Thesis_Mdanley

Let it Snow: Effects of Persistent Organic Pollutants (POPs)on the uptake of
micro-plastics in marine snow
Meghan Danley
Marine Science, Biology Track
May 13th, 2015
Honors Thesis Submitted to the Life Sciences Standing Honors Committee
Committee members: Dr. Steven Sutton, Dr. Stephan Zeeman, Dr. Markus Frederich

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Abstract
In addition to harmful macro-plastics, degraded forms of plastic or micro-plastics, are on the rise.
Their affinity for agglomeration and leaching harmful additives makes them a great subject for
marine snow studies. In this study, marine snow aggregates were generated with polycyclic
aromatic hydrocarbon (PAH) contaminated plastic micro-beads. The composition of these
aggregates was examined with shape and size analysis and flow-cytometry. Contaminated plastic
containing aggregates were found to form chain-like structures with high plastic content unlike
their uncontaminated plastic counterparts which agglomerated with a high algae content.
Introduction
Plastics are a ubiquitous, detrimental pollutant in the world’s oceans and the
ecological impacts of trash sized plastic pollutants are well studied, but the breakdown of
these large pollutants into micro-sized particles, or “micro-plastics” is poorly studied
(Cole et al 2013, Collignon 2012, Farrell & Nelson 2013). Micro-plastics are defined as
plastic debris smaller than 5mm found throughout the sea surface across the globe (Cole
et al 2013, Farrell & Nelson 2013). Degradation into microscopic form can occur via
photo, chemical or physical breakdown (Mathalon 2014). Estimates at the surface of the
total amount of plastic could be 2.5 times less than the actual amount circulating in the
water column (Farrell and Nelson 2013). This gross underestimation and lack of study
makes micro-plastics an important feature for marine pollution studies.
Persistent Organic Pollutants, referred to as POPs from here on, are often added to
plastics during the manufacturing process. The rapid pace of technological developments
has led to a lack of regulation in these harmful additives until recently (Koelmans 2014,
Engler 2012). POP additives leach from plastics as they degrade into other plastics, cells,

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and the environment (Koelmans 2014, Engler 2012, Bakir et al. 2014). Potentially
harmful POPs commonly occurring in plastics include polychlorinated biphenyls (PCBs),
brominated flame-retardants (BFRs), Bisphenol A (BPA), polycyclic aromatic
hydrocarbons (PAHs) and phthalates (Engler 2012). Recent evidence of carcinogenicity
and other human health issues associated with chemicals like BPA and phthalates have
made POPs a major health and environmental concern (Nielsen et al 1996, Collins 1998,
Petry 1996), since dispersal of POPs is partially dependent on marine plastic circulation
(Mathalon 2014, Engler 2012, Koelmans 2014). Furthermore, studies (Koelmans 2014,
Engler 2012, Bakir et al. 2014) suggest that the large surface area to volume ratio in
micro-plastics could make them more prone to leaching toxins than macro-plastic
counterparts, which increase the possibility of harmful effects on organisms (Engler
2012). This study chose to look at PAHs in particular, due to their well-studied
carcinogenic and reproductive effects, and extreme affinity for materials such as plastic
(Cole 2013, Cole 2011, Teuten 2009).
Despite all of the potential risks to marine organisms, the physiological effects of
micro-plastics are poorly understood (Collignon 2012). One of the most susceptible
groups of organisms is filter-feeding (i.e., suspension-feeding) bivalves. These organisms
are inherent bio-accumulators; as filter feeders, they concentrate well-dispersed small
aggregates into their guts. The trophic interactions between bivalves, such as mussels,
and higher-level invertebrates like crabs has also been shown to facilitate transfer of
micro-plastics from the tissues of M. edulis (blue mussel) to C. maenas (green crab)
(Farrell and Nelson 2013). This trophic transfer could have implications for
bioaccumulation of micro-plastics and POP additives for all levels of the trophic system,

