Potential for Occupational Exposure to Engineered Carbon-Based Nanomaterials in Environmental Laboratory Studies

Background The potential exists for laboratory personnel to be exposed to engineered carbon-based nanomaterials (CNMs) in studies aimed at producing conditions similar to those found in natural surface waters [e.g., presence of natural organic matter (NOM)]. Objective The goal of this preliminary investigation was to assess the release of CNMs into the laboratory atmosphere during handling and sonication into environmentally relevant matrices. Methods We measured fullerenes (C60), underivatized multiwalled carbon nanotubes (raw MWCNT), hydroxylated MWCNT (MWCNT-OH), and carbon black (CB) in air as the nanomaterials were weighed, transferred to beakers filled with reconstituted freshwater, and sonicated in deionized water and reconstituted freshwater with and without NOM. Airborne nanomaterials emitted during processing were quantified using two hand-held particle counters that measure total particle number concentration per volume of air within the nanometer range (10–1,000 nm) and six specific size ranges (300–10,000 nm). Particle size and morphology were determined by transmission electron microscopy of air sample filters. Discussion After correcting for background particle number concentrations, it was evident that increases in airborne particle number concentrations occurred for each nanomaterial except CB during weighing, with airborne particle number concentrations inversely related to particle size. Sonicating nanomaterial-spiked water resulted in increased airborne nanomaterials, most notably for MWCNT-OH in water with NOM and for CB. Conclusion Engineered nanomaterials can become airborne when mixed in solution by sonication, especially when nanomaterials are functionalized or in water containing NOM. This finding indicates that laboratory workers may be at increased risk of exposure to engineered nanomaterials.

Large amounts of engineered nano materials are generated annually, and each possesses its own unique charac teristics. Much work has focused on carbonbased nano materials (CNMs), such as fullerenes and carbon nanotubes, because of their strength, conductivity, and applicability for biomedical applications (Ajayan and Zhou 2001). Consequently, considerable effort has been dedicated to understanding the health effects of these nanomaterials before they are widely used in consumer products where the potential for exposure to the general public would be increased (Helland et al. 2007). This is a proactive approach that has not been applied to some other classes of chemicals in the past, such as asbestos. Environmental researchers are actively examining the fate and effects on CNMs in environmentally relevant systems (Asharani et al. 2008;Farré et al. 2009;Helland et al. 2007;Kennedy et al. 2008;Klaine et al. 2008). Furthermore, researchers at the National Institute for Occupational Safety and Health (NIOSH) and other agencies are examining the potential occupational exposures to, and respiratory effects of, CNMs (Han et al. 2008;Helland et al. 2007;Maynard et al. 2004;Methner 2008).
Currently, no occupational exposure lim its govern workplace exposure to engineered nano materials (Methner 2008). NIOSH recommends using basic safety requirements when handling dried CNMs and other nano materials . Less attention has been devoted to workplace exposure and safety of engineered nanomaterials in liquid suspen sions. CNMs and other nano materials are usu ally placed into liquid suspension for easier delivery to experimental models. Conventional wisdom suggests that nano materials in liquid suspension generally pose lower inhalation risk to workers. However, CNMs and other nano materials often agglomerate in aqueous suspension, requiring continuous mixing or sonication to deagglomerate nanomaterials. It is possible that this common laboratory process results in the release and dispersion of nanoma terials into the air via small water droplets. This may concern scientists in general, but especially eco toxicologists, environmental scientists, and environmental engineers working with nano materials in simulated natural waters. These researchers routinely generate environmentally relevant matrices in the laboratory, including waters with natural organic matter (NOM), which acts as a surfactant that enhances the stability of nanoparticle dispersions (Hyung et al. 2007;Hyung and Kim 2008;Kennedy et al. 2008;Lin and Xing 2008;Saleh et al. 2008;Xie et al. 2008). Thus, sonication of NOMcontaining water can, in theory, result in increased aerosolization of the engineered nanomaterials when compared with the same material sonicated in deionized (DI) water.
