bacteria while also higher relative humidity could reduce the drying rate and a ect 26 microbial inactivation. For example, survival of Salmonella may preventing regrowth of enteric bacteria. Lower moisture be greater when dehydration is gradual.39 Poirier et al.40 contents (<10%) protected bacteria from inactivation while 26 showed that decreasing the rate of change in water potential higher moisture content promoted regrowth. The precise increased the viability of several bacterial cells likely due to optimum moisture content to promote inactivation during associated reductions in water flow rates across cell long-term storage of struvite was not evaluated in the present membranes.40 Down- or upregulation of biological processes, study. Reduced moisture of struvite is important nevertheless such as metabolic rates, DNA repair capabilities, or systems to for viral and helminth inactivation.16

13020 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article

Persistence of Heterotrophic Bacteria in Struvite observed during similar isothermal controls, the moisture Dried under Field Conditions. Struvite produced in the content of struvite samples served as a rough indicator of field is exposed to dynamic, rather than constant drying bacteria inactivation in the fertilizer. conditions. It is known that bacterial inactivation during Recommendations for Enhanced Struvite Production. isothermal drying does not necessarily predict behavior under Struvite moisture content may be a more useful parameter than fluctuating temperature and relative humidity.43,44 elapsed time to evaluate bacterial inactivation during struvite To evaluate the effect of dynamic environmental conditions production because linear relationships were observed between (Figure S7−Figure S10) on the efficiency of inactivation of inactivation and moisture content irrespective of drying heterotrophic bacteria relative to controlled drying conditions, condition. The moisture content relationship for HPC was we air-dried struvite produced from both 1 L on nylon filters similar in struvite dried under isothermal conditions in the and 320 L batches captured in cloth bags (Table S3) in the laboratory and under dynamic environmental conditions in the field. The mass of struvite on nylon filters (1 to 4 g initial ww) field. However, greater variability in inactivation was observed was much lower, and the thickness of the cakes was more in struvite produced from pilot-scale struvite reactors under homogeneous (Figure S11), compared to struvite mass fluctuating temperature and relative humidity drying con- captured in cloth filter bags (600−1200 g initial ww). The ditions. Further, the relationship between inactivation and average temperature for the period in which struvite was dried moisture content did not reflect the change in inactivation outside on nylon filters was 21 ± 2 (SD) °C, ranging from 17 mechanism observed between high and low relative humidity to 34 °C. Over this time, the relative humidity averaged 72 ± drying conditions in the laboratory. Tailing of the survivor 11% and ranged from 44 to 91%. The average measured curves occurred at low relative humidity despite attainment of temperature for struvite dried in cloth filter bags was 16 ± 6 low moisture content, and low levels of heterotrophic bacteria (SD) °C and ranged from 5 to 43 °C. The plastic cover used to persisted in all drying conditions. The relationships between protect the cloth filters from rain may have increased the inactivation and moisture content also differed by bacterial temperature measured nearby the struvite bags relative to type. Consequently, the moisture content relationship may fail ambient daily temperatures, which reach a maximum of 35 °C at low moisture contents and under dynamic drying conditions. in Durban during the drying period.45 Average relative humidity Increased homogeneity of struvite cakes produced in the field was 64 ± 22% over the drying period for cloth filter bags and could reduce variability in observed inactivation. This could be ranged from 12 to 98%. achieved, for example, through the application of flat cotton Despite diurnal fluctuations in temperature and relative filters in an automated pilot-scale struvite reactor developed in humidity, the moisture content of struvite cakes decreased eThekwini.46 Such a reactor yields a thinner struvite cake rapidly (Figure 4), and the decline was within the range of the relative to the cloth filter bags and likely more consistent drying rates from controlled drying tests (Figure S4). As moisture across the retained struvite during treatment. expected from the differences in struvite mass captured by each Conversely, increased homogeneity of the struvite cake may filter, the moisture content declined more rapidly for struvite affect the accumulation of bacteria from urine into struvite via dried on nylon filters than for struvite in cloth bags, and modified straining of bacteria in the struvite cake during its remained low in both following the declines. However, HPC formation. concentrations declined slowly, and slopes of the log−linear While the utilization of nonlethal temperatures for struvite inactivation curves were frequently not significantly different drying provided a treatment benefit, drying at elevated from zero, especially for struvite dried in larger batches in the temperature was beneficial in terms of bacterial inactivation. cloth filter bags (Table S3). Similarly to controlled tests, It may be possible to treat struvite at elevated temperatures heterotrophic bacteria remained detectable throughout the without reducing the struvite quality. Mass loss of struvite experiments.Thetimeforfirst log reduction (δ)of heated to 55 °C may be minimal, for example, if the rate and heterotrophs in the field was generally greater than that time of heating is controlled.42,47 This increased temperature is determined in the laboratory. From the application of the likely to have significant treatment benefits. Heating to 55−60 Weibull model (fixed p = 0.38), δ ranged from 9 to 48 days in °C, for example, was recommended to inactivate eight struvite filtered and dried on nylon filters and from 26 to 201 nonspore forming bacterial pathogens in water.29 For thermal days for heterotrophs in struvite captured and dried in cloth treatment of sewage sludge, the U.S. EPA requires a minimum filter bags (Table S4). This is in contrast to δ values of 11 days temperature of 50 °C as time−temperature relationships at or less for HPC bacteria inactivation in struvite dried under any lower temperatures are uncertain; a minimum of 55 °C must be controlled condition in the laboratory (Table 1). maintained for three consecutive days for within-vessel Notwithstanding some differences in the inactivation rates composting of sewage sludge.48 Additionally, given the between the laboratory and the field, the relation between observation of sustained inactivation at higher relative humidity, inactivation and moisture content was rather consistent. For it may be beneficial to treat struvite with elevated moisture comparison to inactivation in isothermal conditions, a linear levels prior to desiccation. Low temperature, short duration regression was also performed to assess the relation between steaming (e.g., at 50 °C for 3 min) has been recommended as a θ inactivation (log10 C/C0) and moisture content (log10 g) in the nonchemical alternative for soil pasteurization that also requires field (Figure 4). While the coefficients of determination are less energy than a typical procedure of 30 min treatment at 70 low, as may be expected for environmental conditions, the °C.49 Elevated temperature treatment with moisture retention slopes of the log inactivation vs log moisture plots are can be achieved with little additional cost: temperatures of 45− consistent for HPC bacteria in struvite dried in the laboratory 60 °C are common in soil solarization in Mediterranean or field conditions and similar to that of both HPC bacteria and regions.35,49 Enterococcus spp. in struvite dried under isothermal conditions Because some bacteria are more susceptible to dehydration, (Table 2). Therefore, while average drying temperature or and desiccation is important for inactivation of viruses and relative humidity in field conditions do not reflect inactivation helminths in struvite, reducing the moisture content of the solid

13021 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023 Environmental Science & Technology Article following any wet treatment remains important. As observed in and Air Stripping in Vietnam. Clean: Soil, Air, Water 2011, 39, 1099− biosolids, regrowth of pathogens may occur when moisture 1104. content is high (or when solids are rewetted) and other bacteria (8) Grau, M. G. P.; Rhoton, S. L.; Brouckaert, C. J.; Buckley, C. A. that inhibit regrowth due to competition are absent.50 Proper Evaluation of an Automated Struvite Reactor to Recover Phosphorus storage of struvite following drying should be therefore from Source-Separated Urine Collected at Urine Diversion Toilets in − conducted to prevent later fluctuations in moisture content. Ethekwini. Water SA 2015, 41, 383 389. (9) Rhoton, S.; Grau, M.; Brouckaert, C. J.; Gounden, G.; Buckley, C. ■ ASSOCIATED CONTENT A. Field Operation of a Simple Struvite Reactor to Produce Phosphorus Fertiliser from Source-Separated Urine in eThekwini. In *S Supporting Information WISA Biennal Conference May 25−28, 2014; Mbombela, Mpumalanga, The Supporting Information is available free of charge on the South Africa, 2014; pp 1−5. ACS Publications website at DOI: 10.1021/acs.est.6b03555. (10) Mihelcic, J. R.; Fry, L. M.; Shaw, R. Global Potential of Contains method details, struvite moisture content Phosphorus Recovery from Human Urine and Feces. Chemosphere 2011, 84, 832−839. evolution, chemical parameters measured in urine, (11) World Health Organization. Guidelines for the Safe Use of model results, climactic data for drying experiments, Wastewater, Excreta and Greywater, Vol. 4: Excreta and Greywater Use in and experimental setup photos (PDF) Agriculture; WHO Press: Geneva, Switzerland, 2006; Vol. 4. (12) Larsen, T. A.; Udert, K. M.; Lienert, J. Source Separation and ■ AUTHOR INFORMATION Decentralization for Wastewater Management; IWA Publishing: London, UK, 2013. Corresponding Author (13) Bischel, H. N.; Özel Duygan, B. D.; Strande, L.; McArdell, C. S.; *Phone: +41 21 693 0891; fax: +41 21 693 47 40; e-mail: fl Udert, K. M.; Kohn, T. Pathogens and Pharmaceuticals in Source- tamar.kohn@ep .ch. Separated Urine in eThekwini, South Africa. Water Res. 2015, 85,57− ORCID 65. Tamar Kohn: 0000-0003-0395-6561 (14) Escher, B. I.; Pronk, W.; Suter, M. J. F.; Maurer, M. Monitoring the Removal Efficiency of Pharmaceuticals and Hormones in Different Author Contributions ∥ Treatment Processes of Source-Separated Urine with Bioassays. S.S. and M.S. contributed equally. Environ. Sci. Technol. 2006, 40, 5095−5101. Notes (15) Ronteltap, M.; Maurer, M.; Gujer, W. The Behaviour of The authors declare no competing financial interest. Pharmaceuticals and Heavy Metals during Struvite Precipitation in Urine. Water Res. 2007, 41, 1859−1868. ■ ACKNOWLEDGMENTS (16) Decrey, L.; Udert, K. M.; Tilley, E.; Pecson, B. M.; Kohn, T. Fate of the Pathogen Indicators Phage ΦX174 and Ascaris Suum Eggs This material is based upon work supported by the National during the Production of Struvite Fertilizer from Source-Separated Science Foundation under Grant No. 1159225. The study was Urine. Water Res. 2011, 45, 4960−4972. undertaken in the framework of the Valorisation of Urine (17) Berney, M.; Hammes, F.; Bosshard, F.; Weilenmann, H.-U.; Nutrients in Africa (VUNA) project funded by the Bill & Egli, T. Assessment and Interpretation of Bacterial Viability by Using Melinda Gates Foundation (www.vuna.ch,GrantNo. the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry. OPP1011603). Funding was also provided by the Swiss Appl. Environ. Microbiol. 2007, 73, 3283−90. National Science Foundation (grant no. 200021_146829/1). (18) Mafart, P.; Couvert, O.; Gaillard, S.; Leguerinel, I. On We thank Teddy Gounden and the EWS staff, Franziska Calculating Sterility in Thermal Preservation Methods: Application Bosshard, Bastian Etter, Max Grau, Sara Rhoton, Nicola Rodda, of the Weibull Frequency Distribution Model. Acta Hortic. 2001, 566, and Bruce Ndlovu for laboratory and field site access as well as 107−114. assistance with struvite production and sample analysis. (19) Geeraerd, A. H.; Valdramidis, V. P.; Van Impe, J. F. GInaFiT, a Freeware Tool to Assess Non-Log-Linear Microbial Survivor Curves. − ■ REFERENCES Int. J. Food Microbiol. 2005, 102,95 105. (20) Van Boekel, M. A. J. S. On the Use of the Weibull Model to (1) Doyle, J. D.; Parsons, S. A. Struvite Formation, Control and Describe Thermal Inactivation of Microbial Vegetative Cells. Int. J. − Recovery. Water Res. 2002, 36, 3925 3940. Food Microbiol. 2002, 74, 139−159. (2) Le Corre, K. S.; Valsami-Jones, E.; Hobbs, P.; Parsons, S. A. (21) Goosse, P. 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13023 DOI: 10.1021/acs.est.6b03555 Environ. Sci. Technol. 2016, 50, 13013−13023

PART IV

QUANTIFYING MICROBIAL RISKS OF FIELD-SCALE STRUVITE PRODUCTION FROM SOURCE-SEPARATED URINE USING FIRST PERSON VIDEOGRAPHY EXPOSURE ASSESSMENT

ABSTRACT

Quantifying microbial risks of field-scale struvite production from source-separated urine using first- person videography exposure assessment

1,2Bischel H.N., 3Caduff L., 1Schindelholz S., 1Kohn T., 3Julian T.J. 1School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland 2Department of Civil and Environmental Engineering, University of California, Davis 3Eawag: Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf, Switzerland

This project quantifies time-dependent occupational exposures to pathogens in urine during the collection of urine and its subsequent processing into struvite fertilizer from indirect pathogen exposure. The study was conducted in Durban, South Africa, where a network of over 80,000 urine-diverting toilets is in place and the opportunity to collect urine for nutrient recovery at scale is ripe. The microlevel activity time series (MLATS) method was selected for the exposure assessment. First person videography of urine collection and processing was coded into high-resolution data yielding the frequency, duration, and chronology of contacts between each study participant hand and each surface. Measured concentrations of E.coli in urine and on surfaces were used to calculate urine volume equivalents on surfaces. Rotavirus contamination on surfaces was calculated from urine volume equivalents and expected concentrations in urine. A model was constructed to simulate the concentrations of Rotavirus on hands through time. Monte carlo simulation was performed to incorporate model parameter uncertainty into the results.

Study participants contacted visibly wet, urine-contaminated surfaces more frequently during the production of struvite than during the collection of urine (e.g., 10 vs. 3 wet-surface contacts/hr). The urine volume equivalent on surfaces was determined to be up to 0.3 ml urine / 100 cm2. However, the modeled risk of pathogen infection for municipality staff was greater during urine collection than during struvite production. This counterintuitive result points to the need for personal safety equipment to be worn even when risks of infection may be perceived as lower (during urine collection as compared to struvite production).

A tracer study was conducted to validate hand contact with presumably urine-contaminated surfaces during struvite production, revealing that 0.04 – 50 ml of urine was contacted per batch of struvite produced. Nevertheless, the probability of Rotavirus infection associated with single dose events from presumed hand- to-mouth contacts was somewhat low during struvite production. For example, for 10K simulations of 61 intermittent hand-to-mouth contacts occurring throughout a day of struvite production, the median probability of infection [reported 95% confidence] was 1.5×10-4 [1.4×10-5 , 5.4×10-4]. Such exposure may be unacceptable if multiple hand-to-mouth contacts occur during a day of work. The model of struvite production was sensitive to the transfer coefficients used for wet surfaces, indicating further research needed regarding the transfer of pathogens from liquids to skin.

In the context of resource reuse, source-separated urine is often falsely considered “sterile” or nearly so. This study demonstrates that urine handling is a potential source of microbial health risk and should be considered as important during risk management planning. Urine treatment via storage can reduce pathogen concentrations in struvite reactor influent to reduce exposure during struvite production. Additionally, fertilizer production techniques that reduce human contact with urine during processing such as automated reactors should be favored. The study is also unique in its approach to accurately model the time-dependent concentration of pathogens on hands due to urine contamination and to quantify the consequent exposure potential using videography, fecal contamination surface swabs and a tracer study. The methods are broadly useful for assessments of indirect exposures to pathogens in liquids.

SUMMARY IMPLICATIONS

The recognition of waste as a resource has the potential to transform business as usual approaches for waste management, encouraging capture of nutrients rather than release into the environment and more financially sustainable sanitation systems. Independent businesses that valorize human urine as part of the sanitation value chain are now appearing around the globe and will look to established agencies like eThekwini Water and Sanitation in adopting their respective waste handling processes and treatment techniques. The thorough consideration of human and environmental health risk factors is thus imperative in the production and use of fertilizers from human urine collected in eThekwini.

Within the resource recovery field, a myth has propagated that source-separated urine is sterile. Our work demonstrated that fecal pathogens as well as viruses excreted in urine are consistently detected in urine collected from urine-diverting toilets. The findings signify that in addition to optimization of nutrient recovery processes, urine resource recovery processes should consider the human and environmental health risks and risk-reduction strategies of urine fertilizer production and reuse scenarios.

The health hazards identified are not insurmountable challenges. Technologies developed to recover nutrients from urine for the production of fertilizers can be optimized to ensure microbially safe endproducts. Our in- depth evaluations of biological nitrification and chemical precipitation of struvite from urine facilitated nuanced recommendations for enhanced treatment of pathogens based on mechanistic understandings of pathogen inactivation. Modeling of microbial risks allowed quantification of the potential impact of changes in urine handling processes and treatment technologies on reducing human health risks associated with fertilizer production from urine. Ultimately, these studies together will aid in successful implementation of urine nutrient recovery and fertilizer production at scale.

A final personal note: My work as a post-doctoral researcher at EPFL conducted over the past four years has been extremely important in formulating my approach to research that brings scientifically rigor to important applied environmental challenges. I will now be joining the faculty of the Department of Civil & Environmental Engineering at the University of California, Davis as an Assistant Professor. In working towards tenure, I intend to continue to develop research on the topic of waste resource recovery, with the ultimate goal of protecting human health while promoting environmental sustainability through innovative reuse. I plan to do this in both developing countries and highly developed regions. In particular, I plan to develop collaborations in California and across the United States to bring concepts acquired from my work in Switzerland and South Africa to the country to advance knowledge on this important topic. My postdoctoral work represents and important contribution to implementing change in the way we manage our waste to consider it as a safe resource.

HEATHER N. BISCHEL

Postdoctoral Information: Personal Information: Environmental Chemistry Laboratory Email Address: [email protected] EPFL-ENAC-IIE-LCE Station 2, CM1-119 Address: Davis CA 95616 1015 Lausanne, Switzerland Date of Birth: 14.09.1983 [email protected] Married; 1 child born 20.08.2015

EDUCATION Stanford University: Doctor of Philosophy, Environmental Engineering and Science 2011 Program, Department of Civil and Environmental Engineering Stanford University: Master of Science, Environmental Engineering and Science Program, 2007 Department of Civil and Environmental Engineering University of California, Berkeley: Bachelor of Science, Department of Civil and 2005 Environmental Engineering with Environmental Emphasis, Highest Honors APPOINTMENTS Assistant Professor, Department of Civil & Environmental Engineering Starts University of California, Davis April 2017 Scientific Collaborator, École Polytechnique Fédérale de Lausanne (EPFL) 11/12-02/17 • Quantifying microbial health risks during urine collection from dry toilets and fertilizer production in eThekwini, South Africa; supported by the Swiss Federal Institute of Aquatic Science and Technology (Eawag) with Timothy Julian and Tamar Kohn; • Supported students and researchers at the University of KwaZulu-Natal (UKZN, South Africa) in evaluating pathogen inactivation in field-scale urine nutrient recovery technologies as Visiting Researcher at UKZN in collaboration with Nicola Rodda; • Assessed human and ecological health risk determinants in source-separated urine intended for fertilizer production in eThekwini, South Africa in collaboration with the Valorisation of Urine Nutrients in Africa (VUNA) project and Tamar Kohn. Post-Doctoral Research Scientist, Engineering Research Center for Reinventing the 10/11-9/12 Nation’s Urban Water Infrastructure (ReNUWIt), Stanford, CA • Evaluated wastewater reuse for habitat enhancement and stream renewal in collaboration with Stanford University and the University of California, Berkeley; • Compiled and presented research progress to the US National Science Foundation for center reporting and site visits during Year 1 of ReNUWIt activities. Environmental Engineering Graduate Student Researcher, Stanford University, CA 01/06-09/11 • Assessed recycled water program implementation in California via survey instrument; • Evaluated perfluoroalkyl acid interactions with proteins using mass spectrometry; • Advised by Richard G. Luthy in the Environmental Engineering and Science Program; • Dissertation: Emerging Contaminants in Ecosystems: New challenges for water reuse implementation and mechanisms of perfluorochemical bioaccumulation. Summer Student Fellow, Woods Hole Oceanographic Institution, MA 06/04-08/04 • Assessed microbial degradation of petroleum hydrocarbons in coastal marsh sediments; • Advised by Christopher Reddy and Helen White in Dept. of Marine Chem. & Geochem. Research Intern, University of California, Berkeley 03/04-06/04 • Measured sediment mercury contamination in Tomales Bay; • Advised by Bryce E. Johnson in Dept. of Civil & Environmental Engineering.

Curriculum Vitae: February 2017, Page 1 Heather N. Bischel

TEACHING EXPERIENCE Course Assistant and Guest Lecturer, Environmental Chemistry Spring Undergraduate course in Environmental Sciences and Engineering, EPFL. Semesters, 2013-2016 Guest Lecturer, Environmental Earth Sciences 14/06/12; Undergraduate course in Earth and Planetary Science, University of California, Berkeley. 10/07/12 Water-team advisor, Sustainable Cities Undergraduate/Masters level course in Urban Planning, Stanford University. Supervised 03/12-06/12 activities of a service-learning team (3 students). Guest Lecturer, A.P. Environmental Science 15/12/11 High school course, Kipp Collegiate School, San Jose, CA. Subsequently recruited and secured a position for Talia Arbit (High School Teacher) in the US National Science Foundation Research Experiences for Teachers program at Stanford University. Course Instructor, Chemical Transformation of Environmental Organic Contaminants 03/11-06/11 Graduate-level course in Civil & Environmental Engineering (CEE), Stanford University. Developed new curriculum with two co-instructors; specifically planned and taught introductory and aquatic photochemistry units (13 students). Course Instructor, Design for a Sustainable World 03/09-06/09 Upper-level undergraduate/Masters level course in CEE, Stanford University. Designed new curriculum with co-instructor; initiated and developed partnerships with international nonprofit organizations for service-learning design projects (29 students). Teaching Assistant Mentor, Teaching Civil & Environmental Engineering 09/07-12/07 Graduate course in CEE, Stanford University. Mentored new teaching assistants; guided discussions; updated curriculum (11 students). Teaching Assistant, Movement & Fate of Organic Contaminants in Water 09/06-12/06 Graduate course in CEE, Stanford University. Led review sessions; lectured in absence of the course instructor; graded; held office hours. Environmental Sciences Teaching Program Mentor/Teacher, UC Berkeley 2003-2005 Mentored/taught minority high school students in summer environmental science program.

ADVISING Note: Advising is recorded below when I was the primary direct supervisor of the student or research assistant activities; the overseeing faculty mentor during the period of advising is listed in parentheses. Nicolas Bochaton, Masters student Internship, Summer Session at EPFL 2016 Yoan Pétremand & Baptiste Gex, Masters Design Project, EPFL 2016 Agnès Novotny and Marie Christelle Horisberger, Masters Design Project, EPFL 2015 Simon Schindelholz, Research Assistant / Civil Service (7 months) at EPFL (T. Kohn) 2014-2015 Ariane Schertenleib, Masters Thesis, Fall Semester at EPFL (T. Kohn) 2013-2014 Ariane Schertenleib, Masters Semester Project, Spring Semester at EPFL (T. Kohn) 2013 Sara Oppenheimer, Masters Semester Project, Spring Semester at EPFL (T. Kohn) 2013 Hugues Oke, Undergraduate (UN Omaha), ReNUWIt/NSF-REU at Stanford (R. Luthy) 2012 Haical Haddad, Masters student, Fall quarter at Stanford (R. Luthy) 2011 Sophie Egan, Research Assistant (1 year, part time), Bill Lane Center for the American West 2010 (J. Christensen) and CEE (R. Luthy) at Stanford Rachel Bush, Undergraduate, Winter quarter at Stanford (R. Luthy) 2009

Curriculum Vitae: February 2017, Page 2 Heather N. Bischel

Linda Tseng, Masters Student, Summer Quarter research at Stanford (R. Luthy) 2008 Advised undergraduates for the Northern California Scholarship Foundation (NCSF): Jacky 2007- Wu (UC Berkeley, 2007-2012); Karina Buggy (University of Oregon, 2011-2015); Amy Present Arend (University of Idaho, 2011-2015)

GRANT CONTRIBUTIONS ACTIVE PARTICIPATION Virus transfer at the skin/liquid interface and associated microbial risks: A case study of urine collection in Durban, South Africa Funding Level: CHF 331’922 (11/2014-03/2017) Source of Support: Eawag/EPFL Collaborative Grant Principle Investigators: Timothy R. Julian and Tamar Kohn Grant Objective: To quantify virus transfer at the liquid/skin interface under different environmental conditions to inform exposure risks; to apply these data to a case study of viral risk associated with urine collection and processing in South Africa. Principle Responsibilities: Field data collection and model development for a quantitative microbial risk assessment of urine collection and nutrient recovery in eThekwini, South Africa.

COMPLETE IRFP: Inactivation of pathogens and fate of pharmaceuticals in novel treatment processes for source-separated urine in Durban, South Africa Funding Level: $164,355 (11/2012-03/2016) Source of Support: US National Science Foundation / Office of International & Integrative Activities / International Research Fellowship Program (IRFP Award # 1159225) Principle Investigator: Heather N. Bischel International Hosts: Tamar Kohn (EPFL) and Chris Buckley (UKZN) Grant Objective: To assess the safety and optimize design parameters of urine recycling systems to inactivate pathogens and to remove trace organic chemicals of concern in South Africa.

