Limnology and Oceanography Letters 2019

© 2019 The Authors. Limnology and Oceanography published by Wiley Periodicals, Inc.
on behalf of Association for the Sciences of Limnology and Oceanography.
doi: 10.1002/lol2.10138

SPECIAL ISSUE-LETTER

The effect of urban point source contamination on microplastic levels in

water and organisms in a cold-water stream
Claire B. Simmerman ,1* Jill K. Coleman Wasik2
1
Soil, Water, and Climate Department, University of Minnesota, Falcon Heights, Minnesota; 2Plant and Earth Science
Department, University Wisconsin River Falls, River Falls, Wisconsin

Scientific Significance Statement

It is well understood that significant point sources of microplastics (MPs) to aquatic systems originate from urban areas. However,
measurements of MP levels in water and organisms from urban and nonurban areas do not always reflect the expected effect of
urban point source contamination. Despite the potential for different types of MPs originating from urban and nonurban areas,
limited information regarding the size distribution of particles in different ecosystem compartments is provided. We found signifi-
cant MP contamination in the water of an agriculturally dominated, small, cold-water stream. We found additional effects of
urbanization on MP contamination in both water and organisms in stream reaches within and downstream of an urban area.
Additionally, we found significant differences in the dominant size of MPs among water, macroinvertebrates, and trout.

Abstract
We examined the influence of point and nonpoint source contamination on microplastic (MP) levels in water,
macroinvertebrates, and trout in a small, cold-water stream in western Wisconsin, U.S.A. We collected samples
along an urbanization gradient centered around a 6-mile corridor that receives numerous MP inputs from
stormwater outfalls and a wastewater plant. We digested samples using a wet peroxide oxidation method. Parti-
cles filtered from digestates were stained with Nile Red dye, then viewed using fluorescence microscopy. We
quantified fluorescing MPs using ImageJ software. MP concentrations in water increased significantly from
upstream to downstream reaches and appeared to influence organism MP levels. Macroinvertebrates and trout
collected upstream of the city had significantly lower MP levels than macroinvertebrates and trout collected
within the city, and for trout, downstream of the city. MP particle lengths were significantly longer in

*Correspondence: simme090@umn.edu

Author Contribution Statement: C.B.S. and J.K.C.W. designed the described study, conducted field work, and drafted the manuscript together.
C.B.S. developed laboratory methods, analyzed all of the samples, performed software analysis of sample images, and created the GIS map of study site.
J.K.C.W. conducted statistical analyses, reported results, and added to data interpretation.

Data Availability Statement: Data are available in the Mendeley Data repository at 10.17632/p8v4t7sfkj.1

Associate editor: Susanne Brander

Additional Supporting Information may be found in the online version of this article.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.

This article is an invited paper to the Special Issue: Microplastics in marine and freshwater organisms: Presence and potential effects
Edited by: Dr Elise Granek, Portland State University, Dr Susanne Brander, Oregon State University, and Dr Erika Holland, California State University, Long Beach

