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Adapting OECD Aquatic Toxicity Tests for Use with Manufactured
Nanomaterials: Key Issues and Consensus Recommendations
Elijah J. Petersen,
†
Stephen A. Diamond,
‡
Alan J. Kennedy,*
,§
Greg G. Goss,
∥
Kay Ho,
⊥
Jamie Lead,
#
Shannon K. Hanna,
†
Nanna B. Hartmann,
∇
Kerstin Hund-Rinke,
○
Brian Mader,
◆
Nicolas Manier,
¶
Pascal Pandard,
¶
Edward R. Salinas,
Δ
and Phil Sayre
◇,+
†
Biosystems and Biomaterials Division, Material Measurement Laboratory, National Institute of Standards and Technology,
Gaithersburg, Maryland 20899, United States
‡
Midwest Division, NanoSafe, Inc., Duluth, Minnesota 55802, United States
§
Environmental Laboratory, U.S. Army Engineer Research and Development Center, Vicksburg, Mississippi 39180, United States
∥
Department of Biological Sciences and National Institute of Nanotechnology, National Research Council, University of Alberta,
Edmonton, Alberta, Canada T6G 2E9
⊥
Office of Research and Development, National Health and Environmental Effects Research Laboratory−Atlantic Ecology Division,
United States Environmental Protection Agency, Narragansett, Rhode Island 02882, United States
#
Center for Environmental Nanoscience and Risk, Department of Environmental Health Sciences, Arnold School of Public Health,
University of South Carolina, Columbia, South Carolina 29036, United States
∇
Department of Environmental Engineering, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark
○
Fraunhofer Institute for Molecular Biology and Applied Ecology, D-57392 Schmallenberg, Germany
◆
Environmental Laboratory, 3M, St. Paul, Minnesota 55144, United States
¶
Institute National de l’Environnement Industriel et des Risques (INERIS), Parc Technologique ALATA, F-60550 Verneuil
en-Halatte, France
Δ
Experimental Toxicology and Ecology, BASF SE, D-67056 Ludwigshafen, Germany
◇
Office of Pollution Prevention and Toxics, United States Environmental Protection Agency, Washington, D.C. 20460, United States
*
SSupporting Information
ABSTRACT: The unique or enhanced properties of manufactured nanomaterials
(MNs) suggest that their use in nanoenabled products will continue to increase. This
will result in increased potential for human and environmental exposure to MNs during
manufacturing, use, and disposal of nanoenabled products. Scientifically based risk
assessment for MNs necessitates the development of reproducible, standardized hazard
testing methods such as those provided by the Organisation of Economic Cooperation
and Development (OECD). Currently, there is no comprehensive guidance on how
best to address testing issues specific to MN particulate, fibrous, or colloidal properties.
This paper summarizes the findings from an expert workshop convened to develop a
guidance document that addresses the difficulties encountered when testing MNs using
OECD aquatic and sediment test guidelines. Critical components were identified by
workshop participants that require specific guidance for MN testing: preparation of
dispersions, dose metrics, the importance and challenges associated with maintaining
and monitoring exposure levels, and the need for reliable methods to quantify MNs in
complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss
critical considerations where expert consensus recommendations were and were not achieved and provide specific research
recommendations to resolve issues for which consensus was not reached. This process will enable the development of
prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure
during the bioassays used to determine hazard values.
■INTRODUCTION
The rapidly accelerating development and implementation of
nanotechnology has inspired vigorous debate about the
adequacy of current regulatory frameworks for assuring the
safe deployment of manufactured nanomaterials (MNs) in the
Received: February 25, 2015
Revised: June 15, 2015
Accepted: July 16, 2015
Published: July 16, 2015
Critical Review
pubs.acs.org/est
© 2015 American Chemical Society 9532 DOI: 10.1021/acs.est.5b00997
Environ. Sci. Technol. 2015, 49, 9532−9547
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commercial marketplace.
1−4
A critical aspect of these debates is
whether standard test protocols currently used in risk
assessment are fully adequate for testing the hazard potential
of MNs.
5,6
Standardized testing protocols, and the guidance
documents that describe them, are a critical component of risk
assessment and regulatory processes that enable placement of
chemical substances on the market. These test protocols
describe specific techniques and methods for the collection and
analyses of data with the goal of quantitatively describing, under
controlled laboratory conditions, the release, fate, transport,
transformation, exposure, and toxicity of chemical substances.
The Organisation for Economic Cooperation and Development
(OECD) has promulgated internationally accepted test guide-
lines (TGs) that are used for these purposes. A subset of these
TGs focus on toxicity in aquatic, sediment, and soil organisms
and constitute the OECD’s Test Guidelines Section 2: “Effects
on Biotic Systems”.
7−10
Several recent publications focused on aquatic and sediment
ecotoxicity assay methods commonly used in regulatory testing
suggest that these methods are generally adequate for testing of
MNs but discuss the need for additional guidance to improve
their applicability for hazard assessment of MNs.
8−14
The
critical issue is that aquatic ecotoxicity testing with MNs
involves exposure of test organisms to colloids or particle−
sediment mixtures rather than solely to dissolved chemicals, for
which the OECD TGs were originally intended. MNs in test
media typically undergo extensive agglomeration, settling,
particle dissolution, and transformations during exposure and
medium-renewal periods.
9,15
These transformation processes
depend in part on the intrinsic properties of the MN, the
concentration of the MN, and the composition of the medium.
The resulting variability in exposure presents unique challenges
for exposure−response estimation. Alternate dose metrics
based on particle number, surface area, or body burden in
addition to mass concentration might be informative; however,
metrics other than mass concentration are not generally
considered within current risk assessment frameworks.
Dissolution and ion release from MNs during testing, as
often observed for silver and zinc oxide MNs,
16,17
further
complicate dosimetry because the resulting exposures poten-
tially involve both MNs and dissolved species. Concentration-
dependent MN agglomeration, settling, and dissolution also
present significant measurement and monitoring challenges,
both logistically and methodologically. These MN behaviors
often alter exposure levels beyond ±20% of the initial
(measured) or nominal concentration during an aquatic
bioassay, a specification in many TGs hereafter termed the
“20% exposure specification”. While MNs released from
nanoenabled products may differ substantially from their as-
produced form (e.g., CNTs released to the environment from
polymer nanocomposites may be partly or fully encased in
component polymers
18−21
), the focus in this review is on as-
produced MNs.
Herein we discuss the findings of a workshop focused on
drafting an OECD guidance document (GD) on Aquatic (and
Sediment) Toxicology Testing of Nanomaterials, which
provides necessary amendments to existing OECD aquatic
toxicity test methods and is an OECD project approved in
2013. This meeting, held at the U.S. Environmental Protection
Agency (EPA) in Washington, DC, in July 2014, was attended
by 23 experts from seven countries. We discuss in depth the key
limitations of current aquatic bioassay study designs for testing
of MNs and knowledge gaps that preclude or hinder the
development of prescriptive, broadly applicable aquatic toxicity
standard tests for MNs, and we suggest research to address
these issues. Each of the following topics raised at the meeting
is critically discussed: key considerations for testing the aquatic
toxicity of MNs; the feasibility of conducting tests with MNs
that meet the 20% exposure specification; dosimetry and
interpretation concerns for MNs; and challenges with testing of
MNs in sediments. We highlight issues where consensus was
and was not reached during the workshop and subsequent
discussions with workshop participants and recommend
research to resolve topics where consensus was not reached.