An investigation of virus inactivation in stored urine and urine treatment processes Funding Level: CHF 185’800 (04/2013-03/2015) Source of support: Swiss National Science Foundation, Division II Principle Investigators: Tamar Kohn (PI); Kai Udert (co-PI) Grant Objective: To develop a better understanding of virus inactivation by ammonia in general and in stored urine in particular, and to determine virus fate during nutrient recovery processes. Principle Responsibilities: Experimental design and field protocol development; student supervision for pathogen inactivation studies.

Le recyclage des effluents pour produire des engrais à Durban/Recycling human waste for fertilizer production in Durban Funding Level: $10,000 (09/2012-08/2013) Source of Support: Rotary Foundation Global/District Grants Principle Investigator: Heather N. Bischel Grant Objective: To engage in human-centered design and to identify needs in sustainable sanitation through community engagement; to promote equitable access to sanitation through public awareness; to build collaborations and community relationships in Switzerland and South Africa.

Engineering Research Center for Re-inventing America’s Urban Water Infrastructure Funding Level: $18,560,000 (08/2011-07/2016, Estimated) Source of Support: US National Science Foundation / Division of Engineering Education and Centers (Award #: 1028968)

Curriculum Vitae: February 2017, Page 3 Heather N. Bischel

Principle Investigators: Richard G. Luthy (PI), David Sedlak (co-PI), Jorg Drewes (co-PI), Nirmal Khandan (co-PI) Grant Objective: Established a 10-year (pending renewal) National Science Foundation Engineering Research Center to advance new strategies for water/wastewater treatment and distribution that will eliminate the need for imported water, recover resources from wastewater, and generate rather than consume energy in the operation of urban water infrastructure while simultaneously enhancing urban aquatic ecosystems. Principle Responsibilities: As a Postdoctoral Researcher in the Natural Water Infrastructure Systems and Urban Water Systems sections during Year 1 (09/2011-08/2012), assessed opportunities and design challenges for water reuse for streamflow augmentation and ecosystem enhancement; formulated plan for the center’s student advisory board; and contributed to research project compilations and reporting to the NSF as well as posters during site visits.

Decision Making in Recycled-Water Project Implementation: Symmetry in Scientific Knowledge and Political Economy Funding Level: $199,587 (07/2008-06/2011) Source of Support: Stanford University Woods Institute for the Environment / Environmental Ventures Projects Principle Investigators: Richard G. Luthy and David Brady Grant Objective: To examine how scientific uncertainty, economic incentive and regulatory pressures influence decisions to implement water-recycling projects for agricultural irrigation and ecosystem restoration in Northern California. Principle Responsibilities: As a PhD student, assessed factors influencing the implementation of recycled water programs and managers in Northern California via an online survey. Conducted structured interviews with water recycle system managers and interpreted information from various databases.

Biodynamic Modeling of Perfluorochemical Bioaccumulation to Assess the Use of Recycled Wastewater for Urban Stream Flow Augmentation and Habitat Restoration Funding Level: $50,000 (2006-2008) Source of Support: UPS Foundation Principle Investigators: Richard G. Luthy, Martin Reinhard, David Epel Grant Objective: To model the bioaccumulation of perfluorinated compounds in aquatic organisms as may occur in streams augmented with recycled water. Principle Responsibilities: As a PhD student, conducted studies to assess the uptake of perfluorinated chemicals (PFCs) the freshwater mollusk, Anodanta californiensis and evaluated mechanisms of perfluorochemical bioaccumulation. Developed and tested a protein binding model for PFCs.

ADDITIONAL PARTICIPATION - INVOLVEMENT DURING POST-FUNDING PHASE (COMPLETE) Valorisation of Urine Nutrients in Africa (VUNA)- Activity 1.4: Hygienic and ecotoxicological aspects Principle Investigators: Kai Udert and Teddy Gounden (Co-PIs); Linda Strande (Activity Leader) Source of support: Bill and Melinda Gates Foundation ($69,750) Grant Objective: To establish, with nutrient recovery from urine, a sanitation system for the poor in the developing world that produces valuable and affordable local fertilizers, lowers costs for sanitation, and reduces pollution of water resources. The objective of Activity 1.4 is to produce end-products that are safe to use, provide adequate human and environmental health protection, and are of adequate quality to ensure optimal market value. Principle Responsibilities: Collaborator in Years 2-4 of VUNA project; organized meetings of hygiene and ecotoxicology group; contributed to project reporting to the Gates Foundation; presented at VUNA annual meetings; produced public outreach documents.

Perfluorinated Organic Compound Biotransformation, Fate, and Availability in the Environment Funding Level: $398,989 (07/2002-07/2006) Curriculum Vitae: February 2017, Page 4 Heather N. Bischel

Funding Agency / Program: US National Science Foundation / Division of Chemical, Bioengineering, Environmental, and Transport Systems (Award # 0201955) Principle Investigators: Richard G. Luthy (PI), Craig Criddle (Co-PI) Grant Objective: To examine the availability, biotransformation, and fate of perfluorinated compounds in aquatic sediments. Principle Responsibilities: As a Masters student, received training and assisted in studies assessing the biological accumulation of perfluorinated compounds from sediments (2006).

SERVICE, PUBLIC OUTREACH & WORK EXPERIENCE Contributor, VUNA public brochure: Pathogens in urine 2013-2015 Produced by the Valorisation of Urine Nutrients in Africa (VUNA) project and the Swiss Federal Institute of Aquatic Science and Technology (Eawag). Available at: vuna.ch/ Documentary Film Participant: Behind the Data 04/13 Produced by The Department of Water and Sanitation in Developing Countries (Sandec) at the Swiss Federal Institute of Aquatic Science and Technology (Eawag). Available at: https://www.youtube.com/watch?v=GPaDrRoflCs Presenter/Organizer, Rotary Foundation Scholar Presentation Series: Le recyclage des 2012-2013 effluents pour produire des engrais à Durban. Presented at Rotary Clubs in Lausanne (Switzerland) and Annecy (France); Short-form project introductions given at Rotary Clubs in Auburn and Palo Alto (California, USA) and North Durban (South Africa). Volunteer, Youth Education/Outreach in Science & Engineering (examples provided) Ongoing • Presented L’ingénerie des toilettes for “Journée des gymnasiens” at EPFL, introducing my research project to students at high school outreach day (08/05/13) • Developed and taught public course “Grey water, Green Energy...Blue Skies!” with co-instructor on green strategies at home, for Grades 7-12 at “SPLASH!”, a large teaching and learning event at Stanford University (18/10/08). • Developed and led demonstration at the San Francisco Bay Model (Sausalito, CA) for Expanding Your Horizons (EYH), an event to introduce girls to STEM fields. • Conducted water treatment demonstration with R. Luthy at Stanford University E- day, a large public event to introduce engineering to alumni and their families. Scholarship Advisor, Northern California Scholarship Foundations (NCSF) 2007-Present NCSF aids ambitious public high school graduates in securing a higher education. National Board of Directors member and Chair of Finance Committee, 12/09-06/11 Engineers for a Sustainable World (ESW-USA, 501-3C) Appointed as the student representative on the board. ESW mobilizes university students and professionals through education and collaborative engineering service projects to foster environmental, social and economic sustainability. National Student Advisory Board member (ESW-USA). Organized strategic planning 06/09-09/09 retreat at Stanford University for Advisory Board members and select student participants. Stanford Chapter Leader, Engineers for a Sustainable World (ESW-Stanford) 2006-2009 President (06/08-09/09), Vice President (06/07-09/08), Treasurer (06/06-09/07) Extreme Affordability Agricultural Design Project, Stanford and International 03/08-06/08 Development Enterprises (IDE)-Myanmar. Competitive entrance for course and need- finding field visit. Included Spring Break field work in Burma. Hurricane Katrina Service-Learning Project, Stanford, CA and New Orleans, LA 01/07-03/07 Competitive entrance for course participation.10-week course on relief & recovery efforts; included Spring break volunteer recovery work. Curriculum Committee, Dept. of Civil & Environmental Engineering, UC Berkeley 2004-2005

Curriculum Vitae: February 2017, Page 5 Heather N. Bischel

Provided student input on undergraduate curriculum and course loads for faculty committee. Flood Control Engineering Intern, Contra Costa County Public Works, CA 6/03-9/03 Conducted hydrology analysis; compiled rainfall and stream flow data. UC Berkeley Chapter Leader, American Society of Civil Engineers 2001-2005 Internal Vice President (03-04), Secretary (04-05), Membership Chair (02-03), Intern (01-02) Student Intern, Placer County Air Pollution Control District, Auburn, CA 07/01 Assisted in gas station inspections and data collection; shadowed air quality engineers.

INVITED PRESENTATIONS AND REPORT CONTRIBUTIONS Bischel, H.N., Towards Sustainable Sanitation: Mitigating microbial health risks in the production of urine- derived fertilizers. Department of Civil and Environmental Engineering, University of California, Davis, March 17, 2016 (Invited Seminar). Bischel, H.N., Water and Waste Reuse to Enhance Environmental and Human Health. Department of Civil and Environmental Engineering, University of California, Berkeley, February 18, 2015 (Invited Seminar). Bischel, H.N., Towards sustainable sanitation: Mitigating microbial health risks in the production of urine- derived fertilizers in South Africa. Environmental Engineering Seminar Series. Ecole Polytechnique Fédérale de Lausanne. November 11, 2014. (Invited Presentation) Bischel, H.N., K. Wigginton, Treatment and Reuse: Engineering Applications. Environmental Sciences: Water, Gordon Research Seminar. Holderness, NH. June 21-22, 2014. (Invited Discussion Leader) Bischel, H.N., with input from K. Udert, L. Strande, E. Tilley. Valorisation of Urine Nutrients in Africa (VUNA). High Level Forum for Water and Sanitation for all in Africa. Abidjan, Ivory Coast. November 21, 2013. (Invited Presentation) This forum was hosted by Water and Sanitation in Africa (WSA) for government ministers and industry representatives. I presented on opportunities for resource recovery in sanitation and business models. I was invited by Patrick Apoya as a resource person for the Africa Sanitation Think Tank, an initiative of WSA, and met with the think tank staff in discussion of their development of three WASH initiatives. Bischel, H.N., Valorisation of Urine Nutrients in Africa (VUNA). Presented as representative of VUNA, with input from K. Udert, T. Gounden, B. Etter, L. Strande, C. Buckley, and E. Tilley. The Good News and Bad News of WASH. Workshop hosted by Clarissa Brocklehurst and Rick Johnston at the UNC Water and Health Conference. UNC-Chapel Hill, NC. October 18, 2013. (Invited Panelist) Bischel, H.N, Ecological water and wastewater management and mechanisms of perfluochemical bioaccumulation. University of Nevada, Reno, February 28, 2013 (Invited Seminar) US Environmental Protection Agency, 2012 Guidelines for Water Reuse. (Report contributor) Available at: http://water.epa.gov/infrastructure/sustain/availability_wp.cfm Bischel, H.N., Water Panel Speaker/Participant, Department of International Studies, University of San Francisco. Multi-disciplinary panel for public (30-50 participants), February 2012 (Invited Presentation) Bischel, H.N., G. Simon, T. Frisby, and R.G. Luthy, A survey of northern California water reuse managers: Learning from the past to inform the future. Bay Area Clean Water Agencies Recycled Water Committee, EBMUD Headquarters, Oakland, CA, December 1, 2010 (Invited Presentation). Bischel, H. Course and Project Model for Student-Initiated Engineering Service Learning. 20/20 Vision: Focusing on Innovation in Sustainable Design, Engineers for a Sustainable World National Conference. Purdue University, West Lafayette, IN. October 9, 2010. (Invited Presentation) Additional invited presentations or posters: Celebrating Sustainability at Stanford University (Panelist, 2012); Energy and Environment Affiliates Program Conference at Stanford University (Poster, 2011); Leading Matters: Bay Area (Stanford University Alumni event, Poster, 2010); United States Geological Survey: Water Resources Discipline Seminar Series (Presentation, 2009); Curriculum Vitae: February 2017, Page 6 Heather N. Bischel

Stanford University Environmental Engineering & Science Program Seminar Series (Presentation, 2009); American Water Works Association CA/Nevada Section Conf. (Luthy Invited, Bischel Presented, 2009); Dialogue on Water in the West at Stanford University (Rapporteur, 2008); Mass Spectrometry Users Meeting / Workshop at Stanford University (Presentation, 2008); Woods Hole Oceanographic Institution Seminar Series (Presentation, 2004).

CONFERENCE PRESENTATIONS (FIRST AUTHOR ONLY) Bischel, H.N., L. Caduff, S. Schindelholz, T. Kohn, T. Julian, A quantitative comparison of microbial health risks during urine collection and struvite production from urine using the microlevel activity time series (MLATS) method. IWA Water Microbiology Conference. UNC-Chapel Hill, May 2017. (Presentation) Bischel, H.N., S. Schindelholz, A. Schertenleib, M. Schoger, L. Decrey, A.K. Pitol, K.M. Udert, T. Julian, T. Kohn. Mechanisms and modeling of pathogen fate in pilot scale nutrient recovery reactors. ACS, San Francisco, April 2017. (Presentation) Bischel, H.N., A.K. Pitol, T. Kohn, T. Julian, Quantifying microbial risks of field-scale struvite production from source-separated urine using first-person videography exposure assessment. UNC Water and Health Conference. UNC-Chapel Hill, NC. October 15, 2016. (Poster, presented by T. Julian) Bischel, H.N., T. Kohn, et al, Towards Sustainable Sanitation: Mitigating microbial health risks in the production of urine-derived fertilizers. NextProf Future Faculty Workshop. University of Michigan, September 28, 2016. (Poster) Bischel, H.N., S. Schindelholz, M. Schoger, T. Kohn, Hygiene considerations during the collection, storage and processing of source-separated urine into a marketable fertilizer. IWA Conference on Small Water and Wastewater Systems & Resources-Oriented Sanitation. Athens, Greece. September 15, 2016. (Presentation) Bischel, H.N., A. Schertenleib, A. Fumasoli, K. Udert, T. Kohn, Hygiene assessment of decentralized sanitation nutrient recovery: Pathogen occurrence and inactivation during urine nitrification. UNC Water and Health Conference. UNC-Chapel Hill, NC. October 15, 2014. (Presentation) Bischel, H.N., A. Schertenleib, T. Kohn, Hygiene assessment of decentralized sanitation nutrient recovery: Pathogen occurrence in source-separated urine and inactivation during urine nitrification. Environmental Sciences: Water, Gordon Research Conference and Seminar. Holderness, NH. June 21-27, 2014. (Poster) Bischel, H.N., T. Kohn, Assessment of diarrheal pathogens in source-separated urine and urine recycling systems in Durban, South Africa. UNC Water and Health Conference. UNC-Chapel Hill, NC. October 15, 2013. (Poster) Bischel, H.N., L. Decrey, T. Kohn, Presence and inactivation of bacteria and viruses in urine storage and recycling systems in Durban, South Africa. International Water Association: WaterMicro Conference. Florianopolis, Brazil. September 16, 2013. (Presentation) Bischel, H.N., J.E. Lawrence, B.J. Halaburka, M.H. Plumlee, A.S. Bawazir, J.P. King, J.E. McCray, V.H. Resh, R.G. Luthy, Renewing urban streams with recycled water for streamflow augmentation: hydrologic, water quality, and ecosystem services management. Environmental Sciences: Water, Gordon Conference. Holderness, NH. June 25, 2012. (Poster) Bischel, H.N., L.A. Macmanus-Spencer, R.G. Luthy. Interactions of medium and short-chain perfluoroalkyl acids with serum albumin. Society of Environmental Toxicology and Chemistry. November 10, 2010 North America Conference. Portland, OR. (Presentation, Platform Presentation award received) Bischel, H., T. Frisby, G. Simon, R.G. Luthy. Drivers and challenges for nonpotable water reuse implementation in northern California and the emerging role of constituents of concern. Environmental Sciences: Water, Gordon Conference. Holderness, NH. June 22, 2010. (Poster) Bischel, H., L.A. Macmanus-Spencer, R.G. Luthy. Noncovalent interactions of long-chain perfluoroalkyl acids with serum albumin. Environmental Protection Agency PFAA Days III (Perfluoroalkyl Acid Research Workshop). Research Triangle Park, North Carolina. June 10, 2010. (Poster) Curriculum Vitae: February 2017, Page 7 Heather N. Bischel

Additional presentations or posters presented as lead author at: Woods Institute for the Environment Chi Conference (Interdisciplinary Conference, Poster, 2010); Environmental Ventures Projects Symposium (Poster, 2009); Gordon Conference on Environmental Sciences: Water (Poster, 2008); Society of Environmental Toxicology & Chemistry National Conf. (Presentation, 2008); Society of Environmental Toxicology & Chemistry National Conf. (Poster, 2007 with travel award); American Society of Mass Spectrometry workshop (Poster/Presentation, 2008); American Association for the Advancement of Science (AAAS) National Conference (Presentation, Honorable Mention awarded, 2003).

LANGUAGE PROFICIENCY English: Native French: Upper Intermediate (CEFR Level B2)

ADDITIONAL INFORMATION • Research article and report peer reviews conducted for: Environmental Science & Technology Letters, Global Public Health, Environmental Health Perspectives, California Ocean Science Trust, Environmental Toxicology & Chemistry, African Journal of Environmental Science & Technology, International Journal of Environmental Health Research; Water Science and Technology; Environmental Science: Water Research & Technology • Member of UC Berkeley and Stanford Club Tennis teams (2003-2007); Recreational sports instructor: Teaching Assistant for Stanford Windsurfing Club (2007) and Recreational Swim Team Coach and Tennis Instructor in Auburn, CA (2000-2002) • Analytical experience: Extraction of persistent organic pollutants from environmental matrices; liquid chromatography; mass spectrometry (ESI-MS/MS, nanoESI-Q-TOF), microbiology/molecular biology techniques (membrane filtration, spread plate, dry plate, double-layer agar methods; DNA/RNA extraction; real-time/reverse transcription/multiplex end-point PCR).

AWARDS & HONORS Selected for NextProf Engineering Workshop for Future Professors (University of Michigan) 2016 National Science Foundation International Research Fellowship 2012-2014 Rotary Foundation Palo Alto University District Scholar 2012-2013 Rising Environmental Leader, Stanford Woods Institute for the Environment: 2012 Selected to attend a 10-day environmental policy workshop in Washington, D.C. Society of Environmental Toxicology & Chemistry, Best Platform Presentation (3rd Place) 2010 UNESCO/Daimler Mondialogo Award (Silver Award; 10,000 Euros) for the Tsunami 2009 Evacuation Infrastructure Project Team of the Stanford Chapter of Engineers for a Sustainable World and the University of Andalas (Padang, Indonesia), received while serving as Stanford Chapter President and project contributor Dean’s Outstanding Achievement Award for the Stanford Chapter of Engineers for a 2009 Sustainable World while serving as Chapter President National Science Foundation Graduate Fellowship 2008-2010 (Estimated $80K for two years tuition and salary), Awarded 2005 National Defense Science and Engineering Graduate Fellowship 2005-2008 (Estimated $180K for three years tuition and salary) Honorary Stanford Graduate Fellowship (Note: declined monetary award) 2005-2008 University of California, Berkeley, Bechtel Achievement Award: highest honor awarded to 2005 one graduating senior in the College of Engineering for outstanding scholastic achievement and service to the College, campus and community. University of California, Berkeley, Civil and Environmental Engineering Department 2005 Citation: Student with the highest Grade Point Average (GPA) of graduating seniors in the department California Certified Engineer in Training (EIT) 2004 University of California, Berkeley, University and Community Partners Environmental 2004 Curriculum Vitae: February 2017, Page 8 Heather N. Bischel

Stewardship Award received as part of the Environmental Sciences Teaching Program AAAS National Conference, Platform Presentation Education Division Honorable Mention 2003 Tau Beta Pi Engineering Honor Society: Inducted 2003 2003 Chi Epsilon Engineering Honor Society: Inducted 2003 2003 County Engineers Association of California Student Award ($2000) 2003 Barry M. Goldwater Scholarship: the most prestigious national awards for undergraduate 2003-2004 students in the fields of science, mathematics or engineering Northern California Scholarship Foundation: Selects awardees from the top two candidates 2001-2005 from each public high school in Northern California. Additionally received honors from NCSF for the highest GPA of all first and second year awardees. California Alumni Association Leadership Scholarship: J. Michael Walford Scholar 2001-2005

Curriculum Vitae: February 2017, Page 9

LIST OF PUBLICATIONS

15. Bischel, H.N., L. Caduff, S. Schindelholtz, T. Kohn, T. Julian, Quantifying microbial risks of field-scale struvite production from source-separated urine using first-person videography exposure assessment. Manuscript in Preparation. 14. Bischel, H.N., S. Schindelholtz, M. Schoger, L. Decrey, C. Buckley, K. Udert, T. Kohn, Bacteria inactivation during the drying of struvite fertilizers produced from stored urine. Environ. Sci. & Tech. 50.23 (2016): 13013-13023. 13. Bischel, H.N., , B. D. Özel Duygan, L. Strande, C. McArdell, K. M. Udert, T. Kohn. Pathogens and pharmaceuticals in source-separated urine in eThekwini, South Africa. Water Research, 2015, 85, 57-65. http://www.sciencedirect.com/science/article/pii/S0043135415301731 12. Bischel, H.N., A. Schertenleib, A. Fumasoli, K. Udert, T. Kohn, Inactivation kinetics and mechanisms of bacterial and viral pathogen surrogates during urine nitrification. Environmental Science: Water Research & Technology. 2015, 1, 65-76. http://pubs.rsc.org/en/content/articlelanding/2015/ew/c4ew00065j/unauth 11. Nelson, R., H.N. Bischel, B.Thompson, R.G. Luthy. Issues of governance, policy and law in managing urban-rural and groundwater-surface water connections. In: Grafton, R.Q., K.A. Daniell, C. Nauges, J.D. Rinaudo, M.B. Ward, N.W.W. Chan, (eds.) Understanding and managing urban water in transition (Global Issues in Water Policy). Springer. 2015. (Peer-Reviewed Book Chapter) 10. Lawrence, J.E., C.P.W. Pavia, S. Kaing, H.N. Bischel, R.G. Luthy, V.H. Resh, Recycled water for augmenting urban streams in mediterranean-climate regions: a potential approach for riparian ecosystem enhancement. Hydrological Sciences Journal. 2014, 59(3-4), 488-501. http://www.tandfonline.com/doi/full/10.1080/02626667.2013.818221#.Um_YHxA8qIk 9. Halaburka, B.J., J.E. Lawrence, H.N. Bischel, J. Hsiao, M.H. Plumlee, V.H. Resh, R.G. Luthy, Economic and ecological costs and benefits of streamflow augmentation using recycled water in a coastal environment. Environmental Science & Technology. 2013, 47(19), 10735-10743. http://pubs.acs.org/doi/abs/10.1021/es305011z 8. Bischel, H.N., J.E. Lawrence, B.J. Halaburka, M.H. Plumlee, A.S. Bawazir, J.P. King, J.E. McCray, V.H. Resh, R.G. Luthy, Renewing urban streams with recycled water for streamflow augmentation: hydrologic, water quality, and ecosystem services management. Environmental Engineering Science. 2013, 30(8), 455- 479. http://online.liebertpub.com/doi/full/10.1089/ees.2012.0201 7. Bischel, H.N., G. L. Simon, T. M. Frisby, R.G. Luthy, Management experiences and trends for water reuse implementation in Northern California. Environmental Science & Technology. 2012, 46(1), 180-188. http://pubs.acs.org/doi/abs/10.1021/es202725e 6. Bischel, H.N., L.A. MacManus-Spencer, C. Zhang, R.G. Luthy, Strong associations of short-chain perfluoroalkyl acids with serum albumin and investigation of binding mechanisms. Environmental Toxicology & Chemistry. 2011, 30(11), 2423-2430. http://onlinelibrary.wiley.com/doi/10.1002/etc.647/full 5. Bischel, H.N. and E.R. Sundstrom, 5-Year evaluation of a course model for student-initiated engineering service learning. International Journal of Service Learning in Engineering. 2011, 6(1), 1-13. http://library.queensu.ca/ojs/index.php/ijsle/article/view/3211 4. Boehm, A.B. and H.N. Bischel. Oceans and Human Health. In: Nriagu J.O. (ed.) Encyclopedia of Environmental Health. Burlington: Elsevier. 2011, Volume 4, 223-230. (Not Peer-Reviewed) 3. Frysinger, G. S., G.J. Hall, A.L. Pourmonir, H.N. Bischel, E.E. Peacock, R.N. Nelson, C.M. Reddy. Tracking and Modeling the Degradation of a 30 Year Old Fuel Oil Spill with Comprehensive Two- Dimensional Gas Chromatography. In: International Oil Spill Conference Proceedings (IOSC). American Petroleum Institute. 2011(1), 428. http://www.ioscproceedings.org/doi/abs/10.7901/2169-3358-2011-1-428 2. Bischel, H.N., L.A. MacManus-Spencer, R.G. Luthy, Noncovalent interactions of long-chain perfluoroalkyl acids with serum albumin. Environmental Science & Technology. 2010, 44(13), 5263-5269. http://pubs.acs.org/doi/abs/10.1021/es101334s

1. MacManus-Spencer, L.A, M. Tse, P. C. Hebert, H.N. Bischel, R.G. Luthy. Binding of perfluorocarboxylates to serum albumin: A comparison of analytical methods. Analytical Chemistry. 2010, 82(3), 974-981. http://pubs.acs.org/doi/abs/10.1021/ac902238u

SELECTED ADDITIONAL PUBLICATIONS

My objectives as a researcher, educator and scientist are to identify and address human and environmental health challenges in water management and resource recovery systems. To this end, my research has evaluated opportunities and water quality challenges for reusing wastewater to renew urban streams, mechanisms influencing the biological uptake of persistent anthropogenic chemicals in aquatic environments, and strategies to mitigate microbial health risks in the reuse of nutrients from decentralized sanitation systems. Through high- impact research, engagement in multi-disciplinary collaborations, and as an effective educator, I plan to continue to promote the development of innovative and sustainable water and sanitation systems as critical components of healthy urban environments. The three publications selected below, presented in chronological order, represent a range of my interests, experiences and impact prior to joining the excellent community at EPFL as a postdoctoral researcher.

