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Discovery and quantification of plastic particle pollution in human blood
Heather A. Leslie, Martin J. M. van Velzen, Sicco H. Brandsma, Dick
Vethaak, Juan J. Garcia-Vallejo, Marja H. Lamoree
PII: S0160-4120(22)00125-8
DOI: https://doi.org/10.1016/j.envint.2022.107199
Reference: EI 107199
To appear in: Environment International
Received Date: 21 December 2021
Revised Date: 11 March 2022
Accepted Date: 18 March 2022
Please cite this article as: H.A. Leslie, M. J. M. van Velzen, S.H. Brandsma, D. Vethaak, J.J. Garcia-Vallejo,
M.H. Lamoree, Discovery and quantification of plastic particle pollution in human blood, Environment
International (2022), doi: https://doi.org/10.1016/j.envint.2022.107199
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1
Discovery and quantification of plastic particle pollution in human
blood
Heather A. Leslie1
Martin J. M. van Velzen1
Sicco H. Brandsma1
A. Dick Vethaak1,2
Juan J. Garcia-Vallejo3
Marja H. Lamoree1*
1 Dept. of Environment and Health, Faculty of Science, Vrije Universiteit Amsterdam, De
Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands
2 Deltares, Delft, the Netherlands
3 Cancer Center Amsterdam and Amsterdam Infection and Immunity, Amsterdam University
Medical Center (VUmc location), De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands
*Corresponding authors: marja.lamoree@vu.nl
2
Plastic particles are ubiquitous pollutants in the living environment and food chain but
no study to date has reported on the internal exposure of plastic particles in human
blood. This study’s goal was to develop a robust and sensitive sampling and analytical
method with pyrolysis double shot - gas chromatography/mass spectrometry and apply
it to measure plastic particles ≥700 nm in human whole blood from 22 healthy
volunteers. Four high production volume polymers applied in plastic were identified
and quantified for the first time in blood. Polyethylene terephthalate, polyethylene and
polymers of styrene (a sum parameter of polystyrene, expanded polystyrene,
acetonitrile butadiene styrene etc.) were the most widely encountered, followed by
poly(methyl methylacrylate). Polypropylene was analysed but values were under the
limits of quantification. In this study of a small set of donors, the mean of the sum
quantifiable concentration of plastic particles in blood was 1.6 µg/ml, showing a first
measurement of the mass concentration of the polymeric component of plastic in human
blood. This pioneering human biomonitoring study demonstrated that plastic particles
are bioavailable for uptake into the human bloodstream. An understanding of the
exposure of these substances in humans and the associated hazard of such exposure is
needed to determine whether or not plastic particle exposure is a public health risk.
Keywords: nanoplastic, microplastic, human whole blood, polymers, pyrolysis-GC/MS
3
1. Introduction
Measuring toxic chemicals in human tissues is invaluable in confirming exposure levels and
driving public health protection measures. A human health risk assessment (HRA) for plastic
particle pollution is currently not possible due to lack of data on both toxicological hazard
and human exposure (Leslie and Depledge 2020; Vethaak and Legler 2021). Measurement of
plastic particle exposure is essential for HRA, yet validated methods sensitive enough to
detect trace amounts of especially the small (<10 µm) size fractions of plastic particles in
biological tissues have been lacking.
‘Microplastic’ is a term for plastic particles for which no universally established definition
exists. In the literature, microplastic is often defined as plastic particles up to 5 mm in
dimensions with no defined lower size limit (e.g. Arthur et al. 2009; GESAMP 2015; ECHA
2019). ‘Nanoplastic’ is a term for plastic particles in the submicron range, <1 μm. In the
nanotechnology field, ‘nanoplastic’ may refer to engineered particles <100 nm, i.e. the
nanotechnology application size limit. To circumvent the ambiguity of the terms microplastic
and nanoplastic particles in this article we will refer to ‘plastic particles’ and where
appropriate define the size or size range. Our study was concerned with plastic particles that
can be absorbed across membranes in the human body. Our operationally defined method
targeted particles that could be retained on a filter with pore size of 700 nm, i.e. particles
≥700 nm in dimension. The inner diameter of the needle used for venipuncture (0.514 mm)
can be considered the upper size limit of particles this method could sample.
1.1. Plastic particle pollution
Analytical studies worldwide have established a large dataset of the occurrence of plastic
particles in various matrices including e.g. biota (or gut contents) (Boerger et al. 2010;
4
Karlsson et al. 2017; Ugwu et al. 2021), air (Gasperi et al. 2018; Wright et al. 2021), water
(Koelmans et al. 2019; Danopoulos et al. 2020; Schymanski et al. 2021), sediment
(Thompson et al. 2004; Phuong et al. 2020; Uddin et al. 2021) and foodstuffs (Van
Cauwenberghe et al. 2014; Barboza et al. 2018; De-la-Torre 2020). The majority of available
data is for particles with dimensions above 10 or 50 µm. Submicron sized plastic particles,
such as those reported in seawater (Ter Halle et al. 2017), have been much less studied so far.
As a whole, such data indicate the ubiquitous nature of plastic particles and raise the question
how exposed humans are to such particles, and if exposure actually leads to uptake within the
human body (Vethaak and Leslie 2016).
