B. Paperwork Reduction Act

    The information collection activities in this final action will be
submitted for approval to the Office of Management and Budget (OMB)
under the Paperwork Reduction Act (PRA). The Information Collection
Request (ICR) document prepared by EPA has been assigned EPA ICR No.
1664.14. You can find a copy of the ICR in the docket for this rule,
and it is briefly summarized here. The information collection
requirements are not enforceable until OMB approves them.
    The NCP Product Schedule listing and authorization of use
provisions of the final rule include requirements for submission of
general product information and documentation of information related to
product testing. For this ICR, EPA has estimated an average annual
total burden for respondents of 487 to 1,165 hours per year in the
first three years, and average annual labor and O&M costs of $1,040,969
to $1,088,123. EPA has carefully considered the burden imposed upon the
regulated community by the regulations. EPA believes that the
activities required are necessary and, to the extent possible, has
attempted to minimize the burden imposed. The minimum requirements
specified in the final rule are intended to encourage the development
of safer and more effective spill mitigating products, and to better
target the use of these products to reduce the risks to human health
and the environment.
    Respondents/affected entities: Manufacturers of dispersants, other
chemical and biological agents, other spill mitigating devices and
substances.
    Respondent's obligation to respond: Mandatory if manufacturer
wishes to have a product listed on the NCP Product Schedule (40 CFR
part 300, subpart J).
    Estimated number of respondents: 109 responses by 89 existing
product respondents during year one and two of the ICR period; in
addition, 5 new product responses per year, and 10 sorbent submissions
per year. The overall average number of responses during the ICR period
is 51.
    Frequency of response: Occasional.
    Total estimated burden: 487 to 1,165 hours per year. Burden is
defined at 5 CFR 1320.3(b).
    Total estimated cost: $1,040,969 to $1,088,123 per year.
    An agency may not conduct or sponsor, and a person is not required
to respond to, a collection of information unless it displays a
currently valid OMB control number. The OMB control numbers for the
EPA's regulations in 40 CFR are listed in 40 CFR part 9. When OMB
approves this ICR, the Agency will announce that approval in the
Federal Register and publish a technical amendment to 40 CFR part 9 to
display the OMB control number for the approved information collection
activities contained in this final rule.

C. Regulatory Flexibility Act (RFA)

    I certify that this action will not have a significant economic
impact on a substantial number of small entities under the RFA. The
small entities subject to the requirements of this action are 88
potentially small businesses in the following industries: Support
Activities for Mining; Specialty Trade Contractors; Paper
Manufacturing; Petroleum and Coal Products Manufacturing; Chemical
Manufacturing; Plastics and Rubber Products Manufacturing; Durable
Goods Merchant Wholesalers; Nondurable Goods Merchant Wholesalers; Non-
store Retailers; Warehousing and Storage; Professional, Scientific, and
Technical Services; Administrative and Support Services; Waste
Management and Remediation Services; Repair and Maintenance; and
Religious, Grantmaking, Civic, Professional, and Similar Organizations.
The Agency has determined that up to five of the affected small
entities may experience an impact of 1% to 3% of revenues and up to
five of the affected small entities may experience an impact of greater
than 3% of revenues. Details of this analysis are presented in EPA's
Regulatory Impact Analysis, Final Revisions to the National Oil and
Hazardous Substances Pollution Contingency Plan Regulations (40 CFR
part 300 Subpart J), which is available in the docket for this action.

D. Unfunded Mandates Reform Act

    This action does not contain any unfunded mandate of $100 million
or more as described in UMRA, 2 U.S.C. 1531-1538, and does not
significantly or uniquely affect small governments. This final rule
imposes no new enforceable duty on any state, local, or tribal
governments or the private sector.

E. Executive Order 13132: Federalism

    This action does not have federalism implications. It will not have
substantial direct effects on the States, on the relationship between
the national government and the States, or on the distribution of power
and responsibilities among the various levels of government.

F. Executive Order 13175: Consultation and Coordination With Indian
Tribal Governments

    This action has Tribal implications. However, it will neither
impose substantial direct compliance costs on federally recognized
Tribal governments, nor preempt Tribal law. EPA has concluded that this
action may have Tribal implications because all Tribes can be affected
by oil spills and the subsequent use of oil spill mitigating agents,
such as dispersants and bioremediation agents. Furthermore, CWA section
311(j)(4)(A)(ii) provides for qualified members of federally

recognized Indian Tribes, where applicable, to be members of Area
Committees. Additionally, E.O. 12777 provides that RRTs may include
representatives from Tribal governments.
    EPA consulted with Tribal officials under EPA Policy on
Consultation and Coordination with Indian Tribes early in the process
of developing this regulation to enable them to have meaningful and
timely input into its development. A summary of that consultation is
provided in Regulatory Impact Analysis, Final Revisions to the National
Oil and Hazardous Substances Pollution Contingency Plan Regulations (40
CFR part 300 Subpart J), which is available in the docket for this
action.
    As required by section 7(a), EPA's Tribal Consultation Official has
certified that the requirements of the executive order have been met in
a meaningful and timely manner. A copy of the certification is included
in the docket for this action.

G. Executive Order 13045: Protection of Children From Environmental
Health Risks and Safety Risks

    This action is not subject to Executive Order 13045 because it is
not economically significant as defined in Executive Order 12866, and
because EPA does not believe the environmental health or safety risks
addressed by this action present a disproportionate risk to children.
The Agency has concluded that the effect of the requirements codified
in this final rule will mitigate the adverse effects of environmental
and socio-economic damage that could otherwise result from major oil
spills. This final action will therefore not have a disproportionate
adverse effect on children.

H. Executive Order 13211: Actions That Significantly Affect Energy
Supply, Distribution or Use

    This action is not a ``significant energy action'' because it is
not likely to have a significant adverse effect on the supply,
distribution, or use of energy. The requirements specified in the final
rule are intended to encourage the development of safer and more
effective spill mitigating products, and to better target the use of
these products to reduce the risks to human health and the environment;
thus, the rule will result in greater overall environmental protection.
The final rule will not cause reductions in the supply or production of
oil, fuel, coal, or electricity; nor will it result in increased energy
prices, increased cost of energy distribution, or an increased
dependence on foreign supplies of energy.

I. National Technology Transfer and Advancement Act

    This rulemaking involves technical standards. Therefore, the Agency
conducted a search to identify potentially applicable voluntary
consensus standards. However, EPA identified no such standards for
efficacy and toxicity testing, and none were brought to the Agency's
attention in comments. Therefore, EPA developed the Baffled Flask
Efficacy Test; the Dispersant Toxicity Test; the Standard Acute
Toxicity Testing for Surface Washing Agents, Bioremediation Agents,
Herding Agents, and Solidifiers; and the Bioremediation Efficacy Test
provided in Appendix C of this final rule.
    Additionally, EPA has decided to use voluntary consensus standards
for several product property data points, such as pH, flash point, and
pour point. The product toxicity testing relies on existing protocols
that are universally accepted. The Agency has removed the incorporation
by reference of specific standards to determine physical and chemical
properties and replaced this with a requirement for a citation of the
current applicable standard methodology used to determine these values.
EPA believes that citing the current applicable standard methodology
used to determine the required values is sufficient in lieu of
specifying commonly recognized standard methodologies. Furthermore, EPA
did not incorporate by reference specific test methodologies in the
regulation to avoid the administrative burden of updating the NCP every
time a test methodology is updated to a newer version.

J. Executive Order 12898: Federal Actions To Address Environmental
Justice in Minority Populations and Low-Income Populations

    Executive Order 12898 (59 FR 7629, February 16, 1994) directs
federal agencies, to the greatest extent practicable and permitted by
law, to make environmental justice part of their mission by identifying
and addressing, as appropriate, disproportionately high and adverse
human health or environmental effects of their programs, policies, and
activities on minority populations (people of color and/or Indigenous
peoples) and low-income populations.
    The EPA believes that the human health or environmental conditions
that exist prior to this action result in or have the potential to
result in disproportionate and adverse human health or environmental
effects on people of color, low-income populations and/or indigenous
peoples. Discharges of oil from facilities regulated by this action
likely pose disproportionate risks to historically marginalized
communities.
    The EPA believes that this action is likely to reduce existing
disproportionate and adverse effects on people of color, low-income
populations and/or indigenous peoples. EPA has concluded that the
regulatory requirements will advance fair treatment of those
populations by reducing the disproportionate damages that oil
discharges might otherwise inflict on those populations. EPA has
concluded that the requirements codified in this final rule will
mitigate the adverse effects of environmental and socio-economic damage
that could otherwise result from major oil spills and are likely to
reduce existing disproportionate and adverse effects on people of
color, low-income populations and/or indigenous peoples. EPA has
concluded that the regulatory requirements will advance fair treatment
of those populations by reducing the disproportionate damages that
major oil spills might otherwise inflict on those historically
marginalized populations.
    The focus of this action is to modernize and update Subpart J of
the NCP. Nonetheless, the EPA identified environmental justice concerns
associated with the final rule and qualitatively assessed whether the
requirements codified in this final rule will mitigate the adverse
effects of environmental and socioeconomic damage that could otherwise
result from oil spills. EPA has concluded that, while the changes in
this rule were independent of environmental justice considerations, the
regulatory requirements will advance fair treatment of those
populations by reducing the disproportionate damages that discharges
might otherwise inflict on those historically marginalized populations.
Specifically, EPA has concluded that:
     The amended requirements to add new listing criteria and
revise efficacy and toxicity testing protocols emphasize development
and listing of ``greener'' oil spill mitigating products and will
increases public transparency on chemical and biological agent
composition.
     The amended requirements for authorization of use,
notifications, and data reporting better target agent use to reduce
risks to human health and the environment. The amended requirements
will increase both public awareness on chemical and biological

agent preparedness planning and response activities, including
potential engagement opportunities, and access to information on the
components for any chemical and biological agent listed on the NCP
Product Schedule. EPA expects the final rule requirements will also
enhance EPA's ability to address area- and regional-specific concerns
and provide greater public awareness of chemical and biological agent
use during a response through public notification.
     EPA expects that the final action's emphasis on developing
safer and more effective spill mitigating products, and on better
targeting their use, will reduce the risks to human health and the
environment when chemical and biological agents are used during oil
spill responses in these newly developed areas.
    The information supporting Executive Order 12898 review is
contained in the Regulatory Impact Analysis, Final Revisions to the
National Oil and Hazardous Substances Pollution Contingency Plan
Regulations (40 CFR part 300 Subpart J), which includes an
environmental justice analysis and is available in the docket for this
action.

K. Congressional Review Act

    This action is subject to the CRA, and the EPA will submit a rule
report to each House of the Congress and to the Comptroller General of
the United States. This action is not a ``major rule'' as defined by 5
U.S.C. 804(2).

List of Subjects

40 CFR Part 110

    Environmental protection, Oil pollution, and Reporting and
recordkeeping requirements.

40 CFR Part 300

    Air pollution control, Area contingency planning, Bioremediation,
Chemicals, Dispersants, Environmental protection, Hazardous materials,
Hazardous substances, Intergovernmental relations, Natural resources,
Oil spills, Oil spill mitigating devices, Regional response teams,
Sorbents, and Surface washing agents.

Michael S. Regan,
Administrator.

    For the reasons set out in the preamble, the Environmental
Protection Agency amends 40 CFR parts 110 and 300 as follows:

PART 110--DISCHARGE OF OIL

0
1. The authority citation for part 110 continues to read as follows:

    Authority:  33 U.S.C. 1251 et seq., 33 U.S.C. 1321(b)(3) and
(b)(4) and 1361(a); E.O. 11735, 38 FR 21243, 3 CFR parts 1971-1975
Comp., p. 793.

0
2. Revise Sec.  110.4 to read as follows:

Sec.  110.4  Chemical or biological agents.

    The addition of any chemical or biological agent, or any other
substance, to oil to be discharged that would circumvent the provisions
of this part is prohibited.

PART 300--NATIONAL OIL AND HAZARDOUS SUBSTANCES POLLUTION
CONTINGENCY PLAN

0
3. The authority citation for part 300 continues to read as follows:

    Authority:  33 U.S.C. 1251 et seq.; 42 U.S.C. 9601-9657; E.O.
13626, 77 FR 56749, 3 CFR, 2013 Comp., p. 306; E.O. 12777, 56 FR
54757, 3 CFR, 1991 Comp., p. 351; E.O. 12580, 52 FR 2923, 3 CFR,
1987 Comp., p. 193.

Subpart A--Introduction

0
4. Amend Sec.  300.5 by:
0
a. Adding in alphabetical order definitions of ``Bioaccumulation'',
``Bioconcentration'', ``Biodegradation'', ``Biological agents'', and
``Bioremediation'';
0
b. Revising the definitions of ``Bioremediation agents'', ``Burning
agents'', ``Chemical agents'', ``Dispersants'';
0
c. Adding in alphabetical order the definition of ``Herding agents'';
0
d. Removing the definition of ``Miscellaneous Oil Spill Control Agents
(MOSCA)'';
0
e. Adding in alphabetical order the definition of ``Products'';
0
f. Revising the definition of ``Sinking agents'';
0
g. Adding in alphabetical order the definition of ``Solidifiers'';
0
h. Revising the definition of ``Sorbents'';
0
i. Removing the definitions for ``Surface collecting agents'' and
``Surface washing agent''; and
0
j. Adding in alphabetical order the definition of ``Surface washing
agents''.