The discussions and viewpoints expressed by the workshop
participants are summarized and inform but are not binding
toward the development of the OECD GD described above.
The workgroup participants agreed to define MNs broadly as
solid-phase substances having one dimension between 1 and
100 nm. While there are more detailed definitions (e.g., the
European Commission-proposed definition
22
), our intent is to
avoid limiting the workgroup findings to current MN
definitions that may change. The more specific terminology
used here (e.g., particle size, dissolution, agglomeration,
aggregation, etc.) generally follow OECD documents on
MNs.
23
■KEY CONSIDERATIONS RELATED TO AQUATIC NM
TOXICITY TESTING
The Importance of Standard Terminology. Workshop
participants strongly agreed on the importance of using precise
terminology when describing results from nanoecotoxicity tests.
The absence of terminology in ecotoxicology TGs specificto
(nano) particles, colloids, dispersions, and suspensions further
complicates the conduct of standard aquatic ecotoxicity tests
with MNs.
24
For example, MN suspensions have been
erroneously called dissolved MNs rather than dispersed or
suspended MNs. The operational definition of “dissolved”
substances varies significantly among different fields, and there
are environmental and mechanistic definitions that are partially
related to the operational definitions;
25
a more detailed
discussion of this topic is available in the Supporting
Information. It is thus critical to make a distinction among
the terms “suspension”and “dispersion”versus “solution.”As
the term “solution”suggests that the MNs are dissolved in the
aqueous test medium, the terms “suspension”and “dispersion”
are favored. This is especially important because “true”
dissolution of MNs into their component ions is an important
process in environmental fate and ecotoxicology. For instance,
some dispersed or suspended MNs will subsequently fully or
partly dissolve to their constituent ions over the exposure time
of nanoecotoxicity tests, and this must be taken into account in
interpreting data. Consistent use of terminology can therefore
minimize misinterpretation of reported results.
For the past two decades, guidance for aquatic toxicity testing
for hazard assessment has included a distinction in the
terminology used to describe adverse effects. Intrinsic toxicity
is derived from exposure to dissolved molecules and is distinct
from adverse physical effects.
26
Physical effects can be
manifested as attachment of insoluble material to the exterior
of an organism as micelles, aggregated particles, or a flocculent
and lead to adverse effects from fouled respiratory surfaces,
impaired mobility, and feeding (daphnids) or light attenuation
(algae). Intrinsic toxicity is the focus of aquatic hazard
assessment based on the concept that the dissolved molecule
represents the most relevant exposure condition for aquatic
Environmental Science & Technology Critical Review
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toxicity testing and undissolved material is excluded from tests
to avoid physical effects.
27,28
Since aquatic exposures to MNs
may include both dissolved and solid phases, additional effort is
required to distinguish “intrinsic”toxicity from physical effects.
In tests with MNs, particulate uptake has the potential to exert
toxic effects that are not solely physical. Carefully designed
control experiments are essential for making a distinction and
avoiding misinterpretations
29
and need to be incorporated into
future work, including evaluations of how and when to include
the hazard from physical effects into aquatic risk assessment.
In addition, the use of terms related to an “equilibrium”
being reached among multiple phases including organism
Figure 1. Examples of changes in nanoparticle stability (transformations) in environmentally relevant test media, with gray regions representing
±20% of the original value. (A) Different settling rates and stable concentrations of carbon nanotubes with different surface modifications and
natural organic matter (NOM). A concentration of 100 ppm indicates 100 mg/L. (B, C) Impact of greater ionic strength in the medium on (B) the
nanosilver concentration and (C) the hydrodynamic diameter. (D) Increase in the dissolved concentration of nanosilver with time at different
temperatures. (E, F) Impact of test organisms on nanoparticle stability: while graphene settling is relatively low in absence of test organisms (E), the
presence of Daphnia magna increases settling (F). Error bars in (C), (E), and (F) represent standard deviations of triplicate measurements, while the
data points indicate the mean values. Panel (A) was reprinted with permission from ref 61. Copyright 2008 SETAC. Panels (B) and (C) were
reprinted from ref 166. Panel (D) was reprinted from ref 16. Copyright 2010 American Chemical Society. Panels (E) and (F) were reprinted from
ref 53. Copyright 2013 American Chemical Society.
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9534
tissues (i.e., bioconcentration factor, bioaccumulation factor,
biota−sediment accumulation factor, etc.) is discouraged
9
or, at
a minimum, needs to be better qualified. The use of these terms
may result in an inaccurate comparison between organism
accumulation of MNs and hydrophobic organic contaminants
(HOCs) or dissolved metals. Bioaccumulation of HOCs is
related to passage through biological membranes via passive
diffusion or active uptake through ion channels or carrier-
mediated transport.
30
For MNs, however, results show that
absorption into organism tissues is typically limited. For
example, ingestion of carbon-based MNs by aquatic organisms
often leads to high ingested concentrations present only in the
gut tract with nondetectable absorption into systemic
circulation,
18,31,32
while many HOCs are concentrated in the
lipid fraction of organisms.
33−36
In addition, changes in the
octanol−water partition coefficients were not shown to
correlate with changes in accumulation of multiwall carbon
nanotubes (MWCNTs) by a benthic organism (Lumbriculus
variegatus) or an earthworm (Eisenia fetida).
37
An OECD
document on sample preparation and dosimetry indicated that
the OECD TG for octanol−water partition coefficients is
unlikely to be directly applicable for use with MNs,
23
a
conclusion also reached by others.
38
MN Behavior in Test Systems. The behaviors of MNs in
aqueous media impact the accuracy and reproducibility of
results derived from OECD ecotoxicity methods in that they
are more dynamic and not predictable by traditional methods
of partitioning and bioavailability. MNs are similar in concept
to solid particulate chemicals or mixtures described as “difficult
substances”.
27
For example, MNs may agglomerate, settle from
suspension, and/or dissolve
18,39
(Figure 1). Moreover, these
behaviors are greatly influenced by the test medium and other
factors such as the MN number concentration. Media with
higher ionic strength, and especially higher concentrations of
divalent and trivalent metal ions, result in higher rates of
agglomeration and settling of MNs from suspension, with
stabilization mechanisms playing a role.
40
Silver nanoparticles
(AgNPs) provide an example of an MN that undergoes
transformations in aqueous media; AgNPs may form silver
chloride or silver sulfide particles if the medium contains
chloride or sulfur, respectively, and these modified particles can
be significantly less toxic than unmodified AgNPs.
15,41,42
Silver
nanoparticles also interact with natural organic material
(NOM), oxidize, and dissolve,
15,29
which influences their
surface chemistry, dissolution, aggregation, and toxicity.
43−46
Formation of AgNPs from reduction of ions can also occur in
aquatic media.
47,48
Agglomeration and settling cause increased
heterogeneity in the test vessel, with higher mass concen-
trations toward the bottom of the container. The procedure
used to disperse MNs in the aqueous medium and the MN
concentration dispersed can also impact the general dispersion
stability and heterogeneity in the test container as well as the
rate of agglomeration.
49
Thus, the assay results for MNs are
often more sensitive to the dispersion and mixing steps than
those for dissolved metals or HOCs. Additionally, washing
procedures to purify MNs can influence their chemistry and
behavior when the coating is weakly bound to the MN
surface.