Title: Noncovalent interactions of long-chain perfluoroalkyl acids with serum albumin. Description and Impact: Perfluoroalkyl substances (PFASs) are a class of extremely persistent compounds commonly used to render products such as clothing, cookware, and food-contact paper non-stick, waterproof, and/or stain resistant. Because of their widespread use, PFASs have recently been detected in the blood of nearly all Americans tested. This work demonstrated strong associations of PFASs with serum albumin, a protein abundant in human blood and other body compartments, at low PFAS:protein mole ratios. The results supported the hypothesis that proteins play an important role in the bioaccumulation of PFASs and were subsequently used by others to model explain the unexpectedly strong bioaccumulation of PFASs. My Role: As a PhD student, I designed experiments, performed laboratory analysis and completed the manuscript with contributions from co-authors. As shown in my List of Publications, I led and contributed to two additional studies on this subject. Working on this topic, I also became interested in understanding the challenges that water reuse system managers face as new persistent and toxic chemicals like PFAS are detected in water systems.

Title: Management experiences and trends for water reuse implementation in Northern California. Description and Impact: In this study, semi-quantitative and qualitative methods from social science were applied to collect and evaluate experiences of water and wastewater general managers in rural and urban regions of Northern California. The data were used to develop an understanding of the factors influencing the implementation of recycled water projects throughout Northern California. The survey also revealed an often overlooked opportunity: that of recycled water to directly benefit ecosystems. This work contributed significantly to the formation of the NSF Engineering Research Center for Re-inventing the Nation’s Urban Water Infrastructure (ReNUWIt), directed by Dr. Richard Luthy. My Role: As a PhD student, I conducted semi-structured interviews and case-studies of recycled water projects in order to design a survey instrument, with feedback from collaborators. I implemented the online survey instrument for data collection, performed data analysis, and was the lead author of the manuscript.

Title: Renewing Urban Streams with Recycled Water for Streamflow Augmentation: Hydrologic, Water Quality, and Ecosystem Services Management. Description and Impact: Recycled water is a reliable water source that can be used both directly and indirectly to renew degraded urban stream ecosystems. In this review, aspects of hydrology, water quality, and ecosystem services in relation to water reuse for urban stream renewal are evaluated to identify research needs and design considerations for new systems. Use of recycled water for streamflow augmentation in urban areas remains largely unexplored scientifically, despite its potential widespread applications among water and wastewater utilities. My Role: As a Post-doctoral researcher, I led this critical review, working with researchers at four universities and in industry. Following this review, I initiated a field study to assess the public benefits of using reclaimed wastewater for streamflow augmentation by coupling tools from environmental economics, water quality, and stream ecology. This subsequent study led to the publication entitled, Economic and ecological costs and benefits of streamflow augmentation using recycled water in a coastal environment, as shown in my List of Publications.

Environ. Sci. Technol. 2010, 44, 5263–5269

long (3.1-4.4 years) in human blood (8). Species-specific Noncovalent Interactions of differences in PFAA distribution patterns and retention may Long-Chain Perfluoroalkyl Acids with be influenced by active uptake by organic anion transporters (9). Studies assessing PFAA-protein interactions (10-16) may Serum Albumin shed light on the tissue distribution patterns, bioaccumu- lation, and in vivo bioavailability of these chemicals. Protein Binding. The binding of PFAAs to proteins was HEATHER N. BISCHEL,† first reported in the 1950s, when PFAAs were investigated for LAURA A. MACMANUS-SPENCER,‡ AND their ability to aid in protein precipitation (17). In the 1960s, RICHARD G. LUTHY*,† organofluorine compounds were first detected in human Department of Civil and Environmental Engineering, Stanford blood serum (18). Serum albumin, the most abundant protein University, Stanford, California 94305, and Department of in blood plasma (35-50 g/L) (19), binds a variety of Chemistry, Union College, Schenectady, New York 12308 endogenous and exogenous ligands including fatty acids, amino acids, metals, and pharmaceuticals (20) and was Received April 23, 2010. Revised manuscript received May reported as the major binding protein for PFOA in blood 26, 2010. Accepted May 27, 2010. (11). PFAA-protein interactions result from the unique surfactant nature of PFAAs. The highly hydrophobic per- fluorocarbon tail paired with a strongly polar carboxylate or sulfonate headgroup resembles the structure of fatty acids Preferential distribution of long-chain perfluoroalkyl acids and facilitates both hydrophobic and ionic interactions with (PFAAs) in the liver, kidney, and blood of organisms highlights proteins. In fact, PFOA binds to liver- and kidney-fatty acid the importance of PFAA-protein interactions in PFAA tissue binding proteins (13), and PFAAs may interfere with the distribution patterns. A serum protein association constant may normal binding of fatty acids or other endogenous ligands be a useful parameter to characterize the bioaccumulative to liver-fatty acid binding protein (14). However, the rigidity potential and in vivo bioavailability of PFAAs. In this work, of the perfluorocarbon tail differs from the relatively more association constants (Ka) and binding stoichiometries for PFAA- fluid hydrocarbon tail (21), limiting extrapolation of fatty albumin complexes are quantified over a wide range of PFAA: acid-albumin binding results to their fluorinated counterparts. albumin mole ratios. Primary association constants for Studies of PFAA-albumin interactions using spectroscopic - 19 perfluorooctanoate (PFOA) or perfluorononanoate (PFNA) with methods (15, 22 24), electrophoresis (22), F NMR (11, 15), dialysis (17, 25), and surface tension (22, 23) report a wide the model protein bovine serum albumin (BSA) determined 2- 6 -1 6 -1 range of association constants (10 10 M ) with most values via equilibrium dialysis are on the order of 10 M with one - suggesting relatively weak binding (<104 M 1). However, to three primary binding sites. PFNA was greater than 99.9% PFAAs are highly bound to proteins in rat, monkey, and bound to BSA or human serum albumin (HSA) at a physiological human plasma (16), suggesting stronger, specific binding. -3 PFAA:albumin mole ratio (<10 ), corresponding to a high protein- Many studies have used relatively high PFAA:albumin mole water distribution coefficient (log KPW > 4). Nanoelectrospray ratios; results from such studies are difficult to extrapolate ionization mass spectrometry (nanoESI-MS) data reveal PFAA- to physiological protein and substrate concentrations and BSA complexes with up to eight occupied binding sites at a may explain weaker observed binding. 4:1 PFAA:albumin mole ratio. Association constants estimated In this study, bovine serum albumin (BSA) serves as a by nanoESI-MS are on the order of 105 M-1 for PFOA and model protein for characterizing PFAA-protein interactions. 4 -1 BSA is widely used as a model protein in evaluating PFNA and 10 M for perfluorodecanoate and perfluorooctane- - sulfonate. The results reported here suggest binding through protein ligand interactions, as the sequences of human and bovine serum albumins are highly conserved (20, 26, 27). specific high affinity interactions at low PFAA:albumin mole ratios. Here association constants (Ka) and stoichiometries for PFAA- albumin complexes are quantified over a range of PFAA: Introduction albumin mole ratios via equilibrium dialysis and automated nanoelectrospray ionization mass spectrometry (nanoESI- Used throughout the past half-century in a variety of MS), with the specific goal of providing quantitative binding industrial and commercial applications, perfluoroalkyl acids data at low ligand:protein mole ratios. Additional tests are (PFAAs) are a class of environmentally persistent anionic performed with human serum albumin (HSA) for comparison surfactants detected globally in water, air, sediment, and biota (1, 2). Field and laboratory studies indicate that to results for PFAA-BSA interactions. Quantification of PFCA- perfluorooctanesulfonate (PFOS) and perfluorocarboxylates BSA association constants at physiologically relevant PFAA: protein mole ratios using equilibrium dialysis, the most (PFCAs) with greater than seven fluorinated carbons bio- - accumulate and biomagnify in aquatic food webs (2-4). thermodynamically sound and straightforward protein ligand Tissue distribution studies show PFAA concentrations are analysis technique (28), has not been reported. Prior equi- librium dialysis studies (17, 25) reporting PFOA-albumin greatest in body compartments high in protein content, such > as the liver, kidney, and blood of organisms (5, 6). Typical affinities at relatively high PFAA:albumin mole ratios ( 3) concentrations of perfluorooctanoate (PFOA) and PFOS in used less sensitive and selective analytical methods (e.g., the serum of nonoccupationally exposed humans are 4-7 surface tension and spectrophotometry) to measure PFOA and 25-46 ng/mL, respectively (7). The half-life of PFOA in concentrations. serum varies widely by species and sex and is considered Analysis via automated nanoESI-MS provides comple- mentary information about PFAA-BSA interactions at low PFAA:albumin mole ratios and is explored as a technique to * Corresponding author phone: (650)721-2615; fax: (650)725-8662; e-mail: [email protected]. more rapidly evaluate the binding affinities and stoichiom- † Stanford University. etries. Automated nanoESI-MS overcomes some disadvan- ‡ Union College. tages of conventional ESI-MS such as irreproducibility due

10.1021/es101334s  2010 American Chemical Society VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5263 Published on Web 06/11/2010 to nonautomated sample introduction. This method, with Individual stock solutions of PFAAs (1 mM) were prepared nL/min flow rates, is gentler than conventional ESI-MS as with the ammonium acetate buffer in polypropylene bottles. complexes are transferred from solution to the gas phase, A sonicating bath (∼38 °C) was used to assist in the dissolution reducing disruption of intact protein-ligand complexes (29). of PFAAs. Stock solutions of BSA (100 µM) were prepared Analysis of PFOA with rat serum albumin (RSA) (11) and fresh daily in 9 mM ammonium acetate (pH 7) at room rat-specific proteins (13) via electrospray ionization mass temperature and dialyzed overnight, with exchange of the spectrometry (ESI-MS) demonstrated intact complexes. same buffer in an external reservoir. BSA solutions were However, challenges of noisy mass spectra due to additional transferred to polypropylene microcentrifuge tubes, spiked protein adducts (13) and limits in instrument sensitivity and with a PFAA in buffer, and diluted with buffer to a final BSA reproducibility (10) were reported in conventional ESI-MS concentration of 50 µM. PFAA-BSA solutions were prepared PFAA-protein binding studies. over a range of ligand:protein mole ratios (0:1, 0.1:1, 0.5:1, Although PFAAs are consistently detected in protein-rich 1:1, 2:1, 4:1, and 8:1) and were allowed to equilibrate for one tissues, this concept has yet to be incorporated into PFAA or more hours before same-day analysis. Samples tested after bioaccumulation models. Models utilizing a lipid partitioning approximately 1 month indicated no change in equilibrium. paradigm may underestimate the bioaccumulative potential Analysis of noncovalent PFAA-BSA interactions was con- of PFAAs (4), although this remains to be further tested. PFAA- ducted by automated nanoESI-MS. Information on instru- protein association constants may thus contribute to im- ment conditions and data processing is available in the proved predictive models for the bioaccumulation of these Supporting Information. Unless otherwise stated, statistical chemicals. comparisons were conducted using a student’s two-tailed t test assuming equal variance. Materials and Methods Binding Model. The binding of ligand, L, to a protein, P, Materials. Essentially fatty acid free human serum albumin be can be described by a series of stepwise equilibria, with (HSA, 96%), Cohn Fraction V protease free, essentially a total of n binding sites. The average number of bound j γ-globulin free bovine serum albumin (BSA, 99%), and ligands per protein molecule, ν, can be expressed in terms ammonium acetate were from Sigma-Aldrich, Inc. (St. Louis, of the association constant, Ka, the free ligand concentration, MO). Fraction V fatty acid-free BSA (99.9%) was from EMD [L], and the total number of binding sites on the protein, n Biosciences, Inc. Standards of perfluorooctanoic acid (PFOA, (33). The full form of this equation, shown in the Supporting 96%) and perfluorodecanoic acid (PFDA, 98%) were from Information, can be simplified by considering classes of Aldrich Chemical Co. (Milwaukee, WI). Perfluorononanoic binding sites with similar affinities (34). For ionic surfactants acid (PFNA, 97%) and potassium perfluorooctane sulfonate such as fatty acids binding to water-soluble proteins, high (PFOS, 98%) were from Fluka through Sigma-Aldrich (St. and low affinity binding sites may be considered, with high- 13 affinity interactions dominant at low [L]:[P] mole ratios Louis, MO). The internal standard [ C5] PFNA was from 13 (19, 35, 36). Thus, for two classes of binding sites the equation Wellington Laboratories (Guelph, ON, Canada), and [ C2] PFOA was from Perkin-Elmer Life Sciences (Boston, MA). becomes Internal standards had purities greater than 98%, as reported n K [L] n K [L] by the suppliers. j) 1 a,1 + 2 a,2 ν + + (1) Equilibrium Dialysis. Purity-corrected stock solutions of 1 Ka,1[L] 1 Ka,2[L] PFOA and PFNA were prepared in 50 mM sodium phosphate buffer (pH 7.4) in polypropylene containers. A sonicating where νj is equivalent to the ratio of the bound PFAA bath (∼38 °C) was used to assist in the dissolution of PFAAs concentration to the total protein concentration. Over a without the use of an organic cosolvent. Stock solutions of narrow range of low [L]:[P] mole ratios, where high affinity BSA were prepared fresh daily in the sodium phosphate buffer binding dominates, data may be best described with a one- for dialysis experiments. Spectra/Pore dialysis membrane class model. tubing with 6000-8000 Da molecular weight cutoff (Spectrum Laboratories, Inc., Rancho Domingo, CA) was cut into 7.5- Results and Discussion cm pieces, soaked in deionized water for 30 min, and rinsed Equilibrium Dialysis: PFNA-Albumin Binding at Low [L]:[P] with deionized water followed by Milli-Q water. Dialysis tests Mole Ratios. Equilibrium dialysis provides direct measure- were prepared with either 500 µM albumin (human or bovine) ment of free and bound PFAA concentrations, minimizing exposed to a single concentration of PFNA or 1 µM BSA uncertainty from indirect methods such as fluorescence exposed to a range of PFOA or PFNA concentrations. For all spectroscopy (15). To quantitatively assess the binding of tests, a known volume of BSA or HSA solution was added to PFNA to albumin at physiologically relevant [L]:[P] mole the dialysis bag, spiked with a PFAA solution prepared in the ratios, equilibrium dialysis was conducted with 500 µM HSA same buffer, and brought to 3 or 10 mL. Molecular weights or BSA and 0.24 ( 0.08 µM or 0.32 ( 0.07 µM PFNA, of 66430 Da (30) and 66248 Da (31) were used to calculate respectively, in 50 mM sodium phosphate buffer. Based on the final concentrations of BSA and HSA, respectively. The measured equilibrium concentrations, at low [L]:[P] mole dialysis bag, which is impermeable to the protein and its ratios (10-3-10-4) greater than 99.9% of PFNA was bound to complexes but freely permeable to PFAAs, was equilibrated both HSA and BSA (Table 1). The percent bound was also in 300 or 500 mL of the sodium phosphate buffer in high for BSA dialysis tests in 50 mM sodium phosphate buffer polypropylene dialysis reservoirs at laboratory temperatures with an additional 9 g/L NaCl (representative of physiological (approximately 21 °C). Controls were prepared using a buffer- salinity) although the high salt concentration slightly reduced only solution in dialysis bags with a PFAA spike, and blanks the fraction of bound PFNA. Further investigation into the were prepared using a BSA solution with no PFAA spike. Free effects of ionic strength and the presence of competing and bound PFAA concentrations were determined via liquid ligands, such as fatty acids, is needed. chromatography tandem mass spectrometry (LC-MS/MS). Debruyn and Gobas (27) utilize BSA and HSA binding Details on sample preparation and LC-MS/MS analysis are data to assess the sorptive capacity of animal protein for a available in the Supporting Information. Instrumentation range of neutral hydrophobic organic contaminants (HOCs) and operating parameters were previously reported (32). by calculating distribution coefficients between BSA or HSA Nanoelectrospray Ionization Mass Spectrometry. A9 and water. Though representation of PFAA-protein binding mM ammonium acetate buffer (pH 7) was selected to by a distribution coefficient may be acceptable only at very minimize interfering adducts during electrospray ionization. low solute concentrations when the binding isotherm tends

5264 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010 TABLE 1. Percent Bound and log KPW for PFNA Binding to 500 µM Albumin Determined by Equilibrium Dialysis and LC-MS/MS

a a,b a albumin conditions percent bound log KPW [ligand]:[protein] mole ratio HSA 50 mM sodium phosphate, pH 7.4 99.95 ( 0.01% 4.93 ( 0.05 5 ( 1 × 10-4 BSA 50 mM sodium phosphate, pH 7.4 99.92 ( 0.01% 4.74 ( 0.05 6 ( 1 × 10-4 BSA 50 mM sodium phosphate, pH 7.4; 9 g/L NaCl 99.89 ( 0.01% 4.56 ( 0.05 10 ( 1 × 10-4 a Errors represent 95% confidence intervals and were calculated from the root mean squared error of all results conducted at a 500 µM albumin concentration (n ) 13). b Means and error calculations performed on the log-transformed data.

FIGURE 1. Equilibrium dialysis results for PFOA (a) and PFNA (b) where νj is the average number of PFAA molecules bound per albumin. Data represent averages of triplicate measurements from each test reservoir or dialysis bag. PFOA and PFNA data from experiments conducted with 1 µM BSA were fit using eq 1. toward linearity, the calculation of such a distribution bag PFAA concentrations ranged from 1.6 µM to 2700 µM coefficient may be useful in PFAA bioaccumulation models. prior to equilibration in reservoirs. Dialysis bags reached The ratio of analyte concentration in the bound phase to equilibrium with external reservoirs within 48 h (see the that in the aqueous phase (KPW) is determined by the protein Supporting Information, Figures S3 and S4), when final concentration, [P], the fraction bound to protein (fbound), and samples were taken. The average relative standard deviations the partial specific volume of protein in aqueous solution for final bag and reservoir concentrations ranged from 4-6% (Falbumin): for PFOA and PFNA. Initial bag concentrations, which generally required additional dilution of samples prior to C f ) P ) bound analysis, had higher relative standard deviations (9% for PFOA KPW F · - (2) CW albumin [P](1 fbound) and 11% for PFNA) than those for final bag concentrations. Average mass balance results for control experiments (94%, ) ) Log KPW values for the low PFNA:albumin mole ratio tests n 5 for PFOA and 107%, n 7 for PFNA) indicate that are presented in Table 1. A partial specific volume of 0.733 PFAAs do not significantly bind to the dialysis membrane or mL/g was used in calculations for BSA and HSA (19). The reservoir vessels. measured value of KPW for PFNA is greater than all protein Equilibrium dialysis results for PFOA and PFNA with 1 distribution coefficients presented by Debruyn and Gobas, µM BSA are displayed in Figure 1 along with 500 µM BSA and where log KPW ranged from less than 0.1 to 3.5 (27). Log KPW HSA results for PFNA. Although anionic surfactants may cause for PFNA is greater than an experimentally determined protein denaturation, this is not expected to occur over the octanol-water distribution coefficient (log KOW ) 2.57 ( 0.07) concentration range tested (38), as reservoir concentrations (37), highlighting the potential influence of interactions with were well below the critical micelle concentrations (CMCs) nonlipid materials on PFNA distribution in organism tissues. of PFOA and PFNA (8.7-10.5 mM and 2.8-5.6 mM, respec- Log KPW for neutral HOCs was also generally greater than tively) (39). Total PFOA and PFNA concentrations, repre- Log KOW for less lipophilic compounds (log KOW < 2) (27). senting the sum of free and bound PFAA concentrations, Confirming results obtained for PFNA with essentially fatty were measured inside dialysis bags at equilibrium. Final bag acid free BSA (99.93 ( 0.01% bound for a 6 ( 1 × 10-4 [L]:[P] concentrations were greater than reservoir concentrations mole ratio, corresponding to a log KPW of 4.80 ( 0.08) suggest for all tests, indicating that PFAAs were bound to BSA and that trace fatty acids in the essentially γ-globulin free BSA that PFAA-BSA complexes were retained in the dialysis bags. used in this and subsequent tests had little influence on the Osmotic dilution of the retentate was assumed to be binding affinities. negligible. Bound PFAA concentrations were calculated from Equilibrium Dialysis: PFAA-BSA Binding over a Wide concentrations measured inside the dialysis bag in excess of Range of [L]:[P] Mole Ratios. Equilibrium dialysis was used free concentrations. to quantify free and albumin-bound PFAAs in an equilibrated Association constants and binding stoichiometries for system over a wide range of PFNA and PFOA concentrations PFOA- and PFNA-BSA complexes determined via equilibrium and 1 µM BSA; [L]:[P] mole ratios ranged from 0.02 to 120 dialysis with 1 µM BSA are reported in Table 2. Data were in these experiments. Concentrations in all initial reservoir fit both over the full range of test concentrations using eq samples (0 h) were below the detection limit, and a null 1 and up to a 5:1 PFAA:BSA mole ratio using a one-class value was used for mass balance calculations. Initial dialysis binding model. Further details of the fitting approaches and

VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5265 TABLE 2. Association Constants (Ka) and Binding Stoichiometries (n) for PFOA and PFNA Binding to BSA Determined by Equilibrium Dialysis

-1 2 compound Ka,1 (M ) n1 R [ligand]:[protein] range model PFOA 0.20 ((0.14) × 106 4.3 ((2.0) 0.772 0.04-5 one-class PFOA 1.4 ((1.9) × 106 1.4 ((0.5) 0.913 0.04-70 two-class PFNA 1.1 ((0.2) × 106 4.6 ((0.3) 0.943 0.02-5 one-class PFNA 3.3 ((3.2) × 106 2.9 ((0.7) 0.953 0.02-120 two-class

TABLE 3. Summary of PFCA-Albumin Association Constants (Ka) and Binding Stoichiometries over a Range of Ligand:Protein Mole Ratios ([L]:[P])

-1 ligand protein method Ka (M ) n [L]:[P] range ref PFOA BSA dialysis 1.4 ((1.9) × 106 1.4 ( 0.5 0.04-70 this papera BSA nanoESI-MS 1.3 × 105 up to 8 0.1-4 this paper HSA -potentialb 2.4 × 104 - ∼1.5 (38) RSA micro-SECc 2.8 ((0.6) × 103 7.8 ( 1.5 1-4(11) HSA micro-SEC 2.6 ((0.3) × 103 7.2 ( 1.3 1-4(11) HSA dialysisd 3.12 × 104 13 ∼4-30 (25) BSA 19F NMR 6.3 ((0.8) × 102 M-1 - 72-200 (15) RSA 19F NMR 3.4 ((1.2) × 103 - 8-400 (11) BSA dialysis 3.2 × 102 85 ∼3 - 110 (17) HSA ion-selective electrode 1.44 × 105 1414 ( 28 ∼180-520 (36)e HSA ion-selective electrode 3.17 × 104 2565 ( 52 ∼520-1100 (36)e PFNA HSA dialysis 2.1 ( 0.3 × 106 - 5 ( 1 × 10-4 this paperf BSA dialysis 1.4 ( 0.4 × 106 - 6 ( 1 × 10-4 this paperf BSA dialysis 3.3 ((3.2) × 106 2.9 ( 0.7 0.02-120 this papera BSA nanoESI-MS 2.6 × 105 up to 8 0.1-4 this paper BSA 19F NMR 8 ((5) × 103 - 15-100 (15) HSA 19F NMR 2 ((3) × 104 - 8-32 (15) a b Primary association constant listed. Electrophoretic mobility measured at pH 10; Ka calculated from ∆G )-25 kJ/mol. c Micro-size exclusion chromatography. d Conducted at 37 °C. e Hill binding constants reported for two binding regimes f with positive cooperativity. Ka calculated for one PFAA:albumin mole ratio assuming n ) 3. results are available in the Supporting Information. To reduce interactions of small molecules with proteins (29, 40, 41). the number of parameters being simultaneously solved in During the transfer of ions from the liquid to gas phase, even eq 1 and thus reduce the error in the solved parameters, weak interactions are largely preserved (41). Exposure of BSA association constants of 630 M-1 and 8000 M-1 were employed to PFAAs causes a shift in the observed m/z depending on for Ka,2 for PFOA and PFNA, respectively. These values were the number of bound PFAA molecules. The charge state of determined under the same buffer conditions but at higher peaks and number of bound ligands is determined by PFAA:albumin mole ratios (15-200) using 19F NMR (15). An + · + · i+ increased error was observed at higher reservoir concentra- m [MWBSA j MWPFAA i H] ) (3) tions when samples were diluted into the analytical range of z i the LC-MS/MS (see the Supporting Information, Figure S6). Due to a strong influence of the two highest-concentration where MWBSA and MWPFAA are the molecular weights of BSA PFOA data points, which also indicated weak binding at a and the PFAA, respectively, j is the number of PFAAs bound high mole ratio, these data points were excluded from the per BSA molecule, and i is the charge state of the protein in fit presented in Table 2 for the PFOA two-class model. The the gas phase, produced in positive ion mode. Results show effects of applying various weighting factors for the full PFOA that multiple PFAAs are bound per BSA molecule at a 1:1 and PFNA data sets were tested and yielded similar results PFAA:BSA mole ratio, with peaks present at j ) 1 and j ) 2. to those in Table 2. The primary association constants This pattern is repeated over a range of charge states and is determined are similar for PFOA and PFNA, on the order of most intense at i ) 16 and 17 (see the Supporting Information, 106 M-1 with binding stoichiometries of one to four or five. Figure S8). BSA had 150 ( 20 secondary binding sites for PFOA and 31 Representative nanoESI-MS spectra for the most intense ( 2 secondary binding sites for PFNA. A modest effect of charge state (+16) of 50 µM BSA exposed to a range of PFOA albumin concentration on calculated PFAA association and PFNA concentrations are shown in Figure 2. Represen- constants, in which increased protein concentration de- tative spectra for PFDA-BSA and PFOS-BSA complexes are creased affinities, has been previously observed (15). This included in the Supporting Information (Figure S9). Decon- effect was not evaluated in detail in the present study. volution of results yields spectra with peaks at m ) MWBSA However, applying a one-class binding model to the results + j × MWPFAA (e.g., see the Supporting Information, Figure at a single PFNA:albumin mole ratio with 500 µM BSA or S10). Results for PFOA and PFNA suggest a maximum of HSA, and assuming n ) 3, yields similar PFNA-albumin eight PFAA molecules bound per BSA molecule at a 4:1 PFAA: association constants (on the order of 106 M-1) to those from BSA mole ratio. At higher ligand concentrations (8:1 PFAA: 1 µM albumin tests (Table 3). BSA, data not shown), the number of bound PFAAs detected NanoESI-MS Results. In nanoESI-MS experiments, soft continues to increase. Han et al. detected up to six binding ionization maintained protein-ligand complexes in the gas sites at an 8:1 mol ratio of PFOA to rat serum albumin using phase such that PFAA-BSA complexes were distinctly ob- conventional ESI-MS (11). The use of nanoESI-MS here may served relative to free BSA at a 0.5 ligand:protein mole ratio have reduced disruption of PFAA-albumin complexes during and greater. ESI-MS is widely used to study noncovalent transfer to the gas phase, resulting in enhanced detection of

