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Research Plan: Novel Technologies
Project
Acting Project Leader: Lidia S. Watrud
Authors: Watrud, L.S., Andersen, C.P., Johnson, M.G.,
Lee, E.H., Olszyk, D.M., Pfleeger, T., Reichman, J.R.,
Rygiewicz, P.T., Schumaker, N.
DRAFT
This document is a preliminary draft. It has not been formally released by the U.S.
Environmental Protection Agency and should not be construed to be Agency policy. It is
being circulated for comments on its technical merit and policy implications. Do not
release. Do not quote or cite.
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TABLE OF CONTENTS
Pages
Overall Executive Summary i - iii
Authors iv
Acknowledgements iv
Chapter 1 - EFFECTS OF CHEMICAL PESTICIDES ON PLANTS 1-26
Chapter 2 - ECOLOGICAL EFFECTS OF GENE FLOW FROM
TRANSGENIC CROPS 27-55
Chapter 3 - GIS-BASED FRAMEWORK TO ASSESS EFFECTS OF
EXPOSURE TO TRANSGENE FLOW AND TO
HERIBICIDE DRIFT 56-65
Chapter 4 - A SPATIALLY-EXPLICIT MODEL OF PESTICIDE
IMPACT ON WILDLIFE 66-80
Chapter 5 - ECOLOGICAL EFFECTS OF NANOTECHNOLOGY
PRODUCTS 81-160
Chapter 6 - PROJECT MANAGEMENT AND QUALITY ASSURANCE 161-167
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AUTHORS
Lidia S. Watrud, Acting Project Leader, Novel Technologies
Chapter 1: David Olszyk, Thomas Pfleeger
Chapter 2: Lidia S. Watrud, Jay Reichman
Chapter 3: E. Henry Lee
Chapter 4: Nathan Schumaker
Chapter 5: Christian Andersen, Mark Johnson, Paul Rygiewicz
ACKNOWLEDGEMENTS
Joan Hurley and Karen Gundersen are thanked for their capable assistance in the
final assembly and formatting of this research plan.
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Executive Summary
NOVEL TECHNOLOGIES PROJECT
FY 08-FY 13
The Novel Technologies Project is comprised of the following sub-projects:
1. Ecological Effects of Chemical Pesticides
2. Ecological Effects of Gene Flow
3. A GIS-Based Framework to Assess Effects of Exposure to Transgene Flow and to
Herbicide Drift
4. A Spatially-Explicit Model of Pesticide Impacts on Wildlife
5. Ecological Effects of Nanotechnology Products
Following outside peer review of their respective plans in 2002, the first four projects
were initiated in 2003. The research proposed herein thus builds upon scientific progress
made during FY 03-FY 07. The fifth project represents a new effort to examine the
potential ecological effects of nanotechnology products. All five sub-projects are linked
by having a common regulatory client i.e. the Office of Prevention Pesticides and Toxic
Substances (OPPTS). The research will support OPPTS by developing laboratory,
greenhouse or field methods and models to measure or predict the ecological effects of
chemical, biological or physical stressors such as herbicides, genes from GM crops or
manufactured nanomaterials. Each of the sub-projects shares potential similarities and
thereby synergies in methodology to measure and predict ecological effects of diverse
types of stressors. The proposed research addresses Agency needs described the Multi-
Year Plan (MYP) Long Term Goal (LTG) 2 and the Safe Pesticides/Safe Communities
(SP2) Plan LTG 3. The primary statutory authorities for the proposed research are the
Federal Insecticide Fungicide and Rodenticide Act (FIFRA) and the Toxic Substances
and Control Act (TSCA). The following sub-project chapters each have their own more
detailed executive summary. The individual chapters will provide in greater detail the
rationale, background, and specific scientific questions that are being addressed. They
will also describe the proposed technical approaches and anticipated outputs for each sub-
project. Highlights from the respective executive summaries for each of the five sub-
projects are presented below.
A. Ecological Effects of Chemical Pesticides
The proposed integrated experimental, modeling and molecular studies will address
information gaps identified as crucial to carrying out risk assessments of the ecological
effects of drift levels of herbicides on non-target plants, especially native plants. The
experimental studies will be geared to providing data for a simplified model of plant
community structural and functional responses to environmental stress. Based on
availability of resources, the methodologies will be used to determine the effects of other
pesticide classes (e.g., fungicides) on terrestrial and possibly aquatic plant community
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structure and function. The major focus will be on empirically evaluating in greenhouse
and field studies, the effects of herbicides on reconstructed Willamette Valley prairie
plant communities. An additional focus will be on identifying molecular indicators of
exposure to drift levels of chemical herbicides, based on the use of custom microarrays
for selected classes of chemical herbicides.
B. Ecological Effects of Gene Flow
In Phase I of Gene Flow Research at USEPA NHEERL WED methods were developed
that resulted in documentation of gene flow from creeping bentgrass on a landscape level
i.e., to a distance of 21 km. These results changed the paradigm for how far viable pollen
could move and thereby provide exposure to GM genes (Watrud et al. 2004, PNAS 101:
14533-14538). Phase I research has also resulted in the first documentation in the US, of
establishment of GM crop/wild (Reichman et al., 2006, Mol. Ecol. 15:4243-4255). In the
Phase II research proposed herein the focus of Gene Flow research shifts from
developing methods for measuring gene flow to developing methods and models for
measuring and predicting the environmental consequences of gene flow from crops that
may contain one or multiple GM traits. Research is proposed at several levels: (a)
molecular laboratory studies to confirm introgression of GM genes in wild populations
and identifying any resultant changes to wild populations (b) greenhouse and large
outdoor mesocosm studies to measure gene flow and its consequences on the ecological
fitness of advanced hybrid and backcross progeny and on the structure of constructed
plant communities and (c) models to predict the ecological consequences of gene flow on
non-agronomic plant communities.
C. A GIS-Based Framework to Assess Effects of Exposure to Transgene Flow and to
Herbicide Drift
The risks of GM crops vary spatially and temporally. Typically, they are evaluated at
least initially, on a case-by-case basis. Currently, most of the US acreage of GM
herbicide resistant canola (Brassica napus) is found in North Dakota. However, with
increasing interest in B. napus not only as an oilseed crop for human consumption, but
also as a biodiesel crop, increasing acreage of canola is anticipated in other US
geographic areas including the Midwest, south central states and the Pacific Northwest.
We propose a multi-year field study in the upper Midwest to determine the ecological
impacts of hybridization and introgression of selectable markers for herbicide resistance
transgenes (e.g., CP4 EPSPS for resistance to glyphosate or bar for resistance to
glufosinate) from fields of GM canola to compatible wild populations of bird's rape
mustard (B. rapa). A GIS-based framework is thus proposed to integrate predictions of
the effects of chemical herbicide drift and ecological data on transgene flow for
resistance to glyphosate herbicide, into wild populations of compatible cruciferous plants.
D. A Spatially-Explicit Model of Pesticide Impacts on Wildlife
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This research will help the Agency to address wildlife population endpoints as a function
of terrestrial habitat quantity, quality and distribution, and as affected by multiple
stressors across many temporal and spatial scales. The proposed modeling effort will
generate tools and databases to meet the needs of our Program Office clients in four
specific problem areas. First, the Scientific Advisory Panel for the Federal Insecticide,
fungicide, and Rodenticide Act (FIFRA) specifically recommended that the Office of
Pesticide Programs conduct probabilistic assessments of risks to ecosystems associated
with pesticide use. Second, the Office of Prevention, Pesticides, and Toxic Substances
needs efficient methods, including models, to review, register, and regulate thousands of
chemicals in a timely fashion. Third, the Office of Water has a need for improved
methodology for probabilistic assessment of the impact of habitat alteration on aquatic-
dependant terrestrial wildlife. Finally, the Office of Solid Waste and Emergency
Response has similar needs for assessing contaminant effects in terrestrial systems. In
addition, this research will contribute to the ECO MYP through examination of the
spread of wildlife diseases that have implications for human health.
E. Ecological Effects of Nanotechnology Products
This proposal establishes a framework for the Western Ecology Division to begin
assessing the effects of various engineered nanoparticles on terrestrial, and in some cases
aquatic ecosystems. The research is divided into three linked phases. Phase I examines
the applicability of a subset of traditional OPPTS testing protocols to determine whether
such tests are adequate to assess the toxicity of nanoparticles. In Phase II, the toxicity
results from Phase I will be explored to elucidate mechanisms of action and the
relationship between responses and the structure and activity of studied nanoparticles
(QSARs). Phase II will also involve research to evaluate novel approaches based on
proteomics and genomics for rapidly assessing toxicity. In addition, in Phase II some
aspects of ecosystem complexity will be added to the toxicity testing to evaluate the
effect of multiple stressors on individual response. Finally, Phase III will examine the
degree to which nanoparticles released into reconstructed ecosystems represents a risk to
ecosystem structure or function. The unique aspect of Phase III is to shift the endpoint of
interest from individuals to groups of individuals and system level responses. In each
phase, empirical data from molecular, whole-organism, population, community and
ecosystem responses to nanoparticle exposure will be used to predict and identify
possible effects of nanoparticles on ecosystem structure and function. The products of
this research will assist the EPA with pre- and post-registration product assessment,
monitoring and enforcement granted to it under the statutory authorities of TSC A and
FIFRA.
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CHAPTER 5
ECOLOGICAL EFFECTS OF NANOTECHNOLOGY PRODUCTS
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Executive Summary
Nanotechnology has the potential to influence virtually every aspect of society. As of
June 2007, there are over 500 products containing engineered particles are already on the
market, including clothing, cosmetics, medicines and machines. The Office of
Prevention, Pesticides and Toxic Substances (OPPTS) is reviewing pre-manufacture
notifications for a number of nanomaterials that have been received under TSCA (Toxic
Substances Control Act) and FIFRA (Federal Insecticide, Fungicide and Rodenticide
Act). It is likely that other EPA offices will also become involved, such as the Office of
Air and Radiation (OAR), and the Office of Transportation and Air Quality (OTAQ)
which has already received an application to register a diesel additive containing nano-
sized cerium oxide. Yet there is little information on the environmental fate, transport,
exposure or effects of nanomaterials. This proposal establishes a framework for the
Agency to begin assessing the effects of various engineered particles on terrestrial, and in
some cases aquatic ecosystems.
The research is divided into three linked phases. Phase I examines the applicability of a
subset of traditional OPPTS testing protocols to determine whether such tests are
adequate to assess the toxicity of nanoparticles. In Phase II, the toxicity results from
Phase I will be explored to elucidate mechanisms of action and the relationship between
responses and the structure and activity of studied nanoparticles (QSARs). Phase II will
also involve research to evaluate novel approaches based on proteomics and genomics for
rapidly assessing toxicity. In addition, in Phase II some aspects of ecosystem complexity
(e.g., competition, water stress, etc.) will be added to the toxicity testing to evaluate the
effect of multiple stressors on individual response. Finally, Phase III will examine the
degree to which nanoparticles released into reconstructed ecosystems represent a risk to
ecosystem structure or function. The unique aspect of Phase III is to shift the endpoint of
interest from individuals to groups of individuals and system level responses.
To address the issues in the planned research project, we will use empirical data from
molecular, whole-organism, population, community and ecosystem responses to
nanoparticle exposure to predict and identify possible effects of nanoparticles on
ecosystem structure and function. Early experiments will employ highly controlled
laboratory conditions to examine the effects on individual organisms such as plants,
microbes and invertebrates, with subsequent studies imparting more complexity of
various kinds to enhance realism both in the trophic structure and scale of tested systems.
WED has extensive research facilities able to accommodate the different levels of control
and complexity required to conduct the range of proposed experiments. The most
challenging and important aspect of the proposed research will be to scale exposure-
response data from "individuals" to higher levels of biological organization, and
eventually to that of ecosystems.
'The products of this research will assist the EPA with enforcing regulatory requirements,
including pre- and post-registration product assessment and monitoring done by OPPTS
granted to it under the statutory authorities of the TSCA and FIFRA. In addition, the
results will allow other Agency offices to identify potential environmental concerns
resulting from nanoparticle exposure via various pathways. Finally, the results will
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enable the Agency to evaluate its current risk assessment framework developed for use
under FIFRA and TSCA, and may lead to the development of a new assessment
framework should the current approaches used by OPPTS be inadequate for
nanoparticles. The acronym that we propose to describe our research approach for
characterizing the ecological effects of nanotechnology particles is "NERA":
Nanotechnology and Environmental Risk Assessment.
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"Nanotechnology is no longer a scientific curiosity. It is in the workplace, the
environment and the home. But if people are to realize nanotechnology's benefits-
in medicine, communications, and energy production- the federal government
needs a master plan for identifying and reducing potential risks. This plan should
include a top-down risk research strategy, sufficient funding to do the job, and the
mechanisms to ensure that resources are used effectively." —Dr. Andrew
Maynard, Project on Emerging Nanotechnologies, Woodrow Wilson
International Center for Scholars.
I. Introduction
Nanotechnology has potential applications in virtually every sector of the American
economy, including consumer products, health care, transportation, energy, agriculture
(Theodore and Kunz, 2005), and national security (Ratner and Ratner, 2004). In addition,
nanotechnology presents new opportunities to improve how we measure, monitor,
manage, and minimize contaminants in the environment (Masciangioli and Zhang, 2005;
U.S. EPA, 2007). Although the U.S. Environmental Protection Agency (EPA) is
interested in researching and developing the possible benefits of nanotechnology, the
EPA also has the obligation and mandate to protect human health and safeguard the
environment by better understanding and addressing potential risks from exposure to
nanoscale (for the purposes of the various uses of such materials, "nano" generally is
used to describe particles that are less 100 nm in their greatest dimension) materials and
products containing nanoscale materials (both referred to here as "nanomaterials") (U.S.
EPA, 2007).
It is speculated that the widespread application of nanomaterials is in its infancy, and
natural nanoparticles, e.g., aerosols, are pervasive in the environment. While products
containing natural and engineered nanoparticles are already commonplace globally, only
a handful of studies have been conducted to determine their inherent toxicity to biological
systems (Nel et al., 2006). This project addresses the question of potential risks of
nanomaterials at several scales - ranging from toxicity to individual organisms to the
structure and function of terrestrial ecosystems - and, as resources may allow, aquatic
ecosystems. The fact that individual organisms may respond differently when grown in
isolation than when grown in populations, in communities or as members of ecosystems,
is of particular interest to this project since an aim is to understand how to scale properly
the impacts of nanoparticles and nanomaterials from toxicological responses of
individuals. The overarching goal of the proposed research is to develop tools to
assess, monitor, predict and model changes in the condition of terrestrial ecosystems
in response to these novel materials. The first important question to address is whether
or not existing traditional toxicology protocols used by EPA's Office of Prevention,
Pesticides and Toxic Substances (OPPTS) for pesticide testing and registration are
sufficient for evaluating the ecological effects of nanoparticles (U.S. EPA, 2007). If not,
then research will be conducted to develop new testing protocols and risk assessment
frameworks to assess ecological/ecosystem effects.
Initial and ongoing efforts being done in other research laboratories of the EPA also
comprise the larger nanomaterials/nanoparticles research effort of the EPA Office of
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Research and Development (ORD). Their goal is to examine fate, transport, and
exposure (FTE) in the environment (U.S. EPA, 2007). Until better information is
available on these issues, it will be more difficult to conduct realistic effects research to
assess the ecological consequences of natural and engineered nanoparticles released into
the environment. Our research proposed in this plan is integrated sequentially into this
greater research effort coordinated by ORD. Our research will build upon and
compliment the results from the FTE research being done at other laboratories as part of
the overall ORD effort.
A. Brief Overview of Nanoparticles, Nanomaterials and Nanotechnology
Nanoparticles fall into three categories: natural, incidental, and engineered (Vicki Colvin,
cited in Goldman and Coussens, 2005). Natural nano-sized particles occur in nature and
are found in, for example, volcanic ash, ocean spray, secondary minerals, and
magnetotactic bacteria. Incidental particles are those that are produced as a byproduct of
industrial processes. For example, nanoparticles are often byproducts of incomplete
combustion. Engineered nanoparticles are those that are intentionally fabricated or
synthesized. These materials are further classified by their composition or use. These
include: metals, metal oxides, nanoclays, nanotubes, quantum dots and semiconductors
(Goldman and Coussens, 2005). Nanomaterials also fall into "wet" and "dry" categories.
The "wet" category pertains to the nano-sized components of biological systems that
exist primarily in a water environment. These components include: genetic material,
organelles, membranes, and enzymes. The "dry" category focuses on the fabrication of
nanomaterials out of carbon, silicon, titanium and other inorganic materials. An
additional category is computational nanotechnology, which provides a means for
modeling nano-scale materials and their interactions with other materials, be it substrates
for catalysis or semi-conductors for electronics applications. Computational
nanotechnology can speed the development and production of both "wet" and "dry"
nanomaterials (Richard M. Smalley Institute for Nano Science and Technology,
http://cohesion.rice.edu/centersandinst/cnst/index.cfm).
Nanometer-sized materials have been used for a long time. For example, carbon black
(nano-sized carbon particles) has been used to strengthen rubber in car tires for more than
a century (Shelley, 2006). Similarly, nanoparticles have been used in photography and in
medieval ceramics and stained-glass. It is known that the same compounds will behave
dramatically differently when their size decreases from micrometers to nanometers,
where quantum effects begin to apply. So while nanoparticles have been employed by
humans for considerable time, what distinguishes the older applications from the products
and applications of modern nanotechnology is that the properties at the nanometer scales
are better understood today and are being engineered and exploited in novel forms. In
fact, it is now possible for the unique properties to be modeled and fine-tuned, to
manufacture products intended for highly-specific, and unique purposes.
While the allure of the myriad products of nanotechnology (see sub-sections below), the
potential discoveries of nanoscience research, and the results of nano-manufacturing are
driving financial investments, extensive speculation for profits, the hopes to increase
national security and to clean up the environment, there may be resultant unintended
negative consequences associated with producing and using such products and
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technologies. While the future of nanotechnology appears very bright, the precautionary
principal may also need to be applied to ensure that the projected benefits emerging from
nanotechnology are not outweighed by either unforeseen or avoidable negative
consequences (Wiesner et al., 2006).
B. Commercial/Industrial Operations and Products
Nanotechnology provides a wide array of opportunities for the manufacture of
commodities that may be utilized in end-user products (e.g., fabrics, sporting goods; see
http://www.nanotechproiect.org/index of the Woodrow Wilson International Center for
Scholars for a current listing of products containing nanomaterials) or for the production
of services (e.g., energy production). Fundamental to nanotechnology is the assembly or
synthesis of materials from nanoparticles that impart unique or specialized functions due
to their composition, structure or surface properties. It is the almost limitless
combination of composition, size, and surface properties of nanoparticles that endows
nanomaterials and nanotechnology with the vast range of real and potential applications.
Nanotechnology is already making important contributions to the electronic, optical,
magnetic, catalytic, and medical-therapeutic fields (Theodore and Kunz, 2005), and the
possibilities and uses of nanotechnology seem to be limited only by the human
imagination.
The properties of nanomaterials can lead to examples of extreme engineering at the
nanoscale. For example, carbon nanotubes (CNTs) have properties that surpass other
materials. With a tensile strength 50 times that of stainless steel, and heat conductivity 5
times that of copper, CNTs become an obvious choice for inclusion in various polymer
matrices for developing new composite materials with advanced properties. In addition
to companies that produce CNTs, other companies are modifying the surface properties
of CNTs so that they integrate explicitly into new materials in such a way as to boost the
electrical, mechanical, chemical or thermal properties of the host material by perhaps
orders of magnitude over traditional fillers ( e.g., www.zyvex.com).