Sec.  300.5  Definitions.

* * * * *
    Bioaccumulation is the process of accumulation of chemicals in the
tissue of organisms through any route, including respiration,
ingestion, or direct contact with the ambient or contaminated medium.
    Bioconcentration is the accumulation of chemicals in the tissues of
organisms from water alone.
    Biodegradation is a process by which microorganisms metabolically
decompose contaminants into biomass and smaller molecular compounds
such as carbon dioxide, water, and end products.
    Biological agents are microorganisms (typically bacteria, fungi, or
algae) or biological catalysts, such as enzymes, that can enhance the
biodegradation of a contaminated environment.
    Bioremediation is the process of enhancing the ability of
microorganisms to convert contaminants into biomass and smaller
molecular end products by the addition of materials into a contaminated
environment to accelerate the natural biodegradation process.
    Bioremediation agents are biological agents and/or nutrient
additives deliberately introduced into a contaminated environment to
increase the rate of biodegradation and mitigate any deleterious
effects caused by the contaminant constituents. Bioremediation agents
include microorganisms, enzymes, and nutrient additives such as
fertilizers containing bioavailable forms of nitrogen, phosphorus, and
potassium.
    Burning agents are additives that, through physical or chemical
means, improve the combustibility of the materials to which they are
applied.
* * * * *
    Chemical agents are elements, compounds, or mixtures designed to
facilitate the removal of oil from a contaminated environment and to
mitigate any deleterious effects. Chemical agent categories include
burning agents, dispersants, herding agents, solidifiers, surface
washing agents, and bioremediation agents that consist of nutrient
additives.
* * * * *
    Dispersants are substances that emulsify, disperse, or solubilize
oil by promoting the formation of small droplets or particles of oil in
the water column.
* * * * *
    Herding agents are substances that form a film on the water surface
to control the spreading of the oil to allow for oil removal.
* * * * *
    Products are chemical or biological agents or other substances
manufactured using a unique composition or formulation.
* * * * *
    Sinking agents are substances introduced into an oil discharge for
the purpose of submerging the oil to the bottom of a water body.
* * * * *
    Solidifiers are substances that through a chemical reaction cause
oil to become

a cohesive mass, preventing oil from dissolving or dispersing into the
water column. Solidifiers are generally collected and recovered from
the environment.
    Sorbents are inert and insoluble substances that readily absorb
and/or adsorb oil or hazardous substances, and that are not combined
with or act as a chemical agent, biological agent, or sinking agent.
Sorbents may be used in their natural bulk form or as manufactured
products in particulate form, sheets, rolls, pillows, or booms.
Sorbents are generally collected and recovered from the environment.
Sorbents consist of:
    (1) Natural organic substances (e.g., feathers, cork, peat moss,
and cellulose fibers such as bagasse, corncobs, and straw);
    (2) Inorganic/mineral compounds (e.g., volcanic ash, perlite,
vermiculite, zeolite, clay); and
    (3) Synthetic compounds (e.g., polypropylene, polyethylene,
polyurethane, polyester).
* * * * *
    Surface washing agents are substances that separate oil from solid
surfaces, such as beaches, rocks, metals, or concrete, through a
detergency mechanism that lifts and floats oil. Product and oil are
generally to be collected and recovered from the environment with
minimal dissolution, dispersion, or transfer into the water column.
* * * * *

Subpart J--Use of Dispersants, and Other Chemical and Biological
Agents

0
5. Revise the heading of Subpart J as set out above.

0
6. Amend Sec.  300.900 by revising paragraphs (a) and (c), and by
adding paragraph (d) to read as follows:

Sec.  300.900  General.

    (a) Section 311(d)(2)(G) of the Clean Water Act (CWA) requires EPA
to prepare a schedule identifying dispersants, other chemicals, other
spill mitigating devices and substances, if any, that may be used in
carrying out the NCP; and the waters and quantities in which they may
be used safely. This subpart establishes a schedule that includes the
NCP Product Schedule identifying chemical and biological agents, the
Sorbents Product List, and the authorization of use procedures that,
when taken together, identify the waters and quantities in which such
dispersants, other chemicals, or other spill mitigating devices and
substances may be used safely.
* * * * *
    (c) This subpart applies to the use of chemical and biological
agents as defined in Subpart A of this part, or other substances that
may be used to remove, control, or otherwise mitigate oil discharges.
    (d) [Reserved]

Sec.  300.905  [Removed]

0
7. Remove Sec.  300.905.

0
8. Revise Sec.  300.910 to read as follows:

Sec.  300.910  Authorization for agent use.

    Use of chemical or biological agents in response to oil discharges
must be authorized by the OSC in accordance with the provisions of this
section.
    (a) Use of agents identified on the NCP Product Schedule or use of
burning agents on oil discharges addressed by a preauthorization plan.
Area Committees and RRTs shall address, as part of their planning
activities, whether preauthorization of the use of chemical and
biological agents listed on the NCP Product Schedule or the use of
burning agents on certain oil discharges is appropriate. Area
Committees and RRTs shall, as appropriate, include applicable approved
preauthorization plans in ACPs and RCPs. When a preauthorization plan
is approved in advance for the use of certain agents under specified
discharge situations, then the OSC may authorize the use of agents
listed on the NCP Product Schedule, or the use of burning agents, for
the purpose for which they were specifically listed without obtaining
the incident-specific concurrences and without the natural resource
trustees consultations described in paragraph (b) of this section.
    (1) Preauthorization plan development. For discharge situations
identified where such agents may be used, the preauthorization plan
must, at a minimum, specify limits for the quantities and the duration
of use, and use parameters for water depth, distance to shoreline, and
proximity to populated areas. In meeting the provisions of this
paragraph, preauthorization plans should document how regional factors
are addressed including likely sources and types of oil that might be
discharged, various potential discharge scenarios, the existence and
location of environmentally sensitive resources or restricted areas
that might be impacted by discharged oil, and logistical factors
including inventory, storage locations and manufacturing capability of
available agents, availability of equipment needed for agent use,
availability of adequately trained operators, and means to monitor
agent use in the environment. Preauthorization plans are to be
developed by the Area Committees or the RRT in consultation with the
Area Committee(s).
    (2) Preauthorization plan approval. The EPA representative to the
RRT, the Department of Commerce and the Department of the Interior
natural resource trustees and, as appropriate the RRT representative
from the state(s) with jurisdiction over waters and adjoining
shorelines within the preauthorization plan area shall review and
either approve, approve with modification, or disapprove the
preauthorization plans. The Area Committees and RRTs shall address the
withdrawal of approval from a preauthorization plan, and the RRT shall
notify the NRT of the status of the preauthorization plan within 30
days from any such withdrawal.
    (3) Preauthorization plan reviews. The RRT in consultation with the
Area Committee(s) must review, and revise, as needed, approved
preauthorization plans. These reviews must be conducted following a
regular timeframe, established by the RRT and documented in the plan,
to address changes that may impact the conditions under which the use
of chemical and biological agents have been preauthorized. Reviews must
also be conducted in any affected region, at a minimum, after a major
discharge or after a Spill of National Significance (SONS) relevant to
the preauthorization plan area; to address revisions of the NCP Product
Schedule impacting chemical or biological agents that may be
individually listed within a preauthorization plan; and to reflect new
listings of threatened and/or endangered species applicable to the
preauthorization plan area. The EPA RRT representative, the Department
of Commerce and Department of the Interior natural resource trustees,
and the RRT representative from the state(s) with jurisdiction over the
waters of the area to which a preauthorization plan applies shall
review and either approve, approve with modification, or disapprove any
revisions to the preauthorization plans.
    (b) Use of agents identified on the NCP Product Schedule or use of
burning agents on oil discharges not addressed by a preauthorization
plan. For discharge situations that are not addressed by a
preauthorization plan developed pursuant to paragraph (a) of this
section, the OSC may authorize the use of chemical or biological agents
identified on the NCP Product Schedule on an oil discharge, or the use
of burning agents, for the specific purpose for which they were listed
with the concurrence of the EPA RRT representative and, as appropriate,
the

concurrence of the RRT representatives from the state(s) with
jurisdiction over the waters and adjoining shorelines threatened by the
release or discharge, and in consultation with the Department of
Commerce and Department of the Interior natural resource trustees. In
meeting the provisions of this paragraph, the OSC must consider and
document for their authorization request to the RRT, at a minimum, the
parameters for the use of agents including the quantities requested to
be authorized, the duration of use, the depth of water, the distance to
shoreline and proximity to populated areas, and should consider and
document factors such as environmentally sensitive resources or
restricted areas that might be impacted, agent inventory and storage
locations, agent manufacturing capability, availability of equipment
needed for agent use, availability of adequately trained operators and
appropriate means to monitor agent use in the environment.
    (c) [Reserved]
    (d) Temporary exception. In circumstances to prevent or
substantially reduce an imminent threat to human life that cannot be
immediately addressed by other procedures or provisions of the NCP, the
OSC may authorize the provisional use of any chemical or biological
agent, whether it is identified or not on the NCP Product Schedule,
without obtaining the concurrence of the EPA RRT representative and, as
appropriate, the RRT representatives from the state(s) with
jurisdiction over the waters and adjoining shorelines threatened by the
release or discharge, and without consultation with the Department of
Commerce and the Department of the Interior natural resource trustees.
This exception shall not be used as a substitute for compliance with
Sec.  300.150 of this part, including the use of personal protective
equipment, or when there is sufficient time to seek authorization in
accordance with paragraphs (a) or (b) of this section. If an agent is
authorized for use pursuant to this paragraph, the OSC shall notify as
soon as possible the EPA RRT representative and as appropriate, the RRT
representatives from the affected state(s) and the Department of
Commerce and Department of the Interior natural resource trustees. The
OSC shall document the circumstances and the reasons for use of the
agent authorized pursuant to this paragraph. Agent use for individual
circumstances under this exception shall be in accordance with
paragraphs (a) or (b) of this section no later than 24 hours after
initial application.
    (e) Prohibited agents or substances. The OSC may not authorize the
use of the following:
    (1) Sinking agents, or any other chemical agent, biological agent,
or any substance that is used to directly sink the oil to the bottom of
a water body.
    (2) [Reserved]
    (f) Storage and use of agents listed on the NCP Product Schedule.
(1) The OSC may authorize for use only products listed on the NCP
Product Schedule that are documented and certified by the responsible
party or its representative to have been stored under the conditions
provided by the submitter under Sec.  300.915(a)(6), and whose date of
use does not exceed the expiration date listed on the container's label
unless otherwise specified for expired products as provided in Sec.
300.910(f)(2), at the time of the incident.
    (2) The OSC may authorize for use products listed on the NCP
Product Schedule that exceed their expiration date after the
responsible party or its representative documents and certifies that
the expired product has been stored under the conditions provided by
the submitter under Sec.  300.915(a)(6) and still meets the applicable
efficacy and toxicity listing provisions under Sec.  300.915, based on
testing of representative samples within the previous 12 months.
    (g) Supplemental testing, monitoring, and information. The RRT may
require, for both planning and response, including authorization of
use, supplemental toxicity and efficacy testing, or submission of
available data and information that addresses site, area, and
ecosystem-specific concerns relative to the use of any chemical or
biological agent. The product manufacturer or responsible party shall
provide, upon request of the RRT or OSC, additional monitoring or
testing data and information to inform chemical or biological agent use
decisions specific to a response.
    (h) Recovery of chemical agents and other substances from the
environment. The responsible party shall ensure that removal actions
adequately contain, collect, store, and dispose of chemical agents and
other substances that are to be recovered from the environment, unless
otherwise directed by the OSC. Chemical agents and other substances to
be recovered include solidifiers, surface washing agents, and sorbents.
The OSC should, at a minimum, consider factors such as the safety of
response personnel and harm to the environment in making determinations
pursuant to this paragraph.
    (i) Reporting of agent use. (1) The authorizing OSC shall provide
the RRT the following information on chemical and biological agents
used in response to an oil discharge: product name, product category,
quantity and concentrations used, duration of use, location(s) of use,
any available data collected, and any available analyses of efficacy
and environmental effects. This information must be provided within 30
days of completion of agent use. This information may be submitted in
accordance with the OSC reporting provisions under Sec.  300.165 of
this part, as applicable, subject to the 30-day timing requirement.
    (2) In support of sections 300.135(n) and 300.155(a) and (b) of
this part, the authorizing OSC shall provide for notification to the
public, updated during a response as appropriate, the following
information on chemical and biological agents used in response to an
oil discharge: product name, product category, quantity and
concentrations used, duration of use, and location(s) of use.

0
9. Revise Sec.  300.915 to read as follows:

Sec.  300.915  Data and information requirements for listing on the NCP
Product Schedule or Sorbent Product List.