5266 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010 intact complexes. At 1:1 and 2:1 PFAA:BSA mole ratios, up [PLj] R ) (4) to two to four bound PFOA and PFNA molecules were j [P] detected. Binding observed by nanoESI-MS was not cat- egorized into primary or secondary binding classes, although Rj was in turn used to calculate stepwise association equilibrium dialysis results suggest one to four or five primary constants binding sites may be occupied at similar concentration ratios. Overlap of ligand-bound protein charge states limits the utility R ) j of nanoESI-MS results at elevated ligand concentrations. Ka,j (5) R - [L] ESI-MS may also be used to quantify protein-ligand j 1 association constants. Association constants determined via The free ligand concentration at equilibrium, [L], can be ESI-MS are in good agreement with solution-based methods written as a function of the known total protein and ligand for a variety of protein-ligand complexes with affinities concentrations, [P] and [L] , and the measured abundance ranging from 102 to 106 M-1 (29, 40, 42). However, limitations o o ratios, as shown in the Supporting Information, to yield exist in extrapolating results from gas-phase measurements to solution-phase behavior. In particular, obtaining high R resolution for proteins in the high-mass range is difficult, K ) j (6) a,j [P] (R + 2R + ... + nR ) and nonspecific binding may be observed at high ligand: - o 1 2 n Rj-1([L]o + + + + ) protein mole ratios (40) as solution-phase equilibrium 1 R1 R2 ... Rn changes during the evaporation and droplet fission processes (41). The detection of solution phase specific and nonspecific Applying eq 6 to deconvoluted spectra for 0.5, 1, and 2 binding is desired, while binding that may occur during PFOA:BSA, PFNA:BSA, and PFDA:BSA mole ratios yields transfer of molecules to the gas phase is not. association constants for three binding sites reported in the Mathematically deconvoluted spectra of native and Supporting Information (Tables S5-S8). For PFOS, decreased ligand-bound BSA were used to quantitatively analyze PFAA- resolution limited calculation of association constants at protein complexes. Because the ligand is small compared to higher concentration ratios; only the 0.5 PFOS:BSA results the protein (tested PFAAs are less than 1% of the molecular are displayed. Calculated PFOA-, PFNA-, and PFDA-BSA 4 -1 weight of BSA) the surface properties of the ligand-bound association constants (Ka,1-Ka,3) range from 0.9 × 10 M to and free protein are expected to be similar. As such, the 5.1 × 105 M-1, 1.4 × 104 M-1 to 3.9 × 105 M-1, and 1.3 × 104 -1 4 -1 ionization and detection efficiencies of the free and ligand- M to 4.5 × 10 M , respectively. For PFOA and PFNA, Ka,1, bound proteins are assumed to be similar, so the concentra- Ka,2, and Ka,3 are not statistically different from one another. tion ratio of complex to free protein, R, is equivalent to the The relative similarity among the three Ka values suggests a abundance ratio of bound and free protein in the gas phase single binding class. Additionally, Ka,1 results for PFOA, PFNA, (43). Deconvoluted spectra were used to determine the and PFDA collected at the 0.5 PFAA:BSA mole ratio were not abundance ratio and calculate Rj for each bound complex statistically different. Two tests were conducted with cone

FIGURE 2. Mass spectra for the +16 charge state of 50 µM BSA in 9 mM ammonium acetate (pH 7) with PFOA (left) and PFNA (right). The number of PFAA molecules associated with BSA and the mole ratio of PFAA to BSA concentrations, [L]:[P], are indicated.

VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 5267 voltage at 100 V and one test at 130 V. Data collected at a values for a one-class binding model reported in Table 3, we cone voltage of 130 V yielded statistically lower Ka,1 and Ka,2 calculate that greater than 98% of PFAAs are bound to values for each PFCA-BSA as compared to data collected at albumin. 100 V (one-tailed t test, p < 0.1). A higher cone voltage was Significance. A standard solution-based method (equi- used to improve peak resolution, but resulting in-source librium dialysis) was compared with a modern mass spec- dissociation of bound ligands could lead to calculation of trometric approach (nanoESI-MS), providing complementary artificially low association constants. In future studies, information about the strength of PFAA-protein binding solution additives may be used to stabilize complexes for interactions and number of binding sites at low ligand:protein prevention of in-source dissociation (43). mole ratios. Results presented, together with previously Results collected at 100 V to limit in-source dissociation published data, suggest stronger specific associations at low and low mole ratios to limit nonspecific binding, which may PFAA:albumin mole ratios and weaker nonspecific associa- occur as ligand concentrations increase, are most appropriate tions at higher mole ratios. Equilibrium dialysis yields primary ∼ 6 -1 in this study for comparison to solution-based results. The association constants of 10 M for PFOA and PFNA, for a class of one to five high affinity binding sites. A high protein- average Ka,1 values for data collected at 100 V and a 0.5 mol - - water partition coefficient for PFNA (log K > 4) relative to ratio are 1.3 × 105 M 1 for PFOA and 2.6 × 105 M 1 for PFNA. PW neutral HOCs supports the characterization of specific Although these results must be interpreted with caution given binding at low ligand concentrations. the wide range of values, results are within an order of NanoESI-MS is a useful technique for more rapidly magnitude of association constants calculated via equilibrium characterizing PFAA-protein interactions. However, a wide dialysis. range of calculated association constants and sensitivity of Binding Regimes. Binding of PFOA and PFNA at con- complexes to instrument conditions limit the utility of centrations spanning several orders of magnitude and 1 µM nanoESI-MS as a fully quantitative method. Stoichiometry BSA was fit well by a two binding class equation, whereas a values obtained from mass spectrometry demonstrate up to one-class equation was applicable for a narrower range of eight bound PFAAs per BSA molecule at a 4:1 mol ratio. results at lower PFAA concentrations. A companion study Binding constants from nanoESI-MS experiments are on the investigating the binding strength of PFCAs with BSA and order of 105 M-1 for both PFOA and PFNA, lower but in HSA using fluorescence spectroscopy and 19F NMR over a qualitative agreement with solution-based values determined range of PFCA concentrations (100 nM-2 mM) suggests via equilibrium dialysis. ∼ 5 -1 2 -1 PFCA-BSA association constants of 10 M and 10 M for Because KOW may underestimate the bioaccumulative primary and secondary binding sites, respectively (15). Taken potential of PFAAs, a serum protein association constant or together with available literature data and results presented protein-water distribution coefficient may be useful in here (Table 3), the data sets suggest two major binding characterizing the bioaccumulative potential and in vivo regimes: strong specific associations at low PFCA:albumin bioavailability of long-chain PFAAs. However, as proportions mole ratios and weaker nonspecific associations at higher of various proteins vary among species and in time, and mole ratios. Specific interactions may be similar to those likely also have different affinities for PFAAs, further analysis proposed for fatty acids. Albumin is known to have a highly is required to test the ability of protein partitioning to enhance flexible conformation in which hydrophobic “pockets” hold perfluoroalkyl acid bioaccumulation models. the hydrocarbon tails of fatty acids, and charged residues contribute an electrostatic component to the binding (35). Acknowledgments Although albumin has a net negative charge in solution, it This work was supported by the National Defense Science generally has a greater affinity for small, negatively charged and Engineering Graduate Fellowship, the Stanford Uni- hydrophobic molecules (20). versity UPS Foundation and Woods Institute for the Envi- PFOA-HSA interactions studied via zeta-potential mea- ronment, and the National Science Foundation Graduate surements and ion selective electrodes also demonstrate Research Fellowship Program. We thank Pavel Aronov and specific interactions at low ligand concentration, where Allis Chien from the Stanford University Mass Spectrometry almost all PFOA molecules are bound to HSA (36). Initial Laboratory. binding to high affinity sites, where Gibbs energies of interaction are at a minimum, stabilizes the HSA structure Supporting Information Available (22). The interaction was more favorable at lower concentra- Analytical and experimental details, binding model deriva- ∼ > tions ( 1 mM) and increased at saturation ( 10 mM PFOA tions and fitting approaches, and additional and summary with 20 µM HSA), where the hydrophobic effect predominated results from nanoESI-MS. This material is available free of (38). Using electrophoretic mobility, Blanco et al. showed charge via the Internet at http://pubs.acs.org. binding to high-energy sites for low PFOA concentrations R with three proteins of varying size and helix contents (44). 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Chem. 2003, 22 remained bound to 80 µM BSA after 60 min of extensive (1), 189–195. extraction with organic solvents. This was attributed to (4) Conder, J. M.; Hoke, R. A.; De Wolf, W.; Russell, M. H.; Buck, covalent binding of the carboxylate headgroup to protein R. C. Are PFCAs bioaccumulative? A critical review and sulfhydryl groups. Isolated albumins normally contain comparison with regulatory lipophilic compounds. Environ. - Sci. Technol. 2008, 42 (4), 995–1003. 0.5 0.7 mols of free SH per mole of protein molecule (19). (5) Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane For comparison to prior results, at a 1.25 mol ratio of free sulfonate in wildlife. Environ. Sci. Technol. 2001, 35 (7), 1339– PFOA and PFNA to BSA (80 µM) and using equilibrium dialysis 42.

5268 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 13, 2010 (6) Vanden Heuvel, J. P.; Kuslikis, B. I.; Vanrafelghem, M. J.; Peterson, (24) Chen, Y. M.; Guo, L. H. Fluorescence study on site-specific R. E. Tissue distribution, metabolism, and elimination of binding of perfluoroalkyl acids to human serum albumin. Arch. perfluorooctanoic acid in male and female rats. J. Biochem. Toxicol. 2009, 83 (3), 255–261. Toxicol. 1991, 6 (2), 83–92. (25) Wu, L. L.; Gao, H. W.; Gao, N. Y.; Chen, F. F.; Chen, L. Interaction (7) Olsen, G. W.; Huang, H. Y.; Helzlsouer, K. J.; Hansen, K. J.; of perfluorooctanoic acid with human serum albumin. BMC Butenhoff, J. L.; Mandel, J. H. Historical comparison of per- Struct. Biol. 2009, 9,7. fluorooctanesulfonate, perfluorooctanoate, and other fluoro- (26) Gelamo, E. L.; Silva, C.; Imasato, H.; Tabak, M. Interaction of chemicals in human blood. Environ. 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Method for stabilizing fluorooctanoate in aqueous solutions. J. Phys. Chem. B 2005, protein-ligand complexes in nanoelectrospray ionization mass 109 (32), 15566–15573. spectrometry. Anal. Chem. 2007, 79 (2), 416–425. (23) Messina, P.; Prieto, G.; Dodero, V.; Cabrerizo-Vilchez, M. A.; (44) Blanco, E.; Ruso, J. M.; Prieto, G.; Sarmiento, F. On relationships Maldonado-Valderrama, J.; Ruso, J. M.; Sarmiento, F. Surface between surfactant type and globular proteins interactions in characterization of human serum albumin and sodium per- solution. J. Colloid Interface Sci. 2007, 316 (1), 37–42. fluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism. Biopolymers 2006, 82 (3), 261–271. ES101334S

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pubs.acs.org/est

Management Experiences and Trends for Water Reuse Implementation in Northern California † ‡ † Heather N. Bischel, Gregory L. Simon, Tammy M. Frisby,§ and Richard G. Luthy*, † Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305-4020 ‡ Department of Geography & Environmental Sciences, University of Colorado Denver, Denver, Colorado 80217-3364 §Department of Political Science and Hoover Institution, Stanford University, Stanford, California 94305-6044 bS Supporting Information

ABSTRACT: In 2010, California fell nearly 300,000 acre-ft per year (AFY) short of its goal to recycle 1,000,000 AFY of municipal wastewater. Growth of recycled water in the 48 Northern California counties represented only 20% of the statewide increase in reuse between 2001 and 2009. To evaluate these trends and experiences, major drivers and challenges that influenced the implementation of recycled water programs in Northern California are presented based on a survey of 71 program managers conducted in 2010. Regulatory requirements limiting discharge, cited by 65% of respondents as a driver for program implementation, historically played an important role in motivating many water reuse programs in the region. More recently, pressures from limited water supplies and needs for system reliability are prevalent drivers. Almost half of respondents (49%) cited ecological protection or enhancement goals as drivers for implementation. However, water reuse for direct benefit of natural systems and wildlife habitat represents just 6À7% of total recycling in Northern California and few financial incentives exist for such projects. Economic challenges are the greatest barrier to successful project implementation. In particular, high costs of distribution systems (pipelines) are especially challenging, with $1 to 3 million/mile costs experienced. Negative perceptions of water reuse were cited by only 26% of respondents as major hindrances to implementation of surveyed programs.

’ INTRODUCTION reuse, as it is traditionally conceived, may be counterproductive to addressing issues such as inadequate institutional arrange- California is at the forefront of recycled water use, treating 17,18 municipal wastewater to a high enough degree that it can be ments. Given that water reuse can simultaneously address returned to the water supply for a variety of beneficial uses both water supply and wastewater disposal needs, how water 1À3 4,5 reuse agencies perceive, and manage, recycled water À as a form including landscape irrigation, agriculture, ecosystem À enhancement,6 industrial cooling and processing,2,7 ground- of waste or an alternative source for water remains open to À water recharge, and indirect potable reuse.7 9 From 1970 to question. Research regarding economics of water reuse and 2001, reuse of municipal wastewater more than doubled in strategic cost recovery schemes is also limited. Considering the California from 175,000 acre-ft per year (AFY, 11.7 m3/s) to promise of recycled water for augmenting water supplies in the approximately 525,000 AFY (20.5 m3/s). Yet this growth fell West and pressing water supply concerns related to dramatic short of the state’s goal to reuse 700,000 AFY by 2000.10,11 population changes and climate change, assessment of past and ’ current experiences in water reuse implementation is needed to California s goal to increase reuse by 2 million acre-feet (AF) ff by 2030 over 2002 levels12 will require a portfolio of projects more e ectively promote, evaluate, and implement water reuse for a range of beneficial uses. Given multiple failures to attain programs. The present study contributes to this task by evaluat- statewide recycling goals (Figure 1), questions remain as to ing the experiences and perspectives of water reuse managers in the sources of such difficulties as well as the feasibility of Northern California to understand major issues confronting ’ recycled water projects in the region. reaching near-term goals described in California s State Water fi Board Strategic Plan Update of 2008À2012.13 Speci cally, our study seeks to document water reuse program Despite efforts to encourage and support water reuse pro- growth and assess historical goals relative to actual performance in grams at the state and federal levels10,12 not all projects are Northern California based on statewide surveys and policy-derived successfully implemented, and nonpotable reuse projects fre- benchmarks; evaluate the roles of regulatory requirements that may quently fall short of planned delivery goals.14,15 Exploration of reasons for failure of water reuse programs is incomplete. Public Received: August 5, 2011 opposition has led to the suspension or abandonment of several Accepted: November 22, 2011 large water reclamation projects for indirect potable reuse in Revised: November 17, 2011 10,16 California, but a focus on lack of public acceptance of water Published: November 22, 2011

r 2011 American Chemical Society 180 dx.doi.org/10.1021/es202725e | Environ. Sci. Technol. 2012, 46, 180–188 Environmental Science & Technology POLICY ANALYSIS

of drivers and barriers experienced in program implementation. To assess relationships between categorical variables, chi square analysis was conducted on two by two contingency tables constructed from frequency results of specific drivers (Table 1) and hindrances (Table 2) to program implementation. The lists of specific drivers and hindrances were also consolidated into eight and nine categorical variables, respectively, and chi square analysis was performed (see Tables S1 À S8 for full results). Representative respondent quotes, extracted primarily from responses to two questions À the single most important driver or hindrance to implementation (Q10 and Q14, Table S12) À are presented (italicized in quotations) to provide context for the diversity of experiences evident throughout the results. Respondent Information and Survey Limitations. A total of 71 distinct agencies, a 53% response rate, are represented by Figure 1. 50-year timeline of major statewide water recycling goals and 2010 Survey responses. Because some parent utilities represent production volumes, drought periods, and water recycling policies in multiple recycled water facilities, a total of 81 unique production California. Refer to the Supporting Information for a description of major facilities are represented by responses; however, most agencies laws and policies. (83%) represent only one recycled water production facility, and another 7% represent a unique distribution facility coupled to a limit discharge relative to water supply and reliability needs in driving production facility. Respondents consist of internal public agency recycled water implementation through time; assess the importance managers or utility staff. The survey completion rate, indicating of ecosystem enhancement or protection goals in water reuse the percentage of invited respondents who submitted a fully decision-making; and elucidate the relative importance of economic complete survey, was 40%. Therefore, the response fractions factors among challenges to nonpotable water reuse implementation reported for each question indicate values for that particular experienced by managers in Northern California. question. Respondent agencies for the 2010 Survey were dis- tributed widely across Northern California, with weaker repre- ’ METHODOLOGY sentation for agricultural programs when compared to the 2009 Survey data (Figure S1 and Table S9). The median year of Data Sources. Primary data on water reuse agencies, practices, recycled water program implementation, based on self-reported and management experiences were collected via an online implementation dates for 56 respondents, was 1991, with the questionnaire of Northern California water reuse managers earliest reported implementation occurring in the early 1960s. conducted for the present study in 2010 (2010 Survey). Addi- tional data on water reuse agency characteristics were obtained ’ ANALYSIS OF WATER REUSE IN CALIFORNIA from the California State Water Resources Control Board (SWRCB) 2001 Water Recycling Survey released in 2002 Recycled Water Distribution Falls Short of Statewide (2001 Survey),11 the National Database of Water Reuse Facilities Goals. Figure 1 displays a timeline of statewide water recycling À (National Database),19 and the 2009 California Municipal Waste- goals and production volumes.10,11,15,20 22 According to the water Recycling Survey, a follow-up survey from the SWRCB 2009 Survey, recycled water used in California in 2001 included released in April 2011 (2009 Survey).20 Municipal water recy- 491,992 AFY (19.2 m3/s) from municipal facilities (private cling agencies in Northern California (defined as the 48 counties facilities excluded).20 The newest data from the California northward of the southern boundaries of Monterey, Kings, Tulare, SWRCB indicate California municipal wastewater facilities re- and Inyo counties) listed on the publicly available National cycled a total of 723,845 AFY (28.3 m3/s) in 2009.20 This Database and the 2001 Survey were invited to participate in represents an increase of more than 230,000 AFY (9.0 m3/s) the 2010 Survey. from levels in 2001 yet once again falls short of goals for recycling Fieldwork Administration and Questionnaire. Data were set by the State of California by nearly 300,000 AFY (12 m3/s, collected online from February to April 2010 using electronic Figure 1 and Table S10). Although the SWRCB 2009 Survey may surveys sent to general managers or water/wastewater directors underrepresent current reuse volumes due to a low survey from 134 agencies. The questionnaire, which is described further response rate, the results underline a need to identify continuing in the Supporting Information and Table S12, was developed challenges associated with implementation of recycled water based on case study research, literature review, and site visits at programs, evaluate strategies to develop new programs, and agencies with water reuse programs for agriculture, landscape expand existing distribution networks. In California, where irrigation, industrial power plant cooling, and ecosystem en- applied freshwater usage was 39.2 million AF in 2005, recycled hancement. Respondents were asked a number of questions water represents a relatively small portion of dedicated or developed related to the drivers and challenges experienced in implement- water supplies (e.g., water used for agriculture or urban uses).23 ing their agency’s water reuse program as well as responses to However, only about 10% of available treated effluent was recycled in recent recycled water policy in California. 2001, indicating important growth potential for this water source.10 Categorization and Statistical Tests. The analyses con- Northern California Context. Our analysis shows that only 20% ducted for 2010 Survey results provide quantitative confirmation of the observed statewide increase in reuse between 2001 and 2009 of trends previously discussed and valuable insights into the occurred in the Northern 48 counties of California, where 120 characteristics of water reuse in Northern California. Results municipal agencies recycled 127,000 AF (1.6 Â 108 m3) in 2001 and represent response data and are supported by qualitative descriptions 143 agencies recycled 173,000 AF (2.1 Â 108 m3) in 2009. Recycled

181 dx.doi.org/10.1021/es202725e |Environ. Sci. Technol. 2012, 46, 180–188 Environmental Science & Technology POLICY ANALYSIS

a Table 1. Percent of Respondents Indicating a Specific Factor As a Driver or One of the Three Most Important Drivers

categorized factor most impt. driver driver specific factor most impt. driver driver

wastewater discharge requirements 51% 65% wastewater discharge volume requirements 51% 65% water supply needs 49% 69% water shortages due to reduced supply 42% 65% water shortages due to increased demand 17% 42% seawater intrusion 5% 6% local, regional, or state policy and mandates 45% 68% basin plan water quality objectives 25% 43% regional or local recycled water policy goals or mandates 20% 42% state recycled water policy goals or mandates 14% 31% climate change adaptation plans 0% 5% institutional control 29% 58% need for reliable water supply 26% 52% need for increased institutional control of water 3% 20% economic/financial incentives 26% 51% availability of federal/state grants or loans 18% 32% cost of alternative freshwater sources 9% 32% ecological goals or requirements 18% 51% ecological protection or enhancement goals 12% 49% ecological protection or enhancement requirements 6% 20% influential stakeholders 11% 34% large volume user(s) 6% 28% citizen initiative 5% 12% technological advancements 3% 22% technological advancements 3% 18% other 18% 18% other 18% 18% a Responses (N = 65) were further categorized as shown and are sorted from top to bottom by the highest frequency categorized Most Important Driver.

a Table 2. Percent of Respondents Indicating a Specific Factor As a Hindrance or One of the Three Most Important Hindrances

most impt. most impt. categorized factor hindrance hindrance specific factor hindrance hindrance

economic/financial disincentives 87% 94% capital costs for construction of recycling plant facilities 56% 85% costs for pipeline construction 48% 80% ongoing operations and maintenance cost recovery 26% 61% availability of federal/state grants or loans 24% 54% cost of alternative freshwater sources 7% 26% perceptions and social attitudes 26% 61% perceived human or environmental health risks 13% 48% due to constituents of emerging concern social attitudes/public perception 13% 33% perception that recycled water will lead to more development 4% 22% perception that recycled water will reduce property value 4% 6% who pays system costs 20% 59% issue of who pays for program capital or operating costs 20% 59% regulatory constraints 15% 52% complexities/conflicts of water law and/or regulation 9% 37% slow regulatory process in permitting 7% 30% water quality impacts 13% 48% downstream water quality impacts/NPDES constraints 7% 31% detection of constituents of emerging concern 4% 33% effluent residuals (e.g., brine) disposal 2% 11% user acceptance 9% 37% user acceptance 9% 37% institutional issues 11% 30% institutional coordination 9% 28% loss of projected users 2% 6% technical issues/treatment 7% 31% technical issues/treatment processes 7% 31% uncertainty over future recycled water uses 4% 13% uncertainty over future recycled water uses 4% 13% other 9% 11% other 9% 11% a Responses (N = 54) were further categorized as shown and are sorted from top to bottom by the highest frequency categorized Most Important Hindrance. water programs in Northern California are generally smaller in AFY (22 m3/s) of water in 2009 by 104 agencies (Figure 2 and volume (median = 347 AFY or 0.014 m3/s in 2009) than programs Figure S2). Water reuse programs are frequent across rural Northern - in the ten Southern California counties (median = 1064 AFY or California and agricultural areas in the Central Valley (Figure 2), 0.042 m3/s in 2009), where 82 municipal agencies recycled 365,000 typically at much lower volumes than urban areas. Historically, AFY (14 m3/s) of water in 2001, increasing to a total of 551,000 agricultural water reuse predominated in California (Figure 3),

