Page 2 of 13 Executive Summary

MRID 477287-01 is a leaching study that was conducted by HeiQ to measure the release of silver nanoparticles from textiles treated with their proposed product, AGS-20 Silver Additive. The testing was conducted by agitating fabric samples in room temperature ultrapure water for 24 hours. The wash water was analyzed for silver ions usi ng an ion specific electrode (ISE) and for particles using scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDX). According to HeiQ, no ionic silver was detected using ISE and no silver particles were detected using SEM/EDX. There were other particles detected but they were confirmed to be non-silver using EDX. AD has reviewed the original study and supplemental information provided by HeiQ in

MRID 478391-01 and determined that it cannot be concluded that very few if any nanoparticles of silver were released. This is because the magnification used, which was at the micron scale, was insufficient to detect the nanosilver particles which ar e 1-10 nm in diameter. It is also not known if the silver nanoparticles would have aggregated and become visible at lower magnifications and there are issues associated with the sensitivity of the EDX method.

Additional supplemental information was provided by HeiQ in MRID 478391-02 which is a prepublication version of a research study that was later published (Geranio et al., 2009). During this study 9 textiles treated with silver, includi ng two treated with HeiQ AGS-20, were machine washed using an International Standards Organization (ISO) wash-test method to determine the effect of pH, surfactants and bleaching agents. The HeiQ samples were polyester fabrics treated with either a surface coating (NP-PES-SURF) or by incorporation into the polyester fiber during manufacturing (NP-PES). Samples of the wash water were analyzed as ions before and after filtration using 0.45 micron filters and 30 kDa membranes (~5 nm) to separate large and small particulate silver from silver ions. As shown in Table 1, the amount of silver released was greater for the surface treated fabric. The analysis of th e filtered and unfiltered samples indicated that for the NP-PES-SURF fabric 80 percent of the released silver was composed of particulate >450 nm while for the NP-PES fabric approximately greater than 50 percent of the silver was composed of particulate. The authors suggest that the mechanical stresses related to the ISO wash-test method, which uses steel balls, were a major cause of particulate release.

Table 1 - Silver Released During Washing of Polyester Fabric Treated with AGS-20

Silver Released (ug/gm) Designation in

Geranio et al. 2009 Designation in HeiQ, 2009 Treatment Type Silver Content (ug/gm)

1st

Wash 2nd

Wash Bleach Cycle

NP-PES-SURF

NP-PES Sample #2 Sample #3 Surface Coating Incorporation 29

99 10.1

1.3 N/A

0.35 N/A

2.7

AD has reviewed the Geranio et al. study and conc luded that HeiQ treated textiles that were subjected to machine washing with detergents and bleach released silver in the form of coarse particulate, but is it not known if this particulate c onsists of nanosilver particles in fiber fragments, aggregates of silver nanoparticles or precipitates of ionic silver. AD recommends that an additional leaching study be done in the manner of Geranio et al. with the addition of electron microscopy to characterize the particulate. The characterization is needed to determine if nanoparticles could be released from the particulate.Page 3 of 13 Section I – Summary of MRID 477287-01 “Are silver particles released from textiles treated with HeiQ AGS-20 Silver Additive?”

This study was conducted by HeiQ to measure the release of silver nanoparticles from textilestreated with AGS-20 Silver Additive. As described in Egger et al., 2009, AGS-20 is a novel silversilica nanocomposite that has silver nanoparticles of 1 to 10 nm embedded within a matrix of amorphous silica particles. In consultation with the EPA, HeiQ based their study protocol on a leaching study published by Benn, 2008. HeiQ tested sa mples of polyester fabric that was untreated (Sample 1), treated with AGS-20 applied as a surface coating (Sample 2), or treated with ACG-20 incorporated during fiber manufacture (Sample 3). They also tested fabric from a commercial nanosilver sock (Sample 4).

