Science of the Total Environment 793 (2021) 148453

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Science of the Total Environment

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Is there an urban pesticide signature? Urban streams in five U.S. regions

share common dissolved-phase pesticides but differ in predicted
aquatic toxicity
Lisa H. Nowell a,⁎, Patrick W. Moran b, Laura M. Bexfield c, Barbara J. Mahler d, Peter C. Van Metre d,1,
Paul M. Bradley e, Travis S. Schmidt f, Daniel T. Button g, Sharon L. Qi h
a
U.S. Geological Survey, California Water Science Center, Placer Hall, 6000 J St., Sacramento, CA 95819, United States of America
b
U.S. Geological Survey, Washington Water Science Center, 934 Broadway, Suite 300, Tacoma, WA 98402, United States of America
c
U.S. Geological Survey, New Mexico Water Science Center, 6700 Edith Blvd NE, Bldg E, Albuquerque, NM 87113, United States of America
d
U.S. Geological Survey, Oklahoma-Texas Water Science Center, 1505 Ferguson Lane, Austin, TX 78754, United States of America
e
U.S. Geological Survey, South Atlantic Water Science Center, 720 Gracern Rd., Suite 129, Columbia, SC 29210, United States of America
f
U.S. Geological Survey, Wyoming-Montana Water Science Center, 3162 Bozeman Ave., Helena, MT 59601, United States of America
g
U.S. Geological Survey, Ohio-Kentucky-Indiana Water Science Center, 6460 Busch Blvd., Suite 100, Columbus, OH 43229, United States of America
h
U.S. Geological Survey, Cascades Volcano Laboratory, 1300 SE Cardinal Ct, Vancouver, WA 98683, United States of America

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Urban centers across the USA shared a

common 16-compound “urban pesti-
cide signature.”
• Pesticide occurrence was affected more
strongly by basin urbanization than by
region.
• Potential toxicity varied by region/
urban center but was driven by same
pesticides.
• Potential toxicity at urban sites was high
for invertebrates, lower for plants & fish.
• Potential invertebrate toxicity was due
to imidacloprid, fipronil & organo
phosphates.

a r t i c l e i n f o a b s t r a c t

Article history: Pesticides occur in urban streams globally, but the relation of occurrence to urbanization can be obscured by re-
Received 5 April 2021 gional differences. In studies of five regions of the United States, we investigated the effect of region and urban-
Received in revised form 1 June 2021 ization on the occurrence and potential toxicity of dissolved pesticide mixtures. We analyzed 225 pesticide
Accepted 10 June 2021
compounds in weekly discrete water samples collected during 6–12 weeks from 271 wadable streams; develop-
Available online 15 June 2021
ment in these basins ranged from undeveloped to highly urbanized. Sixteen pesticides were consistently de-
Editor: Damia Barcelo tected in 16 urban centers across the five regions—we propose that these pesticides comprise a suite of urban
signature pesticides (USP) that are all common in small U.S. urban streams. These USPs accounted for the major-
ity of summed maximum pesticide concentrations at urban sites within each urban center. USP concentrations,

Abbreviations: a.i., active ingredient; ALB, aquatic-life benchmark; BQ, benchmark quotient; CACC, California Stream Quality Assessment; Cmax, maximum concentration; dbRDA,
distance-based redundancy analysis; EC50, median effective concentration; LC50, median lethal concentration; LC-MS/MS, liquid chromatography with tandem mass spectrometry;
MW, Midwest Stream Quality Assessment study; NE, Northeast Stream Quality Assessment study; NWALT, National Water Quality Assessment Wall-to-Wall Anthropogenic Land Use
Trends database; NWQN, National Water Quality Network; OP, organophosphate; PNW, Pacific Northwest Stream Quality Assessment study; PNW-OR, PNW Oregon subregion; PNW-
WA, PNW Washington subregion; PTI, Pesticide Toxicity Index; RSQA, Regional Stream Quality Assessment studies; SEP, Southeast (Piedmont) Stream Quality Assessment study; TC, tox-
icity concentration; USGS, U.S. Geological Survey; USP, urban signature pesticide.
⁎ Corresponding author.
E-mail address: lhnowell@usgs.gov (L.H. Nowell).
1
Deceased May 8, 2021.

https://doi.org/10.1016/j.scitotenv.2021.148453
0048-9697/© 2021 Published by Elsevier B.V.
L.H. Nowell, P.W. Moran, L.M. Bexfield et al. Science of the Total Environment 793 (2021) 148453

Keywords: mixture complexity, and potential toxicity increased with the degree of urbanization in the basin. Basin urbani-
Pesticide mixtures zation explained the most variability in multivariate distance-based models of pesticide profiles, with region al-
Urbanization ways secondary in importance. The USPs accounted for 83% of pesticides in the 20 most frequently occurring 2-
Invertebrate toxicity compound unique mixtures at urban sites, with carbendazim+prometon the most common. Although USPs
Imidacloprid
were consistently detected in all regions, detection frequencies and concentrations varied by region, conferring
Carbendazim
Fipronil
differences in potential aquatic toxicity. Potential toxicity was highest for invertebrates (benchmarks exceeded
in 51% of urban streams), due most often to the neonicotinoid insecticide imidacloprid and secondarily to organ-
ophosphate insecticides and fipronil. Benchmarks were rarely exceeded in urban streams for plants (at 3% of
sites) or fish (<1%). We propose that the USPs identified here would make logical core (nonexclusive) constitu-
ents for monitoring dissolved pesticides in U.S. urban streams, and that unique mixtures containing imidacloprid,
fipronil, and carbendazim are priority candidates for mixtures toxicity testing.
© 2021 Published by Elsevier B.V.

1. Introduction Historically, more attention has focused on monitoring herbicides

The value of healthy urban streams to the vitality and quality of life
in urban and peri-urban areas is gaining recognition. About 82% of the
U.S. population and 55% of the global population (2018 data) (United
Nations, 2019) live in urban areas where healthy urban streams have
social value, providing green spaces for recreation, improving esthetics,
and adding to quality of life and even to home values (Sarvilinna et al.,
2017; Tsakaldimi and Tsitsoni, 2015). A healthy urban stream is a
green corridor that provides habitat and breeding grounds for numer-
ous species, and biodiversity is among the ecosystem services provided
by such streams. Urbanization, however, adversely affects the chemis-
try, habitat, and ecology of streams, disrupts biological functions, and
causes loss of sensitive species (Allan, 2004; Coles et al., 2012; Cuffney
et al., 2010; Pickett et al., 2011; Utz et al., 2011; Walsh et al., 2005;
Walsh et al., 2007). These effects are part of the “urban stream syn-
drome,” which includes increases in the number and concentration of
contaminants in runoff, higher water temperatures, flashier stream
flows, bank destabilization and sedimentation, and altered biological
communities (Walsh et al., 2005).
The urban environment includes homes, yards, gardens, public
parks, golf courses, public and commercial buildings, and rights-of-
way. Pesticides are associated with all of these land uses, and are applied
by government, industrial, and commercial organizations, professional
applicators, and homeowners. Common outdoor nonagricultural
(urban) applications include use on lawns and gardens; ornamental
plants; commercial landscaping and turf; foundations, construction ma-
terials, and building facades; and roadways, railways, and fences
(Hoffman et al., 2000; Shamim et al., 2014). In 2012, 42 million kg of
conventional pesticides were used in nonagricultural applications in
the U.S., consisting of 25, 12, and 5 million kg of herbicides, insecticides,
and fungicides, respectively (Atwood and Paisley-Jones, 2016). This
constitutes about 11% of total conventional pesticide use in the U.S.,
with the remainder used in agriculture (Atwood and Paisley-Jones,
2016). On a per area basis, however, pesticide use rates in urban areas
may exceed those in agricultural applications (Haith and Duffany,
2007; Hoffman et al., 2000).
Pesticides can be delivered to hydrologic systems by surface runoff,
spray drift, atmospheric deposition, and direct application to aquatic en-
vironments (Gilliom et al., 2006). In urban areas, impervious surfaces
contribute to pesticide loss rates to streams that exceed those in agricul-
tural areas (Blanchoud et al., 2004; Wittmer et al., 2011), and delivery of
runoff to urban streams is exacerbated by stormwater drainage systems
(Walsh et al., 2005). The percentage of area that is impervious (pave-
ment, asphalt, buildings) ranges from 20–65% of surfaces in residential
areas (with lots of 1/8 to 1 acre) to 70–85% for commercial and
industrial areas (U.S. Department of Agriculture, 1986). As a result,
pesticides are widespread in urban streams globally (Bradley et al.,
2017; Gilliom et al., 2006; Meftaul et al., 2020; Rippy et al., 2017;
Stehle et al., 2019).
and insecticides than fungicides, especially at large spatial scales
(Malaj et al., 2014; Rippy et al., 2017; Stone et al., 2014b). Herbicides
are of interest because they are used in large quantities, most are highly
water soluble so are prone to runoff from terrestrial environments
(Fairchild, 2011), they are frequently detected in both agricultural and
urban streams (Stehle et al., 2019; Stone et al., 2014b), and some are
being investigated for potential effects on endocrine and immune func-
tion in vertebrates (Fairchild, 2011; Farruggia et al., 2016). Insecticides
have been widely monitored in streams, including in urban areas,
largely because of ecological concerns (Carpenter et al., 2016; Gilliom
et al., 2006), with multiple insecticide classes having shown toxicity to
aquatic invertebrates in ambient toxicity tests (Anderson et al., 2017;
Bailey et al., 2000; de Vlaming et al., 2000; Holmes et al., 2008;
Weston and Lydy, 2016). Fungicides have rarely been monitored at
large spatial scales, although studies have targeted and found fungicides
at a limited number of sites in urban streams (Bradley et al., 2017;
Metcalfe et al., 2016), wastewater (Bollman et al., 2014a; Kahle et al.,
2008), and storm water (Bollman et al., 2014b).
Pesticides by design are bioactive and they are associated with ad-
verse effects on nontarget organisms in stream communities, including
direct toxicity, indirect effects on prey, predators, and competitors, and
effects on community structure and function (Gibbons et al., 2015;
Peters et al., 2013; Schäfer et al., 2012). For herbicides, the most suscep-
tible nontarget taxa to direct toxicity are vascular and nonvascular
plants. Direct effects may be structural (e.g., biomass, chlorophyll a,
and species composition) or functional (e.g., photosynthesis rates). In
field situations, herbicides temporarily inhibit growth of non-target
algae and macrophytes, but populations typically recover once expo-
sure is reduced (Fairchild, 2011). However, effects on community struc-
ture and function may occur if high herbicide concentrations are
sustained (Farruggia et al., 2016). For insecticides, which are intended
to kill or control insects, invertebrates are the most sensitive taxa to di-
rect toxicity, but indirect effects can occur at other trophic levels
(Hasenbein et al., 2016; Miller et al., 2020; Rogers et al., 2016). Multiple
insecticide classes have been detected in urban streams at concentra-
tions predicted or observed to cause direct toxicity to invertebrates
(Bailey et al., 2000; Budd et al., 2020; Ensminger et al., 2013; Phillips
et al., 2010; Stone et al., 2014a). Fungicides are toxic to a wide range
of nontarget organisms because they act on basic biological processes
and have direct and indirect effects on microorganisms, macrophytes,
invertebrates, and vertebrates (Zubrod et al., 2019). Fungicides have
been shown to adversely affect community functional processes such
as litter decomposition (Elskus et al., 2016).
Despite the widespread occurrence of pesticides in urban settings,
relatively little is known about similarities and differences in occurrence
and potential toxicity of pesticides across a wide range of urban centers
and types and degree of urban development. Because the same kinds of
nonagricultural pesticide applications are common in most urban
centers (e.g., ornamentals, turf, structural pest control), the pesticides

