EPA/600/R-09/065
September 2009
Review of OECD/OPPTS-Harmonized and
OPPTS Ecotoxicity
Test Guidelines for Their Applicability to
Manufactured Nanomaterials
by
Steve Diamond, Workgroup Chair, USEPA Office of Research and Development,
National Health and Environmental Effects Research Laboratory, Mid-Continent
Ecology Division
Dennis Utterback, Workgroup Organizer, USEPA Office of Science Policy
Christian P. Andersen, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Western Ecology Division
Robert Burgess, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Atlantic Ecology Division
Seishiro Hirano, National Institute for Environmental Studies, Japan
Kay Ho, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Atlantic Ecology Division
Chris Ingersoll, Columbia Environmental Research Center, U.S. Geological Survey
Mark G. Johnson, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Western Ecology Division
Alan J. Kennedy, U.S. Army, Engineer Research and Development Center Environmental
Laboratory
David R. Mount, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Mid-Continent Ecology Division
John Nichols, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Mid-Continent Ecology Division
Pascal Pandard, INERIS, France
Paul Rygiewicz, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Western Ecology Division
Janeck J. Scott-Fordsmand, National Environmental Research Institute, Denmark
Kath Stewart, AstraZeneca UK Limited, UK
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Mid-Continent Ecology Division
Duluth, MN 55804-2595
Recycled/Recyclable
printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine free.
-------
EPA/600/R-09/065
September 2009
Review of OECD/OPPTS-Harmonized and
OPPTS Ecotoxicity
Test Guidelines for Their Applicability to
Manufactured Nanomaterials
by
Steve Diamond, Workgroup Chair, USEPA Office of Research and Development,
National Health and Environmental Effects Research Laboratory, Mid-Continent
Ecology Division
Dennis Utterback, Workgroup Organizer, USEPA Office of Science Policy
Christian P. Andersen, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Western Ecology Division
Robert Burgess, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Atlantic Ecology Division
Seishiro Hirano, National Institute for Environmental Studies, Japan
Kay Ho, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Atlantic Ecology Division
Chris Ingersoll, Columbia Environmental Research Center, U.S. Geological Survey
Mark G. Johnson, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Western Ecology Division
Alan J. Kennedy, U.S. Arrny, Engineer Research and Development Center Environmental
Laboratory
David R. Mount, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Mid-Continent Ecology Division
John Nichols, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Mid-Continent Ecology Division
Pascal Pandard, INER1S, France
Paul Rygiewicz, USEPA Office of Research and Development, National Health and
Environmental Effects Research Laboratory, Western Ecology Division
Janeck J. Scott-Fordsmand, National Environmental Research Institute, Denmark
Kath Stewart, AstraZeneca UK Limited, UK
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Mid-Continent Ecology Division
Duluth, MN 55804-2595
Recycled/Recyclable
Printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine free.
-------
Notice
The information in this document has been funded in part by the U.S. Environmental
Protection Agency. It has been subjected to review by the National Health and
Environmental Effects Research Laboratory and approved for publication. Approval does not
signify that the contents reflect the views of the Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
Acknowledgements
The authors thank Dr. Christopher A. Impellitteri and Dr. Kim Rogers for their thoughtful
and insightful review of an earlier draft of this report and Mary Ann Starus for her thorough
and expert assistance with grammar and formatting.
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EXECUTIVE SUMMARY
Assessing the environmental risk of manufactured nanomaterials (MNs) presents a
significant and growing challenge for environmental regulators. These materials, defined as
having at least one physical dimension between 1 and 100 nm are being developed, produced,
and incorporated into a broad range of commercial, medical, environmental, and other
products (USEPA 2007). The challenge for regulators derives in part from this rapid pace of
MN development, but also because early evidence suggests that nanomaterials can be created
in nearly unlimited variations in size, form, elemental composition, and the addition of
functional groups (Colvin 2003, USEPA 2007). While the regulatory challenge of ever-
growing numbers of substances is not unique relative to soluble chemicals, MNs also present
several additional and novel challenges due to their particulate or fibrous properties. First, at
sizes smaller than approximately 100 nm, MNs begin to exhibit behaviors and properties that
are not apparent in their bulk forms, including electrical conductivity, elasticity, greater
strength, different color, and greater reactivity (Parak et al. 2005). These novel properties are
due to quantum effects that become dominant at the nanometer scale (most likely the lower
range, approximately 10 to 20 nm). Smaller particle size enhances this phenomenon
indirectly because total surface area for a given volume of material increases as a square
function of decreasing particle size. Hence any quantum effects, assuming they are related to
surface area, will have a higher probability of altering biological systems as surface area
increases. It should also be noted that increased surface area will increase the probability of
interactions that are related to bulk material properties as well, aside from any quantum
effects.
A more immediate concern for regulators is what sort of guidance should be given to
potential registrants on how nanomaterials should be tested. Generally, that guidance is
provided by standard test guidelines within the USEPA, Office of Prevention, Pesticides and
Toxic Substances (OPPTS), Series 850 Ecological Effects Test Guidelines
(http://www.epa.gov/opptsfrs/pubIications/OPPTS_Harmonized/850_EcologicaI_Effects_Tes
^Guidelines/). However, given the unique properties of nanomaterials, the applicability and
adequacy of these test guidelines is questionable (Crane et al. 2008, Hansen 2009). To
address that issue a workgroup was formed to evaluate the Series 850, as well as other,
similar test guidelines, for their adequacy for testing nanomaterials. The workgroup was
comprised of 14 international scientists with expertise in ecotoxicity testing and
nanotechnology. This report summarizes the results of the review process, identifies specific
areas where test guidelines are adequate or inadequate, and provides some recommendation
for regulatory testing of nanomaterials.