The testing was conducted by immersing the 2 gram fabric samples in 38 ml of room temperature ultrapure water in glass jars and agitating for 24 hours using an orbital shaker. This process was repeated for times with the water and the jar being changed each time. The wash water was analyzed for silver ions using an ion specific electrode (ISE) calibrated to a detection limit of 160 ug/liter (page 9) or 800 ug/liter (page 20). The wash water was analyzed for particles using scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDX). The SEM/EDX method was tested on a positive control sample of reference nanosilver powder (<100 nm in diameter) dispersed in ultrapure water and it was determined that the powder could be detected.

According to HeiQ, the test samples with the HeiQ AGS-20 silver material inside a coating (Sample 2) and directly inside the fiber (Sample3) both show no release of silver nanoparticles after washing in water for 24 hours. No ionic silver wa s detected using ISE and no silver particles weredetected using SEM/EDX. There were other particles detected but they were confirmed to be nonsilver using EDX.

AD reviewed this study and generated a list of issues and questions that was included in a July 24, 2009 letter to HeiQ. HeiQ’s responses to these issues and questions are discussed in Section II.Page 4 of 13 Section II - MRID 478391-01 Supplement to MRID 477287-01 “Are silver particles released from textiles treated with HeiQR AGS-20 Silver Additive?” Response to EPA Letter of July 24, 2009.

In this supplement, HeiQ responds to points and questions posed by AD. A listing of the points, HeiQ’s response and the AD’s evaluation of HeiQ’s response is included below:

Point #1 – Uncertainty regarding the detection of free and SiO 2 associated silver nanoparticles in laundry water.

AD had questions regarding the number of samples (i.e. replicate runs) that should be conducted to ensure repeatability in the results and how many TEM fields per sample should be examined to accurately determine the number of free nanoparticles. AD mentioned that the NIOSH Method 7402 “Asbestos by TEM”, which is used to analyze air sampling filters for asbestos fibers, has specifications regarding the number of fields (i.e. grids) that need to be examined. This number depends on the fiber density on the filter and ranges from 40 grids for lightly loaded samples (<5 fibers per grid opening) to 6 grids for heavily loaded samples (>25 fibers per grid opening) with the stipulation that at least 100 fibers should be c ounted. The NIOSH method also specifies that 200 mesh grids should be used and that the magnification should be 10,000X.

HeiQ Response

HeiQ compared their method with the NIOSH method and concluded that the two methods are very similar. With respect to the number of fields examined, HeiQ states that they examined all of the fields within the area of the 5 mm evaporated droplet . They claim that this area is 240 times greater than the area that would be examined if the NIOSH method for lightly loaded samples were followed and 40 grids were examined. They also mention that the fabric samples contained approximately 1.5 x 10 20 particles per gram (assuming a particle size of 5 nm and a fabric loading of 100 ppm silver) which would result in concentration of 7.7 x 10 24 silver particles per m 3 of washwater if all of the particles leached out.

AD Evaluation of HeiQ Response

AD concurs that the method used by HeiQ is similar to the NIOSH method and that a much larger sample area (i.e. more grids) was examined than would be required by the NIOSH method. AD also concurs that density of particles per unit area is much greater than what would be found for an air sample. If it is assumed that most of the particles in the 2ml aliquot that was removed from the 38 ml leachate sample and centrifuged at 14,000 RPM ended up in the drop that was dried onto the slide, the residue with a diameter of 5 mm would contain approximately 4 x 10 23 particles. By contrast, a typical air sample of 1200 liters coll ected from ambient air containing 0.01 fibers/cc

would contain 1.2 x 10 3 fibers deposited on a filter that is 25 mm in diameter.

