Assessment of ﬁ ne particles released during paper printing and shredding processes †

In this study, we investigated the airborne particles released during paper printing and paper shredding processes in an attempt to characterize and di ﬀ erentiate these particles. Particle characteristics were studied with real time instruments (RTIs) to measure concentrations and with samplers to collect particles for subsequent microscopy and cytotoxicity analysis. The particles released by paper shredding were evaluated for cytotoxicity by using in vitro human lung epithelial cell models. A substantial amount of particles were released during both the shredding and printing processes. We found that the printing process caused substantial release of particles with sizes of less than 300 nm in the form of metal granules and graphite. These released particles contained various elements including Al, Ca, Cu, Fe, Mg, N, K, P, S and Si. The particles released by the paper shredding processes were primarily nanoparticles and had a peak size between 27.4 nm and 36.5 nm. These paper particles contained elements including Al, Br Ca, Cl, Cr, Cu, Fe, Mg, N, Na, Ni P, S and Si, as determined by scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDS) and single-particle inductively coupled plasma-mass spectroscopy (SP-ICP-MS) analysis. Although various metals were identi ﬁ ed in the paper particles, these particles did not elicit cytotoxicity to simian virus-transformed bronchial epithelial cells (BEAS2B) and immortalized normal human bronchial epithelial cells (HBE1). However, future studies should investigate other cytotoxicity e ﬀ ects of these paper particles in various types of lung cells to identify potential health e ﬀ ects of the particles.


Introduction
Many employees work 8 h per day, and some spend more than one-third of the day in various indoor settings such as manufacturing industries, offices, and laboratories in the workplace.However, most workers are not aware of either the indoor air quality or their potential exposure to hazardous substances in the workplace.4][5][6][7] With the accelerated development of technology, printers have now become common equipment at home and in the workplace.Karrasch et al. have investigated the effects of laser printer device particle release on human subjects in lowlevel and high-level exposures and have reported 15 symptoms related to laser printer release in 37 subjects. 6Many factors contribute to particle release, especially from printers; these factors include the temperature, speed of printing, toner components and fuser. 8,9Toner, a potential major source of printer particle release, consists of various components, including thermoplastic polymers and styrene-acrylate copolymers.These substances are xed onto paper in a process called 'fusing' during printing. 10This process raised concerns, so toxicological studies have been performed using bronchoalveolar lavage uids (BALF) cells and alveolar macrophages in mice exposed to toner particles to evaluate the effects.An increase in total BALF cell number and a decrease in body weight have been observed during the recovery phase (9, 28, 56, and 84 days) aer exposure in mouse models. 10Pirela et al. have shown that exposure to printer released particles elicits biological responses in human cell lines, such as substantial damage to membrane integrity and increased release of pro-inammatory cytokines. 11sai et al. have investigated the airborne particles emitted inside the shredder basket during paper shredding. 12A substantial amount of particles containing various elements such as C, Pt, Si, and Ca, and ranging in size from nanometers to micrometers, were found inside the shredder.
1][12][13][14][15][16][17] The knowledge of the potential biological effects associated with the toxicity of these particles and its concentration levels in the air are necessary, but have not yet attracted the sufficient public attention needed to support further investigation.This research aimed to characterize the particle release from the printing and shredding of plain and printed paper, and to investigate the potential toxicity of the released paper particles by using in vitro cytotoxicity assays.

Materials and methods
The study comprised four parts: (1) evaluation of printer particle release, (2) evaluation of particle release from the shredding of plain and printed paper, (3) microscopy analysis of the released particles and (4) evaluation of the in vitro cytotoxicity of paper particles released during shredding.