The general conclusion of the workgroup is that none of the current ecotoxicological test
guidelines reviewed are adequate for testing MNs. The breadth of the review, inclusion of
selected, non-Series 850 guidelines, and the nature of the inadequacies, suggests that this is
true of essentially all test guidelines (no colloid-specific toxicity test methods were
identified). This is not to say that many aspects of the guidelines aren't adequate, but rather
that any hazard testing undertaken with full adherence to the current guidance and without the
addition of many critical nanomaterial-specific measurements and exposure approaches will
yield data insufficient to reliably assess the hazard of nanomaterials. All of the inadequacies
identified by the reviewers are directly related to the fact that MNs are generally particulate
or fibrous and occur as colloidal suspensions in aqueous exposure media (including
suspensions generated for wet application to, or mixing with, non-aqueous exposure media).
iii
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Methods and approaches for preparing exposure media, as well as measuring and
characterizing materials both prior to testing and in prepared exposure media are absent in all
test guidelines. Much of the terminology used in current test guidelines is specific to soluble
chemicals and is either wholly inadequate for particulate and fibrous substances or not fully
descriptive. An excellent example of this is the use of terms such as dissolved, solution, and
concentration. The latter term, in particular, is used to describe exposure levels and exposure
response; it is probable that endpoint responses will relate directly to additional factors such
as surface area, particle size and count, and other nanomaterial properties. This also suggests
that the terminology and metrology of exposure-effects relationships (e.g., LC50, EC50,
NOEC, LOEC) are also not applicable (without some modification) for nanomaterials. The
term dissolved is of specific concern because some nanomaterials are known to exist in media
both as particles, and as ions that truly dissolve from the particles (e.g., nano-scale silver,
[Benn and Westerhoff 2008]). However, the particles themselves are best described as being
in suspension, so use of the term dissolved could lead to errors in interpretation of actual
material exposure levels.
The review workgroup found that two aspects of current test guidelines are fully adequate
for testing MNs. The first aspect is the toxicological principles inherent in all test guidelines,
including use of healthy, viable organisms, incorporation of appropriate control treatments,
selecting exposure levels, etc. The second aspect is the endpoints targeted in the test
guidelines, and the species tested. In general, these endpoints, including survival,
reproduction, growth, and others, are integrative of multiple mechanisms of toxicity, and
should be as reflective of MNs toxicity as they are of soluble chemicals and formulations. It
should be noted however that exploratory research may reveal nanomaterial-specific
endpoints that, to be incorporated into regulatory testing, might require modification of
existing, or drafting of new, test guidelines.
Specific, interim suggestions of the review group are:
1) Development of a nanomaterials-specific guideline document that would address the
inadequacies common to all, or most guidelines. As discussed in the review, the
Organization for Economic Co-operation and Development (OECD) guidance
document on testing difficult substances (Guidance Document on Aquatic Toxicity
Testing of Difficult Substances and Mixtures [ENV/JM/MONO(2000)6]) provides an
excellent framework for development of such a document.
2) Existing guidelines could be used to define required tests, but the inadequacies would
need to be identified and approaches to addressing them stipulated to registrants. This
represents an interim solution similar to generating the separate guidance document
suggested above and would necessitate a case by case approach, but could be done
immediately whereas generation of the new guidance document would require a
significant period of development time.
3) It is likely that initial testing on MNs will require a more exploratory approach. For
example, minor variation in test water chemistry and methods used to generate
suspensions (including serial dilution) can cause significant variation in the as-tested
properties of MNs. For these reasons, it is recommended that some investigation and
quantitation of these effects be required.
IV
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4) Some consideration should also be given to material availability, which may be
limited and preclude the use of flow-through or large-volume exposure approaches.
Also, some brief discussion of the unique health and safety issues (e.g., their
dustiness, potential ability to pass through commonly used laboratory gloves,
ventilation from laboratory hoods, etc.) associated with nanomaterials should be
included.
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INTRODUCTION
Manufactured nanomaterials (MNs) present unique challenges for toxicity testing
compared with most soluble chemicals and substances. NM are particles or fibers and when
placed in wet media typically form colloidal suspensions (of varying stability). Despite the
fact that nanomaterials (particles having at least one dimension between 1 and 100 nm) might
pass through a 0.45 jim filter, and thus meet the widely-accepted definition of a soluble
substance, their behavior is clearly distinct from a "truly" soluble substance, e.g., a metal ion
or an organic molecule (Lead and Wilkinson 2006). Many suspended nanoparticles also have
a strong tendency to agglomerate or aggregate, resulting in the formation of larger particles,
or clusters of particles that rapidly settle out of suspension. In addition to the issue of
exposure consistency during testing, particle-size variation may also alter the toxic potency
of materials, in part because available surface area is rapidly reduced as agglomeration or
aggregation occurs, but also because unique, quantum effects can predominate at sub-100 nm
sizes (Parak et al. 2005). These characteristics are an inherent function of the material itself,
but are also strongly affected by very small changes in ionic strength (perhaps to ionic
composition as well), pH, dissolved organic matter (French et al. 2009, Domingos et al.