Scientists from ORD were asked to review the study and comment on the feasibility of scanning the entire 5 mm diameter droplet residue area with sufficient magnification to see 1-10 nm particles. The following specific comments were provided by ORD in regards to this question and additional general comments are included in Attachment (1):Page 5 of 13 C.A. Impellitteri 1 - A 5 mM 2 area can easily be scanned for “hot spots” for a particular element (please see the example in the attached general comments). However, to distinguish specific 1-10 nM particles in impure media (e.g. fibers, soil, sediments etc.) using SEM would be a very difficult and perhaps impossible task. This is especially true for EDX analyses where you need approx. concentrations (w/w or, for example, 1000 mg target analyte/kg media) of 0.01% - 1.0 % (100-1000 mg/kg respectively) in the target area, for EDX to detect the target analyte. Thus, samples where you have high concentrations (or aggregates) of Ag nanoparticles can be detected in a scan of a relatively large area (relative to the small spots chosen in the manufacturers study). We provide an example of this in the attached General Comments.

M. Nadagouda 1

- Reply: Yes it is possible to scan entire 5 mm area but to visualize 1-10 nm particles one needs higher magnification.

Point #2 – Method for detecting silver ions in laundry water is not adequate

The ion specific electrode method used in the HeiQ study had a high detection limit (800 ug/liter) and as a result no ionic silver was detected in the laundry water samples. AD recommended that a

more sensitive method be used to increase the confidence in the results obtained from electronic microscopy.

HeiQ Response

HeiQ states that the ion specific electrode method th ey used is directly analogous to the method that was used in the Benn study that was recommended by the EPA. They mention that the detection limit reported in the study is actually a calibration limit derived from the calibration curve and the detection limit for the electrode is 10 ug/liter. They then provide an extrapolation of the calibration curve beyond the measured zone to achieve an illustrative detection limit of 15 to 80 ug/liter. They also mention that they do not understand why the detection limit for silver ions has any bearing the confidence of the results obtained from electron microscopy.

AD Evaluation of HeiQ Response

AD concurs that the ion specific method used by HeiQ is similar to the method used by Benn and Westerhoff and that the main purpose of the study is to assess the release of silver nanoparticles.Given the uncertainties inherent in using electron microscopy to detect and quantifiy silver nanoparticles within an impure matrix, however, the use of a more sensitive analytical method for silver ions would increase the confidence in the results obtained from electron microscopy. It is acknowledged that the silver nanoparticles are intended to release silver ions and that the detection of silver ions would not necessary mean that silver nanoparticles had been detected. However it is also understood that the HeiQ treatment is intended to provide for a slow release silver ions and therefore it is likely that the release rate of ions from nanoparticles retained within the textile would be very low if the treatment is functioning as intended. In addition, it will be necessary to assess the risk of exposure to silver ions using the toxicological endpoints for ionic silver (i.e. the oral RfD of 0.003 mg/kg/day based on argyria).

1. ORD - National Risk Management Research Lab ., 26 West Martin Luther King Dr., Cincinnati, OH 45268Page 6 of 13 Point #3 – Additional questions to assess leaching from textiles

This section addresses the several questions that were included on the 2 nd page of the EPA memo.

Question 1 – What is the leaching potential when AGS-20 is used to treat cotton and polyester

fibers?

HeiQ Response – HeiQ states that the chemical can be applied either by incorporation during the fiber melt spinning or by a textile coating. For fabrics containing natural fibers such as 100% cotton and poly cotton blends, however, the only method available for applying the AGS-20 particles is to use the surface coating approach (similar to Sample 2 in the submitted leaching study). Like many textile finish products, it is more difficult to achie ve a high degree of fixation on polyester compared to other textiles such as cotton. Because sample 2 was a polyester fabric that was treated using a coating process HeiQ believes that it represents the worst case scenario.

EPA Evaluation of HeiQ Response - EPA concurs with this response.

Question 2 – What is the effect of hot water and bleach on the leaching rate?

HeiQ Response – HeiQ claims that the surface coating would not be affected by high temperature and bleach and cites two papers to substantiate this claim. The first paper by Impellitteri et al. investigates the effect of bleach, detergents and NaCL solutions on the chemical composition of silver nanopowder and commercial socks containing silver nanoparticles. The paper reports that silver chloride is readily formed from contact w ith bleach, either via direct transformation of the nanoparticles or released silver-ions reacting with chloride ions. The authors also state that silver nanoparticles remain inside the fibers and may be directly converted to silver chloride. The second paper by Geranio et. al. was submitted by HeiQ as Supplemental volume 2 and will be discussed in

Section II of this review.

