EMERGING CONTAMINANTS IN ECOSYSTEMS: NEW CHALLENGES FOR WATER REUSE IMPLEMENTATION AND MECHANISMS OF PERFLUOROCHEMICAL BIOACCUMULATION A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Heather Nicole Bischel August 2011
206
Embed
EMERGING CONTAMINANTS IN ECOSYSTEMS: …zs920pn1994/...EMERGING CONTAMINANTS IN ECOSYSTEMS: NEW CHALLENGES FOR WATER REUSE IMPLEMENTATION AND MECHANISMS OF PERFLUOROCHEMICAL BIOACCUMULATION
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
EMERGING CONTAMINANTS IN ECOSYSTEMS:
NEW CHALLENGES FOR WATER REUSE IMPLEMENTATION AND
MECHANISMS OF PERFLUOROCHEMICAL BIOACCUMULATION
A DISSERTATION
SUBMITTED TO THE DEPARTMENT OF
CIVIL AND ENVIRONMENTAL ENGINEERING
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Heather Nicole Bischel August 2011
http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/zs920pn1994
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Richard Luthy, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Martin Reinhard
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Laura MacManus-Spencer
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.
iii
iv
v
Abstract Technological innovations developed in response to pressing water supply needs in
populated arid regions have led to the recovery of municipal wastewater for beneficial
reuse worldwide. At a time when rapid urbanization, severe droughts, and public
concern introduce complex management challenges for water reuse, the persistence of
residual and byproduct pharmaceutical and industrial chemicals in treated municipal
effluent gives rise to new technological hurdles. Bioaccumulation of synthetic organic
chemicals in environments downstream of wastewater effluent discharge and recycled
water use poses an ecological risk and introduces a potential pathway of human
exposure to these contaminants. This dissertation assesses management challenges for
water reuse implementation in Northern California; identifies opportunities of water
reuse for ecosystem enhancement; explores the bioaccumulation of one class of
persistent and toxic unregulated chemical contaminants, perfluoroalkyl acids (PFAAs);
and evaluates mechanisms of bioaccumulation via an in-depth study of PFAA
interactions with a model serum protein.
Chapter 1 provides background and outlines research objectives governing this
dissertation. In Chapter 2, major factors that influenced the implementation of
nonpotable water reuse in Northern California are presented based on a survey of
program managers. Capturing experiences of managers in urban and peri-urban regions
of California provides context for the historical developments of water reuse and the
sources of barriers to implementation. Results demonstrate that in recent times, water
reuse is driven more often by water supply needs rather than by wastewater discharge
limitations. From a management perspective, economic issues stand as the largest
hindrance to successful project implementation, while negative perceptions of water
reuse less frequently inhibit nonpotable water reuse projects. Analysis conducted in
Chapter 3 indicates that while ecosystem protection goals are frequently drivers of
water reuse programs, few water reuse projects have been implemented in California
explicitly for ecosystem enhancement or wildlife habitat creation. Augmentation of
degraded wetlands with recycled water represents an opportunity for expansion of
inland water reuse programs. However, detection of persistent, unregulated
vi
contaminants in recycled water presents management challenges for these projects.
The ability to accurately predict the bioaccumulative potential of chemicals in
aquatic organisms is an essential component to assessing the human health and
ecological risk of trace micropollutants. The bioaccumulation of PFAAs presents a
particularly intriguing case. Unlike other persistent organic pollutants, PFAAs do not
preferentially accumulate in lipids and fatty tissue but rather in body compartments with
high protein content, including the liver, kidneys, and serum. In Chapter 4, PFAA
concentrations detected in the livers of white sturgeon from the San Francisco Bay are
presented as a brief study on the environmental prevalence and bioaccumulation of
these chemicals. A fugacity-based approach that utilizes protein-water distribution
coefficients (KPW) based on interactions with model proteins is introduced as a useful
parameter to characterize the bioaccumulation and in vivo bioavailability of PFAAs.
Based on this modeling paradigm, noncovalent interactions of long-chain perfluoroalkyl
acids with bovine and human serum albumins (BSA and HSA, respectively) are
characterized in Chapter 5. Results suggest binding through specific high affinity
interactions at low PFAA:albumin mole ratios. In an effort to reduce the
bioaccumulation of PFAAs in humans and wildlife, fluorochemical manufacturers have
recently shifted production to shorter chain-length compounds. In Chapter 6,
associations of perfluoroalkyl carboxylates (PFCAs) with 2 to 12 carbons (C2 – C12) and
perfluoroalkyl sulfonates with 4 to 8 carbons (C4, C6, and C8) with BSA and
physiochemical binding mechanisms are evaluated at physiologically relevant
PFAA:albumin mole ratios and various solution conditions using equilibrium dialysis,
nanoelectrospray ionization mass spectrometry, and fluorescence spectroscopy.
Measured log KPW values for C4 to C12 PFAAs confirm that protein associations as
characterized in this model scenario prove to be greater in magnitude for PFAAs than
lipid-based partitioning coefficients. Association constants determined for
perfluorobutanesulfonate and perfluoropentanoate with BSA are on the order of those
for long-chain PFAAs, suggesting that physiological implications of strong binding to
albumin may be important for short-chain PFAAs.
In the final chapter, conclusions are drawn for research objectives outlined initially,
and future research directions are identified. The presented evaluation of management
vii
challenges for water reuse implementation provides context for issues surrounding
chemicals of emerging concern (CECs) in recycled water. However, uncertainty
regarding bioaccumulation of CECs from recycled water used for direct habitat
enhancement or creation remains a concern. Investigation of mechanisms influencing
PFAA bioaccumulation provides insight into one class of CECs now detected in
sensitive aquatic ecosystems. As the elimination of one unsafe chemical does not
guarantee the safety of its commercial replacements, presented findings further
contribute to ongoing efforts to characterize the physiochemical properties and
anticipated environmental fate of compounds used to replace long-chain perfluorinated
chemicals.
viii
Acknowledgement First, I thank my advisor Richard Luthy for thoughtful mentoring, consistent
guidance, and for always challenging each of his students to keep the big picture in
mind. Dick is living reassurance that kindness and compassion go hand-in-hand with
effectiveness and impact – a wonderful role model to have. A special thanks to Laura
MacManus-Spencer for invaluable cross-coast collaboration (including many hours on
the phone and conference-roommate chats) and for a delightful friendship. I also thank
Martin Reinhard, Jim Leckie, and Buzz Thompson, for joining my dissertation
committee.
One of the most enjoyable components of my work has been the opportunity to
interact with and learn from so many extraordinary people. Many thanks to the entire
Luthy Research Group for plenty of support and encouragement; to: Chris, Laura, and
Pam for helping me get started in the lab; to YeoMyoung and Eunah – organic
chemistry extraordinaires – from whom I learned so much as teachers and teaching
partners; Sarah for office-time advice; Jay for mini-debriefs; Aude for her work and
grounded perspectives on water reuse for ecosystems; Jeanne and Diana for a bit of
tennis; Chinghong, Lilli, Sungwoo, Yuan, and Yongju for research advice with a smile;
and Niveen for summertime chats as I finished up.
I am also tremendously grateful to: Gregory Simon and Tammy Frisby for diving
right in with recycled water field trips and helping me view our work from different
perspectives; Sophie Egan for hours of meticulous work, and contagious enthusiasm;
the many water reuse professionals who volunteered their time to participate in our
project and answer our phone calls; the professors at Cal who helped set me on this path
(Go Bears!) and the ones at Stanford who opened up even more; my best bud (at
Stanford), Liv Walter, for giving me the keypad code and keeping her door unlocked;
my dear friend Blythe Layton for countless hugs and laughs; the LCMS csars of the past
for keeping it running; the EES moles– too numerous to name – for advice and beer;
Royal, of course, who always lent a helpful hand; EFML nerds, especially Liv, Joel,
Sarah, Erin, Jaime, and Yacoub for crossing the great divide for many meals and a few
hydrophobicity, perfluorinated compounds with greater than seven fluorinated carbons
bioaccumulate and biomagnify in aquatic food webs (18, 25, 38). Rather than
partitioning to adipose tissue, PFAAs are detected predominantly in protein-rich
compartments such as the liver, kidney and blood (39-42). The bioconcentration factor
(BCF) of PFOS, relating ambient water concentrations to measured tissue
concentrations, ranges from approximately 1,000 to more than 5,000 for bluegill and
rainbow trout fish species, depending on the method of determination and organ
considered (30). The structures of perfluoroalkyl carboxylates (PFCAs) and
perfluoroalkyl sulfonates (PFSAs), two homologue groups of PFAAs, resemble those of
fatty acids and hydrocarbon-based detergents, but the perfluorinated tail renders the
compounds both hydrophobic and oleophobic (16, 33). The nature of PFAA structure
and bioaccumulation suggests an importance of protein interactions (43). However, the
sorptive capacity of animal protein is rarely incorporated in biouptake models to
improve estimations of chemical distribution and bioaccumulation of persistent organic
pollutants (44).
6
1.2 Research Objectives and Précis
Amongst a broad array of challenges, negative public perception of health risks,
actions of influential stakeholders in a changing regulatory environment, and limited
availability of financial assistance may affect the implementation of water reuse
programs. High costs, including those for treatment facilities and distribution systems,
may be exacerbated by limited financial and technological capacity to eliminate trace
contaminants. In the face of new knowledge surrounding chemicals of emerging
concern, coupled with advanced analytical techniques to evaluate the presence of
chemicals, future challenges associated with water reuse programs in California may be
different than historical practices and experiences. Work presented in Chapter 2,
Management experiences and trends for water reuse implementation in Northern
California,1 assesses the greater context of water reuse in California and contains
results from a survey of water reuse managers and professionals in Northern California.
Chapter 3, Water reuse for ecosystem enhancement: Matching opportunity with need,
delves into the role of water reuse for ecosystems, describing existing programs and
broadly identifying opportunities for new enhancements. The analysis draws on
responses from the previously discussed survey and complementary databases to outline
specific challenges for habitat enhancement using tertiary treated wastewater. Together,
these chapters seek to provide insight on the following questions:
• What are the major drivers and barriers to water reuse in Northern
California, and how have these factors evolved through time?
• To what extent has water reuse been applied for the direct benefit of
ecosystems, and what major challenges are associated with the
implementation of water reuse for ecosystem enhancement?
Despite an indication that positive environmental impact is a benefit of water reuse
projects, relatively few projects have been implemented for ecosystem enhancement in
California.
1 The results presented in this chapter are submitted as a Research Article by Heather N. Bischel, Gregory Simon, Tammy M. Frisby, and Richard G. Luthy for the journal Environmental Science & Technology.
7
One potential challenge associated with the implementation of water reuse for
ecosystem enhancement is uncertainty regarding the bioaccumulation of unregulated
chemical contaminants. Chapter 4, Exposure of perfluorinated chemicals to San
Francisco Bay white sturgeon and mechanisms of bioaccumulation, sets the stage for
understanding the role of bioaccumulation of chemicals of emerging concern in
ecosystems as well as mechanisms associated with PFAA bioaccumulation. PFAA
concentrations detected in white sturgeon fish livers from organisms in the San
Francisco Bay are presented as a case study. As perfluorinated chemicals receive
increasingly more attention and are more carefully examined for their potential
ecosystem effects, bioaccumulation processes based on biologically relevant
mechanisms are considered. This chapter asks:
• What dominant processes govern the bioaccumulation of PFAAs, and how
can these processes be captured in bioaccumulation models?
As mentioned, PFAAs do not preferentially accumulate in lipids and fatty tissue but
rather in body compartments with high protein content, including the liver, kidneys, and
serum. Such observations bring question to the appropriateness of using octanol-water
partition coefficients (Kow), which are commonly applied for modeling the
bioaccumulation of persistent organic pollutants, to describe the environmental behavior
of PFAAs.
For PFAAs, molecular interactions with proteins likely contribute to PFAA
bioaccumulation mechanisms. As such, quantitatively determined associations between
perfluorinated chemicals and proteins may be useful parameters to more accurately
describe observed bioaccumulation. In Chapters 5 and 6, processes influencing the
bioaccumulation of these unique compounds are evaluated through associations of
PFAAs with proteins, utilizing bovine serum albumin as a model protein. These
chapters seek to address the following questions:
• How do long-chain PFAAs associate with the model protein, serum albumin,
at physiologically relevant PFAA:albumin mole ratios?
• What analytical tools are appropriate for quantitatively determining PFAA-
albumin associations?
• Given a shift in production of fluorinated compounds to shorter-chain length
8
compounds, how will a reduction in perfluoroalkyl chain length affect
protein-water distribution coefficients?
• What physiochemical mechanisms govern interactions of PFAAs with serum
albumin?
In Chapter 5, Investigating associations with a model protein: Noncovalent
interactions of long-chain perfluoroalkyl acids with serum albumin, 1 association
constants (Ka) and binding stoichiometries for PFAA-albumin complexes are quantified
over a range of physiologically relevant PFAA:albumin mole ratios. Binding
interactions between PFAAs with eight to ten perfluoroalkyl carbons and the model
protein bovine serum albumin (BSA) are studied using equilibrium dialysis with liquid
chromatography tandem mass spectrometry and nanoelectrospray ionization mass
spectrometry. Chapter 6, Strong associations of short-chain perfluoroalkyl acids
(PFAAs) with serum albumin and investigation of binding mechanisms2, expands on
the previous chapter to evaluate associations of PFCAs with 2 to 12 carbons (C2 – C12)
and PFSAs with 4 to 8 carbons (C4, C6, and C8) with BSA at physiologically-relevant
PFAA:albumin mole ratios. Protein-water distribution coefficients (KPW) are quantified,
providing interpretation of hydrophobicity, steric hindrances, and electrostatic effects
on interactions with albumin. This work comprises a thorough evaluation of molecular
interactions of PFAAs with albumin using several analytical tools, a wide range of
ligand and substrate concentrations, a series of fluorochemical chain lengths and two
anionic head group moieties, and varied solution conditions. Chapter 7, Conclusions,
contains final remarks on the research objectives as well as a discussion of research
needs.
1 The results presented in this chapter originally appeared as a Research Article in the journal Environmental Science & Technology: (45) Bischel, H. N.; MacManus-Spencer, L. A.; Luthy, R. G. Noncovalent interactions of long-chain perfluoroalkyl acids with serum albumin. Environ. Sci. Technol. 2010, 44 (13), 5263-5269. 2 The results presented in this chapter are in press as a Research Article for the journal Environmental Toxicology & Chemistry by Heather N. Bischel, Laura A. MacManus-Spencer, Chaojie Zhang, and Richard G. Luthy.
9
Chapter 2
Management experiences and trends for
water reuse implementation in Northern
California
2.1 Introduction
California is at the forefront of recycled water use, treating municipal wastewater to
a high enough degree that it can be returned to the water supply for a variety of
beneficial uses including landscape irrigation (46-48), agriculture (49, 50), ecosystem
enhancement (9), industrial cooling and processing (47, 51), groundwater recharge and
indirect potable reuse (51-53). From 1970 to 2002, reuse of municipal wastewater more
than doubled in California from 175,000 acre-ft per year (AFY) to approximately
525,000 AFY. Yet this growth fell short of the state’s goal to reuse 700,000 AFY by
2000 (2, 3). California’s goal to increase reuse by 2 million acre-feet by 2030 over 2002
levels (54) will require a portfolio of projects for a range of beneficial uses. Given
multiple failures to attain statewide recycling goals (Figure 2.1), questions remain as to
10
the sources of such difficulties as well as the feasibility of reaching near-term goals
described in California’s State Water Board Strategic Plan Update of 2008-2012 (55).
Figure 2.1. Timeline of statewide water recycling goals and production volumes, major
drought periods, and select water recycling laws and policies in California during the
implementation period for survey respondents. Refer to the Supporting Information for
a description of major laws and policies.
Despite efforts to encourage and support water reuse programs at the state and
federal levels (e.g., (2) and (54)), not all projects are successful, and nonpotable reuse
projects frequently fall short of planned delivery goals (56, 57). Public opposition has
led to the suspension or abandonment of several large water reclamation projects for
indirect potable reuse in California (2, 7). Considering the promise of recycled water for
augmenting water supplies in the West and pressing water supply concerns related to
dramatic population changes and climate change, assessment of past and current
experiences in water reuse implementation will aid in more effectively promoting,
evaluating, and implementing water reuse. This paper contributes to this task by
evaluating the experiences and perspectives of current water reuse project managers in
Northern California to understand recent developments and major issues confronting
11
recycled water projects in the region.