Equipment
Direct reading real time instruments (RTIs), including a Nano-Scan scanning mobility particle sizer (NanoScan SMPS) (model 3910, TSI, Shoreview, MN, USA) and an optical particle sizer (OPS) (model 3330, TSI, Shoreview, MN, USA) were used in this study.The NanoScan SMPS measures particle size ranges of 10-420 nm, as monitored in NanoScan manager soware (version 1.0.0.19).The OPS measures a particle size range of 0.3-10 mm, as monitored in aerosol instrument manager soware (version 9.0.0.0).Both instruments recorded data with a 1 min response time; the NanoScan SMPS was operated at a ow rate of 0.9 L min À1 , and the OPS was operated at a ow rate of 1.0 L min À1 .
A Tsai diffusion sampler (TDS) was used to collect particles in the respirable and nanometer size ranges. 18The TDS uses a transmission electron microscopy (TEM) copper grid (400 mesh with SiO 2 lm coating, SPI, West Chester, PA, USA) attached to the center of a 25 mm-diameter polycarbonate membrane lter (0.22 mm pore size, Millipore, Billerica, MA, USA) as the sampling substrate to collect particles and it was operated at a ow rate of 0.3 L min À1 . 18One polycarbonate lter and one TEM grid were used to sample particles for each experiment.
( Particle release was studied during the printing 1000 sheets of paper and was compared with the release in control experiments, running 1000 blank sheets of paper.To improve measurements, a custom-made hood compartment was attached to the printer exhaust port, as shown in Fig. 1a, to contain the released particles within the hood space for The hood was an approximately 160 angled cone hood with a 0.115 m (4.5 in) inlet diameter, 0.305 m (12 in) outlet diameter and 0.18 m (7 in) duct length.The measurements were taken approximately 0.14 m horizontally from the center of the printer exhaust port, and the average air velocity at this sampling location was approximately 0.3 m s À1 .The entire surface of the hood was wiped with isopropyl alcohol before and aer each experiment.
Tubing 0.45 m in length (Tygon, Saint-Gobin, Malvern, PA, USA) was connected to the RTIs and between the sampling pump and TDS to reach the sampling location at the center of the custom-made hood.The total air ow rate of the RTIs and samplers was approximately 2.205 L min À1 .The paper was printed with a total of 806 words in 10 point font per sheet with black toner only.The RTI measurements were collected for a 10 min background reading at the beginning of the experiment, an approximately 40-43 min reading during the printing portion of the experiment (including paper relling and toner replacement time) and a 10 min post-experiment background reading.During the paper relling and toner replacement, the printer was at rest with the motor stopped.These periods are marked as gray highlighted areas in Fig. 2a-f and are denoted as 'resting time' in this study.The printing process was repeated three times, but the duration of resting time varied depending on the condition of the printer, such as the presence of a paper jam.All trials were performed under the same operating conditions with the same number of papers printed.The variations among repeated experiments were due to the resting time needed to clear paper jams and replace toners.
(2) Paper particles released from shredding activities.Shredding was performed in a glove box (Series 100, Terra Universal, Fullerton, CA, USA) equipped with ultra-ltered clean air with the RTIs placed outside the glove box, as shown in Fig. 1b and c.The temperature inside the glove box was 20-22 C, and the relative humidity was between 8.6% and 15%.The average air velocity blown into the glove box at the lter inlet face, located on the ceiling of the glove box, was 2.7 m s À1 and the average outlet face velocity was 1.4 m s À1 .The air velocity range in which samplers were located was less than 0.05 m s À1 .
The shredder was placed on top of a box (0.25 m Â 0.30 m Â 0.20 m), and 0.9 m-long tubes were used to connect the RTIs to reach the sampling locations for measurements.Fig. 1b shows the top view of the experiment with the location of each device.Measurements were taken at approximately 15 cm above the center line of the shredder, as shown in Fig. 1c.Each device was located on each side of the box to avoid ow interruption, and the total air ow rate was the same as that in the printer particle release tests (2.205 L min À1 ).The shredding experiments were performed with 40 sheets of (1) printed paper and (2) plain paper.The printed papers were obtained from the printer View Article Online particle release test and shredded at 30 s intervals for this evaluation.Each shredding experiment was repeated three times.The RTI data were exported into Excel and analyzed for particle number concentration and size distribution.The glove box was wiped clean with de-ionized water before and aer each experiment, and the shredder was also wiped with isopropyl alcohol to remove any contamination before and aer each experiment.All data were summarized to compare the released particle concentration and size during printing and shredding activities in the experiments.
(3) Microscopy analysis.Aer each experiment, the particles collected on TDS polycarbonate lters and copper grids were analyzed through electron microscopy.Small pieces of polycarbonate lters were coated with 10-15 nm gold and analyzed using scanning electron microscopy (SEM) (JSM-6500F, JOEL, Peabody, MA, USA) and energy dispersive spectroscopy (EDS) (model 51-XMX1015, Concord, MA, USA) at 15 kV.The grids were analyzed using TEM (JEM-2100F, JOEL, Peabody, MA, USA) and EDS (model 51-XMX1058, Concord, MA, USA) at 200 kV.These microscopy analyses were necessary for substances in micrometer to nanometer size range, to understand the morphological characteristics, sizes, and elemental compositions of the studied particles.The analyzed results will allow for identication of typical particles and their constituents which might be exposure risks for humans.