EPA Evaluation of HeiQ Response - EPA concurs with the finding that silver chloride is formed

from contact with bleach. EPA does not concur w ith the finding that silver nanoparticles remain inside the fibers because the alleged nanoparticles of silvers in the commercial socks (Xstatic) are not actually nanoparticles but are instead micron sized particles.

Question 3 – What is the leaching potential when AGS-20 treated fabrics are exposed to tap

water and or simulated sweat.

HeiQ Response - HeiQ states that tap water and simulated sweat both contain various electrolytes such as chloride ions and that the surface coating films and fibers containing AGS-20 are not affected by these ions and would not have increased particle leaching potential.

EPA Evaluation of HeiQ Response - EPA concurs with this response.Page 7 of 13 Question 4 – How much nanosilver is left in the treated fabric after washing.

HeiQ Response - HeiQ states the leaching study indicates that the nanosilver particles are thoroughly retained in the textile fabric after washing.

EPA Evaluation of HeiQ Response - The literature study provided in Supplemental volume 2 indicates that a substantial amount of the applied silver is released from the fabric as coarse particulate. This issue is discussed in Section III.

Question 5 – What is the size of the treated load?

HeiQ Response - HeiQ states that the leaching study used 2 gram portions taken from larger fabric pieces.

EPA Evaluation of HeiQ Response - EPA concurs with this response.

Question 6 – What are the temperatures of the different wash waters?

HeiQ Response - HeiQ states that the leaching study used wash water at room temperature (ca. 20 o c) and that a maximum temperature of 40 C is recommended for washing silver treated textiles.

EPA Evaluation of HeiQ Response - EPA concurs with this response.

Question 7 – How much fiber is shed during wash loads?

HeiQ Response - HeiQ states that the proportion of fibers shed from a textile during laundry is very small and estimates that a one hundred gram tshirt would lose one gram after 100 washes.

EPA Evaluation of HeiQ Response - EPA does not concur with this response because it is known that fibers are released as lint during drying to such an extent that they can be a fire hazard if not properly vented out of the dryer. According to literature cited in MRID 478391-02, a textile that is disposed at the end of its use phase has lost about 10% of its weight through abrasion during washing and usage (Koehler et al., 2008).Page 8 of 13 Section III - MRID 478391-02 Supplement to MRID 477287-01 “Are silver particles released from textiles treated with HeiQR AGS-20 Silver Additive?” Behavior of silver nanoparticles during washing.

This submission is the prepublication version of a research study that was later published in the Journal of Environmental Science and Technology (Geranio et al., 2009). During this study 9 textiles treated with silver, including two treated with HeiQ AGS-20, were either immersed in solutions with gentle mixing or machine washed to determine the effect of pH, surfactants and bleaching agents. The HeiQ samples were polyester fabrics treated with either a surface coating (NP-PES-SURF) or by incorporation into the polyester fiber during manufacturing (NP-PES).

During the immersion experiment, textiles were immersed in SDS surfactant solution at pH 10 with agitation at 100 to 150 RPM and 0.1M or 0.5M peracetic acid (PAA) was added after 120 and 180 minutes, respectively. Silver ion content of the immersion water was monitored using an ion specific electrode (ISE) and aliquots of the immers ion water were analyzed using ICP-OES with and without filtration using 0.45 micron filters. For the NP-PES fabric, the silver ion concentration in

the immersion water remained below the limit of detection of 10 -7 M (i.e. 10.7 ug/liter) during the entire experiment and for the NP-PES-SURF fabric, the silver ion concentration rose to 10 -6 M (i.e. 107 ug/liter) during the first 50 minutes and continued on a slight rise until the end of the experiment at 240 minutes. The rate of increase was not affected by the addition of 0.1 M PAA and was only slightly affected by the addition of 0.5M PAA. The analysis of the immersion water with and

without filtration indicated that most of the silver released from the NP-PES-SURF fabric was ionic while most of the silver from the NP-PES fabric was particulate >450nm.