1
Simmerman and Coleman Wasik
macroinvertebrates compared to water and trout, perhaps indicating preferential accumulation of longer parti-
cles from the water column.
Microplastics (MPs) are plastic particles < 5 mm in size.
MPs originate from primary and secondary sources. Primary
MPs are engineered materials, including plastic production
pellets, microbeads in personal care products, and decorative
materials including sequins and glitter. Secondary MPs
include plastic litter and materials that fragment into smaller
pieces, including synthetic textiles and clothing that shed
plastic fibers while being worn or washed (Eerkes-Medrano
and Thompson 2018). MPs are an emerging contaminant of
concern in freshwater ecosystems because of their ubiquitous
presence and poorly quantified risks to human health and the
environment (Eerkes-Medrano and Thompson 2018).
MPs may impact aquatic organisms and food webs through
chemical and physical mechanisms (Li et al. 2018; Wagner
and Lambert 2018). The hydrophobic surface of plastic can
sorb hydrophobic contaminants from the surrounding envi-
ronment (McNeish et al. 2018), or leach toxic chemicals used
in their production (Li et al. 2018). This makes MPs a poten-
tial pathway for other contaminants to enter and
bioaccumulate in food webs. Additionally MPs have been
shown to physically bioaccumulate through gut retention and
trophic transfer in freshwater and marine organisms (Farrell
and Nelson 2013; Batel et al. 2016). Retained MPs can lead to
abrasions, inflammation, and gut blockages (Eerkes-Medrano
and Thompson 2018; McNeish et al. 2018). There is still
uncertainty as to the exact mechanisms whereby MPs impact
aquatic organisms (Windsor et al. 2018). A recent meta-
analysis of 43 studies by Foley et al. (2018) found negative
effects of MPs on consumption and growth for most taxo-
nomic groups of aquatic organisms, and neutral effects on
reproduction and survival of organisms larger than zooplank-
ton. The authors highlighted the difficulty of comparisons in
their analysis because methods and reporting varied from
study to study (Foley et al. 2018).
Land use is considered an important factor influencing MP
abundance in water and aquatic organisms. Some studies
show positive relationships between population density and
MP abundance in water and organisms, suggesting that urban
areas contribute notable loads of MPs into freshwaters
(Eriksen et al. 2013; Horton et al. 2016; Peters and Bratton
2016; Cable et al. 2017; Tibbetts et al. 2018), whereas other
studies found no connection between urban point sources
and MP abundance (Dikareva and Simon 2019). Research in
nonurban and remote aquatic systems has also uncovered
unexpectedly elevated levels of MP contamination (Free et al.
2014; Bergmann et al. 2017; Li et al. 2018; Allen et al. 2019).
We measured MPs in the water column, aquatic
macroinvertebrates, and trout in a small, cold-water stream in
western Wisconsin. Our research questions were: (1) Do urban
point sources influence MP levels in water and organisms
2
The effect of urban point source contamination
similarly in an agricultural stream? (2) Is there a difference in
the size distribution of MP among water and organisms along
an urbanization gradient? To answer these questions, we col-
lected samples above, within, and below the city of River Falls,
the only city in the otherwise predominantly agricultural
watershed. Because the potential for plastic particles to enter
food webs increases with decreasing size (Mrowiec 2018), we
used analytical methods that allowed for the detection of par-
ticles down to ~10 μm in length.
Methods
Study site
Samples were collected from the Kinnickinnic River, a
groundwater-fed, 5th-order, cold-water stream in western
Wisconsin, U.S.A. The Kinnickinnic originates in a wetland
complex and flows southeast 35 km through a predominantly
agricultural watershed (Fig. 1). In the city of River Falls
(population ~ 15,000), the Kinnickinnic receives inputs from
numerous stormwater outfalls and effluent from the city’s
1 million gallon d−1 wastewater treatment plant (WWTP).
There are two run-of-river dams within city limits that provide
hydropower to the surrounding community. Baseflow in the
Kinnickinnic upstream of River Falls is approximately
1.56 m3 s−1 and increases to approximately 2.83 m3 s−1 down-
stream of the city.
Sample collection
Samples were collected upstream of city influences, within
city limits (but above the WWTP), and downstream of city
limits. Water samples were collected under base-flow condi-
tions in January 2019. Bulk water samples were collected from
the thalweg of the river at each location using 1-liter glass
jars. Jars were rinsed 3× with stream water before collection
and kept at 4 C until processed (Barrows et al. 2018).
Macroinvertebrates were collected in January 2019
according to WI DNR protocols (WI DNR 2000). Sampling
occurred along three transverse transects selected using a ran-
dom number generator within a 10-m reach that started at
the head of a riffle in upstream, city limits, and downstream
river reaches. A 600-μm D-shaped kick net was held firmly
against the bottom of the stream and the upstream substrate
was disturbed to dislodge macroinvertebrates. Three kicks,
covering approximately 1 m2 each, were pooled for each tran-
sect. Macroinvertebrates were sorted and classified into fami-
lies. Although many families were present in variable
numbers at different sites, the mayflies (Heptageniidae),
caddisflies (Hydropsychidae), and amphipods (Gammaridae)
were the only families used in this study because they were
present at each site in sufficient numbers for analysis.
Simmerman and Coleman Wasik The effect of urban point source contamination