It was with this premise that research ers at the U.S. Army Engineer Research and Development Center's Environmental Laboratory (ERDCEL) volunteered to be part of a nationwide field study of poten tial occupational exposure to nanomaterials, currently being conducted by the NIOSH Nanotechnology Research Center (NTRC). The specific research objective of the pres ent study was to investigate the potential for the release of airborne CNMs due to research involving the handling and mixing of CNMs with environmentally relevant matrices. The NIOSH NTRC field research team evaluated two laboratory processes: a) transfer of CNMs from storage containers to a weighing balance, and b) sonication. We used both quantitative and qualitative methodologies in this range finding study to determine the presence and concentrations of airborne nanoparticles.

Chemicals.
We purchased fullerenes (≥ 99.5% purity) from SES Research (Houston, TX). Raw multi walled carbon nanotubes bacKgrOunD: The potential exists for laboratory personnel to be exposed to engineered carbonbased nanomaterials (CNMs) in studies aimed at producing conditions similar to those found in natural surface waters [e.g., presence of natural organic matter (NOM)]. Objective: The goal of this preliminary investigation was to assess the release of CNMs into the laboratory atmosphere during handling and sonication into environmentally relevant matrices. MethODs: We measured fullerenes (C60), underivatized multiwalled carbon nanotubes (raw MWCNT), hydroxylated MWCNT (MWCNT-OH), and carbon black (CB) in air as the nanomaterials were weighed, transferred to beakers filled with reconstituted freshwater, and sonicated in deionized water and reconstituted freshwater with and without NOM. Airborne nanomaterials emitted during processing were quantified using two hand-held particle counters that measure total particle number concentration per volume of air within the nanometer range (10-1,000 nm) and six specific size ranges (300-10,000 nm). Particle size and morphology were determined by transmission electron microscopy of air sample filters. DiscussiOn: After correcting for background particle number concentrations, it was evident that increases in airborne particle number concentrations occurred for each nanomaterial except CB during weighing, with airborne particle number concentrations inversely related to particle size. Sonicating nanomaterial-spiked water resulted in increased airborne nanomaterials, most notably for MWCNT-OH in water with NOM and for CB. cOnclusiOn: Engineered nanomaterials can become airborne when mixed in solution by sonication, especially when nanomaterials are functionalized or in water containing NOM. This finding indicates that laboratory workers may be at increased risk of exposure to engineered nanomaterials. (MWCNT) (outer diameter, 10-20 nm; length, 10-30 µm; > 95% purity) and func tionalized MWCNT (i.e., hydroxylated; MWCNTOH) (outer diameter, 20-30 nm; length, 10-30 µm; > 95% purity) were pur chased from Cheap Tubes, Inc. (Brattleboro, VT). Carbon black (CB; amorphous carbon, average primary particle size of 15 nm) from Printex 95 was purchased from Evonik North America (formerly Degussa; Parsippany, NJ). NOM from the Suwannee River was purchased from the International Humic Substance Society (Atlanta, GA).
Laboratory processes evaluated. The first laboratory process ( Figure 1A) we evaluated was weighing 4-200 mg of each of the differ ent CNMs on an electronic balance and trans ferring the CNMs to a beaker of water stirring atop a Corning magnetic mixing plate (Cole Palmer, Vernon Hills, IL). This procedure was performed inside a laboratory safety hood with the air flow turned off temporarily and the sash halfway open. This was done because the hood air velocity (meas ured at 100 ft/min at the face) was high enough to result in loss of nanomate rial from the spatula during the transfer from the material container to the analytical balance. The second laboratory process ( Figure 1B) we evaluated was probe sonication (50 W; 40% duty cycle) of 100 mg/L previously mixed CNMs for 20 min inside an unventilated soni cation enclosure (Branson Sonifier model 450; Branson Ultrasonic, Danbury, CT). CNMs were sonicated in DI water or hard recon stituted water with and without 100 mg/L NOM. Personal protective equipment worn by workers when performing weighing and trans fer tasks and sonication processes consisted of a cotton laboratory coat, latex gloves, and an N95 filtering facepiece respirator.