During the washing machine experiment, textiles were machine washed using the ISO method for wash-tests two times for 30 min at 40 degrees C and using either detergent only or detergent with perborate/TAED bleach. Samples of the wash wate r were analyzed as ions using ISE and ICP-OES before and after filtration using 0.45 micron filters and 30 kDa membranes (~5 nm) to separate large and small particulate silver from silver ions. As shown in Table 1, the amount of silver released was greater for the surface treated fabric. The analysis of the filtered and unfiltered samples as shown in Figure 1 indicated that for the NP-PES-SURF fabric 80 percent of the released silver was composed of particulate >450 nm while for the NP-PES fabric approximately greater than 50 percent of the silver was composed of particulate. The exact fractions for the NP-PES fabric could not be

determined because the total amount released was at the limit of detection for some of the fractions. The authors suggest that the mechanical stresses related to the ISO wash-test method, which uses steel balls, were a major cause of particulate release.

Table 2 - Silver Released During Washing of Polyester Fabric Treated with AGS-20

Silver Released (ug/gm) Designation in

Geranio et al. 2009 Designation in HeiQ, 2009 Treatment Type Silver Content (ug/gm)

1st

Wash 2nd

Wash Bleach Cycle

NP-PES-SURF

NP-PES Sample #2 Sample #3 Surface Coating Incorporation 29

99 10.1

1.3 N/A

0.35 N/A

2.7
Source: Geranio et al., 2009.Figure 1 – Size Fractionation of Silver Released After One Washing

Source: Geranio et al., 2009.

Page 9 of 13Page 10 of 13 Section IV – Conclusions

HeiQ Leaching Study

The information provided in the HeiQ leaching study (MRID 477287-01) and the supplemental information provided in MRID 478391-01 does not support HeiQs conclusion that there was no release of silver nanoparticles from the treated textiles that were subjected to the gentle agitation protocol. This is because the magnification used, which was at the micron scale, was insufficient to detect the nanosilver particles which are 1-10 nm in diameter. It is also not known if the silver nanoparticles would have also aggregated and beco me visible at lower magnifications and there are issues associated with the sensitivity of the EDX method. These points are highlighted in the following comments received from ORD in response to questions asked by AD regarding the use of the images to support the conclusion that no particles were released.

AD Questions for ORD : Can we determine this from the images included in the report? The magnification of these images is not very high. Would we expect silver nanoparticles released from the fabric to agglomerate and thus be detectable at the magnifications used?

ORD Comments: C.A. Impellitteri- No, not from the images. It is partially a magnification issue, but the more important aspect is that Ag nanoparticles can simply go undetected by EDX due to the concentration issues discussed previously. Based on the Geranio et al. preprint provided, some Ag is released (and tends to be particles >450 nM). These particles could consist of aggregates, but aggregates of pure silver should be detectable. It is more probable that the larger particles are fibers. If the fibers do not have the minimum concentration of silver in the EDX target area,

the silver will be undetectable.

M. Nadagouda - Not necessarily true that Ag particles aggregate all the time. Magnifications of Ag images provided are in micron scale and it is not possible to see 1-10 nm particles at that magnification. The EDX data was obtained at differe nt spots rather than the whole area. This questions the ability of SEM for the detection of few nanoparticles selected at different location rather than the whole area. Other alternate method would be digesting the samples with HNO 3 and run ICP analysis wherein one can detect up to ppb level concentrations.

Geranio Leaching Study

Based on the Geranio et al. study, it can be concluded that HeiQ treated textiles that were subjected to machine washing with detergents and bleach released silver in the form of coarse particulate, but is it not known if this particulate c onsists of nanosilver particles in fiber fragments, aggregates of silver nanoparticles or precipitates of ionic silver. There were also limit of detection issues that prevented the accurate size fractionation of the NP PES textile sample.