2009), and even the rate at which dilution media is added to more concentrated media to
produce the concentration range necessary for exposure-response analysis (Former et al.
2006).
The purpose of this brief introduction is not to present an in-depth overview of
nanomaterials and their toxicity. Rather it is intended to describe how the unique nature of
nanomaterials presents problems for regulatory ecotoxicity testing. It should be clear from the
brief discussion above, that test guidelines that limit the discussion of test media preparation
to truly soluble substances cannot be expected to provide sufficient guidance for the
preparation of stable colloidal suspensions. The common thinking among toxicologists,
reflected in all of the reviewers' comments, is that nanomaterial toxicity is likely to be
strongly related to particle size, surface area, possibly surface charge, bulk concentration, and
additional factors that will likely be revealed in exploratory research (Handy et al. 2008,
Klaine et al. 2008). Aside from bulk concentration, these factors are not considered in
assessing risk for soluble substances, nor are they recognized in current test guideline
language. In many cases, measurement techniques have yet to be developed (e.g., surface
area in wet media) or the currently-used methods yield different results (e.g., electron
microscopy and light scattering techniques often yield significantly different particle size
values). Conversations among scientists working in this nascent field often begin with a
discussion of what is meant by the term soluble: a sub-0.45 um particle, or an ion that
actually dissolves from a larger particle; or whether an agglomerate is a particle, or is
comprised of individual, discrete, as-produced particles.
In this summary we discuss these and other MN toxicity testing difficulties, and whether
current test guidelines adequately address these issues. The reviews on which this summary is
based were undertaken with the understanding that the specific goal was to address the issue
of test guideline adequacy and to identify specifically where and how the guidelines might be
inadequate. No effort was made to suggest specific alterations of guidelines to improve their
adequacy.
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TEST GUIDELINES REVIEWED
Two broadly overlapping sets of guidelines, listed in Tables 1 and 2, were reviewed in
two distinct phases. The first set was comprised of 24 ecotoxicity test guidelines promulgated
by the Organization for Economic Co-operation and Development (OECD) and closely
harmonized with test guidelines within EPA's OPPTS Series 850. The OECD guidelines
were reviewed by an international group of scientists at the request of the OECD's Working
Party on Manufactured Nanomaterials (WPMN). The harmonization of OECD and OPPTS
guidelines has resulted in nearly identical method descriptions; thus the OECD reviews were
directly applicable to many OPPTS guidelines. In the second phase of the process, 25 OPPTS
guidelines were reviewed by a group of U.S. scientists from EPA, U.S. Geological Survey,
and the U.S. Army Corp of Engineers (Engineer Research and Development Center). The
participation of several of the reviewers in both phases of the process contributed to the
continuity and comparability of reviews and the summary process. The review of the OECD
test guidelines was summarized in a final report delivered to the WPMN in March of 2008.
That report comprises a large portion of the present document, with the addition of findings
and observations unique to the OPPTS test guidelines.
In addition to the 24 OECD test guidelines, reviewers in the first phase also evaluated an
OECD document on testing difficult substances (Guidance Document on Aquatic Toxicity
Testing of Difficult Substances and Mixtures [ENV/JM/MONO(2000)6]) and selected test
guidelines from Environment Canada, The Ministry of the Environment, Japan, and the
International Organization for Standardization (ISO). The documents were added to the
review process to provide a survey of non-OECD guidelines that might provide a framework,
terminology, or guidance more directly applicable to nanomaterials. The review of these
additional documents is summarized near the end of this report.
Review Process
The OECD and OPPTS (as well as the additional documents) provide guidance for testing
substances for adverse effects on biota. These test guidelines examine effects in all
environmental media (aquatic, terrestrial, sediments, and sludges). They address a variety of
vertebrate, invertebrate, and microbial taxa, and include both acute and chronic tests. The
tests also include both mortality and non-lethal endpoints, e.g., growth, plant vigor, and
respiration. These guidelines have each been evaluated by at least one reviewer and in many
cases by two or three reviewers. The review process involved initial development of a
template for review. This template was simply a section-by-section document that provided
space for reviewers to describe inadequacies (for testing nanomaterials) of each test guideline
section. Subsequent to completion of the OECD reviews, the review group evaluated the
OECD's guidance document on testing difficult substances (Guidance Document on Aquatic
Toxicity Testing of Difficult Substances and Mixtures [ENV/JM/MONO(2000)6]). This
additional review was undertaken in response to a common finding in the test guideline
reviews — that guidance on delivery of substances to test systems was, in all cases, inadequate
for nanomaterials. One approach to addressing this shortcoming is to modify or develop a
single document that describes approaches for delivering nanomaterials in a variety of media
and test systems. A brief review and suggestions for modification, of the Difficult Substances
document is presented at the end of this document. The OECD review group also briefly
reviewed five non-OECD test guidelines in an effort to identify documents that might inform
the nanomaterial-specific test guideline revision or development process. These reviews are
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also summarized at the end of this document. The review of OPPTS test guidelines was
completed using the same procedure.
Table 1. Reviewed OECD ecotoxicity test guidelines.