Airborne particle detection. We used two directreading, realtime instruments to deter mine whether CNM emissions occurred dur ing these laboratory processes. The sampling inlet of each instrument was positioned as close as possible to the suspected point of emission for a given process (indicated by arrows in Figure 1). We used an HHPC6 handheld particle counter (ART Instruments, Grants Pass, OR) to determine the airborne particle number concentration based on optical count ing principles using laser light scattering. This instrument measured the total number of par ticles per liter (particles/L) of air across six spe cific size cut points: 300, 500, 1,000, 3,000, 5,000, and 10,000 nm. The second instru ment used was a TSI model 3007 handheld condensation particle counter (CPC; TSI, Inc., Shoreview, MN), operated as described by Methner (2008). The CPC unit measures particles in the size range of 10-1,000 nm, with data expressed as the total number of particles per cubic centimeter (particles/cc) of sampled air. The upper limit of detection for the HHPC6 and CPC are 70,000 particles/L and 100,000 particles/cc, respectively. Because the size and degree of particle agglomeration were unknown at the time of this evaluation, we determined that using these particlesizing instruments would provide a semi quantitative indication of the relative size range and mag nitude of potential emissions for each process. Ambient/background particle number con centration measurements were collected inside each laboratory before each task/process and used to adjust the processspecific measure ments via subtraction. Additionally, two gen eral area air samples were collected before and after the laboratory processes at an area away from the processes, but in the same room, to serve as an indicator of background concentra tions not related to specific processes.
Transmission electron microscopy (TEM). In addition to directreading instrumenta tion, filterbased air samples were collected to qualitatively determine whether engineered nano materials were emitted during the labora tory processes. The air sampling filters were positioned as close as possible to the suspected emission source (i.e., slightly above the ana lytical balance during weighing of material) ( Figure 1) for the duration of the task or pro cess to increase the probability of capturing nano materials and to simulate extreme case scenarios for laboratory personnel. This type of sampling strategy should not be interpreted as representative of fullshift worker exposure, yet it does provide an indication of potential worker exposure due to inadequate air sampling instrumentation that can be worn by work ers to estimate CNMs in a worker's personal breathing zone (Methner 2008). Sampling times ranged from 25 to 186 min (air volume, 175-1,300 L) and were dependent on the time necessary to complete the task being evaluated. The filterbased air samples were collected using Leland Legacy pumps (SKC Inc., Eighty Four, PA) that were operated at a sampling rate of 7.0 L/min. Pumps were calibrated before and after each day of sampling. Air samples were collected on 37mm diameter, 0.8µm pore size, openface mixed cellulose ester membrane filters. Additionally, one general area air sample was collected at an area away from the process, but in the same room, to serve as an indicator of background concentrations not related to specific processes. Sample filters were then ana lyzed using TEM with energy dispersive spec troscopy and a digital image system for particle sizing and elemental composition. TEM allows the microscopist the ability to identify par ticles in the nanometer size range and the mor phology of the particles (size, shape, degree of agglomeration). The sample filters were pre pared by direct preparation in accordance with NIOSH Method 7402 (NIOSH 1994) using acetone vapor to collapse the filter media onto a copper TEM grid. A bulk sample of each material handled was deposited onto blank mixed cellulose ester filter media and prepped in a manner identical to other air samples. The bulk material was used by the microscopist to identify each nanomaterial of interest. At least 20 random grid openings per sample were examined via TEM. If the nano material of interest was found, a digital image of the struc ture was captured. If no nano material of inter est was observed on the grids, the result for the sample was "none detected."

Results
Airborne particle detection. The goal of this study was to determine the potential for occupational exposure to CNMs when using environmentally relevant matrices to simulate environmental systems, such as streams, riv ers, ponds, and reservoirs. These water bodies contain varying concentrations of NOM, a naturally acting surfactant that improves the aquatic suspension of hydrophobic chemicals such as organic pollutants and agglomerated CNMs (Chefetz and Xing 2009; Hyung et al. 2007; Kennedy et al. 2008). Figure 2 dem onstrates that sonication of water containing 100 mg/L NOM resulted in the aerosolization of water droplets. This water droplet plume was generated during almost every sonication pulse. The cumulative effect over the course of the sonication process may result in substan tial aerosolization of water droplets. This may be of concern when working with CNMs in NOMcontaining waters because of the poten tial presence of CNMs in the water droplets. The particle number concentrations meas ured for each of the eight CNMs and laboratory tasks/processes (i.e., weighing/ handling CNMs and sonicating