Recommendations for Additional Studies

AD recommends that an additional leaching study be done in the manner of Geranio et al. with the addition of electron microscopy to characterize th e particulate. The characterization is needed to determine if nanoparticles could be released from the particulate.Page 11 of 13 References

Benn Troy M. & Westerhoff Paul., 2008. Nanoparticle Silver Released into Water from Commercially Available Sock Fabrics. Environmental Science and Technology 42 (2): 4133-4139.

Egger et al., 2009. Antimicrobial Properties of a Novel Silver-Silica Nanocomposite Material, Applied and Environmental Micribiology, May 2009, p. 2973-2976.

Geranio et al., 2009. The Behavior of Silver Na noparticles during Washing. Environmental Science and Technology. Published online September 24, 2009.

Impellitteri et al., 2009 Speciation of Silver Nanoparticles in Antimicrobial Fabric Before and After Exposure to a Hypochlorite/Detergent Solution, Christopher A. Impelliteri, Thabel M. Tolymat and Kirk G. Scheckel, J. Environ. Qual. 38: pp 1528-1530

Koehler et al., 2008. Studying the Potential Release of Carbon Nanotubes Throughout the Application Lifecycle. Journal Cleaner Production 2008, 16, 927-937.Attachment (1)

- ORD General Comments Provided by C.A. Impellitteri 1

1) It is unclear from reviewing the documents whether or not the issue for EPA is Ag nanoparticles, Ag ion, or Ag total (including nanoparticles, ions, aggregat es, fibers containing Ag etc.).

2) Using an ion selective electrode (ISE) is biased in that the detection limit is not low enough to account for the presence of silver ions which can have an effect on organisms.

3) Research in our labs has shown repeatedly that Ag ions and Ag nanoparticles (depending on capping/coating agents) readily adhere to surfaces and can go undetected if great car e is not taken in transferring stock suspensions and/or samples for analysis. For this study it would be best to esta blish some sort of mass balance by first digesting a fabric sample in nitric acid (e.g. EPA SW-846 Method 3051A) to establis h a total Ag concentration in the fabric. Then subject another sample of the same fabric to some facsimile of a wa shing procedure. The washed fabric should be digested and analyzed.

The next step is critical. Rather than analyzing the resultant liquid from the washing procedure via ISE, the ENTIRE volume of the liquid should be transferred to a digestion vessel, the sides of the beaker (or whatever container) washed/rinsed, in triplicate, with 5-10 mL aliquots of concentrated HNO 3 , the acid rinses transferred to the digestion vessel along with the initial sample, and the entire volume digested in an appropriate amount of HNO 3 . The digestate should then be analyzed via ICP-MS or Graphite Furnace Atom ic Absorption. This will provide a total Ag concentration leached, in one form or another, from the fabric.

The applicability of this process again depends on whether or not ionic Ag or total Ag is desirable for EPA’s purposes. Regardless, total silver leached should be established prior to any work on speciation (e.g. Ag ion vs. Ag particle).

4) The SEM image presented in Figure 1 is from Ag nanopa rticles equilibrated with Kaolinite particles (30 mg Ag nanoparticles/10 g Kaolinite. This equates roughly to 3000 mg Ag/kg kaolinite). The entire image area underwent EDX analyses (courtesy of Dr. M. Nadagouda). Target analytes, in this case Ag, can be highlight ed in the SEM/EDX software by coloration (in this case red = Ag). There are obvious ar eas where Ag aggregates or concentrates. As noted previously, any areas that are not red do not necessarily mean that Ag is not pres ent, it may simply not be concentrated enough in those areas for EDX to detect. SEM/EDX is , at best, a semi-quantitative method of analysis.

5) It is evident from the Geranio et al. paper that some Ag is coming out of these fabrics. It is also documented in several other papers that Ag in other fabrics readily l eaches. This is in contrast to the study provided by the manufacturer. I conclude that the study by the manufacturer is biased towards establishing the conclusion that NO silver is leaching from the fabrics in question.