A number of proposed applications of nanotechnology are intended to improve local and
global environmental conditions (Dionysiou, 2004). For example, utilizing
nanotechnology to create more efficient membranes for filtering water, and using
nanomaterials to remove toxic metals or organics from drinking water have been
suggested. Also proposed is the use of solar-activated nanomaterials to remove
pollutants from air (Shelley, 2006) and nanoparticles of iron specifically to absorb and
destroy toxic organics in water (Zhang, 2003). Nanomaterials may have a key role in the
transition from a fossil carbon-based energy economy to one that is hydrogen-based
(Cheng et al., 2001) and may even help make current energy production more efficient
and less polluting. Many nanomaterial-based sensors have been proposed and include
monitors for cellular-level processes and air pollutants. Environmentally benign
nanocomposites are envisioned for use in construction, electronic devices and computers.
A number of commercially available products are comprised of nanomaterials , with
more such products expected in the future. Nanomaterials are used in a number
sunscreens and cosmetics. They are used to change the porosity of fabrics to impart
water, stain, or perspiration resistance. These fabrics are used to make clothes, bedding,
upholstery, toys and shoes. They are even being used to impart a specific scent to leather
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and other fabrics. "Self-cleaning" windows and bathroom fixtures utilize nanomaterial
coatings (Shelley, 2006). Nanomaterials in composites are used in automobiles and
airplanes as the composites are stronger and lighter than their metallic predecessors.
Nano-scale catalysts improve oil refining and petrochemical synthesis. Nanotechnology
has led to a number advances in information technology via miniaturizing electronic
devices, yielding increased processor speed and data storage capacities. Nano-sized
calcium phosphate is being used in dentistry to replace tooth enamel. Nanomaterials
have also been proposed as catalysts for use in energy production and as a fuel additive
for automobiles and trucks. For example, cerium oxide nanoparticles are being used in
the UK as fuel additives in both on- and off-road diesel vehicles to reduce emissions
(U.S. EPA, 2007). While the intended effect is reduced fuel use, the consequences of
extensive release of cerium oxide to the environment has not been examined.
C. Cosmetics and Medical Applications
As of June 2007, more than 500 products containing nanoparticles were available on the
open market in throughout the world, many of which are cosmetics or related products
(http://www.nanotechproiect.org/index.php?id=44&id=44&action=view&p=0Y The
cosmetic industry has been quick to embrace the use of nanotechnology in their products
and currently cosmetic companies hold the largest number of nanoparticle patents.
Nanoparticles can be found in toothpaste, shampoo, sunscreen, hair conditioners, lipstick,
eye shadow, aftershave, moisturizers and deodorants, and anti-aging creams. The
particles impart unique and beneficial qualities to cosmetics. For example, 'nanospheres'
or 'nanoemulsions' increase penetration into the skin. L'Oreal uses polymer
nanocapsules to deliver vitamin A to the deeper layers of the skin. Similar
nanoemulsions are used to carry active ingredients deeper into the hair follicles in
shampoos. Sunscreens using nanoparticles are more transparent, less greasy and more
absorbable, making them more effective. In addition, the small particles are more
effective at deflecting certain wavelengths of UV light. In Japan, one company adds
nanosized crystals of hydroxyapatite to toothpaste, which is a key component of tooth
enamel. When toothpaste containing nano-sized hyrdoxyapatite is used, it forms a
protective film on the enamel, and can even restore the surface in damaged areas.
Nanoparticles also have very important applications in the medical industry. One area
where nanotechnology products are being developed is in medical diagnostics. These
include 'Lab on Chip' technologies as well as quantum dots and nanosized markers.
Nanoparticles can be used for early detection of cancer cells and the creation of miniature
diagnostic laboratories. Quantum dots have unique spectral characteristics, similar to a
'bar code' that allows them to be tracked very precisely when introduced into the body.
Another important area where nanoproducts are being developed is drug delivery
systems. By attaching specific chemicals to nano-sized particles, they can be delivered to
specific sites in the body. Their small size allows them to evade the body's immune
system, making delivery more effective.
A third area where nanotechnology is being used in the medical industry is in tissue
regeneration, growth and repair. For example, nanoparticles are being used in prosthetic
devices because of their high strength-to-weigh ratios and biocompatibility. They also
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show promise in cellular manipulations, such as in neural regeneration and re-growth of
body parts, however, these developments are not likely to be realized for 5-10 years.
Nonetheless, nanoparticles hold tremendous potential for future medical applications.
Despite the exciting application of this technology to improve the quality of human life,
caution is needed to ensure that products are safe as industry rushes to market these
products. While the immediate concern in most cases is whether there is any risk to
humans, there is also concern regarding possible environmental exposure. Unlike in the
medical applications, which are regulated by strict testing prior to FDA approval, most
cases in the cosmetic industry are relatively unregulated, even with respect to labeling
products that contain nanoparticles. Widespread human use may result in environmental
exposure through a number of pathways. For example, to what extent are sunscreens and
other cosmetics washed from the skin and transported to waste water treatment plants? In
this case, particles may end up in sludge, which is applied to agricultural and forest lands.
Drug delivery systems might be expected to follow a similar route to be deposited on the
same kind of lands, despite the fact that concentrations may be much lower than in the
case of cosmetics. It is important to consider the likely pathways of release of
nanoparticles to the environment in order to anticipate the likely targets of exposure.
D. Nanoscience and National Security
Interest in nanoscience by the U.S. Department of Defense (DOD) extends back to the
late 1970s when its Ultrasubmicron Electronics Research (USER) program began (Ratner
and Ratner 2004). Achieving size scales below micron was considered highly ambitious,
and the results of such success were still largely unknown. The prefix "ultrasub" was
coined, the usage implying that there would be a presumed simple extrapolation from the
more understood world of the micron scale to the submicron scale. The prescient visions
of President Kennedy and physicist Richard Feyman set the political and scientific tone
for the future of nanosciences through responding to perceived threats and the needs of
the Cold War. In 1959, Feymann described the futuristic new field of manipulating and
controlling things on a small scale. He predicted that by the year 2000 such capabilities
would be easily accessible allowing for work to be done at the atomic and molecular
scales. The early linkage between the realm of U.S. national defense and security with
nanosciences, in all its forms, still remains a driving force for the development of a
substantial portion of the field of nanoparticles and nanomaterials.
The USER program was followed in the 1980s by several programs to develop and utilize
the new tools of scanning electron microscopy and atomic force microscopy, which are
used today to measure and manipulate nanoparticles. The vision of Feyman materialized
considerably by the early 1990s when the DOD initiated programs to exploit
nanostructures. For example, the Defense Advanced Research Projects Agency
(DARPA) began the ultra fast, ultra dense (ULTRA) electronics program, an early
contributor in exploring technology options that the electronics industries currently use as
they continue to miniaturize electronics components into the nanometer size range.
Another example can be found in the Office of Naval Research when it began a program
in nanostructured coatings that by 2003 led to improved mechanical components onboard
ships. It is estimated that savings of up to $100M per year will be realized by the Navy
due to use of these new nanostructures. Several other defense and security related
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entities of the U.S. government showed ongoing interest in nanoscience such that in 1997
the DOD designated nanotechnology as a "strategic research area".
Such strategic decisions, and the initiating of development programs by governments in
the name of defense and national security, only begins to hint at the extent to which
nanoparticles may be released into the environment, through what is generally not
considered strictly commercial uses. It is likely that such releases will occur without the
full knowledge of the citizenry due to the sensitive intelligence nature of the applications.
Goals include achieving lower casualty rates of combatants in full-fledged warfare or
during military "police actions"; providing much greater real-time information
acquisition, manipulation, storage and connectivity; continuing to miniaturize and
reconfigure electronic devices so that computing surfaces can be formed into any shape
including flexible surfaces (e.g., data storage and manipulations surfaces can be applied
to walls, tank exteriors, light posts, etc.); deploying flexible and paper thin displays for
the military and police (e.g., direct write of information on the retina); and developing
prolific unattended sensors and uninhabited, automated surveillance vehicles and drones
to provide constant high data streams on local situations. The marriage of
semiconductors and biology will provide physiological monitors for alertness,
chemical/biological (CB) agent threat, and casualty assessment. Nanofiber protectants
will guard against CB threats while they also minimize heat burns and provide dynamic,
chameleon-like color adaptation for camouflage. Large scale dispersals of nanoparticles
as obscurants will render forces invisible; however, by remaining airborne for extending
periods of time, they may be distributed over long distances into the environment.
At this point, the future uses of nanoscience products for military applications are
seemingly endless, which raises a note of caution. The EPA's program to assess the
risks to ecological relationships and ecosystem function of producing and releasing
nanoproducts for commercial uses will be augmented by the substantial challenge to
assess and balance potential risks of poorly defined, and quite likely largely unknown,
releases of nanoparticles and nanomaterials in the pursuit of personal security through
defense and national security activities.
In summary, regardless of the application, e.g., commercial/industrial, cosmetic, medical,
or military, the potential future use of nanomaterials will be extremely high. The brief
review presented above of the expected applications of nanoparticles suggests that
manufactured particles, at unknown concentrations and in unknown structural forms, are
likely to enter every compartment of the biosphere (Wiesner et al., 2006) and as the
quantity and kinds of nanomaterials used by society increase, so does the potential for
unintended consequences (Colvin, 2003).
II. Statutory Authority
The products of this research will assist the EPA with enforcing regulatory requirements,
including pre- and post-registration product assessment and monitoring performed by
EPA's Office of Pollution Prevention and Toxic Substances (OPPTS) performed under
the statutory authorities of the Toxic Substances Control Act (TSCA) and the Federal
Insecticide, Fungicide and Rodenticide Act (FIFRA). OPPTS is already reviewing pre-
manufacture notifications for a number of nanomaterials that have been received under
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TSCA. In addition to OPPTS, the products will be of interest to EPA's Office of Air and
Radiation (OAR) and the Office of Transportation and Air Quality (OTAQ), specifically
with regard to nanomaterials registration applications. For example, OAR/OTAQ has
already received an application to register a diesel additive containing cerium oxide. The
results or our research also will be important for the Office of Solid Waste and
Emergency Response (OSWER). It is possible that nanomaterials will fall under
additional regulatory frameworks, such as the Clean Air Act, but additional on transport
and fate, as well as effects, will be needed to determine the full extent to which EPA
offices and legislation may be involved.
ORD is embarking on a new research effort to assess risk of stressors to ecosystems
through the development of its new ECO Multi-year Plan (ECO MYP). It is envisioned
that through the outputs resulting from the research done under this plan, EPA would be
able to embark on building a new framework for assessing risk to the environment by
explicitly linking ecology with the expectations and needs that humans have for their
continued existence. The goal of the new ECO MYP is to provide EPA with human-
centric ecological risk assessment tools and approaches that evaluate and value changes
in bundles of ecosystem services as EPA conducts its regulatory oversight, e.g.,
numerous permitting activities, establishing and overseeing "cap and trade" programs,
etc. It is possible that this new risk assessment framework eventually may influence the
activities of all of EPA's regulatory activities. While we are conducting the research of
this project we will keep apprised of the work being done in the ORD ECO MYP to
develop the new ecosystems services assessment framework. In fact, WED will be one
the "placed-based centers" for conducting the research of the new ECO MYP. It has
been identified that the initial overarching and program-wide critical ecosystem services
to consider will include, but not be limited to, water control, N/nutrient control and
carbon sequestration. This will be fortuitously strategic given the recent U.S. Supreme
Court ruling on Massachusetts vs EPA concerning regulating CO2, and the several
pending pieces of Congressional legislation because knowing the fate of carbon in the
biomes of the U.S. will be paramount to establishing and managing any cap and trade
program. Similarly, for assessing the effects of nanoparticles and potentially for all
activities and stressors of interest to EPA, it may come to pass that understanding the
sequestration of carbon will be a fundamental and critical ecosystem service in need of
consideration. As we approach the later phases of this project that involve assessing
ecosystem effects of nanoparticles, we will consider how to conduct our research so it
would be in support of and complementary to the emerging ECO MYP work on the new
ecosystem services risk assessment framework. Doing so would predispose this project's
efforts to develop an ecological risk assessment framework for nanoparticles that could
be linked with the ecosystem services risk assessment framework.
III. Scientific Rationale
Because of the unique physical characteristics of nanomaterials, they may affect aquatic
or terrestrial organisms differently than larger particles of the same materials (Colvin,
2003). However, assessing nanomaterial toxicity is extremely complex and multi-
factorial, and potentially is influenced by a variety of physicochemical properties such as
size and shape, and surface properties such as charge, area, and reactivity (Wiesner et al.,
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2006). Furthermore, use of nanomaterials in various products may result in novel
byproducts or degradation products that also may pose risks once they reach the
environment. Although the routes of transport and the ultimate fate of nanoparticles
reaching the environment are not well understood, work should begin now to assess the
toxicity of various particles as a first step in understanding the effect/toxicity component
of risk (Nel et al., 2006).
By comparing physical-chemical properties of nanoparticles to those of larger molecules
of the same material, it may be possible to estimate the tendency of nanomaterials to
cross cell membranes and bioaccumulate. Until now, current studies have been limited to
a very small number of nanomaterials and target organisms (Maynard, 2006). Similarly,
existing knowledge may allow us to predict how certain natural materials may behave at
the nano-scale in the environment (e.g., organic carbon); however, this knowledge may
not be sufficient to predict the behavior of a wide range of intentionally produced
nanomaterials (Murashov, 2006).
Several chemical and physical characteristics of nanoparticles control their uptake and
fate in organisms and systems. Molecular weight (MW, Zitko, 1981; Opperhuizen et al.,
1985; Niimi and Oliver, 1988; McKim et al., 1985) and effective cross-sectional diameter
of particles (Opperhuizen et al., 1985; Zitko, 1981) control their uptake across the
gastrointestinal (GI) tract of aquatic and terrestrial organisms and the gill membranes of
aquatic organisms. Uptake of neutral particles mediated by passive diffusion is low for
particles within the range of 600-900 molecular weight, a range containing the molecular
weights of nanomaterials (n-C6o fullerene is about 720, and for C84 carbon nanotube, >
1000). Published data indicate the limit for passive diffusion through gills ranges from 0.
95 to 1.5 nm, well within the variation found among nanoparticles. Charge also
contributes to both uptake and allocation of nanoparticles. For example, surface charge
of model wax nanoparticles alters the integrity and permeability of the blood-brain barrier
(Lockman et al., 2004). Other chemical and physical as well as biotic characteristics may
need to be considered when predicting accumulation and toxicity of nanoparticles in
aquatic systems. When calculating bioaccumulation factors for highly hydrophobic
neutral organic compounds the US EPA Office of Water, Oceans and Wetlands
(OWOW) uses water chemistry (e.g., dissolved organic carbon and particulate organic
carbon) and lipid content and the trophic levels of the target organisms (U.S. EPA, 2003).
Unfortunately, there is even less experimental evidence for nanoparticles in terrestrial
systems than for aquatic systems and humans. This is particularly concerning due to the
myriad life histories, genetics, species richness, and different system structural and
biological complexities found in terrestrial systems compared with these characteristics
and the work done on humans and aquatic systems.
Because the properties of nanomaterials are likely to result in uptake and distribution
phenomena that differ from conventional chemicals, it is critically important to conduct
mechanistic studies that will provide a solid understanding of these phenomena with a
range of nanomaterials and species. By understanding mechanisms related to
physicochemical uptake and distribution, there is potential to predict the toxicity for new
particles as they are developed. Studies related to human health effects assessments will
provide an important foundation for understanding mammalian exposures and some
cross-species processes (e.g., ability to penetrate endothelia and move out of the gut and
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into the organism). However, mammalian models of toxicity are limited compared to the
broad range of physiological differences among animal classes, most notably with respect
to respiratory physiology (e.g., gills in aquatic organisms, and air sacs and unidirectional
air flow in birds) and GI physiology (e.g., ruminents and hindgut fermentors). In
addition, plant and invertebrate (terrestrial and aquatic) models of toxicity may be even
more limited as they have even greater physiological differences among them as well as
different physiological functions compared with mammals. It is likely that understanding
toxicity to plants and invertebrates will require the development of different test models,
methods and approaches compared with those developed and being developed for
mammals. Perhaps the most challenging aspect of studying nanoparticle toxicity is
related to size, since the uptake and distribution of nanoparticles may follow pathways
not normally considered in the context of conventional materials (e.g., pinocytosis,
facilitated uptake, and phagocytosis).
A. Aquatic Ecosystem Effects
Few studies have been published on the effects of nanomaterials on aquatic ecosystems
and those that are published generally focus on specific life stages and processes in a
limited number of aquatic systems. We are not aware of any chronic or full life-cycle
studies being reported. Oberdorster (2004a,) studying effects of fullerenes in the brain of
juvenile largemouth bass concluded that C6o fullerenes induce oxidative stress, based on a
trend toward reduced lipid peroxidation in the liver and gill, significant lipid peroxidation
in the brain, and depleted levels of glutathione-S-transferease (GST) in the gill.
However, changes in fish behavior were not observed. Due to concerns about dosing
levels, the effects could have been due to random variation in individual fish. Using
standard EPA protocols, Oberdorster (2004b) tested non-coated, water soluble, colloidal
fullerenes (nC6o) and estimated a Daphnid 48-h lethal concentration for 50% of the
sample (LC50) at 800 ppb, and Lovern and Klaper (2006) tested titanium dioxide (Ti02)
and non-coated C6o fullerenes using Daphnia magna. Toxicity of TiC>2 and fullerenes
differed by an order of magnitude; fullerene solutions (particle clumps of 10-20 nm
diameter) had an LC50 of 460 ppb, and TiC>2 (10-20 nm) an LC50 of 5.5 ppm. How
particles are handled during preparation for use also affects toxicity. Particles of less
than 100 nm, obtained by filtering, had an LC50 of 7.9 ppm while if present at all, toxicity
was below detection limits for the larger lumps. Daphnia exhibited behavioral responses
to filtered fullerenes with juveniles unable to swim down from the surface. Adults were
disoriented and swam sporadically (Lovern and Klaper, 2006).
It would be prudent at this early stage in the collective research effort not to discount the
potential for differences in toxicity between natural, and engineered or manufactured
nanomaterials. Carbon black and suspended clay particles may have low toxicity to
aquatic organisms considering work of the EPA OPPTS based on toxicity studies and
quantitative structure-activity relationships (QSARs). Some suspended natural
nanoparticles have low toxicity to aquatic organisms, with ranges in thresholds for
observed effects from tens to thousands ppm. The limited preliminary work done with
engineered, manufactured nanomaterials substantiates this conclusion. Embryos of
zebrafish exposed to aggregates of single-walled carbon nanotubes (SWCNTs) had
hatched at reduced rates at 72 hrs; by 77 hrs post fertilization, all embryos hatched
(Cheng and Cheng 2005). However, SWCNTs have greater pulmonary toxicity than
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carbon black nanoparticles (Lam et al., 2004). Some nanomaterials have injured organs
of test animals by novel mechanisms such as the unusual inflammatory responses to
specific nanomaterials found in mammals reported by Shvedova et al., (2005).
Nanomaterials (C6o aggregates) may be bactericides against gram positive and negative
bacteria in growth media (Fortner et al., 2005). Work is needed to determine if reduced
growth and respiration can be replicated under more realistic conditions. Such findings
may have substantial implications on the efficacy of waste water treatment plants, the
structure and function of microbial communities in sewage sludge effluents and natural
waters, as well as on the functioning of soils.
B. Terrestrial Ecosystem Effects
There have been very few studies conducted to assess potential toxicity of nanomaterials
to terrestrial species (plants, wildlife, soil invertebrates, or soil microorganisms), and no
studies examining broader, integrated ecosystem effects. Toxicity test data on rats and
mice obtained for human health risk assessments are some of the few available to
consider as surrogates for terrestrial mammals. Studies using lungs found that ultrafine
or nano-sized particles are more toxic than larger particles of identical chemical
composition when expressed on a mass basis (Oberdorster et al., 1994; Li et al., 1999;
Hohr et al., 2002). However, some nanomaterials display unique toxicity unexplained by
particle size alone (Lam et al., 2004; Warheit et al., 2004). Toxicity to mammalian
epidermal cells in culture has also been reported (Shvedova et al., 2003).