Specifically, our study reveals the following: (1) In Northern California, water reuse
programs are widely distributed across 48 counties and, though more numerous than
programs in the 10 Southern California counties, are often smaller in the volumes of
reclaimed water delivered annually. This finding highlights the importance of capturing
experiences of managers in rural regions of California, which likely differ from
experiences in highly urbanized centers. (2) Regulatory requirements that limit
discharge played an important role in motivating many water reuse programs in
Northern California. However, a trend away from reuse as a wastewater disposal issue
is documented in Northern California, as water supply and reliability become more
prevalent drivers of water reuse. (3) Although ecosystem enhancement or protection
goals are frequently cited as drivers of water reuse, such goals are rarely the most
important drivers for reuse programs. Few water reuse programs in California have
been implemented for the purpose of ecosystem enhancement. (4) Negative perceptions
of water reuse were not frequently major hindrances to implementation of water reuse
programs in Northern California. Public perception of water reuse may be positively
influenced by a shift in view of recycled water towards that of a valuable resource and
as public knowledge of water supply challenges increases. (5) Economic issues stand as
the largest hindrance to successful project implementation from a management
perspective. In particular, smaller water reuse programs are less frequently incentivized
by federal or state grants and loans, while larger programs have somewhat greater
challenges associated with distribution system (pipeline) costs.
2.2 Methodology
Data sources. Primary data on water reuse agencies, practices, and management
experiences were collected via an online questionnaire of Northern California water
reuse managers conducted for the present study in 2010 (2010 Survey). Additional data
on water reuse agency characteristics were obtained from the California State Water
Resources Control Board (SWRCB) 2001 Water Recycling Survey released in 2002
(2001 Survey, (3)), the National Database of Water Reuse Facilities (National Database,
(58)), and the 2009 California Municipal Wastewater Recycling Survey, a follow-up
12
survey from the SWRCB released in April 2011 (2009 Survey, (59)). Municipal water
recycling agencies in Northern California (defined as the 48 counties northward of the
southern boundaries of Monterey, Kings, Tulare, and Inyo counties) listed on the
National Database and the 2001 Survey were invited to participate in the 2010 Survey.
Fieldwork administration and questionnaire. Data were collected online from
February to April 2010 using electronic surveys sent to general managers or
water/wastewater directors from 134 agencies in 41 Northern California counties using
a distribution list compiled from the SWRCB 2001 Survey and the publicly available
National Database (58). The questionnaire, which is described further in the Supporting
Information, was developed based on case study research, literature review and site
visits at water and wastewater facilities and agencies with programs implemented for
agriculture, landscape irrigation, industrial power plant cooling, and ecosystem
enhancement. Respondents were asked a number of questions related to the drivers and
challenges experienced in implementing their agency’s water reuse program with
additional survey components addressing responses to recent recycled water policy in
California and future expectations for programs in development. Prior to distribution,
survey testing by several consultants and project staff was conducted for usability and
content feedback.
Categorization and statistical tests. The analyses conducted for 2010 Survey
results provide quantitative confirmation of trends that have been previously discussed
and valuable insights into the characteristics of water reuse in Northern California.
Results represent quantitative response data and are supported by qualitative
descriptions of drivers and barriers experienced in program implementation. Chi square
analysis was conducted on two by two contingency tables constructed from frequency
results of specific drivers (Table 2.1) and hindrances (Table 2.2) to program
implementation to assess relationships between categorical variables. For simplicity in
additional analysis and discussion, the list of specific drivers and hindrances was
consolidated into eight and nine categorical variables, respectively. Chi square analysis
was also performed on these data, and categories were used to contextualize qualitative
responses to survey questions (See Tables 2.3S – 2.10S for full results). The
presentation of representative respondent quotations, extracted primarily from responses
13
to two questions – the single most important driver or hindrance to implementation –
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 2010 Survey responses. Because some parent
utilities represent multiple recycled water facilities, a total of 81 unique production
facilities are represented by responses; however, most agencies (83%) represent only
one recycled water production facility, and another 7% represent a unique distribution
facility coupled to a production facility. Respondents consist of internal public agency
managers or utility staff. The survey completion rate was 40% of invited participants.
Therefore, the response fractions reported for each question indicate values for that
particular question. Respondent agencies for the 2010 Survey were distributed widely
across Northern California though survey representation appears somewhat weak for the
number of agricultural programs relative to the 2009 Survey data (Figure 2.5S and
Table 2.11S). The median year of recycled water program implementation, based on
self-reported implementation dates for 56 respondents, was 1991, with the earliest
reported implementation occurring in the early 1960’s.
2.3 Analysis of Water Reuse in California
Recycled water distribution falls short of statewide goals. Figure 2.1 displays a
timeline of statewide water recycling goals and production volumes (2, 3, 13, 57, 59,
60). According to the 2009 Survey results, recycled water used in California in 2001
included 491,992 AFY from municipal facilities, with the additional volume attributed
to private facilities (59). The newest data from the California SWRCB indicates
California municipal wastewater facilities recycled a total of 723,845 AFY in 2009 (59).
This represents an increase of more than 230,000 AFY from levels in 2001, yet once
again falls short of goals for recycling set by the State of California by nearly 300,000
AFY (Figure 2.1 and Table 2.12S). Although the SWRCB 2009 Survey may
underrepresent current reuse volumes due to the low survey response rate, the results
underline a need to identify continuing challenges associated with implementation of
water reuse programs and to evaluate strategies to develop new recycled water
programs and expand existing distribution networks.
14
Northern California context. Our analysis shows that only 20% of the observed
state-wide increase in reuse between 2002 and 2009 occurred in the Northern 48
counties of California, where 120 municipal agencies recycled 127,000 AF in 2002, and
173,000 AF was produced from 143 agencies in 2009. Recycled water programs in
Northern California are generally smaller in volume (median = 347 AFY in 2009) than
programs in the ten Southern California counties (median = 1064 AFY in 2009), where
82 municipal agencies recycled 365,000 AFY of water in 2002, increasing to a total of
551,000 AFY of water in 2009 by 104 agencies (Figure 2.2 and Figure 2.6S). Water
reuse programs are frequent across rural Northern California and agricultural areas in
the Central Valley (Figure 2.2), typically at much lower volumes than urban areas
generating larger volumes of wastewater. Though reuse in Northern California
represents a lesser fraction of overall reuse in the state, challenges associated with the
implementation of smaller, rural programs are important to consider in developing the
total portfolio of state projects. Several larger programs have been implemented over
the last decade in Northern California, and more are likely to be developed in large
urban centers. However, recycled water program size has remained relatively stable on
average in Northern California.
15
Figure 2.2. A snapshot of water reuse facilities in California from the National
Database of Water Reuse Facilities (Annual Production, reported as Facility Production
Average Annual Actual in million gallons) and the California 2009 Municipal Water
Recycling Survey (Annual Reuse, reported as Total Reuse for 2009 in AFY). In the
inset box plot, the boundary of the box indicates the upper and lower quartiles; a line
within the box indicates the median; whiskers above and below the box demarcate 1.5
times the interquartile distance with outlying points also shown.
16
2.4 Drivers of Water Reuse Implementation in Northern CA
Various social, economic, and environmental factors have been identified as drivers
of water reuse by governments and stakeholders globally (2, 8, 56, 61). These driving
forces include: drought, demand due to population and economic growth, wastewater
management, ecological protection, availability near urban areas, and availability of
proven treatment technologies (8, 56). To establish a forum for free-form responses
regarding principal driving forces behind recycled water implementation in Northern
California, respondents first considered the relative importance of several broad
categories of drivers. The fraction of respondents indicating each broad category as a
very important driver or a driver, respectively, was: regulatory requirements (0.59,
0.27), water shortages (0.49, 0.34), economic 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 water
reuse in Northern California, respondents were asked to select factors that drove
program implementation. Amongst a list of 19 specific factors (Table 2.1), 63% of
respondents indicated “wastewater discharge volume requirements” as a driver of
implementation, with 49% 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 80% of all
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.”
17
Table 2.1. Percent of respondents indicating a specific factor as a Driver or one of the
three Most Important Drivers. Responses (n = 65) were further categorized as shown
and are sorted from top to bottom by the highest frequency categorized Most Important
Driver.
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%
18
Figure 2.3. Beneficial uses of recycled water in Northern California in 2001 and 2009.
See Supporting Information for a description of categories.
In addition to specific regulatory requirements, state recycled water policy goals or
mandates were selected as a driver by nearly a third (29%) of 2010 Survey respondents
and as one of the three most important drivers by 13% of respondents. Additionally,
24% of respondents selected basin plan water quality objectives as one of the three most
important drivers of implementation. Such objectives may relate to discharge volume
requirements: one respondent who described Basin Plan Water Quality Objectives as
the single most important driver of their program’s implementation stated, “Reducing
our volume discharged to surface water helps us to meet increasingly more stringent
effluent discharge loading requirements.” Drivers of implementation, other than those
shown in Table 2.1, identified by respondents (n = 12 total) often reflected site-specific
conditions including a need for a specific effluent disposal method or location, the cost
of disposal, a water conservation Executive Order, and the need for replacement water.
Notably, “Ecological protection or enhancement goals” were drivers for the
implementation of many programs (49%) but were rarely the most important drivers for
these programs (12%). In 2001 and 2009, only 6-7% of reuse was for natural
system/wildlife enhancement (Figure 2.3).
Controlling wastewater discharge and the role of regulation.
“We needed a method [to] dispose of treated effluent. The only viable
alternative was recycling.”
Results demonstrate that regulatory requirements, such as those limiting discharge
19
of wastewater, have historically played an important role in driving the implementation
of water reuse in Northern California. The California Department of Public Health
establishes state public health criteria for wastewater reclamation via Title 22 for
bacterial quality, treatment types and levels, and facility reliability. Individual Regional
Water Quality Control Boards (RWQCBs) and local water and health agencies may also
develop more stringent policies and programs related to recycled water use (2). In free-
form responses, respondents who cited regulatory requirements as a very important
category of drivers (n = 28) noted a range of specific regulatory pressures that drove the
implementation of their program (see Supporting Information for details). Various
agencies were mandated or recommended to reduce percolation and increase reuse, cap
discharge flows despite population growth, and eliminate point source discharges or
meet dilution requirements in receiving waters during a particular time period (e.g.,
summer months).
Transitioning from wastewater discharge control to recycled water as a
resource.
“The original driver is not the current driver. Currently water supply and
reliability is the most important driver.”
Water shortages are commonly experienced throughout California, with several
severe droughts throughout the period of implementation represented by survey
responses (Figure 2.1). California’s elaborate system of dams, canals, aqueducts,
groundwater basins, and levees mediates the dichotomy between the state’s water
sources and demand centers, where 75% of the state’s precipitation falls north of
Sacramento, and 75% of demand occurs in the population and farming centers to the
south (62). Because of the interconnectedness of water infrastructure in the state and the
dependence of the largest urban centers on imported water, Northern California is not
immune to challenges associated with limited water supplies. The growing awareness
and response to water supply challenges are reflected in agency experiences. Programs
implemented after 1990 were more likely to cite water shortages due to increased
demand as a driver than older programs (p < 0.01) and were somewhat more likely to
indicate water shortages due to reduced supply as a driver (0.1 < p < 0.2, Figure 2.4).
Conversely, wastewater discharge volume requirements were more frequently indicated
20
as one of the three most important drivers of implementation by agencies with reported
implementation dates before 1991 (p < 0.05), suggesting that early implementation of
water reuse in the region was driven more frequently by such regulatory requirements.
Newer recycled water programs were also more likely to cite the need for reliable water
supply as an important driver of implementation (p < 0.01).
Figure 2.4. Results of χ2 analyses by implementation date for specific factors indicated
as one of the Three Most Important Drivers (top) or more generally a Driver of
implementation (bottom).
When expanding on the role of water shortages in driving program implementation
21
(n = 25), respondents frequently cited recycled water as a replacement source for
potable water supplies, where there may be a site-specific water need or shortage (e.g.,
for golf courses or parks) or a cap on freshwater source allocations (e.g., through
externally controlled piped sources). Increased demand without additional supplies was
also evident in several cases of overdraft of groundwater systems leading to degraded
water quality. Water shortages and reliability planning resulting from droughts were
noted separately as driving forces. For example, the 1976-1977 drought was followed
by the adoption of the Policy and Action Plan for Water Reclamation in California by
the SWRCB and subsequent increased funding recycled water planning studies (60).
Such opportunistic funding support strategies may continue to be important to capitalize
on increased incentive for water reuse implementation during periodic drought periods
in the region. Interestingly, only 5% of respondents in the present survey indicated
climate change adaptation plans as a driver of recycled water program implementation.
However, guidance by the California Natural Resources Agency (63) and Department
of Water Resources (5) incorporate recycled water as a drought-proof and sometimes
energy efficient water management strategy to complement climate change adaptation
measures. As these goals filter from state planning to local practices, state policies for
climate change adaptation will likely become more influential in recycled water
implementation.
Although water shortages were not directly an issue during project implementation
for some older projects, anticipated water shortages and need for long-term reliable
sources are now critical issues, especially following the 2007 – 2009 drought in
California. Projects that were implemented initially due to wastewater requirements
may expand or find new benefits of reuse due to water supply challenges. One
respondent illustrated this changing paradigm, stating:
“Fifteen years ago when we started our program, public acceptance was
an issue. People did not understand recycled water, and we spent a lot of
time educating potential customers and marketing recycled water. There
was some 'fear factor' slowing the expansion. However, things have
changed completely with the worsening drought, delta water problems,
climate change awareness, and the public's desire to be 'green' and
22
recycle everything now. We currently cannot get the water out to
customers fast enough.”
Implementation to increase reliability of potable water supplies (e.g., by sustaining
groundwater supplies for drinking), supplement water supply needs, or free up
freshwater entitlements for use elsewhere were described as other drivers of
implementation.
2.5 Challenges for Water Reuse Implementation in Northern
CA
Challenges for water reuse projects include a need for public education, lack of
available funding, recovery of capital costs for dual distribution systems, a need for
improved documentation of economic benefits of water reuse, political support, a need
for additional research for innovative technologies, public perception, flawed or
unevenly applied regulations and standards, and concerns and liability over the
unknown long-term health effects of chemical contaminants (2, 8). When asked to
select factors that hindered program implementation at the respondent’s site from a list
of 20 specific options, 87% of respondents cited financial or economic challenges as
one of the three most important hindrances to water reuse implementation (Table 2.2).
One respondent commenting on the single most important hindrance to implementation
simply stated, “These projects are big ticket items outside the range of a rate base.”
Specific hindrances from the financial or economic challenges list included: availability
of federal/state grants or loans, capital costs for construction of recycling plant facilities,
cost of alternative freshwater sources, costs for pipeline construction, and ongoing
operations & maintenance cost recovery. Together, these factors dominated the
selection of the most important challenges relative to other categories shown in Table
2.2 consistently through time. Despite various sources of policy and financial support
for water reuse in California, lack of sufficient funding may be the main factor
preventing recycling goals from being achieved (59).
23
Table 2.2. Percent of respondents indicating a specific factor as a Hindrance or one of
the three Most Important Hindrances. Responses (n = 54) were further categorized as
shown and are sorted from top to bottom by the highest frequency categorized Most
Important Hindrance.
Categorized Hindrance
Most Impt. Hind.
Hind. Specific Hindrance Most Impt. Hind.
Hind.
Economic/ financial disincentives
87% 94% capital costs for construction of recycling plant facilities
56% 85%
costs for pipeline construction 48% 80% ongoing operations & 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 due to constituents of emerging concern
13% 48%
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%
4% 13% uncertainty over future recycled water uses
4% 13%
Other 9% 11% other 9% 11%
24
Challenges in the next most-cited category, public perception and social attitudes,
were indicated as an important hindrance by only 26% of respondents. Specific
hindrances categorized under public perception and social attitudes challenges were:
constituents of emerging concern, perception that recycled water will lead to more
development, perception that recycled water will reduce property value, and the more
general factor of social attitudes/public perception. In addition to those in Table 2.2,
other factors hindering program implementation identified by individual respondents
included soil salinity, lack of seasonal storage, and overcoming opposition from
influential stakeholders.
Economic constraints and financial implications of challenges.
“Generally in the industry and specifically for us, the cost of pipelines is
really the only reason we haven't been recycling more.”
Several examples of recycled water programs in Northern California provide
context for the expected costs of recent treatment facilities and distribution systems. For
16 projects seeking regional federal funding as part of the San Francisco Bay Area
Recycled Water Coalition, the total costs ranged from $220/AF to $3400/AF, with a
$1200/AF median value, assuming a 20-year period for recycled water generated at the
initial project yield (Table 2.13S, (64)). Recycled water deliveries expected for these
projects range from 115 AFY initially to up to 28,000 AFY in the future. A City of Palo
Alto analysis indicates an annualized cost of $2700/AF (over 30 years, in March 2008
dollars) expected for expansion of distribution facilities. This compares with a projected
cost of $1,600/AF by 2015 for wholesale purchase of potable water from the San
Francisco Public Utilities Commission (SFPUC) (65). An earlier phase of the Palo Alto
project completed in 2009 came to approximately $3.4 million/mile of pipeline for
construction base contract of approximately 5 miles of pipeline along US Highway 101
to the neighboring City of Mountain View (66). A project under analysis by the SFPUC
estimates $9.4 million (including a 30% contingency) for approximately 6.5 miles of
pipeline construction costs as part of a $153 million recycled water treatment and
distribution system (67, 68).