    If you are submitting an application for listing a product to the
NCP Product Schedule or Sorbent Product List, you must provide EPA the
information required under Sec.  300.955. Technical product data
submissions are not required for burning agents. Your submission for
each product must contain:
    (a) General information for any product category. (1) Your name,
physical address, email, and telephone number;
    (2) Your identity and documentation of that identity, as the
manufacturer of the product, vendor, importer, distributor of the
product, and/or a designated agent acting on behalf of the
manufacturer.
    (3) All name(s), brand(s), and/or trademark(s) under which the
product is to be sold;
    (4) Names, physical addresses, emails, and telephone numbers of the
primary distributors, vendors, importers and/or designated agent acting
on behalf of the manufacturer;
    (5) The Safety Data Sheet (SDS) for the product;
    (6) The maximum, minimum, and optimum temperature, humidity, and
other relevant conditions for product storage and a brief description
of the consequences to performance if the product is not stored within
these limits;

    (7) The anticipated shelf life of the product at the storage
conditions noted in paragraph (a)(6) of this section and documentation
for this determination;
    (8) A sample product label for all name(s), brand(s), and/or
trademark(s) under which the product is to be sold that includes
manufacture and expiration dates, and conditions for storage. You may
use an existing label provided it already contains the required dates
and storage information;
    (9) The chemical or biological agent category under which you want
the product to be considered for listing on the NCP Product Schedule,
including detailed information on the specific process(es) through
which the product affects the oil, and the specific environment(s) on
which it is intended to be used (e.g., waters and/or adjoining
shorelines). If your product meets the definition of more than one
chemical or biological agent category, you must identify all applicable
categories and provide the test data to meet the listing criteria
appropriate to each;
    (10) Recommended product use procedures, including product
concentrations, use ratios, types of application equipment, conditions
for use, any application restrictions; and, as applicable, procedures
for product and oil containment, collection, recovery, and disposal.
These procedures must address, as appropriate, variables such as
weather, water salinity, water temperature, types and weathering states
of oils or other pollutants. The procedures must include supporting
documentation and current applicable standard methods used to determine
them;
    (11) Available information on environmental fate, including any
known measured data, methodologies, and supporting documentation, on
the persistence, bioconcentration factor, bioaccumulation factor, and
biodegradability of the product and all of its components in the
environment;
    (12) The physical and chemical properties of the product, as
appropriate, and a citation for the current applicable standard methods
used to determine them, including:
    (i) Physical state and appearance;
    (ii) Vapor pressure;
    (iii) Flash point;
    (iv) Pour point;
    (v) Viscosity;
    (vi) Specific gravity;
    (vii) Particle size for solid components; and
    (viii) pH;
    (13) The identity and concentration of all components in the
product, including each specific component name; corresponding Chemical
Abstract Service (CAS) Registry Number; the maximum, minimum, and
average weight percent of each component in the product; and the
intended function of each component (e.g., solvent, surfactant);
    (14) For products that also contain microorganisms, enzymes, and/or
nutrients, provide the following along with a citation or a description
of the methodology used to determine:
    (i) The name of all microorganisms by current genus and species,
including any reclassifications, and any physical, chemical, or
biological manipulation of the genetic composition and the weight
percent of each genus in the product;
    (ii) The name of all enzymes and their International Union of
Biochemistry (I.U.B.) number(s); Enzyme Classification (EC) code
numbers; the source of each enzyme; units; and specific oil-degrading
activity;
    (iii) The name(s), maximum, minimum, and average weight percent of
the nutrients contained in the product; and
    (iv) Data, methodology, and supporting documentation, for the
levels of bacterial, fungal, or viral pathogens or opportunistic
pathogens including, but not limited to: enteric bacteria such as
Salmonella, fecal coliforms, Shigella, coagulase positive
Staphylococci, and beta hemolytic Streptococci and enterococci;
    (15) Data, methodology, and supporting documentation for the levels
of the following:
    (i) Arsenic, cadmium, chromium, copper, lead, mercury, nickel,
vanadium, zinc, and any other heavy metal reasonably expected to be in
the product;
    (ii) Cyanide;
    (iii) Chlorinated hydrocarbons;
    (iv) Pesticides;
    (v) Polychlorinated Biphenyls (PCBs); and
    (vi) Polycyclic aromatic hydrocarbons (PAHs).
    (16) Certification, including data, methodology, and supporting
documentation, indicating that the product does not contain any of the
prohibited agents or substances identified in Sec.  300.910(e);
    (17) Information about the accredited laboratory that conducted the
required tests, including:
    (i) Name of the laboratory, address, contact name, email, and phone
number; and
    (ii) The national and/or international accreditations held by the
laboratory that are applicable to the test(s) performed;
    (18) All test data and calculations, including:
    (i) Raw data and replicates, including positive controls;
    (ii) Notes and observations collected during tests;
    (iii) Calculated mean values and standard deviations;
    (iv) Reports, including a summary of stock solution preparation;
    (v) Source and preparation of test organisms;
    (vi) Test conditions; and
    (vii) Chain of custody forms;
    (19) An estimate of the annual product production volume, the
average and maximum amount that could be produced per day, and the time
frame needed to reach that maximum production rate in days;
    (20) Recognition received from EPA's Design for the Environment
(DfE) or Safer Choice programs, as applicable; and
    (21) International product testing or use data or certifications,
if available, informing the performance capabilities or environmental
impacts of the product.
    (b) Dispersant testing and listing requirements--(1) Dispersant
efficacy test and listing criteria. Test the dispersant product for
efficacy using the Baffled Flask Test (BFT) method in Appendix C to
part 300. To be listed on the NCP Product Schedule, the dispersant must
demonstrate for each temperature a Dispersant Effectiveness (DE) at the
95% lower confidence level (LCL95) greater than or equal to:
    (i) >=70% for Strategic Petroleum Reserve Bryan Mound at 5 [deg]C;
    (ii) >=75% for Strategic Petroleum Reserve Bryan Mound at 25
[deg]C;
    (2) Dispersant toxicity tests and listing criteria. Use the methods
specified in Appendix C to part 300 to test the dispersant alone, and
the dispersant mixed with Strategic Petroleum Reserve Bryan Mound for
acute toxicity, using Americamysis bahia and Menidia beryllina. Use the
methods specified in Appendix C to part 300 to test the dispersant
alone for developmental toxicity using Strongylocentrotus purpuratus or
Arbacia punctulata and for subchronic effects using Americamysis bahia
and Menidia beryllina. To be listed on the NCP Product Schedule, the
dispersant alone must demonstrate:
    (i) A median lethal concentration (LC50) at the lower
95% confidence interval greater than 10 ppm;
    (ii) An inhibition concentration for 50% of the test species
(IC50) at the lower 95% confidence interval greater than 1
ppm; and
    (iii) A subchronic No Observed Effect Concentration (NOEC) greater
than 1 ppm.

    (3) Limitations. A dispersant may only be listed on the NCP Product
Schedule for use in saltwater environments for which it meets the
efficacy and toxicity listing criteria.
    (c) Surface washing agent testing and listing requirements--(1)
Surface washing agent efficacy test and listing criteria. To be listed
on the NCP Product Schedule, using an applicable standard methodology,
the surface washing agent must meet an efficacy of greater than or
equal to 30% in either freshwater or saltwater, or both, depending on
the intended product use.
    (2) Surface washing agent toxicity test and listing criteria. Using
the toxicity test methodology in Appendix C to part 300, test the
surface washing agent for acute toxicity against freshwater species
Ceriodaphnia dubia and Pimephales promelas, or saltwater species
Americamysis bahia and Menidia beryllina, or both, depending on the
intended product use. To be listed on the NCP Product Schedule, the
surface washing agent must demonstrate an LC50 at the lower
95% confidence interval greater than 10 ppm in either freshwater or
saltwater for all tested species.
    (3) Limitations. Surface washing agent listing would be for use
only in freshwater and/or saltwater environments for which it was
tested and for which it met the efficacy and toxicity listing criteria.
    (d) Bioremediation agent testing and listing requirements--(1)
Bioremediation agent efficacy test and listing criteria. To be listed
on the NCP Product Schedule, a bioremediation agent must successfully
degrade both alkanes and aromatics as determined by gas chromatography/
mass spectrometry (GC/MS) in freshwater or saltwater, or both,
depending on the intended product use, following the test method
specified in Appendix C to part 300. The percentage reduction of total
alkanes (aliphatic fraction) from the GC/MS analysis must be greater
than or equal to 85% at day 28, based on the ninety-fifth (95th)
percentile Upper Confidence Limit (UCL95) for both
freshwater and saltwater. The percentage reduction of total aromatics
(aromatic fraction) must be greater than or equal to 35% at day 28 for
both saltwater and freshwater based on the UCL95.
    (2) Bioremediation agent toxicity test and listing criteria. The
bioremediation agent must be tested for acute toxicity in freshwater or
saltwater, or both, depending on the intended product use, following
the method specified in Appendix C to part 300. To be listed on the NCP
Product Schedule, the bioremediation agent must demonstrate an
LC50 at the lower 95% confidence interval greater than 10
ppm in either freshwater or saltwater for all tested species.
    (3) Limitations. Bioremediation agent listing would be for use only
in the freshwater and/or saltwater environments for which it was tested
and for which it met the efficacy and toxicity listing criteria.
    (4) Generic listing. If the product consists solely of: ammonium
nitrate, ammonium phosphate, ammonium sulfate, calcium ammonium
nitrate, sodium nitrate, potassium nitrate, synthetically-derived urea,
sodium triphosphate (or tripolyphosphate), sodium phosphate, potassium
phosphate (mono- or dibasic), triple super phosphate, potassium
sulphate, or any combination thereof, no technical product data are
required. The product will be generically listed as non-proprietary
nutrients on the NCP Product Schedule, and no further action is
necessary.
    (e) Solidifier testing and listing requirements. (1) Solidifiers
must be tested for acute toxicity in freshwater or saltwater, or both,
depending on the intended product use, following the method specified
in Appendix C to part 300. To be listed on the NCP Product Schedule,
the solidifier must demonstrate an LC50 at the lower 95%
confidence interval greater than 10 ppm in either freshwater or
saltwater for all tested species.
    (2) Limitations. Solidifier listing would be for use only in the
freshwater and/or saltwater environments for which it was tested and
for which it met the toxicity listing criteria.
    (f) Herding agent testing and listing requirements. (1) Herding
agents must be tested for acute toxicity in freshwater or saltwater, or
both, depending on the intended product use, following the method
specified in Appendix C to part 300. To be listed on the NCP Product
Schedule, the herding agent must demonstrate an LC50 at the
lower 95% confidence interval greater than 10 ppm in either freshwater
or saltwater for all tested species.
    (2) Limitations. Herding agent listing would be for use only in
freshwater and/or saltwater environments for which it was tested and
for which it met the toxicity listing criteria.
    (g) Sorbent requirements. Known sorbent materials and products will
be identified on a publicly available Sorbent Product List for the use
of such products when responding to an oil discharge as follows:
    (1) For sorbent products that consist solely of the following
materials, or any combination thereof, no technical data are required
to be submitted for listing on the Sorbent Product List, and no further
action is necessary for use as a sorbent:
    (i) Feathers, cork, peat moss, and cellulose fibers such as
bagasse, corncobs, and straw;
    (ii) Volcanic ash, perlite, vermiculite, zeolite, and clay; and
    (iii) Polypropylene, polyethylene, polyurethane, and polyester.
    (2) If the product consists of one or more natural organic
substances, inorganic/mineral compounds, and/or synthetic compounds not
specifically identified in paragraph (g)(1) of this section but you
believe the product meets the definition of a sorbent then, as
applicable under Sec.  300.955(a) and (b), you must submit the
following information for consideration for listing it as a sorbent on
the Sorbent Product List:
    (i) The information required under paragraphs (a)(1) through
(a)(8), and paragraph (a)(13) through (a)(15) of this section;
    (ii) The certification required under paragraph (a)(16) of this
section; and
    (iii) Information, including data, to support the claim your
product meets the sorbent definition under Sec.  300.5.

Sec.  300.920  [Removed]

0
10. Remove Sec.  300.920.

0
11. Add Sec.  300.950 to read as follows:

Sec.  300.950  Submission of Proprietary Business Information (PBI).

    (a) Except as provided in paragraph (b) of this section, all
product information submitted to EPA as required under Sec.  300.915
and Sec.  300.955 will be available for public disclosure upon
submission, without further notice to the submitter.
    (b) You may only claim as PBI the concentration; the maximum,
minimum, and average weight percent; and the units of each component as
identified in Sec.  300.915(a)(13) and (14) and as applicable. EPA will
handle such claims in accordance with 40 CFR part 2, subpart B
Confidentiality of Business Information.
    (1) You must make your PBI claim at the time you submit your
information to EPA to be listed on the NCP Product Schedule or Sorbent
Product List.
    (2) You must separate the PBI from all other submitted information.
Include all PBI separately with your submission package, marking it as
``Proprietary Business Information'' and placing it in a separate inner
envelope labeled with ``PROPRIETARY BUSINESS INFORMATION--TO BE OPENED
BY

THE PRODUCT SCHEDULE MANAGER ONLY.''

0
12. Add Sec.  300.955 to read as follows:

Sec.  300.955  Addition of a product to the NCP Product Schedule or
Sorbent ProductLlist.