As a starting point, it is reasonable to consider that many of the same chemical, physical
and biotic characteristics of nanomaterials that modulate the fundamental mechanisms of
uptake by aquatic organisms may similarly influence the uptake systems of individual
plants, fungi, bacteria and soil mesofauna, especially when studied in vitro. However,
more than in aquatic systems, bioaccumulation in intact or reconstructed terrestrial
systems is affected by a very highly complex and species rich trophic structure that is
further influenced by the varying, complex physical and chemical environment within the
soil. It is unlikely that data derived from comparatively well-mixed aquatic systems will
be useful for making direct projections on effects in and to terrestrial systems.
Because many industrial nanomaterials are designed to have strongly reactive surfaces, it
is possible that mechanisms for toxicity may exist independent of uptake (e.g., by
disrupting respiratory epithelial structure/function or other surface cell structures and
functions). It also is possible that the highly reactive surfaces of nanomaterials may
reduce toxicity once the particles are in soils due to strong sorption kinetics on soil
surfaces. Yang and Watts (2005) reported that alumina nanoparticles (13 nm) slowed
root growth in a soil-free exposure medium of the agronomic species used in toxicity
tests to conduct risk assessments of pesticides: corn {Zea mays), cucumber (Cucumis
sativus), soybean (Glycine max), cabbage (Brassica oleracea), and carrot (Daucus
corota). They also reported that coating the alumina nanoparticles with an organic
compound (phenanthrene) reduced inhibition of root elongation. Larger alumina
particles (200-300 nm) did not alter root growth, suggesting that alumina was not causing
the toxicity. The authors hypothesized that the surface charge on the alumina
nanoparticles may have decreased plant root growth. Further, Murashov (2006) noted
limitations of this report related to any discussion of phytoxoicity of alumina, and
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increased solubility of nanoscale alumina that may have increased concentrations of
alumina species. Depending on the soil type, substantial amounts of complex and highly
diverse organic materials can be present in soil that result from decomposition of net
primary production products. Any extrapolations of results from soil-free experimental
conditions to field situations must be done judiciously for reasons discussed above. In
addition, individual ecosystem responses to stresses are known to be idiosyncratic
especially when considered without full appreciation of the condition of the ecosystem.
Understanding the interacting web of ecosystem processes is essential to reducing
uncertainties around projections of nanoparticle effects, especially when starting with
data derived from lab studies that use systems of low biological complexity.
C. Nanoarticles of Interest in this Research Effort: Ti02 and Single Walled
Carbon Nanotubes (SWCNTs)
As points of entry into the characterization of the potential environmental effects of
nanomaterials the EPA has selected nano-sized titanium dioxide (Ti02) and single-walled
carbon nanotubes (SWCNTs). Nano Ti02 is a metal-based material that is used widely
in a variety of products, most notably in modern sunscreens. SWCNTs represent the
family of carbon-based nanomaterials that includes fullerenes (C6o) and muti-walled
carbon nanotubes. SWCNTs impart added strength to plastics and polymers used in
automobile and airplane parts as well as a variety of other products. Both nano-Ti02 and
SWCNTs have potential for use in environmental remediation of contaminated sites
(Lovern and Klaper, 2006). A few papers have reported on the environmental effects of
nano-Ti02. Lovern and Klaper (2006) reported that exposure to either nano-Ti02 or C6o
fullerene increased the mortality of Daphnia magna, with the Ceo fullerene having higher
levels of toxicity at lower concentrations than nano-Ti02. In a study evaluating the
effects of 5 types of nanoparticles (multi-walled carbon nanotubes, aluminum, alumina,
zinc and zinc oxide) on seed germination and root growth of higher plants, only nano-
zinc and nano-zinc oxide had any effect (Lin and Xing, 2007). Hund-Rinke and Simon
(2006) reported that Ti02 had toxic effects on algae (Desmodesmus subspicatuas) and
Daphnia magna. The toxicity was enhanced when the experimental units were exposed
to UV-illumination since Ti02 has photocatalytic properties. In one of the first reports on
the effects of nano-materials on soils, exposure of soil to Ceo fullerene had no significant
effects on the structure and function of the soil microbial community or microbial
processes (Tong et al., 2007). These authors suggested that the Ceo fullerene partitioned
into the soil organic matter which decreased its solution-level bioavailability. It is
unknown what the effects would be in soils with low organic matter or if the
bioavailability of metal-based nanomaterials would also be reduced by the native soil
organic matter. Surface modification of nanomaterials may also affect these outcomes.
Since a major problem associated with nanomaterials is their inherent insolubility and
nanoparticle-to-nanoparticle interactions that cause particles to aggregate, the surfaces of
nanoparticles are being functionalized to increase their solubility and to reduce inter-
particle interactions (Wiesner et al., 2006). These modifications may make
functionalized nanomaterials more mobile in the environment thereby increasing the
probability for exposure to a wide spectrum of organisms in many types of systems
[human,marine, aquatic and terrestrial (natural and managed)]. In a recent study where
the solubility of SWCNTs was increased by coating them with lysophospholipids (LPP),
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Daphnia magna ingested the modified SWCNTs through normal feeding behavior and
then digested the LPP which led to acute toxicity (Roberts et al., 2007). This is the first
report of in vivo biomodification of a nanomaterial by an aquatic organism. The
biomodification changed the physical properties of the initial nanomaterial resulting in
increased toxicity of the core nanomaterial. The potential for biomodification of
nanomaterials needs to be included as the environmental fate and toxicity of
nanomaterials are considered.
IV. Objectives and Science Questions
The primary objective of this project is to conduct research on terrestrial organisms and
terrestrial ecosystems that will assist in the overall ORD research effort to support the
regulatory work done by OPPTS as they seek to address emerging questions and evaluate
the effectiveness of their current toxicity risk assessment frameworks to assess
nanoparticles and nanomaterials. Our contribution will be: 1) to evaluate a limited set of
current OPPTS toxicological screening tests to determine if they are appropriate for
nanomaterials, and 2) to develop methods, recommendations and information to help
them determine risks of such particles and materials on ecological relationships and
ecosystem structure and function, primarily for terrestrial ecosystems.
The resultant overarching science and regulatory question in this project is:
Do nanoparticles and nanomaterials, in their various forms, impart toxicity to
individual organisms, alter ecological relationships, or change ecosystem structure
or function?
Because of the importance of providing timely information to OPPTS via interim
research products and updates, and the longer time-frame that is essential for conducting
research on ecological relationships/ecosystems, we have organized the research into
three phases. Our involvement in the phases will overlap somewhat in time, and their
purposes and goals are not mutually exclusive. However, in general, the three phases
consist of reasonably distinct goals and objectives that, when all are completed, will
provide a substantial body of knowledge to OPPTS about the ecological risks of two
types of nanoparticles. This body of knowledge about effects on ecological relationships
and ecosystems will serve as part of the larger understanding that personnel in OPPTS
will attain and use as their guide to making decisions about establishing the EPA's
overall human and ecological risk assessment frameworks for nanomaterials.
A. Phase I
The primary objectives of Phase I include 1) identifying, and establishing as needed,
appropriate facilities at WED to conduct the research, and then 2) conducting traditional
OPPTS toxicity tests with plants and soil invertebrates to evaluate their suitability for
nanoparticles (Figure 1). The set of 3 traditional toxicity tests selected will be from
among those used or recommended by OPPTS in its current risk assessment framework
for pesticides
(http://www.epa.gov/opptsfrs/publications/OPPTS Harmonized/850 Ecological Effects
Test Guidelines/Drafts/Drafts.html). The 3 tests we have selected evaluate a particular
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cross-section of organisms or organism processes characteristic of terrestrial and aquatic
systems. These tests were selected because they evaluate a subset of key processes in
terrestrial and aquatic ecosystems, e.g., photosynthesis, cell division and differentiation.
In particular, the organisms studied in these tests will be among those with which we are
familiar, i.e., we either know or can learn quickly how to work with them, or we already
have a highly informed understanding of their physiology and ecology. As a lesser goal,
there will be some limited work conducted in Phase I to serve as a basis to identify
toxicity mechanisms, i.e., mainly to establish whether or not a given nanoparticle is taken
up by the test organism. Finally, there will be the opportunity to begin exploring the use
of or developing novel testing methods, novel both within the larger scientific research
effort and novel to OPPTS's current established risk assessment framework, e.g.,
involving proteomic and genomic analyses. Exploring the use of such novel testing
methodologies will be advantageous especially in cases where existing traditional
protocols are deemed ineffective or inconclusive, and can also contribute to identifying
toxicity mechanisms. Results from Phase I will be important for OPPTS regarding the
suitability of using its existing risk assessment framework and bioassays, and they also
will be the foundation for the research to be done in Phase II.
Contributions made in Phase I will address several key questions:
• Are established EPA toxicity testing protocols applicable for testing
nanop articles?
• What methods are appropriate for challenging organisms with nanoparticles in
ways that are congruent with the media and the chemical/physical forms in
which actual ecological conditions are likely to occur?
• What are the appropriate methods for testing the toxicity/hazard of
nanomaterials to ecological receptors (terrestrial invertebrates, plants,
microbes) ?
• What are other testing methodologies to consider that might be more
appropriate and useful to determining toxicity of terrestrial ecosystem
organisms to nanoparticles?
B. Phase II
Research in Phase II explores why we did or did not observe a response using the
traditional toxicity tests during Phase I (Figure 2). We also will take initial steps to
increase test system complexity, both by increasing stressor complexity (applying
multiple stressors) and by increasing biological complexity. We will expand our metrics
of interest by measuring additional response variables (e.g., biochemical pathways,
responses that develop over longer time periods, etc.) to be more comprehensive in our
toxicity assessments. We will continue to explore novel testing methodologies by
undertaking laboratory work with nanoparticles to link results of novel methodologies
with our research to increase system complexity. Overall, for Phase II, we are interested
in obtaining a mechanistic understanding of the responses observed or not observed
during Phase I, particularly as biological outcomes relate to size (agglomeration), charge,
and composition of the nanoparticle. Even though our research will focus on a limited
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number of nanoparticles it will be critical in that it will contribute to a longer-term goal of
EPA to develop an appropriate type of QSAR for nanomaterials/nanoparticles. Also, it
will aid us in moving our research work on the two nanoparticles into Phase III. The
focus through out Phase II, as during Phase I, will be the response of individual
organisms, but in this Phase not when the organisms are living in isolation; in other
words, Phase II begins to explore how organisms respond individually as members of a
community. Lastly, a central connecting thread through out Phase II will be to use the
information obtained on mechanisms to formulate hypotheses and design experiments
with more complexity (biologically and edaphically) that will be undertaken as part of
Phase III.
Contributing to the following questions of interest to EPA will be of central focus in
Phase II:
• What are the mechanisms related to any toxicity observed during the traditional
testing approaches?
• Are there unique toxicity mechanisms or effects associated with exposure to
nanoparticles?
• What are the effects (local and systemic, acute and chronic) resulting from
exposure to nanoparticles or their byproducts?
• What are the underlying modes of toxicity and how do they vary across an array
of ecological receptors?
• What are the absorption/adsorption, distribution, metabolism, elimination
(ADME) parameters for various nanoparticles using an array of ecological
receptors?
• Are there initial indications that developing molecular level QSARs may be
possible (or worthwhile) at some point for nanoparticle stresses on terrestrial
organisms, to provide primary screening information for ecological risk
assessments?
• What predictive tools and methods may be relevant to address the enormous
range of nanoparticles that will likely be developed?
• What are the appropriate exposure and/or dose metrics for comparing potency
among nanoparticles (e.g., particle size, charge, geometry) as we move beyond
the simplified traditional toxicity testing approach) ?
• Might it possible to further modify ADME and mode of action information into
QSAR) and other related predictive indicators?
C. Phase III
Phase III builds on results from Phases I and II, and involves examining responses to
nanomaterials at higher levels of biological diversity and system complexity, focusing on
both responses of individuals and on more integrated system responses (Figure 3). By
this we mean that the responses of individuals are not the only endpoints of interest;
rather, the goal is to understand how previously determined individual responses affect
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organism interrelationships and hence the function of ecosystems. To do this we will
conduct experiments across different scales and complexities to examine ecosystem
trophic diversity, changes in fluxes and pool sizes of major resources and nutrients, and
ultimately identify if ecosystem function and/or structure are/is altered by exposure to
nanoparticles. Reconstructed ecosystems will include native soil and plant species to
reflect trophic structure and diversity characteristic of natural ecosystems. The results
from Phase III research will allow for model development and verification at larger
scales, and hopefully while considering both shorter- and longer-term response times.
Phase III work will help evaluate any extrapolations that were made, or could have been
made, originating from earlier EPA ORD work on fate, transport and exposure. In
addition, the intention is that results from Phase III will provide a basis for extrapolating
toxicity to other (untested) nanoparticles based on their unique chemical or surface
characteristics.
The following questions will be addressed in Phase III:
• Will nanoparticles interact with other stressors or the physical environment in
ways that create unanticipated and perhaps novel risks?
• Are there important interactions between nanoparticles and microbial, plant, or
animal populations and their interrelationships in natural or managed systems?
• Do nanoparticles alter trophic structure in ecosystems, leading to changes in
pools or fluxes of important material resources?
• Can the ecological consequences of nanoparticle exposure be adequately
assessed with existing ecological models?
V. Experimental Approach
We will address the questions listed above in the three phases using empirical data from
molecular, whole-organism, population, community and ecosystem responses to
nanoparticle exposure to predict and identify possible effects of nanoparticles on
terrestrial ecosystem structure or function. Early experiments will employ highly
controlled laboratory conditions to examine the effects on individual organisms, with
subsequent studies imparting more complexity of various kinds to enhance realism both
in trophic structure and scale of tested systems. The most challenging aspect of the
proposed research will be to scale exposure-response data from individuals to higher
levels of biological organization, e.g., eventually to that of ecosystems.
Since research on most aspects of the ecosystem effects of nanoparticles is in its infancy,
this project will initially focus on developing safety protocols for working with the
nanoparticles, and building and/or improving facilities to conduct this research while
minimizing exposure of research staff. We also will do initial evaluations of potential
biological activity of the nanoparticles selected for study. Initial empirical research will
focus on 3 currently used, traditional OPPTS toxicity test protocols for toxicity to
terrestrial, aquatic and marine plants, and soil invertebrates. We will then make initial
recommendations to OPPTS on whether the traditional toxicity testing framework is
appropriate or whether it may need to be modified in its entirety or perhaps altered by
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considering including procedures derived from novel methodologies to assess risks of
these nanoparticles. As effects on individual species are identified in the earlier phases of
the project, nanoparticle effects research at community and ecosystem scales will be
developed. Finally, through developing appropriate hypotheses resulting from the earlier
research done in the project, integrated ecosystem level studies using reconstructed
ecosystems will be conducted in a state-of-the-science controlled environment facility
located at WED.
A. Phase I Research Activities
Several components will comprise Phase I activities (Figure 5-1). Our initial effort will
aim to develop safe protocols for handling nanoparticles. The procedures will be based
on the most recent NIOSH guidelines and recent publications as they become available.
We will consult other laboratories already conducting nanoparticle research to become
familiar with the procedures they employ. A Health and Safety Plan will be developed in
cooperation with WED's Health and Safety Specialist, and will include safe handling
procedures for all aspects of the research, from purchasing materials to their disposal.
Since worker exposure is perhaps the most important consideration in handling
nanoparticles, it is critical that contained facilities be used for storage, transfer and
disposal of nanoparticles. One dedicated lab will be set up for this purpose. The lab will
have an air flow hood, into which a HEPA filter will be installed to hinder release of
particles and reduce the likelihood of particles contaminating WED and exposing any of
the on-site staff. In addition, special containers will be set up and labeled for
nanoparticle waste. The lab will be labeled and locked when not occupied so personnel
at WED are aware of the presence of nanoparticles and the purpose of the lab. In
addition to establishing appropriate laboratory facilities and developing safety protocols,
we will begin to modify as necessary on-site growth chambers, glass houses and other
exposure facilities.
We noted in a section above that our research will focus initially on two nanoparticles:
Ti02 and single-walled carbon nanotububes (SWCNTs). There are significant
overarching challenges to studying the toxicology of these two particles. One challenge
deals with identifying an aqueous carrier that is non-toxic, and which also allows the
particles to remain in suspension without significant agglomeration. For example,
SWCNTs are not soluble in water unless they are functionalized, which affects their
toxicity (Jaclyn Canas, pers. comm.) and likely their reactivity within the environment.
Any carrier used should maintain the unique surface charge characteristics of the particle.
Therefore, initial research related activities will be focused on identifying appropriate and
effective carriers. One possible source of Ti02 is Dr. Bellina Veronesi, of the
Neurotoxicology Division of NHEERL in Research Triangle Park, NC. She has
extensive experience with Ti02 in mammalian systems and may be able to help us with
initial characterization and localization after cellular uptake. We also are discussing
options with materials science specialists at Rice University and Batelle labs as possible
sources for these particles. Our intent is to establish a memorandum of understanding
(MOU), a cooperative agreement or a contract with the appropriate organizations who
can supply us with a consistent source of Ti02 and SWCNT suspensions, of known
concentration and particle size distribution.
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Another challenge is determining the rate of application of nanoparticles for the
traditional toxicity tests; determining representative exposures will be of concern for all 3
phases of the project. Initially, relatively high doses will be used to increase the
likelihood of a response. However, since virtually all chemicals are potentially toxic to
cells at some level, it will be important to conduct subsequent tests at concentrations
expected to occur in the environment (Colvin, 2003). An additional complicating factor
related to selecting the proper exposure rates of nanoparticles is that many of them are
imbedded in products that also have their own chemical and physical characteristics. For
example, HO2 is added to sunscreens to provide protection from harmful UV radiation.
At the same time it is being considered for treatment of water in waste water treatment
plants because its photocatalytic properties enhance the photodegradation of organics in
waste water (Hund-Rinke and Simon, 2006). Therefore, one primary route of
environmental exposure is through water, especially through waste water. Water, waste
water and any waste treatment plant sludge likely will reach the soil. Such an indirect
pathway makes it extremely difficult to assess appropriate application rates for toxicity
assessments. Also, it would be difficult at this time to determine which of the myriad
forms of the particles reaching the unconstrained environment might be worthy of
investigation. For both nanoparticles, we will start our research using particles in their
native, unadulterated forms.
Unfortunately, there is almost no information on exposure levels for most particles
currently being marketed, and the ORD effort on the fate, transport and exposure of
nanomaterials also is in its early stages. At the point we begin our research on toxicity
we will rely on what others within or outside ORD have found.
1. Traditional OPPTS Toxicity Tests
Once we have identified a consistent source of particle suspensions, three existing
OPPTS toxicity tests will be examined for their efficacy in testing the Ti02 and SWCNT
nanoparticles. The first toxicity test employed will be the Terrestrial Plant Toxicity Test
(OPPTS 850.4100; see Appendix A). A similar test looking at germination and root
elongation was recently run with 5 nanoparticles and will be used as a starting point for
our tests (Lin and Xing, 2007). The second toxicity test we will examine is the Algal
Toxicity Test (OPPTS 850.5400; Appendix B). A recent study looked at the ecotoxic
effects of nanosized Ti02 on the algae Desmodesmus subspicatus. This report provides
useful information on how to set this test up. The third toxicity test to be examined will
be the Earthworm Subchronic Toxicity Test with the earthworm Eisinia foetida (OPPTS
850.6200; Appendix C). Dry-runs of each toxicity test will be conducted prior to use of
any particles to identify problems with test organisms and ensure consistency in results.
2. Terrestrial Plant Toxicity Test (Appendix A)
This test was selected because it examines a critical life stage of plants involving cell
initiation, differentiation, elongation and development, and maturation. Seeds of 10 plant
species (see Table 1, Appendix A) will be collected and germinated according to standard
procedures. Prior to germination, filter paper in each Petri dish will be treated with
nanoparticles and germinating roots will be examined after a standard test period for
indications of toxicity. Two controls will be employed, one consisting of distilled water
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treatment only, and a second consisting of the aqueous carrier only. Because earlier tests
using this protocol with other particles have found that some carriers used to suspend
nanoparticles may exhibit toxicity (Jaclyn Canas, pers. comm.), we may need to test
additional carriers for toxicity. If carrier toxicity is identified in early runs, we will test
additional carriers in the absence of nanoparticles until a non-toxic carrier is identified.