Guideline
Identification
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
224
227
Description of Test
Alga, Growth Inhibition Test
Daphnia sp. Acute Immobilisation Test
Fish, Acute Toxicity Test
Fish, Prolonged Toxicity Test
Avian Dietary Toxicity Test
Avian Reproduction Test
Earthworm, Acute Toxicity Tests
Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test
Activated Sludge, Respiration Inhibition Test
Fish, Early-Life Stage Toxicity Test
Daphnia magna Reproduction Test
Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages
Honeybees, Acute Oral Toxicity Test
Honeybees, Acute Contact Toxicity Test
Fish, Juvenile Growth Test
Soil Microorganisms: Nitrogen Transformation Test
Soil Microorganisms: Carbon Transformation Test
Sediment- Water Chironomid Toxicity Using Spiked Sediment
Sediment- Water Chironomid Toxicity Using Spiked Water
Enchytraeid Reproduction Test
Lemna sp. Growth Inhibition Test
Earthworm Reproduction Test (Eisenia fetida'Eisenia andrei)
Determination of the Inhibition of the Activity of Anaerobic Bacteria Reduction of Gas
Production from Anaerobically Digesting (sewage) Sludge
Terrestrial Plant Test: Vegetative Vigour Test
Table 2. Reviewed OPPTS ecotoxicity test guidelines. Guidelines that were not reviewed by
the OPPTS reviewers because of their comparability with previously-reviewed OECD
guidelines are indicated with the comparable OECD identification. Those guidelines that
were not reviewed are indicated with NR.
Guideline
Identification
850.1000
850.1010
850.1020
850.1025
850.1035
850.1045
850.1055
850.1075
850.1085
850.1300
850.1350
850.1400
850.1500
850.1710
850.1730
Description of Test
Special Considerations For Conducting Aquatic Laboratory Studies
Group A — Aquatic Fauna Test Guidelines
Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids
Gammarid Acute Toxicity Test
Oyster Acute Toxicity Test (Shell Deposition)
Mysid Acute Toxicity Test (Shrimp, s.w.)
Penaeid Acute Toxicity Test (Shrimp)
Bivalve Acute Toxicity Test (Embryo Larval)
Fish Acute Toxicity Test, Freshwater and Marine
Fish Acute Toxicity Mitigated By Humic Aid
Daphnid Chronic Toxicity Test
Mysid Chronic Toxicity Test
Fish Early-Life Stage Toxicity Test
Fish Life Cycle Toxicity
Oyster BCF
Fish BCF
OECD
Review
**
202
^3
211
210/212
204/215
OPPTS
Review
X
X
X
X
X
X
X
X
X
l__
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Guideline
Identification
850.1735
850.1740
850.1790
850.1800
850.1850
850.1900
850.1925
850.1950
850.2100
850.2200
850.2300
850.2400
850.2450
850.2500
850.3020
850.3030
850.3040
850.4000
850.4025
850.4100
850.4150
850.4200
850.4225
850.4230
850.4250
850.4300
850.4400
850.4450
850.4600
850.4800
850.5100
850.5400
850.6200
850.6800
850.7100
Description of Test
Whole Sediment Acute Toxicity Invertebrates, Freshwater
Whole Sediment Acute Toxicity Invertebrates, Marine
Chironomid Sediment Toxicity Test
Tadpole/Sediment Subchronic Toxicity Test
Aquatic Food Chain Transfer
Generic Freshwater Microcosm Test, Laboratory
Site-Specific Aquatic Microcosm Test, Laboratory
Field Testing for Aquatic Organisms
Group B — Terrestrial Wildlife Test Guidelines
Avian Acute Oral Toxicity Test
Avian Dietary Toxicity Test
Avian Reproduction Test
Wild Mammal Acute Toxicity
Terrestrial (Soil-Core) Microcosm Test
Field Testing for Terrestrial Wildlife
Group C — Beneficial Insects and Invertebrates Test Guidelines
Honey bee Acute Contact Toxicity
Honey Bee Toxicity of Residues On Foliage
Field Testing For Pollinators
Group D—Nontarget Plants Test Guidelines
Background — Nontarget Plant Testing
Target Area Phytoloxicity
Terrestrial Plant Toxicity, Tier I (Seedling Emergence)
Terrestrial Plant Toxicity, Tier I (Vegetative Vigor)
Seed Germination/Root Elongation Toxicity Test
Seedling Emergence, Tier II
Early Seedling Growth Toxicity Test
Vegetative Vigor, Tier II
Terrestrial Plants Field Study, Tier III
Aquatic Plant Toxicity Test Using Lemna spp. Tiers 1 and 11
Aquatic Plants Field Study, Tier III
Rhi:obium-Legume Toxicity
Plant Uptake and Translocation Test
Group E — Toxicity to Microorganisms Test Guidelines
Soil Microbial Community Toxicity Test
Algal Toxicity, Tiers I and II
Group F — Chemical-Specific Test Guidelines.
Earthworm Subchronic Toxicity Test
Modified Activated Sludge, Respiration Inhibition Test for
Sparingly Soluble Chemicals
Group G — Field Test Data Reporting Guidelines.
Data Reporting for Environmental Chemistry Methods
OECD
Review
218/219
218/219
218/219
218/219
205
205
206
213/214
213/214
227
227
208/227
208/227
227
208/227
208/227
208/227
221
216/217
201
220/222/2
07
209/224
OPPTS
Review
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
NR
** Some similarity to OECD Difficult Substances document.
Organization of Reviews Summary
The greatest concern of reviewers is that guidance on preparation, delivery, measurement,
and metrology in all of the test guidelines is currently insufficient for testing of
nanomaterials. As this opinion applied equally across all tests, independent of endpoint,
media, target organisms, or duration, it seemed most expedient to summarize the reviews on a
test component basis, as opposed to a test-by-test, or section-by-section summary. These test
components include 1) toxicological principles, 2) terminology, 3) lest endpoints, 4) material
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characterization, and 5) media preparation, delivery, exposure quantification, and are
discussed in that order in the following section.