2010 Survey respondents were asked to characterize, as quantitatively as possible,
the impact of cited hindrances to implementation in terms of program cost, scope, and
25
timing. Respondents indicated that hindrances led to a change in program cost (n = 9),
reduced program scope (n = 5), delay of implementation (n = 14), project cancellation
(n = 7), or other (n = 1). For a subset of these responses, estimated costs associated with
impacts (n = 21) ranged from $50,000 to almost $100 million per agency. Estimates by
respondents for changes in program cost represented construction cost increases over
time, costs for additional studies, increased staff time, additional testing “beyond
reasonable needs,” “huge” impacts from years of delay, costs to upgrade to tertiary
treatment, costs for new processes, and a combination of changes in program scope,
changes in design, addition of professional consultants or a combination of conveyance
pipes, distribution piping, tanks and pressure stations.
Issues related to institutional coordination were also noted for increasing project
costs. Nearly a third of respondents indicated institutional coordination as a hindrance
to implementation. One example described:
“While water agencies need recycled water to help them with long term
supply issues, they cannot justify the increased costs and thus tend to be
unsupportive. Water agencies are also concerned about loss of revenue
with recycled water projects. If the water agency is not the same as the
recycled water agency (as in our area), implementation of recycled water
projects means a loss of revenue for the water district as customers are
shifted to the recycled water agency. This means that the potable water
agency must raise rates for the remaining customer base, which is very
difficult in today's economic climate.”
Limited role of negative perceptions.
“In 1984, the biggest hindrance was the negative perception by
landowners next to the farms scheduled to receive recycled water today.
Today the biggest hindrance is cost.”
Since the 1970s, a significant amount of research has investigated reasons for public
resistance to recycled water (69, 70). Although public perceptions of risks are identified
as key impediments in the adoption of indirect potable water reuse (71-73), nonpotable
water reuse programs generally receive public support (56). Thus, opposition
surrounding high-profile indirect potable reuse is likely unrepresentative of the
26
landscape of challenges faced by managers of nonpotable reuse programs distributed
throughout California cities and rural areas. A notable contrary case developed when
homeowners actively opposed the use of recycled water for landscape irrigation in
Redwood City, CA (46). While utilities and consultants have developed more
appropriate modes of communicating with the public, some members of the public
remain skeptical about the safety of the practice, especially as projects are proposed in
their community and the likelihood of human contact increases (74-76). Organizational
trust correlates with intended behavior towards using recycled water and may be an area
of further focus for institutional practices to increase public acceptance (77), and
principles of fairness and equity are significant to people’s decision-making (69).
Analyses emphasize the importance of public engagement early during project
conception and continuously throughout planning, design, and construction (2, 56).
The primary drivers of water reuse programs may also influence public opposition
or acceptance. An early public opinion study in California indicated that those who
believed water supply augmentation was necessary in California were somewhat less
likely to be opposed to reclaimed water for drinking than those who did not believe that
water was scarce (70). Consequently, public education efforts to effectively
communicate the need for water reuse are important. In the present study, respondents
who cited wastewater discharge volume requirements as a driver of implementation
were somewhat more likely to also cite a specific factor within the category of public
perceptions and social attitudes as a hindrance (0.2 > p > 0.1). As freshwater supply and
distribution agencies experience increased demands and pressures on existing resources,
greater public awareness of augmentation needs may reduce challenges associated with
public perceptions. Conversely, in communities where the drivers of recycled water are
discharge-based, rather than supply driven, public perception problems may arise more
readily.
“Perceived human or environmental health risks due to constituents of emerging
concern” was cited as a hindrance to implementation by almost half of respondents.
Yet, this factor was not correlated to program implementation date, reminding us that
unknown or unregulated contaminants change in specific definition with time, but have
challenged managers for decades. Concern for residuals in recycled water has been
27
expressed in various forms. In the 1970’s and 80’s, issues of public perception were
difficult to overcome, as recycled water was relatively unfamiliar and long-term safety
of reuse for high-contact uses was unproven. Today, CECs are a topic for technological
research and a source of concern for recycled water managers (13). Noting this issue as
an additional challenge to cost hindrances, one respondent commented that “opponents
are also trying to use the issue of emerging constituents as a way to portray the project
in a negative light.” Public perception of recycled water continues to be an important
non-technical challenge for water reuse implementation, especially with regards to
CECs. However, the present study finds that economic issues, rather than public
perception, stand as the largest hindrance to nonpotable reuse implementation for
Northern California programs.
Responses to recycled water policy. In 2009, the California State Water Resources
Control Board adopted a California Recycled Water Policy “to increase the use of
recycled water from municipal wastewater sources.” Providing statements towards the
beneficial uses of recycled water, the State Water Board “strongly supports recycled
water as a safe alternative to potable water for such approved uses.” Despite the
policy’s stated objectives, whether the water reuse policy will actually accelerate efforts
to develop and maintain new recycled water projects remains unclear. The legislation
itself takes on a hopeful tone by striving for, among other items, increased use of
recycled water “over 2002 levels by at least one million acre-feet per year (AFY) by
2020 and by at least two million AFY by 2030” (54).
Recycled water managers were questioned about their expectations concerning how
the California Recycled Water Policy of 2009 will facilitate or hinder the
implementation of new recycled water programs. Survey responses reveal both support
and trepidation towards the policy, with a greater number of respondents voicing
concern that the policy will hinder project implementation. According to respondents, a
perceived beneficial impact of the policy stems from standardized and consistent
guidance for recycled water projects. For example, the water reuse policy established a
Blue Ribbon Panel for evaluating contaminants of emerging concern that will apply to
all projects across California and also contains language endorsing water reuse under
the California Environmental Quality Act (CEQA) (13). Second, many respondents
28
viewed the policy favorably due to its singular management structure. The
establishment of an overarching permitting process, and of salt and nutrient
management requirements, in particular, drew positive reviews. As one manager put it,
“The standardization of salinity and nutrient management provisions among the various
regional boards should facilitate reuse and make it easier for some projects to get
permitted.” Thus, for water reuse project managers, the provision of administrative,
legal and scientific continuity across state, regional and local agencies was perceived as
the most beneficial aspect of the policy.
Much of the skepticism expressed for the 2009 policy may be traced to funding
issues. A majority of respondents (19 of 30 question responses) felt the policy would
obstruct new projects through onerous regulatory and cost requirements. According to a
number of managers, while statewide project streamlining and standardization is
important, ultimately the fate of projects will depend on adequate funding support. A
common refrain amongst respondents was a concern over added administrative layers
that will arise with new oversight and reporting requirements. In sum, the perceived
presence of additional financial costs and administrative requirements have led nearly 2
of every 3 survey respondents to suggest the 2009 water reuse policy will in some way
hinder new project implementation. From a management perspective, results suggest
that the 2009 policy has done little to alter the perceived drivers and hindrances of water
reuse project implementation for managers in Northern California.
2.6 Significance
A diverse body of responses from the 2010 Survey illuminates a number of
influential drivers of water reuse implementation, including the protection of
ecosystems, meeting wastewater discharge requirements, and needs for water supply
and reliability. We continue to detect manifestations of the intrinsic links between water
supply and quality: threats of long-term diminished water quality (e.g., seawater
intrusion) necessitates new water conservation and reuse measures, while new water
supplies of altered quality may galvanize community opposition. Although water supply
agencies increasingly face challenges associated with population growth and drought,
wastewater agencies have traditionally approached recycled water as an issue of
29
disposal. This push/pull duality that either push implementation forward (via regulatory
requirements for wastewater discharge) or pull agencies into recycled water programs
(by increased demand for water) is apparent. Results provide evidence of changing
perspectives towards recycled water management, from a waste disposal issue towards a
water supply resource opportunity.
Failure to meet statewide reuse goals results largely from lack of sufficient funding
for water recycling, as the cheapest recycled water opportunities have already been
exploited (2). Following three years of drought and recent passage of the Safe, Clean,
and Reliable Drinking Water Supply Act of 2010 by the State of California that
included $1.25 billion general obligation bond proposal for Water Recycling and Water
Conservation, the physical and political climates may be ripe for aggressive
implementation of new water reuse programs, where financially viable, socially
accepted, and technically sound. Yet the legislature’s 2010 decision to postpone the
water bond initiative for at least two years (62) is testament to the realities of financial
limitations for new water infrastructure in California.
Supporting Information Available. Contains (1) methodological details, (2) a
brief description of recycled water policy and regulation in California, and (3)
additional analysis and summary tables.
Acknowledgement. We thank the numerous participants in the project and survey
respondents for their generous donation of time and thoughtful contributions. This work
was funded by the Stanford University Woods Institute for the Environment
Environmental Venture Projects, the Bill Lane Center for the American West, the NSF
Graduate Research Fellowship Program, and the NSF Engineering Research Center for
Re-inventing Urban Water Infrastructure (UrbanWaterERC.org). We especially thank
Sophie Egan for detailed technical assistance.
Publication Information. Reproduced with permission from Environmental
Science & Technology, submitted for publication. Unpublished work copyright 2011
American Chemical Society.
30
2.7 Supporting Information
Description of data sources. Invited and respondent agency locations for the
present study (2010 Survey) are shown by county in Figure 2.5S. The California State
Water Resources Control Board (SWRCB) 2001 Water Recycling Survey (2001
Survey, (3)) compiled data on planned direct reuse of treated municipal wastewater in
California, excluding industrial reuse. The 2009 California Municipal Wastewater
Recycling Survey (2009 Survey) was conducted by the SWRCB Water Recycling
Funding Program (WRFP), compiling volumetric data for municipal wastewater
recycling facilities to determine progress towards goals set in 2008 and 2009 by the
California State Water Board Strategic Plan Update and the Recycled Water Policy.
Although the 2009 Survey represents the best available information on current water
recycling volumes in California, the survey itself was completed initially by only 18%
of agencies invited to participate (118 agencies responding). SWRCB staff collected
additional data through recycled water annual reports, agency websites, telephone
communication, and carry-over of data from the 2001 Survey, assuming volumes
reported in 2001 remained the same in 2009. Updates in the beneficial use categories
noted in the 2009 Survey as compared to the 2001 Survey include: Golf Course
Irrigation was separately quantified from Landscape Irrigation in the 2009 Survey;
Wildlife Habitat & Miscellaneous Enhancement in the 2001 Survey was labeled Natural
Sys. Restoration, Wetlands, Wildlife Habitat in the 2009 Survey; and Wastewater
Treatment Plant uses were not specified in the 2009 survey (included as Other for 2001
Survey results in manuscript Figure 2.3). Recreational Impoundments was 0% in 2009
and <1% in 2001. The 2001 Survey included private agencies, which were excluded in
the 2009 Survey. Annual flows reported in Northern and Southern CA for the 2001 and
2009 Surveys are compared in Figure 2.6S.
The National Database of Water Reuse Facilities (National Database, (58)) is
maintained by the WateReuse Association. The database was initially populated in 2006
and may be updated manually by agency representatives (78). The National Database
was accessed for the California Query in January 2010 for the 2010 Survey. Private
facilities and individuals from the 2001 Survey were excluded from the initial
31
distribution list for the 2010 Survey, and surveys were not sent to several agencies that
requested exclusion when compiling contact information. Additional information on
survey respondent characteristics was collected from the member-accessible portion of
the National Database in July-August 2010. Data for average annual production
volumes (million gallons) reported by water reuse facilities in California were accessed
from the National Database on August 16, 2010. Facility locations for mapping
purposes were obtained from zip codes available in the National Database or from
online searches for the facility location. Data from the 2010 Survey are not intended to
update or compare directly with the 2009 Survey results. 2010 Survey respondent
characteristics are described to assess representation of surveyed agencies compared to
agencies in the sampling frame.
Questionnaire. For the 2010 Survey, the invited participant was asked to complete
the survey or designate an individual familiar with the implementation of the water
reuse program to complete the survey. Coding entries for respondent “Position” shows
that 72% of respondents were directors/managers of the agency/utility (e.g., Public
Works Director, General Manager, Deputy Director, Division Manager), 14% of
respondents were engineers (e.g., Chief Engineer, Senior Engineer), 10% were
operators or other technical positions (e.g., Chief Plant Operator, Operations Director),
and 4% of respondents did not list a position. In the final dataset, one response per
agency was retained. Three agencies submitted two responses, so the more complete
survey was retained. One respondent did not input agency identification information;
responses from this individual were not excluded. In statistical analysis, quantitative
results were omitted for two respondents who indicated that their facilities do not
produce recycled water; these responses were retained for qualitative analysis purposes.
In the questionnaire, reclaimed or recycled water were noted to be synonymous and
defined as: “water that is used more than one time before it passes back into the natural
water cycle, or wastewater that has been treated to a level that allows for its reuse for a
beneficial purpose.” Driving forces and barriers to water reuse program implementation
in the region were examined through several qualitative and quantitative components.
Respondents rated the importance of five broad categories of potential drivers
(economic concerns, regulatory requirements, recycled water policy, water shortages,
32
and influential stakeholder groups) and five broad categories of hindrances (cost
recovery, human health or water quality concerns, institution/management issues,
influential stakeholder groups, and salt and nutrient management) for program
implementation on a three-point scale (Not a Driver/Hindrance, Driver/Hindrance, or
Very Important Driver/Hindrance). Additionally, from lists of specific factors
(manuscript Tables 2.1 and 2.2) respondents selected all factors that were considered
drivers/hindrances of program implementation and three factors considered to be the
most important drivers/hindrances for the implementation of their agency’s recycled
water program. In several cases, respondents marked a specific factor as one of the three
most important drivers/hindrances without selecting this same factor as a
driver/hindrance. In these cases, responses were updated such that all most important
drivers/hindrances were necessarily considered drivers/hindrances. Respondents were
given opportunity to insert other categorical and specific factors. To encourage free-
form descriptive responses and for clarification, respondents were further invited to
describe components of the broad categories previously marked as very important.
Subsequent questions and focus sections on the role of influential stakeholders,
constituents of emerging concern, and ecosystem enhancements will be the subject of
future analysis.
Categorization and statistical tests. Response data were categorized by self-
reported values of total reclaimed water use and date of program implementation such
that an equal number of respondents were in each category. Thus, recycled water
program size was operationally categorized as programs with self-reported total annual
reclaimed water deliveries greater or less than the median value (990 AFY), and the
date of implementation was categorized as either before or after the median date of
implementation, 1991. Analysis was omitted when the frequency in any contingency
table bin was less than five and p > 0.1 or when the frequency in any bin was equal to
zero. When the frequency of a bin was between 1 and five and p < 0.1 for the
association test, the results were listed in parentheses. Chi square analyses are
summarized in Table 2.3S to 2.10S.
33
Characteristics of 2010 Survey respondent agencies. Based on cross checking of
categorizations available in the member-accessible National Database, which lists all
respondent agencies in the 2010 Survey, respondent agencies represent Public Utilities
(45%), Private Utilities (6%), or Special Districts (23%). The remaining 23% of
respondent agencies were not categorized in the National Database. Most agencies
(76%) conduct “Both Production and Distribution” of recycled water, with 7%
categorized as “Only Production” and 7% as “Only Distribution;” 6% of respondents
represent other agencies (that may have been involved by funding, management of
construction, etc.), and 4% (3 agencies) are not categorized. Non-zero total annual
reclaimed water deliveries reported for 63 respondent agencies ranged from 6 AFY to
28,000 AFY, with a median value of 920 AFY. With the inclusion of delivery rates
from the 2009 Survey for three agencies where a value was not entered and data were
available in the SWRCB survey, the median reclaimed water delivery value was 990
AFY. Responses captured a larger flow range than reported in the 2009 Survey and
yielded a larger median annual recycled water volume (Figure 2.7S).
Historically, agricultural water reuse predominated in California, occurring in the
Central Valley in places where farmland was located adjacent to wastewater treatment
facilities (2). 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. However, significant population growth, particularly in the Central Valley,
creates challenges for new or increased wastewater discharge in largely agricultural
areas, especially for environments with limited assimilative capacity (55). Beneficial
uses of recycled water reported in the 2009 and 2010 Surveys are displayed in Table
2.11S. Additionally, between 2002 and 2009, fourteen agencies in Northern California
reported reduced volumes, averaging 61% of 2002 levels recycled in 2009, including
two null values reported in 2009 for previously recycling agencies. From 1970 to 1977
and 1977 to 1987 various agencies were dropped from the latter surveys of water reuse
in California due to: (1) discontinued reuse, (2) treatment plants shut down or converted
to a wet weather plant with the construction of regional facilities, (3) changes in
reporting criteria or interpretation, (4) name changes, or (5) inadvertent omission (60).