50
All of these changes to the MN distribution could
lead to inaccurate or inconsistent organism exposure.
29
Monitoring and Quantifying MN Exposure. The current
lack of widely available routine measurement methods with
known accuracy, precision, and method performance require-
ments for quantifying the mass concentration and dispersion
state of MNs in test media further complicates MN testing.
While quantitative measurements of the distribution of MNs in
the test containers throughout bioassays are critical for
understanding variable test results, such measurements are
rarely performed (exceptions include refs 51−54). When
nonstandardized methods are used, they are often experimental
in nature and not easily implemented by testing laboratories.
Describing quantification methods for each type of MN is
beyond the scope of this paper but has been considered
elsewhere.
55−58
Quantifying the MN concentration in the test
suspension is most difficult for lower MN concentrations (i.e.,
μgL
−1) with most methods; while a promising recent study
used atomic force microscopy to produce concentrations down
to micrograms per liter,
59
this process has not yet been
standardized and is not available to most ecotoxicology
laboratories for routine analysis. It is possible to measure
aqueous-phase concentrations of carbon nanomaterials
(CNMs) greater than 1 mg L−1using techniques such as
UV/vis absorption spectroscopy
60,61
and gravimetric anal-
ysis.
31,62,63
While some methods for quantifying lower CNM
concentrations are described in the literature, these methods
detect only specific types of carbon nanotubes (CNTs)
64
or
additional work is needed to standardize the methods.
65−67
Metal and metal oxide MNs can be quantified in bulk by
elemental analysis (e.g., by inductively coupled plasma mass
spectrometry (ICP-MS)) at low concentrations. Separation
methods such as ultrafiltration, centrifugation, and dialysis
membrane techniques can be used to distinguish between
unagglomerated, agglomerated, and dissolved MNs but have
not yet been standardized.
16,29,68,69
The applicability and
reproducibility of these separation methods will be assessed
by an OECD group developing a test guideline for measuring
MN dissolution. Emerging techniques such as single-particle
ICP-MS
70−75
and liquid nebulization/differential mobility
analysis
76
can distinguish among some of these different
transformations for metal-containing MNs. However, they
require standardization and have MN-dependent limitations
because their lowest measurable MN sizes are above 1 nm, and
thus, their practical application for routine hazard testing has
not yet been demonstrated. Recently, Mader et al.
76
addressed
this issue by providing a framework for evaluating the
performance of new MN measurement methods.
The Role of Standardized Hazard Testing in MN Risk
Assessment. The different behaviors of MNs in comparison
with soluble chemicals such as HOCs and dissolved metals have
raised questions about the common practice of separately
assessing hazard and exposure. While significant progress has
been made toward understanding the environmental fate and
transformation of MNs
15,77−80
and obtaining the basic
information required to estimate exposure,
81
work is still
ongoing to develop models to predict the fate and hazard of
MNs on the basis of their composition and physicochemical
characteristics.
82,83
This knowledge, which informs and
simplifies hazard testing for dissolved chemicals, is rarely
available for MNs, suggesting that fate and exposure testing
may need to be incorporated into hazard testing guidance for
MNs. For example, the environmental relevance of testing the
aquatic toxicity of MNs that rapidly settle out of suspension
with pelagic organisms was debated during the workshop. The
ongoing efforts at OECD to develop TGs and a GD on MN
dissolution, dispersion stability, and environmental fate will
inform these decisions, while the TG on MN sorption to
activated sludge that is also currently under development will
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enable more realistic estimates of surface water and terrestrial
nanomaterial concentrations. At a minimum, the toxicity of the
corresponding dissolved bulk material (if available) should be
determined for a complete interpretation of aquatic hazard data
generated for MNs.
84
Limit Testing. While the concept of limit testing is
described in many OECD TGs, its applicability to MNs was
not explicitly discussed during the workshop. The use of limit
testing to assess the hazard of MNs is complicated by many of
the exposure issues described here for concentration−response
(multiple exposure concentration) testing. Limit tests employ a
recommended maximum exposure concentration to determine
whether a substance has hazard potential within reasonable
limits. The goal is to identify a single high concentration of the
test substance at which no effects are observed, eliminating the
need for further testing. OECD TGs 218 and 219 (sediment-
water Chironomid testing with spiked sediment
85
or water
86
)
describe the limit-test concentration as “...sufficiently high to
enable decision makers to exclude possible toxic effects of the
substance, and the limit is set at a concentration which is not
expected to appear in any situation.”OECD 218 sets this
concentration at or below 1000 mg/kg of sediment. Applicable
aquatic TGs
93,101,130
recommend limit tests be set at 100 mg
L−1(or the highest soluble concentration, whichever is lower)
for water-only tests. For substances that form stable dispersions,
an existing OECD GD
27
(that does not specifically consider
MNs) recommends a limit concentration of 1000 mg L−1or the
dispersibility limit, whichever is lower. The application of limit
testing based solely on mass concentration is potentially
problematic for MNs, as the particle number concentration and
surface area vary significantly for a given mass of material
present at mean sizes between 1 and 100 nm. Other issues
include varying MN transformation rates (i.e., dissolution,
agglomeration) at different concentrations and the potential for
nanomaterial atypical dose−response curves.
Potential Modifications to Test Procedures. Adjusting
Medium Composition. A number of potential modifications to
standard testing were considered for MN ecotoxicity testing to
address the behaviors of MNs described above. One of these
modifications is to prescribe a single test medium for each
commonly used test organism for use with each bioassay
method. Current TGs typically allow for flexibility in selection
of the bioassay medium in recognition of variability among
various testing facilities. However, for MNs this flexibility can
lead to difficulty in comparing test results and potentially a lack
of agreement among laboratories that are using the same basic
test method. Diluting the test medium (i.e., reducing the ionic
strength) or adjusting the pH of the medium away from the
point of zero charge of the MN may reduce the rate of
agglomeration and settling for many MNs
87
but may be
physiologically stressful for test organisms.
88
Thus, in selecting
the standard test medium, there is a potential trade-offbetween
maintaining organism health and vitality and minimizing the
MN agglomeration and transformation rates. For example,
Daphnia magna growth and reproduction are typically raised
with greater water hardness,
89
but this leads to greater rates of
MN agglomeration for charge-stabilized MNs, resulting in
lower or less consistent exposure. Choosing an alternate
daphnid test species adapted to softer waters (e.g., Daphnia
pulex
88
) may be a viable alternative. Any modifications to the
standard methodology that may alter the physiological stress
responses of the test organism should be validated with a
positive control experiment such as a reference toxicant test,
which can be found in OECD method validation studies and
the open literature.
129
In addition, some MNs may yield
acceptable assay variability in standard test media, and altering
standard and historically used test media would limit relative
comparisons to previous data generated using OECD
ecotoxicity TGs. For MNs where dissolved metal ions may
impact the toxicity (e.g., ZnO and AgNPs
17,29
), it is important
to exclude metal chelators such as EDTA as described in
previous OECD documents for metal toxicity testing (e.g.,
algae testing
90
). While some studies have used chelators such as
cysteine to eliminate the impact of released ions to highlight the
impact of an MN itself, interactions between the chelators and
the MN surface may impact MN behaviors and trans-
formations.
91,92
Standardizing Test Vessels and Systems. The selection of
test vessels can also impact ecotoxicological results.