182 dx.doi.org/10.1021/es202725e |Environ. Sci. Technol. 2012, 46, 180–188 Environmental Science & Technology POLICY ANALYSIS occurring where farmland was located adjacent to wastewater ’ DRIVERS OF WATER REUSE IMPLEMENTATION IN treatment facilities.10 Agricultural reuse was more common for NORTHERN CA older respondent agencies in the 2010 Survey, as determined by a Various social, economic, and environmental factors have been chi square test with implementation dates before or after 1991 (p < identified as drivers of water reuse by governments and stakeholders 0.05). Significant population growth, particularly in the Central globally.10,14,24,25 These driving forces include the following: drought, Valley, creates challenges for new or increased wastewater discharge demand due to population and economic growth, wastewater in largely agricultural areas, especially for environments with limited 13 management, ecological protection, availability near urban areas, assimilative capacity. Though reuse in Northern California repre- and availability of proven treatment technologies.14,25 To estab- sents a lesser fraction of overall reuse in the state, challenges lish a forum for free-form responses regarding principal driving associated with the implementation of these programs are important forces behind recycled water implementation in Northern Cali- to consider in developing the total portfolio of state projects. Several fornia, respondents first considered the relative importance of larger programs have been implemented over the past decade in several broad categories of drivers. The fraction of respondents Northern California, and more are likely to be developed in large indicating each broad category as a very important driver or a urban centers. However, recycled water program size has remained driver, respectively (Q8, Table S12), was as follows: regulatory relatively stable on average in Northern California. requirements (0.59, 0.27), water shortages (0.49, 0.34), econom- ic concerns (0.28, 0.37), recycled water policy (0.23, 0.49), and influential stakeholders (0.21, 0.33). To further gauge the extent to which a range of specific factors motivated program implementation, respondents were asked to select from a list of 19 specific factors (Table 1). Altogether, 65% of respondents indicated “wastewater discharge volume require- ments” as a driver of implementation, with 51% of respondents selecting this factor as one of the three most important drivers. “Water shortages due to reduced supply” was cited as a driver by 65% of respondents and by 42% of respondents as one of the three most important drivers of implementation. Together, these two factors were cited by 86% of respondents. Expressing a common experience for the most important driver of program implementation, one respondent described that their “initial recycled water program was established as a wastewater disposal option out of concern for discharge capacity... Expansions to the recycled water system since 2005 were based on prudent use of water resources and extending the limited potable supply.” In addition to specific regulatory requirements, state recycled water policy goals or mandates were selected as a driver by nearly a third (31%) of 2010 Survey respondents and as one of the three Figure 2. A snapshot of water reuse facilities in California from the most important drivers by 14% of respondents. Additionally, National Database of Water Reuse Facilities (Annual Production, reported 25% of respondents selected basin plan water quality objectives as Facility Production Average Annual Actual in million gallons) and the as one of the three most important drivers of implementation. California 2009 Municipal Water Recycling Survey (Annual Reuse, re- Such objectives may relate to discharge volume requirements: ported as Total Reuse for 2009 in AFY). In the inset box plot, the boundary one respondent who described Basin Plan Water Quality Objec- of the box indicates the upper and lower quartiles; a line within the box tives as the single most important driver of their program’s indicates the median flows (347 AFY and 1064 AFY); whiskers above and implementation stated, “Reducing our volume discharged to surface below the box demarcate 1.5 times the interquartile distance with outlying ffl points also shown. The distributions of flows from the 2009 Survey differed water helps us to meet increasingly more stringent e uent discharge significantly between Northern and Southern California (MannÀWhitney loading requirements.” Notably, “Ecological protection or en- ” U = 10080.5, n1 = 143, n2 = 104, P < 0.0001, two-tailed). hancement goals were drivers for the implementation of many

Figure 3. Beneficial uses of recycled water in Northern California in 2001 and 2009. 2001 Survey data shown include private agencies, which were excluded in the 2009 Survey; see the Supporting Information for a description of categories. In recent years, agricultural reuse volumes have remained relatively stable, becoming a smaller fraction of total reuse as new industrial and commercial uses are developed.

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Figure 4. Results of χ2 analysis by implementation date for specific factors indicated as one of the Three Most Important Drivers (top) or more generally a Driver of implementation (bottom) with p-values indicated (*estimated p < 0.05, see the Supporting Information). programs (49%) but were rarely the most important drivers for Transitioning from Wastewater Discharge Control to Recycled these programs (12%). Recognition of ecological benefits from Water As a Resource recycled water implementation exemplifies how issues may “The original driver is not the current driver. Currently water generate common ground between stakeholders, even if they supply and reliability is the most important driver.” are not of high priority to any one group. However, in 2001 and Water shortages are commonly experienced throughout 2009, only 6À7% of reuse was for direct natural system/wildlife California, with several severe droughts throughout the period enhancement (Figure 3), indicating a missed opportunity for of implementation represented by survey responses (Figure 1). recycled water usage. Controlling Wastewater Discharge and the Role of Regulation Recognizing such challenges, the California State Legislature enacted, for example, the Water Recycling Act of 1991 following “We needed a method [to] dispose of treated effluent. The only a 5-year period of drought, setting new recycling goals from those viable alternative was recycling.” established in 1977.26,27 California’s elaborate system of dams, Results demonstrate that regulatory requirements, such as those canals, aqueducts, groundwater basins, and levees mediates the limiting discharge of wastewater, have historically played an dichotomy between the state’s water sources and demand important role in driving the implementation of water reuse in centers, where 75% of the state’s precipitation falls north of Northern California. The California Department of Public Health Sacramento and 75% of demand occurs in the population and establishes state public health criteria for wastewater reclamation farming centers to the south.28 Because of the interconnected- via Title 22 for bacterial quality, treatment types and levels, and ness of water infrastructure in the state and the dependence of facility reliability. Individual Regional Water Quality Control the largest urban centers on imported water, Northern California Boards (RWQCBs) and local water and health agencies may also is not immune to challenges associated with limited water supplies. develop more stringent policies and programs related to recycled The growing awareness and response to water supply challenges water use.10 In free-form responses, respondents who cited are reflected in agency experiences. Programs implemented after regulatory requirements as a very important category of drivers 1990 were more likely to cite water supply needs including water (n = 28) noted a range of specific regulatory pressures that drove shortages due to increased demand as drivers than older pro- the implementation of their program (see the Supporting Infor- grams (p < 0.01, Figure 4 and Table S3). Conversely, wastewater mation for details). Various agencies were mandated or recom- discharge volume requirements were more frequently indicated mended to reduce percolation and increased reuse, cap discharge as one of the three most important drivers of implementation by flows despite population growth, and eliminate point source agencies with reported implementation dates before 1991 (p <0.05, discharges or meet dilution requirements in receiving waters Figure 4), suggesting that early implementation of water reuse during a particular time period (e.g., summer months). in the region was driven more frequently by such regulatory

184 dx.doi.org/10.1021/es202725e |Environ. Sci. Technol. 2012, 46, 180–188 Environmental Science & Technology POLICY ANALYSIS requirements. Newer recycled water programs were also more Table 2, other factors hindering program implementation identified likely to cite the need for reliable water supply as an important by individual respondents included soil salinity, lack of seasonal driver of implementation (p < 0.01). storage, and overcoming opposition from influential stakeholders. Only 5% of respondents in the present survey indicated climate Economic Constraints and Financial Implications of change adaptation plans as a driver of recycled water program im- Challenges plementation. However, guidance by the California Natural Re- “Generally in the industry and specifically for us, the cost of sources Agency29 and Department of Water Resources30 incorporate pipelines is really the only reason we haven’t been recycling more.” recycled water as a drought-proof and sometimes energy efficient Several examples of recycled water programs in Northern California water management strategy to complement climate change adapta- provide context for the expected costs of recent treatment facilities tion measures. As these goals filter from state planning to local and distribution systems. For 16 projects seeking regional federal practices, state policies for climate change adaptation may become funding as part of the San Francisco Bay Area Recycled Water more influential in recycled water implementation. Coalition, the total costs ranged from $220/AF to $3400/AF, Although water shortages were not directly an issue during with a $1200/AF median value, assuming a 20-year period for project implementation for some older projects, anticipated recycled water generated at the initial project yield (Table water shortages and need for long-term reliable sources are now S11).31 Recycled water deliveries expected for these projects critical issues, especially following the 2007À2009 drought in range from 115 AFY (0.0045 m3/s) initially to up to 28,000 AFY California. Projects that were implemented initially due to (1.1 m3/s) in the future. A City of Palo Alto analysis indicated an wastewater requirements may expand or find new benefits of annualized cost of $2700/AF (over 30 years, in March 2008 reuse due to water supply challenges. One respondent illu- dollars) expected for expansion of distribution facilities. This strated this changing paradigm, stating the following: compared with a projected cost of $1,600/AF by 2015 for “Fifteen years ago when we started our program, public wholesale purchase of potable water from the San Francisco acceptance was an issue. People did not understand recycled Public Utilities Commission (SFPUC).32 An earlier phase of the water, and we spent a lot of time educating potential customers Palo Alto project completed in 2009 came to approximately $3.4 and marketing recycled water. There was some ‘fear factor’ million/mile of pipeline for construction base contract of slowing the expansion. However, things have changed comple- approximately 5 miles of pipeline along US Highway 101 to tely with the worsening drought, delta water problems, climate the neighboring City of Mountain View.33 A project under change awareness, and the public’sdesiretobe‘green’ and analysis by the SFPUC estimates $9.4 million (including a 30% recycle everything now. We currently cannot get the water out to contingency) for approximately 6.5 miles of pipeline construc- customers fast enough.” tion costs as part of a $153 million recycled water treatment and distribution system.34,35 To assist and encourage user connection Increasing reliability of potable water supplies (e.g., by sustaining to the recycled water system, agencies may provide financial groundwater supplies for drinking), supplementing water supply incentives for recycled water use via a recycled water rate structure needs, or freeing up freshwater entitlements for use elsewhere were discounted from potable water rates (e.g., by 20% to 80%) or by described as other drivers of implementation. offering services for establishing connections, retrofits, training, permit review, and testing.36 However, in describing an important strategy to overcome cost recovery challenges (Q15, Table S12), one ’ CHALLENGES FOR WATER REUSE IMPLEMENTA- respondent described, “Recycled water must be treated as a TION IN NORTHERN CA commodity and a utility rate charged to completely recover costs.” Challenges for water reuse projects include the following: a Additional strategies adopted by agencies to address funding need for public information, education, and outreach; lack of challenges are described in the Supporting Information. System available funding; recovery of capital costs for dual distribution cost recovery is often supplemented by grants and loans, includ- systems; political support; a need for additional research for ing Title XVI through the US Bureau of Reclamation as well as innovative technologies; flawed or unevenly applied regulations Proposition 50 Grant and the State Revolving Fund loan Program and standards; and concerns and liability over the unknown long- for the State of California.26,36 term health effects of chemical contaminants.10,25 When asked to 2010 Survey respondents were asked to characterize, as select factors that hindered program implementation at the quantitatively as possible, the impact of cited hindrances to im- respondent’s site from a list of 20 specific options, 87% of plementation in terms of program cost, scope, and timing. respondents cited financial or economic challenges as one of Respondents indicated that hindrances led to a change in the three most important hindrances to water reuse implementa- program cost (n = 9), reduced program scope (n = 5), delay of tion (Table 2). One respondent commenting on the single most implementation (n = 14), project cancellation (n = 7), or other important hindrance to implementation simply stated, “These (n = 1). For a subset of these responses, estimated costs projects are big ticket items outside the range of a rate base.”. Specific associated with impacts (n = 21) ranged from $50,000 to almost hindrances from the category of financial or economic disin- $100 million per agency. Estimates by respondents for changes in centives shown in Table 2 dominated the selection of the most program cost represented construction cost increases over time, important challenges relative to other categories consistently costs for additional studies, increased staff time, additional testing through time. Despite various sources of policy and financial “beyond reasonable needs”, “huge” impacts from years of delay, support for water reuse in California, lack of sufficient funding costs to upgrade to tertiary treatment, costs for new processes, may be the main factor preventing recycling goals from being and a combination of changes in program scope, changes in achieved.20 Challenges in the next most-cited category, public design, addition of professional consultants or a combination perception and social attitudes, were indicated as an important of conveyance pipes, distribution piping, tanks, and pressure hindrance by only 26% of respondents. In addition to those in stations.

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Issues related to institutional coordination were also noted for rather than supply driven, public perception problems may arise increasing project costs. Nearly a third of respondents indicated more readily. Organizational trust correlateswithintendedbehavior institutional coordination as a hindrance to implementation. One toward using recycled water and may be an area of further focus for respondent described the following: institutional practices to increase public acceptance.48 “Perceived human or environmental health risks due to “While water agencies need recycled water to help them with constituents of emerging concern” was cited as a hindrance to long term supply issues, they cannot justify the increased costs implementation by almost half of respondents. Yet this factor was and thus tend to be unsupportive. Water agencies are also not correlated to program implementation date, reminding us concerned about loss of revenue with recycled water projects. If that unknown or unregulated contaminants change in specific the water agency is not the same as the recycled water agency (as definition with time but have challenged managers for decades. in our area), implementation of recycled water projects means a Concern for residuals in recycled water has been expressed in loss of revenue for the water district as customers are shifted to various forms. In the 1970s and 1980s, issues of public perception the recycled water agency. This means that the potable water were difficult to overcome, as recycled water was relatively agency must raise rates for the remaining customer base, which is unfamiliar and long-term safety of reuse for high-contact uses very difficult in today’s economic climate.” was unproven. Today, chemicals of emerging concern (CECs) are a topic for technological research and a source of concern for Limited Role of Negative Perceptions recycled water managers.22 Noting this issue as an additional “In 1984, the biggest hindrance was the negative perception by challenge to cost hindrances, one respondent commented, landowners next to the farms scheduled to receive recycled water “opponents are also trying to use the issue of emerging constituents today. Today the biggest hindrance is cost.” as a way to portray the project in a negative light.” Public perception Since the 1970s, a significant amount of research has investigated of recycled water continues to be an important nontechnical reasons for public resistance to recycled water.37,38 Although public challenge for water reuse implementation, especially with regards perceptions of risks are identified as key impediments in the to CECs. However, the present study finds that economic issues, À adoption of indirect potable water reuse,39 41 nonpotable water rather than public perception, stand as the largest hindrance reuse programs generally receive public support.14 Thus, opposition to nonpotable reuse implementation for Northern California surrounding high-profile indirect potable reuse is likely unrepresen- programs. Responses to Recycled Water Policy. In 2009, the SWRCB tative of the landscape of challenges faced by managers of non- “ potable reuse programs. A notable contrary case developed when adopted a California Recycled Water Policy to increase the use of recycled water from municipal wastewater sources”. The homeowners actively opposed the use of recycled water for land- “ scape irrigation in Redwood City, CA.1 Analyses emphasize the policy strongly supports recycled water as a safe alternative to potable water for such approved uses”. Despite the policy’s stated importance of public engagement early during project concep- objectives, whether the water reuse policy will actually accelerate tion and continuously throughout planning, design, and con- efforts to develop and maintain new recycled water projects struction.10,14 While utilities and consultants have developed remains unclear. The legislation itself takes on a hopeful tone by more appropriate modes of communicating with the public, striving for, among other items, increased use of recycled water some remain skeptical about the safety of the practice, especially “over 2002 levels by at least one million acre-feet per year (AFY) as projects are proposed in their community and the likelihood of ” 12 42À44 by 2020 and by at least two million AFY by 2030 . human contact increases. Nearly two-thirds of 2010 Survey Recycled water managers were questioned about their expec- respondents (61%) cited perceptions or social attitudes as tations concerning how the California Recycled Water Policy of hindrances to program implementation, though these factors 2009 will facilitate or hinder the implementation of new recycled were less frequently considered among the most important water programs. Survey responses reveal both support and challenges to overcome. Forms of public communication (e.g., trepidation toward the policy, with a greater number of respon- signage, symbols, and terminology) can influence consumer dents voicing concern that the policy will hinder project im- intentions to use, and willingness to pay for, recycled water,45,46 plementation. According to respondents, a perceived beneficial and the media may play a role in constructing positive or negative impact of the policy stems from standardized and consistent perceptions of recycled water.17,47 guidance for recycled water projects. For example, the water The primary drivers of water reuse programs may also influence reuse policy established a Blue Ribbon Panel for evaluating CECs public opposition or acceptance. An early public opinion study in that will apply to all projects across California and also contains California indicated that those who believed water supply augmen- language endorsing water reuse under the California Environ- tation was necessary in California were somewhat less likely to be mental Quality Act (CEQA). Second, many respondents viewed opposed to reclaimed water for drinking than those who did not the policy favorably due to its singular management structure. believe that water was scarce.38 Consequently, public education The establishment of an overarching permitting process, and of efforts to effectively communicate the need for water reuse are salt and nutrient management requirements, in particular, drew important. In the present study, respondents who cited wastewater positive reviews. As one manager put it, “The standardization of discharge volume requirements as a driver of implementation were salinity and nutrient management provisions among the various somewhat more likely to also cite a specific factor within the regional boards should facilitate reuse and make it easier for some category of public perceptions and social attitudes as a hindrance projects to get permitted.”. Thus, for water reuse project managers, (0.1 < p < 0.2). As freshwater supply and distribution agencies the provision of administrative, legal, and scientific continuity experience increased demands and pressures on existing resources, across state, regional, and local agencies was perceived as the greater public awareness of augmentation needs may reduce most beneficial aspect of the policy. challenges associated with public perceptions. Conversely, in com- Much of the skepticism expressed for the 2009 policy may be munities where the drivers of recycled water are discharge-based, traced to funding issues. A majority of respondents (19 of 30

186 dx.doi.org/10.1021/es202725e |Environ. Sci. Technol. 2012, 46, 180–188 Environmental Science & Technology POLICY ANALYSIS question responses) felt the policy would obstruct new projects or state grants and loans, while larger programs have somewhat through onerous regulatory and cost requirements. According greater challenges associated with distribution system costs. to a number of managers, while statewide project streamlining Failure to meet statewide reuse goals results largely from lack and standardization is important, ultimately the fate of projects of sufficient funding for water recycling, as the cheapest recycled will depend on adequate funding support. A common refrain water opportunities have already been exploited.10 Following among respondents was a concern over added administrative three years of drought and recent passage of the Safe, Clean, and layers that will arise with new oversight and reporting require- Reliable Drinking Water Supply Act of 2010 by the State of ments. In sum, the perceived presence of additional financial California that included $1.25 billion general obligation bond costs and administrative requirements have led nearly 2 of every proposal for Water Recycling and Water Conservation, the 3 survey respondents to suggest the 2009 water reuse policy will physical and political climates may be ripe for aggressive im- in some way hinder new project implementation. From a plementation of new water reuse programs, where financially management perspective, results suggest that the 2009 policy viable, socially accepted, and technically sound. Yet the legisla- has done little to alter the perceived drivers and hindrances of ture’s 2010 decision to postpone the water bond initiative for at water reuse project implementation for managers in Northern least two years28 is testament to the realities of financial limita- California. tions for new water infrastructure in California.

’ ASSOCIATED CONTENT ’ SIGNIFICANCE A diverse body of responses from the 2010 Survey illuminates bS Supporting Information. (1) Methodological details, (2) a a number of influential drivers of water reuse implementation, brief description of recycled water policy and regulation in California, including the protection of ecosystems, meeting wastewater and (3) additional analysis and summary tables. This material is avai- discharge requirements, and needs for water supply and relia- lablefreeofchargeviatheInternetathttp://pubs.acs.org. bility. Throughout the analysis, we detect manifestations of the intrinsic links between water supply and quality: threats of long- ’ AUTHOR INFORMATION term diminished water quality (e.g., seawater intrusion) necessi- Corresponding Author tates new water conservation and reuse measures, while new *Phone: 650.721.2615. Fax: 650.725.8662. E-mail: luthy@ water supplies of altered quality may galvanize community opposi- tion. Although water supply agencies increasingly face challenges stanford.edu. associated with population growth and drought, wastewater agencies have traditionally approached recycled water as an issue ’ ACKNOWLEDGMENT of disposal. This push/pull duality that either push implementa- We thank the numerous participants in the project and survey tion forward (via regulatory requirements for wastewater dis- respondents for their generous donation of time and thoughtful charge) or pull agencies into recycled water programs (by increased contributions. This work was funded by the Stanford University demand for water) is apparent. Results provide evidence of Woods Institute for the Environment Environmental Venture changing perspectives toward recycled water management, Projects, the Bill Lane Center for the American West, the National from a waste disposal issue toward a water supply resource Science Foundation Graduate Research Fellowship Program, and opportunity. the NSF Engineering Research Center for Re-inventing Urban Specifically, our study reveals the following: 1) In Northern Water Infrastructure (UrbanWaterERC.org). We especially thank California, water reuse programs are widely distributed across 48 Sophie Egan for detailed technical assistance. counties and, though more numerous than programs in the 10 Southern California counties, are often smaller in annual reclaimed ’ water delivery volumes. This finding highlights how management REFERENCES experience across both urban and rural regions of Northern (1) Ingram, P. C.; Young, V. J.; Millan, M.; Chang, C.; Tabucchi, T. California differ from the predominance of highly urbanized From controversy to consensus: The Redwood City recycled water – centers in the south. 2) Regulatory requirements that limit experience. Desalination 2006, 187, 179 190. (2) Rosenblum, E. Selection and implementation of nonpotable discharge played an important role in motivating many water “ ” reuse programs in Northern California. However, a trend away water recycling in Silicon Valley (San Jose area) California. Water Sci. Technol. 1999, 40 (4À5), 51–57. from reuse as a wastewater disposal issue is documented, as water (3) Hermanowicz, S. W.; Diaz, E. S.; Coe, J. Prospects, problems and supply and reliability become more prevalent drivers of water reuse. pitfalls of urban water reuse: A case study. Water Sci. Technol. 2001, 43 3) Although ecosystem enhancement or protection goals are (10), 9–16. frequently cited as drivers of water reuse, such goals are rarely the (4) Sheikh, B.; Cort, R. P.; Kirkpatrick, W. R.; Jaques, R. S.; Asano, T. most important drivers for reuse programs. Few water reuse Monterey waste-water reclamation study for agriculture. Res. J. Water programs in California have been implemented for the purpose Pollut. Conrol Fed. 1990, 62 (3), 216–226. of ecosystem enhancement. 4) Negative perceptions of water (5) Nakamoto, M., Watsonville recycled water project story: Pre- reuse were not frequently major hindrances to implementation of serving agriculture in the Pajaro Valley. In WateReuse California Annual water reuse programs in Northern California. Public perception Conference, San Diego, CA, 2010. of water reuse may be positively influenced by a shift in view of (6) Gearheart, R. A. Use of constructed wetlands to treat domestic waste-water, City of Arcata, California. Water Sci. Technol. 1992, 26 recycled water toward that of a valuable resource and as public (7À8), 1625–1637. knowledge of water supply challenges increases. 5) Economic (7) Levine, B.; Reich, K.; Sheilds, P.; Suffet, I. H.; Lazarova, V. Water issues stand as the largest hindrance to successful project imple- quality assessment for indirect potable reuse: A new methodology for mentation from a management perspective. In particular, smaller controlling trace organic compounds at the West Basin Water Recycling water reuse programs are less frequently incentivized by federal Plant (California, USA). Water Sci. Technol. 2001, 43 (10), 249–257.

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ENVIRONMENTAL ENGINEERING SCIENCE Volume 30, Number 8, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/ees.2012.0201

Renewing Urban Streams with Recycled Water for Streamflow Augmentation: Hydrologic, Water Quality, and Ecosystem Services Management

Heather N. Bischel,1,2 Justin E. Lawrence,1,3 Brian J. Halaburka,1,2 Megan H. Plumlee,1,4 A. Salim Bawazir,1,5 J. Phillip King,1,5 John E. McCray,1,6 Vincent H. Resh,1,3 and Richard G. Luthy1,2,*

1Engineering Research Center for Re-Inventing the Nation’s Urban Water Infrastructure (ReNUWIt), National Science Foundation, Stanford, California. 2Department of Civil & Environmental Engineering, Stanford University, Stanford, California. 3Department of Environmental Science, Policy, and Management, University of California, Berkeley, California. 4Kennedy/Jenks Consultants, San Francisco, California. 5Department of Civil Engineering, New Mexico State University, Las Cruces, New Mexico. 6Department of Civil & Environmental Engineering, Colorado School of Mines, Golden, Colorado.