Human feces were previously analysed with Fourier Transform Infrared spectroscopy
(FTIR), providing evidence that micro-sized plastic particles can be excreted via the
gastrointestinal tract (Schwabl et al. 2019; Zhang et al. 2021). Plastic particles were also
detected in human colectomy specimens with FTIR (Ibrahim et al. 2020). Raman
microspectroscopy has been recently applied to image and identify three polypropylene
particles between 5 and 10 µm in human placental tissue (Ragusa et al. 2020).
1.2. The blood compartment
Blood as a compartment makes up 6-7% of body weight in humans. It irrigates the body’s
organs and is the transport pathway for oxygen, nutrients and potentially also plastic particles
around the body to other tissues and organs. The ultimate fate of plastic particles depends on
whether they can be eliminated by e.g. renal filtration or biliary excretion, or deposited in
either the liver, the spleen, or in other organs via fenestrated capillaries and sinusoids. A
particle’s size, shape, surface chemistry and charge govern its interactions with biological
systems, including the formation of a protein corona on the particle surface (Kihara et al.
2020). Blood’s role as transport pathway coupled with the feasibility of accessing samples
5
directly from the body, without contact with plastic materials, makes it a suitable matrix for
human biomonitoring of plastic particles and for the present study.
The degree of mixing within a bloodstream as a whole is considered to be high in healthy
individuals, with environmental contaminants being distributed over different phases
(aqueous, lipid, protein) throughout the circulatory system. Environmental microcontaminant
levels measured in venous blood samples are assumed to be indicative for the entire
bloodstream, including the microvascular system. Considering capillaries are typically only
5-8 µm in diameter, this forms a limit to particle sizes that can be expected in circulation in
these microvessels, and any particles present would likely have an impact on microvascular
fluid dynamics. In a well-mixed bloodstream, or in subsamples of a well-mixed blood
sample, there are many open questions regarding how plastic particles of different sizes might
be distributed. Some are likely to be localized in immune cells, while others may be adhered
to proteins, lipid particles, other plastic particles or the vascular endothelium. While mass
concentrations in a given sample may be detectable, the particles may be agglomerated, or the
number of particles themselves may be present at dilute concentrations in the matrix. This
gives rise to the possibility of observing non-detects and detects in duplicate samples
especially for small sample intake volumes. However, there are both ethical and practical
reasons for small blood sample volumes.
Because of the variety of interferences and non-plastic particles that could be present in a
given blood sample, it is important to develop methods that can confirm both the polymer
types and the concentrations present. In the advanced field of air pollution and human risk
assessment (HRA), the concentrations of particulate matter ≤2.5 µm or ≤10 µm (PM2.5 and
PM10, resp.) are sum parameters of particles that are collected through operationally defined
6
sampling methods, quantified, and then mass concentrations per unit air volume are reported.
One approach to quantifying plastic particles in the nascent field of plastic particle HRA is
based on mass concentrations of polymers from plastic present as particles, analogous to
PM10 for instance.
1.3. Analytical approaches
Many publications report abundance of particles identified as plastic with spectroscopy
techniques such as attenuated total reflection-FTIR and µFTIR (Veerasingam et al.
2020), and Raman (Anger et al. 2018) or stimulated Raman (SRS) (Zada et al. 2018). Particle
imaging provides information about particle sizes. A full characterization of particles in terms
of particle size, shape, chemistries, surface charge, degree of weathering, protein corona in a
given matrix are all legitimate parameters that can strengthen our understanding and the HRA
process. However, for real-world biological matrices methods are currently still under
development and measurement of all such parameters concomitantly is something for the
future. Promising approaches include the use of the above-mentioned approaches but also e.g.
time-of-flight secondary ion mass spectrometry (Jungnickel et al. 2016) and photoinduced
forced microscopy (Ten Have et al. 2021), among others (Ivleva 2021), to characterize the
smallest particles, requiring sensitivity and selectivity of low and submicron particles that are
expected in biological matrices. No one method fits all, therefore a combination of methods
will be required to capture all possible information. Meanwhile, more and more laboratories
are exploring thermal desorption mass spectrometry-based techniques to identify and quantify
the mass of individual polymers in a sample (Fries et al. 2013; Duemichen et al. 2019;
Ribeiro et al. 2020). Particle counting techniques and mass determination of polymers are
useful, complementary approaches. While waiting for other methods to reach technical
readiness, which is expected to take several years, we can already start building datasets for
7
human exposure to plastic particles based on mass concentrations, analogous to air pollution
particulate matter datasets based on particulate mass.
The present study focused on the analytical method development and measurement of human
blood to identify and quantify the mass of five high production volume polymers applied in
plastic materials: poly(methyl methylacrylate) (PMMA), polypropylene (PP), materials
containing polymerized styrene (PS), polyethylene (PE) and polyethylene terephthalate
(PET). These polymers have applications in food contact materials, textiles and a wide range
of other products humans come into daily contact with. PE and PP are the highest in demand
worldwide, followed by PET and polymers containing styrene such as polystyrene, expanded
polystyrene (EPS) and acrylonitrile butadiene styrene (ABS) (PlasticsEurope 2020). PMMA
has the lowest production volume in the test set, though it was selected because it is used in
various applications inside the human body, such as in dental work (Frazer et al. 2005). For
the present study, great emphasis was placed on method development and validation, running
large numbers of blanks and other quality control measures in order to achieve sufficient
method sensitivity and prevent false positives.