2
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
detected in urban streams may be more uniform than in agricultural
streams, where the pesticides used depend on the local crops. However,
pesticide inputs to streams can be affected by regional factors, such as pest
pressures, environmental conditions, background land use within the ur-
banized or urbanizing areas, and land- and water-management practices.
Here we evaluate the occurrence and potential toxicity of dissolved
pesticides in U.S. urban streams and investigate whether there is a com-
mon “urban pesticide signature.” We collected weekly water samples
from 271 streams across five major U.S. regions over a 6–12 week period
and measured dissolved concentrations of 108 pesticide active ingredi-
ents (a.i.) and 117 pesticide degradates. Land use in the 271 drainage
basins ranged from undeveloped to highly urbanized. Our objectives
were to characterize occurrence of dissolved pesticides and their mix-
tures in urban streams and in particular to identify pesticides that
were consistently detected; to assess the degree and likely drivers of po-
tential toxicity from pesticides in urban streams; to determine whether
pesticide occurrence and potential toxicity varied with the degree of ur-
banization in the drainage basin; and to evaluate differences and simi-
larities within and among regions. To do this, we set criteria for
identifying an urban pesticide signature and we used a variety of uni-
variate and multivariate techniques to assess the relations of basin ur-
banization and region to dissolved pesticide occurrence and potential
toxicity. We tested the representativeness of our data for urban streams,
both spatially and temporally, by comparing them with data from addi-
tional U.S. urban streams located outside the RSQA regions and with
long-term pesticide data available for a subset of RSQA sites. Finally, to
ascertain whether common urban pesticides were unique to urban set-
tings, we compared their urban occurrence to that in agricultural
streams within the same regions.
2. Methods
2.1. Study design
Samples for pesticide analysis were collected as part of the U.S.
Geological Survey (USGS) Regional Stream Quality Assessment studies
(RSQA) (https://webapps.usgs.gov/rsqa/#!/), which targeted streams
in five U.S. regions: the Midwest (MW) in 2013, Southeast Piedmont
(SEP) in 2014, Pacific Northwest (PNW) in 2015, Northeast (NE) in
2016, and the California Central Coast (CACC) in 2017. (Fig. 1; Supple-
mental Information (SI) Figs. A1–A6; SI Table A.1). Weekly water
Fig. 1. Study area boundaries for the five U.S. Geological Survey regional stream-quality assessment studies.
3
Science of the Total Environment 793 (2021) 148453
samples were collected at 75 to 100 wadable streams (SI Figs. A2–A6,
SI Table A.2) in each region during 6 to 12 weeks of the stated year, de-
pending on the region (SI Table A.1). Most RSQA sampling sites were lo-
cated along a gradient of the dominant land use in the region, either
agriculture (in MW) or urban (in the other regions), with additional
sites selected to represent other land uses characteristic of the region.
Study areas are further described elsewhere (Coles et al., 2018;
Garrett et al., 2017; Journey et al., 2015; May et al., 2020; Sheibley
et al., 2017).
2.2. Sampling sites and geospatial data
Stream drainage basins ranged in size from 21 to 1650 km2 (median
43 km2) and were characterized by land use and related variables (SI
Table A.2). Most land-use data were from the National Water-Quality
Assessment Wall-to-Wall Anthropogenic Land Use Trends (NWALT) da-
tabase (Falcone, 2015). Imperviousness was from the 2011 National
Land Cover Database (U.S. Geological Survey, 2014; Xian et al., 2011),
golf course area was from ESRI (2019), and population and housing
data were from the 2010 U.S. Census (Falcone, 2018). We characterized
32 variables in each basin, representing land use, population, and infra-
structure (defined in SI Table A.10). Seventeen of these variables were
urban-related: 7 were general urban variables (e.g., total urban land
use, imperviousness) and 10 represented specific types of urban land
use (e.g., commercial services, low-medium density residential, golf
course area).
Each RSQA site was assigned to one of seven land-use categories (de-
fined in SI Table A.11): Urban, subdivided into low, medium and high;
Agricultural, subdivided into low and high; Mixed (containing agricul-
tural and urban land); and Undeveloped. Of the 442 RSQA sites, 271 rep-
resented a gradient of urbanization from undeveloped to highly
urbanized and are the primary focus of this paper. The remaining, agri-
culturally influenced sites are beyond the scope of this paper, although
summary statistics for all land-use site categories are provided in Sup-
plemental Information (SI Tables B.1–B.5).
The urban gradient can be represented using continuous variables
(e.g., total urban land or imperviousness) or categorically (sites in the
Urban-high, -medium, and -low and Undeveloped categories). For
urban-gradient sites, urban land within the basin was <5% for Undevel-
oped sites, 5–25% for Urban-low, 25–75% for Urban-medium, and
75–99% for Urban-high sites; also, urban-gradient sites had minimal
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
agricultural influence (<5% cropland and <25% total agricultural land in
the basin). For analyses requiring a strong urban signal we used the sub-
set of Urban-medium and Urban-high (Med/high urban) sites (n =
143).
Urban-gradient sites were assigned to urban centers based on Met-
ropolitan Statistical Areas from the U.S. Census Bureau (2018) (SI Part
A.6, SI Table A.2). Sixteen urban centers (those that had 5 or more
urban-gradient sites, including 3 or more Med/high urban sites) were
used in some analyses. One of the 16 urban centers was the MW,
where urban-gradient sites were combined into a single “urban center”
to represent this region, although technically these sites belonged to dif-
ferent Metropolitan Statistical Areas. The urban gradient in the MW
(consisting of 2 Undeveloped and 7 Urban-high sites) was less extensive
than those for the other RSQA studies (each with 60–74 sites).
2.3. Sample collection and analysis
Discrete water samples were collected weekly during the spring-
summer (when streams were wadable) for 12 weeks in MW, 10
weeks in SEP and PNW, 9 weeks in NE, and 6 weeks in CACC. About
15% of sites were sampled only during the last 4 weeks of the study;
these were mostly in Undeveloped basins, where pesticide occurrence
was expected to be low. Water samples were collected and processed
by an isokinetic, equal-width increment method following USGS proto-
cols (U.S. Geological Survey, 2006).
Water samples were syringe-filtered (0.7-μm pore-size glass-fiber)
in the field and analyzed for 225 pesticides and degradates using a
broad-spectrum, direct aqueous-injection liquid chromatography with
tandem mass spectrometry (LC-MS/MS) method (Sandstrom et al.,
2015) at the USGS National Water Quality Laboratory in Denver, Colo-
rado. The filtration step excludes pesticides that are sorbed to particu-
lates (>0.7 μm); this targets bioavailable (dissolved) concentrations,
as sorption reduces the bioavailability of hydrophobic compounds in
the water column (Spurlock et al., 2005). The highest reporting level
used by the laboratory during the five-year study was 2–100 ng/L for
92% of analytes (SI Table A.3). Laboratory reagent spikes indicated that
median recoveries were in the acceptable range (70–130%; SI
Table A.5). Some results were reported as less than a raised reporting
level (RRL) because of issues resulting in uncertainty in identification
or quantification of the compound. Nondetections reported with a RRL
greater than a certain magnitude were excluded from the dataset be-
cause of an increase in false negative results (Bexfield et al., 2020).
Data were censored in a few sample records (0.03%) based on field or
laboratory blank samples to minimize bias from potential incidental
contamination (SI Parts A.4, A.5). The analytical method, quality control
(QC) measures, and data review and processing are further described in
SI Parts A.3–A.5 and QC data are summarized in SI Tables A.4–A.9.
2.4. Screening for potential toxicity
Three types of screening tools were used to assess potential toxicity
of pesticides and their mixtures. Aquatic-life benchmarks (ALB) from
the U.S. Environmental Protection Agency (2018) and the Pesticide
Toxicity Index (PTI) were used to assess potential toxicity of individual
pesticides and of pesticide mixtures, respectively, to fish, invertebrates,
and(or) plants (SI Table A.3). A Pesticide Rank Index was used to
identify pesticides that approached but did not exceed an ALB.
Acute ALBs for fish, invertebrates, and plants were compared with
concentrations of individual pesticides in each sample. Chronic bench-
marks for fish and invertebrates were compared to the highest 60- or
21-day (respectively) average pesticide concentration occurring during
the study, following U.S. Environmental Protection Agency (2017b) de-
terministic risk-assessment procedures. A benchmark quotient (BQ)
was computed by dividing the environmental concentration by the ap-
propriate ALB and exceedance was reported if the BQ was >1. ALB com-
parisons are minimum values because non-detections were assumed
4
Science of the Total Environment 793 (2021) 148453
equal to zero, benchmarks were available for only 103 of the 108 pesti-
cides and 26 of the 117 pesticide degradates analyzed in this study, and
the weekly sampling design in these studies underestimates acute ex-
posures (Norman et al., 2020). In addition, the reporting level was
higher than the lowest ALB value available for nine insecticides and
one herbicide (for which the lowest ALBs were invertebrate and plant
benchmarks, respectively); in these cases, the applicable benchmark ex-
ceedance rates may be biased low.
The Pesticide Toxicity Index (PTI) was used to evaluate the potential
toxicity of pesticide mixtures in each sample. The PTI is a screening-
level, concentration addition (CA) model used to assess potential cumu-
lative toxicity of mixtures, and is available for three taxonomic groups:
fish, cladocerans, and benthic invertebrates (Munn et al., 2006; Nowell
et al., 2014). CA is technically appropriate only for compounds with a
common mode of action. A CA model may overestimate the toxicity of
pesticide mixtures with multiple modes of action, but typically by a fac-
tor of 2–3 or less (Belden et al., 2007; Deneer, 2000; Faust et al., 2003;
Warne, 2003). To compute the PTI for a sample, the measured concen-
tration of each pesticide was divided by its toxicity concentration (PTI-
TC), which is an acute median lethal concentration (LC50) or median ef-
fect concentration (EC50) for the specified taxonomic group; the
resulting compound-specific toxicity quotients, or PTI toxic units (TU),
then were summed. PTI values were calculated here using sensitive
PTI-TCs (i.e., 5th percentile or minimum toxicity value, depending on
the quantity of available data) and resulting in a Sensitive-PTI (hence-
forth, PTI) score from Nowell et al. (2014) (SI Part A.7). Above a PTI
threshold of 1, there is a high likelihood of acute toxicity because the un-
derlying toxicity values are LC50s (Nowell et al., 2014).
A Pesticide Rank Index, consisting of the weighted percentage of
samples approaching U.S. Environmental Protection Agency (2018)
ALBs (further described in SI Part A.7.2), was calculated for samples col-
lected at Med/high urban sites during the last 6 weeks of the study pe-
riod, by region. This was modified from the Ranking Index of
Kuzmanovic et al. (2019), which was in turn based on a prioritization
approach by von der Ohe et al. (2011). The Rank Index can range from
0 (if all sites have mean concentrations less than 1% of the ALB) to 100
(if all sites have mean concentrations that exceed the ALB).
Acute toxicity toward invertebrates was predicted for a site if the
Acute-Invertebrate BQ was >1 for any individual pesticide, or if the
PTI score for the pesticide mixture was >1 for either cladocerans or ben-
thic invertebrates, in one or more samples. Chronic toxicity at a site was
predicted if the 21-d average concentration of any pesticide exceeded
the Chronic-Invertebrate ALB at any time during the study period. Pre-
dictions were made similarly for fish, except that Chronic-Fish ALBs
were compared with 60-d average concentrations (or with the average
over the sampling period, if this was <60 d) and fish PTI scores were
used. For aquatic plants, acute toxicity was predicted for a site if one
or more individual pesticide concentrations exceeded the lower of the
applicable Acute-Nonvascular or Acute-Vascular Plant ALBs in one or
more samples. A sample mixture resulted in a cumulative acute toxicity
prediction only if the PTI score was >1 and all individual pesticide BQs
were <1. Mixtures were not assessed for chronic toxicity as part of
site-specific toxicity predictions, although Chronic-Fish, Chronic-
Invertebrate, and Acute Nonvascular Plant BQs were summed to assess
the relative contributions of individual pesticides to potential toxicity
for individual samples (Section 3.5).
2.5. Data analysis
Analyses use urban-gradient sites (Undeveloped to Urban-high, n =
271), Med/high urban sites (n = 143), or Urban sites (low/med/high; n
= 205), depending on the objective. Urban-gradient sites are used to
examine relations of pesticide results to degree of urbanization in the
basin. To equally weight sites with different numbers of samples, the
urban-gradient dataset was restricted to samples collected during the
last 4 weeks of each study. To compare urban pesticide results among
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
regions or urban centers, the Med/high urban dataset (n = 143 sites)
was used to provide a strong, clean urban signal. That dataset was re-
stricted to samples collected during the last 6 weeks of each study pe-
riod, the maximum available for all Med/high urban sites. To identify
consistent urban pesticides and to assess benchmark exceedances,
data for all samples from all Urban sites (n = 205) were used to capture
as many detections and exceedances as possible.
Because weekly discrete samples represent a snapshot of pesticide
occurrence and therefore underestimate pesticide exposure in flowing
waters (Crawford, 2004; Norman et al., 2020), we focused much of
our analyses on the maximum concentration detected (Cmax) at each
site during the stated number of weeks. Most analyses (e.g., mixtures
and multivariate analyses) were limited to pesticide a.i., although
benchmark exceedance calculations encompassed degradates also, to
fully characterize pesticide exposure. Counts of pesticide a.i. always in-
cluded two degradates (carbendazim and 4-hydroxychlorothalonil) as
surrogates for their parents, thiophanate-methyl and chlorothalonil, re-
spectively, for which we did not have data.
Multivariate analysis was done using PRIMER (version 7) (PRIMER-
e, 2015), and unique mixtures were determined with the USGS program
qwmixtures.pl, run using Perl programming language (version 5.30.1)
on Windows 10 Enterprise version 1809 (Scott et al., 2013). Otherwise,