93−95
Increasing the consistency of the test vessel dimensions
(material, size, aspect ratio, internal surface area) for each test
type and species is expected to reduce differences in the rate of
MN agglomeration, settling, dissolution, or sorption, although
it should be considered that a single type of test vessel may not
always be suitable for all types of MNs. A consistent test vessel
for each test type and species should be selected from common
commercially available products. Assay-specific modifications
should also be considered, such as the impact of the agitating
mode for the algae test on MN behaviors and the grazing on
the bottom of the vessel for the Daphnia magna test.
90,96,97
Furthermore, interlaboratory comparison testing can be used to
evaluate specific TG accuracy and precision among laborato-
ries.
98−100
Preparing Initial MN Dispersions. There are multiple
approaches for preparing MN dispersions for aquatic toxicity
testing, such as the use of deionized (DI) water stock
dispersions for spiking test media, sonication of MNs in test
media, and the use of stabilizing agents. The approaches
described in this section relate to the preparation of dispersions
in DI water prior to mixing with the test medium. It is often
easier to produce stable dispersions of MNs in DI water as a
result of the lower ionic strength and thus reduced
agglomeration and settling rates. There are several potential
approaches to disperse MNs in DI water that can be used
individually or in combination: (1) use of commercial
dispersants, capping agents, or solvents; (2) use of NOM;
and (3) sonication of unmodified MNs.
Many MNs are not stable in aqueous media in the absence of
surface coatings or dispersants. When commercial MNs are
synthesized with a dispersant or capping agent, it should be
considered an integral part of the MN; control experiments can
be conducted if it is important to elucidate the impact
(stimulatory or inhibitory) of the dispersant or capping agent
on the assay results.
29
Workshop participants discouraged use
of additional synthetic organic solvents or dispersing agents,
such as tetrahydrofuran (THF) or sodium dodecyl sulfate
(SDS), when dispersing MNs because of their high potential to
confound the results, as thoroughly discussed in previous
papers.
12,19,101−103
However, if commercial products use
synthetic solvents or dispersing agents in the MN formulation,
then the bioassay should be conducted with the product as
produced.
63
Thus, in these cases carefully designed control
experiments (as described in ref 29) are needed to elucidate the
toxicity mechanism and avoid artifacts.
Ubiquitous natural dispersants such as NOM may be
considered with the recognition of their potential to
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significantly alter the MN dispersion stability and toxic-
ity.
31,32,67,104,105
Environmentally relevant concentrations
should be considered;
106,107
however, to maintain a con-
servative approach for hazard assessment, only the lowest
concentration necessary to achieve a stable dispersion should
be used. Workshop participants discussed whether a standard
NOM could be identified or used, but no consensus was
reached. However, it was agreed that control experiments are
essential for understanding the influence of NOM on toxicity.
This topic and discussion are covered in greater detail in the
Supporting Information. Guidance on evaluating the effects of
NOM on polymer toxicity
27
and an existing U.S. EPA
guideline
108
may be of use in addressing this issue for MN.
Dispersion by sonication is implemented in the OECD TG
on MN dispersibility and dispersion stability that is under
development, but sonication is known to generate oxidative
species in solution as well as pyrolysis conditions. A variety of
sonicator types and models exist and differ in power
transformation efficiency and in the way in which the energy
is delivered to the sample (e.g., sonication probes, bath
sonication, and cup-horn sonication). The potential effect of
sonication on the MN surface chemistry and size should be
evaluated, as this procedure has been shown to destroy or
damage CNTs
109,110
if an ice−water bath is not used.
Importantly, sonication may degrade molecules coating
MNs,
111
and in some cases, the sonication process may alter
the toxicity of surface coatings
29,112
or add metal contamination
through disintegration of the sonicator tip.
113
However,
sonication may provide only short-term dispersion of some
MNs, as agglomeration may reoccur after sonication ceases and
during the bioassay.
Different approaches exist for dosing test media with MNs,
such as creating a working stock dispersion for spiking test
media and performing a serial dilution to create test
concentrations or direct addition of the test substance to the
medium to individually prepare each test concentration. If the
agglomerate state of the MNs is not impacted by serial dilution,
the stock dispersion approach may be appropriate; if the state
of the MNs is impacted by dilution, individual preparation of
each concentration should be considered. While the approaches
described thus far relate to the production of a stock MN
dispersion, it may be advisible to follow a different approach if
an MN has more than one potentially toxic component. This
approach, which is typically used for testing of chemical
mixtures because the various components may be present at
differentratiosatdifferent concentrations, involves the
preparation of a separate dispersion for each concentration.
27
One example of MNs with multiple toxic components is CNTs
that release toxic metals from the residual metal catalysts. If a
stock dispersion is made, the concentration of released metal
impurities will be higher in the stock dispersion because
dispersed and settled CNTs will both release toxic metals.
Dilutions made from the stock dispersion to obtain different
dispersed CNT concentrations would have different CNT to
metal ion ratios than if a separate dispersion was made for each
concentration. If the primary toxic effect is driven by the
dissolved metal impurity, a dilution series prepared from this
stock dispersion may produce an acceptable dose−response
curve; however, the effect may be erroneously attributed to the
CNT rather than the impurity. Preparing separate dispersions
for each test concentration helps to distinguish effects due to
the MN from those due to impurities. However, preparing
separate dispersions at low concentrations (<1 mg L−1) could
lead to higher variability in assay results due to the inaccuracy
of weighing small masses.
Preparing Dispersions in Assay Chambers for Organism
Exposure. After stock dispersions or dispersions for each test
concentration are prepared using the procedures described in
the proceeding section, it may be necessary to add the
dispersions to the test medium. If the dispersibility and
dispersion stability TG is used to prepare the dispersion, it is
important to note that the TG is designed to test the stability of
MNs in different aquatic media and not to prepare the best
dispersion for ecotoxicity testing using other OECD methods.
After the addition of dispersed MNs to the test medium,
there are multiple options regarding when to test the
ecotoxicity of the resulting suspension. One approach is to
immediately add the dispersed MN to the test medium. This
approach may minimize the variability among laboratories in
the initial MN dispersion to which the organisms are exposed if
the dispersion procedure is robust. However, the MN settling
rate during the course of the ecotoxicity assay may be quite
variable as a result of factors such as different test media.
An alternative option for unstable MNs is to first add the
dispersed MN to the test medium or to sonicate the sample in
the test medium and then to monitor the MN suspension
stability over time to determine whether, and wait until, a
pseudosteady state is established, at which point the settling
rate has reached a minimum (or acceptable level) or there is no
longer any detectable settling.
27
The MN suspension that has
reached a pseudosteady state could be transferred to test vials
to start the bioassay. However, no consensus was reached in the
workshop on a recommended maximum time limit to reach the
pseudosteady state. Measurements may be needed to assess
whether transferring the suspension causes additional agglom-
eration, settling, and sorption to test containers, resulting in
reduced exposure. Settled material included in bioassays may
also act as a source of dissolved materials or resuspended
particles and potentially alter the system chemistry (e.g.,
oxidation or reduction states).
114
The approach described
above is conceptually similar to water-accommodated fraction
(WAF) methods frequently used in petroleum testing.
28,115,116
Some similarities are that energy is first added to the system
(e.g., by sonication for MNs and by blender mixing or slow
stirring for petroleum) followed by a period of settling for MNs
or separation for petroleum and then collection of the MN
dispersion or WAF, leaving behind the unsuspended material.