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including humans (Koelmans 2014, Engler 2012, Farrell & Nelson 2013). Bivalve
feeding mechanisms are not efficient at capturing particles much smaller than 4 μm
(Ward and Shumway 2004). The inability to retain such small particles in bivalve feeding
would prevent a wide range of micro-plastics from being ingested directly however, a
large portion of bivalve diets consists of marine aggregates, or “marine snow” (Newell et
al. 2005, Ward and Shumway 2004, Lyons et al. 2005). Marine snow consists of detritus,
bacteria, and organic/inorganic accumulations. These aggregations can also potentially
harbor harmful POP containing plastics, creating a direct transport of micro-plastics from
the water to bivalve tissues (e.g., Kach and Ward 2008).
Methods
The study of plastic micro-plastics specifically in marine snow has little previous
literature (other pollutants have been studied, e.g. Lyons et al. 2005), so a conglomerate
of methods for this project was developed using techniques from Dr. Evan Ward of
UCONN’s marine snow research, PAH sorption techniques from plastic leaching studies
(Velzeboer 2014, Engler 2012, Cole 2013), and flow cytometric analysis of the resulting
aggregates. In addition, a novel method for analyzing aggregate shape and size was
developed using Image J image measuring software.
Choosing a micro-bead
To determine whether the micro-plastics are in the water or within the marine
snow they need to be visible with either fluorescent dyes, or brightly colored dyes. This
project used Cospheric’s 10 to 45 μm, fluorescent red polyethylene microspheres. The

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size and range of the beads was selected to be more representative of actual pollutants.
The fluorescent color was selected to differentiate the beads from the natural aggregate
material to be detectable by flow cytometry.
Plastic Stock Preparation
See Protocol 1 in appendix for additional details. The beads are highly
hydrophobic and require a surfactant in order to disperse them in water. Rather than use a
chemical surfactant a natural “weathered state” method adapted partially from Velzeboer
2014 and Adams 2007 was used to better mimic the natural environmental conditions
(Velzeboer 2014, Adams et al. 2007). The PAH additive selected was Accustandard’s
PAH Mix containing 18 different PAHs at various concentrations in acetonitrile. A full
list with the concentrations of each can be found in the Appendix (Table 1). The PAH
mix was added to create a concentration of 150μg/L of the lowest concentration PAH in
the water and ensure the plastics would have ample PAH mix available for sorption.
The beads were added to 400 mLs of 0.2 μm filtered seawater, and spiked with
150 μLs of either acetonitrile, or PAH mix dissolved in acetonitrile to create two stock
solutions at 350mg/L each. Each stock solution was aged for two weeks in a temperature
controlled environmental chamber on a shaker table at 100 rpm to agitate the stock
mixture (Velzeboer 2014, Adams et al. 2007). After the two-week period when the beads
were fully submerged and evenly distributed, they were used to spike the rolling bottles
to the desired concentration of 1 million beads per liter. An additional “Blank” bottle of
filtered seawater and equivalent volume of acetonitrile, to account for biocide effects
(Adams et al. 2007), was prepared and used to spike the non-plastic containing bottles.

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Solvent effects were negligible because the acetonitrile content was less than 0.22% of
the total volume (Velzeboer 2014).
Rolling experiments
The rolling experiment method was based on a one developed by Evan Ward at the
University of Connecticut where the experiments were carried out. See Protocol 1 in the
appendix for additional details. Rolling experiments create marine snow through water
motion generated by rolling bottles for several days. A standard rolling table set up is
shown in Figure 1.
Figure 1. A standard rolling table set up.
Raw seawater was collected locally at least 1 week prior to any experiment. It was passed
through a 210μm sieve to remove zooplankton and other organisms that could interfere
with aggregation. Water was then frozen in three acid-washed glass carboys until each
experiment. Each carboy had water quality analysis to ensure they were statistically
equivalent. The results for these analyses can be found in the appendix.

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The “blank” (non-plastic containing) was run first two weeks prior to the plastic
experiments, and the two bead-containing experiments were run on the same day to
ensure equal treatment of both plastic-stock types. On the day of an experiment, six
bottles were filled in alternating 300mL aliquots of thawed seawater, spiked with stock
solution and set on the rolling tables. In addition to the “rolled” bottles, two spiked
“unrolled” bottles were set aside to settle naturally without aggregate formation to
compare the behavior of the plastics. The rolling table rotated the bottles about twenty
times per minute for three days, to create differential settling and collision of particles
without breaking up aggregations.
After removal from the table, the bottles were set aside for one hour to allow
aggregates of larger sizes to settle. The unrolled bottles were inverted gently three times
and also left to settle. After the settling time, aggregates visible without magnification
were collected with a glass Pasteur pipette with as little water as possible and transferred
into clean glass scintillation vials. A final water sample of 20 mL (after collection of
aggregates) was taken after vigorously shaking the bottles. If the plastics are incorporated
in the aggregates, then presumably the majority of the particles can be collected. Particles
that are not incorporated in the aggregates will be found in the final water sample.
Image J
Image J software is an image tool used for video and photo analysis. In this project the
“Particle Analysis” toolbox was used to determine the shape and size of aggregates in
samples. A web tutorial provided by Ruzicka 2014 was adapted to be able to measure
individual aggregates, remove non-aggregates, and quantify physical characteristics of

