Gaining a Critical Mass: A Dose Metric Conversion Case Study

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Gaining a critical mass: A dose metric conversion case study using silver nanoparticles Alan James Kennedy, Matthew S. Hull, Stephen Diamond, Mark A Chappell, Anthony J Bednar, Jennifer G. Laird, Nick Melby, and Jeffery A Steevens Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03291 • Publication Date (Web): 16 Sep 2015 Downloaded from http://pubs.acs.org on September 22, 2015

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Gaining a critical mass: A dose metric conversion case study using silver nanoparticles

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Alan J. Kennedy†*, Matthew S. Hull‡§, Stephen Diamond§, Mark Chappell†, Anthony J.

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Bednar†, Jennifer G. Laird†, Nick Melby†, Jeffery A Steevens†

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* U.S. Army Engineer Research and Development Center

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Environmental Laboratory, Building 3270, EP-R

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3909 Halls Ferry Rd

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Vicksburg, MS, 39180

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Voice: 601-634-3344

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Fax: 601-634-2263

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* Corresponding author: [email protected]; phone: 601-634-3344; fax: 601-634-2263

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† U.S. Army Engineer Research and Development Center, Environmental Laboratory, Vicksburg, MS, USA

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‡ Virginia Tech Institute for Critical Technology and Applied Science (ICTAS), Blacksburg, VA, USA

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§NanoSafe, Inc., Blacksburg, VA, USA

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Mass concentration is the standard convention to express exposure in ecotoxicology for

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dissolved substances. However, nanotoxicology has challenged the suitability of the

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mass concentration dose metric. Alternative metrics often discussed in the literature

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include particle number, surface area and ion release (kinetics, equilibrium).

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unlikely that any single metric is universally applicable to all types of nanoparticles.

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However, determining the optimal metric for a specific type of nanoparticle requires

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novel studies to generate supportive data and employ methods to compensate for

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current analytical capability gaps. This investigation generates acute toxicity data for

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two standard species (Ceriodaphnia dubia, Pimephales promelas) exposed to five sizes

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(10, 20, 30, 60, 100 nm) of monodispersed citrate and polyvinylpyrrlidone coated silver

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nanoparticles. Particles were sized by various techniques to populate available models

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for expressing the particle number, surface area and dissolved fraction.

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indicate that the acute toxicity of the tested silver nanoparticles is best expressed by ion

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release, and is relatable to total exposed surface area.

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relatable to the observed acute silver nanoparticle effects.

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It is

Results

Particle number was not


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Introduction

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Dose metrics based on total or dissolved chemical mass continue to be the standard

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after

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nano(eco)toxicology has challenged the efficacy of the mass-only paradigm. Reviews

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[1,2,3] and documents [4] on the toxicology of nanoparticles (NPs) have discussed the

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applicability of the mass-only metric without consensus.

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recognized the need for alternative dose metrics (particle count, surface area, charge,

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shape) to express the implications of NPs [5,6,7,8,9,10,11,12,13]. Most aquatic ecotoxicology

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studies continue to express NP toxicity primarily by mass [14], with recent exceptions

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[15,16,17,18,19]. This is due in part to the intensiveness of characterizing all needed NP

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attributes (size, surface area, number, agglomeration, dissolution) in aquatic exposures,

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changes during the assay, lack of standardized guidance on how to apply alternative

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dose metrics and analytical limitations (low µg/L) at environmentally and toxicologically

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relevant concentrations [20]. However, the large variability in ecotoxicological endpoints

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for the same type of NP [21] provides evidence that total NP mass is not predictive of

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biological effects.

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The ecotoxicology community has considered size [22,23,24] and shape [25,26,27] effects on

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toxicity; such work mainly involved comparison to mass-expressed toxicity.

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dependent toxicity of silver nanoparticles (AgNPs) was observed in many [16,17,18,28,29,30],

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but not all [31,32] aquatic ecotoxicology studies.

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toxicity of AgNPs may relate to differences among studies, such as variation in

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dispersion conditions, overlapping size ranges, polydispersity, dissolution, handling and

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storage [18,30,33]. Particle number or surface area may be more expedient dose metrics

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than size and shape [3,34].

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Recognizing characterization constraints in bioassays, Hull et al. [14] provided

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calculations to convert mass-based NP data to particle number concentration (NC), total

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exposed surface area (TESA) and the dissolved fraction (Df), using size and mass as

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inputs.

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monodispersed spherical particles; thus, applying them to non-ideal, polydispersed and

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dynamic particle suspensions (prevalent in the literature) creates uncertainty [13].

decades

These

of

environmental

calculations

toxicology

assume

research.