(4) In vitro cytotoxicity assays of paper particles released by shredding activities.This analysis has been widely used as an indicator of potential biological toxic effects by measuring the viability of cells aer exposure to studied particles.
Generation and collection of paper particles for cytotoxicity assays.The same shredding method as in (2) paper particles released by shredding activities was used with slight modications to collect airborne paper particles for cytotoxicity evaluation.A total of 200 sheets, printed in black, with one sheet fed every 30 s, were shredded into a 44 gallon bag placed underneath the shredder, and paper particles inside the bag were collected.The NIOSH manual of analytical methods (NMAM) 0500 method was slightly modied and used to collect particles in 37 mm cassettes with a polycarbonate lter at a ow rate of 1 L min À1 , instead of a PVC lter.The cassettes were located inside the bag.Aer shredding, the bag lled with shredded paper was shaken for 2 h for additional particle collection.The average total mass concentrations calculated based on the collected paper dust mass and sampling air volume were 1.35 Â 10 À4 and 2.08 Â 10 À4 mg mL À1 for black and plain paper respectively.
Preparation for cytotoxicity assays.The particles collected through the NIOSH NMAM 0500 method were weighed to obtain the mass, and the particles were then suspended in Dulbecco's modied Eagle's medium (DMEM) at a concentration of 10.77 mg mL À1 , which was the highest concentration (100%) exposed to cells in this experiment.The dose was determined to model the extreme scenarios for cell viability response on biomarkers caused by the exposure.The highest dose (100%) used was approximately 80 000 to 50 000 times higher than the average total mass concentration of paper particles collected using NMAM 0500 method.Several dilutions (e.g., 100%, 50%, and 25%) of particle suspended media samples were prepared for variations on cell treatment.The prepared particle suspensions in media were further sonicated with a sonic dismembrator (model 100, Fisher Scientic, Hampton, NH, USA) for 20 min in an ice bath.
Cytotoxicity assays used the following human bronchial epithelial cell line models: simian virus-transformed bronchial epithelial cells (BEAS2B, CRL-9609, ATCC, Manassas, VA, USA) and immortalized normal human bronchial epithelial cells (HBE1, a kind gi from Dr Reen Wu's laboratory, University of California, Davis, CA, USA) for treatments of 24-48 h.BEAS2B cells were isolated from normal human bronchial epithelium obtained from autopsies collected from individuals without cancer, [19][20][21][22][23][24] and HBE1 cells were obtained from a 60 year-old female donor with idiopathic pulmonary brosis. 25Thus, both cell lines are non-transformed.
In vitro cytotoxicity assays.HBE1 and BEAS2B cells were grown to conuence in 96 well plates (15705-066, VWR, Radnor, PA, USA) before paper particle exposure.Serum deprivation was then initiated 24 h before paper particle treatment for the BEAS2B cells and was followed by treatment with two types of paper particles (plain and printed) for 24-48 h.CellTiter 96 AQueous One Solution Cell Viability assays (MTS assay, Promega, Madison, WI, USA) were then performed to detect cytotoxicity according to the manufacturer's protocol.HBE1 cells were treated with the particles in their medium which were serum-free, similarly to the cells in BEAS2B medium, and cytotoxicity was tested 24-48 h aer treatment.
Plates were read at 490 nm with a microplate reader (Innite 200 PRO NanoQuant Microplate Reader, Tecan, Morrisville, NC, USA).Each concentration was assessed in three replicates per experiment, and the experiments were repeated three times.The cell viability results of three replicates were calculated to determine the standard error of the mean and were standardized by calculating the percentage change relative to control (set at 100%) for each plate.
Statistical analysis.Statistical analysis was conducted with the SPSS statistical analysis soware package (version 1.0.0.1126,IBM, Armonk, NY, USA).The cell viabilities of two cell lines (BEAS2B and HBE1) treated with various concentrations of paper particles were assessed and evaluated for statistical signicance with one-way analysis of variance.At a 95% condence level, p-values < 0.05 were considered statistically signicant.
(5) SP-ICP-MS analysis.A portion of particle-containing media prepared for cell exposure was analyzed for elemental composition using single particle inductive coupled plasmamass spectrometry (SP-ICP-MS) with a NexION 350D mass spectrometer (PerkinElmer, Bradford, CT, USA) connected to a self-aspirating nebulizer (PFA-ST nebulizer) (Elemental scientic, Omaha, NE, USA) and a Peltier (PC3x, Elemental Scientic, Omaha, NE, USA) controlled quartz cyclonic spray chamber (Elemental Scientic, Omaha, NE, USA) set at 2 C. Samples were centrifuged to remove agglomerates and were diluted 100 times with 2% HNO 3 .Samples were introduced with auto-dilution equipment (prepFAST SC-2 autosampler) (Elemental Scientic, Omaha, NE, USA).Before analysis, the nebulizer gas ow and quadrupole ion deector were optimized for maximum indium signal intensity.A daily performance check was also performed to ensure that the instrument was operating properly and that a CeO + to Ce ratio less than 0.025 and a Ce ++ to Ce ratio less than 0.030 were obtained.Aer suspending the particles in the media, the liquid suspension was injected into the SP-ICP-MS; the detection the sizes and elements of nanoparticles.This test was repeated three times.The differences in elemental composition were compared to the blank medium and analyzed with Syngistix's soware (PerkinElmer, Bradford, CT, USA).