CNMs in aqueous suspensions) are presented in Table 1. After adjusting for background par ticle number concentrations, it was evident that increases in the airborne particle num ber concentration occurred during each process for almost all the CNMs exam ined. Airborne particle number concentra tions were inversely related to particle size, with the size distribution of particles skewed toward those CNMs with an aero dynamic diameter < 1 µm. During handling of hydro phobic C60 and raw MWCNT, the highest airborne particle number concentrations were seen at the 300nm size {53,119 particles/L for C60 and 123,403 particles/L for raw MWCNT [above the upper limit of detection (70,000 particles/L) for the HHPC6], fol lowed by the 500nm size (3,884 particles/L for C60 and 34,446 particles/L for raw MWCNT)}. When analyzed at the 10-1,000 nm scale, airborne C60 and raw MWCNT particle number concentrations were higher than background particle number concen trations and approximately the same particle number concentrations (~ 1,500 particles/cc). Similar handling effects were seen by Maynard et al. (2004) when gentle air currents in the laboratory produced airborne singlewalled carbon nanotube particles. Sonication caused aerosolization of C60 in a DI water suspen sion and raw MWCNT in a hard reconsti tuted water suspension containing 100 mg/L NOM. Sonication produced airborne C60 and MWCNT at concentrations approxi mately onehalf and onethird, respectively, of those observed during the weighing process (23,856 particles/L for C60 and 42,796 particles/L for raw MWCNT in the   a Average background number concentration was computed from two measurements obtained inside the room before material handling began and two measurements obtained after handling ceased. b If the difference between the measured particle number concentration and the average background particle number concentration was less than zero, the adjusted particle number concentration was reported as zero. c Particles in the range of 300-10,000 nm were quantified with the HHPC, and particle concentrations are given as particles/L. d Particles in the 10-1,000 nm range were quantified with the CPC, and particle concentrations are given as particles/cc. e Particle counts exceed the upper limit of quantification for the HHPC (70,000 P/L) or the CPC (100,000 P/cc). f Because of a change in background particle number concentration, a new average background particle number concentration was calculated for these tasks.
volume 118 | number 1 | January 2010 • Environmental Health Perspectives 300nm range). We observed a similar trend to the handling process during sonication, where highest particle number concentrations were in the 300 and 500nm size ranges. However, sonication increased airborne C60 and raw MWCNT particle number concen trations in the 10-1,000 nm size range (2,176 and 2,776 particles/cc, respectively) compared with weighing and handling dry CNMs. We observed a slightly different trend with MWCNTOH and CB, two functional ized, watersoluble forms of CNMs. Airborne concentrations of MWCNTOH and CB were very low during weighing and transfer ring, with the highest particle number con centrations detected in the 500nm range (3,065 particles/L for MWCNTOH and 1,428 particles/L for CB). This was confirmed in the 10-1,000 nm size range as well (676 and 0 particles/cc, respectively). However, soni cation of MWCNTOH in a moderately hard reconstituted water suspension with 100 mg/L NOM and CB in DI water suspension resulted in dramatically higher airborne particle num ber concentrations compared with handling dry CNMs. The highest particle number con centrations were in the 300nm size range [144,623 particles/L for MWCNTOH and 156,336 particles/L for CB; both of these val ues exceeded the upper limit of quantification of the HHPC6 (70,000 particles/L), followed by the 500nm range (65,402 particles/L for MWCNTOH and 54,242 particles/L for CB]. In the 10-1,000 nm size range, there was no change in particle number concen trations between handling and sonicating MWCNTOH, but there was an increase in particle number concentration when sonicat ing CB (1,057 particles/cc).
TEM. Filterbased air samples were col lected during each of the laboratory tasks and processes. TEM images verified the morphol ogy and rela tive sizes of particles captured during the laboratory processes ( Figure 3). All samples were collected as shortduration, pro cessspecific area samples and were not in the breathing zone of the workers. The background sample image shows amorphous particles that were identified as not being engineered CNMs ( Figure 3A). C60 particles were agglomerated during handling but partially deagglomerated when sonicated ( Figure 3B and C, respec tively). Figure 3D, E, and F represent raw airborne MWCNT during weighing, sonica tion in DI water, and sonication in moderately hard reconstituted water containing 100 mg/L NOM, respectively. Note that typical tubu lar structures are missing from raw MWCNT during the handling process. However, we observed tubular structures during sonication in both types of suspension, with more tubes aerosolized and captured on the filter when in water containing NOM. The raw MWCNT agglomerates featured in the TEM images for both suspensions were approximately 500 nm in diameter. MWCNTOH was highly agglomerated when handled, with a diameter of > 1,000 nm ( Figure 3G). Airborne CB was somewhat agglomerated during handling and more highly agglomerated when sonicated in DI water ( Figure 3H and I, respectively).