In both cases, the goal is to produce repeatable water column
exposures. However, in both cases, physical effects or continued
release of toxic components from the separated material are
excluded from the hazard assessment. For example, physical
effects of petroleum can be significant in oil spills, and Park et
al.
117
demonstrated that removal of settled particles reduced the
toxicity of Ag MNs to D. magna but not Oryzias latipes. Due in
part to the many uncertainties associated with this approach, a
consensus was not reached on the application of WAF
approaches for MN hazard testing. However, it was noted
that WAF approaches are suggested for some difficult-to-test
substances in existing guidance documents.
26
Potential MN Artifacts. When testing the potential
ecotoxicological effects of MNs, a significant complication is
that the MNs themselves may cause artifacts or misinter-
pretations in ecotoxicology assays.
29,118−120
A comprehensive
discussion of the potential artifacts and misinterpretations
inherent in bioassay testing of MNs is provided in a recent
publication
29
and is beyond the scope of this review. Briefly,
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issues such as the use of control experiments, evaluation of
nutrient depletion caused by MNs, interference of the MN with
the assay measurement (e.g., algal density), and inaccurate
dosimetry quantification and metrics need attention in order to
achieve consistent toxicological results. MNs may confound
toxicity measurements by limiting the applicability of common
approaches. For example, a recent study showed that Coulter
counter and hemocytometer measurements of algal density
after exposure to titanium dioxide or gold nanoparticles were
impeded as a result of heteroagglomeration between the algae
cells and the MN; fluorometric methods were found to be the
most suitable.
119
Overall, multiple methods (e.g., Coulter
counter and fluorometric analysis of algae), ideally using
promulgated or standard test methods, should be utilized when
available and careful consideration of relevant control experi-
ments is critical.
■CONSIDERATIONS FOR APPLYING THE 20%
EXPOSURE SPECIFICATION TO TESTING OF MNS
OECD harmonized aquatic toxicity TGs discuss acceptable
limits of variation in water column concentrations and provide
suggestions for approaches to maintain these limits. These are
invariably set at 80% to 120% of the nominal or initial
(immediately upon dosing) measured water column concen-
trations. The TGs vary in specifying whether changes in water
column concentration should be relative to nominal or
measured values. Further, TGs vary in their prescription of
what should be done if the 20% exposure specification is
exceeded. In some TGs, this outcome simply determines
whether exposure−response analyses and reporting can be
based on nominal rather than measured concentration
values.
97,121
In others, the need for more frequent substance
quantification is discussed,
90,122
but neither a specific schedule
for these analyses nor an approach to determine the rate of
concentration change is provided. In other TGs, it is suggested
thattheexposuresystembepreconditioned(tolimit
adsorption), medium renewal intervals be shortened, or
continuous renewal (or flow-through) systems be employed.
It seems implicit in the TGs that variation in excess of ±20%
does not constitute test failure as long as diligent efforts were
made to attempt to maintain consistent exposure, the exposure
was quantified on the basis of measured values, and
measurements were made frequently during a test or medium
renewal period. Beyond the TGs, there are documents
27,123
that
provide some guidance when the 20% exposure specification is
exceeded. These GDs state that if the concentrations remain
within ±20% then the results may be based on nominal or
mean measured values and that if the concentrations deviate by
more than ±20% then the results must be reported on the basis
of measured values (geometric or time-weighted mean). It is
also important to recognize that among these TGs and GDs,
substance losses are generally attributed to their elimination
from test systems (e.g., by volatilization and chemical
degradation processes). In TGs and GDs where substance
losses from the water column (but not from the test system)
are observed (e.g., by settling or physical separation), it is
recommended that insoluble components be removed by
filtration, centrifugation, or other separation methods;
26,27
this
is potentially applicable to MNs on a case-specific basis to
ensure that the worst-case, most conservative hazard result is
generated, but consensus on this approach was not reached by
the workshop participants.
Some advantages and disadvantages of the ±20% exposure
specification are summarized in Table 1. On the basis of the
literature and the experience of workshop participants, it was
concluded that for many MNs, maintaining water column
concentrations within ±20% of the initial concentration during
ecotoxicity assays with or without medium renewal and without
the use of dispersants or solvents is likely to be difficult if not
logistically infeasible, especially at higher concentrations (e.g.,
mg L−1). Even if a stable dispersion is initially prepared, it may
not be possible to maintain consistent exposure if changes in
the state of agglomeration, particle dissolution, and/or some
other transformation of the particles continue to occur during
the bioassay. Examples of rapid decreases in MN concentration
and increases in agglomeration are shown in Figure 1. Clearly,
it is important to consider whether the 20% exposure
specification should be applied to MNs, and this suggests a
need for guidance on how MN losses should be addressed and
reported. Unfortunately, it is unclear from TGs what the basis
or rationale for setting the level at ±20% is, other than the goals
of maintaining stable exposures, facilitating end-point calcu-
lation, and avoiding overlapping exposure concentrations
among treatment levels within a concentration series. Hence,
Table 1. Arguments for and against Implementing the ±20% Test Specification for Aquatic Bioassays Testing Nanomaterials
That Are Not Inherently Stable in Bioassay Test Media; It Was Generally Agreed That Attempts Should Be Made To Maintain
the Concentration
advantages of the 20% test specification challenges related to applying the 20% test specification with MNs
Maintaining high and stable concentrations of
nanomaterials will lead to more reproducible
test results and agreement among laboratories.
Attempting to maintain stable concentrations of MNs that are inherently unstable in water lowers the environmental
relevance and does not account for MN transformation. The worst-case scenario is not achieved if the transformation
product is more toxic than the parent material (e.g., dissolution of metals). It is generally not recommended that the
toxicity of a parent material be tested if its half-life is less than 12 h.
26
Maintaining relatively stable exposure concen-
trations is consistent with the existing risk
paradigm of assessing hazard independently
from exposure. In this paradigm, hazard values
are often interpreted in context with natural
factors that affect fate and exposure.
It is difficult or impossible to maintain the stability of nanomaterials that are not stable in test media. Even if the
concentration is maintained, the state of agglomeration and/or dissolution of the particles would likely change. The use of
dispersants that would assist in maintaining the stability is generally not favored.
9,26
Maintaining stable concentrations facilitates the
calculation of toxicity end points without the
need for weighted averages (or other methods).
Additional logistics added to maintain the stability of unstable MNs (e.g., frequent water exchanges, flow-through conditions,
agitation) are more labor-intensive and expensive, are not tailored to particle delivery (e.g., clogging of tubing), and may
result in repeated tests and increased costs.
Water-accommodated fraction approaches are already recommended for difficult-to-test substances such as partially miscible
petroleum products.
26
This involves testing of the stabilized fraction that is more relevant to water column testing; testing
of the stabilized fraction is expected to allow for a more consistent exposure concentration and thus should better facilitate
the calculation of end points. Excluding settled particles from bioassays may reduce the variability by avoiding confounding
physical effects. However, excluding the settled particles may remove the physical effects and may not facilitate a worst-
case determination of the toxicity.
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it is difficult to assess whether this exposure specification would
be more or less applicable to MNs compared with soluble
chemicals. Regardless of the specific level of acceptable change
in the aqueous concentration, the critical issue is how the MN
concentration (and other metrics such as particle size, particle
count, or surface) should be quantified during testing.
