Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles
SUPPORTING INFORMATION
Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles
Zong-ming Xiu, Qing-bo Zhang, Hema L. Puppala, Vicki L. Colvin, Pedro J. J. Alvarez*
a
Dept. of Civil & Environmental Engineering, Rice University, Houston, TX 77005 b
Dept. of Chemical Engineering, Rice University, Houston, TX 77005
* Corresponding Author; Email: [email protected], PHONE: (713)348-5903
9 pages in total, with 3 figures and 2 tables
1
AgNPs and Chemicals Glycol-thiol coated AgNPs (PEG-AgNPs: 3, 5 and 11 nm) were synthesized in the lab
1
and transferred to an anaerobic chamber for storage. Commercial polyvinylpyrrolidone-coated AgNPs of three different sizes (PVP-AgNPs: 18, 51 and 72 nm), which have been widely used in previous studies,
2-6
were obtained from NanoAmor (Houston, TX). The PVP-AgNPs powders
were suspended in DI water and homogenized by an ultrasonic cleaner (5510, Branson, CT). The sizes and zeta-potential (ζ) of the six particles (Table S1) were characterized in the exposure medium (2 mM sodium bicarbonate buffer), using JEM 2100F TEM (JEOL 2100 Field Emission Gun Transmission Electron Microscope, Japan) and dynamic light scattering with a Malvern Zetasizer (ZEN 3600, Malvern Instrument, UK) respectively. The morphologies of the PVPAgNPs are provided in Figure S1. AgNO3 and HNO3 (~69.0%) was obtained from Sigma-Aldrich (St. Louis, MO); NaCl, LB (Luria-Bertani) broth, NaHCO3 and H2O2 (30%) were all obtained from Fisher Scientific (Fair Lawn, NJ). All chemicals used were reagent grade or better unless otherwise specified. Table S1. Characterization of PEG- and PVP-AgNPs stock suspensions PEG-AgNPs
Sample 1
Sample 2
Sample 3
Particle size
2.8 ± 0.5 nm
4.7 ± 0.2 nm
10.5 ± 0.6 nm
Zeta-potential (mV)
-16.1 ±1.2
-13.5 ± 0.5
-17.1 ± 2.1
Stock concentration
44.5 mg/L
70.3 mg/L
82.0 mg/L
(Dissolved Ag+)
(0.31 mg/L)
(0.38 mg/L)
(0.28 mg/L)
PVP-AgNPs
Sample 1
Sample 2
Sample 3
Particle size
17.5 ± 2.9 nm
51.4 ±18.7 nm
71.5 ± 20.3 nm
Zeta-potential (mV)
-27.5 ± 1.4
-35.8 ± 0.6
-37.1 ± 0.6
Stock concentration
8,200 mg/L
5,700 mg/L
6,700 mg/L
(Dissolved Ag+)
(2.93 mg/L)
(7.90 mg/L)
(7.15 mg/L) 2
(a)
(b)
(c)
Figure S1. TEM characterization of the commercial PVP-AgNPs (a) PVP-18nm (17.5 ± 2.9 nm), (b) PVP-51nm (51.4 ±18.7 nm) and (c) PVP-72nm (71.5 ± 20.3 nm).
Bacteria E. coli strain K12 (ATCC 25404) was chosen as a model microorganism for inactivation experiments. This facilitates comparison with numerous other related antimicrobial studies, and (because E. coli is facultative) allows for testing under both aerobic and anaerobic conditions. E. coli exhibits equal susceptibility to silver ions under aerobic and anaerobic conditions (Figure S2) 7
. The detailed bacteria stock suspension preparation method was provided in our previous paper
7
. Sodium bicarbonate (2 mM), which is commonly used as exposure medium 8, was chosen to
avoid ligands present in complex growth media that could bind with Ag+/AgNPs and affect silver bioavailability and/or promote precipitation or other confounding effects. All AgNPs/Ag+ toxicity assays were below the Ag2CO3 precipitation potential (Ksp=0.81×10-12).
3
Figure S2. E. coli strain K12 (ATCC 25404) exhibits equal susceptibility to the silver ion (which is the definitive molecular toxicants) under aerobic and anaerobic conditions. Reproduced with permission from reference 7. Copyright 2011 American Chemical Society.