34
California recycled water policy and regulation. Several milestones in recycled
water policy and regulation have been implemented in California since the Porter
Cologne Act of 1969 that formulated state and regional water quality control regulatory
bodies (e.g., Table 2.12S). As required by Assembly Bill No. 331 passed by the
California Legislature and signed into California law on October 7, 2001, a Recycled
Water Task Force was convened by the California Department of Water Resources
(DWR) in 2002 to address issues related to the impediments or constraints related to
increasing water recycling. Six issues areas were identified: funding/CALFED
coordination; public information, education, and outreach; plumbing code/cross-
connection control; regulations and permitting; economics of water reclamation; and
science and health issues of indirect potable reuse (2). In February 2009, the California
SWRCB adopted a Recycled Water Policy to encourage expanded reuse in California
(54). In a progressive move to address issues of trace contaminants in recycled water,
the policy led to a series of recommendations on chemicals of emerging concern,
published in 2010 (13).
Funds have been made available through state and federal financial assistance and a
series of bond initiatives from 1978 – 2002, totaling close to $132 million in planning
and construction grants and $509 million in low interest loans for water recycling
projects distributed by the State Water Board from 1978 to 2006 (79). An additional
$275 million in construction grants and $11.5 million in planning grants was distributed
by the U.S. Bureau of Reclamation Title XVI program through 2007 in California (79).
The 2003 Recycled Water Task Force estimated an $11 billion dollar investment was
required at the time to reach 2030 water recycling goals (2), and the 2008-2012 State
Water Board Strategic Plan Update estimates $300 million in annual grants and loans
are required (55).
2010 Survey respondents noted specific sources of regulatory requirements,
mandates, or strong recommendations that influenced program implementation,
including: National Pollutant Discharge Elimination System (NPDES) permits,
Regional Water Quality Control Boards (RWQCBs) and the SWRCB, Basin Plans, the
Porter Cologne Act, Waste Discharge Requirements, waste production located outside
of city limits for a treatment system (e.g., for a college campus), a water right
35
requirement to move golf courses off river water to recycled water, construction
approvals for new stand-alone subdivisions requiring production of recycled water, and
California Energy Commission recommendations to use recycled water for power plant
cooling water. Several other responses inserted by respondents as very important drivers
(n = 9), including flow limits on discharges, need for wastewater disposal, and RWQCB
requirements, supported the theme of regulatory requirements as important drivers of
implementation.
Small- versus large-volume recycled water programs. A chi square analysis
indicated that respondents representing the lower 50% of reuse programs by volume
(small-volume reuse programs) were somewhat more likely to cite wastewater
discharge volume requirements as a driver for implementation (0.1 < p < 0.2). At the
same time, small-volume programs were somewhat less likely to indicate the
availability of federal and state grants and loans as a driver of program implementation
(0.1 < p < 0.2) than programs with total annual reclaimed wastewater deliveries greater
than the median reported value. One respondent noted, “In rural foothill communities,
regulations tend to push agencies towards recycling because there are no viable
alternatives.” An effort to coordinate funding proposals was successfully implemented
by the San Francisco Bay Area Recycled Water Coalition (BARWC), a partnership of
17 agencies formed to secure federal funding under Title XVI of the 1992 Reclamation
Wastewater and Groundwater Study & Facilities Act. Typical project costs and the
federal shares of funding for the BARWC are shown in Table 2.13S (64). This
exemplifies the potential of collaborative funding efforts. Such efforts to pool resources
regionally, rather than compete individually for funding, may prove beneficial for rural
communities seeking facility upgrades for implementation of smaller-volume programs.
Respondents representing small-volume programs also provided somewhat greater
recognition of the role of influential stakeholders in driving program implementation,
citing either citizen initiatives or large volume users as drivers of implementation more
frequently than respondents from large volume programs (0.1 < p < 0.2). Larger volume
programs were more likely than smaller volume programs to be implemented due to a
need for reliable water supplies (0.1 < p < 0.2).
36
Impacts of hindrances. Cost impacts estimated by respondents for project delays
caused by cited hindrances specifically ranged from $50,000 to $25,000,000 due to time
delays, increased material costs, California Environmental Quality Act (CEQA)
documentation, staff time to contact interested parties, and for additional storage ponds
required for groundwater recharge. “Delays in customer connections caused by slow
regulatory approval and issues related to customer acceptance” led to lost revenues
estimated at $1,000,000 for one project. Another respondent noted that lack of local
project funding led to the loss of state grant funding and the cancellation of a project.
One respondent described a revenue challenge experienced in transitioning to recycled
water, “Recycled water is also typically discounted by 20 to 25% in Northern California
to encourage customers to connect, so even if the water and recycled water agency is
the same, the agency loses revenue by connecting recycled water customers while also
having to pay for the expensive recycled water infrastructure required.” A contingency table chi square analysis indicated that respondents who identified at
least one negative impact of stated hindrances to implementation were somewhat more
likely to cite costs for pipeline construction as one of the three most important
challenges as compared to those agencies who did not experience significant negative
impacts (p < 0.1). Programs recycling more than the median flow were also somewhat
more likely to cite costs for pipeline construction as a hindrance than smaller projects
(0.1 < p < 0.2), underlining the need for support of distribution system costs as recycled
water receiving sites are located further from recycled water production facilities.
37
Figure 2.5S. Distribution of survey invitations and responses collected from Northern
California counties. The number of respondents from a given county is displayed, with
the number of agencies invited to participate in a county indicated in parentheses.
38
Figure 2.6S. Agencies binned by annual recycled water flow (AFY) in 2001 and 2009.
39
1
10
100
1000
104
105
2010 Survey 2009 Survey
Tota
l R
euse (
AF
Y)
Figure 2.7S. Representation of total annual reclaimed water deliveries reported from
the 2010 Survey of Northern California agencies1 (n = 64) and the State Water
Resources Control Board 2009 Municipal Wastewater Recycling Survey (n = 143).
Both plots exclude zero-volume survey responses.
1 Includes reclaimed water deliveries values from the 2009 Survey for three agencies in which a value was not entered in the 2010 Survey and data was available in the SWRCB 2009 Survey.
40
Table 2.3S. C
hi square analysis of drivers of program im
plementation by self-reported date of im
plementation. O
lder and recent
implem
entation dates were operationally defined as before and after the reported m
edian value (1991), respectively. 1
D
river M
ost Important D
river
n = 54
Statistical tests (df = 1)
n = 54 Statistical tests
(df = 1)
Older
impl.
date, %
Recent
impl.
date, %
χ2
p O
lder im
pl. date, %
Recent
impl.
date, %
χ2
p
need for reliable water supply
17%
39%
8.45 0.004
9%
19%
1.98 0.16
water shortages due to increased dem
and 9%
30%
8.13
0.004 2%
13%
(4.94)
(0.03) w
astewater discharge volum
e requirements
37%
28%
1.98 0.16
33%
19%
4.59 0.03
water shortages due to reduced supply
22%
44%
(8.47) (0.004)
15%
26%
2.23 0.14
basin plan water quality objectives
26%
19%
1.29 0.26
19%
7%
(3.56) (0.06)
ecological protection or enhancement goals
26%
24%
0.13 0.72
7%
4%
regional or local recycled water policy goals or
mandates
19%
19%
0.01 0.93
4%
11%
state recycled water policy goals or m
andates 15%
15%
0.01
0.94 6%
7%
availability of federal/state grants or loans
6%
22%
(6.72) (0.01)
2%
11%
(3.84) (0.05)
cost of alternative freshwater sources
7%
24%
(6.14) (0.01)
2%
7%
1 A
nalysis is omitted w
hen the frequency in any contingency table bin was less than five and p > 0.1 or w
hen the frequency in any bin was equal to
zero. When the frequency of a bin w
as between 1 and five and p < 0.1 for the association test, the results are listed in parentheses.
41
Tab
le 2
.4S.
Chi
squ
are
anal
ysis
of h
indr
ance
s to
pro
gram
impl
emen
tatio
n by
sel
f-re
porte
d da
te o
f im
plem
enta
tion.
Old
er a
nd re
cent
impl
emen
tatio
n da
tes w
ere
oper
atio
nally
def
ined
as b
efor
e an
d af
ter t
he re
porte
d m
edia
n va
lue
(199
1), r
espe
ctiv
ely.
1
H
indr
ance
M
ost I
mpo
rtan
t Hin
dran
ce
n
= 44
St
atis
tical
test
s (d
f = 1
) n
= 44
St
atis
tical
test
s (d
f = 1
)
Old
er
impl
. da
te, %
Rec
ent
impl
. da
te, %
χ
2 p
Old
er
impl
. da
te, %
Rec
ent
impl
. da
te, %
χ
2 p
avai
labi
lity
of fe
dera
l/sta
te g
rant
s or l
oans
20
%
34%
2.
21
0.14
7%
14
%
user
acc
epta
nce
16%
27
%
1.59
0.
21
7%
2%
dow
nstre
am w
ater
qua
lity
impa
cts/
NPD
ES c
onst
rain
ts
11%
20
%
1.19
0.
28
0%
5%
dete
ctio
n of
con
stitu
ents
of e
mer
ging
con
cern
11
%
20%
1.
19
0.28
2%
0%
co
sts f
or p
ipel
ine
cons
truct
ion
39%
48
%
30%
25
%
0.88
0.
35
perc
eive
d hu
man
or e
nviro
nmen
tal h
ealth
risk
s due
to
cons
titue
nts o
f em
ergi
ng c
once
rn
23%
32
%
0.78
0.
38
5%
9%
issu
e of
who
pay
s for
pro
gram
cap
ital o
r ope
ratin
g co
sts
27%
36
%
0.73
0.
39
7%
11%
co
mpl
exiti
es/c
onfli
cts o
f wat
er la
w a
nd/o
r reg
ulat
ion
16%
23
%
0.48
0.
49
5%
2%
capi
tal c
osts
for c
onst
ruct
ion
of re
cycl
ing
plan
t fac
ilitie
s 41
%
45%
30
%
27%
0.
42
0.52
so
cial
atti
tude
s/pu
blic
per
cept
ion
18%
18
%
0.05
0.
82
5%
9%
slow
regu
lato
ry p
roce
ss in
per
mitt
ing
16%
16
%
0.04
0.
84
2%
5%
ongo
ing
oper
atio
ns &
mai
nten
ance
cos
t rec
over
y 30
%
32%
0.
00
0.94
14
%
14%
0.
03
0.85
pe
rcep
tion
that
recy
cled
wat
er w
ill le
ad to
mor
e de
velo
pmen
t 11
%
14%
0.
03
0.86
2%
2%
te
chni
cal i
ssue
s/tre
atm
ent p
roce
sses
5%
23
%
(6.3
8)
(0.0
1)
2%
0%
inst
itutio
nal c
oord
inat
ion
5%
20%
(5
.13)
(0
.02)
2%
7%
ef
fluen
t res
idua
ls (e
.g. b
rine)
dis
posa
l 2%
11
%
(2.6
9)
(0.1
0)
0%
2%
1 Ana
lysi
s is o
mitt
ed w
hen
the
freq
uenc
y in
any
con
tinge
ncy
tabl
e bi
n w
as le
ss th
an fi
ve a
nd p
> 0
.1 o
r whe
n th
e fr
eque
ncy
in a
ny b
in w
as e
qual
to
zero
. Whe
n th
e fr
eque
ncy
of a
bin
was
bet
wee
n 1
and
five
and
p <
0.1
for t
he a
ssoc
iatio
n te
st, t
he re
sults
are
list
ed in
par
enth
eses
.
42
Table 2.5S. C
hi square analysis of categorized drivers of program im
plementation by self-reported date of im
plementation. O
lder and
recent implem
entation dates were operationally defined as before and after the reported m
edian value (1991), respectively. 1
D
river M
ost Important D
river
n = 54
Statistical tests (df = 1)
n = 54 Statistical tests
(df = 1)
O
lder im
pl. date, %
Recent
impl.
date, %
χ2
p O
lder im
pl. date, %
Recent
impl.
date, %
χ2
p
Institutional control 19%
41%
8.39
0.004 11%
20%
1.80
0.18 W
ater supply needs 24%
44%
(7.10)
(0.01) 15%
33%
6.02
0.01 Influential stakeholders
9%
20%
2.77 0.10
6%
2%
Wastew
ater discharge requirements
37%
28%
1.98 0.16
33%
19%
4.59 0.03
Economic/financial incentives
20%
30%
1.35 0.24
7%
17%
Other policy and regulation
33%
33%
0.02 0.88
22%
20%
0.13 0.72
Ecological goals or requirements
26%
26%
0.01 0.91
11%
6%
1 A
nalysis is omitted w
hen the frequency in any contingency table bin was less than five and p > 0.1 or w
hen the frequency in any bin was equal to
zero. When the frequency of a bin w
as between 1 and five and p < 0.1 for the association test, the results are listed in parentheses.
43
Tab
le 2
.6S.
Chi
squ
are
anal
ysis
of c
ateg
oriz
ed h
indr
ance
s to
pro
gram
impl
emen
tatio
n by
sel
f-re
porte
d da
te o
f im
plem
enta
tion.
Old
er
and
rece
nt im
plem
enta
tion
date
s wer
e op
erat
iona
lly d
efin
ed a
s bef
ore
and
afte
r the
repo
rted
med
ian
valu
e (1
991)
, res
pect
ivel
y.1
H
indr
ance
M
ost I
mpo
rtan
t Hin
dran
ce
n
= 44
St
atis
tical
test
s (d
f = 1
) n
= 44
St
atis
tical
test
s (d
f = 1
)
O
lder
im
pl.
date
, %
Rec
ent
impl
. da
te, %
χ
2 p
Old
er
impl
. da
te, %
Rec
ent
impl
. da
te, %
χ
2 p
Wat
er q
ualit
y im
pact
s 14
%
27%
2.
53
0.11
2%
5%
U
ser a
ccep
tanc
e 16
%
27%
1.
59
0.21
7%
2%
Pu
blic
per
cept
ion
and
soci
al a
ttitu
des
36%
32
%
1.19
0.
28
11%
16
%
0.24
0.
62
Econ
omic
/fina
ncia
l dis
ince
ntiv
es
48%
50
%
0.93
0.
33
43%
45
%
Who
pay
s for
pro
gram
cos
ts
27%
36
%
0.73
0.
39
7%
11%
R
egul
ator
y co
nstra
ints
25%
30
%
0.08
0.
78
7%
7%
Tech
nica
l iss
ues/
treat
men
t pro
cess
es
5%
23%
(6
.38)
(0
.01)
2%
0%
In
stitu
tiona
l iss
ues
7%
20%
(3
.42)
(0
.06)
5%
7%
1 A
naly
sis i
s om
itted
whe
n th
e fr
eque
ncy
in a
ny c
ontin
genc
y ta
ble
bin
was
less
than
five
and
p >
0.1
or w
hen
the
freq
uenc
y in
any
bin
was
equ
al to
ze
ro. W
hen
the
freq
uenc
y of
a b
in w
as b
etw
een
1 an
d fiv
e an
d p
< 0.
1 fo
r the
ass
ocia
tion
test
, the
resu
lts a
re li
sted
in p
aren
thes
es.
44
Table 2.7S. C
hi square analysis of drivers of program im
plementation by self-reported total annual reclaim
ed water use. Sm
all and
large volume program
s were operationally defined as agencies w
ith total reclaimed w
ater use less than and greater than the reported
median value, respectively. 1
D
rivers T
hree Most Im
portant Drivers
n = 65
Statistical tests (df=1)
n = 65 Statistical tests
(df=1)
Sm
all V
olume
%
Large
Volum
e %
χ
2 p
Small
Volum
e %
Large
Volum
e %
χ
2 p
availability of federal/state grants or loans 8%
23%
6.79
0.01 3%
14%
5.11
(0.02) need for increased institutional control of w
ater 14%
5%
3.91
(0.05) 2%
2%
large volume user(s)
18%
9%
3.03 0.08
6%
0%
need for reliable water supply
20%
31%
2.60 0.11
11%
15%
0.60 0.44
wastew
ater discharge volume requirem
ents 35%
28%
2.09
0.15 29%
22%
1.87
0.17 regional or local recycled w
ater policy goals or m
andates 23%
15%
1.89
0.17 11%
6%
state recycled water policy goals or m
andates 11%
18%
1.65
0.20 6%
6%
w
ater shortages due to reduced supply 29%
34%
0.37
0.54 20%
20%
0.01
0.92 cost of alternative freshw
ater sources 17%
15%
0.12
0.73 5%
5%
technological advancem
ents 8%
9%
0.08
0.78 2%
2%
ecological protection or enhancem
ent goals 23%
25%
0.02
0.90 5%
8%
basin plan w
ater quality objectives 20%
20%
0.01
0.92 14%
11%
0.42
0.52 w
ater shortages due to increased demand
20%
20%
0.01 0.92
9%
6%
1 A
nalysis is omitted w
hen the frequency in any contingency table bin was less than five and p > 0.1 or w
hen the frequency in any bin was equal to
zero. When the frequency of a bin w
as between 1 and five and p < 0.1 for the association test, the results are listed in parentheses.