Environmental

Aerosol toxicologists have

Size-

Inconsistencies in size-dependent

adequate

characterization


and

stable,

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Particle number is recommended in the aerosol science community [5,12,13] and may be

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representative of particle interactions with biological membranes or dietary uptake [35].

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Surface area was reported useful in aerosol and microbial toxicology [7,8,9,10,11,12].

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While the dissolved fraction has less novelty, it must be considered for soluble NPs to

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avoid confounding other dose metrics since desorption kinetics and particle dissolution

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are faster for smaller particles [36,37,38].

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To gain acceptance, a suitable dose metric must be amenable to commonly available

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measurement capability, logical for setting regulatory threshold values and provide a

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predictive improvement over mass-based standards. Our objectives were to compare

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the suitability of mass versus alternative dose metrics (N, TESA, Df) for monodispersed

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AgNPs in aquatic bioassays.

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(citrate, polyvinylpyrrlidone (PVP)), and different test species (Ceriodaphnia dubia,

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Pimephales promelas) on dosimetry conversions was considered. Ceriodaphnia dubia

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was selected as a filter-feeder (direct particle ingestion) and P. promelas was selected

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as a predator that may be exposed to particles passively at the gill. We hypothesized

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that total surface area would be the best predictor of AgNP toxicity, based on previous

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results that the Df was relatable to the acute biological effects [18,39,40,41,42].

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Methods

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Test materials and preparation

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Citrate- and PVP-AgNPs (10, 20, 30, 60, 100 nm) were obtained from NanoComposix

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(San Diego, CA).

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ultrapure water (Milli-Q Plus, 18.2 mΩ/cm, Billerica, MA) while bath sonicating for five

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minutes (Fisher Scientific model FS-60, 130W, Pittsburgh, PA), according to Coleman

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et al. [43]. Suspensions were used in fate or bioassay experiments within 30 min of

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preparation, since storage increases dissolution and toxicity [18]. The AgNP toxicity was

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compared to dissolved Ag (AgNO3, Sigma Aldrich, 204-390-50G, St. Louis, MO).

The impact of measurement techniques and coating

Working stocks (nominally 10 mg/L) were prepared in 100 mL



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Particle characterization and analytical chemistry

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Characterization was as previously described [18,44,45].

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determined by transmission electron microscopy (TEM; Zeiss 10CA, 60kV, Oberkochen,

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Germany for AgNPs except for 60 nm citrate-AgNPs; a JOEL JEM 1400 for the 60 nm

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citrate-AgNPs) through measurements of individual particles (≥200, 12 images), using

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ImagePro® (v7, Media Cybernetics Inc., Bethesda, MD). Hydrodynamic diameter (HD)

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in suspension was determined by dynamic light scattering (DLS; 635 nm laser; 90

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Plus/BI-MAS, Brookhaven Instruments, Holtsville, NY) by intensity-weighting and by

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Field Flow Fractionation (PostNova F-1000 symmetrical flow FFF, Salt Lake City, UT)

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interfaced to an Inductively Coupled Plasma Mass Spectrometer (ICP-MS, Elan DRC-II,

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Perkin-Elmer, Waltham, MA).

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aliquots 1 cm below the water surface. Total silver samples (2 mL) were acidified

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without further modification. Dissolved silver ( 93-100%. Data were summarized as the mean, and one standard deviation. The geometric mean between TEM and HD for each particle is provided to more heavily weight primary particle size.

Coating

Nominal size (nm)

Measured primary size (TEM images)

Citrate

10

9±4 (1342)

18.1 ± 0.6 (10 – 32)

Citrate

20

Citrate

30

Citrate

60

Citrate

100

PVP

10

PVP

20

PVP

30

PVP

60

PVP

100

19 ± 5 (534) 18 ± 5 (612) 39 ± 11 (143) 102 ± 12 (186) 32 ± 10 (567) 21 ± 4 (868) 31 ± 6 (324) 70 ± 11 1 (717) 122 ± 24 2 (204)

27.8 ± 0.2 (17 – 46) 49.7 ± 0.1 (27 - 89) 63.8 ± 0.6 (43 – 93) 99.7 ± 0.4 (44 – 148) 26.8 ± 0.1 (14 – 52) 32.3 ± 0.4 (18 - 57) 38.9 ± 0.1 (28 – 54) 73.3 ± 0.4 (43 – 122) 98.6 ± 0.2 (63 – 154)

1 2

Geometric Mean (nm)

Nominal Surface area 2 (m /g)

Calculated surface area 2 (m /g)