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the aggregates for statistical comparison. Several photographs were taken of each rolling
bottle immediately after it was removed from the rolling table, prior to it being allowed to
settle. Aggregates were suspended with slight agitation and photos taken from several
angles to ensure all aggregates were accounted for. These photos were uploaded and
converted to black and white 8-bit images, and set to color-fill specific color ranges in
each image. The resulting “silhouette” images shown in Figure 2 are the subject of the
size and shape analysis.
Figure 2. 8-bit conversion of aggregate photograph for PAH-spiked bottle 1.
Particles of all circularities were accepted but a specific range of sizes was set to remove
outliers from the results. Numbered outlines of the accepted particles were over-layed to
check that non-aggregates were not selected and all aggregates were accounted for in the
Excel data sheet that was generated.
If non-aggregates were included in the overlay, their corresponding number and data was
removed from the excel sheet. Conversely, if aggregates were missing or improperly
outlined, they were re measured and added to the data set. This method provided an
assigned number, area or size in mm2, a solidity value and a circularity value.

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The circularity of a particle is measured without units as 4𝜋 ×
𝐴𝑟𝑒𝑎
𝑃𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟
on a scale of 0
to 1, where 1 is a perfect circle. The Solidity of a particle is the
𝐴𝑟𝑒𝑎
𝐶𝑜𝑛𝑣𝑒𝑥 𝐴𝑟𝑒𝑎
. The convex
area is the area of an imaginary hull around the particle. Essentially solidity is the
“ruffled-ness” of a particle. A high solidity indicates a full, spherical shape, and a low
solidity a jagged or irregular surface. It is also measured on a unit-less scale from 0 to 1.
These measures characterize the overall shape of the aggregates with numerical values
that can then be compared.
Flow Cytometry
Flow cytometry was ideal for assessing aggregate composition because it is able to
differentiate between particles of different colors. The aggregates were made of a
composite of differently colored particulates of a variety of sizes, but could also be
broken up after collection to examine this composition. No literature method was used.
To prepare each sample, 100 μL of Tween-80, a non-biocide surfactant recommended by
Cospheric for use with the beads, was added to the aggregate and water sample vials and
then vortexed until the aggregates were dispersed into particulate form and evenly
distributed throughout water. 50 μL of the sample/Tween mixture was added to 150μL of
Milli-Q clean water in a 96-well plate. To prevent crossover of plastics, wells between
lanes of different sample types were filled with detergent and clean water to wash off the
stirring mechanism and capillary tube. The results were plotted as measures of green
fluorescence versus yellow fluorescence to show both algae and plastic in each sample.

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Results
Image J
Figure 1. Area of aggregates plotted over circularity (unit-less) for two treatments of micro-beads.
A circularity of 1 indicates a perfect circle, and 0 an elongated ellipse. VPE demonstrates more
even and tighter distribution, where PAH distribution varies greatly. Low circularity can be
correlated with larger size for PAH aggregates,but not in VPE. p values <0.05 for both size and
circularity.
The PAH-spiked (polyethylene with PAH). aggregates demonstrated a less even
distribution of sizes and low circularity values for larger particles. In the bottles large
stringy particles as well as much smaller varied aggregate sizes were observed, which
corresponds with the data. In general the Virgin polyethylene (plastic, no PAH)
aggregates were more uniform in size, and very circular. No strings or chain-like shapes
0
5
10
15
20
25
30
35
40
45
50
55
60
65
0 0.2 0.4 0.6 0.8 1
Area(mm^2)
Circularity
VPE
0
5
10
15
20
25
30
35
40
45
50
55
60
65
0 0.2 0.4 0.6 0.8 1
Area(mm^2)
Circularity
PAH