data analysis was done in R (version 3.6.3) (R Core Team, 2020) using R
Studio (version 1.3.1093) (RStudio Team, 2020).
2.5.1. Criteria for urban signature pesticides
Selection criteria were developed to identify the most consistently
detected pesticide a.i. in filtered water samples from urban streams dur-
ing the study periods. Filtration at 0.7 μm excludes the major portion of
hydrophobic pesticides (such as pyrethroid or organochlorine insecti-
cides) that may be present in whole water (Hladik and Kuivila, 2009).
In the RSQA studies, therefore, we relied on bed sediment sampling to
identify hydrophobic pesticides present in the sampled streams because
their dissolved concentrations were typically low; results for sediment
are published elsewhere (Mahler et al., 2020; Moran et al., 2020;
Moran et al., 2017). Thus, the selection criteria for consistent urban pes-
ticides pertain to dissolved-phase pesticides only, and did not identify
hydrophobic pesticides that are likely to be present in association with
particulate matter.
A dissolved pesticide was classified as Tier A if it met all of the fol-
lowing criteria: it was detected in filtered water in all 5 RSQA studies
and in all 16 urban centers, had a median detection frequency of >10%
of samples across the 5 regions, and was ranked in the top 10 com-
pounds by use group (i.e., fungicide, herbicide, or insecticide) for both
Cmax and detection frequency. Tiers B and C had more relaxed criteria,
but in different ways. Tier B pesticides met the same criteria, except that
detection was required in only 13 or more of the urban centers (>80%);
this meant that Tier B USPs were not detected in 1–3 of the 16 urban
centers. Tier C pesticides were detected in all 5 RSQA studies and in 15
or more of the urban centers (>90%) and had a median detection fre-
quency among regions >10%, but they were not required to be ranked
among the top 10 per use group; thus, Tier C USPs may be present at
lower concentrations and(or) detection frequencies relative to other
pesticides in their use group but still must be detected in all five regions
and in at least 15 urban centers. We propose that pesticides in Tiers A, B,
and C constitute an “urban signature.” The urban signature pesticides
(USPs) are not a comprehensive list of dissolved pesticides that were
detected in multiple urban streams—for example, any pesticide that
was detected in only 4 of the 5 RSQA studies (e.g., methoxyfenozide)
or was detected in 5 RSQA studies but in only 12 urban centers
(e.g., sulfentrazone) did not meet the stringent criteria for a USP (SI
Table B.6).
2.5.2. Evaluation of urban pesticides at other sites
To ascertain whether the USPs were unique to Urban sites, their de-
tection frequencies and concentrations at Med/high urban sites were
5
Science of the Total Environment 793 (2021) 148453
compared to those at Agricultural sites also sampled during the RSQA
studies.
USP results from RSQA also were compared to results from another
USGS study (the National Water Quality Network [NWQN]) to meet
two objectives: to validate the characterization of USPs and to provide
temporal context for RSQA results. For the NWQN, water samples
were collected year-round at 10 urban and 9 agricultural streams during
2013–2017 using the same sampling procedures and analyzed for pes-
ticides using the same analytical method (Covert et al., 2020) as in the
RSQA studies. Five NWQN urban sites were located outside of the
RSQA regions and USP occurrence at these NWQN sites was investigated
during April–July (similar to the RSQA study period) each year to test
the hypothesis that USPs are likely to occur in U.S. urban streams.
These five sites were classified as Urban-high (n = 2), Urban-medium
(n = 2) or Urban-low (n = 1), and each was sampled between 2 and
4 times per month during April–July. In addition, 10 NWQN sites (5
Urban and 5 Agricultural) were among those sampled by RSQA and
were used to assess the representativeness of the results from the
RSQA sampling year. Concentrations of atrazine measured by RSQA
and NWQN were compared at the ten Agricultural or Urban NWQN/
RSQA sites; although atrazine is not a USP, it has well-documented sea-
sonal occurrence (Gilliom et al., 2006; Thurman et al., 1991), so it was
selected to illustrate variations in seasonality among sites and sampling
years. In addition, concentrations of eight example USPs measured by
RSQA were compared to those in year-round NWQN samples during
the rest of the five-year period (2013–2017) to provide temporal con-
text for RSQA findings at the five Urban RSQA/NWQN sites.
2.5.3. Relations to urbanization and among regions
2.5.3.1. Individual pesticides and pesticide metrics. Relations to urbaniza-
tion were evaluated at RSQA urban-gradient sites for commonly de-
tected pesticides and for pesticide metrics, defined as summed
concentrations by pesticide class (e.g., OP insecticides) or use group
(e.g., total herbicides) (SI Table A.12). Relations to urbanization within
a region were assessed as significant if at least one of three statistical
analyses had significant results: (1) the Cmax values for a pesticide at
urban-gradient sites were significantly correlated (Spearman rank)
with more than one-half (9+) of 17 urban-related variables (examples
below); (2) the linear least-square regression between log-transformed
Cmax at urban-gradient sites and the percentage of urban land use was
significant; and(or) (3) there was a significant difference (Wilcoxon
rank sum test) between concentration distributions at Med/high
urban and Undeveloped sites.
For selected pesticides, potential sources associated with urban land
uses were explored by identifying which of 32 land use-related
geospatial variables (SI Table A.10) was most strongly correlated to
Cmax (i.e., higher Spearman rho). A possible source relation was in-
ferred if a specific land-use type (e.g., residential land use or golf course
area) had the highest rho value for a given pesticide in three or more re-
gions. No inferences were made about possible sources if a general
urban variable (e.g., total urban land use or imperviousness) had the
highest rho value or if there was no consistency among regions.
Significant differences in mixture complexity (number of pesticide
compounds per sample) among regions were assessed at Med/high
urban sites using the Kruskal-Wallis rank sum test, followed by Tukey's
multiple comparison test for differences among means. Censored data
techniques were used to compare concentration distributions at Med/
high urban sites among regions using the NADA package in R (Lee,
2020). Specifically, significant differences in concentration distributions
for common pesticides among regions were analyzed using the Peto-
Prentice test, followed by the Wilcoxon test between each pair of re-
gions. A significance level of p < 0.05 was used in statistical tests.
2.5.3.2. Multivariate analyses. Multivariate techniques were used to eval-
uate the importance of urbanization and to assess similarities and
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
differences in pesticide profiles among regions, where pesticide profile
is defined as the assemblage of Cmax values for pesticide a.i. at individ-
ual sites. Multivariate analyses used a dataset of log-transformed
Cmax+0.01 values for the pesticide a.i. (n = 91) detected at urban-
gradient sites during the last 4 weeks of the study for each region, and
a similar dataset for USPs only (n = 16). To test for significant differ-
ences among pesticide profiles in each region, the non-parametric anal-
ysis of similarity (ANOSIM routine) was done on Euclidean distance
measures of the pesticide concentrations at each site—this is a multivar-
iate analog of ANOVA. The SIMPER routine was then applied, which uses
the Euclidean distance measure to analyze the contribution of each pes-
ticide to the average squared distance (dissimilarity) between sites
within a group (in this case, region). SIMPER also analyzes the contribu-
tion of pesticides to the dissimilarities between sites in different regions
by comparing all possible pairs of regions (here, 15 inter-region pairs for
six regions). The SIMPER routine identifies pesticides that best discrim-
inate between sites, within a region and between regions, thus provid-
ing an independent approach to determine which pesticides constitute
an urban signature.
Distance-based linear modeling was used to determine whether
land-use variables for each basin could explain the pesticide concentra-
tion profiles in the regions, analogous to multiple linear regression but
using distance measures representing multivariate space. The DISTLM
routine in Primer 7 was used on both Euclidean and Manhattan
distance-based measures of the pesticide profile. This routine asks
how much variation a linear predictor (or predictors) can explain of
the multivariate (in this case multi-pesticide) data cloud measured
across the sites, as summarized by a distance measure of choice, here
the Manhattan distance. We tested seven predictors: region, percent-
ages of four categories of NWALT urban land use in the basin (commer-
cial services, industrial/military, recreational, and low-medium density
residential) (Falcone, 2015), percentage of imperviousness in the
basin (U.S. Geological Survey, 2014), and golf course area in the basin
(ESRI, 2019) (SI Table A.10). Distance-based redundancy analysis
(dbRDA) was used to visualize the best DISTLM solution as a plot in 2-
dimensional space.
2.5.4. Unique mixtures
To characterize the most common pesticide mixtures in urban
streams, unique mixtures of pesticide a.i. were identified at Med/high
urban sites in each of the six regions. A unique mixture was defined as
a specific combination of two or more pesticides, regardless of other
pesticides present. The number of mixtures in a sample was the set of
all pairwise combinations, triplets, quadruplets, etc., up to the number
of detected pesticides in that sample. Samples evaluated were those col-
lected during the last 6 weeks of each study to equally weight all sites.
Mixtures detected three or more times in a region were reported. Pesti-
cide degradates, although frequently detected in RSQA samples (Mahler
et al., 2021), were not included because the number of mixtures would
be unfeasibly large and we were interested in identifying co-occurring
bioactive compounds. The top 20 mixtures (the 20 that occurred most
frequently) for each number of combinations (i.e., 2, 3, 4 compounds,
etc.) were identified for each region; ties were included.
3. Results
3.1. Urban pesticide occurrence
At least one pesticide compound was detected in 96% of samples from
Med/high urban sites, and 76 pesticides and 66 degradates were detected
at least once (SI Tables B.1–B.5; (Morace et al., 2020)). Twenty-eight
pesticide a.i. (counting carbendazim and 4-hydroxychlorothalonil as
surrogates for their parent pesticides) were detected in all five RSQA
studies. Within the PNW study, pesticide occurrence was substantially
higher in Oregon than Washington (Fig. 2); the PNW therefore was
divided into the subregions PNW-OR and PNW-WA for subsequent
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Science of the Total Environment 793 (2021) 148453
regional comparisons. Henceforth “RSQA study” will be used to
refer to the original five regional studies, and “region” will refer to
the six regions/subregions (considering PNW-OR and PNW-WA
separately). The most frequent detections at Med/high urban sites
tended to occur in the MW and SEP regions and the lowest in
PNW-WA (Fig. 2).
3.1.1. The urban pesticide signature
A suite of 16 pesticide a.i. qualified as one of Tiers A–C (Table 1) and
are proposed as USPs. Eleven USPs were detected in all 16 urban centers
and five in 13–15 urban centers. An additional 11 pesticide a.i. were de-
tected in all 5 RSQA studies and in ≥50% of urban centers; although they
did not meet the stringent criteria for USPs (Tiers A-C), these urban pes-
ticides are labeled as Tier D in SI Table B.6.
The 16 USPs accounted for two-thirds or more of the summed me-
dian Cmax concentrations at Med/high urban sites in each urban center
(Fig. 3A). The summed median Cmax is the sum of the median Cmax
values (one for each pesticide) and is a summary indicator of the high
end of concentrations at the majority of Med/high urban sites within
an urban center. The summed median Cmax was determined for an
urban center by taking (for each pesticide) the Cmax at each Med/
high urban site, then selecting the median Cmax for all Med/high
urban sites within that urban center; the median Cmax values for all
pesticides were then summed for that urban center. Herbicides com-
prised more than 50% of the summed median Cmax in most urban
centers.
3.1.2. Are urban signature pesticides unique to urban settings?
To evaluate whether the USPs were specific to urban streams, we
compared USP occurrence at Med/high urban sites and Agricultural
sites sampled as part of the larger RSQA effort. The USPs dominate the
summed median Cmax at Med/high urban sites (Fig. 3A) but make a
negligible contribution at Agricultural sites in all regions except PNW-
OR, where USPs accounted for about one-third of the total summed me-
dian Cmax (Fig. 3B). Additionally, 7 of the 16 USPs had higher detection
frequencies at Med/high urban sites than Agricultural sites in all regions
(SI Figs. B.1–B.3) and 9 had higher detection frequencies at Med/high
urban sites than at Agricultural sites in 4 or 5 regions, with CACC or
PNW-OR being the occasional exceptions. These results support the
concept that the urban pesticide signature is not shared across all land
uses, although individual USPs were detected at Agricultural sites in
some regions (Fig. 3B, SI Fig. B.4).
3.1.3. Other urban centers
Given the consistent detection of USPs at urban centers in the RSQA
studies, we hypothesized that the USPs would occur in other urban cen-
ters in the U.S. This hypothesis was tested by evaluating USP occurrence
at five (non-RSQA) NWQN urban sites at a comparable time of year
(April–July) during 2013–2017. Of the 16 USPs, 14 were detected at all
5 NWQN urban sites. The two exceptions, 4-hydroxychlorothalonil
and tebuconazole, were detected at four of the five NWQN sites. Further,
the mean detection frequency of 15 of the 16 USPs at the NWQN sites
(i.e., mean percentage of samples with detections during April–July for
all years combined) was greater than or within 1 percentage point of
the mean detection frequency at RSQA Med/high urban sites. The ex-
ception was fipronil, which was detected more frequently at RSQA
Med/high urban sites (48%) than at the NWQN urban sites (mean 36%,
range 7–97%). The summed median Cmax values at the five NWQN
sites (Fig. 3C) resembled those at RSQA Med/high urban sites and
were within the range of values observed at RSQA urban centers
(Fig. 3A).
3.1.4. Representativeness of RSQA data across years and seasons
To assess how the RSQA sampling period (April–July) compared to
the rest of the year and whether the RSQA sampling year was typical
of other years, we used year-round NWQN data collected from 2013
L.H. Nowell, P.W. Moran, L.M. Bexfield et al. Science of the Total Environment 793 (2021) 148453