Fig. 1. The Kinnickinnic River and sample collection locations over a changing land use gradient with point sources of pollution in River Falls, Wisconsin,
U.S.A. The white point represents where only water was collected (D3). The purple point represents where only trout were collected (U5). Lime green
points represent where water and trout were collected; water was collected at the lime green points (U1–U3, U6, C1, C2, C4, D1, and D4), and trout
were collected in segments of stream between lime green points, including the purple point where only trout were collected (U1–U3, U5–U6, C1–C4),
and at lime green points D1 and D4. The yellow point represents where water and macroinvertebrates were collected (C3). Red points represent where
macroinvertebrates were collected (U4 and D2). Navy blue points represent stormwater outfalls, and the black star represents the local WWTP. The anno-
tation U stands for upstream, C stands for city limits, and D stands for downstream. GIS data obtained from the WI DNR Open Data Portal (State of Wis-
consin Department of Natural Resources (WI DNR), and Bureau of Technology Services 2017), and from the City of River Falls Engineering Department.

Specimens were kept in glass jars and preserved with 95% eth-
anol until ready to be processed.
Trout were collected sporadically in the general open sea-
sons between October 2017 and September 2018. Recreational
anglers caught individual trout using inline spinners and clas-
sified samples as either brown (Salmo trutta) or brook
(Salvelinus fontinalis). Total length was measured to the nearest
0.02 cm from snout tip to the end of the caudal fin. Samples
were transported on ice. The entire gastrointestinal tract (GIT)
was dissected from each sample within 1 h after collection
using a DIH2O-rinsed metal knife and marble cutting board.
Tissues were immediately frozen and stored at −20 C until
digestion.
MP analysis and quantification
Detailed descriptions of sample processing, analysis, quan-
tification, QAQC practices, and the statistical treatment and
analysis of our data are included in the Supporting
Information.

3
Simmerman and Coleman Wasik The effect of urban point source contamination

Fig. 2. Series of images displaying visual method (a1, b1, c1, d1, e1, and f1), Nile Red method (a2, b2, c2, d2, e2, and f2), and associated images
analyzed in ImageJ (a3, b3, c3, d3, e3, and f3). Image rows a and b (fragments and fibers) are MPs from water samples, rows c and d (fragments) are
MPs from trout, and rows e (film) and f (bead) are MPs from macroinvertebrates.

Briefly, suspended solids filtered from water column sam- samples were subjected to a wet peroxide oxidation digestion
ples (retained on 0.4-μm polycarbonate track-etch membrane (0.05 mol L−1 FeSO4 + 3 mL H2SO4 + 30% H2O2) (McNeish
filters), dried and ground trout GIT, and macroinvertebrate et al. 2018). Samples containing lipid-rich tissues were further