2. Methods
The analytical method developed, validated and applied here measures individual polymer
mass concentrations in the sample (not particle counts) using pyrolysis double shot - gas
chromatography/ mass spectrometry (Py-GC/MS). This semi-quantitative technique
quantifies thermal degradation products of the plastic particles present in the samples (i.e. a
destructive analysis).
8
2.1. Sample collection
Whole blood was obtained by venipuncture from 22 anonymized, healthy, non-fasting adult
volunteers who signed an informed consent under the rules and legislation in place within the
Netherlands and maintained by the VU Medical Center Medical Ethical Committee. Blood
was collected in 10-ml glass heparinized vacutainer (BD Biosciences, Plymouth UK) tubes.
The vacutainer was sealed by a rubber seal that is delivered with the glass vacutainers. The
vacutainers remained sealed during the entire sampling procedure and storage period.
Attenuated Total Reflectance-FTIR spectroscopy was used to identify the rubber seal
material as isobutylene-isoprene rubber, a copolymer type that is not targeted in this analysis.
Importantly, the vacutainer system is a closed device that allows blood withdrawal avoiding
any contact with plastic tubing or reservoirs. Venipuncture was done by means of a surgical-
grade sterile stainless-steel 21G needle (Becton Dickinson and Company, USA) that was
connected to the glass vacutainer under vacuum, such that blood was drawn from the donor’s
vein directly into the glass vacutainer. The entire sampling system (including needle and
vials) was tested for background contamination (field sampling blank, n=5) (see Quality
control section). The blood samples were stored in vacutainers in the freezer at -20 °C until
analysis.
2.2. Extraction method
The blood samples in this study were extracted and measured in duplicate (sample volume
permitting, n=18), in consecutive series, with multiple procedural blanks in each series to
control for background contamination and correct measured concentration data in blood for
the average procedural blank (see Quality control section). (Two aliquots were taken from
each vacutainer for the duplicate analysis.) Throughout recovery experiments and the sample
measurement series, a large number of procedural blanks were run.
9
After thawing, the blood samples in the vacutainers were mixed on a roller bank (CAT RM5,
Zipperer, Germany) for 1 h. Per analysis, approximately 1 ml of whole blood was weighed
and quantitatively transferred to a 20 ml glass scintillation vial (12383317, Yell, Germany)
that was pre-rinsed with analytical grade MilliQ® water (Millipore, Burlington MA, USA).
After adding 15 ml of TRIS-HCl buffer (400 mM Tris-HCL, pH 8, 0.5 % SDS, Trizbase
T6791, HCl H1758, Sigma, Schnelldorf, Germany) the vials were heated in a water bath at 60
°C for 1 h to denature proteins. To digest the proteins present in the whole blood, 100 µl of
the Proteinase K (1 mg/ml, 3.0-15.0 unit/mg, T. album, P8044 Sigma, Schnelldorf, Germany)
was added together with 1 ml of 5 mM CaCl2 (12095, Riedel-de Haën, Seelze, Germany) and
the vials were incubated for 2 h at 50 °C. The CaCl2 prevents autolysis of Proteinase K and
enhances thermal stability and substrate binding. Finally, the vials were shaken on a shaking
table for 20 min. at room temperature and heated once more at 60 °C for 20 min.
The samples were then filtered over a mm GF/F glass fiber filter, diameter 25 mm, mesh size
700 nm (1825-025, Whatman, Maidstone, United Kingdom). To ensure removal of any
plastic contamination present, filters were always heated in a 500 °C muffle oven purged with
nitrogen prior to filtration. For filtration a special glass setup (crafted by the glass workshop
of the University of Amsterdam, Amsterdam, the Netherlands), was adapted from a setup
previously used (Karlsson et al. 2017) to concentrate all the filtered sample in the center of
the filter within a surface diameter of 8 mm. The filter collected particles that could not pass
through the 700 nm mesh of the filter. The sample residue on the filter was rinsed with 10 ml
of a 30% H2O2 solution (Merck, Darmstadt, Germany) and rinsed with 15 ml MilliQ® water.
The inner 8-mm circle of the filter containing the analyte residue was then sliced out of the
center of the whole filter using a custom-made ring-shaped blade. The 8-mm filter was small
10
enough to later fit into a pyrolysis cup (which has a volume of ca. 80 µl). Before transferring
to a pyrolysis cup, the filter was placed in a pre-cleaned glass Petri dish (4 cm x 1.2 cm,
41042006, Karl Hecht, Sondheim, Germany) with a glass cover and dried in an oven (Binder,
Emergo, Landsmeer, the Netherlands) at 45 °C for 4 h, to complete dryness.