Fig. 2. Detection frequencies, by region, at Med/high urban sites of the most commonly detected (A) herbicides, (B) insecticides, and (C) fungicides during the study period. Urban
signature pesticides are in bold. The top of the bar represents the total (uncensored) detection frequency (see Supplemental Information A.5.2), and the colored segments of the bar
represent detection frequencies at common assessment thresholds of 10 and 100 ng/L.

7
L.H. Nowell, P.W. Moran, L.M. Bexfield et al. Science of the Total Environment 793 (2021) 148453

Table 1
Urban signature pesticides (USP), the number of urban centers with detections, ranks in use group by maximum concentration and detection frequency, and regions with significant urban
relations via at least one of three statistical methods. All USPs were detected at urban-gradient sites in all five Regional Stream Quality Assessment (RSQA) studies.

Urban signature pesticide Tier Use group No. of urban centers with detectionsa Rank in use group Regions with significant urban relationb

Cmax Detection frequency

4-Hydroxychlorothalonil A F 16 1 4 CNS
Azoxystrobin A F 16 6 2 M N Por S
Carbendazim A F 16 2 1 C M N Por Pwa S
Propiconazole A F 16 4 3 C N Por Pwa S
2,4-D A H 16 1 2 C M N Por S
Diuron A H 16 2 3 C M N Por Pwa S
Prometon A H 16 8 1 C M N Por Pwa S
Triclopyr A H 16 3 8 M N Por Pwa S
Fipronil A I 16 4 1 C N Por S
Imidacloprid A I 16 1 2 C M N Por S
Tebuconazole B F 14 3 5 M N Por S
Bromacil B H 13 5 7 M N Por S
Sulfometuron-methyl B H 15 4 5 C M N Por S
Carbaryl B I 14 2 3 N Por S
Hexazinone C H 15 13 10 N Por
Tebuthiuron C H 16 11 4 C M N Por Pwa S
a
Of 16 urban centers in RSQA studies.
b
Significance: via one or more of three statistical methods (Section 2.5.3.1; SI Table B.9). Regions: C, CACC; M, MW; N, NE; Por, PNW-OR; Pwa, PNW-WA; S, SEP.