Identifying new carriers will be done in collaboration with materials science specialists to
ensure that we are not selecting a carrier that inherently alters the structure or properties
of the particles.
If either nanoparticle significantly inhibits germination or radicle growth for any species
during the test, we will move on to Tier II testing for that species-particle combination in
order to develop exposure-response relationships. To do this, the test will be repeated
using a range in concentrations of nanoparticle suspensions. Radial emergence and
overall growth over a standard measurement period will be recorded.
3. Algal Toxicity Test (Appendix B)
This test was selected because it involves a aquatic plant species, and are green plants in
their mature form undergoing the important process of photosynthesis. Ribulose
bisphosphate carboxylase (Rubisco), the enzyme responsible for chemically fixing CO2
into organic molecules through the process of photosynthesis, is arguably the most
important enzyme on earth. If nanoparticles affect Rubisco or other important
photosynthetic enzymes or organelles, it would indicate the potential for significant
ecosystem effects. Similar to the Terrestrial Plant Toxicity Test, we will conduct dry
runs of the experiment during the first year while lab facilities are being updated in order
to ensure standardization of the test.
One unique challenge in running this test is the introduction of the nanoparticle
suspension into aqueous (and marine) nutrient environments. Algal species selection will
be based on commercial availability and published use in prior toxicity testing with other
compounds. As with the Terrestrial Plant Toxicity Test, carrier toxicity will be tested
using a deionized water control. Regardless of the nanoparticle carrier being used, there
is a high potential for binding of the nanoparticle in the algal growth medium. If this
occurs, it is likely that the toxicity of the particles will be diminished. In Phase I we will
not attempt to identify the reasons for a negative test resulting from particle binding; this
will be examined in Phase II. If algal toxicity is observed in Phase I under our test
conditions, Tier II tests may be performed also.
4. Earthworm Subchronic Toxicity Test (Appendix C)
Earthworms (Eisinia foetida) are critically important organisms in the decomposition of
soil organic matter, subsequently releasing nutrients and building soil structure. As a soil
invertebrate, it may be a good indicator species for identifying toxic effects on other soil
invertebrates. Earthworms may be particularly susceptible to nanoparticles since each
day they ingest several times their body weight in soil. In addition to direct ingestion,
earthworms may be susceptible to dermal uptake.
As in the other toxicity tests, carrier toxicity will need to be examined, and dry runs will
be performed to optimize the growing conditions for earthworms. Two controls will be
run, one with carrier alone and one with deionized water only. As with the Algal
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Toxicity Test, there is high potential for particle binding with the growth medium, in this
case, the soil substrate used to grow the earthworms. We will test at least two soil media
for their possible use. Endpoints of interest will include growth, biomass, and mortality.
If toxicity is observed, Tier II testing will be conducted to develop exposure-response
relationships. If no toxicity is observed, the possible reasons for this negative result will
be examined more thoroughly in Phase II.
5. Particle Uptake
As noted above, if we observe a negative response (e.g., toxicity is not observed) in any
of the above tests, the response provides little information on the applicability of the test
for assessing nanoparticle toxicity. It could result from either a lack of uptake, for
example, due to particle binding, surface adhesion, agglomeration, etc. or it could
indicate the particle is not toxic to the test organism at the concentrations tested. In order
to help elucidate the meaning of a negative result, it is important to determine whether or
not the particle is taken up (adsorbed and/or absorbed), and further, where in the cell it
exists. While answering this question begins to address issues of mechanisms, and is to
be conducted as one of the primary objectives of Phase II, there will be some need to
examine uptake in Phase I to examine or develop novel test procedures.
Characterizing uptake is not an easy task, and several methods will be examined to help
elucidate uptake and location; also see Phase II section below for details on techniques
not discussed here. One method would be to use 14C labeled SWCNTs in the tests
followed by employing techniques of micro-autoradiography (http://www.leica-
microsvstems.com/website/lms. nsf?opendatabase&path=/Website/Applicat.nsf/(ALLIDs)
/E580CEAEDF96882CC1256DA20046BA28). This procedure involves exposing the
test organism to the labeled nanoparticle, allowing sufficient time for uptake, and then
detecting the location of the labeled particle visually via the decay of the isotope. We
will be able to determine if any uptake occurred and if so, whether it was strictly
adsorption or also included absorption. If absorption occurred, then the intracellular
locations can be determined. While 44Ti is radioactive
(http://www.aanda.org/images/stories/PressRelease/PRaa200614/praa200614 print.pdf)
it exists as ejecta of supernovas and thereby requires exceptionally high temperatures to
be produced (Ahmad et al., 1999), doing micro-autoradiography of TiC>2 nanoparticles
may not be possible as we are not aware of readily accessible sources of this isotope.
However, several stable isotopes of Ti (MW 46 through 50) exist that might be exploited
in other detection methodologies (http://www.americanelements.com/tiox49.htmn. WED
is no longer licensed to use radioisotopes, so micro-autoradiography work would need to
be conducted at Oregon State University, or we could issue a contract to a licensed
researcher to do this work.
A second detection/localization method involves using nanoparticles known as quantum
dots (http://en.wikipedia.org/wiki/Quantum dots), which are spectrophotometrically
unique in their signature. In this method, we would work with a materials science
specialist to chemically generate quantum dots, and attach them to both Ti02 and
SWCNTs. Care must be taken not to alter the unique properties or surface characteristics
of each nanoparticle by attachment to the quantum dots. Following exposure, confocal
microscopy can be used to identify uptake and location in the cell. Oregon State
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University has confocal microscopy capabilities. This technique is not without
shortcomings in that the nature of the structure and chemical composition of the quantum
dot may also impart a toxic response in the subject organism. Since quantum dots may
alter the inherent toxicity of the particles we will first examine the toxicity of the dots
before including them in our tests.
A third method for assessing uptake and location of nanoparticles in cells involves
electron microscopy and tomology to provide 3-D maps of particle location (Porter et al.,
2006; also see Phase II section below). This is relatively new technology, and will be
possible for us to do only if we develop cooperative agreements with another
organization. The various electron microscopy methods likely represent the best
diagnostic tools to use as in some cases, 3-dimensional maps of particle location can be
constructed, but such techniques are also the most expensive to gain access to, or to
obtain the facilities.
Although all of the above methods give information on nanoparticle uptake and location,
none of them will yield good information on the quantity of particles taken up. The
segment of nanosciences dealing with quantification and characterization is rapidly
changing, and it is likely that new tools will be available within the timeframe of this
project. Therefore, we will continue to examine the literature and identify state-of-the-art
technologies that will help us to characterize responses to these two nanoparticles.
Nonetheless, it is likely that the majority of routine effects studies will continue to rely on
'exposure' concentrations rather than 'dose', since dose implies uptake in the test
organism. Our current inability to quantify particle uptake may impair our assessment of
risk to ecosystems in the long-term (Phase III), but it should not impair our ability to
examine mechanisms in the short-term (Phase II).
6. Novel Testing Methodologies
Results from evaluating the traditional OPPTS toxicity tests done in Phase I may reveal
important information that will allow us to suggest alternative, and perhaps develop,
novel testing methods. For example, if our carriers are interacting with the growth media
and prohibiting uptake, we may be able to modify the growth media to eliminate the
interaction. Likewise, altering the carrier or the mode of exposure (dry vs. wet) may
enable us to improve the existing protocols for use with nanoparticles. However, until we
have a better and fuller mechanistic understanding of the uptake and responses to
nanoparticles, we cannot fully comment on developing modified or new testing protocols.
Since most of the mechanistic studies will be conducted in Phase II, there will be limited
ability to suggest other alternative testing protocols based solely on Phase I results.
We will begin initial work in this phase on identifying and evaluating potential novel
methodologies as alternatives to the traditional OPPTS toxicity testing procedures. One
alternative test methodology that holds great promise involves using organisms whose
genomes are well characterized. For example, by using a collection of mutants with
systematically produced gene deletions involving non-essential genes ("knock-outs"), we
may be able to expose large numbers of organisms to nanoparticles and look for
responses at the gene level along with whole-organism level responses (see sub-section
Development and Assessment of Novel Testing Methodologies in Phase II section below
on yeast knockout (YKO) collections for more details). Some advantages of using such
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an approach are that screening is rapid, and more importantly, that a positive response
can readily aid in gaining a mechanistic understanding of toxicity since the affected genes
are identified. This genomic-based information would be extremely valuable in
characterizing other organisms that may be susceptible to nanoparticle exposure. We will
begin to explore the availability and adaptability of such rapid screening protocols during
Phase I, and will do so more fully in Phase II.
7. Developing a Conceptual Framework for Ecosystem Effects
The final component of Phase I will involve developing a conceptual framework,
presented in a white paper for publication, concerning assessing risk of possible effects of
nanoparticles on ecosystems. In this effort we will review the literature to date on fate,
transport, exposure and toxicity of various nanoparticles and nanomaterials, evaluate the
strengths and weaknesses of employing the risk assessment testing framework of the EPA
as used currently, and the use of various novel methodologies, linking all of the above to
well known concepts of ecology and terrestrial ecosystem sciences. Through doing this,
we intend to propose the core elements that are needed to evaluate the ecological risks
arising from the myriad products of the emerging nanotechnology field. The proposed
risk assessment framework will focus on diversity and ecosystem complexity, identifying
possible ways that nanoparticles may disrupt ecosystem structure or function. Given our
expertise, we will be interested in any possible roles that nanoparticles may play in
altering the function of keystone species in terrestrial ecosystems. Of particular concern
will be soil biota populations and plant-soil biota interactions because of the critical
importance of soil biota in cycling nutrients and other resources, and maintaining the
overall trophic structure of systems.
B. Phase II Research Activities
Phase II research will build on results from Phase I primarily by 1) further developing our
understanding of mechanisms related to any responses, or lack of responses, observed
when conducting the traditional toxicity tests in Phase I, and 2) starting to increase the
stressor and biological complexity of tests to instill more ecological realism in developing
a risk assessment framework for nanoparticles (Figure 5-2). We will increase system
complexity both in terms of stressors (multiple stressors) and biology while also
measuring different response variables. We will continue to explore various novel testing
methodologies involving proteomics and genomics. We are interested in obtaining a
more complete mechanistic understanding of the responses observed, particularly related
to the size, charge and composition of the nanoparticle involved. This information will
be critical in that it will contribute to any broader and longer-term effort done by EPA to
develop QSARs. The focus throughout Phase II, as in Phase I, is the response of
individual organisms, but in the latter stages of Phase II, not when living in isolation; in
other words, Phase II begins to explore how organisms respond individually as members
of a community.
Research in Phase II will be organized along three areas: Mechanisms of Response,
Development and Assessment of Novel Testing Methodologies, and Stressor and
Biological Complexity.
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1. Mechanisms of Action
Conducting research in Phase I will be critical for determining exactly which organisms
and methods are useful to begin understanding mechanisms of action (MOA) as part of
Phase II research. In Phase I, we anticipate both negative and positive response to
traditional OPPTS toxicity tests. A positive response suggests toxicity. In contrast, a
negative response does not necessarily indicate non-toxicity. One overall goal of the
MOA research to be conducted in this phase is to understand the underlying reasons for
the responses observed in Phase I in hopes of being able to extrapolate these responses to
other organisms. This work also will be necessary to evaluate the likely success or
failure of applying traditional OPPTS testing protocols to nanoparticles. Perhaps just as
important to the issue above, the MOA research will be required to understand the
relationship between organism responses and the nature of the nanoparticles resulting in
response, perhaps eventually leading EPA to the development of QSARs. Since we will
only be testing two particles here initially, research towards developing QSARs will need
to proceed in conjunction with other divisions in NHEERL, and with research done
domestically and internationally at other organizations. If initial results from assessing
the standardized toxicity tests warrant it, we will coordinate with the larger EPA program
to select additional nanoparticles to contribute to building a body of knowledge relating
to QSARs.
Regardless of whether toxicity is observed it will be instructive to know whether the
nanoparticle was retained in the apoplastic region or was absorbed into the symplasm,
and if any chemical or biochemical transformation and/or other changes occurred to the
nanoparticle. If taken up, the location within the symplasm, i.e., retained in the
cytoplasm, or affixed to or further transported across membranes of organelles, will be
determined. While particle location in the cell was initially discussed, and will be a focus
of research in Phase I, in Phase II we will attempt to link location to organelle function.
The field of localizing nanoparticles in biological systems is developing rapidly [e.g., the
National Institute of Standards and Technology programs on Biomaterials
(http://www.cstl.nist.gov/proiects/fy05/bio05batteas.pdf). and Health and Medical
Technologies (http://www.cstl.nist.gov/proiects/fy05/health05zeissler.pdf). We are
aware of some promising techniques for Ceo fullerenes, in this case with the ability to
construct 3-dimensional maps of cells and the location of the nanoparticles; energy-
filtered transmission electron microscopy (EFTEM) and scanning transmission electron
microscopy (STEM)-based electron tomography. Porter et al. 2006 demonstrated the
ability to obtain 3-dimensional distributions when they studied the uptake of Ceo
fullerenes by human monocyte macrophages using EFTEM and STEM-based electron
tomography and were able to visualize various cell structures such as membranes,
mitochondria, ribosomes and the nucleus without the need for traditional staining. They
found the fullerenes were in several sub-cellular compartments; among them secondary
lysosomes, along the outer and nuclear membranes and most notably in the nucleus.
Another technique we may consider to locate particles within tissues is laser-confocal
microscopy (http://www.phvsics.emory.edu/~weeks/confocal/). As we approach these
needs of the project we will assess the then-current state of the sciences, our budget, the
personnel available and their expertise at WED, the confocal facilities in Corvallis and
any capabilities and facilities existing within NHEERL and ORD, and form the necessary
collaborative or contractual relationships. Alternatively, we may form collaborations
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external to EPA or Corvallis with materials science specialists, physical chemists, cell
biologists and microscopists.
An important second step after linking particle location with organelle function is to
measure whether the expected physiological function of cells, organelles or organisms is
altered by the uptake of the nanoparticle. For example, if HO2 or SWCNTs are taken up
by chloroplasts, is photosynthesis affected? If taken up by mitochondria, is ATP
production or respiration affected? It will be important to link the observed effects with
location to document the direct and indirect effects of nanoparticles, and also to help
evaluate the validity of the toxicity tests (be they traditional or novel). It also is possible
that nanoparticles may associate with biomolecules (see discussion in Introduction) and
alter biochemical pathways in cells. Much work is being done in the medical field with
the intention of creating nanoparticle-DNA linked biomolecules for treatment of a variety
of diseases (Han et al. 2007). However, unintended consequences may arise from
interactions between nanoparticles and DNA. For example, Hirakawa et al. (2004) found
that photo-irradiated Ti02 particles (being included more and more frequently as a
component of sunscreens) catalyze copper-mediated site-specific DNA damage via the
formation of hydrogen peroxide. They conclude that this DNA-damaging mechanism
may participate in the photo-toxicity of Ti02. It is reasonable to wonder if the
demonstrated potential for nanoparticles to bind to nucleic acids and to cause damage to
DNA may result in unforeseen negative effects. For example, fundamental principles
concerning fate, transport and exposure of nanoparticles, and production of nanomaterial
degradation molecules, suggest that there is potential for movement of nanoparticles
through all the compartments of the biosphere. It is unknown to what extent a release
into the environment of particles, that occurs after the intended initial use of the product
is completed, will bind to and/or catalyze oxidative damage to DNA and thereby affect
target and non-target organisms via propagating errors in DNA replication and
reproduction.
We will explore the use of test organisms where gene mutations have been developed
(see below for details). Briefly, there are available four systematic yeast knockout
collections (YKOs), e.g., a mutant collection of Saccharomyces cerevisiae produced from
the Genome Deletion Project (Giaever, et al., 2002) that collectively represents various
haploid and diploid essential (lethal) and non-essential (non-lethal) gene deletions. Use
of such mutant collections and resultant testing methods have advantages both as
potential rapid screening approaches for nanoparticles (see section below Development
and Assessment of Novel Testing Methodologies), but also as a means quickly to identify
mechanisms of response. We will explore gene microarrays (see below), which also have
the advantage of linking mechanisms with response.
The exact approach to elucidating mechanisms and determining the validity of tests is
particularly challenging given the rapidly expanding field of nanosciences and
nanotechnology. We anticipate that success in this portion of the research will require
collaborations among several research groups given the potential complexity of responses
and the breadth of possible methodological approaches. As noted above we will
continually assess the state of the sciences and any capabilities existing within NHEERL
and elsewhere to form collaborations to accomplish these tasks.
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2. Development and Assessment of Novel Testing Methodologies
Reaching consensus for nanoparticles on how, if at all, to adopt the current standardized
OPPTS toxicity tests may be even less straightforward than when implementing the
initial procedures for assessing traditional pesticides and chemicals. This is due, in part,
to the great variation in composition, and the unknown physical and chemical behavior of
nanoparticles and nanomaterials. Also, there is substantial variation in the types and uses
of nanoparticles and nanomaterials produced by the government and industrial sectors,
and variability from batch to batch of an industrial nanoparticle within a company. Such
diversity and variability will challenge establishing a framework to assess ecological
risks. In addition, quantum chemistry and physics relationships prevail at the nanoscale,
and that complexity, as assessed by most any metric, is great across the spectrum of
nanoparticles envisioned to be produced; it likely will be necessary to consider these
substances using different assessment principles than those used for traditional (larger-
sized) particles and chemicals. Understanding surface chemistry, size distribution,
composition, and agglomeration tendencies, fundamentally appear to be essential to
understanding the presence and absence of modes of action. In particular, assessing
solubility, and ion exchange capacity leading to nanoparticles binding onto a variety of
substrates are crucial to assessing toxicity. In addition, solubility often depends on
whether or not the nanoparticle is functionalized and this process may alter the toxicity,
e.g., some studies report that functionalized carbon-based nanomaterials are less toxic
than non-functionalized versions while other studies suggest otherwise (Roberts et al.,
2007). The potential for high diversity, complexity and variation in many attributes of
nanoparticles and nanomaterials may make it difficult to compare data among traditional
standardized tests to reach consensus on whether particular nanoparticles, or a particular
'variant' of a nanoparticle, are toxic or what might be estimates for toxic concentrations
in the environment.
Regardless of the results of the traditional OPPTS toxicity tests used in Phase I, we plan
to explore novel testing methodologiess to 1) improve applicability of test responses to
ecological responses, 2) improve the speed, consistency and quality control of testing
among various organizations, and 3) increase the applicability of test results to other taxa.
As noted above in the previous sub-section, the YKO collections provide a unique
opportunity for rapid throughput testing to examine potential toxic effects of
nanoparticles. It appears possible to develop high throughput genotypic profiling using
the mutants of the YKO collections, grown individually in the presence of a nanoparticle,
while analyzing the optical density of each developing culture (Weiss, et al. 2004; Alan
Bakalinsky, pers. comm.). Additionally, one can grow all the mutants of an YKO
collection simultaneously in a mixed culture, and then quantify the abundance of each
mutant by hybridization to an oligonucleotide array of the complimentary nucleic acids
tagged with the appropriate "bar code" detection sequence (Giaever, et. al., 2002).
Relative abundances of affected mutants would be expected to drop out compared with
abundances of the entire mutant collection, thereby providing information on susceptible
genes.
Another molecular testing methodology we will explore is microarrays. While YKO
collections deal with whole organisms and their DNA, the focus of microarrays is on
DNA, RNA and proteins. Two standard approaches of existing genomic technology can
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be readily applied to detect molecular perturbations of cell function following exposure to
nanoparticles. First, microarray chips containing oligonucleotide features that are
complementary to unique RNA transcripts are now commercially available for several
different potential test species of interest in this project. For example, standardized
Agilent Affymetrix 3' expression arrays have been produced for Arabidopsis, Bacillus
subtilis, Escherichia coli, Hordeum sp., Caenorhabditis elegans, cotton, maize,
medicago, rice, soybean, tomato, wheat and yeast. These microarrays allow analyses of
expression of tens of thousands of genes simultaneously and are especially useful for
detecting changes in the expression of individual genes that correlate with observed
phenotypic differences between individuals (or samples).