    (a) Submission. Submit your complete package to: U.S. Environmental
Protection Agency, 1200 Pennsylvania Ave. NW, Mail Code: 5104A, Room
1448, William J. Clinton North, Washington, DC 20460, Attention:
Product Schedule Manager.
    (b) Package contents. Your package shall include, as applicable, in
this order:
    (1) A cover letter on company letterhead signed and dated by you
certifying that:
    (i) All testing was conducted on representative product samples;
    (ii) Testing was conducted at a nationally or internationally
accredited laboratory in accordance with the methods specified in
Appendix C to part 300, and other applicable methods as appropriate;
and
    (iii) All test results and product technical data and information
are true and accurate.
    (2) A page numbered Table of Contents showing the information and
data submitted under Sec.  300.915(a) through (g), as applicable;
    (3) All required data and information arranged in the same order as
specified in Sec.  300.915(a) through (g); and
    (4) A separate envelope containing and labeled Proprietary Business
Information as specified in Sec.  300.950(b), if applicable.
    (c) EPA Review. EPA shall, within 90 days of receiving a submission
package:
    (1) Review the package for completeness and compliance with all
data and information requirements in Sec. Sec.  300.915, 300.950, and
this section; verify information; and request clarification or
additional information, including testing as necessary;
    (2) Make a product listing determination based on a technical
evaluation of all data and information submitted in accordance with the
requirements for each product category, relevant information on impacts
or potential impacts of the product or any of its components on human
health or the environment, and the intended use of the product; and
    (3) Notify you in writing of its decision to list the product on
the NCP Product Schedule or the Sorbent Product List, or of its
decision and supporting rationale to reject the submission. If your
submission is rejected:
    (i) You may revise and resubmit a complete package to address test
results, data, or information deficiencies.
    (ii) EPA's 90-day review will not start until a complete package is
resubmitted.
    (d) Request for review of decision. If your product is rejected for
listing on the NCP Product Schedule or the Sorbent Product List, you
may request that the EPA Administrator or designee review the
determination. Your request must be in writing within 30 days of
receipt of notification of EPA's decision not to list the product on
the NCP Product Schedule or the Sorbent Product List. Your request must
contain a clear and concise statement with supporting facts and
technical analysis demonstrating why the product meets the listing
requirements.
    (1) The EPA Administrator or designee may request additional
information from you and may offer an opportunity for you to meet with
EPA.
    (2) The EPA Administrator or designee will notify you in writing of
the decision within 60 days of receipt of your request, or within 60
days of receipt of requested additional information.
    (e) Changes to a product listing--(1) Administrative change. You
must notify EPA in writing within 30 days of any changes to information
submitted under Sec.  300.915(a)(1) through (8) and Sec.
300.915(a)(19) through (21) for a product on the NCP Product Schedule.
In the notification, you must detail the specific changes, the reasons
for such changes and supporting data and information. EPA may request
additional information and clarification regarding these changes.
    (2) Reformulation. If you change the components and/or
concentrations, you must retest the reformulated product according to
the requirements for the product category and submit a new complete
package under a new, distinct name in accordance with Sec.  300.955(b)
for review and consideration for listing on the NCP Product Schedule or
Sorbent Product List by EPA.
    (f) Transitioning Listed Products to the New NCP Product Schedule
or Sorbent Product List. All products on the current NCP Product
Schedule as of December 11, 2023 will remain conditionally listed until
December 12, 2025 at which time all products that have not been
submitted and listed in the new NCP Product Schedule based on the
amended test and listing criteria will be removed. Your product will be
transitioned from the current NCP Product Schedule to the new NCP
Product Schedule prior to December 12, 2025 after you submit a new
complete package in accordance with Sec.  300.955(b), and EPA makes a
determination to list the product on the new NCP Product Schedule. All
products previously identified as sorbents by EPA will remain available
for use until December 12, 2025, at which time all sorbent products
must have submitted information as applicable under Sec.  300.955(a)
and (b) and be listed in the new Sorbent Product List.

0
13. Add Sec.  300.965 to read as follows:

Sec.  300.965  Mandatory Product Disclaimer.

    The listing of a product on the NCP Product Schedule does not
constitute approval or recommendation of the product. To avoid possible
misinterpretation or misrepresentation, any label, advertisement, or
technical literature for products listed on the NCP Product Schedule
must display in its entirety the disclaimer shown below. The disclaimer
must be conspicuous and must be fully reproduced on all product
literatures, labels, and electronic media including website pages.
Disclaimer
    [PRODUCT NAME] is listed on the National Contingency Plan (NCP)
Product Schedule. This listing does NOT mean that EPA approves,
recommends, licenses, or certifies the use of [PRODUCT NAME] on an oil
discharge. This listing means only that data have been submitted to EPA
as required by Subpart J of the NCP. Only a Federal On-Scene
Coordinator (OSC) may authorize use of this product in accordance with
Subpart J of the NCP in response to an oil discharge.

0
14. Add Sec.  300.970 to read as follows:

Sec.  300.970  Removal of a product from the NCP Product Schedule or
Sorbent Product List.

    (a) The EPA Administrator or designee may remove your product from
the NCP Product Schedule or the Sorbent Product List for reasons
including, but not limited to:
    (1) Statements or information that are misleading, inaccurate,
outdated, or incorrect regarding the composition or use of the product
to remove or control oil discharges made to any person, or private or
public entity, including on labels, advertisements, technical
literature, electronic media, or within the product submission to EPA;
or
    (2) Alterations to the components, concentrations, or use
conditions of the product without proper notification to EPA as
required by Sec.  300.955(e); or
    (3) Failure to print the disclaimer provided in Sec.  300.965 on
all labels, advertisements, technical literature, or electronic media
for products listed on the NCP Product Schedule; or

    (4) New or relevant information not previously considered
concerning the impacts or potential impacts of the product to human
health or the environment.
    (b) EPA will notify you in writing, at your address of record, of
its reasons for deciding to remove the product from the NCP Product
Schedule. If EPA receives no appeal from you in 30 days, the product
will be removed from the NCP Product Schedule without further notice to
you.
    (c) You may appeal the decision to remove your product from the NCP
Product Schedule within 30 days of receipt of EPA's notification. Your
appeal must contain a clear and concise statement with supporting facts
and technical analysis demonstrating why the product should not be
removed. The EPA Administrator or designee will notify you in writing
of the decision within 60 days of your appeal, or within 60 days of
receipt of any requested additional information.

0
15. Revise Appendix C to Part 300 to read as follows:

Appendix C to Part 300--Requirements for Product Testing Protocols and
Summary Test Data: Dispersant Baffled Flask Efficacy and Toxicity
Tests; Standard Acute Toxicity Test for Bioremediation Agents, Surface
Washing Agents, Herding Agents, and Solidifiers; and Bioremediation
Agent Efficacy Test

Table of Contents

1.0 Applicability and Scope
2.0 Baffled Flask Dispersant Efficacy Test (BFT)
3.0 Dispersant Toxicity Testing
4.0 Standard Acute Toxicity Testing for Surface Washing Agents,
Bioremediation Agents, Herding Agents, and Solidifiers
5.0 Bioremediation Agent Efficacy Test Protocol

Illustrations

Figure Number

1. A Baffled Trypsinizing Flask

Tables

Table Number

1. Constituent Concentrations for GP2 Artificial Seawater
2. Test Oil Characteristics
3. Stock Standard Solution Preparation
4. Dispersant Calibration Example for Test Oil
5. Sample Calculation With ANS
6. Toxicity Testing Requirements for Dispersants
7. Summary of Test Conditions--Dispersant Toxicity
8. Toxicity Testing Requirements for Surface Washing Agents, Herding
Agents, Bioremediation Agents and Solidifiers
9. Summary of Test Conditions--Surface Washing Agents, Herding
Agents, Bioremediation Agents and Solidifiers Toxicity
10. Artificial Seawater Nutrient Concentrations
11. Artificial Seawater Nutrient Concentrations for Bioremediation
Agents Having No Nutrients Included
12. Constituent Concentrations for Artificial Freshwater (Bushnell-
Haas)
13. Freshwater Nutrient Concentrations
14. Artificial Freshwater Nutrient Concentration for Bioremediation
Agents Having No Nutrients Included
15. Bioremediation Efficacy Test--Summary of Experimental Setup
16. Bioremediation Efficacy--Summary of Analytical Procedures
17. QA/QC Checks

Standard Operating Procedures Tables

SOP 3-1 Amount of Stock Solutions Required To Make the Working
Standards
SOP 4-1 Ions Associated With Retention Time Groups
SOP 4-2 Instrumental Conditions for Crude Oil Analysis
SOP 4-3 Ion Abundance Criteria for DFTPP
SOP 4-4 Target Compound List

    1.0 Applicability and Scope. This Appendix establishes
laboratory protocols required under Subpart J (Use of Dispersants
and Other Chemical and Biological Agents) of 40 CFR part 300
(National Oil and Hazardous Substances Pollution Contingency Plan)
to make listing determinations for the Product Schedule. The
protocols apply, based on product type, to dispersants,
bioremediation agents, surface washing agents, herding agents, and
solidifiers as defined in Subpart A (Introduction) of 40 CFR part
300.
    2.0 Baffled Flask Dispersant Efficacy Test (BFT)
    2.1 Summary. This laboratory protocol establishes procedures to
evaluate the degree to which a product effectively disperses oil
spilled on the surface of seawater, using a modified 150-mL screw-
cap trypsinizing flask (an Erlenmeyer flask with baffles) with a
glass and Teflon[supreg] stopcock near the bottom to allow removal
of subsurface water samples without disturbing the surface oil
layer. The efficacy of a dispersant is measured using one reference
oil, Strategic Petroleum Oil Reserve Bryan Mound at two temperatures
(5 [deg]C and 25 [deg]C). Six replicates and one method blank are
required at each temperature. A layer of oil is placed on the
surface of artificial seawater, and the dispersant is added to the
slick at a dispersant:oil ratio (DOR) of 1:25 (4%) by volume. A
standard orbital shaker table provides turbulent mixing at a speed
of 250 revolutions per minute (rpm) for 10 minutes, immediately
after which it is maintained stationary for 10 minutes to allow non-
dispersed oil to rise to the water's surface. An undisturbed water
sample is removed from the bottom of the flask through the stopcock,
extracted with dichloromethane (DCM), and analyzed for oil content
by UV-visible absorption spectrophotometry at wavelengths ranging
between 340 and 400 nm.
    2.2 Apparatus. All equipment must be maintained and calibrated
per standard laboratory procedures.
    2.2.1 Modified Trypsinizing Flask. A modified 150 mL glass
screw-capped Erlenmeyer flasks with baffles (e.g., Wheaton No.
355394 or equivalent) fitted with a 2 mm bore Teflon[supreg]
stopcock and glass tubing, the center of which is no more than 1.3
cm from the bottom, as shown in Figure 1.

[GRAPHIC] [TIFF OMITTED] TR12JN23.078

Figure 1. A Baffled Trypsinizing Flask

    2.2.2 Orbital Shaker Table. An orbital shaker table with a
variable speed control unit capable of maintaining 250 rpm. The
orbital diameter must be approximately 1.0 inch (2.5 cm) +/-0.1 inch
(0.25 cm).
    2.2.3 Spectrophotometer. A UV-visible spectrophotometer capable
of measuring absorbance between 340 and 400 nm (e.g., Shimadzu UV-
1800, Agilent 8453, or equivalent). Use standard transmission-
matched quartz 10-mm path length rectangular cells with PTFE cover
for absorbance measurements.
    2.2.4 Glassware. Including: 25-ml graduated mixing cylinders (a
graduated cylinder with a ground glass stopper); 50- and 100-ml
graduated cylinders; 125-mL separatory funnels with Teflon
stopcocks; 10-ml volumetric flasks; 30-ml crimp style glass serum
bottles; 1-, 2-, 5-mL pipettes; other miscellaneous laboratory
items.
    2.2.5 Micropipettor. Use a micropipettor capable of dispensing 4
[micro]L of dispersant and 100 [micro]L of oil (e.g., Brinkmann
Eppendorf repeater pipettor with 100 [micro]L and 5 mL syringe tip
attachments or equivalent).
    2.2.6 Syringes. 25-, 100-, 250-, 1,000-, 2,500-, 5,000-[micro]l
gas-tight syringes.
    2.2.7 Constant temperature rooms or incubators to hold the
shaker at 5 [deg]C and 25 [deg]C.
    2.2.8 Analytical Balance.
    2.2.9 Chemical fume hood.
    2.3 Reagents.
    2.3.1 Artificial seawater. Use the artificial seawater GP2
formulation shown in Table 1 of this Appendix.
    2.3.2 Test oil. Use the EPA standard reference oil Strategic
Petroleum Reserve Bryan Mound. To obtain this oil at no charge
(except for a minimal shipping fee), see the instructions at http://www.epa.gov/emergencies/content/ncp/index.htm. Selected properties
are summarized in Table 2 of this Appendix.
    2.3.3 Dichloromethane (DCM) (also known as methylene chloride),
pesticide quality.
    2.4 Container Handling and Storage.
    2.4.1 Glassware. If the glassware has been used with oil before,
rinse with DCM to remove as much of the oil adhering to the sides of
the flask as possible; waste DCM may be used. Soak in warm water
with detergent and individually wash with bristled brushes. First
rinse with tap water, then follow with two de-ionized water rinses.
Dry either on a rack or in a 110 [deg]C drying oven. After drying,
rinse with fresh DCM (use sparingly).
    2.4.2 Serum bottles and other non-volumetric glassware. Bake for
at least 4 hours in a muffle furnace at 450 [deg]C.
    2.5 Calibration Curve for the UV-visible spectrophotometer.
    2.5.1 Stock Standard Solution Preparation. Stock standard
solution concentrations are based on the mass measurements after
each addition and density determinations of the oil/dispersant/DCM
solution using a density bottle or a 1-mL gas tight syringe. An
example calculation is given in Table 3 of this Appendix according
to the following equation:
[GRAPHIC] [TIFF OMITTED] TR12JN23.079