Printer particle release tests
The measured particle concentrations obtained from RTIs were analyzed and are presented in two types of graphs showing (1) the total particle number concentration changes throughout the entire experiment and (2) the size-fractionated particle number concentration.
The total concentration of each experiment is presented separately in Fig. 2a-c for running 1000 sheets (control) and in Fig. 2d-g for printing 1000 sheets due to the inconsistent resting periods.The experimental periods (pre-experiment, during printing, during resting time and post-experiment) were noted.The average total concentrations and standard deviations throughout the experimental periods were calculated and presented in Table 1.Concentration changes during printing were calculated in this section by subtracting the preexperiment concentration, and the background concentration, to adjust the particle concentrations from the environment in the laboratory room.During the printing periods for printing 1000 sheets, the total net concentration of particles smaller than 420 nm (NanoScan SMPS data) increased by approximately 98 600 particles per cm 3 from 1400 particles per cm 3 to 100 000 particles per cm 3 .However, the net increase of running 1000 sheets was approximately 500 particles per cm 3 from 3100 particles per cm 3 to 3600 particles per cm 3 , thus indicating a 200-fold difference between printing and running 1000 sheets.The laboratory where this experiment was performed has the average background concentration of 7000 particles per cm 3 for particle size less than 420 nm and 7 particles per cm 3 for particle size range from 0.3 to 10 mm.The particles released during printing 1000 sheets had 14 and 7 times higher average concentrations than the laboratory background, in particle sizes less than 420 nm and in a range of 3-10 mm respectively.Printing 1000 sheets resulted in a substantial increase in concentration, as also seen through the comparison to the resting time values indicated in gray highlights in Fig. 2d-g.This increase caused by printing paper was apparent on sub-micrometer sized particles measured by NanoScan SMPS but not on larger particles measured by OPS.Currently, there are no health guidelines or standards for particulate number concentrations in the U.S.However, the contribution of high number particle concentration by printing processes to the indoor environments may become a concern.
In the control experiments presented in Fig. 3a and b, the dominant particle size generated from running 1000 sheets peaked at 27 nm, as determined through NanoScan SMPS, and the average concentration was approximately 6000 particles per cm 3 during the experiment.Although this corresponding mode size had a relatively higher concentration than the pre-and post-experiment concentrations, the mode peaked at 27 nm throughout the entire experiment.As discussed previously, when the toner was used for printing of 1000 sheets, the mode size appeared to be smaller (15 nm), with a concentration of approximately 300 000 particles per cm. 3 Thus, the printing process generated substantially more particles with smaller sizes within 10-420 nm than were generated by running the printer.The amount of released particles with larger than sub- micrometer diameters, as measured by OPS (0.3-10 mm), did not differ between printing and the control process of running the printer, as presented in ESI Fig. S1a and b. † The OPS mode sizes were 337 nm for both experiments, with a concentration range of 150-350 particles per cm 3 .