Received: May 18, 2012 Accepted in revised form: September 13, 2012

Abstract As demands for freshwater withdrawals continue to escalate in water-stressed regions, negative consequences of alterations to natural systems will become ever more severe. Habitat restoration projects may mitigate some of these challenges, but new strategies will be needed to maintain or enhance ecosystem health while simulta- neously meeting human needs. Recycled water is a reliable water source that can be used both directly and indirectly to renew degraded urban stream ecosystems. In this review, aspects of hydrology, water quality, and ecosystem services in relation to water reuse for urban stream renewal are evaluated to identify research needs and design considerations for new systems. Use of recycled water for streamflow augmentation in urban areas remains largely unexplored scientifically, despite its potential widespread applications among water and wastewater utilities. To move this innovative concept toward implementation, experimental studies in stream microcosms are needed to examine ecological response to coupled modification of both hydrology and water quality. Appropriate methods for selecting potential sites for urban stream renewal should be identified, along with ecological and economic metrics for evaluating success. Examples of projects in California, Japan, Israel, and Spain are used to identify different management scenarios. However, design criteria from both successful and unsuccessful case studies require additional review and synthesis to develop robust guidelines for recycled water use in urban stream renewal. Motivations for past stream renewal projects include regulatory require- ments for water quality improvement and endangered species protection, although these motivations alone may not be enough to facilitate widespread adoption of reusing wastewater for ecosystem enhancement. Conse- quently, future project designs should include more detailed ecosystem service valuations to describe broader societal benefits and attract the attention of government agencies and private organizations that ultimately make the choice between environmental perturbation or enhancement.

Key words: critical review; ecosystem renewal; natural system enhancement; recycled water; streamflow augmentation

Introduction pecially severe in arid and semiarid regions (Patten, 1998; Brooks et al., 2006). However, the recognized need to ensure ncreases in water use associated with urban growth and long-term ecological integrity of riverine systems, which are development have led to dramatic, negative impacts on I highly degraded in many urban areas, has resulted in a sig- aquatic ecosystems (Paul and Meyer, 2001; Groffman et al., nificant increase in the undertaking of stream restoration 2003; Walsh et al., 2005). The consequences observed are es- (Bernhardt et al., 2005; Bernhardt et al., 2007; Palmer et al.,2007). With continuing improvements in wastewater treatment technologies and escalating water demands, recycled water is *Corresponding author: Department of Civil and Environmental Engineering, Stanford University, Yang & Yamazaki Environment & a resource that can be used to a greater extent to benefit eco- Energy Building, 473 Via Ortega, Rm. 191, Stanford, CA 94305-4020. systems (Jackson et al., 2001; Sala and Serra, 2004). Success Phone: (650) 721-2615; Fax: (650) 725-9720; E-mail: [email protected] stories of recycled water use in managed natural systems,

455 456 BISCHEL ET AL. predominately for constructed wetlands, demonstrate that evaluations of ecosystem responses to altered flow regimes, effective planning of water reuse projects at local and regional water quality in effluent-dominated streams, and ecosystem scales can enhance aquatic habitat, improve water quality, service provisions identified from past natural system resto- and provide public value (Vymazal et al., 2006; Vymazal, ration projects. Experiences from a particularly relevant 2007; Rousseau et al., 2008). However, the intentional use of stream renewal project are subsequently highlighted in a recycled water for stream renewal is an often-overlooked detailed case study of Calera Creek in Pacifica, California. opportunity. Reasons for this include an absence of regulatory After the review and case study, we identify barriers to im- guidance or prohibitory regulations in some cases, presence of plementation, outline research needs, and envision how suc- competing demands for recycled water use, and lack of ap- cess may be achieved in the future. The analysis represents a propriate water quality standards and riparian habitat indi- part of a larger research effort coordinated through the U.S. cators to monitor and demonstrate benefits (Latino and National Science Foundation’s Engineering Research Center Haggerty, 2007; Bischel et al., 2012; Plumlee et al., 2012). for Reinventing the Nation’s Urban Water Infrastructure Nevertheless, with integrated water resource management, (ReNUWIt) (Sedlak et al., 2013). recycled water has the potential to serve as a viable resource for renewal of water-stressed streams (i.e., rivers, creeks). Review Ecosystem renewal in this context refers to replenishing Water reuse for ecosystem enhancement in practice and regenerating natural systems to guide them toward a future dynamic state in which the abiotic and biotic compo- Wastewater treatment plants (WWTPs) often discharge nents are within the historic range of natural variability for secondary-treated effluent directly into streams and fresh- that location or region. This definition is similar to that which water wetlands (Tchobanoglous et al., 2003; Carey and is often used in the literature for ecosystem enhancement, Migliaccio, 2009). This practice is especially common for in- restoration, or rehabilitation (Grenfell et al., 2007). The term land facilities, whereas many coastal facilities discharge di- ecosystem renewal is adopted for the purpose of this review rectly into the ocean. In some cases, effluent discharged under and is intended as a general phrase that includes any resto- low-flow stream conditions may incidentally serve to protect ration, rehabilitation, or enhancement project that incorpo- the integrity of aquatic ecosystems downstream (Brooks et al., rates a return of water, including recycled water available 2006), especially when industrial pollution inputs to the from wastewater reclamation, back to a stressed ecosystem. wastewater and river systems are low. While wastewater ef- Stream renewal assumes that flow augmentation occurs fluent requires specified treatment for disposal requirements, alongside other objectives, as flow augmentation by itself may recycled water is the end product of wastewater reclamation not be adequate to meet the objectives of a recycled water that meets additional and appropriate water quality require- project designed for ecosystem enhancement. The objective ments and is produced with the intent of being used for was not a restoration of a system to the exact conditions at a beneficial purposes (Levine and Asano, 2004; NRC, 2012). specific point in the past; we are not suggesting an approach Because the quality of recycled water used for stream renewal to outperform nature or force an oppressively fixed structure would be no lower, and likely higher, than that of traditional onto a system that requires dynamism. Rather, a successful wastewater effluent, wastewater effluent discharge to surface project will maintain or increase the resilience and resistance waters represents a worst-case scenario for identifying po- of the biological communities (Stanley et al., 1994). tential ecological effects of using recycled water for stream- The motivations for stream enhancement and restoration flow augmentation (NRC, 2012). Discharge of wastewater are diverse, and include moral, regulatory, political, and with low levels of treatment into streams has resulted in economic concerns (Wu et al., 2003; Clewell and Aronson, ecosystem degradation in many cases, and as a result, nega- 2006; Corsair et al., 2009). Moral arguments recognize that tive perception of the effects of wastewater on the environ- stream ecosystems are part of our natural heritage and that ment is widespread (Carey and Migliaccio 2009; Grantham our duty as stewards of the environment is to protect them et al., 2012). (Lee and Roth, 2003). Likewise, federal and state governments Alternatively, when carefully managed for augmentation have regulations in place to protect the environment, such as in natural systems, recycled water, a term here used synon- the Endangered Species Act and the Clean Water Act, which ymously with reclaimed water, can renew urban streams ei- water utilities must adhere to in their effluent-release proce- ther directly or indirectly in a variety of planned scenarios dures (Rosan, 2000; MacDonnell, 2009). However, regulations (Table 1). Direct augmentation as practiced in Calera Creek and moral arguments are often not enough to provide an (Pacifica, CA) and the San Antonio River (San Antonio, TX) adequate level of protection (Moyle and Yoshiyama, 1994; entails identifying opportunities to supplement streamflow in Costanza et al., 1997). Analyses of the economic benefits that ecologically critical stream reaches as well as securing existing intact ecosystems provide, which are often described as eco- or new sources of recycled water. As an alternative to existing system services, may increasingly provide the informa- centralized recycled water facilities, distributed treatment tion necessary to motivate institutional change (Postel and plants could be constructed to produce recycled water at the Thompson, 2005; Goldman et al., 2007). location of an intended augmentation. The water quality re- The objective of this review is to assess the potential for quired for stream renewal can also be achieved in some lo- water reuse to renew urban stream ecosystems, with a focus cations by using the intrinsic filtration capabilities of natural on both the opportunities and concerns for management systems, such as through unit-process wetland treatment, in relation to hydrology and ecology, water quality and before streamflow augmentation (Sala and Tejada, 2008; Jas- treatment, and ecosystem services. Because research specifi- per et al., 2012). In these systems, unit-process wetlands could cally addressing the intentional application of recycled water serve as the tertiary treatment needed to produce recycled for urban stream renewal is limited, we drew broadly from water from secondary-treated wastewater. A stream reach RENEWING URBAN STREAMS WITH RECYCLED WATER 457

Table 1. Management Scenarios for Recycled Water to Augment Streamflow and Case Studies

Example design or management Scenario Concept Example projects challenges

Wastewater effluent- Treated effluent is discharged San Luis Obispo Creek, San Status quo approach lacks dependent streams routinely to streams with Luis Obispo, CA (DiSimone, active streamflow manage- regulatory-driven ambient 2006; Asano et al., 2007) ment and introduces water quality protection and Hadera River, Israel hydrological and water may provide habitat for (Hophmayer-Tokich quality challenges. aquatic species. and Khot, 2008) Direct streamflow Recycled water may be designed Salado Creek and San Antonio Insufficient guidelines exist augmentation to flow directly from the River, San Antonio, TX (Dean for project development engineered treatment system and Shih, 1975; Crook, 2004; and implementation. Per- into the designated stream Eckhardt, 2004; USEPA, mitting processes may be reach. 2004) complex and lengthy or Nobidome and Tanagawa regulations may not allow Streams, Tokyo, Japan stream augmentation. Po- (Ohgaki and Sato, 1991; tential recycled water qual- Maeda et al., 1996) ity issues include excess Bell Creek, City of Sequim, WA nutrients, chlorine residual, (Latino and Haggerty, 2007) and elevated temperature. Calera Creek, City of Pacifica, CA (see case study) Unit-process wet- Recycled water passes through Tossa de Mar Creek, Tossa de Requires land for wetland land treatment a constructed wetland before Mar, Spain (Sala and Tejada, cells and treatment reliabil- system augmenting the targeted 2008) ity. Efficacy of long-term stream reach, creating habitat, Danshui River, Taipei, Taiwan treatment by wetlands and and receiving additional (Cheng et al., 2011) mechanisms are uncertain. treatment. River Dommel, Boxtel, The Netherlands (Kampf and Claassen, 2004) Petaluma River, City of Petalu- ma, CA (USEPA, 2004) In-stream treatment Recycled water passes through a Natural attenuation of trace Uncertain treatment capacity zone designated in-stream treatment organics in Santa Ana River, in hyporheic zone, river- zone upstream of the targeted CA (Lin et al., 2006) and banks, or macrophytes. augmentation stream reach. Trinity River, TX (Fono et al., Potential degradation of 2006) upstream reach. Indirect augmenta- Recycled water percolates into Subsurface discharge through Requires land for infiltration; tion or discharge groundwater and indirectly a 300-ft perforated pipe requires groundwater flow augments streamflow. The adjacent to the Columbia and temperature modeling subsurface may be used to cool River in Dallesport, WA to establish streamflow water or attenuate contami- (Dallesport, 2007) connection and expected nants when recycled water Pilot-scale infiltration wetland water quality changes. exceeds limits protective of at the Pudding River, City in-stream biota. of Woodburn, OR (Stewart, 2010) Agricultural return Recycled water used to irrigate Landscaping at polishing Return flow water quality flow agricultural fields is collected wetlands irrigated with may be compromised from and treated before runoff into recycled water at Petaluma agricultural chemical inputs adjacent streams. Recycled River, City of Petaluma, CA (e.g., nutrients and water could also be used to (USEPA, 2004) pesticides). irrigate stream banks during Agricultural return flow treated native vegetation germination in constructed wetland before periods to simulate floods that stream discharge in Kongju no longer occur. City, Korea (Maniquiz et al., 2012) Water savings/ Water conservation or savings Water conservation and recy- Requires appropriation of indirect benefits from water reuse can indirectly cling for more natural flows in-stream flows or commit- provide environmental benefit in the Russian River, Sonoma ments to reduce requests for by allowing maintenance of County Water Agency further diversions. flows. (Dickinson et al., 2011) 458 BISCHEL ET AL. with unique substrate characteristics and location in relation where water is transported over great distances to urban to the groundwater table (i.e., upwelling or down-welling centers. These experiences highlight the importance of proper areas), rather than a constructed wetland, could also be site selection based on both historical data and modeling ef- identified as a natural treatment zone upstream of the target forts to understand baseline hydrologic regimes and water stream reach. quality characteristics. Nonetheless, some streams were pe- Alternative streamflow management scenarios may also be rennial historically and are now nonperennial as a result of achieved indirectly using recycled water. Infiltration basins urbanization, and these streams certainly warrant further in- and subsurface wetlands can offer a filtration function vestigation as potential augmentation sites (BARWRP, 1999). (Stewart, 2010; Jasper et al., 2012; Lawrence et al., 2012), pro- Additionally, climate change will reduce flows in some viding soil aquifer treatment for wastewater discharged to a streams, which could be mitigated through water reuse ap- stream via a hydraulically connected groundwater aquifer. plications. Better understanding of the science, the motiva- Another benefit of indirect discharge may be temperature tions, and the institutional impediments of water reuse for mitigation—for example—by attenuation of wastewater ecosystem renewal is needed to determine to what extent this temperature via the subsurface (made possible by the large will be a viable approach in the future. heat capacity of aquifer materials) as modeled for the Pudding River in Oregon (Stewart, 2010) and demonstrated in Dalle- sport, WA adjacent to the Columbia River (Dallesport, 2007). Hydrology and ecology Over 300 WWTPs indirectly discharge treated wastewater Flow modifications in urban environments. Freshwater through rapid infiltration to nearby surface waters (Crites biota are highly adapted to the local components of the nat- et al., 2000). Recycled water can also renew stream ecosystems ural flow regime, which include the magnitude, frequency, through its application toward uses that would otherwise duration, timing, and rate of change of flows over time draw water from streams. Water resource managers in the (Richter et al., 1996; Poff et al., 1997; Tharme, 2003). The natural San Francisco Bay Area of California, for example, recog- flow regime in water-stressed regions often includes season- nize that using recycled water for agriculture could reduce the ally predictable wet and dry periods (Fig. 1). The adaptations need for imported water and diversions from the sensitive San of freshwater biota to the natural flow regime are especially Francisco Bay-Delta ecosystem (BARWRP, 1999). Likewise, pronounced in water-stressed areas, such as deserts and recycled water applications to vineyards in Sonoma County, Mediterranean climate regions (Gasith and Resh, 1999). Mi- California, may indirectly augment streamflows through re- gratory fish (Bunn and Arthington, 2002; Jager and Rose, duced groundwater abstractions (Lawrence et al., 2011) and 2003), benthic macroinverterbrates (Mendez and Resh, 2008), increased return flow. Lastly, reduction of degradation asso- and riparian vegetation (Merritt et al., 2010) all show key ciated with excessive wastewater effluent discharge in one inter-relationships with flow. Moreover, the characteristics of location via redistribution of the water for stream renewal the natural flow regime are essential to maintaining com- where needed at an alternative location has the potential munity structure and food-web dynamics in these complex to improve the ecosystem health through strategic flow ecosystems (Power et al., 2008). management. Flow has been called the ‘‘master variable’’ (Power et al., Upgrades of existing WWTPs to meet stricter regulatory 1995) and the ‘‘maestro that orchestrates pattern and process requirements for effluent discharge will in some cases blur the in rivers’’ (p. 85, Walker et al., 1995). The 1960s mantra, ‘‘the line between intentionally planned and unintentional cases of solution to pollution is dilution,’’ illustrates how water quality ecosystem renewal. For example, if wastewater is discharged and flow are related; however, many studies have shown that through constructed wetlands for additional polishing before even dilute concentrations of pollutants can have deleterious river discharge (Green et al., 1996), river water quality is im- effects on biota. Flow also affects habitat quality (e.g., tem- proved, even though the design may lack additional elements perature magnitude and fluctuations, shading provided by that would further promote stream renewal (e.g., manage- the riparian canopy, substrate characteristics, and diversity of ment of flows to mimic natural hydrographs). Ecosystem re- pool and riffle habitats). Furthermore, the interconnectedness newal should entail a holistic evaluation of stream needs of flows and ecosystems among streams, hyporheic zones, related to factors, including managing flow, water quality, groundwater, wetlands, and estuaries is of great importance geomorphology, and habitat in concert, distinguishing opti- [as exemplefied by Christensen et al. (1996), Slocombe (1998), mally designed streamflow augmentation from traditional and Granek et al. (2010)]. An understanding of the effects of wastewater discharge. human disturbance on these interconnected natural flow re- Streamflow augmentation using recycled water will likely gimes, and mediating these effects through innovative solu- provide the greatest rejuvenating effect in urban streams that tions involving both natural systems and engineered systems, are most severely degraded in hydrology, ecology, and water is a major goal of integrated water resource management and quality (Ponce and Lindquist, 1990). Augmentation will not is essential for preserving biodiversity (Jewitt, 2002; Draper be universally applicable, however. For example, many urban et al., 2003). streams actually have too much water as a result of impor- Urban streams have dramatically altered hydrology as a tation from outside of the drainage basin, which can cause dry result of human development. For example, groundwater weather flows. Excess water can turn historically non- withdrawals, water diversions, and WWTP discharges can perennial streams into perennial streams, which can have modify all of the components of the natural flow regime (e.g., adverse effects on indigenous flora and fauna that are adapted decreasing flood magnitudes, altering the frequency of high to the natural seasonal cycles of wetting and drying (Gasith flow events, and/or changing the timing of flows). Urban and Resh, 1999). These problems are especially prevalent in streams tend to be flashier than natural streams in the sense the semiarid or Mediterranean climates, such as in California, that the streamflow rises much more quickly and to a higher RENEWING URBAN STREAMS WITH RECYCLED WATER 459

FIG. 1. Flow regimes for Coyote Creek from USGS gage #11170000 showing the (A) natural flow regime and (B) altered flow regime. Coyote Dam and LeRoy Anderson Dam were constructed upstream of this gage in 1936 and 1950, respectively. The natural flow regime of Coyote Creek (A) is representative of a typical perennial stream in a Mediterranean-climate region with predictable seasonality in wet-season and dry-season flows. In the altered flow regime (B), wet-season floods are decreased dramatically in both magnitude and frequency, and summer low flows are severely reduced or eliminated. magnitude during storm events (Konrad and Booth, 2005; and what components of that regime are most important Walsh et al., 2005). This flashiness is primarily a result of the ecologically (Stoddard et al., 2006; Poff et al., 2010). Manage- relatively high coverage of impervious surfaces in the urban ment of the magnitude, timing, and variability of flows environment, such as paved roads, parking lots, and roofs should take higher priority than just providing a minimum on buildings, which alter hydrologic flow paths (Arnold flow, which has been the standard management practice in and Gibbons, 1996; Paul and Meyer, 2001). These higher- many regions of the United States (Postel and Richter, 2003). magnitude streamflows also increase flooded areas in Fortunately, this minimal-flow approach is being recon- downstream wetlands and estuaries, and can increase incision sidered as a result of new environmental flow policies in in stream channels, which can both scour the hyporheic zone many U.S. states (MacDonnell, 2009). and drain the local groundwater reservoir (Henshaw and In many cases, it will not be possible or desirable in cases Booth, 2000; Groffman et al., 2003). where it increases flooding risks, to return a streamflow re- Natural flow regimes in semiarid regions are often char- gime to a historic condition, but significant benefits could still acterized by a low-flow period that occurs in the dry season be realized by providing a new flow regime that captures (Fig. 1). During this part of the year, the effects of urbanization some ecologically important aspects of the natural hydro- on freshwater biota are magnified, because any changes in graph (Naiman et al., 2002). This was accomplished at Putah flow have a larger effect on the total volume [see Smakhtin Creek in California, where targeted changes to the flow re- (2001) for a review of low-flow hydrology]. Urban streams gime below a dam re-established native fishes and reduced can have reduced base flows relative to the natural flow abundances of alien fishes (Kiernan et al., 2012). Such hydro- regime from water diversions, or if the groundwater reservoir logic modifications may also prove beneficial to establishing is depressed as a result of withdrawals from wells, reduced native riparian flora. For example, along the Rio Grande River infiltration during rainfall events, or stream-channel incision in the southwestern United States, non-native riparian plants (Paul and Meyer, 2001; Konrad and Booth, 2005). In contrast, such as salt cedar (Tamarix spp.) outcompete native plants urban streams can have higher base flows during this water- such as cottonwoods (Populus spp.) and willows (Salix spp.), stressed period of the year caused by augmentations, altered because the annual floods have been largely eliminated by microclimates, irrigation return flow, hydropower releases, upstream dams and water withdrawals (Stromberg et al., recreational flow releases for boaters or anglers, WWTP dis- 2007). Although recycled water volumes are insufficient to charges, and landscaping applications, as well as from in- restore the natural floods along this river to their full magni- creased groundwater recharge from septic tank discharge tude, the banks of the active channel and the floodplain could and/or leaks in underground potable or nonpotable water be irrigated seasonally with recycled water to simulate aspects lines [examples have been reported by Lerner (2002) and of the natural flood cycle while preventing damaging floods Garcia-Fresca and Sharp (2005)]. on private property. This process could allow native plants to Many urban streams are highly degraded, signifying great regain a competitive stronghold against invasive species. potential for creative engineering solutions to renew these Managed streamflow augmentation using recycled water ecosystems through new approaches to the design and offers important opportunities to renew streams in urban and operation of urban water infrastructure. For these ecosystem periurban environments where other sources of water have benefits to be realized, it must be determined how the flows in been allocated for competing uses (e.g., residential, agricul- these urban streams are altered from the natural flow regime tural, or industrial), and either flows are reduced relative to 460 BISCHEL ET AL. the natural flow regime, or there is an opportunity to mitigate responses over the course of several months (Resh and Jack- aquatic habitat loss elsewhere in the urbanized watershed. son, 1993; Resh, 1994). In contrast, fishes may require multiple For example, a historically nonperennial stream could be years (Karr, 1981; Aparicio et al., 2011), and riparian forests made perennial if many of the perennial stream habitats in the can take decades to respond (Hierl et al., 2008; McClain et al., region have been eliminated by human development. Table 2 2011). Microbial diversity is also a fundamental indicator of provides examples of hydrologic metrics for evaluating stream ecological health and is influenced by changes in streamflow relative to historic conditions and effects of aug- geomorphology and exposure to certain water quality con- mentation. Selected metrics should include low-flow condi- stituents [such as those reported by Davy-Bowker (2006) and tions when possible, because augmentation with recycled Feris et al. (2003)], but is rarely used for quantifying ecosystem water will likely have the most impact under these conditions. benefits or degradation. In terms of comparative advantages, In addition to such hydrologic indices, multiple biological, benthic macroinvertebrates respond in a timeframe that is physical, and chemical parameters should be utilized to assess most amenable to scientific evaluation, and they are very how alternative water management scenarios for recycled sensitive to a variety of disturbances (Resh, 2008). water might impact ecosystem integrity (Richter et al., 1996). The evaluation of whether a change in benthic fauna with Although many studies have documented declines in recycled water flow augmentation is a net benefit that can be freshwater biota in response to altered flow regimes [see Poff performed using a multitude of structural and functional and Zimmerman (2010) for a review], very few have examined metrics, including those that describe abundance, composi- the ecological response to intentionally augmented flow [see tion, diversity, and biological traits. Multivariate statistical Ponce and Lindquest (1990) for the most recent review]. Based metrics that predict the proportion of taxa observed relative to on observations of natural systems and either unintentional or those that would be expected under reference conditions, of- indirect flow augmentations, managed streamflow augmen- ten determined using a RIVPACS-type predictive model [for tations clearly have demonstrated a potential to benefit eco- examples, see Hawkins et al. (2000) and Ostermiller and systems not only in the water column and benthos of streams, Hawkins (2004)], may also be useful in some cases. The se- but also in the hyporheic zones [as reported by Kasahara and lection of evaluation metrics should be tailored to the site Hill (2007)]. Moreover, groundwater reservoirs, wetlands, es- under examination in that the choice should make the best tuaries, and riparian zones to which these streams are inextri- possible use of the historical biological information available cably connected may also benefit (Wolff et al., 1989; Debano and should be sensitive to the unique taxa that are present. and Schmidt, 1990; Henszey et al., 1991; Troendle et al.,2001; Species composition, diversity, and trait metrics are widely Robinson et al., 2003; Huertas et al., 2006; Zou et al., 2010). applied for aquatic ecosystem assessments and may be ap- plied to quantify benefits of flow augmentation. For example, Evaluating benefits. The type of fauna used to evaluate as a composition metric, the proportion of the individuals or ecosystem integrity will dramatically affect the duration of species present in the three insect orders—Ephemeroptera, flow augmentation required to evaluate changes (Resh and Plecoptera, and Trichoptera—relative to the total benthic Jackson, 1993; Carlisle and Clements, 1999) where recycled macroinvertebrate community is widely considered to be in- water is used for stream renewal. For example, diatoms can dicative of water quality, and thus an increase in the value of show changes on the order of hours (Winter and Duthie, 2000; this metric can be considered a benefit (Carter et al., 2009; Smucker and Vis, 2011), but benthic macroinvertebrates show Purcell et al., 2009). Other composition metrics, such as

Table 2. Examples of Hydrologic Metrics for Monitoring the Effects of Streamflow Augmentation

Component of the natural flow regime Metric codea Description of metric How to calculate

Magnitude ML22 Specific mean annual Calculate the mean of annual minimum flows and divide minimum flow by the drainage area. Frequency FL1 Low flood pulse count Calculate the average number of flow events with flows below a threshold equal to the 25th percentile value for the entire flow record and determine the average number of events. Duration DL18 Number of zero flow Calculate the number of zero-flow days for the entire flow days. record and determine the mean number per year. Timing TL3 Seasonal predictability Divide years into 2-month periods, count the number of of low flow events with flows £ 5-year flood threshold in each period over the entire flow record, and calculate the maximum number of these events in any one period divided by the total number of such events. Rate of Change RA3 Fall rate (m3/day) Calculate the change in flow for days in which the change is negative for the entire flow record and determine the mean of these values.