ADEQUACY OF TEST GUIDELINES
Toxicological Principles
All reviewers agreed that the basic toxicological practices on which these test guidelines
are based are adequate for testing nanomaterials. These include, in part, assuring that test
organisms are healthy and viable prior to exposure, use of reasonable dilution series based on
needs for statistical analyses of exposure-response relationships, and full control of all
preparation and exposure variables including positive controls for population responses to
stress. However, review of all ecotoxicity test guidelines revealed common inadequacies
relative to their use in testing nanomaterials. Specifically, their guidance on reporting the
properties of substances, the delivery of substances to test systems, exposure quantification,
and dose metrics are not adequate for nanomaterials.
Terminology
All of the current test guidelines reviewed use terminology that is primarily applicable to
chemical substances. In many cases, the term substance is used rather than the term chemical;
however neither term is fully descriptive of, or specific to, the particulate or fibrous nature of
nanomaterials. It should be noted however, that the use of the term chemical, by itself does
not preclude the applicability of test guidelines to nanomaterials. Other terms that are not
applicable to nanomaterials are listed below. These inadequacies are more than semantic;
they define, in the case of the term concentration, the specific metric that will be used in
estimation of effect levels, or dose-response relationships. It is the opinion of the reviewers
that such terminology will need to be revised to be both more specific to nanomaterials and to
assure that test outcomes accurately reflect the potential hazard of nanomaterials, based on
the most predictive properties of nanomaterials. The issues associated with these terms are
discussed in more detail below.
Chemicals
If test guidelines are to be used for both chemical and nanomaterial substances, then the
term nanomaterial should be defined and incorporated into all descriptions of their handling
and testing. Many OECD test guidelines refer to the testing of preparations or formulations
(e.g., OECD 213 and 214). This concept may be particularly applicable to some
nanomaterials which may be dependent on surface treatments and coatings or specific
solvents and emulsifiers to maintain their nano-scale characteristics.
Solution/solubility
Nanomaterials are generally in particulate or fibrous forms and their preparation and
delivery is best described in terms of preparations or suspensions, rather than solutions.
Some thought should also be given to the use of closely related terms such as soluble, solvent,
or dissolved, which might be interpreted as precluding the testing of suspensions of
nanomaterials. Terms such as suspension agents or matrices and suspension are more
descriptive of nanomaterials. Such terminology might be interpreted as precluding the testing
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of suspensions of nanomaterials. The importance of terminology in this case is exemplified
by work with silver nanoparticle formulations, which typically consist of suspensions of
elemental nano-sized cores, variably associated or bound silver ions, and free silver ions in
solution. In this case, the particles are suspended (as opposed to dissolved) but may, to some
extent, be soluble and release free silver ions into solution. An additional point is that the
bulk concentration (see the next section) may remain the same yet the proportion of free ionic
silver is very likely to be the major contributor to toxicity or potency of the mixture or
formulation.
Concentration
For soluble chemicals the term concentration is definitive and is a direct measure of
exposure. This is not true for suspensions of nanomaterials unless particle size (and size
distribution), surface area, and other properties are quantified. This is of particular concern
where effect levels are discussed. Current knowledge of the toxicity of nanomaterials
suggests that particle size, surface area, or surface charge may be more accurate predictors of
adverse effects. For these reasons, other terminology will be used when discussing exposure
levels and their relationship to observed adverse effects.
EC50, LC50, NOEC, LOEC, etc.
The corollary to the above comments concerning the use of the term concentration is that
predictive exposure-response relationships will also require terminology that is not dependent
on concentration. Effect-level metrics may need to be developed to incorporate several
properties specific to the biological activity of nanomaterials, including, but not limited to,
particle size, surface area, or surface charge.
Test Endpoints
There is little evidence to suggest that the majority of endpoints described in the current
test guidelines are not applicable to the testing of nanomaterials. These endpoints generally
involve whole-organism responses that integrate many possible modes of toxicity and are
thus likely to be indicators of potential adverse effects of nanomaterials. In some cases, for
example, respiration or gas production in microbial communities, the endpoints are also
integrative of adverse effects across taxa and at the microbial community level.
Future research may reveal that nanomaterials have modes of action that are unique,
relative to chemical stressors. For example, nanoparticles are of a scale that suggests possible
interaction with DNA or RNA, resulting in effects that might be revealed only in multi-
generation tests, and possibly involving novel endpoints. Early testing has suggested that
some nanomaterials may have adverse effects that involve physical smothering of exterior
surfaces, physical blockage of digestive processes, or physical inhibition of motility, e.g.,
coating of appendages in cladocerans. Because nanomaterials are particles or fibers,
exposures and uptake are likely to involve processes not typical for soluble chemicals. This
suggests that test endpoints may be developed that are more predictive of adverse effects
compared with the current test endpoints addressed by ecotoxicity guidelines. In addition,
because nanomaterials are currently in the early stages of development it is difficult to predict
their fate or pathways of exposure for biota. The current state of knowledge concerning
nanomaterial toxicity, as well as possible routes of exposure, precludes the reviewers from
making specific recommendation for the development of such new test guidelines.