A second approach using microarrays is a newer generation of Agilent microarray
platforms know as ChlP-on-Chip (chromatin immunoprecipitation-on-chip). The general
function of ChlP-on-Chip technology (now produced for Arabidopsis, S. cerevisiae, S.
pombe, C. elegans, and others) is to analyze how regulatory proteins interact with the
genome of living cells. Applications include (but are not limited to) elucidating modes of
action and activities of compounds relevant to pathophysiological states, and identifying
biomarker responses to protein-DNA binding events to serve as bioassays or toxicant
signatures (http://www.affymetrix.com/products/arrays/index.affx). Technical assistance,
hardware needed for microarray data collection, and bioinformatics analyses are available
to WED researchers through the US EPA Toxicogenomics Core Facility located in the
NHEERL Research Triangle Park, NC facility and through the Oregon State University
Center for Genome Research and Biocomputing.
One concern regarding any molecular testing methodology is the degree to which the
results are relevant at larger spatial and temporal scales characteristic of ecosystems.
Unfortunately, even though the proteomics and genomics fields are rapidly expanding, at
this time platforms for all organisms of key importance to this project that represent a
cross section of ecosystems are not available commercially. Fortunately, in the case of
the YKOs and microarrays, there is relevance to larger scales since fungi are critical,
often keystone components of virtually all terrestrial ecosystems. We recognize that
yeasts are not the filamentous fungi extensively present in soils, but they do represent a
reasonable model system for screening of nanoparticles that is commercially available.
Plant microarrays that are available represent primarily angiosperms, however, we are
aware that work is being done with gymnosperm microarrays (Blinker et al., 2004). We
plan to explore the availability and suitability of gymnosperm microarrays for use in
Phase II in order to expand the number of species examined.
Developing novel methodologies based on directly assessing genes and/or their
expression will offer insights at basic physiological scales into putative ecological-scale
toxicity responses. If such links can be identified while extrapolating responses across
scales of system complexity, it may ameliorate some of the concerns posed by the
traditional OPPTS toxicity testing framework (see above discussion). Besides providing
information about the mechanisms of toxicity, such results will aid in directing and
focusing toxicity assessments at scales beyond those of individuals or communities.
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3. Stressor and Biological Complexity
Acute, especially lethal, responses of individuals determined from traditional OPPTS and
novel toxicity tests represent relatively simple assessments of risk of nanoparticles. In
reality, individuals in ecosystems are exposed to a host of biotic and abiotic factors or
stresses that determine their response to environmental or anthropogenic triggers. Up to
this point, we have focused on both lethal and sublethal effects, however, sub-lethal,
especially chronic responses of individuals pose additional considerations when scaling
risk from responses of individuals to more complex systems. This complexity consists of
individuals growing in intra- and interspecific groups that influence their responses, as
well as complexity in the number and timing of influencing stresses. There is also the
consideration of indirect effects of stresses when evaluating complexity. For example,
nanoparticle deposition onto leaves of plants may influence soil microbial communities
via alterations in carbon allocation to roots and subsequent translocation to the soil as a
result of decreased photosynthesis. In this example, decreases in photosynthesis and any
growth of the plants may be insignificant compared to the longer-term influence on
decreased carbon flow to soil and its effect on biological structure and function. In Phase
II, we will begin to address some of these issues of complexity by dealing with multiple
stresses and use of two-species mixes rather than focusing on individuals growing in
isolation as was done in Phase I. The focus will continue to be on the response of the
individuals, measuring different endpoints than were examined in the traditional toxicity
tests. If possible, we will attempt to measure endpoints based on results from prior
mechanistic work including any information we obtain from the, YKO, proteomics and
genomics methodologies.
We envision using a small number of bipartite associations (both plant-plant, and plant-
mycobiont) that we believe can serve as good models for initial experiments to
characterize complexity present in terrestrial biomes. Likely candidates for examining
interactions among plants in plant-plant associations might include agronomic plants
competing in single pots. Likely candidates for plant-mycobiont associations might
include an agricultural crop plant in symbiosis with an endomycobiont, or a woody
species in symbiosis with an ectomycobiont. Given the well-known, substantial roles that
mycobiont associations play in regulating flows of materials in terrestrial ecosystems,
multiple stresses employed for these bipartite systems might include alterations in water,
nutrients and light. We also need to consider the temporal aspects of response,
particularly for chronic, sublethal effects that may take extended times to develop. The
temporal component is very important to consider when comparing responses in annual
versus perennial plant systems. In perennial systems, carbon and nutrient storage are
critical for over winter maintenance of plants and other organisms. For example,
Andersen et al. (1991) found that air pollution stress affected root growth and
carbohydrate concentrations in tree seedlings the year following exposure, in the absence
of additional exposure to the pollutant. The possible carry-over (legacy effects) and
confounding effects of long-term exposure to nanoparticles is a challenging aspect of this
research that will be dealt with more carefully in Phase III.
Scientists at WED have developed an experimental system (mycocosm) whereby
individuals growing in bipartite associations can be monitored individually (Figure 4,
Rygiewicz and Andersen, 1994). The unique aspect of the mycocosm system is the
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ability to monitor the response of each individual while maintaining the physiological
integrity of each partner in the association. Using this system we envision using two
routes of nanoparticle exposure: rooting medium drench of nanoparticle suspensions, and
leaf application of particle suspensions. We will be able to monitor particle uptake,
retention and translocation by and within each partner, as well as transfer of the particles
between partners, while assessing the response variables as discussed above.
Regarding additional endpoints, we will increase the length and timing of exposure in
Phase II experiments to help identify subtle responses that may occur over longer time
frames; such responses might be missed in the shorter-term traditional toxicity tests, and
particularly, in the genomics and proteomics work.
Results of Phase II will in large part help determine the overall specifics of how to
conduct experiments in Phase III. And they will also help explain how to interpret
responses observed in the more complex systems that we will investigate as described
below.
C. Phase III Research Activities
Phase III will require a substantially different approach than those employed in Phases I
and II, concerning the methods used, endpoints measured, and the nature of responses
(i.e., acute vs. chronic, lethal vs. sub-lethal) (Figure 3). The goal in Phase III is to
determine if nanoparticles in their various forms alter the structure and function of
ecosystems. This may occur through changes in trophic interactions, or in disrupting the
function of key species responsible for important ecosystem processes. In Phases I and
II, individual responses to nanoparticles were of interest, often at acute levels but
sometimes also extending to chronic responses. These included responses at the whole
organism, cellular, biochemical, physiological, and molecular levels. In Phase III, the
emphasis shifts from individual responses to interactions among organisms primarily
addressing chronic and variable, in terms of lethal and sub-lethal effects, and includes
interactions at different spatial and temporal scales.
An area of particular concern regarding nanoparticle effects on terrestrial ecosystems
involves potential effects on soil processes. One anticipated route of exposure of
nanoparticles to soil is through sludge application or waste waters containing
nanoparticles from waste treatment plants. Organisms comprising the soil food web are
responsible for processing plant-derived carbon compounds and soil-derived nutrients,
the fluxes of both being essential for the maintenance of a functional and stable below-
ground ecosystem and consequently the entire terrestrial ecosystem (de Ruiter et al.,
1998; Wolters, 1998). Processing of carbon residues leads to the formation of soil
organic matter, which influences soil physical, chemical and biological properties. Soil
food web organisms are responsible for development of soil properties such as porosity,
aggregate structure, water holding capacity and cation exchange capacity. It is these
properties that are fundamental to establishing and maintaining terrestrial ecosystems and
all of the organisms comprising them.
Another route of nanoparticle exposure is through air deposition to plants, soils or
inhalation of particles by wildlife. We will not deal with inhalation aspects of exposure
in this research plan. In the case of plant deposition and uptake, it may alter net primary
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productivity (NPP) or partitioning of resources within plants. One concern is the indirect
effect of altering soil function via the amount of carbon that is translocated and released
below-ground. It is highly likely that any change in the quality or quantity of carbon
movement from plants to soils, or from plants to the organisms that feed on them, could
rapidly be propagated through other levels of the food web (de Ruiter et al., 1998; Cardon
et al., 2001). Food webs are highly integrated systems that have a regular trophic
structure consisting of three or four transfers along most food chains (Pimm & Lawton,
1977; Pimm, 1982; Moore, et al., 1993). Well developed food webs with long trophic
'loops' are thought to be stable as a result of weak links between predator and prey
organisms (Neutel et al., 2002). Despite apparent stability resulting from these weak
links, it is not known which soil processes and to what extent these links may be
disrupted as a result of nanoparticle deposition.
Predicting the effect of nanoparticles on ecosystems is especially difficult because
currently we have limited information on the specific role of specific organisms in the
trophic structure of most ecosystems (de Ruiter et al., 1998). Naeem et al. (1994)
manipulated species diversity while maintaining trophic structure in model ecosystems,
and found resultant changes in soil N, K and P availability, but the responses were
idiosyncratic with respect to what trophic groups were being manipulated. These results
show that specific ecosystem processes may respond uniquely and rapidly to loss of
species diversity but in unpredictable ways. In addition, alteration of certain soil
organisms, such as mycorrhizal fungi, may have a greater impact on ecosystem
productivity than others (Moore et al., 1993; van der Heijden et al., 1998). It has been
hypothesized that the functional groups represented by the least total biomass and often
found at the more distant (from the primary producers) trophic levels, exert the greatest
effect on system function upon their elimination via strong negative feedback loops.
Clearly, species diversity affects soil processes and hence ecosystems structure and
function (Naeem et al., 1994), but the idiosyncratic nature of reactions by soil food web
organisms makes it difficult to predict how nanoparticles will affect ecosystems.
Another aspect of Phase III will be to examine how nanoparticles affect interactions of
organisms in systems exposed to other stresses concurrently. Andersen et al. (2001)
found that tropospheric ozone at moderate levels did not significantly affect Ponderosa
pine growth in a mesocosm experiment. However, when Ponderosa pine was grown in
competition with a native grass species, ozone significantly affected plant biomass. This
study illustrates the potential interactions that can occur when organisms are exposed to
multiple stresses simultaneously. There are many examples in the literature that show
negatively synergistic effects of combined stresses on organisms, and also when
organisms are growing together in communities.
Phase III experiments will be conducted initially in growth chambers, later in
glasshouses, and ultimately in the Terrestrial Ecophysiology Research Area (TERA) at
WED. As research proceeds from facility to facility, system complexity will be
increased, the types and rigor of edaphic control will vary, longer time frames will be
examined, and systems of greater complexity will be employed, increasing the relevance
of results. The TERA facility is one of the few facilities globally that allows for
imparting and tracking natural diurnal and seasonal variation in environmental conditions
while maintaining linkages between above and below ground components (Tingey et al.,
Ill
-------
1996). It is a system that allows near real-ecosystem conditions in a state-of-the-art
facility that, with modification, can achieve high levels of confinement. This facility is
especially unique because it will allow us to track fluxes and pools sizes of key elements
at a system level to create resource budgets of the reconstructed ecosystems. Because the
exact experiments to be conducted at each level of control and complexity, i.e., in each of
the three types of facilities, will be determined after Phases I and II, we will present now
a framework for conducting experiments at each level but details will not be available
until Phases I and II are near completion.
1. Growth Chamber Experiments
In the growth chamber experiments, mini systems will be constructed in pots using native
soil and 1-2 plant species in combination. Because of the ability to control light and
temperature, we will focus on interactions of these two stresses on nanoparticle toxicity
to plants and soils. The addition of components a natural soil, including a full
complement of soil microorganisms, will enable us to examine how nanoparticles
influence mycorrhizal symbioses and microbial populations. For example, by using
mycocosms developed at WED (Rygiewicz and Andersen, 1994), we can examine uptake
of particles from the soil to the plant, as well as responses of each of the individuals in
the symbiosis (Figure 5-4). The growth chamber work will allow us to tease apart
specific responses that may be harder to identify once we move to glasshouses and TERA
by providing a link between the mechanisms identified in Phases I and II, and the more
complex and intractable holistic nature that represents intact ecosystems. In addition, the
growth chamber experiments will occur over relatively short time frames compared to the
other scales of complexity and scope.
We will conduct both above-ground and below-ground exposures in chambers. Initially
we will not be able to expose plants or soils to dry deposition of particles, even though
dry deposition may be an important route of exposure for ecosystems, because we lack
the confinement facilities. To conduct above-ground exposures, we will apply aqueous
nanoparticle suspensions directly to leaf surfaces. The concentration and carrier used
will be determined according to the results found in Phases I and II, and appropriate
controls will be included to address potential carrier effects. For below-ground studies,
we will apply a soil drench of nanoparticle suspensions, addressing the same concerns
with concentrations, controls and carriers discussed above. These studies will be limited
to one growing season or less, and variable light levels and temperatures will be
examined to identify possible interactions with these edaphic variables.
Responses or endpoints of interest in the growth chamber studies will focus on processes
at both the individual as well as at system levels. Some examples of individual responses
include photosynthesis and respiration, nutrient status, particle uptake and translocation,
and plant phenology and primary production. Depending on the results from Phase II
with proteomics and genomics work, we also may be able to focus on genetic markers of
responses in the growth chamber studies. Examples of system level responses include
nutrient turnover, leaching, soil CO2 efflux, and microbial community structure.
112
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2. Glasshouse Experiments
Glasshouse experiments will mimic growth chamber experiments in terms of species
used and variables measured, but will allow us more time and space to examine
responses. The goal in moving to the glasshouse as the next stage of experiments is to
increase edaphic variability, to lengthen the experiments, and to increase biological and
system complexity. It is possible that chronic effects of nanoparticle exposure may not
be observable for relatively longer time frames, particularly at lower levels of exposure.
For example, we will be able to grow communities of plants and plant mixtures in order
to examine the influence of competition on responses to particles. Because of the ability
to run experiments over longer time frames, we can follow responses over multiple
growing seasons. In this regard, we can look at both carry-over effects and chronic
effects of longer-term exposures on individuals and complex systems. We will be able to
look closely at soil-plant interactions, especially changes in soil chemistry or physical
characteristics that take longer to develop. In the glasshouse, we can expose ecosystems
to natural diurnal and seasonal variation in light quality and quantity. While responses of
individuals will be observed, endpoints of interest will shift away from individual
responses to system level responses, such as changes in soil nutrient levels, or whole
system productivity.
One major goal of the glasshouse experiments is to integrate the responses we observed
in the more simplified systems into a framework that allows us to focus our next set of
hypotheses that we will develop to address the most pressing issues in the TERA facility.
3. Terrestrial Ecophysiology Research Area (TERA)
The TERA facility (Figure 5-5) has been used in the past to conduct multiyear, 2-by-2
factorial experiments. We can also do experiments with other treatment designs but there
are limitations in possible designs given that there are only 12 chambers in the facility.
Previously in TERA, we have conducted two highly successful, sophisticated
experiments involving multiple stresses and integrated ecosystem responses (Tingey, et
al., 1996; Rygiewicz, et al, 2000; Tingey, et al., 2007). We are confident that we will be
able to use TERA in a similar fashion to assess effects of nanoparticles on responses of a
reconstructed plant-soil ecosystem. Our concern with using TERA involves augmenting
the chambers to provide sufficient confinement capabilities to reduce any potential
release of nanoparticles.
We know from past experiments done in TERA that we can use a native soil and it can be
reconstructed by horizon in the lysimeters (below-ground soil compartments) of the
exposure chambers and will mimic the biological and other processes of the source soil.
A plant species, or mix of species, will be selected based on the overall successes of
using and developing all the response measurement techniques and methodologies
discussed previously. Similarly, the experimental design will arise from the work done
previously on the project and likely will involve some combination of a nanoparticle
treatment and edaphic condition treatment. Responses at both the individual level (for
some members of the reconstructed ecosystem), and at the ecosystem level will be
observed. Figure 5-6 illustrates the extensive nature of the responses we have observed
in previous experiments conducted in TERA. Making observations and taking samples
can range from continuous to periodic, depending on the nature of the response. Another
113
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benefit of the TERA facility is the capability it provides to link responses between the
above-ground and below-ground components of an ecosystem. Figure 5-7 illustrates a
monitoring design as well as a scheduled frequency for sampling which was used
previously to understand responses in one component of the reconstructed ecosystem in
relation to those occurring in the other component.
4. Field Testing and Modeling
The final aspect of Phase III involves field testing and model parameterization to
extrapolate the results across broader spatial and temporal scales. The field testing
approach will build on the results of the TERA experiments, and will consider the results
of the fate, transport and exposure work being done in the larger ORD project. For
meaningful field testing, we need to consider the mode of transport, the concentration and
ultimate form of particles reaching the environment. At this time, we do not have
adequate information to design a meaningful field test. Similarly, models able to
characterize the fate, transport and toxicity of particles will be evaluated as part of the
larger NHEERL ecosystem effects research effort, and applied to our results as
appropriate.
114
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IV. Potential Collaborators
Rice University, Qilin Li, Civil and Environmental Engineering,
(http://www.cohesion.rice.edu/engineering/ceve/people detail.cfm?facultv id=38) -
Characterization of nanoparticles and perhaps supplying characterized particles to us.
University of Tennessee, Robert Compton, Department of Chemistry - Characterization
and manipulation of nanoparticles.
DOE-PNNL, Nanoparticles Risk Assessment, and Ecotoxicology groups - consultancy
about particle behavior in solutions, proteomics and genomics, ecosystem exposure
effects, characterization and production of functionalized particles.
Texas Technical University, Jaclyn Canas, The Institute of Environmental & Human
Health - Exposure and effects, characterization and quantification of particles.
Oregon State University
Robert Tanguay - Quantification and localization of particles
Alan Bakalinsky - Yeast mutant genomic responses to nanomaterial exposure
Jennifer Field, Environmental and Molecular Toxicology - Analytical chemistry
of fullerenes
Center for Genome Research and Biocomputing - comprehensive genomic analyses
facility.
Oregon Nanoscience and Microtechnologies Institute (ONAMI) - Experimental facilities,
particle characterization (http://www.onami.us/).
University of Oregon, Jim Hutchinson, Center for Advance Materials Characterization in
Oregon (CAMCOR) (http://darkwing.uoregon.edu/~chem/camcor.htmn - Designer
nanomparticles (made to our specification); particle characterization.
Neurotoxicology Division, NHEERL, Bellina Veronesi, characterization and localization
ofTi02
115
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Figure 5-1. Critical path of anticipated research activities for Phase I.
Phase I
Develop Research
Prepare White Paper to
potential Eco Effects
Develop Health and
Safety Protocol
Helps form basis for
Phase
Identify particles to be
studied in Phase I
(NHEERL-Wide)
Chemical and physical
material
characterization for
tracing and measuring
particles
>
Identify possible
collaborators
Toxicity testing
using 3-5 standard
protocols with
different organisms
J
Identify
Particle
new test
^
is not
methods
taken up
Toxicity
detected
Report to
OPPTS
No
toxicity
detected
Particle
is taken
up
Phase
116
-------
Figure 5-2. Critical path of anticipated research activities for Phase II.
Phase II
Phase I
Report to
OPPTS
Mechanistic understanding-
-Location in ceil?- uptake?
-Location on surface?
-Chemical transformation
-Biochemical changes
Observed
toxicity
No observed
Uptake
toxicity
No uptake
Cell Surface
chemistry and
binding;
agglomeration
V
Test additional
particles- different
surface charges
and size
Develop
hypotheses
and contribute
to development
of SAR's
Examine
different
response vars.
Examine longer-
term responses
Begin Increasing
system complexity
Propose
novel test
methods
Interactions
with other
stressors
Phase III
117
-------
Figure 5-3. Critical path of anticipated research activities in Phase III.