4
Simmerman and Coleman Wasik
Fig. 3. Boxplots show the mean, median, interquartile range, and 10th and 90th percentiles of MPs detected in water samples (a), macroinvertebrates
(b), and trout (c) from upstream, city limits, and downstream reaches in the Kinnickinnic River. The letters a, b, and c indicate statistically significant dif-
ferences in the means ( ) among locations at the level p < 0.05 as determined by Wilcoxon Rank Sum tests.
digested using a 5% Alcojet detergent solution to completely
breakdown remaining lipids (Crichton et al. 2017). Digested
solutions were filtered, transferred to a glass Petri dish, and
particles were stained with Nile Red (NR) dye according to
Erni-Cassola et al. (2017). The NR dye method has been vali-
dated in the literature as a fast, unbiased, and cost-effective
way to identify MP (Shim et al. 2016; Erni-Cassola et al. 2017;
Maes et al. 2017; Mason et al. 2018) and can increase the
probability of particle detection over visual methods (Lavers
et al. 2016; Supporting Information Figure S1). Samples were
viewed under a fluorescence microscope and fluorescent parti-
cles were verified as plastic using a simple hot needle test
according to Kapp and Yeatman (2018) and Hendrickson et al.
(2018). Digital images of each sample were captured and
ImageJ software was used to count the fluorescing plastic parti-
cles in each image. ImageJ was also used to determine the area
of each fluorescing particle. To estimate particle size distribu-
tions, particle length was calculated by taking the square root
of particle areas (Erni-Cassola et al. 2017). Method blanks com-
prised 10–15% of samples (Supporting Information Table S1).
Results
MPs were present in 100% of water (n = 11) and
macroinvertebrate (n = 9) samples, and in 98% of trout
(n = 42) samples. Fragments and fibers were the dominant MP
type found in all samples (Fig. 2). The range of MP concentra-
tions found in all water, macroinvertebrate, and trout samples
were 545–3622 particles L−1, 1.2–33.2 particles g−1 dry weight,
and 0–1242 particles fish−1, respectively. The range of particle
5
The effect of urban point source contamination
lengths found in all water, macroinvertebrate, and trout sam-
ples were 14–1494 μm, 15–1910 μm, and 12–5569 μm, respec-
tively. Particle lengths differed significantly among water,
macroinvertebrate, and trout samples. The smallest particles
were present in water samples and the largest were present in
macroinvertebrate samples (Supporting Information Table S2;
p < 0.0001 for each pair, Wilcoxon).
Mean and median MP concentrations were lowest in
water samples collected from upstream reaches and highest
in samples from downstream reaches (Fig. 3). Statistical dif-
ferences in MP concentrations were not found when water
data were pooled by location (p = 0.23, Kruskal-Wallis); how-
ever, linear regression showed a significant positive correla-
tion between particle concentration and distance from
headwaters (p < 0.05, adjusted R2 = 0.35). Although
mean and median particle lengths in water samples were
similar among locations (Supporting Information Table S2),
significant differences among all locations were found
(p < 0.0001, Kruskal-Wallis), likely as a result of the large
numbers of particles recorded in each sample (p < 0.001 for
each pair, Wilcoxon). At all locations 75–90% of particles
were estimated to be smaller than ~ 50 μm in length (Fig. 4).
Linear regression on particle length data showed no correla-
tion with distance from headwaters (p > 0.05, adjusted
R2 = 0.09).
Mean and median MP concentrations in macroinvertebrate
samples were lowest in upstream reaches and highest within
the city limit reaches (Fig. 3). Macroinvertebrates within the
city limits had significantly higher MP than upstream
macroinvertebrates (p < 0.05, Wilcoxon). There were no
Simmerman and Coleman Wasik The effect of urban point source contamination

Fig. 4. Percentage of particles found in four different length classes in water samples (a), macroinvertebrates (b), and trout (c) from upstream, city
limits, and downstream reaches in the Kinnickinnic River.

significant differences between MP in macroinvertebrates the city (p = 0.14, Wilcoxon). No significant differences in MP
upstream and downstream of the city (p = 0.3, Wilcoxon), nor concentrations were found among macroinvertebrate families
between samples collected in city limits and downstream of for pooled location data (p = 0.26, Kruskal-Wallis; Fig. 3) or

Fig. 5. Boxplots show the mean, median, interquartile range, and 10th and 90th percentiles of MP detected in caddisflies (Hydro.), mayflies (Hept.), and
amphipods (Gamm.) (a), and brook and brown trout (b) from upstream, city limits, and downstream reaches in the Kinnickinnic River. The letters a, b,
and c indicate statistically significant differences in mean MP concentrations among locations at the level p < 0.05 as determined by Wilcoxon Rank Sum
tests.