45
Tab
le 2
.8S.
Chi
squ
are
anal
ysis
of h
indr
ance
s to
pro
gram
impl
emen
tatio
n so
rted
by to
tal a
nnua
l rec
laim
ed w
ater
use
. Sm
all a
nd la
rge
volu
me
prog
ram
s wer
e op
erat
iona
lly d
efin
ed a
s age
ncie
s with
tota
l rec
laim
ed w
ater
use
less
than
and
gre
ater
than
the
repo
rted
med
ian
valu
e, re
spec
tivel
y.1
H
indr
ance
s T
hree
Mos
t Im
port
ant H
indr
ance
s
n
= 52
St
atis
tical
test
s (d
f = 1
) n
= 52
St
atis
tical
test
s (d
f = 1
)
Sm
all
Vol
ume
%
Lar
ge
Vol
ume
%
χ2
p Sm
all
Vol
ume
%
Lar
ge
Vol
ume
%
χ2
p
cost
of a
ltern
ativ
e fr
eshw
ater
sour
ces
8%
19%
2.
38
(0.1
2)
2%
6%
dete
ctio
n of
con
stitu
ents
of e
mer
ging
con
cern
10
%
21%
2.
07
0.15
0%
4%
sl
ow re
gula
tory
pro
cess
in p
erm
ittin
g 10
%
21%
2.
07
0.15
2%
6%
pe
rcep
tion
that
recy
cled
wat
er w
ill le
ad to
mor
e de
velo
pmen
t 6%
15
%
2.00
(0
.16)
2%
2%
cost
s for
pip
elin
e co
nstru
ctio
n 33
%
46%
1.
72
(0.1
9)
19%
29
%
0.73
0.
39
user
acc
epta
nce
13%
25
%
1.63
0.
20
4%
6%
inst
itutio
nal c
oord
inat
ion
10%
17
%
0.84
0.
36
8%
2%
2.55
(0
.11)
pe
rcei
ved
hum
an o
r env
ironm
enta
l hea
lth ri
sks d
ue to
co
nstit
uent
s of e
mer
ging
con
cern
19
%
27%
0.
36
0.55
6%
6%
capi
tal c
osts
for c
onst
ruct
ion
of re
cycl
ing
plan
t fa
cilit
ies
40%
44
%
29%
27
%
0.82
0.
37
dow
nstre
am w
ater
qua
lity
impa
cts/
NPD
ES c
onst
rain
ts
13%
17
%
0.05
0.
82
4%
2%
ongo
ing
oper
atio
ns &
mai
nten
ance
cos
t rec
over
y 27
%
33%
0.
03
0.86
10
%
17%
0.
84
0.36
is
sue
of w
ho p
ays f
or p
rogr
am c
apita
l or o
pera
ting
cost
s 27
%
33%
0.
03
0.86
10
%
10%
0.
07
0.79
com
plex
ities
/con
flict
s of w
ater
law
and
/or r
egul
atio
n 17
%
19%
0.
02
0.89
6%
4%
so
cial
atti
tude
s/pu
blic
per
cept
ion
15%
17
%
0.01
0.
93
6%
8%
1 Ana
lysi
s is o
mitt
ed w
hen
the
freq
uenc
y in
any
con
tinge
ncy
tabl
e bi
n w
as le
ss th
an fi
ve a
nd p
> 0
.2 o
r whe
n th
e fr
eque
ncy
in a
ny b
in w
as e
qual
to
zero
. Whe
n th
e fr
eque
ncy
of a
bin
was
bet
wee
n 1
and
five
and
p <
0.1
for t
he a
ssoc
iatio
n te
st, t
he re
sults
are
list
ed in
par
enth
eses
.
46
Table 2.9S. C
hi square analysis of categorized drivers of program im
plementation by self-reported total annual reclaim
ed water use.
Small and large volum
e programs w
ere operationally defined as agencies with total reclaim
ed water use less than and greater than the
reported median value, respectively. 1
D
rivers T
hree Most Im
portant Drivers
n = 65
Statistical tests (df = 1)
n = 65 Statistical tests
(df = 1)
Sm
all V
olume
%
Large
Volum
e %
χ
2 p
Small
Volum
e %
Large
Volum
e %
χ
2 p
Wastew
ater discharge requirements
35%
28%
2.09 0.15
29%
22%
1.87 0.17
Influential stakeholders 20%
12%
1.99
0.16 11%
0%
8.09
(0.004) Econom
ic/financial incentives 20%
29%
1.87
0.17 11%
14%
0.26
0.61 O
ther policy and regulation 29%
34%
0.37
0.54 23%
18%
0.74
0.39 Institutional control
26%
31%
0.37 0.54
12%
17%
0.55 0.46
Ecological goals or requirements
23%
26%
0.14 0.71
8%
11%
0.34 0.56
Technological advancements
11%
9%
0.14 0.71
2%
2%
1 A
nalysis is omitted w
hen the frequency in any contingency table bin was less than five and p > 0.1 or w
hen the frequency in any bin was equal to
zero. When the frequency of a bin w
as between 1 and five and p < 0.1 for the association test, the results are listed in parentheses.
47
Tab
le 2
.10S
. Chi
squ
are
anal
ysis
of h
indr
ance
s to
pro
gram
impl
emen
tatio
n by
sel
f-re
porte
d to
tal a
nnua
l rec
laim
ed w
ater
use
. Sm
all
and
larg
e vo
lum
e pr
ogra
ms
wer
e op
erat
iona
lly d
efin
ed a
s ag
enci
es w
ith t
otal
rec
laim
ed w
ater
use
les
s th
an a
nd g
reat
er t
han
the
repo
rted
med
ian
valu
e, re
spec
tivel
y.1
H
indr
ance
s T
hree
Mos
t Im
port
ant H
indr
ance
s
n =
52
Stat
istic
al te
sts (
df =
1)
n =
52
Stat
istic
al te
sts (
df =
1)
Sm
all
Vol
ume
%
Lar
ge
Vol
ume
%
χ2
p Sm
all
Vol
ume
%
Lar
ge
Vol
ume
%
χ2
p
Reg
ulat
ory
cons
train
ts
19%
33
%
1.88
0.
17
6%
10%
U
ser a
ccep
tanc
e 13
%
25%
1.
63
0.20
4%
6%
0.
08
0.77
In
stitu
tiona
l iss
ues
10%
19
%
1.39
0.
24
8%
4%
Publ
ic p
erce
ptio
n an
d so
cial
atti
tude
s 25
%
35%
0.
55
0.46
10
%
15%
0.
41
0.52
W
ater
qua
lity
impa
cts
19%
27
%
0.36
0.
55
8%
4%
1.15
0.
28
Who
pay
s for
pro
gram
cos
ts
27%
33
%
0.03
0.
86
10%
10
%
0.07
0.
79
Tech
nica
l iss
ues/
treat
men
t pro
cess
es
13%
15
%
0.00
0.
96
4%
4%
1 Ana
lysi
s is o
mitt
ed w
hen
the
freq
uenc
y in
any
con
tinge
ncy
tabl
e bi
n w
as le
ss th
an fi
ve a
nd p
> 0
.1 o
r whe
n th
e fr
eque
ncy
in a
ny b
in w
as e
qual
to
zero
. Whe
n th
e fr
eque
ncy
of a
bin
was
bet
wee
n 1
and
five
and
p <
0.1
for t
he a
ssoc
iatio
n te
st, t
he re
sults
are
list
ed in
par
enth
eses
.
48
Table 2.11S. R
epresentation of recycled water beneficial uses from
the 2010 Survey of Northern C
alifornia (n = 69) agencies and the
State Water R
esources Control B
oard 2009 Municipal W
astewater R
ecycling Survey (n = 143). Volum
es for each beneficial use were
not collected in the 2010 Survey.
A
gricultural Irrigation
Landscape
Irrigation1
Industrial
Wildlife
Habitat
Enhance-m
ent. 2
Com
mercial/
Residential
Buildings 3
Groundw
ater R
echarge R
ecreational Im
poundment
Geotherm
al/ E
nergy Production
Other
2009 Total R
euse (AFY
) 4 91,360
22,556 13,975
12,071 7,371
2,500 0
12,665 10,049
2009 Frequency Percent
60.8%
32.9%
7.7%
4.9%
2.8%
0.7%
0.0%
0.7%
9.1%
2010 Frequency Percent
42.0%
52.2%
24.6%
21.7%
7.2%
11.6%
7.2%
N/A
24.6%
1 Landscape Irrigation and G
olf Course Irrigation w
ere combined from
the 2009 Survey results in this table. 2 Labeled as N
atural Sys. Restoration, Wetlands, W
ildlife Habitat in 2009 Survey results.
3 Labeled as Com
mercial in 2009 Survey results.
4 No agencies in N
orthern California indicated Seaw
ater Intrusion Barrier, Surface Water Augm
entation, or Indirect Potable Reuse as beneficial uses in the 2009 Survey results.
49
Table 2.12S. Milestones for California water reuse and statewide recycling goals.1
Year Name (Ref.) Description 1967 California Legislature supports
policies and laws to promote water recycling
Declared that “the state undertake all possible steps to encourage development of water reclamation facilities…to help meet the growing water requirements of the state.” (California Water Code, Section 13512)
1969 Porter-Cologne Water Quality Control Act (62)
Established State Water Resources Control Board and nine Regional Water Quality Control Boards (California Water Code, Division 7).
1974 Water Reuse Law of 1974 Enacted with the mission that “the primary interest of the people of the State in the conservation of all available water resources requires the maximum reuse of reclaimed water in the satisfaction of requirements for beneficial uses of water.” (California Water Code, Section 461)
1977 Policy and Action Plan for Water Reclamation in California and Executive Order B-36-77
Encouraged water reclamation and funding to support legislative directives. Established Office of Water Recycling and goal “to make available an additional 400,000 acre-feet by 1982.”
1991 California Water Recycling Act of 1991
Established statewide goal to recycle 700,000 AFY by 2000 and 1,000,000 AFY by 2010. (California Water Code, Section 13577)
1994 Statement of Support for Water Reclamation
Joint statement signed by State Water Resources Control Board, the USEPA, California Conference of Environmental Health Directors, Department of Water Resources, U.S. Bureau of Reclamation, and Water Reuse Association of California to pursue and develop policies and regulations that reduce constraints and promote water reclamation.
2000 Water Recycling in Landscaping Act
Required local public or private entities that produce or will provide recycled water to notify the local agency and required local agencies in turn to adopt and enforce a recycled water ordinance requiring recycled water use. (Senate Bill 2095)
2001 Recycled Water Task Force (2) Assembly Bill 331 required the Department of Water Resources to convene a Recycled Water Task Force "to identify opportunities/constraints to increase the industrial and commercial use of recycled water."
2006 Assembly Bill 371 (2) Included statement that agencies should take appropriate steps to implement the Recycled Water Task Force recommendations to meet the goal of recycling one million acre-feet per year of water by 2010. Required installation of piping for landscape irrigation if recycled water will be provided.
2008 State Water Board Strategic Plan Update (55)
Stated goal to recycle 1,250,000 AFY by 2015.
2009 Recycled Water Policy (54) Established goal to recycle 1,525,000 AFY by 2020 and 2,525,000 AFY by 2030.
2010 Science Advisory Panel report on Monitoring Strategies for Chemicals of Emerging Concern (CECs) (13)
A Science Advisory Panel was established by the 2009 Recycled Water Policy “to provide guidance for developing monitoring programs that assess potential CEC threats from various water recycling practices.”
1 Includes Water Recycling Laws and Policies summarized in (79) Water Recycling Funding Program Division of Financial Assistance Strategic Plan; California State Water Resources Control Board: Sacramento, 2007. Excludes summary of funding sources (e.g., Bond Laws).
50
Table 2.13S. San Francisco Bay Area Recycled Water Coalition 2011 Project Summary
(64)
Project Yield
(AFY), Project
Yield (AFY), Future
Total Cost
Federal Share of
Cost
Cost per ac-ft1
South Bay Water Recycling Phase 1.d.
2000 3000 $39.2 M $9.8 M $980
South Santa Clara County Recycled Water Project
1790 2440 $28 M $4.2 M $780
Antioch Recycled Water Project 490 850 $12.5 M $0.875 M $1300 South Bay Advanced Recycled Water Treatment Facility
6720 28000 $53 M $5 M $390
Central Contra Costa Sanitary District (CCCSD)-Concord Recycled Water Project
255 255 $7.2 M $1.8 M $1400
Contra Costa County Refinery Recycled Water Project
5600 22500 $25 M $6.25 M $220
Central Redwood City Recycled Water Project
1075 3170 $32 M $8 M $1500
Central Dublin Recycled Water Distribution and Retrofit Project & other projects
215 215 $4.6 M $1.15 M $1100
Delta Diablo Sanitation District (DDSD) Recycled Water Advanced Treatment and Expansion Project
3900 12500 $25 M $6.25 M $320
Dublin San Ramon Services District (DSRSD) Recycled Water Expansion Project
350 3250 23.85 M $5.96 M $3400
Hayward Recycled Water Project 3760 3760 27 M $6.75 M $360 Ironhouse Sanitary District Recycled Water Project
910 1320 26 M $6.5 M $1400
Palo Alto Recycled Water Pipeline Project
1000 1500 33 M $8.25 M $1700
Petaluma Recycled Water Project, Phases 2A, 2B and 3
1610 3280 24 M $6 M $750
Pleasanton Recycled Water Project 440 1840 20 M $5 M $2300 Yountville Recycled Water Project 115 400 3 M $0.75 M $1300
1 Assumes 20-years of Project yield.
51
Chapter 3
Water reuse for ecosystem enhancement:
Matching opportunity with need
3.1 Introduction
Water and wastewater treatment systems were developed during the twentieth
century as two separate systems that served mutually excusive goals of water supply
and protection of the integrity of receiving waters. Upgrades of wastewater treatment
facilities to meet more expansive regulatory requirements improved ambient water
quality. Yet increase in urban water demand has come at the expense of aquatic
ecosystems. Approximately 91% of historical California wetlands, including 85% of
saline wetlands and 92% of freshwater tidal wetlands, have been lost due to
urbanization (80). Urban and peri-urban development, and a traditional emphasis of
engineers on the prevention of floods and disposal of wastewater, has adversely
impacted urban hydrology and damaged aquatic ecosystems. In California, more than
half (62%) of estuarine wetlands exhibit medium to poor health due to modification of
physical structure, including levees and transportation infrastructure that have changed
52
the shape and reduced the size of wetlands (80). In these regions, non-natural tidal and
freshwater hydrology couple with excessive sediment supplies to reduce physical
complexity and wetland health.
Effective management of urban water can benefit aquatic habitat. Given the
availability of tertiary treated recycled water within the San Francisco Bay Area (81)
and potentially throughout California, the question arises whether some portion of
available highly treated recycled water can be used for beneficial wetland enhancement
and creation or stream augmentation. Redesign of urban hydrology in a manner that
enhances existing aquatic habitat has the potential to provide new sources of water
storage, while restoring the integrity and improving the aesthetics of watersheds and the
urban environment.
In Chapter 2, the growth of water reuse in Northern and Southern California was
documented using statewide survey data and ground-truthing to evaluate major trends in
the size, location, and form of projects implemented over the past half-century. The
objectives of the present chapter are to characterize existing and potential cases of water
reuse for natural system enhancement in California and to outline perceived challenges
associated with the implementation of water reuse projects for ecosystem enhancement.
Projects in California in which environmental enhancement drove the project design,
distinct from discharge of highly treated wastewater with incidental environmental
benefit, are identified. Opportunities for new projects are evaluated based on responses
to the previously described survey of water reuse managers (2010 Survey), an
assessment conducted in the San Francisco Bay Area by a regional coalition of
municipalities, and a statewide projection of wetland condition. Lastly, general issues
and challenges associated with wetland creation and enhancement as well as stream
augmentation with recycled water are discussed.
3.2 Few Existing Examples of Water Reuse for Direct
Ecosystem Enhancement in California
Several databases were queried and assessed to compile data on the use of
wastewater for the benefit of ecosystems: the California State Water Resources Control
53
Board (SWRCB) recycled water surveys (3, 59) conducted in 2001 (2001 Survey) and
2009 (2009 Survey), the National Database of Water Reuse Facilities (National
Database, (58)), the Treatment Wetland Database (TWDB), and the 2010 Survey
conducted as part of this research. Brief descriptions of identified projects that are
located in Northern California are given in Table 3.1.