Phase
Increasing level of complexity
Laboratory
experiments with
reconstructed
ecosystems
Mesocosm
experiments in
reconstructed
ecosystems
Field testing
Complex
systems
response using
particles
identified in
Phases I & II
Response variables:
-individual growth and
development
-Trophic diversity
-Fluxes and pools
Report to OPPTS;
other offices
Scaling, model
development and
parameterization
Propose novel
test methods
118
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Figure 5-4. A root mycocosm colonized by a Ponderosa pine seedlings and the
mycobiont Hebeloma crustuliniforme. The mycocosm allows for the two symbionts to
be grown in symbiosis while physiological integrity is maintained. The mycocosm
consists of the central compartment containing both symbionts, and two side
compartments where only the mycobiont is allowed entrance. The inset is a close up
view of the border area between the two compartments showing ectomycorrhizas at the
boundary, and only extramatrical hyphae being allowed to enterthe side chamber.
119
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Terrestrial Ecophysiology Research
Area (TERA)
Figure 5-5. Picture of the Terrestrial Ecophysiology Research Area (TERA) at WED.
TERA consists of 12 exposure chambers whose aboveground component (approx. 1 to
1.3 m high) is enclosed by Teflon film, and a soil lysimeter (1 x 2 m footprint x 1 m
deep). The facility replicates ambient climatic conditions that are assessed by the
meteorology tower seen in the background. The chamber field is supported by a
glasshouse containing mechanical, electrical, computer, and other exposure, monitoring
and support capabilities and is located outside the bounds of the figure to the right.
120
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Shoot Carbon and
Water Fluxes
Fluxes
Canopy C02/H20
Whole Tree H20
Branch/Needle C02/H20
Stoma+al Conductance
PAR
Needle Temperature
Dark Respiration
System Nutrients
Plant <5t Litter
C, N, Cations, Anions
- Total
- Soluble
Rjor hem istrv
Soil
C, N, Cations, Anions
- Total
- Extractable
pH
Soil Solution
Cations, Anions, pH
Root Growth and Phenology
Dynamics (video) Demographics
Appear/Disappear Root Size Classes
- Roots Mycorrhizae
- Mycorrhizae - Morphotypes
- Hyphae - Colonization
m
m
m
Litter Layer
Soil Biology
Enzymes
C, N, P Processing
Foodweb
¦ Bacteria/Fungi
Nematodes/Protozoa
Gases
Profile
Headspace
Mass Loss
Chemical Composition
Fauna (Micro/Meso)
Mycorrhizae
Genetic Diversity
Micro/Ultra Structure
Morphotypes
Stable Isotopes
13C, 15N (Litter, Soil, Roots)
180, 13C (H20, C02)
Shoot Growth
and Phenology
Stem Height and Diameter
Terminal Shoot and Bud Length
Branch Count
Bud Count
Needle Area/Dry Weight
Tree Needle Area
System Water
Chamber
Evapotranspiration
Irrigation
Humidification
Plant
Transpiration
Soil
Moisture Content
Drainage
Soil Organic Matter
Resource Ratios
Budgets
Biologically Relevant Fractions
15N and 13C of Fractions
DOC
Figure 5-6. Diagram of web of possible ecosystem measures that can be assessed in
TERA. Sampling capabilities in TERA are extensive and include biological and edaphic
variables. Estimates of fluxes and pool sizes are such that budgets of major resources can
be assessed in each of the 12 enclosed chambers. For example, in a previous experiment
done in the facility, carbon, nitrogen and water resources were evaluated for both the
above-ground and belowground components of a reconstructed plant-soil ecosystem.
121
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Sampling and Monitoring Based on Above- and Below/ground Phenological Events
-------
Agency Framework for Addressing Nanoparticle Ecological Risk
Assessment
APGs
WED Project
NHEERL Program
Protocols for
effects testing
QSARs for
predicting
effects across
substances
Estimation of
where effects
would be most
likely to occur
ORD MYP
To be
determined
Terrestrial
Ecological
Effects
• tox.
tests
compl
ex
syste
ms
•
ecolo
gical
proce
sses
Figure 5-8. Diagram depicting how WED research fits into the EPA framework for
nanotechnology ecological risk assessment.
Agency Need
Appropriate
tests or
approaches for
OPPTS when
assessing
potential
human and
environmental
risks of mfg
nanomaterials
Estimate of
probability of
effects in
natural
systems
123
-------
07
08
09
10
11
12
13
APG 1.1.07
Develop
research plan
for assessing
the ecological
effects of
nanoparticles
and have it peer
reviewed.
APG 1.1.08
Prepare white
paper for
potential
ecological
effects of
nanoparticles
APG 1.2.08
Develop
health and
safety
protocols as
per NIOSH
recom-
mendations
APG 1.1.10
Report results
of toxicity tests
to OPPTS for
testing
nanomaterials
in traditional
ecotoxicology
tests.
APG 1.1.11
Report on use
of novel
methodologies
for testing
toxicity of nano
products
APG 1.2.11
Contribute to
ORD effort to
develop QSARs
for
nanoparticles
APG 1.1.12
Report on
individual
organism
responses
under
increased
system
complexity.
APG 1.1.13
Report on
ecosystem
effects of
nanoparticles
Figure 5-9. Draft Annual Performace Goals (APGs) for NERA Project. APGs to meet
EPA Long Term Goal (LTG) #1: whether nanomaterials, in particular free nanoparticles
released into the environment, pose significant risks to humans and ecosystems.
124
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Evans, B.-K. Ku, D. Ramsey, A. Maynard, V.E. Kagan, V. Castranova, P. Baron. 2005.
Unusual Inflammatory and Fibrogenic Pulmonary Responses to Single Walled Carbon
Nanotubes in Mice. Am. J. Physiol. Lung Cell Mol. Physiol,
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129
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1 Appendix 5-A. OPPTS 850.4100 Terrestrial Plant Toxicology, Tier I
(Seedling Emergence)
UnitedStates Prevention, Pesticides EPA712-C-96-153
Environmental Protection and Toxic Substances April 1996
Agency (7101)
SEPA Ecological Effects Test
Guidelines
OPPTS 850.4100
Terrestrial Plant Toxicity,
Tier I (Seedling
Emergence)
"Public Draft'
130
-------
Introduction
This guideline is one of 11 series of test guidelines lhu1 have been
developed by llie Office ul Prevention Pesticides and Toxie Substances.
Untied Stales F.nviroiimenfal Protection Agency lor use in the testing of
pesticides and toxic substances, and the development of test data that nuts!
be submitted to the Agency tin review under I edetal regulations.
The Office of Prevention. Pesticides and Toxic Substances (< >PPTS)
hits developed thin guideline through a process of harmonization that
blended the testing guidance and lequirements thai existed in the Office
of Pollution Prevention and Toxics (< >P1T) and appealed in Title 40,
Chuptet 1, Subchapter R of the Code of Federal Regulations tCTR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Ieehnieal Infoimalum Service fN'I'IS) and the guidelines pub-
lished hv the Organization for T'coiiomic Cooperation and IXnelopment
(OPCD),
The purpose of harmoni/ina these guidelines into a single set of
OPPTK guidelines is to minimise variations among the testing procedures
ihrtl imrnt be performed to meet the data requirements of the LI. S. Knviron-
mental Protection Agency under the Toxic Subslanees Control Act <15
U.S.C 2<»01) and the Federal Insecticide. Fungicide and Rodentietde Act
(7 U.S.C. 136. et-ieq.'f.
Public Draft \ccess Information: Ihis dial! ijuidclinc i-. pari oi a
series of related harmonized guidelines that need to be considered as a
unit. Fur copies; These guidelines are available electronically from the
EPA Public Access Gopher (gophcr.epa.gov) under the heading 'iimiron-
mentai Test Methods and Guidelines'" or in paper by contacting the OPP
Public Docket at t,703) 305 5805 or In e-mail,
gu idol i n eft a 'epani a i l.epa. gov,
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section Office
of Pesticide Programs, field Operations Division (75(J6C)_ Fnvironmetital
Protection Agency. 401 M St. SW., Washington. DC 20460. In person:
bring to: Rm. 1132. Crystal Mull -2. 1921 Jefferson Davis Highway, Ar-
lington. VA. Comments may also be submitted electronically b\ sending
electronic mail (e-mail) to: yuidolmestf'cpainnil.epa gov.
Final Guideline Release: This guideline is available from the U.S.
(iovemment Printing Office. Washington. DC 20402 on I'he t-'ederal Ilui-
lelm Board, By modem dial 2(12 512 1387, telnet and ftp,
t«ibbs.access gpo.gov (IP 162.140.64.19), or call 202' 512' (#135 for disks
or paper copies. This guideline is also available electronicalty in ASCII
and PDF (portable document format) from the I'PA Public Access liopher
{gopher.epa.gov) undei the heading "F'nvironmcntal 'fest Methods and
Guide! ines,"
i
131
-------
OPPTS 850.4100 Terrestrial plant toxicity, Tier i (seeding emer-
gence).
(a) Scope (1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide. Fungicide, and
Rodetilieide Ac! (T1FRA) (7 I I.S C. Iet .wtj. ) and the "1 oxie Substances
Control Ac! f I'SCA) (I 5 I i.S.C. 2601)
t2) Background. The source material used in developing this har-
monized UMTS test guideline is. < >M' 122 1 Seed Germination Seedling
Fmeigence and Vegetative Vigor (Tie! 1) (Pesticide Assessment (iitide-
lines. Subdivision J Ua/;ird lnahtalioir, Nonlarucl Plains) l;PA report
540'iW-82-020. 1982.
O) I est objective. litis guideline should be used it) conjunction with
UPl'l S guideline 850,4000, Background N on target plant testing, which
provides general information and overall guidance lor the mwitarjiet plants
test guidelines.
(i) < iencr»J. Seedling emergence studies are designed to prtnide
phytotoxieit\ data on a pesticide. These phytotoxicity data are needed to
evaluate the elTeet of the level of pesticide exposure to mmtamet ant! ter-
restrial plants and to assess the impact of pesticides ott endangered and
threatened plants as noted under the Endangered Speetes Ac I. The prelimi-
nary level (Tier I) study evaluates the eftec-t of the maximum exposure
level. Where a ph\toto.xio effect is noted in one or more plants, further
studies may he required. These studies are required by 40 ITR 158 130
to support the registration of miv pesticide intended for outdoor use under
FIFRA. as amended,
i ii i Ohjwtivc ut' scMliing eim-rtfonev test. 1 ier I. i \ j f he objective
of the Tier I seedling emergence lest is to determine if a pesticide exerts
a detrimental effect to plants during early critical stages in their develop-
ment, The test is performed on species from a cross-section of the
nontarget terrestrial plant population that have been historically used for
this type of testing and. therefore, have known types of responses. This
is a maximum dose test designed to evaluate the phytoUTsic effects of
the pesticide quickly at the one dose.
(B) The terrestrial mm target plant phytotoxicity seedling emergence
lest is a greenhouse or growth chamber test. The test organisms are three
required .species com, soybeans, and a root crop, plus seven other spc
cies. usually tomato, cucumber, lettuce, cabbage, oat. ryegrass, and onion
('six species of at least four families of dieots and four species of at least
two families of monocoLs1) The soil or plant .surface is treated with lest
chemical (typical end use product (IFF)) at a concentration comparable
to the maximum label application rate or at a concentration 3> the esti-
mated en \ iron mental concentration. Results are reported in grams or
pounds of active ingredient (AI) per acre and arc expressed as the percent
1
132
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of detrimental effect growth compared to the control after at least 14 da\s.
Parameters measured include plant height, plant da weight, and percent
ph\foto\icity, The results are used to establish uculc toxicity levels to indi-
cate if farther testing at a higher tier is necessary.
tb) Test standards. In addition to the general 'est standards set lot tit
in OPPTS 850.4000. the following standards for the seedling emergence
studies nppK:
(1) Test substance. Refer to 40 CFR part I5K ft»r information re-
quired on the test substance.
(2) Species, Kadi report should include the following information:
(it Identilication of the sis dicot)Icduneao spo.ies and four
monoeotvlcdoneae species with fainiK identification
(A) The. \i\ dieot.s are to he ol at least four different families and
the tmmocots of at least two families. Soybeans.. corn and a dieot mot
erop like eurrot are the required speeies The proposed species and families
are given below and uie acceptable for the seed lint; emergence test:
Table 1 .—Species and families acceptable for the seedling emergence test
Family
Species
Common name
Lycopersicon esculentum
Cucumis sativ
Solanaceae
Cucurbit aceae Cucumis sativ us
Compositae Lactuca sativa
Leguminosae 1 Glycine max
Cruciferae Brassica oteracea
Umbelliferae Daucus carota
Gramineae Avena sativa
Gramineae ' Lolium perenm
Gramineae Zea mays
Amaryllidaceae Allium cepa
Tomato
Cucumber
Lettuce
Soybean
Cabbage
Carrot
Oat
Perennial ryegrass
Corn
Onion
1 Innocuiation with Rhizobium japonicum is unnecessary
jB) Seeds of plants with a low or variable germination potential
should be avoided fot the seedling emergence study When selecting plant
species other than com, soybean, and a rout erop. the Agency encourages
the use of sensitive plants other than crop plants weeds, nnthe species,
perennial sptx-ies ele. The Agency also encourages testing of more than
It) plant species.
(ill Identification of the cultivars of the plant species or assignment
ol an identification number to the eultivar used and seed or plant sotttee.
(lii I Identilication of the number of replicates and the number of
plants per replicate per dose.
133
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(iv i Identification of ihc dale- of planting, date of pesticide application
and height of plants at application, and date of phytotoxicity ruling or har-
vest and analysis
(3) Application levels. One concentration level equal to no less limn
maximum label rale should be tested. 11* it can be determined that the maxi-
mum quantity that will he present in the noutarget area is significantly
less than the maximum label rate, a concentration equal to no less lhan
3>. (hat maximum quantity may be tested. The phrase *'Ihc maximum label
rate" means the maximum recommended amount of A1 in the rec-
ommended minimum quantity of carrier such as water to be used per land
area.
(4) Number of plants. At least three replicates, each with 10 plants,
should be tested per dose level for the seedling emergence tests. Larger
population* and more replicates may be needed to increase the statistical
significance of (he test.
t5) Site, The seedling emergence studies should be conducted tinder
controlled conditions m growth chambers, gieenhouses. or in small Held
plots.
(6) Duration. Scedltug emergence should be obscned weekly, or
more frequently. for at least 2 weeks aftei germination.
Reporting. In addition to the information required in < tPPTS
850.4000. the test report should include the following information.
f 1) The number of seeds tested and the number emerged pel- dosage
level foi each replicate.
(2) Descriptions of the appearance and the growth and development
of the seeds and emergent plants, indicating any abnormalities and expres-
sions of phytotoxicity,
3
134
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(3) lobulation of the results indicating the percentage effect level for
each species as compared to untreated control plants,
(•IS Data on dry weights and heights, or other growth parameters are
required to be submitted.
te) Tier progression. II the results of the seedling emergence test
htne indicated sin adverse effect greater than 25 percent on one 01 more
pl;mi species, the seedling emergence tests at the Tier II lex el are required
(see t JPPTS X5U.42W!). If less than a 25 percent detrimental effect or re-
sponse. is noted for the seedling emergence test, no additional testing of
the respective tests at higher tiers is ordinarily icqtitred. The Agenc\. alter
review of the data, mu> rvquitc eeitam additional tests to determine a more
definite nondisccmible effect level
(f) Data reporting. (I i The registniut's report on pielmiinm seedling
emergence studies should inclndu all in limitation neeessan to provide:
(1) A complete and accurate description of the laboraton greenhouse'
field treatments and procedures
(ii i Sampling data and phvtoto\icit\ mtmg,
fiii) Data on storage of the plant material if so performed.
(iv) An\ chemical anuhsis of the plant material as to chemical con-
tent.
i \) Reporting of the data, rating system, and statistical analysis
O h Qualm control measures precautions taken to ensure the fidelity
of the operations
(2) Each laboratory greenhouse small lietd plot seedling emergence
report should include the following information:
(t) General, (A) Cooperator oi researcher (Mine and address),test lo-
cation (count) and state, country. if outside of the United Slates), and
date of study.
(Bl Nome (and signature), title, organization, nddiess. and telephone
number of the persons responsible for planning .supervising- monitoring.
fC) Trial identification number.
(1)) Qualify assurance indicating control measures precautions fal-
lowed to ensure the !idelit\ of the phytoto\ic»t\ determinations, record-
keeping procedures, and availability of logbooks; skill of the laboraton
personnel, equipment status of the laboratory oi greenhouse: degree of ad-
herence to good laboratorv practices: and degree of adherence to good
agricultural practices in maintaining hoalthly plants.
4
135
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(li) Other information the registrant considers appropriate and relaxant
to provide a complete aucl thorough description of the lest procedures and
results.
(it) Test substance (pesticide). (A) Identification of the lest pesticide
A1 including chemical name. common name tANSi, HSi. ISO, WSSA).
and compwn developmental experimental name.
(IVt A1 percentage. plus any inerts and adjmants in lest material.
(O Solvent used to dissolve and apph the pesticide il" the pesticide
is insoluble in water or other intended earner.
(D) Dose latex in terms of A! per area of land or of leaf (if icaf-
area-index is provided!.
(F) Dow rates in terms of the maximum label rate, or i! the registrant
has .shown that the nm\imum quantity that will He present m the uontarget
area is the maximum label rate, the dose equal to or no less than three
times that maximum environmental quantity (environmental qualih cal-
culations).
tT) Method of application including equipment type.
(.Ci i Number of applications.
(iii) Site <>t' the lost. (A) Site description of the seedling emergence
and vegetative \igor studies such an the l\pe of growth chamber. green-
house, or field plot.
(B> Location of the test site.
(C) ChmatotoHkul data during the test (records of applicable condi-
tions for the type of sile, i.e.. temperature, thermoperiod. rainfall or water
regime, light regimen intensity and qaalitv. relative humiditv„ wind
speed).
(D> Plant dcnsit> and container t\pcs
if:'t Cultural practices such as cultivation, pest control, and irrigation
practices (frequency of watering and method used met head vs. bottom
watering).
(Ft Substrate characteiistics (name'designation of soil hpe and its
physical and chemical properties, including pi 1 and percent organic mat-
ter).
fi\ i Results. (A) Reporting of percent emergence, plant height, plant
drs weights, toot dr\ weights, root length, dead plants, or other growth
parameters that mas have been measured to ascertain toxic effects of the
5
136
-------
pesticide upon the plants with dates of observation (Root measurements
are only needed if lire test chemical is a root inhibitor*.
(B) Phytotovicity rating (including u description ot the rating s\stem)
for each plum or population in Ihc test,
(O Statistical anahsis <.ii* the results including an environmental or
o (Teethe concentration effect (KC) value, (Note, for Tier I, there will be
only u percent clVcet level «t a spec-ilk concentration which is then com-
peted to 25 percent ol the growth (mass or rate] of (he control.)
(\) Evaluation, ti) For Tier 1 studies, determination at. to whether
Tier II studies would he required due to phytotoxte effects noted in one
or more of the tested species
(ji) Rrfcri'iici's. lite following references should be consulted for ad-
ditional background tiiateriyl on this test jiuideltnc
11) fruekn-e, B.. ed.. Rewanh llethodx in Weed Sin'inv. Southern
Weed Science Society, Auburn Printing. Auburn. At f 1977),
(2) |Re,sencdj
6
137
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Appendix 5-B. OPPTS 850.5400 Algal Toxicity, Tiers I and II
United States Prevention, Pesticides EPA712-C-96-164
Environmental Protection and Toxic Substances April 1996
Agency (7101)
EPA Ecological Effects Test
Guidelines
OPPTS 850.5400
Algal Toxicity, Tiers I and
"Public Draft"
138
-------
Introduction
This guideline is one of 11 series of test guidelines lhu1 have been
developed by llie Office ul Prevention Pesticides and Toxie Substances.
Untied Stales F.nviroiimenfal Protection Agency lor use in the testing of
pesticides and toxic substances, and the development of test data that nuts!
be submitted to the Agency tin review under I edetal regulations.