Metric values at a site should fall within the regional range of natural variability for the five components of the natural flow regime (Richter et al., 1996), which can be determined using historical USGS streamflow gauge data. aKennen et al. 2007. Sources: Olden and Poff (2003); Kennen et al. (2007). RENEWING URBAN STREAMS WITH RECYCLED WATER 461 regional indices of biotic integrity (Ode et al., 2005; Rehn et al., and Renofalt, 2008; Canobbio et al., 2009), especially when 2007; Lunde and Resh, 2012), are also useful. Likewise, as a wastewater comprises a majority of streamflow. Yet, treated biodiversity metric, taxa richness is widely accepted as an effluent has also long been recognized for its ecological value indicative of ecosystem health, and an increase in the value of in supporting riparian and aquatic habitats in effluent- this metric would also be considered a benefit (Bonada et al., dependent ecosystems in the arid West (USEPA, 1992). For 2006; Resh, 2008). Two other diversity metrics, Shannon- example, improved water quality and endangered species Wiener diversity and Simpson’s diversity, are also widely presence were recorded after the discharge of tertiary-treated used, and increases in their values could be considered a wastewater to San Luis Obispo Creek, an effluent-dependent benefit (Resh, 1994; Pires et al., 2000). creek in California (Arnold, 2000; DiSimone, 2006). For Traits metrics describe the functional adaptations of or- streamflow augmentation using recycled water, water quality ganisms to their environment and as such can be useful to variables that significantly influence ecosystem processes track biotic responses to specific physical changes in the en- should be identified for the planned flow conditions (Poole vironment, such as in temperature, streamflow, and nutrient et al., 2004; Palmer et al., 2005; Grantham et al., 2012). availability (Beˆche and Resh, 2007; Lawrence et al., 2010). As a The recycled water quality will be an improvement com- relatively newer biological monitoring tool, trait databases are pared to baseline stream conditions for some water quality still in development, and the methods for their application are parameters, but treatment technologies must be selected and being tested (Usseglio-Polatera et al., 2000; Vieira et al., 2006). designed to lower concentrations of other constituents before Several of these are potentially useful in evaluating benefits of the recycled water is added. In Coyote Creek (San Jose, augmented stream flow (Table 3). California), where flow augmentation using recycled water In the inevitable tradeoff among selective advantages was under consideration for a demonstration project, metals conferred by different traits, managed streamflow augmen- and pathogen contamination in the creek were expected to tation will certainly benefit some taxa and hinder others, and improve from dilution with the better-quality recycled water. these changes can be hypothesized and examined using our However, increased temperatures from the warmer recycled knowledge of each taxon’s set of traits (Table 3). For example, water were a concern, as were perfluorochemical concentra- benthic macroinvertebrates that are filter feeders should in- tions that were higher in the recycled water than the receiving crease proportionally over time under higher flows as a result stream waters and associated groundwater (Plumlee et al., of the increased suspension of organic matter in the water 2011). Although this demonstration project was not im- column (James et al., 2008; Fuller et al., 2010). Likewise, plemented, management of the recycled water release volume diatoms, a higher-quality food source for many macro- and timing was planned to mitigate temperature issues. invertebrates and fish, should replace the lower-food-quality Riverine environmental flow management often lacks ex- filamentous algae (Robson et al., 2008). In terms of restoration plicit consideration of natural water quality variability in of fauna and flora, if the streamflow augmentation mimics the space and time, prioritizing water quantity over water quality natural flow regime, the benefits should accrue mostly to the parameters (Nilsson and Renofalt, 2008; Olden and Naiman, native taxa as opposed to the non-native taxa. Evolutionarily, 2010). However, when water quality variables (e.g., tempera- the native taxa have acquired their unique set of traits through ture, dissolved oxygen, ammonia, biological oxygen demand, natural selection under historical flow conditions, whereas and trace metals) fluctuate beyond acceptable ranges, eco- invasive species tend to be opportunistic and are favored in logical status may decline even when specified flow condi- modified and disturbed environments. tions are met. For example, in a stream mesocosm study, Obvious changes, such as general increases in abundance increased secondary-treated wastewater discharge volumes with augmented recycled water, may have to be examined and corresponding altered concentrations of ammonium, more closely. For example, abundance metrics can be indic- dissolved oxygen, chloride, soluble phosphate, and sulfate ative of improved conditions when a stream that is depau- negatively impacted the composition and diversity of the perate shows an increase in the total number of organisms. resident aquatic invertebrate community (Grantham et al., Nonetheless, if this increase in abundance is dominated by 2012). Given the interrelated nature of biogeochemical and non-native, invasive, or pollution-tolerant organisms, then aquatic chemistry processes (Nimick et al., 2011), it can be the increase should not be considered a benefit. An increase in difficult to predict how stream ecosystems will react to chan- the abundance of a species that is listed as endangered is ges in contaminant loading from the addition of reused water. certainly a benefit, and such benefits once achieved are pro- Water quality design parameters for streamflow augmenta- tected under the Endangered Species Act in the United States tion programs and appropriate treatment technologies for [see Good et al. (2007) for example]. Ideally, multiple met- enhancing in-stream biological integrity are necessarily site rics should be used so that the benefits (or harms) can be specific such that water quality guidelines will be difficult to quantified and evaluated using multiple lines of evidence, apply broadly. To better assess stream renewal using recycled and if the time and resources are available, metrics based on water, additional comparative mesocosm experiments are other fauna such as fish, amphibians, reptiles, algae, zoo- needed to evaluate changes in biotic integrity with recycled plankton, and riparian vegetation could be included in the water that undergoes tertiary processes such as biological analysis to provide a more comprehensive benefit assessment nutrient removal and alternative disinfection strategies. (as demonstrated in Table 4). From an operations perspective, project managers must avoid exceeding predetermined concentration limits, or maintain water quality above minimum thresholds for key Water quality and treatment technologies parameters (such as those shown in Table 4). In some cases, Effluent-dominated streams have often raised water qual- the discharge volumes may be reduced to achieve levels ity and ecotoxicological concerns (Brooks et al., 2006; Nilsson within an acceptable range based on known natural 462 BISCHEL ET AL.

Table 3. Examples of Aquatic and Semiaquatic Taxa That Could Benefit from Managed Streamflow Augmentation Based on Their Biological Traits

Biological trait category Favored trait Reason References

Benthic macroinvertebrates Body size Large body size Large taxa will benefit from Beˆche et al., 2006; Townsend increased aquatic habitat and Thompson, 2007; Feio and Dole´dec, 2012 Functional feeding Filter-feeders Filter feeders will benefit from Spooner and Vaughn, 2008; group greater suspension of organic Paillex and Dole´dec, 2009; matter Feio and Dole´dec, 2012 Body shape Streamlined body shape Thin-bodied taxa are morphologi- Beˆche and Resh, 2007; Statzner cally adapted to faster-moving and Beˆche, 2010; Feio and water Dole´dec, 2012 Locomotion Greater swimming Strong swimmers would benefit Poff et al., 2006; Tullos et al., 2009; abilities from increased water availability Rice et al., 2010 Fish Body size Large body size Large taxa will benefit from Poff and Allan, 1995; Mims et al., increased aquatic habitat 2010; Olden and Kennard, 2010 Body shape Streamlined body shape Thin-bodied taxa are morphologi- Craven et al., 2010; Haas et al., cally adapted to faster-moving 2010; Schaefer et al., 2011 water Locomotion Greater swimming Strong swimmers would benefit Craven et al., 2010; Olden and abilities from increased water availability Kennard, 2010; Rice et al., 2010 Amphibians and reptiles Body size Large body size Large taxa will benefit from Indermauer et al., 2010; Johans- increased aquatic habitat son et al., 2010; Winne et al., 2010 Locomotion Greater swimming Strong swimmers would benefit Johansson et al., 2010; Kupferberg abilities from increased water availability et al., 2011 Adult longevity Short-lived adult life Less stress on juvenile recruitment Gibbons et al., 2006; Bateman spans because of continuous water et al., 2008 availability Algae Light requirements Lower light Deeper water will have less Schiller et al., 2007; Poulı´ckova´ requirements sunlight penetration et al., 2008; Centis et al., 2010; Torne´s and Sabater, 2010 Substrate preference Suspended in the water Increased availability of habitat Murdock et al., 2004; Suren and column in the water column Riis, 2010 Development rate Fast development Algal taxa develop faster in moving Reynolds, 1994; Culp et al., 2010 water as a result of constant disturbance Zooplankton Body size Large body size Large taxa will benefit from Barnett et al., 2007; Hart and increased aquatic habitat Bychek, 2010 Functional feeding Filter-feeders Filter feeders will benefit from Barnett et al., 2007 group greater suspension of organic matter Riparian vegetation Tissue flexibility Flexible tissues Taxa with flexible tissues could Bornette et al., 2008; Merritt et al., better withstand stress 2010; Bornette and Puijalon, from high flows 2011 Vascular system Arenchymous tissue Taxa with arenchymous tissue Blom and Voesenek, 1996; can better transport oxygen Merritt et al., 2010 when submerged Water demand High water demands Taxa with high water demands Merritt et al., 2010; Stromberg will increase with increased et al., 2010 water availability conditions and biota requirements. Water quality require- with wastewater discharge (e.g., see Calera Creek case study ments for existing augmentation projects, such as those below), but are insufficient for the design of projects aimed at specified in national pollutant discharge elimination system stream renewal. For example, current regulations for con- (NPDES) permits, provide the context for the current level of taminants are limited to addressing known priority contam- regulatory guidance aimed at minimizing harm associated inants with high ecotoxicological and human health risks. Table 4. Important Water Quality Parameters and Typical Limitations for Recycled Water Used in Streamflow Augmentation

Example tertiary treatment Calera Creek treatment options for recycled water Typical recommended plant limitationsb use in streamflow Parameter Remarksa rangea for recycled water augmentationa,c

Chlorine Disinfectant for water treat- Total Cl2 < 0.1 mg/L UV disinfection Dechlorination (residual residual ment, but exhibits toxicity (dechlorinated) appliedd remains); UV disinfec- in many fish species. tion as an alternative TDS Measure of salinity. May be Subject to NPDES Regulated as an inland Electrodialysis; distillation; toxic to aquatic species. compliance freshwater stream ion exchange; nanofiltra- Compliance may be diffi- by the Basin Plan tion; reverse osmosis cult due to high TDS in source water. TSS Conventional water quality < 20 mg/L avg. annual < 10 mg/L (10 NTU Chemical coagulation/ indicator. turbidity instanta- filtration; membrane neous max.) bioreactor; advanced oxidation processes DO Requirements should be DO ‡ 5 mg/L DO ‡ 7.0 mg/L in Discharge methods that based on the most sensi- receiving waters introduce turbulence as tive species. well as modification of flow and stream mor- phology can enhance aeration.

Organic matter Degradation of organic BOD < 20 mg/L BOD5 < 10 mg/L (5-day Carbon adsorption; unit- matter, as measured by BOD at 20C) process wetlands; soil BOD, can deplete DO. aquifer treatment Nutrients May cause eutrophication Total nitrogen < 3 mg/L Ammonia-nitrogen: Selective ion exchange; leading to nuisance algal Ammonia < 2mg < 2mgNH3-N/L, overland flow; biological growth and oxygen NH3-N/L dry season; < 5mg nutrient removal; chemi- depletion. Unionized Total phosphorous NH3-N/L, wet season cal precipitation; unit- ammonia is toxic to < 1 mg/L Basin Plan requires process wetlands aquatic life. unionized ammonia: < 0.025 mg NH3-N/L in receiving waters Temperature Important for sensitive – 2.8C(– 5F) of Regulated in CA Riparian vegetation can fish species. ambient stream Thermal Plane shade stream to lower the water temperature water temperature; sub- surface cooling in pipeline or via infiltration (indirect discharge) to stream. Inorganic and Metals and other priority Varies 0.017 lg/L Hg Advanced oxidation pro- organic trace pollutants are regulated 6.0 lg/L Bis(2-ethyl- cesses; ozonation; carbon constituents by Clean Water Act pro- hexyl)phthalate adsorption; chemical visions (e.g., NPDES per- 3.2 lg/L Pb precipitation; nanofiltra- mits).f The ecotoxicity of 4.5 lg/L CN tion and reverse osmosis; many unregulated trace 10 lg/L Cu unit-process wetlands organic chemicals remains uncertain. Pathogens Bacterial indicators (total 23 coliform MPN/ < 200 MPN/100 mL Chlorine; UV disinfection; and fecal coliform) typi- 100 mL (avg.) 5-sample geometric membrane filtration cally regulated for recrea- 240 coliform MPN/ mean tional waters. May be 100 mL (max.) present in the surface waters due to wildlife.

aAdapted from Asano et al. (2007). bAverage monthly value, unless specified. Effluent or receiving water limitations from the California Regional Water Quality Control Board, San Francisco, Bay Region, Order No. R2-2006-0067, NPDES permit No. CA0038776. cAdapted from USEPA (2004). dChlorine residual prohibited by the U.S. Fish & Wildlife for restored Calera Creek Wetlands. eWater Quality Control Plan for Control of Temperature in the Coastal and Interstate Water and Enclosed Bays and Estuaries of California (SWRCB, 1975). fExample compounds shown for Calera Creek: water quality criteria and water quality objectives for priority pollutants in the receiving waters are based on the Water Quality Control Plan for the San Francisco Bay Basin (Basin Plan), the California Toxics Rule, and the National Toxics Rule. BOD, biological oxygen demand; DO, dissolved oxygen; NPDES, National Pollutant Discharge Elimination System; TDS, total dissolved solids; TSS, total suspended solids; UV, ultraviolet.

463 464 BISCHEL ET AL.

Further research is required to assess the unknown risks Advanced or tertiary-treated recycled water can have as of other potentially toxic wastewater-derived pollutants much as a 20C temperature variation between winter and (Daughton and Ternes, 1999) to develop guidelines for summer (Abdel-Jawad et al., 1999), so temperature may re- recycled water use in stream renewal (Anderson et al., 2012). quire engineered controls to achieve final stream tempera- Development of enhanced stream augmentation project de- tures within an established range, depending on the sign criteria that are based on coupled stream system flow– ecosystem requirements and designated uses. The cost asso- quality interactions would be valuable. ciated with controlling temperature for restoration could be Technology options for tertiary or advanced treatment in a significant, influencing decisions about whether to restore wastewater reclamation treatment process depend on the native species or consider habitat creation for species tolerant form of reuse and the locally identified priority contaminants to a greater thermal range. Recycled water or in-stream tem- (USEPA, 2004). For streamflow augmentation, some pro- peratures may be actively modulated by the use of heat ex- cesses such as nitrification for ammonia removal will be es- changers (Shah et al., 2000) and flow volume control (Plumlee pecially important, whereas others may be avoided, as was et al., 2012) or passively through heat loss in constructed chlorine disinfection in the case of Calera Creek. If the re- wetlands (Steinmann et al., 2003), shading in forested riparian cycled water quality is anticipated to be inadequate based on zones (Osborne and Kovacic, 1993; Sinokrot and Stefan, 1993), initial water quality analysis, additional treatment may be and indirect discharge into the stream via the subsurface considered either at the main treatment facility (i.e., centralized (Crites et al., 2000; Lancaster et al., 2005; Dallesport, 2007; treatment) or near the stream release site (i.e., decentralized Stewart, 2010). For example, a coupled wetland–indirect dis- treatment), although advanced treatment technologies are not charge system under consideration for temperature mitiga- necessarily required for streamflow augmentation projects. tion of treated wastewater discharged to the Willamette River Additionally, facilities developing recycled water programs (Oregon, USA) is predicted to reduce the discharge temper- for other purposes can incorporate ecological enhancement ature by up to 5C in the constructed wetland, followed by up into facility master planning such that treatment processes to 2C in the subsurface, allowing the discharger to meet adopted for planned uses also allow ecosystem renewal as an regulatory temperature limits (Corvallis, 2011). additional option (e.g., leading to selection of ultraviolet [UV] disinfection instead of chlorination). Nutrients. Pollution of surface waters by nitrogen (as NH3, + - - Three common issues and their treatment options are dis- NH4 ,NO3 ,andNO2 ), phosphorous in its soluble and cussed in more detail below: temperature, nutrients, and trace particulate forms (Vitousek et al., 1997; Carpenter et al.,1998; metals and organic contaminants. With respect to water Correll, 1998), and other nutrients in wastewater has had chemistry, these factors are consistently associated with urban widespread ecological impacts. Excess nutrients in effluent- stream degradation (Walsh et al., 2005; Brooks et al., 2006) and dominated ecosystems alter ecosystem dynamics (e.g.,lowered are key potential hindrances to the reuse of water for primary-to-bacterial production ratios) and are a common streamflow augmentation. cause of eutrophication (Vitousek et al., 1997; Carey and Mi- gliaccio, 2009; Waiser et al., 2011b). Nitrogen as ammonia Temperature. The ecological significance of water tem- (NH3) is particularly toxic to aquatic life (Passell et al., 2007). perature is widely recognized (Magnuson et al., 1979; Poole Temperature- and pH-dependent regulation of ammonia is and Berman, 2001; Caissie, 2006). The daily cycle of stream established by the National Ambient Water Quality Criteria, water temperature is modulated by natural processes, in- which accounts for greater sensitivity of freshwater mussels cluding incident solar radiation, atmospheric heat exchange, and fish early-life stages (USEPA, 2009), and state or regional and hyporheic zone processes (Nilsson and Renofalt, 2008). authorities may establish stricter criteria. For example, the San Temperature cycles influence streambed hydraulic con- Francisco Regional Water Quality Control Plan (Basin Plan) ductivity and stream–groundwater exchange, chemical mass- requires nonionized ammonia (NH3) in receiving surface wa- transfer and transformation rates, metabolic processes, ters to remain < 0.025 mg NH3-N/L (RWQCB, 2010). productivity of stream biota, and suitability of habitat for Conventional secondary biological treatment processes do aquatic life (Nimick et al., 2011). not remove total nitrogen (TN) or total phosphorus (TP) to a Due to domestic warm water additions, wastewater ef- significant degree. However, more than 40 alternative bio- fluent is usually higher in temperature than that of the water logical and chemical technologies for nitrogen and phospho- supply or receiving stream (Kinouchi et al., 2007). Most rus removal in municipal wastewater treatment are available, aquatic organisms exhibit tolerance to a specific temperature with annual average concentrations of £ 0.1 mg/L for TP range (Coutant, 1977), and the thermal tolerance of fish is and £ 3 mg/L for TN reliably achievable. Due to technological, typically used to develop criteria and set water quality regulatory, and cost considerations, biological nitrification– standards (Welch and Lindell, 1992). High water tempera- denitrification processes are generally preferred over physical– tures in receiving streams decreases dissolved oxygen solu- chemical nitrogen removal such as air stripping or ion bility, creating adverse habitat conditions for cold-water exchange (USEPA, 2008). Nitrification for ammonia removal fisheries, and can lead to an increase in the presence of warm- can improve the water quality of the effluent before in-stream water predators (Caissie, 2006; Spellman, 2011). Similar to application and lower nitrogenous oxygen demand (Asano environmental flows that attempt to mimic natural flow re- et al., 2007). Installation of nitrification/denitrification systems gimes (Acreman and Dunbar, 2004; Arthington et al., 2006), is increasingly common because of the adverse impacts of recycled water temperature should be managed to maintain nitrogen (Schmidt et al., 2003; Pehlivanoglu and Sedlak, 2004; the riverine thermal regime (which naturally vary within a USEPA, 2008). range) based on species present in the system or that are an- Vegetated buffer strips employed in riparian zones and ticipated to return. wetlands can lower nitrate concentrations from nitrified RENEWING URBAN STREAMS WITH RECYCLED WATER 465 effluent as well as nonpoint sources as a secondary method to the occurrence of other unknown TrOCs. Additionally, envi- source control (Barling and Moore, 1994; Vought et al., 1994; ronmental risk assessments that characterize TrOC hazards, Vymazal, 2007). Use of constructed wetlands for ammonia exposure, and effects can be used to identify specific com- removal has increased, for example, with reliable treatment pounds for potential monitoring and management (Knacker demonstrated for an array of wastewater sources and volumes et al., 2004). In adopting an approach for TrOC management in in diverse climates (Vymazal, 2007). Reconstructed stream recycled water stream augmentation, project managers must features (e.g., gravel bars and re-meandered stream channels) identify water quality criteria that will drive decisions re- have also been shown to significantly lower nitrate concen- garding an appropriate level of treatment for targeted waste- trations in the hyporheic zone (Kasahara and Hill, 2007), al- water-derived organic compounds. However, early adopters though hyporheic zone flow is often negligible compared to of stream renewal will likely encounter unanticipated chal- total stream flow. Additional research on methods to increase lenges regarding water quality, and further research is needed flow through the hyporheic zone may prove to be fruitful. to identify which unregulated contaminants merit additional Attenuation of nutrients may also be achieved via soil aquifer monitoring and potential mitigatory action. Responding to treatment in indirect augmentation (Debroux et al., 2012). such uncertainties regarding the risk of unregulated TrOCs, in this case perfluoro-octane sulfonate detected in the source- Trace metals and organic contaminants. Risk assess- recycled water, a California utility cancelled its research ments of trace metals are complicated in that some metals are demonstration streamflow augmentation project in Coyote essential nutrients for organisms at low levels while toxic at Creek (Plumlee et al., 2008, 2012). A planned recycled water higher concentrations (Reiley, 2007). Further, metal speciation streamflow augmentation project for the Hillsborough River in and complexation with other organic or inorganic ligands Tampa, FL, was similarly cancelled in part due to uncertainty have long been recognized to affect compound bioavailability regarding TrOCs (Latino and Haggerty, 2007). (Tessier and Turner, 1995). Monitoring and control of trace Detection of many TrOCs in effluent-dominated rivers is metals in streams augmented with recycled water will likely dependent on the degree of treatment employed (Ramirez be dictated by regulatory authorities, and are particularly et al., 2009). Some conventional technologies may have cobe- influenced by site-specific stream conditions (Brooks et al., nefits for removal of TrOCs, including pharmaceuticals and 2006). Recently, the Biotic Ligand Model (BLM) has been used personal care products (POSEIDON, 2004), though the effec- to incorporate receiving water body characteristics, such as tiveness of many municipal wastewater treatment technolo- the presence of competing cations and expected metal speci- gies for the removal of wastewater-derived TrOCs remains ation, in developing site-specific water quality criteria, and largely unknown. Tertiary or advanced treatment process predicting metal bioavailability (Reiley, 2007). The BLM was schemes may be selected for removal of TrOCs depending on used, for example, in updating nonregulatory aquatic life- the chemical of interest. For example, endocrine-disrupting ambient freshwater quality criterion recommendations for compounds such as steroid-derivative estrogens are of con- copper (USEPA, 2007). Further research is required to expand cern with respect to the ecotoxicological risk. Some endocrine- the BLM to other metals and to evaluate chronic exposure disrupting compounds may be removed in part via biological toxicity (Reiley, 2007; USEPA, 2007). degradation (in membrane bioreactors or sequencing batch While regulatory measures limit the allowable concentra- reactors [SBR]), advanced oxidation processes, membranes, tions of trace metals and some priority organic contaminants sorption to activated carbon, or electrochemical methods in wastewater (including recycled water) that is discharged to (Basile et al., 2011). surface waters, other unregulated compounds (e.g., trace or- TrOCs may also undergo natural attenuation in effluent- ganic chemicals [TrOCs]) may also pose risks to aquatic life dominated wetlands and rivers due to a combination of (Kolpin et al., 2002; Barber et al., 2006; Brooks et al., 2006; Wang photolysis, microbial degradation, and sorption (Gross et al., et al., 2007; Waiser et al., 2011a). Dickenson et al. (2011) provi- 2004; Gurr and Reinhard, 2006; Fono et al., 2006; Conkle et al., des several examples of urban streams that contain concerning 2008; Pal et al., 2010), although these processes are generally levels of TrOCs. Notably, estrogenic effects detected in fish slower and less effective than natural or engineered subsur- exposed to WWTP effluent were linked to natural and syn- face systems (e.g., soil aquifer treatment) (Drewes et al., 2008). thetic hormones, including 17b-estradiol, 17a-ethynylestra- When considering TrOC removal, processes that lead to de- diol, and estrone in effluent-dominated waters (Purdom et al., struction of the target compounds are preferable to technol- 1994; Desbrow et al., 1998). Gonadal intersex, impaired ovarian ogies that transform the constituent (e.g., chlorination), which and testicular histopathology, and other deleterious effects on may produce more toxic compounds, or that transfer them, fish downstream of a WWTP in Boulder Creek, CO, were at- which leads to disposal challenges (e.g., reverse osmosis). tributed to estrogenic wastewater compounds (Vajda et al., 2008). Many TrOCs remain untested for their potential eco- Ecosystem services toxicological effects, although modeling techniques will be useful to predict wildlife exposure at a river catchment scale While potential ecological benefits are a major driver for without time-intensive field approaches (Sumpter et al.,2006). enhancement of freshwater ecosystems, consideration of Attempts to narrow the suite of compounds that should be economic benefits is highly important in a watershed man- monitored, and possibly treated, in recycled water down to a agement context. Economic benefits provide justification for range of representative, or indicator, compounds or treatment public agencies to allocate limited financial resources toward performance surrogates [such as those reported by Drewes environmental stewardship. Quantification of how much the et al. (2008)] would be valuable for the management of water public is willing to pay for ecosystem enhancement, which reuse for streamflow augmentation. The presence or absence of has not been typically expressed in monetary terms in the a narrow, but diverse, suite of indicators may be used to infer past, can help water resource managers identify both the 466 BISCHEL ET AL. magnitude and distribution of benefits of these projects to the re-regulation of Glen Canyon Dam in Arizona (Loomis, the public. 1998). However, this type of analysis for the use of recycled Ecosystems provide society with services that have value. water for environmental flows has not been thoroughly Although ecosystem services can be defined in different ways explored. [compare those reported by Boyd and Banzhaf (2007), Cost- Recycled water is a market good, although with typically anza et al. (1997), Daily (1997), and Fisher et al. (2009), for ex- lower market value than pristine river water, which is com- ample], the widely utilized Millennium Ecosystem Assessment monly used for a variety of applications, including landscape definition is adopted for use in this review, referring to eco- irrigation, agriculture, groundwater recharge, other non- system services as the benefits humans obtain from ecosystems, potable uses, and indirect potable reuse. The use of recycled including the categories of provisioning, regulation, cultural, water for streamflow augmentation may generate value for and supporting services (Millennium Ecosystem Assessment, society that far exceeds value from either competing uses (e.g., 2005). These services include all processes that are necessary to agricultural irrigation) or, in some cases, complete lack of use sustain human life on a large scale (e.g., nutrient cycling, soil (e.g., via discharge directly to the ocean). However, the ben- stabilization, and climate regulation) and those that affect hu- efits of environmental flows are often distributed broadly mans more directly (e.g., water purification by forested wa- (e.g., to the public), whereas benefits of consumptive uses are tersheds and flood damage mitigation by coastal wetlands) more concentrated (e.g., by private interests or local water (Brauman et al., 2007). In particular, humans derive a number of agencies). Distributed financing mechanisms may be feasible benefits from water-related ecosystem services (Fisher et al., to address this challenge. For example, the local population in 2009). Increasing stream flow in a river or improving water the Segura River Basin in southeastern Spain was willing to quality in a lake can result in improved recreational opportu- almost double the wastewater treatment charge of their water nities—including hiking, fishing, bird watching, and white- utility bill to maintain sufficient stream flow in the Segura water rafting—as well as improved natural aesthetics and River, a component of achieving good ecological status under habitat for threatened or endangered species (Table 5). the European Water Framework Directive (Alcon et al., 2012). While the benefits derived from increasing stream flow or While the Segura River Basin study provides a good example improving lake water quality may be easy to observe (e.g.,as of the economic value of using recycled water for ecosystem an increase in the number of visitors to a lake or stream or in renewal, there is a need for additional studies demonstrating the diversity and abundance of wildlife observed at a lake or the value of water reuse for ecosystems in other management stream), it is more difficult to assign these benefits a dollar contexts and environmental conditions. value for a benefit–cost analysis. However, economists have developed nonmarket valuation techniques to assign dollar Services provided by streamflow augmentation. Ecosystem values to goods and services that cannot be traded in a typical valuation case studies of different projects illustrate the po- market (Wilson and Carpenter, 1999). The most popular tential for increased streamflow to provide value under a techniques used for water-related ecosystem services include variety of circumstances (Table 5). A number of valuable the contingent valuation method (Loomis, 1987; Loomis, freshwater ecosystem end services can be created or renewed 1998), the hedonic price index method (Bark-Hodgins and by augmenting streamflow, including creation of habitat, Colby, 2006), and the travel cost method (Duffield et al., 1992; provision of recreational opportunities, increased water in- Loomis and Creel, 1992; Weber and Berrens, 2006). The con- filtration to aquifers, improved water quality, and enhanced tingent valuation method involves conducting a survey to biodiversity. Quantification of such values suggests potential assess how much individuals are willing to pay for a hypo- mechanisms for financing water supply contracts or recycled thetical environmental improvement. The hedonic price water infrastructure to access new water sources. For exam- method relies on using a market commodity, such as real- ple, based on hedonic-pricing indices, reduction of ground- estate prices, as a surrogate for environmental quality. Lastly, water withdrawals and investment in riparian habitat the travel cost method uses the cost of travel as an approxi- restoration would significantly increase property values near mation for the value of an environmental amenity. riparian corridors in the Sonoran Desert of Arizona, and thus increase property tax revenue (Bark-Hodgins and Colby, Potential benefits of water reuse for ecosystems. Environ- 2006). The economic benefits of increased streamflow for rec- mental flows require allocation of water to streams with an reational opportunities are especially pronounced (Table 5). appropriate magnitude, frequency, duration, timing, and rate Angling and whitewater-rafting activity were found to esca- of change. These flow regimes can include managed flood late as a result of increased dam releases (Duffield et al., 1992; releases to reproduce benefits of floodplains such as nutrient Loomis and Creel, 1992), and unimpaired flows in a pristine cycling and off-channel habitat. Historically, in-stream flow environment were valued at a premium because of aesthetic provisions based on minimum-flow requirements (e.g., 10% of enhancement (Weber and Berrens, 2006). Aesthetics and rec- the mean annual flow) were often used instead of more eco- reational value, quantified using contingent valuation, com- logically based environmental-flow provisions. However, the prised a significant fraction of benefits ($560–$1100 per foot) use of minimum-flow regimes is not typically economically conferred by urban stream restoration in Baltimore, MD efficient. In many cases, the marginal value of environmental (Kenney et al., 2012). The preservation of Mono Lake in Cali- flows in excess of the minimum-flow requirement (or the fornia’s Sierra Nevada Mountains was considered valuable opportunity cost of reduced flows) far exceeds marginal enough for the city of Los Angeles to seek an alternate source economic benefits of competing uses of the water, such as for of water (Loomis, 1987). agriculture (Katz, 2006). Economic valuations of environ- Despite their value proposition, ecosystem services pro- mental flows have led to major policy changes, such as the vided by augmenting streamflow with recycled water are provision of increased flows to Mono Lake in California and often left unquantified. The San Antonio River, whose flow Table 5. Examples of Ecosystem Valuation Studies Conducted for Streamflow Augmentation Projects or Scenarios