In order to reduce the number of depolymerization products of PET in the analysis step, a
reagent was used that results in the formation of a dominant product, dimethyl terephthalate
(DMT) (reactive pyrolysis GC/MS, or RxPy-GC/MS). The dried filters for polymer analysis
were treated with 10 µl of tetramethylammonium hydroxide reagent (25% in MeOH, 334901,
Sigma, Schnelldorf, Germany). The filters were then dried again in the oven at 45 °C for 1.5
h. The dry filters were transferred whole to a pyrolysis cup for analysis with Py-GC/MS.
2.3. Analysis by Py-GC/MS
Analysis was performed using the multishot pyrolysis unit EGA/PY-3030D (Frontier
Laboratories, Saikon, Japan) in “double shot” mode. First, the sample was placed in the
pyrolyzer unit at 100 °C, which was then heated to 300 °C at a rate of 50 °C/min. After the
sample was retracted, the GC/MS measurement started for any volatile compounds present on
the filter, as they thermally desorb between 100 and 300 °C. The GC/MS (Agilent 6890 GC
and 5975C MS, Santa Clara CA, USA) was equipped with a Ultra Alloy-5 column (30 m x
0.25 mm x 0.25 µm, Frontier Laboratories, Saikon, Japan). Measurements were done in SIM
mode (Table S1) and in split mode (1:5 split ratio). The compounds were trapped on the GC
column. The column was programmed from 40 °C (2 min) at a rate of 20 °C/min to 230 °C,
and then 50 °C/min to 320 °C, resulting in a total run time of 13.3 min. After the thermal
desorption step, the pyrolyzer was heated to 600 °C and the filter was again introduced (1
11
min) for the next measurement (pyrolysis). The column was programmed from 40 °C (2 min)
at a rate of 20 °C/min to 320 °C (2 min), resulting in a total run time of 18 min.
The compounds that are desorbed in the first run (‘shot’) are molecules that are volatilized
between 100 and 300 °C and can include unpolymerized monomers, additives and other
sorbed chemicals. Polymerized target analytes such as polystyrene, EPS, ABS, PP, PE,
PMMA are physically unable to volatilize in the ‘first shot’ because the maximum
temperature of 300 °C is too low (therefore they are retained for the second shot). Any
monomers (e.g. styrene) potentially present in the ‘first shot’ run were not used in
determining concentrations of plastic particles, except for PET where the derivatization
product already forms at 300 °C and the results from both the first and second shots were
combined. The pyrolysis second ‘shot’ chromatograms were used for determination of the
other polymer concentrations associated with particles. These were not affected by
interferences from monomers in the first shot. Note that the analysis of additives was outside
the scope of this study. Note also that studies of chemical additives in plastic in whole blood
do not confirm the concomitant presence of plastic particles in the sample, as additives can
desorb (or leach) from plastic materials throughout the lifetime into other hydrophobic phases
such as organic matter and the food chain prior to uptake in the bloodstream, or in some cases
come from non-plastic sources.
Target polymers included PMMA, PP, PS, PE and PET. The pyrolysis products measured in
double-shot Py-GC/MS were methyl methacrylate (for PMMA), 2,4-dimethyl-1-heptene
(PP), styrene (PS), 1-decene (PE) and as mentioned above, dimethyl terephthalate (PET).
Because styrene can be a pyrolysis product of not only polystyrene, but also of EPS and
copolymers of styrene (e.g. acrylonitrile butadiene styrene, styrene/butadiene co-polymer),
12
we quantified styrene as a pyrolysis product of any polymerized styrene from different
styrene-based plastics (abbreviated here as PS).
Quantification of pyrolysis products was performed using a calibration curve containing all
target polymer types at known concentrations. The polymer standards used were: poly(methyl
methylacrylate) (PMPMS-1.2, Cospheric, Santa Barbara, California, USA), polypropylene
(Sigma-Aldrich, Schnelldorf, Germany), polystyrene to represent materials containing
polymerized styrene (PSMS-1.07 Cospheric, Santa Barbara, California, USA), polyethylene
(CPMS-0.96 Cospheric, Santa Barbara, California, USA) and polyethylene terephthalate
(Goodfellow Cambridge Ltd., United Kingdom).
The five target polymer standards were weighed (3 mg each) and transferred to a 22-ml
stainless steel accelerated solvent extraction (ASE) cell containing 23 grams of sea sand
(which had been preheated at 600 °C in a muffle oven purged with nitrogen for 1 hour to
remove plastic residues) for dispersing and assisting in the ASE process. The polymers were
dissolved in dichloromethane (DCM) (Biosolve, 0013796002BS, for Dioxins, Pesti-S,
Furans, PCBs analysis, Valkenswaard, the Netherlands) in an ASE (Thermo ScientificTM
ASETM 350 Accelerator Solvent Extractor 083146, Waltham, MA, USA) at 180 °C, at a
pressure of 1500 psi, with further ASE conditions as follows: static time 5 min, 3 cycles,
rinse volume 80%, purge time 1.25 min, and heating time 9 min. From the resulting solution,
five different volumes were added to pyrolyzer cups and measured to obtain a calibration
curve (linear fit) for the 5 polymer standards present. The concentrations in the calibration
curve for the 5 different standards were between 15 and 400 ng polymer absolute each.