Preparation of air-exposed AgNP stock suspensions To prepare air-exposed AgNPs samples, PEG-AgNPs (3, 5, 11 nm) and PVP-AgNPs (18, 51, 72 nm) stock suspensions (~2g/L) were transferred out of the chamber and exposed to air for 5 days (with pH adjusted to 4.0 to accelerate the oxidation of AgNPs and the release of Ag+). The released Ag+ concentration of each sample was monitored by ICP-OES/MS (Perkin Elmer, Waltham, MA) to avoid complete oxidation of AgNPs. The air-exposed AgNPs suspensions were then transferred to anaerobic chamber and resuspended in NaHCO3 buffer solution to achieve a constant pH (8.1) for Ag+ dissolution equilibration (5 days). The final Ag+ concentration for PVP-AgNPs are 9.5 mg/L, 17 mg/L. 10.5 mg/L respectively. Anaerobic synthesis of the PEG-AgNPs The anaerobic PEG-AgNPs were synthesized by mixing the pre-synthesized PEG-AgNPs suspension with sodium borohydride under vigorous agitation in an anaerobic chamber. All 4
precursors (including AgNPs suspensions and NaBH4 solution) were transferred and equilibrated inside the anaerobic chamber for 24h to allow for oxygen depletion. Excess amount of NaBH4 was added to ensure a complete reduction of all Ag+. The AgNPs were sealed in an Amicon ultra centrifugal filter unit (molecular weight cutoff 10,000, 2~5nm in pore size, Millipore, MA) and moved out of the chamber for centrifugation (J2-MC Centrifuge, Beckman Coulter, CA) to get rid of the impurities (e.g., NaBH4). The centrifuged samples were transferred back to the chamber to collect the filtrate and refilled with 2mM NaHCO3 buffer. This washing process was repeated continuously four times and the filtrates were monitored for dissolved silver with ICP-Ms. The final PEG-AgNPs stock suspensions were re-suspended in 2mM NaHCO3 buffer solution, with no detectable Ag+ (by ICP-Ms, detection limit: 1 µg/L) in supernatants. Preparation of filtered AgNP stock suspensions To purify the AgNPs, PEG- and PVP-AgNPs stock suspensions were washed by DI water and filtered through the Amicon ultra centrifugal filter units by centrifugation. AgNPs stock suspensions were filtered continuously for ~10 times to get rid of the residual Ag+ as much as possible. The filtrates were analyzed for total dissolved silver with an ICP-OES/MS to obtain the concentrations of dissolved silver. After filtration, the retentates were resuspended and transferred to 50-ml corning tubes (Corning, Lowell, MA). The concentrations of the AgNPs stocks were determined by nitric acid/hydrogen peroxide digestion as described earlier 7. These stocks were stored and tested inside the anaerobic chamber to avoid any confounding effects caused by oxidative Ag+ release during the dose-response assays.
5
The minimum lethal concentration (MLC) of the six filtered AgNPs to E. coli was compared to Ag+ to assess their relative toxicity contribution to AgNPs. The MLC is defined as the minimum lethal concentration of AgNPs in the exposure medium (bicarbonate buffer) that causes statistically significant E. coli mortality (p < 0.05) relative to the control set without AgNPs, as we reported earlier 7. Dose-response assay of AgNPs Six fresh and six air-exposed AgNPs stock suspensions were tested for dose-response on E. coli mortality inside the anaerobic chamber following the procedure as showed in Xiu et al 7. Specially, antimicrobial assay of the air-exposed AgNPs was conducted after 5 days of equilibration in anaerobic chamber to ensure a complete Ag+ dissolution. Free Ag+ concentrations were measured in the same medium and toxicity data of each AgNPs suspension was plotted against the free Ag+ concentration in it. E. coli mortality in different treatments was then determined by viable plate counts 9 and was calculated as 1-N/N0 × 100%, where N and N0 are the remaining and initial concentrations of viable bacteria (CFU/mL), respectively. The doseresponse curves of E. coli mortality versus Ag+ concentrations was fitted using a sigmoidal model (Eq. S1)10 (Sigma-Plot v10.0):
ݕൌ ݕ ଵା
ೣషೣబ ష ್
(Eq. S1)