Ecosystem benefit Study (valuation method) Description Valuation Policy change

Montana’s Big Hole Provision of fish for angling Estimated the economic value of Net increase of $10–20 per acre-foot Results indicate that gains may be and Bitterroot Rivers (Travel Cost Method) stream flows for the Big Hole and for stream flows during low flow achieved at many flow levels by (Duffield et al., 1992) Bitterroot rivers. compared to irrigation. reallocating water from con- sumptive use to stream uses. San Joaquin and Stanislaus Provision of fish for angling Survey of California households to Net increase of $70 per acre-foot for Study supports potential to renew Rivers (Loomis and Creel, (Travel Cost Method) determine trip frequency changes stream flow versus consumptive contract for minimum flows from 1992) due to increased flows in San use. the Friant Dam of the Federal Joaquin and Stanislaus Rivers. Central Valley Project. Sonoran Desert Conservation Aesthetic Value (Hedonic Estimated increase in property Property premiums estimated at Results demonstrate that urban Plan, AZ (Bark-Hodgins and Price Indices) values and property tax $126.54–$253.08 million; riparian habitat restoration can be Colby, 2006) associated with healthy riparian $1.23–2.46 million per annum in self-supporting and indicate corridors compared to the cost of incremental property tax revenue. potential benefits to modifying providing flow for water- Cost of supply water is well-spacing rules in Arizona. dependent habitat. $0.54 million annually. 467 Arizona’s Aravaipa Wilderness Recreation and camping Estimated the recreational value of Consumer surplus per visitor day Study indicates that surface water (Weber and Berrens, 2006) (Travel Cost Method) maintaining stream flows to the values estimated at $25.06 and ecosystems have significant Aravaipa Wilderness from cost of $17.31 (in 2003 dollars), for two nonuse value. The public will pay travel to visit site. separate access sites. Total net a premium for the remoteness of a present value of wilderness esti- site. mated at $3.6–4.7 million. Mono Lake in Southern Bird habitat and biodiversity Survey of California households Value of bird habitat estimated at Los Angeles’s water rights California (Loomis, 1987; (Contingent Valuation estimated the value of increase $1.3 billion, over 50 times the cost decreased by half to allow for Loomis, 1998) Method) flows into Mono Lake. of developing an alternative water increased flows into Mono Lake. source. Reregulation of Glen Canyon White-water rafting (Contin- Survey of white-water rafting Increased base flows represented a Study-opened discussion for the Dam, AZ (Loomis, 1998) gent Valuation Method) companies estimated value of potential increase of $2 million eventual re-regulation of Glen increased flow through Glen annually in rafting activities. Canyon Dam to mimic natural Canyon Dam. flow regimes Segura River, Segura River Water Framework Directive Survey of individuals estimated Respondents were willing to Study demonstrates that willingness Basin, Spain (Alcon et al., good ecological status increased willingness to pay for increase their household water bill to pay for increased river flow 2012) (Contingent Valuation using recycled water to augment by an average of e63.72 annually, using recycled water is larger than Method) stream flow in the Segura River. amounting to e24.1 million of the costs of recycled water in the annual benefits. Segura River Basin. 468 BISCHEL ET AL. augmentation using recycled water began in 2000 after a se- plant handles an average daily dry weather flow of 4.0 MGD vere water quality decline in regional streams and litigation to and releases all of this effluent through a constructed wetland maintain flows for endangered species protection, provides a into Calera Creek, which then runs a length of about one-half picturesque setting for a vibrant commercial area in down- mile (*900 m) before reaching the Pacific Ocean (Fig. 2). The town San Antonio known as the Riverwalk (Eckhardt, 2004). City of Pacifica selected the current location for its treatment Yet, no studies have comprehensively evaluated ecosystem plant in 1995, responding to increased capacity needs for services provided by this effluent-dominated system. Simi- wastewater treatment that could not be met by the former larly, the Las Vegas Wash in Nevada, comprising a 12- treatment facility (Pacifica, 1997). Replacement of the treat- mile-long effluent-dominated stream and associated wetlands ment plant was accompanied by restoration of *9 acres (4 ha) flowing from Las Vegas to Lake Mead, sustains numerous of wetlands (below the mean high water level) in a former ecosystem services. Despite major historical changes in eco- rock quarry and *20 acres (8 ha) of additional stream bank system dynamics, the system provides a riparian habitat for and upland buffer areas (RWQCB, 2006; S. Holmes, personal migratory birds and other wildlife, wastewater treatment communication, January 4, 2012). Recycled water releases for capacity through natural processes, and recreational oppor- ecosystem benefits began in 2002 under an allowance for a tunities for residents (Stave, 2001; Adhikari et al., 2011). shallow-water discharge from the local regulatory authority Quantification of such positive externalities can be an im- that eliminated the use of an offshore outfall. The project re- portant tool for incentivizing the maintenance and enhance- sulted in a return of the lower stream channel of Calera Creek, ment of ecosystems threatened by human development and which was formerly channelized and diverted from its his- for examining the economic, social, and environmental tra- torical course, to its natural path. This relatively small-scale deoffs of recycled water application for stream renewal. water reuse project exemplifies a multiuse stream improve- ment project that provides various forms of both environ- Case Study mental and social benefits.

Urban stream renewal in Calera Creek Project planning and design. In initial project planning, (City of Pacifica, CA) navigating the regulatory framework for stream enhancement Project setting. The City of Pacifica’s Calera Creek Water using recycled water presented a challenge, because the cre- Recycling Plant is located just south of San Francisco, CA, and ation of an inland outfall was a new concept for a coastal serves a population of 39,000 people. The tertiary treatment treatment facility (S. Holmes, personal communication,

FIG. 2. The restored Calera Creek meets the Pacific Ocean (bottom) after streamflow mixes with tertiary-treated effluent from the Calera Creek Water Recycling Plant (top). RENEWING URBAN STREAMS WITH RECYCLED WATER 469

January 4, 2012). The tertiary effluent was required to meet degraded stream conditions that existed before the restoration Title 22 standards (CDPH, 2010) for an unrestricted use at the presented an opportunity to recreate a lost habitat for both the point of discharge into Calera Creek, such that polishing endangered San Francisco Garter Snake (Thamnophis sirtalis treatment from the wetland and creek could not be used to tetrataenia) and the threatened California red-legged frog meet the treatment requirement. Nevertheless, improving (Rana arora draytonii). The red-legged frog population has in- water quality by increasing the residence time of water creased dramatically from several adult and juvenile indi- within the wetland ecosystem was an initial project objective viduals recorded in the year 2000 at baseline conditions (Pacifica, 2007), and local geology dictated placement of the (Pacifica, 2000) to high population levels documented in the constructed wetland (S. Holmes, personal communication, year 2009 (e.g., > 40 individuals recorded in a single survey of January 4, 2012). Project planning also involved navigating the ponds onsite) (Pacifica, 2010). Plants have also benefited permitting requirements from four agencies that have juris- from the use of recycled water. Dense growth of native ri- diction over the waters and wetlands impacted by the project: parian trees and shrubs, such as willows (Salix sp.) and the San Francisco Bay Region California Regional Water horsetails (Equisetum arvense), is occurring along the stream. Quality Control Board (Regional Board), the U.S. Army Corps The rapid growth of native plants helps control invasion of of Engineers, the California Department of Fish and Game, non-native plants adjacent to the stream to some extent, al- and the California Coastal Commission (Pacifica, 2010). though weed control has been a challenge (Pacifica, 2007), Using a hydrogeomorphic method, comparable streams particularly because of regulatory requirements that must be and their riverine wetlands along the California coast were met to ensure that no harm is done to the now-resident red- sampled to provide a reference framework for developing legged frogs that are protected under the Endangered Species design criteria for the functions of the restored Calera Creek Act. The San Francisco Garter Snake has not yet been ob- wetland in terms of hydrology, biogeochemistry, plant com- served in this stream ecosystem (Pacifica, 2010), and no native munity, and habitat (Pacifica, 1997). The planners of the res- fish were documented in the stream. However, if a fish pop- toration sought to create a relatively natural flow regime that ulation does become established, particularly of an endan- would accommodate additional discharge from the water gered species, additional regulatory requirements could be recycling facility while establishing a compositionally and imposed at a much higher burden for plant operations and structurally complex ecosystem (Pacifica, 2007). Un- maintenance. Additionally, benthic macroinvertebrate com- fortunately, historical hydrographs were unavailable for this munity analyses were not conducted to document and eval- stream, and thus the natural flow regime can only be ap- uate changes in Calera Creek after augmentation. proximated through inference. A detailed monitoring plan included yearly characterization of stream biological and Ecosystem services. An inland outfall coupled with a physical habitat, vegetation density, and water quality over a restored creek system avoided costly maintenance (ca. > $100 5-year period after implementation (Pacifica, 1996). k/yr) and uncertain performance experienced with an ocean outfall at the former facility. In fact, approval with the local Water quality and treatment. The treatment facility com- City Council was largely an economic decision based on the prises screens at two pump stations, grit removal, SBR for pri- tradeoffs between a new inland outfall and treatment facility mary and secondary treatment and nutrient removal, sand with nutrient removal and UV disinfection versus a replace- filters, and UV disinfection (RWQCB, 2006). A cascade outfall ment offshore pipeline with expanded secondary treatment aerates the effluent as it enters the wetland. Several numeric capacity at the former facility (S. Holmes, personal commu- water quality NPDES permit limits are summarized in Table 4. nication, January 4, 2012). Selection of the appropriate level of SBR was the preferred treatment technology to produce high- treatment for stream enhancement may increase treatment quality effluent and accomplish nutrient removal with a facility costs relative to ocean disposal. However, in this case, mechanically simple process in a minimum number of steps avoided maintenance costs with an outfall were recognized as (Pacifica, 1997). Forested wetlands were also selected for shad- important savings. Invasive plant species (e.g., pampas grass) ing to minimize growth of filamentous algae in the stream from control remains an ongoing cost for wetland maintenance. In high nutrient loads. Despite large energy use, production of addition to serving as an outfall alternative, the newly created tertiary-treated recycled water with UV disinfection eliminates wetlands provide a habitat for locally endangered and risks associated with chlorine residual and the formation of ha- threatened species. An actively used, paved walking/biking logenated disinfection byproducts (RWQCB, 2006; S. Holmes, path now runs alongside the creek and provides a significant personal communication, January 4, 2012). Incomplete dechlo- additional recreational value to the local community. Specific rination as well as refractory inorganic and organic chloramine beneficial uses of the inland stream, including preservation of byproducts formed during chlorination can pose threats to rare and endangered species, creation of freshwater and aquatic life (Jameel and Helz, 1999; Bedner et al., 2004). Further, wildlife habitat, and contact and noncontact water recreation concern regarding safety in transportation and storage of chlo- (RWQCB, 2006), were not explicitly considered in economic rine gas as well as sulfur dioxide, which is commonly used for terms for the project. dechlorination, was expressed during project planning (Pacifi- ca, 1990). UV disinfection can be comparable to chlorination/ Discussion dechlorination in new facilities both in terms of costs and ef- Barriers to water reuse for ecosystems, research fectiveness (Blatchley et al., 1996; Wojtenko et al., 2001). needs, and envisioning success Ecological performance. Although Calera Creek was The comprehensive review of literature and case studies converted from a nonperennial to a perennial stream and is conducted suggests that recycled water use for ecosystem now effluent dominated in the summer months, the highly renewal through streamflow augmentation remains a largely 470 BISCHEL ET AL. unexplored research topic, which could have widespread The added energy and construction costs must be consid- applications in engineering, restoration, and aquatic ecology ered in planning such systems, but much progress is be- practice. Barriers identified for the development of water re- ing made toward producing energy-positive treatment use for ecosystems programs give rise to several core short- plants at relatively low cost (Verstraete et al., 2009). Upstream term and long-term research needs (Table 6), which span unit-process wetlands, or soil aquifer treatment during across diverse disciplines, including aquatic ecology, hy- indirect (subsurface) augmentation of streamflow, can also drology, environmental engineering, environmental man- provide decentralized tertiary treatment capacity with agement and planning, social science, law, and water resource lower energy investments [see Hoppe-Jones et al. (2010) economics. for example]. The motivations for individual stream renewal projects, Although water reuse projects may be driven by regulatory whether moral, regulatory, or financial, will directly inform requirements for wastewater discharge [such as those re- the site-specific design criteria. Both the natural flow regime ported by Bischel et al. (2012)], regulatory constraints and lack and water quality should be used to guide decisions on of incentives for wastewater treatment facilities to play a role whether streamflow augmentation is appropriate for a par- in stream restoration activities may also limit implementation ticular site. In many cases, augmentation may not be appro- of streamflow augmentation using recycled water. For ex- priate or may be appropriate only during particular seasons. ample, it is easier to reach agreement on effluent quality for Flexible designs that allow recycled water releases to be tai- discharge, and to measure chemicals in a pipe for an NPDES lored to natural variability will likely result in the greatest permit, than to achieve consensus on how to plan, monitor, ecosystem benefits. Distributed or satellite treatment plants and evaluate ecosystem status and restoration success. As that process water in lower volumes may allow tailoring both experienced at Calera Creek, the permitting process currently the water quantity and quality to the site (Latino and Hagg- used in California did not particularly lend itself to ecological erty, 2007), and installation of such plants closer to the restoration with recycled water. Current discharge regula- headwaters where many streams are most water-stressed (in tions focus on preventing the harm associated with water contrast to traditional downstream wastewater discharge) quality impacts rather than creating a positive ecological could provide greater benefits (Gengenbach et al., 2010). impact, and there is currently no regulatory framework

Table 6. Barriers to Water Reuse for Ecosystem Renewal and Research Needs

Barrier Short-term research or logistical need Long-term research or logistical need

Few successful water-reuse stream Select potential metrics for both Develop engineering practice guidelines augmentation cases implemented identifying project opportunities for urban stream renewal using recycled worldwide; little regulatory guidance and evaluating project success. water. Address habitat management Identify new opportunities at re- issues that may result from return of gional scales based on historical endangered species. Assess stream re- stream hydrology, water quality newal using recycled water as a miti- and ecosystem needs, and re- gation alternative for changing cycled water availability. population and climactic conditions. Available structural and functional Develop and select biological indi- Apply metrics to evaluate success of biological metrics not widely applied ces that are responsive to manip- streamflow augmentation under a range to relevant scenarios ulations in water flow and quality of environmental conditions. over varied time scales. Uncertainty regarding the impacts of Develop and utilize environmental Develop water quality guidelines specifi- wastewater-derived TrOCs risk assessments to characterize cally for implementing and permitting TrOC hazards, exposure, and ef- recycled water-based stream enhance- fects to identify indicator com- ment projects. pounds for potential monitoring and management. Lack of streamflow augmentation- Conduct proof-of concept demon- Conduct large-scale coupled flow-water controlled environmental flow stration studies to establish quan- quality experiments to provide insight experiments titative relationships between into the treatment needs for stream benthic invertebrate indices and renewal projects and anticipated bio- modified flow regimes. logical responses. Motivations to implement new projects Evaluate and apply appropriate Compare the public value of stream re- lack economic component; ecosystem ecosystem valuation methods to newal as a recycled water portfolio services valuation not applied widely case studies of recycled water for option with competing uses and incor- at the scales of individual projects stream renewal. porate analyses into recycled water master planning. Institutional impediments and uncer- Explore cost recovery mechanisms Establish key institutional partnerships tainties regarding financial sustain- and water rights ownership and and update regulatory processes to ability transfer for streamflow augmen- couple water-recycling production with tation. ecosystem renewal goals.

TrOCs, trace organic chemicals. RENEWING URBAN STREAMS WITH RECYCLED WATER 471 or guideline for implementing and permitting recycled be used in conjunction with water reuse programs to renew water-based stream enhancement projects. and regenerate ecosystems (Curry and Carson, 2008; Tom In addition to improving aquatic life habitat, stream res- et al., 2011). However, neither of these programs used alone toration in urban environments may be implemented to meet can rise to the challenge of addressing the increasing level of storm water total maximum daily load requirements under water scarcity in urban areas. Such programs will require the Clean Water Act and NPDES permits (Kenney et al., 2012), incremental changes in human behavior and views, which insofar as restored streams are better able to assimilate con- will take time and effort to achieve. Success will likely require taminants loads such as nutrients. Such an approach could be that water utilities, academic institutions, government agen- performed in conjunction with flow augmentation using re- cies, environmental nonprofit organizations, and other ad- cycled water. This type of watershed-scale approach for storm vocacy groups engage with one another to create institutional water pollutant reduction, which encourages enhancement of partnerships to garner public support and to fund, design, waterways throughout the watershed over end-of-pipe and implement water reuse for ecosystem benefit. strategies, requires integrated regional management, greater Advanced technologies, including higher levels of treat- institutional capacity, consistent regulatory oversight, and ment, energy capture from wastewater, desalination, and effective funding and market incentives (Roy et al., 2008). sensors that continuously monitor effluent volume and Low-quality wastewater historically released into streams quality could be utilized as components of ecosystem renewal either untreated or after only primary or secondary treatment projects and should be designed for applications in water has had dramatic negative effects on many aquatic ecosys- reuse for ecosystem enhancement. Water reuse could also be tems and has also been the cause of numerous public health used to mitigate the anticipated effects of climate change by concerns (Cooper, 1991; Carey and Migliaccio, 2009; Gran- augmenting altered flow regimes and thereby reducing sea- tham et al., 2012). As a result, a negative public perception has water intrusion. Although ecosystem renewal will require developed toward wastewater in many cases that are often management of hydrology and water quality, potential linked to concerns regarding the quality of the drinking water benefits to the environment and society as a whole could be supply (Robinson et al., 2005; Dolnicar and Scha¨fer, 2009; significant. Dolnicar et al., 2011). Although every case will be unique, typically recycled water use for stream renewal should be at Acknowledgments least tertiary treated (e.g., filtration and nutrient removal) to gain public support as well as to reduce risks associated with This work was supported by the National Science Foun- exposure to pathogens, nitrogen, phosphorous, suspended dation Engineering Research Center for Re-Inventing the solids, organics, and metals. Water temperature should also Nation’s Urban Water Infrastructure (ReNUWIt). We thank be managed to maintain levels at which the native fauna are David L. Sedlak (ReNUWIt and University of California– adapted, which will require some knowledge of historical Berkeley), Alexandria B. Boehm (ReNUWIt and Stanford conditions, or to support known species of concern in the University), Scott Holmes (former Director of Public Works, system. Ecosystem valuation studies could provide incentive Pacifica, CA), and Stephen D. Comello (Stanford University) for water utilities to initiate water reuse for ecosystem pro- for reviewing all or portions of the manuscript. jects, because they would allow water and wastewater man- agers to assess the value of the public benefits. Author Disclosure Statement Recycled water that is used to renew streams will eventu- No competing financial interests exist. ally re-enter the drinking water supply as it passes through the water cycle, though the distance it travels through the References water cycle as well as the amount of natural filtration and dilution that will occur are highly variable depending on the Abdel-Jawad, M., Ebrahim, S., Al-Tabtabaei, M., and Al- particular system (Weiss and Reemtsa, 2008; Vizintin et al., Shammari, S. (1999). Advanced technologies for municipal 2009; Musolff et al., 2010). 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