The Office of Prevention. Pesticides and Toxic Substances (< >PPTS)
hits developed thin guideline through a process of harmonization that
blended the testing guidance and lequirements thai existed in the Office
of Pollution Prevention and Toxics (< >P1T) and appealed in Title 40,
Chuptet 1, Subchapter R of the Code of Federal Regulations tCTR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Ieehnieal Infoimalum Service fN'I'IS) and the guidelines pub-
lished hv the Organization for T'coiiomic Cooperation and IXnelopment
(OPCD),
The purpose of harmoni/ina these guidelines into a single set of
OPPTK guidelines is to minimise variations among the testing procedures
ihrtl imrnt be performed to meet the data requirements of the LI. S. Knviron-
mental Protection Agency under the Toxic Subslanees Control Act <15
U.S.C 2<»01) and the Federal Insecticide. Fungicide and Rodentietde Act
(7 U.S.C. 136. et-ieq.'f.
Public Draft \ccess Information: Ihis dial! ijuidclinc i-. pari oi a
series of related harmonized guidelines that need to be considered as a
unit. Fur copies; These guidelines are available electronically from the
EPA Public Access Gopher (gophcr.epa.gov) under the heading 'iiiniroii-
mentai Test Methods and Guidelines'" or in paper by contacting the OPP
Public Docket at t,703) 305 5805 or In e-mail,
gu idol i n eft a 'epani a i l.epa. gov,
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section Office
of Pesticide Programs, field Operations Division (75(J6C)_ Fnvironmetital
Protection Agency. 401 M St. SW., Washington. DC 20460. In person:
bring to: Rm. 1132. Crystal Mull -2. 1921 Jefferson Davis Highway, Ar-
lington. VA. Comments may also be submitted electronically b\ sending
electronic mail (e-mail) to: yuidolmestf'cpainnil.epa gov.
Final Guideline Release: This guideline is available from the U.S.
(iovemment Printing Office. Washington. DC 20402 on Ilk' FeJcmt llut-
leim Board, By modem dial 2(12 512 1387, telnet and ftp,
fcdbt>s.access gpo.gov (IP 162.140.64.19), or call 202' 512 f»135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the I'PA Public Access Gopher
(gopher.epa.gov) undei the heading "F'nvironmcntal Test Methods and
Guide! ines,"
i
139
-------
OPPTS 850.5400 Aigal toxicity. Tiers I and II.
(a) Scope - -(11 Applicability. This guideline is intended to meet tast-
ing requirements of both the Federal Insecticide, fungicide. and
Rodcntieide Act (TIFRA) (7 U.S.C. B6, ei .«» and the Toxic Substances
Control Act (TSCA)t IS U.S.C, 2M>1).
(2) Background. The source material used in developing this har-
monized OPPTS lesl guideline are 40 CI'R 797,1050 Algal Acute Toxicity
Test; OPP 123 2 (irovvth ami Rcptoduelkm of Aquatic Plants (Tier 2)
I,Pesticide Assessment Guidelines. Subdivision J—Hu/.ard Hvaluation;
N on target Plants) 1 PA report 540TW-82-020. IMX2; and OKCD 20 1. Algal
Growth Inhibition lost.
tht Purpose. This guideline is intended tor use in developing data
on the acute toxicity ot"chemical substances and mixtures tehemteuls) sub-
ject to on iron mental effects test regulations, and was written .specifically
fot Selethntritm cuprwonmtum and Skch'fotH'ma coshttnm (.see paragraph
(dX2xiii) of this guideline) Use of Aiiubaena or ,Y
-------
(ii) At the end of % h. and at the end ('e.g. 2. 4, X. 16, 32, and M mg.l I, Often it is possible to choose
test chemical concentrations based on the anticipated slope of the con-
centration-response curve, and these concentrations should bracket the ex-
pected lest end-points. Algae are to be placed in a minimum of three rep-
licate test containers for each concentration of test chemical and control.
2
141
-------
With the exception of the use of four replicates lor A", pellicuU/xa. more
than three replicates may he required Hi provide sufficient Jala, liach test
chamber should con Iain equal volumes of test solution and approximately
1 > Id'1 St'Iemisimnt- Xmiaila. or. hhilkiena cells per milliliter or 7.?:-' ItV
Skeh'tmienu cells per milliliter of test solution. The chemical concentra-
tions should result m greater than W percent of algal growth being inhib-
ited or stimulated at the highest concentrations of test substance compared
to conttols or that the test concentrations should bracket the expected F.C50
\ulue.
(iii'l Fvery test is to include a control (negative eotttrol) consisting
of the same nutrient medium. conditions, procedures, and algae front the
same etilmre. eseqtl thai none of the lest substance is added. If a carrier
is present u> tins of the test chambers, a separate earner control is required.
(iv) Positive controls using /.inu chloride as a reference chemical
should also he run periodically. The purpose of a positive control with
a reference chemical is to determine that the test algae are responding
to a known chemical in the expected manner. If the algae arc responding
to subsequent reference clientical tests consistently, it is assumed that the
algae will respond to other chemicals consistently. Changes in algal re-
sponse caused bv such factors as |x>oi nutrition, genetic drift, and contami-
nants may not be detected by negative controls, yet may still influence
test results At least three concentration!! of the reference chemical are run
at or tteai the expected median effect lex el.
(v) The lest begins when algae (inoeu)a) from 3 - (o 7--day-old slock
cultures aie placed in the lest chambers containing test solutions having
the appropriate concentrations of the test substance. The mean cell volume
of inoculu should be approximately 35-45 (.igni^ at the onset of testing
Algal growth in controls should reach the logarithmic growth phase by
% h tat which lime the number of algal cells should be approximately
1,5 x lf)'-; tnL for Skek'towma or 3.5 a If^ triL for Sek-nuxtrum If" loga-
rithmic growth cannot be demonstrated, the test is to be repeated. At the
end of 96 h. and, if possible, at the end of 24, 48, and 72 h. the algal
growth response (number or weight of algal cells per milliliter") in all test
containers and controls is to be determined by an indirect
(spectrophotometry. electronic cell counters, dry weight, etc.) or a direct
(.actual microscopic ceil count of al least 4<>0 cells per flask) method. Indi-
icct methods are to be calibrated by a direct microscopic count or data
should be presented that relate electronic counts with microscopic counts.
'Ihe percentage inhibition or stimulation of growth for each concentration.
EC50. and the concentration-response curves are determined from these
counts.
!\i"! A particle cotmtei or microscopic counting cannot he used ibi
, inithietM unless the filaments are broken up and dispersed using a s\-
ritigc, ultrasonic bath, or blender. Limited use of somfieation is allowed
3
142
-------
fro Anabaetht The proucJarc used to break up the filaments should result
in consistent iflamciil lengths across treatments and replicates
Sonitication, ultrasonic bath. blunder, syringe. or any cither methods of
coll separation oilier than manual or rotary shaking mo not allowed foi
Seli'ihisintm, Skelehmenhi, or Viiviciihi
ivii) At flic end of the definitive test, the following additional analyscs
of algal growth response arc to he performed
(A I Determine whethei the altered growth response between controls
and test algae (in highest lest chemical concentrationsj was due to a chance
ni relative cell numbers, eel I sizes, or both. Also note any unusual cell
shapes, color differences, differences in ehluruplast morphology.
Iloeeiilations. adherence of algae to test containers, or aggregation of algal
cells These observations are qualitative and descriptive, and arc not used
in end-poinl calculations, They can be useful in determining additional
effects of tested chemicals,
(BI In test concentrations where growth is maximally inhibited,
alpistatie effects may he diiferentiated I Rim algictdal effects by the follow-
ing two methods,
I / 1 Add i! 5 mL of a II. 1 percent solution t weight- volume) of (.'vans
blue stam lo a 1- ml. aliquot of algal suspension from a control container
and to a I ml aliquot of algae from the test container having the lowest
concentration of test chemical which completely inhibited algal growth (.if
algal growth was not completely inhibited, select an aliquot of algae for
staining front (he test container having the highest concentration of lest
chemical which inhibited algal growths. Wail 10 to 3t.) min. examine mi-
croscopically. and determine the percent of the cells which stain blue (indi-
cating cell mortality)- A staining control is to be performed concurrently
using heat-killed or formaldehyde-preserved algal cells; 100 percent of
these cells should stain blue This method will work for Skc/i'imicma and
poviiblv Ximcttla. bill probably will not work with SclciMUmm or
Attabaena.
(2) Remove 0.5 ml. aliquots of test solution containing growth-inhib-
ited algae from each replicate test container having the concentration of
lest substance evaluated in paragraph (dX-^XviiXBX/> of this guideline.
Combine these aliquots into a new test container and add a sufficient vol-
ume of fresh nutrient medium to dilute the test chemical to a concentration
which does not aiTect growth. Incubate this subculture tinder the environ-
mental conditions used in the definitive lest J'or a period of up to 9 days,
and observe periodically (e.g. every other day) for algal growth (direct
or indirect methods) to determine if the algistatie effect noted alter the
% h test is reversible. This subculture test may be discontinued as soon
as growlIi occurs.
4
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i-4 i \nalv ileal measurements (i) (heinieal. (At Water oi •.utlicieiU
qtiulih (e.g. ASTM i'ype I water) is to be used in the preparation of the
nutrient medium, 'flic pi! of the tcsl solution and controls is to be meas-
ured al ilie beginning and at the aid of the definitive test. ! be concentra-
tion of tcsl chemtertl in the test containers is to be determined at the begin-
ning and end of the definitive test by standard unahtical methods which
have been validated prior to the test. An analytical method is unacceptable
if likely degiadatiixi product* of the chemical, such as hvdrolysis and oxi-
dation products, give positive or negative inteiferenee to the method. To
he acceptable, the analytical method must he corrected for these mtei-
ierences.
(Hi At the end of the. test and after aliquots hnvc been removed for
algal growth-response determinations, microscopic examination, mortal
staining, or subeulturing the replicate test containers for each chemical
concentration may he pooled into one sample. An aliquot of the pooled
sample mu\ then be taken and the concentration of test chemical deter-
mined alter all algal cells luive been removed. In addition, the concentra-
tion of test chemical associated with the algae alone may be determined.
Separate and concentrate the algal cells from the test solution by
ecntrifiigittg 01 tillering the remaining pooled sample and measure the test
substance concentration 111 the algal-cell concentrate
(it) Numerical. Algal growth response las percent of inhibition or
stimulation in the test solutions compared to the controls! is calculated
at the end of the test. Mean and standard deviation should he calculated
and plotted for each treatment and control. Appropriate statistical anakses
(sec pmugraphs (g)(l) and (gX2) of this guidelinei should provide a good-
ness-of-tlt determination for the concentration response curves. The con-
centration response curves are plotted using the mean measured lest solu-
tion concentrations obtained in the test chambers at the end of the test.
Results from the recovery- phase are used to determine the algistatic con-
centration (refer to paiagrnph fgi(3t of this guideline) Various statistical
procedures for modeling continuous toxicitv data are available and can
he used (see paragraph (g)(4) of this guideline).
(e) Test conditions (J ) Test species. Species of algae recommended
u.s test organisms for this lest arc the freshwater green alga, X
cupncomittiim (or R. stihcapihUit). the marine diatom, S, tasmitim. the
freshwater diatom, V. pclliciilosti. and the blue-green alga or
eyanobacterium. .1. flos-thpnw. Algae to be used in acute toxicity tests
may be initially obtained from Commercial sources and subsequent!} cul-
tured using slciilc technique. Toxicity testing should not be performed
until algal cultures are shown to he activch growing ti e capable of loga-
rithmic growth within the test period) m at least two subcultures lasting
7 da\s each pnoi to the start of the definitive test All algae used for
11 particular lest should be from the same source and the same stock cul-
5
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tine. Also, the clone of nil species should be sjieeified 1est algae should
not tune been used in u previous test. either in a treatment or a control.
(2) Facilities (i) Central (A) Facilities needed K> perform this test
include. A gtrmtli chamber or n controlled environment room that can
hold the lest containers and will maintain the air temperature, lighting in-
tensity . and photoperiod .specified 111 this test guideline, apparatus tor ail-
tunng and enumerating algae, a source of natct ol'acceptable quality. and
uppaiatus lor carrying wit analyses of the lest chemical.
-------
(13) Dilution water used tor preparation of nutrient medium and test
solutions should be of sufficient 41ml itv (e.g. ASTM Type 1 Hater), Salt-
water for marine algal nutrient medium anil lest solutions should be pre-
pared by adding a commercial synthetic sea sail formulation or a modified
synthetic seav «ter formulation to disiiiled-deioni/ed water to a conceit tril-
lion of 30 ppt (24 to 35 g kg)
(w) Carriers. Nutrient medium is to he used in making st. 1) for SL'k'hmema. 7 5( + tU )
foi ,VimV«/n. and 7.5(+0,11 lor ,ln
-------
(v J light intensify should be monitored at lite beginning of the test
at the level of the test solution or at each test chamber posit urn. If it is
suspected that light intensity has changed, monitoring more often during
the test v ill be nceeswtn..
tO Reporting. The sponsor must submit to the I 'PA all data devel-
oped by the test including those (hat are suggestive or predictive of acute
phytoto\ich\. 1h addition to the reporting requirements ns specified under
(•PA Good Laboratory Practice Standard*., 40 CI R pat! 7l>2, subpart j.
the following specific information is to be reported:
(ll Detailed information about the test organisms, including the sci-
entific name, method of venlicntion, strain, uiul source,
(21 Contiol charts of growth in the mwlruatment tmd solvent controls
for each !o\teit\ test.
(,3) A description of the test chambers and containers, the volumes
of solution tn the containers, the wav the test was beytttn fc g conditioning,
test substance additions, etc,) the number of replicates, the temperature,
the lighting, and method of incubation. oscillation rates, and type of appa-
ratus Specific modifications in test procedures due to using AnuhaeiM ot
Xavintia must be noted,
(4) 1 he concentration of the lest chemical in the control and in each
treatment at the end of the test and the pi I of the solutions.
(51 The number of algal cells per milliliter in each treatment and con-
trol and the method used to derive these values at the beginning, at 24,
48. and 72 h. and at the end of the test: the percentage of inhibition or
stimulation of growth ickttive to controls, and other adverse effect in the
control and in each treatment,
(b) The 96-h f:C50 values, and when sufficient data have been gen-
erated, the 24 , 48 . and 72 h liCSOs and 95 percent confidence limits,
the methods used to derive these values, the data used to define the shape
of the concentration-response curve and the goodness-ol-fit determination.
Flectmnic data submission (raw data) is encouraged to reduce data entry
tune required to conduct statistical analyses.
(7) Methods and data records of all chemical analyses and lest sub-
stance concentrations, including method validations and reagent blanks.
(X) "fhe results of anahses such as: Microscopic appearance of algae,
si/e or color changes, percent mortality of cells and the fate of subcultured
cells, the concentration of test substance associated with algae and test
solution supcrnate or liltiatc.
If the range-folding test showed that the highest concentration of
the chemical tested (not less than 1.000 nigl. or saturation concentration)
8
147
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had no effect on the alyae. report the results and concentration and a state-
ment thul the chemical is of minimum plniotoxie concern.
(10) If the rangc-findine lest showed greater than a 5»> percent inhibi-
tion of algal growth at a lest concentration at or below the unah tical detec-
tion limit. icpori the results, concentration, and a statement that the chemi-
cal is phytotovic at or below the unah tical dcleclion limit.
(g) References, The following references should be consulted for ad-
ditional background imperial on this lest uuidcline.
(1) American Society for Testing and Materials. ASTM FI21S 2u.
Standard guide for conducting 9(> h toxicii) tests with imetoalgae fn
I'Wl Ammal Book of A.S'1'IVI Standards. Vol 11.04: Pesticides; resource
rceo\erv, hazardous substances and oil spill response, waste disposal; bio-
logical effects, pp 845 856 {1W11,
(2) American Soeiels tor resting and Materials ASTM D 397K X0
Standard practice lor algal growth potential testing with St-k'ihi.\liimi
atprhtmnmtm- In1 Wl Annual Hook of ASTM Standards. Vol, 11.04;
Pesticides, resource recover}, hazardous substances and oil spill response;
waste disposal; biological effects, pp 32 -3b t llWl).
(3) i'auie, A.< i. and R H. Hall A method for measuring algal toxicits
and its application to the safety assessment of new ehemieak pp 171-
1 HO in 1 ,1 , Marking and R.A Kimerle (eds.). Aquatic Toxicology, ASTM
STM 667. American Society for Testing and Materials. Philadelphia. PA
nfmn.
t4t Bruce, RJ). and D.J. Veistevg. A statistical procedure for model-
ing continiious toxicih data. Etivinmmetihil Taxicolagi and ('Ih'mislrx'
if 1485- 144M 11W2S. ~
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3 Appendix 5-C. QPPTS 850.6200 Earthworm Subchronic Toxicity Test
United States Prevention. Pesticides EPA712-C-96-167
Environmental Protection and Toxic Substances April 1996
Agency (7104)
\>EPA Ecological Effects Test
Guidelines
OPPTS 850.6200
Earthworm Subchronic
Toxicity Test
LA
fr
"Public Draft"
149
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Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Preventum. Pesticides mid Toxic Substances,
United States Fnumnmental Protection Agencv lor use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review mulct Federal regulations.
The (>lTioc of Prevention, Pesticides and Toxic Substances lOPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and foxics (OPP'l ) and appeared in Title 4(1,
Chapter 1. Subchapter R of the Code of Federal Regulations fCT'R J. the
Oflicc of Pesticide Progtnms ((>FPi which appealed in publications uf the
National Technical information Sen ice tN'IlS) and the guidelines pub-
lished bv the Organization for f.eonomic Cooperation and Development
(OECDV
The purpose of tatrroom/iiig these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the 11. S. Knv iron-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C 2601) and the federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 13ft, el seq i
Pubfic Dr.ifl \cce^«. fnfinniution: Hits di..tn guideline o, pari ol a
series of related harmonized guidelines that need lo be considered sis a
unit, Fur copies. These guidelines are available electronically from the
I: PA Public Access Gopher (gophet epa.gov) under (he heading "Environ-
mental Test Methods and (uiidelines " or in paper by contacting the OPP
Public Docket at (703) 305-5X05 or b) e-mail'
guidel ines'tf-epamail .epa.gtn
To Submit Comments: Interested persons are invited to submit com-
ments, By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs. 1 ield Operations Division (75(X>C). 1'nvironmentul
Protection Agenev. 401 M St. S\V.. Washington, DC 204fi0. In poison
bring to: Rm 1132, Crystal Mall '2. 1921 Jefferson Davis Highway. 'Ar-
lington, VA, Comments maj also be submitted electronical!) hv sending
electronic mail (e-mail) to- guidelinestfepamail.epa.gov.
Filial Guideline Relfa.se; This guideline is available from the U.S
Government Printing Office. Washington, DC 20402 on f/w t-Wierai liitl-
letm Hi tin/ By modem dial 202 512 1387. telnet and lip:
fedbbs,access,gpo guv (IP 162 140,64.19) or call 202 512 0135 for disks
or paper copies.. This guideline is also available electronically in ASCII
and PDF (portable document format* lrom the 1'1'A Public Access Gopher
tpopher.epa.gov) under the heading "Ftniromnental lest Methods and
Guidelines."'
i
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OPPTS 050.6200 Earthworm subchronic toxicity test.
(a) Scope- (I) Applicability. This guideline is intended to meet test-
ing rcquiiemaits of both the Federal Insecticide. Fungicide, and
Rodenlieide Act tFlFRA) (7 U.S.C. 136. el seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C, 2601),
(2) Background. The source materials used in developing this har-
moni/xJ OPl'TS lest guideline is the UI'PT guideline under 40 CFR
795,150 Earthworm Tu\icit\ Test (proposed in the FH;t-:i8 pcicent of
No 711 mesh silica sand. 20 percent kaolin clay. 10 percent sphagnum
peat moss, mid 2 percent calcium carbonate These ingredients are weighed
and mixed in the above proportions and moistened to 35 percent (bv
weight) with deioni/ed< distilled water.