to 2017 for 10 sites (5 Urban and 5 Agricultural) common to both the
RSQA and NWQN studies. Atrazine, a non-USP that typically shows
peak occurrence during a “spring flush” (Thurman et al., 1991) was
evaluated at the 10 RSQA/NWQN sites (5 Urban and 5 Agricultural). Al-
though the degree of seasonality varied by site, the RSQA data were rep-
resentative of peak atrazine concentrations during the RSQA sampling
year, and similar patterns were seen from year to year (SI Fig. B.5).
USP occurrence measured by RSQA was compared to year-round
samples from the NWQN at the five Urban RSQA/NWQN sites (exam-
ples shown in SI Fig. B.6). The NWQN data demonstrated that concen-
trations were often higher during the spring-early summer growing
season, which includes the RSQA sampling periods, but that seasonal
patterns were consistent across years and that concentrations measured
during the RSQA studies were typical of concentrations measured dur-
ing other years. While seasonal trends were not part of the design of
the RSQA study, some inferences can be made by comparison of detec-
tion frequencies during the RSQA study period (as % samples with de-
tection) with annual detection frequencies at NWQN/RSQA Urban
sites (as % time with detections) reported for the RSQA sampling year
by Stackpoole et al. (2020). Diuron and bromacil had higher detection
frequencies during the entire year than during the RSQA study period
(SI Table B.7), illustrating that RSQA did not capture peak occurrence
for all pesticides. Some USPs showed more consistent seasonal peaks
during the spring-summer (e.g., imidacloprid) than others
(e.g., bromacil), and seasonality was more distinct at some Urban sites
(e.g., Accotink Creek, VA) than others (e.g., Norwalk Creek, CT; SI
Fig. B.6). Carbendazim detection frequencies were high all year long
(96–100% detection frequencies) at four of the five Urban sites (SI
Table B.7) and concentrations appeared to peak after the RSQA study
period (SI Fig. B.6F).
We further hypothesized that the detection of USPs in the RSQA
study during its sampling year was representative of other years and
tested this hypothesis using NWQN data from the five urban sites com-
mon to both studies. We considered NWQN data collected during
2013–2017 “off years” (i.e., excluding the RSQA sampling year for each
RSQA study). Thirteen of 16 USPs were detected in at least 2 of 4 off
years at every site, and usually during all 4 years; USPs were detected
in all 4 off years in 72% of the 80 cases, i.e., 16 USPs × 5 sites (SI
Table B.8). One USP (hexazinone) was never detected at one RSQA/
NWQN site (Sope Creek in Atlanta, GA) during either RSQA or NWQN
samplings. Although hexazinone was detected at 5 of 12 Med/high
urban sites in the Atlanta urban center, Sope Creek was not one of
them—illustrating that a USP may be commonly detected in an urban
center, but not always detected in every Med/high urban stream within
that urban center.
3.2. Relation to urbanization
The relation between basin urbanization and pesticide concentra-
tions was investigated at RSQA urban-gradient sites; relations between
individual USPs and pesticide metrics were tested with univariate com-
parisons and pesticide a.i. profiles (i.e., the assemblage of Cmax values
for pesticide a.i. at individual sites) were tested in multivariate space.
3.2.1. Urban signature pesticides and pesticide metrics
All 16 USPs were significantly related to urbanization by at least one
statistical method (Section 2.5.3.1) at urban-gradient sites in 2–6 re-
gions. The most consistent relations were for carbendazim, diuron,
prometon, and tebuthiuron (significant in all 6 regions), followed by
2,4-D, imidacloprid, propiconazole, sulfometuron-methyl, and triclopyr
(in 5 regions) (SI Table B.9). Hexazinone had the weakest relation to ur-
banization (significant in only NE and PNW-OR). There were significant
urban relations with 15–16 USPs in three regions (NE, SEP, and PNW-
OR); the fewest significant relations (6 USPs) were for PNW-WA.
PNW-WA also had the lowest pesticide detection frequencies at Med/
high urban sites (Fig. 2, SI Tables B.1–B.5).
Cmax for most pesticide metrics was significantly related to multiple
measures of urbanization for urban-gradient sites (SI Table B.9). Eight of
10 pesticide metrics were significantly correlated with 9 or more of the
17 urban variables in at least 5 regions: total fipronil, sulfonyl urea and
urea herbicides, triazine herbicides, acid herbicides, total fungicides,
total insecticides, total herbicides, and the median number of pesticide
compounds per sample. The largest number of pesticide metrics with
significant urban relations occurred in NE and PMW-OR (where all 10
metrics were significantly correlated with at least 9 of 17 urban vari-
ables). The smallest number occurred in the MW region, but this region
also had the fewest number of urban-gradient sites (n = 9) and lacked
mid-range urban sites. The land use variables with the strongest corre-
lations (highest rho) are specified for each USP and region in SI
Table B.10.
3.2.2. Multivariate models of pesticide profiles
Pesticide profiles at urban-gradient sites, each consisting of the as-
semblage of Cmax values of the 91 pesticide a.i., were strongly related
to measures of urban land use, as determined with distance-based lin-
ear modeling. This urban trend was best explained by the commercial

8
L.H. Nowell, P.W. Moran, L.M. Bexfield et al. Science of the Total Environment 793 (2021) 148453

Fig. 3. Contribution of the 16 urban signature pesticides (USP) to the sum (for all pesticides detected) of median values of the maximum concentrations (Cmax) and of various maximum
benchmark quotients (BQmax). The USPs dominate the summed median Cmax at (A) Med-high urban Regional Stream Quality Assessment (RSQA) sites but make little or no contribution
at (B) Agricultural RSQA sites. The USPs similarly dominate the summed median Cmax at (C) Urban sites from the National Water Quality Network (NWQN) sampled during April–July
2013–2017. After concentrations are toxicity-weighted (as benchmark quotients), the USPs dominate the summed median BQmax at Med/high urban RSQA sites for (D) Chronic-
Invertebrate and (E) Chronic-Fish, but not for (F) Acute-Nonvascular Plants. na, no applicable sites.

9
L.H. Nowell, P.W. Moran, L.M. Bexfield et al. Science of the Total Environment 793 (2021) 148453

services variable; imperviousness performed nearly as well. Region was
a significant variable in all models but was always secondary in impor-
tance to the top urban variable. The overall model that maximized the
adjusted (adj)-R2 value used all seven predictor variables to explain al-
most half (adj-R2 = 0.47) of the variability in Cmax (SI Table B.11). A
model with three explanatory variables (commercial services, low-
medium density residential, and region) performed nearly as well
(adj-R2 = 0.43). In the model containing all seven explanatory vari-
ables, region had the least power to separate among groups (lowest
pseudo-F), although it was the second variable added to the model
after commercial services.
Results for models of USP-only profiles were similar to those for the
full 91-pesticide profiles. Commercial services again explained the most
variance, followed by imperviousness, and region again was the lowest-
performing single variable. The best-fit model again contained all seven
explanatory variables (adj-R2 = 0.54) (Fig. 4). Variability among pro-
files is primarily driven by urbanization, and in particular impervious-
ness, commercial services, and golf course area (reflected in the
horizontal spread of sites and the vectors with the greatest horizontal
length in Fig. 4). Region plays a secondary role (reflected in the vertical
separation of sites in Fig. 4). A four-variable model (commercial ser-
vices, recreation, low-medium density residential, and region) per-
formed nearly as well (adj-R2 = 0.52), and a model using just two
variables, commercial services and region, explained 45% of the varia-
tion in the dataset (adj-R2 = 0.45).
3.3. Differences among regions
Although many pesticides, and the USPs in particular, were detected
in urban streams, their detection frequencies and concentrations dif-
fered by region. Detection frequencies and concentrations at Med/high
urban sites were generally higher in MW and SEP for herbicides
(Fig. 2A) and in MW, SEP, and NE for insecticides and fungicides
(Fig. 2B, C). Detection frequencies and concentrations at Med/high
urban sites were lowest in PNW-WA and CACC for herbicides and
fungicides, and in PNW-WA for insecticides. A few pesticides were de-
tected predominantly in one or two regions only, such as the herbicides
acetochlor in MW and chlorsulfuron in MW and PNW-OR and the insec-
ticides methoxyfenozide in CACC and ethoprop in PNW-OR (SI
Tables B.1–B.5).
Cmax distributions for the 16 USPs differed significantly among
some regions, and Cmax for imidacloprid was significantly different
among all 6 regions (SI Fig. B.7). Cmax distributions were typically
highest in MW and lowest in PNW-WA. Concentration distributions
for 11 of the 16 USPs were significantly different between PNW-OR
and PNW-WA, which contributed to the decision to treat these sepa-
rately in inter-regional analyses.
Pesticide profiles in multivariate space, like individual USPs, dif-
fered significantly among regions, based on multivariate ANOSIM
tests. The Cmax for the 91 pesticides detected at urban-gradient
sites were significantly different from one another in 12 of 15
inter-region pairs (i.e., in comparisons between all possible combi-
nations of two regions). Only SEP and PNW-OR (significance level
of 65%), CACC and PNW-WA (62%), and NE and PNW-WA (26%)
were not significantly different. Pesticide profiles at sites within
PNW-WA were the most similar to each other, based on the
SIMPER analysis, and profiles within MW were the most dissimilar.
This dissimilarity among sites within a region (average squared dis-
tance of 133–405) was lower than dissimilarity between regional
pairs (368–1024), indicating that within-region differences were
generally less than between-region differences. When the top five
pesticides contributing to within-region differences in all six regions
were combined, there were eight pesticides identified: seven USPs
were important in 2–6 regions each (2,4-D, triclopyr, carbendazim,
diuron, 4-hydroxychlorothalonil, imidacloprid, and sulfometuron-
methyl) and one non-USP (chlorsulfuron) was important only in
MW. Additionally, the 16 USPs were among the 20 largest contribu-
tors to the summed dissimilarities between the 15 inter-region pairs
and together they accounted for 65% of the total summed dissimilar-
ity between sites across all regions.

Fig. 4. Two-dimensional distance-based redundancy analysis plot of the best fit model from DISTLM results explaining pesticide Cmax values for the 16 urban signature pesticides (USP) at
the 271 urban-gradient sites in the six regions during the last 4 weeks of the study period. The model used the Manhattan distance measure of the pesticide profile. Each symbol represents
an urban-gradient site, color-coded by region. The symbol size for each site represents the percent imperviousness in the basin, overlaid on the plot. Percentages of total and fitted variation
of the pesticide profile explained by each axis are shown. The vectors (within the large circle) indicate the direction and degree of influence for each predictor variable in the model. The
much greater variability explained by the x-axis than the y-axis indicates the importance of the variables with large horizontal dimensions to their vectors, especially commercial services
and imperviousness.