    Use the reference oil and the specific dispersant being tested
for a particular set of experimental test runs. Prepare the stock
standard solution of dispersant-oil mixture in DCM, starting with 2
ml of the oil, then adding 80 [micro]l of the dispersant followed by
18 ml of DCM.
    2.5.2 Six-point Calibration Curve. For the reference oil, add
specific volumes of its stock standard solution (given in Table 4 of
this Appendix) to 30 ml of artificial seawater in a 125 ml
separatory funnel. Extract the oil/dispersant water mixture with
triplicate 5 ml volumes of DCM. Follow each DCM addition by 15
seconds of vigorous shaking, carefully releasing the initial
pressure inside the separatory funnel by partially removing the

glass stopper inside a fume hood after the first few shakes. Then,
allow a 2-minute stationary period for phase separation for each
extraction. Drain the extracts into a 25-mL graduated mixing
cylinder. Release any entrained bubbles of DCM from the water layer
by sideways shaking of the funnel. Use precaution not to drain water
into the DCM extract as it can affect the absorbance readings.
Adjust the final volume of the collected extracts to 25 mL in the
mixing cylinder using DCM. Determine specific masses for oil
concentrations in the standards as volumes of oil/dispersant
solution multiplied by the concentration of the stock solution. An
example calculation is given in Table 4 of this Appendix. One
calibration curve is needed for the reference oil and dispersant
combination.
    2.6 Sample Preparation and Testing. See section 2.7 of this
Appendix for a detailed description of the spectrophotometer's
linear calibration procedure.
    2.6.1 Six replicates of the oil and test dispersant are required
at each temperature plus two additional tests of method blanks
(artificial seawater without oil and dispersant), one at each
temperature. A completed test consists of 14 baffled flask tests (a
total of six replicates for the reference oil/test dispersant
combination at two temperatures (5 [deg]C and 25 [deg]C), plus two
method blanks).
    2.6.2 Attach a 3-inch length of Teflon tubing to the stopcock of
each of the 150-mL baffled flasks. Add 120 mL of artificial seawater
to each flask. Put screw cap on flasks and place them at the
appropriate temperature (either 5 [deg]C or 25 [deg]C) for
equilibration.
    2.6.3 Calibrate and adjust the shaker table to 250
10 rpm.
    2.6.4 Prepare and time separately each baffled flask.
Sequentially add 100 [micro]L of oil and 4 [micro]L of dispersant to
the flask layering them onto the center of the seawater to give a
dispersant-to-oil ratio (DOR) of 1:25. Avoid any oil or dispersant
splashing on the flask walls, as it may reduce efficacy or cause
errors in the calculated results. Discard the sample and repeat the
setup if: (1) any oil or dispersant splashing occurs during the
additions, or (2) the dispersant contacts the water first rather
than the oil. This is especially important for 5 [deg]C work because
of increased oil viscosity.
    2.6.5 For the oil, fill the tip of the pipettor, using a wipe to
remove any oil from the sides of the tip. Holding the pipettor
vertically, dispense several times back into the reservoir to ensure
that the oil flows smoothly. Insert the syringe tip vertically into
the baffled flask and let the bottom of the pipettor rest on the
neck of the flask. Slowly and carefully dispense the oil one time
onto the center of the water's surface. The remainder of the oil can
either be returned to the oil bottle or set aside for use in the
next test flask.
    Note to 2.6.5: If a Brinkmann Eppendorf repeater pipettor is
used for dispensing the oil, attach a 5-mL syringe tip, and set the
dial to 1.
    2.6.6 For the dispersant, use the same procedure as for the oil
to dispense onto the center of the oil slick surface. As the
dispersant first contacts the oil, it will usually push the oil to
the sides of the flask. Replace the screw cap onto the flask.
    Note to 2.6.6: If a Brinkmann Eppendorf repeater pipettor is
used for dispensing the dispersant, attach a 100-[micro]L syringe
tip, and set the dial to 2.
    2.6.7 Carefully place flask securely onto the shaker and agitate
for 10  0.25 minutes at 250  10 rpm.
    2.6.8 Remove the flask from the shaker table and allow a
stationary, quiescent period of 10  0.25 minutes to
allow undispersed and/or recoalesced oil droplets to refloat to the
surface.
    2.6.9 Carefully open the screw cap, then the stopcock at the
bottom, and discard the first several mL of seawater into a waste
beaker to remove non-mixed water-oil initially trapped in the
stopcock tubing. Collect a volume slightly greater than 30-mL into a
50-mL graduated cylinder. Adjust the collected volume to the 30-mL
mark by removing excess with a disposable glass Pasteur pipette. A
web-like emulsion may form at the solvent/water interface during the
water sample extraction. Avoid pulling any emulsion phase into the
DCM extract as it may cloud the DCM-extract, leading to error.
    2.6.10 Transfer the water-oil sample from the graduated cylinder
into a 125-mL glass separatory funnel fitted with a Teflon stopcock.
    2.6.11 Add 5 mL DCM to the separatory funnel. Start shaking,
releasing pressure into the fume hood by loosening the glass
stopper. Shake vigorously at least 20 times for 15 seconds.
    2.6.12 Allow the funnel to remain in a stationary position for 2
minutes to allow phase separation of the water and DCM.
    2.6.13 Drain the DCM layer from the separatory funnel into a 25
mL mixing cylinder. Avoid pulling any emulsion phase into the DCM
extract as it may cloud the DCM extract.
    2.6.14 Repeat the DCM-extraction process two or three additional
times until the DCM is clear. Collect each extract in the graduated
cylinder. After the final extraction, lightly shake the separatory
funnel sideways once or twice to dislodge entrained bubbles of DCM
and drain.
    2.6.15 Adjust the final volume to a known quantity, 25 mL, in
the mixing cylinder. Using a syringe, dispense 2.5 mL or 5.0 mL of a
reference oil sample into a 10-mL volumetric flask, and fill with
DCM to make either a 1:4 or 1:2 dilution, respectively.
    2.6.16 If analysis cannot be conducted immediately, store the
extracted DCM samples at 4  2 [deg]C until time of
analysis. Glass-stoppered mixing cylinders may be used for short-
term storage or prior to bringing the extracts up to volume. After
bringing to volume, transfer the DCM extracts to 25-30 ml crimp-
style serum vials with aluminum/Teflon seals.
    2.6.17 Complete all analysis within 10 consecutive days from
when the sample was collected.
    2.7 UV-Visible Spectrophotometer Linear Stability Calibration
    2.7.1 A six-point calibration of the UV-visible
spectrophotometer is required at least once per day for each oil.
The stability calibration criterion is determined with the six oil
standards identified in Table 4 of this Appendix.
    2.7.2 Turn on spectrophotometer and allow it to warm up for at
least 30 minutes before beginning analysis. Blank the instrument for
the wavelengths between 340 and 400 nm with DCM.
    2.7.3 If refrigerated, allow all extracts, standards, and
samples to warm to room temperature.
    2.7.4 Determine the absorbance of the six standards between the
wavelengths of 340 and 400 nm. This can be done by either one of the
following methods:
    <IP_ADDRESS> Trapezoidal Rule. Program the spectrophotometer to take
readings every 5[lambda] or 10[lambda] and calculate the area under
the curve using the Trapezoidal rule:
[GRAPHIC] [TIFF OMITTED] TR12JN23.080

where N + 1 = number of absorbance measurements to delineate N
equally spaced sections of the curve, and H = the distance
([lambda]) between each reading. For H = 5, N + 1 = 13 measurements,
for H = 10, N + 1 = 7. The following formula illustrates readings
taken every 10[lambda].
[GRAPHIC] [TIFF OMITTED] TR12JN23.081

    When using readings taken every 5[lambda], each absorbance sum
is multiplied by 5.
    <IP_ADDRESS> Automatic Integration. Program the spectrophotometer to
automatically integrate the area under the curve between 340 nm and
400 nm.
    <IP_ADDRESS> If the wavelengths must be manually set on the
spectrophotometer, the older method of only measuring at
340[lambda], 370[lambda], and 400[lambda] may be used. Then
calculate using the trapezoidal rule for N + 1 = 3, H = 30. While
the resulting area count with the older method is less accurate, the
final results are similar since the inaccuracy is systematic.
    2.7.5 After determining the area count for each standard,
determine the response factor (RF) for the oil at each concentration
using the following equation:
[GRAPHIC] [TIFF OMITTED] TR12JN23.082

    2.7.6 Spectrophotometer stability for the initial calibration is
acceptable when the RFs of the six standard extracts are less than
10% different from the overall mean value for the six standards, as
calculated in Equation 5 of this Appendix and depicted in the
example in Table 4 of this Appendix.
[GRAPHIC] [TIFF OMITTED] TR12JN23.083

    2.7.7 If this criterion is satisfied, begin analysis of sample
extracts. Absorbances greater than or equal to 3.5 are not included
because absorbance saturation occurs at and above this value. If any
of the standard oil extracts fails to satisfy the initial-stability
criterion, the source of the problem (e.g., preparation protocol for
the oil standards, spectrophotometer stability, etc.) must be
corrected before analysis of the sample extracts begins.
    2.7.8 Determine the slope of the calibration points by using
linear regression forced zero intercept:
[GRAPHIC] [TIFF OMITTED] TR12JN23.084

    2.8 Spectrophotometric Analysis and Calculations
    2.8.1 Once a successful calibration curve for the reference oil
has been created and verified, measure experimental replicates for
the reference oil at each temperature followed by a standard check
sample.
    2.8.2 Determine the area for the absorbance values obtained for
the experimental samples by using Equation 2 of this Appendix and
illustrated by Equation 3 of this Appendix.
    2.8.3 Calculate the Total Oil dispersed and the percentage of
oil dispersed (%OD) based on the ratio of oil dispersed in the test
system to the total oil added to the system, as follows:
[GRAPHIC] [TIFF OMITTED] TR12JN23.085

where:

VDCM = final volume of the DCM extract (mL)
Vtw = total seawater in Baffled Flask (120 mL)
Vew = volume seawater extracted (30 mL)
[GRAPHIC] [TIFF OMITTED] TR12JN23.086

where:

rOil = density of the specific test oil, mg/mL and
VOil = Volume (mL of oil added to test flask (100
[micro]L = 0.1 mL))

    2.8.4 The %ODs for the six replicates within a particular
treatment are then subjected to an outlier test, the Grubb's Test

or Maximum Normal Residual test (6). A convenient internet-based
calculator of a Grubbs outlier may be found at: http://www.graphpad.com/quickcalcs/Grubbs1.cfm. If an outlier is detected
(p < 0.05), analyze an additional replicate to obtain the required
six replicates.
    2.8.5 Report the Dispersion Efficacy value for each oil and each
temperature, which is the lower 95% confidence level of the 6
independent replicates (DELCL95) for each oil/temperature
combination. Error bars are not needed as reporting the lower
confidence level computationally takes the variability of the
replicates into account as shown in Equation 9 of this Appendix.
[GRAPHIC] [TIFF OMITTED] TR12JN23.087

where %OD%OD = mean percentage oil dispersed for the n = 6
replicates, S = standard deviation, and
t(n-1,1-[alpha]) = 100 * (1-[alpha])th
percentile from the t-distribution with n-1 degrees of freedom. For
6 replicates, tn-1,1-[alpha] = 2.015, where [alpha] =
0.05. An example of the calculations is given in Table 5 of this
Appendix.

    2.9 Performance Criterion
    The dispersant product tested will remain in consideration for
listing on the NCP Product Schedule if the dispersant efficacy
(DELCL95), as calculated in section 2.8.6 of this
Appendix, is:

------------------------------------------------------------------------
                                                     Temp    DELCL95 (%)
                       Oil                         ([deg]C)
------------------------------------------------------------------------
Bryan Mound......................................        5         >=70
Bryan Mound......................................       25         >=75
------------------------------------------------------------------------

    2.10 Quality Control (QC) Procedures for Oil Concentration
Measurements
    2.10.1 Absorbance readings. Perform at least 5% of all UV-
visible spectrophotometric measurements in duplicate as a QC check
on the analytical measurement method. The absorbance values for the
duplicates must agree within 5% of their mean value.
    2.10.2 Method blanks. Analytical method blanks involve an
analysis of artificial seawater blanks (artificial seawater without
oil or dispersant in a baffled flask) through testing and analytical
procedures. Analyze method blanks with a frequency of at least two
per completed test. Oil concentrations in method blanks must be less
than detectable limits.
    2.10.3 Accuracy. Determine accuracy by using a mid-point
standard calibration check after each set of replicate samples
analyzed. The acceptance criterion is based on a percent recovery of
90-110% using the following equation:
[GRAPHIC] [TIFF OMITTED] TR12JN23.088

    2.10.4 Calibration QC checks. Before analyzing samples, the
spectrophotometer must meet an instrument stability calibration
criterion using the oil standards. The instrument stability for
initial calibration is acceptable when the RFs (Equation 5 of this
Appendix) for each of the six standard concentration levels are less
than 10% different from the overall mean value.