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were observed. The virgin-plastic aggregates tended to resemble the non-plastic
containing or “Blank” marine snow, besides color. Photographs of the blank samples
were not taken because the image analysis portion of the project had not been considered
at the time of the experiment. The low circularity of the PAH-spiked samples suggests a
chain forming agglomeration, which may be more unstable and prone to breakdown into
smaller aggregates, which may account for the larger number of small aggregates
compared to the Virgin-plastic sample.
Figure 2. Area of aggregates plotted over solidity (unitless) for two treatments of microbeads. A
solidity of 1 indicates a rounder edge, and a 0 more “ruffled” edges. VPE demonstrates more even
and tighter distribution, where PAH distribution varies greatly. Low circularity can be correlated
with larger size for PAH aggregates,but not in VPE. p values <0.05 for both size and circularity
(see appendix).
0
5
10
15
20
25
30
35
40
45
50
55
60
65
0 0.2 0.4 0.6 0.8 1
Area(mm^2)
Solidity
VPE
0
5
10
15
20
25
30
35
40
45
50
55
60
65
0 0.2 0.4 0.6 0.8 1
Area(mm^2)
Solidity
PAH

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The circularity and the solidity are related, so the expected result was to see low solidity
where there was low circularity. As seen in the circularity, there is a more uniform and
closer distribution of solidity for the Virgin-plastic but a wider range of values for the
PAH. This suggests that the PAH-spiked aggregates are less stable and less compact than
the Virgin plastic aggregates.
Flow Cytometry
Figure 3. Side and Forward scatter results for (from left to right) a) Blank b) Virgin plastic c)
Spiked. A higher forward scatter value indicates a larger size. A larger Side scatter value
indicates higher internal complexity of the particle.
The size range of the particles in each sample was wide for all three sample types: Blank
(no plastic), Virgin polyethylene (plastic, no PAH), and PAH-spiked (polyethylene with
PAH). The uneven size distribution was expected because the size range of the beads was
from 10 to 45 μm. The range of the blank sample was broader than the plastic samples
due to varying algae and sediment particulates that were not affected by plastic. The
larger range and number of side scatter values in the blank sample shows the algae not
obscured by plastic. That is, the higher “internal complexity” as indicated by the side-
scatter corresponds to the high internal complexity of an algae cell, rather than a low

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complexity for the micro beads. This is what causes the bunching of low complexity in
the spiked-plastic samples: the micro-beads dominate the sample. In the Virgin-plastic
sample however, there is still a higher level of this internal complexity that is more
representative of something like the blank sample. The Virgin-plastic samples appear to
be similar to the blank samples with high algae content, despite that there should not be
significantly more algae in these samples. The reason for this is demonstrated with the
fluorescence plots where a theorized “grouping” model was created.
Figure 4. Yellow versus red fluorescence for (from left to right) a) Blank b) Virgin plastic c)
Spiked. Results show non-plastic (not red) particles in each sample. Points farther to the right or
farther up indicate higher sizes at the fluorescence it is nearest to.
The blank sample results can be used as a control, points that match between it and the
plastic samples can be identified as algae or similar composition. The Virgin-plastic
sample shows a large cluster of green particles that are larger than the algae in the blank
sample, but are not plastic. That is, the Virgin-plastic sample contains both the same
particles as the blank which is to be expected, but also has larger algae “groupings”.
These groupings (aggregates) registering as large algae particles are actually algae
attached to beads, in such a way the algae is obscuring the plastic itself. An example of
the way this clumping is occurring is shown in the figure below. The opposite

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phenomenon is seen in the PAH-spiked samples. There is an absence of the algae signal
seen in the Virgin-plastic and the blank samples around 100 -101 on both axis, but there is
still a mixed group of particles that also appears on the Virgin-plastic plot. The green
fluorescent plot better supports evidence of the PAH-spiked postulated “grouping”.
Figure 5. The postulated clumping mechanism of plastics and algae generated with GeoGebra
design software. These groupings appear as single particles on the plots with larger sizes than the
cell and plastic components within them.
Figure 6. Green versus yellow fluorescence for (from left to right) a) Blank b) Virgin Plastic c)
Spiked. Results show red particles (mainly plastics). Points farther to the right or farther up
indicate higher sizes at the fluorescence it is nearest to.
The green fluorescence plots should not have had any points in the blank sample, as only
the red polyethylene spheres were expected to appear. The blank sample still had red
particulates, which are believed to be red algae fluorescing at a different wavelength than
the beads, but still show up as red particles on the plot. The green fluorescence peaks
below also show the fluorescence of the blank and the plastic is different. A similar trend