Where y is the E. coli mortality rate, y0 is the baseline mortality rate without silver addition, x is silver concentration (expressed as released Ag+), and x0 is the Ag+ concentration that results in 50% mortality (EC50). All tests were conducted in triplicate and repeated at least three times to ensure reproducibility. Statistical Analyses
6
Whether differences between EC50 values for different treatments were statistically significant was determined using Student’s t-test at the 95% confidence level. T-tests were also used to determine MLC values (i.e., the lowest concentrations that resulted in a significant (p < 0.05) decrease in cell viability, assessed by plate counts) as well as the significance of hormesis effect relative to unexposed controls. For the dose-response assays, E. coli mortality data for each type of air-exposed AgNPs (expressed as their corresponding released Ag+ concentrations) and for the AgNO3 treatment were fitted with the Sigmoidal model (Eq. S1) using non-linear regression. The SAS PROC NLIN procedure and R software, version 2.9.2 (R Foundation for Statistical Computing, Vienna, Austria) was used for this purpose.11 Whether differences in dose-response trends were statistically significant were determined by the F-test at the 95% significance level using the SAS/STAT version 9.2 software (SAS Institute Inc., Cary, NC). This analysis (Table S2) inferred that the sigmoidal curves were statistically indiscernible and thus, that the antibacterial activity of AgNPs could be fully explained by the toxicity of the released Ag+. Table S2. Dose-response assay statistical analysis results F-test p value
Ag+ control
PEG3 nm
PEG5 nm
PEG11 nm
PVP18 nm
PVP51 nm
PVP72 nm
0.80
0.71
0.56
0.79
0.65
0.22
Ag+ release at low pH found near a cell membrane under the influence of the proton motive force The air-exposed PVP-AgNPs (three types in total) were transferred into the anaerobic chamber and each type of AgNPs was added to different media with different pH values. The lowest pH used (3.0, diluted HNO3 solution) was mimicking the local pH of a cell membrane
7
under the influence of the proton motive force. An intermediate pH (5.5, DI water) was used to represent the inside of a lysosome in eukaryotic cells. The highest pH (8.1, 2mM NaHCO3 solution) is representative of bicarbonate-buffered natural waters and was the same as in the toxicity assays. The released Ag+ concentrations in the supernatants were measured by ICP-Ms after 72-h equilibration. As shown in Figure S2, Ag+ release was affected by the solution pH, with higher Ag+ release at lower pH, suggesting increased release as AgNPs become in contact with these organelles.
35
+
Ag released (mg/L)
30 25
pH 3.0 pH 5.5 pH 8.1
20 15 10 5 0 25 nm
36 nm
86 nm
Figure S3. Dissolution of air-exposed PVP-AgNPs at different pH, as encountered near bacterial cell membranes influenced by the proton motive force (pH 3), or inside lysosomes of eukaryotic cells (pH 5.5) or in bicarbonate-buffered natural water (pH 8.1). Ag+ release increases with decreasing pH.
REFERENCES (1) Hiramatsu, H.; Osterloh, F. E. Chem. Mater. 2004, 16, (13), 2509-2511. (2) Yang, X. Environ. Sci. Technol. 2012. 8
(3) Meyer, J. N.; Lord, C. A.; Yang, X. Y. Y.; Turner, E. A.; Badireddy, A. R.; Marinakos, S. M.; Chilkoti, A.; Wiesner, M. R.; Auffan, M. Aquat. Toxicol. 2010, 100, (2), 140-150. (4) Laban, G.; Nies, L. F.; Turco, R. F.; Bickham, J. W.; Sepulveda, M. S. Ecotoxicology 2010, 19, (1), 185-195. (5) Lara, H. H.; Ayala-Nunez, N. V.; Ixtepan-Turrent, L.; Rodriguez-Padilla, C. J Nanobiotechnology 2010, 8, 1. (6) Beer, C.; Foldbjerg, R.; Dang, D. A.; Autrup, H. Toxicol. Lett. 2011, 205, S44-S44. (7) Xiu, Z. M.; Ma, J.; Alvarez, P. J. J. Environ. Sci. Technol. 2011, 45, (20), 9003-9008. (8) Chen, J.; Xiu, Z.; Lowry, G. V.; Alvarez, P. J. Water Res. 2010, 45, (5), 1995-2001. (9) Adams, L. K.; Lyon, D. Y.; Alvarez, P. J. J. Water Res. 2006, 40, (19), 3527-3532. (10) Poch, G.; Vychodil-Kahr, S.; Petru, E. Int. J. Clin. Pharm. Th. 1999, 37, (4), 189-192. (11) SAS/STAT(R) 9.2 User's Guide, Second Edition, the NLIN Procedure, http://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#nlin_t oc.htm.