     Table 1--Constituent Concentrations for GP2 Artificial Seawater
                     [Based on Spotte et al., 1984]
------------------------------------------------------------------------
                                                       Concentration (g/
                     Constituent                               L)
------------------------------------------------------------------------
NaCl.................................................              21.03
Na2SO4...............................................               3.52
KCl..................................................               0.61
KBr *................................................              0.088
Na2B4O7 x 10H2O *....................................              0.034
MgCl2 x 6H2O.........................................               9.50
CaCl2 x 2H2O.........................................               1.32
SrCl2 x 6H2O *.......................................               0.02
NaHCO2 *.............................................               0.17
------------------------------------------------------------------------
* Use Stock Solution, 1 mL/L GP2 for 100X stock solution for Bromide,
  Borate, and Strontium. 10 mL/L GP2 for bicarbonate--10X stock solution
  as it is not soluble in a 100X solution. Adjust to pH 8.0 prior to
  autoclaving.

                                        Table 2--Test Oil Characteristics
                                             [April 2023 oil assay]
----------------------------------------------------------------------------------------------------------------
                                   Density, mg/mL    API gravity @15    Viscosity @25
               Oil                   @15 [deg]C          [deg]C         [deg]C, (cSt)    Category by API gravity
----------------------------------------------------------------------------------------------------------------
SPR Bryan Mound.................            0.8320              38.6             4.721  Light Oil.
----------------------------------------------------------------------------------------------------------------

  Table 3--Sample Calculation for Preparation of Oil + Dispersant Stock
                            Standard Solution
------------------------------------------------------------------------
                Item                      Identifier          Amount
------------------------------------------------------------------------
Mass of Bottle, g...................                  A           29.498
Mass of Bottle + oil, g.............                  B           31.225
Mass of bottle + disp + oil + DCM, g                   C          54.380
Mass of oil, g (derived)............            F = B-A            1.727
Mass of disp + oil + DCM, g                        G = C-A        24.882
 (derived)..........................
Mass of 1 mL syringe, g.............                  D           14.556
Mass of 1 mL syringe + solution, g..                  E           15.820
Density of solution, g/mL (derived).            H = E-D            1.264
Volume of solution, mL (derived)....            I = G/H           19.687
Conc. of stock solution, mg/mL             J = F*1000/I           87.704
 (derived)..........................
------------------------------------------------------------------------

                     Table 4--Sample Calculations for Oil + Dispersant Six Point Calibration
----------------------------------------------------------------------------------------------------------------
                  Oil + Dispersant Stock Standard Solution Concentration = 87.7 mg/mL (Table 3)
-----------------------------------------------------------------------------------------------------------------
                                    Theoretical    Area (340-400                           Dev. from
    Standard--stock vol. (uL)      conc., mg/mL         nm)           RF        Avg. RF     avg. RF      Slope
----------------------------------------------------------------------------------------------------------------
25..............................           0.088           4.126       0.021       0.021       2.931      48.759
50..............................           0.175           8.757       0.020  ..........       3.017  ..........
100.............................           0.351          16.559       0.021  ..........       2.577  ..........
150.............................           0.526          25.666       0.021  ..........       0.731  ..........
200.............................           0.702          34.142       0.021  ..........       0.500  ..........
250.............................           0.877          43.006       0.020  ..........       1.260  ..........
----------------------------------------------------------------------------------------------------------------

                                                           Table 5--LCL95 Sample Calculation With Test Oil and Example Dispersant `A'
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                 Area (340-             Extract             Mass in   Total oil
                              Rep                                 400 nm)    Dilution   volume   Conc, mg/  30 mL,   dispersed,  Efficiency,   Average    Std.     Variance   Coef. of    LCL95
                                                                              factor    (ml) *      mL.       mg         mg           %                   dev.               variation
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
1..............................................................     32.197          1        25      0.66     16.51       66.03        79.76     81.30      4.46      19.85       5.48     81.30
2..............................................................     35.470          1        25      0.73     18.19       72.75        87.87  ........  ........  .........  .........  ........
3..............................................................     30.260          1        25      0.62     15.52       62.06        74.96  ........  ........  .........  .........  ........
4..............................................................     31.831          1        25      0.65     16.32       65.28        78.85  ........  ........  .........  .........  ........
5..............................................................     33.355          1        25      0.68     17.10       68.41        82.63  ........  ........  .........  .........  ........
6..............................................................     33.791          1        25      0.69     17.33       69.30        83.71  ........  ........  .........  .........  ........
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
* = 25 ml of DCM extract captured oil from 30 ml of aqueous DE test.

    2.11 References for Section 2.0

(1) U.S. Environmental Protection Agency (1994), ``Swirling Flask
Dispersant Effectiveness Test,'' Title 40 Code of Federal
Regulations, Pt. 300, Appendix C, pp 47458-47461.
(2) Sorial, G.A., A.D. Venosa, K.M, Koran, E. Holder, and D.W. King.
2004. ``Oil spill dispersant effectiveness protocol: I. Impact of
operational variables.'' ASCE J. Env. Eng. 130(10):1073-1084.
(3) Sorial, G.A., A.D. Venosa, K.M, Koran, E. Holder, and D.W. King.
2004. ``Oil spill dispersant effectiveness protocol: II. Performance
of revised protocol.'' ASCE J. Env. Eng. 130(10):1085-1093.
(4) Venosa, A.D., D.W. King, and G.A. Sorial. 2002. ``The baffled
flask test for dispersant effectiveness: a round robin evaluation of
reproducibility and repeatability.'' Spill Sci. & Technol. Bulletin
7(5-6):299-308.
(5) Spotte, S., G. Adams, and P.M. Bubucis. 1984. ``GP2 medium is an
synthetic seawater for culture or maintenance of marine organisms,''
Zoo Biol, 3:229-240.
(6) Grubbs, F. 1969. ``Sample Criteria for Testing Outlying
Observations,'' Annals of Mathematical Statistics, pp. 27-58.
    3.0 Dispersant Toxicity Testing
    3.1 Summary. This laboratory protocol includes testing for: (1)
dispersant standard static acute toxicity tests for the mysid
shrimp, Americamysis bahia (48-hr duration) and the inland
silverside, Menidia beryllina (96-hr duration); (2) dispersant-oil
mixture static acute toxicity tests for Americamysis bahia and
Menidia beryllina (48-hr and 96-hr duration, respectively); (3)
dispersant developmental assay for Strongylocentrotus purpuratus or
Arbacia punctulata, (72-hr duration); and (4) dispersant 7-day
static subchronic tests with Americamysis bahia and Menidia
beryllina (Table 6 of this Appendix).

                                                 Table 6--Toxicity Testing Requirements for Dispersants
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                     Test procedure
---------------------------------------------------------------------------------------------------------------------------------------------------------
                                   96-Hr static acute: Menidia        48-Hr static acute:             72-Hr sea urchin           7-Day subchronic: M.
         Test substance                     beryllina                  Americamysis Bahia           developmental assay          beryllina & A. bahia
--------------------------------------------------------------------------------------------------------------------------------------------------------
Dispersant only.................  yes..........................  yes..........................  yes........................  yes.
Dispersant--Reference Oil         yes..........................  yes..........................  no.........................  no.
 Mixture.
--------------------------------------------------------------------------------------------------------------------------------------------------------

    3.2 Preparation of Stock Solutions
    3.2.1 Dispersant. Prepare a 1000 [mu]L/L primary stock solution
prior to test initiation by adding 1.1 mL of dispersant to 1100 mL
of dilution water consisting of salinity

adjusted uncontaminated natural or artificial seawater, in a glass
vessel. Using a laboratory top stirrer equipped with a stainless-
steel blade, center the stirrer blade in the mixing vessel one inch
off the bottom. Initially mix the resulting stock solution for
approximately five seconds at speeds of <10,000 rpm to avoid
foaming. Thereafter, set the speed to provide a 70% vortex. Using a
glass pipette, remove appropriate aliquots of stock solution from
between the mixing vessel wall and edge of the vortex and place
directly into the dilution water within an exposure vessel. Suspend
mixing of the stock solution after the removal of each aliquot. Base
the preparation of exposure solutions on the nominal concentration
of the stock solution and follow procedures outlined in sections 3.5
and 3.6 of this Appendix.
    3.2.2 Dispersant-Reference Oil(s) Mixtures. Use Strategic
Petroleum Reserve Bryan Mound reference oil. To obtain this oil at
no charge (except for a minimal shipping fee) see https://www.epa.gov/emergency-response/national-contingency-plan-subpart-j#howto. Assessment of dispersant-reference oil mixture (DOM)
toxicity is determined for each reference oil using the aqueous
phase of a chemically enhanced-water accommodated fraction (CE-WAF).
Fit a glass aspirator bottle (approximately 23 L) equipped with a
hose bib at the base with a length of silicon tubing containing a
hose clamp. Fill the bottle with 19L of seawater leaving a 20%
headspace above the liquid, place on a magnetic stir plate then add
and center a stir bar. Add the reference oil at 25 g/L using a
silicon tube attached to a glass funnel that reaches just below the
water surface. Using this method reduces the production of air
bubbles on the oil surface slick. Adjust the stir plate to obtain an
oil vortex of 25% of the total volume of the seawater, then add the
dispersant to be tested at a ratio of 1:10 dispersant:oil (2.5 g/L).
Securely seal the bottle to reduce the loss of volatiles using a
silicon stopper and wraps of Parafilm and stir for 18 hours, then
allow the solution to settle for 6 hours. Maintain the temperature
at 25 [deg]C during stirring and settling. Purge the hose at the
base of the bottle of any material followed by removal of the CE-WAF
(aqueous phase) into a clean glass container without disturbing the
surface oil slick. The CE-WAF should be remixed and 1 to 2 L removed
for chemical analysis of total petroleum hydrocarbons (TPH)
following the procedures outlined in section 3.4 of this Appendix.
The remaining volume will be used for the preparation of exposure
solutions following procedures outlined in section 3.3 of this
Appendix. To reduce time and cost, mix sufficient amounts of
dispersant product-reference oil mixture CE-WAF to allow preparation
of exposure solutions for conducting simultaneous acute tests with
both Americamysis bahia and Menidia beryllina.
    3.3 Preparation of Exposure Concentrations.
    3.3.1 Concentration Selection. Preliminary rangefinder tests may
be necessary using a series of logarithmic concentrations (e.g. 0.1,
1, 10, 100 [micro]l dispersant product/L or mg TPH/L) to determine
the appropriate exposure concentration range necessary to determine
LC50 values and 95% confidence intervals. For definitive
tests, conduct a minimum of five test concentrations using a
geometric ratio between 1.5 and 2.0 (e.g. 2, 4, 8, 16, and 32). Note
that when testing only the dispersant product, the highest test
concentration must not exceed the dispersant's self-dispersibility
limit.
    3.3.2 Exposure Concentrations. Exposure solutions are prepared
by adding the appropriate amount of stock solution directly to
dilution water in each test chamber. Mix each exposure solution
using five rotations in one direction followed by five rotations in
the opposite direction using a solid glass stir rod.
    3.3.3 Reference Toxicants. Separate toxicity tests must be
performed with a reference toxicant for each species tested. Conduct
additional reference toxicity tests any time a change in the
population or source of a test species occurs. Use sodium dodecyl
sulfate (SDS), also known as dodecyl sodium sulfate (DSS), and
sodium lauryl sulfate (SLS) as the reference toxicant for exposures
conducted with Menidia beryllina and Americamysis bahia. Use copper
chloride as the reference toxicant for exposures conducted with the
sea urchin developmental test. Use reagent grade quality SDS and
copper chloride for tests. Information on procedures for conducting
reference toxicant tests with these species can be found in the
specific EPA methods documents cited in sections 3.5.1, 3.6.1, and
3.7.1 of this Appendix.
    3.4 Chemical Analysis of Stock Solutions. Add the 1 L sample of
CE-WAF (Section 3.2.2 of this Appendix) solutions directly to amber
glass bottles with Teflon[supreg]-lined cap. Collect a replicate
sample in the event of accidental loss or if reanalysis of the stock
solution becomes necessary. Adjust sample to a pH=2 using 50%
hydrochloric acid, immediately refrigerate and analyze within 48
hours of collection. Analyze samples for C9-C32 TPH by gas
chromatography-flame ionization detection (GC-FID) following EPA SW-
846, Method 8015B-DRO (4). Report TPH concentration of stock
solutions as milligrams TPH/L and use in the calculation of exposure
concentrations for all toxicity tests conducted with CE-WAF.
    3.5 Static Acute Tests with M. beryllina and A. bahia
    3.5.1 General. Use EPA's Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to Freshwater and Marine
Organisms (EPA-821-R-02-012) (1) for testing each species separately
with dispersant product or a mixture of dispersant product and
reference oil (DOM).
    3.5.2 Test Solutions. Modify procedures in EPA-821-R-02-012
specifically dealing with the handling and toxicity testing of
effluents or receiving water samples as follows: Prepare stock
solutions following section 3.2 of this Appendix and exposure
concentrations following section 3.3 of this Appendix.
    3.5.3 Number of Treatments, Replicates and Organisms. Conduct a
minimum of three replicates of at least five exposure treatments
plus a minimum of three replicate dilution water controls. Expose
ten organisms per replicate treatment.
    3.5.4 Exposure Period. Test duration is 48-hr for Americamysis
bahia and 96-hr for Menidia beryllina. Mortality must be recorded at
each 24-hour period of each test.
    3.5.5 Test Acceptability. For each test performed, survival of
control animals must be >90% and test results must allow
determination of statistically valid LC50 and 95%
confidence interval values except in cases where the LC50
is >1000 [mu]l/L or is determined to be greater than the limits of
water solubility of dispersibility.
    3.5.6 Static Acute Test Summary. A summary of required test
conditions is provided in Table 7 of this Appendix.
    3.6 Sea Urchin Developmental Test with Dispersant Product
    3.6.1 General. Use Section 15, ``Purple Urchin,
Strongylocentrotus purpuratus and Sand Dollar, Dendraster
excentricus Larval Development Test Method'' of EPA's Short-Term
Methods for Estimating the Chronic Toxicity of Effluents and
Receiving Waters to West Coast Marine and Estuarine Organisms (EPA/
600/R-95-136) (2). Alternatively, the development of the urchin
Arbacia punctulata may be tested (see Table 7).
    3.6.2 Test Organism. Tests of dispersant products are to follow
methods for the purple urchin only. Tests with the sand dollar are
not required.
    3.6.3 Test Solutions. Modify procedures in EPA/600/R-95-136,
Section 15 specifically dealing with the handling and toxicity
testing of effluents or receiving water samples as follows: Prepare
stock solutions following section 3.2.1 of this Appendix and
exposure concentrations following section 3.3 of this Appendix.
    3.6.4 Number of Treatments and Replicates. Conduct a minimum of
four replicates of five exposure treatments plus a minimum of four
replicate dilution water controls.
    3.6.5 Exposure Duration and Test Endpoint. Examine the effects
of the dispersant product on normal development of sea urchin
embryos over a period of 72 hours. An IC50 (the exposure
concentration at which normal development is inhibited in 50% of the
embryos) with 95% confidence intervals are to be determined in place
of an IC25. The concentration of dispersant causing
inhibition of development in 50% of exposed embryos
(IC50) with the lower and upper 95% confidence intervals
(LCI95 and ULCI95) must be calculated at the
end of the exposure period. Mortality determinations are not
required.
    3.6.6 Test Acceptability. Requirements of the assay are: (i)
>=80% normal larval development in the control treatment, (ii) the
minimum significant difference (MSD) that can be statically detected
relative to the control is <=25%, iii) test results which support
the determination of a statistically valid IC50 and 95%
confidence interval unless the LC50 is >1000 [mu]l/L or
is greater than the limits of water solubility of dispersibility.
    3.6.7 Urchin Developmental Test Summary. A summary of required
test conditions is provided in Table 7 of this Appendix.
    3.7 Seven-day Subchronic Tests with M. beryllina and A. bahia