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from green plots to the red plots is the lack of algae visibility on the PAH-spiked
samples, but much more algae visible in the Virgin-plastic-plastic samples. There are a
greater number of large red particles in the PAH-spiked samples, but still shared overlap
with Virgin polyethylene that indicates a mixture of individual beads and algae cells. The
generally larger size of the particles in the PAH-spiked plot supports the aggregate model
proposed. These larger particles are actually the groupings of mainly plastics that obscure
the algae. In addition to the support for the PAH grouping model, the Virgin-plastic
model is also supported. The fluorescence includes algae, has less of these large plastic
only groups and has a much more similar green fluorescence to the blank sample which is
again indicative of algae attaching around plastics.
Figure 7. Green fluorescence histograms for (from left to right) a) Blank b) Virgin plastic c)
Spiked. Results show standard curve for number of particles of green fluorescence.
The similarity between the green fluorescence value for the Virgin-plastic and the blank
sample is likely due to algae fluorescence. The lower count for the Virgin polyethylene
can explained by it having beads obscured which are then not counted as green, but also
having less algae visible when it is obscured by beads.
Discussion
It was theorized at the beginning of this project that like would attract like: PAH would
attract PAH (Velzeboer et al. 2014, Engler 2012). This appears to be the case for the

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PAH sample, where long chains mostly plastic were created as the plastics agglomerated.
The virgin-plastics however, still attached other particles but instead of attracting plastic
to plastic, it attracted the algae to the plastic, which obscured the plastic’s fluorescent
signal. This could be due to the method used to weather the plastics. While the
weathering was a success and the plastics were dispersed, it is possible that the reason for
each surfactant behavior was not the same. The Virgin polyethylene may have created a
natural biofilm that did not damage the plastics surface (Cole 2013, Adams et al. 2007).
The PAH however may have damaged the surface of the beads or coated them which
created a surfactant effect, but did not allow for a biofilm to form (Adams et al. 2007).
When each was introduced to organic matter in the seawater, the PAH-spiked beads were
more attracted to one another, but the Virgin beads with biofilm were more attracted to
the algae and other natural materials in the water. The exact cause for attraction is a
subject for a different more in depth study, but the evidence this behavior is clear from
the results of this project.
The ecological implications for micro-plastic pollution are well studied for free-floating
micro-plastics but not as well studied for aggregations. The leaching of POPs from
plastics is also well studied (Cole 2013, Koelmans 2014, Cole 2011, Teuten 2009,
Mathalon 2014) and the effects of those POPs on organisms are as well. The next
consideration is the unavoidable aggregation that will occur as microplastics move
throughout the water and collide (Ward and Shumway 2004) and how the chemicals that
are likely moving from plastics to plastic affect the way these plastics interact (Koelmans
2014).

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The results from this study show that when PAHs are introduced to plastics, the
plastics develop an affinity for one another and gather around algae particles, which
prevents the algae from aggregating with other algae. This type of aggregation behavior
could be even more harmful to marine ecosystems than having even uncontaminated
micro-plastics. With this behavior, smaller but more plastic laden aggregates will be more
ubiquitous and widespread than large stable particles. If these smaller more numerous
aggregates were not contaminated with carcinogenic chemicals, it may not be as
problematic but studies show that these chemicals that cause this behavior can cause
many complications for organisms that ingest them (Cole 2013, Cole 2011, Collignon
2012, Browne et al. 2013).
Besides their introduction to the environment, the interactions of contaminated
plastics and cells are concerning. Many of the chemicals used as additives are lipophilic
and therefore are far more likely to be drawn out of the plastics into cells where they can
interfere with cellular processes (Cole 2013). This can result in cell death, cancer and
bioaccumulation and bio magnification of the chemicals. As these chemicals are dosed
into cells in small amounts over time the organism accumulates them. The bio
magnification occurs as organisms low on the trophic ladder are ingested by larger
organisms that consume multiple contaminated prey, thus receiving a much higher
amount of the contaminant. This pattern can continue up to humans who are huge
consumers of marine invertebrates such as shellfish (lobster, bivalves etc.) and fish. The
main consumers of marine snow are bivalves and zooplankton, which are highly likely to
be vectors for bioaccumulation. They are at the highest risk for ingesting PAHs and

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plastic, and the aggregates sizes generated in this study are well within the range of
capture size for these animals to ingest (Ward and Shumway 2004).
Conclusion
Marine snow is essentially agglomerations of detritus, which is as large a base of the
trophic pyramid as phytoplankton. These pollutants are already widespread, and as shown
in this study, are determining the composition, size, and stability of marine snow, which
has serious implications for the health of the oceans and humans. Further studies can be
done to explore the exact nature of the POP plastic interaction within marine snow using
microscopy to examine the exact shape of the groupings discussed in this paper.
Organismal studies are ideally the next step to confirm if the transfer of POPs is actually
occurring.