2.4. Quality control
13
Measures taken to ensure data quality were performing a spiking experiment, reduction and
control of background contamination during sampling, extraction and analysis through the
inclusion of a large number of blanks (sampling blanks, procedural blanks for the spiking
experiment and donor blood analyses), reporting limit calculations and performing duplicate
analyses.
2.4.1. Recovery experiment. To determine the recoveries of the polymers in blood with this
method, a spiking experiment was performed. A mixture of the five polymer standards was
spiked into blood subsamples (of a large sample of a single donor) at low analyte
concentrations, in eight-fold. This was repeated for a mixture of the five polymer standards
but at higher concentrations, also in eight-fold. Eight unspiked blood samples were added to
the series. All 24 blood samples in the recovery experiment were measured following the
procedure for extraction and Py-GC/MS analysis as described above.
At the same time, eight procedural blank analyses using analytical grade MilliQ® water were
measured. With these blanks, we were able to correct for background polymer concentrations
potentially introduced during sample preparation and analysis of the unspiked blood, in order
to determine which polymers were already present in the blood used for the recovery
experiment.
For the lower concentration spike experiment, 10 µl of the multiple standard solution was
added to each of the blood subsamples (n=8). For the higher concentration spike experiment,
75 µl of the standard solution was added to each of the blood samples (n=8). After
measurement, the spiked blood samples were corrected using the data for the unspiked blood
samples (note: without correction for the MilliQ® procedural blank), and the recoveries were
14
calculated for the 5 individual polymers. Recovery (%) was calculated by dividing the
measured polymer concentration (corrected for the blank) by the nominal spiked
concentration x 100%.
2.4.2 Controlling for background contamination during sampling. Sampling blank analyses
(n=5) were performed on the glass BD vacutainers used for sample collecting by rinsing the
vacutainers thoroughly with MilliQ®, collecting the residue in the filtration setup used for
samples followed by analysis with the same Py-GC/MS method. If a blank signal is found,
this step makes it possible to make corrections for background contamination inside the
vacutainer or needle.
2.4.3. Controlling for background contamination during sample preparation and analysis.
Procedural blanks (n=31) were performed using analytical grade MilliQ® water during
measurement of the donor blood samples. Procedural blanks underwent sample pretreatment
and analytical steps identical to whole blood samples. The procedural blank measurements
were plotted in Shewart charts for each polymer type. The analyte concentrations reported for
the blood samples were corrected for the corresponding average blank value from the
Shewart chart.
2.4.4. Limits of detection and quantification. The limit of detection (LOD) was calculated as
3 × the standard deviation of the average long-term value for each analyte’s procedural blank
signal. The <LOD samples contained unknown concentrations of the analytes between zero
and the LOD. The limit of quantification (LOQ) was calculated as 3.3 × LOD. The LOQ is
the lowest concentration of analyte that produces signals that can be quantitatively
15
determined with appropriate precision and accuracy. Only values >LOQ were used for
assessment of concentrations of plastic particles in blood.
2.4.5. Duplicate measurements of donor blood. Observing the level of agreement between
duplicate measurements can indicate the degree of analytical precision, sensitivity, but also
the potential heterogeneity of the analytes’ distribution throughout the blood sample. Each
blood sample was measured in duplicate in consecutive series with the exception of donors 6,
9, 15, and 18.
3. Results and Discussion
The sensitivity and performance of the method was demonstrated via the recovery experiment
and the control of background contamination throughout sampling and analytical procedures,
as described in section 3.1. The attention to quality control was key to ensuring the accuracy
of the measured concentrations in blood (section 3.2) was sufficient to support the
conclusions of this study.
3.1 Quality control
3.1.1. Recovery experiment. The background contamination in the eight MilliQ® blanks in
the recovery experiment are given in Table 1. The blood used for the recovery experiment
contained very low but still slightly elevated levels of PE and PS compared to the blanks; no
other analytes appeared to be present in the unspiked blood (Table 1).
Recoveries for the high spike experiment ranged from 64% to 114%, with low coefficients of
variation, CV, (between 8% and 29%) (Table 2), indicating an adequate performance of the
16
method at this concentration level. When PS was quantified with either styrene or the styrene
trimer, the recoveries were 79% and 68% respectively. Similarly, for PE the recoveries were
similar whether 1-decene (108% recovery) or 1-undecene (114% recovery) was used for
quantification (Table 2). The recoveries are acceptable for this concentration range, which
roughly corresponds to the range of concentrations quantified in actual blood samples from
the donors. This part of the work demonstrated that the method could extract, identify and
quantify low ppm concentrations of major polymers applied in contemporary plastics (Table
3).
Table 1. Amounts of polymers (ng absolute) for procedural blank with MilliQ® analytical
grade water, for unspiked blood and for low-spiked blood. s.d. standard deviation; CV,
coefficient of variation; LOD, limit of detection.
Analyte (and
monomer for
quantification)
MilliQ®
blank
mean (s.d.)
(ng)
CV
(%)
Unspiked
blood mean*
(s.d.)