6
Simmerman and Coleman Wasik
when macroinvertebrate data were analyzed by family for
each location (p > 0.07, Kruskal-Wallis; Fig. 5).
Mean and median particle lengths in macroinvertebrate
samples varied somewhat among locations (Supporting
Information Table S2). At all locations, 30–50% of particles
were estimated to be 100–330 μm in length (Fig. 4). No signifi-
cant differences were found among locations for pooled fam-
ily data (p = 0.06, Kruskal-Wallis). When macroinvertebrate
data were analyzed by family, some significant differences
were found, but no clear patterns emerged (Fig. 5).
Mean and median MP concentrations in trout samples
were lowest in upstream reaches and highest within city limits
and downstream reaches (Fig. 3). Upstream trout had statisti-
cally lower MP levels in their GIT than trout found within
and downstream of the city (p < 0.05 each pair, Wilcoxon).
No significant difference was found in MP concentrations
between trout within city limits and those downstream of the
city (p = 0.69, Wilcoxon). No significant differences in MP
concentrations were found between trout species when spe-
cies data were pooled across locations (p = 0.57, Wilcoxon),
nor where species were compared at specific locations (Fig. 5).
Statistically significant differences in particle lengths were
found among all locations for trout samples when species data
were pooled for each location (p < 0.001, Kruskal-Wallis). At all
locations, 30–50% of particles were estimated to be smaller
than ~ 50 μm in length (Fig. 4). Particles were shortest in
upstream samples relative to city limit (p < 0.001, Wilcoxon)
and downstream (p < 0.05, Wilcoxon) samples. Particle lengths
in city limit trout GIT were significantly longer than particles
in downstream trout (p < 0.001, Wilcoxon). When trout data
were divided by species for different sampling locations, more
significant differences found and but no clear patterns emerged
(Fig. 5). Brook and brown trout lengths overlapped (brook:
15–24 cm, n = 7; brown: 15–30 cm, n = 35). No clear associa-
tions were found between trout length and MP abundance.
Discussion
Influence of MP point sources along an urbanization
gradient
The Kinnickinnic River provides a natural experimental
design wherein upstream reaches are predominantly agricul-
tural (Fig. 1) and MP point sources are confined to the city of
River Falls. The prevalence of MPs in our samples is consistent
with other studies performed on rivers impacted by urban areas
(Mani et al. 2015; Nel et al. 2017; Pazos et al. 2017; Silva-
Cavalcanti et al. 2017; McNeish et al. 2018). We found a signif-
icant correlation between MP concentrations in the water col-
umn and distance downstream of headwaters. Mean MP
concentrations within city limits and downstream of the city
were 2–3× the mean MP concentration found upstream (Fig. 3,
Supporting Information Table S2). MP burdens in both
macroinvertebrates and trout generally followed the pattern of
MP concentrations in water samples, with macroinvertebrates
7
The effect of urban point source contamination
and trout having significantly lower MP at upstream sites rela-
tive to city limit sites and, for trout, downstream sites (Fig. 3,
Supporting Information Table S2). This suggests that increased
exposure to MP from urban point sources, such as stormwater
outfall and wastewater effluent, increases the MP burden of
aquatic organisms in the Kinnickinnic River.
Despite the increasing trend in water MP concentrations
and the accumulation of more point source inputs from city
limits to downstream reaches, macroinvertebrates and trout
collected downstream of the city generally had lower MP
burdens than those collected within city limits (Fig. 3),
Supporting Information Table S2). This pattern was not sta-
tistically significant, but may have resulted from a change in
the dominant MP source to the river between city limit and
downstream sampling locations. All city limit samples were
collected upstream of two small, run-of-river dams. Most
stormwater inputs to the river enter at or upstream of the
first structure. The WWTP discharges its effluent between the
dams and downstream of most stormwater outfalls. It may
be possible that a greater proportion of stormwater MP, rela-
tive to WWTP MP, is trapped and buried in the impound-
ment sediments preventing their movement into
downstream reaches (Ballent et al. 2016; Watkins et al.
2019). Flow dilution may also explain the absence of a trend
(Windsor et al. 2018). River discharge increases by 40%
between the city limit and downstream reaches. Groundwa-
ter inputs contribute the greatest proportion to this increase,
but likely do not contribute substantially to MP loading. The
result would be a dilution of MP, despite WWTP inputs, and
could lead to similar MP concentrations at city limit and
downstream sites; however, MP concentrations in water sam-
ples do not support a dilution mechanism. Furthermore,
mean MP lengths found in downstream water samples were
significantly shorter than MP at upstream and city limit sites
(Supporting Information Table S2), suggesting either a
changing MP source or preferential entrainment of smaller
particles and/or loss of larger particles as water moves
through the impoundments.
Although urban point sources were influential on particle
concentrations for all sample types in this study, it is evident
from our data that unidentified, nonpoint MP sources impact
the upper reaches of the river. The Kinnickinnic is a popular