10
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
3.4. Pesticide mixtures
Two or more pesticide compounds (i.e., mixtures) were detected in
91% of samples from Med/high urban sites. Mixture complexity was
represented by the pesticide count (defined as the number of pesticide
compounds per sample, including degradates). The median pesticide
count ranged from 2 pesticide compounds in PNW-WA to 43 in MW
(2–15 for pesticide a.i. only) and differed more between RSQA studies
than among major centers within a RSQA study, except in PNW,
where Portland, Oregon, and Seattle, Washington, were very different
(SI Fig. B.8). The pesticide count increased along the urban gradient
(SI Fig. B.9) and was significantly related to the percentage of urban
land in the basin at urban-gradient sites in all regions (Fig. 5) and in
all urban centers (not shown).
Unique mixtures of pesticide a.i. were identified at Med/high urban
sites during the last 6 weeks of the study period and compared among
regions. Over 1.91 million unique mixtures of pesticide a.i. were identi-
fied, each containing between 2 and 17 compounds; the most complex
mixtures are shown in SI Table B.12. The number of unique mixtures
Fig. 5. For urban-gradient sites within each region, the number of pesticide compounds detected per sample (mixture complexity) increased with the percentage of urban land in the
drainage basin. (A) MW, Midwest, (B) SEP, Southeast Piedmont, (C) NE, Northeast, (D) PNW-OR, Pacific Northwest-Oregon, (E) PNW-WA, Pacific Northwest-Washington, (F) CACC,
California Central Coast. m = slope.
11
Science of the Total Environment 793 (2021) 148453
was an exponential function of the total number of compounds detected
in that region (p < 0.01, adj R2 = 0.90), with the highest number in SEP
(1.58 million, with 2–17 compounds per mixture) and the fewest in
PNW-WA (275, with 2–6 compounds per mixture).
There were 661 unique 2-compound mixtures at Med/high urban
sites (SI Table B.13), with carbendazim+imidacloprid the most fre-
quent. After adjusting for unequal sample size among regions, the 2-
compound mixture with the highest mean occurrence frequency was
carbendazim+prometon. In the most common unique 2-, 3-, 4-, and
5-compound mixtures (SI Table B.14), combinations of fungicides and
herbicides occurred frequently in all regions, and insecticides occurred
frequently in SEP, NE, and CACC.
The occurrence of a pesticide in the top-20 mixtures for a region was
directly related to its detection frequency in samples from that region,
and consequently the USPs occurred frequently in these mixtures. Com-
bining the top-20 unique 2-compound mixtures from each region
(ranked by the number of occurrences and including ties) results in a
list of 67 mixtures, of which 66 contained at least 1 USP and 45
contained 2 USPs (SI Table B.13). Thirteen of the 16 USPs were in
L.H. Nowell, P.W. Moran, L.M. Bexfield et al. Science of the Total Environment 793 (2021) 148453

these most common mixtures (including all Tier A USPs) and the USPs
accounted for 83% of the pesticides in these top-20 mixtures.
Carbendazim and prometon occurred in the top 20 mixtures for all 6 re-
gions, and 2,4-D, diuron, and fipronil for 5 regions each (SI Fig. B.10).

3.5. Potential aquatic toxicity

Potential toxicity varied by taxonomic group, degree of urbanization

in the basin, and region. At RSQA Urban sites, benchmark exceedances
occurred more frequently for invertebrates (at 51% of sites) than for
plants (3% of sites) or fish (0.5% of sites). There was at least one exceed-
ance of an Acute-Invertebrate benchmark at 15% of Urban sites, and at
least one exceedance of a Chronic-Invertebrate benchmark at another
36% of Urban sites. At 14% of Urban sites, multiple pesticides reached
concentrations exceeding their invertebrate benchmarks (acute,
chronic, or both) during the study period, indicating that organisms at
these sites were exposed to potentially toxic concentrations of between
2 and 6 different pesticides within a short period of time (6 to 12
weeks). The pesticides most often responsible for invertebrate bench-
mark exceedances at Urban sites were imidacloprid, followed by OP in-
secticides and fipronil (SI Table B.15). For plants and fish, benchmark
exceedances occurred relatively infrequently at Urban sites: pesticides
exceeded Acute-Plant benchmarks for the USPs sulfometuron-methyl
(at 4 sites) and diuron (1 site) and the non-USPs atrazine (2 sites) and
pyraclostrobin (1 site). Only one pesticide exceeded an Acute-Fish
benchmark: the non-USP pyraclostrobin (in a single sample at an
Urban-low CACC site).
The percentage of sites with pesticide compounds exceeding one or
more invertebrate benchmarks increased with the percentage of urban
land in the basin (Fig. 6), from 1.5% at Undeveloped sites to about 75% at Fig. 7. Pesticides responsible for invertebrate benchmark exceedances at Med/high urban
Urban-high sites. Of Med/high urban sites, 65% had one or more pesti- sites by region: (A) number of Acute-Invertebrate benchmark exceedances divided by the
cide compounds that exceeded an invertebrate benchmark (18% of number of samples and (B) number of sites with exceedances (by 21-d average
concentrations) of Chronic-Invertebrate benchmarks divided by the number of sites.
sites for acute and 47% additional sites for chronic benchmarks), and
19% had exceedances by multiple pesticides. Urbanization had a clear
relation to the potential toxicity of pesticide mixtures. Four pesticide- the summed Chronic-Invertebrate BQ) were significantly different
mixture toxicity metrics were significantly related to urban land in the among regions and among urban centers.
basin (p < 0.01) at urban-gradient sites in all regions: the PTI for benthic Despite regional differences in the magnitude of predicted inverte-
invertebrates, cladocerans, and fish, and the summed Chronic- brate toxicity at Med/high urban sites, similar pesticides were responsi-
Invertebrate BQ. ble in most regions. The frequency of invertebrate benchmark
The magnitude of potential invertebrate toxicity varied among re- exceedance (summed for all pesticides) was highest in MW, SEP, and
gions (Fig. 7) and urban centers (Fig. 3D)—for which comparisons NE, where imidacloprid and OP insecticides were responsible for most
were made at Med/high urban sites to get a strong urban signal for acute benchmark exceedances by sample (Fig. 7A) and imidacloprid
each region. One or more invertebrate benchmarks were exceeded at and fipronil for most chronic exceedances by site (Fig. 7B). PNW-WA
4% (PNW-WA), 22% (PNW-OR), 26% (CACC), 86% (MW), 90% (NE), had low exceedance rates and was the only region without imidacloprid
and 98% (SEP) of Med/high urban sites. The four pesticide-mixture tox- exceedances.
icity metrics (the PTI for benthic invertebrates, cladocerans, and fish and A single USP dominated potential toxicity to invertebrates
(imidacloprid) and fish (carbendazim) and was a major contributor in
potential toxicity to plants (diuron) at Med/high urban sites—although
the magnitude of potential toxicity varied substantially by taxonomic
group (Fig. 3D–F). Here potential toxicity is represented by the summed
median BQmax, which was determined as described previously for the
summed median Cmax (Section 3.1.1) except using BQ values instead
of concentrations; thus, it reflects the high end of BQ values (because
it entails taking the BQmax for each site) yet is representative of all
Med/high urban sites within the urban center (because it takes the me-
dian BQmax for those sites). For invertebrates, the imidacloprid contri-
bution to the summed median Chronic-Invertebrate BQmax values is
substantially >1 (i.e., exceeds a potential toxicity threshold) in 13 of
the 16 urban centers (Fig. 3D). However, these values should be
interpreted with caution because they were calculated using BQmax
values in individual samples, whereas chronic effects are more appro-
priately based on 21-d average concentrations (U.S. Environmental
Protection Agency, 2017b). USPs, and imidacloprid in particular, also
Fig. 6. The percentage of sites with one or more pesticide compounds exceeding an Acute-
or Chronic-Invertebrate benchmark, for site categories with increasing amounts of urban
dominated the summed median Chronic-Invertebrate BQmax at the
land in the basin: Undeveloped (<5% urban land), Urban-low (5–25%), Urban-medium NWQN urban streams located outside the RSQA study area (SI
(25–75%), Urban-high (>75%). Fig. B.11B).