9
Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles
Zong-ming Xiu, Qing-bo Zhang, Hema L. Puppala, Vicki L. Colvin, Pedro J. J. Alvarez*
a
Dept. of Civil & Environmental Engineering, Rice University, Houston, TX 77005 b
Dept. of Chemical Engineering, Rice University, Houston, TX 77005
* Corresponding Author; Email: [email protected], PHONE: (713)348-5903
9 pages in total, with 3 figures and 2 tables
1
AgNPs and Chemicals Glycol-thiol coated AgNPs (PEG-AgNPs: 3, 5 and 11 nm) were synthesized in the lab
1
and transferred to an anaerobic chamber for storage. Commercial polyvinylpyrrolidone-coated AgNPs of three different sizes (PVP-AgNPs: 18, 51 and 72 nm), which have been widely used in previous studies,
2-6
were obtained from NanoAmor (Houston, TX). The PVP-AgNPs powders
were suspended in DI water and homogenized by an ultrasonic cleaner (5510, Branson, CT). The sizes and zeta-potential (ζ) of the six particles (Table S1) were characterized in the exposure medium (2 mM sodium bicarbonate buffer), using JEM 2100F TEM (JEOL 2100 Field Emission Gun Transmission Electron Microscope, Japan) and dynamic light scattering with a Malvern Zetasizer (ZEN 3600, Malvern Instrument, UK) respectively. The morphologies of the PVPAgNPs are provided in Figure S1. AgNO3 and HNO3 (~69.0%) was obtained from Sigma-Aldrich (St. Louis, MO); NaCl, LB (Luria-Bertani) broth, NaHCO3 and H2O2 (30%) were all obtained from Fisher Scientific (Fair Lawn, NJ). All chemicals used were reagent grade or better unless otherwise specified. Table S1. Characterization of PEG- and PVP-AgNPs stock suspensions PEG-AgNPs
Sample 1
Sample 2
Sample 3
Particle size
2.8 ± 0.5 nm
4.7 ± 0.2 nm
10.5 ± 0.6 nm
Zeta-potential (mV)
-16.1 ±1.2
-13.5 ± 0.5
-17.1 ± 2.1
Stock concentration
44.5 mg/L
70.3 mg/L
82.0 mg/L
(Dissolved Ag+)
(0.31 mg/L)
(0.38 mg/L)
(0.28 mg/L)
PVP-AgNPs
Sample 1
Sample 2
Sample 3
Particle size
17.5 ± 2.9 nm
51.4 ±18.7 nm
71.5 ± 20.3 nm
Zeta-potential (mV)
-27.5 ± 1.4
-35.8 ± 0.6
-37.1 ± 0.6
Stock concentration
8,200 mg/L
5,700 mg/L
6,700 mg/L
(Dissolved Ag+)
(2.93 mg/L)
(7.90 mg/L)
(7.15 mg/L) 2
(a)
(b)
(c)
Figure S1. TEM characterization of the commercial PVP-AgNPs (a) PVP-18nm (17.5 ± 2.9 nm), (b) PVP-51nm (51.4 ±18.7 nm) and (c) PVP-72nm (71.5 ± 20.3 nm).
Bacteria E. coli strain K12 (ATCC 25404) was chosen as a model microorganism for inactivation experiments. This facilitates comparison with numerous other related antimicrobial studies, and (because E. coli is facultative) allows for testing under both aerobic and anaerobic conditions. E. coli exhibits equal susceptibility to silver ions under aerobic and anaerobic conditions (Figure S2) 7
. The detailed bacteria stock suspension preparation method was provided in our previous paper
7
. Sodium bicarbonate (2 mM), which is commonly used as exposure medium 8, was chosen to
avoid ligands present in complex growth media that could bind with Ag+/AgNPs and affect silver bioavailability and/or promote precipitation or other confounding effects. All AgNPs/Ag+ toxicity assays were below the Ag2CO3 precipitation potential (Ksp=0.81×10-12).
3
Figure S2. E. coli strain K12 (ATCC 25404) exhibits equal susceptibility to the silver ion (which is the definitive molecular toxicants) under aerobic and anaerobic conditions. Reproduced with permission from reference 7. Copyright 2011 American Chemical Society.