According to the 2009 Survey, water reuse for ecosystem enhancement totaled
27,849 AFY, representing 4% of total wastewater reuse in the state. A total of 17
programs listed either “Wildlife habitat or misc. enhancement” on the 2001 Survey or
“Natural System Restoration, Wetlands, Wildlife Habitat” on the 2009 Survey. Eight of
these projects are located in the northern 48 counties of California, and the remaining
projects are located in the ten southernmost counties. The average size of Northern
California projects listed on the 2009 Survey was 1,700 AFY. The National Database
notes a total of six projects, three of which are in Northern California, with “Natural
System Restoration – Wetlands” as a beneficial use category. Five of the National
Database listings are represented on the 2001 or 2009 Surveys. Other agencies with
wildlife enhancement beneficial uses noted on the 2001 and 2009 Surveys are listed in
the National Database without beneficial use categorizations.
Wetlands ecosystems that serve as polishing for secondary treated wastewater may
also be considered water reclamation systems. Due to their ability to accept large
quantities of effluent, their partly oxic and partly anoxic soils, and resilient aquatic plant
species, wetlands are particularly suitable for wastewater purification (82). The TWDB
contains system descriptions and performance data on pilot and full-scale constructed
wetlands (83). At the time of access (May 2011), the database lists 11 unique systems in
California, with several additional pilot systems: Arcata Treatment Marsh, Gustine,
Hayward, Hemet/San Jacinto, Kelly Farm, La Franchi, Las Gallinas Sanitary District,
Manila Community Treatment Plant, Mt. View Marsh, Richmond, and Sacramento
Demonstration Wetland. Except for the Hemet/San Jacinto, all of these systems are in
Northern California and several overlap with systems identified via the 2001 and 2009
Surveys. Wetlands as treatment systems are an attractive options for small communities
that may be disproportionately affected by the construction, operation, energy, and labor
costs associated with centralized “concrete and steel” water pollution control facilities
54
(84).
Additional data regarding wastewater reuse for ecosystem enhancement were
collected via an online questionnaire of Northern California water reuse managers
(2010 Survey), which was discussed in detail in Chapter 2. If “Wildlife habitat
enhancement” was selected as a direct beneficial use of the treated wastewater in the
initial background section of the survey, respondents were asked to list the type (e.g.,
wetland enhancement or restoration, stream augmentation, freshwater marsh, etc.),
location, and volume of recycled water for existing uses of recycled water from their
agency’s program for ecosystem enhancement purposes. Fourteen respondents indicated
“Wildlife habitat enhancement” as a direct beneficial reuse utilized by their agency, and
seven of these respondents provided further information on the systems. Descriptions
listed in Table 3.1 from the 2010 Survey were included only if the system was also
listed on either the 2001 Survey, the 2009 Survey, the National Database, or the TWDB.
As a result, three project descriptions, the Dow Wetlands Preserve in Antioch supported
with approximately 0.1 MGD by the Delta Diablo Sanitation District, a Moss Landing
research project using 0 to 8 MGY, and habitat enhancement projects coupled to
development near the Sutter Creek Wastewater Treatment Plant in Amador County,
were described by respondents but not included in Table 3.1.
55
Table 3.1. Projects in Northern California utilizing recycled water for ecosystem
enhancement or treatment wetlands for wastewater effluent polishing.
Name Agency Noted in 2001 or
2009 Survey
Noted in 2010 Survey
Noted in TWDB
Arcata Enhance-ment Wetlands
City of Arcata Y2 Y Y A free-surface constructed wetland operating year round with continuous loading of secondary treated chlorinated effluent, the Arcata Enhancement Wetlands became fully operational in 1986 at a cost of just over $500,000 (1986 base year of capital cost). With an approximately 15 ha footprint, three wetland cells (Allen, Gearheart, and Hauser Marshes) operate in series for tertiary treatment of solids, organics, and nutrients as well as for habitat creation/enhancement, recreation, research, and acting as nature preserve (83). Additional references: (9, 85).
Calera Creek Wetlands
City of Pacifica Y N N The Calera Creek wetland restoration was conducted to improve riverine waters and wetland ecosystem function and to create habitat for the threatened California Red-Legged Frog and endangered San Francisco Garter Snake. Pacifica used a Hydro Geomorphic model for planning their wetland restoration projects. The treatment facility utilizes ultraviolet disinfection (83). In 1999, Pacifica delivered an average of 2,020 AFY (1.8 MGD) to existing wetlands (81). The City of Pacifica reported 3,280 AFY of recycled water use for Natural System Restoration, Wetlands, Wildlife Habitat in the SWRCB 2009 Survey (59).
Emily Renzel Wetlands
City of Palo Alto N2 Y N The Emily Renzel Wetlands restoration project in the Palo Alto Baylands comprises a 15-acre freshwater pond that receives 1 to 2 million gallons per day of pumped reclaimed water from the nearby Palo Alto Regional Water Quality Control Plant. When completed in 1992, it was one of only three projects in the State using reclaimed wastewater to develop freshwater marshes for birds. In 1999, Palo Alto delivered an average of 280 AFY (0.25 MGD) to existing wetlands (81). The City of Palo Alto does not report any use of recycled water for ecosystem enhancement on the 2002 or 2009 SWRCB Surveys, but the wetland enhancement project is listed on the National Database of Water Reuse Facilities.
Kelly Farm Santa Rosa N Y Y A small free surface constructed wetland (4 ha; 5 cells) that receives advanced secondary treated effluent, Kelly Farm became fully operational in 1990 (83). The marsh uses about 20 million gallons of water per year from the City of Santa Rosa Laguna Treatment Plant. This treatment facility also supports riparian revegetation projects that may use 20,000 gallons per day in the dry season.1
Hayward Marsh
Union Sanitary District (USD) Y2 Y Y The original two-phase implementation completed in 1980 and 1988 restored nearly 400 acres of the 1800 acres of Hayward shoreline (12). The 5-cell free surface wetland treatment system (~60 ha) receives conventional secondary effluent for year-round operation (83). In 1999, USD delivered an average of 11,000 AFY (10 MGD) to existing wetlands (81). In 2009, USD reported its total recycled water use, 3,493 AFY, as that for natural system restoration, wetlands, or wildlife habitat (59).
Gustine constructed wetlands
City of Gustine N N Y Fully operational in 1988 at a total cost of $882,000, the 9.6 ha free-surface constructed wetland utilizes 24 marsh cells to manipulate hydraulic detention time after receiving effluent from up to 11 oxidation ponds operated in series (83, 84). The City of Gustine only reports water reuse for irrigation in the 2002 or 2009 SWRCB Survey.
1 Additional information from 2010 Survey response. 2 “National System Restoration – Wetlands” beneficial use also listed on the National Database of Water Reuse Facilities.
56
Name Agency Noted in 2001 or
2009 Survey
Noted in 2010 Survey
Noted in TWDB
Las Gallinas, San Rafael
Las Gallinas Sanitary District (LGSD) N N Y The 20-acre free surface constructed freshwater marsh/pond was designed with varying depths and vegetation in a single unit to incorporate different wildlife habitat types (86). An additional 40 acres of storage ponds are used to irrigate pasture. Including land acquisition, the total cost of the reclamation system was $8.6 million, with state and federal Clean Water Grant funds covering 87.5% of the costs. LGSD reports 378 AFY for agricultural irrigation on the 2009 SWRCB Survey but does not indicate reuse for an environmental purpose (59).
La Franchi Santa Rosa N N Y La Franchi became fully operational as a free-surface constructed wetland (0.1 ha) treating low rate pond secondary-treated agricultural and animal waste in 1991 (83, 87). Because this recycling does not occur from a municipal wastewater treatment facility, the listing is not reported on the SWRCB Surveys.
Manila wetlands
Manila Comm. Treatment N N Y The free surface constructed wetland in Manila, CA operates year round with continuous loading after passing through a low-rate pond for secondary treatment in Manila, CA (83).
Moorhen Marsh and McNabney Marshes
Mt. View Sanitary District (MVSD) Y Y Y Moorhen Marsh is a 21-acre constructed wetland that is 100% fresh water and effluent dominated. MVSD cites the marsh as the first to use conventional secondary treated effluent as its primary water source. The adjacent McNabney Marsh (formerly known as Shell Marsh) is estuarine and seasonally saline, in total consisting of 130-acre restored, seasonally tidal wetland (88). The free-surface constructed wetland, reported as 3 cells and 37 ha in the TWDB, became operational in 1974 at a system cost of $90,000 and annual operation and maintenance cost of $20,000 (1978 base year of costs) (83). In 1999, MVSD delivered an average of 1,000 AFY (0.9 MGD) to existing wetlands (81).
Sacramento Demon-stration Wetlands
Sacramento Regional WWTP N N Y Treatment of municipal disinfected secondary effluent via a full scale free surface constructed wetland (8.9 ha) that utilizes 10 cells began in 1994, operating at about 1 MGD (83, 89), The Sacramento Regional County Sanitation District did not report reuse on the SWRCB 2009 Survey (59).
Wetland Enhance-ment / Restoration
Sonoma County Water Agency Y Y N The Sonoma County Water Agency includes four wastewater treatment facilities. Of these, two report use of recycled water for natural system enhancement or restoration on the SWRCB 2009 Survey: the Sonoma Valley Treatment Plant reported 100 AFY for wildlife of 1,600 AFY total reuse in 2009, and the Russian River Treatment Plant reported 90 AFY for wildlife of 150 AFY total reuse in 2009 (59).
Many 2010 Survey respondents considered the major beneficiaries from the
implementation of recycled water programs to include environmental groups in addition
to natural habitats. Examples cited of environmental and public benefits were: less
reliance on Delta Water, stakeholders concerned with protection of water quality in
Clear Lake, restoring water levels at Lake Merced in San Francisco (e.g., Cal Trout and
Natural Heritage Institute), and reduced discharge flows to Monterey Bay. Beneficiaries
cited also included general environmental advocacy groups (e.g., in San Jose/Santa
Clara) as well as natural habitat and recreational users (e.g., birders).
57
3.2 Identifying Opportunities for Ecosystem Enhancement
Although opportunities to reuse reclaimed water may be gleaned by quantification
of water needs for various applications (1), the needs of ecosystems are less practically
quantified. However, the system hydrology and measurable ecosystem characteristics
are intricately linked. Urbanized estuaries tend to have lower wetland health due to
hydrologic and biotic community structures (80). Water source, velocity, flow rate,
renewal rate, and inundation frequency influence the chemical and physical properties,
and thus biological structure, of wetland substrate (84). A major recommendation of the
Surface Water Ambient Monitoring Program (SWAMP) includes the need to increase
the size of estuarine wetlands to reduce the effects of stressors such as terrestrial
predators (80). Water movement through wetlands tends to have positive impacts on the
ecosystem, promoting increased regional production (84). For streams that have
experienced significant flow reductions due to anthropogenic influences, augmentation
using highly treated recycled water may beneficially impact the stream via increased
summer flows, improved water quality, support of healthier riparian areas, lowered
stream temperatures, enhanced fish and wildlife habitat, and improved aesthetics (90).
Based initially on this premise, artificial augmentation of wetlands, including riparian
corridors that have experienced significant flow reductions and altered hydrologic
regimes, represents potential opportunities for habitat restoration.
California assessment to match opportunity with need. Rapid assessment
methods represent a potential cost effective and consistent mechanism to monitor
relative wetland and riparian health, evaluating complex ecological condition using
observable field indicators. The California Rapid Assessment Method (CRAM) was
developed to assess the health of California wetlands along a continuum of conditions
based on attributes and metrics identified from a literature review and selected for
appropriate accuracy, precision, robustness, ease of use, and cost (91). The analysis
assumes that ecosystem condition, and the ability to support wildlife, may be measured
by structural characteristics and increases with complexity and size. Seven wetland
classifications were selected for the CRAM: riverine and riparian, estuarine, lacustrine,
depressional, wet meadows, vernal pools, and playas. The goal of the CRAM
58
assessment is to “provide rapid, scientifically defensible, standardized, cost-effective
assessments of the status and trends in the condition of wetlands and related policies,
programs and projects throughout California.” Subsequent validation of the CRAM
methodology indicates that the score corresponds with multiple independent
assessments of condition for avian diversity, plant community composition, and benthic
macroinvertebrate indices (92).
Four attributes, landscape context, hydrology, physical structure, and biotic
structure, are each characterized semi-quantitatively based on narrative analyses on a
series of metrics that are further assigned an ordinal or interval score relative to a
pristine condition (91). The hydrology attribute incorporates three metrics: water
source, hydroperiod, and hydrologic connectivity (92). External stressors (e.g.,
anthropogenic influences or natural disturbances to the wetland) are documented
separately from the wetland condition. The CRAM score, expressed as percent possible
ranging from 25 to 100 (80), summarizes the condition, or health, of a wetland or
riparian habitat relative to its maximum achievable condition based on a field visit by
two trained individuals. CRAM scores falling between 25 and 44 are indicative of poor
estuarine wetland health while 44 to 63 indicates medium to poor health. Other widely
used wetland assessment methods include the Hydrogeomorphic Method (HGM), the
Index of Biotic Integrity (IBI), and the Habitat Evaluation Procedure (HEP). These
methods are generally much more time and cost intensive and thus are generally
unavailable at a statewide level (91). For the present assessment, CRAM data were
obtained in July and August 2010. The CRAM scores categorize water bodies as
Estuarine Saline, Estuarine Non-saline, Riverine Confined, or Riverine Non-confined.
59
Figure 3.1. Distribution of California Rapid Assessment Method (CRAM) overall
wetland scores, included for estuarine (saline and non-saline) and riverine (confined and
non-confined), and wastewater facilities with tertiary treatment capacity.
Monitoring wetlands on a broad scale provides general information about
opportunities for wetland enhancement using recycled water. For this purpose, the
locations of tertiary treatment facilities were identified for proximity to wetland
ecosystems under stress. In Figure 3.1 the location of California tertiary treatment
facilities is overlaid with CRAM scores (93). As a conservative approach, only
wastewater facilities that currently utilize tertiary treatment, as indicated by the National
Database, are included. Typically tertiary treated recycled water for general purpose
irrigation comprises additional steps of coagulation, filtration and disinfection beyond
60
secondary treated wastewater. According to the National Database, advanced/tertiary
treatment technologies used by water reuse facilities in California include carbon
adsorption, ion exchange, disk filters, media filtration, ultrafiltration, nanofiltration,
reverse osmosis, and chemical precipitation. The treatment facility locations shown in
Figure 3.1 were generated from the zipcode of the facilities. Further refinement with
GIS mapping and system features are underway. Analysis of the location of tertiary
treatment facilities as generated from the zipcode was compared to the location of the
lower 50% of CRAM scores as a proxy for distance to wetlands. There were 27 low-
quality wetland sites within 10 miles of a wastewater treatment plant. Additional
evaluations at an ecoregion level can inform prioritization of restoration projects.
San Francisco Bay regional assessment. Of California’s 44,456 acres of perennial
tidal estuarine wetlands, 77% are located in the San Francisco Bay Estuary (80). The
1999 Bay Area Water Recycling Master Plan (BAWRMP) contains the most
comprehensive assessment of opportunities for wetland and stream augmentation using
tertiary treated recycled water for the San Francisco Bay region (81). The goals of the
Bay Area Regional Water Recycling Program environmental enhancement committee
were to identify potential environmental enhancement projects, use a watershed context
to evaluate environmental issues, and identify and develop action plans to address
regional environmental issues in recycled water implementation. The March 1999
Baylands Ecosystems Habitat Goals Report was utilized to frame the analysis of
potential for ecosystem enhancement using recycled water. The team evaluated 16
potential wetland restoration locations and 13 possible stream augmentation sites for
ecosystem enhancement, totaling of 13,000 AFY and 19,000 AFY, respectively (81).
The evaluation was not comprehensive, and further identification of potential sites
should be conducted, especially as new wetland assessment techniques are developed
and implemented. The application of recycled water for environmental enhancement
requires further investigation to determine the value of this option as well as appropriate
water quality criteria.
The BAWRMP evaluation of wetland sites involved potential site identification
from an initial market assessment, evaluating wetland water demand based on acreage
and a wetland water application rate (10 AF/acre based on wetland biological
61
requirements), and site evaluation of potential benefits and impacts, as well as
implementation strategies, for 16 potential sites (81). Possible site benefits included
habitat, species, and habitat diversity and management benefits, as well as the potential
to intercept non-point source pollution runoff and improve aesthetics. Potential adverse
impacts considered included the conversion of an existing valuable habitat, impact to
existing habitat or special status species, inconsistency with habitat management plans,
possible bioaccumulation of pollutants based on potential design, and impact on
biological resources from pipeline infrastructure.