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Material Characterization
This component, in all of these test guidelines, is currently inadequate for nanomaterial
testing. The particulate or fibrous nature of nanomaterials limits the usefulness of solubility
or nominal or measured concentrations as properties useful for describing exposure-response
relationships. Current research suggests that particle count, size distribution, surface area,
surface charge, and other surface characteristics might be better predictors of toxicity, and
that their incorporation into exposure metrics will be necessary for accurate statistical
determination of dose-response relationships. While concentration may be a useful parameter
in modeling toxic effects, its usefulness will depend on knowledge of the state of the particles
contributing to nominal or measured concentrations; e.g., ten 1-mg particles may be far more
toxic than four 2.5-mg particles, given equal suspension volumes and yielding equal
concentrations.
An OPPTS work group has reviewed current physical-chemical test guidelines to assess
their applicability to nanomaterials (Utterback et al 2008). As part of that process the
workgroup made recommendations for characteristics that should be incorporated into new or
existing physical-chemical test guidelines. We recommend that these reviews and suggestions
for nanomaterial-specific physical-chemical guidelines be carefully considered as the current
ecotoxicity test guidelines are modified, or newly developed. In addition, some physical-
chemical properties currently described should be removed if the test guideline is to be used
specifically for testing of nanomaterials. For example, several guidelines include vapor
pressure as one of few identified physical-chemical properties to be identified for test
substances; this property is unlikely to be applicable to nanomaterials (see Utterback et al.
2008). It is also expected that new research on the ecotoxicity of nanomaterials will also
guide the process of the revision of test guidelines.
The physical-chemical characteristics of nanomaterials have also been identified as being
a primary concern relative to the other major test guideline components, discussed below.
Media Preparation, Delivery, Exposure Quantification
The test guidelines related to ecotoxicity involve several media, including soils,
sediments, water, food, and direct application (Bee Test, OECD 213, albeit by application of
suspensions). Testing in each of these media presents unique problems relative to the
properties of nanomaterials. Concerns specific to water exposures include factors that can
strongly affect nanomaterial aggregation and agglomeration, including pH, ionic strength,
and concentration of dissolved organic matter. Some test guidelines (e.g., OPPTS 1055)
recommend the use of natural seawater, which could introduce considerable variability in
exposures between laboratories, and even between tests due to small differences in water
quality variables. In some test guidelines (e.g., OPPTS 1020), it is suggested that water
quality factors vary month-to-month by no more than 10%. However, some factors, such as
hardness or particulate concentrations might significantly alter agglomeration/aggregation
behavior of nanomaterials even over this narrow range. Early.testing has also demonstrated
that the characteristics of suspended nanomaterials can vary significantly (and predictably in
some cases) depending on mixing method, e.g., stirring versus sonication, and even the rate at
which a diluent is added to working suspensions (Handy et al. 2008, Fortner et al. 2008). The
presence of dissolved organic matter and suspended natural substances can affect the physical
properties of nanomaterials, as well as the stability of suspension.
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These concerns apply directly to test sediments and soils that are prepared using
suspensions in water. Dry application of nanomaterials will preclude these suspension-related
issues, however, the effect of soils and sediment composition and physical-chemical
properties will affect the characteristics of nanomaterials. Similarly, when nanomaterials are
mixed into food, the method of mixing and the composition of the food matrix will affect
their characteristics. It should be noted here that dry application of nanomaterials may
involve significant exposure hazards for lab personnel; this issue should be addressed.
None of the test guidelines related to ecotoxicity provides information on how to measure,
control for, or otherwise address these exposure preparation variables. SG4-2 recommends
that such guidance be added to modified or newly-developed test guidelines to assure their
applicability to nanomaterials.
Many existing test guidelines make specific recommendations about volumes of exposure
media, organism loading rates, and the necessity of flow-through exposures. These
recommendations may need to be reconsidered for nanomaterials that are particularly
expensive or difficult to obtain in large quantities.
Some test guidelines (e.g., OPPTS 1020) recommend filtration of samples prior to their
analyses for concentration of test substances. In the case of OPPTS 1020, the recommended
pore size for filtration is 0.45 urn; this is a pore size that is very likely to remove particles
from some nanomaterial preparations.
Stability and Consistency
All of the exposure preparation and delivery issues discussed above are complicated by
the stability and consistency of the properties of nanomaterials in the various exposure
matrices used. In general, the current test guidelines do not provide adequate direction for
monitoring the characteristics of nanomaterials over the duration of tests, nor for
documenting the consistency of materials obtained from different sources or production runs.
Many nanomaterials agglomerate or aggregate and settle from solution. Generally, achieving
a fully stable suspension is not possible. Variability of exposure levels can occur with
chemical test substances as well, and many test guidelines describe allowable limits for
chemical stability in test chambers. However, both the frequency of analysis, and specific
characteristics to be analyzed, are inadequate for nanomaterials. Additionally, some
consideration should be given to how representative test media are of nanomaterial-specific
fate processes that might occur in natural systems. The suggestions made by the OPPTS
workgroup that reviewed physical-chemical guidelines (Loux et al. 2008) should also be
incorporated into guidance on quantifying and characterizing exposure stability and
consistency.