Morphology and elemental composition analysis of printer release
The printer released particles collected through TDS were in various shapes and sizes.The typical shapes of the particles were granular, irregular and layered.These particles were consistently found through microscopy analysis of samples collected during printing (Fig. 4a-d, 5a and ESI S2a-f †).The sizes of the particles observed under TEM and SEM were in the sub-micrometer range, which corresponded to the RTI measurements of 1 mm or less.Fig. 4a-d shows the results of granular and irregular shaped particles in TEM analysis and Fig. 4e shows the elemental composition of the particles in EDS analysis.For these particular particles, each granule ranged in size between 1 and 10 nm, and the major elemental composition comprised C, Cu, P, and S. Regardless of whether copper grids were used, Cu was found to account for a major portion of the particle composition.The blank copper grid in EDS analysis showed a 1 : 5 ratio between the La-shell and Ka-shell, whereas the copper-containing granular particles displayed a stronger peak ratio between the La-shell and Ka-shell (1 : 3 ratio or higher; Fig. 4e), compared with the other types of particles (Fig. S3 †).
Another typical observed particle was irregular and layered (Fig. 5a).To identify the characteristics of this type of particle, we used TEM line prole analysis to measure the distance between layers.The interlayer space measurement of the particle was 0.34 nm, a typical carbon bond length.This structural property is commonly identied as graphite, a multi-layer form of carbon, through the established analysis method. 26ther than the granular and layered particles, the irregular shaped particles observed under TEM were also analyzed with  On the basis of our ndings, multiple factors can contribute to the constituents of released particles from printing, such as metal-containing parts inside the printer and the heat generated during printing.The high level of nanoparticle release from a printer can cause respiratory problems and indoor air quality issues in similar environments, such as commercial printing rooms or any locations where printers are in use.As also stated in the NIOSH Pocket Guide to Chemical Hazards, copper may be associated with adverse health effects, such as acceleration of mutation in respiratory tract, skin, liver and kidney cells.Different printers may emit particles with different characteristics and airborne concentrations.