The BAWRMP goals for 2010 were to develop an additional 11,000 AFY for
streamflow augmentation and 9,500 AFY for wetland enhancement or creation (81).
This was expected at a total cost of approximate $15 million for wetlands and $0.9
million for streams (discounted using 6.875% nominal discount rate; reported in 1997
dollars). Sites included in these goals included stream augmentation projects at San
Francisquito Creek by the City of Palo Alto facility, San Mateo Creek by South Bay
Systems Authority, Pillarcitos Creek by the San Francisco International Airport facility,
and the Guadalupe River by San Jose/Santa Clara (SJSC) among several other sites.
Based on the database analysis, many of the projects identified by the BAWRMP as
potential enhancement sites have likely not been implemented. The relatively large
number of projects expected for environmental uses by 2010 may have been a result of
low costs of additional treatment expected in the modeling scenarios. At the time of the
BAWRMP, the San Jose Coyote Creek study was underway, with note that additional
treatment beyond tertiary filtration may be a conclusion of that study. As will be
described in more detail later, this project was canceled in 2008 following extensive
water quality analysis that showed the presence of perfluorinated chemicals.
Additional opportunities in Northern California. In addition to identifying
existing sites, respondents from the 2010 Survey of Northern California water reuse
facilities were questioned regarding “opportunities to expand the use of recycled water
for restoration or protection of natural environments.” Respondents were asked to list
future opportunities identified to use or expand use of recycled water from their
recycled water program for ecosystem enhancement purposes. Several agencies noted
future opportunities for wetland or salt pond restoration, creation, or expansion:
62
• South Bay wetland creation (1-5 MGD) and bittern ponds habitat restoration
(5-15 MGD), City of San Jose;
• Napa Salt Marsh wetland reclamation (up to approximately 5 MGD), Napa
Sanitation District;
• Duer and Irwin Creek (estimated to use about 20,000 gallons per day each),
City of Santa Rosa;
• Wildlife habitat, Sacramento Regional County Sanitation District;
• Salt ponds restoration/wetland enhancement, Sonoma County Water
Agency;
• Carmel River Lagoon, Carmel Area Wastewater District;
• Possible wetland enhancement, Laurel Pond, Mammoth Community Water
District;
• Expansion of Hayward Marsh (north and south of existing marsh), East Bay
Quality assurance and data analysis. Solvent blanks were run every six samples
to monitor instrument background. To monitor ion suppression and enhancement,
matrix spike (MS %) recoveries were determined for each extract by performing a
second analysis of each extract, spiked with a known concentration of PFAA analyte.
Tissue concentrations are reported for MS % recoveries that were between 70 and
130%. Tissue samples were analyzed in triplicate; relative standard deviations of PFOS
replicates averaged 5% for white sturgeon samples. In several instances, tissue PFAA
concentrations were at or close to the LOQ, yielding at least one replicate above and
one below the LOQ. In these cases, the tissue concentration was reported as the average
of the measured concentration for replicates above the LOQ and the LOQ for
concentrations below the LOQ. The method detection limits (MDL) determined from
the extraction of replicate fish liver tissue samples (n = 12) are displayed in Table 4.1.
The MDL was calculated as the product of the relative standard deviation of sample
replicates and the student’s t-statistic (99% confidence level) for 11 degrees of freedom.
77
PFOA was spiked into dried tissue whereas other values were determined from analytes
already present in the extracted tissue. A least-squares regression linear fit with
corresponding coefficient of determination (R2) was calculated and displayed for each
correlation plot. Data were analyzed in Microsoft Office Excel (Microsoft Corporation;
Redmond, WA) and Kaleidagraph (Synergy Software Systems; Dubai, United Arab
Emirates).
4.4 Results and Discussion
White sturgeon PFAA tissues concentrations in the San Francisco Bay. White
sturgeon liver PFAA concentrations (ng/g wet weight) are displayed in Figure 4.2.
PFOS was detected in 14 of 15 samples, ranging in concentration from 14 ng/g ww to
180 ng/g ww. PFOS was also detected in the striped bass (83 ng/g ww) and leopard
shark (37 ng/g ww) samples. PFOS was below the LOQ in four white sturgeon muscle
tissue samples also analyzed as well as in side-by-side extracted blanks. For
comparison, concentrations of PFOS were 180-680 ng/g ww in livers of polar bears
from Alaska, up to 2570 ng/mL in blood plasma of bald eagles less than 200 days old,
and as high as 300 ng/g ww in fish (24). PFDS was detected in 13 of 15 white sturgeon
fish liver samples, ranging from 4.1 ng/g ww to 16.8 ng/g ww. PFDA was detected in 6
samples (0.9 – 8.2 ng/g ww), though five additional samples exhibited low PFDA MS%
recoveries and are thus not included. PFNA was greater than the LOQ in 12 of 15 white
sturgeon fish liver samples, ranging from 2.2 ng/g ww to 20.1 ng/g ww. However, 10 of
these samples were less than the MDL. PFNA concentrations above the LOQ, but not
necessarily above the MDL, are displayed in Figure 4.2. PFOA concentrations were
near or below the LOQ for all samples analyzed. Additionally, because PFOA was
detected in one of five blank samples (at a level near the LOQ), tissue concentrations
are not reported for this analyte. Few samples exhibited PFOA concentrations higher
than the LOQ in a global study of birds, fish, and marine mammals (24). PFOS
exhibited relatively high concentrations with wide variability; thus further analysis
regarding correlations with physiological and ecological parameters is limited to
comparisons with PFOS.
78
Figure 4.2. Measured PFAA concentrations (ng/g ww) in white sturgeon fish livers (n =
15). The tissue samples were archival specimens from animals taken from North San
Francisco Bay in December 1999 and January 2000. The boundary of the box indicates
the 25th and 75th percentile; a line within the box marks the median; whiskers above
and below the box indicate the maximum and minimum concentrations; outlying points
(>1.5 times the upper quartile) are shown as open circles. Number of detects for each
PFAA are also shown; samples with concentrations below the LOQ were excluded from
the plot.
An ecological perspective on contaminant variability: Influence of trophic level
and feeding location. Stable isotope ratios for nitrogen (δ15N) and carbon (δ13C) act as
naturally occurring intrinsic tracers by providing integrated measures of trophic
relationships and feeding locations along a salinity gradient. Isotope results are
presented as deviations from standard reference materials, where: δX = [Rsample/Rstandard
– 1] × 103. Here, X is 13C or 15N and R is 13C/12C or 15N/14N. Because 15N becomes
enriched with increasing trophic level (by 2.5 – 5% between prey and predator) without
varying along a salinity gradient in lower trophic level organisms (bivalves and
zooplankton), this ratio can serve as a quantitative measure of trophic position (108). A
higher δ15N for an individual within a given species may indicate consumption of higher
trophic level biota, due to the introduction of more prey-predator trophic steps from
79
baseline organism nitrogen signatures to the higher trophic position of the individual
under consideration.
Figure 4.3a displays PFOS white sturgeon concentrations with trophic position,
measured by muscle and liver δ15N. White sturgeon have enriched δ15N over lower
trophic organisms, such as the clams that are found as a dominant food items in
sturgeon digestive tracts (108). A wide range of PFOS concentration occurs with little
variability in δ15N. Notably, the highest liver PFOS concentration corresponds to the
highest trophic position white sturgeon. PFOS concentrations increase with trophic
position, as concentrations in predatory animals exceeded concentrations in their diets
(24). The high trophic position of this individual may result from increased
consumption of higher trophic level biota through a piscivorous dietary pattern rather
than more typical clam-based consumption. Additionally, this sturgeon was second to
the smallest, by length, of all white sturgeon considered, and PFOS concentrations
decreased with increasing fish length (Figure 4.3c). Although Martin et al. suggest that
the half-life for PFAAs in trout may be much longer for a larger fish of the same species
(37), growth dilution can be an important determinant of concentration for a substance
with slow uptake or clearance rates (107). The striped bass and leopard shark samples
are included in Figure 4.3 and, as expected, do not reflect the length trend for white
sturgeon. The high trophic level of the striped bass individual corresponds with a
relatively high liver PFOS concentration, whereas the leopard shark PFOS
concentration falls below the average PFOS concentration despite its slightly higher
trophic position.
Stable carbon isotope ratios can identify contributions of different foods in a diet by
tracking distinct isotopic signatures of food types. In estuaries, δ13C shows little to no
enrichment with trophic level but is enriched in algae with increasing salinities due to
the influence of δ13C in dissolved inorganic carbon that is incorporated into algae. As
these distinct isotopic signatures are incorporated into the base of the food web, the δ13C
of consumers will reflect their predominant foraging location, as determined by the
salinity gradient (108). Figure 4.3b displays the relationship between PFOS
concentration and foraging location, increasing on the ordinate from the freshwater
eastern reaches of the estuary to the more saline Suisun Bay. When excluding the high
80
trophic position outlier, PFOS liver concentrations decrease with increasing salinity.
Thus, fish spending a greater time feeding in more saline environments appear to
exhibit lower PFOS liver concentrations. Little is known regarding the affect of changes
in salinity on PFAA accumulation and toxicity, however an increase in distribution
coefficient with increasing water salinity suggests that long-chain PFAAs may “salt-
out” onto particles. This led to an increase in bioaccumulation in Pacific oysters
(Crassostrea gigas), filter-feeding bivalves that accumulate contaminants through
ingestion of contaminated particles (112). For organisms in which aqueous uptake is
more important than dietary accumulation, such as rainbow trout in which the blood-
water interface of gills is a major route of uptake and clearance (37), a decrease in
accumulation with increased salinity may be postulated. Additionally, freshwater
sources of PFAAs, such as wastewater treatment plant effluent (27), likely influence site
specific accumulation of PFAAs.
The relationship of total mercury concentration (organic and inorganic Hg) with
PFOS liver concentrations is displayed in Figure 4.3d. With the exception of the
sturgeon outlier, there appears to be a slight inverse relationship between these two
contaminant concentrations. Although both mercury (in its methylated form) and PFOS
are organic contaminants, PFOS sorption behavior does not typically follow the
paradigm of hydrophobic, lipophilic organic contaminants because of its surfactant
properties. The weak relation between Hg and PFOS is confounded by analysis of
different fish organs, with potentially different sorption properties. The physiochemical
behavior of PFAAs cannot be expected to predictably mimic other organic
contaminants (43). The influence of the unique PFAA chemical properties on
mechanisms controlling bioaccumulation requires further study. No significant
relationship was evident between selenium and PFOS concentrations.
81
Figure 4.3. Stable isotopes, fish length, and muscle Hg concentration plotted with white
sturgeon, striped bass, or leopard shark liver PFOS concentrations on the ordinate. (a)
Muscle and liver δ15N serve as measures of organism trophic position. A high trophic
position outlier was excluded from the linear regression. (b) δ13C represents integrated
measure of organism feeding location, indicating foraging location along a salinity
gradient. Liver PFOS concentration decreases with increasing salinity gradient. (c) An
increase in fish length corresponded to a decrease in liver PFOS concentration. (d) With
the exception of the high trophic position sturgeon outlier, PFOS liver concentration
(ng/g ww) yields and inverse relationship with total muscle mercury concentration (µg/g
dw).
Additional factors contributing to PFAA bioaccumulation. In addition to fish
size, trophic position, and foraging location, other factors not considered in this analysis
influence the exposure and retention of PFOS in white sturgeon tissue. For example,
82
fish age and sex may influence elimination rates of contaminants (37). PFAA source
locations in relation to foraging location are important; PFOS concentrations in
relatively industrialized regions may be several times greater than those in isolated areas
(24). Further, although PFOS is metabolically inert (37), precursors to this compound
are not – yielding additional sources of PFOS and other PFAAs in organisms. The role
of precursor compounds as sources of PFAAs has been studied extensively. N-ethyl
perfluorooctane sulfonamidoethanol (N- EtFOSE), produced directly and attached to
phosphate esters in paper coatings, degrades to N-ethyl perfluorooctane sulfonamido
acetic acid (N-EtFOSAA) in wastewater treatment processes (22). PFOS is the terminal
metabolite of this microbial degradation pathway. N-EtFOSE may also be stripped to
the atmosphere from treatment facilities (22) and oxidized to PFCAs and PFSAs (123).
N-EtFOSAA has been detected in natural San Francisco Bay sediments at levels often
exceeding PFOS (27) and oxidizes to perfluorooctane sulfonamide (FOSA) and PFOA
in hydroxyl-mediated photolysis (23). Recent studies elucidate the biotransformation
from such biologically labile precursor compounds to PFAAs as terminal metabolites.
N-EtFOSAA appears to undergo biotransformation to PFOS in an aquatic oligochaete
(116), likely contributing to organism PFOS body burden. PFCAs may form from
fluorotelomer alcohols (FTOHs) via biotransformation in rats and other organisms (124,
125), oxidation in the atmosphere (21), and indirect photolysis (126). 8:2 FTOH (127)
and fluorotelomer acrylates (128) biotransform to PFOA in rainbow trout.
Depuration and elimination rates of PFAAs from fish vary with PFAA chain length
(a proxy for hydrophobicity) as well as head group type (sulfonates or carboxylates)
(37). Such chemical properties may influence the extent of accumulation within a
certain organ, with blood concentrations in rainbow trout exhibiting higher
concentrations than the kidney and liver (114). Chemical properties influence the
interaction of PFAAs with gill membranes, an important site for elimination of
contaminants in fish (37). Controls of contaminant elimination on a cellular level may
contribute to PFOS bioaccumulation. For example, organic anion transporters play a
role in pharmacokinetics of PFAAs (129). Additionally, greatest inhibition of cellular
efflux transporter activity, which serves as a first line of defense against toxic
compounds (130), occurred with exposure to the longer chain acids, PFNA and PFDA,
83
for the marine mussel Mytilus californianus (122).
4.5 Significance
Perfluorinated compounds are bioaccumulative and ubiquitous among
environmental samples. In order to better understand the accumulation and variability of
concentrations measured within a species collected over a limited spatial and temporal
range, ecological and physiological processes must be considered. Wide variability of
concentrations of perfluorooctane sulfonate can occur within a single species type.
PFOS concentrations decreased for sturgeon feeding primarily in more saline
environments. Although correlations such as those presented are useful in developing an
assessment of the fate of PFOS in an ecosystem, a complete understanding of the
ecological and physiological diversity that influences contaminant concentrations
requires analysis of mechanistic processes such as elimination and uptake rates,
compound specific properties, and ecosystem dynamics such as contaminant sources
and transport processes. Even simplistic Tier 1 screening measures for evaluating the
bioaccumulative potential of new chemicals, a necessity for effective decision-making,
generally do not incorporate expected bioaccumulation mechanisms relevant to PFAAs.
In the following two chapters, the binding of perfluoroalkyl acids to a model protein,
BSA, is explored. In Chapter 5, commonly utilized dissociation constants relating free
chemical concentrations to protein-bound concentrations are determined for PFOA and
PFNA. Analytical methods are applied over a wide range of concentrations to assess the
contribution of different binding regimes at varied concentrations and for comparison to
literature values. In Chapter 6, a protein-water partition coefficient is quantified for C5 –
C10 PFCAs as well as C4, C6, and C8 PFSAs, and physiochemical mechanisms of
interactions are explored.
Acknowledgment. This work was conducted while funded by the National Defense
Science and Engineering Graduate Fellowship. Thanks to Christopher P. Higgins and
Laura A. MacManus-Spencer for laboratory guidance and feedback. We thank Robin
Stewart for provision of tissue samples and insights on analysis and interpretation.
84
85
Chapter 5
Investigating binding to a model protein:
Noncovalent interactions of long-chain
perfluoroalkyl acids with serum
albumin1
5.1 Introduction
Used throughout the past half-century in a variety of industrial and commercial
applications, perfluoroalkyl acids (PFAAs) are a class of environmentally persistent
anionic surfactants detected globally in water, air, sediment, and biota (20, 25). Field
1 Reproduced (with modifications) with permission from Bischel, H. N.; MacManus-Spencer, L. A.; Luthy, R. G. Noncovalent interactions of long-chain perfluoroalkyl acids with serum albumin. Environ. Sci. Technol. 2010, 44 (13), 5263-5269. Copyright 2010 American Chemical Society.