(ng)
CV
(%)
Low
spike
mean **
(s.d.) (ng)
CV
(%)
PMMA (methyl
methacrylate)
5.2 (3.6)
70
4.5 (1.6)
35
-
16 (3.7)
22
PP (2,4-dimethyl-1-
heptene)
43 (3.7)
9
54 (16)
29
43 (11)
25
PS (styrene)
PS (styrene trimer)
40 (9.1)
8.3 (2.8)
23
33
111 (18)
<LOD
16
<LO
D
33 (20)
24 (8.6)
59
36
PE (1-decene)
PE (1-undecene)
72
65
40
38
150 (30)
206 (52)
20
25
120 (104)
151 (101)
87
67
17
PET (dimethyl
terephthalate)
8.8
31
14 (11)
76
33 (27)
83
*unspiked blood data shown here as not corrected for procedural blank; **corrected for unspiked
blood that is uncorrected for MilliQ® blank
The low spike experiment recoveries were lower, with recoveries under 50% for most
analytes except PP and PE (Table 2). This could be expected because the low spike samples
were close to the LOQ. The recovery experiments showed that the method performed well in
terms of recoveries of spiked analytes and CV (%) of the analysis of eight replicates in the
quantifiable analyte concentration range of the samples.
Table 2. Analyte recoveries from a) high spike and b) low spike blood samples, mean
recovery corrected for analyte concentrations in unspiked blood, standard deviation of the
mean (s.d.), coefficient of variation (CV).
a) High spike (n=8)
b) Low spike (n=8)
Analyte (and
monomer for
quantification)
Mean
recovery
(%)
s.d.
CV (%)
Mean
recovery
(%)
s.d.
CV (%)
PMMA (methyl
methacrylate)
73
0.13
17
27
0.06
22
PP (2,4-dimethyl-1-
heptene)
100
0.11
11
60
0.15
25
PS (styrene)
PS (styrene trimer)
79
68
0.06
0.10
8
14
41
32
0.24
0.11
59
36
PE (1-decene)
PE (1-undecene)
108
114
0.10
0.11
9
10
164
201
1.43
1.34
87
67
PET (dimethyl
terephthalate)
79
0.08
10
40
0.33
83
18
3.1.2. Controlling for background contamination during blood sampling. It is essential to
control for background plastic contamination throughout sampling and analysis of plastic
particles. No background contamination of any of the target analytes could be detected from
the glass BD vacutainers (all values <LOD), therefore no corrections were made for
background from the sampling procedure.
3.1.3. Controlling for background contamination during sample pretreatment and analysis.
During the method development and validation stage of the study, we performed a large
number of procedural blank analyses to ensure we could achieve low limits of detection of
the method through sampling pretreatment to analysis. The data for procedural blanks (n=31)
performed during the measurement of the blood sample series were sufficiently low for this:
the average ng absolute (on column) and coefficients of variance (CV) were 7.1 ng PMMA
(CV 86%); 75 ng PP (CV 61%); 36 ng PS (CV 63%); 95 ng PE (CV 43%); 12.5 ng PET (CV
72%). All blank data fell within ±2σ control limits, with the exception of one outlier for
PMMA and 2 outliers for PET (i.e. 1.6% of 186 measured blank data points). The large
number of blank analyses built the evidence that the values measured in real samples are not
false positives.
Background contamination during sample preparation and analysis measured via blanks
(n=31) were sufficiently low to enable detection and quantification of four polymers. The
measured concentration data in blood were corrected for the average procedural blank for
each analyte.
19
3.1.4. Limits of detection and quantification. The LOD and LOQ for each analyte are given in
Table 3.
Table 3. Limits of detection (LOD) and limits of quantification (LOQ) of the method.
Polymer
type
LOD
(µg/ml)
LOQ
(µg/ml)
PMMA
0.10
0.33
PP
0.68
2.3
PS
0.34
1.1
PE
0.61
2.0
PET
0.13
0.43
Values between LOD and LOQ are displayed in the supplementary information (Table S2)
with an asterisk, though they cannot be reliably quantified because of the difficulty of
calibration near detection limits. Such values can be regarded as being above the lowest
concentration of each polymer that can be detected though without guarantee of precision.
Values under the LOD after blank correction are reported as less than the value of the LOD
for that sample (Table S2).
3.1.5. Duplicate measurements of donor blood. In the 18 donors for which duplicate analyses
were performed, it was rare for duplicates samples to show both a non-detect (value <LOD)
and a value >LOQ: of all duplicate measurements, no cases of this are reported for PP or
PMMA, one case for PS and PE, and three cases for PET (Fig. 1 and Table S2). Because of
the samples with low analyte concentrations around the LOD, it was more common to find
one duplicate measurement to be a non-detect and the other to be between LOD and LOQ:
one case for PMMA, four cases for PP and PS, seven cases for PE and six cases for PET.
Unlike dissolved and sorbed micromolecules that passively diffuse and partition among
20
phases in the matrix, these target analytes are present in particulate form and may have very
different particle masses or form agglomerates. Inhomogeneity of samples may explain some
of the differences in duplicate measurements, though analytical sensitivity likely plays a role.
Many measurements were close to the LOQ and duplicate measurements were often both just
above and just below LOQ in the same donor (Table S2). Replicate analyses of samples is
useful for this analytes-sample matrix combination at this stage of methodological maturity.
This is the first study of its kind to use Py-GC/MS for plastic particle analysis of whole
human blood, a highly complex matrix, and we expect the sensitivity of the next generation
of this method to improve, as with all new methodologies.