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References
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Hydrophobic Organic Compounds in Aquatic Environments. Environmental
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Bakir A., Rowland S.J., Thompson R.C., 2014. Enhanced desorption of persistent organic
pollutants from micro plastics under simulated physiological conditions.
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Cole M., et al., 2011. Microplastics as contaminants in the marine environment: A
review. Marine Pollution Bulletin 62: 2588–2597.
Cole M., et al., 2013. Microplastic ingestion by zooplankton. Environmental science and
Technology 47: 6646−6655.
Collignon A., et al. 2012. Neustonic microplastic and zooplankton in the north western
Mediterranean sea. Marine Pollution Bulletin 61: 861-864.
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Some Polycyclic Aromatic Hydrocarbons and Polycyclic Aromatic Hydrocarbon
Derivatives. Regulatory Toxicology and Pharmacology 28(1): 45-54.
Engler R.E., 2012. The Complex Interaction between Marine Debris and Toxic
Chemicals in the Ocean. Environmental Science and Technology. 46:
12302−12315.
Farrell P., Nelson K., 2013. Trophic level transfer of microplastic: Mytilus edulis (L.) to
Carcinus maenas (L.). Environmental Pollution 177: 1-3.
Koelmans A.A., Besseling E., Foekema E.M., 2014. Leaching of plastic additives to
marine organisms. Environmental Pollution 187: 49-54

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Lyons M.M., Ward J.E., Smolowitz R., Uhlinger K.R., Gast R.J., 2005. Lethal marine
snow: Pathogen of bivalve mollusc concealed in marine aggregates. Limnology
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Appendix
Protocol1: Rolling
Collection
 Acid wash 3 carboys and 2 collection flasks or beakers
o 10% HCl, 2 rinses with DI, 1 with MQ. Dry with loose foil caps.
 Collect at incoming tide, not after wind event or rain.
 Check that salinity is between 28 and 30ppt with YSI.
 Collect in 2 or 4L flask and pass through 210um mesh. Fill each carboy 1/3 and
switch filter to next carboy so that water is evenly filtered and distributed. Pass
off flasks to collect while the other person pours.
 Cap carboys with foil and freeze in -20 room, at an angle on towels/cardboard.
Concentrations and calculations
 Make up 350mg/L stock with surfactant Type 1C. Run 4 samples at a 1.5:25mL
dilution (dilution factor=17.67) through coulter counter to determine size
distribution and average particle number per mL. Use dilution factor to calculate
number of beads per mL for the 350mg/L stock. Back-calculate to find beads per
mg.
350
𝑚𝑔
𝐿
= 12,250
𝑏𝑒𝑎𝑑𝑠
𝑚𝐿
𝐵𝑒𝑎𝑑𝑠
𝑚𝑔
…..
12,250 𝑏𝑒𝑎𝑑𝑠
𝑚𝐿
×
1000 𝑚𝐿
1𝐿
×
1 𝐿
350 𝑚𝑔
=
12,250,000 𝑏𝑒𝑎𝑑𝑠
350 𝑚𝑔
= 𝟑𝟓, 𝟎𝟎𝟎
𝒃𝒆𝒂𝒅𝒔
𝒎𝒈
o Use 35,000 beads/mg for all conversions for stocks
 Make a stock in filtered SW as described below.
 Rolling bottle concentrations will be 1 million bds/L (100 x104 bds/mL),
28.57ppm.
 970mLs fits into bottle comfortably. 1L is right to brim.
 Start with 940mLs of water in each bottle. Add spike of 50mLs to bring to 990
mLs at 100 x104 bds/mL. After an initial 20mL water sample, 970mLs of the
proper concentration in each bottle.
Rolling
Prior:
 Thaw carboy to be used in cold room for 3 days. Swirl to break up periodically.
 Prep the seawater stocks (PAH Spike, PAH Blank, Plastic Blank) to age for 2
weeks:
o Collect fresh seawater
o Filter down to 0.2um
o 3 bottles, each with the following composition:
 PAH-PE Stock: 400mLs filtered seawater, 0.2828g 10-45um
LDPE beads, 150uLs PAH-mix.