Paper particles released from shredding activities
The particle concentrations measured from shredding activities were compared among the pre-experimental background, the shredding process and the post-experimental background, and the changes in particle concentration and size distribution were observed (Fig. 6a-c).As presented in Fig. 6a, the paper particle concentration in the 10-420 nm size range (NanoScan SMPS) increased at the beginning of the shredding, and the concentration in the 0.3-10 mm size range (OPS) increased at the end of the shredding experiment.Table S2 † summarizes the average concentrations of various particle sizes on both instruments by the rstand second-half (10 min) of shredding time (20 min).There was no indication of a mode size change between the rstand second-half of shredding for OPS measurements.The mode size on OPS was determined to be the same as 337 nm on both the rstand second-half of the shredding period, and as expected the overall concentrations of all sizes on second-half of shredding period were higher than the rst half.The mode size of NanoScan SMPS measurements varied from 20.5 to 36.5 nm  and the concentrations did not give a clear indication of concentration increase as seen on OPS measurements.The released paper particles had similar average concentrations of 77-82 particles per cm 3 , on the basis of OPS measurements, regardless of whether plain (control) or printed paper was shredded.However, Table 2, the ndings regarding released particles less than 420 nm (NanoScan SMPS) showed that shredding printed paper released three times fewer particles than shredding plain paper.This result was notable in terms of the size distributions and upper standard deviations (Fig. 6b and c).The mode size of the released printed paper particles was 27 nm with a concentration of 10 000 particles per cm 3 , whereas plain paper had a mode size of 37 nm with a concentration of 26 000 particles per cm 3 , a value 2.6 times greater.The high variations of standard deviations (Fig. 6b) are due to the loosely structured plain paper that was not pressed with the toner through the heating process.More small particles were released from plain paper than printed paper due to the structural alteration, as conrmed by the TEM and SEM analyses.