Metrics and Measurement
As mentioned above, the particulate or fibrous nature of nanomaterials will require new
approaches to estimating and predicting levels of effects based on biota exposure. Current test
guidelines recommend dose-response metrics based on substance concentration (EC50,
EC50, NOEC, LOEC, etc.). While concentration may remain as a major component in
expression of exposure for nanomaterials, it is likely that other metrics including (amongst
others) particle size, surface area, and surface charge may be essential for the development of
predictive exposure metrics. Specific nanomaterial properties that might be critical for
development of these metrics are identified in the work of the OPPTS workgroup that
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reviewed physical-chemical guidelines (Loux et al. 2008). It is the recommendation of the
work group that these characteristics, as well as results of current toxicological research, be
considered for the revision of existing, or the development of new, test guidelines.
ADDITIONAL REVIEWS
OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and
Mixtures
As described above, the reviews of ecotoxicology test guidelines indicated that their
inadequacies for testing of nanomaterials are consistently related to material characterization
and properties, and metrology. This finding suggests that, rather than extensive modification
of all test guidelines, these nanomaterial-specific issues might be addressed in a single
document that would provide guidance on how existing test guidelines could be used in
testing nanomaterials. This approach has been applied to other substances that are deemed,
"difficult substances" in OECD's Guidance Document: Aquatic Toxicity Testing of Difficult
Substances and Mixtures (ENV/JM/MONO(2000)6). The goal of this document is to describe
the preparation, delivery, and measurement of substances that would not be adequately tested
if existing test guidelines were used. This document was reviewed to determine if the
guidance provided would be adequate for nanomaterials, and might address some of the
issues identified in the test guideline reviews. Reviewers were also charged with making
recommendations for modification of the Difficult Substances document, or a similar
guidance document directed at nanomaterial testing.
A summary of the review findings are enumerated below:
a. The document provides a good framework for developing guidance for the aquatic
toxicity testing of nanomaterials. Such specific guidance could be incorporated into
the existing document or developed as a similar, but separate document. It should be
noted, however, that the guidance is specifically for testing in aquatic systems.
Similar guidance may be necessary for terrestrial testing as well;
b. As with the review comments above for the OECD Ecotoxicity Test Guidelines, the
"Difficult Substances" document lacks sufficient guidance for the characterization of
nanomaterials. The guidance does describe procedures for characterizing traditional
test substances, including their stability, as well as media preparation and sampling.
However, many of the properties defined are unlikely to be applicable to
nanomaterials (e.g., volatility), and many that are presumptively critical for
nanomaterials (e.g., agglomeration and aggregation), are not mentioned. Specific
nanomaterial properties to be measured or documented, and methods to do so will
need to be described; and
c. Many physical and chemical properties that make substances difficult to test are
described, as are approaches for overcoming these difficulties in toxicity testing, and
some of this guidance might be applicable to nanomaterials. However, many
properties specific to nanomaterials will also need to be addressed; e.g., particle size,
surface area, agglomeration potential or rate, as well as how to prepare and maintain
stable suspensions or distribution of nanomaterials.
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Recommendation of reviewers on the "Difficult Substances" document:
Guidance very similar to that provided by the Difficult Substances document, but specific
to nanomaterials toxicity testing, should be either added to the existing document or
developed as a new guidance document. Such guidance could provide a means to rapidly
advance toxicity testing of nanomaterials using existing test guidelines; an outcome that
would also provide critical information necessary for development of nanomaterial-specific
test guidelines. To serve these purposes, the guidance would need to address the issues just
discussed. Key considerations would include the physical-chemical properties of
nanomaterials identified by the OPPTS P/Chem workgroup.
Non-OECD/OPPTS Test Guidelines for Ecotoxicity
Five non-OECD biological testing guidelines (Table 2) were briefly reviewed. This was
an effort to identify sources of guidance that might be directly applicable to nanomaterial
testing, and thus inform the revision or rewriting of OECD test guidelines. As it was not
possible for the workgroup to review a large number of additional test guidelines, a small
putatively representative sample was selected based on environmental media (water,
sediment, or soil) and compartment (pelagic or sediment). These abbreviated reviews
involved scanning the guidelines to identify descriptions or terminology adequate for
nanomaterials. These were not intensive, section-by-section reviews as was undertaken with
the OECD/OPPTS test guidelines.
Table 3. Reviewed non-OECD/OPPTS ecotoxicity test guidelines.
Guideline
Identification
Environment Canada
EPS1/RM/45E
Environment Canada
EPS1/RM/11E
Japan, Ministry of the
Environment
International Standard
ISO 11 267: 1999
International Standard
ISO 634 1:1996
Description of Test
Test for Measuring Emergence and Growth of Terrestrial Plants
Exposed to Contaminants in Soil
Acute Lethality Test Using Daphnia spp.
Algal Growth Inhibition Test, Daphnia Acute Immobilization
Test, and Fish Acute Toxicity Test
Soil quality — Inhibition of Reproduction of Collembola
(Folsomia Candida} by Soil Pollutants
Water quality — Determination of the Inhibition of the Mobility
of Daphnia magna Straus (Cladocera, Crustacea) — Acute
Toxicity Test
None of the non-OECD test guidelines provided guidance that addressed the inadequacies
identified by the workgroup in the OECD/OPPTS test guidelines. This is not surprising given
the unique nature of nanomaterials and the fact that new test guidelines are typically based on
existing, well-vetted guidelines. In the case of OECD, EU Testing Methods, and U.S.
EPA/OPPTS Test Guidelines, the harmonization process has led to identical language in most
cases.
Observations on Specific OPPTS Guidelines
(850.5400) Algal toxicity, Tiers I and II: Methods described for estimating algal
population growth might be confounded by the presence of nanoparticles, especially where
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agglomeration or aggregation results in particle sizes that overlap the size of tested lifestages.