Preparation of air-exposed AgNP stock suspensions To prepare air-exposed AgNPs samples, PEG-AgNPs (3, 5, 11 nm) and PVP-AgNPs (18, 51, 72 nm) stock suspensions (~2g/L) were transferred out of the chamber and exposed to air for 5 days (with pH adjusted to 4.0 to accelerate the oxidation of AgNPs and the release of Ag+). The released Ag+ concentration of each sample was monitored by ICP-OES/MS (Perkin Elmer, Waltham, MA) to avoid complete oxidation of AgNPs. The air-exposed AgNPs suspensions were then transferred to anaerobic chamber and resuspended in NaHCO3 buffer solution to achieve a constant pH (8.1) for Ag+ dissolution equilibration (5 days). The final Ag+ concentration for PVP-AgNPs are 9.5 mg/L, 17 mg/L. 10.5 mg/L respectively. Anaerobic synthesis of the PEG-AgNPs The anaerobic PEG-AgNPs were synthesized by mixing the pre-synthesized PEG-AgNPs suspension with sodium borohydride under vigorous agitation in an anaerobic chamber. All 4
precursors (including AgNPs suspensions and NaBH4 solution) were transferred and equilibrated inside the anaerobic chamber for 24h to allow for oxygen depletion. Excess amount of NaBH4 was added to ensure a complete reduction of all Ag+. The AgNPs were sealed in an Amicon ultra centrifugal filter unit (molecular weight cutoff 10,000, 2~5nm in pore size, Millipore, MA) and moved out of the chamber for centrifugation (J2-MC Centrifuge, Beckman Coulter, CA) to get rid of the impurities (e.g., NaBH4). The centrifuged samples were transferred back to the chamber to collect the filtrate and refilled with 2mM NaHCO3 buffer. This washing process was repeated continuously four times and the filtrates were monitored for dissolved silver with ICP-Ms. The final PEG-AgNPs stock suspensions were re-suspended in 2mM NaHCO3 buffer solution, with no detectable Ag+ (by ICP-Ms, detection limit: 1 µg/L) in supernatants. Preparation of filtered AgNP stock suspensions To purify the AgNPs, PEG- and PVP-AgNPs stock suspensions were washed by DI water and filtered through the Amicon ultra centrifugal filter units by centrifugation. AgNPs stock suspensions were filtered continuously for ~10 times to get rid of the residual Ag+ as much as possible. The filtrates were analyzed for total dissolved silver with an ICP-OES/MS to obtain the concentrations of dissolved silver. After filtration, the retentates were resuspended and transferred to 50-ml corning tubes (Corning, Lowell, MA). The concentrations of the AgNPs stocks were determined by nitric acid/hydrogen peroxide digestion as described earlier 7. These stocks were stored and tested inside the anaerobic chamber to avoid any confounding effects caused by oxidative Ag+ release during the dose-response assays.
5
The minimum lethal concentration (MLC) of the six filtered AgNPs to E. coli was compared to Ag+ to assess their relative toxicity contribution to AgNPs. The MLC is defined as the minimum lethal concentration of AgNPs in the exposure medium (bicarbonate buffer) that causes statistically significant E. coli mortality (p < 0.05) relative to the control set without AgNPs, as we reported earlier 7. Dose-response assay of AgNPs Six fresh and six air-exposed AgNPs stock suspensions were tested for dose-response on E. coli mortality inside the anaerobic chamber following the procedure as showed in Xiu et al 7. Specially, antimicrobial assay of the air-exposed AgNPs was conducted after 5 days of equilibration in anaerobic chamber to ensure a complete Ag+ dissolution. Free Ag+ concentrations were measured in the same medium and toxicity data of each AgNPs suspension was plotted against the free Ag+ concentration in it. E. coli mortality in different treatments was then determined by viable plate counts 9 and was calculated as 1-N/N0 × 100%, where N and N0 are the remaining and initial concentrations of viable bacteria (CFU/mL), respectively. The doseresponse curves of E. coli mortality versus Ag+ concentrations was fitted using a sigmoidal model (Eq. S1)10 (Sigma-Plot v10.0):
ݕൌ ݕ ଵା
ೣషೣబ ష ್
(Eq. S1)