Analysis of paper surface and elemental composition
To determine the structures of printed paper and plain paper, the surfaces of paper pieces and elemental composition analysis were conducted by using SEM and EDS (Fig. 7a and c) and TEM (ESI Fig. S4 †).The surface of the plain paper (Fig. 7a) showed entangled bers with nano-to micro-meter sized particle agglomerates.The surface of the printed paper (Fig. 7c) had a melted appearance similar to basalt, possibly as a result of the heat pressing during printing with the toner.The composition analysis showed that the printed paper contained Al, Ca, Cl, S and Si (Fig. 7d) and Na, Mg, and P elements were found from both the printed paper and plain paper (Fig. 7b).

Cytotoxicity effects of released paper particles and elemental composition analysis
In the cytotoxicity study, two human lung cell lines (BEAS2B and HBE1) were treated with paper particles, and the cytotoxicity responses of the cells to the particles were measured (Fig. 8a and  b).The cytotoxicity varied substantially and yielded inconsistent results according to statistical analysis (p-value > 0.05, determined at 95% condence level, for exposed concentration levels in all types of experiments).For the treated BEAS2B cells, the cytotoxicity results aer exposure to various concentrations did not show signicance among different concentrations of paper dust treatment for both plain paper particles (p-value of 0.866) and printed paper particles (p-value of 0.603).Similarly, for the treated HBE1 cells, the cytotoxicity responses aer exposure to plain paper particles (p-value of 0.324) and printed paper particles (p-value of 0.732) at various concentrations did not show signicance.In summary, treatment with all concentrations in both cell lines did not yield signicant changes in cell viability and appear to increase cell number; thus, there is no evidence that the various levels of paper particle concentrations to which the BEAS2B and HBE1 cells were exposed had signicant toxicity effects in terms of cellular response.
Investigating the effect of paper particle exposure on human lung cells is important because of the exposure possibilities to humans in various indoor environments.Although the cytotoxicity results of paper particle exposure showed inconsistency across various concentrations, and no signicant differences were observed, this result may not represent the response from human exposure.The cytotoxicity response in humans may differ depending on the particle size, an individual's physical and medical status and susceptibility to the constituents in the paper particles.For example, individuals with asthma may be more susceptible to these particles due to potential co-exposure effects, 27,28 the focus of future studies.In addition, many toxicants are not cytotoxic yet still exert biological effects in the human body; for example, some polycyclic aromatic hydrocarbons can lead to inammatory mediator production at nontoxic doses in lung epithelial cells. 29Regardless of the uncertainty of cytotoxicity, the nanometer-sized particles released by the shredding process are still of concern because of their deposition in the alveolar region aer inhalation and their potential to enter the bloodstream.
Additionally, the medium alone and the particle suspended medium used for cell treatment were analyzed with SP-ICP-MS to identify the potential elements affecting cytotoxicity.Fig. 9a and  b show the average intensity of each element (as the average relative intensity difference relative to blank media from three replicates with standard errors).The original SP-ICP-MS measurement report is presented in ESI Table S1.† The elements Br, Ca, Fe and P were identied from both plain and printed paper, and Al, Cu, and Ni were additionally found from only plain paper.A comparison of SP-ICP-MS and EDS analysis indicated that the elements Al, Ca, and P were commonly identied from plain paper, and Ca from printed paper, on both instruments.However, the remaining elements identied from SP-ICP-MS, such as Br, Cu, Fe, and Ni from plain paper, and Br, Fe, and P from printed paper, did not overlap with the EDS results.Cl, Mg, Na, S, and Si, the other elements identied in EDS analysis, were not detected in SP-ICP-MS analysis.The elemental composition analysis of paper particles showed limited overlapping constituents in SP-ICP-MS and EDS analysis.This discrepancy may be explained by the sample preparation process for SP-ICP-MS analysis, such as centrifuging, dilution, and removal of some paper particles to form evenly suspended solution, the variation of instrument detection limits and operating sensitivity.The EDS analyzes samples directly on particle or paper without processing any treatment or further laboratory procedures.As observed from the results, the use of different analytical instrument may alter the composition of samples due to the sample preparation procedures.

Conclusions
In conclusion, this study showed substantial particle release from printer printing and that those particles contained various elements.Shredding of printed paper released fewer particles than shredding of plain paper.A review article regarding indoor air qualities of PM 2.5 and PM 10 has reported the particle concentrations measured at various locations (homes, schools, offices and aged care facilities).Comparisons of the measurements using different instruments are challenging because some instruments measure particle in aerodynamic size or mobility size.Our measurements using NanoScan SMPS and OPS have shown the particle number concentrations in a range of hundreds to thousands of particles per cubic centimeters for particles less than 2.5 mm, which represents PM 2.5.Other studies have shown the PM 2.5 measurements in the range from 1200 to 1.2 Â 10 6 particles per cm 3 at various indoor locations. 30he contribution of particles released from printer and shredder use to the indoor air in such environments 30 will add to the indoor particles and may become of concern, especially for susceptible people.The cytotoxicity tests on BEAS2B and HBE1 cells exposed to paper particles showed no toxicity; thus, the ndings are inconclusive regarding additional potential health effects.However, the metal elements found on paper pieces and particles are known to have adverse health effects aer excessive exposure; the health outcomes from such exposure may vary depending on an individual's susceptibility and health condition.Additional cellular endpoints, such as inammatory mediators and wound healing, will need to be further evaluated in the future.
) Printer particle release test.Experiments were conducted in a NanoHood (Labconco, Kansas City, MO, USA).The high-efficiency particulate air (HEPA) ltration of the Nano-Hood was always in operation during the experiments.The atmospheric temperature and relative humidity during each experiment were measured with a VeloCalc air velocity meter (model 9515, TSI, Shoreview, MN, USA); the average relative humidity was approximately 51.3%, and the average temperature was 20-21 C. Particle release tests were conducted to assess the release and its constituents related to the toner use (TN420, Brother, Bridgewater, NJ, USA) during paper printing.The printer exhaust fan (D06K-24TU, Nidec Corporation, St. Louis, MO, USA) xed in a monochrome laser printer (HL-2270DW, Brother, Bridgewater, NJ, USA) had a maximum air ow rate of 0.63 m 3 min À1 .The same monochrome laser printer, toner, and paper (multipurpose copy paper, 8 1 2 00 Â 11 00 , #513096, Staples, Framingham, MA, USA) were used for all experiments.