In addition, some nanomaterials may agglomerate with algal cells or cause cell-to-cell
clumping. The methods discussed include optical particle counting, gravimetry, and
spectroscopy. The test guideline also includes the requirement that the accuracy of these
approaches be confirmed using some form of microscopy. These measurement approaches,
including confirmatory microscopy, will need to be evaluated, and possibly modified for use
in nanomaterial testing.
(850.1055) Bivalve acute toxicity test (embryo-larval): Endpoints should be evaluated
with the recognition that early lifestages of some mollusks may have size ranges that overlap
the size range of the particles being tested, particularly where agglomeration/aggregation are
expected or observed. Adverse effects of such an overlap in size might include direct
physical interference with movement, respiration, feeding, etc., and could make
quantification of effects on the end-point stages (veliger larvae) difficult. It is also notable
that this is the only reviewed test guideline that specifically suggests that ultrasonic
dispersion is an acceptable method for dispersing test substances. No specific guidance is
provided on how ultrasound should be employed, e.g., energy intensity, duration of
sonication, or whether probe or bath systems are acceptable.
(850.1085) Fish acute toxicity mitigated by humic acid: This guideline describes an
approach for examining the effect that hurnic substances might have on the toxicity of
traditional chemicals, a question that is equally important for assessing the hazard and risk of
nanomaterials. The guideline provides an excellent framework for examining these effects,
but will require considerable modification to address the terminology, metrology, and other
issues associated with the particulate or fibrous nature of nanomaterials that are described
here for all other test guidelines.
(850.1850) Aquatic food chain transfer: This is a very brief guideline that provides
limited and very general guidance on examining food chain transfer of soluble chemicals.
The guideline could serve as a framework for nanomaterial testing (albeit limited and very
general), but would need a few key modifications. Most notable among these is that, as
currently written, the need for this testing is based on water solubility and log Kow values.
The former of these would essentially exclude nanomaterials (due to their colloidal
character), whereas the applicability of the latter has yet to be determined for nanomaterials.
Issues associated with Kow approaches are discussed relative to OPPTS 850.1730, below.
(850.1730) Fish BCF: This guideline describes methods for determining bioconcentration
factors and the rates of uptake and depuration for contaminants. All of the issues associated
with defining and using the term concentration apply to this guideline, with the added
complication that the concept of proportionate concentrations, e.g., octonol/water, as a
surrogate for lipophylicity, are not yet defined or well-investigated for most particulate or
fibrous materials. The critical unknowns are how particle size and level of aggregation or
agglomeration should be incorporated into these concentration metrics.
(850.4800) Plant uptake and translocation test, and (850.4600) Rhizobium-iegame
toxicity: These guidelines describe methods for collecting data on rates of uptake and
translocation of chemical substances, and toxicity to rhizobium-legumes, respectively. There
are three unique concerns relative to applying these guidelines to nanomaterials testing. The
first is the requirement for nutrient addition to growth media, either dry or wet, that is likely
to strongly influence rates and levels of aggregation and agglomeration. The second concern
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is the assumption that substances can be added to stable solutions that will infiltrate sand-
based media. As well as the previously-mentioned concern with suspension issues, it is also
very likely that nanomaterials will interact with sand surfaces, and the interactions will vary
significantly depending on the specific nanomaterial being tested. The third concern is the
requirement for illumination that optimizes plant growth and vigor. The photo reactivity of
some nanomaterials, e.g., the anatase form of TiC>2, has been shown to increase their toxicity.
This reactivity is likely to be wavelength dependent suggesting that additional guidance on
how to incorporate or address this factor in testing will need to be developed.
OPPTS (850.1000) Special considerations for conducting aquatic laboratory studies:
This guideline discusses many of the basic principles of aquatic toxicity testing and could
provide a framework for incorporation of guidance specific to nanomaterials testing, or
development of a similar document focused on nanomaterials. Many of the principles
discussed are as applicable to nanomaterials as to soluble substances. There is some
discussion of the presence of colloids in test preparations; however, the focus is on removing
them by centrifugation, a procedure that would certainly remove nanoparticles from
suspension (with rates dependent upon particle size, surface charge, and a variety of media
characteristics). Of particular interest is the following section:
(3) Current policy allows chemicals that are poorly soluble (solubility <100
ppm) or dispersible in water to be tested up to the maximum water solubility
or dispersibility limit obtainable for the given test conditions employed,
provided that certain prerequisites apply:
(i) Concentrations of test chemical in test media are measured at
appropriate intervals and from appropriate test chambers of all test levels are
determined from centrifuged supernatant or other appropriate separation
(e.g., filtrate). Self-dispersing industrial chemicals (e.g., surfactants,
detergents, or charged polymers) should be sampled directly.
Given some modification, in particular discussion of recommended particle characterization,
this section could be adapted to address nanomaterials specifically.
OPPTS 850.1950, 850.2400, 850.2500, 850.3040, 850.4300, 850.4450 (generally, field-
testing guidelines): These test guidelines describe approaches to testing for effects of
pesticides in natural settings or media. They may be generally more adequate for
nanomaterials due to the lack of detailed methods, and thus lack of terminology that
precludes testing of suspensions or colloids. In some cases, the intent is to test formulations,
rather than pure compounds, a goal that is likely to apply equally to nanomaterials used as
field pesticides, which would also be likely to involve formulations of emulsifiers, stabilizers,
solvents, etc.
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