Discussion
This case study served as a rangefinding sur vey of airborne nanomaterials emitted dur ing common tasks performed in a laboratory that investigates environmental risks of engi neered nano materials. In addition, this study allowed the NIOSH NTRC field team to test analytical equipment and methodolo gies under various laboratory conditions to evaluate potential occupational exposure to engineered nano materials. Specifically, this research effort combined semi quantitative air borne particle number concentrations with qualitative TEM imaging to provide a weight ofevidence evaluation of whether engineered nano materials were released during laboratory tasks. To our knowledge, this is the first study to suggest that engineered nano particles may be released from aqueous suspensions during sonication. Results of this study imply that the commonly held belief that engineered nanomaterials in suspension during sonica tion pose low risk of inhalation exposure may need some reconsideration. This is especially true with regard to results from a recent inter national survey of nano material firms and laboratories; in that study, Conti et al. (2008) found that many workers in the field think nanomaterials pose no risk.
After accounting for background par ticle counts, we detected increased particle number concentrations during the handling of dry CNMs and also during the sonication of CNM suspensions (Table 1). An inter esting observation during the present study was the differential behavior between hydro phobic and hydro philic CNMs with regard to different laboratory processes. During material handling and weighing, we observed higher airborne particle number concentra tions of the hydrophobic CNMs (C60 and raw MWCNT) compared with hydrophilic CNMs. Lower particle number concentra tions of aerosolized CNMs at 300 and 500 nm were noted during sonication, yet cumula tive particle number concentrations in the 10-1,000 nm size range were elevated com pared with the handling process. This finding was more pronounced when raw MWCNTs were sonicated in moderately hard recon stituted water containing 100 mg/L NOM, suggesting that sonication of CNM suspen sions may increase the number of smaller sized CNM agglomerates (i.e., < 300 nm)-as would be expected with sonication-that were not detected by the HHPC6 particle counter. An opposite pattern was observed when the hydrophilic CNMs (MWCNTOH and CB) were compared. Very low particle number concentrations were detected during handling of hydrophilic CNMs, yet sonicating these hydrophilic CNMs, whether in a DI water suspension or a moderately hard reconsti tuted water suspension with 100 mg/L NOM, resulted in dramatically higher airborne par ticle number concentrations. From this find ing, along with visual evidence provided by TEM examination of the airsampling filters, we hypothesize that CNM agglomerates are being emitted to the laboratory atmosphere in water droplets. These data demonstrate that care should be exercised when handling dry hydrophobic CNMs and also when sonicating wet CNMs in suspension. A similar pattern of emissions and potential exposure was observed by Methner et al. (2007) during a study of nanomaterial polymer laboratory workers.
In the present study, all filterbased air samples collected during weighing and transfer processes, with the exception of raw MWCNT, showed the presence of the engineered nano material handled. Likewise, all samples col lected during sonication, regardless of the nano material in suspension, showed visual evi dence of the presence of the engineered nano material when analyzed by TEM. The majority of the images presented in Figure 3 indicate that single spheres or nanotubes are more the exception than the rule; most particles showed clear evidence of agglomeration. However, this may be due to the current methodology that uses 0.8µm filter membranes, which may allow small, individual CNMs to pass through and thus be unavailable for analysis. The images shown in Figure 3 clearly provide strong visual evidence that emissions from specific tasks and processes can occur. No evidence of engineered nano material was present on the background air filter sample collected.
Our data indicate that although suspen sions may minimize aerosolization of CNMs relative to their dry form, sonication of such suspensions outside protective enclosures can result in aerosolization and thus potential exposure to nanosized particulates (Figure 4). If sonication occurs outside an enclosure, as often occurs in laboratory settings, the prox imity of the researcher's breathing zone may result in inhalation of CNM particulates in water droplets and/or mists. Similarly, air borne water droplets can be generated by standard aquaria that use air stones or other air supplies to aerate test waters during long term aquatic toxicology studies. The airborne CNMs in water droplets have the potential to cause pulmonary effects similar to those described for particulate matter, singlewalled carbon nanotubes (SWCNT), and MWCNTs (Laks et al. 2008;Lam et al. 2006;MaHock et al. 2009;Mitchell et al. 2007;Shvedova et al. 2008). The mass balance of CNMs col lected during the laboratory processes were not determined, so the mass of airborne CNMs is unknown, making it difficult to compare with CNM inhalation toxicity studies or to occupa tional exposure limits for carbonbased materi als such as respirable graphite or particulate matter. However, Conti et al. (2008) found that organizations that used nano materials in suspensions or embedded in matrices were less likely to make recommendations for respira tory protection. With sonication being a criti cal component of nano material synthesis and deagglomeration, this survey result suggests that inhalation exposure may be an overlooked safety component during this commonly used laboratory process.