86
and laboratory studies indicate that perfluorooctanesulfonate (PFOS) and
perfluorocarboxylates (PFCAs) with greater than seven fluorinated carbons
bioaccumulate and biomagnify in aquatic food webs (25, 37, 113). Tissue distribution
studies show PFAA concentrations are greatest in body compartments high in protein
content, such as the liver, kidney and blood of organisms (24, 40). Typical
concentrations of perfluorooctanoate (PFOA) and PFOS in the serum of non-
occupationally exposed humans are 4 – 7 and 25 – 46 ng/mL, respectively (131). The
half-life of PFOA in serum varies widely by species and sex and is considered long (3.1
– 4.4 years) in human blood (132). Species-specific differences in PFAA distribution
patterns and retention may be influenced by active uptake by organic anion transporters
(41). Studies assessing PFAA-protein interactions (42, 133-138) may shed light on the
tissue distribution patterns, bioaccumulation, and in vivo bioavailability of these
chemicals.
Protein binding. The binding of PFAAs to proteins was first reported in the 1950s,
when PFAAs were investigated for their ability to aid in protein precipitation (139). In
the 1960s, organofluorine compounds were first detected in human blood serum (140).
Serum albumin, the most abundant protein in blood plasma (35 – 50 g/L) (141), binds a
variety of endogenous and exogenous ligands including fatty acids, amino acids, metals,
and pharmaceuticals (142) and was reported as the major binding protein for PFOA in
blood (42). PFAA-protein interactions result from the unique surfactant nature of
PFAAs. The highly hydrophobic perfluorocarbon tail paired with a strongly polar
carboxylate or sulfonate head group resembles the structure of fatty acids and facilitates
both hydrophobic and ionic interactions with proteins. In fact, PFOA binds to liver- and
kidney-fatty acid binding proteins (135), and PFAAs may interfere with the normal
binding of fatty acids or other endogenous ligands to liver-fatty acid binding protein
(136). However, the rigidity of the perfluorocarbon tail differs from the relatively more
fluid hydrocarbon tail (143), limiting extrapolation of fatty acid – albumin binding
results to their fluorinated counterparts.
Studies of PFAA-albumin interactions using spectroscopic methods (137, 144-146),
Equilibrium dialysis: PFAA-BSA binding over a wide range of [L]:[P] mole
ratios. Equilibrium dialysis was used to quantify free and albumin-bound PFAAs in an
equilibrated system over a wide range of PFNA and PFOA concentrations and 1 µM
BSA; [L]:[P] mole ratios ranged from 0.02 to 120 in these experiments. Concentrations
in all initial reservoir samples (0 hours) were below the detection limit, and a null value
was used for mass balance calculations. Initial dialysis bag PFAA concentrations ranged
from 1.6 µM to 2700 µM prior to equilibration in reservoirs. Dialysis bags reached
equilibrium with external reservoirs within 48 hours (see Supporting Information,
Figures 5.6S and 5.7S), when final samples were taken. The average relative standard
deviations for final bag and reservoir concentrations ranged from 4 – 6% for PFOA and
PFNA. Initial bag concentrations, which generally required additional dilution of
samples prior to analysis, had higher relative standard deviations (9% for PFOA and
11% for PFNA) than those for final bag concentrations. Average mass balance results
for control experiments (94%, n = 5 for PFOA and 107%, n = 7 for PFNA) indicate that
PFAAs do not significantly bind to the dialysis membrane or reservoir vessels.
Equilibrium dialysis results for PFOA and PFNA with 1 µM BSA are displayed in
Figure 5.1 along with 500 µM BSA and HSA results for PFNA. Although anionic
1 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). Means and error calculations for Log KPW were performed on the log-transformed data.
97
surfactants may cause protein denaturation, this is not expected to occur over the
concentration range tested (157), as reservoir concentrations were well below the
critical micelle concentrations (CMCs) of PFOA and PFNA (8.7 – 10.5 mM and 2.8 –
5.6 mM, respectively) (16). Total PFOA and PFNA concentrations, representing the
sum of free and bound PFAA concentrations, were measured inside dialysis bags at
equilibrium. Final bag concentrations were greater than reservoir concentrations for all
tests, indicating that PFAAs were bound to BSA and that PFAA-BSA complexes were
retained in the dialysis bags. Osmotic dilution of the retentate was assumed to be
negligible. Bound PFAA concentrations were calculated from concentrations measured
inside the dialysis bag in excess of free concentrations.
Figure 5.1. Equilibrium dialysis results for PFOA (a) and PFNA (b) where
!
" 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 Equation 5.4.
Association constants and binding stoichiometries for PFOA- and PFNA-BSA
complexes determined via equilibrium dialysis with 1 µM BSA are reported in Table
5.2. Data were fit both over the full range of test concentrations using Equation 5.4 and
up to a 5:1 PFAA:BSA mole ratio using a one-class binding model. Further details of
the fitting approaches and results are available in the Supporting Information. To reduce
the number of parameters being simultaneously solved in Equation 5.4 and thus reduce
the error in the solved parameters, association constants of 630 M-1 and 8000 M-1 were
98
employed for Ka,2 for PFOA and PFNA, respectively. These values were determined
under the same buffer conditions but at higher PFAA:albumin mole ratios (15 – 200)
using 19F NMR (137). An increased error was observed at higher reservoir
concentrations when samples were diluted into the analytical range of the LC-MS/MS
(see Supporting Information, Figure 5.9S). Due to a strong influence of the two highest-
concentration PFOA data points, which also indicated weak binding at a high mole
ratio, these data points were excluded from the fit presented in Table 5.2 for the PFOA
two-class model. The effects of applying various weighting factors for the full PFOA
and PFNA data sets were tested and yielded similar results to those in Table 5.2. The
primary association constants determined are similar for PFOA and PFNA, on the order
of 106 M-1 with binding stoichiometries of one to four or five. BSA had 150 ± 20
secondary binding sites for PFOA and 31 ± 2 secondary binding sites for PFNA. A
modest effect of albumin concentration on calculated PFAA association constants, in
which increased protein concentration decreased affinities, has been previously
observed (137). This effect was not evaluated in detail in the present study. However,
applying a one-class binding model to the results at a single PFNA:albumin mole ratio
with 500 µM BSA or HSA, and assuming n = 3, yields similar PFNA-albumin
association constants (on the order of 106 M-1) to those from 1 µM albumin tests (Table
5.3).
Table 5.2. Association constants (Ka) and binding stoichiometries (n) for PFOA and
PFNA binding to BSA determined by equilibrium dialysis.
1 Primary association constant listed 2 Electrophoretic mobility measured at pH 10; Ka calculated from ΔG = -25 kJ/mol 3 Micro size exclusion chromatography 4 Conducted at 37 °C 5 Ion-selective electrode 6 Hill binding constants reported for two binding regimes with positive cooperativity 7 Ka calculated for one PFAA:albumin mole ratio assuming n = 3
105
Results presented here demonstrate that PFNA is highly bound to BSA (>99%) at
low [L]:[P] mole ratios (< 10-3). Vanden Heuvel et al. (134) determined that 80% of 100
µM PFDA remained bound to 80 µM BSA after 60 minutes of extensive extraction with
organic solvents. This was attributed to covalent binding of the carboxylate head group
to protein sulfhydryl groups. Isolated albumins normally contain 0.5 – 0.7 moles of free
SH per mole of protein molecule (141). For comparison to prior results, at a 1.25 mole
ratio of free PFOA and PFNA to BSA (80 µM) and using equilibrium dialysis values
for a one-class binding model reported in Table 5.3, we calculate that greater than 98%
of PFAAs are bound to albumin.
5.4 Significance
A standard solution-based method (equilibrium dialysis) was compared with a
modern mass spectrometric approach (nanoESI-MS), providing complementary
information about the strength of PFAA-protein binding interactions and number of
binding sites at low ligand:protein mole ratios. Results presented, together with
previously published data, suggest stronger specific associations at low PFAA:albumin
mole ratios and weaker nonspecific associations at higher mole ratios. Equilibrium
dialysis yields primary association constants of ~106 M-1 for PFOA and PFNA, for a
class of one to five high affinity binding sites. A high protein-water partition coefficient
for PFNA (log KPW > 4) relative to neutral HOCs supports the characterization of
specific binding at low ligand concentrations.
NanoESI-MS is a useful technique for more rapidly characterizing PFAA-protein
interactions. However, a wide range of calculated association constants and sensitivity
of complexes to instrument conditions limit the utility of nanoESI-MS as a fully
quantitative method. Stoichiometry values obtained from mass spectrometry
demonstrate up to eight bound PFAAs per BSA molecule at a 4:1 mole ratio. Binding
constants from nanoESI-MS experiments are on the order of 105 M-1 for both PFOA and
PFNA, lower but in qualitative agreement with solution-based values determined via
equilibrium dialysis.
Because Kow may underestimate the bioaccumulative potential of PFAAs, a serum
protein association constant or protein-water distribution coefficient may be useful in
106
characterizing the bioaccumulative potential and in vivo bioavailability of long-chain
PFAAs. However, as proportions of various proteins vary among species and in time,
and likely also have different affinities for PFAAs, further analysis is required to test
the ability of protein partitioning to enhance perfluoroalkyl acid bioaccumulation
models.
Supporting Information. Contains (1) analytical and experimental details, (2)
results of fitting approaches, and (3) additional and summary results from nanoESI-MS.
Acknowledgment. This work was supported by the National Defense Science and
Engineering Graduate Fellowship, the Stanford University UPS Foundation and Woods
Institute for the Environment, and the National Science Foundation Graduate Research
Fellowship Program. We thank Pavel Aronov and Allis Chien from the Stanford
University Mass Spectrometry Laboratory.
107
5.5 Supporting Information
Figure 5.4S. Structures and names of perfluoroalkyl acids (PFAAs) used in this study.
Equilibrium dialysis results fitting approach. As previously described, a
nonlinear curve fit was applied to equilibrium dialysis results using the two-class
binding equation:
! =n1K
a,1[L]
1+Ka,1[L]
+n2K
a,2[L]
1+Ka,2[L]
(5.16S)
where
!
" is average number of bound ligands per protein molecule, L is the free ligand
concentration, Ka,1 and Ka,2 and are the association constants and n1 and n2 are the total
number of binding sites for each class of binding sites. For results presented in Tables
5.4S and 5.5S, association constants of 8000 M-1 for PFNA and 630 M-1 for PFOA were
inserted for Ka,2 in Equation 5.16S. Initial guess values for n2 were 30 and 100 for
PFNA and PFOA data, respectively. A range of initial guess values for n2 was tested for
the non-weighted PFNA and PFOA data fits in Tables 5.4S and 5.5S and did not
influence the fit in these cases. Parameters determined for Equation 5.16S without
insertion of values for Ka,2 showed greater error on determined binding stoichiometries
and association constants for n2 and Ka,2 and are presented in Table 5.6S. For these fits,
initial guess values were Ka,2 = 630 M-1 and n2 = 100 for PFOA and Ka,2 = 8000 M-1 and
n2 = 30 for PFNA. The results for PFAA bound to albumin are obtained by subtracting
the measured free (reservoir) PFAA concentrations from the corresponding total (final
bag) concentrations, displayed in Figure 5.8S. The error of the bound concentrations is
thus a propagation of error of both the free PFAA concentration and the total PFAA
concentration. The standard deviations of bound concentrations (Sbound) may be
calculated from the standard deviation of the reservoir triplicate samples for a single
108
point (Sfree) and the standard deviation of the final bag samples (Stotal) as:
Sbound = (Sfree )2+ (Stotal )
2 (5.17S)
These deviations were linearly correlated with the free and total PFAA
concentrations, such that at higher concentrations, the error of measured values
increases. This correlation is displayed in Figure 5.9S for measured free PFNA
concentrations in tests with 1 µM BSA. Consequently, a weighting factor may be
applied using the Kaleidagraph software when fitting the data to Equation 5.16S.
However, because standard deviations were determined only from triplicate samples at
each point, weighting bound concentrations in the fitted equation by the standard
deviation for that point was not performed. Alternatively, fits were tested with
weighting factors inversely proportional to the free, total, or bound measured PFAA
concentrations or the square of these values. Although fit parameters for PFOA (Table
5.4S) and PFNA (Table 5.5S) changed with different weighting factors applied, results
were consistently of the same order of magnitude for primary association constants and
number of primary binding sites. For these fits, initial guess values for n2 were 100 for
PFOA and 30 for PFNA, unless otherwise noted. In some cases for PFOA the fit did not
converge or yield physiologically relevant results for a fitted parameter, so the initial
guess was modified and data refit. Due to the large standard deviation and strong
influence on the fitted parameters of measurements at the two highest free PFOA
concentrations, these two data points were excluded from the non-weighted PFOA fits
presented in Tables 5.4S and 5.6S. Non-weighted fits for the full PFOA dataset did not
yield physiologically relevant results.
PFOA and PFNA data sets span several orders of magnitude, and as expected, are
not fit well by a model that represents only one binding class (including only Ka,1 and
n1). However, a subset of the experimental results (data for PFAA:albumin mole ratios
less than 1 or 5) was generally fit well by a one class model (Figure 5.10S), although
several outliers reduced the R2 for the fit of the non-weighted PFOA data. Fits utilizing
a one-class model yielded primary association constants similar to those obtained with a
two-class model applied to the full PFAA:albumin mole ratio range (Table 5.7S). Errors
in Tables 5.4S through 5.7S represent the standard error calculated by the Kaleidagraph
software for each parameter.
109
Statistical comparisons for nanoESI-MS results. Statistical comparisons of
results presented in Tables 5.8S – 5.11S were conducted using a student’s two-tailed t-
test assuming equal variance. PFOA and PFNA, Ka,1, Ka,2, and Ka,3 values are not
statistically different from one another (p > 0.1). For PFDA, pooled results for Ka,1 from
all exposure concentrations collected at 100 V are significantly greater than Ka,2 (p <
0.1) and Ka,3 (p < 0.05), indicating a somewhat stronger first binding site. However, all
PFDA-BSA measured affinities are on the order of 104 M-1, and Ka,2 and Ka,3 are not
statistically different. Ka,1 for PFDA was significantly less than Ka,1 for PFOS (p < 0.05)
although both values are on the order of 104 M-1. Pooled results for Ka,1 from data
collected at three concentration ratios and 100 V were compared between each PFAA.
Results for PFDA Ka,1 were significantly less than that for PFOA (p < 0.1) , PFNA (p <
0.05), and PFOS (p < 0.05). No other comparisons for Ka,1 determined at 100 V were
statistically significant. Further, Ka,1 results for PFOA, PFNA, and PFDA collected at
the 0.5 PFAA:BSA mole ratio and 100 V or 130 V were not statistically different. For
PFDA and PFOS, the averages of Ka,1 calculated at a PFAA:BSA mole ratio of 0.5 and
100 V are 3.5 × 104 M-1 and 7.4 × 104 M-1, respectively.
110
Figure 5.5S. Samples taken prior to equilibration in the reservoir from control bags
containing only buffer and the PFAA spike are compared to samples taken from test
bags containing 1 µM BSA with the same PFAA spike. Points fall along the 1:1 line
(plotted), indicating minimal effects from the BSA matrix in LC-MS/MS sample
analysis. The average relative standard deviation of initial bag samples from tests was
9% for PFOA and 11% for PFNA. Error bars represent 95% confidence intervals for
triplicate samples from the same dialysis bag.
111
Figure 5.6S. Reservoir samples taken over time in a PFNA equilibrium dialysis test
indicate equilibrium of the system after 24 hours. At equilibrium, control bag and
reservoir sample concentrations were also equivalent (data not shown). Error bars
represent 95% confidence intervals for triplicate samples from the same reservoir.
Figure 5.7S. Reservoir samples taken over time in a PFOA equilibrium dialysis test
suggest equilibrium of the system after approximately 48 hours. Samples were taken
after 48 hours for all PFOA tests. Error bars represent 95% confidence intervals for
triplicate samples from the same reservoir.
112
0.01
0.1
1
10
100
1000
0.01 0.1 1 10 100
Tota
l [P
FO
A] (!
M)
Free [PFOA] (!M)
0.1
1
10
100
1000
0.01 0.1 1 10 100
Tota
l [P
FN
A] (!
M)
Free [PFNA] (!M)
Figure 5.8S. Measured total and free PFOA and PFNA concentrations taken at
equilibrium from dialysis bag and reservoir samples, respectively. Results for bound
PFAA are obtained by subtracting the measured free PFAA concentrations from the
corresponding total concentrations.
113
Figure 5.9S. Standard deviations of triplicate measurements of bound PFAAs (Sbound)
were linearly correlated with free PFAA concentrations, as shown above for PFNA in
1µM BSA equilibrium dialysis tests. Consequently, a weighting factor may be
employed to account for larger error at higher measured concentrations.
114
Table 5.4S. Association constants (Ka,1) and binding stoichiometries (n1 and n2) for
PFOA binding to 1 µM BSA as determined by equilibrium dialysis for a range of
applied weighting factors. In these fits, 0.00063 µM-1 was inserted for Ka,2 in Equation
5.16S. Fits did not converge or yield physiologically relevant results for the non-