Figure 1 – SEM Slide

1. These comments were provided in an Email of 03/29/2010, 10:18 AM forwarded by Dennis Utterback of ORD to Timothy Dole of AD, Re: Leaching Study Review.

Page 12 of 13Page 13 of 13 Attachment (2) – Comments Provided by Dr. Kevin Dreher 1

I have read the documents that were sent to me regardi ng the analyses conducted by HeiQ related to nanoAg and ionic Ag release from fabrics treated with their nanocomposite composed of nanoAg/micron Si. I do have very serious concerns about both the sensitivity and specificity of the studies performed by HeiQ in their assessment to detect Ag release and my comments will directly address OPP's questions:

Question for ORD? Do the images provided in the report support these conclusions? How much magnification would be needed to see 1 to 10 nm particles? Question for ORD? Could someone actually scan the en tire 5 mm diameter droplet residue area with sufficient magnification to see 1-10 nm particles?

I agree with OPP's concerns regarding the analyses perfo rmed by HeiQ using SEM/EDX and ion selective electrode methods to detect either nanoAg and/or ionic Ag released from fabric treated with HeiQ nanocomposite of nanoAg/micronSi. My concerns are specifically:

1. SEM resolution at the 1 - 20 micron levels used by HeiQ will not detect nanoAg, or nanoscale aggregates of this element, released from fabrics that has a primary diameter of 1 - 10 nm as characterized by S. Egger et al. Applied and Environmental Microbiology 75(9), 2973 - 2976, 2009. This is due to the lack of the ability of SEM to adequately resolve material at the nano size range which requires th e use of high resolution Transmission Electron Microscopy (TEM);

2. EDX sensitivity - since HeiQ cannot detect Ag in the 1 - 10 nm size they cannot with any degree of certainty use EDX to qualitatively detect an Ag signal. In addition, even if detected, the EDX silver signal is very weak and cannot be correlated with the Si signal due to the high leve ls of Si background generated by their SEM matrix;

3. It is not clear why SEM resolution at the 1 - 20 micron le vels were employed in HeiQ's analyses when they were capable of performing these studies at the 100 nm resolution le vel, see Figure 1 in reference S. Egger et al. Applied and Environmental Microbiology 75(9), 2973 - 2976, 2009;

4. HeiQ DID NOT prepare or analyze their samples accord ing to Benn and Westorhoff, Environmental Science and Technology 42(2): 4133 - 4139, 2008, and as indicated in HeiQ Study #PT-019-01, Section 3.3 Protocol Comparison, Step 3 Sample Preparation and Detection. The Benn a nd Westorhoff procedure employed drying down or lyophylization of samples with subsequent TEM analysis whereas HeiQ centrifuged samples at a centrifugal force incapable of recovering 1 - 10 nm Ag particles or nanoscale aggregates of nanoAg, thus potentially losing them in the sample preparation prior to their SEM analysis. In addition, lyophylization would be the preferred sample preparation to eliminate loss of nanoscale Ag aggregates. In addition, TEM, as employed by Benn and Westorhoff, would be the preferred level of resolution required to detect 1 - 10 nm Ag or aggregates of this nanomaterial;

5. It is not clear why ICPMS was not simply used to analy ze the leachate or a lyophylized sample of the leachate as this

would have been the most sensitive method to detect eith er ionic Ag, fibers containing nanoAg, or nanoAg itself.

Based on these concerns I do not agree with HeiQ's conclusion that nanoAg and/or ionic Ag is not released from fabrics by leaching based on inadequate sample preparation and analytical methods employed to assess this endpoint.

Thank you,

Kevin

************************************************** Kevin Dreher, M.S., Ph.D., Environmental Public Health Division (B143-01) National Health and Environmental Effects Research Laboratory U.S. Environmental Protection Agency, 109 TW Al exander Drive, Research Triangle Park, NC 27711 USA

1. These comments were provided in an Email of 03/ 30/2010, 11:25 AM from Kevin Dreher of EPA RTP to Ginger

Moser of EPA RTP.