Percent Dissolved (P. promelas) in biological test media

Percent Dissolved (C. dubia) in biological test media

19.5

13

57.2

63.6

18.9 ± 9.2

20.3 ± 1.6

27.1

23

28.6

30.1

6.5 ± 1.1

5.0 ± 0.8

40.2

28

19.1

31.8

12.1 ± 9.5

3.7

83.4

53

9.5

14.7

6.3 ± 5.5

0.7 ± 0.0

131.9

108

5.7

5.6

1.6 ± 0.8

0.8 ± 0.1

47.4

34

57.2

17.9

3.6 ± 0.6

1.9

30.0

26

28.6

27.2

3.9 ± 1.8

4.0

39.6

35

19.1

18.5

1.2 ± 0.2

0.9

86.7

75

9.5

8.2

2.6 ± 3.2

0.5

127.3

117

5.7

4.7

0.4 ± 0.0

0.2 ± 0.1

Hydrodynamic Diameter in ultrapure water (DLS, nm)

Hydrodynamic Diameter in ultrapure water (DLS, nm)

Second batch used in P. promelas retest (66 ± 8, N = 776) Second batch used in P. promelas retest (115 ± 21, N = 374)



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. Table 2. Summary of toxicity reference values for Ceriodaphnia dubia. Lethal median effect metrics are expressed as total measurable silver (LCT50), total exposed surface area (LSA50) and the dissolved fraction (LCD50). Confidence intervals (95%) are provided in parentheses. Median effects concentrations for particle number are not provided since this metric was not predictive of the study material toxicity. Coefficients of variation (CV) for each dose metric are provided. NA = not applicable; n/a = not available # previously excluded as outlier Test material

Nominal size (nm)

Total measureable LCT50 (µg/L)

LSA50 2 (mm /L)nominal size

LSA50 2 (mm /L) TEM Size

LSA50 2 (mm /L) FFF size

LSA50 2 (mm /L) DLS size

AgNO3

Dissolved

NA

NA

NA

NA

NA

Citrate

10

Citrate

20

Citrate

30

Citrate

60

Citrate

100

1.5 (1.3 – 1.6) 16.0 (12.8 – 19.9) 13.7 (11.4 -16.6) # 60.0 (46.1 – 78.2) 32.3 (24.4 – 42.9) 91% 23.5 (19.1 – 29.0) 19.7 (14.8 – 26.2) 29.6 (23.0 – 38.0) 33.5 (26.7 – 42.0) 66.8 (56.6 – 78.9) 54%

84 (76 – 93) 456 (366 – 568) 262 (216 – 316) 572 (439 – 745) 185 (139 – 245) 64% 1346 (1091 – 1680) 563 (422 – 750) 564 (439 – 724) 318 (250 – 404) 382 (323 – 451) 65%

93 (84 – 103) 480 (386 – 598) 436 (361 – 527) 880 (676 – 1147) 181 (137 – 241) 74% 420 (341 – 519) 536 (402 – 715) 545 (424 – 701) 272 (214 – 346) 313 (265 – 370) 30%

43 (39 – 48) 337 (270 – 419) 195 (162 – 236) 412 (316 – 536) 140 (106 – 186) 66% 284 (230 – 351) 375 (261 – 500) 427 (332 – 548) 220 (173 – 280) 300 (254 – 355) 25%

46 (42 – 51) 328 (264 – 409) 158 (131 – 191) 538 (413 – 701) 185 (140 – 246) 75% 502 (407 – 620) 348 (261 – 465) 435 (338 – 558) 260 (204 – 330) 388 (328 – 458) 24%

CV (%) PVP

10

PVP

20

PVP

30

PVP

60

PVP

100

CV (%)


Dissolved LCD50 (µg/L) 0.3 (0.3 – 0.4) 0.2 (0.2 – 0.3) 0.8 (0.7 – 1.0) 0.1 (0.1 – 0.2) 0.4 (0.3 – 0.5) 0.3 (0.2 – 0.4) 75% 0.9 (0.8 – 1.0) 0.8 (0.6 – 0.9) 0.6 (0.6 – 0.7) NA/ND NA/ND 7436

2047 (1745 – 2401) 2066 (1775 – 2405) 4322 (3941 – 4739) 2428 (2084 – 2830) 1270 (1225 – 1316) 47% 5465 (4486 – 6659) 3951 (3462 – 4510) 7574 (6660 – 8613) 4 >6373

945 (805 – 1108) 1448 (1244 – 1686) 1935 (1765 – 2122) 1135 (974 – 1323) 982 (947 – 1018) 32% 3690 (3029 – 4495) 2766 (2423 – 3157) 5929 (5214 – 6743) >5146

1019 (869 – 1194) 1412 (1213 – 1643) 1565 (1427 – 1716) 1484 (1274 – 1730) 1299 (1253 – 1346) 16% 6526 (5357 – 7951) 2569 (2251 – 2933) 6036 (5308 – 6865) >6086