Where y is the E. coli mortality rate, y0 is the baseline mortality rate without silver addition, x is silver concentration (expressed as released Ag+), and x0 is the Ag+ concentration that results in 50% mortality (EC50). All tests were conducted in triplicate and repeated at least three times to ensure reproducibility. Statistical Analyses
6
Whether differences between EC50 values for different treatments were statistically significant was determined using Student’s t-test at the 95% confidence level. T-tests were also used to determine MLC values (i.e., the lowest concentrations that resulted in a significant (p < 0.05) decrease in cell viability, assessed by plate counts) as well as the significance of hormesis effect relative to unexposed controls. For the dose-response assays, E. coli mortality data for each type of air-exposed AgNPs (expressed as their corresponding released Ag+ concentrations) and for the AgNO3 treatment were fitted with the Sigmoidal model (Eq. S1) using non-linear regression. The SAS PROC NLIN procedure and R software, version 2.9.2 (R Foundation for Statistical Computing, Vienna, Austria) was used for this purpose.11 Whether differences in dose-response trends were statistically significant were determined by the F-test at the 95% significance level using the SAS/STAT version 9.2 software (SAS Institute Inc., Cary, NC). This analysis (Table S2) inferred that the sigmoidal curves were statistically indiscernible and thus, that the antibacterial activity of AgNPs could be fully explained by the toxicity of the released Ag+. Table S2. Dose-response assay statistical analysis results F-test p value
Ag+ control
PEG3 nm
PEG5 nm
PEG11 nm
PVP18 nm
PVP51 nm
PVP72 nm
0.80
0.71
0.56
0.79
0.65
0.22
Ag+ release at low pH found near a cell membrane under the influence of the proton motive force The air-exposed PVP-AgNPs (three types in total) were transferred into the anaerobic chamber and each type of AgNPs was added to different media with different pH values. The lowest pH used (3.0, diluted HNO3 solution) was mimicking the local pH of a cell membrane
7
under the influence of the proton motive force. An intermediate pH (5.5, DI water) was used to represent the inside of a lysosome in eukaryotic cells. The highest pH (8.1, 2mM NaHCO3 solution) is representative of bicarbonate-buffered natural waters and was the same as in the toxicity assays. The released Ag+ concentrations in the supernatants were measured by ICP-Ms after 72-h equilibration. As shown in Figure S2, Ag+ release was affected by the solution pH, with higher Ag+ release at lower pH, suggesting increased release as AgNPs become in contact with these organelles.
35
+
Ag released (mg/L)
30 25
pH 3.0 pH 5.5 pH 8.1
20 15 10 5 0 25 nm
36 nm
86 nm
Figure S3. Dissolution of air-exposed PVP-AgNPs at different pH, as encountered near bacterial cell membranes influenced by the proton motive force (pH 3), or inside lysosomes of eukaryotic cells (pH 5.5) or in bicarbonate-buffered natural water (pH 8.1). Ag+ release increases with decreasing pH.
REFERENCES (1) Hiramatsu, H.; Osterloh, F. E. Chem. Mater. 2004, 16, (13), 2509-2511. (2) Yang, X. Environ. Sci. Technol. 2012. 8
(3) Meyer, J. N.; Lord, C. A.; Yang, X. Y. Y.; Turner, E. A.; Badireddy, A. R.; Marinakos, S. M.; Chilkoti, A.; Wiesner, M. R.; Auffan, M. Aquat. Toxicol. 2010, 100, (2), 140-150. (4) Laban, G.; Nies, L. F.; Turco, R. F.; Bickham, J. W.; Sepulveda, M. S. Ecotoxicology 2010, 19, (1), 185-195. (5) Lara, H. H.; Ayala-Nunez, N. V.; Ixtepan-Turrent, L.; Rodriguez-Padilla, C. J Nanobiotechnology 2010, 8, 1. (6) Beer, C.; Foldbjerg, R.; Dang, D. A.; Autrup, H. Toxicol. Lett. 2011, 205, S44-S44. (7) Xiu, Z. M.; Ma, J.; Alvarez, P. J. J. Environ. Sci. Technol. 2011, 45, (20), 9003-9008. (8) Chen, J.; Xiu, Z.; Lowry, G. V.; Alvarez, P. J. Water Res. 2010, 45, (5), 1995-2001. (9) Adams, L. K.; Lyon, D. Y.; Alvarez, P. J. J. Water Res. 2006, 40, (19), 3527-3532. (10) Poch, G.; Vychodil-Kahr, S.; Petru, E. Int. J. Clin. Pharm. Th. 1999, 37, (4), 189-192. (11) SAS/STAT(R) 9.2 User's Guide, Second Edition, the NLIN Procedure, http://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#nlin_t oc.htm.
9