trout-fishing and kayaking destination in the region. There
are numerous bridges that cross the river and ditches littered
with plastic trash that connect to the stream. Atmospheric
deposition of MP is also very likely because the Kinnickinnic
watershed is downwind of a large metropolitan area (Dris
et al. 2016). Agriculture is a major land use in the upper water-
shed, and terrestrial application of sewage sludge or use of MP
containing agricultural products may occur (Ng et al. 2018;
Accinelli et al. 2019). Given their potential to negatively
impact aquatic organisms, better quantification of nonpoint
sources of MP in rural areas are necessary to understand over-
all MP contamination in streams generally considered to be
Simmerman and Coleman Wasik
unimpacted by this type of pollution (Alimi et al. 2017;
Eerkes-Medrano and Thompson 2018; Allen et al. 2019).
Size distribution of MP in water and organisms in a
cold-water stream with urban point source inputs
In this study, very small particles were found to make up
large proportions of the MP in our samples. In water samples,
75–90% of particles were estimated to be smaller than
~ 50 μm in length (Fig. 4, Supporting Information Table S4),
an important cutoff size for many MP studies (Li et al. 2018).
This size class did not dominate the particle distributions in
macroinvertebrate and trout samples; however, it is still an
important and overlooked consideration for studying the con-
nections between landscape scale sources of MPs, the
observed concentrations in water, and potential levels of MP
exposure experienced by aquatic organisms. Also, it should be
noted that there can be error introduced when quantifying
particles < 50 μm in length, and future research in method
development focused on this size class is needed in order to
more accurately quantify and validate very small MP.
Although organisms are exposed to very high levels of
small MP in this river, the significant differences in median
particle lengths between organisms and the water column
suggest that organisms may somehow “fractionate” MPs based
on particle size (e.g., through differential gut retention, parti-
cle avoidance, and/or other mechanisms). Median particle
lengths in macroinvertebrates were ~ 1.5–3× median particle
lengths in trout at the same location (Supporting Information -
Table S2). Between 35% and 50% of particles in
macroinvertebrates fell within the 100–330 μm size class
(Fig. 4, Supporting Information Table S4). This could suggest
some preferential accumulation of certain MP sizes within
macroinvertebrates. If larger MPs (or certain shapes) pass more
slowly through organism GITs, then they may accumulate rel-
ative to smaller size MP, and thereby constitute a greater pro-
portion of MP burdens within organisms. In many studies,
MP burdens in aquatic organisms appear to be highly variable
and do not always correlate with expected MP sources.
Slootmaekers et al. (2018) found that 8–96% of samples were
contaminated with MP in a small survey of studies of MP
prevalence in fish from urban streams. If the dominant parti-
cle sizes/shapes in the environment are easily passed or
egested (or not analyzed), then there may be a mismatch
between known sources and observed burdens.
It should be noted that trout samples were collected over a
very different time frame from water and macroinvertebrate
samples. Our reliance on recreational anglers meant that sam-
ples were collected sporadically throughout the season (early
May to mid-October) as anglers were able to fish. This would
increase the variability inherent in our sample population
because flow conditions, land use, and feeding behaviors
change throughout the course of a year.
This study quantified MP contamination in water,
macroinvertebrates, and trout in a small, cold-water stream.
8
The effect of urban point source contamination
Our findings agree with previous studies in that we found
strong evidence that urban point sources increase MP levels in
the water column and aquatic organisms in the Kinnickinnic
River. It also appears that unidentified nonpoint sources are
contributing to elevated MP levels in river water and organ-
isms in nonurban reaches. Although atmospheric and agricul-
tural nonpoint sources are important to quantify, more
research into contributions from recreational activities, road-
ways, and ditches will be important to understanding non-
point MP sources to water and organisms in this particular
river. Our study also suggests that it is important to quantify
particle sizes over as broad a range as possible because particle
sizes vary by ecosystem compartment (i.e., water, prey, preda-
tor). This finding highlights the ongoing needs for the
research community to develop standardized analytical
methods and agreement around reporting for better compari-
sons within and across study systems.
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Acknowledgments
We thank the University of Wisconsin—River Falls Office of Undergraduate
Research, Scholarly and Creative Activity for providing funding to conduct
this research, the editors and anonymous reviewers who provided
thoughtful constructive criticism on our manuscript, Kevyn Juneau
for ImageJ guidance and manuscript feedback, Heather Sumner Davis for
providing laboratory facilities, Eric Simmerman and Ron Schmidt for
catching all trout analyzed in this study, and Esri for the ArcGIS® and
ArcMap™ software used to make the map in this manuscript.
Submitted 04 March 2019
Revised 10 December 2019
Accepted 11 December 2019