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 Virgin-PE Stock: 400mLs filtered seawater, 0.2828g 10-45um
LDPE beads, 150uLs acetonitrile.
 Blank Stock: 400mLs filtered seawater, 150uLs acetonitrile
 Note: Checked a paper that says <0.22% of solvent has negligible
effects. 0.11% AcCN is under threshold.
o Age for 2 weeks until biofilm is formed.
Day of
 Move carboy from cold room to warm water bath in sink
 Thaw plastic stock or blank
 Lay out bottles and label
 Have foil lined cap scin vials ready for initial samples
 Distribute 900mLs exactly to each bottle in 300mL aliquots, pouring 300 for all 6
bottles, then the next 300 for all etc….
 Take 200mL for DOC and set aside
 Take 200mL for Alkalinity and set aside
 Take 3L for TSS and set aside
 Spike individual bottles with 10mLs of plastic stock. Invert or stir bottles to mix
particles
 Take initial samples
 To cap, place a small piece of clean foil on bottle top. Screw cap over foil and
tighten. (PAHs won’t be lost to aluminum, but could be lost to plastic caps)
Tested this and did not see leaks after a few hours.
 Invert 3 times. Place on rolling table and roll for 3 days.
Accustandard PAH Mix M-8310-QC-ATI contents:
Analyte CAS Conc Conc.in Stock(µg /L)
Acenaphthene 83-32-9 1000 µg/mL 1500
Acenaphthylene 208-96-8 2000 µg/mL 3000
Anthracene 120-12-7 100 µg/mL 150
Benz(a)anthracene 56-55-3 100 µg/mL 150
Benzo(a)pyrene 50-32-8 100 µg/mL 150
Benzo(b)fluoranthene 205-99-2 200 µg/mL 300
Benzo(k)fluoranthene 207-08-9 100 µg/mL 150
Benzo(g,h,i)perylene 191-24-2 200 µg/mL 300
Chrysene 218-01-9 100 µg/mL 150
Dibenz(a,h)anthracene 53-70-3 200 µg/mL 300
Fluoranthene 206-44-0 200 µg/mL 300
Fluorene 86-73-7 200 µg/mL 300
Indeno(1,2,3-cd)pyrene 193-39-5 100 µg/mL 150
Naphthalene 91-20-3 1000 µg/mL 1500
Phenanthrene 85-01-8 100 µg/mL 150
Pyrene 129-00-0 100 µg/mL 150
1-Methylnaphthalene 90-12-0 1000 µg/mL 1500
2-Methylnaphthalene 91-57-6 1000 µg/mL 1500

23
Total Suspended Solids (TSS) and Alkalinity
Alkalinity plots
0
2
4
6
8
10
0
0.02
0.04
0.06
0.08
0.1
0.12
0 0.5 1 1.5 2 2.5 3
pH
GranFunctionf(V)
Acid Volume (mL)
Blank
f(V)
pH
0
2
4
6
8
10
0
0.02
0.04
0.06
0.08
0.1
0.12
0 0.5 1 1.5 2 2.5 3
pH
GranFunctionf(V)
Acid Volume (mL)
PAH
0
2
4
6
8
10
0
0.02
0.04
0.06
0.08
0.1
0.12
0 0.5 1 1.5 2 2.5 3
pH
GranFunctionf(V)
Acid Volume (mL)
VPE

24
Alklainity Values
Calculated using the gran function (F(v)method.
Alk = (x-intercept) * (Normality of titrant)/ (sample volume in liters)
Alkalinity values (equivalents per liter):
Blank 1.75
VPE 1.77
PAH 1.76
Total Suspended Solids
Table 2.
P values for Total Suspended Solids
Blank/VPE VPE/PAH PAH/Blank
Total solids 0.580906684 0.27192912 0.643187718
Inorganic solids 0.628327623 0.268480841 0.4735534
All greater than 0.05=no significant difference
Image J results:
p-values for measures
Area 0.000460069
Circularity 0.0000962
Solidity 0.0000002
Calcluated with a 2-tailed T-Test with unequal variance. Even when outliers are removed,
all less than 0.05=significantly different.

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