3.2. Measured concentrations in blood
Data for concentrations in blood were generated for PMMA, PP, PS, PE and PET,
demonstrating that 77% of donors (n=17 out of 22) carried a quantifiable (>LOQ) mass of
plastic particles in their blood (Fig. 1 and Table S2). The patterns of polymer types and
concentrations varied per sample. PET was the most widely encountered (>LOQ values in
50% of all tested donors), followed by PS (36%), PE (23%) and PMMA (5%). No PP >LOQ
could be measured in any donor. The three polymers most frequently measured >LOQ were
also present at the highest concentrations. The maximum concentration of PET analysed in a
blood sample was 2.4 µg/ml, for PS this was 4.8 µg/ml, for PE this was 7.1 µg/ml. Up to
three different polymer types in a single sample were measured (Fig. 1). To make a
conservative estimate of the quantifiable sum polymer concentrations in the blood of donors
in this study, we summed all analyte values >LOQ per sample and took the mean of the
duplicate measurements per donor. Where values were <LOQ, we conservatively assumed
21
these to be zero. The mean of the sum concentrations for each donor was 1.6 µg total plastic
particles/ml blood sample (s.d. 2.3). This can be interpreted as an estimate of what might be
expected in future studies, and a helpful starting point for further development of analytical
strategies for human matrices research.
Figure 1. Concentrations of plastic particles by polymer type in whole blood samples of 22
donors (duplicates a and b, except for No. 6, 9, 15 and 18). All values >LOQ.
The duplicate measurements of blood aliquots from the same donor show better agreement
when values between LOD and LOQ are observed along with >LOQ values (Table S2) rather
than solely the values >LOQ (Fig. 1). Reasons for this include the high percentage of data
between LOD and LOQ compared to the data >LOQ. We also considered the possibility of
patchy distribution of the particles in the whole blood matrix. When determining the
concentrations in an individual donor it may be important to make use of replicate
measurements, considering the possible patchiness of the analytes and the challenges of
acquiring a single representative sample for analysis. As method sensitivity improves in next
22
generation analyses, lower LOQs may significantly enhance duplicate measurements in this
concentration range.
While this study provides evidence that plastic particles are present in the human
bloodstream, we found a high frequency of non-detects, and the percentage of donors for
which values >LOQ were measured varied per polymer type. No values >LOQ were
measured for PP in any donor, and only one donor tested >LOQ for PMMA. For the three
most detected polymers, the number of donors with values >LOQ were 5 for PE (23% of all
donors), 8 for PS (36%) and 11 for PET (50%) (Table S2). The frequency of non-detects
measured also varied per polymer type. The percentage of all donors for whom the analyte
was consistently <LOD in any measurement was 91% for PMMA, 82% for PP, 27% for PE,
and 9% for PET, and 5% for PS. Donor 18 was the only donor in which all analytes were
<LOD (this was one of the four donors not measured in duplicate).
The relatively high frequency of non-detects and the lower frequency of >LOQ values led to
the decision to present the data in Table S2 showing all values >LOQ, <LOD, and between
LOD and LOQ. The data in this pilot dataset should be interpreted as a clear signal that such
polymers can be present in human blood, as evidenced by the quantifiable concentrations
after blank correction, rather than an in-depth assessment of internal exposure in individuals.
For HRA many more data need to be collected and the science will benefit in further
improvements to the sensitivity of analysis in ongoing work in this field.
The particle size range targeted in this study was between 700 and 500,000 nm due to the
filtration step prior to analysis and the inner diameter of the needles used for the blood draw.
Because of the individual polymer concentrations detected (not exceeding 7.1 µg/ml),
23
particles in the blood samples were likely in the low- or submicron range. A sample with e.g.
a polymer concentration of 1.6 µg/ml could hypothetically be achieved by the presence of a
single spherical plastic particle with diameter of around 125 - 150 µm, or multiple smaller
particles. However, such large particles are less likely to be present in real blood samples due
to their lower bioavailability for uptake. With thermal desorption techniques such as Py-
GC/MS, we can report the particle masses in blood per polymer type, though not the number
of particles. Human exposure studies of air particulates also report particulates in µg/m3 air,
and not in particle numbers. The Py-GC/MS technique is suited to cases where the mass
concentrations of contaminants are needed, e.g. mass balance modeling, pharmacokinetics
studies and comparisons among individuals in a population.
3.3. Plastic’s biological fate?
The fate of plastic particles in the bloodstream needs further study to answer questions
regarding the potential accumulation in the general population and occupationally exposed
workers, the environmental factors contributing to the internal exposure and toxicological and
human health effects that may result from different exposure scenarios (Wright et al. 2017;
Leslie and Depledge 2020; Vethaak and Legler 2021). It is scientifically plausible that plastic
particles may be transported to organs via the bloodstream. The human placenta has been
shown to be permeable to 50, 80 and 240 nm polystyrene beads (Wick et al. 2010) and likely
also to microsized polypropylene (Ragusa et al. 2021). In a study of acute lung exposure to
nanopolystyrene spheres (20 nm) in rats, the translocation of plastic particles to placental and
fetal tissues was demonstrated (Fournier et al. 2020). Bioaccumulation of small polystyrene
micro-particles in the liver, kidney and gut was observed after oral administration in mice in
vivo (Deng et al. 2017; Lu et al. 2018).