6.5 (5.5 – 7.6) 4.1 (3.4 – 4.8) 4.5 (3.7 – 5.4) 1.3 (1.0 – 1.6) 5.8 (4.6 – 7.2) 3.4 (3.0 – 3.7) 43% 11.7 (9.2 – 14.9) 5.8 (4.7 – 7.2) 4.5 (3.9 – 5.2) >2.4

PVP

10

PVP

20

PVP

30

PVP

60

32.2 (27.5 – 37.8) 68.6 (59.0 – 79.9) 136.0 (124.0 – 149.1) 165.6 (142.1 – 193.0) 226.4 (218.5 – 234.7) 61% 305.8 (251.0 – 372.5) 145.1 (127.1 – 165.6) 410.5 (361.0 – 466.8) 3 >780

PVP

100

>1100

>6292

>5157

>4942

>6381

NA

84%

101%

75%

79%

81%

83%

CV (%)

CV (%) 3

Extrapolate higher concentration complete kill 774 (644 – 929) µg/L

4

Extrapolate higher concentration complete kill 6707 (5585 – 8054) mm /L

2



. Figure 1. Total and dissolved concentrations of citrate- and PVP-silver nanoparticle (AgNP) dispersions in light and dark conditions. Data are presented as (a) total measureable silver for citrate-AgNPs, (b) dissolved silver for citrate-AgNPs, (c) total measureable silver for PVP-AgNPs, (d) dissolved silver for PVP-AgNPs. Solid points and lines represent the dark condition while open points and dashed lines represent the light condition. Points with different letter designations were statistically significant (lowercase for dark condition, capital letters for light condition). When only one set of letters was provided for a particle size, light condition was not a significant factor in determining concentration.


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. Figure 2. Size dependent toxicity of citrate- and PVP-silver nanoparticles (AgNPs). Data are summarized for (a) Ceriodaphnia dubia and (b) Pimephales promelas exposures. In panel (a), the 60 nm citrate-AgNPs were excluded (solid line) and included (dashed line) in the regression. The 60 nm citrate-AgNPs were modeled as an outlier since its toxicity was less than the 100 nm citrate-AgNP; the toxicity of the 100 nm citrate-AgNP supported by Kennedy et al [18]. In panel (b) data were not plotted for PVP-AgNPs since no LC50 could be generated up to the highest exposure concentrations). (a)

(b)



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. Figure 3. Comparison of dose-response curves for Pimephales promelas exposed to citrate-silver nanoparticles. Data are expressed as (a) total mass, (b) total exposed surface area, (c) particle number concentration. Relationships for both species, both capping agents and all metrics can be found in the supporting information (Figure S1-4, SI). (a)

(b)

0.8 0.6 0.4 0.2 0.0

1.0

Proportion surviving

10 nm 20 nm 30 nm 60 nm 100 nm

0.8 0.6 0.4 0.2 0.0

10

100

10 nm 20 nm 30 nm 60 nm 100 nm

100

1000

1000

Total surface area (mm2/L) - DLS

Concentration (ug/L)

(c) 1.0



1.0

10 nm 20 nm 30 nm 60 nm 100 nm

0.8 0.6 0.4 0.2 0.0

1e+7

1e+8

1e+9

1e+10

Particle number concentration (N/mL) - DLS


10000

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. Figure 4. Dose response curves related to the dissolved silver concentration measured in exposures. Data are summarized for (a) Pimephales promelas exposed to citratesilver nanoparticles (AgNPs), (b) P. promelas exposed to PVP-AgNPs, (c) Ceriodaphnia dubia exposed to citrate-AgNPs and (d) C. dubia exposed to PVP-AgNPs. C. dubia were also exposed to supernatants taken from select AgNP suspensions. (b)

1.0



(a)

0.8 0.6 0.4 0.2 0.0

10 nm 20 nm 30 nm 60 nm 100 nm AgNO3

0.01

0.1

1

10

100

1.0 0.8 0.6 0.4 0.2 0.0

10 nm 20 nm 30 nm 60 nm 100 nm AgNO3

0.01

Concentration (µg/L), dissolved

0.1

1

10 nm 20 nm 30 nm 60 nm 100 nm AgNO3

0.8 0.6

10 nm (supernatant) 20 nm (supernatant) 60 nm (supernatant)

0.4 0.2 0.0

1

10

100



(d)

1.0

0.1

100


(c)

0.01

10

1000

0.8

20 nm (supernatant) 60 nm (supernatant)

0.6 0.4 0.2 0.0

0.01


10 nm 20 nm 30 nm AgNO3

1.0

0.1

1

10

100



1000

Gaining a Critical Mass: A Dose Metric Conversion Case Study

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