    3.7.1 General. Use Section 13, Method 1006.0, ``Inland
Silverside (Menidia beryllina) Larval Survival and Growth Method,''
and Section 14, Method 1007.0, ``Mysid (Mysidopsis [renamed
Americamysis] bahia) Survival, Growth, and Fecundity Method'' of
EPA's Short-Term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Marine and Estuarine Organisms
(EPA-821-R-02-014) (3) for testing of dispersant product.
    3.7.2 Test Solutions. Modify procedures in EPA-821-R-02-014,
sections 13 and 14 specifically dealing with the handling and
toxicity testing of effluents or receiving water samples as follows:
Prepare stock solutions following section 3.2.1 of this Appendix and
exposure concentrations following section 3.3 of this Appendix.
Exposure solutions should be renewed every 24 hours for the duration
of the test.
    3.7.3 Number of Treatments, Replicates and Organisms. (i)
Menidia beryllina: Conduct a minimum of four replicates of at least
five exposure treatments plus a minimum of four replicate dilution
water controls. Expose ten M. beryllina per replicate treatment.
(ii) Americamysis bahia: Conduct a minimum of eight replicates of at
least five exposure treatments plus a minimum of eight replicate
dilution water controls. Expose five A. bahia per replicate
treatment.
    3.7.4 Exposure Duration and Test Endpoint. The test duration is
seven days for both species. Test endpoints for Menidia beryllina
are survival and growth (dry weight) and for Americamysis bahia is
survival, growth (dry weight) and fecundity. Calculate an
LC50 and 95% confidence interval for survival and
IC25 and IC50 with 95% confidence intervals
for growth (and fecundity for A. bahia only). Report the lowest
observed effect concentration (LOEC) and no observed effect
concentration (NOEC) for each endpoint.
    3.7.5 Test Acceptability. Requirements of the assay are: (i)
>=80% survival in the control treatment for each species, (ii) dry
weights must meet the specific requirements as stipulated in Method
1006.0 for Menidia beryllina and Method 1007.0 for Americamysis
bahia.
    3.7.6 Subchronic Test Summary. A summary of required test
conditions for each species is provided in Table 7 of this Appendix.
    3.8 Laboratory Report. The laboratory must include, for each
toxicity test report, all applicable information, data and analyses
as follows:
    3.8.1 Test Objective: protocol title and source, endpoint(s);
    3.8.2 Product Information: product name, manufacturer contact
information, lot number, production date, date received/chain of
custody;
    3.8.3 Contract Facility: contact information;
    3.8.4 Dilution Water: source, pretreatment, physical and
chemical characteristics (pH, salinity);
    3.8.5 Test Conditions: date and time of test (start and end),
test chambers type and volume, volume of solution per chamber,
number of organisms per chamber, number of replicate chambers per
treatment, feeding frequency, amount and type of food, test
concentrations, test temperature (mean and range), test salinity
(mean and range);
    3.8.6 Test Organisms: common and scientific name, source contact
information, age and date purchased, acclimation conditions (e.g.,
temperature, salinity, both mean and range), age at test start;
    3.8.7 Reference toxicant: date received, lot number, date of
most recent test, results and current Cumulative Sum Chart, dilution
water used, physical and chemical methods used;
    3.8.8 Quality Assurance: verification of laboratory
accreditation, including subcontractor facilities;
    3.8.9 Test Results: raw data in tabular and graphical form,
daily records of affected organisms in each concentration replicate
and controls, table of required endpoints (i.e., LC50
with 95% confidence interval (CI), IC25 and
IC50 with 95% CI, LOEC and NOEC), statistical methods
used to calculate endpoints, summary tables of test conditions and
QA data;
    3.8.10 Analytical Results: method summary including Limit of
Detection (LOD)/Limit of Quantitation (LOQ), deviations and reasons
if any, sample summary, results including chromatograms and data
qualifiers, QA summary including calibration curves, method blank
and surrogate recovery, analytical results summary; and
    3.8.11 Conclusions: Relationship between test endpoints and
threshold limit.

                                                Table 7--Summary of Test Conditions--Dispersant Toxicity
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                                Development S.
                                       Acute M. beryllina        Acute A. bahia          Subchronic M.       Subchronic A. bahia       purpuratus/A.
                                                                                beryllina                                     punctulata
--------------------------------------------------------------------------------------------------------------------------------------------------------
Test type..........................  Static non-renewal....  Static non-renewal....  Static renewal         Static renewal         Static non-renewal.
                                                                                (daily).               (daily).
Test duration......................  96 hours..............  48 hours..............  7 days...............  7 days...............  72  2
                                                                                hours.
Salinity...........................  20          20          20         20         34
                                      2[permil].              2[permil].              2[permil].             2[permil].             2[permil].
                                    ----------------------------------------------------------------------------------------------
Temperature........................       25  1 [deg]C. Test temperatures must not deviate (maximum minus minimum      15  1
                                                           temperature) by for than 3 [deg]C during the test.                       [deg]C.
                                    --------------------------------------------------------------------------------------------------------------------
Light quality......................                                            Ambient laboratory illumination.
Light intensity....................                                                  10-20 [mu]E/m\2\/s.
Photoperiod........................                            16 h light, 8 h darkness, with phase in/out period recommended.
                                    --------------------------------------------------------------------------------------------------------------------
Test chamber size \1\..............  250 mL................  250 mL................  600 mL-1 L...........  400 mL...............  30 mL.
Test solution volume \1\...........  200 mL................  200 mL................  500-750 mL...........  150 mL...............  10 mL.
Age of test organism \2\...........  9-14 days.............  1-5 days..............  7-11 days............  7 days...............  1 hr old fertilized
                                                                                eggs.
No. organisms per test chamber.....  10....................  10....................  10...................  5....................  25 embryos per mL.
No. of replicate chambers per        3.....................  3.....................  4....................  8....................  4.
 concentration.
                                    ----------------------------------------------------------------------------------------------
Feeding regime.....................               Refer to specific feeding procedures provided in each test method.               None.
                                    --------------------------------------------------------------------------------------------------------------------
Aeration...........................               None, unless DO falls below 4.0 mg/L, then aerate all chambers. Rate: <100 bubbles/minute.
Test concentrations................                              5 exposure concentrations and a control (minimum required).
                                    --------------------------------------------------------------------------------------------------------------------
Test acceptability (required)......  >=90% survival in       >=90% survival in       For controls: >=80%    For controls: >=80%    >=80% normal shell
                                      controls.               controls.               survival; average      survival; average      development in
                                                                                dry weight >=0.5mg     dry weight >=0.20 mg.  controls.
                                                                                where test starts
                                                                                with 7 day old
                                                                                larvae, or >=0.43 mg
                                                                                for larvae preserved
                                                                                for <=7days.
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\ Recommended minimum value.
\2\ Less than or equal to 24-hr range in age.

    3.9 References for Section 3.0

(1) U.S. EPA. 2002. Methods for Measuring the Acute Toxicity of
Effluents and Receiving Waters to Freshwater and Marine Organisms.
Fifth Edition. U.S.

Environmental Protection Agency, Washington, DC (EPA-821-R-02-012).
(2) U.S. EPA. 1995. Short-Term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to West Coast Marine and
Estuarine Organisms. First Edition. U.S. Environmental Protection
Agency, Washington, DC (EPA/600/R-95-136)
(3) U.S. EPA. 2002. Short-Term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Marine and Estuarine
Organisms. Third Edition. U.S. Environmental Protection Agency,
Washington, DC (EPA-821-R-02-014).
(4) U.S. EPA. 2008. Test Methods for Evaluating Solid Waste,
Physical/Chemical Methods U.S. Environmental Protection Agency,
Washington, DC (SW-846) http://www.epa.gov/osw/hazard/testmethods/sw846/online/index.htm.

    4.0 Standard Acute Toxicity Testing of Surface Washing Agents,
Bioremediation Agents, Herding Agents, and Solidifiers.
    4.1 Summary. This laboratory protocol includes testing for: (1)
saltwater standard static acute toxicity tests for test products
with the mysid shrimp, Americamysis bahia (48-hr duration) and the
inland silverside, Menidia beryllina (96-hr duration); and (2)
freshwater standard static acute toxicity tests for test products
with the daphnid, Ceriodaphnia dubia (48-hr duration) and the
fathead minnow, Pimephales promelas (96-hr duration) (see Table 8 of
this Appendix).

  Table 8--Toxicity Testing Requirements for Surface Washing Agents, Herding Agents, Bioremediation Agents and
                                                   Solidifiers
----------------------------------------------------------------------------------------------------------------
                                                                  Test procedure
                                 -------------------------------------------------------------------------------
     Application environment         96-hr Static        48-hr Static        96-hr Static        48-hr Static
                                    acute: Menidia          acute:         acute: Pimephales        acute:
                                       beryllina      Americamysis bahia       promelas       Ceriodaphnia dubia
----------------------------------------------------------------------------------------------------------------
Saltwater only..................  yes...............  yes...............  no................  no.
Freshwater only.................  no................  no................  yes...............  yes.
Freshwater and saltwater use....  yes...............  yes...............  yes...............  yes.
----------------------------------------------------------------------------------------------------------------