12
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
The fungicide degradate carbendazim dominated potential toxicity
toward fish, and together the USPs accounted for 50–90% of the
summed median Chronic-Fish BQmax in most RSQA urban centers
(Fig. 3E) and NWQN urban streams (SI Fig. B.11C). The summed median
Chronic-Fish BQmax values were substantially less than a potential tox-
icity threshold of 1 on a per-sample basis but were within a factor of 10
of that threshold (>0.1) in two urban centers (Midwest and Atlanta, GA;
Fig. 3E). Although no Med/high urban sites had any fish benchmark
exceedances, Pesticide Rank Index values for fish were high for
carbendazim in the MW (32%), SEP (19%), and NE (10%) during the
last 6 weeks of sampling (SI Fig. B.12), indicating that concentrations
were approaching the benchmark at a substantial number of sites.
The USP diuron accounted for the largest part of the USP contribu-
tion to potential plant toxicity (Fig. 3F). Together, the USPs generally
accounted for one-half or less of the summed Acute-Plant BQmax (non-
vascular) in most urban centers, with other (non-USP) herbicides ac-
counting for most of the remainder. Summed median Acute-Plant
BQmax were substantially below a toxicity threshold of 1.
4. Discussion
4.1. Urban pesticide signature
An urban pesticide signature emerged from the five RSQA studies,
with 16 pesticides (Table 1) meeting the criteria for consistent detection
in urban streams. These 16 USPs accounted for most of the summed pes-
ticide concentrations and potential toxicity at Med/high urban stream
sites in the 16 urban centers where samples were collected (Fig. 3A,
D–F). The existence of an urban pesticide signature was supported by
data for streams in other urban areas from the USGS NWQN study.
The presence of a common urban pesticide signature in U.S. urban
streams likely reflects the relatively homogeneous nature of urban pes-
ticide applications, which are dominated by turf and ornamentals, land-
scape maintenance, structural pest control, and rights-of-way. The
consistency among USPs demonstrates that there is a large degree of
commonality in urban pesticide contamination across time and space
in small urban U.S. streams. This consistency is all the more notable
given that the five RSQA studies were relatively short-term, collected
weekly discrete samples, and were done in different years and in
areas of the U.S. with contrasting climates and demographics.
Having a relatively short list of compounds (Table 1) that cause
much of the soluble pesticide contamination in U.S. urban streams in di-
verse settings (Fig. 3A) could have important implications for manage-
ment and research. The suite of USPs identified here, especially Tier A
compounds, would make a logical core of target analytes for studies
monitoring dissolved pesticides in urban streams. Such a list could be
supplemented with other pesticides that reflect local use, have some
potential toxicity (e.g., Fig. 7, SI Table B.15), and(or) have high regional
occurrence (e.g., SI Table B.6). The USPs are not an exclusive list of urban
dissolved-phase pesticides but represent the most consistently detected
pesticides in urban streams across these five regional studies. By
tweaking the selection criteria for USPs applied to the data summarized
in SI Table B.6, one could flag a correspondingly smaller or larger group
of urban pesticides of interest. Tier D compounds could be considered as
supplemental candidates for monitoring in urban areas; these were
found in all regions and in >50% of urban centers, but less consistently
than the USPs (Tiers A–C).
The 16 USPs identified here each have been detected in past studies
of urban stream contaminants, although these studies sampled far
fewer urban streams and urban centers than the RSQA study and none
identified a common pesticide occurrence signature across the U.S.
The eight USP herbicides were detected in U.S. urban streams in one
or more studies that collected samples between 2002 and 2014
(Baldwin et al., 2016; Bradley et al., 2017; Ensminger et al., 2013;
Metcalfe et al., 2016; Sprague and Nowell, 2008), and three (prometon,
diuron, and tebuthiuron) were among the most frequently detected in
13
Science of the Total Environment 793 (2021) 148453
U.S. urban streams during 1987–2014 (Stehle et al., 2019). The USP in-
secticide carbaryl has been one of the most common insecticides de-
tected in U.S. urban streams from 1992 to the present (Gilliom et al.,
2006; Stone et al., 2014b). Starting in the 2000s, fipronil began to be
widely detected in U.S. urban streams and concentrations increased
during 2000–2008 (Ryberg et al., 2010). By 2009, imidacoprid was the
largest selling insecticide in the world, and it has been frequently re-
ported in urban streams (Hladik and Kolpin, 2015; Silvanima et al.,
2018; Stehle et al., 2019). The USP fungicides each have been detected
at urban stream sites (Bradley et al., 2017; Metcalfe et al., 2016;
Wittmer et al., 2010), in stormwater (Bollman et al., 2014b; Masoner
et al., 2019), or in municipal wastewater (Bollman et al., 2014a; Kahle
et al., 2008). The recognition of their consistent co-occurrence in
urban streams from across the contiguous U.S., however, is a novel find-
ing of our study.
It is likely that additional pesticides will join the USP list in the fu-
ture, as pesticide use continues to change in response to regulations,
pesticide resistance, availability of new pesticides, and health and eco-
logical concerns. A prime example is the shift in dominant urban insec-
ticides from organochlorines during the 1960s and 1970s (Schmitt et al.,
1990; U.S. Environmental Protection Agency, 1983), to OPs during the
1990s (Gilliom et al., 2006), to fipronil and pyrethroid and
neonicotinoid insecticides during the 2000s (Hladik and Calhoun,
2012; Hladik and Kuivila, 2012; Holmes et al., 2008; Kuivila et al.,
2012; Ryberg et al., 2010; Silvanima et al., 2018; U.S. Environmental
Protection Agency, 2016; Weston et al., 2005). Future changes in
urban pesticide occurrence are anticipated as urban use patterns con-
tinue to evolve. Newer or increasing-use pesticides with nonagricultural
applications, such as chlorantraniliprole and dinotefuran (California
Department of Pesticide Regulation, 2020), may be good supplementary
candidates for future urban monitoring.
Our identification of USPs and urban mixtures focused on pesticide a.
i. in the dissolved phase. To be common in the dissolved phase, pesti-
cides must be at least moderately water soluble and have at least slight
mobility in soil. The 16 USPs were universally non-volatile, ranged in
soil mobility from slightly mobile to mobile, had water solubilities be-
tween 3.78 and 33,000 mg/L at 20 °C, and had log Kow values between
−0.82 to 3.75 (SI Table B.16) (University of Hertfordshire, 2021). Of the
USPs, fipronil stood out as having the lowest water solubility and the
highest log Kow and therefore moderate hydrophobicity; indeed,
fipronil was found both in the dissolved phase in water samples (as a
USP) as well as in bed sediment samples in RSQA (Mahler et al.,
2020). In contrast, the pyrethroids bifenthrin and permethrin are
strongly hydrophobic (with respective water solubilities of 0.001 and
0.2 mg/L and log Kow values of 6.6 and 6.1) (University of
Hertfordshire, 2021); if present in whole water in the sampled streams,
their concentrations would be greatly reduced by removal of particu-
lates during filtration (Hladik and Kuivila, 2009; Liu et al., 2004;
Spurlock et al., 2005).
Comprehensive urban monitoring also might target hydrophobic
pesticides in sediment, and glyphosate and pesticide degradates in
water. Although the hydrophobic pyrethroid insecticides are not effi-
ciently determined in the dissolved phase, they have been frequently
detected in sediment, suspended particulates, and whole water from
urban streams and outfalls in scores of studies across the U.S. (Budd
et al., 2020; Ding et al., 2010; Hladik and Kuivila, 2012; Kuivila et al.,
2012; Tang et al., 2018; Weston et al., 2009), and in many cases pyre-
throids (especially bifenthrin) in stream sediment were associated
with reduced survival to the amphipod Hyalella azteca in 10-d ambient
sediment toxicity tests (Amweg et al., 2006; Hintzen et al., 2009;
Holmes et al., 2008; Phillips et al., 2010; Weston et al., 2005). Although
this paper addresses dissolved pesticides only, the pyrethroid bifenthrin
was a common contaminant in bed sediment in RSQA urban streams
(Mahler et al., 2020; Moran et al., 2020; Moran et al., 2017). Glyphosate
was not included here because it was not amenable to the broad-
spectrum LC-MS/MS analytical method used by RSQA. However,
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
glyphosate was one of the top two herbicides (with 2,4-D) used nation-
ally in nonagricultural applications in 2012 (Atwood and Paisley-Jones,
2016) and it has been frequently detected in urban streams and waste-
water (Kolpin et al., 2006; Medalie et al., 2020), including urban streams
in the MW sampled by RSQA (Mahler et al., 2017). Finally, degradates of
the USPs also may warrant inclusion as target analytes, especially those
that were frequently detected in RSQA urban streams (SI Tables B1–B5)
and have comparable toxicity to the parent, such as several fipronil
degradates (Miller et al., 2020; Weston and Lydy, 2016).
4.2. Relative importance of land use and region
Urbanization within the basin had a stronger influence on the pesti-
cides present in streams along the urban gradient than did region. The
distance-based linear model for pesticide Cmax profiles at the 271
urban-gradient sites (illustrated in Fig. 4 for USPs) demonstrated that
basin urbanization was more important than region in explaining the
difference between sites, further supporting commonality of urban
pesticide occurrence across regions. This is consistent with the idea
that similar pesticides are applied in geographically diverse urban
settings—although quantities and timing likely differ—despite differ-
ences in climate, topography, and demographics. It further suggests
some commonality in the supply of these pesticides that are
generally available for use by professional applicators and(or) for
over-the-counter sales.
The largest contributions to dissimilarities in Cmax values among
urban-gradient sites within each region were made by USPs. Because
USP concentrations were significantly related to urban land in the
basin, we hypothesize that within-region variations in USP Cmax values
correspond to differences in the degree of urbanization within a region.
The USPs also contributed about 65% of the total summed dissimilarity
between inter-region pairs (i.e., differences between sites in all possible
pairs of regions). It is logical that USPs would be key compounds that
differentiate pesticide profiles within and between regions because
the USPs by definition are among the most frequently detected com-
pounds and they account for most of the summed median Cmax
(Fig. 3A). If a compound is rarely or never detected, its occurrence
among sites and among regions is relatively similar.
A strong and consistent (among regions) correlation between some
pesticides, or pesticide metrics, with specific urban land uses provided
an indication of potential sources within the urban landscape (SI
Table B.10). Residential land use variables had the strongest correlations
(highest rho) in 3–6 regions for fipronil, total fipronil, carbendazim, and
total insecticides. The strong relations for fipronil may reflect its use in
outdoor residential applications (e.g., household dwellings, lawns, orna-
mentals, control of termites and fire ants) (U.S. Environmental
Protection Agency, 2011). Golf course areas had the highest rho in
four regions for 4-hydroxychlorothalonil, which is consistent with the
use of its parent chlorothalonil on turf grass/golf courses (U.S.
Environmental Protection Agency, 2010). Carbendazim residues in res-
idential areas could result from the use of its parent, thiophanate-
methyl, on turf and ornamentals (California Department of Pesticide
Regulation, 2020; U.S. Environmental Protection Agency, 2014) and
(or) from use of carbendazim as a microbiocide in exterior paints, ce-
ments, and building materials. Carbendazim has been observed to
leach from building facades during wash-off experiments (Burkhardt
et al., 2009; Müller et al., 2020). Total fungicides had the highest rho
for residential land uses in three regions and for recreational urban
(golf courses, cemeteries, parks) in the other three regions, consistent
with the use of USP fungicides on turf grass, golf courses, ornamentals,
and shade trees (SI Table B.17). Land-use associations for other USPs
and pesticide metrics are less specific. The eight USP herbicides, most
herbicide classes, the USP carbaryl, and OP insecticides were most
strongly related either to dense urban land or to general urban variables.
Such land uses provide little indication of specific pesticide sources but
suggest a possible association with denser urban landscapes.
14
Science of the Total Environment 793 (2021) 148453
Most urban variables were significantly correlated with one another,
although rho values varied widely. For example, population density was