24
Further supporting evidence for the translocation of plastic particles comes from drug
delivery sciences, where polymeric carriers of pharmaceuticals have been dosed in
mammalian test systems (Yee et al. 2020). The polymeric nanosized carriers are able to
deliver drugs across the blood brain barrier (Han et al. 2018). The typical residence time of
plastic particle in the bloodstream is at present unknown, as is the fate of these particles in the
human body. From polymeric nanocarrier research, we expect the residence time to vary with
particle chemistries, surface charges, shapes and sizes (Bertrand et al. 2012; Rabanel et al.
2012, 2019; Fullstone et al. 2015). In preclinical experiments in drug delivery it is known that
a phenomenon termed accelerated blood clearance (Dams et al. 2000) acts to reduce
residence time upon repeated (chronic) exposure to polymeric nanoparticles in the
bloodstream.
The uptake routes of plastic particles detected in human bloodstream are likely to be via
mucosal contact (either ingestion or inhalation). Dermal uptake of fine particles is unlikely
except if the skin is damaged (Schneider et al. 2009). Airborne particles between 1 nm and 20
µm are considered respirable. Ultrafine (<0.1 µm) inhaled particles may become absorbed
and accumulate in the lung, while most larger particles are expected to be coughed up and
eventually swallowed, and have a second chance of absorption via the gut epithelium (Wright
et al. 2017).
The plastic particle concentrations reported here are the sum of all potential exposure routes:
sources in the living environment entering air, water and food, but also personal care products
that might be ingested (e.g. PE in toothpaste, PET in lip gloss), dental polymers, fragments of
polymeric implants, polymeric drug delivery nanoparticles (e.g. PMMA, PS), tattoo ink
residues (e.g. acrylonitrile butadiene styrene particles).
25
4. Conclusion
The quality-controlled measurements of plastic particles as mass concentrations using Py-
GC/MS in blood demonstrated in this study provide a unique dataset that supports the
hypothesis that human exposure to plastic particles results in absorption of particles into the
bloodstream. This indicates that at least some of the plastic particles humans come in contact
with can be bioavailable and that the rate of elimination via e.g. the biliary tract, kidney or
transfer to and deposition in organs is slower than the rate of absorption into the blood. HRA
requires measured internal exposure data, and these must be empirically collected. Without
such measured exposure data, no absorption models can be validated and no statements about
risk or no risk can be made (Leslie and Depledge 2020; Vethaak and Legler 2021). It remains
to be determined whether plastic particles are present in the plasma or are carried by specific
cell types (and to which extent such cells may be involved in translocating plastic particles
across mucosa to the bloodstream). If plastic particles present in the bloodstream are indeed
being carried by immune cells, the question also arises, can such exposures potentially affect
immune regulation or the predisposition to diseases with an immunological base?
CRediT authorship contribution statement
H. A. L.: Conceptualization, methodology, validation, formal analysis, resources, data
curation, writing – original draft, visualization, supervision, project administration, funding
acquisition. M. J. M. v. V.: Methodology, validation, investigation, writing – review and
editing. S. H. B.: Methodology, writing- review and editing. A. D. V.: Conceptualization,
writing - review and editing. J. J. G.V.: Conceptualization, resources, writing – review and
26
editing, funding acquisition. M. H. L.: Methodology, supervision, writing – review and
editing.
Declaration of Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was made possible by grants from the Common Seas Foundation, London, United
Kingdom and from ZonMw (Netherlands Organisation for Health Research and
Development) in The Hague, the Netherlands. The funding bodies provided funding only,
and did not play any other role in the scientific work or manuscript publication process. We
would like to thank the anonymous volunteer blood donors, the Amsterdam University
Medical Center (location VUmc) staff for the blood drawing, Dr. M. Brits for his comments
on the data, Prof. J. de Boer for his assistance with the acquisition of the pyrolysis unit used
in this study.
Supplementary Information is available for this paper.
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Leslie et al. Plastic in Blood manuscript 2022
CRediT authorship contribution statement
H. A. L.: Conceptualization, methodology, validation, formal analysis, resources, data
curation, writing – original draft, visualization, supervision, project administration, funding
acquisition. M. J. M. v. V.: Methodology, validation, investigation, writing – review and
editing. S. H. B.: Methodology, writing- review and editing. A. D. V.: Conceptualization,
writing - review and editing. J. J. G.V.: Conceptualization, resources, writing – review and
editing, funding acquisition. M. H. L.: Methodology, supervision, writing – review and
editing.
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Figure S1. Schematic to accompany the paper (optional graphical abstract).
Leslie et al. Discovery and quantification of plastic particle pollution in human blood
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Leslie et al. Discovery and quantification of plastic particle pollution in human blood
Highlights
A method was validated for polymer mass concentrations in human whole blood
Polymers from plastics were detected and quantified in human blood
Polymers in human blood represent several high production volume plastics
Blood donors were from general public
Quality control of background plastic throughout sampling and analysis is key
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐ The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