    4.2 Dilution Water. Use Section 7 of EPA's Methods for Measuring
the Acute Toxicity of Effluents and Receiving Waters to Freshwater
and Marine Organisms (EPA-821-R-02-012) [1] for preparation of the
appropriate dilution water for each species tested. Use of clean
natural or synthetic seawater for tests conducted with saltwater
species is acceptable.
    4.3 Preparation of Stock Solutions.
    4.3.1 Liquid Surface Washing Agents and/or Herding Agents.
Prepare a 1000 [micro]L/L stock solution prior to test initiation by
adding 1.1 mL of test product to 1100 mL of dilution water in a
glass vessel. Place on a magnetic stir plate then add and center a
stir bar and adjust the stir plate to obtain a vortex of 25% of the
total volume of the liquid. Mix the resulting stock solution for
approximately five minutes at room temperature. Using a glass
pipette, remove appropriate aliquots of stock solution from between
the mixing vessel wall and edge of the vortex and place directly
into the dilution water within an exposure vessel. Base the
preparation of exposure solutions on the nominal concentration of
the stock solution and follow procedures outlined in sections 4.6
and/or 4.7 of this Appendix, as appropriate.
    4.3.2 Bioremediation Agents. For products consisting of two or
more liquid and/or solid components, prepare the product following
the manufacturers recommended procedure and ensure the test product
mixture is completely blended. Prepare a 1000 [micro]L/L stock
solution prior to test initiation by adding 1.1 mL of the test
product mixture to 1100 mL of dilution water in a glass vessel.
Place on a magnetic stir plate then add and center a stir bar and
adjust the stir plate to obtain a vortex of 25% of the total volume
of the liquid. Mix the resulting stock solution for approximately
five minutes at room temperature. Using a glass pipette, remove
appropriate aliquots of stock solution from between the mixing
vessel wall and edge of the vortex and place directly into the
dilution water within an exposure vessel. Base the preparation of
exposure solutions on the nominal concentration of the stock
solution and follow procedures outlined in sections 4.5 and/or 4.6
of this Appendix, as appropriate.
    4.3.3 Solid Phase Products. Assessment of the toxicity of
solidifiers and other solid phase products are determined using the
aqueous phase of water-accommodated fractions (WAFs) of the test
product. Fit a glass aspirator bottle (approximately 23L) equipped
with a hose bib at the base with a length of silicon tubing
containing a hose clamp. Fill the bottle with 19L of dilution water
leaving a 20% headspace above the liquid, place on a magnetic stir
plate then add and center a stir bar. Add the test product at 25 g/L
and securely seal the bottle using a silicon stopper and wraps of
parafilm. Adjust the stir plate to obtain a vortex of 25% of the
total fluid volume, stir for 18 hours then settle for 6 hours.
Maintain the temperature at 25 [deg]C during stirring and settling.
Purge the hose at the base of the bottle of any material followed by
removal of the WAF (aqueous phase) into a clean glass container
without disturbing the product on the surface. The WAF should be
remixed and used for the preparation of exposure solutions following
procedures outlined in section 4.4 of this Appendix.
    4.4 Preparation of Exposure Concentrations.
    4.4.1 Concentration Selection. Preliminary rangefinder tests may
be necessary using a series of logarithmic concentrations (e.g. 0.1,
1, 10, 100 [micro]l test product/L) to determine the appropriate
exposure concentration range necessary to determine LC50
values and 95% confidence intervals. For definitive tests, conduct a
minimum of five test concentrations using a geometric ratio between
1.5 and 2.0 (e.g. 2, 4, 8, 16, and 32). Note that when testing the
product, the highest test concentration should not exceed the test
product's self-dispersibility limit.
    4.4.2 Exposure Concentrations. Exposure solutions are prepared
by adding the appropriate amount of stock solution directly to
dilution water in each test chamber. Mix each exposure solution
using five rotations in one direction followed by five rotations in
the opposite direction using a solid glass stir rod.
    4.4.3 Reference Toxicants. Separate toxicity tests must be
performed with a reference toxicant for each species tested. Conduct
additional reference toxicity tests any time a change in the culture
population or source of a test species occurs. Use reagent grade
quality sodium dodecyl sulfate (SDS), also known as dodecyl sodium
sulfate (DSS), and sodium lauryl sulfate (SLS) as the reference
toxicant. Information on procedures for conducting reference
toxicant tests with these species can be found in section 4 of EPA's
Methods for Measuring the Acute Toxicity of Effluents and Receiving
Waters to Freshwater and Marine Organisms (EPA-821-R-02-012) (3).
    4.5 Saltwater Static Acute Tests with Menidia beryllina and
Americamysis bahia
    4.5.1 General. Use EPA's Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to Freshwater and Marine
Organisms (EPA-821-R-02-012) (1) for testing each species separately
with the test product.
    4.5.2 Test Solutions. Modify procedures in EPA-821-R-02-012
specifically dealing with the handling and toxicity testing of
effluents or receiving water samples as follows: Prepare stock
solutions following the appropriate sections (4.3.1, 4.3.2, or
4.3.3) of this Appendix and exposure concentrations following
section 4.4 of this Appendix.
    4.5.3 Number of Treatments, Replicates and Organisms. Conduct a
minimum of three replicates of at least five exposure treatments
plus a minimum of three replicate dilution water controls. Expose
ten organisms per replicate treatment.
    4.5.4 Exposure Period. Test duration is 48-hr for A. bahia and
96-hr for M. beryllina.

Mortality must be recorded at each 24 hour period of each test.
    4.5.5 Test Acceptability. For each test performed, survival of
control animals must be >90% and test results must allow
determination of statistically valid LC50 and 95%
confidence interval values except in cases where the LC50
is >1000 [micro]l/L or is determined to be greater than the limits
of water solubility or dispersibility.
    4.5.6 Static Acute Test Summary. A summary of required test
conditions is provided in Table 9 of this Appendix.
    4.6 Freshwater Static Acute Tests with Pimephales promelas and
Ceriodaphnia dubia
    4.6.1 General. Use EPA's Methods for Measuring the Acute
Toxicity of Effluents and Receiving Waters to Freshwater and Marine
Organisms (EPA-821-R-02-012) (1) for testing each species separately
with the test product.
    4.6.2 Test Solutions. Modify procedures in EPA-821-R-02-012
specifically dealing with the handling and toxicity testing of
effluents or receiving water samples as follows: Prepare stock
solutions following the appropriate sections (4.3.1, 4.3.2, or
4.3.3) of this Appendix and exposure concentrations following
section 4.4 of this Appendix.
    4.6.3 Number of Treatments, Replicates and Organisms. P.
promelas: Conduct a minimum of three replicates of at least five
exposure treatments plus a minimum of three replicate dilution water
controls. Expose ten organisms per replicate treatment. C. dubia:
Conduct a minimum of four replicates of at least five exposure
treatments plus a minimum of four replicate dilution water controls.
Expose five organisms per replicate treatment.
    4.6.4 Exposure Period. Test duration is 48-hr for C. dubia and
96-hr for P. promelas. Mortality must be recorded at each 24 hour
period of each test.
    4.6.5 Test Acceptability. For each test performed, survival of
control animals must be >90% and test results must allow
determination of statistically valid LC50 and 95%
confidence interval values except in cases where the LC50
is >1000 [micro]l/L or is determined to be greater than the limits
of water solubility of dispersibility.
    4.6.6 Static Acute Test Summary. A summary of required test
conditions is provided in Table 9 of this Appendix.
    4.7 Laboratory Report. The laboratory must include, for each
toxicity test report, all applicable information, data and analyses
as follows:
    4.7.1 Test Objective: protocol title and source, endpoint(s);
    4.7.2 Product Information: product name, manufacturer contact
information, lot number, production date, date received/chain of
custody;
    4.7.3 Contract Facility: contact information;
    4.7.4 Dilution Water: source, pretreatment, physical and
chemical characteristics (pH, salinity);
    4.7.5 Test Conditions: date and time of test (start and end),
test chambers type and volume, volume of solution per chamber,
number of organisms per chamber, number of replicate chambers per
treatment, feeding frequency, amount and type of food, test
concentrations, test temperature (mean and range), test salinity
(mean and range);
    4.7.6 Test Organisms: common and scientific name, source contact
information, age and date purchased, acclimation conditions (e.g.,
temperature, salinity, both mean and range), age at test start;
    4.7.7 Reference toxicant: date received, lot number, date of
most recent test, results and current Cumulative Sum Chart, dilution
water used, physical and chemical methods used;
    4.7.8 Quality Assurance: verification of laboratory
accreditation, including subcontractor facilities;
    4.7.9 Test Results: raw data in tabular and graphical form,
daily records of affected organisms in each concentration replicate
and controls, table of required endpoints (i.e., LC50,
95% CI, inhibited concentration for 50% of the species
(IC50), lower observed effect concentration (LOEC) and no
observed effect concentration (NOEC)), statistical methods used to
calculate endpoints, summary tables of test conditions and QA data;
and
    4.7.10 Conclusions: Relationship between test endpoints and
threshold limit.

     Table 9--Summary of Test Conditions--Surface Washing Agents, Herding Agents, Bioremediation Agents and
                                              Solidifiers Toxicity
----------------------------------------------------------------------------------------------------------------
                                  Saltwater acute M.  Saltwater acute A.   Freshwater acute    Freshwater acute
                                       beryllina             bahia            P. promelas          C. dubia
----------------------------------------------------------------------------------------------------------------
Test type.......................  Static non-renewal  Static non-renewal  Static non-renewal  Static non-
                                                                                renewal.
Test duration...................  96 hours..........  48 hours..........  96 hours..........  48 hours.
Salinity........................  20      20      NA................  NA.
                                   2[permil].          2[permil].
                                 -------------------------------------------------------------------------------
Temperature.....................    25  1 [deg]C. Test temperatures must not deviate (maximum minus
                                            minimum temperature) by more than 3 [deg]C during the test.
Light quality...................                         Ambient laboratory illumination.
Light intensity.................                              10-20 [micro]E/m\2\/s.
Photoperiod.....................          16 h light, 8 h darkness, with phase in/out period recommended.
                                 -------------------------------------------------------------------------------
Test chamber size \1\...........  250 mL............  250 mL............  250 mL............  30 mL.
Test solution volume \1\........  200 mL............  200 mL............  200 mL............  15 mL.
Age of test organism \2\........  9-14 days.........  1-5 days..........  1-14 days.........  <24 hours.
No. organisms per test chamber..  10................  10................  10................  5.
No. of replicate chambers per     3.................  3.................  3.................  4.
 concentration (minimum).
                                 -------------------------------------------------------------------------------
Feeding regime..................        Refer to specific feeding procedures provided in each test method.
Aeration........................    None, unless DO falls below 4.0 mg/L, then aerate all chambers. Rate: <100
                                                                  bubbles/minute.
Test concentrations.............            5 exposure concentrations and a control (minimum required).
Test acceptability (required)...                            >=90% survival in controls.
----------------------------------------------------------------------------------------------------------------
\1\ Recommended minimum value.
\2\ Less than or equal to 24-hr range in age.

    4.8 References for Section 4

(1) U.S. EPA. 2002. Methods for Measuring the Acute Toxicity of
Effluents and Receiving Waters to Freshwater and Marine Organisms.
Fifth Edition. U.S. Environmental Protection Agency, Washington, DC
(EPA-821-R-02-012).

    5.0 Bioremediation Agent Efficacy Test Protocol
    5.1 Summary. This protocol quantifies changes in weathered
Alaska North Slope (ANS) crude oil composition of alkanes and

aromatics resulting from the use of a bioremediation agent in either
artificial seawater or freshwater. The manufacturer may test either
one or both freshwater or saltwater, depending on the product's
intended use. Biodegradation of the alkanes and aromatics is
monitored for 28 days at 20-23 [deg]C. Product flasks at Day 28 are
compared to Day 0 flasks to determine reductions in alkanes and
aromatics. A positive control of a known oil-degrading bacterial
consortium supplied by EPA is tested. A negative, sterile control is
also set up containing exposure water, weathered crude oil, product,
and a sterilant, sodium azide. The purpose of the negative, killed
control is to make sure the disappearance of the oil constituents at
day 28 is due to biodegradation and not some physical loss such as
volatilization. The day 28 GC/MS results from the killed control
must not be less than 90% of the day 0 results. The sample
preparation procedure extracts the oil phase into the solvent
dichloromethane (DCM) (also known as methylene chloride) with a
subsequent solvent exchange into hexane. The hexane extracts are
analyzed by a high-resolution gas chromatograph/mass spectrometer
(GC/MS) operated in the selected ion monitoring mode (SIM) at a scan
rate of >5 scans per second.
    Note to 5.1: Alaska North Slope (ANS) crude oil is artificially
weathered by distillation at 521 [deg]F (272 [deg]C) to remove the
low molecular weight hydrocarbons to approximate natural weathering
processes that occur after a spill.
    5.2 Apparatus. All equipment must be maintained and calibrated
per standard laboratory procedures.
    5.2.1 Assorted flasks and other glassware;
    5.2.2 Graduated cylinders (100 mL);
    5.2.3 Deionized water;
    5.2.4 250 mL borosilicate glass Erlenmeyer flasks;
    5.2.5 250 mL separatory funnels with stopcocks
    5.2.6 Pasteur pipettes;
    5.2.7 Multichannel pipettor (5-50 mL and 50-200 mL);
    5.2.8 Autoclave; environmental room or incubator;
    5.2.9 Balance accurate to 0.1 mg;
    5.2.10 Orbital shaker table with clamps sized to hold flasks
securely;
    5.2.11 GC/MS instrument equipped with a DB-5 capillary column
(30 m, 0.25 mm ID, and 0.25 mm film thickness) or equivalent, and a
split/splitless injection port operating in the splitless mode, such
as an Agilent 6890 GC/5973 MS (or equivalent) equipped with an auto-
sampler for testing multiple samples; and
    5.2.12 Fixed Rotor Centrifuge.
    5.3 Reagents and consortium medium.
    5.3.1 Stock Seawater Preparation. Prepare the artificial
seawater GP2 (modified from Spotte et al., 1984) following the
procedures in section 2.3 of this Appendix, to obtain the final
concentration of the salts listed in Table 1 of this Appendix,
except for the sodium bicarbonate (NaHCO3) which is
prepared separately. Autoclave the artificial seawater. Filter
sterilize the concentrated solution of sodium bicarbonate through a
0.45 [mu]m membrane filter and add to the autoclaved and cooled
artificial seawater GP2 to obtain the final concentration listed in
Table 1 of this Appendix.
    5.3.2 Seawater for the positive control flasks. Prepare sodium
triphosphate (a.k.a., sodium tripolyphosphate)
(Na5P3O10), potassium nitrate
(KNO3), and ferric chloride hexahydrate (FeCl3
[middot] 6H2O) as a concentrated solution. Filter
sterilize through a 0.45 [mu]m membrane filter and add to autoclaved
artificial seawater to obtain the final nutrient concentrations
listed in Table 10 of this Appendix. Calibrate the pH meter at room
temperature (approximately 20-23 [deg]C) using commercial buffers of
pH 4.0, 7.0, and 10.0, as appropriate, prior to use. Adjust the pH
of the artificial seawater with concentrated hydrochloric acid (HCl)
or 10 normality sodium hydroxide (10 N NaOH), as appropriate.