Fig. 1
Fig. 1 Illustration of the release test experimental setup.(a) Front view of the printer release experiment setup.(b) Top view of the shredding experiment setup.(c) Three-dimensional view of the shredding experiment setup.

Fig. 2
Fig. 2 Real time instrument (RTI) data for particle release tests from running 1000 plain paper sheets and printing 1000 sheets.(a)-(c) Total concentrations from three repeated experiments of running 1000 sheets each, as measured by RTIs.(d)-(f) Total concentrations from three repeated experiments printing 1000 sheets each, as measured by RTIs.Note: Gray highlighted areas represent 'resting time' in Fig. 2, which the printer was at rest with the motor stopped for paper refilling and toner replacement.

Fig. 3
Fig. 3 NanoScan SMPS data of released particle size distribution.(a) Average particle size distributions of three repeated experiments running 1000 sheets each, as represented in Fig. 2a-c.(b) Average particle size distributions of three repeated experiments printing 1000 sheets each, as represented in Fig. 2d-f.

Fig. 4
Fig. 4 Microscopy analysis (SEM/TEM/EDS) of printer released particles.(a) SEM image of printer released particles on a TDS polycarbonate filter.(b) TEM image showing collected printer released particles on a TDS copper grid at low magnification.(c) TDS TEM image of printer released particles, with many attached granular particles, at high magnification.(d) TEM-EDS image of analyzed particles with attached granules, as observed through TDS.(e) EDS quantitative analysis of the image in (d) and qualitative analysis indicated by color.Note: Gold (Au) was used as a coating, which was excluded in this analysis.

Fig. 5
Fig. 5 Graphene TEM lattice analysis.(a) Representative TEM image of graphene particles released from the printer.(b) The intensity line profiles of the selected area from (a).

Fig. 6
Fig. 6 RTI [OPS (0.3-10 mm) and NanoScan SMPS (10-420 nm)] data for shredding 40 sheets of plain and printed paper.(a) Area total particle concentration, as measured by RTIs.(b) Paper particle size distribution with standard deviations, as determined by NanoScan SMPS.(c) Paper particle size distribution with standard deviations, as determined by OPS.

Fig. 7
Fig. 7 Microscopy analysis of paper particles from shredding plain and printed paper.(a) SEM images of plain paper at low and high magnification.(b) EDS quantitative and qualitative analyses of image (a), plain paper.(c) SEM images of printed paper at low and high magnification.(d) EDS quantitative and quantitative analysis of image (c), printed paper.Note: Gold (Au) was used as a coating in this analysis and was excluded.

Fig. 8
Fig. 8 Cell viabilities in two different cell lines (BEAS2B and HBE1) after paper particle exposure for 24-48 h.The mean values of each concentration are presented in bar graphs as a percentage with respect to the control in each cell line not exposed to paper particles.Error bars are standard error of the mean.(a) Changes in viability in the BEAS2B cell line.(b) Changes in viability in the HBE1 cell line.

Fig. 9
Fig. 9 Average element intensities of paper particles in media used for cytotoxicity assays, with standard error bars of each mean measured by SP-ICP-MS, representing the net intensity difference after subtraction of the blank sample.(a) Overall intensity results at a scale up to 15 000 mg g À1 .(b) Intensity results at a scale less than 700 mg g À1 of the highlighted area in (a).

Table 1
Average total concentrations from printer particle release tests, as measured by NanoScan SMPS and OPS a

Table 2
Average total concentration from shredding experiments, as measured by NanoScan SMPS and OPS.Standard deviations are presented in parentheses a a Standard deviations are presented in parentheses.This journal is © The Royal Society of Chemistry 2019 Environ.Sci.: Processes Impacts, 2019, 21, 1342-1352 | 1349 Paper Environmental Science: Processes & Impacts