most strongly correlated with housing density (rho = 1.0), impervious-
ness (0.96), and commercial service (0.93), and less so to recreational
urban (0.66) or golf course area (0.43). One socioeconomic variable,
mean adjusted household income, was weakly correlated with semi-
developed land uses, recreational urban, golf course area, and low-
medium density residential, but not with general urban variables
(e.g., total urban land, imperviousness, dense urban; variables are de-
fined in SI Table A.10). The household income variable was significantly
(positively) correlated with a few USP fungicides (azoxystrobin, 4-
hydroxychlorothalonil, carbendazim, propiconazole) and insecticides
(imidacloprid and fipronil) in 2–3 regions, implying higher use in higher
income areas—however, these USPs had stronger correlations to urban
variables than they did to household income. Altogether, correlations
among urban-related variables limit our ability to ascribe particular
land-use-related sources to pesticide occurrence.
Of the USPs, all except prometon and sulfometuron-methyl are reg-
istered for use in some agricultural applications (SI Table B.17)—so how
much did agricultural use contribute to their detection at RSQA Urban
sites? For most USPs in most regions, detection frequencies were higher
at Med/high urban sites than at Agricultural sites, except for 8 USPs in
PNW-OR, and 6 USPs in CACC (SI Tables B.1–B.5). For those USPs with
substantially higher occurrence (e.g., by >10%) in Agricultural streams
than Urban streams within a region, the possibility cannot be ruled
out that partial residues in Urban streams may derive from agricultural
use in some parts of the basin, or via atmospheric transport from
nearby agricultural areas (Hoffman et al., 2000)—examples include
azoxystrobin and imidacloprid in PNW-OR and CACC, and diuron,
hexazinone, 4-hydroxychlorothalonil, propiconazole, and tebuconazole
in PNW-OR. Small amounts of cropland in MW urban-gradient sites
probably contributed to higher levels found there of the (non-USP) her-
bicides atrazine, metolachlor, and acetochlor—which are intensively
used on corn and soybeans in the Midwest—than occurred at urban-
gradient sites in other regions (SI Fig. B.1). Agriculture likely plays
more of a role in MW, PNW-OR, and CACC because urban and cropland
are in close proximity and urbanization has included conversion of agri-
cultural land (Garrett et al., 2017; May et al., 2020; Sheibley et al., 2017).
4.3. Unique mixtures
Unique mixtures of pesticides have been only rarely identified
(Gilliom et al., 2006; Smiley et al., 2014). Here we go beyond reporting
the pesticide count and identify the most common unique mixtures of
pesticide a.i. at Med/high urban sites in each region and establish that
USPs play a dominant role in these mixtures. The sheer number of pes-
ticides co-occurring in environmental waters (we identified 1.91 mil-
lion unique mixtures of 2–17 compounds) render infeasible the direct
toxicity testing of all potential mixtures (Belden et al., 2007). However,
documentation here of the most common pesticide mixtures from U.S.
urban streams can guide research on mixture toxicity testing and
mesocosm experiments by identifying the most common “real world”
mixtures.
Of the most common 2-compound mixtures at urban sites, the relative
intrinsic toxicity to invertebrates (assessed by averaging the chronic
benchmarks of both pesticides) was highest for fipronil+imidacloprid,
followed by carbendazim+fipronil and carbendazim+imidacloprid. For
fish, the common mixtures with the highest relative intrinsic toxicity
were carbendazim+fipronil, followed by carbendazim+atrazine and
atrazine+fipronil. For plants, the common mixture atrazine+diuron
had the highest relative intrinsic toxicity (assessed by averaging the
Acute-Nonvascular Plant benchmarks of both pesticides). Although high
relative intrinsic toxicity does not necessarily imply potential toxicity of
mixtures in the hydrologic system, which depends on the concentrations
of co-occurring pesticides—concentrations of the above pesticides
reached or approached toxic thresholds at some RSQA Urban sites
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
(SI Tables B.15, SI Fig. B.12). Environmental exposure and relative intrinsic
toxicity thus provide guidance on selecting mixtures to test in toxicity
bioassays or mesocosm experiments.
4.4. Potential adverse effects on aquatic biota
The USP neonicotinoid insecticide imidacloprid was a nationally im-
portant stressor for invertebrates in RSQA Urban streams, dominating
the summed median Chronic-Invertebrate BQmax in almost all urban
centers (Fig. 3D). Secondary in importance were fipronil and OP insecti-
cides, which each exceeded invertebrate benchmarks at multiple Urban
sites in four regions. Although OP insecticides were rarely detected
(usually in <1 to 5% of urban samples in any one region), most are
very highly toxic to invertebrates (U.S. Environmental Protection
Agency, 2017a; U.S. Environmental Protection Agency, 2018). An ex-
treme case was the OP dichlorvos, which exceeded its Chronic-
Invertebrate benchmark in all 26 samples in which it was detected. Al-
though the OPs were detected far less often and at lower concentrations
in RSQA than during the 1990s (Bailey et al., 2000; Gilliom et al., 2006),
OPs still exceeded invertebrate benchmarks at 11% of Med/high urban
RSQA streams overall. Because their occurrence was sporadic, however,
the OPs did not make substantial contributions to the summed median
Chronic-Invertebrate BQmax (Fig. 3D), which was based on median
values within an urban center. Diflubenzuron and permethrin, which
exceeded benchmarks at Urban and(or) Mixed sites in three regions
each (SI Table B.15), may have more than local importance. Otherwise,
isolated benchmark exceedances by additional pesticides (SI Table B.15)
indicate that concentrations reached potentially toxic levels locally but
there is no evidence from the RSQA weekly discrete sampling results
that these pesticides were important stressors at a larger scale.
What role did mixtures play in potential invertebrate toxicity in
urban streams? Use of the PTI to predict cumulative potential toxicity
of mixtures to invertebrates did not add appreciably to site-specific pre-
dictions of toxicity, as only 2% of sites had a PTI >1 without also exceed-
ing Acute-Invertebrate benchmarks for individual pesticides. However,
29 Urban sites (14%) had multiple pesticides that exceeded their inver-
tebrate benchmarks during the study period, suggesting that exposure
to co-occurring pesticides has a temporal element that is missed when
mixture effects are evaluated only on a per-sample basis. Biota in
these 29 streams were exposed to mixtures of pesticides, in which at
least 2–6 pesticides reached potentially toxic levels during the relatively
short duration of the RSQA studies (6 to 12 weeks, depending on the re-
gion). Mixture effects would depend on the organisms' ability to avoid
or recover from multiple exposures during this time period. The USP
imidacloprid was one of the pesticides responsible at all 29 sites, and
the USP fipronil was another in half of them.
The aquatic-toxicity predictions are screening-level estimates and
are likely biased low. First, weekly discrete sampling underestimates
acute exposure to pesticides in small streams (Norman et al., 2020),
and samples collected following runoff tend to have higher concentra-
tions than those collected at regular intervals (Bundschuh et al., 2014;
Xing et al., 2013). This underestimation is particularly true for insecti-
cides (Stehle et al., 2013), which are often transient (Norman et al.,
2020) and tend to be highly toxic to invertebrates. Second, not all pesti-
cide compounds have ALBs and some compounds have ALBs that were
below the analytical reporting levels in this study (SI Table A.3), so
exceedances (SI Table B.15) may be underestimated. Furthermore, the
ALBs and PTIs are based on single-species toxicity tests, so the extent
to which these are broadly protective of aquatic communities will de-
pend on the availability of toxicity test data for a given pesticide with
a range of sensitive taxa. The current toxicity test gap is especially prob-
lematic for fungicides, because standard toxicity test organisms
(e.g., daphnids and green algae) are not necessarily the most sensitive
taxa toward fungicides, and single-species LC50 values may not reflect
ecosystem processes such as litter decomposition (Elskus, 2014;
Zubrod et al., 2019; Zubrod et al., 2015). Third, aquatic communities
15
Science of the Total Environment 793 (2021) 148453
may be adversely affected at lower pesticide concentrations than ex-
pected based on single-species laboratory bioassays (Liess and
Beketov, 2011; Miller et al., 2020; Schäfer et al., 2012). Therefore, the
screening-level estimates of potential toxicity provided here are likely
to be minimum estimates but serve to put the measured pesticide con-
centrations in an ecological health context.
5. Conclusions
The analysis presented here demonstrates that dissolved pesticides
occur frequently in urban centers and that pesticide occurrence and po-
tential aquatic toxicity increase along the urban gradient. Sixteen pesti-
cides were consistently detected in all or most urban centers in
contrasting regions of the United States, comprising an urban pesticide
signature. Although the urban signature pesticides (USP) were found in
all regions, their detection frequencies and concentrations varied by
region, conferring differences in potential aquatic toxicity. Degree of ur-
banization in the basin was the best predictor of pesticide concentra-
tions at urban-gradient sites, and region was secondary. The 16 USPs
accounted for about 75% of summed median Cmax concentrations and
>80% of pesticides in the most common mixtures in streams with
>25% urban land use in the basin. Potential toxicity of the observed pes-
ticide mixtures was highest for invertebrates (predicted at 51% of Urban
sites), and low for aquatic plants (3% of Urban sites) and fish (<1% of
Urban sites). The USPs contributed the largest share of potential toxicity
of pesticide mixtures to invertebrates (dominated by imidacloprid and
to a lesser extent fipronil) and fish (dominated by carbendazim), and
a substantial part of the potential toxicity to plants (especially diuron)
—although the magnitudes of potential toxicity to fish and plants were
generally low. We propose that the USPs identified here would make
a logical core set of (nonexclusive) target analytes for monitoring
dissolved pesticides in U.S. urban streams, and that unique mixtures
containing imidacloprid, fipronil, and carbendazim would be good
candidates for determining the effects of pesticide mixtures on inverte-
brates. These results add new specificity to our understanding of the
urban stream syndrome in U.S. streams, and the identification of an
urban pesticide signature may aid those tasked with remediating
urban streams.
CRediT authorship contribution statement
Lisa H. Nowell: Conceptualization, Methodology, Formal analysis,
Investigation, Writing – original draft, Writing – review & editing, Visu-
alization. Patrick W. Moran: Conceptualization, Methodology, Formal
analysis, Investigation, Writing – original draft, Writing – review &
editing, Visualization. Laura M. Bexfield: Formal analysis, Writing –
review & editing. Barbara J. Mahler: Conceptualization, Investigation,
Writing – review & editing, Visualization. Peter C. Van Metre: Concep-
tualization, Investigation, Writing – original draft, Writing – review &
editing, Visualization, Project administration. Paul M. Bradley: Concep-
tualization, Investigation, Writing – review & editing. Travis S. Schmidt:
Conceptualization, Investigation, Writing – review & editing. Daniel T.
Button: Data curation, Writing – original draft. Sharon L. Qi: Data
curation, Formal analysis, Visualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This research was conducted and funded by the U.S. Geological
Survey National Water Quality Program, Regional Stream Quality
Assessment. The authors thank Mark Sandstrom, Megan Shoda, Laura
L.H. Nowell, P.W. Moran, L.M. Bexfield et al.
Medalie, and Wesley Stone (U.S. Geological Survey) for their analysis of
laboratory and field quality control data for National Water Quality
Laboratory schedule 2437, and Megan Shoda and Wesley Stone for
sharing data from the National Water Quality Network study. We thank
Sarah Stackpoole (U.S. Geological Survey) and two anonymous
reviewers for their helpful reviews of the manuscript, and Emerson Gusto
(U.S. Geological Survey) for his work on illustrations. The photograph in
the graphical abstract was taken by Alan Cressler (U.S. Geological Survey).
Any use of trade, firm, or product names is for descriptive purposes only
and does not imply endorsement by the U.S. Government.
Appendix A. Supplementary data
The raw data for this article are summarized in Supplemental
Information and are publicly available in ScienceBase (Morace et al.,
2020; Qi and Nakagaki, 2020a; Qi and Nakagaki, 2020b). Supplementary
data to this article can be found online at https://doi.org/10.1016/j.
scitotenv.2021.148453.
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