Despite being housed in an enclosure dur ing this experimental process evalua tion, the sonicator has the potential to emit engineered nano materials when the enclosure door is opened after the sonication process is com plete. If this occurs, airborne CNMs may be inhaled by laboratory workers. In addition, if the sealing gasket around the perimeter of the enclosure door is damaged or other wise breached, release of aerosolized droplets to the laboratory atmosphere may result. Furthermore, these airborne CNMcontaining water droplets have the potential to deposit on other surfaces within the sonication cabi net and in the laboratory. Once dried, CNM may become resuspended if disturbed and potentially result in exposure via inhalation. Finally, these nano materials may be available for dermal deposition if laboratory workers unknowingly contact contaminated surfaces with unprotected skin (e.g., hands, forearms). Currently, there are no occupational exposure limits specific to engineered nano materials (Methner 2008); however, basic precaution ary procedures and control equipment can dramatically reduce airborne releases of nano materials (NIOSH 2009). Therefore, environ mental scientists should implement a general or nanospecific environmental, health, and safety program at their organizations (Conti et al. 2008), use personal protective equip ment, and develop standard operational pro cedures to minimize potential hazards when working with engineered nano materials in environmentally relevant laboratory systems.
Although this preliminary research has generated some interesting and relevant find ings, specific uncertainties associated with experimental design and implementation need to be addressed. First, this singlecase study was designed to determine the relative mag nitude of airborne nano material emissions associated with tasks and materials used in environmental laboratory experiments. We used only a single data point for each of the tasks and materials during this first assessment. Thus, the data presented here are not statisti cally based. These data should be viewed as an indicator of the need for additional stud ies that focus on a robust statistically based experimental design, experimental variables, specific engineered nano materials, and sample collection. Second, the data interpretation can be confounding because of the two different particle counters used to measure airborne nanosized particles. The two particle coun ters use different counting principles, count ing efficiencies, and size ranges, so the data are not directly convertible to identical units. Our intentions were to show the size ranges and rela tive number concentrations on a task specific basis. This way, a reader can examine the data separately according to task and deter mine which task emitted nano materials. Thus, these data should be interpreted as relative indicators of CNM release, especially since the data were adjusted by subtracting background particle number concentrations. Furthermore, because the directreading, realtime instru mentations are not material specific (e.g., MWCNTs or CB only) and cannot identify the chemical composition of the particles detected (e.g., MWCNT vs. background par ticulate matter or water droplets), we cannot definitively conclude that increases in particle number concentrations for a specific opera tion are due to a release of particulate material from that process. However, because the par ticle number concentrations in the lower size ranges were higher than background and the results of the TEM analyses yielded visual evi dence of the engineered nano materials, we can conclude that a release occurred and that the potential for exposure exists.

Conclusions
Care must be taken when conducting labo ratory studies using CNMs in environmen tally relevant matrices. Sonicating hydrophobic CNMs in DI water suspensions results in air borne particle number concentrations lower than when handling dry CNMs. In contrast, sonicating hydrophilic CNMs in a moderately hard reconstituted water suspension containing natural surfactants dramatically increases air borne CNM particles compared with handling of dry CNMs. Thus, researchers using these environmentally relevant matrices should use appropriate protective equipment (respiratory and dermal protection) in addition to employ ing adequate engineering controls to minimize CNM aerosolization during preparation and experimental usage. Although we examined lab oratory processes in an environmental research laboratory, similar results are also possible in other laboratories that use similar materials (e.g., functionalized CNMs), similar tasks (e.g., sonication), and similar dispersive agents (e.g., surfactants). Additional research is needed to better characterize CNM emissions and worker exposure during handling and sonication to corroborate the results of this case study.