Approaches for calculating toxicity end points if there is a
greater than 20% decrease in the aqueous-phase concentration
are discussed in the Supporting Information.
■DOSIMETRY AND INTERPRETATION
Dosimetry. An inherent hypothesis in nanotoxicology is
that the size-specific properties that make MNs useful for
technology applications will also be important for determining
biological effects.
39,124−131
However, a consensus on the
particle-specific or unique effects that consistently apply to
specific classes of MNs has yet to be reached.
132,133
Various
studies in the ecotoxicology literature have reported higher
toxicities for smaller particles,
134−137
although size-related
toxicity is not always observed.
138,139
It is widely recognized
that the standard mass-only dose metric paradigm used in
toxicology for traditional substances may not adequately
represent exposure−response relationships for MNs.
39,140,141
The mass-only paradigm is further compromised by decreasing
suspended MN concentrations during bioassays, a scenario
where a time-weighted averaging approach more accurately
reflects exposure concentrations but is seldom used in practice.
There are numerous alternative dose metrics for MNs other
than mass; the most commonly discussed are total available
particle surface area and particle number concentration.
140
For
example, Van Hoecke and co-workers
142,143
reported that the
available surface area (m2L−1) of CeO2and SiO2MNs was
better correlated with growth inhibition of algal cells than was
the mass concentration. For some soluble metal MNs (e.g., Ag,
Cu), the dissolved fraction (and dissolution kinetics) in test
media also must be considered in dosimetry determina-
tions.
137,144−146
While some studies have reported that the
toxicological response is correlated with certain MN properties,
it has been difficult to confirm these trends across toxicological
investigations. This is likely in part a result of poor
understanding of how the state of MN exposure differs (e.g.,
different states of polydispersity) among investigations because
of challenges associated with measuring polydisperse MN
suspensions in test media. Furthermore, size-unique effects are
suggested to be most likely to occur below 30 nm,
147
and
therefore, studies focusing on size-related effects above 30 nm
may not isolate particle-specificeffects.
The aerosol science literature has addressed alternative dose
metrics for particles (e.g., see refs 148−151), and several recent
ecotoxicology studies have reported improved dose−response
expression by surface area,
134−136,152
ion release,
136,152
or
particle number.
153
However, the development of a stand-
ardized alternative dose metric for MNs for hazard assessments
is encumbered for a number of reasons: (1) it is unlikely that
any one alternative dose metric will provide an improvement
over mass for all MNs in all test systems; (2) it is more difficult
to directly measure surface areas and particle numbers
compared with mass concentrations at bioassay-relevant
concentrations in bioassay media,
140
although methods are
becoming available;
59
(3) unless size distribution data are
known or measurable, polydisperse particle suspensions in test
media will further complicate the interpretation of exposure
relative to effect; and (4) dynamic changes in dispersion
stability or consistency (suspended concentration, agglomer-
ation, and dissolution) confound concise interpretation and
render dose metric conversions from size and mass less
accurate. Unless the particle number concentration and/or size
distribution are directly measured,
59
the uncertainty in the
surface area and MN number concentrations will be
substantially higher than those based on mass concentrations.
In this context, OECD recommended that particle number,
surface area, and mass should all be measured when feasible to
allow calculation of alternative dose metrics.
23
These measure-
ments should be monitored throughout the test at all test
concentrations to account for concentration-specific changes in
dispersion characteristics.
Interpretation. Bioassays involving exposure to suspended
MNs need to be interpreted on the basis of multiple factors:
their relevance and appropriateness for assessing the tested
MN, the consistency of the exposure (stable concentration,
agglomeration, and dissolution), whether maintaining a
consistent exposure is possible in the bioassay-method-specific
test system, the accuracy of the representation of the exposure
(e.g., whether the frequency of characterization measurements
was sufficient to capture changes in exposure during the
bioassay), whether nanospecific bioassay acceptability criteria
(e.g., sufficiently consistent exposure concentration with respect
to agglomeration and dissolution) are met, and whether the
characterization and monitoring data during the bioassay are
amenable to expressing the data in terms of an alternative dose
metric. If the suspended MNs cannot be maintained within
20% of the starting value within the water phase (with respect
to concentration, agglomeration, and dissolved fraction), it is
difficult to employ any dose metric without complicated and
potentially inconsistent conversions,
150
and a time-weighted
mass approach may be a more expedient option to express
dosimetry. While challenging calculations may be feasible in
research, a more straightforward approach is needed for hazard
and risk assessments. However, most of the historical literature
used to determine regulatory hazard concerns for chemicals are
mass-based and provide a critical benchmark against which to
compare the toxicity of new MNs.
■SEDIMENT TESTING
Many of the considerations previously discussed for water
column testing are relevant to sediment tests, with the notable
exception that there is no need to remove insoluble test
material according to standard assay protocols.
85,154
While the
latter is a major conceptual difference between tests of MN and
traditional chemicals with pelagic organisms, it is not an issue in
sediment testing. Some added complications are that MN
interactions in sediments can significantly alter the MN
properties, and methods for quantifying concentration or
other MN characteristics in sediments are very limited.
However, in view of the fact that most MN suspensions are
generally not stable in environmentally relevant water
chemistries (Figure 1), there was consensus from the expert
workshop that consideration of sediment exposure and hazard
is relevant and in many cases more representative of
environmental exposure than aqueous tests. Current sediment
toxicity standard methods acknowledge significant uncertainty
regarding test substance homogeneity, exposure, bioavailability,
and synergisms. Thus, poorly understood bioavailability issues
are commonplace in sediment testing and are not unique to
nanoecotoxicology. An evaluation of available standardized
sediment bioassay methods (OECD, EPA, ASTM, etc.)
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Table 2. Summary of Major Issues Discussed by Workshop Participants Where Consensus Was Reached or Was Not Reached and Research Recommendations To Fill
Knowledge Gaps That Prevented Consensus
issue consensus items from the workshop items lacking consensus key research recommendations to address items lacking consensus
Feasibility of
considering
hazard and
exposure sep-
arately for
MNs
The focus of the guidance document is to increase
the consistency of bioassay results used for hazard
assessment. However, dispersion stability must be
considered in bioassay method selection and
monitoring. Effort should be made to maintain a
consistent MN concentration when logistically
feasible.
Designating a limit of acceptable exposure variability
either at 20% (the ±20% test specification) or some
other level over the duration of the bioassay.
Approaches for maintaining MNs in suspension (e.g., frequent medium renewal, flow-through delivery,
and test medium modifications) should be studied. Testing of flow-through systems should consider the
potential for increased MN concentrations in the test system resulting from settled material not removed
from chambers. It should be determined whether maintaining stable concentrations reduces variability in
test results when agglomeration and dissolution cannot be avoided. Time-weighted averaging and more
complex approaches to express variable exposures should be investigated. The extent to which settled
MNs influence ecotoxicity results should be determined.
Research could also focus more broadly on quantifying the uncertainties that arise when exposure varies
beyond specific thresholds (including ±20%).
Dispersion
methods
It is acceptable to disperse MNs in either working
stocks (for spiking biological media) or to disperse
MNs directly in the test media. Working stocks
should be used only if there is a single substance in
the MN that exerts toxicity. The optimal method
will be contingent on target concentration,
medium, and bioassay method selection.