Behavioral .symptimis are indicators of toxicitv to earthworms such
that a distinct dilVetenee in position in the test container can be identified,
e.g.. below surface or on the surface; writhing on the surface: stiffened
and shortened on the surface or elongated and pulsing: or inactive below
surface in a. bull.
CUtellum means a glandular portion of the .interior epidermis, appear-
ing as saddle-shaped or annular, usually differentiated externally by color.
('ninth.- means the animals which are raised on-site or maintained
under controlled conditions to produce test organisms through reproduc-
tion.
F.( 'SO means that test substance concentration calculated
fiomc\'petimetitalh-derived growth or sublethal effects data that has af-
fected 50 percent ot a test population during continuous exposure over
a specified period of time
Lt"3t> means that experimentally derived concentration of test sub-
stance that is estimated to kill 50 percent of a test population dining con-
tinuous exposute over a specified pemxi of tunc.
1
151
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Lowest uh.senvdeffect emuentrattoit (1 C >KC) means the lowest treat-
ment (i.e., lest concentration) of a test .substance that is statistically dif-
ferent in adverse effect on a specific population of lest oigunisms from
that ohKened in controls.
Mature or adult worritt means a condition of the worm exhibiting
a elilellum in the anterior of the body
,1 ft truth tv means the lack of (mnemetu by the test organism in re-
sponse to a definite tactile stimulus to (lie anterior end Also, because
earlhwotms tend to disinte»iate rapidly alter death, the absence of orga-
nisms in the enclosed soil test container is considered to mean death has
occurred.
\'o ohwrsvd effect triiH t-nimiion tNOFC) means the highest treat-
ment (i.e.. lest concentration) of a lest substance that shows no stalislieal
diJYerenec in adverse effect on a specific population of test utganisms trom
that observed in controls.
I'tilhttlogwal swiptom* means toxic effects, such as sm face lesions
and midsey mental swellings or general ulcerated areas on the surface of
the eai thw orm.
I'est mixtnrv means the test substance,-artificial soil mixtures which
the earthworms are exposed to during the test.
Test sukxhvtce means any compound used in artificial soils spiked
for laboratory testing of toxicity.
(ds lest prwi'tliirt's (!> Summary of (he test mi Icsl eli.imbeis
are filled with appropriate amounts of test mixtures,
(ii) This toxicity test may be done by placing earthworms in test
chambers containing test mixtures oil J allowing earthworms to mgest this
lest mixture soil ad libitum.
(hi) Acclimated earthworms are introduced into the test and control
chambers bj stratified random -assignment.
(iv) Farthworms in the test and control chambers should be observed
ever})' 7 days and the findings should be iecorded and dead carlhwotms
removed.
(\ i The pi I. temperature, and the coiieentiation of the test mixtures
should be measured at 7 da\ intenais in each test chamber
(vi) Initial weight of earthworm should be between 300 to (>()() g per
container.
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(vii) Coneen tratioo-response curves. LC50. IC50. I (!K(J. NOHC val-
ues, and 95 percent confidence intervals for the test substance are devel-
oped from the- data collected during the test.
(2) Range-finding tost (it If (lie toxicity of the test substance is not
alrejuh known, a range-finding test should ho jwformcd to determine the
range. This lest is designed to determine a concentra-
tion-mortality curve at 28 days and estimate the respective I C50. HC50,
I (>FC, NOliC values and l>5 percent confidence intervals,
lii) If data permit, the concentration-response curves. LC50. EC50.
I.OI'C. NOl.C values, and <>5 pejcesi! confidence interval also should be
determined for 7. 14, and 21 days.
tiii) This toxicity test uses earthworms which are maintained in direct
contact with an artificial sod allowing earthvvonns to ingest contaminated
soil tic/ h'bilum.
(iv) A minimum of *0 earthworms exposed to each of 5 or more
test concentrations and a control should He tested.
(v) Test concentrations should he chosen in a geometric series in
which I he ratio is between 1.5 and 2 0 mg'kg (eg.. 2 4. 8, 10. 32. and
64 mc ki>). All test concentrations should be based on milligram of test
chemical (100 percent acthe ingredient) per kilogram of artificial soil (air-
dry weight).
(vii '1 on earthworms per container of 200 g (dry weight) artificial
soil should be placed in three replicates for each concentration and control.
The distribution of individual earthworms among the lest chambers should
be randomized. Test concentrations in artificial soil should be anah/.ed
for test chemical concentrations piior to the stall of (he test and at days
7, 14. 21. and 28 as minimum.
(vii| The living earthworms should be placed on the surface of the
medium and the jar capped mid secured without making an airtight seal.
3
153
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(viii) Any changes in soil temperature should not exceed 3 C per day
or 11 C pur hour. Earthworms should be held for a mini mum of 7 dsns
at the test temperature prior to testing-
fix) Hveiy test should include n negative control consisting ul'
iinemit.-tminafed artificial soil, conditions, procedures, and earthworms
tVum the same group used in the definitive test as shown, except that none
of the lest substance is added,
(x i The test duration is 2K dm s.
(4) 'lest results, (it Death is the primary criterion used in this test
guideline to evaluate the loxieih of the test substance
(ii) In addition to death, weight loss, behavioral symptoms and patho-
logical symptoms should he recorded
(iii I bach lest and control chamber should be cheeked tor dead or
afl'ected earthworms and observations recorded ?. 14. 21, and 28 days after
the beginning of the test or within 1 hour of the designated times, Missing
earthworms should be considered to have died.
(iv) Mortality is assessed by emptying the test medium on a glass
or other inert surface, sorting earthworms from the lest mixture and testing
their reaction to si gentle mechanical stimulus, Any adverse effects (eg.
weight loss, behavioral or pathological symptoms) are noted and should
be reported. 1 he medium is returned to each container.
tv) The 28 day test result is he mi acceptable if
(A) More than 20 percent of control organisms die: or
(B) The total mean weight of the earthworms in the control containers
declines significantly during the lest (i.e.. by 30 percent).
(vij Mortality is checked and recorded at days 7. 14, 21. and 28.
t,vii) The mortality data should be used to calculate I C5n values and
their 95 percent confidence limits, and Us plot eoneeiitiation-resfxmse
curves at days 7, 14. 21. and 28
(viii) The sublethal effects and growth (t e„ fresh weight) data should
he used to plot concentration-response curses, calculate HC50 values, and
determine LuI'C and NOliC values, Approptiate statistical methods (e.g,.
one-way analysis of variance and multiple comparison test) should be used
to test for significant dilferwices between treatment means and determine
1 DI.C and NO!
15) Analytical measurements (ii Artificial soil analysis. During the
test, the temperature and pfi should be mcasuied in the aitificial soil at
the beginning of the lest(/ert> hourt. and every 7 days thereafter,
4
154
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(ii) Measurement of test substance
(A) The concentration of lest substance in artificial soil should be
measured at a minimum in each test chamber at the beginning 1 zero hour,
before earthworms are added) and even 7 d«\s thereafter,
(Bl The uiinKtical methods used to measure the amounl of lust sub-
stance in a sample should be validated befoic beginning the lest The accu-
racy of a method should be perilled In a method such as using known
additions This involves adding a known umount of the test substance In
three samples of artificial soil taken front the test chamber and (he saute
number of earthworms as arc used in the test. The measured concentration
of the test .substance in those samples should span the concentration range
lo be used in the test. Validation of the analytical method should be per-
formed on at least two separate Ja\s ptior to starling the test.
I'O Ait Ileal method is not acceptable if likeh degiadation prod-
ucts of the test substance give positive or negative interferences. unless
it is shown that such degradation products ate not present in the test cham-
bers during the tesl
(D) In addition to anoh/.ing samples of artificial soil, at least one
reagent blank, containing all reagents used, should also he analv/ed
(K") The measured concentration of the test substance in artificial .soil
in am chamber dining the tost should not van' more than 50 percent from
the measured concentration prior to initiation of the tesl: concentration
measurements should be as. described In Ncuhauscr el a)., in paragraphs
(g)(5) and of tins guideline, or an equivalent method.
(f") The mean measured eoncentration of lest substance in artificial
soil (dr\ weight) should be used to plot all concentration-response curves
and to calculate all LC50. l:C5fl. Lul.'C. and NOKC values
((!) The total carbon (TO should be determined as measured by the
method of Plumb described in paragraph (g'X? t of this guideline, or an
equivalent method.
(lii) Numerical. The statistical methods recommended for use in cal-
culating the I C50 and Ft"50 values include prolnt. logit. moving average,
and binomial.
ic I I est eomlifiims i I i 1 est-.pedes 11 > Selection. I he lest species
for this test is the earthworm iii.wnht fetiiia andrei ilkmche). The species
identity of the test organism should be verified using appioptuite ta\o-
nomie keys as described In fender in paiagraph (g)(2) of this guideline,
or an equivalent method.
(ii) Age and condition of earthworm*. (A) Adult earthworms. 300
MR) mg. are to be used to start the tesl.
5
155
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(]i) f'arttmontts used in toxieiu tests should he purchased from a
commercial source lhut can veiify the species. Once v en lied, cultures
should he maintained at the lest facility Records should be kepi regarding
the source of the initial stock and culturing techniques. All organisms used
for a particular test should have originated from the same population (cul-
ture*.
(C f All newly acquired c:ti1h\\oims should he quarantined and ob-
served lor at least 14 days prior to use in a test.
(1)'t Kurthworms should not be used if they have been under stress
from too much or a luck of moisture as dcscubcd by Reinecke and Venter
in paragraph te)(8i of this guideline. 01 an equivalent method, excessive
or inadequate food or temperature us described by Tom hit and Miller in
paragraph (g¥l 1) of' this guideline or an equivalent method: pi I variation
as described by Satchel! and Dortie in paragraph of this jandetme.
or an equivalent method; or crowding. Any of these conditions will
produce earthworms that mm not be healthy,
Itiit Preparation. Stitiieient numbers of earthworms .should be hat-
vested and soiled to insure that health)' individuals are used lor the test.
Anv animals that appear |t> be injured should not be used 111 the test and
must be discarded.
(t\) Acclimation of fest earthsurms. Adah earthworms should lie
handled with care. Harthworms should be held tor a minimum of 7 days
in uncon laminated soil at the test temperature prior to testing.
(v) Feeding, (A) Substrate food lor entering Eiwniit felidu andrei
should be saturated ("water) alfalfa (' \!edicag<> saliva\ pellets.
(B) The earthworms are not fed during the test period.
(2) Facilities (ii General. Facilities needed to perform this test in-
clude;
(A) Apparatus for providing continuous lighting.
(R) Chambers for exposing test earthworms to the test substance.
(C) A mechanism for controlling and maintaining the artificial soil
temperature and relative humidity during the holding, acclimation, and test
periods.
(ri) Construction materials. (A) Construction materials and equip-
ment that contact test mixtures should not contain substances that can be
leached or dissolved into artificial soil in quantities that can affect the
test results. Material and equipment that contact test mixtures should be
chosen to minimize sorption of test substances. Hard glass jars arc pret-
6
156
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etable ami should he healed in an asking oven between tests; soft glass
jars should he used only once,
(Bt Polycthvlene containers (retangular dish pans measuring
32.5/27 5/ 12 5 em'i for ctilUiring earthworms, a mechanism te.g , envi-
ronmental chamber) liir maintaining temperature and relative humidity of
the cultures during cultiiring. an J separate facilities for testing are required,
I'D Testing containers (e.g. I pt glass canning jars) and lids, and suit-
able balances to measure soil mixtures and sample weights should also
he used,
ID.) Relative humidity should he maintained above 85 percent, An
open pun of water can be used lor this purpose to prevent moisluto loss
from the cunlaistas.
(iii) Test chambers. (A) Glass tanning jars, 1-pt capacity, or (licit
equivalent, should be used for testing,
(Bl fhe lids should he te\ ersed sic. turned upside down i. loosely
capped and scented without making an airtight seal to I educe evaporation
and permit air exchange.
(iv) Cleaning of test system, fhe test chambers should be cleaned
before each test following standard laboratory procedures If soft glass is
lo be used it must only be used once and then thrown awav
(v) Medium preparation, (At For each concentration tested and con-
trols, enough artificial soil must be prepared by recipe lo yield 270 g of
artificial soil (wet weights per replicate. A dry weight mixture of 68 per-
cent of No. 7(1 mesh silica sand, 20 percent kaolin clav. and 10 percent
sphagnum peat moss are mixed until c\ enh distributed,
(lit Up to 2 percent pulverized calcium carbonate mav be added to
adjust the soil pH to 6.5 x(>,5.
(C) An appropriate amount of high punty water (e.g. 70 g, per 200
g of dry soil l is added to the artificial soil and mixed with the artificial
soil lo raise the artificial soil moisture kvei to 33 percent by weight to
yield a total weight of X10 g artificial soil at 35 percent moisture.
tj)) Appropriate portions of fhe artificial soil are mixed thoroughly
with appropriate amounts of test .substance io yield three replicates for
each lest concentration, Each test mixture is divided into three equal quan-
tities of about 270 g as determined by weight, Fach portion is placed into
a separate 1 - pi jar and represents one replicate for exposing 10 earthworms
at the same concentration, fhree replicates for negative and. if necessary,
solvent controls are prepaicd from untreated portions of the aititicial soil
mixture,
7
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(I-'j If a solvent is used, the opened ehambei•» tire placed in a hood
for 24 hours to evapotate the solvent prior to addma the earthworms.
fi t 1'riot to the addition of earthworms, u If) g simple should he
removed from each replicate to measure pi I and test concentrations.
(3) Test parameters (it I.oatliuji. The number of earthworms
placed in a test chamber should not he so great as to affect the results
of the test, The weight of the individual earthworms xhould be between
300 nia and rag each The earllnvotms are selected front the culture
randomlv into groups of 10. These groups are then randomh assigned to
lite lest containers mid then weighed such that they do not differ more
than - 10 percent aiitoaa, tile replicates.
(li) Temperature, (A) The test soil icmperutuie should be 22 ±2 C
as described by Edwards in paragraph fgK 1) of this guideline, or using
an equivalent method.
tlii fomperuhtre should be measured and re|x>ited at the beginning
of the test and on davx 7. 14. 21. and 28. The temperature should he
measured al least hourh in one test container.
fiii) (A) Replicates shook! lie illuminated continuously with
incandescent or fluorescent lights as described b\ hdwards in paragraph
(g)( I.) ot'this guideline or using an equivalent method.
(B| I ight mtensitv should be about 400 Is measured al the artilieial
soil surface.
(Cl Light intensity should be measured at least once during the test
at the surface of I he container and cheeked weekly in the test chambers.
(f) Reporting, (1) The sponsot should submit all data developed by
the test thai are suggestive or predictive of toxicity and all concomitant
giosx toxicoloyieai manifestations. I he reporting of test data should in-
clude the following information;
(i) Test Background including the name of the sponsor, testing labora-
tory- principal investigator, and dates of testing
Mi) A detailed description of the test chemical including its chemical
identification (CAS Regis!rv No., trade name, common name i. source, lot
number, composition (jdentih and concentration or major ingredients and
major impurities), known phvsieal and chemical properties, empirical foi-
mula, water solubiiitv. vapor pressure, manufacturer, method of applica-
tion, and ain carriers oi other additives used and their concentrations 1'he
volume or mass of any eairiers should be reported An e\aet description
of how the lest substance has been mixed into the artificial soil,
8
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(iii'l Detailed information about the earthworms used as brood stock,
including the .scientific name and method of verification. age. source, treat-
ments. feeding history. and culture method.
(iv) A description of the le.si situation, especial h if there was a devi-
ation fiom this text guideline us described abo\e in soil preparation (para-
graph (et(2)(v)(Ai of this guide!met addition of the chemical, culuiring
of the test species, lighting. pH. temperature. replicates, or the number
of organisms per cvmtumet.
(v) A description of the tesi ct>riturner u>cd, its size. volume and
weight of soil used in each container, mini her of test organisms pci con-
tainer. number of lost containers per conceMiution, conditioning of the test
container, description of the method of lest chemical introduction into the
test medium (eg . as a powder), stock solution used 01 not, and time be-
tween mixing of the stock solution and introduction of the earthworms.
(\it The concentration in aitificial soil at the beginning of the test
and the actual concentrations of the test chemical (if measured) in the
soil he lot e (day 0). during t Jus 7, 14. 21) and upon the conclusion of
the test (du\ 2X) and the dales the ansthses were performed
(\ it) The total organic carbon (T< >C) ot the soil mixture.
|2) The reported results should include:
(i) The number and percentage of organisms that were killed or
showed any adverse elleets at each test concentration, including controls,
in each test jar at each observation period.
(ii > Concentration response curves fitted to mortality data al 7, 14,
21. and 2K -day periods, A statistical text of goodness-of-lit should be per-
formed and reported
(iii) The J.C5U/FC50 \allies and the 95 percent confidence limits
using the mean measured test concentration and the methods used to cal-
culate both the I.C50'KC50: also the l.OKC and NOKC values and the
conltdenee intervals by the Trimmed Speatmun- Koiher method as de-
scribed by Hamilton et al,. in paragraph of this guideline, or an
equivalent method. The probii technique should follow the methods de-
scribed by Weber et ul.. in paiagraph (g)( 12) of this guideline, or an equiv-
alent method. Appropriate statistical methods (e.g., one-way analysis of
variance and multiple comparison test) .should he used to test lor signifi-
cant differences between treatment and determine the LOKC and NOJiC,
(i\) All chemical analyses of tost material including methods, method
validations, and reagent blanks,
(v) The data lecords for the culture and lighting,
f
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I v ii Moisture content for the test mixture at start of test.
I'vii) "1 he pi I and temperature values at start of lest and on da\s 7,
14. 21. and 28 uf the test.
(viii) Any deuation from this test guideline and atnlhmsj unusual
about the test fe jj.. equipment failure. llueliutioiis iti temperature, pit. or
other environmental conditions)
(g) References, lor additional background information on this test
guideline the following references should be consulted:
(1) Fdwurds. C.A. Report of the second shtge in development of a
xhmiktrdized Itthriralor)' method jar assessing the toxicity of chemical sub-
stances to I'l/rllm'tirim I he Artificial Soil Test IX) XI Al l82 43. Revi-
sion 4 119841
(2) Fender. WM fuir/hwurms of lite IS'estem I 'titled Stales, Part I.
Lumhrieidae. Slegadrilogku. 4. 93 -129 t 1985).
(3) Hamilton. MA. et al. Primmed Xpearman-Kurber method tor esti-
mating median lethal concentrations in toxicity Noassavs Environmental
Science and I'micokm". 11(7);714 717 (19771 Correction: Ibid 12; 417
(1978).
(4 t i lartenstein, R et al. Reproductive potential of the earth worm
fiVirn; foetidu . 43 CJecahigut. 329 340 (197')).
(5) Neuhauscr, li.F. et al Contact and artificial soil tests usinp earth-
worms to evaluate the impact of wastes in soils. In: Hazardous and Indus-
trial Soil Waste I'estiitg: Fourth Sunposktm, ASTM K I P 886 J.K Pcfros,
Jr. and R A. Con\\j\, eds., (American Society for Testing and Materials.
Philadelphia. PA I9Kf>)pp. 192 203.
16) Neuhiiiisor. li.F. et ul. The toxieih of selected organic chemicals
to the earthworm Eisetiia feiida. Journal of Environmental OimUH', 14.
383 388(1985).
17) Plumb. R.H., Jr. Procedures for handling and chemical an a I v.sis
of sediment and water sampler. Technical Repot! l.'PA CP 81 I. prepared
by Great Lakes Patxiiaton. State Ilimersih College at Buffalo, ButVaki,
NY,, for the U.S. Pnvironmental Protection Age»ie> Corp. ol Engineers
Technical Committee on Criteria for Diedged and Pill Material, IJ S, Atm\
Pngineer Waterways experiment Station. CP, Vickshurg, MS ( 1981).
(8) Reinecke. A J mid Venter. J M Moisture piefeiences, growth and
reproduction of the compost worm i'tsettia feiultt (Oltgoehaeuii, ttioltnfx1
of Fertility Soils, 3: 135 141 (19871,
1#
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