Addition of
substances to
enhance MN
dispersion
Dispersants should not be used to prepare nanoma-
terial suspensions for biological testing unless they
are present in the (commercial) product formula-
tion. Natural organic matter (e.g., humic acid) may
be used as a dispersant; however, control experi-
ments are essential to understand the influence of
NOM on toxicity.
9
The type of natural organic matter to recommend. Impacts of different types of natural organic matter on MN stability and toxicity testing results should be
investigated.
Modifications of
methods to
address MN
instability
Water column bioassays should be conducted to
maintain consistency with chemical hazard assess-
ment practices. However, alternative water column
bioassay designs should be considered for very
unstable MNs.
Whether to allow particle agglomeration, settling, and
dissolution kinetics to come to equilibrium before
adding test organisms. It was agreed this could be
presented as an option for nondispersible materials
along with caveats.
Whether effects such as inducing turbulence and flow-
through systems should be employed to maintain
particle concentration.
The reproducibility of test results when initial suspensions versus pseudosteady-state suspensions are
tested, the relative impact of chemical versus physical effects on MN toxicity, and the impact of
approaches (turbulence and flow-through systems) to maintain particle concentration on MN toxicity
should be assessed.
Standard test
media and test
chambers
One standard exposure chamber and test medium for
each OECD test method/organism should be
recommended for MNs to maximize test consis-
tency. If the test medium is modified (relative to
current practice), a positive control test with a
reference toxicant in the modified medium is
recommended.
Whether it is acceptable to modify standard media to
increase particle stability and ultimately maintain MN
concentration. pH adjustments away from the iso-
electric point (within biological limits) are more
acceptable. However, there was concern that ionic
strength dilutions could impact animal health and
decrease comparability with historic data sets.
Research to support the development of a single test medium for each TG that would lead to the most
reliable ecotoxicity results for MN testing should be carried out. Studies should quantify acceptable
thresholds for maintaining organism health and environmental relevance.
Different types of test containers (size, type of material, geometry) should be tested to assess the
robustness of the different TGs with regard to this parameter. The impact of the agitating medium should
be evaluated for tests requiring agitation, such as the algae growth inhibition test.
90
While using standard
exposure chambers may increase hazard data consistency, the utility of chamber modifications for the
purpose of environmental risk assessment needs further consideration.
Expressing and
interpreting
dosimetry
Preliminary testing is recommended to determine
particle stability in the specific test system and
biological test medium prior to organism testing to
inform test design, characterize the monitoring
frequency, and reduce animal use by reducing the
number of unsuccessful or unacceptable tests.
Establishing a standard dose metric and reliable
analytical techniques for monitoring MNs. Without
readily available direct measurement methods, it will
be difficult to relate dose response to surface area or
particle number metrics for heterodispersed suspen-
sions of MNs that are unstable in biological media
over time.
It is important to develop, validate, and standardize analytical methods to directly measure particle
number concentrations and size distributions in aqueous samples at toxicologically relevant
concentrations (sometimes low μgL
−1). Best practices for calculating exposure−response values also need
to be developed.
Sediment toxic-
ity testing
Sediment toxicity tests are most relevant for MNs
that are unstable in the medium.
Whether very unstable MNs should be tested only in
sediments (i.e., no water column testing).
Characterization methods for particles in the complex sediment matrix should be developed, especially for
carbon-based MNs. For metal and metal oxide MNs, the development of methods to differentiate
between MNs, dissolved metal ions, and MN agglomerates is needed. Dosing directly to the sediment
versus indirectly dosing the sediment through the overlying water (for a surficial sediment exposure) and
the associated impacts homogeneity and toxicological results should be investigated.
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suggested that the test end points assessed in these methods
will contribute valuable MN hazard information.
13
While it may
not be currently feasible to rigorously characterize many types
of MNs present in sediment, the consistency of sediment
toxicity bioassays can still be generally improved by
implementing standards for particle preparation, dispersion,
spiking, and equilibration in sediment.
11
Further, the use of a
standardized (e.g., OECD) freshwater sediment in MN spiking
studies would reduce variability in bioassay results relative to
the use of field-collected sediments because sediment-specific
factors (e.g., organic carbon concentration) that can influence
toxicity assay results are controlled. This discussion is divided
into different important topics for MN sediment toxicity
testing: (1) methods for consistently spiking sediment, (2)
equilibration time, and (3) sampling and analysis of MNs in
sediments during and after the test.
Methods for Spiking and Determining Homogeniza-
tion. Spiking of aquatic sediments is generally expected to be
more consistent in terms of homogeneity if the materials are
predispersed into relevant water according to standardized
methods rather than adding dry MNs to sediment.
12,23
This is
related to general difficulties regarding homogenizing chemicals
into sediments.
155
If a MN is added to sediment in powder
form (undispersed), it is likely that substantial clumping of
particles within the sediment would occur, resulting in greater
heterogeneity and therefore greater variability between bioassay
test replicates.
11
As previously discussed, the use of a standardized sediment
in MN spiking studies would likely lead to more comparable
results than the use of field-collected sediments. Two
alternative MN spiking methods have been discussed and
used for sediment MN toxicity testing: (1) direct addition of
dispersed MNs to the sediment followed by homogeniza-
tion
37,156,157
and (2) indirect addition of MNs to the overlying
water, followed by subsequent settling of the MN to the
surficial sediment.
12,158,159
In the literature, the direct addition
method is much more frequently used. Selection of one (or
both) of these methods may relate to the test objectives, study
system, or functional ecology of the organism used in the test
or at the site of concern. For instance, a testing laboratory may
elect to use the direct addition method for an infaunal deposit-
feeding organism, which will feed on sediment below the
sediment surface, while the indirect method may be desirable
for an epibenthic surface-deposit-feeding or filter-feeding
organism, which will interact to a substantially larger degree
with the sediment directly below the water−sediment interface.
Research is needed to determine how to most consistently
spike sediments (e.g., mixing method, duration) by these two
spiking strategies so that particles are dispersed throughout the
sediment as homogeneously as practical to increase the inter-
replicate reliability. Additionally, research is needed to better
understand how water exchanges, which are typically performed
during longer-term sediment toxicity tests, may impact MN
concentrations and distributions within or on the surface of the
sediment.
Equilibration Time To Reach a Pseudosteady State
after Spiking with MNs. It is well-known that the time
required to reach a quasi-steady state by equilibrium
partitioning for spiked sediment studies is important for
determining bioavailability, especially for hydrophobic com-
pounds that take a long time period (weeks to months) to
approach pseudoequilibrium in sediments.
155
Thus, 2 weeks
160
to 4 weeks
161,162
on a roller mill is a typical equilibration time
to allow interactions between the spiked compound and ligands
to approach some level of steady state. However, currently
available OECD sediment spiking methods recommend 48 h of
equilibration.
85,154,163
As reflected by recommended ASTM and
EPA equilibration mixing times, a 48 h duration, while
convenient, does not allow adequate equilibration-reaction of
metals in spiked sediment
164
but may provide a worst-case
scenario in terms of greater MN bioavailability. While selection
of equilibration times may be contingent on experimental
objectives, research is needed to determine how interactions of
MNs with sediment may change over time in order to
determine the optimal equilibration time prior to test organism
addition and exposure.
Sampling and Analysis. While current gaps in methods
for MN characterization may limit the determination of particle
characteristics following spiking into sediment, certain measure-
ments may still be performed, such as the use of ICP-MS to
determine the total elemental concentration for metal and
metal oxide MNs. It is practical to take samples for such
measurements from the whole sediment, sediment porewater,
and overlying water at test initiation and termination, as
recommended in current OECD sediment testing guidance;
however, MN-specific modifications of porewater separation
methods may be needed to yield accurate results.
■WORKSHOP FINDINGS
While the findings discussed in this workshop primarily pertain
to issues related to the applicability of OECD aquatic toxicity
TGs, many of the findings also more widely apply to test
methods for other documentary standards agencies (e.g., ISO
and ASTM) and for terrestrial organism testing, academic
research, and regulatory decisions. The discussion of the
workshop participants led to both convergent and divergent
opinions on how the major issues impacting the consistency,
environmental relevance, and accuracy of aquatic bioassay
results should be handled in aquatic toxicity testing. To the
extent possible, it is desirable to minimize the amount of
developmental work performed by commercial testing
companies, such as assessing which procedure should be used
to disperse MNs in the test medium or designing a complicated
system to comply with the ±20% test specification. A summary
of issues for which workshop participants both achieved and
failed to achieve consensus is summarized in Table 2; where
consensus was not achieved, targeted research studies are
recommended in the table. The proposed research is designed
to support the development of precise guidance for conducting
OECD aquatic toxicity tests that will simplify this process for
commercial testing laboratories and to help regulators interpret
the results through the OECD aquatic toxicity testing GD to be
developed following this paper.
The workshop participants agreed that it can be acceptable to
disperse particles in either working stocks (for spiking test
media) or dispersing MNs directly into test media, as described
above. The optimal method will be contingent on the
physicochemical properties of the MN, the target concen-
tration, the medium, and the bioassay method selected, and
preliminary data should be gathered prior to decision making.
Synthetic dispersants should not be used to prepare MN
suspensions for aquatic toxicity testing; however, if they are part
of the (commercial) product formulation, then the bioassay
should be conducted with the as-produced material. This
recommendation aligns with previous aquatic toxicity test
guidances.
26,123,165
Natural dispersants such as dissolved
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organic carbon (i.e., humic acid) may be relevant, but their
impact on the toxicity of MNs should be considered (e.g., for
metal MNs); the total organic carbon concentration should be
within the range of surface waters. Additionally, while particle
stability is likely to be an issue, water column bioassays should
be conducted with the goal of maintaining exposure
consistency to abide by chemical hazard assessment practices
(e.g., REACH
28
). However, alternative water column bioassay
designs or sediment exposures should be considered for very
unstable MNs, adapting guidance described in the difficult
substances document.
27
For aquatic toxicity bioassays with
MNs, an exposure chamber with consistent dimensions and
one test medium for each OECD test method/organism is
desirable for MNs to increase test consistency. Standard testing
end points and numbers of test replicates should be applicable
to MN testing. Some preliminary but nonexhaustive exper-
imentation to determine particle stability in the test medium
prior to organism testing would be informative for test design
and reducing animal use in unsuccessful tests.
While the workshop participants did not come to consensus
on whether the 20% test specification in the water column can
be consistently applied for MNs, the group agreed that an effort
should be made to maintain the concentration when logistically
feasible. Consensus was not reached on whether turbulence or
flow-through systems should be employed to maintain the
particle concentration. Also, no consensus was reached on
whether to allow particle agglomeration, settling, and
dissolution kinetics to come to equilibrium before adding test
organisms, as related to WAF testing. While some workshop
participants agreed that a pseudosteady state (or constant
concentration) was likely to lead to greater test reliability and
repeatability, there were divergent opinions on allowing a
pseudosteady state to be reached and removal of the settled
fraction of particles, as they may not offer a worst-case scenario;
it should be noted that a pseudosteady state may not occur in
the aqueous phase for some MNs (e.g., complete settling from
suspension or continual ion release due to adsorption to
container or ligand surfaces). No consensus was reached on
whether altering standard media to increase the particle stability
and ultimately maintain the concentration was acceptable.
While pH adjustments away from the isoelectric point (within
biological limits) were generally more acceptable, there was
concern that ionic strength dilutions would impact animal
health and decrease comparability with historic data sets. While
consensus was not reached on these items, suggestions for
future research to help resolve the lack of consensus are
provided in Table 2. Additional suggestions for future research
to support more definitive suggestions for modifications to
OECD aquatic toxicity test methods are provided in Table S1
in the Supporting Information; the research topics in Table S1
are categorized by section of the review, while those in Table 2
are provided for each area for which consensus was not
reached.
Following the consensus in Table 2 will help to substantially
improve the reliability and data quality of nanoecotoxicology
research and provide substantive improvements for regulatory
testing. Facilitating the aquatic toxicity testing of MNs using
standardized methods will help MN risk assessments to be
conducted more efficiently. This will potentially allow MN-
enabled products to reach the market in a shorter time period,
allow registrants to improve the quality of data for fulfilling
regulative information requirements, and promote green
product design by identifying MNs with potentially significant
toxicological effects or with the potential to design more benign
alternatives early in the development stages.
■ASSOCIATED CONTENT
*
SSupporting Information
Supplemental discussions of definitions and measurements of
“dissolved”substances, which type of NOM to recommend for
aquatic toxicity testing, and the impact of calculating toxicity
end points where the 20% specification is not achievable and a
table describing key additional research topics for each section
of this review. The Supporting Information is available free of
charge on the ACS Publications website at DOI: 10.1021/
acs.est.5b00997.
■AUTHOR INFORMATION
Corresponding Author
*E-mail: Alan.J.Kennedy@usace.army.mil;phone:601-634-
3344; fax: 601-634-2263.
Notes
The views expressed in this article are solely those of the
authors and do not reflect official policies or positions of the
Department of Defense, the U.S. Environmental Protection
Agency, the National Institutes of Standards and Technology,
or the Organization for Economic Cooperation and Develop-
ment. Certain commercial equipment, instruments, or materials
are identified in this paper to foster understanding. Such
identification does not imply recommendation or endorsement
by the National Institute of Standards and Technology or the
U.S. Environmental Protection Agency, nor does it imply that
the materials or equipment identified are necessarily the best
available for the purpose.
The authors declare no competing financial interest.
+
Retired from the EPA. E-mail: phil.sayre@verizon.net.
■ACKNOWLEDGMENTS
We acknowledge the significant contribution of workshop
attendees who did not contribute to the writing of this paper:
Zachary Collier, Teresa Fernandez, JeffGallagher, Richard
Handy, Dale Hoff, Amuel Kennedy, Laura Nazef, Jeffery
Steevens, Norishisa Tatarazako, David Tobias, Doris Voelker,
and Kathrin Schwirn. Frank von der Kammer is thanked for
giving a presentation at the workshop. Permission to publish
this material was granted by the U.S. Army Corps of Engineers
Chief of Engineers and by the U.S. EPA Office of Pollution
Prevention and Toxics. A portion of this work was funded
through the Army Environmental Quality and Installations
(EQI) Technology Research Program (Dr. Elizabeth Ferguson,
Technical Director) for the U.S. Army Engineer Research &
Development Center (ERDC). J.L. acknowledges financial
support from the SmartState Center for Environmental
Nanoscience and Risk. G.G.G. acknowledges financial support
from Environment Canada, the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC), and the National
Institute of Nanotechnology. N.B.H. received funding from the
Danish Environmental Protection Agency.
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